Pulmonary Circulation 1:3 2011 by Pulmonary Vascular Research Institute

COVER PHOTO: Visual signatures of the morphological changes undergone by a single healthy human pulmonary artery smooth muscle cell moving across a fibrillar type I collagen thin film substrate. Data captured via time-lapse phase-contrast microscopy over 14 hours, and analyzed and visualized with custom geometric algorithms within 3-D architectural graphics software. In addition to the cell’s shape changes over time, quantitative parameters of cell motility are also visually represented, such as the furthest reach from the cell’s centroid, pictured as gray circles and magenta lines. Credit: Sabin+Jones LabStudio, University of Pennsylvania; Erica S. Savig, Mathieu C. Tamby, Jenny E. Sabin and Peter L. Jones; Illustration Credit: Erica S. Savig

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Pulmonary Circulation

ISSN 2045-8932, E-ISSN 2045-8940

An official journal of the Pulmonary Vascular Research Institute Editors-in-Chief

Senior Editor

Jason X.-J. Yuan, MD, PhD (Chicago, USA) Nicholas W. Morrell, MD (Cambridge, UK) Harikrishnan S., MD (Trivandrum, India)

Ghazwan Butrous, MD (Canterbury, UK)

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)

Editors

Executive Editor

Harikrishnan S., MD (Trivandrum, India)

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)

Scientific Advisory Board

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)

Robert F. Grover, MD, PhD (Denver, USA) Joseph Loscalzo, MD (Boston, USA) Charles A. Hales, 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. Cornfield, 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. Forfia, 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 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

Editorial Staff

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

Nikki Krol (London, UK), nkrol@imperial.ac.uk Karen Gordon (Chicago, USA), gordonk@uic.edu Paul Soderberg (Phoenix, USA), paulsoderberg@hotmail.com

Pulmonary Circulation | July-September 2011 | Vol 1 | No 3

Pulmonary Circulation

| July-September 2011 | Vol 1 | No 3 |

An official journal of the Pulmonary Vascular Research Institute

CONTENTS Chromosome

Hypotension

Increased RVEDP

LEFT TO RIGHT: 328, 349, 371, 394

Histone modification

Nucleosome

Reduced RV coronary blood flow miRNA

RV ischemia

mRNA

RNA interference

mRNA Protein Me

Decreased cardiac output DNA methylation

General Information

inside front cover

Editors and Board Members

Editorial

The world of pulmonary vascular disease

Jason X.-J. Yuan, Nicholas W. Morrell, S. Harikrishnan, and Ghazwan Butrous

303

Review Articles The genetics of pulmonary arterial hypertension in the post-BMPR2 era

305

COPD/emphysema: The vascular story

320

Surgical treatment of pulmonary hypertension: Lung transplantation

327

Apelin and pulmonary hypertension

334

Epigenetic mechanisms of pulmonary hypertension

347

MicroRNAs-control of essential genes: Implications for pulmonary vascular disease

357

Joshua P. Fessel, James E. Loyd, and Eric D. Austin

Norbert F. Voelkel, Jose Gomez-Arroyo, and Shiro Mizuno

Jason Long, Mark J. Russo, Charlie Muller, and Wickii T. Vigneswaran

Charlotte U. Andersen, Ole Hilberg, SĂ¸ren MellemkjĂŚr, Jens E. Nielsen-Kudsk, and U. Simonsen

Gene H. Kim, John J. Ryan, Glenn Marsboom, and Stephen L. Archer

Sachindra R. Joshi, Jared M. McLendon, Brian S. Comer, and William T. Gerthoffer

Research Articles Blood flow redistribution and ventilation-perfusion mismatch during embolic pulmonary arterial occlusion K. S. Burrowes, A. R. Clark, and M. H. Tawhai

365

Pulmonary Circulation | July-September 2011 | Vol 1 | No 3

CONTENTS continued

Pulmonary hemodynamic responses to inhaled NO in chronic heart failure depend on PDE5 G(-1142)T polymorphism Thibaud Damy, Pierre-François Lesault, Soulef Guendouz, Saadia Eddahibi, Ly Tu, Elisabeth Marcos, Aziz Guellich, Jean-Luc Dubois-Randé, Emmanuel Teiger, Luc Hittinger, and Serge Adnot

Pharmacogenomics in pulmonary arterial hypertension: Toward a mechanistic, target-based approach to therapy Sami I. Said and Sayyed A. Hamidi

Idiopathic and heritable PAH perturb common molecular pathways, correlated with increased MSX1 expression

Eric D. Austin, Swapna Menon, Anna R. Hemnes, Linda R. Robinson, Megha Talati, Kelly L. Fox, Joy D. Cogan, Rizwan Hamid, Lora K. Hedges, Ivan Robbins, Kirk Lane, John H. Newman, James E. Loyd, and James West

S1P4 receptor mediates S1P-induced vasoconstriction in normotensive and hypertensive rat lungs Hiroki Ota, Michelle A. Beutz, Masako Ito, Kohtaro Abe, Masahiko Oka, and Ivan F. McMurtry

Fenfluramine-induced gene dysregulation in human pulmonary artery smooth muscle and endothelial cells Weijuan Yao, Wenbo Mu, Amy Zeifman, Michelle Lofti, Carmelle V. Remillard, Ayako Makino, David L. Perkins, Joe G. N. Garcia, Jason X. J. Yuan, and Wei Zhang

Neurogenic responses in rat and porcine large pulmonary arteries Daniel J. Duggan, Detlef Bieger, and Reza Tabrizchi

377

383

389

399

405

419

Case Report Isolated large vessel pulmonary vasculitis as a cause of chronic obstruction of the pulmonary arteries Guy Hagan, Deepa Gopalan, Colin Church, Doris Rassl, Chetan Mukhtyar, Trevor Wistow, Chim Lang, Pasupathy Sivasothy, Susan Stewart, David Jayne, Karen Sheares, Steven Tsui, David P. Jenkins, and Joanna Pepke-Zaba

Snapshot Survival in pulmonary arterial hypertension: A brief review of registry data

PASMC3

(c)

PASMC1

PASMC2

PAEC

Sunil Pauwaa, Roberto F. Machado, and Ankit A. Desai

430

(c)

LEFT TO RGHT: 402, 409, 415, 427

Pulmonary Circulation | July-September 2011 | Vol 1 | No 3

425

iii

Corrigenda In the last issue, the Snapshot by Desai A and Machado R (Drugs currently used for treatment of PAH) presented a column labeled â&#x20AC;&#x153;Companyâ&#x20AC;? for each drug used in PAH.Â One aim of this column was to provide a name of a representative company for each drug available in the US, which is accurate in its depiction in the table. This column, however, does not comprehensively capture the breadth, complexity, and dynamic nature of the various companies which own or have rights to produce, distribute, market the drugs listed on a global scale. To address this inadvertent error, the authors have requested to omit this section. The remainder of the contents of the table, however, accurately reflect the purpose of the table, which is to provide a concise profile of the current drugs used to treat PAH. iv

Pulmonary Circulation | July-September 2011 | Vol 1 | No 3

Edi t ori al

The world of pulmonary vascular disease The world of pulmonary vascular disease (PVD) is lopsided in this sense: those nations that have the highest numbers of PVD researchers have the lowest number of PVD patients. We ran a PubMed search for PVD researchers and found that, of all the authors of published articles on PVD, 90.3% were from the United States, Canada, the United Kingdom, France, Germany and Japan, while those from all the rest of the world totaled only 9.7% (Fig. 1). Meanwhile, schistosomiasis is very rare in North America, but is the most common cause of pulmonary hypertension worldwide.[1] In addition, the burden of HIV-related pulmonary hypertension is likely to be far greater in Africa.[2] The world’s 193 nations protect their borders, and the movement of people between nations is carefully controlled. But diseases ignore borders and nationalities, and “view” the world in their own terms. Idiopathic pulmonary hypertension (IPAH), for example, ignores nationality and focuses on gender, predominantly affecting young women. Happily, knowledge is just like disease in that sense, that it ignores national boundaries, makes a graveyard of ignorance, and improves public health.

This journal, Pulmonary Circulation, was the first-ever peer-reviewed journal dedicated to PVD, and at present it remains the only journal devoted to the field of pulmonary circulation publishing original research articles, review articles, case reports and perspectives in PVD. But we are only part of “the Voice,” which is also being expressed around the world in various ways, notably at the annual meetings of professional associations. For example, just in 2011 so far, there have been PVD conferences in Panama and in Dubai, as well as two in China and two in India, all hosted by the Pulmonary Vascular Research Institute, the PVRI, publisher of Pulmonary Circulation– over and above important meetings that have been held Paper on PVD

Global distribution of researchers studying pulmonary vascular disease

Perhaps the most basic lesson from the whole history of Medicine is that diseases thrive in a “climate” of ignorance, and are defeated by knowledge. Yellow fever decimated the world until Cuban doctor Carlos Finlay proposed, in 1881, that its vector was the female of the mosquito species Aedes aegypti, which American doctor Walter Reed confirmed in 1900. Likewise, pulmonary vascular disease is flourishing today in some parts of the world (and indeed in some parts of the so-called “developed” world) in part because of the ignorance that still surrounds it. But, again happily, that ignorance is being reduced by what can be called “the Voice of the World of PVD.”

70 60 50 40 30 20 10

1990 1995 2000 2005 2010 Time (year)

(a)

Africa

Oceana

S. America

Europe

Middle East

Number of researchers 1 760

Asia

900 600 300 0 1 2 3 4

N. America

Population Researcher (million)

(b)

(c)

Figure 1: The global distribution of researchers studying pulmonary vascular disease. (a) Intensity map depicting the number of researchers in different countries represented by relative color intensity. (b) The number of papers published on pulmonary vascular disease each year since 1990. (c) The regional distribution of researchers. The data were compiled from a PubMed-wide search by Abigail Drennan and Amy Zeifman in the University of Illinois at Chicago. Pulmonary Circulation | July-September 2011 | Vol 1 | No 3

303

throughout North America and Europe.[3] Meanwhile, educational efforts for the general public are also a critical part of the Voice of PVD, notably those being undertaken by the Pulmonary Hypertension Association, whose former board member, Gail Boyer Hayes, recently wrote, “If a village is required to raise a child, surely the whole world is needed to cure PH.”[4]

Pulmonary Circulation is therefore not merely a repository of research findings. In sharing important knowledge among the world’s physicians, physician scientists, investigators, and even patients–both in print and online–this journal is slowly but surely helping to reduce the ignorance that PVD needs in order to keep killing people. As its editors, we keep “the big picture” firmly in mind. The goal is not merely to produce a medical periodical. The goal is to empower our profession by facilitating the information exchange that will lead to new therapies, new strategies, and ultimately even cures. Our goal is not merely a world-class publication, but nothing less than a world without PVD.

When vascular and cardiopulmonary diseases no longer exist, this journal will have achieved its purpose. Until that day, we welcome the addition of your voice to ours, through your submission of manuscripts describing your best work, which will then help the work of others throughout the lopsided World of PVD.

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

REFERENCES 1. 2. 3.

Ghazwan Butrous. Schistosomiasis pulmonary hypertension: The forgotten disease. Egyptian J Bronchology 2008;2:143-6. Barnett CF, Hsue PY, Machado RF. Pulmonary hypertension: An increasingly recognized complication of hereditary hemolytic anemias and HIV infection. J Am Med Assn 2008;299:324-31. The 2011 conferences hosted by the PVRI: an international meeting in Panama (3-5 February); the 4th Annual Joint Pulmonary Hypertension Conference in Dubai (19-22 April); 2 international meetings in China (28-30 July in Shenyang and 13-14 August in Beijing); and 2 in India (21-23 September in Leh, and 1-2 October in Trivandrum). For details of these 6 conferences, visit www.pvri.info. Other important meetings were held in San Francisco, California (Neonatal and Childhood Pulmonary Vascular Diseases, 11-12 March); in Virginia (the 2011 PH Professional Network Symposium, 22-24 September); and in Colorado (the Grover Conference, 7-11 September), in addition to many other side meetings at the annual conferences of the American College of Cardiology, the American Thoracic Society, the European Society of Cardiology, and the European Respiratory Society. Gail Boyer Hayes. Pulmonary Hypertension: A Patient’s Survival Guide. US Pulmonary Hypertension Association; 2004. Access this article online Quick Response Code:

Website: www.pulmonarycirculation.org DOI: 10.4103/2045-8932.87291 How to cite this article: Yuan JX, Morrell NW, Harikrishnan S, Butrous G. The world of pulmonary vascular disease. Pulm Circ 2011;1:303-4.

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 journal’s website. 304

Pulmonary Circulation | July-September 2011 | Vol 1 | No 3

Review Ar ti cl e

The genetics of pulmonary arterial hypertension in the post-BMPR2 era Joshua P. Fessel1, James E. Loyd1, and Eric D. Austin2 1

Departments of Medicine, Division of Allergy, Pulmonary, and Critical Care Medicine, 2Pediatrics, Division of Allergy, Pulmonary, and Immunology Medicine, Vanderbilt University, Nashville, Tennessee, US

ABSTRACT Pulmonary arterial hypertension (PAH) is a rapidly progressive and fatal disease for which there is an ever-expanding body of genetic and related pathophysiological information on disease pathogenesis. The most common single culprit gene known is BMPR2, and animal models of the disease in several forms exist. There is a wealth of genetic data regarding modifiers of disease expression, penetrance, and severity. Despite the rapid accumulation of data in the last decade, a complete picture of the molecular pathogenesis of PAH leading to novel therapies is lacking. In this review, we attempt to summarize the current understanding of PAH from the genetic perspective. The most recent PAH demographics are discussed. Heritable PAH in the post-BMPR2 era is examined in detail as the most robust model of PAH genetics in both animal models and human pedigrees. Important downstream molecular pathways and modifiers of disease expression are reviewed in light of what is known about PAH pathogenesis. Current and emerging therapies are examined in light of genetic data. The role of genetic testing in PAH in the post-BMPR2 era is discussed. Finally, directions for future investigations that ideally will fulfill the promise of novel therapeutic or preventive strategies are discussed. Key Words: BMPR2, heritable pulmonary arterial hypertension, idiopathic pulmonary arterial hypertension, pulmonary arterial hypertension, right ventricle

INTRODUCTION Pulmonary arterial hypertension (PAH) is a disease of the pulmonary vasculature that is pathologically characterized by progressive neointimal proliferation leading to vasoocclusive lesions as well as to dropout and pruning of the smallest pulmonary arteries.[1] This drives the clinically apparent disease, characterized by progressive dyspnea, exercise intolerance, increasing pulmonary vascular resistance, and ultimately right ventricular failure and death.[2] Under the current classification system, WHO Group 1 PAH is divided into disease subgroups that include heritable (HPAH, formerly familial PAH or FPAH), idiopathic (IPAH), and PAH associated (APAH) with a variety of other systemic diseases or drug/toxin exposures.[3]

Untreated, PAH results in death from right heart failure in less than 3 years for most patients.[4] Despite advancements in therapies, as evidenced by FDA approval Address correspondence to:

Dr. Joshua P. Fessel Vanderbilt University Medical Center, Department of Medicine, Division of Allergy, Pulmonary and Critical Care Medicine, Suite T-1218, Vanderbilt Medical Center North, Nashville, TN 37232-2650 Email: joshua.p.fessel@vanderbilt.edu

Pulmonary Circulation | July-September 2011 | Vol 1 | No 3

of 7 drugs specifically for the treatment of PAH, the currently available therapies are, for most patients, only partially or temporarily effective. No therapies tested to date have shown any significant ability to reverse the disease, and despite suggestions in epidemiologic studies of improvement there has been no conclusive demonstration in large clinical trials of the ability of currently available therapies to prolong survival, although patients who have a good initial response to therapy may derive some survival benefit.[5-10] Much has been learned about the genetic underpinnings of PAH since its initial descriptions as primary pulmonary hypertension by Dresdale and colleagues in 1951.[11] This line of inquiry expanded to include the recognition of PAH as a familial disease in some cases, with the initial description in 1954 of a family in which PAH was identified Access this article online

Quick Response Code:

Website: www.pulmonarycirculation.org DOI: 10.4103/2045-8932.87293 How to cite this article: Fessel JP, Loyd JE, Austin ED. The genetics of pulmonary arterial hypertension in the post-BMPR2 era. Pulm Circ 2011;1:305-19.

305

Fessel et al.: PAH genetics post-BMPR2

in a mother and in her son and sister.[12] Intense study of the inheritance and genetic patterns of familial PAH ultimately led to the identification of altered transforming growth factor beta (TGF-β) signaling via the bone morphogenetic protein receptor Type 2 (BMPR2) as being the key heritable risk factor for development of PAH, [13,14] with other primary (e.g., mutations in ALK1 or ENG) and modifier genetic risk factors having been identified as well. The evolution of the current understanding of the genetics of PAH, as well as existing questions, is the topic of this review.

DISCUSSION

PAH: Definition, incidence, and demographics

For the purposes of clinical and research definition, PAH is defined as a mean pulmonary artery pressure > 25 mmHg at rest,[15] confirmed by right heart catheterization, and in the absence of other conditions known to elevate pulmonary vascular pressures, such as pulmonary embolism, left-sided heart disease, other lung diseases, and various conditions associated with PAH.[16] HPAH is a subset of PAH in which patients either belong to a pedigree known to be affected by PAH in multiple family members or are found to have a mutation in a gene known to associate with PAH (most commonly BMPR2) in what was previously thought to be a case of sporadic or IPAH. It was this latter group of patients that prompted the official change in classification from familial PAH (FPAH) to HPAH. [3] Because the mutations that drive HPAH show reduced penetrance, there can be multiple unaffected generations in a family pedigree, and thus family history can be apparently negative for pulmonary vascular disease. It is only with the advent of genetic testing for particular mutations that these patients have become apparent. In addition, genetic testing has allowed for the identification of what may be de novo mutations driving disease in patients previously classified as IPAH patients.[17]

The overall incidence of PAH is difficult to determine with accuracy, as very few studies have actually reported incidence and prevalence data, and clinical underrecognition of the disease has been a challenge until recent years. One of the most recent studies to assess incidence was a large multicenter French study looking at 17 university hospitals across France during a 1-year period from October 2002 to October 2003.[18] In this study, 18% (121 of 674) of the cases were new diagnoses during the period of the study. The low end of the range of estimated incidence was 2.4 cases per million adult population per year. Estimated prevalence was 15 cases per million, with an estimated 5.9 cases of IPAH per million adults. Within the cohort, 39.2% were classified as IPAH, and 3.9% were classified as familial. In the initial report of their study, the 306

investigators described a less severe clinical presentation for the familial PAH patients compared to IPAH. None of the familial patients presented with NYHA class IV heart failure, and as a group the familial patients had a better 6-minute walk distance than the IPAH patients. However, hemodynamics were similar in both groups. Subsequently, these same investigators compared BMPR2 mutation carriers (28 familial and 40 idiopathic PAH patients) to non-carriers (155 IPAH patients) and found that mutation carriers are diagnosed and die approximately 10 years earlier and with worse hemodynamics (e.g., higher mean pulmonary artery pressure, lower cardiac index, and lower mixed venous oxygen saturation) compared to non-carriers. Mutation carriers also have shorter times from diagnosis to death or lung transplantation, but the overall survival is similar between mutation carriers and non-carriers.[19] Following this, these investigators have examined the clinical presentation in HPAH caused by mutations in the activin A receptor Type II-like kinase-1 (ACVRL1 or ALK1), a receptor in the TGF-β receptor family. Mutations in ALK1 are associated with PAH and with hereditary hemorrhagic telangiectasia (HHT). Comparing a small group (32 patients) of ALK1 mutation carriers to both BMPR2 mutation carriers and IPAH non-carriers, the ALK1 mutation carriers presented at a younger age than even the BMPR2 mutation carriers. Despite better hemodynamics at the time of diagnosis and despite similar PAH therapies, the ALK1 mutation carriers had shorter survival times compared to BMPR2 carriers and to noncarriers.[20] Overall, then, the data from the French Registry and others suggest that while histopathologically identical, there may be subtle differences between HPAH and IPAH that impact the clinical presentation and progression of disease in the two groups.[21] The most recent and largest observational cohort study of PAH to date has been compiled by the Actelionsponsored Registry to Evaluate Early and Long-term PAH Disease Management (REVEAL) database. The registry was designed to enroll 3,000 prevalent and/or incident patients from 54 centers in the United States with WHO Group 1 PAH and to study their baseline characteristics as well as to examine their clinical progression and responses to therapy in a prospective way.[22] The baseline characteristics on the first 2,525 adult patients enrolled between March 2006 and September 2007 have been reported.[23] The proportions of PAH defined as IPAH and familial PAH were 46.2% and 2.7%, respectively, in line with what was described for the French Registry initially. Of note, subgroups of WHO Group 1 PAH were determined by the clinician enrolling the patient in the registry, based upon his/her assessment of what was the most likely etiology of the patient’s PAH. Thus, genetic testing was not a necessary criterion, and the FPAH patients defined as such are not identical to the HPAH patients that have Pulmonary Circulation | July-September 2011 | Vol 1 | No 3

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been described in the later analyses from the French Registry. Some of the salient differences between REVEAL and the French Registry as well as the 1987 NIH Registry have recently been summarized.[24] No studies from the REVEAL Registry comparing FPAH to IPAH have been forthcoming.

One of the most interesting findings to come out of the REVEAL Registry to date that may have a major impact on the future understanding of PAH disease and diseasemodifying genetics has been the finding that 79.5% of the adult PAH patients in the registry are female. A female predominance has been noted in previous studies both in the U.S. and in the French Registry, with the female predominance being even more pronounced among blacks in these studies. The female-to-male ratio of 4.1:1 is much higher than that reported in the 1987 NIH registry (1.7:1)â&#x20AC;&#x2030;[4] or in the French Registry (1.9:1), but is in keeping with what was observed in the Surveillance of Pulmonary Hypertension in America Registry (4.3:1).[25] The prevalent hypothesis is that estrogen or its metabolites have an influence on development of disease and/or survival, and indeed recent studies have supported this notion (see below: Modifiers of BMPR2-mediated PAH, subsection Steroid hormones).

Heritable PAH and BMPR2

Dresdale first recognized a familial or heritable component to PAH in 1954, soon after the initial description in the literature of the disease itself.[12] In the 30 years that

followed, a total of 13 families were described in the English literature with features of a heritable disease fitting the description of PAH. In 1984, these 13 families were reviewed and the pedigrees expanded by the PAH group at Vanderbilt, and a 14th family was added with 6 PAH deaths (all in women) already identified at that time.[26] We have continued to follow this family (Fig.â&#x20AC;Ż1), which now has 36 members diagnosed with HPAH (29 females and 7 males) and at least another 48 members who are unaffected but who are obligate carriers of what has been identified as a mutation in the ligand-binding domain of BMPR2. Prior to the identification of BMPR2 as the major heritable risk factor for HPAH, there were a number of things known about the genetic behavior of HPAH, not all of which have been explained since the discovery of the culprit gene. The inheritance pattern in HPAH is best described as autosomal dominant with reduced penetrance.[26,27] The penetrance is highly variable, from 20%-80%. This strongly suggests the presence of modifying factors, genetic or environmental, that confer increased or decreased risk. Age of onset or diagnosis of the disease is highly variable, with HPAH being diagnosed any time from infancy out to 70 years of age. As mentioned above, there is a female predominance in PAH that is seen in all forms of WHO Group 1 PAH including HPAH. Interestingly, there have been several reports of HPAH occurring at earlier and earlier ages in subsequent generations, a phenomenon known as genetic anticipation.[28]

Figure 1: Updated pedigree of the 14th family reported in the literature with HPAH. Symbols are standard for pedigree analysis and are explained in the figure caption. This family carries a mutation in the ligand-binding domain of BMPR2. Pulmonary Circulation | July-September 2011 | Vol 1 | No 3

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There are two major genetic mechanisms by which anticipation is known to occur. The first is trinucleotide repeat expansion, first described for Fragile X syndrome and now recognized to be causative or contributory in 40 or more neurologic diseases, most of which exhibit genetic anticipation.[29] The second mechanism is progressive telomere shortening, as has been described to explain the genetic anticipation observed with dyskeratosis congenita.[30] This is a disease characterized by mutations in telomerase reverse transcriptase, which is one of the enzymes responsible for maintaining telomere length in humans. Both of these mechanisms have been investigated in HPAH, and to date neither has proven to be the explanation for the apparent genetic anticipation in families with PAH.[31] There is a single report in the literature from Uziel et al. describing apparent genetic anticipation of neurologic disease associated with the mitochondrial mutation T8993G in 6 pedigrees. In this relatively small study, anticipation of symptoms correlated with increasing degree of heteroplasmy for the mitochondrial mutation. [32] This potential mechanism has not been investigated in HPAH, and to date no mutations in the mitochondrial genome itself have been associated with HPAH. An important alternative explanation to consider is statistical artifact or bias, particularly ascertainment bias. With progressive improvements in genetic and clinical screening in families with PAH, it could simply be that family members are diagnosed earlier or with a greater frequency at younger ages than they would have been in prior generations. Or, sufficient time has not passed to allow for additional “older age” diagnoses to occur in more recent generations. However, given the particularly poor survival in HPAH, the temporal distance between diagnosis and death is short enough that profound differences in age at diagnosis according to ascertainment bias are less likely. Regardless, until a biological mechanism can be demonstrated to explain the anticipation phenomenon observed in HPAH, the possibility of statistical artifact will remain. With this background information, two teams working independently first began to localize the gene responsible for the majority of HPAH in the mid-1990s. Using linkage analysis referenced to short tandem repeats and to other microsatellite markers, both groups identified chromosome 2q31-33 as the region of interest and published their findings in 1997.[33,34] Again working independently, both groups subsequently found that the mutated gene responsible was BMPR2. Deng et al. used a modified candidate gene approach, examining 3 genes by denaturing high performance liquid chromatography (DHPLC) and identifying 5 nonsense and 2 missense mutations in BMPR2, which were not present in 196 control samples.[35] Lane et al. used positional cloning of patient genomic samples and a candidate gene approach to examine 17 possible genes in the region of 2q33 and 308

identified 2 frame-shift, 2 nonsense, and 3 missense mutations in BMPR2.[36] Subsequent to these reports in 2000, there have been numerous studies that have identified nearly 300 different mutations in BMPR2 using methods as diverse as direct sequencing, melting curve analysis, DHPLC, Southern blotting, and multiplex ligation-dependent probe amplification.[17] Mutations in BMPR2 account for 75%-80% of the cases of HPAH, with a small percentage of cases attributed to known mutations in other TGF-β family members (e.g., ACVRL1/ ALK1 or endoglin, ENG). The remaining cases of HPAH that are negative for known mutations may well have as yet unidentified alterations in genes in the TGF-β pathway, as exemplified by a case report describing a Smad 8 gene mutation in a patient with sporadic PAH.[37]

At the time of its identification as the gene responsible for the majority of cases of HPAH, BMPR2 possessed biological plausibility. It was already known that mutations in ACVRL1/ALK1 or in ENG were causative in HHT and were associated with pulmonary arterial hypertension.[38] ACVRL1/ALK1 is a Type I TGF-β receptor, and endoglin is its co-receptor. Moreover, altered TGF-β signaling had already been described in remodeling pulmonary arteries in PAH.[39] Finally, TGF-β signaling was known to influence cell proliferation and survival, tissue growth and repair, and inflammation,[40] and all of these processes seemed to be dis-regulated in PAH. Surprisingly, despite identification of BMPR2 and implication of the TGF-β pathway, and despite the subsequent development of powerful investigative tools, a clear mechanistic connection between altered BMPR2 and/or TGF-β signaling and PAH has remained elusive. More unfortunately, knowing the genetic underpinning of HPAH has not yet allowed the development of novel therapeutics that target the underlying molecular pathogenesis of the disease. A number of important questions were immediately logical follow-ups to the identification of the gene responsible for HPAH. These were questions that were raised and addressed with very little knowledge of the function of the gene product or its molecular pathogenesis. The first of these was to ask whether BMPR2 mutations were responsible for sporadic PAH in addition to HPAH, perhaps even serving as a common cause of all forms of PAH. Very soon after the report of BMPR2 as the causative gene in familial PAH, Thomson et al. reported the detection of germline BMPR2 mutations in 11 out of a sample of 50 unrelated patients with IPAH (sporadic IPAH without family history or known genetic association to explain PAH).[41] These mutations encompassed the same spectrum of mutation types (frame shift, missense, and nonsense) as that described for familial PAH. Other groups have subsequently reported BMPR2 mutations in cases previously identified as sporadic PAH.[42,43] The Pulmonary Circulation | July-September 2011 | Vol 1 | No 3

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prevalence of BMPR2 mutations among sporadic PAH patients is estimated to be between 11% and 40%, with the most recent report from the French Registry at 14.8% (49 of 332 patients) as of April 2009.[20] BMPR2 mutations have also been examined in other forms of PAH. In a series of 106 children and adults with congenital heart defects of various types, BMPR2 mutations were detected in 6 patients.[44] Most of these were atrioventricular canal or septation defects, and this was in keeping with prior studies in animal models showing that BMP signaling is important for normal cardiac septation and outflow tract formation in a BMPR2 hypomorph mouse model. [45] In small studies of less than 25 patients each, BMPR2 mutations were not found in patients with PAH associated with scleroderma or in HIV-infected patients with PAH. [46,47] In a larger series of 103 patients with chronic thromboembolic PAH, perhaps not surprisingly, no BMPR2 mutations were detected.[48] Taken together, the data suggest that BMPR2 mutations are responsible for the majority of HPAH and for a significant subset of sporadic PAH patients initially identified as IPAH.

Molecular mechanisms underlying BMPR2 mutations

The next step, following the identification of a culprit gene for a complex disease, would be to ask what types of mutations are present and how those mutations affect the gene product—e.g., haploinsufficiency, loss of function, gain of function, dominant negative, etc. From the initial investigations, frame shift, missense, and nonsense mutations were identified in the BMPR2 coding region.[35,36] On further investigation of intron/exon boundaries, splice site single nucleotide mutations have been identified.[49] In addition, larger disruptions of the BMPR2 gene, including both small and large rearrangements (exon or partial gene deletions or insertions and duplications) have been described.[50] It has subsequently been demonstrated that at least some of the nonsense mutations identified result in haploinsufficiency through the process of nonsensemediated decay (NMD). [50,51] This is a feature of cellular quality control that exists between transcription and translation whereby nonsense mutations that result in significantly truncated transcripts are identified and degraded before they ever undergo translation.[52] In a study of 45 families with HPAH caused by BMPR2 mutations, 24 of the families were found to have a mutation that results in NMD. Other studies have estimated that the rate of NMD-causing BMPR2 mutations in HPAH may be as high as 70%. Other BMPR2 mutations have been shown to act in what seems to be a dominant negative fashion, and these mutations tend to have a more severe clinical phenotype. The major mechanism for dominant negative mutations is thought to be due to failed trafficking of the mutant receptor to the cell surface and the formation of nonfunctional intracellular heteromers composed of Pulmonary Circulation | July-September 2011 | Vol 1 | No 3

mutant and wild-type receptors, which effectively traps the wild-type receptor in the cytoplasm. [53] The BMPR2 gene is comprised of 13 exons that encode for 4 major functional domains of the receptor—the extracellular ligand binding domain (exons 2-3), the transmembrane domain (exon 4), the serine/threonine kinase domain (exons 5-11), and a long cytoplasmic tail (exons 12- 13) that is unique amongst the TGF-β receptor family members for its length. Mutations have been identified in all of these functional domains. Perhaps not surprisingly, the extracellular domain, kinase domain, and cytoplasmic tail domain are the sites of the vast majority of diseasecausing BMPR2 mutations, representing 187 out of 210 distinct mutations. Of these 187 mutations, 105 were found to affect the kinase domain.[49] Before engaging in a detailed biochemical investigation of how mutations affect BMPR2 signaling and how this leads to PAH, a final question to ask from the standpoint of HPAH genetics is whether BMPR2 mutation has any detectable impact on the clinical presentation or behavior of disease. As mentioned above, the French Registry examined BMPR2 mutation carriers and noncarriers and compared their clinical disease course. The mutation carriers were younger at diagnosis, had worse hemodynamic parameters at diagnosis, progressed faster to death or lung transplantation, and died at younger ages, but their overall survival was the same as for noncarriers. BMPR2 mutations have also been associated with decreased or absent vasoreactivity in PAH. Elliott et al. examined 67 unrelated patients with PAH (52 idiopathic, 15 familial) for BMPR2 mutations. Non-synonymous BMPR2 mutations were found in 16 of the idiopathic patients and 11 of the familial patients. Vasoreactivity at right heart catheterization was demonstrated for only 1 of the patients with non-synonymous BMPR2 variations, compared to 14 of the 40 patients who did not have nonsynonymous BMPR2 changes (P=0.003).[54] Rosenzweig et al. looked at vasoreactivity in a larger cohort of 147 patients, comprised of 69 adults and 78 children, with 114 IPAH patients and 33 familial PAH patients. Of the 147 patients, 23 were positive for a BMPR2 mutation. The mutation carriers in this study were found to be much less likely to exhibit acute vasoreactivity at right heart catheterization (4% vs. 33% for non-carriers, P<0.003). Patients with BMPR2 mutations also had lower mixed venous oxygen saturations and lower cardiac index, similar to what was described in the French Registry.[55] Taken together, these findings suggest that BMPR2 mutation carriers are less likely to exhibit acute vasoreactivity and are more likely to have worse hemodynamics at right heart catheterization. This suggests more severe disease at the time of diagnosis. Thus far, however, this has not seemed to translate into excess mortality in the studies that have been reported to date. 309

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Identification of BMPR2 as the gene responsible for HPAH has allowed for a number of subsequent informative inquiries into the nature of BMPR2 mutations and how these affect the clinical presentation of disease. It has also allowed for widespread targeted genetic testing for HPAH. However, the central point to investigate following the identification of the culprit gene(s) for any complex disease is the central point of molecular pathogenesis. By elucidating how a mutant gene mechanistically leads to disease, the hope is that novel and specific therapeutic targets can be identified and pharmacologically engaged with the eventual goal of developing more effective treatments, and perhaps even curative or preventative therapies.

TGF-β/BMP signaling

Much was known about the TGF-β superfamily at the time of the discovery of BMPR2 as the gene responsible for HPAH. The cytokine ligands in the TGF-β family are encoded by 42 open reading frames in humans. These ligands function as dimers and interact with heterotetrameric complexes consisting of 2 Type I receptors (7 subtypes identified) and 2 Type II receptors (5 subtypes identified, including BMPR2). The Type II receptor then phosphorylates the Type I receptor’s kinase domain, activating it. The Type I receptor’s kinase domain phosphorylates and activates 1 or more of the R-Smad proteins (Smads 1, 2, 3, 5, and 8). The phosphorylated R-Smads then interact with the Co-Smad, Smad 4. This complex then translocates to the nucleus to affect the transcription of target genes (Fig. 2). Regulation of TGF-β signaling is very complex, with negative regulators present at the level of ligand binding (e.g., ligand binding traps, decoy receptors), of intracellular receptor-binding proteins, and of Smad complexes (e.g., the I-Smads, which are Smads 6 and 7). TGF-β signaling has multiple roles in cellular differentiation during development, TGFBR2

TGFβ ALK5

BMPR2

BMP ALK1/2/3/6

c-Src Smad 2/3

Smad 1/5/8 Smad 4

P P

Gene expression

Cytoskeletal dynamics

Tctex-1 LIMK1

XIAP

Cofilin

XIAP

Ub Ub Ub

Cell survival

Figure 2: Simplified canonical TGF-β/BMP signaling and non-canonical signaling unique to BMPR2. The dashed line delineates canonical TGF-β signaling (on the left) from some of the pathways interacting with BMPR2 via its long cytoplasmic tail. 310

determination and maintenance of cell fate, apoptosis, cell proliferation, and regulation of inflammation.[17,40] In general, TGF-β signaling has a negative effect on cell growth in the post-development period, and loss of TGF-β signaling has been linked to tumorigenesis.[56]

Central to the pathology of PAH are the findings of muscularization of the smallest pulmonary arteries, occlusion of small pulmonary arteries by aggregations of cells of unclear lineage, and loss of the smallest branches of the pulmonary vascular tree (so-called “pruning”). Given the neointimal and smooth muscle proliferation and the vaso-occlusive plexiform lesions seen in histologic sections of PAH lungs, as well as findings of altered cellular proliferation and apoptosis in cells isolated from PAH patients, the TGF-β pathway seems optimally positioned to mediate these effects. BMPR2 is expressed in smooth muscle cells and endothelial cells in the human pulmonary vasculature, though its effects appear to be opposing in these two cell types.[57-59] In pulmonary artery endothelial cells, loss of BMP responsiveness confers increased sensitivity to apoptosis with abnormal repopulation/ repair responses. In pulmonary artery smooth muscle cells, decreases in BMP signaling result in a loss of growth restriction and abnormal smooth muscle proliferation. [60] These phenotypes are likely mediated not so much by widespread and severe loss of BMP signaling but rather by more subtle changes in receptor multimer formation and makeup that shift the overall phenotype in the directions described. BMPR2 participates in TGF-β signaling at the level of a Type II receptor. The most obvious hypothesis to link BMPR2 mutation and PAH would be through altered Smad signaling. While there has been some evidence linking defective Smad signaling to abnormalities in pulmonary vascular homeostasis that are similar to what is seen in PAH,[59,61-63] the molecular pathogenesis is more complicated than this. Mutations in the cytoplasmic tail domain of BMPR2 that have been identified in HPAH patients have been shown to have essentially intact Smad signaling when expressed in vitro.[64,65] The unusually long cytoplasmic tail of BMPR2 is thought to participate in Smad-independent signaling and has been shown to interact with a number of other proteins, including Tctex-1 (a dynein light chain), LIMK-1 (involved in regulation of actin cytoskeletal dynamics), XIAP (a negative regulator of apoptosis), p38 MAPK, c-Src, and RACK-1 (Fig. 2).[65-72]

The BMPR2 transgenic mouse model of PAH

One of the best tools available for studying the molecular pathogenesis of any complex disease is a robust animal model. For laboratory research purposes, this often implies a rodent model, as rodents possess the necessary organ system complexity to more accurately reproduce human Pulmonary Circulation | July-September 2011 | Vol 1 | No 3

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disease while still being easy to work with and maintain as well as being genetically tractable. This is worth mentioning in light of the fact that there are strains of fowl and of cattle that develop pulmonary hypertension. [73,74] Prior to the identification of BMPR2 in HPAH, several rodent models of PAH existed, including monocrotaline treatment, chronic hypoxia with or without treatment with the VEGF receptor antagonist Sugen 5416, extrarenal overexpression of mouse renin in the Ren-2 rat, and endothelial nitric oxide synthase (eNOS) knockout plus hypoxia. The major animal models of PAH in current experimental use have been recently reviewed elsewhere. [75] The fundamental problem with all of these models is that they required a condition, either an environmental exposure or a genetic condition, which was not present in patients with PAH. While this is a problem common to many animal models of complex diseases, and while subsequent studies have described altered BMPR2 expression and signaling in at least some of these models, the discovery of the culprit gene in HPAH allowed for the development of much more robust transgenic animal models. This, however, was not as straightforward as it initially appeared. Because TGF/BMP signaling plays a large role in development, terminal differentiation, and determination of cell fate, animals with severe germline loss of BMPR2, such as BMPR2 hypomorphs or homozygous deletion of BMPR2, exhibit an embryonic lethal phenotype.[76] Homozygous deletion leads to growth arrest at the early gastrulation/egg cylinder stage, and BMPR2 hypomorphs have failure of septation of the cardiac outflow tract, as mentioned above.[45] BMPR2+/- heterozygotes show mildly elevated right ventricular systolic pressure (RVSP) and modest muscularization of small pulmonary arterioles in some studies, but not in others.[77,78] To attempt to achieve a more dramatic and consistent phenotype, a transgenic mouse was made that expresses a dominant negative BMPR2 allele only in smooth muscle, under the control of the Sm22 promoter. These animals showed more consistent elevation of RVSP and some muscularization of the small pulmonary arterioles, but many of the features of human PAH were not present.[79] Hong et al. subsequently demonstrated that conditional knockout of BMPR2 in pulmonary endothelial cells resulted in elevated RVSP with associated RV hypertrophy, muscularization of the distal pulmonary arteries, and perhaps some evidence of early “onion skin” lesions.[80] Though still not a true mimic of the human disease, these studies are important for demonstrating that neither the pulmonary endothelial cell nor the vascular smooth muscle cell is clearly the dominant cell type in PAH, as both seem to drive disease in these animal models. Further progress in the creation of a robust animal model of HPAH was realized when West et al. created a double Pulmonary Circulation | July-September 2011 | Vol 1 | No 3

transgenic mouse that expresses rtTA under the control of the Sm22 promoter and expresses a BMPR2 receptor with a dominant negative truncation mutation in the cytoplasmic tail (R899X) under the control of the TetO(7) promoter. These mice thus express the mutant BMPR2 only in smooth muscle cells and only when exposed to doxycycline. Not only does this circumvent the problems associated with a BMPR2 mutation expressed from conception, but these animals also exhibit elevated RVSP, muscularization of distal pulmonary arteries, and larger pulmonary vascular structural changes.[81] Importantly, the particular mutation expressed in these animals is the most common BMPR2 mutation in the cohort of HPAH patients followed at Vanderbilt University. A further refinement to the model has been the creation of a transgenic mouse that expresses the same BMPR2 mutation only in the presence of doxycycline, but expresses the mutant gene in every cell in the body.[82] This is actually a better representation of what occurs in the human disease, as BMPR2 and its mutations exhibit widespread tissue expression. Moreover, it avoids the inherent bias of restricting expression of the mutant gene to one particular cell type. Finally, these animals recapitulate nearly every aspect of the human disease (Table 1). In addition to elevated RVSP and muscularization of the distal pulmonary arteries, these animals show plexiform lesions, influx of perivascular inflammatory cells, increase in oxidative stress, and alterations in actin cytoskeletal dynamics. Finally, and perhaps most interestingly, these animals show reduced penetrance of the disease phenotype. While perhaps a frustrating aspect of the animal model in one sense, this suggests that this transgenic animal model is the most robust available for the study of the molecular pathogenesis of HPAH. Table 1: Comparison of changes observed in PAH patients and in mice expressing mutant BMPR2 Finding in late-stage human PAH

Present in BMPR2 mutant mice?

Increase in RVSP

Yes – measured directly via catheter Yes – particularly RV dilatation

Abnormal echocardiographic findings Increase in total body mass Pruning of small pulmonary vessels Presence of complex vascular lesions Muscularization of pulmonary arterioles Widespread gene expression changes

Yes – with mice showing 20- 30% increase compared to wild-type Yes – measured by fluorescence microangiography and by histology Yes – though variable and well after increase in RVSP Yes – most prominent in vessels <25 μm in diameter Yes – in pathways similar to those altered in PAH patients

RVSP: right ventricular systolic pressure; PAH: pulmonary arterial hypertension

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Modifiers of BMPR2-mediated PAH

Steroid hormones As mentioned above, the reduced penetrance of BMPR2 mutations in HPAH strongly suggests the presence of modifiers that increase and/or decrease disease risk. It is likely that these modifiers represent both genetic and environmental factors, as evidenced by the fact that the most robust transgenic mouse models are incompletely penetrant and should have very similar or identical genetic backgrounds from one individual to the next. Nonetheless, the evidence is strongest for genetic modifiers. The earliest identified modifier, noted in all of the epidemiologic studies of PAH discussed above, is female gender, which increases the incidence of the development of disease up to 4-fold. This would seem to be due either to a detrimental effect of estrogen or its metabolites or to a protective effect of testosterone or its metabolites, or perhaps a combination of these. It is also possible that more complex changes at the chromosomal level, such as aberrant X-inactivation, may play a role, though there is only scant data to support this idea.[61] West et al. used expression arrays to examine EBV-immortalized B lymphocytes from HPAH patients with BMPR2 mutations, from family members who were mutation carriers but had not developed disease, and from non-carrier family members, in an attempt to discover modifier genes not previously identified. Overall, the study concluded that pathway analysis was more informative than single gene changes. However, changes in the expression level of CYP1B1, an estrogen metabolizing enzyme, were highly correlated with disease penetrance in female but not in male PAH patients in the study. CYP1B1 showed 10-fold lower expression levels in female patients compared to controls.[83] This enzyme metabolizes estrogens to 2-hydroxy and 4-hydroxy metabolites.[84] This reaction is in direct competition with other cytochrome P450 enzymes that metabolize estrogens to 16-alpha-hydroxy metabolites, which have been shown to possess significant mitogenic activity and to be pro-tumorigenic.[85,86] Austin et al. followed up the expression array findings by examining CYP1B1 polymorphisms in 140 BMPR2 mutation carriers. Of the 140 subjects, 92 had HPAH (62 of whom were female) and 48 were unaffected mutation carriers (24 of whom were female). Genotyping for a CYP1B1 polymorphism (N435S mutation, previously associated with breast, endometrial, and prostate cancers) was done, and from the female subjects, a nested case-control study examining the urinary metabolites of estrogen (2-OHE and 16α-OHE1 metabolites) in 5 female HPAH patients and 6 female unaffected mutation carriers. Among female mutation carriers, there was a 4-fold higher penetrance of disease for those homozygous for the N/N CYP1B1 allele compared to those who were heterozygous (N/S) or homozygous (S/S) for the polymorphism (P=0.005 for 312

Chi-squared analysis). In the nested case-control portion of the study, the 2-OHE/16α-OHE1 ratio was 2.3-fold lower (0.65 vs. 1.48 ng/mg creatinine/mL, P=0.006) in the 5 female HPAH patients compared to the unaffected mutation carriers.[87] Taken together, these data strongly establish CYP1B1 as a potentially important modifier of BMPR2-mediated PAH, at least in female patients. The influence of estrogen and estrogen metabolites is likely a bit more complex, as estrogen has a number of metabolic fates in vivo. For example, estradiol is converted to 2-OHE by CYP1A1/CYP1B1, which is converted to 2-methoxyestradiol (2-ME) by catechol-Omethyltransferase (COMT). 2-ME has been demonstrated in at least some animal models of PAH to have potent antimitogenic and overall beneficial therapeutic effects, and it is possible that the overall balance of estrogen and its metabolites is the biologically relevant variable. [88,89] However, it is likely that not all of the enzymes involved in estrogen synthesis and metabolism are equally important. The critical control point may still be CYP1B1, as there are no published reports linking PAH and COMT polymorphisms or COMT inhibitors. Whether diagnosis of PAH and propensity to survive after diagnosis reflect distinct processes is unclear. Reports from the two large epidemiologic registries of PAH in France and in the US found reduced survival among males, although it was unclear if this was due to effects on the pulmonary vasculature, effects on the ability of the right ventricle to respond to stress, or both.[90,91] Furthermore, any survival disadvantage for males may be specific to men over the age of 60 years. This was the case for the REVEAL study in terms of men over age 60 compared with younger men and compared with women at any age. Detailed prospective studies with validation will be necessary to fully appreciate the degree to which gender participates in survival, if at all.

A logical extension of the sex hormone discussion is to androgens. To date, though studies have indicated a potential protective role for testosterone via its actions as a pulmonary vasodilator,[92,93] no investigations have revealed an association between polymorphisms in any of the androgen synthesis, signaling, or metabolic machinery and increased or decreased penetrance of HPAH in males or females. However, a study conducted by Roberts et al. examining genetic risk factors (independent of BMPR2 or the serotonin transporter, SERT) for the development of portopulmonary hypertension in males and females did find that 2 polymorphisms in the gene for aromatase, which is the rate-limiting step in the conversion of androgens to estradiol, conferred an increased risk for developing portopulmonary hypertension. The increased risk, correlated with increasing levels of estradiol in a Pulmonary Circulation | July-September 2011 | Vol 1 | No 3

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dose-dependent fashion, controlled for gender.[94] One possible explanation for this finding is a change in the balance of estradiol and its metabolites, but another possibility is a decreased protective effect of androgens due to increased conversion to estradiol. This study did not measure androgens or estrogen metabolites, so the question remains an open one.

Variation in the TGFb/BMP/BMPR2/Smad axis Interestingly, one modifier of disease expression has been described involving the BMPR2 gene itself. It has been estimated that as many as 70% of the total mutations in BMPR2 would be predicted to result in truncated transcripts that would likely be subject to nonsense-mediated decay. [49] Indeed, NMD has clearly been demonstrated for some BMPR2 mutations, which is predicted to result in functional haploinsufficiency. However, even amongst subjects harboring NMD+ mutations, disease penetrance is not uniform, and only 20% of mutation carriers will go on to develop HPAH. Hamid et al. hypothesized that variations in the expression of the wild-type BMPR2 allele in these individuals may be an important disease-modifying factor by contributing to variable degrees of haploinsufficiency. To test this, they examined EBV-immortalized B lymphocytes from members of 4 families, each family with a different NMD+ BMPR2 mutation. From each family, immortalized lymphocytes from both HPAH patients and related unaffected mutation carriers were analyzed for expression levels of wild-type BMPR2. Compared to unaffected family members, HPAH patients had significantly lower expression levels of the wild-type BMPR2 allele (P<0.005), a finding that was independent of the specific NMD+ mutation in the other allele.[95]

Just as variations in the BMPR2 receptor cause or contribute to disease, variations in the gene for at least one of the ligands in the TGF-β/BMP pathway, TGFβ1, have been shown to influence disease penetrance. TGFβ1 SNPs were separated into least active, intermediate active, or most active groups and examined in 81 HPAH patients and 39 unaffected BMPR2 mutation carriers. In the context of NMD-resistant BMPR2 mutations, the relative activity of TGFβ1 mutations present influenced both age of disease onset and penetrance. More active TGFβ1 SNPs correlated with earlier age at disease onset; and the least, intermediate, and most active SNP groups showed penetrances of 33%, 72%, and 80%, respectively (P<0.003). There also appeared to be a dose effect, as those with 0-1, 2, or 3-4 active SNPs had penetrances of 33%, 72%, and 75%, respectively (P<0.005). Phosphorylated Smad-2 was shown to be increased on immunohistochemical analysis of lung sections from HPAH patients, though this was not clearly a difference in expression but more likely a difference Pulmonary Circulation | July-September 2011 | Vol 1 | No 3

in activity of the TGF-β pathway.[96] Interestingly, there appear to be only two reports in the literature of genetic alteration of any Smad gene in a patient with PAH. Both are case reports of alterations in the Smad-8 gene. One patient had a germline BMPR2 mutation and a somatic deletion on chromosome 13 that involved loss of the gene for Smad8.[61] The other had a nonsense mutation reported in the Smad-8 gene as mentioned above.[37]

Other modifiers and gene arrays Alterations in a number of genes outside the BMPR2/TGF-β pathway have been associated with HPAH or IPAH. Most of these have been examined on the basis of perceived biological plausibility, as they primarily are in pathways that contribute to the regulation of vascular tone (including the renin/angiotensin pathway, the prostacyclin pathway, potassium channels, endothelial nitric oxide synthase, carbamyl phosphate synthetase 1 through its influence on nitric oxide production, and vasoactive intestinal peptide through vasodilator action that is at least partially dependent upon nitric oxide) and/or to cell proliferation (e.g., the serotonin transport pathway).[97-107] Several issues should be considered when interpreting the findings from these various studies. Many of these are relatively small studies, making it more difficult to detect weaker associations and increasing the likelihood of statistical artifact. For those candidate risk factors that have been examined in multiple studies, such as polymorphisms in the serotonin transporter (SERT or 5-HTT), the data are conflicting, with some studies reporting an association with SERT polymorphisms and PAH while others have not shown any relationship.[108-110] A number of these potential risk factors have been shown to associate with varieties of PAH in the pediatric population (such as ACE polymorphisms and persistent pulmonary hypertension of the newborn or CPS-1 polymorphisms and pulmonary hypertension following surgical correction of congenital cardiac defects) but not in the adult PAH population. Polymorphisms in several of these candidate genes have been shown to modify the expression of adult pulmonary hypertension in very specific circumstances, such as pulmonary hypertension associated with COPD, chronic thromboembolic pulmonary hypertension, exerciseinduced pulmonary hypertension, and portopulmonary hypertension. The best available data suggest that the pathologies involved in these various settings are quite different from one another and from HPAH or IPAH, so the ability to generalize these findings is in question. For the most part, each of these has been examined as an independent risk factor or contributor, as opposed to being a modifier for the expression of disease mediated by other known heritable risk factors such as BMPR2 mutations. One larger study by Machado et al. did examine SERT polymorphisms in PAH patients, a subgroup of whom had known BMPR2 mutations, and was unable to detect 313

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any modifying influence of SERT polymorphisms on the expression of disease (e.g., severity, age of onset, clinical course, etc.).[109] These and other proposed modifier genes are summarized in (Table 2).

While the examination of polymorphisms in biologically plausible modifiers for HPAH and IPAH has proven to be only minimally informative, the increasing availability of gene array studies has allowed for the identification of potential modifier genes or pathways previously unsuspected. In particular, changes in pathways involved in actin cytoskeleton regulation, stress response, cellular metabolism, and inflammation have all been identified as potential contributors to IPAH and/or HPAH. [111] These studies have been performed in mouse models of

disease, in human lung samples, and in circulating cells from human patients and controls. Mouse models have the disadvantage of not representing the actual human disease but only an approximation, and have the additional possibility of representing pathways that are only relevant in rodents. Studies of human patients are inherently biased because PAH has already affected the person to a degree severe enough to permit clinical diagnosis, and it can therefore be difficult to know what is contributory and what is response. The caveats and limitations germane to these studies, as well as some of the common important findings, have been recently reviewed.[112] Despite the known limitations, gene array studies have been and will continue to be very informative for ongoing inquiry into the pathogenesis of PAH.

Table 2: Proposed modifier genes, their hypothesized mechanisms of action, and the observed effect on human PAH phenotype Modifier gene

Hypothesized mechanism

Effect on PAH phenotype

SERT

Increased vascular tone and increased cell proliferation via increased serotonin availability

Prostacyclin synthase

Increased vascular tone via decreased prostacyclin production

eNOS

Increased vascular tone via decreased nitric oxide production

Angiotensin converting enzyme type 1

Increased vascular tone and increased cell proliferation via increased angiotensin signaling

Angiotensin II receptor type 1

Increased vascular tone and increased cell proliferation via increased angiotensin signaling Increased vascular tone via decreased substrate availability for nitric oxide synthase

Variable, with some studies reporting a positive association between SERT polymorphisms and PAH and others failing to confirm such an association No consistent association with PAH, though reported association with CTEPH No reported association with PAH, but eNOS polymorphisms have been associated with pulmonary hypertension related to COPD Polymorpisms associate with pulmonary hypertension in type 1 Gaucher’s disease, high altitude pulmonary hypertension, and some cases of CTEPH, but no association with PAH One polymorphism associated with later age at diagnosis in IPAH

Carbamoyl phosphate synthetase 1

Smooth muscle potassium channels (KCNQ, KCNA5)

TGFβ1

CYP1B1

Increased vascular tone via direct effects on smooth muscle cell membrane potential and increased cell proliferation/ decreased apoptosis Enhanced activation of TFGβ receptors coupled with reduced activity of mutated BMPR2 promotes dysfunctional Smad signaling balance; causes increased cell proliferation/ decreased apoptosis Unbalanced estradiol metabolism promotes increased cell proliferation/decreased apoptosis via genomic effects

Polymorphism associated with increased pulmonary artery pressures in children after repair of congenital heart defects, no known associations with HPAH or IPAH Some polymorphisms associate with IPAH, though small numbers of patients Two polymorphisms associated with enhanced classic TGFβ signaling found in one study to modify age at diagnosis and penetrance of PAH among BMPR2 mutation heterozygotes Polymorphism in CYP1B1 associated with penetrance of PAH among female BMPR2 mutation heterozygotes in one study; supported by urine metabolite data

SERT: serotonin transporter; eNOS: endothelial nitric oxide synthase; TGFβ: transforming growth factor β; CYP1B1: cytochrome P450 1B1; PAH: pulmonary arterial hypertension; IPAH: idiopathic pulmonary arterial hypertension; HPAH: heritable pulmonary arterial hypertension

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Current and future therapies for PAH

The current therapies for PAH largely reflect the older notion of the disease as primarily a perturbation of pulmonary vasoreactivity. Current FDA approved therapies include prostacyclin analogs, endothelin-1 receptor antagonists, and phosphodiesterase Type V inhibitors (which potentiate nitric oxide’s actions by inhibiting the intracellular breakdown of cyclic GMP). In addition, calcium channel antagonists can be highly effective pulmonary vasodilators in a small subset of patients who have a very robust acute pulmonary vasodilator response, but these drugs do not have FDA approval for this use. These therapies were developed prior to the confirmation of the importance of BMPR2 and the TGF-β pathway in PAH. Unfortunately, of the hundreds of interventional clinical trials currently registered with clinicaltrials.gov that have “pulmonary arterial hypertension” as a key search term, only a very small number are specifically designed to investigate possible interventions in adult PAH that do not directly impact upon the pathways targeted by existing therapies. There are registered trials of administration of endothelial progenitor cells; trials examining the tyrosine kinase inhibitors imatinib and nilotinib; a small phase IV trial examining the effect of pioglitazone in PAH patients with insulin resistance; a trial of ranolazine to improve angina from RV ischemia related to PAH; a trial of dichloroacetate sodium in IPAH, HPAH, or anorexigen-associated PAH; and a trial of administration of coenzyme-Q10, an important intermediate in the mitochondrial electron transport chain that also has intrinsic antioxidant properties. Other compounds have shown promise in pre-clinical studies, including fasudil (a Rho-kinase inhibitor), vasoactive intestinal peptide, and angiotensin converting enzyme Type 2.[113-117] However, none of these has been tested in large clinical trials in PAH patients. Clearly, there remains a significant gap between genetic investigations of PAH to date and an understanding of the pathophysiology of the disease that is detailed enough to permit identification and investigation of novel therapeutic targets in humans.

