Textbook of Pulmonary Vascular Disease
Jason X.-J. Yuan Joe G.N. Garcia Charles A. Hales Stuart Rich Stephen L. Archer John B. West ●
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Editors
Textbook of Pulmonary Vascular Disease
Editors Jason X.-J. Yuan, MD, PhD Professor of Medicine Institute for Personalized Respiratory Medicine Department of Medicine University of Illinois at Chicago Chicago, IL
[email protected] Charles A. Hales, MD Professor of Medicine Massachusetts General Hospital Harvard Medical School Boston, MA
[email protected] Stephen L. Archer, MD Harold Hines Jr. Professor of Medicine Chief of Cardiology Department of Medicine University of Chicago Chicago, IL
[email protected] Joe G.N. Garcia, MD Earl Banes Professor of Medicine Vice Chancellor for Research University of Illinois at Chicago Chicago, IL
[email protected] Stuart Rich, MD Professor of Medicine Department of Medicine University of Chicago Chicago, IL
[email protected] John B. West, MD, PhD, DSc Distinguished Professor of Medicine and Physiology Department of Medicine University of California, San Diego La Jolla, CA
[email protected] ISBN 978-0-387-87428-9 e-ISBN 978-0-387-87429-6 DOI 10.1007/978-0-387-87429-6 Springer New York Dordrecht Heidelberg London Library of Congress Control Number: 2011920684 © Springer Science+Business Media, LLC 2011 All rights reserved. This work may not be translated or copied in whole or in part without the written permission of the publisher (Springer Science+Business Media, LLC, 233 Spring Street, New York, NY 10013, USA), except for brief excerpts in connection with reviews or scholarly analysis. Use in connection with any form of information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed is forbidden. The use in this publication of trade names, trademarks, service marks, and similar terms, even if they are not identified as such, is not to be taken as an expression of opinion as to whether or not they are subject to proprietary rights. Printed on acid-free paper Springer is part of Springer Science+Business Media (www.springer.com)
Preface
The major function of the lungs is gas exchange and it does this using a low resistance circulation. The pulmonary circulation (or the pulmonary vasculature) is a unique system that differs dramatically from the systemic circulatory system (e.g., coronary, cerebral, renal arteries) in structure, function, and regulation. A typical example of functional differences between the pulmonary and systemic vasculature is that hypoxia causes pulmonary vasoconstriction but systemic vasodilation. Furthermore, in patients with systemic arterial hypertension (e.g., essential hypertension), pulmonary arterial pressure is normal, while in patients with idiopathic pulmonary arterial hypertension (previously referred to as primary pulmonary hypertension), systemic arterial pressure is usually within the normal range. The divergent vascular responses to hypoxia and the alternative existence of systemic or pulmonary arterial hypertension in patients indicate that the pulmonary vasculature or the pulmonary circulation is unique in terms of its anatomic and histological structure, physiological and pharmacological properties, genetic and epigenetic development as well as cellular and molecular determinants for vasoconstriction, vascular-wall remodeling, and embolus formation. Therefore, the pathogenic mechanisms of pulmonary vascular diseases are rather different from those of systemic circulatory disorders. Development of therapeutic approaches and improvement of clinical management for patients with pulmonary vascular diseases should be directed by understanding the unique physiological and pathological features of the pulmonary vasculature at organ, tissue, cell, and molecular levels. Although many books have addressed clinical aspects of cardiovascular diseases, systemic arterial hypertension and basic science progress about structural and functional studies on systemic arteries (e.g., coronary, cerebral, and other peripheral arteries and microcirculation), very few books have focused on the pulmonary circulation and pulmonary vascular disease. Given the significant differences between the pulmonary and systemic vasculature and between systemic and pulmonary vascular diseases, it is urgent to have a comprehensive reference book specifically designated to describe a) basic structure and function of the pulmonary vasculature or the pulmonary circulation, b) pathophysiology of the pulmonary circulatory system, and c) clinical aspects (diagnosis, treatment, and prevention) of pulmonary vascular diseases. Textbook of Pulmonary Vascular Disease is therefore designed for and is of special interest to a) clinicians (pulmonologists, cardiologists, intensive care physicians, cardiothoracic and vascular surgeons, and emergency physicians), b) physician-scientists and basic-science researchers in the fields of cardiopulmonary and critical care medicine, vascular physiology and pathophysiology, translational medical research, and bioengineering, c) healthcare workers in cardiopulmonary and critical care medicine, and d) clinical and research fellows as well as residents, medical and graduate students. Textbook of Pulmonary Vascular Disease combines basic scientific concepts and knowledge on the pulmonary circulation with clinical diagnosis and treatment on pulmonary vascular diseases. Textbook of Pulmonary Vascular Disease is unique in that no book currently available i) focuses on elucidating the cellular and molecular regulation of normal pulmonary vasculature and the pathogenic mechanisms of pulmonary vascular diseases, ii) includes advanced techniques and technology for basic and clinical research, and iii) includes conventional and molecular approaches currently available for v
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diagnosis and treatment of patients with pulmonary vascular diseases. Another feature of the book is the inclusion of surgical approaches for treatment of pulmonary vascular diseases, which have not been well described in previously published books. Textbook of Pulmonary Vascular Disease is divided into five parts. Part 1 (Structure, Function and Regulation), consisting of nine sections and twenty-nine chapters, is designated for basic knowledge and recent findings related to pulmonary vascular structure, function, and regulation at levels of molecule, cell, tissue, organ and system. Part 2 (Methodological Approaches for Research) is composed of six sections and sixteen chapters that are designed to provide a basic knowledge and spectrum on the techniques and technology that are commonly used to study genetic, molecular, cellular, systemic and pathophysiological aspects of the pulmonary vasculature. Part 3 (Pathology and Pathobiology) includes four sections and nineteen chapters that discuss the potential mechanisms or sequence of events involved in the initiation and progression of abnormalities in the pulmonary vasculature in patients with pulmonary vascular disease. Part 4 (Pulmonary Vascular Diseases) consists of nine sections and thirty-two chapters devoted to describe pathogenesis, epidemiology and pathophysiology of almost all of the pulmonary vascular diseases identified so far by the World Health Organization (WHO). One of the sections is specifically designated to describe pulmonary vascular disease in pediatric patients. Part 5 (Diagnosis and Treatment) includes three sections and twenty chapters designed to illustrate, in details, the diagnostic and therapeutic procedures currently used for patients with pulmonary vascular disease. Textbook of Pulmonary Vascular Disease is written by more than 220 experts in the field including physicians, surgeons, epidemiologists, bioinformaticians, nurses, physician scientists and investigators. All of the contributors are actively involved in clinical, physiological, and pathophysiological studies on the pulmonary circulation and pulmonary vascular diseases. The vast majority of authors are recognized experts in the research area of the topic on which the chapter is based with many contributors also Fellows of the Pulmonary Vascular Research Institute, a not-for-profit, international scientific association focused on the pulmonary circulation and pulmonary vascular disease. Founded in 2007, the Pulmonary Vascular Research Institute (PVRI) has assembled clinical, epidemiological, translational and basic scientists from around the world to perform research and advance education regarding pulmonary vascular disease and right heart failure. There is a focus on performing research in and providing education and treatment to underserved populations of the world. The unique strength of the PVRI is that it brings together a multidisciplinary faculty from around the world within a single focused institute. PVRI members have the expertise to conduct basic, translational, and clinical research at a level that no single academic institution can offer. The publication of an authoritative textbook on pulmonary vascular disease was an initial goal of the Institute. Textbook of Pulmonary Vascular Disease will not only serve as a reference book for physicians, surgeons, private practitioners, translational medical researchers, clinical and research fellows, and medical and graduate students, but also can be used as a guidance manual for technical and marketing personnel in pharmaceutical and biotechnological companies, that are interested in clinical and basic science research in cardiopulmonary diseases, pulmonary vascular diseases, vascular biology, and lung/heart transplantation. We hope that this book will also allow readers to foster new concepts and new collaboration and cooperation among clinicians, physician scientists and investigators so as to further understand the pathogenic mechanisms of pulmonary vascular disease and develop novel therapeutic approaches for the disease. La Jolla, California May 9, 2010
Jason X.-J. Yuan, MD, PhD Joe G.N. (Skip) Garcia, MD Charles A. Hales, MD Stuart Rich, MD Stephen L. Archer, MD John B. West, MD, PhD, DSc
Acknowledgements
The book could not have been completed without the support and encouragement of our families (Ayako Makino, Sue Garcia, Mary Ann Hales, Kathie Doliszny, Andrea Rich, and Penelope West) as well as our colleagues and students at the University of California, San Diego, the University of Chicago, the Massachusetts General Hospital at Harvard Medical School (Boston, MA) and the University of Illinois at Chicago. We are especially grateful to Ms. Melissa Ramondetta at Springer for her encouragement and support in compiling the book, to Dr. Carmelle V. Remillard for her diligence in preparing the figures and editing the text, to Ms. Portia Bridges for tirelessly going through all the details, and to all the contributors for their patience and conscientiousness in writing the manuscripts. In addition, we thank the students, research fellows, and staff of our laboratories who have made considerable contributions to collecting data, selecting representative figures, making schematic diagrams, and reading through the manuscript for grammatical and typographic errors. We sincerely thank Amy Zeifman, Michael Song, Frank Kuhr and Adriana Zimnicka for their diligent work on proofreading and copyediting the galley proofs. Finally, we would like to take this opportunity to thank the publisher, Springer, for its interest in developing such a comprehensive reference book for the field (and readers) of pulmonary circulation and pulmonary vascular disease.
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Contents
Part I Structure, Function and Regulation The Human Pulmonary Circulation 1 The Human Pulmonary Circulation: Historical Introduction........................... John B. West
3
Structure and Function of the Pulmonary Circulation 2 Microcirculation of the Lung: Functional and Anatomic Aspects..................... Joan Gil
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3 Pulmonary Vascular Development........................................................................ Rosemary C. Jones and Diane E. Capen
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4 Pulmonary Vascular Function............................................................................... Robert Naeije and Nico Westerhof
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5 Pulmonary Vascular Mechanics............................................................................ Alejandro Roldán-Alzate and Naomi C. Chesler
73
6 Modeling of the Pulmonary Vasculature.............................................................. Merryn H. Tawhai and Kelly S. Burrowes
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7 Metabolic and Clearance Function at the Pulmonary Microvascular Endothelial Surface in Pulmonary Hypertension...................... Stylianos E. Orfanos and David Langleben
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Pulmonary Vasomotor Tone, Vascular Reactivity, and Vascular Permeability 8 The Influence of the Major Vasoactive Mediators Relevant to the Pathogenesis of Pulmonary Hypertension................................. Margaret R. MacLean and Yvonne Dempsie 9 The Normal Fetal and Neonatal Pulmonary Circulation.................................... Steven H. Abman and Robin H. Steinhorn 10 Excitation–Contraction Coupling and Regulation of Pulmonary Vascular Contractility.................................................................... Jeremy P.T. Ward and Greg A. Knock
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11 Endothelial Regulation of Pulmonary Vascular Tone......................................... Stephen Y. Chan and Joseph Loscalzo 12 Acute Lung Injury: The Injured Lung Endothelium, Therapeutic Strategies for Barrier Protection, and Vascular Biomarkers........ Eddie T. Chiang, Ting Wang, and Joe G.N. Garcia
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Signal Transduction 13 Ion Channels and Transporters in the Pulmonary Vasculature: A Focus on Smooth Muscle.................................................................................... Amy L. Firth and Jason X.-J. Yuan 14 Receptor-Mediated Signal Transduction and Cell Signaling.............................. Fiona Murray, Jason X.-J. Yuan, and Paul A. Insel
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15 Role of Calcium as a Second Messenger in Signaling: A Focus on Endothelium........................................................................................ Donna L. Cioffi, Christina J. Barry, and Troy Stevens
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16 Caveolae and Signaling in Pulmonary Vascular Endothelial and Smooth Muscle Cells.................................................................. Geerten P. van Nieuw Amerongen, Richard D. Minshall, and Asrar B. Malik
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Oxygen Sensing and Reactive Oxygen Specials in the Lung 17 The Chemistry of Biological Gases........................................................................ D. Jeannean Carver and Lisa A. Palmer
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18 Role of Oxygen-Derived Species in the Regulation of Pulmonary Vascular Tone.................................................................................. Michael S. Wolin, Mansoor Ahmad, and Sachin A. Gupte
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19 Mitochondrial Reactive Oxygen Species and Redox State in Pulmonary Vascular O2 Sensing........................................................................ Stephen L. Archer and John J. Ryan
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Cell Proliferation and Apoptosis 20 Cellular and Molecular Mechanisms of Pulmonary Vascular Smooth Muscle Cell Proliferation......................................................... Tamara Tajsic and Nicholas W. Morrell
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21 Role of Ca2+ in Vascular Smooth Muscle Gene Expression and Proliferation................................................................................. Karen M. Lounsbury and Patricia C. Rose
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22 Biochemistry and Cellular Mechanisms of Apoptosis in Vascular Smooth Muscle and Endothelial Cells.............................................. Oliver Eickelberg and Fotini M. Kouri
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Coagulation, Thrombosis, and Fibrinolysis 23 The Coagulation Cascade and Its Regulation...................................................... James T.B. Crawley, Jose R. Gonzalez-Porras, and David A. Lane
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24 Platelets in Pulmonary Vascular Physiology and Pathology............................... Michael H. Kroll
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25 Lysis and Organization of Pulmonary Thromboemboli...................................... Timothy A. Morris and Debby Ngo
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Interactions of Pulmonary Vascular Cells with Circulating Blood Cells 26 Interactions of Leukocytes and Coagulation Factors with the Vessel Wall................................................................................... Scott Visovatti, Takashi Ohtsuka, and David J. Pinsky 27 Interaction of the Plasminogen System with the Vessel Wall.............................. Riku Das and Edward F. Plow 28 Endothelial Apoptosis and Repair in Pulmonary Arterial Hypertension............................................................................................. Rohit Moudgil, Manoj M. Lalu, and Duncan J. Stewart
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The Systemic Circulation in the Lung 29 Bronchial Arterial Circulation in the Human...................................................... Gabor Horvath and Adam Wanner
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Part II Methodological Approaches for Research In vivo and Genetic Animal Models 30 Animal Models of Pulmonary Hypertension........................................................ Jocelyn Dupuis and Norbert Weissmann 31 Transgenic and Gene-Targeted Mouse Models for Pulmonary Hypertension................................................................................. James D. West 32 Animal Models of Increased Lung Vascular Permeability................................. Sara Hanif Mirza, M. Kamran Mirza, and Asrar B. Malik
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Morphological, Functional, and Regulatory Approaches 33 Isolation and Culture of Pulmonary Vascular Smooth Muscle and Endothelial Cells................................................................................. Carmelle V. Remillard, Ayako Makino, and Jason X.-J. Yuan
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34 Conventional Patch Clamp Techniques and High-Throughput Patch Clamp Recordings on a Chip for Measuring Ion Channel Activity................... Carmelle V. Remillard and Jason X.-J. Yuan
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35 Measurement of Pulmonary Vascular Structure and Pulmonary Blood Distribution by Multidetector-Row Computed Tomography and Magnetic Resonance Imaging Techniques........... Sara K. Alford and Eric A. Hoffman
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Molecular Biological Techniques 36 Quantification of DNA, RNA, and Protein Expression....................................... Fiona Murray, Jason X.-J. Yuan, and Paul A. Insel
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37 Gene Cloning, Transfection, and Mutagenesis..................................................... Ellen C. Breen and Jason X.-J. Yuan
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38 Approaches for Manipulation of Gene Expression.............................................. Ying Yu and Jason X.-J. Yuan
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Genomic, Proteomic and Bioinformatical Approaches 39 Bioinformatics, Genomics, and Functional Genomics: Overview...................... Ali Torkamani, Eric J. Topol, and Nicholas J. Schork
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40 Genomic Applications to Study Pulmonary Hypertension................................. Todd M. Bull and Mark W. Geraci
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41 Proteomics and Functional Proteomics................................................................. Dayue Darrel Duan
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Stem Cells 42 Maintenance, Propagation, and Differentiation of Human Embryonic Stem Cells and Induced Pluripotent Stem Cells.............................. Zoë L. Vomberg, Megan Robinson, Thomas Fellner, and Karl H. Willert
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43 Identification of Adult Stem and Progenitor Cells in the Pulmonary Vasculature...................................................................... Amy L. Firth, Weijuan Yao, and Jason X.-J. Yuan
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44 Differentiation of Embryonic Stem Cells to Vascular Cell Lineages................. Andriana Margariti, Lingfang Zeng, and Qingbo Xu
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Clinical Research Approaches 45 Statistics and Clinical Data Analysis: A Reference Guide.................................. Michael C. Donohue, Tanya Wolfson, and Anthony C. Gamst
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Part III Pathology and Pathobiology Hypoxia and Hyperoxia in the Development of Pulmonary Hypertension 46 Hypoxic Pulmonary Vasoconstriction................................................................... E. Kenneth Weir, Jesús A. Cabrera, Douglas A. Peterson, Saswati Mahapatra, and Zhigang Hong 47 Pathogenic Roles of Ca2+ and Ion Channels in Hypoxia-Mediated Pulmonary Hypertension....................................................................................... Jian Wang, Dandan Zhang, Carmelle V. Remillard, and Jason X.-J. Yuan 48 Roles of Endothelium-Derived Vasoactive and Mitogenic Factors in the Development of Chronic-Hypoxia-Mediated Pulmonary Hypertension....................................................................................... Rajeev Malhotra and Kenneth D. Bloch
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49 Oxygen-Sensitive Transcription Factors and Hypoxia-Mediated Pulmonary Hypertension....................................................................................... Louise Østergaard, Vinzenz H. Schmid, and Max Gassmann
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50 Developmental Regulation of Pulmonary Vascular Oxygen Sensing................. David N. Cornfield
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51 Pulmonary Vascular Remodeling by High Oxygen............................................. Rosemary C. Jones and Diane E. Capen
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Pulmonary Vasoconstriction and Vascular Remodeling in Pulmonary Hypertension 52 Pulmonary Vascular Remodeling: Cellular and Molecular Mechanisms................................................................................... Kurt R. Stenmark and Maria G. Frid
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53 Carbon Monoxide and Heme Oxygenase in the Regulation of Pulmonary Vascular Function and Structure.................................................. Stella Kourembanas
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54 Shear Stress, Cell Signaling, and Pulmonary Vascular Remodeling................. Shampa Chatterjee and Aron B. Fisher
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55 Pulmonary Hypertension and the Extracellular Matrix..................................... Marlene Rabinovitch
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56 Role of Progenitor Cells in Pulmonary Vascular Remodeling............................ Kurt R. Stenmark, Susan M. Majka, and Maria G. Frid
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57 Receptor Signaling in Pulmonary Arterial Hypertension................................... Patricia A. Thistlethwaite, Robin N. Leathers, Xioadong Li, and Xiaoxue Zhang
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Endothelium and Thrombosis in the Development of Pulmonary Hypertension 58 Role of Endothelium in the Development of Pulmonary Hypertension............. Bryan Ross and Adel Giaid
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59 Coagulation and the Vessel Wall in Pulmonary Embolism................................. Irene M. Lang
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Pulmonary Edema and Acute Lung Injury 60 Alveolar Epithelial Fluid Transport in Lung Injury........................................... Hans G. Folkesson
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61 High-Altitude Pulmonary Edema.......................................................................... Erik R. Swenson
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62 Statins and Acute Lung Injury.............................................................................. Amit K. Mahajan and Jeffery R. Jacobson
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63 Genomics of Acute Lung Injury and Vascular Barrier Dysfunction................. Roberto F. Machado and Joe G.N. Garcia
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64 Ventilator-Induced Mechanical Stress and Lung Vascular Dysfunction........... Konstantin G. Birukov
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Part IV Pulmonary Vascular Diseases Nomenclature, Classification and Epidemiology of Pulmonary Hypertension 65 Classification of Pulmonary Hypertension: History and Perspectives.............. David B. Badesch
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66 Epidemiology of Pulmonary Arterial Hypertension............................................ Jess Mandel and Darren B. Taichman
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67 Air Pollution and the Pulmonary Vasculature..................................................... Melissa L. Bates and Rebecca Bascom
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Pulmonary Arterial Hypertension 68 Idiopathic Pulmonary Arterial Hypertension...................................................... Jane E. Lewis and Richard N. Channick
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69 Genetics of Familial and Idiopathic Pulmonary Arterial Hypertension........... Eric D. Austin, James E. Loyd, and John A. PhillipsIII
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70 Pulmonary Arterial Hypertension Related to Scleroderma and Collagen Vascular Diseases............................................................................. 1011 Paul M. Hassoun 71 Pathology and Management of Portopulmonary Hypertension......................... 1023 Michael J. Krowka 72 Pathobiology and Treatment of Pulmonary Hypertension in HIV Disease................................................................................. 1033 Michael H. Ieong and Harrison W. Farber 73 Pulmonary Arterial Hypertension Secondary to Anorexigensand Other Drugs and Toxins........................................................ 1043 Kim Bouillon, Yola Moride, Lamiae Bensouda-Grimaldi, and Lucien Abenhaim 74 Pulmonary Arterial Hypertension Related to Gaucher’s Disease, Sarcoidosis, and Other Disorders........................................................... 1061 Judd W. Landsberg and Jess Mandel Pulmonary Arterial Hypertension in Pediatric Patients 75 Pediatric Pulmonary Hypertension: An Integrated View from Pediatric Subspecialists....................................................................... 1083 Judy L. Aschner, Candice D. Fike, Eric D. Austin, Frederick E. Barr, and J. Donald Moore 76 Persistent Pulmonary Hypertension of the Newborn: Mechanisms and Treatment................................................................................... 1109 Steven H. Abman, Robin H. Steinhorn, and Judy L. Aschner
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77 The Pulmonary Circulation in Congenital Heart Disease................................... 1119 Thomas J. Kulik and Mary P. Mullen 78 Pulmonary Hypertension Secondary to Congenital Systemic-to-Pulmonary (Left-to-Right) Shunts................................................... 1139 Antonio A. Lopes 79 Surgical Evaluation of Congenital-Heart-Disease-Associated Pulmonary Hypertension....................................................................................... 1153 Clive J. Lewis and Andrew A. Klein Pulmonary Venous Hypertension 80 Pulmonary Veno-occlusive Disease........................................................................ 1169 Peter F. Clardy and Jess Mandel 81 Left Ventricular Diastolic Heart Function and Pulmonary Hypertension........ 1183 Stuart Rich and Mardi Gomberg-Maitland Pulmonary Hypertension Associated with Hypoxia and Hypoxemia 82 Pulmonary Hypertension Associated with Chronic Obstructive Pulmonary Diseases........................................................................... 1189 Norbert F. Voelkel, Catherine Grossman, and Herman J. Bogaard 83 Pulmonary Hypertension Associated with Interstitial Lung Disease................. 1197 Mary E. Strek and Julian Solway 84 High-Altitude Pulmonary Hypertension.............................................................. 1211 Fabiola León-Velarde and Francisco C. Villafuerte 85 Pulmonary Hypertension and Congenital Heart Defects at High Altitude......................................................................................... 1223 Alexandra Heath de Freudenthal, Franz-Peter Freudenthal Tichauer, Carmen R. Condori Taboada, and Janne C. Lopes Mendes Pulmonary Hypertension due to Chronic Thrombotic and/or Embolic Disease 86 Pulmonary Embolism and Deep Vein Thrombosis.............................................. 1231 Paul F. Currier and Charles A. Hales 87 Pulmonary Hypertension Due to Pulmonary Embolism and Thromboembolic Obstruction of Proximal and Distal Pulmonary Arteries.............................................................................. 1239 Peter F. Fedullo, Kim M. Kerr, and William R. Auger 88 Risk Factors for Chronic Thromboembolic Pulmonary Hypertension............. 1253 Diana Bonderman and Irene M. Lang 89 Evaluation of Small-Vessel Arteriopathy in Chronic Thromboembolic Pulmonary Hypertension......................................................... 1261 Nick H. Kim
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90 Hemolytic-Anemia-Associated Pulmonary Hypertension: Sickle-Cell-Disease- and Thalassemia-Associated Pulmonary Hypertension..................................................................................... 1269 Elizabeth S. Klings and Mark T. Gladwin Pulmonary Hypertension due to Disorders Directly Affecting the Pulmonary Vasculature 91 Pulmonary Hypertension Due to Schistosomiasis.............................................. 1283 Ghazwan Butrous and Angela P. Bandiera 92 Pulmonary Hypertension Due to Capillary Hemangiomatosis........................ 1297 Mourad Toporsian, David H. Roberts, and S. Ananth Karumanchi Right Heart Dysfunction and Pulmonary Hypertension 93 Molecular Basis of Right Ventricular Hypertrophy and Failure in Pulmonary Vascular Disease....................................................... 1305 Yuichiro J. Suzuki 94 Right Ventricular Dysfunction in Pulmonary Hypertension............................ 1313 Francois Haddad, Mehdi Skhiri, and Evangelos Michelakis Other Disorders of the Pulmonary Circulation 95 Large Vessel Pulmonary Arteritis....................................................................... 1333 Kim M. Kerr 96 Tumors of the Pulmonary Vascular Bed............................................................. 1343 Eunhee S. Yi 97 Cor Pulmonale....................................................................................................... 1355 Mardi Gomberg-Maitland, Thenappan Thenappan, John J. Ryan, Ankush Goel, Nicole Cipriani,Aliya N. Husain, Amit Patel, Savitri E. Fedson, and Stephen L. Archer 98 Pregnancy and Contraception in Patients with Pulmonary Arterial Hypertension........................................................................................... 1377 Barbara A. Cockrill and Charles A. Hales Part V Diagnosis and Treatment Diagnostic Approaches for Pulmonary Vascular Diseases 99 Cardiac Catheterization in the Patient with Pulmonary Hypertension.......... 1387 Christopher Barnett and Ori Ben-Yehuda 100 Imaging of Pulmonary Vascular Diseases........................................................... 1403 Michael A. Bettmann 101 Histological and Pathological Diagnosis of Pulmonary Hypertension: Pathological Classification of Pulmonary Vascular Lesions.................................................................................................... 1413 Brian B. Graham, Li Zhang, and Rubin M. Tuder
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102 Echocardiography in Pulmonary Vascular Disease........................................... 1425 Paul R. Forfia Therapeutic Interventions for Pulmonary Vascular Diseases 103 Calcium Channel Blockers in the Treatment of Pulmonary Arterial Hypertension.................................................................. 1447 Stuart Rich and Mardi Gomberg-Maitland 104 Prostacyclin and Prostaglandins.......................................................................... 1451 Horst Olschewski 105 Endothelin Receptor Antagonists for the Treatment of Pulmonary Arterial Hypertension.................................................................. 1465 Victor F. Tapson and Robyn J. Barst 106 Phosphodiesterase Inhibitors in the Treatment of Pulmonary Hypertension................................................................................. 1477 Lan Zhao, Zhenguo Zhai, John Wharton, and Martin R. Wilkins 107 Nitric Oxide for Children..................................................................................... 1487 Judy L. Aschner, Candice D. Fike, Eric Austin, J. Donald Moore, and Frederick E. Barr 108 The Serotonin System as a Therapeutic Target in Pulmonary Hypertension................................................................................. 1501 Serge Adnot 109 Combination Therapy for Pulmonary Arterial Hypertension......................... 1509 Ioana R. Preston and Nicholas S. Hill 110 Thrombolytic and Anticoagulant Therapy for Pulmonary Embolism and Chronic Thromboembolic Pulmonary Hypertension.............. 1521 Paul F. Currier and Charles A. Hales 111 Nursing Care of Patients with Pulmonary Arterial Hypertension................... 1531 Christine Archer-Chicko Interventional and Surgical Approaches to Treatment of Pulmonary Vascular Diseases 112 Atrial Septostomy.................................................................................................. 1559 Julio Sandoval, Jorge Gaspar, and Héctor Peña 113 Evaluation of Patients with Chronic Thromboembolic Pulmonary Hypertension for Pulmonary Endarterectomy.............................. 1567 William R. Auger, Peter F. Fedullo, and Kim M. Kerr 114 Pulmonary Endarterectomy................................................................................ 1575 Michael M. Madani and Stuart W. Jamieson 115 Evaluation of Patients with Pulmonary Hypertension for Lung Transplantation..................................................................................... 1591 Gordon L. Yung
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116 Lung Transplantation for Pulmonary Hypertension......................................... 1597 Ashish S. Shah and John V. Conte Jr. 117 Living-Donor Lobar Lung Transplantation for Pulmonary Arterial Hypertension................................................................ 1601 Hiroshi Date and Takahiro Oto 118 Results of Lung Transplantation......................................................................... 1611 Janet R. Maurer Index................................................................................................................................. 1625
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Contributors
Lucien Abenhaim, MD, PhD (Ch: 73) Professor, London School of Hygiene and Tropical Medicine, London, UK Steven H. Abman, MD (Ch: 9, 76) Professor, Department of Pediatrics, University of Colorado, The Children’s Hospital, Denver CO, USA Serge Adnot, MD, PhD (Ch: 108) Professor, Department of Physiology, Henri Mondor Hospital, Creteil, France Mansoor Ahmad, MD, PhD (Ch: 18) Research Associate, Department of Physiology, New York Medical College, Valhalla, NY, USA Sara K. Alford, MS (Ch: 35) Predoctoral Research Fellow, Department of Radiology, University of Iowa, Iowa City, IA, USA Stephen L. Archer, MD (Ch: 19, 97) Harold Hines Jr. Professor and Chief of Cardiology, Department of Medicine, University of Chicago, Chicago, IL, USA Christine Archer-Chicko, MSN, CRNP-BC (Ch: 111) Program Coordinator, Pulmonary Vascular Disease Program, Penn Presbyterian Medical Center, Philadelphia, PA, USA Judy L. Aschner, MD (Ch: 75, 76, 107) Department of Pediatrics, The Monroe Carell Jr. Children’s Hospital, Vanderbilt University School of Medicine, Nashville, TN, USA William R. Auger, MD (Ch: 87, 113) Division of Pulmonary Critical Care Medicine, University of California, San Diego, CA, USA Eric D. Austin, MD (Ch: 69, 75) Assistant Professor, Department of Pediatrics, The Monroe Carell Jr. Children's Hospital, Vanderbilt University, Nashville, TN, USA David B. Badesch, MD (Ch: 65) Professor, Department of Medicine, University of Colorado, Denver, Aurora, CO, USA
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Angela P. Bandiera, MD, PhD (Ch: 91) Consultant Cardiologist, Department of Cardiology, Health Sciences Centre, PROCAPE – University of Pernambuco and Memorials S. Jose Hospital, Recife, Brazil Christopher Barnett, MD, MPH (Ch: 99) Assistant Professor, Department of Cardiology, University of California, San Francisco, CA, USA Frederick E. Barr, MD (Ch: 75, 107) Professor and Division Chief, Department of Pediatrics, The Monroe Carell Jr. Children’s Hospital, Vanderbilt University, Nashville, USA Christina J. Barry, BS (Ch: 15) Graduate Student, Center for Lung Biology, University of South Alabama, Mobil, AL, USA Robyn J. Barst, MD (Ch: 105) Professor, Columbia University College of Physicians and Surgeons, New York, NY, USA Rebecca Bascom, MD, MPH (Ch: 67) Professor, Department of Pulmonary Allergy and Critical Care Medicine, Milton S. Hershey Medical Center, Hershey, PA, USA Melissa L. Bates, PhD (Ch: 67) Postdoctoral Fellow, Department of Pediatrics, University of Wisconsin, Madison, WI, USA Ori Ben-Yehuda, MD, FACC (Ch: 99) Professor, Department of Medicine, University of California, San Diego, San Diego, CA, USA Lamiae Bensouda-Grimaldi, PharmD (Ch: 73) Doctor, Department Paris Santé Cochin, Inserm/La-ser, Paris, France Michael A. Bettmann, MD (Ch: 100) Professor and Vice Chair for Interventional Services, Department of Radiology, Wake Forest School of Medicine, Winston-Salem, NC, USA Konstantin G. Birukov, MD, PhD (Ch: 64) Assistant Professor, Department of Medicine, University of Chicago, Chicago, IL, USA Kenneth D. Bloch, MD (Ch: 48) William Thomas Green Morton Professor, Departments of Anesthesia and Medicine, Massachusetts General Hospital, Harvard Medical School, Boston, MA, USA Herman J. Bogaard, MD, PhD (Ch: 82) Assistant Professor, Department of Internal Medicine, Virginia Commonwealth University, Richmond, VA, USA Diana Bonderman, MD (Ch: 88) Assistant Professor, Department of Cardiology, Medical University of Vienna, Vienna, Austria Kim Bouillon, MD, MPH (Ch: 73) Doctor, Paris Santé Cochin, La-ser, Paris, France Ellen C. Breen, PhD (Ch: 37) Associate Research Scientist, Department of Medicine, University of California, San Diego, La Jolla, CA, USA
Contributors
Contributors
Todd M. Bull, MD (Ch: 40) Associate Professor, Department of Medicine, University of Colorado, Denver, Aurora, CO, USA Kelly S. Burrowes, PhD (Ch: 6) Postdoctoral Research Fellow, Oxford University, Oxford, UK Ghazwan Butrous, MB, ChB, PhD (Ch: 91) Professor, Division of Cardiopulmonary Sciences, University of Kent, Canterbury, UK Jesús A. Cabrera, MD, PhD (Ch: 46) Assistant Professor, Department of Surgery, University of Minnesota, Minneapolis, MN, USA Diane E. Capen, BA (Ch: 3, 51) Department of Anesthesia, Critical Care and Pain Management, Massachusetts General Hospital, Harvard Medical School, Boston, MA, USA D. Jeannean Carver, MD (Ch: 17) Associate Professor, Department of Pediatrics, University of Virginia, Charlottesville, VA, USA Stephen Y. Chan, MD, PhD (Ch: 11) Instructor, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, MA, USA Richard N. Channick, MD (Ch: 68) Associate Professor and Director, Pulmonary Hypertension Program, Department of Pulmonary and Critical Care Medicine, Massachusetts General Hospital, Harvard Medical School, Boston, MA, USA Shampa Chatterjee, PhD (Ch: 54) Research Assistant Professor, Institute for Environmental Medicine, University of Pennsylvania, Philadelphia, PA, USA Naomi C. Chesler, PhD (Ch: 5) Associate Professor, Department of Biomedical Engineering, University of Wisconsin, Madison, WI, USA Eddie T. Chiang, MA (Ch: 12) Senior Research Technician, Department of Medicine, University of Illinois at Chicago, Chicago, IL, USA Nicole Cipriani, MD (Ch: 97) Fellow, Department of Pathology, University of Chicago, Chicago IL, USA Peter F. Clardy, MD (Ch: 80) Clinical Instructor, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, USA Barbara A. Cockrill, MD (Ch: 98) Clinical Director, Pulmonary Vascular Disease Center, Brigham and Women's Hospital, Harvard Medical School, Boston, MA, USA Donna L. Cioffi, PhD (Ch: 15) Assistant Professor, Center for Lung Biology, University of South Alabama, Mobile, AL, USA John V. Conte, Jr. MD (Ch: 116) Professor and Director, Heart Transplantation and Mechanical Circulatory Support, Johns Hopkins University School of Medicine, Baltimore, MD, USA David N. Cornfield, MD (Ch: 50) Department of Pediatrics – Pulmonary Medicine, Lucile Salter Packard Children’s Hospital at Stanford, Stanford, CA, USA James T. B. Crawley, PhD (Ch: 23) Department of Haematology, Imperial College London, Hammersmith Hospital, London, UK
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Paul F. Currier, MD, MPH (Ch: 86, 110) Associate Program Director, Department of Medicine, Massachusetts General Hospital, Harvard Medical School, Boston, MA, USA Riku Das, PhD (Ch: 27) Postdoctoral Fellow, Department of Molecular Cardiology, Lerner Research Institute, Cleveland Clinic, Cleveland, OH, USA Hiroshi Date, MD (Ch: 117) Professor, Department of Thoracic Surgery, Kyoto University, Kyoto, Japan Yvonne Dempsie, PhD (Ch: 8) Faculty of Biomedical and Life Sciences, University of Glasgow, Glasgow, UK Michael C. Donohue, PhD (Ch: 45) Assistant Project Scientist, Division of Biostatistics and Bioinformatics, University of California, San Diego, La Jolla, CA, USA Dayue Darrel Duan, MD, PhD (Ch: 41) Professor, Department of Pharmacology, University of Nevada School of Medicine, Reno, NV, USA Jocelyn Dupuis, MD, PhD (Ch: 30) Associate Professor, Department of Medicine, Montreal Heart Institute, Montreal, Quebec, Canada Oliver Eickelberg, MD (Ch: 22) Professor, Institute of Lung Biology and Disease, Comprehensive Pneumology Center, Neuherberg/Munich, Bavaria, Germany Harrison W. Farber, MD (Ch: 72) Professor, Pulmonary Center, Boston University, Boston, MA, USA Savitri E. Fedson, MD (Ch: 97) Assistant Professor and Medical Director of Cardiac Care Unit, University of Chicago, Chicago, IL, USA Peter F. Fedullo, MD (Ch: 80, 87, 113) Division of Pulmonary Critical Care Medicine, University of California, San Diego, CA, USA Thomas Fellner, PhD (Ch: 42) Assistant Director, Human Embryonic Stem Cell Core Facility, University of California, San Diego, La Jolla, CA, USA Candice D. Fike, MD (Ch: 75, 107) Professor, Department of Pediatrics, The Monroe Carell Jr. Children’s Hospital, Vanderbilt University, Nashville, TN, USA Amy L. Firth, PhD (Ch: 13, 43) Postdoctoral Fellow, Laboratory of Genetics, The Salk Institute for Biological Studies, La Jolla, CA, USA Aron B. Fisher, MD (Ch: 54) Professor and Director, Institute for Environmental Medicine, University of Pennsylvania, Philadelphia, PA, USA Hans G. Folkesson, PhD (Ch: 60) Department of Physiology and Pharmacology, Northeastern Ohio Universities College of Medicine, Rootstown, OH, USA
Contributors
Contributors
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Paul R. Forfia, MD (Ch: 102) Professor and Director, Institute for Environmental Medicine, Department of Medicine/Cardiology, University of Pennsylvania, Philadelphia, PA, USA Franz-Peter Freudenthal Tichauer, MD (Ch: 85) Department of Interventional Cardiology, Kardiozentrum Medical Specialty Center, La Paz, Bolivia Maria G. Frid, PhD (Ch: 52, 56) Instructor, Department of Pediatric Critical Care, University of Colorado, Denver, Aurora, CO, USA Anthony C. Gamst, PhD (Ch: 45) Associate Professor, Division of Biostatistics and Bioinformatics, University of California, San Diego, La Jolla, CA, USA Joe G. N. Garcia, MD (Ch: 63, 12) University of Illinois at Chicago, Chicago, IL, USA Jorge Gaspar, MD (Ch: 112) Chief, Interventional Cardiology Department, Ignacio Chávez National Institute of Cardiology, Mexico City, Mexico Max Gassmann, DVM (Ch: 49) Professor, Vetsuisse Faculty, Institute of Veterinary Physiology and Zurich Center for Integrative Human Physiology, University of Zurich, Zurich, Switzerland Mark W. Geraci, MD (Ch: 40) Professor and Division Head, Department of Medicine, University of Colorado, Denver, Aurora, CO, USA Adel Giaid, MD, PhD (Ch: 58) Department of Cardiology, McGill University Health Centre, Montreal, Quebec, Canada Joan Gil, MD (Ch: 2) Professor,Department of Pathology, Mount Sinai School of Medicine, New York, NY, USA Mark T. Gladwin, MD (Ch: 90) Professor and Chief, Division of Pulmonary, Allergy, and Critical Care Medicine, University of Pittsburgh, Pittsburgh, PA, USA Ankush Goel, MD (Ch: 97) Fellow, Department of Medicine, University of Chicago, Chicago, IL, USA Mardi Gomberg-Maitland, MD, MSc (Ch: 97, 103, 81) Associate Professor and Director of Pulmonary Hypertension, Department of Medicine/Cardiology, University of Chicago, Chicago, IL, USA Jose R. Gonzalez-Porras (Ch: 23) Department of Haematology, Imperial College London, Hammersmith Hospital, London, UK Brian B. Graham, MD (Ch: 101) Fellow, Department of Medicine, University of Colorado Hospital, Denver, CO, USA Catherine Grossman, MD (Ch: 82) Assistant Professor, Department of Internal Medicine, Virginia Commonwealth University, Richmond, VA, USA
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Sachin A. Gupte, MD, PhD (Ch: 18) Assistant Professor, Department of Biochemistry and Molecular Biology, University of South Alabama, Mobile, AL, USA Francois Haddad, MD (Ch: 94) Attending Cardiologist, Department of Medicine, Stanford University, Palo Alto, CA, USA Charles A. Hales, MD (Ch: 86, 98, 110) Professor, Pulmonary and Critical Care Unit, Massachusetts General Hospital, Harvard Medical School, Boston, MA, USA Sara Hanif Mirza, MD (Ch: 32) Internal Medicine Resident, Department of Medicine, University of Illinois at Chicago, Chicago, IL, USA Paul M. Hassoun, MD (Ch: 70) Professor and Director of Pulmonary Hypertension Program, Department of Medicine, Johns Hopkins University, Baltimore, MD, USA Alexandra Heath de Freudenthal, MD (Ch: 85) Head of Pediatric Cardiology, Kardiozentrum Medical Specialty Center, La Paz, Bolivia Nicholas S. Hill, MD (Ch: 109) Professor and Chief of Pulmonary, Division of Pulmonary, Critical Care and Sleep Medicine, Tufts Medical Center, Boston, MA, USA Eric A. Hoffman, PhD (Ch: 35) Professor, Department of Radiology, University of Iowa, Iowa City, IA, USA Zhigang Hong, MD, PhD (Ch: 46) Assistant Professor, Department of Medicine, University of Minnesota, VA Medical Center, Minneapolis, MN, USA Gabor Horvath, MD, PhD (Ch: 29) Associate Professor, Department of Pulmonology, Semmelweis University, Budapest, Hungary Aliya N. Husain, MD (Ch: 97) Professor, Department of Pathology, University of Chicago, Chicago, IL, USA Michael H. Ieong, MD (Ch: 72) Department of Allergy, Pulmonary, and Critical Care Medicine, Boston University Medical Center, Boston, MA, USA Paul A. Insel, MD (Ch: 14, 36) Professor and Vice Chair, Departments of Pharmacology and Medicine, University of California, San Diego, La Jolla, CA, USA Jeffrey R. Jacobson, MD (Ch: 62) Assistant Professor, University of Illinois at Chicago, Illinois, Chicago Stuart W. Jamieson, MD (Ch: 114) Distinguished Professor and Director of Cardiothoracic Surgery Division, Department of Surgery, University of California, San Diego, San Diego, CA, USA Rosemary C. Jones, PhD (Ch: 3, 51) Department of Anesthesia, Critical Care and Pain Management, Massachusetts General Hospital, Harvard Medical School, Boston, MA, USA
Contributors
Contributors
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S. Ananth Karumanchi, MD (Ch: 92) Associate Professor, Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, USA Kim M. Kerr, MD (Ch: 87, 95, 113) Associate Professor, Department of Medicine, University of California, San Diego, La Jolla, CA, USA Nick H. Kim, MD (Ch: 89) Division of Pulmonary and Critical Care Medicine, University of California, San Diego, La Jolla, CA, USA Andrew A. Klein, MBBS (Ch: 79) Consultant Anaesthetist, Transplant Unit, Papworth Hospital NHS Trust, Cambridge, UK Elizabeth S. Klings, MD (Ch: 90) Pulmonary Center, Boston University, Boston, MA, USA Greg A. Knock, PhD (Ch: 10) Division of Asthma, Allergy and Lung Biology, King’s College London, London, UK Stella Kourembanas, MD (Ch: 53) Clement A. Smith Professor and Chief, Harvard Division of Newborn Medicine, Children’s Hospital, Harvard Medical School, Boston, MA, USA Fotini M. Kouri, PhD (Ch: 22) Department of Neurology, Northwestern University, Chicago, IL, USA Michael H. Kroll, MD (Ch: 24) Professor and Chief, Department of Benign Hematology, University of Texas, MD Anderson Cancer Center, Houston, TX, USA Michael J. Krowka, MD (Ch: 71) Professor and Vice-Chair, Division of Pulmonary and Critical Care Medicine, Mayo Clinic, Rochester, MN, USA Thomas J. Kulik, MD (Ch: 77) Senior Associate in Cardiology, Department of Cardiology, Children’s Hospital Boston, Boston, MA, USA Manoj M. Lalu, MD, PhD (Ch: 28) Resident Physician, Department of Anesthesiology, Ottawa Hospital, Ottawa, ON, Canada Judd W. Landsberg, MD (Ch: 74) Assistant Professor and Medical Director of Intensive Care Unit, VA San Diego Healthcare System, University of California, San Diego, La Jolla, CA, USA David A. Lane, PhD (Ch: 23) Professor, Department of Haematology, Imperial College London, Hammersmith Hospital, London, UK Irene M. Lang, MD (Ch: 59, 88) Professor, Department of Cardiology, Medical University of Vienna, Vienna, Austria David Langleben, MD (Ch: 7) Professor and Director, Center for Pulmonary Vascular Disease, Department of Cardiology, Jewish General Hospital, McGill University, Montreal, Quebec, Canada Robin N. Leathers, BS (Ch: 57) Staff Research Associate, Division of Cardiothoracic Surgery, University of California, San Diego, San Diego, CA, USA
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Fabiola León-Velarde, DSc (Ch: 84) Professor, Department of Clinical Biology and Physiology, Universidad Peruana Cayetano Heredia, Lima, Peru Clive J. Lewis, MB, BChir, PhD (Ch: 79) Consultant Cardiologist, Transplant Unit, Papworth Hospital NHS Trust, Cambridge, UK Jane E. Lewis, MD (Ch: 68) Postdoctoral Fellow, Department of Medicine, University of California, San Diego, La Jolla, CA, USA Xioadong Li, MD, PhD (Ch: 57) Project Scientist, Division of Cardiothoracic Surgery, University of California, San Diego, San Diego, CA, USA Antonio A. Lopes, MD (Ch: 78) Professor, Department of Pediatric Cardiology and Adult Congenital Heart Disease, Heart Institute, University of São Paulo School of Medicine, São Paulo, Brazil Joseph Loscalzo, MD, PhD (Ch: 11) Bersey Professor and Chairman, Department of Medicine, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA, USA Karen M. Lounsbury, PhD (Ch: 21) Associate Professor, Department of Pharmacology, University of Vermont, Burlington, VT, USA James E. Loyd, MD (Ch: 69) Rudy W. Jacobson Professor, Department of Medicine, Vanderbilt University, Nashville, TN, USA Roberto F. Machado, MD (Ch: 63) Assistant Professor, Department of Medicine, University of Illinois at Chicago, Chicago, IL, USA Margaret R. MacLean, PhD (Ch: 8) Mandy MacLean College of Medical, Veterinary and Life Sciences, University of Glasgow, UK Michael M. Madani, MD (Ch: 114) Associate Professor, Department of Surgery, University of California, San Diego, San Diego, CA, USA Amit K. Mahajan, MD (Ch: 62) Fellow, Department of Medicine, University of Chicago, Chicago, IL, USA Saswati Mahapatra, MS (Ch: 46) Assistant Scientist, Department of Medicine, University of Minnesota, Minneapolis, MN, USA Susan M. Majka, PhD (Ch: 56) Assistant Professor, Department of Cardiovascular Pulmonary Research, University of Colorado, Denver, Aurora, CO, USA Ayako Makino, PhD (Ch: 33) Assistant Professor, Department of Medicine, University of Illinois at Chicago, Chicago IL, USA Rajeev Malhotra, MD (Ch: 48) Fellow, Massachusetts General Hospital, Harvard Medical School, Division of Cardiology, Boston, MA, USA
Contributors
Contributors
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Asrar B. Malik, PhD (Ch: 16, 32) Distinguished Professor and Head, Department of Pharmacology and Center for Lung and Vascular Biology, University of Illinois, Chicago, IL, USA Jess Mandel, MD (Ch: 66, 74, 80) Associate Professor, Division of Pulmonary and Critical Care Medicine, University of California, San Diego, La Jolla, CA, USA Andriana Margariti, PhD (Ch: 44) Cardiovascular Division, King’s College London, Coldharbour Lane, London, UK Janet R. Maurer, MD, MBA, MBC (Ch: 118) Vice President, Medical Director, Department of Provider Services and Coaching Effectiveness, Health Dialog Service Corporation, Desert Hills, AZ, USA Janne C. Lopes Mendes (Ch: 85) Master of Public Health/Epidemiology, General Medicine, Kardiozentrum Medical Specialty Center, La Paz, Bolivia Evangelos Michelakis, MD (Ch: 94) Professor, Division of Cardiovascular Medicine, University of Alberta, Edmonton, Canada Richard D. Minshall, PhD (Ch: 16) Associate Professor, Departments of Pharmacology and Anesthesiology and Center for Lung and Vascular Biology, University of Illinois at Chicago, Chicago, IL, USA M. Kamran Mirza, MD, PhD (Ch: 32) Postdoctoral Research Associate, Department of Pharmacology, University of Illinois at Chicago, Chicago, IL, USA J. Donald Moore, MD (Ch: 75, 107) Assistant Professor, Division of Cardiology, Vanderbilt Children’s Hospital, Nashville, TN, USA Yola Moride, PhD (Ch: 73) Associate Professor, Faculty of Pharmacy, University of Montreal, Montreal, Quebec, Canada Nicholas W. Morrell, MD (Ch: 20) Professor, Department of Medicine, University of Cambridge, Cambridge, UK Timothy A. Morris, MD (Ch: 25) Professor, Department of Medicine, University of California, San Diego, San Diego, CA, USA Rohit Moudgil, MD, PhD (Ch: 28) Postdoctoral Fellow, Department of Medicine, Ottawa General Hospital, Ottawa, Ontario, Canada Mary P. Mullen, MD, PhD (Ch: 77) Associate in Cardiology,Department of Cardiology, Children’s Hospital Boston, Harvard Medical School, Boston, MA, USA Fiona Murray, PhD (Ch: 14, 36) Postdoctoral Fellow, Departments of Medicine and Pharmacology, University of California, San Diego, La Jolla, CA, USA Robert Naeije, MD, PhD (Ch: 4) Professor, Laboratory of Physiology, Erasme Hospital, Brussels, Belgium
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Debby Ngo, MD (Ch: 25) Postdoctoral Fellow, Department of Medicine, University of California, San Diego, La Jolla, CA, USA Geerten P. van Nieuw Amerongen, PhD (Ch: 16) Assistant Professor, VU University Medical Center, Institute for Cardiovascular Research, Department of Physiology, Amsterdam, The Netherlands Takashi Ohtsuka, MD (Ch: 26) Research Fellow, Department of Internal Medicine, University of Michigan, Ann Arbor, MI, USA Horst Olschewski, MD, PhD (Ch: 104) Professor, Department of Internal Medicine, University Hospital, Giessen, Germany Stylianos E. Orfanos, MD, PhD (Ch: 7) Associate Professor, 2nd Department of Critical Care, University of Athens Medical School and Pulmonary Hypertension Clinic, Attikon Hospital, Haidari Athens, Greece Louise Østergaard, PhD (Ch: 49) Zurich Center for Integrative Human Physiology, Vetsuisse Faculty, Institute of Veterinary Physiology, University of Zurich, Zurich, Switzerland Takahiro Oto, MD, PhD (Ch: 117) Senior Assistant Professor and Head of Lung Transplant Program, Department of Cancer and Thoracic Surgery, Okayama University, Okayama, Japan Lisa A. Palmer, PhD (Ch: 17) Associate Professor, Departments of Pediatrics and Anesthesiology, University of Virginia, Charlottesville, VA, USA Amit Patel, MD (Ch: 97) Assistant Professor and Director of Cardiac Magnetic Resonance, Department of Medicine, University of Chicago, Chicago, IL, USA Héctor Peña, MD (Ch: 112) Staff, Cardiopulomonary Department, Ignacio Chávez National Institute of Cardiology, Mexico City, Mexico Douglas A. Peterson, MD, PhD (Ch: 46) Assistant Professor, Department of Medicine, VA Medical Center, University of Minnesota, Minneapolis, MN, USA John A. Phillips III, MD (Ch: 69) Professor and David T. Karzon Chair; Director, Division of Medical Genetics, Vanderbilt University, Nashville, TN, USA David J. Pinksy, MD (Ch: 26) J. Griswold Ruth, MD & Margery Hopkins Ruth Professor; Chief, Division of Cardiovascular Medicine; Director, Cardiovascular Center, Department of Internal Medicine, Cleveland Clinic, Cleveland, OH, USA Edward F. Plow, PhD (Ch: 27) Professor and Chairman, Department of Molecular Cardiology, Lerner Research Institute, Cleveland Clinic, Cleveland, OH, USA Ioana R. Preston, MD (Ch: 109) Assistant Professor and Co-Director, Division of Pulmonary, Critical Care and Sleep Medicine, Pulmonary Hypertension Center, Tufts Medical Center, Boston, MA, USA
Contributors
Contributors
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Marlene Rabinovitch, MD (Ch: 55) Dwight and Vera Dunlevie Professor, Department of Pediatrics; Research Director, Vera Moulton Wall Center for Pulmonary Vascular Disease, Stanford University, Stanford, CA, USA Carmelle V. Remillard, PhD (Ch: 33, 34, 47) Assistant Project Scientist, Department of Medicine, University of California, San Diego, La Jolla, CA, USA Stuart Rich, MD (Ch: 81, 103) Section of Cardiology, University of Chicago Medical Center, Chicago, IL, USA David H. Roberts, MD (Ch: 92) Clinical Director, Division of Pulmonary, Critical Care and Sleep Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, USA Megan Robinson, BS (Ch: 42) Staff Research Associate, Human Embryonic Stem Cell Core Facility, University of California, San Diego, La Jolla, CA, USA Alejandro Roldán-Alzate, PhD (Ch: 5) Research Associate, Department of Biomedical Engineering, University of Wisconsin, Madison, WI, USA Patricia C. Rose, PhD (Ch: 21) Postdoctoral Fellow, Department of Pharmacology, University of Vermont, Burlington, VT, USA Bryan Ross, BScH (Ch: 58) Department of Cardiology, McGill University Health Centre, Montreal QC, Canada John J. Ryan, MD (Ch: 19, 97) Cardiology Fellow, Department of Medicine, University of Chicago, Chicago, IL, USA Julio Sandoval, MD (Ch: 112) Professor and Chief, Cardiopulmonary Division, Ignacio Chávez National Institute of Cardiology, Mexico City, Mexico Vinzenz H. Schmid, PhD (Ch: 49) Zurich Center for Integrative Human Physiology, Vetsuisse Faculty, Institute of Veterinary Physiology, University of Zurich, Zurich, Switzerland Nicholas J. Schork, PhD (Ch: 39) Professor and Director of Research, Scripps Genomic Medicine; Director of Biostatistics and Bioinformatics, Scripps Translational Science Institute; Department of Molecular and Experimental Medicine, Scripps Research Institute, La Jolla, CA, USA Ashish S. Shah, MD (Ch: 116) Assistant Professor and Director of Lung Transplantation, Department of Surgery, Johns Hopkins University, Baltimore, MD, USA Mehdi Skhiri, MD (Ch: 94) Fellow Heart Transplant and Heart Failure, Department of Medicine, Stanford University, Palo Alto, CA, USA Julian Solway, MD (Ch: 83) Walter L. Palmer Distinguished Service Professor and Associate Dean for Translation Medicine; Vice Chair for Research, Department of Medicine, University of Chicago, Chicago, IL, USA
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Robin H. Steinhorn, MD (Ch: 9, 76) Professor and Division Head of Neonatology, Department of Pediatrics, Children’s Memorial Hospital, Northwestern University, Chicago, IL, USA Kurt R. Stenmark, MD (Ch: 52, 56) Professor, Department of Pediatric Critical Care, University of Colorado Denver, Aurora, CO, USA Troy Stevens, PhD (Ch: 15) Professor and Director, Center for Lung Biology and Departments of Medicine and Pharmacology, University of South Alabama, Mobile, AL, USA Duncan J. Stewart, MD (Ch: 28) Professor, Ottawa Hospital Research Institute, Ottawa, ON, Canada Mary E. Strek, MD (Ch: 83) Associate Professor, Department of Medicine, University of Chicago, Chicago, IL, USA Yuichiro J. Suzuki, PhD (Ch: 93) Professor, Department of Pharmacology, Georgetown University, Washington, DC, USA Erik R. Swenson, MD (Ch: 61) Professor, Department of Medicine, University of Washington, VA Puget Sound Health Care System, Seattle, WA, USA Carmen R. Condori Taboada, MD (Ch: 85) Master of Public Health/Epidemiology, General Medicine, Kardiozentrum Medical Specialty Center, La Paz, Bolivia Darren B. Taichman, MD, PhD (Ch: 66) Assistant Professor and Associate Director, Pulmonary Vascular Disease Program, University of Pennsylvania, Philadelphia, PA, USA Tamara Tajsic, MD (Ch: 20) Department of Medicine, Addenbrooke’s Hospital, Cambridge, UK Victor F. Tapson, MD (Ch: 105) Professor and Director, Pulmonary Vascular Disease Center, Duke University, Durham, NC, USA Merryn H. Tawhai, PhD (Ch: 6) Associate Professor, Auckland Bioengineering Institute, University of Auckland, Auckland, New Zealand Thenappan Thenappan, MD (Ch: 97) Fellow, Department of Medicine, University of Chicago, Chicago, IL, USA Patricia A. Thistlethwaite, MD, PhD (Ch: 57) Fellow, Division of Cardiothoracic Surgery, University of California, San Diego, San Diego, CA, USA Eric J. Topol, MD (Ch: 39) Professor and Director, Scripps Translational Science Institute; Chief Academic Officer, Scripps Health, The Scripps Research Institute; Senior Consultant, Division of Cardiovascular Diseases, Scripps Clinic, La Jolla, CA, USA Mourad Toporsian, PhD (Ch: 92) Instructor, Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, USA
Contributors
Contributors
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Ali Torkamani, PhD (Ch: 39) Research Scientist, Scripps Translational Sciences Institute, The Scripps Research Institute, La Jolla, CA, USA Rubin M. Tuder, MD (Ch: 101) Professor, Department of Medicine, University of Colorado, Denver, CO, USA Francisco C. Villafuerte, DPhil (Ch: 84) Department of Clinical Biology and Physiology, Universidad Peruana Cayetano Heredia, Lima, Perú Scott Visovatti, MD (Ch: 26) Clinical Lecturer, Department of Medicine, University of Michigan, Ann Arbor, MI, USA Norbert F. Voelkel, MD (Ch: 82) Professor and Director, Victoria Johnson Center and Department of Internal Medicine, Virginia Commonwealth University, Richmond, VA, USA Zoë L. Vomberg, BS (Ch: 42) Staff Research Associate, Human Embryonic Stem Cell Core Facility, University of California, San Diego, La Jolla,CA, USA Jian Wang, PhD (Ch: 47) Professor, First Affiliate Hospital, Guangzhou Medical University, Guangzhou, Guangdong, China Ting Wang, PhD (Ch: 12) Postdoctoral Fellow, Department of Medicine, University of Illinois at Chicago, Chicago, IL, USA Adam Wanner, MD (Ch: 29) Joseph Weintraub Professor, Division of Pulmonary and Critical Care Medicine, University of Miami, Miami, FL, USA Jeremy P. T. Ward, PhD (Ch: 10) Professor, Division of Asthma, Allergy and Lung Biology, King’s College London, London, UK E. Kenneth Weir, MD (Ch: 46) Professor, Department of Medicine, VA Medical Center, University of Minnesota, Minneapolis, MN, USA Norbert Weissmann, PhD (Ch: 30) Professor, Excellence Cluster Cardio-Pulmonary System, Justus-Liebig University Giessen, Giessen, Germany James D. West, PhD (Ch: 31) Assistant Professor, Department of Medicine, Vanderbilt University, Nashville, TN, USA John B. West, MD, PhD, DSc (Ch: 1) Distinguished Professor, Department of Physiology, University of California, San Diego, La Jolla, CA, USA Nico Westerhof, PhD (Ch: 4) Professor, Department of Pulmonary Diseases, VU University Medical Center, Amsterdam, The Netherlands John Wharton, PhD (Ch: 106) Department of Experimental Medicine and Toxicology, Imperial College Healthcare NHS Trust, London, UK
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Martin R. Wilkins, MD (Ch: 106) Department of Experimental Medicine, Imperial College London, Hammersmith Campus, London, UK Karl H. Willert, PhD (Ch: 42) Assistant Professor, Human Embryonic StemCell Core Facility, University of California, San Diego, La Jolla, CA, USA Tanya Wolfson, MA (Ch: 45) Principal Statistician, Computational and Applied Statistics Laboratory, San Diego Supercomputer Center, University of California, San Diego, La Jolla, CA, USA Michael S. Wolin, PhD (Ch: 18) Professor, Department of Physiology, New York Medical College, Valhalla, NY, USA Qingbo Xu, MD, PhD (Ch: 44) Professor and BHF John Parker Chair, Cardiovascular Division, King’s College London, London, UK Weijuan Yao, PhD (Ch: 43) Associate Professor, Department of Physiology and Pathophysiology, Peking University, Beijing, China E.S. Yi, MD Professor, Department of Anatomic Pathology, Mayo Clinic, 200 First Street SW, Rochester, MN 55905, USA Ying Yu, MD, PhD (Ch: 38) Postdoctoral Fellow, Department of Medicine, University of California, San Diego, La Jolla, CA, USA Jason X.-J. Yuan, MD, PhD (Ch: 13, 14, 33, 34, 36, 37, 38, 43, 47) Professor, Departments of Medicine and Pharmacology, The Institute for Personalized Respiratory Medicine, University of Illinois at Chicago, Chicago, IL, USA Gordon L. Yung, MB, BS (Ch: 115) Professor and Medical Director of Lung Transplant Program, Department of Medicine, University of California, San Diego, San Diego, CA, USA Lingfang Zeng, PhD (Ch: 44) Lecturer, Cardiovascular Division, King’s College London, London, UK Zhenguo Zhai, MD, PhD (Ch: 106) Associate Professor, Beijing Institute of Respiratory Medicine and Beijing Chaoyang Hospital, Capital Medical University, Beijing, China Dandan Zhang, PhD (Ch: 47) Research Scientist, Respiratory Department, Guangzhou Medical College, Guangzhou, Guangdong, China Li Zhang, MD (Ch: 101) Professional Research Assistant, Department of Medicine, University of Colorado, Denver, CO, USA Xiaoxue Zhang, MD (Ch: 57) Staff Research Associate, Division of Cardiothoracic Surgery, University of California, San Diego, San Diego, CA, USA Lan Zhao, MD, PhD (Ch: 106) Lecturer, Department of Experimental Medicine and Toxicology, Imperial College Healthcare NHS Trust, London, UK
Contributors
Part I
Structure, Function and Regulation
Chapter 1
The Human Pulmonary Circulation: Historical Introduction John B. West
Abstract The history of our gradual understanding of the pulmonary circulation is a fascinating topic, partly because it involves some of the key figures in the development of cardiopulmonary physiology, but also because various misconceptions continue to surface from time to time and are seen even today. Of course, the story has been told before and this account follows the same sequence as one of earlier assays. Naturally, a history like this covering such a vast area must be both derivative and selective, and I apologize in advance to any reader who feels that his contributions have been overlooked. Keywords Pulmonary circulation • History
1 Introduction The history of our gradual understanding of the pulmonary circulation is a fascinating topic, partly because it involves some of the key figures in the development of cardiopulmonary physiology, but also because various misconceptions continue to surface from time to time and are seen even today. Of course, the story has been told before and this account follows the same sequence as one of my earlier essays [1]. Naturally, a history like this covering such a vast area must be both derivative and selective, and I apologize in advance to any reader who feels that his contributions have been overlooked.
2 Galen’s Scheme The Greco-Roman physician Galen (ca 130–ca 201) and his school elaborated a very comprehensive scheme for blood flow within the body that included a reference to the pulmonary J.B. West () Department of Physiology, University of California, San Diego 9500 Gilman Drive, La Jolla, CA 92093-0623, USA e-mail:
[email protected] c irculation. One of the remarkable features of this theory (Fig. 1) was the extraordinary influence it had for the next 1,400 years or so. In fact, when William Harvey was at Cambridge University in the late 1500s, part of the instruction included Galen’s writings [2], and indeed Galen’s teachings on bloodletting, for example, were followed for another 200 years. In Galen’s scheme, food underwent “concoction” in the gut and reached the liver, where the blood was formed and imbued with “natural spirit.” It then flowed to the right ventricle, from where some entered the lungs via the pulmonary artery to nourish them, and the remainder of the blood reached the left ventricle through “invisible pores” in the interventricular septum. The existence of these so-called pores was a puzzle for anatomists for over a 1,000 years, but it was a necessary feature of the Galen scheme because it was not appreciated that a large amount of blood flowed from the lung to the heart. In the left ventricle, the blood was mixed with “pneuma” or “soul” from the air that was inhaled, and the result was the formation of “vital spirit,” which was distributed throughout the body by the arterial blood. Some of this reached the brain, where it received “animal spirit,” which was then distributed throughout the body via the nerves, which were thought to be hollow. The generation of vital spirit in the left ventricle gave rise to fuliginous (sooty) waste products that were thought to travel back to the lung through the pulmonary vein. From there they were exhaled in the expired air. Of course, Galen’s scheme seems very fanciful to us now, but in those early times there was no notion of the continuous circulation of the blood nor the fact that energy-producing metabolism occurred in the peripheral tissues. In fact, the belief that all the metabolic changes occurred in the lungs persisted until the end of the 1700s and was espoused by the great Antoine Lavoisier (1734–1794), not receiving its death knell until almost 100 years later as a result of the work of Eduard Pflüger (1829–1910). One of the reasons why Galen’s scheme lasted so long was that European science fell to a low ebb following the sack of Rome in 401. In these so-called Dark Ages, knowledge was mainly preserved by Arabian scholars and it was from one of these that the first intimations of the next advance in understanding the pulmonary circulation came about.
J.X.-J. Yuan et al. (eds.), Textbook of Pulmonary Vascular Disease, DOI 10.1007/978-0-387-87429-6_1, © Springer Science+Business Media, LLC 2011
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air, pass through the arteria venosa (pulmonary vein) to reach the left chamber of the heart and there form the vital spirit....” This was a remarkably perceptive statement and in the thirteenth century was way ahead of its time. Ibn alNafis was a polymath who, although born in Damascus, worked mainly in Cairo, and he contributed to a large variety of disciplines, including physiology and medicine, but also Sunni theology and law. His writings on the pulmonary circulation were essentially unknown to the Western world until 1924, when the manuscript was discovered in a Berlin library by a young Egyptian physician [3]. This being so, Michael Servetus (1511–1533) is credited with independently discovering the pulmonary transit when he wrote that blood passed from the right ventricle to the left from the pulmonary artery to the vein, not “as is commonly believed” through the interventricular septum. He also said that the color of the blood changed when it went through the lung. The statement was made in the theological book Christianismi Restitutio (“The Restoration of Christianity”), which was considered to be heretical because of Servetus’s views on the both the Trinity and infant baptism and as a result of which he was burned at the stake in Geneva (Fig. 2). Visitors to the Geneva Medical School should look for the small, insignificant memorial near the school which refers to
Fig. 1 The cardiopulmonary system according to Galen and his school. This had an enormous influence for some 1,400 years. (Reproduced with permission [58])
3 The Transit of Blood Through the Lung As we have seen, Galen’s view was that some blood entered the lung from the right ventricle, but there was no realization that it returned to the heart. In fact, it was believed that the products of combustion in the left ventricle moved retrogradely along the pulmonary veins to the lungs, where they could be exhaled. The first person to suggest the pulmonary transit of blood was the Arab scholar Ibn al-Nafis (1213–1288). He was a physician from Damascus and developed the idea that blood from the pulmonary artery reached the lung, where it mixed in some way with the air and then passed through the pulmonary veins to the heart. Writing in a commentary on the works of Avicenna, a Persian physician, Ibn al-Nafis (ca 980–1037), stated “...the blood from the right chamber of the heart must arrive at the left chamber but there is no direct pathway between them. The thick septum of the heart is not perforated and does not have visible pores as some people thought or invisible pores as Galen thought. The blood from the right chamber must flow through the vena arteriosa (pulmonary artery) to the lungs, spread through its substances, be mingled there with
Fig. 2 Michael Servetus (1511–1553). He described the transit of blood through the pulmonary circulation but was burned at the stake for religious heresy. (From Stirling [59])
1 The Human Pulmonary Circulation: Historical Introduction
the execution on one side but has an apology from the citizens of Geneva on the other. What was thought to be the last copy of Servetus’s heretical book was chained to his leg when he was burned, although in fact three copies survived. A few years later in the sixteenth century, Realdus Columbus (1516–1559) of Cremona, but working in Padua, also described the transit of blood through the lung, although since the circulation of the blood was not understood, there was no notion of the continuous flow at this time. Columbus was working as an assistant to the great sixteenth-century anatomist Andreas Vesalius (1514–1564), whose magnificent publication De Humani Corporis Fabrica (“Mechanisms of the Human Body”), produced when he was only 18, marked a watershed in the progress of science from its dominance by the Galenical school to the new thinking of the Renaissance. Any reader who is not familiar with the splendid woodcuts in this book should get hold of a copy forthwith! Nevertheless, Vesalius continued to be influenced by Galen to a large extent although he certainly questioned the existence of interventricular pores between the right and left ventricles, which was a cardinal feature of the Galenical scheme (Fig. 1). Vesalius looked carefully for the pores and, of course, found none. At about the same time, another Italian physician, Andreas Caesalpinus (1519–1603), who was a professor of medicine at Pisa, also described the pulmonary transit.
4 Discovery of the Circulation of the Blood There was bound to be confusion about the nature of blood flow in the body until its continuous circulation was first described by William Harvey (1578–1657) in his watershed book Exercitatio Anatomica de Motu Cordis et Sanguinis in Animalibus (“An Anatomical Study of the Motion of Heart and Blood in Animals”) published in 1628. In fact, his extant lecture notes show that he actually conceived the idea some 10 years earlier [2]. Unlike the scientists named so far, Harvey was an Englishman, but in fact he had to travel to Italy, where the scientific Renaissance was in full swing, to reach the cutting edge of the new medical science, and he spent two of his most influential years in Padua at the same university where both Columbus and Vesalius taught. Harvey initially went to Cambridge University but this was weak in science at the time, whereas at Padua the faculty included the anatomist Fabricius ab Aquapendente (1537–1619) and also the great Galileo Galilei (1564–1642). There is no evidence that Harvey and Galileo met, but there is a nice symmetry about the fact that just as Galileo made his epochal discovery that the Earth circulated around the Sun, Harvey made the equally important discovery that the blood circulated around the body. Harvey was greatly influenced by the fact that
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Aquapendente had described the valves in the veins and indeed part of Exercitatio Anatomica de Motu Cordis et Sanguinis in Animalibus is devoted to this. It is interesting that in spite of Harvey’s great insights into the circulation of blood around the whole body, he did not initially understand the pulmonary circulation, or indeed the nature of respiration. It is clear that for some time the role of the pulmonary circulation was a puzzle to him, and he wrote, “it is altogether incongruous to suppose that the lungs need for their nourishment so large a supply of food, so pulsatorily delivered” [4]. Remarkably, it was not until near the end of his life that he wrote a letter to his friend Slegel in which he described experiments in which the passage of blood from the pulmonary artery to the left ventricle was conclusively demonstrated [4]. However, Harvey had no way of knowing how the blood moved from the arteries to the veins in the peripheral tissues or from the pulmonary artery to the pulmonary veins. Indeed this was the next major advance. Although the concept of peripheral metabolism remained elusive, Harvey argued that the function of the circulation of the blood was to provide nutrition to the peripheral tissues. For example, in some of his lecture notes that are still extant he stated, “It is certain from the experiment . . . that there is a passage of the blood from the arteries to the veins. And for this reason it is certain that the perpetual movement of the blood in a circle is caused by the heartbeat. Why? Is it for the sake of nutrition, or is it rather for the preservation of the blood and of the limbs by means of the infused heat? And the blood by turns heating the limbs and when it is cold is warmed by the heart” [2].
5 Discovery of the Pulmonary Capillaries The name Marcello Malpighi (1628–1694) is usually associated with Bologna, although he spent periods at other Italian universities (Fig. 3). He was one of the first people to use the newly discovered microscope to make extensive studies on both animal and plant tissues. His most famous observations from our point of view were set out in two letters to his friend Giovanni Borelli (1608–1679), who was professor of mathematics at Pisa, but like so many Renaissance scientists had interests in many fields. Malpighi began very modestly by referring to “a few little observations that might increase the things found out about the lungs” [5]. But he then went on to describe the discovery of both the alveoli and the pulmonary capillaries using his microscope to look at the surface of frog lungs. He described the alveoli in his first letter as “an almost infinite number of orbicular bladders just as we see formed by wax plates in the walls of the honeycomb cells of beehives.” The second letter includes the discovery of the pulmonary capillaries, where Malpighi states that the network
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Fig. 3 Marcello Malpighi (1628–1694). Left: he discovered the pulmonary capillaries. Right: his drawing of the capillaries in a frog lung is shown at the top. (From Stirling [59])
of tiny vessels occupies not only the walls but also the floors of the alveoli, and that as the blood flows through the tortuous vessels it is not “poured into spaces but always works through tubules.” Remarkably, he also reported on the appearance of the red blood cells within the capillaries, although he thought that these were fat globules. These two letters make delightful reading even today. Malpighi’s discoveries of the pulmonary alveoli and capillaries completes the circle of the pulmonary circulation and finally resolves much of the confusion that we saw earlier. There is a nice symmetry in the fact that Malpighi was born in 1628, which was the same year as Exercitatio Anatomica de Motu Cordis et Sanguinis in Animalibus was published.
6 First Measurements of Blood Pressure One of the first measurements of blood pressure was made by Stephen Hales (1677–1761), who inserted a tube into the femoral artery of a horse and was astonished to see that the blood rose to the height of about 9 ft. Although he did not measure the pressure in the pulmonary circulation, he made many other important discoveries in that area. For example, in his book Haemostaticks [6] he calculated that the blood spent a little over 2 s in the pulmonary capillary, which was a remarkably accurate estimate since the presently accepted figure in man under resting conditions is about 0.75 s. He also obtained a value of about 17 mm (converted from his measurement in inches) for the diameter of a pulmonary capillary, and again this was a good estimate since we now know the value is about 5–10 mm. He noted that the flow in the pulmonary veins was pulsatile and he was well aware of the enormous area of the blood–gas barrier when he calculated
the surface area of the interior of a calf’s lung to be 30 m2. The area of the blood–gas barrier in the human lung is now given as about 50–100 m2. For the diameter of the alveoli in the calf lung, he calculated a figure of about 250 mm, and the current value for the human lung is about 300 mm. All these measurements were made using very primitive techniques, but Hales prided himself on his meticulous numerical approach using careful measurements, which explains his term “staticks” and “statistical” essays. Stephen Hales is one of my heroes partly because his investigative energy is so well documented in his very readable books, and partly because his place of work is still extant. He carried out his scientific work while a full-time minister of the parish of Teddington in west London and visitors to that city would be rewarded by a visit to his church, St. Alban’s, which still has the tower that he built, a nice stained glass window celebrating his ministry, and his grave with its fine commemorative stone which was recently refurbished with partial support from the American Physiological Society. Hales also introduced the U-tube manometer, but this was not for measuring blood pressure, but the pressure caused by the rising of sap in plants. Like many scientists of his day, he was equally interested in plants and animals and one of his bestknown books is Vegetable Staticks [7]. In fact, Hales is equally celebrated by plant and animal physiologists. The French physician Jean Poiseuille (1799–1869) was apparently the first person to accurately measure systemic blood pressure and he did this by means of a mercury U-tube manometer. Poiseuille’s name is best known for his law describing the pressure-flow relations in narrow tubes under laminar flow conditions. He recognized that the resistance to the flow in such a tube depends on the fourth power of the radius, a very important concept in respiratory physiology for both blood and air flow.
1 The Human Pulmonary Circulation: Historical Introduction
Of course, it was one thing to measure the blood pressure in a systemic artery but quite another to measure the pressure in the far less accessible pulmonary circulation. Beutner, a student of the great German physiologist Carl Ludwig (1816– 1895), was the first to record the pressure in the pulmonary arteries and he did this in several animals, including cats, dogs, and rabbits [8]. The figure he reported for the mean pressure was between 14 and 23 mmHg depending on the species, and this is within the range that is accepted today. He therefore recognized that the pressures in the pulmonary circulation were a factor of 4 or 5 less than in the systemic circulation, which was a key advance. We now recognize that whereas the systemic circulation needs a high pressure to perfuse the raised arms, for example, the pressure in the pulmonary circulation under resting conditions needs only to be sufficient to raise blood to the top of the lung. The fact that the pulmonary circulation has such a low pressure has many implications, including the thin walls of the pulmonary blood vessels. The measurements made in the Ludwig laboratory were done by opening the chest of animals and cannulating the pulmonary artery. However, this is a very unphysiological situation because the normal negative intrapleural pressure is abolished and some other means of ventilating the lung has to be used. It was therefore an important advance in 1862 when Jean-Baptiste Chauveau (1827–1917) and Etienne Marey (1830–1904) in France conceived the idea of introducing long thin catheters into blood vessels and they did this for both the jugular vein and the carotid artery [9]. In this way they were able to measure the pressures in the right and left ventricles, respectively, and they thus confirmed the large differences between the pressures in the pulmonary and systemic circulations, and the remarkably low pressures in the former. As mentioned above, opening the chest of an animal causes a very striking change in the physiological conditions, and related to this, there ensued controversy for a long time on the effect of the respiratory movements on pulmonary pressures and flows. We now know that during inspiration, for example, venous return is initially increased, but it then falls off. In fact, various respiratory maneuvers, such as the Valsalva maneuver, can have very complicated effects on pulmonary pressures and flows. Interestingly, as early as the eighteenth century, Albrecht von Haller (1708–1777) showed that pulmonary blood flow decreased when the lungs were ventilated with an increased pressure. He did this by perfusing frog lungs with dye.
7 Measurement of Pulmonary Blood Flow A key advance in our understanding of the pulmonary circulation was the introduction of the Fick principle to measure total pulmonary blood flow. Furthermore, because the whole of the output of the right ventricle goes through the lungs, this value is therefore the same as the cardiac output. Adolf
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Fig. 4 Adolf Fick (1829–1901). His “Fick principle” for measuring pulmonary blood flow has been used extensively for nearly 140 years. (Reproduced with permission [1])
Fick (1829–1901) was a student in the Ludwig laboratory referred to earlier and the first mention of his “Fick principle” was a brief note in the proceedings of a medical scientific society in Würzburg, Germany [10]. Fick (Fig. 4) is remembered eponymously in another context because he described the law of diffusion of gases in fluids, for example, across the blood–gas barrier, when he was only 26 years old. However, it must be said that this very general law of diffusion was previously known to physical chemists. The Fick principle is basically a statement of the conservation of mass, that is, that the amount of oxygen taken in (or carbon dioxide given out) at the mouth must be equal to the amount taken up (or given out) as the blood moves through the lung. Nevertheless, this simple statement gave a great boost to the increase in knowledge of the pulmonary circulation. Admittedly, actual application of the principle is complicated by the fact that the oxygen concentration (for example) in mixed venous blood is required, and this necessitates placing a catheter in the pulmonary artery. This was done by Grehant and Quinquaud in 1886 to measure the pulmonary blood flow in dogs [11], but it was not until many years later that sampling blood from the pulmonary artery became possible in humans. Until this became possible in man, other techniques for esti mating the concentration of gases in the pulmonary artery were
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introduced, and these are referred to as indirect Fick methods. Along these lines, Loewy and von Schrötter [12] pointed out in 1903 that if an airway to part of the lung was blocked, the Po2 and Pco2 of the gas distal to the block would become the same as those of mixed venous blood [12]. Later, rebreathing techniques were introduced to obtain equilibration between the partial pressures of gases in the mixed venous blood and a rebreathing bag, although an objection to these techniques is that the respiratory maneuvers required for rebreathing may alter cardiac output. Another technique was not to wait for full equilibration but to infer the Pco2 of mixed venous blood from an extrapolation procedure [13]. Other methods of determining total pulmonary blood flow have been introduced that do not depend on the Fick principle but depend on the uptake of a foreign gas. For example, Markoff et al. [14] and Krogh and Lindhard [15] used nitrous oxide to measure pulmonary blood flow by measuring the rate at which it was taken up from the alveolar gas by the blood. A variant of this technique was introduced by Grollman [16], who used acetylene, which has a much higher solubility in blood than nitrous oxide and is therefore removed more rapidly. Techniques such as these are particularly valuable in unusual environments where the Fick principle cannot be used, and, for example, the nitrous oxide uptake procedure was successfully employed in Spacelab to measure the cardiac output in orbiting astronauts [17].
8 Cardiac Catheterization In retrospect, it is perhaps remarkable that it took so long to develop the technique of human cardiac catheterization. As we saw earlier, Chauveau and Marey in 1863 passed long catheters
Fig. 5 André Cournand (1895–1988) and Dickinson Richards (1895–1973). Together with Werner Forssmann (1904–1979), they were awarded the Nobel Prize in Medicine and Physiology in 1956 for the introduction of cardiac catheterization. (Reproduced with permission [1])
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through the jugular vein and carotid artery, respectively, in unanesthetized horses, thus entering the right and left sides of the heart. However, there was a general feeling that a procedure like this was too dangerous to be used in humans. In fact, when Werner Forssmann (1904–1979) introduced a ureteric catheter into his own right atrium, the medical establishments scorned the procedure. Forssmann was a young German physician who had the idea of injecting drugs directly into the cavity of the heart via a catheter through a vein rather than by a needle through the chest wall to treat emergencies [18]. He persuaded a surgical nurse to provide the necessary sterile ureteric catheter 65 cm long, he inserted this into the antecubital vein of his left arm after local anesthesia, and with the catheter dangling free, he walked down a flight of stairs to the X-ray department, where he was able to advance it into his right atrium [19]. When his chief, Professor Sauerbruch, heard about it, his comment was to the effect that they ran a clinic there not a circus! Forssmann subsequently abandoned cardiology and was astonished when he was selected as one of the recipients for the Nobel Prize in Medicine and Physiology in 1956. Twelve years later, André Cournand (1895–1988), Dickinson Richards (1895–1973), and their collaborators at Bellevue Hospital in New York developed cardiac catheterization in man (Fig. 5) Beginning in 1941 they reported the use of modified ureteric catheters introduced via a peripheral vein to measure first the pressure in the right atrium, then the right ventricle, and finally, in 1944 the pulmonary artery [20–24]. These studies laid the basis for modern interventional cardiology and they, together with Forssmann, were awarded the Nobel Prize in 1956. A further important advance was when Dexter and colleagues showed that it was possible to wedge the catheter in a small pulmonary artery and thereby obtain a measurement that was approximately equal to the pulmonary venous pressure [25].
1 The Human Pulmonary Circulation: Historical Introduction
Building on these studies, it soon became possible to insert catheters into the left side of the heart via a peripheral systemic artery. These procedures were combined with angiography using radio-opaque contrast material, and a key advance occurred in 1958 when Mason Sones accidentally injected contrast material into the ostium of the right coronary artery of a patient and was astonished to record the first coronary angiogram; the patient recovered successfully [26].
9 Advances During the Last 50 Years There has been an enormous increase in knowledge of various aspects of the pulmonary circulation in the last 50 years and space does not permit description of these in detail. Many of these advances will be alluded to in subsequent chapters in this book. The following is a selection that is bound to reflect the author’s interest.
9.1 D istinction Between Alveolar and Extra-Alveolar Vessels of the Pulmonary Circulation Macklin [27] made an important advance when he injected a latex suspension into the blood vessels of excised lungs and showed a difference in behavior between the small vessels and the large vessels when the lungs were inflated or deflated. He reported that when the lungs with inflated with positive pressure, the caliber of the larger vessels increased, whereas the capillaries were compressed. He concluded from this that the act of breathing helped to move blood through the lung. A much more extensive study was later carried out by Howell et al. [28] when they filled the pulmonary blood vessels with kerosene. Because of surface tension effects, this liquid does not penetrate into the capillaries and other small vessels, and thus the investigators were able to separate the effects of lung inflation on the two types of vessels. To their surprise, they found that when the lungs were inflated with positive pressure, that is, the alveolar pressure was raised with respect to vascular pressures, the larger, kerosene-containing vessels increased their caliber, whereas, as they had expected, the small vessels, not filled with kerosene, were compressed. As a result, they divided the pulmonary vasculature into “compressed” and “expanded” compartments [29]. An interesting related finding was that although the change in volume of the larger, “expanded” vessels was quite small, they were capable of developing surprisingly large pressures. Somewhat later, Mead and Whittenberger [30] named the smaller and larger vessels “alveolar” and “extraalveolar,” respectively, because the former were clearly
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exposed to alveolar pressure and the latter were not. This nomenclature remains in use and the distinction is very important in understanding changes in pulmonary blood flow under a variety of conditions.
9.2 Starling Resistor Effect The critical importance of alveolar pressure in pulmonary hemodynamics became clearer when it was shown that if alveolar pressure exceeds pulmonary venous pressure, pulmonary blood flow is determined by the difference between pulmonary arterial and alveolar pressure rather than the expected pulmonary arterial pressure and pulmonary venous pressure difference. One of the first clear demonstrations of this was by Banister and Torrance [31] in 1960, although a number of previous investigators had recognized the importance of transmural pressure in determining flow through collapsible tubes [32–34]. One of the most extensive studies was then carried out by Permutt et al. [35], who showed that if the pulmonary arterial pressure was held constant in a lung preparation and pulmonary venous pressure was gradually raised, there was no effect on pulmonary blood flow until the venous pressure exceeded the alveolar pressure. In other words, as stated above, flow is independent of venous pressure as long as this is less than alveolar pressure. The phenomenon has been referred to as the “Starling resistor effect” because the English physiologist Ernest Starling used a collapsible tube to maintain the outflow pressure constant in a heart–lung preparation, but it is also called the “vascular waterfall” or “sluice effect” because in both instances the flow over the waterfall or sluice is independent of the pressure in the water below.
9.3 T opographical Distribution of Pulmonary Blood Flow As long ago as 1887, the German pathologist Johannes Orth suggested that the apex of the lung might have a reduced blood flow under certain conditions because of the weight of the blood, and he speculated that this could be related to the apical development of pulmonary tuberculosis [36]. Some 60 years later when the first measurements of vascular pressures on the right side of the heart were made in humans, as described in Sect. 8, William Dock argued that the pulmonary arterial pressure was so low that it might not be sufficient to raise blood to the apex of the upright lung [37]. However, the gradation in the topographical distribution of blood flow down the upright human lung was first demonstrated when it was shown that the rate of removal of inhaled oxygen-15-labeled carbon dioxide during a short period of
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breath holding was very slow at the apex of the upright lung and gradually increased toward the base [38]. A little later, a very similar distribution of blood flow was reported using radioactive xenon [39]. It was quickly shown that the topographical distribution was very sensitive to posture, exercise (which increased the pulmonary arterial pressure), and acceleration in a centrifuge [40], so gravity was clearly the cause. Then extensive studies using an isolated lung preparation where careful control over the pulmonary arterial, venous, and alveolar pressures was possible showed how the lung could be divided into various zones depending on the magnitudes of the pulmonary arterial, venous, and alveolar pressures [41]. Lung volume was also shown to have an important role, especially at very low states of lung inflation [42]. Subsequently, it has become clear that the topographical distribution of blood flow plays an important role not only in physiological conditions but also in many pathophysiological conditions.
9.4 N ongravitational Factors Affecting the Distribution of Pulmonary Blood Flow Gravity has a striking effect on the topographical distribution of blood flow in the lung as discussed in the previous section. Blood flow is much higher at the bottom of the upright human lung than at the apex, and indeed some earlier studies found no blood flow at all at the extreme apex [39]. However, it was never contended that blood flow at one particular level in the lung was completely uniform. For example, an early study by Glazier et al. [43] using rapid freezing to look at the histological characteristics of isolated perfused dog lungs clearly showed considerable inequality of red cell filling in adjacent alveolar walls. This inequality was later analyzed theoretically using networks of pulmonary capillaries and it was shown that if there were preferential channels of flow for some reason such as some capillaries being wider than others, blood flow inequality was bound to develop as the perfusion pressure was gradually increased [44]. More recently, the role of nongravitational factors has been increasingly emphasized. For example, Glenny [45] and Glenny and Robertson [46] injected small microspheres into the pulmonary circulation of animals, inflated and fixed the lungs, diced them into small portions, and showed considerable inequality of blood flow between these pieces. Other workers have shown differences in regional vascular conductance in dog lungs [47], and also stratified inequality of blood flow along terminal lung units [48, 49]. Furthermore, in measurements made on astronauts during sustained microgravity in orbital flight, there was clear evidence that the topographical inequality of blood was
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reduced, but some inhomogeneity of pulmonary perfusion remained [50].
9.5 L ongitudinal Distribution of Vascular Resistance in the Pulmonary Circulation The distribution of vascular resistance along the pulmonary circulation from the pulmonary arteries to the veins has been the subject of numerous studies and there is still not universal agreement on the results. However, there is strong evidence that the major site of vascular resistance is not in the small pulmonary arteries as is the case in the systemic circulation, where the arterioles are highly muscularized and act as throttles. In fact, as we saw earlier, the pulmonary circulation has a very much lower resistance than its systemic counterpart, and the small pulmonary arteries have relatively little vascular smooth muscle, and as a result, they are easily mistaken for pulmonary veins. Some of the most convincing studies have come from the cannulation of small pulmonary blood vessels in experimental animals [51]. The overall message is that the pressure in the pulmonary capillaries is probably about halfway between pulmonary arterial and venous pressures, and that much of the pressure drop in the pulmonary circulation occurs in the capillary bed. This makes sense because the primary function of the pulmonary circulation is to allow the exchange of gas across the pulmonary capillaries, and it is not necessary to direct blood flow to different parts of the lung as is the case in the systemic circulation, where blood flow has to be diverted, for example, to exercising muscles. The result is that the pressure drop in the pulmonary circulation is much more uniform than it is in the systemic circulation, where much of the high aortic pressure is lost in the highly muscularized arterioles. A topic which has resulted in considerable confusion in the past is to what extent the pulmonary venous pressure rises during exercise. At rest, the pressure at the downstream end of the pulmonary veins must be close to left atrial pressure, which is about 5 mmHg in man. However, there is considerable evidence that the pulmonary venous pressure rises substantially on exercise when the cardiac output is increased. Most of this evidence comes from measurements of pulmonary artery wedge pressure, although some direct measurements of left atrial pressure by means of catheters have been made in experimental animals. In one study on normal humans at high levels of exercise, the pulmonary artery wedge pressure increased from 3.4 to 21.1 mmHg, and this was associated with an increase in pulmonary arterial pressure from 13.2 to 37.2 mmHg [52]. Another study on exercising humans reported similar values [53]. Galloping racehorses develop extremely high pulmonary wedge or left
1 The Human Pulmonary Circulation: Historical Introduction
arterial pressures, up to 50 mmHg or more [54, 55]. These animals have been selectively bred for hundreds of years to develop very high maximal oxygen consumptions and these are associated with very high cardiac outputs that necessitate high filling pressures for the left ventricle.
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uniquely suited to modifying blood-borne substances such as angiotensin I, bradykinin, serotonin, norepinephrine, some prostaglandins, and leukotrienes. Indeed a substantial fraction of all the vascular endothelial cells in the body are located in the lung. These metabolic functions of the pulmonary circulation are critically important in a number of diseases as discussed later in the book.
9.6 Hypoxic Pulmonary Vasoconstriction As long ago as 1946, von Euler and Liljestrand [56] ventilated anesthetized cats with 10% oxygen in nitrogen and showed that there was a sudden increase in pulmonary arterial pressure compared with air breathing. When the animals were returned to air breathing, the pressure fell. They argued that the hypoxia had a direct action on part of the pulmonary circulation, and the mechanism of the vasoconstriction has been an intense topic of study ever since. In fact, several of the chapters in this book are related in some way to this. There seems no point in reviewing the research here except to say that the constriction apparently involves voltage-gated potassium channels. Many investigators have pointed out that this mechanism has some value in lung disease, for example, asthma, because it results in some blood flow being shunted away from poorly ventilated lung units. However, the chief evolutionary pressure for hypoxic pulmonary vasoconstriction is likely to be the events that occur at birth. The fetal lung has only about 15% of the cardiac output passing through it, with the rest being diverted through the patent ductus arteriosus. At birth, there is a sudden transition from placental to air breathing, which necessitates a very large increase in blood flow through the lung, and this is brought about by a dramatic fall in pulmonary vascular resistance caused in part by the loss of hypoxic vasoconstriction, and in part by inflation of the lung. The high vascular resistance of the fetal lung is assisted by the fact that the walls of the pulmonary arteries contain large amounts of vascular smooth muscle. After birth, considerable involution of the smooth muscle takes place, and the distribution also becomes patchy [57]. The result is that in the adult, hypoxic pulmonary vasoconstriction is uneven, and this is an important factor in some diseases, such as highaltitude pulmonary edema.
9.7 N onrespiratory Functions of the Pulmonary Circulation Again this topic is addressed in several subsequent chapters in this book and there is little point in reviewing the history here. Suffice to say that since the lung is the only organ except the heart that receives the whole circulation, it is therefore
References 1. West JB (1996) Respiratory physiology: people and ideas. Oxford University Press, New York 2. Keynes G (1978) The life of William Harvey. Clarendon Press, Oxford, p 15 3. Mohyi (1924) Der Lungenkreislauf nach el-Koraschi. Dissertation, Freiburg 1924 4. Harvey W (1989) The works of William Harvey (trans: Willis R). University of Pennsylvania Press, Philadelphia 5. Young J (1929) Malpighi’s De Pulmonibus. R Soc Med Proc 23:1–11 6. Hales S (1733) Statistical essays containing haemastaticks. Innys & Manby, London 7. Hales S (1727) Vegetable staticks. Innys & Innys, London 8. Beutner A (1852) Über die Strom- und Druckkrätfte des Blutes in der Arterie und Vena pulmonalis. Z Rat Med N F 2:97–126 9. Chauveau JBA, Marey EJ (1862) De la force déployée par la contraction des différentes cavités du coeur. Coll R Soc Biol (Paris) 4:151–154 10. Fick A. Ueber die Messung des Blutquantums in den Herzventrikeln. S B Phys Med Ges, Würzburg, July 9, 1870 11. Grehant N, Quinquaud CE (1886) Recherches expérimentales sur la mesure du volume du sang qui traverse le poumon en un temps donne. C R Seances Hebd Soc Biol 5:159 12. Loewy A, von Schrötter H (1903) Ein Verfahren zur Bestimmung der Blutgasspannungen, der Kreislaufgeschwindigkeit und des Herzschlagvolumens am Menschen. Arch Anat Physiol Abt 394 13. Kim TS, Rahn H, Farhil E (1966) Estimation of true venous and arterial Pco2 by gas analysis of a single breath. J Appl Physiol 21: 1338–1344 14. Markoff I, Muller F, Zuntz N (1911) Neue Methode zur Bestimmung der in menschlichen Körper umlaufenden Blutmenge. Z Balneol 4:373–411 15. Krogh A, Lindhard J (1912) Measurements of the blood flow through the lungs of man. Skand Arch Physiol 27:100 16. Grollman A (1932) The cardiac output of man in health and disease. Thomas, Springfield 17. Prisk GK, Guy HJB, Elliott AR, Deutschman RA III, West JB (1993) Pulmonary diffusing capacity, capillary blood volume and cardiac output during sustained microgravity. J Appl Physiol 75:15–26 18. Forssmann W (1929) Die Sondierung des rechten Herzens. Klin Wochenschr 8:2085 19. Forssmann W (1974) Experiments on myself. St. Martin’s Press, New York 20. Cournand AF, Ranges HS (1941) Catheterization of the right auricle in man. Proc Soc Exp Biol Med 46:462–466 21. Cournand AF, Lauson HD, Bloomfield RA, Breed ES, Baldwin E (1944) Recording of right heart pressures in man. Proc Soc Exp Biol Med 55:34–36 22. Cournand AF, Riley RL, Breed ES, Baldwin ED, Richards DW, Lester MS, Jones M (1945) Measurement of cardiac output in man using the technique of catheterization of the right auricle or ventricle. J Clin Invest 24:106–116
12 23. Cournand A, Bloomfield RA, Lauson HD (1945) Double lumen catheter for intravenous and intracardiac blood sampling and pressure recording. Proc Soc Exp Biol Med 60:73–75 24. Richards DW (1945) Cardiac output by the catheterization technique in various clinical conditions. Fed Proc 4:215–220 25. Dexter L, Haynes FW, Burwell CS, Eppinger EC, Sagerson RP, Evans JM (1947) Studies of congenital heart disease. II. The pressure and oxygen content of blood in the right auricle, right ventricle, and pulmonary artery in control patients, with observations on the oxygen saturation and source of pulmonary capillary. J Clin Invest 26:554–560 26. Hurst JW (1985) History of cardiac catheterization. In: King SB III, Douglas JS (eds) Coronary arteriography and angioplasty. McGrawHill, New York, pp 5–6 27. Macklin CC (1946) Evidence of increase in the capacity of the pulmonary arteries and veins of dogs, cats and rabbits during inflation of the freshly excised lung. Rev Can Biol 5:199–233 28. Howell JBL, Permutt S, Proctor DE, Riley RL (1961) Effect of inflation of the lungs on different parts of pulmonary vascular bed. J Appl Physiol 16:71–76 29. Permutt S, Howell JBL, Proctor DF, Riley RL (1961) Effect of lung inflation on static pressure volume characteristics of pulmonary vessels. J Appl Physiol 16:64–70 30. Mead J, Whittenberger JL (1964) Lung inflation and hemodynamics. In: Fenn WO, Rahn H (eds) Handbook of physiology, respiration. American Physiological Society, Washington 31. Banister J, Torrance RW (1960) The effects of the tracheal pressure upon flow: pressure relations in the vascular bed of isolated lungs. Q J Exp Physiol 45:352–367 32. Duomarco JL, Rimini R (1954) Energy and hydraulic gradients along systemic veins. Am J Physiol 178:215–220 33. Rodbard S (1955) Flow through collapsible tubes: augmented flow resistance produced by resistance at the outlet. Circulation 11:280–287 34. Holt JP (1941) The collapse factor in the measurement of venous pressure. Am J Physiol 134:292–299 35. Permutt S, Bromberger-Barnea B, Bane HN (1962) Alveolar pressure, pulmonary venous pressure and the vascular waterfall. Med Thorac 19:239–260 36. Orth J (1887) Atiologisches und Anatomisches über Lungensch windsucht. Hirshwald, Berlin 37. Dock W (1947) Reasons for the common anatomic location of pulmonary tuberculosis. Radiology 48:319–322 38. West JB, Dollery CT, Naimark A (1960) Distribution of blood flow and ventilation perfusion ratio in the lung, measured with radioactive CO2. J Appl Physiol 15:405–410 39. Anthonisen NR, Milic-Emili J (1966) Distribution of pulmonary perfusion in erect man. J Appl Physiol 21:760–766 40. Glaister DH (1967) The effect of positive centrifugal acceleration upon the distribution of ventilation and perfusion within the human lung, and its relation to pulmonary arterial and intraoesophageal pressures. Proc R Soc Lond B 168:311–334
J.B. West 41. West JB, Dollery CT, Naimark A (1964) Distribution of blood flow in isolated lung: relation to vascular alveolar pressures. J Appl Physiol 19:713–724 42. Hughes JMB, Glazier JB, Maloney JE, West JB (1968) Effect of lung volume on the distribution of pulmonary blood flow in man. Respir Physiol 4:58–72 43. Glazier JB, Hughes JMB, Maloney JE, West JB (1969) Measurements of capillary dimensions and blood volume in rapidly frozen lungs. J Appl Physiol 26:65–76 44. West JB, Schneider AM, Mitchell MM (1975) Recruitment in networks of pulmonary capillaries. J Appl Physiol 39:976–984 45. Glenny RW (1988) Blood flow distribution in the lung. Chest 114:8s–16s 46. Glenny RW, Robertson HT (1990) Fractal properties of pulmonary blood flow: characterization of spatial heterogeneity. J Appl Physiol 69:532–545 47. Beck KC, Rehder K (1986) Differences in regional vascular conductances in isolated dog lungs. J Appl Physiol 61:530–538 48. Wagner PD, McRae J, Read J (1967) Stratified distribution of blood flow in the secondary lobule of the lung. J Appl Physiol 22: 1115–1123 49. West JB, Maloney JE, Castle BL (1972) Effect of stratified inequality of blood flow on gas exchange in liquid-filled lungs. J Appl Physiol 32:357–361 50. Prisk GK, Guy HJB, Elliott AR, West JB (1994) Inhomogeneity of pulmonary perfusion during sustained microgravity on SLS-1. J Appl Physiol 76:1730–1738 51. Bhattacharya J, Staub NC (1980) Direct measurement of microvascular pressures in the isolated perfused dog lung. Science 210: 327–329 52. Wagner PD, Gale GE, Moon RE, Torre-Bueno JR, Stolp BW, Saltzman HA (1986) Pulmonary gas exchange in humans exercising at sea level and simulated altitude. J Appl Physiol 61:260–270 53. Groves BM, Reeves JT, Sutton JR, Wagner PD, Cymerman A, Malconian MK, Rock PB, Young PM, Houston CS (1987) Operation Everest II: elevated high-altitude pulmonary resistance unresponsive to oxygen. J Appl Physiol 63:531–539 54. Jones JH, Smith BL, Birks EK, Pascoe JR, Hughes TR (1992) Left atrial and pulmonary arterial pressures in exercising horses. FASEB J 6:A2020 55. Manohar M (1993) Pulmonary artery wedge pressure increases with high-intensity exercise in horses. Am J Vet Res 54:142–146 56. von Euler US, Liljestrand G (1946) Observations on the pulmonary arterial blood pressure in the cat. Acta Physiol Scand 12:301–320 57. deMello D, Reid LM (1991) Arteries and veins. In: Crystal RG, West JB (eds) The lung: scientific foundations. Raven, New York, pp 767–777 58. Singer C (1957) A short history of anatomy and physiology from the Greeks to Harvey. Dover, New York 59. Stirling W (1902) Some apostles of physiology. Waterlow, London
Chapter 2
Microcirculation of the Lung: Functional and Anatomic Aspects Joan Gil
Abstract The fixed pulmonary vascular anatomy differs from the systemic anatomy in the arrangement and shape of the capillary segments, but an even more striking peculiarity of the lung is that it needs to adapt itself to three different pressures, which leads to considerable adaptations and changes in morphology. Because of the pressure changes required by respiratory mechanics, the morphology differs both at the level of the capillaries and at the level of the extraalveolar small arteries and veins as a function of the existing mechanical conditions, adapting them in a way that is best suited to fulfill their respective functions. The configuration of the pulmonary capillary network markedly differs from that in the systemic capillary bed. The gas exchange needs are different in the systemic and in the pulmonary capillaries. In the periphery, the capillaries are longitudinal and their number and density in the tissue reflect local needs; in the lung, their purpose is to be capable of picking up from the outside as much oxygen as possible to fulfill the most extreme conceivable needs for gas exchange (the “diffusing capacity”). This capacity is normally not reached and the capillaries tolerate recruitment, de-recruitment, and changes in configuration that support variable quantitative levels of gas exchange. Keywords Gas exchange • Small pulmonary arterioles • Capillary • Flow kinetics • Microvascular circulation
1 Introduction Long gone are the times when the pulmonary circulation was absurdly referred to as the lesser circulation. No other organ circulation, not even when the systemic circulation is studied as a whole, raise issues as complicated and unexpected as those encountered in the lung. This is not limited to the J. Gil () Department of Pathology, Mount Sinai School of Medicine, New York, NY 10029, USA e-mail:
[email protected] biochemical, cellular, and reactive properties of the vessels, but includes the fixed and functional vascular anatomy [1–6]. The fixed anatomy differs from the systemic anatomy in the arrangement and shape of the capillary segments, but an even more striking peculiarity of the lung is that it needs to adapt itself to three different pressures, which leads to considerable changes in morphology. Because of the pressure changes required by respiratory mechanics, the morphology differs both at the level of the capillaries and at the level of the extraalveolar small arteries and veins as a function of the existing mechanical conditions, adapting them in a way that is best suited to fulfill their respective functions. A pulmonary-specific structure required to understand the process is the connective tissue (“fibrous”) continuum [7] (Fig. 1), which inserts itself proximally in the hilum, which is relatively fixed like a fulcrum. At the distal end, however, the visceral pleura is not mechanically fixed, as it moves in solidarity with the parietal pleura and the chest wall only by virtue of the existing subatmospheric air pressure without physical contacts between both of them. While several elements contribute to the generation of recoil pressure of the lung, only the pleural pressure helps to keep the healthy organ open. The connective tissue continuum connects both visceral pleura and hilum, becomes tensed during inhalation, and distributes subatmospheric pressure throughout all interstitial points of the lung. This has many consequences. Nowhere else in the body are capillaries normally exposed to changes in pericapillary pressures. More important yet, the configuration of the capillary network markedly differs from that in the systemic capillary bed. The gas exchange needs are different in the systemic and in the pulmonary capillaries. In the periphery, the capillaries are longitudinal and their number and density in the tissue reflect local needs; in the lung, their purpose is to be capable of picking up from the outside as much oxygen as possible to fulfill the most extreme conceivable needs for gas exchange (the “diffusing capacity”). This capacity is normally not reached and the capillaries tolerate recruitment, derecruitment, and changes in configuration and in velocity of the fluid that support variable quantitative levels of gas exchange.
J.X.-J. Yuan et al. (eds.), Textbook of Pulmonary Vascular Disease, DOI 10.1007/978-0-387-87429-6_2, © Springer Science+Business Media, LLC 2011
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Fig. 1 The fibrous continuum of the lung. a The fibrous continuum extends from the hilum to the visceral pleura extending into all the alveolar wall. b Hexagonal capillary network in the alveolar septum supported by an incomplete layer of elastic and collagenous fibrils and fibers. Occasional interstitial cell elements are not shown. (Reproduced with permission [7])
The lung exhibits two different circulations [1]: (1) a s ystemic component arising mostly from the aorta and its intercostal branches that supplies the bronchial walls and the pleura and provides them with nourishment and oxygen and (2) the narrowly designated pulmonary circulation originating in the right ventricle which exchanges gas (oxygen intake and CO2 elimination) to serve the entire body and exhibits the complex properties described by others in this book. Remarkably, both components distally anastomose at the level of their venules in the wall of the small bronchioles and to a lesser extent in the pleura, which creates a permanent right-to-left shunt of variable flow intensity. Pulmonary shunts are about the most perplexing problems of the pulmonary vascular morphology [8].
2 T echnical Fixation Problems: Vascular Perfusion The lung does not have a rigid, really fixed shape or size: it is a semiliquid organ. Its shape depends in part on the container. It is not easy to determine its true outside anatomy after removing the lung from the chest and placing it on the dissecting table, but the difficulty doubly applies to the internal configuration, raising a complex methodological issue [3] discussed by many authors, as the original morphology is lost when the subject dies and the chest is opened. What should be done to preserve the real anatomy? Supravital microscopy through a chest wall window is appealing but is limited and precludes study of deeper areas [9]. Many in the past [10–12] tried to visualize the “true” in vivo morphology by applying a rapid freezing technique [13]. Liquid CO2 was the medium of choice. Liquid nitrogen is generally not desirable, as it forms a mantle of boiling fluid and insulating gas around the tissue, resulting in slow cooling and poor fixation. In both
cases, unless the lung has been pretreated with a cryoprotector, which frequently defeats the purpose, the slow penetration of the cold temperature results in an unacceptable number of artifacts, in particular large ice crystals and torn membranes, which cast doubt on the quality of the preservation and the dependability of the observations. Generally, only a thin peripheral layer of tissue will be preserved for interpretation, which renders the possibility of representative sampling impossible and limits all observations to the surface. Chemical fixation [3, 6, 7] is a more desirable approach to the preservation of the lungs for light- or electron-microscopic study because it allows unrestricted sampling of wellpreserved tissue. Buffered aldehyde (formalin for light microscopy, glutaraldehyde for electron microscopy) is rapidly instilled into the trachea or a stem bronchus following pneumothorax. The use of a controlled instillation pressure in the fluid, some 20 cm H2O [7, 14], is advisable and results in the adjustment of a reproducible and well-defined lung volume, corresponding to the plateau of the pressure–volume curve of the fluid-filled lung. Morphologically, this simple approach yields outstanding results, requires little preparation and modest training, and it is currently the method of choice for most morphology studies, for instance, of lung injury. But airway flooding with fixatives is clearly unsuitable for correlative studies of morphology with pulmonary mechanics or lung circulation [3, 6]. At the very least, the alveolar surface tension is abolished and the pressure–volume curves of air- and water-filled lung are very different, so the morphology must also be suspected of being different. For the purpose of this chapter, we are naturally interested in the air-filled natural condition. As repeatedly shown by us [3, 4, 15], this preservation can be achieved only by inflating the lung with a known air volume after an O2-wash-induced atelectasis, full air inflation, subsequent deflation, and adjustment and stabilization of the desired final pressure. This is followed by flush of the pulmonary
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Fig. 2 a Rabbit, fluid-filled lung fixed by vascular perfusion. Note the empty capillary spaces and the undulated appearance of the surface. b Rabbit, air-filled lung fixed by vascular perfusion under zone II conditions. Note the smooth surface and the rectangular internal capillary luminal outlines. (Reproduced with permission [4])
circulation through a catheter inserted in the pulmonary main artery and perfusion of the fixative also at a well-known and controlled pressure. Most workers perfuse with buffered, isotonic glutaraldehyde as the fixative, followed by block immersion of the entire or a large fragment of lung and very careful dicing. We [3, 4, 15] believe that a perfusion with 1% buffered OsO4 yields superior results, but it must be warned that osmium emanates highly toxic, dangerous fumes and handling of large volumes of that substance requires training, exhaust equipment, precautions, and great care. Most of our studies and descriptions rely on findings in lungs fixed by vascular perfusion. Figure 2 shows three alveolar walls of two perfusion-fixed rabbit lungs (the capillaries are empty) photographed at the same magnification. The lung in Fig. 2a has been filled with saline, whereas the lung in Fig. 2b is filled with air under zone II conditions (to be discussed later). The difference is striking.
