Pulmonary Circulation 1:4 2011 by Pulmonary Vascular Research Institute

COVER PHOTO: : Illustration of a portion of an anatomically-based computational model indicating preferential distribution of pulmonary emboli within pulmonary arterial vessels. Vascular models are created using a combination of image data and computational algorithms using the software platform CMISS. Computational predictions of blood low are used to determine the most likely location of embolus occlusion (colours range from red=most likely to dark blue=least likely) and the impact on resultant pulmonary function. This image was digitally created using the open-source visualisation software Cmgui (www.cmiss.org/cmgui). Publication in previous issue, refer: Blood low redistribution and ventilation-perfusion mismatch during embolic pulmonary arterial occlusion. Burrowes KS, Clark AR, Tawhai MH. Pulm Circ. 2011 Jul;1(3):365-76. Credit: Kelly S Burrowes, Alys R Clark, and Merryn H Tawhai, University of Oxford and Auckland Bioengineering Institute; Illustration Credit: Kelly S Burrowes.

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Addresses Editorial Ofϔice Chicago, USA Pulmonary Circulation Editorial Of ice University of Illinois at Chicago Ms. Christina N. Holt Email: chris88@uic.edu Website: www.pvri.info Journal website: www.pulmonarycirculation.org Published by Medknow Publications and Media Pvt. Ltd. B5-12, Kanara Business Centre, Off Link Road, Ghatkopar (East) Mumbai – 400075, India Phone: 91-22-66491818 Email: publishing@medknow.com Website: www.medknow.com Printed by Dhote Offset Technokrafts Pvt. Ltd. Jogeshwari, Mumbai, India.

Pulmonary Circulation

ISSN 2045-8932, E-ISSN 2045-8940

An official journal of the Pulmonary Vascular Research Institute Editors-in-Chief Jason X.-J. Yuan, MD, PhD (Chicago, USA) Nicholas W. Morrell, MD (Cambridge, UK) Harikrishnan S., MD (Trivandrum, India)

Senior Editor

Executive Editor

Ghazwan Butrous, MD (Canterbury, UK)

Harikrishnan S., MD (Trivandrum, India)

Editors Kurt R. Stenmark, MD (Denver, USA) Kenneth D. Bloch, MD (Boston, USA) Stephen L. Archer, MD (Chicago, USA) Marlene Rabinovitch, MD (Stanford, USA) Joe G.N. Garcia, MD (Chicago, USA)

Stuart Rich, MD (Chicago, USA) Martin R. Wilkins, MD (London, UK) Hossein A. Ghofrani, MD (Giessen, Germany) Candice D. Fike, MD (Nashville, USA) Werner Seeger, MD (Giessen, Germany)

Sheila G. Haworth, MD (London, UK) Patricia A. Thistlethwaite, MD, PhD (San Diego, USA) Chen Wang, MD, PhD (Beijing, China) Antonio A. Lopes, MD, PhD (Sao Paulo, Brazil)

Scientiﾏ進c Advisory Board Robert F. Grover, MD, PhD (Denver, USA) E. Kenneth Weir, MD (Mineapolis, USA) Charles A. Hales, MD (Boston, USA)

Joseph Loscalzo, MD (Boston, USA) John B. West, MD, PhD, DSc (San Diego, USA) Magdi H. Yacoub, MD, DSc, FRS (London, UK)

Editorial Board Steven H. Abman, MD, USA Serge Adnot, MD, France Vera D. Aiello, MD, Brazil Almaz Aldashev, MD, PhD, Kyrgyz Republic Diego F. Alvarez, MD, PhD, USA Robyn J. Barst, MD, USA Evgeny Berdyshev, PhD, USA Michael A. Bettmann, MD, USA Jahar Bhattacharya, MD, PhD, USA Konstantin G. Birukov, MD, USA Murali Chakinala, MD, USA Navdeep S. Chandel, PhD, USA Richard N. Channick, MD, USA Hunter C. Champion, MD, USA Shampa Chatterjee, PhD, USA Xiansheng Cheng, MD, China Naomi C. Chesler, PhD, USA Augustine M.K. Choi, MD, USA Paul A. Corris, MD, UK David N. Corn ield, MD, USA Michael J. Cuttica, MD, USA Hiroshi Date, MD, PhD, Japan Regina M. Day, PhD, USA Steven M. Dudek, MD, USA Raed A. Dweik, MD, USA Yung E. Earm, MD, PhD, Korea Jeffrey D. Edelman, MD, USA Oliver Eickelberg, PhD, Germany C. Gregory Elliott, MD, USA Serpil Erzurum, MD, USA A. Mark Evans, PhD, UK Karen A. Fagan, MD, USA Barry L. Fanburg, MD, USA Harrison W. Farber, MD, USA Jeffrey A. Feinstein, MD, USA Jeffrey Fineman, MD, USA Patricia W. Finn, MD, USA Sonia C. Flores, PhD, USA Paul R. For ia, MD, USA Robert Frantz, MD, USA M. Patricia George, MD, USA Mark W. Geraci, MD, USA Stefano Ghio, MD, Italy Mark N. Gillespie, PhD, USA

Reda Girgis, MD, USA Mark T. Gladwin, MD, USA Mardi Gomberg-Maitland, MD, USA Andy Grieve, PhD, Germany Alison M. Gurney, PhD, UK Elizabeth O. Harrington, PhD, USA C. Michael Hart, MD, USA Paul M. Hassoun, MD, USA Abraham G. Hartzema, USA Akiko Hata, PhD, USA Jianguo He, MD, China Jan Herget, MD, PhD, Czech Republic Nicholas S. Hill, MD, USA Marius M. Hoeper, MD, Germany Eric A. Hoffman, PhD, USA Yuji Imaizumi, PhD, Japan Dunbar Ivy, MD, USA Jeffrey R. Jacobson, MD, USA Roger Johns, MD, PhD, USA Peter L. Jones, PhD, USA Naftali Kaminski, MD, USA Chandrasekharan C. Kartha, MD, India Steven M. Kawut, MD, USA Ann M. Keogh, MD, Australia Nick H. Kim, MD, USA Sung Joon Kim, MD, PhD, Korea James R. Klinger, MD, USA Stella Kourembanas, MD, USA Michael J. Krowka, MD, USA Thomas J. Kulik, MD, USA R. Krishna Kumar, MD, DM, India Steven Kymes, PhD, USA David Langleben, MD, Canada Timothy D. Le Cras, PhD, USA Normand Leblanc, PhD, USA Fabiola Leon-Velarde, MD, Peru Irena Levitan, PhD, USA Jose Lopez-Barneo, MD, PhD, Spain Wenju Lu, MD, PhD, China Roberto Machado, MD, USA Margaret R. MacLean, PhD, UK Michael M. Madani, MD, USA Ayako Makino, PhD, USA Asrar B. Malik, PhD, USA

Jess Mandel, MD, USA Michael A. Matthay, MD, USA Marco Matucci-Cerinic, MD, PhD, Italy Paul McLoughlin, PhD, Ireland Ivan F. McMurtry, PhD, USA Dolly Mehta, PhD, USA Marilyn P. Merker, PhD, USA Barbara O. Meyrick, PhD, USA Evangelos Michelakis, MD, Canada Omar A. Minai, MD, USA Liliana Moreno, PhD, USA Timothy A. Morris, MD, USA Kamal K. Mubarak, MD, USA Srinivas Murali, MD, USA Fiona Murray, PhD, USA Kazufumi Nakamura, MD, PhD, Japan Norifumi Nakanishi, MD, PhD, Japan Robert Naeije, MD, Belgium Viswanathan Natarajan, PhD, USA John H. Newman, MD, USA Andrea Olschewski, MD, Austria Horst Olschewski, MD, Austria Stylianos E. Orfanos, MD, Greece Ronald J. Oudiz, MD, USA Harold Palevsky, MD, USA Lisa A. Palmer, PhD, USA Myung H. Park, MD, USA Qadar Pasha, PhD, India Andrew J. Peacock, MD, UK Joanna Pepke-Zaba, MD, UK Nicola Petrosillo, MD, Italy Bruce R. Pitt, PhD, USA Nanduri R. Prabhakar, PhD, USA Ioana R. Preston, MD, USA Tomas Pulido, MD, Mexico Soni S. Pullamsetti, PhD, Germany Goverdhan D. Puri, MD, India Rozenn Quarck, PhD, Belgium Deborah A. Quinn, MD, USA J. Usha Raj, MD, USA Amer Rana, PhD, USA Thomas C. Resta, PhD, USA Ivan M. Robbins, MD, USA Sharon I. Rounds, MD, USA

Nancy J. Rusch, PhD, USA Tarek Safwat, MD, Egypt Sami I. Said, MD, USA Julio Sandoval, MD, Mexico Maria V.T. Santana, MD, Brazil Bhagavathula K. Sastry, MD., India Anita Saxena, MD, India Marc J. Semigran, MD, USA Ralph T. Schermuly, MD, Germany Dean Schraufnagel, MD, USA Paul T. Schumacker, PhD, USA Pravin B. Sehgal, MD, PhD, USA James S.K. Sham, PhD, USA Steven D. Shapiro, MD, USA Larisa A. Shimoda, PhD, USA Robin H. Steinhorn, MD, USA Troy Stevens, PhD, USA Duncan J. Stuart, MD, Canada Yuchiro J. Suzuki, PhD, USA Victor F. Tapson, MD, USA Merryn H. Tawhai, PhD, New Zealand Dick Tibboel, MD, PhD, The Netherlands Christoph Thiemermann, MD, PhD, UK Mary I. Townsley, PhD, USA Richard C. Trembath, MD, UK Rubin M. Tuder, MD, USA Carmine D. Vizza, MD, Italy Norbert F. Voelkel, MD, USA Peter D. Wagner, MD, USA Wiltz W. Wagner, Jr., PhD, USA Jian Wang, MD, USA Jian-Ying Wang, MD, USA Jun Wang, MD, PhD, China Xingxiang Wang, MD, China Jeremy P.T. Ward, PhD, UK Aaron B. Waxman, MD, USA Norbert Weissmann, PhD, Germany James D. West, PhD, USA R. James White, MD, USA Sean W. Wilson, PhD, USA Michael S. Wolin, PhD, USA Tianyi Wu, MD, China Lan Zhao, MD PhD, UK Nanshan Zhong, MD, China Brian S. Zuckerbraun, MD, USA

Editorial Staff Christina N. Holt (Chicago, USA), chris88@uic.edu Nikki Krol (London, UK), nkrol@imperial.ac.uk Karen Gordon (Chicago, USA), gordonk@uic.edu Pulmonary Circulation | October-December 2011 | Vol 1 | No 4

Pulmonary Circulation

| October-December 2011 | Vol 1 | No 4 |

An official journal of the Pulmonary Vascular Research Institute

CONTENTS LEFT TO RIGHT: 444, 451, 458

General Information

inside front cover

Editors and Board Members

Editorial No doctor is an island Jason X. -J. Yuan, Nicholas W. Morrell, S. Harikrishnan, and Ghazwan Butrous

435

Guest Editorial Re lections on the rise and fall of PVD, medical nanotechnology and Australia covered with house lies Paul Soderberg

437

Review Article Obesity and pulmonary arterial hypertension: Is adiponectin the molecular link between these conditions? Ross Summer , Kenneth Walsh, and Benjamin D. Medoff

440

Research Articles FDG PET imaging can quantify increased cellular metabolism in pulmonary arterial hypertension: A proof-of-principle study

Guy Hagan, Mark Southwood, Carmen Treacy, Robert MacKenzie Ross, Elaine Soon, James Coulson, Karen Sheares, Nicholas Screaton, Joanna Pepke-Zaba, Nicholas W. Morrell, and James H. F. Rudd

448

Signi icant intrapulmonary Schistosoma egg antigens are not present in schistosomiasis-associated pulmonary hypertension Brian B. Graham, Jacob Chabon, Angela Bandeira, Luciano Espinheira, Ghazwan Butrous and Rubin M. Tuder

456

High-altitude pulmonary hypertension in cattle (brisket disease): Candidate genes and gene expression pro iling of peripheral blood mononuclear cells John H. Newman, Timothy N. Holt, Lora K. Hedges, Bethany Womack, ShaĎ&#x201D;ia S. Memon, Elisabeth D. Willers, Lisa Wheeler, John A. Phillips III, and Rizwan Hamid ii

462

Pulmonary Circulation | October-December 2011 | Vol 1 | No 4

CONTENTS continued

Quantitative estimation of right ventricular hypertrophy using ECG criteria in patients with pulmonary hypertension: A comparison with cardiac MRI Kevin G. Blyth, James Kinsella, Nina Hakacova, Lindsey E. McLure, Adeel M. Siddiqui, Galen S. Wagner, and Andrew J. Peacock

470

475

487

Case Report Cardiopulmonary hemodynamic clues for pulmonary vein stenosis diagnosis Mateo Porres-Aguilar, Genaro Fernandez, and C. Gregory Elliott

499

History and Whoâ&#x20AC;&#x2122;s Who Lofty goals at high altitude: The Grover Conferences, 1984â&#x20AC;&#x201C;2011 E. Kenneth Weir, Wiltz W. Wagner Jr., and Stephen L. Archer

501

Author Index, 2011

508

Title Index, 2011

510 Si

Scientific Abstracts

LEFT TO RIGHT: 459, 464, 482 Pulmonary Circulation | October-December 2011 | Vol 1 | No 4

iii

Author Institution Mapping (AIM)

Please note that not all the institutions may get mapped due to non-availability of the requisite information in the Google Map. For AIM of other issues, please check the Archives/Back Issues page on the journalâ&#x20AC;&#x2122;s website. iv

Pulmonary Circulation | October-December 2011 | Vol 1 | No 4

Edi t ori al

No doctor is an island “A single conversation across the table with a wise man is better than ten years mere study of books.” —Henry Wadsworth Longfellow (1807-1882) What the 17th-Century English poet said about people in general, that “No man is an island, entire of itself; every man is a piece of the continent, a part of the main,”[1] is especially true for doctors and all the other people in all the medical professions. And on that “continent” called Medicine, the single most crucial medical tool is: conversations. Spoken words are like oxygen, so common as to be ignored but essential for life. A recent study found that, on average, people speak about 16,000 words per day.[2] But speaking is not at all the same as having a conversation. Professional organizations’ meetings often feature speakers—one person talking, multiple people listening. Conversations, in contrast, are interactive, everyone having the opportunity both to speak and to listen. In Medicine, the best meetings and conferences are conversation-based, not speakerbased. It is for precisely that reason that the Grover Conference is today the world’s most in luential scienti ic meeting focusing on the pulmonary circulation, gas exchange, and pulmonary vascular diseases (PVD). As E. Kenneth Weir, W. Wagner Wiltz Jr. and Stephen L. Archer point out in this issue of Pulmonary Circulation, in their historical review of the 15 Grover Conferences held in Colorado since the irst one in 1984, the Conference is named for a man who was, and remains, not only a pioneer and innovator in the profession, but also a master of the art of conversation: a mentor. Robert F. Grover “was also an inspirational mentor and the Conference celebrates both his scienti ic interests and his tradition of mentorship.” Signi icantly, the attendees at each Grover Conference include both leaders and beginners, both the “big names” of today and the young, promising investigators who, thanks in large part to conversations like those at such conferences, will be the big names of tomorrow. We welcome and salute the newest venue for the conversation-based advancement of our ield, the Pulmonary Hypertension Society of Australia and New Zealand (PHSANZ), whose members held their inaugural scienti ic meeting in Sydney on 25 November 2011. Signi icantly, 2 of the PHSANZ’s 4 mission goals have to do with people speaking and listening to each other: ongoing pulmonary hypertension education, to Pulmonary Circulation | October-December 2011 | Vol 1 | No 4

“expand the knowledge base of PH and the complexities of diagnosis and management of this group of patients”; and lobbying power, “to create a force that will have levels of in luence on many facets of PH management in Australia and New Zealand.”[3] (See the PHSANZ abstracts published in this issue of Pulmonary Circulation). How such societies come into existence is nicely illustrated by the case of the publisher of this journal, the Pulmonary Vascular Research Institute, the PVRI. It was in fact the result of a year-long conversation, on the need for such an organization, that began in London early in 2006, continued in San Diego in May 2006, and concluded in Malta in January 2007. At the original chat were 7 friends and colleagues, including Ghazwan Butrous, Ardeschir Ghofrani, Fritz Grimminger, Nick Morrell, Stuart Rich, and Martin Wilkins; they were joined by still more people in San Diego; and at Malta there were 25 experts from around the world. Since coming into existence, the PVRI has sponsored meetings throughout the world — India, China, Brazil, and nations in Africa and the Middle East — and has held annual conferences in Spain, Mexico, Portugal, Panama, and in 2012, Cape Town, South Africa. The success and ever -increasing popularity of all these meetings and conferences is due very largely to the fact that they feature discussions and debates, meaning conversations rather than speeches.The international collaboration initiated by the PVRI in supporting PVDrelated educational programs and research in the Third World, where the vast majority of people af licted with PVDs live, has assumed vital signi icance in controlling these diseases. By January 2007, in Malta, when the idea of the original conversation, the PVRI, had become a reality, a new conversation began. Led by Jason Yuan, the attendees discussed and debated the necessity of the PVRI having its own “voice” in print. This led irst to the launching in 2008 of the PVRI Review and then, only 3 years later, to the publication both of this journal, Pulmonary Circulation, aimed speci ically at the pulmonary circulation and Access this article online Quick Response Code:

Website: www.pulmonarycirculation.org DOI: 10.4103/2045-8932.93540

How to cite this article: Yuan JX, Morrell NW, Harikrishnan S, Butrous G. No doctor is an island. Pulm Circ 2011;1:435-6.

435

pulmonary vascular diseases, and a 1,690-page Textbook of Pulmonary Vascular Disease.[4] This trio of publications illustrate the crucial point that many of Medicine’s most important conversations occur in print. Indeed, the point was made literally in the “Welcome” editorial in the inaugural issue: “Pulmonary Circulation: A new venue for communicating your indings, ideas and perspectives,” the key word being “communicating.” Look at the references listed for each article in this journal, and what you are seeing, in fact, are the speakers to whom the article’s authors listened to before they began to speak in print themselves. The wonder and magic of such conversations is that they are not bound by time. For one example, all articles ever published on DNA may be viewed as contributions to the conversation started by James Watson and Francis Crick in their single-page article published 58 years ago.[5] For another example, it happens that 3 of the articles in this issue of Pulmonary Circulation have to do with high altitudes (Newman et al. on high-altitude PH in cattle; Pham et al. on high-altitude pulmonary edema; and the historical review by Weir et al., “Lofty goals at high altitude: The Grover Conferences, 1984–2011”), as do 7 abstracts (Abstracts 1.2, 2.3, 2.14, 2.17, 2.18, 2.19 and 2.20). In a mostly symbolic but actually profound sense, these 10 articles and abstracts are contributions to a conversation that began 351 years ago: in London on the evening of 12 November 1660, in what turned out to be the very irst meeting of what would become England’s most prestigious scienti ic institution, the Royal Society, one of the topics the 12 attendees discussed and debated— that is conversed about — was

436

whether a pendulum clock high on a mountain told the same time as one at sea level.[6] To facilitate further conversion, we present the Abstracts segment in this issue of Pulmonary Circulation. Submissions were received by physicians, physician scientists and investigators from the 2011 Grover Conference, the 5th Scienti ic Workshops and Debates of the Pulmonary Vascular Research Institute (PVRI), and the Inaugural Scienti ic Meeting of the Pulmonary Hypertension Society of Australia and New Zealand (PHSANZ). The international conversation among our scholars from different disciplines and across multiple geographic locations will contribute greatly to the global awareness of Pulmonary Vascular Disease.

Jason X. -J. Yuan, Nicholas W. Morrell, S. Harikrishnan, and Ghazwan Butrous Email: jxyuan@uic.edu

REFERENCES 1. 2. 3. 4.

5. 6.

Donne J. “Meditation XVII,” Devotions upon Emergent Occasions.1624. Mehl MR, Vazire S, Ramírez-Esparza N, Slatcher RB, Pennebaker JW. Are women really more talkative than men?Science2007;317:82. The new society’s other 2 mission goals are workforce sustainability and collaborative research. Available from: http://phsanz.com.au/. Yuan JX, Garcia JG, Hales CA, Rich S, Archer SL, West JB, editors. Textbook of Pulmonary Vascular Disease, New York: Springer Publishing; 2011. Watson JD, Crick FH. Molecular structure of nucleic acids: A structure for deoxyribose nucleic acid.Nature1953;171:737-8. Soderberg P. Chapter 3, Young Scientist Journeys,Canterbury UK: The Butrous Foundation;2010. p. 30.

Pulmonary Circulation | October-December 2011 | Vol 1 | No 4

Guest Edi t o r i al

Reflections on the rise and fall of PVD, medical nanotechnology and Australia covered with houseflies From a cultural anthropology perspective, the very near future of pulmonary vascular diseases (PVDs) seems as fantastic as their ancient past: They did not exist before, and they soon will cease to exist – possibly. Cultures all over the world had legends of the golden ages in which people had extreme lifespans. We are told that Devraha Baba of India was 513 years old when he died, that Methuselah of Zion lived 969 years, that Tiresias of Greece lived “more than” 600 years and PengZu of China 800 years, that Abdul al-Habashi of Arabia lived 674 years and 100 days, and so on around the world – all according to such legends. As Norris McWhirter, certainly an authority on extreme claims (he and his brother Ross authoring the irst Guinness Book of Records in 1955), wrote, “No single subject is more obscured by vanity, deceit, falsehood and deliberate fraud than the extremes of human longevity.”[1] What is interesting, however, is not the question of whether those extreme ages were literal or igurative years, but simply the fact that it was universally believed that people used to live far longer than they do now, which presumably would mean the absence of killer diseases, including PVDs (e.g., idiopathic pulmonary arterial hypertension, persistent pulmonary hypertension in the newborn, pulmonary embolism, chronic thromboembolic pulmonary hypertension and pulmonary hypertension associated with congenital heart defects, to name only a few). Intriguingly, there were numerous other legends about the arrival of diseases on this planet, such as the Greek one of Pandora: She was given a beautiful sealed jar and told never to open it. As curious as a scientist, she opened it, and all diseases and evils were released into the world. As might have been true in the ancient past, so might be true in the near future: The absence of PVD and other killer diseases enabling extreme human lifespans. There is this signi icant difference, though: For the past, the authorities were tribal mythmakers and shamans, but for the future the authorities are – not will be, but are – scientists, engineers and physicians. Sandhiva et al. state that devices enabled by nanotechnology “can be used to probe cellular movements and molecular Pulmonary Circulation | October-December 2011 | Vol 1 | No 4

changes associated with pathological states. Nanodevices like carbon nanotubes to locate and deliver anticancer drugs at the speci ic tumor site are under research. Nanotechnology promises construction of arti icial cells, enzymes and genes. This will help in the replacement therapy of many disorders that are due to de iciency of enzymes, mutation of genes or any repair in the synthesis of proteins.”[2] All this is quite recent. Only 13 years ago, Robert A. Freitas Jr., now a Senior Research Fellow at the Institute for Molecular Manufacturing in Palo Alto, California, authored the world’s irst detailed technical design study of a “respirocyte,” an arti icial red blood cell that will be able to store and transport 236-times more oxygen than natural red blood cells thus preventing ischemia, for example.[3] Freitas and others have since also begun work on “microbivores,” arti icial white blood cells that will attack pathogens far more effectively than do natural white blood cells. Clearly, in other words, “nanotechnology is spreading its wings to address the key problems in the ield of medicine.”[2] But, it has already “ lown the coop,” so to speak, into popular culture. On 27 December 2011, a thriller titled 77 Shadow Street was published: Dean Koontz’s “Pandora” version of a world in which medical nanodevices have run amok. Before the horrors crescendo in the novel, two characters discuss the new ield: “A great many scientists and ‘futurists’ believed that the day was fast approaching when human biology and technology would merge, when all diseases and genetic maladies would be cured and the human lifespan vastly extended by Biological Micro Electron Mechanical Systems (BioMEMS). These tiny machines, as small as or smaller than a human cell, would be injected by the billions into the bloodstream Access this article online Quick Response Code:

Website: www.pulmonarycirculation.org DOI: 10.4103/2045-8932.93541 How to cite this article: Soderberg P. Reflections on the rise and fall of PVD, medical nanotechnology and Australia covered with houseflies. Pulm Circ 2011;1:437-9.

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to destroy viruses and bacteria, to eliminate toxins, and to correct DNA errors, as well as to rebuild declining organs from the inside out. . . . They’re predicting nanoroboticaugmented blood by 2025, maybe 2030 at the latest. You know what’s going to happen if the lifespan of people goes up like to 300 years or something?”[4] Of course, the point is that, no, we do not know what would happen –and we should know. A single example from the insect world makes the point in a particularly graphic way that extending lifespans, in this case that of the common house ly, can have ghastly consequences. “If a pair of house lies mated and all their descendants lived and bred without any losses to predators, then within a single summer season there would be a million, million, million, million lies. That would be enough lies to cover the whole of Australia 11 m (36 ft) deep in lies.”[5] In, and of itself, longevity is a truly fascinating and complex topic. Why, for example, does a may ly rarely live more than 24 h? Why does one ocean creature, the worm-like gastrotrich, live a maximum of 3 days, while another ocean creature, the quahog (a bivalve mollusk) live up to 410 years? And, does the ultimate secret of longevity lie not in lesh and blood but in cellulose and chlorophyll, given the fact that the oldest living thing on Earth at present is a bristlecone pine tree in California, nicknamed “Methuselah,” which today has a veri ied age of 4,844 years? But more pressing, it seems, are accurate answers to this simple question: What might be the long-term consequences of tampering with a natural lifespan? In the United States today, you can hardly build a fence without irst compiling a detailed Environmental Impact Statement. Why are not detailed Social Impact Statements required for things like new devices that will “merge human biology with technology” so as to alter human lifespans? Perhaps the Ancient Roman adage for doctors, Primum non nocere, “First, do no harm,” should be amended after all these years to recognize nanotechnology: “First, do no harm. Second, think about the harm not doing harm might do.” In his introduction to Young Scientist Journeys, a book for scientists aged 12–20 years, Ghazwan Butrous coined the term “the Great Unknown” as the place where everything that remains to be discovered now exists, including “technological innovations that will make today’s cuttingedge marvels seem like blunt Stone Age implements.”[6] Today, those marvels seem to be doing precisely that at breakneck speed, which is why the world’s most proli ic science writer, Isaac Asimov (more than 500 books written 438

or edited), uttered this warning: “The saddest aspect of life right now is that science gathers knowledge faster than society gathers wisdom.”[7] The latter, society’s wisdom regarding science, is, ultimately, also the responsibility of scientists, which is excellent news. All that is required is for scientists to turn back the clock to circa 1960, or to regain their status in those days. Very brie ly, in the past 50-some years, scientists have gone, in the eyes of the nonscienti ic public, from Santas to suspects, from the bringers of great gifts for everyone, to the bearers of bad news for everyone; from the marvels of space light, Te lon and Tang, the just-add-water orange juice, to the experts who keep saying that this causes cancer, that is bad, this is destroying that, these things cause obesity, we will soon be entering an Ice Age, we are now in the Age of Global Warming, etc. ad nauseam. This transmogri ication, fueled by the media’s preference for bad news (“If it bleeds it leads”), has done two unfortunate things: It has turned the general public’s total trust in scientists into widespread distrust and it has given rise to “scientist impersonators,” a.k.a. “pseudo-scientists.” Thus, for example, in the minds of far too many people, a religious leader’s views on any given scienti ic issue or a conspiracy buff are just as valid as a Nobel Laureate’s. That is precisely why the Royal Society in 2002 hosted a forum on the topic: “Do We Trust Today’ Scientists?” The resounding answer then and since then has been: “No.” One expert, after studying North America, the UK, New Zealand and Japan stated: “It has been said that public trust in scientists, and indeed in science, is dwindling. People seemingly rely on beliefs rather than fact and in any case do not trust authoritative information. Instead they rely on information presented to them by groups of individuals with whom they share beliefs or ideologies.”[8] Another expert, after studying the United States, stated: “Nationally representative surveys conducted in 2008 and 2009 found signi icant declines in Americans’ climate change beliefs, risk perceptions and trust in scientists.”[9] In theory, nothing could be simpler: For scientists to regain their former authority on science issues requires only that all the scientist impersonators lose their authority. Yes, that simple theory would be complex to implement. But, scientists generally and medical experts speci ically thrive on solving complex problems. After all, they just came up with a microscopic robot blood cell that can store and transport 236-times more oxygen than a natural red blood cell.

REFERENCES 1.

McWhirter N, McWhirter R, editors. The Guinness Book of Records, London: Random House Publishing Group;1986. See also Young RD,

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2. 3.

4. 5. 6.

Desjardins B, McLaughlin K, Poulain M, Perls TT. Typologies of extreme longevity myths.CurrGerontolGeriatrRes2010;2010:423087. Sandhiya S, Dkhar SA, Surendiran A. Emerging trends of nanomedicine— an overview. FundamClinPharmacol2009;23:263-9. Freitas RA Jr. Exploratory design in medical nanotechnology: A mechanical artificial red cell. Artif Cells Blood SubstitImmobilBiotechnol1998;26: 411-30. Koontz D. 77 Shadow Street, New York: Bantam Books;2011. p.330-1. Wonderfully weird’s “Information on a fly”. Available from: Http://www. amazingflygun.com/facts.asp[Last accessed on 2011 Dec 28]. Butrous G. The Journeys Trilogy.Young Scientist Journeys, Canterbury UK: The Butrous Foundation;2010. p.11.

7. 8. 9.

Soderberg P. Your journey and the future,Young Scientist Journeys, Canterbury UK: The Butrous Foundation;2010. p.75. Jensen P. Public Trust in Scientific Information, Institute of Science and Technology Foresight, 14 September 2000. Leiserowitz AA, E.W. Maibachb, C. Roser-Renoufb, N. Smitha, E. Dawsonc. Climategate, Public Opinion, and the Loss of Trust.

Paul Soderberg Consulting Editor Email: paulsoderberg@hotmail.com

“Quick Response Code” link for full text articles The journal issue has a unique new feature for reaching to the journal’s website without typing a single letter. Each article on its first page has a “Quick Response Code”. Using any mobile or other hand-held device with camera and GPRS/other internet source, one can reach to the full text of that particular article on the journal’s website. Start a QR-code reading software (see list of free applications from http://tinyurl.com/ yzlh2tc) and point the camera to the QR-code printed in the journal. It will automatically take you to the HTML full text of that article. One can also use a desktop or laptop with web camera for similar functionality. See http://tinyurl.com/2bw7fn3 or http://tinyurl.com/3ysr3me for the free applications. Pulmonary Circulation | October-December 2011 | Vol 1 | No 4

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Review Ar ti cl e

Obesity and pulmonary arterial hypertension: Is adiponectin the molecular link between these conditions? Ross Summer 1, Kenneth Walsh2, and Benjamin D. Medoff 3,4 1

The Pulmonary Center, Boston University School of Medicine, Boston, MA, 2Whitaker Cardiovascular Institute/Molecular Cardiology, Boston University School of Medicine, Boston, MA, 3Center for Immunology and Inflammatory Diseases, 4Pulmonary and Critical Care Unit, Massachusetts General Hospital and Harvard Medical School, Charlestown, MA, USA

ABSTRACT Pulmonary arterial hypertension (PAH) is a condition of unknown etiology whose pathological features include increased vascular resistance, perivascular inflammatory cell infiltration and pulmonary arteriolar remodeling. Although risk factors for PAH are poorly defined, recent studies indicate that obesity may be an important risk factor for this condition. The mechanisms leading to this association are largely unknown, but bioactive mediators secreted from adipose tissue have been implicated in this process. One of the most important mediators released from adipose tissue is the adipokine adiponectin. Adiponectin is highly abundant in the circulation of lean healthy individuals, and possesses well-described metabolic and antiinflammatory actions. Levels of adiponectin decrease with increasing body mass, and low levels are directly linked to the development of PAH in mice. Moreover, overexpression of adiponectin has been shown to protect mice from developing PAH in response to inflammation and hypoxia. Based on the findings from these studies, it is suggested that the effects of adiponectin are mediated, in part, through its antiinflammatory and antiproliferative properties. In this review, we discuss the emerging evidence demonstrating a role for adiponectin in lung vascular homeostasis and discuss how deficiency in this adipocyte-derived hormone might explain the recent association between obesity and PAH. Key Words: adipocyte, adipokine, inflammation, metabolism, treatment of pulmonary hypertension

INTRODUCTION Pulmonary arterial hypertension (PAH) is a severe medical condition associated with a sustained elevation of pulmonary arterial pressure. PAH occurs as either a sporadic disease without identi iable risk factors or develops in association with other preexisting medical conditions such as connective tissue diseases, chronic infections (e.g., human immunode iciency virus) and cirrhosis.[1] To date, the pathogenesis of PAH remains poorly understood, but it is generally accepted that imbalances in vasodilator and vasoconstrictor substances and altered immune, growth and proliferative processes contribute signi icantly.[1] There are surprisingly few risk factors known to be associated with the development of PAH, but recent epidemiological data suggest that increased body mass Address correspondence to: Dr. Benjamin D. Medoff Pulmonary and Critical Care Unit, Massachusetts General Hospital, 55 Fruit Street, Bulfinch 148, Boston, MA 02114, USA Email: bmedoff@partners.org 440

index in luences the development of this condition.â&#x20AC;&#x2030;[2] While the mechanisms linking obesity to PAH are not well established, there is emerging data to suggest a pathogenic role for the adipocyte-derived hormone adiponectin in this process.[3-7] This review highlights the recent data related to adiponectin in lung vascular homeostasis and discusses the potential mechanisms by which hypoadiponectinemia might in luence the development of PAH.

Obesity as a risk factor for vascular disease

Obesity is a major public health problem, not least because of its association with increased mortality. The major cause of increased mortality in obese subjects is cardiovascular disease, as obese individuals are at Access this article online Quick Response Code:

Website: www.pulmonarycirculation.org DOI: 10.4103/2045-8932.93542 How to cite this article: Summer R, Walsh K, Medoff BD. Obesity and pulmonary arterial hypertension: Is adiponectin the molecular link between these conditions?. Pulm Circ 2011;1:440-7.

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greater risk for developing hypertension, atherosclerotic heart disease and stroke.[8,9] Despite the well-recognized association between obesity and systemic vascular diseases, evidence has only recently emerged for a similar relationship between obesity and diseases of the pulmonary circulation. These data suggest that increased body mass index is linked to the development of both acute and chronic pulmonary vascular diseases. For example, clinical studies have identi ied increased body mass index as a risk factor for the development of acute lung injury; a condition that results, in a large part, from a loss of endothelial cell barrier function. [10] In addition, several lines of evidence now suggest that obesity plays a pathogenic role in the development of PAH. Speci ically, autopsy studies indicate a higher prevalence of hypertensive changes in blood vessels of the pulmonary circulation of obese subjects when compared with nonobese historical controls.[11] Moreover, indings from the REVEAL registry, the largest pulmonary hypertension database in the United States, indicate a higher prevalence of overweight and obese individuals among those with idiopathic forms of PAH.[2] Notably, this association appears independent of conditions associated with the development of PAH (e.g., diastolic dysfunction, obstructive sleep apnea). Together, these indings suggest that obesity is a condition that globally disrupts vascular homeostasis and predisposes to the development of systemic and pulmonary vascular diseases.

Obesity and adipokines

Over the last several decades, there have been many important discoveries regarding the mechanisms that mediate the health-related consequences of obesity. One key inding has been the observation that obesity is a chronic in lammatory condition, and that persistent low-grade in lammation contributes signi icantly to the pathogenesis of obesity-related diseases. Adipose tissue is now recognized to be an important endocrine organ through its release of bioactive mediators, called adipokines, and chronic low-grade in lammation develops from obesity-driven imbalances in the secretion of proand antiin lammatory adipokines. In lean organisms, these molecules regulate biological processes important to energy homeostasis, in lammation and tissue remodeling. However, excess accumulation of body fat, as occurs in obesity, is associated with adipocyte dysfunction and the altered secretion of these hormones, which in turn contributes directly or indirectly to the development of obesity-related diseases.

Adiponectin is a multi-functional adipokine

Adiponectin is arguably the most important adipokine secreted from adipose tissue because of its pleiotropic actions in metabolism, immune regulation and vascular homeostasis. As suggested by its name, adiponectin is Pulmonary Circulation | October-December 2011 | Vol 1 | No 4

produced almost exclusively by adipocytes and is secreted into the plasma at a high concentration, where it is present at 3–30 μg/mL and accounts for up to 0.01% of the total plasma protein.[12] Circulating forms of adiponectin exist as high molecular weight oligomers, whose molecular weight can exceed 300 kDa, as well as hexamer and trimer structures.[13] The abundance of adiponectin and its ability to form several oligomeric fractions provide a possible explanation for why adiponectin has multiple functions. Circulating adiponectin levels are decreased in obesity,[12] type 2 diabetes,[14] metabolic syndrome[15] and a variety of cardiovascular diseases. [16-20] Conversely, plasma adiponectin levels are elevated by weight loss, [14] treatment with thiazolidinediones[21] and dietary ish oils.[22] At a cellular level, adiponectin production by adipocytes is impaired by oxidative and endoplasmic reticulum stress and activation by in lammatory cytokines that are prevalent in the adipose tissue of the obese.[23] However, it should be noted that adiponectin levels are elevated, rather than decreased, in a number of chronic in lammatory and autoimmune diseases.[24] The reason for this paradoxical behavior is unknown, but it could be the result of the compensatory regulatory mechanisms or development of an adiponectin-resistant state.[25] Relevant to this review, adiponectin functions to protect against the development of both metabolic and vascular diseases. The most convincing studies employ mouse genetic models that lack a functional adiponectin gene or transgenically overexpress adiponectin acutely or chronically. In addition, some studies have employed recombinant adiponectin protein preparations, but these experiments are confounded by variations in the quality and structural features of the adiponectin protein preparation obtained from a different source.[26] Overall, studies suggest that when compared with wild-type mice, adiponectinde icient mice are prone to metabolic and vascular diseases. For example, adiponectin-de icient mice develop a more severe insulin-resistant state when fed a high-calorie diet.[27,28] On the other hand, transgenic overexpression of adiponectin in obese ob/ob mice leads to a normalization of glucose despite a massive increase in fat depot size.[29] With regard to vascular disease, the majority of studies have focused on the systemic circulation. In these studies, adiponectin has been found to be protective in models of myocardial ischemia–reperfusion injury,[30] pressure overload cardiac hypertrophy,[31] cardiac remodeling associated with heart failure, [32,33] hypertension, [34] peripheral artery disease[35,36] and stroke.[37] At a cellular and molecular level, adiponectin appears to exert these effects by acting on a variety of cell types (Fig. 1). For example, adiponectin will confer an antiin lammatory phenotype in macrophages [38] and 441

Summer et al.: Obesity, PAH and adiponectin

vascular endothelial cells,[39] and stimulate catabolic pathways in liver and muscle.[40,41] These actions are mediated, in part, through the ability of adiponectin to activate intracellular signaling pathways via its interaction with the cell surface receptors AdipoR1 and AdipoR2.[42] However, as noted previously, adiponectin circulates at unusually high levels and it undergoes multimerization into high molecular weight complexes. These features suggest an atypical ligand–receptor interaction. In this regard, it has been recognized that adiponectin will bind to the GPI-anchored cell surface protein T-cadherin that localizes adiponectin to the vascular endothelium.[43] Denzel et al. recently showed in murine genetic models that T-cadherin is essential for adiponectin-mediated cardioprotection.[44] In addition, the structural similarity of adiponectin with the collectin family of proteins suggests that it may be functionally similar to this class of proteins as well. Consistent with this hypothesis, it has been shown that adiponectin can suppress in lammation by facilitating the uptake of apoptotic cell debris by macrophages, a property that is shared by other collectin proteins.[45]

Pathophysiological mechanisms of pulmonary arterial hypertension

The pathogenic mechanisms that lead to PAH are complex, but ultimately result in functional and structural changes to the pulmonary vasculature. Notably, in most forms of the disease, there is accumulation of immune and vascular cells (endothelial cells and pulmonary artery smooth muscle cells) within the arterial lumen, and this is associated with vascular remodeling and changes in vascular tone.[46] How adiponectin potentially modi ies these processes is discussed in the following sections.

Adiponectin is a modulator of vascular tone

Classically, PAH was thought to develop from an imbalance in vasodilator and vasoconstrictor substances. Although this is no longer thought to be the only pathogenic mechanism of PAH, increased vascular tone is often an important feature of this condition. In addition, most current therapies are directed at decreasing vascular resistance through either augmenting vasodilator activity or inhibiting the activity of vasoconstrictor substances. Adiponectin is known to have direct vasodilator activity [47- 49] and adiponectin de iciency is associated with the development of systemic hypertension and impaired vasodilation.[50] Adiponectin-de icient mice have also been found to have reduced levels of endothelial cell nitric oxide in the vascular wall and develop an age-dependent increase in pulmonary artery pressure when compared with wild-type mice.[51] These data strongly suggest that adiponectin de iciency may be associated with impaired vasoreactivity; however, there are no studies speci ically examining the effects of adiponectin-de iciency on the vasoreactivity of the pulmonary vasculature. 442

Figure 1: Effects of adiponectin on key cellular targets.

Adiponectin is a suppressor of inflammation There is a growing appreciation that pulmonary vascular in lammation is an important stimulus for the pathologic changes seen in various types of PAH in both human and animal models.[1,3,46,52] A role for in lammation in the pathogenesis of PAH is suggested by studies demonstrating the presence of increased levels of cytokines in patients with PAH[53,54] and the accumulation of macrophages and T cells in and around the remodeled vasculature of the lung. [55- 57] In addition, a recent study has demonstrated a strong correlation between cytokine levels and survival in PAH.[58] It is believed that chronic low-grade in lammation, as occurs in obesity, contributes to the development of PAH through promoting vascular remodeling. Activation of in lammatory pathways has been shown to stimulate endothelial cell activation, to promote pulmonary artery smooth muscle proliferation and to activate antiapoptotic pathways.[59,60] Indeed, the ability of in lammation to directly induce PAH has been demonstrated in animal models in which chronic in lammation alone was shown to promote pulmonary vascular remodeling and lead to elevated pulmonary artery pressures.[61-65] Overall, these studies support the hypothesis that in lammation is an important component of the pathogenesis of PAH and suggest that processes that suppress in lammation could have a therapeutic role in treating this disease. One important function of adiponectin is to tonically suppress vascular in lammation. This is exempli ied in adiponectin-de icient mice, which develop a spontaneous phenotype characterized by activated lung endothelium, age-dependent increases in perivascular in lammatory cell in iltration and elevated pulmonary artery pressures.[5] In addition, these mice develop an exaggerated eosinophilic vascular response to allergic lung in lammation, which Pulmonary Circulation | October-December 2011 | Vol 1 | No 4

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is associated with increased pulmonary artery pressure and muscularization of the pulmonary vasculature.[4] Interestingly, elimination of eosinophils in this model prevents the development of PAH in the adiponectinde icient mice.[6] Thus, indings in these studies provide further support for the concept that in lammation is an important stimulus for PAH and that adiponectin’s ability to modulate in lammatory responses may be in luential in the development of this condition.

Adiponectin is a suppressor of growth and proliferation

As previously discussed, a characteristic pathological feature of PAH is narrowing and/or obliteration of the vessel lumen due to thickening of the vascular wall. In large muscular arteries, this is usually secondary to medial hypertrophy, while smaller pulmonary arteries and precapillary vessels can be obliterated by plexiform lesions. [46] These changes have been attributed to increased proliferation and migration of mesenchymal cells that stain positive for a-smooth muscle cell actin (-SMA), indicating that there is dysregulation of local smooth muscle cells (SMCs) or myo ibroblast growth.[1] Alternatively, these cells could derive from circulating progenitor cells, but data supporting this hypothesis is less robust.[66,67] Regardless of the cellular origin, these cells are presumably stimulated to divide in response to mitogenic stimuli. Currently, the particular mitogenic substances that are most important in mediating the progression of PAH have yet to be identi ied. However, clinical studies have shown that concentrations of multiple growth factors are increased in lung biopsy specimens of patients with PAH. [68-72] For example, transcript and protein levels for platelet-derived growth factor (PDGF), epidermal growth factor (EGF) and vascular endothelial growth factor (VEGF) are increased in distal pulmonary arteries of patients with PAH. Moreover, various experimental studies have shown these factors to be important in promoting SMC proliferation and in augmenting resistance through their effects on cell survival.[68-72] While each of these factors is likely to be important, the search for other mitogenic substances that contribute to the development of PAH is ongoing. Although adiponectin is not a well-described factor in growth regulation, there is a growing appreciation for its effects on tissue remodeling.[73-75] In vitro, adiponectin suppresses vascular SMC proliferation and migration,[76] and in vivo, adiponectin-de icient mice have increased accumulation of SMCs in vessel walls following vascular injury.[77] In addition, adiponectin-de icient mice have increased cardiac remodeling with pressure overload and larger infarct size following cardiac ischemia when Pulmonary Circulation | October-December 2011 | Vol 1 | No 4

compared with wild-type mice.[74,78] As mentioned above, adiponectin-de icient animals develop more prominent pulmonary vascular remodeling in the setting of pulmonary vascular in lammation, and similar indings have also been reported in a model of hypoxia-induced PAH. These indings suggest a general and robust effect of adiponectin on pulmonary artery remodeling.[79] The mechanisms mediating adiponectin’s inhibitory actions on cell proliferation are poorly understood; however, these actions appear to be independent of its effects on in lammation.[7] In a recent study, it was demonstrated that overexpression of adiponectin reduced pulmonary vascular remodeling in an in lammation-induced model of PAH without reducing vascular in lammation.[7] In addition, adiponectin has been shown to directly affect several signaling pathways in SMCs important for cell proliferation and growth. The intracellular signaling mechanisms that regulate the SMC phenotype are poorly understood, and likely involve several different signaling cascades.[80] Growth factors such as PDGF, EGF and ibroblast growth factor (FGF) stimulate SMC proliferation in part through phosphorylation of PKB (AKT1) and effects on the mTOR pathway, which stimulates cell growth and proliferation. [81,82] Adiponectin has been shown to inhibit growth factor-mediated activation of mTOR via AMPK activation.[75,83] In addition, adiponectin has been shown to directly bind to growth factors thus modulating their activity by controlling their bioavailability at a prereceptor level.[76,84] Overall, these data suggest that adiponectin may suppress vascular remodeling via a complex set of mechanisms. The increased numbers of muscle cells seen in pulmonary hypertension are likely derived from existing SMCs that migrate around the vessel, proliferate and then form new muscle.[85,86] For this to occur, the SMC must irst dedifferentiate into a highly proliferative phenotype prior to migration and proliferation, and then later differentiate into a contractile phenotype to form new muscle. Transcription factors such as GATA6, MEF2 and the serum response factor–serum response element (SRF-SRE) pathway regulate the SMC phenotype. SRF is a phylogenetically conserved MADs-box transcription factor that binds a 10-base pair DNA sequence (CC[A/T] 6GG or CArG box) and mediates the rapid transcriptional response to growth and differentiation signals. SRF is one of the major regulators of SMC growth, migration, survival and differentiation,[87-90] controlling the expression of more than 200 genes, nearly half of which are involved in cytoskeletal dynamics and cellular contractility. [91] Deletion of SRF in mice is lethal,[92] and SMC-speci ic deletion leads to impaired vascular SMC 443

Summer et al.: Obesity, PAH and adiponectin

differentiation in the embryo.[93] Consistent with this, expression of SMC-speci ic genes, such as SM-myosin heavy chain, SM - and -actin, SM22, SM-myosin light chain kinase, calponin, -actinin and smoothelin-A, are downregulated in the absence of SRF. [94,95] Because the SRF-SRE pathway is the major regulator of SMC growth and differentiation,[87,88] factors that affect it are likely to have a profound effect on the differentiation state of SMCs and may in luence vascular remodeling in the lung. Recent data has demonstrated that adiponectin can reduce SRF-SRE activity, [7] and thus could work to limit SMC growth and proliferation by effecting the differentiation of SMCs into a proliferative phenotype. These data suggest that adiponectin may also inhibit pulmonary vascular remodeling by modulating the growth phenotype of SMCs.

Adiponectin is a metabolic hormone

Several indings suggest that metabolism may in luence the pathogenesis of pulmonary hypertension. Patients with pulmonary hypertension have reduced expression of PPAR in the lung, a receptor that regulates adiponectin and insulin resistance. [96] In addition, mice with a targeted deletion of PPAR in SMCs spontaneously develop pulmonary hypertension with muscularization of distal pulmonary arteries. [97] Furthermore, apoEde icient mice on a high-fat diet develop PAH. It has also been shown that insulin resistance and dyslipidemia are more common in women with PAH and that insulin resistance was associated with worse outcomes in these patients.[98] Adiponectin is an important modulator of metabolism leading to improved insulin sensitivity and decreased glucose and free fatty acid levels in the plasma. Speci ically, adiponectin stimulates β-oxidation and downregulates the expression of mediators of lipid synthesis. [99] Furthermore, in animal models, ectopic expression of adiponectin promotes metabolic function independent of body mass.[100] At least some of the bene icial effects of the PPAR agonist class of drugs seems to be related to their ability to increase adiponectin levels.[99] In fact, a recent study has demonstrated that the cardioprotective effects of PPAR agonists are dependent on their ability to increase adiponectin levels.[101] In accordance with this, male apoE-de icient mice on a high-fat diet do not upregulate adiponectin, but develop insulin resistance and PAH.[96] However, female apoE-de icient mice on a high-fat diet had increased adiponectin levels at baseline and did not develop insulin resistance and had less PAH. Treatment of these mice with the PPAR agonist rosiglitazone (which increases adiponectin levels) attenuates the PAH,[96] suggesting that the link between metabolism and PAH may relate in part to changes in adiponectin levels. 444

CONCLUSION AND CLINICAL IMPLICATIONS There is increasing data suggesting that obesity may be a risk factor for PAH independent of its effects on systemic vascular disease and obesity–hypoventilation syndrome. [102] Although the data does not support a primary role for obesity in causing PAH, the data does suggest that the effects of obesity on metabolism and vascular inflammation could contribute to the development of pulmonary vascular remodeling and PAH. Thus, it is possible that obese patients with PAH from other causes could have more rapid progression and more severe disease than lean patients. Furthermore, as PAH is associated with reduced exercise capacity, many patients may become obese overtime due to an inability to exercise and thus could have accelerated disease due to the effects of obesity on the pulmonary vasculature. Clearly, more research is needed on the exact role obesity may have on the pathogenesis of PAH. Given the known effects of adiponectin on vascular in lammation and remodeling (Fig. 2), it seems likely that relative de iciency of adiponectin as seen in obesity could be an important mechanistic link between obesity and PAH. Animal models demonstrate that adiponectin can modulate pulmonary vascular in lammation and remodeling, which then directly in luences the development of PAH. Whether there is a similar effect in humans with PAH is unknown at this time, but the data does suggest that measures to augment adiponectin levels could have therapeutic value in patients with PAH, especially those with obesity and insulin resistance. It should be noted that such therapy is already available with the thiazolidinedione class of antidiabetic drugs (such as pioglitazone), which increase adiponectin secretion by stimulating PPAR. Based on the

Figure 2: Effects of adiponectin in the pulmonary vasculature. Pulmonary Circulation | October-December 2011 | Vol 1 | No 4

Summer et al.: Obesity, PAH and adiponectin

available data, a trial of these agents in PAH patients with obesity may be warranted.

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Source of Support: Nil, Conflict of Interest: None declared.

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Research Ar t i cl e

FDG PET imaging can quantify increased cellular metabolism in pulmonary arterial hypertension: A proof-of-principle study

Guy Hagan1, Mark Southwood1, Carmen Treacy1, Robert MacKenzie Ross1, Elaine Soon2, James Coulson3, Karen Sheares1, Nicholas Screaton4, Joanna Pepke-Zaba1, Nicholas W. Morrell2, and James H. F. Rudd2 1 Pulmonary Vascular Disease Unit, Papworth Hospital, Papworth Everard, 2Division of Cardiovascular Medicine, University of Cambridge, Cambridge, 3Wales Heart Research Institute, Cardiff University, Cardiff, 4Department of Radiology, Papworth Hospital, Papworth Everard, United Kingdom

ABSTRACT The past decade has seen increased application of 18-flurodeoxyglucose positron emission tomography (18FDG-PET) imaging to help diagnose and monitor disease, particularly in oncology, vasculitis and atherosclerosis. Disordered glycolytic metabolism and infiltration of plexiform lesions by inflammatory cells has been described in idiopathic pulmonary arterial hypertension (IPAH). We hypothesized that increased 18FDG uptake may be present in the lungs, large pulmonary arteries and right ventricle of patients with pulmonary hypertension, and that this uptake would be related to markers of immune activation. We imaged the thorax of 14 patients with pulmonary hypertension (idiopathic and chronic thromboembolic) and six controls by 18FDG-PET/computed tomography (CT) and measured uptake in the lung parenchyma, large pulmonary arteries and right ventricle. 18FDG uptake in the lungs and pulmonary arteries was normalized for venous blood activity to give a target-to-background ratio (TBR). Blood was contemporaneously drawn for high-sensitivity CRP - C-reactive protein (CRP) (hsCRP), N-Terminal Probrain natriuteric peptide (NT-ProBNP) and other inflammatory cytokines. IPAH patients had significantly higher lung parenchymal TBR (P =0.034) and right ventricle FDG uptake (P =0.007) than controls. Uptake in the main pulmonary arteries was similar in chronic thromboembolic pulmonary hypertension, IPAH and controls. There were no correlations between 18FDG uptake and hsCRP or inflammatory cytokine levels. NT-ProBNP correlated with RV uptake in those with pulmonary hypertension (r=0.55, P =0.04). In this pilot study, we found increased 18FDG uptake in the lung parenchyma and right ventricle of subjects with IPAH. The lung uptake might be useful as a surrogate marker of increased cellular metabolism and immune activation as underlying mechanisms in this disease. Further evaluation of the impact of targeted therapies in treatment-naïve patients and the significance of right ventricular uptake is suggested. Key Words: inflammation, pulmonary artery, pulmonary arterial hypertension, positron emission tomography, right ventricle

INTRODUCTION Idiopathic pulmonary arterial hypertension (IPAH) is a condition associated with endothelial dysfunction, small pulmonary artery smooth muscle cell and ibroblast proliferation, and in situ thrombosis. These changes result in an elevated pulmonary vascular resistance (PVR), which may progress to failure of the right ventricle and death. A large contribution to the PVR comes from small precapillary “resistance” arteries.[1] The etiology of IPAH Address correspondence to: Dr. James H. F. Rudd Box 110, Level 6, ACCI, Addenbrooke’s Hospital, Hills Road, Cambridge, CB2 0QQ, United Kingdom Email: jhfr2@cam.ac.uk 448

is unclear, with abnormalities found in many cellular pathways[2] as well as heritable cases being caused by mutations in the bone morphogenetic protein receptor type II (BMPR- II) gene.[3] Pulmonary hypertension may be associated with other conditions, such as chronic thromboemboli in the Access this article online Quick Response Code:

Website: www.pulmonarycirculation.org DOI: 10.4103/2045-8932.93543 How to cite this article: Hagan G, Southwood M, Treacy C, Ross RM, Soon E, Coulson J, et al. 18FDG PET imaging can quantify increased cellular metabolism in pulmonary arterial hypertension: A proof-ofprinciple study. Pulm Circ 2011;1:448-55.

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Hagan et al.: PET imaging in PAH

pulmonary arteries (chronic thromboembolic pulmonary hypertension (CTEPH)).[4]

patients, with the uptake ascribed to increased vascular endothelial glycolytic activity.

There is mounting evidence that immune dysregulation and possibly in lammation play a role in IPAH. Patients with IPAH have elevated serum levels of the proin lammatory cytokines interleukin (IL)-1 and IL6[5] (5) and C-reactive protein[6] in comparison with healthy controls. Recently, elevated levels of ILs 6, 8, 10 and 12p70 have been shown to predict survival in patients with pulmonary arterial hypertension.[7] The source of production of these cytokines in pulmonary hypertension is largely unknown, but may be the lungs. [8] In iltrates of T-cells, B-cells and macrophages are seen in the plexiform lesions often found in IPAH.[9] The role of immune dysregulation in CTEPH is less clear. Raised serum levels of the proin lammatory cytokines IL-10 and tumor necrosis factor alfa (TNFα) have been described in CTEPH.[10]

The past decade has seen the development of imaging techniques able to detect and track changes in vascular in lammation.[11,12] There has been particular interest in the use of 18- lurodeoxyglucose positron emission tomography (18FDG-PET), an imaging technique that is sensitive to cellular processes that metabolize glucose. 18FDG is taken up into cells via glucose transporter proteins, and phosphorylated to 18FDG-6-phosphate, which cannot be metabolized further along the glycolytic pathway and accumulates within cells in proportion to their glucose uptake (and hence metabolic activity). While commonly used in oncology to identify primary and metastatic tumor cells, several other cell types may also demonstrate high levels of 18FDG accumulation, including activated leucocytes,[13] lymphocytes[14] and ibroblasts. [15] Increased 18FDG uptake has been demonstrated in animal models of in lammation[16] as well as a range of human in lammatory conditions including abscesses,[17] tuberculosis,[18] sarcoidosis[19] and large vessel vasculitis. [20] 18 FDG uptake in atherosclerosis of the aorta and carotid and vertebral arteries at PET imaging correlates with the presence of vascular symptoms,[21] risk factor load [22] and in lammatory biomarker levels.[23] Such imaging might have a role in monitoring the response of atherosclerosis to therapy.[24] Several human cancer cell lines have high mitochondrial membrane potentials and low expression of the Kv1.5 potassium channel, which may lead to an apoptosisresistant phenotype and a switch to glycolytic metabolism;[25] these energy-handling abnormalities have also been described in IPAH.[26,27] Increased glycolytic metabolism is present in endothelial cells derived from IPAH-transplant explants;[28] this study also described higher 18FDG lung uptake on PET scan than normal control Pulmonary Circulation | October-December 2011 | Vol 1 | No 4

FDG-PET imaging of the heart in PAH shows excessive FDG uptake within the wall of the pressure-overloaded right ventricle, with the degree of uptake correlating with pulmonary hemodynamics.[29]

Given the possible pathological role of in lammation in idiopathic PAH and CTEPH, the presence of deranged endothelial cell bioenergetics and the recent development of techniques allowing the imaging of vascular in lammation, we hypothesized that increased 18FDG uptake in the large “conduit” pulmonary arteries, lung parenchyma and right ventricle would be present in subjects with pulmonary hypertension compared with controls. We also determined whether 18FDG uptake correlated with systemic in lammation as evaluated by levels of relevant in lammatory cytokines.

MATERIALS AND METHODS Study population

Outpatients attending our national pulmonary hypertension referral service at Papworth Hospital, Cambridge, UK, with a diagnosis of IPAH or CTEPH with a distal distribution of disease were recruited. All patients met recognized diagnostic criteria.[30] Exclusion criteria were a diagnosis of connective tissue, diabetes, intercurrent infection or in lammatory lung disease (e.g., bronchiectasis, emphysema). All patients gave written informed consent and the study was approved by the Cambridgeshire Regional Ethics Committee. The comparator group was comprised of a group of healthy ex-smokers (median smoking history of 10 pack years) without chronic obstructive pulmonary disease or pulmonary hypertension from a recent imaging study. [31] Sample sizes were based on previous vascular PET/ computed tomography (CT) studies and powered to show differences in uptake between vascular beds.[31,32]

FDG-PET/CT

Imaging was performed using a PET/CT system (GE Discovery 690). All subjects fasted for 6 h before imaging; 222-239 MBq (average 232 MBq) of 18FDG was injected intravenously after which subjects rested in a quiet room. PET/CT acquisition began 85–96 min after injection. PET acquisition was for 20 min over the mediastinum to include the pulmonary arteries and right ventricle. Low-dose CT of the thorax was performed immediately prior to the PET to allow anatomic colocalization and attenuation correction of PET data. No CT contrast was administered. Attenuation-corrected PET images 449

Hagan et al.: PET imaging in PAH

were iteratively reconstructed using “Time of Flight” information.[33] PET/CT image analysis was performed using Osirix version 3.7.1 (Osirix Imaging Software, Geneva, Switzerland). The ascending aorta, main pulmonary arteries and pulmonary trunk, lung parenchyma and right and left ventricles were analyzed, described in detail below. Regions of interest were placed by one reader blinded to the diagnosis (GH) and reviewed independently by two readers prior to the inal analysis. Mean and maximum body weight-corrected standardized uptake values (SUV) were calculated from the pixel activity within each region of interest (ROI). Where appropriate, SUVs were normalized to blood 18FDG activity by dividing the average SUV by the uptake of superior vena cava (SVC) blood to give a blood-corrected SUV, known as a target-to-background ratio (TBR), a validated technique for vascular PET imaging.[34]

Ascending aorta

The ascending aorta was identi ied from anatomic landmarks on CT. As previously described, [31] the maximum SUV was measured from a circular ROI placed around the lumen on each transaxial slice (approximately 10 per subject), from which TBR was derived.

Large pulmonary arteries

We were unable to ind published methods for determining 18FDG uptake in the pulmonary arteries on PET/CT. We therefore adapted methods validated in atherosclerosis imaging studies.[23,24] The pulmonary trunk and right and left main pulmonary arteries were included in the analysis and identi ied from anatomic CT landmarks. The region bordered inferiorly by the right ventricular out low tract to the superior limit of the pulmonary trunk was included, and ROIs drawn around the pulmonary trunk to the distal limit of the main pulmonary arteries on each transaxial slice. An average SUV max and TBR were derived.

Lung parenchyma

ROIs of radius 1 cm were drawn in peripheral lung locations, away from obvious pulmonary vessels in the anterior, middle and posterior sections of each lung (to minimize any effects of gravitational pooling of the blood pool and lung heterogeneity) on a transaxial slice taken at the level of the carina. ROI placement was validated by a thoracic radiologist blinded to the patient group (NS). SUV max was measured from which the TBR was derived as per published methods.[35,36]

Right ventricle

As previously described,[29] we placed a number ( ive to 14) of circular ROIs on each of the RV and LV free walls of 450

a transaxial image to obtain an averaged mean SUV. We compared 18FDG uptake in the right and left ventricles using the ratio method of Kluge.[37]

Biomarkers

Immediately prior to PET/CT imaging, venous blood was drawn for high-sensitivity C-reactive protein (hsCRP), N-Terminal Probrain natriuteric peptide (NT-ProBNP) and in lammatory cytokine analyses. NT-ProBNP was analyzed using a Siemens Dimension Clinical chemistry system and hsCRP using a Dade Behring immunonephelometry assay. Blood for in lammatory cytokines was centrifuged for 10 min at 3,000 revolutions per minute, and the serum supernatant removed and stored at -85°C for analysis. Serum levels of in lammatory cytokines (IL-1β, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12, IL-13, interferon [IFN]-γ and TNFα) were measured using a high-sensitivity cytokine multiplex assay and a multiplex analyzer following the manufacturer’s protocols.

Statistics

One-way ANOVA was used for comparisons among the three groups. When a P-value under 0.05 was found, Tukey’s post test comparison was performed between individual groups. For comparison between two groups, an unpaired Student’s t test was used if the data were normally distributed; otherwise, the Mann–Whitney test was used. Associations between variables were tested using Pearson’s correlation for normally distributed values and Spearman’s rank correlation for nonnormally distributed data. The intraclass correlation coef icient (ICC) was used to ascertain intrareader reproducibility. Analysis was performed using GraphPad Prism software (Version 5.02). Statistical signi icance was set at the 5% level.

Intrareader reproducibility

Six scans were reread after 3 months to estimate the intrareader variability. There was no signi icant difference between results, and the TBR values were highly reproducible (intraclass correlation coef icient for aorta: 0.95, large pulmonary arteries: 0.99).

RESULTS Demographic, imaging and biochemical data for all three groups are summarized in (Table 1). Eight IPAH patients, six CTEPH patients and six comparator subjects were enrolled: Seven/eight idiopathic PAH and ive/six distal CTEPH patients were receiving PAH-targeted therapy (phosphodiesterase-5 inhibitor, endothelin antagonist or prostanoid). There was no difference in the body mass index or 18FDG circulation time among the three groups. No IPAH patients carried a BMPR2 mutation. Pulmonary Circulation | October-December 2011 | Vol 1 | No 4

Hagan et al.: PET imaging in PAH

PET and fused PET/CT images of the right ventricle and the pulmonary trunk of an IPAH subject are shown in (Fig. 1).

parenchymal TBR max than the comparator group (P=0.034). There were no overall differences between the CTEPH group and either the comparator or the idiopathic PAH group (Fig. 4).

Large vessel uptake

LV uptake, RV uptake and RV/LV ratio

There were no signi icant differences in 18FDG uptake (expressed as TBR) in the ascending aorta (Fig. 2) or in the main pulmonary arteries between the IPAH, CTEPH and comparator groups (Fig. 3).

There was no difference in LV uptake among the three groups. Both RV uptake (Fig. 5) and the RV/LV ratio were signi icantly higher in idiopathic PAH patients than in the comparator group (P=0.007 for RV, P=0.017 for RV/LV). There were no differences between the CTEPH group and the other two groups.

Lung parenchymal uptake

Idiopathic PAH patients had a signi icantly higher lung

Table 1: Demographics, blood and positron emission tomography results for the three groups n Male (%) Age (mean±SD) NYHA class (I/II/III/IV) NT-ProBNP (pg/mL) hsCRP (mg/L) Cytokines (pg/mL) IL-1β IL2 IL4 IL5 IL6 IL7 IL8 IL10 IL12p70 IL13 IFN-γ GMCSF TNF-α PET/CT uptake Ascending aorta TBR (max) Pulmonary artery TBR (max) Lung parencymal TBR (max) Right ventricle mean SUV

IPAH

CTEPH

Comparators

8 3 (37.5) 40.4±5.5 1/3/4/0 1272.49±951.95 1.86±1.63

6 3 (50) 67.3±7.7 0/3/3/0 1044.23±984.12 1.71±1.26

6 6 (100) 65.3±5.7 N/A N/A N/A

0.06±0.11 0.23±0.34 1.83±4.17 0.2±0.31 3.85±3.52 5.42±2.6 3.99±1.4 14.06±10.09 2.79±5.39 0.06±0.09 1.99±2.2 0.3±0.61 8.8±10.06

0.03±0.05 0.02±0.04 1.21±2.24 0.04±0.07 3.25±3.03 4.89±1.98 3.68±0.89 8.4±2.53 0.13±0.28 0.03±0.06 0.09±0.1 0.03±0.03 6.02±3.37

N/A N/A N/A N/A

2.32±0.24 2.27±0.35 0.67±0.12* 4.78±2.41†

2.14±0.21 2.06±0.22 0.54±0.09 2.91±1.62

2.4±0.42 2.28±0.2 0.53±0.1 1.29±0.39

Values given as mean±SD; *P<0.05 compared with the comparator group; †P<0.01 compared with the comparator group; IPAH: idiopathic pulmonary arterial hypertension; CTEPH: chronic thromboembolic pulmonary hypertension; PET/CT: positron emission tomography/ computed tomography; TBR: target-to-background ratio; SUV: standardized uptake values

Figure 1: Positron emission tomography (PET) and fused PET/computed tomography of the right ventricle and pulmonary trunk of an idiopathic pulmonary arterial hypertension subject. Pulmonary Circulation | October-December 2011 | Vol 1 | No 4

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Figure 2: Ascending aorta target-to-background ratio. No significant difference is present among the three groups.

Figure 3: Large pulmonary artery target-to-background ratio. No significant difference is present among the three groups.

Figure 4: Lung parenchymal max target-to-background ratio. Idiopathic pulmonary arterial hypertension lung had a significantly higher uptake compared with controls.

Figure 5: Right ventricle standardized uptake values. Idiopathic pulmonary arterial hypertension patients had significantly higher right ventricular uptake than controls.

SVC venous blood uptake

did not correlate with lung parenchymal or large vessel (aorta or pulmonary) uptake in either group. There was no correlation between large pulmonary artery TBR, lung parenchymal TBR or RV uptake and hsCRP or any of the in lammatory cytokines in either group.

There was no signi icant difference between venous blood uptake among the three groups.

Correlation between uptake in RV, pulmonary arteries and lung parenchyma The RV uptake did not correlate with lung parenchymal uptake or pulmonary artery uptake in any group. Pulmonary artery uptake correlated with lung parenchymal uptake for both the IPAH group (r=0.81, P=0.0153) and the combined IPAH and CTEPH groups (r=0.71, P=0.0039) and also in the comparator group(r=0.91, P=0.012).

Correlation with inflammatory cytokines and NT-ProBNP Cytokine measurements were not available for the comparator group. The levels of hsCRP, cytokines or NT-ProBNP did not differ signi icantly between the IPAH and the CTEPH groups. There was an association (r=0.55, P=0.04) between NT-ProBNP and RV uptake for the combined IPAH and CTEPH groups. NT-ProBNP levels 452

DISCUSSION To our knowledge, this is the irst study to attempt to measure endothelial metabolism, in lammatory cell activity or vascular remodeling using the surrogate of 18FDG uptake in the pulmonary arteries in pulmonary hypertension. In this pilot prospective imaging study, we demonstrated that lung 18FDG uptake, expressed as TBR, is higher in patients with idiopathic PAH than the comparator or CTEPH group. RV uptake was also higher in our IPAH patients than comparators. Correlations between lung parenchymal uptake and pulmonary artery uptake were present. There was no difference in the uptake in the large pulmonary arteries or aorta among the three groups. The absence of signi icantly increased uptake in Pulmonary Circulation | October-December 2011 | Vol 1 | No 4

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the large pulmonary arteries suggests that these processes are con ined to the distal vessels in this disease. Our results are concordant with previously published studies. Increased lung parenchymal 18FDG uptake in IPAH was reported in a smaller study (n=4).[28] A positive correlation between adverse pulmonary hemodynamics and RV 18FDG uptake has been described.[29] The RV uptake in our pulmonary hypertension group (IPAH and CTEPH) correlated with NT-ProBNP. We did not examine correlations between right heart catheter or echocardiographic indices and 18FDG uptake as there was a time interval of a few months between the hemodynamic investigations and the PET/CT scan for most patients. We found no correlation between 18FDG uptake in any region and in lammatory cytokine or hsCRP levels. As RV uptake in IPAH correlates with hemodynamics,[29] and little or no correlation exists between in lammatory cytokines or hsCRP and hemodynamics,[5,6,7] a correlation between RV uptake and in lammatory cytokines may not be expected. The reasons for the increased lung parenchymal TBR in IPAH are unclear. Increased glycolytic metabolism of pulmonary artery endothelial cells has been proposed. [28] Rapidly dividing cells, including leucocytes [13] and ibroblasts,[15] have also increased glucose uptake and, therefore, lung TBR may be a surrogate marker of pulmonary vascular remodeling. In lammatory cells in plexiform lesions may also represent a source of 18FDG uptake. The correlation between lung parenchymal TBR and pulmonary artery TBR may be due to the same underlying changes in glycolytic metabolism, or those patients with a more severe small resistance pulmonary artery vasculopathy having more in lammatory or ibroblast cells present in the larger conduit pulmonary arteries. The correlation was also seen in the comparator group, although numbers were small. The absence of signi icantly increased uptake in the large pulmonary arteries is also of interest. While some earlier reports suggest the presence of atherosclerotic-like lesions in the pulmonary arteries, [38] these reports predate the modern treatment era of pulmonary hypertension. The majority of our IPAH patients were clinically stable patients under long-term follow-up at our center. Therefore, treatment-naïve incident patients or unstable patients were underrepresented. Markers of in lammation (hsCRP and in lammatory cytokines) in our IPAH pulmonary hypertension cohort were lower than those in the published literature. Quarck et al.[6] described a mean hsCRP of 4.4 mg/L (con idence interval 3.5–5.4) in treatment-naïve idiopathic PAH patients, which was signi icantly higher than their control group who had a mean hsCRP of 2.3 mg/L (con idence interval 1.9–2.7), whereas our idiopathic PAH cohort had a mean hsCRP of Pulmonary Circulation | October-December 2011 | Vol 1 | No 4

only 1.86. Our cytokine values were also lower than that of the study of Soon et al.[7] This data suggest that our pulmonary hypertensive patients had well-controlled stable disease. Even though no differences in large pulmonary artery uptake were found between the IPAH group and the comparator group, the IPAH group had a few “outliers” that could be representative of a different IPAH phenotype. The two patients with the highest large pulmonary artery TBR values included a 33-yearold male with rapidly progressive IPAH listed for lung transplantation and a 45-year-old female with relatively stable disease. They both also had hsCRP values above the mean for the IPAH group. There were some limitations to this study. The number of patients was small, although we did show signi icant differences between the groups. Lack of simultaneous hemodynamic measurements precluded direct correlation with 18FDG uptake, although NT-ProBNP levels were measured. Our imaging protocol was designed for maximizing uptake in vascular structures, not the right ventricle; therefore, caution is advised in the interpretation of the RV uptake results. A dedicated PET/CT study of the right ventricle would require ECG gating and clamping of glucose levels. Similar to 18FDG PET, there are other vascular imaging techniques that may have a role in answering the questions raised in this study. Animal models suggest that macrophage-targeted CT contrast agents may measure in lammation in atherosclerosis.[39] Ultra-small superparamagnetic iron oxide particles accumulate in macrophages in carotid artery plaques, which then can be quanti ied using high-resolution magnetic resonance imaging.[40] It would be of interest to apply these novel methods of disease assessment in pulmonary arterial hypertension, particularly for assessing the impact of targeted therapy on treatment-naïve patients and identi ication of novel drugs at early stages of development. Further studies may also clarify the signi icance of RV 18 FDG uptake in pulmonary hypertension of different etiologies with measurement of contemporaneous hemodynamics.