Before undertaking clinical genetic testing for BMPR2 mutations, patients should receive professional genetic counseling from trained individuals prior to testing. This ensures that all involved understand the possible results of the testing and what these results might imply for both the patient and his or her family members. A brief discussion of the possible outcomes of genetic testing is in order. For the asymptomatic offspring of a BMPR2 mutation carrier, there is an overall pre-test probability of developing disease of approximately 1 in 10, or 10%, because there is an approximately 50% chance that the mutation was inherited, modified by the approximately 20% chance that the mutation would actually cause disease (because of reduced penetrance). If clinical genetic testing is undertaken in this asymptomatic person and found to be negative for BMPR2 mutations, the predicted risk of developing PAH drops to the level observed in the general population, which is a risk of less than 1 in 500,000 and which represents a 50,000-fold risk reduction. If this asymptomatic person is found to have inherited the mutant BMPR2 allele, the risk of developing PAH increases to approximately 20%, the known incidence of PAH in BMPR2 mutation positive individuals. This represents only a modest, approximately 2-fold increase in the risk of developing disease. This logic is depicted schematically in (Fig. 3). Thus, for these individuals, a negative genetic screen is extremely reassuring, and a positive test only modestly increases the risk of developing HPAH. The question of whether to test a patient with IPAH for mutations in BMPR2 is worth consideration as well. Approximately 20% of incident IPAH is thought to be driven by mutations in BMPR2, suggesting that a significant portion of IPAH may in fact have a heritable component. Though detection of a BMPR2 mutation does not guarantee that the mutation is a germline mutation, detection of the mutant allele in cells/tissues distant from

Genetic testing for PAH

Genetic testing for known mutations in BMPR2, ALK1, and ENG are currently available from several laboratories in North America and Europe. Unless there is a known family history of HHT or a strong clinical suspicion for the disease, clinical genetic testing specific to PAH should focus on testing for BMPR2 mutations. There is no current indication to incorporate testing for common genetic variants into the clinical testing approach. Likewise, there are no other pulmonary hypertension-specific genetic variants for other forms of the disease that support routine clinical genetic testing. However, testing for underlying causative diseases or syndromes is appropriate when clinically indicated. Pulmonary Circulation | July-September 2011 | Vol 1 | No 3

Asymptomatic, pretest probability of disease is 1 in 10

Negative

Asymptomatic, post-test probability of disease is < 1 in 500,000

BMPR2 Mutation Testing

Positive

Asymptomatic, post-test probability of disease is ≥ 1 in 5

Figure 3: Diagram of pre- and post-test probabilities for developing HPAH in an unaffected family member, and the mathematical impact of testing for BMPR2 mutation. 315

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the site of disease (e.g., peripheral blood mononuclear cells or a buccal wash/swab) strongly suggests such. Detection of a BMPR2 mutation is often surprising, disappointing, and anxiety-provoking for a patient who previously thought that he or she had a “sporadic” disease, particularly if the individual has children. This is often the first time that increased risk in the patient’s family members is perceived. There can be significant emotional stress both for the patient, who can experience what has been termed the “guilt of heritability,” as well as for other family members (if informed of the result), who can have significant difficulty with the inherent uncertainty of HPAH and its genetic underpinnings.[118] These issues thus highlight the importance of genetic counseling prior to testing.[119] The decision of whether or not to test a subject for BMPR2 mutations or other single gene variants related to PAH is a complicated one, and particular caution should be used with children. Clinical genetic testing should only be considered at this time for children with diagnosed PAH, or for healthy children within a family affected by HPAH or IPAH. Several factors further complicate this issue in particular for children, including potentially profound psychological effects (e.g., seeing oneself as “sick” or “diseased” at a period of vulnerability during psychological development); concerns about future insurability and employer discrimination; and the uncertainty caused by the reduced penetrance and variable disease expressivity noted above, which can be especially difficult for both children and parents.

The question then arises of how to manage individuals who have tested positive for a mutation but who do not yet have any symptoms or evidence of clinical disease. To date there have been no studies specifically designed to investigate the best strategy for screening and early detection of clinically significant disease. The most current recommendation for these asymptomatic, mutation positive individuals is to have clinical and non-invasive echocardiographic screening every 3-5 years.[120] Those members of HPAH families who do undergo genetic testing and are found to be negative do not require future screening. The further question of the optimal time to start therapies that can be expensive, complicated, disruptive to normal routines, and associated with sometimes significant side effects, has not been adequately addressed. With specific regard to PAH in families, given the vast number of potential mutations in the large BMPR2 gene, screening for mutations best starts with the patient, so that if present the specific mutation in the family can be identified.[17] The current cost in U.S. dollars of genetic testing ranges between $1,000 and $3,000 to screen the entire BMPR2 gene, with mutation-specific testing then 316

costing approximately $300 to $500 once a mutation is known. Genetic testing should only be provided in concert with professional genetic counseling by experienced counselors for the reasons noted above.[119,120]

Summary and future directions

There are fundamental questions that stem from the current knowledge of PAH genetics that remain to be answered. It is not clear how or why a disease caused by a mutation in a gene expressed widely in many tissues has its primary manifestations only in the pulmonary vasculature (and perhaps the right ventricle). It is known that the pulmonary vasculature differs from systemic vascular beds in many ways, but whether any of these differences alone or in combination explains the phenotype of PAH is not known. There is mounting evidence that there are detectable abnormalities outside of the heart and lungs in PAH. It is possible that these are changes secondary to the known pathophysiology of PAH. However, it is also entirely possible that PAH is truly a systemic disease, with systemic manifestations that are fundamental to the development of elevated pulmonary vascular resistance and right heart failure, and that “pulmonary arterial hypertension” is a misnomer derived from the most easily observed and life-limiting manifestations of the disease. A systemic disease might actually make more intuitive sense given the expression of BMPR2 in many tissues outside the heart and lungs. As discussed above, the reduced penetrance of BMPR2 mutation continues to represent a large gap in our understanding of the pathophysiology of PAH. More broadly stated, the fundamental understanding of how a mutation in this gene leads to PAH is far from complete or what would even be described as robust. The canonical signaling pathways downstream from the receptor do not explain the development of PAH, and indeed, in many animal and human studies do not even seem to be directly related to the disease. Investigation of genetic modifiers has begun to yield insights into how penetrance is modified in HPAH, and thus how reduced penetrance in other “single gene” diseases might be investigated. Given the similarities between HPAH and IPAH in particular, a better understanding of the genetic and molecular pathogenesis of HPAH should further inform IPAH and all types of PAH, and thus make the study of HPAH critical to the community of PAH patients, families, and researchers.

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

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Review Ar ti cl e

COPD/emphysema: The vascular story Norbert F. Voelkel1, Jose Gomez-Arroyo1, and Shiro Mizuno2 1

Department of Internal Medicine, Victoria Johnson Laboratory for Obstructive Lung Disease Research, Virginia Commonwealth University, Richmond, Virginia, USA, 2Department of Pulmonary Medicine, Kanazawa Medical University, Kanazawa, Japan

ABSTRACT In this perspective, we review published data which support the concept that many or most chronic and progressive lung diseases also involve the lung vessels and that microvascular abnormalities and endothelial cell death contribute to the pathobiology of emphysema. Lung vessel maintenance depends on Vascular Endothelial Growth Factor signaling and both are compromised in the emphysematous lung tissue. Although hypoxic pulmonary vasoconstriction has been considered as an important factor contributing to the vascular remodeling in chronic obstructive pulmonary disease (COPD) (COPD/emphysema, it is now clear that inhaled cigarette smoke can damage the lung vessels independent of the lung vascular tone. We propose that a “sick lung circulation” rather than the right heart afterload may better explain the cardiac abnormalities in COPD patients which are usually summarized with the term “cor pulmonale.” The mechanisms and causes of pulmonary hypertension are likely complex and include vessel loss, in situ thrombosis, and endothelial cell dysfunction. Assessment of the functional importance of pulmonary hypertension in COPD requires hemodynamic measurements during exercise. Key Words: cor pulmonale, exercise, lung endothelial cells, pulmonary hypertension, pulmonary vascular remodeling

INTRODUCTION We owe the first description of emphysema to Laenec[1] and the first thorough description of the lung vessel pathology in chronic obstructive pulmonary disease (COPD) to Liebow,[2] and we find the 1963 statement of G.W. Wright[3] that the injury in emphysema was of a “vasculonecrotic nature” remarkable. From the vantage point of the first decade of the 21st Century the concept that lung vessels are involved in most, if not all, chronic and progressive lung diseases is still perhaps more intuitive than accepted knowledge. The topic of the vascular involvement and pulmonary hypertension (PH) in emphysema/ COPD has been previously reviewed and appears and disappears periodically on the radar screen of investigators interested in new treatment strategies for patients with COPD/emphysema. Whereas, understandably, the overwhelmingly large segment of COPD publications addresses issues of airway mechanics and airway pathology, the fact that the improved survival of COPD patients shown in the long-term oxygen treatment trial was associated with a small reduction in the pulmonary artery pressure[4] gives clinicians pause and repeatedly raises the question whether the pulmonary hypertension in COPD patients should be treated—and if so, with what drugs? The topic of pulmonary vascular involvement and Address correspondence to:

Dr. Norbert Voelkel 1220 E Broad St MMRB, 6th Floor, Richmond, USA Email: nvoelkel@mcvh-vcu.edu 320

pulmonary hypertension has many fascinating aspects and has been recently reviewed,[5-7] yet many questions remain unresolved. More recently systemic disease components in COPD are being discussed; however, earlier studies have already begun to address issues of hypercoagulability and deep venous thrombosis in patients with COPD.[8,9] Here we review recent data and concepts of the pulmonary vascular pathophysiology and pathobiology, introduce the more general concept of a “sick lung circulation” in integrated systems biology of COPD and propose new mechanistic concepts for the condition that we call cor pulmonale.

EMPHYSEMA

Emphysema is defined as “an anatomic alteration of the lung characterized by an abnormal airspace enlargement distal to the terminal bronchioles accompanied by destructive changes of the alveolar walls”[10] and is a variable component of the syndrome COPD which is now understood to have also extrapulmonary systemic manifestations.[11] Laennec, who described emphysema Access this article online

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Website: www.pulmonarycirculation.org DOI: 10.4103/2045-8932.87295

How to cite this article: Voelkel NF, Gomez-Arroyo J, Mizuno S. COPD/ emphysema: The vascular story. Pulm Circ 2011;1:320-6.

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in 1838, entertained the hypothesis that emphysema was the result of chronic bronchitis, which he called “catarrh.” The modern view that chronic airway inflammation causes emphysematous airspace destruction and that cigarette smoking drives and maintains airway inflammation is dominant and complemented by the “chronic infection” hypothesis. [12,13] One unanswered question is: How does airway inflammation (bronchiolitis) cause the disappearance of the surrounding alveoli? One hypothesis is that there is a gradient of proteases which is released from neutrophils and macrophages. This protease gradient is responsible for the “digestion” of alveolar septae surrounding the small airways. A second question is why there are patients (never smokers) who develop severe emphysema without significant airflow limitation, and a third is why individuals with the genetic α1-antitrypsin deficiency usually do not develop emphysema—unless they smoke cigarettes. The definition of “destructive changes of the alveolar walls” includes the loss of lung capillaries, but not the loss of precapillary arterioles which is apparent on pulmonary angiography of patients with emphysema. Of interest, a mutation in a gene encoding a copper transporter protein is responsible for the congenital emphysema in Menkes disease[14] which finds it correlate in the emphysema of the “blotchy mouse”,[15] and emphysema has been described in patients with coeliac disease,[16] pointing towards malnutrition or autoimmunity, or both, as etiologies of emphysema in these non-smoking patients. An animal model of autoimmune emphysema has recently been described.[12] This model is based on antibodies and T lymphycytes which cause lung endothelial cell apoptosis.

COPD and pulmonary hypertension

Vascular pathology The topic of pulmonary hypertension in patients with COPD, with and without cor pulmonale has been reviewed previously.[17-22] New clinical imaging technology to study COPD is being developed ,[23,24] however, a detailed study of the lung vessels still requires invasive angiography. Histological and morphometric studies of the lung vessels require lung tissue samples which become available only after lung cancer surgery, lung volume reduction or lung transplantation. The Lung Division of the NIH HLB Institute has established a COPD lung tissue repository; here lung tissue can be requested for cellular and molecular studies.[25] Pulmonary vascular remodeling: The role of cigarette smoke and chronic hypoxia Our concepts of pulmonary vascular remodeling in COPD/ emphysema have evolved to a large extent because of the work of the group of Joan Albert Barbera in Barcelona. Their histological examination of lung tissue Pulmonary Circulation | July-September 2011 | Vol 1 | No 3

resected from patients with cancer and nearly normal lung function revealed lung arteriolar abnormalities of muscularization and significant intima fibrosis that had to be attributed to cigarette smoking and not to hypoxia; the patients were not hypoxemic and Barcelona is at sea level. Thus, these findings have challenged the previous mechanistic explanation that hypoxia vasoconstriction was the root cause of the pulmonary vascular changes observed in the lungs from COPD patients. Indeed animal experiments of chronic cigarette smoke exposure have reproduced some of these pulmonary vascular changes. [26] In severe, end-stage COPD in patients that undergo lung transplantation most certainly, large regions of the lung tissue are hypoxic and we can propose that hypoxia will contribute to the lung vascular remodeling in these patients. Taken together, it is likely that early in the COPD syndrome development there are direct toxic effects of cigarette smoke on the lung vessels, and perhaps in later stages of COPD, there is hypoxia-induced lung vessel remodeling. Many investigators, even today, find it difficult to understand how inhaled cigarette smoke can injure lung vessels. They believe that the lung airway compartment is sufficiently separated from the vascular compartment and ask, “How does the cigarette smoke get to the lung vessels?” Without a doubt particles of the cigarette smoke reach the alveoli and volatile components can diffuse from the terminal respiratory bronchioles into the surrounding tissue areas, but we also know that stable volatile components of the cigarette smoke, for example the very aggressive aldehyde acrolein, reach the systemic circulation and their endothelial cells. With this information in mind, we understand that lung vessels are exposed to cigarette smoke components both from the outside and inside. Pulmonary resistance vessels, the arterioles, become muscularized after a period of chronic hypoxia and we can interpret this phenomenon as a “meaningful,” adaptive response of the lung vessels in order to match lung blood flow to ventilation, to adjust to high-altitude living, and to protect the lung capillaries against flooding due to increased precapillary pulmonary arterial pressure. This hypoxia-induced pulmonary vessel muscularization can be explained by vascular smooth muscle cell hypertrophy and hyperplasia but it is probably more complex. The muscularization can also be in part explained, at least in rodent studies—by the participation of endothelial cells and cells arriving from the systemic circulation via the vasa vasorum.[27,28] (Fig. 1) distinguishes lung vascular remodeling as an adaptive response to wall stress, i.e., muscularization from the response to endothelial cell (EC) injury and apoptosis and the loss of the EC monolayer integrity. The latter response can be extremely complex and involve 321

Voelkel et al.: Lung vessels in emphysema

a local immune response (perivascular accumulation of inflammatory cells) recruitment of bone marrowderived precursor cells, phenotypic alterations of EC and VSMC via endothelial cell to mesenchymal cell transition (EMT)[29] and the deposition of an altered matrix which is produced by the phenotypically altered vascular cells. In this schematic we attempt a synopsis of the most relevant mechanisms of lung vessel remodeling.

As mentioned, we owe the original description of the vascular pathology in emphysematous lungs to A. Liebow.[2] The most frequently listed vascular abnormalities, some of their underlying mechanisms and their potential consequences, are shown in (Fig. 2).

The modern view of pulmonary vascular remodeling is that in addition to the fluid-mechanical effects of hypoxic vasoconstriction—and thus increased shear stress of the resistance vessels, during hypoxia— there is a role for HIF-1α-dependent mechanisms. Here it is important to remember that hypoxia is linked via HIF-1α to “inflammation” and the innate immune response. [30,31] To summarize: pulmonary arteriolar muscularization, once understood as a consequence of smooth muscle contraction, has now become the result of complicated actions and interactions of transcription factors [32,33] and genes encoding growth factor proteins and proteins encoding multiple enzymes involved in the control of cell energy metabolism. [23,24,34] The studies of Johns et al.[35] and Daley et al. [36] indicate that hypoxia activates lung macrophages to release FIZZ1 (also called RELMα or hypoxia-induced mitogenic factor [HIMF]) which promotes pulmonary arteriolar SMC growth. One of the early experimental studies linking chronic hypoxiainduced lung vessel remodeling and inflammation is the study by Ono et al.[37] In the lungs from patients with COPD/emphysema we find evidence of endothelial cell apoptosis[38] and endothelial cell dysfunction;[39,40] there is a loss of a number of proteins expressed in normal endothelium [20] which can explain both endothelial cell loss and dysfunction (Fig. 2). The schematic (Fig. 2) pays tribute to the modern ideas about hypoxia and lung vessel remodeling; this figure also illustrates that the loss of the expression of enzyme proteins like prostacyclin synthase and nitric oxide synthase and loss of the expression of Vascular Endothelial Growth Factor (VEGF) and VEGF receptor proteins, may lead to EC dysfunction, EC apoptosis, and intima fibrosis. The events which lie upstream are unclear, but oxidant and endoplasmic reticulum stress (ERS) are candidates. A critical controller of lung endothelial cell growth and survival is VEGF.[25,41] How endothelial cell dysfunction causes the intima to become a thrombogenic surface is not yet understood, but in situ thrombosis [6] and 322

Figure 1: Comparison of adaptive pulmonary vascular remodeling with the complex cellular changes which can be a consequence of the damage of the endothelial cell (EC) monolayer.

Figure 2: This diagram highlights the components of pulmonary vascular alterations observed in COPD/emphysema lungs and some of their proposed mechanisms. Hypoxia and vasoconstriction likely independently affect the lung vessels and promote remodeling. Remodeling affects the three layers of the vessels: intima, media and adventitia. Endothelial cell apoptosis may be critically important and lead to both intima fibrosis and muscularization. The large box symbolizes an endothelial cell and illustrates alterations in gene expression which affects endothelial cell function.

bronchial artery thrombosis have been described in the COPD/ emphysematous lungs.

The topic of bronchial endothelial cell dysfunction has been recently reviewed.[42] Upstream triggers for both pulmonary vascular and bronchial vascular endothelial cell damage and dysfunction are likely oxidative and endoplasmatic reticulum stress (ERS)[43,44] and activation of immune responses. Our understanding of how immune cells and their cytokines shape pulmonary vascular remodeling is still in its infancy.[45,46]

Effect of cigarette smoke on pulmonary vessels Altered pulmonary vascular morphology in emphysema attributable to cigarette smoking has been reported by several investigators,[47-49] and these findings are supported Pulmonary Circulation | July-September 2011 | Vol 1 | No 3

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by animal studies and experiments demonstrating the effect of cigarette smoke extracts on cultured EC. Yamato et al.[26] reported that cigarette smoke exposure of guinea pigs induced emphysema associated with a diffusely reduced lung capillary density, and Wright et al. showed that cigarette smoke increases the expression of vasoactive mediators in pulmonary arteries[50] and causes rapid changes in gene expression in the pulmonary arteries. [51] Lee et al.[52] reported induction of endothelin-1 in pulmonary artery EC by cigarette smoke extract and Nana Sinkam et al. reported loss of prostacyclin synthase expression.[53] Cigarette smoke extract (CSE) can induce superoxide in EC, which inactivates NO and generates peroxynitrite[54] and this oxidative and nitrosative stress inactivates VEGFR2 signaling; [43] CSE induces p53dependent pulmonary EC apoptosis[55] and sildenafil can protect against CSE-stimulated EC apoptosis.[56] Pulmonary hypertension We have known since the pioneering studies of Benjamin Burrows that the degree of pulmonary hypertension in patients with COPD at rest and during exercise is very variable,[57] and these early findings have been confirmed by a number of larger studies,[58] in particular by the work of E. Weitzenblum and his group in Strassbourg, France.[59] Whereas most patients with COPD/emphysema have small elevations of the pulmonary artery pressure at rest, a small subgroup of the COPD patients (around 1%) presents with pulmonary artery pressures at rest that are in the range usually observed in patients with idiopathic forms of PH.[59] The pathobiology of the PH in these patients remains unexplored and lung histological studies have not been reported. Because echocardiographic evaluation of patients with hyperinflated lungs is problematic, hemodynamic assessment of these patients is necessary. It should become the standard procedure to evaluate exercise hemodynamics in these patients, in particular if the clinician wants to rule out PH as a cause of dyspnea. Obesity, airtrapping and autoPEEP effects, a limited venous return and cardiac output, [60] are other reasons for dyspnea in these patients (Fig. 3). Still today, the interplay of mechanical aspects of impaired lung function (stretch) and the cellular and molecular functional consequences of inflammation and oxidative stress for pulmonary vascular tone regulation and cardiac performance (see below) are largely unexplored. The extent of daily physical activities in patients with COPD/ emphysema decreases with the severity of lung function impairment (GOLD Classification II-IV) and the degree of hyperinflation.[29] The desire to predict the presence and severity of PH in COPD/emphysema by using noninvasive lung function and/or blood gas variables is understandable, yet the assumptions are that COPD is Pulmonary Circulation | July-September 2011 | Vol 1 | No 3

Figure 3: How exercise affects the lung circulation.

a homogeneous disease and that patients do not have also thromboembolic or left heart disease. Correlations between the systolic pulmonary artery pressure PAP and the PaO2[59] and the mean Pap and the O2 saturation[61] have been published and the large scatter of the data points makes it impossible to predict the Pa pressure in the individual patient. COPD patients living at altitude and those with the diagnosis of sleep apnea are more likely to have PH.

Cor pulmonale

The late David Flenley used to say that, “COPD patients die with cor pulmonale, not of cor pulmonale”.[17,18] It is unclear whether this statement is correct given the more recent epidemiological data which demonstrate that most COPD/emphysema patients die not from respiratory failure but from cardiovascular causes. Cor pulmonale is also an interesting topic of discussion because we do not really understand the pathobiology[17,18] and because some clinicians believe that it (cor pulmonale) has disappeared as a consequence of supplemental oxygen treatment of patients with COPD.

Whereas we distinguish cor pulmonale, the involvement of the heart in the setting of chronic lung diseases, from right ventricular failure in the setting of severe forms of lung vascular diseases, the pathobiology and pathohistology of the lung vessels may determine the cardiac response. We have recently demonstrated experimentally that chronic elevation of the right ventricular afterload alone generates adaptive RV hypertrophy but not RV failure. The right ventricle failed when there was angioobliterative lung vessel involvement, but not when the lung vessels were muscularized but patent.[62] Right heart failure in the setting of chronic left ventricular failure, or associated with angioproliferative PAH, causes death and is a strong predictor of survival.[58] We have recently proposed that a “sick lung circulation” (lung vascular endothelial cell apoptosis and/or proliferation) affects the heart. This “bad lung humor” hypothesis posits that information from the sick lung vessels is carried to the myocardium resulting in cardiac capillary rarefaction and fibrosis.[63] Information carriers could be cytokines, microparticles and microRNA contained in the blood exiting from the pulmonary veins and entering the coronary circulation. The schematic (Fig. 4) is an attempt to illustrate this concept. 323

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patients with severe emphysema, a reduction of expressed HDAC2, HIF-1α, and VEGF proteins, as well as a reduction in the expressed phosphoAkt. Recognizing that not a single protein, but a pattern— or signature — characterizes the destruction of the lung vessel maintenance program, where chromatin structure modification and growth factor signaling are all impaired (Fig. 5), it appears that there may not be a single molecular target for therapy.

0.8 0.4

0 COPD VEGF

Mild

Severe

(a) P<0.01 P<0.01

2 pAkt/Akt

1.5 1 0.5 0 COPD pAkt

Mild

Severe

(b) P<0.01 P<0.01

1.2 HDAC2/Lamin B

Can the lung vasculature be or become a target for the treatment of COPD/emphysema? [67] And, if so, then could it be that the treatment may not be restricted to the lung vessels but aimed at the improvement of general endothelial cell dysfunction? Yasuo et al. [25] demonstrated, after analyzing lung samples from

P<0.001

1.2

Treatment of pulmonary hypertension in COPD

Supplemental oxygen therapy, now well established in the treatment plan of many COPD patients, may reduce oxygen-and endoplasmic reticulum stress and improve EC function.[39,40,44] Interestingly such a hypothesis has not been explored to date.

P<0.001

1.6

VEGF/β actin

At present, we have no histological or molecular studies that would refute such a concept, and the discovery of microparticles and circulating microRNAs[64] now makes it attractive to begin to investigate this hypothesis. Products released from a sick lung circulation would be expected to affect — via the coronary circulation — both ventricles of the heart, and indeed, several studies over the years reported impaired left ventricular function in patients with COPD; we refer the reader to a recent publication by the Vienese group which had shown left ventricular diastolic dysfunction also in COPD patients without PH.[60,65] Diastolic dysfunction can be mechanistically explained as a consequence of endothelial cell to mesenchymal transition (EMT). At least in mice TGF-β can transform cardiac capillary EC into myofibroblasts resulting in cardiac fibrosis and greater stiffness of the ventricles, as shown by Zeisberg et al.[66] In short, the “sick lung circulation” hypothesis postulates that the endothelial cell disease of the lung causes endothelial cell dysfunction and capillary loss in the heart.[58]

0.8 0.4

0 COPD HDAC2

Mild (c)

P<0.01

HIF-1α/Lamin B

2.5 2 1.5 1 0.5 0

Figure 4: Comparison between pulmonary hypertension in the setting of COPD/emphysema (left) and pulmonary hypertension due to left ventricular dysfunction (right). Both conditions affect the lung vessels and can lead to right heart failure. The loss of myocardial capillaries is emphasized. *Diastolic dysfunction as a consequence of myocardial fibrosis. 324

Severe

COPD HIF-1α

Mild

Severe

(d)

Figure 5: Analysis of lung tissue samples from patients with normal lung function (no COPD) and samples from patients with mild and severe COPD. Western blot protein expression data reproduced with permission.[21] Pulmonary Circulation | July-September 2011 | Vol 1 | No 3

Voelkel et al.: Lung vessels in emphysema

The data shown in Figure 5 likely reflect a generalized impairment of lung cellular repair — not only the impairment of vascular maintenance. Examples of strategies which may protect the lung microvessels are simvastatin treatment, which protected against experimental cigarette smoke exposure-induced emphysema and PH, [68] the protective effect of the prostacyclin analog beraprost [69] or of endothelin receptor 1 blockade.[70] The report of a reduction in the cardiovascular mortality of COPD patients treated with the anticholinergic tiotropium (UPLIFT trial)[71] raises perhaps the question whether long-acting anticholinergic agents also have a vascular protective effect.

CONCLUSIONS

Pulmonary vascular involvement is part of many chronic and progressive lung parenchyma diseases and this includes COPD/emphysema and chronic left heart failure. Oswald-Mammosser et al.[53] reported 15 years ago that the survival of COPD patients was worse in patients older than 63 and having a mean Pap >25 mmHg. Both the pathobiology of PH in patients with COPD/emphysema and the consequences for cardiac performance are incompletely understood. New concepts for both have been proposed in this review and these may encourage studies which make use of the disease-relevant tissues and modern cell biology and molecular tools.

ACKNOWLEDGMENTS

This work has been supported by the Victoria Johnson Laboratory for Obstructive Lung Disease Research. The authors want to thank Mrs. Leslee Key for her expert help with the preparation of this manuscript.

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

Pulmonary Circulation | July-September 2011 | Vol 1 | No 3

Review Ar ti cl e

Surgical treatment of pulmonary hypertension: Lung transplantation Jason Long1,2, Mark J. Russo1, Charlie Muller3, and Wickii T. Vigneswaran1 2

1 Department of Surgery, Section of Cardiac and Thoracic Surgery, University of Chicago Medical Center, Chicago, Illinois, Department of Surgery, Section of Thoracic Surgery, University of Michigan, Ann Arbor, Michigan, and 3Pritzker School of Medicine, University of Chicago, Chicago, Illinois, USA

ABSTRACT Pulmonary hypertension (PH) is a serious and progressive disorder that results in right ventricular dysfunction that lead to subsequent right heart failure and death. When untreated the median survival for these patients is 2.8 years. Over the past decade advances in disease specific medical therapy considerably changed the natural history. This is reflected in a threefold decrease in the number of patients undergoing lung transplantation for PH which used to be main stay of treatment. Despite the successful development of medical therapy lung transplant still remains the gold standard for patients who fail medical therapy. Referral for lung transplant is recommended when patients have a less than 2-3 years of predicted survival or in NYHA class III or IV. Both single and bilateral lung transplants have been successfully performed for PH but outcome analyses and survival comparisons generally favor a bilateral lung transplant. Key Words: pulmonary vascular disease, surgical procedure, lung transplant

INTRODUCTION Pulmonary hypertension (PH), an abnormal elevation in pulmonary artery pressure, is defined as a mean pulmonary artery pressure ≥ 25 mmHg at rest or ≥ 30 mmHg with exercise, with a pulmonary capillary wedge pressure ≤ 15 mmHg as measured by cardiac catheterization.[1] PH was traditionally divided into primary and secondary but this classification system has since been replaced by a system proposed by the World Health Organization in 1998 and most recently updated at Dana Point, California in 2008.[2] The current classification system categorizes PH into five major categories with further subdivisions in each category allowing patients to be placed in groups sharing similarities in clinical presentation, pathophysiology, and therapeutic approaches (Table 1). Regardless of its etiology, PH is a serious and progressive disorder that results in right ventricular dysfunction and impairment in activity tolerance that can lead to subsequent right-heart failure and death.

Pathobiology

PAH has a multifactorial pathophysiology.[3] Abnormalities in molecular pathways regulating the pulmonary vascular Address correspondence to:

Dr. Wickii T. Vigneswaran Section of Cardiac and Thoracic Surgery, University of Chicago Medical Center, 5841 S. Maryland Avenue, MC 5040, Chicago, IL 60637, USA Email: wvignesw@surgery.bsd.uchicago.edu Pulmonary Circulation | July-September 2011 | Vol 1 | No 3

endothelial and smooth muscle cells have been described as underlying PAH with perturbations in vasoconstriction, smooth-muscle cell and endothelial-cell proliferation, and thrombosis. This includes inhibition of the voltageregulated potassium channel,[4] mutations in the bone morphogenetic protein-2 receptor,[5] increased serotonin uptake in the smooth muscle cell,[6] increased angiopoietin expression in the smooth muscle cells,[7] and excessive thrombin deposition related to a procoagulant state.[8] As a result, there appears to be a loss of apoptosis of the smooth muscle cells allowing their proliferation, and the emergence of apoptosis-resistant endothelial cells that can obliterate the vascular lumen. Vasoconstriction, remodeling of the pulmonary vessel wall, and thrombosis contribute to increased pulmonary vascular resistance in PAH. Pulmonary vascular remodeling occurs at all levels of the vessel wall. Endothelial cells, smooth muscle cells, and fibroblasts as well as inflammatory cells and platelets may play a significant role in PAH. Access this article online

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Website: www.pulmonarycirculation.org DOI: 10.4103/2045-8932.87297 How to cite this article: Long J, Russo MJ, Muller C, Vigneswaran WT. Surgical treatment of pulmonary hypertension: Lung transplantation. Pulm Circ 2011;1:327-33.

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Table 1: Revised World Health Organization classification of pulmonary hypertension, Dana Point, California Group 1: Pulmonary arterial hypertension Idiopathic (primary) Familial Related conditions: Collagen vascular disease, congenital systemic-to-pulmonary shunts, portal hypertension, HIV infection, drugs and toxins (e.g., Anorexigens, rapeseed oil, L-tryptophan, methamphetamine, and cocaine); other conditions: Thyroid disorders, glycogen storage disease, Gaucher’s disease, hereditary hemorrhagic telangiectasia, hemoglobinopathies, myeloproliferative disorders, splenectomy Associated with significant venous or capillary involvement Pulmonary veno-occlusive disease Pulmonary-capillary hemangiomatosis Persistent pulmonary hyptertension of the newborn Group 2: Pulmonary venous hypertension Left-sided atrial or ventricular heart disease Left-sided valvular heart disease Group 3: Pulmonary hypertension associated with hypoxemia Chronic obstructive pulmonary disease Interstitial lung disease Sleep-disordered breathing Alveolar hypoventialtion disorders Chronic exposure to high altitude Developmental abnormalities Group 4: Pulmonary hypertension due to chronic thrombotic disease or embolic disease Thromboembolic obstruction of proximal pulmonary arteries Thromboembolic obstruction of distal pulmonary arteries Pulmonary embolism (tumor, parasites, foreign material) Group 5: Miscellaneous Sarcoidosis, pulmonary Langerhans’-cell histiocytosis, lymphangiomatosis, compression of pulmonary vessels (adenopathy, tumor, fibrosing mediastinitis) Adapted from Simonneau et al.

Pulmonary vasoconstriction is believed to be an early component of the pulmonary hypertensive process and may be related to abnormal function of potassium channels and endothelial dysfunction. [4] Endothelial dysfunction leads to chronically impaired production of vasodilators such as nitric oxide and prostacyclin along with overexpression of vasoconstrictors such as endothelin. [9] Recent genetic and pathophysiologic studies have emphasized the relevance of several mediators in this condition, including prostacyclin,[10] nitric oxide,[11] endothelin,[12] angiopoietin,[7] serotonin,[13] and members of the transforming growth factor superfamily (TGF)-b.[5]

Pathophysiology

The pathophysiology of PH can be understood as a lethal cycle of increased pulmonary vascular resistance (as a result of any of the causes listed in the WHO classification scheme) leading to increased right ventricular performance and oxygen consumption with resultant right ventricular hypertrophy and dilatation, leading to decreased cardiac output and eventual right ventricular failure (Fig. 1).[14,15] In response to an increase in resistance within the pulmonary circulation, the right ventricle responds by increasing right ventricular systolic pressure as necessary to preserve cardiac output. Over time, the pulmonary vascular system responds with progressive remodeling that sustains and promotes PH. 328

Hypotension

Increased RVEDP

Reduced RV coronary blood flow

RV ischemia

Decreased cardiac output

Figure 1: The downward spiral of pulmonary hypertension (adapted from Vigneswaran et al.).

The degree to which the right ventricle responds to such changes is dependent upon the age of the patient and rapidity of onset of PH. A large acute pulmonary embolism can result in right ventricular failure and shock whereas chronic thromboembolic disease of equal severity may result in only mild exercise intolerance. The right ventricle is well designed to adapt to wide variations in preload owing to its anatomical structure and geometry, however these features are not suited to adequately deal with increases in afterload. One of the Pulmonary Circulation | July-September 2011 | Vol 1 | No 3

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key features to right ventricular adaptation to chronic pressure overload is hypertrophy due to increased wall stress (Laplace’s Law).[15] Hypertrophy is greatest in the right ventricular outflow tract and worse in patients with decompensated function.[15] In the setting of increased afterload, right ventricular stroke volume decreases linearly with increasing resistance leading to eventual ventricular dilatation and consequent decreased right ventricular coronary blood flow at a time when oxygen consumption is increased.[16] Furthermore, right ventricular dilatation shifts the interventricular septum to the left, decreasing left ventricular preload and compliance and thus cardiac output. Recent data also suggests that hypoxemia may impair the ability of the right ventricle to make compensatory changes. These studies suggest that right ventricular failure occurs in PH when the myocardium becomes progressively ischemic due to excessive demands and inadequate right ventricular coronary blood flow.[16] The onset of peripheral edema and other clinical manifestations of right heart failure usually portend a poor outcome.[17]

Presenting signs and symptoms

Patients with PH may present with a myriad of cardiopulmonary symptoms however exertional dyspnea is the most frequent symptom and unexplained dyspnea should always raise suspicion. PH may be asymptomatic in the early stages and may be an incidental finding on echocardiogram. Chest pain and syncope are usually late symptoms. Patients may present signs and symptoms of right heart failure such as peripheral edema or ascites. A family history of PH, use of fenfluramine appetites suppressants, cocaine or amphetamines, prior history of deep vein thrombosis (DVT) or pulmonary embolism (PE), chronic liver disease or portal hypertension, HIV, thyroid disease, splenectomy, and sickle cell disease should be sought in all patients suspected to have PH. Physical exam findings include increased jugular venous pressure, a reduced carotid pulse, and a palpable right ventricular impulse. Most patients have an increased pulmonic component of the second heart sound, a rightsided fourth heart sound, and tricuspid regurgitation. Peripheral cyanosis and/or edema tend to occur in later stages of the disease.

Diagnosis and assessment of functional status

The goals of work-up in PH include confirmation of diagnosis, establishing an underlying cause, and quantifying severity with hemodynamics and functional impairment. All patients who appear to have PH after noninvasive testing should undergo right heart catheterization confirm the diagnosis, quantify the degree of hypertension (measurement of pulmonary artery pressure, cardiac output, and left ventricular filling pressure, underlying cardiac shunt) and undergo acute vasodilator testing. Pulmonary Circulation | July-September 2011 | Vol 1 | No 3

Acute vasodilator testing, during catheterization defines the extent of pulmonary vasodilator reactivity and dictates prognosis and therapy. The majority of centers use inhaled nitric oxide (NO) as a pulmonary vasodilator (10-80 ppm). [18] A positive vasodilator response is defined as a decrease of at least 10 mmHg in mean PAP and achieving a mean PAP <40 mmHg, an increase or no change in cardiac output with no significant fall in blood pressure. [19] Patients who respond to acute vasodilator therapy can often be treated with calcium channel blockers and have a more favorable prognosis.[20] Echocardiography is helpful in confirming the diagnosis and excluding left-sided lesions as the cause of PH but is not specific enough to confirm a diagnosis of PH alone. Overall functional status assessed by symptoms (New York Heart Association [NYHA] functional class/World Health Organization functional class [WHO-FC]) and functional capacity assessed primarily by the six-minute walk test (6-MWT) – a very simple and reproducible submaximal exercise test that can be performed by a patient not tolerating maximal exercise tests. The 6-MWT has been the cornerstone functional test to evaluate treatment efficacy both in clinical trials and in daily clinical practice. In unselected patients (treated or not), a 6-MWT of less than 332 meters is associated with a worse prognosis in iPAH and has a strong independent association with mortality in patients with type 1 PH.[21,22]

Prognosis

The natural history of PH is dismal, with a reported median survival rate of 2.8 years when untreated.[23] Functional class remains a strong predictor of survival, with patients who are in NYHA functional class IV having a mean survival of less than six months. The cause of death is usually right ventricular failure, which is manifest by progressive hypoxemia, tachycardia, hypotension, and edema. Over the past decade, advances in medical therapy considerably changed the prognosis of the disease. Most expert centers discuss the notion of transplantation early after diagnosis and closely follow patients’ symptoms, functional status – including the six minute walk test distance, and hemodynamics.[24] Benza et al. analyzed data from 2716 patients with PAH in the US Registry to Evaluate Early and Long-Term PAH Disease Management (REVEAL) study to determine factors that may predict 1-year survival in patients with PAH awaiting transplantion. The authors found in a multivariable analysis that factors independently associated with increased mortality included pulmonary vascular resistance >32 Wood units, PAH associated with portal hypertension, a modified NYHA/WHO functional class IV, men >60 years of age, and family history of PAH. [25] Their data also confirmed an increased mortality risk in patients with renal insufficiency or any pericardial effusion on echocardiogram.[25] 329

Long et al.: PH and lung transplantation

Medical management of PH

Of all the conditions for which lung transplantation is performed, PH is the only one in which there have been significant strides made in medical management. This is seen by the ever-decreasing number of patients with PH who ultimately undergo transplantation. In 1990, approximately 10.5% of all lung transplants were for patients with PH whereas in 2001 only 3.6% of all lung transplants were performed in patients with this condition and most recently, 3.3% as reported by the ISHLT Transplant Registry in 2010.[26,27] Treatment of PH is individualized, based upon severity of functional impairment. As summarized in Table 2, there are currently eight FDA-approved therapies for WHO group 1 PAH.[28] These medications include endothelin receptor antagonists (ERAs) (bosentatn and ambrisentan), phosphodiesterase-5 inhibitors (PDE5-I) (sildenafil and tadalafil), and prostanoid derivatives (epoprostenol, trepostinil, and iloprost). The latter group—the prostanoids introduced in the 1990s—have been the most important advance in the management of patients with PH. Chronic intravenous epoprostenol therapy leads to an improvement in exercise tolerance, hemodynamic measures, as well as survival in iPAH.[29,30] Initially intended to serve as a bridge to transplantation, with experience it has been realized that the need for transplantation can be averted in some cases. [31- 34] Additionally, a select number of patients who have substantial reductions in pulmonary arterial pressure in response to short-acting vasodilators at the time of cardiac catheterization should be treated initially with calcium channel blockers.[20] Patients who respond favorably usually have dramatic reductions in pulmonary artery pressure and pulmonary vascular resistance associated with improved symptoms, regression of right ventricular hypertrophy and improved survival. [35] Anticoagulation with warfarin has been shown to provide a survival benefit in PH, even without documented thromboembolism. [36] Additionally, diuretic therapy relieves peripheral edema and may be useful in reducing right ventricular end diastolic pressure (RVEDP). Supplemental oxygen should be provided to alleviate dyspnea and right ventricular ischemia as hypoxemia is a potent pulmonary vasoconstrictor. A more comprehensive list of medical therapies is available in the most recent ACCP guidelines.[37]

Indications for lung transplantation

Despite the successful development of disease-specific medical therapies for PH which has reduced patient referral for lung transplant programs,[38] transplantation remains the gold-standard for patients who fail medical therapy. Survival among patients requiring treatment with intravenous prostacyclin is approximately 63% at 3 years[39,40] and up to 25% of patients with iPAH may fail 330

to improve on disease-specific therapy and the prognosis of patients who remain in WHO-FC III or IV is poor.[32,33] McLaughlin et al. describe a treatment algorithm based on a risk assessment based on various clinical variables (Table 3). Patients at highest risk should be considered for intravenous therapy as first-line therapy and immediate assessment for lung transplantation. Patients as lower risk are candidates for oral therapy and should be followed closely, and response to therapy reassessed in several months if treatment goals are not met.[41]

In general, referral for transplantation assessment is advisable when patients have a less than 50%, 2- to 3-year predicted survival or NYHA class III or IV level of function, or both.[24] With regards to PAH, most experts recommend transplantation early after diagnosis based on patients’ symptoms, functional status—including the 6-MWT distance—hemodynamics.[24] The decision to list for transplant is made when functional status and hemodynamics decline to the point where survival without transplantation is likely to be compromised[24] (Table 4).

Type of transplantation

The appropriate surgical procedure for patients with Table 2: US Food and Drug AdministrationApproved medications for pulmonary hypertension Name

Approved NYHA class

Bosentan (Tracleer)

NYHA II, III, IV

Ambrisentan (Letairis)

NYHA II, III, IV

Sildenafil (Revatio)

NYHA II, III, IV

Epoprostenol (Flolan)

NYHA III, IV

Treprostinil (Remodulin) Tadalafil (Adcirca)

NYHA II, III, IV NYHA I, II, III, IV

Iloprost (Ventavis)

NYHA III, IV

Treprostinil (Tyvaso)

NYHA III

NYHA: New York Heart Association

Table 3: Determinants of risk in patients with pulmonary hypertension Lower

Determinants of risk

Higher

Clinical evidence of RV failure Progression WHO class 6 minute walk distance Echocardiographic findings

Yes

Gradual II, III Longer (>400 m) Minimal RV dysfunction Normal/near normal RAP and CI

Hemodynamics

Rapid IV Shorter (<300 m) Pericardial effusion Significant RV dysfunction High RAP, low CI

Adapted from McLaughlin et al.

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Long et al.: PH and lung transplantation

Table 4: ISHLT guidelines for lung transplantation Persistent NYHA class III or IV on maximal medical therapy Low (<350 meter) or declining 6-MWT Failing therapy with intravenous epoprostenol, or equivalent Cardiac index of less than 2 liters/min/m2 Right atrial pressure exceeding 15 mmHg NYHA: New York Heart Association; ISHLT: International Society for Heart and Lung Transplantation

iPAH and secondary PAH has been a topic of longstanding debate. Single lung transplant (SLT), bilateral lung transplant (BLT), and heart-lung transplant (HLT) are all used in the treatment of PH. HLT was originally the standard procedure for patients with PH until the 1990’s and is still the preferred treatment for PH patients with irreversible heart disease. However the number of adult HLT have declined significantly in recent years[42-50] to about 70 to 90 per year. It has largely been replaced by bilateral lung transplant, now the most commonly used form of transplantation PH patients. This change was multifactorial: results with SLT or BLT for PH are comparable to or better than HLT, right heart dysfunction improves following SLT or BLT due to right ventricular recovery with the rapid reduction in pulmonary vascular resistance after lung transplantation, as well as ethical and pragmatic concerns centered on donor shortages and organ distribution.[51] In 2008, according to the Official Lung and Heart-Lung Transplant Report of the Registry of the International Society for Heart and Lung Transplantation reported 73 heart-lung transplants and 2,769 lung transplant procedures. Among these, approximately one-quarter of heart-lung transplants were performed with PH as the primary diagnosis and while less than 5% of isolated lung transplant procedures were performed for PH.[52] Among patients undergoing isolated lung transplantation, BLT is used much more frequently–accounting for 91% of transplants performed for PH and the remaining 9% consisting of SLT.[52]

Outcomes following isolated lung transplantation for pulmonary hypertension

For all lung transplant recipients, overall unadjusted survival rates were 79% at one year, 63% at three years, 52% at five years, and 29% at 10 years.[52] Survival rates at three months after lung transplantation were lowest for iPAH (76%) as well as idiopathic pulmonary fibrosis (85%) and highest for cystic fibrosis (90%) and chronic obstructive pulmonary disease (91%).[52] In contrast, among patients surviving at least one year, diagnoses of iPAH, cystic fibrosis, sarcoidosis, and emphysema had significantly better survival at five and 10 years following lung transplantation than those with chronic obstructive pulmonary disease and idiopathic pulmonary fibrosis. Pulmonary Circulation | July-September 2011 | Vol 1 | No 3

The evidence regarding the optimal transplant procedure for PH is remains weak. Available data is largely composed of single center studies. Both SLT and BLT have been performed successfully for PH however outcome analyses and survival comparisons generally favor BLT. There is a trend towards a survival benefit with BLT in the ISHLT registry, however it does not reach statistical significance.

Those who support SLT argue that SLT is easier to perform technically, has less morbidity and mortality versus BLT and HLT, less ischemic time and bypass time with resultant better early graft function, has improved early survival, and will allow more patients to receive lung transplants. Hemodynamics following SLT for PH are characterized by a rapid and sustained drop in pulmonary artery pressures, substantial improvement in right ventricular function, and a preponderance (>90%) of pulmonary blood flow to the allograft as a result of high pulmonary vascular resistance in the remaining native lung.[53-55] This leads to a postoperative situation in which ventilation is evenly divided between the allograft and native lung while perfusion is almost entirely directed to the allograft. Any complication in the allograft (e.g., pneumonia, primary graft dysfunction, rejection) can result in severe ventilation-perfusion mismatch and hypoxemia.

Most centers favor BLT because of the physiologically increased functional reserve, making patients less prone to respiratory insufficiency with subsequent insult. [47,56] Proponents of BLT have argued that bilateral lung transplants result in less ventilation perfusion (V/Q) mismatches, are easier to care for in the perioperative period, will enable more “marginal lungs” to be utilized, will provide better overall lung function, are protective against the physiologic manifestations of bronchiolitis obliterans, and have a better long-term survival. In its most recent publication, the ISHLT registry reported a significantly better survival in the bilateral lung transplantation group as compared with the single lung transplant in patients with iPAH.[52] The Pittsburgh group compared recipients of SLT versus BLT with PH and found similar functional status and postoperative recovery (hemodynamics, duration of mechanical ventilation, intensive care unit/hospital length of stay, and incidence of acute/chronic rejection).[43] They did, however, report that SLT recipients had a significantly lower PaO2/FiO2 at one hour and higher mean pulmonary artery pressure at 12 and 24 hours.[43] Bando et al. concluded that despite significantly shorter cardiopulmonary bypass duration in their cohort, SLT recipients with underlying PH had significantly less functional recovery (less reduction in mean pulmonary artery pressure and increase in cardiac index) and higher graft mortality compared with BLT and HLT recipients.[45] The Hopkins experience evaluated 15 patients with iPAH and found BLT to have a highly 331

Long et al.: PH and lung transplantation

significant survival advantage compared with SLT at 4 years of follow-up.[51] The most recent ISHLT registry reported 788 iPAH lung transplant recipients of which 91% were BLT, highlighting the continuing trend at most centers favoring BLT versus SLT for iPAH.[27]

10.

According to the 2009 ISHLT registry report comparing the outcome of HLT with BLT for pulmonary hypertension demonstrated similar survival between the two groups. Of 2712 adult patients who underwent heart-lung transplantation between 1982 and 2007 survival rates of 72 percent at three months and 64 percent at one year. Survival for HLT was less than with lung transplantation alone (89% and 79%, respectively, in the era from 1994 to 2007). However, overall survival five years and ten years was 43% and 28%, which was comparable to isolated lung transplantation. Among HLT patients who survived one year, there was a low but steady mortality rate with a survival conditional half-life (contingent on survival to one year) of 9.2 years.[57]

14.

Outcomes following heart-lung transplantation for pulmonary hypertension

CONCLUSIONS

Lung transplant is an effective form of treatment for patients with PH who have exhausted all medical therapy. BLT is the current choice of transplantation in this patient population.

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pulmonary hypertension. J Thorac Cardiovasc Surg 1993;106:229-307. 47. Birsan T, Zuckermann Z, Artermiou O, Senbaklavci O, Taghavi S, Wieselthaler G, et al. Bilateral lung transplantation for pulmonary hypertension. Transplant Proc 1997;29:2892-4. 48. Whyte RI, Robbins RC, Altinger J, Barlow CW, Doyle R, Theodore J, et al. Heart-lung transplantation for primary pulmonary hypertension. Ann Thorac Surg 1999;67:937-42. 49. Mikhail G, Al-Kattan K, Banner N, Mitchell A, Radley-Smith R, Khaghani A, et al. Long-term results of heart lung transplantation for pulmonary hypertension. Transplant Proc 1997;29:633. 50. Ueno T, Smith JA, Snell GI, Williams TJ, Kotsimbos TC, Rabinov M, et al. Bilateral sequential single lung transplantation for pulmonary hypertension and Eisenmenger ’s syndrome. Ann Thorac Surg 2000;69:381-7. 51. Conte JV, Borja MJ, Patel CB, Yang SC, Jhaveri RM, Orens JB. Lung transplantation for primary and secondary pulmonary hypertension. Ann Thorac Surg 2001;72:1673-80. 52. Taylor DO, Edwards LB, Aurora P, Christie JD, Dobbels F, Kirk R, et al. The registry of the International Society for Heart and Lung Transplantation: Twenty-fifth official adult lung and heart-lung transplant report—2008. J Heart Lung Transplant 2010;27:943-56. 53. Griffith BP, Hardesty RL, Armitage JM, Hattler BG, Pham SM, Keenan RJ, et al. A decade of lung transplantation. Ann Surg 1993;218:310-20. 54. Katz W, Gasior TA, Quinlan JJ, Lazar JM, Firestone L, Griffith BP, et al. Immediate effects of lung transplantation on right ventricular morphology and function in patients with variable degrees of pulmonary hypertension. J Am Coll Cardiol 1996;27:384-91. 55. Pasque MK, Trulock EP, Kaiser LR, Cooper JD. Single-lung transplantation for pulmonary hypertension. Three-month hemodynamic follow-up. Circulation 1991;84:2275-9. 56. Levine SM, Jenkinson SG, Bryan CL, Anzueto A, Zamora CA, Gibbons WJ, et al. Ventilation-perfusion inequalities during graft rejection in patients undergoing single lung transplantation for primary pulmonary hypertension. Chest 1992;101:401-5. 57. Afshar K, Weill D, Valentine V, Dhillon G. Comparison of double-lung and heart-lung transplantation for pulmonary hypertension: Analysis of the UNOS database. J Heart Lung Transplant 2009;28:S310. Source of Support: Nil, Conflict of Interest: None declared.

Author Help: Online submission of the manuscripts Articles can be submitted online from http://www.journalonweb.com. For online submission, the articles should be prepared in two files (first page file and article file). Images should be submitted separately. 1) First Page File: Prepare the title page, covering letter, acknowledgement etc. using a word processor program. All information related to your identity should be included here. Use text/rtf/doc/pdf files. Do not zip the files. 2) Article File: The main text of the article, beginning with the Abstract to References (including tables) should be in this file. Do not include any information (such as acknowledgement, your names in page headers etc.) in this file. Use text/rtf/doc/pdf files. Do not zip the files. Limit the file size to 1 MB. Do not incorporate images in the file. If file size is large, graphs can be submitted separately as images, without their being incorporated in the article file. This will reduce the size of the file. 3) Images: Submit good quality color images. Each image should be less than 4096 kb (4 MB) in size. The size of the image can be reduced by decreasing the actual height and width of the images (keep up to about 6 inches and up to about 1800 x 1200 pixels). JPEG is the most suitable file format. The image quality should be good enough to judge the scientific value of the image. For the purpose of printing, always retain a good quality, high resolution image. This high resolution image should be sent to the editorial office at the time of sending a revised article. 4) Legends: Legends for the figures/images should be included at the end of the article file. Pulmonary Circulation | July-September 2011 | Vol 1 | No 3

333

Review Ar ti cl e

Apelin and pulmonary hypertension Charlotte U. Andersen1, Ole Hilberg2, Søren Mellemkjær3, Jens E. Nielsen-Kudsk3, and U. Simonsen1 1

Department of Biomedicine, Aarhus University, Departments of 2Allergology & Respiratory Diseases, and 3Cardiology, Aarhus University Hospital, Denmark

ABSTRACT Pulmonary arterial hypertension (PAH) is a devastating disease characterized by pulmonary vasoconstriction, pulmonary arterial remodeling, abnormal angiogenesis and impaired right ventricular function. Despite progress in pharmacological therapy, there is still no cure for PAH. The peptide apelin and the G-protein coupled apelin receptor (APLNR) are expressed in several tissues throughout the organism. Apelin is localized in vascular endothelial cells while the APLNR is localized in both endothelial and smooth muscle cells in vessels and in the heart. Apelin is regulated by hypoxia inducible factor -1α and bone morphogenetic protein receptor-2. Patients with PAH have lower levels of plasma-apelin, and decreased apelin expression in pulmonary endothelial cells. Apelin has therefore been proposed as a potential biomarker for PAH. Furthermore, apelin plays a role in angiogenesis and regulates endothelial and smooth muscle cell apoptosis and proliferation complementary and opposite to vascular endothelial growth factor. In the systemic circulation, apelin modulates endothelial nitric oxide synthase (eNOS) expression, induces eNOSdependent vasodilatation, counteracts angiotensin-II mediated vasoconstriction, and has positive inotropic and cardioprotective effects. Apelin attenuates vasoconstriction in isolated rat pulmonary arteries, and chronic treatment with apelin attenuates the development of pulmonary hypertension in animal models. The existing literature thus renders APLNR an interesting potential new therapeutic target for PH. Key Words: apelin, aPJ, Apelin and the apelin receptor, pulmonary hypertension

INTRODUCTION Pulmonary hypertension

Pulmonary arterial hypertension (PAH) is a severe disease with a median survival of 2.8 years if left untreated.[1] Over the past two decades, novel drugs with a pulmonary vasodilator action and a possible additional inhibitory effect on vascular cell proliferation have been developed, but even after the introduction of such compounds the chance of survival remains poor, with a 3-year survival less than 60%.[2]

PAH is characterized by a mean pulmonary arterial pressure (MPAP) above 25 mmHg at rest and an increased pulmonary vascular resistance (PVR) in combination with a normal pulmonary capillary wedge pressure (PCWP). [3] PAH eventually leads to right ventricular pressure overload and compensatory hypertrophy followed by dilatation and failure of the right ventricle,[4,5] which is the most common cause of death.[6] The current therapeutic Address correspondence to:

Dr. Charlotte Andersen Department of Biomedicine, Pulmonary and Cardiovascular Pharmacology, Aarhus University, Wilhelm Meyers Alle 4, Dk-8000 Aarhus C, Denmark Email: cua@farm.au.dk 334

drugs are primarily pulmonary vasodilators such as endothelin-1 (ET-1) receptor antagonists, prostacycline analogues and phosphodiesterase-5 inhibitors that aim to correct for abnormalities in the secretion of endotheliumderived vasoactive mediators. Nevertheless, no current therapy against PAH is sufficient to cure or stop the disease progression. Consequently, there is a need for new therapies.

Pathophysiological mechanisms of PAH

Multiple genetic, cellular and molecular functions are involved in the pathophysiology of PAH. These have recently been reviewed extensively. [7] A number of pathophysiological mechanisms involved in PAH are relevant in relation to the subject of this paper. For example, normoxic activation of hypoxia-inducible factor (HIF-1α), normally exerting the physiologic Access this article online

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Website: www.pulmonarycirculation.org DOI: 10.4103/2045-8932.87299 How to cite this article: Andersen CU, Hilberg O, Mellemkjær S, Nielsen-Kudsk JE, Simonsen U. Apelin and pulmonary hypertension. Pulm Circ 2011;1:334-46.

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Andersen et al.: Apelin and PH

hypoxic vasoconstriction, can occur in cells prior to the spontaneous development of PAH in fawn-hooded rats and is thought to be a possible contributor to the development of PAH.[8] Furthermore, genetic aspects play a role. One of the most prominent genes involved in PAH is the bone morphogenetic protein receptor 2 (BMPR-2), in which mutations occur in 70% of patients with familial PAH and in 25% of patients with idiopathic PAH.[7] Abnormal apoptosis and proliferation of vascular endothelial and smooth muscle cells, [7,9] is involved in the remodeling process of the pulmonary arteries, development of plexiform lesions, and loss of the microvasculature. Numerous humoral factors, including vascular endothelial growth factor (VEGF), are involved in this response. [9] Furthermore, the function of the endothelium is altered in PAH, resulting in an imbalance between endothelium-derived vasoconstrictors and proliferative agents such as ET-1 and thromboxane, and vasodilators with antiproliferative effects including nitric oxide (NO) and prostacyclin. [10] In addition to contributing to the remodeling process, it results in decreased vasorelaxation of the pulmonary vascular bed. Angiotensin-II also induces vasoconstriction and mitogenesis in PAH, while enhanced expression of the angiotensin-II converting enzyme 2 (ACE2) has been found to have a beneficial effect in animal models of pulmonary hypertension.[11,12] The right ventricle is subjected to pressure-induced alterations in PAH. Compensatory hypertrophy and fibrosis of the right ventricle develops, followed by decreased systolic function and dilatation. [4] Among other mechanisms, ischemia and apoptosis are central players in this process, [4] and have increased the interest to investigate whether drugs directly targeting mechanisms in the right ventricle may improve the course of PAH.