3 S ystemic Component of Circulation in the Lung: Bronchial and Pleural Circulation The bronchial arteries arise from the descending thoracic aorta and from the posterior intercostal arteries [16]. They supply with oxygen and nutrition the mediastinal bronchi and the following conducting airways all the way to the terminal bronchioles. In the wall of the large bronchi they form two plexuses, one in the incomplete muscle layer and the other in the submucosa. The bronchial veins are much smaller and have a lower flow volume. Some of them are deep veins that arise parallel to the bronchial arteries but run in the opposite direction, increasing in volume only modestly because they drop much of their blood in the abundant anastomoses, with the
pulmonary veins returning it to the left atrium. Other veins are superficial and arise in the pleural surface. The bronchial venous blood is drained in the azygos or hemiazygos veins. The lymphatics are also a vascular component. There are none in the alveolar walls, because the overflow compartment of the capillaries is the alveolar space. The pulmonary lymphatics do not arise from closed sacks, as they do in the systemic beds, but instead arise from connective tissue spaces in the periarterial sleeves, partly lined by boundary cells that appear to be either fibroblasts or endothelial cells [17] (Fig. 3). The periarterial lymphatic vessels are barely visible unless the lung is edematous. Lymph nodes are frequently found in the subpleural and peribronchial parenchymal areas.
4 R ole of the Connective Tissue Continuum of the Lung in Relation to the Capillaries A connective tissue continuum of connective tissue of fibers, ground substance, and a few cells extends from the visceral pleura and septa to the hilum [7]. The continuum is fundamental in pulmonary mechanics [18]. Throughout the body, blood vessels and nerves are embedded in the connective tissue stroma of the organ, a filling and supporting component which might be compared to a soft tissue skeleton. In the alveolar walls, however, the pulmonary stroma exhibits peculiar characteristics. The connective tissue continuum (Fig. 1) mostly consists of elastic and collagen fibers and fibrils, generally of type 3, is resistant to traction but is thin and malleable, and fulfills the function of connecting the hilum with the visceral pleura [1, 7].
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Fig. 3 a Rat lung fixed by instillation of fixative fluids into the airways following pneumothorax. Note that blood vessels are filled with blood. A represents air spaces, C is a capillary, AV is an extraalveolar vessel surrounded by its own thin sheath of connective tissue, and B is an interstitial cell, probably a fibroblast of an
endothelial cell giving rise to a lymphatic of an incomplete wall. b Rat lung fixed by vascular perfusion under zone II conditions. Note that the extraalveolar vessel (bottom) contains a lymphatic (ly) and a muscular medium. A represents alveolar spaces. (Reproduced with permission [17])
During inhalation, the subatmospheric pressure generated in the pleural space pulls on the visceral pleura and causes an expansion of the lung parenchyma. The pull counteracts the effects of the alveolar surface tension and any connective tissue resistance due to overstretching and results in the generation of an interstitial subatmospheric pressure throughout the organ. The opposite happens during expiration. The extraalveolar arteries and veins, like the alveolar capillaries, are embedded within the continuum, although the arrangement and consequences are markedly different in both of them.
bronchioles, arteries, despite the smooth muscle which serves different purposes, lack surface tension to actively retract but the surrounding connective tissue sheath with its changes of pressure allows them to participate in pulmonary mechanics in association with the respiratory movements. The important characteristic (Figs. 3, 5), however, is that unlike the alveoli arteries and veins are never directly exposed to air pressure. During inflation, the alveolar walls radially inserted in the periphery of the connective tissue sheaths are pulled out of the sheath’s perimeter and cause an enlargement of the vascular diameter and therefore an increase of their volume. During expiration, as the alveolar volume is reduced, the radial alveolar wall anchors are somewhat relaxed and the diameter and the volume of the extraalveolar blood vessels accordingly decrease [12]. As we shall see later, this result is exactly the opposite of what happens to the alveolar capillaries, whose volume decreases as the alveolar air pressure compresses them during inspiration. During inspiration, arteries and veins therefore become a blood reservoir.
5 B lood Vessels Inside the Lung but Outside the Alveolar Parenchyma: Extraalveolar Arteries and the Connective Tissue Continuum We have already described how the pulmonary arteries become surrounded with adipose and connective tissue upon their entering the hilum. Structurally, they continue to differ from systemic arteries of comparable caliper only in the smaller thickness of their medial walls. Because of this they stay functionally and anatomically separated from the alveolar wall (Fig. 3), which is occupied mostly by capillaries (Fig. 4) (blood vessels consisting only of an endothelium without a muscle cell layer). Therefore, a major difference exists between the environment in which arteries and veins function and the situation surrounding alveolar capillaries. Unlike the alveoli and the
6 P ulmonary Circulation: Conducting Arteries 6.1 Arteries Outside the Lung The main pulmonary artery arises directly from the conus arteriosus of the right ventricle. It and its main branches are of the elastic type. This means that the medium is made up of parallel
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Fig. 4 a Dog lung fixed by instillation of fixatives into the airways and photographed with the electron microscope at very low power. b Highpower electron micrograph of two air-wall barriers of lung fixed by instillation of the airways. Note the dark erythrocytes at the bottom, and
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the thin endothelial and epithelial layers containing many pinocytotic vesicles. EI, epithelial cell; EN, endothelial cell (Reproduced with permission [7])
Fig. 5 The difference between extraalveolar and alveolar vessels during respiratory movements, deflation a and inflation b
elastic lamellae connected to each other by smooth muscle cells inserted obliquely to the lamellae but parallel to each other. As in the systemic circulation, smaller branches become muscular, with a solid medium made up of smooth muscle and
a well-developed intimal layer. The most peculiar characteristic of pulmonary arteries is the relatively thin medial wall when compared with the luminal diameter, which reflects the small prevailing pulmonary pressure. Once they penetrate into the
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pulmonary parenchyma and the hilum, the pulmonary arteries and veins become surrounded by a loose adipose connective tissue which frequently contains lymph nodes. An arterial branch that runs in parallel to the bronchial walls is named an axial artery. Evidently, there are as many generations of axial arteries as there are of conducting airways, on average 18 in the human lung [14]. Most parenchymal lymphatics arise in the connective tissue sleeve that surrounds the artery rather than the bronchi. In the bronchiolar peripheral territory, the connective tissue sheaths are frequently separated from the bronchiolar sheaths, but are close to them. Additionally to their dynamic changes of volume in association with mechanics, the periarterial sheaths are also the site of the earliest accumulation of interstitial lung edema [6, 18]. The axial arteries are not the only extraalveolar arteries. Hislop and Reid [19] demonstrated that although they are frequently ignored, numerous supernumerary arteries additionally exist and exceed in number the axial ones. Supernumerary arteries branch out of an axial artery and run into the parenchyma surrounded by a connective tissue sheath and enter the alveolar wall and the alveolar capillaries in a way that has never been clarified but probably approaches the scheme shown in Fig. 6a, feeding the hexagonal network of capillary segments in the area of the corner vessels as demonstrated in Figs. 6b and c and 7, at intervals. In the systemic circulation, arterioles (small arteries where the endothelium is surrounded by a single layer of smooth muscle cells) are located between the last arteries and the capillaries and they are responsible for most of the resistance to blood flow and the drop of pressure. Such anatomically distinct arterioles are absent in the lung circulation, which raises the issue of the location of the main site of resistance to flow. In some cases it may well be the capillary corner capillaries (see later), which, if this assumption is correct, would represent the universal entry point of the arterial blood into the alveolar capillaries. At any rate, in many cases of pulmonary hypertension small extraalveolar vessels of minimal caliper with an abnormal layer of smooth muscle cells can be recognized. The veins inside of the lung rarely receive much attention, but they are also extraalveolar vessels because they are surrounded by connective tissue. They typically originate in the subpleural areas and are located in the interlobular septa, where they increase in size and advance toward the hilum (Fig. 7). Their wall is still thinner than that of the arteries of the same caliper and is frequently less well organized. Finally, the existence of numerous anastomoses between the pulmonary and the bronchopleural systemic circulation needs to be pointed out. They are located at the venular origins of the bronchiolar and the pleural circulations. The existence and possible reversibility of blood flow through these anastomoses is credited with the relative rarity of lung infarcts when compared with the frequency of embolisms.
J. Gil
Fig. 6 a The hexagonal capillary network of an alveolar wall shown as a continuum being drained and fed at intervals by small arteries and veins. b Flat view of the alveolar wall (comparable to a) where capillaries have been filled with a red stain. c Scanning electron micrograph of alveolar walls showing protruding filled capillaries. P is a pore of Cohn. Compare with a and b. (Reproduced with permission [7])
7 I nside the Alveolar Septa: Static Capillary Alveolar Microcirculation 7.1 The Hexagonal Capillary Network The small systemic arteries branch out into arterioles which split into a brush-like bundle of capillaries. Their blood is immediately collected by a mirrorlike assembly of venous capillaries soon assembled into a venule that leads to a drainage vein. This is not so in the pulmonary circulation.
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Fig. 7 Longitudinal section through an alveolar duct showing the relation of axial and supernumerary arteries to alveolar walls and the difference between primary and secondary alveolar walls. Not shown is the fact that arteries and veins are connected by the corner vessels which act as shunts
The first realistic description of the arrangement of the alveolar capillary segments in the alveolar wall was offered by Weibel [14] in his seminal book Morphometry of the Human Lung. Determined to quantify everything quantifiable, Weibel, needing a realistic model, pointed out the major difference between the systemic and the pulmonary capillary networks: the capillaries are in the form of a dense anastomosing mesh of capillary segments, which for modeling purposes approach hexagons (Fig. 6). Weibel [7, 20] later revealed the extraordinary size of the capillary endothelial surface, slightly smaller than the epithelial surface, in an average human lung some 120 m.2 In referring to surface areas, however, it is necessary to consider fractal effects. In other words, the size of an area always depends on the magnification at the time of the measurement
7.2 P rimary Versus Secondary Alveolar Walls Before discussing the intraseptal capillary morphology, we need to point out another anatomic characteristic. Because more than 90% of the alveolar walls is occupied by pulmonary capillaries, it follows that the architecture, geometry, and relationships of alveolar walls must have a decisive influence on the geometry of alveolar capillary networks. In Fig. 7, note two important details: (1) only a small part of the alveolar capillaries are located close to the bronchioles, where the front of bulk air transport normally ends and where gas exchange should logically reach its maximum and (2) there are two different types of alveolar walls [21] — the primary septum at the bottom of alveoli, which separates alveoli open to different ducts (and sometimes possibly to different bronchioles), and the secondary septum, which
rises from the primary septum to separate two neighboring alveoli belonging to the same duct. At the end of the last duct, whenever the final insertion of the alveolar ducts into the pleura or a septum is near, the secondary walls taper off slowly and disappear, simplifying the ductal morphology and transforming alveolar ducts into cones or cylinders [21]. Considering the capillary flow, on a flat section one could be tempted to view the secondary alveolar wall as collateral of the primary wall: the blood that penetrates it will have to rise and fall back, causing considerable turbulence inside the hexagonal capillary segments. On the other hand, if the flow is viewed from a cross section of the duct, the impression would be that of a complex single layer of capillaries flowing constantly from the bronchiolar end toward the pleura, describing a zigzag motion in the secondary septa and a relatively straight line in the primary septa. Both alternatives are difficult to analyze from the hydrodynamic point of view.
7.3 Corner Capillaries Corner capillaries are those located in the corners of alveolar walls, where three septa meet. The word “corner” in this case is based on two-dimensional flat sections (Fig. 7). In the undisturbed three-dimensional space, they are likely to represent long lines at the junctions between primary and secondary septa. They have been observed by multiple authors when attempting to fix the dry lung at different degrees of inflation with high intraalveolar air pressures [21–28]. As we shall discuss below, high-pressure alveolar inflation (with air pressures above the local pulmonary arterial pressure) invariably results in the total collapse of the septal capillaries, which are mounted on the connective tissue continuum (Figs. 8, 9a, b) but lack any rigid supporting protective
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Fig. 8 Rat lung fixed by vascular perfusion under zone II conditions. To the right note septal pleat and open capillaries, to the left note a dilated corner vessel opening into a capillary
structure. At the same time, as discussed already, the extraalveolar arteries and veins dilate because of the radial pull of the periarterial alveolar walls. This results in the alveolar blood being squeezed out of the septa and pushed into storage in the extraalveolar vessels. Yet this situation would appear to be unstable as it might conceivably prevent the blood from returning to the veins and the left atrium. It is therefore necessary to have a mechanism capable of maintaining the patency of the lung even when the air pressure collapses most of the alveolar network. This is the function of the corner vessels (Figs. 8, 9a, b). They frequently appear dilated, which enables them to carry an increased flow volume possibly at higher velocity [29] and they are not affected by the high air pressure because of their corner location. The alveolar spaces acquire a more or less rounded structure that leaves the corner capillaries well protected in dead spaces. Corner capillaries are the most striking anatomic characteristic of West’s zone I.
7.4 Corner Pleats Gil and Weibel [4, 5, 15] first described in rat lungs fixed by vascular perfusion of chemical fixatives the existence of a reversible alveolar wall feature named “corner pleat” (Figs. 8, 9c) also located, like the corner vessels, in alveolar junctions. This caused confusion, as it was originally interpreted as a mechanism of pulmonary mechanics permitting the relaxation of alveolar corners tensed during inflation [5] and therefore the reduction in volume of the alveolar space. This interpretation is attractive because there is nothing in the alveolar wall to suggest that it might be particularly extensible and because areas of microatelectasis morphologically look like progressions of these pleats with the same structure. Pleats consist of nicked alveolar walls with capillaries folded over themselves and minute gaps filled
Fig. 9 Rabbit lungs fixed by vascular perfusion a, b under zone I, c under zone II, and d under zone III conditions. (Reproduced with permission [4])
with a surfactant-like fluid. It was later observed that alveolar pleats only appear under what is called zone II in West’s nomenclature, which is when the arterial pressure exceeds the air pressure and flow is driven by the difference between air and venous pressures. In this situation many alveolar
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Fig. 10 Summary of the findings in the three zones
walls are also collapsed and it can be assumed that the alveolar pleats fulfill a function similar to that of the corner vessels. In collapsed lumps it can be observed that pleats serve as needs for microatelectasis. Presumably the presence of surfactant reduces the retraction force arising from such areas.
8 F unctional Adaptation of Alveolar Capillary Networks to Respiratory Movements In all organs of the body except the lung, vascular anatomy is generally fixed. Even if the number or the diameter of open capillaries varies as a function of the local functional or oxygen requirements, the structural arrangements remain unchanged. The reason for that difference is twofold: first, the very limited connective tissue support available to the alveolar structures, which renders them vulnerable to the effects of pressures, and secondly, the existence, in addition to the arterial pressure Pa and the venous pressure Pv, of a third player, the alveolar air pressure PA. It was West who first pointed out three different pressure combinations (named by him “zones”) that result in major changes of the alveolar and capillary configurations. Neither the connective tissue support nor the thin epithelial and
endothelial cells confer any stiffness. Here is a summary of the conditions and the findings [29] (Figs. 9, 10). In all cases it is understood that the alveolar pressure PA is identical to the pleural subatmospheric pressure: Zone I: PA > Pa > Pv (Figs. 9a, 10), where the air pressure is higher than the arterial pressure, which itself is higher than the venous pressure. In the systemic circulation the arterial flow is driven by the difference between arterial and venous pressure, but here the driving force for the circulation is the difference between PA and Pa. The air pressure (including that generated by the alveolar surface tension) overwhelms any plastic tissue or intracapillary adaptations related to the perfusate and causes an ironing out or flattening of the entire alveolar surface. The exception is, of, course the alveolar corners, where a hidden space remains between the three rounded-off alveolar spaces, occupied by dilated capillaries named “corner vessels,” which most likely are equivalents of arterioles. These corner vessels are hidden from the alveolar pressure and will remain open and permit flow at any extreme alveolar air pressures (for instance, when using the abdominal press or during forced expiration), therefore ensuring a patent path between the right and left chambers of the heart. These corner vessels have similarities with the corner pleats discussed below. Zone II: Pa > PA > Pv (Figs. 2b, 9b, c, 10, 11a, b), where the arterial pressure is the highest and the air pressure (including pressure generated by the surface tension) is intermediate
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Fig. 11 a Zone II rat lung showing at the bottom left pleated capillaries. b Zone II lung remarkable for a large, dilated corner vessel (top right)
between the arterial and venous pressures. Here the appearance of the tissue and vessels is the result of the balance between the arterial and the air pressure. There are areas where the arterial pressure prevails slightly and the alveolar septa are therefore perfused. Other alveolar septa, however, contain only collapsed capillaries. The alveolar epithelial surface remains smooth and the outline of the open capillaries is rectangular, not round (Fig. 10). This finding may result from the small difference between the values of the arterial and air pressures. Even small variations of intracapillary pressures seem to be able to turn the balance toward perfusion of the alveolar wall (when Pa prevails) or total collapse (when PA is locally higher than Pa). It is remarkable that an entire alveolar wall will show either empty capillaries or open capillaries but never isolated open capillaries in the middle of the wall. This suggests that folds at the intersection of septa may act as flood gates for an entire side of an alveolar wall. Finally, single, dilated, corner vessels are generally absent, but their place is taken by small corner pleats, infoldings of septal walls containing capillaries that acquire the appearance of a bundle and fill the space between the three rounded alveolar walls. The narrow gaps between folded-over alveolar walls are filled with a fluid thought to be alveolar surfactant. We [14] reported in a reconstruction of sections of rabbit lungs fixed by perfusion with osmium that these corner pleats can be seen connected to both an artery and a vein, making it clear that they represent shunt mechanisms aimed, like the corner vessels, at permitting the passage of blood regardless of high alveolar air pressure. The morphology of this zone is important because it corresponds to the situation prevailing in intubated patients with positive end-expiratory pressure. It is relevant to note that the alveolar wall owing to the alternative stitching of the basement membranes of epithelial and endothelial cells to the epithelium of both sides has little capability for distension. The small spaces between capillaries contain only connective tissue and some rare cells, such as pericytes or fibroblasts, which may predispose them to folding. It is impossible to determine if these pleats are related or not to the hexagonal pattern. All the above applies only to
air-filled lungs. If the alveolar spaces are flooded with edema fluid, or surfactant the smoothening of the alveolar epithelial surface by alveolar pressure does not happen regardless of pressure relationships, as the pressure is equally transmitted to all points of the surface as described by the principles of solid mechanics [4]. Zone III: Pa > Pv > PA (Figs. 9d, 10), where both the arterial and the venous pressure exceed the alveolar air pressure. This is the case when the alveolar morphology approaches the textbook description of the pulmonary circulation and the situation encountered in systemic organs, with the flow driven by the difference between Pa and Pv. Since Pv exceeds PA, the alveolar surface is undulated, no longer ironed out, with each alveolar capillary protruding into the alveolar space. No corner vessels and no alveolar pleats can be recognized and all available capillaries appear completely filled. In summary, the study of the morphological equivalents of the three zones of West is difficult and can be achieved only in lungs fixed by vascular perfusion of the fixative (preferably osmium). It is not known whether pressure alone is responsible for all observations described here or whether other factors, for instance, contractile cells also play a role. The observations appear to have the capability of profoundly affecting the functionality of the circulation, but also appear relevant to pulmonary mechanics. In the perspective of a dense hexagonal network of capillary segments fed and drained at regular intervals with a maximal endothelial surface of some 120 m2, without terminal vessels the corner pleats of zone II and the corner vessels seem to represent the secure and uncollapsible entry and exit points of the arterial and venous pulmonary circulations, whereas in zone III the entire morphological capillary network is available to exercise its gas-exchanging and metabolic functions without interference from the alveolar air pressure. Of interest are also the hydrodynamic properties and singularities of the system as described as it concerns flow velocity, pressure distribution and turbulence, and particularly the interplay with pulmonary mechanics. The capillary pulmonary pressure is believed to be of the order of 10 mmHg
2 Microcirculation of the Lung: Functional and Anatomic Aspects
[13, 30–34]. Years ago, there was a popular discussion between the followers of the tube flow theory under Poiseuillian conditions and the theorists of the sheet flow [9, 11, 13, 33, 34], where alveolar flow would be compared to circulation inside a parking garage with a low ceiling interrupted by posts (the center of the hexagons). The sheet flow theory seems to accommodate certain findings in zone II, whereas the tube flow theory suits bests the mainstream zone III circulation pattern.
9 Gas Exchange Few physiological functions are as dependent on physical properties as gas exchange. The morphometric basis of diffusing capacity has been repeatedly described [7, 20]. A simple way to approach the problem is that a tissue barrier or membrane is interposed between gas mixtures of different concentrations (Fig. 4). This barrier consists of thin layers of alveolar epithelium, interstitium, and capillary endothelium. This always applies, although one should additionally consider the distance to the front of inspired air. We have described three different configurations of the relevant gasexchanging structures and it is reasonable to assume that all three of them support this essential function. The most striking characteristic of zone I is that only corner vessels are open. Lamm [26, 27] showed that zone I lungs are capable of gas exchange. From our point of view, we note that the gas-exchanging membrane that surrounds the corner vessels is locally as thin as in other alveolar capillaries. Corner vessels are shunts that connect the arterial to the venous circulation, bypassing all septal vessels. It stands to reason that if they absorb all the circulation in the corners, additionally to some widening they need to be associated with a higher flow velocity of the blood. When all capillaries are open (Zone III) are would assume that their location is the site of capillary drainage. Similar considerations can be made with regard to zone II, which is particularly important in medicine. As described, the alveolar walls, if dry, that is, without alveolar edema, are smooth. Some alveolar walls will include patches of closed and open capillaries which typically have a quadrangular profile, whereas most of the alveolar wall capillaries are compressed and closed. The patches always extend from corner to corner, but we cannot explain the reason for this configuration. These are evidently suitable for gas exchange under all conditions. Regarding the corner pleats which represent a more or less circumferential (in two dimensions) convoluted bundle of capillaries inside folded alveolar walls, we have shown that they represent arteriovenous shunts which evidently allow some blood to escape and penetrate into the alveolar septa [15]. They have at least superficially some similarity with the glomerular capillaries in the kidney
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which produce only minimal resistance to perfusion and are therefore suitable for the function. The considerations presented for the corner vessels apply fully: the cluster of capillaries on several sides offers very thin air–blood barriers suitable for gas exchange. Finally, zone III, where the flow is driven only by the pressure difference between arteries and veins, is the model of the commonly shown pulmonary anatomy: the alveolar walls no longer appear flattened out because of the capillary bulges and all capillaries are open and it is also configuration where the diffusing capacity would reach its maximum. One should add that whenever alveolar edema exists and the air spaces are filled with fluid, the surface tension is abolished and the configuration is always that of zone III regardless of the pressures. This is shown in Fig. 12, which shows salinefilled rabbit lungs fixed under pressure conditions identical to those in the air-filled lungs shown in Figs. 3, 5, 8, 9.
Fig. 12 Counterpart to the micrographs of air-filled rabbit lungs shown in Fig. 9, but this time, prior to the perfusion with fixatives, the alveolar spaces of the lungs having been filled with saline, thus simulating the conditions of alveolar edema. (Reproduced with permission [4])
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References 1. Gil J (1990) The normal pulmonary circulation. In: Fishman AP (ed) The pulmonary circulation, normal and abnormal. University of Pennsylvania Press, Philadelphia 2. Gil J (1978) Morphologic aspects of alveolar microcirculation. Fed Proc 37:2462–2465 3. Gil J (1990) Controlled and reproducible fixation of the lung for correlated studies. In: Gil J (ed) Models of lung disease: methods in microscopy. Dekker, New York 4. Gil J, Bachofen H, Gehr P, Weibel ER (1979) Alveolar volume surface area relation in air and saline filled lungs fixed by vascular perfusion. J Appl Physiol 47:990–1001 5. Gil J, Weibel ER (1972) Morphological study of pressure-volume hysteresis in rat lungs fixed by vascular perfusion. Respir Physiol 15:190–213 6. Gil J (1988) The normal lung circulation: state of the art. Chest 93:805–825 7. Weibel ER, Gil J (1977) Structure function relationships of the alveolar level. In: West JB (ed) Engineering aspects of lung. Dekker, New York 8. Lovering AT, Stickland MK, Eldridge MW (2006) Intrapulmonary shunt during normoxic and hypoxic exercise in healthy humans. Adv Exp Med Biol 588:31–45 9. Lamm WJ, Bernard SL, Wagner WW, Glenny RW (2005) Intravital microscopic observations of 15-micron microspheres lodging in the pulmonary circulation. J Appl Physiol 98:2242–2248 10. Fung YC, Sobin SS (1977) Pulmonary alveolar blood flow. In: West JB (ed) Bioengineering aspects of the lung. Dekker, New York, pp 267–359 11. Fung Y, Yen RT (1986) A new theory of pulmonary blood flow in zone 2 condition. J Appl Physiol 60:1638–1650 12. Howell JBL, Permutt S, Proctor DF, Riley RL (1961) Effect of inflation of the lung on different parts of the pulmonary vascular bed. J Appl Physiol 16:71–76 13. Sobin SS, Fung YC (1992) Response to challenge to the Sobin-Fung approach to the study of pulmonary microcirculation. Chest 101: 1135–1143 14. Weibel ER (1963) Morphometry of the human lung. Springer, Berlin 15. Ciurea D, Gil J (1996) Morphometry of capillaries in three zones of rabbit lungs fixed by vascular perfusion. Anat Rec 244:182–192 16. Warwick R, Williams PL (eds) (1975) Gray’s anatomy, 35th edn. Longman, London 17. Gil J, McNiff JM (1981) Interstitial cells at the boundary between alveolar and extraalveolar connective tissue in the lung. J Ultrastr Res 76:149–157 18. Lai-Fook JS (1993) Mechanical factors in lung liquid distribution. Annu Rev Physiol 55:155–179 19. Hislop A, Reid L (1973) Pulmonary arterial development during childhood: branching pattern and structure. Thorax 28:129–135 20. Waehrli P, Burri PH, Gil J, Weibel ER (2007) Ultrastructure and morphometry of the human lung. In: Shields TW (ed) General thoracic surgery. Lee and Febiger, Philadelphia
J. Gil 21. Ciurea D, Gil J (1989) Morphometric study of human alveolar ducts based on serial sections. J Appl Physiol 67:2512–2521 22. Gil J, Ciurea D (2004) The functional structure of the pulmonary circulation. In: Peacock AJ, Rubin LJ (eds) Pulmonary circulation, diseases and their treatment, 2nd edn revised. Arnold, London 23. Guntheroth WG, Luchtel DL, Kawabori J (1992) Functional implications of the pulmonary microcirculation. An update. Chest 101:1131–1134 24. Bachofen H, Wagensteen D, Weibel ER (1982) Surfaces and volumes of alveolar tissue under zone II and zone II conditions. J Appl Physiol 53:879–885 25. Koyama S, Hildebrandt J (1991) Air interface and elastic recoil affect vascular resistance in three zones of rabbit lungs. J Appl Physiol 70:2422–2431 26. Lamm WJ, Obermiller T, Hlastala MP, Albert RK (1995) Perfusion through vessels open in zone 1 contributes to gas exchange in rabbit lungs in situ. J Appl Physiol 79:1895–1899 27. Lamm WJ, Kirk KR, Hanson WL, Wagner WW Jr, Albert RK (1991) Flow through zone I lungs utilizes alveolar corner vessels. J Appl Physiol 70:1518–1523 28. Conhaim RL, Rodenkirch LA (1998) Functional diameters of alveolar microvessels at high lung volume in zone II. J Appl Physiol 85:47–52 29. Naeije R (2004) Pulmonary vascular function. In: Peacock AJ, Rubin LJ (eds) Pulmonary circulation. Arnold, London 30. Pellett AA, Johnson RW, Morrison GG, Champagne MS, DeBoisblanc BP, Levitzky MG (1999) A comparison of pulmonary arterial occlusion algorithms for estimation of pulmonary capillary pressure. Am J Respir Crit Care Med 160:162–168 31. Ehrhart IC, Granger WM, Hofman WF (1986) Effect of arterial pressure on lung capillary pressure and edema after microembolism. J Appl Physiol 60:133–140 32. Gaar KA Jr, Taylor AE, Owens LJ, Guyton AC (1967) Pulmonary capillary pressure and filtration coefficient in the isolated perfused lung. Am J Physiol 213:910–914 33. Zhuang FY, Fung YC (1983) Yen RT analysis of blood flow in cat’s lung with detailed anatomic and elasticity data. J Appl Physiol 55:1341–1348 34. Sobin SS, Fung YC, Tremer HM, Rosenquist TH (1972) Elasticity of the pulmonary alveolar microvascular sheet in the cat. Circ Res 30:440–450
Additional Reading 35. West J, Dollery C, Naimark A (1964) Distribution of blood flow in isolated lungs; relation to vascular and alveolar pressures. J Appl Physiol 19:713–724 36. Staub NC, Storey WF (1962) Relation between morphological and physiological events in lung studied by rapid freezing. J Appl Physiol 17:381–390
Chapter 3
Pulmonary Vascular Development Rosemary C. Jones and Diane E. Capen
Abstract The vascular beds of the adult lung, the pulmonary and bronchial circulations, are formed by extensive branching systems of large and small arteries and veins, and capillary networks. The focus of this chapter is the development of the pulmonary circulation in the normal lung as this evolves through the embryo/fetus to birth and the postnatal stage; and its continued development through childhood to the adult. In principle, the central large pulmonary arteries and pulmonary veins have a wall structure that reflects their role as conduits of deoxygenated or oxygenated blood. In addition to this function, the distal vascular loops of small thin-walled pulmonary (precapillary) arteries, capillaries, and (postcapillary) veins, which together form the lung’s microcirculation, serve as part of a gas-exchange surface for blood transiting this complex network. Blood vessels form and assemble networks in a series of elegant and intricate steps. Since the lung’s vasculature cannot develop in isolation from its airways, the mechanisms of morphogenesis discussed include, in brief, ones regulating specification of the lung primordium, and the early formation of the lung bud and airways. Mechanisms of alveologenesis, as these pertain to capillary bed formation, are also considered. Keywords Lung vasculogenesis • Angiogenesis • Alveolo genesis • Pulmonary artery • Capillary • Vein
1 Overview of Lung Vascular Development The vascular beds of the adult lung, the pulmonary and bronchial circulations, are formed by extensive branching systems of large and small arteries and veins, and capillary networks. The focus of this chapter is the development of the pulmonary circulation in the normal lung as this evolves through R.C. Jones (*) Department of Anesthesia, Critical Care and Pain Management, Massachusetts General Hospital, Harvard Medical School, Boston, MA 02114, USA e-mail:
[email protected] the embryo/fetus to birth and the postnatal stage; and its continued development through childhood to the adult (Fig. 1a). In principle, the central large pulmonary arteries and pulmonary veins have a wall structure that reflects their role as conduits of deoxygenated or oxygenated blood. In addition to this function, the distal vascular loops of small thin-walled pulmonary (precapillary) arteries, capillaries, and (postcapillary) veins, which together form the lung’s microcirculation, serve as part of a gas-exchange surface for blood transiting this complex network (Fig. 1b). Blood vessels form and assemble networks in a series of elegant and intricate steps. This account of pulmonary vascular development is based on a series of reviews and original publications by investigators who addressed both the larger picture and specific details of cellular and molecular events. Since the lung’s vasculature cannot develop in isolation from its airways, the mechanisms of morphogenesis discussed include, in brief, ones regulating specification of the lung primordium, and the early formation of the lung bud and airways. Mechanisms of alveologenesis, as these pertain to capillary bed formation, are also considered. The development of bronchial vessels (which supply nutrients to large vascular and airway structures) [1, 2], and of their closely associated lymphatics (which drain lung liquid and plasma proteins into the systemic circulation), is discussed elsewhere [3–8]. These systems must develop appropriately with the pulmonary vascular bed and airways to maintain adequate lung function. Following lung organogenesis, fetal vascular development, growth, and maturation to the time of birth, and in the perinatal period, proceeds in a series of time-related steps: a pseudoglandular, canalicular, and saccular phase followed by one of septation/alveologenesis. Although major changes in the overall template (blueprint) regulate lung vascular development in the fetus and neonate, growth and maturation then continue mainly by expansion in the child and through adolescence, until thoracic growth ceases in the adult [9–16] (Table 1). Because of the focus on the mouse in genetic regulation studies of lung development, the timing for this species is given together with that for the human lung (Table 1). The sequence of vascular patterning is constant, but the timing of
J.X.-J. Yuan et al. (eds.), Textbook of Pulmonary Vascular Disease, DOI 10.1007/978-0-387-87429-6_3, © Springer Science+Business Media, LLC 2011
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Fig. 1 Arteriograms and polymer cast of the pulmonary vascular bed. (a) Pulmonary arteriograms showing the human lung at three ages (prepared after injecting the pulmonary arterial bed with a barium sulfate–gelatin mixture), i.e., a newborn (upper left), an 18-month old infant (lower left), and an adult (right). The pre-acinar artery distribution (complete by 28 weeks in the fetus) is present at each time point shown, and the dense background haze, representing the growth of small intra-acinar arteries, increases with age [11]. (b) Photomicrograph of a lung injected with silicone polymer to show the dense capillary networks surrounding each alveolus in the normal pulmonary circulation; polymer perfused (at 50 mmHg infusion pressure) and fixed (2 cm-thick) tissue slices, from an adult lung following acute myocardial infarction, were cleared by incubation in 50, 75, 85, and 100% glycerol (each for 24 h) and examined with a Wild M8 stereomicroscope. Original magnification: (a) ×0.3 (the three lungs are photographed at the same magnification); (b) ×56. (a From Reid [11] copyright American Thoracic Society. (b) Reprinted from [272] with permission)
each phase varies with species [17–19]. These events are summarized in a broad timetable for airway, alveoli, and blood vessel development [20] (Table 2) and are illustrated in a schematic form [21] (Fig. 2). Airway morphogenesis is increasingly recognized as modulated by pulmonary vascular development [22]. For normal vascular development to proceed, the template at each stage must successfully generate vessels appropriate for size, wall structure, and location in the lung; and for age and stage of thoracic growth. Whether catch-up growth is possible will depend on when the normal development program is interrupted or fails. The lung anlage develops as an outpouch (ventral diverticulum) at the caudal end of the laryngotracheal groove from the embryonic foregut, and is vascularized by ingrowth of a vascular plexus from the heart/aortic sac and dorsal aorta.
From its connection to the right ventricle of the heart, the main pulmonary artery splits into a right and a left branch, and thereafter one branch remains in close association with each airway branch as these divide to supply the lung lobes and subunits; the central veins connect to the left side of the heart (Fig. 3a). These vessels form as angioblasts (cells committed to an endothelial lineage) and these develop into endotheliallike cells that assemble into tubes: capillary-like structures give rise to small-vessel networks, which increase in complexity by destabilization of existing structures and loop formation. The pulmonary arteries run centrally through the lung and end by supplying capillary segments in the intra-acinar region and pleura (save at the lung hilum). Blood draining from the capillaries enters the pulmonary veins at the periphery of lung units, the most distal veins lying at the edge of the
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Table 1 Timing of structural events in human lung development: organogenesis, pseudoglandular, canalicular, saccular, and alveolar stages (names describing fetal lung development as suggested by the Commission on Embryology Terminology in 1970). Some overlap in the timing of these structural events is recognized. The timing of stages in the mouse lung is included for comparison: the structural changes will be similar but species-specific (i) Organogenesis (human ~3–5 weeks, mouse E9.5–E14.2a) Ventral outpouching of endodermal cells from anterior foregut into surrounding mesenchyme Ventral diverticulum forms primitive trachea and primary bud divides into two lung buds (endodermal cells forming an inner layer of epithelium, mesenchymal cells a middle layer, and mesothelial cells a thin outer layer) and ventral trachea separates from dorsal esophagus First of six pairs of aortic arch arteries appear and sixth aortic arch branches to lung buds (origin of main pulmonary arteries) a (ii) Formation of proximal airways and vessels – pseudoglandular stage (human ~5–16 weeks, mouse E14.2–E16.6 ) Lung buds undergo repetitive dichotomous branching to form main and segmental bronchi, bronchioli, and terminal bronchioli – epithelial cells are undifferentiated (especially in the early phase). No respiration possible – airways are hollow blind-ending tubes The sixth left aortic arch develops into the main pulmonary artery and gives rise to the left and right pulmonary arteries Vascular branching parallels airways In human fetal lung, in the child and adult, and in the lungs of several other species studied, the number of arterial venous branches exceeds that of airways. Vessel branches accompanying airways are termed “conventional” and ones that do not accompany them are termed “supernumerary” (iii) Formation of distal airways and vessels – canalicular stage (human ~16–26 weeks, mouse E16.6–17.4a) Airway branching is complete and epithelial cell differentiation advances – ciliated epithelium lines airways and distal epithelium differentiates to “flattened” respiratory type – starting the formation of respiratory bronchioli and the acinar region Cells expressing epithelial type 2 markers herald the onset of surfactant synthesis Main arterial and venous pathways within a segment and side branches are present but are small relative to lung size Peripheral vascular network has developed (iv) Start of formation of acinus – saccular stage (human ~24 weeks to term, mouse E17.4–E19a) Additional respiratory bronchioli and saccules form and terminal saccules give rise to alveolar ducts and saccules Saccule epithelium attenuates to form the septal wall The number of epithelial type 2 cells increases Fetal pulmonary fluid is abundant in potential air spaces The density of small vessels increases and capillaries reorganize to form an air–blood interface At birth alveolar ducts and saccules, in close apposition to capillary networks, form functional gas-exchange units. (v) Alveolarization (human from birth to ~8 years, mouse from birth to ~6 weeks) At birth, a critical transition from fetal life (in utero) to air-breathing – thin-walled alveoli and saccules are still immature structures After birth, alveoli and capillary structures continue to form and increase in size and number Initial phase – septation of original saccules (2 weeks in mouse, several months in human) Second phase – formation of additional alveoli (4 weeks in mouse, 7–8 years in human) Growth by expansion continues until thoracic growth is complete in the young adult Data from [19, 48, 75, 203, 227, 249, 270] a Embryonic days Table 2 Laws of lung development Law I – airway: The airways (i.e., bronchi and bronchioli) are present by the 16th week of intrauterine life Law II – alveoli: Alveolar structures appear before birth and increase in number, size, and complexity during growth Law III – vessels: The pre-acinar branches of the pulmonary artery (i.e., those accompanying bronchi or bronchioli), and pre-acinar venous tributaries, appear at the same time as the accompanying airways. Intra-acinar vessels appear as the alveoli grow; muscularization lags behind the appearance of new arteries Data from [271]
acinus: venous tributaries arise within alveolar walls and ducts, bronchial walls, the pleura, and connective tissue sheaths, and drain to axial veins that increase in size between the distal lung and hilum [10]. How the developing vascular units are regulated spatially within the surrounding mesenchyme is not known, although Hislop and Reid [13] have shown familial patterns of branching in the human lung, indicating a genetic component for the central vessels. Much still needs to be
understood of the “patterning rules” [23] that first determine formation of the lung’s vascular and microvascular networks, as well as the less ordered (but still hierarchical) vascular networks that form in the lung in injury or disease [24]. A remarkably simple and elegant process is proposed for the airways, for which developmental patterning data indicate three local “modes” of branching orders [25, 26]. Both regular and irregular dichotomous branching systems arise as vascular networks evolve from the expanding central arterial and venous structures by secondary budding, branching, and arborization, and by outgrowth of smaller vascular offshoots. The main arteries accompany airway branches – which branch first in a repetitive, stereotypic pattern (for 16 generations) and then in a nonstereotypic pattern (to generation 23). As the vascular networks increase in size and complexity with lung growth, they are simultaneously pruned by the regression of unperfused units until appropriate for the stage of development (endothelial cell apoptosis likely being triggered by signals related to change in blood flow patterns).
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Fig. 2 Summary of stages of fetal and postnatal lung development and growth, and their timing. Note the overlap between stages, particularly between the alveolar stage and the stage of microvascular maturation. Open-ended bars indicate uncertainty as to exact timing. (Reproduced from Burri [21] with permission from McGraw-Hill companies [21])
Fig. 3 Development of main pulmonary artery branches, and conventional and supernumerary artery branches. (a) Arrangement of the main pulmonary vessels in relation to bronchi: the pulmonary arteries arising from the right ventricle (RV) penetrate into the lung alongside the bronchi (left), whereas the main stems of the pulmonary veins leave each lung and enter the left atrium (LA, right). [273] (b) A broncho-arterial bundle illustrating conventional and supernumerary artery branches. Conventional arteries arise at acute angles to
the main axis and supply the respiratory region at the end of the axial pathway. Supernumerary arteries are short and have differing diameters. They arise at right angles to the main axis and supply adjacent air spaces, ultimately providing collateral circulation to a respiratory unit via the backdoor [11, 20, 27]. (a Reproduced from Weibel and Taylor [273] with permission from McGraw-Hill companies. (b) Reproduced from Jones and Reid [248] with permission from Elsevier)
Although the central arteries branch along with airways, the lung has more arterial than airway branches; at all levels these extra branches, termed “supernumerary” arteries, outnumber the “conventional” artery branches dividing with the airways [27, 28] (Fig. 3b). Typically, the supernumerary branches run a short course to supply the adjacent alveolar region. They appear to have their own vasoregulatory pathways, vasoconstriction being greater than for conventional arteries and the response to nitric oxide is different [29]. Conventional and supernumerary
veins are also present. Distally, small arteries and veins join an extensive capillary network, individual capillary segments being interconnected and running across several alveolar walls [30, 31]. It is proposed that the capillary bed represents two sheets of flat membranes, connected by interstitial tissue posts, which allow blood to flow readily across their surface [32, 33]. The size of the capillary network increases as alveolar structures develop. The total number of alveoli is generally accepted to be about 20 million at birth and to have reached the adult
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number of approximately 300 million by 8 years of age. [12, 34–37] In the adult lung, a total alveolar surface area of approximately 70 m2 is invested with a myriad of small arteries and veins and capillary segments. The capillary networks, for example, are calculated to increase in density four-fold between birth and adulthood, when their surface area is approximately 126 m2 [33, 38–40]. As the template changes, the remodeling of artery and vein walls is a striking feature of normal vascular growth and development [9, 10, 14]. In the fetus, vascular cells derive from the mass of mesenchymal cells surrounding the developing airway buds forming from endodermal cells. These mesenchymal cells give rise to a variety of vascular cell types that include endothelial cells and perivascular cells [smooth muscle cells (SMCs), pericytes, and fibroblasts]. Thereafter, these cell types interact to achieve the structural and functional heterogeneity of lung vessels and capillaries. The convention is to identify (class) lung vessels by their wall structure. When SMCs in vessel (artery or vein) walls are enveloped by numerous elastic laminae, as in the main pulmonary vessels and their large branches, these are termed elastic (Fig. 4a). More peripherally, as the number of laminae and SMCs decrease, vessels are termed transitional, and as the number of laminae decrease until SMCs are present between only an internal and an external lamina, they are termed muscular. Although this principle holds true for most segments, often the double laminae (and on occasion even a single lamina) are incomplete in small muscular veins. Distally in the lung, at the entrance to or within the acinus, the SMC layer thins to a few cells; within the acinus, as these cells change pitch to form a spiral, the
Fig. 4 Characterization of segmental and distal (pre- and post-capillary) artery profiles. (a) Pulmonary arteriogram (from a lung injected with barium sulfate–gelatin mixture) showing an anterior basal segmental artery in a (39-year old) normal man, the region distal to the arrow representing a secondary lobule (left). Enlarged tracings of the same anterior basal segmental artery (#8 by standard nomenclature, as referenced by Elliott and Reid [27]), illustrating its wall structure, by the distribution along its length of elastic, transitional, and muscular regions (right). (b, 1–3) The wall structure of a distal pulmonary artery segment (i.e., pre-capillary segment within the acinus) showing the arrangement of smooth muscle cells (SMCs). In tissue sections examined by light microscopy (after injection of the pulmonary arterial bed with barium sulfate–gelatin mixture), and in cross-sectional profiles, the muscular artery segment has a complete medial smooth muscle coat, the partially muscular segment a crescent of medial smooth muscle, and the non-muscular segment no medial smooth muscle (see b1). Since the transition from muscular (to partially muscular) to non-muscular does not occur at the same point along each pathway, a particular wall structure cannot be predicted by the diameter or location (branch level) of a segment. Morphometric analysis of a population of artery profiles provides a useful way to establish the normal distribution and assess change in disease. For this, wall structure is noted; artery size
Fig. 4 (continued) (external diameter) is assessed by measuring the distance between the two edges of the external elastic laminae (EEL, see b2); and the medial thickness is measured as the distance between the internal elastic lamina (IEL) and the external one (see b3). Wall thickness is similarly measured but also includes endothelium. From the two values, i.e., external diameter and medial thickness/wall thickness, the percent medial thickness, or the percent wall thickness, is calculated to relate the proportion of the wall consisting of smooth muscle to vessel size. To assess the position of an artery profile in the branching pattern, the accompanying airway is used as a landmark, i.e., bronchiolus or terminal bronchiolus (pre-acinar), respiratory bronchiolus, alveolar duct or alveolar wall (intra-acinar). The distal pulmonary veins, which also have a muscular, partially muscular and non-muscular segment, are similarly measured to analyze their population distribution. By transmission microscopy (after injection of a fixative into the pulmonary circulation), SMCs are identified by their characteristic organelles and location in the walls of muscular and partially muscular arteries (dense shading). In non-muscular arteries, and in the non-muscular region of partially muscular ones, intermediate cells are present in normal lung (stippled shading); these are cells with a phenotype midway between a pericyte and a SMC, i.e., a population of precursor cells that rapidly express a SMC phenotype in response to a growth stimulus triggered by injury and/or change in transmural pressure [42, 149, 150, 174]. Original magnification: (a), left ×1, right ×2/3. (a Modified from Elliott and Reid [27] with permission. (b) Modified from Hislop and Reid [16] and Jones et al. [272] with permission)
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vessels are termed partially muscular and where the cells disappear from the wall, they are termed non-muscular. The walls of the non-muscular vessels typically consist only of endothelium and a single lamina (Fig. 4b). Additional precursor SMCs are present beneath the endothelium in some of these vessels in the normal lung (Fig. 4b), and these cells play an important role in the development of new SMCs in response to injury and disease (see Chapter 42). Capillary walls consist of endothelial cells and basement membrane, with or without pericytes present as supporting perivascular cells [41]. The alveolar–capillary membrane in its thinnest region (1–2 mm) consists of capillary walls encased by epithelial cell processes, with or without an occasional perivascular cell between; in thicker regions, the interstitium of the membrane contains fibroblasts, myofibroblasts, migratory cells, collagen fibers and fibrils, and elastin [41]. Mainly in regions where several capillary segments converge, small vessels (arteries and veins) are embedded in the intervening matrix [42]. The normal distribution for a vessel population, and any change in the population in development or in disease, is revealed by a morphometric analysis of vessel profiles (i.e., vessel segments viewed in cross section) by their wall structure, external diameter, medial or wall thickness (Fig. 4b), and location (level in the branching pattern). Profiles associated with bronchi, bronchioli, or terminal bronchioli are termed preacinar; ones associated with respiratory bronchioli, alveolar ducts, or within the alveolar wall are termed intra-acinar. The acinus, defined as the respiratory unit supplied by a terminal bronchiolus, includes the respiratory bronchioli, alveolar ducts, and alveolar sacs, or alveoli. In the adult lung, the acinus is approximately 1 cm in diameter and consists of many alveoli. The lobule represents a group of three to five acini at the end of an airway. In the adult, the segment from artery to vein is usually short (less than 1 cm in length) and consists of vessels less than 200 mm in diameter. At all levels within this loop, lumen obliteration, occlusion, or restriction by wall thickening is usually the basis of a maintained rise in pulmonary artery pressure (see Chapter 43). The degree of muscularity of vessel walls is related to vessel size, and is proportional to blood flow and transmural pressure [43], save in the small intra-arteries positioned close to the entrance to the acinus, where wall thickness is normally high for vessel diameter. These are the “resistance” arteries of the lung. This local increase in wall thickness encroaches on the lumen rather than increasing the vessel diameter. Changes to the vascular template at birth reflect the lung’s adaptation from the high pressure/low flow system in utero to a low pressure/high flow system [11]. The relatively sparse number of SMCs present in pulmonary artery walls reflects the low resistance and pressure normally present, and is in contrast to the more muscular walls of systemic arteries where the pressure is normally six times greater. For morphometric analyses, the pulmonary vascular bed is prepared under standard conditions, the arterial or venous
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bed being distended with a (radio-opaque) barium sulfate– gelatin mixture at a hypertensive (100 cm H2O) pressure, and the airways are simultaneously distended with fixative at constant (23 cm H2O) pressure. This permits assessment by angiography and gives satisfactory lung fixation for vessels when quantified by brightfield microscopy [44]. When distending vessels greater than 15 mm in diameter, the barium injection medium normally does not cross the capillaries, thereby distinguishing the arteries from the veins. Pulmonary vessels more than 160 mm in diameter are distinctly outlined in the angiogram [45], and smaller vessels are discernible as a background haze (Fig. 1). Typically, the central region of the normal adult lung is characterized by relatively few, but large, conducting arteries or veins on the angiogram, whereas distally the density reflects the presence of small distributing vessels. Sparse in the fetal lung, these increase in size and number during normal growth (Fig. 1). The following provides an overview of lung vascular morphogenesis and signaling systems that regulate vascular development, maturation, and growth (Section 2). The vascular templates of the normal human lung at different ages are then described (Section 3), followed by an account of aberrant patterns of vascular development that result in an adequately functioning lung or ones in which vascular patterning is irreversibly changed (Section 4).
2 Regulation of Lung Vascular Morphogenesis As the lung anlage develops, its formation and remodeling patterns are regulated by a host of transcription and growth factors, the former integrating genetic instructions with growth factor mediated signaling [46–55]. Other factors that influence lung vascular growth and maturation in important ways include physical factors such as intra-thoracic space, lung liquid volume and pressure, amniotic fluid volume, and hormones released by endocrine glands (pituitary, adrenal, and thyroid) [56]. Extracellular matrix signals (particularly basement membrane components such as laminins, entactin, type IV collagen, perlecan, SPARC, and fibromodulin) interact to regulate development [57–63]. Oxygen signaling pathways also play a role in the formation of the lung and its vasculature [64, 65], a low level of oxygen (hypoxia) enhancing distal airway branching and a high level of oxygen inhibiting it and inducing mesenchymal cell apoptosis [66]. Genetic studies in mice continue to reveal the function of a variety of highly regulated factors such as thyroid transcription factor-1 (also known as Nx.1), Gli family genes, retinoid receptors, Smad, N-myc, hepatic nuclear factor-3b, (HNF-3β), helix forkhead-4 (HFH-4), glucocorticoid receptor, cyclic AMP response element binding protein, and CCAAT/enhancer binding proteins, which control the expression of genes encoding
3 Pulmonary Vascular Development
growth factors (which stimulate growth), and of morphogens (which regulate growth responses by forming gradients) in the developing lung [46, 49, 50, 53, 55]. Several of these have dual roles, regulating cell specification in early lung development and the specialized functions of differentiated airway and vascular cells. Each early growth phase in the lung depends on specific interactions between endodermal and mesenchymal cells mediated by growth factors, and morphogens. These include fibroblast growth factor (FGF), transforming growth factor (TGF)-b, epidermal growth factor, vascular endothelial growth factor (VEGF), placental growth factor, platelet-derived growth factor (PDGF), angiopoietin (ANG), and sonic hedgehog (SHH) [46, 49, 50, 53, 55]. Factors known to regulate pulmonary vascular development in the lung are the focus of the present account. In considering the molecular basis of lung vessel formation, Stenmark and Abman [65] included a comprehensive list of molecules likely involved at different stages. Since the factors regulating cell proliferation, migration, differentiation, and survival are not lung-specific, the exquisite and complex architecture of the lung’s vascular bed is determined by their expression patterns and interactions, as demonstrated by investigators in the field [49, 50, 52, 53, 55].
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Genetic modifications in mice illustrate examples of failed lung development [48] (Fig. 5a). Once the lung buds form, the trachea extends. The presence of bronchial mesenchyme is
required for airway development to proceed from the trachea by appropriate branching [67]. For example, lung buds cultured without mesenchyme, or with tracheal mesenchyme, fail to branch; and when cultured with the mesenchyme from stomach, gut, or liver, the buds form gastric glands, villi, and hepatic chords, respectively [67]. The lobes form as the developing structures are subdivided by the visceral pleura (Fig. 5b). Left–right (L–R) lung asymmetry, and antero-posterior and proximal–distal patterning are instructed early. Lefty-1, Lefty2, nodal, and Pitx-2 determine L–R asymmetry: [48, 52, 54, 68] transient expression (E8–E8.5) on the left side of prospective floor plate and lateral plate mesoderm indicates that specification of the branching pattern and lobation occurs 24–36 h before the primordial bud appears. Abrogation of Lefty-1 results in bilateral expression of Lefty-2, nodal, and Pitx2 (indicating that Lefty-1 normally restricts Lefty-2 and nodal expression to the left side and leads to left-sided expression of Pitx2). L–R determining genes are expressed when foregut retinoic acid (RA) synthesis/utilization is high and thus are RA targets. HFH-4−/− mice, in which left/right dynein (a protein critical for nonrandom visceral orientation) is abrogated, demonstrate random visceral L–R laterality and lack cilia. These determinants of symmetry/asymmetry are superimposed on branching mechanisms effected by signaling between epithelium, mesenchyme, and mesothelium [48, 52, 54, 68]. Early restriction of epithelial progenitor/precursor cells [69] results in non-overlapping cell lineages being established for conducting airways (trachea and bronchi) and distal airways (bronchioli, acini, and alveoli) before the definitive lung buds form (at E9–E9.5). Two days later (at E11.5),
Fig. 5 Early lung formation: unsuccessful development in null mutant phenotypes and a dominant negative misexpression phenotype. (a) The HNF3b−/− phenotype is a severe early embryonic lethal phenotype in which the primitive foregut endoderm fails to close into a tube [274]. In the compound null Gli2−/−/Gli3−/− mutant phenotype the primitive lung anlage completely fails to arise from the primitive foregut endoderm [275]. Gli2−/− null mutation and Gli2 gene dosage reduction result in abnormalities of lung lobation. The fibroblast growth factor (FGF)-10−/− phenotype results in the larynx and trachea forming and separating dorso-ventrally from the esophagus, but the primary bronchial branches completely fail to arise from the trachea [276]. The morphologically similar Shh (Nkx2.1) null mutant phenotypes result in the trachea failing to separate dorso- ventrally from the esophagus, forming a tracheoesophageal tube from
the sides of which grossly hypoplastic and dysplastic epithelial bags arise. In the Nkx2.1 null mutant, epithelial differentiation is arrested prior to the expression of peripheral epithelial markers; by contrast, in the Shh null mutant, peripheral lung epithelial markers are expressed [277–279]. Finally, the phenotype in transgenic mice that misexpress a dominant negative, tyrosine kinase deleted FGF receptor under the control of the surfactant protein C promoter/enhancer causes bronchial morphogenesis abrogation distal to the primary bronchi [280]. (b) Successive stages in human lung development, at 5 weeks (left), at 6 weeks (center), and at 8 weeks (right). Note the formation of lung lobes by invagination of the visceral pleura (shown in red). (a From Warburton et al. [48], copyright 2000, with permission from Elsevier [48]. b Reprinted from Sadler [281] with permission)
2.1 Early Pre-specification Patterns in Lung Assembly
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labeled precursors of peripheral lung are restricted to a few bronchial tube cells and to cell clusters in bronchial tips and lateral buds: these cells subsequently undergo expansion to form the alveolar surface. It is proposed that the repetitive branching patterns that characterize the development of preacinar airway generations result from a series of complex interactions between signaling molecules in tissue zones termed “morphogenetic signaling centers”: FGF-10/FGF receptor-2 signaling, for example, appears key and the temporal–spatial patterns of expression for selected regulator molecules (such as SHH, TGF-b, bone morphogenetic protein 4, and mSpry) are consistent with their role as modulators of FGF signaling [50, 70]. It is not established if central vascular branching is achieved in a similar way.
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The lung vasculature forms by integrated mechanisms of sprouting angiogenesis and vasculogenesis (Fig. 6). These structures are remodeled in a process termed intussusception (i.e., of itself) whereby the capillary wall invaginates to form a contact zone between opposing endothelial cells. The formation of an intraluminal connective tissue post at the site of endothelial cell contact splits the capillary structure into two segments. Capillary networks continue to expand by intussusceptive microvascular growth [33] as well as simple expansion (Fig. 7). In addition, endothelial cells may remodel the forming capillary segments by their “guided migration” over existing capillary endothelial cells [71]. Small arteries and veins subsequently evolve from the capillary networks by intussusceptive arborization and branch remodeling [72–74] (Fig. 8). On the basis of the analysis of Mercox casts of mouse lung, and scanning and transmission microscopy, deMello et al. described steps in lung vascular formation [75, 76]. The growth of central arterial and venous trunks and their branches proceeds by angiogenesis (Fig. 6c), offshoots of endothelial sprouts at the growth points of these vessels invading the mass of mesenchymal cells surrounding the airway tubes. The solid
tubelike sprouts first formed organize into open tubules as the endothelial cells polarize around a lumen. These tubelike segments continue to elongate by cell migration and proliferation and branch, or fuse (at their blind end) to another vascular sprout to form a new loop. The central artery branches accompany the intrapulmonary airways by E10 in the mouse; [75] by E11.5 there is significant secondary branching and development of further bronchial structures, and by E12.5 arborization is extensive as the central units expand in diameter and length and offshoots grow by expansion and irregular dichotomous branching [75]. At about this time, the segmental pulmonary arteries connect to the central vessels [75]. The vascular side branching pattern of the pulmonary arterial tree, which accounts for up to 40% of the vascular endothelium in wild-type mice, is reduced in transgenic mice highly expressing matrix Gla protein, an inhibitor of bone morphogenetic protein [77]. Such changes are thought to occur in response to reduced levels of phosphorylated Smad5 or Smad8 in alveolar epithelium, and epithelial expression of the activin-like kinase receptor 1 and VEGF (being critical for vascular formation) may lead to loss of coordination (cross talk) between the developing vascular and airway cells [77]. In a separate but concurrent process, vascular segments in the peripheral (sub-pleural) zone of the developing mouse lung form by vasculogenesis [75], the angioblasts forming nests of cells that assemble into capillary-like structures enclosing hemangioblasts (future blood cells) (Fig. 6a, b). Transmission microscopy studies [75] reveal, as early as when the first airway branches form (at E9), abundant blood-filled channels in the peripheral sub-pleural mesenchyme. These lack connections to the central arteries. The intercellular spaces forming the lumen of the first channels arise by loss of vesicles from the apical membranes of mesenchymal cells. By thinning their processes, and reforming their apical membranes, mesenchymal cells evolve to endothelial-like cells (Fig. 6b). The separately developing central and distal vascular systems fuse in the mid fetal stage to complete the vascular circuit (E13–E14) [75]. The extent of growth in the normal mouse lung at this time (E14 and E15) provides a striking example of change in the vascular template (Fig. 9). Although connections between the central pre- and post-capillary vessels and distal vascular networks are sparse by the end of the pseudoglandular stage (E14.2–E16.6), in the canalicular stage (E16.6–E17.4) the arterial–capillary–venous circuit is well
Fig. 6 (continued) an endothelial lineage, and associate into blood islands; developing contacts, the angioblasts assemble into capillary-like structures that subsequently connect and retain hemangioblasts (hematopoietic precursor cells) within their lumen. (c) Transmission electron micrographs of a mouse lung (E9) showing densely packed mesenchymal cells containing cytoplasmic vesicles surrounding the developing lung bud (top left), and intercellular spaces forming (which appear to result from discharge of intracytoplasmic vesicles, leaving a ruptured cell membrane)
between plump, densely packed mesenchymal cells (top right). Thinning of lung mesenchymal cells adjacent to intercellular spaces, in a later fetus (E10), results in endothelial-like cells (lower left). Hematopoietic precursor cells are present in some luminal spaces (lower right). Original magnification: (c) top left ×2,800; top right ×8,750; lower left and lower right ×5,250. (a, b Reprinted from Schoefl [282] with permission from WileyBlackwell. (c) Redrawn and reproduced from deMello et al. [75] with permission, copyright American Thoracic Society [75])
2.2 Lung Vessel Morphogenesis 2.2.1 Endothelial Tube Formation (Vessels and Capillaries)
3 Pulmonary Vascular Development
Fig. 6 Capillary assembly by angiogenesis and vasculogenesis. (a) Capillary formation by angiogenesis: endothelial cells and their processes, released from the constraint of a basement membrane, and in response to a signal triggering growth, invade the surrounding tissue (sprout in the direction of the growth signal) to form cell-lined channels (top left and top right). Wall destabilization, with focal degradation of the endothelial basement membrane and surrounding matrix, is followed by the formation of a spur as endothelial pseudopodia extend through the gap. Endothelial cell migration in the direction of the growth spur forms a sprout [282, 283]. The delicate microspikes at the leading edge of migrating endothelial cells forming the tip of the sprout lack basement membrane. Rather than migrating singly, they move as a shifting sheet
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of cells [282]. Typically, proliferating cells lying behind the growing tip increase the length of the sprout [283], although sprouts can develop in the absence of endothelial cell proliferation (as in inflammation): an increasing cell population is needed, however, for sustained growth [283, 284]. Although still connected at their origin to a patent vessel or capillary, sprouts continue to elongate and to branch or fuse to an adjacent sprout at their blind end to form a new loop and establish a contiguous network (lower left and lower right). Development of a slit-like lumen followed by the entry of plasma and blood cells completes the formation of a continuous channel. (b) Capillary formation by vasculogenesis: aggregates of undifferentiated mesenchymal cells [281] differentiate in situ into angioblasts, i.e., precursor cells committed to
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Fig. 7 Capillary remodeling and growth: intussusceptive microvascular growth (IMG), and expansion. (a) Steps of intussusception: the capillary lumen is divided as opposing walls protrude to create a contact zone between endothelial cells. Following central perforation of the cellular bilayer, the fused endothelial cells form a transluminal cuff, which is later invaded (and strengthened) by myofibroblasts or pericytes. (b) Growth of an existing vascular bed occurs by simple expansion, by the addition of wall cells and by increase in lumen diameter and segment length (a Reproduced from Kurz et al. [74] with permission. (b) Reprinted from Schoefl [282] with permission from Wiley-Blackwell)
established and connections are widespread [75]. The report of even earlier connections between the two vascular systems in the mouse lung indicates continuity from the time of inception [78]. Other concepts have been proposed, with the vasculature formed, on the one hand, by distal angiogenesis throughout the mouse lung by peripheral vessels connected to the embryonic circulation [79], and, on the other, by vasculogenesis throughout the lung of the chick embryo [80]. The extent to which the central and distal lung vessels develop by processes of angiogenesis and/or vasculogenesis is still subject to debate. Studies demonstrating that endothelial cell function and molecular phenotypes are vessel-specific, however – those of large vessels having a distinct and different molecular phenotype from microvascular ones, small-vessel endothelial cells arising from within the mesenchyme and large-vessel endothelium originating from the pulmonary truncus by angiogenesis – support the concept that the lung’s vasculature develops by combined processes [81–83]. Although, in general, it has been accepted that capillaries form by angiogenesis and vasculogenesis before birth, and by angiogenesis after birth, their formation by vasculogenesis may also occur postnatally [84]. Of great current interest is the potential role in the growing (and injured) lung of endothelial
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cells, and endothelial cell precursors, derived from the bone marrow and circulating in blood [85, 86]. Circulating hematopoietic progenitor/precursor cells have been reported to contribute to capillary growth and repair in the developing and adult lung [87, 88], and have been detected in the walls of pulmonary arteries in lung disease [89]. The possible population of blood-borne cells fulfilling this role is likely heterogeneous and a true “vascular” progenitor remains to be defined [85]. Studies describing the molecular basis of endothelial channel formation increasingly provide insights into structural events [90–96]. Growth factors such as the FGF family first induce angioblast and hemangioblast formation in the mesenchyme. The homodimeric glycoprotein VEGF is then central in regulating blood vessel formation, maintaining angioblast differentiation and survival via its receptor tyrosine kinases (RTKs), i.e., VEGF-R1 (Flt-1), VEGF-R2 (Flk-1/KDR), and VEGF-R3 (Flt-4). VEGF-A and VEGF-D are abundant in developing lung, VEGF-A being detected in the lung mesenchyme throughout fetal development and VEGF-D from E13.5 [97]. Alternative splicing produces homodimeric VEGF-A isoforms – VEGF-A120/121, VEGF-A164/165, VEGF-A189, and VEGF-A206. Of these, VEGF121 and VEGF165 act as survival factors for endothelial cells, and before E14 these soluble isoforms predominate in the developing lung [97]. VEGF is induced by SHH and is highly expressed by epithelial cells localizing within the matrix at the leading edge of branching airways [96, 98]. The VEGF signals controlling angiogenesis in the lung thus arise from adjacent epithelial cells, stimulating cognate RTKs on endothelial cells. VEGF-R1 and VEGF-R2, in particular, play critical roles in both angiogenesis and vasculogenesis, being required for endothelial cell proliferation, tubule assembly, and wall stabilization [99–101]. Clusters of VEGF-R2+ (Flk-1+) vascular precursors, localized in the mesenchyme close to developing epithelium from E11 to birth, demonstrate that vascularization is initiated, and endothelial cell lineage established, when the lung anlage first develops. VEGF-R1 expression is required by angioblasts to assemble into vascular channels, including tubule formation and sprouting (Fig. 10); VEGF-R2, expressed by endothelial precursors as they differentiate to endothelial cells, results in proliferation [92, 100, 102–105] (Fig. 10). During the differentiation of cells within the mesenchyme there is an ordered progression of surface markers, with the expression of VEGF-R2 being followed by that of vascular endothelial cadherin, platelet-endothelial capillary adhesion molecule (PECAM-1), and CD34 [106]. Yamamoto et al. [107] characterized developing endothelial cells from undifferentiated mesenchymal cells by their expression of VEGF-R2/VEGF-R1, and PECAM-1, and the shift in temporal–spatial expression patterns of these molecules as capillary structures form in the lung’s pseudoglandular and canalicular phase. Early in development (E9.5–E10.5)
3 Pulmonary Vascular Development
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Fig. 8 Capillary and vessel remodeling and intussusceptive microvascular growth IMG, intussusceptive arborization (IAR), and intussusceptive branch remodeling (IBR). (a) Insertion of transluminal pillars (arrowheads) results in rapid expansion of the capillary plexus by IMG. IAR generates feed vessels from the capillary plexus by vertical pillar formation in rows (a arrows), which demarcate future vessels. (b, c) Narrow tissue septa (arrows) formed by pillar reshaping and pillar fusions segregate the new vascular entity. (d) Formation of horizontal
pillars and folds (arrowheads) separate the newborn feeding vessels from the capillary plexus. (e–g) IBR finally adapts the deepness of the branching angle and the diameters of daughter vessels in the newly formed supplying and draining vessels by insertion of transluminary pillars at branching points (arrows). (f–h) Moreover, IBR leads to vascular pruning by repetitively eccentric formation, augmentation, and fusion of pillars (arrowheads). (Reproduced from Djonov et al. [72] with permission from Lippincott, Williams & Wilkins)
n umerous undifferentiated mesenchymal cells and VEGF-R2+ cells, and a few PECAM-1 expressing cells, surround the developing distal airway bud, whereas VEGF-R1+ cells are virtually absent. Subsequently (E11.5–E13.5), fewer undifferentiated mesenchymal cells and VEGF-R2+ cells are present, PECAM-1+ cells increase in number, and small numbers of VEGF-R1+ cells appear. Later, in the pseudoglandular phase (E14.5–E16.5) an organized layer of undifferentiated mesenchymal cells, and few VEGF-R2+ cells surrounding the airway, are in turn now surrounded by an organized layer of PECAM-1 cells, whereas the numbers of VEGF-R1+ expressing cells increase. Finally, in the canalicular/saccular phase (E17.5), VEGF-R1+ and VEGF-R2+ cells are sparse, and a well-organized layer of PECAM-1+ cells surrounds alveolar epithelial cell tubules. Different ligands determine cell differentiation: for example, whereas VEGF promotes VEGF-R2 expressing cells to differentiate to endothelial cells, PDGF-B promotes their differentiation to SMCs and pericytes [108].
While endothelial cells develop in VEGF−/− mice, the vessels formated are abnormal. Loss of VEGF164 and VEGF188 isoforms leads to failure to develop a normal distal vasculature and impairs alveolarization [109] (Fig. 11). VEGF expression in (surfactant protein C–VEGF) transgenic mice increases lung blood vessel growth and distorts branching morphogenesis [110]. VEGF-R1−/− mice have normal progenitor cells and abundant endothelial cells that migrate and proliferate but do not assemble into tubules or form functional vessels, whereas VEGF-R2−/− mice have failed vasculogenesis and blood island formation and do not develop endothelial cells, possibly because hemangioblasts fail to differentiate. VEGF stabilizes developing vessel walls by accelerating the development of supporting perivascular cells [111, 112]. Immature vessels that lack perivascular cells need VEGF to prevent their endothelial cells from detaching and undergoing apoptosis, but during postnatal life endothelial cells become VEGF-independent – at least from paracrine signaling
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Fig. 9 Mercox casts illustrating normal pulmonary vascular growth patterns. (a) Scanning electron micrograph of the vascular bed of a mouse (at E14). Note the presence of occasional small capillary buds and branches (arrowheads), intersecting at right angles with the lumen of larger adjacent capillary segments. (b–d) Scanning electron micrographs of the vascular bed of a mouse (at E15). Extensive connections between the central and peripheral systems are now present, revealing a complex peripheral network. (c–d) At higher magnification
(see area indicated in b, arrow) blind-ending pre-capillary branches are seen to approach the future capillary network at right angles. Residual constrictions suggest sites of coalescence (fusion). It seems likely that the vessel upstream from the arrow is central, whereas the more irregular vessels below the arrow are derived from the peripheral system. Original magnification: (a) ×320; (b) ×69; (c) ×320; (d) ×1,200. (Reprinted from deMello et al. [75] with permission, copyright American Thoracic Society [75])
p athways – although autologous VEGF signaling (independent of paracrine VEGF signaling) is likely maintained for endothelial cell homeostasis [113]. Gebb and Shannon [96] have shown that endothelial expression of VEGF-R2 within the mesenchyme requires the presence of epithelium, demonstrating a regulatory loop between the two systems; and pulmonary vascular development has been shown to have a rate-limiting role in epithelial branching [114]. The divergent homeobox gene Hex is expressed in the distal lung mesenchyme where primitive blood islands appear and is a specific and early marker of endothelial cell precursors. Unlike VEGF-R2, it is downregulated as endothelial cell differentiation begins. Hex disruption, unlike the disruption of VEGF-R2, has no effect on the presence of hematopoietic precursor cells or vascular network formation [115, 116].
Tie-1 and Tie-2 are a second family of RTKs expressed by endothelial cells (Fig. 10); ANG1, the major ligand for Tie2, is expressed by mesenchymal cells. As yet, no ligand has been identified for Tie-1, which modulates transcapillary fluid exchange. ANG2 is a negative Tie-2 ligand [91, 92, 117–123]. Tie-2 expression modulates VEGF activity and is required for endothelial sprout formation [119, 120, 124]. Although Tie-2−/− mice have normal numbers of endothelial cells, these assemble into immature channels that lack branching networks and the presence of large and small vessels. ANG1−/− mice have vascular defects similar to those of Tie2−/− mice and die of dilated/impaired vessel branching (at E10.5). ANG1 together with VEGF enhances vascular density, whereas ANG2 and VEGF produce longer sprouts, indicating a role for ANG2 in vessel formation in addition to regression
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Fig. 10 Regulation of vascular morphogenesis, maintenance, and remodeling by receptor tyrosine kinases (RTKs) and their ligands. A model for regulation of vascular endothelium is demonstrated by the prototypical angiogenesis factor vascular endothelial growth factor (VEGF) and the class of angiogenic regulators angiopoietin-1 (ANG1) and angiopoietin-2 (ANG2). All three ligands bind to RTKs that have similar cytoplasmic signaling domains, yet their downstream signals elicit distinct cellular responses. Only VEGF binding to VEGF-R2 (Flk1) sends a classic proliferative signal. When first activated in embryogenesis, this interaction induces the birth and proliferation of endothelial cells. In contrast, VEGF binding to VEGF-R1 (Flt1) elicits endothelial cell–cell interactions and capillary tube formation, a process
that closely follows proliferation and migration of endothelial cells. ANG1 binding to Tie-2 RTK recruits and likely maintains association of peri-endothelial support cells (pericytes, SMCs, myocardiocytes), thus solidifying and stabilizing newly formed blood vessels. ANG2, although highly homologous to ANG1, does not activate Tie-2 RTK; rather, it binds and blocks kinase activation in endothelial cells. The ANG2 negative signal causes vessel structures to become loosened, reducing endothelial cell contacts with matrix and disassociating periendothelial support cells. This loosening appears to render the endothelial cells more accessible and responsive to the angiogenic inducer VEGF (and likely to other inducers). (Reprinted from Hanahan [92] with permission from AAAS [92])
Fig. 11 Mercox casts illustrating abnormal pulmonary vascular growth patterns. (a–f) Scanning electron micrographs show different growth patterns in VEGF 120/120 littermate fetal mice (E17): wild-type (a, d), heterozygous (b, e), homozygous (c, f). In the homozygous fetus, there
are fewer small peripheral vessels and they are of larger caliber than those in heterozygous or wild-type littermates. Magnification: Bar (a–c) 152 mm and (d–f) 50 mm. (Reprinted from Galambos et al. [109] with permission, copyright American Thoracic Society [109])
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[124]. In the absence of VEGF and in the presence of ANG2, vessels destabilize as endothelial cells undergo apoptosis. TGF-b1−/− mice have poor endothelial cell differentiation and hematopoiesis and die at mid-gestation (E10.5) [125, 126]. TGF-b1 binds TbRII and activin-receptor-like kinases, ALK1 and ALK5 – the ALK5 (TbRI) receptor phosphorylating Smad2 and Smad3: these complex with Smad4, which translocates to the nucleus and activates transcription target genes [127]. ALK5 (TbRI)−/− mice form blood islands, and their vessels initially are normal, but they develop defects similar to TGF-b1 and TbRII mutants, their vessels lacking hierarchical organized branching and being fragile because supporting perivascular cells fail to differentiate, and the mice die (at E10.5–E11.5) [128]. ALK1−/− mice have fused, dilated major vessels (at E9.5); they fail to form capillary networks, and die (at E11.5). The vascular malformations in these (and endoglin−/−) mice (increased numbers of dilated vessels and hemorrhage) are characteristic of vascular abnormalities in human hereditary hemangiotalactasia [129]. Hoxa-3−/− mice die shortly after birth with heart defects and enlarged pulmonary veins. The walls of their vessels, which normally consist of endothelial cells, SMCs, and an outer layer of cardiac cells, are altered, suggesting that ablation of this gene leads to impaired venous wall formation and vein enlargement resulting from limited cardiac cell migration. Wnt signals are key regulators of cell proliferation and differentiation, and within this growth factor family, signaling of the gene Wnt7b regulates cell proliferation and growth in lung mesenchyme. Analysis of Wnt7blacZ−/− neonatal mice reveals vessel rupture and hemorrhage, indicating a critical role for Wnt7b in lung mesenchymal growth and vascular development [130]. Hox genes regulate lung airway development and can serve to induce an angiogenic phenotype in endothelial cells
[131]. For example, Hoxd-3 colocalizes with avb3 integrin in vessels formed in response to FGF-2 (increasing components of the angiogenic cascade), Hoxb-3 (a Hoxd-3 paralogue) promotes lung capillary formation (controlling expression of ephrin A1/EphA2, see later), whereas Hoxb-7 expressed in fetal lung upregulates angiogenic stimuli (FGF-2, VEGF, and matrix metalloproteinases). Yet other Hox genes suppress an angiogenic phenotype, perhaps to maintain endothelial cells in a fully differentiated state, as in the mature lung. Hoxd-10 is an example of this, being expressed in the adult but not the developing lung, with its expression greater in quiescent endothelial cells, where it blocks FGF-2 dependent migration. As an antiangiogenic factor (inhibiting vessel growth in corneal and Matrigel models), the cytokine-like molecule endothelial monocyte activating polypeptide II (EMAPII) is proposed to negatively regulate lung vascularization. As the lung evolves from a poor to a richly vascularized tissue (E14– E18.5/E19), messenger RNA/protein expression decreases five-fold. Postnatal EMAPII expression levels generally remain low [132, 133]. EMAPII induces endothelial cell apoptosis in adult lung, and its localization to perivascular regions of large arteries may inhibit their branching while the peripheral vasculature continues to develop [132, 133].
Fig. 12 Distribution and sites of action of ephrin B2 and EphB4, a ligand– receptor pair for endothelial cell specification, in arteries and veins of a primary capillary plexus and in a maturing vascular network. [94] They are presumed to interact between opposing arterial and venous endothelial cells in a cis manner (left). During later maturation and remodeling of the
primary plexus (right), by interdigitation, branching, and differential growth of vascular segments, they remain localized to arterial and venous units (cis interactions) but may also interact at the interdigitating surfaces of large vessels (trans interactions). (Reprinted from Yancopoulos et al. [93], copyright 1998, with permission from Elsevier)
2.2.2 Arterial and Venous Specification and Differentiation Arteriovenous relationships are likely established and maintained by the expression of the ligand ephrin B2 and its receptor EphB4 [93–95] (Fig. 12). Functional vessels are first reported in the distal mesenchyme in the earliest phase of lung development in the mouse (E11.5), blood flow being
3 Pulmonary Vascular Development
indicated by the delivery of intravascular fluorescein-labeled lectin; later (by E14.5), such vessels extend to the epithelial interface (in response to VEGF signals from epithelial cells) [134]. Despite the evidence of flow, however, the emerging pulmonary vasculature was found to lack arteriovenous specification as determined by ephrin B2 and EphB4 expression [134]. Additional studies are needed to determine the timing of such specification in line with the development of dual pathways reported in structural studies. Notch receptors and ligands are expressed in and around the developing vasculature [135] and this signaling appears to establish the initial arterial and venous identity/fate upstream of EphB4/ ephrin B2 signaling. Notch1–Notch4 interact with five ligands – Delta-like 1, Delta-like 3, Delta-like 4, Jagged1, and Jagged2. Notch1 and Notch4, Delta-like 4, Jagged1, and Jagged2 are each expressed in arterial endothelial cells [135] but only in low amounts, and if at all in venous ones. Notch3 is expressed in arterial but not venous SMCs, and Jagged1 is expressed in arterial endothelial cells and SMCs. Notch1 and Jagged1 appear in larger vessels in the embryonic lung before being expressed in the smaller developing networks [135]. The forkhead box (Fox)f1 transcription factor, which belongs to a family of transcription factors critical to the development of the lung’s microvasculature, may mediate the effects of Notch signaling [136, 137]. EphB4/ephrin B2 signaling is critical for appropriate vascular development, possibly being required for “like” vessels to fuse. The endothelial cells of developing arteries specifically express ephrin B2, and ones of developing veins specifically express EphB4, before the presence of a functional circulation. Ephrin B2−/− and EphB4−/− mice die (at E9.5 and E11.5) with defects in both arteries and veins. Postnatally, EphB4 acts as a negative regulator of blood vessel remodeling and network formation, switching the vascular program from sprouting angiogenesis to circumferential vessel wall growth [138]. Additionally, ephrin B2 and EphB4 are expressed in mesenchymal cells and play a role in endothelial and mesenchymal cell interactions [95].
2.2.3 Vessel and Capillary Wall Maturation (Stabilization) The endothelial channels forming in the lung are stabilized as perivascular cells and develop from within the mesenchyme [17, 91, 92, 117, 139–143]. Their close apposition to endothelial basement membrane triggers differentiation, likely in response to endothelial expression of TGF-b, which also inhibits endothelial cell movement and proliferation [117]. Within the walls of developing vessels, perivascular cells differentiate to SMCs and adventitial fibroblasts, and within capillaries into pericytes. Pericytes develop particularly in those capillary segments that arise directly from a vessel wall, and around postcapillary venules [144–146]. Around forming endothelial channels, they serve to bridge gaps between adja-
39
cent endothelial cells and between the leading edges of merging endothelial sprouts [147, 148]. In some vessel segments adjacent to capillaries (Fig. 4b) the perivascular cells develop into intermediate cells [42, 145, 149, 150] (Fig. 4b). Although the mesenchyme represents an important source of perivascular precursors [142, 151, 152], perivascular cells are also reported to develop from bronchial SMCs and endothelial cells [153], and to express smooth muscle proteins similar to the cells that are mesenchymal in origin [153]. Furthermore, data on the origin of SMCs indicate that embryonic endothelial cells can become mesenchymal cells expressing smooth muscle proteins, and so are also a potential source of these cells [154– 156]. The proteins forming the cytoskeletal and contractile filament networks of the perivascular cells appear sequentially, a-smooth muscle actin (aSMA) followed by calponin, h-caldesmon, a-tropomyosin, and metavinculin, with the smooth muscle myosin heavy chain (SM-MHC) SM1 isoform appearing during the fetal period and the SM-MHC SM2 isoform appearing after birth [142, 157–164]. The contractile filaments of differentiated SMCs confer tensile strength on the vessel wall and an ability to contract [165, 166]. In all but the smallest venules, SMCs are surrounded by basement membrane and are characterized by extensive filaments, fusiform dense bodies, and attachment plaques. To proliferate or migrate, these cells readily disassemble their filaments networks (dedifferentiate); and they may revert to a contractile phenotype by their reassembly. Distally, at the level of the respiratory bronchiolus and proximal alveolar duct, the SMC processes penetrate their surrounding basal lamina to form lateral (SMC–SMC) contacts and the basal lamina of adjacent endothelial cells to form junctional complexes [150]. As vessels approach capillaries, the numbers of endothelial cell contacts increase, and their processes penetrate their basal lamina and the internal elastic lamina to contact SMCs [150]. More distally, at the level of the alveolar duct and alveolar wall, intermediate cells, which have now lost their basal lamina, contact adjacent endothelial cells by their projections [150]. The outermost layer of vessel walls is formed by circumferentially aligned adventitial fibroblasts. Their number, and that of SMCs, determines oxygen diffusion (which becomes restricted once the perivascular tissue cuff is 100 mm or more thick). Although these fibroblasts typically express vimentin, actin isoforms, and nonmuscle myosin, under certain conditions they express two SMCspecific proteins, aSMA and desmin [167]. They are characterized by arrays of microfilaments (4–6 nm in diameter), intermediate-sized filaments, pinocytotic vesicles, and by abundant extensive rough endoplasmic reticulum. Basement membrane is absent, and collagen fibers and fibrils form along their adluminal and abluminal cell margins. Capillary pericytes share the endothelial basement membrane and normally lack extensive filament networks, dense bodies, or attachment plaques [145–147, 168]. Their processes often extend to contact the endothelial cell and rare gap junctions allow nucleotides
40
to pass between the cells [147, 168]. During endothelial sprout formation, they prevent plasma from escaping into interstitial tissue [147, 168]. Possessing cyclic-GMP-dependent kinase, actin, desmin, vimentin, a-tropomyosin, and myosin, capillary pericytes are reported to contract or relax in response to vasomediators [169]. Their ability to express the proteins typical of contractile muscle cells, such as SM-MHC, aSMA, and desmin, however, varies greatly [170–172]. Evidence that interstitial fibroblasts (cells normally present within the interstitium of the alveolar–capillary membrane) are a further source of perivascular cells, and acquire the phenotype of a pericyte or SMC, comes from studies of mature vascular beds, i.e., dorsal and mesenteric capillaries [145, 147, 173], and from studies of distal vessel remodeling in the adult lung in pulmonary hypertension [42, 174–177]. SMCs separate from adjacent endothelial cells and adventitial fibroblasts within the vessel wall as internal and external elastic laminae form along their inner and their outer margin. The essential regulation of smooth muscle growth by elastin in the normal lung is demonstrated by obstructive intimal hyperplasia in (and the death of) mice that lack the elastin gene [178]. In the developing lung, each vascular cell type (endothelial cell, SMC, and fibroblast) is elastogenic [179], although the relative contribution of each to lamina formation is unknown. The process of lamina assembly, as currently understood, derives from data for other sites [118, 178, 180–189]. Expression levels of tropoelastin (a major component of the forming lamina) fall in artery walls and increase in vein walls in the lung after birth, a reverse of the fetal pattern [179]. The presence of endostatin (an inhibitor of endothelial cell proliferation) within matrix and elastic laminae of large vessels may restrict further vascular sprouting from the wall [190]. Homotypic and heterotypic contacts are important for perivascular cell recruitment [191, 192]. At the molecular level, PDGF and in particular (of the genes identified, e.g., PDGF-A, PDGF-B, PDGF-C, and PDGF-D) PDGF-B signaling via its cognate RTK (PDGF-Rb), have a significant role [151, 152, 191, 193, 194]. Typically perivascular cells, including ones in lung vessels, express PDGF-Rb [191, 192, 195, 196] and arterial and capillary endothelial cells express PDGF-B [191, 192, 195, 196]. This suggests that the endothelial cell targets (recruits) the perivascular cell, driving wall formation. Complex shifts in the gradients formed by changing expression patterns of these molecules, however, likely determine this interaction in the developing lung as in the adult [195, 196]. The lack of pericytes in mice deficient in the PDGF-B gene results in capillary dilatation, microaneurysms, and vascular leak and hemorrhage; the absence of the PDGF-Rb gene in mice elicits a similar response (the mice dying at E17.5–E18.5 and at birth, respectively). PDGF-B promotes mesenchymal cell proliferation, initiates their progression through the cell cycle, and induces chemotaxis [191,
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192]; and SMCs may use PDGF-B to suppress differentiation (assembly of contractile filaments and networks), enter the cell cycle, and stimulate self-replication via synthesis of PDGF-A [197, 198]. PDGF-B also enhances wall stabilization by inducing perivascular cell expression of VEGF [199, 200]. PDGF retention during vascular development is essential for normal conduit vessels and capillaries to form [201]. Actin reorganization and membrane ruffling (each essential for cell migration within vessel walls) are induced by PDGF-AB and PDGF-B, and therefore via PDGF-Rb. In addition to PDGF-B/PDGF-Rb signaling, ANG and its RTK (Tie-1) are required for perivascular cells to be recruited (Fig. 10). The cells express ANG1 and ANG2 ligands and endothelial cells express their (Tie-1 and Tie-2) receptors. Endoglin (TGF-b RIII), endothelial differentiation gene (Edg-1)/sphingosine isophosphate, and TGF-b1 signaling are also required for appropriate perivascular cell development. Endoglin−/− mice have arrested remodeling of endothelial networks with poor SMC development, dilatation, and hemorrhage (and die at E11.5), whereas Edg-1−/− mice have deficient SMCs/pericytes (and die at E12.5–E14.5). TGF-b1 signaling triggers perivascular cells to express a SMC phenotype. Prx-1 and Prx-2 also are expressed in developing lung [202], regulating patterning of blood vessels and promoting SMC differentiation, and controlling SMC-specific gene expression by enhancing binding of serum response factor to CarG elements (a DNA motif present in many SMC-specific gene promoters).
2.2.4 Vessel and Capillary Network Formation in Distal Lung The lung’s respiratory surface starts to form just before birth and in the immediate period after. Proliferating and organizing endothelial cells expand its capillary and small-vessel networks significantly at this time [32, 203]. A saccule (a primitive structure or sac) first forms at the end of the airway: this is also referred to as a terminal saccule (terminal sac) or primary septum. It is proposed that it forms (a) as a sleeve of inhibition develops around the airway tube to prevent random branching while promoters (such as FGF-10) enhance branching at the tip – the tube initially expanding and then branching under the direction of two such growth-promoter zones separated by a cleft of inhibition – and/or (b) by increased intraluminal pressure, secondary to fluid secretion into the airway (also regulated by FGF-10), which causes expansion of the terminal bud between inhibitory points (of highly resistant tissue areas) created by myofibroblasts making elastin [204]. The terminal saccules are supplied by small vessels and a double capillary system at birth (Fig. 13); during septation the capillary network remodels into a single system. Septation occurs as secondary crests protrude from the primary septa, and as secondary
3 Pulmonary Vascular Development
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Fig. 13 Fetal and postnatal development of the pulmonary capillary bed. (a) In the pseudoglandular stage the epithelial tubes push into the mesenchyme, which contains a loose capillary network (top left). The amount of intervening mesenchyme is reduced and capillaries arrange around epithelium. At sites of epithelium–capillary contact, portions of the air–blood barrier begin to thin as epithelial type 1 and type 2 cells form (top right), whereas, at the periphery, the still cuboidal epithelium allows for further growth and branching. Perinatally, secondary septa are developing (lower left, arrowheads) from primary septa: at this stage, all septa are of the primitive type, containing a double capillary network and a central layer of connective tissue. In the mature lung (lower right), inter-alveolar septa contain a single capillary network meandering through the interstitium. (b) A secondary septum forming from a primary septum by lifting of one
of the two capillary layers (arrows). Elastin deposition (dark regions) is necessary for the formation of alveoli. (c) Photomicrograph of mouse alveolar–capillary membrane (at post-natal day 3): the primary septal wall illustrated is seen to consist of the cytoplasmic extensions of alveolar epithelial (type 1) cells (arrows) separated from capillary endothelial cell processes and the interstitial layer by a continuous basal lamina. The three capillaries present contain erythrocytes in their lumen (e, cl); they form a double capillary layer, together with their basal lamina, ground substance and a portion of an interstitial cell (g, isc). Magnification × 10,000. (c) Bar 1 mm. (a Reprinted www.annual reviews.org from Burri [203] with permission, copyright 1984 Annual Reviews. (b) Reproduced from Burri [203] with permission from McGraw-Hill companies [21]. (c) Reprinted from Amy et al. [205] with permission from Wiley-Blackwell)
septa form, at these points, the primary septum (terminal saccule) divides into alveoli [21, 205, 206]. The secondary septum forms in regions where the capillaries and primary septum can be folded up: fibroblasts proliferating at the sites of origin of the secondary crest and lengthening the secondary septum as it projects (at a right angle) into the alveolar space. Myofibroblasts assemble in the tips of the secondary crests adjacent to elastic fibers. The septal walls of the newly forming alveoli then thin as the intervening tissue regresses [207, 208], with interstitial fibroblasts undergoing apoptosis as the capil-
laries fuse [21]. Further insight into the mechanism of septation is provided by studies showing that PDGF-A/ PDGF-Ra signaling is required [151, 152, 209, 210]. In wild-type mice, the distal mesenchyme generates PDGFRa positive myofibroblasts (considered SMC progenitors) in response to PDGF-A, which develop in a cluster around the terminal ends of epithelial buds; in the canalicular stage, these cells spread to surround the prospective terminal sacs, downregulate PDGF-Ra expression, and upregulate tropoelastin expression to start the process of septal formation
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Fig. 14 Model of alveolar SMC development. (a) At the pseudoglandular stage, alveolar SMC progenitors (blue) originate as clustered plateletderived growth factor-receptor a positive (PDGF-Ra+) mesenchymal cells located around the epithelial buds. (b) In conjunction with the canalicular stage, these cells normally spread to acquire positions surrounding the prospective terminal sacs; and (c) during the terminal sac to alveolar stages, the cells downregulate PDGF-Ra expression and upregulate tropoelastin expression (black), leading to septation. (d) In PDGF-A−/− lungs, the distal spreading of PDGF-Ra+ progenitors does not occur; (e) consequently, in the absence of alveolar septal elastin deposition, alveogenesis fails. (Reproduced from Lindahl et al. [151] with permission)
forming until approximately 40 days after birth, supports an additional growth process [206] and this appears linked to the action of RA. A high concentration of cellular retinol binding-protein 1 is present in the lung during the period of septation [216]. Furthermore, retinol administration induces alveolus formation, and retinol-storing cells appear first diffusely through the lung (where alveoli form in the septation period at birth) and later only in the subpleural region (where additional alveoli form after septation) [206, 217]. RA receptor (RAR)-b inhibits septation, and in RAR-b−/− mice, septation starts early and alveoli initially form twice as fast as in wild-type mice: later, septation occurs at the same rate as in wild-type mice, supporting the concept of two mechanisms regulating alveolar formation [206]. RA/RAR interaction(s) may regulate septation via cell proliferation, migration, and temporal differentiation by upregulating RA response elements (which modulate other gene expression patterns) [206]. Both RA and RA response elemens increase alveolarization by stimulating proliferation of lung fibroblast progenitors via a PDGF-mediated autocrine mechanism.
3 Vascular Templates in Normal Human Lung at sites along the saccule wall (Fig. 14). PDGF-A mice have −/−
reduced numbers of PDGF-Ra+ myofibroblast progenitor cells, and cells that fail to spread, proliferate, and synthesize elastin, resulting in impaired septation [151, 152, 209, 210] (Fig. 14). The critical role of tropoelastin/elastin deposition and myofibroblast migration for alveolar formation is clearly demonstrated in the neuraminidase-1 (Neu1) null mouse, which is unable to survive [211]. The expression in the mesenchyme of Robo (a receptor involved in repellant signaling in developing neuronal systems) and Slit (its ligand) may regulate mesenchymal cell organization [212, 213], and Robo−/− mice lack septation [204]. From this, an intricate mechanism for septal wall formation is proposed in which myofibroblast migration progresses to the points where septa form (a source of high PDGF-A expression) and elastin synthesis is induced along a “morphogen gradient” of PDGF-A signals from epithelial cells: subsequent invagination of the saccule wall and formation of the secondary septum occurs as capillaries and myofibroblasts following epithelial cells (expressing Robo) are repulsed into the airway space (from a Slit source) [204]. As the distance of these cells from the repulsive signal increases, formation of the septal wall ceases [204]. An alternative form of alveolar septation to that at the time of birth is indicated by the finding that septation of the number of primary saccules present at birth accountsonly for approximately one third of the number of alveoli present at postnatal day 6 and approximately one quarter by day 14 [206, 214, 215]. This discrepancy, and the presence of alveoli
The main features that define the changing vascular template in the normal human lung are the number, diameter, and wall structure of the preacinar and intra-acinar vessels [9, 10, 13, 14, 16, 20, 27, 78, 153, 203, 218–223].
3.1 Fetal Lung Vascular networks begin to form at the onset of organogenesis in the fourth week of gestation: the central pre- and post- capillary vessels arise as outgrowths from the great vessels of the heart, the pre-capillary vessels being derived from the fourth aortic arches, and the post-capillary vessels from the primitive atrium. Distal blood lakes appear, in advance of central vascular branches being established, as early as the sixth week (32–44 days) of gestation in the primitive mesenchyme surrounding the lung bud at the neck, and by the seventh week (50.5 days) they are abundant in the sub-pleural mesenchyme [223]. The adult pattern of central vascular and airway structures, consisting of lobar and segmental branches, is present by the sixth week of gestation (42 days) [9, 10, 15, 218, 224– 227]. At this stage the pulmonary artery accompanies airways only to the third or fourth generation [223]. By the eighth week (about 56.5 days), the branching pulmonary artery, a thickwalled blind-ended tube, lags behind the branching airway by two to three generations. Later, at 12–14 weeks, an extensive
3 Pulmonary Vascular Development
capillary network now surrounds the distal airway buds, although they are separated from the buds by subpleural mesenchyme. By the 20th week, the full number of preacinar pulmonary vessels has formed in each segment. By 22–23 weeks, the capillary network closely approaches alveolar epithelium, and the pulmonary artery accompanies each airway branch. In their study, deMello and Reid [223] reported the central vessels of the human lung forming by angiogenesis, and the peripheral vascular network by vasculogenesis (as described in their earlier study of vascular development in mouse lung, see Section 2). The first connection between developing distal and proximal networks appears between peripheral lakes and a thin-walled hilar vein, the venous drainage system of the lung thus being established before the pulmonary artery. Studies of insulin-like growth factor expression (a potent angiogenic factor), demonstrating distinct populations of endothelial cells that arise from diverse genetic lineages in the human lung, support the concept of vascular development occurring by both angiogenesis and vasculogenesis [228]. The additional concept that all pulmonary arteries and veins in the human lung arise by vasculogenesis, however, is further proposed from detailed analyses of the lung’s developing vascular (arterial and venous) beds [153, 222]. Although vessel size, length, and diameter increase in the fetal lung, density does not change [9, 10, 16]. Since vessels at the hilum grow faster than ones at the periphery, the gradient of vessel diameter relative to length increases with fetal age. As each airway develops in the fetal lung so do the accompanying conventional arteries with supernumerary arteries. From 18 weeks of gestation onward, the numbers of airways and conventional artery branches are similar, and numerous supernumerary arteries are present [9, 16] (Fig. 15). The ratio of supernumerary to conventional arteries present along the pathway is the same as in the adult (even before airway branching is complete). Analysis of the branching pattern of a lateral pathway in fetal lung (the seventh lateral branch of the axial pathway illustrated in Fig. 15) shows 11 airway generations accompanied by 11 conventional arteries and 40 supernumerary arteries [9]. Additional measurements along the posterior basal segment of a right lung at 40 weeks of age confirmed the same patterns described for the left lung, indicating a similar arterial branching pattern throughout the fetal lung [9]. Conventional and supernumerary veins first appear at a time similar to that for the arteries, developing in a progressive fashion from the hilum to the lung periphery. From 20 weeks of gestation, the number of preacinar conventional veins is within the adult range for the intrasegmental airway number. Although the number of conventional veins in a segment equals that of the airways and conventional arteries, there are more supernumerary veins than supernumerary arteries [10]. By 28 weeks of gestation, a difference is evident in the arrangement of the supernumerary veins: type 1
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Fig. 15 Distribution of arteries (conventional and supernumerary) by size in fetal lung. Conventional and supernumerary artery branches are shown by diameter, relative to the diameter of the axial arterial pathway from which they arise (see the black line), in a fetus (19 weeks gestation). In this (and subsequent images) each branch is seen as a vertical line whose height represents the diameter. From the lobar hilum to the terminal bronchiolus, the 25 airway generations are accompanied by 21 conventional arteries, and there are 58 supernumerary arteries. For clarity, the first eight conventional and 11 supernumerary arteries are shown separately (top image) from the remaining conventional and supernumerary ones (lower image). The asterisk indicates the point of overlap between the top and lower image. Note the change in scale of the vertical axis. At the hilum, the diameter of the main pathway is 1,300 mm. Conventional arteries have a mean diameter of 282.7 mm, and the supernumerary arteries are smaller, with a mean diameter of 62.6 mm. (Reprinted from Hislop and Reid [9] with permission from WileyBlackwell)
supernumerary veins drain lung tissue immediately surrounding an axial vein and receive only occasional tributaries whereas the larger type 11 supernumerary veins drain the more distal capillary bed and are joined by several postcapillary tributaries [10]. Reconstruction of the same pathway in the lung up to 38 weeks of age shows that the branching patterns are more or less the same to the level of the terminal bronchiolus, and are as present in the adult lung. However, many small arteries (and veins) develop late in fetal life and continue to increase at the time of birth – their density (per unit area of lung) increasing as the respiratory region of the lung develops with the appearance of respiratory bronchioli and saccules (Fig. 16). An elastic wall structure extends to the seventh generation along the axial arterial pathway by 19 weeks of gestation [9]; from here on, an elastic structure is present to the same level
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Fig. 16 The acinus at six stages of development. At all ages, airway generations are drawn the same length so that increase in length represents an increase in generations. A given generation may be traced vertically, permitting its structural remodeling to be followed. The actual increase in size is shown by the length from the terminal bronchiolus to the pleura. Note that the changing length of the terminal bronchiolus (the last airway segment that does not give rise to alveoli) is not drawn to scale but is noted numerically. At 16 weeks gestation, the airways end in a tubule close to the pleura. By 19 weeks gestation, the epithelium in the last generation of the airways has thinned and now forms the first respiratory bronchiolus: branching has led to a second-generation respiratory bronchiolus. By 28 weeks gestation, further branching has given rise to a total of three generations of respiratory bronchioli and one generation of transitional ducts, the latter giving rise to primitive saccules. By birth, the number of generations of saccules has increased to three, the last forming the terminal saccule. No true alveoli are present at this time although a few indentations representing future alveoli appeared just before birth. By 2 months,
alveoli have developed from the walls of the respiratory bronchioli, from transitional ducts and from saccules, which transforms these last two structures into alveolar ducts. Alveoli also open into the terminal saccules. By 6 years, several changes have occurred – the remodeling of respiratory bronchioli and alveolar ducts may occur in several ways: along some pathways a bronchiolus may transform into an extra generation of respiratory bronchioli by centripetal alveolarization; distal respiratory bronchioli may transform into alveolar ducts; further branching of another one or two alveolar duct generations may occur; or there may be no change. Certainty for some of the above changes is not possible because of variation between acini and cases. At the distal end, however, the terminal sac has probably transformed into the adult atrium, a short, wide passage lined by alveoli, which has given rise to a number of alveolar sacs, formed by budding rather than branching, and each lined by alveoli. This pattern is similar to that seen in the adult: there is probably little further development save an increase in size. (Reproduced from Hislop and Reid [227] with permission from BJM Publishing Group Ltd)
in both the axial and the conventional arteries. These are considered supportive arteries in the fetal lung because of their high tensile strength and ability to maintain vessel patency. The level at which a transitional structure gives way to a muscular structure (Fig. 4a) is established midway through gestation, and the point where a muscular structure gives way to partially muscular and/or non-muscular structures occurs in vessels with a similar diameter throughout fetal life.
Calculation of the percent wall thickness reveals little change with increase in diameter in larger arteries, and that in smaller arteries the percent wall thickness increases rapidly as the diameter decreases, the most rapid increase occurring in arteries less than 200 mm in diameter (Fig. 17). Phenotypically distinct populations of SMCs in the wall of large developing pulmonary arteries raise the possibility of different lineages:[229]: when present in the fetal lung, the SMCs are
3 Pulmonary Vascular Development
Fig. 17 Percent arterial medial thickness at different developmental stages. The percent medial thickness related to the external diameter of arteries (ranging in size from below 200 mm to more than 600 mm) is illustrated for the fetus, a 3-day old infant, and a 4-month old child to adulthood. In the 3-day old infant, arteries over 200 mm in diameter still show the (thicker) fetal pattern of medial thickness, whereas those less than 200 mm in diameter show the (thinner) childhood and adult state. (Reprinted from Hislop and Reid [16], copyright 1981, with permission from Elsevier)
immature. The pulmonary veins are relatively free of muscle: at 20 weeks of gestation, even the largest veins lack muscle, and although muscle fibers are identifiable within vein walls by 28 weeks, these form a continuous muscle layer only at term [10, 16]. At this time, SMCs extend into veins as small as 80 mm but unlike the arrangement in the arteries they are thin-walled and no elastic laminae are present [10, 16]. In a population count of vessels in the normal human lung at term, most arteries above 150 mm in diameter have a muscular wall, and most ranging from approximately 90 mm to approximately 75 mm in diameter have a partially muscular wall. Arteries with a non-muscular wall overlap in size with partially muscular arteries (Fig. 18); below 37 mm, all arteries are non-muscular. Throughout fetal life the shift from muscular arteries to a structurally mixed arterial population occurs in the pre-acinar region (proximal to the terminal bronchiolus). The relationship between vessel size and wall structure is similar at all fetal ages [9]. Although the smallest muscular artery, and the range for partially muscular and non-muscular arteries by size, is the same in the fetal and the adult lung, the wall thickness of an artery (of given diameter) is twice as thick in the fetus as in the adult (Fig. 17) Even at birth, however, little muscle is found in the wall of arteries beyond the terminal bronchiolus (Fig. 19). Thus, the fetal lung has a more muscular pulmonary circulation than the adult lung if judged by wall thickness of an artery of given size, but it is less muscular when judged by the presence of muscle in arterial walls, at any level in the branching pattern within the acinus [9]. A striking finding, within hours of birth, is the increased compliance (by wall thinning) of the smallest muscular resistance arteries [11, 218, 230] – associated with a fall in resistance at this time – as the lung transitions from a low-flow/high pressure system to a high flow/low pressure system.
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Fig. 18 Population distribution (count) of vessels in neonatal lung identified by size and wall structure. The percentage of the total number of vessel profiles with a given structure (muscular, partially muscular, or non-muscular) in the human lung at 40 weeks of age is shown for size ranges between 25 and 200 mm in diameter. Smooth muscle is seen to develop in vessel walls in relation to vessel size. In all artery profiles close to 200 mm in diameter, the wall is muscular; below 200 mm, partially muscular artery profiles appear and increase until, at about 100– 75 mm in diameter, they form 68% of the artery population. Below 37 mm all artery profiles are non-muscular, and these then range in size between 125 and 100 mm. (Reprinted from Hislop and Reid [9] with permission from Wiley-Blackwell)
Fig. 19 Extension of arteries with smooth muscle in their wall with age (in the fetus, child, and adult). In the human fetus no arteries with smooth muscle are seen within the acinus (i.e., distal to the terminal bronchiolus). The arteries with smooth muscle extend gradually into the acinus with age, but even by 11 years they are not present within the alveolar wall: only in the adult (19 years) is smooth muscle present in arteries within the alveolar wall. (Reproduced from Hislop and Reid [14] with permission from BJM Publishing Group Ltd)
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3.2 Neonatal and Child Lung At 36 weeks to term, the preacinar pattern of arteries and veins is complete [9, 10]. The lung can now support air breathing but, in terms of its structure, as Reid elegantly described in her study, it is not the adult lung in miniature since, along with need for central vessel growth, its alveolar region is still being formed [231]. At this stage the alveolar region has four generations of respiratory bronchioli and two alveolar ducts proximal to the saccules [226]. It must evolve from this small simple structure into an efficient gas-exchange surface to match functional needs. It responds by a burst of vascular growth: existing vascular units continue to expand in diameter and length and many new intra-acinar units are added as the gas-exchange surface forms [9, 16–18, 227]. From arteriograms of the human lung (between 35 and 48 weeks of gestational age), and the analysis of tissue sections, Frey et al. [232] determined arterial diameter as a function of generation number, and showed that the branching properties of pulmonary arteries in the pre- and post-natal phases of human lung development are unchanged (the dimensions between mother and daughter branches being relatively constant from generation to generation). This demonstration of relatively constant branching ratios is consistent with the fractal pattern/properties of the pulmonary vascular tree [232]. As intra-acinar arterial structures grow and multiply, alveoli develop. Alveoli increase in number but become smaller
Fig. 20 Distribution of arteries (conventional and supernumerary) by size, relative to the diameter of the axial arterial pathway from which they arise (see the black line) in the infant and child lung. (a) In the pre- and intra-lobular arteries of a 4-month old child, the horizontal axis represents the last 5.1 mm of an axial pathway. The acinus is small with only five conventional and six supernumerary arteries. (b) In the pre- and intra-lobular arteries of a 5-year old child, the horizontal axis represents the last 12.6 mm of an axial pathway. The length of the acinus and lobule has increased with age as have the number of conventional and supernumerary arteries (especially the latter). All vessels have also increased in size with age. (Reproduced from Hislop and Reid [14] with permission from BJM Publishing Group Ltd)
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in size as newly forming alveoli divide existing structures. The greatest increase in alveolar number by multiplication occurs between 4 months and 3 years of age. Later, between 4 and 10 years of age, as multiplication slows, alveoli increase in size. In the first 4 months of postnatal life, the number of arteries per unit area of lung, and the density of capillary networks, increase [203]. Within the acinus, an increase in the diameter of the proximal vessels compared with distal ones reflects the burst of small vessels developing at the periphery. After 18 months, the number of new vessels forming slows along with alveolar growth [9, 16]. Between 4 months and 4 years, the number of intra-acinar arteries (up to about 200 mm in diameter) increases per unit area of lung. Although the ratio of their number to the number of alveoli remains similar throughout childhood, their concentration per unit area of lung falls after 5 years of age as alveoli increase in size and separate adjacent arteries [14, 16]. The veins grow at the same time as airways and arteries and, whereas the preacinar drainage pattern is complete halfway through fetal life, the intraacinar venous pattern develops during childhood. Reconstruction of the venous branching system in the lungs of a 3-year-old and a 10-year-old child identifies distal veins arising from the saccular respiratory region [10]. Arteries and veins develop alongside new airway outgrowths in the postnatal lung, with both conventional and supernumerary vessels continuing to appear (Figs. 20, 21). Conventional arteries accompany new airway branches up to
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Fig. 21 Distribution of veins (conventional and supernumerary) by size, relative to the diameter of the axial vein from which they arise (see the black line) in the lung of a 3-year old child. The drainage pattern at the lung periphery is illustrated. The supernumerary veins
18 months of age; supernumerary arteries reach the adult number by 5 years of age, and new ones continue to form up to 8 years [14, 16] (Fig. 20). At all ages the conventional arteries are smaller than the parent artery at their point of origin. The mean diameter of these vessels, and that of the supernumeraries, increases with age. Overall, supernumeraries are smaller in diameter than their adjacent conventional arteries [14]. Conventional and supernumerary venous tributaries are found along the length of the axial vein. Conventional veins run in their own connective tissue sheath, enter the axial vein at an acute angle, are of size similar to that of the axial vein, and lie at some distance from the capillary bed they drain [10]. Some supernumerary veins drain the lung tissue immediately around the axial vein (as in the fetus), having no collagen sheath but passing directly through the main vein sheath to the axial vein; others receive postcapillary tributaries and are surrounded by a collagenous sheath continuous with the sheath of the axial vein [10]. Along the axial pathway, the conventional veins are equivalent in number to airway generations and the arteries accompanying them. Both conventional and supernumerary veins become more frequent toward the periphery (Fig. 21). At birth, as in the fetal lung, the percent wall thickness of arteries is still greater overall than later in childhood [14]. After 4 months of age, the wall thickness of larger vessels falls to match that in the adult lung and increases in smaller ones (less than 200 mm in diameter). During childhood, the number of large arteries with an elastic or transitional wall remains constant. Between 4 and 10 months, as vessels increase in size, muscle development lags. Although the wall structure along any axial artery pathway is established midway through fetal life, the distribution of intra-acinar vessels shifts significantly in childhood, more partially muscular and non-muscular arteries than muscular ones now being present than in the fetal or the adult lung [14] (Fig. 22). By 3 days after birth, the distribution of arteries by size and wall structure is similar to that at birth, partially muscular and non-muscular
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include two types: type I and type II (the type II veins being larger on average than the type I veins). The actual length of the pathway is approximately 11 mm. (Reproduced from Hislop and Reid [10] with permission from BJM Publishing Group Ltd)
Fig. 22 Population distribution of artery profiles by size and wall structure (muscular, partially muscular, and non-muscular) in (a) the normal newborn lung and (b) the lung of an 11-year old child. Partially and nonmuscular structures are now present in larger arteries. (a Reprinted from Kitagawa et al. [285] with permission from Wiley-Blackwell. (b) Reproduced from Davies and Reid [12] with permission from BJM Publishing Group Ltd)
arteries being as large as 180 and 150 mm in diameter and ones below 40 mm in diameter are non-muscular; indicating
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that muscle growth keeps pace with increase in artery size at this time. The distribution of muscle is then similar in the lung at 18 months of age and at 3, 4, 5, and 11 years of age [14], the largest partially muscular artery being 300 mm and the largest non-muscular one 245 mm. Muscular arteries smaller than 185 mm in diameter were not found. In another lung, at 10 years of age the distribution pattern was similar to that of the adult, indicating a watershed in wall maturation by this time, alongside some degree of individual variation. Muscle gradually extends into arteries within the acinus (to the level of respiratory bronchioli and alveolar ducts) but not distally into the arteries within alveolar walls (Fig. 19). The presence of a high population of thin-walled intra-acinar arteries may provide children with an advantage, since ones as large as 100 mm (or 200 mm) in diameter (with a capillarylike wall structure) can contribute to oxygen transfer [14]. The wall structure of the veins is more developed in the child lung than in the fetal lung: whereas the walls of small veins still consist only of endothelium, some of the larger veins have SMCs [10].