CONCLUSION In this pilot study, we demonstrated increased 18FDG uptake in the lungs of patients with IPAH, and higher RV uptake in pulmonary hypertensive subjects compared with controls. Increased lung uptake may be due to changes in glycolytic metabolism in endothelial cells in IPAH or an increased amount of cells involved in in lammation. Further imaging studies in treatment-naïve patients, to measure the impact of targeted therapies on 453

Hagan et al.: PET imaging in PAH

the lung, 18FDG signal and dedicated imaging of the right ventricle uptake may provide validation of this approach.

ACKNOWLEDGMENTS This research was partly supported by the NIHR Cambridge Biomedical Research Centre. M. Southwood holds an NIHR Healthcare Scientist Fellowship. We are grateful to Addenbrooke’s radiologist HK Cheow for governance reading of PET/CT images.

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Source of Support: Nil, Conflict of Interest: None declared.

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Research Ar t i cl e

Significant intrapulmonary Schistosoma egg antigens are not present in schistosomiasisassociated pulmonary hypertension Brian B. Graham1, Jacob Chabon1, Angela Bandeira2, Luciano Espinheira3, Ghazwan Butrous4 and Rubin M. Tuder1 Department of Medicine, Program in Translational Lung Research, University of Colorado Denver, Denver, CO, USA 2Department of Cardiology, Memorial S. Jose Hospital, Universidade de Pernambuco in Recife, Brazil, 3Department of Pathology, Hospital Prof. Edgard Santos, Universidade Federal da Bahia, Salvador, Brazil, 4University of Kent, Kent, UK

ABSTRACT Schistosomiasis-associated pulmonary arterial hypertension (PAH) is one of the most common causes of pulmonary hypertension worldwide. A potential contributing mechanism to the pathogenesis of this disease is a localized immune reaction to retained and persistent parasite-derived antigens. We sought to identify Schistosoma-derived egg antigens present in the lungs of individuals who died of the disease. We obtained 18 lung samples collected at autopsy from individuals who died of schistosomiasis-associated PAH in Brazil. A rabbit polyclonal antibody was created to known Schistosoma mansoni-soluble egg antigen (SEA). Histologic assessment and immunostaining of the human tissue was performed, along with immunostaining and immunoblotting of lung tissue from mice experimentally infected with S. mansoni. All 18 lung samples had evidence of pulmonary vascular remodeling with plexiform lesions and arterial medial thickening, but no visible eggs were seen. The anti-SEA antibody detected S. mansoni egg antigens in visible eggs in mouse lung and human intestine specimens, but did not identify a significant amount of egg antigen in the human lung specimens. In mouse granulomas containing degraded eggs, we observed colocalization of egg antigens and macrophage lysosomes. In conclusion, there is unlikely to be a significant amount of persistent parasite-derived antigens within the lungs of individuals who die of schistosomiasis-associated PAH. This suggests that retained and persistent parasite proteins are not contributing to a localized immune response in the pathogenesis of this disease. Key Words: pulmonary arterial hypertension, pulmonary hypertension, schistosomiasis

INTRODUCTION Schistosomiasis affects over 200 million people in 74 countries, where it causes more than 250,000 deaths and up to 4.5 million disability-adjusted years lost annually. [1] Approximately 10% of those chronically infected with Schistosoma mansoni develop hepatosplenic schistosomiasis, a syndrome of preportal fibrosis and portocaval shunting. [2] Approximately 10â&#x20AC;&#x201C;20% of those with hepatosplenic disease, or 2â&#x20AC;&#x201C;5 million people worldwide, develop pulmonary arterial hypertension (PAH), a progressive and fatal pulmonary vascular disease.â&#x20AC;&#x2030; [3,4] The pathology of

schistosomiasis-associated PAH is similar to other forms of PAH, with smooth muscle cell hypertrophy and intimal thickening.[5] The pathogenic mechanism by which this parasitic infection results in pulmonary vascular remodeling is unclear. Potential contributing factors include portal hypertension with resulting portopulmonary hypertension and/or egg embolism, and a host immune responses that may be systemic and/or locally directed at parasite antigens in the lung. Such a Access this article online Quick Response Code:

Address correspondence to: Dr. Brian B. Graham Program in Translational Lung Research, Division of Pulmonary Sciences and Critical Care Medicine, University of Colorado, Denver, Research 2-9th Floor, Mail Stop C-272, 12700 East 19th Avenue, Aurora, CO 80045, USA Email: brian.graham@ucdenver.edu 456

Website: www.pulmonarycirculation.org DOI: 10.4103/2045-8932.93544 How to cite this article: Graham BB, Chabon J, Bandeira A, Espinheira L, Butrous G, Tuder RM. Significant intrapulmonary Schistosoma egg antigens are not present in schistosomiasisassociated pulmonary hypertension. Pulm Circ 2011;1:456-61

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localized inflammatory response in particular could be directed at persistent antigens or, once initiated, could continue despite clearance of the inciting antigenic material.

rodents were approved by the Animal Care and Use Committees at the University of Colorado Denver.

Histologic examination of lung tissue from individuals with schistosomiasis-associated PAH reveals a dark pigment that is often located adjacent to sites of vascular remodeling, the nature of which is unclear, and which has historically been variously speculated to be derived from red blood cells,[6] “bile pigment,”[7] a component of scar tissue [7,8] or remnants of the parasite.[6,8-10] To clarify the nature of this pigment and potentially identify antigens that could be the target of a localized host inflammatory response, we sought to detect parasite egg antigens in the lung tissue from individuals who had died of schistosomiasis-associated PAH.

A rabbit polyclonal antibody was prepared to hold S. mansoni -soluble egg antigen (SEA). The SEA was prepared using a published protocol.[13] The S. mansoni eggs were of the NMRI strain provided by the BRI. The antibody was produced by GenicBio Limited, Shanghai, China. Sera from two rabbits were collected before and after immunization to SEA.

MATERIALS AND METHODS Sources of human tissue

Tissue from patients who died of schistosomiasisassociated PAH was obtained from two centers in Brazil: Memorial S. Jose Hospital, Universidade de Pernambuco in Recife, Pernambuco; and Hospital Prof. Edgard Santos, Universidade Federal da Bahia, Salvador. This tissue had been previously collected at autopsy and was formalin ixed and paraf in embedded. As the material was derived from deceased persons, no Institutional Review Board approval was required.

Sources of mouse tissue

We developed an experimental mouse model of schistosomiasis-associated pulmonary hypertension. [11] Briefly, wild-type C57Bl6/J mice (Taconic) receive 5,000 S. mansoni eggs injected intraperitoneally, followed 2 weeks later by challenge with 5,000 S. mansoni eggs injected intravenously. The eggs had been purified from the homogenized livers of S. mansoni - infected mice provided by the Biomedical Research Institute (BRI, Rockville, MD, USA); the strain of S. mansoni, the NMRI strain, was originally from Puerto Rico.[12] One week after intravenous challenge, the mice are sacrificed and the lung tissue is harvested and analyzed. The blood is flushed out of the lungs, the right bronchus sutured and 2% agarose instilled into the left lung through a transtracheal catheter. The left lung is removed, formalin fixed and processed for paraffin embedding, and the right lung is removed and frozen for subsequent protein analysis. All mice were bred and housed under specific pathogen-free conditions in an American Association for the Accreditation of Laboratory Animal Care-approved facility. All experimental procedures in Pulmonary Circulation | October-December 2011 | Vol 1 | No 4

Po l y c l o n a l a n t i b o d y p r o d u c t i o n a n d immunoblotting

The ability of the generated antibody to detect proteins in SEA was tested by probing a Western blot of puri ied SEA. Pre- and post-immunization serum was applied to the Western blot membrane at a concentration of 0.1 ug/mL overnight at 4°C. The immunoblot secondary antibody was HRP-labeled goat anti-rabbit (Vector, Burlingame, CA, PI-1000), used at a concentration of 1:5,000 for 1 h at room temperature, and detected using enhanced chemiluminescence (GE Healthcare, Little Chalfont, UK, RPN2106, RPN2106). Mouse whole lung lysates prepared by macerating and sonicating samples of the frozen right lung tissue in buffer containing antiproteases were also probed using the antiSEA antibody.

Tissue immunostaining

Sections of large intestine and lung from individuals with schistosomiasis-associated PAH and sections of lung from mice infected with S. mansoni were stained using the antiSEA antibody. The sections were heated at 100°C in citrate buffer for 20 min (Vector H-3300), blocked with 10% horse serum in phosphate-buffered saline (PBS) for 1 h, followed by the antibody (either preimmunization serum as a negative control or the postimmunization serum containing anti-SEA antibodies) applied at a concentration of 100 μg/mL overnight at 4°C and a secondary antibody of AF594-labeled donkey anti-Rabbit (Invitrogen A21207) diluted 1:200 applied for 1 h. Sections of infected mouse lung tissue were stained with both the rabbit anti-SEA antibody and a rat antiMac-3 antibody to identify macrophage lysosomes.[14] The sections were heated at 100°C in Borg buffer for 20 min (Biocare #BD1000G1); blocked with a mixture of 10% horse serum, 10% goat serum, 40% Superblock (ScyTek AAA5000) and 40% of 5% bovine serum albumin reconstituted in PBS for 1 h; the combination of anti-SEA antibody (either preimmunization serum as a negative control or the postimmunization antibody) at a concentration of 5 μg/mL and anti-Mac-3 antibody (BD Pharmingen #550292) or rat IgG (negative control) at a dilution of 1:50 applied for 1 h at room temperature; and 457

Graham et al.: Pulmonary antigens in schistosomiasis-associated PH

a combination of secondary antibodies of AF594-labeled donkey anti-rabbit (Invitrogen A21207) and AF488labeled goat anti-rat (Invitrogen A11006) each diluted 1:200 applied for 1 h. Human and mouse immuno luorescence-stained sections were counterstained with DAPI and imaged with a Nikon Eclipse E800 microscope with either a color camera (Nikon) or a black and white CCD camera (Photometrics). The double immuno luorescence stain of anti-SEA and anti-Mac-3 was imaged using a Zeiss LSM 510 META confocal microscope system with a 100Ă&#x2014; oil-immersion objective.

RESULTS We obtained and analyzed pulmonary tissue from 18 patients who had died of schistosomiasis-associated PAH. Histologically, all lung samples had evidence of pulmonary vascular remodeling (Fig. 1). All the samples (100%) contained plexiform lesions, and 16 of 18 (89%) had evidence of arterial medial thickening. Only four of the 18 (22%) samples had granulomas. Although described by others,[15,16] we did not observe any intact S. mansoni eggs in the pulmonary samples, even within granulomas. The distribution of severity of histopathologic findings is listed in (Table 1). All specimens contained a granular pigment, which was often located adjacent to vascular lesions (Fig. 1b), but appeared indistinguishable from anthracotic pigment. To identify antigenic material derived from S. mansoni eggs, we created a polyclonal rabbit antibody targeted to known soluble SEA. Two rabbits were inoculated and the serum samples from the two rabbits gave identical

results. The ability of the antibody to detect proteins in SEA was con irmed by immunoblot and immunostain. Western blots of puri ied SEA were probed with the serum collected from the rabbit preimmunization (serving as a negative control) or postimmunization (Fig. 2a). Only the anti-SEA antibodies in the postimmunization serum detected proteins in the SEA, across a wide range of molecular weights. We had previously developed an experimental mouse model of schistosomiasis-associated pulmonary hypertension, which utilizes intraperitoneal sensitization with S. mansoni eggs followed by an intravenous challenge with S. mansoni eggs. [11] Anti-SEA antibody in the postimmunization serum detected egg antigens present in immunoblots of whole-lung lysates of S. mansoni-infected mice (Fig. 2b). Immunostaining the infected mouse tissue with the anti-SEA antibodies in the postimmunization serum readily identi ied visible eggs in the mouse tissue, while the preimmunization serum failed to detect the eggs (Fig. 3a and b). Table 1: Range of histopathologic findings in 18 autopsy specimens from patients who died of schistosomiasis-associated PAH Severity*

Number of samples with observed histopathology Plexiform lesions

+++ ++ + 0

4 11 3 0

Arterial Granulomas Visible medial eggs thickening 1 10 5 2

1 2 1 14

0 0 0 18

*Severity scores: (+++) severe, (++) moderate, (+) mild, (0) none

Figure 1: Representative pulmonary pathology of schistosomiasis-associated pulmonary arterial hypertension. (a) Plexiform lesion (white arrow) in close proximity to concentric (onion-skinned) intimal thickening (black arrow). (b) Increased medial thickness (black arrowheads) adjacent to dark pigment (white arrowheads). (c) A perivascular granuloma (white star). All stains are hematoxylin and eosin. Scale bars are 100 Îźm. 458

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Graham et al.: Pulmonary antigens in schistosomiasis-associated PH

(a)

(b)

Figure 2: Immunoblots using anti-Schistosoma mansoni-soluble egg antigen (SEA) antibody identifies egg antigens in vitro and in vivo. (a) Serum collected from a rabbit before immunization to SEA fails to detect purified SEA proteins, while serum collected after the same rabbit has been immunized to SEA detects multiple proteins in the SEA at many molecular weights. (b) Postimmunization serum detects multiple antigenic proteins in whole-lung lysates of S. mansoni infected mice (each lane is a single animal).

Figure 3: Anti-Schistosoma mansoni-soluble egg antigen (SEA) antibody detects S. mansoni eggs in infected mouse lung and human intestine. (a and b) Mice with S. mansoni eggs administered intravenously develop peri-egg granulomas. The eggs can be visualized with the polyclonal anti-SEA antibody in postimmunization serum, but no significant signal is seen when antibodies in preimmunization rabbit serum are used. (c and d) Similarly, postimmunization serum but not preimmunization serum detects S. mansoni eggs in infected human intestines. Eggs are marked with a â&#x20AC;&#x153;*â&#x20AC;?; blue is DAPI; all scale bars are 100 Îźm.

We performed anti-SEA immunostaining of human tissue to detect S. mansoni antigens present in situ. One of the tissue specimens from a patient who had died of schistosomiasis-associated PAH had visible S. mansoni eggs in the colonic submucosa, and the anti-SEA antibody in the postimmunization serum readily detected SEA present in these eggs, while the preimmunization serum did not identify the eggs (Fig. 3c and d). We then probed human lung specimens with the anti-SEA antibody. No significant staining was observed throughout the lung tissue using either Pulmonary Circulation | October-December 2011 | Vol 1 | No 4

the post- or preimmunization serum (Fig. 4). We did identify rare fragments of S. mansoni eggs in human lung specimens using the anti-SEA antibody, which were only present within granulomas, and were not apparent by conventional staining. However, there was no significant staining adjacent to the pulmonary vascular lesions. We sought to localize the degraded egg fragments within granuloma cells. Costaining the mouse lung tissue for Mac-3/CD107b (a marker of macrophage plasma membranes and lysosomes[14]) and anti-SEA revealed that in granulomas encompassing degraded eggs, there were macrophage lysosomes containing intracellular egg antigens (Fig. 5).

DISCUSSION We sought to identify parasite proteins within the lungs of individuals who had died of schistosomiasisassociated PAH using a polyclonal antibody generated to known soluble SEA. The antibody was validated by identifying egg antigens in vitro and in mouse lung tissue and human intestinal tissue. We did not find evidence of egg antigens in the human lung tissue except for a very small amount within visible granulomas, which are presumably eggs being degraded by the host immune system. In particular, the commonly seen black granular pigment did not contain apparent schistosomal antigen, and given its identical appearance to anthracotic pigment, may represent carbon fragments from environmental smoke inhalation. 459

Graham et al.: Pulmonary antigens in schistosomiasis-associated PH

Although unlikely, we cannot entirely exclude the possibility that there may be a more extensive distribution of parasite-derived antigens, such as entirely insoluble proteins (not included in the soluble egg antigen inoculation of the rabbits) or nonegg parasite proteins left behind during the initial passage of the schistosomula through the lungs in acute schistosomiasis infection (Katayama syndrome).[17] Similarly, the dark pigment frequently observed adjacent to pulmonary vascular lesions in schistosomiasis-associated PAH, and which has been speculated to be derived from the parasite,[6,8-10] is unlikely to be parasite derived.

Figure 4: Anti-Schistosoma mansoni-soluble egg antigen (SEA) antibody identifies only a small amount of egg antigen within human lung granulomas, and not adjacent to vascular lesions. (a and b) Although there is no visible egg by hematoxylin and eosin stain, the anti-SEA antibodies in the postimmunization serum identify a small fragment of egg antigen (white arrow) present in a granuloma. (c-f) No Schistosoma egg antigens are found adjacent to pulmonary vascular lesions, including increased medial thickness (white arrowheads in c and d), a plexiform lesion (black arrow in e and f) and a dilated or angiomatoid lesion (black arrowhead in e and f). All scale bars are 100 μm.

Numerous investigators have postulated that an underlying cause of pulmonary vascular remodeling is in lammation. [18,19] Historically, Schistosoma ova were frequently seen in the lungs of individuals who died of schistosomiasis-associated PAH. For example, in 1954, de Faria reported histologically visible eggs present in 18 of 18 (100%) autopsy cases, with the number of eggs seen in a single 2.5 cm2 section ranging from four to approximately 250.[16] The report by Crosby et al. that mice chronically infected with schistosomiasis develop PH only when eggs are present in the lung, and that treatment with the anti-helmenthic praziquantel prevents or reverses this PH,[20] suggests that the localized parasite antigens trigger in lammation-mediated vascular remodeling, which stops once the antigenic material is eliminated. In comparison with the historical reports, it is striking that we saw no intact intrapulmonary ova in similarly

Figure 5: Immunofluorescence staining demonstrates colocalization of an anti-Schistosoma mansoni-soluble egg antigen (SEA) antibody and anti-Mac-3 antibody in infected mouse lung. (a–h) The anti-SEA antibody colocalizes with Mac-3 within many macrophages (white arrowheads) within granulomas surrounding largely degraded S. mansoni eggs; (e–h) imaged using a confocal microscope with a 100x oil-immersion objective. (i and j) Costaining with isotype controls does not demonstrate significant colocalization. All scale bars are 50 μm. 460

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Graham et al.: Pulmonary antigens in schistosomiasis-associated PH

sized sections from any of our 18 samples. A potential explanation for this difference is the introduction of antihelmenthic therapies such as praziquantel, which was developed in the late 1970s.[21] However, although praziquantel is effective at killing the parasite (and thereby preventing further eggs from being laid), clinically, praziquantel is largely ineffective at treating the vascular remodeling in those who have developed PAH after chronic and repeated infection with S. mansoni.[22] Similarly, our inding of vascular remodeling in patients who died of this disease, despite the absence of immunohistochemically identi ied antigenic material, suggests that after an initial acute in lammatory response, vascular lesions are established and follow a course of persistence or even progression. Beyond this “point of no return,” processes such as altered cellular bioenergetics[23] or clonal proliferation[24] may drive the lesions to continue.

5. 6. 7. 8. 9. 10. 11.

12.

13.

14.

Although alternatively activated (M2) macrophages have been described to be present in the in lammatory granulomas surrounding Schistosoma eggs,[25] to our knowledge, it has not been previously directly demonstrated that some of the macrophages contain degraded S. mansoni egg antigens. The speci ic colocalization of egg antigens to macrophage lysosomes suggests that macrophages phagocytose and digest the fragments of parasite after degradation by extracellular proteins secreted by the host immune response. Overall, there is unlikely to be a signi icant amount of persistent parasite-derived antigens within the lungs of individuals who die of schistosomiasis-associated PAH. This suggests that retained and persistent parasite proteins are not driving a localized immune response contributory to the pathogenesis of pulmonary vascular remodeling.

15. 16.

17. 18. 19.

20.

21. 22.

23. 24.

REFERENCES 25. 1. 2. 3.

Chitsulo L, Loverde P, Engels D. Schistosomiasis. Nat Rev Microbiol 2004;2 (1):12-3. Warren KS. Hepatosplenic schistosomiasis: A great neglected disease of the liver. Gut 1978;19:572-7. de Cleva R, Herman P, Pugliese V, Zilberstein B, Saad WA, Rodrigues JJ, et al. Prevalence of pulmonary hypertension in patients with hepatosplenic Mansonic schistosomiasis–prospective study. Hepatogastroenterology 2003;50;2028-30. Lapa M, Dias B, Jardim C, Fernandes CJ, Dourado PM, Figueiredo M,

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et al. Cardiopulmonary manifestations of hepatosplenic schistosomiasis. Circulation 2009;119:1518-23. Tuder RM. Pathology of pulmonary arterial hypertension. Semin Respir Crit Care Med 2009;30;376-85. Sadigursky M, Andrade ZA. Pulmonary changes in schistosomal cor pulmonale. Am J Trop Med Hyg 1982;31:779-84. Macieira-Coelho E, Duarte CS. The syndrome of portopulmonary schistosomiasis. Am J Med 1967;43:944-50. Girgis B, Guirguis S, Mowfaty R, El-Katib H. Bilharzial cor pulmonale: A clinicopathological report of two cases. Am Heart J 1953;45:190-200. Andrade ZA, Andrade SG. Pathogenesis of schistosomal pulmonary arteritis. Am J Trop Med Hyg 1970;19:305-10. Gelfand M. Cor-pulmonale and cardiopulmonary schistosomiasis. Trans R Soc Trop Med Hyg 1957;51:533-40. Graham BB, Mentink-Kane MM, El-Haddad H, Purnell S, Zhang L, Zaiman A, et al. Schistosomiasis-induced experimental pulmonary hypertension: Role of interleukin-13 signaling. Am J Pathol 2010; 177:1549-61. Lewis FA, Stirewalt MA, Souza CP, Gazzinelli G. Large-scale laboratory maintenance of Schistosoma mansoni, with observations on three schistosome/snail host combinations. J Parasitol 1986;72:813-29. Boros DL, Warren KS. Delayed hypersensitivity-type granuloma formation and dermal reaction induced and elicited by a soluble factor isolated from Schistosoma mansoni eggs. J Exp Med 1970;132:488-507. Chen JW, Murphy TL, Willingham MC, Pastan I, August JT. Identification of two lysosomal membrane glycoproteins. J Cell Biol 1985;101:85-95. Andrade ZA. Pathology of human schistosomiasis. Mem Inst Oswaldo Cruz 1987;82:17-23. de Faria JL. Cor Pulmonale in Manson’s Schistosomiasis I. Frequency in Necropsy Material; Pulmonary Vascular Changes Caused by Schistosome Ova. Am J Pathol 1954;30:167-93. Ross AG, Vickers D, Olds GR, Shah SM, McManus DP. Katayama syndrome. Lancet Infect Dis 2007;7:218-24. Dorfmuller P, Perros F, Balabanian K, Humbert M. Inflammation in pulmonary arterial hypertension. Eur Respir J 2003;22:358-63. Pullamsetti SS, Savai R, Janssen W, Dahal BK, Seeger W, Grimminger F, et al. Inflammation, immunological reaction and role of infection in pulmonary hypertension. Clin Microbiol Infect 2011;17:7-14. Crosby A, Jones FM, Southwood M, Dunne DW, Morrell NW. Praziquantel prevents progression of right ventricular hypertrophy in a mouse model of Schistosomiasis. Am J Respir Crit Care Med 2010;181: A4895. Gonnert R, Andrews P. Praziquantel, a new board-spectrum antischistosomal agent. Z Parasitenkd 1977;52:129-50. Richter J. Evolution of schistosomiasis-induced pathology after therapy and interruption of exposure to schistosomes: A review of ultrasonographic studies. Acta Trop 2000;77:111-31. Dromparis P, Sutendra G, Michelakis ED. The role of mitochondria in pulmonary vascular remodeling. J Mol Med (Berl) 2010;88:1003-10. Tuder RM, Radisavljevic Z, Shroyer KR, Polak JM, Voelkel NF. Monoclonal endothelial cells in appetite suppressant-associated pulmonary hypertension. Am J Respir Crit Care Med 1998;158:19992001. Wilson MS, Mentink-Kane MM, Pesce JT, Ramalingam TR, Thompson R, Wynn TA. Immunopathology of schistosomiasis. Immunol Cell Biol 2007;85:148-54.

Source of Support: This study was funded by a research grant from the Pulmonary Vascular Research Institute (PVRI; BBG), NIH/NHLBI 1K08HL105536-01A1 (BBG), a Parker B Francis Career Development Grant (BBG), and a grant from the Cardiovascular Medical Research Fund (CMREF; RMT), Conflict of Interest: None declared.

461

Research Ar t i cl e

High-altitude pulmonary hypertension in cattle (brisket disease): Candidate genes and gene expression profiling of peripheral blood mononuclear cells John H. Newman1, Timothy N. Holt2, Lora K. Hedges3, Bethany Womack3, Shafia S. Memon , Elisabeth D. Willers1, Lisa Wheeler1, John A. Phillips III3, and Rizwan Hamid3,4 3

Departments of 1Medicine, 3Pediatrics, and 4Cancer Biology, Vanderbilt University School of Medicine, Nashville, TN, 2 College of Veterinary Medicine, Colorado State University, Fort Collins, CO, and USA

ABSTRACT High-altitude pulmonary hypertension (HAPH) is a consequence of chronic alveolar hypoxia, leading to hypoxic vasoconstriction and remodeling of the pulmonary circulation. Brisket disease in cattle is a naturally occurring animal model of hypoxic pulmonary hypertension. Genetically susceptible cattle develop severe pulmonary hypertension and right heart failure at altitudes >7,000 ft. No information currently exists regarding the identity of the pathways and gene(s) responsible for HAPH or influencing severity. We hypothesized that initial insights into the pathogenesis of the disease could be discovered by a strategy of (1) sequencing of functional candidates revealed by single nucleotide polymorphism (SNP) analysis and (2) gene expression profiling of affected cattle compared with altitude-matched normal controls, with gene set enrichment analysis (GSEA) and Ingenuity pathway analysis (IPA). We isolated blood from a single herd of Black Angus cattle of both genders, aged 12–18 months, by jugular vein puncture. Mean pulmonary arterial pressures were 85.6±13 mmHg STD in the 10 affected and 35.3±1.2 mmHg STD in the 10 resistant cattle, P<0.001. From peripheral blood mononuclear cells, DNA was hybridized to an Affymetrix 10K Gene Chip SNP, and RNA was used to probe an Affymetrix Bovine genome array. SNP loci were remapped using the Btau 4.0 bovine genome assembly. mRNA data was analyzed by the Partek software package to identify sets of genes with an expression that was statistically different between the two groups. GSEA and IPA were conducted on the refined expression data to identify key cellular pathways and to generate networks and conduct functional analyses of the pathways and networks. Ten SNPs were identified by allelelic association and four candidate genes were sequenced in the cohort. Neither endothelial nitric oxide synthetase, NADH dehydrogenase, TGinteracting factor-2 nor BMPR2 were different among affected and resistant cattle. A 60-gene mRNA signature was identified that differentiated affected from unaffected cattle. Forty-six genes were overexpressed in the affected and 14 genes were downregulated in the affected cattle by at least 20%. GSEA and Ingenuity analysis identified respiratory diseases, inflammatory diseases and pathways as the top diseases and disorders (P<5.14×10-14), cell development and cell signaling as the top cellular functions (P<1.20×10-08), and IL6, TREM, PPAR, NFkB cell signaling (P<8.69×10-09) as the top canonical pathways associated with this gene signature. This study provides insights into differences in RNA expression in HAPH at a molecular level, and eliminates four functional gene candidates. Further studies are needed to validate and refine these preliminary findings and to determine the role of transcribed genes in the development of HAPH. Key Words: brisket disease, microarray analysis, hypoxia

INTRODUCTION High mountain disease (brisket disease) is right heart Address correspondence to: Prof. John H. Newman Division of Allergy, Pulmonary and Critical Care Medicine, Vanderbilt University School of Medicine, Nashville, TN 37232, USA Email: john.newman@vanderbilt.edu 462

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Website: www.pulmonarycirculation.org DOI: 10.4103/2045-8932.93545 How to cite this article: Newman JH, Holt TN, Hedges LK, Womack B, Memon SS, Willers ED, et al. High-altitude pulmonary hypertension in cattle (brisket disease): Candidate genes and gene expression profiling of peripheral blood mononuclear cells. Pulm Circ 2011;1:462-9.

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Newman et al.: Gene search bovine PH

failure due to hypoxic pulmonary hypertension in cattle residing at high altitudes.[1-3] Hypoxia is the most potent stimulus for pulmonary hypertension, and the hypoxia of high altitude (>7,000 ft) is a well known cause.[4-8] Some cattle (Bos taurus) possess a heritable susceptibility to severe high-altitude pulmonary hypertension (HAPH). [9- 12] While most cattle thrive at high altitudes, susceptible cattle develop pulmonary hypertension that is suf icient to cause right heart failure, edema of the brisket and death. Experiments conducted by Grover and Reeves in cattle susceptible and resistant to HAPH suggest an autosomal-dominant mode of inheritance, or inheritance related to a few major genes. [11-15] No information currently exists on the identity of these genes. HAPH in cattle occurs in about 20% (10–50%) of animals brought to high altitudes (>7,000 ft) to replenish herds,[8,14-16] suggesting that the gene of interest may be a relatively common polymorphism only expressed with the stimulus of altitude hypoxia. There is no known phenotype of the gene at low altitude. We hypothesized that the insights into pathogenesis of brisket disease may be gained through two approaches: (1) allelic association analysis of single nucleotide polymorphism (SNP) differences; and (2) a comparative gene expression analysis of hypertensive versus resistant cattle. Therefore, we analyzed DNA and RNA from 20 cattle of the same herd; 10 with severe pulmonary hypertension and 10 with normal pressures. We used an Affymetrix 10K Bovine Gene SNP array and sequenced four genes of interest. Expression data were then analyzed for genes and canonical pathways that were statistically different between the two groups. Gene set enrichment and Ingenuity map analyses were performed to further re ine the data.[16-19] Collectively, the results of this study provide molecular and cellular observations in possible genes of interest in HAPH leading to brisket disease.

MATERIALS AND METHODS Selection of study animals and blood samples

We obtained blood from a single herd of black Angus cattle of both genders, aged 12–18 months, residing at 8,500 feet, by jugular vein puncture on the same day of right heart catheterization screening for HAPH by one of us (Timothy N. Holt, hereinafter TH). The project was approved by the Vanderbilt Medical Center IACUC. Right heart catheterization is standard practice at altitude to discover hypertensive animals so that they can be shipped down to a low altitude.

DNA and RNA preparation and analysis

DNA was isolated from whole blood using QIAmp DNA mini-kit as directed by the manufacturer’s instructions Pulmonary Circulation | October-December 2011 | Vol 1 | No 4

(Qiagen, Valencia, CA, USA) and subsequently quanti ied using a spectrophotometer. Total cellular RNA was prepared from blood on silica-gel membranes with oncolumn DNAse treatment (RNeasy Total RNA Isolation Kit; Qiagen). RNA integrity was assessed via agarose gel electrophoresis and samples showing evidence of degradation were discarded.

Affymetrix bovine 10K SNP genotyping and bioinformatic analysis

For SNP comparison, genomic DNA was extracted as described above and hybridized to Affymetrix - GeneChip Bovine Mapping 10K SNP chip according to manufacturer’s instructions (Affymetrix, Santa Clara, Calif., USA). Following bovine genotype collection, we remapped all of the SNP loci represented on the bovine 10K array using the Batch query within dbSNP online (NCBI) in conjunction with the latest bovine genome assembly (Btau 4.0). In addition, SNP loci present on the array more than once (duplications) were identi ied, and all duplications were removed. Likewise, SNPs with multiple chromosomal assignments were also lagged and subsequently removed. At the conclusion of our bioinformatic analyses, 8,011 unique bovine SNPs with single chromosomal assignments and updated bovine genome annotation were available for genome-wide association (GWA) analysis.

Statistical approach to SNP microarray

Because the precise mode of inheritance related to HAPH in cattle has not been fully elucidated, we used several standard case–control GWA approaches in a preliminary effort to identify new potential candidate genes. All statistical tests were performed using formulae incorporated within the software program HelixTree 6.4.2 (Golden Helix Inc., Bozeman, MT, USA). In the irst approach, we performed a simple allelic association test across all unique SNP loci for 10 cattle exhibiting severe HAPH (n=20 alleles) and 10 cattle de ined as “resistant” at altitude (n=20 alleles). Thereafter, we also performed standard genotypic association tests in an effort to evaluate whether SNPs implicated by a simple allelic test would systematically assemble into disparate genotypic classes for cattle with severe HAPH, as compared with altitude-matched HAPH-resistant controls. Raw P-values for all tests were corrected by either full-scan permutation and/or the Bonferroni procedure implemented within HelixTree 6.4.2.

Sequencing SNP candidates

TG-interacting factor-2 (TGIF2), endothelial nitric oxide synthetase (eNOS) and NADH coding regions were sequenced using a cDNA-based approach. Brie ly, we performed irst strand cDNA synthesis using the Superscript First-Strand System (Invitrogen Life 463

Newman et al.: Gene search bovine PH

Technologies, Carlsbad, CA, USA) with 3 mg of total RNA and an oligo-dT primer. We then used one-tenth volume of the irst strand reaction as a template for polymerase chain reaction (PCR) ampli ication using the Elongase Ampli ication System (Invitrogen Life Technologies). The forward and reverse primers corresponded to sequences located in the 5’ and 3’ untranslated mRNA sequence of BMPR2, eNOS and TGIF-2 (primer sequences and conditions available upon request). We then carried out individual PCR reactions and visualized all amplicons on a 2% agarose gel. Thereafter, we used 2 μl ExoSAP-it (USB) per 5 ul of PCR reaction (37C for 15 min, followed by 80C for 15 min) to eliminate residual primer and dNTPs. Sequencing was accomplished using the Big Dye Terminator cycle sequencing technology (Applied Biosystems, Carlsbad, Calif., USA) in10 μl reactions containing 2 μl Big Dye, 2 μl Big Dye Buffer, 50 ng template and 2.5 ul of primer. Thermal cycling parameters were as follows: 95C for 5 min; 30 cycles at 95C for 30 s, 55C for 10 s and 60C for 4 min. After ethanol precipitation, the products were resuspended in 10 μl formamide and analyzed on a 3100 Genetic Analyzer (Applied Biosystems).

Microarray and gene set enrichment analysis analyses Pulmonary tissue was not available from these animals. It is also possible that the lung tissue expression signature might be overwhelmed by adaptive cellular responses. This would then make the interpretation of the expression data particularly susceptible to falsepositive and false-negatives.[16-19] Our solution to this was to avoid disease effector cells and to use peripheral blood mononuclear cells (PBMCs) in our studies. Peripheral blood mononuclear cells are genetically the same as lung tissue intraindividuals, and are genetically different among hypertensive and resistant cattle and are likely to be free of end-stage disease effects. RNA was extracted from PBMCs. After the RNA was isolated, it was used to probe the Affymetrix GeneChip Bovine Genome Array (900561). Raw data were then analyzed by the Partek software package at the Vanderbilt Functional Genomics Shared Resource to produce expression intensities for each probe set. To study pathway-level differences between groups, GSEA was conducted on the microarray gene expression data.[1] We used the Gene Ontology Biological Process (c5; from the MSigDB database) as our gene set database.

used to query a global molecular network developed from information contained in the Ingenuity Pathways Knowledge Base. IPA generates models of gene interactions called networks that are presented graphically to show relationships between genes and the pathways they regulate. These networks are ranked according to a score calculated via a right-tailed Fisher’s exact test. In network graph, proteins encoded by genes are represented as nodes and their relationships as edges (links). All edges are supported by references from the literature. The functional analysis of a network identi ied the biological functions and/or diseases that were most signi icant to the genes in the network. The network genes associated with biological functions and/or diseases in the Ingenuity Pathways Knowledge Base were considered for further analysis. Fisher’s exact test was used to calculate a P-value, determining the probability that each biological function and/or disease assigned to a network is by chance alone. Canonical pathways analysis identi ied the pathways from the IPA library of canonical pathways that were most signi icant to the data set. The signi icance of the association between the data set and a canonical pathway was measured in two ways.[1] A ratio of the number of genes from the data set that map to the pathway divided by the total number of genes that map to the canonical pathway is displayed.[2] Fischer’s exact test was used to calculate a P-value determining the probability that the association between the genes in the dataset and the canonical pathway is explained by chance alone.

RESULTS Study animals

Pulmonary arterial (PA) pressures were measured by jugular vein puncture at the time of right heart catheterization. Mean PA pressures were 86.10±5 mmHg STD in the affected and 31.20±0.7 mmHg STD in the resistant cattle, P<0.0001, by two-tailed Upaired t test (Fig. 1). A mean PA pressure of 30–40 mmHg is normal for

In silico functional analysis HPAH signature using ingenuity pathways analysis We used IPA in the Ingenuity System to generate networks and conduct functional analyses of the HAPH signature. A data set containing gene identi iers was uploaded into the application. These genes, called focus genes, were 464

Figure 1: Mean pulmonary arterial pressures in the 10 cattle with pulmonary hypertension at altitude versus 10 unaffected at the same altitude. Pulmonary Circulation | October-December 2011 | Vol 1 | No 4

Newman et al.: Gene search bovine PH

cattle residing at altitude for at least 12 months.[3,8] A mean PA pressure greater than 49 mmHg denotes a high risk for development of brisket disease.[8] Inclusion criteria were animals with a mean pulmonary artery pressure (mPAP) of 72–116 mmHg, thereby considered “affected,” and those with a mPAP of 32–37 mmHg, thereby considered “resistant” at altitude.

GWA using the affymetrix geneChip bovine mapping 10K array Among the bovine cohort investigated, 6,344 of the 8,011 unique bovine SNPs with single chromosomal assignments and updated bovine genome annotation were diallelic, thereby making them potentially informative for subsequent analyses. The most robust allelic association was detected on BTA10 (0.0000028 ≤ praw ≤ 0.0000451), with weaker signals detected on additional bovine chromosomes. As expected, full-scan permutation and/or Bonferroni correction for multiple testing revealed a much weaker BTA10 signal for both approximate and exact tests (0.0476 ≤ pcorrected ≤ 0.2856), respectively. Summary data for the top 10 bovine SNPs implicated by an allelic GWA test are presented in Table 1. Additionally, bovine genes proximal to each SNP are also given, with relevant human–bovine comparative data. Examination of relevant genotypes determined for the top 10 SNP loci elucidated by an allelic association test revealed six corresponding loci whereby severe bovine HAPH cases were ixed for a genotype underrepresented in our altitude-matched HAPH-resistant controls (Table 1). However, genotypic association tests for all variable loci (n=6344) revealed no statistically signi icant associations after correction for multiple testing. Relevant bovine genotypic data and corresponding distributions are summarized in (Table 1).

Because of its role in human PAH[20-23] and its place in the TGF-b family of growth and repair receptors, the bovine BMPR2 gene was an obvious initial functional candidate for HAPH. We screened 10 severely affected and 10 unaffected cattle with a set of bovine microsatellites proximal to bovine BMPR2 to evaluate whether local genetic variation exhibited any evidence of segregation with the brisket disease phenotype (HAPH). This microsatellite analysis revealed a total of seven BMPR2 alleles, with no signi icant differences in the allelic distributions between the HAPHaffected and -resistant cattle, as determined by Fisher’s exact test. No microsatellite allele was found uniformly among severely hypertensive cattle or resistant cattle thus providing no evidence of linkage disequilibrium between any particular allele and the HAPH phenotype. These data suggest, but do not prove, that variation within bovine BMPR2 is not responsible for modulating HAPH in cattle. Two other high-priority functional candidates were screened by cDNA-based sequence analysis, including eNOS and TGIF2.[24,25] Low NO production has been implicated in the pathogenesis of high-altitude pulmonary edema (HAPE),[26,27] while TGIF2 is a homeobox transcriptional repressor and an important downregulator of BMPR2 and TGF-b signaling.[27,28] Sequence comparison of the eNOS and TGIF2 coding regions of HAPH-affected and -resistant cattle did not result in the identi ication of any statistically signi icant variants predictive of HAPH in our samples.

Statistical analysis of microarray data reveals altered expression in HAPH cattle

Gene expression pro iles were determined by oligonucleotide microarray analyses using total RNAs from the PBMCs cells as described in the Methods section. In order to identify candidate genes involved in HAPH or protection from HAPH, we used a hierarchical clustering analysis to analyze the expression signature from the

Table 1: Top six bovine SNPs implicated by allelic association tests for a small bovine cohort consisting of severe HAPH cases and altitude-matched HAPH-resistant controls Bovine SNPa

Bovine chrom.b

Proximal bovine gene(s)c

Human chrom.d

Human Mb rangee

rs29024744

BTA10

NRXNIII/DIO2

BTA24

BRUNOL4

rs29024519

BTA29

FOXR2

rs29016420

BTA1

MYH15

rs29024944

BTA24

NDUFV2/LOC781824

rs29013210

BTA3

LOC100138920/FKBP1A

79,733,622– 79,748,2 33,077,828– 33,391,941 55,666,558– 55,668,483 109,581,906– 109,730,859 9,092,725– 9,124,313 1,297,622– 1,321,753

rs29025872

Relevant human genef Deiodinase RNA binding protein Forkhead box R2 Myosin heavy chain 15 NADH dehydrogenase FK binding protein 1A

SNP: single nucleotide polymorphism; HAPH: high-altitude pulmonary hypertension; NADH: Nicotinamide adenine dinucleotide phosphate

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PBMCs. A combination of relative level (fold-change) of expression and statistical signi icance (Student’s t-test) was used to distinguish these genes. This analysis identi ied 675 genes that were signi icantly upregulated and 320 genes that were downregulated (the HAPH signature) in affected animals. The top 15 candidates are listed in Table 2. Interestingly, the gene with the highest expression in affected animals is important for antigen presentation while the gene with the lowest expression is important for vessel elasticity as well as regulation of the TGF-beta pathway (Table 2). Genes related to disease process are shown in (Table 3).

DISCUSSION HAPH with right heart failure was irst described by Glover and Newsome in 1915 as “Brisket disease: Dropsy of high altitude.”[1] The link of altitude to hypoxic pulmonary hypertension was made after the discovery of hypoxic pulmonary vasoconstriction by von Euler and Liljestrand in 1946.[28] The exact mechanism causing acute hypoxic vasoconstriction remains elusive, although much is

known about modifying in luences.[29-31] Chronic hypoxia is a powerful stimulus for pulmonary hypertension, which involves not only vasoconstriction but also remodeling[32] of the pulmonary arteries. K-channel function has a central role, as does oxidant stress,[33,34] with Rho–Rho kinases involved in transduction of the hypoxic pressor response.[35-39] To date, no major genetic variants have been identi ied that modify the strength of the hypoxic pressor response. [37] Some observations have been made regarding genetic associations with pulmonary responses to altitude in humans.[30-40] For example, eNOS and tyrosine kinase gene variants were overrepresented in a Japanese cohort of individuals susceptible to HAPE as compared with healthy controls at altitude.[27,40] Eddahibi and colleagues detected an association between the LL genotype of the human SLC6A4 (formerly SERT) with pulmonary artery pressure in a cohort of patients with chronic obstructive pulmonary disease as compared with controls.[41] Recent studies implicate HIF2a polymorphism in Tibetans versus Han as a possible difference in successful high-altitude

Table 2: The top 15 up and down genes expressed in peripheral blood mononuclear cells comparing highaltitude pulmonary hypertension with resistant cattle at 8,500 ft altitude Name

Fold-change

Molecular function

Functions in cells

+2.05 +2.01 +1.93 +1.93 +1.78 1.77 −2.03 −1.6 −1.2 −1.15 −1.13 −1.01

MHC Class II receptor Notch binding and calcium ion binding Sodium symporter Voltage-gated calcium channel IgA binding GTPase binding Unknown binds MLX, MLXIP Uridine phosphorylase Glycerol-3-phosphate dehydrogenase [NAD+] activity Calcium ion biding, GTPase activity G protein-coupled receptor DNA binding Zymosan binding Transcription factor Chemotaxis, immune response Extracellular matrix, regulation of TGF-beta Inhibitory MHC Class I receptor Class I MHC Zinc and metal ion binding Calcium ion binding, unfolded protein response Zinc-binding transcription factor

−0.97 −0.89 −0.82 −0.78 −0.75 −0.74 −0.67 −0.66 −0.65

T-cell receptor GTP binding, GTPase activity Transcription factor Actin binding MHC Class I Transcription factor T-cell receptor Myosin complex Endothelial growth factor

Antigen processing and presentation Differentiation, notch signaling Vascular tone Response to hypoxia, calcium transport Immune response, respiratory burst Golgi to plasma membrane transport Unknown Growth NADH metabolic process, oxidation reduction Differentiation, protein modification Proliferation, adhesion, calcium response DNA binding Inflammatory response, +NOS process Immune function, particularly T cells Immune response Muscle and heart development Regulation of immune response Antigen processing, immune response T cell function and proliferation Endoplasmic reticulum chaperone protein Mesoderm development, lymphocyte development Immune response GTP signaling, protein transport Apoptosis Actin function Immunity, calcium-mediated signaling Apoptosis, differentiation Immune response Muscle function Angiogenesis, positive regulation of endothelial function

HLA-DQA2 DNER SLC28A3 RYR1 FCAR RAB3IP ARRDC4 UPP1 GPD2

+4.95 +2.97 +2.88 +2.43 +2.36 +2.25 +2.21 +2.17 +2.10

TGM3 EMR1 HIST1H1C PTX3 NFIL3 DEFB4A FBN1 KIR3DL1 BOLA CRIP3 CLGN IKZF3 TCRB RAB30 EBF1 PHACTR1 CD8A BCL11A TCRA MYO5C VEGFB

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Table 3: Top 10 disease processes associated with high-altitude pulmonary hypertension in cattle Category

P-value

Respiratory disease

1.61E-117.01E-03

Inflammatory response

1.02E-107.81E-03

Inflammatory disease

1.66E-087.01E-03

Connective tissue disorders

1.07E-074.28E-03

Skeletal and muscular disorders

1.07E-076.72E-03

Immunological disease

2.05E-075.92E-03

Genetic disorder

1.33E-066.72E-03

Hematological disease Cardiovascular disease

2.51E-06-

Metabolic disease

6.05E-056.83E-03

1.12E-

Genes HIST1H1C, SOCS3, TREM1, ACVRL1, ARG2, CD164, HRH1, HMOX1, NFIL3, GPR77, CXCR2, F5, MXD1, RYR1, FOSL2, LTBR, PDXK, FCGR3A, PTX3, IL15, IL1R1, TLR4, CSF2RB, HP, NCF1, S100A9, IL1RN, CEBPD, FBN1, IL1B, CD14, IL2RA, CHI3L1 FCAR, KIR3DL1, SOCS3, TREM1, IGSF6, OLFM4, ETS2, CASP4, BCL6, IL1R2, CD164, HMOX1, HRH1, NFIL3, CD47, GPR77, SOD2, HLA-A, CXCR2, F5, S100A8, LTBR, CCL16, FCGR3A, TNFSF13B, PTX3, CCR1, MGST1, ATF3, IL15, ALOX5AP, IL1R1, CSF2RB, TLR4, AQP9, NCF1, HP, S100A9, DUSP1, IL1RN, CEBPD, IL1B, CD14, IL2RA, SIRPA SOCS3, KIR3DL1, DPYD, ARG2, MAPK13, BCL6, IL1R2, HRH1, GPR77, SOD2, GPD2, FOSL2, DYSF, LTBR, SLCO4C1, TNFSF13B, ACSL6, MYOF, STX2, ACVR1B, CSF2RB, CLIC2, NCF1, AQP9, IL1RN, DUSP1, FBN1, CD14, RNF149, HLA-DQA2, SIRPA, MCTP2, TREM1, RBM47, CASP4, HMOX1, WDFY3, HLA-A, CXCR2, F5, WLS, MXD1, S100A8, C9ORF150, FCGR3A, CCR1, KCNK17, DCK, DNER, IL15, ALOX5AP, IL1R1, TLR4, HRSP12, P2RY13, HP, S100A9, FAM129A, IL1B, IL2RA, C15ORF48, EMR1, CHI3L1, TCF7L2, MAOA SOCS3, DPYD, MAPK13, BCL6, IL1R2, HMOX1, SOD2, HLA-A, GPD2, CXCR2, ANTXR2, S100A8, LTBR, TNFSF13B, SLCO4C1, FCGR3A, CCR1, KCNK17, DNER, IL15, ALOX5AP, IL1R1, MYOF, ACVR1B, TLR4, CSF2RB, P2RY13, NCF1, HP, CLIC2, AQP9, S100A9, DUSP1, IL1RN, FAM129A, IL1B, FBN1, IL2RA, RNF149, CHI3L1, HLA-DQA2, SIRPA, TCF7L2 SOCS3, DPYD, SGK1, MAPK13, BCL6, NPL, IL1R2, HRH1, GPCPD1, SOD2, GPD2, PDXK, DYSF, LTBR, SLCO4C1, TNFSF13B, MYOF, ACVR1B, KAT2B, CSF2RB, NCF1, AQP9, CLIC2, IL1RN, DUSP1, CD14, FBN1, RNF149, TBXAS1, MTDH, HLA-DQA2, SIRPA, MCTP2, ETS2, GNG7, HMOX1, HLA-A, CXCR2, MXD1, F5, SNX10, RYR1, S100A8, FCGR3A, CCR1, KCNK17, DCK, DNER, IL15, ALOX5AP, IL1R1, TLR4, P2RY13, HP, S100A9, FAM129A, IL1B, IL2RA, CHI3L1, TCF7L2, SOCS3, KIR3DL1, DPYD, MAPK13, FNDC3B, BCL6, IL1R2, HRH1, GPD2, LTBR, DYSF, SLCO4C1, TNFSF13B, MYOF, ACVR1B, CSF2RB, NCF1, AQP9, DUSP1, IL1RN, CD14, FBN1, RNF149, HLA-DQA2, ELL2, SIRPA, TREM1, CASP4, ID1, HMOX1, CD47, HLA-A, WDFY3, CXCR2, S100A8, FCGR3A, CCR1, OAS1, KCNK17, DCK, DNER, PSTPIP2, IL15, IL1R1, TLR4, HRSP12, P2RY13, HP, S100A9, FAM129A, IL1B, IL2RA, CHI3L1, TCF7L2 SOCS3, DPYD, KCNJ2, SGK1, KLF6, ACVRL1, ARG2, FNDC3B, BCL6, NPL, IL1R2, GPCPD1, HRH1, SOD2, GPD2, FOSL2, DYSF, ALPK1, PDXK, LTBR, TNFSF13B, SLCO4C1, MGST1, ATF3, ACSL6, MYOF, VLDLR, STX2, SERINC2, KAT2B, CSF2RB, NCF1, IL1RN, DUSP1, FBN1, CD14, RFX2, MTDH, TBXAS1, HLA-DQA2, SIRPA, MCTP2, LITAF, TREM1, RBM47, ETS2, CASP4, VAT1L, EIF4E, GNG7, HMOX1, ID1, CD47, HLA-A, WDFY3, CXCR2, MXD1, F5, WLS, ADAM19, SNX10, ANTXR2, RYR1, S100A8, C9ORF150, MARCKS, AGPAT9, RNF144B, RAB8B, FCGR3A, CCR1, MOCS1, KCNK17, DCK, DNER, IL15, DGAT2, ALOX5AP, IL1R1, PPP1R3B, MARCKSL1, TLR4, HP, S100A9, FAM129A, IL1B, IL2RA, EMR1, C15ORF48, CHI3L1, TCF7L2, ISG20, MAOA SOCS3, TREM1, MAPK13, BCL6, HMOX1, HRH1, CD47, SOD2, GPD2, F5, RYR1, FOSL2, LTBR, FCGR3A, ATF3, DCK, IL15, TLR4, CSF2RB, S100A9, IL1RN, IL1B, CD14, IL2RA, SIRPA SOCS3, DPYD, KCNJ2, KLF6, ACVRL1, ARG2, BCL6, NPL, HRH1, SOD2, GPD2, ALPK1, DYSF, PTX3, MYOF, VLDLR, KLF4, CSF2RB, NCF1, AQP9, IL1RN, CD14, FBN1, SIRPA, RBM47, ETS2, VAT1L, HMOX1, CD47, F5, ANTXR2, ADAM19, RYR1, S100A8, KCNK17, PSTPIP2, IL15, DGAT2, ALOX5AP, IL1R1, TLR4, MARCKSL1, HP, S100A9, IL1B, IL2RA, CHI3L1, ISG20, MAOA ID1, HMOX1, CD47, SOD2, IL1RN, DUSP1, CEBPD, CD14, IL1B, BCL6, KLF4

adaptation.[42] Many other signaling pathways possess potential candidate genes.[43] Relevant to the present study, the risk of acquiring brisket disease in low-altitude cattle brought to very high altitude (10,000 ft) is about 10–50%.[31,44] Notably, the prevalence of Pulmonary Circulation | October-December 2011 | Vol 1 | No 4

brisket disease among herds chronically residing and bred above 2100 m (c. 7,000 ft) has been reduced to about 1%, presumably because of selective loss of susceptible cattle to heart failure and to removal from herds after HAPH has been identi ied.[3] Previously, Reeves, Weir and Grover bred a cohort of cattle with HAPH whose mean PA pressure was 467

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50mmHg (n=8) and a group of resistant animals whose mean PA was 29mmHg (n=11), all residing at 10,000 ft.[11,14] The 19 cattle were initially taken down to an altitude of 4,916 ft, where the cattle with HAPH recovered, and then the cattle were bred within each phenotypic class. Bulls and cows were equally represented. First-generation offspring were studied at both low and high altitude. Mean PA pressures were 27±4 (STD) at low altitude. After residing at 10,000 ft for 2 months, the offspring of cattle with HAPH (brisket disease) had a mean PA pressure of 87±7 (SE), and those from resistant stock had a mean PA of 44±3 mmHg. Second-generation breeding (breeding experiments not overtly clear) within these two cohorts of calves yielded the same pattern of susceptibility and resistance. Pressures continued to diverge over time at altitude. Proof that the stimulus to pulmonary hypertension was hypoxia and not “altitude” was subsequently demonstrated by exposures in a hypoxic chamber at low altitude, whereby similar pulmonary hypertensive responses were recorded. Arterial blood gas measurements in susceptible and resistant cattle revealed similar severity of hypoxemia, similar alveolar ventilation (de ined by the arterial carbon dioxide partial pressure, PC02) and only small differences in hematocrit, not considered suf icient to cause viscosity effects. The mode of inheritance of HPAH currently remains unclear.[8] Nevertheless, what does remain clear among several studies is that HAPH in cattle is heritable, which indicates that cattle may be selected for resistance or susceptibility, and that the major gene(s) involved are likely to be discovered using large-scale genomic approaches followed by ine mapping. Our initial screen using four functional candidate genes (selected because they are involved in human PAH) was negative and suggested that a candidate gene approach may not be ef icient. We then employed a 10K bovine SNP array for a GWA aimed at unearthing new potential candidate genes. This analysis revealed six or more genes of interest proximal to bovine SNPs exhibiting the most disparate distributions between severe HAPH and resistant cattle. Of the genes located near the SNPs of interest, NADH dehydrogenase (ubiquinone) lavoprotein 2 (NDUFV), myosin heavy chain 15 (MYH15) and the myocardial signaling protein (FKBP1A) were candidates possibly involved in pulmonary hypertension. NADH dehydrogenase is no different between affected and resistant cattle, and additional studies are needed to further evaluate candidate genes. Furthermore, the recent availability of higher density bovine SNP arrays[44,45] provides a natural progression to more thorough GWA, which are likely to identify additional candidate genes due to greatly enhanced genomic coverage. The use of these new higher density bovine SNP arrays with larger cohorts of severe HAPH and resistant cattle has the potential to maximize the probability of detecting SNPs that display a nonrandom relationship with either severe HAPH or 468

putative resistance. It is likely that the major gene(s) responsible for bovine HAPH will have identi iable human ortholog(s) that may also modify human pulmonary hypertension related to hypoxia (altitude, emphysema, hypoventilation syndromes), and possibly in other conditions that lead to pulmonary hypertension.

Biological significance of HAPH signature

To gain biological insights into resistance or susceptibility to HAPH, we analyzed the expression data using the Gene Ontology and Ingenuity Map database. Our goal was to identify the disease processes, physiological, cellular and molecular functions and the canonical pathways that are enriched in HAPH. These analyses showed that the top disease process re lected in our data is respiratory disease (Table 2); the top three physiological processes are organism survival, hematological system and immune cell traf icking; the top three cellular and molecular pathways are cellular movement, cell signaling and small molecule biochemistry; and the top canonical pathways enriched in HAPH are IL-10 signaling, RXR activation,and, interestingly, immune and endothelial cell function (data not shown but available on request). We then used the IPA application (IPA version 8.7 ) to examine the biological context and interactions of our signature. The IPA network analysis identi ied 10 networks with high P-values (1.0×10-21) (data not shown but available upon request). The top three networks had many genes (MAP13, STAT5a/b, ERK1/2, Cyclins, VEGF, PDGF, IL1B, ID1, CREB, p38MAPK, IL15, etc.) with a documented role in endothelial and immune cell function through their effects on cellular pathways such as TGF-beta, BMP and MAPK (important in human PAH) and molecular functions such as cell death, survival, in lammatory response and cell morphology. Function annotation of all the networks showed that the top canonical functions associated with these 10 networks are BMP, TGF-beta, PPAR, IL6, endothelial cell function, NOS, TREM1, VEGF, glucocorticoid, ERK/MAPK, NF-kB and HIF1a signaling (data not shown but available upon request). Not surprisingly, many of these pathways are also known to be important in human pulmonary hypertension. In summary, herein, we have provided the results of the irst molecular interrogation of HPAH, including a low-density SNP search, sequencing of four candidate genes and analysis of expression arrays to seek clusters of genes that may be a signature of hereditary bovine HAPH. Our hope is that this initial study of HPAH will spur further discovery and investigation of the molecular mechanisms behind HPAH. Our next challenge will be to attempt whole genome sequencing on the hypothesis that a polymorphism responsible for an autosomal-dominant Pulmonary Circulation | October-December 2011 | Vol 1 | No 4

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phenotype at altitude will emerge from the similar background genomes of the Angus herds.

25.

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Source of Support: Pulmonary Hypertension Association Scientific Award, Newman PI. Elsa S. Hanigan Chair, Dr. Newman. Dr. Hamid is supported by 1R01HL102020-01, Conflict of Interest: None declared.

469

Research Ar t i cl e

Quantitative estimation of right ventricular hypertrophy using ECG criteria in patients with pulmonary hypertension: A comparison with cardiac MRI Kevin G. Blyth1, James Kinsella2,3, Nina Hakacova3, Lindsey E. McLure2, Adeel M. Siddiqui4, Galen S. Wagner3, and Andrew J. Peacock2 1

Southern General Hospital, 2Scottish Pulmonary Vascular Unit, Glasgow, UK, 3Department of Cardiology, Duke University, Durham, USA, 4The Aga Khan University Hospital, Karachi, Pakistan

ABSTRACT In patients with pulmonary arterial hypertension (PAH), right ventricular mass (RVM) correlates linearly with pulmonary artery pressure, and decreases with successful treatment. Accurate measurement of RVM currently requires cardiovascular magnetic resonance (CMR) imaging. We therefore tested the relationship between RVM and a simple, 12 lead ECG-derived value, the Butler-Leggett (BL) score. This has previously been validated in patients with RV hypertrophy (RVH) due to mitral stenosis. We also tested the diagnostic accuracy of the BL score in detecting RVH. The Scottish Pulmonary Vascular Unit database was reviewed retrospectively. Twenty-eight patients with PAH were identified, in whom CMR and ECG data had been recorded no more than 28 days apart. All had completed a comprehensive clinical assessment, including right heart catheterization. CMRderived absolute RVM and RV mass index (RVMI=RV mass/LV mass) were correlated against BL score. The ability of this score to detect RVH was tested using 2 x 2 contingency tables. RVM and RVMI correlated with BL score (r=0.77, P<0.001 and r=0.78, P<0.001, respectively). A BL score >0.7 mV proved a highly specific but insensitive indicator of RVH, based on either absolute RVM (sensitivity 74%, specificity 100%) or a high RVMI (sensitivity 61%, specificity 100%). The BL score, which can be defined using a standard 12-lead ECG, correlates with RVM and RVMI in patients with PAH. A score >0.7 mV was a highly specific but insensitive indicator of RVH in these patients. Key Words: ECG, magnetic resonance image, pulmonary hypertension, right ventricular hypertrophy

INTRODUCTION In the normal adult heart, the right ventricle (RV) is a thin-walled, low-pressure pump that is poorly adapted to cope with a high afterload.[1] In patients with pulmonary arterial hypertension (PAH), elevated pulmonary vascular resistance (PVR) results in a rise in RV afterload. This typically occurs gradually, allowing a compensatory increase in RV mass (RVM), helping to maintain stroke volume and cardiac output. RVM is, therefore, an important measurement in PAH patients. Previous studies have shown that RVM decreases with successful treatment[2] and has a strong relationship with mean pulmonary artery pressure in PAH. This is true when RVM is recorded in Address correspondence to: Prof. Andrew J. Peacock Scottish Pulmonary Vascular Unit, Regional Heart and Lung Centre, Glasgow G81 4HX, UK Email: apeacock@udcf.gla.ac.uk 470

isolation,[3] but the correlation is more powerful when RVM is related to left ventricular (LV) mass in the same patient (known as RV mass index (RVMI)).[4] The current â&#x20AC;&#x153;gold standardâ&#x20AC;? method of directly measuring RVM is cardiovascular magnetic resonance (CMR) imaging. However, the high cost and limited availability of this technology restricts its clinical utility. We were interested in using the standard 12-lead ECG to describe RVM in PAH patients, given its clinical utility for this purpose in earlier Access this article online Quick Response Code:

Website: www.pulmonarycirculation.org DOI: 10.4103/2045-8932.93546 How to cite this article: Blyth KG, Kinsella J, Hakacova N, McLure LE, Siddiqui AM, Wagner GS, et al. Quantitative estimation of right ventricular hypertrophy using ECG criteria in patients with pulmonary hypertension: A comparison with cardiac MRI. Pulm Circ 2011;1:470-4.

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studies. Al-Naamani et al.[5] used the ECG as a screening test for PAH; however, the criteria tested, including the dimensions of rightward and anterior QRS waveforms and QRS axis orientation, demonstrated insuf icient levels of sensitivity and speci icity. More recently, Henkens et al. used a vectorcardiographic (VCG) adaptation of the 12-lead ECG to more speci ically study RVM in PAH patients.[6] Using a locally developed computer program for calculating ventricular gradient, which considered both the depolarization and the repolarization of the RV through analysis of the QRS complex and T wave, they were able to distinguish between normal RVM, mild RV hypertrophy (RVH), moderate RVH and severe RVH. However, this method requires a speci ic electronic program to analyze the ECG, which is not widely available. We tested a simpler ECG method based on the previously described Butler-Leggett (BL) criteria derived from a standard 12-lead ECG. Butler et al. showed that this method could be used to identify RVH in patients with increased pulmonary resistance due to mitral stenosis and cor pulmonale.[7,8] The BL criteria are based on the principal that activation of the LV free wall produces forces directed posteriorly and leftward (PL). These forces are opposed by those generated by the RV free wall, which are directed anteriorly (A) and rightward (R).[9] This concept can be expressed by a formula (A + R – PL), incorporating each of these forces, as measured on a standard 12-lead ECG. This results in a continuous variable (the BL score) for each ECG recording. In the normal situation, the LV forces dominate, but if the mass of the RV myocardium increases, as may occur in PAH, there is a net increase in rightward and anterior forces resulting in a higher BL score. The current study was performed to test the hypothesis that the BL score would correlate closely with RVM in PAH patients. We suspected that any correlation would be stronger with RVMI than with absolute RVM, as this took into account the opposing contribution of the LV in any given patient. We also tested the diagnostic sensitivity of the BL score as a means of detecting RVH as a consequence of PAH.

MATERIALS AND METHODS Case notes and clinical details from a prospectively recorded database were reviewed retrospectively. All patients who had attended the Scottish Pulmonary

Vascular Unit between August 2003 and May 2008 for diagnostic assessment were considered for inclusion. Only patients with a diagnosis of PAH, who had undergone both CMR imaging and 12-lead ECG within a 30-day period, were considered for inclusion. In addition, a copy of the ECG had to be available in the case notes, as ECGs were not recorded digitally at that time. Patients were excluded if they had a history of signi icant left heart disease or if their ECG showed complete right or left bundle branch block or ST depression (preventing accurate measurement of the S wave in leads I, V1 or V6). Twenty-eight patients met these criteria. All were subjected to rigorous diagnostic evaluation, including right heart catheterization, echocardiography, high-resolution computed tomography (CT) thorax, CT pulmonary angiography and pulmonary function testing. Six control subjects were recruited and underwent CMR to establish a normal range for RVMI (normal have not previously been published). All gave informed written consent. None had any history of cardiorespiratory disease. The mean age of the control population was 37 (±10) years and all were male. Systemic blood pressure was normal in these individuals (systolic 114 [±8], diastolic 73 [±6], mean 87 [±6]).

ECG analysis

The QRS duration was measured using handheld calipers. Patients with QRS duration above 110 ms were reviewed by two observers (authors James Kinsella and Nina Hakacova, hereinafter JK and NH) for the presence of complete left or right bundle branch block. A modi ied version of the BL criteria was used as shown in Table 1. The terms “positive waveform” or “negative waveform” replaced designations of speci ic waveforms to remove the ambiguity of differentiation between the two QRS waveforms, which both indicate that the balance of forces is either toward (R and R’ waves) or away from (Q and S wave) the positive pole of a particular ECG lead. JK and NH performed the waveform measurements using handheld calipers (to the nearest 0.1 mV). Disagreements in measurements were reviewed and consensus achieved. Anterior forces (A) were designated as the largest positive waveform in either lead V1 or V2. Rightward forces (R) were designated as the largest negative waveform in either lead I or V6, and posterior–lateral forces (PL) as the negative waveform in V1. These measurements were

Table 1: Original and modified methods of defining the Butler-Leggett score Original Modified

Anterior forces

Rightward forces

Posterolateral forces

Formula

Maximal R or R’ in V1 or V2 Maximal positive waveform in V1 or V2

Maximal S in I or V6 Maximal negative waveform in I or V6

Minimal S in V1 or R in I or V6 Maximal negative waveform in V1

A+R-PL A+R-PL

The modified method was used in the current study; A: anterior forces; R: rightward forces; PL: posterolateral forces

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combined into the BL formula through the equation: A + R – PL=BL score (mV).

Magnetic resonance image acquisition

Magnetic resonance image (MRI) scans were performed using a 1.5 Tesla scanner (Sonata Magnetom, Seimens, Germany). Fast imaging with steady-state precession sequences were used to generate the initial axial scout images required to localize the heart within the thoracic cavity and all subsequent cine images. Vertical and horizontal-long axis (VLA and HLA) cines were planned and acquired based on the scout images. The irst of a series of short axis (SA) cines was then planned on image 1 of the HLA cine, intersecting the atrioventricular valve roots on this view. The SA imaging plane was then propagated apically, covering both ventricles with 8-mm SA imaging slices, separated by a 2 mm interslice gap. The traditional LV SA cine stack is used for the acquisition of both RV and LV images.

CMR image analysis

All CMR images were analyzed by a single operator (KGB) using the Argus analysis software (Siemens, Erlangen, Germany). The endocardial and epicardial borders of the image in end-diastolic and -systolic phases of the cycle at each slice position within the SA stack were de ined by manual planimetry, including trabeculae and papillary muscles. These methods have been published previously. [10,11] RV and LV mass were determined as the product of the difference between the end-diastolic and end-systolic volume for each ventricle and the quoted density of cardiac muscle (1.05 g/cm3). RVM was determined as RV free wall mass, with the interventricular septum considered part of the LV. RVMI was calculated by dividing RVM by LV mass.

Statistical analysis

For all variables, a normal distribution was veri ied using Kolmogorov-Smirnov tests. Pearson’s correlation method was used to assess the relationship between BL score and both RVM and RVMI derived from MRI. Contingency tables (22) were used to calculate the sensitivity and speci icity of the various BL scores as a means of detecting RVH. RVH was de ined by two different methods to allow comparison. By method 1, RVH was de ined as an RVM two standard deviations above the previously published normal mean RVM (35 [±8] g).[12] By this method, RVH was de ined by a measured RVM >51 g. By method 2, RVH was de ined by the upper 95% con idence interval of the mean RVMI (0.5002) of our control subjects. This de ined RVH by an RVMI >0.582. The BL score with the greatest diagnostic accuracy (that which produced the lowest number of false-positive and false-negative results) was chosen. Negative and positive predictive values (PPV and NPV) for results either side of this threshold were 472

then calculated. The implications of an above-threshold result were quanti ied by likelihood ratios and Fisher’s exact test. All data are presented as mean (±SD), unless otherwise stated.

RESULTS Results of right heart catheterization in the 28 patients with PAH are shown in (Table 2). Twenty were female, and the mean age of the population was 51 (±17) years. Nine had connective tissue disease-associated PAH (CTD-PAH) and 19 had idiopathic PAH (IPAH); 26/28 had CMR imaging and ECG within 8 days, one had CMR imaging 18 days after ECG and one had CMR imaging 23 days after ECG.

ECG and CMR imaging results

The mean BL score among PH patients was 1.12±1.51. Mean RVM, LV mass and RVMI were 86.2 (±36.0) g, 92.1 (±31.3) g and 0.95±0.3, respectively.

Correlation between BL score and RVM and RVMI

The correlation between RVM and BL score was r=0.77, P<0.001 (Fig. 1a). The correlation between RVMI and BL score was r=0.78, P<0.0001 (Fig. 1b).

Detection of RVH

The BL score performed best at the 0.7 mV threshold for detecting RVH by either of the methods studied (elevated absolute RVM or elevated RVMI). Using a de inition of RVH as an RVM >51 g (method 1), this threshold yielded no false-positives, but yielded six false-negatives. This produced a sensitivity of 74% (95% con idence interval 52–90%) and a speci icity of 100% (95% con idence interval 48–100%). NPV and PPV were 45% and 100%, respectively. Based on the de inition of RVH as an RVMI>0.582 (method 2), a BL score >0.7 mV yielded no false-positives but 10 false-negatives. This resulted in a sensitivity of 61% (95% con idence interval 41–79%) and a speci icity of 100% (95% con idence interval 81–100%). NPV and PPV were 9% and 100%, respectively. Table 2: Right heart catheterization data Mean±SD Systolic PAP (mmHg) Diastolic PAP (mmHg) Mean PAP (mmHg) PA wedge pressure (mmHg)

83 29 50 8

(±26) (±14) (±17) (±4)

Results of right heart catheterization in 28 patients with pulmonary arterial hypertension; PAP: pulmonary artery pressure; PA: pulmonary artery

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Blyth et al.: ECG criteria in estimation of RVH

(a)

(b)

Figure 1: Right ventricular mass (RVM) and RV mass index (RVMI) were calculated from the cardiac magnetic resonance images acquired in 28 patients with pulmonary arterial hypertension. These data were correlated against Butler-Leggett (BL) scores derived from standard 12-lead ECG recordings. (a) Relationship between BL score and RVM (r=0.77, P<0.001). (b) Relationship between BL score and RVMI (r=0.78, P<0.001). On both scatter plots, the vertical lines indicate the upper limits of normal for RVM (51 g) and RVMI (0.6); values to the right of this line indicate RV hypertrophy (RVH). The horizontal lines on each figure indicate the threshold of BL score (0.7 mV) that proved most accurate in identifying RVH.

