Pulm Circ Apr-Jun 2012

www.PulmonaryCirculation.org Volume 2, Number 2 (April‐June 2012)

Pulmonary Circulation • Volume 2 • Issue 2 • April-June 2012 • Pages 137-270

Pulmonary Circulation

ISSN: 2045-8932 E-ISSN: 2045-8940

Three-dimensional reconstructions of the left and right ventricles in a patient with pulmonary hypertension (please see COVER PHOTO inside)

A Journal of the Pulmonary Vascular Research Institute

COVER PHOTO: Three-dimensional reconstructions of the left and right ventricles in a patient with pulmonary hypertension. Comparative analyses of right ventricular structure and function in subjects with and without pulmonary hypertension using three-dimensional cardiac reconstructions of two dimensional echocardiography formed the basis of this project. Cardiac Imaging Research Lab, Department of Cardiology, University of Washington. Credits: Peter J. Leary and Florence H. Sheehan.

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

Pulmonary Circulation

ISSN 2045-8932, E-ISSN 2045-8940

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

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

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

Robert F. Grover, MD, PhD (Denver, USA) E. Kenneth Weir, MD (Mineapolis, USA) Charles A. Hales, MD (Boston, USA)

Executive Editor

Harikrishnan S., MD (Trivandrum, India) 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)

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

Editorial Board

Steven H. Abman, MD, USA Serge Adnot, MD, France Vera D. Aiello, MD, Brazil Almaz Aldashev, MD, PhD, Kyrgyz Republic Micheala Aldred, PhD, USA 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 Akiko Hata, PhD, USA Jianguo He, MD, China Jan Herget, MD, PhD, Czech Republic Nicholas S. Hill, MD, USA Marius M. Hoeper, MD, Germany Eric A. Hoffman, PhD, USA Yuji Imaizumi, PhD, Japan Dunbar Ivy, MD, USA Jeffrey R. Jacobson, MD, USA Roger Johns, MD, PhD, USA Peter L. Jones, PhD, USA Naftali Kaminski, MD, USA Chandrasekharan C. Kartha, MD, India Steven M. Kawut, MD, USA Ann M. Keogh, MD, Australia Nick H. Kim, MD, USA Sung Joon Kim, MD, PhD, Korea James R. Klinger, MD, USA Stella Kourembanas, MD, USA Michael J. Krowka, MD, USA Thomas J. Kulik, MD, USA R. Krishna Kumar, MD, DM, India Steven Kymes, PhD, USA David Langleben, MD, Canada Timothy D. Le Cras, PhD, USA Normand Leblanc, PhD, USA Fabiola Leon-Velarde, MD, Peru Irena Levitan, PhD, USA Jose Lopez-Barneo, MD, PhD, Spain Wenju Lu, MD, PhD, China Roberto Machado, MD, USA Margaret R. MacLean, PhD, UK Michael M. Madani, MD, USA Ayako Makino, PhD, USA Asrar B. Malik, PhD, USA Jess Mandel, MD, USA Michael A. Matthay, MD, USA

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

Editorial Staff

Julio Sandoval, MD, Mexico Maria V.T. Santana, MD, Brazil Bhagavathula K. Sastry, MD., India Jean-Pierre Savineau, MD, France 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 Yunchao Su, MD, PhD, USA 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 Yong-Xiao Wang, MD, PhD, USA 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

Christina N. Holt (Chicago, USA), chris88@uic.edu Nikki Krol (London, UK), nkrol@imperial.ac.uk Karen Gordon (Chicago, USA), gordonk@uic.edu

Pulmonary Circulation | April-June 2012 | Vol 2 | No 2

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Pulmonary Circulation

| April-June 2012 | Vol 2 | No 2 |

An official journal of the Pulmonary Vascular Research Institute

CONTENTS LEFT TO RIGHT: 141, 159, 159, 166

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inside front cover

General Information

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Editors and Board Members

Editorial Science is an endless frontier: Encouraging translational research in pulmonary vascular disease Jason X.-J. Yuan, Nicholas W. Morrell, S. Harikrishnan, and Ghazwan Butrous

Review Articles Clinical perspectives with long-term pulsed inhaled nitric oxide for the treatment of pulmonary arterial hypertension Robyn J. Barst, Richard Channick, Dunbar Ivy, and Brahm Goldstein

Severe pulmonary hypertension: The role of metabolic and endocrine disorders Harm J. Bogaard, Aysar Al Husseini, Laszlo Farkas, Daniela Farkas, Jose Gomez-Arroyo, Antonio Abbate, and Norbert F. Voelkel

Evaluation of patients with chronic thromboembolic pulmonary hypertension for pulmonary endarterectomy William R. Auger, Kim M. Kerr, Nick H. Kim, and Peter F. Fedullo

Diagnosis and management of pulmonary hypertension associated with left ventricular diastolic dysfunction Vinicio A. de Jesus Perez, Francois Haddad, and Roham T. Zamanian

Research Articles Mesenchymal stem cell-mediated reversal of bronchopulmonary dysplasia and associated pulmonary hypertension

Georg Hansmann, Angeles Fernandez-Gonzalez, Muhammad Aslam, Sally H. Vitali, Thomas Martin, S. Alex Mitsialis, and Stella Kourembanas

137

139

148

155

163

170

Group V phospholipase A2 increases pulmonary endothelial permeability through direct hydrolysis of the cell membrane

182

Furegrelate, a thromboxane synthase inhibitor, blunts the development of pulmonary arterial hypertension in neonatal piglets

193

Nilda M. Muñoz, Anjali Desai, Lucille N. Meliton, Angelo Y. Meliton, Tingting Zhou, Alan R. Leff, and Steven M. Dudek

Dinesh K. Hirenallur-S., Neil D. Detweiler, Steven T. Haworth, Jeaninne T. Leming, John B. Gordon, and Nancy J. Rusch

ii

Pulmonary Circulation | April-June 2012 | Vol 2 | No 2

CONTENTS continued

Metabolomic analysis of bone morphogenetic protein receptor type 2 mutations in human pulmonary endothelium reveals widespread metabolic reprogramming Joshua P. Fessel, Rizwan Hamid, Bryan M. Wittmann, Linda J. Robinson, Tom Blackwell, Yuji Tada, Nobuhiro Tanabe, Koichiro Tatsumi, Anna R. Hemnes, and James D. West

Leptin levels predict survival in pulmonary arterial hypertension

Adriano R. Tonelli, Metin Aytekin, Ariel E. Feldstein, and Raed A. Dweik

201

214

Mast cell number, phenotype, and function in human pulmonary arterial hypertension

220

Activation of the unfolded protein response is associated with pulmonary hypertension

229

Distinct responses to hypoxia in subpopulations of distal pulmonary artery cells contribute to pulmonary vascular remodeling in emphysema

241

Samar Farha, Jacqueline Sharp, Kewal Asosingh, Margaret Park, Suzy A. A. Comhair, W. H. Wilson Tang, Jim Thomas, Carol Farver, Fred Hsieh, James E. Loyd, and Serpil C. Erzurum

Michael E. Yeager, Monica B. Reddy, Cecilia M. Nguyen, Kelley L. Colvin, D. Dunbar Ivy, and Kurt R. Stenmark

L. S. Howard, A. Crosby, P. Vaughan, A. Sobolewski, M. Southwood, M. L. Foster, E. R. Chilvers, and N. W. Morrell

Case Reports Partial anomalous pulmonary venous return presenting with adult-onset pulmonary hypertension Edmund H. Sears, Jason M. Aliotta, and James R. Klinger

Massive dilatation of the pulmonary artery in association with pulmonic stenosis and pulmonary hypertension Sejal Morjaria, Dan Grinnan, and Norbert Voelkel

250

256

History and Who’s Who The herd shot “round the world” Robert F. Grover

258

Letter to Editor Critical care rehabilitation—is it the answer for reducing morbidity in ARDS survivors?

Regarding “Acute respiratory distress syndrome: A clinical review” Abraham Samuel Babu, and Lenny T. Vasanthan

Pulmonary Circulation | April-June 2012 | Vol 2 | No 2

LEFT TO RIGHT: 197, 252, 259

265

iii

Author Institution Mapping (AIM)

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

Pulmonary Circulation | April-June 2012 | Vol 2 | No 2

Edi t ori al

Science is an endless frontier: Encouraging translational research in pulmonary vascular disease Like a frontier, science is vast; it’s endless. And like a pioneer entering a new land, when a scientist gazes across that expansive space, he sees opportunity and he sees freedom to pursue his thoughts and interests. Broadly speaking, there are two main types of scientists: the “basic” scientist, and the clinician. The basic scientist is most like the pioneer in that he has the most range to pursue his work, but unfortunately not always the most opportunity.

The clinician is like the town doctor; he has access to all the clinical material, but not always the freedom or the knowledge to pursue the research necessary. But what happens when the basic scientist collaborates with the clinician? Results are found, science’s unanswerable questions are solved, and clinical diagnosis and treatment of disease can be improved.

Translational research has numerous definitions, all of which have two ideas in common: application of results and collaboration between different fields of research.

Because the basic scientist devotes his career to research, he develops the breadth of knowledge needed to assist humanity (for example, the intricacies of disease biology or the development of a drug to manipulate the disease process). And because the clinician devotes his career to the patient, he has insights into what is needed to control or cure a disease, but doesn’t always have the time or the know-how to pursue that notion.

That is why we need to encourage basic scientists across all ages, career levels, and geographic locations to look for a way to take their invaluable research and, if at all possible, find a way to apply it to the cure of pulmonary vascular disease. Likewise, we encourage all clinicians to collaborate and share their thoughts with basic scientists in order to identify the genetic and pathogenic mechanisms of pulmonary vascular disease (Fig. 1).

Of course, like anything worth pursuing, there are barriers to translational research, the most poignant of which is funding. Many funding agencies typically encourage either basic research or clinical trials; they are somewhat resistant or reluctant to support moving discovery from wet laboratory research to patient care via drug development. Additionally, very few basic scientists have the opportunity to learn about drug development, disease pathogenesis, clinical management of a disease, and very few have the access to clinical data and patient’s samples and specimens needed to engage in translational research.

Moreover, most basic scientists work in the world of academia where translational research may be encouraged, but is not a major focus, for tenure and promotions. And because of these challenges, collaboration between the basic scientist and the clinician is trying; without that key element of collaboration, translational research may cease to exist.

Access this article online

Quick Response Code:

Website: www.pulmonarycirculation.org DOI: 10.4103/2045-8932.97585 How to cite this article: Yuan JX, Morrell NW, Harikrishnan S, Butrous G. Science is an endless frontier: Encouraging translational research in pulmonary vascular disease.

Pulm Circ 2012;2:137-8.

Figure 1: Collaboration between clinical and basic science.

Pulmonary Circulation | April-June 2012 | Vol 2 | No 2

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For inspiration, look to the work of Dr. Daria Mochly-Rosen who initiated SPARK at Stanford University, a program designed to help strengthen the appeal of translational research and especially to encourage young investigators to consider its potential. The driving forces behind SPARK include five main ideas: (1) direct funding tied into the achievement of milestones; (2) seminars and handson programs on drug and diagnostic development; (3) biweekly progress reports; (4) access to facilities; and (5) mentorship. For more detailed information on the SPARK program, please visit http://sparkmed.stanford.edu/.

The promotion of translational research will only be successful if there is an increase in collaboration; the Pulmonary Vascular Research Institute (PVRI) has set an example in this setting. The PVRI is a forum for interaction between basic and clinical scientists; Pulmonary Circulation, for example, is a medium where both the clinician and the basic scientist meet in print. In the developing world, where the problem of pulmonary vascular disease is all too rampant, translational research is almost non-existent.

There are many reasons for this (the number of scientists working in “basic sciences� is too few, lack of funding, lack of

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interest from funding agencies, lack of adequate laboratory equipment, clinicians have too many patients to tend, etc.), but forums like PVRI and Pulmonary Circulation may help inspire action.

Basic scientists and clinicians everywhere have the potential to increase awareness and to possibly control pulmonary vascular diseases; they do not, however, always have the knowledge or the opportunity needed to complete these actions. The basic scientist must again look across that vast space, but this time he must look not only for the pursuit of research, but he must discover or create opportunity where it lies. And the clinician must take his knowledge about his patients and help the basic scientist grab hold of that opportunity. And together they must apply their minds and their work, and break down the barriers to achieve their ultimate goal, a cure for pulmonary vascular disease.

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

Pulmonary Circulation | April-June 2012 | Vol 2 | No 2

Review Ar ti cl e

Clinical perspectives with long-term pulsed inhaled nitric oxide for the treatment of pulmonary arterial hypertension Robyn J. Barst1, Richard Channick2, Dunbar Ivy3, and Brahm Goldstein4 1

Department of Pediatrics and Medicine, Columbia University, New York, New York, USA, 2Massachusetts General Hospital, Boston, Massachusetts, USA, 3Department of Pediatrics, University of Colorado School of Medicine, Children’s Hospital Colorado, Aurora, Colorado, USA, 4Ikaria, Inc., Hampton, New Jersey, USA

ABSTRACT Pulmonary arterial hypertension (PAH) is a chronic, progressive disease of the pulmonary vasculature with a high morbidity and mortality. Its pathobiology involves at least three interacting pathways – prostacyclin (PGI2), endothelin, and nitric oxide (NO). Current treatments target these three pathways utilizing PGI2 and its analogs, endothelin receptor antagonists, and phosphodiesterase type-5 (PDE-5) inhibitors. Inhaled nitric oxide (iNO) is approved for the treatment of hypoxic respiratory failure associated with pulmonary hypertension in term/near-term neonates. As a selective pulmonary vasodilator, iNO can acutely decrease pulmonary artery pressure and pulmonary vascular resistance without affecting cardiac index or systemic vascular resistance. In addition to delivery via the endotracheal tube, iNO can also be administered as continuous inhalation via a facemask or a pulsed nasal delivery. Consistent with a deficiency in endogenously produced NO, long-term pulsed iNO dosing appears to favorably affect hemodynamics in PAH patients, observations that appear to correlate with benefit in uncontrolled settings. Clinical studies and case reports involving patients receiving long-term continuous pulsed iNO have shown minimal risk in terms of adverse events, changes in methemoglobin levels, and detectable exhaled or ambient NO or NO2. Advances in gas delivery technology and strategies to optimize iNO dosing may enable broad-scale application to long-term treatment of chronic diseases such as PAH. Key Words: drug, hypertension, inhalation administration, nitric oxide, pulmonary arterial hypertension, pulmonary circulation, pulmonary hypertension, pulmonary/physiopathology, pulse therapy, vasodilator agents

Pulmonary arterial hypertension (PAH) is a chronic, progressive disease of the pulmonary vasculature resulting in right ventricular failure and death, if untreated.[1,2] PAH is defined by the following: a resting mean pulmonary arterial pressure (mPAP) ≥25 mmHg; pulmonary capillary wedge pressure or left ventricular end diastolic pressure ≤15 mmHg; and pulmonary vascular resistance (PVR) ≥3 Wood units. [2] PAH can be idiopathic, heritable, or associated with other conditions, such as connective tissue diseases (CTDs).[3,4] The prevalence of PAH was estimated as 26–52 cases per million from the Scottish epidemiological study; a more conservative lower-bound estimate from the French PAH Registry reports 5–25 cases per million.[5,6] Prevalence is greater in high-risk groups, such as patients with CTDs, congenital heart disease (repaired and unrepaired), human immunodeficiency virus, and portal hypertension.[7-9]

Address correspondence to: Dr. Robyn J. Barst 31 Murray Hill Road Scarsdale, NY 10583, USA Email: robyn.barst@gmail.com

Pulmonary Circulation | April-June 2012 | Vol 2 | No 2

Pulmonary arterial hypertension: Mortality and unmet medical need

The mortality with PAH remains high despite treatment advances over the past several decades. In the 1980s, the 5-year survival rate for idiopathic PAH (IPAH; formerly termed “primary pulmonary hypertension”) was 34% in the National Institutes of Health (NIH) Registry; although 5-year survival has increased to ≈60% using currently available drugs, the mortality remains unacceptable.[4] Patients in the NIH registry in the 1980s were treated with the conventional therapy available at the time, including diuretics, digoxin, supplemental oxygen, warfarin, and Access this article online

Quick Response Code:

Website: www.pulmonarycirculation.org DOI: 10.4103/2045-8932.97589 How to cite this article: Barst RJ, Channick R, Ivy D, Goldstein B. Clinical perspectives with long-term pulsed inhaled nitric oxide for the treatment of pulmonary arterial hypertension. Pulm Circ 2012;2:139-47.

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Barst et al.: Long-term pulsed iNO for treatment of PAH

calcium channel blockers (if clinically indicated).[4] Prior to 1995, no drugs were approved for PAH. However, there are currently eight drugs approved for the treatment of PAH: intravenous (IV) epoprostenol, IV/subcutaneous (SC) treprostinil, inhaled treprostinil, inhaled iloprost, oral bosentan, oral ambrisentan, oral sildenafil, and oral tadalafil.

A meta-analysis of all randomized, controlled PAH trials published through 2008 suggested that with these available PAH-specific treatments, mortality has decreased 43% (RR: 0.57; 95% CI: 0.35–0.92; P=0.023).[10] Despite these improvements in survival rates, a significant unmet medical need remains: PAH continues to progress with no cure.

Patients with PAH also report severe impairment of healthrelated quality of life (HRQOL), including poor general and emotional health, and impaired physical functioning. [9] These impairments to HRQOL with PAH are comparable and not infrequently greater than those reported in patients with severely debilitating conditions such as spinal cord injury or cancers unresponsive to therapy.[9] Improvement in HRQOL scores has been reported (e.g., increased exercise capacity and physical functioning) utilizing the currently available PAH-specific drugs.[11-13]

Pathobiology

The postulated pathobiology of PAH involves interactions between the prostacyclin (PGI2), endothelin (ET-1), and nitric oxide (NO) pathways, in addition to a host of other pathways (Fig. 1).[2,14-16] Specific mechanisms responsible for the development and progression of PAH include the following: reduced PGI2 synthase; increased ET-1 expression; decreased NO synthase; elevated plasma levels and low platelet 5-hydroxytryptamine levels; downregulation of potassium channels of pulmonary vascular smooth muscle cells; activity of autoantibodies and proinflammatory cytokines; and prothrombotic states arising from endothelial, coagulation, and fibrinolytic cascade/platelet dysfunction.[16] These changes give rise to a complex process of pathobiologic changes in the pulmonary vascular bed, including endothelial dysfunction, vasoconstriction, vascular remodeling, and in situ thrombosis.[2]

Pharmacologic targets of currently approved treatments for PAH

Current PAH treatment approaches include PGI 2 and its analogs, ET-1 receptor antagonists (ERAs), and phosphodiesterase type-5 (PDE-5) inhibitors. [17] Combination trials have demonstrated additive and/or synergistic benefit by targeting more than one pathway.[17] Prostanoid monotherapy (epoprostenol, treprostinil, and iloprost) improves symptoms, exercise capacity, and hemodynamics.[17] Increased survival was also demonstrated in IPAH/heritable PAH (HPAH) with IV epoprostenol.

However, common side effects with prostanoids include headache, flushing, nausea, jaw pain, diarrhea, skin rash, 140

Figure 1: Pathways involved in the development and maintenance of pulmonary arterial hypertension. AA: arachidonic acid; ET: endothelin; eNOS: endothelial NO synthase; PS: prostacyclin synthase; ECE: endothelinconverting enzyme; PGI2: prostaglandin I2 (prostacyclin); ETRA: endothelin receptor agonist; GTP: guanylate triphosphate; GC: guanylate cyclase; ATP: adenosine triphosphate; AC: adenylyl cyclase; CCB: calcium channel blocker; cGMP: cyclic guanylate monophosphate; cAMP: cyclic adenosine monophosphate; PDE5: phosphodiesterase-5; PDE5i: PDE5 inhibitor.

Reprinted from The Lancet, Vol. 358, Jocelyn Dupuis, Endothelin-receptor antagonists in pulmonary hypertension, pages no. 1113–1114, Copyright (2001), with permission from Elsevier[15] and with permission from Mayo Clinic Proceedings, Volume 84, Michael D. McGoon and Garvan C. Kane, pulmonary hypertension: diagnosis and management, pp 191–207, Copyright Mayo Foundation for Medical Education and Research (2009).[16]

and musculoskeletal pain. Treatment with PGI2 and its analogs often requires continuous intravenous parenteral infusion, which can cause blood stream infections and/or thromboembolic events that can be life threatening.[2]

Endothelin-1 exerts vasoconstrictor and mitogenic effects, whereas ERAs (i.e., bosentan and ambrisentan) improve exercise capacity, functional class, and hemodynamics.[2,8] Adverse effects include acute hepatotoxicity, anemia, and fluid retention. Additionally, ERAs may cause testicular atrophy and male infertility. Use of bosentan requires monthly liver function tests and two modes of birth control, as it has been shown to cause severe fetal toxicity in animal studies.[2,18]

In three randomized trials, the PDE-5 inhibitors sildenafil and tadalafil improved exercise capacity and hemodynamics (either as monotherapy or as add-on therapy).[8,11,17] Both agents cause pulmonary vasodilation.[8] Side effects include headache, flushing, and dyspepsia and are generally related to systemic vasodilation. Epistaxis has also been reported with sildenafil use in PAH.[8,11] Prostacyclin analogs, ERAs, and PDE-5 inhibitors are the mainstays of current PAH treatment; however, all have systemic effects in addition to their pulmonary effects that can cause untoward side effects.[19] An optimal agent for PAH therapy remains to be identified.[17] Pulmonary Circulation | April-June 2012 | Vol 2 | No 2

Barst et al.: Long-term pulsed iNO for treatment of PAH

Inhaled nitric oxide Inhaled nitric oxide (iNO) is a selective pulmonary vasodilator that can acutely decrease pulmonary artery pressure (PAP) and pulmonary vascular resistance (PVR) in neonates with hypoxic respiratory failure associated with pulmonary hypertension.[20] Nitric oxide regulates vascular smooth muscle tone and increases blood flow to regions of the lungs with normal ventilation/perfusion ratios by dilating pulmonary vessels in better-ventilated areas.[21] After inhalation, NO is absorbed systemically, with the majority of NO traversing the pulmonary capillary bed and combining with 60–100% oxygen-saturated hemoglobin.[20] The effect of iNO is localized to the lung, as once absorbed iNO is rapidly oxidized by hemoglobin to form nitrite, which interacts with oxyhemoglobin, leading to the formation of nitrate and methemoglobin (metHb). [20,22] This metabolic production of metHb is a potential toxic effect of iNO treatment. While doses <100 ppm most often result in insignificant metHb levels in adults and children, methemoglobinemia has been reported with 80 ppm when exposure was >18 hours.[23] Inhaled NO is currently indicated for the treatment of term/ near-term neonates (>34 weeks gestation) with hypoxic respiratory failure associated with pulmonary hypertension (PH). The recommended dose is 20 ppm delivered via constant concentration during inspiration for up to 14 days or until hypoxia has resolved.[20]

Inhaled NO has also been used as an agent for acute vasodilator testing (AVT) as part of the evaluation of PAH patients; doses of 20–80 ppm for 5–10 minutes are typically used.[2,24] Detecting an acute response with AVT is useful in selecting patients who should be considered

(A)

for initial treatment with high-dose oral chronic calcium channel blockade; AVT response may also be helpful in predicting long-term prognosis with medical therapy and following surgical interventions, such as heart or heart–lung transplantation.[24] Administered as continuous inhalation via face mask, iNO can selectively decrease PAP and PVR without reducing cardiac index or systemic vascular resistance.[24] Inhaled NO has also been used in other contexts, such as perioperatively for cardiac surgery,[25-33] right heart failure after insertion of the left ventricular assist device,[34-37] cardiogenic shock due to right ventricle myocardial infarction,[38] and pulmonary ischemia-reperfusion injury.[39-44]

Because the pulmonary vasodilator effects of NO are transient, it is administered continuously during inspiration, with careful monitoring of NO and NO2 concentrations.[20] Nitric oxide gas can be safely administered in both intubated and nonintubated patients.[45] The pulmonary selectivity of iNO may render it useful as an adjunct to other therapies that are dose limited by their systemic effects.

Inhaled NO has also been administered long term via pulsed nasal delivery (ml/breath/h) in clinical trials; this method has been studied for continuous long-term outpatient as well as short-term inpatient treatment (Fig. 2).[46-49] This ambulatory administration method delivers a set, pulsed volume of NO at the beginning of each breath via a nasal cannula connected through a NO demand valve to a cylinder of up to 200 ppm NO in N2.[46- 48] Both the continuous face mask and pulsed delivery via nasal cannula have comparable hemodynamic effects. [50] A potential theoretical advantage of iNO, in contrast to IV vasodilators, is its

(B)

Figure 2: Examples of pulsed inhaled nitric oxide delivery systems used in clinical studies: (A) Ambulatory system. Reproduced with permission from the American College of Chest Physicians, Chest, Volume 109, Richard N. Channick, John W. Newhart, F. Wayne Johnson, Penny J. Williams, William R. Auger, Peter F. Fedullo, and Kenneth M. Moser, pulsed delivery of inhaled nitric oxide to patients with primary pulmonary hypertension: an ambulatory delivery system and initial clinical tests, pp 1545–1549, Copyright (1996), American College of Chest Physicians;[48] (B) Hospital system. Reprinted with permission from Internal Medicine, Volume 41, Osamu Kitamukai, Masahito Sakuma, Tohru Takahashi, Jun Nawata, Jun Ikeda, and Kunio Shirato, hemodynamic effects of inhaled nitric oxide using pulse delivery and continuous delivery systems in pulmonary hypertension, pp 429–434, Copyright The Japanese Society of Internal Medicine (2002).[49] iNO: inhaled nitric oxide NO2: nitrogen dioxide Pulmonary Circulation | April-June 2012 | Vol 2 | No 2

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pulmonary selectivity (due to rapid hemoglobin-mediated inactivation).[22] Although prostanoids administered via inhalation appear to have less ventilation–perfusion mismatching than when administered intravenously/ subcutaneously or orally, some degree of ventilation– perfusion mismatching persists; in addition, systemic spillover can result in untoward systemic effects.[51-53]

Clinical application of inhaled nitric oxide AS long-term treatment for PAH

Long-term (>1 month) pulsed iNO dosing appears to favorably affect pulmonary hemodynamics findings[46-48,54,55]

which, with other types of therapy, appear to correlate with benefit (Table 1).

In a study of eight patients with IPAH, Channick et al. reported decreased mean PAP (mPAP), mean right atrial pressure (mRAP), and PVR (P ≤ 0.01) with short-term iNO treatment using an ambulatory NO delivery system via nasal cannula (Table 1).[48] No adverse symptoms and no changes in metHb levels were reported. One patient was discharged home on chronic pulsed iNO and reported no adverse effects after 9 months of treatment.

Ivy et al. also reported that in 26 children and young adults with PAH (short-term therapy, n=24; long-term therapy, n=2) constant concentration and pulsed delivery of NO (via nasal cannula) were equally effective in decreasing

Table 1: Inhaled long-term nitric oxide use for treatment of pulmonary arterial hypertension Study

Study design

N

Age, diagnosis

Route of administration: Dose

Duration

Channick et al., 1996[48]

Open label

8

NR, IPAH

Pulsed iNO via cannula: 80 ppm 0.1 sec pulse at 10 L/min

15 min (n=8); 24 h (n=1); 9 mo (n=1)

Ivy et al., 2003[46]

Open label, controlled

26

1–24 y, PAH

Pulsed iNO via cannula: 100 ppm, alveolar concentration = 20 ppm

15 min (n=24), 7 mo (n=1), 2 y (n=1)

Adult: 15–60 mL NO/ breath, flow rate = 10 L/ min

31–78 y, severe PAH

Pediatric: 3–10 mL NO/ breath, flow rate = 2 L/min Continuous iNO via face mask: 20 ppm

Pérez-Peñate et al., 2008[47]

Open label, uncontrolled

11

Snell et al., 1995[55]

Case report, open label

1

40-year-old female, endstage PAH

Pulsed iNO via face mask, then transtracheal Scoop™ catheter: Mean: 50.4±23 ppm

Pérez-Peñate et al., 2001[54]

Case report, open label

1

32-year-old male, severe PAH

Pulsed iNO via nasal cannula: 80 ppm

1 y (n=9)

68 d

1y

Flow rate = 0.9 L/min

Hemodynamic findings Decreased mean PAP (51–43 mm Hg), RAP (9–6.6 mm Hg), and PVR (790–620 dyne·s·cm-5) (P≤0.01) Marked reductions in mPAP (>20%) and PVR (>30%) (n=3) Pulsed: Decreased mean PAP (54–41 mm Hg), PVR (13.6 to 9.4 U· m2), and RPSVR (0.62–0.41) (P<0.05) Continuous: Decreased mean PAP (53 to 39 mm Hg), PVR (12.7–8.8 U· m2), and PVR/SVR (0.58–0.38) (P<0.05)

Decreased mean PAP (64–58 mm Hg), PVR (1195–1016 dyne·s·cm-5), and increased CI (2.1–2.2 liters/min/m2) (P≤0.04) Also improved dyspnea, BNP level, and 6-min walk distance (P≤0.02) Increased mean systemic BP (73–87 mm Hg), stabilized central venous pressure (21 mm Hg) and O2 saturation (92%) at 660 minutes Decreased mean PAP (78– 72 mm Hg), PVR (1145–890 dyne·s·cm-5) and increased CO (4.4–5.3 L/min) Also improved dyspnea, renal function, and edema after 20 d Improvement to NYHA Class II with no edema at 1 y

BNP: brain natriuretic peptide; CI: cardiac index; CO: cardiac output; iNO: inhaled nitric oxide; NR: not reported; PAP: pulmonary arterial pressure; PVR: pulmonary vascular resistance; PVR/SVR: ratio of pulmonary to systemic vascular resistance; RAP: right atrial pressure.

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PAP and PVR (P<0.05 vs. baseline; Table 1; Fig. 3).[46] Adult and pediatric devices were studied, and the adult device delivered 15–60 ml NO per breath at a flow rate of 10 l/ min while the pediatric device delivered 3–10 ml per breath at a flow rate of 2 l/minute. Two patients were discharged home on iNO using a pulsed device; 1 for 7 months and 1 for 2 years with no reported adverse events including no reports of syncope or near syncope.

Long-term treatment with pulsed iNO was evaluated in 11 patients (7 with PAH and 4 with chronic thromboembolic PH) in an uncontrolled, open-label study. The study design included the addition of PDE-5 inhibitor (dipyridamole or sildenafil) for clinical worsening; this was suggested as a means to “stabilize and potentiate the effects of iNO” and to “potentially serve as rescue therapy in severe PH” (Table 1).[47] After 1 month of an ambulatory iNO system via nasal cannula, patients had an improvement in World Health Organization functional class concomitant with improvements in 6-minute walking distance (P=0.003), and brain natriuretic peptide (BNP) level (P=0.02; Fig. 4).[47] One patient died from refractory right heart failure at month 8; 7 of the 11 patients had a PDE-5 inhibitor added at 6–12 months due to symptomatic deterioration. At the 1-year follow-up, 9 of the 11 patients reported durability of effect as observed after 1 month of therapy with associated significant improvements in mPAP, PVR, and CI. In addition, the significant improvements in 6-minute walking distance (P=0.003) and BNP levels (P=0.02) were maintained at the 1-year follow-up. There were no reports of NO air contamination, changes in metHb levels, adverse reactions, NO toxicity, or rebound PH from sudden withdrawal.[47] Two case reports have also examined long-term iNO administration in PAH patients, including its use as a “bridge to heart-lung or lung transplantation” (Table 1).

A 40-year-old woman presented with end-stage IPAH and experienced severe dyspnea, right ventricular angina, oliguria, and syncope despite treatment with dopamine infusion and with prostacyclin. The patient then initiated treatment with pulsed iNO, initially via face mask and then transtracheal catheter, until she underwent heart–lung transplantation after 68 days of therapy.[55] The patient’s condition appeared to stabilize on iNO treatment, although she had a hypotensive bradycardic event after 53 days, requiring reinitiation of intravenous prostacyclin. While iNO was administered, she was able to move about her room independently and participate in a physiotherapy exercise program. The explanted lungs revealed no evidence of NO toxicity.[55]

Another case reported the effects of 12 months of iNO administration in a 32-year-old man with IPAH (Table 1). [54] The patient presented with exertional dyspnea and Pulmonary Circulation | April-June 2012 | Vol 2 | No 2

Figure 3: Correlation between mean pulmonary arterial pressure during mask delivery and pulsed nasal nitric oxide delivery. PAP: pulmonary artery pressure.

Reprinted from The American Journal of Cardiology, Vol 92, D. Dunbar Ivy, Donna Parker, Aimee Doran, Donna Parker, John P. Kinsella, and Steven H. Abman, acute hemodynamic effects and home therapy using a novel pulsed nasal nitric oxide delivery system in children and young adults with pulmonary hypertension, pages no. 886–890, Copyright (2003), with permission from Excerpta Medica, Inc.[46]

Figure 4: World Health Organization functional class and brain natriuretic peptide levels (mean±SD) at baseline compared with 1 month and 1 year after onset of iNO treatment. *In Patients 1 and 2, the measure was taken at 6 months. BNP: brain natriuretic peptide. Reprinted from The Journal of Heart and Lung Transplantation, Vol 27, Gregorio Miguel Pérez-Peñate, Gabriel Juliá-Serdà, Nazario Ojeda-Betancort, Antonio García-Quintana, Juan PulidoDuque, Aurelio Rodríguez-Pérez, Pedro Cabrera-Navarro, Miguel Angel Gómez-Sánchez, Long-term inhaled nitric oxide plus phosphodiesterase 5 inhibitors for severe pulmonary hypertension, Pages No. 1326–1332, Copyright (2008), with permission from the International Society for Heart and Lung Transplantation.[47]

anasarca, and was treated with long-term iNO monotherapy via an ambulatory system with nasal cannula. After 20 days, there was an improvement in dyspnea and gas exchange, and a resolution of the anasarca. After 12 months of continuous iNO, the patient remained clinically stable, with maintained hemodynamic improvement and no signs of toxicity or tachyphylaxis.[54] 143

Barst et al.: Long-term pulsed iNO for treatment of PAH

Ivy et al. reported that short-term pulsed nasal delivery utilizing constant concentration was as effective in lowering PAP and PVR as mask delivery in the acute setting in eight children with PAH (Fig. 5).[50] Based on the results of this study, the authors concluded that the practicality of long-term iNO therapy via pulsed flow nasal delivery is potentially dependent on four factors: (1) maintenance of sufficient iNO delivery; (2) improvement of hemodynamic derangements by nasal cannula at low flow rates; (3) effective delivery of nasal NO with minimal release of gas into the environment; and (4) minimized consumption of NO gas.[50] Aside from the reports involving long-term use summarized in Table 1, the practicality of pulsed delivery of iNO for improvement in oxygenation with less NO consumption and less environmental contamination has been demonstrated in several other studies.[56-58]

roles for long-term INHALED NITRIC OXIDE in the TREATMENT of pulmonary arterial hypertension

Potential uses of pulsed, long-term iNO treatment in PAH patients include the following: use as a bridge to transplantation; a means of deferring transplantation; and as an add-on therapy to currently approved PAH drugs[2,45] with potential additive or synergistic effects.[59,60] It is important to note that NO synthase 3 (NOS3) has been reported to be decreased in PAH patients; in uncontrolled observational studies, PAH has been associated with impaired NO release, at least in part, due to reduced expression of NOS3 in the vascular endothelium of pulmonary arterioles.[61] As a result, long-term administration of iNO may serve both as a selective pulmonary vasodilator and as NO replacement therapy, making it a logical choice for clinical evaluation as add-on therapy.

Safety considerations

A potential safety concern with iNO treatment is rebound PH upon its sudden discontinuation after longer-term (days) use[20,62]; this phenomenon is well known and has been well documented in neonates and in postoperative cardiac surgery patients. Such patients include cardiac transplant recipients, children undergoing surgery for congenital heart disease, and adults with mitral and/or aortic stenosis. Gradual weaning of iNO has been shown to minimize the potential for rebound PH in the acute ICU setting.[45] Davidson et al. presented a method to safely withdraw iNO in infants treated for hypoxic respiratory failure, recommending the gradual weaning of iNO down to 1 ppm prior to treatment discontinuation.[63] Further research has implicated the rapid degradation of smooth 144

(A)

(B)

Figure 5: Delivery of inhaled NO by continuous mask or pulsed nasal cannula was equally effective in lowering mean pulmonary artery pressure (A) and pulmonary vascular resistance index (B). PAP: mean pulmonary artery pressure; PVRI: pulmonary vascular resistance index; iNO: inhaled nitric oxide; BL: baseline. Reprinted from The Journal of Pediatrics, Vol 133, D. Dunbar Ivy, Jeffrey L. Griebel, John P. Kinsella, and Steven H. Abman, acute hemodynamic effects of pulsed delivery of low flow nasal nitric oxide in children with pulmonary hypertension, Pages No. 453–456, Copyright (1998), with permission from Mosby, Inc.[50]

muscle intracellular cyclic guanosine monophosphate (cGMP) by local phosphodiesterases (PDEs) as a primary mechanism for this rebound effect. As a result, initial approaches focused on the use of dipyridamole, a PDE-5 inhibitor, as a means for reducing rebound PH after iNO withdrawal. Ivy et al. first demonstrated this concept in a prospective study of 23 children treated with iNO after surgery for congenital heart disease. [64] Later studies examined the role of sildenafil, another PDE-5 inhibitor, in the context of rebound PH, showing that its introduction prior to withdrawal of iNO resulted in facilitation of iNO weaning, as well as prevention/amelioration of rebound PH effects in infants and children with PH after congenital heart disease surgery, persistent PH of the newborn, and other abnormalities. [65-68] As with any approach, it is important to consider patient characteristics and treatment familiarity, availability, and contraindications, as well as optimal ventilation and supplemental vasodilators, when initiating treatment for rebound PH.[66]

A review of the published literature on long-term iNO dosing in PAH patients has not revealed any reports of rebound PH crises or associated symptoms (e.g., syncope, systemic arterial oxygen desaturation, systemic hypotension, bradycardia, or cardiac arrest).[46-48,54,55] It may be that more acute initial rise in PAP is associated with a greater likelihood and severity of a rebound effect occurring with acute iNO withdrawal. This may explain why the rebound phenomenon has been observed in the acute care setting (e.g., neonates with persistent pulmonary hypertension of the newborn and high risk postoperative cardiothoracic surgical patients) and not observed in the more chronic setting of PAH or chronic obstructive pulmonary disease.[69] Pulmonary Circulation | April-June 2012 | Vol 2 | No 2

Barst et al.: Long-term pulsed iNO for treatment of PAH

Cytotoxicity is another possible concern with iNO and its oxidized derivatives (principally NO2). Nitric oxide may be directly toxic to alveolar and vascular tissue; therefore, it has been proposed that NO be stored in combination with nitrogen and blended with oxygen at the time of administration to prevent oxidation to toxic products, in addition to maintaining NO2 levels <5 ppm.[23,70]

eleven patients who led a nonsedentary life were able to leave their home daily, with four returning to work while on long-term iNO therapy.

In summary, uncontrolled observational studies of long-term use (>1 month) of continuous pulsed iNO (as monotherapy or as part of combination therapy) in a total of 14 patients with PAH across five studies[46-48,54,55] have reported no significant adverse events, no elevated metHb levels, and no detectable exhaled or ambient NO or NO2. In one study, a patient experienced three episodes of severe epistaxis over two years while on a combination of pulsed iNO and epoprostenol.[46] In a case report of a patient awaiting heart–lung transplantation, the patient experienced hypotensive bradycardia upon an attempt to wean from iNO therapy. In addition, a recurrence in hypotensive bradycardia resulted in the increase of iNO dose (40–106 ppm), followed by a decrease to 70 ppm (along with administration of bicarbonate and reintroduction of prostacyclin) after increasing metabolic acidosis.[55]

ACKNOWLEDGMENTS

Conclusions and future directions

There is evidence that pulsed delivery may allow utilization of lower NO concentrations compared with continuous face mask administration, potentially minimizing the risk of associated adverse events as well as resulting in a more practical delivery system.[49]

The consensus on treatment for PAH encompasses numerous goals, the most important being to improve overall quality of life by decreasing symptoms while minimizing treatment-related side effects.[2] Additional goals include enhancing functional capacity, i.e., exercise capacity, improving hemodynamic derangements (lowering PVR and PAP, and normalizing RAP and CO), and preventing, if not reversing, disease progression. Finally, improving survival, although certainly desirable, is rarely an end point in trials examining PAH treatment.[2] The availability of novel treatments and the improvement in survival rates have allowed the goals of PAH therapy to expand from improving survival and preventing disease progression to also improving HRQOL. [71] Potential advances in long-term PAH treatment, such as ambulatory iNO administration, may allow for greater improvements in HRQOL. Pérez–Peñate et al. observed that ambulatory pulsed iNO treatment did not diminish quality of life beyond the consequences of the disease itself.[47] Eight of Pulmonary Circulation | April-June 2012 | Vol 2 | No 2

An ideal drug-device for long-term PAH treatment should emphasize portability and safety features for outpatient use. Advances in iNO gas delivery technology and strategies to optimize dosing should allow for randomized controlled trials of iNO and, hopefully, may lead to broad-scale application of iNO in the treatment of chronic diseases such as PAH.[45]

The authors thank Michael Morren, RPh, of Peloton Advantage, LLC, for providing medical writing and editorial assistance, which was funded by Ikaria, Inc., during the preparation of this manuscript. No author received an honoraria or other form of financial support for the preparation of this manuscript.

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A randomized trial of inhaled nitric oxide to prevent ischemia-reperfusion injury after lung transplantation. Am J Respir Crit Care Med 2003;167:1483-9.

Meade M, Granton JT, Matte-Martyn A, McRae K, Cripps PM, Weaver B, et al. A randomized trial of inhaled nitric oxide to prevent reperfusion injury following lung transplantation. J Heart Lung Transplant 2001;20:254-5.

Thabut G, Brugiere O, Leseche G, Stern JB, Fradj K, Herve P, et al. Preventive effect of inhaled nitric oxide and pentoxifylline on ischemia/reperfusion injury after lung transplantation. Transplantation 2001;71:1295-300.

Fullerton DA, Eisenach JH, McIntyre RC Jr., Friese RS, Sheridan BC, Roe GB, et al. Inhaled nitric oxide prevents pulmonary endothelial dysfunction after mesenteric ischemia-reperfusion. Am J Physiol 1996;271:L326-31.

Bloch KD, Ichinose F, Roberts JD Jr., Zapol WM. Inhaled NO as a therapeutic agent. Cardiovasc Res 2007;75:339-48.

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Perez-Penate GM, Julia-Serda G, Ojeda-Betancort N, Garcia-Quintana A, Pulido-Duque J, Rodriguez-Perez A, et al. Long-term inhaled nitric oxide plus phosphodiesterase 5 inhibitors for severe pulmonary hypertension.

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Kitamukai O, Sakuma M, Takahashi T, Nawata J, Ikeda J, Shirato K. Hemodynamic effects of inhaled nitric oxide using pulse delivery and continuous delivery systems in pulmonary hypertension. Intern Med 2002;41:429-34.

Ivy DD, Griebel JL, Kinsella JP, Abman SH. Acute hemodynamic effects of pulsed delivery of low flow nasal nitric oxide in children with pulmonary hypertension. J Pediatr 1998;133:453-6.

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Perez-Penate G, Julia-Serda G, Pulido-Duque JM, Gorriz-Gomez E, CabreraNavarro P. One-year continuous inhaled nitric oxide for primary pulmonary hypertension. Chest 2001;119:970-3.

Snell GI, Salamonsen RF, Bergin P, Esmore DS, Khan S, Williams TJ. Inhaled nitric oxide used as a bridge to heart-lung transplantation in a patient with end-stage pulmonary hypertension. Am J Respir Crit Care Med 1995;151:1263-6.

Heinonen E, Nyman G, Merilainen P, Hogman M. Effect of different pulses of nitric oxide on venous admixture in the anaesthetized horse. Br J Anaesth 2002;88:394-8.

Heinonen E, Merilainen P, Hogman M. Administration of nitric oxide into open lung regions: delivery and monitoring. Br J Anaesth 2003;90:338-42.

Heinonen E, Hogman M, Merilainen P. Theoretical and experimental comparison of constant inspired concentration and pulsed delivery in NO therapy. Intensive Care Med 2000;26:1116-23.

Lepore JJ, Maroo A, Bigatello LM, Dec GW, Zapol WM, Bloch KD, et al.

Hemodynamic effects of sildenafil in patients with congestive heart failure and pulmonary hypertension: Combined administration with inhaled nitric oxide. Chest 2005;127:1647-53.

Lepore JJ, Maroo A, Pereira NL, Ginns LC, Dec GW, Zapol WM, et al.

Effect of sildenafil on the acute pulmonary vasodilator response to inhaled nitric oxide in adults with primary pulmonary hypertension. Am J Cardiol 2002;90:677-80.

Giaid A, Saleh D. Reduced expression of endothelial nitric oxide synthase

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in the lungs of patients with pulmonary hypertension. N Engl J Med 1995;333:214-21.

62. Miller OI, Tang SF, Keech A, Celermajer DS. Rebound pulmonary hypertension on withdrawal from inhaled nitric oxide. Lancet 1995;346:51-2.

63. Davidson D, Barefield ES, Kattwinkel J, Dudell G, Damask M, Straube R, et al. Safety of withdrawing inhaled nitric oxide therapy in persistent pulmonary hypertension of the newborn. Pediatrics 1999;104:231-6.

64. Ivy DD, Kinsella JP, Ziegler JW, Abman SH. Dipyridamole attenuates rebound pulmonary hypertension after inhaled nitric oxide withdrawal in postoperative congenital heart disease. J Thorac Cardiovasc Surg 1998;115:875-82.

65. Atz AM, Wessel DL. Sildenafil ameliorates effects of inhaled nitric oxide withdrawal. Anesthesiology 1999;91:307-10.

66. Raja SG. Treatment of rebound pulmonary hypertension: why not sildenafil?

Anesthesiology 2004;101:1480.

67. Namachivayam P, Theilen U, Butt WW, Cooper SM, Penny DJ, Shekerdemian LS. Sildenafil prevents rebound pulmonary hypertension after withdrawal of nitric oxide in children. Am J Respir Crit Care Med

2006;174:1042-7.

Lee JE, Hillier SC, Knoderer CA. Use of sildenafil to facilitate weaning from inhaled nitric oxide in children with pulmonary hypertension following surgery for congenital heart disease. J Intensive Care Med 2008;23:329-34.

69. Vonbank K, Ziesche R, Higenbottam TW, Stiebellehner L, Petkov V, Schenk P, et al. Controlled prospective randomised trial on the effects on pulmonary haemodynamics of the ambulatory long term use of nitric oxide and oxygen in patients with severe COPD. Thorax 2003;58: 289-93.

70. Mizutani T, Layon AJ. Clinical applications of nitric oxide. Chest 1996;110:506-24.

71. Chen H, De Marco T, Kobashigawa EA, Katz PP, Chang VW, Blanc PD. Comparison of cardiac and pulmonary-specific quality of life measures in pulmonary arterial hypertension. Eur Respir J 2011;38:608-16.

68.

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

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147

Review Ar ti cl e

Severe pulmonary hypertension: The role of metabolic and endocrine disorders Harm J. Bogaard1, Aysar Al Husseini2, Laszlo Farkas2, Daniela Farkas2, Jose Gomez-Arroyo2, Antonio Abbate2, and Norbert F. Voelkel2 1 Department of Pulmonary Medicine, VU University Medical Center, Amsterdam, The Netherlands, 2Pulmonary and Critical Care Medicine Division and Victoria Johnson Laboratory for Lung Research, Virginia Commonwealth University, Richmond, Virginia, USA

ABSTRACT Pulmonary arterial hypertension (PAH) is a multi-factorial condition and the underlying pulmonary vascular disease is shaped by the combined action of genetic, epigenetic and immune-related factors. Whether and how gender, obesity and the metabolic syndrome modify PAH and associated right heart failure is under intense investigation. Estrogens may enhance the process of pulmonary angioproliferation, but may also protect the right ventricle under pressure. Obesity may affect the pulmonary circulation via interactions with inflammatory cells and mediators, or via alterations in endocrine signaling. Obesity is a major risk factor for pulmonary hypertension in patients with elevated pulmonary venous pressure and preserved LV ejection fraction. Given the overlap between PAH and autoimmune diseases, hypothyroidism in patients with PAH is commonly considered a consequence of an autoimmune thyroiditis. In the clinical setting of hyperthyroidism, severe pulmonary hypertension may develop due to a hyperdynamic circulation, but a more complex situation presents itself when hyperthyroidism is associated with PAH. We recently showed in a relevant animal model of severe PAH that thyroid hormone, via its endothelial cell-proliferative action, can be permissive and drive angioproliferation. Signaling via the integrin αvβ3 and FGF receptors may participate in the formation of the lung vascular lesions in this model of PAH. Whether thyroid hormones in euthyroid PAH patients play a pathobiologically important role is unknown- as we also do not know whether the commonly diagnosed hypothyroidism in patients with severe PAH is cardioprotective. This brief review highlights some recent insights into the role of metabolic and endocrine disorders in PAH. Key Words: pulmonary hypertension, right heart failure, epigenetics, thyroid, obesity, gender

Pulmonary hypertension (PH) has been clinically classified or categorized, and mechanistically the four descriptors introduced by Paul Wood: vasoconstrictive, vasoobstructive, hyperkinetic, and passive (left heart failure) continue to be useful.[1] Although the clinical classification distinguishes between pulmonary arterial hypertension (PAH; Group 1), pulmonary venous hypertension (Group 2), and pulmonary hypertension associated with thrombotic or embolic disease (Group 4), PAH (Group 1) combines at least three of the pathogenetically important mechanisms: vasoconstriction, hyperkinesis and vasoobliteration. Hyperkinesis plays an etiological role in PAH associated with congenital cardiac shunt abnormalities, porto-pulmonary hypertension, hyperthyroid disease-associated PAH and PAH associated with sickle cell anemia are all hyperkinetic, high cardiac

Address correspondence to:

Prof. Norbert F. Voelkel Division of Pulmonary and Critical Care Medicine Victoria Johnson Laboratory for Lung Research Virginia Commonwealth University 1200 East Broad Street, MMRB, 6th Floor Richmond, VA 23298, USA Email: nvoelkel@mcvh-vcu.edu 148

output forms of severe PAH.[2-4] Clearly PAH, as classified, is a group of diseases with different long-term survival likely requiring different treatments.[5] Not only is PAH not a single disease, but it is also increasingly appreciated that disease-specific pathobiologically important mechanisms and neuroendocrine factors shape the disease and determine outcome in individual patients. For example, autoimmunity/ inflammation plays an important role in patients with systemic lupus erythematosus-associated pulmonary hypertension and in HIV/AIDS-associated PAH.[6,7] Both genetic susceptibility factors and epigenetic influences need to be investigated for a more complete understanding of the pathobiology of PAH.[8] Thyroid disease and obesity are likely epigenetic disease modifiers, and if the REVEAL registrydata Access this article online

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Website: www.pulmonarycirculation.org DOI: 10.4103/2045-8932.97592

How to cite this article: Bogaard HJ, Al Husseini A, Farkas L, Farkas D, GomezArroyo J, Abbate A et al. Severe pulmonary hypertension: The role of metabolic and endocrine disorders. Pulm Circ 2012;2: 148-54.

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Bogaard et al.: Severe PH and role of disorders

are representative; then pulmonary hypertension has now become overwhelmingly a disease of menopausal women.[8-11] A large number of publications have confirmed the initial report of an association of hypothyroidism and PAH and a recent review by Biondi et al established a strong link between Graves disease and multinodular goiter and PAH. [2,12-16] According to Biondi et al., more than 80% of patients with hyperthyroid diseases develop pulmonary hypertension. In this review, we wish to put PAH in a wider neuroendocrine disease context and summarize recent experiments which had been designed to investigate a functional role of thyroid hormones in the development of PAH.

Endocrine modifiers of pulmonary hypertension

The association between PAH and hypothyroid disease hypothetically suggests that the patients had at some time experienced an autoimmune thyroiditis, and this association is cited as evidence of a participation of the immune system in the pathogenesis of PAH. [17] However, the broader context may be estrogen and other hormone-dependent mechanisms of disease development or disease modification (Fig. 1). In addition to endocrine factors which may modify the pathogenesis there are neuroendocrine factors which enter the stage as right heart failure develops; these are hyperaldosteronism and sympathetic overdrive.[19] Obesity, which is now understood as an inflammatory disease, may be a cotrigger of PAH or may worsen the

inflammatory contribution of chronic pulmonary vascular diseases.[20,21] Sweeney et al. in a survey of 88 patients with PAH found that 25% had a body mass index (BMI) of 30 kg/ m2 or greater. [9] Epidemiological data suggest a significant association between increasing BMI and several cancers and Leung et al. pointed out that obesity was a risk factor for pulmonary hypertension in patients with elevated pulmonary venous pressure and preserved LV ejection fraction (Fig. 2).[22,23] In addition to storing excess calories in the form of lipid, adipose tissue plays an active role in endocrine signaling. Adipocytes generate angiogenic leptin and adipokines and macrophages of the adipose tissue secrete IL-6. Circulating IL-6 levels are correlated with the BMI and adipose tissue is thought to account for 35% of circulating IL-6 in healthy people.[24] Interactions between IL-6 and estrogen may be important in cancer pathobiology.

There is at present no information regarding dietary factors and their potential impact on the lung circulation. Based on strong literature data describing the angiogenic role of copper, a recent study explored copper deficiency and found that a Cu++-depleted diet prevented the development of angioobliterative PAH in the Sugen/chronic hypoxia rat model.[25]

Vitamin D and parathyroid hormone

Vitamins and vitamin deficiency states may also have to be included in the list of modifiers of pulmonary vascular diseases although potential connections between

Hypothalmus

Carrier protein Bound hormone Autocrinesignaling

Pituitary

Endocrine end organ

Pulmonary vasculature

Disease milieu

Myocardium

Juxtacrine and paracrine signaling Bioavailable hormone

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Figure 1: Overview of a general concept of the participation of the endocrine system in pulmonary hypertension and right heart failure. Hormones can be transported by carrier proteins to sites of paracrine signaling receptors or act as in an autocrine fashion. The “sick lung circulation” releases factors which act upon the myocardium [reprinted with permission].[18] 149

Bogaard et al.: Severe PH and role of disorders

Inflammation Obesity Angiogenesis Sleep Apnea

Hypoxic pulmonary vasoconstriction

Left ventricular dysfunction

vitamin deficiencies and pulmonary hypertension remain unexamined. Vitamin D deficiency is a very frequent finding; in fact it has been reported that up to 90% of U.S. adults aged 50–70 are Vitamin D deficient. [26] Vitamin D is important as a controller of cell growth and regulator of immune functions.[27-29] Vitamin D may have an angioproliferative role in cancer mediated via HIF-1α. [30] Calcitriol (1,25[OH2] D3), the active form of Vitamin D, reduces the expression of VEGF and also endothelin. [30] Vitamin D deficiency was found in 49% of patients with the antiphospholipid syndrome and Ulrich et al. described secondary hyperparathyroidism in patients with pulmonary hypertension; [31] they suggest that the hyperparathyroidism is a consequence of vitamin D deficiency.[32] Parathyroid hormone (PTH) elevation may mobilize bone marrow-derived progenitor cells comparable to the effect of G-GSF.[33] PTH increases mesenchymal stem cell proliferation and stimulates VEGF expression in HUVEC.[34,35]

Estrogen

In recent years the “female pulmonary hypertension factor” has received recognition and investigators have begun to develop hypotheses which address the roles of sex hormones in the pathogenesis of pulmonary hypertension. PAH develops in young and in menopausal women; young women have high levels of circulating estrogen, while middle-aged women had a high cumulative exposure to estrogen and/or to estrogen replacement therapy. Sweeney et al. surveyed female patients with PAH and reported that 80% of the women had prior use of hormone therapy whether estrogen is a risk factor for the development of PAH is not clear.[9] It is also not clear whether mechanistically an imbalance between proand-antiangiogenic estrogen metabolites is important, and whether estrogen receptors are involved remains unresolved. [18] However, any mechanistic model of sex steroid hormone impact on the lung circulation and lung vascular remodeling should consider an imbalance between androgens and estrogens. Sweeney et al. had collected serum samples from 70 patients with severe PAH and 18 normal control women and analyzed the samples 150

Pulmonary hypertension

Figure 2: This diagram illustrates the connections between obesity, left ventricular dysfunction and pulmonary hypertension which can be pulmonary arterial or pulmonary venous hypertension.

for estrogen metabolites, using mass spectroscopy. This single time point analysis showed that patients of this PAH cohort had a relative 17-β-estradiol deficit when compared to age-matched control women.[36] To the degree that estrogen has cardioprotective effects this deficit may put women with PAH at a disadvantage.

Thyroid disease

The pulmonary hypertension group at the University of Colorado brought attention to an association of PAH with hypothyroidism. [12] Subsequently other investigators around the world have confirmed this association. [9,13- 15] More recently an association between pulmonary hypertension and hyperthyroid conditions, in particular Graves disease and multinodular goiter, have been reported (Table 1).[37-42] In addition, there are several case reports of the development of hyperthyroid disease in patients with idiopathic PAH after treatment with prostacyclin. Remarkably there is no formulated hypothesis which provides a mechanistic explanation for these associations. Given the overlap between PAH and autoimmune diseases, hypothyroidism in patients with PAH is commonly considered a consequence of an autoimmune thyroiditis – as another manifestation of a still ill-defined underlying immune dysregulation.

It is intriguing to speculate that in the lung and in the thyroid gland a common denominator is a disease of the microcirculation: the thyroid gland is hypervascularized in Graves disease but hypovascularized in Hashimoto thyroiditis. [43] Thyroid hormones can affect the cardiopulmonary system by increasing heart rate and blood flow through the lung. Pulmonary hypertension in Graves disease is in part due to a high cardiac output.

Because thyroid hormones stimulate endothelial cell growth [44], we hypothesized that thyroid hormones can also contribute to the development of pulmonary hypertension by promoting angioproliferation. We therefore assessed the contribution of thyroid hormones to pulmonary vascular remodeling in the Sugen / chronic hypoxia rat model of severe PAH. The results of this study were published recently[45] and here we recapitulate the main findings of this study.