3.3 Adult Lung In 1965, Elliott and Reid [27] provided an account of the pulmonary artery branching pattern in the normal human adult lung. In the lungs studied, they obtained arteriograms and identified arterial branches in tissue sections. They described the distribution of elastic and muscular arteries, relating these by their wall structure and size to their location within the pulmonary arterial tree, and determined the relationship of the arterial branching pattern to that of the bronchi. From this study it emerged that the pulmonary artery is a more muscular vessel than had been previously supposed and a striking divergence first appeared between the branching patterns of the arterial and bronchial trees on the basis of the presence of extra (supernumerary) branches with no counterpart in the bronchial tree (see Sect. 1) [27]. The pattern was defined on the arteriogram (Fig. 4a), or in serially prepared tissue sections, by counting the number of branches as generations – which may also be determined by order [27, 233, 234]. A useful convention is to consider a segmental airway as the first generation (the trachea and lobar branches are counted separately). Using this approach, the inferior lingular segment has about 28 generations and the apical lower lobes as few as 15. In adolescence, new vessels continue to form from preexisting ones by angiogenesis, in line with lung growth. Both the absolute and the relative number of supernumerary arteries remain more numerous than conventional ones (Fig. 23): 3 times as numerous as conventional artery branches, the supernumerary arteries at all levels along an axial pathway remain smaller in size than
Fig. 23 Distribution of pre-lobular and intra-lobular (conventional and supernumerary) arteries in adult lung. A pulmonary artery from the segmental hilum to the pleura, with conventional and supernumerary side branches included down to the start of the lobule is illustrated, and the conventional arteries thereafter. The arterial pathway shown here is the same as that illustrated in Fig. 4a. The 15 conventional arteries were numerically designated by reference to the bronchial generation. This part of the artery was 5 cm in length. (Reprinted from Elliott and Reid [27], copyright 1965, with permission from Elsevier)
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their adjacent conventional branches. They comprise one quarter to one third of the potential cross-sectional area of the branch vessels. The conventional veins are larger than the supernumerary veins at any level and arise from a region of division of an airway. They drain the alveolar region immediately adjacent to the venous pathway. In the adult lung, all preacinar arteries have a complete muscle coat and several elastic laminae. The elastic wall of the main pulmonary artery at the lung hilum has a medial muscle layer between internal and external elastic laminae, and seven or more central elastic laminae. Subsequently, it then has four to seven additional central laminae in its transitional segment [27]. The elastic arteries of the adult lung are more than 3,200 mm in diameter, transitional ones are 3,200–2,000 mm in diameter. An elastic or transitional wall structure extends approximately halfway along an axial pathway to about the ninth airway generation. Distal to this, in arteries 2,000 mm or less in diameter, the wall then changes to a muscular structure with a medial muscle layer between internal and external laminae with four or fewer central laminae: almost all lateral branches of the distal axial pathways have a muscular wall. SMCs finally extend into the most distal arteries in alveolar walls (Fig. 16). A population distribution shows that all arteries greater than 150 mm in diameter are muscular; that partially muscular ones are most frequent in the 40–80-mm size range, and that non-muscular ones appear just below 130 mm, increasing in frequency and representing most of the vessel population below 50 mm (Fig. 24). Arteries as small as 30 mm in diameter, however, may have a muscular wall, and even smaller ones approaching the capillary bed can have a single SMC in their wall (Fig. 19). The wall is thickest in muscular arteries that are 200–500 mm in diameter and found immediately pre-acinar or proximally within the acinus, i.e., the
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resistance arteries of the adult lung. The structural features of the intra-acinar pulmonary veins are similar to those of the pulmonary arteries – first a non-muscular segment, then a segment with a spiral of muscle before the muscle coat becomes complete as wall thickness and diameter increase. The large elastic and transitional arteries of the aged lung have fewer elastic fibers than ones in the young adult [219, 220]. This is associated with a series of changes: vessel narrowing, caused by intimal and medial wall cells encroaching on the lumen [219, 220], alveolar wall thinning resulting in large and relatively simple alveolar structures, and an overall fall in lung elastic recoil [219, 235]. The intima of large elastic and transitional arteries may be thickened by acellular deposits, although large muscular arteries appear free of these, and hyaline deposits develop in the walls of small vessels less than 150 mm in diameter [219]. The medial thickness of vessels in all size ranges is greater in the aged lung. Most are twice as thick as in the normal young adult lung, and the increase is nearly threefold in ones approximately 3,000 mm in diameter [219]. The range for wall thickness, narrow in the young adult lung, is wide in the aged lung. The wall thickness of pulmonary arteries is greater than in the young adult lung as is the increase in the wall thickness of resistance arteries. When the number of branches arising from the axial pulmonary artery was counted on arteriograms, and the diameter of successive branches to within 0.5 cm of the pleura and the distance between them were measured, no difference was found between the aged and the normal young lung. The axial pathways appear clearer in the aged lung, however, possibly because of a fall in the number of small blood vessels and capillaries [20].
4 Compromised Lung Vascular Development
Fig. 24 Population distribution of vessels by size and wall structure in the adult lung. In a typical pattern, as vessels increase in size the relative number of muscular artery profiles increases: the relative number of nonmuscular arteries decreases, and most partially muscular ones are found in the mid-size range. (Reprinted with permission from Reid [20])
The template for vascular development, growth, and maturation in the normal lung is altered by failed gene–growth factor interactions and by hormones, vitamins, drugs, toxins, radiation, and other factors. The effect of any given agent, and subsequent viability, will depend on when vascular growth is interrupted, be it during organogenesis and cell specification, during the formation of preacinar or intra-acinar vessels, or as the alveolar region forms. It will further depend on the changes in the cascade of signaling pathways regulating lung tissue organization via cell proliferation, migration, differentiation, and survival. The ability to recover from compromises that permanently alter developmental processes and vascular architecture is minimal such that the earlier the challenge to growth the greater the potential for abnormal development. Dysanaptic (unequal) growth
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describes the pattern in the lung whereby the subsequent growth potential of the central conducting airways and vessels is limited compared with that of the acinar region as, for example, during compensatory growth in the postnatal lung [236–238]. Even when the developmental program is normal in relation to its various stages, restricted fetal growth, or normal fetal growth but premature birth, will result in a small and immature lung in terms of cell specialization and differentiation. This increases the risk of respiratory deficiency and postnatal problems [239]. Important examples of factors associated with a compromised intrauterine environment that restrict fetal development and growth include fetal hypoxemia, reduced substrate supply, and hypercortisolemia [240, 241]. Postnatally, the transition from placental to lung-based respiration at birth, i.e., from a hypoxic environment to a relatively hyperoxic one, exposes the perinatal lung to oxidative stress. Surfactant, enzyme, and nonenzymatic antioxidant syntheses normally match each other to protect the lung during this transition. Factors that especially affect the perinatal lung as it undergoes extensive remodeling during alveolar formation and growth include a high or low environmental oxygen tension, as well as interactions between the oxygen tension and genes regulating growth factors, such as VEGF. Dynamic change in the inspired oxygen levels represents a potential signaling mechanism for the expression, and activation, of redoxsensitive transcription factors, cell fate (apoptosis), and downstream proinflammatory cytokine signaling. Redoxsensitive transcription factors are likely differently regulated by oxygen availability to bind specific DNA consensus sequences and activate expression of genes controlling adaptive vascular homeostasis at the time of birth. Hence, both temporal and spatial variance in oxygen abundance will determine lung cell survival or apoptosis via oxygendependent activation of cell regulators and genes.
4.1 Failure to Develop the Normal Quota of Vascular Units In the following conditions, adaptation to disturbance in the lung’s growth pattern is associated with relatively normal function and good prognosis, although under certain circumstances, as in environmental change or in disease, reduced pulmonary reserve becomes apparent.
4.1.1 Single Lung in Agenesis Interference at the earliest stage of the development program results in a single airway branch that gives rise to a single
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lung which typically grows to fill both thoracic cavities. Quantitative analysis of one such lung in a 3-month old infant confirms that the vascular, mesenchymal, and epithelial components produce twice the normal capillary and alveolar quota [242]. Capillary and alveolar multiplication in response to the increased available thoracic space for the single lung thus results in an alveolar number equivalent to that of two lungs. This pattern is consistent with normal lung function.
4.1.2 Congenital Diaphragmatic Hernia and Lung Repair In congenital diaphragmatic hernia (CDH), reduction of the thoracic space by the upward movement of abdominal contents reduces lung volume and the number of airway branches. This pattern of hypoplastic lung development is based predominantly on the mechanical constraints imposed by lack of space. The gestational age at which the hernia is produced determines the pattern of disturbance in lung and vascular growth [243, 244]. Lung vessels are remodeled to reflect the reduced volume receiving blood supply as well as the reduced number of airways and alveoli. Wall thickening by SMCs occurs in arteries in all size ranges and muscle extends into more peripheral vessels than normal. The prognosis of infants with CDH at birth is strongly influenced by impaired vascular development [245, 246]. After CDH repair, lung volume typically increases to fill the available thoracic space, alveolar size becomes abnormally large, but airway, alveolar, and vascular multiplication do not catch up. A gradient of hypoplasia is typically apparent, the ipsilateral lung (particularly its lower lobe) being most compressed. At least during adolescence, lung function is satisfactory, although physiology studies detect reduced function [247].
4.1.3 Musculoskeletal Disorders In certain musculoskeletal disorders, lung growth is impaired metabolically or mechanically [248]. In some, the metabolic disturbance impairing cartilage and bone development also affects tissues needed for lung growth; in others, a reduced thoracic space restricts lung growth and airway development [249]. For example, in osteogenesis imperfecta, lung volume and airway branching are reduced because of reduced thoracic volume and the arteries are crowded and abnormally large, whereas in camptomelic dwarfism the thorax is small, as is lung volume, but airway and arterial size and branching are more appropriate to volume, suggesting that a metabolic effect is less important [249]. A dissociation between thorax size and lung growth, in the absence of metabolic impairment,
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is illustrated by a case of Jeune’s syndrome (asphyxiating thoracic dystrophy), a rare inherited malformation characterized by disturbance in utero of bone formation, in which the thorax fails to grow, but the lungs grow to normal size by displacing the diaphragm [250]. The small arteries of this lung had remodeled structurally at the time of death (from hypoxia), and although the lung was normal in volume it was abnormal in shape as it conformed to the abnormal thoracic contours.
4.2 Severe Impairment of Alveolar Growth and Abnormal Vascular Growth: Alveolar–Capillary Dysplasia Here the blood–gas barrier of the lung fails to form, leading to the development of severe respiratory distress shortly after birth. Parenchymal and vascular abnormalities are the basis as discussed by Galambos and deMello: typically, dysplastic thin-walled vessels, somewhat larger than capillaries, form centrally rather than abutting the edge of the airspace, resulting in absence of the normal interface between cells forming the gas-exchange surface [76, 251]. The arteries upstream have thick walls, similar to ones in persistent pulmonary hypertension of the newborn (PPHN). These changes are associated with veins that are misaligned, developing centrally within the lobule (alongside the bronchoarterial sheath) rather than at its periphery (within the interlobular septa). Their presence occurs not only in alveolar–capillary dysplasia, however, but also in other conditions such as alveolar proteinosis and congenital pulmonary venous obstruction [76, 251]. These changes in neonates are rarely compatible with survival of more than a few months, even with supportive measures. Why normal alveolar epithelial and capillary endothelial cell fusion events fail is not known. Galambos and deMello [76, 251] pointed out that, given the importance of VEGF signaling [109], alteration in this signaling pathway may be a causative factor in the development of alveolar–capillary dysplasia, the similar impairment in endothelial nitric oxide synthase (eNOS)deficient mice, and mice administered eNOS inhibitors, reflecting a link between eNOS and VEGF (nitric oxide synthase being an upstream activator, and a downstream target, of VEGF). Of note, in congenital acinar aplasia, a form of hypoplasia that stems from a failure to form saccular structures and alveoli (and thus not compatible with survival beyond a few days), extensive capillary networks develop in the lung despite the lack of respiratory/alveolar surface [76, 251]. It may be that a selective reduction in TGF-b, as shown in a study of this condition, impairs epithelial–mesenchymal cell interactions (in the pseudoglandular phase) [251].
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4.3 Vascular Restriction and Wall Remodeling in Developing Vascular Units: Persistent Pulmonary Hypertension of the Newborn The hypertensive state of the pulmonary vascular bed in utero diverts blood from the lung to the placenta. At birth, the pulmonary vascular bed adapts and pulmonary pressure and resistance fall rapidly as arteries dilate (and stretch) and vascular compliance increases [11, 230]. In PPHN this adaptation fails, blood is diverted (via the foramen ovale and ductus arteriosus) to the aorta, and a high pressure persists. Abnormal vascular development, or the remodeling of vascular structures, before birth is the basis of different forms of PPHN [252, 253], which are characterized by (a) dysplasia – abnormal muscularization of intra-acinar arteries associated with increased adventitial collagen formation, and lumen narrowing in preacinar arteries, (b) hypoplasia – the development of too few vessels, and/or vessels that are too small and with abnormally muscular walls, and (c) maladaptation – the failure of small vessels to adapt perinatally [253]. In the idiopathic form, the airways and alveoli develop normally but large numbers of SMCs occur in small arteries, extending to include ones in the alveolar wall [254, 255]., A possible cause of this has been suggested to be relative hypoxia in utero or premature closure of the ductus; however, this currently remains undetermined. The vascular remodeling present in idiopathic PPHN was also found in the lungs of infants with meconium aspiration syndrome (considered a marker rather than a cause of PPHN), who died in the first days of life [256]. In infants with high flow to the pulmonary circulation prior to birth, as in total anomalous pulmonary venous return, and obstructive lesions to the left side of the heart [aortic atresia, aortic stenosis, coarctation of the aorta associated with ventricular septal defect (VSD), and patent ductus arteriosus], similar vascular changes occur and the structural changes are considered secondary to hemodynamic changes [257–260]. In these infants the vein walls are also abnormally thickened by SMCs. Similarly, high pulmonary blood flow before birth resulting from pulmonary (or systemic) arteriovenous malformations, or fistulae, produces severe patterns of arterial wall muscularization. Hypoplasia represents underdevelopment of the lung and is commonly associated with increased muscularization of pulmonary arteries. In the hypoplastic lung that forms in renal agenesis, the cause is metabolic rather than mechanical (as in CDH, for example): pre-acinar arteries and airways are reduced in number, and the alveolar number and size are also reduced [261]. The arteries, although small for their age, are disproportionately large for the lung volume, and more peripheral arteries than normal are muscularized [261]. In pulmonary hypoplasia resulting from phrenic nerve agenesis and diaphragmatic amyoplasia, excessive muscularization
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occurs with typically no change in alveolar and airway development [262]. Although the evidence is indirect, infants with VSD and other congenital heart conditions initially adapt in the perinatal stage, but as lung blood flow increases (with right-to-left shunt), pulmonary hypertension develops. Repair of the heart defect before the age of 2 years results in no residual pulmonary hypertension. In a small group of infants with VSD, however, death occurs in the first year. Their lungs typically have resistance arteries that are small and more thick-walled than in the fetus, indicating impaired or failed adjustment to increased flow in the perinatal period.
4.4 Bronchopulmonary Dysplasia In neonates who encounter significant respiratory distress at the time of birth, distal airways may be disrupted and alveolarization impaired, leading to bronchopulmonary dysplasia [65, 230, 249, 263–266]. The importance of impaired vascular growth is also currently recognized. The earlier the interruption to the existing developmental program, the greater the potential for impaired development or growth. Infants with severe bronchopulmonary dysplasia develop pulmonary hypertension [264] likely based on distal vascular restriction. Restricted vascular and alveolar development, including capillaries and small vessels developing in the forming alveolar–capillary membrane of a preterm lung or term lung at birth, may be exacerbated by administration of supplemental/iatrogenic oxygen. Supplemental (high) oxygen may impair capillary formation (via reduced VEGF signaling) and so alter normal endothelial–epithelial-driven patterns of alveolar formation (see also Chapter 42) [65]. Future function and vascular growth depend on whether catch-up growth is possible.
4.5 Induced Postnatal Growth How to induce, or promote, catch-up growth in the still developing lung and how to induce regenerative growth in the adult lung are important questions. Optimally, the degree of growth will be sufficient to normalize the lung’s vascular system(s). In the developing lung, the question then becomes whether, after correction, appropriate developmental growth and maturation continue to proceed. In a comprehensive review of the mechanisms and limits of induced postnatal growth published by the American Thoracic Society [86], future directions to address the need for growth induction were reviewed. Potential models for stimulated lung growth were discussed in relation to increased alveolarization and vascularization, including the effects of increased lung
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etabolic oxygen demand, mechanical strain, reduced-size m lung transplant, chronic hypoxia or high-altitude exposure, and elastic fiber formation. Induced lung growth in response to mechanical signals (septal strain or strain-induced signal transduction) and nonmechanical signals and mediators (hormones, growth factors, retinoids, and hypoxia) were also considered and the limits imposed by the presence of dysanaptic lung growth carefully set out. The final section of the review emphasized the following key issues pertaining to future directions: (a) the need for studies to define similarities and differences between types of lung growth, since the developing lung differs from the adult (non-growing) lung in mechanical stresses, hormonal/growth factor levels, and cell–cell (epithelial–mesenchymal–endothelial) interactions; (b) the need to develop models of mechanical loading on alveolar capillary growth and remodeling, and to isolate and characterize progenitor/precursor cell subpopulations in the lung; (c) the need to understand issues relating to the regulation of alveolar–capillary architecture, and how septal growth is eventually limited; and (d) the need to understand and develop ways to optimally match alveolar to capillary growth. This review is recommended for a greater understanding of some of the important issues surrounding the question of induced and regenerative lung growth [86]. Based on medical imaging techniques, computational modeling studies of the (adult) human arterial and venous tree, including its supernumerary vessels, are currently of interest as part of a renewed effort to relate vessel branching structure to blood flow distribution [267]. Accurate determination of the spatial relationships between airways, vessels, and supporting tissues in these imaging-derived models allows regional interactions within the lung to be determined [268]. This holds significant potential for differentiating normal and disease-related heterogeneity in regional blood flow [268] and for modeling of a virtual lung [269]. The additional aim of these multiscale modeling studies is to provide an effective bridge between the cellular systems of the lung and whole organ function [269]. This approach may offer additional insight into the vascular template(s) required for lung regenerative studies, and serve as a future determinant of their functional success. Although lung or lobar transplant offers hope for patients with dysfunctional lungs from impaired vascular (and/or airway) growth, the constraints determined by the availability of the organ donor pool often restrict this approach [86]. Alternative approaches focus on methods of lung tissue regeneration, and on correction of vascular growth in the postnatal or adult lung by administration (infusion) of progenitor/precursor cells [85, 86]. Here, outstanding questions focus on (a) the need to generate different vascular cell phenotype(s), including the possibility of cells, e.g., endothelial cells, with a functional memory [81], (b) how to achieve the organization of different cell types into tissue structures appropriate to
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proximal or distal regions of the vascular bed, and (c) the need for an understanding of signaling pathways responsible for the modulation of these cells. Clearly, more than one cell type, and the interaction of several signaling systems, will likely be needed to replicate the exquisite coordination evident in lung vascular development and growth [85, 86] but current small steps in this direction offer the potential for future success.
5 Summary of Normal Pulmonary Vascular Development 1. Transcription and growth factors regulate normal pulmonary vascular development in temporal–spatial patterns to achieve the appropriate distribution of vessels, by size, location, and wall structure, for the age and stage of lung growth. 2. Organogenesis begins as the lung anlage arises from the developing foregut; a single airway tube (trachea) develops and buds into the surrounding mesenchyme to form primitive branching airway structures appropriate to a right and left lung; the lung anlage is vascularized by a vascular plexus derived from the heart/aortic sac and dorsal aorta. 3. Pulmonary arteries and veins continue to develop progressively in the central zone, and form from cells within the mesenchyme surrounding the developing airway buds. 4. Central arteries develop in concert with the airways; the central veins develop (midway) between the forming bronchoarterial structures. 5. Distal vascular structures likely develop into networks independently from the central vessels and connect to them in the mid-fetal stage. 6. The venous system is established (functionally) before the lung’s arterial system. 7. Developing central (conventional) arteries branch with airways, and additional (supernumerary) arterial branches form; similar (conventional and supernumerary) venous branches develop. 8. Vascular (arterial and venous) wall structure develops as perivascular cells in the mesenchyme give rise to (medial) SMCs and (adventitial) fibroblasts that surround the endothelial cell networks. 9. Along vascular pathways (from the lung hilum to periphery) wall thickness relates to vessel diameter, save in small intra-acinar (resistance) arteries where thickness is high: in the newborn lung, these arteries are preacinar. 10. Distal to intra-acinar resistance arteries, precapillary arterial and postcapillary venous units (in the alveolar wall) include a thin-walled muscular segment, a partially muscular segment (with muscle spiral), and a non-
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muscular segment larger in diameter than a capillary but with a similar wall structure; intermediate cells and pericytes represent precursor SMC types in distal (nonmuscular intra-acinar) wall regions. 11. Pulmonary vascular growth before and after birth occurs by an increase in artery and vein size, the number of units, and wall maturation: after birth, this growth occurs during modification to the lung’s alveolar template, and continues by expansion. 12. The overall vascular arrangement described above is similar in all mammalian lungs but vessel size and wall structure, and the position within the branching pattern, are typical within a species and for a given stage of development. Temporal–spatial stages specific to: Fetal lung (mouse E14.2–E19/human 5–38 weeks) 1. Proximal (airways and) vessels form by dichotomous branching (pseudoglandular stage). 2. Distal (airways and) vessels form and central artery and vein branches multiply (canalicular stage). 3. Start of acinus and respiratory region (blood–gas barrier) formation (saccular stage). Neonatal and child lung (mouse birth to 4 weeks/human birth to 8 years) 4. Septation initiates formation of alveoli (alveolarization stage) and alveoli multiply thereafter. Existing vascular structures expand, and new ones form within the enlarging alveolar zone. Adult lung (mouse from 4 weeks/human from 8 years to approximately 22–25 years) 5. Existing vascular structures expand until thoracic growth ceases. Acknowledgements The writting of this chapter by RJ and DC was supported by a grant from the National Institutes of Health (NIH HL 089252). The sterling work of the colleagues and investigators cited here is gratefully acknowledged. Section 2 dealing with regulation of the molecular basis of lung development is based on the author’s (RJ) presentation, and printed handout, for a symposium on genes and lung development, at a plenary session of an international meeting of the US/ Canadian Society of Pediatric Pathology, held in 2004 in Vancouver, British Columbia, Canada.
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Chapter 4
Pulmonary Vascular Function Robert Naeije and Nico Westerhof
Abstract The pulmonary circulation is a low-pressure and high-flow circuit. The low pressure prevents fluid moving out of the pulmonary vessels into the interstitial space, and allows the right ventricle to operate at a low energy cost. The flow is matched to ventilation for pulmonary gas exchange. As a low-pressure system, the pulmonary circulation is very sensitive to mechanical influences, and the thin-walled right ventricle is poorly prepared for rapidly increased loading conditions. The pulmonary circulation is functionally coupled to the right ventricle. Pulmonary pressure–flow relationships are determined by a dynamic interaction between ventricular pump function and mechanical properties of the pulmonary arterial tree. In this respect, it is always important to remember that the pulsatility of the pulmonary circulation is greater than that in the systemic bed. The ratio of pulse pressure over mean pressure in the pulmonary artery is about unity, whereas in the aorta, pulse pressure is about 40% of mean pressure, implying that pulsatile energy to be generated. Keywords Pulmonary vascular function • Pressure-flow relationship • Blood flow • Hemodynamics
1 Introduction The pulmonary circulation is a low-pressure and high-flow circuit. The low pressure prevents fluid moving out of the pulmonary vessels into the interstitial space, and allows the right ventricle to operate at a low energy cost. The flow is matched to ventilation for pulmonary gas exchange. As a low-pressure system, the pulmonary circulation is very sensitive to mechanical influences, and the thin-walled right ventricle is poorly prepared for rapidly increased loading conditions. The pulmonary circulation is functionally coupled to the right ventricle. Pulmonary pressure–flow relationships are R. Naeije (*) Department of Pathophysiology, Free University of Brussels, Campus Erasme CP 604, 808, route de Lennik, Bldg 4. Rm Es.4.108, 1070 Brussels, Belgium e-mail:
[email protected] determined by a dynamic interaction between ventricular pump function and mechanical properties of the pulmonary arterial tree. In this respect, it is always important to remember that the pulsatility of the pulmonary circulation is greater than that in the systemic bed. The ratio of pulse pressure over mean pressure in the pulmonary artery is about unity [1], whereas in the aorta, pulse pressure is about 40% of mean pressure [2], implying that pulsatile energy to be generated by the right side of the heart is relatively more important.
2 Steady-Flow Pulmonary Hemodynamics 2.1 Pulmonary Vascular Resistance The function of a vascular system is defined by pressure difference–flow relationships. In the “steady-flow” hemodynamic approach, pressure and flow waves are summarized by their mean values, and a single-point pressure difference– flow relationship is calculated as a resistance. This provides a single-number description of the resistive properties, or “function,” of the vascular system under consideration. The functional state of the pulmonary circulation can thus be defined by the pulmonary vascular resistance (PVR) calculated as the difference between the pulmonary artery pressure (PAP), taken as an inflow pressure, and the mean left atrial pressure (LAP), taken as the outflow pressure, divided by the mean flow (Q):
PVR = (PAP − LAP) / Q.
(1)
This equation is most often implemented in clinical practice by fluid-filled thermodilution catheters. These catheters are balloon-tipped, allowing for an estimate of LAP by an occluded PAP (PAOP). Sometimes, a measurement of LAP or PAOP cannot be obtained, and the total PVR (TPVR) is calculated as
J.X.-J. Yuan et al. (eds.), Textbook of Pulmonary Vascular Disease, DOI 10.1007/978-0-387-87429-6_4, © Springer Science+Business Media, LLC 2011
TPVR = PAP / Q.
(2)
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Since LAP is not negligible with respect to PAP, TPVR is larger than PVR and is flow-dependent. Thus, TPVR is not a correct characterization of the resistive properties of the pulmonary circulation. A resistance calculation derives from Ohm’s law first derived for electric circuits. The resistance is determined by vessel and fluid properties. A law that governs laminar flows of Newtonian fluids through nondistensible, circular tubes was originally proposed by the French physician Poiseuille and later put into mathematical form by the German physicist Hagen. The law states that the resistance R to flow of a single tube is equal to the product of the length l of the tube and a viscosity constant h divided by the product of fourth power of the internal radius r and p, and can be calculated as a pressure drop DP to flow Q ratio:
R = 8.1h / πr 4 = ∆P / Q.
(3)
The ratio of the pressure drop and flow of an entire vascular bed accounts for the resistances in series and in parallel of the individual vessels The fact that r in the equation is at the fourth power explains why R is mainly located in the smallest arteries and arterioles (the resistance vessels) and is exquisitely sensitive to small changes in the caliber of these small vessels (a 10% change in radius results in almost 50% change in resistance) Accordingly, PVR is a good indicator of the state of constriction or dilatation of pulmonary resistive vessels and is helpful to monitor disease-induced pulmonary vascular remodeling and/or changes in tone. The limits of normal or resting pulmonary vascular pressures and flows as derived from measurements obtained in a total of 55 healthy resting supine young adult healthy volunteers [3–5] are shown in Table 1. In that study population, Q Table 1 Limits of normal pulmonary blood flow and vascular pressures Variables
Mean
Limits of normal flow and pressure
6.4 4.4–8.4 Q (l/min) Heart rate (bpm) 67 41–93 PAP systolic (mmHg) 19 13–26 PAP, diastolic (mmHg) 10 6–16 PAP, mean (mmHg) 13 7–19 PAOP (mmHg) 9 5–13 PCP (mmHg) 10 8–12 RAP (mmHg) 5 1–9 PVR (dyne s cm−5) 55 11–99 SAP, mean (mmHg) 91 71–110 Limits of normal flow and pressure are from the mean minus twice the standard deviation (SD) to the mean plus twice the standard deviation, n = 55 healthy resting volunteers (n = 14 for the measurement of PCP Pulmonary Capillary Pressure) Data from [3–5] Q cardiac output, PAP pulmonary artery pressure, PAOP occluded pulmonary artery pressure, PCP pulmonary capillary pressure (measured by single occlusion), RAP right atrial pressure, PVR pulmonary vascular resistance, SAP systemic arterial pressure
was lower in women, who are smaller than men, and thus PVR was calculated to be higher. However, there were no gender differences in pulmonary hemodynamics after correction for body dimensions. Earlier studies showed that aging is associated with a slight increase in PAP and a decrease in Q, leading to a doubling of PVR over a five-decade lifespan [6–9].
2.2 Pressure–Flow Relationships The inherent assumption of a PVR calculation is that the PAP–Q relationship is linear and crosses the pressure axis at a value equal to LAP, allowing PVR to be constant whatever the absolute level of pressure or flow. Although the relationship between (PAP − LAP) and Q has indeed been shown to be reasonably well described by a linear approximation over a limited range of physiological flows, the zero crossing assumption may be true only in case of well-oxygenated lungs in supine resting subjects, suggesting complete recruitment and minimal distension. Hypoxia and a number of cardiac and respiratory diseases increase both the slope and the extrapolated intercepts of multipoint (PAP − LAP)–Q plots [10]. Although an increase in the slope of a PAP–Q plot is easily understood as being caused by a, generally, decreased radius and thus cross-sectional area of pulmonary resistive vessels, the positive extrapolated pressure intercept has inspired various explanatory models. Permutt et al. conceived a vascular waterfall model made of parallel collapsible vessels with a distribution of closing pressures [11]. At low flow, these vessels would be progressively derecruited, accounting for a low-flow PAP–Q curve that is concave with respect to the flow axis, and intercepts the pressure axis at the lowest closing pressure to be overcome to generate a flow. At higher flow, completed vessel recruitment and negligible distension account for a linear PAP–Q curve with an extrapolated pressure intercept representing a weighted mean of closing pressures. In this model, the mean closing pressure is the effective outflow pressure of the pulmonary circulation. If LAP is lower than the mean closing pressure, it is irrelevant to the flow. In that situation, a PVR calculation becomes misleading because of flow dependency: increased or decreased flow necessarily decreases or increases PVR, respectively, without a change in the functional state in the pulmonary circulation taking place. These concepts are illustrated in Fig. 1, which represents pulmonary vessels as collapsible with tone or owing to chamber pressure within a device called a “Starling resistor.” This device was conceived by Starling and his coworkers to control systemic arterial pressure in their heart–lung preparation. The “waterfall model” of Permutt et al. is also called the “Starling resistor model.”
4 Pulmonary Vascular Function
PAP
CP
Outflow pressure
PAP-LAP 2 CP
PVR = (PAP–LAP) / Q
LAP
Inflow pressure
1
A: LAP > CP B: CP > LAP 1 → 2: vasoconstriction 1 → 3 : no change
A
A
Q
Q
Fig. 1 Starling resistor model to explain the concept of closing pressure within a circulatory system. Flow (Q) is determined by the gradient between an inflow pressure, or mean pulmonary artery pressure (PAP), and an outflow pressure, which is either the closing pressure (CP) or the left atrial pressure (LAP). When LAP > CP, the (PAP − LAP)/Q relationship crosses the origin (A curve) and pulmonary vascular resistance (PVR) is constant. When CP > LAP, the (PAP − LAP)/Q relationship has a positive pressure intercept (B curve), and PVR decreases curvilinearly with increasing Q. Also shown are possible misleading PVR calculations: PVR, the slope of (PAP − LAP)/Q may remain unchanged in the presence of a vasoconstriction (from datapoint 1 to datapoint 2) or decrease (from datapoint 1 to datapoint 3) with no change in the functional state of the pulmonary circulation (unchanged pressure–flow line). (Reproduced with permission [10])
PAP-PAOP (mmHg)
30
E
D
20
B C
10
A
0
1
Resistances Resistances differ are equal
2
3
Vasoconstriction 2
C
Uncertainty
4
2 Uncertainty
1 Vasodilation
Q
B
PVR
PAP-LAP
1
PVR
B
3
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5
Flow (l/min) Fig. 2 PAP versus flow at two levels of pulmonary hypertension is correctly described by a linear approximation over a physiological range of flows (3–5 l/min). The linearized pressure–flow relationships (dashed lines) have extrapolated pressure intercepts (open squares) that are positive, suggesting a CP higher than the LAP occluded PAP. However, the pressure–flow relations are better described by a curvilinear fit (fully drawn lines), which takes into account the distensibility of the pulmonary vessels. In the curvilinear relations as well as the linear relations over the physiological range, PVR calculations are misleading: from A to B, PVR does not seem change, and from C to B and from D to E, i.e., running over a single relation, PVR decreases, and in the presence of aggravated pulmonary hypertension as assessed by higher pressures at a given flow range the slope, and thus the resistance, may differ little (arrows)
However, distensible vessel models have been developed which explain the shape of PAP–Q curves by changes in resistance and compliance [12, 13]. In fact, as illustrated in Fig. 2, PAP–Q curves can always be shown to be curvilinear with
Fig. 3 Pressure–flow diagram for the interpretation of pulmonary hemodynamic measurements. The central point C corresponds to initial mean PAP, LAP, and flow (Q) measurements. A decrease in PAP − LAP at increased Q can only be explained by pulmonary vasodilatation. An increase in PAP − LAP at decreased Q can only be explained by pulmonary vasoconstriction. Rectangles of certainty are extended to adjacent triangles because negative slopes or pressure intercepts of (PAO − LAP)/Q lines are impossible. Arrows indicate changes in measured PAP − LAP and Q, 1 vasodilatation, 2 vasoconstriction. (Reproduced with permission [10])
concavity to the flow axis provided a large enough number of PAP–Q coordinates are generated and subjected to an adequate fitting procedure. However, a derecruitment can be directly observed at low pressures and flows [14]; therefore, it seems reasonable to assume that both recruitment and distension probably explain most PAP–Q curves [15]. According to this integrated view, at low inflow pressure, many pulmonary vessels are closed as a consequence of their intrinsic tone and surrounding alveolar pressure, and those that are open are relatively narrow. As inflow pressure increases, previously closed vessels progressively open (recruitment), and previously narrow vessels progressively dilate (distension). Both mechanisms explain a progressive decrease in the slope of pulmonary vascular pressure—flow relationships with increasing flow or pressure. Whatever the model, PVR determinations are better replaced by multipoint pressure–flow relationships for the evaluation of the functional state of the pulmonary circulation at variable flow. The problem of in vivo pressure–flow relationships in intact animals is to alter flow without affecting vascular tone. Exercise to increase the flow may spuriously increase the slopes of PAP–Q plots, in normal subjects [16] as well as in patients with cardiac or pulmonary diseases, leading to linear fitting of PAP–Q plots with negative extrapolated pressure intercepts [17, 18]. An infusion of low-dose dobutamine to increase flow might be preferable [18], although it is always difficult to exclude a possible flow-induced or b-adrenergic or a-adrenergic receptor mediated change in tone, depending on the dose and the preexisting functional state. Most often only a single PVR determination can be obtained for every given functional state of a pathophysiological condition. It is then advised to reason using a pressure– flow diagram as illustrated in Fig. 3. This diagram shows four possible combinations of flow and pressure changes, with
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certainties about true changes in structure- or tone-determined resistance in only two of them: decreased pressure with increased flow as a true decrease in PVR, and increased pressure with decreased flow as a true increase in PVR, the other combinations remaining in an uncertainty domain.
3 Effects of Exercise Supine exercise is associated with proportional increases in cardiac output and pulmonary vascular pressure gradient, with a slight to moderate decrease in PVR. Upright exercise is associated with a marked initial hyperbolic decrease in PVR. At high levels of exercise, PVR at any given load is independent of body position. The sharp curvilinear decrease in PVR at low levels of upright exercise may be explained by a vascular derecruitment, together with an inherent slight curvilinearity of pressure–flow relationships [19]. High levels of exercise markedly increase pulmonary vascular pressures. In athletes able to increase their cardiac output to 25–35 l/min, PAP may increase to 40–45 mmHg and PAOP increased to 25–35 mmHg [19]. None of the previously reported pulmonary hemodynamic measurements at exercise in normal subjects have included direct measurements of LAP through a left-sided heart catheterization. However, PAOP at exercise is unlikely to overestimate LAP. High levels of cardiac output are associated with a complete recruitment of the pulmonary capillary network, which is the condition for the valid estimation of LAP by a PAOP. On the other hand, the filling pressures of both ventricles, as assessed by directly measured right atrial pressure (RAP) and indirectly measured LAP, rise at exercise in relation to stroke volume and exercise capacity [20]. This suggests that the left ventricle tends to overuse the Frank–Starling mechanism (increase stroke volume by an increased preload, or end-diastolic ventricular volume/pressure) to maximize cardiac output at the highest levels of exercise [19]. As mentioned already, PVR increases with age, so the average slope of PAP–Q plots during exercise is 1 mmHg/l/ min in young adults, but more than doubles, up to 2.5 mmHg/l/ min, in old subjects. Much of the slope of PAP–Q is caused by an increase in PAOP (or LAP) [7, 8, 19]. An earlier and more important increase in LAP in older subjects at exercise could be explained by age-related decreased diastolic compliance of the left ventricle [8]. Although PAP–Q relationships at exercise are generally best described by a linear approximation [19], a sufficient number of measurements at high levels of exercise, above the anaerobic threshold, may disclose an increased slope as the cause of a biphasic “takeoff” pattern on log PAP–log VO2 plots [16]. Because of the tight relationship between Q and VO2, this is to be interpreted as an high level of exerciseinduced pulmonary vasoconstriction, caused by sympathetic
R. Naeije and N. Westerhof
nervous system activation, acidosis, and decreased mixed venous oxygenation. An increased slope of PAP–Q plots above the anaerobic threshold may also be related to an increase in LAP. It is intriguing that the takeoff pattern of PAP–VO2 plots at exercise is not observed in patients with pulmonary vascular disease, who rather show a “plateau” pattern [16]. The reason for the decreased slope of PAP–VO2 relationships at high levels of exercise in patients with pulmonary vascular diseases is not clearly understood.
4 P assive Regulation of Steady-Flow Pulmonary Hemodynamics 4.1 Left Atrial Pressure At a given Q, an increase in LAP is transmitted upstream to PAP in a less than 1:1 proportion, depending on the state of arterial distension and the presence or absence of a closing pressure higher than LAP [10, 15]. In a fully distended and recruited pulmonary circulation, DPAP/DLAP is close to unity.
4.2 Lung Volume An increase in lung volume above functional residual capacity increases the resistance of alveolar vessels, which are the vessels exposed to alveolar pressure, but decreases the resistance of extra-alveolar vessels, which are the vessels exposed to interstitial pressure. A decrease in lung volume below functional residual capacity has the opposite effects. As a consequence, the lowest resultant PVR is observed at functional residual capacity [21].
4.3 Gravity Pulmonary blood flow increases almost linearly from nondependent to dependent lung regions. This inequality of pulmonary perfusion is best demonstrated in an upright lung [22]. The vertical height of a lung is, on average, about 30 cm. The difference in pressure between the extremities of a vertical column of blood of the same size is 23 mmHg, which is quite large compared with the mean perfusion pressure of the pulmonary circulation. Accordingly, the physiological inequality of the distribution of perfusion of a normal lung can be explained by a gravity-dependent interplay between arterial, venous, and alveolar pressures. At the top of the lung, alveolar pressure (PA) is higher than mean PAP
4 Pulmonary Vascular Function
(MPAP) and pulmonary venous pressure (PVP). In this zone 1, flow may be present only during systole, or not at all. Zone 1 is extended in clinical situations of low flow, such as hypovolemic shock, or increased alveolar pressure such as during ventilation with a positive end-expiratory pressure. Further down the lung there is a zone 2 where PAP > PA > PVP. In zone 2, alveolar pressure is an effective closing pressure, and the driving pressure for flow is the gradient between MPAP and PA. As mentioned earlier, such a flow condition can be likened to a waterfall since PVP, the apparent outflow pressure, is irrelevant to the flow as is the height of a waterfall. In zone 3, PVP is higher than PA, so the driving pressure for flow is PAP − PVP. In the most dependent regions of the upright lung, there is an additional region where flow decreases, defining an additional zone, zone 4 [23]. Zone 4 has been attributed to an increase in the resistance of extra-alveolar vessels, because it expands when lung volume is reduced or in the presence of lung edema. Active tone may be an additional explanation for zone 4 as it is also reduced by the administration of vasodilators. The vertical height of lung tissue in a supine subject is, of course, much reduced compared with that of the subject in the upright position, and accordingly, the lung is then normally almost completely in zone 3, with, however, persistence of a still measurable increase in flow from nondependent to dependent lung regions. Three-dimensional reconstructions using single-photonemission computed tomography have shown that there is also a decrease in blood flow from the center of the lung to the periphery [24]. High-resolution methods and fractal modeling of the pulmonary circulation have actually led to the suggestion that the distribution of pulmonary blood flow would be determined as a consequence of the fractal structure of the pulmonary arterial tree, with only secondary minor gravity-dependent adjustments [25]. Subtle differences in arterial branching ratios may indeed influence the flow distribution, with increased heterogeneity as the scale of the inquiry narrows, corresponding to the “what is the length of the coastline” effect. However, the overwhelming evidence remains in favor of the thesis that gravity is the single most important determinant of pulmonary blood flow distribution [26]. Vascular-geometry-related small unit heterogeneity of pulmonary blood flow distribution has not been shown to be relevant to gas exchange.
5 A ctive Hypoxic Regulation of Steady-Flow Pulmonary Hemodynamics There is an active intrapulmonary control mechanism able to some extent to correct the passive gravity-dependent distri-
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bution of pulmonary blood flow: a decrease in PO2 increases pulmonary vascular tone. Hypoxic pulmonary vasoconstriction was first reported by von Euler and Liljestrand [27], who proposed a functional interpretation that can still be considered valid. In lung tissue, PO2 is determined by the ratio between O2 carried to the lung by alveolar ventilation (VA) and O2 carried away from the lung by blood flow (Q):
PO 2 = VA / Q.
(4)
This is in contrast with hypoxic vasodilation in systemic tissue, where local PO2 is determined by the ratio flow of O2 carried to the tissues (Q) and local O2 consumption (VO2):
PO2 = Q / VO2 .
(5)
The hypoxic pulmonary pressor response is universal in mammals and in birds, but with considerable interspecies and interindividual variability. The attributes of hypoxic pulmonary vasoconstriction can be summarized as follows [28]. The response is turned on in a few seconds, fully developed after 1–3 min, and more or less stable thereafter according to the experimental conditions. It is reversed in less than 1 min. It is observed in lungs devoid of nervous connections, and indeed also in isolated pulmonary arterial smooth muscle cells. Hypoxic pulmonary vasoconstriction is enhanced by acidosis, a decrease in mixed venous PO2, repeated hypoxic exposure (in some experimental models), perinatal hypoxia, decreased lung segment size, cyclooxygenase inhibition, nitric oxide inhibition, and certain drugs or mediators which include almitrine and low-dose serotonin. Hypoxic pulmonary vasoconstriction is inhibited by alkalosis, hypercapnia, an increase in pulmonary vascular or alveolar pressures, vasodilating prostaglandins, nitric oxide, complement activation, low-dose endotoxin, calcium channel blockers, b2 stimulants, nitroprusside, and, paradoxically, peripheral chemoreceptor stimulation. The hypoxic pressor response is biphasic, with a progressive increase as PO2 is progressively decreased to approximately 35–40 mmHg, followed by a decrease (“hypoxic vasodilatation”) in more profound hypoxia. The hypoxia-induced increase in PVR is mainly caused by a constriction of precapillary small arterioles [28]. Small pulmonary veins also constrict in response to hypoxia, but this should not normally contribute to more than 20–30% of the total change in PVR [29]. An exaggerated hypoxic constriction of small pulmonary veins could explain the development of pulmonary edema which is observed in a small proportion, of the order of 1–2%, of subjects rapidly taken to high altitudes [5]. Grant et al. [30] used the equations of the control theory and the linear relationships between lobar blood flow and alveolar PO2 (PAO2) found in the coatimundi, an animal with
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a strong hypoxic pressor response, to calculate the efficiency of hypoxic vasoconstriction as a mechanism to stabilize PAO2. They found a maximum gain due to feedback (Gfb) of 0.9 at a PAO2 between 60 and 80 mmHg, falling rapidly off outside these values. A Gfb of 0.9 represents an active correction of 47% of the decrease in PAO2 that would occur in a passive system without hypoxic vasoconstriction. Mélot et al. used the same equations and linear relationships between compartmental blood flow and PAO2 derived from data for elimination of inert gases obtained in healthy volunteers, and found a maximum Gfb of 0.63 at a PAO2 of 60 mmHg, also falling rapidly off at lower and at higher PAO2 [4]. A Gfb of 0.63 represents an active correction by 39% of a decrease in PAO2 that would occur in a passive system without hypoxic vasoconstriction. These studies suggested that the hypoxic pressor response is an only moderately efficient feedback mechanism, acting essentially at PAO2 values higher than are known to occur in severe lung diseases. However, more recent evaluations of the efficiency of hypoxic pressor response using multicompartment lung models [31] fed by biphasic stimulus–response curves using real data [32] led to the conclusions that hypoxic vasoconstriction is really effective in improving gas exchange in severe respiratory insufficiency. A quantification of the efficiency of hypoxic pulmonary vasoconstriction in terms of correction of arterial hypoxemia in chronic obstructive pulmonary disease (COPD) is presented in Fig. 4 [32]. Patients with COPD are hypoxemic because of increased dispersion of the distributions of perfusion and ventilation, with increased perfusion to lung units with a lower than normal VA/Q. Thus, in these patients, altered pulmonary gas exchange can be quantified by the logarithm of the standard deviation of VA/Q dispersion. On the other hand, the strength of hypoxic vasoconstriction can be expressed as PAP in hypoxia divided by PAP in hyperoxia at constant flow. The magnitude of hypoxic vasoconstriction normally ranges from 1 to 4 in the canine and in the human species. It can be seen that, in COPD, arterial PO2 may increase by up to 20 mmHg through the effects of vigorous hypoxic vasoconstriction. The same analysis was performed in patients with acute respiratory distress syndrome (ARDS), who are hypoxemic mainly because of an increased shunt [32]. Thus, in these patients, altered gas exchange can be quantified by intrapulmonary shunt, expressed as a percentage of cardiac output. In ARDS, arterial PO2 could increase by as much as 20 mmHg owing to vigorous hypoxic vasoconstriction. All these predictions are in keeping with the magnitude of decreases in arterial oxygenation observed in patients with ARDS or COPD due to the administration of vasodilating drugs that inhibit hypoxic pulmonary vasoconstriction [32]. The biochemical mechanism of hypoxic pulmonary vasoconstriction remains incompletely understood [28, 33].
R. Naeije and N. Westerhof
Fig. 4 Effects of hypoxic pulmonary vasoconstriction (HPV) in chronic obstructive pulmonary disease (COPD), a lung disease characterized by VA/Q mismatching. LSD logarithmic standard deviation of the log–normal VA/Q ratio distribution. The fraction of inspired O2 ( FIO2) was set to 0.30; other variables were set to normal values and remained unchanged during calculations (barometric pressure 760 Torr, temperature 37°C, hemoglobin 15 g/dl, base excess 0 mmol/l, P50 26.8 Torr, O2 consumption 300 ml/min, CO2 production 240 ml/ min, cardiac output 6.00 l/min, ventilation 7.20 l/min, shunt 0%, dead space 30%). a HPV significantly improved PAO2 at all LSD values and improved SaO2 when it was most decreased. b At PAO2 of 40 Torr (LSD = 2.2), HPV decreased blood flow by 30% in hypoxic lung units (VA/Q 100
SMC, EC SMC, EC SMC, EC SMC, EC SMC, EC SMC, EC SMC, EC EC SMC SMC, EC SMC, EC – EC SMC, EC SMC, EC SMC, EC SMC, EC SMC SMC, EC –
6 1
>100 0.8
– ND
Store depletion, PLC pathway DAG Store depletion, DAG Store depletion? La3+, calmidazolium Store depletion? La3+, NO DAG, PIP3, Ca2+, 20-HETE Store depletion, DAG Translocation? ADP-ribose/AA/NAD/H2O2/Ca2+ Osmotic cell swelling/store depletion? Ca2+/voltage/PIP2 Ca2+/voltage/PIP2/heat Translocation ATP/H+/phosphorylation/PIP2 Cool/menthol/icilin/pH/PIP2 Heat/vanilloids/anandamide/PIP2/camphor Heat/osmotic cell swelling/exocytosis Warm/menthol/PUFAs Warm/osmotic cell swelling/5¢6¢-EET Low Ca2+ concentration/hyperpolarizaton/ exocytosis Store depletion, exocytosis Cold/icilin/isothiocuanates/cannabinoids/ bradykinin/DAG/PUFAs pH/Ca2+/proteolytic cleavage
BRP2/Gd/La Ca2+/2-APB 2-APB 2-APB/La Ca2+/La [137–139] Clotrimazale Mg2+ AMP/ATP Spermine Mg2+ Mg2+ Capsazepine Capsazepine DAT La Gd/La Ca2+/Cd Ca2+/Cd Amiloride
[137–139]
[137]
Amiloride? [137] 1 MVC-selective ND 2 – 3 – TRPP 2 Nonselective ND Ca2+/TRPP1/EGF/PIP2/actin cytoskeleton Flufenamate/Cd/La? [137] 3 4.3 Ca2+ Gd/La/Ni? 5 – – Question marks represent controversial observations.AA arachidonic acid, CaM calmodulin, DAG diacylglycerol, DAT 1,3-di(arylalkyl)thioureas, DVC divalent cation, EC pulmonary artery endothelial cells, PLC phospholipase C, MVC monovalent cation, ND not determined, PIP2 phosphoinositide 4,5-bisphosphate, PIP3 phosphatidyl 3,4,5-trisphosphate, PUFAs polyunsaturated fatty acids, SMC pulmonary artery smooth muscle cell, 2-APB 2-Aminoethoxydiphenyl Borate, 20-HETE 20-Hydroxy Arachidonic Acid, 5'6' EET 5′6′ Epoxyeicosatrienoic acid, EGF Epidermal Growth Factor, BRP2 TRPML
13 Ion Channels and Transporters in the Pulmonary Vasculature: A Focus on Smooth Muscle
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Fig. 5 Structure and function of transient receptor potential (TRP) channels. (a) Planar views of TRPC1, TRPV1, and TRPM1 channel subunits. (b) Representative traces reflecting changes in cytosolic Ca2+ concentration recorded in smooth muscle cells, capacitative calcium entry subsequent to cyclopiazonic acid (CPA)-dependent depletion of the endoplasmic reticulum/sarcoplasmic reticulum (ER/SR) Ca2+ stores (in the absence of extracellular Ca2+). The lower panel shows representative current traces and I–V curves recorded in human PASMCs
overexpressing TRPC6 before and after CPA exposure. (c) A currently proposed functional interaction between TRP channels, STIM, and Orai. Activation of G-protein-coupled receptors (GPCRs) increases inositol 1,4,5-trisphosphate mediated depletion of Ca2+ from the ER/ SR causing STIM dimerization and redistribution to discrete puncta in the ER/SR membrane in closer proximity to the plasma membrane where interactions with Orai (and TRP) channels can occur increasing Ca2+ influx
PASMC proliferation [24, 25]. In addition to forming homomultimeric channels, TRPC1 is also reported to form heteromultimeric channels with TRPC5, TRPC3, and TRPP2; such interaction may be crucial to the trafficking or translocation of TRPC1 to the plasma membrane [26]. TRPC4 is widely expressed and is known to be present in VSMCs [27]; however, its role is more pertinent in the endothelium, where it regulates vascular permeability and vasorelaxation [28, 29]. TRPC1, TRPC4, and TRPC6 protein expression varies with the location in the pulmonary vascular tree: higher expression was observed in the distal pulmonary artery than in the proximal pulmonary artery; this correlated with changes in cytosolic Ca2+ concentration occurring during hypoxic pulmonary vasoconstriction (HPV) [30]. TRPM2–TRPM4, TRPM7, TRPM8, and TRPV1– TRPV4 have all been detected using reverse transcription PCR in the pulmonary artery [31]. For more detail on TRP channels in the pulmonary vasculature, please refer to Firth et al. or Townsley et al. [32, 33].
Ca2+-Release-Activated Ca2+ Channels Recent evidence suggests a significant role for CRACs in voltage-independent Ca2+ influx into cells and it is postulated that this may contribute to SOCs in PASMCs [18]. Orai-1 is the fundamental CRAC pore-forming subunit in the plasma membrane [34]. ER/SR Ca2+ concentration is sensed by stromal interaction molecule 1 (STIM-1) and upon store depletion it oligomerizes, enabling it to redistribute to discrete sites in the ER/SR membrane in close proximity to the plasma membrane [35]. The precise functional association between STIM-1 and Orai-1 is currently unknown and the mechanism of Ca2+ influx mediated by Orai-1 remains to be fully elucidated.
2.2.3 Plasmalemmal Ca2+-ATPase The PMCA is a plasmalemmal P-type Ca2+-ATPase regulated by CaM which extrudes Ca2+ from cells in VSMCs to
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maintain Ca2+ homeostasis. It is encoded by four genes (PMCA-1, PMCA-2, PMCA-3, and PMCA-4), of which PMCA-4 and, to a lesser extent, PMCA-1 are expressed by vascular smooth muscle [36]. Alternative splicing of these four genes has created 20 isoforms of the PMCA. Each PMCA is a ten transmembrane spanning domain with cytosolic N- and C-termini. The CaM-binding site is located in a key regulatory domain in the C-termini along with PKC and protein kinase A (PKA) phosphorylation sites [37]. Interestingly, its activity has been shown to increase in the presence of oxidants, such as hydrogen peroxide (1 mM), and matrix metalloprotease 2 in PASMCs [38]. The pump has recently been purified from bovine PASMCs, which will open the door for more detailed studies on its ion transport mechanisms [39].
2.2.4 Na+–Ca2+ Exchanger The NCX represents the second system functioning to export Ca2+ from intact cells. The NCX has a much larger capacity than the PMCA and its importance as a primary mechanism for Ca2+ extrusion has been acknowledged by four dedicated international conferences. The expression and function of the NCX have been widely documented in both normal and diseased PASMCs and pulmonary artery endothelial cells [40–42]. The NCX operates in parallel with Ca2+ channels and pumps in the plasma membrane and can move Ca2+ either into or out of cells. Its activity is regulated by the electrochemical gradient across the membrane using Na+ gradients as a source of energy. The NCX is noticeably inhibited in PASMCs exposed to mild hypoxia, decreasing the rate of Ca2+ extrusion from the cytosol [43]. There are three currently identified isoforms of the NCX (NCX1–NCX3) and a further four isoforms for a potassiumdependent NCX (NCKX) [44]. The isoforms have a similar overall structure consisting of an intracellular loop, which connects to a five transmembrane domain at the N-terminus and a six transmembrane domain at the C-terminus. Messenger RNA (mRNA) for the NCX1 and NCKX3 isoforms has been isolated from cultured human PASMCs [45]. This study also reported that the NCX could contribute to Ca2+ entry owing to NCX activity in the reverse mode (inhibited by KB-R9756). At this stage it was predicted that blockade of the reversemode NCX may be a possible therapeutic target in the treatment of pulmonary hypertension [45]. More recent evidence, however, suggests that a contractile response, resembling that observed in HPV induced by the removal of extracellular Na+ in intact pulmonary arteries, was not due to inhibition of the NCX and thus may not be supportive of a direct role for the NCX in HPV [46].
A.L. Firth and J.X.-J. Yuan
2.2.5 Functional Interaction of TRPC Channels and NCX in Caveolae Caveolae are cholesterol- and sphingolipid-rich flask-shaped invaginations of the plasma membrane, whose principal structural component is caveolin. In the vasculature, caveolae appear to be more prominent in endothelial cells than in smooth muscle cells. However, the number of caveolae and the expression level of caveolin are significantly increased in PASMCs in patients with idiopathic PAH (Fig. 6a). The “simplest” physiological role of caveolae and caveolin is to centralize, concentrate, and colocalize cooperative receptors, signaling molecules/proteins, and effectors (e.g., ion channels and transporters) within microdomains. A caveolin-scaffolding domain (CSD) on caveolin itself can associate with membrane proteins. The TRPC1 channel proteins contain the C-terminal CSD-consensus binding sequence that allows for their physical and functional interaction with caveolin-1 (cav-1) in the caveolae of PASMCs (Fig. 6b). Cav-1 also binds to an N-terminal cav-1-binding motif (amino acids 322–349) of TRPC1 (Fig. 1a). Truncation of cav-1 (i.e., removal of CSD, membrane anchoring domain, and palmitoylation sites) significantly decreases store-operated Ca2+ entry through TRPC channels and downregulates TRPC1 and cav-1 expression. The presence of TRPC channels and NCX proteins in these caveolar lipid raft domains seems to constitute an important factor in their functional roles in regulating intracellular Ca2+. In addition to functioning as Ca2+ channels, TRPC channels are also permeable to Na+. TRPC-mediated Na+ influx into the cytosol of PASMCs, such as that provoked by activation of G-protein-coupled receptors (GPCRs) (Fig. 6c), is one of the major mechanisms to increase cytosolic Na+ concentration. A rise in cytosolic Na+ concentration (especially in the submembrane area) functionally converts the forward mode of NCX (inward Na+ and outward Ca2+ transportation) to the reverse model (outward Na+ and inward Ca2+ transportation). The inward Ca2+ transportation through the reverse mode of NCX plays an important role in maintaining a high level of cytosolic Ca2+ and refilling the SR/ER with Ca2+ via SERCA (Fig. 6c). By binding with cav-1 in both N- and C-termini, TRPC can be efficiently regulated upon activation of receptors. As shown in Fig. 6c, accumulated ligand (e.g., growth factors, mitogenic and angiogenic ligands, and vasoactive substances) in caveolae activates the receptors (GPCRs or receptor tyrosine kinases) located in caveolae and increases synthesis of inositol 1,4,5-trisphosphate (IP3) and diacylglycerol. By binding with IP3 receptors (IP3R) on the ER/SR membrane, the second-messenger IP3 induces a transient increase in cytosolic Ca2+ concentration owing to Ca2+ release from the SR/ER to the cytosol. Opening of the Ca2+ release channels or IP3R is an efficient way to deplete Ca2+ from the
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Fig. 6 Functional coupling of receptors, TRPC channels, and Na+–Ca2+ exchangers (NCX) in caveolae in PASMCs. (a) Ultrastructure of plasma membrane of PASMCs isolated from normal subjects and patients with idiopathic pulmonary arterial hypertension (IPAH) was assessed by electron microscopy. IPAH PASMCs have increased flask-like invaginations of the plasma membrane consistent with the morphology of caveolae (indicated by arrows) and as determined by the number of caveoli per membrane length. (b, c) The caveolin-1 binding domains in
TRPC1 (b) and the potential mechanisms (c) that are involved in ligandmediated regulation of cytosolic Ca2+ concentration through GPCRs, TRP channels, and NCX in caveolae. CBM caveolin-1-binding motif, PBD protein 4 binding domain, CSD caveolin-scaffolding domain, PM plasma membrane, ER/SR endoplasmic reticulum and sarcoplasmic reticulum, G, G protein, IP3 inositol 1,4,5-trisphosphate, IP3R inositol 1,4,5-trisphosphate receptor, SERCA the sarcoplasmic reticulum/endoplasmic reticulum Ca2+–Mg2+-ATPase, Cav-1 caveolin-1
intracellular stores (i.e., SR or ER). The store depletion then activates TRPC-encoded SOCs, which are located in caveolae, and promotes Ca2+ influx or causes store-operated Ca2+ entry. Since TRPC channels are also highly permeable to Na+, the local accumulation of intracellular Na+ would then activate the reverse mode of Na+–Ca2+ exchange and further enhance Ca2+ entry (Fig. 6c). The proximity of the receptor, the channel, the transporter, and the caveolin allows the ligand-mediated increase in cytosolic Ca2+ concentration to be efficiently and rapidly regulated for signal transduction and for stimulating functional changes of the cell.
nary vasculature are (1) voltage-gated K+ channels (KV), (2) large-, intermediate-, and small-conductance Ca2+-activated K+ channels (BKCa, IKCa, and SKCa respectively), (3) ATPsensitive K+ channels (KATP), and (4) two-pore-domain K+ channels (K2P). The Na+–K+-ATPase (or Na+ pump) is the chief mechanism for Na+ extrusion, transporting both Na+ and K+ against their concentration gradients.
3 Potassium Channels and Transporters Potassium channels are the key regulators of cellular excitability, being fundamentally involved in the regulation of cellular Em. Efflux of K+ after activation of K+ channels leads to membrane hyperpolarization, inhibition of VDCCs, and vasodilation. A small change in K+ channel activity can have a substantial effect on membrane potential and thus factors targeting K+ channels can modulate vessel tone [47]. The main families of potassium channels identified in the pulmo-
3.1 Voltage-Gated K+ Channels Voltage-gated K+ channels, or KV channels, are the most diverse group of K+ channels ubiquitously expressed in VSMCs. There are currently over 40 KV channel members grouped into 12 subfamilies (the properties are summarized in Table 4). KV channels are tetramers of six transmembrane spanning proteins formed from symmetrically arranged KV a subunits. They align in either in a homomultimeric or in a heteromultimeric combination, surrounding a central pore highly selective to the transportation of K+ across the cell membrane (depicted in Fig. 7a) [48]. The voltage sensor is located in the S4 transmembrane domain and contains a preserved region of positive arginine or lysine amino acid residues (repeats of Arg
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Table 4 Expression and function of K+ channels in the pulmonary vasculature Family Subunits Accessory subunits Activators Inhibitors KV
BKCa
KATP
Antagonists
References
Kv1.1–Kv1.7
Kvb1
Depolarization
Hyperpolarization
4-AP
[69, 130, 131, 140–145]
Kv1.10 Kv2.1
Kvb2
PKA
PKC
Kv3.1b/3.3/3.4
Kvb3
Kv4.1.1/4.2 Kv5.1 Kv6.1–Kv6.3 Kv9.1/9.3 Kv10.1 Kv11.1-1.7 Slo1/KCNMA1
Decreased pHcyt Increased cytosolic Ca2+ concentration Cytochrome c Rho kinase Hypoxia
CytochromeP450 inhibitors (clorimazole) Correolide
b1
PKG/PKA
PKC
b2 b3 b4
NO EETS CO AA PKA/PKG
ROS Decreased pHcyt c-Src
CGRP Decreased ATP Pinacidil Depolarization Increased pHcyt PKA
Ca2+-activated PP2B PKC
Kir6.1
SUR2B
Kir6.2
K2PD
Increased ATP
IBTX, ChTX, paxilline, TEA (>1 mM) PDE inhibitors
[142, 146–149]
Glybenclamide, tolbutamide
[76, 143]
TASK1/2 Not yet identified Hyerpolarization Zn2+, anandamide [86–88, 150] THIK-1 Decreased pHex TRAAK TREK-1/2 TWIK-1/2 KCNQ KCNQ1 Not yet identified Depolarization Hyperpolarization [79, 80, 143] 4-AP aminopyridine, AA arachidonic acid, CO carbon monoxide, CGRP calcitonin gene related peptide, EETS epoxyeicosatrinoic acids, NO nitric oxide, PKA protein kinase A, PKC protein kinase C, PKG protein kinase G, PDE phosphodiesterase, PP2B protein phosphatase 2B, ROS reactive oxygen species, SUR2B sulfonylurea receptor subunit 2B, TEA tetraethylammonium, IBTX Iberiotoxin, CHTX Charybdotoxin
Fig. 7 Structure and function of K+ channels. (a) Planar membrane topologies of single potassium channel subunits for KV, BKCa, and Kir channels, respectively. The pore-forming loop and the voltage sensor in transmembrane unit 4 are indicated. (b) Representative current traces recorded in human pulmonary arterial smooth muscle cells indicating diversity in the Kv currents; from left to right, (1) rapidly activating and slowly inactivating IK(V), (2) rapidly activating and noninactivating IK(V), (3) slowly activating and noninactivating IK(V), and (4) rapidly activating and rapidly inactivating IK(V). (b Reproduced with permission [142])
13 Ion Channels and Transporters in the Pulmonary Vasculature: A Focus on Smooth Muscle
or Lys-Xaa-Xaa(7)) essential for depolarization-dependent opening of the channel [49]. Selectivity to K+ is energetically favored through a flexible pore lined by a backbone of carbonyl groups, a sequence highly conserved within the K+ channel family [50]. Although the cytoplasmic C-terminus differs among KV channel a subunits, the cytoplasmic N-terminus is highly conserved throughout the KV channel family. This 120 amino acid domain contains a tetramerization domain (T1-S1) comprising the molecular determinants for the formation of functional tetrameric channels from specific a-subunit assembly [51]. The tetrameric T1 domain exists as a region distinct from the transmembrane channel and, owing to a structural resemblance, is commonly referred to as a ball and chain structure [52]. The T1 domain is fundamental in influencing the channel conformation, gating, and electrophysiological properties [53]. KV channels are activated by depolarization and are central to the regulation of resting membrane potential in PASMCs [54, 55]. Whole-cell currents carried by KV channels, denoted IKv, are determined by the following equation,
I KV = N × Popen × iK ,
where N denotes the total number of KV channels, Popen is the steady-state open probability of a KV channel, and iK is the current amplitude through a single KV channel. Decreased IKv may occur owing to (1) a decrease in the total channel protein due to transcriptional inhibition of the KV genes via transcriptional silencer elements (e.g., the KV1.5 repressor element in the promoter region of the gene) [56, 57], (2) a decrease in the functional surface expression of KV channels due to inhibition of channel trafficking by the KV channel interacting proteins (e.g., KChIP) [58] or due to the inhibition of a–a subunit assembly via the cytoplasmic N-terminal tetramerization domain (T1) [51], (3) association with the regulatory b subunits either decreasing activation and/or increasing inactivation of KV channels [59, 60], (4) inhibition of channel activity by PKC-mediated phosphorylation of the channel a and b subunits [61], (5) pharmacological blockade of the KV channels by selective [e.g., 4-aminopyridine (4-AP), bepridil, and correolide] [62, 63] and nonselective (e.g., nicotine, endothelin-1, serotonin, fenfluramine, acute hypoxia, and dichloroacetate) inhibitors [64–66], and (6) transcriptional/translational and functional inhibition of KV channels by antiapoptotic proteins (e.g., Bcl-2) [67]. In PASMCs, a heterogeneity of KV channel currents exists [68, 69] (Fig. 7b). Different KV channel currents were recorded by Smirnov et al. in PASMCs isolated from conduit pulmonary arteries. Sixty-seven percent of the cell population comprised a large, rapidly activating IKv inhibited by 4-AP but insensitive to tetraethylammonium (TEA) (termed IKv1) and 33% of the cells expressed a smaller, slower activating current which was more sensitive to TEA than to 4-AP (termed IKv2).
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Resistance pulmonary arteries exhibited a third more uniform current resembling that of IKv1, but distinguished by a higher current density, a significantly larger time constant for activation, and more negative IKv activation and inactivation [69].
3.2 Ca2+-Activated K+ Channels Ca2+-activated K+ (KCa) channels are subcategorized on the basis of their conductance; large (BK), intermediate (IK), and small (SK). Like KV channels, BKCa channels also form tetramers which consist of four a and four b subunits. BKCa channels are ubiquitously expressed in VSMCs and can be activated by changes in both membrane potential and intracellular Ca2+ concentration[70]. The a subunit has seven transmembrane domains and features extracellular N-termini and intracellular C-termini [71]. The channel is regulated by the b subunit, which comprises two membrane-spanning domains separated by an extracellular loop. BKCa channels may act as a negative-feedback mechanism in response to depolarization and thus increased cytosolic Ca2+ concentration during vasoconstriction. Cox and Rusch showed that inhibition of Ca2+ efflux decreased the BKCa current and increased the KV current, indicating that cytosolic Ca2+ levels are critical regulators of these channels [72]. BKCa channels are present in PASMCs; however, their overall contribution to the whole cell current has species diversity and varies in different pulmonary artery tree regions [73].
3.3 ATP-Sensitive K+ Channels and Inwardly Rectifying K+ Channels KATP is another ubiquitous class of K+ channels; these channels show little or no voltage dependence and have low open probability under basal conditions. Structurally, KATP channels consist of an octameric complex of four pore-forming inward rectifier K+ (Kir) channel subunits (Kir 6.1 or 6.2) and four sulfonylurea receptors (SURs) [74]. Coexpression of SUR subunit 2B (SUR2B) with Kir 6.1 or 6.2 produces channels with the characteristics of two distinct KATP families, KNDP (named reflecting a primary role for various nucleotide diphosphates in their activation) and KATP (predominantly sensitive to ATP), respectively [75]. The expression of Kir 6.1 with SUR2B has been detected in human PASMCs [76]. Some evidence suggests they may contribute to resting membrane potential in PASMCs, although the regulatory mechanisms are still to be elucidated [76]. Additionally, there is no proposed role in HPV. Robertson et al. suggested that KATP channels are normally closed in the pulmonary arteries and are not activated by the levels of hypoxia that cause a constriction [77].
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3.4 KCNQ Channels KCNQ channels, previously known as members of the KV7 family of voltage-dependent K+ channels, were first identified in cardiac cells where mutations in a chromosomal locus on the KVLQT1 gene encode for an inherited form of long QT syndrome [78]. Five isoforms (KCNQ1–KCNQ5) have been identified and, of these, only KCNQ1 is known to be expressed in PASMCs [79, 80]. Linopirdine and XE991, KCNQ blockers, were demonstrated to be potent and selective pulmonary artery constrictors and data suggest that KCNQ channels could contribute to the regulation of resting membrane potential in PASMCs [79, 80].
3.5 K2P Channels K2P channels have four transmembrane segments (denoted M1–M4), two pore-forming domains (P1 and P2), a short cytoplasmic N-terminus, and a long cytoplasmic C-terminus. In addition, an extracellular loop exists between the M1 and P1 regions (as shown in Fig. 8) [81]. It is believed that two a subunits of K2P are necessary to form functionally active
Fig. 8 Structure and function of two-pore-domain K+ channels (K2P) channels. (a) Membrane topology of a K2P channel subunit featuring two pore regions, P1 and P2, and four transmembrane spanning domains, M1–M4, and cytoplasmic N- and C- termini. (b) Representative trace and I–V relationship recorded in TASK-expressing COS cells at pH 6.1, 7.4, and 8.4. The arrow indicates the zero-current level. (c) Summarized data indicating the pH dependence of TASK activity in COS cells at −50, 0, and +50 mV. (b, c Reproduced from Duprat et al. [89] with permission from Macmillan Publishers Ltd, copyright 1997)
A.L. Firth and J.X.-J. Yuan
channels. K2P channels are expressed in PASMCs and have a noninactivating current, denoted IKN, characterized by a lack of both voltage and time dependency [82]. To date, 14 K2P channels have been identified and these are subcategorized into five structural groups: 1 . TWIK-1 (KCNK1), TWIK-2 (KCNK6), and KCNK7 2. TASK-1 (KCNK3), TASK-3 (KCNK9), and TASK-5 (KCNK15) 3. TREK-1 (KCNK2), TREK-2 (KCNK10), and TRAAK (KCNK4) 4. TASK-2 (KCNK5), TALK-1 (KCNK16), and TALK-2 (KCNK17) 5. THIK-1 (KCNK13) and THIK-2 (KCNK12) Of these channels, TASK-1, TASK-2, TREK-1, TREK-2, and TWIK-2 have been detected in the pulmonary circulation [83–86]. K2P channels are considered proponents of the regulation of resting membrane potential; TASK-1 (TWIKrelated acid-sensitive K+ channel [59]), in particular, is able to regulate membrane potential by 10 mV in both a hyperpolarizing and a depolarizing direction [87]. In PASMCs from rats and rabbits [88] and, most recently from humans [87], the pharmacological profile of IKN has been shown to match TASK-1, being insensitive to glibenclamide, TEA, and Ca2+ channel blockers and having a high sensitivity to external pH but not cytosolic Ca2+ concentration. TASK-1 currents display outward rectification under physiological conditions conforming to the Goldman–Hodgkin–Katz current equation predicting the I–V curve, thus suggesting that TASK-1 is a pure K+-selective leak channel (as shown in Fig. 8) [89]. Inhibition of this channel by acid (H+) is dependent upon membrane voltage and potassium concentration, indicative of a pore-blocking inhibition mechanism. It has been shown that TASK-1 senses changes in pH within a physiological range via protonation of a histidine residue in the P1 pore-forming domain which leads to blocking of the channel [90]. It is also noteworthy that TASK-1, in addition to its acid sensitivity, exhibits sensitivity to hypoxia and, as such, these channels are proposed to be functionally important in mechanisms underlying HPV [87]. There is currently limited evidence suggesting a direct role of TASK in HPV or PAH; recently; however, it has been shown that endothelin-1, a vasoconstrictive agonist known to be upregulated in PAH, was shown to inhibit TASK-1 in human PASMCs [91].
3.6 Na+–K+-ATPase Na+–K+-ATPase, or Na+–K+ pump, is ubiquitously expressed and was the first enzyme to gain recognition as an ion pump. It enables the electrogenic, active transportation of both
13 Ion Channels and Transporters in the Pulmonary Vasculature: A Focus on Smooth Muscle
Na+ and K+. In each “cycle” the pump extrudes three Na+ and the cell accumulates two K+, the net change in charge hyperpolarizes the cell; the basic function of the Na+–K+ATPase is to maintain low cytosolic Na+ concentration and high cytosolic K+ concentration. The pump is a heterodimer formed from a catalytic a subunit and a glycosylated b subunit [92]. The a subunit is functionally important, conferring the binding sites for the physiological ligands (namely, Na+, K+, Mg2+, and ATP) and for ouabain, an inhibitor which displays tissue-specific affinity for the pump. The b subunit acts as a chaperone that ensures correct insertion of the a subunit in the plasma membrane and modulates the affinity of the enzyme for cations. Several distinct isoforms of the a subunit exist; the a1 isoform is ubiquitously expressed, whereas the a2–a4 isoforms are more limited in their distribution. It has recently been shown that the a2b1 and a1b1 isoforms of Na+–K+-ATPase exist in small membrane microdomains known as caveolae in PASMCs, whereas only the a1b1 isoform is detected in the noncaveolae regions of the plasma membrane [93]. The Na+–K+-ATPase utilizes energy derived from ATP hydrolysis to undergo conformational changes to enable binding and release of Na+ and K+ [94]. The pump may also be regulated by phosphorylation; PKA and PKC have been postulated to phosphorylate the a subunit [95]. Recently, small membrane proteins with a characteristic FXYD motif have been shown to associate with the Na+–K+-ATPase and modulate its properties for the transport of Na+ or K+, possibly implicating a role in pathophysiological states (reviewed by Geering [96]). Regulation of the Na+–K+-ATPase has a crucial role in the
Fig. 9 Structure and function of Na+ channels. (a) Planar schematic diagram showing structural arrangement of Na+ channel a and b1 and b2 subunits. (b) Representative currents showing inward INa in human PASMCs; the traces represent depolarizations, from a holding potential of −70 mV to test potentials between −80 and +80 mV in 20-mV increments. The inset indicates the summarized I–V relationship for INa. (c) Representative INa from PASMCs before, during, and after extracellular application of 1 mM tetrodotoxin. (b, c Reprinted from Platoshyn et al. [100] with kind permission from Springer Science+Business Media)
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regulation of the contractility of the vasculature [97] and, indeed, in the canine pulmonary artery, hypoxia inhibits the Na+–K+ ATPase [98].
4 Sodium Channels and Transporters 4.1 Voltage-Dependent Na+ Channels A rapidly activating and inactivating inward current (Iin) in rabbit PASMCs, dependent upon extracellular Na+, was first described by Okabe et al. [99]. The current has since been fully characterized in cultured human PASMCs [100]. Although the presence of Na+ channels is certain, their functional role in the pulmonary artery is not clear, especially as PASMCs have a resting membrane potential more positive than that at which the Na+ channels would be inactivated. Furthermore, tetrodotoxin (TTX), a specific Na+ channel inhibitor, has no effect upon membrane potential, cytosolic Ca2+ release, or proliferation in pulmonary arteries and, therefore, Na+ channels are generally regarded as unimportant in the control of the normal function of these vessels [100]. Na+ channels consist of four internally homologous subunits each containing six transmembrane domains; the S5–S6 transmembrane domains fold to create an internal pore (Fig. 9). These subunits form a single principal pore region: the structural basis of this pore determines the selectivity and conductance properties of the channel and was described in detail by
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Table 5 Expression and function of Na+ channels Species/cell
Subunit (gene)
Accessory subunits (gene)
Electrophysiology
Pharmacology
References
Inactivation TTX-sensitive [100] V0.5 = −75.5 ± 3 mV ta = 0.485 ± 0.06 ms (at 0 mV) th = 2.85 ± 0.41 ms (at 0 mV) Cultured human PASMC ND ND Inactivation 30% TTX-sensitive [102] V0.5 = −75.5 ± 0.8 mV K = 10.73 Primary cultured human ND ND Activation 44% TTX-sensitive [151] PASMC V0.5 = −25 ± 4 mV K = 4.2 ± 0.5 Primary cultured rabbit main ND ND Activation TTX-sensitive [99] PASMC V0.5 = −65 mV K = 5.7 ND not determined, V0.5 half-activation/inactivation potential, K slope factor for activation/inactivation, PASMC pulmonary artery smooth muscle cell,t time constant (dependency) of activation/inactivation, TTX tetrodotoxin Cultured human PASMC
SCN2A–SCN4A, SCN7A–SCN9A, SCN11A
SCN1B, SCN2B
Marban et al. [101]. Currently more than ten voltage-dependent Na+ channel genes (SCN1A–SCN11A) have been identified; of these it is predominantly those forming TTX-insensitive channels (Nav1.5) that have been identified at the mRNA expression level in rabbit PASMCs (Table 5). A TTX-sensitive current has, however, been recorded in rabbit PASMCs, characterized by a very rapid inactivation (less than 10 s) [99]. In cultured human PASMCs, mRNA was detected for Na+ channel genes SCN2A, SCN3A, SCN4A, SCN7A, SCN8A, SCN9A, and SCN11A and accessory b subunits SCN1B and SCN2B (Table 5) [100]. The rapid outward current in cultured human PASMCs was sensitive to submicromolar concentrations of TTX [100, 102]. Figure 9 shows representative Na+ currents recorded from cultured human PASMCs at a series of voltages and confirms the TTX sensitivity of these currents.
4.2 Amiloride-Sensitive Epithelial Na+ Channel The amiloride-sensitive epithelial Na+ channel (ENaC) is most frequently associated with defective chloride transport in patients with cystic fibrosis. With regard to the pulmonary circulation, ENaC is implicated in the progression of highaltitude pulmonary edema, characteristically associated with exaggerated hypoxia pulmonary hypertension. During highaltitude pulmonary edema, hypoxia inhibits reabsorption of Na+ and concomitant fluid from the alveolar space by inhibiting ENaC and Na+–K+-ATPase.
4.3 Na+-Dependent Exchangers In addition to the NCX, discussed earlier in this chapter, two other Na+-dependent exchangers are expressed and are func-
tional in the pulmonary circulation; the Na+–Mg2+ exchanger and the Na+–H+ exchanger (NHE). Such extrusion mechanisms are functionally important in the maintenance of ion homeostasis.
4.3.1 Na+–H+ Exchanger The NHE is electrogenic, exchanging Na+ for H+ and is primarily involved in pH homeostasis in many mammalian cell types, acting alongside several other ion transport systems, such as the Na+-independent Cl−–HCO3 and Na+-dependent Cl−–HCO3 transporters. The NHE is activated by an increase in intracellular H+ concentration and extracellular Na+ is exchanged for intracellular H+ in a 1:1 stoichiometry. Excessive stimulation of the exchanger will ultimately increase the concentration of intracellular Na+, which, in turn, can drive the Na+–K+-ATPase, increasing the energy demands of the cell, and/or activate the NCX, driving Ca2+ into the cell. The NHE has recently been studied in PASMCs and significant roles in apoptosis, proliferation [2, 103], and hypoxia-induced pulmonary hypertension are evident [104, 105]. In PASMCs from 3-week-old hypoxic rats, it was found that pHcyt and NHE1 mRNA expression levels were significantly elevated and that inhibition of the NHE by 5-(N,N-dimethyl) amiloride elevated the rate of apoptosis [103]. Additionally, exposure of mouse PASMCs to chronic hypoxia increased pHcyt, NHE activity, and NHE1 expression [105]; such changes have been confirmed to contribute to the development of pulmonary hypertension [104]. Deficiency of NHE1 in mice prevented the development of hypoxia-induced pulmonary hypertension and vascular remodeling [104]. The NHE has additionally been shown to aid in recovery from acid loads during hypoxia [106]. Since the cloning of the first
13 Ion Channels and Transporters in the Pulmonary Vasculature: A Focus on Smooth Muscle
NHE (NHE1) by Pouyssegur’s laboratory in 1989, nine isoforms of the exchanger have been identified (NHE1–NHE9) [107]. NHE9 is more restricted in its distribution, unlike NHE1, which is ubiquitously expressed in the plasma membrane. The structures of the 815 amino acid proteins are similar; NHE2–NHE4 share 45–65% amino acid homology within the transmembrane domains. The exchangers contain ten to twelve helical hydrophobic membrane-spanning domains, an approximately 500 amino acid N-terminus functionally responsible for catalyzing the amiloride-sensitive NHE and an approximately 300 amino acid C-terminus acting as a regulatory domain [108].
4.3.2 Na+–Mg2+ Exchanger The Na+–Mg2+ exchanger has been widely studied in erythrocytes and in VSMCs, with a pertinent role in systemic hypertension having been identified; however, is yet to be cloned and extensively studied in the pulmonary vasculature. The Na+–Mg2+ exchanger presents a predominant mechanism for Mg2+ extrusion from the cell and has been identified in a variety of tissues, including sheep ruminal epithelial cells [109], VSMCs [3], and erythrocytes [110]. Interestingly, activation of the NHE has been shown to modulate Na+–Mg2+ transport in VSMCs and increased activity of NHE could influence the change in Na+dependent regulation of cytosolic Mg2+ concentration observed in spontaneously hypertensive rats [111]. A role for intracellular Mg2+ in the regulation of KV channels in rat PASMCs has recently been shown and presents a potential for the activity of the Na+–Mg2+ exchanger to contribute to the development of HPV and/or PAH [112].
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5 Chloride Channels and Transporters Despite chloride being the most abundant intra- and extracellular ion, until recently there was very little data on the presence and regulation of Cl− channels in the pulmonary vasculature. Activation of Cl− channels results in the movement of Cl− ions along their electrochemical gradient from inside the cell to the extracellular environment, resulting in a net positive current in the cell (i.e., inside the cell becomes more positive and the cell depolarizes). Cl– ions can be actively accumulated in the cell via one of three uptake mechanisms: the Na+–K+–2Cl− cotransporter, the Cl–HCO3 exchanger, or the relatively unknown “pump III” [113]. In the pulmonary circulation, the most extensively studied Cl– channel current is stimulated by an elevation of cytosolic Ca2+ concentration, commonly referred to as the calcium-activated chloride current (IClCa). In addition, voltage-gated and volume-sensitive Cl− channels exist, though they are not as well characterized in the pulmonary vascular system (Table 6). Chloride transportation is involved in cell-volume regulation and is implicated in essential cellular functions such as progression of the cell cycle, cell proliferation, and contraction, thus having a pertinent role in the development of pulmonary edema and potentially pulmonary vascular remodeling.
5.1 Ca2+-Activated Cl− Channels Calcium-activated chloride channels are stimulated by an elevation of cytosolic Ca2+ concentration,due to either intracellular store (SR) release of Ca2+ or influx of Ca2+ ions. In the pulmonary artery of rabbits and rats it has been shown that
Table 6 Expression and function of Cl− channels Ca -activated (ICl(Ca))
cGMP-activated (ICl(cGMP,Ca))
160–600 nM ~74 nM 1–4 pS I−>Br−>Cl− Below 1 mM at resting cytosolic Ca2+ concentration 2–20 mM 15–250 mM Independent
No effect 3–6 mM
2+
EC50 cytosolic Ca2+ EC50 cGMP Conductance Halide permeability Voltage/time dependence
Niflumic acid IC50 DIDS IC50 Zn2+ IC50 TFP IC50 Protein tyrosine kinase inhibition
Br−>I−, Cl − or Cl−>I− Independent
No effect at 100 mM No effect at 200 mM ~2–6 mM
Gene Bestropohin-3 References [152] [152] DIDS 4 4′-diisothiocyanato-stilbene-2,2′-disulfonic acid, TFP trifluoperazine
Ca2+/CaMKII-activated (ICl(CaMKII))
Volume-sensitive (ICl(swell))
~430 nM Not sensitive 25–30 pS I−>Br−»Cl−>>gluconate Voltage-dependent and voltage-independent Ca2+ binding Not sensitive
5.7 mM Tyrphostin B46 (50 mM), genistein (100 mM) [153]
[154]
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both norepinephrine and histamine (GPCR agonists) activate IClCa [114, 115]. Furthermore, activation of the reverse mode of the NCX can also activate IClCa, a potential mechanism in the development of pulmonary hypertension [116]. Many investigators have characterized the biophysical and pharmacological properties of IClCa in human and animal PASMCs. In a study on rat PASMCs, it was shown that depolarization or agonist-mediated elevation of cytosolic Ca2+ concentration elicited a time-dependent outward Cl− current that reversed near to the equilibrium potential for Cl−, approximately 0 mV (Fig. 10a), and that was inhibited by the Cl− channel blocker niflumic acid (10–50 mM) [117]. Cl− channel inhibition also attenuated serotonin-induced membrane depolarization and agonist-induced pulmonary artery contraction [117]. Although the electrophysiological and pharmacological properties of IClCa are relatively well defined (Fig. 10b), the molecular correlate for IClCa in the pulmonary artery is not precisely known. The ClCa channels are heterogeneous with several different characteristics; currently they can be divided into three distinct families: (1) channels activated directly by Ca2+, (2) channels requiring Ca2+/CaM-dependent protein kinase II, and (3) cyclic GMP (cGMP)-dependent channels. A group of proteins, known as CLCA, and controversially associated with ICaCl, are the bestrophin family [118]. Expression of bestrophin proteins (Best-1 to Best-4) in different cell types correlates with the appearance of a Cl− conductance. The cGMP-dependent Ca2+activated Cl− current was recently characterized in several vascular beds, including the pulmonary artery. This current had a high cytosolic calcium sensitivity Ca2+ concentration sensitivity, a linear voltage dependence, and permeability to anions were found [118, 119]. With use of small interfering RNA, Best-3 was shown to be a critical component of the endogenous ICaCl in smooth muscle cells of intact pulmonary artery walls [118]. Although Best-3 may encode a “nonclassical” Ca2+-activated Cl− current owing to the lack of voltage sensitivity, it is the “classical” Ca2+-activated Cl− current that is expressed at its highest level in the pulmonary artery. In a recent breakthrough study, Caputo et al. provided evidence that the membrane protein TMEM16A is an intrinsic component of the voltage-sensitive Ca2+-dependent Cl− channel; its expression in the pulmonary vasculature remains to be investigated [120]. TMEM16A is an 840 amino acid, eight transmembrane spanning protein with cytoplasmic N- and C-termini; it undergoes alternative splicing, generating a variety of isoforms with significant sequence homology (e.g., TMEM16b, approximately 60% sequence homology). The TMEM16 proteins may represent subsets of chloride channels with a biophysical profile that remains to be characterized. Bovine pulmonary endothelial cells also have ICaCl; elevation of cytosolic Ca2+ concentration activated an outwardly rectifying Cl− current for which the reversal potential was dependent upon extracellular Cl− concentration and the current inactivated rapidly at negative membrane potentials and
A.L. Firth and J.X.-J. Yuan
Fig. 10 Structure and function of Cl− channels. (a) A representative family of traces for a Ca2+-activated Cl− current in PASMCs elicited by a series of test potentials from −60 to +60 mV in 20-mV increments (from a holding potential of −70 mV). The cells are superfused with modified Kreb’s solution including 1.8 mM Ca2+. (b) Representative traces of Ca2+-activated Cl− currents recorded in the presence of either 3 mM ATP (left) or AMP-PNP 5′-adenylyl-beta,gamma-imidodiphosphate (right) at various intracellular Ca2+ concentrations. The voltage protocol is featured below the traces. (a Reproduced with permission [117]. (b) Copyright 2006 Angermann et al. [156])
had a single channel conductance of 7.9 ± 0.7 pS. Furthermore, strong calcium chelation with 1,2-bis(2-aminophenoxy) ethane-N,N,N¢,N¢-tetraacetic acid prevented ATP-induced activation of ICaCl, and 4,4¢-Diisothiocyanatostilbene-2,2¢disulfonic acid (DIDS) and niflumic acid inhibited it in a voltage-dependent manner [121].
13 Ion Channels and Transporters in the Pulmonary Vasculature: A Focus on Smooth Muscle
5.2 Voltage-Gated Cl− Channels The CLCN family of voltage-gated chloride channels has nine identified members. Although a significant similarity in sequence homology exists, there is a distinct diversity in function. CLCN1–CLCN7, CLCNKa, and CLCNKb are the currently identified voltage-sensitive chloride channel genes. CLCN channels characteristically have ten transmembrane spanning regions with intracellular C- and N-termini and it is suggested that functional channels arise from dimerization of genes. The expression of CLCN3 has been shown in the human fetal lung and in smooth muscle cells and endothelial cells of the large and small pulmonary arteries, pulmonary epithelial cells, and bronchial smooth muscle cells [122]. The first functional evidence demonstrated an upregulation of the ClCn-3 gene in both rat pulmonary artery in the monocrotalineinduced model of pulmonary hypertension and PASMCs cultured from canines and incubated with inflammatory mediators. Furthermore, overexpression of CLCN3 improved resistance of these cells to reactive oxygen species [123], potential role in the pathogenesis of pulmonary hypertension.
5.3 Volume-Sensitive Cl− Channels Volume-sensitive chloride channels are ubiquitously expressed and activated in response to cell swelling. Yamazaki et al. demonstrated that functional volume-sensitive chloride channels were present in canine PASMCs [124]. Exposure of cells to hypotonic solutions increased Cl− conductance, a current exhibiting outward rectification in a symmetrical Cl− gradient. This channel was sensitive to inhibition by DIDS, ATP, and tamoxifen. It is currently not known whether this current is carried by a homomeric channel or CLCN3 in heteromeric association [124].
6 Aquaporins Aquaporins are a family of water-transporting membranebound proteins. To date 13 mammalian aquaporin homologues have been detected; several of these are expressed and functional in the human lung. Aquaporins are six transmembrane spanning proteins with five interconnecting loops and intracellular N- and C-termini. Two hydrophobic loops, the cytoplasmic B loop and the extracellular E loop, each containing a signature motif consisting of a tripeptide sequence of asparagine, proline, and alanine, are proposed to from the aqueous pore region of the channel [125]. Aquaporins are important for water permeability across microvessels in intact lungs; more specifically, in knockout mice, aqua-
239
porin-1 was demonstrated to provide an important route for osmotic and hydrostatic water movement between the air space, interstitial compartment, and microvessels [126, 127]. Aquaporins are most influential in regulating pulmonary fluid homeostasis and, in a bleomycin-induced pulmonary edema rat model, the expression of aquaporin-1 has been shown to be significantly reduced. It is postulated that alterations in aquaporin expression may lead to pathophysiological conditions [126, 128].
7 Interaction of Ion Channels During Homeostasis and Disease Ion channels form a complex signaling system functioning to maintain a cellular homeostasis: a balance between apoptosis and proliferation, between contraction and relaxation, between cell shrinkage and cell swelling, and control of cellular excitability and resting membrane potential. Alterations in the expression and function of ion channels are a significant factor in the mechanisms leading to the development and persistence of PAH. To avoid repetition with other chapters in this book, the involvement of ion channels in the development and progression of pulmonary vascular disease will only be discussed very briefly here using K+ channels as an example.
7.1 Hypoxic Regulation of KV Channel Expression and Function Potassium channels are key regulators in cell signaling in response to hypoxia and, therefore, the development of acute HPV and chronic hypoxia-induced pulmonary hypertension. Post et al. were the first to observe that a decrease in oxygen tension to hypoxic levels directly inhibited K+ currents in concert with membrane depolarization in isolated canine PASMCs, an effect not present in canine renal arterial smooth muscle cells [129]. Subsequently, Archer et al. identified KV1.5 and KV2.1 as the potential molecular correlates for the slowly inactivating, 4-AP-sensitive, charybdotoxin-insensitive delayed rectifier currents in resistance artery PASMCs [130]. Furthermore, acute hypoxia has been shown to depolarize PASMCs by inhibiting K+ currents, and extended exposure to hypoxia (more than 72 h) is known to decrease KCNA5 gene expression in these cells [66]. A distinct heterogeneity in KV channel sensitivity to hypoxia exists, and is postulated to be due to differential expression and functional sensitivity of the KV channels to hypoxia [131]. Reviews by Mauban et al. and Weir and Olschewski convey the key aspects of the role of ion channels in HPV [132, 133].
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7.2 Role of Plasmalemmal K+ Channels in Apoptosis and Proliferation in PASMCs Initiation of apoptosis is recognized by cell shrinkage, a process regulated by a change in the ionic composition of the extracellular and intracellular environments of the cell. Apoptotic volume decrease is an early apoptotic event in which K+ efflux through open plasmalemmal K+ channels creates a positive potential outside the cell; in turn, Cl− leaves the cells along its concentration gradient. An osmolarity imbalance is created and, in an effort to maintain the osmotic balance, water leaves the cell via aquaporins and cells shrink. Several studies have confirmed the highly influential role of K+ channels in the process of apoptosis. In PASMCs, overexpression of KCNA5, encoding KV channel isoform KV1.5, enhanced apoptosis, whereas inhibition of K+ channels by 4-AP reduced staurosporine-induced apoptotic volume decrease and apoptosis [62]. Furthermore, overexpression of antiapoptotic oncogene Bcl-2 causes a downregulation of KV channel a subunit mRNA and decreases KV channel activity [67].
8 Summary Ion channels are fundamental to the maintenance of cellular homeostasis and to the excitability of cells in response to a variety of stimuli. Alterations in the expression and function of ion channels are key features in the development and pathogenesis of pulmonary vascular disease and thus ion channels and consequential signaling cascades are potential sites for therapeutic intervention. Acknowledgements The authors acknowledge the California Institute of Regenerative Medicine and the NIH for supporting their research.
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13 Ion Channels and Transporters in the Pulmonary Vasculature: A Focus on Smooth Muscle 107. Sardet C, Franchi A, Pouyssegur J (1989) Molecular cloning, primary structure, and expression of the human growth factor-activatable Na+/H+ antiporter. Cell 56:271–280 108. Noel J, Pouyssegur J (1995) Hormonal regulation, pharmacology, and membrane sorting of vertebrate Na+/H+ exchanger isoforms. Am J Physiol 268:C283–C296 109. Schweigel M, Park HS, Etschmann B, Martens H (2006) Characterization of the Na+-dependent Mg2+ transport in sheep ruminal epithelial cells. Am J Physiol Gastrointest Liver Physiol 290:G56–G65 110. Rivera A, Ferreira A, Bertoni D, Romero JR, Brugnara C (2005) Abnormal regulation of Mg2+ transport via Na/Mg exchanger in sickle erythrocytes. Blood 105:382–386 111. Touyz RM, Schiffrin EL (1999) Activation of the Na+-H+ exchanger modulates angiotensin II–stimulated Na+-dependent Mg2+ transport in vascular smooth muscle cells in genetic hypertension. Hypertension 34:442–449 112. Firth AL, Yuill KH, Smirnov SV (2008) Mitochondria-dependent regulation of Kv currents in rat pulmonary artery smooth muscle cells. Am J Physiol Lung Cell Mol Physiol 295:L61–L70 113. Chipperfield AR, Harper AA (2000) Chloride in smooth muscle. Prog Biophys Mol Biol 74:175–221 114. Hogg RC, Wang Q, Helliwell RM, Large WA (1993) Properties of spontaneous inward currents in rabbit pulmonary artery smooth muscle cells. Pflugers Arch 425:233–240 115. Wang Q, Large WA (1993) Action of histamine on single smooth muscle cells dispersed from the rabbit pulmonary artery. J Physiol 468:125–139 116. Leblanc N, Leung PM (1995) Indirect stimulation of Ca2+-activated Cl− current by Na+/Ca2+ exchange in rabbit portal vein smooth muscle. Am J Physiol 268:H1906–H1917 117. Yuan XJ (1997) Role of calcium-activated chloride current in regulating pulmonary vascular tone. Am J Physiol 272:L959–L968 118. Matchkov VV, Larsen P, Bouzinova EV et al (2008) Bestrophin-3 (vitelliform macular dystrophy 2-like 3 protein) is essential for the cGMP-dependent calcium-activated chloride conductance in vascular smooth muscle cells. Circ Res 103:864–872 119. Sun H, Tsunenari T, Yau KW, Nathans J (2002) The vitelliform macular dystrophy protein defines a new family of chloride channels. Proc Natl Acad Sci U S A 99:4008–4013 120. Caputo A, Caci E, Ferrera L et al (2008) TMEM16A, a membrane protein associated with calcium-dependent chloride channel activity. Science 322:590–594 121. Nilius B, Prenen J, Szücs G et al (1997) Calcium-activated chloride channels in bovine pulmonary artery endothelial cells. J Physiol 498:381–396 122. Lamb FS, Clayton GH, Liu B-X, Smith RL, Barna TJ, Schutte BC (1999) Expression of CLCN voltage-gated chloride channel genes in human blood vessels. J Mol Cell Cardiol 31:657–666 123. Dai Y-P, Bongalon S, Hatton WJ, Hume JR, Yamboliev IA (2005) ClC-3 chloride channel is upregulated by hypertrophy and inflammation in rat and canine pulmonary artery. Br J Pharmacol 145:5–14 124. Yamazaki J, Duan D, Janiak R, Kuenzli K, Horowitz B, Hume JR (1998) Functional and molecular expression of volume-regulated chloride channels in canine vascular smooth muscle cells. J Physiol 507:729–736 125. Fu D, Lu M (2007) The structural basis of water permeation and proton exclusion in aquaporins. Mol Membr Biol 24:366–374 126. Bai C, Fukuda N, Song Y, Ma T, Matthay MA, Verkman AS (1999) Lung fluid transport in aquaporin-1 and aquaporin-4 knockout mice. J Clin Invest 103:555–561 127. Carter EP, Olveczky BP, Matthay MA, Verkman AS (1998) High microvascular endothelial water permeability in mouse lung measured by a pleural surface fluorescence method. Biophys J 74:2121–2128
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Chapter 14
Receptor-Mediated Signal Transduction and Cell Signaling Fiona Murray, Jason X.-J. Yuan, and Paul A. Insel
Abstract The maintenance of low resistance, pressure, and tone in the pulmonary circulation is dependent on the interaction of circulating and locally produced vasomodulatory regulators; many such vasoactive mediators act via receptormediated signaling pathways. Many types of receptors that regulate pulmonary vascular tone are expressed on the plasma membrane of cells, although vasomotor activity is also influenced by intracellular receptors, such as calcium-release receptors and receptors that regulate transcription (e.g., steroid hormone receptors). The pulmonary vasculature expresses a wide variety of receptor classes and subtypes that facilitate the interaction of cells of the pulmonary circulation with their extracellular environment (hormones, neurotransmitters, and other factors in the extracellular milieu play key roles in modifying blood flow under both physiological and pathophysiological conditions). One can identify such receptors by assessing the binding of radioligands, molecular cloning and expression studies, antisense approaches, and/or by conducting studies with transgenic or knockout animals. Receptormediated signaling in the pulmonary circulation changes with development and disease, is highly species specific, cell-type specific, and often depends on an intact endothelium. Moreover, the accessibility of plasma membrane receptors for neurotransmitters and hormones from the extracellular environment makes them excellent drug targets. The major classes of membrane receptors that regulate pulmonary vascular tone are G-protein-coupled receptors, ligand-gated ion channels, and receptor protein kinases [receptor tyrosine kinase and serine/threonine kinase receptors]. This chapter provides an overview of signaling by cell-surface receptors in the pulmonary circulation and highlights mediators whose activation regulates pulmonary vascular development, tone, and permeability. Keywords Signal transduction • Intracellular signaling cascades • Molecular pharmacology • G protein-coupled receptor • Tyrosine kinase receptor F. Murray (*) Departments of Medicine and Pharmacology, University of California at San Diego, BSB 3073, 9500 Gilman Drive, La Jolla, CA, 92093-0636, USA e-mail:
[email protected] 1 Introduction The maintenance of low resistance, pressure, and tone in the pulmonary circulation is dependent on the interaction of circulating and locally produced vasomodulatory regulators; many such vasoactive mediators act via receptor-mediated signaling pathways [1–3]. Many types of receptors that regulate pulmonary vascular tone are expressed on the plasma membrane of cells, although vasomotor activity is also influenced by intracellular receptors, such as calcium-release receptors and receptors that regulate transcription (e.g., steroid hormone receptors). The pulmonary vasculature expresses a wide variety of receptor classes and subtypes that facilitate the interaction of cells of the pulmonary circulation with their extracellular environment (hormones, neurotransmitters, and other factors in the extracellular milieu play key roles in modifying blood flow under both physiological and pathophysiological conditions). One can identify such receptors by assessing the binding of radioligands (i.e., radiolabeled agonists or antagonists), molecular cloning and expression studies, antisense approaches, and/or by conducting studies with transgenic or knockout animals. Receptor-mediated signaling in the pulmonary circulation changes with development and disease, is highly species specific, cell-type specific, and often depends on an intact endothelium [1–4]. Moreover, the accessibility of plasma membrane receptors for neurotransmitters and hormones from the extracellular environment makes them excellent drug targets. The major classes of membrane receptors that regulate pulmonary vascular tone are G-protein-coupled receptors (GPCR), ligand-gated ion channels, and receptor protein kinases [receptor tyrosine kinase (RTK) and serine/threonine kinase receptors]. This review provides an overview of signaling by cell-surface receptors in the pulmonary circulation and highlights mediators whose activation regulates pulmonary vascular development, tone, and permeability. Receptor-dependent signaling cascades contribute to the progression of diseases such as pulmonary arterial hypertension (PAH) and pulmonary edema; however, these topics will be discussed in more detail in subsequent chapters and have been reviewed elsewhere [1–3].