DISCUSSION The primary result of this study is that a BL score ≥0.7 mV proved a highly speci ic indicator of RVH (as de ined by either RVM or RVMI). However, this threshold was relatively insensitive (sensitivity 74% and 61% for RVM and RVMI, respectively). The BL score also correlated linearly with RVM and RVMI in PAH patients. Although the BL score is the electrical equivalent of RVMI, which re lects the relative mass of the ventricles, its relationship with RVMI was not any more powerful than that with absolute RVM. Our indings are consistent with the results of an earlier PH screening study that demonstrated insuf icient diagnostic sensitivity using different ECG criteria.[5] We used a slightly modi ied version of the original BL criteria (Table 1). This was necessary to avoid confusion when considering the two negative waveforms (Q and S) and the two positive waveforms (R and R’). This modi ication is in accordance with methods used in a recent study by Siddiqui et al., in which the BL criteria were used in patients with RV volume overload.[13] The exclusion of R wave amplitudes in leads I or V6 is valid as analysis of the formation of the ECG has shown that these waveforms do not re lect postero–lateral forces; this revision of the original BL methodology has been included in subsequent reprints of the formula.[14] The BL score used in the current study also differs from the VCG method used by Henkens et al.[6] That method incorporates information regarding both ventricular depolarization and repolarization by analyzing both QRS complexes and T waves. The authors were able to show that their VCG method is a highly accurate means of identifying increased RV afterload and differentiating Pulmonary Circulation | October-December 2011 | Vol 1 | No 4

between subgroups of patients with mild-to-moderate and severe RV pressure overload. They showed a moderate inverse correlation between RVM and VCG projection on the x-axis, as determined by their method.[6] The strength of this correlation (r=0.323, P=0.048) was lower than that detected in the current study using BL criteria (r=0.77, P<0.001). Clearly, these two methods are not directly comparable and we did not look for any relationship between RV afterload and BL score in the current study. Nevertheless, it would be interesting to directly compare the performance of the two methods in a future study. Although the VCG method is complicated by the requirement for a noncommercially available program to derive the ECG information, this could be overcome with appropriate software, and both approaches may have future clinical applications. Most of the patients in the current study had IPAH (19/28); the remainder had CTD-PAH (9/28). Recent echocardiographic and CMR imaging studies have shown that RV diastolic and systolic function and RV pulmonary arterial coupling are adversely affected in CTD-PAH in comparison with IPAH patients, at equivalent levels of pulmonary hypertension.[15] Despite this, we found no difference in the extent of RVH on CMR imaging between these disease groups and no difference in their BL scores. Although it is possible that this re lects the relatively small sample size in our study, we do not believe that this factor signi icantly altered our conclusions. Because the BL score correlates strongly with RVM, and a score ≥0.7 mV was a highly speci ic indicator of RVH, this method may be useful in referral centers with no access to CMR imaging, or in the outpatient PH clinic. Although a high BL score is a strong indicator that RVH 473

Blyth et al.: ECG criteria in estimation of RVH

is present (PPV 100%), a normal score does not exclude RVH, given the low sensitivity (<70%) and NPV (<42%) of this method. As RVM has been shown to decrease with effective treatment,[2] the BL score may also decrease. This might prove useful in the noninvasive monitoring of treatment effect, but this hypothesis would need to be tested in a prospective study. Interestingly, a recent study has demonstrated that alterations in QRS axis, P wave amplitude in lead II and T wave axis correlate well with hemodynamic improvements on PAH therapies.[16] The number of patients in the current study was relatively small. It would therefore be inappropriate to propose that the speci ic BL threshold (0.7 mV) for RVH that we report be applied more widely in PAH populations. CMR imaging does provide, however, an extremely accurate and reproducible gold standard for RVM, limiting the number of patients required for such studies. ECGs and CMR imaging studies were not acquired contemporaneously in all patients. This introduces a source of potential error in the results described, although the likelihood and magnitude of any change in RVM, even over the maximal interval between studies (23 days), are both likely to be small.

CONCLUSION The BL score, which can be de ined using a standard 12lead ECG, correlates linearly with both RVM and RVMI in patients with PAH. A score of >0.7 mV was a highly speci ic but relatively insensitive indicator of RVH in these patients.

REFERENCES 1.

Naeĳe D. Pathophysiology of Pulmonary Arterial Hypertension. ERS Respiratory Monograph - Pulmonary Vascular Pathology: A Clinical Update. 2004. p. 191-203. Wilkins MR, Paul GA, Strange JW, Tunariu N, Gin-Sing W, Banya WA,

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et al. Sildenafil versus Endothelin Receptor Antagonist for Pulmonary Hypertension (SERAPH) study. Am J Respir Crit Care Med 2005;171:1292-7. Katz J, Whang J, Boxt LM, Barst RJ. Estimation of right ventricular mass in normal subjects and in patients with primary pulmonary hypertension by nuclear magnetic resonance imaging. J Am Coll Cardiol 1993;21:1475-81. Saba TS, Foster J, Cockburn M, Cowan M, Peacock AJ. Ventricular mass index using magnetic resonance imaging accurately estimates pulmonary artery pressure. Eur Respir J 2002;20:1519-24. Al-Naamani K, Hĳal T, Nguyen V, Andrew S, Nguyen T, Huynh T. Predictive values of the electrocardiogram in diagnosing pulmonary hypertension. Int J Cardiol 2008;127:214-8. Henkens IR, Mouchaers KT, Vonk-Noordegraaf A, Boonstra A, Swenne CA, Maan AC, et al. Improved ECG detection of presence and severity of right ventricular pressure load validated with cardiac magnetic resonance imaging. Am J Physiol Heart Circ Physiol 2008;294:H2150-7. Butler PM, Leggett SI, Howe CM, Freye CJ, Hindman NB, Wagner GS. Identification of electrocardiographic criteria for diagnosis of right ventricular hypertrophy due to mitral stenosis. Am J Cardiol 1986;57:639-43. Behar JV, Howe CM, Wagner NB, Leggett SI, Hinohara T, Moser KF, et al. Performance of new criteria for right ventricular hypertrophy and myocardial infarction in patients with pulmonary hypertension due to cor pulmonale and mitral stenosis. J Electrocardiol 1991;24:231-7. Cowdery CD, Wagner GS, Starr JW, Rogers G, Greenfield JC Jr. New vectorcardiographic criteria for diagnosing right ventricular hypertrophy in mitral stenosis: Comparison with electrocardiographic criteria. Circulation 1980;62:1026-32. Lorenz CH, Walker ES, Morgan VL, Klein SS, Graham TP Jr. Normal human right and left ventricular mass, systolic function, and gender diﬀerences by cine magnetic resonance imaging. J Cardiovasc Magn Reson 1999;1:7-21. Sievers B, Kirchberg S, Bakan A, Franken U, Trappe HJ. Impact of papillary muscles in ventricular volume and ejection fraction assessment by cardiovascular magnetic resonance. J Cardiovasc Magn Reson 2004; 6:9-16. Hudsmith LE, Petersen SE, Tyler DJ, Francis JM, Cheng AS, Clarke K, et al. Determination of cardiac volumes and mass with FLASH and SSFP cine sequences at 1.5 vs. 3 Tesla: A validation study. J Magn Reson Imaging 2006;24:312-8. Siddiqui AM, Samad Z, Hakacova N, Kinsella J, Ward C, White M, et al. The utility of modified Butler-Leggett criteria for right ventricular hypertrophy in detection of clinically significant shunt ratio in ostium secundum-type atrial septal defect in adults. J Electrocardiol 2010;43:161-6. Wagner G. Marriott’s Practical Electrocardiography, 10th ed. Philadelphia: Lippincott Williams & Wilkins; 2001. Kowal-Bielecka O, Delcroix M, Vonk-Noordegraaf A, Hoeper MM, Naeije R. Outcome measures in pulmonary arterial hypertension associated with systemic sclerosis. Rheumatology. 2008;47 Suppl 5:39-41. Henkens IR, Gan CT, van Wolferen SA, Hew M, Boonstra A, Twisk JW, et al. ECG monitoring of treatment response in pulmonary arterial hypertension patients. Chest 2008;134:1250-7.

Source of Support: KG Blyth was funded by a project grant from Chest, Heart & Stroke, Scotland, Conflict of Interest: None declared.

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Research Ar t i cl e

Pulmonary artery endothelium resident endothelial colony-forming cells in pulmonary arterial hypertension Heng T. Duong, Suzy A. Comhair, Micheala A. Aldred, Lori Mavrakis, Benjamin M. Savasky, Serpil C. Erzurum, and Kewal Asosingh Department of Pathobiology, Lerner Research Institute, Genomic Medicine Institute, Respiratory Institute, Cleveland Clinic, Cleveland, Ohio, USA

ABSTRACT Proliferative pulmonary vascular remodeling is the pathologic hallmark of pulmonary arterial hypertension (PAH) that ultimately leads to right heart failure and death. Highly proliferative endothelial cells known as endothelial colony-forming cells (ECFC) participate in vascular homeostasis in health as well as in pathological angiogenic remodeling in disease. ECFC are distinguished by the capacity to clonally proliferate from a single cell. The presence of ECFC in the human pulmonary arteries and their role in PAH pathogenesis is largely unknown. In this study, we established a simple technique for isolating and growing ECFC from cultured pulmonary artery endothelial cells (PAEC) to test the hypothesis that ECFC reside in human pulmonary arteries and that the proliferative vasculopathy of PAH is related to greater numbers and/or more proliferative ECFC in the pulmonary vascular wall. Flow cytometric forward and side scatter properties and aggregate correction were utilized to sort unmanipulated, single PAEC to enumerate ECFC in primary PAEC cultures derived from PAH and healthy lungs. After 2 weeks, wells were assessed for ECFC formation. ECFC derived from PAH PAEC were more proliferative than control. A greater proportion of PAH ECFC formed colonies following subculturing, demonstrating the presence of more ECFC with high proliferative potential among PAH PAEC. Human androgen receptor assay showed clonality of progeny, confirming that proliferative colonies were single cell-derived. ECFC expressed CD31, von Willebrand factor, endothelial nitric oxide synthase, caveolin-1 and CD34, consistent with an endothelial cell phenotype. We established a simple flow cytometry method that allows ECFC quantification using unmanipulated cells. We conclude that ECFC reside among PAEC and that PAH PAEC contain ECFC that are more proliferative than ECFC in control cultures, which likely contributes to the proliferative angiopathic process in PAH. Key Words: endothelium, endothelial colony forming cell, endothelial progenitor cell, pulmonary arterial hypertension, angiogenesis

INTRODUCTION Pulmonary arterial hypertension (PAH) is a poorly understood disease that carries a devastating prognosis. Increased pulmonary vascular tone in PAH leads to right heart failure and death. In addition to elevated pulmonary artery pressure, PAH is characterized by increased and dysfunctional angiogenesis of the pulmonary circulation.[1,2] The pathogenesis of these vascular lesions is not well understood.

many disease processes.[3-5] Though endothelial cells are normally quiescent, injury, in lammation, hypoxia and other factors can initiate angiogenesis through mobilizing pro-angiogenic cells from the bone marrow and activating local endothelial cells to proliferate.[2,3,6,7] These mobilized pro-angiogenic cells are myeloid in origin, while in contrast a population of true endothelial cells found in the vessel wall called endothelial colony-forming Access this article online Quick Response Code:

Dysregulation of angiogenesis is a notable feature of Address correspondence to: Dr. Kewal Asosingh, 9500 Euclid Ave, Department of Pathobiology, NC22 Cleveland OH 44195 USA Email: asosink@ccf.org Pulmonary Circulation | October-December 2011 | Vol 1 | No 4

Website: www.pulmonarycirculation.org DOI: 10.4103/2045-8932.93547 How to cite this article: Duong HT, Comhair SA, Aldred MA, Mavrakis L, Savasky BM, Erzurum SC, et al. Pulmonary artery endothelium resident endothelial colony-forming cells in pulmonary arterial hypertension. Pulm Circ 2011;1:475-86.

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Duong et al.: ECFC in pulmonary artery endothelium and PAH

cells (ECFC) are believed to contribute to angiogenesis through cell proliferation.[8] While sprouting angiogenesis can be an appropriate response to vascular injury and hypoxia, increased and dysregulated angiogenesis is a hallmark of many disease states such as cancer,[8,9] macular degeneration[10,11] and pulmonary arterial hypertension.[1,2] Identifying the key cellular players and how they interact in healthy and pathologic angiogenesis are important goals in understanding the pathogenesis of numerous diseases.

and grow ECFC would yield clonal populations of cells. In this study we describe the characterization of ECFC derived from the cultured endothelial cells from the human pulmonary artery and differences in these cell populations between healthy control and PAH patients.

ECFC, also termed late outgrowth endothelial cells or blood outgrowth endothelial cells, are proliferative endothelial cells residing in the vessel wall and found rarely in the peripheral blood.[7,12] ECFC were identi ied by Ingram and colleagues using a single cell in vitro colonyforming assay.[8,12] Although identi ied in the search for an endothelial progenitor cell, it is unclear whether ECFC represent a unipotent or multipotent stem cell or are just fully differentiated endothelial cells with high proliferative potential. ECFC express endothelial cell-speci ic surface markers, such as CD34, CD146, CD31, Flk-1 and CD105, but there are currently no markers that distinguish ECFC from other endothelial cells.[8,12] ECFC meet rigorous tests of endothelial cell function, including possessing the capacity to form functional vessels in vivo when implanted in immunode icient mice.[13] ECFC therefore demonstrate the capacity to form structural cells of mature vessels.

PAH and control PAECs were obtained from explanted PAH human lungs and donor lungs not used in transplantation, respectively. All patients were female for purposes of clonality analysis (see HUMARA assay below). Pulmonary arteries were dissected down to the distal small arterioles, longitudinally cut, and incubated with collagenase type II to detach endothelial cells. Cells were grown in MCDB107 (Sigma, St. Louis, Mo.) on ibronectin-coated tissue culture plates. Tissue culture plates were pre-coated with 1 mL of bovine serum ibronectin (Calbiochem, La Jolla, Calif.) diluted in phosphate-buffered saline (PBS) to 50Îźg/mL for 20-30 minutes. PAEC were passaged at 70% to 80% con luence by dissociation with 0.25% trypsinethylenediaminetetraacetic acid (Invitrogen, Carlsbad, Calif.) with trypsin reaction stopped with MCDB-107 media containing serum. Endothelial cell phenotype was con irmed by immunocytochemistry for the endothelial cell-speci ic markers CD31 (1:30 dilution; Dako, Glostrup, Denmark) and von Willebrand factor (vWF; 1:200 dilution; Dako, Glostrup, Denmark), and luorescence-activated cell sorting (FACS) analyses for CD31 and VEGFR2 expression (Becton Dickinson, San Jose, Calif.). Immunohistochemical analysis of cultured cells identi ied that >95% were CD31-positive and >99% vWF-positive. FACS analysis con irmed that >95% of the cells were CD31- and VEGFR2positive.[17,21] Primary cultures at passage 5 were used in experiments. DNA from original PAEC cultures was analyzed for mutations and chromosomal abnormalities known to be associated with PAH. Cultured PAEC were harvested by manual scraping with a cell scraper and DNA was extracted using the Qiagen DNA Mini kit (Qiagen, Valencia, Calif.) according to the manufacturerâ&#x20AC;&#x2122;s recommended protocol. BMPR2 mutation analysis was performed by direct sequencing and multiplex ligationdependent probe ampli ication as previously described.[17] To detect genome-wide copy number changes, DNA was hybridized to single nucleotide polymorphism arrays, as previously described.[22]

Although originally isolated from peripheral blood where they comprised only 1 in 108 plated human mononuclear cells, ECFC have since been identi ied in several different vascular beds, including the human umbilical vein and aorta, as well as the rat pulmonary artery.[8,13,14] A hallmark of ECFC is their remarkable proliferative potential, having been documented to achieve over 100 population doublings in vitro.[8,12] The role of ECFC in disease has been suggested by studies in diabetes and pulmonary arterial hypertension (PAH) that suggest that ECFC function is compromised in these illnesses. Circulating ECFC demonstrate impaired angiogenic tube formation in both diabetes and PAH.[15,16] While there have been several studies investigating the role of circulating pro-angiogenic hematopoietic cells in PAH, the importance of ECFC in the genesis of the vascular lesions of PAH is less well studied.[16-20] Given the angiogenic and proliferative capacities of ECFC and the exuberant angiogenesis that occurs in PAH, it is possible that differences in either proliferative capacity or numbers of these cells may contribute to the vasculopathy of PAH. We hypothesized (1) ECFC reside in the human pulmonary arteries in PAH and controls and (2) that PAH PAEC will contain a greater number or more proliferative ECFC. We further sought to validate that the assay used to isolate 476

MATERIALS AND METHODS Pulmonary artery endothelial cell isolation and culture

Endothelial colony-forming cell assay

We adapted the ECFC assay originally described by Ingram and colleagues for the culture of PAEC.[8] The principle is to sort single endothelial cells into the wells of a 96well plate and subsequently culture and expand dividing Pulmonary Circulation | October-December 2011 | Vol 1 | No 4

Duong et al.: ECFC in pulmonary artery endothelium and PAH

cells. Through the use of single-cell sorting, the range of clonogenic potentials of PAEC can be assessed using this assay. ECFC that possess high proliferative potential form secondary colonies upon replating. PAEC at passage 5 from frozen stock stored in liquid nitrogen in 10% DMSO in fetal bovine serum (FBS) were brought to room temperature and cultured in complete endothelial growth media-2 (EGM-2; bullet kit, Lonza, Walkersville, Md.) in a ibronectin-coated 100 mm tissue culture plate. Cells were grown in 9 mL of EGM-2 supplemented with 10% FBS, 2% penicillin/streptomycin and 0.25 μg/mL amphotericin B (EGM-2 complete) and incubated in a humidi ied incubator at 37°C with 5% CO2. At 50-90% con luency, PAEC were subsequently rinsed twice with PBS and trypsinized with 1 mL of warm trypsin-EDTA. Trypsinization was stopped with 4 mL of EGM-2 complete and cells were harvested and spun down at 233 g for 5 minutes at 20° C. Media was aspirated and cells were resuspended with 1-3 mL of EGM-2 complete and then iltered with a 40 μm ilter for single-cell sorting into 96 well plates. Suspended cells were kept on ice to reduce aggregation. Ninety-six-well plates and all subsequent plates used for culture of ECFC were coated with 5 μg/cm2 of rat tail collagen type I (BD Biosciences, San Jose, CA) in 0.02N acetic acid prior to adding cells. Stock solution of 0.02N acetic acid was prepared from glacial acetic acid (17.4N) diluted in Milli-Q-puri ied water (Millipore, Billerica, Mass.) and iltered with a 0.22 μm vacuum ilter under a laminar low tissue culture hood. Prior to preparation of plates for sorting, collagen type I solution was made fresh by adding the appropriate amount of collagen type I to the acetic acid stock to make a 50 μg/mL collagen type I (collagen-I) solution. After determining the surface area of each well, enough collagen-I solution was added to cover the plating area with 5 μg/cm2 of collagen-I. Plates were coated 2 hrs. to overnight in a humidi ied incubator at 37°C with 5% CO2. Collagen-I solution was then aspirated and wells were rinsed twice with PBS. Two hundred μL of EGM-2 complete was added to each well of a 96-well plate. For subculturing, larger culture vessels received appropriate volumes of EGM-2 complete. PAEC suspended in complete EGM-2 were sorted by FACS using a FACSAria II low cytometry cell sorter (BD Biosciences, San Jose, Calif.) to place a single cell in each well of a 96-well tissue culture plate. Cells were sorted by forward scatter and side scatter (FSC/SSC) gated for the cellular fraction with aggregate correction to ensure that multiple cells were not sorted into each well. One hundred cells were sorted into the irst well on each 96-well plate to assist with focusing of the microscope during visualization. PAEC were sorted into 3 96-well Pulmonary Circulation | October-December 2011 | Vol 1 | No 4

plates for each patient. Media was changed on Days 4 and 8. After 2 weeks, wells were examined for formation of ECFC colonies by an inverted microscope. Cell counts were performed by visual inspection where possible or otherwise with a hemacytometer. To detach cells for subculturing and counting the supernatant overlying, each well of a 96-well plate was removed and the well was rinsed once with 70 μL PBS. Twenty μL of warm trypsin was added to the well and incubated for 2.5 minutes at 37° C. Trypsinization was stopped by adding 30 μL EGM-2 complete. Plates were put on ice after this step to prevent aggregation. Where counting was achieved with a hemacytometer (>50 cells), 10 μL of the cell suspension was loaded onto a hemacytometer and the 4 corner ields were counted and averaged. A colony was de ined by the presence of 2 or more cells in a well. ECFC were de ined as single cells giving rise to a colony containing 50 cells or greater.[8]

Replating of ECFC After the number of cells/colony was counted, ECFC colonies were subcultured in 600 μL of complete EGM-2 in a 24-well tissue culture plate pre-coated with 5 μg/ cm2 rat tail collagen-I and examined for secondary colony formation or con luence after 1 week. Wells in which there was colony formation were subsequently subcultured in 2 mL complete EGM-2 to a 6-well tissue culture plate precoated with 5 μg/cm2 rat tail collagen-I in complete EGM-2 and allowed to grow to con luence. Con luent wells were then subcultured in a 100 mm tissue culture plate precoated with 5 μg/cm2 rat tail collagen-I in complete EGM-2.

Immunohistochemistry ECFC which were expanded to 6-well plates and 100 mm tissue culture dishes were analyzed for endothelial cell-speci ic markers to con irm endothelial phenotype: vWF; endothelial nitric oxide synthase (eNOS); caveolin-1 (cav-1); CD31; and CD34. Cells were trypsinized with 1 mL of warm trypsin and transferred onto 4-well chamber slides pre-coated with rat tail collagen type I and grown in complete EGM-2 as previously described. After overnight incubation at 37° C, media was removed and cells were ixed using the following methods speci ic to the staining protocol for each antibody: vWF—4.0% paraformaldehyde/PBS/0.2% Triton 100 for 10 minutes at room temperature; eNOS—methanol for 10 minutes at -20° C; CD31—4.0% paraformaldehyde/PBS/0.2% Triton 100 for 10 minutes at room temperature; caveolin-1— acetone-methanol (1:1) for 10 minutes at -20° C; and CD34–acetone for 10 minutes at -20° C. After ixation, cells were washed 3 times with PBS and blocked with 3.0% normal goat serum (vWF, eNOS, CD31) or horse serum (cav-1, CD34). Following aspiration of blocking serum, cells were incubated with 200 μL of the following primary 477

Duong et al.: ECFC in pulmonary artery endothelium and PAH

antibodies at the listed dilutions at room temperature for 90 minutes with gentle agitation: polyclonal rabbit anti-human: vWF at 1:1200 (Dako, Glostrup, Denmark); polyclonal rabbit anti-human NOS3 (eNOS) at 1:500 (Santa Cruz Biotechnology, Santa Cruz, Calif.); polyclonal rabbit anti-CD31 prediluted at 1:4 (Abcam, Cambridge, Mass.); monoclonal mouse anti-caveolin 1 at 1:200 (BD Transduction Laboratories, San Jose, Calif.); and monoclonal mouse anti-human CD34 Class II at 1:25 (Dako, Glostrup, Denmark). A negative control which was incubated with blocking serum only instead of primary antibody was used for each PAEC sample or ECFC clone. Cells were then washed with PBS/0.05% Tween 20 three times for 5 minutes and the appropriate secondary antibody was added and incubated at room temperature for 30 minutes. For vWF, eNOS, and CD31, goat anti-rabbit IgG (Vector Laboratories, Burlingame, Calif.) was used and for cav-1 and CD34, horse anti-mouse IgG (Vector Laboratories, Burlingame, Calif.) was used. Cells were then washed with PBS/0.05% Tween 20 three times for 5 minutes with gentle agitation and avidin/biotin complex was added and incubated for 30 minutes at room temperature. Cells were washed again with PBS/0.05% Tween 20 three times for 5 minutes. Following wash step, microchambers were removed and visualization was achieved with ImmPACT DAB peroxidase substrate for 2-10 minutes. Slides were rinsed with running tap water, counterstained with hematoxylin (Vector Laboratories, Burlingame, Calif.) for 2-3 minutes, and rinsed again with running tap water until rinse water was colorless. Slides were destained with one quick dip in 1.0% HCl in 70% ethanol, rinsed with running tap water, and dipped 10-20 times in bluing solution (Richard Allan Scienti ic, Kalamazoo, Mich.). Following a inal rinse with running tap water, slides were dehydrated using an ethanol and xylene series and mounted with Permount.

Acetylated LDL uptake

ECFC which grew to populate a 100 mm tissue culture dish were trypsinized and cultured in a 2-well chamber slide pre-coated with rat tail collagen-I as previously described. ECFC were incubated with 20 μg/mL with DiI-acetylated-low-density lipoprotein (DiI-Ac-LDL; Invitrogen, Carlsbad, Calif.) for 2 hrs. in EGM-2 complete at 37°C. Cells were washed twice with complete EGM-2 and examined for DiI-Ac-LDL uptake using a Leica DM IRB inverted microscope.

Electron microscopy

For ECFC that expanded to populate a 100 mm tissue culture dish, we con irmed endothelial phenotype by examining for caveolae and Weibel-Palade bodies with electron microscopy. Samples were trypsinized and grown in a 2-well chamber slide pre-coated with collagen-I. ECFC were trypsinized, transferred to chamber slides, 478

and grown overnight in 1.6 mL complete EGM-2 in a 5% CO2 humidi ied incubator at 37° C. Media was aspirated and samples were ixed with 2.5% glutaradehyde and 4% paraformaldehyde with 0.2M cacodylate buffer and kept overnight at 4° C. Samples were washed with sodium cacodylate buffer (0.2M, pH 7.3) 3 times for 5 min. each. Cacodylate buffer was removed and 1% osmium tetroxide (in H2O) was added and incubated for 60 min. at 4° C. Samples were washed again with sodium cacodylate twice, 5 min. each, and rinsed with maleate buffer (pH 5.1) 1 for 5 min. Staining was then performed by adding 1% uranyl acetate in maleate buffer and stained for 60 min. Uranyl acetate was removed and samples were washed with maleate buffer 3 times. Samples were subsequently dehydrated by rinsing with increasing concentrations of cold ethanol (50-95%), 5 min. each, followed by 3 rinses with 100% room temperature ethanol. Following this, samples were rinsed with propylene oxide 3 times for 15 min. each. Propylene oxide was removed and replaced with 1:1 propylene oxide/eponate 12 medium (Ted Pella Inc., Redding, Calif.) at room temperature overnight. Subsequently, media was aspirated and pure eponate 12 medium was added for 4-6 hrs at room temperature. Polymerization was allowed to progress for 24 hrs., after which ultra thin sections of 85 nm were cut with diamond knife, stained with uranyl acetate and lead citrate, and then observed with a Philips CM12 electron microscope operated at 60 kV.

HUMARA assay

We assessed the clonality of ECFC that proliferated to populate a 100 mm tissue culture dish by examining the X-inactivation distribution of ECFC and the parent PAEC culture with the human androgen receptor assay (HUMARA). HUMARA uses the methylation status of a highly polymorphic CAG repeat region lanking the human androgen receptor gene as a surrogate for X-inactivation status.[23] The inactive X chromosome is methylated in this region while the active X chromosome is not. X-inactivation is normally random in a human female; however, clonal populations of cells demonstrate inactivation of the same X-chromosome. HUMARA was performed as previously described.[17] ECFC in 100 mm tissue culture dishes were trypsinized and resuspended in EGM-2 and DNA was extracted using the Qiagen DNA Mini kit (Qiagen, Valencia, Calif.) according to the manufacturer’s recommended protocol. One hundred ng of DNA was digested 16 h with RsaI and 100 ng DNA was digested with RsaI+HhaI. NEB Buffer 4 and 1X bovine serum albumin (BSA; New England BioLabs, Ipswich, Mass.) was used for both digestions. After digestion, restriction enzymes were inactivated at 80° C for 20 min. For HUMARA PCR, 1 μL or 4 μL of the diluted digestion product (RsaI and RsaI+HhaI, respectively) was added to Pulmonary Circulation | October-December 2011 | Vol 1 | No 4

Duong et al.: ECFC in pulmonary artery endothelium and PAH

2 μL of ABgene buffer (Applied Biosystems, Carlsbad, Calif.), 2 μL dNTPs (2 mM), 2 μL custom synthesized luorescentlylabeled primers (Integrated DNA Technologies, Coralville, Iowa), 0.5 μL of dimethyl sulfoxide, 0.2 μL ABgene Taq polymerase, and the remainder with distilled water to achieve a inal volume of 20 μL. PCR reaction was run for 36 cycles on a thermal cycler. Ten μL of the PCR products were run on a 1.25% agarose gel to check for ef icient ampli ication. Two μL of the remaining product was diluted 1:10-1:100 in distilled water, as judged from the intensity of the band on the agarose gel. 1 μL of diluted PCR product was loaded with 8.75 μL of HiDi formamide (Applied Biosystems, Carlsbad, Calif.) and 0.25 μL of a luorescent ladder on an Applied Biosystems AB3730XL luorescent sequence using the “genotyping” module. Data were analyzed with Gene Mapper v4.0 software (Applied Biosystems, Carlsbad, Calif.). The raw peak height values of the digested samples were corrected for ampli ication ef iciency by using the average of the 2 samples digested with RsaI alone.[23] Results for ECFC were compared to HUMARA analysis of the original PAEC culture from which ECFC were derived. HUMARA PCR primer sequences were as follows: Forward—5’-GTT TCC AGA ATC TGT TCC AGA GCG TGC-3’; and Reverse—5’-6-FAM/ATG GGC TTG GGG AGA ACC ATC CTC-3’. 6-FAM is 6-carboxy luorescein, a luorescent PCR primer tag.

Statistical analysis

All data were analyzed using the JMP 9.0 software program. The Wilcoxon test was used for comparison of nonparametric data and the Wilcoxon signed rank test was used for comparison of paired nonparametric data, as appropriate. P values of less than 0.05 were considered signi icant. Mean ± standard error of mean for each group is shown.

RESULTS Patient selection PAEC were isolated from donor lungs not used in transplantation (n=4) and explanted lungs from PAH patients at the time of transplantation (n=11). Mean age of patients was 36.3±4.9 for control versus 48.8±3.5 for PAH, P=0.11. All patients were Caucasian females. The PAH group included patients with different subtypes of PAH and were genetically characterized for chromosomal or mutational changes known to be associated with PAH.[22] These genetic alterations included bone morphogenetic protein receptor 2 (BMPR2) mutations and a proportion of cells harboring mosaic deletion of the X-chromosome. Also included were PAH patients with no known genetic abnormalities (Table 1).

Single-cell sorting of PAEC PAEC were isolated from control and PAH lungs as outlined in the “Methods” section [Fig. 1]. Cultured PAEC at passage 5 were grown to between 50%-90% con luency and sorted using a BD FACsAria II low cytometer to put a single cell in each well of a 96-well tissue culture plate pre-coated with rat tail collagen-I. Aggregate exclusion was performed as described in “Methods.” Visual inspection of colonies with eosin staining 24 hrs. after sorting con irmed that 97% of wells received a single cell that was able to become adherent. Adherent cells included healthy-looking cells, pyknotic cells, and cells already undergoing division. Thus low cytometric techniques were effectively employed to achieve single-cell sorting of unstained PAEC (Fig. 2).

ECFC among control and PAH PAEC A modi ied version of the ECFC assay described by

Table 1: Patient demographics Patient

Age

Sex

Race

Disease

Genetic characterization

Ctrl 1 Ctrl 2 Ctrl 3 Ctrl 4 PAH 1 PAH 2 PAH 3 PAH 4 PAH 5 PAH 6 PAH 7

n/a 28 36 45 60 34 30 59 50 32 56

F F F F F F F F F F F

Caucasian Caucasian Caucasian Caucasian Caucasian Caucasian Caucasian Caucasian Caucasian Caucasian Caucasian

Donor Donor Donor Donor IPAH IPAH IPAH IPAH FPAH FPAH Pulmonary veno-occlusive disease

PAH PAH PAH PAH

62 49 49 56

F F F F

Caucasian Caucasian Caucasian Caucasian

IPAH IPAH APAH PAH

n/a* n/a n/a n/a No abnormalities No abnormalities No abnormalities No abnormalities BMPR2: (Deletion of exons 4,5) BMPR2: (Point mutation: 961C→T) BMPR2: (966A→T, causing alternative splicing of exon 7) X-deletion X-deletion X-deletion X-deletion

8 9 10 11

*n/a. Information is not available. Some donor lungs occasionally came from outside institutions and lacked this information; PAH: pulmonary arterial hypertension; IPAH: idiopathic pulmonary arterial hypertension; APAH: associated pulmonary arterial hypertension; BMPR2: bone morphogenetic protein receptor 2

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479

Duong et al.: ECFC in pulmonary artery endothelium and PAH

Ingram and colleagues was used to isolate and culture ECFC as described.[8] We determined the quantity of ECFC contained in cultured PAEC, the numbers of cells contained in each ECFC colony, and the capacity for ECFC to form secondary colonies following subculture into larger culture vessels. Our indings indicate that altogether, 216/1140 (18.9%) cells sorted from control PAEC formed colonies, while 820/3040 (27.0%) cells from sorted PAH PAEC formed colonies. The remainder of the cells did not divide and remained as single cells. Treating data from each patient as independent events, 18.0±4.6% of control versus 25.7± 2.4% of PAH PAEC gave rise to colonies (P=0.17, Wilcoxon test). In several cases, colonies containing greater than 50 cells when seen under light microscopy were trypsinized for counting with a hemacytometer, but no cells were subsequently visualized on the hemacytometer. We imputed a value of 50 cells/colony for these cases since the colonies were seen to be there. There was a broad range of colony sizes in both groups, ranging from cells that divided only once to cells that formed colonies containing over 3,000 and 10,000 cells in the control and PAH groups, respectively. The median number of cells per colony in controls was 55±5 cells versus 209±57 cells in the PAH group (P=0.14). Eight out of 11 patients from the PAH PAEC demonstrated a median number of cells per colony greater than the highest value in the control group. Although not statistically signi icant, PAH PAEC tended to give rise to colonies with larger numbers of cells. We found that dividing cells from both control and PAH PAEC contained similar percentages of cells that gave rise to colonies of ≥50 cells (ECFC); (73.0±5.3% control vs. 75.0±5.3% PAH, P=0.40). However, PAH PAEC contained a larger proportion of highly proliferative ECFC and contained the more proliferative ECFC (Fig. 3). The median number of cells in each ECFC colony was 144±36 cells in control versus 355±64 cells PAH, P=0.03 (Fig. 4a). Thus, PAH PAEC give rise to larger ECFC colonies. There was not a statistically signi icant difference in the number of ECFC between the 2 groups. In each 96-well plate sorted, there were 14±5 ECFC in control versus 20±3 ECFC in PAH PAEC (P=0.17). This number ranged between 7 and 27 ECFC per 96-well plate in controls versus 9 and 34 ECFC/96-well plate in PAH. Both control and PAH PAEC contain ECFC supporting the hypothesis that ECFC are resident cells of the pulmonary artery endothelium. Together the data indicate that PAH pulmonary artery endothelium harbors more proliferative ECFC. ECFC are classi ied as possessing either high proliferative potential (HPP) or low proliferative potential (LPP) based on ability to form secondary colonies upon subculturing 480

ECFC

Figure 1: Schematic of ECFC assay. Cultured control and PAH PAEC were single-cell sorted into 96-well plates by FACS based on forward and side scatter properties and allowed to grow for two weeks after which wells were scored for the formation of colonies. ECFC are defined as single cells that gave rise to a colony containing ≥50 cells. ECFC were subsequently subcultured into progressively larger culture vessels. Those colonies that retain the ability to give rise to colonies upon secondary culturing were considered high proliferative potential (HPP) ECFC. ECFC which lacked this capacity are considered low proliferative potential (LPP) ECFC. The most proliferative ECFC were assessed for endothelial cell phenotype and for clonal origin.

Figure 2: Flow cytometric single cell sorting. PAEC suspended in EGM-2 were sorted into 96-well plates by FACS. FSC-A/SSC-A dot plot was used to define gate for endothelial cells. In order to prevent the sorting of >1 endothelial cell in each well aggregate correction was performed. Aggregates lead to increased SSC-W and FSC-W due to prolonged scatter of the laser due to their increased size. Use of aggregate exclusion led to 97% of wells receiving a single endothelial cell that was able to adhere to the collagen matrix when assessed 24 hrs. after sorting.

Figure 3: Distribution of number of cells per ECFC colony in PAH and control PAEC. Horizontal axis represents number of cells per ECFC colony. Vertical axis represents frequency per sample. ECFC derived from PAH PAEC gave rise to the most proliferative colonies arising from a single cell.

(Fig. 5).[8] We investigated whether there was a greater proportion of HPP ECFC in PAH PAEC compared to control. We found that 24.9±5.6% of control ECFC versus Pulmonary Circulation | October-December 2011 | Vol 1 | No 4

Duong et al.: ECFC in pulmonary artery endothelium and PAH

the leukocyte common antigen CD45, consistent with a putative role as the source of dividing endothelial cells during angiogenesis.[6-8]

Figure 4: PAH PAEC gave rise to ECFC that generate larger colonies and a higher proportion of HPP ECFC: (a) Median number of cells per ECFC colony from control and PAH PAEC. Vertical axis represents cells per ECFC colony. Each circle represents the median number of cells per ECFC colony from an individual patient. Open circles represent control and filled circles represent PAH. (b) ECFC were subcultured into 24-well plates to assess for HPP ECFC based on ability to form colonies. Vertical axis is percentage of HPP ECFC as a fraction of total ECFC.

Figure 5: HPP ECFC. ECFC colonies from 96-well plates were subcultured into 24-well plates and assessed for colony formation or confluence after 1 week. Representative images are shown. Secondary ECFC colonies ranged in size from small (A), medium (B), or large/confluent (C). Scale bar is 200 μm.

41.2±2.9% PAH ECFC were HPP ECFC, P=0.04 (Fig. 4b). HPP ECFC formed larger colonies in 96-well plates than LPP ECFC. The median number of cells per colony for LPP ECFC was 732±79 cells fewer than HPP ECFC at the 96well stage (869 cells per colony in PAH vs. 137 in controls, P<0.01), suggesting that the capacity for ECFC to form secondary colonies is directly linked to their proliferative potential. Furthermore, the most proliferative ECFC were also derived from PAH PAEC. Out of 4,180 single cells plated into 96-well plates, 15 grew to form subsequent colonies greater than 100,000 cells. All of these most proliferative ECFC were derived from PAH ECFC. Thus PAH PAEC give rise to a greater percentage of HPP ECFC and yield the more proliferative ECFC compared to control. This corroborates previous evidence demonstrating a hyperproliferative/apoptosis resistant phenotype among PAH PAEC and suggests more proliferative ECFC may contribute to this phenotype.

ECFC express endothelial cell markers

ECFC isolated from the circulation as well as other vascular beds have been demonstrated to be of endothelial origin as assessed by cell surface markers, functional assays, and gene expression pro ile.[8,12,13,24] 16ECFC express several endothelial cell-speci ic markers and do not express Pulmonary Circulation | October-December 2011 | Vol 1 | No 4

We chose to assess for the expression of CD31, vWF, cav-1, eNOS, and CD34. CD31 is expressed by platelets, endothelial cells, and macrophages and is used frequently used to stain for endothelial cells in immunohistochemical staining.[25] Von Willebrand factor, a protein that binds Factor VIII and is important for platelet adhesion, is highly expressed by endothelial cells.[26] eNOS is expressed in endothelial cells, and reduction of nitric oxide levels is implicated in the increased pulmonary vascular tone.[27] PAH PAEC express more eNOS than endothelial cells from the systemic circulation. Cav-1 is a protein that localizes to caveolae, invaginations of the plasma membrane which are found in most cells and is particularly important for vesicular traf icking in endothelial cells.[28] Cav-1 also regulates eNOS activity and may play a role in PAH pathogenesis.[27] CD34 is a cell surface protein implicated in cell migration and halting of differentiation that has been documented to be expressed in ECFC and PAEC, and which has served most commonly as a hematopoietic stem cell marker.[8,29] Our indings indicate that ECFC are endothelial cells. They express CD31, vWF, cav-1, eNOS, and CD34, consistent with the expression pattern expected of endothelial cells and corroborating previous reports on ECFC surface marker expression.[8,9] Staining of PAEC cultures prior to ECFC sorting also demonstrated expression of these markers (Fig. 6). The staining pattern was similar in both PAEC and ECFC groups, suggesting expression of CD31, vWF, Cav-1, eNOS, and CD34 is maintained in highly proliferative as well as less proliferative endothelial cells. ECFC also demonstrated uptake of acetylated LDL, a functional property of endothelial cells (Fig. 7). Endothelial cell ultrastructure is typi ied by the presence of caveloae invaginations of the plasma membrane, as well as by Weibel-Palade bodies. ECFC demonstrated the presence of both these elements as visualized by electron microscopy (Fig. 8). Thus we found strong evidence that ECFC exhibit endothelial cell phenotype and are not colonies of any potential contaminating cells in the primary PAEC cultures.

Clonality of expanded ECFC confirmed by HUMARA

The ECFC assay allows the clonogenic potential of individual endothelial cells to be assessed. In order to further verify that this methodology could isolate cells that could undergo signi icant clonal expansion, we assessed the clonality of 3 of the most proliferative clones using HUMARA. Our data suggest that the expanded ECFC were clonal in origin (Fig. 9). For PAH 3, PAEC 481

Duong et al.: ECFC in pulmonary artery endothelium and PAH

Figure 6: ECFC express endothelial cell-specific markers. Representative images are shown. Both PAEC and ECFC demonstrate positive staining for CD31, vWF, cav-1, eNOS, and CD34. vWF stains intracellularly in both PAEC and ECFC, as expected for endothelial cells. CD31 exhibits staining localized to the membrane, particularly at sites of cell-cell contact, and in the cytosol. The cell-junctional staining for CD31 staining in ECFC is not as pronounced owing to cells not being confluent. eNOS staining is present in both PAEC and ECFC, particularly in the perinuclear region. Cav-1 stains both PAEC and ECFC. There is also CD34 staining in both PAEC and ECFC in both control and PAH groups (arrows). There are no significant differences in staining pattern between PAH and control groups. Scale bar is 50 μm.

Figure 7: ECFC take up acetylated LDL. ECFC were incubated with 20 μg/mL DiI-acetylated LDL for 2 hrs. at 37°C and assessed for uptake with fluorescence microscopy. (A) Phase contrast ECFC image taken with inverted microscope (B) Strong uptake of the DiI-labeled acetylated LDL was demonstrated by ECFC. Scale bar is 100 μm.

demonstrated random X-inactivation. After methylationspeci ic digestion, both a 226 bp and 223 bp peak were ampli ied in roughly equal proportions. However, in 2 ECFC clones derived from PAH 3, there was a loss of the 223 bp peak following methylation-speci ic digestion, suggesting that the same X chromosome is inactivated in all cells, implying a common clonal origin. Similarly, in 482

Figure 8: ECFC show ultrastructural properties typical of endothelial cells. (A) ECFC demonstrate the presence of numerous caveolae along the plasma membrane (arrowheads), consistent with endothelial cell morphology. Scale bar is 500 nm. (B) ECFC also demonstrate the presence of Weibel-Palade bodies, which manufacture vWF (arrow). Scale bar is 200 nm.

PAH 11, which contains a mosaic X-chromosome deletion, the ECFC clone contained a single X-chromosome in all cells, indicating that it derived from a single cell with the X-deletion. Furthermore, the single X-chromosome was active in all cells. This veri ies the ability of the ECFC assay to isolate clones and potentially allow for studies of the most proliferative endothelial cells contained in culture. Pulmonary Circulation | October-December 2011 | Vol 1 | No 4

Duong et al.: ECFC in pulmonary artery endothelium and PAH

BMPR2 mutation display a hyperproliferative phenotype and have impaired in vitro tube-forming capacity.[16] However this work did not establish whether circulating ECFC were derived from the pulmonary arteries. Thus, it is not known whether these indings represent widespread endothelial dysfunction or a PAEC-speci ic pathology. ECFC isolated from the pulmonary vasculature itself has previously only been well-characterized in the rat lung, where ECFC were found in rat PAEC and were enriched in the pulmonary microvascular endothelial cells.[14] While this study offered important insight into the presence, distribution, and biology of ECFC in the pulmonary circulation, it did not establish a role for these cell populations in human disease. In the human pulmonary artery, the presence, quantity, and potentials of ECFC are largely unknown. The preseny study represents the irst attempt to address these questions.

Figure 9: X-inactivation analysis. Allele patterns at the androgen receptor locus are shown before (left panel) and after digestion with the methylationsensitive restriction enzyme HhaI (right panel). Alleles are labeled with their size in basepairs. (a) Patient PAH 3. PAEC DNA shows an identical pattern before and after HhaI digestion, indicating random X-inactivation, whereas the two ECFC show a single peak after digestion, consistent with clones derived from a single cell. (b) Patient PAH 11 has a known mosaic X-chromosome deletion, with loss of the 241bp allele in approximately 50% of cells. HhaI digestion reveals significant skewing of X-inactivation in the â&#x20AC;&#x153;parentâ&#x20AC;? PAEC culture. The ECFC clone contains a single 232bp allele, indicating that it is derived from a cell carrying the X-chromosome deletion. Furthermore, there was no amplification following HhaI digestion, suggesting that the single X-chromosome is active in all cells.

DISCUSSION ECFC are proliferative endothelial cells that demonstrate in vitro and in vivo angiogenic capacity, are structural cells of the endothelium, and are present but rare in the circulation.[8,12,30,31] They have been isolated from cultured endothelial cells from the human umbilical vein and aorta, as well as the rat pulmonary arteries and microvessels.[12,14] ECFC possess signi icant clonal proliferative potential and are implicated in both physiologic and pathologic angiogenesis. For example, ECFC demonstrate the ability to participate in vascular repair in mouse models of hind limb ischemia.[32,33] Conversely, circulating ECFC dysfunction has been demonstrated in diseases of dysregulated angiogenesis, such as diabetes mellitus and PAH.[15,16] This combination of proliferative capacity, in vivo angiogenic function, and evidence of dysfunction in diseases of aberrant angiogenesis all argue for a crucial role of ECFC in angiogenesis, perhaps as the source of endothelial cells required for new vessel formation. A role for ECFC in PAH pathogenesis has been suggested from work done by Toshner and colleagues, who documented that circulating human ECFC in PAH with Pulmonary Circulation | October-December 2011 | Vol 1 | No 4

In this study, we demonstrated a simple method to isolate ECFC using low cytometry to sort single endothelial cells into 96-well plates based on forward and side scatter with aggregate exclusion. We con irmed single-cell sorting by visual inspection with eosin staining and by indirect assessment of clonality (HUMARA). Using this method, we identi ied the presence of ECFC in cultured human PAEC derived from control and PAH populations. Our methodology notably avoided the use of retroviral vectors to introduce GFP into endothelial cells as described in the literature, which has the potential to introduce unforeseen changes to cell growth as well as adding to the biohazard for the manipulation of these cells.[8,12] We observed that ECFC among PAEC are not rare, representing between 5-30% of all sorted endothelial cells in the healthy and PAH subject samples that we studied. There was a wide range of proliferative capacities among ECFC, with some giving rise to over a million cells while others proliferated to 50 cells and were unable to form colonies after subculturing. We found that PAH PAEC tend to give rise to larger ECFC colonies than controls and also contained more HPP ECFC, suggesting PAH ECFC proliferate more or are more apoptosis-resistant than control. Although there was not a statistically signi icant difference in the number of ECFC between control and PAH groups, the number of cells per ECFC colony was higher in PAH and the most proliferative cells after subculturing of ECFC were also found in the PAH group. Sorted PAH PAEC demonstrated a skew towards a more proliferative phenotype compared to control. Overall these observations corroborate a previous report by our group demonstrating PAH PAEC in culture exhibit a hyperproliferative/apoptosis-resistant phenotype,[21] as well as indings by Toshner and colleagues that circulating ECFC are hyper-proliferative in PAH.[16,21] Our indings indicate that more proliferative ECFC are present among PAH PAEC and may be linked to the hyper-proliferative phenotype of these cells in relation to control. 483

Duong et al.: ECFC in pulmonary artery endothelium and PAH

Our indings differ somewhat from previous studies on ECFC in the systemic circulation. We overall observed less ECFC proliferation than was reported in previous studies of ECFC. For example, Ingram and colleagues noted that human cord blood-derived ECFC could achieve over 100 population doublings and human umbilical vein endothelial cells (HUVEC) and human aortic endothelial cells (HAEC) could be passaged to 40 or more population doublings.[8,12] In our study 2 ECFC clones isolated from a single PAH patient (PAH 9) each achieved at least 20 population doublings. A smaller percentage of sorted PAEC divided (18.0Âą4.6% of control and. 25.7Âą 2.4% of PAH) compared to previous reports by Ingram et al. demonstrating >50% of cells divided among HUVEC and HAEC. Reasons that a sorted, adherent cell would not divide include it undergoing apoptosis or exiting the cell cycle and becoming quiescent. Re lecting this, the discrepancy between our indings and those of other groups may be multifactorial. Ingram and colleagues studied commercially available HAEC and HUVEC at passage 3, while this study included PAEC from passage 5.[12] Cells from later passages might be expected to possess either diminished proliferation, increased apoptosis, or contain a greater proportion of quiescent cells. We were unable to assess what proportion of non-dividing cells exhibited each of these traits. The amount of time cells are frozen and GFP overexpression may also affect the number of cell divisions they can undergo. Furthermore, as has been reported in rats, it is possible that in humans more proliferative ECFC reside in the pulmonary microvasculature compared to the pulmonary arteries.[14] Nonetheless, the identi ication of ECFC with signi icant proliferative potential in the pulmonary circulation mirrors what has been reported in the systemic circulation. Taken with the fact that ECFC have also been isolated from HUVEC and rat pulmonary microvasculature, it suggests that ECFC are general structural cells of the endothelium. The ability to use the ECFC assay to clonally isolate and characterize pulmonary artery-derived ECFC can add to the growing body of knowledge regarding endothelial cell heterogeneity in the lung. Endothelial cell heterogeneity in the systemic circulation is well established, and there is increasing understanding of differences in pulmonary endothelial cell phenotype,[34] including segmentally dependent variations in nitric oxide production, barrier properties, surface antigen expression, and proliferative potential.[14,34,35] The majority of this knowledge is based on rat studies and thus much work remains to be done in humans.[35,36] Elucidating PAEC heterogeneity could be important to broadly understanding how the pulmonary vasculature responds to stress, and speci ically PAH pathogenesis. PAH demonstrates segment-speci ic pathology.[37] For example, plexiform lesions arise in the 484

distal precapillary circulation and are characterized by minimal muscularization while more proximally, lesions are luminal and concentric in nature, with exaggerated smooth muscle hyperplasia and invasion.[37] The ECFC assay can allow for expansion of distinct clonal populations of cells permitting a detailed study of endothelial heterogeneity that cannot be achieved simply through analysis of cells in bulk culture. It is possible, for example, that even within the same segment of the pulmonary artery only a small subset of cells produce factors that mobilize pro-angiogenic progenitors, or stimulate other endothelial cells to divide. These cells could be isolated and expanded by the ECFC assay. Additionally, in light of recent work by Aldred and colleagues demonstrating chromosomal disturbances in PAH PAEC, the ECFC assay also allows for detailed genetic analyses of whether multiple genetic hits identi ied in culture arise from separate hits in different cells or multiple hits within the same cell.[22] Thus the ECFC assay could be used to identify clonal genetic changes in PAEC that may confer a selective growth advantage. More broadly, the ECFC assay allows for the characterization of the endothelial cells with the most proliferative potential. In the setting of PAH, there is widespread PAEC dysfunction, including a reduction in nitric oxide production, metabolic switch to glycolysis, and upregulation of HIF and HIF-inducible factors. [19,38] While these characteristics are noted of PAEC in culture, it is not known whether they are present in the most proliferative cells. The clonal expansion and subsequent characterization of cells in the ECFC assay could allow for determination of whether there is correlation between endothelial dysfunction and proliferative potential of a cell. This knowledge may help elucidate whether increased proliferation and other types of endothelial dysfunction are intrinsically linked. Many additional questions remain unanswered. The in vivo functionality of the ECFC was not assessed, and thus, their relevance to angiogenesis could not be directly ascertained. Furthermore, where in the pulmonary tree the most proliferative ECFC were derived is not known, since the preparation process does not distinguish between segments of the pulmonary arteries. This hampers a segmental-analysis of PAEC heterogeneity. It is possible that, given plexiform lesions in PAH are comprised of monoclonal cell proliferations,[39] ECFC may give rise to these lesions seen in end-stage disease. Our study was also limited in sample size, and did not allow for statistically meaningful comparisons between different subclasses of PAH. Lastly, an existing challenge in current ECFC understanding is that it is not known what distinguishes the more proliferative from less proliferative ECFC or whether they Pulmonary Circulation | October-December 2011 | Vol 1 | No 4

Duong et al.: ECFC in pulmonary artery endothelium and PAH

are truly phenotypically distinct from other endothelial cells. The differences in proliferation potential can currently only be assessed by the ECFC assay. No markers distinguish ECFC from endothelial cells or predict their proliferative capacity.[8] Whether ECFC represent a true progenitor cell is also not known, as no studies have shown whether they exhibit asymmetric division or have the ability to differentiate into non-endothelial cells.

11.

12.

13.

The ECFC assay allows for the proliferative capacity of individual endothelial cells to be interrogated and may be a potential tool to help elucidate functional and genetic heterogeneity among endothelial cells. In this study we reported development of a simpli ied method for ECFC assay using non-manipulated endothelial cells. Using this method we showed that numerous ECFC are present in the pulmonary artery endothelium. PAH pulmonary artery endothelium exhibit high proliferative ECFC compared to healthy controls. The methods and indings described herein represent another step towards unraveling the mechanisms by which PAEC contribute to this devastating disease.

14.

15.

16.

17.

18.

ACKNOWLEDGMENTS

19.

The authors thank Denise Hatala (immunohistochemistry), Mei Yin (electron microscopy) and Dr. John Peterson (light microscopy) in the Lerner Research Institute Digital Imaging Core. In the Flow Cytometry core, we thank Moneen Morgan, Sage O’Bryant and Cathy Shemo for technical assistance with instrument operation. We also thank Dr. Amy Nowacki in the Quantitative Health Sciences Department for advice on statistical analysis.

20.

21.

22.

23.

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AC133-positive cells suggests a possible role of endothelial progenitor cells in the formation of choroidal neovascularization. Invest Ophthalmol Vis Sci 2006;47:1642-5. Thill M, Strunnikova NV, Berna MJ, Gordiyenko N, Schmid K, Cousins SW, et al. Late outgrowth endothelial progenitor cells in patients with agerelated macular degeneration. Invest Ophthalmol Vis Sci 2008;49: 2696-708. Ingram DA, Mead LE, Moore DB, Woodard W, Fenoglio A, Yoder MC. Vessel wall-derived endothelial cells rapidly proliferate because they contain a complete hierarchy of endothelial progenitor cells. Blood 2005;105:2783-6. Yoder MC, Mead LE, Prater D, Krier TR, Mroueh KN, Li F, et al. Redefining endothelial progenitor cells via clonal analysis and hematopoietic stem/ progenitor cell principals. Blood 2007;109:1801-9. Alvarez DF, Huang L, King JA, ElZarrad MK, Yoder MC, Stevens T. Lung microvascular endothelium is enriched with progenitor cells that exhibit vasculogenic capacity. Am J Physiol Lung Cell Mol Physiol 2008;294: L419-30. Tan K, Lessieur E, Cutler A, Nerone P, Vasanji A, Asosingh K, et al. Impaired function of circulating CD34(+) CD45(-) cells in patients with proliferative diabetic retinopathy. Exp Eye Res 2010;91:229-37. Toshner M, Voswinckel R, Southwood M, Al-Lamki R, Howard LS, Marchesan D, et al. Evidence of dysfunction of endothelial progenitors in pulmonary arterial hypertension. Am J Respir Crit Care Med 2009; 180:780-7. Asosingh K, Aldred MA, Vasanji A, Drazba J, Sharp J, Farver C, et al. Circulating angiogenic precursors in idiopathic pulmonary arterial hypertension. Am J Pathol 2008;172:615-27. Montani D, Perros F, Gambaryan N, Girerd B, Dorfmuller P, Price LC, et al. C-kit-positive cells accumulate in remodeled vessels of idiopathic pulmonary arterial hypertension. Am J Respir Crit Care Med 2011; 184:116-23. Farha S, Asosingh K, Xu W, Sharp J, George D, Comhair S, et al. Hypoxiainducible factors in human pulmonary arterial hypertension: A link to the intrinsic myeloid abnormalities. Blood 2011;117:3485-93. Diller GP, van Eĳl S, Okonko DO, Howard LS, Ali O, Thum T, et al. Circulating endothelial progenitor cells in patients with Eisenmenger syndrome and idiopathic pulmonary arterial hypertension. Circulation 2008;117:3020-30. Masri FA, Xu W, Comhair SA, Asosingh K, Koo M, Vasanji A, et al. Hyperproliferative apoptosis-resistant endothelial cells in idiopathic pulmonary arterial hypertension. Am J Physiol Lung Cell Mol Physiol 2007;293:L548-54. Aldred MA, Comhair SA, Varella-Garcia M, Asosingh K, Xu W, Noon GP, et al. Somatic chromosome abnormalities in the lungs of patients with pulmonary arterial hypertension. Am J Respir Crit Care Med 2010;182:1153-60. Amos-Landgraf JM, Cottle A, Plenge RM, Friez M, Schwartz CE, Longshore J, et al. X chromosome-inactivation patterns of 1,005 phenotypically unaffected females. Am J Hum Genet 2006;79:493-9. Hirschi KK, Ingram DA, Yoder MC. Assessing identity, phenotype, and fate of endothelial progenitor cells. Arterioscler Thromb Vasc Biol 2008;28:1584-95. Woodfin A, Voisin MB, Nourshargh S. PECAM-1: A multi-functional molecule in inflammation and vascular biology. Arterioscler Thromb Vasc Biol 2007;27:2514-23. Wang JW, Eikenboom J. Von Willebrand disease and Weibel-Palade bodies. Hamostaseologie 2010;30:150-5. Mathew R, Huang J, Gewitz MH. Pulmonary artery hypertension: Caveolin-1 and eNOS interrelationship: A new perspective. Cardiol Rev 2007;15:143-9. Majkova Z, Toborek M, Hennig B. The role of caveolae in endothelial cell dysfunction with a focus on nutrition and environmental toxicants. J Cell Mol Med 2010;14:2359-70. Nielsen JS, McNagny KM. CD34 is a key regulator of hematopoietic stem cell trafficking to bone marrow and mast cell progenitor trafficking in the periphery. Microcirculation 2009;16:487-96. Yoder MC, Ingram DA. The definition of EPCs and other bone marrow cells contributing to neoangiogenesis and tumor growth: Is there common ground for understanding the roles of numerous marrow-derived cells in the neoangiogenic process? Biochim Biophys Acta 2009;1796:50-4. Duong HT, Erzurum SC, Asosingh K. Pro-angiogenic hematopoietic progenitor cells and endothelial colony-forming cells in pathological angiogenesis of bronchial and pulmonary circulation. Angiogenesis 2011;14:411-22.

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Jonigk D, Golpon H, Bockmeyer CL, Maegel L, Hoeper MM, Gottlieb J, et al. Plexiform lesions in pulmonary arterial hypertension composition, architecture, and microenvironment. Am J Pathol 2011;179:167-79. Xu W, Koeck T, Lara AR, Neumann D, DiFilippo FP, Koo M, et al. Alterations of cellular bioenergetics in pulmonary artery endothelial cells. Proc Natl Acad Sci U S A 2007;104:1342-7. Lee SD, Shroyer KR, Markham NE, Cool CD, Voelkel NF, Tuder RM. Monoclonal endothelial cell proliferation is present in primary but not secondary pulmonary hypertension. J Clin Invest 1998;101:927-34.

Source of Support: American Heart Association 11SDG4990003, American Thoracic Society/Pulmonary Hypertension Association grant (PH07-003), and National Institutes of Health grants RC37 HL60917 and R01 HL098199. KA is a scholar of the International Society for Advancement of Cytometry. HD is a Howard Hughes Medical Institute Medical Research Fellow (Grant ID 57006973), Conflict of Interest: None declared.

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Research Ar t i cl e

Hypoxia modulates the expression of leucine zipper-positive MYPT1 and its interaction with protein kinase G and Rho kinases in pulmonary arterial smooth muscle cells Dev K. Singh, Joy Sarkar, Aarti Raghavan, Sekhar P. Reddy, and J. Usha Raj Department of Pediatrics, Division of Developmental Biology and Basic Research, University of Illinois at Chicago, Childrenâ&#x20AC;&#x2122;s Hospital University of Illinois, Chicago, IL, USA, 1st & 2nd Author contributed equally

ABSTRACT We have shown previously that acute hypoxia downregulates protein kinase G (PKG) expression and activity in ovine fetal pulmonary vessels and pulmonary arterial smooth muscle cells (SMC). Here, we report that acute hypoxia also reduces the expression of leucinezipper-positive MYPT1 (LZ+ MYPT1), a subunit of myosin light chain (MLC) phosphatase, in ovine fetal pulmonary arterial SMC. We found that in hypoxia, there is greater interaction between LZ+MYPT1 and RhoA and Rho kinase 1 (ROCK1)/Rho kinase 2 (ROCK2) and decreased interaction between LZ+MYPT1 and PKG, resulting in increased MLC20 phosphorylation, a higher pMLC20/MLC20 ratio and SMC contraction. In normoxic SMC PKG overexpression,LZ+MYPT1 expression is upregulated while PKG knockdown had an opposite effect. LZ+MYPT1 overexpression enhanced the interaction between PKG and LZ+MYPT1. Overexpression of a mutant LZ-MYPT1 isoform in SMC mimicked the effects of acute hypoxia and decreased pMLC20/MLC20 ratio. Collectively, our data suggest that hypoxia downregulates LZ+MYPT1 expression by suppressing PKG levels, reduces the interaction of LZ+MYPT1 with PKG and promotes LZ+MYPT1 interaction with RhoA or ROCK1/ROCK2, thereby promoting pulmonary arterial SMC contraction. Key Words: hypoxia-induced pulmonary hypertension, signal transduction, cGMP, pulmonary vasoconstriction

INTRODUCTION Acute hypoxia leads to a rapid reversible physiological response of pulmonary vasoconstriction (reviewed in[1]). Myosin light chain (MLC) phosphorylation is a critical step that leads to activation of myosin ATPase and subsequent smooth muscle cell (SMC) contraction. Myosin light chain 20 (MLC20) is phosphorylated by MLC kinase (MLCK) and de-phosphorylated by MLC phosphatase (MLCP). Although a recent study has shown that MAP kinase phosphorylates MLC independent of MLCK in urinary bladder SMC,[2] MLCK is still considered to be the main enzyme responsible for phosphorylation of MLC20 and MLC phosphatase is the main enzyme involved in regulation of MLC20dephosphorylation and vasodilatation.[3,4] In general, the contractile state of the SMC re lects the ratio of activities of MLCK/MLCP and pMLC20/MLC20. Address correspondence to: Dr. Dev K. Singh Department of Pediatrics, Division of Developmental Biology and Basic Research, University of Illinois at Chicago, Chicago, IL 60612, USA Email: devkaram@yahoo.com Pulmonary Circulation | October-December 2011 | Vol 1 | No 4

MLCP is a hetero-trimeric protein composed of a catalytic subunit PP1Cď ¤ (see References 5 and 6 for review), a regulatory subunit also known as myosin phosphatase targeting unit (MYPT) and a 20 kD protein of unknown function. Regulatory subunits of the MYPT family are the central targeting proteins thatinteract with a number of other signaling molecules such as protein kinase G (PKG), Rho kinase 1 (ROCK1) and Rho kinase 2 (ROCK2) (reviewed in Reference 7). There are fourdifferent types of MYPT1 monomers produced endogenously by splicing, i.e. MYPT1 with or without central insert (CI), and both these forms are further spliced with (LZ+MYPT1) or Access this article online Quick Response Code:

Website: www.pulmonarycirculation.org DOI: 10.4103/2045-8932.93548 How to cite this article: Singh DK, Sarkar J, Raghavan A, Reddy SP, Raj JU. Hypoxia modulates the expression of leucine zipper-positive MYPT1 and its interaction with protein kinase G and Rho kinases in pulmonary arterial smooth muscle cells. Pulm Circ 2011;1:487-98.

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without (LZ-MYPT1) LZ, a leucine zipper at the c-terminal. The speci icity of MLCP is primarily determined by the interaction of PP1cä with the targeting protein MYPT.[8,9] It has been reported that the sensitivity of smooth muscle to cGMP-induced relaxation correlates with the relative expression of LZ+MYPT1 and LZ-MYPT1 isoforms, as overexpression of LZ+ MYPT1 or LZ- MYPT1 isoforms in cultured SMC modulates cGMP-mediated MLC20 dephosphorylation. [10-12] Smooth muscle contractility is modulated by the ratio of phosphorylated MLC 20 to unphosphorylated MLC20 (pMLC20/MLC20), and this ratio is regulated by the relative activities of MLCK/ MLCP.[3,4]

in DMEM containing 10% heat-inactivated fetal bovine serumand antibiotics (Invitrogen, Carlsbad, Calif., USA). Primary ovine FPASMC were con irmed to beSMCs by their typical “hill and valley” morphology and by α-smooth muscle actin immuno luorescent staining. Contamination with endothelial cells is ruled out by negative immuno luorescent staining with an anti-von Willebrand factor VIII antibody. All experiments were performed with cells at passages 4–6.

Hypoxia exposure

MATERIALS AND METHODS

Subcon luent FPASMC were serum-starved (0.5% serum) for 16 h and then exposed to 4 h of hypoxia or normoxia at 37°C, as previously described.[13] Brie ly, cells were placed in a specialized hypoxia chamber into which a gas mixture of 0% O2, 5% CO2 and balance nitrogen was lowed in at a rate suf icient to keep O2 concentration within the chamber at 3%. The concentration of O2 within the chamber was constantly monitored and controlled with an O2 controller (Model ProOxC, Biospherix, Lacona, NY, USA).[13] The PO2 in the cell media during hypoxia was 30–40 Torr and during normoxia was 100 Torr. For normoxia experiments, cells were incubated in a humidi ied incubator with a constant supply of 5% CO2 at 37°C.

Reagents

siRNA transfection

We tested the hypothesis that hypoxia regulates the level of LZ+MYPT1 and LZ-MYPT1 isoforms, and thereby SMC contractility, in cultured ovine fetal pulmonary arterial SMC (FPASMC). We report that hypoxia downregulates the expression of LZ+MYPT1, a predominant isoform in vascular smooth muscle in a PKG-dependent manner, reduces the interaction between LZ+MYPT1 andPKG and promotes its interactions with ROCK1/ROCK2, resulting in increased MLC phosphorylation and SMC contraction.

Trolox [(+)-6-hydroxy-2,5,7,8-tetramethyl-chroman-2carboxylic acid], N-acetyl cysteine (NAC) and all other chemicals (unless otherwise speci ied) were obtained from Sigma-Aldrich (St. Louis, Mo., USA). 8-Bromo-cGMP (8-Br-cGMP) was purchased from Axxora LLC/Biolog Life Science Institute (San Diego, Calif., USA).

Animals

Pregnant ewes carrying single or twin fetuses (140 days of gestation; term being 147 days, either sex) were obtained from Nebeker Ranch in Lancaster, California (altitude 300 m.; arterial PO2 (PaO2): 102 +2 Torr). After the fetuses were delivered, each ewe was euthanized with T-61 euthanasia solution (Hoechst-Roussel, Somerville, NJ, USA). All procedures and protocols used in the present study were approved by the Animal Research Committees of Loma Linda University, Los Angeles Biomedical Research Institute at Harbor-UCLA and by the University of Illinois at Chicago.

Tissue preparation and cell culture

Fourth-generation pulmonary arteries (outside diameter: 1.5–2.5 mm) were dissected free of parenchyma and kept in ice-cold modi ied Krebs-Ringer bicarbonate buffer (composition in mM: 118.3 NaCl, 4.7 KCl, 2.5 CaCl2, 1.2 MgSO4, 1.2 KH2PO4, 25.0 NaHCO3 and 11.1 glucose) and primary ovine FPASMC were isolated from pulmonary arteries as described earlier.[13] Cells were maintained 488

We used PKG-speci ic small interfering ribonucleic acid (siRNA) to knockdown PKG expression in FPASMC.[13] A nonsilencing oligonucleotide sequence (nonsilencingsiRNA) that does not recognize any known homology to mammalian genes was used as a negative control (Qiagen, Valencia, Calif., USA cat. no. 1022563). FPASMC were transfected with siRNAs using Lipofectamine 2000 reagent (Invitrogen). Fresh medium was added after 6 h of transfection and the cells were further cultured for 24 hbefore exposing them to hypoxia or normoxia for 4 h. PKG1 expression level and activity was determined as described earlier.[13] FPASMC were treated with 210-4M Trolox (an ONOO- scavenger) or 110-3M NAC for 30 min before exposure to 4 h hypoxia.

Transient transfections To overexpress PKG1, we used the Lenti-X overexpression system (Clontech, Mountain View, Calif., USA) as recommended by the manufacturer. In brief, Lent-X vector containing a full-length PKG1α tagged with green luorescent protein (GFP) was co-transfected along with a Lenti-X HT Packaging Mix into 293 T cell line using Lipofectamine 2000. Lentiviral supernatant produced by the transfected packaging cells was then used to infect FPASMC along with polybrene (4g/mL). FPASMC overexpressing PKG1 were selected by Puromycin (1.5 g/mL). Lentiviral negative control cells were included in all the experiments. After 24 h of transfection, Pulmonary Circulation | October-December 2011 | Vol 1 | No 4

Singh et al.: Hypoxia modulation and interaction

cells were serum-starved overnight before exposure to hypoxia or normoxia for 4 h. A full-length MYPT1 (+LZ) construct with all the functional domains in MYPT1 and a MYPT1 (-LZ) construct lacking the LZ domain, gifts from F.V. Brozowich,[14] were puri ied and their sequence veri ied before use in experiments. Cells weretransfected with MYPT1+LZ and MYPT-LZconstructs using lipofectamine 2000 (Invitrogen) and subsequently exposed to normoxia. Empty vector, pcDNA3.1, was transfected as a negative control. Transfection ef iciency was analyzed by immunoblot analysis using appropriate antibodies.

Western blot analysis

After each experimental exposure, FPASMC were harvested in RIPA buffer (20 mMTris-HCl–pH 7.5, 150 mMNaCl, 1 mM EDTA, 1 mM EGTA, 1% IGEPAL, 2.5 mM sodium pyrophosphate, 1mM β-glycerophosphate) containing protease and phosphatase inhibitor cocktails (Sigma-Aldrich) and protein concentration was determined using the Bradford protein assay kit (Biorad, Hercules, Calif., USA). Equal amounts of total protein (10–50 μg) from cell lysates were subjected to sodium dodecyl sulfate—polyacrylamide gel electrophoresisand blotted onto nitrocellulose membrane and probed with primary antibodies according to the manufacturer’s instructions. Horseradish peroxidase (GE Life Sciences, Piscataway, NJ, USA) was used as secondary antibody at concentrations from 1:2,000 to 1:20,000. Immunoreactive bands were detected using SuperSignal West Pico Chemiluminescent Substrate (Pierce , Rockford, Ill). The relative intensities of immunoreactive bands were quanti ied by densitometry using -actin band as a reference. The primary antibodies used for this study include antiMYPT1, anti-pMYPT1 (Ser695) and anti-MLC20 (Santa Cruz Biotech, Santa Cruz, Calif.); anti-pMLC20 (Ser18/Ser19) from Cell Signaling, Danvers, Mass., USA; anti-PKG1α and anti-actin antibodies from Calbiochem, San Diego, CA, USA; and MYPT1 monoclonal antibody (BD) and LZ+MYPT1 antibody (custom antibody from Af inity Bioreagent, Rockford, Ill., USA). The amount of PKG-mediated MLCP phosphorylation was determined by measuring phosphorylation of MYPT1 at Ser695 using pMYPT1Ser695 antibodies (Santa Cruz Biotechnology, Santa Cruz, Calif., USA). Also, phosphorylation of MLC at Thr 18/Ser 19 and MLC20 was evaluated by immunoblotting with the respective antibodies (Santa Cruz Biotechnology).

Immunoprecipitation

Total proteins from FPAMSC were extracted in RIPA buffer after exposure to acute hypoxia or normoxia for 4 h, as described earlier. One microgram of polyclonal anti-RhoA, ROCK 1, ROCK2 or PKG1α antibody was prebound with Ultralink immobilized protein A/G (Pierce) by gentle Pulmonary Circulation | October-December 2011 | Vol 1 | No 4

shaking for 1 h at room temperature. The prebound primary antibody and protein A/G agarose complex was then added to each sample (200 μg protein) and incubated at 4°C overnight. The agarose beads were washed in tris buffered saline (TBS) three-times and boiled in 1x sample buffer (Bio-rad). Western blot analysis was performed as described above.

Statistical analysis

Statistical analysis of the data was performed using a standard two-sample Student’s t-test assuming unequal variances of the two data sets and one-way ANOVA for the comparison of multiple experimental groups. Statistical signi icance was determined using a two-tailed distribution and was set at the 5% level (P<0.05).

RESULTS Hypoxia downregulates PKG and LZ+MYPT1 expression in FPASMC

Cells at 75% confluence were exposed to hypoxia or normoxia for 4 h, cell lysates prepared and separated on acrylamide gels. Membranes were probed with anti-PKG, anti-LZ +MYPT1 or anti-â-actin antibodies. Bands were quantified by densitometry using â-actin as a reference. As shown in Figure 1, hypoxic exposure significantly decreased (by 20–35%) the expression levels of PKG (Fig. 1 a and b) and LZ+MYPT1 (Fig. 1 c and d) in FPASMC.

PKG Knockdown mimics the effects of acute hypoxia on LZ+MYPT1 expression in FPASMC

Next, we determined whether lower expression levels of PKG would mimic the effects of acute hypoxia on LZ+MYPT1 expression. FPASMC were transfected with siRNA speci ic for PKG (si-PKG) or nonsilencingsiRNA that does not recognize any known homology to mammalian genes. PKG and LZ+MYPT1 expression was analyzed by Western blot analysis. There was a 45–60% decrease in PKG expression in siPKG-transfected cells compared with control cells (Fig. 2a and b). There was no signi icant change in the expression level of PKG and LZ+MYPT1 between mocktransfected FPASMC and nonsilencingsiRNA-transfected control cells (Fig. 2a–d). PKG knockdown caused decreased LZ+MYPT1 expression (Fig. 2c and d). This result suggests that hypoxia downregulates LZ+MYPT1 expression by reducing PKG expression.