Apelin and the apelin receptor

The peptide apelin and the apelin receptor (APLNR) are present in the heart,[13,14] the systemic and pulmonary vasculature, and the expression of apelin and APLNR is regulated by HIF-1α[15] and BMPR-2.[16] Furthermore, the apelin-APLNR system is involved in normal vascular development [17] and regulation of apoptosis, [16] and has been shown to be involved in regulation of NOdependent vasodilatation[15,18] and to improve cardiac contractility. [19] Most studies on apelin have addressed the systemic circulation, but the characteristics revealed by these studies have drawn attention to apelin-APLNR as a potential new target in pulmonary hypertension. Recently, support for a role of apelin and APNLR in pulmonary hypertension has emerged through experiments in animal models of pulmonary hypertension. Pulmonary Circulation | July-September 2011 | Vol 1 | No 3

The purpose of this paper is to introduce the apelinAPLNR system, to review the associations between PAH pathophysiology and apelin based on existing studies, to touch upon apelin as a potential biomarker for PAH, and to point out the regulating effects of apelin on vascular endothelial and smooth muscle cell proliferation, the vasodilatory effect and positive inotropic effect that renders modulation of APLNR an interesting therapeutic target in PAH.

DISCUSSION

Apelin and APLNR

Structure APLNR was discovered in 1993 by O’Dowd and colleagues who were looking for subtypes of the vasopressin receptor. The APLNR was first named APJ and is a G-protein coupled 7 trans-membrane receptor coded for on chromosome 11 band 12, and shares 54% homology in the transmembrane region with the AT1 receptor. It does not, however, bind angiotensin-II.[20] Human APLNR shares 74% homology with the rat APLNR.

In 1998, apelin was isolated by Tatemoto et al.[21] They showed that the active forms of apelin are peptides cleaved from the C-terminal of the 77 amino acids long preproapelin. Fragments of 36, 17, 13 including the N-terminal pyroglutamated apelin-13 (Pyr 1 apelin-13), exist in vivo and have biological activity, but also synthetic fragments of the 12 amino acids from the C-terminal can activate the receptor.[22] It has been suggested that Pyr1-apelin-13 is the final active product of apelin, which is more resistant to enzymatic cleavage. [23,24] Biological differences between apelin fragments have been shown[25] (e.g., apelin-13, and -17 binds the receptor with higher potency), whereas it is proposed that apelin-36 dissociates slower from the APLNR.[26-28] ACE 2 cleaves a single amino acid from the c-terminal of apelin-13 by hydrolysis[29] (Fig. 1a). It is not fully elucidated whether this inactivates the protein, since studies investigating apelin-17 with amino-acids deleted from the c-terminal found that such peptides still bound to the receptor.[30] However, the study suggested that receptor activation was altered, in that internalization did not take place.[19] So far, ACE 2 is the only known enzyme metabolizing apelin. Nevertheless, it seems that the halflife of apelin is short. A half time less than 8 minutes has been documented in humans.[27]

In short, the difference between the apelin-peptides of different lengths is not fully determined, and furthermore, the pharmacokinetics of the peptides are largely unknown. 335

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Apoptosis

Gln Arg pro Arg Leu Ser His LysGlyProMet Pro Phe

Gi Gi PI-3k Akt

N AMP kinase Kruppel-like factor

eNOS

D: Cardiomyocyte Gi

Contraction Apoptosis

Gq Contraction

PKC MLCK

PLC Na2+/H+E Na2+/Ca+E PI-3K PKC Ca2+ sensitivity Ca2+ AKT

C: Smooth muscle cell

PLC

ACE 2

A 13

B: Endothelial cell

GC cGMP

Figure 1: Proposed signaling pathways for apelin and APLNR. The figure summarizes data obtained from both systemic and pulmonary vasculature and both left and right ventriclet. A: the structure of apelin-13 showing the site for ACE-2 mediated hydrolysis. B: known pathways in endothelial cells, C: vascular smooth muscle cells, and D: cardiomytes, blue →: Stimulates activity; red →: Decreases activity. Na2+/Ca2+ E: Na2+/Ca2+ exchanger, Na2+/ H+ E: Na2+/Ca2+ exchanger.

Apelin-APLNR interaction and signaling There is evidence that the APLNR is coupled to an inhibitory Gi-protein because apelin inhibits forskolin induced 3’5’ cyclic adenosine monophospate (cAMP) production in Chinese hamster ovary (CHO) cells[21,26,31] and because extracellular acidification in CHO cells can be blocked by pertussis toxin.[26] Furthermore, apelin also works by stimulating the Na 2+/H+ exchanger in CHO cells, [26] and apelin has been shown to activate extracellular signal-regulated kinase (ERK).[28,32] Some effects of apelin have been shown to be mediated by the phophoinositol-3-phosphokinase (PI-3K)/AKT activated pathway.[33] Although Masri et al. reported that apelin could not induce increments of inositol phosphate in CHO cells and concluded that the APLNR did not bind a Gq/G11 protein,[32] the positive inotropic effects of apelin are dependent on the function of phospholipase C (PLC), protein kinase C (PKC), the Na2+/H+ and the Na2+/ Ca2+ exchanger,[19] and an increase in intracellular Ca2+ transients.[34] These findings are consistent with coupling of the APLNR to a Gq-protein[35] (Fig. 1).

A 2004 study found that the APLNR was internalized into the cytoplasm and subsequently, the nucleus, upon apelinAPLNR interaction in CHO cells, and it was observed that only apelin fragments able to induce internalization exerted a biological in vivo response.[30] However, other G-protein coupled receptors such as the angiotensin-II receptor AT1 are able to elicit biological responses both through activation of the G-protein, but also to act on intracellular pathways after internalization.[36] Hence, this coupling between internalization and effect requires further investigation. To date, apelin has not been proven to bind to other receptors,[31,37,38] and no subtypes of the APLNR have been 336

found. Also, no other endogenous ligands for the APLNR are known, but mice lacking the APLNR are different, as described below, from mice lacking the apelin gene, which suggests that other endogenous substances influence the activity of APLNR.[39] For example, the APLNR has been shown to form heterodimers and oppose the effect of the angiotensin-II receptor independently of the presence of apelin.[40]

Localization of APLNR and apelin Early studies showed that apelin and APLNR mRNA was expressed in numerous tissues of the rat,[26,41] including several areas of the brain, adipose tissue, heart and lungs. The expression in the lungs was greater than in other tissues, except for a very high expression of apelin in the mammary gland.[41] In the cardiovascular system, APLNR immunoreactivity was present in both endothelial and smooth muscle cells in the vasculature, in endocardium and myocardium. Apelin immunoreactivity was present in the myocardium, endocardium, and in endothelial cells of the large conduit vessels. In the lungs, apelin immunoreactivity was demonstrated only in the endothelium of small pulmonary arteries, not in the epithelium or vascular smooth muscle cells.[14] Apelin immunostaining was localized to vesicle-like structures in the endocardial endothelium and in structures associated to the nuclear surface in endothelial cells.[13] The APLNR has been demonstrated in nuclear fragments of cells.[42] On the basis of these observations, it has been suggested that apelin is secreted by endothelial cells[13] and that the APLNR may affect regulation of gene transcription.[42] In human tissue, the relative expression of apelin differs from the rat as the mRNA expression of preproapelin and the apelin receptor in the spleen and central nervous system seems higher than in the lungs.[43] Apelin was also shown to be present in the endothelium of small and larger pulmonary vessels in human tissue.[44] Accordingly, the location of apelin in the pulmonary endothelium with receptors in both endothelium and smooth muscle makes this system interesting in relation to pulmonary vascular disease.

Regulation of apelin and APLNR

Factors regulating apelin and APLNR are summarized in Figure 2.

Hypoxia Vast evidence supports the assertion that apelin expression is regulated by hypoxia. In mice, an increase in total pulmonary apelin mRNA was reported to take place after 1 week of hypoxia,[45] and in another study in mice, approximately a doubling of apelin mRNA and an increase of apelin protein of about 25% was found after 5 hours Pulmonary Circulation | July-September 2011 | Vol 1 | No 3

Andersen et al.: Apelin and PH

Hypoxia Disrupted oxygen sensing

Heart failure Mechanical stress

HIF-1α ?

Angiotensin-II

Apelin / APLNR

Insulin TNF-α Growth hormone

BMPR-2 Figure 2: Factors influencing expression of apelin. Blue →: Stimulates expression; Red →: Decreases expression.

of hypoxia.[46] In rats, however, total pulmonary apelin content was unchanged after exposure to hypoxia for 2 weeks.[47] An explanation of this apparent incongruence was demonstrated in cultured pulmonary endothelial cells, where apelin mRNA levels were up-regulated after 8 hours of hypoxia, but fell after further hypoxic exposure.[46] In a study made on mice by Chandra et al., a similar tendency was shown in right ventricular tissue. Here, apelin and APLNR mRNA was four- and three fold increased after one week of hypoxia, but unchanged after 3 weeks.[48] An initial up-regulation followed by a decrease is also found after glucose deprivation in cultured cardiomyocytes.[33] The hypoxia-induced regulation of apelin and the APLNR was also found in cultured human hepatocytes, stellate cells[49] and adipocytes,[15,50] where APLNR and apelin were up-regulated within 24 hours of hypoxic exposure. Similarly, secretion of apelin to extracellular medium is increased by short-time hypoxia in cultured cardiac myocytes[51] and adipocytes.[50] The regulation induced by hypoxia was shown to be mediated by HIF-1α in adipocytes.[15,50] In short, it seems that hypoxia regulates the apelin pathway in various tissues, and that there is a biphasic response of apelin and the APLNR to hypoxia with an initial upregulation followed by a normalization or decrease. Interestingly, in the context of pulmonary hypertension, HIF-1α is a mediator for the hypoxia induced regulation of apelin and APLNR.

Heart failure—mechanical strain or hypoxia? Apelin and APLNR are altered in heart failure. In an animal model of ischemic heart failure, total heart apelin protein was up-regulated 6 weeks after induction of myocardial infarction.[52] A study in Dahl salt-sensitive hypertensive rats showed that apelin and APLNR mRNA expression was unaltered in a phase of compensated left heart hypertrophy, but decreased in manifest heart failure,[53] and in a model of isoprenaline-induced heart failure, apelin and APLNR were found to be downregulated.[54] Pulmonary Circulation | July-September 2011 | Vol 1 | No 3

In one human study, APLNR mRNA and apelin protein levels were up-regulated in left ventricular tissue, but lower in atrial tissue from patients with heart failure compared to healthy controls.[55] Other studies addressing plasma levels of apelin in heart failure patients have reported down regulation of the apelin system.[44,56,57] In line with the proposed biphasic response to hypoxia, it is also possible that apelin and APLNR are regulated in opposed directions during different phases of heart failure. This is supported by a study by Chen et al., who showed that apelin plasma levels were in fact higher in patients with low NYHA class heart failure than in healthy controls, but that the levels declined in more advanced functional classes. [58] Interestingly, cardiac APLNR mRNA rose in patients after implementation of a left ventricular assist device.[58] This could point towards a regulatory effect of mechanical strain on the heart or other factors involved in heart failure e.g., humoral mediators. To support this, apelin and APLNR were down-regulated in the right ventricle of monocrotaline rats with pulmonary hypertension[23] and in mice subjected to aortic banding.[59] On the other hand, tissue hypoxia may also be present in these types of heart failure, and the hypoxia stimulus attenuated by improving left ventricular function. This topic therefore needs more investigation. Apelin levels were also shown to be decreased in patients with ischemic heart disease,[60-62] and again both hypoxia and mechanical stress may be involved. BMPR-2, angiotensin-II, insulin and other regulatory factors A link between the BMPR-2 and apelin was recently recognized. [16] It was shown that disrupted BMPR2 signaling mediated through Peroxisome proliferatoractivated receptor gamma (PPARγ) / β-catenin resulted in decreased apelin expression and increased endothelial cell apoptosis. In addition, the authors showed that apelin secreted from pulmonary endothelial cells inhibited proliferation of pulmonary arterial smooth muscle cells. The authors suggested that apelin is a downstream protein from the BMPR-2 receptor signaling involved in pulmonary vascular homeostasis. Treatment with angiotensin-II has been shown to upregulate APLNR expression in cultured hepatocytes. [49] In models of decompensated heart failure APLNR expression was decreased,[53,63] and in one study, this was prevented by treatment with an angiotensin-II receptor antagonist. [53] A β-adrenoceptor agonist failed to do so, even though the two drugs improved the systolic function of the left ventricle equally.[53] These results are apparently contradictory, but indicate a regulatory relationship between the apelin and angiotensin-II systems. In adipose tissue, insulin up-regulates the expression of apelin in obese mice and insulin levels are related to 337

Andersen et al.: Apelin and PH

plasma apelin levels in obese individuals.[64] Furthermore, growth hormone and tumor necrosis factor –α are proposed as regulators of apelin in adipose tissue.[65,66]

Overlap between apelin regulation and factors involved in PAH Accordingly, apelin and APLNR are regulated by hypoxia, which is an important factor in pulmonary vasoconstriction. In addition, HIF-1α, BMPR-2 and angiotensin-II that have all been linked to the pathophysiology of pulmonary hypertension also regulate apelin/APLNR expression. Furthermore, it is possible that mechanical strain and cardiac overload, that are also features of pulmonary hypertension, affects apelin-APLNR levels. Two studies have shown that serum apelin levels in patients with PAH are lower than in controls,[44,48] and also a decreased content of apelin in pulmonary arterial endothelial cells in patients with PAH has been demonstrated.[16] This supports a role of apelin and APLNR in pulmonary hypertension. The regulation by insulin makes it interesting to investigate the role of apelin in obesity-related pulmonary hypertension.

Apelin as a plasma biomarker

The changes in plasma apelin levels in heart failure[44,55-58] and pulmonary hypertension[44,48] have brought forward the idea of apelin as a possible biomarker for these diseases. In heart failure patients,[55] the concentration of apelin was 200 fold higher in atrial tissue than in left ventricular tissue. Plasma apelin concentrations correlated to atrial apelin levels, and it was therefore suggested that atrial apelin might be an important source of apelin in plasma.[55]

0.5

1.0

1.5

2.0

In short, it is not fully established, which organs are most important in determining the plasma levels of apelin, but the widespread distribution of apelin probably reduces the use of the peptide as a marker for functional change in a specific organ. At least apelin does not seem to be a lung-derived biomarker for pulmonary hypertension, and, extrapolating the results from studies in patients with left heart disease, the BNPs may be expected to be more specific than apelin in detecting right ventricular failure secondary to pulmonary hypertension.

0.5

Plasma apelin ng/ml (a)

1.0

1.5

0.0

0.5

RVSP (mmHg) 40 60

1.0

1.5

In pulmonary hypertension, apelin was proposed as a potential lung-derived biomarker, because of the high expression of apelin in the lungs.[44] If pulmonary apelin was reflected in plasma, it would be a very interesting biomarker yielding information about the small pulmonary artery endothelium given the presence of apelin here.[13] Nevertheless, an animal study in chronic hypoxic rats with pulmonary hypertension showed that apelin content in the lungs were not reflected in plasma samples and that plasma apelin levels did not correlate to right ventricular pressure[47] (Fig. 3). On the other hand plasma apelin levels correlated weakly (r2 = 0.33) to changes in right ventricular apelin levels (Fig. 3).

R 2 = 0.04, p=0.4

R 2 = 0.3, p=0.01

-0.5

RV apelin concentration (ng/mg protein)

2 1.5 1 .5

R 2 = 0.5, p=0.001

Lung apelin concentration (ng/mg protein)

However, studies have compared apelin with brain natriuretic peptide (BNP) and the split product

N-terminal BNP (NT-proBNP) as biomarkers in heart failure. It has been established that the main source of circulating BNP and NT-proBNP is the heart,[67] and in patients with myocardial infarct, apelin-36 was inferior to BNP in order to detect decreased left ventricular systolic function.[68] Similarly, other studies report that NT-proBNP is superior to apelin in correlation with heart failure severity[69] and prediction of mortality in acute heart failure.[70]

2.0

0.5

1.0

1.5

Plasma apelin ng/ml

(b)

(c)

2.0

Figure 3: Original figure adapted from data in Andersen et al. (a): In chronic hypoxic rats, lung concentrations of apelin were not altered in the same direction as plasma apelin by hypoxia and sildenafil treatment. (b): Right ventricular apelin concentrations correlated weakly with plasma apelin levels; (c): Plasma apelin levels were not correlated to right ventricular systolic pressure. RVSP: Right ventricular systolic pressure. ■ = Normoxic rats; ● = Hypoxic rats, ▲ = Hypoxic rats treated with sildenafil 25 mg/kg/day. [47]

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Effect of apelin in angiogenesis and vascular cell homeostasis

Angiogenesis In 1996 the APLNR was found to be present in endothelial cells and precursor cells of the embryonic vasculature.[17,71] Disrupting the apelin/APJ pathway resulted in reduction or loss of intersegmental vessels in frog embryos,[72] and the authors also showed that apelin was a mitogen and migration factor for endothelial cells. Mice lacking the apelin gene are viable, and postmortem analyses have shown no major anatomical or histological abnormalities. However, APLNR deficient mice show abnormalities in regulation of blood pressure, and in the cardiovascular development. Furthermore, the offspring of APLNR heterozygous mice did not follow a Mendelian order, showing a significantly smaller number of APLNR-/- mice than expected.[39] This suggests that the APLNR plays a more pivotal role than apelin per se, and as mentioned previously, that the APLNR may be activated by other ligands. Apelin has also been shown to be an angiogenic factor in retinal cells.[73-77] Lack of apelin has been shown to decrease the hypoxia-induced regeneration of vessels in zebra fish,[46] and in accordance, a study from 2010 showed that lack of apelin worsens necrosis in a model of hind limb ischemia in mice. Furthermore, apelin administration was able to increase the number of larger vessels and decrease necrosis in a model of hind limb ischemia in mice.[78]

Apelin and VEGF—similarities and differences Like vascular endothelial growth factor (VEGF), apelin inhibits apoptosis in endothelial cells.[16,79] There are conflicting results about the effect of apelin on apoptosis and proliferation in vascular smooth muscle cells. Some studies found that apelin inhibits apoptosis in umbilical vein smooth muscle cells,[80] and stimulates proliferation in vascular smooth muscle cells from rat thoracic aortas[81,82] evoked by activation of the PI3-K/ AKT pathway.[80,81,83] However, a study investigating the effects in pulmonary artery smooth muscle cells found that apoptosis was increased by apelin. [16] Another characteristic of apelin similar to VEGF is a stimulatory effect on tumour and micro-vessel growth in relation to cancer.[80,83,84]

In contrast to apelin or APLNR deficient mice, homozygous VEGF knockout mice die mid-gestational due to severe developmental abnormalities of the circulatory system. [85] This suggests that VEGF is more important in vascular development than apelin. The case seems to be an interrelationship between apelin and VEGF in that apelin stimulated the expression of VEGF-A in cell culture.[49] Moreover, VEGF and apelin combined has been shown to more effectively establish new vessels in animal models of limb ischemia;[78] and, interestingly, apelin counteracted Pulmonary Circulation | July-September 2011 | Vol 1 | No 3

the increased vascular permeability of new vessels induced by VEGF.[78] Increased vascular leakage may be involved in the pathobiology of PAH,[7] and apelin has been shown to reduce lipopolysaccharide-induced vascular leakage in rat lung tissue.[86]

PAH, apelin and VEGF In PAH patients, endothelial cells have been shown to be abnormal in that proliferation and migration is enhanced, while angiogenesis is less effective in order to generate structured networks,[87] and other studies also point toward a disordered angiogenesis in PAH.[88] The role of VEGF in pulmonary hypertension is ambiguous since the lack of VEGF clearly worsens the severity of pulmonary hypertension development in animal models, [89] due to apoptosis of normal endothelial cells. This leads to recruitment of poorly differentiated apoptosis-resistant endothelial cells forming plexiform lesions, [90-92] and furthermore, smooth muscle cells proliferate abnormally. [16] On the other hand, it seems that excessive VEGF in advanced stages of PAH contributes to the abnormal endothelial cell proliferation and generation of plexiform lesions.[93] So far apelin seems to be favorable in pulmonary hypertension. The study by Chandra et al. showed more severe pulmonary hypertension and a more pronounced loss of microvessels in the pulmonary vasculature in chronic hypoxic mice lacking the apelin gene.[48] This, taken together with the attenuating effect of apelin on pulmonary endothelial cell apoptosis,[16] may suggest that apelin could modulate the angiogenesis in a positive way in PAH. In pulmonary hypertension related to scleroderma and pulmonary fibrosis, a decreased vascular capacitance is of particular importance,[94,95] and the effects of apelin in these diseases are worthy of further elucidation.

Effects of apelin on vascular tone and blood pressure

Isolated arteries In isolated systemic artery[96,24] and vein[97] preparations, apelin reduces vascular tone by 25%-50%, and vasoconstriction in response to angiotensin-II has been shown to be inhibited by incubation of arteries with apelin.[98,99] Other studies[100] confirm the modulatory effect of apelin on angiotension-II induced vasoconstriction, including a study in APLNR deficient mice that had increased pressor response to angiotensin-II.[38] However, it was observed that apelin exerted vasoconstriction in denuded vena saphena magna preparations.[37] In keeping with this, apelin phosphorylates myosin light chain kinase dependent on PKC in vascular smooth muscle.[101] This suggests that the apelin induced vasodilatation is endothelium-dependent, and that apelin has the opposite effect when acting directly on the smooth muscle cells (Fig. 1). 339

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Blood pressure One of the first proven physiological effects of apelin was the ability to temporarily lower blood pressure after injection in rats,[18] and several studies have confirmed this effect,[24,25,30,38,102] including studies showing vasodilation in human volunteers and heart failure patients[27,103] (Table 1). However, a single study found that apelin given intravenously resulted in an increment of blood pressure in conscious rats [105] (Table 1). Furthermore, apelin injected or overexpressed in the rostral ventrolateral medulla of the central nervous system results in blood pressure increments.[106,107]

The results suggest that the effects of apelin on blood pressure is not straightforward, and that the net result may depend on the apelin fragment, way of administration, conscious state of the experimental animal, and the propensity for compensatory increases in heart rate and cardiac output.

Apelin and the endothelial NO synthase Inhibition of prostaglandin synthesis has been shown to both reduce[24] and be without influence[97] on the effects of apelin, while numerous studies show that the vasodilatory effect of apelin is inhibited by inhibition of endothelial NO synthase (eNOS).[16,27,38,96,97] It is also evident that apelin stimulates the activity of eNOS. [38,98,99] Apelin near-normalized the eNOS phosphorylation in in vitro preparations of the aorta[99] and renal artery[98] in diabetic mice with endothelial dysfunction. On the contrary, apelin-induced vasodilatation was reduced in hepatic arteries from patients with liver cirrhosis

compared to mesenteric arteries from healthy donor patients,[96] and this was suggested to be due to a decreased function of eNOS in the arteries from liver cirrhotic patients. However, caution should be taken interpreting these data, since two different vessel types are compared. In short, it is controversial whether apelin normalises impaired eNOS function and improves endothelial function in disease states, or if apelin looses its effects in case of endothelial dysfunction because of decreased eNOS function. AKT,[99] AMP kinase[48] and kruppel-like factor 2[48] have been suggested to link stimulation of APLNR to eNOS (Fig. 1). Effect of apelin on pulmonary arteries and pulmonary pressure in vitro and in vivo The effect of apelin in pulmonary arteries has been examined in only a few in vitro studies.[47,108] One group found that only very high concentrations of apelin were able to transiently decrease the vascular tone with maximally 17%. In a Chinese study, apelin decreased tone by maximally 11%, and the vasorelaxation was abolished by removal of the endothelium and treatment with the eNOS blocker L-NAME[108] in line with the findings in the systemic circulation. Furthermore, the Chinese group found that the relaxation was decreased by 60% in isolated pulmonary arteries from hypoxic animals. Accordingly, it was shown that in isolated pulmonary resistance arteries from normoxic control rats, treatment with apelin inhibited vasoconstriction to endothelin-1,[47] while this effect was absent in hypoxic rats with pulmonary hypertension. There was no down-regulation of the APLNR in the arteries from hypoxic rats,[47] suggesting that the impairment was downstream to the apelin receptor in pulmonary hypertension. This favors the hypothesis that

Table 1: Effects of apelin on blood pressure in the systemic circulation Reference

Model

Lee et al[18] Charles et al[102]

Anaesthetized rat Conscious sheep

El Messari[30] Ishida et al.[38] Lee et al.[25]

Japp et al.[27] Cheng et al. [104] Kagiyama et al[105]

Effect

Duration (in minutes)

BP 10-13.0 mmHg ↓ 3-4 12 mmHg↓ followed by ↓:2 12 mmHg↑ in MAP ↑: 4 to 15 Anaesthetized rat MAP 13 mmHg ↓ 2 Anaesthetized rat, MAP 5-9 mmHg ↓ wistar / spontaneously Effect larger in SHY hypertensive rats than Wistar rats Anaesthetized SHY rat MAP 60% ↓ 6 minutes, but sustained 20% reduced MAP 30% ↓ 6 Anaesthetized wistar rat MAP 30% ↓ 6 MAP 15% ↓ Forearm blood flow in Blood flow 3 fold ↑ human volunteers Conscious rat MAP 20 mmHg ↓ Conscious rat MAP 13 mmHg ↑ -

APLNR ligand Apelin-13 Apelin-13 Apelin-17 Pyr1-apelin-13 Apelin-13 Apelin-12 Apelin-13 Apelin-12 Pyr1-apelin-13 Apelin-36 Apelin-12 Apelin-12

Dose 1-2 ug/300g i.v. 10, 100, 1000 ug i.v. 3 nmol/kg i.v. 2.4 -10 nm/l i.v. 15 ug/kg i.v.

15 ug/kg i.v. 0.1 -30 nmol/min i.v. 20-40 umol/kg i.v. 20-40 umol/kg i.v.

Studies showing blood pressure reducing effect of apelin in the systemic circulation. MAP: mean systemic arterial blood pressure; BP: blood pressure; i.v.: intravenously; ↓: decrease; ↑: increase

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vasodilation induced by apelin is decreased in disease states.

In vivo experiments with dogs subjected to acute pulmonary embolism, showed that apelin injected as a single dose of 20 mg/kg slightly reduced mean pulmonary arterial pressure for approximately 2 minutes. However, the ratio of pulmonary to systemic vascular resistance was not changed.[109] Long-term studies of apelin in animal models of pulmonary hypertension have shown that it has an attenuating effect on the pulmonary pressure.[16,23,110] It is, though, difficult to say if this is due to direct vasodilatation, to counteraction or down-regulation of constrictive mediators,[23] to decreased loss of endothelial cells[16] and microvasculature,[48] or to inhibition of smooth muscle cell proliferation.[16] PAH, angiotensin-II and apelin Angiotensin-II has been proposed to contribute to the increased pressure and remodeling in PAH,[12] and an attenuating effect of apelin on angiotensin-II mediated vasoconstriction in isolated pulmonary arteries from normoxic rats has been observed[47] in accordance with findings in the systemic circulation. In hypoxic rats with pulmonary hypertension, the inhibitory effect was not present, but this does not exclude that long-term apelin treatment could modulate the effect of angiotensin-II. For example, in cardiomyocytes, apelin blocked angiotensinII-induced activation of the Rho-A kinase pathway,[100] which plays a pivotal role in pulmonary hypertension;[7] and, furthermore, apelin has been shown to antagonize angiotensin-II-induced gene transcription. [40] The significance of apelin and angiotensin-II sharing ACE 2 as a part of their degradation pathway remains enigmatic in relation to pulmonary hypertension. Exogenous activation of ACE 2 with a synthetic activator has been shown to prevent monocrotaline-induced pulmonary hypertension in rats, [111] because of an increased breakdown of angiotensin-II to the vasodilatory angiotensin(1-7). How this beneficial effect agrees with increased hydrolysis of apelin by ACE 2 is yet unexplored.

Cardiac effects of apelin-APLNR

Effect on cardiac contractility An important role for apelin in cardiac function was shown by results from Kuba et al. who observed that apelindeficient mice developed impaired cardiac contractility with age, unlike their wild type littermates. [59] Apelin has also been proven to be a potent positive inotropic agent. This was shown for the first time by Szokodi et al. using an isolated perfused heart preparation. [19] This study showed that apelin was able to increase contractility (by 60% of developed tension) independently of angiotensin-II receptors, endothelin-1 receptors or α- and β- adrenoceptors, and independently of eNOS. Pulmonary Circulation | July-September 2011 | Vol 1 | No 3

Furthermore, they showed that the positive inotropic effect was reduced by the inhibition of PLC and PKC as well as the Na2+-H+ exchanger and Na2+-Ca2+ exchanger (Fig. 1). In this study, very low concentrations of apelin-16 (0.01-10 nM) were used, and thus apelin was characterized as one of the most potent positive inotropic agents known. The inotropic effect has been confirmed by other studies,[34,52,54,112] and interestingly, in normal wild type mice, two weeks infusion of apelin resulted in increased cardiac output, but with no evidence of cardiac hypertrophy.[113]An interesting report from Japp et al. showed increased coronary blood flow, decreased blood pressure, increased contractility and increased cardiac output following acute apelin administration in patients with heart failure.[103] Some groups have found that the increased contractility was associated with an increment of intracellular Ca2+,[34] while other studies have shown no increases in Ca2+ transients, but a possible higher myofilament sensitivity towards Ca2+.[114] Cardioprotective effect of apelin Improved systolic cardiac function[115] and reduction in infarct size[115,116] have been observed in isolated rat hearts after ischemic insult and reperfusion. This was associated to decreased apoptosis, [33,115] and in the majority of studies,[33,115,117] the PI3/AKT pathway has been found to mediate this effect (Fig. 1). Another cardioprotective role of apelin is the ability to reduce angiotensin-II-induced cardiac fibrosis.[100]

Right ventricle, apelin and PAH The findings of improved contractility without concomitant hypertrophy, and cardioprotective abilities are intriguing in the setting of pulmonary hypertension, because the final consequence of the disease is right ventricular hypertrophy, fibrotic remodeling, and failure followed by death. It has been shown that apelin increases contractility in failing right ventricular myocardium from animals with pulmonary hypertension.[34] A study addressing the effect of apelin in monocrotaline induced pulmonary hypertension found that apelin was able to normalize contractility indices (dP/dtmax and dP/dtmin) of the right ventricle measured in vivo compared to control animals. [23] However, it is uncertain whether this finding is due to a decreased pulmonary vascular resistance and right ventricular pressure in the apelin-treated rats or is a direct effect on the right ventricular myocardium.

Therapeutic potential of apelin-APLNR in pulmonary hypertension Apelin—a new modulator of pulmonary vascular resistance? The acute vasodilatory effect of apelin on pulmonary arteries [47,108] and pulmonary pressure [109] is modest (10%-17%), and is attenuated in pulmonary hypertension. However, this does not exclude the possibility that 341

Andersen et al.: Apelin and PH

vasodilatation might occur over a longer period with chronic administration. Apelin could potentially modify secretion of endothelial derived vasoactive factors, as shown in cardiomyocytes. [23] At any rate, current vasodilatory drugs available in the treatment of PAH are not sufficient to control the disease,[10] and focus in the management of PAH is now directed towards the basic mechanisms leading to proliferation and pulmonary vascular remodeling. The pressure-reducing effect seen in the long-term experimental models of pulmonary hypertension with chronic administration of apelin[16,23,110] may possibly be attributed to the stabilizing effect of apelin on endothelial cells,[16] and the prevention of loss of microvasculature.[48] Apelin may also affect inflammatory processes, although results are so far sparse and somewhat controversial.[40,118-121]

A new positive inotropic agent? The benefits of the positive inotropic effect of apelin are uncertain. In heart failure caused by a compromised left ventricle, there are still no proven beneficial effects of any positive inotropic agents on survival despite many clinical studies with several drugs.[122] Actually, worse outcomes have been observed with β-adrenoceptor stimulating drugs and phosphodiesterase 3 inhibitors,[123] probably because of increments in energy expenditure and an increased propensity for cardiac arrhytmias, due to increased cAMP in the cardiac conductivity cells.[124] Even so, the search for useful positive inotropic agents is still ongoing, and the key to a successful drug may lie within the mechanism of which contractility is induced.[122] The known inotropic mechanisms for apelin differs from those of β-adrenoceptor agonists and phosphodiesterase 3 inhibitors, in that apelin has not been shown to increase levels of cAMP and levels of intracellular calcium may be unaffected. Speaking in favor of apelin as a promising positive inotropic agent in chronic heart failure are the attenuating effects of angiotensin-II-induced cardiac fibrosis[100] and the fact that it prevents cardiac apoptosis in relation to ischemia.[33,115,117]

Potential adverse effects and pharmacological challenges Apelin increases electrical conduction velocity in the heart[114] and apelin levels are changed in patients with cardiac arrhytmias. [125-127] Of 10 sheep injected with apelin, 4 developed some degree of atrio-ventricular block or other abnormalities of the electrocardiogram. [102] Therefore, the effects of apelin on cardiac conductivity and arrhytmias need closer human testing before clinical application. Furthermore, obvious challenges for apelin as a drug for treatment of pulmonary hypertension are the 342

short half life and the systemic vasodilatory effect, because PAH patients are already susceptible to symptomatic hypotension.[128] This may be overcome by pharmacological methods, e.g., chemical modification of the peptide and targeted delivery to the lungs by inhalation. Synthetic forms of apelin with the same biological activity as apelin-13 already exist,[129] and further development of such analogues may make the modulation of APLNR easier in the future, and allow different routes of administration.

CONCLUSIONS

In summary, apelin and APLNR are present in the pulmonary vasculature, are regulated by factors involved the pathogenesis of PAH, and are down-regulated in pulmonary arterial endothelial cells in patients with PAH. It may not, however, be suited as a specific biomarker for PAH. Apelin and APLNR play a role in regulation of endothelial and smooth muscle cell homeostasis, and have effects similar and opposite to VEGF. Apelin modulates eNOS expression, induces eNOS-dependent vasodilatation in the systemic and pulmonary circulation, counteracts angiotensin-II-induced vasoconstriction, and has positive inotropic and cardioprotective effects (Fig. 4).

Thus in the laboratory, the peptide has many intriguing characteristics, but so far most studies have investigated effects in the left heart and systemic circulation, and the peptide has been administered to human subjects in only a very few studies. It should, however, be emphasized that the existing studies in experimental models of pulmonary hypertension point toward a beneficial effect of the drug (Table 2). Further research is needed to clarify the pharmacodynamics, pharmacokinetics and safety of apelin in pulmonary hypertension. A: Endothelial cell

Apelin

Apoptosis

eNOS Abnormal proliferation NO & angiogenesis

Shear stress, inflammation, hypoxia C: Cardiomyocyte Apoptosis Fibrosis

Apelin

Contractility

B: Smooth muscle cell Endothelin-I

Apelin Apelin vasodilatation Proliferation↑ Angiotensin-II Remodelling

Figure 4: Factors in PAH pathogenesis opposed by apelin, A: In endothelial cells, B: In smooth muscle cells, and C: Right ventricle. Blue →: Stimulates activity/ expression. Red →: Decreases activity/expression. Pulmonary Circulation | July-September 2011 | Vol 1 | No 3

Andersen et al.: Apelin and PH

Table 2: Studies on apelin in isolated pulmonary arteries, PAH patients or PAH animal models Reference

Model

Main results

APLNR ligand

Dose

Goetze et al,[44]

PAH patients

–

Dai et al,[34]

Chronic hypoxic rats

Apelin-12

10-70 nM

Falcao-Pires et al,[23]

Monocrotaline rat

Pyr1-apelin-13

200 ug/kg/day i.p 17 days

Andersen et al,[47]

Chronic hypoxic rats Isolated arteries

PAH patients have lower apelin levels than healthy controls Apelin increases contractility in right ventricular tissue Apelin treatment induces: RVSP ↓ Cardiac fibrosis ↓ RV expression of ET-1 and ANG-II ↓ Contraction to ANG-II and ET-1↓ In pulmonary arteries from normoxic rats MPAP ↓ RVSP/LV+ S ratio ↓

Apelin-13

CRC 10-10-3x10-6 M

Pyr1- apelin-13

MPAP ↓ RVSP/LV+ S ratio ↓ in 10 nMol/kg group Extravasation ↓

Pyr1- apelin-13 Apelin-13

10 nMol/ kg/day, S.C., 28 days 5-10 nMol/kg/day, S.C. 28 days 2 mg/kg i.p.

MPAP ↓ (<5 mmHg, duration 3 minutes)

Apelin-13

10 and 20 ug/kg i.v.

Apelin-deficient mice: RVSP ↑ Muscularized pulmonary arteries ↑ Loss of microvessels ↑ Plasma apelin in iPAH patients < controls Vascular tone 10% ↓ in normoxic rats

–

Apelin

CRC 0.01-100 nM

Apelin reverses mild pulmonary hypertension Disruption of BMPR-2 decreases apelin expression

Apelin

200 ug/kg i.p.

Mao et al,

[130]

Chronic hypoxic rats

Fan et al,[110]

Chronic hypoxic rats

Petrescu et al,[86] Feng et al,[109]

LPS induced pulmonary edema Acute pulmonary embolism in anaesthetized dogs Apelin deficient mice exposed to chronic hypoxia

Chandra et al,[48]

Huang et al,[108] Alastalo et al,[16]

Chronic hypoxic rats. Isolated pulmonary arteries TIE2CrePPARγfl/fl mice

14 days

Plasma apelin in iPAH patients < controls Studies on apelins effect in isolated pulmonary arteries, PAH patients and PAH animal models. RVSP: right ventricular systolic pressure; RV: right ventricle; CRC: concentration-response curves; ET-1: endothelin-1; ANG-II: angiotensin-II; MPAP: mean pulmonary artery pressure; RV/LV+S: right ventricular weight/left ventricular weight + septum; LPS: lipopolysaccharide; i.p.: intraperitoneally; S.C.: subcutaneously; ↓: decrease; ↑: increase

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prognosis. J Thorac Oncol 2010;5:1120-9. Carmeliet P, Ferreira V, Breier G, Pollefeyt S, Kieckens L, Gertsenstein M, et al. Abnormal blood vessel development and lethality in embryos lacking a single VEGF allele. Nature 1996;380:435-9. Petrescu BC, Gurzu B, Iancu RI, Indrei A, Dumitriu I, Chelaru L, et al. Apelin effects on lipopolysaccharide-increased pulmonary permeability in rats. Rev Med Chir Soc Med Nat Iasi 2010;114:163-9. 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. Tuder RM, Chacon M, Alger L, Wang J, Taraseviciene-Stewart L, Kasahara Y, et al. Expression of angiogenesis-related molecules in plexiform lesions in severe pulmonary hypertension: Evidence for a process of disordered angiogenesis. J Pathol 2001;195:367-74. Taraseviciene-Stewart L, Kasahara Y, Alger L, Hirth P, Mc MG, Waltenberger J, et al. Inhibition of the VEGF receptor 2 combined with chronic hypoxia causes cell death-dependent pulmonary endothelial cell proliferation and severe pulmonary hypertension. FASEB J 2001;15:427-38. Sakao S, Taraseviciene-Stewart L, Lee JD, Wood K, Cool CD, Voelkel NF. Initial apoptosis is followed by increased proliferation of apoptosisresistant endothelial cells. FASEB J 2005;19:1178-80. Sakao S, Taraseviciene-Stewart L, Wood K, Cool CD, Voelkel NF. Apoptosis of pulmonary microvascular endothelial cells stimulates vascular smooth muscle cell growth. Am J Physiol Lung Cell Mol Physiol 2006;291:L362-8. Sakao S, Taraseviciene-Stewart L, Cool CD, Tada Y, Kasahara Y, Kurosu K, et al. VEGF-R blockade causes endothelial cell apoptosis, expansion of surviving CD34+ precursor cells and transdifferentiation to smooth muscle-like and neuronal-like cells. FASEB J 2007;21:3640-52. Tuder RM, Cool CD, Yeager M, Taraseviciene-Stewart L, Bull TM, Voelkel NF. The pathobiology of pulmonary hypertension. Endothelium. Clin Chest Med 2001;22:405-18. Farkas L, Gauldie J, Voelkel NF, Kolb M. Pulmonary hypertension and idiopathic pulmonary fibrosis: A tale of angiogenesis, apoptosis, and growth factors. Am J Respir Cell Mol Biol 2011;45:1-15. Manetti M, Guiducci S, Ibba-Manneschi L, Matucci-Cerinic M. Mechanisms in the loss of capillaries in systemic sclerosis: angiogenesis versus vasculogenesis. J Cell Mol Med 2010;14:1241-54. Salcedo A, Garijo J, Monge L, Fernandez N, Luis Garcia-Villalon A, Sanchez Turrion V, et al. Apelin effects in human splanchnic arteries. Role of nitric oxide and prostanoids. Regul Pep. 2007;144:50-5. Gurzu B, Petrescu BC, Costuleanu M, Petrescu G. Interactions between apelin and angiotensin II on rat portal vein. J Renin Angiotensin Aldosterone Syst 2006;7:212-6. Zhong JC, Huang Y, Yung LM, Lau CW, Leung FP, Wong WT, et al. The novel peptide apelin regulates intrarenal artery tone in diabetic mice. Regul Pep. 2007;144:109-14. Zhong JC, Yu XY, Huang Y, Yung LM, Lau CW, Lin SG. Apelin modulates aortic vascular tone via endothelial nitric oxide synthase phosphorylation pathway in diabetic mice. Cardiovasc Res 2007;74:388-95. Siddiquee K, Hampton J, Khan S, Zadory D, Gleaves L, Vaughan DE, et al. Apelin protects against angiotensin II-induced cardiovascular fibrosis and decreases plasminogen activator inhibitor type-1 production. J Hypertens 2011;29:724-31. Hashimoto T, Kihara M, Ishida J, Imai N, Yoshida S, Toya Y, et al. Apelin stimulates myosin light chain phosphorylation in vascular smooth muscle cells. Arterioscler Thromb Vasc Biol 2006;26:1267-72. Charles CJ, Rademaker MT, Richards AM. Apelin-13 induces a biphasic haemodynamic response and hormonal activation in normal conscious sheep. J Endocrinol 2006;189:701-10. Japp AG, Cruden NL, Barnes G, van GN, Mathews J, Adamson J, et al. Acute cardiovascular effects of apelin in humans: Potential role in patients with chronic heart failure. Circulation 2010;121:1818-27. Cheng X, Cheng XS, Pang CC. Venous dilator effect of apelin, an endogenous peptide ligand for the orphan APJ receptor, in conscious rats. Eur J Pharmacol 2003;470:171-5. Kagiyama S, Fukuhara M, Matsumura K, Lin Y, Fujii K, Iida M. Central and peripheral cardiovascular actions of apelin in conscious rats. Regul Pep. 2005;125:55-9. Yao F, Modgil A, Zhang Q, Pingili A, Singh N, O’Rourke ST, et al. Pressor effect of apelin-13 in the rostral ventrolateral medulla: Role of NAD(P)H oxidase-derived superoxide. J Pharmacol Exp Ther 2011;336:372-80. Zhang Q, Yao F, Raizada MK, O’Rourke ST, Sun C. Apelin gene transfer into the rostral ventrolateral medulla induces chronic blood pressure

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elevation in normotensive rats. Circ Res 2009;104:1421-8. 108. Huang P, Fan XF, Pan LX, Gao YQ, Mao SZ, Hu LG, et al. Effect of apelin on vasodilatation of isolated pulmonary arteries in rats is concerned with the nitric oxide pathway. Zhongguo Ying Yong Sheng Li Xue Za Zhi 2011;27:1-5. 109. Feng JH, Li WM, Wu XP, Tan XY, Gao YH, Han CL, et al. Hemodynamic effect of apelin in a canine model of acute pulmonary thromboembolism. Peptides 2010;31:1772-8. 110. Fan XF, Wang Q, Mao SZ, Hu LG, Hong L, Tian LX, et al. Protective and therapeutic effect of apelin on chronic hypoxic pulmonary hypertension in rats. Zhongguo Ying Yong Sheng Li Xue Za Zhi 2010;26:9-12. 111. Ferreira AJ, Shenoy V, Yamazato Y, Sriramula S, Francis J, Yuan L, et al. Evidence for angiotensin-converting enzyme 2 as a therapeutic target for the prevention of pulmonary hypertension. Am J Respir Crit Care Med 2009;179:1048-54. 112. Berry MF, Pirolli TJ, Jayasankar V, Burdick J, Morine KJ, Gardner TJ, et al. Apelin has in vivo inotropic effects on normal and failing hearts. Circulation 2004;110:II187-93. 113. Ashley EA, Powers J, Chen M, Kundu R, Finsterbach T, Caffarelli A, et al. The endogenous peptide apelin potently improves cardiac contractility and reduces cardiac loading in vivo. Cardiovasc Res 2005;65:73-82. 114. Farkasfalvi K, Stagg MA, Coppen SR, Siedlecka U, Lee J, Soppa GK, et al. Direct effects of apelin on cardiomyocyte contractility and electrophysiology. Biochem Biophys Res Commun 2007;357:889-95. 115. Zeng XJ, Zhang LK, Wang HX, Lu LQ, Ma LQ, Tang CS. Apelin protects heart against ischemia/reperfusion injury in rat. Peptides 2009;30:1144-52. 116. Kleinz MJ, Baxter GF. Apelin reduces myocardial reperfusion injury independently of PI3K/Akt and P70S6 kinase. Regul Pep. 2008;146:271-7. 117. Simpkin JC, Yellon DM, Davidson SM, Lim SY, Wynne AM, Smith CC. Apelin-13 and apelin-36 exhibit direct cardioprotective activity against ischemia-reperfusion injury. Basic Res Cardiol 2007;102:518-28. 118. El-Shehaby AM, El-Khatib MM, Battah AA, Roshdy AR. Apelin: A potential link between inflammation and cardiovascular disease in end stage renal disease patients. Scand J Clin Lab Invest 2010;70:421-7. 119. Hashimoto T, Kihara M, Imai N, Yoshida S, Shimoyamada H, Yasuzaki H,

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et al. Requirement of apelin-apelin receptor system for oxidative stresslinked atherosclerosis. Am J Pathol 2007;171:1705-12. Leeper NJ, Tedesco MM, Kojima Y, Schultz GM, Kundu RK, Ashley EA, et al. Apelin prevents aortic aneurysm formation by inhibiting macrophage inflammation. Am J Physiol Heart Circ Physiol 2009;296:H1329-35. Pitkin SL, Maguire JJ, Kuc RE, Davenport AP. Modulation of the apelin/ APJ system in heart failure and atherosclerosis in man. Br J Pharmacol 2010;160:1785-95. Metra M, Bettari L, Carubelli V, Bugatti S, Dei CA, Del MF, et al. Use of inotropic agents in patients with advanced heart failure: lessons from recent trials and hopes for new agents. Drugs 2011;71:515-25. Packer M, Carver JR, Rodeheffer RJ, Ivanhoe RJ, DiBianco R, Zeldis SM, et al. Effect of oral milrinone on mortality in severe chronic heart failure. The PROMISE Study Research Group. N Engl J Med 1991;325:1468-75. Burt JM, Spray DC. Inotropic agents modulate gap junctional conductance between cardiac myocytes. Am J Physiol 1988;254:H1206-10. Ellinor PT, Low AF, MacRae CA. Reduced apelin levels in lone atrial fibrillation. Eur Heart J 2006;27:222-6. Falcone C, Buzzi MP, D’Angelo A, Schirinzi S, Falcone R, Rordorf R, et al. Apelin plasma levels predict arrhythmia recurrence in patients with persistent atrial fibrillation. Int J Immunopathol Pharmacol 2010;23:917-25. Kallergis EM, Manios EG, Kanoupakis EM, Mavrakis HE, Goudis CA, Maliaraki NE, et al. Effect of sinus rhythm restoration after electrical cardioversion on apelin and brain natriuretic Peptide prohormone levels in patients with persistent atrial fibrillation. Am J Cardiol 2010;105:90-4. Schannwell CM, Steiner S, Strauer BE. Diagnostics in pulmonary hypertension. J Physiol Pharmacol 2007;58 Supp. 5:591-602. Hamada J, Kimura J, Ishida J, Kohda T, Morishita S, Ichihara S, et al. Evaluation of novel cyclic analogues of apelin. Int J Mol Med 2008;22:547-52. Mao SZ, Hong L, Hu LG, Fan XF, Zhang L, Guo YM, et al. Effect of apelin on hypoxic pulmonary hypertension in rats: Role of the NO pathway. Sheng Li Xue Bao 2009;61:480-4.

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

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Review Ar ti cl e

Epigenetic mechanisms of pulmonary hypertension Gene H. Kim, John J. Ryan, Glenn Marsboom, and Stephen L. Archer Department of Medicine, University of Chicago, Chicago, Illinois, USA

ABSTRACT Epigenetics refers to changes in phenotype and gene expression that occur without alterations in DNA sequence. Epigenetic modifications of the genome can be acquired de novo and are potentially heritable. This review focuses on the emerging recognition of a role for epigenetics in the development of pulmonary arterial hypertension (PAH). Lessons learned from the epigenetics in cancer and neurodevelopmental diseases, such as Prader-Willi syndrome, can be applied to PAH. These syndromes suggest that there is substantial genetic and epigenetic cross-talk such that a single phenotype can result from a genetic cause, an epigenetic cause, or a combined abnormality. There are three major mechanisms of epigenetic regulation, including methylation of CpG islands, mediated by DNA methyltransferases, modification of histone proteins, and microRNAs. There is substantial interaction between these epigenetic mechanisms. Recently, it was discovered that there may be an epigenetic component to PAH. In PAH there is downregulation of superoxide dismutase 2 (SOD2) and normoxic activation of hypoxia inducible factor (HIF-1α). This decrease in SOD2 results from methylation of CpG islands in SOD2 by lung DNA methyltransferases. The partial silencing of SOD2 alters redox signaling, activates HIF-1α) and leads to excessive cell proliferation. The same hyperproliferative epigenetic abnormality occurs in cancer. These epigenetic abnormalities can be therapeutically reversed. Epigenetic mechanisms may mediate gene-environment interactions in PAH and explain the great variability in susceptibility to stimuli such as anorexigens, virus, and shunts. Epigenetics may be relevant to the female predisposition to PAH and the incomplete penetrance of BMPR2 mutations in familial PAH. Key Words: CpG islands, DNA methyl transferases, histone acetylation, small inhibitor RNA, superoxide dismutase 2

INTRODUCTION This review focuses on the emerging recognition of a role for epigenetics in the development of pulmonary arterial hypertension (PAH). Epigenetics refers to changes in phenotype mediated by altered gene expression, which are not the result of alterations in DNA sequence. Epigenetic mechanisms can be acquired and/or heritable and constitute a means by which gene-environment interactions occur. To date there are few examples of epigenetics contributing to PAH;[1] however, there are many unexplained observations in PAH that may have an epigenetic component. For example, although most cases of familial PAH are associated with mutations of the bone morphogenetic protein receptor (BMPR2),[2,3] it is still unclear why only 20% of BMPR2 carriers develop PAH during their lifetime. Typically this reduced penetrance is attributed to gender, genetic modifiers and/or the environment. Address correspondence to:

Prof. Stephen L. Archer Section of Cardiology, Department of Medicine, University of Chicago, 5841 South Maryland Avenue, (MC6080), Chicago, Illinois, 60637. Phone: 773/702-1919, Fax: 773/702-1385 Email: sarcher@medicine.bsd.uchicago.edu Pulmonary Circulation | July-September 2011 | Vol 1 | No 3

While genetic studies search for modifier genes that may be important determinants of penetrance, [4] experiments in fawn-hooded rats, a strain with heritable, spontaneously developing PAH, suggest an important role for environmental factors in determining the time to onset and severity of PAH. Exposing fawn-hooded pups to small reductions in inspired oxygen that does not affect other rat strains leads to a more rapid onset of PAH.[5] Environmental factors also determine the prevalence of pulmonary hypertension in broiler fowl (a strain of chicken intercrossed for meat production).[6] Cold temperatures and differences in diet and increased rates of weight gain have been described to worsen the onset of PAH in broiler fowl.[7] Such environmental factors could predispose to PAH by as yet undiscovered epigenetic mechanisms. Access this article online

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Website: www.pulmonarycirculation.org DOI: 10.4103/2045-8932.87300 How to cite this article: Kim GH, Ryan JJ, Marsboom G, Archer SL. Epigenetic mechanisms of pulmonary hypertension. Pulm Circ 2011;1:347-56.

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In this review, we first give a general overview of the exponential increase in scientific articles in epigenetic research. We then review the epigenetic mechanisms of gene regulation, with examples from the fields of oncology and neurodevelopment. Finally, we highlight recent publications that study epigenetics in pulmonary hypertension.

Trends in biomedical epigenetic research publications

There has been a recent, near exponential increase in the number of research articles that discuss epigenetics (Fig. 1a). Since 1997, the first year with more than 100 published research articles with either “epigenetic” or “epigenetics” in the title or abstract, publication numbers have increased annually, with more than 2,000 in 2010. Based on the publications to date in 2011 (2,099 as of 18 August 2011), this trend continues. When reviews and editorials are included in the analysis, it becomes obvious that the scientific community is interested in epigenetics. Arguably there is more interest than information, with review articles (such as this one) accounting for approximately one-third of all publications with the key words “epigenetic” or “epigenetics” in their title or abstract (Fig. 1b). Epigenetics research is currently focused on cancer (70% of published research articles in 2010) with some interest in stem cells and neuronal disease and much less attention to cardiopulmonary diseases (including asthma) (Fig. 1c).

Mechanisms of epigenetic regulation

There is a large body of evidence that epigenetic modifications are involved in the pathological mechanisms of many diseases including cancer,[8,9] asthma,[10] and several human syndromic disorders, such as Prader-Willi, Angelman, Silver-Russell, and Beckwith-Wiedermann syndromes.[11-13] # Research Articles

2500

Relevant to PAH, many of these conditions have both genetic and epigenetic mechanisms which can independently produce the same phenotype. PraderWilli syndrome (hypotonia, insatiable appetite, obesity, developmental impairment) and Angelmann’s syndrome are neurobehavioral syndromes resulting from paternal or maternal imprinted genes or genetic deletions within the chromosomal 15q11-q13 region.[14] While deletions of chromosome 15q11-q13 can give rise to Prader-Willi syndrome, so too can uniparental disomy and imprinting mutations.[14] The epigenetic or “imprinting” disorders create a syndrome that is phenotypically indistinguishable from genetic deletion. However, in patients where uniparental disomy (two copies of a chromosome or segment thereof from one parent) causes Prader-Willi syndrome, there is no DNA sequence abnormality.[11] Epigenetic and genetic mechanisms targeting the same gene can yield a similar phenotype. As an example, there is an imprinting center (epigenetic target) near the promoter for the small nuclear ribonucleoprotein N (SNRPN) gene. This is a gene which, if deleted, causes Prader-Willi syndrome.[11] In a further variation, Beaudet et al. have pointed out that there are Prader-Willi patients with imprinting defects in whom a small deletion in the imprinting center results in a larger epigenetic defect. [11] Thus Prader-Willi syndrome provides an important precedent when one considers the interplay between genetic and epigenetic causes of PAH. It demonstrates that an abnormality which can be genetically encoded can also be epigenetically mediated and that either or both mechanisms may be at play in a given patient.