J.X.-J. Yuan et al. (eds.), Textbook of Pulmonary Vascular Disease, DOI 10.1007/978-0-387-87429-6_14, © Springer Science+Business Media, LLC 2011
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2 G-Protein-Coupled Receptors Over 30% of all prescription drugs, in addition to many hormones and neurotransmitters, act via GPCRs. GPCRs are a superfamily of receptors that have a similar general structural arrangement: seven transmembrane spanning domains, which are seven a-helices, linked by three alternating intraand extracellular loops. GPCRs are the largest receptor family in the human genome, constituting approximately 3% of the genes [5–7]. Activation of these receptors occurs when ligands bind to the receptors (depending on the GPCR, to extracellular or transmembrane binding pockets), thereby altering receptor confirmation and promoting coupling, in particular by the third intracellular loop and proximal portion of the C-terminal tail, to intracellular heterotrimeric (G) proteins, which comprise a guanosine diphosphate (GDP)-bound Ga subunit and a Gbg complex (Fig. 1). In mammalian cells at least 15 genes encode for the Ga subunits, five for the Gb subunits, and 12 for Gg subunits, but in addition, there are
Fig. 1 G-protein-coupled-receptor (GPCR)-mediated signaling in the pulmonary artery smooth muscle cell (PASMC). GPCR activation promotes the exchange of the G-protein-bound GDP for GTP, thereby inducing a conformational change and dissociation of Ga from the Gbg and thus initiating downstream signaling. The Gbg subunits can directly activate phosphatidylinositol 3-kinase (PI3K) and phospholipase Cb (PLCb). PLCb, activation (by Gbg and Gaq) promotes the hydrolysis of phosphatidylinositol 4,5-bisphosphate and yields the intracellular messengers 1,2-diacylglycerol (DAG) and inositol 1,4,5-trisphosphate. DAG remains membrane-bound and promotes the translocation of protein kinase C from the cytoplasm to the membrane and its subsequent activation. Ga12/13-dependent pathways (guanine nucleotide exchange factors for Rho activate the small G protein RhoA followed by Rho kinase) and Gai-dependent pathways (inhibition of adenylyl cyclase decreases cyclic AMP accumulation) lead to PASMC vasoconstriction and proliferation. In contrast Gas stimulates the production of cyclic AMP via activation of adenylyl cyclase, which in turn decreases PASMC proliferation and enhances vasodilation
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many splice variants of each [8]. Ligand binding triggers the exchange of guanosine triphosphate (GTP) for GDP on the a-subunit, thus facilitating dissociation from the Gbg and usually from the receptor as well. This classic model has recently been challenged, on the basis of evidence suggesting that a conformational change within the G protein may not necessarily induce subunit dissociation and that such dissociation may not be obligatory for the activation of G-proteinregulated effector molecules [9]. Inactivation of Ga, which occurs when GTP is hydrolyzed to GDP, determines the rate and length of the signal derived from GPCR-promoted activation of heterotrimeric G proteins. Intrinsic GTPase activity of the Ga subunits can be promoted by GTPase-activating proteins, such as regulator of G protein signaling (RGS) proteins, or even by effector molecules, such as phospholipase Cb (PLCb); specific RGS proteins terminate the activation of particular G proteins [10]. To date, limited information is available regarding the role of particular RGS proteins in cells from the pulmonary circulation; however, since knockdown of specific RGS family members, such as RGS2, can lead to systemic hypertension by amplifying signaling responses to vasoactive mediators, this may be an interesting topic for future research and may represent a novel way to manipulate GPCR signaling in the pulmonary circulation [11]. G proteins are divided into four main classes according to their a subunit, Gas, Gai, Gaq/11, and Ga12/13, which each lead to the activation/inactivation of signaling pathways that control the production of second messengers and gene expression: Gas stimulates the membrane-associated enzyme adenylyl cyclase (AC) which increases the level of cyclic adenosine 3¢,5¢-monophosphate (cAMP), and regulates Ca2+ channels; Gai inhibits AC activity, decreasing the level of cAMP, and also regulates K+ and Ca2+ channels; Gaq/11 stimulates membrane-bound PLCb, which cleaves phosphatidylinositol 4,5-bisphosphate into inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG); Ga12/13 regulates GTPase RhoA, a low molecular weight G protein. Gbg can act as a single entity to activate both concordant and disconcordant signals, for example, by directly regulating Kir3 K+ channels, PLCb, AC, voltage-gated calcium channels, and even other proteins, such as PDZ-domain-containing proteins [5]. The stoichiometric relationship of GPCR signaling pathways, the absolute concentrations and relative proportions of each component, is important for defining the efficiency of each pathway. Data in a number of cell types for the b-adrenergic receptor (AR) pathway indicate that the ratio of receptors (fewer than 10,000 per cell) to G proteins (about 1,000,000 per cell cell) to ACs (about 30,000 per cell) is approximately 1:100:3, suggesting that although receptor activation of Gas is important for amplification of signaling, AC is the critical component that limits maximal b-AR response [12]. Limited data are available for the stoichiometric ratios of other classes of G proteins and their effectors; defining the critical components in Gaq/11, Ga12/13, and for Gbg subunits will be important
14 Receptor-Mediated Signal Transduction and Cell Signaling
for determining which components determine potency (concentration dependence of agonists) and efficacy (maximal response) of their signaling pathways. Stoichiometry cannot fully explain the efficient activation and amplification of response to GPCR agonists: such observations have provided a rationale for the assessment of compartmentation (i.e., colocalization) of GPCRs and their downstream effectors via “scaffolding” proteins or in specific plasma membrane domains [13–15]. The spatial and temporal organization of GPCR signaling is regulated by scaffolding and GPCR interacting proteins such as b-arrestin, A-kinase anchoring proteins (AKAPs), and PDZ domain proteins that colocalize membrane proteins in specific subcellular domains: PDZ domains primarily bind proteins containing a C-terminal S/TXV(L/I) motif, which is present in some GPCRs such as the b2-AR, in which that domain facilitates binding to a Na+–H+ exchanger regulatory factor [16]. In addition to such adaptor proteins there are membrane microdomains, such as clathrin-coated pits, lipid (membrane) rafts, and caveolae (a subset of lipid rafts that contain the structural protein caveolin) that serve as cellular platforms and bring receptors together with downstream mediators, thereby facilitating receptor-, tissue-, and cellspecific signal transduction. The movement of receptors and their effectors into and out of these complexes contributes to both the qualitative (i.e., as a determinant of “preferred” signaling partners) and quantitative (e.g., rapidity and reversal) regulation of cell signaling. A number of GPCRs localize in membrane rafts/caveolae either before (e.g., b-AR subtypes) or after (e.g., bradykinin receptors) stimulation by agonists [15]. Caveolae are highly expressed in the pulmonary circulation – indeed, the pulmonary vascular endothelium is the most enriched source of caveolae in the body and appears to be functionally important and also contributes to disease: moreover, increased expression in PASMC occurs in PAH; the role of microdomains in pulmonary function is discussed in detail in subsequent chapters [15, 17]. Further levels of complexity have recently emerged regarding GPCR signaling. For example, GPCRs were previously thought to couple preferentially to only one G protein; however, coupling to more than one G protein can occur; for example, b2-AR can activate both Gas and Gai [18]. In addition, cross talk between GPCR pathways occurs, such that agonists that activate Gaq can potentiate stimulation of GaS [19]. In addition “ligand-directed” signaling appears to occur, i.e., particular agonists that bind to the same receptor can stimulate distinct G protein signaling cascades [20, 21]. The large number of isoforms of downstream mediators, such as AC and phospholipase C (PLC), or the fact that multiple active receptor conformations exist, may be important for this complex regulation. Assembly of individual GPCRs into homodimers or heterodimeric combinations also can occur, although the precise physiological role of such multimers is not yet well defined [22].
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2.1 GPCRs and the Pulmonary Circulation Individual cells can express up to 100 different GPCRs so it is likely that the full range of GPCRs in the pulmonary circulation have yet to be fully defined. Recent work profiling GPCR expression indicates that the lung expresses previously unappreciated GPCRs, many of which may have physiological and pathological roles [23]. Given this caveat, we speculate that key GPCRs involved in regulation of the pulmonary circulation may not have yet been studied. Endogenous mediators acting via specific GPCRs can have differing effects depending on the predominant receptor subtype in the vessel, the presence or absence of endothelium, and on the initial tone of the pulmonary circulation [1– 4]. For example, angiotensin II (Ang II), endothelin (ET)-1, or 5-hydroxytryptamine (5-HT) antagonists do not vasodilate the normal pulmonary circulation but they attenuate hypoxic pulmonary vasoconstriction, and under normoxic conditions some vasodilators have little effect on pulmonary tone [1, 24, 25]. Together, these data imply that when vascular tone is increased, the lung is more tightly under the control of vasoactive mediators than in the normal pulmonary circulation. Because effects of agonists and antagonists can be influenced by the experimental conditions employed, caution should be taken when interpreting data from isolated pulmonary arteries (PAs) on which tone is added, or even cultured isolated pulmonary cells that are typically grown in enriched nutrients and O2 without the interaction of other cells. Thus, the response of a mediator in vitro does not necessarily define its response in the pulmonary circulation in vivo.
2.1.1 Mechanisms of G-Protein-Mediated Pulmonary Arterial Vasodilation GPCR agonists that vasodilate the pulmonary circulation act via increasing the intracellular concentration of the second messengers cAMP in the case of b2-AR and cyclic guanosine 3¢,5¢-monophosphate (cGMP) [e.g., for agonists that increase the levels of nitric oxide (NO)] in PA smooth muscle cells (PASMCs; Fig. 1). Agonists that activate Gas stimulate the production of cAMP via activation of AC. Nine membranebound isoforms of mammalian ACs have been characterized, each with their own tissue distribution and regulation; AC6, which is Gas-coupled, is highly expressed in both PASMCs and PA endothelial cells (PAECs) [26, 27]. The intracellular level of cAMP is also dependent on its hydrolysis by a family of 11 enzymes, phosphodiesterases (PDEs). The basal activity of PDEs (PDE1, PDE3, and PDE4 appear to control cAMP degradation in normal PASMCs and PAECs) maintains a high level of intracellular cyclic nucleotides and thus a low level of vasomotor tone. cAMP acts via a number of downstream effectors, the most well characterized
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being cAMP-dependent protein kinase (PKA) [28]. PKA phosphorylates serine and threonine residues on targets such as myosin light chain kinase (MLCK), resulting in vasodilation. In addition cAMP can also act via PKA-independent mechanisms, in particular cyclic-nucleotide-gated (CNG) channel, and the more recently identified effector Epac (exchange protein directly activated by cAMP; Epac-1 and Epac-2 are known), a guanine nucleotide exchange factor (GEF) for the a low molecular weight G protein Rap-1, the role of which in the pulmonary circulation is as-yet poorly defined [29]. The activation of the specific cAMP effectors may be GPCR-dependent, for example, in airway smooth muscle cells b2-AR agonists decrease proliferation preferentially via Epac [30]. AKAPs also aid in coordinating the biological effects of cAMP by targeting cAMP effectors to distinct compartments in the cell and bringing them in close proximity to specific intracellular mediators, in particular by facilitating effects of PKA activation [31]. cAMP not only produces relaxation of the PA but also can reduce PASMC proliferation and alter gene expression. For example, cAMP (via PKA) modulates expression of genes by transcriptional activation of CREB, the cAMP response element binding protein. CREB alters gene expression through its interaction with cis-regulatory cAMP-responsive DNA elements, which are located in cAMP/PKA-target genes, such as cyclooxygenase-2, an increase in activity of which persists long after the cAMP has been degraded [32, 33]. In addition, cAMP acts via PKA to protect PAEC barrier function by promoting cell–cell and cell–matrix association through enhanced phosphorylation of particular proteins [34]. GPCR stimulation, for example, via ET-1, on PAECs can also lead to vasodilation through effects on PASMCs: activation of Gaq receptors on PAECs raises the intracellular Ca2+ concentration ,which stimulates the production of NO and prostacyclin (PGI2), which diffuse to PASMCs and raise, respectively, the intracellular concentrations of cGMP and cAMP. NO stimulates soluble guanylyl cyclase, which catalyzes the hydrolysis of GTP to cGMP. NO can also have cGMP-independent effects, such as downregulating the number and binding activity of Ang II receptors, thereby blunting the vasoconstrictor effect of Ang II and activating K+ channels [35, 36]. The intracellular concentration of cGMP can also be increased via atrial natriuretic peptide (ANP) acting on natriuretic peptide receptors (NPR-A and NPR-B) on PASMCs; natriuretic peptide receptors are sometimes termed “enzyme receptors,” because of their intrinsic guanylyl cyclase activity. Interestingly, ANP is released in response to acute hypoxia, presumably as part of a negative-feedback loop that helps keep pulmonary vascular tone low [37]. As with cAMP, cGMP levels are also determined by the activity of PDEs: PDE1 and PDE5 appear to be the main cGMP PDEs in the pulmonary circulation [28]. Increasing the intracellular cGMP concentration in PASMCs activates protein kinase G (PKG), which reduces the intracellular Ca2+ concentration by inhibiting voltage- and receptor-operated calcium
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channels, resulting in the uncoupling of the contractile apparatus by activating myosin light chain phosphatase and can also promote hyperpolarization of the membrane by activating K+ channels [38]. In PAECs cGMP can directly activate CNG ion channels, which mediate membrane depolarization following activation of store-operated calcium entry [39]. Thus, an intact endothelium is essential for low basal tone of the pulmonary circulation, since many of the endogenous mediators that stimulate release of NO and PGI2 from PAECs can lead to vasoconstriction if they directly act on PASMCs.
2.1.2 Mechanisms of G-Protein-Mediated Vasoconstriction and Vascular Cell Proliferation Activation of GPCRs that link to Gai, Gaq/11, and Ga12/13, e.g., by 5-HT on PASMCs, leads to vasoconstriction and in many cases increased cellular (in particular PASMC) proliferation (Fig. 1). Stimulation of Gai decreases the intracellular cAMP concentration by inhibiting the activity of specific ACs; AC2 and AC8 are the main Gai-regulated ACs in the PA [26, 27]. Agonist stimulation of Gaq increases the activity of PLCb, which by elevating IP3 levels increases intracellular Ca2+ concentration by opening receptor-operated Ca2+ channels and inducing Ca2+ mobilization from the sarcoplasmic reticulum; IP3 also opens store-operated Ca2+ channels directly or indirectly by store depletion to further increase intracellular Ca2+ concentration. Intracellular Ca2+ can bind to calmodulin or activate intracellular enzymes, such Ca2+–calmodulindependent protein kinase (CAMKII) and MLCK, leading to phosphorylation of the myosin light chain. That phosphorylation stimulates myosin ATPase and promotes hydrolysis of ATP, thereby generating energy for the cycling of the myosin cross-bridges with the actin filament. In parallel, DAG, which is also derived from the action of PLCb, binds protein kinase C (PKC) and promotes its association with the cell membrane. PKC, several isoforms of which coexist in PASMCs (PKCa, PKCb, PKCd, PKCe, and PKCz), can activate mitogen-activated protein kinases (MAPK), and phosphorylate a number of substrates involved in the contractile process [40]. Gaq can also activate RhoA (a low molecular weight monomeric G protein) and Rho kinase (ROCK), which mediate Ca2+ sensitization and can promote sustained vasoconstriction [41]. Activation of Ga12/13, for example, by thrombin, promotes vasoconstriction of the PA and proliferation of PASMCs, primarily by activating Rho GEFs (p115 RhoGEF, PDZRhoGEF) that are linked to the activation of Rho. Rho, in turn, can activate ROCK, inhibit cadherin-mediated aggregation, activate or inhibit one or more Na+–H+ exchangers, and activate phospholipase Ce (PLCe) [42, 43]. Ga12/13 can also directly activate PLCe, AKAP 110, and heat shock protein 90 [43]. Interaction of Ga12/13 and other G proteins, in particular Gaq, can produce overlapping biological responses such as sustained Ca2+ influx and Rho activation;
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Table 1 G-protein-coupled receptors (GPCR) expressed on pulmonary vascular cells Endogenous ligand GPCR Cell type G protein Angiotensin II Endothelin-1
Norepinephrine/epinephrine
Acetylcholine
Bradykinin Vasopressin Adrenomedullin Vasoactive intestinal polypeptide Calcitonin gene-related peptide Substance P Histamine Urotensin II Adenosine ATP, ADP, UTP, UDP 5-Hydroxytryptamine Prostacyclin Thromboxane A2 Sphingosine Thrombin
AT1 ETA ETB ETB b2-AR a1-AR a2-AR M1 M3 M3 B2 V1 CRLR/RAMP VPAC-2 CGRP CGRP NK1 NK2 H1 UT
PASMC PASMC PASMC PAEC PASMC PASMC PAEC PAEC PAEC PASMC PAEC PAEC PASMC PAEC PASMC PASMC PAEC PAEC PASMC PASMC PASMC
Gaq Gaq Gaq Gaq Gas Gaq/Ga12/13 Gaq Gaq Gaq Gaq Gaq Gaq Gas Gaq Gas Gas Gaq Gaq Gaq Gaq Gaq
A2B P2Y2, P2Y4, and P2Y6 5-HT1B 5-HT2A IP FP and TP S1P1 S1P3 PAR1 PAR1/PAR2
PASMC PASMC PAEC PASMC PASMC PASMC PASMC PAEC PAEC PAEC PASMC
Gas Gaq Gaq Gai Gaq/Ga12/13 Gas Gaq Gai Gai/Gaq/Ga12/13 Gaq/Ga12/13 Gaq/Ga12/13
Response Proliferation/vasoconstriction [160] Proliferation/vasoconstriction Proliferation/vasoconstriction Vasodilation [77–85] Vasodilation [48–56] Vasoconstriction Vasodilation Vasodilation [58–61] Vasodilation Vasoconstriction Vasodilation [161] Vasodilation [162] Vasodilation [163] Vasodilation Vasodilation [164] Vasodilation/antiproliferation [165] Vasodilation [166] Vasoconstriction Vasoconstriction [167] Vasoconstriction large pulmonary artery [168] Vasodilation [63–66] Vasoconstriction [63, 64, 68–73] Vasodilation Both proliferation/vasoconstriction [92–98] Vasodilation/anti-proliferation [104] Proliferation/vasoconstriction [109] PAEC barrier protection PAEC barrier dysfunction [115–118] PAEC barrier dysfunction [111–114] Proliferation/vasoconstriction
AR adrenergic receptor, PAEC pulmonary artery endothelial cell, PASMC pulmonary artery smooth muscle cells
coordinated action of these proteins occurs with pulmonary smooth muscle contraction [44, 45]. Gbg can recruit phosphatidylinositol 3-kinase (PI3K), a potent mitogen, to the cell membrane and activate PI3K by direct interaction with its catalytic subunit (p110, Fig. 1). PI3K occurs in several subtypes and can also be activated by phosphorylation by RTKs, non-receptor protein tyrosine kinases of the src family, and focal adhesion kinase. Several downstream targets of PI3K have been identified, including phosphoinositide-dependent kinase-1, Akt, mammalian target of rapamycin (mTOR), and p70 ribosomal protein S6 kinase (p70S6K), all of which can play a role in cell proliferation [5, 46]. A direct role for PI3K in Ang-II-induced DNA synthesis and proliferation occurs in PA adventitial fibroblasts in response to hypoxia [47]. The main GPCRs in the pulmonary circulation, their preferred G protein, and their functional response on ligand binding are summarized in Table 1; a number of which will be discussed in more detail in the following.
2.1.3 Adrenergic Receptors Sympathetic nerves help maintain basal pulmonary vascular tone via both a-ARs and b-ARs, and their multiple subtypes, although this contribution is lower than the sympathetic tone that exists in numerous peripheral vascular beds. In the normal pulmonary circulation, low tone is maintained predominantly via b-ARs since in response to sympathetic stimulation a-antagonists lead to vasodilation, whereas b-AR antagonists enhance vasoconstriction [48]. In the PA of humans, rats, and mice b1-AR and b2-AR (but not b3-AR) on PASMCs mediate vasodilation, although the contribution of each receptor is species-specific [48–51]. Functional studies have shown b2-AR predominates in human, murine, and rat PA. b2-AR stimulation leads to activation of the Gas–cAMP pathway and PASMC relaxation [50, 51]. Depending on the size of the PA investigated and the initial level of precontraction, b-AR-mediated relaxation can be either dependent on or independent of an intact endothelium [50].
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Sympathetic nerve stimulation regulates vascular tone and maintains an adequate ventilation/perfusion matching under physiological conditions. However, excessive stimulation of a1-ARs produces vasoconstriction and proliferation; the pulmonary circulation expresses both a1-ARs and a2ARs on PASMCs [49, 52, 53]. Agonists for a-AR such as norepinephrine, and phenylephrine promote pulmonary vasoconstriction and proliferation via activating Gaq or Ga12/13, and as outlined earlier, increase intracellular Ca2+ concentration [54]. Furthermore, a1-ARs block membrane K+ ion channels via PKC activation, thereby leading to influx of Ca2+ through voltage-dependent channels secondary to membrane depolarization [55]. In contrast, a2-ARs on the surface of PAECs, via production of NO, can stimulate vasodilation of PAs [56]. Eckhart et al. (1996), showed that hypoxia increased the de novo synthesis of a1-ARs in smooth muscle cells both in vivo and in vitro, shifting the balance further in favor of vasoconstriction and proliferation [57].
2.1.4 Muscarinic Cholinergic Receptors The pulmonary circulation receives parasympathetic input but this parasympathetic influence is less than that derived from the sympathetic system. Five muscarinic cholinergic receptor isoforms (M1–M5) have been identified; M1 and M3 are most important for the regulation of the pulmonary circulation [58]. The effects of acetylcholine (Ach), an endogenous muscarinic receptor agonist, on the pulmonary circulation differ depending on the relative number of receptors on the PAECs and PASMCs, the integrity of the endothelium, and the initial level of pulmonary vascular tone [59–61]. Under basal tone, Ach increases PA pressure, which is enhanced by removal of the endothelium, whereas under increased tone, Ach relaxes the PA [61]. In human PAs Gaqcoupled M1 and M3 receptors located on the endothelium are responsible for Ach-induced relaxation, whereas Achinduced contraction is mediated by Gaq-coupled M3 receptors on PASMCs: M3-receptor activation on PAECs leads to release of both NO and PGI2 in an IP3/Ca2+-dependent manner. Unlike in human PA, M1 receptors mediate pulmonary vasoconstriction in vivo in the rabbit pulmonary circulation, thus highlighting species-specific effects of receptor activation in the pulmonary circulation [62].
2.1.5 Purinergic Receptors: GPCRs and Ligand-Gated Ion Channels Purinergic receptors are comprise adenosine (P1) receptors and the P2 receptors, which respond to nucleotides, including ATP, ADP, UTP, and UDP [63]. The P2 receptors are divided into two families: P2Y, which are GPCRs, and P2X, which
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are ligand-gated ion channels. Pharmacological profiling has subdivided P1 receptors into four subtypes (A1, A2A, A2B, and A3), P2X into seven subtypes (P2X1, P2X2, P2X3, P2X4, P2X5, P2X6, and P2X7), and P2Y into eight subtypes (P2Y1, P2Y2, P2Y4, P2Y6, P2Y11, P2Y12, P2Y13, and P2Y14). Ectonucleotidases in the circulation and on cell surfaces act to rapidly degrade extracellular ATP into ADP, AMP, and adenosine, thereby terminating signaling at P2 receptors but potentially promoting signaling at P1 receptors. Purinergic signaling influences normal vascular tone in both PAECs and PASMCs [63, 64]. Adenosine, the breakdown product of ATP, inhibits PASMC proliferation and induces pulmonary vasodilation in an endothelium-independent manner by raising the intracellular cAMP concentration via binding to Gas-dependent A2 receptors: A2B adenosine receptors mediate P1-mediated vasodilation [64, 65]. Adenosine attenuates the vasoconstrictor responses of 5-HT in intact endothelium and denuded rabbit PAs [66]. However, adenosine can also promote apoptosis of PAECs, which appears to depend on the generation of adenosine from extracellular ATP by ectonucleotideases and on its cellular uptake [67]. UTP, UDP, and ATP act via P2Y receptors but only ATP acts via P2X to modulate vascular tone. The specific P2 receptors in the pulmonary circulation, their distribution, and species specification is not yet entirely clear [63, 64]. In both large and small rat PA, P2 receptor agonists enhance vasoconstriction via P2 receptors on PASMCs at resting tone but lead to relaxation via PAEC P2 receptors when the tone is raised [68, 69]. Molecular and pharmacological approaches have implicated Gaq-coupled P2Y2, P2Y4, and P2Y6 receptors as potentially important regulators of the function of PA [68, 70]. In PASMCs stimulation of P2Y receptors raises the intracellular Ca2+ concentration, leading to contraction, whereas in PAECs an increase in intracellular Ca2+ concentration leads to NO- and PGI2-promoted vasodilation [71, 72]. ATP stimulates proliferation of both PASMCs and PAECs, a response that appears dependent on increased PI3K activity, phosphoERK1/2 (extracellular signal-regulated kinase) phosphoAkt, and p70S6K, all known to be associated with the proliferation [73]. ATP binding to ligand-gated P2X receptors facilitates the nonselective passage of cations across the plasma membrane, resulting in an increase in intracellular Ca2+ concentration and depolarization of the cell membrane [74]. P2X receptors contain two transmembrane-spanning domains with intracellular hydrophobic C- and N-termini and a large extracellular loop with ATP-binding sites. The presence of P2X1, P2X2, P2X3, P2X4, P2X5, and P2X7 has been established in human PA; P2X1 receptors most likely mediate the vasoconstriction of PASMCs [68, 69, 73–75]. Shear stress stimulates PAECs to release ATP, which acts in an autocrine fashion to activate Ca2+ influx via P2X4 receptors; in the absence of P2X4 Ca2+ influx is blunted, less NO is released, and vasodilatation is
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prevented [76]. Extracellular ATP thereby protects PAECs exposed to oxidant stress and enhances vasodilation, thus emphasizing a key role of purinergic receptors in maintaining normal pulmonary vasculature function.
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increased circulating levels of ET-1 are found in the lung of patients with PAH [91].
2.1.7 5-HT Receptors 2.1.6 ET Receptors ET-1 is a 21 amino acid polypeptide produced by PAECs and one of the most potent endogenous vasoconstrictors. ET-1 acts via two ET receptor subtypes; both ETA and ETB are expressed on PASMCs but only ETB is expressed on PAECs [2, 77–79]. ET receptors in PAECs clear ET from the circulation and induce release of endogenous NO and PGI2 via Gaq-dependent pathways; the contribution of ETB receptors on PAECs to pulmonary vascular tone appears low as removal of the endothelium does not significantly enhance ET-1-induced pulmonary vasoconstricton [78–81]. ET-1 acting on PASMCs causes a concentration-dependent contraction in isolated PAs in vitro and increases pulmonary vascular resistance in vivo [78, 81, 82]. The relative expression of each type of ET receptor varies depending on the size of the PA studied, which is likely due to the distribution of phenotypically distinct PASMCs: in human PAs the ETA receptor is predominant in larger vessels, with an increased proportion of ETB receptors in more distal branches [78, 83]. ET receptor distribution differs between species in that ETA and ETB receptors coexist in both large and small rabbit PAs [84]. Hypoxia can lead to the redistribution of the ET receptor subtypes, increasing ETA-mediated vasoconstriction in both large and small rat PAs [85]. McCulloch et al. (1998), demonstrated that the ET-1 response in pulmonary resistance vessels after hypoxic exposure could only be blocked by a mixed ETA/ETB antagonist [78, 85]. Thus, ET receptor subtype distribution differs among branches of the PA and species and can be altered by various stimuli. Both ETA and ETB receptors couple to Gaq-dependent pathways, leading to increased concentrations of intracellular Ca2+, PKC activation, ERK1/2 activation, and induction of c-fos and c-jun [82, 86]. ETA receptors also can enhance intracellular Ca2+ concentration by activating nonselective calcium channels on the surface of smooth muscle cells and depolarize human PASMCs via PKC activation and TASK-1 phosphorylation (two-pore domain K+ channel) [87, 88]. Furthermore, ET-1, via Rho/ROCK activation, has been implicated in agonist-stimulated Ca2+ sensitization of 20-kDa myosin light chain phosphorylation and contraction in smooth muscle [89]. ET-1, via ETA receptors, can also act as a co-mitogen in PASMC and pulmonary fibroblasts, in part as the result of the rapid activation of the prosurvival PI3K/ Akt signaling pathway, a mechanism that appears to depend on p38 MAPK [89, 90]. Since ET-1 is a potent pulmonary vasoconstrictor and mitogen, it is perhaps not surprising that
Another vasoactive mediator that has a key role in pulmonary vasoconstriction and proliferation of PASMCs is 5-HT, also known as serotonin [92]. 5-HT is synthesized from L-tryptophan by tryptophan hydroxylase and is released from PAECs. There are at least 17 different 5-HT receptors, including 5-HT1A-F, 5HT2A-C, 5-HT3, and 5-HT4 [93]. In the pulmonary circulation vasoconstriction by 5-HT is mediated by 5-HT1B, 5-HT2A, and 5-HT2B receptors, with their various contributions depending on the level of pre-existing tone and the species [94, 95]. In the absence of tone, 5-HT2A mediates the vasoconstrictive effect of 5-HT in bovine and rat PAs, whereas in the presence of tone, 5-HT elicits contraction via 5-HT1B receptors [96–98]. However, in small and large human PAs, 5-HT1B receptors promote vasoconstriction, even in the absence of tone; this is important since this role of 5-HT1B receptors contrasts with most of the effects of 5-HT in the systemic arteries, which occur via 5-HT2A [96, 97]. 5-HT acting by 5-HT1B receptors induces contraction via a Gai-dependent mechanism, thereby decreasing the level of cAMP by negatively coupling to AC, whereas 5-HT2A receptors activate Gaq-dependent pathways, increasing intracellular Ca+ concentration and activating PKC: Gaq and Gai pathways are thought to synergize to enhance the contractile effect of 5-HT. The 5-HT transporter (SERT), which is located on PAMSCs, appears key for the mitogenic effects of 5-HT since selective SERT inhibitors prevent the remodeling of the pulmonary vasculature [99]. Intracellular accumulation of 5-HT via SERT appears to induce reactive oxygen species (via activation of monoamine oxidase or NADPH oxidase), which leads to the phosphorylation of ERK1/2 and the activation of transcription factors such as GATA4 [100]. As with ET-1, 5-HT can activate Rho/ROCK, thus providing another mechanism by which increased contraction and proliferation is achieved [101]. Sweeney et al. (1995), demonstrated that increasing the level of cGMP could inhibit the ability of 5-HT to constrict PAs [102]. Such results imply that high circulating levels of cyclic nucleotides in the pulmonary circulation, by attenuating the effect of vasoactive mediators, help in maintaining a low pulmonary vascular tone.
2.1.8 Prostanoid Receptors Arachidonic acid (AA) is metabolized through the cyclooxygenase and lipoxygenase pathways to form prostaglandins, thromboxanes, and leukotrienes, which can promote
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constriction or relaxation in PA [1, 103]. Predominant eicosanoids produced by the pulmonary circulation are PGI2 and prostaglandin E2 (PGE2), which both cause vasodilation, and prostaglandin F2a (PGF2a) and thromboxane A2 (TXA2), which are vasoconstrictors: many eicosanoids are cleared by the lung. Prostanoid receptors are classified into five main types: DP, EP, FP, IP, and TP. PGI2 synthase predominates in PAECs and directs metabolism toward PGI2, which is both a very potent pulmonary vasodilator and antiproliferative [104]. PGI2 binds to Gas-coupled IP receptors on PASMCs, leading to an elevation of the cAMP level and a subsequent decrease in the intracellular Ca2+ level, helping to keep vasomotor tone low. PGI2 and its analogues inhibit DNA synthesis and cell proliferation in distal human PASMCs, whereas proximal human PASMCs are comparatively unresponsive [105]. Such results imply that regional heterogeneity and variation in IP receptor expression occurs in the pulmonary arterial tree. Transgenic mice overexpressing PGI2 synthase do not show PASMC hypertrophy following exposure to hypoxia, and PGI2 has been shown to be increased 2.7-fold after 7 days of hypoxia, suggesting that PGI2 has a protective effect in the pulmonary circulation [104, 106]. In fact, synthetic stable analogues of PGI2, such as iloprost and beraprost, are used in the treatment of PAH [107]. In contrast, the eicosanoids PGF2a and TXA2 are both potent vasoconstrictors that bind to FP and TP receptors, respectively, on PASMCs, leading to activation of Gaqdependent pathways and an influx of Ca2+ and release of Ca2+ from store-operated channels. TXA2, acting via PKCz, inhibits voltage-dependent K+ channels, leading to depolarization and activation of L-type Ca2+ channels, thereby constricting the PA [108, 109]. Since a balance of eicosanoids is key in the control of vascular tone under physiological conditions, altered eicosanoid production, possibly via endothelial dysfunction, could shift the balance to the vasoconstrictors and be detrimental to pulmonary function.
2.1.9 Endothelial Barrier Function: Role of Protease-Activated Receptor-1 and Sphingosine 1-Phosphate An intact endothelium is vital for maintaining a low tone in the pulmonary circulatory bed. In addition to growth factors (which are discussed below), certain GPCR agonists, in particular thrombin and sphingosine 1-phosphate (S1P) play an integral role in the regulation of endothelial cell barrier function; thrombin reduces whereas S1P enhances barrier integrity [110]. Adherens junctions, which are composed of vascular endothelial cadherin (VE-cadherin), a-catenin, b-catenin, and p120 catenin, determine the stability of the connections between endothelial cells. The serine protease thrombin directly acts on PAECs, changing their shape and forming gaps; thrombin acts by cleaving
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the extracellular N-terminal domain of the protease-activated receptor (PAR1-4 have been identified): both PAR1 and PAR2, which activate Gaq- and Ga12/13-dependent pathways, mediate the vascular effects of thrombin [111–114]. Thrombin binding to PAR1 acts via Gaq to activate PLCb, thus increasing intracellular Ca2+ concentration and activating kinases and phosphatases that lead to the dephosphorylation of VE-cadherin and b-catenin and phosphorylation of p120 catenin [112]. Gaq- and Ga12/13-dependent pathways also stimulate RhoA via its interaction with p115RhoGEF, activating ROCK and LIM kinase; both pathways increase the numbers of F-actin stress fibers and actomysin contraction. Cytoskeletal rearrangement leads to disruption of the endothelial barrier, thereby increasing albumin permeability. Inhibition of MLCK and ROCK inhibits thrombin-dependent barrier dysfunction [113]. Intercellular gaps that form as a result of PAEC contraction facilitate the ability of thrombin to access and bind to protease-activated receptors on PASMCs, thereby increasing contraction and proliferation; recent data indicate that PASMCs express high levels of PAR1 and PAR2 [114]. Activation of PAR1 can stimulate S1P1-signalling pathways, thus highlighting another protective mechanism of the pulmonary circulation, in this instance to keep PAEC barrier function intact [111]. In contrast, S1P (primarily released by platelets) acts via S1P receptors on PAECs to enhance barrier function. S1P1 and S1P3 receptors are highly expressed in the pulmonary circulation: S1P1 couples to Gai, whereas S1P3 couples to Gai, Gaq, and Ga12/13 [115]. Reverse transcription PCR profiling has revealed that expression of SIP1 receptors is highest in the lung compared with 40 other tissues of the body [23]. S1P1 receptors promote cytoskeletal rearrangement and adherens junction assembly in a Rac1-dependent manner by increasing the recruitment of PI3K and increasing the concentration of intracellular Ca2+ and thereby enhancing polymerization of F-actin and myosin light chain phosphorylation [116]. Inhibition of Gai, PLC, or IP3 prevents the S1P-activated increase in Ca2+ concentration, Rac activation, adherens junction assembly, and subsequent endothelial barrier enhancement [117]. The levels of S1P to which the PAECs are exposed are crucial: high levels (more than 5 mM) can activate S1P3 receptors and mediate RhoA-dependent barrier disruption [118]. How an increase in intracellular Ca2+ concentration both enhances and disrupts the barrier function of PAECs depending on the agonist and agonist concentration is not well understood; however, compartmentalization of the receptor and its effectors may, at least in part, explain such effects. S1P recruits S1P1 receptors to lipid-rich microdomains in PAECs in a PI3K-dependent manner; altering these domains (by depleting them of cholesterol) inhibits S1P signaling and barrier enhancement [119]. Such findings may help explain why caveolin-1 knockout mice (which have a loss of caveolae) develop PAH in association with a disruption of the endothelial layer [17, 119].
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3 Receptor Tyrosine Kinases RTKs, of which there are 90 in the human genome, are receptors that have intrinsic enzyme activity [120, 121]. RTKs are composed of N-terminal extracellular ligand-binding domains and C-terminal intracellular tyrosine kinase domains, which, upon ligand binding, catalyze the transfer of a phosphate residue from ATP to tyrosine residues in the substrates, thereby altering their activity, subcellular location, and stability. RTK ligands include growth factors such as basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), plateletderived growth factor (PDGF), vascular endothelial growth factor (VEGF), and insulin-like growth factor-1 (IGF-1). Growth factors acting via RTKs trigger the activation of several MAPKs, the best characterized of which include the extracellular-signal-regulated kinase (ERK1/2, also known as p42/p44 MAPK), the c-Jun N-terminal kinase (JNK), p38, and ERK5 [120, 121]. MAPK cascades are involved in cellular proliferation, differentiation, and migration [122]. Activation of MAPK involves three sequentially activated kinases: MAPK kinase kinase (MKKK) phosphorylates and activates MAPK kinase (MKK), which in turn phosphorylates and activates MAPK (Fig. 2). Specific MKKK and MKK upstream mediators induce each MAPK subfamily. MAPK phosphatases (MKPs) dephosphorylate the threonine and tyrosine residues of MAPKs both in vitro and in vivo: MKP-1 and MKP-2 are widely distributed and induced in response to robust growth factor stimulation, suggesting that they negatively feed back on MAPK activity [123]. Most RTKs exist as monomers but form dimers following ligand binding; such dimers initiate the autophosphorylation of the RTK, which facilitates the binding of enzymes (Src, PLCg, Shp-2 and PI3K), adaptor proteins (Grb2 and Shc), or docking proteins (IRS, FRS and Gab/Dok) that contain Srchomology 2 (SH2) or phosphotyrosine binding (PTB) domains [120, 121]. The interaction of RTK with adaptors and docking proteins promotes the recruitment of mediators and activation of numerous signaling cascades. For ERK1/2, RTKs promote the activation of the low molecular weight G protein Ras through the recruitment of Grb2/Sos complexes, which activates Ras, thereby recruiting Raf-1 to the plasma membrane through its interaction with the N-terminal domain of Ras (Fig. 2) [124]. Raf-1 can phosphorylate MEK (MAPK/ERK Kinase), which in turn phosphorylates ERK1/2. Downstream effectors of ERK1/2 include cytoplasmic proteins such as cytosolic phospholipase A2 and transcription factors such as c-Myc and c-Fos, which contribute to alterations in gene expression. RTK activation can also trigger PLC- and PI3K/Akt-dependent signaling. MAPK scaffolds have been identified that include the kinase suppressor of Ras, which bring all the components of the specific MAPK cascade together either at the plasma membrane or at other subcellular compartments, thereby
Fig. 2 Receptor tyrosine kinase (RTK)-mediated signaling in the PASMC. Activation of RTKs leads to signaling by three kinases that are connected in series: a mitogen-activated protein kinase (MAPK) kinase kinase that phosphorylates and activates a MAPK kinase, which in turn activates MAPK. For the extracellular-signal-regulated kinase (ERK) pathway, Ras activation occurs through the recruitment of Grb2/Sos complexes, which leads to the recruitment of Raf-1, which phosphorylates MEK, which in turn phosphorylates ERK1/2. ERK1/2 can activate transcription factors such as Elk-1, c-Myc, and c-Fos, therefore altering gene expression. RTK can also activate the PI3K/Akt pathway in PASMCs, which in turn increases transcription of antiapoptotic and pro-proliferative genes
determining the strength, duration, and location of ERK signaling [120]. Furthermore, as with GPCRs, the localization of RTKs in membrane microdomains, such as rafts/caveolae, facilitates activation: for example, the EGF receptor (EGFR) contains a 60 amino acid region that mediates targeting to the raft/caveolae microdomain [15, 125]. MAPKs are not only activated by growth factors but also by GPCR agonists and cytokines and thus can serve as a point of convergence and cross talk among multiple signaling pathways; receptor cross talk is discussed in more detail in the following.
3.1 RTKs in the Pulmonary Circulation A number of growth factors, including PDGF, bFGF, EGF, and VEGF, have been implicated in the development and maintenance of the pulmonary circulation [126]. Different growth factors can function at different stages of the cell cycle, highlighting the importance of multiple signals operating in concert. Excessive production of growth factors and/ or increased expression of their receptors can also play a role in pulmonary vascular disease, such as PAH [1–3].
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A number of studies have shown that VEGF-A is highly expressed in lung and is important for vascular development and maintenance of PAEC function and survival. VEGF is essential for embryonic lung development: knocking out a single allele is embryonically lethal and inhibition of signaling mediators prevents development in neonatal rats [127]. VEGF, which acts via VEGF receptor-1 (FIT) and VEGF receptor-2 (KDR), increases NO and PGI2 formation from PAECs; VEGF binding leads to c-Src activation, which, via PLCg, DAG, and IP3, leads to PKC activation and increased intracellular Ca2+ concentration, and in parallel Akt activation via PI3K, both of which can activate NO synthase [128, 129]. Interestingly VEFG overexpression protects against monocrotaline-induced PAH, presumably by protecting endothelial integrity [130]. PDGF, bFGF, IGF-1, and EGF, via RTK activation, also contribute to the development of the pulmonary arterial tree, for example, by aiding tube formation [126, 131]. These mitogens also have a key role in pulmonary vascular remodeling in response to hypoxia or vascular injury: inflammatory cells, platelets, and cytokines can all stimulate their release [1–3]. Inhibition of PDGF receptor and EGFR can reverse the progression of pulmonary arterial remodeling [132, 133]. PDGF-mediated PASMC proliferation, via PI3K/Akt signaling, has been shown to be associated with c-Jun/STAT3induced upregulation of transient receptor potential channel 6 expression, an increase in capacitative Ca2+ entry, nuclear export, and proteasomal degradation of CREB [134, 135]. Recently, PDGF was shown to regulate PASMC proliferation and phenotype by inducing miR-221, a noncoding RNA that plays a role in negative posttranscriptional regulation of genes [136]. Furthermore, fibroblast growth factor 2 (FGF2) acting via fibroblast growth factor receptor 1 in PAECs, is linked to the pathogenesis of PAH by altering endothelial function; FGF2 increases the expression of the antiapoptotic proteins XIAP and BCL-X via PKC [137]. It is important for the pulmonary circulation to tightly regulate growth factor expression after development since chronic exposure can lead to the muscularization of the small PAs and increased PA pressure.
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members act via a receptor complex of type I and type II serine/threonine kinase receptors (seven type I and five type II have been identified), which comprise a cysteine-rich extracellular domain, a single transmembrane domain, and a conserved intracellular Ser-Thr kinase domain. Upon ligand binding, the preformed dimers of receptor type II phosphorylate the preformed dimers of type I receptor on serine or threonine residues, which can then recruit and phosphorylate receptor-regulated Smads (R-Smads; Smad1, Smad2, Smad3, Smad5, and Smad8) in a receptor-dependent manner (Fig. 3). These activated R-Smads form complexes with Smad4, which allows translocation to the nucleus and regulation of gene transcription such as of c-myc and cyclin D1, chromatin-remodeling complexes, and histone-modifying enzymes. The activation of R-Smads can be inhibited by inhibitory Smads (Smad6 and Smad7), which lead to degradation or phosphorylation of the complex, thereby attenuating signaling. In addition, nonSmad-dependent signal pathways downstream of TGF-b receptors have been proposed: these include interaction with MAPK pathways; for example, TGF-b receptors associate with TRAF6, which recruits TAK1 to activate JNK/p38
4 Receptor Serine/Threonine Kinases Transforming growth factor (TGF)-b family members are multifunctional cytokines: over 30 members that include TGF-b isoforms, activins, and the protein nodal, bone morphogenetic proteins (BMPs), and growth and differentiation factors are present in mammals [138, 139]. These factors are pleiotropic, playing vital roles during embryogenensis and maintaining normal tissue function by coordinating cell proliferation and differentiation. TGF-b family
Fig. 3 Receptor serine/threonine kinase mediated signaling in the PASMC. The transforming growth factor b (TGF-b)/bone morphogenetic proteins (BMP) signaling pathway is initiated after the formation of a heterotetrameric complex of type I and type II receptors at the cell membrane. TGF-b signaling can activate Smad2/Smad3, whereas BMP signaling induces Smad1/Smad5/Smad8 activation. Activated Smads form complexes with Smad4, which allows translocation to the nucleus and regulation of gene transcription, such as of c-myc and cyclin D1. The activation of receptor-regulated Smads can be inhibited by inhibitory Smads (Smad6 and Smad7) that shut off signaling
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[140]. TGF-b signals in most cells via TGF type II receptor and ALK5, activins via activin receptor types IIA and IIB and ALK4, and BMPs via BMP type II receptor (BMPR-II). Depending on the cell type, the specific ligand, and the environment, binding to these receptors can have different functional outcomes.
4.1 Receptor Serine/Threonine Kinases in the Pulmonary Circulation BMP signaling is essential for development of the lung and maintenance of the pulmonary vasculature since altered expression can lead to death during early embryogenesis [141]. BMP-2, BMP-4, and BMP-7 exert their effects by interacting with BMP receptor IA (ALK3) or BMP receptor IB (ALK6) and their coreceptor, BMPR-II. Ligand binding to BMPR-II, which are expressed in PAECs and PASMCs, results in the formation of a complex with BMP type I receptor that activates Smad1, Smad5, and Smad8, whereas in parallel TGF-b acts via an ALK5/Smad3dependent pathway [142]. The endogenous expression of the BMP inhibitors noggin and Smurf1 blunts BMP signaling and is crucial in pulmonary development by the inhibition of branching caused [143]. The tight regulation of BMP signaling may be key in preventing pulmonary remodeling and keeping tone low since mutations in BMPR-II that lead to reduced BMPR-II expression and Smad1/ Smad5 phosphorylation are associated with the development of PAH [144, 145]. Smad1 is essential for both the BMP-2- and the BMP-4dependent survival of PAECs and growth suppression of PASMCs; however, p38 and ERK1/2 are required for their mitogenic response in PASMCs [142, 145, 146]. BMPs are either proliferative or antiproliferative in PASMCs depending on the branch of the pulmonary arterial tree from which the smoothe muscle cells are isolated [145, 146]. Other effectors of BMP signaling have recently been noted, for example, the antiproliferative effect of BMP-2 on PASMCs has been shown to involve activation of the transcription factor peroxisome proliferator-activated receptor g (PPARg), independent of Smad1/Smad5/Smad8 and correlated with reduced nuclear phospho-ERK and apoE RNA expression [147]. BMPR-II can also recruit the canonical Wingless (Wnt) signaling pathway via ERK1/2 to promote PAEC survival, while in parallel BMPR-II can also stimulate the noncanonical Wnt pathway via Smad1 to activate RhoA and Rac1 to induce migration [148]. These data highlight the complexity of the signaling of the pulmonary circulation with respect to the distribution of receptors and effectors in different cells or even the same cell type from different regions of the pulmonary arterial tree.
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5 Receptor Cross Talk and Convergence It is well established that intracellular signaling pathways from different classes of receptors can both positively and negatively modulate each other’s activity and thereby govern their biological response. Such interactions can occur either from the spatial organization of the signaling pathways via multifunctional adaptor proteins or through convergence among receptors: such associations may be important in the homeostasis of the pulmonary circulation. “Cross talk” between signaling pathways initiated by GPCRs and those by RTKs have been widely studied, with GPCRs or components of GPCR-induced pathways being either upstream or downstream of RTKs: for example, ET-1 can activate EGFR in the absence of growth factors [149]. On the basis of as-yet indirect data, it appears that activation of certain G–proteins, for example, Gai, can induce the extracellular activity of a transmembrane metalloproteinase causing ectodomain shedding of a transmembrane RTK ligand precursor, such as heparin-binding EGF, which in turn activates the EGFR [150]. GPCRs can also activate cytoplasmic tyrosine kinases, such as c-Src and Pyk, which phosphorylate RTK, or by formation of multimeric complexes via interaction of components of GPCR-mediated signaling, such as b-arrestin and GRKs (G-protein coupled receptor kinases), can integrate signaling pathways (the latter phenomenon is termed “transactivation”) [151]. For example, association of b-arrestins with GPCRs can promote the recruitment of signaling molecules such as adaptor protein complex 2, clathrin, Src, Hck, and c-Raf-1, several of which are important for ERK activation [151]. Novel convergence mechanisms of GPCRs with other receptors have recently been shown, e.g., a GPCR/RhoA/ Cyr61/integrin pathway contributes to sustained GPCR responses and b-arrestins can interact with components of Wnt pathways (e.g., Dvls and Axins), thereby allowing GPCRs to recruit components of the canonical Wnt pathway [152, 153]. Such receptor cross talk may play a critical role in the normal homeostasis of the pulmonary circulation as well as in the development of disease. For example, Gas activation, via increasing the intracellular concentration of cAMP and PKA activation, antagonizes mitogenic pathways as a consequence of phosphorylation of Raf-1 on Ser-43 and Ser-621, thereby attenuating smooth muscle cell proliferation [154]. cAMP can blunt PI3K-dependent pathway activation as part of its antiproliferative effects [155]. In addition, ANP, which increases cGMP levels, inhibits proliferation of PASMCs, in part, by preventing TGF-b -induced Smad2 and Smad3 nuclear translocation and increases in extracellular matrix expression [156]. Moreover, cGMP via PKG can activate RGS2, thereby attenuating Gaq-coupled vasoconstriction of smooth muscle; such an interaction may help explain some of the beneficial effects of the PDE5 inhibitor sildenafil in treating PAH [11].
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These data suggest that receptor-mediated pathways can also work as “physiological antagonists,” i.e., by having actions that oppose one another in the pulmonary circulation. In contrast, in the setting of disease, receptor-mediated signaling can act synergistically to enhance constriction and proliferation. For example, 5-HT through the activation of ERK1/2 and phosphorylation of the linker region of Smad1 can antagonize the BMP pathway [157]. In addition, decreased levels of cGMP can enhance 5-HT-promoted vasoconstriction of PASMCs [102]. Decreased PKA activity via Gai activation enhances ERK1/2-mediated signaling in PAECs, thereby contributing to cytoskeletal reorganization and barrier dysfunction [158]. In addition, TGF-b1 can downregulate the expression of AC1, AC2, and AC4, increase Gai protein levels, and reduce IP receptor messenger RNA expression, thereby impairing PGI2 signaling in PASMCs [159]. Such effects work together and contribute to vascular remodeling in response to these mediators: these data imply that cross talk of signaling can regulate disease progression via the action of multiple signaling components.
6 Summary The maintenance of low tone in the pulmonary vascular tree is the result of a tight balance of endogenous vasoconstrictors and vasodilators. Receptor-mediated signaling plays a pivotal role in coordinating cellular functions: receptor cross talk can either attenuate or augment changes in the vasculature. Receptormediated downstream pathways in the PA are complex: they are species- and cell-specific and dependent on an intact endothelium and on basal tone. Such complexity implies that studies of receptor function in PASMCs derived from the proximal pulmonary arterial vasculature may not be representative of the full range of effects in vivo. It remains unclear why the response of certain receptor ligands can alter dramatically when tone is increased and why blockade of certain receptors has little or no effect on basal tone. Thus, a comprehensive understanding of the normal regulation of pulmonary function by receptors and signaling pathways is necessary before researchers and clinicians can fully appreciate the complexity (and exploit information) regarding abnormalities that accompany and may lead to disease and that may be therapeutic targets. With increased understanding of receptors and postreceptor components involved in signal transduction mechanisms and the development of molecular tools to interrogate such receptors and signaling components, future studies will likely assess the role of previously unappreciated receptors (especially GPCRs that have not previously been studied) and their effectors in the pulmonary circulation. We hypothesize that newly recognized receptors may prove to be more important than those highlighted above. Recent data have indicated that
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the profiling of GPCR expression can cluster tissues that comprise classical physiological systems (cardiovascular, gastrointestinal, etc.), thus implicating such receptors in homeostatic regulation. Despite the large number of physiological responses that are regulated by cell-surface receptors, especially GPCRs, only a small percentage are currently targeted therapeutically; therefore, identification of “novel” (normally expressed but not previously recognized) receptors and their function in the pulmonary circulation could be promising for the discovery of new targets for drugs for the treatment of pulmonary vascular disease. Acknowledgements This work was supported by grants from the National Heart, Lung, and Blood Institute of the National Institutes of Health (5K99HL091061-02), the Leukemia & Lymphoma Society (7332-06), and the Ellison Medical Foundation (AG-SS-1662-06).
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Chapter 15
Role of Calcium as a Second Messenger in Signaling: A Focus on Endothelium Donna L. Cioffi, Christina J. Barry, and Troy Stevens
Abstract Endothelial cells line blood vessels and lymphatic vessels, and in so doing separate circulating solutes, macromolecules, and cells from the underlying tissues. In this capacity, endothelium fulfills a gatekeeper role, responding to mechanical and chemical signals in the blood and tissues to regulate the directed transport of molecules and cells. The endothelial cell barrier is established by cell–matrix interactions that connect endothelium to the basement membrane, and by cell–cell junctions that connect adjacent endothelial cells together. The strength of both the cell–matrix and the cell–cell junctions is adjusted dynamically, modulated in part by cytosolic calcium concentrations. Whereas basal cytosolic calcium concentrations are maintained at low nanomolar levels, inflammatory first messengers increase cytosolic calcium concentrations. This increase in calcium concentration disrupts cell–matrix and cell–cell adhesion, inducing interendothelial cell gaps that provide a paracellular transport pathway. Calcium channels on the cell membrane allow for calcium entry that mediates this change in cell shape. However, recent advances have identified that endothelial cells express many different calcium channels, with quite distinct cellular functions. This chapter reviews how calcium homeostasis is controlled in tissue compartments, how cells such as endothelial cells compartmentalize calcium, and how endothelial cells utilize calcium signals as a second messenger to regulate their shape. Keywords Lung microvascular endothelium • Endothelial cells • Signaling cascades • Vascular permeability • Barrier function
1 Introduction Endothelial cells line blood vessels and lymphatic vessels, and in so doing separate circulating solutes, macromolecules, and D.L. Cioffi (*) Center for Lung Biology, University of South Alabama, MSB 2316, 5851 USA Drive N., Mobile, AL 36688, USA e-mail:
[email protected] cells from the underlying tissues [1, 2]. In this capacity, endothelium fulfills a gatekeeper role, responding to mechanical and chemical signals in the blood and tissues to regulate the directed transport of molecules and cells. The endothelial cell barrier is established by cell–matrix interactions that connect endothelium to the basement membrane, and by cell–cell junctions that connect adjacent endothelial cells together. The strength of both the cell–matrix and the cell–cell junctions is adjusted dynamically, modulated in part by cytosolic calcium concentrations. Whereas basal cytosolic calcium concentrations are maintained at low nanomolar levels, inflammatory first messengers increase cytosolic calcium concentrations [3, 4]. This increase in calcium concentration disrupts cell–matrix and cell–cell adhesion, inducing interendothelial cell gaps that provide a paracellular transport pathway. Calcium channels on the cell membrane allow for calcium entry that mediates this change in cell shape. However, recent advances have identified that endothelial cells express many different calcium channels, with quite distinct cellular functions. This chapter reviews how calcium homeostasis is controlled in tissue compartments, how cells such as endothelial cells compartmentalize calcium, and how endothelial cells utilize calcium signals as a second messenger to regulate their shape.
2 The Origins of Calcium Calcium is an alkaline-earth metal that occurs in either an ionized or a combined state. It is the fifth most abundant element in the earth’s crust. In 1808 Sir Humphry Davy first discovered calcium by electrolyzing a mixture of lime and mercuric oxide [5]. A critical role for calcium in mammalian physiological function was established in 1883 when Sydney Ringer demonstrated that isolated frog hearts require calcium in the bathing solution to continue beating [6]. Since its discovery, calcium has been found to play significant roles in numerous biological functions [7], including egg fertilization [8], tissue development [8], conduction of nerve impulses to muscle [9], cell adhesion, metabolism, proliferation, secretion, and learning and memory [7].
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3 Calcium Homeostasis in the Blood We now recognize that calcium is a mineral that is necessary to sustain life. Calcium can be found in many food sources, such as milk, cheese, and broccoli. Once calcium has been consumed, stomach acid converts inorganic calcium into a soluble, ionized state to be absorbed in the small intestine. Calcium absorption by the small intestine consists of a passive paracellular pathway and an active transcellular pathway [10–12]. The paracellular mechanism is a diffusional process that functions throughout the length of the intestine [13]. On the other hand, the transcellular mechanism predominantly occurs in the duodenum and upper jejunum and is regulated by vitamin D, or the hormonal form 1,25-dihydroxyvitamin D3 [14]. The transcellular mechanism is a three-step process. First, calcium is driven by the electrochemical gradient across the brush border through the transient receptor potential vanilloid (TRPV) calcium channels TRPV5 and TRPV6 [15–18]. Calcium then diffuses toward the basolateral membrane, and is extruded into blood via the plasma membrane calcium ATPase 1b (PMCA1b) and sodium/calcium exchanger [19]. Blood calcium concentrations are highly regulated, and are maintained at approximately 10 mg/dL (5 mEq/L, 2.5 mmol/L). Deviations from the normal calcium range result in either hypercalcemia or hypocalcemia, which can lead to disease. To maintain strict blood calcium concentrations, the small intestine, bones, and kidneys supply or remove calcium from the blood (Fig. 1). Dietary calcium is absorbed in the small intestine, which contributes to regulation of blood calcium concentration by adjusting the rate of calcium absorption. For example, when dietary calcium intake is low, calcium absorption by the transcellular process is func-
Fig. 1 Regulation of blood calcium levels. When calcium concentration in the blood is low, the body responds by stimulating secretion of the parathyroid hormone, which in turn results in increased production of vitamin D. Vitamin D enhances intestinal absorption of calcium. The parathyroid gland and vitamin D increase the release of calcium from bone and decrease the renal excretion of calcium
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tionally important [20, 21]. On the other hand, when dietary calcium intake is high, calcium absorption by the transcellular process is reduced and calcium is predominantly absorbed by the paracellular process [21]. Bone serves as a vast calcium reservoir, storing 99% of the body’s calcium [22, 23]. When blood calcium levels are low, osteoclasts release calcium from the bone in an effort to restore blood calcium concentrations [24, 25]. In contrast, when blood calcium concentrations are high, osteoblasts store calcium in bone [24, 25]. The kidney filters calcium [25, 26]. Under normal physiological conditions, 98–99% of filtered calcium is reabsorbed, preserving blood calcium levels [27]. Approximately 60% of the reabsorption occurs in the proximal tubules and the remainder in the ascending limb of the loop of Henle and the distal tubule. However, when blood calcium concentrations are high, the kidney tubular calcium reabsorption decreases, allowing more calcium to be excreted [25, 27]. Thus, the small intestine, bones, and kidneys are critical components of calcium homeostasis and are the main organs that regulate calcium blood concentrations. In addition to the small intestine, bone, and kidney, calcium homeostasis is regulated by hormonal control systems. The parathyroid hormone, vitamin D, and calcitonin are three hormones that control blood calcium concentrations. The parathyroid hormone, also known as parathormone and parathyrin, is a peptide hormone secreted by the parathyroid in response to a low blood calcium level [25, 28]. Parathormone increases the blood calcium level by three main mechanisms. First, it increases the number of osteoclasts that break down bone matrix and release calcium into the blood. Second, parathormone maximizes tubular calcium reabsorption within the kidney. Third, it stimulates kidney vitamin D production to increase calcium absorption in the intestines. Vitamin D increases calcium blood concentrations [29]. Vitamin D is a steroid hormone that promotes intestinal calcium absorption. Vitamin D can be found in food or, under normal sunlight exposure, can be synthesized by the skin. As mentioned previously, parathormone converts vitamin D to its active form and facilitates small intestine calcium absorption. In concert with parathormone, vitamin D releases calcium from bone. Calcitonin is a polypeptide secreted by parafollicular cells of the thyroid; it reduces blood calcium levels [23, 25, 29]. Calcitonin suppresses renal tubular calcium reabsorption, and therefore enhances calcium excretion. Calcitonin also inhibits calcium removal from bone by osteoclasts, promoting bone formation. By inhibiting calcium removal, calcitonin minimizes calcium flux from bone to blood. Parathormone, vitamin D, and calcitonin each contribute to blood calcium homeostasis.
15 Role of Calcium as a Second Messenger in Signaling: A Focus on Endothelium
4 Calcium Homeostasis in the Cell
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Calcium functions in biological systems when it is in an uncomplexed form, as well as when it is in complexed forms. In the free form, calcium is an ion with a +2 oxidation state. Whereas low millimolar calcium concentrations are found in blood and interstitium, cells maintain just nanomolar calcium concentrations, establishing an approximately 20,000:1 concentration gradient across the plasma membrane. Although cellular calcium can exist in an uncomplexed state, its lifetime as a free ion is very short, on the order of 3 × 10−5 s, compared with buffered calcium, which exhibits a lifetime of approximately 1 s [30]. The short free calcium lifetime is largely a reflection of the abundance of negatively charged proteins and molecules that exist within the cell. Indeed, there are numerous calcium-binding proteins with binding affinities that cover a wide range. The higher the binding affinity, the more tightly the protein holds on to calcium, whereas a protein with a weak binding affinity will be more apt to release calcium. Calcium-binding proteins play variable roles within the cell, including calcium buffering, transcription, ion channel regulation, cell proliferation, and homeostasis. Although there are thousands of known calcium-binding proteins, they generally fall into one of several categories. First is the wellknown superfamily of EF hand calcium-binding proteins. The EF hand motif is one of the most common domains encoded by the human genome. This is a small domain comprising 29 residues in the form of a helix–loop–helix structure (Fig. 2a) [31]. The loop, which is 12 or 14 amino acids long, is the actual calcium-binding moiety (reviewed in [32, 33]). Most EF hand proteins contain adjacent pairs of the motif that are connected by a variable-length linker [32, 33].
There are more than 120 proteins within the EF hand superfamily [33]. Some of the more widely studied include calmodulin, calcineurin, calreticulin, and the S100 subfamily. Calmodulin is a ubiquitously expressed calcium-binding protein that is conserved throughout evolution. Calcium binding to calmodulin changes the conformation of calmodulin, and enables calmodulin to regulate the function or activity of binding partners [34]. Calmodulin has two globular domains forming a dumbbell shape, each of which has two EF hand motifs [35], and thus can bind a total of four calcium ions (Fig. 2b). Within each motif there are six key amino acids which play an important role in calcium binding [31]. Although the calcium-binding sites exhibit similarity to one other, they nonetheless exhibit quite distinct binding affinities as well as distinct conformational changes induced by calcium binding [31]. Calmodulin exhibits another interesting feature. In addition to binding calcium, it also binds to proteins that bear a calmodulin-binding domain. When calmodulin is not bound to a protein, its binding to calcium is positively cooperative between the two EF hands within each of the globular domains, yet there is no cooperativity between the domains. However, when calmodulin is bound to peptides expressing calmodulin-binding domains, there appears to be cooperativity between all four calcium-binding sites [34]. Annexins comprise another superfamily of calcium-binding proteins. Annexins are perhaps best known for their role in apoptosis. However, with the identification of more than 1,000 proteins within this superfamily, it is believed that the annexins have numerous other functions (reviewed in [36]). The identifying feature of annexins is that they are not only calcium-binding proteins, but that they are also phospholipid-binding proteins (reviewed in [37]). The annexin repeat, which is in all annexins described to date, contains four repeating units, each with approximately 70 amino acids (Fig. 2c). The repeats contain up to five a-helices and usually have a “type 2” calcium-binding
Fig. 2 Calcium-binding proteins. a. The basic calcium-binding structure of the EF hand motif is a helix–loop–helix (reprinted by permission of MacMillan Publishers Ltd from Burgoyne [31] copyright 2007). b Calmodulin exhibits two globular domains each of which has two EF hand motifs which can cooperatively bind two calcium ions (yellow spheres) (reprinted from Valeyev et al. [35]). c The annexin
core contains four repeats (blue, yellow, green, and red), each of which has five a-helices. Each annexin repeat can bind one calcium ion. The red spheres denote oxygen atoms that are involved in calcium binding. Importantly, annexin association with membrane surfaces is mediated through bound calcium (reprinted by permission of MacMillan Publishers Ltd from Gerke et al. [143] copyright 2005)
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motif [36]. Indeed, with its positive charge, calcium regulates the interaction between annexin and the negatively charged phospholipids within the plasma membrane.
4.2 Intracellular Calcium Compartments Both prokaryotic and eukaryotic cells rely on the precise regulation of free calcium in a temporal and concentrationdependent manner. The cell has multiple ways in which it controls the free calcium concentration, including different cell compartments, transporters, and channels and various calcium handling modes. Different cell compartments often act as calcium “stores” in which they contain a much higher calcium concentration as compared with the cytosol. The cytosol typically has the lowest concentration of free calcium, in the unactivated state, which is on the order of 100 nM for both aortic and pulmonary artery endothelial cells [38, 39]. Importantly, the cytosol is a calcium-storage compartment. Other compartments are formed by organelles that include the endoplasmic/sarcoplasmic reticulum, mitochondria, and even the nucleus. Intracellular vesicles also act as calciumstorage compartments. There are different types of calciumstorage vesicles. Calciosomes are small vacuoles that contain, in addition to calcium, calcium-binding proteins similar to calsequestrin [40]. Calsequestrin is found within the lumen of junctional sarcoplasmic reticulum of cardiac and skeletal muscle, and functions to bind calcium in a high capacity, yet with moderate affinity [41]. Calciosomes were first described by Volpe et al. [42], who postulated that they might be the intracellular target of inositol 1,4,5-trisphosphate (InsP3). In non-muscle-cell calciosomes, the calsequestrin-like protein was later identified as calreticulin [43]. Another type of calcium-storage vesicle is characterized by acidic lumenal pH, similar to the acidocalcisomes of bacteria and unicellular eukaryotes [40]. In mammalian cells, these acidic calciumstorage depots come in multiple forms, such as secretory granules, endosomes, lysosomes, and the trans-Golgi network (reviewed in [44]). For example, human platelet dense granules are acidic compartments which contain calcium and inorganic polyphosphate and pyrophosphate [45]. In conjunction with other similarities with the bacterial acidocalcisomes, it is suggested that these organelles are evolutionarily conserved from bacteria to humans. Polyphosphates, which are polymers of inorganic phosphates linked through high-energy phosphoanhydride bonds, act as strong cation chelators and can thus serve as a calcium-storage polymer (reviewed in [46, 47]). Indeed, compartmental calcium storage is a key regulator of cytosolic calcium concentration. Given the physiological importance of precisely regulating cytosolic calcium concentration, it is not surprising that the cell possesses several different calcium regulatory mechanisms.
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4.3 Calcium Transporters Calcium transporters and ion channels are another way in which the cell regulates its free calcium concentration. Calcium transporters in general move the ion against its concentration gradient, thus requiring energy input in the form of ATP hydrolysis. There are numerous calcium transporters in mammalian cells. On the plasma membrane, an ATP-dependent calcium pump, known as the calcium-ATPase or the plasmalemmal calcium pump, is expressed and is responsible for extruding calcium from the cytosol into the extracellular space. This pump, which acts via active transport, exhibits high calcium affinity (reviewed in [48]). Interestingly the calcium-ATPase has been shown to be localized to caveolae along with InsP3receptor-like protein, thus implicating caveolae in control of cytosolic calcium concentration [49]. In support of this idea, plasma membrane calcium-ATPase function is inhibited and calcium extrusion is reduced in caveolin-1 knockout mice [50]. The sodium/calcium exchanger similarly extrudes calcium from the cell. Although calcium is still moved against its concentration gradient, this transporter does not require ATP hydrolysis. Instead, calcium movement out of the cell is coupled with sodium movement into the cell, down its concentration gradient (reviewed in [51]). Although this antiporter system displays a high calcium transport capacity, it has only a weak calcium affinity (reviewed in [48]). Different cells express different types and amounts of calcium transporters. The sodium/calcium exchanger will compete with other calcium transport pathways, and the importance of the sodium/calcium exchanger depends upon the abundance of other transport pathways. In some tissues, e.g., the liver, its importance is low, whereas in other tissues, e.g., kidney, smooth muscle, brain, and heart, its importance is high (reviewed in [51]). Although calcium storage and calcium extrusion represent the principal ways of lowering cytosolic calcium concentration, the cell also possesses mechanisms whereby it can increase cytosolic calcium concentration. There are two key mechanisms to increase cytosolic calcium concentration, including calcium release from internal stores and calcium entry through channels in the plasma membrane.
4.4 Calcium Release Calcium mobilization from internal stores and external calcium entry are critically dependent upon the action of the calciummobilizing second messenger, InsP3. InsP3 couples external hormone signals to intracellular calcium release signals [52, 53]. InsP3 is a small water-soluble molecule generated at the plasma membrane, from where it detaches and rapidly diffuses through the cytosol. InsP3 then binds to specific receptors on
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intracellular calcium stores, such as the endoplasmic reticulum [54]. Once InsP3 has bound to specific receptors, it induces calcium store release and quickly raises the cytosolic calcium concentration [55, 56]. InsP3 is generated by phospholipase C (PLC) cleavage of lipid phosphatidylinositol 4,5-bisphosphate (PIP2). Two pathways activate PLC and ultimately generate InsP3 [53]. The first pathway is a G-protein-coupled-receptor pathway, specifically the Gq pathway, which activates PLCb1. Activated PLCb1 cleaves PIP2 to generate two products, InsP3 and diacylglycerol. InsP3 diffuses through the cytosol and binds to its receptor on the calcium store. Diacylglycerol, on the other hand, remains embedded in the membrane, and can either be further cleaved to release arachidonic acid or specifically activate the serine/threonine protein kinase referred to as PKC. The second pathway that generates InsP3 is the tyrosine kinase pathway [53, 57]. Tyrosine kinase receptors are unified by the presence of a single membrane-spanning domain and an intracellular tyrosine protein kinase catalytic domain. In general, a ligand binds to the tyrosine kinase receptor, resulting in receptor dimerization. Receptor dimerization activates the kinase activity of both intracellular chains, resulting in cross-phosphorylation of several tyrosine residues. Once the tyrosine kinase receptor has been phosphorylated, the receptor is activated. The activated receptors stimulate PLCg1, which in turn hydrolyzes PIP2 to InsP3 and diacylglycerol. Once InsP3 has been generated, it has a half-life of just a few seconds [58]. The average InsP3 half-life is approximately 9 s [59]. InsP3 is inactivated by either phosphorylation to inositol 1,3,4,5-tetrakisphosphate or dephosphorylation to inositol 1,4-bisphosphate, which is rendered an ineffective ligand at the InsP3 receptor [57]. InsP3 is also subject to rapid metabolism by InsP3 3-kinase isoforms [57, 60] and membrane-associated InsP3 5-phosphatase [61]. Because InsP3 possesses a short half-life, most InsP3 receptors are located close to the plasma membrane. InsP3 is unique to the various inositol phosphates and phospholipids in that it is the only molecule that has a channel as a target molecule [62]. The InsP3 receptor is a tetrameric channel embedded in the membrane of the endoplasmic reticulum and functions as a calcium release channel [63– 65]. The InsP3 receptor is located on the endoplasmic reticulum close to the plasma membrane and PLC complexes. This prime receptor location is well situated to respond to locally generated InsP3. The InsP3 receptor has an N-terminal InsP3binding domain and a C-terminal channel forming a sequence separated by a large intervening regulatory domain. The InsP3 receptor consists of three different isoforms that share a 60–70% amino acid sequence homology. Most cells express more than one InsP3 isoform. Once InsP3 has activated its receptor, calcium is rapidly released from the store into the cytosol [58].
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The InsP3 receptor itself is under the dual control of InsP3 and calcium [53, 66]. Low levels of calcium activate the receptor, whereas a cytosolic calcium concentration above 300 nM inhibits the receptor [67–70]. However, the ability of calcium to promote its own release is regulated by InsP3, such that in the absence of InsP3, calcium has little effect on the InsP3 receptor [53]. As the concentration of InsP3 increases, the receptor’s sensitivity to calcium increases [53]. Whereas InsP3 receptor activation is the principal calcium release mode in endothelial cells, other cells rely on ryanodine receptors and cyclic ADP ribose mechanisms as well. Indeed, the ryanodine receptor is responsible for the calciuminduced calcium release mechanism that is critically important for skeletal muscle contraction [71, 72], and cyclic ADP ribose dependent calcium release may contribute to the second messenger response to hypoxia [73].
4.5 Calcium Entry The activation of Gq-linked agonists or receptor tyrosine kinases, as described earlier, leads to generation of InsP3 and subsequent release of calcium from the endoplasmic reticulum through the InsP3 receptor. In many cell types, especially nonexcitable cells such as endothelial cells, depletion of stored calcium, i.e., in endoplasmic reticulum, opens channels in the plasma membrane to allow for calcium influx into cells (Fig. 3). These particular ion channels, which are opened in response to store depletion, are known as storeoperated entry channels (reviewed in [74]). It has been a little over 20 years since Putney first described this store-operated entry channel process [75]. Since his original description of this phenomenon, numerous groups worldwide have committed substantial time and resources to try to answer two critical questions. First, what is the signal(s) that links store depletion to channel activation and, second, what is the molecular identity of the channel(s) itself? Interestingly, 20 years later, the answers to both questions remain unsettled. From the literally thousands of experiments that have been performed trying to identify the signal(s) linking store depletion to channel activation, three principal hypotheses have been formulated (reviewed in [76]). One hypothesis promotes the idea that the InsP3 receptor in the endoplasmic reticulum is directly and physically coupled to the store-operated entry channel in the plasma membrane. In this model it is proposed that when calcium is released from the endoplasmic reticulum through the InsP3 receptor, the proteins that constitute the receptor change conformation. As the InsP3 receptor is believed to be physically tethered to the store-operated entry channel, a change in receptor conformation will induce a subsequent change in store-operated entry channel conformation,
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Fig. 3 Activation of Gq or receptor tyrosine kinases promote storeoperated channel (SOC) entry. Agonists which bind to G-proteincoupled receptors or tyrosine kinase receptors activate phospholipase C (PLC). PLC, in turn, cleaves phosphatidylinositol 4,5-bisphosphate to produce the second messengers inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG). SOC entry involves diffusion of IP3 to the endoplasmic reticulum (ER), where it binds to IP3 receptors. The IP3 receptor is itself a calcium channel which when activated releases calcium from the ER store. Depletion of the calcium store triggers, by an unknown mechanism, opening of SOCs on the plasma membrane. Receptoroperated channels can be activated by a number of second messengers, including DAG, without requiring calcium store depletion. Adapted from Li et al. [145]
and in doing so gate open the channel. A variant of this model proposes a physical link between the InsP3 receptor and the store-operated entry channel, but not via direct interaction. The second hypothesis proposes that the signal linking store depletion to store-operated entry channel activation is a soluble factor that is released from the endoplasmic reticulum. This soluble factor is believed to diffuse through the cytosol and bind to and activate the store-operated entry channel. Although much work has been devoted to this hypothesis, to date, no single protein or molecule has been identified which fulfills the criteria of the soluble factor. The third and final hypothesis is the vesicle-insertion model. Here, the storeoperated entry channels are believed to reside in vesicles that directly underlie the plasma membrane. Upon store depletion, the vesicles are stimulated to move to and insert themselves into the plasma membrane, thus exposing the store-operated entry channels to the extracellular medium. Although the quest to identify store-operated entry channel proteins has been difficult, some progress has been made. The first identified candidate for a store-operated entry channel was the transient receptor potential (TRP) protein found in the Drosophila melanogaster eye [77]. As the Drosophila phototransduction cascade was known to follow the path of activated PLC and generation of InsP3, the TRP became a candidate for a store-operated entry channel. Indeed, when expressed in vitro, the Drosophila TRP protein can be activated by store depletion, i.e., it acts as a store-operated entry channel, whereas the related TRP “leak” (TRPL) protein is constitutively active [78]. Homologues of Drosophila TRP proteins have been identified in mammals, Caenorhabditis elegans, and zebrafish, and now TRP proteins constitute a superfamily (reviewed in [79]). Interestingly, the Drosophila TRP was cloned at roughly the
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same time as Putney described store-operated entry channels (reviewed in [80]. Indeed, this early observation set the stage for experiments over the following 20 years. The mammalian TRPC (canonical) subfamily most closely resembles the Drosophila TRP (reviewed in [81]). They are six transmembrane-spanning-domain proteins with intracellular C- and N-termini. On the basis of homology with voltage-gated potassium channels, the functional TRPC channel likely comprises four subunits that coalesce and form a pore through the transmembrane 5 and 6 regions. Seven members of the TRPC subfamily (TRPC1–TRPC7) have been identified to date. Of these seven, TRPC1, TRPC3, TRPC4, and TRPC5 have been shown to be able to perform in a store-operated capacity [82–92]. Although numerous studies have determined that the TRPC1, TRPC3, TRPC4, and TRPC5 proteins fulfill the criteria for storeoperated entry channels, there is a problem. For as many reports supporting the idea that these TRPCs are storeoperated, there are just as many refuting the idea. Indeed, the concept that TRPC proteins can contribute to storeoperated entry channels has become quite controversial. At present, this issue remains unresolved, although it appears that cell-specific channels can form from variable combinations of TRPC proteins, perhaps contributing to the difficulty in data interpretation. Just recently, another protein, orai1, was introduced as a potential store-operated entry channel. Unlike TRPC proteins, orai1 is a four-transmembrane-spanning-domain protein. Similar to the Drosophila TRP, orai1 was originally identified as a potential store-operated entry channel when a naturally occurring mutation surfaced. It was observed that patients with a particular form of severe combined immunodeficiency syndrome exhibited a single point mutation, R91W, in T cells [93]. Further, these cells lacked the originally described store-operated entry channel current, ICRAC, leading to the hypothesis that orai1 proteins form the ICRAC channel. Considerable excitement was generated within the scientific community when the heterologous expression of orai1 was found to restore ICRAC to cells naturally deficient in this current. However, not all cells expressing orai1 possess an ICRAC [94–97], and recently Liao et al. [98, 99] and Chang et al. [100] independently recognized that orai1 interacts with TRPC proteins, where it functions to link calcium store depletion to activation of calcium entry through the TRPC channel. It will be interesting to follow the progression of this story over the next few years. Indeed, only when the molecular identity of a store-operated entry channel has been accurately described will we then be able to determine how the channel is gated. Although there is still much uncertainty regarding the identity and regulation of store-operated entry channels, significant progress in the field has been made. Endothelial cells possess a specific store-operated entry channel current, referred to as ISOC, that has been described by Vaca and
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Kunze [78], Fasolato and Nilius [101]; Wu et al. [102, 103]. ISOC is a calcium-selective channel, meaning that it prefers to conduct calcium even in the presence of other ions. This characteristic is reflected in the reversal potential of the current, which is approximately +40 mV. ISOC is a small current, on the order of 1–1.5 pA/pF and is inwardly rectifying. The molecular identity of the ISOC channel that underlies ISOC has been partially unraveled. In the first set of studies, TRPC1 expression was inhibited using small interfering RNA [103]. TRPC1 knockdown decreased store-operated channel (SOC) entry and inhibited ISOC, but did not effect a left-shift in the reversal potential. The observation that there was only a partial inhibition of ISOC, and not a complete abolishment, led to the idea that TRPC1 contributes at least one subunit to the channel, but it is not the critical subunit that somehow senses store depletion. Thus, there must be at least one other type of subunit in the ISOC channel. The second observation, i.e., no shift in the reversal potential, revealed that TRPC1 does not play a role in determining the calcium selectivity of the channel, again suggesting that there is at least one other type of subunit in the channel’s molecular makeup. The second set of studies focused on the contribution of TRPC4 to the ISOC. Here, a TRPC4 knockout mouse was developed [104]. In aortic endothelium, not only was the ISOC reduced to a negligible current, the reversal potential was left-shifted to zero. These results were exciting because they suggested critical roles for TRPC4 in ISOC activation (or sensing store depletion), and in determination of the calcium-selective nature of this channel. If indeed the ISOC is formed by four subunits, then we can be confident of at least two, TRPC1 and TRPC4. Although the identity of the other two subunits is uncertain, on the basis of the molecular identity of other tetrameric ion channels, it is likely that the remaining two subunits exhibit structures homologous to those of TRPC1 and TRPC4. Thus, we can hypothesize that the remaining two subunits are likely to be some combination of TRPC1, TRPC4, and/or other TPRC homologues. Since orai1 has only four transmembrane-spanning domains, and as such does not exhibit a structure homologous to the structures of the TRPC proteins, we predict that it does not contribute to the pore-forming part of the channel structure. As the molecular identity of ISOC has been partially resolved, so too has the mechanism that underlies channel activation. Early work in platelets and endothelial cells revealed that the cytoskeleton plays a critical role in activation of store-operated entry channels. The cytoskeleton constitutes a molecular framework which provides structural integrity to the cell. It is composed of numerous proteins, including actin filaments, intermediate filaments, and microtubules. In some cell types, including endothelial cells, this spectrin–actin meshwork contributes to the formation of a unique structure directly underlying the plasma membrane, the membrane skeleton. In the well-studied erythrocyte (reviewed in [105, 106]), the membrane skeleton has been
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Fig. 4 The membrane skeleton. Filamentous actin (yellow) is crosslinked by spectrin (brown) to form a scaffolding network that provides membrane support. Protein 4.1 (blue) and ankyrin (green) link transmembrane proteins to the membrane skeleton; additionally protein 4.1 forms a ternary complex with spectrin and actin. (Reprinted with permission from [3])
shown to provide structure and support to the plasma membrane, important for cell shape; additionally, the erythrocyte membrane skeleton appears to limit movement of integral membrane proteins, phospholipids, and cholesterol. Although much less is known about specific membrane skeleton functions in nonerythroid cells, proteins which contribute to erythrocyte membrane skeleton composition have also been identified in nonerythroid cells [107–113]. The proteins spectrin and actin represent principal components of the membrane skeleton (reviewed in [106]), with spectrin cross-linking actin filaments [114] to form a molecular meshwork underlying the plasma membrane (Fig. 4). The membrane skeleton physically interacts with the plasma membrane, providing structural support to the cellular membrane. Protein 4.1 and ankryin are principally responsible for linking the membrane skeleton to the membrane, by simultaneously binding to spectrin in the membrane skeleton and transmembrane proteins in the plasma membrane (reviewed in [115]). Initial studies examining whether an intact actin cytoskeleton was important for activation of SOC entry supported the physical coupling model, in that disruption of filamentous actin prevented activation of SOC entry [116–118]. Importantly, calcium release from the store as well as basal cytosolic calcium levels were not affected by filamentous actin disruption. However, these studies utilized pharmacological agents that grossly stabilized (e.g., with jasplakinolide) or disrupted (e.g., with cytochalasin D) filamentous actin; thus, specific protein–protein interactions within the cytoskeleton important for channel activation could not be determined. Subsequent studies in endothelial cells focused on the role of the membrane skeleton in store-operated entry channel activation, and further sought to determine whether specific protein–protein interactions within the membrane skeleton were important for channel activation. To address these questions, more refined experiments were designed. Antibodies that targeted and disrupted specific protein–protein interactions were microinjected into endothelial cells and SOC entry was measured [102]. An antibody to the protein 4.1–spectrin interaction decreased the overall SOC entry and completely inhibited the ISOC channel, whereas an antibody that disrupted
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the actin–spectrin interaction had no effect. These observations led to the exciting hypothesis that protein 4.1 functionally links the ISOC channel to the membrane skeleton, supporting the conformational coupling model of store-operated entry channel activation. Subsequent sequence analysis of TRPC1 and TRPC4 revealed that TRPC4 contains a conserved protein 4.1 binding domain [119], and protein 4.1 coimmunoprecipitates with TRPC4 [74], indicating that these two proteins physically interact. The protein 4.1 binding domain on TRPC4 is located on the cytosolic C-terminus approximately 50 amino acids downstream of where the last transmembrane domain exits the membrane. As the pore region of the channel is predicted to form by coalescence of the five to six transmembrane domains of the four subunits (reviewed in [120]), this would place the protein 4.1 binding domain in direct proximity to the pore region. If protein 4.1 binds at its binding domain on TRPC4, then it would be in close proximity to the pore region of the channel, and as such, in a manner analogous to the ball-and-chain model of voltage-gated channels, it is conceivable that protein 4.1 acts as the “ball” and gates the ISOC channel. Indeed, this idea represents a novel function of protein 4.1. Although SOC entry represents a principal mechanism of calcium entry into nonexcitable cells, it is not the only mechanism. For instance, multiple other channels have been identified in pulmonary endothelial cells, including receptoroperated channels, cyclic-nucleotide gated channels, TRPV (vanilloid)-containing channels, and T-type calcium channels. These channels are distinct from store-operated entry channels in that activation is not triggered by store depletion per se. Channels that are formed from TRPV4 subunits can be activated by several different factors, including elevated temperature, mechanical stress, and arachidonic acid metabolites (reviewed in [121]). Cyclic-nucleotide-gated channels are directly activated by cyclic AMP and/or cyclic GMP (reviewed in [122]). Pulmonary artery endothelial cells possess a cyclicnucleotide-gated channel that is a nonselective cation channel which can be activated downstream of SOC entry [123]. Receptor-operated calcium channels were originally described as voltage-insensitive channels that are activated by receptor agonists such as norepinephrine, acetylcholine, and prostaglandins (reviewed in [124]). Subsequently, with the discovery of the TRPC subfamily of proteins, the term “receptor-operated channel” almost became synonymous with TRPC3, TRPC6, and TRPC7. The receptor-operated TRPC channels exhibit similarities to SOCs. Receptors coupled to Gq/11 or receptor tyrosine kinases activate both channel types, through generation of InsP3 and diacylglycerol. Whereas InsP3 diffuses through the cytosol to bind to its receptor on the endoplasmic reticulum, as described previously, diacylglycerol remains associated with the plasma membrane, where it activates receptor-operated channels (Fig. 3). In general, receptor-operated calcium channels exhibit nonselective currents. Given that store- and receptor-operated
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currents are both activated by similar agonists, it can be difficult to differentiate their independent contributions to shifts in cytosolic calcium concentration. Fortunately, studies of SOCs have been facilitated by the discovery of thapsigargin. Thapsigargin is a plant alkaloid that blocks the calcium-ATPase pump on the endoplasmic reticulum [78, 125]. It functions by preventing calcium reuptake into the endoplasmic reticulum, and as the endoplasmic reticulum exhibits a slow, constitutive calcium leak, in the presence of thapsigargin it will soon become depleted of its calcium content. This, in turn, activates SOCs. Use of thapsigargin allows one to follow events triggered solely by store depletion, without the confounding effects of additional signal transduction events that occur with Gq-linked agonists. The receptor-operated community has relied on the use of diacylglycerol analogues, e.g., oleylacylglycerol, to specifically activate receptor-operated channels without concomitantly activating SOCs. To date, in pulmonary endothelial cells, receptor-operated calcium entry has not been as intensely studied as store-operated calcium entry. Another calcium entry pathway that has been described in pulmonary endothelium is the T-type voltage-gated channel. This discovery was a surprise, considering that endothelial cells are considered to be nonexcitable cells that do not express voltage-gated ion channels. However, in the pulmonary vasculature, pulmonary microvascular endothelial cells express the T-type channel, whereas pulmonary artery endothelial cells do not (reviewed in [126]). The T-type channel in microvascular endothelial cells possesses an a1G subunit, and exhibits a window current in the range of voltages between −60 and −30 mV. The window current range of voltages lies between the two peaks of the bimodal resting membrane potential of the microvascular endothelial cells (reviewed in [127]). These peaks are at −60 to −70 mV and −40 to −10 mV. The relationship between the resting membrane potential and window current range of voltages is believed to be physiologically important with respect to activation of the T-type channel. Inflammatory agonists such as thrombin, and other Gq-linked agonists, cause an initial transient hyperpolarization which is then followed by a sustained depolarization. Either hyperpolarization from the −40 to −10 mV peak or depolarization from the −60 to −70 mV peak could transition the membrane potential into the window current range of voltages and thereby activate the T-type channel allowing for calcium entry. As the different calcium entry pathways were described in the pulmonary endothelium, an interesting observation was made. Not all subsets of pulmonary endothelial cells express the same calcium entry pathways. For instance, both the T-type calcium channel and TRPV4 are found in pulmonary microvascular endothelial cells but not pulmonary artery endothelial cells [126, 128, 129]. The TRPC1 and TRPC4 proteins of the ISOC channel are expressed in both pulmonary artery and microvascular cells, and indeed ISOC can be
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activated in both cell types [130]. However, ISOC activation is differentially regulated among cells. Whereas thapsigargin alone can activate ISOC in pulmonary artery endothelial cells, it is insufficient to activate ISOC in microvascular cells.
5 Physiological Functions of Calcium Entry For most of its scientific history, endothelium was recognized as an inert, squamous epithelial layer [1, 2, 131]. More recently, the systematic study of structure and function has begun to resolve the highly dynamic and complex nature of endothelium. From this work has come a realization that considerable heterogeneity exists in endothelium, not only when comparing cells between different organs, but perhaps most provocatively when comparing cells along the vascular tree within an organ [132, 133]. The lung is no exception, as striking endothelial cell heterogeneity is seen along the artery–capillary–vein axis within this organ. The study of calcium channel expression and function has greatly aided our understanding of pulmonary endothelial cell heterogeneity. Activation of store-operated calcium entry channels increases endothelial cell permeability [134–136]. More specifically, ISOC activation appears to provide a calcium source that is uniquely coupled to cytoskeletal reorganization, disruption of cell–cell adhesions, and intercellular gap formation [130, 137]. Use of thapsigargin has been pivotal in resolving these mechanisms both in vitro and in the intact circulation. In the intact circulation, thapsigargin application increases extra-alveolar permeability, resulting in perivascular cuff formation that impairs lung compliance [138]. Rolipram is a type 4 phosphodiesterase inhibitor that increases the level of cyclic AMP. Rolipram prevents thapsigargin from increasing extra-alveolar permeability, but reveals a thapsigargin-induced capillary leak site [130]. Thus, although the mechanisms responsible for ISOC activation in extra-alveolar and capillary endothelium are different, the result is the same, as calcium permeation through this channel results in gap formation and increased permeability. Whereas thapsigargin typically increases permeability in extra-alveolar vessels, it fails to do so in animals with heart failure. Indeed, Alvarez et al. [139] placed an aortocaval fistula in rats causing development of heart failure. As heart failure developed, the permeability response to thapsigargin was abolished. This functional response to heart failure was accompanied by the downregulation of TRPC1, TRPC3, and TRPC4 in extra-alveolar endothelium. Although the thapsigargin-induced increase in permeability was abolished in these animals, the permeability response to a different calcium agonist, 14,15-epoxyeicosatrienoic acid, was retained, bringing into question how the response to one agonist, thapsigargin, could be abolished whereas the response to a second agonist, 14,15-epoxyeicosatrienoic acid, was not.
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14,15-Epoxyeicosatrienoic acid activates a calcium channel belonging to the TRPV proteins, TRPV4 [140]. Endothelial cell TRPV4 expression in the pulmonary circulation is concentrated in the capillaries, and this expression pattern is not changed in animals with heart failure [129, 141]. Hence, channel activation results in a calcium response that increases capillary permeability, but does not increase extraalveolar permeability [138, 141]. Unlike the activation of SOC entry, TRPV4 activation does not result in endothelial cell gap formation, but causes loss of cell–matrix tethering. The mechanisms allowing for store-operated entry channels to control cell–cell adhesion and the TRPV4 channel to control cell–matrix adhesion are still incompletely understood. Not all calcium channels regulate endothelial cell barrier function. Wu et al. recently discovered that lung capillary endothelium expresses a T-type calcium channel [142]. Activation of this channel does not increase endothelial cell permeability, but rather is responsible for the P-selectin upregulation that is important for neutrophil trafficking [141]. Thus, while T-type channel activation is not responsible for increasing permeability, it is involved in the endothelial cell response to inflammation. The systematic study of these three calcium channels, the ISOC channel, TRPV4, and the T-type calcium channel, has greatly advanced our understanding of lung endothelial cell heterogeneity and basic cell biology. Whereas ISOC activation induces interendothelial cell gaps in all vascular segments, TRPV4 activation decreases cell–matrix interaction in capillaries. Whereas the TRPV4 channel controls permeability in capillaries, the T-type calcium channel controls P-selectin surface expression in this vascular segment. A major challenge is now to determine how these discrete intracellular calcium pools are coupled to their physiologically relevant effectors with such high fidelity, how these mechanisms are engaged in the course of inflammation, and how such diverse mechanisms are coordinated to elicit an appropriate inflammatory response.
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Chapter 16
Caveolae and Signaling in Pulmonary Vascular Endothelial and Smooth Muscle Cells Geerten P. van Nieuw Amerongen, Richard D. Minshall, and Asrar B. Malik
Abstract Caveolae exist in most cell types (with certain exceptions, e.g., erythrocytes, lymphocytes, and neurons) and are particularly abundant in endothelial cells (ECs) and smooth muscle cells (SMCs) of blood vessels. It is clear that the major plasmalemma vesicle structure in ECs and SMCs is caveolae as opposed to clathrin-coated vesicles. The number of caveolae is high in continuous endothelium (e.g., 73 caveolae per square micrometer in ECs of intramuscular capillaries) and low in fenestrated or discontinuous endothelium. Caveolae have a lipid composition similar to that of membrane rafts, but in addition, they possess other proteins, including the organellespecific structural protein caveolin and the more recently identified cavin. Caveolae appear to represent a specialized form of membrane raft domain, where caveolin-1, glycosphingolipids, and cholesterol are preferentially concentrated. Instead of assembling these structures in the plasma membrane, cells may in fact build caveolae in the Golgi apparatus and then send them to the plasma membrane proper, where they are incorporated, a process involving Na/K-ATPase. This chapter describes the structure and function of caveolae and caveolin proteins and their interaction with membrane receptors, transporters and signaling proteins that are related to the pulmonary vascular permeability and pulmonary hypertension. Keywords Caveolae • Caveosome • Caveolin • Clathrin • Lung microvascular endothelial cell • Vascular permeability • Signal transduction
1 Introduction 1.1 Structure and Function of Caveolae Caveolae exist in most cell types (with certain exceptions, e.g., erythrocytes, lymphocytes, and neurons) and are G.P. van Nieuw Amerongen (*) VU University Medical Center, Institute for Cardiovascular Research, Department of Physiology, van der Boechorststraat 7, 1081 BT Amsterdam, The Netherlands e-mail:
[email protected] p articularly abundant in endothelial cells (ECs) and smooth muscle cells (SMCs) of blood vessels. It is clear that the major plasmalemma vesicle structure in ECs and SMCs is caveolae as opposed to clathrin-coated vesicles (Fig. 1) The number of caveolae is high in continuous endothelium (e.g., 73 caveolae per square micrometer in ECs of intramuscular capillaries [1]) and low in fenestrated or discontinuous endothelium. On the basis of electron microscopy data, caveolae exist luminally, abluminally, and as free cytoplasmic vesicles, with the largest number in perijunctional zones between endothelia [2]. One issue often unappreciated is that caveolae number, quantified by electron microscopy, decreases 10- to 1,000-fold in cultured ECs (0.1–9 per square micrometer of plasma membrane), although ample caveolin-1 protein expression is detected in cultured cells [3]. Caveolae have a lipid composition similar to that of membrane rafts, but in addition, they possess other proteins, including the organelle-specific structural protein caveolin [4, 5] and the more recently identified cavin [6, 7]. Caveolae appear to represent a specialized form of membrane raft domain, where caveolin-1, glycosphingolipids, and cholesterol are preferentially concentrated [4, 8, 9]. Instead of assembling these structures in the plasma membrane, cells may in fact build caveolae in the Golgi apparatus and then send them to the plasma membrane proper, where they are incorporated, a process involving Na/K-ATPase [10]. Caveolae were originally described by Palade during electron microscopy analyses of capillary ECs [11] and somewhat later in gall bladder epithelium [12]. In collaboration with Simionescu [13, 14], Palade demonstrated the participation of caveolae in macromolecular transport across ECs by transcytosis. Recent dynamic intravital fluorescence microscopy has revealed that caveolae operate effectively as pumps, moving antibodies within seconds from blood across the endothelium into lung tissue, even against a concentration gradient [15]. In larger blood vessels, medial SMCs likewise display abundant caveolae [16] (Fig. 2) and various stimuli and ligands have been reported to induce caveolae internalization in SMCs. Recent studies have confirmed that caveolae are directly involved in the internalization of membrane constituents, extracellular ligands and macromolecules, bacterial toxins,
J.X.-J. Yuan et al. (eds.), Textbook of Pulmonary Vascular Disease, DOI 10.1007/978-0-387-87429-6_16, © Springer Science+Business Media, LLC 2011
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Fig. 1 (a) Transmission electron microscopy micrograph of small muscularized mouse artery showing predominance of caveolae vesicular structures in a smooth muscle cell (SMC) and an endothelial cell (EC) (scale bar 2 mm). (b) Higher magnification of the EC in (a) showing numerous caveolae along the luminal plasma membrane above the nucleus (scale bar 0.5 mm). (c) A ×10 zoom of EC caveolae open to the lumen (solid arrow) (scale bar 200 nm). (d) Higher magnification of (a) showing SMC caveolae attached to the plasma membrane. (e) A ×10 zoom of SMC caveolae attached to basal membrane (scale bar 200 nm). (Courtesy of Oleg Chaga, Center for Lung and Vascular Biology, University of Illinois at Chicago)
Fig. 2 Fluorescent micrographs of caveolin-1 and 4¢,6-diamidino-2phenylindole nuclear immunostaining of mouse artery. Note abundant green caveolin-1 staining along the plasma membrane of ECs aligned with the direction of blood flow, and relatively weaker caveolin-1 staining in SMCs aligned perpendicular to the flow. (Courtesy of E.C. Eringa, Institute for Cardiovascular Research, VU University Medical Center)
and nonenveloped viruses [17–19]. The internalized substances have been suggested to be delivered, whether directly or indirectly, to various intracellular compartments, such as the Golgi apparatus and the endoplasmic reticulum (ER) [20]. The caveolae-mediated pathway is considered to be distinct from the clathrin-mediated internalization pathway [21, 22]. In contrast to cargo sorting and vesicle formation by clathrin,
COPI, and COPII coats, caveolae contain permanently stabilized membrane-integrated coats that do not undergo cyclic association and dissociation. Efficient sorting is achieved by releasing cargo in response to local cues received in a particular compartment, such as low pH in endosomes [19]. The interaction and association between these two internalization pathways, if any, remains largely unknown. The term “caveosome” has been proposed to identify a caveolin-containing multivesicular structure thought to mediate sorting in a manner akin to, but independent of, endosomes in the clathrin-coated-pit/clathrin-coated-vesicle endocytic pathway [22, 23]. However, recent data indicate that caveosomes correspond to late endosomal compartments, wherefore this term no longer should be used [24]. This review will not deal with lipid transport/cholesterol homeostasis (not so much related to signaling function of caveolae).
1.2 Lessons from Cav-1-/- and Cavin-/- Mice Studies revealed that caveolin-1-null (Cav-1-/-) mice show (1) lack of caveolae, (2) downregulation (degradation) of caveolin-2 but normal expression of caveolin-3, (3) vascular
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dysfunction with reduced endothelium-dependent (NO) relaxation, contractility, and myogenic tone, and (4) lung abnormalities with thickened alveolar septa [25–28]. It was further observed that caveolin-1/caveolin-3 double-knockout mice lack caveolae both in muscle and in nonmuscle cells and develop a severe cardiomyopathy with myocyte hypertrophy, inflammation, and interstitial fibrosis [29]. Although the phenotypes of caveolin-1-deficient animals appear less serious than expected: the findings stress the importance of caveolin proteins and caveolae in the cardiovascular system. The primary observations from Cav-1-/- mice and small interfering RNA (siRNA)-mediated caveolin-1-depleted mice for the purposes of this review are (1) absence of caveolae in capillary endothelium, (2) persistence of caveolae in cardiomyocyte and vascular SMC sarcolemma, and (3) increased thickness of alveolar septa [27] and capillary basement membrane [30]. Cav-1-/- mice exhibit disruption of EC barrier properties and edema formation, particularly in the lung. Studies in Cav-1-/- models do not distinguish between the actions of caveolins within or outside of caveolae. The absence of the caveolin protein not only results in the lack of caveolae as a structure but also a total lack of interaction and modulation of activity of enzymes/molecules (e.g., endothelial nitric oxide synthase, eNOS) to which caveolin binds (whether inside or outside caveolae). Cavin, another caveolae-specific protein localized to the cytosolic face of plasma membrane adipocyte caveolae [31], colocalizes with caveolin-1 [32, 33]. It is highly expressed in adipocytes, lungs, heart, and colon, in lesser amounts in the thymus, spleen, kidney, and testis, and is weakly expressed in liver and brain [32, 33]. Animals deficient in cavin do not have caveolae in any cell type and, interestingly, also lack significant expression of all three caveolin isoforms even though caveolin messenger RNA expression is normal. Cavin-/- mice are viable and of normal weight but have higher circulating triglyceride levels, significantly reduced adipose tissue mass, glucose intolerance, and hyperinsulinemia – characteristics that constitute a lipodystrophic phenotype [6, 7] also observed in Cav1-/- mice [34].
2 Signaling 2.1 Caveolin-1 as a Scaffolding Molecule One of the most important structural components of caveolae is caveolin-1, a protein of 21–24 kDa. Caveolin-1 is a member of the caveolin family, which comprises caveolin-1, caveolin-2, and caveolin-3 [35–37]. (Fig. 3) Caveolin-1 and caveolin-2 are found in most cells, whereas caveolin-3 is
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Fig. 3 Phosphorylation map of the caveolin family of proteins. Caveolins are highly homologous and conserved proteins. All isoforms contain membrane-spanning, oligomerization and caveolin-scaffolding domains. (a) The caveolin-1a isoform consists of 178 amino acids with phosphorylation sites at Tyr14 and Ser80. The caveolin-1b isoform lacks the first 31 amino acids, and thus it does not contain the tyrosine phosphorylation site. Residues 75–158 (part of scaffolding domain, membrane-spanning domain, and most of the C-terminus) are involved in binding dynamin 2. Scaffolding and membrane-spanning domains of caveolin-1 also participate in homo-oligomerization and formation of heteroligomers with caveolin-2. (b) Caveolin-2 is approximately 50% homologous to caveolin-1, and the caveolin-2a isoform contains 162 amino acids with phosphorylation sites at Tyr19, Tyr27, Ser23, and Ser36. The caveolin-2b isoform, produced by alternative splicing of messenger RNA, is truncated by 13 N-terminal amino acids, whereas no information is yet available about the phosphorylation of the caveolin-2g isoform. (c) Caveolin-3 is a muscle-specific isoform, which contains 151 amino acids and is very homologous to caveolin-1. No phosphorylation sites have been demonstrated for caveolin-3 to date. (Modified with permission [37])
muscle-specific (most strongly expressed in skeletal and heart muscle cells). In smooth muscles, even in the same cell, caveolin-1 and caveolin-3 show different distributions, suggesting that the two caveolins form distinct caveolae, and that caveolin-1 and caveolin-3 serve different functions [38]. Two isoforms of caveolin-1 exist in the lung: caveolin-1a is expressed predominantly in ECs and caveolin-1b is expressed in epithelial cells [39]. Deletion of the caveolin-1 gene in mice resulted in cardiac hypertrophy and lung hypercellularity and matrix deposition [27], indicating its importance in cardiac and lung development. Expression of caveolin-1 induces the formation of caveolae-like structures [40, 41] and, hence, it appears to play a pivotal role in the biogenesis of caveolae. Coexpression of caveolin-1 and caveolin-2 facilitates the formation of deep caveolae [38, 41]. Recent studies have begun to identify the functions of caveolin-1. Caveolin-1 assembles as 12–18-mers in a stoichiometric complex with cholesterol [42]. Caveolin-1 appears to regulate caveolar internalization by stabilizing caveolae at the plasma membrane rather than controlling the shape of the membrane invagination [35]. Internalized caveolin-1 also colocalized with endocytosed transferrin in
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Rab5-labeled, Rab4-labeled, or early endosome antigen-1labeled compartments where caveolin-1 was phosphorylated; it then moved to a Rab11-associated compartment. Immunogold electron microscopy also revealed that internalized caveolin-1 colocalized with Rab5 or Rab4 in vesicles larger than caveolae. These results suggest that the internalized caveolae interact with early endosomes [43, 44]. Three palmitoylation sites in the C-terminal region next to the transmembrane domain contribute to anchoring caveolin proteins to the membrane. The N-terminal region of all caveolins contains a conserved FEDVIAEP motif that has been defined as the “caveolin signature” sequence, and has been suggested to be important for the binding of caveolin to cholesterol and glycosphingolipid-rich membrane domains [45, 46]. Caveolin-1, caveolin-2, and caveolin-3 molecules form homoand hetero-oligomers in the membrane and interact directly with cholesterol. They also interact with a multitude of signaling molecules (via a caveolin-binding motif in the latter) identified by proteomic methods [47–50]. As of yet, a definitive list of proteins (which may be cell-type-specific and context-dependent) that interact with caveolin-1 has not been established as outlined by Springer and Horrevoets [51]. The central segment of caveolin proteins contains the scaffolding domain (CSD), which allows oligomerization of caveolin monomers and direct interaction with other proteins, presumably regulating their activity [52]. Because caveolin-1 is a scaffolding protein, it has also been hypothesized to function as a “master regulator” of signaling molecules in caveolae [53]. In the endothelium, caveolin-1 regulates signaling by binding to and inhibiting eNOS. Increased cytosolic Ca2+ concentration or activation of the kinase Akt leads to eNOS activation and its dissociation from caveolin-1 [54]. The CSD (residues 82–101) has been considered to be a single functional domain for some time. In the last few years, however, it has been proposed that the CSD could be functionally divided into two regions, the former (82–95) mediating signaling protein (such as eNOS) inhibition and the latter (95–101) involved in cholesterol and membrane binding. Notably, the association with caveolin seems in most cases (eNOS, Ras, Wnt, and Erk1 and Erk2 cascades) to be inhibitory and to maintain the signaling molecules in an inactive state [53, 55]. Patel et al. [56] proposed therefore “that interaction of signaling proteins with the CSD regulates signal transduction events by sequestering components away from their signal transduction partners, such that in the ‘basal’ state signaling is inhibited, but colocalization in caveolae facilitates the interaction of components upon activation of the signaling pathway.” In addition, caveolin proteins contain several serine and tyrosine residues within their intracellular domains that are substrates for a variety of kinases and become phosphorylated in response to different stimuli [57, 58] (Fig. 3). Caveolin-1 was originally identified as a substrate of v-Src tyrosine kinase and, in fact, is heavily phosphorylated in v-Src-transformed cells [59]. The activation of temperature-sensitive v-Src
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kinase induced rapid tyrosine phosphorylation of caveolin-1 followed by a marked redistribution of phosphorylated caveolin-1 into cytoplasmic vesicular compartments [60]. Tyrosine phosphorylation of caveolin-1 and endocytosis of caveolae are also induced by oxidative stress or phosphatase inhibitors, such as hydrogen peroxide, okadaic acid, sodium orthovanadate, and pervanadate [43, 61–64]. Tyrosine phosphorylation of caveolin-1 was also reported in cells stimulated by insulin, insulin-like growth factor-1, epidermal growth factor, and albumin [62, 63, 65–69]. Although earlier work suggested that phosphorylated caveolin-1 (pY14Cav1), which comprises less than 1% total caveolin-1 under basal conditions, localizes mainly to focal adhesions and is largely absent from caveolae [57, 70], more recent work has indicated that the presence in focal adhesions likely reflects an artifact of the antibody used [71].
2.2 G-Protein-Coupled Receptors and Receptor Tyrosin Kinases in Caveloae Caveolae are rich sites of a large number of G-protein-coupled receptors (GPCRs; including the bradykinin, angiotensin, muscarinic, endothelin, TNFa, and b-adrenergic receptors), heterotrimeric G proteins, G-protein-regulated effectors, and receptor tyrosine kinases [vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF), epidermal growth factor] [56]. As implied by the caveolin signaling hypothesis, caveolae bring downstream effectors near to receptors (e.g., GPCRs) to help initiate receptor-, tissue-, and cellspecific signal transduction. These effectors are thought to reside in caveolae owing to direct interaction with caveolins (via the CSD) or by other caveolae-associated proteins. Palmitoylation may aid or enhance caveolar localization of proteins; the reversibility of palmitoylation may help regulate the movement of molecules into and out of caveolae in response to stimulation by agonists. In spite of the localization of certain GPCR and postreceptor signaling components in rafts or caveolae (see below), the precise determinants of this localization are not known. Certain GPCR and postreceptor signaling components show cell-selective patterns of localization in caveolae microdomains, but a generally accepted explanation for such patterns is as of yet not available. Certain non-receptor tyrosine kinases, such as members of the Src family (c-Src, Fyn, Lyn), are enriched in caveolae owing to N-terminal myristoylation and formation of complexes with caveolin [72]; palmitoylation of caveolin-1 at Cys156 is essential for caveolae/c-Src interaction [73]. Caveolin-1, owing to interaction via the CSD, suppresses the activity of c-Src and Fyn [58, 74]. Tyrosine phosphorylation of caveolin-1 (Tyr14) and caveolin-2 (Tyr19) facilitates recruitment of SH2-domain-containing proteins, such as Grb7 and matrix metalloproteinases [57, 59]. Caveolin-1 mediates
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the association of integrins with Src kinases, which can activate extracellular-signal-regulated kinase (ERK) and promote cell cycle progression [75, 76].
2.3 Caveolae and Ca2+ Handling In ECs, a number of molecules responsible for Ca2+ handling have been shown to be compartmentalized in caveolae, including a d-myoinositol 1,4,5-trisphosphate receptor like protein [77], dihydropyridine-sensitive Ca2+ channels [78], a Ca2+-ATPase [77], and Trp1 channels involved in capacitive Ca2+ entry [79]. Some of the proposed endothelium-derived hyperpolarizing factors (EDHFs), such as the epoxyeico satrienoic acids (EETs), seem to interact directly or in a membrane-delimited fashion with these Ca2+-regulatory proteins. Saliez et al. [80] demonstrated that expression of caveolin-1 is required for EDHF-related relaxation by modulating membrane location and activity of TRPV4 channels and connexins, which are both implicated at different steps in the EDHF-signaling pathway. Furthermore, caveolin-1 interaction can directly modulate the open probability of some channels such as the “big conductance” Ca2+-dependent potassium channels (BK), a potential end effector of EDHFmediated relaxation. Moreover, it has been demonstrated recently that the gap junction protein connexin (Cx43) and caveolin-1 partially colocalize in cells where they are endogenously expressed, mainly at junctional membranes of contacting cells. Electron microscopy studies also showed that caveolae are in close proximity to the ER [81]. The functional importance of caveolae with regard to Ca2+ release and reuptake was assessed [82]. It was demonstrated that caveolae are the preferred sites of Ca2+ entry when ER Ca2+ stores are depleted. Caveolae are thus the compartment involved in regulating store-operated Ca2+entry [83]. Patel et al. [84] recently showed that increased caveolin-1 expression in SMCs from patients with idiopathic pulmonary arterial hypertension was associated with enhanced Ca2+ entry in response to Ca2+ store depletion, indicating that increased caveolin-1 expression contributes to Ca2+ influx in SMCs. Cav-1 knockout (Cav-1-/-) mouse studies showed that loss of caveolin-1 expression in ECs abrogated Ca2+ entry due to Ca2+ store depletion [85]. Moreover, reexpression of wild-type Cav-1 in Cav-1-/- ECs rescued Ca2+ entry [85], indicating the role of caveolin-1 in regulating Ca2+ entry in ECs. We have shown that CSD peptide markedly reduced ER Ca2+ store depletion as well as store-depletion-activated Ca2+ entry in response to thrombin in ECs [86]. We also observed the interaction of the CSD with TRPC1 C-terminal residues 781-789 [87] and that the CSD interacts with inositol 1,4,5-trisphosphate receptor 3 and TRPC1 to regulate the Ca2+-store-releaseinduced Ca2+ entry in ECs [88].