Overexpression of PKG abrogates the effect of acute hypoxia on LZ+MYPT1 expression

To determine whether overexpression of PKG can abrogate the effect of acute hypoxia on LZ+MYPT1 expression, a plasmid encoding a full-length PKG1α (PKG-GFP) tagged with a GFP was transfected into FPASMC exposed to 489

Singh et al.: Hypoxia modulation and interaction

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* 75 50 25 0 Normoxia

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Figure 1: Effect of hypoxia on protein kinase G (PKG) and LZ+MYPT1 expression.Fetal pulmonary arterial smooth muscle cells were exposed to acute hypoxia (4 h) and cell lysates were prepared and probed with anti-PKG1á, anti-LZ+MYPT1 and â-actin antibodies. (a and c) Western blot analysis of PKG and LZ+MYPT1, respectively.(b and d) Quantification of PKG and LZ+MYPT1 expression. Bands were quantified and normalized to that of â-actin bands. The values are shown as percent of normoxia control after normalizing the data to respective â-actin values. Data represent means±SE, from at leastthree3 independent experiments. *P<0.05 compared with normoxia.

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80 60 40

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Figure 2: The effect of protein kinase G (PKG) knockdown on LZ+MYPT1 expression. Fetal pulmonary arterial smooth muscle cellsFPASMC were transfected with siRNA specific for PKG or non-silencings iRNA, as a negative control. Cell lysates were isolated and probed with antibodies specific for PKG, LZ+MYPT1, and ↓-actin. (a) Representative Western blot analysis of PKG expression. (b) Quantification of PKG expression. Bands were quantified and normalized to that of ↓-actin bands. (c and d) Analysis of LZ+MYPT1 expression in PKG-–siRNA siRNA-transfected cells. Samples as in panel A, were probed with antibodies specific for LZ+MYPT1 and ↓-actin. (d) Densitometry quantification of LZ+MYPT1 expression. Data represent means±SE from at least three to six3-6 independent experiments. *P<0.05 compared with the respective control (un-transfected cells). 490

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normoxia or hypoxia for 4 h and the expression of PKG1á and LZ+MYPT1 was analyzed by immunoblot analysis. Overexpression of PKG1α (Fig. 3 a and b) signi icantly increased LZ+MYPT1 expression in normoxic cells and also blocked the inhibitory effects of hypoxia on LZ+MYPT1 expression (Fig. 3c and d). These data further support our contention that hypoxia by reducing PKG1á expression suppresses LZ+MYPT1 levels in FPASMC.

Reactive oxygen species, by reducing PKG expression, decrease LZ+MYPT1 levels in hypoxia

Reactive oxygen species (ROS) generated in hypoxia(23, 28-30) have been shown to play a role in the downregulation of PKG expression.[15] To test whether ROS are directly involved in the downregulation of LZ+MYPT1 expression, FPASMC were pretreated with ROS scavengers, N-acetyl cysteine (NAC) or trolox (scavenger of peroxynitrite) before exposing them to hypoxia. There was no change in the expression level of PKG or LZ+MYPT1 in cells preincubated with trolox or NAC under normoxia (Fig. 4). However, trolox or NAC blocked (a)

the downregulation of PKG and LZ+MYPT1 expression by hypoxia (Fig. 4a–d).To determine whether loss of LZ+MYPT1 expression in hypoxia-exposed cells is attributable to decreased levels of PKG expression or enhanced levels of ROS production, cells were transfected with PKG-siRNA and then exposed to hypoxia in the presence or absence of troloxor NAC. Under these conditions, trolox or NAC could not restore hypoxia-induced reduction in LZ+MYPT1 expression in PKG-siRNA transfected cells (Fig. 5a–g).This result suggests that ROS regulate LZ+MYPT1 expression only indirectly by modulating PKG expression in hypoxia.

Role of PKG and/or ROS in hypoxia-mediated decrease in MLCP activity

To further determine the mechanism for inactivation of MLCP in hypoxia, FPASMC transfected with PKG-siRNA or nonsilencingsiRNA were exposed to hypoxia in the presence and absence of trolox or NAC. Cell extracts were isolated, blotted onto membrane and probed with native MYPT1 or phospho-speci ic (Ser695) MYPT1 antibodies. The (b)

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Figure 3: Overexpression of protein kinase G (PKG) inhibits LZ+MYPT1 down-regulation by hypoxia. Fetal pulmonary arterial smooth muscle cells FPASMC were transfected with a plasmid encoding a full-length PKG1α tagged with green fluorescent protein (GFP). PKG-–GFP GFP-expressing cells were exposed to acute hypoxia (4 h) and then expression levels of PKG (a) and LZ+MYPT1 (c) were analyzed. Panels b and d are the quantified levels of PKG and LZ+MYPT1 expression analyzed by immunoblot analysis. Data represent means + SE (n=3). *P<0.05 compared with respective control (normoxia). Pulmonary Circulation | October-December 2011 | Vol 1 | No 4

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Figure 4: Hypoxia decreases LZ+MYPT1 expression via reactive oxygeb species-dependent protein kinase G (PKG) reduction. Cells were exposed to acute hypoxia (4 h) in the presence or absence of trolox (100 ìM) or 1 mM N-acetyl cysteine. (a, c and e) Representative immunoblots showing the levels of PKG, LZ+MYPT1 and pMYPT1 (Ser695), respectively. (b, d, f and g) Quantitative analysis of PKG (B) LZ+MYPT1 (D), pMYPT1 (Ser695, F) and pMYPT1/LZ+MYPT1 (g). The values are represented as percent of control (normoxia) after normalizing the data to the respective â-actin. Data represent means+SE from at least three independent experiments. *P<0.05 compared with the respective control (normoxia) groups. 492

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Figure 5: Hypoxia decreases LZ MYPT1 expression via reactive oxygen species-dependent protein kinase G (PKG) reduction. Cells were transfected with siRNA specific for PKG or nonsilencings iRNA and then exposed to acute hypoxia (4 h) in the presence or absence of trolox (100 ìM) or 1 mM NAC. (a, c and e) Representative immunoblots showing the levels of PKG, LZ+MYPT1 and pMYPT1 (Ser695), respectively. (b, d, f and g) Quantitative analysis of PKG (b) LZ+MYPT1 (D), pMYPT1 (Ser695, F) and pMYPT1/LZ+MYPT1 (g). The values represented as percent of control (normoxia) after normalizing the data to respective â-actin. Data represent means + SE from at least three independent experiments. *P<0.05 compared with the respective control (normoxia) groups. +

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phosphorylation of Ser695 represents the activated state of MYPT1. We found decreased (two-fold) levels of MYPT1 phosphorylation at Ser695 in both acute hypoxia (Fig. 4a–g) and PKG-siRNA transfected cells treated with trolox or NAC (Fig. 5a–g), but not in trolox- or NAC-treated cells not transfected with PKG-siRNA (Fig. 4e and f). This result suggests that hypoxia inactivates MLCP by decreasing PKGmediated MYPT1 phosphorylation at Ser695.

Overexpression of MYPT1 lacking LZ motif (LZMYPT1) mimics the effects of hypoxia on PKGmediated LZ+MYPT and MLCP phosphorylation

FPASMC that were transfected with a MYPT1 ( -LZ)

construct lacking the leucine zipper showed signi icantly decreased levels of endogenous LZ+MYPT1 in normoxia, similar to the decreased levels following hypoxia (4h). Likewise, there was a signi icantly decreased (50% of control values) level of Ser695 phosphorylation in MYPT1 in ( -LZ) transfected cells as compared with control untransfected cells in normoxia. Because the MYPT1 (-LZ) construct lacks the leucine zipper, PKG was unable to bind and phosphorylate it at Ser695 (Fig. 6a and c). There was a two-fold increase in the relative ratio of pMLC20/ MLC20 in MYPT1 (-LZ)-transfected cells as compared with untransfected cells (Fig. 6a and d), indicating decreased levels of MLCP activity. There was no signi icant change

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Figure 6: Deletion of the LZ motif in MYPT1 mimics hypoxia and decreases protein kinase G-mediated MYPT1 and MLC phosphatase phosphorylation. Fetal pulmonary arterial smooth muscle cells were transfected with MYPT1 expression vector without leucine zipper (-LZ) or with Leucine zipper (+LZ). Empty vector was used for mock transfections and untransfected cells as control. Cell lysates were immunoprobed with anti-LZ+MYPT1, phospho (Ser695) specific-MYPT1, native MYPT1, MLC20, pMLC20 and â-actin antibodies. (a) Representative Western blot probed with antibodies as indicated is shown. (b) Quantification of Western blot data for LZ+MYPT1 relative to control (untransfected cells). (c) Quantitative analysis of pMYPT1 (Ser695)/MYPT1 levels. values are represented as percent of control ratios between pMYPT1 (Ser695) and total MYPT1 after normalizing to their respective â-actin. (d) Normalized ratios of pMLC20/MLC20 as percent of control (untransfected cells) after normalizing the data to respective â-actin. Data represent means + SE from at least three independent experiments, *P<0.05 compared with the respective control (untransfected cells). 494

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in the pMLC20/MLC20ratio (Fig. 6a and d) between mocktransfected cells and untransfected cells (Fig. 6a and d). These results indicate that the LZ domain of MYPT1 is important for PKG-mediated phosphorylation (Ser695) of MYPT1, which subsequently activates MLCP, leading to dephosphorylation of pMLC20, a step required for smooth muscle cell relaxation.

Hypoxia increases the interaction between RhoA or ROCK and LZ+MYPT1, and overexpression of MYPT1 (-LZ) mimics hypoxia

To determine the interaction between RhoA or ROCK and LZ+MYPT1 in hypoxia, FPASMC were exposed to normoxia or hypoxia for 4 h, cell lysates prepared and immunoprecipitated with anti-PKG, anti-RhoA, antiROCK1 or anti-ROCK2 antibodies and then Western blotted with LZ+MYPT1 antibodies. There was a twofold increase in the interaction between LZ +MYPT1 and RhoA or ROCK1/ROCK2 and decreased LZ+MYPT1 interaction with PKG in FPASMC exposed to hypoxia (Fig. 7). In MYPT1 (-LZ) overexpressing FPASMC, there was a signi icant decrease in interaction between PKG and LZ+MYPT1 (Fig. 8a and b), whereas an increase (~two to four-fold) in interaction between LZ+MYPT1 and RhoA, or ROCK1/ ROCK2, was observed (Fig. 8c–f). In cells transfected with MYPT1 (+LZ) expression vector,

there was enhanced (~2.5-fold) interaction between PKG and LZ+MYPT1 (Fig. 8a and b), accompanied by decreased levels of MYPT1 (+LZ) interaction with RhoA or ROCK1/ ROCK2 (Fig. 8c–f).

DISCUSSION Our data show that acute hypoxia downregulates the expression levels of LZ+MYPT1 by reducing PKG expression, decreases the interaction of LZ+MYPT1 with PKG and promotes its interaction with ROCK1/ROCK2 in ovine FPASMC. These changes are accompanied by a decreased ratio of pMLC20/MLC20,indicative of increased smooth muscle contraction. Possible explanations for the change in binding af inity of PKG with LZ+MYPT1 in acute hypoxia may be: (1) differential downregulation of PKG and LZ+MYPT1, (2) increased af inity of RhoA, compared with PKG, to bind with LZ+MYPT1; and/or (3) posttranscriptional modi ication of PKG such that its af inity to bind with the LZ domain of MYPT1 is decreased. The irst two possibilities were examined by studying the effects of MYPT1 (LZ+) or MYPT1 (-LZ) overexpression on hypoxia-induced effects in SMC. Overexpression of LZ+MYPT1 or its known upstream activator, PKG, reversed hypoxia-induced effects, whereas LZ-MYPT1

(a) IP with ROCK-1 and IB with LZ+MYPT1

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Figure 7: Hypoxia promotes interaction between RhoA or Rho kinase (ROCK) and LZ+MYPT1. To determine the interactions between protein kinase G (PKG) and LZ+MYPT1, fetal pulmonary arterial smooth muscle cells were exposed to normoxia or hypoxia for 4 h and cell lysates (200 ìg) were immunoprecipitated and probed with the indicated antibodies. (a) Representative Western blot data showing reduced interaction between PKG and LZ+MYPT1 and increased interaction between LZ+MYPT1 with RhoA or ROCK1/ROCK2 following hypoxia exposure. (b) Quantitative analysis of Western blots from three independent experiments and values represented as percent of control (normoxia). Data represent means ± SE, *P<0.05 compared with control (normoxia). Pulmonary Circulation | October-December 2011 | Vol 1 | No 4

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Figure 8: Overexpression of MYPT1 (-LZ) mimics hypoxia-induced effects on protein kinase G (PKG) and LZ+MYPT1 interactions. Cells were transfected with MYPT1 expression vector with (+LZ) or without (-LZ) leucine zipper. Empty vector was used as a control. Cell lysates (200 ĂŹg) were immunoprecipitated with PKG, Rho kinase 1 (ROCK1) or Rho kinase 2 (ROCK2) antibodies and then immunoblotted with LZ+MYPT1 antibody. Panel a: Western blot showing the interaction between PKG and LZ+MYPT1. Cell lysates were immunoprecipitated with PKG antibody and then immunoblotted with LZ+MYPT1 antibody. Panel b: Quantification of data in Panel a. Panel c: Western blot probed with LZ+MYPT1 antibody showing interactions between LZ+MYPT1 and ROCK1. Cell lysates were immunoprecipitated with ROCK1 antibody and then probed with LZ+MYPT1 antibody. Panel d: Quantitative analysis of immunoblots in Panel c. Panel e: Representative immunoblot probed with LZ+MYPT1 antibody to detect interaction between LZ+MYPT1 and ROCK2 after immunoprecipitation with anti-ROCK2 antibodies. Panel f: Quantification of the data from panel e. Data represent means + SE from three to five independent experiments, *P<0.05 compared with the respective control (untransfected cells).

overexpression mimicked the effects of hypoxia. Based on these observations and those from our earlier studies,[13,15,16] we propose that hypoxia causes the downregulation of PKG expression, thereby leading to reduced LZ+MYPT1 expression (see schematic, (Fig. 9)). This leads to suppression of PKG binding to LZ domain of MYTP1, 496

decreased phosphorylation of MYPT1 at Ser695 and the inactivated state of MLCP. Lack of MLCP activation results in increased levels of pMLC20 and contraction of SMC. Previous studies have shown that ROS generated by hypoxia[17-20] may be responsible for both decreased PKG Pulmonary Circulation | October-December 2011 | Vol 1 | No 4

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binding of PKG to MYPT1. [22] Another group[23] used multiple biophysical techniques to fully characterize the interaction of the C-terminal region of MYPT1 with the N-terminal LZ+ motif of PKG. Their data showed that under physiological conditions, the LZ+ motif of PKG binds to the LZ+ motif of MYPT1 to form a heterodimer. When the LZ+ motif of MYPT1 is absent, the PKG LZ+ binds to the coiled-coil region and upstream segments of MYPT1 with the formation of a heterotetramer; however, there is no phosphorylation at Ser695 and no activation of MLCP.[22]

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Figure 9: Schematic representation of signaling in smooth muscle cell in normoxia and hypoxia.

and LZ+MYPT1 expression. Our studies show that ROS modulates LZ +MYPT1 expression indirectly through modulation of PKG protein and activity levels. Although we found that hypoxia downregulates PKG expression in a ROS-dependent manner, PKG overexpression alone restored the levels of MYPT1 ( +LZ) in cells exposed to hypoxia, and ROS scavengers failed to restore the expression levels of LZ +MYPT1 in PKG-de icient (knockdown) cells. These results suggest that hypoxia downregulates LZ + MYPT1 expression by reducing PKG levels through ROS, and not directly through ROSmediated effects. Furthermore, our data also suggest that PKG-mediated activation of MLCP is required for the expression of LZ+MYPT1, as overexpression of MYPT (-LZ) mimics the effects of acute hypoxia on the expression of endogenous LZ+MYPT1. SMC contractility is modulated by the ratio of phosphorylated MLC 20 to unphosphorylated MLC 20 (pMLC 20/MLC 20), and this ratio is regulated by the relative activities of MLCK/MLCP. [3,4] Activation and inactivation of MLCP depends on the ratio of site-speci ic phosphorylation of MYPT1 (Ser695) to unphosphorylated MYPT1 (pMYPT1 Ser695/MYPT1). In our study, hypoxia increased the pMLC20/MLC20 ratio, as expected. Increased pMYPT1/MYPT1 ratio and MYPT1 ( + LZ) and PKG expression were associated with increased MLCP activity and smooth muscle relaxation in normoxia. Our data also showed that hypoxia decreases the interaction of PKG with MYPT1 and increases the interaction between LZ+MYPT1 and ROCK1/ROCK2. Surks et al. irst reported that the binding of PKG to MYPT1 is mediated by the LZ+ motifs located at the N- and C-termini of the two proteins, respectively. [21] However, others have reported that the LZ + motif of MYPT1 is not required for the Pulmonary Circulation | October-December 2011 | Vol 1 | No 4

Regulation of expression levels of MYPT1 (+LZ) via the NO–cGMP–PKG pathway plays a key role in smooth muscle relaxation under a number of physiological and pathophysiological conditions.[14] For instance, in rats between postnatal days 6 and 12, expression of MYPT1 in portal veins switches from LZ (+) to LZ (-) isoforms,[11] which is concordant with a switch from cGMP-sensitive vascular relaxation to insensitivity to cGMP.[11,21,24,25] In the rat model of congestive heart failure, decreased relaxation of the aorta is associated with decreased expression of MYPT1 ( +LZ). [7,24,26] Earlier studies have shown that continuous exposure of pulmonary vessels to nitric oxide leads to NO tolerance with decreased cGMP-induced PKG activity.[27,28] In these studies, it was reported that increased accumulation of cGMP led to downregulation of PKG protein expression and activity in a negative feedback manner. A similar study demonstrated that a reduction in MYPT1 (+LZ) expression, downstream of the NO–cGMP–PKG pathway, is involved in the development of NO tolerance and that this process is in part due to proteasome-dependent degradation of LZ+MYPT1.[27,29] Earlier studies have also shown that preservation of MYPT1 (+LZ) expression could prevent the decrease in cGMP-mediated vasodilatation in chronic heart failure.[24,26]

CONCLUSIONS In summary, data presented in this study support our hypothesis that hypoxia stimulates multiple signaling pathways that act in a concerted manner to regulate SMC contraction. MYPT1 subunit of MLCP is the key regulatory protein in SMC contraction and relaxation, and dysfunctional MYPT1 signaling can contribute to the pathophysiology of a number of diseases;e.g., hypertension, gastrointestinal dysmotility, vasospasm and congestive heart failure.[7,30-32] Thus, we propose that strategies to restore MYPT1 (+LZ) expression, by selective inhibition of MYPT1 (+LZ) degradation and/or over expression of PKG or MYPT1 (+LZ), may provide a promising approach for the treatment of diseases linked to SMC dysfunction. 497

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ACKNOWLEDGMENTS The authors would like to thank V. Brozovich, Mayo Clinic, Rochester, Minnesota, for providing full-length MYPT1 (+LZ) and MYPT1 (-LZ) constructs. They would also like to thank R. Ramchandran, G. Zhou, Q. Yang and D. Gou for helpful feedback and Laura Bach for technical assistance.

17.

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monophosphate-dependent protein kinase in fetal pulmonary vascular smooth muscle cell through generation of reactive oxygen species and promotes development of pulmonary hypertension. Chest 2005;128: 577S-8S. Roede JR, Stewart BJ, Petersen DR. Decreased expression of peroxiredoxin 6 in a mouse model of ethanol consumption. Free RadicBiol Med 2008;45:1551-8. Ward JP. A twist in the tail: Synergism between mitochondria and NADPH oxidase in the hypoxia-induced elevation of reactive oxygen species in pulmonary artery. Free RadicBiol Med 2008;45:1220-2. Waypa GB, Marks JD, Guzy R, Mungai PT, Schriewer J, Dokic D, et al. Hypoxia triggers subcellular compartmental redox signaling in vascular smooth muscle cells. Circ Res 2010;106:526-35. Wu W, Platoshyn O, Firth AL, Yuan JX. Hypoxia divergently regulates production of reactive oxygen species in human pulmonary and coronary artery smooth muscle cells. Am J Physiol Lung Cell MolPhysiol2007;293: L952-9. Surks HK, Mochizuki N, Kasai Y, Georgescu SP, Tang KM, Ito M, et al. Regulation of myosin phosphatase by a specific interaction with cGMPdependent protein kinase Ialpha. Science 1999;286:1583-7. Huang D, Hinds TR, Martinez SE, Doneanu C, Beavo JA. Molecular determinants of cGMP binding to chicken cone photoreceptor phosphodiesterase. J BiolChem2004;279:48143-51. Lee E, Hayes DB, Langsetmo K, Sundberg EJ, Tao TC. Interactions between the leucine-zipper motif of cGMP-dependent protein kinase and the C-terminal region of the targeting subunit of myosin light chain phosphatase. J Mol Biol 2007;373:1198-212. Chen FC, Ogut O, Rhee AY, Hoit BD, Brozovich FV. Captopril prevents myosin light chain phosphatase isoform switching to preserve normal cGMP-mediated vasodilatation. J Mol Cell Cardiol2006;41:488-95. Huang QQ, Fisher SA, Brozovich FV. Unzipping the role of myosin light chain phosphatase in smooth muscle cell relaxation. J BiolChem2004;279:597-603. Karim SM, Rhee AY, Given AM, Faulx MD, Hoit BD, Brozovich FV. Vascular reactivity in heart failure: Role of myosin light chain phosphatase. Circ Res 2004;95:612-8. Dou D, Ma H, Zheng X, Ying L, Guo Y, Yu X, et al. Degradation of leucine zipper-positive isoform of MYPT1 may contribute to development of nitrate tolerance. Cardiovasc Res 2010;86:151-9. Gao Y, Dhanakoti S, Trevino EM, Wang X, Sander FC, Portugal AD, et al. Role of cGMP-dependent protein kinase in development of tolerance to nitric oxide in pulmonary veins of newborn lambs. Am J Physiol Lung Cell MolPhysiol2004;286: L786-92. Ma H, He Q, Dou D, Zheng X, Ying L, Wu Y, et al. Increased degradation of MYPT1 contributes to the development of tolerance to nitric oxide in porcine pulmonary artery. Am J Physiol Lung Cell MolPhysiol2010;299: L117-23. Hata T, Soga J, Hidaka T, Idei N, Fujii Y, Fujimura N, et al. Calcium channel blocker and Rho-associated kinase activity in patients with hypertension. J Hypertens2011;29:373-9. Neppl RL, Lubomirov LT, Momotani K, Pfitzer G, Eto M, Somlyo AV. Thromboxane A2-induced bi-directional regulation of cerebral arterial tone. J BiolChem2009;284:6348-60. Ohama T, Hori M, Ozaki H. Mechanism of abnormal intestinal motility in inflammatory bowel disease: How smooth muscle contraction is reduced? J Smooth Muscle Res 2007;43:43-54.

Source of Support: This study was supported in part by NIH/NHLBI Grants HL-059435 and HL-075187 o J. Usha Raj, Conflict of Interest: None declared.

Pulmonary Circulation | October-December 2011 | Vol 1 | No 4

C ase Repor t

Cardiopulmonary hemodynamic clues for pulmonary vein stenosis diagnosis Mateo Porres-Aguilar1, Genaro Fernandez2, and C. Gregory Elliott3 Department of Internal Medicine, Division of Hospital Medicine, Texas Tech University Health Sciences Center/Paul L. Foster School of Medicine, El Paso, TX, 2Department of Internal Medicine, Division of Hospital Medicine, McKay Dee Regional Hospital, Ogden, UT, 3Division of Pulmonary and Critical Care Medicine, Intermountain Medical Center, Murray, Utah, and University of Utah School of Medicine, Salt Lake City, UT, USA

ABSTRACT Pulmonary vein stenosis (PVS) post radiofrequency ablation for chronic atrial fibrillation poses a diagnostic challenge for the clinician. PVS presents with nonspecific symptoms, signs and radiographic features, and may be associated with significant pulmonary vascular involvement. Interestingly, others have described variation of the pulmonary artery wedge pressure between sites of the lung as a clue to pulmonary veno-occlusive disorders. We report, to the best of our knowledge, the first case that describes the regional loss of V waves while recording the mean pulmonary artery wedge pressure (mPawp) as well as the difference in pulmonary artery wedge pressure gradients as the main diagnostic clues for PVS. Key Words: atrial fibrillation, radiofrequency ablation, pulmonary vein stenosis, pulmonary hypertension

INTRODUCTION Radiofrequency ablation (RFA) procedures for chronic atrial ibrillation (AF) are being performed with increasing frequency. Pulmonary vein stenosis (PVS) following RFA for symptomatic AF occurs in 1â&#x20AC;&#x201C;3% of the current series.[1,2] The clinical presentation of PVS varies widely. Symptoms, signs and radiographic indings of PVS are nonspeci ic and can be attributed incorrectly to a primary lung process (e.g., pneumonia, acute pulmonary embolism, interstitial lung disease or lung cancer).[3] For this reason, identi ication of clues to the diagnosis of PVS is important. We report a case of PVS with pulmonary hypertension (PH), where the presence and location of PVS was suggested by the observation of regional loss of the V waves during the measurement of the mPawp. To the best of our knowledge, this is the irst descriptive case of this cardiopulmonary inding of PVS or occlusion.

CASE REPORT A 65-year-old male was diagnosed with AF and underwent transcatheter RFA twice in 2007, followed by thoracoscopic Address correspondence to: Dr. Mateo Porres-Aguilar Department of Internal Medicine, Division of Hospital Medicine, Texas Tech University Health Sciences Center, Paul L. Foster School of Medicine, 4800 Alberta Avenue, El Paso, TX 79905, USA Email: mateo.porres@ttuhsc.edu Pulmonary Circulation | October-December 2011 | Vol 1 | No 4

pulmonary vein isolation in 2008. He was seen in the emergency room on several occasions with dyspnea on exertion, for which he was treated for a suspected pneumonia with antibiotics. He presented to our pulmonary clinic with persistent symptoms and intermittent dry cough. Transthoracic echocardiography (TTE) showed mild mitral regurgitation without signs of PH. Cardiac magnetic resonance imaging (MRI) showed an atretic and thin left inferior pulmonary vein of 2 mm in diameter, suggestive of severe PVS. He underwent right heart catheterization (RHC) for cardiopulmonary hemodynamic assessment, which showed the tracings depicted below (Fig. 1aâ&#x20AC;&#x201C;d).

DISCUSSION The development of PH with PVS has been rarely reported, and could represent complex and severe PVS with progressive pulmonary vasculopathic changes in the long term.[4-6] Our patient presented with persistent symptoms Access this article online Quick Response Code:

Website: www.pulmonarycirculation.org DOI: 10.4103/2045-8932.93549 How to cite this article: Porres-Aguilar M, Fernandez G, Elliott CG. Cardiopulmonary hemodynamic clues for pulmonary vein stenosis diagnosis. Pulm Circ 2011;1: 499-500.

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Figure 1: (a) Pulmonary hemodynamic tracing showing a mean right ventricular pressure (mRVP) of 42 mmHg. (b) Pulmonary hemodynamic tracings showing a mean pulmonary arterial pressure (mPAP) of 42 mmHg. (c) Pulmonary hemodynamic tracings showing a right mean pulmonary artery wedge pressure (mPawp) of 22 mmHg with the presence of tall V waves (arrow). (d) Pulmonary hemodynamic tracings showing a left mean pulmonary artery wedge pressure (mPawp) of 34 mmHg. Note the flattening of the V waves.

despite having involvement of only one vessel. Additionally, he had marked elevation in his left mPawp. He also demonstrated a loss of the V wave during the measurement of the left mPawp. The V wave represents the venous illing of the left atrium during ventricular systole when the mitral valve is closed.[7] Our patientâ&#x20AC;&#x2122;s tall peaked V waves noted when measuring the right mPawp may be explained by his mitral regurgitation. However, the tall V wave was not noted in the left mPawp tracing due to the lack of pressure transmission. To the best of our knowledge, this is the irst description of this hemodynamic inding in the context of PVS. An additional clue suggesting PVS was the difference in the pressure gradients between the right and left mPawp (right mPawp of 22 mmHg versus left mPawp of 34 mmHg), emphasizing that equalization of pressures occurred between the right mPawp and the left atrium. Sharkey described the absence of V waves in acute pulmonary embolism. The A and V waves frequently disappear from the wedge tracing as abnormal pulmonary vasculature does not allow retrograde transmission of these pressure waves from the left atrium to the distal catheter lumen.[7]

CONCLUSION Signi icant PH represents a rare complication of PVS, indicating the development of advanced pulmonary vascular disease as the stenosis progress to complete occlusion, particularly when more than two pulmonary veins are involved. Regional loss of V waves recorded 500

during mPawp measurement represents a clue to the diagnosis and location of PVS. The difference in pressure gradients between right and left mPawp could suggest the diagnosis and location of PVS. However, we understand that given the absence of previous reports in the literature, these indings must be con irmed with reliable reproducibility in future PVS hemodynamic studies.

REFERENCES 1.

Holmes DR, Monahan KH, Packer D. Pulmonary vein stenosis complicating ablation for atrial fibrillation. J Am Coll Cardiol Interv 2009;2:267-76. Barrett CD, Di Biase L, Natale A. How to identify and treat patients with pulmonary vein stenosis post atrial fibrillation ablation. Curr Opin Cardiol 2008;24:42-9. Ernst S, Ouyang F, Goya M, Lober F, Schneider C, HoďŹ&#x20AC;mann-Riem M, et al. Total pulmonary vein occlussion as a consequence of catheter ablation for atrial fibrillation mimicking primary lung disease. J Cardiovasc Electrophysiol 2003;14:366-70. Nehra D, Liberman M, Vagefi PA, Evans N, Inglessis I, Kradin RL, et al. Complete pulmonary venous occlusion after radiofrequency ablation for atrial fibrillation. Ann Thorac Surg 2009;87:292-5. Robbins IM, Colvin EV, Doyle TP, Kemp E, Loyd JE, McMahon WS, et al. Pulmonary vein stenosis after catheter ablation of atrial fibrillation. Circulation 1998;98:1769-75. Arentz T, Weber R, Jander N, Burkle G, von Rosenthal J, Blum T, et al. Pulmonary haemodynamics at rest and during exercise in patients with significant pulmonary vein stenosis after radiofrequency catheter ablation for drug resistant atrial fibrillation. Eur Heart J 2005;26:1410-4. Sharkey SW. Beyond the wedge: Clinical physiology and the Swan-Ganz catheter. Am J Med 1987;83:111-22.

Source of Support: Nil, Conflict of Interest: None declared.

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Hi st or y and W h o ’s W h o

Lofty goals at high altitude: The Grover Conferences, 1984–2011 E. Kenneth Weir1, Wiltz W. Wagner Jr.2, and Stephen L. Archer3 1

VA Medical Center 111C, 1 Veteran’s Drive, Minneapolis, MN 55417, 2Department of Pharmacology and Center for Lung Biology, College of Medicine, University of South Alabama, Mobile, AL, 36688, 3Department of Medicine, The University of Chicago, 5841 South Maryland Avenue, Chicago, IL 60637, USA.

ORIGIN The irst Grover Conference was held at the Lost Valley Ranch Conference Center in Deckers, Colorado, in 1984. Both the concept of the conference and the choice of the site were initiatives of John T. “Jack” Reeves MD (Fig. 1). At the time, there was no major conference dedicated to pulmonary circulation or pulmonary hypertension and a PubMed search for articles with the following key words detected <80 publications/year: pulmonary circulation, hypoxic pulmonary vasoconstriction, and pulmonary hypertension. By 2010, there were >900 publications/year with those keywords in their title (Fig. 2). Although the biannual Grover Conferences cannot take all the credit for this new interest in lung circulation, they have certainly contributed. The impact of the conference has been enhanced by the careful selection of new topics that are identi ied as areas of emerging opportunity (Table 1). Thus, new science is always on the “menu.” Moreover, the invitation of scientists to a ranch that is literally in a “Lost Valley,” with only a pay phone for communication with the outside world, has meant that these established and emerging scienti ic leaders are fully engaged. In addition, enduring camaraderie is derived from sharing cabins and enduring the terror of novices on horseback. The hypoxic majesty of the Pike National Forest at elevations of >7000 feet focuses the mind on hypoxia (especially as one pants up the inclines to cabins with names like “Huff n Puff ”). A remarkable transformation of the intellectual elite occurs when they are wrapped in denim and plaid giving a talk using a ishing pole as a pointer. If that did not generate suf icient humility, Jack Reeves was ever ready with a deceptively simple question that would humble the arrogant and point out the need for more research. Address correspondence to: Dr. E. Kenneth Weir, Veterans Administration Medical Center, One Veterans Drive, Minneapolis, MN 55417, USA E-mail: weirx002@umn.edu Pulmonary Circulation | October-December 2011 | Vol 1 | No 4

Dr. Reeves and his colleagues, Drs. Wiltz Wagner, Norbert Voelkel, Ivan McMurtry and Ken Weir (Fig. 3), recognized the need for an ongoing international, scienti ic conference devoted to the pulmonary circulation. The meeting in 2011 was the 15th in the series. Those who study the pulmonary circulation do not need to be reminded of its unique characteristics. However, the great majority of the non-medical public are still in the pre-Harveian era and do not know that there is an entirely separate circulation of blood to the lungs. Few of those who are medically trained, other than cardiologists and pulmonologists, remember the complex embryology that gives rise to the proximal, capacitance, pulmonary arteries. Many have forgotten that the pulmonary vasculature is a low-pressure system, accommodating the entire cardiac output and interfacing with the airways down to the level of the alveoli, with the bronchial circulation and with the lymphatics. Those who work in the area know that the lungs provide a large area of contact between the blood and endothelium and consequently, the lungs’ metabolic activity is enormously important. Many endogenous substances are activated, such as angiotensin l, or inactivated, such as bradykinin; and yet others, such as nitric oxide and prostacyclin, are generated by the endothelium of the pulmonary vasculature. In most respects, the pulmonary circulation is quite different from the systemic circulation, as illustrated in terms of acute reactivity by hypoxic pulmonary vasoconstriction, in altered function by the potentially fatal condition, high-altitude pulmonary edema, and in chronic disease by idiopathic pulmonary arterial hypertension. It is clear from even a brief summary of Access this article online Quick Response Code:

Website: www.pulmonarycirculation.org DOI: 10.4103/2045-8932.93550 How to cite this article: Weir EK, Wagner WW, Archer SL. Lofty goals at high altitude: The Grover Conferences, 1984-2011. Pulm Circ 2011;1:501-7.

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Table 1: The Grover Conferences on the Pulmonary Circulation, 1984-2011 1984 1986

Pulmonary Vascular Reactivity The Role of Lipid Mediators

1988

The Control of Cellular Proliferation

1990

The Diagnosis and Treatment of Pulmonary Hypertension The Pulmonary Circulation and Gas Exchange The Role of Ion Flux The Role of Radicals The Pathogenesis and Treatment of Pulmonary Edema The Fetal and Neonatal Pulmonary Circulations The Blood and the Pulmonary Circulation Proinflammatory Signaling Mechanisms in the Pulmonary Circulation Genetic and Environmental Determinants of Pulmonary Endothelial Cell Function Rho Family GTPases in Pulmonary Vascular Pathophysiology Membrane Receptors, Channels and Transporters in Pulmonary Circulation Risk Factors in Pulmonary Hypertension

1991 1992 1994 1996 1998 2000 2002

2004

2006 2008 2011

Publisher

Conference Director

Chest 88: 199S-272S, 1985 Am. Rev. Respir. Dis. 136: 196-224; 782-788, 1987 Am. Rev. Respir. Dis. 140: 1093-1135; 1446-1493, 1989 Futura, New York, NY

E.K. Weir, I.F. McMurtry, J.T. Reeves E.K. Weir, J.T. Reeves

Futura, New York, NY

W.W. Wagner, E.K. Weir

Plenum Press, New York, NY Futura, New York, NY Futura, New York, NY

E.K. Weir, J. R. Hume, J.T. Reeves E.K. Weir, S.L. Archer, J.T. Reeves E.K. Weir, S.L. Archer, J.T. Reeves

Futura, New York, NY

E.K. Weir, S.L. Archer, J.T. Reeves

Futura, New York, NY

E.K. Weir, H.L. Reeve, J.T. Reeves

Humana Press, Totowa New Jersey, NJ

J. Bhattacharya

http://www.groverconference.org/ grover_past_conft.htm

T. Stevens

http://www.groverconference.org/ grover_past_conft.htm Humana Press-Springer, New York, NY Adv. Exp. Med. Biol. Vol. 661 Pulm. Circ. Vol. 1-2, 2011-2012

K. Fagan, I.F. McMurtry

E.K. Weir, J.T. Reeves E.K. Weir, S.L. Archer, J.T. Reeves

J.P.T. Ward, J.X.-J. Yuan M.R. MacLean, N.W. Morrell, K. Fagan

PVRI Pulmonary Vascular Research Institute

Publications/year

1000 800 600 400

1st Grover Meeting st Grover Lost1Valley Ranch Conference Dekkers, CO

2nd World Symposium on PH (EVIAN)

NIH Registry Publication

200 0 1970 1975 1980 1985 1990 1995 2000 2005 2010

Year Figure 2: Key meetings in the development of the study of pulmonary circulation. Although association is not causality, the Grover meeting in 1984 clearly occurred at the fi rst defl ection point in the fi eld, as evident by the subsequent growth in what had been a niche area of research. Many other important events occurred that also stimulated interest, including the Evian meeting in which a modern classification of pulmonary hypertension was developed, the publication of the NIH’s PPH registry and, most recently, the creation of an international PVRI, which engages scientists from countries around the world in pulmonary vascular research (HYPERLINK “http://www.pvri.info” www.pvri.info).

Figure 1: (Left, right and bottom) Bob Grover, Jack Reeves, and the Lost Valley Conference Center at Sedalia, Colorado where the Grover Conferences are held since 1984. 502

some of these features of the pulmonary circulation that a dedicated, ongoing conference was, and remains, necessary. Pulmonary Circulation | October-December 2011 | Vol 1 | No 4

Weir et al.: History of the Grover Conferences

Figure 3: Bob Grover and his colleagues, Wiltz Wagner, Norbert Voelkel, Ivan McMurtry, and Ken Weir.

The conference is named for Robert F. Grover MD PhD in recognition of his many contributions to our understanding of the physiology and pathophysiology of the pulmonary circulation. His studies of chronic hypoxic pulmonary hypertension in cattle (brisket disease) were among the irst describing this condition (1963). [1] He instigated or performed many studies on factors that in luence acute and chronic hypoxic pulmonary hypertension, differences between species (1963),[2] sympathetic activity (1968),[3] genetic differences (1974), [4] endotoxins (1974), [5] prostaglandins (1976),[6] L-type calcium channels (1976),[7] acetylcholine (1976),[8] platelets (1976),[9] ethyl alcohol (1978)[10] and cold (1978).[11] Dr. Grover was one of the irst to demonstrate a component of reversible vasoconstriction in patients with congenital heart disease. [12] He performed the irst measurements of pulmonary arterial pressure in normal North American residents of high altitude.[13] He was involved in studies of pulmonary vascular reactivity in subjects who had had an episode of high-altitude pulmonary edema,[14] and also in pregnant women.[15] All these studies demonstrate the breadth of his interests in the pulmonary circulation and also illustrate many of the puzzles that continue to stimulate our research.

CONFERENCE EVOLUTION

Dr. Grover was also an inspirational mentor and the conference celebrates both his scienti ic interests and his tradition of mentorship. In the irst three decades of its existence the Grover Conference has become a retreat to which mentors return to see their mentees shepherding a new generation of scientists in the search to understand the lung circulation and its diseases. The sense of community and the excitement for discovery are perhaps 2 of the greatest bene its of this meeting, both very consistent with Dr. Grover’s legacy. At the 2011 Conference, he gave a stirring summary of the high-altitude adventures that led him and his protégé, Jack Reeves, to understand the basis for brisket disease as both excessive HPV and

In the 27 years since the irst Grover Conference there have been enormous changes in our understanding of the pulmonary circulation but, Our improved basic science understanding has not been translated to cost-effective treatment. It is fascinating to read the description given by Robert Furchgott (co-winner of the 1998 Nobel Prize in Physiology or Medicine), at the 1984 conference, of endothelium-derived relaxing factor (EDRF) [16] (Fig. 5). He reported that EDRF was released from intrapulmonary arteries by acetylcholine and bradykinin causing vasodilatation, probably through an increase in cyclic GMP. He demonstrated that hemoglobin would inhibit EDRF and speculated about the identity of EDRF,

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exuberant remodeling of the pulmonary vasculature. In Figure 4a, the young friends, Reeves and Grover, are seen on the slide while the adventure of research is evident in Figure 4b, which summarizes cardiac catheterization of normal teenagers at high altitude, showing they too may have pulmonary hypertension. The talk ended with an emotional standing ovation (Fig. 4c), both for the talk and for the legacy of mentorship. The conference’s proceedings, published in a series of books and journal issues, bear witness to many discoveries which have changed our understanding of hypoxic pulmonary vasoconstriction, the pulmonary vascular effects of high altitude, the causes and treatment of pulmonary arterial hypertension, and the latest information of the role of gender in pulmonary hypertension. The publications arising from the conference are listed in Table 1. However, even more compelling are the areas of research that were begun and the collaborations and friendships that were launched during these conferences.

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Figures 4: (a) Bob Grover at the 2011 meeting, summarizing the pioneering work he and Jack Reeves, then a Cardiology Fellow at the University of Colorado, performed in discovering the basis for brisket disease (b) Bob Grover describing how the work in cattle was quickly replicated in normal volunteers in the high-altitude community of Leadville, Colorado (c) A tribute to a fine after-dinner talk and an even finer scientist and mentor.

smooth muscle. Sami Said discussed vasoactive peptides in the lung and, in particular, the vasodilator activity of vasoactive intestinal peptide (VIP). Among many papers on pulmonary vasoconstriction, David Harder reported that hypoxia caused membrane depolarization in pulmonary artery smooth muscle cells, and Lew Rubin reviewed the use of calcium channel blockers in the treatment of primary pulmonary hypertension (now called IPAH, idiopathic pulmonary arterial hypertension).

Figure 5: (Left to right) Jack Reeves, future Nobel Laureate Robert Furchgott, and E. Kenneth Weir, at the inaugural (1984) Grover Conference.

including “the free radical nitric oxide” among other candidates. Other presentations in the initial conference included Kees Wagenvoort describing fenestrations in the internal elastic lamina of small pulmonary vessels seen on electron microscopy which he suggested might facilitate communication between the endothelium and 504

Most relevant to the 2011 Conference was the paper by Jimmy Sylvester on “Prostaglandins and Estradiol-induced Attenuation of Hypoxic Pulmonary Vasoconstriction.”[17] In the isolated perfused sheep lung he found that estradiol pre treatment reduces the normoxic control pulmonary vascular resistance by a mechanism unrelated to the production of prostaglandins. Further, estradiol reduces HPV by a mechanism that does involve prostaglandins. Meanwhile, an important presentation by Milton Packer cast doubt on the ef icacy of vasodilator therapy in the treatment of primary pulmonary hypertension.[18] This was an early shot in the continuing debate over the role of vasoconstriction in the etiology of IPAH. The ensuing discussion of his paper re lected the recognition that the Pulmonary Circulation | October-December 2011 | Vol 1 | No 4

Weir et al.: History of the Grover Conferences

response to vasodilator agents would be determined by the underlying histology, being poor in those patients with plexiform lesions. The titles of the subsequent conferences are given in Table 1. The fact that the third conference (1988) was devoted to the control of cellular proliferation in the pulmonary circulation indicates the early understanding that the effective treatment of pulmonary hypertension would include the inhibition of cellular proliferation. The ifth conference (1991), directed by Wiltz W. Wagner, Jr., was the most memorable in a historical sense. Many of the pioneers of research into the pulmonary vasculature described their personal development and work in the ield (Fig. 6). To mention a few highlights, Bob Grover spoke eloquently of his introduction to physiology, the history of research into the effects of high altitude on the pulmonary circulation, and his early work with Estelle Grover and Wiltz Wagner.[19] Donald Heath described his induction into the world of pulmonary vascular pathology and the histology of the small pulmonary vessels of men and animals at high altitude.[20] Peter Harris gave a talk, illustrated by many of his own line drawings, reporting experiments on sheep, goats, cattle and yak in the Himalayas.[21] Ewald Weibel talked about the insights that morphometry, and electron microscopy in particular, provide to our understanding of lung physiology.[22] In the course of their lectures, both Johannes Piper and Kees Wagenvoort described their experiences as young men during the Second World War. The publications derived from the Grover Conferences stand as a unique record of the progress of research into the pulmonary vasculature and pulmonary hypertension.

Over the course of the conference, the scienti ic reports have appeared in books, published initially by Steven Korn with Futura and more recently as articles in the journal Pulmonary Circulation, edited by Dr. J. X.-J. Yuan, Dr. Harishrishnam S. and Dr. N.W. Morrell (who helped organize the 2011 Conference). The idea that there would be a journal dedicated to pulmonary circulation was unthinkable when the irst Grover Conference occurred, and bears testimony to the maturation of the ield.

LOGISTICS All the Grover Conferences have been held at the Lost Valley Ranch Conference Center. It provides the ideal environment for such meetings, fostering opportunities for formal and informal discussion between those attending. This is a rare conference in which forced isolation creates focus and creative collegiality. Along the winding dirt road to the Lost Valley, “Big City” scientists lose their suits, put on jeans and are soon sharing communal bunk houses, speculating on grizzly bear traf ic in the valley and commiserating about the saddle sores they acquired during the daily horseback ride. In the beginning, the conference was a “do-ityourself” enterprise and guests from around the world were greeted at the old Stapleton Airport by Rosann McCullough and driven up the mountain by faculty and fellows from the University of Colorado’s legendary CVP lab. The JVL™ Lost Valley Ranch is an intrinsic part of the meeting. The Foster family who owns and operates this working ranch have embraced the city slickers who return to the ranch with a welcoming Ooh-Ah every 2 years. The Grover Conferences have been a catalyst for

Figure 6: Three generations of scientists at the 5th Grover meeting (1992). Front row (left to right): Robert A. Klocke (on one knee), James R. Snapper, Mark N. Gillespie, Wiltz W. Wagner Jr., Almas Aldechev, David J. Riley, Anne Clark, Gwenda R. Barer, Troy Stevens, Grant deJ. Lee (looking down), Tawfic S. Hakim, John N. Evans, Robert F. Grover, Norman C. Staub, Lynne M. Reid, J. Michael Kay, Michael R. T. Yen, Vaclav Hampl. Middle row (left to right): Robert E. Forster II, Claire Doerschuk, J. Usha Raj, John B. West, Aubrey E. Taylor, Solbert Permutt, Christopher A. Dawson, Roy G. Brower, J. T. Sylvester, C. A. Wagenvoort, Donald Heath, Serge Adnot, Gerald Simonneau, Phillipe Herve, Peter D. Wagner, John T. Reeves, David Badesch, Barbara O. Meyrick, Norbert Voelkel, A.N. Other, E. Kenneth Weir, Bertron M. Groves. Back row (left to right): Albert L. Hyman, Stephen Archer, Inder Anand, Peter Harris, Nicholas S. Hill, John A. Linehan, A.N. Other, Robert G. Presson, Stephen J. Lai-Fook, Robert L. Johnson, Jr., Robert Capen, Leonard Latham, John Butler, Lorna Moore, Gregory J. Redding, Ewald R. Weibel, Y. C. Fung, Walker Long, Keith Horsfield, John H. Newman, Thomas Jacobs. Pulmonary Circulation | October-December 2011 | Vol 1 | No 4

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Figure 7: Grover attendees 2011. Taken in front of the ranch house where science and life are discussed.

established scientists. It has also been an inspiration for new scientists. It holds the memories of friends departed and the promise of new friends and colleagues, as evident in the 2011 photograph (Fig. 7). There are between 33 and 43 speakers and chairs, with a total of up to 90 participants at each meeting. The fact that the conference site is isolated encourages everyone to be present throughout the four days of the meeting. Almost all of the conferences have received grant support from the National Heart, Lung and Blood Institute (NHLBI), and most have been supported by the Cardiopulmonary and Critical Care Council of the American Heart Association, the American Thoracic Society and the Pulmonary Circulation Foundation. Additional funding has been received from many pharmaceutical, instrumentation and publishing companies. The process of selecting a subject for the next meeting initially rested with Drs. Weir, Wagner, McMurtry, Voelkel and Reeves. The stewardship for the meeting passed to the Leadership Committee of the Pulmonary Circulation Assembly of the American Thoracic Society in 2005. This logistic formula has provided a cost-effective method of achieving vigorous discussion of topical issues, from basic science, through translational studies, to current clinical treatment. The publications that contain the proceedings of the Grover meetings (Table 1) are a unique record of progress in pulmonary vascular research. The legacy of the Grover meeting continues to be written but the chapters recorded to date are rich with the best traditions of science. Lectures honor igures who have 506

been key to the spirit of the meeting, including the Estelle Grover Lecture, The John T. Reeves Lecture and the Terry Wagner Lecture. As each new generation of scientists picks up the torch, the stories of conferences past are shared. At the Lost Valley Ranch, curiosity, mentorship and camaraderie fuel the pursuit of better understanding of the pulmonary circulation, pulmonary hypertension and oxygen sensing. It seems that lofty goals are best pursued at high altitude.

REFERENCES 1. 2.

5. 6.

Grover R, Reeves J, Will D, Blount SJ. Pulmonary vasoconstriction in steers at high altitude. J Appl Physiol 1963;18:567-74. Grover R, Vogel J, Averill K, Blount SJ. Pulmonary hypertension. Individual and species variability relative to vascular reactivity. Am Heart J 1963;66:1-3. Silove E, Grover R. Eﬀects of alpha-adrenergic blockade and tissue catecholamine depletion on pulmonary vascular response to hypoxia. J Clin Invest 1968;47:274-85. Weir E, Tucker A, Reeves J, Will D, Grover R. The genetic factor influencing pulmonary hypertension in cattle at high altitude. Cardiovasc Res 1974;8:745-9. Reeves J, Grover R. Blockade of acute hypoxic pulmonary hypertension by endotoxin. J Appl Physiol 1974;36:328-32. Weir EK, McMutry IF, Tucker A, Reeves JT, Grover RF. Prostaglandin synthetase inhibitors do not decrease hypoxic pulmonary vasoconstriction. J Appl Physiol 1976;41:714-8. McMurtry IF, Davidson AB, Reeves JT, Grover RF. Inhibition of hypoxic pulmonary vasoconstriction by calcium antagonists in isolated rat lungs. Circ Res 1976;38:99-104. McMurtry I, Weir E, Reeves J, Will D, Grover R. Prostaglandin synthesis does not mediate the pulmonary vasodilator eﬀect of acetylcholine. Prostaglandins 1976;11:63-9. Weir E, Mlczoch J, Seavy J, Cohen J, Grover R. Platelet antiserum inhibits hypoxic pulmonary vasoconstriction in the dog. J Appl Physiol 1976;41:211-5.

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Weir et al.: History of the Grover Conferences

10.

11. 12. 13.

14.

15.

16. 17.

Doelkel R, Weir E, Looga R, Reeves J, Grover R. Potentiation of hypoxic pulmonary vasoconstriction by ethyl alcohol in dogs. J Appl Physiol 1978;44:76-80. Will D, McMurtry I, Reeves J, Grover R. Cold induced pulmonary hypertension in cattle. J Appl Physiol 1978;45:469-73. Grover RF, Reeves JT, Blound SG Jr. Tolazoline hydrochloride (Proscoline): An eﬀective pulmonary vasodilator. Am Heart J 1961;61:5-15. Vogel J, Weaver W, Rose R, Blount SJ, Grover R. Pulmonary hypertension on exertion in normal man living at 10,150 feet (Leadville, CO). Med Thorac 1962;19:461-77. Hultgren H, Grover R, Hartley L. Abnormal circulatory responses to high altitude in subjects with a previous history of high altitude pulmonary edema. Circulation 1971;44:759-70. Weir EK, Greer BE, Smith SC, Silvers GW, Droegemuller W, Reeves JT, et al. Bronchial constriction and pulmonary hypertension during abortion induced by 15-methyl prostaglandin F2alpha. Am J Med 1976;60:556-62. Furchgott R, Martin W. Interactions of endothelial cells and smooth muscle cells of arteries. Chest 1985;88:210-3S. Sylvester J, Gordon J, Malamet R, Wetzel R. Prostaglandins and estradiolinduced alternuation of hypoxic pulmonary vasoconstriction. Chest 1985;88:252-4S.

18. 19.

20.

21.

22.

Packer M. Does pulmonary vasoconstriction play an important role in patients with primary pulmonary hypertension? Chest 1985;88:265-8S. Grover R. Pulmonary hypertension: The price of high living. In the Pulmonary Circulation and Gas Exchange, In: Wagner W Jr., Weir E, editors. Armonk, New York: Futura Publishing Company, Inc.; 1994. p. 317-41. Heath D. Pulmonary vascular disease in Sheﬃeld, the Andes, Tibet and Tanzania. In: Wagner W Jr., Weir E, editors. The Pulmonary Circulation and Gas Exchange. Armonk, New York: Futura Publishing Company, Inc.; 1994. p. 265-82. Harris P. The Pulmonary circulation of some domestic animals at high altitude. In: Wagner W Jr., Weir E, editors. The Pulmonary Circulation and Gas Exchange. Armonk, New York: Futura Publishing Company, Inc.; 1994. p. 283-315. Weibel E. Exploring the structural basis for pulmonary gas exchange. In: Wagner W Jr., Weir E, editors. The Pulmonary Circulation and Gas Exchange. Armonk, New York: Futura Publishing Company, Inc.; 1994. p. 19-45.

Source of Support: None declared, Conflict of Interest: None declared.

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Aut hor Index , 2 0 1 1 Abe K see Ota H et al. Abman S see del Cerro MJ et al. Adatia I see del Cerro MJ et al. Adatia I see Lammers AE et al. Adnot S see Damy T et al. Aldred MA see Duong HT et al. Andersen CU, Hilberg O, Mellemkjær S, Nielsen-Kudsk JE, Simonsen U. Apelin and pulmonary hypertension 334 Archer SL see Kim GH et al. Archer SL see Weir EK et al. Asosingh K see Duong HT et al. Austin E see Lane KL et al. Austin ED see Fessel JP et al. Austin ED, Menon S, Hemnes AR, Robinson LR, Talati M, Fox KL, Cogan JD, Hamid R, Hedges LK, Robbins I, Lane K, Newman JH, Loyd JE, West J. Idiopathic and heritable PAH perturb common molecular pathways, correlated with increased MSX1 expression 389 Bandeira A see Graham BB et al. Barst RJ. Classification of pediatric pulmonary hypertensive vascular disease: Does it need to be different from the adult classification? 134 Beutz MA see Ota H et al. Bieger D see Duggan DJ et al. Blackwell T see Lane KL et al. Bloch KD see Malhotra R et al. Blyth KG, Kinsella J, Hakacova N, McLure LE, Siddiqui AM, Wagner GS, Peacock AJ. Quantitative estimation of right ventricular hypertrophy using ECG criteria in patients with pulmonary hypertension: A comparison with cardiac MRI 470 Burg ED see Kuang T et al. Burrowes KS see Tawhai MH et al. Burrowes KS, Clark AR, Tawhai MH. Blood flow redistribution and ventilation-perfusion mismatch during embolic pulmonary arterial occlusion 365 Butrous G see Graham BB et al. Butrous G see J. Yuan JX et al. Butrous G see Yuan JX et al. Butrous G. The PVM History Initiative of the PVRI 125 Chabon J see Graham BB et al. Champion HC see George MP et al. Chesler NC see Wang Z et al. Church C see Hagan G et al. Clark AR see Burrowes KS et al. Clark AR see Tawhai MH et al. Cogan JD see Austin ED et al. Comer BS see Joshi SR et al. Comhair SA see Duong HT et al. Coulson J see Hagan G et al. Dahal BK, Heuchel R, Pullamsetti SS, Wilhelm J, Ghofrani HA, Weissmann N, Seeger W, Grimminger F, Schermuly RT. Hypoxic pulmonary hypertension in mice with constitutively active platelet-derived growth factor receptor-β 259 Damy T, Lesault P, Guendouz S, Eddahibi S, Tu L, Marcos E, Guellich A, Dubois-Randé J, Teiger E, Hittinger L, Adnot S. Pulmonary hemodynamic responses to inhaled NO in chronic heart failure depend on PDE5 G(-1142)T polymorphism 377 del Cerro MJ see Lammers AE et al. del Cerro MJ, Abman S, Diaz G, Freudenthal AH, Freudenthal F, Harikrishnan S, Haworth SG, Ivy D, Lopes AA, Raj JU, Sandoval J, Stenmark K, Adatia I. A consensus approach to the classification of pediatric pulmonary hypertensive vascular disease: Report from the PVRI Pediatric Taskforce, Panama 2011 286 Desai A, Machado R. Drugs currently used for treatment of PAH 299 Desai AA see Pauwaa S et al. Desai AA, Machado RF. Diagnostic and therapeutic algorithm for pulmonary arterial hypertension 122 Diaz G see del Cerro MJ et al. Diaz G see Lammers AE et al. Donahoe M. Acute respiratory distress syndrome: A clinical review 192 Donahoe MP see Stamm JA et al. Doughty NJ see Soon E et al. Dubois-Randé J see Damy T et al. Duggan DJ, Bieger D, Tabrizchi R. Neurogenic responses in rat and porcine large pulmonary arteries 419 Duong HT, Comhair SA, Aldred MA, Mavrakis L, Savasky BM, Erzurum SC, Asosingh K. Pulmonary artery endothelium resident endothelial colony-forming cells in pulmonary arterial hypertension 475 Eddahibi S see Damy T et al. Elliott CG see Porres-Aguilar M et al. Erzurum SC see Duong HT et al. Espinheira L see Graham BB et al. Fantozzi I see Firth AL et al. Fernandez G see Porres-Aguilar M et al. Fessel JP see Lane KL et al. Fessel JP, Loyd JE, Austin ED. The genetics of pulmonary arterial

508

hypertension in the post-BMPR2 era 305 Fike C see Lane KL et al. Firth AL, Remillard CV, Platoshyn O, Fantozzi I, Ko EA, J. Yuan JX. Functional ion channels in human pulmonary artery smooth muscle cells: Voltage-dependent cation channels 48 Forfia PR see Roberts JD et al. Fox KL see Austin ED et al. Freudenthal AH see del Cerro MJ et al. Freudenthal AH see Lammers AE et al. Freudenthal F see del Cerro MJ et al. Freudenthal F see Lammers AE et al. Frid MG see Yeager ME et al. Garcia JG see Yao W et al. George MP, Champion HC, Pilewski JM. Lung transplantation for pulmonary hypertension 182 Gerthoffer WT see Joshi SR et al. Ghofrani HA see Dahal BK et al. Ghofrani HA see Thamm M et al. Gladwin MT see Stamm JA et al. Gomez-Arroyo J see Voelkel NF et al. Gopalan D see Hagan G et al. Graham BB, Chabon J, Bandeira A, Espinheira L, Butrous G, Tuder RM. Significant intrapulmonary Schistosoma egg antigens are not present in schistosomiasis-associated pulmonary hypertension 456 Green DE, Sutliff RL, Hart CM. Is peroxisome proliferator-activated receptor gamma (PPARγ) a therapeutic target for the treatment of pulmonary hypertension? 33 Grimminger F see Dahal BK et al. Grimminger F see Thamm M et al. Guellich A see Damy T et al. Guendouz S see Damy T et al. Gupta K see Kumar B et al. Hagan G, Gopalan D, Church C, Rassl D, Mukhtyar C, Wistow T, Lang C, Sivasothy P, Stewart S, Jayne D, Sheares K, Tsui S, Jenkins DP, Pepke-Zaba J. Isolated large vessel pulmonary vasculitis as a cause of chronic obstruction of the pulmonary arteries 425 Hagan G, Southwood M, Treacy C, Ross RM, Soon E, Coulson J, Sheares K, Screaton N, Pepke-Zaba J, Morrell NW, Rudd JH. 18FDG PET imaging can quantify increased cellular metabolism in pulmonary arterial hypertension: A proof-of-principle study 448 Hakacova N see Blyth KG et al. Hamid R see Austin ED et al. Hamid R see Newman JH et al. Hamidi SA see Said SI et al. Harikrishnan S see del Cerro MJ et al. Harikrishnan S see J. Yuan JX et al. Harikrishnan S see Lammers AE et al. Harikrishnan S see Yuan JX et al. Hart CM see Green DE et al. Haworth SG see del Cerro MJ et al. Haworth SG see Lammers AE et al. Hedges LK see Austin ED et al. Hedges LK see Newman JH et al. Hemnes AR see Austin ED et al. Hemnes AR see Lane KL et al. Hess D see Malhotra R et al. Heuchel R see Dahal BK et al. Hilberg O see Andersen CU et al. Hittinger L see Damy T et al. Holt TN see Newman JH et al. Huang X see Kuang T et al. Ito M see Ota H et al. Ivy D see del Cerro MJ et al. Ivy D see Lammers AE et al. J. Yuan JX see Firth AL et al. J. Yuan JX, Morrell NW, Harikrishnan S, Butrous G. Pulmonary Circulation: A new venue for communicating your findings, ideas and perspectives 1 Jafri S, Sivasothy P, Wells F, Morrell NW. Clinical demonstration of efficiency and reversibility of hypoxic pulmonary vasoconstriction in a patient presenting with unilateral incomplete bronchial occlusion 119 Jason XJ see Kuang T et al. Jayne D see Hagan G et al. Jenkins DP see Hagan G et al. Johnson JA see Lane KL et al. Joshi SR, McLendon JM, Comer BS, Gerthoffer WT. MicroRNAs-control of essential genes: Implications for pulmonary vascular disease 357 Kim GH, Ryan JJ, Marsboom G, Archer SL. Epigenetic mechanisms of pulmonary hypertension 347 Kinsella J see Blyth KG et al. Ko EA see Firth AL et al. Kuang T, Wang J, Zeifman A, Pang B, Huang X, Burg ED, Jason XJ, Wang C.

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Author index

Combination use of sildenafil and simvastatin increases BMPR-II signal transduction in rats with monocrotaline-mediated pulmonary hypertension 111 Kumar B, Puri GD, Manoj R, Gupta K, Shyam KS. Severe pulmonary artery hypertension following intracardiac repair of tetralogy of Fallot: An unusual finding 115 Lammers AE, Adatia I, del Cerro MJ, Diaz G, Freudenthal AH, Freudenthal F, Harikrishnan S, Ivy D, Lopes AA, Raj JU, Sandoval J, Stenmark K, Haworth SG. Functional classification of pulmonary hypertension in children: Report from the PVRI pediatric taskforce, Panama 2011 280 Lane K see Austin ED et al. Lane KL, Talati M, Austin E, Hemnes AR, Johnson JA, Fessel JP, Blackwell T, Mernaugh RL, Robinson L, Fike C, Roberts II LJ, West J. Oxidative injury is a common consequence of BMPR2 mutations 72 Lang C see Hagan G et al. Lee JE see Sehgal PB et al. Lendeckel F see Thamm M et al. Lesault P see Damy T et al. Lewis GD see Malhotra R et al. Lofti M see Yao W et al. Long J, Russo MJ, Muller C, Vigneswaran WT. Surgical treatment of pulmonary hypertension: Lung transplantation 327 Long L see Yang X et al. Lopes AA see del Cerro MJ et al. Lopes AA see Lammers AE et al. Loyd JE see Austin ED et al. Loyd JE see Fessel JP et al. Machado R see Desai A et al. Machado RF see Desai AA et al. Machado RF see Pauwaa S et al. Makino A see Song MY et al. Makino A see Yao W et al. Malhotra R, Hess D, Lewis GD, Bloch KD, Waxman AB, Semigran MJ. Vasoreactivity to inhaled nitric oxide with oxygen predicts long-term survival in pulmonary arterial hypertension 250 Manoj R see Kumar B et al. Marcos E see Damy T et al. Marsboom G see Kim GH et al. Mathier MA see Stamm JA et al. Mavrakis L see Duong HT et al. McLendon JM see Joshi SR et al. McLure LE see Blyth KG et al. McMurtry IF see Ota H et al. McVerry BJ see Stamm JA et al. Medoff BD see Summer R et al. MellemkjĂŚr S see Andersen CU et al. Memon SS see Newman JH et al. Menon S see Austin ED et al. Mernaugh RL see Lane KL et al. Mizuno S see Voelkel NF et al. Morrell NW see Hagan G et al. Morrell NW see J. Yuan JX et al. Morrell NW see Jafri S et al. Morrell NW see Soon E et al. Morrell NW see Yang X et al. Morrell NW see Yuan JX et al. Mu W see Yao W et al. Mukhtyar C see Hagan G et al. Muller C see Long J et al. Newman JH see Austin ED et al. Newman JH, Holt TN, Hedges LK, Womack B, Memon SS, Willers ED, Wheeler L, Phillips JA, Hamid R. High-altitude pulmonary hypertension in cattle (brisket disease): Candidate genes and gene expression profiling of peripheral blood mononuclear cells 462 Nielsen-Kudsk JE see Andersen CU et al. Oka M see Ota H et al. Ota H, Beutz MA, Ito M, Abe K, Oka M, McMurtry IF. S1P4 receptor mediates S1P-induced vasoconstriction in normotensive and hypertensive rat lungs 399 Pang B see Kuang T et al. Pauwaa S, Machado RF, Desai AA. Survival in pulmonary arterial hypertension: A brief review of registry data 430 Peacock AJ see Blyth KG et al. Pepke-Zaba J see Hagan G et al. Pepke-Zaba J see Soon E et al. Perkins DL see Yao W et al. Phillips JA see Newman JH et al. Pilewski JM see George MP et al. Platoshyn O see Firth AL et al. Porres-Aguilar M, Fernandez G, Elliott CG. Cardiopulmonary hemodynamic clues for pulmonary vein stenosis diagnosis 499 Pullamsetti SS see Dahal BK et al.

Pulmonary Circulation | October-December 2011 | Vol 1 | No 4

Puri GD see Kumar B et al. Raghavan A see Singh DK et al. Raj JU see del Cerro MJ et al. Raj JU see Lammers AE et al. Raj JU see Singh DK et al. Rassl D see Hagan G et al. Reddy SP see Singh DK et al. Remillard CV see Firth AL et al. Remillard CV see Yao W et al. Reynolds PN see Yang X et al. Risbano MG see Stamm JA et al. Robbins I see Austin ED et al. Roberts II LJ see Lane KL et al. Roberts JD, Forfia PR. Diagnosis and assessment of pulmonary vascular disease by Doppler echocardiography 160 Robinson L see Lane KL et al. Robinson LR see Austin ED et al. Ross RM see Hagan G et al. Ross RM see Soon E et al. Rudd JH see Hagan G et al. Russo MJ see Long J et al. Ryan JJ see Kim GH et al. Said SI, Hamidi SA. Pharmacogenomics in pulmonary arterial hypertension: Toward a mechanistic, target-based approach to therapy 383 Sandoval J see del Cerro MJ et al. Sandoval J see Lammers AE et al. Sarkar J see Singh DK et al. Saul MI see Stamm JA et al. Savasky BM see Duong HT et al. Schermuly RT see Dahal BK et al. Screaton N see Hagan G et al. Seeger W see Dahal BK et al. Seeger W see Thamm M et al. Sehgal PB, Lee JE. Protein trafficking dysfunctions: Role in the pathogenesis of pulmonary arterial hypertension 17 Semigran MJ see Malhotra R et al. Sheares K see Hagan G et al. Sheares K see Soon E et al. Shyam KS see Kumar B et al. Siddiqui AM see Blyth KG et al. Simonsen U see Andersen CU et al. Singh DK, Sarkar J, Raghavan A, Reddy SP, Raj JU. Hypoxia modulates the expression of leucine zipper-positive MYPT1 and its interaction with protein kinase G and Rho kinases in pulmonary arterial smooth muscle cells 487 Sivasothy P see Hagan G et al. Sivasothy P see Jafri S et al. Soderberg P. Reflections on the rise and fall of PVD, medical nanotechnology and Australia covered with houseflies 437 Song MY, Makino A, Yuan JX. STIM2 contributes to enhanced store-operated Ca2+ entry in pulmonary artery smooth muscle cells from patients with idiopathic pulmonary arterial hypertension 84 Soon E see Hagan G et al. Soon E, Doughty NJ, Treacy CM, Ross RM, Toshner M, Upton PD, Sheares K, Morrell NW, Pepke-Zaba J. Log-transformation improves the prognostic value of serial NT-proBNP levels in apparently stable pulmonary arterial hypertension 244 Southwood M see Hagan G et al. Stamm JA, McVerry BJ, Mathier MA, Donahoe MP, Saul MI, Gladwin MT. Doppler-defined pulmonary hypertension in medical intensive care unit patients: Retrospective investigation of risk factors and impact on mortality 95 Stamm JA, Risbano MG, Mathier MA. Overview of current therapeutic approaches for pulmonary hypertension 138 Stenmark K see del Cerro MJ et al. Stenmark K see Lammers AE et al. Stenmark KR see Yeager ME et al. Stewart S see Hagan G et al. Summer R, Walsh K, Medoff BD. Obesity and pulmonary arterial hypertension: Is adiponectin the molecular link between these conditions? 440 Sutliff RL see Green DE et al. Tabrizchi R see Duggan DJ et al. Talati M see Austin ED et al. Talati M see Lane KL et al. Tawhai MH see Burrowes KS et al. Tawhai MH, Clark AR, Burrowes KS. Computational models of the pulmonary circulation: Insights and the move towards clinically directed studies 224 Teiger E see Damy T et al. Thamm M, Voswinckel R, Tiede H, Lendeckel F, Grimminger F, Seeger W, Ghofrani HA. Air travel can be safe and well tolerated in patients with clinically stable pulmonary hypertension 239 Tiede H see Thamm M et al. Toshner M see Soon E et al.

509

Author and title index

Treacy C see Hagan G et al. Treacy CM see Soon E et al. Tsui S see Hagan G et al. Tu L see Damy T et al. Tuder RM see Graham BB et al. Upton PD see Soon E et al. Vigneswaran WT see Long J et al. Voelkel NF, Gomez-Arroyo J, Mizuno S. COPD/emphysema: The vascular story Voswinckel R see Thamm M et al. Wagner GS see Blyth KG et al. Wagner WW see Weir EK et al. Walsh K see Summer R et al. Wang C see Kuang T et al. Wang J see Kuang T et al. Wang Z, Chesler NC. Pulmonary vascular wall stiffness: An important contributor to the increased right ventricular afterload with pulmonary hypertension Waxman AB see Malhotra R et al. Weir EK, Wagner WW, Archer SL. Lofty goals at high altitude: The Grover Conferences, 1984-2011 Weissmann N see Dahal BK et al. Wells F see Jafri S et al. West J see Austin ED et al. West J see Lane KL et al. Wheeler L see Newman JH et al. Wilhelm J see Dahal BK et al.

320

212 501

Willers ED see Newman JH et al. Wistow T see Hagan G et al. Womack B see Newman JH et al. Yamamura A, Yamamura H, Zeifman A, Yuan JX. Activity of Ca2+-activated Cl- channels contributes to regulating receptor- and storeoperated Ca2+ entry in human pulmonary artery smooth muscle cells 269 Yamamura H see Yamamura A et al. Yang X, Long L, Reynolds PN, Morrell NW. Expression of mutant BMPR-II in pulmonary endothelial cells promotes apoptosis and a release of factors that stimulate proliferation of pulmonary arterial smooth muscle cells 103 Yao W, Mu W, Zeifman A, Lofti M, Remillard CV, Makino A, Perkins DL, Garcia JG, Yuan JX, Zhang W. Fenfluramine-induced gene dysregulation in human pulmonary artery smooth muscle and endothelial cells 405 Yeager ME, Frid MG, Stenmark KR. Progenitor cells in pulmonary vascular remodeling 3 Yuan JX see Song MY et al. Yuan JX see Yamamura A et al. Yuan JX see Yao W et al. Yuan JX, Morrell NW, Harikrishnan S, Butrous G. No doctor is an island 435 Yuan JX, Morrell NW, Harikrishnan S, Butrous G. Our journey continues 133 Yuan JX, Morrell NW, Harikrishnan S, Butrous G. The world of pulmonary vascular disease 303 Zeifman A see Kuang T et al. Zeifman A see Yamamura A et al. Zeifman A see Yao W et al. Zhang W see Yao W et al.