Epigenetic mechanisms of altered gene expression

Epigenetic modifications provide a mechanism that allows the stable propagation of gene expression states from one generation of cells to the next.[15] Mechanisms of epigenetic

2000 1500 1000 500 0 1960

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2000 2010

Editorials Reviews Research articles (b)

Cancer Stem Cells Neuronal disease Cardiopulmonary disease (c)

Figure 1: Trends in biomedical epigenetic research publications. PubMed citations containing the words epigenetic or epigenetics in the title or abstract were counted (www.ncbi.nlm.nih.gov/sites/entrez?db=PubMed). (a) Research articles, excluding review articles and editorials, were counted for each year up to 2010. (b) All publications covering epigenetic(s) are categorized to editorials, reviews and research articles. (c) Distribution of research articles published in 2010 for cancer, stem cells and their differentiation, neuronal diseases and cardiopulmonary diseases including asthma. 348

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regulation include DNA methylation, histone modification, and RNA interference represented in (Fig. 2).[16]

DNA methylation

In the context of epigenetics, DNA methylation refers to the covalent attachment of a methyl group to the C5 position of cytosine residues in CpG dinucleotide sequences that are called CpG islands. DNA islands are often in the promoter or enhancer regions of genes and methylation of these sites can alter gene transcription. DNA methylation is involved in normal cellular control of gene expression and is dynamically regulated.[17] However, changes in DNA methylation are also relevant to disease. Hypermethylation of CpG islands can lead to silencing of tumor-suppressor genes, promoting the development of cancer;[9,18] conversely, hypomethylation can lead to gene overexpression, which may also promote cancer.[19-23] CpG methylation is an important mechanism to ensure the repression of transcription of repeat elements and it plays a crucial role in imprinting and X-chromosome inactivation.[24] Transcriptional gene silencing by CpG methylation also restricts the expression of some tissuespecific genes during development and differentiation by repressing them in non-expressing cells, thus providing another layer of important temporal-spatial control of expression. Throughout the genome, areas of increased CpG dinucleotides, or CpG islands, in the promoter regions of many genes have been intensively studied for changes in methylation status.[25] Generally, it has been found that hypermethylation of CpG islands is associated with epigenetic silencing.[26] CpG methylation can suppress transcription by several mechanisms. First, the presence of the methyl group at a specific CpG dinucleotide site may directly block DNA recognition and binding by some transcription factors.[27] In other instances, some proteins may preferentially bind to methylated DNA, thereby blocking transcription factor access to these regulatory elements.[28] Taken together, DNA methylation represents an important mechanism in the regulation of gene expression. Critical to understanding this means of regulating gene expression is the mechanism by which DNA is methylated. A family of DNA methyltransferase enzymes (DNMTs) is involved in de novo DNA methylation and the maintenance of methylation. Thus, epigenetic control of gene expression by cytosine methylation is facilitated by the activity of DNMTs. The regulation of the expression of the DNMTs (whether mediated transcriptionally or by post-translational means) represents an additional mechanism of epigenetic control.[29] Cell- and tissuespecific activation of DNMTs may lead to localized changes in gene expression. In brief, DNMTs recognize CpGs within double-stranded DNA as substrates. Maintenance DNA methylation is generally performed by DNMT1 Pulmonary Circulation | July-September 2011 | Vol 1 | No 3

Chromosome

Histone modification

Nucleosome

miRNA

mRNA

RNA interference

mRNA Protein Me

DNA methylation Me

FigureÂ 2: Schematic of the mechanisms of epigenetic regulation. DNA methylation, histone modifications, and RNA-mediated gene silencing constitute three distinct mechanisms of epigenetic regulation. DNA methylation is a covalent modification of the cytosine (C) that is located 5â&#x20AC;&#x2122; to a guanine (G) in a CpG dinucleotide. Histone (chromatin) modifications refer to covalent post-translational modifications of N-terminal tails of four core histones (H3, H4, H2A, and H2B). The most recent mechanism of epigenetic inheritance involves RNAs. Reproduced with permission from Z. Herzeg.[16]

and occurs in step with DNA replication. Thus, one may consider one of the major roles of DNMT1 as the passing on epigenetic control of gene expression to daughter cells.[30] Interestingly, DNMT3a and DNMT3b do not exhibit a substrate preference between hemimethylated and unmethylated DNA and therefore appear to be critical for de novo methylation.[31] During embryogenesis, de novo methylation is performed by DNMT3A and DNMT3B. Although some studies suggest an ongoing role for DNMT3A and DNMT3B in maintaining methylation status in some cell types, the ubiquitously expressed DNMT1 is predominantly responsible for maintaining cellular levels of CpG methylation.[31]

Histone modification

Genetic information is packaged into higher order structures by nucleosomes. Nucleosomes package approximately 146 base pairs of DNA wrapped around an octamer of core histone proteins. Each core nucleosome consists of two of each histone protein: H2A, H2B, H3, and H4. Aside from the organizational function of DNA in the nucleus, nucleosomes play a critical role in regulating gene activity by controlling their accessibility and therefore their transcriptional activity. Histones can be posttranslationally modified to restructure chromatin in many ways, including acetylation, methylation, phosphorylation, ubiquitination, poly-ADP-ribosylation, biotinylation, and sumoylation. 349

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Acetylation is one of the most frequent epigenetic modifications. Histone acetylation occurs at the many lysine residues in both histones H3 and H4 (Table 1). Increased levels of histone acetylation are highly correlated with increased transcriptional activity, whereas decreased levels of acetylation repress gene expression. This process is catalyzed by histone acetyltransferases (HATs) that utilize acetyl-CoA as a cofactor. Recruitment of these HATs to promoters is generally associated with activated transcription. Unlike histone acetylation, histone methylation can activate or inhibit transcription, depending on where the modification occurs. The methylation of histones is carried out by a large family of histone methyltransferases (HMTs). Just like DNMTs, HMTs utilize S-adenosylmethionine (SAM) as a cofactor to methylate their target amino acids and produce S-adenosylhomocysteine (SAH) as a byproduct (Table 1).

kingdoms. To date, miRNAs have been shown to inhibit translation or decrease mRNA stability by binding to specific sites usually in the 3’ untranslated region (3’UTR) of target messages, thus providing another layer of control of gene expression. MicroRNAs are initially transcribed as primary microRNAs by endogenous RNA polymerases and undergo a series of processing steps and incorporation into the RNA-induced silencing complex (RISC). [33] MicroRNAs interact with mRNA through sequence specific interactions with key areas of sequence homology at the 5’ end of the microRNA, particularly at bases 2-8 which is termed the “seed” region. This control is sequence specific, and changes in just a single base within the miRNA target site can abolish this regulation. It is this region which forms the basis for virtually all computational algorithms predicting mRNA targets. However, beyond Watson–Crick base pairing, the efficiency of repression depends on the number and configuration of mismatches between the miRNA and the target mRNA, the secondary structure of the surrounding region, and the number of target sequences on the mRNA.[34] Up to one-third of human genes are predicted to be regulated by one or more microRNAs. Despite this vast regulatory network and hundreds of microRNAs, few targets have been validated.[35]

Acetylation and methylation of histone tails are not permanent modifications. Histone acetylation and deacetylation are dynamic processes determined by the balance between histone acetyltransferases and histone deacetylases (HDACs). Similarly, histone demethylases remove methylation from histones. Regulation of chromatin structure directly affects the transcriptional process. The extensive combinatorial post-translational modifications to histones represent yet another layer of complexity to understanding the “histone code.” More importantly, this dynamic nature of acetylation and methylation provides flexibility to the epigenetic control of gene expression.[32]

RNA interference

MicroRNAs (miRNAs) are small RNA molecules, approximately 22 nucleotides long that can negatively control their target gene expression posttranscriptionally. First described in Caenorhabditis elegans in 1993, miRNAs have now been identified throughout the plant and animal

Table 1: Summary of epigenetic histone modifications Tri-methylation of histone H3 lysine-4 Methylation of lysine-36 and lysine-79 of histone H3 Tri-methylation of histone H3 lysine-9 Methylation of histone H3 lysine 27 H3K9 acetylation Trimethylation at H3K4, H3K36, or H3K79 Methylated H3K9 provides a binding site for the chromodomain-containing heterochromatin protein 1 (HP1) H3K4 demethylation through histone demethylase LSD1 350

As the functions of individual microRNAs are being studied, it has become clear that microRNAs can be regulated by epigenetic mechanisms including DNA methylation and histone modification such as let-7a, miR- 9, miR-34a, miR-124, miR-137, miR-148 and miR-203. [36- 40] Conversely, another subset of miRNAs controls the expression of important epigenetic regulators, including DNA methyltransferases and histone deacetylases.[41] MicroRNA-29b can reduce the expression of DNMT enzymes and thereby affect global methylation status.[42] This complex network between miRNAs and epigenetic pathways appear to form an epigenetics–miRNA regulatory circuit intertwined with the transcriptional

Associated with transcriptional activation and creates a binding site for proteins containing chromodomain that recruits HATs[74] Associated with active chromatin and transcriptional activation[32] Transcriptional silencing by recruiting heterochromatin protein (HP1) and triggers the formation of heterochromatin[74] Associated with transcriptional repression and maintaining silent chromatin through recruiting the Polycomb complex (PRC1)[75] Associated with chromatin decondensation and the formation of chromatin loops. These loops separate out actively transcribed genes from more compact chromosome territories[76] Associated with an open chromatin configuration which is characteristic of euchromatin[32] Induces transcriptional repression and heterochromatinization[32] Results in to transcriptional inactivation[32] Pulmonary Circulation | July-September 2011 | Vol 1 | No 3

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and post-transcriptional pathways known to regulate epigenetic modulators and to organize the whole gene expression profile.[41] Though not yet demonstrated, one can conceive of situations in which potential microRNA target binding site accessibility is influenced by the methylation of the target region.

Examples of epigenetic regulation of PAH

Superoxide dismutase 2 Superoxide dismutase 2 (SOD2), a candidate tumorsuppressor gene,[43] is silenced in several malignancies.[44,45] In multiple myeloma and pancreatic carcinoma, the epigenetic silencing of SOD2 is caused by hypermethylation of CpG islands within the promoter for SOD2. [45] SOD2 is subject both to DNA methylation and histone acetylation.[46,47] Demethylation of SOD2 in cancer restores SOD2, increases H2O2 production and decreases cell proliferation and tumor growth. Production of H2O2 is a critical link between SOD2 expression and regulation of proliferation. SOD2 is found in the mitochondria where it regulates production of H2O2 (produced physiologically from mitochondrial superoxide during respiration). H2O2 is less toxic than superoxide and its greater diffusion radius allows it to serve as a signaling molecule. H2O2 modulates the activity of transcription factors such as HIF-1α (which it inhibits) [48] and sulfhydryl rich proteins, including the voltage-gated potassium channel Kv1.5 (which it activates). SOD2 deficiency has been identified in the pulmonary arteries and plexiform lesions of PAH[49,50] and activation of HIF-1α is also evident.[1,49-52] However, the mechanism by which these two abnormalities was linked had been unclear. We found that fawn-hooded rats, the development of PAH is preceded by downregulation of SOD2. Interestingly, in the fawn hooded rat, PAH is heritable and yet sequencing has shown that the SOD2 gene has no mutations.[1,50] However, several regions of the SOD2 promoter and intronic regions contained CpG islands, which could be targets for epigenetic regulation via CpG methylation. We discovered that the selective hypermethylation of CpG islands in the SOD2 gene reduces its expression ~50% compared to PASMCs from genetically matched consomic rats[50] and this contributes to the proliferative, antiapoptotic phenotype in pulmonary artery smooth muscle cells (PASMC) of the FHR.[1]

To demonstrate this, the promoter of SOD2 and the first 2kb after the transcriptional start site were surveyed using genomic sodium bisulfite sequencing (Fig. 3). As shown in other tissues, the region immediately 5’ to the transcriptional start site was completely unmethylated. However, differentially methylated CpG dinucleotides within intron 2 (an enhancer region) were found. This methylation pattern was tissue specific as the SOD2 methylation in intron 2 was not seen in aortic smooth muscle cells of FHRs. Moreover, this same region is methylated in various Pulmonary Circulation | July-September 2011 | Vol 1 | No 3

tumors. Interestingly, treatment with the DNMT inhibitor, 5-azacytidine (5-AZA) resulted in a dose dependent increase in SOD2 expression.[1] Both DNMT1 and DNMT3b are significantly upregulated in lung tissue and pulmonary arterial smooth muscle cells compared to control (Fig. 4).[1]

Reversing the methylation status of the SOD2 gene using 5-AZA not only restored SOD2 expression, it also decreased proliferation and slightly increased apoptosis in the abnormally hyperproliferative FHR PASMCs. Likewise, increasing SOD2 in the PASMC, either by administering adenovirus carrying the SOD2 transgene, or by giving a SOD analog (MnTBAP) inactivated HIF-1α (i.e. achieved similar effects as demethylating the gene). The similarities in therapeutic effects of SOD supplementation and 5-AZA in cellular experiments suggests an important role for decreased SOD2 in the mechanism of PAH. In vivo studies of MnTBAP, given to FHR with established PAH for 4-weeks, showed a decreased mean pulmonary artery pressure and increased exercise capacity, while reducing the medial thickness of precapillary resistance arteries (Fig. 5). This study demonstrated that SOD2 methylation is important in the development of PAH and contributes to HIF-1α activation and the development of an apoptoticresistant state with marked proliferation. [1] From a therapeutic standpoint it is also exciting because of the potential to increase SOD2 either through DNMT inhibition (5-AZA) or administering MnTBAP. It is noteworthy that similar abnormalities in SOD2 expression are epigenetically mediated and are similarly reversible with a resultant decrease in cell proliferation in several cancers.[44,47] Maternal restrictive diet The Barker hypothesis suggests that fetal stressors can lead to cardiovascular disease in adults.[53] While the effect of poor maternal diet and low birth weight on the lifetime development of coronary artery disease is recognized,[54] the effects of maternal malnutrition on the right ventricle and pulmonary hypertension has only recently been considered. Rexhaj et al. assessed the pulmonary vascular responsiveness in offspring of pregnant mice fed with a restrictive diet in both normoxic and hypoxic conditions. [55] To detect reversible epigenetic changes, the authors also administered the histone deacetylase inhibitor trichostatin A to male offspring of pregnant mice that had been fed a restrictive diet.

In the offspring of restrictive diet pregnancy, there was exaggerated hypoxic pulmonary hypertension and right ventricular hypertrophy. An epigenetic change was demonstrated by the increased update of radioactive methyl groups in the offspring of the pregnant diet-restricted mice. Of note, the pulmonary 351

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Figure 3: Methylation of SOD2 in FHR PASMC is reversible by 5-AZA. (a) Schematic of the CG dinucleotide percentage and CpG islands within the SOD2 promoter and first 2 kb after the transcriptional start site. Seven amplicons were surveyed within the SOD2 gene. Their approximate locations are represented by solid horizontal lines. The positions of individual CpG dinucleotides are shown as vertical tick marks below the amplicon map. No methylated CpG pairs were identified in amplicons 3 to 6. *Differentially methylated CpG dinucleotides in FHRs within amplicon 7 vs BN1 tissue. (b) Corresponding methylation percentage of the differentially methylated CpG in intron 2. Results are expressed as a frequency of cytosine methylation in PAs from consomic control BN1 rats (n=2), FHRs (n=3), and FHRs treated with 5-AZA (FHR-Tx; n=3). (c) Representative sequencing traces of genomic DNA from cultured PASMCs. Only methylated cytidines are protected against bisulfite-mediated deamination of cytidine into uridine (which is recognized as thymidine when the polymerase chain reaction product is amplified). As indicated by the arrow, the cytidine in FHR PASMCs was methylated (and therefore remains a cytidine; top left); this is reversed by 5-AZA. The site is not methylated in FHR aortic SMCs or in SDR PASMCs. The bar graph shows the mean data indicating the reversibility and tissue specificity of this SOD2 methylation in intron 2 in cultured PASMCs. (d) FHR PASMCs have lower SOD2 mRNA levels vs consomic PASMCs. 5-AZA causes a dose-dependent increase in SOD2 expression. Reproduced with permission from Archer et al.[1]

Lung

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0 DNA MT1

DNA MT3A (a)

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Figure 4: DNA methyltransferase expression is increased in FHR lung and PASMCs. (a) DNA MT1 and 3B mRNA are increased in FHR vs SDR lungs (n=12 each). *P<0.05, **P<0.01. (b) In low-passage (3 to 4) PASMCs (n=8 in each group), FHRs had higher DNA MT3B expression and a trend toward increased DNA MT1. Reproduced with permission from Archer et al.[1] Black bars = FHR; White bars = SDR 352

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20 10 0

.20 .16 .12 .08 .04

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Figure 5: Administration of the SOD analog, MnTBAP, regresses PAH in FHRs. (a) MnTBAP reduces mean PA pressure measured by Doppler (lengthens PA acceleration time [PAAT]) and decreases right ventricular (RV) thickness in FHRs treated for 4 weeks (n=5 per group). *P<0.05. (b) MnTBAP therapy reduces mean pulmonary artery pressure (PAP) and total pulmonary resistance (TPR). (c) FHRs treated with MnTBAP exercise longer on a graded treadmill (n=15 per group). (d) Lung sections were stained for von Willebrand factor (vWF; red), a-smooth muscle cell (SMC) actin (green), and DAPI (blue). Note the fully muscularized (white arrows), partially muscularized (yellow arrows), and nonmuscularized blood vessels (red arrows). Bottom, A representative fully muscularized PA in a vehicle-treated FHR (left) vs MnTBAP (right). The percent medial thickness of precapillary resistance PAs was reduced and the number of nonmuscularized resistance PAs was increased by MnTBAP. **P<0.01 vs control. Reproduced with permission from Archer et al.[1]

DNA methylation induced by dietary restriction, and the epigenetic changes, were reversed by the administration of butyrate and trichostatin A, inhibitors of histone deacteacylation (HDAC inhibitors). In fact, butyrate administration to male offspring prevented the transmission of pulmonary vascular dysfunction to their own offspring, again suggesting reversal of the epigenetic effects. Tempol, a membrane-permeable radical scavenger, administered to the mothers in conjunction with their restrictive diet during pregnancy prevented endothelial dysfunction, RVH and hypoxic pulmonary hypertension implying a key role of oxidative stress in mediating the epigenetic effects of restrictive diets. Primary pulmonary hypertension in the newborn Primary pulmonary hypertension in the newborn (PPHN) is defined as a failure of pulmonary vascular Pulmonary Circulation | July-September 2011 | Vol 1 | No 3

resistance to fall at birth and results in severe hypoxemia and PAH. [56] PPHN can be primary or secondary to a variety of clinical conditions, including asphyxia, sepsis, pneumonia, meconium aspiration syndrome and antenatal exposure to non-steroidal anti-inflammatory drugs. Additional intrauterine environmental factors such as large for gestation age and maternal asthma might be also important risk factors for PPHN. [57] Endothelial nitric oxide synthase (eNOS) levels has been increasingly implicated in the development of PPHN[58] and thus the expression of eNOS has been studied from an epigenetic perspective. In a study by Xu et al., the authors demonstrated a 6-fold upregulation of eNOS expression in pulmonary vascular endothelial cells derived from a neonatal rodent PPHN model induced by intrauterine exposure to hypoxia and indomethacin between the 19th and 21st day of gestation. 353

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In this model, NOS upregulation was associated with increased H3 and H4 histone acetylation in the eNOS promoter.[59] They also noted a mild decrease in eNOS methylation. However, this research did not have a reversal protocol which would be required to definitively demonstrate the role of these epigenetic abnormalities in the pathogenesis of PPHN.

Epigenetics also explains the cellular localization of eNOS expression. It has been shown that endothelial-specific expression of eNOS is regulated by epigenetics.[60] Chan et al. demonstrated that the nine CpG dinucleotides in the promoter region of the eNOS gene were unmethylated or lightly methylated in human endothelial cells.[61] In contrast, in vascular smooth muscle cells, the eNOS promoter was found to be almost completely methylated. In addition to changes in methylation of the eNOS promoter, the eNOS core promoter is highly enriched in acetylated histone H3/K9 and H4/K12, and methylated H3/K4 in endothelial cells.[60] Furthermore, HDAC inhibitor, trichostatin A, may induce eNOS expression in non-endothelial cells, and small RNA may suppress eNOS expression by altering histone acetylation and DNA methylation in endothelial cells.[62] Taken together, the dysregulation of endothelial eNOS, and thereby the development of this particular pulmonary vascular disease, appears to be epigenetically controlled. Epigenetics and RV hypertrophy Epigenetic changes occurring in the right ventricle have only recently been studied. Marked changes in the right ventricular morphology coincide with changes in the metabolic profile.[63] Though much focus remains on the changes in the pulmonary vasculature, the adaptive changes in the right ventricle may be even more important a predictor of survival.[64]

Adults with PAH and children with congenital heart disease compensate for RV pressure overload by developing right ventricular hypertrophy (RVH). Why some patients develop RVH that is compensatory while others develop a maladaptive RVH and rapidly develop RV dilatation and failure is uncertain. Whereas left ventricular hypertrophy (LVH) can be regressed with angiotensin converting enzyme (ACE) inhibitors resulting in therapeutic benefit, the benefit of reducing RVH are less clear, as is the optimum way to do this. Cho et al. studied the effects of an HDAC inhibitor, sodium valproate on RVH in monocrotaline (MCT) rats and pulmonary artery banding (PAB) rats. MCT rats develop a maladaptive form of RVH and tend to die within 6 weeks with RV failure; conversely, PAB rats develop an adaptive form of PH and are much less prone to premature death or RV failure.[65] HDAC inhibition in 354

PAB rats significantly decreased RVH. Pulmonary artery flow acceleration was significantly reduced in PAB rats treated with sodium valproate, suggesting a functional as well as anatomical improvement with epigenetic modification. HDAC inhibition also reduced myocardial fibrosis. The involvement of HDACs in these response was demonstrated by the increased acetylation of histone H3 in the RV in animals that received sodium valproate versus controls. As would be expected with sodium valproate there was no change in HDAC1, HDAC2, HDAC 3 and HDAC8. Similar improvements in RVH were observed in MCT rats treated with sodium valproate. ACE inhibitor therapy did not show this benefit in either models in terms of RVH or fibrosis.

However, not all studies suggest benefits from HDAC inhibition in RVH. In a study by Bogaard et al., [66] a different HDAC inhibitor, trichostatin A (TSA), was administered intraperitoneally four weeks after PAB. TSA-treated rats developed decreased cardiac output and signs of RV failure. In contrast to the Cho study in RVH and the beneficial effects of HDAC inhibition on LV hypertrophy in aortic banding models, trichostatin A actually increased RVH. TSA-treated PAB rats also demonstrated increased RV fibrosis, capillary rarefaction and rates of cell death. These negative results contrast with the findings of benefit in studies by Cho and Rexhaj, as well as the beneficial effects of HDAC inhibition in LV hypertrophy. [67] Do these differences reflect the different HDAC inhibitors, differences in the models or (in the case of LVH) differences in the RV versus the LV transcriptome response to pressure-overload?[68] There are several classes of HDAC inhibitors and further study is clearly required to determine whether a subclassspecific inhibition of a specific HDAC family is beneficial or harmful.

CONCLUSIONS

The study of the epigenetic regulation of development, cancer, and other diseases has increased at an exponential rate. In the area of pulmonary hypertension, the number of studies remains small, but is on the rise. The evolving technology to study epigenetics is accelerating.[69,70] High throughput means of studying the methylation status of the genome, or the “methylome,” are emerging and will likely uncover an extensive network of epigenetically regulated genes. The application of methylomics to the field of pulmonary hypertension may also provide insight into new molecular targets or regulatory pathways not previously recognized. Epigenetic regulation of gene expression may link known risk factors for the development of pulmonary Pulmonary Circulation | July-September 2011 | Vol 1 | No 3

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REFERENCES

PAH

Environment Phen-fen Amphetamines Diet Altitude Hypoxia

Epigenetic modification Histone acetylation CpG methylation RNA interference

Comorbidities Cirrhosis CREST Congenital heart disease Thrombosis HIV, PVOD, PCH, OSA

Genetics BMPR2 mutations Ion channel SNPs ALK1 mutations SERT overexpression

Cell signaling abnormalities HIF-1α cGMP/NO Ca2+ channel function K+ channel function RhoA/ROK activity

Figure 6: Schematic of pathophysiologic mechanisms leading to the development of PAH.

hypertension and contribute to the net risk of developing overt disease (Fig. 6). Epigenetic changes may be the missing link between genomic sequence variation, comorbid disease states, environmental exposure, and cell signaling events. Epigenetics could also play a role in the phenotypic variability of PAH (severity, time to disease onset). The accumulation of epigenetic “hits” en route to overt disease has been observed in cancer formation.[71] In a similar way, the influences of gender and toxin exposure, on top of genetic polymorphisms and disease conditions may change the methylation status of a network of disease modifying genes and lead to pulmonary hypertension.

Epigenetic modifications may be reversible[72] and it is possible that epigenetic modifications could be targeted by pharmacological intervention,[73] suggesting novel therapeutic options for experimental testing. Inhibition of DNMTs or HDACs have shown both promise and harm. The lack of specificity for epigenetic treatments is an important issue and warrants cautious application given the concerns for “off-target” effects of HDAC inhibition or demethylating agents. Interestingly, changes in the activity or expression of DNMTs have been noted to affect relatively specific outcomes in terms of site-specific methylation and regulation of specific genes in certain tissues. Current models suggest that the specificity of DNMT activity can depend on their expression levels or their interaction with other epigenetic regulators. While the development of selective HDAC inhibitors may have more utility, a more comprehensive understanding of the intricate intersecting pathways between DNA methylation, miRNAs, and chromatin remodeling will be critical. Similarly, investigations into the epigenetic control of right ventricular gene expression and adaptive hypertrophy will be required to understand a disease process which is not isolated to the pulmonary vasculature. Pulmonary Circulation | July-September 2011 | Vol 1 | No 3

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Source of Support: This work is supported by NIH K08-HL098565-01 (GHK), NIH-R01-HL071115 (SLA) and 1RC1HL099462-01 (SLA), the American Heart Association (AHA) and the Roche Foundation for Anemia Research (SLA)., Conflict of Interest: None declared.

Pulmonary Circulation | July-September 2011 | Vol 1 | No 3

Review Ar ti cl e

MicroRNAs-control of essential genes: Implications for pulmonary vascular disease Sachindra R. Joshi, Jared M. McLendon, Brian S. Comer, and William T. Gerthoffer Department of Biochemistry and Molecular Biology, University of South Alabama, Mobile, Alabama, USA

ABSTRACT During normal lung development and in lung diseases structural cells in the lungs adapt to permit changes in lung function. Fibroblasts, myofibroblasts, smooth muscle, epithelial cells, and various progenitor cells can all undergo phenotypic modulation. In the pulmonary vasculature occlusive vascular lesions that occur 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 defined. Advances in molecular biology have shown that multiple classes of ribonucleic acid (RNA) collaborate to establish the set of proteins expressed in a cell. Both coding Messenger Ribonucleic acid (mRNA) and small noncoding RNAs (miRNA) 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 defined in the pulmonary vasculature. Recent progress in defining miRNAs that modulate vascular cell phenotypes is reviewed to illustrate both functional and therapeutic significance of small noncoding RNAs in pulmonary arterial hypertension. Key Words: hypertension, Krueppel-like factor 4, myocardin, smooth muscle, translation, vascular remodeling, vascular injury

INTRODUCTION During development of the airways and the pulmonary circulation, multiple cell types adapt to changing chemical and physical signals to establish appropriate lung structure and function. Progenitor cells of various vascular cell types proliferate, migrate and reorient themselves to form nascent vessels and airways that eventually mature to the adult phenotype. During disease development there are also adaptive changes in function and structure, some of which are maladaptive and some of which oppose the disease process. Functional adaptations are manifest in the pulmonary circulation in pulmonary hypertension syndromes as hypercontractility. Structural adaptations are manifest as vascular pruning, medial thickening, leukocyte invasion and development of a variety of occlusive lesions. The initial triggers or stimuli for these remodeling events are under Address correspondence to:

Dr. William T. Gerthoffer Department of Biochemistry and Molecular Biology, University of South Alabama, 5185 USA Drive N., Mobile AL 36688 USA Email: wgerthoffer@usouthal.edu Pulmonary Circulation | July-September 2011 | Vol 1 | No 3

intense investigation, as are the pathways and proteins altered during pulmonary diseases. Structural studies suggest multiple cell types including lung fibroblasts, myofibroblasts, smooth muscle, epithelial, endothelial and progenitor cells all undergo varying degrees of phenotypic modulation during disease development. Blood vessel remodeling events include matrix remodeling, secretion of numerous cell signaling molecules, cell and tissue hypertrophy, and hyperplasia. In all hollow organs, including pulmonary blood vessels, smooth muscle cells undergo dynamic changes in gene expression and protein composition to adapt to changes in the local environment. When such changes are long-lasting, they are described as being due to â&#x20AC;&#x153;cellular plasticity.â&#x20AC;? The set of proteins expressed is determined by multiple parallel signal Access this article online

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Website: www.pulmonarycirculation.org DOI: 10.4103/2045-8932.87301 How to cite this article: Joshi SR, McLendon JM, Comer BS, Gerthoffer WT. MicroRNAs-control of essential genes: Implications for pulmonary vascular disease. Pulm Circ 2011;1:357-64.

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transduction pathways that ultimately regulate one or more events in transcription, translation, mRNA half-life and protein degradation. Transcriptional controls have been studied extensively in vascular cells, but epigenetic mechanisms contributing to smooth muscle phenotype are not as well defined. Dynamic changes in methylation of CpG sites in key promoters, histone modifications and microRNA-induced gene silencing are subjects of intense study in cardiovascular physiology. These phenomena are not nearly as well defined in smooth muscle cells as they are in cardiac muscle cancer cells and the immune system.[1-3]

The goal of this review is to summarize emerging knowledge of the microRNA (miRNA) class of small, noncoding RNAs in vascular smooth muscle cell phenotypes in normal tissue and in pulmonary diseases. We will focus on miRNAs with validated targets that are relevant to smooth muscle contractility and vascular development. The reader interested in miRNAs in endothelial cells and stem cells is referred to several excellent recent reviews of the subject and a recent review of miRNAs in pulmonary hypertension.[4-8] Our narrow approach to the topic in this review is justified by the compelling need to identify novel, druggable targets for modifying vascular remodeling. There is certainly continuing need to define sets of miRNAs that control both conserved (proliferation and cell survival) and unique (smooth muscle contractile protein expression) processes in all vascular cells and all cells in the lung. There is also an appealing opportunity to capitalize on current knowledge of miRNA-induced gene silencing in developing novel therapeutic approaches to pulmonary hypertension. To that end, we describe examples of RNAi-based therapy of animal models of cardiovascular and respiratory diseases. These studies provide an exciting proof of principle for RNAi therapy of lung diseases including pulmonary hypertension.

Smooth muscle cell phenotypes

Smooth muscle cells in vitro are highly plastic cells that are easily manipulated by altering culture conditions to favor a more contractile phenotype or a proliferative, secretory and migrating phenotype. Contractile characteristics are promoted by culturing at high density and in reduced serum concentrations in the presence of soluble factors including insulin, retinoic acid, and transforming growth factor beta 1 (TGF-β1). Some soluble factors are clearly derived from or promoted by endothelial cells, which, in coculture, promote differentiated, contractile pulmonary artery smooth muscle cells.[9] Contractile smooth muscle cells are defined as cells expressing smooth musclerestricted contractile and cytoskeletal proteins that contract in response to physiological agonists (e.g., norepinephrine, serotonin, histamine, enthothelin-1). There are several well-defined smooth muscle-restricted 358

contractile proteins including myosin II heavy chain, α and γ smooth-muscle actins, h-caldesmon, h1-calponin, smooth muscle tropomyosins, SM22 (transgelin) and smoothelin.[10,11] The contractile proteins are typically downregulated by conditions promoting proliferation. In culture proliferation, cell migration and secretion of mediators of inflammation can be induced by serumcontaining medium with the trophic growth factors epidermal growth factor and fibroblast growth factor. The gene expression profile of proliferative and migratory vascular smooth muscle cells is not as well defined as the contractile phenotype. It is frequently used in the context of cultured cells that proliferate in serum-containing medium, express a chemotactic response to platelet derived growth factor (PDGF), and secrete a variety of proteins. Secreted proteins include type I collagen, cytokines, chemokines and growth factors. Growth, migration and proliferation in vitro are thought to recapitulate organogenesis during fetal and neonatal development.

There are differing views of the two “phenotypes” of vascular smooth muscle. One view holds that switching from proliferating/migrating cells to contractile cells is a stable, mutually exclusive condition—a binary phenomenon.[11] An alternate view is that the phenotype of smooth muscle cells is graded with cells in a tissue having a mosaic pattern of contractile protein gene expression. [10,12,13] In either case, gene expression programs in smooth muscle appear to be highly adaptable depending on tissue type, culture conditions and disease processes. Current progress in epigenetic mechanisms controlling gene expression strongly suggests part of the adaptability of vascular smooth muscle and other vascular wall cells is due to dynamic changes in gene expression. One topic of great interest is the influence miRNAs might have on networks of target genes that are important in disease progression. Defining the miRNAs and their targets in pulmonary vascular smooth muscle phenotype switching should add novel therapeutic targets for anti-remodeling drugs. Progress on this topic will have high impact on translational research aimed at developing novel treatments of pulmonary hypertension.

MiRNA biogenesis and miRNA-induced silencing

MiRNA biogenesis and the mechanisms of miRNA-induced gene silencing have been well described, and the basic steps appear to be highly conserved among various cell types. The current consensus on biogenesis is illustrated in Figure 1, and the interested reader is directed to recent reviews of the topic for more detailed description of the process.[14,15] Many miRNA genes are hosted within other genes distributed throughout mammalian genomes. They are often located in introns and sometimes in exons, and some are in intergenic Pulmonary Circulation | July-September 2011 | Vol 1 | No 3

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Figure 1: MicroRNA biogenesis and RNA-induced gene silencing. Transcription of primary micro RNA (Pri-miRNA) from miRNA genes is followed by cleavage to precursor mRNA (Pre-miRNA) by the Drosha nuclear RNase III. The Pre-miRNA is then exported to the cytoplasm by exportin via nuclear pore. In the cytoplasm, Pre-miRNA is further processed by RNase activity of Dicer to the mature micro RNA duplex. The duplex loads onto Argonaut ribonucleases in the RISC complex and separates. One of the mature miRNA strands (red strand) mediates small interfering RNA silencing by degrading the target mRNA or interfering with translation. The outcome of RISC formation varies with the degree of complementarity of the seed sequence of miRNA and 3’ untranslated regions (UTR) of the target mRNA.

regions rather than within a host gene. Clusters of coexpressed, polycistronic primary transcripts are common and many miRNA genes have multiple copies in the human genome. Hypoxia-regulated miRNAs are a good example of coexpressed clusters of miRNAs relevant to pulmonary vascular diseases.[16] MiRNAs hosted by protein-coding genes are under control of Pol II promoters and familiar transcription factor families that control expression of mRNAs. However, some miRNA genes have independent promoters. A few miRNA genes are transcribed by Pol III, which transcribes tRNA, 5S rRNA and small nuclear RNA genes. Primary miRNA transcripts are capped and polyadenylated then cropped to a ~70 nucleotide precursor (Pre-miRNA) by the nuclear ribonuclease Drosha. After export from the nucleus a cytoplasmic RNase (Dicer) cleaves the loop structure of the pre-miRNA yielding a mature 21~24 nucleotide miRNA duplex (Fig. 1). These processing steps are necessary for proper smooth muscle development based on studies of smooth-muscle restricted knockout of Dicer in mice. Knocking out Dicer is known to inhibit blood vessel maturation and intestinal tract development.[17,18] MiRNA processing is also under control of functionally important extracellular signals in vascular smooth muscle. For example, TGF-β family proteins have profound effects on processing of miR-21 in human pulmonary artery cells in culture.[19] Gene silencing mediated by the mature miRNAs then occurs by two somewhat different mechanisms that Pulmonary Circulation | July-September 2011 | Vol 1 | No 3

both require the mature miRNA to complex with several proteins including Argonaut family members Ago-1 and Ago-2. The mature dsRNA duplex loads into RNA-induced silencing complexes (RISCs) that mediate posttranscriptional silencing by reducing mRNA stability or by translational block depending on the degree of complementarity of the miRNA seed sequence (nucleotides 2-8) with the target sequence (Fig. 1). MiRNA tends to be cleaved by Ago-2 when complementarity is perfect, although this is not universally true. The transcript can then be further modified by uridinylation and decapping, and then completely degraded by exonuclease cleavage. When complementarity is imperfect a variety of miRNA/mRNA/RISC structures can form that block initiation, cause premature termination, and induce dissociation of ribosomes. MiRNA degradation then occurs following deadenylation, decapping and exonuclease action. Translation of sets of target proteins is thereby reduced. For the purposes of this review we are interested in miRNA-mediated gene silencing, but the reader should be aware that instances of miRNA-mediated translation enhancement have also been reported. [20] The remainder of the review focuses on particular miRNAs that target genes important in vascular smooth muscle development, contraction and lifespan. The aim is to show how miRNA-induced silencing could alter smooth muscle progenitor differentiation, smooth muscle restricted contractile protein expression, smooth muscle proliferation, and proinflammatory mediator synthesis. Each of these processes participate in vessel wall remodeling that contributes to pathogenesis of pulmonary vascular diseases.

MiRNAs and vascular smooth muscle plasticity

The role of miRNA-mediated gene silencing in vascular smooth muscles was first described in 2007. The number of studies describing miRNAs expressed vascular tissues under a variety of conditions is growing exponentially. There are now reports of miRNAs necessary for normal vascular development as well as miRNAs that are altered in vascular diseases including vascular damage, atherosclerosis and pulmonary hypertension. These miRNAs are sorted into functional groups in Table 1 to illustrate the miRNAs known to contribute to smooth muscle cell fate.

The earliest reports linking miRNAs and vascular smooth muscle remodeling were studies of miRNAs upregulated during injury. Zhang and coworkers discovered miR-21 levels increased following carotid artery injury.[21] They then went on to establish that miR-21 promotes vascular smooth muscle proliferation by silencing expression of phosphatase and tensin homolog (PTEN) and increasing 359

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Table 1: MicroRNAs regulating smooth muscle cell fate miRNA regulating SMC phenotype Contractile proteins miR-1 miR-25[32] miR-133a[60] miR-143~145[23,26] [31]

Synthetic functions

Differentiation

miR-24 miR-25[32] miR-26a[61]

miR-10a[58] miR-143~145[4,7,59] miR-155[62,63]

[36]

miRNA regulating SMC proliferation, migration and survival Proliferation

Apoptosis/Survival

miR-1 miR-146a miR-21[21] miR-204[44] miR-26a[65] miR-221[21,37] [64]

[33]

miR-21

[21]

expression of B-cell leukemia/lymphoma 2 (Bcl2). Paradoxically, miR-21 was subsequently shown by Davis et al.[19] to promote contractile protein expression induced by TGF-β family proteins in cultured pulmonary artery vascular smooth muscle by silencing programmed cell death 4 (PDCD4). TGF-β family proteins enhanced processing of the miR-21 primary transcript to the mature miRNA, and increased miR-21 was found to enhance smooth-muscle restricted contractile protein expression.[19] These findings are important because they were the first example of growth-factor regulation of miRNA processing in smooth muscle, and they showed that one miRNA (miR-21) under different conditions promotes either contractile or proliferative phenotypes. It remains to be seen whether other miRNAs also exert dual effects on smooth muscle phenotype, but the early studies point to the complexity and potential duplicity of miRNA targets and physiological effects.

The initial studies of miR-21 in vascular remodeling were quickly followed by a series of landmark studies of the miR-143~145 cluster. Neointimal lesion formation is associated with downregulation of miR145 as well as downregulation of contractile protein expression and increased proliferation of neointimal cells. [22] Downregulation of miR-143~miR-145 and downregulation of contractile protein expression was then shown to be cause-and-effect in a series of loss of function studies by Cordes et al.[23] Studies in cultured vascular smooth muscle cells and knockout mice have defined a pathway for reciprocal control of Kruppel-like factor 4 (KLF4) and myocardin expression by miR-145 as shown in Figure 2. Knockout mouse studies have corroborated the initial cell culture studies and have verified that the miR-143~145 cluster is a dominant regulator of smooth muscle differentiation. The miR143~145 cluster enhances contractile protein expression required for contractility and proper blood pressure regulation.[24-26] It also has a profound effect: to promote differentiation of stem cells to smooth muscle cells.[4] MiR-145 can directly 360

Migration/Cytoskeletal proteins miR-143~145[24]

Figure 2: MicroRNAs regulating smooth muscle restricted contractile protein expression. Multiple miRNAs modulate the key transcriptional co-regulators myocardin and KLF4, which are positive and negative regulators of SRFdependent smooth muscle gene expression. Current evidence shows miR-1, miR-25, miR-133a, miR-146a and miR-145 all modulate expression of either KLF4 or myocardin to influence contractile protein expression. The red lines indicate silencing of protein expression or inhibition of miRNA expression by pathway components. The green arrows indicate activation or upregulation of the pathway component.

silence expression of KLF4 and can indirectly upregulate myocardin expression (Fig. 2), which contributes to TGF-β1 enhancement of serum response factor (SRF)dependent contractile protein expression.[27] SRF regulates a loosely coordinated set of smooth muscle contractile, cytoskeletal and matrix protein genes with CArG boxes in the 5’ untranslated region.[24,28,29]

In addition to regulating contractile protein expression, miR-143~145 also influences expression of proteins involved in matrix remodeling and cell migration. Downregulation of miR-143~145 upregulates formation of podosomes and upregulates expression of PDGF receptor, protein kinase C (PKC) epsilon and the actin bundling protein fascin. [30] Podosomes are discrete sites of matrix remodeling necessary for invasive migration of vascular smooth muscle cells during vascular wall remodeling. Whether podosome formation is necessary for development of arteriopathy in pulmonary vascular diseases is unknown, but seems plausible given the extensive structural remodeling that Pulmonary Circulation | July-September 2011 | Vol 1 | No 3

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occurs in humans and in animal models of pulmonary hypertension.

In addition to the miR-143~145 cluster, other miRNAs can modulate smooth muscle gene expression by altering KLF4 expression (Fig. 2). KLF4 is a direct target of miR-1 in stem cell differentiation,[31] miR-25 in airway smooth muscle,[32] and miR-146a in vascular smooth muscle.[33] Regulation of KLF4 by miR-146a involves a feedback loop in which miR-146a silences KLF4 which competes with KLF5 to reduce transcription of the miR-146a gene. Neointima formation is thereby enhanced by smooth muscle cell proliferation and migration due in part to increased KLF4 expression. The proximal signals that activate the miR146a-KLF4/KLF5 pathway are not defined in vascular smooth muscle. However, in airway smooth muscle expression of primary-miR-146a expression is activated by nuclear factor kappa beta signaling and primary-miR146a processing is regulated by MEK-1/2 and JNK-1/2. [34] Mature miR-146a is also induced by stretch in C2C12 myoblast cells.[35] Defining the trigger and upstream transduction pathways in vascular smooth muscle might identify high-value targets for anti-remodeling therapy in PAH.

The peptide growth factors PDGF and TGF-β1 are known to regulate smooth muscle phenotype and to mediate vascular development and remodeling in PAH. PDGFBB promotes the proliferative/migratory/secretory phenotype in culture and is necessary for proper formation of new blood vessels during development. In contrast, TGF-β family proteins often enhance the contractile phenotype via Smad-dependent signaling. Although they can produce opposing effects on smooth muscle phenotype, both proteins signal changes in primary miRNA transcription and processing. Recent evidence points to signaling convergence of these factors that explain functional antagonism in smooth muscles. PDGF-BB induces expression of miR-24 which directly silences expression of Tribbles-like protein 3 (Trb3) and indirectly decreases Smad1 levels. [36] Overexpression of miR-24 reduces Smad2 and Smad3 expression and reduces TGF-β-mediated activation of Smad2. Therefore, miR-24 is a point of antagonistic signaling convergence for PDGF-BB and the TGF-β family members in vascular smooth muscle. This suggests that miR-24 might be an interesting target to alter vascular remodeling. However, the timing and effect of any intervention with a miR-24 antagonist is difficult to predict given the complex interplay between BMP and TGF-β family members during pathogenesis of pulmonary hypertension. Empirical tests in animal models of pulmonary hypertension at various stages of disease development are needed to establish an effective therapeutic strategy. Pulmonary Circulation | July-September 2011 | Vol 1 | No 3

The PDGF signaling pathway in vascular smooth muscle also induces expression of miR-221 which might also contribute to neointimal proliferation. [37] MiR- 2 21 is upregulated in a variety of cancers, and miR-221 silences expression of p27Kip1 during skeletal muscle differentiation. [38] In cultured human pulmonary artery smooth muscle, silencing p27Kip1 by miR-221 overexpression promotes proliferation.[37] In a separate study of rat aorta, smooth muscle miR-221 and miR-222 expression was induced with PDGF, which also decreased p27Kip1 and p57Kip2 expression.[39] MiR-221 and miR222, like miR-21, are good examples of miRNAs that are conserved in many cells and have consistent effects on cell cycle control proteins in vascular smooth muscle cells. It will be important to determine whether modifying the highly conserved process of cell cycle transit with RNAibased therapy can be an effective anti-remodeling strategy. The timing of such treatments during development of pulmonary vascular remodeling will be important as was suggested above for modifying growth factor signaling with miR-24 antagonists.

MiRNAs and pulmonary hypertension

Studies of miRNAs in vascular remodeling during development of pulmonary hypertension must consider the multifocal, multicellular nature of the changes in vascular structures. Vascular lesions involving multiple cell types are observed in humans and in animal models of pulmonary arterial hypertension.[40] It seems likely that phenotype modulation of endothelial cells, smooth muscle cells, fibroblasts and both resident and immigrating progenitor cell types occurs.[41] Therefore understanding miRNA expression patterns in each cell type as a function of disease development and degree of severity is vital for designing novel therapeutic strategies.

To this point we have focused on miRNAs in vascular smooth muscle cells; but there is also a significant literature on miRNAs in endothelial cells and various types of pluripotent cells that is highly relevant. Comprehensive discussion of this issue is beyond the scope of this review, but some key observations are worth making. A number of miRNAs described in smooth muscle cells (e.g., miR-21 and miR-221 in Figure 3) have conserved functions in differentiation, proliferation and survival of endothelial cells and other vascular mural cells.[6] The initial studies of miRNAs in the cardiovascular system cited above suggest some likely targets for RNAi-based antagonism of remodeling—e.g., miR-21, miR-145 and miR-221.[42] However, until recently it was not clear which miRNAs were altered during development of pulmonary arterial hypertension (PAH). MiRNAs that promote arterial muscularization, that increase cell survival or proliferation and promote endothelial to mesenchymal transitions are clearly of great interest. 361

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miR-21

miR-221

PTEN Bcl2

PIP3

miR-204

SHP2 p27Kip1

Akt/PKB Apoptosis

Src Stat 3

Proliferation

Figure 3: Signal transduction pathways implicated in pathogenesis of vascular remodeling relevant to pulmonary hypertension. Panel A: miR-21 may increase vascular smooth muscle cell number by targeting proteins that regulate cell proliferation (PTEN) and apoptosis (Bcl2). Changes in miR-21 expression have been observed in lung tissues and in vascular smooth muscle in animal models of pulmonary hypertension. Panel B: miR221 promotes vascular smooth muscle cell proliferation by silencing the cell cycle inhibitor p27Kip1. Panel C: miR-204 downregulated in pulmonary hypertension in animals and in human leukocytes can indirectly promote cell proliferation. Derepression of SHP2 expression activates a Src/Stat3 cascade that promotes vascular smooth muscle proliferation.

In the earliest published study of miRNAs in pulmonary hypertension, Caruso et al.[43] surveyed miRNA expression in total lung extracts from two rat models (chronic hypoxia and monocrotaline model) of PAH. MiR-21 was downregulated in the monocrotaline model and in lung samples from humans with PAH. In the same study miR451 was upregulated in both models but no differences were detected in lung samples from control and PAH human subjects. This initial report showed that there are disease-related changes in miRNAs associated with development of PAH in both animals and humans. A subsequent study by Courboulin et al.[44] found miR-204 was also downregulated in humans with PAH and in rat models of PAH. Delivery of a miR-204 mimic to rat lungs reduced the severity of the disease, providing an exciting proof of principle for rescuing vascular smooth muscle phenotype in vivo with RNAi-based therapy. Courboulin et al.[44] also showed downregulation of expression of miR204 in mononuclear cells from in blood of PAH patients. This raises the possibility that changes in miRNAs in plasma and leukocytes might be useful biomarkers of PAH pathogenesis. In the animal models of PAH Courboulin et al.[44] identified potential targets for miR-204. They found Stat3 activation was increased upon attenuation of miR-204 expression and that miR-204 directly regulates SHP2 by targeting its 3’UTR. They developed the signaling model shown in Figure 3 where decreased miR-204 increases SHP2, which by activating Src increases Stat3 activation. Stat3 is hypothesized to promote smooth muscle proliferation and pulmonary vessel wall thickening. This landmark study provides solid proof of principle that “rescue” of low 362

miRNA expression can prevent progression of established PAH.[44] Rapid advances are being made in RNAi therapy of several vascular diseases in which target miRNAs have been identified, some target proteins and processes have been identified, and some demonstration of effective drug delivery has been presented.[45-47]

The study of miR-204 in PAH[44] is unique because roles for miR-204 in myogenesis and in other vascular diseases have not been described before. MiR-204 may have a particular set of functions in the pulmonary circulation that differ qualitatively or quantitatively from its function in other vascular beds. It seems reasonable to speculate that miR204 is one of several miRNAs that promote differentiation of vascular smooth muscle, and that downregulation of miR-204 might occur during pathogenic vascular remodeling in atherosclerosis and restenosis. However, there are no reports of a strong association or causeeffect in these other vascular diseases. Nevertheless, targets of miR-204 have been validated in other cell types. Some of the target proteins have important roles in smooth muscle cell physiology and vascular diseases: TGF-β receptor 2, [48] epidermal growth factor (EGF) receptor signaling,[49] forkhead box C1 (FOXC1),[50] and runt-related transcription factor 2 (Runx2).[51] It is not clear whether these other targets of miR-204 are also contributing to arteriopathy in pulmonary hypertension, but these are target proteins worthy of further study.

CONCLUSIONS AND FUTURE DIRECTIONS

MiRNA expression surveys have yielded several candidate molecules that could contribute to disease development involving remodeling of smooth muscles. The results of unbiased expression surveys and hypothesis-driven biochemical and functional validation studies have provided important insights into disease mechanisms and potential targets of new treatments of vascular remodeling (Table 1). Some conserved miRNAs have been described in vascular smooth muscle tissues and cells in culture that are known from the cancer literature. MiRNAs controlling cell proliferation and cell survival (miR-21, miR-221 and miR-222) are altered in a variety of diseases. Some miRNAs appear to serve important roles in smooth muscle differentiation unique to this cell type. Novel findings relevant to miRNAs in differentiation were led by investigations of vascular remodeling in disease models and in humans. The best example is the prominent role of the miR-143~145 cluster in regulating KLF4 and myocardin in smooth muscle differentiation. Further investigation is needed to define how miRNAs such as miR145 can control a set of highly smooth-muscle restricted Pulmonary Circulation | July-September 2011 | Vol 1 | No 3

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genes and yet in other settings act as tumor suppressors and regulators of pluripotency.

A number of important questions are raised by the recent surge in knowledge about miRNAs in vascular diseases. One issue is related to the general question of the significance of dynamic changes in epigenetic mechanisms of gene expression in pulmonary vascular diseases. Do pulmonary vascular smooth muscle cells respond to environmental inputs by altering epigenetic factors such as DNA methylation patterns in CpG regions of promoters? Are there diagnostic or prognostic modifications of histones that prime particular genes for expression that is subsequently modulated by the miR-143~145 cluster? Will new assays of miRNA expression and miRNA processing during various stages of disease development illuminate some new biomarkers or new candidates for inhibiting pathological vascular remodeling? Can RNA mimics and antagonists be effective anti-remodeling drugs in vivo?

There are reasons for optimism that RNAi therapy might be a useful anti-remodeling approach. One of the earliest examples of RNAi “therapy” in animals was intranasal delivery of antisense oligonucleotides against a respiratory syncytial virus protein to the lungs of mice inhibited virus replication.[52,53] RNAi therapy can also be scaled up for use in primates. A locked nucleic acid modified miR-122 when administered IV to green monkeys inhibits cholesterol synthesis.[54] Recently RNAi therapies targeting smooth muscle remodeling have also been shown to effective in animal models. Pulmonary hypertension and asthma in animal models are both responsive to lung-restricted delivery of RNAi drugs that rescue (miR-204),[44] or antagonize (miR-145)[55] miRNAs altered by the disease. There is also hope that atherosclerotic plaque stability might be susceptible to manipulation via systemic delivery of RNAi-based drugs. [46,56,57] To address the question of effective RNAibased anti-remodeling therapy, novel RNAi-based drugs and novel delivery methods must be developed in animal models and then rapidly moved to first-in-human trials. It is now clear that chemically stabilized antisense ribonucleotides and modified miRNAs can be effective “therapy” in animal models of vascular and respiratory diseases. With ongoing development in RNAi drug design and delivery, these approaches should soon be applied to humans.

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

Pulmonary Circulation | July-September 2011 | Vol 1 | No 3

Research A r t i cl e

Blood flow redistribution and ventilationperfusion mismatch during embolic pulmonary arterial occlusion K. S. Burrowes1, A. R. Clark2, and M. H. Tawhai2 1

Department of Computer Science, University of Oxford, UK, 2Auckland Bioengineering Institute, University of Auckland, New Zealand

ABSTRACT Acute pulmonary embolism causes redistribution of blood in the lung, which impairs ventilation/perfusion matching and gas exchange and can elevate pulmonary arterial pressure (PAP) by increasing pulmonary vascular resistance (PVR). An anatomically-based multi-scale model of the human pulmonary circulation was used to simulate pre- and post-occlusion flow, to study blood flow redistribution in the presence of an embolus, and to evaluate whether reduction in perfused vascular bed is sufficient to increase PAP to hypertensive levels, or whether other vasoconstrictive mechanisms are necessary. A model of oxygen transfer from air to blood was included to assess the impact of vascular occlusion on oxygen exchange. Emboli of 5, 7, and 10 mm radius were introduced to occlude increasing proportions of the vasculature. Blood flow redistribution was calculated after arterial occlusion, giving predictions of PAP, PVR, flow redistribution, and micro-circulatory flow dynamics. Because of the large flow reserve capacity (via both capillary recruitment and distension), approximately 55% of the vasculature was occluded before PAP reached clinically significant levels indicative of hypertension. In contrast, model predictions showed that even relatively low levels of occlusion could cause localized oxygen deficit. Flow preferentially redistributed to gravitationally non-dependent regions regardless of occlusion location, due to the greater potential for capillary recruitment in this region. Red blood cell transit times decreased below the minimum time for oxygen saturation (<0.25 s) and capillary pressures became high enough to initiate cell damage (which may result in edema) only after ~80% of the lung was occluded. Key Words: computational model, embolic pulmonary hypertension, pulmonary embolism, pulmonary hemodynamics

INTRODUCTION Acute pulmonary embolism (APE) is characterized by full or partial occlusion of one or more pulmonary arteries, resulting in a redistribution of blood to the non-occluded vessels. This leads to ventilation/perfusion (V/Q) matching abnormalities which includes extremely high or infinite V/Q values in the embolized region(s) but also potentially decreased V/Q units in the non-occluded tissue, hence impaired gas exchange, hypoxemia and hypocapnia can result.[1] Tsang et al.[2] and Altemeier et al.[3] concluded that the changes in V/Q after APE are determined primarily by the redistribution of pulmonary blood flow, with a minor contribution from ventilation redistribution; however this blood flow redistribution has not been studied in detail. It is Address correspondence to:

Dr. Kelly Burrowes Department of Computer Science, University of Oxford, Wolfson Building, Parks Road, Oxford, OX1 3QD, UK Phone: +44 1865 610807, Fax: +44 1865 283531. Email: jelly.burrowes@comlab.ox.ac.uk Pulmonary Circulation | July-September 2011 | Vol 1 | No 3

not clear what happens to V/Q, or oxygen levels, within the non-occluded region alone, where the lung must attempt to achieve sufficient oxygenation in a reduced volume. The degree of vascular recruitment, distension, and other changes in hemodynamics in these non-occluded regions are also not well described in the literature. APE can also result in pulmonary hypertension (PH) which is defined clinically by a mean pulmonary artery pressure (mean PAP) ≥25 mmHg at rest[4] (normal resting mean PAP is ~14 mmHg with an upper limit of normal of ~20 mmHg[5]). While it is apparent that thromboembolic occlusion increases pulmonary vascular resistance (PVR) Access this article online

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Website: www.pulmonarycirculation.org DOI: 10.4103/2045-8932.87302 How to cite this article: Burrowes KS, Clark AR, Tawhai MH. Blood flow redistribution and ventilation-perfusion mismatch during embolic pulmonary arterial occlusion. Pulm Circ 2011;1:365-76.

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via mechanical obstruction of distal vascular beds, there is evidence to suggest that additional mechanisms are required to increase pulmonary pressures to levels observed in patients presenting with APE. Subjects with inert (non-thromboembolic) vascular occlusion of ~50% of the lung (as observed in patients undergoing unilateral pneumonectomy) often have normal pulmonary arterial pressures; however patients with thromboembolic occlusions of ~25% of the lung may experience PH.[6,7] The contribution of vasoconstriction via neural reflexes and the release of humoral factors (i.e., serotonin, endothelin-1, thromboxane-A2) in APE is relatively well established in animals.[6,8] Data are more difficult to obtain within human subjects; however, some studies have shown preliminary evidence for endothelin abnormalities and the role of vasoconstriction in human APE.[9-12] Hypoxic pulmonary vasoconstriction resulting from hypoxemia has also been suggested as a mechanism for the increase in PVR in APE.[6] In this study, we used a structure-based computational model of the human pulmonary circulation[13] to determine how cardiac output is accommodated post-occlusion, and what impact this would have on V/Q matching and oxygen transfer from air to blood if ventilation distribution remained unchanged post-embolus. A secondary question was to determine whether direct obstruction of a blood vessel by itself can be sufficient to elevate PAP to a level that is indicative of PH. The model used here incorporates an anatomically-based geometry of the extra-acinar blood vessels, the effect of axial and radial tissue tethering on vascular geometry, and accounts for the zonal flow mechanisms[14] that occur in the micro-circulation.

MATERIALS AND METHODS

This study uses a previously published multiscale computational model of blood flow through the distensible vessels of the full pulmonary circulation (arteries, veins, and intra-acinar circulation) coupled to parenchymal tissue deformation.[13] Only a brief description of the methods of this model will be given here: additional detail may be found in the Multiscale Model given below (following our Conclusions, and before the Acknowledgments), as well as in the corresponding references.

The perfusion model

Specific details of the components of the model can be found in references.[13,15-17] A summary of the most important model features is provided here and in the Multiscale Model. To summarize, this model includes the following. First, it includes anatomically-based geometry of the lung surface and central blood vessels, [18,19] and 366

computationally-generated, morphometrically-consistent models of the “accompanying” arterial and venous vessels (i.e., not including supernumerary vessels) to the level of the acini.[20] There are approximately 64,000 each arterial and venous branches in the model, and each terminal vessel supplies a single pulmonary acinus (31,800 in the whole lung model). Diameters were defined using a Strahler-diameter ratio.[13,15]

Second, it includes an intra-acinar circulation model[16] linking each of 9 symmetric branches of arterioles and venules via a “sheet” flow model of the capillaries[21] that crosses between each generation of arteriole and venule, forming a ladder-like configuration.

Third, it includes a model of parenchymal tissue deformation under gravity, [19] to which the vascular networks are tethered. Tissue deformation influences perfusion via shift in the vessel locations (the “Slinky” effect,[22]) by elastic recoil pressures acting to distend the extra-capillary vessels, and by the effect of alveolar inflation on capillary sheet distensibility.[13,15] Tissue deformation was simulated for a supine lung at functional residual capacity (FRC).

Steady blood flow through the full model was simulated after applying boundary conditions for pressure or flow at the “inlet” and “outlet” vessels: this corresponded to an inlet flow (QRV) of 5 l/min. at the pulmonary trunk, and left atrial pressure (LAP) of 5 mmHg. These values were used for all simulations unless otherwise stated. By solving equations for Poiseuille resistance including gravity, conservation of mass, vessel elasticity, and a microcirculatory model (see Multiscale Model and Clark et al.,[13]) predictions were obtained for the regional distribution of blood flow, blood and transmural pressures, capillary recruitment, red blood cell (RBC) transit time, and vessel radius. An output of the simulation was mean pulmonary arterial pressure (PAP), and therefore pulmonary vascular resistance (PVR=(PAP-LAP)/QRV) was calculated across the full circulation.

Simulating occlusions

Post-occlusion simulations were performed by setting vessel radius at the occlusion site to 10% of its initial radius which, in general, was sufficient to reduce flow to ~5% of baseline in the occluded region. Emboli vary in size and shape, and an embolus traveling into the lung via the pulmonary trunk could deposit in numerous locations. Studying the relationship between embolic arterial obstruction, PAP, and hemodynamics therefore first requires assessment of the likely distribution of emboli. All potential sites and their probability of occlusion were calculated for three sizes of emboli (5, 7, or Pulmonary Circulation | July-September 2011 | Vol 1 | No 3

Burrowes et al.: Pulmonary blood flow after occlusion

10 mm radius). The probability of occlusion was assumed to be proportional to the baseline flow rate to the occluded vessel (i.e., regions of higher flows more likely to occlude). The emboli were assumed to occlude the first vessel that they encountered that was smaller in radius than the embolus, and they did not deform within the artery. Flow distribution was simulated for each site of occlusion.