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2.4 Regulation of eNOS/Akt eNOS-derived NO production is tightly regulated by direct interaction with caveolin-1 and is activated during caveolaemediated endocytosis [54]. Caveolar localization of eNOS in ECs was identified independently by two laboratories [89, 90]. eNOS localization is tightly regulated by a variety of transcriptional, posttranscriptional, and posttranslational mechanisms such as dimerization, phosphorylation, and protein–protein interactions. The activity of eNOS is determined by its localization and by phosphorylation at Ser1177 by several serine/threonine kinases, such as Akt. Several proteins residing in or recruited to caveolae serve as regulators of eNOS. Some of the proteins that modulate eNOS activity in addition to caveolin-1 include heat shock protein (HSP90), cationic amino acid transporter 1 (arginine transporter), Ca2+–calmodulin, ion channels, Ca2+ pumps, soluble guanylate cyclase, bradykinin B2 receptor, and VEGF receptor 2 [82, 91–95]. The localization of signaling molecules within caveolae provides the proximity necessary for a rapid and efficient propagation of signals to downstream targets. A novel mechanism of eNOS activation and NO production in ECs was recently described in which eNOS phosphorylation, dissociation from caveolin-1, and activation was induced by caveolae-mediated endocytosis and activation of caveolae transport machinery [54]. Inhibition of Gbg activation and downstream Src, Akt, and phosphatidylinositol 3-kinase pathways blocked caveolae-mediated endocytosis of albumin as well as NO production induced by the activation of albumin-binding protein gp60 in caveolae [54, 63]. In response to various stimuli such as VEGF and shear stress, eNOS binds to HSP90 which facilitates calmodulininduced displacement of caveolin-1 from eNOS, illustrating that the interrelationship of caveolin-1 and eNOS is a dynamic one [96, 97]. Recently, internalization of eNOS via caveolae has been shown to be a mechanism involved in the regulation of plateletactivating factor (PAF)-induced endothelial permeability, suggesting the novel concept that eNOS internalization into the cytosol is a signaling mechanism that leads to the onset of microvascular hyperpermeability in inflammation [98].
3 Regulation of Vascular Function by Caveolae Acute vascular inflammation [99] and progression of lung fibrosis [100] in mice were prevented by systemic administration of the CSD peptide, presumably by sequestering signaling proteins required for the response. These and many other studies strongly argue that caveolin-1, through its scaffolding domain, regulates EC and vascular SMC signal transduction, and thereby, vascular function.
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3.1 Differences Between EC and SMC Caveolae
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ECs and SMCs. Because SMCs also express caveolin-3 and thus caveolae still form in SMCs of caveolin-1 null mice (whereas they do not in ECs from these mice), it seems likely that caveolae and caveolin-1 may have differing functions in vascular regulation and homeostasis.
EC caveolae are thought to play an important role in transcellular transport and regulate basal endothelial permeability, oncotic pressure, and the delivery of hormones, drugs, and immunoglobulins [17]. In SMCs, caveolae have also been shown to mediate the uptake of solutes, although it seems less likely that they mediate a transport or transcellular permeability function. Rather, there is an emerging role of the caveolins in organizing and modulating SMC functions, including growth and proliferation, contraction, and metabolic support systems that support these functions [101]. In both cases, caveolae, via the scaffolding function of caveolin-1 and protein sequestering function of lipid raft domains, serve to concentrate signaling molecules and thereby control the kinetics of receptor-mediated signaling events, including Ca2+ influx and eNOS activation [62]. It remains unclear as to whether cytoskeleton–caveolae interactions are similar between ECs and SMCs and if this provides equivalent control of vesicular trafficking and domain-specific signaling events. Thus, it remains to be determined whether molecular signaling events specific to ECs and SMCs are regulated similarly by caveolin-1 or whether caveolin-1 controls critical cellular functions such as migration, proliferation, activation or reactivity, endocytosis, and exocytosis (secretion) of peptide hormones and adhesion molecules in a similar fashion in
Although caveolae form in vascular SMCs, they likely do not have a significant transport function, but rather are likely used for internalization of fatty acids and steroid hormones bound to albumin as well as for insulin-dependent signaling and glucose uptake [101]. Signaling pathways mediating the formation and release of caveolae from the plasma membrane are poorly understood [22]. Phosphorylation events are likely important since caveolar fission is increased by phosphatase inhibition and decreased by kinase inhibition [37, 61]. Caveolin-1 is phosphorylated by Src family kinases [102] on Tyr 14 [59], suggesting a relationship between tyrosine kinase activity and release of caveolae from the membrane [37, 63, 65, 74, 103] (Fig. 4). Immunoprecipitation studies showed that the 60-kDa albumin-binding glycoprotein (gp60) associates with caveolin-1 in ECs after gp60 activation [65]. In fact, both proteins are tyrosine-phosphorylated during caveolaemediated endocytosis of albumin [63, 65, 74]. In addition,
Fig. 4 Model of caveolae-mediated endocytosis in endothelial cells. Caveolin-1 plays a central role because it serves a scaffolding function for components of the “caveolar release complex,” Gi and Src, the signaling machinery responsible for endocytosis. Stimulation of G proteincoupled receptors or tyrosine kinase receptors, for example upon engagement and clustering of albumin binding protein gp60 (1, 2), activated Src phosphorylates tyrosine residues on caveolin-1 (Y14) and
caveolin-2 (Y19, Y27) (3) and also the GTPase dynamin-2 (Y231, Y597) which is thought to help recruit and activate dynamin-2 at the plasma membrane (4). The caveolar release complex thus engaged (i.e., phosphorylated caveolin, dynamin-2, and Src) activates vesicle fission. Src-dependent phosphorylation events may be the trigger that activates caveolar fission by decreasing the rigid structure of the caveolar coat and activating dynamin pinchase function. (Modified with permission [37])
3.2 Endocytosis Versus Transcytosis
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inhibition of G protein signaling with pertussis toxin or Gaiantagonist mini-gene peptide, as well as dominant-negative Src, blocked albumin-activated endocytosis in ECs [65]. Because both gp60 and Src activation are required for albumin endocytosis, Minshall et al. [65] addressed the role of Src phosphorylation of caveolin-1 and dynamin-2 in ECs in signaling the fission of caveolae and their release into the cytosol. Endocytosis of fluorescently tagged albumin or cholera toxin subunit B in ECs was blocked by filipin and methylb-cyclodextrin [65, 104], sterol binding agents that disassemble cholesterol-rich caveolae [5, 105]. Coincident with the endocytosis of albumin (within 1 min after gp60 activation), caveolin-1 and dynamin-2 were tyrosine-phosphorylated at residues 14 and 597, respectively [63, 103]. In both cases, pretreatment of cells with Src kinase inhibitor PP1 or PP2 abolished the phosphorylation. The functional importance of these events with respect to caveolae-mediated endocytosis was investigated in pulmonary microvessel ECs stably expressing non-Srcphosphorylatable caveolin-1 or dynamin-2 mutants. Expression of either Y14F caveolin-1 or Y597F dynamin-2 abolished albumin and cholera toxin subunit B endocytosis, indicating Src phosphorylation of these residues is required for signaling caveolar-mediated endocytosis [63, 103]. Furthermore, association between caveolin and dynamin was increased when dynamin was phosphorylated at Tyr597 and reduced by the nonphosphorylatable dynamin mutant [63, 103]. More recent data provide further support for the novel concept that a large component of pulmonary vascular hyperpermeability induced by activation of polymorphonuclear neutrophil (PMNs) adherent to the vessel wall is dependent on signaling via caveolin-1 and increased caveolae-mediated transcytosis. Thus, it is important to consider the role of the transendothelial vesicular permeability pathway that contributes to edema formation in developing therapeutic interventions against PMN-mediated inflammatory diseases such as acute lung injury [72]. Whether this also applies in SMCs is not known, but it at least seems plausible that agonists such as VEGF and insulin or engagement of cell adhesion molecules in SMCs may also regulate caveolae formation and fission. Caveolin-1-independent pinocytosis and transcellular transport has received some attention recently as a small pool of vesicles, speculated to be less than 1% of the caveolar vesicular population in wild-type mouse ECs [28], were observed in caveolin-1 knockout ECs and were shown by transmission electron microscopy to be similar in size to or slightly larger than (100–120-nm diameter) caveolae resembling vesicular-vacuolar organelles [28, 106]. Thus, assembly of these cellular structures does not require the presence of caveolin-1. This finding, reported by the three independent groups that generated the Cav1-/- mice [25, 26, 28], indicates there may be an additional pool of vesicles in ECs that
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is neither clathrin-coated nor caveolin-1-coated. As clathrincoated vesicles do not compensate for the loss of caveolae in Cav1-/- ECs, a proposed raft-mediated mechanism of endocytosis may predominate in the absence of caveolin-1 [107, 108]. The role of this endocytic pathway in transcellular transport, nutrient delivery, and receptor signaling in vascular ECs and SMCs is only now being investigated.
3.3 Paracellular Permeability Several lines of evidence indicate that caveolin-1 not only is essential for caveolae-mediated transcellular permeability, but also affects paracellular permeability. In addition to defective endocytosis of albumin, studies carried out in caveolin-1 knockout mice showed increased junctional (paracellular) permeability of the endothelial barrier [109, 110], providing a compensatory mechanism responsible for survival of the mice. Indeed, ultrastructural studies indicated opening of interendothelial junctions in caveolin-1 null mice [110] and following siRNA-mediated caveolin-1 depletion in vivo [109]. Remarkably, caveolin downregulation also contributes to leakiness of tumor vessels [111] and structural abnormalities of the tumor vasculature. Furthermore, inhibition of caveolae internalization by blocking caveolar scission with a dominant-negative dynamin mutant (K44Adynamin) inhibited PAF-induced hyperpermeability to fluorescein isothiocyanate–dextran-70 [98] as well as albumin [63]. However, thrombin-induced myosin light chain phosphorylation and stress fiber formation were not altered in caveolin-1-depleted cells, suggesting that caveolin-1 is not indispensable for all forms of vascular permeability [112]. There are several possible mechanisms by which caveolae/caveolin-1 might affect the paracellular or junctional endothelial barrier. Perhaps the most striking observation regarding the phenotype of Cav1-/- mice was the fivefold increase in plasma NO levels [27] thought to mediate opening of the junctional or paracellular pathway. This effect of NO may be due to enhanced phosphorylation of vascular endothelial cadherin (VE-cadherin) or b-catenin in the junctional complex, resulting in NO-mediated disassembly of the adherens junctions. Another possible mechanism of integrating caveolae-mediated transport and endothelial paracellular permeability involves the recently described role of the intersectin family of scaffolding proteins [113–115]. These proteins are present in abundance in ECs, where they regulate the activity of dynamin and mediate Cdc42-dependent actin polymerization. Studies showed that siRNA-induced knockdown of the long form of intersectin-2 resulted in decreased caveolae-mediated internalization but at the same time increased inter-EC dimensions, thereby regulating the paracellular permeability pathway. Alternatively, increased
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VEGF receptor 2 phosphorylation and decreased association with the adherens junction protein VE-cadherin [116] might also contribute to decreased endothelial barrier function in Cav1-/- mouse lungs. No direct evidence has been provided to indicate whether caveolin-1 interacts with the VE-cadherin complex, although it is known that b-catenin can be recruited to caveolae and that caveolin-1 binding to b-catenin blunts Wnt/lef-1 signaling by preventing interactions with glycogen synthase kinase-3b and the nuclear targeting of b-catenin [55]. Depletion of caveolin-1 in mouse brain microvascular ECs also reduces the expression of adherens junction proteins b-catenin and VE-cadherin as well as tight junction proteins ZO1 and Occludin [117, 118]. Additional studies are needed to determine how caveolin-1 regulates endothelial barrier function, how elevated NO levels perturb junctional integrity, and how caveolin-1 keeps ECs in a contact-inhibited state to maintain proper tissue organization in organs. Understanding the mutual interactions between junctional and caveolae-mediated permeability will likely be important in delineating how lung fluid balance is controlled and vascular homeostasis is maintained.
3.4 Mechanosensation and Shear Stress ECs and SMCs concentrate transmembrane receptors, and signaling proteins that transduce chemical and mechanical signals into the cell and mediate, for example, Rho GTPase activation [106, 119, 120]. In vascular SMCs, stretch-induced activation of focal adhesion kinase, Erk1/2, and Rho does not require caveolin-1 [121]. In ECs, Akt activation is insensitive to stretch, but is activated by shear stress in a caveolin-1dependent fashion. These data suggest that endotheliumdependent sensing of shear stress is primarily associated with caveolin-1-dependent Akt phosphorylation, whereas stretch sensing by vascular SMCs involves rapid mitogenactivated protein kinase and slow Rho–cofilin signaling. Exposure of mesangial cells to 10% equal biaxial cyclic mechanical strain caused Rho activation that was dependent on Rho–caveolin-1 interactions and was abrogated by disruption of caveolae using filipin or methyl-b-cyclodextrin treatment [122]. Stretch-induced activation of both RhoA and Rac1 through caveolae was reported in cardiomyocytes [123]. It was suggested that in these cell types, activation of RhoA and Rac1 localized in caveolae was essential for sensing externally applied forces and transducing these signals to the actin cytoskeleton and for Erk1/2 translocation [75]. A role for caveolin-1 or caveolae in shear-mediated EC responses has also been suggested. Caveolae have been proposed to mediate EC responses to flow in vitro [124, 125]. In mice, caveolin-1 expression is required for dilation and Akt signaling in response to acute changes in shear, as well as
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long-term remodeling after carotid artery ligation and resultant reduced blood flow [121, 125]. eNOS activation is enhanced in caveolae isolated from lungs exposed to flow, supporting the concept that mechanosignaling can occur via proteins found in caveolae [124]. Furthermore, Cav1-/- mice in the ApoE-/- background show decreased atherosclerosis [126]. However, caveolin-1 is also a critical negative regulator of eNOS, and Cav1-/- ECs have constitutively active eNOS and elevated NO production [127, 128]. Thus, the effects of caveolin-1 deletion might be explained in part by the dysregulation of eNOS; alternatively, Cav1-/- mice have an elevated high density lipoprotein level, reduced hepatic low density lipoprotein (LDL) production, and decreased LDL transcytosis across ECs [129]. Thus, a role for caveolin-1 in mechanotransduction per se has not been established.
3.5 Vascular Tone In ECs, caveolin-1 acts as a scaffolding protein to cluster lipids and signaling molecules within caveolae and, in some instances, regulates the activity of proteins targeted to caveolae. Specifically, putative mediators of EDHF-mediated relaxation are located in caveolae and/or regulated by the structural protein caveolin-1, such as potassium channels, calcium regulatory proteins, and connexin 43, a molecular component of gap junctions. Comparing relaxation in vessels from caveolin-1 knockout mice and their wild-type littermates, we observed a complete absence of EDHF-mediated vasodilation in isolated mesenteric arteries from caveolin-1 knockout mice. The absence of caveolin-1 is associated with an impairment of calcium homeostasis in ECs, notably, a decreased activity of Ca2+permeable TRPV4 cation channels that participate in NO- and EDHF-mediated relaxation. Moreover, morphological characterization of caveolin-1 knockout and wild-type arteries showed fewer gap junctions in vessels from knockout animals associated with a lower expression of connexins 37, 40, and 43 and altered myoendothelial communication. Finally, we showed that TRPV4 channels and connexins colocalize with caveolin-1 in the caveolar compartment of the plasma membrane. Expression of caveolin-1 is required for EDHF-mediated relaxation by modulating membrane location and activity of TRPV4 channels and connexins, which are both implicated at different steps in the EDHF-signaling pathway [80]. In pulmonary artery SMCs from patients with idiopathic pulmonary arterial hypertension, an increase in caveolin-1 expression is associated with increased formation of caveolae, enhanced capacitive Ca2+ entry, and accelerated SMC proliferation [84]. Furthermore, disruption of caveolae was shown to inhibit pulmonary vasoconstriction [130]. For a review of the role of caveolin-1 in pulmonary arterial hypertension, see Mathew et al. [131] and Chap. 69, where the role of caveolae in pulmonary hypertension is described.
16 Caveolae and Signaling in Pulmonary Vascular Endothelial and Smooth Muscle Cells
3.6 SMC Mitogenic Signaling The role of caveolin and caveolae functions in SMC physiologic and pathophysiologic processes is not yet known. Studies thus far suggest that caveolae are less abundant in synthetic than in contractile SMCs [132] and are less abundant in proliferating than in nonproliferating cells [115, 132]. Furthermore, Peterson et al. [134] used cultured human coronary vessel SMCs to study (1) how PDGF, a potent SMC mitogen and an important factor in atherogenesis [114, 133, 134], affects caveolin-1 expression, and (2) how overexpression of caveolin-1 influences the response to PDGF [133, 135]. The results demonstrate that exposure of serum-starved SMCs to PDGF caused a dose-dependent reduction in caveolin-1 levels as determined by immunoblotting. Electron microscopy analysis further showed that the loss of caveolin-1 was accompanied by a decrease in the number of cell-surface caveolae. In vivo confirmation of these findings was demonstrated in that the decrease in caveolin-1 protein in the PDGF-treated cultures was paralleled by a distinct increase in caveolin-1 messenger RNA content. Hence, caveolin-1 downregulation in response to PDGF could not be ascribed to an inhibition of caveolin-1 gene transcription. On this basis, it was inferred that the PDGF-induced reduction in caveolin-1 was due to enhanced degradation, via either lysosomes or the ubiquitin-proteasome pathway. Overexpression of caveolin-1 in SMCs did not interfere with early events in PDGF signal transduction (Erk1/2 activation), but blocked entrance into the G1 phase of the cell cycle (cyclin D1 synthesis) and progression into S phase (DNA synthesis), thereby shifting the cells into an apoptotic program [136]. Taken together, these findings suggest that stretch-induced growth signaling in vascular SMCs is dependent on cholesterol-rich plasma membrane domains such as caveolae. These findings indicate that the transition of SMCs from a contractile to a synthetic phenotype involves a marked decline in the number of plasma membrane caveolae. In parallel, caveolin is internalized and redistributed to Golgi-associated vesicles in the perinuclear cytoplasm [132].
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Caveolae appear to provide a strategic pathway worthy of targeting. They contain intravenously accessible, tissue-specific proteins that mediate active transport of macromolecules into the tissue, overcoming the restrictive EC barrier. The caveolae gateway may therefore provide a selective, speedy shuttle service for imaging and therapeutic applications [15, 138]. Although mutations in caveolin-3 have been reported to cause autosomal dominant limb-girdle muscular dystrophy and caveolin-1 mutations occurring during the early stages of mammary transformation have been suggested to be critical upstream/initiating events leading to increased ERa levels, no such mutations have been associated with cardiovascular disease. Inhibition of caveolar endocytosis may help to limit vascular hyperpermeability associated with inflammation [72, 98]. The study by Saliez et al. [80] emphasizes the role played by caveolin-1 in EDHF-related signaling and raises the possibility of targeting caveolae to improve vascular relaxation in the context of coronary or peripheral ischemic diseases characterized by deficient endothelium-dependent relaxation. Mutations in caveolin-1 have been observed in 16% of human breast cancers [115, 139], and many human tumors have reduced levels of caveolin-1 expression [114]. For some tumors, however, caveolin-1 expression appears to be related to cell survival and growth [140, 141]. Thus, depending on the tumor origin, caveolin-1 can function either as a tumor suppressor or as a tumor promoter. The molecular mechanisms underlying these cell-type-specific growth-regulatory behaviors are currently unknown. Dysregulation of caveolin-3 results in muscular defects, reflecting its restricted tissue distribution to muscle cells. Patients with Duchenne muscular dystrophy show increased levels of caveolin-3 and caveolae at the sarcolemma. The opposite situation occurs in limbgirdle muscular dystrophy, where mutations in caveolin-3 result in severe reduction of caveolin-3 expression and in the number of caveolae in muscle. Alterations in caveolin-3 have also been described for hereditary rippling muscle disease, which is linked to a locus that spans Cav3.
References 4 Concluding Remarks Caveolinopathy needs to be considered as a differential diagnosis in a range of clinical situations, including patients who do not have clinical symptoms. Mutations in the caveolin-3 gene (CAV3) can lead to a broad spectrum of clinical phenotypes. Five different mutations were identified. Phenotypes that have so far been associated with primary caveolin-3 deficiency include limb-girdle muscular dystrophy, rippling muscle disease, distal myopathy, and hyperCKemia [137].
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16 Caveolae and Signaling in Pulmonary Vascular Endothelial and Smooth Muscle Cells 127. Ju H, Zou R, Venema VJ, Venema RC (1997) Direct interaction of endothelial nitric-oxide synthase and caveolin-1 inhibits synthase activity. J Biol Chem 272:18522–18525 128. García-Cardeña G, Martasek P, Masters BS et al (1997) Dissecting the interaction between nitric oxide synthase (NOS) and caveolin. Functional significance of the NOS caveolin binding domain in vivo. J Biol Chem 272:25437–25440 129. Frank PG, Pavlides S, Cheung MW, Daumer K, Lisanti MP (2008) Role of caveolin-1 in the regulation of lipoprotein metabolism. Am J Physiol Cell Physiol 295:C242–C248 130. Schach C, Firth AL, Xu M et al (2008) Regulation of pulmonary vasoconstriction by agonists and caveolae. Exp Lung Res 34:195–208 131. Mathew R, Huang J, Gewitz MH (2007) Pulmonary artery hypertension: caveolin-1 and eNOS interrelationship: a new perspective. Cardiol Rev 15:143–149 132. Thyberg J, Roy J, Tran PK, Blomgren K, Dumitrescu A, Hedin U (1997) Expression of caveolae on the surface of rat arterial smooth muscle cells is dependent on the phenotypic state of the cells. Lab Invest 77:93–101 133. Raines EW, Ross R (1996) Multiple growth factors are associated with lesions of atherosclerosis: specificity or redundancy? Bioessays 18:271–282 134. Peterson TE, Guicciardi ME, Gulati R et al (2003) Caveolin-1 can regulate vascular smooth muscle cell fate by switching plate-
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Chapter 17
The Chemistry of Biological Gases D. Jeannean Carver and Lisa A. Palmer
Abstract Today there are at least four biological gases: oxygen (O2), nitric oxide (NO), carbon monoxide (CO), and hydrogen sulfide (H2S). Except for molecular oxygen, many of these gases were originally known for their detrimental physiological effects. However, the current consensus suggests that these gases play a role in signal transduction, modulating physiological function. The roles of these gases are complex as each is dependent on the rate of production, concentration, chemical reactivity, and availability of target proteins. Moreover, many of these gases share the same molecular targets, and thus are able to modify the responses to each other. This chapter discusses the chemistry of each of these gases, their biological production, potential interactions, and physiological consequences. Keywords Reactive oxygen species • Radicals • Oxidant • Superoxide • Nitric oxide • Carbon monoxide • Hydrogen sulfide • Nitrosylation
1 Introduction Today there are at least four biological gases: oxygen (O2), nitric oxide (NO), carbon monoxide (CO), and hydrogen sulfide (H2S). Except for molecular oxygen, many of these gases were originally known for their detrimental physiological effects. However, the current consensus suggests that these gases play a role in signal transduction, modulating physiological function. The roles of these gases are complex as each is dependent on the rate of production, concentration, chemical reactivity, and availability of target proteins. Moreover, many of these gases share the same molecular targets, and thus are able to modify the responses to each other. This chapter will discuss the chemistry of each of these
D.J. Carver (*) Department of Pediatrics, University of Virginia, Charlottesville, VA, USA e-mail:
[email protected] gases, their biological production, potential interactions, and physiological consequences.
2 Oxygen and Reactive Oxygen Species Oxygen (O2) makes up 21% of the air we breathe. It is carried by hemoglobin in red blood cells, which deliver oxygen to and remove carbon dioxide from tissues in the body. Changes in oxygen levels can act as a molecular switch; turning on ion channels [1], altering blood flow [2], and modifying the activity or expression of transcription factors [3–5]. With respect to the pulmonary vasculature, inhaled oxygen is a prompt vasodilator and the classic therapy for acute pulmonary hypertension. O2 is also necessary in the production of energy by being the terminal electron acceptor in the mitochondrial transport chain and in the process generating reactive oxygen species. These radical and reactive oxygen species produced from oxygen are, in normal physio logical conditions, necessary for normal redox-mediated vascular signaling. In disease states where they are overproduced or poorly controlled, however, they are likely counterproductive, with resultant proliferation of vascular smooth muscle and increased vascular tone.
2.1 Reactive Oxygen Species Biologically, molecular oxygen generates cellular respiration through its mitochondrial reduction to water in a series of oneelectron transfers. During this process, reactive oxygen species are also produced, including superoxide radical (O2•−), hydrogen peroxide (H2O2), hydroxyl radical (HO•), and peroxynitrite (in the presence of radical nitric oxide). Radicals are chemical entities with unpaired electrons in an otherwise open-shell configuration, i.e., all valence electrons have not been shared in forming chemical bonds. These electrons are highly “reactive” in that they are quickly engaged in forming new chemical bonds. Most radicals, therefore, have short half-lives (Fig. 1).
J.X.-J. Yuan et al. (eds.), Textbook of Pulmonary Vascular Disease, DOI 10.1007/978-0-387-87429-6_17, © Springer Science+Business Media, LLC 2011
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Fig. 1 Biosynthesis of oxygen radicals and other reactive oxygen species
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Fig. 3 NADPH oxidase and the biosynthesis of reactive oxygen species. NADPH oxidase is activated by a number of luminal stressors, with resultant liberation of a free electron necessary for oxygen radical formation
Fig. 2 Enzymes, catalysts, and cofactors in the biosynthesis of reactive oxygen species
Peroxide and hypohalous acids, such as HOCl and HOBr, although not radicals per se, are potent and “reactive” oxidizing molecules that serve as intermediate molecules in the conversion of one radical oxygen species to another. The production of oxygen radicals may be catalyzed by a number of tissue- or cell-specific enzymes or by metals, especially iron and copper (Fig. 2).
2.2 Superoxide The major source of superoxide radical is the one-electron reduction of oxygen in the electron transport chain of mitochondria and in the endoplasmic reticulum. Additionally, O2•− is produced in the vascular wall primarily by the constitutively active enzyme NADPH oxidase found in endothelium, vascular smooth muscle cells, and adventitial fibroblasts [6, 7]. Catalysts of vascular superoxide production include NADPHdependent oxidases, xanthine oxidases, lipoxygenases, and mitochondrial oxidases (Fig. 2). Studies have identified NADPH oxidase as the most important source of superoxide anion in the vasculature, playing a significant role in the vascular remodeling and endothelial dysfunction found in cardiovascular diseases [8] (Fig. 3). Likewise, superoxide may be produced during the conversion of hypoxanthine to xanthine by xanthine oxidase, and by nitric oxide synthase (NOS) when l-arginine and tetrahydrobiopterin are limiting [9]. Once formed, superoxide radical is typically converted to significantly more reactive and biologically dangerous oxygen species through (1) the slower metal-catalyzed Haber– Weiss reaction to hydroxyl radical (Fig. 4) or (2) interaction with nitric oxide to peroxynitrite (ONOO−) (Fig. 1).
Fig. 4 The Haber–Weiss reaction. The Haber–Weiss reaction is the iron-catalyzed transformation of superoxide and hydrogen peroxide to the hydroxyl radical. The second step of it is referred to as the Fenton reaction
2.3 Hydrogen Peroxide H2O2 is not a classic radical as it has no unpaired electrons and may have a longer half-life, diffusing easily through cell membranes and to distant locations from its site of origin. It is, however, an important intermediate product in the conversion of the superoxide radical (O2•−) to the more toxic hydroxyl radical (HO•) and is produced in microsomes, mitochondria, and phagocytic cells. H2O2 is produced from superoxide nonenzymatically, or by superoxide dismutase, whereupon it may react with ferrous iron (Fe2+) or copper (Cu+) to produce the hydroxyl radical (Fig. 5). Additionally, H2O2 can be produced by monamine oxidase, galactose oxidase, xanthine oxidase, and amino acid oxidase [10]. In addition to the hydroxyl radical, H2O2 can be oxidized by eosinophil-specific peroxidase and neutrophil-specific peroxidase from halide substrates (Cl− or Br−) to form potent oxidant hypohalous acids (HOX) and other reactive halogenating species. Furthermore, H2O2 can inactivate enzymes through oxidation of essential thiol groups. In opposition to the damaging effects of H2O2, the enzyme catalase neutralizes it to water and diatomic oxygen.
17 The Chemistry of Biological Gases
Fig. 5 The influence of superoxide dismutase activity on the vasculature
Fig. 6 The production of the hydroxyl radical from hypochlorous acid
2.4 Hydroxyl Radical The hydroxyl radical is a more dangerous compound than superoxide because it reacts with an oxidizable compound faster than it can undergo enzymatic detoxification. In fact, most of the damage produced by superoxide and peroxide in vivo is due to the production of HO•, although its high reactivity limits its actions locally to the site of initiation. The hydroxyl radical may be generated by the Haber–Weiss reaction (Fig. 4) or by the one-electron reduction of hypochlorite by ferrous iron or superoxide (Fig. 6). It can oxidize, disabling proteins, carbohydrates, nucleic acids, and lipids, resulting in injury to the cell membrane, DNA mutation, and protein signaling dysfunction. Fortunately, abundant endogenous antioxidants such as glutathione reduce its toxicities in the normal physiological state.
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mitochondrial electron transport chain) generates a diffusible mediator (i.e., reactive oxygen species) to regulate an effector protein (i.e., voltage-gated potassium and calcium channels). An analogous mechanism for regulating oxygen diffusion and circulation also exists in other specialized mammalian O2-sensitive tissues, such as the carotid body and ductus arteriosus [14]. Reactive oxygen species influence other physiological processes as well, including host immunity, hormone biosynthesis, and cell signaling [15]. The physiologic regulatory effects of reactive oxygen species specific to vascular function include modulation of cell growth, apoptosis, immune cell migration, inflammation, secretion, and extracellular matrix protein production [7]. However, in disease states where pro-oxidants may overwhelm antioxidant capacity, an imbalance in the redox state may result, whereby reactive oxygen species contribute to vascular dysfunction and remodeling through oxidative damage, or “stress.” This may alter a number of redox-sensitive signaling pathways involved in the functional and structural vascular changes associated with various abnormalities, including hypertension, hyperlipidemia, atherosclerosis, diabetes, chronic renal failure, and pulmonary hypertension [7, 16, 17] (Fig. 7). Several recent studies have provided evidence that reactive oxygen species produced specifically by the activities of NADPH oxidase may contribute significantly to alterations in vascular tone in disease states associated with chronic or intermittent hypoxia-induced pulmonary hypertension, such as bronchopulmonary dysplasia, persistent pulmonary hypertension of the newborn, and obstructive sleep apnea [18–22]. A better understanding of the role played by radical and reactive oxygen species in pulmonary vascular function may provide significant opportunities for the therapy of pulmonary hypertension.
2.5 Physiology Oxygen and its radicals play important roles in the regulation of vascular function, both for normal physiological function but also in disease, regulating endothelial NOS (eNOS) and nitric oxide (NO) [11–13]. Under physiological conditions, low concentrations of intracellular oxygen radicals play an important role in the normal redox signaling of vascular function and integrity. One example is hypoxic pulmonary vasoconstriction (HPV), the homeostatic and widely conserved vasomotor response of resistance pulmonary arteries to alveolar hypoxia. HPV balances ventilation and perfusion, diverting pulmonary blood flow away from unoxygenated lung, and optimizing systemic pO2 by reducing intrapulmonary shunt. It is a phenomenon intrinsic to the lung, modulated by endothelium, but mechanistically occurring in the smooth muscle cell. The “redox theory” for the mechanism of HPV proposes the synchronized action of a redox sensor (the proximal
Fig. 7 Reactive oxygen species and vascular cell function. Reactive oxygen species influence vascular cell function through a variety of pathways, including ion channels, transcription factors, metalloproteinases, posttranslational modifications by nitric oxide, and classic kinase signaling cascades
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mediated through interactions with oxygen and reactive oxygen species, transition metals, and thiols.
3 Nitric Oxide and Reactive Nitrogen Species Nitric oxide (NO) is a small diatomic molecule. Historically, it was known for its adverse effects on the environment, including the generation of smog, depletion of ozone, and in the formation of acid rain. In the late 1970s, NO was found to increase the activity of guanylate cyclase and relax smooth muscle [23, 24], and an endothelium-derived factor, named endothelium-derived relaxing factor (EDRF), was found to relax isolated vascular strips [25]. In 1987, EDRF was identified as NO [26, 27]. Since that time, NO has been found to be involved in a variety of biological functions, including regulation of vascular tone, platelet function, cellular adhesion, extravasation of leukocytes into tissue, neuronal transmission, immune system function, mitochondrial respiration, and regulation of iron bioavailability [28].
3.1 Nitric Oxide Biosynthesis NO is produced endogenously from l-arginine by the action of NOSs, Eq. (1).
L - Arginine + NADPH + O 2 → L - citrulline + NO + NADP +
(1)
NOSs exist in three isoforms: neuronal (or type I) NOS (nNOS), inducible (or type II) NOS (iNOS), and eNOS (or type III). Each enzyme is a homodimer requiring three cosubstrates (l-arginine, NADPH, and O2) and five cofactors (FAD, FMN, calmodulin, heme, and tetrahydrobiopterin) for activity. Each NOS is composed of an N-terminal oxygenase domain, a calmodulin binding domain, and a C-terminal reductase domain. The oxygenase domain binds heme, tetrahydrobiopterin, and l-arginine, which forms the active site where NO synthesis takes place. The reductase domain binds FMN, FAD, and NADPH. During NO synthesis, the reductase flavins acquire electrons from NADPH and transfer them to the heme iron, permitting it to bind and activate O2, catalyzing the formation of NO [29]. NOS abundance is regulated by transcriptional, translational, posttranslational, and biochemical means [30]. NO production is controlled by (1) the availability of substrate l-arginine via the activity of arginase [31], (2) the availability of cofactors [32], and (3) the presence of the endogenous inhibitors such as asymmetric dimethyl l-arginine [33]. The varied functionality of NO is due to its wide range of chemical reactivity and number of potential reactive targets (lipids, proteins, nucleic acids, and sugars). Chemical interactions of NO are determined by the local redox environment which determines its chemical form: free radical (NO.), nitrosyl anion (NO−), and nitrosonium cation (NO+). Chemically, the actions of NO are
3.2 Nitric Oxide Reactions with Oxygen/ Reactive Oxygen Species The effects of NO can be mediated through the formation of reactive nitrogen oxide species from reactions of NO with either O2 or O2−. Reactions with oxygen, as represented by Eqs. (2–4) are slow at physiological concentrations: nanomo lar concentrations of NO; micromolar concentration of oxygen.
2NO + O 2 → 2NO 2
(2)
NO 2 + NO → N 2 O3
(3)
N 2 O3 + H 2 O → 2HNO3
(4)
However, under pathological conditions where the production of NO is elevated or in microenvironments (membranes or in the hydrophobic core of a protein), these reactions may be more relevant. NO and O2− rapidly combine to form peroxynitrite (ONOO−, Eq. (5)), which has been proposed to play a central role in physiological processes involving NO and pathological changes caused by NO. H
+
NO + O − → ONOO − − ONOOH →·NO +·OH (5) 2 2 This reaction occurs close to the diffusion-controlled limit, with an average rate constant of 1010 M−1s−1 [34, 35]. In healthy cells, the levels of NO are regulated by the activity of NOS and the presence of oxyhemoglobin, whereas the levels of O2− are regulated by the activity of superoxide dismutase [36]. Thus, peroxynitrite production in biological systems is determined by the rates of production of O2− and NO, their relative concentrations, and their proximity to one another. Peroxynitrite is a transient species with a biological halflife of 10–20 ms [37]. Although peroxynitrite anion is relatively stable, peroxynitrous acid decays rapidly as it homolyzes to form nitrogen dioxide (NO2) and hydroxyl radical, Eq. (5). However, in the absence of a target, the main product from peroxynitrite decay is nitrate. Peroxynitrite reacts directly with certain amino acids, the most susceptible being the sulfur-containing amino acids (cysteine and methionine) and the aromatics (tryptophan, tyrosine, phenylalanine, and histidine) [38]. Peroxynitrite can also react with carbon dioxide to form nitrogen dioxide and carbonate radicals [39], Eq. (6) This reaction occurs with a second-order rate constant of 4.6 × 104 M−1s−1 at pH 7.4 and 37°C [40].
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(6)
ONOO − + CO 2 →·NO 2 + CO3.−
Transition metal centers (heme and non-heme iron, copper, and manganese ions) are also targets of peroxynitrite [38], forming a secondary oxidizing species at the metal center plus nitrogen dioxide, as described by Eq. (7) ONOO
−
+M
n+
→ ONOO
−
−M
n+
→·NO 2 +
−.
O−M
n+
(7)
Hydrogen peroxide does not act directly with nitric oxide. However, interactions can occur between hypervalent metal–oxo complexes following reactions between hemes and peroxides. These hypervalent metal–oxo complexes are integral components to the catalytic cycle of peroxidases [glutathione peroxidase, catalase, heme oxygenase (HO)-1, myloperoxidase] and thus have the potential to alter signaling mediated by NO. NO interactions with hypervalent metal–oxo complexes are discussed next.
3.3 Nitric Oxide Reactions with Metals Nitric oxide can interact with a variety of transition metals. Direct interactions of these metal centers can form metal nitrosyl complexes with no net change in redox state, Eq. (8).
Fe (II )(H 2 O )6
2+
+ NO → Fe (II )(H 2 O )5 NO 2 + + H 2 O (8)
These nitrosyl complexes can be formed by the process of nitrosation through NO+ donation or through oxidative nitrosation where the thiol is oxidized to a radical prior to the addition of NO. NO can interact directly with metal–oxygen complexes. One good example of this reaction is the interaction of NO with oxyhemoglobin to form methemoglobin and nitrate Eq. (9). This is considered to be one of the primary ways in which NO radical is consumed as the reaction is rapid (3 × 107 M−1s−1) and oxyhemoglobin is at a high concentration (4–8 mM) [28].
Hb (Fe − O 2 ) + NO → Met Hb (Fe3+ )+ NO3−
(9)
NO can interact with high-valence metal complexes formed from the oxidation of metal species or metal–oxygen complexes by hydrogen peroxide, Eqs. (10, 11). A reduction of the hypervalent complexes to a lower-valence state protects tissues from peroxide-mediated damage.
Fe (II ) + H 2 O 2 → Fe (IV )O + NO → Fe (II ) + NO 2 − (10) Fe (III ) + H 2 O 2 → Fe (V )O + NO → Fe (III ) + NO 2 − (11)
3.4 Nitric Oxide Reactions with Thiols Traditionally, nitric oxide synthase is thought to generate NO radical (NO.). NO. diffuses into target cells and signals through the activation of guanylate cyclase. In 1992, S-nitrosothiols (SNOs) were discovered within a biological system [41]. Since that time, numerous proteins have been found to be S-nitrosylated. These include hemoglobin [42], hypoxia-inducible factor-1a [43–46], SP-1 [47], eNOS [48–58], and cycloxygenase-2 [51], to name a few. In addition, genetic and biochemical data [52–55] suggest a role of SNOs in mediating many of the guanylate cyclase independent actions of NOS. SNOs can be synthesized, transported, and metabolized. Synthesis can occur through a variety of ways. SNOs can be formed by a direct interaction with thiol radicals or thiols to form SNOs or SNO radicals. SNO radicals may be stabilized through the loss of an electron or through protonation [55, 58]. In addition, SNOs can be produced through the oxidation of NO to NO+ followed by a reaction with thiols [55–58]. Oxidation of NO can be catalyzed by oxygen, aromatic residues, and transition metal ion complexes. Both iron (hemoglobin [59]) and copper (ceruloplasmin [57]) catalyze the formation of SNOs. Hemoglobin can be viewed as an S-nitrosothiol synthase [60] where nitrite is the substrate. Under physiological concentrations and time constraints, nitrite in the presence of deoxyhemoglobin forms an intermediate with the properties of Fe3+NO which is converted to SNO–hemoglobin upon oxygenation. Thus, hemoglobin, converts NO into SNOs through an allosterically modulated heme–NO redox reaction. Ceruloplasmin can also serve as an enzyme to synthesize S-nitrosoglutathione (GSNO), an endogenously produced SNO. In this reaction, copper serves as the electron acceptor, and NO+ is transferred to the thiolate of glutathione. Transport of GSNO into cells can be mediated by g-glutamyl transpeptidase (gGT), the L-AT transport system, or protein disulfide isomerase [61–64]. One enzyme that plays a physiological role in the catabolism of SNOs is S-nitrosoglutathione reductase (GSNO-R) [65, 66]. This ubiquitously expressed enzyme is responsible for the breakdown of GSNO to oxidized glutathione and ammonia. GSNO-R does not directly affect SNO–proteins; however, these SNO–proteins may be affected indirectly through altered transnitrosation equilibria with GSNO. Besides GSNO-R, other enzyme systems have been implicated in the catabolism of SNOs in vivo. These include thioredoxin/thioredoxin reductase [67], Cu/Zn superoxide dismutase [68–70], and gGT [61–63]. Physiologically, SNOs have been found to play a role in signaling hemoglobin desaturation [71], signaling hypoxic ventilatory drive [61], and regulating hypoxic gene regulation [72].
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3.5 Tyrosine Nitration Tyrosine nitration is mediated by reactive nitrogen species (ONOO−) and nitrogen dioxide (NO2) formed as secondary products of NO metabolism in the presence of oxidants which include superoxide, hydrogen peroxide, and transition metal centers [73]. In vivo, different nitration pathways can contribute to tyrosine nitration. Most involve free-radical biochemical reactions with carbonate radials and/or oxo– metal complexes oxidizing tyrosine to tyrosyl radicals followed by a diffusion-controlled reaction with .NO2 to yield 3-nitrotyrosine.
3.6 Relationship Between Nitric Oxide and Oxygen The relationship between oxygen and nitric oxide is complex. Nitric oxide affects arteriolar tone and blood flow, thus affecting oxygen delivery to cells and tissues. Yet, nitric oxide release by NOS depends on oxygen concentration. The Km values for oxygen for each of the three NOS isoforms are significantly different: 350 mM for nNOS, 130 mM for iNOS, and 4 mM for eNOS [74]. Release of nitric oxide in the form of S-nitrosothiols from hemoglobin has been connected with oxygen saturation [2] and is thought to optimize ventilation/perfusion within the lung [71]. Nitric oxide can also alter cytochrome c oxidase activity, thus regulating mitochondrial respiration and tissue oxygen consumption. Lastly, nitric oxide can enhance the stabilization of hypoxia-inducible factor-1a [44–46], altering hypoxic signaling and potentially leading to the development of disease such as pulmonary hypertension [72]. Taken together, interactions between nitric oxide and oxygen can alter the bioavailability of both molecules, through a variety of mechanisms.
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transferred to acceptor proteins via transnitrosation reactions from hemoglobin [76, 77]. In the nitrite reductase model, NO signaling is related to nitrite reduction by deoxygenated hemoglobin. The conversion of nitrite to NO is allosterically regulated: increases in oxygen binding increase the rate at which nitrite is converted to NO [78]. Stepwise, nitrite reacts with deoxygenated hemogloblin to make methemoglobin and NO. It is proposed that to prevent scavenging of NO by resident oxyhemoglobin within the red blood cell, an intermediate species is formed in the nitrite reductase reaction. This intermediate diffuses out of the red blood cell and decomposes into NO or a chemical species with NO-like bioactivity. One potential candidate for this intermediate is N2O3 [79]. The formation of N2O3 could occur through the formation of an adduct with Fe2+–NO2− character from the interaction of methemoglobin and nitrite. The radical nature of this adduct would mean the adduct would quickly react with NO, forming N2O3, which can homolyze into NO and NO2., affecting vascular smooth muscle tone (Fig. 8). In this second model, red blood cells are a vehicle for O2-regulated delivery of NO signals in the form of S-nitrosothiols (Fig. 9). Hemoglobin binds NO at cysteine b93 and converts it to bioactive SNO as a function of blood O2 saturation [77, 80, 81]. The binding of O2 changes the conformation of hemoglobin from the deoxygenated state to the oxygenated state [77, 81]. This permits the transfer of nitrosonium (NO+) equivalents from the heme (Fe2+) to the thiol (cysteine b93) [77, 82, 83]. Upon deoxygenation, NO+ equivalents are transferred from cysteine b93 to a receptor cysteine in the cytosolic N-terminus of anionexchange protein 1 (AE1) [82, 83] on the red blood cell membrane. The NO+ equivalents located on AE1 are then exported from the red blood cell by a mechanism that is not well defined, but may occur directly through cell–cell transfer or indirectly through the formation of a serum intermediate such as S- GSNO or S-nitrosocysteine [83]. Subsequent entry of GSNO into cells can be mediated by gGT or other transporters [64].
3.7 Hemoglobin and Nitric Oxide Bioavailability
4 Carbon Monoxide
Hemoglobin plays a number of roles in regulating NO bioavailability. Initial observations demonstrated that hemoglobin is a NO sink owing to its rapid reactions with both oxygenated and deoxygenated hemoglobin, limiting the presence of NO in the blood. This catabolic reaction is exemplified in (11), where NO reacts with oxyhemoglobin to form methemoglobin and nitrate. However, in recent years, NO bioactivity has been proposed to be generated through interactions with hemoglobin either as a nitrite reductase [75] or
Carbon monoxide (CO) is an endogenously produced gas generated by enzymatic heme metabolism. It is colorless with low water solubility but chemically stable under physiological conditions. The reaction is catalyzed by HO in the reticuloendothelial system of the spleen and liver [84]. The biological effects of CO are dependent on its ability to bind to heme proteins, which are related to the concentration of CO, the concentration of O2, and the availability of reduced transition metals.
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Fig. 8 Nitrite- and hemoglobin-mediated N2O3 export from the red blood cell. Nitrite reacts with deoxyhemoglobin to make methemoglobin and nitric oxide. Most of the nitric oxide binds to the heme moiety of deoxyhemoglobin or forms nitrate and methemoglobin from oxyhemoglobin. Methemoglobin binds nitrite to form an
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adduct with Fe2+–NO2 character (hemoglobin–NO2.). This adduct can rapidly react with NO to form N2O3. N2O3 can diffuse out of the red cells, homolyze to NO and NO2., allowing for NO export. (Reprinted with permission from Macmillan Publishers Ltd [79]. Copyright 2007)
4.1 Carbon Monoxide Biosynthesis There are three specific isoenzymes of HO: HO-1, HO-2, and HO-3. HO-1 is the inducible isoform, whereas HO-2 and HO-3 are constitutive isoforms. The distribution of these enzymes is tissue-specific and linked to specific biological actions. Mechanistically, HO forms a complex with NADPH-dependent flavoprotein reductase (cytochrome P450 reductase) and biliverdin reductase on the endoplasmic reticulum. HO breaks the a-methylene carbon bond of the porphyrin ring using NADPH and molecular O2, releasing equimolar amounts of biliverdin, iron, and CO (Fig. 10). NADPH and oxygen are required cofactors in this reaction. The end products, biliverdin and bilirubin, are powerful endogenous antioxidants, whereas CO is a signaling molecule.
4.2 Carbon Monoxide Chemistry
Fig. 9 Oxygen-regulated delivery of NO in the form of S-nitrosothiols. S-Nitrosothiol binding, formation and release is allostericallly regulated by red blood cell hemoglobin and oxygen saturation. At high pO2, hemoglobin is in the R-state configuration. In this configuration, the cysteine 93 residue of the b chain is protected in a hydrophobic pocket. With deoxygenation, the conformation of hemoglobin changes to the T state, exposing the cysteine 93 residue of the b chain. In the T state, a subpopulation of hemoglobin reacts with NO., NO2−, or low-mass S-nitrosothiols to produce a nitrosylated heme in the b chain. The change in configuration to the R state, brings the cysteine b93 residue close to the nitrosylated heme. This is followed by a nitrosylation reaction between the NO heme and the cysteine b93 residue. (Reprinted with permission [77])
Carbon monoxide itself is unreactive in biological systems. Transition metals are effective in promoting the reduction of CO [85]. CO binds iron only in its reduced state – Fe(II) – which is in contrast to what is seen with NO. Transition metals are effective in promoting the reduction of CO [85]. CO readily forms metal carbonyls, which are susceptible to attack of the CO oxygen atom by electrophiles. Once formed in the body, metal carbonyls are relatively stable until CO is displaced by molecular oxygen. However, most CO is bound to hemoglobin as carboxyhemoglobin, which is carried to the lungs for excretion.
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Fig. 10 Heme oxygenase mediated formation of carbon monoxide. (Reproduced with permission [84])
4.3 Biological Function of Carbon Monoxide The biochemical effects of CO depend on its ability to bind heme proteins or proteins that contain other transition metals at their active site, thus altering protein function. CO can activate guanylate cyclase, influencing vascular tone, gene regulation, and neurotransmission. CO binds tightly to hemoglobin ferrous hemes, interfering with the oxygencarrying capacity of hemoglobin. This results in a reduction of the oxygen content of arterial blood, hinders the release of oxygen, and ultimately lowers tissue pO2 [86]. CO can also bind to the ferrous heme a3 of cytochrome c oxidase, the terminal electron acceptor of the mitochondrial respiratory chain. This results in a slower rate of cellular respiration, an increase in complex III peroxide leak rates, and a limited turnover of adenosine diphosphate [86]. The increase in peroxide level may affect the stabilization of hypoxia-inducible factor 1, cell proliferation, and mitochondrial biogenesis [86]. Lastly, the ability of CO to bind to iron may limit iron availability for the participation in redox cycling. This attribute may be an important antioxidant mechanism that can limit superoxide-driven Fenton reaction within a cell. This has been proposed to be the mechanism for the protective effects of CO in cerebral malaria [86].
4.4 Carbon Monoxide/Nitric Oxide Interactions There is known interplay between CO and NO. The affinity of NO for Fe(II) is 1,500 times greater than that of CO [87]; however, the dissociation constant for CO is greater than that for NO or for O2 [88]. Both CO and NO bind iron in the heme moiety of heme proteins, share downstream signaling pathways, and have similar regulatory functions. For instance, low concentrations of CO stimulate NO release in blood platelets and vascular cells [89], which is proposed to be due to redistribution of NO in the cells.
5 Hydrogen Sulfide Another naturally occurring and biologically relevant gas is hydrogen sulfide (H2S). Known widely as an environmental pollutant with extreme toxicity at high levels, it has recently gained new notoriety as a naturally produced gas of significant physiological influence, with major effects on vascular tone. When applied exogenously in very small doses, it also has provided remarkable protective effects from the cell level to the whole-animal level.
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Fig. 11 Biosynthesis of hydrogen sulfide. Biosynthesis of hydrogen sulfide requires the activity of cystathionine a-lyase and/or cystathionine b-synthetase on a cysteine substrate
5.1 Hydrogen Sulfide Biosynthesis H2S formation is widespread in mammalian cells and occurs through the activity of two pyridoxal phosphate-dependent enzymes on cysteine and homocysteine substrates (Fig. 11): cystathionine a-lyase – abundant in peripheral tissue – and cystathionine b-synthetase – abundant in brain tissue and liver.
5.2 Hydrogen Sulfide Catabolism H2S is a labile and easily diffusible gas with a half-life of minutes. H2S is rapidly metabolized (Fig. 12) through sequestration by hemoglobin binding, or reacting chemically with oxyradicals, peroxynitrite, hypochlorous acids, and homocysteine, or rapid enzymatic oxidation to thiosulfate and sulfite, and is finally eliminated in the urine mainly as sulfate in free and conjugated forms. All details of its oxidation to thiosulfate are not known, and it takes place with more efficiency in some tissues than in others. Sulfide oxidase, glutathione, and heme-containing compounds have all been proposed as catalysts for this oxidative conversion. It can also undergo methylation to CH3SH, but the importance of this is debated.
5.3 Hydrogen Sulfide Biochemistry H2S, known for its rotten egg odor, is colorless, flammable, and has a structure similar to that of water. O H
H
However, sulfur is not nearly as electronegative as oxygen, so hydrogen sulfide is also not nearly as polar as water. Because of this, comparatively weak intermolecular forces exist for H2S as compared with water. H2S is soluble and a weak acid in water or plasma, with a pKa at 37°C of 6.76.
H 2S ↔ HS− + H + Both sodium hydrosulfide (NaHS) and H2S may be dissolved in physiological liquid solution of pH 7.4, forming approximately 18.5% H2S and 81.5% hydrosulfide anion (HS−).
NaHS ↔ HS− + Na + In more acidic conditions, the percentage of H2S is higher.
S H
Fig. 12 Catalysis of hydrogen sulfide. Sulfide is metabolized by hemedependent nonenzymatic and enzymatic reactions, then excreted mostly in the urine
H
NaHS + H + ↔ H 2S + Na +
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The potential for biological activity is also enhanced by its highly lipophilic properties, and ability to freely penetrate cell membranes. High-concentration H2S (millimolar range) exhibits toxicity by its ability to inhibit mitochondrial cytochrome c oxidase, carbonic anhydrase, and monamine oxidase. It is made endogenously, however, in several normal tissues, including plasma and brain, in the 50–100-mM range, where it has many important physiological regulatory properties. Circulating blood levels have been described in the 10–100-mM range [90]. These measurements have been classically spectrophotometric, likely measuring not H2S per se, but total sulfide concentration. The H2S physiological functions described below may occur through a variety of chemical activities of the gas. Sulfides can bind proteins in vitro, presumably as persulfides, then subsequently be liberated by the chemical reaction of dithiothreitol [91]. This binding may alter protein activity. H2S also reacts with many metals to produce the corresponding metal sulfides. It is known to react with (and be sequestered by) hemoglobin to form sulfhemoglobin [91], having the potential to modulate activities of heme-containing enzymes. H2S, a thiol with strong reducing properties, may also be important as an antioxidant molecule, similar to other small thiols such as cysteine and glutathione. It can interact and neutralize a number of species abundant in tissues, including superoxide radical (O2•−), hydrogen peroxide (H2O2), peroxynitrite (−ONOO), and /or hypochlorite (ClO−). These interactions may result in some of the cytoprotective effects of H2S at physiological concentrations.
2H 2S + 2O 2• − ↔ O 2 + 2OH − + HS − SH ↔ SO32 − + H 2 O 8H 2S + 8H 2 O 2 ↔ S8 + 16H 2 O H 2S + − ONOO ↔ NO 2 − + H 2S = O H2S may also counteract cellular injury caused by hypochlorous acid, an oxidant product of peroxide (H2O2 + Cl → HOCl + OH−).
5.4 Physiological Effects of Hydrogen Sulfide 5.4.1 Blood Pressure Control H2S was shown to affect vascular tone in both aortic rings and the whole animal in a biphasic dose-dependent manner. At higher doses, it activated ATP-sensitive K+ channels to vasodilate, but at low H2S concentrations, NO was scavenged, resulting in vasoconstriction [92]. This endothelial NO-dependent vasoconstrictor effect was also hypothesized by others to be due to eNOS inhibition [93]. However, a very recent study of mutant mice deficient in the enzyme that
generates H2S from l-cysteine displayed pronounced s ystemic hypertension and decreased endothelium-dependent vasorelaxation. The vasorelaxation thus attributed to H2S was felt to be consistent with that of other endotheliumderived relaxing factors, such as NO, and not to changes in reactive oxygen species or glutathione activity [94]. 5.4.2 Hypoxic Pulmonary Vasoconstriction and Pulmonary Hypertension Elevated serum levels of H2S achieved through daily intraperitoneal NaHS dosing in hypoxic rats attenuated the hypoxic pulmonary vasoconstrictor response and increased the HO-1 level in the pulmonary vascular smooth muscle cells [95]. However, the vascular effect of sulfide is dramatically dependent on the ambient oxygen concentration and pH [96, 97].
5.4.3 Suspended Animation H2S is also a potent reversible inhibitor of complex IV (cytochrome c oxidase) in oxidative phosphorylation, having the powerful potential to control cellular metabolism. Mice treated with 80 ppm H2S had a significant and dramatic reduction in metabolic rate along with induction of a suspended-animation-like state. After a 6-h treatment, VO2 was reduced by 90%, body temperature decreased to 2°C above ambient, and respiratory rate decreased from 120 to ten breaths per minute, with no apparent adverse effects [98]. Investigators have shown that the reduction in metabolic rate caused by H2S was independent of body temperature, and was more likely due to inhibition of the sinus node and therefore heart rate, with an associated increase in vascular resistance [92]. 5.4.4 Neutralization of Reactive Species Multiple studies have demonstrated that H2S neutralizes a variety of reactive species, including oxyradicals, peroxynitrite, hypochlorous acid, and homocysteine. Several reports also noted that micromolar levels of H2S can upregulate endogenous antioxidant systems and enhance known antioxidants, such as glutathione, N-acetylcysteine, and superoxide dismutase [91].
5.4.5 Anti-inflammatory Effects Multiple studies have also demonstrated a number of antiinflammatory effects, such as decreased immune cell adhesion and infiltration, decreased edema following injury,
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decreased coronary artery restenosis following balloon angioplasty, fibrosis pathway inhibition, and upregulation of a number of anti-inflammatory and proangiogenic genes, such as HO-1, vascular endothelial growth factor, insulinlike growth factor receptor, and transforming growth factorb receptor pathway genes.
5.4.6 Cytoprotective Effects H2S improved myocardial preconditioning and cell survival of injury due to ischemia/reperfusion, b-agonist therapy, and cardiopulmonary bypass. One report suggests that the beneficial effects of garlic on cardiovascular disease are due to its role in the biological production of H2S leading to vasorelaxation [99].
5.4.7 Hypoxia Tolerance A 20-min pretreatment of mice with the gas resulted in survival from near anoxia for up to 3.5 h, presumably due in part to the reduction in metabolic rate [100]. The suggested hypothesis was that prior exposure to H2S reduced oxygen demand, making it possible for mice to survive extreme hypoxia, with cytochrome c oxidase playing a major role as the place where supply and demand meet in aerobic cells. The investigators further speculated that H2S decreases temperature through decreased mitochondrial function to generate heat in brown fat, and H2S decreases respiratory rate by inhibition of mitochondrial function in the brainstem or lung chemoreceptors.
5.4.8 Hemorrhagic Shock Sulfides improved survival following lethal hemorrhage.
6 Summary Biological gases play important roles in the physiological process and pathological changes of the pulmonary vasculature. The influence of each of the gases is dependent on the relative abundance, location (subcellular distribution, cell type, organ type), and presence of target molecules. Cross talk between these gases regulates metabolism, signaling cascades, as well as gene expression. In health, the interplay between these gases may be important in finetuning the physiological responses to luminal and/or alveolar conditions. In disease, this interdependence may be disrupted.
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Chapter 18
Role of Oxygen-Derived Species in the Regulation of Pulmonary Vascular Tone Michael S. Wolin, Mansoor Ahmad, and Sachin A. Gupte
Abstract Early studies detected potentially important roles for reactive oxygen-derived species (ROS) in influencing pulmonary vascular tone in models of lung injury and in responses of the pulmonary vasculature to hypoxia. In addition, observations on the inhibitory effect of superoxide on endothelium-dependent relaxation and its association with producing relaxation through stimulating the soluble, or heme-containing, form of guanylate cyclase (sGC) in pulmonary arteries were major contributing factors to Lou Ignarro’s Nobel-prize-associated work in identifying the role of nitric oxide (NO) as an endothelium-derived relaxing factor. Studies on the roles of ROS and their interaction with NO (generating reactive NO-derived species, NOx) evolved into evidence that these species may have major roles in influencing vascular function under various physiologically adaptive and pathophysiological situations to which the pulmonary circulation is exposed. For example, ROS and their interaction with NO appear to be part of the progressive adaptation of the pulmonary circulation to changes in flow, pressure, and inflammation. Although this chapter emphasizes interactions of ROS (and NOx) with signaling systems that acutely control vascular force generation or tone, it is important to note that some of the signaling systems considered are also likely to be major factors in the adaptive remodeling of the pulmonary circulation. Keywords Oxygen radicals • Guanylate cyclase • Nitric oxide • Pulmonary vasoconstriction • Endotheliumderived factors
1 Introduction: Where Are Reactive Oxygen-Derived Species Important in the Regulation of Pulmonary Vascular Tone? Early studies detected potentially important roles for reactive oxygen-derived species (ROS) in influencing pulmonary vascular tone in models of lung injury [1, 2] and in responses of the pulmonary vasculature to hypoxia [3–8]. In addition, observations on the inhibitory effect of superoxide on endothelium-dependent relaxation and its association with producing relaxation through stimulating the soluble, or heme-containing, form of guanylate cyclase (sGC) in pulmonary arteries were major contributing factors to Lou Ignarro’s Nobel-prize-associated work in identifying the role of nitric oxide (NO) as an endothelium-derived relaxing factor [9]. Studies on the roles of ROS and their interaction with NO (generating reactive NO-derived species, NOx) evolved into evidence that these species may have major roles in influencing vascular function under various physiologically adaptive and pathophysiological situations to which the pulmonary circulation is exposed. For example, ROS and their interaction with NO appear to be part of the progressive adaptation of the pulmonary circulation to changes in flow [10], pressure [11], and inflammation [12]. Although this chapter emphasizes interactions of ROS (and NOx) with signaling systems that acutely control vascular force generation or tone, it is important to note that some of the signaling systems considered are also likely to be major factors in the adaptive remodeling of the pulmonary circulation.
2 Overview of the Role of ROS in Vascular Function M.S. Wolin () Department of Physiology, New York Medical College, Valhalla, NY 10595, USA e-mail:
[email protected] Various stimuli to which the pulmonary vasculature is exposed, such as those highlighted in Fig. 1, may directly alter the activity of oxidases which generate ROS. The species
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3 Regulation of the Sources of ROS Generation Fig. 1 Model showing how exposure of the pulmonary vasculature to various physiological and pathophysiological conditions modulates the production of reactive oxygen species by oxidases that interact with redox-regulated components of signaling mechanisms regulating vascular reactivity and remodeling Table 1 Processes activating oxidases Oxidases Activation mechanisms Nox1 & Nox2
Receptors and cellular stress promote p47phox binding by a PKC-mediated phosphorylation and cytosolic subunit aggregation Receptors and oxidants stimulate rac1 binding by a Src kinase, EGF receptor, PI3-kinase pathway Nox4 Basal activity may participate in oxygen sensing? Nox5 Direct calcium binding plus PKC phosphorylation Increased mitochondrial NADH and electron Mitochondria (electron density (complex I) transport chain) Increased coenzyme Q semiquinone (complex III) Opening mitochondrial K+ channels and mild depolarization Inhibition by severe depolarization/uncoupling May participate in oxygen sensing? Nitric oxide Signaling associated with activation of NO synthase production plus oxidation/loss of tetrahydrobiopterin cofactor Xanthine oxidase Activation of p38 MAP kinase, thiol oxidation, and proteolysis Cytochrome P450 Receptor signaling? PKC protein kinase C, PI3-kinase phosphatidylinositol 3-kinase, EGF epidermal growth factor, MAP mitogen-activated protein kinase
produced influence vascular force through redox signaling mechanisms that influence the release of endotheliumderived vasoactive mediators and/or directly control vasodilator or vasoconstrictor mechanisms within vascular smooth muscle. In some situations ROS can function as tissue hormone-like mediators that regulate vascular force. The activities of oxidases are controlled by very specific signaling mechanisms (Table 1). There is emerging evidence for a high level of organization of signaling mechanisms within vascular cells which appear to be associated with compartmentalization of signaling based on the activities of ROS metabolizing systems and the ability to alter redox systems linked to the metabolism of the species generated. The specific mechanisms regulating the activities of oxidases and their subcellular colocalization with redox-controlled signaling systems allow individual ROS to have either vasodilator or vasoconstrictor activity in the same regions of the circulation in a manner which is determined by the stimuli-promoting responses mediated through ROS.
The lung seems to contain most of the oxidases that have been associated with regulating vascular function (Table 1). Vascular smooth muscle in pulmonary arteries has been shown to contain NAD(P)H oxidases (Nox), including Nox2 and Nox4 [13], and mitochondria [14], which appear to be active generators of ROS under physiological conditions. Mice deficient in the gp91phox subunit of Nox2 show decreased levels of superoxide in pulmonary arterial smooth muscle [15]. Although this defect does not seem to alter the acute response to hypoxia [15], it appears to suppress the progression of changes associated with the development of pulmonary hypertension elicited by chronic hypoxia [16]. There is evidence that Nox are significant sources of ROS in pulmonary vascular endothelium, and that pathophysiological conditions cause ROS generation by endothelial nitric oxide synthase (NOS) [10]. Other systems which potentially generate ROS, including Nox1, xanthine oxidase, cyclooxygenase, and cytochrome P450, are also thought to be associated with the pulmonary vasculature, but less is known regarding their role as a source of vasoactive ROS. Table 1 contains a list of many of the subunit components and signalinglike mechanisms that potentially control ROS production by the vascular-associated oxidases, but only limited studies have been reported on the function of these signaling mechanisms in the pulmonary vasculature. There is substantial evidence that hypoxia can modulate ROS production by Nox [8] and mitochondria [6, 7] in a manner that modulates pulmonary arterial force. Pathophysiological conditions appear to increase ROS generation by Nox and NOS uncoupling in endothelium in a manner which influences the release of endothelium-derived vasoactive mediators [10]. For example, there is substantial evidence that changes in laminar and oscillatory flow through systemic arteries have major effects on vascular function through mechanisms associated with increased Nox activity in the endothelium [17, 18]. In addition, ROS production by NOS and mitochondria may also be a contributing factor in flow-elicited responses [10, 19]. Although there is evidence that some vasoactive stimuli, cytokines, and growth factors can increase the levels of mitochondrial ROS [19], little is known regarding the direct influence these alterations in mitochondrial function have on the control of vascular contracting and relaxing mechanisms. However, changes in expression of subunits influencing the activities of oxidases by flow, pressure, cytokines, and other contributors to vascular pathophysiological function appear to be important factors in the progression of pulmonary vascular diseases which are associated with alterations in vascular reactivity [20].
18 Role of Oxygen-Derived Species in the Regulation of Pulmonary Vascular Tone
3.1 NAD(P)H Oxidases Studies on the oxidases in vascular tissue have detected evidence for the presence of Nox1, Nox2, Nox4, and Nox5 [21, 22]. These oxidases differ in their subunit content and cellular and subcellular localizations, and Nox5 seems to be expressed primarily in humans. Nox1 was initially termed Mox1, and its expression was associated with cellular growth and proliferation. Nox2 was actually the first characterized enzyme in this group as the gp91phox oxidase present in neutrophils and related phagocytic cells. It appears that Nox1 and Nox2 are generally associated with cell membrane structures and they seem to have similar mechanisms for activation. Stimulation of phosphorylation of a cytosolic p47phox subunit by protein kinase C (PKC) seems to be one of the best known mechanisms of initiating activation of these oxidases. Other subunits, including p67phox and p40phox, may bind to the p47phox subunit and these normally cytosolic proteins then bind to the membrane-bound Nox1 or Nox2 subunits which are already associated with a p22phox subunit. Although a Nox01 subunit can replace the phosphorylated p47phox subunit and a NoxA1 subunit can substitute for the p67subunit, little is known about the roles of these alternative subunits in pulmonary vascular regulation. Endothelium appears to contain the p67phox and p40phox subunits; however, there is little evidence for their importance in pulmonary vascular smooth muscle. There is a second mechanism of activating Nox1 and Nox2 which appears to enhance and prolong the initial p47phox binding-associated activation through a subsequent binding of rac1 to the membrane-bound Nox subunits. One pathway controlling rac activation appears to originate from Nox-derived ROS initiating a c-Src/epidermal growth factor receptor kinase signaling mechanism mediated through stimulating phosphatidylinositol 3-kinase [23]. Nox4 appears localized to internal cellular structures, and this oxidase is known to consist of the membrane-bound Nox4 and p22phox subunits. Currently, changes in Nox4 activity seem to be closely associated with the levels of expression of its Nox4 and p22phox subunits, and the enzyme does not appear to be regulated by signaling mechanisms controlling Nox1 and Nox2. Peptide mediators associated with the progression of pulmonary hypertension such as urotensin II have been associated with increased expression of Nox4 and activation of vascular smooth muscle proliferation-associated signaling [24]. Nox5 is different from the other Nox in that it has a calcium binding site that may be regulated through a PKC mechanism, and it lacks a requirement for the p22phox subunit or other cytosolic subunits known to influence the activity of Nox. Although Nox5 appears to be expressed in human vascular tissue, little is known about if it has a role in regulating pulmonary vascular function. Our studies suggest that increasing passive force on pulmonary
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arteries by stretching them causes an acute activation of Nox2 associated with enhancing their reactivity to vasoconstrictors [25] and increasing the expression of Nox4 by organ-culturing pulmonary arteries with transforming growth factor b causes a hypoxia-reversible depression of force mediated by an increased level of hydrogen peroxide [26]. Thus, the activities of Nox2 and Nox4 may have important roles in regulating vasoconstrictor and vasodilator signaling mechanisms in pulmonary arterial smooth muscle.
3.2 Mitochondria Mitochondria are present in essentially all types of cells potentially involved in vascular regulation, and studies suggest that they have important roles in regulating force in pulmonary arterial smooth muscle through their influence on mechanisms associated with both energy metabolism and the generation of ROS [6, 7, 27]. Mitochondrial function is very sensitive to energy-consumption-associated processes, metabolic conditions, hypoxia, and some cellular signaling mechanisms such as changes in intracellular calcium levels. Studies on the properties of mitochondria suggest that complex I and complex III in the electron transport chain appear to have sites which can transfer electrons to oxygen, generating superoxide anion [6– 8]. It appears that high levels of electron density in complex I are associated with increased superoxide generation in the mitochondrial matrix, and rotenone appears to increase the generation of superoxide from this site. The conditions controlling the production of superoxide by complex III are less well understood. It is thought that the one-electron-reduced semiquinone form of coenzyme Q reacts with oxygen to generate superoxide. Superoxide appears to be generated in both the matrix and the intermembrane space when it is produced by the complex III site. Increased electron flow into complex III seems to promote superoxide production, and this has been suggested to occur in vascular tissue when it is exposed to mitochondrial K+ channel opening drugs such as diazoxide or under conditions of a partial depolarization of the mitochondrial membrane potential by H+-transporting agents known as protonophores or mitochondrial uncouplers [28]. Rotenone inhibits electron flow from complex I into complex III in a manner which attenuates superoxide generation at this site, and the interaction of antimycin with complex III increases superoxide generation at the complex III site [7, 8]. There is a possibility that pulmonary vascular mitochondria may have different properties because both rotenone and antimycin appear to inhibit ROS generation [14, 29]. Recent studies have suggested that metabolic conditions increasing the level of mitochondrial NADH may be a factor in increasing mitochondrial superoxide generation in vascular tissue, and exposure to
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conditions which strongly depolarize mitochondria seems to be associated with decreased superoxide production [30]. Owing to the actions of manganese superoxide dismutase (SOD) in the matrix of mitochondria and Cu,Zn-SOD in the region between the inner and outer membranes of mitochondria, hydrogen peroxide may be the main ROS released from this organelle. Hypoxia appears to be a major regulator of mitochondrial ROS production, and there is convincing evidence that hypoxia can both increase [7] and decrease [6] mitochondrial ROS production in pulmonary arterial smooth muscle. On the basis of recent work on systemic arteries [30], the effects of hypoxia under the conditions studied on mitochondrial NADH redox may be a major factor in determining if hypoxia increases or decreases mitochondrial ROS generation.
3.3 Nitric Oxide Synthase The NOS enzyme in endothelium produces NO from l-arginine when a variety of stimuli, such as the shear force of flow, stimulate signaling mechanisms activating electron flow from NADPH into the flavoprotein–heme region of this protein. Although endothelial cell calcium is a key factor is stimulating NOS activity, extensive studies (generally in systemic vascular tissue) have identified several other protein binding interactions, phosphorylation signaling systems, and other posttranslational modifications that have major roles in influencing the function of this NO-producing enzyme [31]. It seems that the availability of tetrahydrobiopterin is one of the most influential factors in maintaining NO biosynthesis once the activity of this enzyme is stimulated. When this cofactor is oxidized or not available, the NOS reaction undergoes what is described as “uncoupling,” and it can become a major source of superoxide production in the endothelium. Although many of the regulatory processes influencing NOS activity, including the availability of l-arginine, seem to influence NOS uncoupling, the significance of these mechanisms in functional vascular tissue is not well understood. A tetrahydrobiopterin-controlled NOS uncoupling seems to be a major factor in the loss of endothelium-derived relaxation by NO seen in multiple vascular diseases [32], and uncoupled NOS has been reported to be a source of endothelium-derived hydrogen peroxide, the levels of which control vascular contractile function. The uncoupling of NOS promotes the formation of reactive NO-derived species which may also contribute to changes in endothelial function. A more dominant source of quantities of NO that promote formation of reactive NO-derived species in amounts that directly influencing vascular function seems to be associated with increased expression of the inducible form of NOS by inflammatory mediators and other stimuli.
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3.4 Other Oxidases There are a variety of other oxidases which potentially influence the pulmonary vasculature, but there is only minimal documentation of their influence on vascular contractile function. Although cytochrome P450 has been demonstrated to be a potential source of endothelium-derived relaxing levels of hydrogen peroxide in systemic arteries [33], the role of this process in pulmonary vascular regulation is not well documented. Lung injury is associated with activation of xanthine oxidase and the gp91phox (Nox2) in phagocytic cells in models where these sources of ROS produce pulmonary edema potentially associated with vasoconstriction [34, 35].
4 Metabolic Redox Processes Controlling the Concentrations of Vasoactive Species and Interactions with Signaling Systems 4.1 Metabolic Control of ROS Levels The activities of oxidases, mitochondria, and NOS discussed in Sect. 3 are the most dominant factors influencing the levels of ROS in the subcellular environments of cells. In addition, the levels of enzymes metabolizing ROS have major roles in determining the local concentrations of individual vasoactive species such as superoxide, peroxide, and peroxynitrite, which have important roles in controlling signaling mechanisms and regulating vascular force and reactivity. (Fig. 2) The concentrations of Mn-SOD in the mitochondrial matrix and Cu,ZnSOD in other intracellular regions probably maintain superoxide concentrations in the picomolar range. The efficient
Fig. 2 Model showing how the competition between superoxide dismutase and NO for reaction with superoxide (O2.–) generated by oxidases regulated by signaling mechanisms described in Table 1 goes on to modulate signaling mechanisms controlling vascular force described in the text and Table 2 through subsequent chemical and enzymatic reactions of peroxynitrite (ONOO–) and hydrogen peroxide (H2O2). Some actions of ONOO and its derived nitrogen dioxide (.NO2) include the oxidation and/or nitrosation/nitration of thiols (RSH), including iron (Fe–S) and zinc (Zn–S) binding sites, unsaturated fatty acids resembling arachidonic acid, and heme. The efficient metabolism of peroxide by enzymes including catalase, glutathione peroxidases, peroxiredoxins, and heme peroxidases in the cellular regions exposed to peroxide are thought to cause localized redox effects which influence signaling mechanisms
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intracellular conversion of superoxide into hydrogen peroxide by the SOD enzymes then generates fluxes of peroxide which are continually being metabolized by the peroxide-metabolizing systems present in subcellular regions. Although glutathione (GSH) peroxidases are probably the major consumers of peroxide in most subcellular regions of vascular tissue, other peroxide-consuming enzymes, including catalase and peroxiredoxins, are also likely to be significant consumers of peroxide in the poorly documented regions where these proteins are found. The availability of reducing cofactors, including GSH and thioredoxin for GSH peroxidase and peroxiredoxins, both controls the abilities of these enzymes to consume peroxide and generates oxidized forms of GSH (GSSG) and thioredoxin that are potentially involved in regulating the signaling systems discussed in the next section. Metabolic sources of NADPH generation in subcellular compartments, such as glucose 6-phosphate dehydrogenase (G6PD) in the pentose phosphate pathway of glucose metabolism, seem to be key factors in maintaining the availability GSH and thioredoxin for the metabolism of peroxide. These metabolizing systems are thought to maintain intracellular peroxide concentrations in the low nanomolar range. As superoxide levels increase, superoxide begins to efficiently scavenge NO in the region where it is produced. Since NO reacts with superoxide at a rate which is three- to fourfold faster than the rate superoxide reacts with SOD, NO will become a significant consumer of superoxide when its concentrations approach the local levels of SOD. Under these conditions, the generation of peroxynitrite (ONOO–) becomes a significant factor. Vascular smooth muscle secretes an extracellular Cu,Zn-containing form of SOD, which seems to have a major role in protecting NO from being consumed by superoxide in the extracellular environment of systemic and pulmonary arteries.
4.2 Metabolic Interactions of ROS with Signaling Systems Direct chemical reactions of ROS with components of signaling systems and redox changes resulting from the metabolism by enzymes such as peroxidases seem to contribute vascular regulatory processes. Owing to the low levels of superoxide resulting from its consumption by SOD, there seems to be only minimal evidence for direct interactions of superoxide with components of signaling systems beyond its reaction with NO and conversion to peroxide. The scavenging of NO by superoxide attenuates the direct interactions of NO with sites such as the ferrous heme of sGC. When significant amounts of ONOO are generated in subcellular regions, it appears to become a major factor in biological regulation as a result of its chemical reactivity with thiols, unsaturated fatty acids, and other biological molecules. Although the chemical
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reactions of ONOO are complex, and may involve species such as nitrogen dioxide (.NO2) and bicarbonate radical, its formation is likely to be a major pathway through which superoxide is involved in biological regulation beyond its role in generating peroxide. The consequences of these reactions include thiol oxidations that disrupt iron and zinc binding sites, the formation of protein–protein and protein–GSH (S-glutathionation) containing disulfides, protein tyrosine nitration, heme oxidation, and fatty acid oxidation and/or nitration which potentially have important roles in vascular regulatory mechanisms and the progression of pathophysiological processes. Since the fastest rates of reactions of peroxide with biological molecules seem occur with peroxidases, including the key enzymes which metabolize peroxide, these processes should be the most dominant route for peroxide to influence signaling systems. There are several enzymes with which peroxide reacts that appear to have important roles in biological regulation. A component of the mechanism through which peroxide initiates prostaglandin production by cyclooxygenase is its metabolism by the heme peroxidase produced by reaction of this enzyme, an oxidation process needed for the production of prostaglandin G2 [36]. The peroxide prostaglandin G2 (and/or hydrogen peroxide) then sustains generation of prostaglandin H2 by cyclooxygenase based on the availability of arachidonic acid. Peroxide metabolism by catalase appears to be able to stimulate sGC [4, 37]. The metabolism of peroxide through GSH peroxidases generates GSSG, and the shift in the redox status of GSH to oxidation promotes reversible protein thiol oxidations through glutaredoxin and protein–disulfide isomerase reactions. The metabolism of peroxide by peroxiredoxins seems to be linked to the oxidation of thioredoxin, which also appears to function as a reversible regulator of protein thiol redox. Although these thiol modifying systems seem to be very logical pathways for the redox regulation of signaling, very little is currently known about how these systems function, and it is often assumed regulation occurs through the direct reactions of peroxide with thiols on proteins being regulated [38]. The direct rates of reaction of peroxide with thiols on proteins such as protein phosphatases appear to be very slow, and the oxidase generating peroxide would have to colocalize in the immediate vicinity of the thiol being oxidized for regulation to occur through chemical reactions. The redox status of NADPH in subcellular regions is likely to be a major factor in determining the activities of thiol redox-controlled systems because NADPH-dependent enzymes including GSH and thioredoxin reductases maintain the redox status of GSH and thioredoxin. Thus, the balance of interactive of systems controlling redox in subcellular regions is a major factor in determining the behavior of redox-controlled processes. The biological chemical reactions of ONOO and the species it generates such as nitrogen dioxide (.NO2) and bicarbonate radical under physiological conditions seem complex and are
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not well understood [39–41]. However, some of the products of reactions of these reactive NO-derived species (NOx) detected in biological systems appear to influence vascular function in a manner which provides evidence for potentially important regulatory roles. Although thiols seem to function as scavengers of NOx, there is evidence that these reactions result in nitrosation (RSNO) and nitration (RSNO2) of thiols, and oxidation reactions such as disulfide (RSSR) formation which may have roles in regulating signaling systems [39–41]. For example, RSNOx are potent vasodilators, and they may be a tissue source of NO that is released over time. In addition, the reactions of protein thiols with NOx seem to be important contributors to the regulation of systems controlled by thiol redox. It is well established that ONOO results in nitrotyrosine formation, and this has been hypothesized to also be a contributor to biological regulation. For example, prostaglandin I2 synthase appears to be readily inactivated by tyrosine nitration, and this can shift prostaglandin-mediated regulation from vasodilation to vasoconstriction as a result of the loss of prostaglandin I2 and increased availability of prostaglandin H2. Unsaturated fatty acids, such as arachidonic acid, also directly react with NOx to generate nitrated and oxidized fatty acids with potent effects on vascular reactivity [42].
Table 2 Redox mechanisms regulating vascular force Mechanism Signaling systems Autocoids and endotheliumderived vasoactive factors
NO-mediated relaxation Prostaglandins
5 Roles for ROS in Controlling Signaling Pathways Regulating Force in the Pulmonary Vasculature The various ways oxidases are regulated and the availability of multiple signaling mechanisms with which individual ROS can potentially interact in subcellular regions helps provide explanations for why these species can be involved in both vasoconstrictor and vasodilator mechanisms that potentially regulate pulmonary vascular function. This section considers some of the documented interactions of ROS and redox with signaling systems listed in Table 2 that are potentially involved in regulating pulmonary vascular function. It is also important to consider that the level of tone in the pulmonary vasculature may enhance the expression of constrictor mechanisms at low tone and vasodilator mechanisms when pressure is increased by vasoconstriction of the pulmonary circulation. For example, peroxide can stimulate vasoconstriction under basal conditions in the saline-perfused rabbit lung through thromboxane A2 generation [2], and vasodilation is observed when tone is elevated in the rabbit lung preparation with the thromboxane A2/prostaglandin H2 receptor agonist U46619.
Action
Superoxide scavenges NO preventing relaxation Peroxides stimulate vasodilator or vasoconstrictor prostaglandin production ONOO and other oxidants inactivate PGI2 synthase converting vasodilation by PGI2 to vasoconstriction by PGH2 Other endothelium-derived factors Decreased level of NO promotes vasoconstriction through endothelin release Systems regulated by NADPH oxidation promotes the NADPH oxidation is associated with increased SR Ca2+ uptake by the cytosolic NAD(P)H redox coordination of multiple systems SERCA pump, decreased intracellular Ca2+ release, and influx that promote relaxation through lowering intracellular Ca2+ level Relaxation through increased Stimulation of cGMP generation by sGC is activated by NO, peroxide metabolism by catalase, and other cGMP signaling sGC mechanisms sGC stimulation by NO is inhibited by superoxide, and oxidation of its heme and thiol sites Stimulation of PKG cGMP stimulates PKG Peroxides promote a thiol-oxidation-mediated subunit dimerization that directly activates PKG Ion channels K+ channels Oxidant mechanisms open K+ channels, promoting relaxation through hyperpolarization Increased levels of cytosolic Ca2+ Many redox-controlled interactive systems promote or inhibit the release promoting vasocontriction and/or reuptake of intracellular Ca2+ Protein kinases promoting ERK Peroxide and thiol oxidants stimulate vasoconstriction PKC Receptors activate PKC, stimulating oxidant signaling which promotes vasoconstriction Rho Stimulated by Nox2-derived peroxide PGI2 prostaglandin I2, PGH2 prostaglandin H2, SR sarcoplasmic reticulum, SERCA sarcoplasmic/endoplasmic reticulum Ca2+-ATPase, sGC soluble guanylate cyclase, cGMP cyclic GMP-dependent protein kinase, ERK extracellular-regulated kinse, PKC protein kinase C
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5.1 Autocoids and Endothelium-Derived Vasoactive Factors 5.1.1 Prostaglandins One of the most potent effects of exposing microvascular preparations to ROS-generating systems seems to be the responses mediated through peroxide stimulating the generation of vasoactive prostaglandins [1, 43]. As mentioned already, one of the earliest responses reported [1] in the pulmonary circulation of rabbits exposed to ROS was vasoconstriction that appeared to be mediated through increased thromboxane A2 generation. Although the cellular sources of ROS-stimulated prostaglandin generation which influence pulmonary vascular function are not well understood, it has been established that peroxide stimulates phospholipase A2 to release arachidonic acid in pulmonary vascular endothelium [44]. Peroxide stimulation of arachidonic metabolism by cyclooxygenase to generate prostaglandin H2 is a fundamental property of the cyclooxygenase enzyme [36]. In addition, ROS may shift the profile of prostaglandins produced toward vasoconstrictors, especially under conditions where an inhibition of vasodilator biosynthesis by enzymes such as prostaglandin I2 synthase is observed.
5.1.2 Nitric Oxide It is well established that endothelium-derived NO often functions to depress the expression of vasoconstriction in the pulmonary circulation. Attenuation of the influence of NO by its reaction with superoxide is often a major factor in many aspects of physiological and pathophysiological aspects of pulmonary vascular regulation, especially under conditions where the vasculature is exposed to high levels of flow, pressure, and inflammation-related processes. Since NO is transported as a membrane-permeable dissolved gas, it will be removed by a combination of processes including diffusion into the blood of the circulation or the airway region and by reaction with any intracellular and extracellular source of superoxide in the vessel wall such as endothelium, smooth muscle, fibroblast, and inflammatory-cell-associated-oxidases. Increased expression of the inducible-inflammatory form of NOS can also be a factor in depressing pulmonary vascular force through NO or in the generation of ONOO. The complex chemical reactions of ONOO and its interactions with signaling systems make it difficult to predict most aspects of its role in biological regulation. It is likely that ONOO may initially have signaling effects which directly regulate vascular force, but as the vasculature is exposed to ONOO enzymes become inactivated, and ONOO may have effects resembling
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other forms of oxidative stress. Thus, exposure of the pulmonary vasculature to increased levels of ONOO is likely to be a major factor in the progression of many adaptive and pathophysiological processes.
5.1.3 Other Endothelium- and InflammatoryCell-Derived Vasoactive Factors Endothelin is often released from the endothelium under conditions where NO levels are decreased by processes often associated with oxidative stress. Many stressful conditions, and the autocoids and cytokines they generate, have the potential to increase oxidant signaling in the vessel wall through stimulating oxidases, changing the expression of subunits which influence the activities of activities, or altering the expression of ROS-metabolizing enzymes. Most of the studies published in this area focused on examining the progression of pulmonary vasculature adaptation related to disease processes. The adaptation process potentially influences both ROS-dependent and redox-independent mechanisms regulating pulmonary vascular tone and reactivity.
5.2 Redox-Controlled Mechanisms Regulating Vascular Smooth Muscle Force Force generation by vascular smooth muscle is a combination of the activity of signaling mechanisms promoting contraction and relaxation, and the interactions between these mechanisms. The topics will be discussed on the basis of mechanisms that seem to be redox-regulated, without separating them into processes promoting vasodilation and constriction because removal of an existing vasoactive process will elicit the opposite response.
5.2.1 Systems Regulated by Cytosolic NADP(H) Redox Studies on the actions of inhibitors of G6PD have resulted in evidence that glucose metabolism by the pentose phosphate pathway maintains the levels of cytosolic NADPH, and that its oxidation or increasing the ratios of cytosolic NADP/NADPH appears to coordinate multiple processes that lower intracellular calcium levels associated with vascular relaxation [45, 46]. These processes appear to include stimulation of calcium uptake into the sarcoplasmic reticulum by the sarcoplasmic/endoplasmic reticulum Ca2+ATPase (SERCA) pump, and inhibition of intracellular
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calcium release and calcium influx. Owing to the existence of multiple ion transport systems and channels sites that could be regulated by redox and the interactive nature of the systems controlling intracellular calcium levels around the sarcoplasmic reticulum and plasma membrane, the actual sites regulated by NADPH redox remain to be defined. Although K+ channels appear to have only a minor role in the NADPH-oxidation-associated relaxation of coronary arteries by inhibitors of G6PD [45], the Kv channel has a prominent role in the relaxation of rat pulmonary arteries exposed to these inhibitors [47]. Thus, the metabolic control of cytosolic NADPH can be a factor controlling force, and it has been suggested that increased levels of NADPH in pulmonary arteries from processes such as decreased peroxide generation and metabolism under hypoxia can be a factor in regulating pulmonary arterial tone [3, 48]. In addition, it appears that pulmonary arteries maintain higher levels of cytosolic NADPH than coronary arteries as a result of increased expression of G6PD [13]. The increased levels of cytosolic NADPH in pulmonary arteries have been proposed to maintain the production of vasodilator levels of peroxide by Nox. Thus, hypoxia can cause vasoconstriction by depressing the consumption of NADPH needed for both the generation and the metabolism of peroxide. Since NADPH redox potentially has a major influence on other redox systems, such as the processes controlling thiol redox in subcellular regions discussed in Sect. 4, it is likely to be a factor in most of the redox mechanisms regulating vascular smooth muscle force that are considered next.
5.2.2 Cyclic GMP Signaling The cytosolic form of guanylate cyclase, or sGC, is a major site of redox regulation: it promotes vascular relaxation through generating cyclic GMP (cGMP) and stimulating cGMP-dependent protein kinase (PKG) activity. Many processes including multiple systems regulating the level of intracellular calcium and its influence on force generation by vascular smooth muscle seem to be directly controlled by PKG [49]. Since NO directly stimulates sGC by binding its Fe2+ heme, it is a major target for the influence of superoxide and other factors that control the bioavailability of NO in the region of vascular smooth muscle. The heme of sGC seems to be readily oxidized by ROS and ONOO, and this also seems to be a factor in the expression of NO-mediated vasodilation [50]. Thus, the oxidant stress associated with most vascular pathophysiology is a major factor in attenuating NO-mediated vasodilation through its effects on sGC–cGMP signaling. There are other redox factors that potentially contribute to the regulation of sGC–cGMP signaling. Peroxide can stimulate
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relaxation of pulmonary arteries through cGMP signaling [37]. One process may involve the direct stimulation of sGC activity resulting from peroxide metabolism by catalase, and this mechanism is sensitive to inhibition by superoxide [37]. Another process may involve peroxide promoting a disulfideelicited dimerization of the PKG protein subunits which directly stimulated PKG-mediated vasodilation [51]. Cytosolic NADPH is potentially a factor in influencing all of the mechanisms discussed in this section as a result of its role in controlling NO biosynthesis, Nox activity, peroxide metabolism, and protein thiol and heme redox. Peroxide stimulation of sGC appears to be suppressed by NO [52], and it is likely that NADPH redox influences the thiol-oxidation-elicited stimulation of PKG. Thus, the redox regulation of sGC and that of PKG are potentially major factors in the coordination of multiple processes promoting vascular relaxation that are controlled by cGMP-associated signaling. Although there is general agreement on the role of superoxide attenuating NO-mediated relaxation in pulmonary vascular pathophysio logical processes, the roles of most other aspects of redox regulation of cGMP signaling in regulation of the pulmonary circulation remain to be better defined.
5.2.3 Ion Channels Interest in the mechanism of hypoxic pulmonary vasoconstriction has resulted in studies and hypotheses on the redox regulation of potassium channels and processes controlling intracellular calcium levels in pulmonary arterial smooth muscle. Although this topic has been debated in many recent reviews [6–8], the sites directly regulated by redox remain to be more precisely defined. This is because most potassium channels and processes controlling intracellular calcium levels have sites such as cysteine thiols which can be regulated by redox. In addition, many of the processes regulating calcium and potassium channels influence each other through redoxindependent interactions. Redox-regulated systems such as cGMP may also control the function of many of the ion channels suggested to be redox-regulated. Some of best documented aspects of redox-regulated ion channels in pulmonary and systemic vascular smooth muscle include potassium channels and the SERCA pump. Weir and Archer have reported the findings of studies that show that plasma membrane K+ channels are opened by oxidant-related conditions that are associated with hyperpolarization [3, 14, 53]. Hyperpolarization promotes vascular relaxation through processes which include the closing of voltage-regulated calcium channels. One of best documented redox-regulated ion transport systems is the SERCA pump. Thiol oxidation of sites on the SERCA pump that are thought to occur under physiological conditions seem to stimulate calcium uptake into the sarcoplasmic reticulum [54]. Filling of the sarcoplasmic
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reticulum with calcium is thought to inhibit the influx of calcium through the plasma membrane through processes associated with the capacitive regulation of calcium. This interactive system may be part of mechanisms which then coordinate the lowering of intracellular calcium level and relaxation. It could also be hypothesized that the regulation of K+ channels and hyperpolarization may be part of this mechanism through effects such as sarcoplasmic reticulum filled with calcium closing calcium-regulated potassium channels and TRP channels associated with calcium influx. Hyperpolarization would then also close voltage-regulated calcium channels. In addition, the SERCA pump is also stimulated by PKG, allowing cGMP signaling to regulate this interactive system by lowering the cytosolic calcium level in a manner similar to oxidants.
5.2.4 Protein Kinases Promoting Vasoconstriction There are several protein kinases which appear to be redoxrelated and generally function to enhance the actions of calcium in promoting contraction through mechanisms such as the inhibition of myosin phosphatase. Some of the best documented systems include the Src kinase, PKC, extracellularregulated kinase (ERK), and Rho kinase systems [25, 55–58]. Although the precise redox-controlled process regulating each of these kinase systems is not well understood, it is likely that a step involving inhibition of protein phosphatases by oxidants is often involved. Since the PKC and Src kinase systems participate in the activation of Nox [23], they may also contribute to feed-forward mechanisms where oxidants increase the production of ROS. The ERK system appears to be activated in pulmonary arteries by stretch through mechanisms that appear to involve increasing the generation of Nox-derived peroxide, Src kinase activation, and a thiol-oxidation-mediated process [25]. Although its activation causes enhancement of the force generation by contractile stimuli, ERK activation does not appear to participate in the bovine pulmonary artery contractile response elicited by an acute exposure to hypoxia that was studied [25]. Oxidants have been shown to activate Rho kinase in vascular tissue [56, 58]. Rho kinase activation seems to be an important contributing factor to changes in pulmonary artery reactivity that occur when pulmonary arteries are exposed to prolonged periods of hypoxia [55]. Sorting out what is occurring with the PKC and Rho kinase systems is complicated because they may also be activated and regulate force by redox-independent mechanisms associated with signaling activated by contractile agents. In addition, cGMP–PKG signaling may inhibit systems such as Rho kinase [49]. Thus, poorly understood aspects of the redox regulation of several different protein kinases may be important factors in the control of pulmonary vascular function.
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6 Coordination, Integration, and Changes in Pathophysiological Aspects of ROS Signaling Smooth muscle of the pulmonary vasculature is probably normally exposed to low levels of NO which is derived from the endothelium and other sources in the lung in amounts that partially stimulate relaxation through cGMP. In addition, if the concept that pulmonary arterial smooth muscle is exposed to vasodilator levels of peroxide derived from sources within such as Nox and mitochondria under basal normoxic conditions is correct, it is also probably exposed to sources of peroxide generation that activate vasodilation through processes which might involve mechanisms such as the stimulation of sGC and thiol redox changes influencing ion channels and perhaps PKG. Since most of the redox-regulated vasoconstrictor mechanisms in the pulmonary circulation seem to involve enhancing the sensitivity of the contractile apparatus to calcium when the vasculature is exposed to vasoconstrictors, these mechanisms may have only minor roles in influencing the pulmonary circulation under the minimal levels of basal tone that appear to exist under normoxic physiological conditions. Pathophysiological conditions influencing the pulmonary circulation generally increase the generation of ROS and activate adaptive responses that are poorly understood. Although stimuli such as changes in the shear of flow, pressure, and inflammation, and exposure to hypoxia, vasoactive mediators, cytokines, and other regulatory factors have been reported to influence the generation of ROS and their interaction with NO, there is generally a lack of a consensus on which oxidases are involved and how they influence redox signaling. Some observations suggest ROS and redox-signaling processes are part of the progressive adaptation of the pulmonary circulation to pathophysiological conditions [11, 16, 59]. In addition to structural changes in the vasculature, there are a diversity of reports of changes in the expression of components of signaling systems associated with ROS and redox regulation. Studies examining vascular reactivity under these conditions have in some cases detected a loss of endothelium-derived relaxation associated with superoxide scavenging NO, changes in the function of relaxing and contractile mechanisms, and the profile of prostaglandins and other eicosanoids which either have been demonstrated or could be hypothesized to be linked to the direct influence of ROS signaling on these processes [1, 11, 12, 55, 59]. For example, an increased role for Rho kinase enhancing vasoconstriction which could originate from changes in many different redoxregulated processes seems to be an important factor in the expression of pulmonary hypertension [56]. Although these processes could be influenced by acute therapies that target ROS, a more important factor may be reversing the adaptive
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effects of stimuli promoting dysfunction. It is interesting to note that the pulmonary circulation generally shows little evidence of contributing to the oxidant-associated pathological process of circulating factors in systemic vascular diseases such as hypertension and diabetes. Although the reasons for this are not understood, some factors such as angiotensin II increasing the expression of endothelial NOS through activation of pulmonary endothelial angiotensin type 2 receptors [60] could be a factor. The many facets of ROS redox-regulated processes originating from the subcellular localization of multiple distinct signaling pathways allow these systems to have many different roles in the sequential progression and expression of pulmonary vascular disease processes which remain to be defined.
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18 Role of Oxygen-Derived Species in the Regulation of Pulmonary Vascular Tone 37. Burke TM, Wolin MS (1987) Hydrogen peroxide elicits pulmonary arterial relaxation and guanylate cyclase activation. Am J Physiol 252:H721–H732 38. Forman HJ, Fukuto JM, Torres M (2004) Redox signaling: thiol chemistry defines which reactive oxygen and nitrogen species can act as second messengers. Am J Physiol Cell Physiol 287:C246–C256 39. Wolin MS (2000) Interactions of oxidants with vascular signaling systems. Arterioscler Thromb Vasc Biol 20:1430–1442 40. Pacher P, Beckman JS, Liaudet L (2007) Nitric oxide and peroxynitrite in health and disease. Physiol Rev 87:315–424 41. Pryor WA, Houk KN, Foote CS et al (2006) Free radical biology and medicine: it’s a gas, man! Am J Physiol Regul Integr Comp Physiol 291:R491–R511 42. Balazy M, Iesaki T, Park JL et al (2001) Vicinal nitrohydroxyeicosatrienoic acids: vasodilator lipids formed by reaction of nitrogen dioxide with arachidonic acid. J Pharmacol Exp Ther 299:611–619 43. Wolin MS, Rodenburg JM, Messina EJ et al (1987) Oxygen metabolites and vasodilator mechanisms in rat cremasteric arterioles. Am J Physiol 252:H1159–H1163 44. Chakraborti S, Gurtner GH, Michael JR (1989) Oxidant-mediated activation of phospholipase A2 in pulmonary endothelium. Am J Physiol 257:L430–L437 45. Gupte SA, Arshad M, Viola S et al (2003) Pentose phosphate pathway coordinates multiple redox-controlled relaxing mechanisms in bovine coronary arteries. Am J Physiol Heart Circ Physiol 285:H2316–H2326 46. Gupte SA, Wolin MS (2006) Hypoxia promotes relaxation of bovine coronary arteries through lowering cytosolic NADPH. Am J Physiol Heart Circ Physiol 290:H2228–H2238 47. Gupte SA, Li KX, Okada T et al (2002) Inhibitors of pentose phosphate pathway cause vasodilation: involvement of voltage-gated potassium channels. J Pharmacol Exp Ther 301:299–305 48. Wolin MS, Ahmad M, Gao Q et al (2007) Cytosolic NAD(P)H regulation of redox signaling and vascular oxygen sensing. Antioxid Redox Signal 9:671–678 49. Lincoln TM, Dey N, Sellak H (2001) Invited review: cGMPdependent protein kinase signaling mechanisms in smooth muscle: from the regulation of tone to gene expression. J Appl Physiol 91:1421–1430
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50. Stasch JP, Schmidt PM, Nedvetsky PI et al (2006) Targeting the heme-oxidized nitric oxide receptor for selective vasodilatation of diseased blood vessels. J Clin Invest 116:2552–2561 51. Burgoyne JR, Madhani M, Cuello F et al (2007) Cysteine redox sensor in PKGIa enables oxidant-induced activation. Science 317:1393–1397 52. Mohazzab HK, Fayngersh RP, Wolin MS (1996) Nitric oxide inhibits pulmonary artery catalase and H2O2-associated relaxation. Am J Physiol 271:H1900–H1906 53. Olschewski A, Hong Z, Peterson DA et al (2004) Opposite effects of redox status on membrane potential, cytosolic calcium, and tone in pulmonary arteries and ductus arteriosus. Am J Physiol Lung Cell Mol Physiol 286:L15–L22 54. Tong X, Ying J, Pimentel DR et al (2008) High glucose oxidizes SERCA cysteine-674 and prevents inhibition by nitric oxide of smooth muscle cell migration. J Mol Cell Cardiol 44:361–369 55. Jernigan NL, Walker BR, Resta TC (2008) Reactive oxygen species mediate RhoA/Rho kinase-induced Ca2+ sensitization in pulmonary vascular smooth muscle following chronic hypoxia. Am J Physiol Lung Cell Mol Physiol 295:L515–L529 56. Gupte SA, Kaminski PM, George S, et al (2009) Peroxide generation by p47phox-Src activation of Nox2 has a key role in protein kinase C-induced arterial smooth muscle contraction. Am J Physiol Heart Circ Physiol 296:H1048–H1057 57. Thakali K, Davenport L, Fink GD et al (2007) Cyclooxygenase, p38 mitogen-activated protein kinase (MAPK), extracellular signal-regulated kinase MAPK, Rho kinase, and Src mediate hydrogen peroxide-induced contraction of rat thoracic aorta and vena cava. J Pharmacol Exp Ther 320:236–243 58. Jin L, Ying Z, Webb RC (2004) Activation of Rho/Rho kinase signaling pathway by reactive oxygen species in rat aorta. Am J Physiol Heart Circ Physiol 287:H1495–H1500 59. Jankov RP, Kantores C, Pan J et al (2008) Contribution of xanthine oxidase-derived superoxide to chronic hypoxic pulmonary hypertension in neonatal rats. Am J Physiol Lung Cell Mol Physiol 294:L233–L245 60. Olson SC, Dowds TA, Pino PA et al (1997) ANG II stimulates endothelial nitric oxide synthase expression in bovine pulmonary artery endothelium. Am J Physiol 273:L315–L321
Chapter 19
Mitochondrial Reactive Oxygen Species and Redox State in Pulmonary Vascular O2 Sensing Stephen L. Archer and John J. Ryan
Abstract Mammals possess a homeostatic O2-sensing system that comprises the resistance pulmonary arteries, ductus arteriosus, carotid body, neuroepithelial body, systemic arteries, fetal adrenomedullary cell, and fetoplacental arteries. Together these specialized tissues form a homeostatic system that increases the organism’s ability to survive hypoxia, whether encountered during development, at altitude, or during disease. Thus, the homeostatic O2-sensing system optimizes O2 uptake and delivery. One important part of the homeostatic O2-sensing system is hypoxic pulmonary vasoconstriction (HPV), a vasomotor response of resistance pulmonary arteries to alveolar hypoxia, that optimizes ventilation/perfusion matching and optimizes systemic O2 tension. The core mechanisms of hypoxic pulmonary vasoconstriction resides in the smooth muscle cell, although it is modulated by the endothelium. This chapter explores and updates the redox theory for the mechanism of hypoxic pulmonary vasoconstriction. Keywords Oxygen sensing • Hypoxic pulmonary vasoconstriction • Redox signaling • Reactive Oxygen Species (ROS)
1 Introduction There is now considerable evidence that HPV results from the coordinated action of a redox sensor (possibly the proximal mitochondrial electron transport chain, ETC) which generates a diffusible mediator (a reactive oxygen species, ROS) that regulates effector proteins [voltage-gated K+ (Kv) channels]. The controversy whether HPV results from hypoxic increases in the production of superoxide anion or from hypoxic withdrawal of ROS was discussed in a recent published debate [2, 3]. Although the identity of the sensor (mitochondria vs. NADPH oxidase, NOX) and its function (to increase the level of superoxide or decrease the level of S.L. Archer (*) Department of Medicine, University of Chicago, Chicago, IL 60637, USA e-mail:
[email protected] H2O2) remains controversial, there is better (although imperfect) consensus regarding the downstream effector pathway (K+ channel inhibition, Ca2+ channel activation). It appears that hypoxia ultimately elicits vasoconstriction by inhibiting Kv channels (e.g., Kv1.5 and Kv2.1) in the PASMC which depolarizes the PASMC, thereby activating voltage-gated Ca2+ channels, increasing Ca2+ influx and causing vasoconstriction. HPV is sustained and enhanced by capacitative calcium entry, activation of Rho kinase, which mediates calcium sensitization, and also by a variety of paracrine factors, such as endothelin. A role for cyclic ADP ribose as a redox-regulated mediator of intracellular calcium release has been proposed. Here we discuss the redox theory for HPV. The redox theory for HPV is increasingly recognized to be relevant to a variety of diseases. Disorders of the O2 sensing may contribute to the proliferative, apoptosis-resistant phenotype of PASMCs in pulmonary arterial hypertension and cancer. Prior to discussing identification of a redox sensor for HPV, it is critical to define some of the characteristics of HPV (which must be satisfied by any putative sensor). HPV is a homeostatic vasomotor response of small, muscular “resistance” PAs to alveolar hypoxia. HPV actively diverts perfusion to optimally ventilated lung segments. The central mechanism of HPV involves a redox-based O2 sensor (e.g., mitochondria or NOX), which alters the production of a diffusible redox mediator (e.g., superoxide, H2O2, or redox couples such as reduced/oxidized glutathione of NADH) in direct or inverse proportion to changes in inspired O2 concentration. Our group has reported that hypoxia creates a reduced environment characterized by an increased ratio of reduced glutathione to oxidized glutathione and decreased levels of superoxide and H2O2; other groups, who otherwise subscribe to the redox theory of O2 sensing, find diametrically opposed redox signaling (hypoxia increasing the level of superoxide). In either case, the resulting alteration of the redox environment must inhibit certain Kv channels in PA SMCs (PASMCs). Upstream of this pathway, the magnitude of HPV is impacted by the endothelium (e.g., enhanced by endothelin, inhibited by nitric oxide); downstream it is modulated
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Fig. 1 Initial (1986) redox model for hypoxic pulmonary vasoconstriction (HPV). The concept is that redox state, determined by metabolism (in the mitochondria and elsewhere), regulates sulfhydryl balance in the membranes. This controls pulmonary artery smooth muscle cell K+ flux and membrane potential and thereby regulates the L-type Ca2+ channel and thus the cytosolic Ca2+ level. This pathway was also proposed as a means of regulating insulin secretion in pancreatic b cells. (Reproduced with permission [1])
by Rho kinase through sensitization of the contractile apparatus to calcium. This chapter, extracted from two recent reviews [4, 5], presents a critical appraisal of the redox theory for the mechanism of HPV (Figs. 1, 2).
2 Homeostatic Oxygen-Sensing System Before discussing the mechanism of HPV, it is useful to review the homeostatic oxygen-sensing system, to which HPV is a contributor. Mammalian cells depend on appropriate amounts of oxygen for survival. There are a group of specialized tissues that contain these specialized cells which detect and govern responses to changes in oxygen tension all to optimize systemic oxygen delivery. These specialized cells are situated within the carotid body, ductus arteriosus, PAs, systemic arteries, and neuroepithelial cells [6] in the lungs. Fetoplacental arteries [7] and the fetal adrenomedullary cells [8] also contain specialized tissue that senses local oxygen tension [9].
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Fig. 2 Updated redox theory of HPV. In this model the mitochondrial electron transport chain senses hypoxia and decreases reactive oxygen species (ROS) production. A decrease in ROS (superoxide anion and/or H2O2) production inhibits the effector Kv1.5 and Kv2.1 channels. Kv channels depolarize the membrane and activate Ca2+ entry via L-type Ca2+ channels. Other theories, which are not mutually exclusive, include NADPH oxidase as a sensor and source of ROS, cyclic adenosine diphosphate ribose as a redox-activated mediator that causes intracellular Ca2+ release by activating ryanodine receptors [80] in the sarcoplasmic reticulum, and changes in the function of adenosine monophosphate kinase. In addition, activation of Rho kinase may enhance HPV by sensitizing the contractile apparatus to Ca2+
2.1 Carotid Body This organ, derived from neural crest cells, is located at the bifurcation of the carotid arteries. The carotid body plays a key role in oxygen sensing and maintaining oxygen homeostasis by stimulating the brainstem respiratory center at times of hypoxia. It is through this mechanism that a decline in arterial oxygen level results in a compensatory increase in ventilation (and our sense of “huffing and puffing” at altitude). The carotid body has a considerable blood supply from branches of the external carotid body. The carotid body contains glomus cells which determine the levels of hypoxia in the capillaries which flow through the carotid body. In turn, the afferent sensory fibers from the carotid body join the glossopharyngeal nerve, which signals the hypoxia to the respiratory center of the brain. Efferent activity is likely mediated through autonomic neurons. Hypoxia activates neurosecretion by inhibiting potassium channels in the glomus cells [10], a mechanism that is (more or less) conserved in all specialized O2-sensitive tissues.
19 Mitochondrial Reactive Oxygen Species and Redox State in Pulmonary Vascular O2 Sensing
2.2 Ductus Arteriosus In utero, the ductus arteriosus allows blood from the PAs to bypass the lungs, travel through the aorta, and undergo oxygenation in the placenta. At birth, when the neonate breathes in air and the lungs expand, there is an increase in arterial oxygen tension. As a response, the PAs dilate and there is a large increase in blood flow into the lungs. Concurrently, the ductus arteriosus responds to this increase in oxygen level in the exact opposite manner by constricting and closing. When the ductus arteriosus contracts, the infant experiences the switch from fetal circulation to neonatal circulation. Blood exiting the right ventricle into the PA now is forced to enter the lung to undergo oxygenation. Closure of the ductus arteriosus happens within hours after birth (by active vasoconstriction) and the lumen is subsequently permanently closed by a process of remodeling (mediated by SMC migration and fibrosis). In the ductus SMCs, inhibition of K+ channels also initiates acute constriction in response to the rise in PO2 that occurs with the first breath [11, 12].