Ti t l e I ndex, 2 0 1 1 18 FDG PET imaging can quantify increased cellular metabolism in pulmonary arterial hypertension: A proof-of-principle study A consensus approach to the classification of pediatric pulmonary hypertensive vascular disease: Report from the PVRI Pediatric Taskforce, Panama 2011 Activity of Ca2+-activated Cl- channels contributes to regulating receptor- and store-operated Ca 2+ entry in human pulmonary artery smooth muscle cells Acute respiratory distress syndrome: A clinical review Air travel can be safe and well tolerated in patients with clinically stable pulmonary hypertension Apelin and pulmonary hypertension Blood flow redistribution and ventilation-perfusion mismatch during embolic pulmonary arterial occlusion Cardiopulmonary hemodynamic clues for pulmonary vein stenosis diagnosis Classification of pediatric pulmonary hypertensive vascular disease: Does it need to be different from the adult classification? Clinical demonstration of efficiency and reversibility of hypoxic pulmonary vasoconstriction in a patient presenting with unilateral incomplete bronchial occlusion Combination use of sildenafil and simvastatin increases BMPR-II signal transduction in rats with monocrotaline-mediated pulmonary hypertension Computational models of the pulmonary circulation: Insights and the move towards clinically directed studies COPD/emphysema: The vascular story Diagnosis and assessment of pulmonary vascular disease by Doppler echocardiography Diagnostic and therapeutic algorithm for pulmonary arterial hypertension Doppler-defined pulmonary hypertension in medical intensive care unit patients: Retrospective investigation of risk factors and impact on mortality Drugs currently used for treatment of PAH Epigenetic mechanisms of pulmonary hypertension Expression of mutant BMPR-II in pulmonary endothelial cells promotes apoptosis and a release of factors that stimulate proliferation of pulmonary arterial smooth muscle cells Fenfluramine-induced gene dysregulation in human pulmonary artery smooth muscle and endothelial cells Functional classification of pulmonary hypertension in children: Report from the PVRI pediatric taskforce, Panama 2011 Functional ion channels in human pulmonary artery smooth muscle cells: Voltage-dependent cation channels High-altitude pulmonary hypertension in cattle (brisket disease): Candidate genes and gene expression profiling of peripheral blood mononuclear cells Hypoxia modulates the expression of leucine zipper-positive MYPT1 and its interaction with protein kinase G and Rho kinases in pulmonary arterial smooth muscle cells Hypoxic pulmonary hypertension in mice with constitutively active platelet-derived growth factor receptorÎ˛ Idiopathic and heritable PAH perturb common molecular pathways, correlated with increased MSX1 expression Is peroxisome proliferator-activated receptor gamma (PPARÎł) a

510

448 286 269 192 239 334 365 499 134 119 111 224 320 160 122 95 299 347 103 405 280 48 462 487 259 389

therapeutic target for the treatment of pulmonary hypertension? Isolated large vessel pulmonary vasculitis as a cause of chronic obstruction of the pulmonary arteries Lofty goals at high altitude: The Grover Conferences, 1984-2011 Log-transformation improves the prognostic value of serial NT-proBNP levels in apparently stable pulmonary arterial hypertension Lung transplantation for pulmonary hypertension MicroRNAs-control of essential genes: Implications for pulmonary vascular disease Neurogenic responses in rat and porcine large pulmonary arteries No doctor is an island Obesity and pulmonary arterial hypertension: Is adiponectin the molecular link between these conditions? Our journey continues Overview of current therapeutic approaches for pulmonary hypertension Oxidative injury is a common consequence of BMPR2 mutations Pharmacogenomics in pulmonary arterial hypertension: Toward a mechanistic, target-based approach to therapy Progenitor cells in pulmonary vascular remodeling Protein trafficking dysfunctions: Role in the pathogenesis of pulmonary arterial hypertension Pulmonary artery endothelium resident endothelial colony-forming cells in pulmonary arterial hypertension Pulmonary Circulation: A new venue for communicating your findings, ideas and perspectives Pulmonary hemodynamic responses to inhaled NO in chronic heart failure depend on PDE5 G(-1142)T polymorphism Pulmonary vascular wall stiffness: An important contributor to the increased right ventricular afterload with pulmonary hypertension Quantitative estimation of right ventricular hypertrophy using ECG criteria in patients with pulmonary hypertension: A comparison with cardiac MRI Reflections on the rise and fall of PVD, medical nanotechnology and Australia covered with houseflies S1P4 receptor mediates S1P-induced vasoconstriction in normotensive and hypertensive rat lungs Scientific Abstracts Severe pulmonary artery hypertension following intracardiac repair of tetralogy of Fallot: An unusual finding Significant intrapulmonary Schistosoma egg antigens are not present in schistosomiasis-associated pulmonary hypertension STIM2 contributes to enhanced store-operated Ca 2+ entry in pulmonary artery smooth muscle cells from patients with idiopathic pulmonary arterial hypertension Surgical treatment of pulmonary hypertension: Lung transplantation Survival in pulmonary arterial hypertension: A brief review of registry data The genetics of pulmonary arterial hypertension in the post-BMPR2 era The PVM History Initiative of the PVRI The world of pulmonary vascular disease Vasoreactivity to inhaled nitric oxide with oxygen predicts long-term survival in pulmonary arterial hypertension

33 425 501 244 182 357 419 435 440 133 138 72 383 3 17 475 1 377 212 470 437 399 508 115 456 84 327 430 305 125 303 250

Pulmonary Circulation | October-December 2011 | Vol 1 | No 4

Scientific Abstracts The 2011 Grover Conference 5th Scientific Workshops and Debates of the PVRI Pulmonary Hypertension Society of Australia and New Zealand (PHSANZ)

Pulmonary Circulation | October-December 2011 | Vol 1 | No 4

Scientific Abstract Author Index 1.14 - Genetic risk factors for portopulmonary hypertension in patients with advanced liver disease

The 2011 Grover Conference 1.1 - Apelin and pulmonary hypertension Andersen CU

1.2 - Alterations in estrogen metabolism: Implications for higher penetrance of FPAH in females Austin ED, West JD, Hamid R, Hemnes AR, Cogan JD, Parl FF, Phillips JA III, and Loyd JE

Sitbon O S1

1.5 - microRNAs-control of essential genes: Implications for pulmonary vascular disease Gerthoffer WT

1.6 - The role of medical therapy in non-PAH pulmonary hypertension Hoeper MM

1.8 - Update and overview of PAH genetics Loyd JE

MacLean MR, Johansen AK, Wallace E, Campbell A, Dempsie Y, White K

1.13 - Dehydroepiandrosterone and experimental pulmonary hypertension Oka M, Alzoubi A, Toba M, Fagan KA, and McMurtry IF Sii

1.19 - Aerobic capacity and pulmonary arterial hypertension Duggan N, Stevenson C, Ciuclan L, Bonneau O, Rowlands D, Roger J, Koch L, Britton S, Walker C, Westwick J, and Thomas M

1.20 - Estradiol metabolites and progestins in experimental pulmonary hypertension S7

Vidal-Puig A

Voelkel NF, Hussani AA, Abbate A, Farkas L, and Bogaard HJ

1.23 - Adiponectin and pulmonary hypertension

1.12 - Gene expression pro iling of peripheral blood mononuclear cells from cattle with high altitude pulmonary hypertension (brisket disease) Newman JH, Holt TN, Hedges LK, Womack B, West J, and Hamid R

1.18 - Novel loci interacting epistatically with BMPR2 cause FPAH

1.22 - Endocrine determinants of severe pulmonary arterial hypertension

1.11 - Progenitor cells in pulmonary arterial hypertension Morrell NM

1.21 - Adipose tissue expandability, lipotoxicity and the metabolic syndrome

1.10 - Fat, ire and muscle: The effects of adiponectin on pulmonary vascular in lammation and remodeling Weng MQ, Combs TP, Scherer PE, Bloch KD, and Medoff BD

Stenmark KR, Li M, Riddle SR, Flockton AR, Barajas C, Yeager ME, Frid MG, Anderson AL, McKenzie T, and El Kasmi KC

Tofovic SP

1.9 - The in luence of serotonin and estrogen in the development of pulmonary arterial hypertension

1.17 - Chronic hypoxia leads to emergence of proin lammatory pulmonary adventitial ibroblasts capable of inducing proin lammatory and pro ibrogenic activation of monocytes/macrophages: Amelioration by HDAC inhibitors

Stewart WCL, Ho YY, Loyd J, Chung W, and Greenberg DA S2

1.7 - Epigenetics, sex, and cardiovascular function with a focus on exchange Huxley VH

1.16 - Risk factors and responsiveness to therapies in the modern era of PAH treatment

1.4 - Persistent pulmonary hypertension of the newborn: Serotonin as a risk factor Delaney C

1.15 - VIP is a physiological modulator of pulmonary arterial hypertension Said SI

1.3 - Etiologies and comorbidities in PH: Insight from the REVEAL Registry Badesch DB, Raskob GE, Elliott CG, Krichman AM, Farber HW, Frost AE, Barst RJ, Benza RL, Liou TG, Turner M,Giles S, Feldkircher K, Miller DP, and McGoon MD

Roberts KE, Fallon MB, Krowka MJ, Brown RS, Trotter JF, Peter I, Tighiouart H, Knowles JA, Rabinowitz D, Benza RL, Badesch DB, Taichman DB, Horn EM, Zacks S, Kaplowitz N, and Kawut SM

Summer R and Walsh K

1.24 - Sildena il-cGMP-PKG-PPAR-y signaling pathway inhibits angiotensin-II-induced transient receptor potential canonical 6 expression in pulmonary arterial smooth muscles cells Wang J, Yang K, Zhang Y, Lai N, Chen M, and Lu W

1.25 - The estrogen metabolite 16-OHE exacerbates BMPR2-related PAH, associated with defects in receptor traf icking West JD

Pulmonary Circulation | October-December 2011 | Vol 1 | No 4

1.26 - Biomarkers for pulmonary arterial hypertension—Can they be used to predict response to treatment? Wilkins MR

1.39 - Induced pluripotent stem cells as a model for heritable pulmonary hypertension S9

1.27 - Pathological role of TRPC channels in idiopathic pulmonary arterial hypertension Yuan J X-Y

1.28 - Cardiac sympathetic activity evaluated by 123 I-MIBG myocardial scintigraphy in patients with right ventricular dysfunction associated with pulmonary arterial hypertension Abe K, Nagao M, Hirooka Y, Kishi T, Yonezawa M, Higo T, Ide T, and Sunagawa K

S10

1.30 - Large conductance calcium-activated potassium (BK) channel activation causes enhanced vasodilatation in hypoxic lung microvasculature Vang A, Li A, and Choudhary G

S10

1.31 - Compensatory regulation of the BMP-targets ID1 and ID3 in hypoxic pulmonary hypertension Lowery J, Frump A and de Caestecker M

S11

1.32 - Effects of NMD status on susceptibility to pulmonary hypertension and endothelial dysfunction in mice carrying different germ line BMPR2 mutations Frump A and de Caestecker M

S11

S12

1.35 - Evidence of BMPR2 alternative splicing as a novel genetic modi ier of HPAH penetrance Hamid R, Hedges L, Austin E, Womack B, and Cogan JD

S12

1.36 - Urokinase plasminogen activator receptor is a marker of pulmonary venous smooth muscle cells Hunt J

S12

1.37 - Cytochrome P450 1B1 in luences the development of pulmonary arterial hypertension Johansen AK, White K, Morecroft I, Mair K, Nilsen M and MacLean MR

S12

1.38 - 17-Estradiol (E2) attenuates hypoxia-induced pulmonary hypertension through an estrogen receptor-dependent mechanism that involves decreased ERK1/2 activation Lahm T, Albrecht M, Fisher A, Patel N, Justice M, Presson R, and Petrache I S13 Pulmonary Circulation | October-December 2011 | Vol 1 | No 4

Pillay T and Pϔister S

S13

Ryan JJ, Fang YH, Rich JD, Thenappan T, and Archer SL

S14

1.42 - Calpain activates intracellular TGF-1 in pulmonary vascular remodeling of pulmonary hypertension Ma W, Han W, Greer PA, Tuder RM, Wang KKW, and Su Y

S14

1.43 - Downregulation of SERCA2a in maladaptive but not adaptive RVH Thenappan T, Fang YH, Piao L, and Archer SL

S14

1.44 - The cell-penetrating homing peptide CAR selectively enhances pulmonary effects of systemically coadministered vasodilators in a preclinical model of severe pulmonary arterial hypertension S14

1.45 - Possible involvement of endothelial mesenchymal transition in the formation of plexiform lesions in a preclinical model of severe pulmonary arterial hypertension Toba M, Alzoubi A, McMurtry IF, and Oka M

S15

1.46 - Reversion of vascular remodeling in pulmonary hypertension—Impact of AMD-1

1.34 - Dual blockade of IL4 and IL13 in schistosomiasis-associated pulmonary hypertension Graham B, Chabon J, and Tuder R

1.40 - A novel mechanism to explain enhanced pulmonary artery vasoconstriction in female rabbits

Toba M, Abe K, Urakami T, Komatsu M, Jarvinen TAH, Mann D, Ruoslahti E, McMurtry IF, and Oka M

1.33 - mTOR Complex 2 regulates energy levels, proliferation and survival of vascular smooth muscle cells in pulmonary arterial hypertension Goncharov D, Krymskaya V, and Goncharova E

S13

1.41 - QTc prolongation round in animal models of pulmonary hypertension is a marker of survival and is reversible by the fatty acid oxidation inhibitor, trimetazidine

1.29 - Somatic chromosome abnormalities in PAH lungs—Modi ier or bystander? Drake KM and Aldred MA

Majka S, Chow K, Omari A, Jean JC, Bilousova A, Hedges L, Kotton D and Austin E

Weisel FC, Roth M, Kloepping C, Wilhelm J, Pichl A, Seeger W, Ridge KM, Weissmann N, and Kwapiszewska G

S15

1.47 - A critical role for calpain in the development of pulmonary hypertension Zaiman AL, Swaim M, Cingolani O, Damico R, Blanco I, Undem C, Maylor J, Tuder RM, Shimoda LA, and Dietz H

S15

5th Scientific Workshops and Debates of the Pulmonary Vascular Research Institute (PVRI) 2.1 - Clinical characteristics and outcome of pulmonary hypertension among admitted heart failure patients Karaye KM, Yahaya I, Sa’idu H, and Bala MS

S16

2.2 - Heart rate recovery after 6-minute walk test in patients with pulmonary arterial hypertension Minai OA and Gudavalli R

S16 Siii

2.3 - Does high altitude protect against irreversible pulmonary hypertension? Heath A, von Alvensleben I, Graham B, Tuder R, Brockman C, and Perez E. Kardiozentrum

2.14 - Pulmonary hypertension at moderate altitude in children: Importance of the hyperreactivity of pulmonary vascular tree S16

2.4 - Long-term follow-up of closure of atrial septal defects in older children and adults with severe pulmonary arterial hypertension Harikrishnan S, Sonney JP, Randeep S, Venkiteswaran S, Krishnamoorthy KM, Sivasankaran S, Titus T, and Jaganmohan T

Birukov KG, Fu P, Sarich N, and Birukova AA S17

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Chaouat A

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2.9 - Platelet function and morphology in idiopathic pulmonary hypertension Ramakrishnan S, Senguttuvan NB, Lakshmy R, Saxena R, Wadhwa S, Khunger JM, Bhargava B, Kothari SS, Saxena A, and Bahl VK

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2.11 - A review of catheterization data for determining operability in congenital heart disease with severe pulmonary hypertension Juneja R, Ramakrishnan S, Anju, Gupta S, Kothari SS, and Saxena A

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2.20 - Causes of pulmonary arterial hypertension in the pediatric population in a tertiary care center in Saudi Arabia Banjar H

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Savai R, Pullamsetti SS, Kolbe J, Bieniek E, Ghofrani HA, Weissmann N, Fink L, Klepetko W, Voswinckel R, Banat GA, Seeger W, Grimminger F, and Schermuly RT S22

2.22 - Nonresponse to acute pulmonary vasodilator challenge in idiopathic pulmonary arterial hypertension is associated with nonrecruitment of pulmonary functional capillary endothelial surface area Langleben D, Orfanos SE, Giovinazzo M, Hirsch A, Sotiropoulou Ch, Armaganidis A, and Catravas JD

Díaz G, Ruiz AI, Acherman R, Montealegre A, Ome L, and Marquez A

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2.24 - Sildena il plasma concentrations in two HIV patients with pulmonary arterial hypertension treated with ritonavir-boosted protease inhibitors Chinello P, Cicalini S, Pichini S, Paciϔici R, Tempestilli M, and Petrosillo N

2.13 - Nocturnal hypoxemia in patients with Eisenmenger syndrome Ramakrishnan S, Juneja R, Sharma AK, Bardolei N, Shukla G, Guleria R, Bhatia M, Kalaivani M, Kothari SS, Saxena A, and Bahl VK

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2.23 - Utility of the brain natriuretic peptide as a marker in the diagnosis of patients with persistent pulmonary hypertension of the newborn

2.12 - Predictors of adverse clinical outcome in Eisenmenger syndrome Ramakrishnan S, Kukreti BB, Juneja R, Bhargava B, Kothari SS, Saxena A, and Bahl VK

Pandey P, Mohammad G, and Pasha Q

2.21 - Involvement of immune/in lammatory cells to pathology of idiopathic pulmonary arterial hypertension

2.10 - Prevalence of pulmonary hypertension among 6270 asymptomatic school children in India: The Rheumatic Heart Echo Utilization and Monitoring Actuarial Trends in Indian Children Study Saxena A, Ramakrishnan S, Roy A, Seth S, Krishnan A, Misra P, Kalaivani M, Bhargava B, Flather M, and Poole-Wilson P

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2.19 - Interkinase communiqué: The P Code

2.8 - Blockade of TGF- prevents schistosomiasisassociated pulmonary hypertension Graham B, Chabon J, and Tuder R

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2.18 - Vasoactive mediators and ROS interactions under hypobaric hypoxia Ali Z, Mishra A, Mohammad G, and Pasha Q

2.7 - Prognostic value of exercise cardiac index in idiopathic, heritable and anorexigen-associated pulmonary arterial hypertension

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2.17 - Oxygen sensing pathway: Revealing and documenting human adaptations at high altitude Mishra A, Thinlas T, Mohammad G, and Pasha Q

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2.16 - Atrial natriuretic peptide improves Staphylococcus aureus-induced lung in lammation and vascular barrier function Birukova AA, Xing J, and Moldobaeva N

2.6 - Overview of pulmonary hypertension registries and quality standards Pittrow D

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2.15 - Novel roles of radiation protective compound amifostine in attenuation of acute lung injury

2.5 - Pulmonary hypertension in a tertiary health institution: A retrospective review of 24-months echocardiography registry Sani MU, Mijinyawa MS, Ishaq NA, and Shehu MN

Díaz GF

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2.25 - Estimation of pulmonary artery pressure in patients with sickle cell anemia in Ibadan, Nigeria: An echocardiographic study Pulmonary Circulation | October-December 2011 | Vol 1 | No 4

Enakpene EO, Adebiyi A, Ogah OS, Olaniyi JA, Aje A, Adebayo AK, Ojji DB, Adeoye MA, Ochulor KC, Oladapo OO, and Falase AO

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2.26 - Epidemiology of pulmonary heart disease in Nigeria: Insight from the Abeokuta Heart Failure Registry Ogah OS, Falase AO, Stewart S, and Sliwa K

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2.28 - Markers of left and right ventricular remodeling in a native African hypertensive cohort Ojji D, Lacerda L, Lecour S, Adeyemi Billyrose M, and Sliwa K

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2.29 - Serotonin passes through myoendothelial gap junctions to promote pulmonary arterial smooth muscle cell differentiation Gairhe S, Gebb SA, and McMurty IF

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2.30 - Doppler echocardiographic assessment of pulmonary artery pressure in apparently healthy Nigerian primary school children in Ibadan (Western Nigeria) Udo PA, Orimadegun AE, Omokhodion FO

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2.32 - Long-term endothelin receptor antagonist therapy may predispose to carcinogenesis in patients with pulmonary arterial hypertension Safdar Z and Qureshi H

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2.37 - The behavior of the right ventricular dysfunction by echocardiography and clinical transthoracic Murillo C Pulmonary Circulation | October-December 2011 | Vol 1 | No 4

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2.41 - LC/MS/MS method for simultaneous analysis of arachidonic acid and its endogenous eicosanoid metabolites and prostaglandins in rodent lung tissue Sagliani K, Hill NS, Fanburg BF, Dolnikowski G, Levy BL, and Preston IR

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2.42 - Determination of endogenous bioactive lipid pro ile in experimental pulmonary hypertension Sagliani K, Hill NS, Fanburg BF, Warburton RW, Dolnikowski G, Levy BL, and Preston IR

Sagliani K, Preston IR, Roberts KR, Fanburg BF, Dolnikowski G, Levy BL, and Hill NS

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2.44 - Is bronchus-associated lymphoid tissue readily evident in lung sections from patients with PAH and in rates with monocrotaline-induced PH? S30

2.45 - Echocardiographic prediction of pulmonary vascular resistance Opotowsky AR, Clair M, Landzberg MJ, Waxman AB, Arkles JS, Rogers F, Prasanna V, Moko L, Maron B, Fields A, and ForĎ&#x201D;ia PR

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2.46 - The use of cardiac MRI in understanding PAH in complex CHD: Case reports S31

2.47 - Therapeutic potential of HDAC inhibitors in pulmonary hypertension S31

2.48 - Atrial lutter and atrial ibrillation in patients with chronic pulmonary hypertension Olsson KM, Nickel N, Tongers J, and Hoeper MM

2.36 - Subcellular mechanisms in IPAH: Coordinate dysfunctions of the Golgi-ER-mitochondrial axis Sehgal PB

Kulkarni S, Jadhav M, Garekar S, and Rao S

Zhao L, Chen CN, Hajji, Oliver E, Huang TJ, Wang D, Li M, McKinsey T, Stenmark KR, and Wilkins MR

2.35 - Pulmonary hypertension and pulmonary vascular remodeling in mouse models of schistosomiasis Crosby A

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2.40 - Use of combination therapy in postoperative persistent pulmonary arterial hypertension in children

Kappanayil M

2.34 - Pulmonary hypertension among patients with sickle cell disease in Africa Wonkam A

Tiede H, Felix J, Wilkins M, Steyerberg E, Seeger W, Grimminger F, and Ghofrani A

Yeager M

2.33 - Noninvasive evaluation of pulmonary hypertension and its correlates among adult patients with sickle cell disease Mbakwem AC and Kehinde MO

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2.43 - Lipidomic analysis of plasma from patients with pulmonary arterial hypertension: Determination of circulating arachidonic acid metabolites

2.31 - Adverse events of pulmonary hypertension pharmacotherapy in children Roldan T and Cerro MJ

Ă&#x2DC;stergaard, L

2.39 - A practicable risk score for pulmonary hypertension

2.27 - Pulmonary hypertension associated with sickle cell anemia: A review of epidemiology, pathophysiology and management Ogah OS, Falase AO, Stewart S, and Sliwa K

2.38 - Single and combination therapy with erythropoietin and sildena il on hypoxia-induced PAH

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Pulmonary Hypertension Society of Australia and New Zealand (PHSANZ) 3.1 - Pulmonary arterial hypertension in patients with systemic sclerosis is independent of highresolution computed tomography indings of interstitial lung disease Sv

Tuppin M, Chambers D, Slaughter R, Mohammed O, and Kermeen F

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3.2 - Imatinib for the treatment of pulmonary arterial hypertension and pulmonary capillary hemangiomatosis Nayyar D, Muthiah K, Kumarasinghe G, Hettiarachchi R, Macdonald P, Kotlyar E, Hayward C, and Keogh A

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3.6 - Survival and predictors of mortality in Australian patients with connective tissue diseaseassociated pulmonary arterial hypertension Ngian GS, Stevens W, Byron J, Tran A, Roddy J, Minson R, Hill C, Chow K, Sahhar J, Proudman S, and Nikpour M

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3.13 - Treatment for pulmonary arterial hypertension complicating congenital heart disease in adults: Results from a national registry S36

3.14 - Serum ICAM-1 levels are related to the presence of interstitial lung disease in systemic sclerosis Thakkar V, Patterson K, Stevens W, Byron J, Moore O, Roddy J, Zochling J, Sahhar J, Nash P, Tymms K, Youssef P, Proudman S, Hissaria P, and Nikpour M

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3.15 - Predictors of six-minute walk distance in patients with systemic sclerosis associated pulmonary hypertension S37

Harris JE, Seale HE, McKinnon K, Cornwell PL, Morris N, and Kermeen FD

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3.17 - The hidden risk of a 6-minute walk test in pulmonary arterial hypertension Seale H, Harris J, Hall K, and Kermeen F

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3.18 - Symptoms to de initive diagnosis of pulmonary arterial hypertension Strange G, Keogh A, Stewart S, Carrington M, Kermeen F, Williams T, and Gabbay E

3.9 - Lung disease, particularly pulmonary arterial hypertension, is the major cause of death in the Australian Systemic Sclerosis Cohort Study Stevens W, Thakkar V, Moore O, Byron J, Proudman S, Zochling J, Roddy J, Sahhar J, Nash P, Youssef P, Major G, Tymms K, Hill C, Sturgess A, Schrieber A and Nikpour M

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3.16 - Stretching the boundaries: The effectiveness of Tai chi in PAH

3.8 - The demographics of pulmonary arterial hypertension associated with congenital heart disease: Results from a national registry Strange G, Rose M, Kermeen F, King I, Vidmar S, Grigg L, Celermajer D, and Weintraub R (on behalf of the ANZ CHD-PAH Registry)

Thakkar V, Patterson K, Stevens W, Byron J, Moore O, Roddy J, Zochling J, Sahhar J, Nash P, Tymms K, Youssef P, Proudman S, Nikpour M, and Hissaria P

Tuppin M, Chambers D, Slaughter R, Mohammed O, and Kermeen F

3.7 - Chronic thromboembolic pulmonary hypertension: MRI predictors of functional and haemodynamic outcomes with pulmonary endarterectomy Ng BJH, Slaughter RE, Strugnell WE, Yerkovich ST, McNeil K, Dunning JJ, Hopkins PMA, and Kermeen FD

Tuppin M, Chambers D, Slaughter R, Mohammed O, and Kermeen F

Rose M, Strange G, Kermeen F, King I, Vidmar S, Grigg L, Weintraub R, and Celermajer D (on behalf of the ANZ CHD-PAH Registry)

3.5 - N-Terminal pro-Brain Natriuretic Peptide (NT-proBNP) levels predict incident pulmonary arterial hypertension in systemic sclerosis (SSc) in the Australian Scleroderma Cohort Study (ASCS) Thakkar V, Stevens W, Priorv D, Byron J, Patterson K, Hissaria P, Moore O, Roddy J, Zochling J, Sahhar J, Nash P, Tymms K, Youssef P, Proudman S, and Nikpour M

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3.12 - Novel biomarkers of dysregulated angiogenesis are not speci ic to pulmonary arterial hypertension in systemic sclerosis

3.4 - An Australian tertiary referral centre experience of the management of CTEPH Maliyasena VA, Hopkins PMA, Thomson BM, Dunning J, Wall DA, Ng BJH, McNeil KD, and Kermeen FD

Cicovic A, McWilliams T, Coverdale HA, Whyte K, Stewart C, and Wasywich CA

3.11 - Neovascularity in patients with idiopathic pulmonary arterial hypertension

3.3 - Abnormal pulmonary artery stiffness in pulmonary arterial hypertension: In vivo study with intravascular ultrasound Lau EMT, Iyer N, Ilsar R, Bailey BP, Adams MR, and Celermajer DS

3.10 - Evolution of a pulmonary hypertension clinic

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3.19 - Pulmonary hypertension is a common disease: The Armadale echo study S35

Strange G, Playford D, Stewart S, Kent A, Deague J, and Gabbay E

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Pulmonary Circulation | October-December 2011 | Vol 1 | No 4

Abstr ac t s

The 2011 Grover Conference

Risk Factors in Pulmonary Hypertension: Gender, Sex Hormones, Obesity, Novel Genetic Influences and Risk Factors Sedalia, Colorado, USA 7-11 September, 2011

1.1

Apelin and pulmonary hypertension Andersen CU Department of Biomedicine, Aarhus University, Aarhus, Denmark

The peptide apelin and the G-protein-coupled apelin receptor (APLNR) are expressed in several tissues throughout the organism, and are highly expressed in the lungs. In accordance, apelin and the APLNR have proven to in luence many physiological functions such as food and water intake and regulation of insulin in obesity. However, some of the irst established effects of apelin and the APLNR were regulatory roles in vascular and cardiac tissues. In the cardiovascular system, apelin is localized in endothelial cells in agreement with an endothelial synthesis, while the APLNR is localized in both endothelial and smooth muscle cells in the heart and vessels. Apelin plays a role in angiogenesis, and there is evidence from experimental and human studies that apelin exerts nitric oxide (NO)-dependent vasodilatation in the systemic circulation and modulates the expression and activity of endothelial NO synthase (eNOS). In addition, apelin has proven to be a potent positive inotropic agent. In patients with heart failure, including patients with pulmonary hypertension (PH), plasma levels of apelin are altered. On the basis of apelinâ&#x20AC;&#x2122;s localization and effects in the systemic circulation, interest in the role of the peptide in PH has emerged. Apelin has subsequently been investigated as a potential lung-derived biomarker and a possible new therapeutic for PH. A study has shown that apelin levels in the lungs are decreased in animals with PH. This decrease was, however, not re lected in plasma, which does not support that plasma apelin measurements give information about changes in the pulmonary vascular endothelium in the setting of PH. Apelin modulates vasoconstriction in isolated rat pulmonary arteries, and chronic treatment with apelin has been demonstrated to reduce pulmonary pressure in experimental models of monocrotaline and hypoxia induced PH. In agreement, a recent study shows that apelin de iciency worsens the development of PH. The mechanisms involved were downregulation of eNOS and loss of smallsize arteries. The existing literature thus renders APLNR a potential new therapeutic target for PH that needs more investigation. 1.2

Alterations in estrogen metabolism: Implications for higher penetrance of FPAH in females Austin ED, West JD, Hamid R, Hemnes AR, Cogan JD, Parl FF, Phillips JA III, and Loyd JE Department of Medicine, Vanderbilt University Medical Center, Nashville, TN, USA

Female gender is the strongest and best-established risk factor for pulmonary arterial hypertension (PAH), with a female to male ratio of ~2 to 4:1 in many etiologies of PAH, including the idiopathic (IPAH) and heritable (HPAH) forms. However, until recently mechanistic details behind the female predominance were unknown. Complicating the gender dimorphism, recent reports from the two large epidemiological registries Pulmonary Circulation | October-December 2011 | Vol 1 | No 4

of PAH in France and in the United States found improved survival among females, with males over 60 years perhaps the most disadvantaged. The female predominant incidence and prevalence suggest a role for sex hormones in disease pathogenesis, implicating estrogens, androgens, and their metabolic products as a potential cause. However, sex hormonesâ&#x20AC;&#x2122; impact upon the pulmonary vasculature is complex, operating by both acute and chronic effects: acutely protective, some estrogens may be detrimental on a chronic basis. In fact, we and others have hypothesized that while absolute sex hormone levels are important, it is the relative ratio of their metabolites that in luence disease riskâ&#x20AC;&#x201C;especially among those with a genetic predisposition due to a BMPR2 gene mutation. Subjects within families affected by PAH associated with BMPR2 gene mutations (BMPR2-PAH) provide the opportunity to study patients, as well as those at genetic risk of disease, to determine factors which may in luence risk. Parent compound estrogens (e.g., estradiol, E2) in both men and women are predominantly metabolized through hydroxylation at the 2- or 16- position. In vitro, 16-estrogens are more mitogenic and may also be more genotoxic via the formation of unstable DNA adducts, although in most women the precise ratio 2- to 16-estrogens is of minimal concern. However, in the context of a BMPR2 mutation perturbed estrogen metabolism may in luence disease penetrance: Women who have a BMPR2 mutation and preferentially metabolize E2 into 16-estrogens may be at higher risk of disease, while those who preferentially metabolize E2 into 2-estrogens may be protected. CYP1B1 is a cytochrome P450 enzyme critical to estrogen metabolism, although its activity generates 2-estrogens and 4-estrogens preferentially over the 16-estrogens from E2. An association between low CYP1B1 mRNA expression and disease penetrance has been demonstrated, and it was subsequently shown that functional promoter polymorphisms in CYP1B1 are associated with risk of PAH among female BMPR2 mutation carriers, which both imply causation. Furthermore, female BMPR2-PAH patients have a low ratio of 2-estrogens to 16-estrogens in their urine compared to matched-unaffected BMPR2 mutation carriers. These indings suggest that a low 2-estrogen:16estrogen ratio is a risk factor for BMPR2-PAH penetrance and perhaps an exacerbating factor in established PAH of other types. Thus, factors that restore the 2-estrogens, or those that block 16-estrogens, may prevent disease penetrance and progression among BMPR2 mutation carriers and perhaps other subjects at risk. Interestingly, 2-estrogens have been used to prevent and treat monocrotaline-induced rodent pulmonary hypertension, and 16-estrogens appear to exacerbate pulmonary vascular resistance in a rodent model of BMPR2-PAH. Although much work remains to fully understand the complex in luences of estrogens, estrogen metabolites, and other sex hormones on the pulmonary vasculature, mounting data suggest that they are relevant to PAH pathogenesis. 1.3

Etiologies and comorbidities in PH: Insight from the REVEAL Registry Badesch DB, Raskob GE, Elliott CG, Krichman AM, Farber HW, Frost AE, Barst RJ, Benza RL, Liou TG, Turner M, Giles S, Feldkircher K, Miller DP, and McGoon MD University of Colorado Health Sciences Center, Denver, CO, USA S1

Abstracts

The Registry to Evaluate Early And Long-term pulmonary arterial hypertension disease management (REVEAL Registry) was established to provide updated characteristics of patients with pulmonary arterial hypertension (PAH) and to improve diagnosis, treatment, and management. Fifty-four US centers enrolled consecutively screened patients with World Health Organization group I PAH who met expanded hemodynamic criteria of mean pulmonary arterial pressure (PAP) >25 mmHg at rest (30 mmHg with exercise), pulmonary capillary wedge pressure (PCWP) ≤18 mmHg, and pulmonary vascular resistance ≥240 dynes · s · cm−5. Patients meeting the traditional hemodynamic de inition (PCWP≤15 mmHg) were compared with those with a PCWP of 16-18 mmHg. Between March 2006 and September 2007,; 2,967 patients enrolled. Among 2,525 adults meeting traditional hemodynamic criteria, the mean age was 53±14 years, and 2,007 (79.5%) were women. Fortysix percent had idiopathic pulmonary arterial hypertension (IPAH), and 51% associated PAH. Of those with associated PAH, 50% had connective tissue disease (CTD), 20% had congenital heart disease, and 10% had drug- and toxin-related PAH. The mean duration between symptom onset and diagnostic catheterization was 2.8 years, and 1,008 (41.3%) patients were treated with more than one pulmonary vascular-targeted medication. Comorbidities included systemic hypertension, obesity, depression and thyroid disease. Compared with patients meeting the traditional hemodynamic de inition of PAH, patients with a PCWP of 16 to 18 mmHg were older, more obese, had a lower 6-minute walk distance, and had a higher incidence of systemic hypertension, sleep apnea, renal insuf iciency, and diabetes. Patients in the REVEAL Registry are older and more often female than in previous descriptions. Delays between symptom onset and diagnostic catheterization persist. Many treatment regimens are fundamentally empirical, and data will be required to determine outcomes, improve risk strati ication, and develop and validate more precise prognostic tools. Patients with PCWP of 16-18 mmHg differ in a number of important respects from those meeting the traditional hemodynamic de inition of PAH. 1.4

Persistent pulmonary hypertension of the newborn: Serotonin as a risk factor Delaney C Department of Pediatrics, University of Colorado Denver, Denver, CO, USA

Persistent pulmonary hypertension of the newborn (PPHN) is a clinical syndrome characterized by the failure to achieve or sustain the normal decrease in pulmonary vascular resistance (PVR) after birth. Recently, epidemiological studies have linked the use of selective serotonin reuptake inhibitors (SSRIs) for the treatment of maternal depression to a 6-fold increase in the incidence of PPHN when taken during the second half of gestation, suggesting that altered serotonin (5-HT) signaling may contribute to the risk for PPHN. However, very little is known about the roles of 5-HT in the normal development of lungs or in the pathobiology of PPHN. In addition, the direct effects of SSRIs on the fetal pulmonary circulation are unknown, and insights into these observations may lead to a greater understanding of the pathophysiology and treatment of PPHN. In the adult pulmonary circulation, 5-HT is a potent vasoconstrictor and stimulates smooth muscle cell growth and proliferation. Pharmacological or genetic strategies that augment or inhibit 5-HT signaling worsen or improve the severity of pulmonary hypertension in adult animal models, respectively. In striking contrast to the effects of maternal SSRI use on the fetal lung, recent studies suggest that SSRI therapy reduces pulmonary hypertension in adult animal models and in the clinical setting. This apparent disparity between the effects of SSRI further suggests striking developmental differences in 5-HT signaling; however, data on mechanisms of 5-HT signaling pathways in the developing lung or in models of PPHN are limited. We hypothesize that 5-HT plays a key role in maintaining high pulmonary vascular resistance (PVR) in the fetus, and that fetal exposure to SSRIs increases 5-HT activity and causes pulmonary hypertension. We studied the hemodynamic effects of 5−HT, S2

5-HT receptor antagonists, and SSRIs in both chronically prepared fetal sheep and in an ovine model of PPHN. In the chronically prepared ovine fetus, brief infusions of 5−HT (3-20 g) increased PVR in a dose-related fashion. Ketanserin, a 5-HT 2A receptor antagonist, caused pulmonary vasodilation and inhibited 5-HT induced pulmonary vasoconstriction. In contrast, intrapulmonary infusions of GR127945 and SB206553, 5HT 1B and 5HT 2B receptor antagonists, respectively, had no affect on basal PVR or 5HT-induced vasoconstriction. Pretreatment with fasudil, a Rho kinase inhibitor, blunted the effects of 5-HT infusion. Brief infusions of the SSRIs, sertraline and luoxetine, caused potent and sustained elevations of PVR. SSRI-induced pulmonary vasoconstriction was reversed by infusion of ketanserin, and did not affect the acute vasodilator effects of acetylcholine. In the lamb with experimental PPHN, SSRIs and 5-HT cause further elevation of pulmonary vascular without any evidence for vasodilation. We conclude that 5−HT causes pulmonary vasoconstriction and contributes to maintenance of high PVR in the normal fetus through stimulation of 5-HT 2A receptors and Rho kinase activation, and mediates the hypertensive effects of SSRIs. The effect of SSRIs on the developing pulmonary circulation in neonatal pulmonary hypertension differs from their effect in adult models of pulmonary hypertension. This may be due to developmental differences in 5-HT receptor or transporter expression, or unique effects on utero-placental circulation. 1.5

microRNAs-control of essential genes: Implications for pulmonary vascular disease Gerthoffer WT Departments of Biochemistry and Molecular Biology, University of South Alabama, Mobile, AL, USA

During lung development and disease pathogenesis structural cells in the lungs adapt to permit changes in lung function. Fibroblasts, myo ibroblasts, smooth muscle, epithelial cells, and various progenitor cells can all undergo phenotypic modulation. In the pulmonary vasculature occlusive vascular lesions occurring in severe pulmonary arterial hypertension are multifocal, polyclonal lesions containing cells presumed to have undergone phenotypic transition resulting in altered proliferation, cell lifespan or contractility. Dynamic changes in gene expression and protein composition that underlie processes responsible for such cellular plasticity are not fully de ined. Advances in molecular biology have shown that multiple classes of RNA collaborate to establish the set of proteins expressed in a cell. Both coding (mRNA, rRNA) and noncoding RNAs (snoRNA, snRNA, miRNA and lncRNA) act via multiple parallel signaling pathways to regulate transcription, mRNA processing, mRNA stability, translation and possibly protein lifespan. Rapid progress has been made in describing dynamic features of miRNA expression and miRNA function in some vascular tissues. However posttranscriptional gene silencing by microRNA-mediated mRNA degradation and translational blockade is not as well de ined in the pulmonary vasculature. Recent progress in de ining miRNAs that modulate vascular cell phenotypes will be reviewed to illustrate both functional and therapeutic signi icance of small noncoding RNAs in pulmonary arterial hypertension. 1.6

The role of medical therapy in non-PAH pulmonary hypertension Hoeper MM Hannover Medical School, Hannover, Germany

The somewhat copious term non-pulmonary arterial hypertension (PAH) pulmonary hypertension (non-PAH PH) is used to subsume groups 2, 3, and 4 of the current classi ication of PH, i.e., PH due to left heart disease, PH due to lung disease and/or hypoxia, and Pulmonary Circulation | October-December 2011 | Vol 1 | No 4

Abstracts

chronic thromboembolic pulmonary hypertension (CTEPH). Among these, CTEPH stands out as the only entity where even severe PH may be potentially curable. Surgical pulmonary endarterectomy is the treatment of choice for these patients. Medical therapy with “PAH drugs,” i.e., prostanoids, endothelin receptor antagonists (ETRA), or phosphodiesterase-5 (PDE-5) inhibitors, can be considered for inoperable patients but the evidence to support this recommendation remains limited and none of these drugs have received FDA approval for treating CTEPH. PH due to left heart disease or lung disease is much more common than PAH or CTEPH, respectively, but they have been less well evaluated. For both groups there is considerable evidence showing that the development of PH has profound effects on exercise capacity and survival. The pathophysiology of these forms of PH, however, differs substantially from that of PAH and it is unclear whether these patients (or a subpopulation) may bene it from treatment with “PAH drugs.” Several randomized, controlled clinical trials with ETRA have been performed in patients with left heart diseases but have failed to show bene icial effects in these patients. The same is true for patients with chronic obstructive pulmonary disease. ETRA were also investigated in patients with pulmonary ibrosis as experimental data have suggested anti- ibrotic effects of endothelin receptor blockade. However, four randomized clinical trials with ETRA in patients with pulmonary ibrosis were negative and another clinical trial with an ETRA in patients with idiopathic pulmonary ibrosis (IPF) and pulmonary hypertension was early terminated after a futility analysis. PDE-5 inhibitors have also not yet proven to have clinical value in patients with chronic lung disease and PH. A recent randomized controlled clinical trial with sildena il in patients with IPF did not meet the primary endpoint but gave some positive signals on secondary endpoints, such as changes in oxygen saturation and change in DLCO. The clinical implications of these indings are uncertain. In patients with left heart disease and PH, PDE-5 inhibitors hold promise as experimental data suggest that these substances may not only act as pulmonary vasodilators but may also improve the systolic and diastolic function of the left ventricle. Taken together, for all forms of non-PAH PH there is a lack of robust evidence to support any form of targeted medical therapy and much more research is needed to de ine which therapeutic approach will be best suited for which patient. 1.7

Epigenetics, sex, and cardiovascular function with a focus on exchange Huxley VH National Center for Gender Physiology, University of Missouri School of Medicine, Columbia, MO, USA

Epigenetics, in its purest form, investigates heritable changes in gene expression caused by mechanisms other than alterations in DNA sequence. Those mechanisms fall into three categories: the irst being an environmental signal; the second being a cellular response that localizes to the affected chromosomal location; and the third being a signal that results in a chromatin change that transmits phenotypic changes in subsequent generations. These changes result in a wide variety of cell phenotypes that help in accounting for the wider variety of normal and disease states than can be accounted for by genetics alone. It is currently being further appreciated that genomic sex (XX or XY), independent from the actions of the reproductive hormones, is a factor requiring consideration in the study of cardiovascular function in health and disease. The interactions of epigenetics and sex have received attention in the ields of neuroscience, cancer, and mental health; much less focus on the combination of factors has been paid in the areas of cardiovascular and pulmonary function or disease. Drawing on examples from recent research into how males and females regulate vascular exchange similarly and differently, this talk should focus on why epigenetics and sex requires consideration in the clinical and laboratory settings. Pulmonary Circulation | October-December 2011 | Vol 1 | No 4

1.8

Update and overview of PAH genetics Loyd JE Department of Medicine, Vanderbilt University Medical Center, Nashville, TN, USA

Pulmonary arterial hypertension (PAH) in families (FPAH) has been known for most of the last century, but progress in understanding its molecular basis is just beginning. Heritable PAH (HPAH) is a useful term adopted to encompass either FPAH or idiopathic (IPAH) patients who are found to carry a responsible mutation (10-40%) despite negative family history. HPAH should not replace FPAH, but should instead expand our PAH vocabulary. One PAH family in Tennessee has suffered 18 patients from 1950 to 2000, and another 18 patients from 2000 to 2011. Another six American families have each experienced 10 or more PAH patients. Familial PAH is also reported from Western Europe (France, Germany, Italy, UK) and Asia (Japan, China). Heterozygous mutation in BMPR2 accounts for the majority of HPAH (75%), but is confounded by variable age of onset and decreased penetrance. The lifetime penetrance of symptomatic PAH in BMPR2 mutation carriers is about 20%, so only a small minority ever develop PAH, and it may begin at any age. PAH patients with BMPR2 mutation have a more severe hemodynamic pro ile at the time of diagnosis, but it remains unproven whether survival differs. Mutation in other TGF-β family members, including ACVRL1 or ALK1, endoglin, and SMAD 8, are rarely the basis of HPAH. Prospective studies of PAH outcome related to mutation status are few, but one such study in France suggested similar PAH survival of BMPR2 mutation versus not, while ALK1-mutant patients showed far worse survival. Some genetic modi iers for clinical expression of BMPR2 mutation were identi ied by a functional candidate approach (TGF-β) and others by comparison of expression arrays in extremes of phenotype (CYP1B1). The latter inding led to follow-up studies which demonstrate a role for estrogen and its metabolism in the female disparity. Studies in BMPR2 mutations which have nonsensemediated decay demonstrate that the expression level of the normal allele relates to the clinical development of disease. Novel linkage studies have identi ied 4 loci which hold promise for identi ication of modi iers for clinical expression of BMPR2 mutation. Next generation sequencing including whole exome sequencing is expected to identify other genes responsible for PAH families without BMPR2 mutation (>40 families known in the US). Importantly for the several thousand asymptomatic mutation carriers in the 120 families in the US with BMPR2 mutation, early detection of disease is not currently possible. No tests are known to detect the vascular disease itself, and extensive pulmonary vascular loss (~60%) occurs unrecognized prior to onset of symptoms, thus the disease process is already far advanced when the irst symptom occurs. The translation of current knowledge to develop new diagnostic strategies and new therapies is drastically needed for this tragic disease. 1.9

The influence of serotonin and estrogen in the development of pulmonary arterial hypertension MacLean MR, Johansen AK, Wallace E, Campbell A, Dempsie Y, White K Institute of Cardiovascular and Medical Sciences, University of Glasgow, Glasgow, UK

Serotonin has been implicated in the development of clinical and experimental pulmonary arterial hypertension (PAH). The development of hypoxia- and dexfen luramine-induced is ablated in mice devoid of tryptophan hydroxylase 1 (TPH1) and hence requires peripheral serotonin. Mice that over-express the human gene for the serotonin transporter (SERT+mice) develop elevated pulmonary pressures and exaggerated hypoxia-induced PAH. We recently demonstrated that only female SERT+ mice demonstrate this phenotype and only at 5-6 months of age. Ovariectomy of these mice abolishes the PAH phenotype and S3

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subsequent replacement of 17-estradiol restores the PAH phenotype. Dexfen luramine-induced PAH is also only observed in female mice and abolished by ovariectomy. In hPASMCs, 17-estradiol-induced proliferation is abolished by inhibitors of the serotonin system and 17-estradiol can increase expression of TPH1, SERT and the 5-HT1B receptor. Microarray studies show that several genes are elevated in female SERT+ mice that may account for the PAH phenotype. For example, CEBPB, CYP1B1 and FOS are increased in SERT+ female mice. Serotonin and 17β estradiol increased CEBPB, CYP1B1 and FOS protein expression in hPASMCs and in addition, CEBPB, CYP1B1 and FOS mRNA and protein expression are increased in PASMCs derived from IPAH patients. As the activity of CYP1B1 has been shown to be altered in patients with fPAH, we examined the development of PAH in CYP1B1/-mice and demonstrated that PAH ablated in these mice. Likewise the CYP1B1 inhibitor TMS ablated the development of hypoxia-induced PAH. Of the estrogen metabolites examined, 16-hydroxyestrone induced profound proliferation in hPASMCs. We also showed that 1.5 mg/kg 16-hydroxyestrone I.P. for 21 days can induce PAH in mice. Hence we propose that serotonin and estrogens interact to facilitate the development of PAH. We propose that the activity of CYP1B1 and estrogen metabolites play a key role in in luencing the PAH phenotype. This work was funded by the British Heart Foundation and the BBSRC, UK. 1.10

Fat, fire and muscle: The effects of adiponectin on pulmonary vascular inflammation and remodeling Weng MQ, Combs TP, Scherer PE, Bloch KD, and Medoff BD Pulmonary and Critical Care Unit and Anesthesia Center for Critical Care Research, Massachusetts General Hospital, Harvard Medical School, Boston, MA, USA; Department of Nutrition, University of North Carolina, Chapel Hill, North Carolina USA; Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, TX, USA

Accumulating evidence demonstrates that obesity can increase both the incidence and severity of in lammatory diseases such as atherosclerosis, diabetes, and pulmonary hypertension (PH). PH results from a heterogeneous group of diseases, many of which demonstrate characteristic pathologic changes of pulmonary vascular in lammation and remodeling. Adipocytes secrete multiple bioactive mediators that can in luence in lammation and tissue remodeling, suggesting that adipose tissue may directly in luence the pathogenesis of PH. One of these mediators is adiponectin, a protein with a wide range of metabolic, anti-in lammatory, and anti-proliferative activities. Interestingly, individuals with obesity have low plasma adiponectin levels, suggesting that decreased adiponectin may contribute to the increased prevalence of disease seen in obesity. We recently reported that de iciency of adiponectin exacerbated PH in a mouse model, and was associated with enhanced eosinophil recruitment and pulmonary artery smooth muscle cell (PASMC) proliferation. In more recent experiments, we utilized eosinophil-de icient mice and adiponectin-overexpressing mice in a murine model of PH induced by allergic in lammation to demonstrate that adiponectin suppresses pulmonary vascular remodeling via effects on eosinophil recruitment and PASMC proliferation. In support of a pathogenic role of eosinophil recruitment, elimination of eosinophils in the lung signi icantly attenuated pulmonary arterial muscularization and reduced right ventricular systolic pressure (RVSP) in this model of PH. In addition, we found that the extracts of eosinophil granules were able to promote the proliferation of PASMCs in vitro and induce mitogenic signaling in these cells. Furthermore, expression levels of the eosinophil-speci ic chemokines CCL11 and CCL24 were increased in macrophages isolated from adiponectin-de icient mice in the model compared to macrophages from wild-type mice. These data suggest that adiponectin may modulate PH in this model in part by affecting eosinophil recruitment into the lung. In separate experiments, we demonstrated that transgenic mice that overexpress adiponectin had reduced PH and pulmonary vascular remodeling compared to wild-type S4

mice, but developed similar levels of pulmonary vascular in lammation in the model. Consistent with this result, adiponectin directly suppressed pulmonary artery smooth muscle cell proliferation in vitro, and reduced activity of several pro-proliferative molecular pathways in these cells. Overall, these data suggest that eosinophilic in lammation is necessary for pulmonary vascular remodeling in this model, and that adiponectin regulates remodeling through effects on eosinophil accumulation and direct effects on pulmonary artery smooth muscle cells. 1.11

Progenitor cells in pulmonary arterial hypertension Morrell NM Department of Medicine, University of Cambridge, Cambridge, UK

Recent interest has focused on the potential role of endothelial progenitor cells (EPCs) in pulmonary arterial hypertension (PAH). Circulating cytokines stimulate the bone marrow to release progenitor cells, which home to areas of vascular injury. Within the circulation, EPCs can be identi ied by the expression of cell-surface markers such as CD34, CD133, cKit, and vascular endothelial growth factor receptor (VEGFR). The true endogenous role of these progenitor cells is currently unknown. Recent studies have measured the levels of putative endothelial progenitor cells (EPCs) in the circulation of individuals with PAH. EPCs, as de ined by the expression of CD133, CD34, and VEGFR2, were elevated in the peripheral circulation of individuals with idiopathic PAH and those with the heritable form of pulmonary hypertension with mutations in BMPR2. While another group has reported similar results, others found decreases in these markers, and the role of EPCs in pulmonary vascular repair remains unclear. In addition to counting cells in the peripheral circulation, the researchers have looked for the expression of EPC markers in the tissues from patients with pulmonary hypertension. CD133 was faintly expressed in normal peripheral pulmonary arteries, more highly expressed in concentric intimal lesions, and very highly expressed in plexiform lesions. In addition, the major homing signal for EPCs is upregulated in plexiform lesions, raising the possibility that that cells that express EPC markers may be involved in the formation of these lesions. Therapeutically, EPCs, particularly early (E)-EPCs, have been reported to both prevent and reverse the pulmonary hypertensive phenotype in the monocrotaline rat model. These indings led to an ongoing trial in humans, which is producing encouraging preliminary data. Further research using human cells in an athymic nude rat model has indicated that late outgrowth (L-EPCs) do not have the same effect as E-EPCs in rescuing pulmonary hypertensive phenotypes. This research also found that E-EPCs maintain this therapeutic action despite being retained within the lung for only a short period of time (<24 h). A repetition of this study in nude rats in which natural killer cells had been ablated resulted in a longer retention time for EPCs. However, removing the natural killer cells removed the therapeutic bene it of E-EPCs, suggesting an important immune interaction between these cell types. Further studies are required to elucidate the role of these cells as therapeutic agents in PAH. 1.12

Gene expression profiling of peripheral blood mononuclear cells from cattle with high altitude pulmonary hypertension (Brisket disease) Newman JH, Holt TN, Hedges LK, Womack B, West J, and Hamid R Departments of Medicine, Pediatrics, and Cancer Biology, Vanderbilt University School of Medicine, Nashville, Tennessee USA; College of Veterinary Medicine, Colorado State University, Fort Collins, CO, USA

High altitude pulmonary hypertension (HAPH) is a consequence of chronic alveolar hypoxia, leading to hypoxic vasoconstriction and remodeling of the pulmonary circulation. HAPH in cattle is a naturally occurring animal model of hypoxic pulmonary hypertension. Genetically Pulmonary Circulation | October-December 2011 | Vol 1 | No 4

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susceptible cattle develop severe pulmonary hypertension and right heart failure at altitudes >7,000 ft. No information currently exists regarding the identity of the pathway(s) and gene(s) responsible for HAPH or in luencing severity. We hypothesized that initial insights into the pathogenesis of the disease could be discovered by a strategy of gene expression pro iling of affected cattle compared to altitudematched normal controls, combined with gene set enrichment analysis and Ingenuity Pathway analysis. We isolated blood from a single herd of Black Angus cattle of both genders; age 12-18 months, by jugular vein puncture. Mean PA pressures were 85.6±13 mmHg STD in the 10 affected and 35.3±1.2 mmHg STD in 10 resistant cattle, P<0.001. RNA was isolated from peripheral blood mononuclear cells and used to probe an Affymetrix Bovine genome array. Data were analyzed by the Partek software package to identify sets of genes with expression that was statistically different between the two groups. Gene Set Enrichment (GSEA) and Ingenuity Pathways analyses were then conducted on the re ined expression data to identify key cellular pathways and to generate networks and conduct functional analyses of the pathways and networks. A 60-gene signature was identi ied that differentiated affected from unaffected cattle. Forty-six genes were overexpressed in affected and 14 genes were downregulated in the affected cattle by at least 20%. GSEA and Ingenuity analysis identi ied respiratory diseases, in lammatory diseases and pathways as the top diseases and disorders (P<5.14×1014 ), cell development and cell signaling as the top cellular functions (P<1.20×10-08), and IL6, TREM, PPAR, NFkB cell signaling (P<8.69×10-09) as the top canonical pathways associated with this gene signature. This study provided insights into the pathogenesis of HAPH at a molecular level and suggests that HAPH shares cellular pathways and processes with pulmonary hypertension in humans. Further studies are needed to validate and re ine these preliminary indings and to determine their role in the development of human pulmonary hypertension. 1.13

Dehydroepiandrosterone and experimental pulmonary hypertension Oka M, Alzoubi A, Toba M, Fagan KA, and McMurtry IF Departments of Pharmacology and Medicine, University of South Alabama, Mobile, Alabama USA

Dehydroepiandrosterone (DHEA), a C-19 steroid synthesized from cholesterol mainly by the adrenal cortex, circulates primarily in its sulfated form, DHEAS. DHEA and DHEAS are the most abundant steroids in human thee bloodstream and are precursors to both androgens and estrogens. Epidemiological observations indicate that DHEA has a wide variety of bene icial effects, including prevention of obesity, diabetes, cancer, and cardiovascular diseases. However, little is known about its role in patients with pulmonary hypertension. Plasma levels of DHEA are very low in rodents, but pharmacological doses of exogenous DHEA exert a broad range of vasculoprotective activities, including vasodilatory, antiproliferative, anti-in lammatory, and antioxidant effects. DHEA or DHEAS effectively prevents and reverses chronic hypoxia-induced pulmonary hypertension in rats by upregulating K+ channels and improving endothelial function. In addition, DHEA treatment of leftpneumonectomized monocrotaline-injected rats inhibits the upregulation and activation of lung tissue RhoA/Rho kinase signaling, completely arrests progression of pulmonary hypertension, and increases survival from 30% to 100%. How DHEA treatment suppresses activation of Rho kinase is unclear, but possibilities include inhibition of HMG-CoA reductase and upregulation of soluble guanylate cyclase, both of which can lead to inhibition of RhoA-mediated activation of Rho kinase. Its antioxidant/antiin lammatory effects and conversion to estrogen may also play a role. We have recently observed that oral treatment with DHEA arrests progression of severe pulmonary arterial hypertension and normalizes cardiac output in SU5416/hypoxia/normoxia-exposed rats. Considering that DHEA is a relatively safe and orally active drug, and that it or one or more of its metabolites modulate various cellular/molecular signaling pathways that Pulmonary Circulation | October-December 2011 | Vol 1 | No 4

are involved in the pathogenesis of pulmonary hypertension, DHEA or a metabolite might be useful for the treatment of pulmonary hypertension. 1.14

Genetic risk factors for portopulmonary hypertension in patients with advanced liver disease Roberts KE, Fallon MB, Krowka MJ, Brown RS, Trotter JF, Peter I, Tighiouart H, Knowles JA, Rabinowitz D, Benza RL, Badesch DB, Taichman DB, Horn EM, Zacks S, Kaplowitz N, and Kawut SM Pulmonary Vascular Complications of Liver Disease Study Group, University of Pennsylvania, Philadelphia, PA, USA

Portopulmonary hypertension occurs in 6% of liver transplant candidates. The pathogenesis of this complication of portal hypertension is poorly understood. To identify genetic risk factors for portopulmonary hypertension in patients with advanced liver disease, we performed a multicenter case-control study of patients with portal hypertension. Cases had a mean pulmonary artery pressure >25 mmHg, pulmonary vascular resistance >240 dynes/s/cm5, and pulmonary capillary wedge pressure ≤15 mmHg. Controls had a right ventricular systolic pressure <40 mmHg (if estimated) and normal right-sided cardiac morphology by transthoracic echocardiography. We genotyped 1,086 common single nucleotide polymorphisms in 94 candidate genes in each patient. The study sample included 31 cases and 104 controls. Twenty-nine single nucleotide polymorphisms in 15 candidate genes were associated with the risk of portopulmonary hypertension (P<0.05). Multiple single nucleotide polymorphisms in the genes coding for estrogen receptor 1, aromatase, phosphodiesterase 5, angiopoietin 1, and S100A4 were associated with the risk of portopulmonary hypertension. The biological relevance of one of the aromatase single nucleotide polymorphisms was supported by an association with plasma estradiol levels. Genetic variation in estrogen signaling and cell growth regulators is associated with the risk of portopulmonary hypertension. These biologic pathways may elucidate the mechanism for the development of portopulmonary hypertension in certain patients with severe liver disease. 1.15

VIP is a physiological modulator of pulmonary arterial hypertension Said SI Pulmonary and Critical Care Medicine, Stony Brook University, Stony Brook, NY, USA

Pulmonary arterial hypertension (PAH) is characterized by a combination of structural and functional abnormalities in the pulmonary circulation: (1) Pulmonary vasoconstriction; (2) pulmonary vascular thickening; (3) lung in lammation; and (4) RV hypertrophy (RVH). As a means of exploring the physiological role of the VIP gene in the pulmonary circulation, we examined the effects of targeted deletion of the VIP gene in mice. There were two sets of complementary indings, both largely unexpected. First: a spontaneous expression of a PAH phenotype, in the absence of hypoxia or any other “second hit.” This phenotype included: PAH, pulmonary vascular thickening, lung in lammation, and RVH. Second: gene array analysis of lungs from these mice con irmed the absence of the VIP gene, and showed a wide range of additional gene expression alteration: overexpression of most vasoconstrictor, proproliferative and pro-in lammatory genes, and underexpression of most vasodilator, antiproliferative genes. These results demonstrate that: (1) the mere lack of the VIP gene alone is suf icient to generate a PAH-like phenotype in mice; and (2) the VIP gene exerts a powerful, wide-ranging in luence on the expression of a majority of genes and transcription factors controlling the structure and function of the pulmonary circulation. Having identi ied the multiple genes and pathways working in concert with VIP to modulate PAH, we searched for the dominant S5

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pathway that might explain this regulatory in luence of VIP. Evidence so far strongly suggests that VIP modulates PAH pathogenesis mainly by inhibiting the calcineurin-NFAT pathway (Nuclear Factor of Activated T cells). Activation of this transcription factor can elicit all features of PAH: vascular smooth muscle proliferation, in lammation, and cardiac myocyte proliferation. Calcineurin-NFAT activation, as evidenced by nuclear localization, has been demonstrated in VIP-/-mice and two other experimental models of PH, as well as in IPAH patients. In all three PH models, treatment with VIP suppressed this activation, much like VIVIT, the selective NFAT inhibitor. 1.16

Risk factors and responsiveness to therapies in the modern era of PAH treatment Sitbon O Departments of Respiratory and Intensive Care Medicine, University Paris Sud-11, Clamart, France

The last decade has witnessed a remarkable increase in the number of effective treatment options available for the management of patients with pulmonary arterial hypertension (PAH). The advent of PAHspeci ic pharmacological treatments has offered hope to patients with a debilitating, progressive disease and a poor prognosis. In this regard, agents belonging to the therapeutic classes that speci ically target the prostacyclin, endothelin and nitric oxide pathways have shown the greatest ef icacy in clinical studies to date. These various drug treatments have individually been shown to confer improvements in symptoms, exercise capacity, pulmonary hemodynamics and possibly survival in different patient subgroups. However, despite these substantial improvements, long-term survival remains poor in patients with PAH, particularly in those with the most severe functional impairment (New York Heart Association (NYHA)/World Health Organization (WHO) functional class (FC) III-IV). With greater availability of therapies, strategies aimed at optimizing patient outcomes have also evolved. Among them, combination therapy and a “treat-to-target” approach are two current treatment strategies that are strongly linked. PAH is characterized by dysregulation of a variety of pathways. As a consequence, there is increasing interest in the use of treatment combinations in order to target multiple targets with the aim of restoring normal pulmonary vascular function in order to improve clinical status. Combined drug treatment offers improved bene its over monotherapy, and current European treatment guidelines for PAH (European Respiratory Society and European Society of Cardiology) recommend a sequential add-on approach to combination therapy for patients in New York Heart NYHA/ WHO FC II–IV. Alternatively, irst-line combination therapy could be considered in FC III-IV PAH patients. Recent data from our center suggest that upfront triple combination therapy with epoprostenol, bosentan and sildena il dramatically improve symptoms and hemodynamics in particularly severe patients (fall in pulmonary vascular resistance by 70% after 4-month triple combination therapy). For PAH patients who continue to show inadequate clinical response despite optimal medical therapy, or where medical treatments are unavailable, balloon atrial septostomy and/or lung transplantation can be indicated. “Treat-totarget” or “goal-oriented” approaches, in which appropriate treatment goals are established for individual patients, are crucial in maximizing the bene its of pharmacotherapy in PAH. These strategies involve continual monitoring and assessment in order to evaluate patients’ response to therapy. With respect to prespeci ied treatment targets, decisions can be made based on whether the response to therapy is satisfactory or inadequate. Goal-oriented therapy determines the timing of treatment escalation by inadequate response to known prognostic indicators. These include NYHA/WHO FC 6-minute walk distance (6MWD) as well as a number of hemodynamic variables (right atrial pressure, cardiac output, pulmonary vascular resistance). Based on the measurement of such parameters, patients can be classi ied as “stable and satisfactory,” “stable and not satisfactory” or “unstable and deteriorating.” Patients described S6

as “stable and not satisfactory” or “unstable and deteriorating” are considered to have an inadequate response to therapy, and an escalation of their treatment should be considered. Close monitoring of patients (every 3-6 months) aids the early identi ication of inadequate response, so that treatment can be escalated promptly and before the patient’s condition can further deteriorate. Existing treatment goals are based on baseline values of prognostic indicators, but it is vital to identify risk factors that are both relevant during treatment and can be assessed during follow-up appointments. Data from different PAH etiologies indicate that NYHA/ WHO FC is the most appropriate prognostic marker, with 6-minute walk distance and several hemodynamic parameters representing alternatives. Future re inement of goal-oriented therapy could include the use of multiple prognostic markers, while additional large clinical trials will answer questions concerning choice and combination of treatment goals. 1.17

Chronic hypoxia leads to emergence of proinflammatory pulmonary adventitial fibroblasts capable of inducing proinflammatory and profibrogenic activation of monocytes/macrophages: Amelioration by HDAC inhibitors Stenmark KR, Li M, Riddle SR, Flockton AR, Barajas C, Yeager ME, Frid MG, Anderson AL, McKenzie T, and El Kasmi KC University of Colorado Denver, Aurora, CO, USA

A persistent accumulation of monocytes/macrophages in the adventitia of pulmonary arteries of animals and humans with pulmonary hypertension (PH) is well recognized. Chronic in lammation correlates positively with ibro-proliferative changes in the vessel wall. However, the cellular mechanisms contributing to chronic in lammatory responses remain unclear. Pro ibrotic cells can originate from circulating CD14+ monocytes and include ibrocytes and activated macrophages. Although accumulation of ibrocytes has been implicated in the pulmonary vascular remodeling process, little information exists regarding activation of macrophages by adventitial ibroblasts. We hypothesized that the pathogenesis of hypoxic PH involves emergence of adventitial ibroblasts with a persistently activated proin lammatory phenotype capable of recruiting and activating macrophages towards a proin lammatory and pro ibrogenic phenotype. We further hypothesized that the proin lammatory ibroblast phenotype is the result of epigenetic changes and particularly abnormal activity of histone-modifying enzymes, speci ically, class I histone deacetylases (HDACs). Our data demonstrate that, compared to control ibroblasts, PHFibs expressed an activated pro-in lammatory phenotype characterized by signi icantly increased mRNA and protein levels of CCL2/MCP-1, CXCL12/SDF-1, GM-SCF, RANTES, IL-6, and IL-1, but not TNF-β or IL-4 or IL-13. The proin lammatory phenotype of PH-Fibs was associated with epigenetic alterations as evidenced by increased activity of type I HDACs, and the indings that class I HDAC inhibitors markedly decreased cytokine/chemokine mRNA expression and protein levels in these cells. PH-Fibs also induced increased adhesion of THP-1 monocytes, and produced soluble factors that induced increased migration of THP-1 and murine bone marrow-derived macrophages (BMDMs). Further, since increased numbers of CD163+ macrophages were observed in vivo in the pulmonary vascular adventitia of chronically hypoxic animals we examined the effect of PH-Fibs on unstimulated monocytes/macrophages. Supernatants from PH- ibs, but not CO-Fibs, stimulated increases in mRNA levels of TLR-signaling dependent (IL-6) and in lammasome signaling dependent (IL-1, IL-18) pro-in lammatory cytokines as well as NF-κB dependent pro ibrogenic mediators (MMP2,9, Col-1a, TIPM1, 3) in both THP-1 monocytes and BMDMs. Intriguingly, expression of canonical markers for classical M1 activation of macrophages was low (NOS2) or undetectable (IL-23, IRF5). Class I HDAC inhibitors markedly reduced the ability of PH-Fibs to induce proin lammatory activation. BMDMs incubated with supernatants from PH-Fibs, but not CO-Fibs, also exhibited mRNA expression of STAT3 dependent functional markers of alternative Pulmonary Circulation | October-December 2011 | Vol 1 | No 4

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(M2) activation: Arginase-1 and IL-4RA. Importantly, expression of STAT6 dependent M2 markers YM1 and FIZZ1 was not detected. Expression of above markers was unaltered in MyD88-/- and TLR4-/- BMDMs. The in vivo relevance of these indings was con irmed in vivo where administration of Class I HDAC inhibitors both inhibited and reversed hypoxic-induced PH and vascular remodeling. In conclusion, we propose a novel concept for the pathogenesis of chronic hypoxic PH, in which persistently activated proin lammatory adventitial ibroblasts play a key role in promoting TLR, in lammasome and STAT3-dependent signaling pathways leading to the induction of a functionally distinct monocyte and macrophage phenotype tailored to initiate and propagate vascular in lammation and ibrosis. 1.18

Novel loci interacting epistatically with BMPR2 cause FPAH Stewart WCL, Ho YY, Loyd J, Chung W, and Greenberg DA Columbia University, New York, NY, USA

The genetic locus for familial pulmonary hypertension (FPAH) was irst identi ied in 1997. Then, 3 years later, BMPR2 was positionally cloned and shown unequivocally to increase risk for FPAH. Thereafter, the vast majority of all known carriers were shown experimentally (or are expected on the basis of exonic sequence) to have mutations that dramatically reduce BMPR2 gene expression. However, it is estimated that only 20-30% of all carriers go on to develop FPAH. As a result, we hypothesize that some of the reduced penetrance of BMPR2 may be due to genetic variation at interacting loci (aka modiϔiers). To increase our power to detect modi iers, we analyzed the genomes of 197 carriers obtained from 37 FPAH families. The families ranged in size from 2 to 5 generations with 4 to 23 members per family with the carrier status of most individuals determined experimentally by exon sequencing. To further increase power and precision we used EAGLET–our fast and lexible new method of analysis, to extract the maximum amount of information for association and linkage from the available dense single nucleotide polymorphism (SNP) data. Relative to several competing methods, EAGLET has been shown to increase power and to yield accurate and narrow con idence intervals for disease gene location. Our results are consistent with the established fact that BMPR2 in luences FPAH in Caucasians (maximum eLOD=6.44), but our results also suggest that there may only be a small number of modi ier genes with moderate genetic effects. Furthermore, these modi iers may differ across Caucasian subpopulations. For example, in 18 families that originate from an ongoing study at Vanderbilt University, we found strong evidence for a modi ier locus on chromosome 5 (P<0.01). Similarly, in 19 families that originate from a Columbia University study, we found suggestive evidence for a modi ier locus on chromosome 3 (P=0.11). Moreover, the only genomic location where both studies showed strong evidence for linkage was BMPR2. This suggests that there are both shared and distinct genetic factors in luencing FPAH, with BMPR2 being the shared factor that potentially interacts with distinct factors on chromosomes 3 and 5. Undoubtedly a complete genetic description of FPAH remains unclear, but the success of our powerful new approach may inspire others to unite data sets with new methods of analysis to advance our overall understanding of the genetics of this life-threatening disease. 1.19

Aerobic capacity and pulmonary arterial hypertension Duggan N, Stevenson C, Ciuclan L, Bonneau O, Rowlands D, Roger J, Koch L, Britton S, Walker C, Westwick J, and Thomas M Novartis Institutes for BioMedical Research, Horsham, UK; Centre for Integrative Mammalian Physiology and Pharmacology (CIMPP), Imperial College, London, UK; Department of Physical Medicine and Rehabilitation, University of Michigan, Ann Arbor, MI, USA Pulmonary Circulation | October-December 2011 | Vol 1 | No 4

Despite modern therapies, pulmonary arterial hypertension (PAH) remains a progressive, fatal disease where mortality is closely associated with right ventricular hemodynamic function, and exercise limitation. The potential bene it of exercise to increase aerobic capacity as an adjunct to conventional PAH therapies remains controversial. Here, we review clinical and preclinical literature around exercise, aerobic capacity and PAH within the context of emerging data revealing the role of altered cardiomyocyte metabolism in the failing right ventricle. These principles are further explored in novel studies comparing chronic hypoxia-induced PAH pathologies in rats selectively bred for divergent aerobic capacity - might the demonstrated linkage between low aerobic capacity and susceptibility to disease also feature in PAH, and what mechanisms govern such an association? We exposed high capacity runner (HCR) and low capacity runner (LCR) rats to a 10% O2 hypoxic environment for 21 days. While all rats exposed to 10% oxygen developed hypertensive-like changes relative to normoxic controls, hypoxia-exposed LCR rats developed more severe changes than their HCR counterparts, as measured by right ventricular pressure, Fulton index, echocardiographic indices and vessel remodeling. Lung microarray analysis of the lungs revealed perturbed expression of elements in the BMPR2/TGF-β axis in hypoxic LCR animals. Biomarker analysis revealed a ~250% increase in plasma serotonin levels in the hypoxic LCR group, but remained unchanged in all other groups, suggesting that the mechanism of enhanced responsiveness to chronic hypoxia may be via a dysregulated serotonin pathway. Daily treatment with an inhibitor of tryptophan hydroxylase (pCPA), the rate-limiting step in 5-HT synthesis, ablated 5-HT levels and reduced all PAH pathologies in all groups. However, subtractive analysis revealed no impact on the exacerbated vessel remodeling observed in LCR animals, and only a partial effect on RV pressure. RV mass and echocardiographic measures of RV function; however, were fully reversed by pCPA treatment. Gene array analysis in the hearts of hypoxic LCR versus HCR rats  5-HT ablation offer clues to the mechanisms by which aerobic capacity in luences PAH susceptibility. The impact of altered oxidative metabolism, aerobic capacity and the serotonin pathway on PAH therapies are discussed. 1.20

Estradiol metabolites and progestins in experimental pulmonary hypertension Tofovic SP Center for Clinical Pharmacology, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA