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Table 1: Hyperthyroidism and pulmonary hypertension in clinical trials and case reports Number of patients

Diagnosis

Hyperthyroidism diagnosis before PH diagnosis

114 (43% with PH) 75 (46% with PH) 47 (34% with PH) 33 (41% with PH) 23 (65% with PH) 25 (44% with PH)

47 = Graves; 67 = MNG 30 = Graves; 35 = MNG Hyperthyroidism (unspecified) Hyperthyroidism (unspecified) 22 = Graves; 1 = MNG 7 = Graves; 18 = MNG

PASP before hyperthyroidism treatment (mmHg)

PASP after hyperthyroidism treatment

Yes

27±6 (e)

Yes

48±1.2 (e)

Yes

26±12 (e)

Yes

36±12 (e)

Yes

36±8 (e)

Yes

30±8 (e)

<25 (e) (4 weeks) 34±2 (e) (24 weeks) 23±10 (e) (12 weeks) 29±8 (e) (56±32 weeks) 26±5 (e) (36 weeks) 24±5 (e) (24 weeks)

Improvement after treatment

Authors (references)

Yes

Marvisi et al.

Yes

Siu et al.

Yes

Guntekin et al.

Mercé et al.

Yes Yes Yes

Armigliato et al.

Yazar et al.

e: echocardiography; PH: pulmonary hypertension; MNG: multinodular goiter

The Sugen/chronic hypoxia model of PAH

The role of thyroid hormone in the Sugen/hypoxia model of PAH

This model is based on the combination of chronic VEGF receptor blockade and chronic hypoxia. The underlying concept is that two hits are required to generate severe angioobliterative PAH in the rat: Sugen 5416 (semaxinib) binds with high affinity to the intracellular tyrosine kinase part of VEGFR1 (flt1) and VEGFR2 (KDR) and thus inhibits the signal transduction of these two receptors while chronic hypoxia increases the resistance to blood flow via pulmonary vasoconstriction. Sugen 5416-induced VEGFR blockade causes loss of alveolar septal cells by inducing lung endothelial cell apoptosis.[46,47] These emphysematic changes are accompanied by mild PAH. [46] Chronic hypoxia, in addition to causing an increase in pulmonary vascular shear stress, also activates inflammatory mechanisms and causes homing of precursor-or stem cells to the lung.[48,49] We showed in our original description of this two-hit-model of PAH that the Sugen 5416-induced pulmonary endothelial cell apoptosis is necessary for the subsequent development of angioproliferation and Sakao et al. showed in a cell model of HUVEC subjected to high flow shear stress that Su5416 induced HUVEC apoptosis followed subsequently by lumen-filling proliferation of the HUVEC that had survived the initial apoptosis.[50,51] It is probably of interest and worth mentioning that umbilical cord endothelial cells are fetal cells which contain also pluripotent precursor cells. The pulmonary arteriolar lesions in this model have been characterized to some extent and Abe et al. have reported that with passage of time these lesions resemble the plexiform lesions in the lungs from patients with severe PAH.[52,53] The animals treated with Sugen 5416 and exposed to hypoxia for 4 weeks develop right heart failure and the PAH is refractory to treatment.[54-56]

To address the question whether thyroid hormones contribute to the formation of the pulmonary angioobliteration we subjected in the first set of experiments thyroidectomized male rats to the standard Sugen/chronic hypoxia protocol.[45] Subtotal thyroidectomy decreased plasma thyroxin (T4) to about 20% of the levels in nonthyroidectomized animals. The animals also had a lower heart rate and a lower cardiac output when compared to the Sugen/chronic hypoxia animals – and importantly the mean pulmonary artery (mPAP) approached the mPAP observed in normal control rats not treated with Sugen or exposed to hypoxia (Fig. 3). Whereas approximately 40% of the precapillary arterioles were completely obliterated in the Sugen/chronic hypoxia animals only 8% of these vessels were obliterated in the thyroidectomized Sugen/ chronic hypoxia animals (Fig. 4).[45]

Pulmonary Circulation | April-June 2012 | Vol 2 | No 2

We next implanted T4 pellets s.c. in thyroidectomized rats in order to prove that indeed it was lack of the thyroid hormone that had prevented the pulmonary vasoobliteration. The T4-reconstituted thyroidectomized rats were then subjected to the standard Sugen/chronic hypoxia animal. These animals had slightly higher plasma levels of T4 then the normal control rats and when exposed to sugen and chronic hypoxia the degree of pulmonary hypertension was indistinguishable from that of nonthyroidectomized Sugen/chronic hypoxia rats; however, the number of fully obliterated lung vessels was smaller than in the nonthyroidectomized Sugen/hypoxia rats. Thus, in the aggregate the data support the conclusion that thyroid hormone plays a role in the development of angioproliferative PAH in this model. Next we used a different and pharmacological approach and inhibited T4 production in the rats with propylthiouracil (PTU).

This treatment reduced plasma T4 hormone levels, and 151

Bogaard et al.: Severe PH and role of disorders

(A)

(B)

A

B

C

D

E

F

Figure 4: Lung tissue sections (HE staining) from an euthyroid normal control rat lung (A), higher magnification (B) a lung tissue section from a rat treated with Sugen 5416 and exposed to chronic hypoxia for 4 weeks (C, D) and a thyroidectomized rat exposed to Sugen 5416 and chronic hypoxia (E, F).

In animals with a partial thyroidectomy no angioproliferative lesions were observed.

reduced the mPAP in Sugen/chronic hypoxia animals and the number of fully obliterated pulmonary arterioles.

There was a positive correlation between the RVSP and the number of obliterated vessels. Thus the PTU treatment data support the data obtained with thyroidectomized rats.

Important control experiments were performed to find out whether thyroid hormone per se was sufficient to cause pulmonary angioproliferation: rats were injected with Su5416 (but not exposed to hypoxia) and T4 pellets were implanted s.c. and the animals were studied at four weeks after continuous T4 treatment.[45] Because the combination 152

Figure 3: The effect of propylthiouracil (PTU) treatment or thyroidectomy (THx) on the mean pulmonary arterial pressure (mPAP) of rats exposed to the Sugen 5416/ chronic hypoxia (SuHx) protocol. The mPAP was measured in anesthetized rats.

Both PTU treatment and thyroidectomy reduced to the mPAP. One group of the thyroidectomized animals exposed to the SuHx protocol had thyroxin (T4) pellets implanted at the onset of the 4 weeks chronic hypoxia (SuHx-THx-T4). *P<0.05 different when compared with euthyroid controls. *P<0.05 different when compared with SuHx animals.

of Su5416+T4 did not cause angioproliferation, we conclude that T4 in the Sugen/chronic hypoxia model is permissive and that Su5416-induced lung cell apoptosis by itself without accompanying hypoxia is not sufficient as a driver of exuberant T4-induced endothelial cell proliferation.

Putative mechanism of thyroid hormone related pulmonary angioobliteration in the rat model of PAH

Thyroid hormones activate membrane-and nuclear receptor pathways. Davis et al. described in elegant studies endothelial cell growth stimulated by T4 via the αvβ3 integrin and signaling via ERK1/2 and STAT3.[44, 57-59] T4 can activate VEGF production as well as FGF-2 production. Because in the Sugen/ chronic hypoxia model two of three VEGF receptors are blocked, we wondered whether the T4-associated pulmonary angioproliferation could occur with the participation of the αvβ3/FGF-2/ERK1/2 signaling. Indeed, using IHC we could show a strong expression of αvβ3, FGF-2, FGF-receptor and ERK1/2 proteins in the vasoobliterative lesion cells – these signals were not expressed in the thyroidectomized Sugen/ chronic hypoxia rats.[45] These results provide the first hint that signaling via the integrin αvβ3 and FGF receptors may participate in the formation of the lung vascular lesions in this model of PAH. Experiments are underway to target these proteins using blocking antibodies.

CONCLUSIONS

As the WHO classification of PH illustrates, PAH is multifactorial and it is almost certain that in addition to the known genetic factors like BMPR2 mutations mediators of inflammation, immune system abnormalities and a host of epigenetic factors shape the pulmonary vascular disease in the individual patient.[1] Whether and how female factors, Pulmonary Circulation | April-June 2012 | Vol 2 | No 2

Bogaard et al.: Severe PH and role of disorders

obesity and the metabolic syndrome modify PH is under intense investigation. Gender plays an important role in the pathobiology of heart diseases. The incidence of heart disease increases in women after menopause indicating that sex hormones play an important role in the development of heart disease.[60] This general statement likely applies to the right ventricle when under pressure in PAH.[19] Estrogen can be angioproliferative and protects cardiac myocytes against apoptosis.[60] As much as estrogen may enhance the process of pulmonary angioproliferation it may protect the myocardium of the right ventricle under pressure. To what extent thyroid hormones affect the pulmonary vascular/right heart axis in PAH is understood only in part in the clinical setting of hyperthyroidism, with its dramatic manifestations of tachycardia and increased cardiac output. A more complex situation presents itself when hyperthyroidism is associated with PAH. We have illustrated in a relevant animal model of severe PAH that thyroid hormone, via its endothelial cell-proliferative action, can be permissive and drive angioproliferation.[45] Whether thyroid hormones in euthyroid PAH patients play a pathobiologically important role is unknown – as we also do not know whether the commonly diagnosed hypothyroidism in patients with severe PAH is cardioprotective.

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28. Brewer LC, Michos ED, Reis JP. Vitamin D in atherosclerosis, vascular disease, and endothelial function. Curr Drug Targets 2011;12:54-60.

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30. Ben-Shoshan M, Amir S, Dang DT, Dang LH, Weisman Y, Mabjeesh NJ. 1alpha,25-dihydroxyvitamin D3 (Calcitriol) inhibits hypoxia-inducible factor-1/vascular endothelial growth factor pathway in human cancer cells.

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31. Agmon-Levin N, Blank M, Zandman-Goddard G, Orbach H, Meroni PL, Tincani A, et al. Vitamin D: An instrumental factor in the anti-phospholipid syndrome by inhibition of tissue factor expression. Ann Rheum Dis 2011;70:145-50.

32. Ulrich S, Hersberger M, Fischler M, Huber LC, Senn O, Treder U, et al.

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Parathyroid hormone effectively induces mobilization of progenitor cells without depletion of bone marrow. Exp Hematol 2008;36:1157-66.

34. Rashid G, Bernheim J, Green J, Benchetrit S. Parathyroid hormone stimulates the endothelial expression of vascular endothelial growth factor. Eur J Clin Invest 2008;38:798-803.

35. Di Bernardo G, Galderisi U, Fiorito C, Squillaro T, Cito L, Cipollaro M, et al. Dual role of parathyroid hormone in endothelial progenitor cells and marrow stromal mesenchymal stem cells. J Cell Physiol 2010;222:474-80.

36. Chhatwani L, Xu X, Veenstra T, Voelkel N, Sweeney L. Serum estrogen metabolites in severe pulmonary arterial hypertension. Am J Crit Care Med 2010;181:A4860.

37. Marvisi M, Zambrelli P, Brianti M, Civardi G, Lampugnani R, Delsignore R. Pulmonary hypertension is frequent in hyperthyroidism and normalizes after therapy. Eur J Intern Med 2006;17:267-71.

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Armigliato M, Paolini R, Aggio S, Zamboni S, Galasso MP, Zonzin P, et al. Hyperthyroidism as a cause of pulmonary arterial hypertension: A prospective study. Angiology 2006;57:600-6.

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52. Bogaard H, Mizuno S, Guignabert C, Al Hussaini A, Farkas D, Ruiter G, et al. Copper-dependence of angioproliferation in pulmonary arterial hypertension. Am J Respir Cell Mol Biol 2011 Dec 28. [Epub ahead of print].

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54. Oka M, Homma N, Taraseviciene-Stewart L, Morris KG, Kraskauskas D, Burns N, et al. Rho Kinase-mediated vasoconstriction is important in severe occlusive pulmonary arterial hypertension in rats. Circ Res 2007; 100:923-9.

55. Lemon DD, Horn TR, Cavasin MA, Jeong MY, Haubold KW, Long CS, et al. Cardiac HDAC6 catalytic activity is induced in response to chronic hypertension. J Mol Cell Cardiol 2011;51:41-50.

56. Taraseviciene-Stewart L, Scerbavicius R, Choe KH, Cool C, Wood K, Tuder RM, et al. Simvastatin causes endothelial cell apoptosis and attenuates severe pulmonary hypertension. Am J Physiol Lung Cell Mol Physiol 2006;291:L668-76.

57. Barrera-Hernandez G, Zhan Q, Wong R, Cheng SY. Thyroid hormone receptor is a negative regulator in p53-mediated signaling pathways. DNA Cell Biol 1998;17:743-50.

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

Pulmonary Circulation | April-June 2012 | Vol 2 | No 2

Review Ar ti cl e

Evaluation of patients with chronic thromboembolic pulmonary hypertension for pulmonary endarterectomy William R. Auger, Kim M. Kerr, Nick H. Kim, and Peter F. Fedullo Division of Pulmonary and Critical Care Medicine, University of California, San Diego, California, USA

Abstract Pulmonary hypertension as a result of chronic thromboembolic disease (CTEPH) is potentially curable with pulmonary endarterectomy surgery. Consequently, correctly diagnosing patients with this type of pulmonary hypertension and evaluating these patients with the goal of establishing their candidacy for surgical intervention is of utmost importance. And as advancements in surgical techniques have allowed successful resection of segmental-level chronic thromboembolic disease, the number of CTEPH patients that are deemed suitable surgical candidates has expanded, making it even more important that the evaluation be conducted with greater precision. This article will review a diagnostic approach to patients with suspected chronic thromboembolic disease with an emphasis on the criteria considered in selecting patients for pulmonary endarterectomy surgery.

Key Words: chronic thromboembolic pulmonary hypertension, pulmonary endarterectomy, chronic thromboembolic disease, pulmonary hypertension

Several prospective studies have reported that between 0.57% and 4.6% of acute pulmonary embolic survivors will develop symptomatic, chronic thromboembolic pulmonary hypertension (CTEPH).[1-3] As it is also reported that from 42% to 63% of patients with the established diagnosis of chronic thromboembolic disease have no previously documented acute venous thromboembolism, [4-6] the prevalence of CTEPH cases exceeds those estimates that have resulted from following patients with known thromboembolic events. The importance of correctly establishing the diagnosis of CTEPH is underscored by the understanding that, without appropriate therapy, patients with this disorder typically experience profound functional disability with a relatively poor long-term survivorship.â&#x20AC;&#x2030;[7,8] However, for selected CTEPH patients, pulmonary endarterectomy (PEA) surgery offers the potential for reversing the debilitating pulmonary hypertension and right heart failure that characterizes this disease.[9] The evaluation of patients with suspected chronic thromboembolic disease has the principal goal of identifying Address correspondence to: Dr. William R. Auger Division of Pulmonary and Critical Care Medicine University of California, San Diego 9300 Campus Point Drive La Jolla, CA 92037, USA Email: augers@cox.net

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those who are candidates for an endarterectomy. It is meant to (1) confirm the diagnosis of chronic thromboembolic disease and define the extent of surgically accessible chronic thromboembolic residua; (2) to establish the degree of pulmonary hypertension and cardiac compromise; (3) to delineate the comorbidities that might limit the benefits expected with an endarterectomy; and (4) to estimate the extent of coexisting, small-vessel pulmonary vascular disease, which might similarly impact the anticipated hemodynamic benefit with surgery.

Clinical Presentation

Especially early in the course of the disease, the clinical presentation of CTEPH can be subtle. This subtlety contributes to the delay in diagnosis, making it necessary to maintain a high index of suspicion in those patients presenting with exertional dyspnea without apparent cause Access this article online

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Website: www.pulmonarycirculation.org DOI: 10.4103/2045-8932.97594 How to cite this article: Auger WR, Kerr KM, Kim NH, Fedullo PF. Evaluation of patients with chronic thromboembolic pulmonary hypertension for pulmonary endarterectomy.

Pulm Circ 2012;2:155-62.

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or without a prior history of venous thromboembolism.

Atypical chest pain, episodic hemoptysis, a nonproductive cough, and palpitations are rarely presenting complaints.

Evidence of right heart dysfunction such as peripheral edema, severe exercise limitation and associated chest discomfort, exertional dizziness, or syncopal episodes can be manifest late in the disease.

Physical examination findings can be equally deceptive early in the natural history of CTEPH. However, with advancing pulmonary hypertension, clinical presentation and examination findings are similar to that seen in other forms of pulmonary hypertension: discernible right ventricular impulse, a split second heart sound with accentuation of the pulmonic component, varying degrees of tricuspid regurgitation, and a right ventricular S4 gallop. As right ventricular failure develops, jugular venous distension, peripheral edema, hepatomegaly, ascites, and a rightsided S3 may become evident. The presence of pulmonary flow murmurs or bruits is often a physical examination finding that can be used to distinguish small vessel from large vessel variants of pulmonary hypertension.[10] An auscultatory finding in approximately 30% of patients with CTEPH, the bruit results from turbulent flow across partially obstructed, medium- to large-sized pulmonary vessels. They have not been described in pulmonary hypertensive disorders arising from the microvasculature.

However, they are not unique to patients with chronic thromboembolic disease, having been described in other disease states which involve large pulmonary arteries, such as congenital branch stenosis or large vessel pulmonary arteritis. Additional examination findings in the CTEPH patient might include peripheral cyanosis, alerting the clinician to the possibility of a right-to-left shunt through a patent foramen ovale. Examination of the lower extremities may disclose superficial varicosities and venous stasis skin discoloration in those individuals who have experienced prior venous thrombosis.

Diagnostic Evaluation

Defining a procoagulant state in patients evaluated for CTEPH has important implications as certain thrombophilias such as the presence of antiphospholipid antibodies might warrant a more intense level of chronic anticoagulation to prevent thrombosis. Moreover, these patients are at greater risk for thrombosis postendarterectomy. Antithrombin III, Protein C, and Protein S deficiencies, as well as Factor II (prothrombin) and Factor V Leiden mutations, are among the hereditary thrombophilic states which should be pursued in the evaluation of the CTEPH patient. However, in a large study investigating this issue, Wolf and colleagues showed that hereditary thrombophilia was not more prevalent in samples analyzed in 46 CTEPH patients or 156

64 patients with idiopathic pulmonary hypertension compared to control subjects (N=100). The same study, however, demonstrated that 20% of patients diagnosed with CTEPH exhibited antiphospholipid antibodies.[11] Subsequent studies have revealed similar results, with the presence of antiphospholipid antibodies frequently associated with chronic thromboembolic disease. [12,13] Bonderman and colleagues also showed increased levels of Factor VIII in 41% of 122 patients with CTEPH, levels that were substantially higher than those in control subjects and patients with nonthrombotic pulmonary hypertension.[14] In the evaluation of the patient suspected of having CTEPH, “routine” laboratory testing may be helpful in defining the severity of disease. For those with severe right ventricular dysfunction and coexisting liver congestion, elevation of transaminase and bilirubin levels can be expected. In the same subgroup of patients where renal blood flow and glomerular perfusion may be compromised, either from a low cardiac output or the use of diuretics (or both), elevation of serum creatinine and blood urea nitrogen may result.

Pulmonary function testing is most useful in evaluating for coexisting parenchymal lung disease or airflow obstruction.

Approximately 20% of CTEPH patients with parenchymal scarring from prior lung infarction, a mild to moderate restrictive defect may be detected.[15] Similarly, a modest reduction in single breath diffusing capacity for carbon monoxide (DLco) may be present in some CTEPH patients, though a normal value does not exclude the diagnosis.[16] A severe reduction in DLco should raise concerns that the distal pulmonary vascular bed is significantly compromised, making it imperative that an alternative diagnosis other than CTEPH be considered. Furthermore, CTEPH patients will frequently exhibit some degree of hypoxemia, and if measured, elevated dead-space ventilation, [17] both worsening with exercise. These findings reflect a moderate ventilation–perfusion mismatch in CTEPH and an inadequate cardiac output response to exercise resulting in a low mixed venous oxygen saturation. [18] Marked hypoxemia at rest implies severe right heart dysfunction or the presence of a considerable right-to-left shunt, such as through a patent foramen ovale.

The chest radiograph in patients with chronic thromboembolic disease may be deceptively unremarkable early on. With disease progression and the development of pulmonary hypertension, enlargement of the proximal pulmonary vascular bed typically occurs. With chronic thromboembolic involvement of the main or lobar pulmonary arteries, this central PA enlargement can be asymmetric. This is not a radiographic finding in those patients with small-vessel disease pulmonary hypertension. [19] As the right ventricle adapts to the rise in pulmonary vascular resistance, Pulmonary Circulation | April-June 2012 | Vol 2 | No 2

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radiographic signs of chamber enlargement such as obliteration of the retrosternal space and prominence of the right heart border, can be observed.[20] Without coexisting parenchymal lung disease, interstitial-alveolar markings within the lung fields are atypical. However, relatively avascular lung regions can be appreciated if an organized thrombus has compromised blood flow to that area. In these poorly perfused lung regions, the sequela of lung injury such as peripheral alveolar opacities, linear scar-like lesions, and pleural thickening may be found.

Following an episode of pulmonary embolism, routine cardiopulmonary screening has a low yield in the detection of CTEPH.[21] Frequently, the first objective indication as to the presence of elevated pulmonary pressures or right ventricular compromise is provided with transthoracic echocardiography. Current technology allows for estimates of pulmonary artery systolic pressure (using Doppler analysis of the degree of tricuspid regurgitation), along with cardiac output and RV performance.[22] Enlargement of the right heart chambers, tricuspid regurgitation as a result of this chamber enlargement, flattening or paradoxical motion of the interventricular septum, encroachment of an enlarged right ventricle on the left ventricular cavity, and impaired left ventricular diastolic dysfunction not the result of primary left ventricular or valvular heart disease are findings in patients with significant pulmonary hypertension. [23,24] Contrast echocardiography using intravenous agitated saline can detect the presence of an intracardiac shunt, such as a patent foramen ovale or a previously undetected septal defect. If detected preoperatively, the atrial septal defect can be surgically repaired at the time of an endarterectomy. Though not specifically studied in patients with CTEPH, should an echocardiogram obtained at rest demonstrate minimally elevated pulmonary artery pressures or only modest right ventricular compromise in a patient experiencing significant cardiopulmonary symptoms with exertion, exercise echocardiography may demonstrate a substantial rise in pulmonary artery pressures or dilatation of the right ventricle.

V/Q scanning with CT angiography in 227 pulmonary hypertensive patients, there was a sensitivity of 97.4 % for V/Q scanning compared to 51% for CT angiography in the detection of chronic thromboembolic disease.[25] In a more recent study of 12 CTEPH patients, Soler and colleagues demonstrated that SPECT perfusion scintigraphy was more sensitive in detecting obstructed vascular segments when compared to CT pulmonary angiography, with a sensitivity of 62+4.1% versus 47.8+2.9%, respectively.[26] Furthermore, the interpretation of an abnormal perfusion pattern can assist in the differentiation between disorders involving the central or proximal vascular bed from those primarily affecting the peripheral pulmonary circulation. In chronic thromboembolic disease, at least one, but more commonly several, segmental or larger mismatched perfusion defects are present (Fig. 1). For those patients with small-vessel pulmonary vascular disease, perfusion scans either are normal or exhibit a “mottled” appearance characterized by nonsegmental defects.[27,28] Exceptions include cases of pulmonary veno-occlusive disease or pulmonary capillary hemangiomatosis in which multiple, larger mismatched defects have been reported.[29,30] Equally important has been the observation that a relatively normal perfusion pattern on V/Q scan excludes the diagnosis of surgically accessible chronic thromboembolic disease.

It has also been established that the magnitude of perfusion defects exhibited by CTEPH patients with operable disease may understate the degree of pulmonary vascular obstruction determined by angiography.[31] The plausible explanation for this finding is that during the process of thrombus organization, proximal vessel thromboemboli may recannalize, or narrow the vessel in such a manner that radiolabeled macroaggregated albumin may traverse the area of partial obstruction, creating gray zones or regions of relative hypoperfusion. Therefore, chronic thromboembolic

Ventilation–Perfusion Scan

To a large extent, computed tomographic (CT) angiography of the pulmonary vessels has replaced ventilation–perfusion (V/Q) scintigraphy in the evaluation of patients with suspected acute pulmonary embolic disease. However, the V/Q scan continues to provide essential information in the evaluation of the pulmonary hypertensive patient.

In these patients, the V/Q scan can often be the first indication that chronic thromboembolic disease should be considered, and serve as a valuable screening test for this disease. In a single-center, retrospective survey comparing Pulmonary Circulation | April-June 2012 | Vol 2 | No 2

Figure 1: Lung ventilation-perfusion scan showing large, bilateral unmatched perfusion defects; no perfusion to right middle and lower lobes.

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disease should be considered and further evaluation for operable disease should proceed even if the V/Q scan demonstrates a limited number of mismatched perfusion defects, especially when accompanied by hypoperfused lung regions in a patient with pulmonary hypertension.

Assessment of Pulmonary Hemodynamics

Right heart catheterization in the evaluation of patients with suspected CTEPH objectively defines the severity of pulmonary hypertension and the degree of cardiac dysfunction at rest.

This hemodynamic assessment is important in discussions with patients regarding perioperative risks should they prove to be surgical candidates. Available data would suggest that patients with severe pulmonary hypertension (pulmonary vascular resistance >1,000 dyn/s/cm-5) bear a greater perioperative mortality risk.

Hartz et al. reported that a preoperative PVR over 1,100 dyn/s/cm-5 was associated with 41% mortality, compared to less than 6% if PVR was less than 1,100 dyn/s/cm-5. [32] Dartevelle et al. reported an increased postoperative mortality of 20% for patients with preoperative PVR over 1,200 dyn/s/ cm-5 compared to 4% mortality if the preoperative PVR was less than 900 dyn/s/cm-5.[33] Similarly, examining outcomes in 743 patients between 1999 and 2004, Thistlethwaite and colleagues reported a perioperative mortality rate of 10.8% in those patients with a preoperative PA systolic pressure of 100 mmHg (PVR 1299.0 + 532.6 dyn/s/cm-5), compared to 4.2% if the preoperative PA systolic pressure was less than 100 mmHg (PVR 546.4 + 365.1 dyn/s/cm-5).[34] Additionally, for symptomatic CTEPH patients with modest pulmonary hypertension at rest, exercise hemodynamic measurements may be obtained. In these cases, it is likely that the normal compensatory mechanisms of recruitment and dilation of the pulmonary vasculature have been overcome, and with exercise, a linear elevation in pulmonary artery pressure as cardiac output increases can be observed. This hemodynamic information provides objective evidence to explain an individual’s symptoms, and likely reflects a clinically relevant stage in the development of severe CTEPH in which there is coexisting, small-vessel hypertensive changes.

Furthermore, right heart catheterization has the potential to provide objective data in analyzing the degree of this smallvessel disease, information which may help predict outcomes following endarterectomy. “Partitioning” the different elements (proximal vs. distal) of pulmonary vascular resistance in CTEPH has been investigated. In a small series of 26 CTEPH patients, Kim and colleagues, utilizing pulmonary artery occlusion waveform analysis, demonstrated excellent 158

inverse correlation between the percent upstream resistance and postoperative mean pulmonary artery pressure and pulmonary vascular resistance. In addition, all four deaths in this series occurred in patients in whom the upstream resistance was less than 60%.[35] If future investigations validate these preliminary observations, this information may identify a subgroup of CTEPH patients who might be excluded from surgical consideration.

Conventional Pulmonary Angiography

Prior to the availability of CT angiography and magnetic resonance imaging of the chest, conventional pulmonary angiography was the principal means of confirming the diagnosis of chronic thromboembolic disease and assessing the proximal extent of disease in evaluating patients for pulmonary endarterectomy surgery. In most respects, it remains the “gold standard” for achieving these diagnostic goals and in providing a “map” for surgery against which other modalities are to be measured. Conventional pulmonary angiography can be safely performed, when taking proper precautions, even in severe pulmonary hypertensive patients.[36] In terms of technique, multiple, selective injections are not required. A single injection of nonionic contrast into both proximal pulmonary arteries, the volume and injection rate adjusted based on cardiac output, appears to be sufficient. As little as 15–20 ml of contrast may be required for each pulmonary artery injection. Ideally, biplane acquisition provides optimal anatomic detail, the lateral projection providing more definition of lobar and segmental anatomy than can be achieved with an anterior–posterior view alone. Under essentially all circumstances, a properly performed biplane angiogram will provide sufficient information on which to base a decision regarding chronic thrombus location, and as a result, surgical accessibility.

The angiographic appearance of chronic thromboembolic disease bears little resemblance to that of the well-defined, intraluminal filling defects of acute pulmonary embolism.

Instead, the angiographic patterns encountered in chronic thromboembolic disease reflect the complex patterns of organization and recanalization that occur following an acute thromboembolic event. Several angiographic patterns have been described in chronic thromboembolic disease which correlate with the material removed at the time of surgery.[37] These include “pouch defects,” pulmonary artery webs or bands, intimal irregularities, abrupt, frequently angular narrowing of the major pulmonary arteries, and complete obstruction of main, lobar, or segmental vessels at their point of origin (Fig. 2). In the majority of CTEPH patients, two or more of these angiographic findings are present, typically involving both lungs.

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CT Angiography of the Chest With the advances in CT angiography of the chest, and greater availability and use of this technology in assessing the pulmonary vascular bed, CT is playing an increased role in the evaluation of the pulmonary hypertensive patient for chronic thromboembolic disease. There are a number of CT findings which have been described in patients with chronic thromboembolic disease. These include (1) mosaic perfusion of the lung parenchyma; (2) enlargement of the central pulmonary arteries and right heart chambers; (3) variability in the size of lobar and segmental-level vessels with a reduction in vessel caliber of those involved with chronic thrombi; and (4) peripheral, scar-like lesions in poorly perfused lung regions. With contrast enhancement of the pulmonary vasculature during CT imaging, organized thrombus can be seen to line the pulmonary vessels, often in an eccentric manner (Fig. 3). Associated narrowing of pulmonary arteries, web strictures, “pouch defects,” and other irregularities of the intima may also be appreciated[38,39] (Fig. 4); these CT findings are distinct from the intraluminal filling defects of acute thromboemboli and primary pulmonary vascular tumors.[40] And with

A

A

appropriate timing of the intravenous contrast bolus for CTA, opacification of the pulmonary and systemic circulations is possible. In addition to the pulmonary vascular bed, this allows examination of a number of cardiac features including cardiac chamber size, position, and shape of the interventricular septum, the presence of congenital cardiac abnormalities, anomalous pulmonary venous drainage, and the size and distribution of collateral vessels arising from the systemic arterial circulation (bronchial arteries off the aorta, coronary vessels).[41]

What remains unvalidated is the utility of CT angiography in determining operability in certain subgroups of CTEPH patients. This is particularly important as operative techniques allow for resection of chronic thromboembolic material at the segmental vessel level (Fig. 5). Additionally, clinical experience has demonstrated that the absence of lining thrombus or thickened intima of the central vessels on CT does not exclude the diagnosis of chronic thromboembolic disease or the possibility of surgical intervention. Studies directly comparing CT with pulmonary angiography are limited. In one such study, CT and digital angiography were nearly equivalent in terms of identifying complete vessel occlusion at the segmental level. However,

Figure 2: (A) PA and lateral right pulmonary angiogram of the patient whose V/Q scan is shown in Figure 1; complete obstruction of the right interlobar vessel. (B) PA and lateral left pulmonary angiogram, showing a “pouch” occlusion of the descending pulmonary vessel beyond the superior segment; appreciated on the lateral view is a small lingular artery which is difficult to discern on AP view.

B

B

Figure 3: (A) Accompanying CT angiogram to the studies in Figures 1 and 2. Lining and occlusive chronic thromboembolic material observed in the right interlobar and descending PA; lining thrombus involving the left descending PA. (B) Semiorganized and chronic thromboembolic material endarterectomized from the patient.

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A

B

Figure 4: CT angiogram demonstrating the evolution of an acute thrombus to an intravascular chronic “web” in the proximal right descending PA at the level of the right middle lobe take-off. The time interval between the left (A) and right (B) images is 1 year.

for nonocclusive changes, CT was significantly inferior to angiography.[42] Accuracy of CT scanning has improved with technological advances and the introduction of 64-detector row scanners. In a preliminary study involving 27 patients with suspected CTEPH, sensitivity of 64-dectector row CT was 98.3% at the main and lobar level and 94% at the segmental level when compared to digital subtraction angiography.[43]

There is considerable value for CT in detecting disorders of the pulmonary parenchyma and mediastinum. For CTEPH patients with coexisting interstitial lung disease or emphysema, CT will be able to define the extent and location of the parenchymal lung process. Reperfusion of diseased lung parenchyma following an endarterectomy may result in an undesirable postoperative outcome, and thereby exclude a patient from surgical consideration. And for those patients whose V/Q scan demonstrates the absence or near complete absence of perfusion to an entire lung, CT is an essential study to rule out extrinsic pulmonary vascular compression from mediastinal adenopathy, fibrosis,[44] or neoplasm.[45]

Magnetic Resonance of the Chest Experience using magnetic resonance (MR) imaging and magnetic resonance angiography (MRA) to visualize the pulmonary vascular system in patients with chronic thromboembolic pulmonary hypertension is expanding.[46] For centers where conventional pulmonary angiography is either unavailable or felt to be too risky to perform, the evolving information on MR imaging as an alternative means to determine surgical candidacy for CTEPH patients is encouraging. At Papworth Hospital, UK, MRA has replaced conventional angiography in establishing the pulmonary vascular “map” for patients evaluated for endarterectomy surgery.[47] Kreitner and colleagues have shown that contrast-enhanced MRA is able to demonstrate the vascular changes typical for CTE disease. In a study of 34 CTEPH patients, wall-adherent thromboembolic material involving the central pulmonary arteries down to 160

A

B

Figure 5: Two images from a CT angiogram demonstrating irregular intraluminal chronic thrombus and vessel narrowing of the segmental vessels.

These lesions proved to be surgically resectable.

the segmental level could be demonstrated; intraluminal webs and bands, as well as abnormal vessel tapering and “cutoffs” were also detected. Furthermore, they showed that MRA was superior to digital subtraction angiography in determining the proximal location of resectable chronic thromboembolic material. [48] An additional study comparing magnetic resonance techniques with conventional contrast angiography involved 29 patients with either CTEPH or idiopathic pulmonary arterial hypertension (IPAH). Nikolaou and colleagues showed that the combined interpretation of MR perfusion imaging and MR angiography led to a correct diagnosis of IPAH or CTEPH in 26 (90%) of 29 patients when compared to the reference diagnosis based on V/Q scintigraphy, digital subtraction angiography, or CT angiography. The interpretation of MR angiography alone had a sensitivity of 71% for wall adherent thrombi, 50% for webs and bands, and between 83% and 86% for detection of complete vessel obstruction and free-floating thrombi when compared to DSA or CT angiography.[49] More recently, in a retrospective study of 53 patients with chronic thromboembolic pulmonary hypertension, the diagnostic accuracy of contrast-enhanced MR angiography (CE-MRA) and unenhanced proton MR imaging was compared to CT pulmonary angiography.

The sensitivity and specificity of CE-MRA in establishing proximal and distal CTE was 98% and 94%, respectively.

The sensitivity for central vessel disease rose from 50% to 88% when analysis was performed with unenhanced proton MRA. Keeping in mind that this study was comparing MR technology to CT angiography, the detection of stenotic lesions, poststenotic dilatation, and occlusive lesions was better achieved with CE-MRA.[50]

As with CT, there are other features of magnetic resonance imaging that have been demonstrated to be useful in the evaluation of CTEPH patients. Cine imaging allows an assessment of RV and LV function, providing data on endsystolic and end-diastolic volumes, ejection fraction, and muscle mass.[51,52] Furthermore, phase contrast imaging may be used to measure cardiac output, along with pulmonary and systemic arterial flow. In CTEPH patients undergoing Pulmonary Circulation | April-June 2012 | Vol 2 | No 2

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PEA, this technique has been used to measure changes in aortic and pulmonary arterial blood flow before and after surgery.[48,53]

Summary: Selection of Surgical Candidates

In the evaluation of patients with CTEPH, the goals are to establish whether or not pulmonary endarterectomy is feasible, and then to determine whether or not surgery is appropriate. And despite the advancements in imaging techniques, and the forward strides in surgical capabilities, there remains a subjective element, which to a large extent is influenced by experience, in determining surgical candidacy for any one CTEPH patient. The interpretion of conventional angiographic patterns, CT abnormalities, or MR findings that are felt to be consistent with operable CTE disease is not simply based on training and experience but needs to be viewed in the context of the capabilities of the surgical team. This is especially relevant given the greater ability and success in the resection of segmental level chronic thromboembolic disease. More difficult to predict are the factors that influence perioperative mortality and postoperative outcomes. The impact of an individual’s age and comorbid medical conditions on surgical risk is always difficult to assess. And for any individual patient, the level of cardiopulmonary and functional limitation experienced by them is an important consideration in the decision to proceed with surgery, which needs to be balanced against the anticipation that a hemodynamic benefit will result from an endarterectomy. If a meaningful reduction in pulmonary pressures seems unlikely, proceeding with surgery is ill-advised. This prediction of hemodynamic benefit is often based on the degree of coexisting, small-vessel disease and whether the extent of surgically accessible chronic thromboembolic disease is disproportionate to the level of pulmonary hypertension and RV dysfunction experienced by the patient. To date, this assessment is to a large extent subjective and based on the experience of the evaluation team, making it increasingly important to develop evaluative techniques to make it less so. Ongoing research and careful clinical observations are required to make this decision as precise as possible.

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

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Review Ar ti cl e

Diagnosis and management of pulmonary hypertension associated with left ventricular diastolic dysfunction Vinicio A. de Jesus Perez1,2, Francois Haddad3, and Roham T. Zamanian1,2 1

Division of Pulmonary and Critical Care Medicine, 2The Wall Center for Pulmonary Vascular Medicine, and 3Division of Cardiology, Stanford University Medical Center, Stanford, California, USA

Abstract Pulmonary hypertension (PH) is commonly seen in patients who present with left ventricular diastolic dysfunction (LVDD) and is considered a marker of poor prognosis. While PH in this setting is thought to result from pulmonary venous congestion, there is a subset of patients in which pulmonary pressures fail to improve with appropriate management of diastolic heart failure and go on to develop a clinical picture similar to that of patients with pulmonary arterial hypertension (PAH). Despite the utility of Doppler echocardiography and exercise testing in the initial evaluation of patients with suspected PH-LVDD, the diagnosis can only be confirmed using right heart catheterization. Management of PH-LVDD centers on both optimizing fluid management and afterload reduction to reducing left ventricular diastolic pressures and also increase pulmonary venous return. To date, there is no clear evidence that addition of PH-specific drugs can improve clinical outcomes, and their use should only be considered in the setting of clinical trials. In conclusion, PH-LVDD remains a challenging clinical entity that complicates the management of left ventricular dysfunction and significantly contributes to its morbidity and mortality. Determination of the optimal diagnostic and treatment strategies for this form of PH should be the goal of future studies.

Key Words: congestive heart failure, pulmonary hypertension, hemodynamics, echocardiography, therapeutics

Pulmonary hypertension (PH) is often associated with left heart failure.[1] The 2008 revised WHO classification recognizes PH associated with left heart disease as a unique disease category (WHO Class II) that is distinct from other forms of PH such as those associated with pulmonary arterial hypertension (WHO Group I), hypoxic lung (WHO Group III), and chronic thromboembolic diseases (WHO Group IV).[2,3] Awareness of this form of PH is relevant to practitioners as heart failure is the most common cause of PH in the United States. For instance, it has been estimated that over five million people in the United States suffer from heart failure and over 500,000 cases are newly diagnosed each year.[4,5] Congestive heart failure (CHF) is also the most common admission diagnosis among the elderly population and a growing source of significant morbidity and mortality for this age group[6] Given the progressive rise in cases of CHF over the last decade, it is likely that PH associated with heart failure will become the most prevalent form of PH seen in the clinical setting.

Address correspondence to: Dr. Vinicio A. de Jesus Perez Division of Pulmonary and Critical Care Medicine 300 Pasteur Drive Stanford, CA 94305, USA Email: vdejesus@stanford.edu

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While most cases of heart failure are thought to be due to depressed systolic function, about 40â&#x20AC;&#x201C;50% of symptomatic patients have preserved ejection fractions and are diagnosed with heart failure with preserved ejection fraction (HFPEF) or left ventricular diastolic dysfunction (LVDD)[7-9] In contrast to systolic heart failure, the diagnosis of LVDD is challenging as no clear diagnostic criteria or definite noninvasive tests to assess diastolic function are currently available; thus, the impact of the current management strategies on mortality is questionable.

Among known risk factors, aging is strongly correlated with development of LVDD. [6,10,11] With normal aging, there is progressive development of ventricular stiffening and reduced relaxation,[12,13] which may predispose to development of LVDD in patients who also suffer from Access this article online

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Website: www.pulmonarycirculation.org DOI: 10.4103/2045-8932.97598 How to cite this article: de Jesus Perez VA, Haddad F, Zamanian RT. Diagnosis and management of pulmonary hypertension associated with left ventricular diastolic dysfunction. Pulm Circ 2012;2:163-9.

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chronic conditions such as ischemic cardiomyopathy, hypertension, or diabetes. However, as the epidemic of diabetes and systemic hypertension continues to grow and the population ages, it is likely that incidence and prevalence of LVDD will also continue to increase.[6]

A recent study by Lam and colleagues has demonstrated that the prevalence of PH in the setting of LVDD is high. Using Doppler echocardiography in a group of 244 patients with LVDD, it was found that the prevalence of PH (defined as pulmonary artery systolic pressure or PASP of >35 mmHg) was estimated to be 83% with a median PASP of 48 mmHg. Moreover, just as with post myocardial infarction[14,15] and idiopathic dilated cardiomyopathy,[16] the presence of PH in this group was a shown marker for increased mortality[14] (Fig. 1). In addition, it has been proposed that there is a subset of these patients who exhibit PH “out of proportion” to the degree of left ventricular dysfunction and suffer from a form of PH which resembles pulmonary arterial hypertension (WHO Group I); whether these patients have a worse prognosis or respond differently to heart failure therapy, however, is unknown.[15,17,18]

An understanding of the pathophysiology of PH in the setting of LVDD is mandatory prior to deciding on the optimal diagnosis and treatment strategies. In this review, we will summarize the current evidence for the various diagnostic studies and therapies available for PH and LVDD and will present an approach that will hopefully facilitate the proper management of patients with these conditions.

PATHOPHYSIOLOGY OF PH-LVDD

PH-LVDD is a consequence of abnormally elevated left ventricular diastolic pressures (LVDP) and pulmonary venous congestion.[19] In LVDD, the curve for LV diastolic pressure in relation to volume is shifted upward and to the left with a resultant increase in diastolic LV filling.

Elevation of LVDP is the result of both abnormal active relaxation and increased passive stiffness of the left ventricle.[6,8,11,15,18,20] The face that both mechanisms play an active role in the pathophysiology of elevated LVDP was demonstrated in a study in which, despite correction of slow relaxation, LV pressures failed to normalize as a result of increased chamber stiffness. [21] Given the decreased pressure gradient between the left ventricle and the pulmonary venous circulation, venous return is reduced and pulmonary venous pressures increase resulting in progressive distension and damage to the pulmonary veins.[22,23] In this setting, patients can progress to pulmonary edema and alveolar hemorrhage unless the LVDP is reduced with targeted therapy. Moreover, just as with idiopathic dilated cardiomyopathy[16] and post 164

Figure 1: Survival of patients with PH-LVDD is inversely correlated to degree of pulmonary artery systolic pressure (PASP) elevation (Adapted from Lam et al[14]).

myocardial infarction[14,15], the presence of PH in this group was a shown marker of increased mortality (Fig 1).[14]

While most cases of PH-LVDD result from passive venous congestion, a subset of patients suffer from a more severe form of PH-LVDD characterized by findings of increased vascular tone and abnormal pulmonary artery remodeling. Vasoconstriction is thought to be secondary to endothelin release from the pulmonary endothelium in response to venous congestion and subsequent smooth muscle contraction in the neighboring medial layer. While potentially reversible if treated early, vasospasm may progress to a “remodeling” of the pulmonary arteries possibly leading to irreversible vascular disease. The vascular pathology in these cases can be indistinguishable from WHO Class I PAH and is postulated to occur from changes in the elastic fibers of the pulmonary arterial wall, intimal fibrosis, and medial hypertrophy. In addition, abnormal thickening of the veins and neointima formation can also be seen in these cases. In contrast to WHO Class I patients, there is great potential for reversibility of these changes in PH-LVDD with specific treatment of LVDD and improvement of pulmonary venous hypertension.[15,18,24]

DIAGNOSIS OF PH-LVDD

Clinically, patients with PH-LVDD usually present with worsening dyspnea, weight gain, and progressive limitation in exercise capacity. In contrast to patients with PAH, patients with PH-LVDD may also complain of orthopnea and paroxysmal nocturnal dyspnea. The physical examination may reveal signs of fluid retention such as peripheral edema, ascites, and inspiratory crackles; additionally, the cardiac examination typically will reveal presence of an S3, distented jugular veins, and hepatojugular reflux. Further clinical clues that can help differentiate PAH from PH-LVDD include the presence of left atrial enlargement, left rather than right ventricular hypertrophy on EKG, and the finding Pulmonary Circulation | April-June 2012 | Vol 2 | No 2

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of Kerley B lines, pleural effusion, and pulmonary edema on a chest X-ray.[22]

Despite the wealth of information that can be obtained through the use of history, physical examination, and CXR, it can still be difficult for the clinician to differentiate between systolic and diastolic left ventricular impairment without further investigation. Among the imaging studies available for the evaluation of patients with possible PH-LVDD, transthoracic echocardiography (TTE) provides invaluable insight on the anatomy and function of the left ventricle and can help differentiate between systolic and diastolic failure (Fig. 2). Current diagnostic criteria for diastolic dysfunction center on estimating left ventricular filling pressures or abnormal diastolic filling profile. The most useful criteria include left atrial volume, mitral inflow filling profile and tissue Doppler annular velocities profiles. In addition, TTE can provide an estimate of the pulmonary pressures via measurement of right ventricular systolic pressure (RVSP), and can also help identify cardiac valve problems that can also impact left ventricular function.[3,8,15,24] Using the information provided by TTE, several groups have attempted to reach a consensus definition for LVDD. In 1998, for example, the European Society of Cardiology proposed the following criteria for the diagnosis of LVDD: (1) presence of signs and symptoms of CHF; (2) normal or mildly abnormal left ventricular systolic dysfunction defined as EF>45%; and (3) evidence of abnormal left ventricular relaxation, filling, distensibility, or diastolic stiffness.[25] While these criteria attempt to resolve the confusion surrounding the diagnosis of LVDD, there is still an ongoing debate on whether cases of combined systolic and diastolic dysfunction can be reliably differentiated and whether TTE alone is enough to predict left ventricular dysfunction. For this reason, some experts have advocated for routine measurements of left ventricular diastolic filling to determine the presence of diastolic dysfunction. This can be done using the E/A ratio, a measure that incorporates passive left ventricular filling during diastole (E) and active filling following left atrial contraction (A). In normal hearts, the E/A ratio can be between 0.7 and 1.4; however, in the

A

B

setting of diastolic dysfunction, this ratio reverses and values become less than 0.7.[4] Finally, there are studies showing that even the single presence of an abnormally elevated left atrial pressure[26] can serve as a reliable indicator for LVDD.

Despite both its ability to visualize cardiac function noninvasively in real time and also its availability, there are significant limitations to the diagnostic potential of TTE in PH-LVDD. These limitations are due to the fact that TTE is both operator dependent and its reproducibility may vary from center to center. In addition, because pulmonary hypertension itself produces diastolic filling abnormalities in the LV, Doppler echocardiography cannot be relied on to distinguish between Class 1 and 2 PH. Furthermore, proper visualization of the left ventricle in some patients may be technically difficult and severely limit measurement of functional parameters.[27] Thus, given the technical limitations of TTE, confirmation of the diagnosis of PHLVDD will require further testing.

Since its discovery in 1988, brain natriuretic peptide (BNP) has been shown to be both a sensitive and specific biomarker of altered left ventricular structure and function that increases under of atrial and/or ventricular pressure overload and with cardiomyocyte hypertrophy. [28-32] While its role of patients with systolic CHF has been demonstrated in multiple studies, the use of plasma BNP in the evaluation and management of LVDD is less clear.

A major barrier to the routine use of BNP in this setting is the lack of an appropriate threshold value that can help distinguish between left ventricular hypertrophy and diastolic dysfunction or failure. This was shown in a European study where 1,678 patients were screened for diastolic dysfunction using measurements of serum BNP. In this study, despite its high negative predictive value (99.9%), plasma BNP in the detection of diastolic dysfunction had an estimated sensitivity and specificity of 61 and 55%, respectively.[33] The poor performance of the BNP test is likely related to a number of confounding factors within the target population such as presence of

C

Figure 2: Echocardiographic findings in PH-LVDD. (A) Measurement of passive left ventricular filling during diastole (E) and active filling following left atrial contraction (A). In the setting of LVDD, ventricular filling by left atrial contraction becomes prominent and the E/A ratio is reversed (B) Distended left atrium and (C) increased tricuspid regurgitation jet in a patient with PH-LVDD. Pulmonary Circulation | April-June 2012 | Vol 2 | No 2

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LVH and age. However, while age alone can increase plasma BNP, elderly patients with isolated diastolic dysfunction are likely to have higher BNP levels with advanced filling abnormalities on TTE than those with milder forms of diastolic dysfunction. [34] While use of plasma BNP measurements alone may be of limited diagnostic utility, its combination with other modalities can result in an increased diagnostic yield. Studies have shown positive correlation between BNP and echocardiographic measures of diastolic filling (i.e., E/A ratio, see above) at rest and at peak exercise.[35,36] Thus, while alterations in BNP seem to correlate with LVDD, the role of plasma BNP measurements in diagnosis need to be established further.

In recent years, more centers have started to use cardiopulmonary exercise testing (CPET) in the diagnosis of both systolic and diastolic LV failure. Since most patients with PH-LVDD will experience worsening symptoms with exercise, the CPET provides a unique opportunity to study cardiopulmonary interactions, measure left ventricular reserve, and determine whether a pulmonary vascular limit to exercise is present. Furthermore, in patients with no obvious diastolic filling abnormalities at rest, the CPET may help confirm the diagnosis and reveal the contribution of pulmonary, hematologic, musculoskeletal, and neurologic components to the patient’s clinical picture.[18,37-39] Despite the advantages offered by the CPET, it is a labor-intensive study that few centers can perform routinely and most patients will need referral to more specialized clinics, an event that can delay the diagnosis of this condition.

Currently, the gold standard for diagnosis of PH and LVDD is right heart catheterization. In the presence of an elevated pulmonary capillary wedge pressure (PCWP) and a normal ejection fraction, a diagnosis of pulmonary venous hypertension secondary to LVDD should be strongly considered (Fig. 3). In patients in which determination of a PCWP is either technically difficult or when the presence of other conditions such as atrial myxoma, mediastinal fibrosis, or pulmonary vein stenosis may be creating false positive results, direct measurement of left ventricular end diastolic pressure (LVEDP) should be performed. Finally, it should be pointed out that changes in intrathoracic pressures affect PCWP and LVEDP measurements and reliable values can only be obtained during end expiration.[2,3,15,24]

While an elevated PCWP and LVEDP in the setting of a preserved ejection fraction argues strongly in favor of LVDD, some patients can present with symptoms of LVDD without evidence of elevated PCWP and LVEDP. In these patients, differentiation of pulmonary venous from arterial hypertension is very difficult and will require the use of additional diagnostic studies to elicit evidence of 166

A

B Figure 3: Hemodynamic profile of a patient with PH-LVDD. (A) Patient demonstrates a mean pulmonary artery pressure (in magenta) of approximately 70 mmHg in the setting of a pulmonary capillary wedge pressure (in magenta) of approximately 20 mmHg (B).

diastolic dysfunction such as an exercise, fluid, or inotropic challenge. [15,40,41] Under these conditions, patients with PAH may increase their cardiac output without a significant change in the PCWP while those with PH-LVDD will show both an increase in cardiac output and PCWP supporting the presence of pulmonary venous hypertension.

As alluded to previously, there appears to be two types of patients with PH-LVDD: those whose PH is explained solely by the increase in pulmonary venous pressures; and those who, despite improvement of LV function and fluid status, will demonstrate evidence of persistent PH. In the first group, PH is a compensatory response to increase the pressure gradient between the pulmonary veins and the left ventricle, while in the second group there appears to be a combination of pulmonary arterial and venous hypertension. [14] Despite the absence of firm diagnostic criteria, it may be possible to distinguish these two disease phenotypes by using the transpulmonary gradient (TPG), defined as the difference between the mean pulmonary artery pressure (mPAP) and the PCWP. In patients with PH-LVDD secondary to passive venous congestion, the TPG will be <10 given that both mPAP and PCWP increase to a similar degree. In contrast, patients with PH-LVDD with a superimposed pulmonary arterial component will display TPG that can range between 25 and 35 mmHg, an increase that is considered to be “out of proportion” to what is expected from pulmonary venous hypertension alone (Fig. 3).[15,18] Given that a proposed component in the pathology of elevated TPG is pulmonary vasoconstriction, some experts have recommended the routine performance of vasodilator testing in these patients; however, whether the presence of a vasodilator response in this group of patients would carry the same prognostic and therapeutic implications as in cases of PAH is unclear at this time.

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TREATMENT OF PH-LVDD Despite the high prevalence of LVDD in the general population, few therapies have been systematically studied and their impact survival remains unproven. Initial management is centered on improving symptoms related to volume overload with the use of oral and/or intravenous diuretics. Given that most patients with LVDD suffer from systemic hypertension, aggressive control of blood pressure should be attempted with a goal of achieving less than 130/85 mmHg. Medications such as the ACE inhibitors, angiotensin receptor blockers (ARBS), beta blockers and calcium channel blockers can induce regression of left ventricular hypertrophy and improve diastolic relaxation and ventricular filling.[4,6,23]

The presence of PH has been shown to be a marker of poor prognosis and increased mortality in patients with heart failure.[18,42,43] For those patients in whom the PH fails to improve despite aggressive management of LVDD as well as for those who suffer from the â&#x20AC;&#x153;out of proportionâ&#x20AC;? form of PH-LVDD, the optimal management remains unclear.

Currently, there are no FDA-approved medications for the management of PH-LVDD, and the efficacy and safety of PAH specific therapies for this indication has not been critically evaluated. Available studies using prostanoid agents to treat patients with CHF have revealed conflicting data that limit enthusiasm for their routine use in this patient population.

For example, one study found that, in patients with PH and end-stage CHF preparing for cardiac transplantation, inhaled iloprost was as safe and effective as nitric oxide in reducing pulmonary pressures preoperatively.[44] Several studies using IV epoprostenol have shown acute improvement in left ventricular ejection fraction,[45,46] but the duration of this effect in the long-term has not been evaluated.

While these studies suggest that prostanoids can improve cardiac function in CHF patients, there is also evidence for significant adverse events in the wake of their use. The Flolan International Randomized Survival Trial (FIRST) was designed to study the long-term impact of epoprostenol (i.e., flolan) therapy on patients with refractory CHF. This trial was stopped prematurely as a result of an increased mortality in the treatment arm which was thought to be a consequence of epoprostenol-induced increased venous hypertension and worsening PCWP.[47] While most patients in the previously mentioned studies were diagnosed with systolic CHF, it is possible that these adverse effects could also occur in PHLVDD patients in which prostanoid therapies are introduced.

Therefore, at this time, we advise against using prostanoids to treat PH in the setting of systolic and/or diastolic heart failure until further characterization is undertaken.

Endothelin receptor antagonists (ERAs) and phosphodiesterase 5 (PDE5) inhibitors are two classes of oral therapies used in the management of patients Pulmonary Circulation | April-June 2012 | Vol 2 | No 2

with WHO Class 1 PAH. As with prostanoids, there have been a few studies that have looked at their impact on left ventricular performance in patients with heart failure. The Endothelin A Receptor Antagonist Trial in Heart Failure Trial recruited 642 patients who had LVEF less than 35% and persistent symptoms of CHF despite use of standard CHF therapies and randomized them to either placebo or the ERA darusentan. After 24 weeks, there was no evidence of regression in left ventricular remodeling nor improvement in left ventricular function.[48] Another study using bosentan to treat patients with severe CHF was terminated early as a result of increased liver toxicity and failure of the drug to demonstrate any clinical benefit.[48] At present, a study looking at the safety and efficacy of bosentan in patients with diastolic heart failure and secondary PH (BADDHY trial, NCT00820352) is currently enrolling participants and should hopefully help clarify the role of ERAs in PH-LVDD. Of all the FDA-approved therapies for treatment of PAH, the PDE5 inhibitors hold the most promise for management of PH-LVDD. In a small case series, patients with PH and left heart disease treated with the PDE5 inhibitor sildenafil demonstrated a reduction in pulmonary artery pressures and an increase in exercise capacity without changing cardiac index or PCWP.[49] A more recent study by the same group showed that in a group of 45 patients with stable systolic failure treated with sildenafil for one year, there was evidence for improved diastolic filling and regression of LV hypertrophy and left atrial distension.[50,51] While these data are very encouraging, more systematic studies are required prior to recommending the routine use of PDE5 agents on PH-LVDD. Of note, a Phase II/III randomized double-blind clinical trial looking at the impact of sildenafil in pulmonary hemodynamics in patients with PH-LVDD over a 1-year period (NCT01156636) has been completed and the data are currently being analyzed.

CONCLUDING REMARKS

PH-LVDD is a unique form of PH that is associated with poor prognosis and for which the optimal management strategy remains to be determined. Evaluation and approach to management of these patients are further complicated by the existence of patients whose PH is a consequence of elevated pulmonary congestion and those who may have both pulmonary arterial and venous pathology, leading to more severe PH, and possibly worse outcomes.

While the early diagnostic approach can include a careful history, physical examination, performance of a TTE and/ or exercise study, the only way to firmly establish the diagnosis of PH-LVDD is by right heart catheterization.

Initial management of PH-LVDD should focus on optimizing blood pressure and fluid status followed by reassessment of pulmonary pressures to gauge the impact of these 167

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interventions on pulmonary hemodynamics. At this time, the use of PH-specific therapies in patients who fail to demonstrate improvement in PH with CHF management is undetermined. Use of these agents (prostanoids, ERAs, and PDE5 inhibitors) should only be done as part of clinical studies designed to evaluate their efficacy in controlling PH with LVDD.