To quantify the relationship between volume of obstructed tissue and hemodynamic outcomes in the model, emboli of a single size (either 5, 7, or 10 mm radius) were added cumulatively (in random order) and flow distribution recalculated, until there were emboli in blood vessels that feed nearly all capillary beds. The number of acini distal to the occlusion and PAP were recorded. Lung occlusion was defined as the percentage of acinar units distal to occlusions (relative to total number of acini). To assess the impact of obstruction on microcirculatory flow in the unobstructed tissue, for the 10 mm simulations the capillary sheet flow-rate, capillary pressures, and capillary recruitment were calculated. To quantify the relationship between PAP and cardiac output in the PE model, simulations were repeated for increased cardiac output.

Estimating alterations in oxygen transfer

To estimate alterations in regional oxygen transfer post occlusion, a 3%/cm linear gradient of ventilation was assumed along the gravitational axis (compared with 2.75.2 %/cm in the supine posture for healthy humans[23-25]) with total alveolar ventilation of 5 l/min. such that mean V/Q=1. Oxygen transfer from air to blood was estimated using a simple model based on Kapitan and Hempleman[26] describing oxygen (O2) partial pressure balance in each acinus. VI ⋅ PI O2 − VA ⋅ PAO2 = Q( PC O2 − Pv O2 ), (1)

where VI is the inspired ventilation (l/min.); VA is expired or alveolar ventilation (l/min.); Q is blood flow to the acinar compartment (l/min.); and PkO2 is the O2 partial pressure (mmHg) of oxygen in humidified inspired air (k=I), alveolar air (k=A), end-capillary blood (k=C), or the mixed venous blood that enters the lungs from the systemic circulation (pulmonary arterial blood) (k=v). PIO2 was 150 mmHg (for all cases), and PvO2 was set to 40 mmHg (baseline level). For post-occlusion simulations retaining PvO2 at 40 mmHg assumes that oxygen uptake in the systemic circulation adapts (reduces) and so this will likely overestimate oxygen partial pressures. More likely, oxygen consumption by the body remains close to baseline levels and PvO2 reduces; therefore, lower values of 30 mmHg and 20 mmHg were also considered in simulations. The perfusion model and ventilation distribution gives Q and V (=VI=VA) in each acinus, with unknowns PAO2 and Pulmonary Circulation | July-September 2011 | Vol 1 | No 3

PCO2. Assuming that blood remains in the capillaries for long enough for O2 to equilibrate between alveolar air and capillary blood, then at end inspiration PAO2=PCO2. This assumption is likely to be valid except at very high levels of occlusion (as discussed later). These assumptions reduce equation (1) to PC O2 = PAO2 =

(VI ⋅ PI O2 + Q ⋅ PV O2 ) (VA + Q )

. (2)

Equation (2) was solved for each acinus to predict PCO2 and PAO2. The ventilation-weighted sum of PAO2 was calculated as an estimate of expired O2 partial pressures from the full lung. As most capillary O2 is bound to Hemoglobin, to calculate arterial (pulmonary venous) O2 partial pressures PCO2 was converted to O2 content (CCO2) and an acinar perfusion-weighted sum of CCO2 was calculated before conversion back to partial pressure units. To do this we used the formula

CC O2 = (15 × 1.34 × ρ ( PC O2 ) + 0.03PC O2 )/ 100, (3)

where ρ(PCO2) is the oxygen saturation—a function of PCO2. The first term on the right hand side represents the O2in hemoglobin, and the second term represents dissolved O2 concentration in blood plasma. Hemoglobin has an oxygen binding capacity of 1.34 ml O2 per gram of hemoglobin, and 15 g of hemoglobin per 100 ml of blood. Dissolved O2 concentration was calculated from Henry’s Law (for each mmHg of partial pressure there is 0.03 ml of O2 in 100 ml of whole blood). Oxygen saturation was in turn calculated using the MonodWyman-Changeux (MWC) model[27] ρ ( PC O2 ) =

LK T σ PC O2(1 + K T σ PC O2 )3 + K Rσ PC O2(1 + K Rσ PC O2 )3 , (4) L(1 + K T σ PC O2 )4 + (1 + K Rσ PC O2 )4

where KT=10×103 L mol−1, KR=3.6×106 L mol−1, L=171.2×106, and the O2 solubility, σ is 1.4×10−6 M mmHg−1.[28]

RESULTS

The baseline lung model

A full comparison of the baseline lung model to literature data is given in Clark et al.[13] In brief, PAP for the baseline model was 15.8 mmHg (a PVR of 2.2 mmHg/l/min.) in the upright posture, 16.8 mmHg (a PVR of 2.4 mmHg/l/min.) in the prone posture and 18.2 mmHg (a PVR of 2.6 mmHg/l/ min.) in the supine posture. In the supine posture (used in this study) a mean RBC transit time of 1.76±0.53 sec. (range 0.30-9.89 sec.) was calculated in the model at baseline. 367

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Predicted variability in the sites of occlusion

Sites of occlusion, their probability of occlusion, and all distal vessels that are affected by a single 5, 7, or 10 mm radius embolus are illustrated in the supine lung model in (Fig. 1). The number (and percentage of total) of acinar units affected by each occlusion are given in (Table 1) (column 2). The 10 mm emboli occluded lobar level arteries, the 7 mm emboli predominantly occluded segmental vessels, and the 5 mm emboli were distributed within the segmental and sub-segmental arterial level. A slight preference for occlusion of the gravitationally dependent regions was predicted, consistent with the flow distribution. Emboli had an almost identical probability of occluding the left (49.5%) or right (50.5%) lung. For the 5 mm emboli, the probability of lobar deposition was 19.2%, 5.0%, and 26.3% for the right upper, mid, and lower lobes, respectively; and 23.4% and 26.2% in the left upper and lower lobes, respectively. This anatomical distribution of emboli compares well with measurements in humans.[29]

occluded flow to larger volumes of tissue and therefore resulted in greater mean increases in PAP and PVR (Table 1). The gravitationally dependent flow gradient decreased after all occlusions, regardless of occlusion size or location.

Relationship between vascular obstruction and PAP

The probability of occlusion had a strong linear correlation with the volume of tissue distal to the occlusion (not shown). This is expected as larger regions of tissue will typically receive a higher flow of blood. The larger emboli

The effect of cumulative vascular occlusions on PAP is plotted in (Fig. 2) for each embolus size, with emboli added sequentially until almost all acinar units were occluded (simulations are not conducted for 100% occlusion). The clinical definition for PH (mean PAP ≥25 mmHg) was not reached until approximately 55% of the vascular tissue was occluded. Both clinical[30] and animal studies[31] have shown that a mean PAP of approximately 40 mmHg is the maximum pressure that an initially non-hypertrophied right ventricle can sustain acutely before progressing to right ventricular failure (RVF)— this pressure was not reached in the model until ~90% of the lung was occluded. These results were consistent for the different sized emboli in this study. Response to embolization in human and animal studies is typically heterogeneous. It has been shown that only 25-30%

Figure 1: Visualization of all potential occluded regions for emboli of (a) 10, (b) 7, or (c) 5 mm in radius. Images are oriented in the supine posture and show right (upper panel) and left (lower panel) lung side views. Color range indicates the probability of each occlusion (vessels distal to occlusion have the same color). The color spectrum ranges from most likely (red) to least likely (dark blue) occlusion (ranges: 0.31 to 0.19, 0.19 to 0.014, and 0.059 to 0.011 for 10, 7, and 5 mm emboli). Non-occluded, central vessels shown in black. Flow solution is obtained in the supine posture at FRC.

Figure 2: The effect of increasing the amount of vascular obstruction on mean PAP (mmHg) shown for 5, 7, and 10 mm emboli. Pulmonary hypertension (PH) is defined clinically as PAP >25 mmHg,[4] and right ventricular failure (RVF) is considered likely at pressures >40 mmHg[30,31].

Table 1: Mean hemodynamic outcomes after a single occlusion from the range of occlusion locations

with emboli of 10, 7, and 5 mm in radius PE radius (mm) Baseline 10 7 5

No. occlusion locations

% of total acini occluded

PAP (mmHg)

PVR (mmHg/l/min.)

Flow gradient (%/cm height)

N/A 4 12 35

0 25.0±4.4 8.3±4.4 2.9±1.8

18.2 20.1 (10.4)±0.4 18.8 (3.3)±0.4 18.4 (1.1)±0.09

2.6 3.0 (15.4)± 0.08 2.8 (7.7)±0.08 2.7 (3.8)±0.02

-8.56 -8.32±0.04 -8.48±0.06 -8.53±0.02

Results are the number of potential occlusion locations, the % of total acini occluded, PAP and PVR (shown in both absolute values and % increase from baseline) across the full circuit after occlusion. Values are mean across the number of potential occlusion locations±SD. Baseline data are also provided for comparison

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total vascular occlusion can lead to hypertension, with hypoxemia, embolus location, and post-embolus release of vasoactive mediators each playing a role in the heterogeneity of response.[7,30,32] Inert occlusion studies have demonstrated that more than half of the lung (~6070%) may be occluded before PH is reached.[6,8,33] The model is representative of an inert (nonhematogenous) obstruction—given that no vasoconstriction is included in the model—and, given the slightly higher than average baseline PAP, predicts a level of occlusion very similar to these measurements.

Contribution of capillary recruitment, and capillary pressures

Capillary sheet flow-rate, pressure, and recruitment (Fig. 3a-c respectively) are shown as a function of gravitationally-dependent height for cumulative levels of occlusion with 10 mm emboli (other embolus sizes gave similar results, so are not shown). Values are averaged within 10 mm iso-gravitational slices. Each hemodynamic measure increased as the proportion of occluded tissue

increased, because the same cardiac output (constant QRV) was accommodated through an effectively smaller vascular circuit. Mean overall capillary recruitment increased from 70% at baseline to 73.2, 77.2, and 89% recruited after 25.2, 48.7, and 80.5% of the tissue was occluded. Note that for the 80.5% occlusion, the model was only perfused in the right upper lobe hence the solution does not extend to the full gravitationally dependent height (maximum height ~92.5% of the total dorso-ventral height). Experimental studies have shown that the endothelium begins to break at transmural pressures of approximately 24 mmHg (24-40 mmHg for as little as 4 min. leads to cell rupture)[34] and the level of damage is increased at higher lung volumes.[35] The model predicts that once perfusion is occluded to ~20% of the tissue (without additional vasoconstriction) capillary pressures begin to reach a high enough level to cause this destruction which would result in fluid filtration and alveolar edema. However, it is not until perfusion is occluded to ~80% of the tissue that

Figure 3: Micro-circulatory results at different levels of occlusion (0%, 25.2%, 48.7%, and 80.5%) with emboli 10 mm in radius. (a) Capillary flow rate (ml/ min.), (b) capillary pressure (mmHg), (c) % capillary sheet recruited. Each plot displays mean values in 10 mm isogravitational slices plotted with respect to gravitationally-dependent lung height (dorsoventral axis, %). In (b) the pressure (~24 mmHg) when the blood gas barrier may start to become compromised[35] is included. (d) RBC transit time distribution for each occlusion level. All occluded capillary ‘sheets’ were removed from this analysis. Pulmonary Circulation | July-September 2011 | Vol 1 | No 3

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a significant proportion of capillaries reach this limit (as illustrated in Fig. 3b).

Relationship between PAP and cardiac output, pre- and post-occlusion

The relationship between PAP and cardiac output (flow) is shown in (Fig. 4) at baseline and for two levels of occlusion (~48% and ~80%) using emboli of 7 mm in radius. For the 48% occlusion the PAP remained below the PH level up to a QRV of ~8 l/min. When vascular obstruction was ~80% the PH level was reached at ~3 l/min. A slightly curvilinear relationship between PAP and Q is predicted due to vessel distension (limited by the distensibility parameter α, see Multiscale Model Equation A1) and the potential for increased capillary surface area via recruitment (R, see Equation A6 in the Multiscale Model) under increased pressure/flow.

Redistribution of blood flow within nonoccluded regions Blood flow was found to redistribute preferentially to non-dependent lung regions following occlusion. (Fig. 5a) shows the ratio of post-occlusion to baseline flow (Qoccluded/Qbaseline) in the full lung model, for a single embolus occlusion of radius 10 mm. (Fig. 5c) shows the same ratio within acini (averaged within 10 mm slices) as a function of gravitationally-dependent height for each of the four possible occlusion sites for 10 mm embolus. For all occlusions, regardless of the location (and size, data not shown), the flow was preferentially redistributed to the gravitationally non-dependent region of the lung via increased capillary recruitment (Fig. 5d). The gravitationally non-dependent tissue is the region where

Figure 4: Relationship between flow into the pulmonary circuit and resultant mean pulmonary arterial pressure (PAP) at baseline (0% obstruction) and two levels of vascular obstruction (~49% and ~80% vascular obstruction using emboli of size 7 mm in radius). At the standard flow used in this study (5 l/min.) PH (PAP >25 mmHg) is not present with ~48% of vascular bed occluded, but is present when flow exceeds ~3 l/min. with 80% vascular obstruction. 370

there is the most potential for increased flow through the capillary bed (as has previously been shown in an isolated acinar model[16]) due to low baseline recruitment (Fig. 3c). The relationship used to determine capillary recruitment as a function of capillary pressure is illustrated in (Fig. 5b) (from Equation A6 in the Multiscale Model and Equation 6 in Clark et al.[13]) The relationship was derived from the experimental measurements of Godbey et al.,[36] who measured the percentage of capillary pathways perfused in five dog lungs, with intact thorax, perfused over a range of capillary pressures. (Fig 5b) illustrates that in lower pressure regions (such as in the non-dependent lung) the same increase in pressure results in a proportionately larger increase in capillary recruitment than in higher pressures (gravitationally dependent) regions. This gravitationally influenced redistribution of flow has implications on V/Q matching and gas exchange after occlusion (discussed below).

Oxygen transfer

A common clinical outcome of APE is some degree of hypoxemia and most (>90%) of APE patients present with a higher than normal alveolar-arterial oxygen partial pressure difference (P(A-a)O2).[1] (Fig. 6a) shows the reduction in arterial (blood leaving the lungs) oxygen partial pressures (PaO2) with increasing occlusion. Only the 7 mm radius emboli are shown, as similar results were found for 5 and 10 mm emboli. These results are presented for three different values of PvO2 (40, 30, and 20 mmHg); PvO2 has been found to range between 21-35 mmHg in APE[37] compared to 40 mmHg in normals, suggesting that to maintain sufficient oxygen transfer in the systemic circulation PvO2 must drop from baseline levels. At the higher level of PvO2, PaO2 in the model did not fall below the normal range (reported to be between 80-100 mmHg) [38] until ~49% of the lung was occluded. However, when PvO2 was dropped to the lower level measured in APE (20 mmHg) PaO2 was reduced below normal when as little as 12% of the lung was occluded. This prediction is more aligned with measurements that have shown that PaO2 may be depressed with as little as 13% of lung tissue occluded. [7] The P(A-a)O 2 difference provides a measure of the efficiency of gas exchange and is found to be ~8- 12 mmHg in normal healthy subjects.[39] This difference is attributable partly to venous admixture of shunted blood and partly to V/Q mismatch, if diffusional limitation is assumed to be negligible. A normal shunt of 2% of cardiac output accounts for 4 mmHg of the difference,[40] this amount was added explicitly to the value of P(A-a)O2 calculated by the model in this study. P(A-a)O2 increased notably with embolization, with values exceeding the normal limit with as little as 10% tissue occlusion (Fig. 6b). It is known that APE can result in bimodal distributions of V/Q ratios[41,42] and so by inference must have an impact on Pulmonary Circulation | July-September 2011 | Vol 1 | No 3

Burrowes et al.: Pulmonary blood flow after occlusion

Figure 5: Flow redistribution after occlusion with 10 mm emboli. (a) Supine model with 1 occlusion (PE, 1), occluded vessels in black, color spectrum represents Qoccluded/Qbaseline (red=1.3, dark blue=1.0). (b) Plot of the capillary recruitment function (Eqn A6). (c) Acinar flow values (Qoccluded/Qbaseline) and (d) capillary recruitment post-occlusion normalized by baseline values (Roccluded/Rbaseline). (b-d) show all 4 potential occlusions. (c,d) Values averaged in 10 mm slices, plotted with respect to gravitationally dependent height (%). All occluded capillary ‘sheets’ removed from this analysis.

Figure 6: (a) The reduction in arterial (blood leaving the lung) oxygen partial pressures (PaO2) and (b) the difference between alveolar and arterial oxygen partial pressures (P(A-a)O2, mmHg). Both plotted with respect to increasing occlusion indicating three different levels of mixed venous oxygen partial pressure (PvO2=40, 30, or 20 mmHg) for emboli of 7 mm in radius. PaO2 reduced and P(A-a)O2 increased with increasing occlusion and decreasing PvO2.

oxygen partial pressure at the alveolar level. (Fig. 7) shows a transverse view through the supine lung model at FRC, for 40% vascular occlusion resulting from the insertion Pulmonary Circulation | July-September 2011 | Vol 1 | No 3

of multiple 7 mm emboli. Each acinus in the section is colored to show the distribution of (A) Q, (B) V/Q, and (C) PAO2 (=PcO2). Baseline distributions of PAO2 in the upright 371

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theoretically occur with relatively small levels of vascular occlusion.

DISCUSSION

This study used an anatomically-based, integrated functional model of blood flow through the full pulmonary circuit to investigate the redistribution of pulmonary blood flow, elevation of PAP, and micro-circulatory hemodynamics after occlusions of arteries of various size and location. The main outcomes from this study are: the prediction that blood flow preferentially redistributes to the (gravitationally) non-dependent regions after vessel occlusion; evidence and explanation that the effect of mechanical occlusion alone does not increase PAP to hypertensive levels without significant vascular occlusion; and evidence that significant (particularly localized) hypoxia could occur as a direct result of mechanical occlusion.

Physiological consistency of the model

Figure 7: Acinar values of (a) blood flow (Q, mm3/s), (b) V/Q matching, and (c) PAO2 (mmHg), shown in a transverse section (caudal view) through a supine lung model at FRC with 40% tissue occlusion by multiple 7 mm emboli. The color spectrum ranges are indicated to the right of each image. V/Q >1 in (b) and PAO2<80 mmHg in (c) are colored black to highlight regions of poor perfusion and regions where hypoxic pulmonary vasoconstriction could theoretically be occurring, respectively.

model (not shown) were consistent with estimates from the literature (~89 mmHg at the base and ~132 mmHg in the apex of the lung.[40]) The post-occlusion results in the supine model show a clear bimodal distribution of PAO2 (Fig. 7c), with a shift to high PAO2 in most of the left (occluded) lung, and a shift to low PAO2 in the right lung. PAO2 < 80 mmHg developed in the posterior of the right lung, with some heterogeneity in its distribution. These low values for PAO2 indicate regions where hypoxia could theoretically develop. The emergence of this distribution of PAO2 is explained by the V/Q distribution in (Fig. 7b): regions of V/Q > 1 develop high PAO2 due to flow occlusion (Fig. 7a) and therefore low O 2 uptake by the blood; redistribution of Q to the non-occluded tissue results in a lower than baseline V/Q, and the preferential gravitational redistribution establishes lowest V/Q in the dependent tissue such that the O2 uptake to the blood is enhanced, thereby reducing PAO2 to low levels. In the absence of any V redistribution to optimize the matching of postocclusion V and Q, it is clear that localized hypoxia could 372

Comparison of baseline model outputs with physiological measurements and a detailed sensitivity analysis of all model parameters has previously been conducted by Clark et al.[13] This prior study demonstrated that the model predicts physiologically consistent distributions of blood flow and PAP under “normal” conditions.

Oser et al.[29] observed that emboli were distributed anatomically, with 53% in the right lung (16% in the upper, 9% in the mid, and 25% in the lower lobe) and 47% in the left lung (14% in the upper and 26% in the lower lobe). Similar distributions were predicted in the current model, suggesting that emboli preferentially distribute in proportion to flow rates. In the human experimental studies the emboli could have lodged while the subject’s lung was in any position, whereas here we studied only the supine lung. However for both the upright and the supine lung the flow was preferentially distributed to the lower lobes, and this was also the region of greatest probability for embolus location in both the experiments and the model.

Relationship between proportion of vascular occlusion and PAP

Pulmonary hypertension triggered by PE is believed to primarily be a result of the combined effect of mechanical obstruction and exacerbation in the presence of underlying cardiopulmonary disease. Another factor that has been found to play an important role in the immediate response to embolus occlusion in animal studies is pulmonary vasoconstriction, initiated by neural reflexes or via the release of vasoactive mediators from platelets or plasma (for example, serotonin and thromboxane A2.[9,10,32,41]) Several studies have demonstrated that larger portions of Pulmonary Circulation | July-September 2011 | Vol 1 | No 3

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the lung need to be occluded by inert occlusions (such as balloon catheters) to induce the same rise in pulmonary arterial pressure seen during embolic occlusion,[6-8,33,43] which is indicative of vasoconstriction playing a role in elevating PVR. The extent and course of this constriction, however, does not appear to have been characterized in detail and is still a topic of debate. Vasoconstriction was neglected in this study; therefore the results are indicative of outcomes induced by “passive” occlusions such as balloon catheters or glass beads. Model results indicate that ~55% of the vascular tissue needs to be occluded before hypertensive pressure levels are reached—this is in agreement with inert occlusion studies that obstructed an entire lung before PH developed.[6,32] McIntyre and Sasahara [7,30] estimated the degree of obstruction in multiple patients with no prior cardiopulmonary disease and with variable levels of tissue obstruction using selective pulmonary angiography. The data showed a reasonably good correlation between mean PAP and obstruction level, however a fairly large amount of heterogeneity was evident across subjects. Mean PAP values in that study were, on average, slightly higher than the model for similar embolic obstructions. The clinical data indicates a rapid rise in PAP with occlusion size which may be a result of the additional effect of pulmonary vasoconstriction; the level of this response may vary across subjects which could partly explain the variability of values. Indeed the oxygen transfer model presented here suggests that localized hypoxia post-embolus is a potential contributor to increased PVRs.

Relationship between PAP and cardiac output, pre- and post-occlusion

Both experimental[41,42,44] and modeling[45] studies have assessed the effect of embolization on the relationship between PAP and flow. Each study has demonstrated an increase in the gradient of pressure against flow and the linearly interpolated pressure intercept following embolization. The current model predicts behavior that is consistent with these previous studies, showing that increasing the size of the occluded region results in an increase in both the gradient and pressure intercept (Fig. 4). The model did not predict a strictly linear relationship between PAP and Q, but instead displayed a slight curve as a result of vessel distension and recruitment during increased flow. This type of curved relationship has also been measured in murine lungs.[46]

Redistribution of blood flow within nonoccluded regions

Preferential redistribution of blood flow to the (gravitationally) non-dependent regions was predicted, regardless of embolus size or location. This was demonstrated via decreased gradients of blood flow (Table 1, column 6), and the ratio of post-occlusion flow Pulmonary Circulation | July-September 2011 | Vol 1 | No 3

to baseline flow with respect to gravitationally dependent height (Fig. 5a,c). This pattern of redistribution occurred because of the greater potential for capillary recruitment (Fig. 5b) in the non-dependent tissue. The additional flow was therefore distributed more evenly to the nonoccluded region than if it were distributed in proportion to the baseline flow.

Oxygen transfer

Measurements have shown that, under normal conditions, there is a gravitationally-dependent gradient of V/Q, both in animals[47] and in humans,[48] whereby V/Q decreases from (>1) in non-dependent to (<1) in dependent regions. This is because of the relatively steeper gradient of blood flow compared to air. This distribution results in a gradient of PAO2;[40] the model of oxygen transport employed here was consistent with these findings at baseline.

Assuming that ventilation distribution is unchanged postembolization[3,49] we can estimate the effect of emboli on V/Q and gas exchange from the predictions of blood flow redistribution in this study. In each case considered, embolization reduced the gas exchange capacity of the lung. In occluded regions high V/Q ratios predominate due to minimal blood flow distal to the occlusion. Where there is complete vascular occlusion these distal regions would have an infinite V/Q ratio and would contribute to physiological dead space. As little oxygen is taken up from air to blood in these regions, they develop higher than normal alveolar oxygen partial pressures (PAO2, Fig. 7c).

The blood flow that would otherwise have supplied the occluded region has to be redistributed to the non-occluded tissue (Fig. 7a). Figure 7c shows a significant reduction in PAO2 in the non-occluded regions due to increased oxygen uptake by the blood; the level of reduction (to < 80 mmHg) could lead to localized hypoxia, even when whole lung gas exchange is sufficient to maintain arterial blood gases. This is a particularly important consideration as hypoxic pulmonary vasoconstriction (HPV) in these regions has the potential to increase overall PVR, although it has been observed that the potential for HPV may be reduced in PE.[9] The model has also shown that it is possible for the arterial (pulmonary venous) oxygen level to reduce significantly post-embolus, before the development of hypertension.

Model limitations

We have followed the paradigm of using as simple a model as possible for our study; the model used in the current study is considerably more complex (in particular with the extra-acinar vessel representation) than previous models used to study PE, however this has been necessary to enable predictions about the regional variation in perfusion parameters both at the macro- and micro-scale 373

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level within the pulmonary vascular circuit that have not previously been possible. Steady-state, laminar blood flow is assumed: turbulent flow is unlikely to occur in any but the largest pulmonary blood vessels. Most other models of pulmonary perfusion also use this approximation, and it is considered sufficient for studies of PVR and perfusion distribution.[50] The model represents vessels as 1D ‘elements’ so it cannot account for alterations in 3D flow profiles arising from different geometries of emboli. This is most likely to have a localized effect, with minimal influence on the redistribution of blood flow. We maintained a constant flow rate into the pulmonary circuit. Cardiac output (CO) may, in reality, be reduced; however, a decrease in CO is unusual without at least a 50% obstruction.[7] If the flow was to reduce postocclusion the same distributions would be predicted but with a smaller magnitude than in the current study. Our oxygen transfer model assumes alveolar-capillary oxygen equilibration. This requires RBC transit times to be > 0.25 s, a condition that is met in most of the lung except during very severe (>80%) occlusion. At this level capillary blood pressures are such that vessel rupture and edema would occur, causing diffusional limitation. In this situation the model would underestimate the impact of embolus occlusion on gas exchange.

CONCLUSIONS

This structure-based computational modeling study provides evidence that passive obstruction down to the level of the sub-segmental pulmonary arteries in the human lung is insufficient to raise PAP to PH levels, until ~55% of the capillary bed is distal to occlusions. Elevation of PAP to a critical level for smaller levels of obstruction is therefore most likely due to vasoconstriction that is initiated by localized hypoxia or via vasoactive mediators. Redistribution of blood flow in the non-occluded regions of the lung model followed a gravitationally-preferential distribution, which could improve V/Q matching in some cases of minor occlusions, and hence be protective of gas exchange.

The Multiscale Model

The following model description summarizes the integrated model for the pulmonary circulation that was presented by Clark et al.[13]

Extra-acinar vessels Each extra-acinar blood vessel was defined using a one dimensional (1D) finite element positioned within the anatomical 3D lung geometry, represented by a centerline and a numeric value for its unstrained diameter (at zero transmural pressure, P tm). The diameters of the left pulmonary artery (14.80 mm) and right pulmonary vein 374

(12.97 mm) were assigned based on measurements by Huang et al.[51] All other arteries and veins down to the level of the acinus were assigned diameters based on a constant rate of decrease in diameter with decreasing vessel order (the Strahler diameter ratio RDS), where RDS was 1.53 in the arterial tree and 1.54 in the venous tree.[13] Intra-acinar vessels Each acinar circulatory ‘unit’ connects a single artery and a single vein. Within each unit the intra-acinar vessels were explicitly represented with nine symmetric bifurcations each of arteries and veins. These intra-acinar vessels are joined at each generation by a capillary bed that covers the alveoli present at that generation, forming a ‘ladder-like’ structure, described previously by Clark et al.[16]

Vessel radius as a function of pressure Radial deformation of the extra-capillary (extra- and intra-acinar) blood vessels is related to Ptm by a linear relationship D = α Ptm + 1 , (A1) D0

where D is the strained vessel diameter and α is a compliance constant (baseline model compliance, α = 1.50 × 10-4 Pa-1.[13]) In the larger blood vessels Ptm≈PbPe (where Pb is the average blood pressure across the length of the vessel and Pe is the elastic recoil pressure acting on the vessel – values are derived from a soft tissue mechanics model.[15,19]) In the smallest vessels (diameter <200 µm) the dominant pressure acting externally to the blood vessel is assumed to be alveolar pressure (Pa), so Ptm≈Pb-Pa (Pa=0 in this study). Capillary sheet thickness is defined using a relationship analogous to Eqn. A1.[16] Eqn. A1 is assumed valid for Ptm < 32 cmH2O, beyond which the vessel is maximally extended in the radial direction. The blood flow equations Blood flow in the extra-acinar vessels was described by the Poiseuille equation incorporating the effect of gravity acting on the blood in the direction of the vessel centerline. Thus flow in an artery or vein was described by

∆P =

128m L Q + rb gL cos Θ, (A2) p D4

where ΔP is the pressure drop along the vessel, µ is the viscosity of blood in the vessel (µ=3.36×10 -3 Pa.s), Q is the volumetric blood flow rate, ρb is the blood density in the vessel (ρb=1.05×10-6 kg.mm-3), g represents gravitational acceleration (9.81 m.s-2), L is the length of the vessel, and Θ is the angle that the vector along the centerline of Pulmonary Circulation | July-September 2011 | Vol 1 | No 3

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the blood vessel makes with the direction of gravity. The gravity vector is oriented either along the craniocaudal axis (upright) in this study. Gravity in the acinar arterioles and venules is neglected; therefore flow in the arterioles and venules was described by Poiseuille’s equation DP =

128m L Q . (A3) p D4

Finally, blood flow in a capillary sheet is described using the sheet flow model of Fung and Sobin[52]

SA H 3dPtm , (A4) mC flC2 ∫

where S is the proportion of alveolar surface area comprised of capillaries (S=0.86 (no units)), A is alveolar surface area (A=73 m 2 – at TLC), µC is the apparent viscosity of blood in the capillaries (µC =1.92×10-3 Pa.s), f is a numerical friction factor (f= 21.6 (no units)), lC is the average path length from an arteriole to a venule through the capillary network (lC= 1186×10-6 m – at TLC) and H is the height of the capillary sheet. In the above equations (A2-4) we solve for ΔP and Q. D (or H) is then updated based on the pressure values and the solution is iterated until convergence is reached.

Mean red blood cell (RBC) transit times (TT) through capillary sheets can also be calculated using the theory of Fung and Sobin[53] using the formula

TT =

mc flc2

2 ∫ H dPtm

. (A5)

The different forms of Equations A4-5 describing Q and TT through the alveolar sheet as a function of alveolar and blood pressures are provided in the Appendix of Clark et al.[16]

Capillary recruitment A model of capillary recruitment is incorporated into the sheet flow model. This model predicts the proportion of capillary bed perfused (R) as a function of capillary blood pressure (Pcap) as follows

2 2 R = 1 − Frec exp( − Pcap / σ rec ), (A6)

where the constants F rec=0.65 and σ rec=22.7 cmH 2O (2.23 kPa) were fitted to the raw data of.[54] Capillary surface area (A in Eqn. A4) was scaled by R in the flow and pressure calculations. Pulmonary Circulation | July-September 2011 | Vol 1 | No 3

ACKNOWLEDGMENTS The authors would like to acknowledge the valuable input of clinical background from Dr. Amy Marcinkowski, Dr. Margaret Wilsher, and Dr. David Milne.

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Pulmonary Circulation | July-September 2011 | Vol 1 | No 3

Research A r t i cl e

Pulmonary hemodynamic responses to inhaled NO in chronic heart failure depend on PDE5 G(-1142)T polymorphism Thibaud Damy1,3,4, Pierre-François Lesault2,3,4, Soulef Guendouz1,3, Saadia Eddahibi3, Ly Tu3, Elisabeth Marcos3, Aziz Guellich3, Jean-Luc Dubois-Randé1,3,4, Emmanuel Teiger2,4, Luc Hittinger1,3,4, and Serge Adnot2,3,4 3

1 Department of Cardiology, 2Department of physiology, all at AP-HP, Groupe Hospitalier Albert Chenevier-Henri Mondor, IMRB,INSERM, U 955 team 08, and 4Faculté de Médecine, Université Paris-Est Créteil ; UPEC, Créteil suburb of Paris, France

ABSTRACT To evaluate the vasoconstrictor component of PH in CHF by investigating the hemodynamic response to inhaled nitric oxide (iNO) and to determine whether this response was influenced by the phosphodiesterase 5 gene (PDE5) G(1142)T polymorphism. CHF patients underwent right heart catheterization at rest and after 20 ppm of iNO and plasma cGMP and PDE5 G(1142)T polymorphism determinations. Of the 72 included CHF patients (mean age, 53±1 years; mean left ventricular ejection fraction, 29±1%; and mean pulmonary artery pressure, 25.5±1.3 mmHg), 54% had ischemic heart disease. Proportions of patients with the TT, GT, and GG genotypes were 39%, 42% and 19% respectively. Baseline hemodynamic characteristics were not significantly different across PDE5 genotype groups, although pulmonary capillary wedge pressure (PCWP) tended to be lower in the TT group (P=0.09). Baseline plasma cGMP levels were significantly lower in the TT than in the GG and GT patients. With iNO, PVR diminished in TT (-33%) but not GG (-1.6%) or GT (0%) patients (P=0.002); and PCWP increased more in TT than in GT (P<0.05) or GG (P<0.003) patients. The PDE5 G(-1142) polymorphism is therefore a major contributor to the iNO-induced PVR decrease in CHF. Key Words: heart failure, nitric oxide, phosphodiesterase

INTRODUCTION Pulmonary hypertension (PH) is common and of adverse prognostic significance in patients with chronic heart failure (CHF).[1,2] PH in CHF patients is often associated with an increase in pulmonary vascular resistance (PVR), which may result from constriction and/or remodeling of the pulmonary vessels. Only those patients in whom the predominant mechanism is pulmonary vasoconstriction can be expected to benefit from vasodilator therapy after heart transplantation.[3-5] A possible mechanism of pulmonary vasoconstriction in CHF is diminished relaxation of the arteriolar smooth muscle. Inhalation of nitric oxide (NO) is now considered Address correspondence to:

Dr. Thibaud Damy Fédération de Cardiologie, Hôpital Henri Mondor, 51 Avenue Maréchal de Lattre de Tassigny, 94010 Créteil, France Phone: 0033149812253, Fax: 0033149812883. Email: thibaud.damy@hmn.aphp.fr Pulmonary Circulation | July-September 2011 | Vol 1 | No 3

useful for assessing the contribution of vasoconstriction to idiopathic PH or PH associated with an underlying disease. Inhaled NO is used to treat patients with PH associated with various conditions including congenital heart disease, valvular heart disease, respiratory failure after heart transplantation, and adult respiratory distress syndrome.[6] Exogenous NO diffuses rapidly through the tissues, increasing the NO exposure of smooth muscle cells in pulmonary resistance vessels and thereby producing a direct vasodilator effect. Similar to intravenous beta-1 agonist or phosphodiesterase 3 inhibitor administration, NO inhalation is often used to assess the PVR response to vasodilation in CHF patients considered for heart Access this article online Quick Response Code:

Website: www.pulmonarycirculation.org DOI: 10.4103/2045-8932.87303 How to cite this article: Damy T, Lesault P, Guendouz S, Eddahibi S, Tu L, Marcos E, et al. Pulmonary hemodynamic responses to inhaled NO in chronic heart failure depend on PDE5 G(-1142)T polymorphism. Pulm Circ 2011;1:377-82.

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transplantation. In CHF patients, the PVR decrease produced by NO inhalation is ascribable to an increase in left ventricular (LV) filling pressure, as opposed to a decrease in pulmonary artery pressure.[7] The mechanisms of this LV filling pressure increase are unknown. However, studies have shown inter-individual variations in the inhaled-NO response that were independent from the cause of PH.[7-11] NO signaling is mainly mediated by cyclic guanosine monophosphate (cGMP) accumulation within the cells as a consequence of increased guanylate cyclase activity. However, cGMP bioavailability is limited by the hydrolytic activity of phosphodiesterases (PDE), most notably PDE5. [12] A recently identified regulatory region of the PDE5 gene (GenBank accession n°AB001615) has two functionally relevant AP1 regulatory sequences,[13] of which one harbors a T/G polymorphism at position 1142.[14] We hypothesized that the PDE5 G(1142)T polymorphism affected inhaled-NO responses in CHF patients, indicating that it contributed to the inter-individual differences in basal pulmonary vasoconstriction across CHF patients.

We prospectively studied 72 consecutive CHF patients to evaluate potential associations linking the PDE5 G(1142)T polymorphism to the pulmonary hemodynamic characteristics and/or inhaled-NO response. We report here for the first time that the PDE5 G(-1142) polymorphism is associated with differences in cGMP levels in CHF patients and that the inhaled-NO response, used as a measure of pulmonary vasoconstriction, varies with the PDE5 genotype.

MATERIALS AND METHODS Inclusion and exclusion criteria

Inclusion criteria were an LV ejection fraction (LVEF) less than 35% and a history of symptomatic CHF. Exclusion criteria were acute heart failure, severe tricuspid regurgitation, severe obstructive lung disease, pulmonary embolism, and valvular disease.

The patients were selected among participants in a genetic study of the causes of PH. It was supported by a publicly funded French research agency (DRRC: Délégation Régionale à la Recherche Clinique, www.drrc.aphp.fr, registration number, P020902) and approved by the ethics committee of the Henri Mondor Teaching Hospital. Written informed consent was obtained from all patients before inclusion.

Hemodynamic measurements

Maximal oxygen consumption (VO2) was determined and echocardiography (Vivid 7, General Electric) performed 24 hrs. before or after right-sided cardiac catheterization. During echocardiography, left and right ventricular 378

functions were assessed as recommended by the American Society of Echocardiography[15] and tricuspid regurgitation was quantified using standard criteria.

All treatments were stopped 12 hrs. before right-sided cardiac catheterization. A Swan-Ganz catheter was introduced up to the pulmonary artery. Heart rate, right atrial pressure, mean pulmonary artery pressure (mPAP), cardiac output (Q) measured by thermodilution, and pulmonary capillary wedge pressure (PCWP) were measured while breathing air and after breathing 20 ppm of NO for 10 min. via a closed facemask. Pulmonary vascular resistance (PVR) was computed as follows: PVR = (mPAP - PCWP)/Q. After catheter removal, patients were monitored clinically for 4 hours.

Assay of cGMP

Venous blood was used for cGMP measurement in all patients. In 12 patients, cGMP was also measured after NO inhalation in blood collected at the distal pulmonary tip of the catheter. After immediate centrifugation at 4°C, the serum was separated and stored at -80°C for future use. A commercially available cGMP assay was used (Biovision, Calif., USA). The cGMP levels in the GG and GT groups were pooled and compared to those in the TT group.

Genetic analysis

Venous blood was collected during the initial evaluation. Genomic DNA was extracted from leukocytes using a DNA extraction kit (QIAamp DNA blood kit, QUIAGEN, Hilden, Germany). The published sequence of the PDE5A 5’-flanking region (AB001615) was analyzed and amplified by polymerase chain reaction (PCR, Ready-To-Go PCR kit, Amersham Pharmacia Biotech, Paris, France) in a total volume of 25 ml using 40 ng of DNA and 50 ng of each primer (upstream, 5’-TTGCTTTTCTTTGGTTGTGGCT-3’; and downstream, 5’GTGTCATCCTTGCTTTGTCTG-3’). Each of the 35 amplification cycles consisted of denaturation at 96°C for 50 s, annealing at 62°C for 50 sec, and elongation at 72°C for 1 min. (DNA Thermal Cycler, Perkin-Elmer/Cetus). An initial denaturation step was carried out at 97°C for 5 min. and a final elongation step at 72°C for 10 min. The PCR products were analyzed by gel electrophoresis using 2% Seakem LE agarose (BioWhittaker Molecular Applications, Rockland, Me., USA).

Statistical analysis

Normally distributed data were described as mean±SEM. Between-group comparisons of quantitative variables were done using ANOVA or the Student t test, as appropriate. Qualitative variables were compared using the χ2 test. Differences between baseline and inhaled-NO values were compared using repeated measures ANOVA with a post hoc analysis using Fisher’s probable least-significant difference test. P values smaller than 0.05 were considered significant. Statistical analyses were performed using Statview software (SAS Institute, Cary, NC, USA). Pulmonary Circulation | July-September 2011 | Vol 1 | No 3

Damy et al.: PDE5 polymorphism and NO pulmonary responses

RESULTS

Neither the clinical characteristics nor the plasma brain natriuretic peptide (BNP) levels differed significantly across the 3 genotype groups (Table 1). Plasma cGMP under basal conditions was significantly lower in the TT group than in the GG or GT group (Fig. 1), suggesting increased PDE5 activity in the TT group.

Study population, baseline characteristics

We studied 72 patients, whose baseline characteristics are listed in (Table 1). All patients were receiving optimal CHF medications. Hemodynamic parameter values on room air are listed in Table 2. Mean values of PCWP and PVR were elevated.

Baseline hemodynamic characteristics were not significantly different across the three genotype groups. A trend toward lower PCWP values was found in the TT group compared to the GT-GG group, despite the absence of significant differences in CHF severity or pulmonary artery pressure (Table 1). The transpulmonary gradient (TPG) normalized for mPAP was significantly higher in the TT group compared to the GG-GT group.

Genotype distribution and interaction with baseline characteristics

Of the 72 patients, 28 (38.9%) had the TT genotype, 30 (41.7%) the GT genotype, and 14 (19.4%) the GG genotype. The G(-1142) allele accounted for 40.3% of all alleles at position 1142.

Table 1: Baseline characteristics of the whole cohorte and divided by PDE5 G(1142)T polymorphism All patients N Genotype frequency (%) Clinical and echocardiographic characteristics Age (yrs) Male (%) NYHA functional class Ischemic heart disease (%) Systolic BP, mmHg Diastolic BP, mmHg Peak VO2, ml·kg-1·min-1 LVEF, % LVEDDind, mm·m-2 TAPSE, mm BNP level, pg·ml-1 cGMP, pmoles·ml-1 Baseline treatment Beta-blocker (%) ACE inhibitor (%) Angiotensin receptor antagonist (%) Aldosterone antagonist (%)

PDE5 genotypes GG

72 -

14 19

30 42

28 39

53±1 81 2.2±0.1 54 106±3 66±1 16±1 29±1 36±1 16±1 407±55 114±4

56±3 93 2.2±0.3 38 106±7 65±4 17±2 28±2 36±1 16±1 433±133 130±7*

53±2 80 2.3±0.2 65 105±5 67±3 16±1 28±2 37±1 16±1 473±95 116±5†,‡

51±2 75 2.2±0.2 49 107±3 66±2 16±1 29±2 35±1 16±1 362±74 103±5

95 80 22 70

100 90 10 64

88 72 29 68

100 83 20 76

NYHA: New York Heart Association; BP: blood pressure; LVEF: left ventricular ejection fraction; LVEDDind: left ventricular end-diastolic diameter indexed to body surface area; TAPSE: tricuspid annular plane systolic excursion; BNP: brain natriuretic peptide; cGMP: cyclic guanosine monophosphate; ACE: angiotensin-converting enzyme. *P=0.003 versus TT, †P<0.08 versus TT, ‡P<0.09 versus GG

Table 2: Hemodynamic characteristics at baseline and after NO inhalation depending on the PDE5 G(1142)T polymorphism Inhaled gas PDE5 (1102) genotype N sPAP, mmHg mPAP, mmHg PCWP, mmHg TPG, mmHg Cardiac output, L·min-1 PVR, wood Heart rate, bpm

Air

All

72 38.3±1.9 25.5±1.3 16.1±1.0 9.3±0.5 4.2±0.1 2.3±0.1 71±2

14 38.4±4.9 26.3±3.7 18.0±3.4 8.4±0.8 4.1±0.3 2.2±0.2 68±3

30 39.6±2.9 26.6±1.9 17.3±1.4 9.3±0.8 4.2±0.2 2.3±0.2 73±3

28 36.8±2.8 23.8±1.8 13.9±1.4 9.9±0.8 4.3±0.2 2.5±0.2 70±2

72 37.1±1.8 25.4±1.3 17.8±1.1*,† 7.6±0.5*,† 4.2±0.1 1.9±0.1*,† 71±2

14 37.4±5.0 25.6±3.5 17.1±3.0 8.5±0.8 4.2±0.4 2.1±0.1 69±3

30 38.6±2.9 27.5±2.2 19.1±1.7 8.4±0.8 4.2±0.2 2.1±0.3 72±3

28 35.3±2.6 23.0±1.7 16.8±1.6* 6.3±0.7* 4.6±0.3 1.5±0.1* 71±2

sPAP: systolic pulmonary artery pressure; mPAP: mean pulmonary artery pressure; PCWP: pulmonary capillary wedge pressure; TPG: transpulmonary gradient; PVR: pulmonary vascular resistance. *NO versus air, P<0.05, †P<0.05, ANOVA for repeated measurements

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Damy et al.: PDE5 polymorphism and NO pulmonary responses

Effect of inhaled NO on hemodynamic parameters according to PDE5 genotype NO was well tolerated, with no effects on heart rate, Q, or pulmonary artery pressure (Table 2). NO inhalation significantly decreased PVR (Table 2) by increasing PCWP and the TPG without decreasing mPAP.

The hemodynamic effect of NO inhalation was greatest in the TT group, where the PCWP increase was largest 0.0026

0.07

DISCUSSION

The main finding from this study is that the PDE5 G(- 1142) polymorphism influences plasma cGMP levels and hemodynamic parameter values under basal conditions and after NO inhalation in patients with CHF.

Baseline characteristics

150

The G(-1142) allele frequency and PDE5 genotype distribution were consistent with a previous report.[14] The only baseline feature that differed significantly across the three genotype groups (TT, GT, and GG) was plasma cGMP concentration, which was lower in the TT

100 50

0.002

GT (n=30)

GG (n=14)

TT (n=27)

PDE5 G(-1142)T genotype Figure 1: Plasma cGMP concentration depending on PDE5 G(1142)T polymorphism. % mPAP (NO-Air)

%PCWP (NO-Air) 0.003 ,4

3 Pulmonary Vascular Resistance (wood)

cGMP (pmoles/ml)

200

(Fig. 2). In TT, the PVR decrease was significant (Fig. 3) and was larger in the group with PH (Fig. 4). After NO inhalation, cGMP increased significantly in the TT and GT groups but not in GG (Fig. 5). Plasma cGMP elevation was not correlated with the PVR decrease.

PDE5G(-1142)T genotype

2,5

GG GT

*, †

1,5

1 0

-,2

Air

% Transpulmonary Gradient (NO-Air) ,4 ,2

Pulmonary Vascular Resistance (wood)

mPAP<25 mmHg

-,2 -,4

-,2

* 0.0012

PDE5G( -1142)T genotype GG

Figure 2: Percent of change between baseline and after inhaled NO in transpulmonary gradient, mean pulmonary artery pressure, pulmonary capillary wedge pressure and cardiac output. 380

Figure 3: Change between baseline and after inhaled NO in pulmonary vascular resistance.

mPAP≥25 mmHg

3,5

0.009

2,5

1,5

0.001

Air

PDE5G(-1142)T genotype GG-GT

Figure 4: Change between baseline and after inhaled NO in pulmonary vascular resistance depending on mean pulmonary artery pressure subgroup. Pulmonary Circulation | July-September 2011 | Vol 1 | No 3

Damy et al.: PDE5 polymorphism and NO pulmonary responses

hearts.[18] Nevertheless, increased PDE5 expression has been found in myocardial specimens from patients with ventricular hypertrophy.[19]

0.038

200

cGMP (pmoles/ml)

160

Effect of inhaled NO on pulmonary artery

0.047 120 80 40 0

Air GG (n=3)

Air

GT (n=5)

Air

TT (n=4)

Figure 5: Plasma cGMP concentration depending on PDE5 G(1142)T polymorphism at baseline and after inhaled NO.

group (Fig. 1), suggesting a functional effect of the PDE5 polymorphism, with the TT genotype being associated with greater PDE5 activity and higher clearance of cGMP. In the TT group, a trend toward a lower filling pressure was noted (P=0.09). A recent in vitro study suggests that different PDE5 conformations involving the H-loop may affect the interaction of PDE5A1 with cGMP. [16] Furthermore, the H-loop variant may have a less critical interaction with PDE5 inhibitors,[16] However, plasma cGMP levels depend not only on cGMP clearance, but also on cGMP synthesis, which involves endogenous NO production (soluble guanylate cyclase) and atrial natriuretic peptides (particulate guanylate cyclase). However, in our study plasma BNP levels were not significantly different across genotype groups (Table 1), suggesting that the lower cGMP level in the TT group may have been due to greater PDE5 activity. PDE5 activity in pulmonary arteries, and therefore plasma cGMP levels, may affect pulmonary vascular tone. Conceivably, the higher cGMP levels in the GG and GT groups may explain the trend toward an increase in baseline PCWP in these genotype groups. Thus, the higher cGMP levels (due to diminished PDE5 activity) in the GG and GT groups may indicate greater pulmonary vasodilation with increased venous blood return to the left cardiac chambers and, therefore, increased basal PCWP in CHF patients. The TT group, in contrast, may have a higher basal pulmonary vessel tone associated with lower capillary pressures. No significant differences were found across genotypes for baseline LV function (LVEF) or right ventricular function (TAPSE) (Table 1), suggesting that PDE5 activity did not affect ventricular function under basal conditions. Similarly, earlier experimental studies found no effect of PDE5 inhibition on basal contractility of human papillary muscle strips[17] or failing canine Pulmonary Circulation | July-September 2011 | Vol 1 | No 3

Our study agrees with a previous report that inhaled NO significantly diminished PVR and the TPG in patients with CHF, by increasing PCWP.[7,20] A possible explanation is that NO inhalation induced pulmonary dilatation with shifts of blood volume from the pulmonary arterial to the pulmonary venous compartment. LV failure causes an increase in pulmonary venous volume and, therefore, in LV filling pressure.[21,22] Here, we found that the PVR decrease in response to inhaled NO response was dependent on the PDE5 G(-1142) polymorphism. The TT genotype was associated with a greater PVR decrease due to larger increase in PCWP and decrease in TPG. NO inhalation increased plasma cGMP concentrations in all genotype groups. However, the effects of NO inhalation depend also on the severity of the hemodynamic abnormalities.[7] Loh et al. showed that the response to inhaled NO was greater in patients with decompensated CHF than in patients with compensated CHF. We included only patients with compensated CHF. Among them, those having mPAP values greater than 25 mmHg exhibited a stronger response to inhaled NO. However, in the TT-genotype patients with mPAP values lower than 25 mmHg, the PVR response remained significantly greater than in the other two genotype groups (Fig. 3). Conceivably, changes in PDE5 activity related to the PDE5 G(1142) polymorphism may affect the regulation of pulmonary artery vasodilation at baseline and after NO inhalation. The TT genotype may be associated with greater PDE5 activity responsible for an increase in basal vascular tone and, therefore, for protection of the LV from excessive filling pressures.

We used a moderate NO dose (20 ppm). The use of higher dosages (40 or 80 ppm) would perhaps have eliminated the differences across genotypes. However, in earlier studies a moderate dose of inhaled-NO was as effective as a higher dose in decreasing PCWP in patients with congestive CHF.[23] Furthermore, most of the inhaled NO response was achieved with only 10 ppm in patients who had secondary or primary PH,[9] and inhaled NO was as effective as vasodilators selectively affecting the pulmonary vasculature.[24] Furthermore, in patients with stable severe CHF, high doses of NO have been reported to induce pulmonary edema.[25] In the 12 patients whose plasma cGMP levels were measured after NO inhalation, we found no correlation between the individual cGMP response to NO and the magnitude of the pulmonary vasorelaxation response, in keeping with a previous study.[8] 381

Damy et al.: PDE5 polymorphism and NO pulmonary responses

Limitations

This study has several limitations. First, the sample size is relatively small for a genetic study. However, our study sample is the largest included to date in an evaluation of the response to NO inhalation, and we studied the functional activity of a genetic polymorphism as opposed to its distribution frequency. Second, we cannot rule out an interaction between the medications taken by our patients and the NO response. In previous studies, the patients were not receiving beta-blockers. We discontinued betablocker therapy only 12 hrs. before right-sided cardiac catheterization to avoid undue risk to the patients. We found no significant effects of the PDE5 polymorphism on baseline hemodynamics, although there was a trend toward lower PCWP values in the TT group. Further studies are needed to evaluate the impact of the PDE5 polymorphism on the effect of PDE5 inhibitors. Recently, oral PDE5 inhibitors approved for the treatment of erectile dysfunction or primary PH were found to benefit patients with or CHF.[26,27] Some studies found that the hemodynamic response to PDE5 inhibitor administration was increased by concomitant NO inhalation.[20,28]

10.

11. 12. 13.

14.

15.

16. 17.

CONCLUSIONS

18.

Our data suggest that the PDE5 G(-1142) polymorphism is functional and may affect pulmonary artery tone at baseline, thus influencing the response to inhaled NO in patients with CHF. Further studies are necessary to determine the clinical and therapeutic implications of this polymorphism in CHF patients.

19.

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

Pulmonary Circulation | July-September 2011 | Vol 1 | No 3

Research A r t i cl e

Pharmacogenomics in pulmonary arterial hypertension: Toward a mechanistic, target-based approach to therapy Sami I. Said and Sayyed A. Hamidi Department of Medicine, State University of New York at Stony Brook, and Department of Veterans Affairs Medical Center, Northport, New York, USA

ABSTRACT Pharmacogenomics is the study of how genetic variations influence the response to drugs, by correlating gene expression with the drug’s efficacy and toxicity. This concept has recently been successfully applied in oncology. To test its applicability to PAH, we examined two experimental models of the disease: mice with deletion of the Vasoactive Intestinal Peptide gene (VIP- /-); and rats injected with monocrotaline (MCT). Since the two models express comparable phenotypic features, we analyzed their particular gene alterations, with special reference to genes related to pulmonary vasoconstriction, vascular remodeling, and inflammation. We then compared the phenotypic and genotypic responses in each model to treatment with the same drug, VIP. In untreated VIP-/- mice there was over-expression of almost all genes promoting vasoconstriction/ proliferation, as well as inflammation, and under-expression of all vasodilator/anti-proliferative genes. As expected, treatment with VIP fully corrected both the key PAH features, and all gene expression alterations. MCT-treated rats showed two distinct sets of alterations. One, similar to that in VIP- /- mice, i.e., tended to promote vascular remodeling and inflammation, e.g., up-regulation of myosin polypeptides, procollagen, and some inflammatory genes. The other was a set of opposite alterations that suggested an effort to modulate the PAH, e.g., up-regulation of the VIP and NOS3 genes. In this model, VIP treatment failed to correct many of the genotypic abnormalities, and, in parallel, incompletely corrected the phenotypic changes as well. This preliminary proof-of-concept study demonstrates the importance of genomic information in determining therapeutic outcome, and thus in selecting personalized therapy. Full validation of the merits of pharmacogenomics must await studies of lungs from patients with different forms of PAH. Key Words: gene analysis, pharmacogenomics, pulmonary arterial hypertension, targeted therapy of pulmonary arterial hypertension

INTRODUCTION What is pharmacogenomics?

Pharmacogenomics is the study of how genetic variations influence the response to drugs, through correlation of gene expression or single nucleotide polymorphisms (SNPs), with the drug’s efficacy and toxicity.[1,2] By probing the gene expression profile of individual disorders within a broad group of disorders, specific therapy can be targeted to that disorder, leading to greater efficacy and reduced toxicity. This approach has been described as leading to “the path to safer and more effective drugs”,[3] and “the path to personalized medicine”.[4] Address correspondence to:

Dr. Sami I. Said T -17-040, HSC Stony Brook University, Stony Brook, NY, USA 11794-8172. Email: sami.i.said@stonybrook.edu Pulmonary Circulation | July-September 2011 | Vol 1 | No 3

The promise of pharmacogenomics is fulfilled in oncology

Over the past decade, the concept of pharmacogenomics has been applied in the field of oncology, often with dramatic success. Notable results have been reported in the treatment of: breast cancer, in relation to ErbB2/ HER2 amplification; [2,5] lung cancer, in relation to EGFR mutations; [5,6] and colorectal cancer in relation to EGRF - targeted therapy.[5,7] Access this article online Quick Response Code:

Website: www.pulmonarycirculation.org DOI: 10.4103/2045-8932.87306 How to cite this article: Said SI, Hamidi SA. Pharmacogenomics in pulmonary arterial hypertension: Toward a mechanistic, target-based approach to therapy. Pulm Circ 2011;1:383-8.

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L i k e c a n c e r, PA H i s a h e t e r o g e n e o u s disorder—should it benefit from application of pharmacogenomics? As with most solid tumors, which may appear morphologically similar but have different and distinct genomic variations, PAH is a heterogeneous disorder, possibly with multiple different genotypes. It is already widely recognized that different clinical groups of PAH exhibit different degrees of responsiveness to the same therapeutic agents, with PAH associated with scleroderma and other inflammatory disorders having a generally poorer response than idiopathic PAH.[8] Such differences in therapeutic response probably reflect differences in underlying gene expression patterns, which, therefore, may benefit from targeted therapies based on unique genomic features.

Hypothesis

This preliminary study is designed as a first step in validating the hypothesis that PAH, much like cancer, is sufficiently diverse as to merit the application of pharmacogenomics, i.e., gene-targeted therapy based on the specific genetic background of each individual case. Such personalized therapy should lead to enhanced survival and minimized side effects.

MATERIALS AND METHODS Animals

Vasoactive Intestinal Peptide (VIP) knockout mice (VIP- /-), backcrossed to C57BL/6 mice,[9] were bred locally as described (10), and genotyped to confirm the absence of the VIP gene.[10] Sprague Dawley (SD) rats, 200-230 g, were from Taconic Labs (Germantown, NY). All experiments and animal care procedures were approved by the Institutional Animal Care and Use Committee and were conducted according to the National Institutes of Health Guide for the Care and Use of Laboratory Animals.