Pulmonary arterial hypertension (PAH) occurs more frequently in women, yet animal studies in classical models of PAH and limited clinical data suggest protective effects of estrogens (the estrogen paradox in PAH). This contradiction may be explained by the complexity of estradiol metabolism and the in luential balance between estradiol and its metabolites on pulmonary vascular homeostasis. One line of evidence strongly suggests that the vascular protective effects of 17β-estradiol (E2) are mediated largely by its downstream metabolites. E2 is metabolized to 2-hydroxyestradiol (2HE) by CYP1A1/CYP1B1, and 2HE is converted to 2-methoxyestradiol (2ME) by catechol-O-methyl transferase. 2ME is extensively metabolized to 2-methoxyestrone (2ME1), a metabolite that lacks biologic activity but may be converted back to 2ME by 17β -hydroxysteroid-dehydrogenase (17β-HSD-1). Previously, we have shown that in male rats 2ME, 2HE and synthetic analog 2-ethoxyestradiol attenuate monocrotaline (MCT)-induced PAH. In female rats with MCT- or bleomycin-induced PAH, 2ME attenuates both the exacerbation of disease and the increased mortality due to ovariectomy (OVX). Also, 2ME1, a largely biologically inactive E2 metabolite, has preventive effects in male MCT-PAH rats, and 2ME and all-trans retinoic acid (an inducer of 17β -HSD-1 and 2ME1-to-2ME conversion) have synergistic effects in ameliorating MCT-induced PAH. This suggests that 2ME-2ME1 interconversion and the 17β-HSD pathway may play a role in the development of PAH. Whereas E2 S7

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and 2ME exert similar effects in other vascular cells, they have divergent effects in endothelium and therefore we have proposed that unbalanced E2 metabolism may lead to the development of PAH. Because the effects of E2 and 2ME in severe PAH, as characterized by obliterative proliferation of endothelial cells, are currently unknown, recently we examined the effects of gender, estrogen de iciency and treatment with 2ME or E2 on the development and progression of disease in rats with severe occlusive/angioproliferative PAH. Adult male, intact female, and OVX female rats were given the VEGF receptor inhibitor SU 5416 (100 mg/kg) and exposed to hypoxia for 21 days. Subsets of F and OVX rats were treated in a preventive (Day 0-21) or therapeutic (Day 10-21) manner with 2ME and E2 (10 μg/kg/h and 1 μg/kg/h respectively, via osmotic minipumps). Male rats developed less severe PAH and lung injury compared to intact females, and 2ME had therapeutic effects in female, but not male PAH rats. Estrogen de iciency (OVX) did not exacerbate the disease. In OVX-PAH rats, 2ME, but not E2, reduced PAH and lung injury, while both 2ME and E2 reduced isolated RV hypertrophy. The effects of progestins in experimental PAH have been poorly studied, despite vascular effects of progesterone and presence of its receptors in intact endothelium and plexiform lesions in PAH patients. Therefore, we examined the effects of progesterone, medroxyprogesterone and tibolone on MCTinduced PAH in ovariectomized female rats. Progesterone attenuated development of PAH and right ventricular hypertrophy and inhibited pulmonary vascular remodeling; medroxyprogesterone showed effects similar to progesterone; and notably, tibolone, a combined progestin/ estrogen compound, prevented development of disease and eliminated MCT-induced mortality. These studies warrant further investigation of the role of estradiol metabolism in pulmonary vascular disease and the development of PAH. 1.21

Adipose tissue expandability, lipotoxicity and the metabolic syndrome Vidal-Puig A Institute of Metabolic Science, University of Cambridge, Cambridge, UK

The link between obesity and Type 2 diabetes is clear on an epidemiological level; however, the mechanism linking these two common disorders is not well de ined. One hypothesis linking obesity to Type 2 diabetes is the adipose tissue expandability hypothesis. The adipose tissue expandability hypothesis states that a failure in the capacity for adipose tissue expansion, rather than obesity per se is the key factor linking positive energy balance and Type 2 diabetes. All individuals possess a maximum capacity for adipose expansion which is determined by both genetic and environmental factors. Once the adipose tissue expansion limit is reached, adipose tissue ceases to store energy ef iciently and lipids begin to accumulate in other tissues. Ectopic lipid accumulation in nonadipocyte cells causes lipotoxic insults including insulin resistance, apoptosis and in lammation. This article discusses the links between adipokines, in lammation, adipose tissue expandability and lipotoxicity. Finally, we will discuss how considering the concept of allostasis may enable a better understanding of how diabetes develops and allow the rational design of new anti diabetic treatments. 1.22

Endocrine determinants of severe pulmonary arterial hypertension Voelkel NF, Hussani AA, Abbate A, Farkas L, and Bogaard HJ Virginia Commonwealth University, Richmond, VA, USA; VU Medical Center, Amsterdam, The Netherlands

Idiopathic pulmonary arterial hypertension (PAH) and also secondary S8

forms of severe PAH occur predominantly in women, and clinically associations with immunological disorders and thyroid diseases are well recognized. Very likely a disordered immune system, female and endocrine factors contribute to the pathobiology of pulmonary vascular remodeling, but very little is known. We postulate that all of these factors participate in in lammation and angiogenesis which are both mechanisms involved in pulmonary vascular remodeling. Because in excess of 80% of patients with Graves disease (hyperthyroidism) develop PAH and because thyroid hormones (T3 and T4) are angiogenic, we hypothesized that thyroid hormones would participate in the development of angioproliferative pulmonary remodeling in the rat Sugen5416/chronic hypoxia model of PAH. We found that thyroidectomy prevented the development of severe angioproliferative PAH, while T4 replacement of thyroidectomized rats treated with Sugen5416 and exposed to hypoxia led to development of severe PAH. When animals with established severe angioobliterative PAH were treated with propylthiouracil (PTU) a decrease in the RVSP and the number of obliterated vessels was observed. Because treatment of rats with Sugen5416 plus T4 did not result in PAH. We conclude that the T4 contributes (is permissive) in the setting of pulmonary hypertension. In this angiogenic action T4 may signal via the integrin αvβ3 and FGF2. Both of these proteins are increased in expression in the lungs from Sugen5416/chronic hypoxia rat lung tissues. 1.23

Adiponectin and pulmonary hypertension Summer R and Walsh K The Pulmonary Center and Whitaker Cardiovascular Institute/Molecular Cardiology, Boston University School of Medicine, Boston, MA, USA

Pulmonary hypertension is a life-threatening condition that develops in association with various medical diseases. Recent clinical studies indicate obesity to be a risk factor for development of pulmonary hypertension; however, the mechanisms leading to this association are unknown. Adiponectin is a circulating factor derived from adipose tissue that is present in high concentration in serum of lean healthy individuals but decreases in obesity. Recent animal studies implicate adiponectin in the pathogenesis of pulmonary hypertension. Most notably, mice de icient in adiponectin develop a spontaneous lung vascular phenotype characterized by upregulation of E-selectin on endothelium, age-dependent increases in perivascular in lammatory cells and elevated pulmonary artery pressures. Moreover, experimental studies showed adiponectin to ameliorate lung vascular remodeling due to hypoxia and to chronic allergic airway in lammation. Emerging evidence indicates adiponectin’s effects are mediated through antiin lammatory and antiproliferative actions on cells in the pulmonary circulation. This review aims to synthesize the existing data related to adiponectin’s effects on pulmonary vascular cells and to discuss how alterations in adiponectin signaling might contribute to the development of pulmonary hypertension in human patients. 1.24

Sildenafil-cGMP-PKG-PPAR-y signaling pathway inhibits angiotensin-II-induced transient receptor potential canonical 6 expression in pulmonary arterial smooth muscles cells Wang J, Yang K, Zhang Y, Lai N, Chen M, and Lu W Guangzhou Institute of Respiratory Disease, Guangzhou Medical University, Guangzhou, People’s Republic of China; Division of Pulmonary and Critical Care Medicine, The Johns Hopkins University School of Medicine, Baltimore, MD, USA

Sildena il, a speci ic type V phosphodiesterase inhibitor that increases cellular cGMP, is recently identi ied as a promising agent for treatment of Pulmonary Circulation | October-December 2011 | Vol 1 | No 4

Abstracts

pulmonary hypertension; however, the underlying mechanisms are not fully understood. In pulmonary arterial smooth muscle cells (PASMCs), Ca2+ in lux through store-operated Ca2+ channels thought to be composed of transient receptor potential canonical (TRPC) proteins is an important determinant of intracellular free calcium concentration ([Ca2+]i) and pulmonary vascular tone. We previously demonstrated that sildena il inhibits chronic hypoxic upregulation of TRPC expression in PASMCs. In this study, we further examined the regulatory effect and signaling of sildena il on angiotensin-II-induced TRPC6 expression in rat distal PASMCs. Treatment with angiotensin-II-induced TRPC6 mRNA expression about 4.5-fold, which was individually blocked by pretreatment with sildena il (1 M), CPT-cGMP (1 mM) or PPAR- agonist GW1929 (1 μM). The blocking effects of sildena il and CPT-cGMP was attenuated by cotreatment with PKG inhibitor Rp8 (1 μM), KT5823 (0.5 μM), or PPAR- antagonist T0070907. Treatment with sildena il or CPT-cGMP alone increased PPAR- activation, which was blocked by pretreatment with PKG inhibitor Rp8, or KT5823. These results provide novel evidence indicating that sildena il negatively regulates TRPC6 expression via cGMP-PKG-PPAR- signaling pathway, which may contribute to the mechanism of its therapeutic effect on pulmonary hypertension. 1.25

The estrogen metabolite 16-OHE exacerbates BMPR2-related PAH, associated with defects in receptor trafficking West JD Division of Allergy, Pulmonary and Critical Care Medicine, Vanderbilt University, Nashville, TN, USA

We have previously shown that decreased CYP1B1 expression and the resulting increase in the estrogen metabolite 16-OHE are associated with disease penetrance in pulmonary arterial hypertension (PAH) patients. The goal of this study was to determine whether this was correlation or causation, and why estrogen, which is bene icial to many models of pulmonary vascular disease, is harmful in the context of PAH. 16-OHE or vehicle were delivered in osmotic pumps to BMPR2 mutant or control mice chronically, followed by detailed hemodynamic, histological, and molecular phenotyping. Estrogen receptor (ER) signaling was examined in BMPR2 mutant or control pulmonary microvascular endothelial cells (PMVEC) through immunohistochemistry and ER sensitive luciferase reporter constructs. Adding 16OHE to BMPR2 mutant mice more than doubles pulmonary vascular resistance, associated with dropout or structural narrowing of resistance level arteries. This accelerates the course of PAH, but has little effect in control mice. BMPR2 mutation causes defects in ER traf icking; addition of exogenous estrogen no longer causes translocation to the nucleus, instead resulting in accumulation of the ER on the cell surface and in a perinuclear structure. This defect in ER traf icking appears to be common to other steroid hormone receptors, although with a different time course. 16-OHE is causative of PAH in the context of BMPR2 mutations. BMPR2 mutation alters steroid hormone receptor traf icking, likely through cytoplasmic tail domain (LIMK or TCTEX-1)-dependent signaling. 1.26

Biomarkers for pulmonary arterial hypertension—Can they be used to predict response to treatment? Wilkins MR Hammersmith Hospital, Imperial College London, London, UK

Biomarker is an inclusive term that refers to any objective measurement that informs on a person’s state of health. It includes physical measurements such as hemodynamics as well as circulating molecules and cells, genetic markers and imaging indings. A biomarker may be used to detect disease susceptibility, diagnose overt disease, categorize Pulmonary Circulation | October-December 2011 | Vol 1 | No 4

disease severity and monitor disease history or therapy response. In the management of pulmonary arterial hypertension (PAH), genotype (e.g., BMPR2 sequence) can inform risk of developing PAH. For diagnosis, hemodynamic measurements are the gold standard. Circulating brain natriuretic peptide (BNP) levels are used in the clinic to follow patients although interindividual variation in levels reduces their value and data have to be interpreted in the context of the sum of available clinical information on each patient. There is a particular interest in using biomarkers to stratify patients and target treatment to individuals. There is considerable potential for this approach in pulmonary arterial hypertension (PAH). The current classi ication of patients with pulmonary hypertension is based on clinical and pathological criteria but this is a blunt instrument. It is well recognized that WHO Group 1 PAH is a heterogeneous collection of diseases. This is re lected in the broad dynamic plasma concentration range seen in many circulating biomarkers. It is also seen in the response to treatment. Less than 10% of patients respond to a calcium antagonist. Not all patients with idiopathic PAH respond to PDE5 inhibitors and for reasons that are not entirely clear, patients with sickle cell disease seem to be poorly tolerant of this drug class. And then there is the differential response of the right ventricle to a pressure-load, allowing some patients to tolerate PAH better than others. The response of the pulmonary circulation to an acute vasodilator challenge, e.g., with nitric oxide, is helpful in predicting the long-term response to an oral calcium antagonist. In this sense, the hemodynamic measurements are acting as a predictive biomarker. While clinically useful, a less invasive test for identifying patients sensitive to calcium channel blockers would be preferred. Careful molecular dissection of well-phenotyped patients on calcium antagonist treatment is overdue. Genomic biomarkers are proving valuable in patient strati ication in oncology. Obtaining disease tissue from patients with PAH, except at transplantation, is not possible but the pulmonary vasculature provides an extensive surface area and damaged tissue will leak cells, proteins and genomic material into the circulation. This could be a rich source of material for molecular phenotyping. Detailed cardiac and molecular imaging provides another potentially powerful approach to understanding cardiac and pulmonary vascular pathology at the individual level. The use of 3-D myocardial imaging and positron emitting tracers to track disease/drug targets is so far underexplored in PAH. The validation of biomarkers is not an insigni icant challenge. This is best addressed by embedding candidate biomarkers in clinical trials. To date, this has only been done routinely with BNP. 1.27

Pathological role of TRPC channels in idiopathic pulmonary arterial hypertension Yuan J X-Y Departments of Medicine and Pharmacology, University of Illinois at Chicago, Chicago, IL, USA

Idiopathic pulmonary arterial hypertension (IPAH), a fatal and progressive disease that predominantly affects young women, is associated with profound pathobiological alternations in small pulmonary arteries. Regardless of the initial etiology, sustained vasoconstriction, excessive vascular remodeling, in situ thrombosis and increased vascular wall stiffness are the major courses for the elevated pulmonary vascular resistance in patients with IPAH and animals with pulmonary hypertension. An increase in cytosolic free Ca2+ concentration ([Ca2+]cyt) in pulmonary artery smooth muscle cells (PASMC) is a major trigger for pulmonary vasoconstriction and an important stimulus for PASMC proliferation that leads to medial hypertrophy and vascular remodeling. A novel class of nonselective cation channels, the transient receptor potential (TRP) channels, have emerged at the forefront of research into cardiovascular diseases. The canonical TRP channels, TRPC channels, are identi ied as molecular correlates for both store-operated and receptor-operated cation channels in different cell and tissue types (1-3), S9

Abstracts

including the pulmonary vasculature. Many TRP isoforms are identi ied at the mRNA and protein expression levels in human lung tissues and pulmonary vasculature. Upregulated TRPC channel expression and increased Ca2+ in lux through TRPC channels are involved in growth factor-mediated PASMC proliferation. In lung tissues and PASMC from patients with IPAH, we previously found that the mRNA and protein expression of TRPC channels (e.g., TRPC3 and TRPC6) was signi icantly greater than in lung tissues and PASMC from normal subjects and normotensive patients. Using DNA samples from normal subjects and more than 250 patients with IPAH, we identi ied a polymorphism (SNP) in the promoter region of the TRPC6 gene (-245C-to-G). The allele frequency of the -254(C-to-G) SNP in IPAH patients (12%) is signi icantly higher than in normal subjects (6%; P<0.01). Genotype data showed that the percentage of -254(C-to-G) homozygotes in IPAH patients was 2.85 times that of normal subjects. Interestingly, the -254(C-to-G) SNP creates a binding sequence for nuclear factor-κB (NF-κB). Functional analyses revealed that the -254(C-to-G) SNP-enhanced NF-κB-mediate promoter activity and stimulated TRPC6 expression in PASMC. Inhibition of NF-κB activity attenuated TRPC6 expression and decreased agonistmediated Ca2+ in lux in PASMC of IPAH patients harboring the -254G allele. These data implicate upregulation of speci ic TRPC isoforms to be associated with increased Ca 2+ in lux in PASMC from IPAH patients, which subsequently causes pulmonary vasoconstriction and vascular remodeling by stimulate PASMC contraction and proliferation, respectively. The -254(C-to-G) SNP in the TRPC6 gene may predispose individuals to an increased risk of developing IPAH by linking abnormal TRPC6 transcription and expression to NF-κB, an in lammatory transcription factor. Targeting TRPC6 transcription, expression and function may lead to the development of novel and effective therapeutic approaches for IPAH. 1.28

Cardiac sympathetic activity evaluated by 123I-MIBG myocardial scintigraphy in patients with right ventricular dysfunction associated with pulmonary arterial hypertension Abe K, Nagao M, Hirooka Y, Kishi T, Yonezawa M, Higo T, Ide T, and Sunagawa K Departments of Advanced Cardiovascular Regulation, Therapeutics, Cardiovascular Medicine, and Clinical Radiology, Kyushu University Graduate School of Medical Sciences, Fukuoka, Japan

Most patients with pulmonary arterial hypertension (PAH) die from right ventricular (RV) heart failure. Recently, it has been reported that patients with severe RV dysfunction associated with PAH might have autonomic nerve dysfunction evaluated by heart rate variability and muscle sympathetic nerve activity. However, noninvasive direct methods to measure the RV sympathetic nerve activity have not been established yet. In this study, we examined whether 123I-metaiodobenzylguanidine (123I- MIBG) myocardial imaging is useful to assess the RV sympathetic nerve activity in patients with RV dysfunction associated with PAH. Patients underwent right heart catheterization and echocardiography to determine RV function. SPECT was performed in the resting state 15 min. (early imaging) and 4 hr. (delayed imaging) after the injection of 123I-MIBG. RV sympathetic nervous function was assessed by the washout ratio of 123I-MIBG in RV-free wall. Patients with severe RV dysfunction showed higher washout ratios and the more extensions of the scintigraphic defects in the RV-free walls compared with the patients with mild RV dysfunction. The washout ratio of 123I-MIBG in RV-free wall was also positively correlated with the plasma level of brain natriuretic peptide (BNP). Our results suggest that 123I- MIBG imaging may be useful to evaluate the degree of RV sympathetic activities in the PAH patients with RV dysfunction. In a future study, it remains to be examined whether 123 I- MIBG imaging will also be useful as a prognostic method in the PAH patients with RV dysfunction. S10

1.29

Somatic chromosome abnormalities in PAH lungs— Modifier or bystander? Drake KM and Aldred MA Genomic Medicine Institute, Cleveland Clinic, Cleveland, OH, USA

Vascular remodeling in pulmonary arterial hypertension (PAH) has been likened to a neoplastic process, with evidence of monoclonal expansion of endothelial cells within plexiform lesions, microsatellite instability, hyperproliferation, apoptosis resistance and mitochondrial abnormalities. Recently we reported somatic chromosome abnormalities in endothelial cells from explant lungs in 5 of 9 PAH patients. All were mosaic abnormalities, present in 10-50% of cells. Many outstanding questions remain, including the timing of these events in the course of the disease process and their role (if any) in disease pathogenesis. We have now extended our analysis and identi ied abnormalities in 10 of 30 cases, including whole or partial deletion of chromosome 13 (2 cases) and X-chromosome deletions in 4 of 24 females. Two cases harbor more than one abnormality. Only one case has a germline BMPR2 mutation. No abnormalities were identi ied among 16 controls. One case of particular interest was a large interstitial deletion of chromosome 13, including SMAD9, present in approximately 70% of endothelial cells. This high level of abnormality has enabled functional studies. The cells proliferated signi icantly faster than controls and were indistinguishable from cells with a germline BMPR2 or SMAD9 mutation. They also showed signi icant attenuation of non-canonical microRNA processing, analogous to the 3 germline mutation cases. In contrast, cytogenetically normal pulmonary artery smooth muscle cells from the same patient showed normal proliferation rates and microRNA processing. While it remains dif icult to determine how early these changes arose in the course of the disease, these results suggest that somatic chromosome changes may act as disease modi iers and contribute to the altered phenotype of lung vascular cells in PAH patients. 1.30

Large conductance calcium-activated potassium (BK) channel activation causes enhanced vasodilatation in hypoxic lung microvasculature Vang A, Li A, and Choudhary G Providence VA Medical Center and Brown University, Providence, RI, USA

Pulmonary vasculature endothelial cells expresses BK channels that upon activation can cause endothelial cell hyperpolarization and vasodilation. However, the effect of BK channel activation on macroand microvasculature in chronically hypoxic lungs is not known. We assessed the effect of BK channel activation in pulmonary arterial (PA) rings and microvasculature ex vivo in lungs isolated from rats exposed to 3 weeks of normoxia (N) or hypoxia (H; 10% FiO2). BK channel  and subunit expression was assessed in pulmonary artery (PAEC) and microvascular (PMVEC) endothelial cells cultured for 10 days in N/H (1% FiO2). Phenylephrine-constricted PA rings from N/H animals were used to assess the dose-dependent effects of acetylcholine on vessel relaxation in the presence or absence of BK channel activator (NS1619) and various inhibitors. In isolated ventilated-perfused rat lungs from N/H animals preconstricted with U46619, we monitored PA pressures upon exposure to NS1619 in presence/absence of various inhibitors (L-NAME, tetraethylammonium-TEA, iberiotoxin). Hypoxia resulted in increased BK 4-subunit expression in PAEC. Increased endotheliumdependent vasodilation, which was attenuated by iberiotoxin and TEA, was noted in the presence of NS1619 in PA rings from normoxic but not in hypoxic rats. In contrast, hypoxia was associated with decreased expression of BK  and 4-subunits in PMVEC. NS1619 caused enhanced vasodilation in lungs isolated from hypoxic compared Pulmonary Circulation | October-December 2011 | Vol 1 | No 4

Abstracts

to normoxic rats. The vasodilation in both normoxic and hypoxicisolated lungs was attenuated by eNOS inhibition, iberiotoxin and TEA. Enhanced vasodilation is noted in hypoxic lungs and not in PA in response to BK channel activation. This may be related to differential effects of hypoxia on the expression of BK channel subunits across the lung vasculature. 1.31

Compensatory regulation of the BMP-targets ID1 and ID3 in hypoxic pulmonary hypertension Lowery J, Frump A and de Caestecker M Departments of Medicine, Cell and Developmental Biology, Vanderbilt University, Nashville, TN, USA

Bone morphogenetic protein (BMP) signaling has been linked to the development of pulmonary hypertension (PH). In previous studies we have shown chronic hypoxia up-regulates BMP2 and BMP4 ligand expression in and around the pulmonary vasculature, and this is associated with increased activating phosphorylation of the downstream mediators of BMP signaling, Smad1/5 and 8. In this study we have explored the downstream effects of BMP signal activation by chronic hypoxia on the regulation and function of inhibitors of differentiation proteins (ID1-4). The ID family proteins are basic helix-loop-helix (bHLH) transcription factors that are downstream targets of the BMP signaling pathway, but the role that ID proteins play in the development of PH is unknown. To address this, we evaluated pulmonary expression of ID proteins in a mouse model of hypoxiainduced PH. There is selective induction of ID1 and ID3 in hypoxic pulmonary vascular smooth muscle cells (VSMCs) in vivo, and ID1 and ID3 expression are increased by hypoxia in cultured pulmonary VSMCs in a BMP-dependent fashion. ID4 protein is barely detectable in the mouse lung, and while ID2 is induced in hypoxic peripheral VSMCs in vivo, it is not increased by hypoxia or BMP signaling in cultured pulmonary VSMCs. In addition, the PH response to chronic hypoxia is indistinguishable between wild type and Id1 null mice. This is associated with a compensatory increase in ID3 but not ID2 expression in pulmonary VSMCs of Id1 null mice. These indings indicate that ID1 is dispensable for mounting a normal pulmonary vascular response to hypoxia, but suggest that ID3 may compensate for loss of ID1 expression in pulmonary VSMCs. Taken together, these indings indicate that ID1 and ID3 expression are regulated in a BMPdependent fashion in hypoxic pulmonary VSMCs, and that suggest that ID1 and ID3 may play a cooperative role in regulating BMP-dependent VSMC responses to chronic hypoxia. 1.32

Effects of NMD status on susceptibility to pulmonary hypertension and endothelial dysfunction in mice carrying different germ line BMPR2 mutations Frump A and de Caestecker M Departments of Medicine, Cell and Developmental Biology, Vanderbilt University, Nashville, TN, USA

The majority of HPAH patients have heterozygous mutations in the Bone Morphogenetic Type 2 receptor (BMPR2). Experimental and clinical evidence indicate that these patients have a primary defect in pulmonary endothelial cells (PECs), and that germ line BMPR2 mutations subject to nonsense-mediated mRNA decay (NMD+ mutations) promote less severe pulmonary hypertension (PH) than BMPR2 mutations that escape NMD (NMD- mutations). However, the mechanisms by which NMD+ and NMD- BMPR2 mutations exert distinct effects on the pulmonary vasculature are unknown. We are addressing this question by studying the biology and processing of endogenously expressed Bmpr2 mutations in PECs derived from Pulmonary Circulation | October-December 2011 | Vol 1 | No 4

mouse mutants that harbor known NMD+ (Bmpr2 +/-) and NMDBmpr2 mutations (Bmpr2Δex2/+, in which there is an in-frame deletion of Bmpr2 exon 2) since we have shown that Bmpr2+/- mice have less severe PH than Bmpr2Δex2/+ mice. PECs from Bmpr2Δex2/+ and wild type mice both express the expected 150 kDa wild type Bmpr2 protein product. However, the Bmpr2Δex2/+ mice also express high levels of a 125 kDa product that is undetectable when probed with an antibody raised against exon 2. This 125 kDa product is also observed in skin ibroblasts from HPAH patients with carrying the same BMPR2 EXON2 deletion mutation, indicating that these mutations are able to escape the NMD pathway and express a mutant protein product. Smad 1/5/8 are activated and phosphorylated in response to BMP ligands in wild-type PECs but this response is markedly reduced in BMPR2Δex2/+ PECs. These indings suggest that the BMPR2Δex2 product is exerting dominant inhibitory effects on BMP signaling. To determine how this occurs we have evaluated the subcellular localization of wild-type and BMPR2Δex2 products in these cells. Cell-surface biotinylation studies indicate that unlike the wild-type allelic product, BMPR2Δex2 does not reach the cell surface. Furthermore, the BMPR2Δex2 mutant band is sensitive to Endo-H endoglycosidase treatment, indicating that it is unable to leave the ER and pass through the Golgi. Taken together, these studies indicate that the BMPR2Δex2 mutant product is expressed at high levels but that it is incorrectly processed and retained in the ER. These indings suggest that dominant inhibitory effects of this mutant allele on BMP signaling result from its sequestration in the ER. Further studies are ongoing to establish the mechanism by which BMPR2Δex2 is retained in the ER and what impact this has on the processing of other BMP signaling pathway components in the ER. 1.33

mTOR Complex 2 regulates energy levels, proliferation and survival of vascular smooth muscle cells in pulmonary arterial hypertension Goncharov D, Krymskaya V, and Goncharova E Pulmonary, Allergy & Critical Care Division, University of Pennsylvania, Philadelphia, PA, USA

Increased proliferation and survival of pulmonary arterial vascular smooth muscle cells (PAVSMC) are key pathophysiological components of vascular remodeling in pulmonary arterial hypertension (PAH). PAVSMC in PAH have metabolic shift to glycolysis, but the molecular link between disturbed metabolism and increased cell proliferation and survival remains elusive. mTOR, a key regulator of cell growth and proliferation, acts through two distinct complexes, rapamycin-sensitive mTORC1 that modulates cell growth via S6K1 and 4E-BP1 and rapamycin-resistant mTORC2 that activates Akt. We previously demonstrated that chronic hypoxia-induced PAVSMC proliferation requires activation of both mTORC1-S6K1 and mTORC2-Akt. Here, we show that PAVSMC from subjects with idiopathic PAH (human PAH PAVSMC) and from rats with chronic hypoxia-induced pulmonary hypertension (PH) have altered cellular ATP levels, elevated proliferation, increased protein levels of antiapoptotic Bcl2 and decreased levels of pro-apoptotic Bim compared to control PAVSMC. Glycolytic inhibitor 2DG, but not mitochondrial inhibitor rotenone, decreased ATP levels, inhibited proliferation and promoted apoptosis in human PAH and rat PH PAVSMC suggesting that PAVSMC proliferation and survival in PAH depend on glycolytic metabolism. Rapamycin-inhibited human PAH and rat PH PAVSMC proliferation, but had little effect on cellular ATP levels and cell survival. Inhibition of mTORC2-Akt by siRNA rictor or speci ic Akt1/2 inhibitor decreased cellular ATP levels, inhibited proliferation and induced apoptosis in human PAH and rat PH PAVSMC while having little effect on mTORC1. Collectively, these data demonstrate that mTORC2-Akt regulates energy levels, proliferation and survival of PAVSMC in PAH and suggest that targeting mTORC2-Akt in addition to mTORC1 may be bene icial to inhibit PAVSMC proliferation, induce apoptosis and attenuate pulmonary vascular remodeling in PAH. S11

Abstracts

1.34

Dual blockade of IL4 and IL13 in schistosomiasisassociated pulmonary hypertension Graham B, Chabon J, and Tuder R Program in Translational Lung Research, University of Colorado, Denver, CO, USA

Schistosomiasis-associated pulmonary arterial hypertension (Sc-PAH) is one of the leading causes of PAH worldwide. We have previously found that IL13 signaling alone is suf icient but not necessary to cause experimental schistosomiasis-associated pulmonary hypertension (Sc-PH) in a mouse model. IL4 is potentially redundant with IL13, and thus we hypothesized that the genetic dual blockade of both IL4 and IL13 would prevent the vascular remodeling in this disease. IL4-/-IL13-/(4/13KO) and wild-type (WT) mice (both with C57BL6/J background; 4-5 per group) were sensitized to Schistosoma mansoni eggs, and then intravenously challenged with 5,000 S. mansoni eggs. One week after injection, right ventricular catheterization was performed, followed by analysis of the lung tissue. Both uninfected WT and uninfected 4/13KO mice had normal right ventricular systolic pressures (RVSP) at baseline, but both infected WT and infected 4/13KO groups had an elevated RVSP. However, by quantitative histological analysis, only infected WT mice had pulmonary vascular remodeling. The 4/13KO mice had smaller peri-egg granulomas and were less effective at clearing the S. mansoni eggs compared to the WT mice. Separately, intravenous egg challenge alone resulted in an elevated RVSP in 4/13KO mice; we have previously shown this is inadequate to cause an elevated RVSP in WT mice. Although mice lacking both IL4 and IL13 develop PH after infection with S. mansoni, they do not have pulmonary vascular remodeling, and the PH appears to be primarily due to vascular obstruction by undegraded eggs. 1.35

Evidence of BMPR2 alternative splicing as a novel genetic modifier of HPAH penetrance Hamid R, Hedges L, Austin E, Womack B, and Cogan JD Vanderbilt University School of Medicine, Nashville, TN, USA

The molecular reasons of why only 20% of BMPR2 mutation carriers develop PAH remain unknown. Here we present data that show that this reduced penetrance is likely due to BMPR2 alternative splicing. BMPR2 gene product is alternatively spliced to produce two splice variants: isoform-A (full-length); and isoform-B (missing exon 12). Several lines of evidence point to the importance of exon 12 in HPAH pathogenesis: (1) deletion of exon 12 is a common HPAH-inducingBMPR2-mutation; and (2) mice overexpressing exon 12-deleted BMPR2 transcripts develop PAH phenotypically similar to human HPAH. Here we analyzed the relative amounts of these isoforms mRNAby real-time PCR analysis in PBMCs of 46 BMPR2 mutationpositive HPAH-patients and 31 BMPR2 mutation-positive unaffectedcarriers. Our data show that affected individuals had relatively higher isoform-B expression compared to isoform-A expression (B/A ratio) than unaffected individuals (P<0.002). We then proceeded to determine the molecular mechanism behind this differential isoform expression. Analysis of exon 12 sequence identi ied a putative exonic splice enhancer whose mutations result in exon 12 skipping leading to increased isoform-B mRNA. Using siRNA and Western blot analysis we identi ied three splicing factors that bind to this splice enhancer and regulate BMPR2 exon 12 skipping. We then conducted a focused drug screen and identi ied FDA-approved pharmacological agents, which alter the activity of this enhancer resulting in alteration of the splice isoform ratio in either direction. In conclusions, penetrance of BMPR2 related HPAH can be predicted by BMPR2 isoform ratio; this isoform ratio is controlled by an exonic splice enhancer and its S12

associated splicing factors; this isoform ratio can be manipulated in either direction by FDA-approved pharmacological agents. 1.36

Urokinase plasminogen activator receptor is a marker of pulmonary venous smooth muscle cells Hunt J Departments of Pulmonary and Critical Care Sciences, University of Colorado Health Sciences Center, Denver, CO, USA

The study of vascular pathophysiology in pulmonary arterial hypertension has primarily focused on the precapillary circulation; comparatively little attention has been afforded to the venous system. Recent evidence, however, suggests that pulmonary veins (PVs) play an important pathophysiological role in severe pulmonary hypertension. One challenge to studying the PVs is an inability to reliably identify them, by anatomical means, in the lung parenchyma. Furthermore, no speci ic molecular markers to PVs have been identi ied. We hypothesized that a systematic molecular characterization of normal PVs would identify distinct molecular markersâ&#x20AC;&#x201C;an important initial step to further research of PVs in pulmonary vascular disease. A mixture of agarose and luorescent beads was retrogradely injected into the pulmonary veins through the left ventricle of mice while the aorta was clamped. Veins (20-200 Îźm) were identi ied under tissue section using luorescent microscopy. Arteries, veins, and parenchyma were dissected using laser capture microdissection and RNA puri ied. qRT PCR arrays tailored to vasculogenic transcripts were used to characterize expression pro iles of veins, arteries, and parenchyma for comparison. A list of candidate transcripts upregulated in veins was generated. Commercial antibodies were purchased and protein expression in paraf in embedded lung tissue was assessed. Pulmonary veins could be successfully and reliably identi ied by retrograde illing with luorescent beads. Importantly, luorescent beads were absent in arteries and airways. Comparison of RNA expression pro iles between arteries and veins identi ied several candidates which were differentially expressed across vessel types. Of these, urokinase plasminogen activator receptor (UPAR) was expressed in pulmonary venous smooth muscle cells, distinguishing veins from arteries or lymphatics. Mounting evidence suggests the PVs play an important pathophysiological role in severe PAH; yet they remain poorly understood and understudied. Using a back- illing technique with luorescent tags, the PVs were reliably identi ied for use in laser-capture microdissection and RNA puri ication. Comparison of RNA expression pro iles between veins, arteries, and parenchyma successfully identi ied candidate markers of the pulmonary veins. Characterization of protein expression using immunohistochemistry-con irmed UPAR as a distinguishing marker of pulmonary venous smooth muscle cells. This research represents an important initial discovery in our understanding of the pulmonary veins in human disease. 1.37

Cytochrome P450 1B1 influences the development of pulmonary arterial hypertension Johansen AK, White K, Morecroft I, Mair K, Nilsen M and MacLean MR Institute of Cardiovascular and Medical Sciences, University of Glasgow, Glasgow, UK

The incidence of idiopathic and heritable PAH is up to three-fold higher in women than men. A mutation in the gene encoding the estrogenmetabolizing enzyme cytochrome P450 1B1 (CYP1B1) is associated with increased incidence of PAH in patients harboring BMPR-II mutations. CYP1B1 expression is increased in pulmonary arterial smooth muscle cells (PASMCs) in experimental and human PAH. Here we investigate the hypothesis that CYP1B1 promotes the development of PAH by assessment of hypoxia-induced PAH in mice de icient of CYP1B1 Pulmonary Circulation | October-December 2011 | Vol 1 | No 4

Abstracts

(CYP1B1-/- mice). Right ventricular systolic pressure (RVSP), right ventricular hypertrophy (RVH) and pulmonary vascular remodeling (PVR) were assessed. WT littermate mice were studied as controls. Following exposure to 2 weeks chronic hypoxia, male CYP1B1-/- mice showed a reduction in RVSP and PVR while both male and female CYP1B1-/- mice exhibited reductions in RVH. Pulmonary arteries from CYP1B1-/- mice had reduced vasoconstriction to serotonin in males and females. Immunohistochemistry staining con irmed increased expression of CYP1B1 in remodeled pulmonary arteries of patients with idiopathic and heritable PAH. 17β estradiol-induced proliferation of human PASMCs (hPASMCs) was inhibited with the selective CYP1B1 inhibitor, 2,3',4,5'-tetramethoxystilbene. Furthermore, the CYP1B1 metabolite 16α-hydroxyestrone stimulated proliferation in hPASMCs. In conclusion, our indings suggest that increased 17β estradiol metabolism via CYP1B1 may in luence PAH pathogenesis. 1.38

17-Estradiol (E2) attenuates hypoxia-induced pulmonary hypertension through an estrogen receptordependent mechanism that involves decreased ERK1/2 activation Lahm T, Albrecht M, Fisher A, Patel N, Justice M, Presson R, and Petrache I Division of Pulmonary, Allergy, Critical care, and Occupational Medicine, Indiana University, Bloomington, IN, USA

We investigated whether the protective effects of E2 in hypoxiainduced pulmonary hypertension (HPH) are mediated by estrogen receptor (ER)activation, or by CYP450- and catechol-O-methyltransferase (COMT)dependent conversion to E2 metabolites. Furthermore, we investigated whether E2 protection is associated with decreased proproliferative and/ or increased antiproliferative signaling. Adult male Sprague-Dawley HPH rats (PO2=380 mmHg; 2 weeks) were treated with E2 alone (75 mcg/ kg/d), or cotreated with E2 and (a) ER-antagonist ICI182,780, (b) CYP450inhibitor ABT, or (c) COMT-inhibitor OR-486. Standard hemodynamic and remodeling endpoints including cardiac output (CO) were assessed. Additional studies were performed in E2-treated hypoxic male wild-type, ER-α-/-, and ER-β-/- mice; with complementary experiments in primary rat pulmonary artery (PA) endothelial cells (PAECs) and smooth muscle cells (PASMCs) in 1% or 21% O2 for 48 hrs. P<0.05 was considered signi icant. E2-treatment attenuated hypoxia-induced increased in RVSP, RVSP/CO, RV/(LV+septum) and hematocrit, and attenuated the hypoxia-induced decrease in CO. E2 decreased hypoxia-induced PA and RV remodeling, inhibited proproliferative ERK1/2-activation in lung and RV, and increased expression of cell cycle-inhibitor p27Kip1 and autophagy marker LC3-II. ER-blockade, but not E2 conversioninhibition, attenuated E2’s effects. E2 protection was attenuated in ER-β-/- HPH mice. In hypoxic (but not in normoxic) PAECs, E2 (1 nM-1 μM) dose-dependently attenuated proliferation (BrdU), ERK1/2activation, and VEGF-secretion, while enhancing p27Kip1-expression and autophagy. ER-blockade attenuated E2 effects on proliferation and ERK1/2-activation. Antiproliferative E2 effects were also observed in hypoxic PASMCs. E2 protection in HPH is mediated by ER-activation without a requirement for metabolite conversion. This is associated with decreased proproliferative signaling, and increased cell cycle-inhibition and autophagy. ER-β appears necessary for E2 protection. Elucidating E2 signaling mechanisms may reveal potential therapeutic targets in HPH. 1.39

Induced pluripotent stem cells as a model for heritable pulmonary hypertension Majka S, Chow K, Omari A, Jean JC, Bilousova A, Hedges L, Kotton D and Austin E Pulmonary Circulation | October-December 2011 | Vol 1 | No 4

University of Colorado, Aurora, Colorado USA; Boston University, Boston, Massachusetts USA; Vanderbilt University, Nashville, TN, USA

Heritable pulmonary hypertension (HPAH) is characterized by profound vascular endothelial (EC) dysfunction and remodeling, accompanied by mutations in the gene encoding the BMP Type 2 receptor (BMPR2). Rodent models to date do not adequately recapitulate the disease pathology. A novel option for understanding the pulmonary vascular dysfunction manifested in HPAH is the use of patient-derived induced pluripotent stem cells (iPSCs). We hypothesized that HPAH patient derived iPSC cells and directed EC differentiation would prove to be an effective model in order to understand the consequences of BMPR2 mutation. To demonstrate that HPAH patient derived iPS would be a valid model to study cell-based mechanisms of disease, in collaboration with Drs. Kotton and Austin, we generated transgene free, karyotypically normal iPSC lines from skin ibroblasts obtained from an HPAH patient. iPSC exhibited an ES cell-like morphology. Sequencing of genomic DNA con irmed the retention of mutation in the BMP3ACr1 iPS cell line. These Hu iPS were subsequently differentiated into multipotent mesenchymal cells. Differentiation of iPSCs into mesenchymal cells was demonstrated by surface marker expression of CD 29, 73, 105, Stro-1 and lack of the hematopoietic markers CD45, CD3 and CD14. Multipotent differentiation to mesenchymal lineages was also represented by bone (alkaline Phosphatase and Von Kossa stain), and (alcian blue and Aggrecan stain) and adipose/fat, oil red O stain. Endothelial differentiation was demonstrated by expression of VE-cadherin (CD144) and PECAM-1 (CD31) and lack of CD45, CD14,CD3. The use of iPS cells derived from HPAH patients with known BMPR2 mutations provides an alternative source of patient-derived vascular cells to model disease. 1.40

A novel mechanism to explain enhanced pulmonary artery vasoconstriction in female rabbits Pillay T and Pfister S Department of Pharmacology and Toxicology, Medical College of Wisconsin, Milwaukee, WI, USA

In women the incidence of certain forms of pulmonary arterial hypertension is 4-fold greater than that observed in men. Mechanisms to explain the female predominance are scarce but likely relate to hormonal changes that contribute to the pathogenesis of the disease. We previously reported that arachidonic acid and the 15-lipoxygenase (LO)-derived arachidonic acid metabolite, 15-hydroxyeicosatrienoic acid (15-HETE) produced more vasoconstriction in pulmonary arteries of female compared to male rabbits. Expression of 15-LO protein was greater in females and 15-LO expression increased in males in response to estrogen treatment. The overall objective of the present study was to determine mechanisms that explain female sex/estrogen modulation of 15-LO expression in the pulmonary vasculature. Because it is known that cytokines increase 15-LO mRNA and protein expression, we incubated pulmonary arteries with 17β-estradiol (1 μM, 18 hrs.) and found that the protein expression of interleukin 4 (IL-4) increased compared to controls. Blockade of IL-4 production with IBMX prevented estrogen-induced 15-LO expression. Additional studies explored the hypothesis that the ability of estrogen to increase cytokine expression results in the activation of the transcription factor, STAT3 that then signals to increase 15-LO gene transcription. To test this, activated STAT3 expression was compared in pulmonary arteries of females and males with and without estrogen treatment. Isolated male pulmonary arteries incubated with estrogen had increased activated STAT3 expression compared to controls. Interestingly, control pulmonary arteries from females also had greater expression of activated STAT3 compared to males. Further de ining the mechanisms of estrogen and 15-HETE interactions in the pulmonary vasculature in models of PAH will likely provide new insights in understanding their role in the pathogenesis of PAH. S13

Abstracts

1.41

QTc prolongation round in animal models of pulmonary hypertension is a marker of survival and is reversible by the fatty acid oxidation inhibitor, trimetazidine Ryan JJ, Fang YH, Rich JD, Thenappan T, and Archer SL Department of Medicine, University of Chicago, Chicago, IL, USA

We hypothesized that prolongation of the QTc interval observed in rodent models of pulmonary hypertension is (a) modi iable with metabolic therapy and (b) also occurs in human PH. In rodent models of pulmonary arterial hypertension (PAH) and pure RVH induced by pulmonary artery banding (PAB), the QTc interval is prolonged, re lecting downregulation of repolarizing Kv channels in RV myocytes. We measured fatty acid oxidation (using a dual isotope technique) and monophasic action potential duration (MAPD) in the RV Langendorff model. In vivo we recorded QTc in PAB versus sham rats. PAB rats were treated with partial inhibitors of fatty acid oxidation (pFOXi-ranolazine and trimetazidine) versus placebo. We measured the expression of the repolarizing cardiac K+ channel, Kv1.5, Hexokinase 2, a mediator of glycolysis, and the fatty acid transporter, FATP/CD36 in the RV by qRT-PCR. We measured the QTc interval and prospectively examined the impact of QTc interval on survival in 202 patients on PAH-speci ic therapy. Both trimetazidine and ranolazine decreased fatty acid oxidation in experimental RVH. In rats having undergone sham surgery the mean QTc was 0.122 s. Four and 8 weeks after PAB, the QTc increased to 0.205 s and 0.213 s, respectively (versus 0.122 s in Sham rats). Treatment with trimetazidine for 8 weeks decreased the QTc to 0.19 s. Ranolazine had no effect on QTc. MAPD was increased in PAB versus sham rats and returned to baseline after treatment with trimetazidine (MAPD50 18 ms in Sham compared with 27 ms in PAB models and 20 ms in PAB + trimetazidine [P<0.01 between PAB MAPD50 and PAB + trimetazidine MAPD50]). RV Kv1.5 mRNA expression was decreased in PAB rats and was partially restored by trimetazidine. Trimetazidine also decreased the expression of HKII and FATP/CD36. Ranolazine did not signi icantly affect HKII but did decrease FATP/CD36 expression. QTc intervals were longer in PH patient versus controls (454.8±29 ms versus 429.8±18 ms, P<0.001). QTc interval did not differ based on PH etiology or therapy. On multivariate analysis, a QTc >480 ms was an independent predictor of mortality (HR 4.21, 95% CI 1.31 –13.49). QTc prolongation is a feature of experimental RVH and human PH. QTc prolongation has prognostic signi icance. In the PAB model, QTc prolongation is amenable to metabolic therapies with pFOXi that are approved for human use for other cardiovascular conditions. 1.42

Calpain activates intracellular TGF-1 in pulmonary vascular remodeling of pulmonary hypertension Ma W, Han W, Greer PA, Tuder RM, Wang KKW, and Su Y Departments of Pharmacology & Toxicology and Medicine, Georgia Health Sciences University, Augusta, Georgia USA; Queen’s University Cancer Research Institute, Kingston, Ontario, Canada; Department of Medicine, University of Colorado, Aurora, Colorado USA; Center of Innovative Research, Banyan Biomarkers Inc., Alachua, FL, USA

In the present study we examined the role of calpain in collagen synthesis and pulmonary vascular remodeling in two models of pulmonary hypertension. We found that calpain inhibition using conditional knockout of calpain-4 prevented calpain activation and increases in right ventricular systolic pressure (RVSP), right ventricular hypertrophy, as well as collagen deposition and thickening of pulmonary arterioles of mice with hypoxic pulmonary hypertension. Moreover, the speci ic calpain inhibitor MDL28170 prevented the progression of RVSP, right ventricular hypertrophy as well as collagen deposition and thickening of pulmonary arterioles of rats S14

with established pulmonary hypertension induced by monocrotaline. Calpain inhibition did not affect increases in PDGF-BB and EGF but ameliorated the increase in active TGF-β1 in hypertensive lungs. Furthermore, inhibition of calpain using MDL28170 or calpain siRNAs attenuated EGF- and PDGF-BB-induced increases in calpain activity, proliferation, and collagen-I synthesis in pulmonary artery smooth muscle cells. EGF and PDGF-BB increased intracellular active TGF-β1 and p-Smad2/3 but not intracellular total TGF-β1 or active TGF-β1 and total TGF-β1 in the culture medium. Cell-impermeable TGF-β neutralizing antibody did not block EGF- and PDGF-BB-induced increases in collagen-I and p-Smad2/3. However, the cell-permeablespeci ic Alk5 inhibitor SB431542 or Smad2/3 inhibitor SIS3 prevented EGF- and PDGF-BB-induced increases in collagen-I and p-Smad2/3 and hypoxic pulmonary hypertension. Additionally, calpain inhibition attenuated EGF- and PDGF-BB-induced increases in intracellular active TGF-β1 and p-Smad2/3. Finally, lung tissues from patients with pulmonary arterial hypertension demonstrated concordant higher levels of calpain activation and intracellular active TGF-β in smooth muscle cells of pulmonary arterioles. Our data provide the irst evidence that calpain mediates EGF- and PDGF-BB-induced collagen synthesis and proliferation of pulmonary artery smooth muscle cells via an intracrine TGF-β1 pathway in pulmonary hypertension. 1.43

Downregulation of SERCA2a in maladaptive but not adaptive RVH Thenappan T, Fang YH, Piao L, and Archer SL Department of Medicine, University of Chicago, Chicago, IL, USA

Patients with pulmonary hypertension can develop either an adaptive right ventricular hypertrophy (RVH) and retain their right ventricular (RV) function or a maladaptive RVH leading to rapid RV failure. The mechanism underlying the transition from adaptive to maladaptive state is unclear. Sarco (endo) plasmic reticulum Ca2+ ATPase (SERCA2a) plays a key role in cardiac contractility by regulating cardiomyocyte calcium. SERCA2a expression and activity is depressed in experimental and clinical left heart failure. However, very little is known about changes in SERCA2a expression and activity in RV failure. We sought to study the expression of SERCA2a in rats with adaptive (pulmonary artery banding (PAB)) or maladaptive (monocrotaline-induced pulmonary hypertension (MCT))RVH. In the PAB model, PAB versus sham surgery was performed in male Sprague Dawley rats (n=6 in each group). In the MCT group, male Sprague Dawley rats were randomized to either a single subcutaneous injection of monocrotaline (60 mg/kg) or sterile water (control, n=5 each group). PAB animals were sacri iced at 8 weeks whereas MCT-treated rats were sacri iced at 4 weeks (at which time they manifested RV failure). The mRNA expression of SERCA2a, quanti ied using qRT-PCR, was signi icantly decreased in MCT versus control RV. However, in PAB-induced RVH there was no statistically signi icant decrease in SERCA2a expression as compared to RVs from the sham animals. SERCA2a expression is signi icantly down-regulated in maladaptive RVH but not in adaptive RVH. The upstream signaling that mediates this down-regulation in maladaptive RVH needs further study. This inding suggests SERC2a gene therapy using adeno associated viral vectors merits investigation. 1.44

The cell-penetrating homing peptide CAR selectively enhances pulmonary effects of systemically coadministered vasodilators in a preclinical model of severe pulmonary arterial hypertension Toba M, Abe K, Urakami T, Komatsu M, Jarvinen TAH, Mann D, Ruoslahti E, McMurtry IF, and Oka M Pulmonary Circulation | October-December 2011 | Vol 1 | No 4

Abstracts

Departments of Pharmacology and Medicine and Center for Lung Biology, University of South Alabama, Mobile, Alabama USA; Vascular BioSciences, Sanford-Burnham Medical Research Institute, La Jolla, CA, USA

A recent study has identi ied a cell-penetrating homing peptide, CARSKNKDC (CAR), which speci ically recognizes the neovasculature of wound tissue and also homes to hypertensive pulmonary arteries. We hypothesized that CAR would selectively augment the pulmonary vasodilatory effect of a coadministered substance by facilitating its transportation into pulmonary vascular tissue. Severe pulmonary arterial hypertension (PAH) was induced in rats by SU5416 injection followed by 3 weeks of exposure to hypoxia and 2 weeks of return to normoxia. Intravenously administered luorescein-labeled CAR accumulated in the remodeled pulmonary arteries, while no or low signal was detected in other organs, except for the kidney where CAR is excreted. A simple coadministration of CAR (without conjugation with the vasodilators) enhanced pulmonary but not systemic vasodilatory effects of the Rho kinase inhibitor fasudil and the tyrosine kinase inhibitor imatinib in catheterized PAH rats. These results suggest that CAR selectively enhanced the pulmonary activity of the coadministered drugs by facilitating their transportation into the hypertensive arteries where CAR homes. The exact mechanism by which the co-administered CAR enhances the pulmonary effects of drugs remains to be determined. Our results suggest a new paradigm in the treatment of pulmonary diseases, using a cell-penetrating homing peptide to selectively augment pulmonary vasodilation by non-selective vascular drugs without potentially problematic conjugation process. 1.45

Possible involvement of endothelial mesenchymal transition in the formation of plexiform lesions in a preclinical model of severe pulmonary arterial hypertension Toba M, Alzoubi A, McMurtry IF, and Oka M

Weisel FC, Roth M, Kloepping C, Wilhelm J, Pichl A, Seeger W, Ridge KM, Weissmann N, and Kwapiszewska G Excellence Cluster Cardio-Pulmonary System (ECCPS), Universities of Giessen and Marburg Lung Center (UGMLC), Giessen, Germany

Pulmonary hypertension (PH) can be induced by chronic alveolar hypoxia which results in a vascular remodeling process. In this study, we investigated whether pulmonary vascular remodeling observed in the mouse model of hypoxia-induced PH could be reversed by reoxygenation (reverse remodeling). Furthermore we sought to identify new genes that may trigger this process. Mice exposed to chronic hypoxia (21 days, 10% O2) were re-exposed to normoxia (for up to 42 days). Reversal of PH during reoxygenation was evident by decreased right heart hypertrophy, right ventricular pressure, and muscularization of small pulmonary vessels compared to hypoxic controls. Microarray analysis from these mice revealed S-Adenosylmethionine decarboxylase 1 (AMD-1) as one of the most downregulated candidates. As AMD-1 was already shown to be important for polyamine synthesis and cell proliferation, we focused on the impact of AMD-1 in the development and the reversal of pulmonary hypertension. In situ hybridization revealed AMD-1 localization in pulmonary vessels. AMD-1 silencing by siRNA decreased proliferation of pulmonary arterial smooth muscle cells and diminished phosphorylation of PLC-γ1. Furthermore, AMD-1+/- mice exhibited attenuated PH when exposed to chronic hypoxia compared to wild-type controls. Promoter analysis revealed that AMD-1 could be regulated by Egr1 as a consequence of EGF stimulation. These indings indicate that in the animal model of hypoxia-induced PH, vascular remodeling can be reversed by reoxygenation. AMD-1 may be involved in both the remodeling of pulmonary arteries during chronic hypoxia and the process of reverse remodeling. 1.47

A critical role for calpain in the development of pulmonary hypertension

Departments of Pharmacology and Medicine and Center for Lung Biology, University of South Alabama. Mobile, AL, USA

Zaiman AL, Swaim M, Cingolani O, Damico R, Blanco I, Undem C, Maylor J, Tuder RM, Shimoda LA, and Dietz H

We have reported that the severe, sustained pulmonary hypertension in a very late stage of the SU5416 plus hypoxia/normoxia-exposed rat is accompanied by the formation of plexiform lesions that are indistinguishable from those observed in human pulmonary arterial hypertension. The major cellular components that form the plexiform lesion are the hyperchromatic and oval-shaped core cells and endothelial cell marker positive lining cells. The origins and characteristics of these types of cells, however, remain unknown. We have performed immunohistochemical analyses of these lesions to further characterize the cellular composition. Adult male Sprague Dawley rats were injected subcutaneously with SU5416 (20 mg/kg; a VEGF receptor blocker), and exposed to hypoxia (10% O2) for 3 weeks followed by a return to normoxia for an additional 10 weeks. At 13 weeks after the SU5416 injection, hemodynamic parameters were measured and lungs were ixed for immunohistchemical staining. All rats developed severe pulmonary hypertension. Immunohistochemical analyses showed the lining cells were positive for mesenchymal as well as endothelial cell markers, while they were negative for the stem cell markers CD133 and c-Kit. Results for the core cells were not uniform—i.e., some but not all core cells were positive for the ibroblast marker S100A4 and α-smooth muscle cell actin and were negative for endothelial and stem cell markers. These results raise the possibility that an endothelial mesenchymal transition may be involved in the formation of the plexiform lesion in this model.

Johns Hopkins University School of Medicine, Baltimore, MD, USA

1.46

Reversion of vascular remodeling in pulmonary hypertension—Impact of AMD-1 Pulmonary Circulation | October-December 2011 | Vol 1 | No 4

We have recently identi ied a critical role for a novel gene family, calpain, in the chronic hypoxia and monocrotaline animal models of PH. The calpains are a family of cytosolic, Ca2+ activated, neutral cysteine proteases, many of which are ubiquitously expressed. When activated, these enzymes cleave a broad spectrum of functionally important targets that regulate cytoskeletal organization, cell proliferation, migration, apoptosis, and platelet aggregation—all key processes in the development of PH. Inhibiting calpain activity attenuated the development of PH. Mice overexpressing calpastatin (CAST), the highly speci ic endogenous and inhibitor of calpain developed less RV hypertrophy, a lower right ventricular pressure, and decreased pulmonary vascular remodeling compared to control animals after exposure to hypoxia for 3 weeks. Inhibition of calpain activity with calpeptin (250 μg/kg, SQ twice daily), a highly permeable small molecule inhibitor of calpain, attenuated the development of monocrotaline-induced PH. Twenty-eight days after MCT injection, the animals developed signi icantly less right ventricular hypertrophy and pulmonary vascular remodeling. Biochemical analysis of lung lysates revealed increased calpain 1 and 2 expression with MCT and a return toward baseline in those animals treated with calpeptin. On a cellular level, the pulmonary artery smooth muscle cells (PASMC) isolated from patients with PH or animal models of PH possess increased migratory and proliferative phenotype. PASMC isolated from CAST overexpressors demonstrated decrease migration compared to PASMC obtained from wild-type C57BL6 when exposed to hypoxia. We are currently evaluating the role of calpain in smooth muscle cell proliferation. S15

Abstr ac t s

5th Scientific Workshops and Debates of the Pulmonary Vascular Research Institute (PVRI) Cape Town, South Africa 7-10 February, 2012

2.1

Minai OA and Gudavalli R

Clinical characteristics and outcome of pulmonary hypertension among admitted heart failure patients

Respiratory Institute, Cleveland Clinic, Cleveland, OH, USA

Karaye KM, Yahaya I, Sa’idu H, and Bala MS Departments of Medicine and Chemical Pathology, Bayero University, Aminu Kano Teaching Hospital, Kano, Nigeria

Heart failure (HF) and pulmonary hypertension (PHT) are common syndromes with signi icant morbidity and mortality globally. However, knowledge gap exists in sub-Saharan Africa regarding the impact of one over the other on morbidity and mortality. The present study, which was part of a larger study on HF, aimed at assessing the clinical characteristics and in-hospital outcome of PHT among HF patients admitted to a tertiary health center in Kano, Nigeria. The study was cross sectional in design, carried out in the medical wards of Aminu Kano Teaching Hospital, Kano, Nigeria. Patients admitted with HF were serially recruited and studied after obtaining informed consent. All patients were clinically evaluated, and had baseline investigations including electrocardiogram and echocardiogram (echo), while some of them had assay for N-terminal pro-B type natriuretic peptide (NT-BNP). PHT was de ined as the presence of mean pulmonary artery pressure (mPAP) of ≥25 mmHg, assessed using continuous wave Doppler echo and Chemla formula. A total of 90 patients were studied, but the results presented here are for the 80 patients who had complete data. Fifty-three patients (66.25%) (Group 1) were found to have PHT while the remaining 27 (33.75%) had normal mPAP (Group 2). When the two groups were compared, mPAP for Group 1 was 38.31±12.23 mmHg while that for Group 2 was 16.39±5.48 mmHg (P<0.001). Two patients in Group 1 and 1 patient in Group 2 were smokers. In addition, Group 1 as compared with Group 2 patients had signi icantly lower left ventricular ejection fraction, and larger left atrium and left ventricle (P<0.05). NT-BNP was assessed in only 37 patients and the concentrations were found to be very high; 26 of them in Group 1 (14, 408±11,449) and 11 in Group 2 (12,266±11,497) (P=0.607). Twelve patients had atrial ibrillation, 8 of them in Group 1 and 4 in Group 2. The in-hospital mortality in Group 1 (8 patients=15.09%) was similar to that for Group 2 (4 patients=14.82%) (P=0.626); higher among females than males (8 versus 4 patients, respectively). The chance of discharge from hospital admission or in-hospital mortality was signi icantly in luenced by the diagnosis of cardiogenic shock (odds ratio=0.069; 95% con idence interval=0.017-0.286; P<0.001). The most common cause of HF was hypertensive heart disease seen in 28 patients, 13 of them in Group 1. Fifteen patients had peripartum cardiomyopathy; 13 of them were in Group 1. These were followed by dilated cardiomyopathy in 13 patients, 9 of them in Group 1; rheumatic heart disease in 8 patients, 7 of them in Group 1; ischemic heart disease in 6 patients, all of them in Group 1; corpulmonale in 5 patients, all in Group 1; and effusive pericarditis in 4 patients, all of them in Group 2. PHT is common in admitted HF patients, and is associated with worse parameters for morbidity. However, its in luence on in-hospital mortality was not apparent. A follow-up study is needed to assess it effect on long-term mortality. 2.2

Heart rate recovery after 6-minute walk test in patients with pulmonary arterial hypertension S16

Attenuated heart rate recovery (HRR) after exercise testing is associated with increased mortality. The purpose of this study was to evaluate early HRR after 6-minute walk test (6-MWT) and the association between abnormal HRR and prognostic markers (WHO class, RV function, 6MWD etc.) in patients with idiopathic pulmonary arterial hypertension. Heart rate at 1-minute post 6MWT was collected routinely at our institute from August 2009. Demographic, clinical, hemodynamic, pulmonary function test and 6-MWT variables were collected in patients who had idiopathic pulmonary arterial hypertension and had a 6-MWT from August 2009 to March 2010. HRR1 was de ined as the difference in the heart rate at the end of 6-MWT and at 1 minute after completion of the 6-MWT. A total of 1,444 patients underwent 6-MWT from August 2009 to March 2010. Of these, 75 patients had a diagnosis of PAH (Group 1.1 and 1.2) and were included in the inal analysis. In our cohort of PAH patients, the mean age was 49.13 years, mean mPAP was 52.25 mmHg, mean plasma BNP was 177.1 pg/ml, mean eRVSP based on transthoracic echocardiogram (TTE) was 80.69 mmHg, mean 6-MWT was 440.6 meters, mean HRR1 was 21.29 bpm. HRR1 had a signi icant negative correlation with age (r= -0.37), RVSP (r= -0.30), and BNP levels (r= -0.61). HRR1 had a signi icant positive correlation with DLCO (r=0.34) and 6MWD (r=0.58). HRR1 was signi icantly lower in patients with WHO Class III/IV symptoms, elevated BNP levels, and moderate to severe RV dysfunction on TTE. Patients with a lower HRR1 (≤15) had a higher risk of hospitalization. HRR1 after 6-MWT correlates with several prognostic markers in PAH (Group 1.1 and 1.2) patients. HRR1 after 6-MWT was signi icantly lower in patients with WHO class III/IV symptoms, moderate to severe RV dysfunction on TTE, and elevated serum BNP levels. 2.3

Does high altitude protect against irreversible pulmonary hypertension? Heath A, von Alvensleben I, Graham B, Tuder R, Brockman C, and Perez E. Kardiozentrum Fundacion Cardioinfantil La Paz, La Paz, Bolivia; Department of Medicine, University of Colorado at Denver, Denver, Colorado USA; Bolivian Medical Centre of Surgery, Cochabamba, Bolivia

High altitude inhabitants with posttricuspidal shunts rarely develop severe pulmonary hypertension, and very late Eisenmenger Syndrome. The patients remain operable, and the pulmonary pressure remains reversible. We hypothesize thick pulmonary vessels are responsible for the stabilization of the vessel, which does not allow for the development of deformations, which are typical for the progressive disease of the vascular bed in pulmonary hypertension. Owing to a continuous vasoconstriction, the muscular media does not allow the high low to the pulmonary vessels, which is typical for this condition. The aim of the clinical study is to assess if the patients with left to right shunts (ductus, VSD, ASD and AV channel) have a protective factor which impairs the development of pulmonary hypertension at early Pulmonary Circulation | October-December 2011 | Vol 1 | No 4

Abstracts

stages of the evolution of the disease at high altitude. Prospective, consecutive, case based a 1-year study with inclusion and exclusion criteria (VSD, PDA, or AV Channel patients older than 5 years with pulmonary hypertension and indication of surgical closure). All patients planned for (1) diagnostic catheter to assess operability and for invasive pulmonary pressure measures, (2), surgical correction of the CHD and lung biopsy, and (3) post-OP catheter for invasive pulmonary pressure measures. Four patients (Patient 1: 12-year-old boy with VSD; Patient 2: 28-year-old man with PDA; Patient 3: 11-year-old girl with VSD; and Patient 4: 10-year-old-boy with VSD) underwent cardiac catheterization with a hyperoxia test to assess operability. All four patients had pulmonary hypertension and the pulmonary pressures dropped with hyperoxia. Patients 1, 2 and 3 are already operated on and recovered well. Histological examination of the lung biopsies was suggestive of increased pulmonary artery medial thickness in all three cases but not more typical changes for advanced disease in the pulmonary vessels. Patient 4 is waiting for the operation. Patient 1 completed the three steps. The post-operative catheterization showed normal values for pressure and vascular resistance. High altitude exposure may be protective against severe irreversible congenital heart disease-associated pulmonary hypertension. The irst study results are encouraging. We are looking forward to completing the series.

some degree of PAH on follow-up (RVSP 42±3.4 mmHg). Comparison of pre-op hemodynamic data between two groups (Group 1: patients with post-operative regression of PVR ≥20%, Group 2: post-operative regression of PVR<20%) revealed a signi icant difference only in Qp:Qs following oxygen inhalation (P<0.05). On multivariate analysis advanced age and basal PVR >9 were signi icant predictors of non-regression of PAH and clinical worsening after ASD closure. ASD closure in patients with signi icant pulmonary arterial hypertension improved functional status, decreased PA systolic pressure and PA mean pressure but did not signi icantly alter the PVR. Complete resolution of PAH is uncommon on long-term follow-up. Patients with advanced age and preoperative PVR >9 WU have signi icant risk of severe PAH, RV dysfunction and CHF on long-term follow-up. 2.5

Pulmonary hypertension in a tertiary health institution: A retrospective review of 24-months echocardiography registry Sani MU, Mijinyawa MS, Ishaq NA, and Shehu MN Department of Medicine, Aminu Kano Teaching Hospital, Kano, Nigeria

2.4

Long-term follow-up of closure of atrial septal defects in older children and adults with severe pulmonary arterial hypertension Harikrishnan S, Sonney JP, Randeep S, Venkiteswaran S, Krishnamoorthy KM, Sivasankaran S, Titus T, and Jaganmohan T Department of Cardiology, Sree Chitra Tirunal Institute for Medical Sciences & Technology, Thiruvananthapuram, Kerala, India

The aim is to study the long-term outcome of older children and adults (>5 years) with pulmonary arterial hypertension (PAH) who underwent surgical closure of atrial septal defects. Retrospective analysis of data of consecutive patients who had PAH and underwent ASD closure following an invasive hemodynamic study. The study included 38 patients (24 females). Mean age at the time of surgery was 24.8±11 years. Four (10%) had sinus venosus (SV) and 34 (90%) had ostium secundum (OS) defects. Preoperative (pre-op) PVR (pulmonary vascular resistance) or PVR Index>4 wood units (WU) was the inclusion criteria. Left-to-right shunt ratio (QP/QS), systemic pressure (Ps in mmHg), pulmonary artery systolic pressure (Pp in mmHg), mean pulmonary artery pressure (PA mean), PVR and ration of PVR to systemic vascular resistance (Rp:Rs) were studied in all patients preoperatively. These parameters were reassessed after inhalation of 100% oxygen for 15 minutes in all patients. In the preoperative study, we saw a signi icant increase in QP/QS ratio (1.76±0.5 to 2.63±1.1), decrease in Pp (82±14.6 to 75.1±14.3) and PVR (8.9±2.8 to 5.23±1.7 WU) from the baseline after oxygen inhalation (P<0.05). The mean follow-up was 12.4±6.3 years. Four patients were lost to follow-up. All the remaining 34 patients had clinical and echocardiographic follow-up, and were assessed for resolution of PAH, RV function and functional class. None of the patients had complete normalization of PA pressure on follow-up. Invasive hemodynamic study was done in 20 patients, at mean 6.2±3.8 years (6 months–12 years) following surgery. In these patients, there was a signi icant reduction in Pp (77.9±16.6 pre-surgery to 61.75±21.43 mmHg, post-surgery), Pp:Ps ratio (0.66±0.0.13 to 0.42±0.14) and PA mean (50.6±12.4 pre-surgery to 41±12 mmHg, post-surgery), but the reduction in PVR was not signi icant (8.59±2.80 to 7.03±3.80). Patients with basal PVR >9 (N=14): ive patients developed severe PAH on follow-up and four developed RV dysfunction and congestive heart failure (CHF). The pre-op response to O2 was not a signi icant predictor of outcome in this group. Patients with basal PVR <9 (N=20): only one patient had worsening of PAH on follow-up. Majority of this group had regression of PAH to some extent, but most of them continued to have Pulmonary Circulation | October-December 2011 | Vol 1 | No 4

Pulmonary hypertension is a complex multidisciplinary disorder. It is a syndrome resulting from restricted low to the pulmonary circulation leading to increased pulmonary vascular resistance and ultimately right heart failure. It is a devastating, progressive disease with increasingly debilitating symptoms and usually, shortened overall life expectancy. The epidemiology of PHT in sub-Saharan Africa has not yet been determined, but limited reports suggest that the incidence is higher than that reported from developing countries, owing to the pattern of diseases prevalent in the region. We set out to determine the primary cardiac diagnosis in patients with pulmonary hypertension found at echocardiography in Aminu Kano Teaching Hospital. Between September 2009 and August 2011 (a 24-month period), we retrospectively reviewed the primary cardiac diagnosis of all patients with pulmonary hypertension in our hospital. All the studies were trans-thoracic echocardiography done using ALOKA SSD-4000 machine. All measurements were done according to the recommendation of the American Society of Echocardiography (ASE). Continuous-wave Doppler was used to interrogate the valves when there was suspicion of any valvular lesion. Pulmonary artery systolic pressure (PASP) was estimated by measuring the Doppler systolic tricuspid regurgitant low velocity and applying the Bernoulli equation and adding the right atrial pressure (RAP) which was assumed to be 10 mmHg. PHT was de ined as mild if PASP was 35-45 mmHg, moderate if 46-60 mmHg and severe when PASP was >65 mmHg. Information obtained from the records included age, gender, clinical diagnosis and echocardiogram indings. Data was analyzed using SPSS version 13.0 software. A total of 1411 echocardiographic examinations were done over the 24-month period. Of these, 256 (18.1%) had pulmonary hypertension, and they were analyzed. There were 117 males and 139 females, giving a male to female ratio of 1:1.2. The causes of pulmonary hypertension were hypertensive heart disease present in 80 patients (31.3 %), followed by cardiomyopathies (dilated and peripartum) seen in 57 patients (22.3%). Rheumatic heart disease and Corpulmonale were the causes in 46 (18.0 %) and 45 (17.5%) patients respectively. Other causes were ischemic heart disease in 10 (3.9%), idiopathic in 7 (2.7 %) and others in 11 (4.3 %). The others include three atrial septal defects and one case each of HIV infection, sickle cell disease, ventricular septal defect, Marfan’s syndrome, thyroid heart disease, mitral valve prolapse, degenerative valve disease and hypertrophic cardiomyopathy. The most common cause of pulmonary hypertension from our echo registry is left heart disease, followed by lung disease i.e., corpulmonale (COPD and tuberculosis). HIV infection, schistosomiasis, chronic hepatitis B and C and Sickle Cell Disease are surprisingly rare causes, perhaps because of the nature of the study. There is need for a prospective registry to understand the epidemiology of this condition in Africa. S17

Abstracts

2.6

Overview of pulmonary hypertension registries and quality standards Pittrow D Institute of Clinical Pharmacology, Technical University of Dresden, Dresden, Germany

Registries are usually designed as multicenter cohorts of patients suffering from a speci ic disease, with long-term follow-up. As for other diseases, in pulmonary hypertension (PH) registries have become an important source of information as they provide data that cannot be captured in any other way (patient characteristics, practice patterns, outcomes and prognosis under everyday care conditions). Major currently active clinical PH registries include, but are not limited to, the REVEAL registry in the US, the CompERA-XL registry in several European countries, cohorts of the French National Registry, or the Spanish REHAB registry. These registries differ considerably in terms of scope, size, inclusion criteria, and length of follow-up. Thus, a global database has repeatedly been called for to unify such efforts. In contrast to clinical trials that usually follow the ICH-GCP guidelines that have been translated in national drug law legislation, for registries there are no generally accepted or mandatory quality standards. Depending on the groups who own or make use of the data, various guidance documents are being used, for example “Good Epidemiological Practice” (GEP), European Medicines Agency “European Network of Centres for Pharmacoepidemiology and Pharmacovigilance (ENCePP) standards,” Strengthening the Reporting of Observational Studies in Epidemiology” (STROBE), or “Cardiology Audit and Registration Data Standards” (CARDS) for registries conducted under the auspices of the European Society of Cardiology. Further, quality assurance elements of clinical trials are regularly applied, for example monitoring with source data veri ication. In pulmonary hypertension (PH) registries have become an important source of information as they provide data that cannot be captured in any other way (patient characteristics, practice patterns, outcomes and prognosis under everyday care conditions). Further standardization of key quality factors (completeness of data, extent of data validation, explicit de initions for variables etc.) would be helpful for PH registries, too.