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

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Research A r t i cl e

Mesenchymal stem cell-mediated reversal of bronchopulmonary dysplasia and associated pulmonary hypertension Georg Hansmann1, Angeles Fernandez-Gonzalez1, Muhammad Aslam1, Sally H. Vitali2, Thomas Martin3, S. Alex Mitsialis1, and Stella Kourembanas1 1 Department of Pediatrics, Division of Newborn Medicine, 2Department of Anesthesia, 3Department of Pediatrics, Division of Respiratory Diseases, Children’s Hospital Boston, Harvard Medical School, Boston, Massachusetts, USA.

G.H. and A.F.G. contributed equally as first authors

ABSTRACT Clinical trials have failed to demonstrate an effective preventative or therapeutic strategy for bronchopulmonary dysplasia (BPD), a multifactorial chronic lung disease in preterm infants frequently complicated by pulmonary hypertension (PH). Mesenchymal stem cells (MSCs) and their secreted components have been shown to prevent BPD and pulmonary fibrosis in rodent models.

We hypothesized that treatment with conditioned media (CM) from cultured mouse bone marrow-derived MSCs could reverse hyperoxia-induced BPD and PH. Newborn mice were exposed to hyperoxia (FiO2=0.75) for two weeks, were then treated with one intravenous dose of CM from either MSCs or primary mouse lung fibroblasts (MLFs), and placed in room air for two to four weeks.

Histological analysis of lungs harvested at four weeks of age was performed to determine the degree of alveolar injury, blood vessel number, and vascular remodeling. At age six weeks, pulmonary artery pressure (PA acceleration time) and right ventricular hypertrophy (RVH; RV wall thickness) were assessed by echocardiography, and pulmonary function tests were conducted. When compared to MLF-CM, a single dose of MSC-CM-treatment (1) reversed the hyperoxia-induced parenchymal fibrosis and peripheral PA devascularization (pruning), (2) partially reversed alveolar injury, (3) normalized lung function (airway resistance, dynamic lung compliance), (4) fully reversed the moderate PH and RVH, and (5) attenuated peripheral PA muscularization associated with hyperoxia-induced BPD. Reversal of key features of hyperoxia-induced BPD and its long-term adverse effects on lung function can be achieved by a single intravenous dose of MSC-CM, thereby pointing toward a new therapeutic intervention for chronic lung diseases.

Key Words: airway hyperresponsiveness, chronic lung disease of infancy, lung vascular pruning, lung injury, hyperoxia

Bronchopulmonary dysplasia (BPD) is a complex chronic lung disease (CLD) with multifactorial etiology, characterized by the arrest of alveolar and vascular growth associated with inflammation and parenchymal fibrosis. [1- 4] Historically, oxygen toxicity and ventilator-induced injury have been the prerequisites for BPD in premature infants born at less than 28-32 weeks gestation with respiratory distress syndrome, but BPD may also occur in immature infants with few signs of initial lung injury. Approximately one in four infants with moderate-to-severe BPD develops pulmonary hypertension (PH) [5] that can be triggered

Address correspondence to: Dr. Stella Kourembanas Division of Newborn Medicine Children’s Hospital Boston Harvard Medical School 300 Longwood Avenue Boston, MA 02115, USA Email: stella.kourembanas@childrens.harvard.edu 170

by inflammation and endothelial dysfunction, and perpetuated through alveolar hypoxia. Importantly, the occurrence of PH in BPD is not only an epiphenomenon or minor secondary event, but appears to greatly increase mortality (estimated death rate is 47% two years after diagnosis of PH).[6] Moreover, with an increasing percentage of extremely immature newborns surviving in the post-surfactant era, BPD is one of the most common primary diagnoses in neonatal follow-up and pediatric Access this article online

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Website: www.pulmonarycirculation.org DOI: 10.4103/2045-8932.97603 How to cite this article: Hansmann G, Fernandez-Gonzalez A, Aslam M, Vitali SH, Martin T, Mitsialis SA et al.

Mesenchymal stem cell-mediated reversal of bronchopulmonary dysplasia and associated pulmonary hypertension. Pulm Circ 2012;2:170-81.

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PH clinics today. Bronchopulmonary dysplasia as a major etiologic factor for PH development has been recognized and, in fact, BPD was assigned its own category in the recently published classification of pediatric pulmonary hypertensive vascular disease (PPHVD Category 4: BPD; PVRI Panama classification, 2011).[7] The saccular stage of murine lung development is completed after two weeks of postnatal alveolarization.

Hence, the developmental stage of the mouse lung at birth resembles that in the human preterm neonate between 24 and 28 weeks gestation, making the newborn mouse an excellent model to study human developmental lung injury. Hyperoxia-induced lung damage in neonatal mice is characterized by rarefication and simplification of alveoli and thickened alveolar septa, inflammation and parenchymal fibrosis, a pattern which is similar to human BPD.[8]

Mesenchymal stem cells, also referred to as multipotent stromal cells (MSCs), have attracted significant attention as potential cell-based therapy for BPD and other severe lung diseases because these multipotent cells exhibit beneficial effects in related animal models through anti-inflammatory, immunomodulatory, pro-survival (endothelial, epithelial), and anti-fibrotic mechanisms.[9,10] We and others have previously shown that administration of bone marrowderived MSCs prevent BPD/CLD of prematurity,[11,12] acute lung injury,[13,14] and pulmonary fibrosis[9] in experimental models despite low engraftment rates. Our previous study revealed that conditioned media (CM) from MSCs was most effective in preserving alveolar architecture in the hyperoxia-BPD mouse model and suggested that bone marrow-derived MSCs have important cytoprotective effects predominantly via paracrine mechanisms.[11]

Although innovative approaches for the prevention of mechanical ventilation, oxidative stress, and ultimately BPD have been developed recently,[15-17] larger studies have failed so far to demonstrate an efficient preventative or therapeutic strategy for BPD in preterm newborn infants.[18] From a clinical perspective, reversal rather than postnatal prevention of oxygen-induced lung injury such as BPD is most relevant, given that (1) BPD probably has prenatal origins (preeclampsia,[19,20] oligohydramnios,[5] chorioamnionitis [21,22]), and (2) hyperoxic lung injury occurs in the first few minutes and hours of life when endotracheal intubation, mechanical ventilation, and oxidative stress cannot be avoided due to respiratory failure.[15,23] While the human umbilical cord or placenta may serve as a source for future clinical MSC applications,[24] we herein used CM from cultured mouse bone marrowderived MSCs as a feasible intravenous treatment in Pulmonary Circulation | April-June 2012 | Vol 2 | No 2

the well-established murine hyperoxia-BPD model. We found that several weeks after injury, MSC-CM-treatment (1) reversed the parenchymal fibrosis and peripheral pulmonary arterial devascularization (PA pruning), (2) partially reversed the alveolar injury, (3) normalized the airway hyperresponsiveness, (4) fully reversed the moderate PH and right ventricular hypertrophy (RVH), and (5) attentuated peripheral PA muscularization that was associated with BPD. Thus, hyperoxia-induced BPD and associated PH can be reversed with MSC-CM-treatment, thereby pointing toward a new therapeutic approach for chronic lung diseases with alveolar and vascular injury.

Importantly, we show that a single intravenous dose of MSC-CM after the acute phase of lung injury in the neonatal period inhibits longterm impairment of lung function, an increasingly recognized serious complication of BPD.

MATERIALS AND METHODS Experimental design

Mouse pups (FVB) were exposed to 75% oxygen from postnatal day 1 (P1) to P14 (Fig. 1, upper panel), or remained in room air from birth (normoxic controls).

On P14, when chronic hyperoxic lung injury is evident in this BPD model,[8,11] the mice were placed in room air, and intravenously injected with concentrated MSC-CM containing 10 µg protein, or the equivalent amount of mouse lung fibroblast-CM (MLF-CM) as control. Two weeks after the end of hyperoxia, i.e., after two weeks recovery in room air at postnatal age four weeks, lung tissue was harvested for histology and immunohistochemistry as described below. Four weeks after the end of hyperoxia, i.e., at postnatal age six weeks and body weight of 18- 2 2 grams, all mice (including normoxic controls) underwent echocardiography and pulmonary function tests. All animal experiments were approved by the Children’s Hospital Boston Animal Care and Use Committee.

Hyperoxia chamber

Neonatal pups were pooled and exposed to 75% oxygen in a plexiglass chamber or to room air beginning at birth and continuing for 14 days. Ventilation was adjusted by an Oxycycler controller (Biospherix, Lacona, N.Y.) to remove CO2 so that it did not exceed 5,000 ppm (0.5%). Ammonia was removed by ventilation and activated charcoal filtration through an air purifier. Dams were rotated from hyperoxia to room air every 48 hours to prevent excessive oxygen toxicity to the adult mice.

Cell isolation, culture, and differentiation

Bone marrow-derived MSCs were isolated from the femurs and tibiae of 5- to 7-week-old FVB mice as previously described.[11,25-27] Briefly, the bone marrow cells were 171

Hansmann et al.: Reversal of BPD and chronic airway disease by MSCs

Figure 1: MSC-CM-treatment partially reverses neonatal hyperoxia-induced alveolar injury, septal thickening, collagen accumulation, myofibroblast infiltration, and inflammation. Upper panel: Experimental Design. Newborn mice were either left in room air, or exposed to hyperoxia (FiO2=0.75) for 2 weeks (P1-14), and then intravenously injected once with CM from either bone marrow-derived MSCs or primary MLFs, and placed in room air for 2 additional weeks, followed by harvest, inflation and fixation of lungs. Lung sections were stained for hemotoxylin & eosin (A, C, E; 100×), and Mason Trichrome (B, D, F; 400×). Inserts were taken at 200× (H&E) and 400× (Mason Trichrome) magnification to illustrate interstitial inflammation (C, arrows), inflammation and fibrosis (D). Severe destruction of the alveolar architecture with overall widened airspaces and interstitial infiltration of inflammatory cells (macrophages, leukocytes) and myofibroblasts was seen in the hyperoxia-exposed/MLF-CM-treated mice (C, D) when compared to normoxic controls (A, B). Enhanced collagen deposition was seen in myofibroblasts, alveolar septa, and perivascular spaces in hyperoxia-exposed/MLF-CM-treated mouse lungs (D, arrows and inset).

These changes were absent or greatly abrogated in MSC-CM-treated mice that had honeycomb-like alveoli (E, F) similar to normoxic controls, with residual emphysema. Quantification of mean linear intercept (Lm) as a surrogate of average air space diameter (G) and collagen content (H) are shown in the lower panels. Mean±SEM, n=4-7 per group, ***P<0.001, **P<0.01, *P<0.05. Scale bar = 100 µm (A, C, E) and 50 µm (B, D, F).

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layered on a Ficoll-Paque (Amersham, Piscataway, N. J.) density gradient, centrifuged, and plated. Plastic adherent cells were maintained in culture with α-MEM changed every two to three days. Following two to three passages, immunodepletion was performed as per published protocols and the International Society for Cellular Therapy (ISCT) guidelines.[28] The cells were negatively selected for CD11b, CD14, CD19, CD31, CD34, and CD45, and CD79α antigens using the appropriate fluorescenttagged antibodies (BD Biosciences, Pharmingen, San Diego, Calif.) in a fluorescence-activated cell sorter (MoFlo, Beckman-Coulter), further propagated, and then positively selected for CD73, CD90, CD105, c-kit, and Sca1 antigens, as described. The differentiation potential of MSC cultures was assessed following published protocols.[26,27] Primary MLFs were derived according to the standard methods.[29]

Preparation of mesenchymal stem cellconditioned media

Bone marrow-derived MSC confluent cultures were incubated in serum-free α-MEM media for 24 hours and the CM from equal numbers of cells in each culture were concentrated 10-fold using an Amicon Ultra Centrifugal Filter Device (Millipore Corporation, Billerica, Mass.) with a molecular weight cut-off of 10 kDa. MLF-CM in the same concentration and volume served as control. Cells were used for derivation of conditioned media between passages 8-10.

Intravenous injection of MSC- and MLFconditioned media A volume of 50 µl of MSC- or MLF-derived CM concentrate, equivalent to a total of 10 µg MSC-CM protein per mouse, was injected via the superficial temporal vein or the jugular vein on P14. Under these conditions, survival of injected pups was greater than 90%.

Echocardiography

Anesthesia was induced with isoflurane 3% and continued at 1–2% to a goal heart rate of 350–450 bpm. Transthoracic echocardiography was performed in anesthesized mice (FiO2 1.0) using the Vevo 2100 machine (Visual Sonics, Toronto, Calif.) and a 40 MHz linear transducer with simultaneous ECG recording. In the anteriorly angulated left parasternal long axis view, PW Doppler was applied to measure the pulmonary artery acceleration time (PAAT) and the PA ejection time (PAET). A short PAAT or small PAAT/PAET ratio indicates high peak systolic PA pressure, as previously described and validated.[30,31] In the right parasternal long axis view, M-mode was applied to determine RV anterior (free) wall thickness (RVWT) using a virtual guideline across the RV free wall and RV cavity that cuts through the mitral valve annulus.

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Pulmonary function tests

To evaluate lung function, mice were anesthetized with 80 mg/ kg pentobarbital sodium i.p. Following tracheostomy, mice were mechanically ventilated at a rate of 150 breaths/minute, a tidal volume of 10 ml/ kg, and a positive end-expiratory pressure (PEEP) of 3 cmH 2 O with a computer-controlled small animal ventilator (Scireq, Montreal, Canada). To evaluate bronchial hyperresponsiveness, normal saline alone and escalating doses of inhaled methacholine (1.6, 5, 16, and 50 mg/ ml) were aerosolized by using an ultrasonic nebulizer (Scireq, Montreal, Canada). Average airway resistance was calculated at baseline and maximal values were recorded after each dose of methacholine. In addition, dynamic lung compliance (Cdyn) was determined at baseline and minimum values after each methacholine dose were recorded.

Lung tissue perfusion and histology

Following terminal anesthesia with 240 mg/kg Avertin i.p., lungs were perfused with PBS through the right ventricle at a constant pressure of 25 cmH2O. Lungs were inflated to a fixed pressure of 15–20 cmH2O with 4% paraformaldehyde and postfixed overnight, subsequently processed, and paraffin embedded for sectioning.

Immunohistochemical staining

Lung tissue sections were deparaffinized in xylene and rehydrated. Immunohistochemical analysis was performed by incubating with the indicated primary antibody at a dilution of 1:200 (von Willebrand factor, vWF; rabbit polyclonal anti human; Dako), 1:125 (alpha smooth muscle actin, α-SMA; mouse monoclonal; Sigma) overnight at 4○C after 20 minutes of blocking at room temperature to reduce nonspecific binding. Endogenous peroxidase activity was inhibited with 0.3% H2O2 in methanol (Sigma).

Secondary antibodies and peroxidase staining was performed according to manufacturer’s instructions (Vector laboratories, Burlingame, Calif.). Slides were counterstained with methyl green.

Lung parenchymal and pulmonary vascular morphometry and quantification

Lung sections were stained by Hematoxylin & Eosin (H&E) and Mason Trichrome (collagen). Ten randomly selected areas from 5 μm H&E stained lung sections were captured at 100× (H&E) and 400× (Mason Trichrome) magnification using a Nikon Eclipse 80i microscope. Calibrations for the images were done by acquiring standard micrometer images using the same magnification. Large airways and vessels were avoided for the lung morphometry. The air space chord length (Lm) was calculated using Metamorph image analysis software (Molecular Devices, Sunnyvale, Calif ). The images were superimposed on a grid with parallel lines 173

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spaced at 58 μm intervals, and the mean length of each chord, defined as the distance between two sequential intersections of the alveolar surface with the test line, was measured. The alveolar septal thickness (µm) was calculated by measuring the fiber breadth (area/length) using Metamorph software. Peripheral vascularization was determined by counting the number of vWF-positive vessels in 15 random images at 200× magnification, stratified by diameter (<50 and 50–100 μm), using Metamorph software. The vessel wall thickness was assessed by measuring α-SMA positive staining in vessels less than 100 μm diameter in 10–15 sections captured at 400× magnification. The wall thickness was measured using Metamorph software and compared between groups using the following equation: Medial thickness index = [(areaext − areaint)/areaext], where areaext and areaint are the areas within the external and internal boundaries of the α-SMA layer, respectively.

Pulmonary artery barium injections

A subset of left lungs (approximately half) were bariuminfused via PA-inserted tubing to label central and peripheral pulmonary arteries for micro-CT-imaging, as previously described.[32] Barium was prepared by mixing 50 g gelatin (Sigma), 400 ml barium powder in 550 ml water, and injected at 60–70°C into the main pulmonary artery.

Computed tomography and 3D reconstruction of PA barium injected mouse lungs

The basic method of micro-CT in vitro imaging has been previously described.[32] For this study, a Micro CAT II CT (Siemens) and RVA software was used. Formalin-fixated left lungs were each placed in an empty 50 ml Falcon tube and centered in the scanner by creating anterior-posterior and lateral X-rays images. A field of view of 768×768 voxels (1 voxel = 0.023 mm) was chosen. The left lungs of six weeks old untreated normoxic mice, hyperoxia-exposed/ MSC-CM-treated, or hyperoxia-exposed/MLF-CM-treated mice were fully scanned ex vivo at 45 µm resolution (binning = 2) and 512 views (62 kVp, 1200 ms single image acquisition time), followed by realtime 3D reconstruction.

Subsequently, Amira 4.1 software was applied on the 3D images. Isosurface rendering at a threshold of 1000 arbitrary units and appropriate positioning allowed display of all lungs in anterior-posterior views with best demonstration of segmental and peripheral branching of the left pulmonary artery.

Statistical analysis

All values were expressed as means±SEM. Comparison between different groups was performed by one-way ANOVA followed by Tukey’s multiple comparison test, or unpaired Student’s t-test, using GraphPad Prism 5.0 software (GraphPad, La Jolla, Calif.). P values <0.05 were considered significant.

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RESULTS MSC-CM-treatment reverses alveolar injury, septal thickening, and myofibroblast infiltration associated with hyperoxia-induced lung Injury Newborn mice exposed to two weeks of hyperoxia followed by a single dose of non-MSC control conditioned media (MLF-CM) showed severe destruction of the alveolar architecture with overall widened airspaces, alveolar simplification, airway remodeling, and interstitial infiltration of inflammatory cells (macrophages, neutrophils) and myofibroblasts, when compared to normoxic mice at four weeks of age (Fig. 1A–F). In the MLF-CM-treated animals, the many myofibroblasts within the alveolar walls had a high collagen content and frequently dendritic extensions elongating along the destructed alveolar remnants thereby creating ellipsoid to round structures (Fig. 1C and D). Collagen deposition, that was practically absent in normoxic mice, was also seen in alveolar septal and perivascular spaces of hyperoxiaexposed/MLF-treated mouse lungs (Fig. 1D). These hallmarks of dysfunctional pulmonary regeneration and fibrosis after hyperoxia in MLF-CM-treated animals were absent or greatly ameliorated in MSC-CM-treated mouse lungs that had honeycomb-like alveoli similar to normoxic controls (Fig. 1E and F). To quantify the effect of MSC-CM on hyperoxia-induced lung damage, septal collagen content, alveolar septal thickening, and Lm, as an approximation of alveolar air space diameter, were determined. MSCCM-treatment after two weeks of hyperoxia decreased the deposition of alveolar septal collagen by 50% when compared to the hyperoxia-exposed/MLF-CM-treated animals (3.75±1.92 vs. 7.51±0.89% collagen staining of total septal area; P<0.01; Fig. 1H). Alveolar septal thickness, a combined variable of interstitial edema, inflammation, and parenchymal fibrosis, was quantified by measuring the fiber breadth (area/length). Septal thickening was evident in hyperoxia-exposed/MLF-CM-treated neonatal mice versus normoxic controls (3.92±0.07 vs. 3.36±0.20 µm; P<0.05), and ameliorated in animals that were treated with a single dose of MSC-CM (3.636±0.05 μm; Fig. 1E and F).

The mean alveolar chord length was approximated

by Lm measurements and was found to be increased by 97% in hyperoxia-exposed/ MLF-CM-treated mice when compared to normoxic controls (Lm 62.4±2.5 vs. 31.7±1.0 μm; P<0.01). Hyperoxia-exposed/MSCCM-treated mice had significantly smaller airspaces (Lm 55.3±1.7 μm; P<0.01) than MLF-CM-treated animals, but moderate residual emphysema four weeks postnatally when compared with normoxic controls (P<0.01; Fig. 1G).

Thus, a single intravenous dose of MSC-CM improved the alveolar simplification, inflammation, and fibrosis associated with hyperoxia-induced BPD resulting in Pulmonary Circulation | April-June 2012 | Vol 2 | No 2

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moderate residual alveolar emphysema but otherwise near-normal lung structure.

MSC-CM improves lung function after hyperoxiainduced lung injury

To determine the functional impact of the histological findings, we performed pulmonary function testing in 6-week-old mice 4 weeks after the end of hyperoxia, and in age-matched normoxic mice. All animals were ventilated at a PEEP of 3 cm H2O. Airway resistance was measured at baseline and after serial methacholine doses in order to quantify bronchial hyperreactivity. At baseline, the airway resistance was not different between the three groups (Fig. 2A). Intriguingly, MSC-CM-treatment fully reversed the abnormal increase in airway resistance response from low to high intratracheal methacholine doses (5, 16, 50 mg/ml) seen in the MLF-CM group to levels not different from normoxic controls (Fig. 2A).

At a dose of 50 mg/ml intratracheal methacholine, dynamic lung compliance was greatly decreased in the hyperoxiaexposed/MLF-CM-treated mice when compared to normoxic controls, but was normal in the hyperoxia-exposed/MSCCM-treated animals (Fig. 2B). Thus, MLF-CM-injected mice exposed to high oxygen concentrations had severe airway hyperresponsiveness to inhaled methacholine, consistent with our histological observations of airway remodeling and myofibroblast infiltration (Fig. 1C and D). However, MSCCM-treated mice had normal dynamic lung compliance, and normal airway resistance (bronchial reactivity) responses to methacholine (Fig. 2), consistent with the histological findings (Fig. 1E and F vs. 1C and D).

M S C - C M-treatment revers e pul m on a r y hypertension and RV hypertrophy in hyperoxiainduced lung injury

To assess the effects of hyperoxia and MSC-CM on both RV mass and PA pressure, M-mode and PW-Doppler echocardiography were applied. Compared with normoxia (Fig. 3A), the PAAT and PAAT/PAET ratios (i.e., surrogates of mean PA pressure) were found to be decreased in hyperoxiaexposed/MLF-CM-treated animals (Fig. 3C), but improved to normoxic values in hyperoxia-exposed/MSC-CM-treated mice (Fig. 3E) (PAAT: 10.87±0.45 vs. 14.92±0.36 vs.

15.22±0.34 ms in normoxic mice, P<0.001; PAAT/PAET ratio: 0.174±0.007 vs. 0.235±0.005 vs. 0.231±0.008 ms in normoxic mice, P<0.001). In accordance with the reversal of PH after MSC-CM-treatment, we found thickening of the RV free wall (i.e., RVH) in the hyperoxia-exposed/MLF-CM-treated mice (Fig. 3D) that was normalized in the MSC-CM-treated animals (Fig. 3F) (RVWT: 0.354±0.008 vs. 0.259±0.016 vs. 0.235±0.013 mm in normoxic mice, P<0.01). Thus, a single dose of MSC-CM reversed the moderate pulmonary hypertension and RV hypertrophy that was associated with hyperoxia-induced BPD (Fig. 3G and H).

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Figure 2: MSC-CM improve lung function after hyperoxia-induced lung injury. Pulmonary function testing was performed in six-week-old mice four weeks after the end of hyperoxia, and in age-matched normoxic control mice (see experimental design shown in Figure 1, and Methods section).

Airway resistance (A) was measured at baseline and under escalating doses of intratracheal methacholine in order to quantify bronchial hyperreactivity.

At baseline, airway resistance was not different between the three groups.

MSC-CM-treatment fully reversed the abnormal increase in airway resistance seen in the MLF-CM group after administration of low to high intratracheal methacholine doses (5, 16, 50 mg/ml), to levels not different from normoxic controls (A). Dynamic lung compliance (Cdyn) was remarkably impaired in the hyperoxia-exposed/MLF-CM-treated mice at a methacholine dose of 50 mg/ml but normal in the MSC-CM-treated mice when compared with normoxic controls, indicating normalized compliance under metacholine stress (B). The corresponding histological findings are shown in Figure 1. Mean±SEM, n=3–4 per group, ** P<0.01, * P<0.05.

M S C - C M a t t e n u a t e h y p e r ox i a - i n d u c e d peripheral pulmonary arterial remodeling

To explore the potential impact of MSC-CM on hyperoxiainduced peripheral pulmonary vascular muscularization, random lung sections were stained for the smooth muscle marker, α-SMA. The hyperoxia-induced enhancement of peripheral PA muscularization was already evident in the Mason Trichrome stained lung sections in the MLFCM group (Fig. 1D) and confirmed by quantification of the SMA staining (Fig. 4). Accordingly, the medial thickness index was 46.3±1.8 versus 17.1±0.8 in normoxic controls (P<0.001; Fig. 4B and D). A single dose of MSCCM at the end of chronic hyperoxia ameliorated the abnormal peripheral PA muscularization seen in the 175

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Figure 3: MSC-CM-treatment reverses pulmonary hypertension and RV hypertrophy in hyperoxia-induced lung injury. Pulmonary artery acceleration time (PAAT; syn. PAT), as a surrogate of mean PA pressure, was echocardiographically measured by PW-Doppler (A, C, E) and found to be shortened in hyperoxiaexposed/MLF-CM-treated animals (C), but normal in hyperoxia-exposed/MSCCM-treated mice (blue line, E), when compared to normoxic controls (A, graphically summarized in G). Similar results were obtained for the PAAT/PAET ratio, where PAET is the pulmonary artery ejection time (syn. PET; data not shown).

The end-diastolic diameter of the RV free wall (RVWT, RV wall thickness) was measured by M-mode echocardiography (B, D, F) and found to be increased in the hyperoxia-exposed/MLF-CM-treated mice but normalized in the MSC-CMtreated animals (H). Thus, a single dose of MSC-CM reversed the moderate pulmonary hypertension (G) and RV hypertrophy (H) that was associated with hyperoxia-induced BPD. See Figure 1 for experimental design. Mean±SEM, n=4 per group, * P<0.05.

MLF-CM-treated mice exposed to high inspiratory oxygen concentrations (medial thickness index 40.8±1.7, P<0.05; Fig. 4C and D).

MSC-CM rescue hyperoxia-induced loss of peripheral pulmonary blood vessels

In order to determine whether the loss of pulmonary vessels with chronic high oxygen exposure can be improved or reversed by a single dose of MSC-CM, quantification of pulmonary blood vessels of less than 50 and 50–100 µm diameter was performed in vWF-stained lung sections.

Hyperoxia led to significant loss of small vessels of less than 50 µm diameter in the MLF-CM group (Fig. 5B and D), whereas MSC-CM injection restored the small (peripheral) vessels two weeks in recovery from chronic exposure to 75% oxygen (P<0.01; Fig. 5C and 5D). There was also a clear trend toward a protective effect of MSC-CM-treatment on the number of moderate-sized vessels (50–100 µm) (MSC-CM vs. MLF-CM; P=0.0534; Fig. 5D). The counts of larger vessel were low and there was no significant difference in the quantity of vessels >100 µm diameter between the groups (data not shown).

176

MSC-CM reverse pulmonary artery pruning in hyperoxia-induced lung injury

To confirm the histological findings on the rescue of pulmonary vessel loss with MSC-CM-treatment, we performed PA barium injections and subsequent CT-3D reconstruction of the PA vascularization. In accordance with the histological evidence of peripheral vessel loss after chronic hyperoxia we found severe rarefication of peripheral pulmonary arteries in lung CT scans of PA barium injected mouse lungs in the hyperoxia-exposed/ MLF-CM-treated (control) group at four weeks recovery in room air (PA pruning; Fig. 6B). Hyperoxia-induced PA pruning was fully reversed by a single intravenous dose of MSC-CM given at the end of hyperoxia at P14, indicating a remarkable angiogenic/vasculogenic effect of MSC-CM (Fig. 6C).

DISCUSSION

Chronic lung diseases such as BPD and pulmonary fibrosis will be the second leading cause of death Pulmonary Circulation | April-June 2012 | Vol 2 | No 2

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pulmonary inflammation and damage. Preterm infants are predominantly affected by the disease because of their underdeveloped airway supporting structures, surfactant deficiency, decreased lung compliance, and decreased antioxidant capacity. In the post-surfactant era, the pathobiology and clinical course of BPD has changed, and the disease is now characterized mainly by (1) impaired alveolarization with fewer, larger, and simplified alveoli, and (2) dysmorphic vasculogenesis, resulting in fewer (small) pulmonary arteries and frequently pulmonary hypertensive vascular disease that impacts survival. Additional features of the “new BPD” include inflammation, bronchial smooth muscle thickening, and interstitial edema.[1-4] Pulmonary hypertensive vascular disease in BPD is characterized by pulmonary arteriolar muscularization, vessel loss, and RV hypertrophy, among other findings.[2,7] Nearly all of these histological features – including excess myofibroblast proliferation and septal collagen deposition – were evident in the murine hyperoxia-BPD model used in this study.

To date, all available interventions for the prevention and/ or treatment of BPD have not been effective in randomized controlled trials or have unacceptable adverse effects (e.g., postnatal glucocorticoids).[18] Prevention of premature birth and elimination of prenatal risk factors for BPD, such as preeclampsia[19,20] and chorioamnionitis,[21,22] is desirable but difficult to achieve. Therefore, finding an effective treatment approach for BPD and associated pulmonary vascular disease is of tremendous clinical importance.

Figure 4: MSC-CM attenuate hyperoxia-induced peripheral pulmonary arteriole remodeling. Lung sections were stained for the smooth muscle marker, a-SMA, and peripheral PA muscularization (<100 µm outer diameter) quantified in random views at 400× magnification as described under Methods.

When compared to normoxic controls (A), hyperoxia exposure induced peripheral PA muscularization in the MLF-CM-treated mouse lungs (B) that was significantly reduced in the MSC-CM-treated lungs (C). Quantification of the a-SMA staining is shown in D. See Figure 1 for experimental design.

Mean±SEM, n=4-7 per group, * P<0.05. Scale bar=25 µm.

worldwide by 2020. [33] BPD is a complex disease of premature infants with multiple pre and postnatal risk factors, including infection, preeclampsia, postnatal oxygen toxicity, and barotrauma, ultimately leading to Pulmonary Circulation | April-June 2012 | Vol 2 | No 2

There is emerging evidence from animal and clinical pilot studies that stem cell and progenitor cell-based therapies modulate disease markers and may be efficient in tissue/organ regeneration.[33,34] Bone marrow-derived MSCs have been shown to be efficient in the repair of heart and lung diseases such as myocardial infarction,[35] pulmonary fibrosis,[9] and LPS-induced lung injury.[9,36] Bone marrow-derived MSCs and MSC-CM from mice[11] and rats,[12] or human cord blood-derived MSCs,[37] when given intravenously or intratracheally in a preventive fashion, have been shown to improve lung architecture in rodent models of hyperoxia-induced BPD.

Previously, we demonstrated that intravenous injection of bone marrow-derived MSCs in newborn mice conferred significant vascular and immunological protection from hyperoxia-induced injury but had limited effect in preserving alveolar architecture.[11] Concentrated MSC-CM, however, prevented both vascular and alveolar hyperoxic injury resulting in normal alveolar number and thin septa, comparable to controls in room air. [11] The results of our previous preventive approach suggested that bone marrowderived MSCs have important cytoprotective effects in the hyperoxia mouse model of developmental lung injury 177

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Figure 5: MSC-CM rescue hyperoxia-induced loss of peripheral pulmonary blood vessels. Lung sections were stained for the endothelial cell marker vWF. vWF-positive vessels between 25 and 200 µm outer diameter were counted at 200× magnification in 10–15 random views as described under Methods. Compared to normoxic controls (A), hyperoxia exposure led to significant loss of small vessels < 50 µm diameter in the MLF-CM group (B), whereas MSC-CM injection (C) restored small vessels after 2 weeks in recovery from chronic exposure to 75% oxygen (see Fig. 1 for experimental design). There was also a clear trend toward MSC-CM-treatment effect on the number of larger vessels (50-100 µm) in mice exposed to hyperoxia (MSC-CM vs. MLF-CM; P=0.0534). Quantification of pulmonary blood vessels of less than 50 and 50–100 µm diameter in vWF-stained lung sections (D) was performed as described under Methods. There was no significant difference in numbers of larger vessels (100-200 µm) between the groups. See Figure 1 for experimental design. Mean±SEM, n=4–7 per group, ** P<0.01, * P<0.05. Scale bar = 50 µm.

mimicking BPD via paracrine mechanisms including the release of immunomodulatory and vasoprotective mediators.[11]

In the current study, we showed that a single intravenous dose of MSC-CM reversed – to a significant degree – hyperoxia-induced BPD and pulmonary vascular disease versus MLF-CM control: MSC-CM-treatment (1) reversed the hyperoxia-induced parenchymal fibrosis and peripheral PA devascularization (PA pruning), (2) partially reversed alveolar injury, (3) normalized lung function (airway hyperresponsiveness, dynamic lung compliance), (4) fully reversed the moderate PH and RVH, and (5) attenuated peripheral PA muscularization associated with hyperoxiainduced BPD.

The paracrine effects of MSCs include the release of immune and growth modulators identified in the CM by mass spectroscopy analysis.[11] These factors in MSC-CM promote signaling pathways of lung repair and include inhibitors of inflammation that are linked to the development of PH and pulmonary fibrosis. An attractive speculation is that the beneficial effect of MSC-CM may be, at least in part, due to activation of endogenous bronchioalveolar stem cells (BASCs), an adult lung stem cell population capable of selfrenewal and differentiation in culture, and proliferation in response to bronchiolar and alveolar lung injury in vivo.

Very recently, Tropea et al.[38] have demonstrated that intravenous treatment of neonatal hyperoxia-exposed mice with MSCs or MSC-CM led to a significant increase in 178

BASCs compared to untreated controls. Treatment of BASCs with MSC-CM in culture resulted in an increase in growth efficiency, suggesting a paracrine effect of MSCs on BASCs. Lineage tracing in bleomycin-treated adult mice showed that CCSP-expressing cells, including BASCs, are capable of contributing to alveolar repair after lung injury. Thus, MSCs and MSC-derived factors probably stimulate BASCs to contribute to the restoration of distal lung cell epithelia in BPD.[38] Several studies have highlighted the existence of different MSC phenotypes in bone marrow, blood, and airways/ lung. MSC phenotypes and function likely depend on maturity (gestational age) and environmental factors such as tissue oxygenation, and are distinctly regulated by the two major pathways of MSC differentiation, i.e., transforming growth factor beta (TGFβ) superfamily and canonical Wnt pathways. [35,39] Along these lines, in vitro treatment of airway MSCs of ventilated preterm infants with recombinant TGFβ1 induced myofibroblast differentiation, whereas adult human bone marrowderived MSCs that were not exposed to high oxygen concentrations failed to undergo such differentiation upon TGFβ1 stimulation.[40] Popova et al. concluded that neonatal lung MSCs demonstrate an expression pattern characteristic of myofibroblastic progenitor cells (mRNAs encoding contractile and extracellular matrix proteins, and expression of α-SMA, MHC, and SM22 protein).

Conditioned media from cultured tracheal aspirate MSCs of preterm infants exposed to hyperoxic stress contain Pulmonary Circulation | April-June 2012 | Vol 2 | No 2

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Figure 6: MSC-CM reverse pulmonary artery pruning in hyperoxia-induced lung injury. Pulmonary artery barium injections and subsequent computed tomography angiograms with 3D reconstruction of the PA vascularization of left lungs were performed ex vivo, as described in the Methods section. When compared to normoxic controls (A), severe rarefication (pruning) of peripheral PAs (arrows) was evident on CT-angiograms of hyperoxia-exposed/MLF-CMtreated mice at 4 weeks recovery in room air (B). Such hyperoxia-induced PA pruning was completely absent in lungs from mice at 4 weeks recovery in room air injected with a single intravenous dose of MSC-CM at the end of hyperoxia exposure (C). For experimental design, see Figure 1.

high protein concentrations of TGFβ1, and it was proposed that autocrine production of TGFβ1 by neonatal lung MSCs drives myofibroblast differentiation;[40] however, it is yet unclear whether increased TGFβ1 signaling and subsequent MSC to myofibroblast transdifferentiation occurs as a direct response to hyperoxia in the human preterm lung.

Importantly, increased expression of TGFβ1 and Wnt has been reported in whole lung tissue of neonatal mice with hyperoxia-induced BPD and is associated with fibrosis in BPD[41-43] and several other CLD models.[44-46] However, alveolar myofibroblasts are required for the formation of secondary septa during normal lung development, a process that is arrested in BPD, indicating that the timing and adequate regulation of MSC differentiation is crucial for normal lung development.

Besides the bone marrow, multipotent MSCs have been shown to reside in the perivascular compartment of many organs,[24] and arise from both small and large arteries[47,48] as well as capillaries.[24] It was proposed that the tracheal aspirate MSCs found in one study in preterm infants who later on develop BPD are derived from the perivascular tissue of pulmonary and bronchial arteries.[49]

We speculate that hyperoxia-induced TGFβ1 expression and secretion from airway/perivascular MSCs and other lung cells switches the phenotype of pulmonary MSCs and other proliferative cells toward pathological myofibroblast transdifferentiation resulting in dysfunctional repair (alveolar collagen accumulation/ fibrosis and simplification, airway and pulmonary vascular remodeling, inflammation), consistent with Pulmonary Circulation | April-June 2012 | Vol 2 | No 2

our histological findings. The report by Popova et al.[40] suggests that adult bone marrow-derived MSCs may be rather resistant to profibrotic stimuli such as hyperoxia and TGFβ1. Our study shows that bone marrow-derived MSCs secrete factors with antifibrotic, antimitogenic, and anti-inflammatory effects that may regulate signaling downstream of the TGFβ receptor and inhibit myofibrogenic transdifferentiaton of neonatal lung MSCs and/or alveolar epithelial type II cells.

This is supported by our previous analysis of the MSC secretome.[11]

Recent experimental and clinical data suggest that hyperoxia-induces BPD in premature newborns by impairing the number and in vivo functionality of circulating (blood), lung, and bone marrow endothelial progenitor cells (EPCs). [50,51] The quantity of circulating EPCs is low at extremely low gestational ages and increases during gestation. Importantly, extremely preterm infants who display lower EPC numbers in blood at birth have an increased risk of developing BPD.[52] Differentiation, release from bone marrow, tissue migration, and the predominant actions of EPCs in peripheral tissues/organs might be regulated by MSCs through paracrine mechanisms, as recently shown for BASCs (see above). In particular, the macrophage-colony stimulating factor (M-CSF) and granulocyte-colony stimulating factor (G-CSF) accelerate neovascularization in vivo[53] and induce differentiation of bone marrow cells into endothelial progenitor cells in vitro.[54] Interestingly, macrophage-CSF has been shown to induce EPC release from the bone marrow into the circulation through the augmentation of vascular endothelial growth factor (VEGF) production in bone marrow cells, especially from myeloid lineage cells.[55] Given that BMSC-CM are enriched in M-CSF,[11] a possible mechanism underlying the therapeutic action of BMSC-CM treatment is the mobilization of EPCs in the recipient animals. The latter would result in augmented neovascularization and the negation of the hyperoxia-induced pruning.

Possible risks of stem cell therapy include the potential for tumor formation and fibrosis. Indeed, fibrocytes, a pool of circulating mesenchymal precursors which can differentiate into myofibroblasts, were reported to be recruited to the lung and contribute to fibrosis[56] as well as pulmonary adventitial remodeling in experimental PH.[57] The human bone marrow, umbilical cord (tissue, blood), or placenta might serve as a source for MSC/ MSC-CM and probably will be used clinically as rescue therapy for BPD and other CLD in the near future, when safety of such interventions can be confirmed and the mechanisms of the important paracrine effects of MSCs are better understood (BPD-MSC Phase I trials are underway: NCT01207869, NCT01297205, under http://clinicaltrial. gov). In particular, in-depth MSC characterization (e.g., surface markers), the composition and properties of 179

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MSC-CM, the immunological responses to autologous or allogeneic MSC transplantation, certain compounds of MSC-CM, and the best source and route of administration need to be explored more comprehensively.

This study shows that a single intravenous dose of bone marrow-derived MSC-conditioned media rescues hyperoxiainduced BPD by reversing lung parenchymal fibrosis, pulmonary hypertension, vessel loss (PA pruning), and RV hypertrophy, significantly decreasing alveolar injury, reversing airway hyperresponsiveness, and normalizing dynamic lung function long-term. While the mechanisms underlying the beneficial effects of MSC-conditioned media will need to be explored in subsequent studies, MSC-derived interventions appear to be a promising treatment option for BPD, pulmonary hypertension, and other chronic lung diseases, and should be investigated in future clinical trials.

ACKNOWLEDGMENTS

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We thank Xianlan Liu for her expert technical assistance with the mouse breeding and oxygen exposures, Sarah Gately for the preparation of the manuscript, and Pat Dunning and Erin Snay for their technical assistance with micro-CT.

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Source of Support: Supported by National Heart Lung and Blood Institute (NHLBI) grants R01 HL055454 (S.K.), R01 HL085446 (S.K.), and P50 HL067669 (S.K. and S.A.M.). M.A. was supported by 5T32 HD007466-12 (S.K., PI). Conflict of Interest: None declared.

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Research A r t i cl e

Group V phospholipase A2 increases pulmonary endothelial permeability through direct hydrolysis of the cell membrane Nilda M. Muñoz1, Anjali Desai2, Lucille N. Meliton3, Angelo Y. Meliton3, Tingting Zhou2, Alan R. Leff 3 , and Steven M. Dudek2 1 Philippine Foundation for Lung Health, Research and Development, Inc. and Research and Biotechnology Division, St. Luke’s Medical Center, Quezon City, Philippines, 2Institute for Personalized Respiratory Medicine, Section of Pulmonary, Critical Care, Sleep, and Allergy, University of Illinois at Chicago College of Medicine, Chicago, Illinois, USA, 3Department of Medicine, Section of Pulmonary and Critical Care Medicine, University of Chicago, Chicago, Illinois, USA

ABSTRACT Acute lung injury (ALI) is characterized by inflammatory disruption of the alveolar—vascular barrier, resulting in severe respiratory compromise. Inhibition of the intercellular messenger protein, Group V phospholipase A2 (gVPLA2), blocks vascular permeability caused by LPS both in vivo and in vitro. In this investigation we studied the mechanism by which recombinant gVPLA2 increases permeability of cultured human pulmonary endothelial cells (EC). Exogenous gVPLA2 (500 nM), a highly hydrolytic enzyme, caused a significant increase in EC permeability that began within minutes and persisted for >10 hours. However, the major hydrolysis products of gVPLA2 (Lyso-PC, Lyso-PG, LPA, arachidonic acid) did not cause EC structural rearrangement or loss of barrier function at concentrations <10 µM. Higher concentrations (> 30 µM) of these membrane hydrolysis products caused some increased permeability but were associated with EC toxicity (measured by propidium iodide incorporation) that did not occur with barrier disruption by gVPLA2 (500 nM). Pharmacologic inhibition of multiple intracellular signaling pathways induced by gVPLA2 activity (ERK, p38, PI3K, cytosolic gIVPLA2) also did not prevent EC barrier disruption by gVPLA2. Finally, pretreatment with heparinase to prevent internalization of gVPLA2 did not inhibit EC barrier disruption by gVPLA2. Our data thus indicate that gVPLA2 increases pulmonary EC permeability directly through action as a membrane hydrolytic agent. Disruption of EC barrier function does not depend upon membrane hydrolysis products, gVPLA2 internalization, or upregulation of downstream intracellular signaling.

Key Words: phospholipase A2, vascular permeability, cytoskeleton, actin, acute lung injury, barrier function

Despite advances in supportive care and ventilator management, the most severe cases of acute lung injury/ acute respiratory distress syndrome (ALI/ARDS) continue to cause unacceptably high mortality rates in afflicted patients.[1,2] Because effective pharmacologic intervention for ALI/ARDS is not available,[3,4] improved understanding of the underlying pathophysiology is needed to develop targeted therapies. A critical early step in the pathogenesis of ALI/ARDS is the disruption of the lung vascular endothelial cell (EC) barrier by inflammatory stimuli, leading to pulmonary edema and subsequent respiratory

Address correspondence to: Dr. Steven M. Dudek Associate Professor of Medicine Section of Pulmonary, Critical Care, Sleep and Allergy University of Illinois at Chicago COMRB 3143, MC 719 909 S. Wolcott Ave. Chicago, IL 60612, USA Email: sdudek@uic.edu 182

compromise.[5] Endothelial barrier function is primarily regulated by the structural arrangement of the EC actin cytoskeleton linkages to the cell membrane and underlying junctional complexes.[6] Investigations into the mechanisms by which inflammatory signals disrupt EC barrier function therefore provide insights into pathways that potentially may be exploited therapeutically.

Secretory phospholipase A2 (sPLA2) lipolytic enzymes catalyze the cleavage of fatty acids from the sn-2 position Access this article online

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Website: www.pulmonarycirculation.org DOI: 10.4103/2045-8932.97604 How to cite this article: Muñoz NM, Desai A, Meliton LN, Meliton AY, Zhou T, Leff AR et al. Group V phospholipase A2 increases pulmonary endothelial permeability through direct hydrolysis of the cell membrane. Pulm Circ 2012;2:182-92.

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Muñoz et al.: gVPLA2 directly disrupts EC barrier function

of phospholipids [7,8] and have been implicated in the pathogenesis of ALI in both animals[9] and patients.[10,11] At least 10 different sPLA2 enzymes with varying tissue distributions and phospholipase activities have been identified in mammals.[7] Recent data implicate a functional role for the 14 kDa secretory group V PLA 2 (gVPLA2) enzyme in ALI pathophysiology. Inhibition of gVPLA2 by specific blocking antibody[12] or pharmacologic inhibition[13] significantly attenuates vascular permeability caused by LPS in mice. In addition, deletion of the gene encoding gVPLA 2 in mice (pla2g5 -/- knockout) blocks increases in multiple indices of lung injury after LPS.[12] Studies performed in vitro using cultured human pulmonary EC have demonstrated that disruption of the endothelial barrier by LPS can be blocked by inhibition of gVPLA2.[14] Moreover, the extracellular application of recombinant gVPLA2 directly increases permeability of cultured human pulmonary EC. [14] Accordingly, prior studies strongly support an important mechanistic role for gVPLA2 in the development of ALI-associated permeability both in vivo and in vitro. However, the mechanism by which gVPLA2 increases EC permeability remains unclear.

The objective of this present study was to further characterize in vitro the potential pathway(s) responsible for disruption of pulmonary EC barrier function by gVPLA 2. We hypothesize that one of three putative mechanisms might account for the development of EC barrier dysfunction caused by gVPLA2: (1) direct outer membrane hydrolysis; (2) secondary effects induced by products of gVPLA2 membrane hydrolysis; and (3) induction of intracellular signaling pathways. It is likely that direct hydrolysis of the EC outer membrane by gVPLA2 physically disrupts its integrity to increase permeability. The second possibility is that the products of membrane hydrolysis generated by gVPLA2 are the primary agents that initiate downstream signaling events that result in EC barrier dysfunction. gVPLA2 activity generates multiple products with potential biologic effects, including free fatty acid, arachidonic acid (AA), lysophosphatidylcholine (lyso-PC), lysophosphatidylglycerol (lyso-PG), lysophosphatidic acid (LPA), and others.[7,8] A final possibility is that gVPLA2 activity at the EC membrane induces intracellular signaling pathways to produce downstream effects (e.g., junctional complex disruption) resulting in barrier dysfunction.

In this study, we now demonstrate that the primary membrane hydrolysis products generated by gVPLA2 do not duplicate the increased permeability caused by gVPLA2 itself in cultured pulmonary EC. In addition, multiple intracellular signaling pathways induced by gVPLA2 in pulmonary EC do not participate in barrier disruption.

Thus, our data indicate that gVPLA2 increases pulmonary EC permeability through direct hydrolytic action at the EC membrane and provide mechanistic insights into an Pulmonary Circulation | April-June 2012 | Vol 2 | No 2

important inflammatory signal that participates in the generation of vascular leak during ALI syndromes.

MATERIALS AND METHODS Reagents

Recombinant human gVPLA2 was purchased from Cayman Chemical (Ann Arbor, Mich.). Arachidonic acid, lyso-PC, lysoPG, LPA were obtained from Avanti Polar Lipids (Alabaster, Ala.). Pharmacologic inhibitors UO126, SB203580, TFMK, and LY294002 were obtained from EMD Chemicals (Gibbstown, N.J.). Heparinase I was obtained from SigmaAldrich Chemical (St. Louis, Mo.). Antibodies were obtained as follows: pan-ERK, phospho-ERK, pan-p38, phospho-p38, pan-AKT, phospho-AKT, pan-gIVaPLA2, phospho-gIVaPLA2 from Cell Signaling (Beverly, Mass.), mouse anti-VEcadherin antibody (Santa Cruz Biotechnology, Santa Cruz, Calif.). Texas-Red phalloidin was obtained from Invitrogen (Carlsbad, Calif.). All other reagents were obtained from Sigma unless otherwise noted.

Cell culture

Human pulmonary artery endothelial cells (HPAEC) and human lung microvascular endothelial cells (HLMVEC) were obtained from Lonza (Walkersville, Md.) and cultured according to the manufacturer’s instructions as previously described.[15] EC (Passages 6–9) were grown in Endothelial Growth Medium-2 (EGM-2) at 37°C in a 5% CO2 incubator.

The medium was changed 1 day prior to experimentation.

Transendothelial monolayer electrical resistance

EC were grown to confluency in polycarbonate wells containing evaporated gold microelectrodes, and Transendothelial monolayer electrical resistance (TER) measurements were performed using an electrical cellsubstrate impedance sensing system (ECIS; Applied Biophysics, Troy, N.Y.) as previously described in detail.[16] TER values from each microelectrode were expressed as normalized resistance and pooled as discrete time points and plotted versus time as the mean±SEM.

Dextran transwell permeability assay

A transendothelial permeability assay was performed as per the manufacturer’s instructions utilizing labeled tracer flux across confluent EC grown on confluent polycarbonate filters (Vascular Permeability Assay Kit, ML) as previously described.[14] Briefly, EC on transwell inserts were exposed to gVPLA2 (500 nM) or lyso-PC (1–30 µM) for two hours.

FITC-labeled dextran (~40 kDa) was added to the luminal compartment for an additional two hours, and FITC-dextran clearance across the filter to the abluminal compartment was measured by relative fluorescence excitation at 485 nm and emission at 530 nm. Data were expressed as arbitrary fluorescence units.

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Immunofluorescence EC were grown on gelatinized cover slips before exposure to various conditions as described for individual experiments.

EC were then fixed in 3.7% formaldehyde for 10 minutes, permeabilized with 0.25% Triton-X100 for five minutes, washed in PBS, blocked with 2% BSA in TBS-T for one hour, and then incubated for one hour at room temperature with the primary antibody of interest. After washing, EC were incubated with the appropriate secondary antibody conjugated to immunofluorescent dyes (or Texas-Red conjugated phalloidin for actin staining) for 1 hour at room temperature. Final washing was performed with TBS-T, and coverslips were mounted using Prolong Anti-Fade Reagent (Invitrogen) and analyzed using a Nikon Eclipse TE2000-s inverted microscope and Adobe Photoshop 7.0.

Immunoblotting analysis

Cultured EC were stimulated with either vehicle control or 500 nM gVPLA2 for 1-15 min at 37°C. Treated EC were subsequently washed with cold Ca2+/Mg-free PBS and lysed with 0.3% SDS lysis buffer containing protease inhibitors (1 mM EDTA, 1 mM PMSF, 1 mM sodium orthovanadate, 1 mM sodium fluoride, 0.2 TIU/ml aprotinin, 10 μM leupeptin, 5 μM pepstatin A). Sample proteins were separated with 4–15% SDS-PAGE gels (Bio-Rad, Hercules, Calif.) and transferred onto Immobilion-P PVDF membranes (Millipore). Membranes were then immunoblotted with primary antibodies (1:500–1000, 4°C, overnight) followed by secondary antibodies conjugated to HRP (1:5000, room temperature, 30 minutes). Protein expression was detected with enhanced chemiluminescence (Pierce ECL or SuperSignal West Dura, Pierce Biotechnology, Rockford, Ill.) on Biomax MR film (Kodak, Rochester, NY).

Multiple blots were scanned and quantitatively analyzed using ImageQuant software (v5.2; Molecular Dynamics, Piscataway, N.J.).

Determination of toxicity on human pulmonary artery endothelial cells

To determine the cytotoxic effect of membrane hydrolysis products on EC, propidium iodide staining was assessed as previously described for eosinophils.[17] Aliquots of 0.5 × 106 EC were incubated for 30 minutes at 37°C with 10–50 µM lyso-PC, lyso-PG, LPA, or arachidonic acid in a total volume of 250 µl. Propidium iodide at 5 µg/ml was added to the medium of drug-treated cells, and the cell suspension was immediately analyzed by flow cytometry, or plated for fluorescent imaging. Red fluorescence intensity was determined by flow cytometry on at least 10,000 cells from each sample, and the percentage of stained cells was analyzed using the Cellquest software.

Statistical analysis

Data were expressed as mean + SEM. Statistical analyses 184

among groups were performed using standard Student’s t-test. Statistical significance in all cases was defined at P<0.05.

RESULTS gVPLA2 membrane hydrolysis products do not induce EC barrier disruption

Prior studies have demonstrated that 0–500 nM recombinant gVPLA2 increases the permeability of cultured microvascular and macrovascular human pulmonary EC in a concentration-dependent fashion.[14] Differential barrier properties of these two classes of EC have been described in some models.[18,19] Our data indicate that gVPLA­2 disrupts human lung microvascular EC (HLMVEC) permeability in a qualitative manner similar to macrovascular human pulmonary artery EC (HPAEC). In this study, we chose to use HPAEC for most analyses because the magnitude of disruption induced by gVPLA2 in general is greater than that observed in microvascular cells. To determine the effects of gVPLA2 and individual gVPLA2 hydrolysis products on human pulmonary EC barrier function, we first measured the transendothelial monolayer resistance (TER), a highly sensitive method for obtaining real-time permeability data.[15,16] As previously observed,[14] 500 nM of gVPLA2 causes a rapid decrease in TER (which correlates with increased permeability and disruption of barrier function) in both HPAEC and HLMVEC by ~30–50% for>10 hours (Fig. 1A-C). In contrast, multiple hydrolysis products known to be produced by gVPLA2 activity at the cell membrane (arachidonic acid [AA], lysophosphatidylcholine [lysoPC], lysophosphatidylglycerol [lyso-PG], lysophosphatidic acid [LPA]) [7,8] did not decrease TER when added individually to HPAEC at concentrations<1 µM (Fig. 1A). Further TER studies revealed that AA, Lyso-PG, and LPA all failed to significantly alter HPAEC barrier function until their concentration was>10 µM (data not shown).

Lyso-PC is expected to be the major lysophospholipid produced by gVPLA2 at the cell surface because mammalian cell membranes are enriched in its precursor, PC, and gVPLA2 demonstrates greater hydrolytic activity for PC than other glycerophospholipids.[20] In additional TER studies, at least 30 µM lyso-PC was required to increase permeability in both HPAEC and HLMVEC (Fig. 1B and C).

To determine the effects of lyso-PC on EC permeability to larger particles, a transwell assay utilizing labeled dextran (~40 kD) was employed. As previously demonstrated for HPAEC,[14] gVPLA2 (500 nM) significantly increased HLMVEC permeability to dextran as a cumulative measurement after two hours of incubation (Fig. 2).

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However, lyso-PC did not increase HLMVEC permeability to larger particles at concentrations less than 30 µM (Fig. 2). Interestingly, 30 µM lyso-PC induced dramatically more permeability in HLMVEC as measured by labeled dextran than by TER (Figures 1C and 2). The cause of this quantitative discrepancy is unclear, but it likely relates to the differential cell properties assessed by each technique.

The TER assay uses the passage of electrical current across the cell monolayer to estimate permeability. This

(A)

(B)

current can pass via paracellular pathways as in the dextran assay, but it also can transit directly through the cells, or pass between the cells and the underlying matrix.

[21] In certain situations, the resistance to current under the cells can dominate the effects of the paracellular compartment so that the overall TER reading reflects this property to a greater extent than the flow of current between the cells, [22] thus potentially leading to a discrepancy with labeled macromolecule assessments of permeability. However, regardless of the assay used, all the data reported here are consistent with the primary observation that lyso-PC produced by gVPLA2 activity is unlikely to be a primary mediator of EC permeability induced by gVPLA2.

Immunofluorescent analysis was performed to assess the effects of lyso-PC on EC cytoskeletal structure.

Consistent with prior observations, [14] incubation of HPAEC with gVPLA 2 (500 nM, 30 minutes) produced increased actin stress fibers, intercellular gap formation, and disruption of peripheral VE-cadherin staining (the major cell-cell junction protein in EC; [23] Fig. 3).

These structural changes are known to cause increased permeability in cultured EC. [6] In contrast, HPAEC incubated with lyso-PC at concentrations<30 µM exhibited stable or increased cortical actin staining and intact intercellular VE-cadherin distribution (Fig. 3), a pattern indicative of intact barrier function. Although 30 µM of lyso-PC resulted in some stress fibers and large gap formation between EC, this high concentration produced cell toxicity not seen with gVPLA 2 alone.

Propidium iodide staining demonstrated significantly increased toxicity in HPAEC after incubation with 30 µM lyso-PC (50% of cells with positive uptake) compared with vehicle controls (20.5%, P<0.05) or those incubated

(C) Figure 1: Effects of gVPLA2 hydrolysis products on EC permeability. Human lung EC were stimulated at time 0 (arrow) and TER measurements taken as follows: (A) HPAEC: vehicle (black), gVPLA2 500 nM (red), 1 µM each of LPA (purple), AA (aqua), lyso-PC (yellow), lyso-PG (green). Mean±S.E. shown for each timepoint. N=3–5 independent experiments. P<0.05 for gVPLA2 vs. all other conditions. (B) HPAEC: vehicle (black), gVPLA2 (red), or lysoPC 5 µM (yellow), 10 µM (aqua), 30 µM (purple). N=4–5. (C) HLMVEC: vehicle (black), gVPLA2 (red), or lyso-PC 5 µM (yellow), 10 µM (aqua), 30 µM (purple). N=3–8.