3 The Pulmonary Circulation and HPV The adult pulmonary circulation is a unique, low-resistance circuit designed for gas exchange. It is perfused by a thinwalled, afterload-intolerant right ventricle. The pulmonary circulation accommodates the entire cardiac output at one fifth the pressure and resistance of the systemic vasculature. The large arteries, which serve as conduits, manifest only weak HPV, and may dilate in response to hypoxia, whereas the small intrapulmonary arteries, which control pulmonary vascular resistance (PVR), are the site of maximal HPV [13, 14]. In most forms of pulmonary hypertension (PHT), the burden of disease is also localized to these small arteries. This anatomical (proximal–distal) and functional (conduit– resistance) distinction extends to the cellular level. SMCs from these PA segments have different cellular electrophysiological characteristics, with the strongest being the oxygen-sensing mechanism evident in small muscular PAs that are less than 100 mM in diameter [15]. Indeed, proximal and distal PAs have different embryological origins. Conduit PAs are derived from the sixth aortic arch, whereas the distal PAs are derived from expansion of the capillary plexus, originating by vasculogenesis from the mesenchymal lung bud [16, 17]. HPV is defined as the increase in PVR that occurs when the alveolar PO2 falls below a threshold level. The first observation of HPV was made by Bradford and Dean [18] in 1894. Von Euler and Liljestrand recognized the potential homeostatic role of HPV in optimizing systemic PO2, noting that it increases the blood flow to better aerated lung areas, which
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leads to improved conditions for the utilization of alveolar air [19]. In atelectasis, when the hypoxia is segmental, HPV is restricted to the vascular segments serving the hypoxic lobe, thereby achieving ventilation/perfusion matching and optimizing systemic PO2, without elevating PA pressure (PAP) [20, 21]. During global hypoxia, as occurs at altitude or in hypoventilation syndromes (such as sleep apnea), HPV increases PAP, causing PHT. HPV is found in virtually all mammals, although its incidence is diminished in races or species that are adapted to life at altitude (Tibetan natives [22], yaks). Likewise chronic hypoxia (10% O2 for more than 3 days) impairs HPV in rats, despite enhanced constriction to other stimuli [23].
3.1 Properties of HPV in Humans The nature of HPV in humans helps us define physiologic hypoxia and delineates the fundamental characteristics of HPV. Results from reductionist models should be judged against these studies because some of the basic science of HPV has been conducted under conditions that may not be relevant to HPV (i.e., using anoxic PO2s, cell lines, conduit arteries, etc.). At the summit of Mount Everest (27,559 ft, barometric pressure 272 mmHg), the mean PaO2 in ambient air was a remarkable 24.6 mmHg [24]. This serves as a practical definition of “physiologically tolerable” hypoxia. To go higher (in altitude) or lower (in PO2) is not sustainable even for those acclimated to hypoxia; yet much of the molecular science of HPV is conducted at PO2s below 30 mmHg with no measure of pH or PCO2. Focusing on literature that uses “physiologically tolerable” hypoxia helps clarify the HPV literature. The onset of HPV is rapid (seconds) [25] and the elevation in PVR is sustained [26]. In humans, the maximum elevation of PVR occurs within 15 min and the magnitude of HPV is not potentiated by repeated hypoxic challenges [27]. Moderate hypoxia (12.5% O2, PO2 ~ 50 mmHg) doubles PVR. In healthy volunteers, 8 h of hypoxia increases PVR from 1 to 3 Wood units within 2 h; thereafter the PVR remained at a stable level of elevation. Systemic vascular resistance decreases in parallel, falling from 19 to 14 Wood units and PVR reverts to normoxic levels upon return to breathing 20% O2 [28]. The intrinsic nature of these opposing responses to hypoxia in pulmonary versus systemic circulations is recapitulated in the isolated, serially perfused, lung–kidney model [29]. This reminds us that any proposed sensor–effector combination should have opposing effects in cells from the pulmonary and systemic circulations. In humans, HPV is significantly impaired by hyperventilation and the resulting respiratory alkalosis; however, HPV is not reduced by inhibition of synthesis of eicosanoids or endothelin receptor antagonism (reviewed in [4]), consistent with endothelium independence of the core mechanism of
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HPV. Support for the proposed role of K+ and Ca2+ channels in HPV can also be found in patients with chronic obstructive pulmonary disease (COPD). HPV is attenuated by 53% in COPD patients by nifedipine, an L-type, voltage-gated Ca2+ channel blocker, and this occurs at doses that do not lower systemic vascular resistance [30].
3.2 HPV in Animal Models In contrast to the clarity of the in vivo and isolated lung research, there is disagreement about the characteristics of HPV in PA rings. Although we find that HPV causes a rapid constriction in resistance PA rings that gradually plateaus and is sustained [31], much as occurs in vivo, several groups have found that hypoxia elicits a biphasic response consisting of an immediate, endothelium-independent constriction which peaks in approximately 10 min (phase I) and a subsequent slowly developing endothelium-dependent contraction peaking at approximately 30–40 min (phase II) [32]. The basis for the discrepancy is uncertain, but likely relates to the duration of the hypoxic challenge (our work focuses on the first 10 min of the response, other groups focus on longer hypoxic exposure), details of tissue selection (resistance versus conduit arteries), and details of tissue handling/ viability (which are often obscure in the methods sections).
4 General Features of HPV Certain features of HPV must be accounted for by any proposed mechanism: 1. HPV begins rapidly, is intrinsic to PAs, and is strongest in resistance PAs. HPV occurs in isolated lungs [33], small PAs [34], and PASMCs [13, 35, 36]. Although most PA segments constrict to hypoxia [37], HPV is strongest in small PAs (greater than the third division, less than 200 mm) [14, 15, 37, 38]. Proximal PAs dilate in response to hypoxia [15, 39]. The restricted occurrence of hypoxic constriction of intrapulmonary PAs (and to a lesser extent veins) reflects the localized abundance of HPV’s molecular apparatus in these segments [40]. 2. HPV is triggered by airway hypoxia. Resistance PAs are surrounded by terminal airways and preferentially respond to moderate alveolar hypoxia, rather than hypoxemia. 3. HPV involves the coordinated inhibition of Kv channels and activation of L-type Ca2+ (CaL) channels. HPV is initiated, at least in part, by the inhibition of a family of O2sensitive Kv channels leading to membrane depolarization, opening of CaL channels, and vasoconstriction [41–43]. Hypoxia dilates systemic arteries, and hypoxic inhibition of whole-cell K+ current (IK) occurs in PASMCs, but not
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in systemic arterial SMCs [41, 43]. The activity of the CaL channel is largely regulated by membrane potential (Em), which is established by K+ channels [20]. However, the CaL channel is also directly O2-sensitive, being activated by hypoxia (independent of Em) [44]. Accordingly, Ca2+ channel blockers (nifedipine) inhibit HPV [33], whereas Kv channel inhibitors (4-aminopyridine, 4-AP [15, 45]), and CaL agonists (BAYK8644) enhance HPV [46, 47]. 4. HPV is reversible. PVR returns to the baseline level rapidly (within 1 min) with discontinuation of hypoxic ventilation. Indeed, perfusion defects that relate to atelectasis from proximal airway obstruction cause chronic HPV that reverses with relief of air obstruction even after years [48]. This reversibility is consistent with the redox theory but is hard to explain if one postulates a major role for Rho kinase activation in HPV.
5 Mechanisms of O2 Sensing in HPV The redox mechanism for HPV was proposed initially in 1986 [1] (Fig. 1), and has been refined subsequently (Fig. 2) (reviewed in [4]). This theory proposes that a drop in alveolar O2 concentration decreases production of a redox mediator virtually instantaneously (less than 30 s). The mediator, in turn, alters the function of one or more effector proteins, which ultimately increases cytosolic Ca2+ concentration, thereby activating the PASMC’s contractile machinery. There are two potential cytochrome-based O2-sensitive sources of ROS which are candidate O2 sensors, the mitochondrial ETC and a family of classical and novel nicotinamide adenine dinucleotide phosphate (NADPH) oxidases (NOX). The final common pathway of all forms of pulmonary vasoconstriction involves activation of the contractile apparatus. Thus, actin and myosin, and their regulatory systems, such as Rho kinase, are very important determinants of the magnitude of HPV. However, since they appear not to be unique to the pulmonary circulation and act downstream from the “O2-sensing unit,” they are not discussed in this chapter. The role of Rho kinase activation in HPV becomes increasingly important as the duration of hypoxic exposure is sustained. Advocates of the theory that hypoxia increases superoxide production note that superoxide anion causes pulmonary vasoconstriction by activating Rho kinase [49, 50].
5.1 Mitochondria as O2 Sensors The mitochondrion’s role as a primary site for O2 consumption during aerobic metabolism makes it an ideal candidate for early detection of changes in PO2. The evidence suggesting that the mitochondrial ETC could be important in O2 sensing is based in part on the concordant effects of certain ETC inhibitors
19 Mitochondrial Reactive Oxygen Species and Redox State in Pulmonary Vascular O2 Sensing
and hypoxia. Inhibitors of the mitochondrial ETC and oxidative phosphorylation cause pulmonary vasoconstriction in isolated blood-perfused lungs [51]. Moreover, inhibitors of complex I (rotenone) and complex III (antimycin A), but not inhibitors of complex IV (cyanide), mimic hypoxia’s hemodynamic effects, causing pulmonary vasoconstriction, dilatation of the human ductus arteriosus [11], and systemic circulation [29]. Rotenone and antimycin not only inhibit subsequent acute HPV but their constrictor potency, like that of hypoxia, is selectively suppressed in chronic hypoxic PHT (CH-PHT), a condition in which constriction to most stimuli (e.g., angiotensin II) is enhanced [52]. The parallels between hypoxia and ETC inhibitors are also evident in nonvascular tissue. ETC inhibitors mimic hypoxia’s effects on the carotid body [53]. The initial phase of the pulmonary vasoconstriction induced by both metabolic inhibitors and hypoxia appears to occur without ATP depletion. ATP is preserved in HPV induced by moderate hypoxia; conversely, anoxia, which does deplete ATP, results in only transient pulmonary vasoconstriction followed by pulmonary vasodilatation [54]. We find that rotenone and antimycin reduce lung and PA ROS production in a manner analogous to that of physiological hypoxia [29, 42, 55]. However, the effects of hypoxia and mitochondrial inhibitors in the pulmonary circulation are controversial and other groups that have similarly concluded that mitochondria are sensors in HPV disagree on the signaling mediator [56, 57]. Notably, Schumacker and Chandel [56, 58] and Ward [50] found that hypoxia increases superoxide production and implicated this as a means by which cytosolic Ca2+ concentration increases in PASMCs, leading to HPV. On a cellular level, the proximal ETC inhibitors also mimic hypoxia. Hypoxia, inhibits K+ current and depolarizes PASMCs, but does not have this effect in systemic arterial SMCs [41, 43]. Like hypoxia, rotenone and antimycin A (and some other ETC inhibitors) inhibit IK in PASMCs [42]. As would be predicted from the redox theory, the ETC inhibitors, which dilate the ductus arteriosus, increase IK in human ductus arteriosus SMCs [11]. In terms of the ROS up versus down controversy, the K+ current in PASMCs is inhibited and the membrane potential is depolarized by reducing agents, not by ROS or oxidants [59]. The PASMC’s O2-sensing mechanism appears to relate to the small proportion of total electron flux involving unpaired electrons and the associated production of intramitochondrial ROS. Teleologically, this uncoupled electron flow provides an early warning of potential risk to the upstream supply of electrons and their originating reducing equivalents which could limit ATP production. Thus, there is some poorly understood but obligatory link between signaling ROS and redox couples. Moreover, the role that compartmentalized redox signaling (cytosolic vs. mitochondrial) plays is unclear. However, new redox probes permit dissection of the various subcellular compartments [60, 61].
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The ETC comprises four megacomplexes (each with many subunits) that mediate transfer of electrons down a redox potential gradient through a series of carriers, resulting in final acceptance of the electron by O2, producing ATP, with water as an end product. In the course of electron transport, H+ ions are translocated, creating a proton gradient that generates the mitochondrion’s extremely negative membrane potential (∆ym). This potential energy is used to produce ATP. Complexes I and III produce most of the mitochondrial ROS [62], although there is tissue heterogeneity in which complex predominates. Complex I, which is inhibited by the isoflavinoid rotenone, is an NADH ubiquinone oxidoreductase where NADH is oxidized to NAD+ as it transfers two electrons to ubiquinone. Complex III is a ubiquinol cytochrome c oxidoreductase that transfers electrons from FADH2 to cytochrome b, and subsequently to the final electron acceptor, cytochrome c. Complex IV, composed of cytochromes a and a3, is a cytochrome oxidase that is inhibited by cyanide. Complex IV accepts electrons from reduced cytochrome c and passes them to O2. Inhibition of a complex proximal to the site of ROS production should decrease ROS production (as we observe with rotenone); conversely, distal inhibition would disrupt electron flow, diverting electrons to react with O2 and generate ROS, a phenomenon we have observed with cyanide [42]. This suggests the site of critical ROS generation is proximal to or at complex III. To detoxify the mitochondrial ROS, the mitochondria uniquely express an inducible superoxide dismutase (SOD) isoform (manganese superoxide dismutase, SOD2). SOD2 transforms toxic superoxide radicals to H2O2. We hypothesize that H2O2 is a diffusible redox mediator connecting the sensor (mitochondria) to the effector (K+ channel), linking redox state and vascular tone [29]. In this redox theory the low normoxic pulmonary vascular tone is due to tonic generation of ROS which maintains the O2-sensitive Kv channels in the PASMC oxidized and open (thereby relaxing the PASMC) (Fig. 2). Conversely, HPV results from the withdrawal of these ROS and Kv channel closure. Differences in mitochondrial ETC function and ROS generation appear to account for the observed heterogeneity in SOD2 expression in PA versus renal artery mitochondria, the levels of ROS and SOD2 being far higher in PASMCs [29]. In general, SOD2 levels are inversely proportional to mitochondrial ROS production. Whereas the redox theory views SOD2 as a H2O2 generator, others view it as an antioxidant-eliminating superoxide (it is in reality both).
5.2 NOX or NOX Isoforms as O2 Sensors Another potential O2-sensor is NAD(P)H oxidase or one of the NOX isoforms. Lung levels of NADPH increase with hypoxia [63]. This flavocytochrome contains two membrane-bound
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subunits (p22phox and gp91phox) and two cytosolic proteins (p47phox and p67phox). NOX (or a variant that preferentially uses NADH as a substrate) produces ROS in proportion to the PO2 and has been suggested as an O2 sensor [64]. Production of ROS by NOX isoforms has been demonstrated in cells derived from several O2-sensitive tissues, including the neuroepithelial body, PASMCs, and carotid body. Much of the evidence favoring NOX as an O2 sensor derives from experiments using diphenylene iodonium (DPI). DPI inhibits NOX and, in several respects, mimics hypoxia. After causing slight vasoconstriction, DPI decreases subsequent pressor responses to hypoxia (reviewed in [4]). Moreover, it reduces normoxic ROS production in the PA and inhibits PASMC IK. Unfortunately, DPI nonspecifically inhibits flavoproteincontaining enzymes, including nitric oxide synthase and complex I of the mitochondrial ETC (reviewed in [4]). Mice deficient in the 91phox-containing NOX (Nox-2) manifest dramatically lower normoxic lung ROS production than backcrossed control mice [55]. However, HPV and the O2-sensitive portion of PASMC IK are preserved [55]. Moreover, rotenone constriction is preserved or enhanced, consistent with the mitochondrial O2-sensor hypothesis [55]. Preserved O2 sensing has also been reported in the type 1 cell of the carotid body from these mice [65]. Overall, these findings argue against the classical NOX system as an O2 sensor in HPV, although they do not exclude a role for other novel oxidases. Moreover, since the NOX is simply another cytochrome-based electron shuttle, it may combine with a mitochondrial signal to regulate K+ channels and vascular tone.
6 Diversity in Redox Signaling Because PAs constrict and systemic arteries relax in response to hypoxia, it appears probable that the redox signals generated in response to hypoxia differ between these vascular beds. ROS levels are higher in normal PAs than in systemic arteries, such as the renal artery, and only the PAs have a ROS signal which is hypoxia-inhibited [29]. The relatively depolarized mitochondria in PASMCs (relative to renal artery SMCs) may account for the higher ROS levels and the hypoxia-sensitive changes in ROS production in PASMCs. PAs also differ from coronary arteries (both endotheliumdenuded arteries) in that the activity of glucose 6-phosphate dehydrogenase, the rate-limiting enzyme generating cytosolic NADPH, is 1.5-fold higher in bovine PAs than in coronary arteries. This may explain why bovine PAs had 4.2-fold higher levels of NADPH and greater superoxide levels, despite similar NOX2 and NOX4 expression [66]. Elevated levels of cytosolic NADPH in PASMCs may drive NOX activity [66]. Thus, differences in mitochondrial function and the pentose phosphate pathway may account for the
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unique occurrence of a hypoxia-induced redox shift in the pulmonary circulation.
7 Effector Mechanisms of HPV: K+ Channels K+ channels are proteins consisting of four transmembranebound a-subunits and four regulatory b-subunits. The ionic pore, which determines the channel’s intrinsic conductance and ionic specificity, is created by the formation of tetramers of a-subunits. The Kv channels also have, in their S4 region, a voltage sensor. b-subunits associate with many K+ channels and alter their expression and kinetics. There are several types of K+ channel a-subunits, including Kv, inward rectifier (Kir), and two-pore (TASK) channels. Inhibitors of Kv channels in general (e.g., 4-AP) [41, 45, 67] and inhibitors of Kv1.x channels (e.g., correolide) cause pulmonary vasoconstriction [40, 45]. Additionally, preconstriction of resistance PAs with the Kv blocker 4-AP eliminates subsequent HPV [40]. Hypoxia inhibits IK and depolarizes EM in canine PASMCs, but not in renal, arterial SMCs [41, 43] and is now widely accepted. In the last decade, significant advances have been made in the molecular identification of the relevant O2-sensitive K+ channels. How do K+ channels control pulmonary vascular tone? In PASMCs, as in most vascular SMCs, closure of Kv channels decreases the tonic efflux of K+ that occurs down the intracellular/extracellular concentration gradient (145 mM/5 mM). Channel closure renders the cell interior relatively more positive (depolarized) and when Em exceeds approximately −30 mV, the open probability of L-type voltage-gated Ca2+ channels increases, and intracellular Ca2+ concentrations rise. K+ channel inhibition increases both cytosolic Ca2+ and K+ concentrations, which not only promotes contraction, but also induces a phenotype that favors proliferation and suppression of apoptosis. Thus, regulation of K+ channel activity, and the subsequent regulation of Ca2+ concentration, may be important for maintaining both low PVR and the pulmonary circulation’s thin-walled vascular morphology. Eleven Kv channel families have been identified. A variety of putative O2-sensitive channels exist in the PASMC (Kv1.2, Kv1.5, Kv2.1, Kv3.1b, and Kv9.3) (reviewed in [4]). Certain K+ channels are specially suited to O2 sensing, by virtue of possessing key cysteine and methionine groups. Reduction or oxidation of sulfhydryl residues in these channels by a redox mediator, such as ROS, can cause conformational changes in the channel, thereby altering pore function and channel gating. Only certain Kv channels appear to be effectors of HPV (e.g., Kv1.5 and Kv2.1) [4]. In PASMCs, oxidants increase IK (e.g., H2O2, diamide, and oxidized glutathione); in contrast, reducing agents (e.g., reduced glutathione) and agents that facilitate electron shuttling (e.g., duroquinone) inhibit
19 Mitochondrial Reactive Oxygen Species and Redox State in Pulmonary Vascular O2 Sensing
IK (reviewed in [4]). Moreover, oxidants (e.g., diamide) dilate the pulmonary circulation (mimicking O2), whereas reducing agents mimic hypoxia. Hypoxia and redox agents may alter the function of K+ channels directly, through modulation of levels of electron donors (NADH) or by modulating ROS production. The role of Kv1.5 and Kv2.1 in HPV can best be seen in two models in which HPV is selectively suppressed, the CH-PHT model and the Kv1.5 knockout mouse. Suppression of acute HPV and the O2-sensitive portion of IK in CH-PHT results, in part, from loss of Kv1.5 expression [52, 68]. Kv1.5 adenoviral gene transfer restores Kv expression, O2-sensitive IK, and HPV in rats with CH-PHT [69]. In mice with targeted Kv1.5 deletion, HPV is also impaired and the size of the O2sensitive IK in PASMCs is reduced [31]. Similar effects on vascular tone of correolide, a selective Kv1.x channel inhibitor [40], and hypoxia suggest that a Kv1.x channel contributes to the mechanism of HPV [40]. In patch–clamp studies, intracellular dialysis of PASMCs with anti-Kv1.5 antibodies (immunoelectropharmacology) depolarizes resistance, but not conduit, PASMCs. Indeed, the combination of anti-Kv1.5 and anti-Kv2.1 causes a large depolarization, and hypoxia has no additional effects [40, 70]. Kv1.5 channels are preferentially expressed in resistance PASMCs (within the vascular segment with the greatest HPV) [40]. Heterologously expressed human Kv1.5, cloned from normal PA, generates an outward Kv current that is active near the resting Em of –65 mV in Chinese hamster ovary cells and which is inhibited by hypoxia [40]. Thus, the consequence of enriched Kv1.5 expression in resistance PAs is relative hyperpolarization of resistance versus conduit PASMCs (about -60mV vs. about −35 mV) and a unique O2 sensitivity that underlies the localized enhancement of HPV in resistance PAs.
8 Controversies Regarding the Role of ROS in HPV Even among groups that agree that there is a redox basis for HPV and that the mitochondria are the source of signaling ROS, there remains controversy as to the nature of the effects of hypoxia on ROS generation (decreased versus increased).
8.1 Hypoxia Decreases ROS Generation During alveolar hypoxia, we and others have found that ROS production falls in proportion to the amount of inspired O2 [42, 71, 72]. Higher normoxic levels of ROS cause physiological oxidation of K+ channels in PASMCs. In support of this
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view, we measured PA ROS production using three different detection methods (lucigenin-enhanced chemiluminescence, Amplex Red H2O2 assay, and 2¢,7¢-dichlorodihydrofluorescein diacetate, DCF). In endothelium-denuded, resistance PA rings, a concordant decrease in ROS levels during hypoxia or exposure to ETC inhibitors was detected by all three methods [29]. Likewise, Paky et al. found that hypoxia reduces lung ROS production, measured using lucigenin [73]. They noted that the SOD-mimetic tiron or antimycin A decreased lung ROS production. Conversely, ROS production in rat lungs or isolated lung mitochondria increases in direct proportion to PO2. Wolin’s group also suggested that it is the withdrawal of normoxic ROS (H2O2) that elicits HPV, although they attributed the source of the ROS to NADH oxidase [74]. In summary, we find that regardless of the technique of ROS measurement, both in isolated lungs and in isolated PA rings, hypoxia consistently and rapidly decreases ROS production prior to the onset of HPV (reviewed in [4]). This fall in ROS production is not an artifact of vasoconstriction as the vasoconstrictors angiotensin II and KCl have no effect [42, 55].
8.2 Hypoxia Increases ROS Generation In contrast, Marshall et al. found an increase in ROS production during hypoxia in isolated PA myocytes, although they attributed this to NOX [75]. In experiments using both isolated perfused lungs and cultured PASMCs, the Schumacker group showed that hypoxia increased intracellular DCF fluorescence in cultured PASMCs and this was blocked by the complex III inhibitor myxothiazol [56]. Many of their measurements of HPV were conducted in isolated PASMCs (passage number unspecified but more than 6 days in culture) using a surrogate for PVR (SMC shortening) [56]. Since cultured cells rapidly lose ion channels and also have diminished O2 sensitivity, at least in the ductus arteriosus [11], we believe that conclusions about O2 sensing should be derived from experiments in freshly isolated tissue (when possible). Nonetheless, using sophisticated techniques (including redox-sensitive green fluorescent proteins that permit compartment-specific ROS measurement), these investigators continue to find that hypoxia increases ROS [58]. Schumacker recently noted increased ROS production with hypoxia in the cytosol of PASMCs, although he did note a simultaneous reduction in ROS production in the mitochondria (personal communication). The relative importance of the various redox pools to oxygen sensing remains uncertain. Although agreeing that PASMC mitochondria function as O2− sensors, the Schumacker group concluded that increased ROS production triggers HPV. Supporting their conclusions, they observed that a variety of antioxidants (pyrrolidinedithiocarbamate, ebselen, and diethyldithiocarbamate)
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abolished HPV [56]. They measured ROS in cultured PASMCs using DCF. DCF is a suboptimal ROS probe, as discussed in a recent review on controversies in HPV [4]. However, it is unlikely that the use of cultured cells and the use of DCF are the only explanations for the divergent findings among groups because Liu et al. recently reported that hypoxia increases ROS production (measured by DCF fluorescence), and “tended” to increase ROS production (using lucigenin-enhanced chemiluminescence), and variably increased ROS production (measured by electron paramagnetic resonance spectra) [76]. Like the Schumacker group, they found that SOD and catalase reduced HPV in porcine PAs. The latter finding contradicts our previous report that liposomal delivery of SOD plus catalase enhances HPV [77]. Knock et al. concluded that hypoxia increases superoxide generation, although this is inferred from the effects of LY83583, a putative generator of superoxide anion on vascular tone and calcium in small PAs [50]. In that work, ROS were not directly measured and LY83583 did not itself cause relaxation of systemic arterial rings (i.e., did not mimic this aspect of hypoxia). Moreover, 6-anilino-5,8-quinolinequinone (LY83583) is not a specific generator of ROS; it is also an inhibitor of guanylate cyclase [78, 79].
8.3 Points of Agreement and Likely Causes for Disagreements For a review of the controversies of ROS signaling in HPV, the interested reader should consult other sources [2, 3]. Both camps have concluded that ROS produced by the proximal ETC signals the hemodynamic response to hypoxia and ETC inhibitors. Moreover, the isolated lung data from the Schumacker group agree with our observations (i.e., rotenone increases normoxic pressure and inhibits subsequent HPV, whereas cyanide does not) [56, 57]. The Schumacker group found that the proximal ETC inhibitors rotenone (50 ng/mL) and myxothiazole (50 ng/mL) inhibit HPV, whereas antimycin A (1 ng/mL) does not. However they acknowledge that antimycin A at 10 ng/mL does cause pulmonary vasoconstriction and inhibits HPV [56], consistent with our findings [42]. They discount the effects of antimycin A because it also inhibits constriction to U46619 [56]. Interestingly, myxothiazole does not cause pulmonary vasoconstriction, which they interpret as indicating that it targets the relevant ROS generator mediating HPV. Thus, their conclusions about the site of ROS generation are based on the assumption that the relevant “sensor” complex(es) when blocked will selectively inhibit HPV but will not mimic hypoxia by increasing PAP. In contrast, we assume that blocking the sensor should elicit both vasoconstriction and inhibition of subsequent HPV. Since
S.L. Archer and J.J. Ryan
these metabolic inhibitors eventually lower ATP levels, perhaps over the 30 min for which the Schumacker group observed contraction in their cultured cell model, the confounding effects of ATP depletion may have occurred. The resulting KATP channel activation could promote relaxation and might explain the observed reduction in U46619 contraction. In our studies, where ROS and PVR were measured simultaneously, the changes in PVR and ROS occurred within seconds/minutes of administration of hypoxia or ETC inhibitors and ROS levels fell before constriction commenced (as expected if the ROS are indeed a signal). It is likely that the discord in the literature, generated by high-quality investigative teams, relates to divergence in the techniques employed to measure ROS, differences in the timing of the measurement, variations in incubation conditions of the ROS assay (pH, PO2, and PCO2), and differences in the precise tissue preparations studied. We advocate using multiple independent measurement techniques (luminol, lucigenin, L-012, Amplex Red) to measure ROS in freshly isolated, resistance PA rings [29] and isolated perfused lungs [4]. In conclusion, the data indicating HPV is sensed by mitochondria, the sensor is in the proximal ETC, the mediator is a ROS, and the mechanism resides in the PASMC are more concordant than discordant. The redox model (Fig. 2) offers a comprehensive explanation for HPV and is relevant to studies of ROS in other organs. Although there are undoubtedly deficiencies in the detail of the model and areas of controversy remain, this redox hypothesis is testable and we encourage its widespread evaluation with a greater focus on the physiologic attributes of HPV, as observed in vivo.
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Chapter 20
Cellular and Molecular Mechanisms of Pulmonary Vascular Smooth Muscle Cell Proliferation Tamara Tajsic and Nicholas W. Morrell
Abstract Pulmonary arterial hypertension is the end result of a complex series of events termed “vascular remodeling,” which includes proliferation of resident smooth muscle cells in the walls of small muscular pulmonary arteries. This process leads to increased thickness of the smooth muscle component of the vessel wall, which then contributes to reduced lumen diameter and increased contractility. Since smooth muscle cell proliferation contributes significantly to the process of vascular remodeling, it is important to appreciate the main factors driving this process in the hypertensive lung. This chapter will review the basic mechanisms of vascular smooth muscle cell proliferation, focusing on work in pulmonary vascular cells. Keywords Cell proliferation and growth • DNA synthesis • Growth factor • Signal transduction • Pulmonary vascular remodeling
1 Introduction Pulmonary arterial hypertension (PAH) is the end result of a complex series of events termed “vascular remodeling,” which includes proliferation of resident smooth muscle cells (SMCs) in the walls of small muscular pulmonary arteries. This process leads to increased thickness of the smooth muscle component of the vessel wall, which then contributes to reduced lumen diameter and increased contractility. Since SMC proliferation contributes significantly to the process of vascular remodeling, it is important to appreciate the main factors driving this process in the hypertensive lung. This chapter will review the basic mechanisms of vascular SMC (VSMC) proliferation, focusing on work in pulmonary vascular cells.
T. Tajsic (*) Department of Medicine, Addenbrooke’s Hospital, Hills Road, Box 157, Cambridge, CB4 1LL, UK e-mail:
[email protected] 2 The Cell Cycle Cell proliferation involves an ordered sequence of events in which a cell duplicates its content and then divides into two daughter cells each containing chromosomes identical to those of the parental cell. This cycle of duplication and division is known as the cell cycle. Control of the cell cycle is critical for normal development of multicellular organisms and regulation of cell number. In eukaryotes, the cell cycle is composed of four discrete, temporally regulated phases: G1, S, G2, and M phase. The M phase consists of the division of the nucleus, called mitosis, and the division of the cytoplasm, referred to as cytokinesis. The period between two consecutive M phases is referred to as interphase and includes the other three phases: the S phase, during which the cell replicates its DNA content, and two gap periods, G1, between the end of the M phase and beginning of the S phase, and G2, between the end of the S phase and the beginning of the next M phase. During these two gap phases the cell grows, monitors intracellular conditions, and integrates signals from the environment (other cells) to assess if there is a need for the cell to proliferate and if so whether the cell is adequately prepared to commit itself to the demands of DNA replication and cell division. The M phase takes up a relatively small portion of the cell-cycle time as it takes approximately 1 h for most human cells to divide, whereas the interphase can last from several hours up to a whole lifetime in different cell types. The cell cycle is a highly complex, ordered system of biochemical switches. The transition through the cell cycle is regulated by a series of heterodimeric protein kinases that are conserved from yeasts to mammals. The regulatory subunits, cyclins, control cell-cycle events, and their concentration changes as cells progress through the cell cycle. Cyclindependent kinases (Cdks), the catalytic subunits of the protein kinases, have no kinase activity unless they are associated with a cyclin. Each Cdk associates with specific cyclins and the associated cyclin determines which proteins are phosphorylated by a particular cyclin–Cdk complex. Different cyclin– Cdk complexes trigger different steps of the cell cycle. The concentration of each cyclin rises gradually and then falls
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sharply at a specific time in the cell cycle as a result of ubiquitination and proteosome degradation. The proliferation of human cells is controlled by a complex network of signaling pathways that integrate extracellular signals about environmental cues, the identity, and density of neighboring cells. If the growth factors are present through G1, the increase in concentration helps activate the appropriate Cdk, whereas the rapid fall in cyclin concentration returns the Cdk to its inactive state. The proliferation of human cells is principally regulated by a variety of extracellular growth factors. If the growth factors are present through G1, representing the need of the organism for the specific cell type/cell to proliferate, this will stimulate G1 phase cyclin synthesis and transition through the G1 restriction point. Principally, G1 cyclins, cyclins D1,D2, and D3, bind to Cdk4 and Cdk6 and drive progression through the G1 phase. Then, the concentration of the G1/S cyclin, cyclin E, increases and forms a complex with, and thereby activates, Cdk2. This leads to activation of the proteins that regulate initiation and completion of DNA replication (Cdc6, etc.) and their subsequent binding to the origin-recognition complexes: specific nucleotide sequences scattered at various locations along each chromosome. The prereplication complex is thus formed and the cell is now ready to replicate its DNA. The rise in S phase cyclin, cyclin A, concentration, and its binding to Cdk2 triggers the replication process. Additionally, retinoblastoma protein, normally serving as a molecular brake, binding to gene regulatory proteins and preventing them from driving transcription of genes necessary for cell proliferation, is phosphorylated by G1/S and S cyclin–Cdk complexes, altering its conformation and releasing the bound proteins which are now free to activate the genes necessary for proliferation. The cyclin A–Cdk2 complex also prevents re-replication, thus preventing the risk of gene amplification. This complex leads to phosphorylation of the prereplication complex regulatory
Fig. 1 The cell cycle
T. Tajsic and N.W. Morrell
p roteins, which then dissociate from the origin-recognition complexes and are subsequently degraded, ensuring that the replication is not reinitiated in the same cell cycle. Once DNA has been faithfully and accurately replicated, the cell can engage in mitosis. In this step, cyclin B (the M phase cyclin) levels rise, leading to the formation of a complex with Cdk1, which then phosphorylates proteins involved in chromosome condensation, nuclear envelope breakdown, mitotic spindle formation, and mitosis (division of the nucleus) occurs, commonly followed by cytokinesis (division of the cytoplasm). At the end of mitosis, all Cdk activity is reduced to the baseline level, and the M cyclin is degraded. This results in the cell exiting mitosis and re-entering the G1 phase. In the mature adult most human cells are highly or terminally differentiated, and they withdraw from the cell cycle during G1 phase, entering the G0 phase. For most cells there is no need for further replication and the concentration of growth factors is not high enough to drive them through the restriction point in G1. In these cells the molecular brakes are applied and they enter the resting state of G0. In some tissues cells can be stimulated to re-enter the cell cycle and replicate when a need arises or in pathological conditions. The biological processes of molecular brakes are not entirely understood, but the cell-cycle inhibitors (CKIs) seem to play an important role. They block the assembly or activity of certain cyclin–Cdk complexes, leading to cell-cycle arrest. In mammalian cells, two families of CKIs have been identified. The Ink4 family members (p15, p16, p18, p19) specifically bind to and inhibit Cdk4 and Cdk6, thus inhibiting transition through the G1 checkpoint. The members of the Cip/Kip family of CKIs (p21, p27, p57) can bind to Cdk2, Cdk4, and Cdk6, and their complexes with cyclins A, E, and D, and can arrest the cell cycle at the G1 or G2 checkpoint [1–3] (Fig. 1).
20 Cellular and Molecular Mechanisms of Pulmonary Vascular Smooth Muscle Cell Proliferation
Pulmonary artery SMCs (PASMCs) in normal, adult lung blood vessels proliferate at an extremely low rate, and are thus mostly quiescent. In common with other VSMCs, PAMSCs are not terminally differentiated and are able to modulate their phenotype, profoundly and reversibly, and exit their quiescent state in response to changing local environmental conditions that normally regulate phenotype.
3 The Pulmonary Vasculature It has become apparent that PASMCs are not uniform in phenotype throughout the pulmonary circulation, but are heterogeneous both at a single anatomical site and along the vascular bed. Studies of the bovine main pulmonary artery revealed the presence of phenotypically distinct SMC subpopulations throughout the media. These specific populations where differently oriented, as seen by light microscopy, and they exhibited a differential expression of messenger RNA for procollagen and tropoelastin [4]. When conventional histochemical staining techniques and light microscopy are used, the media of the human pulmonary arteries appears homogeneous, but when a panel of antibodies to contractile and cytoskeletal proteins is used, several phenotypes can be discerned, which arise early during development of the arterial media [5]. The subpopulations from bovine arteries exhibit very stable and specific functional capabilities both in vivo and in vitro. They are distinct with regard to their state of differentiation, expression of smooth muscle markers, proliferative response to growth factors, and to hypoxia [4–6]. When isolated in tissue culture, these subpopulations display markedly different rates of proliferation, and matrix protein expression. Thus, in any segment of the vessel wall there may be distinct SMC subpopulations that may play diverse roles in vascular homeostatis and can respond differently to injury and to changes in the environmental cues [4–11]. Furthermore, heterogeneity in SMCs is also observed in cells isolated from different anatomical locations in the lung, i.e., proximal versus peripheral arteries. Human PASMCs from peripheral pulmonary circulation (arteries 1–2 mm in diameter) proliferate more rapidly than cells isolated from the main pulmonary artery and they exhibit different responses to prostacyclin analogs and bone morphogenetic protein (BMP) 4 [12, 13]. There is a possibility that these distinct subpopulations have different embryonic origins, but no fate-mapping analysis of their origin has been undertaken thus far.
4 Factors Involved in PASMC Proliferation As previously noted, in a normal mature blood vessel, VSMCs are contractile and exhibit extremely low rates of proliferation, although growth factors may be present. Furthermore,
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exposure of intact blood vessels to exogenous growth factors, both in vivo and in vitro, does not lead to rapid proliferation [14]. However, VSMCs are stimulated to proliferate mostly in pathological conditions such as a response to injury, or changes in the environment, suggesting that factors exist in the normal vessel wall, which inhibit stimulation of VSMC proliferation by growth factors. Inhibitory signals that prevent VSMC proliferation are thought to be cell–matrix interaction, soluble mediators (such as growth factors), as well as cell– cell adhesion [14–16]. VSMCs in the tunica media of blood vessels are embedded in extracellular matrix (ECM) comprising elastin, collagen (types I, III, IV, VI, VIII, and XIII), proteoglycans, hyaluronan and adhesive glycoproteins (fibronectin and laminin). ECM components are produced by vascular cells. When normal medial VSMCs are placed in culture, within a few days they lose their contractility and myofilaments and develop an extensive rough endoplasmic reticulum and a large Golgi complex, shifting from the normally contractile and quiescent phenotype into a synthetic and proliferative phenotype [17]. Interestingly, laminin, a constituent of normal media, has been shown to inhibit this shift, whereas fibronectin promotes the shift to the “synthetic” phenotype [18]. Culture of VSMCs on polymerized collagen type I or collagen type IV inhibits VSMC proliferation and promotes the quiescent, contractile phenotype [14, 19]. Mutation of one allele of elastin, previously thought to play a purely structural role, is sufficient to induce subendothelial proliferation of VSMCs [20]. Heparan sulfate, produced by VSMCs, has long been recognized as potent inhibitor of smootrh muscle cell proliferation [21]. In addition to direct effects, a number of growth factors bind to the heparin sulfate rich ECM [22], and thus can serve as a local site of storage. Heparin is also critical for oligomerization of fibroblast growth factor (FGF) and subsequent stimulation of endothelial cells and VSMCs [23]. Another group of glycoproteins, termed matricellular or matrix-associated proteins, are a class of secreted proteins that interact with other ECM components, multiple specific cell-surface proteins, and growth factors to modulate cell– matrix interactions. Four members of the group, osteopontin, SPARC, thrombospondin, and tenascin, have been shown to exert common “antiadhesive” functions involved in cell migration and proliferation [24]. In pulmonary arteries, tenascin C localizes to sites of active vascular remodeling, where it colocalizes with proliferating cells and epidermal growth factor (EGF) [25]. Tenascin C augments the proliferative response to growth factors by a mechanism that involves binding of its cell-surface integrin (avb3), leading to a rearrangement of the cytoskeleton and clustering and priming of growth factor receptors (e.g., EGF receptors) [26]. Matrix metalloproteinases (MMPs) are a family of zinccontaining enzymes thought to be responsible for the turnover of ECM components at physiological pH. Increased MMP activity has been demonstrated after injury of systemic blood
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vessels, and therefore it was proposed that MMPs may modulate VSMC proliferation as well as migration [27]. Three synthetic inhibitors of MMPs [28, 29] as well as the adenoviral overexpression of tissue inhibitors of metalloproteinases [30] reduced MMP-induced SMC proliferation. Mechanisms proposed to explain this phenomenon focus on MMPs affecting growth-factor-mediated cell proliferation by regulating their bioavailability and/or activity through cleavage of either matrix or nonmatrix (growth-factor-bound) proteins with the consequent release of active growth factors such as FGF2 [16, 31].
4.1 Growth Factors 4.1.1 Receptor Tyrosine Kinases Receptor tyrosine kinases (RTKs) play an important role in the control of most fundamental cellular processes, including cell proliferation. They are membrane-spanning cell-surface receptors with intrinsic tyrosine kinase activity and they serve as receptors for various growth factors, hormones, and cytokines. All RTKss activate Ras through the exchange of GTP for GDP by the guanine nucleotide exchange factor Sos. The adaptor protein Grb2 forms a complex with Sos, resulting in recruitment of the complex to an activated RTK, thus translocating Sos to the plasma membrane, enabling it to be close to Ras and perform the guanine nucleotide exchange and activate Ras [32]. Once activated, Ras reacts with several effector proteins such as Raf and phosphatidylinositol 3-kinase. Activated Raf stimulates the mitogen-activated protein kinase (MAPK) kinase cascade, eventually phosphorylating extracellular-regulated kinase (ERK), which then translocates to the nucleus, where it phosphorylates and activates various transcription factors [33, 34]. This highly conserved signaling cascade plays an important role in regulating cell cycle, cell proliferation, and other important cellular processes. Phosphatidylinositol 3-kinase activation leads to stimulation of various intracellular responses, a particularly important one being cell survival by phosphorylating the proapoptotic protein BAD, which then blocks its complex with Bcl-2 and Bcl-xl and prevents apoptotic cell death [35]. Several RTK ligands such as platelet-derived growth factor (PDGF), FGF2, insulin-like growth factor (IGF), and EGF had been shown to stimulate PASMC proliferation. The PDGF family consists of PDGF-A, PDGF-B, PDGF-C, and PDGF-D, which are inactive as monomers, but form active homo- and heterodimers (PDGF-AA, PDGF-AB, PDGF-BB, PDGF-CC, PDGF-DD). Most often studied is the PDGF-BB homodimer, which potently stimulates PASMC proliferation [36, 37]. This effect is inhibited by PDGF RTK inhibitors, including imatinib [37]. PDGF is a more potent PASMC mitogen than FGF2, IGF, and EGF.
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4.1.2 Transforming Growth Factor b Superfamily Transforming growth factor b (TGFb) ligands are a large group of cytokines that regulate many cellular functions, including cell growth, adhesion, migration, cell-fate determination, differentiation, and apoptosis. They are also among the key cytokines that control vascular function [38]. Malfunctions in TGFb signaling cause serious human diseases such as cancer, fibrosis, wound-healing disorders [39], and several hereditary conditions such as familial PAH [40], hereditary hemorrhagic telangiectasia [41, 42], and fibrodysplasia ossificans progressiva [43]. The TGFb superfamily of ligands comprises some 30 genes in mammals, including three TGFb isoforms (TGFb1, TGFb2, and TGFb3), and more than 20 BMPs [43]. TGFb superfamily receptors are transmembrane serine/threonine kinases, functionally divided into two classes: type II and type I receptors. There are five type II (TGFbRII, ActRII, ActRIIB, BMPRII, AMHRII) and seven type I (activinreceptor-like kinases, ALK1–ALK7) receptors. Access of ligands to the signaling type I and type II receptors is regulated by soluble ligand binding proteins and by accessory type III receptors. Examples of the latter class that have been most intensively studied are endoglin and b-glycan [44, 45]. These two accessory receptor are highly structurally related transmembrane proteins that facilitate TGFb ligand binding to TGFbRII [46–49]. Endoglin can exert this function only when bound to TGFbRII. However, it is able to bind directly to BMPs [50–52]. Binding of the ligand dimer causes bridging of the preformed dimers of type II receptors and dimers of type I receptors to form heterotetrameric, active receptor complexes. This enables the constitutively active type II receptor to phosphorylate, and thereby activate, the type I receptor in a juxtamembrane region. Once activated, the type I receptor is able to recruit and subsequently phosphorylate receptorregulated Smads (R-Smads) on two serine residues at their extreme C-termini. ALK4, ALK5, and ALK7 recruit, and phosphorylate Smad2 and Smad3, whereas ALK1–ALK3 and ALK6 recruit and phosphorylate Smad1, Smad5, and Smad8. This C-terminal phosphorylation allows the R-Smads to form complexes with the common Smad, Smad4, and, also, to form homomeric complexes [53–56]. These complexes accumulate in the nucleus and regulate gene transcription usually in association with other transcription factors [53–55]. TGFb signaling is negatively regulated by the inhibitory Smads (I-Smads), Smad6 and Smad7. I-Smads are transcriptionally induced in response to TGFb and BMPs in a Smad-dependent manner and are thought to function in signal termination [53–56]. In VSMCs, most TGFb effects appear to be mediated via the TGFbRII/ALK5 complex. Subsequent downstream signaling is complex, not only involving Smad2/Smad3 but also
20 Cellular and Molecular Mechanisms of Pulmonary Vascular Smooth Muscle Cell Proliferation
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Fig. 2 Effects of transforming growth factor b superfamily ligands on pulmonary artery smooth muscle cells (PASMCs)
kinases such as p38 MAPK, p42/p44, and c-Jun N-terminal kinase as well as downstream transcription factors [57, 58]. TGFb1 can attenuate VSMC activation by opposing the effects of mitogenic growth factors, proinflammatory cytokines, and genes that affect vascular remodeling. This antimitogenic effect on VSMCs was found to be ALK5-mediated via signaling pathways dependent on both Smad3 and p38 MAPK [59] (Fig. 2) VSMCs are also influenced by BMPs: BMP2, BMP4, BMP6, and BMP7. BMPs interact with three type 2 receptors, BMPRII, ActRII, and ActRIIB, to activate different type I receptors ALK2, ALK3, and ALK6 [57]. The effect of BMPs depends on the source of VSMCs studied as well as the local environment. The serum-stimulated proliferation of cells harvested from the main or lobar pulmonary arteries tends to be inhibited by BMP2, BMP4, and BMP7. With use of a dominant negative Smad1 construct, the growth inhibitory effects of BMPs have been shown to be Smad1-dependent [12]. Another study demonstrated that BMP2 prevents PDGF-induced VSMC proliferation via induction of the peroxisome-proliferator-activated receptor g/apoE axis [60, 61]. In contrast, in PASMCs isolated from pulmonary arteries of 1–2-mm diameter, BMP2 and BMP4 stimulate proliferation [12, 60, 61]. This pro-proliferative effect of BMPs in peripheral cells is dependent on the activation of ERK1/2 and p38 MAPK. Both Smad and MAPK pathways are activated to a similar extent in cells from both locations, but the integration of these signals by the cell seems to differ [62]. (Fig. 2)
4.2 Calcium and Smooth Muscle Proliferation Contraction is, probably, the most important property of the smooth muscle. It is triggered by a rise in the free cytosolic Ca2+ concentration, because a Ca2+ binding protein,
calmodulin, binds Ca2+ as its cytosolic concentration increases. This complex then activates myosin light chain kinase, which, in turn, phosphorylates the myosin light chain. The phosphorylated myosin light chain stimulates the activity of myosin ATPase, hydrolyzing ATP to release energy for the subsequent cycling of the myosin crossbridges with the actin filament. The formation of these cross-bridges underlies SMC contraction [63]. Cytosolic Ca2+ concentration in PASMCs can be increased by (a) Ca2+ influx through voltage-dependent Ca2+ channels (VDCCs), receptor-operated Ca2+ channels, and store-operated Ca2+ channels, and (b) Ca2+ release from intracellular stores, the most important one being the sarcoplasmic reticulum (SR), via Ca2+-release channels [e.g., inositol 1,4,5- trisphosphate (IP3) receptors and ryanodine receptors]. In contrast, cytosolic Ca2+ concentration in PASMCs can be decreased by (a) Ca2+ expulsion by the Ca2+–Mg2+-ATPase (Ca2+ pump) and by the forward mode of the Na+/Ca2+ exchanger in the plasma membrane, and (b) Ca2+ sequestration by the Ca2+– Mg2+-ATPase in the SR (SERCA) [64–66]. Resting membrane potential in VSMCs normally ranges between −70 and 50 mV [67]. Decreased expression and/or function of K+ channels leads to sustained membrane depolarization and contributes to sustained elevation of cytosolic Ca2+ concentration by (a) activating VDCCs and (b) facilitating the production of IP3, which stimulates the release of SR Ca2+ into the cytoplasm [68]. Free cytosolic Ca2+ concentration is an important determinant not only for VSMC contraction but, also, for its proliferation. Elevated cytosolic Ca2+ concentration leads to a rapid increase in the nuclear concentration of Ca2+, which pushes the cells from quiescence into the cell cycle, where they undergo mitosis, thus promoting their proliferation [69]. Transition through all four phases of the cell cycle appears to be sensitive to Ca2+/calmodulin complex activation. Some of the signaling molecules involved in mitogenic pathways appear to be Ca2+-dependent. MAPK II, an enzyme involved
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in the phosphorylation cascade that transduces the mitogenic signals of various growth factors activated by a rise in cytosolic Ca2+ concentration [70]. It has also been shown that resting cytosolic Ca2+ concentration is significantly elevated in proliferating PASMCs compared with that in growth-arrested cells [71, 72]. Interestingly, sustained elevation in cytosolic Ca2+ concentration due to Ca2+ influx has been shown to upregulate the expression of activating protein-1 (AP-1) family proteins c-Fos and c-Jun [73]. AP-1 is a family of transcription factors composed of Jun (v-Jun, c-Jun, JunB, and JunD), Fos (v-Fos, c-Fos, FosB, Fra1, and Fra2), or activating transcription factor subunits [74]. The target genes for AP-1 are often involved in regulation of cell proliferation, migration, and apoptosis, for example, endothelin (ET)-1, PDGF, and others [75]. Hence, the upregulation of the AP-1 family members c-Fos and c-Jun by the elevated cytosolic Ca2+ concentration could stimulate production of mitogens such as PDGF or ET, which, in turn, stimulate VSMC proliferation [76]. It has also become apparent that the Ca2+ stored in the SR plays an important role in the initiation of DNA synthesis and cell proliferation. Depleting the IP3-sensitive Ca2+ stores with the Ca2+–Mg2+-ATPase inhibitors leads to cell-cycle arrest, whereas Ca2+ rescue via SERCA pumps allows the resumption of the S phase of the cell cycle [77]. Therefore, increased cytosolic and increased SR Ca2+ concentration are both required for PASMC mitosis and proliferation. (Fig. 3)
Fig. 3 Increased cytosolic Ca2+ concentration exerts mitogenic effects on PASMCs through direct stimulation of the cell-cycle transition and through increased expression of the activating protein-1 transcription factor family members c-Fos and c-Jun which stimulate production of the powerful mitogens platelet-derived growth factor and endothelin-1
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4.3 Vasoactive Substances Serotonin (5-hydroxytryptamine, 5-HT) is a monoamine neurotransmitter whose platelet and plasma levels are increased in PAH patients [78, 79]. It is produced by the enterochromaffin cells in the gut, serotoninergic neurons in the central nervous system, and pulmonary neuroendocrine cells and neuroepithelial bodies [80]. Apart from vasoconstriction, 5-HT induces hypertrophy and hyperplasia of human PASMCs [81–85] and also acts as a comitogen since in combination with other growth factors, such as PDGF, FGF, and EGF, the proliferative response is greater than with each mitogen alone. Unlike the vasoconstrictive effects of 5-HT that are believed to be receptor-mediated, proliferative effects require 5-HT internalization via the 5-HT transporter (5-HTT) [82, 83]. Interestingly, 5-HTT is abundantly expressed in the lung, where it is predominantly located in the PASMCs [86]. In PASMCs, mitogenic and comitogenic effects of 5-HT are dose-dependently inhibited by selective 5-HTT inhibitors, but not by 5-HT2A or 5-HT1B/1D receptor antagonists [87]. Once internalized, 5-HT can exert its mitogenic and comitogenic effects on PASMCs, but the underlying intracellular mechanisms are still elusive. It is believed to work through phosphorylation of GTPase-activating protein and through stimulation of MAPK kinase and ERK1/2, which could account for its mitogenic function [88–90].
20 Cellular and Molecular Mechanisms of Pulmonary Vascular Smooth Muscle Cell Proliferation
Endothelin (ET) is a potent vasoconstrictor produced by endothelial cells under pathological conditions. There are three ET isoforms, ET-1, ET-2, and ET-3, ET-1 being the predominant one. ET-1 is initially produced as a 38 amino acid peptide and then processed into a 21 amino acid peptide by the action of ET-converting enzyme. ET can be secreted by PASMCs, airway epithelium, macrophages, and pulmonary artery endothelial cells [91, 92]. ET-1 exerts its cellular functions through two receptor subtypes: ETA and ETB [93]. ET-1 is a potent mitogen for PASMCs. Inhibition of endogenous ET-1 release or its action attenuated sum-stimulated proliferation of PASMCs [93, 94]. Angiotensin II is part of the renin–angiotensin system and is another vasoactive factor that was shown to exert mitogenic effects on PASMCs [95]. Angiotensin II is an active form processed from its precursor angiotensin I by the action of the angiotensin-converting enzyme which is found predominantly in the capillaries of the lung. Thromboxane A2 (TXA2), a metabolite of arachidonic acid, is also a vasoconstrictor and a PASMC mitogen [76, 95]. It is produced in platelets, lungs, and several other tissues. Human TXA2 receptor (TP), a typical G-protein-coupled receptor (GPCR) with seven transmembrane domains, has two splice variants, TPa, and TPb, that vary in their C-terminal regions. TPs communicate mainly with G12/13 and Gq, resulting in phospholipase C activation and RhoGEF activation. Mitogenic effects of TXA2 are believed to be ERK1/2-mediated [96, 97]. Nitric oxide, prostacyclin, and vasoactive intestinal peptide. Nitric oxide (NO) is generated from l-arginine and the reaction is catalyzed by NO synthase (NOS), which has three distinct isoforms [98] NOS-III (i.e., endothelial NOS), which is a Ca2+-dependent constitutive isoform, is expressed basally in vascular endothelial cells, whereas NOS-I (i.e., neuronal NOS) is expressed in neuronal cells. NO generated by NOS-I and NOS-III is responsible for maintaining normal vascular tone and neuronal signal transduction. NOS-II (i.e., inducible NOS), which is expressed in a variety of cells within the body, is a Ca2+-independent isoform, and is induced in response to certain cytokines and endotoxins. NO induces cyclic GMP (cGMP)-mediated vasodilatation and inhibition of PASMC proliferation. Prostacyclin is an important endogenous vasoactive factor that acts through adenylate cyclase and cyclic AMP to induce vascular dilatation and antimitogenic effects on PASMCs. Vasoactive intestinal peptide is a neuropeptide that functions primarily as a neurotransmitter and is, also, a potent vasodilator and PASMC-proliferation inhibitor. It acts through two receptor subtypes (VPAC-1 and VPAC-2) which are coupled to adenylate cyclase and cGMP systems [76, 95, 99].
4.4 Hypoxia Chronic hypoxia leads to the development of PAH, characterized by medial thickening [100, 101]. Hypoxia has
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d istinct effects on PASMCs in different parts of the pulmonary vascular bed, as the cellular composition of the arteries changes along the longitudinal axis of the pulmonary circulation. Data from small animals (rats and mice) suggested that this medial thickening is largely attributable to PASMC hypertrophy and ECM deposition in proximal pulmonary arteries [102–104]. However, data available from proximal arteries of large mammals, including humans, revealed that hypoxia stimulates proliferation of some of the distinct PASMC subpopulations within the media [105, 106]. As described previously, the tunica media of pulmonary and systemic arteries comprises several distinct SMC populations that exhibit different ion-channel and receptor expression and could be derived from different lineages [4–11, 105, 106]. The PASMC subpopulation that exhibits the proliferative response to chronic hypoxia is characterized by a less differentiated or an “undifferentiated” phenotype, as assessed by expression of smooth muscle markers compared with other PASMCs in the vessel media. These data were consistent with those of in vitro studies showing that less differentiated SMCs exhibit a proliferative response to chronic hypoxia, whereas the SMCs with a more differentiated phenotype show a growth-inhibitory response to hypoxia [6, 7, 9]. The mechanisms underlying this proliferative response of the specific proximal PASMC subpopulation have not been elucidated. It has been demonstrated that the proliferation-prone PASMCs show augmented responses to GPCR agonists [6, 7, 9] and to the stimulation of the protein kinase C pathway compared with other PASMC subpopulations [107]. Hypoxia-induced GPCR activation, leading to ERK1/2 activation, has been implicated in the hypoxia-induced proliferation [108]. PASMCs isolated from more peripheral (distal) arteries (1–2 mm in diameter) show a proliferative response to hypoxia. When exposed to chronic hypoxia, distal arteries show profound medial thickening caused by PASMC hyperplasia [100, 104]. However, some researchers have questioned whether the cells that are proliferating in the media of distal arteries in response to hypoxia are actually SMCs or myofibroblasts [103]. The mechanisms underlying hypoxia-induced proliferation of PASMCs could be either direct and/or through hypoxia-induced release of vasoactive factors by endothelial cells and/or platelets. Hypoxia causes intrinsic changes in potassium and calcium homeostasis of PASMCs leading to decreases in KV channel currents, membrane depolarization, elevated intracellular Ca2+ concentration causing vasoconstriction, and PASMC proliferation [72, 109–111]. It seems that Ca2+ influx channels rather than VDCCs mediate these effects [103, 112]. In addition, hypoxia reduces KV channel activity and reduces messenger RNA and protein expression of several pore-forming a-subunits of the K+ channels in rat PASMCs (KV1.1, KV1.5, KV1.6, KV2.1, and KV4.3) [72, 109–111, 113].
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Hypoxia has been shown to reduce the production of prostacyclin and NO [114–118] and to increase the production of ET, 5-HT, and PDGF by endothelial cells [119, 120] and this imbalance may promote mitogenesis of PASMCs. A number of studies have addressed whether hypoxic exposure of cultured PASMCs leads to cell proliferation. Some studies concluded that hypoxia inhibits PASMC proliferation, or is not a direct stimulus [108, 120, 121]. Other studies concluded that hypoxia exerts mitogenic actions on PASMCs [6, 122–129]. The different findings of these studies may arise from (a) a different origin of the cells used for experiments (e.g., proximal versus peripheral arteries), (b) different subpopulations of SMCs isolated, (c) severity of hypoxia – more moderate hypoxia (1–5%) more often had a mitogenic effect than severer hypoxia or anoxia, and (d) the concentration of serum in the culture media – serum concentrations of more than 2% seem to favor the proliferative response to hypoxia [104]. It has also been shown that moderate (5% O2) hypoxia enhances PDGF-AB-, FGF2-, and EGF-driven PASMC proliferation and that this effect is hypoxia-inducible factor (HIF)-1a-dependent and HIF-1bdependent [130].
4.5 Mechanical Stress and PASMC Proliferation VSMCs in the blood vessel media are exposed to circumferential, radial, and axial stress resulting from pulsatile blood flow. This stress is counterbalanced by the vessel wall tone induced by VSMC contraction, elastin, and collagen [131]. Exposure of the vessel wall to stretch leads to deformation of the ECM of the tunica media, which indirectly stimulates cell membrane receptors of the VSMCs embedded within. VSMC membrane cytoskeleton and contractile apparatus are functionally associated; hence, they are all prone to distortion by stretch [131]. Most of the data available on effects of mechanical stretch on VSMCs come from studies on systemic VSMC. Several studies using various levels of stretch (from 5 to 24%) found that it inhibited VSMC proliferation as assessed by thymidine incorporation [132], 5-bromodeoxyuridine incorporation [133], or an increase in p21 expression leading to G1 cell cycle arrest [134]. In contrast to these studies, most other studies have reported an induction of VSMC proliferation by various levels of stretch [135, 136]. The stretch-induced proliferation was initially attributed to the autocrine action of PDGF [136], or stretch-induced phosphorylation of the PDGF receptor a [137]. One of the studies demonstrated the induction of ERK phosphorylation by 20% stretch, leading to an increase in DNA synthesis [138]. More recent studies have clarified the role of focal adhesions and the focal adhesion kinase (FAK) in stretch-induced VSMC
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proliferation in vitro. Focal adhesions are regions of tight, intimate contact of the cells with the underlying ECM that provide a structural link between the actin cytoskeleton and the ECM which enables the cells to sense the mechanical changes in ECM and transduce signals related to cell growth and proliferation [139]. Focal adhesion assembly is regulated by GTP-binding protein Rho. FAK is a cytoplasmic soluble tyrosine kinase that acts as a biomechanical sensor and a signaling switch stimulated by stretch in vitro [140–142]. When VSMCs are subjected to sustained stretch, activation of the Rho-GTPases stimulates cell contractility, which, in turn, leads to the bundling of actin filaments and the aggregation of the ECM receptors, integrins. Clustering of the integrins recruits FAK to focal adhesions, where they bind to talin [139, 143]. FAK is then autophosphorylated at Tyr397, activating an attachment site for c-Src [144, 145]. The FAK/cSrc complex then stimulates MAPKs, such as eERK1/2 and p38 MAPK [146]. ERK1/2 drives proliferation of VSMCs exposed to stretch, whereas p38 MAPK increases apoptosis [147]. In the presence of stretch, ERK1/2 activation in VSMC requires an intact actin cytoskeleton as well as a functioning Rho/Rho kinase pathway. Disruption of the actin cytoskeleton using cytochalasin-D, as well as the use of RhoA and Rho kinase inhibitors, attenuates ERK1/2 activity despite VSMC stimulation using stretch [148]. Several studies showed an increase in DNA synthesis in PASMCs of arteries exposed to static force [149]. One of these studies suggested that mechanical stretch leads to production of VEGF and FGF by PASMCs, with FGF acting as a mitogen [150].
4.6 Inflammation and PASMC Proliferation An inflammatory milieu (i.e., the release of circulating cytokines, chemokines, and growth factors by inflammatory cells) can contribute to PASMC proliferation. Inflammation is thought to contribute to the pathobiological processes in PAH. Fractalkine is a chemokine produced by circulating CD4+ and CD8+ lymphocytes that promotes the chemokine (C-X3-C motif) receptor 1 (CX3CR1)-expressing leukocyte recruitment [151]. Both human and rat PASMCs were shown to express CX3CR1 (the receptor for fractalkine). Fractalkine induced proliferation in cultured rat PASMCs, suggesting that it may act as a growth factor for PASMCs. The level of fractalkine was also shown to be increased in the plasma of PAH patients [152]. Another cytokine, synthesized by vascular cells, is CC ligand 2, formerly called monocyte chemoattractant protein 1. This cytokine is a potent mediator of monocyte/macrophage activation and migration but has also been shown to stimulate PASMC proliferation [153].
20 Cellular and Molecular Mechanisms of Pulmonary Vascular Smooth Muscle Cell Proliferation
Fig. 4 Balance of vasoactive factors. Increase in availability/concentration of the pro-proliferative factors such as 5-hydroxytryptamine, endothelin-1, angiotensin II, and thromboxane A2 with decreased levels of antiproliferative factors such as nitric oxide, prostacyclin, and vasoactive intestinal peptide could contribute to proliferation of PASMCs
5 Conclusions This chapter has reviewed the complex processes known to contribute to SMC proliferation (Fig. 4), one of the key factors in the pathobiological processes in PAH. Although the contributions of numerous pathways have been discussed, there are major connections between pathways and common mechanisms that are crucial to the process of proliferation. In addition, ongoing research needs to determine the hierarchy of these processes. Such knowledge will assist in the development of novel therapies to treat PAH.
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Chapter 21
Role of Ca2+ in Vascular Smooth Muscle Gene Expression and Proliferation Karen M. Lounsbury and Patricia C. Rose
Abstract The importance of Ca2+ in pulmonary smooth muscle physiological function, including events such as contraction, gene transcription, and cell proliferation, is well established. Yet, there is much to learn regarding the complex interplay between Ca2+ signaling, gene expression, and the development of lung diseases such as pulmonary arterial hypertension. Under normal physiological conditions, vascular smooth muscle cells in adult blood vessels show low rates of proliferation and synthetic activity. Mature smooth muscle cells, unlike cardiac muscle cells, are not terminally differentiated, and thus have the unique ability to dedifferentiate and alter their phenotype in response to changes in the extracellular environment. Phenotypical switching is an important survival mechanism in response to injury, and is necessary during vascular development. However, abnormal signal transduction results in the development of a proliferative synthetic phenotype rather than one that is quiescent and contractile, leading to pathologic vascular remodeling. Ca2+ is known to be a key transcriptional regulator of both the onset and the maintenance of many genes associated with vascular remodeling. This chapter focuses on events that underlie changes in gene expression and proliferation that are mediated by Ca2+ signaling in pulmonary smooth muscle. In addition, the role of Ca2+ in the development of lung disease as it relates to cell signaling is discussed in the chapter. Keywords Calcium signaling • Smooth muscle contraction • Cell proliferation • Calcium-sensitive proteins • Pulmonary vasoconstriction • Vascular remodeling
1 Introduction The importance of Ca2+ in pulmonary smooth muscle physiological function, including events such as contraction, gene transcription, and cell proliferation, is well established. K.M. Lounsbury (*) Department of Pharmacology, University of Vermont, Given Building, 49 Beaumont Avenue, Burlington, VT 05452, USA e-mail:
[email protected] Yet, there is much to learn regarding the complex interplay between Ca2+ signaling, gene expression, and the development of lung diseases such as pulmonary arterial hypertension (PAH). Under normal physiological conditions, vascular smooth muscle cells (VSMCs) in adult blood vessels show low rates of proliferation and synthetic activity [1]. Mature smooth muscle cells, unlike cardiac muscle cells, are not terminally differentiated, and thus have the unique ability to dedifferentiate and alter their phenotype in response to changes in the extracellular environment [1, 2]. Phenotypical switching is an important survival mechanism in response to injury, and is necessary during vascular development. However, abnormal signal transduction results in the development of a proliferative synthetic phenotype rather than one that is quiescent and contractile, leading to pathologic vascular remodeling [3]. Ca2+ is known to be a key transcriptional regulator of both the onset and the maintenance of many genes associated with vascular remodeling. This chapter will focus on events that underlie changes in gene expression and proliferation that are mediated by Ca2+ signaling in pulmonary smooth muscle. In addition, the role of Ca2+ in the development of lung disease as it relates to cell signaling will be discussed.
2 Sources of Ca2+ in Pulmonary Artery Smooth Muscle Cells The classic pathway for modulation of Ca2+ in arterial smooth muscle relies on membrane depolarization to stimulate the opening of L-type voltage-dependent Ca2+ channels on the plasma membrane. This influx promotes activation of multiple Ca2+-regulated enzymes, including calmodulin (CaM)dependent protein kinases and phosphatases, Raf kinase, phosphoinositol 3-kinase, and phospholipase C (PLC). The Ca2+ signal is amplified by PLC-mediated production of inositol 1,4,5-trisphosphate (IP3) that activates Ca2+ release from the sarcoplasmic reticulum (SR). If the Ca2+ stores are substantially depleted, store-operated Ca2+ channels on the plasma membrane are opened to restore SR Ca2+ levels and
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Fig. 1 Changes in Ca2+ signal regulation during vascular smooth muscle cell (VSMC) phenotype switching. Maintenance of the differentiated phenotype is promoted by L-type voltage-dependent Ca2+ channel (VDCC) function and large-conductance Ca2+-activated K+ channel (BKCa) activation by Ca2+ sparks released from ryanodine receptors (RyRs). The proliferative phenotype is associated with downregulation of VDCC, BKCa, RyR, and SERCA2a. The proliferative phenotype is associated with an upregulation of transient receptor
potential channels (TRPCs)/Orai channels, inositol 1,4,5-trisphosphate receptors, and SERCA2b, which leads to store-operated Ca2+ entry as the primary Ca2+ signal. GFR growth factor receptors, Gq-R Gq-coupled receptor, PLC phospholipase C, SOS son-of-sevenless, Raf Ras-activated kinase, MEK mitogen-activated protein kinase/ extracellular-signal-regulated kinase kinase, ERK extracellular-signalregulated kinase, CaMK Ca2+/CaM-dependent protein kinase, ROK Rho kinase, CaN calcineurin
further amplify the Ca2+ signal. The Ca2+ signal is attenuated by hyperpolarizing K+ channel activity, Ca2+-binding proteins (buffering), and Ca2+ pumps on the SR and plasma membranes. The amplitude and duration of the Ca2+ signal is important for both muscle contraction and gene transcription. Dysfunctional regulation of any of these steps could disrupt Ca2+ homeostasis, thus leading to vasoconstriction and vascular remodeling. The following section details the individual Ca2+ regulatory components and their role in pulmonary artery smooth muscle cell (PASMC) gene expression and proliferation, with an overall summary depicted in Fig. 1.
(SMMHC) [5]. This loss is accompanied by an increase in expression of T-type Ca2+ channels that can be activated at the more negative membrane potentials observed in dedifferentiated VSMCs. Although not directly studied in PASMCs, the alteration in expression levels of voltage-gated Ca2+ channels could contribute to a change in the Ca2+ homeostasis that promotes cell growth signaling.
2.1 Voltage-Dependent Ca2+ Channels PASMCs, like most smooth muscle cells, express predominantly L-type voltage-dependent Ca2+ channels, which are activated by high voltage and characterized by large singlechannel conductance and slow voltage-dependent inactivation [4]. Influx of Ca2+ through these channels is the primary mechanism of excitation–contraction and excitation–transcription coupling through Ca2+/CaM signaling, resulting in cell contraction and maintenance of the differentiated phenotype. When the signal is prolonged owing to loss of Ca2+ buffering/extrusion or when combined with effectors that prolong the signal, Ca2+ influx through L-type Ca2+ channels can contribute to activation of growth factor signaling that leads to cell proliferation and vascular remodeling. Loss of functional L-type Ca2+ channels in arterial smooth muscle has been associated with cell dedifferentiation and loss of expression of differentiation markers such as smooth muscle a-actin and smooth muscle myosin heavy chain
2.2 IP3 and Ryanodine Receptors VSMCs, like all eukaryotic cells, use agonist signaling through G-protein-coupled receptors to transduce intracellular signals. Signaling through Gq-coupled receptors activates PLC, which hydrolyzes phosphatidylinositol 4,5-bisphosphate to form the second messengers IP3 and diacylglycerol. IP3 selectively activates IP3 receptor ion channels on the SR membrane, initiating a wave of Ca2+ release from the SR into the cytoplasm. These cytoplasmic Ca2+ signals have been termed “Ca2+ waves” and have been shown to be necessary for many cell functions, including cell migration and proliferation. All muscle cells and many other cell types also express ryanodine receptors (RyRs) on the SR membrane that are activated by a rise in cytoplasmic Ca2+ level. Distinct release events from RyRs have been termed “Ca2+ sparks,” and their signaling function is cell-type specific. In cardiac myocytes, Ca2+ sparks are stimulated by L-type Ca2+ channel activation and serve to augment the Ca2+ influx signal in excitation– contraction coupling and promote cell contraction [6]. In most types of smooth muscle cells (freshly isolated or intact), Ca2+ sparks oppose contraction by directly coupling to Ca2+-activated
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K+ channels [7]. In isolated PASMCs, multiple subtypes of RyRs are expressed, but, unlike in other smooth muscle cells, RyR activity is activated by IP3-receptor-mediated Ca2+ release and coupled with membrane depolarization rather than hyperpolarization [8, 9]. This coupling is similar to what is observed in nonmuscle cells, but the significance with regard to pulmonary artery function is not known. It is not completely clear whether the SR pools of Ca2+ used by IP3 receptors and RyRs are distinct or conjoined. In a study of PASMCs, depleting the RyR pool using caffeine and ryanodine did not alter IP3-mediated Ca2+ transients, suggesting that the two stores are distinct [10]. However, similar studies performed in renal artery VSMCs suggested that the two types of receptors used the same SR Ca2+ pool. The spatial organization of these receptors is further complicated by the finding that culturing PASMCs results in a combined pool utilized by both RyRs and IP3 receptors [11]. It is possible that these changes in SR pool regulation may involve store-operated Ca2+ mechanisms that make it more difficult to detect distinct pools of Ca2+. Alternatively, the pools of Ca2+ may be dynamic in nature and their compartmentalization may change with cell conditions. The VSMC proliferative phenotype has been correlated with a loss of RyR expression and an increase in IP3 receptor activity. Sustained Ca2+ signals necessary to promote proliferation in cultured VSMCs have been attributed to IP3 receptor activity, and IP3 receptor antagonists block VSMC proliferation [12]. RyRs are downregulated when VSMCs are cultured, and their suppression is accompanied by an increased expression of IP3 receptors [13]. Furthermore, in an animal model of atherosclerosis, resting arterial Ca2+ levels and IP3-mediated Ca2+ release are both increased, correlating sustained elevations in the levels of store-released Ca2+ with a proliferative arterial disease state [14]. In summary, channels in the SR membrane are clearly important in smooth muscle phenotype, including PASMCs, and the growth phenotype is characterized by a downregulation of RyRs and an upregulation of IP3 receptors and IP3mediated Ca2+ signals (Fig. 1).
2.3 Store-Operated Ca2+ Entry It has long been known that release of intracellular stores of Ca2+ upon stimulation by numerous ligands is necessary for sustained Ca2+ level increases and important in the regulation of gene expression in many cell types, including smooth muscle. It was also found that release of Ca2+ from IP3sensitive stores activated influx of extracellular Ca2+, but the molecular identity of this store-operated Ca2+ entry (SOCE) channel proved difficult to ascertain. The majority of evidence supported the hypothesis that the calcium-release-
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activated current (Icrac) was due to activation of the nonselective ion channel family of transient receptor potential channels (TRPCs). This hypothesis was strongly supported by the observation that Icrac and SOCE were lost in TRPC4 and TRPC1 knockout mice, respectively [15, 16]. TRPC alterations are also linked to vascular diseases, where spontaneously hypertensive rats have been shown to express elevated levels of arterial TRPC3 when compared with normal control rats, and inhibition of TRPC1 in coronary arteries was found to reduce neointimal growth after balloon injury [17, 18]. It was later discovered that the SR membrane protein stromal interaction molecule 1 (STIM1) and the ion channel Orai1 are the only components necessary to make a functional SOCE unit. The importance of this process in vascular smooth muscle proliferation was demonstrated by the finding that suppression of STIM1 using RNA interference reduces VSMC proliferation in rat carotid arteries following balloon injury [19]. The two seemingly separate hypotheses have been reconciled by the finding that overexpression of TRPC proteins increases Orai1 activity and SOCE and that there is a functional interaction between Orai1, TRPCs, and STIM1 [20]. Thus, although TRPC proteins are not likely the channels that move Ca2+ in SOCE, they are clearly directly critical to SOCE regulation and VSMC function. In PASMCs, there are several lines of evidence that upregulation of TRPCs is linked to increased SOCE, cell proliferation, and PAH [21]. Pulmonary arteries express the TRPC isoforms 1, 3, and 6, with the most evidence for TRPC1 having store-operated function [22]. During proliferation, PASMCs exhibit an increase in TRPC1 and TRPC6 levels following serum or platelet-derived growth factor (PDGF) stimulation, respectively [23, 24]. SOCE has been shown to be important for normal physiological functions related to both contraction and proliferation in PASMCs, however TRPC3 and TRPC6 are dysfunctionally upregulated in arteries from patients with PAH. Furthermore, silencing of TRPC6 using small interfering RNA inhibits proliferation in PASMCs from patients with PAH [25]. Overall, the evidence thus far suggests that PAH is accompanied by an increase in TRPC expression. This induction enhances store-operated Ca2+ signaling through the Orai1 channel and results in sustained cytoplasmic Ca2+ transients that are sufficient to alter gene expression leading to a proliferative phenotype (Fig. 1).
2.4 Ca2+ Pumps Restoration of resting Ca2+ levels is achieved by Ca2+–Mg2+ATPases (PMCA pumps) and Na2+/Ca2+ exchangers on the plasma membrane, as well as sarcoendoplasmic reticulum Ca2+–Mg2+-ATPases (SERCA pumps) on the SR membrane.
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The maintenance of expression and function of these pumps is essential for maintenance of Ca2+ homeostasis to keep cytoplasmic Ca2+ levels low and to refurbish both ryanodineand IP3-sensitive Ca2+ stores in the SR. The PMCA pumps and Na+/Ca2+ exchanger are essential for movement of Ca2+ from the cytoplasm up its concentration gradient to the extracellular space. There are four isoforms of PMCA, with PMCA1 and PMCA4 expressed in most cell types, including VSMCs, and having distinct functions with respect to proliferation. Overexpression of PMCA1 inhibits proliferation of cultured serum-stimulated VSMCs, whereas PMCA4 is upregulated during proliferation. In addition, PMCA1 expression is negatively regulated by c-Myb, which is selectively expressed during G1/S phase of the cell cycle, whereas PMCA4 upregulation is thought to be protective against apoptosis [26]. Thus, PMCA1 is essential for maintaining homeostasis in differentiated smooth muscle, whereas PMCA4 is induced and required for responses to phenotype change or cellular stress [27]. It has been proposed that these diverse functions are related to the decreased sensitivity of PMCA4 to Ca2+ and CaM levels, which would only be obtained during conditions of agonist stimulation, cell proliferation, or stress. In the case of the Na+/Ca2+ exchanger, the proliferative state is associated with a change in both its expression and its function. In cultured PASMCs, the Na+/Ca2+ exchanger is expressed at high levels and functions in reverse mode, promoting sustained Ca2+ influx and cell proliferation. In addition, upregulation of the Na+/Ca2+ exchanger contributes to the enhanced Ca2+ entry in PASMCs from patients with idiopathic PAH (IPAH) [28]. The SR Ca2+ store replenishment is known to be important for growth signals in VSMCs, and selective subtype expression of SERCA pumps has been shown to determine the VSMC proliferative state. Culturing of mesenteric arterial myocytes results in downregulation of SERCA2a expression, upregulation of SERCA2b expression, and an increase in the amplitude of store-operated Ca2+ transients [13]. Overexpression of SERCA2a inhibits proliferation of cultured VSMCs, and in PASMCs, inhibiting the SERCA pumps causes depletion of the SR store that correlates with cell cycle arrest [29]. Thus, although function of SERCA is important in maintaining the differentiated state, selective overexpression of the SERCA2b isoform results in a proliferative phenotype (Fig. 1).
2.5 K+ Channels as Regulators of Ca2+ Because many of the Ca2+ cascades are dependent on voltage-gated Ca2+ influx, K+ channels that regulate the resting membrane potential of the VSMCs are important indirect regulators of Ca2+ influx. Ca2+-activated K+ channels have been shown to be altered in the development of arterial rest-
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enosis, with large-conductance Ca2+-activated K+ (BKCa) channels being replaced by intermediate-conductance Ca2+activated K+ (KCa3.1) channels. A link to their role in phenotypic switching was made by the finding that inhibition of KCa3.1 activity by in vivo local administration of a selective blocker significantly reduces the development of angioplastyinduced restenosis in a porcine carotid artery model [30]. Although the mechanisms involved in phenotype modulation are not yet known, KCa3.1 function results in voltage-independent membrane hyperpolarization which is likely important in enhancing Ca2+ signaling through TRPCs and induction of mitogenic pathways. Kv channels have also been widely studied for their important role in Ca2+ homeostasis [31, 32]. Because the membrane potential set point is dominated by membrane K+ permeability, a loss of Kv channel activity will cause the resting membrane potential to be more depolarized, which would facilitate activation of L-type Ca2+ channels and prolong the Ca2+ signal. The increased level of Ca2+ leads to both pulmonary vasoconstriction and the promotion of PASMC proliferation and remodeling through Ca2+-regulated gene transcription. In proof, the expression and function of Kv channels (Kv1.2, Kv1.5) is reduced in PASMCs from IPAH patients when compared with patients with other forms of hypertension or cardiopulmonary disease [33, 34]. On the basis of these lines of evidence, the proliferative phenotype is exemplified by a reduction in BKCa and Kv channel expression and an increase in intermediate-conductance KCa3.1 channel expression which could have effects on gene expression through changes in membrane potential set point and store-operated Ca2+ signaling (Fig. 1).
3 Ca2+ -Regulated Transcription Factors The importance of Ca2+ in cellular and physiological events such as muscle contraction, proliferation, and transcription is well established. Thus, dysregulated Ca2+ signaling in VSMCs as a consequence of altered cytoplasmic Ca2+ homeostasis can activate numerous Ca2+-sensitive transcription factors that could contribute to disease [35]. Several Ca2+-regulated transcription factors whose activity has been associated with altered Ca2+ signaling in diseases associated with pulmonary arteries will be discussed.
3.1 Nuclear Factor of Activated T Cells Nuclear factor of activated T cells (NFAT) was first characterized as a Ca2+-regulated transcription factor necessary for the regulation of genes that encode cytokines and their receptors in activated T cells [36, 37]. Although NFAT expression
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is critical for the activated T-cell phenotype, it also functions in nonimmune cells such as skeletal, cardiac, and smooth muscle cells [36, 37]. The NFAT family of transcription factors consists of four Ca2+-dependent isoforms, NFATc1, NFATc2, NFATc3, and NFATc4, and one Ca2+-independent isoform, NFAT5 [36–38]. Ca2+-dependent nuclear localization of NFAT defines its level of activation. NFAT is constitutively expressed, but its nuclear localization signal is concealed when it is in its phosphorylated form [38]. Elevation of cytoplasmic Ca2+ level activates the Ca2+/CaM-dependent phosphatase calcineurin, which dephosphorylates NFAT, exposing its nuclear localization signal. Upon nuclear translocation, NFAT binds and activates promoters of genes involved in processes ranging from the immune response and inflammation to smooth muscle cell differentiation marker gene expression [36, 38]. Because of its sensitivity to Ca2+/calcineurin signaling, NFAT can respond quickly to changes in cytoplasmic Ca2+ levels and is affected by the frequency of Ca2+ oscillations [39, 40]. NFAT signaling has been substantiated in both cultured and native arterial smooth muscle cells [41, 42]. In cultured VSMCs, NFAT is required for proliferation and migration in response to thrombin or PDGF through a pathway that includes upregulation of interleukin-6 [43, 44]. In intact cerebral arteries, the vasoconstrictor UTP has been shown to stimulate NFATc3 nuclear accumulation in VSMCs. This signaling requires both L-type Ca2+ channel function and release of Ca2+ from intracellular stores, suggesting that membrane depolarization alone is not sufficient to result in nuclear localization of NFATc3 [42, 45]. Because NFAT nuclear export is stimulated by serine/threonine kinases such as c-Jun amino-terminal kinase (JNK), it is likely that both an increase in Ca2+ level and a decrease in JNK activity are required for optimal NFAT induction. Indeed, in JNK knockout animals, a rise in intracellular Ca2+ levels is sufficient to elicit NFAT nuclear import [46]. Thus, although Ca2+ level elevation is necessary, it may not be the rate-limiting step in NFAT nuclear accumulation in VSMCs. Several isoforms of NFAT have been evaluated in VSMCs and correlated with both maintenance of differentiation and disease, depending on the NFAT isoform. In both aortic VSMCs and PASMCs, the nuclear translocation of NFATc1 correlates with expression of the differentiation marker smooth-musclespecific SMMHC promoter, and blocking NFAT signaling promotes a proliferative phenotype [47–49]. These data suggest that NFATc1 signaling is required for PASMC differentiation. In contrast to this study, NFATc2 activity is upregulated in PASMCs from patients with IPAH when compared with normal subjects [50]. Increased NFATc2 activity in IPAH is associated with a more depolarized membrane potential as a consequence of NFATc2-mediated downregulation of Kv channel expression, increased intracellular K+ levels and entry of Ca2+ through L-type Ca2+ channels [33, 50]. The resulting increased level of intracellular Ca2+ in PASMCs facilitates a
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forward-feeding loop of NFATc2 activity, which may ultimately lead to PASMC hyperplasia. Therapeutic targeting of NFATc2 has thus been proposed for the treatment of IPAH. The NFATc3 isoform has been shown to be necessary for vascular development, regulation of VSMC contraction, and maintenance of a differentiated smooth muscle phenotype [51, 52]. However, abnormal Ca2+ signaling can lead to events that trigger changes in the vasculature through altered NFATc3 regulation. Similar to IPAH, in hypoxia-induced PAH, Kv channel expression is reduced, which results in a depolarized membrane potential and Ca2+ influx through L-type Ca2+ channels [53]. Hypoxia also stimulates NFAT activity and enhanced nuclear translocation of NFATc3 [54]. NFATc3 knockout animals or animals treated with the calcineurin antagonist cyclosporine A do not exhibit hypoxia induction of smooth muscle a-actin or an increase in arterial wall thickness, suggesting that NFATc3 is a key regulator of arterial remodeling during hypoxia-induced PAH [54]. Taken together, these results implicate that selective NFAT isoform expression can direct Ca2+ signals to different outcomes. In the development of PAH, upregulation of NFATc2/NFATc3 and downregulation of NFATc1 are observed, and study of how these changes are associated with expression of different Ca2+ regulatory pathways may lead to treatment strategies that take advantage of these subtype selectivities (Fig. 2).
3.2 Serum Response Factor Serum response factor (SRF) is a member of the MADS box family of transcription factors, and binds to serum response elements (SREs) within the promoters of many genes related to VSMC proliferation and differentiation [55]. As its name implies, SRF was discovered as a growth factor signaling molecule, and was shown to bind SREs within the promoter of immediate early genes such as c-fos and egr-1 [56]. The SRE is also referred to as a CArG box element on the basis of its consensus sequence, CC(A/T)6GG. A connection between SRF and the Ras/mitogen activated protein kinase (MAPK) growth factor signaling pathway was found to be through MAPK-mediated phosphorylation/activation of the Elk-1 transcription factor, which then forms a complex with SRF on the SRE [57]. SRF was also found to be necessary for the induction of muscle-specific genes during early development and differentiation as well as downregulation of these genes in response to injury or inflammation [58, 59]. SRF binding sites have been identified on smooth-muscle-specific genes, including caldesmon, calponin, SM22, smooth muscle a-actin, SMMHC, and smoothelin-A [60–62]. The question becomes: How does a single transcription factor, binding to a consensus DNA response element, participate in two seemingly opposite gene expression profiles and phenotypic outcomes?
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Fig. 2 Transcriptional control of VSMC phenotype switching. Expression of myocardin (Mycd) directs serum response factor (SRF) to gene targets such as smooth muscle a actin and smooth muscle myosin heavy chain and requires L-type VDCC and Rho kinase (ROK) activities. Downregulation of Mycd and repressor element 1-silencing transcription factor leads to a loss of RyR and Kv channels in conjunction with upregulation of Elk-1-directed SRF targets, including TRPCs, and
KCa3.1, resulting in overactive store-operated Ca2+ entry and a proliferative phenotype. GFR growth factor receptors, Gq-R Gq-coupled receptor, PLC phospholipase C, SOS son-of-sevenless, Raf Ras-activated kinase, MEK mitogen-activated protein kinase/extracellular-signal-regulated kinase kinase, ERK extracellular-signal-regulated kinase, CaMK Ca2+/ CaM-dependent protein kinase, CaN calcineurin, CREB Ca2+/cyclic AMP response element binding protein, OPN osteopontin
Signal discrimination in the context of growth versus muscle differentiation is actively under investigation, and current evidence supports a model that includes gene specificity through CArG element degeneracy and due to selective expression of SRF coactivators that target SRF to select gene targets. The presence of a single conserved C or G nucleotide substitution in the A/T-rich central domain of the CArG element is found only in the promoters of smooth-muscle-specific genes, and when these elements are replaced by the c-fos CArG element, the differentiation genes are induced instead of downregulated in response to injury [63]. This mechanism of CArG degeneracy explains the ability of SRF to activate transcription of growth-specific promoters and suggests that under these conditions, the CArG elements controlling smooth-muscle-specific genes are inactive. The hypothesis that transcriptional coactivators might also play a role in SRF specificity was based on the observation that the level of SRF protein was not altered in response to vascular injury, nor did it change during the transcription of smooth-muscle-specific genes [64]. A bioinformatics screen of unknown cardiac and smooth-muscle-specific proteins identified myocardin, which became the first member of a family of myogenic transcriptional coactivators [65]. Myocardin forms a ternary complex with SRF bound to CArG elements on cardiac and smooth-muscle-specific gene promoters [64]. The selectivity of SRF gene targets has also been shown to be Ca2+-sensitive. Ca2+ influx through L-type Ca2+ channels has been shown to increase smooth muscle a-actin, SMMHC,
myocardin, and c-fos messenger RNA (mRNA) levels as well as the level of activity on their specific promoter elements [66]. Again, the question arises: How could the upregulation of genes that are so disparate in their function be simultaneously regulated by Ca2+? Although not completely clear, data suggest that in contrast to c-fos, the regulation of smoothmuscle-specific genes requires both Ca2+ influx through L-type Ca2+ channels and activation of the Rho-dependent protein kinase signaling pathway. Alternatively, regulation of growth-related genes can be induced by Ca2+ signals through L-type Ca2+ channels but also by ligands that activate IP3mediated release of Ca2+ from the SR Ca2+ store [66, 67]. The dependence of cofactor expression on Ca2+ signaling that is modulated by secondary signaling pathways allows for tight regulation of gene expression patterns in VSMCs. Taken together, these studies have revealed the existence of an intimate balance wherein vascular injury causes induction of MAPKs and Elk-1 binding to SRF on growth-related CArG elements, while simultaneously causing repression of myocardin expression to prevent activation of SRF on muscle-specific promoters (Fig. 2). These pathways have not been specifically studied in PASMCs, nor have they been examined in models of PAH or human pulmonary artery. Because these pathways are strongly affected by alterations in expression of Ca2+-regulatory channels and pumps known to be altered in PAH, it is likely that SRF selectivity is an important untested component of pulmonary artery remodeling in response to PAH.
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3.3 Ca2+/Cyclic AMP Response Element Binding Protein Initially identified in neurons, the Ca2+/cyclic AMP (cAMP) response element (CRE) binding protein (CREB) regulates transcription of hundreds of genes in all eukaryotic cells through its interactions with Ca2+/CREs on the promoter of gene targets [68]. CREB is activated through phosphorylation of serine 133 in its kinase-inducible domain, which promotes its binding to CREB-binding protein (CBP300) and other transcriptional coactivators necessary for the activation of transcription [69]. Many kinases are capable of phosphorylating CREB, including cAMP-dependent protein kinase, Ca2+/ CaM-dependent protein kinase II, and p90RSK [70, 71]. Inactivation of CREB through phosphatase activity can also be regulated by Ca2+ through Ca2+/CaM activation of calcineurin which indirectly leads to dephosphorylation and inactivation of CREB [38]. CREB and NFAT are inversely regulated by calcineurin; thus, its activity may define the dominant Ca2+-mediated transcriptional activity. In cultured VSMCs, CREB phosphorylation is induced by multiple Ca2+ signals, including L-type Ca2+ channel activation, agonist-mediated IP3 production, and blocking SERCA pumps to stimulate SOCE [42, 72]. Immediate early genes such as c-fos as well as cell-cycle-specific CRE-containing genes, including cyclins A and E, are upregulated following increases in the Ca2+ level [42, 73]. PDGF-stimulated expression of the migratory protein osteopontin has also been shown to be mediated by CREB-dependent binding to a functional CRE site within the osteopontin promoter [74]. A microarray analysis comparing transcription patterns in human VSMCs following stimulation of different sources of Ca2+ showed regulation of overlapping, yet distinct sets of genes, with SOCE activation of CRE-containing genes that reflected a growth phenotype [75]. In human PASMCs, the mitogenic effects of ATP have been partly attributed to CREB-dependent upregulation of TRPC4 expression and activity that results in an increase in SOCE and basal levels of cytoplasmic Ca2+ [76]. Thus, in VSMCs from different arterial beds, CREB activity correlates with a proliferative and migratory phenotype. Evidence for altered CREB function in intact tissue and arterial diseases has also been reported. Cerebral arteries from hypertensive animals exhibit elevated levels of VSMC Ca2+, phospho-CREB, and c-fos mRNA [77]. Interestingly, these effects are reversed by inhibition of L-type Ca2+ channels after arterial dissection, suggesting that the CREB activation may be a result of a defect in the “off” mechanism. The proliferative response to angiotensin II-induced hypertension or oxidative endothelial injury has also been correlated with increased levels of phospho-CREB and a proliferative response [78]. Furthermore, expression of dominant negative CREB constructs in an angioplasty model reduces neointimal formation and promotes apoptosis [79]. Transient
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in vivo ischemia also results in CREB phosphorylation and an upregulation of CRE-mediated BCL-2 expression [80]. Together, these results suggest a role for CREB in both survival and proliferation of VSMCs following injury. An exception to the correlation between CREB activity and the proliferative phenotype is a group of studies showing that hypoxia results in a loss of CREB [81]. This mechanism for CREB reduction was found to include long-term PDGF signaling through phosphoinositol 3-kinase, nuclear export of CREB, and proteasomal degradation [82]. The differences in these findings when compared with the evidence linking CREB to proliferation can be explained by the timing of CREB activation as it relates to gene regulation and effects on arterial phenotype. It is also possible that although CREB levels are reduced, phospho-CREB activity is still elevated.
3.4 Activating Protein-1 The activating protein-1 (AP-1) family of transcription factors is composed of multiple family members subclassed as Jun (v-Jun, c-Jun, JunB, and JunD), Fos (v-Fos, c-Fos, Fra1, Fra2, and FosB) or activating transcription factor (ATF2, ATF3/LRF1, B-ATF), which combine as homo- or heterodimers and bind to AP-1 binding sites (TGAG/CTCA) within the promoters of multiple genes. These transcription factors are the gene products of classical immediate early genes that are themselves regulated by CRE and AP-1 elements. AP-1 family members are upregulated within minutes of growth factor signaling through Ca2+ transients and/or Ras/extracellular-signal-regulated kinase (ERK) pathway activation and become a self-generating logarithmic on switch through generation of their own transcription. The kinase cascade is stopped by phosphatase activity, and AP-1 family members are rapidly removed by ubiquitin-mediated proteasomal degradation [83]. Although AP-1 sites are found in both growth and tissue-specific gene promoters, elevated levels of AP-1 family members have been directly associated with proliferative disorders, including vascular diseases and cancer (reviewed in [84, 85]). Ca2+ regulation of AP-1 signaling and cell proliferation through agonist and IP3-mediated SOCE has been demonstrated in several arterial cell models. Induction of the proliferation-associated protein osteopontin by UTP, angiotensin II, or PDGF is mediated by an AP-1 site bound by c-Fos and c-Jun in aortic VSMCs [86]. Stimulation of SOCE by depleting SR Ca2+ stores induces ERK activation and c-fos transcription in both cultured and intact cerebral VSMCs, and these activities are dependent on Ca2+ entry [72, 75]. ERK activity is elevated in cerebral arteries from hypertensive animals, and also in carotid arteries following balloon injury [87]. Arterial injury is also associated with increased levels of
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c-fos mRNA and AP-1 binding activity [88]. As with cancer, it is likely that the elevated ERK and AP-1 activity is due to defects in the off switch for the pathway, and thus investigations of Ras inactivation and phosphatase activity will be important for understanding the abnormality and developing therapies. AP-1 signaling in the pulmonary vasculature has both direct and indirect links to Ca2+ signaling, PASMC growth, and vascular remodeling. In PASMCs, upregulation of TRPC6 expression and cell proliferation in response to PDGF stimulation requires induction of Jun and STAT3 transcription factors. The increase in SOCE due to TRPC6 upregulation was found to be necessary for the growth trigger [24, 35]. The response of the pulmonary artery endothelial cells to hypoxia includes Ca2+ influx through SOCE and an increase in AP-1 binding activity. This AP-1 response leads to expression of mitogens by endothelial cells that ultimately influence PASMC growth [89]. These findings indicate a strong relationship between AP-1 signaling and Ca2+-mediated vascular remodeling that could have important implications in the development of pulmonary hypertension (Fig. 2). Pharmacologic agents that block PDGF receptors or MAPKs are currently used in the clinics for treatment of cancer, and these agents may also be important therapeutic agents in the future treatment of pulmonary vascular diseases.
4 Genes Altered in PAH Correlate with Altered Ca2+ Signaling and Cell Proliferation Microarray analysis is a powerful and efficient method to examine genes altered in specific diseases because of its ability to simultaneously measure the expression of thousands of genes from a single cell type or from an intact model with multiple cell types. The limitation is primarily at the analysis level owing to the multiple statistical comparison difficulties, and it is therefore important to validate selected results by other methods. This section will outline the results of four microarray studies performed on PASMCs that link PAH with alterations in ion channel homeostasis and regulation of cell proliferation/survival in human tissue [90, 91] and a mouse model of hypoxia-induced PAH [92, 93]. A microarray analysis performed by the Voelkel group used lung tissue obtained from patients with severe forms of PAH, including sporadic primary pulmonary hypertension (SPPH) and familial primary pulmonary hypertension (FPPH), a disease linked to germline mutations of the bone morphogenic protein (BMP) receptor II [94–96]. The lung tissue was compared with normal lung tissue from patients with other diagnoses for
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changes in gene expression [90]. Several genes were found to be significantly changed, with notable increases in Ca2+-regulated survival factors such as BCL-2 and FYN as well as ion channels, including the inward rectifier K+ channel (GIRK) and the voltage-gated Na+ channel (SCN1B) (Table 1). Downregulation was observed for the IP3 receptor type 3 Ca2+ channel (ITPR3), the voltage-gated K+ channel (KCNA), and caveolin 1 (CAV1) (Table 2). Cluster analysis unexpectedly found that the overall gene expression pattern from the FPPH lungs was more closely related to the normal lung profile than that of primary pulmonary hypertension (PPH) lungs. The limitations of this study were the small number of patient samples and the use of whole lung, which dilutes the pulmonary artery signal, but overall the changes support the hypothesis that severe PAH is associated with an environment that has a depolarized membrane potential and activation of survival signals. A study by the Yuan group compared patterns of gene expression in PASMCs derived from patients with IPAH and PASMCs from normal patients [91]. Because BMP-2 signaling is lost in severe forms of PAH, the authors also tested the effects of BMP-2 treatment on gene expression in PASMCs from control and IPAH sources. This study had the advantage that the tissue source was specific to pulmonary artery; however, the cells were exposed to culture conditions, and the control cells were not isolated, but commercially available cultured PASMCs. The results of the study indicated that BMP-2 has opposing effects on PASMCs from the lungs of subjects with IPAH when compared with normal subjects. BMP-2 was shown to cause a reduction in BCL-2 expression and induction of apoptosis in cultured PASMCs [89]. The upregulation of growth factors, ion channels, and signaling molecules important in Ca2+ signaling pathways, along with the downregulation of voltage-gated K+ channels and the gene that encodes a Ca2+-ATPase pump, strongly supports a role for Ca2+mediated regulation of proliferation and remodeling in pulmonary artery hypertensive disease (Tables 1, 2). There is also the suggestion that Ca2+-regulated transcription factors may be important not only in proliferation but also in the upregulation of antiapoptotic genes, i.e., BCL-2 and IP3 receptor type 3 [90, 91]. Additionally BCL-2 is upregulated in the lungs of subjects with FPPH and SPPH, suggesting a resistance to apoptosis in primary pulmonary hypertension [90]. Finally, there is a strong correlation between the Fantozzi and Geraci studies that suggests that mutations in BMP receptor II leads to altered signaling by BMP-2 and the upregulation of genes in PASMCs that favor the proliferative, antiapoptotic phenotype observed in PAH. Although data obtained from microarray analyses done on lung homogenates reveal important clues that explain some of the phenotypical changes observed in PAH, intrapulmonary arteries accounts for less than 10% of lung tissue. Thus, the
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Table 1 Representation of genes upregulated in different types of pulmonary arterial hypertension Function Gene Hypertension Signaling proteins/ transcription factors
BCL-2 (B-cell lymphoma 2) CALM1 (calmodulin 1) CAV2 (caveolin 2) CBP (CREB-binding protein) CREB (cyclic AMP response element binding protein-1) c-Fos EGR1 FYN (SRC-related oncogene) SRF (serum response factor) ATF3 (activating transcription factor) EDN-1 (endothelin-1) GIRK (inward rectifier K + channel) SCN1B (voltage-gated Na+-channel type Ib polypeptide) 5HT-2B (serotonin receptor subtype 2B) IP3 receptor 1 (inositol 1,4,5-trisphosphate receptor) EGF (GRB2) PDGF-b
Lung model
Reference
SPPH, FPPH IPAH IPAH IPAH IPAH IPAH IPAH SPPH, FPPH HIPH HIPH HIPH SPPH, FPPH SPPH, FPPH IPAH IPAH IPAH HIPH, IPAH
Intact [90] Cultured, BMP-2 [91] Cultured, BMP-2 [91] Cultured, BMP-2 [91] Cultured, BMP-2 [91] Cultured, BMP-2 [91] Cultured, BMP-2 [91] Intact [90] Mouse, hypoxia [92] Mouse, hypoxia [93] Mouse, hypoxia [93] Membrane receptors/ion Intact [90] channels Intact [90] Cultured, BMP-2 [91] Cultured, BMP-2 [91] Growth factors Cultured, BMP-2 [91] Mouse, hypoxia or [91, 92] cultured, BMP-2 TGF-b HIPH Mouse, hypoxia [92] Ca2+ binding/Ca2+ FKBP1a (FK506 binding protein 1A) HIPH Mouse, hypoxia [92] signaling S100A4 HIPH Mouse, hypoxia [92] BMP-2 bone morphogenetic protein-2, FPPH familial form of primary pulmonary hypertension, HIPH hypoxia-induced pulmonary hypertension, IPAH idiopathic pulmonary arterial hypertension, SPPH sporadic primary pulmonary hypertension, CREB cyclic AMP response element binding protein, EGF epidermal growth factor, PDGF-b platelet derived growth factor b, TGF-b transforming growth factor b
Table 2 Representation of genes downregulated in different types of pulmonary arterial hypertension Function Gene Hypertension Signaling proteins/ transcription factors Membrane receptors/ion channels
CAV1 (caveolin 1)
SPPH, FPPH
AGTR2 (angiotensin II type-2 receptor) ITPR3 (inositol 1,4,5-trisphosphate receptor type 3)
IPAH IPAH/SPPH, FPPH
Lung model
Reference
Intact
[90]
Cultured, BMP-2 [91] Mouse, hypoxia or [90, 91] cultured, BMP-2 SPPH, FPPH NT [90] KCNA (voltage-gated, shaker-related K+ channel) Enzymes ATP2A3 (Ca2+-transporting ATPase) IPAH BMP-2 [91] BMP-2 bone morphogenetic protein 2, FPPH familial primary pulmonary hypertension, IPAH idiopathic pulmonary arterial hypertension, NT no treatments, SPPH sporadic primary pulmonary hypertension
data obtained from whole-lung studies may not fully represent altered gene expression in intact PASMCs. Investigators from the Fink group thus used an animal model of hypoxiainduced PAH, followed by dissection of the PASMCs using laser scanning microscopy for microarray analysis [92]. This model has the advantage of specifically studying PASMCs and an increased number of samples and time points, but it has the disadvantage that it is not completely representative of the development of human PAH. The results of the study showed that multiple growth factors, including PDGF-b, as well as the transcription factor SRF are significantly upregulated following chronic hypoxia [92]. The McLoughlin group also used microarray analysis in a hypoxia-induced PAH mouse model, but they analyzed mRNA levels in whole lung and focused on expression patterns of overrepresented transcription-factor-binding sites
after different times of hypoxia exposure [93]. A cluster analysis of genes altered by hypoxia revealed a significant increase in the expression of genes that are regulated by CREB and AP-1 family members (Table 1), and this pattern correlated well with protein levels of phospho-CREB. Surprisingly, myocardin upregulation was also identified in this cluster, suggesting that the loss of myocardin may occur later in the development of PAH. The disadvantage of examining whole lung was countered by the advantage of multiple time points and further comparisons with transcription factor activity in human lung microvascular endothelial cells. ATF3, endothelin-1, and follistatin were the only genes validated, but other CREB targets related to hypertrophy, including eukaryotic translation initiation factors, would be interesting to follow in the future. Overall, this model supports the notion that the development of PAH is associated with
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induction of Ca2+-regulated transcription factors known to regulate VSMC growth. In conclusion, the microarray analyses presented here displayed patterns of gene expression in four different PAH models that favor a proliferative phenotype and are related to remodeling as a consequence of proliferation. The microarray study in cultured PASMCs from PAH patients shows an upregulation of genes that relate to cell growth and proliferation, and, taken together with the altered ion channel expression from the intact lung study, it is tempting to conclude that in PAH, dysregulated gene expression supports an environment that facilitates the activation of Ca2+-regulated transcription factors. It is also interesting to note that the genes altered in the hypoxia mouse models represent changes that would be expected as part of the immediate early gene response (i.e., growth factor alterations), whereas the analysis of human disease represents later effects following the immediate early gene response (i.e., ion channel alterations). A study of longer time points in the animal model and a study of healthy patients with the FPPH genotype may lead to better insights related to the role of Ca2+ signaling in the onset and progression of PAH.
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Chapter 22
Biochemistry and Cellular Mechanisms of Apoptosis in Vascular Smooth Muscle and Endothelial Cells Oliver Eickelberg and Fotini M. Kouri
Abstract Cell death plays a pivotal role in physiological development and tissue homeostasis. There are different ways for cellular death, arising from differential conditions and producing differential effects. The fact that perturbations in the death pathways can lead to diseases such as cancer, Alzheimer’s, atherosclerosis, and pulmonary vascular diseases reveals the essentiality of cell death for tissue homeostasis. This chapter briefly discusses the historical aspects and focus on the mechanisms of programmed cell death, or apoptosis, in pulmonary vascular smooth muscle and endothelial cells, which constitute the pulmonary vessel wall, in the context of health and disease. Keywords Apoptosis • Programmed cell death • Caspase • Apoptotic volume decrease • Pulmonary vascular remodeling • Cytochrome c • Mitochondria
1 Apoptosis, Retrospectively The statement “life would be impossible without death” could not be more appropriate to describe the pivotal role of cell death in physiological development and tissue homeostasis. There are different ways for cellular death, arising from differential conditions and producing differential effects. The fact that perturbations in the death pathways can lead to diseases such as cancer, Alzheimer’s, atherosclerosis, and pulmonary vascular diseases reveals the essentiality of cell death for tissue homeostasis [1]. This chapter will briefly discuss the historical aspects and focus on the mechanisms of programmed cell death in pulmonary vascular smooth muscle and endothelial cells, which constitute the pulmonary vessel wall, in the context of health and disease.
O. Eickelberg (*) Institute of Lung Biology and Disease (iLBD), Helmholtz Zentrum München, Ingolstädter Landstraße 1, 85764, Neuherberg/Munich, Germany e-mail:
[email protected] Apoptosis, or programmed cell death, is a common biological process. It is a kind of cellular suicide occurring when the organ or tissue demands it, leading to cell elimination. In concert with cell division, it is necessary for the regulation of cell number in a multicellular organism. Apoptosis is recognized to be a key mechanism for many physiological processes, especially during development, and its impairment contributes to disease. “Apoptosis,” derived from the Greek words apo, meaning “from,” and ptosis, meaning “falling,” means the “dropping or falling off.” The actual process was first described by the German scientist C. Vogt in 1842, but it was not until the end of the 1960s that programmed cell death was demonstrated by J.F.X. Kerr, while studying liver tissue by electron microscopy [2]. Furthermore, in 1972, Kerr et al. [3] characterized this novel phenomenon of programmed cell death, or apoptosis, which was distinct from the already known cell necrosis. The paper at that time received severe criticism regarding the term chosen to characterize this mode of cell death, since the word “apoptosis” was originally used to describe the falling of leaves. There were several constraints on whether this term was etymologically correct and should be used for this purpose. In addition, the fact that apoptosis was suggested to be a truly controlled process was hardly accepted. The apoptotic event was opposing the already known type of cell death called necrosis, which resulted from a tissue injury and led to plasma membrane breakage and release of intracellular components into the extracellular space, creating an inflammatory response. According to Kerr et al., there was much more to apoptosis than simply another way of cell death. With Kerr’s discovery of apoptosis, a novel field of research was born. Therefore, it did not take long for a vast number of studies to focus on the complexity of apoptotic cell death. To date, it is well established that the morphological signature of apoptosis includes cell shrinkage or apoptotic volume decrease (AVD), chromatin condensation, nuclear fragmentation, and blebbing of the plasma membrane [4]. The hallmark of an apoptotic cell is its DNA fragmentation, which originally was thought to take place at the linker region between the nucleosomes, creating 180 base pair fragments that have
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the characteristic morphology of a “DNA ladder” under gel electrophoresis [5]. In-depth analysis demonstrated that although it may vary among cell types, this is a two-phase process. The first phase includes DNA cleavage into around 50 kilobase pair fragments that undergo further cleavage to 180 base pair fragments in the second phase [6]. The final event of apoptosis is the formation of apoptotic bodies, which are cellular fragments, including parts of the nuclear lamina and the cytoskeleton, surrounded by the cell’s plasma membrane. An early critical feature of the apoptotic cell is that the phosphatidylserine in the plasma membrane, which normally faces the internal part of the cell, now is in contact with the extracellular space, allowing an easier detection of early apoptotic cells. The resulting apoptotic bodies are ingested rapidly either by neighboring cells, something common during embryogenesis, or by components of the innate immune system, such as macrophages and dendritic cells, using mechanisms similar to those used to recognize and remove pathogens, in the absence of an inflammatory response [7–9]. Most of the basic knowledge on apoptosis comes from extensive studies in the field of embryogenesis and development. There is a plethora of developmental processes where apoptosis participates. For example, programmed cell death is vital for correct development of the immune and nervous system. Apoptosis is responsible for 50% of neuronal cell inactivation during development, and one billion immune cells commit suicide on a daily basis in the body [10–12]. Mammary gland development is another example of the significance of apoptosis, where removal of detached epithelial cells from their surrounding extracellular matrix is crucial for the correct development of the gland. During the development of the nematode Caenorhabditis elegans as well, many cells undergo apoptosis frequently. Therefore, C. elegans was used as a genetic model to obtain more insight into the apoptotic machinery, which is finely tuned and highly conserved across species, from nematodes to humans [13].