(r2=0.37, standard error of 95 m and P<0.001) among all other clinical and pulmonary hemodynamic measurements performed at rest and exercise. Similarly, in multiple Cox survival analysis exercise cardiac index was the best predictor of death with a hazard ratio of 7.5 (95% con idence interval 2.3: 24.3, P=0.001). Exercise cardiac index is a better predictor of exercise capacity and survival compared with clinical and pulmonary hemodynamic data at rest in our cohort of patients with PAH. 2.8

Blockade of TGF- prevents schistosomiasisassociated pulmonary hypertension Graham B, Chabon J, and Tuder R Program in Translational Lung Research, University of Colorado Denver, Denver, CO, USA

Schistosomiasis-associated pulmonary arterial hypertension (PAH) is one of the leading causes of PAH worldwide. We have previously found that TGF-β signaling is upregulated in human and experimental schistosomiasis-associated pulmonary hypertension (Sc-PH). TGF-β has been shown to be necessary for PH in monocrotaline and chronic hypoxia models of PH. We hypothesized blockade of TGF-β would prevent experimental Sc-PH. Wild-type mice (C57BL6/J background; 3-6 per group) were sensitized to Schistosoma mansoni eggs, and then intravenously challenged with 240 S. mansoni eggs/gram. A monoclonal pan-TGF-β neutralizing antibody, 1D11, or isotype IgG control, was administered every 3 days starting at the time of IV ova administration. 1D11 and IgG was also administered to uninfected mice in the same dosing schedule. One week after the IV ova administration, right ventricular catheterization was performed, followed by analysis of the lung tissue. Mice treated with IgG and infected with S. mansoni had an elevated right ventricular systolic pressure (RVSP) compared to uninfected IgG and 1D11 treated mice. However, mice treated with 1D11 and infected with S. mansoni did not have an elevated RVSP. Infected mice treated with 1D11 also had less pulmonary vascular remodeling than IgG-treated infected mice. 1D11 treatment had no effect on peri-egg granuloma volumes or the effectiveness of egg clearance. Inhibition of TGF-β may prevent Sc-PH without signi icantly compromising the hostimmune response to the infection.

2.7

2.9

Prognostic value of exercise cardiac index in idiopathic, heritable and anorexigen-associated pulmonary arterial hypertension

Platelet function and morphology in idiopathic pulmonary hypertension

Chaouat A Service de Pneumologie, Hôpital de Brabois, CHU de Nancy, Nancy, France

In a study of incident cases of idiopathic, heritable and anorexigenassociated pulmonary arterial hypertension (PAH) we studied the prognostic value of exercise cardiac index during right heart catheterization. Forty-three patients (22 women) with an age of 55 years (median) [interquartile range (IQR): 46: 68] were included. All patients had a right heart catheterization at rest and during an exercise of approximately 40% of the maximum workload. All patients were treated according to European guidelines and were followed up for from 6 to 144 months. At diagnosis, NYHA functional classes were I/II, III or IV in 7, 21 and 15 patients, respectively. Six-minutes walk test distance was 360 m. [IQR 300: 430]. Main pulmonary hemodynamic data at rest were mean pulmonary artery pressure of 50 mmHg [IQR 43: 58], cardiac index of 1.88 L/min/m2 [1.59: 2.21] and pulmonary vascular resistance of 1067 dyn.s.cm-5 [779: 1394]. Patients with lower exercise cardiac index have a signi icantly higher NYHA functional class and lower 6-minutes walk distance. In a multiple stepwise regression analysis, only age and exercise cardiac index were predictor of the 6-minutes walk distance S18

Ramakrishnan S, Senguttuvan NB, Lakshmy R, Saxena R, Wadhwa S, Khunger JM, Bhargava B, Kothari SS, Saxena A, and Bahl VK All India Institute of Medical Sciences, New Delhi, India

Pathogenesis of idiopathic pulmonary artery hypertension (IPAH) is not clear. Thrombosis and proliferation are the two important pathological features of IPAH. Theoretically, both these processes may be initiated by platelets. Clubbing and hypertrophic osteoarthropathy is currently explained by platelet-endothelium hypothesis. Escape of megathrombocytes from lung circulation due to any right-to-left shunt leads to platelet and endothelial activation in the nailbed that leads to clubbing. A similar barrage of activated platelets in the pulmonary circulation may lead to all the known pathological changes seen in IPAH. Nine consecutive patients of IPAH [median age of 24 years (14–47 years)] were included in the study. Nine patients with rheumatic heart disease with pulmonary hypertension and nine patients of RHD with PAH [median age of 42 years (22–66 years)] were recruited as controls. Patients of IPAH in NYHA Class IV status, decompensated heart failure, history of smoking, diabetes mellitus, coronary artery disease, associated hematological disorder, hepatic or renal dysfunction, and patients who were taking NSAIDs or aspirin in the last 1 week, were excluded from the study. All patients underwent cardiac catheterization. Blood samples Pulmonary Circulation | October-December 2011 | Vol 1 | No 4

Abstracts

were taken from superior vena cava, pulmonary artery, left ventricle and femoral artery. Collagen and Adenosine-di-phosphate were utilized for the assessment of platelet reactivity. Samples of blood from the different sites in each patient were utilized for the analysis of platelet morphology under electron microscopy. The morphology of visualized platelets was utilized for the purpose of classifying them into active, partially active and inactive. The pulmonary artery pressures, right ventricular systolic pressure and pulmonary vascular resistance were signi icantly higher in IPAH group, while the pulmonary capillary wedge pressure was higher in the control group. There was no difference in the proportion of active platelets between IPAH group and the control group at various sites. In the IPAH group, the number of active platelets was signi icantly higher in pulmonary artery as compared to that of femoral artery (P=0.01). In contrast, there was no difference in the number of active platelets between various sites in patients with RHD with PAH. Platelet reactivity at the various sites did not differ signi icantly between IPAH and RHD with PAH groups. There was no signi icant difference in platelet reactivity measured biochemically by collagen and ADP between pulmonary artery and the rest of the sites. We found an increased existence of active platelets in pulmonary circulation as compared to systemic circulation by electron microscopy in IPAH patients. We found no difference in the level of platelet reactivity between pulmonary and systemic circulation biochemically. Patients with IPAH did not show signi icantly high platelet reactivity as compared to the patients with RHD with moderate to severe PAH. 2.10

Prevalence of pulmonary hypertension among 6270 asymptomatic school children in India: The Rheumatic Heart Echo Utilization and Monitoring Actuarial Trends in Indian Children Study Saxena A, Ramakrishnan S, Roy A, Seth S, Krishnan A, Misra P, Kalaivani M, Bhargava B, Flather M, and Poole-Wilson P All India Institute of Medical Sciences, New Delhi, India; Royal Brompton Hospital and Imperial College, London, UK

The prevalence of pulmonary artery hypertension (PAH) among school children in India is not known. Conventionally, auscultation has been used for community screening for heart disease, but echocardiography with Doppler may be more sensitive and speci ic. Hence, in the Rheumatic Heart Echo Utilization and Monitoring Actuarial Trends in Indian Children study designed to identify echocardiographic prevalence of rheumatic heart disease, we estimated the prevalence of PAH. We carried out a cross-sectional survey to diagnose heart defects in asymptomatic school children aged 5-15 years, living in rural areas, using portable echocardiography. The demographic data were collected. After history and physical examination, echo-Doppler was performed, using a bedside portable echocardiography machine. A total of 6,270 asymptomatic children were screened and 52% were male. The mean age was 10.79±2.63 years. Echo-Doppler diagnosed pulmonary hypertension in four cases, giving a prevalence of 0.6/1000 school children (90% CI – 0.1 – 1.1/1000 children). The cardiac lesions associated with PAH identi ied by echo-Doppler included: rheumatic heart disease (two patients), Eisenmenger syndrome (one patient), and one patient of atrial septal defect with mitral regurgitation and PAH. The prevalence of PAH among children in India is higher than reported from the developed world. The common causes of pulmonary hypertension in children are rheumatic heart disease and un-operated congenital heart disease in developing nations. 2.11

A review of catheterization data for determining operability in congenital heart disease with severe pulmonary hypertension Pulmonary Circulation | October-December 2011 | Vol 1 | No 4

Juneja R, Ramakrishnan S, Anju, Gupta S, Kothari SS, and Saxena A All India Institute of Medical Sciences, New Delhi, India

Pulmonary artery hypertension (PAH) complicates the clinical course and outcome of many patients with congenital heart disease (CHD) and is an important determinant of morbidity and mortality in these patients. Surgical correction at the correct time is the only method known to prevent these complications. There are several limitations in the present way of determining operability. We started prospectively collecting data of all patients getting catheterized with a diagnosis of left-to-right shunts with severe PAH. Patients were divided into three categories based on the inal recommendation of the cardiologist/ cardiac surgeon. Group I: likely operable; Group II: de initely inoperable (Eisenmenger); and Group III: borderline operability. Of the 100 patients included (mean age 8.5 years) the majority had ventricular septal defect (67%). Forty-six patients were considered operable (Group 1), 17 patients were considered inoperable, and 27 patients were classi ied as borderline operability. PAH secondary to left-to-right shunts remains a major problem in our country. Basal PVRI, pulmonary blood low and Qp/Qs ratio, and post-oxygen fall in pulmonary diastolic pressure and PVRI help in the determination of operability. 2.12

Predictors of adverse clinical outcome in Eisenmenger syndrome Ramakrishnan S, Kukreti BB, Juneja R, Bhargava B, Kothari SS, Saxena A, and Bahl VK All India Institute of Medical Sciences, New Delhi, India

Clinical course and predictors of adverse events in Eisenmenger syndrome are not well characterized. In this interim analysis, we present the data of 50 patients of Eisenmenger syndrome followed up for a mean duration of 18.2 months (range 2-84 months). Routine clinical examination and 6-minute walk test were done at enrolment and follow-up visits. Fifty patients of Eisenmenger syndrome of mean age 24 10 years were followed up for mean duration of 18 months (range 2-84 months). Mean 6-minute walk distance was 475.23±101.4 meters at enrolment and 486.2 115.5 meters at follow-up. Adverse events (worsening heart failure and death) were seen in seven patients. Adverse events were signi icantly correlated with follow-up 6-minute walk distance 509±90 versus 348 161 meters (P=0.011) and post 6-minute walk heart rate. Worsening of 6-minute walk distance is a useful and powerful predictor of clinical worsening in Eisenmenger syndrome patients. 2.13

Nocturnal hypoxemia in patients with Eisenmenger syndrome Ramakrishnan S, Juneja R, Sharma AK, Bardolei N, Shukla G, Guleria R, Bhatia M, Kalaivani M, Kothari SS, Saxena A, and Bahl VK All India Institute of Medical Sciences, New Delhi, India

Signi icant nocturnal hypoxemia occurs in patients with obstructive pulmonary disease and primary pulmonary hypertension. Nocturnal hypoxemia may also be important in patients of Eisenmenger syndrome (ES), but has not been assessed so far. The objective of the study was to ind the prevalence of sleep-related disturbances in patients with ES. The study included 25 patients with ES (mean age 25.2±9.6 years, 18 male) and 12 patients with cyanotic congenital heart disease (CCHD) with pulmonary stenosis physiology (mean age 20.5±8.5 years, eight male) as controls. All the patients underwent an overnight comprehensive polysomnogram study. An oxygen drop is de ined as S19

Abstracts

any fall in SpO2 greater than 5% lasting for at least 9 seconds. Oxygen desaturation index (ODI) is the number of oxygen desaturations per hour. The patients and controls had signi icant nocturnal hypoxemia in the absence of apnea and hypopnea. The mean ODI in ES group was 9.0±6.2 and in CCHD with pulmonary stenosis group was 8.0±5.9 (P=0.63). The apnea hypopnea index (AHI) was 3.37±5.0 in the ES group and was 2.1±3.6 in CCHD with pulmonary stenosis group. Patients with ODI>10 had signi icantly higher hemoglobin (17.2±1.3% versus 14.4±1.5%, P<0.001) than those with ODI<10. Eisenmenger syndrome patients have signi icant nocturnal hypoxemia unrelated to hypopnea and apnea. Nocturnal desaturation occurred more frequently in patients with greater hemoglobin values. These indings may have important therapeutic implications. 2.14

Pulmonary hypertension at moderate altitude in children: Importance of the hyperreactivity of pulmonary vascular tree Díaz GF National University, Bogota, Colombia

The hypobaric hypoxia gives special characteristics to pulmonary hypertension (PH) at altitude related to biopathogenesis, epidemiology, diagnostic approach and treatment. One aspect related to the biopathogenesis is the hyperreactivity, a characteristic that must be studied with precision in the patients at altitude, having in mind its importance related to prognosis and treatment. Due to the importance of the hypobaric hypoxia and the hyperreactivity of pulmonary vascular tree at altitude, we decided to study the effect of the prolonged hyperoxic test (oxygen with FiO2 >80% for more than 1 hr.) in children living in Bogota, Colombia at 2.640 meters above sea level (moderate altitude). Children with severe pulmonary hypertension diagnosed with echocardiogram con irmed by catheterism were exposed to oxygen (FiO2 >80%) for more than 1 hour (1-24 hrs.) after a basal echocardiogram without oxygen. We measured the pulmonary pressure using the Bernoulli equation after getting an excellent tricuspid regurgitation curve. Of the 15 patients, 13 had idiopathic pulmonary hypertension and 2 had PH and VSD: 1 too small muscular VSD and 1 large VSD. All patients were with oxygen and in functional Class III or IV and 1 was in very critical situation. The median of the pick systolic pulmonary pressure (PSPP) before the proof was 92 mmHg (81-161) and the median of the PSPP after the proof was 61 mmHg. One patient did not respond to the proof and died 4 months later. Eight patients went to live at low altitude, are alive and asymptomatic with a follow-up between 6 months and 28 years (median: 8.2 years) with PSPP between 44 and 56 mmHg. The more critical patient with initial PSPP of 153 mmHg is asymptomatic with a follow-up of 10 years and PSPP of 46 mmHg. The patient with large VSD was rejected for surgery; however, after the positive extended hyperoxic test, the patient went to live at low altitude with sildena il and 6 months later was re-catheterized. We found that the pulmonary resistances were at their lowest, the VSD was closed by surgery and at this moment the patient is asymptomatic with a follow-up of 6 years. One patient with very positive extended hyperoxic proof could not afford to live at low altitude for socioeconomic problems and died 6 months later. The other ive patients could not live at low altitude due to a number of factors. Of them, four continue with severe PH and whilst the ifth patient experienced a rise in the pulmonary pressure and is in a critical clinical situation. The hyper-reactivity of the pulmonary vascular tree is an important characteristic of PH at altitude. As the hypobaric hypoxia is important in the biopathogenesis of PH at altitude, the oxygen is an effective vasodilator of the pulmonary vascular tree. We found that the prolonged hyperoxic test is an important tool to evaluate the hyperreactivity of the pulmonary vascular tree at altitude. The patients with hyper-reactivity of the pulmonary vascular tree at altitude must live at low altitude to increase their chances of survival. S20

2.15

Novel roles of radiation protective compound amifostine in attenuation of acute lung injury Birukov KG, Fu P, Sarich N, and Birukova AA Department of Medicine, University of Chicago, Chicago, IL, USA

Acute lung injury (ALI) and adult respiratory distress syndrome (ARDS) remain serious life-threatening conditions with 30% morality and limited therapeutic treatment opportunities. Acute in lammation and vascular leak are cardinal features of ALI/ARDS. Nonspeci ic tissue in lammation and injury in response to infectious and noninfectious insults (i.e., high tidal volume mechanical ventilation (HTV)) lead to oxidative stress, which further exacerbates tissue injury and in lammatory processes in the lung. Despite an encouraging outcome of antioxidant therapy in animal models of acute lung injury, effective antioxidant agents for clinical application remain to be developed. Amifostine is an FDA-approved radiation protection agent with antioxidant and DNA-protective properties used to prevent surrounding tissue damage during radiation therapy. Amifostine mitigating effects have been also tested in the models of toxic tissue injury. This study tested two hypotheses: (a) concurrent treatment with amifostine may reduce lung endothelial barrier dysfunction and lung in lammation caused by Gram-negative bacteria wall-lipopolysaccharide (LPS) by direct mitigation of LPS-induced oxidative stress; and (b) preconditioning with very low doses of amifostine may be protective against ventilator induced lung injury due to induction of endogenous antioxidant defense mechanisms. Concurrent treatment with amifostine abrogated LPS-induced ROS production leading to inhibition of LPS-induced endothelial permeability and redox-sensitive signaling cascades: p38 and Erk-1,2 mitogen-activated protein (MAP) kinases and the nuclear factor-kappaB (NFkB) pathway. In vivo, concurrent amifostine administration inhibited LPS-induced tissue oxidative stress and reduced vascular leak and neutrophil recruitment to the lungs. In turn, 3-day preconditioning with low doses of amifostine prior to HTV attenuated HTV-induced protein and cell accumulation in the alveolar space judged by BALF analysis, decreased Evans Blue dye extravasation into the lung parenchyma, decreased biochemical parameters of HTVinduced tissue oxidative stress, and inhibited HTV-induced activation of redox-sensitive stress kinases and NF-B in lammatory cascade. These protective effects of amifostine were associated with increased superoxide dismutase 2 (SOD2) expression and increased SOD and catalase enzymatic activities in the animal and endothelial cell culture models of VILI. These results suggest that, besides direct antioxidant effects, amifostine preconditioning activates lung tissue antioxidant cell defense mechanisms and may be a promising strategy for alleviation of VILI in critically ill patients with sepsis subjected to extended mechanical ventilation. 2.16

Atrial natriuretic peptide improves Staphylococcus aureus-induced lung inflammation and vascular barrier function Birukova AA, Xing J, and Moldobaeva N Department of Medicine, University of Chicago, Chicago, IL, USA

Lung inflammation and alterations in endothelial cell (EC) permeability are key events to development of acute lung injury (ALI). This study investigated effects of atrial natriuretic peptide (ANP) on pulmonary endothelial barrier dysfunction caused by components of Gram-positive bacterial cell wall, Staphylococcus aureus-derived peptidoglycan (PepG) and lipoteichoic acid (LTA). C57BL/6J wild type or ANP knockout mice (Nppa-/-) were treated with a combination of LTA and PepG (i/t, 2.5 mg/kg each) with or without ANP (i/v, 2 μg/kg). In human pulmonary endothelial cell culture barrier Pulmonary Circulation | October-December 2011 | Vol 1 | No 4

Abstracts

properties were assessed by morphological analysis, measurements of transendothelial electrical resistance, and phosphorylation profile of signaling proteins. Heat inactivated S. aureus bacterial particles, LTA and PepG increased pulmonary EC permeability, which was associated with activation of Rho, MAP kinase, and NFB signaling, which was further promoted by combined LTA and PepG treatment. Disruptive effects of LTA and PepG were abolished by ANP pretreatment. In vivo, accumulation of protein and cell elements in the bronchoalveolar lavage fluid, tissue neutrophil infiltration, and increased Evans blue extravasation reflecting lung vascular leak caused by intratracheal LTA and PepG instillation were significantly attenuated by intravenous injection of ANP. BAL markers of LTA/PepG-induced lung dysfunction were further augmented in ANP-/- mice. These results strongly suggest a protective role of ANP in the in vitro and in vivo models of acute lung injury associated with Gram-positive infection. Thus, ANP may have important implications in therapeutic strategies aimed at the treatment of sepsis and ALI-induced Gram-positive bacterial pathogens. 2.17

Oxygen sensing pathway: Revealing and documenting human adaptations at high altitude Mishra A, Thinlas T, Mohammad G, and Pasha Q Institute of Genomics and Integrative Biology, Delhi, Department of Biotechnology, University of Pune, Pune, and Department of Medicine, SNM Hospital, Leh, Ladakh, India

The residents of Great Himalayas have evidence of positive selection on genetically based trait variation counteracting the effect of environmentally induced changes. These residents have many visible adaptive changes like higher resting ventilation, low hemoglobin concentration, thin-walled pulmonary vasculature, higher exhaled nitric oxide and blunted hypoxic vasoconstriction response that helped them live reproducibly and successfully for thousands of years. The genes of oxygen sensing pathway have emerged the most favorite candidates for genetic adaptation at HA. For example, the two main components Endothelial PAS domain-containing protein 1 (EPAS1) and HIF-1 Prolyl hydroxylase 2 (EGLN1) of HIF-signaling pathway have shown the evidence of positive selection in recent genome-wide association studies (GWAS). The selected regions of both the genes by exhibiting an association with low Hb concentration indicate their strong functional and regulatory role in maintenance of adaptive homeostasis at HA. The case-control design conducted by us enumerated the selection of EGLN1 with T allele of rs480902 and C allele of rs479200 in HA population (P=4.01E-07). Conversely, the T allele of rs479200 that corresponded to higher expression of EGLN1 was overrepresented in HAPE (P<0.05) when compared with healthy sojourners at the same environment. In addition to EPAS1 and EGLN1, other two candidates of this pathway, namely endothelial nitric oxide synthase (NOS3) and endothelin-1 (ET-1), also deserve description. The 298Glu allele of 298Glu/Asp polymorphism and 4b allele of 4b/4a (27 base pair variable number tandem repeat) of NOS3; longer-repeats of (CT)n–(CA)n repeat and G allele of 2288G/T polymorphism of ET-1 were overrepresented in the natives (P≤0.05) whereas 298 Asp allele of 298Glu/Asp and 4a allele of 4b/4a of NOS3; shorter-repeats of (CT)n–(CA)n repeat and T allele of 2288G/T of ET-1 were found to be associated with HAPE (P≤0.05). Surprisingly, however, GWAS has not shown selection of these two or several other markers as adaptation and mal-adaptation is multigenic trait. The evidence of just one or few signals out of millions of SNP in GWAS de initely suggests of improvement in statistical and computational software currently in use so that complete information can be captured from such an expensive, extensive and exhaustive study. Nevertheless, these studies have clearly documented the evolutionary selection of genetic variants speci ic to high-altitude Tibetans which have helped them achieve oxygen saturation and hematological pro ile similar to normoxic condition. Pulmonary Circulation | October-December 2011 | Vol 1 | No 4

2.18

Vasoactive mediators and ROS interactions under hypobaric hypoxia Ali Z, Mishra A, Mohammad G, and Pasha Q Institute of Genomics and Integrative Biology, Delhi, Department of Biotechnology, University of Pune, Pune, and Department of Medicine, SNM Hospital, Leh, Ladakh, India

The hypobaric hypoxic environment, UV radiation and cold temperature at high-altitude induces many changes like hypoxic pulmonary vasoconstriction response, blunted hyperventillatory response, increased hemoglobin concentration and sympathetic nervous response. This extreme environment is also responsible for the increased production of reactive oxygen species (ROS) and alleviated antioxidant capacity of body. Thus, hypoxia-mediated oxidative stress is central to the development of HA related disorders in both sojourners and permanent residents of HA. ROS is responsible for impairment of redox homeostasis, damage to lipids, proteins, DNA and different organs, and is also a modulator of SMC proliferation, vascular remodeling and dysfunction. It has proliferative roles and act as a secondary messenger in receptor-mediated signaling pathways mediated by potent vasoconstrictors like endothelin-1 (ET1) and 8-iso-prostaglandin F2 (8-iso-PGF2) at HA. ET-1, a 21-amino acid polypeptide produced by vascular endothelial cells has mitogenic effect on SMCs whereas 8-iso-PGF2, produced by the random oxidation of tissue phospholipids by oxygen radicals, is a potent vasoconstrictor. Therefore, both have vasoconstrictory role in the vasculature. ROS decrease the NO mediated vasodilatory action by combining with NO to generate peroxynitrite, a highly reactive nitrogen species (RNS) that further oxidizes low density lipoprotein (LDL) and supplements peroxidative stress at HA. The differential level of NO, ET-1, 8-iso-PGF2 and partial pressure of oxygen (SaO2) in HA natives compared to healthy sojourners at the same altitude depicts the physiological hypoxia in them (P0.01). The inverse correlation of ET-1 and 8-iso-PGF2 with SaO2 suggests the reduced bioavailability of O2, as the consequence of oxidative overload in HA natives (P0.05). Similarly, the positive interaction of NO levels with SaO2 is suggestive of this molecule trying to maintain stability (P0.05); in addition, involvement of some assorted mechanisms in mediating protective response against oxidative damage is not ruled out. Therefore, the higher level of vasoconstrictors and ROS in HA natives advocate that this population of HA living for thousands of years is continually under stress. The exploration of intricate mechanism behind the interaction of these vasoactive mediators and ROS will not only help in alleviating the physiological stress at HA but will also be relevant in numerous other cardiovascular and respiratory disorders sharing similar pathophysiological features. 2.19

Interkinase communiqué: The P Code Pandey P, Mohammad G, and Pasha Q Institute of Genomics and Integrative Biology, Delhi, Department of Biotechnology, University of Pune, Pune, Department of Medicine, SNM Hospital, Leh, Ladakh, India

Kinases, a set of mechanistically and evolutionarily distinct enzymes, involved in the transfer of high-energy phosphate moieties, have become an important target in the discovery of newer drugs. Numerous cellular processes like cell signaling, cell division and growth, development, differentiation, and cell death are orchestrated by interaction between a series of spatially distributed kinases. Although phosphorylation surges govern these interactions in cellular metabolome, the essential regulatory details of these signal conducing moieties remain inexplicable till date. This cascade under stable homeostatic conditions is tightly regulated but under strenuous settings such as oxidative stress it becomes unregulated culminating in the disruption of the intracellular S21

Abstracts

signaling networks. Oxidative stress at high altitude leads to altered kinase functioning as is evident in pulmonary vasculature leading to pulmonary hypertension–a hallmark of high altitude-related disorders. Tyrosine kinases remain the class of kinases most investigated, closely followed by serine and threonine kinases as targets for therapeutic intervention. Eukaryotic kinases have a conserved reaction mechanism of catalysis which enables them to be drugged by several small molecular weight inhibitors. Studies in pulmonary hypertensive mice models have provided a breakthrough; i.e., inhibition of Rho kinase with its speci ic inhibitor fasudil has depicted signi icant alleviating results. However there also exist interindividual differences toward therapies; hence an approach is needed that can explain about the hyper- and hyporesponsiveness among the individuals. In view of this, several candidate kinases majorly implicated in pulmonary hypertension have been picked up by us for analysis in a case-control design. To start with, 10 polymorphisms of Rho kinase have been studied; 6 out of 10 have given signi icant results (P<0.05). A number of circulatory markers involved in the candidate kinase pathway have also been measured and correlated with different clinical characteristics with signi icant correlations (P<0.05). Activity measurement of selected kinases is in progress. Studies have provided a lead in better understanding the kinases but the interaction between these diverse ranges of phosphorylating or P-moieties remains to be elucidated. 2.20

Causes of pulmonary arterial hypertension in the pediatric population in a tertiary care center in Saudi Arabia Banjar H Department of Pediatrics, King Faisal Specialist Hospital and Research Center, Riyadh, Kingdom of Saudi Arabia

Pulmonary arterial hypertension (PAH) has been frequently described in the pediatric population. Approximately 40% of cases of pulmonary arterial hypertension are idiopathic, and 6% are heritable in nature, with the remaining mainly associated with congenital heart disease and few associated with connective tissue diseases, human immunodeficiency virus (HIV), or portal hypertension. Mortality associated with pulmonary arterial hypertension is extremely elevated. Once diagnosis has been con irmed, mean survival among adults is 2.8 years and less than 1 year among children. My objective was to identify the different causes and outcomes of PAH and treatment modalities in referred cases to the pediatric pulmonary clinic in a tertiary care center in Saudi Arabia. Retrospective chart review of all referred patients to pulmonary clinic with documented PAH based on cardiac catheterization and or Echocardiogram during a 1-year period (March 2009-February 2010). A total of 114 patients with con irmed PAH. Mean age at diagnosis 3.1 up to 3.8 years. Of these patients, 55 (48%) were males and 59 (52%) were females. The most common cause of pulmonary arterial hypertension was found to be congenital heart disease (CHD): CHD was diagnosed in 100 patients (87.7%). The most common congenital heart diseases that caused pulmonary hypertension were: atrial septal defect (ASD) in 76 patients (67%); ventricular septal defect (VSD) in 63 patients (55%); common atrio ventricular canal (Common A-V Canal) in 39 patients (34%); patent ductus arteriosus (PDA) in 16 patients (14%); tetralogy of Fallot (TOF) in 11 patients (10%); and total anomalous pulmonary venous return (TAPVR) in 2 patients (1.7%). Other causes of pulmonary arterial hypertension were congenital anomalies in 100 (87.7%), and Down syndrome in 44 (38.5%); and 39 of 44 patients (88%) with Down syndrome had congenital heart disease. Other congenital anomalies were CHARGE association, and skeletal dysplasia, and 32 patients had an unknown syndrome. Eleven patients (10%) had congenital lung anomalies as diaphragmatic hernia in association with lung hypoplasia, and congenital lobar emphysema; 11 patients (10%) had chronic lung disease (CLD); 2 patients were living in high altitude; 2 patients had obesity; 2 patients had Alagile syndrome; and 2 patients S22

had idiopathic PAH. Obstructive sleep apnea (OSA) was detected in 31 patients (27%), asthma in 33 (30%), and recurrent chest infection in 41 (36%). Thirty-two patients (28%) required O2, and 36 (32%) were found to have gastroesophageal re lux (GER). Factors that related to the development of PAH at presentation were found to be: Common A-V Canal P-value =0.3205; and OSA P-value=0.0266, with a female sex P-value=0.05. Sixty of 114 patients (53%) were started on vasodilators. Sildena il (Revatio) was the most common drug used in 35 patients (30%) alone or in combination with Bosentan (Tracleer) or inhaled Ventavis (Iloprost). Bosentan alone or in combination was used in 21 patients (18%) and inhaled Ventavis alone or in combination in 8 patients (4%). At follow-up, 75 patients (66%) continued to have PAH. The factors that contributed to persistence of PAH at follow-up included the presence CHD P-value=0.05, unclosed ASD P-value=0.03. Pulmonary hypertension is a common disease and should be diagnosed and treated early before it becomes irreversible and resistant to vasodilators. Early closure of ASD and CHD defect repair improves PAH. 2.21

Involvement of immune/inflammatory cells to pathology of idiopathic pulmonary arterial hypertension Savai R, Pullamsetti SS, Kolbe J, Bieniek E, Ghofrani HA, Weissmann N, Fink L, Klepetko W, Voswinckel R, Banat GA, Seeger W, Grimminger F, and Schermuly RT Departments of Lung Development and Remodelling, Max-Planck Institute for Heart and Lung Research, Bad Nauheim, Germany; Departments of Internal Medicine and Pathology, University of Giessen Lung Center, Giessen, Germany; and Departments of Thoracic Surgery, University Hospital Vienna, Vienna, Austria

Pulmonary arterial hypertension (PAH) is a devastating disease characterized by abnormal increased vasoconstriction and vascular remodeling. Recent studies have revealed that both immune and in lammation responses play a crucial role in pathogenesis of idiopathic PAH (IPAH). In the present study, we systematically evaluated the distribution and the numbers of immune/in lammatory cells in different categories of pulmonary arteries (20-50 μm, 51-150 μm and >150 μm) from explanted lungs of IPAH patients (versus donors) by using immunohistochemical and morphometric techniques. Quanti ication of the immune/in lammatory cells showed a signi icant increase of macrophage (CD68+), mast cell (toluidine blue +), dendritic cell (CD209+), T-cell (CD3+) and cytotoxic T-cell (CD8+) count in IPAH lungs as compared to controls. Moreover, there was a signi icant accumulation/in iltration of these cells in smaller (20-50 μm) and medium (51-150 μm) size pulmonary arteries. Analysis of T-regulatory (Treg) cells (FoxP3-positive) showed a signi icant decrease in the lungs; furthermore, an absence or low number of Treg cells was observed in remodeled pulmonary vasculature of IPAH patients. Our indings show alterations in immune/in lammatory cell in iltrations in pulmonary vascular lesions of IPAH patients. Thus, targeting immune/in lammatory cells may be a novel approach to treat IPAH and can be used to monitor the disease process. 2.22

Nonresponse to acute pulmonary vasodilator challenge in idiopathic pulmonary arterial hypertension is associated with nonrecruitment of pulmonary functional capillary endothelial surface area Langleben D, Orfanos SE, Giovinazzo M, Hirsch A, Sotiropoulou Ch, Armaganidis A, and Catravas JD Jewish General Hospital, McGill University, Montreal, Quebec, Canada; Attikon Hospital, University of Athens Medical School, Haïdari Athens, Greece; Medical College of Georgia, Augusta, GA, USA Pulmonary Circulation | October-December 2011 | Vol 1 | No 4

Abstracts

Acute pulmonary vasodilator challenge is used in patients with idiopathic pulmonary arterial hypertension (IPAH) as a method of predicting responsiveness to high-dose calcium channel blockers. Based on current criteria, most patients are characterized as nonresponders (i.e., they fail to lower their mean pulmonary arterial pressure by at least 10 mmHg to less than 40 mmHg, under unchanged or higher cardiac output (CO). Many nonresponders depict CO increases resulting in a decreased calculated pulmonary vascular resistance (PVR). It is not known if an acute vasodilator challenge is able to recruit underperfused or nonperfused pulmonary microvessels, and whether the increased blood low seen in nonresponders represents recruitment of microvasculature. It is also unclear how the change in PVR relates to recruitment. Eleven patients with IPAH underwent cardiac catheterization as part of their diagnostic workup. Standard hemodynamic variables were measured before and after an acute vasodilator challenge with intravenous epoprostenol. All subjects were characterized as nonresponders. Applying indicator-dilution type techniques twice, prior to the challenge and at peak epoprostenol dose, we measured irst pass transpulmonary percent metabolism (%M) and hydrolysis (v) of the synthetic and highly speci ic substrate 3H-benzoyl-Phe-Ala-Pro (BPAP) by the pulmonary capillary endothelium-bound angiotensin converting ectoenzyme (ACE), under irst-order reaction conditions. We also measured functional capillary surface area (FCSA), previously termed Amax/Km, normalized to body surface area (BSA). Data are given as mean±SD. With vasodilator challenge, mPAP did not change signi icantly (62±13 mmHg pre versus 60±12 post). CO increased (3.26±1.50 L/min pre versus 4.32±1.88 post, P<0.001). PVR decreased from 18.8±10.5 Wood units pre to 13.1±5.3 post (P=0.04). BPAP % metabolism was reduced from 64.1±13.4% pre, to 55.4±14.2% post (P<0.001), and BPAP hydrolysis (v) decreased from 1.10±0.43 pre to 0.87±0.41 post (P<0.001). However, FCSA/BSA did not change signi icantly (1201±555 mL/min/m2 versus 1246±555). There was a negative linear relationship between percent change in FCSA/BSA and absolute or percent change in PVR (r=-0.754, P<0.01 and r=-0.597, P=0.052, respectively). The acute vasodilator challenge in our IPAH cohort of non-responders failed to recruit FCSA, despite the observed increase in pulmonary blood low and the drop in calculated PVR. This is likely because the degree of upstream structural arteriolar remodeling in these patients was advanced. The absence of FCSA changes along with the observed fall in %M and v post acute challenge suggest that the higher pulmonary blood low passes the lung via already perfused microvessels with a quicker transit time (i.e., enzyme-substrate reaction time) and probably causes some vessel distension, resulting in lower substrate utilization. This study provides a mechanistic explanation for the poor vasodilator response in these patients, despite a fall in calculated PVR. 2.23

Utility of the brain natriuretic peptide as a marker in the diagnosis of patients with persistent pulmonary hypertension of the newborn Díaz G, Ruiz AI, Acherman R, Montealegre A, Ome L, and Marquez A Universidad Nacional de Colombia, Instituto Materno Infantil, Bogotá, Colombia; Children´s Heart Center Las Vegas, Nevada; University of Nevada Las Vegas, NV, USA

The brain natriuretic peptide (BNP) is a hormone secreted in excess by the ventricles in situations of pressure or volume overloud. Our objective was to analyze the utility of BNP as a marker of persistent pulmonary hypertension of the newborn (PPHN). We studied 52 neonates with PPHN diagnosed by clinical indings and echocardiogram. The diagnostic criteria were a pulmonary systolic pressure over 80% of the systemic pressure measured simultaneously, with clinical indings of pulmonary hypertension. Patients with hypotension or congenital heart disease excepting ductus arteriosus were excluded. Nine control neonates were included for comparison. A blood sample for BNP was taken after the echocardiogram. The sample was collected in tubes with EDTA and Pulmonary Circulation | October-December 2011 | Vol 1 | No 4

centrifuged. BNP was determined by immunoradiometric assay using the Triage Meter Plus device (Biosite). The laboratory technician was masked about the clinical situation and the results of the echocardiograms. The statistic analysis was made with the STATA 8.0 software using descriptive statistics, Shapiro-Wilk test for normality, and Kruskal Wallis test for comparison. All patients were treated with Sildena il 2 mg/kg/day. The median of the pretreatment pulmonary systolic pressure (PSP) was 56 mmHg (percentile 25: 52.5 mmHg; percentile 75: 62.5 mmHg; range: 48-91 mmHg). The median of the post-treatment PSP was 25 mmHg (percentile 25: 24 mmHg; percentile 75: 33 mmHg; range: 16-60 mmHg) p: 0.0000 pre versus post-treatment. The median of the basal BNP was 408 pg/ml; (percentile 25: 139 mmHg; percentile 75: 3670 pg/ml; range 5-5501). The median of the BNP post-treatment was 72 pg/ml; (percentile 25: 10.65 pg/ml; percentile 75: 277 pg/ml; range 5-2240). P: 0.012 pre versus post-treatment. In the control group the median of BNP was<5 pg/mL. BNP is a good marker in the diagnosis and follow-up of patients with persistent pulmonary hypertension of the newborn. 2.24

Sildenafil plasma concentrations in two HIV patients with pulmonary arterial hypertension treated with ritonavir-boosted protease inhibitors Chinello P, Cicalini S, Pichini S, Pacifici R, Tempestilli M, and Petrosillo N Second Infectious Diseases Unit and Clinical Biochemistry and Pharmacology Laboratory, “L. Spallanzani” National Institute for Infectious Diseases, Rome, Italy; Department of Therapeutic Research and Medicines Evaluation, Drug Abuse and Doping Unit, Instituto Superiore di Sanità, Rome, Italy

Sildena il is increasingly used for the treatment of pulmonary arterial hypertension (PAH) in HIV-infected patients. However, concerns exist about pharmacokinetic interactions between sildena il and protease inhibitors (PI); in particular, ritonavir has been shown to increase sildena il AUC and Cmax by several fold. Data on the pharmacokinetic of sildena il in HIV-PAH patients treated with antiretroviral therapy (ART) including ritonavir are scant. The aim of our study was to determine the plasma levels of sildena il and PI in two HIV patients with PAH treated with ART including ritonavir-boosted PI. Plasma concentrations of sildena il and N-desmethylsildena il were evaluated before the intake of the 20 mg sildena il dose (T0) and 2 h thereafter (T2h), twice in the same day. Heparinized venous blood samples were collected in 7.5mL tubes and were stored after centrifugation at -80°C until analysis. Sildena il and desmethylsildena il were measured in plasma by a liquid chromatography tandem mass spectrometry published methodology, in-house revalidated for the inclusion of N-desmethylsildena il. Plasma concentrations of lopinavir, amprenavir, and ritonavir were assessed before the intake of the daily PI dose (T0) and 1, 2, 4, and 6 hours thereafter (T1h, T2h, T4h, and T6h). PI concentrations were measured in plasma by a validated high-performance liquid chromatography method. Two hours after the morning assumption of sildena il, Patient 1 had a sildena il Cmax of 655.2 ng/mL, and Patient 2 had a sildena il Cmax of 515.4 ng/mL. For Patient 1, the Cthrough of ritonavir and lopinavir were <39 ng/mL and <117.2 ng/mL, respectively; the Cmax of ritonavir and lopinavir were 184 ng/mL and 6800 ng/mL, respectively. For Patient 2 the Cthrough of ritonavir and amprenavir were <39 ng/mL and 2007 ng/mL, respectively; the Cmax of ritonavir and amprenavir were 147 ng/mL and 3770 ng/mL, respectively. According to the European Medicines Agency review of Revatio, a sildena il plasma concentration between 10 and 100 ng/mL is associated with a signi icant effect on pulmonary artery pressure (PAP) and pulmonary vascular resistance (PVR); maximal reductions in PAP and PVR are obtained at plasma concentrations in the range of 100 ng/mL. Both of our patients experienced Cmax above 500 ng/mL; however, they did not report any adverse reactions to sildena il, such as headache, lushing, dyspepsia or priapism; there was no signi icant in luence on systemic blood S23

Abstracts

pressure, which was measured regularly during the investigations. After a 1-year follow-up, the patients are in satisfying general conditions, without signi icant sildena il-related adverse events. In conclusion, in our patients the coadministration of sildena il and ritonavir-boosted PI resulted in an increase of sildena il plasma concentration above the therapeutic range; however, this increase did not cause signi icant adverse events in the follow-up period. Therapeutic drug monitoring of sildena il should be taken in consideration during therapy in order to avoid over-dosage in patients concomitantly treated with ritonavirboosted PI. 2.25

Estimation of pulmonary artery pressure in patients with sickle cell anemia in Ibadan, Nigeria: An echocardiographic study Enakpene EO, Adebiyi A, Ogah OS, Olaniyi JA, Aje A, Adebayo AK, Ojji DB, Adeoye MA, Ochulor KC, Oladapo OO, and Falase AO Departments of Medicine and Haematology, University College Hospital, Ibadan, Nigeria

Medical advances in the management of patients with sickle cell disease have led to a signi icant increase in life expectancy. Pulmonary hypertension is emerging as one of the causes of morbidity and mortality in adults with sickle cell disease. About 2% of adult Nigerians have sickle cell anemia. The prevalence of pulmonary hypertension in Nigerian adults with sickle cell anemia is unknown. Our objective was to estimate the pulmonary artery systolic and diastolic pressures in subjects with sickle cell anemia seen at the University College Hospital, and to determine the frequency of pulmonary hypertension among them. Ninety patients (38 males and 52 females) with sickle cell anemia in steady state and comparable age and sex-matched normal controls had clinical evaluation and echocardiographic examination. They all had Doppler echocardiographic assessment of pulmonary artery pressures. The mean age of SCA subjects was 26.10±7.989 years while the mean age for the control group was 25.55±7.077 years. The sickle cell anemia subjects had lower diastolic blood pressure as well as body mass index. There was no difference in the LV systolic indices between the two groups. The SCA subjects had larger cardiac volumes compared with the control group. There were higher pulmonary systolic and diastolic pressures among SCA subjects. The frequency of pulmonary hypertension as assessed by a tricuspid regurgitant jet velocity of >2.5 m/s in this study was 12.2%. The patients with pulmonary hypertension in this study had mild pulmonary hypertension as evidenced by the range of tricuspid regurgitant jet velocity of <2.9 m/s. However, two of the subjects had tricuspid regurgitant jet above 2.9 m/s. The values in these SCA subjects were 2.97 m/s and 3.2 m/s, respectively. Larger left ventricular dimensions and volumes, higher stroke volume, and increased left ventricular mass indexed by body surface area, were found to be associated with pulmonary hypertension. A multivariate analysis of the potential predictors of pulmonary hypertension in this study showed that the male sex and lower PCV were independent predictors of pulmonary hypertension in SCA patients. This study has shown that the pulmonary artery systolic and diastolic pressures are higher in SCA subjects than normal controls. Higher age, male sex and low PCV have been shown in this study to be independent determinants of pulmonary arterial pressure in subjects with SCA in Nigeria. 2.26

Epidemiology of pulmonary heart disease in Nigeria: Insight from the Abeokuta Heart Failure Registry Ogah OS, Falase AO, Stewart S, and Sliwa K Department of Medicine, University College Hospital, Ibadan, Nigeria; Preventative Health Baker IDI Heart and Diabetes Institute Melbourne, S24

Australia; Hatter Cardiovascular Research Institute, Faculty of Health Sciences, University of Cape Town, Cape Town, South Africa

Recent data shows that pulmonary heart disease, right heart failure or corpulmonale is becoming an important issue worldwide. This is because the disease is now more frequently diagnosed and attention is now more than ever being focused on the study of right heart function. The true epidemiology of this is disease is unknown. In the US, for example, “among 807,000 patients hospitalized with pulmonary hypertension as one of the diagnoses between 2000 and 2002, 61% were women and 24% were younger than age 65.” The paucity of data is even worse in developing countries. We used the data from the Abeokuta Heart Failure Registry to describe the epidemiology of the disease in Nigeria. The Abeokuta Heart Failure Registry was used to de ine and characterize HF in this southern city of Nigeria. It was chosen to provide a large database that contains patients’ characteristics, etiology, and modes of care, as well as outcome of patients admitted for HF. The study was conducted at the Federal Medical Centre and at the sacred Heart Hospitals in Abeokuta. Eligible subjects were those with new onset HF of decompensated chronic established HF. The Framingham criteria were used for diagnosis of HF and all cases were con irmed by echocardiography. A standardized profoma was used to collect information on demographics, medical history, symptoms, signs, investigations as well as medication and outcome of patients. Diagnosis of pulmonary hypertension was based on standard criteria. During the period of study (2006-09) a total of 41 subjects were admitted with diagnosis of right heart failure or corpulmonale. There were 23 men and 18 women, constituting 56.1% and 43.9%, respectively. The mean age was 63.3±16.3 years (range 23-90 years). The identi ied etiology of corpulmonale from this registry were: COPD/COAD (26,63.4%); sickle cell disease (4,9.8%); pulmonary tuberculosis (2,4.9%); connective tissue disease (2,4.9%); and primary pulmonary hypertension (2,4.9%). Others included adult congenital heart disease (2,4.9%), HIV-associated pulmonary hypertension (2,4.9%), and chest wall deformity (1,2.4%). This registry result shows that chronic obstructive pulmonary disease is the commonest cause of corpulmonale. Readmission rate was high with a 6-month mortality of 12.2%. 2.27

Pulmonary hypertension associated with sickle cell anemia: A review of epidemiology, pathophysiology and management Ogah OS, Falase AO, Stewart S, and Sliwa K Department of Medicine, University College Hospital, Ibadan, Nigeria; Preventative Health Baker IDI Heart and Diabetes Institute, Melbourne, Australia; Hatter Cardiovascular Research Institute, University of Cape Town, Cape Town, South Africa

About 20-25 million people live with sickle cell anemia (homozygous SCD) worldwide, with 12-15 million patients in sub-Saharan Africa. In Nigeria alone, more than 6 million people are affected by the disease. With improvement in the life expectancy in people living with sickle cell anemia, end organ complications associated with this disease, such as pulmonary hypertension, are now being frequently encountered. The purpose of the paper is to review the epidemiology and pathophysiology of pulmonary hypertension in sickle cell anemia. A Medline Search of relevant literature on the topic from 1980 to 2010 was carried out. The true incidence, prevalence and burden of pulmonary hypertension associated with sickle cell anemia are unknown, especially in environments where this disease is rampant. However, available data show that 30% of patients have pulmonary hypertension with mortality rates of 40% at 40 months. It has been shown that chronic hemolysis associated with the disease results in nitric oxide scavenging, endothelial dysfunction, and down-regulation of endothelial adhesion molecules, as well as inhibition of platelet activation. People living with sickle cell anemia are at increased risk of developing pulmonary hypertension. This Pulmonary Circulation | October-December 2011 | Vol 1 | No 4

Abstracts

complication is common and most often silent, under-recognized, and associated with increased mortality. There is need for a global registry of pulmonary hypertension in sickle cell anemia. 2.28

Markers of left and right ventricular remodeling in a native African hypertensive cohort Ojji D, Lacerda L, Lecour S, Adeyemi Billyrose M, and Sliwa K Departments of Medicine and Medical Laboratory Sciences, University of Abuja Teaching Hospital, Gwagwalada, Abuja; and Hatter Institute for Cardiovascular Research in Africa, University of Cape Town Medical School Campus, Cape Town, South Africa

Although hypertension affects every ethnic group, the consequences are said to be more devastating among black patients. One of the main manifestations of hypertension’s end-organ effects, especially in black patients, is hypertensive heart disease which includes left ventricular hypertrophy (LVH), increasing vascular and ventricular stiffness and diastolic dysfunction which ultimately lead to heart failure (HF) if not adequately treated. Although brain natriuretic peptide is recognized as an indicator of the presence and severity of heart failure including hypertensive (HF) in native Africans, the diagnostic value in differentiating hypertensive LVH without HF from hypertensive HF due to systolic and/or diastolic dysfunction is unclear. Our objective necessitated a cohort study in 250 Nigerian patients to study the role of novel biomarkers as well as natriuretic peptides in this cohort. We initiated a prospective cohort study. Echocardiography was performed on all subjects. Measurements taken include left ventricular dimensions and transmitral pulse wave Doppler low. LVH was considered present when left ventricular mass/height 2.7 exceeded 46.7 gm/m 2.7 in women and 49.2 gm/m 2.7 in men. Right ventricular (RV) systolic function was assessed on echocardiography using tricuspid annular plane systolic excursion (TAPSE) method. Plasma NT-pro-BNP was measured using electrochemiluminescence type immunoassay. Preliminary data on a subgroup of 88 patients studied will be presented: 60.2% were male, 39.8% female, and the mean age was 52.0 (10.7) years. There was no signi icant difference in the NT pro BNP levels between hypertensive subjects with LVH and those without. There is, however, a signi icant difference when the NT-pro-BNP levels of hypertensive subjects with LVH but without HF was compared with those with hypertensive HF (430.80 fmol/ml versus 582.39 fmol/ml, P=0.004). In addition, subjects with hypertensive HF have signi icantly worse RV systolic function compared to hypertensive subjects with LVH but without HF, with TAPSE values (23.4 mm versus 16.6 mm, P=0.000). NT-pro-BNP may be a useful biochemical marker in differentiating between hypertensive heart failure and hypertensive left ventricular wall hypertrophy without heart failure in the native African population. The contribution of RV function to progression of disease needs to be further investigated. 2.29

Serotonin passes through myoendothelial gap junctions to promote pulmonary arterial smooth muscle cell differentiation Gairhe S, Gebb SA, and McMurty IF Departments of Pharmacology, Cell Biology and Neuroscience, and Medicine, College of Medicine, University of South Alabama, Mobile, AL, USA

Myoendothelial gap junctional signaling mediates pulmonary arterial endothelial cell (PAEC)-induced activation of latent TGF-β and differentiation of co-cultured pulmonary arterial smooth muscle cells (PASMCs), but the nature of the signal passing from PAECs to PASMCs through the gap junctions is unknown. Because PAECs but not PASMCs synthesize serotonin, and serotonin can pass through gap junctions, we hypothesized that the monoamine is the intercellular signal. Our objective Pulmonary Circulation | October-December 2011 | Vol 1 | No 4

was to determine if PAEC-derived serotonin mediates myoendothelial gap junction-dependent PAEC-induced differentiation of PASMCs. Rat PAECs and PASMCs were monocultured or cocultured with (touch) or without (no-touch) direct cell-cell contact. In all cases, tryptophan hydroxylase 1 (Tph1) transcripts were expressed predominantly in PAECs. Serotonin was detected by immunostaining in both PAECs and PASMCs in PAEC:PASMC touch coculture, but was not found in PASMCs in PAEC:PASMC no-touch coculture or in PASMC:PASMC touch coculture. Furthermore, blockade of gap junctions in PAEC:PASMC touch coculture inhibited serotonin transfer from PAECs to PASMCs. Inhibition of serotonin synthesis pharmacologically or by small interfering RNAs to Tph1 in the PAECs inhibited the PAEC-induced activation of TGF-β signaling and differentiation of PASMCs. The PAEC-derived serotonin in touch cocultured PASMCs was partly colocalized with smooth muscle α-actin. Serotonin synthesized by PAECs is transferred through myoendothelial gap junctions to PASMCs, where it activates TGF-β signaling and induces differentiation. This inding suggests a novel role of intercellular serotonin signaling in PASMC differentiation, and alteration of this signaling pathway may contribute to vascular remodeling in pulmonary hypertension. 2.30

Doppler echocardiographic assessment of pulmonary artery pressure in apparently healthy Nigerian primary school children in Ibadan (Western Nigeria) Udo PA, Orimadegun AE, Omokhodion FO Department of Pediatrics, University of Uyo Teaching Hospital Institute of Child Health, University of Ibadan; College of Medicine, University of Ibadan

Pulmonary hypertension is quite prevalent globally. The existing reference values for pulmonary artery systolic pressure were determined through studies in Caucasians. There is need to examine the values in Nigerian children and to encourage regular pulmonary artery pressure monitoring in the developing world. The objective of this study was to use Doppler echocardiography to assess the pulmonary artery pressure values in apparently healthy Nigerian children (in Ibadan) aged 5-12 years. Our design was a cross-sectional study. A multi-stage sampling method was used to recruit 223 apparently healthy primary school children (96 males, 127 females) aged 5-12 years from randomly selected private and public primary schools in the Ibadan North local government area of Oyo state. The pulmonary artery systolic pressure (PASP) of those that had tricuspid regurgitation was measured using echocardiography. The mean PASP (±1SD) of all the study subjects was 22.17 (5.7) mmHg with a 95% con idence interval of 21.4 – 22.9. There was no signi icant difference in the values of the PASP within each socioeconomic class and among all the classes (F= 0.252, df= 4, P=0.908). There was no signi icant difference in the mean PASP of the subjects within each age and among all ages (F=1.640, df=7, P=0.126). There was no correlation between the mean PASP and age, weight, height, BMI, BSA in this study. There was also no statistically signi icant difference between the PASP of males and that of females in this study. Among the 223 apparently healthy primary school children (aged 5-12 years) in this study, the mean pulmonary artery systolic pressure (PASP) was found to be 22.17 (5.7) mmHg with a 95% con idence interval of 21.4–22.9. The values of the PASP revealed no statistically signi icant gender difference nor any correlation with age, weight, height, BMI or BSA. So PASP could not be predicted by any of these variables. 2.31

Adverse events of pulmonary hypertension pharmacotherapy in children Roldan T and Cerro MJ Department of Pharmacology, Pediatric Pulmonary Hypertension Unit, “La Paz” Children´s Hospital, Madrid, Spain S25

Abstracts

There is limited information about adverse events of pulmonary hypertension drug therapy in the pediatric population. Our objective is to evaluate and characterize the frequency and severity of adverse events of pharmacological treatment of pulmonary hypertension (PH) in pediatric patients. The secondary objective is to identify possible risk factors for these adverse events, through an observational, retrospective, longitudinal, descriptive, single-center case series. We reviewed the medical records of pediatric patients receiving speci ic treatment for pulmonary hypertension (sildena il, bosentan, iloprost, treprostinil) in our pediatric PH clinic and collected variables related to patient (gender, age, diagnosis, and WHO functional class), drug therapy variables (drug or drugs, dose, regimen and duration), and variables related to possible adverse events detected (description of the adverse effect, severity, and if it had caused modi ication or withdrawal of the dose). The data were processed for statistical analysis in SPSS version 11.5. We included 63 pediatric patients, 32 male (50.8%) and 31 female (49.2%). Median age at onset of treatment was 3.4 years (0.1-18). The most common etiology was congenital heart disease with 33 cases (52.4%), followed by lung disease in 8 (12.7%). Most of the patients (76%) were in an advanced functional class (III or IV) at the initiation of therapy. The drug most commonly used in monotherapy was sildena il, (56%), followed by bosentan (23%), and the most common combination therapy was sildena il + bosentan. More than half of the patients (34/63, 53.97%) suffered some kind of adverse events. We recorded 90 episodes with 25 different side effects. The most frequent were gastrointestinal disorders in 14 patients (22.2%) and erections in 7 boys (21.9%), followed by headaches (11.1%), respiratory complications of inhaled iloprost (11.1%), hemorrhagic diathesis (9.5%), and lushing (9.5%). Regarding the severity of adverse events detected, 46.7% (n=42) were mild, 43% (n=39) moderate, and 10% (n=9), severe. In eight cases, the treatment was suspended, and one patient needed hospital admission due to the adverse effect. In most situations (64.8%; n=59), the physician’s decision was to continue treatment, and in 19.8% (n=18) to reduce the drug dose. Five adverse events needed pharmacological treatment: ranitidine, paracetamol and salbutamol. The presence of combined treatment was the only variable related to increased number of adverse effects that reached statistical signi icance (P<0.05). We observed an increased number of side effects with increasing age and more advanced functional class, but with P>0.05. Age also in luenced the type of adverse reactions observed: cefaleas were more common in older children (13.3% to 24% in children 2-8 years, and 24% in children 8-18 years, P<0.05). On the contrary, the incidence of gastrointestinal disorders decreased with increasing age (31.0% for children under 2 years, 33.3% in age group 2-8 years, 10% in children older than 8 years). Although more than half of patients had suffered some adverse reaction, treatment with speci ic drugs for PH in the pediatric population was relatively safe, showing a low rate of serious adverse events, both in monotherapy and in combination that did not need modi ication of the prescribed dose in most cases. Combined treatment was associated with a higher rate of adverse effects. The age of the patients in luenced the type of observed adverse events: headaches were more common in older children and gastrointestinal disorders in the younger ones. 2.32

Long-term endothelin receptor antagonist therapy may predispose to carcinogenesis in patients with pulmonary arterial hypertension Safdar Z and Qureshi H

Hypertension Centre from 01/2005 to 12/2010 that identi ied ive patients who developed a malignancy while on ETRA therapy. During this period approximately 280 PAH patients were treated with an ETRA at this center. Demographic data, NYHA/WHO functional class, six-minute walk distance (6-MWD), type of cancer, echocardiogram, and survival data were collected. The mean age of the ive PAH patients was 55.4±8.6 years (Mean±SD). All were female and duration of PAH was 6.86±1.9 years. Mean duration of ETRA therapy was 4.6±0.89 years. At the time of cancer diagnosis, BNP levels were 117±114 pg/ ml, 6-MWD was 398±67 meters, right ventricular systolic pressure was 83±21 mmHg, RAP was 11±2 mmHg and cardiac index was 2.4±0.3 L/ min/m2. Follow-up duration after cancer diagnosis was 0.74±1.6 years. One patient developed bronchoalveolar lung cancer, two patients had in iltrating ductal cancer, one had malignant melanoma, and one had multiple myeloma. One patient had died while the other four patients were alive at the last follow-up. Carcinogenetic potential of ETRA should be considered in PAH patients who are on long-term ETRA therapy. Other unidenti ied factors may also predispose to carcinogenesis. Further studies are needed to con irm these indings. 2.33

Noninvasive evaluation of pulmonary hypertension and its correlates among adult patients with sickle cell disease Mbakwem AC and Kehinde MO Department of Medicine, University of Lagos, Lagos, Nigeria

Pulmonary hypertension is a relatively common complication in adult patients with sickle cell disease (SCD). Chronic hemolysis and asplenia have been identi ied as the pathological link between hemolytic anemia and pulmonary hypertension. There is paucity of data on the prevalence of pulmonary hypertension in SCD in our environment. We, therefore, evaluated the prevalence of pulmonary hypertension and its correlates in stable adult patients with SCD attending the sickle cell clinic in our institution. Participants were recruited from the adult sickle cell clinics of the Lagos University Teaching Hospital and were eligible if they were 18 years or older of age, had electrophoretic pattern of hemoglobin SS and were in steady state. Clinical data of the subjects were obtained. A transthoracic echo was used to for cardiac evaluation and the peak regurgitant jet velocity was used to estimate the right ventricular pressure gradient making use of the Bernoulli equation. Pulmonary systolic artery pressure was computed by adding the estimated right atrial pressure. Data for 48 subjects (20 males and 28 females) so far evaluated is presented. The mean age of the subjects was 25.85±9.61 years and this was similar for both males and females, P=0.18. The mean pulmonary artery pressure was 13.88±8.74 mmHg and the males had a higher mean pulmonary artery pressure than the females though this was not signi icant, 16.18±10.10 versus 12.56±7.57 mmHg, P=0.20. Pulmonary hypertension was de ined as pulmonary artery pressure ≥25 mmHg, which was seen in seven (14.58%) of the subjects (four males and three females). Pearson’s correlation was positive between the pulmonary artery pressure and age, r=0.290 P=0.05; ECG LVH by Cornell’s criteria, r=0.305 P=0.04 and a trend with amount of blood transfusion, r=0.364, P=0.052. Pulmonary hypertension does exist in SCD patients in Nigeria with no apparent difference in gender distribution. There seems to be a relationship with age and the amount of blood transfusion patients require.

Pulmonary-Critical Medicine, Baylor College of Medicine, Houston, TX, USA

2.34

Endothelin receptor antagonists (ETRAs) are commonly used to treat pulmonary arterial hypertension (PAH). Previous data suggested a carcinogenetic potential of ETRA in animals that were treated for an extended period with high dose ETRA. However, data on carcinogenic potential of long-term ETRA therapy has not been reported. We report our novel inding in ive PAH patients who developed a malignancy while on ETRA therapy. Data review was conducted at the Baylor Pulmonary

Wonkam A

S26

Pulmonary hypertension among patients with sickle cell disease in Africa Faculty of Medicine and Biomedical Sciences, University of Yaoundé, Yaoundé Cameroon; Division of Human Genetics, University of Cape Town, Cape Town, South Africa Pulmonary Circulation | October-December 2011 | Vol 1 | No 4

Abstracts

In sub-Saharan Africa (SSA), sickle cell disease (SCD) occurs at its highest frequency: 5-40% sickle cell trait and up to 300,000 affected babies born each year. Patients who are homozygous for the sickle hemoglobin mutation can present with remarkably different clinical courses, varying from death in childhood to being relatively well even until old age. Abnormal transcranial Doppler velocity and pulmonary hypertension are, respectively, proxy of stroke episodes risk and cause of death of SCD patients. The etiology of pulmonary hypertension is multifactorial, including hemolysis, chronic hypoxemia, throboembolism, prenchymal and vascular injury because of sequestration of sickle enrythrocytes. Studies mostly in the US suggest a prevalence of pulmonary hypertension ranging from 20 to 40% in SCD. There is very little description of these phenotype in African SCD patients living on the African continent, where two-third of the patients live. Studies and programs that could lead to the detection and treatment that may reduce regurgitant jet velocity and potentially reverse the disease process are needed to prevent the increased morbidity and mortality associated with pulmonary hypertension in SCA. 2.35

Pulmonary hypertension and pulmonary vascular remodeling in mouse models of schistosomiasis Crosby A Department of Medicine, University of Cambridge School of Clinical Medicine, Addenbrooke's Hospital, Cambridge, UK

Schistosomiasis is the most common worldwide cause of pulmonary arterial hypertension (PAH) and is particularly prevalent in the developing world. Praziquantel is the drug of choice and has been shown to reverse the liver pathology associated with Schistosoma mansoni in mice. More than 80% of patients with PAH in the Western world have a mutation in bone morphogenetic protein type-II receptor (BMPR-II), which is a member of the transforming growth receptor-beta (TGF-b) superfamily and is important in cell proliferation and differentiation. We sought to determine whether praziquantel could reverse established pulmonary vascular remodeling and pulmonary hypertension in a mouse model of S. mansoni and to determine if mice with a heterozygous null mutation in BMPR-II were more susceptible to schistosomiasis– induced PAH, compared to wild-type littermates. C57/BL6 mice were infected percutaneously with a low dose of S. mansoni. At 17 weeks postinfection mice were either sacri iced or received praziquantel by oral gavage or a vehicle control. Right ventricular systolic pressure (RVSP) and right ventricular (RV) hypertrophy, liver and lung egg counts were measured at speci ied time points. Pulmonary vascular remodeling was assessed by morphometry, following immunohistochemistry. A cytokine array was performed and the degree of infectivity was measured by fecal egg counts. In a separate cohort of animals wild-type C57/BL6 mice and mice with a heterozygous null mutation in BMPR-II (mutant mice) mice were infected percutaneously with a low dose of S. mansoni and measurements (as above) were performed 17 weeks post-infection. In addition, human and mouse pulmonary arterial smooth muscle cells (PASMC) were cultured with S. mansoni eggs for 24 hours. At 24 hours the expressions of cytokines in PASMC were measured by qPCR and cytokine levels in the cell supernatant were measured by ELISA. At 25 weeks post-infection there was a signi icant increase in RVSP and RV hypertrophy between infected and control mice, which was reversed by praziquantel treatment. RVSP was elevated in mice at 25 weeks post-infection but had normalized with praziquantel treatment. There was a signi icant increase in the muscularisation of small pulmonary arteries following 25 weeks of schistosomal infection, which was prevented by with praziquantel treatment. Liver, lung and fecal egg counts were elevated following 25 weeks of schistosomal infection and substantially reduced with praziquantel treatment. At 17 weeks postinfection there was no signi icant difference in RVSP, the degree of RV hypertrophy, liver weight or body weight between wild-type or BMPRII mutant mice. However, 33% of the mutant mice died prematurely. Pulmonary Circulation | October-December 2011 | Vol 1 | No 4

After 24 hours co-culture with eggs both mouse and human PASMC showed an increase in cytokine expression and cytokine release. More speci ically we saw an increase in IL-6, Kc (mouse homologue of IL-8) and IL-13 expression and an increase in IL-6 and Kc secretion. We also saw an increase in PASMC proliferation, determined by Ki67. There was a suggestion that PASMC from mutant mice may display an increase in cytokine response to egg stimulation. These studies have shown that severe pulmonary vascular remodeling accompanied by an increase in RVSP and RV hypertrophy occurred 25 weeks post-infection in a mouse model of S. mansoni infection. Importantly, this study has shown that progression of disease can be prevented by two oral doses of praziquantel. The mechanism thought to underlie the dramatic pulmonary vascular remodeling is a local increase in in lammatory cytokines. In addition we have shown that a heterozygous null mutation in BMPR-II does not predispose to schistosomiasis-induced PAH. We have also shown that PASMC respond to S. mansoni eggs by an increase in gene expression and release of in lammatory cytokines. These may play a part in inducing pulmonary vascular remodeling by stimulating PASMC proliferation. However, this affect was not signi icantly enhanced by BMPR-II mutations. 2.36

Subcellular mechanisms in IPAH: Coordinate dysfunctions of the Golgi-ER-mitochondrial axis Sehgal PB Departments of Cell Biology & Anatomy, and Medicine, New York Medical College, New York, New York USA

In 1977 Smith and Heath reported observing using electron microscopy (EM), the marked cystic dilatation of the endoplasmic reticulum (ER) in PAECs in hypoxic rats with PAH. In 1979, Smith and Heath reported observing, also using EM methods, dilatation of the ER cisternae in cells in vascular lesions of PAH in man. These seminal observations on the subcellular mechanisms in the pathogenesis of PAH have gone largely unnoticed until now. In recent years, we have reported Golgi apparatus enlargement and fragmentation, increased cytoplasmic dispersal of the Golgi tether giantin, and defects in intracellular traf icking leading to global changes in the cell surface landscape of pulmonary vascular cells within PAH lesions. Increased cellular levels of an ER structural protein RTN4/Nogo-B have been reported in endothelial and smooth muscle cells in pulmonary vascular lesions in idiopathic PAH as well as the inability of RTN4-/- mice to develop PAH in response to chronic hypoxia. Other investigators have implicated mitochondrial dysfunction in the pathogenesis of IPAH. In observations that unite data concerning Golgi, ER and mitochondrial dysfunctions into a coordinated pathophysiology in this disease, we demonstrated that knockdown of a particular estrogenand progesterone-responsive STAT protein species in the cytoplasm of HPAECs and HPASMCs leads to Golgi enlargement and fragmentation, cystic dilatation of ER, reduced intracellular traf icking in synergy with knockdown of BMPRII (equivalent to BMPRII haploinsuf iciency), and increased mitochondrial fragmentation with reduced TMRE uptake—all changes that we and others have shown to occur in IPAH. Overexpression of an ER-associated GTPase blocks these changes. The data, in principle, provide new insights into potential second-hit, low-penetrance and sexual dimorphism effects in PAH and suggest new avenues of gene therapy aimed at correcting the subcellular defects irst observed in PAH by Smith and Heath in 1977-1979. 2.37

The behavior of the right ventricular dysfunction by echocardiography and clinical transthoracic Murillo C Unidad Estratégica de Bioprospección, Instituto Nacional de Biodiversidad Santo Domingo de Heredia, Costa Rica S27

Abstracts

The purpose of this study was to describe the behavior of the right ventricular dysfunction by echocardiography and clinical transthoracic (ECTT). It is a non-invasive procedure with high availability, and is rapid, sensitive and reliable. Thromboembolic pulmonary hypertension disease (CTEPH) is a clinical entity characterized by an increase in pulmonary arterial pressure secondary to the presence of thrombi intraluminal organized ibrous stenosis and even obliteration of the pulmonary arteries, which result in the elevation of the pulmonary vascular resistance and changes in the right heart, by pressure overload. CTEPH was long considered a very rare entity. Only 0.5-1% of the EP performed the event, but now its incidence has increased to 3.8%. We studied 10 CTEPH patients diagnosed with admission to the service Cardioneumologia in the months March to December 2009. The diagnosis was established according to the ACC and AHA consensus with the following studies: (V/Q scan), computed tomographic pulmonary disease, and prior ECTT PSAP with a report greater than 40 mmHg. All of them were narrowing the demographic variables, risk factors, 6-minute walk, 12-lead ECG Doppler low shunts and RV ECTT. The variables were obtained from subjects in stable condition. Here, “stable condition” was de ined as at least 1 month without manifestations. We studied 10 patients, 6 men and 4 women, with an average age of 45-51 years. It was observed that most patients were overweight (BMI 25.1 to 29.9 kg/m2). Risk factors related to venous thromboembolic disease were smoking, exposure to wood smoke, and antiphospholipid antibody syndrome (SAF). The only acquired and inherited thrombophilias reported were SAF. During the 6-minutes walk test the clinical manifestations were small (only dyspnea), but the impact on physiological variables was obvious. For the most part the results of laboratory tests were within normal parameters. However, the rise in the numbers of the distribution width erythrocyte (ADE), uric acid, the pro-natriuretic peptide N-terminal (NT pro-BNP), the total bilirubin and times coagulation was interesting, as anticoagulant therapy found seven patients with coumarin, and two patients with unfractionated heparin. The glucose was found in the upper normal limit, while the ratio of indirect and total bilirubin was found decreased. In the surface electrocardiogram only found in more than 50% of the shows, 4 of 11 criteria to demonstrate ventricular enlargement right, being the 2 most representative QRS axis >110 degrees and the sum of the R wave in V1 and the S wave in V5 >10 mm. By echocardiography transthoracic feature: severe pulmonary hypertension, increased right ventricular diastolic diameter and increased thickness of the right ventricular free wall was also found a TAPSE, a fraction and right ventricular Tei index increased. By applying the technique of tissue Doppler velocities we obtained minor contraction in the right ventricular free wall, as in the interventricular septum at the middle segment, and a delay in the interventricular septum contraction with respect to the free-wall VD. The chronic pulmonary embolism produces severe pulmonary arterial hypertension. Pulmonary arterial hypertension causes geometric changes in right ventricular anatomy. The alterations in venous return produced right ventricular dysfunction. The surface ECG is an insensitive method for assessing right ventricular enlargement. ECTT markers with greater sensitivity for the detection of right ventricular dysfunction include the TAPSE and Tei index. The tissue Doppler ultrasound best assessed right ventricular deterioration, and that allows assessment of contractile motility lows and SIV and free-wall VD. 2.38

Single and combination therapy with erythropoietin and sildenafil on hypoxia-induced PAH Østergaard, L Institute of Sports Science and Clinical Biomechanics, University of Southern Denmark, Odense M, Denmark

Pulmonary arterial hypertension (PAH) is a group of diseases characterized by elevated pulmonary pressure. This can lead to right S28

ventricular hypertrophy and if left untreated, patients might die from right heart failure within an average of 3 years. The present study was designed to investigate single and combination therapy with erythropoietin (EPO) and sildena il on hypoxia-induced PAH. Mice were randomized, irst in a normoxic and a hypoxic group and second to receive saline, EPO, sildena il or EPO and sildena il. EPO was injected three times per week (500 IU/kg) and sildena il daily (1 mg/kg). The animals were exposed to 3 weeks of either hypoxia (10% oxygen) or normoxia, after which they underwent the different treatments for an additional 2 weeks under the same conditions. Immunohistochemistry was performed to elucidate changes in pulmonary morphology and of right heart hypertrophy. Plasma levels of cardiotrophin-1 and atrial natriuretic peptide (ANP) were measured as well as ventilatory parameters using whole body plethysmography. The pulmonary pressure was measured using right heart catheterization. The increase in pulmonary pressure after hypoxic exposure was attenuated by either drug alone but signi icantly more by the combination treatment. The hypoxia-induced increase in right ventricular hypertrophy and medial wall thickness of pulmonary arterioles was signi icantly reduced with the combination therapy compared both to the control and to the single treatment groups. Similar results were also observed for cardiotrophin-1 and ANP levels. Animals in the hypoxic group exhibited a deteriorated lung function that could be improved upon treatment. The combination treatment with EPO and sildena il demonstrated an improvement in the clinical outcome in hypoxia-induced PAH in rodents, superior to that observed for either drug given alone. 2.39

A practicable risk score for pulmonary hypertension Tiede H, Felix J, Wilkins M, Steyerberg E, Seeger W, Grimminger F, and Ghofrani A University of Giessen Lung Center, Giessen, Germany; Erasmus University of Rotterdam, The Netherlands; Imperial College London, London, UK