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Figure 2: The effects of Lyso-PC on endothelial permeability to labeled dextran.

HLMVEC were cultured on polycarbonate filters as described in the Materials and Methods section and then stimulated with vehicle, gVPLA2 (500 nM), or Lyso-PC (1–30 µM) for 2 hours. FITC-labeled dextran was added to the luminal compartment, and clearance across the EC monolayer was assayed after 2 hours by relative fluorescence excitation. Data are expressed as arbitrary fluorescence units. N=3 independent experiments. (*P<0.05 vs. all other conditions).

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Figure 3: The effects of Lyso-PC on endothelial structure. HPAEC were incubated for 30 minutes with vehicle, recombinant gVPLA2 (500 nM), or Lyso-PC (1–30 µM as indicated) and then fixed and stained for F-actin (red) or VE-cadherin (green) as described in the Materials and Methods section. The F-actin and VE-cadherin images represent the same cell field for each condition. Arrows indicate intercellular gaps or disruption of intercellular VE-cadherin bands. Images are representative of three to four independent experiments.

Figure 4: High concentrations of LysoPC induce EC toxicity. HPAEC were incubated with vehicle, gVPLA2 500 nM, or Lyso-PC 10–50 µM for 30 minutes and then incubated with propidium iodide as per the Materials and Methods section.

(A) Representative bright field (top) and fluorescent (bottom) images are shown.

The same cell field is depicted for each image pairing (20× magnification). Red indicates propidium iodide incorporation.

(B) Propidium iodide incorporation was quantified by flow cytometry as described in the Materials and Methods section.

Mean±S.E. shown. N=3 independent experiments. *P<0.05 for Lyso-PC 30–50 µM vs. all other conditions.

with 500 nM gVPLA2 (24%, P<0.05; Fig. 4). In addition, three other major products of gVPLA2 hydrolysis (LysoPG, LPA, and arachidonic acid) induced significant 186

toxicity at concentrations at which they increase HPAEC permeability (30–50 µM; Fig. 5). These findings do not support a role for lyso-PC, lyso-PG, LPA, or arachidonic Pulmonary Circulation | April-June 2012 | Vol 2 | No 2

Muñoz et al.: gVPLA2 directly disrupts EC barrier function

acid generation by gVPLA2 as a mechanistic contributor to pulmonary EC permeability induced by gVPLA 2 in vitro.

Figure 5: High concentrations of membrane hydrolysis products induce endothelial toxicity. HPAEC were incubated with vehicle, gVPLA2 (500 nM), Lyso-PG, LPA, or arachidonic acid (30–50 µM) for 30 minutes. Propidium iodide incorporation was quantified by flow cytometry as described in the Materials and Methods section. Mean±S.E. shown for each condition. N=3–9 per condition. *P<0.01 vs. vehicle control.

Intracellular signaling pathways are not required for gVPLA2-induced EC barrier disruption

Previous studies in various cell types have demonstrated that gVPLA 2 stimulates upregulation of multiple downstream signaling cascades, including the ERK, p38, Akt, and cytoplasmic gIVaPLA2 pathways.[24,25] To determine the effects of gVPLA2 on these pathways in HPAEC, Western blot analyses were performed at various timepoints following gVPLA 2 stimulation. Phosphorylated ERK (indicative of ERK activation) was significantly increased within 3 minutes in HPAEC following gVPLA2 stimulation (Fig. 6A), while no activation of p38 or Akt occurs (Fig. 6B and C). Downstream cytoplasmic gIVaPLA2 also was rapidly activated following gVPLA2 (Fig. 6D). Thus, the ERK and gIVaPLA2 pathways are induced in HPAEC by gVPLA2 during the timeframe in which the initiation of permeability occurs (Fig. 1A). Because lyso-PC is the major lysophospholipid produced by gVPLA2, its effects on ERK activation in pulmonary EC were assessed. Lyso-PC (10 µM) rapidly activated ERK in HPAEC within 3–5 minutes, which declined thereafter (Fig. 7). Thus, it is possible that some

Figure 6: The effects of gVPLA 2 on HPAEC intracellular signaling. HPAEC were stimulated with vehicle or gVPLA2 (500 nM) for 1–15 minutes, and then cell lysates were collected and analyzed

by Western blotting as described in the Materials and Methods section for expression of total (pan) and phosphorylated ERK (A), p38 (B), Akt (C), and gIVaPLA2 (D). Representative blots for each condition are shown. Bar graphs represent results of densitometric quantification of multiple independent experiments. N=3–7 per condition. *P<0.05 vs. vehicle control.

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portion of the ERK phosphorylation induced by gVPLA2 is a result of lyso-PC generation.

We next determined if pharmacologic inhibition of any of these signaling pathways affected pulmonary EC barrier disruption by gVPLA2. HPAEC were preincubated with UO126 (ERK inhibitor), SB203580 (p38 inhibitor), LY294002 (PI3 kinase inhibitor), or TFMK (gIVPLA 2 inhibitor) for 30 minutes prior to stimulation with gVPLA2. Inhibitor concentrations were selected based upon preliminary experiments demonstrating their effectiveness in blocking agonist-induced activation (Figure 8 demonstrates that 10 µM UO126 prevents ERK phosphorylation by gVPLA2). Pooled data from multiple TER experiments revealed that HPAEC barrier disruption by gVPLA2 is not dependent on activation of any of these pathways (Fig. 8). In addition, immunofluorescent analysis of EC structure demonstrated that ERK inhibition (UO126, 10 µM) did not block the increased actin stress fibers, intercellular gap formation, and disruption of peripheral VE-cadherin produced by gVPLA2 (500 nM, five minutes; Fig. 9). In fact, HPAEC incubated with both UO126 and gVPLA2 exhibited more pronounced disruption of VE-cadherin staining than that caused by gVPLA2 alone (Fig. 9). These results indicate

Figure 7: The effects of lyso-PC on ERK phosphorylation. HPAEC were stimulated with vehicle or lyso-PC (10 µM) for 1–15 minutes, and then cell lysates were collected and analyzed by Western blotting as described in the Materials and Methods section for expression of total (pan) and phosphorylated ERK. Representative blots are shown. Bar graphs represent results of densitometric quantification of multiple independent experiments. *P<0.05 vs. vehicle control. N=3 independent experiments per condition.

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that pulmonary EC permeability induced by gVPLA 2 in vitro does not require ERK, p38, Akt, or gIVaPLA 2 signaling.

gVPLA2 internalization is not required for EC barrier disruption

gVPLA2 also enters mammalian cells through heparin proteoglycan binding at the outer surface to act intracellularly on the perinuclear membrane. This activation generates bioactive lipid mediators both by cytosolic gIVaPLA2dependent[26,27] and gIVaPLA2-independent mechanisms.[28,29] However, internalization of gVPLA 2 into EC did not cause increased membrane permeability. HPAEC were preincubated for several hours with heparinase I to remove cell surface heparin sulfate glycosaminoglycans as previously described.[30] To ensure adequate removal of the cell surface heparin sulfate moieties, we used significantly higher concentrations of heparinase I in this study (1–10 units/ml) compared with prior reports (15 mU/ml).[30] Baseline HPAEC TER was not affected by incubation with heparinase I (data not shown). Despite pretreatment with these high concentrations of heparinase I, HPAEC barrier disruption by gVPLA2 was not inhibited (Fig. 10). Rather,

Figure 8: Effects of intracellular signaling pathways on permeability induced by gVPLA2. Top: HPAEC were incubated with UO126 (0–10 µM) for 30 minutes before stimulation with gVPLA2 (500 nM) for 10 minutes. ERK phosphorylation was determined by Western. Bottom: HPAEC were incubated for 30 minutes with vehicle, UO126 10 µM (ERK inhibitor), SB203580 10 µM (p38), LY294002 25 µM (PI3 kinase), or TFMK 30 µM (gIVPLA2) and then stimulated with gVPLA2. Pooled TER data from multiple independent experiments are expressed as maximal % change in TER from baseline in 5 hours. *P<0.05 for all conditions vs. vehicle. N=4–9.

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Figure 9: ERK inhibition fails to block the effects of gVPLA2 on endothelial structure. HPAEC were preincubated for 30 minutes with vehicle or UO126 (10 µM). The cells were then stimulated for 5 minutes with either vehicle control or gVPLA2 (500 nM) and then fixed and stained for F-actin (red) or VE-cadherin (green) as described in the Materials and Methods section. The F-actin and VE-cadherin images represent the same cell field for each condition. Arrows indicate intercellular gaps or disruption of intercellular VE-cadherin bands.

Images are representative of three to four independent experiments.

induced by LPS in vitro.[14] Animal studies by other groups further support an important role for gVPLA 2 in the pathophysiology of ALI. Transgenic mice overexpressing gVPLA2 develop fatal respiratory failure shortly after birth that pathophysiologically resembles ALI.[31] Recent reports using the general sPLA2 inhibitor LY374388 to attenuate LPS-induced ALI in mice[13] or acute cardiogenic pulmonary edema in a mouse model[32] suggest that this protective effect is largely due to gVPLA2 inhibition. Thus, gVPLA2 may be a critical target for therapeutic modulation of pathways responsible for the development of pulmonary edema, and further exploration of its mechanistic effects is warranted.

Figure 10: Heparinase fails to attenuate permeability induced by gVPLA2.

HPAEC were cultured on gold microelectrodes, and real-time TER measurements were taken as per the Materials and Methods section. EC were preincubated for 3–4 hours with vehicle or heparinase I at the indicated concentrations (1–10 unit/ml). At time 0 (arrow), EC were then stimulated with vehicle or gVPLA2 500 nM as follows: vehicle only (black line), gVPLA2 only (purple line), gVPLA2 + heparinase I 1 unit/ml (yellow line), gVPLA2 + heparinase I 10 units/ml (aqua line). Mean±S.E. shown for each timepoint.

N=3–5 independent experiments per condition.

there was a trend toward increased barrier dysfunction after gVPLA2 in EC pretreated with the higher concentration of heparinase I (10 units/ml, Fig. 10), suggesting that inhibition of gVPLA2 internalization increases permeability.

DISCUSSION

The objective of this investigation was to characterize in vitro the pathway responsible for disruption of pulmonary EC barrier function caused by gVPLA2. We have reported previously that inhibition of gVPLA2 by specific monoclonal antibody or genetic deletion of gVPLA2 blocks ALI caused by LPS in mice, [12] while gVPLA 2 blocking antibody prevents pulmonary EC barrier disruption Pulmonary Circulation | April-June 2012 | Vol 2 | No 2

We have recently reported that the addition of recombinant gVPLA2 to the culture media rapidly induces sustained disruption of EC barrier function in both macrovascular and microvascular pulmonary EC;[14] however, the mechanism by which gVPLA2 produced this effect was not defined.

Although the structurally related enzyme, gIIaPLA2, has been suggested to participate in induction of ALI,[33] it did not increase pulmonary EC permeability in vitro.[14] gVPLA2 has much higher affinity than gIIaPLA2 for zwitterionic phosphatidylcholine-rich outer plasma membranes and therefore greater ability to generate free fatty acids and lysophospholipids at these surfaces.[34] gVPLA2 is unique among sPLA2 enzymes in having a dual ability binding to bind cell membranes via two mechanisms, through cationic residues at the C-terminus which bind to cell surface heparan sulfate proteoglycan (like gIIaPLA2), and via direct binding with membrane phosphatidylcholine (like gXPLA2).[34] These properties make gVPLA2 a more effective paracrine agent during inflammation than gIIaPLA2 because the former enzyme exhibits greater transcellular lipolytic activity[35] These differences may explain why attempts to modulate the type gIIaPLA2 form with the relatively specific inhibitor LY315920NA/S-5920 failed to improve mortality in a study of 250 patients with severe sepsis[36] and suggest that gVPLA2 may be a better target for ALI therapy.

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Our results now further elucidate and simplify the mechanism by which gVPLA 2 causes pulmonary EC permeability in vitro. We investigated the hypothesis that the products of membrane hydrolysis generated by gVPLA 2 are the primary agents that initiate subsequent events leading to EC barrier dysfunction.

Major products produced by gVPLA 2 activity include arachidonic acid (AA), lysophosphatidylcholine (lyso-PC), lysophosphatidylglycerol (lyso-PG), and lysophosphatidic acid (LPA) [7,8] Lyso-PC is expected to be the major lysophospholipid produced by gVPLA2 at the cell surface because mammalian cell membranes are enriched in its precursor, phosphatidylcholine, and gVPLA 2 demonstrates greater hydrolytic activity for PC than other glycerophospholipids. [20] Prior work by others has demonstrated increased permeability caused by lyso-PC in human dermal microvascular EC and bovine pulmonary microvessel EC,[37] human coronary artery EC,[38] and human gastric epithelial cells.[39] However, in all these studies,>10 µM lyso-PC was needed to increase permeability.

In our study, neither lyso-PC nor any of the other products studied (AA, lyso-PG, LPA) disrupt pulmonary EC barrier function at concentrations similar to gVPLA 2 alone (Fig. 1A). They are able to increase permeability in both macro- and microvascular pulmonary EC only at relatively high concentrations (> 30 µM) (Figs. 1 and 2) that are associated with cell toxicity (Figs. 4 and 5). Lyso-PC concentrations in this range previously have been shown to be toxic to eosinophils.[17] Propidium iodide staining demonstrates significantly increased toxicity in HPAEC after incubation with 30 µM lyso-PC (50% of cells with positive uptake) compared with vehicle controls (20.5%, P<0.05) and 500 nM gVPLA2 (24%, P<0.05) (Fig. 4). Moreover, three of the other major products of gVPLA2 hydrolysis (LysoPG, LPA, and AA) induce significant toxicity within the concentration range at which they begin to increase HPAEC permeability (30–50 µM) (Fig. 5). Because gVPLA2 alone does not induce cell toxicity at the range of concentrations associated with permeability (Fig. 4), it is unlikely that it generates membrane hydrolysis products sufficient to produce barrier dysfunction.

It previously has been reported that albumin and other serum proteins can bind to Lyso-PC to inhibit its bioactivity in assays of EC permeability. [37] In this study, lyso-PC concentrations as low as 2 µM significantly decreased TER in human dermal microvascular EC when the concentration of albumin in the media was lowered.[37] However, our TER experiments were performed in serum free media without albumin, which eliminates any potential inhibitory effect on lyso-PC bioavailability by these proteins. Therefore, human pulmonary EC appear to be less sensitive to barrier disruption by lyso-PC than human dermal EC. Consistent 190

with this observation, lyso-PC infusion failed to increase pulmonary microvascular permeability (assessed by fluid filtration coefficient (Kf)) in isolated perfused dog lungs.[40]

These data do not support a role for lyso-PC or other gVPLA2 membrane hydrolysis products as important mechanistic contributors to pulmonary EC permeability induced by gVPLA2. However, it is important to consider the limitations of our current findings. Given that membrane hydrolysis by gVPLA2 produces dozens of products,[7,8] the possibility exists that some of these other compounds may participate in barrier disruption. It is not feasible to test every possible hydrolysis product. Accordingly, we chose to focus on those likely generated in the highest concentrations (e.g., lyso-PC).

Previous studies in various cell types have demonstrated that gVPLA2 stimulates upregulation of multiple intracellular signaling cascades, including the ERK, p38, Akt, and cytoplasmic gIVaPLA2 pathways.[24,25] Although our results demonstrate rapid activation of ERK and gIVaPLA2 in HPAEC following gVPLA 2 stimulation (Fig. 6), pharmacologic inhibition of these pathways fails to block barrier disruption by gVPLA 2 (Fig. 8) or structural cytoskeletal changes (Fig. 9). Although ERK activation occurs in association with multiple barrier-regulatory agonists in EC, this activity is not always necessary for changes in permeability. For example, pharmacologic ERK inhibition attenuates barrier disruption induced by ADAM-15 in HUVEC,[41] but not permeability increases induced by nocodazole in bovine PAEC,[42] despite both agonists producing significant ERK phosphorylation. Differences in cell type and/or agonistspecific activation by other complementary pathways likely account for this variation in ERK effects. Our data do not support a functional role for ERK signaling in pulmonary EC permeability induced by gVPLA2 (Fig. 8). Activation of cytosolic gIVaPLA2 increases intracellular AA levels leading to the production of eicosanoids and other inflammatory signaling molecules,[43] and pharmacologic inhibition of gIVaPLA2 or its genetic deletion partially protects against ALI in mice.[44,45] However, these protective effects in vivo could be due to inhibition of gIVaPLA2 signaling in PMNs and other immune cells, as little data exist that demonstrate a direct role of gIVaPLA2 in EC permeability. Data presented here argue against a functional role for gIVaPLA2 signaling in pulmonary EC permeability induced by gVPLA2 in vitro (Fig. 8).

Although gVPLA2 can enter mammalian cells in a heparin proteoglycan-dependent manner and act intracellularly on the perinuclear membrane to generate additional bioactive lipid mediators through both cytosolic gIVaPLA 2-dependent [26,27] and gIVaPLA 2-independent mechanisms,[28,29] this cellular uptake does not appear to be necessary for gVPLA2 to cause increased permeability.

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with high concentrations of heparinase I fails to block HPAEC TER decrease by gVPLA2 (Fig. 10).

Our results suggest that gVPLA2 increases pulmonary EC permeability through direct action at the EC membrane that does not require membrane hydrolysis products, gVPLA2 internalization, or downstream intracellular signaling. We previously have noted increased actin stress fiber formation and adherens junction disruption induced by gVPLA2 in human pulmonary EC.[14] One possibility is that direct membrane action of gVPLA2 results in pore formation and subsequent calcium influx which can result in MLCK and/ or Rho activation, both of which can produce stress fiber formation and junctional disassembly.[6] Another potential explanation is that gVPLA2 activity disrupts the cell surface caveolin-enriched microdomains that are necessary for optimal EC barrier function.[46,47] Detailed studies are ongoing to more precisely determine the mechanisms responsible for mediating increased EC permeability caused by gVPLA2 action at the cell membrane.

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

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Research A r t i cl e

Furegrelate, a thromboxane synthase inhibitor, blunts the development of pulmonary arterial hypertension in neonatal piglets Dinesh K. Hirenallur-S.1, Neil D. Detweiler1, Steven T. Haworth2,3, Jeaninne T. Leming2, John B. Gordon2,4, and Nancy J. Rusch1 1

Department of Pharmacology and Toxicology, University of Arkansas for Medical Sciences, Little Rock, Arkansas, USA, 2Zablocki Veterans Administration Medical Center, Research Service 151, Milwaukee, Wisconsin, 3Department of Medicine, USA, and 4 Department of Pediatrics, Medical College of Wisconsin, Milwaukee, Wisconsin, USA

ABSTRACT The development of pulmonary arterial hypertension (PAH) in pediatric patients has been linked to the production of the arachidonic acid metabolite, thromboxane A2 (TxA2). The present study evaluated the therapeutic effect of furegrelate sodium, a thromboxane synthase inhibitor, on the development of PAH in a neonatal piglet model. Three-day-old piglets were exposed to 21 days of normoxia (N; 21% FIO2) or chronic hypoxia (CH; 10% FIO2). A third group of piglets received the oral TxA2 synthase inhibitor, furegrelate (3 mg/kg, 2 or 3 times daily) at the induction of CH. In vivo hemodynamics confirmed a 2.55-fold increase of the pulmonary vascular resistance index (PVRI) in CH piglets (104±7 WU) compared to N piglets (40±2 WU). The CH piglets treated twice daily with furegrelate failed to show improved PVRI, but furegrelate three times daily lowered the elevated PVRI in CH piglets by 34% to 69±5 WU and ameliorated the development of right ventricular hypertrophy. Microfocal X-ray computed tomography (CT) scanning was used to estimate the diameter-independent distensibility term, α (% change in diameter per Torr). Pulmonary arterial distensibility in isolated lungs of CH piglets (α=1.0±0.1% per Torr) was lower than that of N piglets (α=1.5±0.1% per Torr) indicative of vascular remodeling. Arterial distensibility was partially restored in furegrelate-treated CH piglets (α =1.2±0.1% per Torr) and microscopic evidence showing muscularization of small pulmonary arteries also was less prominent in these animals. Finally, isolated lungs of furegrelate-treated piglets showed lower basal and vasodilator-induced transpulmonary pressures compared to CH animals. These findings suggest that pharmacological inhibition of TxA2 synthase activity by furegrelate blunts the development of hypoxia-induced PAH in an established neonatal piglet model primarily by preserving the structural integrity of the pulmonary vasculature.

Key Words: pulmonary arterial hypertension, neonatal pulmonary arterial hypertension, hypoxia, thromboxane A 2, thromboxane synthase, furegrelate, vasoconstriction

Cardiorespiratory pathologies of infancy including congenital heart defects and bronchopulmonary dysplasia may lead to chronic hypoxia (CH) and the development of pulmonary arterial hypertension (PAH).[1-4] An imbalance between vasodilator and vasoconstrictor substances in the pulmonary circulation that favors vasoconstriction contributes to increased vascular tone. [5-8] Therefore, treatment options have focused on reversing this imbalance by administering vasodilator substances or attenuating Address correspondence to: Dr. Nancy J. Rusch Department of Pharmacology and Toxicology College of Medicine University of Arkansas for Medical Sciences 4301 West Markham Street, #611 Little Rock, AR 72205-7199, USA Email: nrusch@uams.edu Pulmonary Circulation | April-June 2012 | Vol 2 | No 2

vasoconstrictor pathways. These therapies include calcium channel blockers, inhaled nitric oxide, intravenous prostacyclin, phosphodiesterase type-5 inhibitors, and endothelin (ET) receptor blockers.[1-4,9,10] However, the high cost, short half-lives, lack of vasodilator response, and serious side effects of these molecules dictate their use primarily as diagnostic or short-term interventions. Those Access this article online

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Website: www.pulmonarycirculation.org DOI: 10.4103/2045-8932.97605 How to cite this article: Hirenallur-S. DK, Detweiler ND, Haworth ST, Leming JT, Gordon JB, Rusch NJ. Furegrelate, a thromboxane synthase inhibitor, blunts the development of pulmonary arterial hypertension in neonatal piglets. Pulm Circ 2012;2:193-200.

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drugs administered chronically to patients only slow disease progression that eventually culminates in right heart failure.

One alternative therapeutic target is the thromboxane A2 (TxA2)-mediated signaling pathway.[11,12] TxA2 is a potent vasoconstrictor, platelet aggregator, and mitogenic factor primarily produced in platelets and endothelial cells. The enzymatic conversion of the arachidonic acid metabolite, endoperoxide PGH2, to TxA2 is accomplished by the enzyme TxA2 synthase.[13,14] The effects of TxA2 are mediated by its binding to the TxA2/PGH2 receptor (TP), a Gq-protein coupled receptor that activates phospholipase 2 to mobilize intracellular calcium.[15] Importantly, children with PAH from congenital heart defects show elevated plasma and urinary levels of TxB2,[5,7] a stable metabolite of TxA2.

Similarly, newborn piglets exposed to hypoxia to induce PAH show increased pulmonary vascular TxA2 levels,[16,17] which correlate with an increased expression of thromboxane synthase in the affected arteries.[17] Fike et al.[18] reported that the TP receptor blocking drug, terbogrel (10 mg/kg, p.o., twice daily), ameliorated the development of PAH in neonatal piglets exposed to three days of CH. Although the long-term efficacy of TxA2 modulation is unknown, potently blocking TxA2 synthesis as an early event may potentially blunt its vasoconstrictor and mitogenic effects and slow the progression of PAH. To test this hypothesis, we treated neonatal piglets exposed to CH for 21 days with furegrelate sodium. Furegrelate is a thromboxane synthase inhibitor with a half-effective inhibitory concentration (IC50) of 15 nmol/l in human plasma.[19] Furegrelate was developed as an antiplatelet agent, but Phase 1 clinical trials in normal male patients revealed that it inhibited TxA2 synthesis in vivo without significantly affecting platelet aggregation.[20,21] Furegrelate also is orally available, has a long half-life of 4.2–5.8 hours in adult humans (compared to several other therapies for PAH including nitric oxide and prostacyclin analogs), and reportedly is highly specific for its target enzyme.[19,22] Considering its encouraging drug profile and its apparent safety in Phase 1 clinical trials in adults, the concept of

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“repositioning” furegrelate as a potential therapeutic agent for neonatal PAH is appealing. Thus in the present study, we designed preclinical studies to evaluate furegrelate as a therapeutic option for the treatment of neonatal PAH using an established piglet model of the disease.

MATERIALS AND METHODS Animals

All animal protocols were approved by the Institutional Animal Care and Use Committees at the Zablocki Veterans Administration Medical Center and the Medical College of Wisconsin, or at the University of Arkansas for Medical Sciences. Briefly, three-day-old piglets were exposed in pairs to 21 days of normoxia (N; 21% FIO2) or chronic hypoxia (CH; 10% FIO2) in environmental chambers.[23] Experimental groups of CH piglets received furegrelate (3 mg/kg, p.o. by syringe) either twice daily (FBID) or three times daily (FTID) with the first dose started just prior to the introduction of CH. The dose of furegrelate was chosen based on the literature[19,21,23] and a pilot study (Fig. 1A) in which we observed that furegrelate at a dose of 3 mg/ kg three times daily attenuated the development of CHinduced PAH, whereas twice daily administration had no therapeutic benefit.

Right ventricular hypertrophy

Right ventricular hypertrophy (RVH) was calculated in dissected hearts as the wet weight ratio of right ventricular free wall to left ventricular wall plus septum (RV/LV + S). [24]

Urinary 11-dehydro thromboxane B2

Urine was withdrawn at the end of hemodynamic studies from a subset of piglets and immediately stored at -80°C. Urinary 11–dehydro thromboxane B2, a stable metabolite of TxA 2, was measured using a commercial enzyme immunoassay kit.[25]

In vivo hemodynamics

In order to evaluate the level of PAH, in vivo hemodynamics

(B)

Figure 1: (A) Values of resting pulmonary vascular resistance index (PVRI) in neonatal piglets exposed for three weeks to normoxia (N), chronic hypoxia (CH) or chronic hypoxia + furegrelate twice daily (CH + FBID) or three times daily (CH + FTID).

Sample sizes were 19, 26, 6 and 13, respectively. *Significant difference (P<0.05) from PVRI of N piglets. †Significant difference (P<0.05) from PVRI of CH piglets. (B) Mean arterial pressure (MAP) in the same groups of animals showed no significant difference between groups. Values are mean±S.E.M. Pulmonary Circulation | April-June 2012 | Vol 2 | No 2

Hirenallur-S et al.: TxA2 synthase inhibition blunts PAH

were measured in anesthetized piglets ventilated with room air (FIO2=0.21) at a rate of 15–18 breaths per minute with a peak airway pressure of 10–15 mmHg and a positive endexpiratory pressure (PEEP) of 3 mmHg.[24] Measurements included pulmonary arterial pressure (PAP), pulmonary capillary wedge pressure (PCWP), and cardiac output (CO) measured by thermodilution. CO was divided by body weight to obtain cardiac index (CI). Pulmonary vascular resistance indexed to body weight was then calculated as PVRI (Modified Wood Units) = (PAP – PCWP)/CI. After completing these measurements, piglets were given an additional dose of pentobarbital, heparinized (5000 units i.v.), and exsanguinated. Lungs or lung lobes were dissected for in vitro studies.

Isolated perfused lungs

Pulmonary vascular reactivity was evaluated in left lungs perfused with a mixture of autologous blood and artificially ventilated at a tidal volume of ~15 ml/kg and at a rate of 20 breaths per minute in normoxic conditions.

Pulmonary artery pressure (PAP), left atrial pressure (PLA), and airway pressure (PAW) were measured on-line and the transpulmonary pressure gradient (DPtp=PAP – PLA) was calculated. In some lungs, baseline PAP was obtained before the addition of nifedipine (10 µmol/l in perfusate) and papaverine (15 mg bolus i.v.) to elicit a maximal vasodilator response.[24]

Morphometric analysis

Tissue cubes (1 cm) were dissected from a mid-sagittal slice of the left or right lower lung lobe. The tissues were embedded in paraffin, sectioned, and stained with hemotoxylin and eosin. The %MT (%MT=2× muscle thickness/external diameter) was measured for arteries ranging from 50 µm to 500 µm in outer diameter using a Nikon E600 microscope and MetaMorph software.

Microfocal X-ray computed tomography imaging

We adapted the rat lung microfocal X-ray CT imaging technique to piglet lungs.[26] After flushing the pulmonary artery with a papaverine-saline mixture to remove all blood and minimize active vascular tone, the lobe was suspended in the imaging field and inflated with a 15% O2, 6% CO2, balance N2 gas mixture at a constant airway distending pressure of 5 mmHg. The papaverine–saline solution was replaced with the radiopaque contrast agent perfluorooctyl bromide (PFOB). Over the range of pressure studied, PFOB does not traverse the capillaries to the veins due to the surface tension at the PFOB-aqueous interface.

Thus, only the arterial tree was filled. The lung lobe was then imaged at four intraarterial pressures (6, 12, 21, and 30 mmHg) referenced to the middle of the lobe. At each pressure, planar images were collected as a seven frame average in 1° intervals over 360° of rotation. The Pulmonary Circulation | April-June 2012 | Vol 2 | No 2

set of images was preprocessed, compensating for spatial distortions introduced by the imaging system, and then reconstructed using a Feildkamp cone-beam algorithm. [26] The reconstructed volume was 497 3 with a isotropic resolution of a typical pixel size of 160 µm. Arterial vessel diameters were identified and measured from the reconstructed volumes. Diameter measurements ranging from ~150 µm to ~3000 µm were made on 90 arterial vessel segments per lung, along the main pulmonary trunk and branches at four pressures (6, 12, 21, and 30 mmHg) referenced to the top of the lung. The actual intravascular pressure relative to atmosphere within each artery was obtained from the measured vessels’ vertical distance from the reference pressure level and the PFOB density. The pressure–diameter (P–D) relationship of each of the 90 arteries was then calculated and the slope was estimated by linear regression. Subsequently, the slope of each P–D curve (β) was plotted against its respective undistended vessel diameter intercept at 0 pressure (Do). The trend relating β to Do was analyzed by linear regression through the origin and the diameter-independent distensibility term, α, (percent change in diameter per Torr) was calculated for each lobe.

Statistical analysis

Data were displayed as mean±S.E.M. Comparison of a single variable between groups was subjected to one-way ANOVA with post hoc multiple comparison test (Student–Newman– Keuls method). Differences were judged to be significant at the level of P<0.05.

RESULTS

Furegrelate blunts the development of neonatal PAH

Table I compares data between N piglets and untreated and furegrelate-treated CH piglets after three weeks in environmental chambers. Furegrelate was administered orally by syringe to take advantage of its oral bioavailability.

Weight, arterial pO2, and arterial pCO2 were not significantly different between the three groups of animals. However, the CH piglets showed a higher hematocrit, RV/LV + S ratio (Table 1) and pulmonary vascular resistance index (PVRI; Fig. 1A) compared to N piglets, indicating the development of PAH. In initial therapeutic studies, the oral administration of 3 mg/kg furegrelate orally twice daily (CH + Fureg, BID) failed to lower the elevated hematocrit and RV/LV + S ratio (Table 1) observed in untreated CH piglets. Similarly, furegrelate BID also failed to blunt the elevated PVRI induced by hypoxia that averaged 128±27 WU in treated piglets and 104±7 WU in untreated CH piglets (Fig. 1A; CH + Fureg). However, CH piglets treated with furegrelate three times daily (TID) showed a markedly reduced PVRI of 69±5 WU compared to untreated CH animals. In addition, the RV/ 195

Hirenallur-S et al.: TxA2 synthase inhibition blunts PAH

LV + S ratio was significantly reduced in CH + FTID piglets (0.57±.04) compared to untreated CH animals (0.66±.02) and hematocrit was partially restored to normal values (Table I). Importantly, there was no change in the systemic mean arterial pressure between N and CH+FTID piglets, suggesting the absence of a pronounced systemic dilator effect of furegrelate (Fig. 1B). Collectively, these findings suggest that oral administration of furegrelate three times daily reduces the clinical signs of PAH in CH piglets without inducing systemic hypotension. Thus, the remainder of our studies used the dosing regimen of furegrelate, 3 mg/kg orally three times daily.

The efficacy of furegrelate (3 mg/kg, p.o., TID) to reduce the synthesis of TxA2 was initially evaluated by enzyme immunoassay (EIA) of TxB2, a stable TxA2 metabolite in plasma of N, CH and CH + FTID piglets. However, due to a very high intra-assay coefficient of variation (>20%) these samples were not used. Subsequently, urine was obtained from the final animals studied and the level of 11-dehyro TxB2, a stable urinary TxA2 metabolite, was evaluated by EIA. The 11-dehydro TxB 2 EIA showed a low intra-assay coefficient of variation (5%) after normalizing to creatinine to account for urine volume.

Average 11-dehydro TxB 2 levels were elevated in CH piglets (2.40±0.36 ng/mg creatinine, n = 8) compared to N piglets (1.83±0.21 ng/mg creatinine, n=6; Fig. 2A-B). The urinary 11-dehydro TxB2 level in CH + FTID piglets was 1.40±0.49 ng/mg creatinine (n=4), showing the lowest average value of the three animal groups (Fig. 2A and B). Thus we obtained initial evidence in this subset of animals that the dosing regimen of furegrelate

we used (3 mg/kg, TID) inhibited the synthesis of TxA2 in CH piglets, although high animal-to-animal variability precluded statistical significance.

Furegrelate attenuates pulmonary vascular remodeling

Hypoxia-induced vascular remodeling is an important feature of PAH that may limit responsiveness to vasodilator therapies.

Thus, we compared pulmonary vascular distensibility and the percent muscular thickness (%MT) of pulmonary arteries between N, CH, and CH + FTID piglets. Microfocal X-ray CT imaging revealed overt vascular remodeling in lungs of CH piglets that was evident as a loss of PFOB-filled pulmonary arteries compared to normoxic lungs (Fig. 3A). Accordingly, the distensibility coefficient (a), an indicator of elasticity of the vasculature, was significantly reduced in the pulmonary arteries of CH piglets (1.0±0.1% per Torr) compared to N piglets (1.5±0.1% per Torr; Fig. 3B). Furegrelate therapy TID (FTID) blunted hypoxia-induced vascular remodeling in CH piglets (Fig. 3A) resulting in an improved a value in CH piglets of 1.2±0.1% per Torr, which was not significantly different than the α value of normoxic lungs (Fig. 3B). These findings suggested that 3 mg/kg furegrelate TID prevented the hypoxia-induced loss of distensibility in the pulmonary circulation, which retained vascular elasticity.

To further corroborate our distensibility findings, we compared the percent muscular thickness (%MT) of pulmonary arteries (o.d., 50–500 µm) between lungs of N, CH and CH + FTID piglets. Lung sections stained with hemotoxylin and eosin revealed thickened arterial walls in untreated CH piglets compared to N piglets. This abnormality

Table 1. Profiles of normoxic (N), chronic hypoxic (CH), and CH piglets treated with furegrelate Weight (kg) pO2 pCO2 Hematocrit (%) RV/LV + S ratio

Normoxia (N)

Chronic hypoxia (CH)

5.42±0.24 (21) 85.19±2.07 (19) 43.71±1.58 (19) 23.71±0.93 (21) 0.32±0.01 (20)

5.66±0.18 (25) 90.47±2.03 (25) 37.16±1.31 (25) * 30.96±0.67 (25) * 0.66±0.02 (25)

CH + Furegrelate (3 mg/kg, p.o.) BID TID 4.13±0.51 (6) 100.64±7.33 (5) 35.90±2.53 (5) * 31.67±0.54 (6) * 0.66±0.06 (6)

6.20±0.25 (13) 89.01±2.35 (13) 37.47±1.64 (13) * † 28.83±0.60 (12) * † 0.57±0.04 (13)

Average values for weight, pO2, pCO2, hematocrit and right ventricle to left ventricle plus septal (RV/LV + S) ratio in neonatal piglets exposed for 3 weeks to normoxia (N), chronic hypoxia (CH) or chronic hypoxia + furegrelate twice daily (CH + FBID) or three times daily (CH + FTID). The dose of furegrelate was 3 mg/kg p.o. *Significant difference (P<0.05) from the same measurement in N piglets. †Significant difference (P<0.05) between CH piglets and CH + FTID piglets for the same measurement. Sample size is indicated in parenthesis

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(B)

Figure 2: (A) Individual values of 11-dehydro TxB2, a stable metabolite of TxA2, in urine samples from neonatal piglets exposed for 3 weeks to normoxia (N), chronic hypoxia (CH) or chronic hypoxia + furegrelate three times daily (CH + FTID). Each symbol represents a single animal.

(B) Average urinary 11-dehydro TxB2 values for the animals in A. Sample sizes were 8, 6 and 4, respectively. Values are mean±S.E.M. Pulmonary Circulation | April-June 2012 | Vol 2 | No 2

Hirenallur-S et al.: TxA2 synthase inhibition blunts PAH

was less evident in arteries of CH + FTID piglets (Fig. 4A). The calculated %MT was 2.3-fold higher in arteries of CH piglets (20.5±0.6%) compared to N piglets (8.82±0.2%) suggestive of extensive pulmonary vascular remodeling in the CH piglets with PAH. The %MT value for small pulmonary arteries was significantly lower (14.2±0.6) in CH + FTID piglets compared to untreated CH animals. Thus, we obtained evidence that oral furegrelate therapy mitigated the development of hypoxiainduced pulmonary vascular remodeling in CH piglets.

Furegrelate improves pulmonary pressure profiles in isolated lungs

We also evaluated the effect of furegrelate (3 mg/ kg, p.o., TID) on the resting and vasodilator-induced transpulmonary pressure gradient (∆P tp) in isolated perfused lungs. The isolated lung avoids the systemic hypotensive effect of vasodilators that can confound the interpretation of hemodynamic measurements in vivo; it thereby allows pulmonary vascular tone to be assessed independently. Similar to our previous finding,[24] control DP tp was profoundly elevated in the isolated lungs of CH piglets (23.46±3.05 mmHg) compared to N piglets (11.80 ± 1.09 mmHg; Fig. 5). In contrast, DPtp in isolated lungs of CH + FTID piglets was significantly lower

(14.81±0.63 mmHg) than DPtp in untreated CH piglets, corresponding to a 74% improvement (Fig. 5). Maximal pulmonary dilator responses were obtained in isolated lungs by adding nifedipine (N, 10 mmol/l) and papaverine (P, 15 mg bolus) to the perfusate for additive block of voltage-gated L-type Ca2+ channels and vascular contractile mechanisms, respectively.[24] The difference between the control ∆Ptp value and the ∆Ptp value after vasodilator challenge was regarded as the active pulmonary vascular tone; the residual tone may relate to structural limitations that confer vascular resistance. Calculated accordingly, 43% of active tone was sensitive to vasodilator challenge in isolated lungs of N piglets, resulting in a residual DPtp of 6.42±0.34 mmHg (Fig. 5). Isolated lungs of CH piglets showed a similar vasodilator-induced fall of 39% resulting in a residual ∆Ptp of 13.71±1.20 mmHg after loss of active tone. This value was significantly (2.1–fold) higher than the residual ∆Ptp of N piglets, suggesting that structural changes in the pulmonary circulation of CH piglets limited a further reduction of vascular resistance. Isolated lungs of CH + FTID piglets responded to the vasodilator challenge

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(A)

(B) Figure 3: (A) Computer-generated three-dimensional images of pulmonary arterial trees from neonatal piglets exposed for 3 weeks to normoxia (N), chronic hypoxia (CH) or chronic hypoxia + furegrelate three times daily (CH + FTID). (B) Comparison of distensibility coefficients between the pulmonary circulations of N, CH and CH + FTID piglets. Sample sizes were 15, 7 and 6, respectively. *Significant difference (P<0.05) between N and CH. †Significant difference (P<0.05) between CH and CH + FTID. Values are mean±S.E.M. Pulmonary Circulation | April-June 2012 | Vol 2 | No 2

(B) Figure 4: (A) Histological sections comparing percent medial thickness (%MT) of small pulmonary arteries in lungs from neonatal piglets exposed for 3 weeks to normoxia (N), chronic hypoxia (CH) or chronic hypoxia + furegrelate three times daily (CH + FTID). (B) Average %MT values for arteries of N, CH and CH + FTID piglets. *Significant difference (P<0.05) between N and CH. † = Significant difference (P<0.05) between CH and CH + FTID. Sample sizes were 103 arteries from 4 piglets (N), 192 arteries from 4 piglets (CH), and 184 arteries from 4 piglets (CH + FTID). Values are mean±S.E.M. 197

Hirenallur-S et al.: TxA2 synthase inhibition blunts PAH

Figure 5: Resting ∆Ptp in isolated lungs of piglets exposed to normoxia (N), chronic hypoxia (CH) or CH + furegrelate three times daily (CH + FTID). Lungs were perfused with control (Con) solution before nifedipine (10 µmol/L) and papaverine (15 mg/kg bolus) were added to induce dilation (N+P). *Significant difference (P<0.05) between N and CH piglets for the same measurement. †Significant difference (P<0.05) between CH and CH + FTID piglets for the same measurement. #Significant difference (P<0.05) between control (Con) and vasodilator challenge (N+P) in the same animal group.

with a 33% fall in DPtp resulting in a residual DPtp value of 9.25±0.65 mmHg, a value significantly lower than the residual DPtp of 13.71±1.20 mmHg in lungs of untreated CH piglets. Collectively, these findings show that furegrelate therapy blunts the development of elevated ∆Ptp in response to 3 weeks of hypoxia. After a strong vasodilator challenge, the elevated ∆Ptp in CH piglets still persisted as evidence of structural remodeling and this vasodilator–resistant component was ameliorated by furegrelate treatment.

DISCUSSION

The treatment of pediatric PAH has focused on correcting the imbalance between vasodilator and vasoconstrictor pathways in the pulmonary circulation that favors the development of anomalous pulmonary vascular tone. [1- 4,9,10] Therapeutic options have attempted to reverse this imbalance by administering vasodilator substances or attenuating vasoconstrictor pathways. Unfortunately, existing therapies are often short acting, have potentially severe side effects, or are ineffective. For example, the use of inhalational nitric oxide to ameliorate the abnormal vasoconstrictor tone of PAH is limited by its extremely short half-life and the potential for rebound hypertension upon discontinuation.[3] Prostacyclin analogs including epoprostenol also are very short-acting therapeutics that additionally cause the off-target effect of systemic hypotension.[2,3] Finally, the endothelin receptor blockers including bosentan, sitaxsentan, and ambrisentan are used on a limited basis in adults, but results from a randomized controlled trial in children with PAH are not yet available. [4,9,10] Since treatment options for pediatric 198

patients with PAH are limited, the potent pulmonary smooth muscle constrictor and mitogen, thromboxane A2, has drawn attention as a potential drug target to develop primary or adjunct therapeutic agents for PAH. Several lines of evidence implicate the thromboxane signaling pathway as a contributor to pediatric PAH. Infants with PAH secondary to meconium aspiration show elevated levels of plasma TxB2, a stable metabolite of TxA2 that positively correlates with pulmonary artery pressure.[27] Newborns with congenital heart disease also show elevated plasma TxB2 and elevated urinary TxB2 levels.[5,7] Additionally, TxB2 is increased in broncho-alveolar lavage samples and in the plasma of infants with persistent PAH. In these patients, TxB2 levels positively correlate with negative outcome after extracorporeal membrane oxygenation.[28] However, despite clinical studies implicating TxA2 in pediatric PAH, preclinical studies to evaluate inhibitors of the TxA2 signaling pathway have been limited, due in large part to the rigorous care of infant piglets that is required to carefully evaluate drug effects in this standard model of neonatal PAH. Only Fike and colleagues[18] recently reported that thromboxane synthase was upregulated in pulmonary arteries of CH piglets after 10 days of hypoxia. Earlier, the same authors showed that oral terbogrel, a TxA2 synthase inhibitor and thromboxane (TP) receptor antagonist, blunted the development of early-stage PAH in piglets exposed to hypoxia for three days.[17] However, terbogrel did not pass Phase 1 clinical trials for use in adults with PAH because it caused the off-target effect of leg pain.[12] Thus, the search continues to identify an inhibitor of the TxA2 signaling pathway that has a positive drug profile and retains the ability to ameliorate the development of neonatal PAH.

With this goal in mind, our study evaluated the effect of furegrelate sodium, a potent thromboxane synthase inhibitor,[19] on the development of hypoxia-induced PAH in the piglet model. Furegrelate was initially developed as an antiplatelet agent, but it failed to improve coagulation parameters in Phase 1 clinical trials in adult volunteers. [20,21] However, furegrelate has an appealing drug profile that includes oral bioavailability, a relatively long half-life of 4.2– 5.8 hours compared to nitric oxide and prostacyclin analogs and high specificity for its target enzyme. Studies failed to detect major off-target effects in preclinical testing in dogs or in Phase 1 clinical trials using healthy adult human subjects.[20-23] These encouraging drug properties suggest that furegrelate may represent a valuable therapeutic agent if “repositioned” to treat PAH. Indeed, our findings provide initial evidence that oral administration of furegrelate blunts the development of hypoxia-induced PAH in neonatal piglets and attenuates the right ventricular hypertrophy and pulmonary vascular remodeling that are key detrimental components of the disease. Thus, we provide experimental evidence that furegrelate may represent a promising early intervention to mitigate pediatric PAH. Importantly, the dosing regimen of furegrelate (3 mg/kg, p.o., TID) Pulmonary Circulation | April-June 2012 | Vol 2 | No 2

Hirenallur-S et al.: TxA2 synthase inhibition blunts PAH

that exerted beneficial pulmonary vascular effects failed to significantly lower systemic blood pressure. This feature may be an additional asset of the drug since other vasodilator drugs used to treat pediatric PAH including the prostacyclin analogs cause systemic hypotension in a subset of young patients as a limiting off-target effect.[3,4]

From a mechanistic standpoint, the ability of furegrelate to partially normalize PVRI in CH piglets may relate to at least two different mechanisms. First, furegrelate-induced block of thromboxane synthase would be expected to reduce circulating and local concentrations of TxA2 to attenuate active arterial tone. Second, TxA2 is a known mitogenic factor that promotes the proliferation of pulmonary VSMCs and structural remodeling. The TxA2 ligand binds to the thromboxane A2 /prostaglandin H2 (TP) receptor to stimulate DNA synthesis, and to promote proto-oncogene expression and actin polymerization.[29-32] Of these two mechanisms, our data suggest that the major impact of furegrelate on the pulmonary circulation of CH piglets may relate more closely to the prevention of structural remodeling for several reasons: (1) the medial thickness (%MT) of small pulmonary arteries in lung sections from CH + FTID piglets was significantly less compared to untreated CH piglets; (2) the pulmonary circulation of CH + FTID piglets revealed an increased distensibility on X-ray CT scans compared to untreated CH animals, reflecting improved pulmonary vascular elasticity; and (3) isolated perfused lungs from furegrelate-treated CH piglets showed a lower transpulmonary pressure (Ptp) compared to similar lungs from untreated CH piglets, and this difference persisted after the pulmonary circulation of both animal groups was subjected to the potent vasodilators, nifedipine and papaverine, to minimize active arterial tone. Thus, in our experimental model, it appears that furegrelate ameliorates the earlier stages of hypoxia-induced structural remodeling that precede the final stages of PAH in which vasodilator responsiveness often converts to a fixed vascular lesion resistant to vasodilator therapies.[1] Future evaluations should include studies in which furegrelate is administered to experimental models of PAH in which the disease has progressed to a more recalcitrant stage.

The findings of our study point to the importance of defining the pharmacokinetic profile of furegrelate in neonates. We based our furegrelate dosing regimen on earlier preclinical studies in which the daily administration of furegrelate (3 mg/kg body weight, p.o.) produced >80% inhibition of TxA2 synthase in platelet-rich plasma of adult rhesus monkeys.[19] Additionally, a single oral dose of furegrelate in adult human subjects (200–1600 mg p.o.) dosedependently inhibited thromboxane synthesis resulting in a 90% decline in the generation of TxB2 in platelet-rich plasma challenged with arachidonic acid.[20] Although these studies administered furegrelate once daily, we initially Pulmonary Circulation | April-June 2012 | Vol 2 | No 2

administered furegrelate twice daily (BID) to the CH piglets of our study since the reported drug half-life was only 4.2 to 5.8 hours in human adults. However, the twice daily dosing regimen did not blunt the development of PAH in 21-day CH piglets (CH + FBID). In contrast, the CH piglets that received furegrelate three times daily (CH + FTID) exhibited a 34% and 37% reduction in elevations of PVRI and Ptp, respectively, compared to values in untreated CH animals. Since we did not try to evaluate the beneficial effects of higher or more frequent doses of furegrelate in CH piglets, we may have underestimated its full therapeutic potential, and follow-up studies designed to define the pharmacokinetic profile of furegrelate will be necessary to optimize outcome. These efforts should also include assays to more accurately evaluate thromboxane synthase activity.

Although we ultimately observed that average urinary levels of the stable TxA2 metabolite, 11-dehydro TxB2, were decreased in CH + FTID piglets compared to untreated CH animals, follow-up studies will be necessary to verify the statistical significance of this observation and ensure its association with beneficial outcome in larger sample sizes of experimental animals.

In conclusion, our study shows that the pharmacological inhibition of TxA2 synthase by oral administration of furegrelate blunts the development of CH-induced PAH in neonatal piglets. Furegrelate also attenuated other abnormalities of PAH including right ventricular hypertrophy, medial hypertrophy of small pulmonary arteries and loss of pulmonary vascular distensibility.

Considering these findings, we propose that furegrelate should be explored further as a potentially effective therapeutic strategy to prevent the development of hypoxia-induced PAH in neonates.

ACKNOWLEDGMENTS

The authors would like to thank the staff of the Veterinary Medical Unit at the Zablocki Veterans Administration Medical Center in Milwaukee and Mr. Terry Fletcher at the University of Arkansas for Medical Sciences (UAMS) in Little Rock for their capable assistance.

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Fike CD, Aschner JL, Slaughter JC, Kaplowitz MR, Zhang Y, Pfister SL. Pulmonary arterial responses to reactive oxygen species are altered in newborn piglets with chronic hypoxia-induced pulmonary hypertension.

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Pharm Res 1989;6:53-7 Wynalda MA, Liggett WF, Fitzpatrick FA. Sodium 5-(3’-pyridinylmethyl) benzofuran-2-carboxylate (U-63557A), a new, selective thromboxane synthase inhibitor: Intravenous and oral pharmacokinetics in dogs and correlation with ex situ thromboxane B 2 production. Prostaglandins 1983;26:311-24.

Hirenallur SD, Haworth ST, Leming JT, Chang J, Hernandez G, Gordon JB, et al. Upregulation of vascular calcium channels in neonatal piglets with hypoxia-induced pulmonary hypertension. Am J Physiol 2008;295:L915-24.

Perneby C, Granstrom E, Beck O, Fitzgerald D, Harhen B, Hjemdahl P. Optimization of an enzyme immunoassay for 11-dehydro-thromboxane B2 in urine: Comparison with GC-MS. Thromb Res 1999;96:427-36.

Molthen RC, Karau KL, Dawson CA. Quantitative models of the rat pulmonary arterial tree morphometry applied to hypoxia-induced arterial remodeling. J Appl Physiol 2004;97:2372-2384;discussion 2354.

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Dobyns EL, Wescott JY, Kennaugh JM, Ross MN, Stenmark KR. Eicosanoids decrease with successful extracorporeal membrane oxygenation therapy in neonatal pulmonary hypertension. Am J Respir Crit Care Med 1994;149:873-80.

Pakala R, Benedict CR. Effect of serotonin and thromboxane A2 on endothelial cell proliferation: Effect of specific receptor antagonists. J Lab Clin Med 1998;131,527-37.

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Source of Support: Funding was provided by R01 HL-083013 (N.J.R. and J.B.G.) and R01 HL-19298 (J.B.G.) from the NIH. Additional support was provided from the UAMS Graduate Student Research Fund to D.K.H.-S., and stipend support was provided to N.D.D. from the UAMS Translational Research Institute supported by UL1 RR029884 from the NIH National Center for Research Resources., Conflict of Interest: None declared.

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Research A r t i cl e

Metabolomic analysis of bone morphogenetic protein receptor type 2 mutations in human pulmonary endothelium reveals widespread metabolic reprogramming Joshua P. Fessel1, Rizwan Hamid2, Bryan M. Wittmann3, Linda J. Robinson1, Tom Blackwell1, Yuji Tada4, Nobuhiro Tanabe4, Koichiro Tatsumi4, Anna R. Hemnes1, and James D. West1 1 Department of Medicine, Division of Allergy, Pulmonary, and Critical Care Medicine, Vanderbilt University, Nashville, Tennessee, USA, 2Department of Pediatrics, Vanderbilt University, Nashville, Tennessee, USA, 3Metabolon, Durham, North Carolina, USA, 4Department of Respirology (B2), Graduate School of Medicine, Chiba University, Chiba, Japan

Abstract Pulmonary arterial hypertension (PAH) is a progressive and fatal disease of the lung vasculature for which the molecular etiologies are unclear. Specific metabolic alterations have been identified in animal models and in PAH patients, though existing data focus mainly on abnormalities of glucose homeostasis. We hypothesized that analysis of the entire metabolome in PAH would reveal multiple other metabolic changes relevant to disease pathogenesis and possible treatment. Layered transcriptomic and metabolomic analyses of human pulmonary microvascular endothelial cells (hPMVEC) expressing two different disease-causing mutations in the bone morphogenetic protein receptor type 2 (BMPR2) confirmed previously described increases in aerobic glycolysis but also uncovered significant upregulation of the pentose phosphate pathway, increases in nucleotide salvage and polyamine biosynthesis pathways, decreases in carnitine and fatty acid oxidation pathways, and major impairment of the tricarboxylic acid (TCA) cycle and failure of anaplerosis. As a proof of principle, we focused on the TCA cycle, predicting that isocitrate dehydrogenase (IDH) activity would be altered in PAH, and then demonstrating increased IDH activity not only in cultured hPMVEC expressing mutant BMPR2 but also in the serum of PAH patients. These results suggest that widespread metabolic changes are an important part of PAH pathogenesis, and that simultaneous identification and targeting of the multiple involved pathways may be a more fruitful therapeutic approach than targeting of any one individual pathway.

Key Words: pulmonary arterial hypertension, BMPR2, Warburg effect, anaplerosis, isocitrate dehydrogenase

Pulmonary arterial hypertension (PAH) is a fatal, progressive disease of the pulmonary vasculature characterized by increasing pulmonary vascular resistance that leads to right heart failure and death.[1,2] The disease exists in several forms in humans, including a heritable form caused primarily by mutations in bone morphogenetic protein receptor type 2 (BMPR2) and an idiopathic form that is clinically and in many ways molecularly indistinguishable from the inherited disease. [3-5] Despite extensive investigations in PAH patients and Address correspondence to: Dr. Joshua P. Fessel Vanderbilt University Department of Medicine, Division of Allergy Pulmonary, and Critical Care Medicine 1161 21st Avenue South, MCN Suite T1218 Nashville, TN 37232, USA Email: joshua.p.fessel@vanderbilt.edu

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in a variety of animal models of PAH, the molecular mechanisms of disease pathogenesis have remained relatively obscure. Multiple converging lines of evidence point to disruption of interdependent metabolic pathways as being central to the molecular pathogenesis of PAH. In expression arrays from Bmpr2 mutant mice, nearly 50% of the significantly altered genes fall into metabolic gene ontology groups, without identification of specific metabolic pathways. [6] Several animal models of PAH Access this article online

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Website: www.pulmonarycirculation.org DOI: 10.4103/2045-8932.97606

How to cite this article: Fessel JP, Hamid R, Wittmann BM, Robinson LJ, Blackwell T, Tada Y et al. Metabolomic analysis of bone morphogenetic protein receptor type 2 mutations in human pulmonary endothelium reveals widespread metabolic reprogramming. Pulm Circ 2012;2:201-13.

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show a shift toward aerobic glycolysis, the so-called “Warburg effect” that has been identified as central to malignant transformation in a number of tumor types.[7-9] Alterations in glucose uptake and utilization, alongside changes in mitochondrial oxidative phosphorylation, have been demonstrated in the pulmonary artery endothelium from patients with PAH.[10,11] More recently, PAH patients not previously known to have diabetes or any other obvious metabolic diseases were found to have measurable increases in hemoglobin A1c compared to age- and BMI-matched controls, suggesting that wholebody glucose homeostasis is impaired in PAH. [12,13] Pulmonary hypertension associated with chronic hypoxia has been directly linked to an imbalance between glycolysis, glucose oxidation, and fatty acid oxidation.[9] Finally, therapies aimed at normalizing glucose oxidation directly (e.g., inhibitors of pyruvate dehydrogenase kinase such as dichloroacetate) or via modulation of the balance between fatty acid oxidation and glucose oxidation (e.g., partial fatty acid oxidation inhibitors such as trimetazidine or ranolazine) have shown great promise in treating PAH and have demonstrated the importance of metabolic disturbances in disease initiation and maintenance.[8,14-18] Indeed, dichloroacetate has entered Phase I trials in humans (ClinicalTrials.gov identifier NCT01083524). Though the weight of evidence suggests that metabolic reprogramming is a key feature of the molecular pathogenesis of PAH, existing data focus mainly on abnormalities of glucose homeostasis, and the full breadth and scope of the altered metabolic pathways in PAH are unknown.

We hypothesized that a broad-based metabolomic analysis of BMPR2 mutations that are known to cause PAH would reveal multiple coexisting and interdependent metabolic abnormalities beyond changes in glucose homeostasis. We quantify several hundred small molecule metabolites in native human pulmonary microvascular endothelial cells (hPMVEC) and in hPMVEC expressing one of two different disease-causing BMPR2 mutations.

Organization of the significantly changed metabolites into known biochemical pathways confirms that multiple interconnected metabolic pathways are deranged in PAH. Gene expression array analysis from these same cells shows that metabolic genes represent the largest single group of significantly changed genes and support the findings from the metabolomic analyses. Using these layered metabolomic and transcriptomic analyses, we then predict alteration of the activity of a specific enzyme in the tricarboxylic acid (TCA) cycle – namely, isocitrate dehydrogenase (IDH) — as a proof of principle and demonstrate increased IDH activity in mutant hPMVEC and in the serum of patients with PAH. 202

Materials and Methods Human pulmonary microvascular endothelial cell culture Human PMVEC were grown in culture as previously described.[19-21] Cells were maintained in Endothelial Cell Growth Medium MV from PromoCell (Heidelberg, Germany) in standard cell culture incubators (37°C, humidified, 5% CO2) and were used at or before the 10th passage.