2 Apoptotic Pathways The apoptotic cell-signaling pathway is responsible for converting a death stimulus, which can originate from the intracellular space (intrinsic pathway) or extracellular space (extrinsic pathway), into cellular effects. Apoptosis is divided into an initiation phase, involving the transmission of a specific death signal, and an execution phase, involving destruction of cellular compartments and structures and final cell death. After extensive studies, it is now known that apoptosis can have two different, well-conserved initiation phases, depending on where the death signal comes from. Each initiation phase leads to the activation of a different transduction pathway, however both lead to death. There is the
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intrinsic pathway, which utilizes mitochondria and the endoplasmic reticulum (ER), and the extrinsic pathway, which utilizes specific transmembrane receptors.
2.1 Intrinsic Pathway The intrinsic pathway is activated by a variety of signals, such as lack of growth factors, hypoxia, toxins, reactive oxygen species (ROS), and DNA damage. Although the exact mechanism downstream of these stimuli is not totally clear, the end effect is the activation of the intrinsic apoptotic pathway and eventually cell death. The central organelle for activation of the intrinsic pathway is the mitochondrion [14]. The loss of stability of the outer mitochondrial membrane leads to the release of proapoptotic factors, such as cytochrome c, which are confined within the inner and outer mitochondrial membranes under physiological conditions. Cytochrome c, except for its role in apoptosis, is also involved in the electron transport chain that leads to ATP synthesis. The electron transport chain consists of several protein complexes (I–V) found on the inner mitochondrial membrane. During the transportation of electrons from one complex to the next, protons are produced and pumped from the mitochondrial matrix to the intermitochondrial space. This causes the formation of an electrochemical membrane potential, known as the mitochondrial transmembrane potential (DYm). Upon release of cytochrome c, DYm is lost. In response to an apoptotic stimulus, due to opening of the permeability transition pore, a nonspecific channel on the outer mitochondrial membrane, there is a mitochondrial permeability transition. The permeability transition pore is composed of three components, the voltage-dependent anion channel (VDAC), the adenine nucleotide transporter, and the cyclophilin D. Much attention has been given to the VDAC because of its diverse functions. The VDAC protein family is composed of the VDAC1, VDAC2, and VDAC3 isoforms, which are located on the outer mitochondrial membrane. The presence of VDAC on the outer mitochondrial membrane is critical, since it regulates the energy synthesis via controlling the metabolite transport across the membrane. Downregulation of this protein can lead to growth arrest, due to reduction in the energy synthesis [15]. Recent studies have implicated the VDAC in the regulation of the apoptotic intrinsic pathway as well. It has been shown that a decrease in the expression levels of VDAC1, by small interfering RNA, is able to attenuate the proapoptotic effect of endostatin on endothelial cells [16,17]. Furthermore, VDAC1 expression levels are increased upon treatment of endothelial cells with endostatin and subsequent induction of apoptosis. In another study, depletion of VDAC1, again by small interfering RNA, reduced the cytochrome c release and
22 Biochemistry and Cellular Mechanisms of Apoptosis in Vascular Smooth Muscle and Endothelial Cells
further apoptosis in response to cisplatin [18]. On the other hand, overexpression of VDAC1 was able to induce apoptosis in a variety of cell types. In particular, in endothelial cells, VDAC1 overexpression induced the activation of caspase-9, increased ROS levels, and the endostatin-induced apoptotic effect [16,19]. Several potential mechanisms have been suggested for the mode of action of the VDAC. According to one, the VDAC undergoes oligomerization and allows the release of cytochrome c. This hypothesis was further substantiated by in vitro experiments [20]. The VDAC was shown to assemble into dimers, trimers, and tetramers [21]. The oligomerization of the VDAC was also shown by atomic force microscopy and NMR spectroscopy [22,23]. Evidence also suggests that the VDAC also has binding sites for members of the Bcl-2 family, Ca2+, as well as hexokinase, which is a glycolytic enzyme. Members of the Bcl-2 superfamily, such as the proapoptotic molecules Bax and Bak, can either interact with the VDAC and lead to the opening of the channel and further apoptosis, or oligomerize by themselves, forming a porelike structure, and translocate to the mitochondria, where they lead to cytochrome c release within minutes after the apoptotic stimulus [24,25]. Although it has been suggested that Bax and Bak form a pore, the actual mechanism remains elusive [26]. It has been shown that the regulation of VDAC activity and permeabilization of the mitochondrial outer membrane by Ca2+ occurs in a VDAC-dependent manner [27]. It is very interesting that the glycolytic enzyme hexokinase by binding to the VDAC is able to block apoptotic cell death [19]. Further evidence suggests that this could be the pathway utilized by cancer cells to escape from cell death and proliferate indefinitely [28]. After cytochrome c has been released from mitochondria, the next pivotal step of the intrinsic pathway is the formation of a receptor known as the apoptosome. The main structural unit of the apoptosome is the cytosolic adaptor protein Apaf-1, which when it senses cytochrome c binds to it. This event induces Apaf-1 oligomerization and formation of a wheel-shaped structure, the apoptosome, which will be used as a platform for signaling and apoptosis progression. The apoptosome formation is a complex process, which requires the presence of cytochrome c and ATP. To understand in depth how the apoptosome forms, it is necessary to understand the nature of Apaf-1 [29,30]. Apaf-1 is a multidomain protein, containing three functional regions. It has an N-terminal caspase-recruitment domain (CARD), a nucleotide-binding and oligomerization domain (NB-ARC), and a series of WD40 repeats at its C-terminal. The CARD region of Apaf-1 is able to bind the CARD region of caspase-9 and hence recruit it to the apoptosome. The WD40 repeats are responsible for the cytochrome c binding, and the NB-ARC region, which is found between the CARD and WD40 regions, controls the apoptosome formation. Studies have shown that the NB-ARC region is com-
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posed of an ATPase domain followed by a winged-helix domain and a superhelical domain. In the absence of an apoptotic signal, Apaf-1 is in a monomeric form, where the WD40 repeats confine it in an autoinhibitory state, by folding back on the rest of the protein. It is speculated that the CARD and NB-ARC regions take up a more compact conformation, inhibiting the protein oligomerization, and ATP is bound to the ATPase domain of the NB-ARC region, maintaining this compact conformation. In the presence of an apoptotic signal, cytochrome c is released from the mitochondria and binds to the WD40 repeat region of Apaf-1. This generates a conformational change of Apaf-1 and initiation of the apoptosome formation. There is release of the CARD and NB-ARC regions from the autoinhibitory state, where the CARD region interacts with the ATPase domain and the winged-helix domain of NB-ARC, hindering the recruitment of caspase-9 as well as the blocking the ATPase domain, which is essential for Apaf-1 oligomerization. The structure of the ATPase domain is homologous to the structures of domains found in the AAA+ family of ATPases, characteristic for their property to form oligomers [31]. The principle of this formation is that one ATPase domain settles next to the neighbor molecule, forming a circular structure; in the case of Apaf-1, this is the apoptosome. Although it is not completely clear, ATP appears to be linked to the conformational changes occurring during the apoptosome formation. This is substantiated by the observation that if the ATP binding site is mutated, the apoptosome cannot be formed in the presence of several different apoptotic signals. After the formation of the apoptosome, capsase-9 is recruited and activated. The activation of caspase-9 does not require proteolytic cleavage, but takes place by dimerization, which is a reversible process [32]. Caspase-9 activation has a domino effect, leading to the activation of caspase-3 and caspase-7, and the final phase of apoptosis, the execution phase.
2.2 Extrinsic Apoptotic Pathway The extrinsic pathway involves signal propagation via specific transmembrane receptors, using caspases as the effector molecules. Different death ligands interact with certain receptors on the cell membrane and lead to initiation of an intracellular signaling cascade, involving the activation of caspases [33]. Death receptors are members of the tumor necrosis factor (TNF) receptor superfamily, which all share structural similarities. The different ligands, among which FasL (or CD95L), TNF-a, and TNF-related apoptosis-inducing ligand (Apo2L or TRAIL) by interacting with the receptors Fas (or CD95), TNF receptor 1 (or TNFR1), death receptor 4 (DR4 or TRAILR1), and death receptor 5 (DR5 or
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TRAILR2), respectively, initiate the apoptotic pathway. All the above-mentioned receptors contain the cytoplasmic domain called the death domain, which is essential for the intracellular signal initiation. Furthermore, they all possess a cysteine-rich extracellular domain. TRAILR1 and TRAILR2 are highly homologous with no distinct functions. The ligand–receptor interaction can be regulated by decoy receptors that block the apoptotic signal transmission, due to lack of the death domain or even the whole cytoplasmic domain. For example, the cellular effects of the FasL–Fas interaction are inhibited by the soluble decoy receptor called DcR3. Furthermore, the cell-bound decoy receptors TRAILR3 and TRAILR4 are a drag on the TRAIL–TRAILR1/TRAILR2 signal transduction [34]. The Fas receptor is the most well described receptor from the family of the TNF receptors, and one of its best known functions is the initiation of apoptosis. Binding of FasL to this receptor, which exists in a homotrimeric complex, sparks off the recruitment of adaptor proteins in the intracellular space, such as the Fas associated via death domain (FADD) and the further requisition of inactive procaspases, such as procaspase-8 and procaspase-10, leading to the formation of the death-inducing signaling complex (DISC). First, the death domain of FADD interacts with the death domain of the different cell receptors and, next, the death effector domain (DED) of FADD recruits the initiator procaspase-8 or procaspase-10. The following step in the process of apoptotic signal transmission is the activation of the procaspases to active enzymes. Two different hypotheses have been suggested over the years concerning the mechanism of caspase activation involved in the extrinsic pathway. The first hypothesis suggests that the close proximity of the inactive procaspases within the DISC induces their autoproteolysis and thus activation [35]. According to the second hypothesis, the close proximity of the procaspases in the DISC causes activation, by dimerization. The activated caspase-8 or caspase-10, in turn, will activate the downstream executioner caspases, and finally provoke cell death. Caspase-8 or caspase-10 can be inhibited by the cellular FLICE-like inhibitory protein cFLIP by DED–DED interaction. cFLIPL cFLIPR, and cFLIPS [36] are different cFLIP isoforms. cFLIPR and cFLIPS have been shown to prevent the caspase-8 activation owing to competition with them for FADD binding. The role of the third isoform, cFLIPL, in caspase activation has not yet been clearly established. Some studies have shown that cFLIPL, like cFLIPR and cFLIPS, restrains caspase activation. On the other hand, it has been suggested that it assists procaspase-8 activation and thus is considered a proapoptotic factor. The latter suggestion is supported by the fact that the cFLIP knockout mice have the same phenotype as caspase-8- and FADD-deficient mice, which is death at embryonic day 10.5, due to heart failure [37]. Experiments using different proteasome inhibitors revealed that cFLIP undergoes proteasomal degradation, and Itch is the ubiquitin
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E3 ligase responsible for this [38]. Itch itself is activated by Jun N-terminal kinase (JNK)-dependent phosphorylation, suggesting that the JNK signaling cascade can have a proapoptotic effect [39]. There are also other regulators of DISC formation, for example, protein kinase C, which can block FADD recruitment [40], mitogen-activated protein kinase [41], and protein kinase B [42]. Once caspase-8 or caspase-10 has been activated, there are two paths it can go through. The first one is to activate the executioner caspase-3 and this leads to cell death. The alternative path is the activation of Bid, a Bcl-2 family member, which will induce the release of proapoptotic factors from the intermediate space of mitochondria and thus bring into play the intrinsic apoptotic pathway as well [43]. Thus, cells are classified as type II and type I according to whether the extrinsic pathway requires the activation of the intrinsic pathway to cause cell death or not. In type I cells, possibly owing to excess activation of caspase-8, the cells do not require the intrinsic pathway for apoptosis. In contrast, in type II cells, the DISC formation is weaker, and it requires effector caspase activation via the mitochondria for the execution of cell death. The extrinsic and intrinsic pathways are connected via Bid, which is a caspase-8 substrate.
3 Apoptic Effector Proteins 3.1 Caspases: The Suicide Proteins Regardless of which initiation pathway is activated, apoptosis is a two-step process leading to the activation of highly specialized proteins, the caspases, which are able to amplify the signal and lead to death. Caspases are cysteine proteases that cleave proteins at specific aspartate residues. The fact that caspases themselves are regulated by proteolytic cleavage ensures their rapid activation under different stress conditions [44]. The role of caspases in apoptosis became evident for the first time with the discovery that ced-3, the prototype gene responsible for the induction of programmed cell death in C. elegans, is homologous to the mammalian caspases, and in particular to ICE/caspase-1, which is the protease responsible for the activation of interleukin-1b (IL-1b) and interferon-g [45]. The role of caspases as the main effector enzymes in apoptosis was extensively studied and substantiated by a series of observations. For example, overexpression of caspases in cell lines can induce apoptosis. Experiments using blocking peptides confirmed that caspases are required for most types of apoptosis. Most of the information available today on caspase function comes from studies in caspase-deficient mice [46] (Table 1). Such studies have shown that although caspases are the main regulators of apoptosis, they also have
22 Biochemistry and Cellular Mechanisms of Apoptosis in Vascular Smooth Muscle and Endothelial Cells Table 1 Effects of specific caspase depletion in transgenic mice Caspase knockout
Phenotype
Caspase-1
No IL-1b and IL-18 activationEffects on cytokine activation Oocyte accumulation Defects in brain development. Mice die 3 weeks after birth Defects in development of heart, muscle, and the hematopoietic system Defects in brain development Effects on the immune system homeostasis Effects on cytokine activation and caspase-1 activation Normal development, reduced skin hydration levels
Caspase-2 Caspase-3 Caspase-8 Caspase-9 Caspase-10 Caspase-11 Caspase-14
apoptosis-independent functions. This is why caspases were classified according to their function as proinflammatory and proapoptotic. In humans, for example, caspase-1, caspase-4, and caspase-5, have no role in apoptosis; instead, they control cytokine maturation, such as IL-1b, and therefore are known as proinflammatory. On the other hand, caspase-3, caspase-6, caspase-7, caspase-8, caspase-9, caspase-10, and caspase-12 are known to be a part of the apoptotic machinery and hence are known as proapoptotic [47]. More than 14 family members have been identified up to now. These proteases are initially synthesized as procaspases, in an inactive form, containing a prodomain and a protease domain. The length of the prodomain determines the class of the caspase. Those caspases with long prodomains belong to the class of initiator caspases. For example, caspase-8 and caspase-10 contain two tandem repeats of the DED in their prodomain, which is essential for their function during extrinsic apoptotic cell death. Another group of caspases with long prodomains, such as caspase-1, caspase-2, caspase-4, and caspase-9, contain a CARD, which also greatly influences their function and activity. The caspases with short prodomains, such as caspase-3, caspase-6, and caspase-7 are considered to be downstream effector or executioner caspases that need the initiator caspases to activate them, by proteolytic cleavage. On the other hand, the initiator caspases do not require a proteolytic cleavage to become activated. Their activation depends on the formation and activity of recruitment platforms, which are made of scaffold proteins. Proinflammatory caspases in a way similar to proapoptotic caspases undergo such a kind of activation, in a recruitment platform, called inflammasome, which will eventually lead to an inflammatory response [48]. The role of executioner caspases is to disable processes responsible for cell homeostasis and damage repair and eventually lead to the complete destruction of the dying cell, by caspase-activated DNases that will enter the nucleus and cleave the DNA, leading to the characteristic morphology of a “ladder” upon gel electrophoresis [49].
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Caspases are signaling proteases that are destined for protein cleavage, rather than protein destruction. Usually they do not destroy a protein’s structure, but rather modify it, by creating at maximum a couple of proteolytic cuts at specific aspartate residues. The best example of the proteolytic action of caspases is the caspase signaling itself, where initiator caspases, by irreversible cleavage, activate their downstream effector caspases. Hence, when the executioner caspases are activated, there is no going back; it is an irreversible process leading to the dismantling and final death of the cell [50].
3.2 Inhibitors of Apoptosis Apart from the proteolytic caspase pathway, caspase activity is also regulated by a number of inhibitors of apoptosis (IAP). Most of the IAP have ubiquitin E3 ligase activity, targeting caspases for proteosomal degradation [51]. The best characterized examples are IAP-1, IAP-2, and XIAP. The latter is the stronger inhibitor for activated effector caspase-3 and caspase-7 as well as for the caspase-9, sequestering it in an inactive state. It has been proposed that XIAP might also regulate caspase activity in a rather indirect manner. More specifically, XIAP catalyzes the polyubiquitination of caspase-3 and thus induces its degradation. Similarly, IAP-2 has been shown to monoubiquitinate caspase-3 and caspase-7, which may not lead to their degradation, but affects their stability, in vitro. Last, but not least, survivin is an apoptotis inhibitor, whose role is to interact with XIAP and enhance its antiapoptotic action. De novo survivin synthesis has been linked to cancer progression. The inhibitory action of IAP can be restrained in apoptotic cells by the mitochondrial proteins Smac/Diablo, which when released from the mitochondria accelerate cell death by interrupting the IAP–caspase interaction.
3.3 The Bcl-2 Superfamily Proteins of the Bcl-2 family are the main regulators of the intrinsic apoptotic pathway [52]. The Bcl-2 family is classified into three subfamilies all sharing four domains, the Bcl-2 homology domains (BH1–BH4). The presence of each of these domains is essential for their function. The first Bcl-2 subfamily comprises the antiapoptotic molecules Bcl-2, Bcl-XL, Bcl-w, Mcl-1, A1, and Bcl-B, all of which have the four BH domains. The next subfamily consists of the proapoptotic molecules Bax, Bak, Bok, and Bcl-rambo, which have BH1, BH2, and BH3 domains. The last subfamily is composed of the proapoptotic proteins Bid, Bim, Bad, Bik, Puma, and Noxa; the members of this subfamily share homology only at the BH3 domain, and this is why they are
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also referred to as “BH3-only proteins.” Each subfamily has a different function, which depends on the presence or absence of the different BH domains, although, all Bcl-2 family members control the release of proapoptotic factors from the mitochondria, each in a different way. The antiapoptotic proteins Bcl-2 and Bcl-XL are found on the outer mitochondrial membrane, stabilizing the mitochondrial integrity. Their function is counteracted by the proapoptotic Bcl-2 family members Bax, Bak, and Bok, whose aim is to destabilize the outer mitochondrial membrane and cause the release of cytochrome c and other proapoptotic factors, such as Smac/Diablo, AIF, heat shock protein 60, and endonuclease G, located in the mitochondrial intermembrane space. Cytochrome c and Smac/Diablo are involved in caspase activation, whereas endonuclease G and AIF participate in caspase-independent apoptosis-induced nuclear changes. The function of the third subfamily of proteins, containing the BH3 domain only, target the antiapoptotic molecules Bcl-2 and Bcl-XL, where they neutralize their action, and thus indirectly activate Bax, Bak, and Bok. Examples of such BH3-only proteins include Puma, Noxa, and Bad. On the other hand, Bid and Bim directly activate Bax and Bak. Thus, when caspase-8 activates Bid by proteolysis, during the extrinsic apoptotic pathway, its cleaved form translocates to the mitochondria and leads to the activation of the proapoptotic Bax and Bak and thus initiation of the intrinsic pathway. The remarkable characteristic of the Bcl-2 family members is that they exert effects on each other by forming stable heterocomplexes. Not so long ago it was discovered that caspase-10 can also activate Bid, but via cleavage at another site, resulting in a smaller cleaved form of Bid. It is not clear though whether this differential Bid cleavage happens after the initiation of the extrinsic pathway, and whether it affects Bid’s mode of interaction with the other proapoptotic molecules.
3.4 p53, DNA Damage, and Apoptosis Apart from the Bcl-2 superfamily of proapoptotic factors, several other factors have been shown to regulate the mitochondrial outer membrane permeabilization; such an example is p53. The transcription factor p53 can induce apoptosis by regulating the expression of the proapoptotic BH3-only protein Puma. Further, it has been shown that p53 can provoke apoptosis, via direct interaction with Bax and Bak or even by binding to Bcl-2 and Bcl-XL, and inhibiting their function [53]. Other interesting findings suggest that the nuclear protein Ku70, for example, apart from DNA repair also translocates to the cytosol and inhibits Bax activation. Furthermore, the nuclear protein TR3 also translocates to the cytoplasmic area and interacts with Bcl-2 and thus promotes apoptosis. It has
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also been reported that histone 1.2 is released from the nucleus after DNA damage, interacts with Bcl-2, and causes mitochondrial outer membrane permeabilization.
4 Ion Channels and Apoptosis As already described, one of the morphological signatures of apoptosis is cell shrinkage or in other terms AVD, a process which anticipates caspase activation and cytochrome c release [54]. The process of cell shrinkage depends on ion trafficking through the plasma membrane, and in particular K+ locomotion. Under basal conditions, cytoplasm contains high concentration of K+ ions, which has to be preserved for the maintenance of the cell’s volume. A high concentration of K+ ions is required for the suppression of caspase activity. The intracellular potassium levels, and therefore the cell volume, are regulated by the Na+–K+-ATPase pump, and the K+ channels, leaving the intracellular space with a high concentration of K+ and a low concentration of Na+. On the other hand, in the extracellular space, there are low levels of K+ and high levels of Na+. The opening of K+ channels will cause efflux of K+ ions to the extracellular space, subsequent Cl- release, and finally water leaves the cell via the aquaporins, to maintain the balance between the intracellular and the extracellular space. This causes AVD and imminent apoptosis. There are four different classes of K+ channels, the voltage-gated K+ (Kv) channels, the Ca2+-activated K+ (KCa) channels, the two-pore-domain K+ (K2P) channels, and the inward-rectifier K+ (KIR) channels. Several studies in different cell types have shown that Kv channels are essential for programmed cell death. Kv channels apart from regulating AVD in the early steps of apoptosis, are also able to inhibit caspase activity immediately after the apoptotic stimulus reaches a cell, since loss of intracellular K+ ions leads to activation of caspases [55]. It is now clear that K+ channels are a target of the antiapoptotic factor Bcl-2. Thus, Bcl-2 apart from acting on Bax and Bid also discourages apoptosis progression, by inhibiting Kv channels. Furthermore, the decrease in the level of K+ leads to an overload of cytoplasmic Ca2+, which leads, for example, to increased vasoconstriction of the pulmonary vascular smooth muscle cell and subsequent overproliferation.
5 Endoplasmic Reticulum and Cellular Apoptosis Many studies have indicated that the ER plays a major role in apoptosis induction as well as in other types of cell death, such as autophagy [56]. The ER is the place where proteins
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are folded and secreted proteins undergo posttranslational modifications. In the presence of ER stress leading to alterations in the ER environment, for example, due to oxidative stress, or accumulation of unfolded and misfolded proteins, the unfolded protein response (UPR) pathway is activated, trying to resolve these changes. Three ER kinases participate in this process, protein-kinase-like ER kinase (PERK), inositol-requiring kinase 1 (IRE1), and activating transcription factor 6 (ATF-6). The UPR is mainly a survival pathway; however, if the problem of protein misfolding remains unresolved, it serves as a proapoptotic machinery. The kinases PERK, IRE1, and ATF-6 are involved in both the antiapoptotic and the proapoptotic pathways. In the latter case, it has been shown that PERK and ATF-6 can induce the expression of C/EBI homologous protein (CHOP), which decreases the expression levels of the antiapoptotic factor Bcl-2, and leads to the upregulation of proapoptotic genes [57,58]. Further studies have shown that IRE1, via TRAF2 activation, is able to induce the JNK signaling cascade, and subsequent apoptosis via phosphorylation of key proteins [59]. In most of the cases, the ER-stress-induced apoptosis is regulated by the Bcl-2 family of proteins [59]. It has been also suggested that a pathway similar to the extrinsic and intrinsic pathways takes place at the ER with key initiator molecules, the murine caspase-12 and the human caspase-4. Briefly, activation of caspase-12 or caspase-4 leads to the activation of caspase-9 or caspase-3 in a mitochondria-independent manner [60]. Thus, it has been shown that the murine caspase-12 is indeed involved in the apoptotic cell death. The same is not valid for the human caspase-12. Evidence suggests that human caspase-12 is involved in immune response [61] rather than apoptosis regulation. On the other hand, human caspase-4 is partially located at the ER and is only activated in response to ER stress [60]. Similar pathways have been shown to activate murine caspase-12 and human caspase-4. It is well known that ER stress causes release of the ER-stored Ca2+, leading to an increase in the cytoplasmic Ca2+ concentration and a later increase in the calpain expression levels. Thus, caspase-12 cleavage and subsequent activation have been correlated to calpain activation, induced by the release of ER-stored Ca2+. Further, it has been shown that calpain inhibition or Ca2+ chelation block caspase-12 activation in a series of different experimental settings [62]. Caspase-7 has also been shown to activate caspase-12. ER stress leads to the recruitment of caspase-7 to the ER, where it forms a complex with caspase-12 and the ER chaperone GRP78. Members of the Bcl-2 superfamily also regulate caspase-12 activity. Independent studies have shown that caspase-12 activation occurs in a Bak/Bax-dependent manner. ER-stresstargeted Bak activates caspase-12 in a caspase-7-independent but Ca2+-dependent mode. Less is known about the caspase-4 activation process; however, it is thought that it follows an activation path similar to that of caspase-12.
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Since caspase-12 and caspase-4 are not ubiquitously expressed, in many cases, ER-stress-induced apoptosis happens in a caspase-9-dependent pathway. It also should be stated that many studies have shown that many ER stressors lead to cytochrome c release and thus involve the intrinsic pathway in apoptosis induction. This has been further supported by the fact that Apaf-1 loss inhibits ER-stress-induced apoptosis.
6 Apoptotic Cell Execution and Clearance Finally, during the execution phase of apoptotic cell death, there is proteolysis of specific proteins, such as inhibitors of apoptosis, or other caspases, as well as proteins, which when cleaved lead to the characteristic morphology of apoptotic cells, such as fragmentation of the DNA, membrane blebbing, or formation of apoptotic bodies. Examples of proteins are ICAD/DFF45, the inhibitor of the nuclease responsible for the DNA degradation, nuclear lamina, as well as cytoskeletal regulators [63]. After understanding how important cellular apoptosis is, as well as the diversity of cellular mechanisms controlling this process, it is equally important to get a deeper insight to the process of clearance of the apoptotic bodies, after the cell has committed suicide [64]. The initiating step in this process is the recognition of the apoptotic cell that needs to be removed by either neighboring cells or macrophages and dendritic cells. Thus, the apoptotic cell undergoes some changes that mark it as ready for removal. After the apoptotic cell has been internalized by phagocytes, it goes through a “maturation process” at highly acidified membrane-bound structures called phagosomes, which eventually will fuse with the highly acidic lysosomal structures and lead to complete apoptotic cell destruction. Studies have shown that defects in the removal process of apoptotic bodies can lead to autoimmune disease, for example. The process of apoptotic cell phagocytosis, also known as efferocytosis, is a rather complex and challenging one. In a more detailed view of it, specific plasma membrane receptors on the mammalian phagocyte, such as BAI1, LRP1, MEGF10, TIM4, and MER, are able to recognize the apoptotic cell, in a not that well understood process. Two different pathways have been discovered in both mammals and C. elegans. One pathway suggests that the brain-specific angiogenesis inhibitor 1 (BAI1) on the phagocyte plasma membrane can bind to phosphatidylserine on apoptotic cells and induce their internalization by forming a complex with the guanine nucleotide exchange factors ELMO and Dock180, and further with the small GTPase Rac, regulating the phagocyte cytoskeleton and resulting in better internalization [65]. The second pathway suggests that the protein careticulin on the apoptotic cells induces apoptotic cell engulfment in an low density lipoprotein receptor-related protein (LRP)-dependent manner.
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Upon engulfment and internalization, the apoptotic cell is surrounded by the phagocyte plasma membrane, forming the phagosome. For the degradation process to start, the phagosome needs to reach a sufficiently low pH so that the acidic proteases, belonging to the cathepsin family, can be activated and proceed to the degradation step. The degradation step is very critical because the degraded parts of the cell will be used for presentation on the phagocyte surface via the major histocompatibility complex class I, a process required for the maintenance of self-tolerance. The acidification process of the phagosome is a two-step process. First, there is a small reduction in the pH, which is followed by a translocation of the V-type ATPases into the phagosome, to cause further acidification of its contents. These proteins seem to come with cathepsins from the trans Golgi network. It has been suggested that V-type ATPases might also have a further role in phagosome maturation; however, what this might be is not completely clear and well understood. Most of the knowledge on apoptotic cell clearance and phagosome maturation comes from genetic studies focusing on the receptor-mediated endocytosis in C. elegans, which is responsible for the internalization of particles smaller than 0.5 mm. It has been suggested that the protein dynamin as well as the members of the Rab GTPase family, RAB-5 and RAB-7, whose activity depends on the presence of GTP, are involved in this process. The final step is the acquisition of lysosomal markers such as lysosome-associated membrane proteins 1 and 2 and subsequent fusion to the lysosome, where complete apoptotic cell degradation takes place.
7 Pulmonary Arterial Hypertension and Apoptosis It should be quite clear by now that apoptosis is a very well controlled process. Tissue homeostasis requires a balance between cellular proliferation and apoptosis. Failure to do so inevitably leads to disease. In the context of the pulmonary vasculature, an imbalance between apoptosis and proliferation has been linked to the development of pulmonary arterial hypertension (PAH). PAH is a rare but devastating disease with an annual incidence, one to two per million of population. The hallmark of PAH is excessive vascular remodeling in the small pulmonary arteries, loss of capillaries, and, in severe PAH, plexiform lesions. The process of vascular remodeling is very well orchestrated, with all the cell types of the vessel wall participating. In particular, there is unconfined pulmonary artery smooth muscle cell (PASMC) proliferation resulting from pulmonary artery endothelial cell (PAEC) injury or dysfunction. Extensive research has taken place aiming to elucidate the causes and pathways leading to
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PAH; however, the pathological mechanism still remains unclear. Substantial studies have indicated that endothelial cell dysfunction might be the trigger for the initiation of this disease. Different stimuli have been suggested to cause endothelial cell dysfunction, such as continuous shear stress and strain, an impaired bone morphogenetic protein (BMP)/ transforming growth factor-b (TGF-b) signaling, the proproliferative protein survivin, KV channels, hypoxia, and ROS. In more detail, the discovery that mutations in the bmpr-2 gene [66,67], a member of the TGF-b receptor superfamily, are found in the majority of familiar PAH cases as well as in some sporadic PAH cases turned the attention of researchers toward the BMP/TGF-b signaling cascade. It was thus demonstrated that the BMP signaling, and in particular that of BMP-4, induces proliferation in PAECs and apoptosis in PASMCs. The presence of mutations in the bmpr2 gene, leading to impaired BMP signaling, provoked PAEC apoptosis in a caspase-8- and caspase-9-dependent manner, and PASMC proliferation, a phenotype resembling the PAH development [68]. There is further evidence that the impaired BMP signaling leads to increased TGF-b response, which is responsible for the uncontrolled PASMC proliferation, in the animal model of monocrotaline-induced pulmonary hypertension [69]. Further evidence suggests that shear stress in the pulmonary vasculature induces endothelial cell apoptosis and thus leads to capillary loss and contributes to uncontrolled PASMC proliferation in the larger vessels. On the basis of such studies, apoptotic endothelial cells release cytokines such as TGF-b1, platelet-derived growth factor, and vascular endothelial growth factor, which regulate PASMC proliferation and apoptosis, respectively, in PAH [70]. In addition, there is convincing evidence that the inhibitor of apoptosis, survivin, is de novo expressed in both the PASMCs and the PAECs in remodeled vessels of PAH patients, indicating that inhibition of apoptosis could be responsible for the uncontrolled proliferation of the vessel wall cells, and thus PAH development [71]. Furthermore, there is a significant decrease in the expression of KV channels in tissue from PAH patients, which induces PASMC proliferation, via the increased intracellular K+ levels, which is known to inhibit caspase activity. As mentioned before, KV channels are important for the volume decrease of apoptotic cells. The decrease of KV channel expression induces efflux of Ca2+ ions, severe vasoconstriction, and increased PASMC proliferation. Hypoxia, which occurs during pulmonary hypertension development, regulates K+ ion concentration by inhibiting several potassium channels [54]. Hence, under chronic hypoxia, reduction of K+ ions and subsequent increase of Ca2+ concentrations leads to inhibition of apoptosis and increased proliferation of PASMCs. Although the role of ROS in these processes is still controversial, a perturbation in ROS signaling also plays a role in disease development owing to increased mitochondrial
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hyperpolarization followed by PASMC depolarization and subsequent increased vasoconstriction and excessive proliferation, a process which is also common in cancer [72].
8 Conclusion Apoptosis is a very well controlled process, essential for cellular and tissue homeostasis. It is clear that disturbances in the apoptotic pathway can lead to disease. In the pulmonary vasculature, for example, an imbalance between cellular proliferation and apoptosis in the vascular cells leads to PAH. As already described, there are several factors that regulate these processes, leading to disease. The next step would be to target such factors and such cellular processes as a therapeutic strategy for pulmonary hypertension.
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Chapter 23
The Coagulation Cascade and Its Regulation James T.B. Crawley, Jose R. Gonzalez-Porras, and David A. Lane
Abstract Under normal conditions the blood circulates freely within the confines of the vascular system, carrying oxygen, nutrients, and hormonal information around the body and removing metabolic waste. If the blood gains access to extravascular sites or if the vasculature becomes pathologically challenged, the processes associated with hemostasis may be activated. Employing finely regulated positive- and negative-feedback loops, hemostasis represents the process responsible for both generating and regulating the plug that prevents blood loss following vessel injury. The components of hemostasis are both complex and heterogeneous, consisting of cell-associated and plasma-borne proteins, glycosaminoglycans, and platelets. To ensure the maintenance of both blood fluidity and yet also vascular integrity, the hemostatic response must be fast, and yet specifically localized to the site of vessel damage. In this way, the likelihood of further challenges being imposed upon the vascular system may be minimized. In the event of vascular injury, circulating platelets are rapidly recruited to the damaged region. These platelets become activated and aggregate, forming an unstable platelet plug (primary hemostasis) that immediately limits the loss of blood. The coagulation cascade is initiated simultaneously. Blood coagulation ultimately results in the deposition of fibrin, a molecular scaffold that consolidates the platelet plug and allows tissue repair mechanisms to act. This chapter focuses on the coagulation system and its regulation. An important protease generated during coagulation is thrombin, a serine protease with numerous substrates. As the physiological end points of coagulation are the fibrin clot and platelet activation, which are both determined by thrombin, regulation of coagulation can be viewed in its simplest form as the control of the generation and activities of thrombin. Keywords Haemostasis • coagulant • thrombin • thrombosis • platelet • pulmonary embolism J.T.B. Crawley (*) Department of Haematology, Imperial College London, Hammersmith Hospital Campus, 5th Floor Commonwealth Building, Du Cane Road, London W12 ONN, UK e-mail:
[email protected] 1 Introduction Under normal conditions the blood circulates freely within the confines of the vascular system, carrying oxygen, nutrients, and hormonal information around the body and removing metabolic waste. If the blood gains access to extravascular sites or if the vasculature becomes pathologically challenged, the processes associated with hemostasis may be activated. Employing finely regulated positive- and negative-feedback loops, hemostasis represents the process responsible for both generating and regulating the plug that prevents blood loss following vessel injury. The components of hemostasis are both complex and heterogeneous, consisting of cell-associated and plasma-borne proteins, glycosaminoglycans, and platelets. To ensure the maintenance of both blood fluidity and yet also vascular integrity, the hemostatic response must be fast, and yet specifically localized to the site of vessel damage. In this way, the likelihood of further challenges being imposed upon the vascular system may be minimized. In the event of vascular injury, circulating platelets are rapidly recruited to the damaged region. These platelets become activated and aggregate, forming an unstable platelet plug (primary hemostasis) that immediately limits the loss of blood. The coagulation cascade is initiated simultaneously. Blood coagulation ultimately results in the deposition of fibrin, a molecular scaffold that consolidates the platelet plug and allows tissue repair mechanisms to act. This chapter focuses on the coagulation system and its regulation. An important protease generated during coagulation is thrombin, a serine protease with numerous substrates. As the physiological end points of coagulation are the fibrin clot and platelet activation, which are both determined by thrombin, regulation of coagulation can be viewed in its simplest form as the control of the generation and activities of thrombin.
2 Overview of Coagulation The vasculature is lined by a single layer of endothelial cells. These cells do not normally support the initiation and propagation of coagulation owing to the presence of anticoagulant
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Fig. 1 Coagulation cascade. Coagulation is initiated upon vessel damage, which leads to the exposure of tissue factor (TF) to plasma clotting factors (1). TF–activated factor VII (FVIIa) can activate factor X (FX) and factor IX (FIX) (2). Activated FX (FXa) activates prothrombin inefficiently (3) leading to the generation of trace amounts of thrombin. Thrombin can then activate factor VIII (FVIII) and factor V (FV) (4 ), which function as nonenzymatic cofactors for activated FIX (FIXa) and FXa, respectively. FIXa-activated FVIII (FVIIIa) catalyzes the conversion of increased quantities of FXa (5 ), which in conjunction with activated FV (FVa) leads to the enhanced generation of thrombin (6 ). Thrombin at the site of vessel damage converts fibrinogen to fibrin, which is the molecular scaffold of a clot (7 ). Abbreviation: ProT, prothrombin
molecules on their surfaces. The extrinsic or tissue factor (TF)dependent pathway of coagulation represents the major route by which thrombin generation is initiated in response to vessel damage (Fig. 1). Coagulation is activated by the exposure of subendothelial TF to the blood. TF is an approximately 45 kDa integral membrane protein, and is the primary initiator of blood coagulation and, unlike most other procoagulant factors, it does not require proteolytic activation [1]. TF is considered to be normally located at extravascular sites that are not usually exposed to the blood (i.e., on adventitial fibroblasts and variably on vascular smooth muscle cells) [2]. Therefore, only at sites of vascular injury or endothelial disruption may the blood encounter TF-presenting cells, and in turn activate the coagulation cascade. TF is also expressed in a cell-specific manner within certain organs (i.e., lungs, brain, heart, testes, uterus, and placenta). TF in these locations may provide further hemostatic protection against vascular injury in these organs. More recently, circulating microparticle-associated TF has also been identified in blood and implicated in hemostatic plug development and in thrombus formation [3, 4]. Following mechanical injury to the vasculature, blood (containing plasma clotting factors) gains access to cellular and
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matrix components that are normally separated from the blood by the endothelial barrier. The inactive zymogen, factor VII (FVII), circulates in plasma at a concentration of approximately 10 nM. A fraction (about 1%) of total plasma FVII circulates in a potentially active form, FVIIa [5]. TF binds FVIIa with high affinity. The interaction between TF and FVIIa occurs across an extensive interface between the two proteins [6]. This binding reaction occurs in a 1:1 stoichiometry and induces an allosteric change in the FVIIa serine protease domain that modulates its activity (rather than its substrate-binding function) [7]. It is for this reason that although FVIIa can bind its substrate, factor X (FX) (at least in vitro), in the absence of TF, activation of FX does not occur [8]. The trace amount of TF–FVIIa that forms following the exposure of TF-presenting cells to the blood activates the extrinsic pathway (Fig. 1). TF–FVIIa activates the plasma zymogens factor IX (FIX) and FX [9]. Additional TF–FVIIa may then be generated locally from inactive TF–FVII complexes (as TF also binds to FVII with high affinity), either by autoactivation or through positive-feedback loops involving activation by activated FX (FXa) or activated FIX (FIXa). The limited quantities of FXa that are generated via this route facilitate the inefficient conversion of trace quantities of prothrombin to thrombin. The low concentrations of thrombin that arise enable the feedback activation of factor VIII (FVIII) and factor V (FV), nonenzymatic cofactors in the tenase and prothrombinase complexes, respectively [10]. The major site of thrombin generation is on the membrane of activated platelets, which provide a negatively charged surface upon which the components of the coagulation cascade assemble. Here, the prothrombinase complex, consisting of FXa, activated FV (FVa), and prothrombin, accelerates the formation of thrombin. FV, which circulates in plasma but is also released locally by activated platelets, must first be activated by thrombin. Once activated and part of the complex, FVa enhances the ability of FXa to generate thrombin by about 300,000-fold, leading to a rapid burst in thrombin generation at the site of vessel damage. Thrombin has many functions that influence both coagulation and the vascular system (see Sect. 5). Its procoagulant functions include (1) inducing the conversion of fibrinogen to fibrin (thrombin is the only clotting enzyme capable of this function); (2) activating platelets, which provide the procoagulant surface for further thrombin generation; (3) proteolytically activating FVIII, the cofactor for FIXa in the tenase complex; (4) conceivably activating factor XI (FXI), which provides additional FIXa for the tenase complex; (5) activating FV, the cofactor for FXa in the prothrombinase complex; (6) directly activating the TF–FVII complex; and (7) activating factor XIII (FXIII), a transglutaminase, which in its active form cross-links fibrin to form an insoluble clot. In addition to these procoagulant functions, thrombin also activates thrombin-activatable fibrinolysis inhibitor (also termed
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Table 1 Plasma clotting factors and inhibitors. Circulating coagulation proteins are listed with their molecular mass, plasma concentration, domain organization, and role in coagulation Molecular Plasma concentraComponent mass (kDa) tion (mM) Domain structure Major role of active component in hemostasis Factor V 330 0.03 A1, A2, B, A3, C1, C2 Cofactor for activated factor X Factor VII 50 0.01 Gla, 2× EGF, SP Complexes TF to activate factor X Factor VIII 330 0.003 A1, A2, B, A3, C1, C2 Cofactor for activated factor IX Factor IX 56 0.09 Gla, 2× EGF, SP Complexes activated factor VII to activate factor X Factor X 59 0.136 Gla, 2x EGF, SP Complexes activated factor V to activate prothrombin Factor XI 160 0.031 2× (4× Apple, SP) Activates factor IX Factor XIII 320 0.031 2× A, 2× B Cross-links fibrin clot Fibrinogen 340 9.09 A, B, C Clot scaffold Prothrombin 72 1.38 Gla, 2× kringle, SP Deposition of fibrin TFPI 43 0.002 3× Kunitz Inhibitor of TF–activated factor VII Antithrombin 58 2.50 Serpin Inhibits thrombin, activated factor IX, and activated factor X Protein C 62 0.065 Gla, 2× EGF, SP Inhibits activated factor VIII and activated factor V Protein S 69 0.35 Gla, 4× EGF, SHBG Cofactor for protein C TFPI tissue factor pathway inhibitor, EGF epidermal growth factor, SP serine protease domain, SHBG sex-hormone-binding-globulin-like domain TF tissue factor
“thrombin-activatable procarboxypeptidase B”; TAFI), which cleaves C-terminal Lys and Arg residues from fibrin – this inhibits the binding of fibrinolytic enzymes and, in turn, protects the fibrin clot from degradation. Finally, thrombin also confers anticoagulant and cell-signaling actions. Many of the clotting factors and inhibitors of coagulation are listed in Table 1 along with their function and domain organization.
3 Structure of Plasma Clotting Factors Many of the circulating plasma clotting factors are synthesized primarily by the liver. They all have a modular domain organization, and many share several common structural features, which are discussed next. Those of the anticoagulant proteins are discussed in the later sections.
3.1 Gla Domains Several of the clotting factors (prothrombin, FVII, FIX, FX – and also anticoagulant proteins, protein C, protein S, and protein Z) belong to the vitamin K dependent family of proteins. This family of proteins contain a homologous N-terminal domain that generally contains around 45 amino acids, of which between nine and 11 are Glu residues and become g-carboxylated prior to secretion – a process that is dependent upon vitamin K [11]. These g-carboxylated Glu (Gla) residues define the structural and functional properties of this domain, referred to as the Gla domain. This domain is essential for conferring the affinity of this family of proteins for phospholipid membranes. The Gla domains of vitamin K dependent proteins are highly conserved and are all thought to fold in a similar manner. Despite their similarity, the precise affinity for phospholipid
varies quite widely among vitamin K dependent proteins. The additional carboxyl group that is added to Gla residues confers strong Ca2+-binding properties on the domain. The binding of Ca2+ ions induces a dramatic conformational change that reveals the phospholipid-binding site on the Gla domain. This includes the exposure of three hydrophobic residues in what is termed the w-loop [11]. The hydrophobic side chains of these residues insert into phospholipid membranes. The Gla-coordinated Ca2+ ions also interact ionically with the polar head groups of the phospholipid surface to strengthen this interaction. The clinical importance of the Gla domain in the function of clotting factors is demonstrated by the use of vitamin K antagonist drugs, such as warfarin. In the presence of warfarin, vitamin K is inhibited, resulting in inhibition of a specific carboxylase. The Glu residues are then not g-carboxylated, resulting in dysfunctional protein that lacks specific phospholipid binding properties. As membrane binding concentrates/localizes clotting factors to the surfaces upon which coagulation occurs, the lack of Gla domain function essentially ablates assembly of functional complexes on the surface of platelets and reduces thrombin generation.
3.2 Epidermal-Growth-Factor-Like Domains Epidermal growth factor (EGF)-like domains are also common features of coagulation proteins, and are found in FVII, FIX, and FX [as well as in protein C, protein S, thrombomodulin (TM), and protein Z]. These domains are named because of their sequence homology to domains contained in EGF. These domains contain three disulfide bonds that are paired in a generally conserved manner, although occasionally an additional bond (i.e., protein C), or alternative pairing of cysteines (i.e., TM) may occur. EGF-like domains participate in protein–protein interactions and therefore help define
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the functional specificity. These domains can also coordinate Ca2+ ions, most utilizing specific Asp or Asn residues that are posttranslationally b-hydroxylated.
3.3 Serine Protease Domains The serine proteases of the coagulation cascade [thrombin, FVIIa, FIXa, FXa, activated FXI (FXIa), and activated factor XII (FXIIa)] all contain protease domains similar to that of chymotrypsin. All of these proteases are secreted as inactive zymogens and require activating before they can fulfill their enzymatic roles. Activation usually requires a single bond cleavage in the protease domain, inducing a change in its conformation. The cleavage may or may not lead to release of an activation peptide. For all proteases except FVIIa (which requires additional binding to TF), cleavage activation exposes residues that form the active site, Ser193, His57, and Asp102 (numbering based on chymotrypsin). The characteristic serine protease fold also results in a conserved Na+-binding loop, Ca2+-binding sites, and often allows unique charged patches that confer specificity for substrate recognition.
3.4 Structure of Clotting Cofactors (FV and FVIII) FV and FVIII are homologous precursors that become functional cofactors when activated by thrombin [12]. FVa and activated FVIII (FVIIIa) dramatically enhance the enzymatic activities of FXa and FIXa in the tenase and prothrombinase complexes, respectively. Both FV and FVIII contain two types of internal repeats (A and C domains) [13]. The N-terminal region of both procofactors comprises of two A domains (A1 and A2) and the C-terminus consists of a further A domain and two C domains (A3, C1, and C2). In both FV and FVIII the A3, C1, and C2 domains are separated from the A1 and A2 domains by a nonhomologous B domain. High-affinity phospholipid-binding sites (nanomolar range) are present in both FV(a) and FVIII(a) in their C2 domains [14]. Structural data have also implicated the C1 domains in this interaction [15]. Activation of these cofactors occurs by the cleavage of at least three peptide bonds (see later). This converts the proteins into two-chain molecules (heavy and light). The heavy chain of FVa (A1 and A2 domains) remains noncovalently associated with the light chain (A3, C1, and C2 domains) via a highaffinity Ca2+-dependent interaction between the A1 and A3 domains [16]. Although FVIIIa is activated in a similar manner, an additional cleavage between the A1 and A2 domains results in a comparatively weak noncovalent association between the A2 domain and the rest of the molecule.
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4 Anticoagulant Pathways The generation of thrombin and the formation of a fibrin clot occur rapidly at sites of vascular injury through the pathways and feedback mechanisms outlined earlier. However, if these processes were to proceed unhindered as described, systemic blood coagulation might otherwise ensue. For this reason, inhibitory mechanisms [namely, TF pathway inhibitor (TFPI), activated protein C (APC), and antithrombin] exist that serve to limit the extent of coagulation and to localize both the generation and the activity of thrombin to the site of vessel damage. The cumulative actions of these anticoagulant mechanisms contribute to the observed biphasic generation of thrombin. The initiation phase, during which limited quantities of thrombin are formed, arises following TF exposure to blood, and is strongly influenced by the inhibitory actions of TFPI. Subsequently, when the positive-feedback loops are activated, thrombin is generated rapidly. As this propagation phase does not depend upon TF–FVIIa to generate FXa, the inhibitory actions of TFPI become less evident. This provides thrombin with the opportunity to fulfill its myriad functions, including the deposition of fibrin. Regulation is then primarily conferred by the combined actions of the protein C and antithrombin pathways, which localize and subsequently diminish the procoagulant response.
4.1 Tissue Factor Pathway Inhibitor The principal inhibitor of TF-mediated initiation of coagulation is TFPI. TFPI binds and inhibits FXa [17]. The resulting TFPI–FXa complex acts in a negative-feedback loop through the binding and inactivation of TF–FVIIa [18]. The initiating procoagulant stimulus can then be switched off. This ensures that a small procoagulant stimulus does not elicit uncontrolled generation of thrombin. TFPI is a 43-kDa Kunitz-type inhibitor [19] that contains three tandemly arranged Kunitz domains. The major site of TFPI production is in endothelial cells [20], which constitutively express the protein under normal conditions. TFPI is also normally expressed by vascular smooth muscle cells, megakaryocytes/platelets, monocytes, fibroblasts, and cardiomyocytes. TFPI secreted by endothelial cells circulates in plasma at a concentration of approximately 2.5 nM [21]. Most of the plasma pool circulates in truncated forms and/or in association with low-density lipoproteins [22], and as a consequence exhibits markedly reduced inhibitory potential. Only approximately 10% of plasma TFPI circulates in a full-length, free 43-kDa form. TFPI also directly associates with the surfaces of cells that express the protein.
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4.2 TFPI Anticoagulant Function The inhibitory activity of TFPI involves the Kunitz domains mimicking the substrate of the target protease. Once bound, the target enzyme fails to proteolyze the P1–P1¢ peptide bond, which confers a tight-binding, competitive inhibition. The first Kunitz domain of TFPI (K1) interacts directly with TF–FVIIa [23]. K1 binds ionically to the active site of FVIIa in this complex [23]. The second Kunitz domain (K2) mediates the binding and inhibition of FXa [24]. The third Kunitz domain (K3) has no described inhibitory function. The anticoagulant function of TFPI involves the FXa-dependent inhibition of TF–FVIIa (Fig. 2). By targeting FXa and FVIIa, TFPI directly inhibits the initiation phase of coagulation. The first stage in the inhibitory function of TFPI involves the reversible inhibition of FXa (Fig. 2). This occurs via the ionic binding of
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FXa to either full-length plasma or cell-associated TFPI. This interaction involves the P1 residue (Arg107) in TFPI K2 interacting with the active site of FXa [23]. As truncated forms of TFPI or proteolyzed TFPI are less potent inhibitors of FXa, it is likely that other parts of the TFPI molecule are also important for the interaction with this serine protease. Once FXa is assembled in the prothrombinase complex with FVa, it is protected from TFPI inhibition [25], largely owing to competition for the active site of FXa with prothrombin, which is present at much higher concentrations than TFPI. The second stage in the inhibition of TF-dependent coagulation involves the binding of the TFPI–FXa complex to TF–FVIIa (Fig. 2). During this step, the P1 residue in TFPI K1 (Lys36) interacts with the active site of FVIIa. The FXa-dependent inhibition of TF–FVIIa by TFPI results in the formation of an inactive TFPI–FXa–TF–FVIIa quaternary complex on a cell membrane. Although, as presented here, this process is frequently described as a twostage process, first involving the binding of free FXa and thereafter binding to TF–FVIIa, kinetic data favor a model in which TFPI interacts with FXa that has not yet been released from the activating TF–FVIIa complex [26]. Consequently, the amount of FXa that escapes inhibition is directly related to the concentration/availability of TF.
4.3 Overview of the Protein C Pathway
Fig. 2 The inhibition of TF-dependent coagulation by TF pathway inhibitor (TFPI ). Following the formation of the TF–FVIIa complex, the circulating zymogen, FX, binds to TF–FVIIa (1) and thereafter becomes activated (2). Once activated, FXa may dissociate (3a) from the activating complex. FXa may then elicit its procoagulant function or, alternatively, become inactivated by cell-associated or plasma TFPI via its second Kunitz domain. This complex can now reassociate with TF–FVIIa (4), with the first Kunitz domain of TFPI binding to the active site of FVIIa. Although frequently described as a two-stage process, kinetic studies favor a model whereby TFPI binds and inactivates TF–FVIIa–FXa prior to FXa release (3b/4). In each case the resulting inactive quaternary complex is the same
A second coagulation inhibitory pathway is the protein C anticoagulant pathway. Protein C bound to endothelial cell protein C receptor (EPCR) is activated on the surface of endothelial cells by thrombin bound to the integral membrane protein, TM [27]. APC, supported by its cofactor, protein S, proteolytically inactivates the nonenzymatic cofactors FVIIIa and FVa [28], which limits the further generation of thrombin. As the intact endothelium (i.e., adjacent to the site of vascular injury) normally expresses TM, the action of thrombin can be “switched” to impart an anticoagulant function. Conversely, at sites of endothelial disruption/dysfunction, the procoagulant actions of thrombin are favored. The action of thrombin can thereby be modulated depending on its location relative to the site of injury. Once released from EPCR and the thrombin–TM complex, APC can proteolytically inactivate FVIIIa and FVa by cleavage of specific peptide bonds. These APC-mediated reactions are augmented by its cofactor, protein S, which aids in both localizing APC to negatively charged membranes and favourably orienting the active site of APC toward its substrates. The following sections describe the components of this pathway in more detail.
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4.4 Thrombomodulin The primary site of TM expression is on the surface of the vascular endothelium, where it functions as a receptor/cofactor for thrombin in the activation of protein C. TM accelerates activation of protein C by thrombin by approximately 20,000-fold, and protein C almost certainly does not become activated physiologically in its absence. TM has a modular structure that is made up of an N-terminal lectin-like domain, a hydrophobic region, six tandemly arranged EGF domains, a Ser/Thr-rich region, a transmembrane region, and a C-terminal short cytoplasmic tail [29]. EGF domains 5 and 6 contain a charged patch that interacts with anion-binding exosite I on the surface of thrombin [30], whereas EGF domain 4 contains a low-affinity protein C binding site [31]. The Ser/Thr-rich domain, located between TMEGF6 and the plasma membrane, contains an attachment site for chondroitin sulfate, a glycosaminoglycan moiety that is strongly negatively charged. TM may be synthesized with or without this modification and this varies between different vascular beds. The presence of chondroitin sulfate enhances thrombin binding affinity for TM by approximately 13-fold, when compared with unmodified TM [32]. This is manifest by an additional site of interaction between the chondroitin sulfate on TM and exosite II on the surface of thrombin [33]. The lectinlike domain and EGF domains 1, 2, and 3 of TM are not required for protein C activation.
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the serine protease domain following cleavage of the Arg169– Leu170 peptide bond by the thrombin–TM complex, resulting in the activation of the enzyme. An important feature of the serine protease domain is an exosite region of positively charged residues [35], similar to exosite I of thrombin. This exosite is important for recognition of FVa during its inactivation. The importance of protein C in the downregulation of thrombin generation is supported clinically by the strong association between protein C deficiency and thrombosis.
4.6 Endothelial Cell Protein C Receptor EPCR is a type 1 transmembrane glycoprotein, with a molecular size of 46 kDa [36]. EPCR is expressed primarily by vascular endothelial cells, but preferentially by those of large vessels [37, 38]. The primary role of EPCR is as a cellsurface receptor for protein C/APC [39], which it binds with high affinity through its Gla domain [40]. EPCR binds both protein C and APC with similar affinity (Kd 30–60 nM) [39]. EPCR binding of protein C serves to concentrate/localize protein C in the plain of the endothelial cell plasma membrane. In this way, EPCR augments the rate of protein C activation by thrombin–TM on the endothelial surface by approximately 20-fold [41]. Binding of APC to EPCR also facilitates signaling of APC through proteolysis of proteaseactivated receptor (PAR)-1.
4.5 Protein C/APC
4.7 Protein S
Protein C is synthesized primarily in the liver and secreted into the plasma, where it circulates at an approximate concentration of 65 nM. As noted already, protein C shares homology with other vitamin K dependent serine proteases of the coagulation cascade [34]. Protein C zymogen is secreted as a 62-kDa protein, comprising a Gla domain (residues 1–45) at its N-terminal followed by two EGF-like domains linked to a chymotrypsin-like serine protease domain. As mentioned earlier, Gla domains undergo Ca2+dependent conformational changes that present the hydrophobic residues of the w-loop essential for phospholipid binding. In the case of protein C/APC, this region is also critical for binding to its cellular receptor, EPCR. The serine protease domain of protein C shares approximately 70% homology with other chymotrypsin-like serine protease family members. The catalytic triad consists of Ser, His, and Asp residues, which are brought into close proximity by the folding of the serine protease domain. APC contains a Ca2+-binding site and a Na+-binding site within the catalytic domain [35]. A 12-residue activation peptide is released from
Protein S is a 69-kDa vitamin K dependent plasma glycoprotein that functions as a cofactor for APC [42]. It is synthesized in hepatocytes, endothelial cells, and megakaryocytes. The plasma concentration of protein S is approximately 350 nM [43]; however, only about 40% of this is free protein S, with the remaining 60% circulating in a noncovalent, high-affinity complex with C4b-binding protein (C4BP). C4BP is a cofactor of serine protease factor I that ultimately regulates the formation of the C4b2a complex in the classic complement pathway. Only free protein S has full APC cofactor activity. Protein S is a modular protein made up of an N-terminal Gla domain, a thrombin-sensitive region (TSR), four tandem EGF-like domains, and a C-terminal sex-hormone-bindingglobulin-like domain that mediates the C4BP interaction [44]. High-affinity membrane binding is a characteristic of protein S and is mediated through its Gla domain [45]. This is a prerequisite for the recruitment and colocalization of APC on activated surfaces upon which the procoagulant complexes that it inactivates assemble. Protein S has one of the highest affinities for phospholipids of all vitamin K dependent proteins
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(Kd ~10 nM, as opposed to protein C Kd ~1 mM) [46]. The APC interaction site on protein S involves multiple residues in the Gla domain, TSR, and EGF domains [47, 48]. Protein S interacts specifically with residues 36–39 in APC [49].
4.8 Activation of Protein C Protein C activation is initiated when thrombin that escapes the site of injury encounters TM on the endothelial surface (Fig. 3). The site of interaction between thrombin and TM involves exosite I on the surface of thrombin [50]. This is a high-affinity interaction (Kd ~4 nM – without the TM chondroitin sulfate moiety). This basic (positively charged) patch binds to EGF domains 5 and 6 of TM. As exosite I is also involved in thrombin’s interaction with fibrinogen, PAR molecules, FV, and FVIII, the binding of thrombin to TM prevents association with these procoagulant substrates, and switches its role to that of an anticoagulant. The chondroitin sulfate attachment to the Ser/ Thr-rich region of TM increases the affinity of thrombin for TM through interactions with exosite II of thrombin.
Fig. 3 The activation of protein C by thrombin–thrombomodulin (TM). The domain structure of TM is shown, including the chondroitin sulfate attachment. The exosites (I and II) on the surface of thrombin (activated factor II) are labeled, as is the basic exosite (+) on protein C (PC). During the first stage, thrombin generated at the site of vascular injury that escapes the developing thrombus can bind to TM on the surface of the adjacent endothelium (1). This interaction involves exosite I binding to epidermal growth factor domains 5 and 6 on TM. The chondroitin sulfate moiety provides an additional point of interaction with exosite II on thrombin. PC binds via its Gla domain on endothelial cell surfaces to endothelial cell PC receptor, which serves to increase the local concentration of PC. Once bound to TM, thrombin loses its specificity for procoagulant substrates, but can now activate PC. PC interacts ionically with TMEGF4 via its basic exosite (2). This brings the activation peptide into close proximity with the active site of thrombin (3). Once cleaved, the activation peptide is released to yield the active serine protease, activated PC (APC ). This can now dissociate (4 ) and exert its anticoagulant functions
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Just as exosite I on thrombin mediates the binding to TM, a homologous positively charged region on the surface of the protein C zymogen facilitates its interaction with EGF domain 4 (and also 5) of TM [51]. This interaction is relatively weak and on its own represents an inefficient mechanism for bringing protein C into proximity with TM for activation. The binding of protein C by EPCR on the endothelial surface augments the recruitment of protein C and presents it for activation by the thrombin–TM complex. Thrombin bound to TM activates protein C by a single cleavage after Arg169 in the serine protease domain. Once the short activation peptide of protein C has been cleaved, a conformational change takes place, which converts the zymogen is into an active serine protease. The APC molecule can then be displaced by another protein C molecule and the activation can be process repeated.
4.9 APC Anticoagulant Function The anticoagulant function of APC is conferred through proteolytic inactivation of activated cofactors, FVa and FVIIIa. In FVa, APC recognizes two sites, Arg306 and Arg506, which are exposed on the surface of FVa (Fig. 4). Cleavage after Arg306 separates the A1 and A2 domains, whereas Arg506 bisects the A2 domain [52]. APC also proteolyzes FVa after Arg679 [53]. However, the physiological importance of this is unknown as it is neither necessary nor sufficient to inactivate FVa. In the absence of protein S, APC inactivation of FVa is biphasic, which represents the different rates of proteolysis at the two scissile bonds [53]. The first, most rapid cleavage occurs at Arg506 and results in a molecule with intermediate cofactor activity (25–40%). The second, slower cleavage is at Arg306 and is necessary for complete ablation of FVa procoagulant function [53]. This cleavage causes the dissociation of the A2 domain from FVa, which is the cause of its complete inactivation. A naturally occurring mutation, FV Leiden (Arg506Gln) [54], found in up to 5% of certain Caucasian populations, results in a FV molecule that exhibits partial resistance to APC inactivation. In individuals carrying the FV Leiden mutation, the initial cleavage at Arg506 cannot occur [54]. Therefore, as only the slower cleavage at Arg306 is possible, thrombin generation is not regulated as efficiently. Carriers of FV Leiden thus have a phenotype that predisposes them to thrombosis. The inhibition of FVa cofactor function by APC can only proceed prior to its assembly into the prothrombinase complex. Once bound to FXa, FVa is protected against inactivation, primarily by preventing the proteolytic cleavage after Arg506.
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Fig. 4 Anticoagulant function of APC. 1 FV (and FVIII) are proteolytically activated by thrombin, causing the dissociation of the B domain. FVa binds to activated platelet surfaces via its C domains, where the prothrombinase complex assembles. FVa is inactivated by APC via specific proteolysis of the Arg506 and Arg306 peptide bonds. The active site of PC is predicted to be located about 94 Å above the membrane surface to which it binds via its Gla domain, causing preferential cleavage of the Arg506 bond. The cofactor function of proteins serves to increase the affinity of APC for membrane surfaces and to reposition the active site of APC to 84 Å over the membrane surface, enhancing the proteolysis at the Arg306 site. 2 proteolysis of Arg506 causes partial inactivation of FVa, whereas the slower proteolysis of Arg306 (and also the noncritical Arg679) results in complete inactivation and dissociation of the A2 domain. 3 APC inactivates the homologous FVIIIa in similar manner through analogous cleavage of the Arg562 and Arg336 bonds. Inactivation of FVIIIa can be further enhanced by protein S in synergy with FV inactivated by APC in a reaction that is not fully understood
The anticoagulant activities of APC are augmented by the cofactor function of protein S [55] (Fig. 4). The interaction between protein S and APC is phospholipid-dependent, and results in complex formation on the surface of activated endothelial cells and platelets [56]. Protein S enhances
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APC-dependent inactivation of FVa in several ways. Owing to its high affinity for phospholipids, in binding APC, protein S increases its affinity for the phospholipid surfaces. Protein S can also counteract the protective effect that bound FXa has on the Arg506 cleavage [57, 58]. Third, it specifically enhances cleavage at Arg306 approximately 20-fold, so both cleavages occur at comparable rates through relocation of the position of the APC active site relative to FVa. The net effect of protein S cofactor function upon APC is that it increases the kcat of FVa inactivation. Inactivation of FVIIIa by APC occurs through proteolysis after Arg336 and Arg562 [59] (Fig. 4). However, unlike FVa, cleavage occurs preferentially at Arg336, and thereafter at Arg562 (as opposed to Arg506 and then Arg306 in FVa). The dissociation of the A2 domain that is rapidly induced following these proteolytic events results in complete loss of FVIIIa cofactor function [59]. Once assembled as part of the tenase complex, FIXa can protect the Arg562 peptide bond from APC-mediated cleavage [60]. Furthermore, as the substrate for the tenase complex, FX, competes with APC for the same binding site on FVIIIa, in this way it specifically protects the cleavage site at Arg362 [61]. Consequently, once a part of the tenase complex, FVIIIa is considerably more resistant to proteolytic inactivation by APC. The effects of protein S can overcome the protective action of FX. The role that protein S plays in its cofactor-mediated enhancement of FVIIIa cleavage is somewhat more complex that for FVa. For example, unlike FVa, the rate of cleavage at Arg336 by APC is unaltered by protein S. APC alone does not inactivate FVIIIa very efficiently and although protein S increases the efficiency slightly (threefold), intact FV is necessary for maximum augmentation (tenfold) to occur [62]. This interaction is not fully understood, but it would seem that FV must first be cleaved by APC at multiple sites, with the cleavage at Arg506 appearing to be of particular importance for the ability of FV to serve as an APC cofactor with protein S [63].
4.10 Overview of Antithrombin Function Thrombin is rapidly inactivated in plasma. This is largely due to the inhibitory action of antithrombin (the third anticoagulant pathway), but also a2-macroglobulin. Antithrombin is a plasma-borne serine protease inhibitor (serpin) that functions with broad specificity by mimicking target enzyme substrates. As target enzymes that are already assembled in their respective complexes are generally less accessible, antithrombin preferentially inhibits free enzymes. Thrombin, FIXa, FXa, FXIa, and FXIIa may all be inhibited [64], although arguably thrombin and FXa represent its most important physiological targets. It is through the inhibition of these serine proteases that antithrombin exerts its primary anticoagulant functions.
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Antithrombin is the most important direct inhibitor of coagulation proteases. Circulating at a concentration in blood of 2.5 mM, it inhibits the activity of thrombin, FXa, FIXa, FXIa, and FXIIa by forming tight equimolar complexes that block the accessibility of respective substrates to the protease active sites [65]. Despite its wide specificity, the inhibition of thrombin is probably most effective at suppressing coagulation. This is because of the key role of thrombin in accelerating its own generation by participating in FV and FVIII activation. Antithrombin activity is greatly accelerated by heparin, a highly negatively charged linear glycosaminoglycan, structurally related to heparan sulfate, which is found as a proteoglycan on the endothelial cell surface. A subpopulation of vascular heparan sulfate accelerates the inhibitory action of antithrombin against coagulation proteases. It is this mechanism of accelerated protease inhibition that forms part of this natural anticoagulant pathway [66]. Antithrombin is member of the serpin superfamily. Its importance as an inhibitor of coagulation is highlighted by the thrombotic problems of individuals with inherited disorders or with deficiency of this serpin [67].
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be cleaved, causing reactive bond separation to opposite ends of the molecule, with the polypeptide N-terminal to Arg393 inserting into the sheet A structure (Fig. 5 – “cleaved and inactive complex”). A similar complete loop insertion is observed in denatured or “latent” antithrombin, which is not cleaved but is functionally inactive. Alternatively, during normal inhibitory function, the substrate reaction proceeds from the Michaelis complex to an acyl intermediate stage. At this point, a conformational change is induced in antithrombin that stabilizes the complex with thrombin. This stabilizing conformational change involves partial, near complete, insertion of the reactive loop into sheet A (Fig. 5 – “stabilized complex”). This inactive complex circulates until it is cleared from plasma by the liver.
4.11 Antithrombin Structure Antithrombin has a highly ordered tertiary structure that is folded as three b-sheets (A–C) and nine a-helices (A–I). Like all serpins [68], the inhibitory action of antithrombin against proteases involves recognition of the reactive-site bond (Arg393–Ser394, also known as the P1–P1¢ bond) by the active site of the protease. In antithrombin, the reactive site is located in a reactive loop that protrudes above the main body of the inhibitor. Loop mobility appears to be crucial for the inhibitory action of all serpins. The reactive loop can adopt different positions, which have been identified by structural analysis. These are represented in cartoon form in Fig. 5. The reactive loop is shown above the main body of the serpin. One of the sheets (sheet A) is depicted by its several strands (arrowed lines) and one of the helices (helix D) protrudes from its side. In Fig. 5, two natural conformations are depicted, one with a partial insertion into sheet A (termed “inactive”), and one with fully expelled loop (“active”). The active conformation is also that adopted when heparin interacts with antithrombin (see later).
4.12 Inhibition of Thrombin by Antithrombin The first step in the interaction between antithrombin and target protease is the formation of a Michaelis complex between the reactive loop and the protease. A substrate reaction then begins in which the protease attempts to cleave the P1–P1¢ bond. The complex then has one of two fates. The serpin may
Fig. 5 Antithrombin structural changes. Antithrombin has several structural forms. These involve a mobile protruding reactive loop containing the reactive-site bond (P1–P1¢), helix D around which the heparin-binding site is centered, and a sheet A structure with five strands (arrowed). Antithrombin has two normal conformational states, one relatively “inactive,” with its reactive loop partially inserted into sheet A, and the other “active,” with a more accessible P1–P1¢ bond. During the inhibition of serine proteases (e.g., thrombin), the protease attempts to cleave the reactive-site bond, causing the reactive loop to insert strand A (cleaved inactive complex). If proteolysis does not occur, the protease–antithrombin complex is stabilized by incorporation of the reactive loop into sheet A (stabilized complex)
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It appears that for all serpin–protease reactions there is competition between loop insertion and the substrate reaction, the outcome of which determines the ultimate fate of the interaction (cleaved, inactive free serpin and protease on the one hand, or stabilized serpin–protease complex on the other) [69]. Inhibition of proteases by antithrombin is slow in relation to other serpin–protease reactions. It has been suggested that lack of reactivity is due to relative inaccessibility of the Arg393 (P1) residue to the protease.
4.13 Role of Heparin and Heparan Sulfate Heparin has an appreciable accelerating effect upon the formation of antithrombin–protease complexes. The formation of the antithrombin–thrombin complex is accelerated at least 2,000-fold under optimum conditions. Rate enhancements are also observed for the inactivation of FXa and FIXa by antithrombin. Heparin promotes the formation of a ternary complex of antithrombin and protease in which the active site of the protease is brought into contact with the reactive site of antithrombin. Assembly of the ternary complex proceeds preferentially through initial formation of a heparin–antithrombin complex, due to the high affinity of antithrombin for heparin. The binding of heparin to antithrombin is a two-step process with an initial low-affinity step (Kd of the order of micromolar) that, in turn, induces higher-affinity interaction (Kd ~ 20 nM). Thrombin and other proteases that bind with quite high affinity to heparin will bind to the heparin chain adjacent to antithrombin on the relatively nonspecific regions of the polysaccharide chain that adjoin the antithrombin-binding pentasaccharide sequence. In this circumstance, a crucial role of heparin seems to be the bringing together of inhibitor and protease, in a process termed “approximation” [70]. A key property of heparin is its induction of a conformational change in the reactive loop of antithrombin: with respect to accelerated thrombin inhibition, this induced conformational change is less important than the approximation effect. Binding to the heparin chain and approximation are essential for thrombin’s accelerated inhibition: binding is mediated by exosite II on the surface of the protease. In contrast, FXa and other proteases that bind only weakly to heparin are inhibited directly by the heparin–antithrombin complex, and approximation of antithrombin and protease on heparin does not need to take place. Rather, the conformational change induced in the reactive loop of the inhibitor by heparin appears to enable the inhibition reaction to proceed more rapidly. Once a stabilized complex has formed between the reactive site of antithrombin and the active site of the
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protease (FXa or thrombin), a further change in conformation in antithrombin (the loop insertion into sheet A) results in a reduced binding affinity of the complex for heparin. This enables its release and participation in additional antithrombin–protease reactions. Although blood is exposed to heparin only following its administration during clinical use, the vasculature is surrounded by large quantities of heparan sulfate, which is a proteoglycan with core proteins intercalated into cell membranes. Heparan sulfate itself is greatly heterogeneous but is structurally and biosynthetically related to heparin. Endothelial cells synthesize small amounts of anticoagulantly active proteoglycan. However, although the luminal surface of the endothelium contains only a small amount of the active heparan sulfate, the subendothelial space contains appreciably more.
5 Thrombin Thrombin is a central enzyme in coagulation with multiple roles that are critical to the hemostatic response (Fig. 6). The inactive thrombin precursor, prothrombin, circulates in plasma at a concentration of approximately 1.4 mM. Prothrombin contains an N-terminal Gla domain, two kringle domains, and a serine protease domain. Following the initiation of the coagulation cascade, thrombin is generated through FXa activation of prothrombin, albeit inefficiently. Subsequently, feedback activation of the coagulation cascade occurs by thrombindependent activation of FV and FVIII (see below). Cleavage of prothrombin by FXa or the prothrombinase complex (FXa– FVa) first occurs at Arg320 to generate the intermediate, meizothrombin, and then at Arg271 to generate thrombin [71]. This proteolytic activation liberates fragment F1 + 2, containing the prothrombin Gla and kringle domains. In prothrombin, these modules enable efficient localization of plasma prothrombin to activated platelet membrane surfaces at the site of vessel damage. This therefore enables its efficient recruitment to the prothrombinase complex. Once prothrombin has been activated, thrombin can escape the activation complex and is then free to attack its different and varied target substrates. Thrombin is unique among the activated coagulation serine proteases in that once it has been formed, the domains important for its initial recognition and activation are lost. Loss of the Gla and kringle domains allows thrombin to diffuse freely to encounter, recognize, cleave, and dissociate from its many substrates. This mobility is central to its ability to interact with its numerous substrates. Activation of prothrombin induces the exposure of three cryptic functional regions [72], including the active site and two charged binding regions, termed exosite I and exosite II, each of which is a critical determinant of thrombin specificity.
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Fig. 6 Multifunctional roles of thrombin. Once activated by the coagulation cascade, thrombin can function in procoagulant, anticoagulant, antifibrinolytic, and pro/anti-inflammatory pathways
5.1 Hemostatic Substrates of Thrombin A primary function of thrombin is to catalyze the conversion of fibrinogen to fibrin [73]. During this reaction, two short fibrinopeptides (FPA – 16 amino acids, FPB – 14 amino acids) are cleaved from the N-terminal ends of the fibrinogen Aa and Bb chains, respectively. Cleavage of FPA occurs first and exposes a cryptic polymerization domain. The fibrin monomer formed spontaneously polymerizes by interaction of the newly exposed polymerization domain with a preexisting complementary polymerization domain on the C-terminal domains of the molecule, to form protofibrils. Cleavage of FPB exposes additional polymerization sites that facilitate lateral aggregation to form an extensive meshwork that surrounds/encases the aggregated platelets at the site of vessel injury. Thrombin activates FV and FVIII by excision of their central B domains. In FV, Arg709, Arg1018, and Arg1545 are cleaved, leaving an A1–A2 domain fragment, ionically associated with the A3–C1–C2 fragment. In FVIII, the corresponding and analogous cleavages are Arg740, Arg1649, and Arg1689, with an additional cleavage at Arg372. This leaves the ionically associated A1–A2 fragment noncovalently bound to the A3–C1–C2 fragment. Thrombin proteolytically activates FXIII in a procoagulant reaction that is enhanced by fibrin. Once thrombin has cleaved the FXIII A subunit after Arg37, an N-terminal activation peptide is released. This exposes the active-site Cys314 residue to generate the active transglutaminase that stabilizes deposited fibrin fibrils by catalyzing covalent cross-linking [73]. Thrombin specifically activates platelets by binding and cleaving PAR molecules [74]. Cleavage of PAR-1 occurs at Arg41. This proteolytic event untethers an intramolecular
ligand that self-associates with its ligand binding site. This induces receptor-mediated intracellular signaling that leads to platelet activation. Thrombin can also proteolytically activate FXI, albeit under rather defined conditions that require anions such as dextran sulfate. However, in the presence of activated platelets, a more likely physiological activation process occurs that results in accelerated cleavage of factor XI after Arg369 [75]. Distinct to the procoagulant activities described above, thrombin also participates in interactions/reactions that elicit anticoagulant function, as described in earlier sections. For example, thrombin that is generated adjacent to endothelial cells lining the vessel lumen or thrombin that escapes the hemostatic plug can bind to TM on the surface of the endothelium in a high-affinity complex. Once thrombin is bound in the thrombin–TM complex, its substrate specificity is redirected from procoagulant to anticoagulant reactions. Procoagulant reactions are impeded largely by the occupancy of exosite I, whereas activation of protein C is greatly enhanced. APC and its anticoagulant roles were described in earlier sections in more detail. The thrombin–TM complex also activates the carboxypeptidase TAFI by specific proteolysis at Arg92 [76]. The rate of this activation is approximately 1,000 fold greater than by thrombin alone. Activated TAFI (TAFIa) stabilizes fibrin clots through proteolytic removal of terminal Lys residues from fibrin. The clipping of these amino acids disrupts the binding sites for fibrinolytic proteins that destabilize the fibrin meshwork. TAFIa also has an anti-inflammatory function in inactivating C5a in the complement cascade. Antithrombin locks thrombin in an irreversible inactive complex and is then cleared from the circulation. This reaction is accelerated by glycosaminoglycan cofactors (i.e., heparin or heparan sulfate proteolglycans), which approximate thrombin
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and antithrombin [70]. The multiple interactions of thrombin outlined above raise interesting questions. First, how can thrombin be a specific protease, when it has so many substrates? Second, how are its activities specifically and spatially coordinated during hemostasis to produce a hemostatic plug only at the site of injury and not over intact endothelium? [77] The questions may be explained by the specific occupancy of its surface exosites (I and II) by the above-mentioned cofactors for many of its reactions [78], and by competition of these cofactors for their respective exosites [77, 79].
5.2 Thrombin Exosites and Cofactors The major initial procoagulant functions of thrombin (i.e., cleavage of fibrinogen, FV, and FVIII) occur without cofactors. Interestingly, all of these reactions involve extended interactions of both exosite I and exosite II on thrombin with the target substrate [80–85]. In addition, the Na+-binding loop and the active site impart further specificity to these reactions. These multiple sites of interaction ensure efficient proteolysis in the absence of cofactors. The importance of exosites and cofactors can now be appreciated by considering the anticoagulant reactions of thrombin. Protein C activation by thrombin requires thrombin to be bound by its endothelial cell surface cofactor, TM. This is partly because TM localizes thrombin (bound by exosite I) and protein C (bound weakly by TM) together. The association of thrombin with TM is further enhanced if TM has been posttranslationally modified with the chondroitin sulfate moiety [86]. This carbohydrate structure increases the affinity of thrombin for TM through specific interactions with exosite II. Not only does the occupancy of exosite I by TM block its procoagulant functions, but it may also induce a conformational change in the active site that favors its interaction with protein C [87]. A second example of the functional role of exosites and cofactors is provided by the role of heparin (or heparan sulfate) in accelerating the interaction of antithrombin with thrombin. The glycosaminoglycan is able to bind both antithrombin and thrombin, bringing them into proximity (“approximation”). In this case, the functional exosite on thrombin is exosite II. Thrombin uses an adaptive strategy of cofactor utilization to extend its effective range of substrates. The next example of a cofactor is fibrin, which accelerates the activation of FXIII by thrombin. In this instance, fibrin behaves much as TM does as a cofactor, using exosite I of thrombin for binding, although the fibrin polymer must first be generated by thrombin before a functional cofactor emerges [79]. It has been suggested that fibrin polymerization brings FXIII (which also binds fibrin) into proximity with thrombin, enhancing activation of this transglutaminase [77].
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5.3 Cofactor Competition for Exosites: A Mechanism for Restricting Thrombin Action Cofactors extend the range of efficient thrombin interactions, but can only use one of two exosites on its surface. This raises the question of what happens when two cofactors are available for thrombin to interact with? This has been studied in the case of fibrin-enhanced FXIII activation by addition of TM: both fibrin and TM bind to residues within exosite I. However, fewer exosite residues are involved in fibrin-enhanced FXIII activation than in TM-enhanced protein C activation by thrombin [77, 79]. Moreover, the affinity of the thrombin interaction with TM is orders of magnitude higher that that with fibrin. Consequently, when both cofactors are present, TM will successfully compete with fibrin for exosite I. It will therefore terminate fibrin-enhanced FXIII activation by thrombin and remove thrombin from the clot. Competition for exosite II can occur in much the same way. Exosite II is used to increase the efficiency of reactions involving heparan sulfate (on the endothelial cell surface) and GpIba (on the platelet surface). As the affinities of thrombin for these cofactors are similar, the abundance of the cofactor becomes an important determinant in the outcome of any competition. Many proteins are susceptible to proteolytic cleavage by thrombin. However, the ability to cleave does not necessarily translate into a clearly defined physiological role. The specificity of thrombin is further determined by the concentration of each substrate in conjunction with the relative affinity of the exosite for that substrate.
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23 The Coagulation Cascade and Its Regulation 6. Banner DW, D’Arcy A, Chène C et al (1996) The crystal structure of the complex of blood coagulation factor VIIa with soluble tissue factor. Nature 380:41–46 7. Dickinson CD, Kelly CR, Ruf W (1996) Identification of surface residues mediating tissue factor binding and catalytic function of the serine protease factor VIIa. Proc Natl Acad Sci USA 93:14379–14384 8. Bom VJ, Bertina RM (1990) The contributions of Ca2+, phospholipids and tissue-factor apoprotein to the activation of human blood-coagulation factor X by activated factor VII. Biochem J 265:327–336 9. Morrison SA, Jesty J (1984) Tissue factor-dependent activation of tritium-labeled factor IX and factor X in human plasma. Blood 63:1338–1347 10. Esmon CT, Lollar P (1996) Involvement of thrombin anion-binding exosites 1 and 2 in the activation of factor V and factor VIII. J Biol Chem 271:13882–13887 11. Huang M, Rigby AC, Morelli X et al (2003) Structural basis of membrane binding by Gla domains of vitamin K-dependent proteins. Nat Struct Biol 10:751–756 12. Kane WH, Davie EW (1988) Blood coagulation factors V and VIII: structural and functional similarities and their relationship to hemorrhagic and thrombotic disorders. Blood 71:539–555 13. Fowler WE, Fay PJ, Arvan DS, Marder VJ (1990) Electron microscopy of human factor V and factor VIII: correlation of morphology with domain structure and localization of factor V activation fragments. Proc Natl Acad Sci U S A 87:7648–7652 14. Rosing J, Bakker HM, Thomassen MC, Hemker HC, Tans G (1993) Characterization of two forms of human factor Va with different cofactor activities. J Biol Chem 268:21130–21136 15. Adams TE, Hockin MF, Mann KG, Everse SJ (2004) The crystal structure of activated protein C-inactivated bovine factor Va: Implications for cofactor function. Proc Natl Acad Sci USA 101:8918–8923 16. Esmon CT (1979) The subunit structure of thrombin-activated factor V. Isolation of activated factor V, separation of subunits, and reconstitution of biological activity. J Biol Chem 254:964–973 17. Huang ZF, Wun TC, Broze GJ Jr (1993) Kinetics of factor Xa inhibition by tissue factor pathway inhibitor. J Biol Chem 268:26950–26955 18. Broze GJ Jr, Warren LA, Novotny WF, Higuchi DA, Girard JJ, Miletich JP (1988) The lipoprotein-associated coagulation inhibitor that inhibits the factor VII-tissue factor complex also inhibits factor Xa: insight into its possible mechanism of action. Blood 71:335–343 19. Wun TC, Kretzmer KK, Girard TJ, Miletich JP, Broze GH Jr (1988) Cloning and characterization of a cDNA coding for the lipoproteinassociated coagulation inhibitor shows that it consists of three tandem Kunitz-type inhibitory domains. J Biol Chem 263:6001–6004 20. Bajaj MS, Kuppuswamy MN, Saito H, Spitzer SG, Bajaj SP (1990) Cultured normal human hepatocytes do not synthesize lipoprotein-associated coagulation inhibitor: evidence that endothelium is the principal site of its synthesis. Proc Natl Acad Sci USA 87:8869–8873 21. Dahm A, Osterud B, Hjeltnes N, Sandset PM, Iversen PO (2006) Opposite circadian rhythms in melatonin and tissue factor pathway inhibitor type 1: does daylight affect coagulation? J Thromb Haemost 4:1840–1842 22. Broze GJ Jr, Lange GW, Duffin KL, MacPhail L (1994) Heterogeneity of plasma tissue factor pathway inhibitor. Blood Coagul Fibrinolysis 5:551–559 23. Girard TJ, Warren LA, Novotny WF et al (1989) Functional significance of the Kunitz-type inhibitory domains of lipoprotein-associated coagulation inhibitor. Nature 338:518–520 24. Petersen LC, Bjørn SE, Olsen OH, Nordfang O, Norris F, Norris K (1996) Inhibitory properties of separate recombinant Kunitz-typeprotease-inhibitor domains from tissue-factor-pathway inhibitor. Eur J Biochem 235:310–316
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Chapter 24
Platelets in Pulmonary Vascular Physiology and Pathology Michael H. Kroll
Abstract Platelets are small, nonnucleated cells that circulate for about 10 days after they are released into the bloodstream by bone marrow megakaryocytes. Platelet production derives from sequential processes termed megakaryopoiesis and thrombopoiesis, the biological basis of which is an intricate nexus of signaling events specified by cytokines and growth factors and organized temporally and spatially via exquisitely fine-tuned nuclear and cytosolic responses. Thrombopoietin (TPO) is the primary regulator of megakaryopoiesis. In conjunction with other cytokines, including stem cell factor, interleukin (IL)-6, IL-11, and erythropoietin, it promotes megakaryocyte lineage commitment from pluripotent hematopoietic stem cells. Counterbalancing these promegakaryopoiesis factors are transforming growth factor b1, platelet factor 4, and IL-4, each of which is considered a negative regulator of platelet production. Megakaryocytes mature into unique platelet-producing cells. Thrombopoiesis begins with a shift in compartmentalization as megakaryocytes migrate from the endosteal stem cell compartment to the vascular zone, where they adhere to the marrow sinusoids. These vascular zone megakaryocytes accumulate genetic material and cytoplasm as they maintain cell cycling that never progresses beyond late anaphase, so they fail to undergo nuclear envelope division and cytokinesis, thus resulting in the typically large irregularly shaped polyploid cells containing up to 64 pairs of chromosomes. As they enlarge, megakaryocytes express a huge surface area derived from an internal reservoir of membrane permeating the cytoplasm – designated the demarcation membrane system – that permits them to remodel their cytoplasm into a series of microtubule-scaffolded extensions termed proplatelets. Finally, via a series of microtubular, cytoskeletal, and contractile responses modulated by hydrodynamic stimuli, the proplatelet elongates and bifurcates, with individual platelets released into the circulation following their being sheared off by the forces of the flowing blood.
M.H. Kroll (*) Department of Benign Hematology, University of Texas, MD Anderson Cancer Center, 1515, Holcombe Blvd, Unit 1464, Houston, TX 77030, USA e-mail:
[email protected] Keywords Coagulation • Thrombosis • Embolism • Thrombopoiesis • Megakaryocyte • Hemolysis • Thrombopoietin
1 Platelet Production Platelets are small, nonnucleated cells that circulate for about 10 days after they are released into the bloodstream by bone marrow megakaryocytes. Humans produce about 1 × 1011 platelets daily, and production can be increased at least 20-fold in states such as acute hemorrhage, acute hemolysis, and inflammation. Platelet production derives from sequential processes termed megakaryopoiesis and thrombopoiesis, the biological basis of which is an intricate nexus of signaling events specified by cytokines and growth factors and organized temporally and spatially via exquisitely fine-tuned nuclear and cytosolic responses. Thrombopoietin (TPO) is the primary regulator of megakaryopoiesis. In conjunction with other cytokines, including stem cell factor, interleukin (IL)-6, IL-11, and erythropoietin, it promotes megakaryocyte lineage commitment from pluripotent hematopoietic stem cells [1]. Counterbalancing these promegakaryopoiesis factors are transforming growth factor b1, platelet factor 4, and IL-4, each of which is considered a negative regulator of platelet production. Megakaryocytes mature into unique platelet-producing cells [2]. Thrombopoiesis begins with a shift in compartmentalization as megakaryocytes migrate from the endosteal stem cell compartment to the vascular zone, where they adhere to the marrow sinusoids. These vascular zone megakaryocytes accumulate genetic material and cytoplasm as they maintain cell cycling that never progresses beyond late anaphase, so they fail to undergo nuclear envelope division and cytokinesis, thus resulting in the typically large irregularly shaped polyploid cells containing up to 64 pairs of chromosomes. As they enlarge, megakaryocytes express a huge surface area derived from an internal reservoir of membrane permeating the cytoplasm – designated the demarcation membrane system – that permits them to remodel their cytoplasm into a series of
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microtubule-scaffolded extensions termed proplatelets. Finally, via a series of microtubular, cytoskeletal, and contractile responses modulated by hydrodynamic stimuli, the proplatelet elongates and bifurcates, with individual platelets released into the circulation following their being sheared off by the forces of the flowing blood [3].
2 Does Thrombopoiesis Occur in the Lungs? Platelets may be released into the circulation from a pulmonary thrombopoietic compartment. Over 40 years ago, data derived from studies of right atrial blood in humans undergoing cardiac catheterization provided the estimate that up to 50% of bone marrow megakaryocytes exit the marrow cavity, enter the blood, and end up in the lung vasculature, where active thrombopoiesis results in the release from the pulmonary capillary bed of nearly one fifth of the circulating platelet mass [4]. These clinical data have been corroborated [5] and contradicted [6], but animal data – principally from the mouse – have fairly consistently shown that megakaryocytes are present and making platelets in the lung [7]. The idea that thrombopoiesis occurs in lungs is particularly interesting when considering that pulmonary fibrosis develops in some patients with a platelet secretory defect in which unpackaged growth factors leak from the megakaryocyte into their surrounding environment (e.g., the gray platelet syndrome) and in some patients with myeloproliferative disorders (see later). One might also speculate that platelet production in the lung is important in maintaining hemostasis within the vast pulmonary microvasculature, estimated to contain over one billion capillaries [8]. It is striking to note that pulmonary hemorrhage is an infrequent manifestation of even extreme disturbances in hemostasis, such as severe underproduction thrombocytopenia and hemophilia.
3 Platelets in Hemostasis and Thrombosis Circulating platelets participate in both physiological hemostasis and pathological thrombosis. Primary hemostasis is defined as the platelet–blood vessel interactions that initiate physiological hemostatic plug formation and prevent superficial microvascular hemorrhage. When the trigger is a pathological event, such as a ruptured atherosclerotic plaque, platelet adhesion to the damaged arterial wall leads to platelet aggregation, resulting in a vasooclusive white thrombus. A platelet-dependent thrombus is the fundamental pathological mediator of arterial ischemia or infarction, such as that causing heart attacks and strokes. Platelets circulate in an inactivated state. They respond to vessel wall injury, alterations in blood flow, or chemical
M.H. Kroll
stimuli with the activation of a functional triad of adhesion, secretion, and aggregation. These linked responses occur via a series of carefully coordinated signals that convert extracellular stimuli into intracellular chemical messengers that direct specific enzymatic reactions leading to changes in cell structure, the expression of a new repertoire of functional adhesion receptors, and the secretion of several proaggregatory and growth-promoting substances. The state of platelet activation is regulated dynamically by the actions of a diverse array of excitatory and inhibitory extracellular stimuli [9]. Platelets are equipped with specific plasma membrane receptors that organize these various stimuli and transform them into biological responses. This transformation occurs via transmembranous signaling that results in the generation of intracellular second messengers. The major activation pathways in platelets are stimulated by collagen, von Willebrand factor (VWF), thrombin, adenosine diphosphate (ADP), and epinephrine. The pathways that are activated downstream of the receptors for these stimuli include phospholipase C, which cleaves membrane phosphatidylinositol 4,5-bisphosphate to from diacylglycerol (which activates protein kinase C) and inositol 1,4,5-trisphosphate (which leads to elevated levels of cytoplasmic ionized calcium); phosphatidylinositol 3-kinase, which phosphorylates phosphatidylinositol 4,5-bisphosphate at the 3-position, leading to phosphatidylinositol 3,4,5-trisphosphate; and phospholipase A2, which hydrolyzes arachidonic acid from the 2-position of membrane phosphatidylcholine. Arachidonic acid is rapidly converted by platelet cyclooxygenase 1 to the proaggregatory and vasoconstricting (and therefore prothrombotic) product thromboxane A2 (TXA2). The major inhibitory signaling pathways are activated by prostacyclin (PGI2) and nitric oxide (NO), two constitutively released endothelial cell (EC) products that serve to maintain blood fluidity principally by maintaining platelets in their basal quiescent state. PGI2 binds to specific cell-surface receptors to activate adenylyl cyclase, whereas NO diffuses through the platelet plasma membrane to activate cytosolic (or soluble) guanylyl cyclase. These convert adenosine triphosphate and guanosine triphosphate into cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP), respectively. cAMP and cGMP cause activation of cAMP- and cGMP-dependent protein kinases, which serve to inhibit platelet activation through pleiotropic downstream effectors.
4 Platelet–Coagulation Factor Interactions Activated platelets are very important in regulating the generation of insoluble fibrin. They release a variety of prothrombotic substances – such as fibrinogen, VWF, and factor
24 Platelets in Pulmonary Vascular Physiology and Pathology
V – and they express receptors for factor XI that enhance its activation by thrombin [10]. Through the “flipping” of phosphatidylserine from the inner to the outer plasma membrane, activated platelets express specific surface binding sites for soluble clotting factors and thereby promote critically important coagulation reactions: the activation of factor X by the activated factor IX–activated factor VIII complex and the activation of prothrombin by the activated factor X–activated factor V complex. In addition to these reactions, activated platelets shed “microparticles” that may promote and disperse procoagulant responses. Activated platelets can also regulate natural anticoagulant mechanisms by promoting the inactivation of activated factor V by activated protein C and by releasing two proteins that inhibit activated factor XI [11].
5 Platelet–Leukocyte Interactions Platelet-dependent hemostasis and thrombosis are directly coupled to leukocyte recruitment and activation [12]. Activated platelets express P-selectin, which engages P-selectin glycoprotein ligand-1 (PSGL-1) on neutrophils and monocytes. This results in leukocyte adherence and activation on and within the developing platelet thrombus. In the leukocyte, PSGL-1-mediated responses are coupled to the upregulation of Mac-1 – which binds to platelet glycoprotein (Gp) Iba – and the activation of CD11b/CD18 – which binds to fibrinogen attached to platelet aIIbb3. This process of “secondary capture” is often followed by leukocyte–EC interactions, the release of inflammatory and procoagulant factors by the platelet–leukocyte aggregates, and amplification of platelet and leukocyte activation, leading to pathological changes in EC permeability and function [13]. Of note, eliminating platelet–neutrophil interactions in a mouse model of acute lung injury (ALI) protects against hypoxemia and enhances survival [14]. Platelet–lymphocyte interactions are directed by chemokines. Megakaryocytes synthesize several chemokines, and these molecules are stored in platelet a-granules and are released upon activation. Platelets contain many chemokines, including the a-chemokines platelet factor 4, growth-regulating oncogene a, platelet basic protein (which is cleaved to b-thromboglobulin), epithelial neutrophil activating protein-78, and IL-8; and the b-chemokines inflammatory peptide-1a, regulated on activation normal T cell expressed and secreted (RANTES), monocyte chemotactic protein-3, and thymus and activation-regulated chemokine. These effect immune responses by binding to specific receptors on lymphocytes or monocytes designated CXCR (for the a-chemokines) and CCR (for the b-chemokines). Platelets themselves express CXCR4, CCR1, CCR3, and CCR4, and these
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r eceptors transduce functionally important “priming” signals when they engage their specific chemokine ligand [15].
6 Platelet Secretion Platelets store or synthesize many other bioactive compounds capable of regulating platelet, coagulation protein, leukocyte, and vascular wall responses. The most prominent of these are pharmacological targets: aspirin-inhibitable TXA2, which is proaggregatory and vasoconstricting, and thieneopyridine-inhibitable ADP, which is proaggregatory [11]. TXA2 is synthesized from arachidonic acid by platelet cyclooxygenase 1 and ADP is stored in dense granules and released upon platelet stimulation. In addition, megakaryocytes synthesize and platelets store a variety of growth factors, cytokines, and adhesive proteins, each of which is capable of influencing short- and long-term tissue responses. Platelet a-granules contain platelet-derived growth factor (PDGF), transforming growth factor-b, and basic fibroblast growth factor, and their release into tissue compartments (both vascular and hematopoietic) may cause profound pathological changes, including myelofibrosis and perhaps even pulmonary fibrosis (see later). Platelets also store vascular endothelial growth factor (VEFG) along with a “panoply of endothelial trophogens” [16]. In the steady state they comprise the tools by which platelets accomplish their task as “guardians of the microvasculature.” When they are deficient, such as in thrombocytopenic states, it may not be possible to maintain hemostatic EC junctions, and the break in these junctions results in mucocutaneous bleeding. When they are secreted in excess, they may trigger neovascularization and angiogenesis, and thereby contribute to tissue repair and remodeling, and perhaps tumor cell growth [17].
7 Platelet–Complement Interactions The complement system provides another link between thrombosis and inflammation. Platelets are primed by several complement proteins and are activated to secrete and aggregate by C3a and C5b-9 [18, 19]. Conversely, activated platelets activate the complement system [20]. Complementactivated platelets may be a relatively rich source of procoagulant microparticles [19], which appear to feed-forward activate the complement system [21]. The congenital deficiency of the complement-regulatory protein factor H is associated with a hemolytic uremic syndrome (HUS)-like thrombotic microangiopathy that almost always involves the kidneys and almost never involves the lungs [22]. The role of complement in other thrombotic microangiopathies and the
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basis for sparse pulmonary vasculature involvement in this and other thrombotic microangiopathies [23] are unknown.
8 Pulmonary Vascular Anatomy and Rheology The pulmonary artery is relatively unaffected by atherothrombosis. It is also relatively resistant to bleeding in the face of extreme hemostatic challenge. The basis for these clinical observations is not clear, but it is likely to emanate from the biophysical properties of the pulmonary vasculature. The pulmonary arteries diverge by 15 branch-orders into a vast network of capillaries whose ECs abut alveolar epithelial cells organized into deformable structures that most closely resemble polyhedrons when the alveolar sac is fully inflated [24]. These capillaries, filled with oxygen-rich blood, reconverge about 15 times into increasingly larger pulmonary veins. Vessels of successive orders are connected in series and vessels within each order are connected in parallel. This circuitry design maintains low resistance (high capacitance) and low pressure in the face of high pulsatile blood flow (the entire right ventricular output) moving through a vast circuit estimated to comprise 300 million arteries, 300 million veins, and several billion capillaries [8]. Afferent arterioles are defined as 100 mm descending to less than 10 mm in diameter, with little subendothelium separating the luminal endothelium from the vascular smooth muscle cell layer. The capillary bed comprises an extensive collateral network of tiny (about 6 mm) thin-walled vessels that function in gas and solute exchange. The pulmonary capillaries comprise continuous (without fenestrations) thin (100–200-nm) ECs. There is one capillary per interalveolar septum [24]. Capillaries then converge into widening branches of the efferent venules, which expand in size from tens to hundreds of microns in diameter. Pulmonary venules lack two features found in almost all other venules, and the lack of these features undoubtedly reflects important hemostatic function. The pulmonary venules lack fenestrae – which permit egress of cells and proteins adluminally in other circuits – and valves – which prevent backflux into the capillary network. Rather than valves, pulmonary venules and veins possess a nearly continuous layer of smooth muscle cells surrounding the endothelium. This smooth muscle layer buttresses the barrier function of the return circuit and actively maintains blood flow out of alveolar interstitium all the way back to the left atrium through sequential sphincterlike contractions [25]. It is reasonable to speculate that these features are anatomic elements essential for protecting the lungs against hemostatic challenges that typically lead to hemorrhage from the venular compartment in other tissues and organs.
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9 Virchow’s Triad and the Pulmonary Vasculature Rheological principles govern pulmonary vascular biological processes, physiological hemostasis, and pathological thrombosis, but blood flow is only one component of a complex nexus of variables that modulate prohemostatic and prothrombotic responses. These complexities have been organized for over 150 years according to the model of the eminent German pathologist Rudolph Virchow. Virchow’s triad reminds us that hemostasis and thrombosis are regulated by the simultaneous interactions between blood (cells and soluble constituent), blood vessel (endothelium, subendothelium, and smooth muscle), and blood flow (related to diameter, branching, turbulence, and obstruction). In considering the variables that regulate platelet-dependent hemostasis and thrombosis in the lungs, one can begin by teasing apart Virchow’s triad to see what it looks like within the pulmonary vascular compartment.
9.1 Blood Flow Shear stress is defined as the force per unit area exerted on blood by blood flowing in layers of differential velocity. Blood flow in tubular structures results in a central stream of highest velocity and lowest shear stress and with the lowest velocity and highest shear stress at the vessel wall. Plateletdependent reactions occur in vascular compartments with high wall shear stress, whereas the soluble coagulation protein reactions assemble and generate fibrin under low-shear conditions. The healthy pulmonary circuit maintains relatively low shear stress throughout its expanse because of its ability to accommodate increased flow without changing its intraluminal pressure. This happens because of vasodilation of both arterial and venous elements, and because of the recruitment of capillary reservoirs during episodes of increased alveolar ventilation. It is only in diseased vessels, such as those in primary pulmonary hypertension, that blood flow in the pulmonary artery is accompanied by elevated pressures and generates pathologically elevated wall shear stress. The healthy pulmonary artery and its branches maintain shear stresses below 30 dyn/cm2. Shear stresses may rise to 60 dyn/cm2 in “feed” arterioles, which bifurcate into branch arterioles at about 200-mm intervals, with declining flow velocities and shear stress as branches narrow (for example, a first branch of 20 dyn/cm2 shear stress and a fourth branch shear stress of 9 dyn/cm2). At branch points there is turbulence, increased resistance, and even backflux as cyclical blood flow overcomes afferent arteriolar autoregulatory vasoconstriction triggered with every cardiac diastolic
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r elaxation. As blood enters the extensively branched capillary bed, shear stress decreases further. There are very few published data that provide one with precise measurements of shear stress – or other rheological parameters – in capillaries, but estimates based on viscosity calculations and flow and pressure measurements at the entrance and exit of capillaries indicate that physiological capillary blood flow has low flow velocity and generates low shear stresses. The branching postcapillary venules then converge into efferent vessels of increasing diameter and capacitance and gradually increasing – but still low – shear stress (less than 5 dyn/cm2). In the pulmonary system, venular backflux is prevented and centripetal flow maintained by smooth muscle “sphincters” in venules 50–100 mm in diameter. Flow turbulence decreases and flow velocity increases as smaller venular branches come together into larger venules and, eventually, form the pulmonary veins.
9.2 Blood 9.2.1 Von Willebrand Factor Pathological shear-stress-dependent platelet adherence in arteries and arterioles is triggered by platelet GpIba binding to plasma or vessel wall VWF. VWF is synthesized by vascular endothelium and by megakaryocytes. It is constitutively released adluminally (into the subendothelium) and abluminally (into the blood) by the endothelium, and it is also stored and secreted following cellular activation (stored in EC Weibel–Palade bodies and in platelets’ a-granules). It is a multimeric protein built up of tens to hundreds of disulfide-bonded multivalent protomeric units. Larger multimers appear to have greater hemostatic and prothrombotic properties. The protomeric subunit comprises two disulfide-linked mature VWF polypeptides, each being divided structurally and functionally into several domains: the A, B, C, and D domains. The A domains are of particular importance because the A1 domain forms the primary GpIba recognition site and binds to type VI collagen, the A2 domain contains the recognition sequence for degradation by the VWF multimer-cleaving protease “a disintegrin and metalloproteinase with thrombospondin type 1 motif ” (ADAMTS)-13, and the A3 domain binds to fibrillar collagens types I and III found in arterial subendothelium, thus allowing soluble VWF to tack down onto the subendothelium of ruptured atherosclerotic plaques. Two other domains of VWF are also important for hemostasis and thrombosis: the C2 domain contains an RGD integrin recognition domain essential for VWF binding to platelet aIIbb3 and an N-terminal D domain binds factor VIII [26].
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9.2.2 Platelet GpIb Complex The GpIb complex is made up four type I proteins: GpIba is disulfide-bound to GpIbb and GpIba/b is noncovalently bound to GpIX. Two GpIba/b–GpIX complexes are noncovalently bound to a single molecule of GpV. The VWF binding site is on a region of the N-terminal extracellular domain of GpIba. The conformation of this region of GpIba is altered by shear stress into a ligand-receptive conformation [27]. Upon ligand binding under high-shear-stress conditions, the cytoplasmic domain of GpIba signals secretion and aIIbb3 activation, possibly through cytoskeletal-based mechanotransduction [28]. VWF binding to GpIba is required for microvascular hemostasis, which appears to be triggered in the injured arteriole – a high-shear-stress microenvironment – by soluble and subendothelial VWF alone or bound to collagen (type VI may be the predominant microvascular collagen) attaching to platelets via GpIba, with the subsequent activation of aIIbb3 to a ligand-receptive conformation. The high shear stress in the arteriole limits fibrin deposition and leukocyte recruitment, and neither soluble coagulation factors nor leukocyte number or function contribute in any clinically important manner to microvascular hemostasis in the epithelium (such as skin, mucous membranes, and the urinary and gastrointestinal tracts). Only the GpIb complex, which forms catch bonds with VWF of very high tensile strength and therefore is capable of withstanding the disrupting effects of elevated shear forces, can capture, adhere to, and accrue platelets in arterioles or stenotic arteries [29]. As shear stress falls in the occlusive hemostatic plug to venular levels, fibrinogen binding to activated aIIbb3 is important for interplatelet cohesion. The importance of fibrinogen is emphasized by clinical observations that fibrinogen or aIIbb3 deficiency causes a severe hemostatic defect. Regional regulation of the VWF-triggered hemostatic plug induced by a bleeding time wound is remarkably fine-tuned, as the platelet-rich thrombi accrue only at the mouths of the transected arterioles, and little or no platelet accumulation occurs within the adjacent arteriole lumen. VWF-dependent arteriolar platelet thrombosis is the hallmark of thrombotic thrombocytopenic purpura (TTP) and HUS. In TTP the primary pathogenetic event may be an acquired deficiency of ADAMTS-13, resulting in persistent “ultralarge” VWF multimers – on EC surfaces and in the blood – effecting GpIb–GpIX–GpV-dependent platelet adhesion, secretion (predominantly dense granules which contain costimulatory ADP), aIIbb3 activation, and aggregation. In diarrhea-associated HUS the primary pathophysiological process may involve an enterotoxin-mediated overstimulation of “ultralarge” VWF multimers from arteriolar endothelial Weibel–Palade bodies. This bolus of large VWF multimers remains attached to the EC surface and thereby
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recruits passing platelets through the shear-dependent binding of platelet GpIba to the EC-associated VWF [30]. A similar effect may occur in low-shear-stress postcapillary venules when rapid EC Weibel–Palade body release is stimulated by calcium ionophore or histamine, suggesting that there may be clinical conditions associated with venular inflammation in which part of the pathophysiological process is caused by VWF–GpIb–GpIX–GpV-mediated platelet recruitment within a low-shear-stress microenvironment.