Several markers that are associated with severity and/or prognosis of disease have been suggested for patients with pulmonary arterial hypertension. We aimed to assess the utility and validity of a new prognostic score for different forms of pulmonary hypertension based on comprehensive databases from the PH centers Giessen and Imperial College in London. Every consecutive patient undergoing right heart catheterization with proven pulmonary hypertension was included in the Giessen registry from 1994 to 2011. Overall 1-, 3-, and 5-year survival rates were 89.9, 76.1and 65.6% respectively. Of the total, 615 patients (35.7%) had PAH, 439 (25.4%) had pulmonary hypertension due to chronic lung diseases (PH-LD, mainly interstitial lung disease and chronic obstructive lung disease), 419 (24.3%) had chronic thromboembolic pulmonary hypertension (CTEPH), 224 (13%) had pulmonary venous hypertension, and 28 (1.6%) had pulmonary hypertension due miscellaneous or unknown causes. Differences in survival between the etiological groups were highly signi icant (P<0.001), with 1-, 3-, and 5-year survival rates of 89.8, 78.1 and 67.2% respectively in PAH opposed to 86.6, 65.4, and 53.7% respectively, in PH-LD. In multivariate analysis, uric acid, urea, leucocyte count, brain natriuretic peptide, heart rate, sodium, 6-minute walk test distance, oxygen saturation during 6-minute walk test, cardiac output and systolic blood pressure at baseline were signi icantly associated with survival. We built a prediction model for all classes of PH with outcome being death from any cause. The equation was validated with the cohort from Imperial College, London, UK. By integration of different predictors in one prognostic model we took the complex pathophysiology of PH into account. A formula has been generated that may help researchers and clinicians to better anticipate the prognosis of an individual patient irrespective of the underlying cause of pulmonary hypertension. Pulmonary Circulation | October-December 2011 | Vol 1 | No 4

Abstracts

2.40

Use of combination therapy in postoperative persistent pulmonary arterial hypertension in children Kulkarni S, Jadhav M, Garekar S, and Rao S Children’s Heart Center, Kokilaben Dhirubhai Ambani Hospital, Mumbai, India

We examined the use of combination therapy in six patients with persistent pulmonary arterial hypertension (PAH) after corrective surgery for congenital heart defects. Combination of sildena il and bosentan was used in six patients for persistent pulmonary arterial hypertension (PAH) after corrective surgery for congenital heart defects. Out of these six patients, four had persistent PAH crisis in the immediate postoperative period and needed inhaled nitric oxide. Combination of sildena il and bosentan was used in six patients for persistent PAH after corrective surgery for congenital heart defects. Out of these six patients, four had persistent PAH crisis in the immediate postoperative period and needed inhaled nitric oxide. Pulmonary artery pressures remained very high after discontinuation of nitric oxide after 48 hours and they could not be extubated. These patients were given combination of sildena il and bosentan while coming off from nitric oxide and then they could be extubated safely. The other two patients had persistently high pulmonary arterial pressures after surgery at the time of discharge. They were initially started only on sildena il with marginal improvement in pulmonary arterial pressures. The addition of bosentan helped with a signi icant reduction in pulmonary arterial pressures at 6 months follow-up. Combination therapy of sildena il and bosentan helps create a signi icant reduction in pulmonary arterial pressures in immediate postoperative period. It can be used effectively on followups if pulmonary artery pressures remain elevated after surgeries for congenital heart defects. 2.41

LC/MS/MS method for simultaneous analysis of arachidonic acid and its endogenous eicosanoid metabolites and prostaglandins in rodent lung tissue Sagliani K, Hill NS, Fanburg BF, Dolnikowski G, Levy BL, and Preston IR Pulmonary, Critical Care and Sleep Division, Tufts Medical Center, Boston, MA, USA; Human Nutrition Research Center of Aging, Boston, MA, USA; Brigham and Women Hospital, Boston, MA, USA

Lipoxygenase (LOX)- and cycloxygenase (COX)- generated lipid mediators are biomarkers of in lammation, cell proliferation, and oxidative stress and possess biological activity in various disease states, including those affecting the pulmonary vasculature. Therefore, their quanti ication may be important in understanding the pathophysiology of pulmonary hypertension, in which oxidative stress via lipid metabolites plays a role. Liquid chromatography with tandem mass spectrometer detection (LC/MS/MS) methods allow a highly selective, sensitive analysis of bioactive lipids. However, the LC/MS/MS methods currently used do not allow for simultaneous separation of the major eicosanoids and prostaglandins without multiple high performance liquid chromatography (HPLC) separations. We developed a LC/MS/MS method which allows for simultaneous separation and quanti ication of the major lipid mediators in healthy rat and mouse lung. The method was validated by analyzing multiple times same samples. Lungs were homogenized, the homogenates were centrifuged at 4,500 rpm for 30 minutes at 4°C. The diluted supernatant was acidi ied on ice to a pH of 4.0 with 1-N HCl. A 20-μL aliquot of internal standard master mix (15, 12, 5, HETE-d8, PGE2-d4, TBX2-d4) was added to each sample. From each sample, 100 μL were used to determine protein concentration using the Bradford assay. For the HPLC separation of arachadonic acid metabolites, we Pulmonary Circulation | October-December 2011 | Vol 1 | No 4

used a Luna-3-μm, phenyl-hexyl, 2×150-mm analytical column. The Agilent 1200 Series HPLC system consisted of a high-performance binary pump, and a 108-well plate autosampler at 4°C, and column compartment was set at 25°C. Targeted pro iling of eicosanoids and prostaglandins was performed using a 5500 QTRAP (ABSciex) hybrid triple quadrupole linear ion trap mass spectrometer equipped with a turbo ion-spray electrospray ionization (ESI) source. The dwell time used for all experiments was 80 msec. The source-dependent MS parameters such as temperature and ion-spray voltage were set at 400°C and -4,500 v, respectively. Results were reported in pg/μg protein as mean±STD. T-test was used to compare the results between species and P<0.05 was considered signi icant. Extraction coef icient was 75%. There is signi icant interspecies difference in the values of: 12-HETE, 8-HETE, PGE2, PGI2 and the ratio of PGI2/TBX2. We found signi icant differences in the normal levels of various arachidonic acid metabolites. This can account for difference in responses to stimuli which cause pulmonary hypertension, such as hypoxia and monocrotaline. This study is the irst to report a sensitive, speci ic, robust and validated LC/MS/MS method that allows simultaneous analysis of arachidonic acid metabolites and can be used in the future to study various disease states such as pulmonary hypertension. 2.42

Determination of endogenous bioactive lipid profile in experimental pulmonary hypertension Sagliani K, Hill NS, Fanburg BF, Warburton RW, Dolnikowski G, Levy BL, and Preston IR Pulmonary, Critical Care and Sleep Division, Tufts Medical Center, Boston, MA, USA; Human Nutrition Research Center of Aging, Boston, MA, USA; Brigham and Women Hospital, Boston, MA, USA

Lipoxygenase (LOX)- and cycloxygenase arachidonate products, including prostaglandins (PGs) and hydroxyeicosatetraenoic acids (HETEs) are known to modulate in lammation within tissues. The role of in lammation and increased oxidative stress in pulmonary hypertension has been recently acknowledged. The importance of lipid mediators in modulating in lammation, cell proliferation and migration, and pulmonary vascular remodeling is highlighted by the use of prostaglandin modulators as treatment options (prostacyclin replacement therapy). We showed previously that in experimental model of hypoxia-induced pulmonary hypertension there is an upregulation of the 12-lipoxygenase and its product 12-HETE stimulates pulmonary artery smooth muscle cell proliferation. Here, we aimed to establish whether there was an imbalance of arachidonic acid products in the lungs of hypoxic rats compared with their normoxic controls and to identify those byproducts that might explain the remodeling and in lammation associated with experimental pulmonary hypertension. Rats were exposed to 2 weeks of hypoxia (11% O2). Normoxic controls were kept in similar conditions, but in room air. Rats with chronic hypoxia and their respective controls underwent right ventricular pressure measurements. Right ventricle/left ventricle+septum were indicators of right ventricular hypertrophy. Lungs were frozen in liquid nitrogen and were subjected to LC/MS/MS method for simultaneous determination of arachidonic acid products. Results were reported in pg/μg protein as mean±STD. T-test was used to compare the results between conditions and P<0.05 was considered signi icant. Rats kept in chronic hypoxia developed elevated RV pressures and RV hypertrophy, compared with their normoxic controls. There was wide variation among levels of different eicosanoids, PGE2 and PGI2 between normoxic and hypoxic lungs. Lungs from hypoxic animals had signi icantly elevated levels of thromboxane 2 (TBX2) and the PGI2/TBX2 ratio was signi icantly decreased in these animals. Chronic hypoxiainduced pulmonary hypertension is associated with a local imbalance favoring the vasoconstrictor and pro-thrombotic product TBX2 over the vasodilator PGI2. This is consistent with indings described in patients with pulmonary arterial hypertension. S29

Abstracts

2.43

Lipidomic analysis of plasma from patients with pulmonary arterial hypertension: Determination of circulating arachidonic acid metabolites Sagliani K, Preston IR, Roberts KR, Fanburg BF, Dolnikowski G, Levy BL, and Hill NS Pulmonary, Critical Care and Sleep Division, Tufts Medical Center, Boston, MA, USA; Human Nutrition Research Center of Aging, Boston, MA, USA; Brigham and Women Hospital, Boston, MA, USA

Prior reports suggest that lungs of patients with pulmonary arterial hypertension (PAH) are under oxidative stress and that bioactive lipid components derived from arachidonic acid are released and may participate in the remodeling of the pulmonary vasculature. In addition, urinary metabolites of prostacyclin (PGI2) and thromboxane (TBX2), and their ratio, are abnormal in these patients, suggesting an imbalance among various products of arachidonic acid, with preference for vasoconstrictor and prothrombotic molecules. We aimed to screen plasma of PAH patients for arachidonic acid products and determine their bioactive lipid pro ile. We obtained plasma from consecutive patients with idiopathic or connective tissue associated PAH (WHO Group I) at the time of their right heart catheterization. Patients were grouped into treatment-naïve and those on PAH-speci ic therapies. Plasma from normal controls was obtained for comparison. Samples were frozen in -80°C until measurements were performed using an LC/MS/MS method. We enrolled 34 patients with PAH and 5 controls. Patients were on average 62±12 years of age, and 21 of them were females. Their WHO functional class at the time of collection was 2.7±0.8. PAH-speci ic therapies included: endothelin receptor blockers, PDE5 inhibitors, prostacyclins, and any combination of them. Signi icant differences were between controls and IPAH-treatment naïve and between IPAHtreatment naïve and IPAH-treated groups with respect to: 5-HETE, 12-HETE, 8-HETE. Levels were higher in non-treated patients and were lowered in the presence of treatment. TBX2 levels were signi icantly higher in IPAH-treatment naïve patients compared with controls (P=0.0049). We established for the irst time signi icant differences in levels of bioactive lipids in patients with two forms of PAH and their changes with treatment. Further research remains to determine the value of these changes. 2.44

Is bronchus-associated lymphoid tissue readily evident in lung sections from patients with PAH and in rates with monocrotaline-induced PH? Yeager M Department of Pathology, University of Colorado Health Sciences Center, Denver, Colorado USA

Pulmonary hypertension (PH) is characterized by in lammatory cell recruitment and altered pro-in lammatory cytokine pro iles. In addition, anti-endothelial cell and ibroblast antibodies have been detected in the sera of patients with PAH. The possibility that an autoimmune program may be operational in these patients has been considered for nearly 30 years. In humans, bronchus-associated lymphoid tissue (BALT) plays important roles in antigen sampling and maintenance of self-tolerance during episodes of infection and chronic in lammation. We reasoned that BALT would be readily evident in lung sections from patients with PAH, and in rats with monocrotalineinduced PH. Using immunohistology, we found increased numbers of BALT that were larger in PH rats compared to controls. We found OX62+ dendritic cells in and around BALT, which were characterized by a CD3+ T cell mantle over a follicular core of B220+ B cells coexpressing the class-switching enzyme AID. Anti-rat IgG antibodies decorated cells in BALT, adventitial compartments in large vessels, and in all three layers S30

of medium- to small-sized vessels in lung sections from control rats. Finally, BALTs in monocrotaline-treated rats were highly vascularized by Aquaporin-1+ high endothelial venules and LYVE-1+ lymphatics. These results support the idea that chronic in lammation in PH may lead to loss of self-tolerance and autoimmunity. 2.45

Echocardiographic prediction of pulmonary vascular resistance Opotowsky AR, Clair M, Landzberg MJ, Waxman AB, Arkles JS, Rogers F, Prasanna V, Moko L, Maron B, Fields A, and Forfia PR Department of Medicine, University of Pennsylvania, Philadelphia, PA, Department of Cardiology, Children’s Hospital Boston, Boston, MA, Department of Medicine, Brigham and Women’s Hospital, Boston, MA, USA

Pulmonary hypertension is de ined by elevated pulmonary artery pressure, but is comprised of a heterogeneous collection of diagnoses with distinct hemodynamic pathophysiology. Identifying patients with elevated pulmonary vascular resistance (PVR), in contrast to those with normal PVR and elevated left-sided illing pressure, is critical for appropriate treatment. We reviewed hemodynamics, echocardiography, and clinical data for 175 patients seen at a referral PH clinic who underwent echocardiography and right-heart catheterization within 1 year of each other. We derived three equations to predict PVR with echocardiography using the ratio of estimated PA systolic pressure (PASP) to RVOT VTI as a basic model, with consideration of additional terms (e.g., RVOT Doppler notching, acceleration time) that may independently predict increased PVR, as well as predictors of left atrial pressure (LAP) in more complex models. We compared these models to a published model based on the ratio of trans-tricuspid low velocity (TTFV) to RVOT VTI:

PVR = 10 ×

TTFV + 0.16 RVOT VTI

(Model 1)(Abbas et al, JACC 2003)

The basic derived pressure-based model was:

PVR = 1.3 ×

PASP RVOT VTI

(Model 2)

The presence of RVOT VTI mid-systolic notching was a signi icant predictor of PVR when added to the linear regression model (P=0.0002), so we added a term to integrate this additional information: PVR =

PASP + 3 if notch present RVOT VTI

(Model 3)

A more comprehensive derived model, including a correction for left atrial pressure, was: PVR = 1.2 ×

PASP-LAP + 1 + 2 if notch present RVOT VTI

(Model 4)

PASP was estimated using the simpli ied Bernoulli equation (PASP=4×TTFV2). For AP LA dimension <3.2 cm, est. LAP=10, if 3.2-4.2 cm then LAP=15, and if >4.2 cm then LAP=20. Average mean PAP was 45.3±11.9, mean PVR was 7.3±5.0 and mean PAWP was 14.8±8.1. PAWP was >15 mmHg in 41.7% and 80% had PVR >3WU. The published PVR prediction model systematically underestimated true PVR, especially at higher levels of PVR. There was no such systematic bias in any of the derived models, and the prediction 95% CI was narrower for all models compared with the published equation. The comprehensive equation had the greatest discriminatory power for elevated PVR>3WU (AUC 0.914), though all models had reasonably high AUC. The sensitivities of predicted PVR>3WU for catheterization derived PVR>3WU for Models 1 through 4 respectively Pulmonary Circulation | October-December 2011 | Vol 1 | No 4

Abstracts

were 65.7, 95.0, 95, and 95.7%, with speci icities of 91.4, 28.6, 62.9, and 45.7%. For Model 1, a cut-off predicted PVR of 1.72 corresponded to sensitivity of 95.7% and speci icity of only 20%. A previously published echo model to predict PVR systemically underestimated PVR in a PHreferral population. Using a quadratic term to re lect the exponential relationship of pressure to low improved agreement between predicted and true PVR, adding a term to account for RVOT VTI notching further improved test characteristics. While including a crude estimate of left atrial pressure provided modest additional value, this may be outweighed by increased complexity. 2.46

The use of cardiac MRI in understanding PAH in complex CHD: Case reports Kappanayil M Pediatric Cardiology, Amrita Institute of Medical Sciences, Kerala, India

Cardiac magnetic resonance (CMR) is emerging as a powerful tool in the assessment of congenital heart diseases, and is especially invaluable in assessing older/grown-up patients with complex structural heart diseases. CMR provides not just detailed anatomical information, but also unique physiological insight. The developing world in particular has a large burden of patients with complex heart diseases which are either unoperated, or partially palliated, or growing up with sequelae that include PAH. Many complex lesions are particularly dif icult to evaluate using the conventional modalities of echocardiography and cardiac catheterization, owing to complex anatomy and/ or physiology. We illustrated three case examples with CHD-PAH where MR imaging helped in the primary detailed diagnosis, as well as physiology. My object was to illustrate the usefulness of cardiac MRI in understanding PAH in complex CHD, through three case examples. Case 1: An 18-year-old girl, suspected to have complex CHD in early childhood. Social/ inancial limitations prevented detailed evaluation until recently. She complained of effort intolerance, NYHA class II, and had an SPO2 of 92%. Gadolinium-enhanced angiography showed pulmonary atresia with a large MAPCA from proximal descending thoracic aorta with an unobstructed, large connection to left lung and a proximally stenotic connection to right lung. Gadolinium-enhanced 3D angiography showed clear evidence of differential vascularity, with the left lung showing peripheral pruning, and the right lung showing rich arborization. Phase-contrast sequences were used to study the lows in the aorta, the MAPCAs to right and left lung, and the pulmonary venous returns from either lung. They showed a Qp/Qs of nearly 2:1. However, nearly 80% of the blood low was to the normally arborized right lung (7.5 L/min), and only 20% o the left (2 L/min). The low/time curves showed early peaking, lower peak velocity, and rapidly descending low patterns (suggestive of PAH) in the left lung blood lows. CMR not only helped in establishing the diagnosis, but also in understanding the nature of PAH, which in this case was an Eisenmegerized left lung with a nearly normal right lung. Case 2: A 16-year-old girl with mild effort intolerance, SPO2 85%, with uncertain diagnosis on previous evaluations (possibly truncus arteriosus). CMRI revealed an anatomical diagnosis of large conoventricular VSD with aortic atresia, hypoplastic ascending aorta, two balanced ventricles, large pulmonary artery overriding the VSD, dilated bilateral branch pulmonary arteries, patent ductus arteriosus shunting right-to-left and sustaining systemic blood low, juxta ductal coarctation. Gadolinium-enhanced 3D angiography revealed reduced arborization and peripheral pruning of bilateral lungs, more prominent in the left lung. This angiographic inding was supported by phase contrast ( low) studies which showed lower lows in the left lung compared to the right, with low/time graphs clearly suggestive of PAH. Qp:Qs (derived through low studies) was nearly 2:1. Operability debatable—most likely Eisenmenger syndrome. Case 3: A 30-yearold Ethiopian male patient presented with features of biventricular failure and PAH, with upper body hypertension, differential cyanosis and differential clubbing. CMRI established a diagnosis of large PDA Pulmonary Circulation | October-December 2011 | Vol 1 | No 4

shunting bidirectional with a tight preductal coarctation of aorta, with severe biventricular dysfunction. Gadolinium-enhanced 3D angiography showed a differential vascularity pattern with peripheral pruning of the right lung vasculature. Flow/time graphs showed a pattern of PAH in both lungs, with lower lows in the right lung, with Qp:Qs of 0.9:1. Flow studies of the PDA showed a systolic right-to-left shunt and a pandiastolic left-to-right shunt, suggesting subsystemic diastolic pressures in the pulmonary arteries (net low being about 0.3L/min right-to-left). This patient is being currently planned for dilatation/stenting of the coarctation and subsequent hemodynamic re-evaluation for suitability for PDA closure. All the above cases reveal the usefulness of CMR in evaluating and understanding complex CHDs associated with PAH, and thereby in planning management. 2.47

Therapeutic potential of HDAC inhibitors in pulmonary hypertension Zhao L, Chen CN, Hajji, Oliver E, Huang TJ, Wang D, Li M, McKinsey T, Stenmark KR, and Wilkins MR Centre for Pharmacology and Therapeutics, Imperial College London, UK; Hammersmith Hospital, London, UK; Department of Pediatrics, University of Colorado Denver, Denver, CO, USA

Pulmonary arterial hypertension (PAH) is characterized by structural remodeling of pulmonary arteries and arterioles, the result, at least in part, of excessive cell proliferation and resistance to cell death. Epigenetic programming, dynamically regulated by histone acetylation, is an important mechanism for controlling cell proliferation and survival. Little is known regarding the contribution of changes in histone deacetylase (HDAC) activity to the changes in cell phenotype that occur during remodeling and overall to the development of PAH. HDAC protein expression levels were measured by Western blotting. Speci ically, HDAC1 and HDAC5 levels were increased in lungs from IPAH patients and in the lungs and right ventricles of rats exposed to hypoxia. Both valproic acid (VPA), a HDAC1 inhibitor, and suberoylanilide hydroxamic acid (SAHA), an inhibitor of class I and II a/b HDACs, mitigated the development and reduced established hypoxia-induced pulmonary hypertension in the rat. Both VPA and SAHA inhibited the “imprinted” highly proliferative phenotype of ibroblasts and “R” cells from pulmonary hypertensive bovine vessels and also inhibited PDGF-stimulated growth of human vascular smooth muscle cells in culture. Exposure to VPA and SAHA was associated with increased levels of p21and FOXO3 and reduced expression of Bcl-2 and surviving. VPA also increased lung expression of BMPR2 and reduced HIF1α levels in rats exposed to hypoxia in vivo. Increased HDAC activity contributes to the vascular pathology of pulmonary hypertension. HDAC inhibitors, VPA and SAHA, may have therapeutic potential in PAH. 2.48

Atrial flutter and atrial fibrillation in patients with chronic pulmonary hypertension Olsson KM, Nickel N, Tongers J, and Hoeper MM Departments of Respiratory Medicine and Cardioloy, Hannover Medical School, Hannover, Germany

Supraventricular tachyarrhythmias (SVTs), especially atrial lutter and atrial ibrillation, are observed with increasing frequency in patients with pulmonary hypertension, but their clinical consequences have not been systematically evaluated. We sought to determine the prevalence and clinical relevance of these SVTs in patients with pulmonary hypertension. In a 5-year, prospective study, we analyzed the occurrence of SVTs in patients with newly diagnosed pulmonary arterial hypertension (PAH) or nonoperable chronic thromboembolic pulmonary hypertension (CTEPH). The enrolment period was 4 years, followed by a minimum S31

Abstracts

1-year follow-up. We assessed the incidence of SVTs as well as their clinical implications, treatment modalities, risk factors and impact on outcome. We enrolled 239 patients, 82 with inoperable CTEPH and 157 with PAH. At baseline, the mean 6-minute walk distance was 335 (IQR, 292-429) m. and the mean PVR at baseline was 734 (IQR, 472-922) dyn.s.cm-5. SVTs of new onset were detected in 48/239 (20%) patients, yielding an annual incidence of 4%. Atrial ibrillation and atrial lutter occurred with equal frequency. According to univariate Cox regression analyses, higher values of mean pulmonary artery pressure (HR 1.03, 95% CI 1.01-1.06, P=0.002), right atrial pressure (HR 1.07, 95% CI 1.05-1.16, P=0.01), and serum bilirubin (HR 1.05, 95% CI 1.02-1.08, P=0.01) as well as a lower cardiac output (HR 1.9, 95% CI 1.1-3.5, P=0.048) were linked to

S32

an increased risk of SVTs. The development of atrial ibrillation or atrial lutter resulted in deteriorations in exercise capacity, functional class and NT-proBNP serum levels. Most patients required hospitalization. A stable sinus rhythm was successfully restored in 21/24 (88%) of patients with atrial lutter and in 16/24 (67%) of patients with atrial ibrillation. Patients in whom sinus rhythm was restored had a signi icantly better survival than patients with persistent SVTs (P=0.01 by log rank analysis). New onset SVTs were observed with an incidence of 4% in patients with pulmonary hypertension and the risk was particularly high in patients with severe hemodynamic impairment. The development of SVTs resulted in impaired exercise capacity, clinical deterioration, and a worsened survival, particularly when sinus rhythm was not restored.

Pulmonary Circulation | October-December 2011 | Vol 1 | No 4

Abstr ac t s

Inaugural Scientific Meeting of the

Pulmonary Hypertension Society of Australia and New Zealand (PHSANZ) Sydney, Australia 25 November, 2011

3.1

Pulmonary arterial hypertension in patients with systemic sclerosis is independent of high-resolution computed tomography findings of interstitial lung disease Tuppin M, Chambers D, Slaughter R, Mohammed O, and Kermeen F School of Medicine, Queensland Center for Pulmonary Transplantation and Vascular Disease, Medical Imaging Department, The Prince Charles Hospital, University of Queensland, Brisbane, Queensland, Australia

Systemic sclerosis associated pulmonary arterial hypertension (SScPAH) may occur as an isolated arteriopathy or concurrently with systemic sclerosis associated interstitial lung disease. However, the extent to which the interstitial lung disease in luences pulmonary arterial pressure (PAP) has not been well described in the literature. Therefore, this study aimed to determine how the high-resolution computed tomography (HRCT) features in patients with SSc-PAH correlate to measures of hemodynamics. A retrospective analysis of patients with a diagnosis of SSc-PAH treated at the Queensland Centre for Pulmonary Transplantation and Vascular Disease between 2002 and 2010 was conducted. Existing HRCT scans were scored for the presence and extent of interstitial lung disease features, and measurements taken of the main pulmonary arteries (MPA). These were then correlated to measures of hemodynamics attained by right heart catheterization. Fifty-seven patients with SSc-PAH (50 female), of mean (SD) age of 60.7 (9.6) years and mean (SD) PAP of 44.3 (14.3) mmHg were analyzed; 44/57 (77.2%) had an MPA greater than 30 mm, while only 14/57 (24.6%) had a pulmonary artery/ascending aorta ratio greater than 1.13. The MPA was most strongly correlated to the diastolic pulmonary artery pressure (r=0.545, P<0.01). There were no signi icant correlations between any of the other HRCT features and hemodynamics. These indings suggest that the development of pulmonary arterial hypertension is probably independent of the existence of interstitial lung disease in patients with systemic sclerosis. The MPA size should be regarded as the best marker of pulmonary hypertension on HRCT in this group of patients. 3.2

Imatinib for the treatment of pulmonary arterial hypertension and pulmonary capillary hemangiomatosis Nayyar D, Muthiah K, Kumarasinghe G, Hettiarachchi R, Macdonald P, Kotlyar E, Hayward C, and Keogh A St. Vincent’s Hospital, Darlinghurst, New South Wales, Australia

The effect of imatinib (a platelet-derived growth factor receptor antagonist) in two patients, one with pulmonary capillary hemangiomatosis (PCH) (Case 1), and the other with pulmonary arterial hypertension (PAH) (Case 2), was reviewed. Medical records Pulmonary Circulation | October-December 2011 | Vol 1 | No 4

and pulmonary hypertension database records were reviewed to obtain details of the clinical presentation, management regimen and clinical outcomes. Case 1 was a 62-year-old woman with PCH who deteriorated rapidly despite treatment with ambrisentan, doxycycline and epoprostenol. She was commenced on imatinib and within 2 days demonstrated signi icant symptomatic improvement (from functional Class IV to Class III). After 6 months of treatment with imatinib, there was a resolution in signs of right-sided heart failure, an improvement in exercise tolerance (increased 6-minute walking distance from 90 to 295 meters), and a reduction in pulmonary artery systolic pressure (83 to 45 mmHg). Case 2 was a 43-year-old woman with a 16-year history of severe idiopathic PAH with notable decline despite the use of combination therapy (iloprost, bosentan and sildena il). In 2008, she was commenced on imatinib, which led to an improvement in her functional class (Class IV to Class III). These cases demonstrated a positive outcome of imatinib treatment in two different etiologies of pulmonary hypertension. Large clinical studies are necessary to mandate its wider use. 3.3

Abnormal pulmonary artery stiffness in pulmonary arterial hypertension: In vivo study with intravascular ultrasound Lau EMT, Iyer N, Ilsar R, Bailey BP, Adams MR, and Celermajer DS Department of Respiratory, Royal Prince Alfred Hospital, Sydney, New South Wales, Australia

There is increasing recognition that both resistance and compliance contribute to right ventricular (RV) afterload in pulmonary arterial hypertension (PAH). However, changes in the stiffness properties of the proximal elastic pulmonary arteries (PA) and the contribution of this to RV afterload have not been well studied. Furthermore, the effect of PAHspeci ic therapy on proximal PA stiffness is unknown. Using intravascular ultrasound (IVUS) and simultaneous right heart catheterization, 20 pulmonary segments in 8 PAH subjects and 12 pulmonary segments in 8 controls were studied to determine their compliance, distensibility, pressure-strain modulus and stiffness index β. PAH subjects underwent repeat IVUS examinations after 6 months of bosentan therapy. At baseline, PAH subjects demonstrated greater stiffness in all measured indices compared to controls; compliance (1.50±0.11×10-2 mm2/mmHg versus 4.49±0.43×10-2 mm2/mmHg, P<0.0001), distensibility (0.32±0.03 %/mmHg versus 1.18±0.13 %/mmHg, P<0.0001), pressure-strain modulus (720±64 mmHg versus 198±19 mmHg, P<0.001), and higher stiffness index β (15.0±1.4 versus 11.0±0.7, P=0.046). Strong inverse exponential relationships existed between mean pulmonary artery pressure and compliance (r2 =0.815, P<0.0001), and also between mean PAP and distensibility (r2 =0.790, P=0.002). Bosentan therapy for 6 months was not associated with signi icant changes in PA stiffness. Increased stiffness occurs in the proximal elastic PAs in patients with PAH, and may be an important contributor to the pathogenesis of RV failure in this condition. S33

Abstracts

3.4

An Australian tertiary referral centre experience of the management of CTEPH Maliyasena VA, Hopkins PMA, Thomson BM, Dunning J, Wall DA, Ng BJH, McNeil KD, and Kermeen FD Queensland Centre for Pulmonary Transplantation and Vascular Disease, the Prince Charles Hospital, Brisbane, Queensland, Australia

The intent was to report the outcome of pulmonary endarterectomy (PEA) surgery performed for chronic thromboembolic pulmonary hypertension (CTEPH) at a single tertiary center. Design, setting, and participants: Prospective study of 35 patients with surgically amenable CTEPH undergoing PEA between September 2004 and September 2010. Functional data [New York Heart Association (NYHA) class, 6-minute walk test distance], hemodynamic data (echocardiography, right heart catheterization, and cardiac MRI), and outcome data (morbidity and mortality), were collected. Following PEA, there were signi icant improvements in NYHA class (pre 2.9±0.7 versus post 1.3±0.5, P<0.0001), right ventricular systolic pressure (pre 77.4±24.8 mmHg versus post 44.6±24.3 mmHg, P=0.0003), 6-minute walk distance (pre 438.0±97.9 meters versus post 520.2±81.4 meters, P=0.0005), Borg score (pre 4.2±1.9 versus post 2.8±1.4, P=0.0123), mean pulmonary artery pressure (pre 42±15.1 mmHg versus post 24±8.8 mmHg, P<0.0001), and cardiac MRI indices (end diastolic volume pre 213.8±49.2 ml. versus post 148.1±34.5 ml., P<0.0001; end systolic volume pre 130.1±41.9 ml. versus post 78.8±25.6 ml., P<0.0001). Mean coronary bypass time was 258.77±26.16 minutes, with a mean clamp time of 110.96±35.26 minutes, a mean rewarming time of 81.76±27.02 minutes, and mean circulatory arrest time of 43.83±18.78 minutes. Mean ventilation time was 4.7±7.93 days (range 0.2-32.7), with a mean intensive care unit stay of 7.22±8.71 days (range 1.1-33.8). Complications included slow respiratory wean (25.7%), pericardial effusion (11.4%), persistent pulmonary hypertension (17.1%), reperfusion lung injury (20%) and cardiac tamponade (5.7%). One-year mortality post-procedure was 11.4%. Pulmonary endarterectomy can be performed safely with relatively low mortality. 3.5

N-Terminal pro-Brain Natriuretic Peptide (NT-proBNP) levels predict incident pulmonary arterial hypertension in systemic sclerosis (SSc) in the Australian Scleroderma Cohort Study (ASCS) Thakkar V, Stevens W, Priorv D, Byron J, Patterson K, Hissaria P, Moore O, Roddy J, Zochling J, Sahhar J, Nash P, Tymms K, Youssef P, Proudman S, and Nikpour M St. Vincent’s Hospital Melbourne, Royal Adelaide Hospital, Royal Perth Hospital, The Menzies Institute, Monash Medical Centre, Sunshine Coast Rheumatology, Canberra Rheumatology, Royal Prince Alfred Hospital, Sydney, New South Wales, Australia

To assess the use of NT-proBNP as a screening biomarker for SSc-PAH. NT-proBNP levels were assayed on patients with normal LV function and eGFR >30 ml/min enrolled in the ASCS, which currently includes over 1,150 patients across Australia. Group 1 (n=20) had de inite PAH with pretreatment sera assayed. Group 2 (n=30) were considered “at risk” for PAH based on (i) sPAP on echo>36 mmHg, or (ii) FVC/DLCO% ≥1.6 and no signi icant ILD, or (iii) DLCO <50%, or (iv) resting mPAP of 20-25 mmHg at RHC. Group 3 (n=19) had ILD but no evidence of PAH on echo or RHC. Group 4 (n=31) were SSc controls. Group 1 (PAH) had signi icantly higher mean NT-proBNP levels than patients in Group 4 (SSc controls; P<0.0001). In addition, patients in Group 2 (“at risk”) had signi icantly higher NT-proBNP levels than those in Group 4 (SSc controls; P=0.008). NT-proBNP was positively correlated with echo S34

parameters (P<0.0001). NT-proBNP was positively correlated with mPAP on RHC (adjusted estimate=0.048, 95% CI: 0.01-0.09, P=0.019), independently of corrected DLCO, FVC/DLCO% ratio and 6MWD. An NT-proBNP cut-point of >189.2 pg/ml had a likelihood ratio of 26.4 for presence of PAH (c-statistic=0.9; sensitivity 85%; speci icity 97%). An NT-proBNP level <82.9 pg/ml had a likelihood ratio of 6.8 for exclusion of PAH (sensitivity 67.7%, speci icity 90%). In the absence of LV dysfunction, NT-proBNP is a useful screening biomarker for PAH in SSc, with levels>189.2 pg/ml and <82.9 pg/ml de ining patients with a high and low likelihood of PAH. 3.6

Survival and predictors of mortality in Australian patients with connective tissue disease-associated pulmonary arterial hypertension Ngian GS, Stevens W, Byron J, Tran A, Roddy J, Minson R, Hill C, Chow K, Sahhar J, Proudman S, and Nikpour M The University of Melbourne; St Vincent’s Hospital, Melbourne; Royal Perth Hospital, Perth; Flinders Medical Centre, Adelaide; The Queen Elizabeth Hospital, Adelaide; Royal Adelaide Hospital, Adelaide; Monash Medical Centre, Melbourne, Victoria, Australia

We sought to determine survival and factors predictive of mortality in connective tissue disease-associated pulmonary arterial hypertension (CTD-PAH). This was a retrospective cohort study of patients with CTDPAH recruited from 6 tertiary hospitals. Patients were identi ied from the Australian Scleroderma Cohort Study and registers of patients receiving pulmonary vasodilator therapy. Records were censored at 31/12/09. Survival was determined using Kaplan-Meier estimates. Univariate and multivariable predictors of survival were determined using log-rank/ Wilcoxon tests, and proportional hazards regression modeling. Among 117 patients (105 female) there were 32 deaths. Mean age at PAH diagnosis was 61.5±11.4 years. SSc was the most common underlying CTD, accounting for 104 patients (88.9%). Forty-eight patients (41.0%) had coexistent interstitial lung disease. Average duration of follow-up from PAH diagnosis was 2.6±1.8 years. Seventy patients (59.8%) received monotherapy, 12 (10.3%) sequential monotherapy, and 34 (29.0%) combination pulmonary vasodilator therapy. One-, 2- and 3-year survival was 94, 89 and 73%, respectively. On multiple regression analysis, higher baseline WHO functional class, higher baseline mRAP, lower baseline 6-minute walk distance, pericardial effusion and absence of warfarin or combination pulmonary vasodilator therapy were independent predictors of mortality. Among patients in this study, 3-year survival is 73%. Independent predictors of survival in our study included warfarin and combination pulmonary vasodilator therapy, neither of which has previously been shown to correlate with survival in CTD-PAH. Our indings suggest that earlier diagnosis, anticoagulation and combination pulmonary vasodilator therapy may improve survival in CTD-PAH. 3.7

Chronic thromboembolic pulmonary hypertension: MRI predictors of functional and haemodynamic outcomes with pulmonary endarterectomy Ng BJH, Slaughter RE, Strugnell WE, Yerkovich ST, McNeil K, Dunning JJ, Hopkins PMA, and Kermeen FD Queensland Centre for Pulmonary Translplantation and Vascular Disease, Centre of Excellence in Cardiovascular MRI, The Prince Charles Hospital, Brisbane, Queensland, Australia; UK Centre for Pulmonary Endarterectomy, Papworth Hospital, Cambridge, UK

The assessment of chronic thromboembolic pulmonary hypertension (CTEPH) with MRI before pulmonary endarterectomy (PEA) is well established. However, monitoring after PEA with the use of CMRI, MRA Pulmonary Circulation | October-December 2011 | Vol 1 | No 4

Abstracts

and MR perfusion has not been studied together. Our objective was to identify the changes (∆) in MRI parameters that predict functional and hemodynamic outcomes with PEA. Between 2004 and 2007, 19 subjects underwent MRI before and after PEA. CMRI, MRA and MR perfusion examined RV remodeling (RVEDV, RVESV, RVEF), pulmonary vasculature abnormalities at a segmental level and disease distribution respectively. During 24-month follow-ups, functional (WHO class, 6MWD) and hemodynamic outcomes (PVR, mPAP, cardiac index) were collected. In our cohort (mean±SE age 57±12 years, WHO class 2.8±0.7, 6MWD 414±103 meters and PVR 702±398 dynes/s/cm5), all but two subjects were in WHO class I/II post-PEA. PEA resulted in reduced PVR (262±133 dynes/s/cm5) that correlated with RVEDV ∆ (r2=0.48, P=0.002) and RVESV ∆ (r2=0.28, P=0.024). Multiple linear regression analysis demonstrated that RVEDV ∆ and MR perfusion ∆ were the strongest predictors of hemodynamic and functional outcomes, respectively. Based on MRA, disease clearance related to WHO class improvement at 6-12 months follow-up (P=0.041) on univariate analysis. MR perfusion ∆ is a strong predictor of functional outcomes after PEA. RV remodeling, as demonstrated by CMRI ∆, is a signi icant predictor of hemodynamic outcomes. Based on MRA, disease clearance of at least four segments results in functional improvement. MRI is an invaluable non-invasive tool in assessing outcomes for patients undergoing PEA. 3.8

The demographics of pulmonary arterial hypertension associated with congenital heart disease: Results from a national registry Strange G, Rose M, Kermeen F, King I, Vidmar S, Grigg L, Celermajer D, and Weintraub R (on behalf of the ANZ CHD-PAH Registry) Royal Children’s Hospital, Melbourne; The Prince Charles Hospital, Brisbane; Royal Melbourne Hospital, Melbourne; Royal Prince Alfred Hospital, Sydney, New South Wales, Australia

Pulmonary arterial hypertension (PAH) frequently accompanies childhood congenital heart disease (CHD) and may persist into adult life. The advent of speci ic PAH therapies for PAH prompted formation of a national ANZ registry in 2010 to document the incidence, demographics, presentation and outcomes for these patients. This multicenter, prospective, web-based registry enrolls patients with CHDassociated PAH being followed in a tertiary center. The inclusion criteria stipulated patient age >16 years, a measured mPAP >25mmHg at rest or echocardiographic evidence of PAH or a diagnosis of Eisenmenger syndrome, and followed since 1/1/2000. A single observer collected standardized data during a series of site visits. So far, 137 patients (61.3% females) have been enrolled. The mean age (SD) at the time of PAH diagnosis or con irmation in an adult center was 28.3 (6.7) years and 41 (29.9%) patients were aged >30 years at this time. The mean duration of follow-up was 8.0 (4.4) years. Thirty-eight (27.7%) patients were in WHO functional Class II and 96 (70.1%) in Class III at the time of diagnosis; 134 of 137 (97.8%) had congenital systemic-pulmonary shunts, and 97 (70.8%) never underwent intervention; 43 (31.4%) had Down’s syndrome. Con irmation of PAH by recent cardiac catheterization was available in 90 (65.7%) subjects. During follow-up a total of 19 (13.9%) patients died or underwent transplantation. CHD associated with PAH in adult life has resulted in a new population with unique needs. This registry will allow documentation of clinical course and long-term outcomes for these patients. 3.9

Lung disease, particularly pulmonary arterial hypertension, is the major cause of death in the Australian Systemic Sclerosis Cohort Study Pulmonary Circulation | October-December 2011 | Vol 1 | No 4

Stevens W, Thakkar V, Moore O, Byron J, Proudman S, Zochling J, Roddy J, Sahhar J, Nash P, Youssef P, Major G, Tymms K, Hill C, Sturgess A, Schrieber A and Nikpour M The University of Melbourne; St Vincent’s Hospital, Melbourne; Royal Perth Hospital, Perth; Flinders Medical Centre, Adelaide; The Queen Elizabeth Hospital, Adelaide; Royal Adelaide Hospital, Adelaide; Monash Medical Centre, Melbourne, Victoria, Australia

The objectives of this study were to determine the cause of death among a cohort of patients with systemic sclerosis (SSc). The Australian Systemic Sclerosis Cohort Study is a national multicenter prospective study of patients with SSc. Comprehensive disease data are captured at least annually on all patients. Deaths and details on the cause of death and other comorbidities are recorded. Since 2007, 1,136 patients have been enrolled. Mean±SD follow up was 2.13±0.88 years. Sixty-nine deaths had been reported. Mean±SD age at death was 67.3±10.1 years. Disease duration at time of death was signi icantly shorter in patients with diffuse SSc than in patients with limited SSc (mean 12.0±13.0 versus 20.0±14.1 years, P=0.006). Cause of death was determined to be SSc related in 39 (57%), unrelated to SSc in 29 (42%). The leading cause of SSc-related death was lung disease, accounting for 43% of total deaths and 76% of SSc related deaths. Among the 30 lung-related deaths, 20 patients had isolated PAH, 3 ILD and 7 had both ILD and pulmonary arterial hypertension (PAH). Other SSc-related deaths were classi ied as multiorgan in 3, sepsis related to SSc 2, GIT involvement 2, renal crisis 1, and SSc myocardial disease 1. The second most common cause of death was cancer, accounting for 14 deaths (20% of total deaths). Cardiovascular events were responsible for 8 deaths (11.6% of total deaths). Despite the advent of new therapies, PAH remains a major cause of death in our patients. Earlier detection and treatment of this complication through screening may improve survival. 3.10

Evolution of a pulmonary hypertension clinic Cicovic A, McWilliams T, Coverdale HA, Whyte K, Stewart C, and Wasywich CA Green Lane Cardiovascular Service, New Zealand Heart and Lung Transplant Service, Auckland City Hospital, Auckland, New Zealand

Pulmonary arterial hypertension (PAH) is associated with a high morbidity and mortality. Since the 1990s PAH-speci ic therapy has been shown to be effective in improving symptoms and survival. Access to therapy has been tightly controlled in New Zealand due to the high cost of therapy. In June 2009, PAH-speci ic drug therapy was funded through Special Authority, and all patients were required to commence sildena il as irst-line therapy. This study describes the population of adult patients followed in the Auckland City Hospital PAH clinic. All patients followed long term at the PAH clinic are entered into a database allowing longitudinal evaluation. Patients within this cohort included those treated before the availability of funded therapy and those treated in the current era. Ninety-seven patients (74 female, 76%) were followed. Mean age at the time of diagnosis was 49 (SD 17) years. Most had PAH due to connective tissue disease (42, 43%) and idiopathic PAH (36, 38%). At diagnosis mean PVR was 9.1 WU (SD 5.5WU), median WHO functional class was 3, and mean 6-minute walk distance 335 m. (SD 131 m.). Patients had been treated with a variety of medications [primarily sildena il (78%), bosentan (28%) and iloprost (24%)], 1/4 were treated with combination drug therapy, 25%. Of patients seen since 2000, 21 of 92 have died (data censored 1/3/2011). This study describes a unique group of PAH patients who were commenced on sildena il as irst line therapy. Escalation to combination therapy is common (due to disease progression/failure of monotherapy). 3.11

Neovascularity in patients with idiopathic pulmonary arterial hypertension S35

Abstracts

Tuppin M, Chambers D, Slaughter R, Mohammed O, and Kermeen F School of Medicine, Queensland Centre for Pulmonary Transplantation and Vascular Disease, Medical Imaging Department, The Prince Charles Hospital, The University of Queensland, Brisbane, Queensland, Australia

The term neovascularity has been previously applied to the presence of micronodular and serpiginous intrapulmonary vessels evident on computed tomography (CT) images. Their presence in patients with idiopathic pulmonary arterial hypertension (IPAH) has not been well described in the literature. A retrospective analysis of patients with a diagnosis of IPAH treated at the Queensland Centre for Pulmonary Transplantation and Vascular Disease between 2002 and 2010 was conducted. Existing CT scans were reviewed for the presence of neovascularity, and correlations were made to measures of hemodynamics attained by right heart catheter and transthoracic echocardiography. Forty- ive patients with IPAH (25 female), of mean (SD) age of 53.5 (16.9) years and mean (SD) PAP of 50.4 (2.2) mmHg, were included in the analysis. Neovascularity was present in 8/45 (17.8%) patients, with half of these patients having this feature in more than 50% of their lungs. Neovascularity was generally distributed throughout the entirety of the lung parenchyma, with no predominance for either the left/right lungs or upper/lower zones. There was a moderate positive correlation to all measures of hemodynamics that was strongest for the diastolic pulmonary arterial pressure (r=0.415, P<0.01), and a positive correlation to the six-minute walk distance (r=0.305, P<0.05). This is the irst study to demonstrate a correlation between neovascularity and both pulmonary arterial pressures and six-minute walk distance in patients with IPAH. The presence of this feature may be evidence of severe and longstanding pulmonary arterial hypertension leading to the development of pulmonary plexogenic arteriopathy. 3.12

Novel biomarkers of dysregulated angiogenesis are not specific to pulmonary arterial hypertension in systemic sclerosis Thakkar V, Patterson K, Stevens W, Byron J, Moore O, Roddy J, Zochling J, Sahhar J, Nash P, Tymms K, Youssef P, Proudman S, Nikpour M, and Hissaria P St. Vincent’s Hospital Melbourne, Royal Adelaide Hospital, Royal Perth Hospital, The Menzies Institute, Monash Medical Centre, Sunshine Coast Rheumatology, Canberra Rheumatology, Royal Prince Alfred Hospital, Sydney, New South Wales, Australia

Dysregulated angiogenesis mediated by cytokines, chemokines and growth factors has been postulated to underlie the pathogenesis of systemic sclerosis (SSc)-related pulmonary arterial hypertension (PAH). We undertook an exploratory study of these factors evaluating whether a single serum measurement could be used as a biomarker in SSc-PAH. Two main clinical groups were selected from the Australian Scleroderma Cohort Study (ASCS): Group 1 (n=20) had de inite PAH de ined by Dana Point criteria on RHC; Group 2 (n=26) were SSc controls with no evidence of cardiopulmonary disease. Serum IL-6, IL-13, FGF-2, VEGF and fractalkine levels were measured with the commercially available Millipore Milliplex MAP Human cytokine/chemokine panel. Patients in Group 1 (PAH) were older at the time of study (62±10.3 versus 48.4±10.1 yrs.) and had a longer disease duration (20.4±2.9 versus 7.6±1.3 yrs.) than patients in Group 2 (controls). The mean echocardiography de ined systolic pulmonary artery pressure (PAP) was 65.3±27.8 mmHg (Group 1) versus 26.3±2.6 mmHg (Group 2). The mean PAP in Group 1 at RHC was 39.5±12.4 mmHg. There were no signi icant differences seen in levels of IL-6 (P=0.23), IL-13 (P=0.97), VEGF (P=0.14), FGF-2 (P=0.34) and fractalkine (P=0.12) in SSc patients with and without PAH. VEGF levels (P=0.05) appear to be higher in diffuse subtype of the disease. We did not ind an association between serum IL-6, IL-13, FGF-2, VEGF, S36

fractalkine and SSc-PAH. While these factors may play a role in the pathogenesis of SSc and SSc-PAH, their serum levels do not appear to correlate with clinical PAH. 3.13

Treatment for pulmonary arterial hypertension complicating congenital heart disease in adults: Results from a national registry Rose M, Strange G, Kermeen F, King I, Vidmar S, Grigg L, Weintraub R, and Celermajer D (on behalf of the ANZ CHD-PAH Registry) The Royal Children’s Hospital, Melbourne; The Prince Charles Hospital, Brisbane; Royal Melbourne Hospital, Melbourne; Royal Prince Alfred Hospital, Sydney, New South Wales, Australia

Pulmonary arterial hypertension (PAH) complicates 5-10% of adult congenital heart disease (CHD). Improving survival in CHD patients has resulted in a new cohort of adults for whom PAH-speci ic therapy has recently become available. We established a nationwide registry for adults with PAH complicating CHD, documenting lesions, treatment patterns and outcomes. This multicenter, prospective, web-based registry enrolls patients with CHD-related PAH being followed in a tertiary center, since 1/1/2000. The inclusion criteria include age >16 years, a measured mPAP >25 mmHg at rest, echocardiographic evidence of PAH, or a diagnosis of Eisenmenger syndrome. Standardized data were collected by a single observer during site visits. There are 137 patients enrolled with 134 (97.8%) being in WHO functional Class II or III at their irst eligible visit. The current mean (SD) age was 38.2 (12.7) years. Of the patients, 134 of 137 (97.8%) had congenital systemic-pulmonary shunts and 97 (70.8%) patients had never undergone intervention. At latest follow-up, a total 89 of 137 (65.0%) patients were receiving a PAH-speci ic therapy, including an endothelin receptor antagonist in 77 (56.2%), a PDE5 inhibitor in 20 (14.6%), a prostanoid in 2 (1.5%) and a calcium channel blocker in 4 (2.9%). Anticoagulants (warfarin or antiplatelet agent) were being used in 31 (22.6%) and diuretics in 30 (21.9%). PAH complicating often complex CHD is an increasing clinical problem. Of symptomatic adults with CHD associated PAH, 35% were not receiving PAH-speci ic therapy. The proportion of untreated patients may well be higher outside tertiary centers. 3.14

Serum ICAM-1 levels are related to the presence of interstitial lung disease in systemic sclerosis Thakkar V, Patterson K, Stevens W, Byron J, Moore O, Roddy J, Zochling J, Sahhar J, Nash P, Tymms K, Youssef P, Proudman S, Hissaria P, and Nikpour M St. Vincent’s Hospital Melbourne, Royal Adelaide Hospital, Royal Perth Hospital, The Menzies Institute Hobart, Monash Medical Centre, Sunshine Coast Rheumatology, Canberra Rheumatology, Royal Prince Alfred Hospital Sydney, New South Wales, Australia

Recent studies have suggested elevated ICAM-1 (intercellular adhesion molecule-1) and VCAM-1 (vascular cell adhesion molecule-1) levels may be markers of pulmonary arterial hypertension in systemic sclerosis (SSc-PAH). Four clinical groups were selected from the Australian Scleroderma Cohort Study (ASCS): Group 1 (n=20) had de inite PAH; Group 2 (n=19) had ILD; Group 3 (n=23) were SSc controls; Group 4 (n=34) were normal healthy controls. Patients with LV dysfunction were excluded. Serum VCAM-1 and ICAM-1 levels were measured using the Millipore Milliplex MAP Human 2-Plex Panel (Millipore Corporation, Billerica, MA, USA). Mean ICAM-1 levels were signi icantly higher in the ILD group compared to the PAH (380.4±168.3 versus 266.4±88.4 ng/ml, P=0.035), SSc control (380.4±168.3 versus 257.3±97.8 ng/ml, P=0.006) Pulmonary Circulation | October-December 2011 | Vol 1 | No 4

Abstracts

and healthy control (380.4±168.3 versus 201.8±57.2 ng/ml, P<0.0001) groups. Notably there was no signi icant difference between the PAH group and SSc or healthy normal controls. Among those with ILD there were no signi icant differences in ICAM-1 levels between those who did or did not have previous cyclophosphamide treatment for SSc-ILD (P=0.36). VCAM-1 levels were shown to be signi icantly higher in SSc patients than normal healthy controls (1420.0±53.4 versus 1125.6±46.9 ng/ml, P=0.0005) but were not speci ic for PAH. ICAM-1 levels are associated with the presence of signi icant SSc-ILD. However, ICAM-1 level does not appear to be a speci ic marker for the presence of PAH. VCAM-1 levels are raised in SSc patients but are not speci ic for PAH. 3.15

Predictors of six-minute walk distance in patients with systemic sclerosis associated pulmonary hypertension Tuppin M, Chambers D, Slaughter R, Mohammed O, and Kermeen F School of Medicine, Queensland Centre for Pulmonary Trasnplantation and Vascular Disease, Department of Medical Imaging, The Prince Charles Hospital, The University of Queensland, Brisbane, Queensland, Australia

The six-minute walk distance (6-MWD) is frequently used as a measure of exercise capacity in patients with systemic sclerosis-associated pulmonary arterial hypertension (SSc-PAH). However, multiple patient factors may have an in luence on the distance achieved. Therefore, this study aimed to evaluate 6-MWD predictors in patients with SSc-PAH. A retrospective analysis of patients with a diagnosis of SSc-PAH treated at the Queensland Centre for Pulmonary Transplantation and Vascular Disease between 2002 and 2010 was conducted. A regression analysis (automatic linear modeling, forward stepwise, information criterion) for 6-MWD using measures of hemodynamics and respiratory function, as well as interstitial lung disease features evident on HRCT, was conducted. Fifty-seven patients with SSc-PAH (50 female), of mean (SD) age of 60.7 (9.6) years and mean (SD) PAP of 44.3 (14.3) mmHg were included in the analysis. The most predictive model had 45.0% accuracy, with a mean (SD) residual of 0.000 (1.011). It retained only the forced vital capacity (B=118.520), diffusing capacity for carbon monoxide (B=19.760), alveolar volume (B=-90.036), interlobular septal thickening (B=-12.879) diastolic pulmonary arterial pressure (B=-2.730) and ground-glass opaci ication (B=-4.225) as signi icant covariates, with an intercept of 303.581. These indings suggest that the HRCT features of groundglass opaci ication and interlobular septal thickening, in addition to respiratory function and hemodyamics, are also important predictors for the 6-MWD. This may be a function of the underlying pathology that is not captured satisfactorily through measures of respiratory and haemodyamics, and should be taken into account when evaluating the 6-MWD in patients with SSc-PAH.

with iPAH or CTD-PAH (NYHA Class II–III) underwent a crossover study involving a 12-week period of “usual activity” followed by a 12-week TC intervention program. Primary outcome measures of 6-MWD, resting and exercise SpO2, BORG scale, heart rate response, Cambridge Pulmonary Hypertension Outcome Review (CAMPHOR), Depression and Anxiety Stress Scale (DASS), and the Spirituality Index of Well-Being (SIWB) were performed at baseline, 12 weeks and 24 weeks. The cohort consisted of 10 females with a mean age of 54 years (SD=13.76). Comparisons between baseline and 12-week data revealed no signi icant differences in 6-MWD (t=0.844, P=0.42), resting SpO2 (t=-0.971, P=0.36), BORG (t=0.31, P=0.76) and heart rate response (t=-1.35, P=0.21), and exercise SpO2 (t=-1.514, P=0.16) and BORG (t=-0.87, P=0.40). Comparison of psychosocial outcomes between baseline and 12-week data revealed no signi icant differences in total scores on the CAMPHOR (t=-0.25, P=0.81), DASS (t=-0.27, P=0.80) and SIWB (t=1.59, P=0.14). Initial results have suggested that the study cohort had stable physical and psychosocial status, with outcomes following TC intervention to be analyzed and presented. 3.17

The hidden risk of a 6-minute walk test in pulmonary arterial hypertension Seale H, Harris J, Hall K, and Kermeen F School of Public Health and Community Medicine, University of New South Wales, Sydney, New South Wales, Australia

The 6-minute walk test (6-MWT) is a standard clinical estimate of functional capacity and is routinely used to evaluate response to speci ic pulmonary arterial hypertension (PAH) therapies. While malignant ventricular arrhythmias are reported to be rare in PAH patients, atrial ibrillation and atrial lutter are equally common and invariably associated with deterioration and right ventricular failure which may potentially increase the risk of performing a 6-MWT. Electrocardiographic monitoring is not routinely recorded during a 6-MWT. Case report of ventricular ibrillation (VF) arrest following a 6-MWT: A 25-year-old female with surgically repaired congenital heart disease with associated pulmonary hypertension on combination PAH therapy; Echocardiography: Severe RV dilatation, moderate RA dilatation, RVSP 61 mmHg, moderate LV dysfunction with LV EF 43%. RHC: mean PAP 37 mmHg, CI 2.1 L/min/m2 PVR 10 WU, WHO class ll. 6-MWT: 610 m, SpO2 nadir 85%. One minute post 6-MWT, a patient suffered VF cardiac arrest. Coronary angiogram and IVUS (intravascular ultrasound) with exercise excluded compression of left main coronary artery from a dilated pulmonary artery as possible cause. Review of de ibrillator and pacemaker rhythm suggestive of polymorphic VT secondary to LV dysfunction as probable cause. Implantable de ibrillator inserted. This case highlights the need for identi ication of patients at risk of cardiac arrhythmias associated with PAH and the need for ECG monitoring while undertaking simple tests of functional capacity.

3.16

Stretching the boundaries: The effectiveness of Tai chi in PAH Harris JE, Seale HE, McKinnon K, Cornwell PL, Morris N, and Kermeen FD Johns Hopkins Medical Institutions, Baltimore, MD, USA

Psychological and cardiopulmonary bene its of Tai chi (TC) have been reported for healthy populations, and in a wide range of patient groups, including hypertension and heart failure. The effectiveness of TC has never been studied in pulmonary arterial hypertension (PAH). The aim of this study was to assess the bene its of TC in relation to psychosocial and cardiopulmonary outcomes, in idiopathic PAH (iPAH) and PAH related to connective tissue disorders (CTD-PAH). Twelve patients Pulmonary Circulation | October-December 2011 | Vol 1 | No 4

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Symptoms to definitive diagnosis of pulmonary arterial hypertension Strange G, Keogh A, Stewart S, Carrington M, Kermeen F, Williams T, and Gabbay E Monash University, Melbourne; St Vincent’s Hospital, Sydney; Royal Perth Hospital, Perth; Lung Institute of Western Australia; University of Notre Dame; Baker IDI, Melbourne; The Prince Charles Hospital Melbourne; The Alfred Hospital, Melbourne; and University of Western Australia, Australia

Diagnosis of pulmonary arterial hypertension (PAH) has historically been delayed. We examined time to diagnosis (TTD) and factors potentially S37

Abstracts

contributing to any delay. We retrospectively enrolled consecutively diagnosed patients with idiopathic PAH (iPAH) from four pulmonary hypertension (PHT) centers (total n=32) throughout Australia (January 2007 and December 2008). All patients underwent right heart catheterization (RHC); other forms of PHT were excluded. Patients were interviewed by an examiner blinded to history prior to review of medical records. Multivariate regression analysis was performed to determine factors correlated with TTD. On RHC, mPAP (43.9±13.6 mmHg), PVR (8.8±5.7 wood units), CI (2.74±0.92) and PCWP (11.74±3.76 mmHg) were consistent with PAH. Mean TTD from symptom onset was 47.1±34.2 months. Functional class (FC) at symptom onset was FC II (95%) FC III (5%), compared to FC II (5%), FC III (90%) and FC IV (5%) at diagnosis. Patients were reviewed 5.0 (IQR 1-10) times by their general practitioner (GP) and consulted an average of three specialists prior to diagnosis of iPAH. TTD in iPAH patients was 3.9 years, suggesting signi icant delays persist. Patients experienced a FC deterioration during this time of delay. Factors signi icantly associated with a delay in diagnosis include older age, number of GP visits, higher systolic BP, and lower HR. 3.19

Pulmonary hypertension is a common disease: The Armadale echo study

Strange G, Playford D, Stewart S, Kent A, Deague J, and Gabbay E Monash University, Melbourne, Australia; Royal Perth Hospital, Western Australia; University of Notre Dame Western Australia; Lung Institute of Western Australia, The Baker IDI, Melbourne, Australia

There have been few studies describing the epidemiological of pulmonary hypertension (PHT). We examined the prevalence of all forms of PHT in a population found to have elevated right ventricular systolic pressures (RVSP) at echocardiography (echo). Armadale is a town of 165,450 people 40 kilometers from Perth, Western Australia. We extracted the estimated RVSP (eRVSP) from 10,314 patients (15,633 studies) in our laboratory between 2003 and 2009. We included all patients with eRVSP >40 mmHg or with insuf icient tricuspid regurgitation for eRVSP. Between 2003 and 2009, 936 patients were found to have PASP >40 mmHg (9%), and 3,321 patents had insuf icient TR to accurately estimate RVSP (32%). We calculated a minimum cumulative prevalence of 3,257 patients/million of the population for all forms of PHT. In an unselected population of patients in a general echo laboratory, PHT is common (9% of patients) with a cumulative prevalence of all forms of PHT at least 3,257 patients/million of the population. In our population, a signi icant proportion of patients had no obvious cause for their PHT (15%).

Announcement

Android App A free application to browse and search the journal’s content is now available for Android based mobiles and devices. The application provides “Table of Contents” of the latest issues, which are stored on the device for future offline browsing. Internet connection is required to access the back issues and search facility. The application is compatible with all the versions of Android. The application can be downloaded from https://market.android.com/details?id=comm.app.medknow. For suggestions and comments do write back to us. S38

Pulmonary Circulation | October-December 2011 | Vol 1 | No 4

Request for Proposal

The Cardiovascular Medical Research and Education Fund (CMREF) is seeking proposals from qualified academic medical centers with the ability to coordinate and conduct a clinical study evaluating right ventricular function in patients with pulmonary hypertension, its adaptive and maladaptive responses, and the effects of therapy. For more information, please go to the CMREF website: www.ipahresearch.org

Studies of the Right Ventricular Response to Therapy in Patients with Pulmonary Arterial Hypertension Introduction The Cardiovascular Medical Research and Education Fund (CMREF) is seeking proposals from qualified academic medical centers with the ability to coordinate and conduct a clinical study evaluating right ventricular function in patients with pulmonary hypertension, its adaptive and maladaptive responses, and the effects of therapy. Background The purpose of this clinical feasibility study is to better characterize the status of the right ventricle in patients with advanced pulmonary hypertension (PH) who are candidates for a new or additional treatment. Research in this field has identified changes in the right ventricle as predictive of survival in chronic PH. Understanding how the right ventricle adapts to pulmonary hypertension, and how that response may be altered with medical therapies appears to be critical. Scientific studies that may be relevant to this project cover a broad spectrum of disciplines that include genomics, imaging, and assessments of RV contractility, hypertrophy and metabolism. It is hoped that knowledge gained from this study will impact clinical trials of future therapies developed to treat advanced pulmonary hypertension. Project Guidelines  Study hypotheses: Maladaptation to the pulmonary hypertensive state is largely responsible for right ventricular failure in chronic pulmonary hypertension. Changes that occur in the right ventricle predict clinical improvements in patients undergoing treatments for pulmonary hypertension. The applicant is requested to propose one or more related hypotheses that will be tested in this study.  The patient inclusion criteria for the study should include Category 1 pulmonary arterial hypertension, preferably from one specific etiology, and advanced disease in which a new or additional medical therapy is clinically indicated.  This can be an open label study where the patients will serve as their own controls, or randomized, placebo controlled.  It is suggested that the number of patients enrolled be adequate to test the hypotheses, rather than powered to show statistically significant drug efficacy.

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 

The follow-up assessments should be over 6 months, and patient enrollment should be completed within a 2 year period. The state of the right ventricle should be characterized with multiple parameters that will provide insight into the adaptive and maladaptive state, and reflect changes that occur from therapy that will further the understanding of how therapies may work. These parameters may include measures of: o Genetics and genomics o Metabolism o Cellular signaling o Imaging (ultrasound, PET and MRI) o Contractility and hypertrophy o Hemodynamics o RV function o Myocardial ischemia and/or perfusion

The applicant is expected to develop a clinical research study plan that will identify several assessments that will be studied in the enrolled patients. While it is desirable for a single center to enroll all patients in this study and perform all of the assessments, this may not be practical or feasible. Because PAH is an uncommon disease, it is understood that multiple clinical centers may need to participate in patient enrollment (a clinical center is one that will enroll patients into the study). In addition, given the uniqueness of some of the promising assessments, it is also understood that multiple centers may need to participate as core laboratories to evaluate specific measures (a core laboratory is one that will perform analyses of specific measured assessments collected at the clinical centers). Thus, the research plan should include an organizational plan that identifies the Principle Center, collaborating clinical centers, and core laboratories. The Principle Center will be responsible for all of the data management, and for providing oversight of the entire conduct of the study. The Principle Center should also serve as a clinical center and a core laboratory. Having other clinical centers also serving as core laboratories to expand the different types of assessments will be considered a strength of the proposal. This award can be made to the Principle Center, with subcontracts to clinical and core centers, or to each participating center directly. The applicant should submit a research plan using the NIH R01 format that will include the collaborating investigators for all phases of the study. Required activities by the applicant include, but are not limited to:  finalizing the clinical protocol and protocol-related documents (e.g. informed consent form, case report forms, etc.);  implementation and conduct of the clinical study in accordance with Federal requirements;  management and oversight of the conduct of the clinical study;  providing clinical research support services;  providing monitoring and safety oversight;  organizing investigator meetings and teleconferences;  recruiting patients;  organizing, conducting, and reimbursing for patient protocol evaluations and tests (including follow-up);  analyzing the data and authoring and submitting papers on the study for publication. Application Guidelines  The deadline for grant submission is July 1, 2012. Funding will begin October 1, 2012. All applicants are requested to submit a letter of intent with a brief description of their proposal, and the collaborating institutions.  The direct award for this study will be limited to $2 million, inclusive of all subcontracts. Overhead of 20% will be added to the amount awarded. The award may not be used to cover healthcare related costs that are otherwise clinically indicated, including but not limited to the costs of the medical therapy to be studied if it is clinically indicated.  Because of the high expense of healthcare costs, the applicant is advised to recruit patients where the preponderance of tests and treatments are otherwise clinically indicated and thus will not need to be covered by the grant.  The project should be constructed to meet the following timelines: o 6 months start-up  Case report form completion  Study protocols completed  Research assessments specified  Consent and IRB approvals

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Pulmonary Circulation | October-December 2011 | Vol 1 | No 4

24 months patient enrollment  Enrollment monitors o 6 months close-out  Data analyses  Publications The NIH R01 research application form should be used, including Specific Aims, Background and Importance and Approach (Research Plan), Human Subjects Section, and Biosketches and description of facilities and resources. The following should be included in the response to the RFP: o The research strategy should include a clear description of the study design, study population, subject eligibility and inclusion/exclusion criteria, recruitment and enrollment plans, and outcome measures.  State concisely the goals of the proposed research and summarize the expected outcome(s), including the impact that the results of the proposed research will exert on the research field(s) involved. The major research question and hypothesis being studied should be clearly stated. o The significance of the proposed clinical trial must be clearly stated. The application should make clear how the results will advance our knowledge of theory and practice in this area and the potential impact of the results of the trial. o Explain how the application challenges and seeks to shift current research or clinical practice paradigms. o A list of subject eligibility and inclusion and exclusion criteria should be provided. A recruitment and enrollment plan, including a discussion of the availability of subjects for the proposed study, the ability of enrollment sites to recruit the required number of subjects, and the timeline for completion of recruitment, should be described. o There should be a detailed description of the assessments to be tested. Potential biases and approaches for minimizing bias should be described. The primary and secondary endpoints, and methods/measures to be used to assess these, should be clearly described. The link between endpoints, outcome measures, and hypotheses should be stated clearly. o Data collection plans and statistical methods appropriate for the particular design proposed should be presented. Methods to be used for data collection, preparation, management, quality control should be thoroughly described. o Data from preliminary or pilot studies which show the need for and the feasibility of the trial should also be presented. Additional supporting data from other research should be included so that the approach chosen is clearly justified. This information will also help to establish the experience and competence of the investigators to pursue the proposed project. o A timetable for completion of the various stages of the trial must be included. o



Data Safety and Monitoring All applications must include a general description of the monitoring plan, policies, procedures, responsible entities, and approaches to identifying, managing and reporting reportable events (adverse events and unanticipated problems), to the applicable regulatory agencies (e.g., Institutional Review Board (IRB), the NHLBI/NIH, the Office of Biotechnology Activities, Office of Human Research Protections (OHRP), the (FDA), and the Data and Safety Monitoring Board (if one is used). Contact information Inquiries, letters of intent, applications or questions should be addressed to: Cardiovascular Medical Research and Education Fund 510 Walnut Street, Suite 500 Philadelphia, PA 19106, USA Phone: 215-413-2414 Fax: 215-592-4663 Email: patt.wolfe@ipahresearch.org Website: www.ipahresearch.org

Pulmonary Circulation | October-December 2011 | Vol 1 | No 4

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NOW ENROLLING A Phase III, Randomized, Double-blind, Placebo-controlled, Multi-centre, Multi-national Study to Evaluate the Efﬁcacy and Safety of a Developmental Compound in: Patients with Symptomatic Pulmonary Arterial Hypertension (PAH).

Main Inclusion Criteria: Male and female patients aged 18–80 years with s

Symptomatic PAH (idiopathic, familial, associated PAH due to connective tissue disease, congenital heart disease, portal hypertension with liver cirrhosis, or due to anorexigen or amphetamine use)

Treatment-naïve patients and patients pre-treated with an Endothelin Antagonist or a Prostacyclin Analogue (except I.V.)

Main Exclusion Criteria: s

All types of pulmonary hypertension except subtypes of Venice Group 1 speciﬁed in the inclusion criteria, severe COPD, uncontrolled arterial hypertension, and left heart failure

The study will progress for a duration of 14 weeks, including screening s

GPs will be notiﬁed if any of their patients participates in the study

I.V.: intravenous COPD: chronic obstructive pulmonary disease

FOR MORE INFORMATION Please see: www.clinicaltrials.gov Study identiﬁer: NCT00810693 Contact: Dr Joanna Pepke-Zaba, telephone +44 1480 364230 or email joanna.pepkezaba@papworth.nhs.uk Response to this advertisement will be recorded but will not indicate any obligation This study is sponsored by Bayer HealthCare. This advertisement has been approved by the NRES Committee East of England – Cambridge East. UK.PH.GM.RIO.2011.300

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September 2011

Pulmonary Circulation | October-December 2011 | Vol 1 | No 4

Main Inclusion Criteria: Male and female patients aged 18–80 years with s

Inoperable CTEPH

Pulmonary hypertension that persists or is recurrent after PEA

Main Exclusion Criteria: s

All types of pulmonary hypertension except subtypes 4.1 and 4.2 of the Venice Clinical Classiﬁcation of Pulmonary Hypertension

The study will progress for a duration of 20 weeks, including screening s

GPs will be notiﬁed if any of their patients participates in the study

PEA: pulmonary endarterectomy

FOR MORE INFORMATION Please see: www.clinicaltrials.gov Study identiﬁer: NCT00855465 Contact: Dr Joanna Pepke-Zaba, telephone +44 1480 364230 or email joanna.pepkezaba@papworth.nhs.uk Response to this advertisement will be recorded but will not indicate any obligation This study is sponsored by Bayer HealthCare. This advertisement has been approved by the NRES Committee East of England – Cambridge East. UK.PH.GM.RIO.2011.300

Pulmonary Circulation | October-December 2011 | Vol 1 | No 4

September 2011

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CONTENTS Editorial No doctor is an island Jason X. -J. Yuan, Nicholas W. Morrell, S. Harikrishnan, and Ghazwan Butrous

435

Guest Editorial Re lections on the rise and fall of PVD, medical nanotechnology and Australia covered with house lies Paul Soderberg

437

Review Article Obesity and pulmonary arterial hypertension: Is adiponectin the molecular link between these conditions? Ross Summer , Kenneth Walsh, and Benjamin D. Medoff

440

Research Articles FDG PET imaging can quantify increased cellular metabolism in pulmonary arterial hypertension: A proof-of-principle study

Guy Hagan, Mark Southwood, Carmen Treacy, Robert MacKenzie Ross, Elaine Soon, James Coulson, Karen Sheares, Nicholas Screaton, Joanna Pepke-Zaba, Nicholas W. Morrell, and James H. F. Rudd

448

456

High-altitude pulmonary hypertension in cattle (brisket disease): Candidate genes and gene expression pro iling of peripheral blood mononuclear cells John H. Newman, Timothy N. Holt, Lora K. Hedges, Bethany Womack, Shaϔia S. Memon, Elisabeth D. Willers, Lisa Wheeler, John A. Phillips III, and Rizwan Hamid

462

470

475

487

Case Report Cardiopulmonary hemodynamic clues for pulmonary vein stenosis diagnosis Mateo Porres-Aguilar, Genaro Fernandez, and C. Gregory Elliott

499

History and Who’s Who Lofty goals at high altitude: The Grover Conferences, 1984–2011 E. Kenneth Weir, Wiltz W. Wagner Jr., and Stephen L. Archer

501

Author Index, 2011

508

Title Index, 2011

510

Scientific Abstracts

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