Generation of stably transfected hPMVEC

Cells were transfected with either empty vector (native) or vector containing BMPR2 with R332X (KD) or 25792580delT (CD) mutations and stably selected using G418S as previously described.[22] Endothelial character of the cells for this study was confirmed by immunohistochemistry for the von Willebrand factor and by analysis of expression arrays for a panel of endothelial markers (Figure S1A, B — Access figure at www.pulmonarycirculation.org).

Transcriptomic analysis

Native and mutant hPMVEC were grown to 80% confluence, transitioned from G418S selection for at least 12 hours, and mRNA was isolated as described.[23] Two Affymetrix HGU133 Plus 2 arrays were run for each condition, with RNA for each array representing a pool of three independently grown plates, for a total of six arrays representing 18 biologically distinct events (three conditions × three plates × two arrays each).

Results were analyzed using dChip and R statistical software. Significantly changed genes were determined using a requirement of a minimum of a 2× change, a minimum difference in expression of at least 200 arbitrary Affymetrix units, and a P<0.01 by a t-test for differences.

Metabolomic analysis

Full details of the methodology for the mass spectrometrybased metabolomic analyses are given in Supplemental Methods and as described previously.[24,25] Briefly, samples (N=7 for each condition) were subjected to methanol extraction, split into aliquots for analysis by ultrahigh performance liquid chromatography/mass spectrometry (UHPLC/MS) in either the positive or negative ion mode or by gas chromatography/mass spectrometry (GC/ MS). Internal standards and controls for signal blank, technical replicates, and instrument performance were spiked into the samples and tracked throughout the analysis. Metabolite concentrations were determined by automated ion detection, manual visual curation, and were analyzed in-line using software developed by Metabolon.

[26] Significance was set at P<0.05 by Welch’s two-sample t-test with correction for multiple comparisons using q-values.[27] Pulmonary Circulation | April-June 2012 | Vol 2 | No 2

Fessel et al.: Metabolic reprogramming in PAH

NADP+-dependent IDH activity assays

IDH activity assays were performed using the BioVision Isocitrate Dehydrogenase Activity Assay Kit (Mountain View, Calif.) according to the manufacturer’s instructions.

For hPMVEC, cells were grown in 6-well plates to 50—60% confluence to yield approximately 500,000 cells per well, harvested, and lysed directly in the assay buffer. Aliquots were used for the IDH activity assay and for protein concentration determination by Pierce BCA assay. For serum, samples were used as undiluted 50 µl aliquots and assayed for IDH activity according to the instructions.

Human subjects

All patients and normal volunteers provided written informed consent to participate in research protocols approved by the institutional review boards of all participating institutions (IRB protocol number 9401).

Blood was drawn by standard venipuncture, centrifuged to collect serum, and serum was stored at −80°C until analysis.

Statistical analyses

Analyses were performed using R statistical software and using GraphPad Prism. Welch’s t-test or two-way ANOVA were used for tests of statistical significance. Box-andwhisker plots represent 25th—75th percentiles with the box, the median with the center line, and Tukey whiskers representing 1.5 times the interquartile range. Scatter plots show individual data points with mean±SEM depicted. For most analyses, significance was set at P<0.05, with P<0.01 being used as the significance threshold for RNA expression microarray analysis.

Results

BMPR2 mutations resulted in widespread changes in endothelial cell gene expression that organized into specific pathways

We sought to compare expression arrays from native hPMVEC to those from hPMVEC expressing one of two mutant BMPR2 constructs, and to organize the significantly different genes into functional pathways.

Human pulmonary microvascular endothelial cells were stably transfected with either R332X mutation in the kinase domain (KD) or a 2579-2580delT mutation in the cytoplasmic tail domain (CD). The CD mutation has been previously shown to dysregulate BMPR2 interaction with and signaling through LIMK-1, c-Src, and Tctex-1;[28-31] the KD mutation also includes dysregulated signaling through the canonical Smad pathway.[32]

Using a requirement of a minimum of twofold change, a minimum difference in expression of at least 200 arbitrary Pulmonary Circulation | April-June 2012 | Vol 2 | No 2

Affymetrix units, and a P<0.01 by t-test for difference, we found 687 probe sets representing 507 unique Entrez IDs, with common changes between both BMPR2 mutants and native hPMVEC, with a false discovery rate (FDR) of zero (determined by scrambling group identifiers).

These data have been deposited in GEO, accession number pending , and a full list of the 507 genes is provided in Supplemental Dataset S1 (Access Dataset S1 at www. pulmonarycirculation.org). Distribution of gene ontology groups was nearly identical to our previously published expression arrays interrogating PMVEC isolated from our Bmpr2 R899X and Bmpr2 delx4+ mouse models. [6,33,34] These included genes involved in apoptosis, proliferation, stimulus response, cytoskeletal organization, and development (Fig. 1). Roughly 40% of the genes changed (216/507) were related to small molecule metabolism.

Heterologous expression of BMPR2 mutations resulted in broad changes in TCA cycle, glycolysis, hypoxiainducible factor (HIF) responsive metabolic elements, and carnitine, fatty acid, and glutamate metabolism compared to expression of native BMPR2. Relatively unaffected pathways include glycan synthesis and metabolism, vitamin/cofactor metabolism (with the exception of folate and single-carbon metabolism), and xenobiotic metabolism. Thus, the affected pathways showed a degree of specificity as opposed to nonspecific whole metabolome dysfunction.

Metabolomic analysis of BMPR2 mutant endothelial cells showed significant alteration of multiple interdependent metabolic pathways

To determine the whole metabolome consequences of disease-causing BMPR2 mutation in endothelial cells, we undertook a simultaneous multiplexed mass spectrometric quantification of several hundred small molecule metabolites in CD and KD mutant hPMVEC and compared these mutations to the native hPMVEC. In this analysis, 267 small molecule metabolites were confidently identified in seven biological replicates for each condition described above (native, CD, and KD, Figure S2 — Access figure at www.pulmonarycirculation. org). Significantly changed biochemicals from the native condition were identified as those biochemicals with a P-value <0.05 based upon Welch’s two-sample t-test, which had a maximum FDR of 3.2% based upon q-values[27] for that set of biochemicals with P-values <0.05. The full dataset is provided in Supplemental Dataset S2 (Access Dataset S2 at www.pulmonarycirculation.org). Compared to the native hPMVEC, the CD mutants showed significant changes in 65% of the metabolites quantified (172/267, with 87 increased and 85 decreased) and the KD mutants showed significant changes in 37% of the metabolites (99/267, with 61 increased and 38 decreased). This represented confident identification of approximately 11% of the database of named compounds available in this analysis, with the 203

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Figure 1: Transcriptomic analysis of human pulmonary microvascular endothelial cells expressing one of two dominant negative BMPR2 mutant constructs. The pie chart depicts the pathways represented by the 507 genes showing significantly altered expression in the CD and KD mutant hPMVEC compared to the native hPMVEC. The largest single major ontology group was metabolic genes, and the heatmap at the right of the figure shows changes in individual genes within this larger group broken down by subsets, with red indicating increased expression and blue denoting decreased expression.

CD mutants showing significant changes in the levels of approximately 7% of the total compounds in the database.

For the KD mutants, a further 14% (38 metabolites) approached statistical significance (0.05<P<0.10 by Welch’s two-sample t-test), though these were not included in the analyses discussed below.

Increased pentose phosphate pathway metabolites and polyamine biosynthesis indicate increased proliferation in BMPR2 mutant endothelial cells

Intermediates in the pentose phosphate pathway for BMPR2 mutant hPMVEC along with changes in the expression of the corresponding genes compared to native hPMVEC are shown (Fig. 2). The pentose phosphate pathway interfaces with multiple other metabolic pathways, including the glycolytic pathway and NADPH synthetic pathways, in addition to providing 5-carbon sugars for nucleotide synthesis. The enzyme primarily responsible for synthesis of NADPH in the pentose phosphate pathway, glucose-6-phosphate dehydrogenase, exhibited significantly reduced expression in both the CD and KD mutants. The upregulation of the pentose phosphate pathway in the BMPR2 mutant hPMVEC thus was apparently driven by upregulation from ribose-5phosphate isomerase downstream that overcame decreases in glucose-6-phosphate dehydrogenase expression and 204

that directly related to increased nucleotide synthesis and salvage. The significantly increased levels of purine and pyrimidine nucleosides downstream from both mutations further supported this conclusion. Finally, intermediates in the terminal portion of the polyamine synthesis pathway in the CD and KD mutants were increased preferentially over urea cycle intermediates (Figure S3 — Access figure at www.pulmonarycirculation.org), further supporting the increased cell proliferation previously observed for these specific mutations.[6]

Multiple energy metabolism pathways were disrupted in BMPR2 mutant endothelium

Several groups have previously shown an increase in aerobic glycolysis (the “Warburg effect”) in PAH,[7-9,35] and increased glucose uptake has been demonstrated in the lungs of PAH patients by positron emission tomography.[10] In this whole metabolome analysis, glycolysis showed significant upregulation as a consequence of BMPR2 mutation, though this was not to the same extent for both mutations (Fig. 3). Glycolytic metabolites were significantly increased in the CD mutants for all of the pathway intermediates down to pyruvate, whereas the KD mutants showed statistically significant increases or trends in increases only for the metabolites earlier in the glycolytic pathway. Consistent with global activation of the Pulmonary Circulation | April-June 2012 | Vol 2 | No 2

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Figure 2: Intermediates in the pentose phosphate pathway specific to nucleotide synthesis are increased in BMPR2 mutant hPMVEC. Major intermediates in the pentose phosphate pathway are shown. In all graphs, native hPMVEC are in white boxes, CD hPMVEC in vertical hatched boxes, and KD hPMVEC in diagonal hatched boxes. Quantities are in arbitrary units specific to the internal standards for each quantified metabolite and normalized to protein concentration.

N = 7 for each box, with whiskers indicating Tukey whiskers and extreme data points indicated by filled circles. *P<0.05 compared to native. Genes coding for the enzymes that catalyze particular steps in the pathway are indicated by their Entrez Gene names, with red indicating significantly increased expression in the transcriptomic analysis and blue indicating significantly decreased expression. The four graphs at the bottom of the figure show quantitation of purine and pyrimidine nucleosides. G6PD, glucose-6-phosphate dehydrogenase; H6PD, hexose-6-phosphate dehydrogenase; RPIA, ribose-5-phosphate isomerase A; RPE, ribulose-5-phosphate-3-epimerase; TKT, transketolase.

glycolytic pathway, expression of the majority of the genes for glycolytic enzymes was increased, including genes coding for glucose uptake transporters. The genes for two of the three regulated enzymes in the pathway, hexokinase, and phosphofructokinase, showed decreased expression in arrays from the BMPR2 mutant hPMVEC, though the third regulated enzyme, pyruvate kinase, showed increased expression for at least one isoform.

The interplay between fatty acid oxidation and glucose utilization has been shown to play an important role in pulmonary hypertension related to chronic hypoxia and in right ventricular hypertrophy and failure induced by pressure overload in a pulmonary artery banding model,[9,18] but this has not been explored in pulmonary Pulmonary Circulation | April-June 2012 | Vol 2 | No 2

arterial hypertension specifically. We thus sought evidence for alterations in the major pathways for fatty acid oxidative metabolism in the context of disease-causing BMPR2 mutations. We found that carnitine and its downstream acyl metabolites were significantly reduced in the CD and KD mutant hPMVEC compared to the native endothelial cells (Fig. 4). Decreased levels of carnitine itself as well as glycine (a by-product of carnitine synthesis) suggested decreased synthesis of carnitine itself. Levels of palmitoylcarnitine, isobutyrylcarnitine, and propionylcarnitine were also significantly decreased. Decreased expression of many of the key genes involved in carnitine/acylcarnitine metabolism and trafficking was also observed, including the two major carnitine palmitoyltransferase genes and one of the major carnitine/acylcarnitine translocases. We 205

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Figure 3: Glycolysis is significantly upregulated in BMPR2 mutant hPMVEC, particularly in cytoplasmic tail domain mutants. The classical glycolysis pathway intermediates are shown. In all graphs, native hPMVEC are in white boxes, CD hPMVEC in vertical hatched boxes, and KD hPMVEC in diagonal hatched boxes. Quantities are in arbitrary units specific to the internal standards for each quantified metabolite and normalized to protein concentration. N = 7 for each box, with whiskers indicating Tukey whiskers and extreme data points indicated by filled circles. *P<0.05 compared to native. Genes coding for the enzymes that catalyze particular steps in the pathway are indicated by their Entrez Gene names, with red indicating significantly increased expression in the transcriptomic analysis and blue indicating significantly decreased expression. SLC2A1 and SLC2A3, solute carrier family 2 (facilitated glucose transporter), members 1 and 3; HK2, hexokinase 2; PFKL and PFKM, phosphofructokinase, liver and muscle isoforms; ALDOA, aldolase A; TPI1, triosephosphate isomerase 1; PGK1, phosphoglycerate kinase 1; PGAM5, phosphoglycerate mutase 5; PKM2, pyruvate kinase, muscle.

also found significantly decreased expression of a number of the acyl-CoA dehydrogenase genes involved in fatty acid oxidation.

Activity of the tricarboxylic acid (TCA) cycle has been shown to be reduced in a variety of different types of cancer, and this has been proposed to be a central advantage exploited by cancer cells, allowing for diversion of TCA cycle intermediates toward macromolecule synthesis while relying on other energy-generating pathways such as glycolysis. [36-38] Although a number of the metabolic features of cancer cells have been observed in PAH, defects in the TCA cycle have not been extensively described. In hPMVEC expressing 206

BMPR2 mutations, there were extensive metabolic defects in the TCA cycle indicating overall decreased activity of the cycle downstream from citrate (Fig. 5). In particular, significant decreases in succinate, fumarate, and malate were present in the CD mutants, whereas much more mild nonsignificant decreases in the mean concentrations of succinate and malate were observed for the KD mutants. In both mutants, concentrations of citrate were significantly increased, and concentrations of pyruvate and lactate were equivalent to the native hPMVEC, suggesting that the defect in the TCA cycle occurred distal to citrate. Moreover, this suggested that alternative catabolic pathways (e.g., fatty acid oxidation, peptide/amino acid catabolism) feeding into the TCA Pulmonary Circulation | April-June 2012 | Vol 2 | No 2

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Figure 4: Carnitine metabolism and fatty acid oxidation are significantly depressed in BMPR2 mutant hPMVEC. Multiple carnitine metabolites and their flow into fatty acid oxidation are shown. Intermediates for which significant differences in one or both mutant conditions were detected are shown. In all graphs, native hPMVEC are in white boxes, CD hPMVEC in vertical hatched boxes, and KD hPMVEC in diagonal hatched boxes. Quantities are in arbitrary units specific to the internal standards for each quantified metabolite and normalized to protein concentration. N = 7 for each box, with whiskers indicating Tukey whiskers and extreme data points indicated by filled circles. *P<0.05 compared to native. Genes coding for the enzymes that catalyze particular steps in the pathway are indicated by their Entrez Gene names, with red indicating significantly increased expression in the transcriptomic analysis and blue indicating significantly decreased expression. CPT1A and CPT2, carnitine palmitoyltransferase isoforms 1A and 2; SLC25A20, carnitine/acylcarnitine translocase; ACADS, ACADM, ACADSB, and ACADVL, acyl-CoA dehydrogenases â&#x20AC;&#x201C; short chain, medium chain, short/branched chain, and very long chain; MLYCD, malonyl-CoA decarboxylase.

cycle were insufficient to support concentrations of succinate, fumarate, and malate, particularly in the CD mutants. The balance of TCA cycle intermediates is normally maintained by the complementary processes of anaplerosis and cataplerosis. Broadly defined, anaplerosis refers to the addition of 4- and 5-carbon intermediates into the TCA cycle (e.g., oxaloacetate, alpha-ketoglutarate, and succinyl-CoA) using pyruvate, aspartate, glutamate, or fatty acids as substrates, to support mitochondrial respiration and to replenish TCA cycle intermediates diverted to biosynthesis. Cataplerosis refers to the removal of these same TCA cycle intermediates to support biosynthetic processes such as gluconeogenesis, glyceroneogenesis, and fatty acid synthesis.[39] Pulmonary Circulation | April-June 2012 | Vol 2 | No 2

Two key specific anaplerotic pathways are the conversion of glutamine to glutamate and then to alpha-ketoglutarate, and the conversion of aspartate to oxaloacetate.[40] Levels of these specific amino acids were quantified and found to be significantly reduced in the CD mutant hPMVEC compared to native cells (Fig. 6). By contrast, the KD mutants showed increased levels of glutamine and glutamate, which likely contributed to the more modest reductions of TCA cycle intermediates in these cells, as there was more glutamine and glutamate available for the anaplerotic synthesis of alpha-ketoglutarate. The differences between the CD and KD mutants may be at least in part attributable to differences in the synthesis of N-acetylaspartylglutamate 207

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Figure 5: TCA cycle intermediates are significantly decreased in BMPR2 mutant hPMVEC, particularly in CD mutants. The TCA cycle and its major intermediates are shown. In all graphs, native hPMVEC are in white boxes, CD hPMVEC in vertical hatched boxes, and KD hPMVEC in diagonal hatched boxes. Quantities are in arbitrary units specific to the internal standards for each quantified metabolite and normalized to protein concentration. N = 7 for each box, with whiskers indicating Tukey whiskers and extreme data points indicated by filled circles. *P< 0.05 compared to native. Genes coding for the enzymes that catalyze particular steps in the pathway are indicated by their Entrez Gene names, with red indicating significantly increased expression in the transcriptomic analysis and blue indicating significantly decreased expression. PDK1-4, pyruvate dehydrogenase kinase 1-4; PDHA1, pyruvate dehydrogenase (lipoamide) alpha 1; PDHB, pyruvate dehydrogenase E1 component, beta subunit; DLAT, dihydrolipoamide S-acetyltransferase; ACO2, aconitase 2; IDH3A/B/G, isocitrate dehydrogenase 3 (NAD+) alpha, beta, and gamma subunits; IDH1, isocitrate dehydrogenase 1 (NADP+); SUCLG2, succinate-CoA ligase, GDP-forming, beta subunit; SDHA/B/C, succinate dehydrogenase complex subunits A (flavoprotein), B (iron-sulfur), and C (15kDa integral membrane protein); FH, fumarate hydratase; MDH2, malate dehydrogenase 2.

(NAAG). The CD mutants showed increased expression of NAAG synthetase and increased concentrations of NAAG, whereas the KD mutants showed decreased expression of NAAG synthetase and a subsequently lower level of NAAG compared to the CD mutants.

It thus is likely that, in the CD mutants, much of the glutamate and aspartate that might otherwise have been used to feed into the TCA cycle was instead being used for the synthesis of NAAG. This was not the case in the KD mutants. Both mutations appeared to drive an overall increase in peptide and amino acid catabolism, as both mutants showed significant increases in the concentrations of dipeptides (Figure S4 — Access 208

figure at www.pulmonarycirculation.org), though this was more pronounced in the CD mutants. In addition, anaplerosis via branched chain amino acid metabolism appeared to be more significantly impaired in the CD mutants compared to the KD mutants, as evidenced by more significant decreases in isobutyrylcarnitine and propionylcarnitine, metabolites that participate in branched chain amino acid metabolism as well as fatty acid metabolism. The CD mutants exhibited a major failure of anaplerosis on multiple levels that was much more mild in the KD mutants, and thus must rely on multiple “salvage” pathways of peptide/amino acid catabolism.

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Figure 6: Glutamine/glutamate and aspartate metabolism, two major anaplerotic pathways, are significantly reduced in CD mutant hPMVEC. Intermediates for which significant differences in one or both mutant conditions were detected are shown. In all graphs, native hPMVEC are in white boxes, CD hPMVEC in vertical hatched boxes, and KD hPMVEC in diagonal hatched boxes. Quantities are in arbitrary units specific to the internal standards for each quantified metabolite and normalized to protein concentration. N = 7 for each box, with whiskers indicating Tukey whiskers and extreme data points indicated by filled circles. *P<0.05 compared to native. Genes coding for the enzymes that catalyze particular steps in the pathway are indicated by their Entrez Gene names, with red indicating significantly increased expression in the transcriptomic analysis and blue indicating significantly decreased expression. GLUD1 and GLUD2, glutamate dehydrogenase 1 and 2; GLS, glutaminase; GLUL, glutamate-ammonia ligase; RIMKLB, N-acetylaspartylglutamate synthetase B.

Predicted differences in IDH1/2 activity were present in mutant hPMVEC and in human PAH patients

We chose to examine the TCA cycle in more detail, as this is a major point of integration of multiple pathways involved in energy production as well as biosynthesis and so might better reflect the summation of alterations in these many different pathways. On closer inspection of the transcriptomic and metabolomic data relevant to the TCA cycle, there was a clear functional change in the BMPR2 mutants compared to the wild-type endothelial cells that occurred somewhere between citrate and succinate. The two most likely enzymatic candidates were aconitase and isocitrate dehydrogenase (IDH). Of these two enzymes, aconitase has been shown to be inactivated by oxidative stress,[41] and oxidative stress is known to be increased Pulmonary Circulation | April-June 2012 | Vol 2 | No 2

in the context of BMPR2 mutations.[22] To remove this as a confounding factor, we chose to examine IDH activity.

While NAD+-dependent IDH activity (corresponding to the IDH3 isoform) did not differ between wild-type and BMPR2 mutant endothelial cells, NADP+-dependent IDH activity (corresponding to IDH isoforms 1 and 2, hereafter IDH1/2) was significantly increased in BMPR2 mutant endothelial cells (Fig. 7A). We then sought to determine if these findings were applicable to patients with PAH. We quantified IDH1/2 activity in serum from controls, from patients with heritable PAH known to have BMPR2 mutations, and from two pooled cohorts of patients with IPAH (one from the United States and one from Japan).

Serum IDH1/2 activity was significantly increased in both HPAH and IPAH patients compared to controls (Fig. 7B).

The variability in IDH1/2 activity observed in the PAH 209

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Figure 7: IDH1/2 activity is increased in mutant hPMVEC and in serum from patients with pulmonary arterial hypertension. (A) Compared to native hPMVEC (1.07±0.04 mU/5×105 cells), the CD and KD mutant-expressing hPMVEC both show significantly increased NADP+-dependent IDH activity in cell lysates (1.56±0.04 and 1.38 +/ 0.07 mU/5×105 cells, respectively). N=6 for each group, mean±SEM indicated, P<0.0001 by one-way ANOVA, **P<0.0001 vs. native, *P<0.003 vs. native. (B) NADP+-dependent IDH activity in the serum from patients with heritable PAH (N=8, 1.83±0.1 mU/mL) and idiopathic PAH (N=17, 1.85±0.09 mU/mL) was significantly increased compared to serum from normal control individuals (N=13, 1.61±0.04 mU/mL), mean±SEM indicated, **P<0.03 vs. control by Welch’s t-test.

patients likely does reflect variability in disease activity, at least in part. We separated the PAH patients for whom data were available into two groups based upon the presence or absence of any prostanoid therapy (intravenous, subcutaneous, or inhaled prostacyclin receptor agonist) and analyzed serum IDH1/2 activity (Figure S5 — Access figure at www.pulmonarycirculation.org). Though only approaching statistical significance (P=0.1 by Welch’s t-test), the mean serum IDH1/2 activity for the prostanoid treated group of PAH patients clearly trended toward being higher than for the nontreated group. As prostanoid therapy is only initiated in more severe disease, the higher serum IDH activity may well be a reflection of disease severity.

Discussion

We present here a whole metabolome analysis of the effects of BMPR2 mutations known to cause pulmonary arterial hypertension. We have constructed an integrated picture of the complex and interdependent metabolic changes that occur downstream from BMPR2 mutations in human pulmonary microvascular endothelial cells, and this integrated view has demonstrated more widespread metabolic defects in PAH than have been previously known. The shift toward aerobic glycolysis that is typified by the Warburg effect in PAH has been known for some time.[7] More recently, the interplay between fatty acid oxidation and control of glycolysis versus glucose oxidation (typified by the Randle cycle) has been demonstrated to be important in hypoxic pulmonary hypertension.[9,42] In addition to confirming these previously identified metabolic defects in PAH, we have identified significant 210

alterations in the TCA cycle and at least some of the pathways that interact with it.

Our analysis reveals a profound failure of anaplerosis present downstream from BMPR2 mutations that has been largely unexplored as a mechanism of disease pathogenesis in PAH. TCA cycle intermediates are depleted through what appears to be a combination of decreased activity of the cycle itself plus abnormal shunting of intermediates from other pathways (e.g., glutamate, glutamine, aspartate, and branched chain amino acids) that could otherwise be used to replenish the intermediates of the TCA cycle. This implies that the metabolic defects in PAH cannot be simply summed up under the umbrella of Warburg metabolism.[35,43] It is possible that diversion of TCA cycle intermediates for biosynthesis and reliance on aerobic glycolysis is actually a feature of decreased BMP signaling, as would be seen in settings of tissue remodeling and repair, for example.

However, these would be settings of temporary loss or reduction of BMP signaling, and what might be adaptive cataplerosis in the short-term becomes pathogenic in the setting of permanently decreased BMP signaling due to mutation. More importantly, the therapeutic implication is that it is very likely that most or all of the errant pathways will need to be targeted to expect a significant therapeutic impact, and current investigational therapies address only certain dysfunctional pathways that largely do not address the failure of anaplerosis.

To demonstrate further the utility of our combined metabolomic and transcriptomic analysis, we chose to quantify NADP+-dependent IDH activity. We predicted that this enzymatic activity would be altered in PAH and then demonstrated the accuracy of that prediction in both Pulmonary Circulation | April-June 2012 | Vol 2 | No 2

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cultured cells and in patients with PAH. Alterations in IDH activity have not been previously described in PAH; however, multiple conceptual links between PAH and cancer have been proposed, and there is a growing body of literature linking altered IDH activity in a causative way to at least certain types of cancer,[24,44-47] so the identification of altered IDH1/2 activity in PAH may in fact be directly related to disease pathogenesis.

A second possibility is that the anaplerotic failure and increased IDH1/2 activities in PAH are driving forces for HIF activation that underlies PAH,[15,48] which then perpetuates increased aerobic glycolysis, apoptosis resistance, and decreased mitochondrial number.[11] HIF can be activated by decreased alpha-ketoglutarate and increased citrate concentrations, both of which activate HIF by decreasing the efficiency of prolyl hydroxylase.[49] Under hypoxic conditions, activation of HIF has very recently been shown to increase IDH2-mediated conversion of glutamine-derived alpha-ketoglutarate to citrate.[50] The resultant hypothesized increase in citrate and decrease in alpha-ketoglutarate brought about by increased IDH2 activity would thus be predicted to further drive HIF activation, setting up something of a vicious cycle. Our data show significant changes in the expression of HIF responsive genes in BMPR2 mutant hPMVEC. Alternatively, HIF activation can be driven by increased oxidative stress and decreased antioxidant defenses, as is seen with epigenetic inactivation of SOD2,[51] and this may be the upstream event that drives IDH activation. HIF activation can drive metabolic reprogramming that leads to increased IDH activity,[50] and IDH may be upregulated in response to oxidative stress to serve as a source of NADPH that is used to maintain reduced glutathione pools intracellularly.[52]

More importantly, IDH activity appears to track with disease activity, as patients with more severe disease (i.e., those treated with a prostacyclin agonist) trend toward higher serum IDH activity. The fact that those patients with disease severe enough to warrant prostanoid therapy still exhibit increased serum IDH activity after the initiation of therapy suggests that our most efficacious class of drugs for PAH still does not correct all of the underlying metabolic defects in PAH and highlights the need for therapies that arise from a deeper understanding of the molecular pathogenesis of PAH. Further investigations are needed to determine if the increased IDH activity described in PAH in this study is truly pathogenic, adaptive, or epiphenomenon.

This type of investigation is not without limitations. The pulmonary endothelial cell clearly plays an important role in the pathogenesis of PAH, but many other cell types, including smooth muscle cells,[53] lung mesenchymal stem Pulmonary Circulation | April-June 2012 | Vol 2 | No 2

cells,[54] and resident immune cells[55] all likely contribute to disease development. It is not possible in most cases in this study to determine what changes are truly causative of disease and which are consequences of the disease, although examination of cells in culture minimizes this problem to some degree. [23] While identification of putative molecular targets is possible and is greatly enhanced by this type of layered analysis, each target must be further validated independently and placed into a context. Still, this study has permitted the identification of previously unrecognized pathways that likely directly contribute to the development of PAH; the identification of promising new biomarkers, of which IDH1/2 activity is but one, to guide diagnosis and therapeutic evaluation; and the recognition of the importance of defining and simultaneously targeting the multiple affected metabolic pathways in future therapeutic development. We hypothesize that this approach would be similarly fruitful in many other complex diseases.

appendix

Supplemental Methods

Metabolomic Analysis: Native and mutant hPMVEC were grown to 80% confluence (yielding approximately 1-3 Ă&#x2014;107 cells per sample), transitioned from G418 sulfate selection to complete media without antibiotic for at least 12 hours, and washed with phosphate-buffered saline and trypsinized to harvest cells. Cells were pelleted, the supernatant removed, and the dry pellet snap frozen in liquid nitrogen and stored at -80C until analysis. The non-targeted metabolic profiling platform employed for this analysis combined three independent platforms: ultrahigh performance liquid chromatography/tandem mass spectrometry (UHPLC/MS/MS2) optimized for basic species, UHPLC/MS/MS2 optimized for acidic species, and gas chromatography/mass spectrometry (GC/ MS). Samples were processed essentially as described previously. [1,2] For each sample, 100L was used for analyses. Using an automated liquid handler (Hamilton LabStar, Salt Lake City, UT), protein was precipitated from the homogenate with methanol that contained four standards to report on extraction efficiency. The resulting supernatant was split into equal aliquots for analysis on the three platforms. Aliquots, dried under nitrogen and vacuum-desiccated, were subsequently either reconstituted in 50L 0.1% formic acid in water (acidic conditions) or in 50L 6.5mM ammonium bicarbonate in water, pH 8 (basic conditions) for the two UHPLC/ MS/MS2 analyses or derivatized to a final volume of 50L for GC/MS analysis using equal parts bistrimethyl-silyltrifluoroacetamide and solvent mixture acetonitrile:dich loromethane:cyclohexane (5:4:1) with 5% triethylamine at 60°C for one hour. In addition, three types of controls 211

Fessel et al.: Metabolic reprogramming in PAH

were analyzed in concert with the experimental samples: aliquots of a well-characterized human plasma pool served as technical replicates throughout the data set, extracted water samples served as process blanks, and a cocktail of standards spiked into every analyzed sample allowed instrument performance monitoring.

Experimental samples and controls were randomized across platform run days.

For UHLC/MS/MS 2 analysis, aliquots were separated using a Waters Acquity UPLC (Waters, Millford, MA) and analyzed using an LTQ mass spectrometer (Thermo Fisher Scientific, Inc., Waltham, MA) which consisted of an electrospray ionization (ESI) source and linear iontrap (LIT) mass analyzer. The MS instrument scanned 99-1000 m/z and alternated between MS and MS2 scans using dynamic exclusion with approximately 6 scans per second. Derivatized samples for GC/MS were separated on a 5% phenyldimethyl silicone column with helium as the carrier gas and a temperature ramp from 60°C to 340°C and then analyzed on a Thermo-Finnigan Trace DSQ MS (Thermo Fisher Scientific, Inc.) operated at unit mass resolving power with electron impact ionization and a 50-750 atomic mass unit scan range.

Metabolites were identified by automated comparison of the ion features in the experimental samples to a reference library of chemical standard entries that included retention time, molecular weight (m/z), preferred adducts, and in-source fragments as well as associated MS spectra, and were curated by visual inspection for quality control using software developed at Metabolon.[3]

For statistical analyses and data display purposes, any missing values were assumed to be below the limits of detection and these values were imputed with the compound minimum (minimum value imputation).

Statistical analysis of log-transformed data was performed using “R” (http://cran.r-project.org/). Welch’s t-tests were performed to compare data between experimental groups. A p-value of < 0.05 was considered statistically significant and multiple comparisons were accounted for by estimating the false discovery rate (FDR) using q-values.[4]

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Source of Support: This work was supported in part by the Vanderbilt Clinical and Translational Science Awards grant UL1 RR024975-01 from the National Center for Research Resources/NIH, by support provided to JPF

by NIH training grant T32 HL094296-02, and by R01 HL095797-01A2 (JW)., Conflict of Interest: None declared.

213

Research A r t i cl e

Leptin levels predict survival in pulmonary arterial hypertension Adriano R. Tonelli1, Metin Aytekin2, Ariel E. Feldstein3, and Raed A. Dweik1,2 Department of Pulmonary, Allergy and Critical Care Medicine, Respiratory Institute, 2Department of Pathobiology, Lerner Research Institute, Cleveland Clinic, Cleveland, Ohio, USA, 3Department of Pediatric Gastroenterology and Nutrition, University of California, San Diego, California, USA

1

Abstract Evidence suggests that leptin is involved in relevant processes in the cardiovascular system. Low serum leptin levels have been associated with increased cardiovascular events and mortality in patients with coronary artery, diabetes, or chronic kidney disease. We hypothesized that leptin is increased in pulmonary arterial hypertension (PAH) and provides prognostic information.

We correlated leptin levels with clinical data and assessed its association with survival. Sixty-seven patients with PAH and 29 healthy controls were studied. Plasma leptin levels were nonlinearly associated with BMI. Leptin level <15 µg/l was associated with higher mortality in PAH patients, with an adjusted (age, gender, BMI, and smoking status) hazard ratio of 3.8 (95% CI: 1.3-11.2), P=0.016. Similarly, PAH patients with leptin/BMI ratio <0.5 µg * m2/kg * l had worse survival than those with a level >0.5 µg * m2/ kg * l (P=0.046 by log-rank test). Two-year mortality in PAH patients was 24%. A receiver operating characteristic curve using leptin/BMI ratio as the test variable and 2-year mortality as the state variable showed an area under the curve of 0.74 (95% CI: 0.62–0.86). A leptin/BMI ratio cut-off of 0.6 had a high sensitivity (94%) and negative predictive value (96%) for predicting death of any cause at 2 years. In PAH, plasma leptin levels are directly associated with BMI. Lower leptin levels, when adjusted by BMI, are associated with an increased overall mortality and leptin/BMI ratio has high negative predictive value for mortality at 2 years.

Key Words: leptin, mortality, obesity, pulmonary hypertension

Leptin is a neuroendocrine peptide released by adipose tissue which enhances metabolism and acts on the hypothalamus suppressing appetite.[1-2] Mounting evidence suggests that, besides its central role in energy homeostasis regulation, leptin is involved in important processes in the cardiovascular system, including sympathetic activation,[3-4] angiogenesis,[5-7] and endothelial nitric oxide (NO) production.[6,8,9] Low serum leptin levels have been associated with increased cardiovascular events and mortality in patients with coronary artery disease,[10] diabetes,[11] or chronic kidney disease,[12] independent of obesity.[10] However, no prior investigations have studied the relationship between plasma leptin levels and disease severity and outcomes in pulmonary arterial hypertension (PAH).

On the basis of these observations, we hypothesized that leptin is decreased in PAH and provides prognostic Address correspondence to: Dr. Raed A. Dweik Cleveland Clinic 9500 Euclid Ave. A-90 Cleveland, OH 44195, USA Email: dweikr@ccf.org 214

information. To test our hypothesis, we prospectively determined plasma leptin levels in patients with PAH and healthy controls. We correlated leptin levels with clinical, echocardiographic, and hemodynamic data and assessed its association with survival.

Materials and methods Study population

Patients were recruited from the Pulmonary Vascular Program at Cleveland Clinic. Consecutive patients were asked at the time of right heart catheterization to donate blood for biobank. For the present study, we included all adult (≥ 18 years) PAH patients (n=67) and healthy controls Access this article online

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Website: www.pulmonarycirculation.org DOI: 10.4103/2045-8932.97607 How to cite this article: Tonelli AR, Aytekin M, Feldstein AE, Dweik RA. Leptin levels predict survival in pulmonary arterial hypertension. Pulm Circ 2012;2:214-9.

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Tonelli et al.: Leptin in pulmonary hypertension

(n=29) who donated blood from January of 2003 to August of 2008. PAH was confirmed by right heart catheterization.

All participants signed a consent form that was approved by the Cleveland Clinic Institutional Review Board (IRB) prior to participation in the study.

Clinical evaluation

As part of their clinical evaluation, all patients underwent a comprehensive testing to exclude other etiologies of pulmonary hypertension. Clinical and outcome information were prospectively collected.

Height and weight were obtained at the time of the plasma leptin determination. We defined “current smoker” as an individual who was smoking at the time of the inclusion in the study, “ex-smoker” as a person who had not smoked for at least three months prior to leptin determination, and “nonsmoker” as a subject who smoked less than 20 packs in his or her lifetime.

Clinical transthoracic Doppler echocardiography was performed with commercially available equipment in the left lateral recumbent position. Experienced operators, blinded to the results of leptin levels, reviewed the echocardiograms and assessed right ventricular size and function subjectively.

Right ventricular systolic pressure was estimated by the Bernoulli equation with addition of right atrial pressure estimated by assessing inferior vena cava size and inspiratory collapse.[13-14] Right heart catheterization was performed in the standard manner using a 7F pulmonary artery catheter.

Pressure measurements were obtained at end-expiration.

Cardiac output (CO) was obtained by the thermodilution method; additionally, transpulmonary gradient (mean PAP – pulmonary artery occlusion pressure) and pulmonary vascular resistance (transpulmonary gradient/CO) were calculated.

Plasma leptin levels

tested associations involving the leptin plasma level using linear regression, adjusted for independent variables known to be associated with this measurement (age, gender, BMI, and smoking status).[15-17] We used binary logistic regression to test whether leptin was different in PAH versus controls, adjusted by age, gender, BMI, and smoking status. We utilized the restrictive cubic spline (three knots with fixed percentiles at 10%, 50%, and 90% of the distribution) and Lowess model to test nonlinear association between leptin level and BMI (Fig. 1).

Overall survival was analyzed by the Kaplan–Meier method.

Time 0 was the date of leptin determination. Censoring was performed at the time of transplantation or end of the study (20 July 2011). The date of death of the study participants was ascertained by reviewing patient records or by querying the U.S. Social Security Death Index. No patient was lost to follow-up. Survival differences between patients with and without PAH were assessed by the log-rank test and Cox regression analysis. For the log-rank test, leptin plasma levels were dichotomized using the median as cut-off. The Cox proportional hazards model was adjusted for age, gender, and smoking status; BMI was included when we used leptin level instead of leptin/BMI ratio. In addition to the aforementioned covariates, we tested Cox models that included etiology of PAH (idiopathic/heritable vs. PAH associated with other diseases), NYHA functional class, hemodynamic (right atrial pressure, cardiac output, pulmonary vascular resistance, or transpulmonary gradient), and echocardiographic (right ventricular systolic function) parameters.

Receiver operating characteristic curves (ROC) were used to determine sensitivity, specificity, and positive and negative predictive values for leptin plasma level at different cut-offs (test variable), with 2-year mortality as the state variable. All patients were followed for at

Fasting venous blood was obtained in the morning of the right heart catheterization. Plasma leptin levels were determined in duplicate by enzyme-linked immunosorbent assay (ELISA; R&D Systems catalog # DY398, Minneapolis, MN, USA). The mean intra-assay variation (standard deviation [SD]) was 3% (4%). Laboratory personnel were blinded to patient characteristics and outcomes.

In 53 patients, brain natriuretic peptide (BNP) was also determined in the same blood sample.

Statistics

Continuous variables were summarized using mean and standard deviation. We compared two-group numerical variables by mean difference, and 95% confidence interval by bootstrapping (2,000 repetitions). We compared categorical variables with Pearson’s chi-square test. We Pulmonary Circulation | April-June 2012 | Vol 2 | No 2

Figure 1: Scatterplot of leptin level and BMI. Both axes in the plot are logged. Restrictive cubic spline (black, solid) and Lowess (gray, dash) lines are displayed.

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least 2 years. All the P values were reported as two tailed.

A P value of <0.05 was prespecified as indicative of statistical significance. The statistical analyses were performed using the statistical package SPSS, Version 20 (IBM; Armonk, N.Y., USA), and R version 2.13.0 (The R Foundation for Statistical Computing).[18]

Results

Baseline data

A total of 67 patients with pulmonary arterial hypertension and 29 healthy controls were studied. All patients had a diagnosis of PAH by right heart catheterization.[19] Of patients with PAH, 35 (52%), 6 (9%), and 26 (39%) had idiopathic, heritable, or PAH associated with a variety of conditions, respectively. Conditions associated with PAH were connective tissue disease (n=19; scleroderma: 10; systemic lupus erythematosus: 5; mixed tissue connective disease: 3; dermatomyositis: 1), congenital heart disease (n=4), drug-induced (n=1), hereditary hemorrhagic telangiectasia (n=1), and portal hypertension (n=1).

Seventy-six percent of the patients (n=51) were receiving PAH-targeted treatment at the time of the blood sample (monotherapy in 39%, dual therapy in 41%, and triple therapy in 20%). New York Heart Association (NYHA) functional class was I, II, III, and IV in 3%, 33%, 52%, and 12% of the patients, respectively. Characteristics of the patients and control groups are shown in Table 1.

Plasma leptin determination

Plasma leptin concentration was higher in patients with PAH (median of 15.3 with an interquartile range [IQR] of 7.9–26 µg/l) than in controls (median 9.7 [IQR: 7.7–25.7 µg/l]), with a mean difference of 9.8 (95% CI: 2.6–18) µ/l (Table 1). Leptin was directly associated with BMI with an R of 0.65 (P<0.001), an association that was seen in controls (R=0.41, P=0.03) as well as patients with PAH (R=0.65, P<0.001). The association between leptin and BMI in PAH patients followed a nonlinear relationship best explained by the restrictive cubic spline model (P for nonlinearity=0.005).

No difference was observed in plasma leptin concentration between PAH patients and controls when adjusted by age, gender, BMI, and smoking status (OR: 0.97; 95% CI: 0.93– 1.01). Similarly, a nonsignificant difference was observed between the leptin/BMI ratio in PAH (0.54 [IQR: 0.28–0.83]) and controls (0.43 [IQR: 0.29–0.79]) with a mean difference of 0.11 (95% CI: -0.09 to +0.30).

The leptin/BMI ratio was not associated with gender, smoking status, NYHA functional class, cause of PAH (idiopathic/heritable vs. associated with other diseases), heart rate, or systemic blood pressure (data not shown).

216

Table 1: Baseline characteristics of PAH patients and control groups Age (years) mean±SD Gender (female)% Race (white)% BMI (kg/m2) mean±SD Smoker Current% Former% Never% Leptin (µg/l) median (IQR) Leptin/BMI (µg*m2/kg*l) median (IQR)

Control (n=29)

PAH (n=67)

P

34.9±6

47.2±14

<0.001

65.5

88.1

0.009

62

79.1

0.08

25.9±3.3

31.4±8.4

<0.001

4 17 79 9.7 (7.7–21.7) 0.43 (0.29–0.79)

13 33 54 15.3 (7.9–26) 0.54 (0.28– 0.83)

0.05 * ¶

*mean difference of 9.8 (95% CI: 2.6–18) and ¶mean difference of 0.11 (95% CI: -0.09 to + 0.30)

BNP measurements did not show a significant association with leptin levels or leptin/BMI ratio (P=0.63 and 0.29, respectively). However, the leptin/BMI ratio was lower in patients receiving IV prostanoids versus PH-targeted oral therapies when adjusted by gender, age, and smoking status (adjusted mean difference: -0.37 [95% CI: -0.66 to -0.08]).

Comparison of leptin with 6-minute walk distance Fifty-one patients completed a 6-Minute Walk Distance within a month of the plasma leptin measurement. The Mean±SD distance walk was 376±129 m or 64.8±19% of predicted.[20] The distance walked in meters or percentage of predicted was not associated with leptin/BMI when adjusted by age, gender, and smoking status (P=0.8 and 0.33, respectively).

Comparison of leptin with echocardiographic parameters All but six patients (n=61) had an echocardiogram performed within two months of the plasma leptin measurement. The Mean±SD left ventricular ejection fraction was 55±6% with right atrial area of 24.5±8 cm2.

The right ventricle (RV) was dilated and dysfunctional in 56 patients (91.8%). Right ventricular dysfunction was normal, mild, moderate, moderate to severe, and severe in 5 (8%), 9 (15%), 17 (28%), 18 (29%), and 12 (20%) of the patients, respectively. The tricuspid jet velocity was 4±0.6 m/s with an estimated right ventricular systolic pressure (RVSP) of 73±20 mmHg. Leptin was not associated with tricuspid jet velocity or estimated RVSP when adjusted by age, gender, BMI, and smoking status (P=0.11 and 0.16, respectively).

The degree of RV dysfunction was not associated with leptin level when adjusted by the same covariates (P=0.78).

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Comparison of leptin with right heart catheterization parameters In PAH patients, means±SD of measured parameters were as follows: right atrial pressure 11±7 mmHg; mean pulmonary artery pressure 52±13 mmHg; pulmonary artery occlusion pressure (PAOP) 11±5 mmHg; CO 4.3±1.6 l/m; and pulmonary vascular resistance (PVR) 11.4±8 Wood Units. Mixed venous oxygen saturation was 63±9%. Right atrial pressure, mean pulmonary artery pressure, PAOP, CO, PVR, and mixed venous oxygen saturation did not correlate with plasma leptin levels when adjusted by age, gender, BMI, and smoking history (P=0.3, 0.3, 0.09, 0.8, 0.9, and 0.4, respectively).

Measures of diagnostic accuracy

At 2 years, the mortality in PAH patients was 24%. The leptin/BMI ratio was higher in alive (0.69 [IQR: 0.07–2.1]) versus deceased (0.28 [IQR: 0.06–0.67]) patients with a mean (95% CI) difference of 0.4 (0.21–0.58). The receiver operating characteristic curve using the leptin/BMI ratio as the test variable and 2-year mortality as the state variable showed an area under the curve of 0.74 (95% CI: 0.62–0.86; Fig. 2 and Table 2).

Table 2: Leptin/BMI diagnostic accuracy Leptin/ BMI* (less or equal to) 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9

Sensitivity (%)

Specificity (%)

PPV (%)

NPV (%)

19 25 56 63 69 94 94 94 100

92 84 78 72 64 54 46 34 26

43 33 45 42 38 40 36 31 30

78 78 85 86 87 96 96 94 100

*Leptin/BMI values are shown as µg * m2/kg * l. PPV: positive predicative value; NPV: negative predicative value

(A) Pulmonary Circulation | April-June 2012 | Vol 2 | No 2

A leptin/BMI ratio of ≤ 0.6 had a high sensitivity (94%) and negative predictive value (96%) for predicting death of any cause at 2 years, with relatively low sensitivity (54%) and positive predictive ability (40%; Table 2). The positive and negative likelihood ratios of this cut-off were 2.04 and 0.12, respectively.

Survival analysis

All control patients were alive at the end of the study. A total of five patients with PAH were transplanted and censored for the analysis. Of the patients with PAH, 23.9% (n=16) died during a mean follow-up of 4.6 years. Causes of death were PAH/RV failure (n=6), respiratory failure (pneumonia [n=1], pulmonary embolism [n=1]), septic shock (n=3), and unknown (n=5). Overall, 1-, 2-, 3-, and 5-year survival was 85%, 76%, 72.9%, and 61.4%, respectively.

When survival was adjusted by age, gender, BMI, and smoking status, a plasma level leptin <15 µg/l was associated with higher mortality, with an adjusted hazard ratio (HR) of 3.8 (95% CI: 1.3–11.2), P=0.016. When leptin was added to the model as a continuous variable, the adjusted HR was 0.96 (95% CI: 0.93-0.99, P=0.024), suggesting that higher levels of leptin appear to be protective in PAH patients.

When we excluded patients that died during the first year to reduce the possibility of reverse causality (i.e., presence of comorbidities leads to low leptin levels and higher mortality[21]), our results were unchanged (HR of 3.8 [95% CI: 1–14.7], P=0.05).

Patients with a leptin/BMI ratio <0.5 µg * m2/kg * l had worse survival with log rank test (P=0.046; Fig. 3, panel A) and Cox regression analysis HR: 3.9, P=0.006 (adjusted by age, gender, and smoking status [95%CI: 1.5 – 10.2; Fig. 3, panel B). Leptin/BMI >0.5 µg * m2 / kg * l remained a significant predictor of survival even after adding to the

(B)

Figure 2: ROC curve of leptin/BMI and dot chart of log leptin/BMI with mortality at 2 years as the classification variable.

(A) leptin/BMI measurements are in µg * m2 / kg * l. Area under the curve: 0.74 (95% CI: 0.62-0.86). (B) Dot chart of log leptin/BMI with line placed at a leptin/ BMI ratio of 0.6. All but one of PAH patients who died at 2 years had a leptin/ BMI ratio <0.6.

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Tonelli et al.: Leptin in pulmonary hypertension

(A)

Cox model the etiology of PAH (idiopathic/heritable vs.

PAH associated with other diseases), NYHA functional class, hemodynamic (RA pressure, PVR, and CO), and echocardiographic (RV function) variables (HR of 5.1 [95% CI: 1.5–17.4], P=0.01).

Discussion

In the present study, we demonstrated that a low plasma leptin concentration (adjusted for BMI and smoking status) is associated with worse survival in PAH patients, independent of other clinical, echocardiographic, or hemodynamic data. In addition, patients on intravenous prostanoids had a lower leptin/BMI ratio than patients only on oral PH-specific treatment.

The plasma level of leptin is proportional to body fat mass in humans and mice,[16-17] a finding that held true in our cohort of patients with PAH. Although PAH patients had higher plasma leptin levels than healthy controls, this difference disappeared when adjusting for BMI, since in our cohort PAH patients had higher BMI than healthy controls.

Mantzoros et al.[15] found that smoking was negatively and independently associated with leptin concentration in healthy men. However, we were not able to find an association between smoking status and plasma leptin levels either in controls or PAH patients. Other factors that inhibit leptin secretion include low energy states with decreased fat stores, fasting, catecholamines, androgens, PPAR-gamma agonists, inflammatory cytokines, and exposure to cool temperature.[22] In our study, all patients were observed at room temperature at time of the blood sample and were not receiving cathecholamines, androgens, or thiazolidinediones.

While we found a lower ratio of leptin/BMI in patients on IV prostanoids versus oral PH-specific therapy as marker of PH severity, we were unable to find an association between 218

(B)

Figure 3: Survival analysis. Kaplan– Meier survival analysis with log-rank test comparing the ratio of leptin/BMI <0.5 and ≥ 0.5 µg * m2 / kg * l (A) and Cox regression analysis using the same cut-off of leptin/BMI adjusted for age, gender, BMI, and smoking status (B).

leptin or leptin/BMI and BNP measurements, 6-Minute Walk Distance, echocardiographic, or hemodynamic parameters.

Similarly, Maruna et al.[23] found no association between preoperative plasma levels of leptin and hemodynamic parameters in patients with chronic thromboembolic pulmonary hypertension.

In the present study, low plasma leptin levels were associated with higher mortality in patients with PAH when adjusted for age, gender, BMI, and smoking status.

Similarly, a simple-to-use ratio of leptin/BMI was inversely associated with mortality, raising the possibility that leptin may have a protective effect in PAH in pathways that are independent of obesity.[22] Supporting our findings, low circulating leptin levels have been associated with increased cardiovascular mortality independent of obesity in patients with chronic stable coronary artery disease,[10] diabetes,[11] or chronic kidney disease.[12] However, other studies have shown a direct and independent association between leptin levels and atherosclerosis, hypertension, or thrombosis, underscoring the need to better elucidate the complex relationship between leptin and vascular disease.[24-28]

One potential explanation for the survival advantage observed in patients with higher leptin (adjusted by BMI) may be related to its known cardioprotective role.

Leptin has been shown to have a cardioprotective function since it reduces cardiac apoptosis via downregulation of caspase-3 and activation of signal transducer and activator of transcription (STAT)-3 responsive antiapoptotic genes (antiapoptotic bcl-2 and surviving gene).[29] Experimentally induced myocardial infarction in ob/ob mice (leptin deficient) was associated with blunted antiapoptotic (STAT- 3) response which led to a significant increase in morbidity and mortality which was reverted by leptin treatment.[30]

We observed that a leptin/BMI ratio cut-off of 0.6 has a high negative predictive value (96%) for death of any cause at 2 years. Leptin/BMI ratio >0.6 was present in 43% of the Pulmonary Circulation | April-June 2012 | Vol 2 | No 2

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patients and only one of them died during the 2-year followup period compared with 39.5% in the group with leptin/ BMI index ≤ 0.6. These results support further investigation to determine whether leptin/BMI can be used to identify PAH patients who will have a more favorable prognosis.

In conclusion, in PAH, lower leptin levels, when adjusted to BMI, are associated with an increased, overall mortality. The leptin/BMI ratio cut-off of 0.6 has high negative predictive value for mortality at 2 years.

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Mantzoros CS, Liolios AD, Tritos NA, Kaklamani VG, Doulgerakis DE, Griveas I, et al. Circulating insulin concentrations, smoking, and alcohol intake are important independent predictors of leptin in young healthy men. Obes Res 1998;6:179-86.

Considine RV, Sinha MK, Heiman ML, Kriauciunas A, Stephens TW, Nyce MR, et al. Serum immunoreactive-leptin concentrations in normalweight and obese humans. N Engl J Med 1996;334:292-5.

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Kelesidis T, Kelesidis I, Chou S, Mantzoros CS. Narrative review: The role of leptin in human physiology: Emerging clinical applications. Ann Intern Med 2010;152:93-100.

Maruna P, Lindner J, Kubzova KM. Leptin and soluble leptin receptor changes after pulmonary endarterectomy: Relations to cortisol and cytokine network. Physiol Res 2009;58:569-76.

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McGaffin KR, Zou B, McTiernan CF, O’Donnell CP. Leptin attenuates cardiac apoptosis after chronic ischaemic injury. Cardiovasc Res 2009;83:313-24.

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Source of Support: Dr. Adriano Tonelli is supported by CTSA KL2 Grant # RR024990 (A.R.T.) from the National Center for Research Resources (NCRR). Dr. Metin Aytekin is supported by 0826095H from American Heart Association (AHA). Dr. Feldstein is supported by NIH Grants (DK076852) and (DK082451). Dr. Raed Dweik is supported by the following grants: HL081064, HL107147, HL095181, and RR026231 from the National Institutes of Health (NIH), and BRCP 08-049 Third Frontier Program grant from the Ohio Department of Development (ODOD)., Conflict of Interest: None declared.

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Research A r t i cl e

Mast cell number, phenotype, and function in human pulmonary arterial hypertension Samar Farha1, Jacqueline Sharp2, Kewal Asosingh2, Margaret Park3, Suzy A. A. Comhair2, W. H. Wilson Tang3, Jim Thomas3, Carol Farver4, Fred Hsieh1,2, James E. Loyd5, and Serpil C. Erzurum1,2 1 Respiratory Institute, 2Department of Pathobiology, Lerner Research Institute, 3Heart and Vascular Institute, 4Anatomic Pathology Cleveland Clinic, Cleveland, Ohio, USA, and 5Allergy, Pulmonary & Critical Care, Vanderbilt University, Nashville, Tennessee, USA

Abstract A proliferation of mast cells around the small pulmonary blood vessels and the alveolar septae has been noted in models of pulmonary hypertension, and in plexiform lesions of pulmonary arterial hypertension (PAH) in patients. Here, we hypothesize that total mast cell numbers and activation are increased in PAH and that they contribute to vascular remodeling through cellular and soluble proangiogenic effectors. To test this, blood and urine were collected from patients with PAH (N=44), asthma (N=18) and healthy controls (N=29) to quantitate biomarkers of total body mast cell numbers and activation (total and mature tryptase, N-methyl histamine, leukotriene LTE4 and prostaglandin PGD-M). Serum total tryptase was higher in PAH than that in controls suggesting greater numbers of mast cells, but indicators of mast cell activation (mature tryptase, LTE4 and PGD-M) were similar among PAH, asthma, and controls. Immunohistochemistry of lung tissues identified mast cells as primarily perivascular and connective tissue chymase+ type in PAH, rather than mucosal phenotype. Intervention with mast cell inhibitors cromolyn and fexofenadine was performed in 9 patients for 12 weeks to identify the influence of mast cell products on the pathologic proangiogenic environment. Treatment decreased total tryptase and LTE-4 levels over time of treatment. This occurred in parallel to a drop in vascular endothelial growth factor (VEGF) and circulating proangiogenic CD34+CD133+ progenitor cells, which suggests that mast cells may promote vascular remodeling and dysfunction. In support of this, levels of exhaled nitric oxide, a vasodilator that is generally low in PAH, increased at the end of the 12-week mast cell blockade and antihistamine. These results suggest that mast cells might contribute to the pulmonary vascular pathologic processes underlying PAH. More studies are needed to confirm their potential contribution to the disease.

Key Words: mast cells, pulmonary arterial hypertension, tryptase, proangiogenic progenitor cells

Pulmonary arterial hypertension (PAH) is a severe condition clinically defined by elevated mean pulmonary artery pressure. Endothelial and smooth muscle proliferation and dysfunction were the pathological defining features of the panvascular arteriopathic remodeling that ultimately lead to right heart failure and death.[1-3] However, treatment strategies have primarily focused on vasodilator drugs which have limited effect on long-term survival and little or no effect on the vascular remodeling.[4] Goals of new strategies are to halt or reverse the vascular remodeling; thus, investigation of pathways that may fuel the proliferative angiopathy is important for design of novel therapies.[5] In this context, mast cells play a central role in angiogenesis. [6,7] Although derived from bone marrow progenitors (as are all hematopoietic cells), differentiation of mast cells occurs Address correspondence to: Dr. Samar Farha Cleveland Clinic 9500 Euclid Avenue, NC22, Cleveland, OH 44195, USA E-mail: farhas@ccf.org 220

only after recruitment of the circulating progenitors into local tissues where the environment dictates the final long-lived phenotype of the cell. Mast cells are mainly localized in mucosa and connective tissues exposed to the outside world. They are critical for activation of innate and adaptive immunity. [8] However, evidence from cancer biology studies now reveal that mast cells are master regulators of angiogenesis through the production of vascular endothelial growth factor (VEGF) and release of proangiogenic proteases.[6,7] In classic pathologic descriptions written over a century ago, Heath et al. identified mast cells as “plentiful” within plexiform lesions of idiopathic PAH Access this article online

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Website: www.pulmonarycirculation.org DOI: 10.4103/2045-8932.97609 How to cite this article: Farha S, Sharp J, Asosingh K, Park M, Comhair SA, Wilson Tang WH et al. Mast cell number, phenotype, and function in human pulmonary arterial hypertension. Pulm Circ 2012;2:220-8.