Chemicals and reagents Monocrotaline (MCT) was from Sigma-Aldrich (St. Louis, Mo.), and VIP was from Bachem Americas Inc. (Torrance, Calif.).

Study design: Validating pharmacogenomics in experimental models of PAH

As an initial effort to test the applicability of pharmacogenomics to PAH, we examined two experimental models of PAH: male VIP-/- mice, 10-12 weeks old; and male 6-8-week-old rats injected with MCT (60 mg/kg) 3 weeks earlier. These two models express comparable phenotypic features, with the MCT model showing generally more severe PAH.[10,11] 384

We analyzed and compared the genetic alterations in the lungs of animals from both models, with special reference to genes directly related to promoting or modulating of pulmonary vascular remodeling or inflammation, and to the extent to which these abnormalities were corrected in response to treatment with the same drug, VIP.

Experimental groups

The mouse groups were: 5 untreated VIP-/- mice; 5 VIP- /mice, treated with VIP (500 µg/kg, i.p., every other day for 3 weeks, for a total of 10 injections); and 5 normal wild-type mice. The rat groups were: 5 SD rats injected with MCT 3 weeks earlier, not otherwise treated; 5 SD rats injected with MCT, then treated with VIP, (500 µg/kg, i.p., every other day, for 3 weeks), beginning 3 weeks after MCT; and 5 normal SD rats, not injected with MCT or VIP.

Statistical analysis

Differences in quantitative data among the experimental groups were analyzed by ANOVA and Tukey post-hoc tests for multiple comparisons. Unpaired students t-test were used to analyze differences between two animal models. A P value of < 0.05 was considered significant.

Measurements

Both the mouse groups and the rat groups were compared with regard to: phenotypic features of PAH (RV systolic pressure, RV hypertrophy, pulmonary vascular remodeling, lung inflammation); gene expression alterations related to PAH; and the degree to which treatment with VIP corrected phenotypic and genotypic alterations. Hemodynamic measurements Animals were anesthetized with ketamine (100 mg/kg) and fentanyl (0.05 mg/kg IP). A 1.4F 3-cm Mikro-Tip catheter (Millar Instruments Inc, Houston, Tex.) was inserted through the right jugular vein and advanced to the right ventricle for digital recording of RV pressure.

Histological examination and morphometric analysis For all histological procedures, the lungs were inflated to full capacity and fixed by intratracheal instillation of 10% neutral buffered formalin, immersed in formalin overnight, and then embedded in paraffin. Sections (4 µm thick) were stained with hematoxylin and eosin or Masson’s trichrome stain for general morphology and morphometric analysis. Pulmonary arteries were analyzed; measurements were taken of 4 separate vessels from each slide and averaged to 1 set of values. Only arteries near smaller bronchi or terminal bronchioles, 50 µm in diameter, were selected for analysis. The Image J program, version 1.34r (http://rsb. info.nih.gov/ij/), was used for measurement of total vessel area (mm2), luminal area (mm2), and inner circumference (mm). Medial area (mm2) was calculated as the difference between total and luminal areas. Standard medial Pulmonary Circulation | July-September 2011 | Vol 1 | No 3

Said and Hamidi: Pharmacogenomics in PAH

thickness (mm) was calculated as the ratio of medial area to inner circumference, as described.[12]

Assessment of lung inflammation Lung sections were examined by a pathologist who was blinded to the identities of the samples. Inflammation was graded 0, 1, 2, 3, or 4, based on the intensity and extent of perivascular and peribronchiolar inflammatory cell infiltrates.

vasoconstrictor/proliferative genes, including myosin heavy and light polypeptides, procollagen peptides, Rho-related genes, angiopoetin, and platelet-derived growth factor receptor; down-regulation of vasodilator/ anti-proliferative genes, including adrenergic receptor ß, GTP cyclohydrolase 1, the rate-limiting enzyme in the biosynthesis of tetrahydrobiopterin (BH4), an essential co-factor for the activity of endothelial nitric oxide (NO) synthase (eNOS or NOS3), prostacyclin synthase, apoplipoprotein E, and adrenomedullin receptor; and up-regulation of inflammatory genes, including tumor necrosis factor, mast cell protease 8, chemokine (C-C motif) receptor 6, and NFATC2 (Table 2). Treatment of these mice with VIP corrected the key features of PAH, including the elevated pulmonary artery pressure, vascular remodeling, right ventricular hypertrophy, and lung inflammation. As well, the treatment reversed the genetic alterations toward normal values (Table 2).

RESULTS

DISCUSSION

Anatomic assessment of RV hypertrophy The heart was isolated and placed under a dissecting microscope. Attached vessels and both atria were dissected and removed. The RV wall was cut out, blotted, and weighed; then the left ventricular wall and septum (LV+septum) were treated the same way and weighed. The RV/(LV/septum) ratio was calculated as an index of RV hypertrophy.

Microarray and RT PCR analysis Lungs from mice and rats were removed from freshly euthanized animals, immersed in RNA later TM (10 ml per mg of tissue; Ambion, Austin, Tex., USA), fresh-frozen in liquid nitrogen and shipped overnight on dry ice to SA Biosciences (Qiagen, Fredrick, Md., USA). Microarray data were collected using the Whole Mouse Genome Oligo Microarray Kit with SurePrint technology (4644K slide format; Agilent Technologies, Palo Alto, Calif., USA). The microarray results were validated using quantitative real time PCR. RNA was examined from the same lungs subjected to microarray analysis. The procedures were carried out and the results were analyzed at Superarray Biosciences. Gene expression data of lungs from the mouse and rat groups were separately analyzed relative to untreated animals, according to the 2 –∆∆C T method.[13] Both models expressed the key phenotypic features of PAH, including RV systolic pressure, pulmonary vascular smooth muscle, right ventricular (RV) hypertrophy, and lung inflammation, with the MCT syndrome being generally more severe (Table 1), and uniformly fatal within 3-5 weeks. Lungs from VIP KO mice uniformly expressed a single set of gene alterations, comprising: up-regulation of

With respect to the MCT model, however, VIP treatment incompletely corrected the phenotypic changes: it significantly but only partially attenuated the pulmonary hypertension, vascular remodeling, and lung inflammation, but only slightly and insignificantly reduced RV hyperterophy. Following VIP treatment, genotypic analysis showed two distinct sets of expression alterations: one, similar to that in VIP -/- mice, i.e., alterations that promote vascular remodeling and inflammation (e.g., up-regulation of myosin polypeptides, procollagen, and of some inflammatory genes); and another set of alterations that suggested an effort to modulate the PAH. (e.g., up-regulation of VIP and NOS3). As with the phenotypic abnormalities, VIP treatment was only partially successful in reversing the genotypic abnormalities (Table 3). As noted above, the objective of this study was to provide preliminary evidence in support of applying the concept of pharmacogenomics in the treatment of PAH. We chose two experimental models of the disease that share major pathophysiologic and pathologic features, though to different degrees of severity, thus mimicking the groups of PAH patients with the same overall clinical diagnosis. The two models have sharply different survival rates,

Table 1: Phenotypic features in MCT rats and VIP-/- mice models of PAH

MCT VIP KO MCT vs. VIP KO (P value)

RV systolic pressure (mm Hg)

Perivascular inflammation (0-4)

Vascular thickening*

RV hypertrophy†

61.4±5.3 29.5±1.1 0.00019

3.00±0.26 2.17±0.24 NS

0.76±0.02 0.68±0.04 NS

0.56±0.03 0.34±0.01 0.00003

*Measured as medial area/total area in pulmonary arterioles 50-75 µM diameter. †Measured as RV/(LV+Septum) weight ratio

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Table 2: Gene expression alterations in VIP-/- mice relative to WT mice: Complete correction with VIP treatment Gene Vasoconstrictor, pro-remodeling genes myosin, heavy polypeptide 1 myosin, heavy polypeptide 8 myosin, light polypeptide 3 procollagen, type V, alpha 1 Rho, GDP dissociation inhibitor (GDI) β platelet derived growth factor receptor β Vasodilator, anti-remodeling genes vasoactive intestinal polypeptide adrenergic receptor, beta 2 apolipoprotein E bone morphogenetic protein receptor 2 nitric oxide synthase 3, endothelial cell GTP cyclohydrolase 1 prostaglandin I2 (prostacyclin) synthase vascular endothelial growth factor C Inflammatory genes nuclear factor of activated T cells chemokine (c-c motif) receptor 6 mast cell protease 8 tumor necrosis factor

Symbol

PCR array KO/WT

PCR array (KO+VIP)/KO

Myh1 Myh 8 Myl3 Col5a1 Arhgdib pdgfrb

↑ ↑ ↑ ↑ ↑ ↑

↓ ↓ ↓ ↓ ↓ ↓

Vip Adrb2 Apoe Bmpr II Nos3 Gch1 Ptgis Vegfc

↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓

↑ ↑ NS ↑ ↑ NS ↑ ↑

NFATc2 Ccr6 Mcpt8 Tnf

↑ ↑ ↑ ↓

NS ↓ NS ↓

Table 3: Gene expression alterations in MCT rats relative to normal rats: Partial correction with VIP treatment Gene Vasoconstrictor, pro-remodeling genes myosin, heavy polypeptide 1 myosin, heavy polypeptide 8 myosin, light polypeptide 3 procollagen, type V, alpha 1 Rho, GDP dissociation inhibitor (GDI) β platelet derived growth factor receptor β Vasodilator, anti-remodeling genes vasoactive intestinal polypeptide adrenergic receptor, beta 2 apolipoprotein E bone morphogenetic protein receptor 2 nitric oxide synthase 3, endothelial cell GTP cyclohydrolase 1 prostaglandin I2 (prostacyclin) synthase vascular endothelial growth factor C Inflammatory genes nuclear factor of activated T cells chemokine (c-c motif) receptor 6 mast cell protease 8 tumor necrosis factor

Symbol

PCR array MCT/Control

PCR array (MCT+VIP)/MCT

Myh1 Myh 8 Myl3 Col5a1 Arhgdib pdgfrb

↑ ↑ ↑ ↑ ↓ ↑

↓ NS ↓ ↓ ↓ ↓

Vip Adrb2 Apoe Bmpr II Nos3 Gch1 Ptgis Vegfc

↑ ↓ ↓ ↓ ↑ ↓ ↑

↓ NS ↓ NS NS NS NS NS

NFATc2 Ccr6 Mcpt8 Tnf

↑ ↓ ↑ ↑

↓ NS NS ↓

with VIP- /- mice surviving approximately 15 months[10] and MCT rats only 4-5 weeks, probably because: MCT is poisonous to several vital organs, including the lungs, liver, and kidneys; absence of the VIP gene results in two lung disorders (PAH, an asthma-like condition, possibly with altered function of other organ systems, but none rapidly fatal); and lack of the VIP gene is at least partially compensated for by the presence of two closely related VIP-like peptides (the Pituitary Adenylate Cyclase Activating Peptides, PACAP-27 and PACAP-38). 386

Our plan was: to analyze gene expression alterations in the two models, with special reference to genes related to pulmonary vasoconstriction, vascular remodeling, and inflammation; to compare the phenotypic responses of the two models to treatment with one and the same drug, VIP, which seems particularly suited to the genetic background of one model (VIP-/- mice), but not to the other (MCT rats); and to correlate the success of therapy in reversing phenotypic abnormalities with the success in correcting the corresponding genotypic alterations. Pulmonary Circulation | July-September 2011 | Vol 1 | No 3

Said and Hamidi: Pharmacogenomics in PAH

VIP therapy was more successful in one model than the other

Both animal models examined were treated with the same dose of VIP per weight, injected i.p. We did not measure serum level of VIP following VIP treatment. This decision was based on earlier experience with successful treatment of VIP-/- mice with VIP,[10] and the facts that VIP has a very short half life in serum or plasma, and its actions are mediated chiefly by binding to receptors in key cells and tissues, which would not be reflected in serum levels.

The fact that deletion of the VIP gene alone resulted in expression of the PAH phenotype, together with marked alterations in the expression of numerous genes that promote the development and aggravation of PAH, clearly identifies the underlying mechanism of pathogenesis of PAH in that model. The additional fact that the administration of VIP fully corrected both phenotypic and genotypic abnormalities is further proof of this conclusion. In this case, therefore, with the knowledge that PAH was strictly attributable to absence of the VIP gene, and the consequent gene expression alterations, it was predictable that VIP treatment would fully correct both phenotypic and genotypic abnormalities.

In the MCT rats, however, since the VIP gene was not under-expressed, it was evident that VIP deficiency was not the primary defect in the MCT model, and thus VIP treatment alone, unlike in the VIP-/- mice, could not be expected to correct either the genotypic or phenotypic abnormalities. The complex and mixed nature of the genotypic picture strongly indicates that effective therapy required multiple therapeutic approaches.

Relationship to gene expression alterations in human PAH

A number of the gene expression abnormalities noted in the VIP-/- mice or the MCT rats resemble those reported in human PAH. These include the following. 1. Reduced BMPR-II expression is a feature in common between both models and of the non-Familial forms of PAH.[14] 2. One consequence of reduced BMPR-II function is the increased expression of pro-inflammatory cytokines and inflammation.[14,15] An inflammatory response is a key feature of human PAH, as well as of the 2 models we examined.[16-18] 3. Numerous other examples exist of shared gene expression abnormalities between the human disease and either or both of the animal models. These include increased expression of vasoconstrictor, proremodeling genes, e.g., angiopoetin and decreased expression of apolipoprotein E, and of voltage-gated potassium channels.[17-22] Pulmonary Circulation | July-September 2011 | Vol 1 | No 3

4. The potential importance of VIP gene mutation in human PAH has not been adequately examined. Two clinical trials have been conducted with VIP in human subjects with PAH. In one trial, the peptide, given to 8 patients with IPAH by inhalation, for up to 24 weeks, resulted in significant improvement in hemodynamics and exercise tolerance.[23] In a more recent study, reported only in abstract form,[24] the results were described as negative. In neither of these trials was any information available on genomic data.

CONCLUSIONS

Despite its limited scope, this proof-of-concept study provides evidence supporting the applicability of the concept of pharmacogenomics in PAH. The two examples of correlating genomic analysis with drug response demonstrate the usefulness of genomic information in selecting appropriate, targeted therapy in PAH, and predicting therapeutic outcome. Complete validation of the importance of pharmacogenomics in human PAH must await similar studies in lung tissues from patients with different forms of PAH. Such tissues, likely from patients undergoing lung transplantation, are available from the Pulmonary Hypertension Breakthrough Initiative (PHBI), at the University of Michigan, Ann Arbor, Michigan, USA. A clinical trial to confirm the importance of pharmacogenomics in human PAH is already underway.[25]

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Evans WE, McLeod HL. Pharmacogenomics–drug disposition, drug targets, and side effects. N Engl J Med 2003;348:538-49. Oesterreich S. Pharmcogenomics in breast cancer treatment. Endocrine News 2010;35:20-2. Roses AD. Pharmacogenetics and drug development: the path to safer and more effective drugs. Nat Rev Genet 2004;5:645-56. Hamburg MA, Collins FS. The path to personalized medicine. N Engl J Med;363:301-4. Stuart D, Sellers WR. Linking somatic genetic alterations in cancer to therapeutics. Curr Opin Cell Biol 2009;21:304-10. Paez JG, Janne PA, Lee JC, Tracy S, Greulich H, Gabriel S, et al. EGFR mutations in lung cancer: correlation with clinical response to gefitinib therapy. Science 2004;304:1497-500. Chang DZ, Kumar V, Ma Y, Li K, Kopetz S. Individualized therapies in colorectal cancer: KRAS as a marker for response to EGFR-targeted therapy. J Hematol Oncol 2009;2:18. Humbert M, Sitbon O, Simonneau G. Treatment of pulmonary arterial hypertension. N Engl J Med 2004;351:1425-36. Colwell CS, Michel S, Itri J, Rodriguez W, Tam J, Lelievre V, et al. Disrupted circadian rhythms in VIP- and PHI-deficient mice. Am J Physiol Regul Integr Comp Physiol 2003;285:R939-49. Said SI, Hamidi SA, Dickman KG, Szema AM, Lyubsky S, Lin RZ, et al. Moderate pulmonary arterial hypertension in male mice lacking the vasoactive intestinal peptide gene. Circulation 2007;115:1260-8. Schermuly RT, Dony E, Ghofrani HA, Pullamsetti S, Savai R, Roth M, et al. Reversal of experimental pulmonary hypertension by PDGF inhibition. J Clin Invest 2005;115:2811-21. Weibel ER. Principles and methods for the morphometric study of the lung and other organs. Lab Invest 1963;12:131-55. Livak KJ, Schmittgen TD. Analysis of relative gene expression data using

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14. 15. 16. 17. 18. 19.

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real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 2001;25:402-8. Morrell NW. Genetics of pulmonary arterial hypertension: Do the molecular findings have translational value? F1000 Biol Rep 2010;2. pii:22. Hagen M, Fagan K, Steudel W, Carr M, Lane K, Rodman DM, et al. Interaction of interleukin-6 and the BMP pathway in pulmonary smooth muscle. Am J Physiol Lung Cell Mol Physiol 2007;292:L1473-9. Dorfmüller P, Perros F, Balabanian K, Humbert M. Inflammation in pulmonary arterial hypertension. Eur Respir J 2003;22:358-63. Geraci MW, Moore M, Gesell T, Yeager ME, Alger L, Golpon H, et al. Gene expression patterns in the lungs of patients with primary pulmonary hypertension: A gene microarray analysis. Circ Res 2001;88:555-62. Hamidi SA, Prabhakar S, Said SI. Enhancement of pulmonary vascular remodelling and inflammatory genes with VIP gene deletion. Eur Respir J 2008;31:135-9. Dewachter L, Adnot S, Fadel E, Humbert M, Maitre B, Barlier-Mur AM, et al. Angiopoietin/Tie2 pathway influences smooth muscle hyperplasia in idiopathic pulmonary hypertension. Am J Respir Crit Care Med 2006;174:1025-33. Du L, Sullivan CC, Chu D, Cho AJ, Kido M, Wolf PL, et al. Signaling molecules in nonfamilial pulmonary hypertension. N Engl J Med

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23. 24. 25.

2003;348:500-9. Morrell NW, Adnot S, Archer SL, Dupuis J, Jones PL, MacLean MR, et al. Cellular and molecular basis of pulmonary arterial hypertension. J Am Coll Cardiol 2009;54:S20-31. Thistlethwaite PA, Lee SH, Du LL, Wolf PL, Sullivan C, Pradhan S, et al. Human angiopoietin gene expression is a marker for severity of pulmonary hypertension in patients undergoing pulmonary thromboendarterectomy. J Thorac Cardiovasc Surg 2001;122:65-73. Petkov V, Mosgoeller W, Ziesche R, Raderer M, Stiebellehner L, Vonbank K, et al. Vasoactive intestinal peptide as a new drug for treatment of primary pulmonary hypertension. J Clin Invest 2003;111:1339-46. Galié N. Effects of inhaled aviptadil (Vasoactive Intestinal Peptide) in patients with pulmonary arterial hypertension (PAH). Am J Respir Crit Care Med 2010;181:A2516. Benza RL, Vido D, Gomberg-Maitland M, Rosenzweig EB, Frost A, Correa A, et al. Pharmacogenomics In Pulmonary Arterial Hypertension. Am J Respir Crit Care Med 2010;181:A4882.

Source of Support: NIH Grant HL-70212 and Department of Veterans Affairs (VA). Conflict of Interest: None declared.

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Research A r t i cl e

Idiopathic and heritable PAH perturb common molecular pathways, correlated with increased MSX1 expression Eric D. Austin3, Swapna Menon4, Anna R. Hemnes1, Linda R. Robinson1, Megha Talati1, Kelly L. Fox3, Joy D. Cogan2, Rizwan Hamid2,3, Lora K. Hedges2,3, Ivan Robbins1, Kirk Lane1, John H. Newman1, James E. Loyd1, and James West1 Departments of 1Medicine, 2Genetics, and 3Pediatrics, Vanderbilt University Medical Center, Nashville, Tennessee, USA, 4School of Computational and Integrative Sciences, Jawaharlal Nehru University, New Delhi, India

ABSTRACT The majority of pulmonary arterial hypertension (PAH) is not associated with BMPR2 mutation, and major risk factors for idiopathic PAH are not known. The objective of this study was to identify a gene expression signature for IPAH. To accomplish this, we used Affymetrix arrays to probe expression levels in 86 patient samples, including 22 healthy controls, 20 IPAH patients, 20 heritable PAH patients (HPAH), and 24 BMPR2 mutation carriers that were as yet unaffected (UMC). Culturing the patient cells removes the signatures of drug effects and inflammation which have made interpretation of results from freshly isolated lymphocytes problematic. We found that gene expression signatures from IPAH patients clustered either with HPAH patients or in a single distinct group. There were no groups of genes changed in IPAH that were not also changed in HPAH. HPAH, IPAH, and UMC had common changes in metabolism, actin dynamics, adhesion, cytokines, metabolism, channels, differentiation, and transcription factors. Common to IPAH and HPAH but not UMC were an upregulation of vesicle trafficking, oxidative/nitrosative stress, and cell cycle genes. The transcription factor MSX1, which is known to regulate BMP signaling, was the most upregulated gene (4×) in IPAH patients. These results suggest that IPAH cases have a shared molecular origin, which is closely related to, but distinct from, HPAH. HPAH and IPAH share the majority of altered signaling pathways, suggesting that treatments developed to target the molecular etiology of HPAH will also be effective against IPAH. Key Words: BMPR2, heritable pulmonary arterial hypertension, idiopathic pulmonary arterial hypertension, PPH

INTRODUCTION Pulmonary arterial hypertension (PAH) is a disease of progressively increasing pulmonary vascular resistance, which leads inexorably to failure of the right ventricle and death. Current therapies improve symptoms and function, but meta-analyses disagree over whether survival is improved. The problem with current therapies is a failure to address underlying molecular etiology.[1] The heritable form of disease (HPAH) is usually associated with mutation in BMPR2.[2] In 10 years of study, the molecular etiology of PAH secondary to BMPR2 mutation has become increasingly clear,[3-5] revealing a host of novel drug targets.[1] Address correspondence to:

Dr. James West Vanderbilt University Medical Center 1161 21st Ave S Suite T-1218 MCN 37232-2650 Nashville, TN, USA Email: j.west@Vanderbilt.Edu

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However, the majority of cases of PAH are not associated with BMPR2 mutation. While a few moderate risk factors for idiopathic PAH (IPAH) have been found through candidate gene approaches,[6,7] major genetic risk factors are still unknown. Because of relatively low patient numbers and a lack of a common genetic etiology, broad genetic approaches are unlikely to be successful, and have not been attempted. There have been several expression array experiments addressing molecular events in IPAH, recently reviewed,[8] using freshly isolated tissue from IPAH patients. These include 3 studies using lung tissue[9-11] Access this article online

Quick Response Code:

Website: www.pulmonarycirculation.org DOI: 10.4103/2045-8932.87308 How to cite this article: Austin ED, Menon S, Hemnes AR, Robinson LR, Talati M, Fox KL, et al. Idiopathic and heritable PAH perturb common molecular pathways, correlated with increased MSX1 expression. Pulm Circ 2011;1:389-98.

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and 3 using freshly isolated peripheral blood mononuclear cells (PBMCs).[12-14] A limitation of this approach is that, since they are taken directly from patients with endstage disease, the data is contaminated by signatures of drug effects and inflammation or other host response, effectively masking or confounding signal derived from the primary pathogenesis and early molecular events which may still be present.

While the ideal would be to study pulmonary vascular tissues obtained prior to the development of disease, this is not feasible in humans for many reasons. We have previously demonstrated success in overcoming some of the confounding influences by studies of cultured lymphocytes. Using this methodology to compare BMPR2 mutation carriers who developed PAH to those who did not develop clinical disease (unaffected mutation carriers, UMC), we discovered that decreased expression of the estrogen metabolism gene CYP1B1 in women correlated to disease penetrance.[15] Follow-up studies found that estrogen metabolites measured in patient urine also correlated closely with disease penetrance in HPAH.[16] This methodology thus has proven valuable in producing robust, clinically relevant results. The theory behind this success is that genetically based alterations in gene expression will be present in any tissue, not just those affected by disease, and culturing the cells removes them both from drug effects and inflammatory effects or other host responses seen in end stage disease.

The goal of the present study is to extend this successful approach to determine genetic risk factors for idiopathic PAH. To accomplish this, we used Affymetrix arrays to probe expression levels in 22 healthy controls, 20 idiopathic PAH patients (BMPR2 mutation excluded), 20 heritable PAH patients, and 24 BMPR2 mutation carriers without clinical disease (Unaffected Mutation Carriers, UMC).

MATERIALS AND METHODS Study population

The Vanderbilt Pulmonary Hypertension Research Cohort contains clinical and biologic specimens collected over 30 years, including detailed family pedigree and medical histories of patients with HPAH and IPAH. BMPR2 mutations have been detected in a large proportion of HPAH subjects tested to date, and several IPAH patients. The BMPR2 mutations vary in type, including nonsense mutations, insertion-deletion mutations that lead to splicing errors, frameshift mutations, and missense mutations.

The majority of patients were diagnosed and treated at Vanderbilt University Medical Center (VUMC). For those 390

patients not diagnosed and treated at VUMC, specialist physicians in their geographic regions identified HPAH patients, and our investigators reviewed all medical records for accuracy of diagnosis. PAH was defined either by autopsy results showing plexogenic pulmonary arteriopathy in the absence of alternative causes such as congenital heart disease, or by clinical and cardiac catheterization criteria. These criteria included a mean pulmonary arterial pressure of more than 25 mmHg with a pulmonary capillary wedge or left atrial pressure of less than 15 mmHg, and exclusion of other causes of pulmonary hypertension in accordance with accepted international standards of diagnostic criteria.[17] Clinical information concerning survival in terms of death or lung transplantation was up to date as of March 2011, the closing date for this study. Vanderbilt Pulmonary Hypertension Research Cohort study subjects were recruited via the Vanderbilt Pulmonary Hypertension Center, the Pulmonary Hypertension Association, and the NIH Clinical Trials website (http:// clinicaltrials.gov). The VUMC Institutional Review Board approved all study protocols. All participants gave informed written consent to participate in genetic and clinical studies and underwent genetic counseling in accordance with published guidelines.[18] Samples were obtained following informed consent at the time of hospitalization, clinic visits, or by mail via a kit for collection of whole blood. Ethylenediaminetetraacetic acid (EDTA) anticoagulated blood was collected from 86 individuals, including 20 idiopathic PAH patients, 22 healthy controls, 20 heritable PAH patients with BMPR2 mutation, and 24 BMPR2 mutation carriers who did not have evident PAH.

Genotyping and genetic analysis

We isolated genomic DNA using Puregene DNA Purification Kits (Gentra, Minneapolis, Minn.) according to the manufacturer’s protocol. BMPR2 gene mutation detection was performed by sequencing exons and exon intron boundaries of genomic DNA and by reverse transcriptase polymerase chain reaction (RT-PCR) analysis as described previously.[19,20] The BMPR2 mutations in this study have been previously reported, and are included in a recent summary of detectable BMPR2 mutations.[21]

Lymphocyte cultures

Lymphocyte cultures were performed as previously described. [15] Lymphocytes were isolated from anticoagulated whole blood within 48 hrs of collection and exposed to Epstein-Barr Virus (EBV) to induce cell immortalization. Two ml blood was diluted with 2 ml PBS, layered on top of 3 ml of Lympho Separation Medium (MP Biomedicals) and centrifuged for 10 minutes at 1,000×g at room temperature. Using a Pasteur pipet, the lymphocytes were removed from the serum/Lympho Pulmonary Circulation | July-September 2011 | Vol 1 | No 3

Austin et al.: Molecular etiology of IPAH

Sep Media interface, washed in 10 ml PBS and then resuspended in 3 ml lymphoblast media (RPMI 1640 media containing L-glutamine, and 20% fetal bovine serum) containing 2 mg/ml cyclosporine. The lymphocytes were then infected with 3 ml Epstein-Barr virus (EBV) and transferred to a T-25 vent capped flask. The cells were incubated at 37°C/5% CO2 and fed weekly with lymphoblast media + cyclosporine until signs of growth occurred.

reverse tetracycline transactivator. Other mice included either the TetO7-Bmpr2R899X or the TetO7-Bmpr2delx4+ transgenes,[5,27] which in combination with the Rosa26rtTA2 allow doxycycline-inducible expression of two different Bmpr2 mutations. All mice were treated with six weeks of doxycycline starting at 8 weeks of age, and thus sacrificed at 14 weeks of age. The Institutional Animal Care and Uses Committee at Vanderbilt University approved the animal studies.

RNA was isolated from lymphocytes using a Qiagen RNeasy mini kit (Valencia, Calif.). First and second strand complimentary DNA was synthesized using standard techniques. Biotin-labeled antisense complimentary RNA was produced by an in vitro transcription reaction. Human Genome U133 Plus 2.0 microarrays (Affymetrix, Foster City, Calif.) were hybridized with 20 µg cRNA. Target hybridization, washing, staining, and scanning probe arrays were done following an Affymetrix GeneChip Expression Analysis Manual. All array results have been submitted to the NCBI gene expression and hybridization array data repository (GEO, www.ncbi.nlm.nih.gov/geo/), as series (pending).

Statistics

Affymetrix arrays

Array analysis

The open source software, R2.13/Bioconductor2.8, was utilized for microarray analyses. Preprocessing of all cell files was carried out using the RMA algorithm, followed by duplicate probe removal to retain probes with higher IQR. The summarized data contained 19,701 features for each of the 59 arrays of HPAH, IPAH and control samples. Differential expression analysis was carried out using the standard moderated t-test procedure in package limma. The function decideTests with method=”global” was used to make statistical tests comparable across probes and contrasts. Genes with an average expression above 7 in the group showing higher expression and having P value above 0.05 were considered significant and selected for further analysis. Heirarchical clustering of both samples and genes was performed using algorithms within dChip,[22] according to established methods.[23] Rows were standardized by subtracting mean and dividing by standard deviation; correlation was used as the distance metric, using the centroid linkage method. Analysis of enriched gene function groups was performed using the 2010 release of Webgestalt,[24] using the hypergeometric test for enrichment of wither Gene Ontology consortium categories[25] or KEGG pathways.[26]

Western blot

Mouse lungs used were tissue archived in -80°C storage from prior experiments. Control mice had the Rosa26rtTA2 transgene, which drives universal expression of the Pulmonary Circulation | July-September 2011 | Vol 1 | No 3

Mouse lungs were homogenized in RIPA buffer (PBS, 1% Ipegal, 0.5% sodium deoxycholate, 0.1% SDS) with proteinase and phosphatase inhibitor cocktails (SigmaAldrich St. Louis, Mo.) immediately upon sacrifice. Protein concentration was determined by Bradford assay. Primary antibodies used for Western blot included MSX1 (Abcam ab73883) and Beta-Actin (Abcam ab8227). Statistical methods for array analysis are described above. Correlation z-tests were performed using the JMP program (SAS, Cary, NC).

RESULTS

Experimental design

The goal of this experiment was to identify a gene expression signature for idiopathic pulmonary arterial hypertension (IPAH), using cultured, unaffected tissues, which are free from confounding effects of end-stage disease or drug effects. To accomplish this, we used Affymetrix arrays to probe expression levels in lymphocyte cell lines created from 22 healthy controls, 20 idiopathic PAH patients (BMPR2 mutation excluded), 20 heritable PAH patients, and 24 BMPR2 mutation carriers without clinical disease (Unaffected Mutation Carriers, UMC). The overall experimental design is depicted in (Fig. 1), with patient population described in methods and in Table 1. Results of this analysis flow are presented below.

The same genes are altered in IPAH and HPAH, differing in magnitude

Comparing Affymetrix expression array data between 20 idiopathic PAH patients and 22 healthy controls, we found 168 genes upregulated and 118 genes downregulated, with P<0.05 (see Methods, above). Comparing Affymetrix expression array data between 20 heritable PAH patients and 22 healthy controls, we found 116 genes upregulated and 110 genes downregulated, with P<0.05 (see Methods, above). The significant lists only overlap by 46 genes up and 27 down between IPAH and HPAH, but this paints an 391

Austin et al.: Molecular etiology of IPAH

Table 1: Patient characteristics Group

Healthy controls IPAH HPAH UMC

Number Female (%)

Age at diagnostic Mean RAP, Mean PAP, Mean PCWP, Cardiac Indexed catheterization mmHg mmHg mmHg (S.D.) output, PVR U·m2 (Pts) or current age (S.D.) (S.D.) L·min (S.D.) (S.D.) (Controls), yrs (S.D.)

22 (100)

44.9 (23.6)

N/A

20 20 24

18 (90) 19 (95) 11 (46)

40.1 (18.0) 34.5 (16.7) 56.7 (17.0)

10.8 (7.0) 10.1 (6.1) N/A

56.3 (18.6) 62.4 (12.8) N/A

10.1 (3.7) 9.3 (4.0) N/A

3.4 (1.0) 3.2 (0.9) N/A

15.7 (7.8) 17.1 (4.5) N/A

Figure 2: Gene expression changes in IPAH and HPAH are closely correlated. Grey diamonds represent genes that are only significantly changed in IPAH, but which are changed to nearly the same degree in HPAH. Open circles represent genes which are only significantly changed in HPAH, but which are changed to nearly the same degree in IPAH. Crosses indicate genes significantly changed in both.

Figure 1: Experimental and analysis flow. The first three analysis steps, RMA preprocessing, Filter for high quality probes, and identification of differentially expressed genes, were performed within R version 2.13, Bioconductor version 2.8.

incomplete picture. In (Fig. 2), filled grey diamonds depict fold change in genes significantly changed in IPAH vs. controls compared to fold change in those same genes in HPAH vs. controls. Correlation is excellent (0.922, P<0.0001 by correlation z-test): the difference 392

is that while these genes are changed in both IPAH and HPAH, the magnitude of the average change in HPAH is half that in IPAH. The grey circles in (Fig. 2) are a plot of genes significantly changed in HPAH vs. controls compared to fold change in these same genes in IPAH vs. controls. Correlation is still very strong (0.88, P<0.0001 by correlation z-test). Once again, the difference is that the average magnitude of the change in IPAH is only a little over 40% of that in HPAH. There is no feature of the study design or analysis that would be expected to predispose to correlation between significant genes in IPAH and HPAH cohorts, let alone a correlation of this strength. This pattern of similar changes, but with relative magnitudes inverted between IPAH and HPAH, implies that the same pathways are being altered, perhaps with a different initiating event. Pulmonary Circulation | July-September 2011 | Vol 1 | No 3

Austin et al.: Molecular etiology of IPAH

There do not appear to be multiple molecular etiologies of IPAH We powered this study to detect 20% average changes in individual genes, under the assumption that there might be multiple molecular etiologies of IPAH, and so average fold changes would be reduced by the fact that any individual gene would be changed in only a subset of the sample. To determine whether there were multiple molecular etiologies, we used unsupervised clustering of the samples, without assigning the samples to groups. The 86 patient samples naturally clustered into four groups (Fig. 3), which roughly corresponded to the 4 known a priori classifications (HPAH, UMC, IPAH, and healthy controls). Of the IPAH patient samples (purple color in top bar): 14/20 clustered in a single group; 1 clustered with the healthy controls; and 5/20 clustered with the HPAH, potentially representing IPAH with BMPR2 mutation not detected by sequencing or MLPA.

There are three primary conclusions that can be tentatively drawn from this pattern. First, while most genes altered in lymphocytes cultured from IPAH patients correlate nearly perfectly with those drawn from HPAH patients (Fig. 2), they are for most patients not identical in specific pattern. This once again implies that the molecular etiology for most IPAH patients ultimately perturbs the same pathways as in BMPR2 mutants, but with a different initiating molecular event. Second, some IPAH patients, even with BMPR2 mutation excluded by sequencing, probably still have a problem with signaling through BMPR2, either through unrecognized BMPR2 mutations or through pathway elements not previously screened (e.g., intracellular or secreted inhibitors). Third, the expression signature of those patients that do not cluster with the HPAH group are molecularly relatively homogenous. Thus, there do not appear to be multiple common molecular etiologies of IPAH. Finally, it is informative that asymptomatic BMPR2 mutation carriers (UMC) primarily cluster with HPAH rather than with the healthy controls. This suggests, first, that the pattern of changes seen are not the consequence of end stage disease or drug effects, since they are also seen in UMC. Second, UMC are not very well separated from HPAH in this dendogram. It is our belief, based both on our BMPR2 mutant animal models[3,5] and on UMC exercise tests,[28] that many UMC may have silent pulmonary vascular disease as well, but under the threshold required to diagnose clinical disease (in fact, three samples collected as UMC developed clinical disease between collection and expression analyses, and so have been included as HPAH for these analyses using an intention to treat approach). Our previous Pulmonary Circulation | July-September 2011 | Vol 1 | No 3

Figure 3: Clustered heat map of all 439 genes significantly changed in IPAH or HPAH as compared to controls. Clustering of patient expression profiles is depicted by the dendogram at top (color coding according to legend at top left). Clustering of expression patterns of dysregulated genes is depicted by the dendogram at left, which can be broken into roughly five groups of expression patterns, labeled I through V. Higher than average expression is depicted as increasing red: lower than average expression is depicted increasing blue.

study using cultured lymphocytes was designed through patient selection to determine gene expression patterns that distinguished UMC from HPAH; [15] the present study was not, and so UMC and HPAH are only poorly separated.

Clustering of gene function suggest vesicle trafficking, oxidative/nitrosative stress, and proliferation/apoptosis as critical risk factors Unsupervised clustering was also performed on the 439 (402 named) genes significantly changed in either IPAH vs. control, HPAH vs. control, or both (Fig. 3). Genes that are clustered together have similar patterns of expression across the 86 patient samples. Genes clustered into five groups (labeled I through V). Group I consists of genes that have increased expression in HPAH, UMC, and IPAH compared to controls. Gene ontology groups abundant in this group include regulation of the actin cytoskeleton (P=0.013 by KEGG), macromolecule metabolic processes (P=0.009 by GO, primarily g-protein 393

Austin et al.: Molecular etiology of IPAH

and lipid metabolism), and transcription regulation (P=0.010 by GO) (Fig. 4, and examples in top row of Fig. 5).

Group II consists of genes that have increased expression in HPAH and IPAH compared to healthy controls, but no change in UMC. These genes may correspond to critical risk factors for the development of PAH. They consist of 136 named genes involved in organelle and vesicle localization (P=0.004 by GO), oxidative and nitrosative stress (P=0.003 by GO), and cell cycle (P=0.0004 by GO). (Fig. 4, examples in Fig. 5 second row).

Groups III and V are similar; they consist of 145 named genes that show decreased expression in HPAH, UMC, and HPAH compared to controls. These include cell adhesion molecules (P=0.00004 by KEGG), cytokines (P=0.00008 by GO), potassium or calcium channels, and differentiationrelated genes (Fig. 4, examples in Fig. 5 third row).

dysregulated in IPAH that is not also dysregulated in HPAH. On a molecular level, IPAH is a subset of HPAH.

It is important to note that the gene ontology groups which include the majority of the genes with altered regulation in IPAH are not new: they have all been extensively associated with BMPR2-related PAH in the past, and the connections between these groups are well established (Fig. 4). A central finding of this study is that most of the research that has been done on BMPR2-related heritable PAH is directly applicable to IPAH.

Group IV consists of 39 named genes downregulated in BMPR2 mutants, whether symptomatic or not, but unchanged in IPAH compared to healthy controls. It thus consists of genes altered by BMPR2 mutation, but probably not relevant to disease development (Fig. 4, examples in Fig. 5 bottom row). This group includes some endoplasmic-reticulum specific genes, which may reflect ER stress due to BMPR2 misfolding.[29] It also includes spliceosome-related genes (P=0.003, KEGG), many of which are markers of nonsense-mediated decay that may be affecting some BMPR2 mutations.[30] One of the most important groups, though, is the one that isn’t there: there does not exist a group of genes

Figure 4: Relationship of gene ontology groups dysregulated in IPAH and HPAH, color coded according to the samples in which they are dysregulated. Groups I and III/V are changed in IPAH, HPAH, and UMC. Group IV is dysregulated only in HPAH and UMC. Group II is dysregulated in IPAH and HPAH, but not in UMC, and probably represents pathways critical for disease development. Arrows indicate interaction between groups indicated in the literature, but are not intended as exhaustive. 394

Figure 5: Example genes dysregulated in each ontology group. For each graph, fold change compared to healthy controls is plotted on the y axis. Symbols used for IPAH, HPAH, and UMC are listed in the lower right, with error bars giving SEM. The expression pattern most strongly corresponding to each group of genes is given in the left column (I-V, see Figs. 3 and 4). Pulmonary Circulation | July-September 2011 | Vol 1 | No 3

Austin et al.: Molecular etiology of IPAH

MSX1 expression may drive other expression changes

Risk for IPAH is not caused by independently altered regulation of the 439 genes described above: rather, these alterations are all probably the consequence of alterations in a very small number of genes. MSX1 (muscle segment homeodomain-like homeobox 1) is a strong candidate as an upstream factor regulating much of the alteration seen in IPAH. It shows by far the strongest upregulation of any gene in IPAH, and as a transcription factor likely regulates the expression of many genes downstream. Roughly two thirds (276) of the 439 genes significantly (P<0.05 by correlation z-test) correlate with MSX1 expression, either positively or negatively. For instance, MSX1 has a correlation coefficient of 0.59 with the transcription factor Musculin (MSC) (Fig. 6a). This correlation is not a result of bias in selecting genes differentially regulated in disease: correlation between these two genes, considering only healthy controls, is a very high 0.57. Moreover, it is possible that many of the 163 genes not

directly correlated with MSX1 could still be regulated by it as a second order effect; for instance, as shown above, MSX1 expression correlates strongly with that of MSC, itself a transcription factor important in muscle development.[31] There are an additional 69 genes that significantly correlate with MSC expression that do not correlate with MSX1 expression. Thus, through a cascade of transcription factors, MSX1 could plausibly be responsible for the majority of the changes seen in IPAH. Note, however, that until it is defined by further experiments (e.g., a transgenic overexpression mouse), this remains only a correlation, not causation.

MSX1 targets may be altered in IPAH

Next, we considered whether the correlations between MSX1 and other genes altered in PAH were subgroup dependent. We found that the correlations between MSX1 and other genes were the same when those correlations considered only healthy controls as when those correlations considered only HPAH patients (Fig. 6b, correlation=0.83, P<0.0001 by correlation z-test). This

Figure 6: Transcription factor MSX1 alterations may be upstream of many other changes found. (a) MSX1 expression pattern correlates significantly with the pattern in most other genes; the transcription factor MSC is one example. Each point indicates the expression in a single patient of MSX1 (X axis) and MSC (y axis). (b) Strong MSX1 correlations with other altered gene expression patterns are not an artifact of how the genes were selected: correlations exist even within groups, not just between groups. Each point represents a gene, with correlation to MSX1 considering only healthy control data plotted on the x axis, and considering only HPAH patients plotted on the y axis. (c) MSX1 target genes may be changed in IPAH. Figure is similar to part B above, with the substitution of IPAH for HPAH on the y axis. Correlation between IPAH and healthy controls is much weaker (0.43) than correlation between HPAH and healthy controls (0.83). (d) F-actin regulator CFL1 expression (y axis) strongly correlates with MSX1 expression (x axis) in IPAH (filled circles) but not in HPAH (open circles). (e) MSX1 protein levels are increased roughly 3× in lungs from mice with a BMPR2 mutation inhibiting SMAD signaling (BMPR2Dx4+), but not in lungs from mice with a BMPR2 mutation in which SMAD signaling is intact (BMPR2R899X). Pulmonary Circulation | July-September 2011 | Vol 1 | No 3

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suggests that, while MSX1 is upregulated in HPAH as compared to controls, it is regulating transcription of the same genes in the same way as in healthy controls. However, when we make the same plot using IPAH rather than HPAH (Fig. 6c), the correlation becomes much weaker. This suggests that MSX1 targets may change, or change from repression to activation, in the context of IPAH. One example of this is the central F-actin regulatory gene, cofilin (CFL1), which is functionally (not transcriptionally) regulated directly by BMPR2. [32] CFL1 expression correlates with MSX1 expression with a strength of 0.84 (Fig. 6d, P<0.0001), but only in IPAH: the overall correlation is -0.03. This suggests that while CFL1 has altered regulation through phosphorylation in HPAH, it may be transcriptionally regulated in IPAH.

MSX1 expression in lung is suppressed by SMAD signaling through BMPR2 Previous studies had suggested that MSX1 expression was regulated by BMP signaling, and in particular that its expression was upregulated by SMAD8 activity.[33] This finding contrasts with the current study, in which, at least in lymphocytes, loss of BMPR2 correlated with increased expression of MSX1.

To directly test the hypothesis that loss of BMP signaling in the lung led to increased MSX1 expression, we used archived frozen lung tissue from our existing BMPR2 mutant mouse models. Western blots were performed using protein from lung tissue from Rosa26-rtTA2 × Bmpr2 R899X, Rosa26-rtTA2 × Bmpr2 Dx4+, and control Rosa26-rtTA2 only mice. These mice have universal doxycycline inducible expression of a mutation that leaves SMAD signaling intact, R899X, or a mutation that destroys SMAD signaling, DX4+.[15,27,34] We found that Msx1 protein levels were nearly 3x increased in lungs from the Rosa26-rtTA2 x Bmpr2 Dx4 mice, but slightly downregulated in lungs from the Rosa26-rtTA2 × Bmpr2R899X mice (which have slightly elevated SMAD signaling, probably compensatory) (Fig. 6e). Pulmonary microvascular endothelial cells cultured from these mice showed a similar pattern of MSX1 protein expression (not shown).

These results show that increased MSX1 is a consequence of loss of SMAD signaling through BMPR2 in mouse lung. The discrepancy between this and earlier reports may relate to different tissue types assayed, or because the prior study’s approach was indirect (they demonstrated SMAD binding to the MSX1 promoter, but never directly tested whether it was a repressor or an activator in a nonoverexpressed setting).[33] 396

DISCUSSION This study presents unique insights into the molecular pathogenesis of IPAH and how it relates to BMPR2 mutation, both with and without PAH. The central results are as follows. First, IPAH shares most altered molecular pathways with HPAH (Figs. 2-5). The exceptions to this are the pathways likely directly influenced by processing a mutant BMPR2, rather than the signaling consequences of mutation. Second, while much of the core molecular etiology is shared, global gene expression in most IPAH is molecularly distinct, both from HPAH and from healthy controls (Fig. 3). This indicates that risk for IPAH is at least partially genetic, but that it is usually not caused by cryptic problems with BMPR2. Third, at least in this sample, there seems to be only one IPAH molecular signature that is distinguishable from HPAH: suggesting there are not multiple common etiologies of adult onset IPAH (Fig. 3, dendogram at top). Finally, these points suggest that IPAH has a molecular origin very closely related to BMPR2 mutation, but is not BMPR2 mutation. One possibility is the transcription factor MSX1, which demonstrates the most prominent upregulation of any gene in IPAH patient samples. The set of pathways dysregulated in IPAH, as presented in (Fig. 4), form a summary of much of the current state of PAH research. Examining these in detail is thus a topic more appropriate for a review article than for this discussion. However the mechanism by which BMPR2 mutation leads to PAH can be summarized as follows. BMPR2 mutation results in defects in SMAD signaling through a kinase domain,[35] and actin dynamics through direct BMPR2 interactors TCTEX1 and LIMK1. [32,36] Decreased SMAD signaling results in alteration in gene transcription, including dedifferentiation of smooth muscle,[4] increased adhesion of inflammatory cells and decreased cell-cell adhesion in endothelial cells,[3,37] and alterations in cell cycle, driving some cells to proliferate and others to apoptose (both mechanisms are increased, probably in different cell types). Altered signaling through TCTEX1 and LIMK1 lead to defects in actin dynamics and cytoskeletal trafficking mechanisms, causing metabolic problems[34] and vesicle trafficking defects,[38] and more cell-cell adhesion defects, among other problems.[5] The combination of these defects leads to increased oxidative and nitrosative stress,[34,39] which cause additional injury in a feedback mechanism that may drive PAH. All of these pathways are also deranged, to a very similar degree, in idiopathic PAH, suggesting a very closely related genetic origin.

One possibility as an initiating risk factor for IPAH is increased expression of the transcription factor MSX1. The MSX1 promoter is directly bound[33] and repressed Pulmonary Circulation | July-September 2011 | Vol 1 | No 3

Austin et al.: Molecular etiology of IPAH

(Fig. 6) by SMAD transcription factors. MSX1 has roughly 20 known polymorphisms in the first kilobase of upstream promoter, but is also regulated by an antisense promoter, with the ratio of sense to antisense transcript determining protein level.[40] It thus has quite complex regulation. Functionally, BMPR2 mutations that decrease SMAD signaling result in increased MSX1 expression in lung (Fig. 6e). Moreover, MSX1 also regulates BMP expression; MSX1 knockouts have impaired BMP signal and blood vessel maturation.[41] Upregulated MSX1 has also been correlated with capillary regression.[42] Thus, MSX1 overexpression is a plausible candidate as a driver of IPAH based on the literature. To test it as a candidate, we plan studies both of MSX1 promoter polymorphisms in IPAH patients and effects of MSX1 overexpression in cell culture and mice.

There are several limitations to our study. First, interpretation of our study relies on the hypothesis that the gene expression differences we see are the functional outcome of genetic differences; that they are a cause of disease, not an effect of disease. This hypothesis is supported both by our previous success in using results of this methodology,[15,16] in the presence of many of the same changes in UMC, which do not have end-stage disease, and in the dissimilarity between these results and results from freshly isolated lymphocytes. Second, because lymphocytes are probably not a disease effector cell, there may be important pathways that can not be interrogated in this cell type. This does not invalidate the pathways discovered, but suggests that there may be additional pathways not able to be seen in this cell type. Third, our patient numbers, while substantially larger than in any previous array study, are sufficiently limited that we may not have seen etiologies associated with less common causes: genes with altered regulation in only one or two of our IPAH samples would not have been detected in our current study design. Further, good tests are not available to detect silent pulmonary vascular disease, so the patient categories can migrate: UMC develop clinical disease and become HPAH. Not all IPAH are clinically identical; for instance, one of the IPAH patients developed disease at age 3, but has survived for 30 years. One must imagine that this patient has protective factors, but once again, our study design cannot distinguish them from normal human variation. Finally, there is the question of scope. We have not yet tested functional consequences of any of these changes, although this is an important future direction.

state of the art in PAH research, indicating that while these pathways may seem separate, they must be part of an indispensable whole. As a practical consequence of these facts, treatments aimed at downstream molecular consequences of HPAH will also be effective against IPAH.

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helix-loop-helix transcription factor that antagonizes the actions of MyoD. Proc Natl Acad Sci U S A 1999;96:552-7. Foletta VC, Lim MA, Soosairajah J, Kelly AP, Stanley EG, Shannon M, et al. Direct signaling by the BMP type II receptor via the cytoskeletal regulator LIMK1. J Cell Biol 2003;162:1089-98. Binato R, Alvarez Martinez CE, Pizzatti L, Robert B, Abdelhay E. SMAD 8 binding to mice Msx1 basal promoter is required for transcriptional activation. Biochem J 2006;393:141-50. Lane K, Talati M, Austin E, Hemnes AR, Johnson JA, Fessel JP, et al. Oxidative injury is a common consequence of BMPR2 mutations. Pulmonary Circulation 2011;1:72-83. West J. Cross talk between Smad, MAPK, and actin in the etiology of pulmonary arterial hypertension. Adv Exp Med Biol 2010;661:265-78. Machado RD, Rudarakanchana N, Atkinson C, Flanagan JA, Harrison R, Morrell NW, et al. Functional interaction between BMPR-II and Tctex-1, a light chain of Dynein, is isoform-specific and disrupted by mutations underlying primary pulmonary hypertension. Hum Mol Genet 2003;12:3277-86. Burton VJ, Ciuclan LI, Holmes AM, Rodman DM, Walker C, Budd DC. Bone morphogenetic protein receptor-II regulates pulmonary artery endothelial cell barrier function. Blood 2011;117:333-41. Sehgal PB, Mukhopadhyay S. Dysfunctional intracellular trafficking in the pathobiology of pulmonary arterial hypertension. Am J Respir Cell Mol Biol 2007;37:31-7. Siddiqui MR, Komarova YA, Vogel SM, Gao X, Bonini MG, Rajasingh J, et al. Caveolin-1-eNOS signaling promotes p190RhoGAP-A nitration and endothelial permeability. J Cell Biol 2011;193:841-50. Babajko S, Meary F, Petit S, Fernandes I, Berdal A. Transcriptional Regulation of Msx1 Natural Antisense Transcript. Cells Tissues Organs 2011;194:151-5. Lopes M, Goupille O, Cloment CS, Lallemand Y, Cumano A, Robert B. Msx genes define a population of mural cell precursors required for head blood vessel maturation. Development 2011;138:3055-66. Kiyono M, Shibuya M. Bone morphogenetic protein 4 mediates apoptosis of capillary endothelial cells during rat pupillary membrane regression. Mol Cell Biol 2003;23:4627-36.

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

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Research A r t i cl e

S1P4 receptor mediates S1P-induced vasoconstriction in normotensive and hypertensive rat lungs Hiroki Ota1, Michelle A. Beutz2, Masako Ito1, Kohtaro Abe1, Masahiko Oka1, and Ivan F. McMurtry1 1

Departments of Pharmacology and Internal Medicine, and Center for Lung Biology, University of South Alabama College of Medicine, Mobile, Alabama, 2Cardiovascular Pulmonary Research Laboratory, University of Colorado and Health Sciences Center, Denver, Colorado, USA

ABSTRACT This study aimed to identify receptors mediating sphingosine-1-phosphate (S1P)-induced vasoconstriction in the normotensive and chronic hypoxia-induced hypertensive rat pulmonary circulation. In isolated perfused lungs from normoxic rats, infusion of S1P caused a sustained vasoconstriction, which was not reduced by combinational pretreatment with the dual S1P1 and 3 receptor antagonist VPC23019 and the S1P2 receptor antagonist JTE013. The S1P4 receptor agonists phytosphingosine-1-phospate and VPC23153, but not the dual S1P1 and 3 receptor agonist VPC24191, caused dose-dependent vasoconstrictions. In hypertensive lungs from chronically hypoxic rats, the vasoconstrictor responses to S1P and VPC23153 were markedly enhanced. The S1P4 receptor agonist VPC 23153 caused contraction of isolated pulmonary but not of renal or mesenteric arteries from chronically hypoxic rats. S1P4 receptor protein as well as mRNA were detected in both normotensive and hypertensive pulmonary arteries. In contrast to what has been reported in the systemic circulation and mouse lung, our findings raise the possibility that S1P4 receptor plays a significant role in S1P-induced vasoconstriction in the normotensive and hypertensive rat pulmonary circulation. Key Words: pulmonary arteries, pulmonary hypertension, S1P4 receptor, sphingosine-1-phosphate

INTRODUCTION Sphingosine-1-phosphate (S1P) is an active lipid mediator with regulatory roles in numerous physiological and pathological processes.[1-5] Extracellular signaling of S1P occurs through five known S1P receptors (S1P1-5) that couple to a variety of Ga proteins. While S1P is known to regulate systemic vascular tone via S1P1-3 receptors,[1,6-9] there has been minimal investigation of its effects on pulmonary vascular tone. [10] Two studies show that relatively high concentrations of S1P cause contraction of isolated rat[11] and porcine[12] pulmonary arteries. A recent study reports that S1P induces pulmonary vasoconstriction in mice, and the constriction is dependent on Rho kinase activation via the S1P2 receptor.[13] Nothing, however, has been reported about receptors mediating S1Pinduced pulmonary vasoconstriction in the hypertensive pulmonary circulation. Given the emerging importance of S1P in systemic vasoregulation under physiological and Address correspondence to:

Dr. Masahiko Oka Center for Lung Biology, University of South Alabama, 3158 Medical Sciences Building, Mobile, Alabama 36688-0002 USA Email: moka@usouthal.edu Pulmonary Circulation | July-September 2011 | Vol 1 | No 3

pathological conditions,[1-5] it is likely that S1P also plays a vasoregulatory role in the normal and hypertensive pulmonary circulation. A better understanding of the pulmonary vasoactive effects of S1P is important in view of the consideration of the phospholipid as therapy for acute lung injury.[14] The purpose of this study, therefore, was to investigate which S1P receptors are responsible for S1Pinduced vasoconstriction in the normotensive and chronic hypoxia-induced hypertensive rat pulmonary circulation.

MATERIALS AND METHODS Animals

All experimental animal procedures were approved by the Animal Care and Use Committee of the University of South Access this article online

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Website: www.pulmonarycirculation.org DOI: 10.4103/2045-8932.87309 How to cite this article: Ota H, Beutz MA, Ito M, Abe K, Oka M, McMurtry IF. S1P4 receptor mediates S1P-induced vasoconstriction in normotensive and hypertensive rat lungs. Pulm Circ 2011;1:399-404.

399

Ota et al.: S1P4 mediates S1P-induced pulmonary vasoconstriction

Alabama. Experiments were performed with two groups of male Sprague-Dawley rats (240- 400 g). The normoxic, pulmonary normotensive group was kept in room air. The chronically hypoxic, pulmonary hypertensive group was exposed to normobaric hypoxia (10% O2) for 3 to 4 weeks.

Right ventricular hypertrophy To demonstrate the presence of pulmonary hypertension in the chronically hypoxic rats, the hearts were dissected and an index of right ventricular hypertrophy was calculated as the ratio of wet weight of right ventricular wall to wet weight of left ventricular wall plus septum (RV/LV+S).

Isolated perfused lungs The techniques of lung isolation, ventilation, and constantflow perfusion with physiological salt solution (PSS) have been described previously.[15] Because preliminary experiments showed that bolus injections of S1P into the lung perfusate caused only transient vasoconstrictions (presumably due to rapid degradation within the pulmonary vascular bed), S1P (Enzo Life Sciences) was infused via the pulmonary artery catheter at a constant rate (8.4 nmol/ min) for 10 minutes with and without combinational pretreatment (added 15 min. prior to S1P infusion) of the dual S1P1 and 3 receptor antagonist VPC23019 (3 mM; Avanti) [16] and the S1P2 receptor antagonist JTE013 (1 mM; Tocris) [17] in normotensive (NL) and hypertensive lungs (HL). In a separate set of experiments, effects of bolus administrations of the dual S1P1 and 3 receptor agonist VPC24191 (1-30 mM; Avanti) and two different S1P4 receptor agonists, pytosphingosine-1-phosphate (0.013 mM; Avanti)[18] and VPC23153 (0.01-3 mM; Avanti)[19] were examined in NL. In addition, pressor responses to VPC23153 were compared between NL and HL. To test if the S1P- and S1P4-receptor agonist-induced pulmonary vasoconstrictions were mediated by similar intracellular signaling mechanisms, we examined the sequential effects of the Rho kinase inhibitor fasudil [15] (10 mM) and the Ca2+ channel blocker SKF93635 (50 mM) [20] on sustained, stable vasoconstrictions to continuous infusions of S1P and VPC23153 in both NL and HL.