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Farha et al.: Mast cells in PAH

(IPAH); particularly, he noted their presence in the early cellular lesions.[9] Other studies have confirmed the greater numbers of mast cells in experimental models of pulmonary hypertension.[10-12] Despite the known presence of abundant mast cells in the pulmonary vascular lesions, their role in the mechanisms of the pulmonary vascular disease is unclear.

In model systems, hypoxia-related vasoconstriction and remodeling of the pulmonary artery wall were ascribed in part to release of factors from mast cells, which degranulate under hypoxic conditions.[13] This led to the speculation that mast cell secretion of a multitude of factors which affect vascular tone or activate the renin-angiotensin system likely contribute to the pathogenesis of pulmonary hypertension.

Classically, mast cells are identified in tissues by their unique positivity for tryptase, a serine proteinase, which has been identified as one of the important proangiogenic factors released by peritumoral mast cells infiltrating cancers that may contribute to neoangiogenesis of malignancies.[14,15] The immature form of tryptase is constitutively released from mast cells, and thus serum total tryptase levels reflect mast cell numbers in the body. When activated, mature tryptase is discharged from mast cells, and this has been used as a marker of mucosal mast cell activation.

Interestingly, reports suggest that mast cells in PAH are of the tryptase+/chymase+ “connective tissue” phenotype.[16,17] The serine protease chymase, independent of angiotensin converting enzymes, can lead to localized production of angiotensin II, and the activation of endothelins and matrix metalloproteases, which collectively govern vasomotor tone and neovascularization.[7,18,19] Thus, the perivascular lesional distribution of mast cells in the PAH lung strongly implies a pathophysiological role for mast cell products in driving the lung-specific vascular hypertension and proliferative vascular remodeling. Indeed, in murine models of pulmonary hypertension associated with left heart disease and in monocrotaline-induced rodent pulmonary hypertension, treatment with a mast cell stabilizer or use of mast cell deficient rats attenuated vascular remodeling.[20] Likewise, the early use of mast cell stabilizer cromolyn, or antagonists of the chemokine receptors involved in recruitment of mast cell progenitors, reduce the development of vascular remodeling and right ventricular hypertrophy in hypoxiainduced rodent pulmonary hypertension.[21,22] The contribution of mast cells in the pathogenesis of human PAH is unknown, but studies identify mast cell progenitors in the circulation of IPAH patients, as well as greater numbers of progenitors and mast cells in the pulmonary arterial lesions.[23] Here, we hypothesize that mast cells contribute to PAH pathogenesis through production of secreted factors which dictate pulmonary hypertension and vascular remodeling. To test this, mast cell numbers, phenotype, and activation in PAH were compared to healthy controls and individuals with asthma, a control disease Pulmonary Circulation | April-June 2012 | Vol 2 | No 2

typified by high numbers and activation of mast cells. To investigate the potential contribution to the pathogenesis of PAH, mast cell degranulation products that promote angiogenesis and the mobilization and recruitment of proangiogenic myeloid progenitors were determined before and after treatment of patients with mast cell blockade cromolyn and the H1 histamine antagonist fexofenadine.

Materials and Methods

Blood and urine samples from individuals with PAH, asthma, and healthy controls were collected to measure mast cell products. Clinical data were collected from tests performed as part of standard of care of patients. A mechanistic investigation of cromolyn and fexofenadine to block mast cells activation was performed in a subgroup of PAH patients to determine whether mast cells contribute to the high levels of vasoactive and proangiogenic factors which contribute to the vascular remodeling of PAH. Inclusion criteria included age of at least 18 years, a diagnosis of pulmonary arterial hypertension (PAH), and stability on current PAH medications. Exclusion criteria included participation in other studies, hepatic insufficiency (transaminase levels >4-fold the upper limit of normal, bilirubin >2-fold the upper limit for normal), renal insufficiency (creatinine level, >2.0 mg/dl), pregnancy, breast feeding, lack of use of safe contraception, acute heart failure, known allergy to any of the study drugs, or history of drug or alcohol abuse within the last 12 months. None of the patients enrolled in the mechanistic study had a diagnosis of asthma.

All patients provided written informed consent under an IRB-approved protocol.

Mast cell blockade-directed therapy

Individuals received treatment for 12 weeks with a mast cell stabilizer, cromolyn 800 mcg administered by a multidose inhaler with spacer by two puffs, four times a day and an antihistamine, fexofenadine, 180 mg daily. Patients were evaluated at baseline (time 0) four weeks prior to medications, and after Weeks 4, 8, and 12 of treatment.

Medications were brought to each visit to check for adherence and dosage. Patients were asked at each visit on accurate use of the inhaler and compliance. Spirometry, lung diffusing capacity, brain natriuretic peptide (BNP), echocardiogram, and 6-minute walk distance were evaluated at each study visit. Blood and urine samples were obtained at each visit for measures of vasoactive and angiogenic factors produced by mast cells, and circulating proangiogenic progenitor cells.

Lung function, diffusing capacity for carbon monoxide and exhaled nitric oxide

Spirometry was performed on each study participant according to American Thoracic Society (ATS) Guidelines using a Viasys Master Screen spirometer (San Diego, Calif., 221

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USA). The single breath carbon monoxide diffusing capacity (DLCO) was performed weekly using a Viasys Master Screen analyzer (San Diago, Calif., USA), and measurements were performed at the same time of day on each visit to minimize the effect of diurnal variations.[24] The single-breath DLCO method by ATS standards was performed in duplicate. DLCO was not adjusted for hemoglobin or carboxyhemoglobin.

Single-breath on-line measurement of fractional nitric oxide (NO) concentration in expired breath (FENO) was measured using the NIOX (Aerocrine, N.Y., USA).[25] All analyzers were calibrated daily and weekly controls were completed to ensure accuracy.

Assessment of mast cell activation by levels of degranulation products: Serum total and mature tryptase and urinary N-methyl histamine, LTE4, and PGD-M

Tryptase, total and mature, was measured in serum using fluorescent enzyme immunoassay. [26] N-methyl histamine (NMH) was extracted from urine using solidphase extraction. The elute is analyzed using liquid chromatography-tandem mass spectrometry and quantified using a stable isotope labeled internal standard. [27] LTE4 and PGD-M were measured in urine. After urine acidification, an extraction step using an Empore C-18 solid-phase extraction column (standard density, 6 ml capacity, 3M, St. Paul, Minn., USA) was completed. The eluate was evaporated under a continuous stream of dry nitrogen, was then dissolved in 100 μl methanol, and was finally filtered using a 0.2 μm Spin-X filter (Corning, Corning, N.Y., USA). LTE4 was measured using ultra pressure liquid chromatography/mass spectrometry. PGD-M levels were measured by mass spectrometry as described previously.[28]

Evaluation of enzyme-linked immunosorbent assay for proangiogenic factors

Erythropoietin (Epo), hepatocyte growth factor (HGF), stem cell factor (SCF), and VEGF were measured in plasma using quantikine enzyme-linked immunosorbent assay (ELISA) (R & D system, Minn., USA).

Mast cells numbers and types in lungs of PAH patients

Lung tissues were obtained from explanted PAH and failed donor lungs for investigation of mast cells. Formalin-fixed paraffin embedded tissue sections were stained with human monoclonal tryptase (Promega G3361, dilution 1/1000) and chymase (Abcam, ab2377, dilution 1/100). Pictures were taken of five random fields from each tissue section at 10× using a Leica DM5000B microscope, equipped with a QImaging Retiga SRY digital camera and using Image Pro v.6.2 with Oasis Turboscan module. To quantify the mast 222

cells, the number of positive cells was counted on each picture in a blinded fashion and the average/field was used as a total number.

Echocardiogram

Two-dimensional echocardiogram and Doppler examinations were performed by an experienced sonographer. Interventricular septal (IVS) thickness in enddiastole, left ventricular end-diastolic dimension (LVEDD), left ventricular end-systolic dimension (LVESD), and posterior wall thickness in diastole were measured from the 2D parasternal long-axis image following American Society of Echocardiography (ASE) guidelines. Left ventricular (LV) mass was determined from 2D measurements using the following formula: LV Mass = 1.04((IVS + PW + LVEDD) 3 - LVEDD3) – 13.6g (Devereaux Regression)

LV ejection fraction was determined by visual assessment, and/or apical biplane volumes. LV end-diastolic and endsystolic volumes were calculated from the apical 4- and 2-chamber views using the modified Simpson method. LV fractional shortening was determined from parasternal 2D analysis as (LVEDD-LVESD/LVEDD × 100). Right ventricular (RV) end-diastolic and end-systolic areas were measured in the apical 4-chamber view by tracing the endocardial border of the RV and the tricuspid annular plane. The RV fractional area change was calculated as follows: RV end-diastolic area minus RV end-systolic area divided by RV end-diastolic area × 100. Right atrial volume was measured in the apical 4-chamber view by using the single-plane area–length method. The peak pulmonary artery systolic pressure (PASP) was estimated from the systolic pressure gradient between the RV and the right atrium by the peak continuous-wave Doppler velocity of the TR jet using the modified Bernoulli equation plus estimated right atrial pressure (RAP). RAP was estimated from the subcostal window approach measuring changes in inferior vena caval size and collapsibility as determined by the respiratory sniff test following ASE guidelines. Echo-Doppler was used to estimate the pulmonary vascular resistance (PVR). The highest Doppler continuous wave tricuspid valve peak velocity jet obtained from multiple views (parasternal long axis, parasternal short axis, apical 4-chamber, subcostal, and apical off-axis imaging) was determined as the maximum tricuspid regurgitant velocity (TRV). The pulsed wave Doppler sample was placed in the right ventricular outflow tract (RVOT) at the level of the aortic valve in the parasternal short-axis view just below the pulmonic valve so that pulmonic valve closure could be identified. The Doppler spectrum was traced to determine the time velocity integral of the RVOT. PVR was estimated as follows: PVR (Wood units) = TRVMAX (m/s) / RVOTTVI (cm) × 10 +0.16. Tricuspid annular plane systolic excursion was obtained from the apical 4-chamber RV focused view with M-mode echocardiography across the TV annulus, measuring the distance of longitudinal annular movement Pulmonary Circulation | April-June 2012 | Vol 2 | No 2

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from end diastole to end systole toward the apex. With the exception of the TR continuous wave maximum velocity, all echo parameters were measured three times and reported as an average.

Flow cytometry evaluation of proangiogenic progenitor cells

Mononuclear cells (2×10 ) isolated from peripheral blood and bone marrow were labeled with antihuman CD34FITC (Becton Dickinson, N.J., USA) and antihuman CD133PE (Miltenyi Biotec, Auburn, Calif., USA) monoclonal antibodies to quantify CD34+CD133+ cells, as described previously. [29] Nonspecific antibody binding was analyzed in parallel with isotype-matched irrelevant antibodies.

Following incubation with antibodies, cell suspensions were washed with PBS/1%BSA/0.02%sodium azide and suspended in FACS flow (Becton Dickinson, N.J., USA). The FACScan flow cytometer (Becton Dickinson, N.J., USA) was used to count 0.5×106 events. Data of at least 0.5×106 events were collected, stored as listmode files, and analyzed using Cell-Quest 3.3 Software (Becton Dickinson, N.J., USA).

6

Statistical analysis

Descriptive measures for quantitative variables consist of means with appropriately derived standard errors in the form “mean±SE.” Comparisons of PAH, asthmatic, and healthy subjects were performed using ANOVA or t-test when two means were compared. When ANOVA was significant, Tukey was performed for pairwise comparison.

Spearman correlation coefficients were used to describe relationships among pairs of quantitative variables in Table 1: Characteristics of all PAH population

IPAH FPAH APAH ANOVA P-value

a manner free of the normality assumption. For the mechanistic intervention, outcomes analyzed were all quantitative in nature. The changes in outcomes between visits were assessed using paired t-tests.

Results

Study population Individuals with PAH (N=44), controls (N=31), and asthma (N=18) provided blood and urine for measurement of mast cell biomarkers in relation to clinical disease. PAH subjects were older (age [years]: PAH [N=44] 45±2; controls [N=31] 36±2; asthma [N=18] 37±3, ANOVA P=0.004). There were more females in the control and PAH groups compared to the asthma group (gender [F/M]: PAH [N=44] 33/11; controls [N=31] 26/5; asthma [N=18] 11/7, ANOVA P=0.03; Table 1). Due to sample limitations, not all assays could be performed in all subjects. The numbers of individuals evaluated for each assay are provided in the text. Separate from the biologic samples of blood and urine, tissue sections of explanted lungs from PAH patients (N=18) undergoing transplant or donor lungs (N=4) not used in transplantation were available for quantification of pulmonary mast cells numbers and phenotype. Nine PAH patients participated in the mast cell blockade study over 16 weeks (Table 2). All participants completed the 16-week study.

Mast cells in pulmonary hypertension

Lung sections were stained for tryptase and chymase to localize, characterize, and quantitate the mast cells in the lungs. There was a higher number of tryptase+ mast cells

Age

Gender (F/M)

BMPR2 mutation +/-

BNP (pg/ml)

RVSP (mmHg)

ERA +/-

Prostacyclin +/-

Sildenafil +/-

41±3 42±5 56±4 0.02

18/4 7/3 8/4 0.6

3/19 2/8 0/9 0.4

107±58 9±3 111±29 0.7

69±5 65±8 79±10 0.5

13/32 6/14 7/17 0.8

19/25 4/16 4/20 0.2

8/14 6/4 6/6 0.4

BMPR2: bone morphogenetic protein receptor2; F/M: female/male; RVSP: right ventricular systolic pressure; ERA: endothelin receptor antagonist; IPAH, FPAH, APAH: idiopathic, familial, associated pulmonary arterial hypertension; BNP: brain natriuretic peptide

Table 2: Mast cell-blockade study sample characteristics Patient 1 2 3 4 5 6 7 8 9

Age 30 27 40 63 29 41 46 51 56

Gender (F/M)

Classification

F F M F F F M M F

IPAH IPAH IPAH APAH IPAH IPAH APAH APAH FPAH

BMPR2 mutation+/-

BNP (pg/ml)

6-minute walk (ft)

RVSP (mmHg)

ERA +/-

Prostacyclin +/-

Sildenafil +/-

unknown + -

9 91 18 29 5 9 34 214 333

2040 1460 1680 800 1440 2170 1850 1855 1349

39 90 85 85 59 79 No TR 113 118

+ + + + -

+ + + + + +

+ + + + -

BMPR2: bone morphogenetic protein receptor2; F/M: female/male; RVSP: right ventricular systolic pressure; ERA: endothelin receptor antagonist; IPAH, FPAH, APAH: idiopathic, familial, associated pulmonary arterial hypertension; TR: tricuspid regurgitation; BNP: brain natriuretic peptide

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in lung from patients with PAH compared to controls (mast cells per high power field: PAH [N=12] 56±7; control [N=3] 28±5; t-test P=0.01). As previously shown in human PAH lung[16,17] mast cells were concentrated around vascular lesions and had a tryptase+/chymase+ proteinase phenotype, suggesting a “connective tissue” mast cell phenotype (Fig. 1). In line with the histological findings, serum total tryptase levels were higher in PAH patients compared to controls (total tryptase [ng/ml]: PAH [N=44] 5.7±0.7; controls [N=29] 3.1±0.4; asthma [N=18] 3.8±0.8; ANOVA P=0.01 and Tukey P<0.05 for PAH compared to controls; (Fig. 2). In subclass analyses, idiopathic PAH (IPAH) patients had higher tryptase levels compared to familial PAH (FPAH) (total tryptase [ng/ml]: IPAH [N=33] 6.3±0.8; FPAH [N=10] 4.1±1.4; P=0.03]. There was a minor but significant correlation between total serum tryptase levels and BNP in PAH patients (Spearman R=0.5, P=0.006; Fig. 2) In contrast, levels of mature tryptase were low in all subjects with no difference between PAH and controls (mature tryptase <1 for all samples). Similarly, urinary N-methyl histamine was not different among PAH, controls, and asthma (urinary N-methyl histamine [ug/g creatinine]: PAH [N=9] 108±26; controls [N=5] 88±8; asthma [N=3] 98±26; ANOVA P=0.8 and all tukey P>0.05]. Urinary LTE4 and PGD-M levels, which are rapidly produced by IgE-sensitized mucosal mast cells activated via specific allergen(s), were also similar among the groups (LTE4 [pg/mg creatinine]: PAH [N=7] 42±7; asthma [N=16] 131±65; controls [N=15] 60±9; ANOVA P=0.4; PGD-M [ng/mg creatinine]: PAH [N=7] 2.8±0.3; asthma [N=16] 2.2±0.3; controls [N=14] 2.4±0.3; ANOVA P=0.5]. Likewise, IgE levels among PAH, asthmatic and control individuals were similar (IgE [IU/ml]: PAH [N=40] 108±43, asthma [N=18] 147±53; controls [N=29] 50±17; ANOVA P=0.3 and all Tukey P>0.05]. Altogether, these findings indicate that PAH lungs contain greater numbers of the nonallergic, connective tissue phenotype of mast cells, but not activated mucosal mast cells.

Mast cell blockade in PAH

Given the elevated numbers of mast cells in PAH lungs and the relationship of tryptase to BNP, a subgroup of PAH patients was placed on mast cell stabilizer cromolyn and antihistamine fexofenadine to evaluate effects on the PAH proangiogenic milieu. Mature tryptase did not change over time; however, total serum tryptase dropped from baseline to Weeks 4 and 12 (tryptase [ng/ml]: baseline Table 3: Proangiogenic factors with mast cell blockade

5.2±0.5; Week 4 4.3±0.4; Week 12 4.2±0.4; all P<0.05).

Likewise, PGD-M did not change significantly, however LTE4 appeared to be lower with treatment at Week 8 (LTE4 [pg/ mg creatinine]: baseline 63±17; Week 8 41±11; P=0.04; Fig. 3). To assess the potential contribution of mast cells for angiogenesis, we measured circulating angiogenic factors, including VEGF, HGF, EPO, SCF. VEGF, a potent proangiogenic factor secreted by mast cells, decreased early after four weeks of treatment (VEGF [pg/ m l]: baseline 400±117; Week 4 335±112; P=0.03; Fig. 3). Other angiogenic factors did not vary over the time of the study (all P>0.05; Table 3). VEGF dictates angiogenesis in part by induction of myeloid progenitor cell mobilization into the circulation and subsequent recruitment to local vascular bed. Consistent with the temporal drop of VEGF at Week 4, circulating CD34 +CD133 + proangiogenic progenitor myeloid cells consequently decreased by Week 4 of treatment (CD34+CD133+ cells (%): baseline 0.11±0.03; Week 4 0.08±0.03; week 12 0.07±0.02, all P<0.05; Fig. 3). Furthermore, numbers of CD34+CD133+ cells were related to the levels of serum VEGF across the time of the study (Spearman R=0.4, P=0.02) but not with the other angiogenic factors (all P>0.05).

Exhaled NO was evaluated over the time of mast cell blockade to detect potential positive vascular functional effects with treatment. NO increased significantly at the end of the study from Weeks 8 to 12 of therapy (NO [ppb]: Week 8 15±2; Week 12 17±2; P=0.02; Fig. 4). Overall, NO was inversely related to VEGF (Spearman R=-0.6; P<0.0001; Fig. 4) and CD34+CD133+ cells (Spearman R=0.4; P=0.02).

Effects of mast cell blockade on clinical parameters

Although the study was aimed to determine effects of mast cell blockade on proangiogenic factors and cells, clinical evaluation of patients were evaluated for potential benefits and/or adverse effects. Lung functions (FEV1, FVC and FEV1/FVC, DLCO) were similar across the time of the interventional study (all P>0.05); likewise, BNP and 6-Minute Walk Distance did not change (all P>0.1).

The tricuspid annular plane systolic excursion (TAPSE) decreased from baseline to Week 12 of the study (TAPSE [cm]: baseline 2±0.2; Week 12 1.8±0.1; P=0.03) as did left ventricular (LV) mass average (LV mass average: baseline 214±17; Week 12 171±18; P=0.02). Serum total tryptase

Proangiogenic factor

Baseline

Week 4

Week 8

Week 12

Erythropoietin (mIU/ml) Vascular endothelial growth factor (pg/ml) Hepatocyte growth factor (pg/ml) Stem cell factor (pg/ml)

15±2 400±117 898±151 1042±122

20±5 335±97 913±165 1117±113

18±5 365±126 971±221 1108±97

23±8 331±97 928±150 1138±106

All P>0.05 except the drop in VEGF from baseline to week 4 (P=0.03)

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Figure 1: Increased numbers of connective tissue type mast cells in PAH lungs. (A–H) Tryptase and chymase immunostaining of explanted control and PAH lungs. (A–E) Tryptase stained lung tissue sections. (F–H) Chymase stained lung tissue sections. Explanted PAH and failed donor (control) lung paraffin embedded tissue sections were immunostained for tryptase and chymase to identify and characterize mast cell phenotype (brown cells). Mast cell numbers were increased in the lungs of patients with PAH compared to controls (P=0.01) and localized predominantly to perivascular regions as opposed to submucosal regions as in control lungs. Mast cells in PAH lungs were tryptase+ and chymase+ consistent with a connective tissue phenotype as opposed to primarily tryptase+ in control lungs. Panels A–B – Control lungs stained for tryptase: (A) Mast cells are seen in the submucosal regions of the airways of control lungs. (B) Mast cells around a blood vessel in a control lung are less compared to PAH lungs. Panels C–H – PAH lungs: (C–E) Tryptase+ mast cells in PAH are in the perivascular adventitia and increased in number. (F–H) Mast cells in the perivascular regions are chymase+. In panels E–G where plexiform lesions are noted, mast cells are seen within the lesions. Magnification: (1) 5×, (2) 10×, (3) 20×, (4) 40×. Scale bar: 25mm.

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(A)

(B)

Figure 2: (A) Serum total tryptase levels are higher in PAH than controls. Total serum tryptase levels are higher in PAH compared to controls indicating greater total numbers of mast cells in PAH. Box plots indicate median values, upper and lower quartiles. (B) Serum total tryptase levels correlate with brain natriuretic peptide (BNP). Total tryptase levels are related to disease severity assessed by BNP.

Discussion

Figure 3: Mast cell blockade for 12 weeks with cromolyn and fexofenadine.

Mast cell activation and proangiogenic biomarkers were measured at baseline Week 0 (prior to start of therapy). Four weeks later medications were started, and patients evaluated after 4, 8, and 12 weeks of therapy. Total tryptase and urinary LTE4 dropped with mast cell blockade therapy. Proangiogenic CD34+CD133+ myeloid progenitors and VEGF also decreased with mast cell blockade. Asterisk significant changes compared to baseline (all P<0.05).

levels were inversely related to clinical parameters, including the 6-Minute Walk Distance (Spearman R=-0.4; P=0.006) and TAPSE (Spearman R=-0.5; P=0.04).

Similarly, LTE-4 was correlated with disease severity over time (6-Min Walk: Spearman R=-0.5; P=0.004, BNP: Spearman R=0.5; P=0.005 and RAP: Spearman R=0.5; P=0.05; Table 3). In addition, 6-Minute Walk was inversely related to several proangiogenic factors, including Epo, HGF, VEGF, and SCF (Epo: Spearman R=-0.6; P<0.0001; HGF: Spearman R=-0.6; P=0.0001; VEGF: Spearman R=0.5; P=0.004; SCF: Spearman R=-0.6; P=0.006). HGF was also related to right atrial pressure and TAPSE (right atrial pressure: Spearman R=0.7; P=0.005 and TAPSE: Spearman R=-0.6; P=0.03).

226

Although mast cells have long been described as abundant in the lungs of patients with PAH,[9] their contribution to the pathophysiology of the disease is unclear. In this report, mast cells are again shown to be abundantly present in PAH. In addition, the mast cell is identified as connective tissue type that expresses both tryptase and chymase proteases. [16,17] The small mechanistic intervention with cromolyn to block mast cell degranulation and fexofenadine to block mast cell histamine vasoactive effects led to a decrease of total serum tryptase and urinary LTE4 in parallel to a drop in proangiogenic CD34+ CD133+ cells and mast cell secreted proangiogenic VEGF. These findings suggest that mast cells may contribute to the vascular pathophysiology.

Mast cell blockade and antihistamine therapy did not translate into clinical improvement. This lack of clinical improvement is most likely multifactorial, possibly due to limited sample size and/or short duration of therapy.

The mechanistic intervention included nine patients who were treated for only 12 weeks and were not powered to detect clinical improvement. Furthermore, cromolyn is a weak mast cell blocker in humans; a more potent mast cell inhibition might have provided an indication of clinical benefit. C–kit inhibitors which affect mast cells, such as imatininb, are being studied in pulmonary hypertension and have shown promising results.[30] Mast cells secrete vasomotor mediators and factors that promote angiogenesis and vascular remodeling. The patients enrolled in the study had advanced PAH and almost certainly had severe remodeling, which might also explain the lack of clinical response, and indeed even worsening clinical status by some echocardiographic parameters. In fact, our findings suggest worsening cardiac function based on TAPSE. This seems to indicate that mast cells blockade affect the Pulmonary Circulation | April-June 2012 | Vol 2 | No 2

Farha et al.: Mast cells in PAH

(A)

(B)

Figure 4: (A) Nitric oxide increases with mast cell blockade. Exhaled NO measured over the course of the study suggests improved pulmonary vascular health near the end of the 12-week therapy. The asterisk represents significant change from week 8 to week 12 (P<0.05). (B) NO levels are inversely correlated to VEGF. The inverse relationship suggests that the decrease of VEGF, which occurs with mast cell blockade, was associated with increasing levels of NO. Points are derived from all time points in the study.

myocardium independently of its effect on the vasculature.

Banasova et al. used cromolyn in a murine hypoxia model of pulmonary hypertension and found that if given during the very early phase of hypoxia exposure, pulmonary hypertension, and vascular remodeling were reduced;[22] if provided late in the model, beneficial responses were not observed.[22] In another study using a rodent model of flow associated pulmonary hypertension, early treatment with cromolyn attenuated vascular remodeling, shown by reduced pulmonary artery wall thickness, muscularization, and wall/lumen ratio. [31]However, the effects on the pulmonary vascular bed were not associated with positive effects on RV hemodynamics; in fact there was no improvement in pulmonary arterial pressures and RV hypertrophy.[31] Similarly, Gambaryan et al. used a CXCR4 antagonist and a CXCR7 antagonist to inhibit recruitment of progenitors, including mast cell progenitor in a murine model of hypoxia-induced PH.[21] They showed that when the drugs are used early on before development of PH (preventive strategy), they prevented vascular remodeling, PH, and the perivascular accumulation of c-kit+ progenitors.

However, when used to treat established PH, the drugs did not abrogate the vascular remodeling, RV hypertrophy, and increased pulmonary artery pressures.[21] These findings and others[10] suggest that mast cell targeted therapy might work best if early in the course of the disease, and that the primary effect may be attenuation of vascular remodeling and/or vasomotor tone.

In this study, exhaled NO increased at the end of the treatment period. Several studies have shown that NO levels are low in PAH[32,33] and mechanistically related to dysfunctional vascular endothelium.[34-36] Furthermore, treatments that decrease pulmonary artery pressures are associated with increase of exhaled NO.[32,33] Thus, although there was only a slight increase of NO at the end of the study and no significant change in pulmonary artery Pulmonary Circulation | April-June 2012 | Vol 2 | No 2

pressures, the rise in NO suggests a possible vascular effect. Unfortunately, the lack of a control group limits firm conclusions regarding the significance of changes in NO. On the other hand, studies in asthma, which is characterized by high levels of exhaled NO due to airway expression of the inducible NO synthase,[37] demonstrate that NO levels are generally unaffected by cromolyn.[38] Other biological markers changed with therapy, but changes were not consistent. While increase in NO was noted at the end of the study, LTE-4 dropped at Week 8 and VEGF at Week 4. These variable effects could be due to several limitations, including the small sample size, poor bioavailability of drug, and consistency of dose delivery to the lung vascular compartment. Finally, the time of treatment may have been inadequate, i.e., a longer exposure to cromolyn might be required for consistent effects to be apparent.

Overall, our findings support prior reports that point to mast cells in the vascular processes leading to PAH.[9,10,17,20- 23] Further studies are needed to determine if mast cell blockade and/or more potent mast cell targeted therapies early in the course of the disease can impact the angiopathic processes and improve patients’ outcome.

Acknowledgments

We thank B. Savasky and J. Hanson for excellent technical assistance; D. Hatala, Dr. J. Drazba, and Dr. A. J. Peterson in the Lerner Research Institute Digital Imaging Core; C. Shemo and S. O’Bryant in the Lerner Research Institute Flow Cytometry Core for technical advice and excellent assistant with instrument operation; and M. Koo for study coordination. We also thank Dr. M. Aldred for BMPR2 analysis and Drs. R. Dweik, C. Jennings and O. Minai for help in patient recruitment.

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Schwartz LB, Bradford TR, Rouse C, Irani AM, Rasp G, Van der Zwan JK, et al. Development of a new, more sensitive immunoassay for human tryptase: Use in systemic anaphylaxis. J Clin Immunol 1994;14:190-204.

Martens-Lobenhoffer J, Neumann HJ. Determination of 1-methylhistamine and 1-methylimidazoleacetic acid in human urine as a tool for the diagnosis of mastocytosis. J Chromatogr B Biomed Sci Appl 1999;721:135-40.

Awad JA, Morrow JD, Roberts LJ 2nd. Simplification of the mass spectrometric assay for the major urinary metabolite of prostaglandin D2.

J Chromatogr 1993;617:124-8.

Asosingh K, Aldred MA, Vasanji A, Drazba J, Sharp J, Farver C et al.

Circulating angiogenic precursors in idiopathic pulmonary arterial hypertension. Am J Pathol 2008;172:615-27.

Ghofrani HA, Morrell NW, Hoeper MM, Olschewski H, Peacock AJ, Barst RJ, et al. Imatinib in pulmonary arterial hypertension patients with inadequate response to established therapy. Am J Respir Crit Care Med 2010;182:1171-7.

Bartelds B, van Loon RL, Mohaupt S, Wijnberg H, Dickinson MG, Boersma B, et al. Mast cell inhibition improves pulmonary vascular remodeling in pulmonary hypertension. Chest 2012;141:651-60.

Ozkan M, Dweik RA, Laskowski D, Arroliga AC, Erzurum SC. High levels of nitric oxide in individuals with pulmonary hypertension receiving epoprostenol therapy. Lung 2001;179:233-43.

Machado RF, Londhe Nerkar MV, Dweik RA, Hammel J, Janocha A, Pyle J, et al. Nitric oxide and pulmonary arterial pressures in pulmonary hypertension. Free Radic Biol Med 2004;37:1010-7.

Xu W, Koeck T, Lara AR, Neumann D, DiFilippo FP, Koo M, et al. Alterations of cellular bioenergetics in pulmonary artery endothelial cells. Proc Natl Acad Sci U S A 2007;104:1342-7.

Xu W, Kaneko FT, Zheng S, Comhair SA, Janocha AJ, Goggans T, et al.

Increased arginase II and decreased NO synthesis in endothelial cells of patients with pulmonary arterial hypertension. Faseb J 2004;18:1746-8.

Giaid A, Saleh D. Reduced expression of endothelial nitric oxide synthase in the lungs of patients with pulmonary hypertension. New Eng J Med 1995;333:214-21.

Guo FH, Comhair SA, Zheng S, Dweik RA, Eissa NT, Thomassen MJ, et al.

Molecular mechanisms of increased nitric oxide (NO) in asthma: Evidence for transcriptional and post-translational regulation of NO synthesis.

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Cowan DC, Hewitt RS, Cowan JO, Palmay R, Williamson A, Lucas SJ, et al. Exercise-induced wheeze: Fraction of exhaled nitric oxide-directed management. Respirology 2010;15:683-90.

Source of Support: This work was supported by the Cleveland Clinic Research Programs Council. Kewal Asosingh is a scholar of the international society for advancement of cytometry., Conflict of Interest: None declared.

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Research A r t i cl e

Activation of the unfolded protein response is associated with pulmonary hypertension Michael E. Yeager1,2, Monica B. Reddy3, Cecilia M. Nguyen1, Kelley L. Colvin1, D. Dunbar Ivy4, and Kurt R. Stenmark1,5,6 1

Department of Pediatrics, Division of Pulmonary, and Critical Care Medicine, 2Department of Bioengineering, University of Colorado Denver, Aurora, Colorado, USA, 3Case Western Reserve University Medical School, Cleveland, Ohio, USA, 4 Department of Pediatric Cardiology, University of Colorado Denver, Aurora, Colorado, USA, 5Cardiovascular Pulmonary Research, 6 Gates Center for Regenerative Medicine and Stem Cell Biology

ABSTRACT Pulmonary hypertension remains an important cause of morbidity and mortality. Although there is currently no cure, descriptions of defective intracellular trafficking and protein misfolding in vascular cell models of pulmonary hypertension have been recently reported. We tested the hypothesis that activation of the unfolded protein response (UPR) would be associated with the development of severe PH. We investigated activation of the UPR in archival tissues from patients with severe PH, and in the monocrotaline-induced rat model of severe PH. We tested the ability of a pharmacologic agent capable of modulating the UPR to prevent and reverse pulmonary hypertension. We found evidence of an active UPR in archival tissue from humans with PH, but not in control lungs. Similarly, monocrotaline-treated rats demonstrated a significant difference in expression of each of the major arms of the UPR compared to controls. Interestingly, the UPR preceded the appearance of macrophages and the development of lung vascular remodeling in the rats. Treatment of monocrotaline rats with salubrinal, a modulator of the PERK arm of the UPR, attenuated PH and was associated with a decrease in lung macrophages. In culture, pulmonary artery smooth muscle cells with UPR induction produced IL-6 and CCL-2/MCP-1, and stimulated macrophage migration. These effects were abolished by pretreatment of cells with salubrinal. These data support the hypothesis that the UPR may play a role in the pathogenesis of inflammatory vascular remodeling and PH. As such, understanding the functional contributions of the UPR in the setting of PH may have important therapeutic implications.

Key Words: pulmonary hypertension, unfolded protein response, vascular remodeling, monocrotaline

Pulmonary hypertension (PH) is distinguished by an increase in pulmonary arterial pressure and resistance. It is defined as pulmonary artery pressure greater than 25 mmHg at rest or pressure greater than 35 mmHg during exercise. This insidiously progressive disease can be familial or sporadic, and may arise alone or secondary to other disease processes.[1] The incidence of idiopathic primary pulmonary hypertension (IPAH) is approximately six cases per million, and the disease presents more frequently in young women.

The vascular changes associated with severe PH have been well documented. Resistance pulmonary arteries remodel all three layers. The smooth muscle cells within the medial layer hypertrophy, and the number of peripheral Address correspondence to:

Dr. Michael E. Yeager Department of Pediatrics, Division of Pulmonary, and Critical Care Medicine 12700 E. 19th Ave., Box B131, University of Colorado Denver Aurora, Colorado 80045, USA Email: michael.yeager@ucdenver.edu Pulmonary Circulation | April-June 2012 | Vol 2 | No 2

arteries decreases.[2] Occlusive neointimal lesions form in smaller pulmonary arteries, and are composed of vascular smooth muscle cells, endothelial cells, and immune cells.[3] In addition, there is increasing evidence of larger pulmonary artery change with respect to wall compliance and stiffening.[4] Imbalances in vasodilators and vasoconstrictors, particularly endothelin-1,[5] genetic mutations, [6-8] extracellular matrix perturbation, [9-10] and a pro-inflammatory milieu [11-12] all contribute to a complex remodeling of the pulmonary vasculature leading to hypertrophy of the right ventricle and cor pulmonale.

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Website: www.pulmonarycirculation.org DOI: 10.4103/2045-8932.97613 How to cite this article: Yeager ME, Reddy MB, Nguyen CM, Colvin KL, Ivy DD, Stenmark KR. Activation of the unfolded protein response is associated with pulmonary hypertension. Pulm Circ 2012;2:229-40.

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The origins of the insult(s) that serve as the genesis of PH pathobiology have long been under intense investigation.

Ingestion of diet pills, hypoxia, viruses, and chronic thromboembolic disease have all been implicated.[13-16] We hypothesized that, regardless of etiology, the lung vasculature is severely and chronically stressed under these conditions. In many circumstances of cell or tissue stress, the unfolded protein response (UPR) is activated and plays a number of important roles in both normal and disease processes. [17] When proteins are created in a cellular environment, they are transmitted to the endoplasmic reticulum (ER) to be folded before they are secreted. In response to a cellular stressor resulting in the accumulation of misfolded proteins, the ER stress response has four components: translational attenuation; enhanced expression of ER chaperones; enhanced expression of the components of endoplasmic reticulum associated degradation (ERAD); and induction of apoptosis. [18] When excessive amounts of abnormally folded proteins accumulate within the ER and cytosol of cells, aggregation occurs and cell death pathways are ultimately activated.[19] ER stress has been investigated in various disease contexts and has been linked to the pathobiology of neurodegenerative disorders, atherosclerosis, alcoholic liver disease, viral infection, rheumatoid arthritis, idiopathic pulmonary fibrosis, [20-22] and most recently, severe pulmonary artery hypertension.[23-25]

We hypothesized that one pathobiological mechanism underpinning severe PH could be an underlying protein folding derangement. We therefore investigated whether the UPR is activated in human IPAH and in the monocrotaline (MCT)-induced rat model of severe PH. We first sought to identify whether UPR activation is evident in tissue derived from human PH patients. We found significant evidence of UPR activation in adult PH patient lung sections particularly in areas of vascular remodeling. Using the rat model, we found a similar UPR activation that was robustly concentrated in and around areas of vascular remodeling.

Disturbances in the three primary pathways of the UPR were observed early (Week 1) in the disease process, preceded the development of remodeling, and was associated with influx of CD68+ macrophages. Pharmacologic modulation of UPR by salubrinal, in both prevention and reversal experiments, resulted in decreased plasma ET-1, decreased plasma pro-inflammatory cytokines, decreased pulmonary artery pressure and vascular remodeling, and a sharply decreased macrophage influx. UPR activation in cultured rat pulmonary artery smooth muscle cells was sufficient to induce a pro-inflammatory phenotype capable of inducing macrophage migration, an effect blocked by salubrinal. We conclude that UPR activation precedes vascular remodeling associated with severe PH, and UPR modulation prevents and attenuates PH by protection against cell stress and by diminution of macrophages in the pulmonary vasculature.

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Materials and Methods Human subjects

Lung samples were obtained from surgical lung biopsies performed for evaluation of pulmonary arterial hypertension. Lung biopsies were evaluated from patients with idiopathic pulmonary artery hypertension (IPAH) and with familial pulmonary artery hypertension (fPAH).

Control lung sections (n ≥ 7) were obtained from four subjects and demonstrated normal lung parenchyma.

Hemodynamic and demographic data confirming PAH for these patients (and lack of PAH in controls) has already been published elsewhere (personal communication, JE Loyd, Vanderbilt University Medical Center, Nashville, Tenn.).

Tissue sections were a kind gift from JE Loyd. We examined tissues (n ≥ 4) from seven patients with IPAH and from eleven patients with FPAH. Bone morphogenetic protein receptor type 2 (BMPR2) mutation status was confirmed for all FAPH.

Animal model of PH

The Institutional Animal Care and Use Committee of the institution approved this study. The characterization of the MCT rat model has been previously summarized[26] and utilized male rats with a single subcutaneous injection of 60 mg/kg at 4–6 weeks of age. Wistar and Lewis strains were used in separate experiments, and no strain differences in response to MCT or salubrinal could be discerned (data not shown).

Lung parenchyma from five animals was obtained for each of the 0-, 1-, 2-, and 3-week time-points. For salubrinal treatment, rats were given MCT as described, [27] plus salubrinal at 1 mg/kg in ≤ 1mL neat DMSO, delivered intraperitoneal, twice weekly commencing either at the time of MCT injection, or two weeks post-MCT. This dose and regimen were selected based on a published regimen for rats[28] and on our own dose—response experiments.

The mean right ventricular systolic pressure (mmHg) was 79 ±5, 59 ±4, 35 ±4, 35 ±6 in rats treated with MCT and then salubrinal twice weekly at 0, 0.5, 1.0, and 5.0 mg/kg body weight, respectively. A salubrinal-only control group was included in all subsequent experiments.

Rat hemodynamics and tissue processing

After completion of the treatment period, rats were anaesthetized and the right ventricular systolic pressure was determined by pressure transducer catheter using a subxiphoid approach. The mean right ventricular systolic pressure (mmHg) was 22 ±3, 31 ±4, 45 ±4, 73 ±6 in rats treated with MCT for 0, 1, 2, and 3 weeks, respectively. The chest was then opened, the right and left cardiac atria were dissected from the heart, and the free wall of the heart was isolated and the ratio of right ventricular weight/left ventricle plus septum weight was determined. The Fulton Pulmonary Circulation | April-June 2012 | Vol 2 | No 2

Yeager et al.: Unfolded protein response in PH

index scores (mass right ventricle/[mass left ventricle + mass septum]) were 0.26±0.03, 0.29±0.03, 0.35±0.03, 0.59±3 for rats treated with MCT for 0, 1, 2, and 3 weeks, respectively. Freshly excised lungs from all animals were inflated with 0.5% low-melt agarose at a constant pressure of 25 cm H2O. Each lung was sectioned transversally through the hilum and included intermediate and peripheral lung tissue. A second sagittal section through the peripheral lung parenchyma was also made. Lung sections were taken from the same lung regions in both the treated and control groups. Frozen and paraffin-embedded tissues were sectioned and prepared for histological analysis.

Immunohistochemistry

Human and rat tissues were sectioned at 5 µm for either hematoxylin and eosin staining or immunohistochemical staining. Activation of UPR was assessed by antibody reactivity to activating transcription factor 6 (ATF6, Imgenex-273 1:100) and C/EBP homologous protein (CHOP, Abcam Ab11419 1:100). Apoptotic cells were identified by immunoreactivity to cleaved caspase-3 antibody (Abcam Ab47131 1:200). The cleaved caspase-3-positive cells were counted and divided by the total number of cells in three randomly chosen high-power field (×400) for each section from each of three animals per group. The mean percentage of caspase-3+ cells for each animal was then obtained by averaging the results of the countings.

Macrophages were identified by staining with antibodies against CD68 (AbBiotec 250594 1:200). Secondary antibodies conjugated to fluorescent dyes (Invitrogen), or biotin combined with immunoperoxidase/avidin-biotin, were as per the manufacturer protocol (Vectastain ABC kit, Vector Labs), and either hematoxylin or Nuclear Fast Red (Vector Labs) was used as a counterstain. Avidin/biotin blocking kits (Vector Labs) were used to block endogenous enzyme activity. Antibody isotype negative controls were included with each sample group. Images were acquired at room temperature using a Zeiss Axiovert S100 fitted with Zeiss 20×0.4 numerical aperture (n.a.) and 10×0.3 n.a. objectives and Axiocam camera. Acquisition of images was

by Axiovision 4.6 software (Zeiss).

RNA analyses of rat lung

Total RNA was isolated from each rat group (n=≥5)

by RNeasy (Qiagen). RNA concentration and relative purity were measured by A260/A280 ratio using a Nanodrop ND-1000 spectrophotometer (NanoDrop Technologies, Inc.) according to the manufacturer’s protocols. cDNA was transcribed with the Superscript III First-Strand Synthesis System (Invitrogen). cDNA yield was determined by measuring A260 on the Nanodrop ND-1000. For real-time-PCR of rat lung, primer sequences were used as previously described,[29-30] for the following gene targets: x-box binding protein-1 (XBP1) spliced (Forward 5’-GACTCCGCAGCAGGTG-3’, Pulmonary Circulation | April-June 2012 | Vol 2 | No 2

R e ve r s e 5 ’ - G C G T C A G A AT C C AT G G G A- 3 ’ ) , X B P 1 unspliced (Forward 5’-CAGACTACGTGCGCCTCTGC-3’, Reverse 5’-CTTCTGGGTAGACCTCTGGG-3’), ATF6 (Forward 5’-GGAAGTTACCAAGGCTTCTTTGAC-3’, R e v e r s e 5 ’ -T G G G T G G TA G C T G G TA ATA G C A - 3 ’ ) , growth arrest and DNA damage-inducible protein (GADD34, Forward 5’-TTTCTAGGCCAGACACATGG-3’, Re ve r s e 5 ’ -TGT TC C T T T T TC C TC C GTG G - 3 ’ ) , a n d hypoxanthineguanine phosphoribosyltransferase (HPRT, (Forward 5’-AAGCTTGCTGGTGAAAAGGA-3’, Reverse 5’-CAAGGGCATATCCAACAACA-3’).

Immunoblot analyses

Lysates from rat tissues (n= ≥5 from each rat group) were isolated by addition of either an ice-cold TB or RIPA buffer in the presence of protease, kinase, and phosphatase inhibitors (HALT, Pierce-Thermo Scientific). Proteins (25 µg) were subjected to electrophoresis on 4–12% gradient Bio-Tris gels (NOVEX, Invitrogen) and transferred to PolyScreen PVDF Transfer membrane (NEN Life Science Products) in a Tris–glycine buffer (NOVEX, Invitrogen) containing 10% methanol. Prestained molecular mass marker proteins (Bio-Rad) were used as standards for the SDS-PAGE. Western blots were visualized using Western Blot Chemiluminescence Reagent (GE Amersham).

Densitometric band normalization of ATF6, CHOP, phosphoeIF2alpha (Cell Signaling 119A11), and total eIF2alpha (Cell Signaling 9722) relied on beta-actin (Novus Biologicals NB600-501B) loading control blots.

Rat ET-1 ELISA

Plasma was prepared by centrifugation of whole blood collected in heparinized tubes. The concentration of ET-1 was then measured according to the manufacturer’s instructions (ADI-900-020A, Enzo Life Sciences).

Rat inflammatory cytokine arrays

Plasma was prepared by centrifugation of whole blood collected in heparinized tubes. The concentrations of cytokines were then measured according to the manufacturer’s instructions (Multi-Analyte ELISArray MER004A, SABiosciences).

Resistance vessel thickness measurements

Images were acquired from smooth muscle actin immunostained lung tissue sections as described above.

Morphometry was conducted on digital images using ImageJ software, http://rsb.nih.gov/ij/) as previously described.[31] Measurements included external and lumen diameter derived from circumference and maximal smooth muscle and intimal thickness. Smooth muscle thickness included both medial and intimal smooth muscle and was normalized by expressing as a percentage of the external diameter. Three sections of each rat lung from five animals per group were analyzed.

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Cell culture

Pulmonary artery smooth muscle cells (PASMC) and pulmonary artery adventitial fibroblasts (PAF, not shown) were isolated from control rats as previously described.[25] Immunofluorescent staining of endothelium (von Willebrand Factor Dako A0082 1:250) and smooth muscle cells (smooth muscle actin Sigma A2547 1:100) was used to assess cell identity. Macrophages were differentiated from peripheral blood with recombinant human macrophage colony-stimulating factor (M-CSF, eBioscience 14-8769-80) at 10 ng/mL as previously described.[32] UPR induction by tunicamycin (Sigma T7765) was performed essentially as described at 1 µg/mL in phosphate buffered saline vehicle for 24 hours. [33] Salubrinal was applied as previously described [34] at 25 µM in dimethylsulfoxide (Sigma D8418) and media for 30 minutes prior to introduction of tunicamycin or vehicle. Rat macrophage migration assays were performed as previously described using a modified Boyden chamber (Corning).[35]

Statistical analyses

All experiments were performed in triplicate, and data were expressed as means ±SE and analyzed by unpaired Student’s t-test for comparison between two groups or one-way ANOVA with post hoc analysis for multiple comparisons. A value of P <0.05 was considered significant.

Results

Induction of the unfolded protein response in patients with PH

The pathogenesis of PH has been linked to hypoxia, viral infection, genetic mutation, and inflammation. The UPR has been shown to play pivotal roles in all of these processes.[18] To establish whether the UPR is activated in lungs from patients with IPAH, we performed immunohistology using antibodies specific for activated ATF6 and the proapoptotic UPR protein CCAAT/-enhancer-binding protein

homologous protein (CHOP). We found ATF6 predominantly in medial and adventitial layers of large (>500 µm internal diameter) and medium sized pulmonary vessels (Fig. 1C) in lung sections from patients with IPAH but not in controls (Fig. 1A). Furthermore, we found ATF6 expressed in plexiform lesions (Fig. 1E) and in muscularized small pulmonary arterioles in IPAH lung (Fig. 1F). CHOP was similarly expressed in medium to small pulmonary vessels (Fig. 1D) from PH lungs, but not in control lung (Fig. 1B). In some IPAH lung sections, airway epithelium strongly expressed ATF6, as did intraluminal white blood cells (Fig. 1G). No differences in UPR expression or localization were observed between patients with IPAH and FPAH.

The monocrotaline-induced rat model of PH demonstrates activation of the UPR in lung cells and in macrophages

The MCT rat model of PH is associated with widespread lung cell apoptosis and an acute perivascular inflammation that develops into chronic inflammation with extensive vascular remodeling.[26-27] To determine if the MCT-induced rat model of PH demonstrates activation of the UPR, we repeated the human immunohistological analysis using rat-specific antibodies and also performed whole lung expression analysis by immunoblot and qPCR. In doing so, we closely examined the time course of the development of PH. We found that by two days after MCT injection, expression of ATF6 and CHOP were localized to medial and adventitial layers of pulmonary vessels and to airway epithelium (Figs. 2C and D), compared to a low level of expression in control lungs (Figs. 2A and B). At 21 days post-MCT, we found exuberant expression of ATF6 in the media and adventitia of medium-to-small bronchovascular regions and less so in airway epithelium (Fig. 2E). CHOP was largely absent by three weeks (localized protein, Fig. 2F; whole lung mRNA, Fig. 2I). Using qPCR on whole lung lysates, we found increased transcripts for ATF6, sXBPFigure 1: (A-B) Bronchovascular region of control lung biopsy sections showing scant immunopositivity for ATF6 (A) or CHOP (B). (C-D) Adult pulmonary hypertension lung sections show pronounced medial layer expression of ATF6 (C) and, to a lesser extent, CHOP (D). (E) Plexiform lesion with ATF6+ cells. (F) Small remodeled vessel with ATF6+ medial cells. (G) A number of PAH lung sections showed significant macrophage (short arrows) and airway epithelium (longer arrow) expression of ATF6. (H) Negative control using isotype matched antibody. Bar=50 µm. Antigen DAB, arrows; hematoxylin counterstain.

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Figure 2: Control rats display limited immunoreactivity for (A) ATF6 and (B) CHOP. Airway epithelial cells and endothelium immunopositive for (C) ATF6 and (D) CHOP appear 2 days postmonocrotaline (arrows). At 3 weeks postmonocrotaline, smooth muscle cells and fibroblasts express (E) ATF6 and (F) CHOP (arrows), and less so in airway epithelium. (G) Active caspase-3 (blue) is evident through all three layers of a pulmonary artery from a rat after 1 week of monocrotaline (arrowheads = endothelial cells, medium arrows = smooth muscle cells, long arrows = adventitial cells). (H) Representative example of negative control using isotype matched antibody. “A” = airway. Black bar = 50 µm. Images are representative of analysis of five fields each from n≥7 lung sections obtained from five rats per group. For all images: Antigen Vector Blue, arrows; fast red counterstain. (I) Monocrotaline rats transcriptionally engage the major arms of the UPR as evidenced by increased ATF6, sXBP-1, GADD34, and also CHOP. Whole lung ATF6 and GADD34 gene expression peaks at Week 1, while CHOP and sXBP-1 levels peak at Week 2. ATF6 and sXBP-1 mRNAs remain elevated after 3 weeks postmonocrotaline, while GADD34 and CHOP levels abate to control levels by Week 3. (J) The % of active caspase-3 positive lung cells increases by 7 days postmonocrotaline, and remains diminishingly so for 3 weeks.

1, CHOP, and GADD34 in lungs of MCT treated rats as early as seven days after injection compared to controls (Fig. 2I). The number of active caspase-3 positive cells in PH lungs increased by Week 1, prior to the appearance of remodeling (Fig. 2J). CD68+ macrophages became increasingly organized and restricted to the adventitial layers of medium-to-large bronchovascular areas (Figs. 3A—D). These data indicate acute lung cell activation in the MCT rat model of PH of two arms of the UPR by protein analysis and three arms of the UPR by transcriptional analysis. Furthermore, the pronounced UPR activation and caspase-3 processing we observed occurred prior to any significant macrophage influx, lung vascular remodeling, or hemodynamic change.

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Modulation of UPR prevents and partially reverses monocrotaline-induced PH Based on our immunohistological and qPCR data, we tested the idea that modulation of the UPR could affect the inflammation and potentially the vascular remodeling observed in MCT-induced PH. We treated rats with the ER stress protectant salubrinal, which modulates the selective translation component of the UPR.[36] We performed dose—response experiments and found that intraperitoneal injections of salubrinal at 1.0 mg/kg twice weekly resulted in a statistically significant reduction in right ventricular systolic pressure in MCT rats in both prevention and reversal regimens (Fig. 4A). These findings were corroborated by evidence of significant attenuation 233

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of pulmonary vascular remodeling and reduced vessel thickening by salubrinal compared to MCT alone (Fig. 4B).

Salubrinal was associated with reduced ET-1 plasma levels (Fig. 4C), which may partially account for the decreased

pulmonary artery pressure by virtue of decreased vasoconstriction. Interestingly, MCT rats given salubrinal for prevention displayed decreased lung airway and vascular cell expression of ATF6, CHOP, but not the reversal

Figure 3: Accumulation of CD68 positive cells into PH rat lung. CD68+ cells (A-D, arrows, Cy3, DAPI nuclei) are sparsely dispersed throughout control lung, but steadily increase with monocrotaline beginning at Week 1. CD68+ cells localize to adventitia of medium to large vessels (B, D) and to media of smaller vessels (C). Note the absence of bronchovascular remodeling in Week 1 compared to Week 3, despite the influx of CD68+ cells. “A” = airway. Red bar = 20 µm. Images are representative of analysis of five fields each from n≥7 lung sections obtained from five rats per group.

Figure 4: Pulmonary hypertension and vascular remodeling are prevented and partially reversed by the ER stress protectant salubrinal. (A) Right ventricular systolic pressure is significantly reduced by administration of salubrinal at Day 0 or Day 14 postmonocrotaline injection. (B) Hematoxylin and eosin staining of frozen tissue sections shows reduction in bronchovascular remodeling and resistance vessel thickening (bar graph) in salubrinal-treated PH rats compared to monocrotaline alone. (C) Plasma ET-1 levels decrease in salubrinal treated rats compared to monocrotaline alone. Sal = salubrinal given just prior to MCT injection; Sal 2wk = salubrinal given at 2 weeks post-MCT injection. Black bar = 50 µm. * P=<0.05.

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group (all images taken at 4 weeks post-MCT injection, Figs. 5A–F). CD68+ cells and UPR activation were much less abundant in salubrinal treatment groups compared to MCT alone (Figs. 5G and H). The increased number of active caspase-3 positive cells in PH lungs was greatly attenuated by both prevention and reversal regimens of salubrinal treatment (Fig. 5I). Immunoblot analysis of MCT rat lung lysate revealed temporal increases in both ATF6 and CHOP and a decrease in peIF2alpha, all of which were reversed in rats given salubrinal (Fig. 6A). These findings were consistent with the immunofluorescence results in Figure 5. Salubrinal treated MCT rats had decreased lung transcripts of ATF6 (Fig. 6B) and sXBP-1 (Fig. 6C) compared to MCT alone.

Increases in plasma pro-inflammatory cytokines in the monocrotaline-induced rat model of PH are attenuated by UPR modulation.

UPR has been linked to inflammation in a variety of pathological settings. Recent studies have shown that pharmacologic modulation of the UPR reduces inflammation and subsequent disease.[37-38] We speculated that plasma from MCT-treated rats would be associated with a pro-inflammatory pattern of cytokines that could be reduced by salubrinal. We found that MCT rats had elevated plasma chemokine (c-c motif) ligand 2/monocyte chemotactic protein-1 (CCL2/MCP-1), granulocytemacrophage colony-stimulating factor (GM-CSF), CCL5/ regulated upon activation, normal T-cell expressed, and secreted (RANTES), interleukin (IL)-6, IL-1b, and IL-2

Figure 5: Treatment with the UPR modulator salubrinal decreases UPR localization in bronchovascular cells (arrowheads) and impairs lung influx of macrophages (arrows) in the monocrotaline-induced rat model of PH. Salubrinal was administered twice per week commencing at either Day 0 (Sal right away {RA} or 14 days after (2 weeks) MCT injection. Rat lung sections were tested for immunopositivity for CD68 and ATF6 (A, C, E, G), or CD68 and CHOP (B, D, F, H). Salubrinal largely prevented the appearance of macrophages, and attenuated numbers of ATF6 and CHOP immunopositive cells when given to rats prior to established PH. When given after 2 weeks of established PH, salubrinal treatment was associated with decreased macrophages but with no difference in ATF6 or CHOP positive cells. (G-H) Representative lung staining of control rat given salubrinal for 4 weeks but without monocrotaline. No bronchovascular remodeling or macrophage influx is apparent. No increases in ATF6 or CD68 positivity were noted. For all images, antigen Cy3 or Alexa 488 as indicated, DAPI nuclei. “A” = airway. Red bar = 20 µm. Images are representative of analysis of five fields each from n≥7 lung sections obtained from five rats per group.

(I) The % of active caspase-3 positive lung cells postmonocrotaline is diminished by salubrinal treatment, either as a prevention, or (less so) as a “treatment.”