Isolated arterial rings Pulmonary (extra-lobar first branches), renal (extrarenal first branches), and mesenteric arteries (~1 mm in diameter) were isolated from chronically hypoxic rats and placed on steel wires attached to a force transducer, and suspended in baths containing 10 ml PSS at 37oC. Resting passive force was adjusted to a previously determined optimal tension (1.5 g for hypertensive pulmonary and 1 g for renal and mesenteric arterial rings).[15,21] Rings were gassed with 21% O2-5% CO2-74% N2, and allowed to equilibrate for 60 min. VPC23153 (0.1-10 mM) was added cumulatively to the organ baths at 15-min. intervals. After 400

the final concentration, all rings were exposed to the nitric oxide synthase inhibitor Nwnitro-L-arginine (200 mM) to test for modulation of contraction by endogenous nitric oxide.

Immunohistochemical staining A standard technique was used[22] with an anti-S1P 4 receptor antibody (1:200; LIFESPAN Biosciences, LSB513) as a primary antibody.

RNA isolation and quantitative RT-PCR Intra-pulmonary arteries (200-400 mm in diameter) were isolated from NL and HL and snap frozen. Standard techniques were used for total RNA isolation and realtime PCR.[23] The sequences of primers were as follows: S1P4 f-primer: 5’-GGA AGG CCA TGA ACA TCA GT-3’, S1P4 r-primer: 5’-TGT AGT GCA GGA CGA TGA GC-3’, GAPDH f-primer: 5’-GGA AGG CCA TGA ACA TCA GT-3’, and GAPDH r-primer: 5’-GCT GGT GCT GAG TAT GTC GT-3’.

Statistical analysis

Values are reported as means±SE. Comparisons between groups were made with Student’s t-test or analysis of variance (ANOVA) with Fisher’s post-hoc test for multiple comparisons. Differences were considered significant at P<0.05.

RESULTS

RV hypertrophy

The presence of pulmonary hypertension in the chronically hypoxic rats was reflected in the RV/LV + S weight ratio, which averaged 0.49±0.01 (n=28) vs. 0.25±0.02 (n=46) in normoxic rats (P<0.05).

Vasoconstrictor effect of continuous infusion of S1P.

Continuous infusion of S1P at 8.4 nmol/min. for 10 minutes caused a sustained and progressive vasoconstriction in isolated PSS-perfused lungs, the magnitude of which was much greater in HL than in NL (Fig. 1a). These vasoconstrictions were not reduced by the combinational pretreatment with the dual S1P1 and 3 receptor antagonist VPC23019 and the S1P2 antagonist JTE013. Pretreatment of the perfused lungs with the nitric oxide synthase inhibitor Nwnitro-L-arginine (200 mM) markedly enhanced the sustained S1P-induced vasoconstrictions in both NL and HL (from 3.8±1.1 to 15.0±7.6 mmHg in NL and from 22.4±3.0 to 52.9±11.4 mmHg in HL, n=3 each), indicating the response was moderated by endogenous nitric oxide.

Effects of S1P receptor agonists

In PSS-perfused NL, bolus injections of two structurally different S1P 4 receptor agonists, VPC23153 and phytoshingosine-1-phosphate, caused dose-dependent and, Pulmonary Circulation | July-September 2011 | Vol 1 | No 3

Ota et al.: S1P4 mediates S1P-induced pulmonary vasoconstriction

9 HL

Pressor response (Δ mmHg)

40 *

* 20 10 0

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40 NL

0 -11 c

(3)

(5)

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100

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-3

100

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-7 -5 Concentration (logM)

0 Con (5)

VPC23153 (5)

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NL (3)

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100

% KCI (80 mM)

Pulmonary (4) Mesenteric (4) Renal (4)

60 40 20 0

-7

-6 -5 VPC23153 (log M)

L-NNA (200 μM)

Figure 1: (a) Effects of combinational pretreatment of VPC23019 (3 mM) and JTE013 (1 mM) or the vehicle (dimethyl sulfoxide, Con) on S1P infusion (8.4 nmol/min.)-induced sustained vasoconstriction in normotensive (NL) and hypertensive lungs (HL). *<0.05 vs. NL. (b) Concentration response curves for bolus injections of VPC23153 (closed circle), phytosphingosine-1-phosphate (PhS1P, open circle), and VPC23019 (closed triangle) in normotensive lungs. *<0.05 vs. VPC23153. (c) Concentration response curves for VPC23153 in normotensive (NL, closed circle) and hypertensive lungs (HL, open circle). *P<0.05 vs. NL. (d) Percent reversal by fasudil (black area) and SKF 96365 (gray area) of S1P infusion- (left panel) and VPC23153 infusion-induced vasoconstriction (right panel) in normotensive (NL) and hypertensive lungs (HL). (e) Concentration-response curves for VPC23153 and effects of Nwnitro-L-arginine (L-NNA) in pulmonary, mesenteric and renal arteries isolated from pulmonary hypertensive rats. Sample size is indicated in parenthesis.

as compared to bolus S1P, more sustained vasoconstrictions. In contrast, the S1P 1 and 3 agonist VPC24191 had no vasoconstrictor effect (Fig. 1b). Similar to the responses to S1P, the vasoconstrictor responsiveness to VPC23153 was markedly enhanced in HL as compared to NL (Fig. 1c). Pulmonary Circulation | July-September 2011 | Vol 1 | No 3

Effects of Rho kinase and Ca2+channel blockers on S1P and VPC23153 responses

Because of the increased vasoconstrictor responsiveness to both S1P and VPC23153 in HL, lower rates of infusion were required to elicit stable, sustained pressor 401

Ota et al.: S1P4 mediates S1P-induced pulmonary vasoconstriction

responses of ~5 mmHg in HL as compared to NL (S1P infusion = 0.6±0.2 nmol/min. in HL and 2.3±0.6 nmol/ min. in NL, and VPC23153 infusion = 0.4±0.0 nmol/min. in HL and 4.9±0.7 nmol/min. in NL). As shown in Figure 1d, the sustained vasoconstrictor responses to both S1P and the S1P4 receptor agonist in both NL and HL were rapidly reversed by 60 to 70% with the Rho kinase inhibitor fasudil (10 mM) and an additional 10 to 20% by the Ca2+ entry blocker SKF93635 (50 mM).

Effects of VPC23153 in isolated pulmonary, renal, and mesenteric arteries

The S1P 4 receptor agonist VPC23153 caused a concentration-dependent contraction in pulmonary but not in either renal or mesenteric arteries (Fig. 1e). NwnitroL-arginine markedly enhanced the S1P4 receptor agonistinduced contraction of pulmonary arteries, suggesting that the contraction was moderated by endogenously produced nitric oxide. The nitric oxide synthase inhibitor did not induce a contractile response to VPC23153 in the systemic arteries.

however, our results show that S1P-induced pulmonary vasoconstriction in either NL or HL was not reduced by the pharmacological blockers of S1P 1, 2, and 3 receptors. In addition, the dual S1P1 and 3 receptor agonist VPC24191 induced little increase in perfusion pressure. These results suggest that receptors other than S1P1, 2, and 3 are involved in the S1P-induced vasoconstriction in both the normotensive and hypertensive rat pulmonary circulation.

In contrast to widespread distribution of the S1P1, 2, and 3 receptors, expression of the S1P 4 and 5 receptors is reported to be relatively restricted.[2,24] S1P4 receptors are found predominantly in lymphoid and hematopoietic tissues, while S1P5 receptors are primarily expressed in the white matter of the central nervous system. S1P4 receptor mRNA has been detected in human and mouse lungs[25,26] and cultured human airway smooth muscle cells.[27] However, little is known about the role of S1P4 receptors in lungs, especially with respect to regulation of

Immunohistochemical analysis

S1P4 receptor protein was expressed in pulmonary arterial media as well as airway epithelial and smooth muscle cells of NL (n=3) (Fig. 2a and b). S1P4 receptor protein was also detected in the thickened pulmonary arterial media of HL (n=4), but the intensity of the staining was not clearly greater than that in NL (Fig. 2b and c). In contrast to the positive staining for S1P4 receptor protein in pulmonary arteries, there was no positive staining in renal arterial media of normal rats (Fig. 2d).

S1P4 receptor mRNA expression

Figure 2: Immnohistochemical staining for S1P4 receptor. (a) A representative low magnification photo of normotensive lung. (b) Representative high magnification photos of small pulmonary arteries from normotensive and (c) hypertensive lungs and (d) renal artery from a normal rat.

DISCUSSION

It is well documented that the S1P1, 2, and 3 receptors are widely distributed in the cardiovascular system, and several studies have shown that S1P2 and 3 receptors are the major subtypes responsible for S1P-induced constriction in various systemic arteries.[1,6-9] In fact, a recent study has shown that the S1P2 receptor is involved in S1P-induced pulmonary vasoconstriction in mice. [13] We found in this study in rats that infusion of S1P caused sustained vasoconstriction in isolated perfused NL, and the constriction was markedly augmented in HL. Surprisingly, 402

S1P4 recpetor/GAPDH

S1P4 receptor mRNA was detected in pulmonary arteries from NL and HL with RT-PCR in 2% agarose gel (Fig. 3a). Quantitative RT-PCR showed no statistically significant difference in S1P 4 receptor mRNA levels between pulmonary arteries from NL and HL, although there was a tendency towards higher expression levels in HL arteries (Fig. 3b).

100 bp

3 2 1 0

Figure 3: (a) S1P4 receptor mRNA expression in pulmonary arteries from normal (NL) and hypertensive lungs (HL) (b) Quantified values of S1P4 receptor mRNA expression in NL and HL. The amount of S1P4 receptor mRNA was normalized to that of GAPDH gene. Pulmonary Circulation | July-September 2011 | Vol 1 | No 3

Ota et al.: S1P4 mediates S1P-induced pulmonary vasoconstriction

pulmonary vascular tone. S1P4 receptors are reported to couple to Gai/0 and Ga12/13 but not to Gaq, and can activate phospholipase C and Rho kinase.[28] Thus, it is possible that activation of S1P4 receptors on pulmonary vascular smooth muscle cells induce contraction through increased cytosolic Ca 2+ levels (via phospholipase C-mediated Ca 2+ mobilization and Ca 2+ influx) and Rho kinasemediated Ca2+ sensitization.[24,29] Our results demonstrate that two structurally distinct S1P4 receptor agonists, phytosphingosine-1-phosphate [18] and VPC23153, [19] caused concentration-dependent vasoconstrictions in isolated NL, and that, as was the case with S1P, the response to VPC23153 was markedly enhanced in HL. Other similarities between the sustained vasoconstrictor responses to infused S1P and VPC23153 in both NL and HL were that they were markedly augmented by inhibition of nitric oxide synthesis and substantially reversed by the inhibitor of Rho kinase fasudil and additionally reduced by the Ca2+ channel blocker SKF93635. In addition to eliciting vasoconstriction in perfused lungs, VPC23153 contracted pulmonary but neither mesenteric nor renal arteries isolated from pulmonary hypertensive rats. Also, our immunohistochemical analysis showed S1P4 receptor protein is expressed in the media of pulmonary arteries from pulmonary normotensive and hypertensive rats but not in that of renal arteries from pulmonary normotensive rats. Finally, we clearly detected S1P mRNA in normotensive and hypertensive pulmonary arteries. Collectively, these results support that S1P4 receptors may play a key role in S1P-induced, Rho kinase- and Ca2+-mediated vasoconstriction in the normotensive and hypertensive rat pulmonary circulation.

Although VPC23153 at the concentrations used in this study has agonistic effects on the S1P1 receptor,[18] it is unlikely this receptor plays a major role in S1P-induced pulmonary vasoconstriction because we found that the dual S1P1 and 3 receptor agonist VPC24191 induced essentially no constriction in isolated NL. It should be noted, however, that the S1P 4 receptor agonists phytosphingosine-1-phosphate and VPC23153 also have some affinity to S1P5 receptors,[17,18] and this study does not rule out the possible involvement of this receptor in the vasoconstriction. In contrast to the previous report that S1P-induced vasoconstriction in mouse lungs is reduced by the S1P2 receptor antagonist JTE013 at a concentration of 10 mM,[30] we did not observe inhibition at the more receptor-selective concentration of 1 mM.[31] We found that vasoconstrictor responsiveness to S1P and the S1P4 receptor agonist was similarly and markedly enhanced in HL isolated from chronically hypoxic rats. Because the nitric oxide synthase inhibitor Nwnitro-Larginine increased S1P-induced vasoconstriction in both NL and HL and did not eliminate the difference between Pulmonary Circulation | July-September 2011 | Vol 1 | No 3

them, the enhanced response in HL cannot be attributed to decreased activity of nitric oxide. It is also unlikely that upregulation of the S1P4 receptor plays a major role in this augmented response, since we did not detect a significant increase in its mRNA or protein expression in hypertensive as compared to normotensive pulmonary arteries. Increased vasoconstrictor and myogenic reactivity is generally a characteristic of the hypoxiainduced hypertensive rat pulmonary vasculature,[32,33] although it is unclear exactly why the responsiveness to S1P is so strikingly augmented in HL.

CONCLUSIONS

In summary, this study demonstrates that S1P is a vasoconstrictor in the normal rat pulmonary circulation, and that the constrictor response is markedly augmented in the chronically hypoxic hypertensive pulmonary circulation. In contrast to what has been observed in various systemic arteries and in the mouse lung, S1P-induced pulmonary vasoconstriction appears to be mediated largely by the S1P4 receptor in the rat. These findings raise the possibility that pulmonary vasoconstriction by endogenous S1P, produced either locally within the pulmonary arterial wall or by circulating platelets and/or erythrocytes,[10] could contribute to the pathogenesis of pulmonary hypertension. If so, and if S1P-induced constriction of human pulmonary arteries is also mediated by the S1P4 receptor, then a S1P4 receptor antagonist[34] might be clinically useful as a selective pulmonary vasodilator.

ACKNOWLEDGMENTS

The authors thank Boniface Obiako and Greg Holberg and Drs.Â Tetsutaro Nagaoka and Noriyuki Homma for technical assistance and Dr. Mark N. Gillespie for helpful editorial comment.

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

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Research A r t i cl e

Fenfluramine-induced gene dysregulation in human pulmonary artery smooth muscle and endothelial cells Weijuan Yao1†, Wenbo Mu2†, Amy Zeifman3, Michelle Lofti4, Carmelle V. Remillard1, Ayako Makino1,3, David L. Perkins1, Joe G. N. Garcia3,5, Jason X. J. Yuan1,3,5,6, and Wei Zhang7,8 1 Department of Medicine, University of California, San Diego, La Jolla, California, 2Department of Bioengineering, 3Department of Medicine, 4Department of Biological Sciences, 5Institute for Personalized Respiratory Medicine, 6Center for Cardiovascular Research, 7 Department of Pediatrics and 8Institute of Human Genetics, University of Illinois at Chicago, Chicago,Illinois, USA † These authors contributed equally to this work.

ABSTRACT Fenfluramine is prescribed either alone or in combination with phentermine as part of Fen-Phen, an anti-obesity medication. Fenfluramine was withdrawn from the US market in 1997 due to reports of heart valvular disease, pulmonary arterial hypertension, and cardiac fibrosis. Particularly, idiopathic pulmonary arterial hypertension (IPAH), previously referred to as primary pulmonary hypertension (PPH), was found to be associated with the use of Fen-Phen, fenfluramine, and fenfluramine derivatives. The underlying mechanism of fenfluramine-associated pulmonary hypertension is still largely unknown. We reasoned that investigating drug-induced gene dysregulation would enhance our understanding of the fenfluramine-associated pathogenic mechanism of IPAH. Whole-genome gene expression profiles in fenfluramine-treated human pulmonary artery smooth muscle (PASMC) and endothelial (PAEC) cells (isolated from normal subjects) were compared with baseline expression in untreated cells. Fenfluramine treatment caused dysregulation in a substantial number of genes involved in a variety of pathways and biological processes. In addition to several common pathways and biological processes such as “MAPK signaling pathway,” “inflammation response,” and “calcium signaling pathway” shared between both cell types, pathways and biological processes such as “blood circulation,” “muscle system process,” and “immune response” were enriched among the dysregulated genes in PASMC. Pathways and biological processes such as those related to cell cycle, however, were enriched among the dysregulated genes in PAEC, indicating that fenfluramine could affect unique pathways (or differentially) in different types of pulmonary artery cells. While awaiting validation in a larger cohort, these results strongly suggested that fenfluramine could induce significant dysregulation of genes in multiple biological processes and pathways critical for normal pulmonary vascular functions and structure. The transcriptional and posttranscriptional changes in these genes may, therefore, contribute to the pathogenesis of fenfluramine-associated IPAH. Key Words: anorexigen, gene expression profile, lysosome, mitochondria, pulmonary hypertension

INTRODUCTION Fenfluramine (Fen, 3-trifluoromethyl-N-ethylamphetamine), a drug in the class of anorectics (appetite suppressants), has been prescribed either alone or in combination with phentermine (Phen) as part of Fen-Phen, an anti-obesity medication. The drug was withdrawn from the U.S. market in 1997 due to reports of valvular heart disease and pulmonary hypertension, including a condition known as cardiac fibrosis. Though the magnitude and prevalence of their deleterious cardiopulmonary effects remain undetermined,

Address correspondence to:

Dr. Wei Zhang Department of Pediatrics, University of Illinois at Chicago, COMRB Rm. 3099 (MC 719), 909 South Wolcott Avenue, Chicago, IL 60612, USA Email: weizhan1@uic.edu Pulmonary Circulation | July-September 2011 | Vol 1 | No 3

the links between these anorectics and valvular heart disease and pulmonary hypertension are clearly established.[1] For example, idiopathic pulmonary arterial hypertension (IPAH), previously referred to as primary pulmonary hypertension (PPH), was found to be associated with the use of Fen-Phen, fenfluramine, and fenfluramine derivatives.[2-6] Fatal cases of pulmonary hypertension and valvular heart disease Access this article online

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Website: www.pulmonarycirculation.org DOI: 10.4103/2045-8932.87310 How to cite this article: Yao W, Mu W, Zeifman A, Lofti M, Remillard CV, Makino A, et al. Fenfluramine-induced gene dysregulation in human pulmonary artery smooth muscle and endothelial cells. Pulm Circ 2011;1:405-18.

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have been reported to be associated with even short-term fenfluramine usage.[2,7] Furthermore, based on a study of more than 5,700 former fenfluramine users, damage to the heart valve (e.g., regurgitant valvulopathy) continued long after discontinuing use of the medication.[8] Previous studies suggested that nitric oxide deficiency could predispose affected individuals to develop anorexigen-associated pulmonary hypertension.[9] In addition, Fen and its active metabolite norfenfluramine were found to, indirectly, activate mitogenic serotonin 2B (5-HT2B) receptors,[10,11] thus potentially leading to the valvular abnormalities (e.g., abnormal valve cell division) found in patients taking fenfluramine.[12] Particularly, in cells expressing recombinant 5-HT2B receptors, norfenfluramine potently stimulates the hydrolysis of inositol phosphates, increases intracellular Ca2+, and activates the mitogen-activated protein kinase (MAPK) cascade, the latter of which has been linked to mitogenic actions of the 5-HT2B receptors.[13] The underlying cellular and molecular mechanism of fenfluramine-associated pulmonary hypertension is still largely unknown. Quantitative gene expression is an important intermediate phenotype that situates in the middle of DNA sequence variation, environmental influences (e.g., exposure to drugs) and other cellular/ whole-body phenotypes/traits [14-17] including the susceptibility to complex diseases (e.g., IPAH). For example, a previous genomic study of the expression profiles in peripheral blood mononuclear cells from patients with pulmonary arterial hypertension and normal individuals demonstrated a significant number of dysregulated genes between the patient cohort and normal individuals, as well as between patients with IPAH and secondary pulmonary hypertension.[18] Distinct gene signatures derived from lung tissues in IPAH and secondary pulmonary hypertension patients were also identified based on genome-wide expression profiling.[19] Therefore, we reasoned that a comprehensive examination of fenfluramine-induced gene dysregulation (upregulation or downregulation of mRNAs after treatment with fenfluramine) in relevant normal lung tissues, i.e., pulmonary artery smooth muscle cells (PASMC) and pulmonary artery endothelial cells (PAEC), could potentially help shed light on the molecular pathogenesis of fenfluramine-associated pulmonary hypertension. In order to examine the fenfluramine-associated gene dysregulation, we profiled transcriptional (mRNA) expression using a whole-genome cDNA array (covering 41,000 unique human transcripts with public domain annotations) in human PASMC and PAEC samples derived from normal individuals. We compared gene expression profiles in the PASMC and PAEC samples after treatment with fenfluramine and their baseline expression profiles. Known pathways from the Kyoto Encyclopedia of Genes and 406

Genomes (KEGG),[20,21] Gene Ontology (GO)[22] categories (i.e., biological processes, molecular function, cellular components), and gene networks were further evaluated among the dysregulated genes between cells treated and untreated with fenfluramine. Our findings suggest that fenfluramine may contribute to the pathogenesis of IPAH through causing differential expression of genes in certain pathways and biological processes that are critical to the normal functions of pulmonary arteries.

MATERIALS AND METHODS

Cell culture, drug treatment and cell morphological experiments Normal human PASMC and PAEC (3 samples for each cell type) were purchased from Lonza (Walkersville, Md.) and maintained in cell growth medium supplemented with 10% fetal bovine serum (FBS) and growth factors. Two days before the treatment, 5 × 105 cells were seeded in a 10-cm plate. The cell growth medium was replaced by the medium with 200 μM fenfluramine based on a previous publication,[23] and culturing was continued for 72 hrs. For cell morphological experiments, the cells were stained with the membrane-permeable nucleic acid stain, 4’, 6’-diamidino-2-phenylindole (DAPI, 5 µM). The blue fluorescence emitted at 461 nm was used to visualize the cell nuclei. The smooth muscle α-actin antibody was used to evaluate expression of α-actin in DAPI-stained cells.

Mitochondrial and lysosome imaging

Normal human PASMC and PAEC samples were cultured in medium supplemented with 10% FBS and growth factors. The cover slips with cells were mounted on glass slides and phase contrast images of cells were taken using an Olympus microscopy system. For mitochondrial staining, the media in 6-well plate were removed and pre-warmed medium containing MitoTracker Green FM (100 nM, Invitrogen, Carlsbad, Calif.) was added. The cells were incubated in the incubator for 15 min. The staining solution was removed and cells were rinsed with prewarmed medium, and cells were visualized with a 100× objective on a fluorescent microscope (Nikon, Japan) coupled to the Solamere Imaging System. For lysosome staining, the media in 6-well plate were removed and a staining solution containing LysoTracker Red (50 nM, Invitrogen, Carlsbad, Calif.) was used. The cells were incubated in the incubator for 30 min. before the staining solution was removed. The cells were then rinsed with pre-warmed medium and observed with a 20× objective on an Olympus microscope. Fluorescent images and phase contrast images, which were taken in the same field, were overlaid using the software included in the Olympus fluorescent imaging system. Pulmonary Circulation | July-September 2011 | Vol 1 | No 3

Yao et al.: Fenfluramine-induced gene dysregulation

RNA isolation and microarray hybridization

Total RNAs from treated and untreated PASMC and PAEC samples were isolated using standard molecular biology protocols. High-quality RNA samples with no signs of DNA contamination and RNA degradation were hybridized on the Agilent Whole Human Genome 4×44K Gene Expression Two-color arrays (Agilent Technologies, Santa Clara, Calif.), which contain 41,000 unique human transcripts (targeting 19,596 Entrez Gene RNAs) supported by public sources including RefSeq,[24] Golden Path Ensembl UniGene Human Genome (Build 33) and GenBank (http://www.ncbi.nlm.nih.gov/ genbank/) databases, according to the manufacturer’s recommended protocol at the UCSD Microarray Core Facility. Two PAEC samples did not pass the quality control of array hybridization. Therefore, in total, 4 samples (3 PASMC and 1 PAEC samples) were included in further analyses.

Microarray data preprocessing

The raw expression data were normalized and summarized with the robust multi-array average (RMA)[25] algorithm using GeneSpring GX v10 (Agilent Technologies, Santa Clara, Calif.). Since the ratio of the 2-color channels is most informative when the intensities are well over background for both the cy3 and cy5 channels, we removed those probesets if both the cy3 and cy5 channels had intensities in the lower quartile across all of the 4 samples. However, a gene could be interesting from a biological point of view, even if it has a meaningless ratio (e.g., a gene expressed in only 1 channel). Therefore, we included those genes with intensities above the cutoff (i.e., lower quartile) in either cy3 or cy5 channel. Only transcripts with unique, unambiguous gene annotations according to the manufacturer’s information (retrieved from the Agilent eArray website at http://earray.chem. agilent.com/earray/) were analyzed. In total, ~18,000 gene-level transcripts were included in the final analysis set.

Identification of dysregulated genes

Differentially expressed genes between fenfluraminetreated samples and untreated controls were identified based on a series of fold-changes (e.g., 1.2, 1.5 and 2.0) in the 3 PASMC samples. Due to the exploratory nature of this study and the small sample size, our choices of statistical approaches were limited, so any genes meeting a cutoff (e.g., fold-change>1.5) in at least 2 PASMC samples were considered differentially expressed. For the 1 PAEC sample, a single relatively stringent cutoff (i.e., fold-change>2) was used to control false positives. The expression patterns (i.e., upregulation or downregulation of genes) were also compared between the two cell types. Pulmonary Circulation | July-September 2011 | Vol 1 | No 3

Western blot validation of microarray data Three dysregulated genes that met fold-change>1.5: MMP1 (encoding metallopepetidase 1) for PAEC, CYCS (encoding cytochrome C, somatic) and VIM (encoding vimentin) for PASMC were selected for Western blot validation. Cells were washed with ice-cold PBS, suspended in lysis buffer (1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate, 100 μg/ml phenylmethylsulfonyl fluoride, phosphatase inhibitors, and protease inhibitors), and incubated for 30 min. on ice. The cell lysates were then sonicated and centrifuged at 12,000 rpm for 10 min., and the supernatant was collected. Protein concentrations were determined by DCTM Protein Assay (Bio-Rad Laboratories, Hercules, Calif.) using BSA as a standard. Samples were applied on SDS-PAGE (4– 2 0%), and proteins were transferred onto nitrocellulose membranes by electroblot. Membranes were blocked in 5% nonfat milk and incubated overnight at 4°C with primary antibodies and then with secondary antibodies. Blots were developed with the SuperSignal West Pico Chemiluminescent Substrate (Pierce Biotechnology, Rockford, Ill.).

Gene ontology and pathway analyses

We further searched the KEGG[20,21] and GO[22] databases for any enriched physiological pathways or biological processes among the dysregulated genes relative to the final analysis set using the Database for Annotation, Visualization and Integrated Discovery (DAVID, http:// david.abcc.ncifcrf.gov/).[26,27] For pathway and GO analyses, genes dysregulated in at least one PASMC or PAEC were included. Significantly enriched pathways or biological processes were determined based on an adjusted P-value after the Benjamini-Horchberg (BH) procedure[28] (e.g., adjusted P-value<0.05) and the size of the gene sets (e.g., a minimum size of 5 or 10 hits). To obtain robust evaluation of enrichment patterns, we focused on those enriched pathways or biological processes across different cutoffs of differential expression.

Gene network analysis

We used the the Cytoscape (http://www.cytoscape. org/) [29] plug-in from the Reactome (http://www. reactome.org/)[30,31] to find gene networks among the fenfluramine-induced dysregulated genes. This plugin accesses the Reactome Functional Interaction (FI) network, a highly reliable, manually curated pathwaybased protein functional interaction (e.g., activation, inhibition) network covering close to 50% of human proteins.[30,31] We also evaluated the relative importance of the interacting genes based on betweenness centrality, a measure of a node’s (i.e., a gene’s) centrality in a network. 407

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RESULTS Cell and mitochondrial morphologies after the treatment of fenfluramine

Cell morphologies of untreated controls and fenfluraminetreated PASMC and PAEC were evaluated. In untreated PASMC and PAEC, the phase contrast images showed that the cells appeared to be flat and have a smooth surface in the cytosplasm, and no intracellular organelle structure could be seen in untreated cells (Fig. 1 left panels). Treatment of the cells with 200 µM of fenfluramine for 72 hrs. seemed to cause significant morphological changes. In fenfluramine-treated cells, the surface membrane of the cytoplasm became rough, while perinuclear organelles became strikingly swollen (Fig. 1, right panels). In addition, we observed significant changes in the mitochondrial morphology in PASMC treated with fenfluramine (Fig. 2). In untreated cells, photomicrographs showed typical images of mitochondria; the mitochondrial structure looked intact and distributed throughout the entire cytoplasm (Fig. 2 left panels). After treatment with fenfluramine, however, the mitochondrial structure was severely damaged; the volume and the tubular frequency were significantly decreased (Fig. 2 right panels). These

Furthermore, we observed the changes in lysosomes in PAEC treated with fenfluramine. We stained the untreated and fenfluramine-treated PAEC with LysoTracker Red to label the lysosomes. In untreated cells, the fluorescence intensity from lysosomes was relatively low; however, in fenfluramine-treated cells, the fluorescence intensity of lysosomes was significantly increased (Fig. 3). By overlaying the phase contrast images with the LysoTracker Red-stained images, it seemed that the swollen perinuclear organelles in fenfluramine-treated PAEC were lysosomes.

Genes dysregulated in fenfluramine-treated pulmonary artery cells In total, 17,877 gene-level transcripts met our criteria Control

Fenfluramine

PAEC

PASMC

Control

data imply that fenfluramine treatment may induce mitochondrial fragmentation in PASMC.

PASMC

Figure 1: Morphological changes in human PASMC and PAEC treated with fenfluramine. Phase contrast images of untreated PASMC (upper left panels) and PAEC (lower left panels), and fenfluramine-treated PASMC (upper right panels) and PAEC (lower right panels). The small images depict an enlarged area of a single cell from each of the multi-cell images for PASMC and PAEC. 408

Figure 2: Mitochondrial morphological changes in human PASMC treated with fenfluramine. Photomicrographs show images of mitochondria in untreated PASMC (left panels) and PASMC treated with 200 µM of fenfluramine for 72 hrs. The enlarged images at the bottom show the perinuclear area of an untreated PASMC (left) and a fenfluramine-treated PASMC. Pulmonary Circulation | July-September 2011 | Vol 1 | No 3

Yao et al.: Fenfluramine-induced gene dysregulation

to be included in the analysis set. These transcripts had expression intensities above the lower quartile in at least 1 channel, as well as unique, unambiguous gene annotations (excluding putative genes encoding hypothetical proteins) according to the manufacturer’s information. Figure 4 shows a general characterization of the dysregulated genes in fenfluramine-treated Fenfluramine

Control

Fenfluramine

Control

Fenfluramine

Overlay

LysoTracker

Phase contrast

Control

PASMC and PAEC samples using different fold-change cutoffs. At fold-change>1.5, 881 genes were found to be dysregulated (497 upregulated and 384 downregulated) in the fenfluramine-treated PASMC samples; and 2,534 genes were found to be differentially expressed in the fenfluramine-treated PAEC sample (1,226 upregulated and 1,308 downregulated) (Fig. 4a). In contrast, at

PAEC

Number of genes

Figure 3: The effect of fenfluramine on the lysosomes in human PAEC. Cells were treated with 200 mM fenfluramine for 72 hrs. and the lysosomes in the cells were stained with LysoTracker Red. The phase contrast images (upper panels) of the cells were overlaid with the fluorescent images of the lysosomes (in red, middle panels) to show the location of lysosomes in untreated and fenfluramine-treated PAEC (bottom panels). The LysoTracker Red-staining is mainly localized in the perinuclear area in fenfluramine-treated cells. 3000

Total changed

2500

All up-regulated

2000

All down-regulated

Up-regulated (>2) Down -regulated (>2)

1500 1000 500 0

PASMC

(a)

PAEC

>2 fold

2000

<1.5 fold

1000

PAEC ↑

PAEC ↓

PAEC ↑ (b)

PAEC ↓

(c)

PASMC3

PASMC ↑ PASMC ↑ PASMC ↓ PASMC ↓

PASMC1

PASMC2

500

PAEC

Number of genes

1.5 -2 fold 1500

Figure 4: A general characterization of the genes dysregulated in PASMC and PAEC after fenfluramine treatment. (a) Numbers of genes that are affected by fenfluramine in PASMC and PAEC. The dysregulated genes are classified into five categories: expression level changed (fold-change>1.5) - “total changed,” upregulated - “all upregulated,” upregulated greater than 2-fold - “upregulated (>2),” downregulated - “all downregulated,” and downregulated greater than 2-fold - “downregulated (>2)” in PASMC (left) and PAEC (right). (b) Numbers of genes that are upregulated in both PASMC and PAEC, upregulated in PASMC but downregulated in PAEC, downregulated in PASMC but upregulated in PAEC, or downregulated in both PASMC and PAEC at different fold-changes (<1.5fold, 1.5- to 2-fold, or >2-fold). (c) A heatmap of the 384 dysregulated genes (fold-change>2.0) in PASMC or PAEC. Pulmonary Circulation | July-September 2011 | Vol 1 | No 3

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fold-change>2.0, there were 122 (74 upregulated and 48 downregulated) and 633 (285 upregulated and 348 downregulated) genes found to be dysregulated in the fenfluramine-treated PASMC and PAEC, respectively (Fig. 4a). Notably, there were more dyregulated genes with the same direction (e.g., upregulated in both cell types) in both PASMC and PAEC than those with different direction (e.g., upregulated in PASMC, but downregulated in PAEC) (Fig. 4b). At fold-change>2.0, there were 39 dysregulated genes (32 upregulated and 7 downregulated) in both

PASMC and PAEC samples (Table 1). Figure 4c shows a heatmap of the 384 differential genes (i.e., dysregulated in at least 1 cell type) in both PASMC and PAEC. Particularly, the PASMC samples were clustered together, showing a similar dysregulation pattern relative to PAEC.

Western blot validation of microarray data

Three dysregulated genes (fold-change>1.5), MMP1, CYCS and VIM, after fenfluramine treatment were selected for Western blot validation in the same PASMC

Table 1: Genes dysregulated in both PASMC and PAEC samples (fold-change>2.0) Dysregulation patterna

Gene symbol

Gene title

PASMC↑ and PAEC↑

C12orf42 CECR1 SC4MOL DKKL1 PPP1R12B SMR3B

MYOZ3 –

Chromosome 12 open reading frame 42 Cat eye syndrome chromosome region, candidate 1 Sterol-C4-methyl oxidase-like Dickkopf-like 1 (soggy) Protein phosphatase 1, regulatory (inhibitor) subunit 12B Submaxillary gland androgen regulated protein 3 homolog B (mouse) Dedicator of cytokinesis 8 Integrin, alpha 2 (CD49B, alpha 2 subunit of VLA-2 receptor) Diffuse panbronchiolitis critical region 1 Chloride channel, calcium activated, family member 3 Zinc finger, FYVE domain containing 16 Ligase IV, DNA, ATP-dependent Zinc finger and SCAN domain containing 4 5-Hydroxytryptamine (serotonin) receptor 2B Galactosylceramidase Ankyrin repeat domain 19 Chromosome 5 open reading frame 29 Phosphoinositide-3-kinase adaptor protein 1 Zinc finger, DHHC-type containing 15 FKBP6-like Kruppel-like factor 17 Coiled-coil domain containing 62 Tumor protein D52-like 3 Olfactory receptor, family 2, subfamily W, member 3 Cordon-bleu homolog (mouse) N-Acetyltransferase 8-like Follistatin-like 5 Chromosome 9 open reading frame 18 N-Acylsphingosine amidohydrolase (acid ceramidase) 1 Interferon regulatory factor 8 Solute carrier family 6, member 15 Dynein, cytoplasmic 2, heavy chain 1 Agmatine ureohydrolase (agmatinase) Wingless-type MMTV integration site family, member 11 3-Oxoacid CoA transferase 2 Angiopoietin-like 4 Chromosome 13 open reading frame 21 Solute carrier family 16, member 14 (monocarboxylic acid transporter 14) Myozenin 3 –

–

DOCK8 ITGA2

PASMC↓ and PAEC↓

PASMC↑ and PAEC↓ PASMC↓ and PAEC↑

DPCR1 CLCA3 ZFYVE16 LIG4 ZSCAN4 HTR2B GALC ANKRD19 C5orf29 PIK3AP1 ZDHHC15 LOC541473 KLF17 CCDC62 TPD52L3 OR2W3 COBL NAT8L FSTL5 C9orf18 ASAH1 IRF8 SLC6A15 DYNC2H1 AGMAT WNT11 OXCT2 ANGPTL4 C13orf21 SLC16A14

Fold-change in PASMCb

Fold-change in PAEC

8.4 3.1 2.1 2.0 4.5 4.3

11.7 2.7 2.7 2.0 2.6 3.0

2.9 2.1

2.1 2.2

2.7 2.1 2.3 2.0 2.9 2.8 2.6 2.4 2.3 3.8 2.5 3.4 2.3 2.6 2.2 2.0 2.2 2.0 3.2 4.4 2.1 4.2 3.0 4.0 −2.9 −2.4 −2.5 −2.7 −2.1 −2.1

2.7 2.2 2.2 2.0 5.4 2.7 2.4 2.3 3.7 4.6 5.8 2.8 3.4 2.0 2.6 2.2 2.3 2.8 2.8 3.4 2.1 5.1 3.1 2.1 −2.5 −2.1 −2.1 −7.6 −3.0 −4.6

−2.1 –

−2.0 –

–

PASMC: pulmonary artery smooth muscle cells; PAEC: pulmonary artery endothelial cells. a: ↑ – upregulation, ↓ – downregulation; b: median fold-change for the PASMC samples

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Enriched pathways and GO biological processes among the fenfluramine-induced dysregulated genes We evaluated the enrichment of KEGG[20,21] pathways and GO[22] biological processes among the genes dysregulated in PASMC and PAEC samples after fenfuramine treatment. Figure 6 compares the top 15 enriched pathways and biological processes (enrichment significant at adjusted P-value<0.001 after the BH procedure;[28] a minimum gene set size of 5 hits) between different cutoffs for differential expression (i.e., fold-change>1.5 and 2.0), as well as between both cell types. A significant number of enriched KEGG[20,21] pathways (Table 2) and GO[22] biological processes (Table 3) were identified using both fold-changes of 1.5 and 2.0 for differential expression. Notably, 14 KEGG[20,21] pathways (e.g., “MAPK signaling pathway,” “calcium signaling pathway,” “cell adhesion molecules”) (Table 2) and 7 GO[22] biological processes (e.g., “inflammatory response,” “response to organic substance,” “regulation of cell proliferation”) (Table 3) were enriched among the dysregulated genes in both PASMC and PAEC. Furthermore, some pathways and biological processes were also found to be enriched specifically among the dysregulated genes in either PASMC or PAEC. For example, the KEGG[20,21] pathways: “dilated cardiomyopathy” and “complement and coagulation cascades” (Table 2), as well as the GO [22] biological processes: “regulation of blood pressure” and “ muscle system process” (Table 3), were enriched among the dysregulated genes in PASMC. In contrast, the KEGG[20,21] pathways: “steroid biosynthesis” and “cell cycle” (Table 2), as well as the GO[22] biological process “cell proliferation” and “cell division” (Table 3), were enriched among the dysregulated genes in PAEC.

Gene network analysis

Some of the dysregulated genes in PASMC or PAEC after fenfluramine treatment were found to be connected with each other through certain FIs (e.g., activation, inhibition). Specifically, the Reactome[30,31] FI database was queried to identify FI relationships among the dysregulated genes after fenfluramine treatment in either PASMC (Fig. 7a) or Pulmonary Circulation | July-September 2011 | Vol 1 | No 3

Cont Fen MMP1 PAEC

GAPDH

Cont Fen CYCS PASMC

GAPDH

Cont Fen Vimentin PASMC

GAPDH

(a)

1.6 MMP1 1.4 1.2 1.0 0.8 1.4 CYCS 1.2

1.0 0.8 1.2 Vimentin * 1.1 1.0 0.9 0.8 Cont Fen (b)

RNA expression level (fold change)

and PAEC samples. The gene dysregulation patterns for these 3 genes were recaptured by the Western blot experiments (Fig. 5a and b). Particularly, the mRNA expression of MMP1 in PAEC after fenfluramine treatment (our microarray data) was upregulated by approximately 200% (Fig. 5c), while the protein expression level was upregulated by 50% (Fig. 5b). The fenfluraminemediated mRNA expression upregulation of CYCS and VIM, determined by the microarray data (Fig. 5c), was also consistent with the fenfluramine-mediated protein expression upregulation determined by Western blot analysis (Fig. 5b).

Protein expression level (normalized to GAPDH)

Yao et al.: Fenfluramine-induced gene dysregulation

2.0 MMP1 1.5 1.0 0.5 0.0 1.5 CYCS 1.0 0.5 0.0 2.0 Vimentin 1.5 1.0 0.5 0.0 Cont Fen (c)

Figure 5: Western blot validation of microarray data. Validation of microarray expression data was performed using Western blot in PASMC and PEAC. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as control. (a and b) Western blot analysis on matrix metallopeptidase 1 (MMP1) in untreated and fenfluramine-treated PAEC, and on somatic cytochrome c (CYCS) and vimentin in untreated and fenfluramine-treated PASMC. The data shown in B are mean±SE; *P<0.05 vs. control (Cont). (c) The fold-changes of mRNA expression levels of MMP1, CYCS and vimentin in untreated and fenfluramine-treated cells as indicated by the microarray data.

PAEC (Fig. 8a). We also evaluated the relative importance of the genes in the identified FI networks based on betweenness centrality, a measure in graph theory for a node’s (i.e., a gene’s) centrality in a particular network (Fig. 7b for PASMC and Fig. 8b for PAEC). Particularly, encoding bone morphogenetic protein receptor, type IB (BMPR1B) was located in the hub based on betweenness centrality in the networks comprised of the dysregulated genes in PASMC, while encoding repulsive guidance molecule A (RGMA) was found to be the most important gene based on betweenness centrality in the networks comprised of the dysregulated genes in PAEC (Figs. 7 and 8).

DISCUSSION

Fenfluramine-associated IPAH is likely a complex phenotype caused by multiple genetic and non-genetic factors, as well as by molecular changes after exposure to the drug. Elucidating the effects of this drug on relevant lung tissues (i.e., PASMC and PAEC) can potentially provide much-needed information on the underlying mechanism of fenfluramine-associated IPAH. In terms of cell and mitochondrial morphologies, there were obvious differences observed in PASMC and PAEC before and after treatment with fenfluramine (Figs. 1-3). These morphological data suggest that fenfluramine treatment not only results in mitochondrial damage (or fragmentation) in PASMC but also causes lysosome swelling in PAEC. However, it is unclear how these 411

We hypothesized that gene expression dysregulation after fenfluramine treatment in normal pulmonary artery tissues might contribute to the pathogenesis of

412

12 6

10 5

effects are related to the development of pulmonary hypertension. Mitosis M phase of mitotic cell cycle

Pancreatic cancer

Oocyte meiosis

TGF-β signaling pathway

M phase

Focal adhesion

Lysosome

0 Calcium signaling

0 DNA replication

Interphase

Regulation of kinase activity

Neuroactive ligand-receptor interaction

(b) PAEC enriched GO terms and pathways

Glycine serine and threonine metabolism

MAPK signaling pathway

Regulation of phosphorus metabolic process

(a) PASMC enriched GO terms and pathways

Regulation of phosphate metabolic process

0 Regulation of actin cytoskeleton

Mitosis

M phase of mitotic cell cycle

Cytokine-cytokine receptor interaction Tight junction

Regulation of actin cytoskeleton

Cell cycle pathway Pathway in cancer MAPK signaling pathway

Fold change 1.5

Cytokine-cytokine receptor interaction

Fold change 2

Wnt signaling pathway

Vascular smooth muscle contraction

TGF-β signaling pathway

Neuroactive ligand-receptor interaction

ECM-receptor interaction

Chemokine signaling pathway

Cell adhesion molecules (CAMs)

Regulation of actincytoskeleton

Axon guidance

Calcium signaling pathway

MAKP signaling transduction Cell surface receptor linked signal transduction

Cytokine-cytokine receptor interaction Pathways in cancer Focal adhesion

Fold change 1.5 8

Cell cycle pathway M phase Pathways in cancer

Jak-STAT signaling pathway

Regulation of blood pressure

Focal adhesion

G protein coupled receptor signaling pathway

Regulation of actin cytoskeleton

Calcium signaling pathway

Pathway in cancer

Cellular chemical homeostasis

Neuroactive ligand-receptor interaction

Ion homeostasis

Cellular cation homeostasis

Hematopoietic cell lineage

Cytokine-cytokine receptor interaction Chemokine signaling pathway Cell surface receptor linked signal transduction

Fold change 2

Yao et al.: Fenfluramine-induced gene dysregulation

(d) PAEC enriched GO terms and pathways

Figure 6: Enriched pathways and biological processes among the dysregulated genes. Top 15 enriched KEGG pathways and GO biological processes for the genes that are changed by fenfluramine in PASMC (a and c) and PAEC (b and d) at fold-change greater than 1.5 (a and b) or 2.0 (c and d).

fenfluramine-associated IPAH. Using a whole-genome cDNA microrray, a comprehensive evaluation of gene expression profiles in PASMC and PAEC samples showed a substantial number of dysregulated genes after fenfluramine treatment (Fig. 4), suggesting a wide range of molecular changes in lung tissues after fenfluramine

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Table 2: Enriched KEGG pathways (FDR<0.001) among the dysregulated genes after fenfluramine treatment Cell type

Pathway

PASMC

hsa00230:purine metabolism hsa04115:p53 signaling pathway hsa04210:apoptosis hsa04512:ECM-receptor interaction hsa04610:complement and coagulation cascades hsa04620:Toll-like receptor signaling pathway hsa04621:NOD-like receptor signaling pathway hsa04630:Jak-STAT signaling pathway hsa04650:natural killer cell mediated cytotoxicity hsa04722:neurotrophin signaling pathway hsa04916:melanogenesis hsa04960:aldosterone-regulated sodium reabsorption hsa05014:amyotrophic lateral sclerosis hsa05414:dilated cardiomyopathy hsa05416:viral myocarditis hsa00100:steroid biosynthesis hsa00260:glycine, serine and threonine metabolism hsa04110:cell cycle hsa04114:oocyte meiosis hsa04142:lysosome hsa04350:TGF-beta signaling pathway hsa04360:axon guidance hsa04914:progesterone-mediated oocyte maturation hsa05212:pancreatic cancer hsa05220:chronic myeloid leukemia hsa03320:PPAR signaling pathway hsa04010:MAPK signaling pathway hsa04020:calcium signaling pathway hsa04060:cytokine-cytokine receptor interaction hsa04062:chemokine signaling pathway hsa04080:neuroactive ligand-receptor interaction hsa04510:focal adhesion hsa04514:cell adhesion molecules hsa04640:hematopoietic cell lineage hsa04670:leukocyte transendothelial migration hsa04810:regulation of actin cytoskeleton hsa05200:pathways in cancer hsa05218:melanoma hsa05222:small cell lung cancer

PAEC

Bothc

Fold enrichment of Pathway Genesa (2.0)

Fold enrichment of Pathway Genesb (1.5)

12.3 25.4 23.6 18.9 26.3 16.9 27.8 18.2 18.2 12.4 15.4 34.7 25.1 17.3 20.4 71.9 50.6 35.2 15.0 15.7 18.8 14.8 18.3 25.2 21.0 21.5/26.7 7.7/11.0 18.1/14.0 32.9/16.4 23.6/10.7 17.0/10.8 14.4/10.9 17.5/15.8 37.7/21.3 16.7/14.8 15.2/14.4 12.0/13.3 22.9/19.7 23.9/21.7

10.4 16.5 14.8 18.8 16.7 10.7 15.5 12.5 13.6 8.9 12.3 13.3 13.2 15.8 16.5 32.4 23.8 25.7 15.5 13.9 18.5 12.5 12.1 14.2 9.8 15.4/17.5 10.9/10.2 13.6/10.5 19.8/11.4 11.6/9.0 10.6/10.3 13.2/11.5 15.1/10.2 17.2/14.0 11.4/13.2 10.9/11.4 12.6/12.4 15.5/14.4 13.6/14.0

PASMC: pulmonary artery smooth muscle cells; PAEC: pulmonary artery endothelial cells; KEGG: kyoto encyclopedia of genes and Genomes. a: among genes dysregulated at least 2-fold; b: among genes dysregulated at least 1.5-fold; c: enrichment fold shown in this category is for PASMC and PAEC, respectively

exposure. For example, 122 and 633 genes were identified to be dysregulated for at least 2-fold in PASMC and PAEC, respectively (Fig. 4). In addition to a number of genes that were dysregulated in both PASMC and PAEC (i.e., upregulated or downregulated in both cell types) (Table 1), the majority of dysregulated genes after fenfluramine treatment showed changes in either PASMC or PAEC only (Fig. 4b), thus potentially indicating a differential effect of fenfluramine on different cell types. Therefore, the microarray results suggested that with the exception of some commonly dysregulated genes in PASMC and PAEC, fenfluramine exposure could cause unique or differential changes in Pulmonary Circulation | July-September 2011 | Vol 1 | No 3

different cell types. Notably, some commonly and cell type-specifically dysregulated genes have been implicated in either pulmonary hypertension or related biological processes, demonstrating the relevance of these genes with fenfluramine-associated IPAH. For example, CECR1 (encoding cat eye syndrome chromosome region, candidate 1), which was upregulated (fold-change>2.0) in both PASMC and PAEC (Table 1), has been implicated in cat eye syndrome, a rare disease that features abnormal pulmonary venous return, potentially leading to pulmonary hypertension.[32] PPP1R12 (encoding protein phosphatase 1, regulatory [inhibitor] subunit 12B), an upregulated (fold-change>2.0) gene in both PASMC and PAEC after fenfluramine treatment (Table 1), is a myosin 413

Yao et al.: Fenfluramine-induced gene dysregulation

Tableâ&#x20AC;Ż3: Enriched GO biological processes (FDR<0.001) among the dysregulated genes after fenfluramine treatment Cell type

Biological process

PASMC

GO:0001558-regulation of cell growth GO:0003012-muscle system process GO:0003013-circulatory system process GO:0006873-cellular ion homeostasis GO:0006874-cellular calcium ion homeostasis GO:0006875-cellular metal ion homeostasis GO:0006928-cell motion GO:0006935-chemotaxis GO:0006952-defense response GO:0006955-immune response GO:0007155-cell adhesion GO:0007166-cell surface receptor linked signal transduction GO:0007186-G-protein coupled receptor protein signaling pathway GO:0007204-elevation of cytosolic calcium ion concentration GO:0007242-intracellular signaling cascade GO:0007610-behavior GO:0007626-locomotory behavior GO:0008015-blood circulation GO:0008217-regulation of blood pressure GO:0008284-positive regulation of cell proliferation GO:0010941-regulation of cell death GO:0019725-cellular homeostasis GO:0019932-second-messenger-mediated signaling GO:0022610-biological adhesion GO:0030003-cellular cation homeostasis GO:0030005-cellular di-, tri-valent inorganic cation homeostasis GO:0030334-regulation of cell migration GO:0030335-positive regulation of cell migration GO:0040012-regulation of locomotion GO:0040017-positive regulation of locomotion GO:0042330-taxis GO:0042493-response to drug GO:0042592-homeostatic process GO:0042981-regulation of apoptosis GO:0043066-negative regulation of apoptosis GO:0043067-regulation of programmed cell death GO:0043069-negative regulation of programmed cell death GO:0043085-positive regulation of catalytic activity GO:0043434-response to peptide hormone stimulus GO:0044057-regulation of system process GO:0044093-positive regulation of molecular function GO:0048878-chemical homeostasis GO:0050801-ion homeostasis GO:0051247-positive regulation of protein metabolic process GO:0051270-regulation of cell motion GO:0051336-regulation of hydrolase activity GO:0051345-positive regulation of hydrolase activity GO:0051480-cytosolic calcium ion homeostasis GO:0055065-metal ion homeostasis GO:0055066-di-, tri-valent inorganic cation homeostasis GO:0055074-calcium ion homeostasis GO:0055080-cation homeostasis GO:0055082-cellular chemical homeostasis GO:0060191-regulation of lipase activity GO:0060548-negative regulation of cell death GO:0000087-M phase of mitotic cell cycle GO:0000278-mitotic cell cycle GO:0000279-M phase GO:0000280-nuclear division

PAEC

Fold enrichment of Fold enrichment of GO Genes (2.0)a GO Genes (1.5)b 4.6 5.6 6.3 4.5 6.3 6.8 3.0 6.0 4.5 4.3 3.5 2.7 3.0 7.6 2.0 3.5 4.1 6.3 7.9 3.3 2.5 3.3 4.7 3.5 5.9 5.5 4.8 6.7 4.5 6.6 5.9 4.2 3.0 2.5 3.8 2.5 3.7 3.4 6.2 3.9 3.0 4.3 4.3 4.1 4.2 3.7 5.7 7.4 7.1 5.2 6.4 5.3 4.4 9.1 3.7 5.2 4.8 5.4 5.3

2.9 4.0 3.9 2.5 3.4 3.3 2.7 3.1 3.1 2.7 2.6 2.1 1.8 4.8 1.8 2.5 2.7 3.9 3.8 2.7 2.1 2.1 2.9 2.6 2.9 2.8 3.5 4.3 3.2 4.1 3.1 2.4 2.0 2.1 2.5 2.1 2.5 2.3 2.5 3.3 2.3 2.5 2.5 2.2 3.2 2.4 2.8 4.4 3.3 2.7 3.3 2.8 2.5 3.8 2.5 3.9 3.5 3.5 4.0 (Continued)

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Tableâ&#x20AC;Ż3: Continued Cell type

Bothc

Biological process GO:0007049-cell cycle GO:0007067-mitosis GO:0008283-cell proliferation GO:0019220-regulation of phosphate metabolic process GO:0022402-cell cycle process GO:0022403-cell cycle phase GO:0042325-regulation of phosphorylation GO:0043549-regulation of kinase activity GO:0045859-regulation of protein kinase activity GO:0048285-organelle fission GO:0051174-regulation of phosphorus metabolic process GO:0051301-cell division GO:0051325-interphase GO:0051329-interphase of mitotic cell cycle GO:0051338-regulation of transferase activity GO:0051726-regulation of cell cycle GO:0006954-inflammatory response GO:0007267-cell-cell signaling GO:0008285-negative regulation of cell proliferation GO:0009611-response to wounding GO:0009719-response to endogenous stimulus GO:0010033-response to organic substance GO:0042127-regulation of cell proliferation

Fold enrichment (2.0)a

Fold enrichment (1.5)b

4.3 5.3 4.6 3.7 4.8 5.8 3.6 4.2 4.1 5.1 3.7 4.0 8.0 7.6 4.0 4.3 5.5/4.0 4.1/3.2 3.5/3.8 4.3/3.1 4.5/3.3 3.1/2.6 3.2/3.3

3.0 4.0 3.8 2.7 3.2 3.6 2.6 2.9 2.8 3.8 2.7 2.9 5.1 4.9 3.2 3.1 3.4/2.6 3.1/2.4 3.0/3.2 3.0/2.7 2.8/3.1 2.3/2.4 2.7/2.7

PASMC: pulmonary artery smooth muscle cells; PAEC: pulmonary artery endothelial cells; GO: gene ontology. a: among genes dysregulated at least 2-fold; b: among genes dysregulated at least 1.5-fold; c: enrichment fold shown in this category is for PASMC and PAEC, respectively

(a)

(b)

(c)

Figureâ&#x20AC;Ż7: Gene networks comprised of the dysregulated genes in PASMC after fenfluramine treatment. A substantial proportion of the dysregulated genes after fenfluramine treatment (fold-change>2.0) are connected with each other through functional interactions. (a) A gene network comprised of the dysregulated genes in PASMC. Red: upregulated genes; Blue: downregulated genes. (b) A gene network comprised of the dysregulated genes in PASMC showing betweenness centrality, a measure for the relative importance of genes in a network. Green: low value; Red: high value; Orange/ Yellow: intermediate value. (c) Bone morphogenetic protein receptor, type IB (BMPR1B) is the hub based on betweenness centrality. AMHR2: Anti-Mullerian hormone receptor, type II; GDF10: Growth differentiation factor 10; STK35: Serine/threonine kinase 35; TLL2: Tolloid-like 2.

phosphatase (also known as MYPT2) that regulates muscle contraction, thus potentially affecting blood pressure.[33] In addition, mutations in BMP family including BMPR1B, an upregulated (fold-change>2.0) gene after fenfluramine treatment in PASMC, have been associated with IPAH.[34,35] In contrast, MYLK (encoding myosin, light chain kinase, transcript variant 1), an upregulated gene (foldchange>2.0) in PAEC, has been implicated in endothelial cell contraction and barrier dysfunction.[36-38] Pulmonary Circulation | July-September 2011 | Vol 1 | No 3

We further examined whether these dysregulated genes were enriched in any biological processes or known physiological pathways, which could help elucidate the underlying mechanism of fenfluramine-associated pathogenesis of IPAH. Similar to the comparison of gene dysregulation, a number of common KEGG[20,21] pathways and GO[22] biological processes were identified among the genes dysregulated in PASMC and PAEC samples, respectively, in addition to other cell type-specific 415

Yao et al.: Fenfluramine-induced gene dysregulation

(a)

(b)

(c) Figure 8: Gene networks comprised of the dysregulated genes in PAEC after fenfluramine treatment. A substantial proportion of the dysregulated genes after fenfluramine treatment (fold-change>2.0) can be connected with each other through functional interactions. (a) A gene network comprised of the dysregulated genes in PAEC. Red: upregulated genes; Blue: downregulated genes. (b) A gene network comprised of the dysregulated genes in PAEC showing betweenness centrality, a measure for the relative importance of genes in a network. Green: low value; Red: high value, Orange/Yellow: intermediate value. (c) Repulsive guidance molecule A (RGMA) is the hub based on betweenness centrality. CHRDL1: Chordin-like 1; ZFYVE16: Zinc finger, FYVE domain containing 16.

pathways and biological processes (Fig. 4, Tables 2 and 3). Notably, the enriched pathways (Table 2) and biological processes (Table 3) were robust across different cutoffs for differential expression (i.e., fold-change>1.5 and 2.0). Particularly, the dysregulated genes in PASMC samples were found to be enriched in a number of GO[22] biological processes including “immune response,” “defense response,” “blood circulation,” and “circulatory system process,” as well as processes related to cell adhesion and cell migration (Table 3). Similarly, a number of KEGG[20,21] pathways were enriched specifically among the dysregulated genes in PASMC, including “dilated 416

cardiomyopathy,” and a number of signaling pathways such as “Toll-like receptor signaling pathway,” and “JAK-STAT signaling pathway” (Table 2). Among the dysregulated genes in PAEC, however, there were enriched pathways and biological processes including “mitotic cell cycle,” “regulation of phosphorylation,” and “TGF-β signaling pathway” (Tables 2 and 3). In addition, there were several pathways and biological processes, including “inflammatory response,” “PPAR signaling pathway,” “MAPK signaling pathway,” and “calcium signaling pathway,” that were enriched among the dysregulated genes in both PASMC and PAEC (Tables 2 and 3). Clearly, some of these enriched pathways and biological processes have been implicated in pulmonary hypertension or related physiological processes. For example, the MAPK signaling pathway has been demonstrated to be activated by norfenfluramine, a derivative of fenfluramine, together with the increase of intracellular Ca2+ and the activation of the hydrolysis of inositol phosphates.[13] Previous studies have also implicated the PAAR signaling pathway in PAH. [39,40] Our pathway analysis results also indicated that genes involved in cell permeability, inflammation and immune response, such as “calcium signaling pathway,” and “cell adhesion,” could play critical roles in fenfluramine-associated pathogenesis. Furthermore, our gene network analysis demonstrated that a substantial proportion of the dysregulated genes in PASMC and PAEC after fenfluramine treatment could be linked with each other through certain functional interactions (e.g., activation, inhibition) (Figs. 7 and 8), suggesting that fenfluramine exposure may cause changes in a series of interacting proteins. Particularly, BMPR1B (Fig. 7c) and RGMA (Fig. 8c) were among the most important genes in the networks comprised of the dysregulated genes in PASMC and PAEC, respectively, based on betweenness centrality, indicating their potentially critical roles in determining the fenfluramineassociated pathogenic process in these cells. Interestingly, repulsive guidance molecule, a BMP co-receptor has been found to alter utilization of bone morphogenetic protein (BMP) type II receptors,[41,42] which have been implicated in pulmonary hypertension.[43]

CONCLUSIONS

In summary, our findings strongly suggest that fenfluramine exposure could cause a wide range of gene dysregulation. Significantly, our results confirmed that fenfluramineassociated IPAH is likely due to a complex pathogenic process that could be caused by genes involved in a variety of pathways and biological processes, including those related to normal functions of blood vessels (e.g., cell permeability, cell adhesion), inflammation response, Pulmonary Circulation | July-September 2011 | Vol 1 | No 3

Yao et al.: Fenfluramine-induced gene dysregulation

immune response, and cell cycle. On the other hand, significant genetic and expression variations exist both within human populations and between normal subjects and patients with cardiopulmonary disease[44-47] that may in turn affect the cellular response to fenfluramine. Future investigations with more samples and populations are necessary to validate our findings. Finally, expanding molecular profiling to include other transcriptional targets such as microRNAs and integrating gene expression with genetic and epigenetic variations in the future could provide a more comprehensive picture of the complex cellular response to fenfluramine exposure as well as the pathogenesis of fenfluramine-associated IPAH or pulmonary arterial hypertension in general.