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Figure 6: Lung gene expression associated with the unfolded protein response is modulated by systemic administration of salubrinal. (A) Immunoblot analysis of UPR protein expression. ATF6 and proapoptotic CHOP increase with progression of monocrotaline-induced PH, but are decreased in salubrinal treated rats. Salubrinal increases the phosphorylation of eIF2alpha, reversing the observed decreases as PH progresses from Weeks 1 to 3. (B) Real-time PCR of rat whole lung reveals that control of the PH-associated increase in ATF6 also occurs at the transcriptional level and is sensitive to salubrinal treatment. (C) The spliced form of XBP-1 increases with PH, but is decreased with salubrinal treatment. * P=<0.05

(Fig. 7). The levels of CCL2/MCP-1, GM-CSF, CCL5, IL-1b, and IL-6 cytokines were reduced in both the prevention and reversal groups of salubrinal treated MCT rats, whereas IL-2 levels remained unchanged.

UPR activation in pulmonary artery smooth muscle cells and adventitial fibroblasts promotes a pro-inflammatory phenotype capable of recruiting macrophages

To confirm the target cell(s) associated with the prevention and attenuation of PH by salubrinal-mediated UPR modulation, we performed culture assays using pulmonary artery smooth muscle cells (PASMC) and pulmonary artery adventitial fibroblasts (PAF, not shown). We chose these cell types for several reasons: (1) the macrophage influx into the lung following MCT injection was most pronounced in the adventitia around medium-to-large bronchovascular structures (Fig. 3D); (2) because of the interest in the pulmonary artery media and adventitia vis-àvis inflammation and pulmonary vascular remodeling; and (3) the UPR immunostaining signature was pronounced in these two cell types in human and rat PH lung sections. We induced the UPR using 1 µg/mL tunicamycin, a dose that did not cause widespread cell death (detailed in materials and methods, data not shown). We cultured PASMC with 25 µM salubrinal for one hour prior to treatment with tunicamycin for 24 hours, a regimen based on the literature [34] and our own dosing experiments (data not shown). We found that tunicamycin-treated PASMC (isolated from control rat lungs) significantly increased the migration of rat 236

peripheral blood-derived macrophages compared to control treated PASMC (Fig. 8A). Pretreatment with salubrinal prevented the increase in UPR-associated macrophage migration. Lysates from tunicamycin treated PASMC showed increases in ATF6 and CHOP, with concomitant diminution of peIF2alpha (Figs. 8B and C). Pretreatment with salubrinal abolished the tunicamycin-induced ATF6, CHOP, and peIF2alpha changes. Using ELISA, we measured IL-6 and CCL2/MCP-1 levels in the PASMC supernatants and found a significant increase in production of both in the tunicamycin-treated cells compared to salubrinalonly treated cells (Fig. 8D). Similar results with regard to cytokine and chemokine production as well as macrophage migration were observed when we used rat PAF, and when we used either PAF or PASMC isolated from rats with MCTinduced PH (data not shown). These results confirm that pulmonary vascular cells are cellular targets for induction of the UPR, and when engaged, may promote inflammatory processes of cytokine and chemokine production and secretion, as well as increased macrophage migration.

Discussion

In this study, we demonstrate an active unfolded protein response in lung sections from patients with IPAH and those derived from the MCT-induced rat model of PH. Our finding of UPR activation prior to widespread macrophage influx, pulmonary vascular remodeling, and the development of pulmonary hypertension strongly suggests that UPR activation is an early pathobiological event, at least in the Pulmonary Circulation | April-June 2012 | Vol 2 | No 2

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Figure 7: Plasma inflammatory cytokines CCL2/MCP-1, GM-CSF, CCL5, IL-6, IL-1b, and IL-2 are increased in monocrotaline treated rats. Cytokines were measured after salubrinal injecitons alone (Sal only) or after 3 weeks of MCT injection with and without the indicated salubrinal treatments (Sal = salubrinal given just prior to MCT injection; Sal 2wk = salubrinal given at 2 weeks post-MCT injection). Levels of these cytokines (with the exception of IL-2 measured after MCT treatment with salubrinal commencing after 2 weeks) decrease in rats treated with salubrinal in prevention and reversal experiments. * P=<0.05.

MCT-induced rat model of PH. Furthermore, modulation of one arm of the UPR by salubrinal experimentally prevented PH and attenuated well-established PH by mechanisms that may be associated with lung influx of macrophages.

Finally, we provide evidence that UPR modulation in lung fibroblasts and smooth muscle cells may contribute to the inflammatory process observed in the MCT rat model of PH, since administration of salubrinal abolishes increased levels of pro-inflammatory cytokines and chemokines, both in vivo and also using rat PAF and PASMC in culture.

Our results in the MCT rat model raise the possibility that derangement of ER homeostasis in the lung may help to establish and maintain the inflammation observed in patients with PH.[39] Admittedly, in this study we did not directly confront the definition of â&#x20AC;&#x153;derangementâ&#x20AC;? of the UPR in the context of MCT-induced lung injury, other than documenting a substantial activation of the system. The unfolded protein response can be triggered by a wide variety of stimuli including hypoxia, oxidative stress, gene mutation (and subsequent protein misfolding and/ or transport dysregulation) and glucose deprivation,[40-48] most of which have been implicated in PH. In the MCT rat model of PH, we found early expression of both ATF6 and CHOP in the pulmonary vasculature and similar results Pulmonary Circulation | April-June 2012 | Vol 2 | No 2

were obtained in primary cell culture. This is consistent with previous reports in this model of detection of apoptosis in whole lung tissue[49] and pulmonary artery endothelial and smooth muscle cells,[50,51] as well as UPR activation in alveolar epithelial and pulmonary artery endothelial cell cells treated with MCT-pyrrole.[52] We noted that ATF6 was predominantly localized to pulmonary artery smooth muscle cells and airway epithelium (Figs.

2C and E), while CHOP localized to nuclei and cytoplasm of airway epithelium and pulmonary artery endothelial cells and smooth muscle cells (Figs. 2D and F). This may reflect an as yet unidentified differential sensitivity to UPR-mediated apoptosis in airway versus vascular compartments in this model, and should be investigated further. Indeed, expression of both ATF6 and CHOP in the airways of rats two days after MCT injection (and prior to macrophage influx) suggests that ER stress and homeostatic disturbance of nonvascular lung cells may contribute to the disease process in the vasculature. An accumulation of misfolded proteins can trigger a cellular survival response in the endoplasmic reticulum, as have been observed in rheumatoid arthritis synoviocytes.[53] The possibility that UPR is a contributing pathway towards the establishment of antiapoptotic/pro-survival lung cell phenotypes in PH is intriguing.

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Figure 8: Activation of the UPR in rat pulmonary artery cells can stimulate rat macrophage migration and production of inflammatory cytokines and chemokines, all of which is reversible by salubrinal. (A) Migration of rat peripheral blood macrophages is increased secondary to tunicamycin-UPR induction in pulmonary artery smooth muscle cells (PASMC), and is prevented by salubrinal. The number of macrophages migrating in minimal media was used as a control and arbitrarily set as 1 (CTL). (B-C) UPR induction in PASMC by tunicamycin as assessed by (B) immunoblot, and (C) immunofluorescent staining for ATF6 (green) and smooth muscle actin (SMA, red). Note that salubrinal treatment increases peIF2alpha expression (B). (D) Tunicamycin-treated PASMC secrete pro-migratory and pro-inflammatory cytokines, the production of which is reversible by salubrinal. DAPI nuclei; original magnification = 100Ă&#x2014;; tunicamycin was used at 1 Âľg/mL for 24 hours.

Why would there be such a robust UPR activation in PH and how might the UPR participate in PH disease mechanisms?

Using time course analysis of lung tissue from the MCT rats, we found that the UPR is engaged prior to vascular remodeling, which suggests at least the potential for causal roles for the UPR. MCT is a toxin that causes pulmonary hypertension and multiorgan pathology in the rat, and has been recently suggested to be a poor model of angioobliterative pathobiological components of the disease.[54] In vascular cell culture, MCT causes activation of IRE1,[52] an endoribonucleolytic effector of XBP-1, which we too found robustly spliced in the MCT rat lung tissue compared to controls (Figs. 2H and 6C). Furthermore, in both the MCT and hypoxia rat models of PH compared to controls, proteomic analyses reveal that increased expression of UPR family proteins ERp29 and ERp57 are part of a lung protein pattern change characterized by differential expression 238

of proteins governing vasoconstriction and vascular remodeling. [55] Apart from MCT or hypoxia, transient expression of mutant bone morphogenetic receptors (using mutations sequenced from patients with PH) fused to green fluorescent protein results in dysregulated trafficking and golgi retention.[24] Taken together with our work here, these studies collectively point to a central role for the endoplasmic reticulum and the UPR in the development of PH in so far as vascular cell insult and cell death (MCT), deranged oxygen tension and oxidative stress (hypoxia), and expression of mutant receptors.

In the MCT rats, we were surprised to find cells expressing ATF6 and/or CHOP that had morphology reminiscent of macrophages (Figs. 1G and 5D). Indeed, in lung sections from MCT rats, we found early appearance and later abundance of CD68+ cells (Fig. 3). UPR in macrophages Pulmonary Circulation | April-June 2012 | Vol 2 | No 2

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can be induced by nitric oxide, [56] neuroendocrine hormones,[57] and increased trafficking of cholesterol[58], all of which correlate with immunostimulatory activities.

We are currently quantitatively and qualitatively assessing phagocytic cells by flow cytometry in both peripheral blood and lung single cell suspensions, (not shown). Interestingly, UPR-activated macrophages in advanced atherosclerotic lesions accumulate large amounts of unesterified cholesterol which leads to both cell death and to lesional necrosis, promoting plaque instability[58]. It has previously been shown that clodronate-mediated depletion of circulating phagocytic cells in hypoxic rats prevented pulmonary vascular remodeling.[59] Similarly, when we administered salubrinal to MCT rats, we observed a dramatic decrease in the number of CD68+ cells to the lung bronchovasculature in the context of lowered right ventricular systolic pressure, reduced plasma ET-1, and blunted vascular remodeling (Figs. 4 and 5). This was associated with increased lung peIF2alpha, decreased ATF6, CHOP, and XBP-1, and a reduction in plasma chemokines and cytokines (Figs. 6 and 7). These results collectively suggest that UPR engagement may directly link inflammation to the vasoconstriction and vascular remodeling associated with PH, perhaps by both resident and nonresident phagocytic cell activities. It has been previously reported that high levels of ET-1 can induce the UPR in pulmonary artery smooth muscle cells and that such stimulation leads to a pro-inflammatory/pro-adhesive phenotype competent to recruit inflammatory cells.[25] Further studies should delineate the mechanistic details of such a paradigm.

In conclusion, our study establishes the UPR as an important associative finding in PH. We also provide the first data to suggest that the UPR might be playing important mechanistic roles in macrophage recruitment in PH and perhaps the establishment of a persistent proinflammatory environment in the bronchovasculature.

Further investigations that more mechanistically describe the contributions of the UPR in specific lung cell types might lead to therapeutic strategies for PH patients that could potentially be combined with current modalities.

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Source of Support: This work was supported by National Institutes of Health Specialized Centers of Clinically Oriented Research Grant HL-084923–02 and National Institutes of Health Program Project Grant HL-014985–35 (K.R.S.), the Brigid Hope Research Fund, and the Leah Bult Pulmonary Hypertension Research Fund. Conflict of Interest: None declared.

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Research A r t i cl e

Distinct responses to hypoxia in subpopulations of distal pulmonary artery cells contribute to pulmonary vascular remodeling in emphysema L. S. Howard1, A. Crosby2, P. Vaughan3, A. Sobolewski2, M. Southwood4, M. L. Foster3, E. R. Chilvers2, and N. W. Morrell2 1

National Pulmonary Hypertension Service (London), Hammersmith Hospital, Imperial College Healthcare NHS Trust, London, 2 Department of Medicine, University of Cambridge School of Clinical Medicine, Addenbrooke’s Hospital, Cambridge, 3 Department of Pathology, AstraZeneca R&D Charnwood, Loughborough, Leicestershire, 4Department of Pathology, Papworth Hospital, Cambridge, UK

Abstract We have shown previously that hypoxia inhibits the growth of distal human pulmonary artery smooth muscle cells (PASMC) isolated under standard normoxic conditions (PASMCnorm). By contrast, a subpopulation of PASMC, isolated through survival selection under hypoxia was found to proliferate in response to hypoxia (PASMChyp). We sought to investigate the role of hypoxia-inducible factor (HIF) in these differential responses and to assess the relationship between HIF, proliferation, apoptosis, and pulmonary vascular remodeling in emphysema. PASMC were derived from lobar resections for lung cancer. Hypoxia induced apoptosis in PASMCnorm (as assessed by TUNEL) and mRNA expression of Bax and Bcl-2, and induced proliferation in PASMChyp (as assessed by 3H-thymidine incorporation). Both observations were mimicked by dimethyloxallyl glycine, a prolyl-hydroxylase inhibitor used to stabilize HIF under normoxia. Pulmonary vascular remodeling was graded in lung samples taken from patients undergoing lung volume reduction surgery for severe heterogenous emphysema. Carbonic anhydrase IX expression in the medial compartment was used as a surrogate of medial hypoxia and HIF stabilization and increased with increasing vascular remodeling. In addition, a mixture of proliferation, assessed by proliferating-cell nuclear antigen, and apoptosis, assessed by active caspase 3 staining, were both higher in more severely remodeled vessels. Hypoxia drives apoptosis and proliferation via HIF in distinct subpopulations of distal PASMC. These differential responses may be important in the pulmonary vascular remodeling seen in emphysema and further support the key role of HIF in hypoxic pulmonary hypertension.

Key Words: hypoxia, hypoxia-inducible factor, pulmonary arterial hypertension

It has been appreciated for some time that vascular remodeling in the pulmonary circulation may complicate a number of hypoxic lung diseases. Although there may be other factors involved in this process, such as inflammation, [1] hypoxia is regarded as a significant contributor. Indeed, hypoxic exposure is used to create animal models of pulmonary hypertension.

Many histological changes develop in the pulmonary circulation in response to chronic hypoxia involving both

Address correspondence to: Dr. L. S. Howard National Pulmonary Hypertension Service (London) Department of Cardiac Sciences Hammersmith Hospital Imperial College Healthcare NHS Trust Du Cane Road London W12 0HS Email: l.howard@imperial.ac.uk

Pulmonary Circulation | April-June 2012 | Vol 2 | No 2

proximal and distal pulmonary arteries.[2-6] While significant remodeling in the proximal pulmonary arteries occurs in chronic hypoxia, the hemodynamic significance of this remains uncertain. The predominant site of pulmonary vascular resistance is the distal pulmonary circulation and it is here that hypoxia regulates vascular tone. Consequently, it is likely that changes seen here are of greatest pathological significance in the generation of elevated “fixed” pulmonary vascular resistance as seen in chronic hypoxic pulmonary hypertension.

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Website: www.pulmonarycirculation.org DOI: 10.4103/2045-8932.97616

How to cite this article: Howard LS, Crosby A, Vaughan P, Sobolewski A, Southwood M, Foster ML et al. Distinct responses to hypoxia in subpopulations of distal pulmonary artery cells contribute to pulmonary vascular remodeling in emphysema. Pulm Circ 2012;2:241-9.

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We have recently demonstrated the existence of two distinct subpopulations of pulmonary artery smooth muscle cell (PASMC) present in the distal pulmonary artery in humans: in one, proliferation occurs under normoxic conditions (PASMCnorm) while in the other, proliferation occurs under hypoxic conditions (PASMChyp).[7] This work has led to the hypothesis that alveolar hypoxia may lead to a selective expansion of the PASMChyp population, which contributes to the remodeling of small peripheral pulmonary arteries.

Hypoxia-inducible factor (HIF) is a key regulator of gene expression in response to hypoxia and we sought to characterize its role in the differential apoptotic/ proliferative response of PASMCnorm and PASMChyp to hypoxia.

We also set out to assess whether such a mixed response could be observed in vivo in patients with emphysema and the relation to HIF stabilization. We showed that hypoxia drives apoptosis in PASMCnorm and proliferation in PASMChyp in vitro and that this effect was mimicked by pharmacological stabilization of HIF. In addition, in lung specimens removed from patients undergoing lung volume reduction surgery for severe heterogenous emphysema we demonstrated both apoptosis and proliferation in adjacent smooth muscle cell (SMC) populations. Furthermore, we demonstrated that the extent of apoptosis and proliferation, as well as HIF activation, correlated with the degree of vascular remodeling. These data suggest that pulmonary vascular remodeling in hypoxic lung disease may involve HIF-dependent expansion of a hypoxia-proliferative subpopulation of SMCs.

Materials AND Methods Tissue samples

Samples of lung tissue for the culture of human pulmonary artery smooth muscle cells were obtained from patients undergoing lobectomy. Approval was obtained from the Cambridgeshire and Glenfield Hospital Local Reseach Ethics Committees and all subjects gave informed consent.

Only lungs without histological evidence of significant pulmonary vascular disease were used, as judged by a thoracic pathologist. For analysis of peripheral lung sections from patients with emphysema, samples were collected from patients undergoing lung volume reduction surgery (emphysema specimens) and matched to those undergoing lobectomy for cancer (control specimens).

Culture of human pulmonary artery smooth muscle cells Peripheral segments of human pulmonary artery were obtained. PASMC were cultured from peripheral arteries (<1–2 mm external diameter) in 20% fetal bovine serum (FBS)/Dulbecco’s modified Eagle Medium (DMEM) with smooth muscle growth supplements (bFGF 2ng/ml, 242

EGF 0.5ng/ml, insulin 5µg/ml) (Promocell, Heidelberg, Germany), as previously described.[7] After the first passage, cells were maintained in 10% FBS/DMEM and used between Passages 3-7. The smooth muscle phenotype of isolated cells was confirmed by positive immunoflourescence with antibodies to a smooth muscle actin and smooth muscle specific myosin (Sigma-Aldrich, Poole, UK), as described previously.[8]

Exposure of cells to hypoxia

PASMCs were grown under standard normoxic conditions in a 5% CO2 incubator, before being exposed to hypoxic conditions. Hypoxia was induced by pregassing cell culture medium (DMEM + 25 mM HEPES) with a gas mixture containing 95% N2/5% CO2 for 30 minutes inside a gastight isolator which had been flushed with N2. FBS was left to equilibrate in the isolator and was added to the pregassed medium prior to use. Plates were then placed inside specially designed perspex chambers (Bellco Glass Inc., Vineland, N.J.), which were gassed with 95% N2/5% CO2 for 20 minutes. The chambers were kept inside an O2/ CO2 incubator (GA156, Leec Limited, Nottingham, UK), preset to provide an atmosphere of 1% O2/5% CO2. The pH (7.35–7.45), PO2 (2–3 kPa) and PCO2 (4–5 kPa) in the medium was checked at the beginning and end of each experiment using a blood gas analyzer (ABL5; Radiometer Ltd., West Sussex, UK). Hypoxic cells were not reoxygenated at any stage during the culture period.

S e l e c t i o n o f t h e h y p ox i a p r o l i f e r a t i v e subpopulation PASMC at Passage 1 were plated in 20% FBS/DMEM/ smooth muscle growth supplements (as above) and 25 mM HEPES in 96-well plates at an approximate density of 10 cells/well and left overnight to adhere. Plates were then placed under hypoxic conditions as described above, and maintained for up to two to three weeks. Viable, proliferating cells (observed in approximately 5% of the wells) were trypsinised and transferred sequentially to 24 then 6-well plates before passaging into T75 flasks, while maintaining hypoxic conditions throughout.

Proliferation assay

The effects of hypoxia on cell proliferation were quantified by the incorporation of [3H]-thymidine as an index of DNA synthesis, as described previously.[7] Cells were plated at 15×103 cells/well in 10% FBS/DMEM in 24-well plates and left to adhere under normoxic or hypoxic conditions (3kPa). Cells were grown to 60% confluence and then quiesced for 48 hours in normoxic or hypoxic 0.1% FBS/ DMEM, unless otherwise specified. At the beginning of the experimental procedure, fresh hypoxic (3kPa) or normoxic medium was added, either alone or with specific pharmacological reagents. [3H]-thymidine (0.5mCi/well) was added after 18 hours for the final six hours. The effect Pulmonary Circulation | April-June 2012 | Vol 2 | No 2

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of HIF stabilization was assessed using a prolyl-hydroxylase inhibitor (100 μM and 1mM dimethyloxalyll glycine, DMOG (Alexis, Nottingham, UK)).

Western immunoblotting

Stabilisation of HIF-1a was assessed by Western blotting, following exposure of PASMC to normoxia or hypoxia. Cells were plated in 60 mm dishes and grown to 90% confluence.

They were quiesced as in the proliferation assay and then exposed to experimental conditions in 0.1% FBS/ DMEM for 24 hours. PASMC were lysed and extracts were boiled in a 1:5 ratio with 5× protein loading buffer for 5 minutes. Samples were loaded onto a 12% gel (HIF-1a) and separated by electrophoresis for one to two hours. The gels were transferred to a nitrocellulose membrane, incubated with blocking buffer, then incubated overnight with a specific mouse antibody to HIF-1a (1:250) (BD Biosciences, CA, USA) at 4°C. Blots were then incubated with an appropriate horseradish-peroxidase-conjugated antibody in a blocking buffer for an hour at room temperature. Blots were developed using enhanced chemiluminesece reagent (Amersham Bioscience, Little Chalfont, UK). Loading controls were performed by incubating the membranes with a specific mouse antibody to HIF-1b (1:200; BD Biosciences, Calif., USA).

Apoptosis assays

A Terminal deoxynucleotidyl Transferase Biotin-dUTP Nick End Labelling (TUNEL) assay was performend to determine the degree of apoptosis. Quantitative polymerase chain reaction (qPCR) for BCl-2 and Bax were also performed.

For both assays PASMC were plated into 60 mm dishes at a density of 250×103 cells per plate. Cells were quiesced for 48 hours in 0.1% FBS/DMEM and after the medium was replaced, the cells were treated for 24 hours. At the beginning of the experimental procedure, fresh hypoxic (3 kPa) or normoxic medium was added, either alone or with the addition of DMOG (100 μM) or staurosporine (50 nM) (Sigma-Aldrich).

For the TUNEL assay, following treatment, the cells were trypsinized and transferred to a poly-L-lysine coated slide at a density of 5,000 cells per slide using a Shandon Cytospin 3 centrifuge (Thermo Fisher Scientific, Mass., USA). The TUNEL assay was performed using the DeadEnd Fluorometric TUNEL System (Promega, Southampton, UK) as described in the manufacturer’s instructions.

Briefly, formaldehyde fixed cells were rinsed in phosphatebuffered saline (PBS) and permeabilized in 0.2% Triton X-100 solution (5 minutes). The slides were then rinsed twice in PBS (5 minutes, RT (room temperature)) and then immersed in equilibration buffer (5–10 minutes).

Recombinant terminal deoxynucleotidyl transferase and nucleotide mix in an equilibration buffer was added to cover the slides and a plastic coverslip applied. Slides Pulmonary Circulation | April-June 2012 | Vol 2 | No 2

were incubated in a humid environment at 37°C for 30 minutes. The reaction was terminated by immersing the slides in 2× sodium chloride/sodium citrate (SSC) (15 minutes, RT). Slides were rinsed three times in PBS (5 minutes, RT). Slides were mounted in Vectashield+DAPI (Vector Laboratories, Calif., USA) to stain the nuclei and examined using a fluorescence microscope (Nikon Eclipse, TE300, Japan). The rate of apoptosis was defined as the proportion of positively labeled nuclei counted in three fields of view (×20 magnification). A positive control was performed by adding ~10 units/ml of DNAse 1 to fixed cells (10 minutes, RT).

For qPCR, DNase-digested total RNA was reverse transcribed using a high-capacity cDNA reverse transcription kit (Applied Biosystems) as described in the manufacturer’s instructions. qPCR reactions were prepared using the SYBR Green Jumpstart Taq Readymix (Sigma) with the relevant Bcl-2 and Bax sense and antisense primers (Quiagen) and 10 nM fluorescein (Invitrogen). Reactions were amplified on an iCycler (Bio-Rad) using the primers above. The relative expression of target mRNAs was normalized to HPRT (Quiagen) and B2M (Quiagen) using the DDCT method[9] and expressed as the fold-change relative to the control.

Immunohistochemistry

Lung sections were immunostained using antibodies to caspase 3 active (R&D Systems AF835, 2.5 µg/ml), Carbonic Anhydrase IX (Abcam 15086, 1 µg/ml), Proliferating Cell Nuclear Antigen (Novocastra XD943, 0.5 µg/ml), Desmin (Dako M0760, 2.3 μg/ml), Vimentin (Dako M0725, 3.6 μg/ m l) and CD31 (Abcam ab9498, 3.75 μg/ml).

Sections were then incubated with a secondary biotinylated antibody, then with the streptavidin-HRP complex (ABC kit, Vector Laboratories). All methods used a chromogenic diaminobenzidine tetrahydrochloride end-point. All tinctoral stains (hematoxylin and eosin, elastic van Gieson (EVG), and picrosirius red) were performed according to standard protocols.[10]

Vascular scoring system

Remodeling of the vessel wall was quantified using Miller’s elastic stain (EVG) and picrosirius red. Phenotyping of the cellular component of the vessel wall was studied using CD31 (endothelium), desmin (vascular smooth muscle) and vimentin (mesenchyme) immunohistochemistry. Sections from control/malignancy (n=9) and lung volume reduction surgery (LVRS) (n=10) patients were assessed using a cumulative score grading system (Table 1). All muscular pulmonary arterioles (n=52 control, n=53 emphysema) on every slide were assessed and graded.

Statistics

Data were presented as mean±standard error unless otherwise stated. Statistical analysis of proliferation and 243

Howard et al.: Hypoxic pulmonary vascular remodelling

The hypoxia-proliferative capacity of the PASMChyp subpopulation is preserved despite reoxygenation, indicating a robust phenotype

We have previously demonstrated the differential effects of hypoxia on the proliferation of PASMChyp and PASMCnorm using both [3H]-Thymidine and direct cell counting.[7] In this study, [3H]-Thymidine incorporation alone was used to confirm proliferation in response to hypoxia in PASMChyp (Fig. 1A). When PASMChyp were returned to normoxic culture conditions for 72 hours, quiesced in normoxia for 48 hours and then re-exposed to hypoxia, they maintained their hypoxia-proliferative phenotype (Fig. 1B).

The PASMC and PASMC phenotype can be differentiated by the response to pharmacological stabilisation of HIF hyp

norm

We chose to examine whether the differential response of these sub-populations of PASMC to hypoxia was HIF dependent. Dimethyloxallyl glycine (DMOG) stabilizes HIF- 1 α by inhibiting its hydroxylation by prolylhydroxylases in the presence of oxygen, thereby preventing its targeting by pVHL for proteosomal degradation.

Using Western blot analysis, the stabilization of HIF1α was examined at different concentrations of DMOG (10 μM, 100 μM and 1 mM), with the effect of 100 μM appearing to match most closely the effect of hypoxia (Fig. 2A). The effects of DMOG on HIF-1α stabilization were similar in both PASMC hyp and PASMC norm (data not shown).

Using [3H]-Thymidine incorporation as a surrogate for proliferation, both hypoxia and DMOG (100 μM) had similar effects in PASMChyp (proliferation) and PASMCnorm (inhibition), suggesting that the differential effects of hypoxia on these two subpopulations was HIF-dependent (Fig. 2B and C). At the higher concentration of DMOG (1 mM), there was an inhibitory effect in both populations, suggesting either that this higher concentration was toxic or reflected higher levels of HIF stabilization more akin to anoxia. Conditioned medium from PASMChyp exposed to hypoxia did not alter proliferation in PASMCnorm, suggesting no role for hypoxia-induced autocrine growth factor release (data not shown).

244

Medial features

Intimal features

Hypertrophy/hyperplasia Radial smooth muscle cells/ reorientation Apoptosis Sclerosis/collagen deposition Leucocytes present throughout vessel wall Indistinct internal elastic lamina Indistinct external elastic lamina

Hypertrophy/hyperplasia Roughened endothelium Luminal thrombus Sclerosis/collagen deposition New internal elastic lamina

Scoring system – point allocation 2 points 1 point 0 points

>50% of vessel wall affected <50% of vessel wall affected Absent

Total vessel score

Grade

0–4 5–8 9–12 >13

Normal – 0 Mild - 1 Moderate – 2 Severe - 3

300 200 100 0

Non-re-oxygenated PASMCHyp [3H]-Thymidine CPM (% of normoxia)

Results

Table 1: Cumulative vessel remodeling grading score

[3H]-Thymidine CPM (% of normoxia)

apoptosis assays was performed using a Student’s t-test.

Differences between the control and emphysema vascular remodeling scores were assessed using a Mann–Whitney test, and presented as median [range]. ANOVA was used to assess caspase 3, PCNA and CA-IX immunostaining in emphysema specimens. All statistical analysis was performed using SPSS v12. Significance was assumed to be at P<0.05.

300

Re-oxygenated PASMCHyp

200 100 0

Normoxia Normoxia Hypoxia Hypoxia (B) (A) Figure 1: The effect of reoxygenation on the hypoxia-induced proliferative capacity of PASMC hyp. DNA synthesis measured by [ 3H]-Thymidine incorporation (expressed as % of normoxia) is increased in hypoxia whether cells were maintained in hypoxia (A) or returned to normoxia (B) for 72 hours prior to the experiment. n=4; * P<0.05 vs.normoxia.

Hypoxia and DMOG induce apoptosis in the PASMCnorm subpopulation Having demonstrated previously (by cell counting [7]) that the increase in [ 3 H]-Thymidine incorporation seen in PASMC hyp reflected cellular proliferation, we proceeded to investigate whether the inhibition of [3H]Thymidine by hypoxia and DMOG in PASMCnorm occurred as a result of apoptosis. Apoptosis was examined using TUNEL in parallel with Bcl-2 and Bax mRNA expression (Fig. 3). Although the overall rates of apoptosis were low, both hypoxia and DMOG (100 μM) increased the rate of PASMC norm apoptosis as assessed by TUNEL (Fig. 3A). Bcl-2 expression decreased significantly in response to DMOG, but showed only a nonsignificant trend to decrease in hypoxia (Fig. 3B). Bax expression increased in hypoxia and showed a nonsignificant trend to increase in response to DMOG (100 µM; Fig. 3C).

In all assays cells treated with staurosporin (50 nM) showed elevated levels of apoptosis compared to control conditions (Fig. 3).

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Norm

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DMOG 1mM 100 µm 10 µm

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Normoxia Hypoxia DMOG 100 µm

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bcl-2 expression (normalised to control)

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200

Bax expression (normalised to control)

%TUNEL +ve cells

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Figure 2: (A) Immunoblot of HIF-1α and HIF1β (loading control) in PASMCnorm. Cells were treated for 4 hours in either hypoxic medium (Hyp) or normoxic medium (Norm) with or without DMOG at concentrations of 10 μM, 100 μM and 1 mM. Hypoxia and DMOG at the two higher concentrations stabilized HIF-1α and the DMOG dose of 100 μM was chosen as being closest to the physiological level of hypoxia. Both hypoxia and DMOG (100 μM) inhibited [3H]-Thymidine incorporation in PASMCnorm (B) and increased [3H]-Thymidine incorporation in PASMChyp (C). n=3; *P<0.05 vs. normoxia.

xia

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Pulmonary arteries in emphysematous lung show increased remodeling and carbonic anhydrase IX expression

The distribution of vascular scores for control (n=9) and emphysema lungs (n=10) are shown in Figure 4. More extensive medial and intimal remodeling was seen in the pulmonary arteries in the emphysema lungs compared with controls (P<0.001). In the majority of emphysema specimens there was intimal hypertrophy and medial hyperplasia in the arteriolar walls. Reorientation of the matrix fibers within the media (to adopt a radial alignment) was observed in the LVRS samples, which resulted in PASMC invasion of the intima. These fibers Pulmonary Circulation | April-June 2012 | Vol 2 | No 2

ia

ox

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Figure 3: Apoptosis of PASMCnorm population assessed by TUNEL (A) and Bcl-2 and Bax (B and C). Hypoxia, DMOG (100 µM) and staurosporine (SP) (50 nM) induced apoptosis when assessed by TUNEL. Hypoxia and SP increased Bax expression and DMOG and SP decreased Bcl-2 expression. n=3; *P<0.05; ** P<0.01 compared with control.

stained positive for desmin and vimentin stained negative for CD31, suggesting a mesenchymal origin for these cells and matrix hyperplasia.

Apoptosis of medial cells, confirmed by active caspase 3, was widely distributed in arteriolar vessels in emphysema specimens, with the apoptotic cells nesting within areas of the vessel wall showing matrix hyperplasia (Fig. 5A and B). The amount of apoptosis as assessed by active caspase 3 and proliferation assessed by an increase in PCNA staining in vessels increased with higher remodeling scores (Fig. 5C, P<0.001). Furthermore, in the vessels with greatest remodeling there were higher levels of carbonic 245

Howard et al.: Hypoxic pulmonary vascular remodelling

anhydrase expression, suggesting an association with HIF stabilization (Fig. 5D and E, P<0.001).

Discussion

We have demonstrated previously that two distinct smooth muscle cell subtypes can be derived from the distal

Figure 4: Distribution of remodeling scores in control (malignancy) and lung volume reduction surgery (LVRS) specimens. Median (range) scores were LVRS 4 (1-9) LVRS vs. control 3 (1-6) for intimal score (P=0.006); LVRS 5 (1-11) vs. control 3 (0-7) for medial score (P<0.001); and LVRS 9 (2-20) vs.

6 (4-11) for total score (P<0.001).

pulmonary artery â&#x20AC;&#x201C; one that grows well under standard normoxic culture conditions (PASMCnorm) and another where growth is selected for by hypoxia (~3 kPa).[7] The present study provides further characterization of these cell types and shows that the hypoxia-selected cells display a robust and enduring phenotype, maintaining their proliferative response to hypoxia even after freeze-thawing and a period of sustained reoxygenation. This response was HIF dependent as competitive inhibition of prolyl hydroxylases by dimethyloxallylglycine (DMOG) mimicked both growth inhibition in the PASMC norm and growth stimulation in PASMChyp. We also show that hypoxia induces apoptosis in PASMCnorm via a HIF-dependent mechanism.

Following this observation, we show the presence and colocalization of both apoptotic and proliferating smooth muscle cells in the pulmonary arteries of emphysematous lung and that the extent of HIF activation increases with worsening severity of pulmonary vascular remodeling.

These results suggest that distinct smooth muscle cell populations present in the distal resistance pulmonary arteries may contribute differentially to vascular remodeling in hypoxic lung disease through apoptosis and proliferation and through HIF-transcribed pathways.

Hypoxic pulmonary vascular remodeling

Perhaps the commonest hypoxic lung disease that leads to pulmonary vascular remodeling is chronic obstructive

A

(C)

B

(E)

D 246

Figure 5: Representative immunohistochemistry of active Caspase 3 (A) and proliferating cell nuclear antigen (PCNA) (B) of muscular pulmonary arterioles taken from emphysema specimens. Active Caspase 3 and PCNA increased with vessel grade (C) (ANOVA, P<0.001).

Representative immunohistochemistry of carbonic anhydrase IX (CA-IX) expression (D), which increased with vessel grade (E) (ANOVA, P<0.001).

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pulmonary disease (COPD). In this condition there are clearly many other influences which may contribute to the vascular remodeling process, such as cigarette smoke and inflammation. Importantly, intimal thickening and an increase in the number of muscularized pulmonary arteries is found in patients with mild COPD and in healthy smokers even in the absence of airflow obstruction or systemic hypoxemia.[11-14] However, in patients with severe COPD,[15,16] the same histological changes are seen as those described in hypoxic animal models [4,5] and native highlanders, suggesting that alveolar hypoxia is a significant contributor to vascular remodeling.

Distinct smooth muscle cell phenotypes, with different proliferative and matrix-producing responses, have also been observed in the proximal bovine pulmonary artery. [17] These cells appear to respond differently during the development of hypoxia-induced pulmonary hypertension, with proliferation being restricted to cells that are negative for the cytoskeletal protein, metavinculin.[18] The neomuscularization seen in previously nonmuscularized arteries has been proposed to arise from the differentiation and proliferation of pericytes and intermediate cells or the recruitment of other cell types, either from the lung (interstitial fibroblasts) or the circulation (mesenchymal precursor cells).[6]

However, we and others have reported major functional differences between PASMCs isolated from the proximal and distal pulmonary circulation.[8,19] In addition, we have shown that two PASMC populations can be isolated from the distal pulmonary artery. One is selected for when freshly isolated cells are grown under “normal” culture conditions and undergo apoptosis on exposure to hypoxia; the other are cells isolated by a process of hypoxic selection when PASMCs are plated at early passage at low density and grown under hypoxic conditions for one to two weeks.[7] These cells subsequently proliferate in hypoxia. It is possible that sparse cells may undergo a functional switch to proliferate more readily under hypoxic conditions, but we show that this phenotype is maintained after freeze/ thawing and reoxygenation, making this appear less likely.

Studies of the hypoxic neonatal calf have shown a mixed phenotype of cells in the distal pulmonary circulation.[20] Scattered “myofibroblast” cells expressing lower levels of α-SM-actin, as well as other cells expressing haematopoeitic (CD45), leukocytic/monocytic (CD11b, CD14) and stemcell (cKit) markers were seen within the pulmonary artery media, and when cells were isolated from the distal pulmonary artery, two populations appeared. The first population was more slow-growing and was phenotypically characteristic of the SMC isolated from normotensive calves. The second appeared later in culture, but was subsequently much faster-growing, had a rhomboidal Pulmonary Circulation | April-June 2012 | Vol 2 | No 2

shape and expressed progenitor cell markers (CD34, CD73 and cKit) and was phenotypically and functionally distinct when compared with fibroblasts. These cells appeared to be mesenchymal in origin, constitutively expressing type I procollagen and with time in culture expressed the myofibroblast marker, α-SM-actin. The proliferation of the SMC population was inhibited by hypoxia as in our study, whereas the rhomboidal population was markedly stimulated by hypoxia.

While some similarities exist between Frid et al.[20] and our own study, there are significant differences in methodology.

Our cells were obtained from macroscopically normal lung and the two sub-populations were isolated under normoxic and hypoxic conditions, whereas the cells in the study by Frid et al.[20] were isolated from pulmonary hypertensive calves and both populations were derived under normoxia.

Both our cell populations had similar morphology unlike the populations derived by Frid et al.[20] which appeared to be progenitor like in origin. Nonetheless, both studies have led to the hypothesis that alveolar hypoxia may lead to a selective expansion of a subpopulation of cells, which in vitro exhibit increased proliferation under hypoxic conditions and that these cells may contribute to the remodeling of small peripheral pulmonary arteries. It is perhaps more important to note in our study that hypoxia also induced apoptosis in the normally resident PASMCnorm population, which further supports the hypothesis that these cells do not seem likely to contribute to remodeling.

The role of hypoxia-inducible factors

The data from the present study suggest that hypoxia, acting via a hypoxia-inducible factor (HIF)-dependent pathway, may be important in pulmonary vascular remodeling. HIF consists of α and β subunits. Under conditions of normoxia, the α subunit is hydroxylated by prolyl hydroxylases and subsequently targeted by von Hippel Lindau protein (pVHL) for proteosomal degradation. In hypoxia, HIF translocates to the nucleus as a heterodimer with the β subunit where it acts as a master regulator of the transcriptional response to hypoxia. The α subunit has three known isoforms – 1, 2 and 3.

HIF-1α ± mice display less muscularization of the distal pulmonary arteries on exposure to chronic hypoxia (three weeks at 10% O 2) as well as significantly less right ventricular hypertrophy compared with wild-type littermates.[21] This has also been replicated in HIF-2α +/mice in association with decreased circulating endothelin-1 (ET-1) and catecholamine levels. [22] Furthermore, an activating HIF-2α mutation has been associated with autosomal dominant erythrocytosis and pulmonary arterial hypertension in man. [23] Finally, mice homozygous for a mutation in the pVHL gene, mimicking the human form of Chuvash polycythaemia, developed spontaneous pulmonary 247

Howard et al.: Hypoxic pulmonary vascular remodelling

hypertension and vascular remodeling, which was suppressed by HIF-2α, but not HIF-1α, heterozygosity. [24] A role for HIF-3α in pulmonary vascular cells has not yet been established.

We have shown that the downstream response to HIF stabilization in hypoxia can differentiate two phenotypically distinct populations of smooth muscle cell. In one population, HIF stabilization results in apoptosis and in the other, proliferation. Both processes were seen in areas of lung affected by emphysema, therefore likely to be hypoxic, and both apoptosis and proliferation increased in prevalence with worsening remodeling. Furthermore, evidence of HIF stabilization, quantified by carbonic anhydrase IX expression, was greater in the more severely remodeled vessels, suggesting an association between HIF-dependent transcription, proliferation/apoptosis and remodeling in vivo. Expression profiling of lasermicrodissected murine intrapulmonary arteries has underlined the importance of HIF in hypoxic pulmonary vascular remodeling by demonstrating that 15 out of 21 upregulated genes after 24 hours of hypoxia carried potential hypoxia response elements.[25]

Although hypoxia may well be the dominant stimulus for HIF-dependent cellular responses, other stimuli may also be involved, both in vitro and in vivo. HIF has been implicated in nonhypoxic pulmonary arterial hypertension with immunohistological evidence of HIF-1α and β and VEGF/VEGFR2 (regulated by HIF) overexpression in plexiform lesions of patients with idiopathic pulmonary arterial hypertension.[26] In COPD, pulmonary vascular remodeling can be identified in patients with mild lung disease, suggesting a direct vascular effect from cigarette smoking or inflammation, independent from hypoxia. [1] Indeed, studies in proximal PASMCs have shown that growth factors may induce higher levels of HIF-1α than hypoxia itself and that they act in synergism with hypoxia to increase HIF-1α.[27,28]

To our knowledge the apoptotic effect of hypoxia on distal PASMC has not been described before. HIF induction in both tumor and nontumor cell lines under conditions of anoxia has been shown to induce apoptosis via BNIP3, a member of the Bcl-2 family;[29] and bovine aortic smooth muscle cells also undergo apoptosis via a HIF-dependent mechanism. [30] Thus, there appears to be consensus that HIF is a key orchestrator of PASMC behavior, either controlled by hypoxia or growth factors, but that its effects are contextspecific, both with regard to the cell type and the severity of hypoxia.

Although debate continues over the importance of alveolar hypoxia vs inflammation in driving vascular changes in chronic lung disease, patients with germline homozygosity 248

for a hypomorphic VHL allele, which results in defective HIF degradation show an exaggerated acute hypoxic pulmonary vasoconstrictor response on a background of increased pulmonary vascular tone. Despite the fact that the precise relationship between hypoxic pulmonary vasoconstriction and chronic pulmonary vascular remodeling has not being established, this observation suggests an important role for hypoxia acting through HIF in pulmonary vascular homeostasis.[31]

conclusion

We have demonstrated that the predominant smooth muscle cell present in the distal pulmonary artery media undergoes apoptosis when subjected to hypoxia, but that a more sparse resident cell population with similar morphology can be isolated from normal distal pulmonary artery media, which displays hypoxia-mediated proliferation. Both these processes are active in the media of vessels undergoing remodeling in hypoxic lung and appear to be under HIF-dependent control both in vitro and in vivo. HIF downregulation, induced either pharmacologically or through treatment with oxygen or growth factor inhibition, may be an important target for the treatment of pulmonary vascular remodeling in hypoxic lung diseases.

Acknowledgment

Dr. Luke Howard was supported by a grant (no. FS/04/057/17496) from the British Heart Foundation.

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Source of Support: British Heart Foundation, Grant no. FS/04/057/17496, Conflict of Interest: None declared.

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C ase Repor t

Partial anomalous pulmonary venous return presenting with adult-onset pulmonary hypertension Edmund H. Sears, Jason M. Aliotta, and James R. Klinger Division of Pulmonary, Sleep and Critical Care Medicine, Rhode Island Hospital, Alpert Medical School of Brown University, Providence, Rhode Island, USA

Abstract Partial anomalous pulmonary venous return (PAPVR) is a rare cause of adult onset pulmonary arterial hypertension (PAH) that can present with a wide spectrum of severity from early childhood throughout adult life. We present two patients with PAH secondary to PAPVR who reflect this range of disease. The diagnosis and treatment of PAPVR and its role in pulmonary vascular disease is discussed. Cardiac and pulmonary physicians should be aware of this entity and its diagnosis and management options.

Key Words: pulmonary arterial hypertension, congenital disease, vascular abnormalities

Partial anomalous pulmonary venous return is an uncommon congenital abnormality in which some, but not all, of the pulmonary veins connect to the right atrium or one of its venous tributaries. We discuss two adult patients who presented with pulmonary hypertension, and evidence of right ventricular hypertrophy and dysfunction.

Case Reports Case 1

A 55-year-old man with no significant past medical history presented to our institution with several months of episodic exertional lightheadedness associated with neck pain and diaphoresis. His outpatient workup included a normal EKG, conventional and stress echocardiograms, and a cardiac event monitor which revealed no arrhythmias.

He was admitted to the hospital as his symptoms were becoming more frequent and was found to be in atrial fibrillation. Laboratory studies, including cardiac enzymes, thyroid function tests, liver function tests, electrolytes, and complete blood count, were all within normal limits.

Echocardiography revealed right ventricular hypokinesis and dilation, pulmonary arterial hypertension with

Address correspondence to:

Dr. Edmund H. Sears Division of Pulmonary, Sleep and Critical Care Medicine Rhode Island Hospital 593 Eddy Street Providence, RI 02903, USA Email: esears@lifespan.org 250

an estimated pulmonary artery systolic pressure of 45–55 mmHg, and a normal left ventricular size and function. These findings were new compared with the echocardiogram performed 18 months prior. Pulmonary function tests, including diffusion capacity of the lung for carbon monoxide (DLco), were normal but a 6-Minute Walk test revealed a fall in oxygen saturation from 97–91% on room air. A CT pulmonary angiogram demonstrated no evidence of thromboembolic disease; however, a pulmonary vein communicating from the left upper lobe to the left brachiocephalic vein was discovered (Fig. 1).

Right heart catheterization was performed revealing mean pulmonary artery pressure (PAM) of 16 mmHg; pulmonary artery systolic pressure (PAS) of 27 mmHg, pulmonary artery diastolic pressure (PAD) of 10 mmHg, and pulmonary capillary occlusion pressure (PAOP) of 12 mmHg. Cardiac output was 6.64 l/minute when measured by thermodilution and 5.46 l/minute when measured using the Fick equation. With exercise, mean pulmonary artery pressure increased to 39 mmHg, with wedge remaining Access this article online

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Website: www.pulmonarycirculation.org DOI: 10.4103/2045-8932.97637 How to cite this article: Sears EH, Aliotta JM, Klinger JR. Partial anomalous pulmonary venous return presenting with adultonset pulmonary hypertension. Pulm Circ 2012;2:250-5.

Pulmonary Circulation | April-June 2012 | Vol 2 | No 2

Sears et al.: PAPVR causing adult pulmonary hypertension

Figure 1: Contrast-enhanced CT scan of the chest demonstrating the presence of an anomalous pulmonary vein (white arrow) arising from the left upper lobe of the lung and connecting to the left brachiocephalic vein.

at 12 mmHg, indicating the presence of exercise-induced pulmonary hypertension.

A cardiac MRI with gadolinium enhancement revealed the presence of the anomalous pulmonary vein arising from the left apical posterior and anterior segments of the left upper lobe and draining into the left brachiocephalic vein (Fig. 2), as well as right ventricular hypertrophy and dilation. The estimated shunt fraction (Qp:Qs), by using volumetric measurements and velocity-encoded imaging, was 1.28:1.

His right ventricular dysfunction and exercise-induced pulmonary hypertension was felt to be the result of the additional blood volume being shunted through this anomalous circuit; however, given his stable symptoms, the patient elected to defer surgery. The patient began sildenafil treatment with good symptomatic response and continues close medical observation.

Case 2

A 33-year-old female with a history of well-controlled seizure disorder presented to a walk-in clinic with a year of increasing exertional dyspnea and was found to have cardiomegaly by chest radiography. She was sent to a local community hospital for further workup where an EKG showed right axis deviation, bundle branch block, and evidence of right ventricular hypertrophy. An echocardiogram confirmed right ventricular dilation, hypertrophy, and reduced function with an estimated peak PA pressure of 80 mmHg increasing to 90–100 mmHg with exercise. PFTs revealed moderate obstruction, but normal volumes and gas exchange. She walked 590 feet in six minutes with a slight decrease in pulse oximetry from 96% at rest to 94% on room air.

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Figure 2: Cardiac MRI with gadolinium enhancement demonstrating the presence of the anomalous pulmonary vein (white arrow) arising from the left apical posterior and anterior segments of the left upper lobe and connecting to the left brachiocephalic vein.

Ventilation/perfusion lung scan showed matched perfusion and ventilation without defects. A CT pulmonary angiogram revealed no evidence of pulmonary embolism, although did note an enlarged azygous and hemiazygous vein with evidence of azygous continuation of the IVC. Right heart catheterization revealed a PAM of 60, PAS of 90, PAD of 45, and PCWP of 10, with a cardiac output by thermodilution of 5.0 l/minute (CI 2.7 l/m2). A diagnosis of idiopathic pulmonary arterial hypertension was made and the patient was referred to our institution for further care.

Continuous intravenous infusion of epoprostenol was suggested, but the patient wished to try oral therapy first and was instead started on bosentan and anticoagulation with good clinical improvement, and her 6-Minute Walk Distance improved to 1,180 feet. However, repeat echocardiography continued to show elevated PAP and RV dilatation. Inhaled iloprost, 5 µg 6 times daily, was added to her bosentan therapy, but she had difficulty completing more than four treatments a day. Following addition of inhaled iloprost, her 6-Minute Walk Distance improved to 1,460 meters and peak PAP measured by echocardiogram decreased to 43 mmHg. However, her RV pressures remained grossly elevated and she began to develop signs of right heart failure and marked elevation of BNP.

She was referred for lung transplant evaluation, but was declined because of occasional tobacco use. Sildenafil was added to her treatment regiment, but her right heart failure did not improve. Repeat right heart catheterization done at our institution revealed PAM of 62 mmHg, PAS of 93 mmHg, PAD of 44 mmHg, and PAOP of 14 mmHg. She again declined intravenous epoprostenol, but agreed to initiation of subcutaneous treprostinil infusion, and inhaled iloprost was discontinued. Despite some initial 251

Sears et al.: PAPVR causing adult pulmonary hypertension

improvement on treprostinil, her condition progressed to overt right heart failure and she was hospitalized for treatment of peripheral edema and ascites. At the time of this admission, a chest radiograph showed a new rightsided aberrant pulmonary artery in a curved “scimitar” shape. Further review of her prior noncontrast chest CT obtained as part of her evaluation for lung transplantation revealed evidence of aberrant drainage of the right lung with a “scimitar vein” (Fig. 3). Cardiac MRI was obtained showing that the abnormal vein originated from aberrant right upper and middle pulmonary veins which drained into a right atrial-hepatic vein at the level of the diaphragm (Fig. 4). Moderate hypoplasia of the right lung was also noted. The Qp:Qs ratio was estimated at 1.45:1. Copies of her original contrast enhanced CT were not able to be obtained, but PAPVR was not reported.

Figure 3: Noncontrast CT of the chest demonstrating right sided “scimitar vein” (white arrow) draining toward diaphragm.

The patient was again referred for lung transplantation and eventually underwent heart-lung transplantation, with visualization of the scimitar vein at the time of surgery.

Discussion

Partial anomalous pulmonary venous return (PAPVR) occurs when some of the pulmonary veins connect to the right atrium or one of its venous tributaries rather than the left atrium. PAPVR has traditionally been associated with atrial septal defects (ASD), found in 80% of patients in one pediatric series.[1] It has also been thought that most anomalous pulmonary veins arise mainly from the right lung; 80% of those studied in the above series had a right-sided anomaly, connecting primarily to the superior vena cava (SVC), less commonly to the right atrium (RA) or inferior vena cava (IVC). Only 3–8% have been reported to originate from the left lung, connecting to the left brachiocephalic in all cases.[1,2] PAPVR is often associated with other cardiac defects and is reported in certain congenital syndromes, such as Turner’s syndrome (monosomy X).[3] The scimitar syndrome, as seen in the second case, is a clinical association of a right-sided anomalous pulmonary vein connecting to the RA or IVC with other anatomic abnormalities, including hypoplasia of the right lung and right pulmonary artery, dextroposition, and/or dextrorotation of the heart.

PAPVR is often clinically silent, and an autopsy series in the 1950s found it in 0.4% of postmortem examinations.[4] More recently, retrospective reviews of computed tomography (CT) series in adults receiving imaging for other indications have identified rates of 0.1–0.2% in the adult population.[5,6] Interestingly in these studies, almost half of the identified anomalies were left sided, and only right upper lobe PAPVR was associated with ASD6. This indicates the pediatric 252

Figure 4: Cardiac MRI demonstrating right sided “scimitar vein” (white arrow) draining into right atrial-hepatic vein.

and adult populations who present with PAPVR may be significantly different.

Embryologically, PAPVR arises from failure of primitive lung drainage to regress properly (Fig. 5). In the human embryo, the primordial lung bud has no connection to the heart, draining venous blood from the pulmonary vascular bed through systemic veins (Days 27–29 of gestation). By Days 32–33, the common pulmonary vein forms from the left atrium, establishing a connection with the pulmonary vascular bed. Once a direct connection with the heart is established, connections with systemic veins begin to disappear (Day 40), allowing venous blood from the developing lung to drain into the common pulmonary vein and left atrium via four individual pulmonary veins. By term, the common pulmonary vein incorporates into the left atrium and the pulmonary veins connect directly to the heart. Failure of one or more of the pulmonary veins to establish a connection with the common pulmonary vein, instead maintaining persistent systemic venous connection, results in PAPVR.[7] Pulmonary Circulation | April-June 2012 | Vol 2 | No 2

Sears et al.: PAPVR causing adult pulmonary hypertension

(A)

(B)

(C)

(D)

Figure 5: Embryology of PAPVR. (A) At post-conceptional Days 27–29, the primordial lung buds drain through a vascular bed to the cardinal veins, which will develop into systemic veins. (B) By Days 32–33, the common pulmonary vein forms from the left atrium and establishes a connection with the pulmonary venous circulation. Pulmonary venous connections to systemic veins begin to regress and pulmonary venous blood drains into the common pulmonary vein.

(C) By Day 40, the primitive connections from the pulmonary vascular bed to the cardinal veins should have regressed, but in PAPVR anomalous connections persist. (D) At term, the anomalous connection will have developed into anomalous pulmonary veins draining most commonly into the SVC on the right, or the brachiocephalic vein on the left.

This persistent systemic venous connection acts similarly to a left-to-right shunt, in that a portion of right ventricular output is continuously recirculated and oxygenated blood is returned to the right heart without traveling to the systemic circulation. Over time, which may be years to decades, the increase in pulmonary blood flow can lead to progressive remodeling of the pulmonary circulation and increased pulmonary vascular resistance. If severe enough, pulmonary arterial hypertension (PAH) and right ventricular volume overload occur, leading to RV failure.[8] Patients can present anytime from infancy to the seventh decade, depending on the size of the shunt, as well as the presence of other cardiovascular anomalies and medical conditions.

Noninvasive imaging and diagnosis of PAPVR continues to be an evolving field. Chest radiography has limited sensitivity, but CT scanning is an extremely effective diagnostic modality, especially when iodinated contrast is used.[6] Transthoracic echocardiography (TTE), which is often obtained in patients being evaluated for cardiac symptoms, cannot reliably delineate pulmonary venous anatomy due to technical limitations. In pediatric patients, approximately one third of cases of PAPVR are missed by TTE[9] and the proportion is likely higher in adults although it has not been specifically studied. Transesophageal echocardiography (TEE), by contrast, is quite sensitive and specific in the hands of experienced operators.[10] Finally, cardiac MRI is increasingly being used as it can identify multiple defects and better define structural abnormalities without ionizing radiation. It has also proven useful in providing noninvasive, but accurate, quantification of shunt volume.[11,12] Shunt volume is expressed as a ratio of the flow through the pulmonary arterial bed to the flow through the systemic arterial bed (Qp:Qs). The “gold standard” for quantifying Qp:Qs has been by determining blood flow using a modified Fick equation during right and left heart Pulmonary Circulation | April-June 2012 | Vol 2 | No 2

catheterizations. However, shunt quantification using MRI phase velocity mapping correlated very well with ventricular volumetric data obtained by MRI as well as oximetry data from cardiac catheterizations.[12] Additionally, cardiac MRI may provide better anatomic definition. A study of 13 patients with PAPVR showed that the diagnosis was made with cardiac MRI in three patients whose previous echocardiographic or catheter studies were either nondiagnostic or misinterpreted.[13]

In pediatric patients, PAPVR is usually treated with surgical correction if large enough to create a significant shunt. In general, patients that have a Qp:Qs of 1:1.5 or more are considered for surgical repair as they are more likely to develop pulmonary hypertension and right ventricular failure,[14] although this cutoff has not been subject to rigorous study. In adult patients, the criteria for surgical repair are less clear cut. Those who have already developed symptoms due to shunting, or have evidence of right-sided volume overload, regardless of the magnitude of the shunt, are also considered for surgery. However, in asymptomatic patients with a low shunt fraction and no clinical or echocardiographic evidence of right heart overload, pulmonary hypertension, or other symptoms, surgery may be unnecessary.[15]

The general principle of surgical repair is to separate the pulmonary venous system from the systemic system. This involves anastomosing the aberrant pulmonary vein or veins either to the left atrium (recreating normal anatomy) or, more commonly, to the right atrial appendage. In the latter case, a GORE-TEX™ graft or intracardiac baffle and ASD would be used to direct blood to the left atrium (Fig. 6).

Surgery is generally effective at correcting the abnormal shunt and its associated symptoms; moreover, the complication rate reported in literature is low. In one of 253

Sears et al.: PAPVR causing adult pulmonary hypertension

F i g u re 6 : S u rg i c a l a p p r o a c h e s f o r correcting PAPVR. For anomalous veins connecting portions of the left lung to the left brachiocephalic vein, the anomalous vein is reconnected directly to the LA. For anomalous veins connecting portions of the right lung to the SVC, the SVC is reconnected to the right atrial appendage and blood from the anomalous vein is shunted, with the help of a pericardial patch, though a newly-created (or enlarged) ASD into the LA. Alternatively, there have been reports of GORE-TEX™ grafts being used to create a conduit crossing the right atrium.

the largest series consisting of 306 patients, both adult and pediatric, who underwent surgical correction of PAPVR, Alsoufi et al. reported no deaths.[16] Four patients required reoperation during the follow-up period, and most patients remained free from late complications including pulmonary vein stenosis (86%) and vena caval obstruction (97.8%).[16] Unfortunately, patients with repaired scimitar syndrome were much more likely to have postoperative pulmonary venous obstruction.

A recent, single-center review of 43 adult patients with isolated partial anomalous venous return (i.e., those without ASD or other anomalies) similarly found very good surgical outcomes in the 28 patients who required surgery, with no deaths reported, and few complications. [15] This study also noted that patients with a single anomalous pulmonary vein often had a benign course, not requiring surgery.

Finally, the majority of patients who did require surgery had improvement in echocardiographic measurements of pulmonary artery pressures and right-sided heart function. [15] Unfortunately, in patients with severe PAH and elevated pulmonary vascular resistance, reparative surgery is unlikely to alter disease course as the extensive vascular remodeling is unlikely to be reversible. In these patients, 254

such as our second case, heart-lung transplant may be the only curative option. Unfortunately, the 10-year survival of heart-lung transplant remains only 30–40%, so the timing of transplant in order to optimize survival remains a difficult decision.[17] There is also increasing interest in various “treat-then-repair” strategies combining medical and surgical management, but little data currently exist.

Some patients may be candidates for catheter embolization of the anomalous vein(s), providing that there is some connection from the anomalous vein to the LA that can accommodate the venous drainage after the anomalous vein has been embolized. Forbess et al. reported significant clinical and hemodynamic improvement in two patients with PAPVR after catheter guided embolization.[18] In both patients, symptoms improved by two New York Heart Association classes with trace to no residual flow through the anomalous connection. Similar results were reported in another study of two patients who underwent catheter embolization.[19] In both pediatric and adult patients there has been increasing interest in medical therapy for those in who surgical repair is high risk. While no prospective, controlled trials have been conducted, small retrospective and observational studies of patients with PAH secondary Pulmonary Circulation | April-June 2012 | Vol 2 | No 2

Sears et al.: PAPVR causing adult pulmonary hypertension

to congenital heart diseases, including PAPVR, or of patients who have shown clinical and hemodynamic improvements with prostaglandins, phosphodiesterase inhibitors, and bosentan, have been conducted.[17] Patients such as our first case, who present late in adult life with minor elevation in PA pressure and preserved exercise capacity, may be candidates for medical therapy with newly approved medications for PAH. However, clinical response to treatment and pulmonary hemodynamics should be monitored carefully in these patients, and surgery should be reconsidered if pulmonary hypertension progresses.

In conclusion, PAPVR is a rare congenital condition which is usually recognized in the pediatric population but may also be diagnosed during adulthood in patients who develop PAH, or in asymptomatic patients undergoing pulmonary vascular studies for other indications.

The widespread use of more sophisticated diagnostic techniques such as CT pulmonary angiography, TEE, and cardiac MRI may be increasing the frequency with which this condition is diagnosed. The disease, as demonstrated by the cases discussed, can present with variable degrees of severity. In asymptomatic patients without signs of significant PAH or right-heart overload, no intervention may be necessary. However, these patients should be followed for signs of progressive pulmonary hypertension. For patients with mild-tomoderate pulmonary hypertension, surgical repair is usually safe and effective, although catheter guided and medical therapies may play an increasing role. Finally, in patients who have progress to severe pulmonary hypertension, lung or heart-lung transplantation may be necessary. Physicians who diagnose and treat adult patients with PAH should consider PAPVR as a potential etiology, particularly in those with a history of ASD or other congenital pulmonary vascular or heart diseases.

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Senocak F, Ozme S, Bilgic A, Ozkutlu S, Ozer S, Saraçlar M. Partial anomalous pulmonary venous return. Evaluation of 51 cases. Jpn Heart J 1994; 35:43-50.

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Ho VB, Bakalov VK, Cooley M, Van PL, Hood MN, Burklow TR, et al. Major vascular anomalies in Turner syndrome: prevalence and magnetic resonance angiographic features. Circulation 2004;110:1694-700.

Healey JE Jr. An anatomic survey of anomalous pulmonary veins: their clinical significance. J Thorac Surg 1952;23:433-44.

Haramati LB, Moche IE, Rivera VT, Patel PV, Heyneman L, McAdams HP, et al. Computed tomography of partial anomalous pulmonary venous connection in adults. J Comput Assist Tomogr 2003;27:743-9.

Ho ML, Bhalla S, Bierhals A, Gutierrez F. MDCT of partial anomalous pulmonary venous return (PAPVR) in adults. J Thorac Imaging 2009;24:89-95.

Fraser RS, Paré PD. Fraser and Paré’s diagnosis of diseases of the chest.

4th ed. Philadelphia: W.B. Saunders; 1999. p. 637-75.

Diller GP, Gatzoulis MA. Pulmonary vascular disease in adults with congenital heart disease. Circulation 2007;115:1039-50.

Wong ML, McCrindle BW, Mota C, Smallhorn JF. Echocardiographic evaluation of partial anomalous pulmonary venous drainage. J Am Coll Cardiol 1995;26:503-7.

Ammash NM, Seward JB, Warnes CA, Connolly HM, O’Leary PW, Danielson GK. Partial anomalous pulmonary venous connection: diagnosis by transesophageal echocardiography. J Am Coll Cardiol 1997;29:1351-8.

Petersen SE, Voigtlander T, Kreitner KF, Kalden P, Wittlinger T, Scharhag J, et al. Quantification of shunt volumes in congenital heart diseases using a breath-hold MR phase contrast technique–comparison with oximetry. Int J Cardiovasc Imaging 2002;18:53-60.

Debl K, Djavidani B, Buchner S, Heinicke N, Poschenrieder F, Feuerbach S, et al. Quantification of left-to-right shunting in adult congenital heart disease: phase-contrast cine MRI compared with invasive oximetry. Br J Radiol 2009;82:386-91.

Prasad SK, Soukias N, Hornung T, Khan M, Pennell DJ, Gatzoulis MA, et al. Role of magnetic resonance angiography in the diagnosis of major aortopulmonary collateral arteries and partial anomalous pulmonary venous drainage. Circulation 2004;109:207-14.

Toyoshima M, Sato A, Fukumoto Y, Taniguchi M, Imokawa S, Takayama S, et al. Partial anomalous pulmonary venous return showing anomalous venous return to the azygos vein. Intern Med 1992;31:1112-6.

Majdalany DS, Phillips SD, Dearani JA, Connolly HM, Warnes CA. Isolated partial anomalous pulmonary venous connections in adults: Twenty-year experience. Congenit Heart Dis 2010;5:537-45.

Alsoufi B, Cai S, Van Arsdell GS, Williams WG, Caldarone CA, Coles JG. Outcomes after surgical treatment of children with partial anomalous pulmonary venous connection. Ann Thorac Surg 2007;84:2020-6.

Gatzoulis MA, Alonso-Gonzalez R, Beghetti M. Pulmonary arterial hypertension in paediatric and adult patients with congenital heart disease.

Eur Respir Rev 2009;18:154-61.

Forbess LW, O’Laughlin MP, Harrison JK. Partially anomalous pulmonary venous connection: Demonstration of dual drainage allowing nonsurgical correction. Cathet Cardiovasc Diagn 1998;44:330-5.

Dahnert I, Riede FT, Kostelka M. Partial anomalous pulmonary venous drainage of the left upper pulmonary vein – catheter interventional treatment is sometimes possible. Clin Res Cardiol 2007;96:511-3.

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

255

C ase Repor t

Massive dilatation of the pulmonary artery in association with pulmonic stenosis and pulmonary hypertension Sejal Morjaria1, Dan Grinnan2, and Norbert Voelkel2 1

Department of Internal Medicine, and 2Department of Pulmonary & Critical Care Medicine, Virginia Commonwealth University Health System, Richmond, Virginia, USA

ABSTRACT Congenital pulmonary valve stenosis has been associated with the development of massive pulmonary arterial (PA) dilatation. Over time, this dilatation may distort surrounding structures and lead to compression of the left main coronary artery (LMCA) or the left mainstem bronchus. In this report, we describe a patient with a history of chronic thromboembolic pulmonary hypertension (CTEPH) and congenital pulmonic stenosis with massive PA dilatation. He develops exertional chest pain, presenting an unusual differential diagnosis. Novel diagnostic testing was performed to help narrow the differential diagnosis, and the patient responded well to pulmonary vasodilator treatment for progressive pulmonary hypertension.

Key Words: massive pulmonary arterial dilatation, left main coronary artery, chronic thromboembolic pulmonary hypertension

Congenital pulmonary valve stenosis has been associated with the development of massive pulmonary arterial (PA) dilatation. Over time, this dilatation may distort surrounding structures and lead to compression of the left main coronary artery (LMCA) or the left mainstem bronchus.[1] Symptoms from such compression may mimic angina or asthma, respectively. In addition, significant pulmonic stenosis may lead to symptoms of right heart failure including dyspnea, lower extremity edema, and chest pain.

Patients with pulmonary valve stenosis do not typically develop severe pulmonary hypertension. In this report, we describe a patient with a history of chronic thromboembolic pulmonary hypertension (CTEPH) and congenital pulmonic stenosis with massive PA dilatation. The case presents an unusual differential diagnosis, and novel diagnostic testing was performed to help narrow the differential diagnosis.

CASE REPORT

A 55-year-old man with history of pulmonic stenosis

Address correspondence to: Dr. Sejal Morjaria Department of Internal Medicine Virginia Commonwealth University Health System 1250 East Marshall Street Richmond, VA 23219, USA Email: smorjaria@mcvh-vcu.edu 256

(postvalvuloplasty in 2004) and chronic thromboembolic pulmonary hypertension due to antiphospholipid antibody syndrome, presented with two months of progressive chest pain and exertional dyspnea. A CT scan of the chest was remarkable for massive dilatation of the proximal PA (Fig. 1) but did not reveal evidence of acute pulmonary embolism. Our subsequent differential diagnosis included progressive pulmonic stenosis with right ventricular failure, progressive pulmonary hypertension with right ventricular failure, LMCA compression from massive PA dilatation, and left mainstem bronchus compression from PA dilatation. These potential diagnoses were evaluated through a series of diagnostic tests. First, a cardiac MRI revealed a bicuspid pulmonic valve with a peak gradient estimated at 18 mmHg and an impaired right ventricular ejection fraction (27%). Subsequently, a right heart catheterization revealed pulmonary arterial pressure of 93/38 (52), pulmonary capillary wedge pressure of 18 mmHg, right atrial pressure of 18, Qp:Qs = 1:1, and a Access this article online

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Website: www.pulmonarycirculation.org DOI: 10.4103/2045-8932.97640 How to cite this article: Morjaria S, Grinnan D, Voelkel N. Massive dilatation of the pulmonary artery in association with pulmonic stenosis and pulmonary hypertension. Pulm Circ 2012;2:256-7.

Pulmonary Circulation | April-June 2012 | Vol 2 | No 2

Morjaria et al.: Dilatation of PA w/PH

A

B

Figure 1: CT images showing massive dilatation of the proximal pulmonary artery on sagittal (A) and axial (B) images, with severe compression of the left mainstem bronchus.

gradient across the pulmonic valve of 15 mmHg without significant regurgitation. Next, as attention shifted to the potential mechanical complications caused by this massive PA dilatation, pulmonary function testing was performed but did not show obstructive lung disease as would be expected if left main bronchus narrowing contributed significantly to his symptoms. Last, to evaluate the LMCA, a CT angiogram of the coronary arteries was performed (Fig. 2). The CT angiogram showed effacement of the LMCA, but no compression or flow limitation. The most significant finding from these diagnostic tests was severe pulmonary hypertension with elevated pulmonary vascular resistance and without either a significant gradient across the pulmonic valve or physiologic evidence of significant mechanical compression of the surrounding structures.

Therefore, we started the patient on bosentan (an endothelin receptor blocker) as his clot burden is distal and not amenable to thromboendarterectomy. Six weeks after the initiation of treatment, the patient had resolution of chest pain, improvement in functional class, a decrease in his BNP to 43 pg/ml, and a decrease in his right ventricular diastolic diameter on transthoracic echocardiogram from 6.2 cm to 5.2 cm. These improvements have sustained over the past year.

DISCUSSION

Our case has several unique aspects. First, an isolated bicuspid pulmonic valve in the absence of other congenital defects is exceedingly rare.[2] While it is associated with maternal rubella infection,[3] our patient had no knowledge of any complication during his mother’s pregnancy. As in patients with bicuspid aortic valves, a bicuspid pulmonic valve tends to calcify over time, leading to significant stenosis.[4] Because of the very low resistance and high compliance of the main pulmonary artery, the “jet” of blood created by pulmonic stenosis can lead to remarkable dilatation of the proximal pulmonary artery (compared with the proximal aorta in patients with aortic stenosis). In Pulmonary Circulation | April-June 2012 | Vol 2 | No 2

Figure 2: CT angiography of the coronary arteries shows the enlarged pulmonary artery (PA) effacing, but not compressing, the left main coronary artery (LMCA) as it leaves the aortic root (A).

our patient, massive enlargement of the PA led to narrowing of the left mainstem bronchus as well as effacement of the LMCA. Because both the size of the proximal pulmonary artery and the extent of pulmonary hypertension are associated with the development of LMCA compression syndrome, and our patient has dramatic PA enlargement and pulmonary hypertension, we felt that he was at significant risk of LMCA compression syndrome.[5] He was reluctant to undergo left heart catheterization, having just had a right heart catheterization. Therefore, CT angiography of the coronary arteries was performed to evaluate for LMCA compression syndrome. This is a novel use of this diagnostic tool and helped us to narrow the patient’s differential diagnosis. In conclusion, we present a patient with massive pulmonary artery dilatation and concurrent CTEPH, presenting a unique and difficult diagnostic challenge which led to the initiation of pulmonary vasodilator treatment and subsequent resolution of his presenting symptoms.

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Hungate RG, Newman B, Meza MP. 51. Left mainstem bronchial narrowing: a vascular compression syndrome? Evaluation by magnetic resonance imaging. Pediatr Radiol 1998;28:527-32.

Jashari R, Van Hoeck B, Goffin Y, Vanderkelen A The incidence of congenital bicuspid or bileaflet and quadricuspid or quadrileaflet arterial valves in 3,861 donor hearts in the European Homograft Bank. J Heart Valve Dis 2009;18:337-44.

Stoermer J, Galal O, Arafa R, Rupprath G, Galal I, Neifer B. A rare combination: persistent ductus arteriosus and pulmonary stenoses. Is there a correlation with rubella embryopathy. Klin Padiatr 1989;201:28-32.

Magnoni M, Turri C, Roghi A, Merlanti B, Maseri A. An inverted location of the bicuspid valve disease: A variant of a variant. Circulation 2011;124:e513-5.

Mesquita SM, Castro CR, Ikari NM, Oliveira SA, Lopes AA. Likelihood of left main coronary artery compression basaed on pulm trunk diameter in patients with pulm hypertension. Am J Med 2004;116:369-74.

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

257

Hi st or y and W h o ’s W h o

The herd shot “round the world” Robert F. Grover Emeritus Professor of Medicine, Department of Medicine, Division of Cardiology, University of Colorado School of Medicine, Denver, Colorado, USA

Colorado is known for its “Fourteeners,” mountain peaks over 14,000 feet tall; there are more than 50 of them.

One is Mount Evans, standing at 14,260 feet conveniently located just 60 miles west of Denver. I’m going to take you back in time so you may witness the remarkable adventure that played out near the summit of that mountain half a century ago.

My story begins on the third day of July in the year 1960.

Early that morning, one could have seen Bill Wilson driving a heavily loaded, 18-wheel cattle truck as he began his ascent up the winding road that leads to the summit of the mountain. However, his destination was Summit Lake Flats (Fig. 1A), a broad stretch of tundra lying just below the peak but well above tree line at an elevation of 12,700 feet. He was carrying a load of 10 yearling Hereford steers, 12 Rambouillet/Suffolk spring lambs, and four tons of hay, straw, and pellet feed (Fig. 1B). Bill had been instructed to deliver his cargo to a portable corral that my colleague Jack Reeves and I had fabricated down in Denver. Estelle Grover, my wife, and Donald Will, a veterinarian and previous collaborator in 1958,[1] were coinvestigators on this project.

Assembling the corral within reach of Summit Lake, we now had a potential source of fresh drinking water for our livestock. Our next challenge was to find a way to establish a continuous supply water from the lake to the water troughs for the next two months; we took on this challenge by laying out 1,200 feet of flexible pipe that literally siphoned water out of the lake and over to the corral (Fig. 2). The process was smooth and efficient, except for the occasional night when it froze up; we hadn’t counted on that. As for electric power, we brought in a portable generator. We completed all of these preparations a few days before the truck arrived; the only thing left was to move the three tons of live cargo and four tons of food from the cattle truck over to the corral.

Using a four-wheel drive pickup truck to move the steers, one at a time, over to the corral gate, we turned them Address correspondence to: Dr. Robert F. Grover 191 Century Lane Arroyo Grande, CA 93420, USA Email: drgrover@charter.net 258

loose into one section of the corral. As for the lambs, we unceremoniously hoisted them over the side of another section of the corral, while the hay, straw, and feed were packed into a separate section (Fig. 3). At the end of the day, we could say that we had successfully launched the high altitude phase of the project.

About now I hope you’re asking yourselves what in the world this is all about, placing cattle on a mountain top.

Well, back in the late 1800s cattlemen moving west traveled to the Rocky Mountains where they discovered broad, intermountain valleys lush with tall grass, such as South Park in the shadow of Pikes Peak. Believing this area to be perfect grazing land, they imported thousands of cattle from low altitude, Texas, Oklahoma, Kansas: but they were soon in for a surprise. Within the first six months of ranching, 5% to 10% of their cattle died! While examining the afflicted animals, they noticed a strange swelling at the base of the neck and under the sternum. Meat-cutters call this area “the brisket of beef,” and so the cattlemen dubbed this ailment “brisket disease” (Fig. 4).

In order to better understand what was wrong with their cattle, they turned to the veterinary college at Colorado State University in Fort Collins, just north of Denver. When two veterinarians by the names of George Glover and Isaac Newsom drove up to South Park to examine the animals, they concluded that the cattle were in congestive heart failure. Furthermore, they were convinced that some aspect of the severe climate at high altitude environment was causing this heart failure. That was very perceptive. And so when they published their paper in 1915,[2] they subtitled it “dropsy of high altitude.” Rather quaint, isn’t it?

Now I want to tell you something absolutely confounding.

For the next 35 years, there was absolutely no increase Access this article online

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How to cite this article: Grover RF. The herd shot “round the world”. Pulm Circ 2012;2:258-64.

Pulmonary Circulation | April-June 2012 | Vol 2 | No 2

Grover: Herd shot “round the world”

A

B

Figure 1: Corral well above timberline at Summit Lake Flats, altitude 12,700 feet on Mt. Evans (A).Steers (and lambs) studied in this portable corral for 2 months (B).

Figure 2: Bob laying out 1,200 feet of flexible pipe to siphon fresh water from Summit Lake to the water troughs in the corral.

Figure 3: Bob standing on bathroom scale measuring body weight of lamb.

(1) the significant and selective enlargement of the right ventricle; and (2) the marked dilatation of the trunk of the pulmonary artery. Combining these observations, Jensen concluded that something must be increasing the resistance to blood flow through the lung, placing an excessive pressure load on the right ventricle causing it to fail. Brisket disease was a form of cor pulmonale. A man of few words, Jensen titled his paper “Right Heart Failure.”[3]

Figure 4: Steer at high altitude with cor pulmonale (brisket disease).

in the understanding of the pathophysiology of brisket disease. Nothing! Not until the early 1950s did Rue Jensen, a veterinary pathologist at Fort Collins, come to a fundamental conclusion while dissecting hearts of brisket animals. Jensen was the first to note two abnormalities: Pulmonary Circulation | April-June 2012 | Vol 2 | No 2

Now, neither Jensen nor his colleagues had a clue as to what was causing this pulmonary hypertension. Was it some toxic substance in the grass that grew in South Park?[1] Was brisket disease actually a disorder in blood coagulation causing the animals to develop pulmonary thromboembolism? Or maybe it was an excess intake of salt from the mountain stream drinking water?[1] I want to place emphasis on this line of speculation because in the mid-1950s the scientific community had absolutely no concept of the physiology of the pulmonary circulation. It was a blank page.

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And now back to my story. This (the mid-1950s) was the time when Jack Reeves and I arrived on the scene. We were at the University of Colorado School of Medicine, Denver, working in the Division of Cardiology diagnostic heart catheterization laboratory, seeing children with various forms of congenital heart disease. We were intrigued by the fact that a number of these patients had significant pulmonary hypertension. One day in 1958, Giles Filley,[4] who worked just down the hall, told us that there were veterinarians just up the road at Fort Collins studying pulmonary hypertension in cattle in South Park.[5] Wow! We didn’t waste any time getting in touch with those fellows, Arch Alexander and Don Will, and in a short period of time we were collaborating with them in South Park,[1,6,7] making the very first measurements of the severe pulmonary hypertension in brisket cattle.

Jack and I were already familiar with published reports on how the inhalation of hypoxic gas mixtures elevate the pulmonary arterial pressure.[8,9] This was only acute hypoxia; nothing was known about chronic hypoxia. So we suggested to our veterinary colleagues that, just possibly, the atmospheric hypoxia at high altitude might be causing brisket disease. To my knowledge, this was the first time that either of these gentlemen had ever heard of the concept of “hypoxic pulmonary hypertension.”

Jack and I were fascinated by the fact that the bovine species, cattle, appeared to develop more severe pulmonary hypertension than other species exposed to chronic hypoxia. Did they have a poor ventilatory response to high altitude (relative hypoventilation) resulting in a more severe hypoxic stimulus? Did cattle have unusually strong pulmonary vasoconstriction in response to alveolar hypoxia? To what extent was the increase in pulmonary vascular resistance due to vasoconstriction compared to structural changes (vascular remodeling) of pulmonary blood vessels? Would pulmonary hypertension develop more rapidly if the cattle were taken to a higher altitude (12,700 feet) than in the 1958 studies in South Park (10,000 feet), i.e., a more severe hypoxic stimulus? To address these questions (and others) we decided to expose a group of young steers to the chronic atmospheric hypoxia at 12,700 feet for two months and document the time course of their pulmonary vascular responses. In addition, we thought it would be interesting to simultaneously study lambs, a species known to tolerate high altitude very well. We selected lambs because Estelle and I had observed first hand sheep and llamas grazing side by side at an altitude of 14,000 feet in the Peruvian Andes during a visit with Alberto Hurtado late in 1959.

Now, just how do you go about collecting hemodynamic data from a 500-pound steer? I’ll tell you. Jack and I decided to adopt techniques used by cattle ranchers, of which we had 260

become familiar with while working with the veterinarians (remember, Jack and I were both “city boys”). The first thing you need is this medieval-looking device known as a “squeeze chute” (Fig. 5A). In case you aren’t familiar with their general disposition, steers do not eagerly volunteer to walk into this contraption, but after some “encouragement” they will. Pulling down on the lever, you will see the side of the chute swing up against the animal’s torso, another contraption closes in around the neck, and now the steer is not going anywhere. Next, you will need to blind-fold him because you don’t want these animals to be spooked by what is going on around them (Fig. 5B). Finally, you restrain the head to one side with a rope halter. Now you are ready to go to work.

Steers have jugular veins the size of your forearm; you can’t miss them. Popping in a 10-gauge needle, a stream of blood spurts out, and then you need to thread in a catheter that goes directly to the right heart. To identify where the tip of the catheter is, you attach it to a pressure transducer which in turn will send a signal to a photographic oscillograph.

When you look into the shielded viewing slot, a bright spot of light will move across a slit lens in response to changes in pressure. As soon as the catheter tip enters the right ventricle, the spot begins swinging back and forth and you know exactly where you are. A little farther on, the pressure contour changes to that of an arterial pulse and you know

A

B Figure 5: Bob and his wife Estelle collecting expired air from steer (A).

Jack (standing) and Bob (squatting) performing heart catheterization on steer restrained in squeeze chute (B).

Pulmonary Circulation | April-June 2012 | Vol 2 | No 2

Grover: Herd shot “round the world”

you’re in the pulmonary artery. That spot of light is focused upon light sensitive photographic paper, pulled by a motor past the lens. To develop this recording, you must take it into a dark room, place it through the developer, the fixer, and the water bath; what you end up with is a very elegant analog tracing of the pulmonary arterial pressure in that steer. All that remains is to take a metric measuring device, sit down and measure the height of the pressures. With a stubby pencil, you write down the numbers in a data book.

There was no such thing as automatic pressure recording during that time; it was all manual.

In addition to the pulmonary artery pressure, you will need to know the pulmonary blood flow at the time the pressure was recorded. The Fick principle[10] says that if you know the amount of oxygen going into the lung, and you know how much the oxygen content of the blood increases as it flows through the lung, then you can calculate the total blood flow or cardiac output. In practice, you will need to collect expired air for oxygen uptake, and for the blood a-v difference across the lung you will need samples from both the pulmonary artery and a systemic artery. But how do you collect expired air from a steer? Obviously, you need a face mask (Fig. 6). To construct one, take a length of inner-tube from a car tire, cement this onto a plastic disk, and attach a high-flow respiratory valve; the fit to the steer’s muzzle is quite snug! Then, you will need to connect the valve to a large rubberized Douglas bag, and with a stop cock and stopwatch you will need to collect expired air for exactly 1 minute (Fig. 7). Then to measure the volume of air you squeeze out the bag through a gas flow meter. During this process you also collect an aliquot sample of air for future analysis. For calculation, you must also know the temperature of the expired air as it was collected. This requires inserting a footlong glass mercury thermometer into the steer just below the base of his tail; that was usually my job!

Figure 6: Bob pulling tight-fitting face mask over the muzzle of steer for gas collection.

Pulmonary Circulation | April-June 2012 | Vol 2 | No 2

For the blood samples, while you already have a catheter in the PA you will still need oxygenated arterial blood.

Jack’s solution was to take a 10-inch long needle and thread it between muscles in the animal’s neck, puncturing the carotid artery. The needle in place, you will need to withdraw the pulmonary arterial and systemic arterial blood samples, simultaneously. Once all of the gas and blood samples from several animals have been collected, one member from your research team will need to put them in his pick-up-truck and drive nine miles down the mountain to a permanent facility operated by the University of Denver at Echo Lake. Here, we had set up a temporary analytical laboratory for our technicians, consisting of VanSlyke machines for analyzing blood samples and a Scholander apparatus for analyzing the composition of expired air.

And now for one more chore: To measure the magnitude of the hypoxic stimulus to which the animals were exposed, we needed to measure the oxygen pressure (tension) in arterial blood (PaO2). Furthermore, this analysis had to be made on-site without delay. And so while Jack had his needle in the carotid artery, he withdrew a second sample of blood and handed it to Estelle. She took it to her mini-laboratory, a mere closet shelter from the elements, where she submerged the syringe of arterial blood in a water bath at the animal’s body temperature. With a lowpower microscope to read calibrations on the syringe, she measured PaO2 by the now-obsolete Riley Bubble technique[11] (Severinghaus blood gas electrodes had yet to be perfected). Our 12 lambs were studied by the same techniques, except that we had to construct a mini-squeeze chute of plywood because it is critical that lambs (and sheep) be studied in their normal standing position; held on their side or back, they will die!

These techniques, while dated now, were all state-of-the art during the 1950s and 1960s. Cardiac Catheterization Laboratories were popping up all over the country, and in

Figure 7: Jack with bag of expired air being pushed through gas meter to measure volume.

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every single one they used exactly these same techniques; very labor intensive but they gave good data. Technology really has come a long way.

Now, what did we learn? Let’s begin with the PA pressure data from the 10 steers (Fig. 8). Each dot represents the mean PA pressure from a single animal so at each time period there is a collection of 10 points. At low altitude in Denver, they clustered around 25 mmHg. But as soon as they were exposed to the hypoxia of high altitude, the pressures began to rise relentlessly. After six weeks the average pressure for the whole group had tripled to 75 mmHg. At the same time, you will notice there is tremendous variation among individuals in how much hypertension they actually developed. One steer’s pressure was only 55 while most steers had pressures up around 60, 70, and 80, and two steers had pressures above 100 mmHg;[12] it’s no wonder that one of them went into heart failure.

Histology of the lungs showed marked thickening of the media of the small muscular arteries, or vascular remodeling, as described previously by Alexander.[13] But if you really want to get the feel of vascular remodeling, look at these arteriograms (Fig. 9).

Having seen these spectacular responses in the cattle, don’t forget that we also had 12 lambs living side by side with the steers, exposed to exactly the same hypoxic stimulus, and they developed absolutely no pulmonary hypertension (Fig. 10). After six weeks, their PA pressures were no higher than they had been at low altitude at the start of the study;[14] I believe this to be one of the most provocative findings from the entire Mount Evans study.

In cattle, lungs had multiple vasoreactive mechanisms, vasoconstriction, together with vascular remodeling, while in lambs these mechanisms appear to be totally absent. What is this all about? Subconsciously we think that because something exists it must therefore serve some purpose, and usually it must do something good for the individual; thus, people speculated on the importance of hypoxic pulmonary vasoconstriction. One school of thought is that it serves to match local perfusion to local ventilation (V/Q). For example, if you have an unventilated region of lung (e.g., lobar pneumonia) and you constrict the arteries within that area, that will divert blood to better oxygenated parts of the lung and the lung becomes more efficient at gas exchange. But when you start applying this to multiple sections of the lung, as in patients with COPD, this response becomes disastrous; it creates heart failure and the patient dies. So that is not such a good explanation, I don’t think. Another school of thought says, no, the purpose of hypoxic vasoconstriction is to minimize perfusion of the fetal lung before it becomes functional. This would reduce the workload on the heart 262

Figure 8: Rapid development of hypoxic pulmonary hypertension in 10 steers at 12,700 feet altitude. Note marked variation among individual steers in the severity of hypertension after six weeks.

Figure 9: Pulmonary arteriograms of normal and pulmonary hypertensive steers. Note extreme narrowing of distal blood vessels causing hypertension.

Prepared by Wiltz Wagner.

of the fetus, and then right after birth when oxygen enters the lung, the vessels relax, perfusion begins, but the mechanism doesn’t go away. So when you find it in the adult, it is a legacy from fetal life. It is excess baggage that we have to carry around for the rest of our lives and it may end up killing us. Where is “the wisdom of the body” that Walter B. Canon[15] spoke of? Maybe Shakespeare had the right idea hundreds of years ago, in that “each of us carries within him the seeds of his own destruction.”[16] Hypoxic pulmonary vasoconstriction seems to be one of those seeds of destruction. We simply have to deal with it, try to understand it, and maybe, just maybe, we can do something about it.

Pulmonary Circulation | April-June 2012 | Vol 2 | No 2

Grover: Herd shot “round the world”

This is where the investigators of recent decades come in.

They are the ones who have explored the pathophysiology of the lung circulation. They are the ones who have discovered an amazing array of mechanisms, the HIF’s, the PDGF’s, Rho/Rho kinase, and now something called an autophagia, all unimaginable back in 1960; we were groping in the dark back then. Once the intimate details of these mechanisms have been worked out, the information can be given to pharmacologists and clinicians who may then proceed to develop effective modes of therapy. A classic example is the recent work of Ivan McMurtry and his colleagues[17] who have shown that Rho kinase inhibitors are more effective pulmonary vasodilators than conventional agents such as nitric oxide, prostacyclin, and nifedipine. Such observations have great potential for therapy of pulmonary hypertension.[18] I feel a great sense of pride when I think of the accomplishments these investigators have produced for us. Thanks to them, we now have an impressive body of knowledge assembled through their scientific acumen and imagination. They have written many chapters on the blank pages of 50 years ago. But sometimes I wonder, if in their scientific careers, they have had as much fun as Jack and I had on that mountain top half a century ago (Fig. 11).

Figure 10: Species variation in pulmonary vascular response to the same hypoxic stimulus, severe hypertension in steers versus virtually no response in lambs.

HISTORICAL NOTE

The results of this 1960 study on Mount Evans were first presented to a scientific audience in 1962.[19] This was a conference titled Normal and Abnormal Pulmonary Circulation, the fifth in a series of Annual Conferences on Research in Emphysema founded by Jack Durrance, Roger S. Mitchell and Giles F. Filley, held in Aspen, Colorado. In 1961 they invited me (RFG) to organize the 1962 conference on the subject of the pulmonary circulation. I had never organized any conference in my life, so I was unencumbered by any previous knowledge of such prosaic matters as funding.

Given free rein, I proceeded to invite experts not only from the United States of America but also from around the world. And I even asked Nobel Laureate Andre Cournand to prepare the summary of the conference! It proved to be a wonderful exchange of information among persons who otherwise might not have had this remarkable opportunity to meet. For example, based on Estelle’s and my visit to Lima, Peru, I invited Dante Penaloza to present his fascinating information on pulmonary hypertension in Peruvian natives living at 15,000 feet in the Andes. It proved to be the very first time this information had been presented outside of South America. Arch Alexander,[13] the veterinary pathologist from Colorado State University in Fort Collins, presented his beautiful work on the pulmonary vascular changes in brisket disease to renowned Pulmonary Circulation | April-June 2012 | Vol 2 | No 2

Figure 11: Jack and Bob still collaborating on high altitude research 30 years after Mt. Evans.

pathologists including Jesse Edwards from the Mayo Clinic, CA Wagenvoort from the Netherlands, Averill Liebow, and Javier Arias Stella from Peru. Geoffrey Dawes from England spoke on the fetal lung circulation. John Severinghaus, John West, Abe Rudolf, Al Fishman – it was a stellar cast.

In his summary, Cournand remarked “This conference, I would say without blushing, is the best that I have ever attended. The program was superb.” It was in this illustrious setting that we fledgling investigators, Bob Grover and Jack Reeves, presented our work. Thomas Karger, president of S. Karger AG, Basel, Switzerland, offered to publish all of the individual papers from this conference in their journal Medicina Thoracalis, Vol. 19, followed by a monograph in 1963;[19] we accepted.

BIOGRAPHICAL NOTES

Robert F. Grover was born in 1924 in Rochester, NY. After three years of active service in the US Army during 263

Grover: Herd shot “round the world”

WWII, he completed his education including a PhD in Physiology in 1951, and an MD in 1955. Following his internship and fellowship in Cardiology, he joined the faculty of the University of Colorado School of Medicine in July 1957 on the first rung of the academic ladder, that of Instructor in the Division of Cardiology. He was assigned to the diagnostic cardiology catheterization laboratory, a component of the broader Cardiovascular Pulmonary (CVP) laboratories. This soon functioned as a combined diagnostic and research facility under Dr. Grover’s direction. In 1965, the research facility became independent as the CVP Research Laboratory with its own research training program that Dr. Grover directed for 19 years until his retirement in 1984.

John T. (Jack) Reeves was born in Hazard, Ky. in 1928.

After completing his studies at MIT and the University of Pennsylvania School of Medicine, he was accepted as a Fellow in Cardiology at the University of Colorado School of Medicine in July 1958. His first assignment was the CVP laboratory where he and Bob Grover met. Thus began a life-long friendship and collaboration between Bob and Jack that lasted 47 years until Jack’s untimely death in 2004. Jack spent four years in Denver until 1961 when he returned to Lexington, Ky., but he and Bob remained in constant contact and collaboration. In 1972 Bob was able to offer Jack a faculty position and he eagerly returned to Colorado where they worked together for the rest of Jack’s life.

Thus, at the time of the study on Mount Evans, Bob had worked in the CVP laboratory just three years and Jack only two. Bob was 36 and Jack 32, hardly “seasoned investigators.”

REFERENCES 1.

Will DH, Alexander AF, Reeves JT, Grover RF. High altitude-induced pulmonary hypertension in normal cattle. Circulat Res 1962;10:172-7.

2.

Glover GH, Newsom IE. Brisket disease (Dropsy of high altitude). Colorado Agric Exp Station Bull 1915;204:4-25.

3.

Jensen R. Right heart failure. Calif Vet 1952;5:18-9.

4. Filley GF, MacIntosh DJ, Wright GW. Carbon monoxide uptake and pulmonary diffusing capacity in normal subjects at rest and during exercise.

J Clin Invest 1954;33:530-9.

5.

Pierson RE, Jensen R. Brisket disease in diseases of cattle. Evanston Ill: Amer.

Veterinary Publications; 1956. pp.717-23.

6.

Alexander AF, Jensen R. Gross cardiac changes in cattle with high mountain (Brisket) disease and in experimental cattle maintained at high altitudes.

Am J Vet Res 1959;20:680-9.

7. Alexander AF, Will DH, Grover RF, Reeves JT. Pulmonary hypertension and right ventricular hypertrophy in cattle at high altitude. Am J Vet Res 1960;21:199-204.

8.

Motley HL, Cournand A, Werko L, Himmelstein A, Dresdale D. The influence of short periods of induced acute anoxia upon pulmonary arterial pressures in man. Am J Physiol 1947;150:315-20.

9.

Von Euler US, Liljestrand G. Observations on the pulmonary arterial blood pressure in the cat. Acta Physiol Scand 1946;12:301-20.

10. Fick A. Ueber die Messung des Blutquantums in der Herzenventrikeln.

Sitzung der. Phys Med Gesell zu Wurzburg. July 9, 1870, p 36.

11. Riley RL, Campbell EJ, Shepherd RH. A bubble method for estimation of PCO2 and PO2 in whole blood. J Appl Physiol 1957;11:245-9.

12. Grover RF, Reeves JT, Will DH, Blount SG Jr. Pulmonary vasoconstriction in steers at high altitude. J App Physiol 1963;18:567-74.

13. Alexander AF, Jensen R. Pulmonary vascular pathology of bovine high mountain disease. Am J Vet Res 1963;24:1098-111.

14. Reeves JT, Grover EB, Grover RF. Pulmonary circulation and oxygen transport in lambs at high altitude. J Appl Physiol 1963;18:560-6.

15. Canon WB. The wisdom of the body. New York: Norton; 1932.

16. Shakespeare W. Macbeth.

17. McMurtry IF, Abe K, Ota H, Fagan KA, Oka M. Rho kinase-mediated vaso-constriction in pulmonary hypertension. Adv Exp Med Biol 2010;661:299-308.

18. Oka M, Fagan KA, Jones PL, McMurtry IF. Therapeutic potential of RhoA/Rho kinase inhibitors in pulmonary hypertension. Br J Pharmacol 2008;155:444-54.

19. Grover RF, Reeves JT. Experimental induction of pulmonary hypertension in normal steers at high altitude in normal and abnormal pulmonary circulation, In: RF Grover, editor. Basel /New York, S. Karger; 1963. pp 351-8.

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

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Let t er t o Ed i t o r

Critical care rehabilitation—is it the answer for reducing morbidity in ARDS survivors? Regarding “Acute respiratory distress syndrome: A clinical review” Sir,

Abraham Samuel Babu1, and Lenny T. Vasanthan2

1

We read with interest the article by Michael Donahoe.

He wonderfully reviewed the various aspects of acute respiratory distress syndrome (ARDS) and its management.

[1]

ARDS poses an expensive burden on young patients who survived, not recovering completely with regard to physical function even after five years.[2] Intensive care unit (ICU) acquired weakness is an important contributor to long-term function and quality of life (QoL) in ARDS survivors.[3] Critical care rehabilitation has now become a key factor in the continued care of a patient in the ICU. Recent studies have shown that patients who have not received rehabilitation tend to have increased morbidity, with regard to poor QoL and functional impairment at the time of discharge.[4] Rehabilitation interventions begun in the ICU show improved functional outcomes at discharge from hospital.

Early mobility in the ICU and critical care rehabilitation has been found to be feasible and safe.[5] Mobilization (namely limb exercises), respiratory and peripheral muscle training, and neuromuscular electrical stimulation are also utilized by physiotherapists in the ICU to help improve functional outcomes.[6]

Rehabilitation algorithms are now available to serve as a guide in identifying suitable patients for mobilization and provide appropriate treatment strategies.[7] However, they may require adaptations and modifications to suit each individual patient. Therefore, this active form of rehabilitation, which is safe, should start within the ICU as soon as possible – even while patients are on mechanical

Pulmonary Circulation | April-June 2012 | Vol 2 | No 2

ventilation in order to improve function and reduce morbidity, as 57% of patients who are ventilated for more than 48 hours, stand a greater chance for requiring assistance for upto one year.[8] More studies are required to assess how ARDS survivors respond to rehabilitation programs in the short term and long term.

Department of Physiotherapy, Manipal College of Allied Health Sciences, Manipal University, Manipal, Karnataka, 2 Department of Physical Medicine and Rehabilitation, Christian Medical College and Hospital, Vellore, Tamil Nadu, India Email: abrahambabu@gmail.com

REFERENCES 1.

2.

3.

4.

5.

6.

7.

8.

Donahoe M. Acute respiratory distress syndrome: A clinical review.

Pulmonary Circulation 2011;1:192-211 Herridge MS, Tansey CM, Matte A, Tomlinson G, Diaz-Granados N, Cooper A, et al. Functional disability 5 years after acute respiratory distress syndrome. N Engl J Med 2011;364:1293-304 Wilcox ME, Herridge MS. Lung function and quality of life in survivors of the acute respiratory distress syndrome (ARDS). Presse Med 2011;40 (12 Pt 2):e595-603 Kortebein P. Rehabilitation for hospital-associated deconditioning. Am J Phys Med Rehabil 2009;88:66-77 Bailey P, Thomsen GE, Spuhler VJ, Blair R, Jewkes J, Bezdjian L, et al. Early activity is feasible and safe in respiratory failure patients. Crit Care Med 2007;35:139-45.

Ambrosino N, Janah N, Vagheginni G. Physiotherapy in critically ill patients.

Rev Port Pneumol 2011;17:283-8.

Hanekom S, Gosselink R, Dean E, van Aswegen H, Roos R, Ambrosino N, et al. The development of a clinical management algorithm for early physical activity and mobilization of critically ill patients: Synthesis of evidence and expert opinion and its translation into practice. Clin Rehabil 2011;25:771-87.

Barnato AE, Albert SM, Angus DC, Lave JR, Degenholtz HB. Disability among elderly survivors of mechanical ventilation. Am J Respir Crit Care Med 2011;183:1037-42.

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Announcing Keystone Symposia’s Pulmonary Vascular Disease Conference Present a poster. Learn about the latest research and clinical trials.

Forge new collaborations.

Pulmonary Vascular Disease and Right Ventricular Dysfunction: Current Concepts and Future Therapies September 10–15, 2012 Portola Hotel & Spa • Monterey, California • USA Scientific Organizers: Georg Hansmann, Stephen L. Archer and Margaret R. MacLean

Session Topics:

> The Role of Stem Cells, Progenitor and Differentiated Blood Cells in Pulmonary Vascular Disease and Repair > Growth Factors, TGF-b/BMP Signaling and Pulmonary Vascular Disease > Metabolic Regulators in Pulmonary Vascular Disease > The Right Ventricle in Pulmonary Hypertension: Cardiomyocyte Function and Hemodynamic Performance > MicroRNAs in Proliferative Vascular Disease > Innovative Clinical PAH Trials > Pulmonary Hypertension in Parenchymal Lung Diseases > Pulmonary Arterial Hypertension – Current Concepts and Future Therapies

Confirmed Speakers:

Stephen L. Archer, University of Chicago Medical Center Andrew H. Baker, University of Glasgow Kenneth D. Bloch, Massachusetts General Hospital Sebastien Bonnet, Université Laval Daniel Burkhoff, Columbia University Peter F. Carmeliet, University of Leuven Stefanie Dimmeler, University of Frankfurt Jeffrey Fineman, University of California, San Francisco H. Ardeschir Ghofrani, Justus Liebig University Giessen Georg Hansmann, Children’s Hospital Boston, Harvard Medical School Paul Hassoun, Johns Hopkins University Marc Humbert, Hôpital Antoine-Béclère, AP-HP Stuart W. Jamieson, University of California, San Diego Medical Center Stella Kourembanas, Children’s Hospital Boston, Harvard Medical School Mark A. Krasnow, Stanford University School of Medicine

Titus Kuehne, German Heart Institute Joseph Loscalzo, Brigham and Women’s Hospital, Harvard Medical School Margaret R. MacLean, University of Glasgow Evangelos D. Michelakis, University of Alberta Jane A. Mitchell, Imperial College London Timothy M. Moore, NHLBI, National Institutes of Health Nicholas Morrell, University of Cambridge Mark Nicolls, Stanford University School of Medicine Andrew Redington, Hospital for Sick Children Stuart Rich, University of Chicago Medical Center Kurt R. Stenmark, University of Colorado Denver Duncan Stewart, Ottawa Hospital Research Institute Bernard Thébaud, University of Alberta Patricia Thistlethwaite, University of California, San Diego Matthew Thomas, Novartis Institutes for BioMedical Research Norbert Voelkel, Virginia Commonwealth University Martin R. Wilkins, Imperial College London

The conference will bring together basic scientists and vascular biologists who are applying metabolomics and proteomics to identify novel biomarkers, clinical trialists who will discuss new designs and preliminary results of clinical trials, clinicians, scientific leaders from industry, and regulatory and funding agencies to improve understanding of pulmonary hypertension. A key goal is to develop future strategies to cure this fatal disease and to present cutting-edge technologies, innovations and discoveries with potential to be translated into clinical practice in the near future.

Program information current as of May 31, 2012 but subject to possible change. Please visit our website at www.keystonesymposia.org/12S1 to register and for the most complete and up-to-date program information.

www.keystonesymposia.org/12S1 | 1.800.253.0685 | 1.970.262.1230

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Request for Proposal

The Cardiovascular Medical Research and Education Fund (CMREF) is seeking proposals from qualified academic medical centers with the ability to coordinate and conduct a clinical study evaluating right ventricular function in patients with pulmonary hypertension, its adaptive and maladaptive responses, and the effects of therapy. For more information, please go to the CMREF website: www.ipahresearch.org

Studies of the Right Ventricular Response to Therapy in Patients with Pulmonary Arterial Hypertension Introduction The Cardiovascular Medical Research and Education Fund (CMREF) is seeking proposals from qualified academic medical centers with the ability to coordinate and conduct a clinical study evaluating right ventricular function in patients with pulmonary hypertension, its adaptive and maladaptive responses, and the effects of therapy.

Background The purpose of this clinical feasibility study is to better characterize the status of the right ventricle in patients with advanced pulmonary hypertension (PH) who are candidates for a new or additional treatment. Research in this field has identified changes in the right ventricle as predictive of survival in chronic PH. Understanding how the right ventricle adapts to pulmonary hypertension, and how that response may be altered with medical therapies appears to be critical.

Scientific studies that may be relevant to this project cover a broad spectrum of disciplines that include genomics, imaging, and assessments of RV contractility, hypertrophy and metabolism.

It is hoped that knowledge gained from this study will impact clinical trials of future therapies developed to treat advanced pulmonary hypertension.

Project Guidelines • Study hypotheses: Maladaptation to the pulmonary hypertensive state is largely responsible for right ventricular failure in chronic pulmonary hypertension. Changes that occur in the right ventricle predict clinical improvements in patients undergoing treatments for pulmonary hypertension. The applicant is requested to propose one or more related hypotheses that will be tested in this study.

• The patient inclusion criteria for the study should include Category 1 pulmonary arterial hypertension, preferably from one specific etiology, and advanced disease in which a new or additional medical therapy is clinically indicated.

• This can be an open label study where the patients will serve as their own controls, or randomized, placebo controlled.

• It is suggested that the number of patients enrolled be adequate to test the hypotheses, rather than powered to show statistically significant drug efficacy.

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The follow-up assessments should be over 6 months, and patient enrollment should be completed within a 2 year period.

The state of the right ventricle should be characterized with multiple parameters that will provide insight into the adaptive and maladaptive state, and reflect changes that occur from therapy that will further the understanding of how therapies may work. These parameters may include measures of: o Genetics and genomics o Metabolism o Cellular signaling o Imaging (ultrasound, PET and MRI) o Contractility and hypertrophy o Hemodynamics o RV function o Myocardial ischemia and/or perfusion

The applicant is expected to develop a clinical research study plan that will identify several assessments that will be studied in the enrolled patients. While it is desirable for a single center to enroll all patients in this study and perform all of the assessments, this may not be practical or feasible. Because PAH is an uncommon disease, it is understood that multiple clinical centers may need to participate in patient enrollment (a clinical center is one that will enroll patients into the study). In addition, given the uniqueness of some of the promising assessments, it is also understood that multiple centers may need to participate as core laboratories to evaluate specific measures (a core laboratory is one that will perform analyses of specific measured assessments collected at the clinical centers). Thus, the research plan should include an organizational plan that identifies the Principle Center, collaborating clinical centers, and core laboratories. The Principle Center will be responsible for all of the data management, and for providing oversight of the entire conduct of the study.

The Principle Center should also serve as a clinical center and a core laboratory. Having other clinical centers also serving as core laboratories to expand the different types of assessments will be considered a strength of the proposal. This award can be made to the Principle Center, with subcontracts to clinical and core centers, or to each participating center directly.

The applicant should submit a research plan using the NIH R01 format that will include the collaborating investigators for all phases of the study. Required activities by the applicant include, but are not limited to: • finalizing the clinical protocol and protocol-related documents (e.g. informed consent form, case report forms, etc.); • implementation and conduct of the clinical study in accordance with Federal requirements; • management and oversight of the conduct of the clinical study; • providing clinical research support services; • providing monitoring and safety oversight; • organizing investigator meetings and teleconferences; • recruiting patients; • organizing, conducting, and reimbursing for patient protocol evaluations and tests (including follow-up); • analyzing the data and authoring and submitting papers on the study for publication.

Application Guidelines • The deadline for grant submission is July 1, 2012. Funding will begin October 1, 2012. All applicants are requested to submit a letter of intent with a brief description of their proposal, and the collaborating institutions.

• The direct award for this study will be limited to $2 million, inclusive of all subcontracts. Overhead of 20% will be added to the amount awarded. The award may not be used to cover healthcare related costs that are otherwise clinically indicated, including but not limited to the costs of the medical therapy to be studied if it is clinically indicated.

• Because of the high expense of healthcare costs, the applicant is advised to recruit patients where the preponderance of tests and treatments are otherwise clinically indicated and thus will not need to be covered by the grant.

• The project should be constructed to meet the following timelines: o 6 months start-up  Case report form completion  Study protocols completed  Research assessments specified  Consent and IRB approvals

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o 24 months patient enrollment  Enrollment monitors o 6 months close-out  Data analyses  Publications The NIH R01 research application form should be used, including Specific Aims, Background and Importance and Approach (Research Plan), Human Subjects Section, and Biosketches and description of facilities and resources. The following should be included in the response to the RFP: o The research strategy should include a clear description of the study design, study population, subject eligibility and inclusion/exclusion criteria, recruitment and enrollment plans, and outcome measures.  State concisely the goals of the proposed research and summarize the expected outcome(s), including the impact that the results of the proposed research will exert on the research field(s) involved. The major research question and hypothesis being studied should be clearly stated. o The significance of the proposed clinical trial must be clearly stated. The application should make clear how the results will advance our knowledge of theory and practice in this area and the potential impact of the results of the trial. o Explain how the application challenges and seeks to shift current research or clinical practice paradigms. o A list of subject eligibility and inclusion and exclusion criteria should be provided. A recruitment and enrollment plan, including a discussion of the availability of subjects for the proposed study, the ability of enrollment sites to recruit the required number of subjects, and the timeline for completion of recruitment, should be described. o There should be a detailed description of the assessments to be tested. Potential biases and approaches for minimizing bias should be described. The primary and secondary endpoints, and methods/measures to be used to assess these, should be clearly described. The link between endpoints, outcome measures, and hypotheses should be stated clearly. o Data collection plans and statistical methods appropriate for the particular design proposed should be presented. Methods to be used for data collection, preparation, management, quality control should be thoroughly described. o Data from preliminary or pilot studies which show the need for and the feasibility of the trial should also be presented. Additional supporting data from other research should be included so that the approach chosen is clearly justified. This information will also help to establish the experience and competence of the investigators to pursue the proposed project. o A timetable for completion of the various stages of the trial must be included.

Data Safety and Monitoring All applications must include a general description of the monitoring plan, policies, procedures, responsible entities, and approaches to identifying, managing and reporting reportable events (adverse events and unanticipated problems), to the applicable regulatory agencies (e.g., Institutional Review Board (IRB), the NHLBI/NIH, the Office of Biotechnology Activities, Office of Human Research Protections (OHRP), the (FDA), and the Data and Safety Monitoring Board (if one is used).

Contact information Inquiries, letters of intent, applications or questions should be addressed to: Cardiovascular Medical Research and Education Fund 510 Walnut Street, Suite 500 Philadelphia, PA 19106, USA Phone: 215-413-2414 Fax: 215-592-4663 Email: patt.wolfe@ipahresearch.org Website: www.ipahresearch.org

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Pulmonary Circulation is an Editors-in-Chief: Jason X.-J. Yuan, MD, PhD Nicholas W. Morrell, MD Harikrishnan S., MD

open-access, peer-reviewed, international journal devoted to research in the fields of the pulmonary circulation, pulmonary vascular disease,

Senior Editor: Ghazwan Butrous, MD

and lung injury. Pulmonary Circulation is published

Executive Editor: Harikrishnan S., MD

quarterly and sponsored by the Pulmonary Vascular

Editors: Kurt R. Stenmark, MD Kenneth D. Bloch, MD Stephen L. Archer, MD Marlene Rabinovitch, MD Joe G.N. Garcia, MD Stuart Rich, MD Martin R. Wilkins, MD Hossein A. Ghofrani, MD Candice D. Fike, MD Werner Seeger, MD Sheila G. Haworth, MD Patricia A. Thistlethwaite, MD, PhD Chen Wang, MD, PhD Antonio A. Lopes, MD Scientific Advisory Board: Robert F. Grover, MD, PhD Charles A. Hales, MD, PhD E. Kenneth Weir Joseph Loscalzo, MD John B. West, MD, PhD, DSc Magdi H. Yacoub, MD, DSc, FRS

Research Institute.

 The first journal dedicated exclusively to the pulmonary circulation and pulmonary vascular disease  Indexed and Accessible on PubMed  Devoted to high quality clinical and basic research in the field  No charge for submission, processing or publication of your manuscripts and for color photographs  Open access  Prompt peer review with online submission and review  Immediate publication on acceptance

Want to learn more? Visit www.pulmonarycirculation.org Ready to submit? Visit http://www.journalonweb.com/PC/ View all Author Instructions at http://www.pulmonarycirculation.org/contributors.asp

CONTENTS Editorial Science is an endless frontier: Encouraging translational research in pulmonary vascular disease

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Clinical perspectives with long-term pulsed inhaled nitric oxide for the treatment of pulmonary arterial hypertension

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

Review Articles

Robyn J. Barst, Richard Channick, Dunbar Ivy, and Brahm Goldstein

Severe pulmonary hypertension: The role of metabolic and endocrine disorders

Harm J. Bogaard, Aysar Al Husseini, Laszlo Farkas, Daniela Farkas, Jose Gomez-Arroyo, Antonio Abbate, and Norbert F. Voelkel

Evaluation of patients with chronic thromboembolic pulmonary hypertension for pulmonary endarterectomy William R. Auger, Kim M. Kerr, Nick H. Kim, and Peter F. Fedullo

Diagnosis and management of pulmonary hypertension associated with left ventricular diastolic dysfunction Vinicio A. de Jesus Perez, Francois Haddad, and Roham T. Zamanian

148 155 163

Research Articles

Mesenchymal stem cell-mediated reversal of bronchopulmonary dysplasia and associated pulmonary hypertension Georg Hansmann, Angeles Fernandez-Gonzalez, Muhammad Aslam, Sally H. Vitali, Thomas Martin, S. Alex Mitsialis, and Stella Kourembanas

Group V phospholipase A2 increases pulmonary endothelial permeability through direct hydrolysis of the cell membrane Nilda M. Muñoz, Anjali Desai, Lucille N. Meliton, Angelo Y. Meliton, Tingting Zhou, Alan R. Leff, and Steven M. Dudek

Furegrelate, a thromboxane synthase inhibitor, blunts the development of pulmonary arterial hypertension in neonatal piglets

Dinesh K. Hirenallur-S., Neil D. Detweiler, Steven T. Haworth, Jeaninne T. Leming, John B. Gordon, and Nancy J. Rusch

Metabolomic analysis of bone morphogenetic protein receptor type 2 mutations in human pulmonary endothelium reveals widespread metabolic reprogramming Joshua P. Fessel, Rizwan Hamid, Bryan M. Wittmann, Linda J. Robinson, Tom Blackwell, Yuji Tada, Nobuhiro Tanabe, Koichiro Tatsumi, Anna R. Hemnes, and James D. West

Leptin levels predict survival in pulmonary arterial hypertension

Adriano R. Tonelli, Metin Aytekin, Ariel E. Feldstein, and Raed A. Dweik

Mast cell number, phenotype, and function in human pulmonary arterial hypertension

Samar Farha, Jacqueline Sharp, Kewal Asosingh, Margaret Park, Suzy A. A. Comhair, W. H. Wilson Tang, Jim Thomas, Carol Farver, Fred Hsieh, James E. Loyd, and Serpil C. Erzurum

Activation of the unfolded protein response is associated with pulmonary hypertension

Michael E. Yeager, Monica B. Reddy, Cecilia M. Nguyen, Kelley L. Colvin, D. Dunbar Ivy, and Kurt R. Stenmark

Distinct responses to hypoxia in subpopulations of distal pulmonary artery cells contribute to pulmonary vascular remodeling in emphysema L. S. Howard, A. Crosby, P. Vaughan, A. Sobolewski, M. Southwood, M. L. Foster, E. R. Chilvers, and N. W. Morrell

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Case Reports Partial anomalous pulmonary venous return presenting with adult-onset pulmonary hypertension Edmund H. Sears, Jason M. Aliotta, and James R. Klinger

Massive dilatation of the pulmonary artery in association with pulmonic stenosis and pulmonary hypertension Sejal Morjaria, Dan Grinnan, and Norbert Voelkel

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History and Who’s Who The herd shot “round the world” Robert F. Grover

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Letter to Editor Critical care rehabilitation—is it the answer for reducing morbidity in ARDS survivors? Regarding “Acute respiratory distress syndrome: A clinical review” Abraham Samuel Babu, and Lenny T. Vasanthan

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