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Source of Support: This work was supported, in part, by grants from the NIH/NHLBI (HL066012 and HL098053 to JX-JY and DK083506 to AM). WZ is supported by the new faculty research start-up from the University of Illinois at Chicago. ML was supported by the NSF ASCEND Program., Conflict of Interest: None declared.

Pulmonary Circulation | July-September 2011 | Vol 1 | No 3

Research A r t i cl e

Neurogenic responses in rat and porcine large pulmonary arteries Daniel J. Duggan, Detlef Bieger, and Reza Tabrizchi Division of BioMedical Sciences, Faculty of Medicine, Memorial University, St. John’s, Newfoundland and Labrador, Canada

ABSTRACT Pharmacological differences between neurogenic sympathetic responses in rat and pig isolated pulmonary arteries were examined in strip preparations. Electrical field stimulation in the range of 0.6 to 40 Hz resulted in frequency-dependent contractions in terms of amplitude and rate of rise. Responses in the rat declined sharply from pulmonary trunk to main artery; in contrast, in the pig they continued into the third-order vessels. Contractions were inhibited in the presence of tetrodotoxin, prazosin or WB-4101 and hence neurogenic in origin. Cocaine enhanced field stimulated contractions in both rat and porcine tissues; however, the effect in the former was of significantly greater magnitude in terms of either area under the mechanogram or height of contraction. In addition, the rate of rise, time to peak and duration of peak were all increased in the rat but less so or not in the pig. Field stimulated contractions were virtually abolished by guanethidine (1×10-6 M) in rat but not in porcine pulmonary arteries in which a ten-fold higher concentration significantly reduced neurogenic contractions and abolished them in 2 out of 4 tissues tested. The effect of guanethidine (1×10-6 M) observed in blood vessels of rat exceeded about five-fold that observed in porcine tissues. Thus, neurogenic responses appear to be entirely mediated by extra-junctional a1-adrenoceptors in both species, and in contrast to the rat, pig tissues seem to have a noradrenaline re-uptake that is either less efficient or operating near saturation. Key Words: adrenergic, alpha1-adrenoceptors, porcine, pulmonary artery, rat, re-uptake, vascular neuroeffector

transmission

INTRODUCTION The sympathetic innervation of the pulmonary artery varies considerably among mammalian species.[1] However, the common picture would suggest that large vessels are richly endowed with adrenergic axon terminals while smaller vessels are sparsely endowed or not innervated. Adrenergic fibers are typically located approximately 5-10 µm from the medial smooth muscle layers.[2] In porcine pulmonary arteries, adrenergic terminals were identified in the tunica media, where they were seen to run around the vessel between elastic laminae and smooth muscle cells.[3] Histochemical data reported in rats are somewhat unclear. According to Zussman,[4] large pulmonary arteries of the rat contain a dense adrenergic innervation with the terminals extending into the media and sub endothelial layer. However, a study by El-Bermani[5] supports the view that a direct adrenergic innervation may be lacking in rat pulmonary artery smooth muscle, noradrenergic fibers having been noted to pass through Address correspondence to:

Dr. Reza Tabrizchi Memorial University, Faculty of Medicine, Division of BioMedical Sciences, Health Sciences Centre, St. John’s, NL, A1B 3V6, Canada Email: rtabrizc@mun.ca Pulmonary Circulation | July-September 2011 | Vol 1 | No 3

the smooth muscle layer of the vessel without forming varicosities. MacLean and colleagues[6] have concluded that adrenergic fibers are absent in rat pulmonary trunk and main arteries proper and occur solely in association with their vasa vasorum, which are formed by branches of bronchial arteries. Costa and Majewski[7] reported an electrically evoked release of [3H]-noradrenaline from spiral strips of rat pulmonary artery. There is a lack of data in the current literature on vasomotor responses to sympathetic nerve stimulation in either rat or porcine pulmonary arteries. Thus it appeared useful to demonstrate and compare such neurally mediated responses in these two species, particularly in light of conflicting neurohistochemical information in the rodent. As shown in this investigation, electrical field Access this article online

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Website: www.pulmonarycirculation.org DOI: 10.4103/2045-8932.87311 How to cite this article: Duggan DJ, Bieger D, Tabrizchi R. Neurogenic responses in rat and porcine large pulmonary arteries. Pulm Circ 2011;1:419-24.

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Tissue preparations

All procedures on animals were carried out in accordance with the guidelines and approval of the Animal Care Committee, Memorial University, St. John’s, Newfoundland and Labrador, Canada.

Male Sprague–Dawley rats (280–310 g) were anaesthetized with sodium pentobarbital (65 mg/kg i.p.). The pulmonary trunk and main (left and right) pulmonary arteries were isolated, dissected free of connective tissue at room temperature in Krebs buffer with the following composition (in mM): NaCl, 130; KCl, 4.0; glucose, 11; MgCl2, 1.2; CaCl2, 2.5; KH2PO4, 1.2; NaHCO3, 12.5; EDTA, 0.1. The pH of the buffer following saturation with a 95% O2: 5% CO2 gas mixture was 7.4 at 36±1°C. Pigs of either sex (2-9 months of age) were sedated with ketamine (22 mg/kg i.m.) and xylazine (2.0 mg/kg i.m.), before being intubated and maintained on isoflurane (1.5%-1.0% in 100% O2). Specimens of pulmonary arterial trunk and of juxta-hilar lung plus main bronchi were excised from animals immediately following thoracotomy and removal of the heart. Segments of trunk, main pulmonary artery and intralobar pulmonary arteries were dissected free of connective tissue at room temperature in Krebs buffer under the same condition as above.

Experimental protocol

After isolation from adjoining structures and removal of surrounding adipose tissues, the rat pulmonary trunk and arteries were divided at the bifurcation into two corresponding segments, before being cut into helical strips approximately 7 to 10 mm in length and 1.5 to 2 mm in width. Vascular preparations made from porcine pulmonary arteries included: transverse strips of the extrapulmonary portion of the main right pulmonary artery and its second order branches and helical strips dissected from third order intrapulmonary branches (approximately 10 to 12 mm in length and 2 to 3 mm in width). Tissues were mounted on a holder armed with two platinum ring electrodes spaced 10 mm apart in organ baths (Radnoti LLC, Calif., USA) at 36-37°C. Preload tension levels employed were as follows: for rats, 0.3 g (trunk) or 0.15 g (right pulmonary arterial strips), and for pigs, 1.0 g. Bath media were gassed continuously with a mixture of 95% O2: 5% CO2. Contractile tone was recorded by force displacement transducers (Model FT03, Grass Instruments Co., Mass., USA) connected to a polygraph (Model 7PCPB, 420

Data and statistical analysis

Traces were digitized and responses to EFS assessed by measuring contraction height and the “area under the mechanogram trace” of the evoked response. The data were normalized as percent of the respective control response in the absence of test drugs. Comparisons employed Student’s t-test, analysis of variance with repeated measure and the Bonferroni test for multiple comparisons. The data are based on replications from at least 3-5 individual animals.

RESULTS

EFS consistently elicited contractions in strips obtained from rat pulmonary trunk but produced no or barely detectable (<2 mg) responses in strips of rat main pulmonary artery. Contractile tension began to rise within 10 seconds of EFS and continued to climb for another 15 to 20 seconds after its off-set before falling to baseline over the next 60 to 100 seconds (Fig. 1). Although some pulmonary trunk preparations (33%) responded at a pulse 1.5x10-7 M Tetrodotoxin

Rat

2 min 100 mg

MATERIALS AND METHODS

Grass Instruments Co., Mass., USA). EFS was delivered by a Grass S88 stimulator generating trains of rectangular monophasic pulses of selected frequency (0.6 to 40 Hz), width (0.5, 1.0 or 2.0 ms) and amplitude (50 V) applied in repetitive train mode (10 s train duration; 100 s train interval) or intermittent mode with 100 or 200 pulses per train delivered at intervals of 4-5 minutes. Ascorbic acid (10-4 M) was routinely added to the bathing medium to prevent the electrochemical oxidation of noradrenaline. [8] Cocaine HCl (1.0 µM), guanethidine monosulphate (1.0 and 10 µM), tetrodotoxin (1.0 × 10-7 M), the nonselective a1-adrenoceptor antagonist, prazosin HCl (1.0 and 3.0 × 10-7 M) and, the a1A/D-adrenoceptor antagonist, WB-4101 (1.0 × 10-9 M to 3.0 × 10-8 M)[9] were added in aqueous solution to the bath fluid after the preparations had reached a reproducible and consistent magnitude of evoked contractions.

10 Hz 1.5x10-7 M Tetrodotoxin

Pig 2 min 700 mg

stimulation (EFS) of pulmonary arterial strips produces neurogenic contractions in both species that have distinct pharmacological properties.

10 Hz

5 Hz

2.5 Hz

10 Hz5 Hz

Figure 1: Effect of tetrodotoxin on contraction elicited by 200 pulse train electrical field stimulation (black bars) in rat pulmonary trunk (width 1.0 ms) and pig main pulmonary artery (width 0.5 ms) at frequencies indicated. Pulmonary Circulation | July-September 2011 | Vol 1 | No 3

Duggan et al.: Pulmonary artery and neurogenic contractions

1x10-7 M Prazosin

100 mg

2 min

10 Hz

(a) Rat 1x10-9 M WB -4101

3x10-9 M WB -4101

200 mg

2 min

5 Hz

(b) Rat 3x10-7 M Prazosin

2 min 200 mg

With increasing pulse frequency in the range of 0.6-40 Hz, EFS resulted in contractions of increasing height and rate of rise with the threshold being apparent at 0.6 Hz and peaks being reached below and above 5.0 Hz for rat and pig tissues, respectively (Fig. 3). In the former, maximal neurogenic contractions elicited at 10 Hz amounted to 12-13% of the maximal tension induced by noradrenaline (1.0 to 10 µM). In the pig, the corresponding value showed proximo-distal variation with a range from 40% (main pulmonary artery and first order vessels) to 10-20% (second to third order vessels). The presence of cocaine enhanced field stimulated contractions in both rat and porcine tissues (Fig. 3); however, the effect in the former was of significantly greater magnitude in terms of either area under the mechanogram (Fig. 4) or height of contraction (not shown). In addition, the rate of rise, time to peak and duration of peak were all increased in the rat but less so or not in the pig (Fig. 3). The cocaine-induced enhancement of neurogenic contractions in rat or pig tissues did not vary significantly with increasing EFS pulse frequency in the range tested. The presence of cocaine significantly decelerated relaxation. Thus in rat tissues at frequencies of 2.5 and 5.0 Hz, half maximal relaxation time increased by 83±6% and 56 ± 16% and in the pig it increased by 59 ± 15% and 28±6% (mean ± s.e.m), respectively.

In the presence of guanethidine (1×10 -6 M) field stimulated contractions were virtually abolished in rat but

10 Hz

1 x10 -8 M WB -4101

2 min 250 mg

width of 0.5 ms, the majority required a pulse width of 1.0 ms to generate a contraction >20 mg in amplitude. In contrast, irrespective of their anatomical origin or size, transverse or helical strips from different segments of porcine pulmonary arteries invariably contracted at a pulse width of 0.5 ms. Evoked contractions were inhibited in blood vessels from rat and pig in the presence of tetrodotoxin (Fig. 1), or prazosin or WB-4101 (Fig. 2) and hence considered neurogenic in origin. The antagonism displayed by WB-4101 was equally effective at 2.5 Hz (not illustrated).

10 Hz

(d) Pig

Figure 2: Effect of α1-adrenoceptor antagonists (prazosin and WB-4101) on field stimulated contraction induced in rat pulmonary trunk (pulse width 1.0 ms) and in pig second order pulmonary artery branch (pulse width 0.5 ms) by 100 pulse train electrical field stimulation (black bars).

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5 Hz

2.5 Hz

1.25 Hz

10 Hz (a) Rat

20 Hz

1.25 Hz 5 Hz

2.5 Hz

10 Hz 20 Hz

40 Hz

(b) Pig

Figure 3: Frequency dependence of neurogenic contractions elicited by 200 pulse train field stimulation (pulse width 1.0 ms; black bars) in the absence and (a) presence of cocaine in the rat pulmonary trunk and (b) pig second order pulmonary artery branch. Pulmonary Circulation | July-September 2011 | Vol 1 | No 3

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a,b n=4

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n=4

a n=4

n=3

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% of Control (Area)

400

2 min

100

10 Hz

1.25

2.5 5 Frequency (Hz)

Figure 4: Effect of cocaine (1.0 × 10-6 M) on neurogenic contraction of rat and pig pulmonary arterial strips. Shown are histograms of area under mechanogram traces. Responses were evoked by trains consisting of 200 rectangular pulses (width 1.0 ms, rat; 0.5 ms, pig). aSignificantly different from control P<0.05; b Significantly different from respective response in pig P<0.05.

were unaffected in porcine pulmonary arteries (Fig. 5). In the latter a higher concentration of guanethidine (1×10-5 M) significantly reduced neurogenic contractions and abolished them in 2 out of 4 tissues tested (Fig. 5). The effect of guanethidine (1×10-6 M) observed in rat tissues exceeded by about five-fold that observed in porcine tissues (Fig. 6).

DISCUSSION

The data presented here demonstrate that the pulmonary arteries of the rat and the pig possess a functional adrenergic vasoconstrictor innervation. Despite basic similarities, including inhibition by tetrodotoxin and α1-adrenoceptor antagonists, the observed neurogenic vasoconstrictor responses were shown to exhibit species dependent differences in (a) anatomical distribution and (b) pharmacological characteristics.

Distribution of EFS-evoked responses

In agreement with available histochemical evidence,[3] the porcine pulmonary arterial strips displayed neurogenic contractions irrespective of the vessel caliber tested; however, in the rat the response appeared to be virtually confined to the pulmonary trunk. If adrenergic terminals supplying the vasa vasorum of rat pulmonary arteries were to constitute the sole source of vasomotor innervation at this level, their involvement in neurogenic contractions would be plausible. Yet the difficulty to detect this response in the main pulmonary artery (i.e., past the bifurcation of the pulmonary trunk) would militate against this idea. In any case our observations are consistent with the presence in the rat 422

1x10-5 M Guanethidine

(b)

Figure 5: Differential effect of noradrenergic neuron blocker, guanethidine, on neurogenic contraction evoked in rat pulmonary trunk (pulse width 1.0 ms) (a) and pig second order pulmonary artery branch (pulse width 0.5 ms) (b) by 100 pulse train electrical field stimulation (black bars). 120 % of Control (Height of contraction)

100

n=5 n=5 n=3

Pig Rat

n=3

n=4 80 60 40

a,b n=4

a a n=4 a,b n=4 n=4 a n=3

20 0

Control 1 2 3 4 5 Number of stimulations

Figure 6: Progressive inhibitory effect of guanethidine (1.0×10-6 M) on height of contraction evoked by 100 pulse trains in rat pulmonary trunk (pulse width 1.0 ms), and lack of inhibition in pig second order pulmonary artery branch (pulse width 0.5 ms). aSignificantly different from control P<0.05; b Significantly different from respective response in pig P<0.05.

pulmonary arterial trunk of a functional sympathetic innervation.

The density of adrenergic innervation in arteries of various species has been correlated with the efficacy of EFS in producing vasoconstrictor responses, i.e. blood vessels with low-density innervation have a shallow frequency response relationship and low relative efficacy expressed against agonist-evoked maximal contractions.[10] In this regard, the rat pulmonary trunk and porcine second to third order strips behaved like sparsely innervated tissues. It is, however, worth noting that a larger pulse width was required to evoke a response in rat as opposed Pulmonary Circulation | July-September 2011 | Vol 1 | No 3

Duggan et al.: Pulmonary artery and neurogenic contractions

to pig pulmonary arterial strips. The greater electrical excitability of porcine pulmonary constrictor nerve terminals would be consistent with a higher innervation density or transmission efficiency in this species. The possibility of the EFS-evoked responses being due to direct activation of muscle fibers can be dismissed in view of the observed full blockade obtained with tetrodotoxin in both rat and pig tissues. The density of the vasoconstrictor nerve terminals is known to vary in the same vascular bed. For example in the rat mesenteric artery, the sympathetic innervation, as assessed by histochemical and functional criteria, gradually decreases from the principal arteries to the terminal arterioles, with the precapillary arterioles apparently being devoid of innervation.[11,12] Based on the present results, the density of adrenergic terminals in the rat pulmonary artery would seem to decrease sharply beyond the bifurcation of the trunk.

Pharmacological characteristics

Since the sympathetic neurogenic responses were abolished by the α1-adrenoceptor antagonists, prazosin and WB-4101, co-transmission by ATP appears to be absent or functionally insignificant in the pulmonary arteries of rat and pig. ATP is considered to play no role as a co-transmitter in vasomotor responses mediated via the activation of sympathetic nerves in veins and pulmonary arteries.[13] In addition, no evidence was obtained for a participation of α2 adrenoceptors at low EFS pulse rate. The latter has been demonstrated by Bao et al.[14] in the rat tail artery. As revealed by the effect of guanethidine and cocaine, there were striking pharmacological divergences in rat and pig pulmonary arterial strips. These could be due to different transmitter kinetics i.e. release and uptake, more specifically, the regulation of the clearance of noradrenaline released neurally. Evidently, the pig pulmonary artery was relatively less sensitive to the pharmacological actions of either guanethidine or cocaine. The inhibitory actions of guanethidine are critically dependent on its uptake via the axolemmal amine transporter into the nerve endings while the potentiating actions of cocaine on neurogenic transmission are largely attributed to inhibition of neuronal uptake. Under conditions where neuronal uptake is inefficiently used or saturated, the pharmacological actions of both guanethidine and cocaine would become compromised. The latter possibility may thus apply to adrenergic neurogenic vasoconstriction in the pulmonary artery of the pig.

Neuronal uptake of noradrenaline plays an important role in controlling the extent of the functional response as well as the duration of action in mammalian tissues, Pulmonary Circulation | July-September 2011 | Vol 1 | No 3

and this transporter has also been cloned. [15] It is also recognized that the role of neuronal uptake for noradrenaline-induced neurogenic responses in vascular smooth muscle remains incompletely defined. Thus, in the mesenteric arteries, the density of sympathetic nerve innervation has been cited as the parameter controlling the effects of cocaine on noradrenaline- and neurallymediated vasoconstriction. [12] In the rat caudal artery, a low concentration of noradrenaline (60 nM) exogenously applied or neurally released has been shown to be inactivated and removed by neuronal uptake.[16] However, according to Webb and colleagues[17] in the same tissue, responses to low (0.1-1.0 Hz) but not high (2.0-16 Hz) frequency stimulation were enhanced by cocaine (1.0 µM), at variance with the present findings and those of Stjärne et al.[18] Nonetheless, the apparent half time of relaxation was found not to be different at 1.0 versus 16.0 Hz in the presence of cocaine.[17] The present data obtained in both rat and pig pulmonary arterial strips are in agreement with the literature in showing that cocaine retarded rate of relaxation irrespective of impulse frequency.

A two-compartment working model was proposed for the removal of neurally released noradrenaline.[18] In that model neuronal uptake near active neuromyal junctions saturates very quickly. In addition, released noradrenaline diffuses into a compartment “surround” which rarely saturates irrespective of nerve activity. Associated with the latter compartment is a selective binding substrate “S” acting as a reservoir for sequestration of noradrenaline that can later be released once neural exocytosis has ceased and hence maintains biological function. [18] Our results obtained in pig tissues are at least partly compatible with the two-compartment model. A quantitative description of the mechanical function in isolated blood vessels has been put forward in line with the experimental observations that the relatively slow decline of response after cessation of sympathetic nerve stimulation is mainly due to the slow rate of removal of noradrenaline from the extracellular space by neuronal reuptake and diffusion into surrounding areas.[19] Under such conditions, the effectiveness of both cocaine and guanethidine could be impaired as seems to be the case with our observations in the porcine pulmonary artery, in striking contrast to the rat pulmonary neurogenic vasoconstriction.

Perspective

Sympathetic nerve stimulation in vivo increases vascular resistance in the pulmonary arterial vasculature. Pace and colleagues[20] reported that in dogs, an increase in impedance occurs in the pulmonary arterial bed as a consequence of sympathetic nerve activation. The elevation in impedance in the pulmonary arterial bed is 423

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accompanied by an increase in oscillatory component in input to hydraulic power, and this occurs independently of either positive chronotropic or inotropic effects arising from the right ventricle. The physiological basis for this event is a stiffening of the main pulmonary artery causing augmentation of pulse pressure for the same volume of flow generated by the right ventricular contraction and a more efficient delivery of blood to the lungs.[20] Similar effects have also been reported to occur in cats due to injections of noradrenaline or sympathetic nerve stimulation.[21] Thus, it is proposed that in a state of low cardiac output, α-adrenergic activation in the pulmonary arterial bed increases the fraction of hydraulic power delivered by the right ventricle, which then allows for more efficient propulsion of blood into the pulmonary vascular bed downstream.[21] Certainly, this concept is supported by denser adrenergic innervation in the larger vessels as noted in our current investigation. Regioselective innervation of the pulmonary arterial vessels may permit a more efficient delivery of blood to the lungs with lower work load for the right ventricle: for instance, during low output or intense physical activity sympathetic neuron driven proximal pulmonary vasoconstriction could play an essential role.

ABBREVIATIONS

ATP: Adenosine triphosphate i.m.: Intramuscular EFS: Electrical field stimulation

REFERENCES 1. 2. 3. 4.

Richardson JB. Nerve supply to the lungs. Am Rev Respir Dis 1979;119: 785-802. Downing SE, Lee JC. Nervous control of the pulmonary circulation. Annu Rev Physiol 1980;42:199-210. Wharton J, Haworth SG, Polak JM. Postnatal development of the innervation and paraganglia in the porcine pulmonary arterial bed. J Pathol 1988;154:19-27. Zussman WV. Fluorescent localization of catecholamine stores in the rat lung. Anat Rec 1966;156:19-29.

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5. 6. 7.

8. 9. 10. 11. 12. 13. 14.

15. 16. 17. 18.

19. 20. 21.

El-Bermani AW. Pulmonary noradrenergic innervation of rat and monkey: A comparative study. Thorax 1978;33:167-74. McLean JR, Twarog BM, Bergofsky EH. The adrenergic innervation of pulmonary vasculature in the normal and pulmonary hypertensive rat. J Auton Nerv Syst 1985;14:111-23. Costa M, Majewski H. Facilitation of noradrenaline release from sympathetic nerves through activation of ACTH receptors, betaadrenoceptors and angiotensin II receptors. Br J Pharmacol 1988;95: 993-1001. Kassay-Farkas N, Wyse DG. Prevention of electrochemical oxidation of norepinephrine caused by transmural electrical stimulation. Blood Vessels 1986;23:160-4. Zhong H, Minneman KP. Alpha1-adrenoceptor subtypes. Eur J Pharmacol 1999;375:261-76. Bevan JA. Response of blood vessels to sympathetic nerve stimulation. Blood Vessels 1978;15:17-25. Furness JB, Marshall JM. Correlation of the directly observed responses of mesenteric vessles of the rat to nerve stimulation and noradrenaline with the distribution of adrenergic nerves. J Physiol 1974;239:75-88. Marshall JM. The effect of uptake by adrenergic nerve terminals on the sensitivity of arterial vessels to topically applied noradrenaline. Br J Pharmacol 1977;61:429-32. Jänig W. The Integrative actions of the autonomic nervous system, Neurobiology of Homeostasis. New York: Cambridge University Press; 2006. p. 267-71. Bao JX, Gonon F, Stjärne L. Frequency- and train length-dependent variation in the roles of postjunctional alpha 1- and alpha 2-adrenoceptors for the field stimulation-induced neurogenic contraction of rat tail artery. Naunyn Schmiedebergs Arch Pharmacol 1993;347:601-16. Pacholczyk T, Blakely RD, Amara SG. Expression cloning of a cocaineand antidepressant-sensitive human noradrenaline transporter. Nature 1991;350:350-4. Wyse DG. Inactivation of neural and exogenous norepinephrine in rat tail artery studied by the oil immersion technique. J Pharmacol Exp Ther 1976;198:102-11. Webb RC, Vanhoutte PM, Bohr DF. Inactivation of released norepinephrine in rat tail artery by neuronal uptake. J Cardiovasc Pharmacol 1980;2:121-32. Stjärne L, Bao JX, Gonon F, Msghina M. Nerve activity-dependent variations in clearance of released noradrenaline: Regulatory roles for sympathetic neuromuscular transmission in rat tail artery. Neuroscience 1994;60:1021-38. Bennett MR, Farnell L, Gibson WG. A quantitative description of the contraction of blood vessels following the release of noradrenaline from sympathetic varicosities. J Theor Biol 2005;234:107-22. Pace JB, Cox RH, Alvarez-Vara F, Karreman G. Influence of sympathetic nerve stimulation on pulmonary hydraulic input power. Am J Physiol 1972;222:196-201. Piene H. The influence of pulmonary blood flow rate on vascular input impedance and hydraulic power in the sympathetically and noradrenaline stimulated cat lung. Acta Physiol Scand 1976;98:44-53.

Source of Support: This work was supported by a grant from Natural Sciences and Engineering Research Council of Canada, Conflict of Interest: None declared.

Pulmonary Circulation | July-September 2011 | Vol 1 | No 3

C ase Repor t

Isolated large vessel pulmonary vasculitis as a cause of chronic obstruction of the pulmonary arteries Guy Hagan1, Deepa Gopalan2, Colin Church3, Doris Rassl4, Chetan Mukhtyar5, Trevor Wistow6, Chim Lang7, Pasupathy Sivasothy8, Susan Stewart4, David Jayne9, Karen Sheares1, Steven Tsui10, David P. Jenkins10, and Joanna Pepke-Zaba1 Pulmonary Vascular Disease Unit, Papworth Hospital, London, 2Department of Radiology, Papworth Hospital, London, Pulmonary Vascular Disease Unit, Golden Jubilee Hospital, Glasgow, 4Department of Pathology, Papworth Hospital, London, 5 Department of Rheumatology, Norfolk and Norwich University Hospital, 6Department of Cardiology, Norfolk and Norwich University Hospital, 7Department of Cardiology, Ninewells Hospital, Dundee, 8Department of Respiratory Medicine 9Vasculitis, Addenbrooke’s Hospital, Cambridge, 10Department of Cardiothoracic Surgery, Papworth Hospital, London, UK 1

ABSTRACT Isolated pulmonary artery involvement by large vessel vasculitis is rare. This case report describes two patients with large vessel pulmonary vasculitis initially thought to have chronic thromboembolic pulmonary hypertension who had their diagnosis revised following pulmonary endarterectomy surgery. Advances in imaging techniques such as positron emission tomography and magnetic resonance imaging have permitted complementary radiological methods of diagnosis and follow up of large vessel disease and these are discussed in conjunction with the immunosuppressive and operative management of these patients. Key Words: large vessel vasculitis, pulmonary artery, pulmonary endarterectomy

INTRODUCTION

CASE REPORT

Chronic thromboembolic pulmonary hypertension (CTEPH) is associated with the obstruction of the pulmonary arteries by organized thrombo-embolic material resulting in restriction of blood flow to affected segmental branches and vascular remodelling in the unobstructed pulmonary arteries. The definitive treatment is pulmonary endarterectomy (PEA), which has proven symptomatic and survival benefit.[1]

Case 1

As the national PEA center, our institution has carried out over 700 PEA operations. Many more patients have been referred for assessment of possible CTEPH or other causes of pulmonary artery obstruction. A small proportion of patients undergoing PEA have had alternative diagnoses made at or following surgery. We describe two patients who had a revised diagnosis of isolated large vessel pulmonary artery vasculitis following attempted PEA surgery. Address correspondence to:

Dr. Joanna Pepke-Zaba Papworth Hospital, Papworth Everard Cambridgeshire, CB23 3RE Email: joanna.pepkezaba@papworth.nhs.uk Pulmonary Circulation | July-September 2011 | Vol 1 | No 3

A 40-year-old Caucasian male was referred with a 2-month history of exertional breathlessness and had New York Heart Association (NYHA) Class II performance status at presentation. Prior to referral, he had been therapeutically anti-coagulated for 5 months with a possible diagnosis of CTEPH based on imaging at his local hospital. Repeat CT Pulmonary Angiogram (CTPA) (Fig. 1) demonstrated extensive proximal narrowing of the right and left pulmonary artery wall with concentric soft tissue thickening, and occlusion of the right upper lobe and left upper lobe apical segmental branches. Full blood count and renal function were normal. C-reactive protein (CRP) Access this article online Quick Response Code:

Website: www.pulmonarycirculation.org DOI: 10.4103/2045-8932.87312 How to cite this article: Hagan G, Gopalan D, Church C, Rassl D, Mukhtyar C, Wistow T, et al. Isolated large vessel pulmonary vasculitis as a cause of chronic obstruction of the pulmonary arteries. Pulm Circ 2011;1:425-9.

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was 77 mg/L with an erythrocyte sedimentation rate (ESR) of 79 mm/hr. Anti nuclear antibodies (ANA) and antineutrophil cytoplasmic antibodies (ANCA) were negative. MRI pulmonary angiogram confirmed the distribution of disease and also demonstrated smooth plaque like focal intimal thickening in both proximal main pulmonary arteries. Due to concerns over the possible diagnosis of a pulmonary artery sarcoma, positron emission

tomography CT (PET/CT) was performed (Fig. 2). This showed intermediate uptake in the distal right main pulmonary artery. Pre-operative RHC demonstrated a mean pulmonary artery pressure (mPAP) of 32 mmHg with a cardiac index of 2 l/min/m2.

Figure 1: Axial CTPA image showing concentric soft tissue thickening around the left pulmonary artery (arrow).

As treatment for his pulmonary artery vasculitis, this patient was commenced on 1 mg/kg of prednisolone

Transaxial

Coronal

He underwent PEA for symptomatic benefit and to provide a histological diagnosis. During surgery, the pulmonary arteries were thick walled and fibrotic, making the operation technically difficult. A limited endarterectomy was performed bilaterally. The mPAP fell to 25 mmHg on Day One post-operation. Histology of the excised material revealed transmural lymphohistocytic infiltration with a giant cell granulomatous vasculitis (Fig. 3). There was no evidence of acid-fast bacilli or fungi. Subsequent HIV testing and syphilis serology were negative. Coronary angiography revealed good flow with no coronary artery aneurysms. Ear nose and throat, renal and ophthalmological evaluations excluded systemic vasculitic involvement.

Transaxial

Coronal

Transaxial

Coronal

Figure 2: Selected images from 18 FDG-positron emission tomographic study demonstrates increased tracer uptake in the right pulmonary artery (red cross bars) in keeping with active phase of the vasculitic process. 426

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and pulsed cyclophosphamide, which was subsequently substituted for azathioprine (1.5 mg/kg). Prednisolone was subsequently tapered down monitoring symptoms and MRI appearance. PET/CT scan 15 months after his PEA showed low-level uptake in the lateral wall of the ascending aorta and the medial wall of the pulmonary artery. A repeat MRI angiogram demonstrated new 3 mm soft tissue thickening in the ascending aorta and the aortic arch, as well as persisting fusiform narrowing of the right main pulmonary artery with proximal segment occlusions of the right upper and left lower lobes, suggesting nonprogression of the pulmonary changes on a background of systemic large vessel involvement. In view of these changes and persistently raised inflammatory markers his immunosuppression was changed to mycophenolate mofetil (MMF) with the subsequent addition of infliximab. His condition has been stable for the last three years with normal ESR and CRP. Subsequent MRI pulmonary angiography continues to show the previously described narrowing and occlusion of the pulmonary arteries, presumably representing “burnt out” vasculitis. He continues on anticoagulation and low dose a combination of infliximab (3 mg/kg 8 weekly) and MMF (3 g/day).

Figure 3: Haematoxylin and eosin stain (×100) showing a dense inflammatory cell infiltrate including acute and chronic inflammatory cells with histiocytes and focal giant cells involving the vascular intima and extending into the rim of media included in the specimen.

Case 2

A 53-year-old woman was referred with a two year history of increasing breathlessness and initial investigations suggested multiple pulmonary emboli with ventilation perfusion (VQ) scintigraphy demonstrating perfusion defects in the right mid zone and left mid and lower zones. There was no significant improvement with anticoagulation and her performance status was NYHA class III. An echocardiogram revealed a dilated right ventricle with impaired function. Right heart catheterisation (RHC) demonstrated a mPAP of 41 mmHg with a cardiac index of 1.7 l/min/m2. CTPA (Fig. 4) demonstrated extravascular soft tissue thickening adjacent to the main pulmonary arteries. In addition, there were proximal stenosis of the right and left main pulmonary arteries with additional soft tissue encasement and occlusion of the proximal lingular and lower lobe arteries. Catheter directed pulmonary angiogram (Fig. 5) confirmed the CTPA findings and also demonstrated a marked stretching of the right main pulmonary artery with concentric soft tissue thickening. Baseline ESR was 25 mm/hr. and autoantibody screen was negative.

A PEA was performed. At operation there was a rigid mass of fibrous tissue encasing the pulmonary trunk and both main pulmonary arteries, extending out into the left sub-segmental vessels. This was dissected out and the central pulmonary arteries enlarged with a bovine pericardial patch. Histology of the excised pulmonary Pulmonary Circulation | July-September 2011 | Vol 1 | No 3

Figure 4: Axial multi-detector CT image showing concentric thickening around the pulmonary artery with a stenosis at the origin of the left main pulmonary artery (arrow).

Figure 5: Still image from catheter pulmonary angiogram demonstrates concentric soft tissue thickening around the right pulmonary artery resulting in stretching of the artery and luminal irregularity (arrow). 427

Hagan et al.: Isolated large vessel pulmonary vasculitis

artery wall demonstrated a chronic inflammatory lymphoplasmacytic mass, with no evidence of giant cells or granulomas, extending into the tunica media, implying a vasculitic process. RHC three months after PEA showed a mPAP of 30 mmHg, with cardiac index of 2.36 l/min/m2. PET/CT scanning undertaken at the same time showed increased uptake in the aorta and left lower lobe which corresponded to an area which could not be removed at operation. A diagnosis of large vessel vasculitis was made. She was commenced on prednisolone (1 mg/kg) and azathioprine (1.5 mg/kg), with subsequent weaning of the prednisolone dose. At 12 month follow up her sixminute walk had improved from 260 m preoperatively to 380 m.

DISCUSSION

We have described two patients with large vessel vasculitis who presented with breathlessness in the absence of systemic vasculitic manifestations, with initial imaging demonstrating isolated pulmonary pathology. Raised inflammatory markers were present at the time of assessment. Both patients were Caucasian and in their fifth or sixth decade of life. The causes of chronic obstruction of the pulmonary arteries can be subdivided into endoluminal disease (such as chronic thromboembolic disease or tumor) or extrinsic compression (such as lymphadenopathy or fibrosing mediastinitis). Large vessel pulmonary arteritis can cause either endoluminal blockage or extrinsic compression and therefore may give a similar radiological and clinical presentation to that of chronic thromboembolic disease.[2]

The incidence of isolated pulmonary artery vasculitis is unknown but is thought to be rare. Takayasu arteritis can produce isolated pulmonary artery vasculitis. [3,4] The incidence of Takayasu arteritis in the UK is 0.8/ million/ year.[5] The incidence of pulmonary artery involvement in Takayasu arteritis ranges from 14.3%[6] to 86%;[7] involvement is often subclinical.[6] Pulmonary artery involvement has also been reported due to giant cell arteritis and Behçet’s disease, although the latter tends to cause pulmonary artery aneurysms or mass lesions rather than vascular obstruction.[8] Radiological features on CTPA that may suggest large vessel pulmonary vasculitis rather than CTEPH include wall thickening and contrast enhancement in the early phases, with mural calcium deposition and luminal stenosis in more chronic phases. Circumferential arterial wall thickening is not present in CTEPH.[8] Aortic wall thickening with mural enhancement on gadolinium contrast MRI in Takayasu arteritis has been described 428

which may correlate with disease activity[9] and MRI was utilized for follow up in Case 1. PET scanning has an established role for diagnosis and is included with MRI in contemporary large vessel vasculitis guidelines,[10] but its role in disease monitoring is less well defined. The evidence base for MRI and PET follow up is mostly in aortitis and it is likely that their role has not been specifically studied in isolated pulmonary vasculitis due to the rarity of the condition.

In cases from Japan the diagnosis is based on certain radiological features and HLA types associated with Takayasu’s.[3,4] It is unclear how applicable this approach is to patients of European background and a requirement for a tissue diagnosis is likely to be present in most cases. We have used PEA (with bovine patch enlargement of the pulmonary artery in Case 2) as the operation for symptom relief and diagnosis. There are case reports of a pericardial patch to enlarge the pulmonary artery stenosis, [3] or the use of a bypass graft.[4]

CONCLUSIONS

In summary, isolated large vessel pulmonary artery vasculitis may mimic CTEPH. Entertaining the diagnosis preoperatively is of benefit as the patient can be informed about the differential diagnosis and the surgeon can plan to undertake a reconstructive procedure of a pulmonary artery as well as PEA. Evidence of a pulmonary arterial vasculitis should lead to examination of systemic large vessels to define the extent and severity of disease. Large vessel vasculitis responds to immunosuppressive therapy which arrests disease progression and may improve pulmonary haemodynamics. Ideally reconstructive surgery should be undertaken when the disease is in remission[10] and so the timing of any surgery could be influenced by disease activity and response to therapy. The possibility of these patients subsequently developing a more widespread vasculitis is important to consider. On PET/CT scan, both of our patients were later found to have asymptomatic aortitis. Finally, the advent of modern imaging techniques such as MRI and PET has provided clinicians with novel tools allowing a greater ability to diagnose and monitor response to treatment in patients with this uncommon condition.

ACKNOWLEDGMENTS

The authors acknowledge the contributions of Prof. David Scott (Norfolk and Norwich University Hospitals) and Prof. Andrew Peacock and Dr. Martin Johnson (Golden Jubilee Hospital, Glasgow) who are involved in the continued care of the patients. Pulmonary Circulation | July-September 2011 | Vol 1 | No 3

Hagan et al.: Isolated large vessel pulmonary vasculitis

REFERENCES

2. 3. 4.

Condliffe R, Kiely DG, Gibbs JS, Corris PA, Peacock AJ, Jenkins DP, et al. Improved outcomes in medically and surgically treated chronic thrombolic pulmonary hypertension. Am J Resp Crit Care Med 2008;177:1122-7. Kerr KM, Auger WR, Fedullo PF, Channick RH, Yi ES, Moser KM. Large vessel pulmonary arteritis mimicking chronic thromboembolic disease. Am J Resp Crit Care Med 1995;152;367-73. Yamazaki I, Ichikawa Y, Ishii M, Hamada T, Kajiwara H. Surgical case of isolated pulmonary Takayasu’s arteritis. Circ J 2005;69:500-2. Nakajima N, Masuda M, Imamaki M, Ishida A, Tanabe N, Kuriyama T. A case of pulmonary artery bypass surgery for a patient with isolated Takayasu pulmonary arteritis and a review of the literature. Ann Thorac Cardiovasc Surg 2007;13:267-71. Watts R, Al-Taiar A, Mooney J, Scott D, Macgregor A. The epidemiology of Takayasu arteritis in the UK. Rheumatology 2009;48:1008-11.

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8. 9. 10.

Sharma S, Kamalakar T, Rajani M, Talwar KK, Shrivastava S. The incidence and patterns of pulmonary artery involvement in Takayasu’s arteritis. Clin Radiol 1990;42:177-81. Yamato M, Lecky JW, Hiramatsu K, Kohda E. Takayasu arteritis: Radiographic and angiographic findings in 59 patients. Radiology 1986;161:329-34. Marthen K, Schnyder P, Schirg E, Prokop M, Rummeny EJ, Engelke C. Pattern-based differential diagnosis in pulmonary vasculitis using volumetric CT. AJR Am J Roentgenol 2005;184:720-33. Choe YH, Han BK, Koh EM, Kim DK, Do YS, Lee WR. Takayasu’s arteritis: Assessment of disease activity with contrast enhanced MR imaging. Am J Roent 2000;175:505-11. Mukhtyar C, Guillevin L, Cid MC, Dasgupta B, de Groot K, Gross W, et al. EULAR recommendations for the management of large vessel vasculitis. Ann Rheum Dis 2009;68:318-23.

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

429

Snapshot

Survival in pulmonary arterial hypertension: A brief review of registry data Sunil Pauwaa, Roberto F. Machado, and Ankit A. Desai University of Illinois at Chicago, Chicago, Illinois, USA

Pulmonary arterial hypertension (PAH) is a severe and fatal disease with a prevalence of 15 cases in a million[1] and characterized by increased pulmonary vascular resistance and right heart failure. PAH can be idiopathic (IPAH), familial (FPAH), or associated with other disorders. In 1984 the National Institute of Health (NIH) compiled the first large registry of PAH patients (all IPAH, FPAH, and anorexigen-associated) confirming poor survival and prognosis.[2] In an era of PAH-specific therapies, subsequent PAH registries (the Pulmonary Hypertension Connection [PHC] registry,[3] the French registry,[4] and the Registry to Evaluate Early and Long-term Pulmonary Arterial Hypertension Disease Management [REVEAL][5]) have demonstrated improved prognosis (Table 1).

Each registry is defined by unique strengths and weaknesses. The NIH registry is a landmark accomplishment, but may not be as applicable to the modern patient on PAH-specific therapy. The PHC validated many of the findings of the NIH registry, including various risk factors, and demonstrated improvements in survival. The REVEAL registry is the largest and, in contrast to other registries, includes associated forms of PAH. For the first time, this last distinction opens the potential applicability of the registry findings to nearly all patients with PAH beyond IPAH, HPAH, and anorexigen-associated PAH. Importantly, although both demonstrate consistency in improvement from the NIH registry, the REVEAL and the French registries have reported a significant difference

Table 1: Incidence and survival of PAH based on different registries Registry NIH (n=194) (1981-1988)

PH connection (n=282) (1991-2007)

French (n=354) (2002-2003)

Population characteristics (%)

Risk factors

IPAH (NA) HPAH (NA) Anorexigen (NA)

↓ DLCO Reynaud phenomenon NYHA Class III or IV ↑mean RAP, ≥20 mmHg ↑mean PAP, ≥85 mmHg ↓CI (<2 L/min/m2)

IPAH (84.7) HPAH (8.2) Anorexigen (7.1)

IPAH (74.6) HPAH (7.3) Anorexigen (18.1)

HR (OR*) 0.97* 2.11* 1.93* 1.99* 1.16* 0.62*

Age at diagnosis, per decade increase Exercise capacity, per 1-MET increase NYHA Class III NYHA Class IV ↑RAP, per 5 mmHg increase ↓CI, per 1-L/min/m2 Low 6 MWD ↓ CO ↑RAP Male sex

1.18 0.86 3.96 5.16 1.37 0.56 0.996 0.746 1.06 1.00

Survival Year

82.9

67.1

58.2

Access this article online Quick Response Code:

Website: www.pulmonarycirculation.org DOI: 10.4103/2045-8932.87314 How to cite this article: Pauwaa S, Machado RF, Desai AA. Survival in pulmonary arterial hypertension: A brief review of registry data. Pulm Circ 2011;1:430-1.

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Pauwaa et al.: Survival in pulmonary arterial hypertension

Table 1: Continued Registry

Population characteristics (%)

Risk factors

REVEAL (n=2716)

IPAH (46.5) HPAH (2.9) CHD (11.8) CTD (23.9) Portal HTN (5.1) Drugs/toxins (4.9) HIV infection (1.9) Other (3.1)

PAH-portal HTN PAH-CTD PAH-Family history Mean age>60 yrs old Renal insufficiency NYHA Class III NYHA Class IV 6 MWD<165 m Mean RAP>20 mmHg PVR>32 Wood units HR>92 bpm Resting SBP<110 mmHg BNP>180 pg/ml % predicted DLCO≤32% Pericardial effusion

(2006)

HR (OR*) 3.6 1.59 2.17 2.18 1.90 1.41 3.13 1.68 1.79 4.08 1.39 1.67 1.97 1.46 1.35

Survival Year

1 - low risk#

>95

1 - average risk#

90-95

1 - moderate high risk#

85-90

1 - high risk#

70-85

1 - very high risk#

<70

IPAH: idiopathic pulmonary arterial hypertension; HPAH: hereditary pulmonary arterial hypertension; DLCO: diffusion capacity of the lung for carbon monoxide; NYHA: New York Heart Association; RAP: right atrial pressure; CI: cardiac index; HR: hazard ratio; OR: odds ratio; *: these values represent OR while others represent HR; MET: Metabolic equivalent; 6MWD: 6 minute walk distance; CO: cardiac output; CHD: congenital heart disease; CTD: connective tissue disease; HTN: hypertension; SBP: systolic blood pressure; BNP: B-type natriuretic peptide; NA: Not available; #: low, average, moderate high, high and very high risks with 1, 2, 3, 4, 5, and 6 risk factors, respectively

in survival data at 1 year (95% and 83%, respectively). This discrepancy may be attributed to multiple factors including the consideration, especially in PAH clinical trials, of the proportion of incident versus prevalent patient populations during the estimation for prognosis. Specifically, given the association of poor survival with a subset of PAH patients with very high risk factors, each registry may have different contributions of survival bias based on the individual representation of these patients within each registry. Finally, although all registries establish risk factors associated with adverse outcomes, they fail to incorporate prognosis information after risk factor modification. Nonetheless, there is considerable overlap and validation of established risk factors by the various reports with new makers of prognosis based on functional status and other clinical markers that extend beyond the three hemodynamic-based parameters established by the NIH registry. The latter registries have also identified factors associated with protection from poor outcomes. Several risk scores (equations) have subsequently been developed based on the associated risk factors and are used to predict mortality in patients with PAH. The REVEAL registry, for example, provides the following

Pulmonary Circulation | July-September 2011 | Vol 1 | No 3

equation to estimate survival in patient’s with PAH: P(t) = [H(t)] A(x,y,z), where x, y, and z represent the values for individual risk factors. Similar scores are provided in the other registries.

REFERENCES 1. 2. 3. 4.

McLaughlin VV, Suissa S. Prognosis of pulmonary arterial hypertension: The power of clinical registries of rare diseases. Circulation 2010;122:106-8. D’Alonzo GE, Barst RJ, Ayres SM, Bergofsky EH, Brundage BH, Detre KM, et al. Survival in patients with primary pulmonary hypertension. Results from a national prospective registry. Ann Intern Med 1991;115:343-9. Thenappan T, Shah SJ, Rich S, Gomberg-Maitland M. A USA-based registry for pulmonary arterial hypertension: 1982-2006. Eur Respir J 2007;30:1103-10. Humbert M, Sitbon O, Chaouat A, Bertocchi M, Habib G, Gressin V, et al. Survival in patients with idiopathic, familial, and anorexigen-associated pulmonary arterial hypertension in the modern management era. Circulation 2010;122:156-63. Benza RL, Miller DP, Gomberg-Maitland M, Frantz RP, Foreman AJ, Coffey CS, et al. Predicting survival in pulmonary arterial hypertension: Insights from the registry to evaluate early and long-term pulmonary arterial hypertension disease management (REVEAL). Circulation 2010;122:164-72.

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

431

PATENT

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 •

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: •

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 •

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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BSP05J11030_Pulmonary_Circulation_Ad_210mmx297mm_V1_AW.indd 1

September 2011

Pulmonary Circulation | July-September 2011 | Vol 1 | No 3

29/09/2011 14:38

CHEST

Main Inclusion Criteria: Male and female patients aged 18–80 years with •

Inoperable CTEPH

•

Pulmonary hypertension that persists or is recurrent after PEA

Main Exclusion Criteria: •

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 •

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 | July-September 2011 | Vol 1 | No 3 BSP05J11030_Pulmonary_Circulation_Ad_210mmx297mm_V1_AW.indd 2

September 2011

433 29/09/2011 14:38

The Pulmonary Vascular Research Institute is proud to present

The 6th Annual General Meeting and 5th Scientific Workshops & Debates 2012, at the Protea President Hotel in Cape Town, South Africa

The 2012 PVRI Conference program includes the following themes: • Pulmonary Vascular Disease in Africa • Clinical Trials in PVD: Ethical and Moral Issues • Translational Medicine: Challenges in the Development of New Drugs for Pulmonary Vascular Disease

TUESDAY, 7 FEBRUARY – FRIDAY, 10 FEBRUARY 2012 FOR THE FULL PROGRAM & MEETING REGISTRATON, PLEASE SEE WWW.PVRI.INFO 434

Pulmonary Circulation | July-September 2011 | Vol 1 | No 3

org/

Jason X.-J. Yuan, MD, PhD Nicholas W. Morrell, MD Harikrishnan S., MD

Cir

Senior Editor Ghazwan Butrous, MD

First P

Executive Editor Harikrishnan S., MD

Editors Kurt R. Stenmark, MD Pulmonary C Kenneth D. Bloch, MD Stephen L. Archer, MD of pulmonary c Marlene Rabinovitch, MD review article þ The first peer-‐reviewed medical journal dedicated pulmonary vascular disease. Joe G.N.to Garcia, MD vascular disea þ Pulmonary Circulation is the only journal dStuart evoted Rich, to the fMD ield of pulmonary circulation publishing original forefront of the Martin R. Wilkins, research articles, review articles, case reports, snapshots, and pMD erspectives in pulmonary vascular disease clinicians in re Hossein A. Ghofrani, MD and lung injury. clinical diagnos Fike, MD between basic scientists and clinicians in þ The new journal stands at the forefront of Candice the critical D. collaboration Seeger, MDand treatment of pulmonary vascular research on the pulmonary circulation system Werner and clinical diagnosis Sheila G. Haworth, MD diseases. First d Patricia A. Thistlethwaite, MD, PhD þ Topics of research articles include clinical Chen trials, clinical research, drug development, basic science research, Wang, MD, PhD epidemiology and biomedical informatics, diagnostic and therapeutic guidelines. Antonio A. Lopes, MD

þ For more information: http://www.pulmonarycirculation.org/ Scientific Advisory þ NBoard o charge for processing your manuscript Clinical trials Robert F. Growver, MD, PhD þ No charge for color photographs Charles A. Hales, MD Clinical researc Editors-in-Chief þ No charge for publication Joseph Loscalzo, MD Jason X.-‐J. Yuan, MD PhD Drug developm Nicholas W. Morrell, MD John B. West, MD, PhD, þ IDSc mmediate publication on acceptance Harikrishnan S., MD Basic science r Magdi H. Yacoub, MD, DSc, FRS þ Open access

Senior Editor Ghazwan Butrous, MD PhD Executive Editor Harikrishnan S., MD Editors Kurt R. Stenmark, MD Kenneth D. Bloch, MD Stephen L. Archer, MD Marlene Rabinovitch, MD First Peer-Reviewed Journal Dedicated to Joe G.N. Vascular Garcia, Disease MD Pulmonary Stuart Rich, MD Martin R. Wilkins, MD Pulmonary Circulation is the only journal devoted to the field research articles of pulmonary circulationHossein publishing original A. Ghofrani, MD , review articles, case reports and perspectives in pulmonary Candice . Fike, Mstands D at the vascular disease and lung injury. TheDnew journal forefront of the critical collaboration basic and Werner between Seeger, Mscientists D clinicians in research on the pulmonary circulatory system and clinical diagnosis and treatment ofG pulmonary vascular Sheila . Haworth, Mdiseases D Patricia A. Thistlethwaite, MD PhD First dedicated journal on the pulmonary circulation Chen Wang, MD PhD Immediate publication on acceptance Antonio A. processing Lopes, Mmanuscript D No charge for No charge for color photographs Clinical trials No charge for publication Scientific Advisory Clinical research Open Board access Drug development Robert F. Grover, MD PhD Basic science research Charles A. Hales, MD Epidemiology and biomedical informatics Joseph Loscalzo, MD Diagnostic and therapeutic guidelines John B. West, MD PhD DSc Submit yourMagdi manuscript at: MD DSc FRS H. Yacoub, http://www.journalonweb.com/PC/

Pulmonary Circulation

Epidemiology a Diagnostic and

þ Submit your manuscript at: http://www.journalonweb.com/PC/

Subm http://

Pulmona is an internatio The Pulmonary Vascular Research Institute (PVRI) is an international non-‐profit medical research organization dedicated to For information see: dedicated to increasing the awareness and more knowledge of pulmonary vascular diseases and facilitating advances in pulmonary v the treatment of affected people worldwide. http://www.pulmonarycirculation.org/ in the tr

Pulmonary Vascular Research Institute (PVRI) is an international, non-profit medical research organization dedicated to increasing the awareness and knowledge of pulmonary vascular diseases and facilitating advances in the treatment of affected people worldwide

CONTENTS Editorial The world of pulmonary vascular disease

Jason X.-J. Yuan, Nicholas W. Morrell, S. Harikrishnan, and Ghazwan Butrous

303

Review Articles The genetics of pulmonary arterial hypertension in the post-BMPR2 era Joshua P. Fessel, James E. Loyd, and Eric D. Austin

COPD/emphysema: The vascular story

Norbert F. Voelkel, Jose Gomez-Arroyo, and Shiro Mizuno

Surgical treatment of pulmonary hypertension: Lung transplantation Jason Long, Mark J. Russo, Charlie Muller, and Wickii T. Vigneswaran

Apelin and pulmonary hypertension

Charlotte U. Andersen, Ole Hilberg, Søren Mellemkjær, Jens E. Nielsen-Kudsk, and U. Simonsen

Epigenetic mechanisms of pulmonary hypertension

Gene H. Kim, John J. Ryan, Glenn Marsboom, and Stephen L. Archer

MicroRNAs-control of essential genes: Implications for pulmonary vascular disease Sachindra R. Joshi, Jared M. McLendon, Brian S. Comer, and William T. Gerthoffer

305 320 327 334

347 357

Research Articles Blood flow redistribution and ventilation-perfusion mismatch during embolic pulmonary arterial occlusion K. S. Burrowes, A. R. Clark, and M. H. Tawhai

Pharmacogenomics in pulmonary arterial hypertension: Toward a mechanistic, target-based approach to therapy Sami I. Said and Sayyed A. Hamidi

Idiopathic and heritable PAH perturb common molecular pathways, correlated with increased MSX1 expression

S1P4 receptor mediates S1P-induced vasoconstriction in normotensive and hypertensive rat lungs Hiroki Ota, Michelle A. Beutz, Masako Ito, Kohtaro Abe, Masahiko Oka, and Ivan F. McMurtry

Neurogenic responses in rat and porcine large pulmonary arteries Daniel J. Duggan, Detlef Bieger, and Reza Tabrizchi

365

377

383

389 399

405 419

425

Snapshot Survival in pulmonary arterial hypertension: A brief review of registry data Sunil Pauwaa, Roberto F. Machado, and Ankit A. Desai

430

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