9.2.3 Coreceptors Although the GpIb–IX–V complex is required for the initiation of microvascular hemostasis and is a primary factor contributing to microvascular thrombosis in TTP and HUS, several additional platelet receptors are also involved in both microvascular physiological processes and microvascular pathological changes [11]. In every case, the coreceptor functions to support platelet-mediated microvascular responses after GpIb–IX–V engages ligands (the coreceptor supports postadhesion responses), and in some cases the coreceptor requires activation or expression downstream of VWF–GpIb–IX–V interactions (aIIbb3 and a1b2 are “activated,” whereas P-selectin is expressed following a-granule secretion). Observations of human and/or mouse deficiencies of aIIbb3 (the human defect is called Glanzmann’s thrombasthenia) reveal that it causes a severe bleeding disorder but protects against macrovascular arterial thrombosis. Similar observations have been made with the protease activated receptors that mediate thrombin-induced platelet activation, the TXA2 receptor; and the P2Y12 receptor that binds ADP, is the target of thienopyridine antiplatelet drugs, and appears to be indispensable to platelet aggregation triggered by VWF binding to GpIb–GpIX–GpV. Similar but lesser effects on hemostasis and macrovascular thrombosis are seen in mice lacking the P2Y1 receptor or P-selectin. In contrast, human deficiencies of the collagen receptors – GpVI and integrin a2b1 – result in a mild bleeding diathesis, whereas GpVI- or a2b1-deficient mice have only a small or no hemostatic defect, respectively. Either GpVI or a2b1 deficiency or pharmacological perturbation results in delayed and decreased ex vivo thrombus formation on type 1 collagen under both arteriolar and low-shear-stress conditions, indicating that GpVI and a2b1 (probably to a lesser extent in comparison with GpVI) are important but secondary mediators of microvascular thrombosis. The prototypical example of a platelet coreceptor that appears to effect hemostasis and thrombosis paradoxically is platelet EC adhesion molecule (PECAM)-1, which is expressed by the microvascular endothelium and platelets. Mice deficient in PECAM-1 have a hemostatic defect not because of a loss of platelet expression, but because they lack homotypic interactions between PECAM-1 on
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adjacent ECs needed to regulate the endothelial component of Virchow’s hemostatic response. In fact, PECAM-1deficient platelets are actually hyperresponsive to both VWF and collagen because PECAM-1 is a negative regulator of GpIb–IX–V- and GpVI-dependent signaling [31].
9.2.4 ADAMTS-13 ADAMTS-13 is the VWF multimer-cleaving protease. It is synthesized in the liver, circulates in blood, and may attach to the vascular endothelium. Although the regional distribution of ADAMTS-13 activity is not yet understood, on the basis of the end-organ pathological features of TTP it appears to play an important role in processing “ultralarge” VWF multimers – released constitutively and secreted following EC stimulation – in the cerebral, mesenteric, myocardial, splenic, renal, pancreatic, and adrenal arterioles. It also appears that arteriole-level shear stress is required for ADAMTS-13-mediated cleavage of “ultralarge” VWF multimers: higher levels of shear stress open up or untangle the multimers and thereby expose the ADAMTS-13 cleavage site in the VWF monomer A2 domain. The mechanism by which ADAMTS-13 deficiency leads to TTP is quite well understood [32]. Under physiological conditions, ADAMTS-13 is synthesized, homes to the arteriole EC, and under arteriole levels of shear stress breaks down prothrombotic “ultralarge” VWF multimers. This maintains blood flow within the microvasculature. When ADAMTS-13 is deficient, arteriole-level shear stress triggers platelet GpIba binding to the unprocessed multimers, thus causing the arterioles to become occluded with both EC-attached platelets and platelet clumps that exceed the diameter of the narrowing arteriolar branches. This leads to ischemia and infarction of many organs. Of note, the lungs are usually spared from the thrombotic complications of TTP, HUS, or other thrombotic microangiopathies despite the pulmonary artery and its branches being lined by ECs that synthesize VWF [23]. It may be that ADAMTS-13 is not involved in pulmonary vascular hemostasis and therefore a systemic deficiency of ADAMTS-13 has no impact in the lungs. It is known that ADAMTS-13 is not expressed in pulmonary tissues [33] and it is possible that ADAMTS-13 produced by the liver does not home or bind to the pulmonary endothelium.
9.2.5 Red Blood Cells Platelet GpIb–IX–V-dependent hemostasis is mainly a physiological arteriolar response to injury. In addition to the blood factors that interact directly with platelets, there are blood elements that modify platelet–vascular interactions
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indirectly. Perhaps most important is the red cell. Under arteriolar flow conditions the red cells flow centrally and push the platelet stream peripherally toward the arteriolar wall. Because blood flow in a tubular arteriole can be considered to be parabolic and composed of an infinite number of infinitesimal laminae, the centripetal movement of platelets exposes them to flow laminae of lowest velocity and highest shear stress, thus slowing them and making wall collisions more efficient (or sticky). As arterioles narrow and branch into capillaries, their luminal diameter narrows, red cells are excluded from the central stream, flow velocity falls, and platelets become evenly dispersed throughout the bloodstream. If the average pulmonary capillary diameter is calculated to be 5.8 mm, and the size of a platelet is 3 mm and that of a red cell is 7 mm, it becomes intuitively apparent that platelets flow relatively better (e.g., faster) than red cells through these capillaries. This facilitates red-cell-mediated oxygen uptake in the pulmonary alveoli and delivery everywhere else. It also keeps platelets from slowing and sticking because platelet–capillary interactions are minimized simply because fewer platelets are at the wall and more platelets are in the central stream with highest flow velocity. Pulmonary capillary beds may be relatively protected against thrombosis because of rheological factors – which are a direct consequence of capillary diameter and red cell size, shape, and deformability – that keep platelets flowing rapidly through the thin central stream of capillary blood. The calculated velocity of platelet flow through a pulmonary capillary is approximately 500 mm/s. A similar rheological environment is found in venules where the hemostatic response is compartmentalized: until red cells that have squeezed their way through the capillary bed gradually queue back into concentric central stream laminae, platelets remain randomly dispersed throughout the venular lumen even as the flowing blood accelerates up to velocities 3–4 times greater than those found in the capillaries. This means that platelet and venule wall collisions leading to attachment are relatively rare. This is another reason why defects in platelet-dependent hemostasis in most tissues are manifested by postcapillary venular bleeding. Recall that lung venules are relatively protected from red cell leakage by smooth muscle cells that wrap around the endothelium to create a hemostatic barrier that can be enhanced by their constriction. The pulmonary venule barrier may be a double-edged sword, however, as the pulmonary postcapillary venule is the site where nondeformable sickled red cells cause vasoocclusion leading to the acute chest syndrome in sickle cell diseases [34]. In fact, the major defect resulting from red cell sickling that leads to vasoocclusion is a perturbation in the natural anticoagulant properties of the intact vascular endothelium. Sickled cells trapped in the postcapillary venules undergo hemolysis and release free hemoglobin, which
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s cavenges NO and stimulates arginase, thereby decreasing the level of arginine, which is the substrate for EC nitric oxide synthase. The sum result is that the pulmonary postcapillary venule suffers a deficiency of vasodilatory and antiplatelet NO, secondarily causing ischemia from imbalanced vasoconstriction and platelet activation. In the long term, these events lead to pathological remodeling of the pulmonary microvasculature associated with pulmonary hypertension in approximately 20% of patients with sickle cell disease [35].
9.3 Blood Vessel The vasculopathy of sickle cell disease is an emphatic example of the importance of an intact vascular endothelium in actively maintaining blood fluidity. A bleeding time wound shows platelet thrombus formation only at the site of arteriolar transection because the adjacent arteriolar, capillary, and venular ECs constitutively secrete or express on their surface molecules that prevent platelet adhesion, secretion, and aggregation. These include PGI2 and NO (both of which inhibit platelet adhesion and activation), the ectoADPase CD39 (which breaks down soluble ADP), thrombomodulin and heparan sulfates (which bind and inactivate thrombin), and urokinase-type plasminogen activator and tissue plasminogen activator (which generate plasmin capable of degrading VWF and fibrinogen). The capillary endothelium is particularly richly endowed with tissue factor pathway inhibitor, which is both secreted into the blood and retained on capillary endothelium, where it binds to and inactivates the tissue factor–activated factor VII–activated factor X complex, thereby eliminating thrombin generation. This suggests that the capillary may be an important gate preventing the initiation of coagulation despite continuous low-level exposure to prothrombotic stimuli. Only urokinase-type plasminogen activator or tissue plasminogen activator deficiency is associated with spontaneous microvascular thromboses, but a deficiency of any one of the vessel-derived natural anticoagulants leads to exaggerated responses to thrombotic stimuli. The exception to this is CD39 deficiency, which causes a severe bleeding diathesis due to compromised hemostasis because elevated blood levels of adenine nucleotides lead to P2Y1-mediated platelet desensitization. This is a useful reminder of the dynamic nature of the complex interactions that occur over time within Virchow’s triad. Subendothelial VWF is most abundant in the macrovasculature large veins and arteries (most large veins > pulmonary artery > cerebral arteries > aorta > coronary arteries > renal arteries > hepatic arteries > pulmonary vein). It is generally less abundant in microvascular subendothelium, and its distribution in the microvasculature is
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noteworthy – venules > arterioles > capillaries, with very little or no VWF observed in any embryo capillary bed (in mice) and with relatively little VWF observed in adult myocardium (in pigs).
10 Pathological Interplay Between Platelets and the Pulmonary Circulation As the pathogenesis of pulmonary vascular diseases becomes increasingly illuminated, one can find many shadows of platelets, but the links that could direct innovations in diagnosis and treatment are missing. This probably reflects normal physiological function: the lungs are endowed with innate protection against several platelet-dependent disorders, such as atherothrombosis [35], microvascular thrombosis [23], and hemorrhage from severe thrombocytopenia or functional platelet disorders [36]. And it is clear that the pulmonary vascular elements within Virchow’s triad that are most important in effecting this protection are its unique rheological characteristics and vessel wall anatomy (see earlier). To eliminate the umbra, therefore, one must shine a light on platelets to try to see what they are doing within Virchow’s triad in pulmonary vascular diseases.
10.1 Hemorrhagic Interplay 10.1.1 Alveolar Hemorrhage Diffuse alveolar hemorrhage (DAH) develops in stem cell transplant (SCT) recipients. Its overall incidence is estimated at 1–21% and it occurs equally frequently in both auto-SCT and allo-SCT [37]. In one series, DAH was the principal diagnosis in 39% of SCT patients referred for bronchoscopy and nearly half of these patients died from respiratory failure or multiorgan failure [38]. Although DAH is clearly due to severe pulmonary microvascular derangements from radiotherapy, chemotherapy, graft-versus-host disease, and superimposed infection, thrombocytopenia is an important contributing factor and a standard therapeutic target [38]. DAH can develop in association with connective tissue diseases, Goodpasture’s syndrome (which leads to the “pulmonary–renal syndrome”), antiphospholipid antibodies, and many other vasculitides [39–41]. DAH is also part of a rare congenital syndrome of “idiopathic pulmonary hemosiderosis” [42] and a similar acquired disorder sometimes associated with primary pulmonary hypertension designated “pulmonary capillary hemangiomatosis” (PCH) [43]. In most cases of DAH, the primary insult is to the microvasculature and presumably the postcapillary venules. This insult leads to venular leakage of red cells despite normal
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hemostatic function (i.e., normal platelet counts and coagulation activity). Of note, hemoptysis in mitral stenosis is often from DAH, indicating that elevated pulmonary venous pressure is transduced back to the microvasculature and causes EC junction failure despite normal platelet counts and normal peristaltic function of pulmonary venules and veins to move blood centripetally. Although there is little evidence of a role for platelet-dependent hemostasis in most cases of DAH outside the SCT setting, cases of severe and often lethal DAH have been associated with platelet GpIIb/GpIIIa inhibitors given to patients with acute coronary syndromes [44].
10.2 Thrombotic Interplay 10.2.1 Pulmonary Embolism Pulmonary embolism (PE) is not a primary pulmonary vascular disease. Nonetheless, a massive PE can cause right ventricular collapse from acute pulmonary hypertension and recurrent PEs cause chronic pulmonary hypertension. A PE is a fibrin-rich thrombus that almost always develops in the lowflow venous circuit. Platelets become a constituent of the thrombus secondarily, and their role in the pathophysiological process and treatment of PE is considered small [45]. There are older experimental [46] and clinical [47] data demonstrating that platelets are activated in venous thromboembolism (VTE). There are also many clinical data – including data from recent randomized prospective trials –demonstrating a small but significant effect of antiplatelet therapy on preventing VTE in high-risk settings [45]. Aspirin is less effective than other pharmacological interventions; and it also causes less bleeding. Because of its ease of use, inexpensiveness, and safety, aspirin is being examined in two large prospective placebo-controlled randomized trials of secondary VTE prevention. The WARFASA trial (a European study of 1,400 persons examining if low-dose enterically administered coated aspirin given for 2 years after 6 months of warfarin treatment will prevent recurrent VTE and cardiovascular events) and the ASPIRE trial (an Australian trial of 3,000 persons examining if 100 mg aspirin prevents recurrent VTE after standard anticoagulation therapy) are expected to provide conclusive data about aspirin and VTE recurrence. They are not, however, likely to yield anything other than pilot data about the effect of inhibiting cyclooxygenase on pulmonary vascular responses to VTE. 10.2.2 Thrombotic Microangiopathies Pulmonary vascular ECs synthesize, store, and release VWF, perhaps more robustly than EC in other circuits. Yet the regulation of VWF processing in the lungs is largely
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unknown, and VWF-induced platelet-dependent pulmonary vascular thrombosis is considered rare. The paradigm for a VWF- and platelet-dependent thrombotic diathesis is TTP which, as described already, does not typically involve the lungs. There are, however, reported cases of lung involvement in primary or de novo TTP due to acquired ADAMTS-13 [23]. The pathological investigation of the lungs reveals microvascular hyaline thromboses – containing platelets and fibrin – in the terminal arterioles and capillaries [48]. This is associated in some cases with “noncardiogenic” pulmonary edema from venular capillary leak. It is interesting to note that there are cases of DAH due to microvascular thromboses in transplanted lungs of patients who developed posttransplant TTP due to ADAMTS-13 deficiency, which is an uncommon cause of posttransplant TTP [49]. This is a reminder that the expression of ADAMTS-13 deficiency in any particular organ or tissue depends on other perturbations to Virchow’s triad that develop in that vascular compartment. The physiological processes regulating pulmonary vascular ADAMTS-13 are (to my knowledge) unknown. It is likely that ADAMTS-13 is synthesized and secreted by the liver, enters the circulation though the hepatic vein, and then enters the lungs through the pulmonary artery. Once in the lungs, ADAMTS-13 must home to the vascular endothelial compartment; but the sites of its homing, its recognition and binding motifs, and how its activity is regulated – in short, almost everything about its presence and function in the pulmonary circulation – remain a mystery. It is likely that elucidating the biological processes and pathological changes in pulmonary vascular ADAMTS-13 will teach us a lot about protecting the lung from thromboses and perhaps even provide potential therapeutic insight into common pulmonary inflammatory processes, such as pneumonia and asthma [50, 51].
10.2.3 Sickle Cell Disease There is systemic platelet activation in sickle cell crises. This is a secondary phenomenon of uncertain pathophysiological consequences. It is due in part to shear-induced platelet activation by flow across a regurgitant tricuspid valve (due to pulmonary hypertension), hemolyzed red-cellderived ADP-induced platelet activation, and the decreased endogenous antithrombotic properties of the vascular endothelium due to NO scavenging by free hemoglobin [52]. VWF also participates in the pathogenesis of sickle cell vasoocclusion, but its effect is independent of platelets and ADAMTS-13 deficiency [53]. Freshly secreted large VWF multimers are involved in the adherence of young red cells (“neocytes”), but not sickled red cells, to the microvascular endothelium.
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The two most important pulmonary complications of sickle cell disease are the acute chest syndrome and chronic pulmonary hypertension. Both are common (affect approximately 30%), due to many different factors (such as infection and necrotic marrow fat embolism), and are not simply the short- and long-term consequences of vasoocclusion [54]. Within this complex pathophysiological milieu, it is difficult to rank the importance of platelet-dependent thrombosis. One unfavorable prognostic indicator of the acute chest syndrome is a drop in platelet count, but this finding does not appear to represent pulmonary sequestration of platelets or any associated platelet thromboses. There are no data available to assess an effect of antiplatelet therapy in the acute chest syndrome and there appears to be no affect of antiplatelet agents on the natural history of sickle cell disease, including the frequency of the acute chest syndrome and the development of pulmonary hypertension [55–57].
10.2.4 Pulmonary Fibrosis In the USA, idiopathic pulmonary fibrosis affects between three and 29 persons per 100,000 and the median survival for those affected is between 3 and 6 years [58]. Its pathogenesis – including the role of platelets, VWF, and ADAMTS-13 – is not understood. There are, however, two theoretical reasons to examine platelets in idiopathic pulmonary fibrosis. The first is that platelets store and secrete upon activation several cytokines and growth factors allegedly involved in the fibrotic response, such as transforming growth factor-b, fibroblast growth factor, and PDGF [59]. The second is more compelling: “secondary” pulmonary fibrosis is associated with inherited storage pool defects due to abnormal packaging of platelet a-granules and dense granules. Congenital a-granule deficiency is a very rare bleeding disorder designated the “gray platelet syndrome.” The human mutation leading to it is unknown and its inheritance and penetrance are variable, but the platelet phenotype and disease expression are thoroughly cataloged [60]. Circulating platelets do not have a-granules owing to a failure of proteins synthesized by the megakaryocyte (such as growth factors) or endocytosed by megakaryocytes or platelets (such as fibrinogen) to traffic into the secretable pool of a-granule constituents. Most patients with the gray platelet syndrome have myelofibrosis caused by the constitutive release into the bone marrow microenvironment of megakaryocyte-derived growth and fibrosing factors that would normally be packaged into a-granules. There is one case of pulmonary fibrosis reported out of a total of 18 well-characterized gray platelet syndrome families [61]. Pulmonary fibrosis is more common in the inherited platelet storage pool deficiency designated “Hermansky– Pudlak syndrome” (HPS). This is another rare bleeding
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d isorder due to homozygous deficiency of one of seven HPS genes involved in the biogenesis of several storage organelles such as platelet dense granules and lysosomes. The clinical expression varies with the specific gene affected, but generally the bleeding disorder is accompanied by oculocutaneous albinism and pulmonary disease. The most common variant is due to a mutation in the HPS1 gene on chromosome 10. A 16-base-pair duplication in exon 15 affects over 400 persons in a small region of northwest Puerto Rico, and other mutations in HPS1 are found in similarly affected people in Europe and Japan. Almost all of these persons develop pulmonary fibrosis by their fourth decade and pulmonary fibrosis is lethal in at least 50% by the fifth decade. None develop myelofibrosis. The mechanisms of pulmonary fibrosis in HPS are not known. One model posits that pulmonary alveolar type II pneumocytes fail to secrete surfactant and that unpackaged lipids accumulate as waxy ceroid deposits in the air spaces. These deposits evoke macrophage-induced lung injury, especially the release of cathepsin L, which stiffens lungs by degrading collagen and elastin [62]. The role of platelets in HPS-associated pulmonary fibrosis has not been investigated and the mechanisms of plateletmediated pulmonary fibrosis in the gray platelet syndrome are uncertain. But the paradigm of myelofibrosis in the gray platelet syndrome should not be overlooked. It is possible that pulmonary fibrosis develops in both HPS and the gray platelet syndrome because the dysfunctional granule biogenesis directly affects megakaryocytes within a pulmonary vascular compartment. In HPS, leakage of unpackaged lysosomal contents may lead to airway and alveolar damage and leakage of dense granule serotonin may lead to smooth muscle cell contraction, proliferation, and neointima formation [63]. In the gray platelet syndrome, leakage of mitogenic, angiogenic, and permeability factors may lead to fibrosing airway and alveolar damage. There is additional evidence in support of a connection between abnormal platelet production and pulmonary pathophysiological processes. For example, biopsy-proven pulmonary interstitial inflammation accompanies immune thrombocytopenia (ITP) and remits along with the ITP [64]. This is germane because megakaryocytopoiesis is almost always abnormal in ITP, with the bone marrow typically showing megakaryocyte hyperplasia, and it is possible that brisker megakaryocytopoiesis in the lungs leads to the inflammatory response. As a second example, there is evidence that patients with idiopathic myelofibrosis develop secondary pulmonary hypertension [65] and that patients with primary pulmonary hypertension develop secondary myelofibrosis [66]. The next section will examine these phenomena and attempt to illuminate where platelets fit into this unexpected bidirectional linkage between the lungs and abnormal hematopoiesis.
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10.2.5 Pulmonary Hypertension The example that underlies the basis for considering platelets relevant in the pathogenesis of pulmonary hypertension derives from a rare case of a poorly understood inherited isolated dense granule deficiency (i.e., not associated with lysosomal or melanosomal abnormalities) associated with pulmonary hypertension [67]. The observations that this patient’s platelets leaked serotonin and that there were elevated levels of plasma serotonin are important components of the foundation for the “serotonin hypothesis” of pulmonary hypertension [68]. Although the role of platelets in pulmonary hypertension appears less certain today than when these observations were reported in 1990, data on their potential mechanisms of effect in this disease shed some light on how platelets and pulmonary vascular diseases might be linked [63]. Pulmonary hypertension is associated with thrombocytosis. In many cases it is a reactive thrombocytosis secondary to lung inflammation developing during the natural history of pulmonary hypertension [68, 69]. However, pulmonary hypertension also occurs in patients with postsplenectomy thrombocytosis [70] and myeloproliferative disease (MPD)associated thrombocytosis [71], and it commonly develops in myelofibrosis with myeloid metaplasia [65]. How do these clinical conditions, each of which imposes unique changes on several elements within Virchow’s triad, lead to pulmonary hypertension? Postsplenectomy or MPD-associated thrombocytosis may contribute to pulmonary hypertension through thrombocyte elaboration of growth factors, such as platelet-derived VEGF and PDGF. In fact, platelet-derived VEGF and PDGF are proposed as mediators of vascular changes developing in idiopathic pulmonary hypertension [72]. In postsplenectomy pulmonary hypertension, red cells are also important in the pathogenesis of pulmonary hypertension. When the spleen is absent, there is abnormal red cell processing which causes many weirdly shaped less deformable red cells (poikilocytes) to circulate. The poikilocytes are trapped, ripped up, and hemolyzed within the pulmonary microvasculature. Resulting free hemoglobin shuts down the endogenous NO antithrombotic property of the pulmonary vascular endothelium, whereas free ADP stimulates the platelets, resulting in a prothrombotic imbalance within Virchow’s triad [70]. In the MPDs – including myelofibrosis – extramedullary hematopoiesis may also contribute to pulmonary hypertension. It develops as stem cells leave the dysfunctional marrow compartment and colonize new compartments, including the lungs. This leads to functional changes similar to those in idiopathic pulmonary hypertension, such as arteriolar and capillary obstruction. It also leads to the elaboration of cytokines and growth factors that cause histopathological changes typical of idiopathic pulmonary hypertension, including intimal hyperplasia, vascular smooth muscle proliferation,
24 Platelets in Pulmonary Vascular Physiology and Pathology
and neovascularization. In support of the extramedullary hematopoiesis model, there is one series of patients with the MPD chronic myelogenous leukemia in which ten of 21 autopsies showed pulmonary vascular megakaryopoiesis and two out of the ten persons with pulmonary megakaryopoiesis had accompanying pulmonary fibrosis [73]. In support of the humoral model of cytokine and growth factor effects, there is one case of MPD-associated pulmonary hypertension in which blood levels of platelet factor 4 (a bioactive platelet a-granule constituent) were elevated [70] and one fairly large series in which 22 patients with myelofibrosis and pulmonary hypertension were evaluated and determined to have a “distinctive angiogenic phenotype” characterized by increased levels of circulating endothelial progenitor cells, increased levels of serum VEGF, and increased microvascular density in the marrow compartment [65]. How else might platelets contribute to pulmonary hypertension? There are ambiguous data about systemic platelet activation in idiopathic pulmonary hypertension and pulmonary hypertension associated with congenital heart diseases causing right-sided overload [74]. More consistent elevations of platelet activation are observed in anorectic medication (such as fenfluramine)-induced pulmonary hypertension. In these cases the common denominator may be cellular activation – leading to serotonin release by platelets and pulmonary vascular smooth muscle cell contraction – by drug-induced blockade of a voltage-sensitive potassium channel [75, 76]. Consistent with the idea that platelets are involved in anorectic drug-induced pulmonary hypertension, effective pulmonary hypertension treatment with the prostaglandin PGI2 usually leads to decreased platelet activation [77]. In all of these cases it is impossible to determine if platelet activation is a cause or an effect of pulmonary vascular changes in flow and structure, but it is most likely an effect of uncertain consequence. Similarly, elevations of plasma VWF levels in association with both primary and secondary pulmonary hypertension are probably epiphenomena of no pathophysiological significance [78]. Compelling data show that pulmonary hypertension is associated with increased levels of platelet TXA2 and decreased levels of endothelial PGI2, but the pathophysiological importance of these data seems negligible in the face of other data showing that treatment with aspirin or a thienopyridine has no effect on the severity or natural history of pulmonary hypertension, and that there is no evidence that pulmonary hypertension is associated with an EC-specific cyclooxygenase 2 inhibitor, such as rofecoxib or celocoxib, which selectively depletes PGI2 [75]. 10.2.6 Myelofibrosis in Pulmonary Hypertension The connection between bone marrow and pulmonary vascular compartments is as intriguing as it is opaque. This is
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because it is bidirectional: just as primary myelofibrosis leads to pulmonary hypertension, primary pulmonary hypertension leads to myelofibrosis [66, 79, 80]. The association is very strong: when first reported, all 17 primary pulmonary hypertension patients who were evaluated had myelofibrosis, including 11 designated as having severe fibrosis [79]. The myelofibrosis is a reactive process (not due to a clonal proliferation of myeloid elements) and it may be clinically important because it is accompanied by anemia and/or thrombocytopenia in most patients [66]. The cause of myelofibrosis is unknown, and there are no pathophysiological elements that are known to bridge lung and marrow fibrosis. Speculation focuses on pathological angiogenesis, and there is one study showing a paucity of microvascular pericytes in the bone marrow of pulmonary hypertension patients with secondary myelofibrosis [80]. This finding implies that myelofibrosis is related to aberrant angiogenesis and suggests the possibility that this aberrancy also underlies idiopathic pulmonary hypertension. The role of megakaryocytes and platelets has not been explored. One study showed that the TPO level is elevated in the right ventricle, pulmonary artery, and left ventricle of patients with primary pulmonary hypertension [81]. This could reflect decreased pulmonary thrombopoiesis (platelets regulate blood TPO levels by binding TPO; they are a TPO “sink”) as changes in the vascular compartment in pulmonary hypertension drive platelet precursors from the lungs into the bone marrow compartment.
10.2.7 Miscellaneous Lung Diseases Platelets may be significant elements in the pathogenesis of several pulmonary diseases, including some that are not obviously vascular diseases. Pulmonary venoocclusive disease (VOD) is an uncommon complication of SCT and an uncommon toxicity of certain chemotherapies, especially bleomycin, mitomycin, and carmustine [82]. It is triggered by postcapillary venular inflammation causing thrombosis. This leads to pulmonary hypertension and right-sided heart failure. Pulmonary VOD rarely leads to pulmonary capillary hemangiomatosis (PCH), which is an angioproliferative response to postcapillary venular occlusion [83]. For pulmonary VOD and PCH, the pathogenetic factors downstream of venular damage are unknown. The role of platelets in thrombosis and maintaining postcapillary venular integrity [16] and the fact that platelets are rich sources of cytokine and growth factors [17] suggests the possibility of their involvement. There is no evidence, however, that antiplatelet therapy (or any therapy) favorably affects the natural history of pulmonary VOD or PCH. Acute lung injury (ALI) is a vague term for a common condition encompassing pneumonia, aspiration, sepsis, trauma,
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and even massive transfusion; its severest manifestation is known as “acute respiratory distress syndrome” [14]. The heterogeneity of causes defines multiple pathogenetic mechanisms, some of which include platelet-dependent abnormalities. For example, experimental data demonstrate that platelets are essential for establishing leukocyte–capillary endothelial interactions and subsequent alveolar inflammatory responses in animals with acid- or sepsis-induced ALI [14]. These data introduce the hypothesis that platelet P-selectin is a potential therapeutic target in ALI [84]. As another example, there is evidence that platelet lipid debris accumulating during bloodbank storage may contribute to transfusion-associated ALI (TRALI) [85]. Most cases of TRALI are from donors’ antibodies binding to recipient leukocytes resulting in activated host neutrophils being sequestered in the pulmonary microcirculation. Ten percent of TRALI cases, however, are not associated with leukocyte antibodies, and it is believed that most of these cases are due to the release of biologically active lipids from platelets during storage [86]. Platelets also appear to play a role in animal models of asthma in which allergen-induced leukocyte recruitment and airway inflammation are reduced when platelets are selectively depleted [87]. There is evidence of platelet and leukocyte activation and circulating leukocyte– platelet aggregates in humans with asthma [87] and even evidence of hyperreactive platelets, activated platelets, and circulating leukocyte–platelet aggregates in patients with cystic fibrosis [88]. Of note, however, is that there is no evidence for a beneficial effect of antiplatelet therapy with aspirin or a thieneopyridine on ALI, TRALI, asthma, or cystic fibrosis. Chronic high-dose ibuprofen may slow the progression of cystic fibrosis, but this is probably not a platelet-mediated effect [89].
11 Summary There is an impressive breadth and depth of information about platelets and how they function within Virchow’s triad to effect physiological hemostasis and pathological thrombosis. A similarly impressive information base – much of it immediately available within this vast tome – encompasses pulmonary vascular biological and pathophysiological processes. Little is known, however, about how platelets function in the lungs under normal and pathological conditions, or about how the lungs affect platelets in hemostasis and thrombosis. Obviously important and intriguing questions are unanswered. Is platelet production in the lungs important? Why does severe thrombocytopenia not cause hemoptysis? How is the pulmonary circulation protected against atherothrombosis and thrombotic microangiopathies? These questions are unanswered but not unanswerable. And although obtaining their answers is inevitable, they are likely to be most expedient if we continue to illuminate the platelet
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within the complexities of Virchow’s triad within the pulmonary compartment. By focusing on the platelet, we will elucidate mechanisms of pulmonary vascular hemostasis and thrombosis and develop hypotheses about innovative therapeutics probably relevant to diseases outside the pulmonary compartment. A great adventure awaits the platelet biologist who explores pulmonary vascular biology and pathology.
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M.H. Kroll were improved by continuous prostacyclin infusion. Circulation 102:2720–2725 78. Lopes AA, Maeda NY, Goncalves RC, Bydlowski SP (2000) Endothelial dysfunction correlates differentially with survival in primary and secondary pulmonary hypertension. Am Heart J 139: 618–623 79. Popat U, Frost A, Liu E et al (2005) New onset myelofibrosis in association with pulmonary arterial hypertension. Ann Intern Med 145:466–467 80. Zetterberg E, Popat U, Hasselbach H et al (2008) Angiogenesis in pulmonary hypertension with myelofibrosis. Haematologica 93:945–946 81. Haznedaroglu IC, Atalar E, Ozturk MA et al (2002) Thrombopoietin inside the pulmonary vessels in patients with and without pulmonary hypertension. Platelets 13:395–399 82. Williams LM, Nelson S, Mason CM et al (1996) Pulmonary venoocclusive disease in an adult following bone marrow transplantation: case report and review of the literature. Chest 109:1388–1391 83. Lantuejoul S, Sheppard MN, Corrin B et al (2006) Pulmonary venoocclusive disease and pulmonary capillary hemangiomatosis. Am J Surg Pathol 30:850–857 84. Kuebler WM (2006) Selectins revisited: the emerging role of platelets in inflammatory lung disease. J Clin Invest 116:3106–3108 85. Silliman CC, Bjornsen AJ, Wyman TH et al (2003) Plasma and lipids from stored platelets cause acute lung injury in an animal model. Transfusion 43:633–640 86. Loonet MR, Gropper MA, Matthay MA (2004) Transfusion-related acute lung injury: a review. Chest 126:249–258 87. Pitchford SC, Yano H, Lever R et al (2003) Platelets are essential for leukocyte recruitment in allergic inflammation. J Allergy Clin Immunol 112:109–118 88. O’Sullivan BP, Linden MD, Frelinger AL III et al (2005) Platelet activation in cystic fibrosis. Blood 105:4635–4641 89. Konstan MW, Byard PJ, Hoppel CL, Davis PB (1995) Effect of high-dose ibuprofen in patients with cystic fibrosis. N Eng J Med 332:848–854
Chapter 25
Lysis and Organization of Pulmonary Thromboemboli Timothy A. Morris and Debby Ngo
Abstract After an acute pulmonary thromboembolism, thrombotic material in the pulmonary arteries either resolves by fibrinolysis or remodels into scars within the pulmonary arterial lumen. This chapter focuses on the balance between these two processes and how perturbations in this fine balance may contribute to the development of permanent vascular obstruction within the pulmonary circulation. Keywords Fibrinolysis • Fibrinogen • Fibrin • Pulmonary embolism • Chronic thromboembolic pulmonary hypertension • Thrombosis
1 Introduction After an acute pulmonary thromboembolism, thrombotic material in the pulmonary arteries either resolves by fibrinolysis or remodels into scars within the pulmonary arterial lumen. This chapter focuses on the balance between these two processes and how perturbations in this fine balance may contribute to the development of permanent vascular obstruction within the pulmonary circulation.
reasonable to expect that erythrocytes entrapped within thrombi would disintegrate within this period. The duration for which fibrin persists within thromboemboli may be inferred by plasma levels of fibrin d-dimer, a principal fragment resulting from fibrinolytic digestion. When patients with pulmonary thromboembolism are treated with anticoagulants to prevent the deposition of new fibrin, elevated blood d-dimer levels can be found in about two fifths of patients at the end of 1 month [4]. However, by 3 months only about one tenth of patients remain positive and about half of those are positive due to recurrence. Residual defects after acute pulmonary thromboembolism commonly persist beyond the expected survival of erythrocytes and fibrin [5]. It follows from these observations that long-term residual defects within the pulmonary arteries would be attributed to processes beyond the initial thrombotic episode. Recent experimental data suggest that early after pulmonary thromboembolism, inflammation and associated intimal hyperplasia occur within the pulmonary arteries (Fig. 1b–d) [6], similar to what is observed with chronic thromboembolic pulmonary hypertension [7, 8]. The extent to which this histological process occurs in actual patients must be inferred from clinical outcome studies.
2 Components of Acute Thromboemboli Acute thromboemboli themselves are impermanent structures. Histologically, they are composed of layers of platelets, fibrin, and erythrocytes [1]. Fibrin-entrapped erythrocytes constitute most of the volume in acute thromboemboli (Fig. 1a). The life expectancy of new erythrocytes within the circulation is between 2 and 4 months [2, 3] and it seems
T.A. Morris (*) Division of Pulmonary & Critical Care Medicine, University of California, San Diego, 200 W. Arbor Drive, San Diego, CA 92103, USA e-mail:
[email protected] 3 Recovery of Perfusion After Acute Pulmonary Thromboembolism 3.1 Normalization of Perfusion In some patients after an acute thromboembolism, lung perfusion is rapidly restored, suggesting that fibrinolysis had been accelerated [9]. More typically, the resolution in the first week is incomplete and is followed by a slow recovery over the next 1–2 months [10, 11]. After this recovery phase, it is likely that residual perfusion defects reflect persistence of material other than fibrin-enmeshed erythrocytes.
J.X.-J. Yuan et al. (eds.), Textbook of Pulmonary Vascular Disease, DOI 10.1007/978-0-387-87429-6_25, © Springer Science+Business Media, LLC 2011
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Fig. 1 Histologic sections from patients with unresolved pulmonary thromboembolism, leading to chronic thromboembolic pulmonary hypertension (CTEPH). Hematoxylin and eosin stained sections of pulmonary thromboemboli at various stages of fibrotic organization. (a) A ×10 view shows an acute thrombus with prominent fibrin, platelets, and
T.A. Morris and D. Ngo
erythrocytes. (b) A ×40 view shows inflammatory cell infiltration into the luminal edge of a thrombus. (c) A ×200 view shows fibrotic organization occurring in a region of the thrombus that had been infiltrated by inflammatory cells. (d) A ×40 view shows thrombus organized into scar tissue with fibroblast infiltration and dense collagen deposition Table 1 Normalization of perfusion after acute pulmonary thromboembolism (PE ) Patients with normalization Time interval after Patients of perfusion lung scan (%) acute PE Study (n)
Fig. 2 Serial lung perfusion scans measure perfusion recovery after 6 months of treatment for acute pulmonary thromboembolism. The patient depicted in the top row resolved 100% of the (34%) perfusion defect he demonstrated upon presentation. In contrast, the patient in the bottom row resolved only 7% of his original (43%) defect
One method of characterizing perfusion recovery after pulmonary thromboembolism (PE) is to examine the proportion of patients who have completely recovered lung perfusion at specific time points (Fig. 2). Several clinical studies disclosed that resolution of perfusion continues throughout the first several months of therapy, but that complete recovery (defined as the development of a scintigraphic perfusion scan with no regional perfusion defects) is far from universal. A retrospective cohort study of 102 patients with first-time submassive PE treated with heparin followed by oral anticoagulation for a minimum of 6 months disclosed complete resolution of defects in 6% of patients (six of 97) at 7–10 days and in only 27% of patients (26 of 96) by 6 months [12]. Another series, in which 74 patients received lung scans at various intervals during the first year after acute PE, reported that complete restoration of perfusion occurred in only two patients [13]. A series reporting serial perfusion scans in 69 patients with confirmed PE demonstrated complete resolution in only 46% of patients (32 of 69) at 4 months [11]. A smaller group
Menendez et al. [12]
96a
Walker et al. [13] Tow et al. [11] Murphy et al. [14] Hvid-Jacobson et al. [15] Palla et al. [16] Winebright et al. [17]
74 69 25 30 69 70
6 27 2 46 60 43
7–10 days 6 months 6 weeks to 1 year 6 months 5 months 6 months
0 3 33 13 34 84 b 75c 27
6 months 5 days 60 days Wartski et al. [5] 157 8 days 3 months UPET [18] 105 1 year Miniati et al. [19] 235 1 year Paraskos et al. [20] 33 1–7 years (mean 29 months) Hall et al. [21] 48 42 1–8 years Sutton et al. [22] 18 56 1–8 years De Soyza et al. [23] 13 69 25–59 months (mean 49 months) a n = 96 patients evaluated for complete resolution at 7–10 days b Near-normal perfusion reported and defined as less than 10% perfusion defect c Near-normal perfusion reported and defined as less than 5% perfusion defect
of patients with angiographically proven PE showed a 60% (15 of 25 patients) incidence of complete resolution at some point during a 5-month follow-up period [14] (see Table 1). There is some discordance among the clinical reports about the prevalence of complete recovery several months after PE. For example, one 6-month follow-up of 30 patients reported complete resolution in 43% of PE patients [15], whereas follow-up for a similar period in another group of 69 PE patients did not identify complete restoration of blood flow in a single patient [16]. However, all studies disclosed a
25 Lysis and Organization of Pulmonary Thromboemboli
significant proportion of patients with incomplete perfusion recovery during the first half year after pulmonary thromboembolism. In one long-term follow-up series, perfusion scans were repeated in 70 acute PE patients (without other lung abnormalities) every 5 days for 1 month, then again at 3 months, 6 months, and up to 16 months later [17]. The frequency of perfusion recovery was only 3% (two of 70 patients) at 5 days, but increased to 33% (23 of 70 patients) by 60 days, without further changes after that point. Although perfusion recovery was not precisely defined, which makes the results difficult to compare with those of the other reports, it was clear that some patients recovered relatively rapidly, whereas the rest recovered slowly or incompletely. Several studies have accepted “near-normal” perfusion on follow-up perfusion scans as evidence of complete recovery. Defining a near-normal perfusion scan as showing an overall perfusion defect of 5% or less, the Tinzaparin ou Heparin Standard: Evaluation dans l’Embolie Pulmonaire Study (THESEE) study group reported complete recovery in 13% of patients (21 of 157) at 8 days and in 34% of patients (53 of 157) at 3 months [5]. When a more forgiving cutoff of less than 10% perfusion defect was used to indicate recovery, the recovery rate only increased to 41% (65 of 157 patients) at 3 months. Another series reported only a 3% frequency of completely normal perfusion after 3 months and near-normal recovery (defined as residual perfusion defects of less than 10.5%) in 33% of patients (24 of 74) [13]. The differences in the reported outcomes may reflect different patient populations and different criteria for detecting residual perfusion defects. However, it was typical in these series that more than half of the patients failed to completely resolve their perfusion defects during the first 6 months of follow-up. Some series have demonstrated that many patients fail to recover their perfusion, even years after their acute PEs. Of 105 patients followed out to 1 year in the Urokinase Pulmonary Thromboembolism Trial (UPET), the perfusion scan was near normal (defined as perfusion defect less than 10%) in 84% of patients (88 of 105), with no significant changes in the proportion of near-normal scans between 3 months and 1 year [18]. Another recent study reported near-normal perfusion in 75% of patients 1 year after PE [19]. However, in a different series, repeat lung scans 1–7 years (mean 29 months) after acute PE disclosed complete perfusion recovery in only 27% of patients (nine of 33) [20]. Another study, limited to patients surviving acute massive PE (defined as the involvement of more than half of the major pulmonary arteries), performed repeat lung scans 1–8 years later and reported complete recovery in only 42% of patients (20 of 48) [21]. Somewhat smaller studies have shown similar results. Repeat perfusion scans in a series of
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18 patients 1–8 years after “minor” or “subacute massive” PE demonstrated recovery in 56% of patients (ten of 18) [22], whereas 13 male patients studied 25–59 months (mean 49 months) after PEs associated with pulmonary hypertension showed a perfusion recovery rate of 69% (nine of 13 patients) [23]. Despite the heterogeneity of the clinical data, several generalizations can be made with respect to the time course of perfusion recovery after acute PE. The percentage of patients experiencing complete scintigraphic resolution during the first week is very low. By 1 month, roughly one fifth to one third of patients on average will go on to complete recovery. Between 1 and 3 months, an additional small percentage of patients may recover. After 3 months, additional patients will recover, although the data vary so widely that it is difficult to draw conclusions regarding the proportion who will eventually recover perfusion completely. The likelihood of complete recovery is influenced by the size of the initial defect as well as the existence of other cardiopulmonary diseases. Larger initial perfusion defects lower the incidence of complete recovery at all time points after PE. Short-term recovery of perfusion occurred in one third of patients with small initial perfusion defects (less than 15%), but only in about one fifth of those with larger defects [17]. Likewise, the frequency of complete resolution 4 months after PE varied from 67% in patients with minimal initial perfusion defects (15% or less) to 20% in patients with severe defects (31–50%) [11]. Walker et al. observed complete recovery in 49% of patients whose initial defects were less than 30% compared with only 14% among those with initial defects of 30% or greater, a statistically significant difference [13]. Pre-existing cardiac or pulmonary disease at the time of PE has been associated with a lower likelihood of complete resolution of perfusion defects. One series reported complete recovery by 3 months in 32% of patients (16 of 51) without heart disease compared with only 5% of patients (one of 19) with heart disease [17]. A longer follow-up of 48 patients surviving acute massive PE reported complete scintigraphic normalization in 46% of patients without concomitant cardiopulmonary disease versus 22% of patients with concomitant cardiopulmonary disease [21]. These observations may reflect slower rates of PE resolution in these patients or may be explained by the effect of the concomitant diseases themselves on lung perfusion.
3.2 Partial Recovery of Perfusion Although complete recovery of perfusion is by no means universal after acute PE, relative recovery occurs in most
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patients and continues at least throughout the first 6 months. Perfusion recovery can be characterized by the average amount of perfusion recovered relative to the initial perfusion defect. Relative perfusion recovery = (initial defect-defect on follow-up)/(initial defect), where defects are expressed as proportions of the normal perfusion to both lungs. Most studies disclosed remarkably similar patterns of relative perfusion recoveries after acute PE. In 69 patients with PE followed with serial lung scans, the mean improvement by 6 months was 59% of the initial perfusion impairment [16]. In a similar group of 33 patients with PE, serial lung scans disclosed a mean relative perfusion recovery of about two thirds by 6 months [24]. Serial scans for 1 year in 61 patients with PE showed a mean relative perfusion recovery of 64%, with most of the recovery within the first 6 months [25]. In a series of 96 PE patients, the number of unperfused lung segments improved by 41% at 7–10 days and by 68% at 6 months [12]. Finally, among the 157 patients from the THESEE group, the estimated vascular obstruction decreased by 41% at 8 days and by 61% at 3 months [5]. An earlier and somewhat smaller study [14] reported faster recovery in 25 patients with angiographically proven PE (63% mean relative perfusion recovery by 1 week, 67% by 2 weeks, 80% by 3 weeks, and 80% by 4 weeks), although these results have not been observed in other studies. The rate of recovery is most rapid during the first month [13, 16, 24, 25], which accounts for about three fourths of the entire recovery that will occur [16, 24]. On average, the relative perfusion recovery during this phase falls somewhere between 30 and 50%. During the time period from 1 to 3 months, additional perfusion is recovered, but this improvement is not as substantial or as rapid as that seen during the first month. After 3 months, additional recovery occurs at an even slower rate, if it occurs at all (see Table 2). Table 2 Relative recovery of perfusion after acute PE Mean relative perfusion Time interval Study Patients (n) recovery (%) after acute PE Palla et al. [16] Prediletto et al. [24] Donnamaria et al. [25]
69 33 61
Menendez et al. [12]
96
Wartski et al. [5]
157
Murphy et al. [14]
25
59 60 59 64 41 68 41 61 63 67 80 80
6 months 6 months 6 months 12 months 7–10 days 6 months 8 days 3 months 1 week 2 weeks 3 weeks 4 weeks
The relative perfusion recovery after PE is typically consistent among patients, regardless of the size of the initial perfusion defect. Three separate studies disclosed no significant differences in the relative recovery of perfusion between patients with large initial defects and those with smaller ones when the subjects had repeat perfusion scans at 3 months [5, 17] or 4 months [11]. It follows that the size of the initial PE-related perfusion defect influences the size of the residual defects during follow-up. Among 29 PE patients without other cardiopulmonary disease, those with initial defects of greater than 30% had an average defect of 18.7% after 6 weeks, compared with an average defect of 9.9% for those with smaller initial defects [13]. Two large multivariate analyses of various factors affecting resolution after PE also identified the extent of the defects at 7–10 days as predictive of the extent of residual defects measured at 6 months [12, 26]. Relative perfusion recovery is influenced by the presence of underlying cardiopulmonary disease. One series reported 50% or more relative perfusion recovery in 58% of patients without cardiovascular disease (23 of 40) compared with 44% of patients with cardiovascular disease (12 of 27) [11]. Another series showed the frequency of 50% or greater relative perfusion recovery at 90 days to be 93% in patients without cardiac disease compared with 58% in patients with cardiac disease [17]. Six months after the initial PE, 35 patients without cardiopulmonary disease had a mean relative perfusion recovery of 70% compared with 45% relative perfusion recovery among 34 patients with cardiopulmonary disease [16]. Among 157 patients from the THESEE study scintigraphically followed out to 3 months, those with associated cardiac or pulmonary disease experienced a relative recovery of 57%, with pulmonary vascular obstruction score improving from 53 to 23%, whereas those without associated cardiac or pulmonary disease experienced a relative recovery of 68%, with pulmonary vascular obstruction score improving from 44 to 14% [5]. One may expect that in acute PE patients with pre-existing cardiopulmonary disorders the degree of perfusion recovery will be about one third less than the degree of recovery in other patients with acute PE. It is likely that underlying cardiopulmonary conditions themselves are responsible for some of the perfusion abnormalities observed during PE, but it is also possible that underlying cardiopulmonary disease predisposes to persistent or recurrent thromboembolism. The contribution of each of these factors is difficult to sort through, since the persistence of a perfusion defect in the same position from one scan to another may reflect the failure of an initial thrombus to resolve, the recurrence of thrombus in the same location, or simply decreased perfusion from parenchymal or cardiac disease.
25 Lysis and Organization of Pulmonary Thromboemboli
Fig. 3 Gross pulmonary thromboendarterectomy specimen from a CTEPH patient with bilateral disease, showing segmental and subsegmental extension
3.3 Clinical Importance of Incomplete Recovery of Perfusion In up to 4% of PE patients, pulmonary artery scarring (Fig. 3) develops into symptomatic chronic thromboembolic pulmonary hypertension (CTEPH). CTEPH, however, may be the extreme manifestation of a much more common phenomenon. For example, up to 15% of acute PE patients remain symptomatically compromised 2 years after treatment [27] and may have abnormal pulmonary gas exchange (O2 gradients, dead space, etc.) as well [24]. Some of the hemodynamic manifestations of pulmonary emboli may also persist beyond the acute episode. Serial echocardiography of PE patients suggests a rapid, but often incomplete phase of recovery in the systolic pulmonary artery pressure (as estimated from the tricuspid regurgitation jet velocities) during the first month, followed by a much more gradual rate of recovery in the subsequent year [28, 29]. High pulmonary artery pressures during the initial phase were associated with increased risk of persistent pulmonary hypertension, further supporting the etiological role of incomplete thrombus resolution in the development of chronic disease. These findings suggest that incomplete clot resolution has clinical manifestations in a significant proportion of acute PE patients. Since the most severe manifestation of this phenomenon is CTEPH, it serves as a model to evaluate the mechanisms of unresolved PE.
4 Pharmacologically Enhanced Fibrinolysis The long-term effect of plasminogen-activating drugs on the resolution of acute PE, and the likelihood of CTEPH, is unclear. Management decisions must be made on the basis of
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indirect information rather than by comparative clinical trials in the appropriate patient population. The Urokinase in Pulmonary Thromboembolism Trial (UPET) [18], the largest trial to randomize patients into thrombolytic or standard heparin therapy, was performed on hemodynamically stable patients. There was a very shortterm hemodynamic benefit: cardiac outputs and pulmonary pressures were improved on the day after thrombolytics. No positive effects on morbidity or mortality were demonstrated, however, nor was the ultimate degree of embolic resolution any greater. Multiple studies have confirmed that these agents promote rapid embolic resolution [30]. However, there does not appear to be a long-term benefit to more rapid (but not necessarily more complete) clot resolution [31]. Patients with massive PE have much higher mortality rates than the patients evaluated in the UPET. The mortality for patients with high right ventricular pressures and high pulmonary artery pressures is about double the mortality for more stable PE patients. The mortality increases exponentially as patients progress to hypotension, shock, and then the need for cardiopulmonary resuscitation [32]. It is likely that the acute hemodynamic benefits of thrombolytics will have a much higher impact on these patients than on the stable patients in the UPET. The outcome benefit of thrombolytics in unstable patients has been studied in uncontrolled, retrospective studies. One study reported a lower mortality in patients treated with thrombolytics [32], whereas another study reported a higher mortality (due to bleeding) [33]. A study of “submassive” PE (i.e., with evidence of right ventricular strain) [34] randomized patients to either thrombolytics plus heparin or heparin plus a placebo. The groups were compared on the basis of a “combined” primary end point including death and escalation of treatment (including subsequent thrombolytic therapy). Although the group receiving thrombolytics had a lower incidence of the combined end point, the only actual “outcome” difference between the groups was that those treated with heparin plus a placebo received more thrombolytic therapy during the follow-up period. No other outcome, such as death and the need for mechanical ventilation or vasopressors, was different between the groups, begging the question of whether “rescue” thrombolytic therapy was necessary. Unfortunately, the results did not answer the question of whether thrombolytic therapy is beneficial initially or even during followup for patients with evidence of right-sided heart strain. Thrombolytics are costly and carry significant risks. A review of clinical trials disclosed a 2.1% intracranial hemorrhage rate and 1.6% fatal intracranial hemorrhage rate when thrombolytics were used for thromboembolic disease [35]. For this reason, thrombolytics should be reserved for management of the patient with massive embolism and
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p ersistent hypotension. Even in this situation, patient selection is difficult and only physicians quite familiar with the drugs should use them.
5 CTEPH as a Model of Unresolved Acute PE CTEPH can be induced following an acute PE in an animal model by inhibition of plasmin-mediated fibrinolysis with tranexamic acid. Similar to e-aminocaproic acid, tranexamic acid inhibits fibrinolysis by attaching to lysine-binding sites on plasminogen, preventing its activation [36]. In the absence of pharmacological inhibition, the canine fibrinolytic system is typically so efficient that acute pulmonary emboli would be nearly undetectable after 30 days [37]. However, dogs treated after PE with tranexamic acid and autopsied 30–40 days later had large amounts of chronic, organized material adherent to the pulmonary arterial wall, similar to the material observed in patients with CTEPH. Furthermore, the animals with large amounts of the intra-arterial organized material had persistent perfusion defects on scintigraphic scanning and significantly elevated pulmonary vascular resistances, also similar to patients with CTEPH. Clinically, incomplete recovery of perfusion may predispose patients to developing CTEPH [38]. The reason why a fraction of patients with PE go on to have CTEPH is unclear. Most patients who present with CTEPH who were previously diagnosed with acute pulmonary emboli had initial presentations (clot burden, clinical stability, etc.) that were indistinguishable from those of patients who went on to resolve their acute clots [39]. A prospective study of patients presenting with PE disclosed that, in addition to the size of the initial PE, factors such as recurrent PE, idiopathic PE, and PE at a young age were associated with a higher risk for CTEPH [38]. These findings raise the possibility that an inherited or acquired predisposition to incomplete clot resolution is present in at least some patients who develop CTEPH. Although the specific causes leading to incomplete thrombus resolution after PE are not fully understood, some insights may be gained by laboratory and clinical investigations into the cause of CTEPH. It stands to reason that delayed fibrinolysis could be implicated in the development of CTEPH if the acute pulmonary thromboembolism created a substantial local nidus for remodeling and intravascular scar formation. The resulting persistent large vessel obstruction combined with various degrees of an incompletely understood small vessel reaction lead to progressively worsening pulmonary hypertension [40]. For these reasons, investigation has focused on two areas: (1) the persistence of acute thromboemboli themselves (e.g., inadequacy of or resistance to fibrinolysis) and (2) mechanisms of thrombus replacement by inflammatory cells and connective tissue.
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6 Fibrinolytic Enzymes and CTEPH Despite extensive studies, no specific abnormalities of fibrinolytic mediators that could disturb the balance between plasminogen activators and their inhibitors, or regulate the conversion of plasminogen to plasmin have been discovered in CTEPH patients [41]. There have been no abnormalities in the plasma levels of plasminogen or a2-antiplasmin (the primary physiological inhibitor of plasmin) associated with CTEPH. A study of 32 CTEPH patients revealed neither high resting plasma levels of plasminogen activator inhibitor 1 (PAI-1; the primary physiological inhibitor of tissue plasminogen activator, tPA) nor blunted response of tPA to venous occlusion [42]. In a separate study to determine if failure to lyse pulmonary thromboemboli might be caused by an abnormality in the endothelial-cell-associated fibrinolytic system, endothelial cells were scrape-harvested from the pulmonary arteries of 13 CTEPH patients during pulmonary thromboembolism surgery [43]. Conditioned medium from primary cultures of these cells was analyzed for levels of tPA and PAI-1; cells harvested from pulmonary arteries of organ donors were used as controls. No significant differences between patients and controls were noted. Moreover, CTEPH patient cells were observed to increase the secretion of tPA and PAI-1 in response to thrombin in a fashion similar to cells from organ donors.
7 Fibrinogen and Susceptibility to Fibrinolysis There is evidence to suggest the fibrin clots themselves are relatively resistant to lysis in patients with CTEPH [44]. This section will explore the components of thrombus structure and fibrinolysis that have been investigated as possible etiological factors responsible for CTEPH. Fibrinogen is a dimeric glycoprotein, composed of three pairs of nonidentical, disulfide-linked polypeptide chains (Aa2Bb2g2) [45]. The thrombin-catalyzed conversion of fibrinogen to fibrin involves the sequential removal of fibrinopeptides A and B from the N-terminal regions of the Aa and Bb chains, respectively (Fig. 4a, b). The appearance of new N-terminal sequences on the a and b chains creates “A knobs” and “B knobs,” respectively, in the central domain of the fibrin molecule. The A knobs and B knobs fit noncovalently into complementary “holes” in a terminal globular domain of an adjacent molecule (Fig. 4c). Fibrin then spontaneously polymerizes into dimers that form half-staggered protofibrils. The protofibrils are then stabilized by covalent cross-links that occur first between the g chains and then between the a chains [46]. The organization of these protofibrils results in a
25 Lysis and Organization of Pulmonary Thromboemboli
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herin (VE-cadherin) [48]. Fibrin induces expression of interleukin-8 and facilitates the migration of repair cells along endothelial cells by interactions with intracellular adhesion molecule-1 (ICAM-1) [49] and the leukocyte adhesion receptor Mac-1 [50] (see Fig. 5).
7.1 Biological Properties of the Fibrin B Chain N-Terminus (B Knob) That May Affect Thrombus Remodeling
Fig. 4 Fibrin polymerization and fibrinolysis. (a) Fibrinogen contains fibrinopeptide A (FPA) and fibrinopeptide B (FPB) on the N-terminal region of the Aa and the Bb chain, respectively. (b) Thrombin-induced cleavage of fibrinopeptides exposes the fibrin “A knobs” and “B knobs.” The B knobs (arrow) are shown in gray. (c) The exposed A and B knobs fit into complementary holes in a terminal globular domain of an adjacent molecule, allowing fibrin to polymerize into thick network of fibers. (d) Plasmin (arrows) degrades the fibrin polymers during fibrinolysis. Remodeling cells represent endothelial and fibroblast cells interacting within the dense fibrin network and forming intraluminal connective tissue
thick network of fibers that form the structural backbone of the acute thrombus. It is this network that is ultimately degraded by the proteolytic action of plasmin (Fig. 4d) Fibrinogen may also be important to pulmonary artery remodeling. Fibrin activates pulmonary artery endothelial cells [47] via the fibrin receptor vascular endothelial cad-
The fibrin B knob has been implicated in a variety of physiological events such as heparin binding [51], cell signaling [48], and angiogenesis [52] that may be involved with thrombusinduced pulmonary artery scarring. Fibroblastic and endothelial cell growth onto fibrin polymers (the first step in organization of the thrombus into scar tissue) is stimulated by peptides found at the B knob (Fig. 4d) [53]. Endothelial cell proliferation can be stimulated by the E fragment of fibrin, which contains the B knob, as well as peptide analogues of the B knob. Fibrin fragment E stimulates the proliferation, migration, and differentiation of microvascular endothelial cells in vitro, both in the presence and in the absence of additional growth factors, including vascular endothelial growth factor and basic fibroblast growth factor (bFGF) (Fig. 5). The N-terminus of the b chain occurs in a very flexible part of fibrinogen, which cannot be resolved by X-ray crystallography [54]. The only human variant found within this region involves substitution of the N-terminal Gly, possibly owing to the persistence of fibrinopeptide B and consequently unexposed B knob [55]. However, fibrinogen modifications remote from the N-terminus itself may also affect its plasminmediated removal. For example, the prothrombotic fibrinogen Nijmegen [56] involves a b44 Arg → Cys substitution that may result in steric interference with plasmin-mediated release of the B knob through disulfide bonding [55]. Much of the lytic action of plasmin occurs at the coiledcoil regions that connect fibrin’s globular domains. However, some of the fibrin fragments that plasmin releases encompass only residues close to the N-terminus of the b chain, which is not involved in the coiled-coil regions (Fig. 4d). Cleavage of the N-terminus of the b chain would not help separate the globular regions of fibrin from each other, but may influence the balance between fibrinolysis and cellmediated thrombus remodeling. Thrombi would also enhance the stimulation of repair cells if their B knobs were more accessible to binding (even if the number of B knobs themselves were normal). Binding experiments with T2G1 antibodies (specific for the fibrin B knob) disclosed that less opaque “fine” fibrin clots, composed of thinner fibers [57], have greatly increased accessibility of the B knobs, compared with “coarser” fibrin clots [58, 59]. It stands
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Fig. 5 The factors implicated in remodeling of thrombi into intravascular scars and the interactions between fibrin and inflammatory cells and mediators involved in pulmonary vascular remodeling (see the text). MCP1 monocyte chemoattractant protein 1, bFGF basic fibroblast growth factor, EC endothelial cell
to reason that abnormally “fine” clots, with more accessible B knobs, would be more likely to stimulate fibroblastic and endothelial-cell-mediated thrombus organization [53] than would normal, coarser clots, regardless of lysis rates.
7.2 Resistance to Fibrinolysis and CTEPH Fibrinogen variants might be involved with CTEPH development. For example, five heterozygous fibrinogen gene mutations (fibrinogensSan Diego 1-5) with abnormalities in fibrin polymer structure and susceptibility to lysis were discovered in a series of 33 CTEPH patients (Fig. 6) [60]. Another variant, fibrinogenBellingham, includes a g275 Arg → Cys substitution that confers a relative resistance to tPA-mediated fibrinolysis [61]. The mechanism of resistance was presumably related to steric interference with the normal polymerization/cross-linking structure owing to the cysteine residue or a structure bound to it. Interestingly, family studies of fibrinogenBellingham demonstrated that the mutation alone was not sufficient to cause CTEPH. Rather, CTEPH required the combination of acute PE (in the reported case, due to knee replacement surgery as well as an additive thrombogenic genetic risk factor) along with resistance to fibrinolysis. Relatives with the g275 Arg → Cys substitution alone did not develop CTEPH, nor do most other individuals with the g275 Arg → Cys substitution. For this reason, the absence of disease in family members of CTEPH patients does not rule out the presence of fibrinogen abnormalities.
Fig. 6 CTEPH-associated mutations: fibrinogens (SD-1–SD-5). The positions of five CTEPH-associated mutations are illustrated on a ribbon model of fibrinogen. In fibrinogen SD-1 and fibrinogen SD-5, negative charges are imparted to regions within fibrin, promoting the formation of “thin” fibrin fibers. In fibrinogen SD-2 (gY114H) and fibrinogen SD4 (AaL69H), amino acid substitutions result in the insertion of a polar side chains within the “helix-permissive” hydrophobic center of the fibrin coiled coil, which would disrupt the molecular structure. In fibrinogen SD1–SD3, the Bb P235L substitution imparts a structural change to a site that is involved in the activation of tissue plasminogen activator by fibrin
8 Acute Thromboemboli, Inflammation, and Remodeling Incomplete thrombus resolution would result in pulmonary vascular scarring after an acute PE only if there was a persistence of ligands within the residual thrombotic material that would stimulate inflammation and remodeling by connective tissue cells. Interestingly, there is a clinical association between chronic inflammatory conditions and the development of CTEPH after acute PE (Table 3) [62].
25 Lysis and Organization of Pulmonary Thromboemboli Table 3 Conditions that have been clinically associated with the development of chronic thromboembolic pulmonary hypertension (CTEPH) after acute PE Conditions associated with CTEPH Clinical • Idiopathic cause of the PE [38] • Right-sided heart strain during acute PE [28, 38, 81] • Age [38] • Previous splenectomy [82–84] • Ventriculoatrial shunts (treatment of hydrocephalus) [62, 82–84] • Chronic inflammatory disorders (e.g., osteomyelitis, inflammatory bowel disease) [62, 82–84] Laboratory • Lupus anticoagulant [85] • Elevated factor VIII levels [83] • Non-O blood groups [84] • Lipoprotein (a) [86] • Heart-type fatty acid binding protein [87]
Several inflammatory mediators may be stimulated by thrombosis, and may be implicated in the connection between acute pulmonary thromboemboli and the vascular scaring associated with CTEPH [63]. Fibrin(ogen) has been implicated in biological processes such as angiogenesis in wound healing, tissue repair, tumorigenesis, and cardiovascular disease. Fibrin has been linked to mediators that facilitate interaction of endothelial cells with each other and extracellular matrix molecules (Fig. 5).
8.1 VE-Cadherin The interaction between fibrin and VE-cadherin can induce biological changes that lead to remodeling in the vascular wall (Fig. 5) [64]. Endothelial cells cultured on a fibrin-coated surface will form a dense monolayer; and if they are subsequently exposed to a fibrin gel, they will rearrange themselves along the gel’s surface into a network of capillary tubes [48]. This property of endothelial cells appears to require interaction with the fibrin B knob. Fibrinogen treated with thrombin (which removes fibrinopeptides A and B) is capable of inducing the endothelial cell reaction, whereas unaltered fibrinogen, fibrinogen treated with Atroxin (which removes only fibrinopeptide A), or fibrin treated with protease III (which removes the peptide Bb1-42 that encompasses the fibrin B knob) does not. Furthermore, the addition of “soluble B knob” (peptide b15-42) to medium influenced the endothelial cells’ morphological organization. These finding suggests that the fibrin B knob is an important stimulus of endothelial-cellmediated vascular remodeling by endothelial cells. The interaction with fibrin is specific to vascular endothelial cells and stimulation of vascular endothelial cells by the fibrin B knob appears to be mediated by VE-cadherin [48,
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64–66]. Adipose-derived endothelial cells expressing only N-cadherin but no VE-cadherin demonstrated no specific binding of fibrin B knobs [64]. VE-cadherin is a transmembrane cell–cell adhesion receptor with a modular structure that includes cytoplasmic, transmembrane, and five extracellular domains, four of which appear to be necessary for interaction with fibrin [65]. VE-cadherin regulates cell–cell contacts at endothelial intercellular junctions, and therefore plays an important role in maintaining endothelium integrity. The normal, highly avid, binding between endothelial cells and fibrin B knobs is disrupted by antibodies against residues 26 to 156 of VE-cadherin [48]. In addition, monoclonal antibodies against the first extracellular domain of VE-cadherin inhibited capillary tube formation and disrupted preformed capillary tube integrity in human umbilical vein endothelial cell monolayers [66]. Endothelial cells will bind to fragments of fibrin II (e.g., with fibrinopeptides A and B removed) that contain only the “N-terminal disulfide knot” (NDSK II), which contains the fibrin B knob. Endothelial cells do not specifically bind to NDSK fragments that do not contain the b15-42 peptide (Fig. 7) [64]. Furthermore, endothelial cell binding to NDSK II can be inhibited by a b15-42/ovalbumin conjugate as well as monoclonal antibodies to VE-cadherin [64]. Binding of VE-cadherin to fibrin analogues requires the B knob to be present in the configuration in which it typically occurs within the NDSK II. Normally, fibrin B knobs reside as closely apposed pairs (one from each peptide b chain) within NDSK II. Studies with recombinant peptides have demonstrated that VE-cadherin will bind to B knob analogues only if they are present in dimeric form, with the ligands at a distance from each other similar to what is observed in NDSK II (e.g., dimerized b15-66 peptides) [65].
8.2 E-Selectin and P-Selectin The role of selectins in the balance between thrombus resolution and vascular remodeling remains a topic of investigation. Some experimental data appear to implicate selectins in both thrombus formation and the subsequent inflammatory response to thrombosis (Fig. 5). Significant increases in P-selectin messenger RNA (mRNA) expression and blood levels of P-selectin have been observed in C57BL/g mice for 2 days after experimental induction of inferior vena cava (IVC) thrombi [67]. A subsequent rise in E-selectin mRNA and protein levels was observed 6 days after the induced thrombosis. No effect was observed in sham-treated subjects. Experiments with knockout mice for P-selectin and E-selectin also further elucidate the role of selectins after experimental thrombosis [67]. Knockout of E-selectin,
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Fig. 7 Accessibility of the fibrin B knob in various derivatives of fibrin(ogen). The fibrin B knob (shown in gray), which has been implicated in endothelial cell binding, is not exposed in fibrinogen because of the presence of the N-terminal FPB. The B knob is exposed (arrows) after the conversion of fibrinogen to fibrin II, by thrombin-mediated fibrinopeptide cleavage. Proteolysis of fibrinogen with cyanogen bromide generates the “N-terminal disulfide knot” (NDSK), which contains the intact N-termini of the Aa, Bb, and g chains, including FPA and FPB. NDSK treated with Atroxin releases FPA to create NDSK I. Neither product contains exposed B knobs. If NDSK is treated with thrombin to create NDSK II, in which FPA and FPB are cleaved, the B knob is exposed. NDSK treated with protease III to cleave the Bb1-42 peptide produces NDSK 325, which lacks the B knob entirely. Only the molecules that contained exposed B knob (fibrin II and NDSK II) are able to bind to endothelial cells (see the text)
P-selectin, or both resulted in a more rapid and more complete resolution of experimentally induced IVC thrombi. Immunohistochemistry confirmed that the thrombi formed in selectin-deficient mice contained significantly less fibrin content 2 and 6 days after thrombosis than thrombi from wild-type mice. P-selectin may have a specific role in thrombosis-related inflammation (Fig. 5) [68–70]. P-selectin is an adhesion molecule expressed on the surface of activated platelets and
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endothelium. It mediates their interaction with neutrophils, monocytes, and other inflammatory cells by binding to its ligands, predominantly the transmembrane sialylated and sulfated glycoprotein PSGL-1. P-selectin expressed on platelet membranes is a mediator of neutrophil and monocyte accumulation onto venous thrombi, which in turn enhances further fibrin deposition on the clot surface [71]. Once expressed on the surface of platelets, some of the P-selectin is released into the circulation. In patients with PE, the levels of both P-selectin on activated platelets and soluble P-selectin in the plasma are elevated [72]. P-selectin activates monocytes to produce tissue factor, further stimulating the clotting cascade [73], and induces neutrophils to activate protein kinases involved in inflammation [74]. P-selectin is also involved in platelet–platelet activation interactions, mediated through the counterreceptor glycoprotein Iba–IX–V [75]. P-selectin antagonism with P-selectin glycoprotein legend (rPSGL-Ig) and low molecular weight heparin significantly decreased the postthrombotic appearance of profibrotic mediators such as interleukin-13, monocyte chemoattractant protein 1, bFGF, and transforming growth factor b in a rat model of thrombosis (Fig. 5) [76]. The decrease in these mediators corresponded to a subsequent decrease in postdeep vein thrombosis vein wall fibrosis, which was independent of the masses of the original thrombi. P-selectin enhances leukocyte–leukocyte, leukocyte–platelet, and leuckocyte– endothelial interactions; it is plausible that these interactions might be implicated in the release of profibrotic mediators. This finding supports the role of P-selectin as a possible mediator of thrombus persistence, through the mechanism of postthrombotic inflammation and thrombosis.
8.3 Heparin, Low Molecular Weight Heparin, and Fondaparinx The role of P-selectin and E-selectin in the persistence of thrombi and the subsequent postthrombotic vascular inflammation may have implications for the pharmacotherapy of thrombosis. Heparin, its derivatives (low molecular weight heparin), and analogues (fondaprinux) are the mainstay of the initial therapy of acute pulmonary thromboembolism, but the mechanism by which these drugs encourage resolution has not been systematically studied. Experimental evidence suggests that, in addition to their anticoagulant properties, heparin-type medications may block selectin and attenuate the inflammation and remodeling that can lead to poor resolution of the thromboemboli within the pulmonary arteries (Fig. 5). However, different formulations of heparin and low molecular weight heparin have markedly different selectinblocking potencies in vitro, influenced largely by their patterns of saccharide sulfation [77]. Furthermore, the
25 Lysis and Organization of Pulmonary Thromboemboli
s electin-blocking potencies of heparin and its derivates appear to correspond with their ability to inhibit selectinmediated cellular growth in vivo [78]. However, the relative effects of these medications on the resolution of pulmonary thromboemboli has not been systematically studied.
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There is some experimental evidence to suggest that interleukin-10 (IL-10), an anti-inflammatory cytokine, may attenuate perithrombotic inflammation and thrombosis (Fig. 5). A rat model of thrombosis followed by local transfection with viral IL-10 demonstrated that local IL-10 expression can decrease leukocyte extravasation into the adjacent vascular wall caused by thrombus-related inflammation. In addition, immunohistochemistry studies demonstrated a lower concentration of P-selectin and E-selectin at endothelial cell borders. ELISA studies demonstrated a corresponding decrease in the cellular expression of P- selectin, E-selectin, and ICAM-1 [79]. IL-10 expression rises in response to thrombus formation, although it is not clear whether the increase in blood levels causes an attenuation of postthrombotic inflammation and remodeling [67]. An immediate rise after thrombosis, presumably due to the release of preformed IL-10, is followed by elevations in IL-10 mRNA and IL-10 protein expression for several days. However, experiments to date have not shown a consistent effect of IL-10 on thrombus resolution, so the clinical importance of this mechanism remains unclear [80].
exchange and elevated pulmonary artery pressures, and, finally, severe pulmonary vascular obstruction and hypertension seen in CTEPH. The pathophysiologic mechanisms related to poor thrombus resolution and its eventual organization into scar tissue are incompletely understood. Incomplete thrombus resolution may be due to resistance to fibrinolysis. Organization of the thrombotic material into scar tissue may be due to fibrin-mediated pulmonary vascular remodeling as well as thrombus-mediated inflammation. Mutations of fibrinogen associated with CTEPH may render it more resistant to fibrinolysis. As the initial clot fails to completely lyse, the surrounding pulmonary vascular system is persistently exposed to thrombotic components such as fibrin and platelets. Fibrin, in particular its B knob fragments, has been implicated in several physiologic events such as heparin binding, cell signaling, and angiogenesis that may be involved in thrombus-induced pulmonary artery scarring. Additionally, experimental data suggest involvement of E-selectin and P-selectin in thrombus formation and inflammatory response to thrombosis. Despite these findings, it still remains unclear why some patients with acute PE have poor thrombus resolution and why a few go on to develop CTEPH. There are many factors underlying the development of CTEPH that perturb the complex balance between coagulation and fibrinolysis and the normal processes of pulmonary vascular remodeling and inflammation. Although it is widely believed that there is a thromboembolic basis in the development of CTEPH, the detailed genetic, molecular, and cellular mechanisms underlying its pathogenesis have yet to be fully explained.
9 Conclusion
References
8.4 Interleukin-10
After an acute PE, thromboemboli resolve to varying degrees by fibrinolysis. In rare cases they develop into scars that cause the permanent pulmonary vascular obstructions seen in CTEPH. Although most patients undergo complete perfusion recovery, many patients fail to resolve perfusion defects even months to years after an acute PE. The degree to which resolution occurs is influenced by clinical factors, including the size of initial thrombus as well as coexistence of cardiopulmonary disease. However, the factors predisposing a patient to poor recovery after acute PE and the development of CTEPH have not been completely elucidated. CTEPH can serve as a model to evaluate mechanisms of unresolved pulmonary thromboemboli. It may represent the extreme clinical manifestation of a more common phenomenon of incomplete thrombus resolution. The spectrum of clinical consequences resulting from incomplete thrombus resolution includes no symptoms, persistent symptomatic compromise associated with abnormal pulmonary gas
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Chapter 26
Interactions of Leukocytes and Coagulation Factors with the Vessel Wall Scott Visovatti, Takashi Ohtsuka, and David J. Pinsky
Abstract Vascular endothelium is a highly specialized, metabolically active organ possessing numerous physiological, immunological, and synthetic functions. As a major capacitance reservoir which must accommodate passage of the entire circulating blood volume every minute, flow regulation and control of blood fluidity in the lung are essential. As a major component of the alveolar–capillary unit, the pulmonary endothelium is vulnerable to injury from multiple sources, including inhaled and circulating noxious agents, reactive oxygen species (ROS), and shear stress. Vascular damage triggers a complex and interconnected series of proinflammatory and procoagulation pathways that reinforce and even amplify one another. For example, in severe sepsis, proin-flammatory cytokines upregulate tissue factor (TF) on endothelium and monocytes, leading to systemic activation of coagulation. Conversely, coagulation proteases such as thrombin can bind to protease-activated receptors (PARs) on endothelial cells, mononuclear cells, platelets, fibroblasts, and smooth muscle cells, resulting in an upregulation of inflammatory mediators such as interleukin (IL)-1. Such interactions between the inflammatory and coagulation systems contribute to the pathogenesis of conditions such as acute lung injury (ALI), ventilator-associated lung injury (VALI), disseminated intravascular coagulation, and pulmonary hypertension. The purpose of this chapter is to explore intrinsic features and external factors that interact with pulmonary endothelium which cause a shift between a baseline antithrombotic/anti-inflammatory phenotype and an activated state of endothelium, characterized by prothrombotic/proadhesive properties. Given the complexities of these systems, each will be discussed separately in this chapter, with emphasis placed on areas of overlap. Keywords Coagulation • Blood-endothelium interaction • Thrombosis • Cell-cell adhesion • Barrier function • Lung injury • Reactive oxygen species • Inflammation
S. Visovatti (*) Division of Cardiology, University of Michigan, 1150 W. Medical Center Drive, Rm 7220, MSRB III, Ann Arbor 48109, MI, USA e-mail:
[email protected] 1 Introduction Vascular endothelium is a highly specialized, metabolically active organ possessing numerous physiological, immunological, and synthetic functions. From one vascular bed to another, endothelial cells exhibit a phenotypic heterogeneity that allows them to provide tailored functions for specific microenvironments [1]. For example, the spacing between endothelial cells can range from the continuous, nonfenestrated configuration that closely regulates fluid and solute transfer in the vasculature of the lungs and heart, to the discontiguous or sinusoidal endothelium found in the liver [1]. Barrier function varies widely between vascular beds, such as those of the brain and testes, where it is highly restrictive to the passage of macromolecules, and those of the dermis, where there is far less restriction. Other aspects of endothelial heterogeneity include variations in the number of clatherin-coated pits and caveolae [2]; expression of MHC I and II molecules, growth factors, chemokines, and integrins; and release of vasoactive molecules [1]. Such heterogeneity of form and function is especially apparent in pulmonary endothelial cells. As a major capacitance reservoir which must accommodate passage of the entire circulating blood volume every minute, flow regulation and control of blood fluidity are essential. As a major component of the alveolar–capillary unit, the pulmonary endothelium is vulnerable to injury from multiple sources, including inhaled and circulating noxious agents, reactive oxygen species (ROS) [3], and shear stress. Vascular damage triggers a complex and interconnected series of proinflammatory and procoagulation pathways that reinforce and even amplify one another. For example, in severe sepsis, proinflammatory cytokines upregulate tissue factor (TF) on endothelium and monocytes, leading to systemic activation of coagulation [4] (Fig. 1). Conversely, coagulation proteases such as thrombin can bind to protease-activated receptors (PARs) on endothelial cells, mononuclear cells, platelets, fibroblasts, and smooth muscle cells [5], resulting in an upregulation of inflammatory mediators such as interleukin (IL)-1 [6]. Such interactions
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Fig. 1 Interactions of inflammatory mediators and coagulation factors with vascular endothelium. The complex intersection between intravascular inflammation and coagulation. ADP adenosine diphosphate, AMP adenosine monophosphate, APC activated protein C, ATIII antithrombin III, CD39 ectonucleoside triphosphate diphosphohydrolase-1, eNOS endothelial nitric oxide synthase, EPCR endothelial protein C receptor,
GPIIb/IIIa glycoprotein IIb/IIIa, HSPG heparan sulfate proteoglycan, ICAM-1 intercellular adhesion molecule-1, IL-1 interleukin-1, NO nitric oxide, PAR protease-activated receptor, PGI2 prostacyclin, PSGL-1 P-selectin glycoprotein ligand-1, ROS reactive oxygen species, TF tissue factor, TFPI tissue factor pathway inhibitor, TM thrombomodulin, TNFa tumor necrosis factor-a, VWF von Willebrand factor
between the inflammatory and coagulation systems contribute to the pathogenesis of conditions such as acute lung injury (ALI), ventilator-associated lung injury (VALI), disseminated intravascular coagulation, and pulmonary hypertension. The purpose of this chapter is to explore intrinsic features and external factors that interact with pulmonary endothelium which cause a shift between a baseline antithrombotic/anti-inflammatory phenotype and an activated state of endothelium, characterized by prothrombotic/proadhesive properties. Given the complexities of these systems, each will be discussed separately, with emphasis placed on areas of overlap.
2 Interactions of Leukocytes with Pulmonary Endothelium: Proinflammatory Factors It has been recognized that endothelial cells orchestrate the immune response by shifting from their quiescent anti-inflammatory phenotype to an activated state characterized by proadhesive properties [7]. Stimulated neutrophils transmigrate along chemotactic gradients into lung tissue across the endothelium en route to sites of pathogen invasion or tissue damage due to metabolic, toxic, or physical insult. A critical event in this transformation is the expression of adhesion
26 Interactions of Leukocytes and Coagulation Factors with the Vessel Wall
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Fig. 2 Effect of ischemia on ICAM-1 expression. Immunohistochemical localization of ICAM-1 antigen expression in the lungs. Sites of binding of a primary mouse monoclonal anti-rat ICAM-1 antibody emit intense green fluorescence when exposed to an excitation wavelength of 475 nm. (a) ICAM-1 immunostaining of fresh (nontransplanted) lung. ICAM-1 appears to be expressed primarily in the structures of the
a lveolar wall. (b) ICAM-1 immunostaining of a lung that had been preserved for 4 h at 4°C, transplanted orthotopically into an isogeneic recipient, and then reperfused for 6 h. The endothelial lining of small pulmonary vessels stained intensely for ICAM-1, with an increased intensity of alveolar staining as well. Magnification ×3,250. (Reprinted from [15] with permission)
molecules on the endothelium surface [8], which serve as cognate ligands for their counterparts on leukocytes.
such as P-selectin glycoprotein ligand-1 (PSGL-1) [14]. E-selectin is rapidly synthesized by endothelium after cell activation by cytokines such as TNF-a and IL-1, or the bacterial wall product, endotoxin [12]. The second phase is firm neutrophil adhesion. This requires the interaction of the b2 (CD18) integrin family (more specifically the CD11/CD18 integrins) expressed on neutrophils, mainly with intercellular adhesion molecule (ICAM)-1, a member of the immunoglobulin superfamily expressed on endothelial cells [12]. ICAM-1 expression on endothelial cells is augmented by inflammatory mediators such as TNF-a, IL-1, g-interferon, and endotoxin. Inhibiting ICAM-1 expression significantly improves lung graft function after lung transplantation [15] (Fig. 2). Oxidant stress promotes neutrophil adhesion [16], and the aerated microenvironment of the lungs, particularly when reperfused, is a highly oxidizing environment. When neutrophils firmly adhere on the pulmonary endothelial layer, they create a microenviroment for injury, mainly via the production of proteases and ROS (i.e., oxidant burst), which induces cell injury and death. Neutrophil adherence to matrix proteins appears to prime the former for a massive burst lasting 1–3 h in response to a stimulus such as TNF-a. Activated endothelium also generates ROS, contributing to maintaining an oxidant-rich environment. In the lungs, therefore, there are multiple cellular sources and a plentiful supply of oxidizing molecules.
2.1 Adhesion Molecules (Intercellular Adhesion Molecule-1, P-Selectin, E-Selectin, Integrin) Pulmonary endothelium–leukocyte interaction is a key step in ALI development since alterations in cell–cell adhesion is an intermediate step in leukocyte migration from the capillaries into the lung parenchyma, and the subsequent inflammatory response. Neutrophils appear to be the key cell type driving pulmonary injury in ALI, although eosinophils and macrophages have also been implicated [9–11]. Neutrophil adhesion to endothelium is a multistage process and is necessary for successful neutrophil migration and extravasation. The initial phase, neutrophils rolling and capture, is mediated by cell adhesion molecules of the selectin family: L-selectin is constitutively expressed on neutrophils, P-selectin is found on platelets and endothelial cells, and E-selectin is expressed solely on endothelial cells [11, 12]. P-selectin is expressed within minutes on the endothelial surface after endothelial activation by a stimulus such as histamine, thrombin, bradykinin, leukotriene C4, or free radicals [13]. P-selectin interacts with neutrophil counterreceptors
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2.2 Cytokines, Soluble Factors (TNF-a , IL-1, IL-8, IL-10) Cytokines are soluble polypeptides serving as chemical messengers between cells. They are involved in processes such as cell growth and differentiation, tissue repair and remodeling, and regulation of the immune response [17]. Endothelial cells are both targets and cytokine producers. In the lungs, TNF-a and IL-1 are mainly produced by activated interstitial and alveolar cells (primarily macrophages), as well as endothelial cells, and have a major role in the early stages of ALI [18]. TNF-a and IL-1 share a number of biological properties, and markedly amplify each other’s biological actions [18]. They act on endothelial cells mainly by inducing a functional program that promotes thrombosis and inflammation [19]. Hypoxia, or tissue ischemia, is a stimulus which induces massive endothelial cell synthesis and release of IL-1a, resulting in an autocrine enhancement in the expression of adhesion molecules [20]. Hypoxia of endothelial cells also leads to synthesis of IL-8, which can recruit and activate neutrophils [21]. When proadhesive, proinflammatory paradigms are triggered, countervailing mechanisms swing into action. IL-4 and IL-10 have been described to have suppressive effects on TNF-a, IL-1, and inducible nitric oxide synthase (iNOS) [22, 23]. Recombinant IL-10 given to IL-10-null mice reduced pulmonary vascular fibrin deposition, and rescued mice from ALI [24].
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tone. NO is a potent vasodilator and inhibitor of hypoxic pulmonary vasoconstriction as well as platelet aggregation. Animal studies showed that augmenting NO and the cyclic GMP (cGMP) pathway in lung transplantation improves lung graft function and recipient survival [26]. However, other studies seem to support the notion that high-output NO from iNOS, in addition to its bactericidal effects, can worsen lung injury, presumably by direct effects of highly reactive and toxic NO metabolites on various proteins and lipids [27]. Prostacyclin is produced downstream of cyclooxygenase by prostacyclin synthase in endothelial cells and, like NO, is also a potent vasodilator and inhibitor of platelet aggregation, acting primarily through cyclic AMP (cAMP) as opposed to NO, which predominantly activates cGMP production [26]. In a study addressing the mechanism of action of prostacyclin and prostaglandin E2 on endothelial cells, it was shown that these compounds enhanced the barrier properties of cultured endothelial cells at the baseline, attenuated thrombin effects, and conferred lung protection in an experimental model of ventilator injury [28]. In lung ischemia– reperfusion injury, the protection conferred by prostaglandin E1 (PGE1) has been attributed to its vasodilator properties, which leads to a better distribution of the preservation solution throughout the lung graft. However, PGE1 also protects lungs from ischemia–reperfusion injury through its ability to reduce neutrophil and platelet accumulation, and maintain endothelial barrier function [29].
2.4 Activated Protein C 2.3 Pulmonary-Endothelium-Derived Vasoactive Mediators (Nitric Oxide, Prostaglandins) Nitric oxide (NO) modulates pulmonary vascular tone and leukocyte–endothelium interactions. NO released from endothelial cells maintains vascular homeostatic properties by relaxing vascular smooth muscle, inhibiting neutrophil adhesivity and platelet aggregation, and maintaining endothelial barrier properties. Endothelial nitric oxide synthase (eNOS), an enzyme abundantly expressed in the lung, constitutively produces NO from l-arginine and oxygen in an NADPH-dependent fashion [25]. A number of physiologic stimuli can contribute to NO production via eNOS, triggered by calcium flux within endothelial cells: endothelial cell stretch and receptor ligation (bradykinin, histamine, and thrombin are classic examples) are proximate triggers of the calcium pulse leading to NO release. NO released in the lungs takes on special significance in that some believe it to be an essential mediator of oxygen release from hemoglobin in distal tissues, and a key modulator of pulmonary vascular
Protein C, a zymogen made by endothelial cells, when activated by thrombin (a process accelerated by thrombomodulin) has distinct anti-inflammatory and anticoagulant properties. Treatment with activated protein C (APC) had been promising in a number of animal studies and its use relies on solid scientific evidence [30]. APC appears to play a major role in sepsis, being an important regulator of the coagulation system (anticoagulant protein C pathway) and, in addition, numerous investigations have provided evidence that APC exerts direct cytoprotective effects on various cell types, and more specifically on endothelial cells. These cytoprotective effects appear mostly related to modulation of gene expression, antiinflammatory and antiapoptotic activity, and endothelium barrier stabilization [30]. Most cytoprotective effects require the activation of PAR-1, whereas the endothelial protein C receptor (EPCR) serves as a c-receptor. Animal studies using APC inhalation documented lung protective effects of the drug [31] despite apparent variations in the mode of APC action probably related to differences in experimental design.
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ALI and the related adult respiratory distress syndrome 3 Interactions of Leukocytes with Pulmonary have an incidence of 200,000 cases per year in the USA [42] Endothelium: Procoagulation Factors Pulmonary artery endothelium is an active participant in regulation of platelet aggregation, coagulation, and fibrinolysis [32]. More than just being barriers that line the interior of vessels which separate circulating blood from prothrombotic subendothelial factors, pulmonary artery endothelial cells are master regulators of hemostasis. Early research advanced the concept of the endothelium existing in either a quiescent, vasodilatory, anticoagulant and antiadhesive phenotype or an activated, vasoconstrictive, procoagulant, proadhesive phenotype [33]. Subsequent work has shown that such an off/on concept of endothelial function is overly simplistic, and that endothelial cells are capable of a spectrum of responses. The role of the endothelium in hemostasis illustrates the ability of endothelial cells to produce a variety of effects and thus tip the balance toward either a procoagulative or an anticoagulative state. The high procoagulant potential of the pulmonary endothelium, observed under conditions of stress, fosters interactions between damaged endothelium and circulating platelets, necessitating the existence of especially robust mechanisms for maintaining blood flow. Quantitative reverse transcription PCR measurements of von Willebrand factor (VWF) messenger RNA in various murine tissues showed a 5–50 times higher concentration of this procoagulant in the lung compared with the kidney and liver [34]. In addition, gene expression of TF, a potent trigger of coagulation, has been shown to be higher in the lung than in the heart and kidney in a murine model of septic shock [35]. Yet at the same time, anticoagulant mechanisms in the lung are highly active – including thrombomodulin expression, NO and prostacyclin generation, and APC synthesis. Seminal work in the areas of ALI and VALI has established a link between these conditions and disruptions of hemostasis [36]. In 1969, Saldeen et al. noted that injection of thrombin produced canine respiratory insufficiency that was linked to emboli in the pulmonary microcirculation [37]. In addition, bronchoalveolar lavage fluid from patients with ALI has shown elevated levels of fibrinopeptide A, factor VII and D-dimer, as well as an increase in the procoagulant plasminogen activator inhibitor (PAI) and a decrease in the thrombolytic urokinase [38]. The importance of active fibrinolytic mechanisms in the lung cannot be overstated, as in the end, the lungs serve to passively capture embolized clot or clot fragments from the peripheral circulation. Under pathologic conditions, native thrombolysis is disrupted. Coagulation also triggers other pathologic processes in ALI, as studies have shown that fibrin contributes to alveolar collapse by inhibiting surfactant production, promotes fibrosis by recruiting fibroblasts, and is a chemoattractant for neutrophils [39, 40]. In addition, coagulation genes have been shown to be among the most highly induced by the mechanical stress of VALI [41].
and a mortality rate of 25–40% [43]. Given that disordered hemostasis contributes to the pathobiological processes of such devastating pulmonary diseases, an understanding of the mechanisms by which coagulation is regulated in the pulmonary vascular bed is vital.
3.1 Platelet Tethering and Activation: The Collagen Pathway The process of coagulation often begins with physical disruption of the endothelium. The resulting exposure of subendothelial collagen and TF leads to an initial tethering of circulating platelets that allows them to attach to the vessel wall despite the shearing force of flowing blood. A new paradigm [44] describes two distinct pathways of platelet tethering and subsequent activation. In the collagen pathway, platelets are first captured through interactions between two sets of ligands: exposed collagen and platelet glycoprotein VI, as well as collagen-bound VWF and the platelet glycoprotein Ib–V–IX complex. Glycoprotein VI subsequently activates platelets and triggers platelet granule release [44]. Collagen types I, II, III, and IV are thought to be the most reactive to platelets, although the shearing forces in the vicinity of tethering may affect the reactivity of each type of collagen [45].
3.2 Platelet Tethering and Activation: The Tissue Factor Pathway The TF pathway of platelet activation is itself dependent upon the extrinsic pathway of coagulation. Exposed TF and activated factor VII (factor VIIa) catalyze the conversion of factor X into its active form (factor Xa), which then cleaves prothrombin to thrombin. In addition to its role in fibrin formation, thrombin activates platelets by cleaving PARs on the platelet surface [44]. Previously hidden epitopes are thereby revealed, and these “tethered ligands” bind to themselves to trigger platelet activation. Although endothelial cells express little or no TF constitutively, cell culture studies have shown that endothelial TF may be induced by stimulation with activated platelets [46], monocytes [47], lipopolysaccharide [48], TNF-a, and IL-1 [49]. The pulmonary vasculature may be especially prone to TF upregulation as evidenced by an in vivo murine model of sickle cell disease which used immunostaining to show that endothelial TF is induced in pulmonary veins and further augmented by hypoxia [50]. This prothrombotic potential is
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Fig. 3 Lung tissue from normoxic mice (a) and mice hypoxic for 6 h (b) was simultaneously immunostained with goat anti-rabbit TF IgG, with TF (pink areas identified by arrows) being more prominent in
hypoxic lung. Magnification ×3,600. (Reproduced from [52] with permission from the American Society for Clinical Investigation)
supported by studies linking TF to ALI. For example, TF-induced alveolar thrombin generation has been documented in a chimpanzee model of endotoxemia [51]. Hypoxia alone is sufficient to trigger TF expression in cells of the lung, as well as microvascular thrombosis [52]. The mechanism by which TF is induced on endothelial cells likely involves activation of at least two promoter regions. The lipopolysaccharide-response element contains binding sites for activator protein-1 and nuclear factor kB, allowing inducibility by inflammatory mediators such as lipopolysaccharide, TNF-a, and IL-1B [53]. The second promoter region, the serum-response region, contains early growth response protein-1 binding sites which mediate TF induction in response to hypoxia, shear stress, low-density lipoprotein, and vascular endothelial growth factor. CD40 ligation on endothelial cells results in a broader activation of the intracellular TF activation pathways, thus providing another example of how the extrinsic milieu can lead to transcriptional regulation of the gene [53]. Recent research has proposed that NO may inhibit TF, although results from various studies are conflicting [54] (Fig. 3).
P-selectin and facilitates adhesion of TF-rich microparticles to activated platelets [63]. The resultant concentration of TF near sites of vascular injury illustrates a putative role for microparticles in the initiation of coagulation [64]. In addition, interactions involving PSGL-1 and CD15 (an adhesion molecule) on microparticles and P-selectin on platelets and endothelial cells may facilitate the transfer of TF from circulating microparticles to platelets [65]. The role of P-selectin in TF-associated thrombus formation is supported by studies showing high levels of circulating of P-selectin associated with increased venous thrombus formation in mice [66], whereas P-selectin-null mice have developed thrombi containing minimal TF and thrombin [63]. It has been proposed that TF on some microparticles exists in an inactive, or cryptic, conformation, which may limit the ability of circulating microparticles to trigger systemic coagulation [44].
3.3 Circulating Tissue Factor In addition to its endothelial-bound form, TF has been found on circulating monocytes, platelets [55], and some microparticles. Microparticles are sub-micron-sized particles composed of cell membrane and associated surface proteins that are shed from activated or injured platelets, leukocytes, erythrocytes, or endothelial cells [56]. Microparticleassociated TF may induce thrombogenesis in diseases such as pulmonary hypertension [57, 58], lupus [59], metabolic syndrome in type 2 diabetes [60], and acute coronary syndrome [61] and in patients with coronary artery disease undergoing bypass surgery [62]. Among other proteins, microparticles express PSGL-1, which binds to platelet
3.4 Platelet Recruitment and Thrombus Growth Following initial platelet adhesion to the endothelium, the platelet aggregation response is amplified and sustained through the effects of mediators including ADP, thrombin, epinephrine, and thromboxane A2 [67]. These agonists activate G-protein-coupled receptors [68] that ultimately facilitate platelet-to-platelet aggregation through VWF and fibrinogen bridges between glycoprotein IIb/IIIa integrins on platelets [69] (Fig. 4) An important example of this type of activation involves the interaction of ADP with its P2Y12-coupled Gi receptor located on the platelet surface. Activation leads to a decrease in the level of intraplatelet cAMP and results in the perpetuation of platelet stimulation. In a model of mesenteric artery injury, P2Y12-null mice were shown to generate only small, unstable thrombi with decreased platelet density and activity [70].
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Fig. 4 (a) Electron micrograph of murine pulmonary vasculature exposed to hypoxia (FiO2 ~ 6%) for 16 h showing endothelium, red blood cells (RBC), platelet clumping (Plt), and platelet-associated fibrin (arrow). (b) Higher magnification demonstrates 22.5-nm periodicity characteristic of fibrin; black bar 112 nm. (Reproduced from [52] with permission from the American Society for Clinical Investigation)
The process of thrombus development requires a complex process of platelet coalescence that is dependent upon interactions between glycoprotein Iba on free-flowing platelets and VWF on the surface of immobilized platelets [69]. Aggregation is a dynamic process during which only a small percentage of platelets that initially tether upon the growing thrombus ultimately adhere [69]. As a thrombus grows, it requires additional stabilization so as to withstand the shearing effect created by blood as it flows with greater velocity at the center of a vessel than near the wall. This difference in velocities creates a shear stress that is greatest near the wall and thus the developing thrombus [71]. Endothelial cells, particularly when activated, secrete from Weibel–Palade bodies a highly thrombogenic form of ultralarge VWF multimers that support stable platelet aggregation [71]. Pulmonary endothelium contributes to global hemostasis in important ways. For example, human adult microvascular endothelial cells have been shown to be an important extrahepatic source of factor VIII, which is vital for factor X activation and the subsequent conversion of prothrombin to thrombin [72]. This may explain why factor VIII levels may increase despite severe liver disease. This could provide a mechanism through which the pulmonary endothelium regulates the common pathway of the clotting cascade.
4 Endothelial Modulation of Coagulation Coagulation occurs after inciting stimuli trigger a cascade resulting in a large fibrin clot. To prevent an explosive spread of clot formation beyond a site of injury, the body relies upon a system of intrinsic anticoagulants to restore the hemostatic balance. Given the central role of the endothelium in the coagulation cascade and the high
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p rothrombotic potential of the pulmonary vasculature, it is not surprising that pulmonary endothelial cells maintain an array of potent anticoagulation mechanisms. In keeping with the concept of endothelial heterogeneity, the distribution of these mediators of hemostasis varies depending on the vascular bed, thus giving pulmonary endothelial cells a relatively unique anticoagulation phenotype. For example, radioimmunoassay has shown much higher thrombomodulin antigen concentrations in the lungs and placenta compared with the heart, liver, or brain [73]. The endothelium modulates coagulation by producing, sequestering, and modifying various coagulation-inhibitory factors. NO, prostacyclin, tissue plasminogen activator (t-PA), and TF pathway inhibitor (TFPI) are produced and released by endothelial cells. The endothelium supports anticoagulant surface proteins including thrombomodulin, the EPCR, the ectonucleotidase CD39, heparan sulfate proteoglycan, and protein S. Finally, the endothelium contributes to anticoagulation by modifying circulating protein C and antithrombin.
4.1 Anticoagulants Elaborated by Vascular Endothelium 4.1.1 Nitric Oxide NO is a ubiquitous free radical that functions as both an intra- and extracellular signaling molecule. NO is synthesized from precursor l-arginine by three isoforms of nitric oxide synthase, including the endothelial- and plateletbound e-NOS. In addition to its function as a regulator of vascular tone, NO prevents platelet glycoprotein IIb/IIIa binding to fibrinogen by stimulating intraplatelet cGMP production [74]. Inhaled exogenous NO has been shown to modulate platelet aggregation in healthy animals [75] and humans with adult respiratory distress syndrome [76]. The combination of effects driven by NO and its distal effector cGMP, vasodilation and inhibition of platelet aggregation, could serve as a powerful means for disaggregating local thrombus and moving fragments downstream. A potent vasodilatory drug which raises the level of cGMP, sildenafil, is in fact used as treatment for pulmonary arterial hypertension, which may have thrombosis as a contributing mechanism.
4.1.2 Prostacyclin Prostacyclin is a labile prostanoid synthesized by endothelial and smooth muscle cells through the arachidonic acid pathway [77]. Prostacyclin works synergistically with NO
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[78] to inhibit platelet activation, secretion, and aggregation by increasing the level of intracellular cAMP [79]. Thromboxane A2, another cyclooxygenase product, triggers platelet aggregation [80]. Of note, antithrombin III has been shown to stimulate prostacyclin production [81], illustrating a method by which one anticoagulant is capable of triggering or amplifying the production of another.
4.1.3 Tissue Plasminogen Activator Thrombin and shear stress induce the production of the serine protease t-PA by the endothelium [82]. In the presence of thrombin, t-PA cleaves plasminogen into plasmin, which subsequently degrades fibrin. Free t-PA can then bind to the endothelium, where it is protected from its inhibitor, PAI-1, itself a product of the endothelium. The net result is lysis of thrombus. This topic is covered in Chap. 24.
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4.2 Membrane-Associated Anticoagulants 4.2.1 Thrombomodulin Thrombomodulin is a transmembrane cofactor on the endothelial surface that forms a reversible, high-affinity complex with thrombin. Estimates of surface expression of thrombomodulin suggest that there are approximately 50,000 active sites per cell [88]. The thrombin–thrombomodulin complex blocks thrombin’s ability to catalyze fibrin formation and activate both factor V and platelets. In addition, thrombomodulin-bound thrombin converts circulating protein C into APC, a potent modulator of coagulation and inflammation. It can therefore be viewed as an important, fixed component of the endothelial anticoagulant machinery. It is interesting to note that upon exposure of endothelial cells to hypoxia or inflammatory mediators expression of thrombomodulin becomes markedly diminished [89].
4.2.2 Protein C Receptor 4.1.4 Tissue Factor Pathway Inhibitor TFPI is an endogenous serine protease inhibitor of coagulation synthesized by the endothelium that forms a quaternary TF–factor VIIa–factor Xa–TFPI complex, thus limiting the amount of factor Xa available to cleave prothrombin into thrombin [83]. Once released, TFPI can bind to either circulating lipoproteins [84] or microvascular glycosaminoglycans capable of later releasing TFPI into the circulation after exposure to heparin [85]. Exposure of blood to TF has been shown to trigger disseminated intravascular coagulation (DIC), the widespread, disseminated occurrence of microvascular thrombosis that compromises blood supply to multiple organs [86]. Again illustrating the overlap that exists between the pathways of coagulation and inflammation, studies have investigated the role of TFPI in regulating the microvascular thrombosis that occurs in the setting of septic shock. Creasey et al. administered lethal intravenous Escherichia coli infusions to baboons and noted that early postinfusion treatment with purified recombinant TFPI resulted in survival of all animals receiving therapy, whereas all control animals died within 40 h. In addition, there was evidence that TFPI attenuated lung, kidney, and adrenal disease [87]. Such benefits were not found in the OPTIMIST trial, which randomized 1,754 patients with severe sepsis and an elevated international normalized ratio to treatment with or without tifacogin, a recombinant TFPI. Tifacogin therapy was shown to have no effect on all-cause mortality and created an increased risk of bleeding [85]. This area of therapy, albeit promising, remains controversial.
In addition to expressing thrombomodulin, endothelial cells are capable of activating protein C through EPCR. This type 1 transmembrane protein binds to protein C and presents it to the thrombin–thrombomodulin complex for activation [90]. EPCR also binds APC, and although this interaction prevents APC from exerting its anticoagulant activity [91], EPCR-bound APC activates cellular signaling leading to endothelial barrier protection [92]. Conceivably, sustenance of the endothelial barrier limits contact between the circulation and the procoagulant vascular substrata.
4.2.3 CD39 Endothelial ENTPDase1 (CD39) is a Ca2+/Mg2+-dependent ectonucleotidase that hydrolyzes extracellular ATP and ADP to AMP [93]. Through interactions with platelet P2X1, P2Y1, and P2Y12 purinergic receptors [94], ADP released from activated platelets both activates platelets and potentiates the anticoagulation signals of other mediators [95]. In addition, CD39-mediated removal of ADP from the intravascular milieu has been shown to inhibit platelet function [96]. The importance of CD39 in maintaining the anticoagulant endovascular phenotype is illustrated by the finding that it maintains vascular fluidity even in the complete absence of prostacyclin and NO [97]. In a murine model of cerebral infarction, CD39-deficient animals were shown to have increased microvascular thrombosis, reduced postischemic perfusion and increased infarct volumes [98]. The administration of a recombinant soluble form of CD39 following infarction
26 Interactions of Leukocytes and Coagulation Factors with the Vessel Wall
was able to rescue the knockout animals, leading to restored cerebral blood flow and markedly decreased infarct volumes [98].
4.2.4 Heparan Sulfate Proteoglycans Heparan sulfate proteoglycan, a linear polysaccharide composed of alternating units of hexuronic acid and d-glucosamine [99], is expressed on the surface of endothelial cells, providing an estimated 50,000 binding sites for antithrombin per cell [100]. This represents an additional anticoagulant defense at the blood–vessel interface.
4.3 Anticoagulation via Endothelial Interaction with Circulating Factors 4.3.1 Protein C and Protein S Protein C is a vitamin K dependent serine protease that is synthesized by the liver as a zymogen. Circulating protein C is activated by the endothelial-bound thrombin–thrombomodulin complex to form APC, which proteolytically inactivates activated factor V (factor Va) and activated factor VIII, thus blocking downstream thrombin generation [32]. Protein S is a vitamin K dependent plasma protein synthesized by the liver and endothelium. Protein S is best known as a cofactor for protein C, and its presence increases the inactivation of factor Va by 20-fold [101], and factor VIIa inactivation by at least threefold [102]. In addition, recent research has identified protein S as a potentially important cofactor of TFPI [103]. APC is an important modulator of both coagulation and inflammation in the lungs. Patients with ALI and decreased plasma levels of APC have higher mortality and more multiorgan system dysfunction [104, 105]. Findings such as these have led to clinical trials involving APC administration in the setting of sepsis. In the landmark PROWESS trial, the administration of recombinant human APC to patients with severe sepsis was associated with a reduction in the relative risk of death of 19.4% and an absolute risk reduction of 6.1% [106]. A trial involving the use of APC in patients with ALI showed no benefit in terms of ventilator-free days, mortality, or lung injury score, although these patients were less critically ill than those in the PROWESS study [43].
4.3.2 Antithrombin Antithrombin III is a plasma serine protease inhibitor that targets activated factor XII, activated factor XI, factor Xa,
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activated factor IX, factor VIIa, and activated factor II (thrombin) [107]. The endothelium plays a role in thrombin inactivation, as it sequesters both thrombin and antithrombin III from the circulation and facilitates formation of an antithrombin–thrombin complex that prevents thrombin activation [32]. Endogenous endothelial-bound or exogenous administered heparin activates antithrombin by facilitating the detachment of a reactive center loop from partial insertion in the A sheet of the molecule [108]. Heparin activation produces a 9,000-fold increase in inhibitory capacity against thrombin and a 17,000-fold increase against factor Xa [109]. A diffuse procoagulant state develops in patients with sepsis [110] as anticoagulant factors are depleted [111] and PAI-1 (which inhibits fibrinolysis) is induced. The KyperSept trial evaluated the use of high-dose antithrombin III administration in severe sepsis. This therapy had no effect on all-cause mortality when administered with heparin, possibly owing to an increased risk of hemorrhage. A subgroup analysis of patients who received antithrombin III without concomitant heparin suggested some benefit [112]. A recent meta-analysis of three randomized controlled trials involving patients with severe sepsis and DIC concluded that the use of antithrombin concentrate for up to 5 days may significantly reduce all-cause short-term mortality [113]. Taken together, these trials suggest a role for novel antithrombotic strategies in treating sepsis or DIC, but much yet remains to be learned.
5 Conclusion The pulmonary endothelium’s unique location at the capillary–alveolar interface makes it vulnerable to injury from a wide variety of sources, including oxygen radicals, sepsis, and barotrauma. Other extracellular signals leading to endothelial cell damage and/or activation include growth factors, cytokines, shear stress, circulating lipoproteins, coagulation factors, and components of the extracellular matrix [107]. The pulmonary endothelium integrates this barrage of external signals into a variety of responses that tip the hemostatic and inflammatory balances in one direction or the other, depending upon the unique and frequently changing requirements of a particular vascular bed. Although the interaction between circulating factors and the endothelium may be the major determinant of pulmonary vascular inflammatory and coagulation phentoypes, it is important to look broadly when considering all the interactive mechanisms involved. For example, environmental factors such as tobacco use, hormone ingestion, air pollution, diet, and medical conditions may modify the mechanisms discussed in this chapter. In addition, intracellular modifiers such as genetic polymorphisms or epigenetic
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changes may alter the interactions between pulmonary endothelium and circulating factors of inflammation and coagulation. For example, inherited deficiencies of protein C [114], protein S [115], and antithrombin, [116] and polymorphisms of factor V Leiden [117] and prothrombin 20210A [118] have long been known to increase the risk of venous thrombotic disease. It is reasonable to suspect that alterations in genes encoding for coagulation factors, thrombomodulin, fibrinogen, platelet glycoproteins, and the fibrinolytic system may also contribute to thrombosis, although multiple polymorphisms may be required for a change in phenotype. For example, a TFPI polymorphism has been identified that predisposes carriers to venous thromboembolism, but only when inherited with factor V Leiden or antiphospholipid antibody syndrome [119]. Other challenges to the identification of additional modifier genes include phenomena such as variable expressivity and incomplete penetrance.
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Chapter 27
Interaction of the Plasminogen System with the Vessel Wall Riku Das and Edward F. Plow
Abstract In this chapter, we use the term “plasminogen system” to designate the pathway that controls the conversion of plasminogen to plasmin and the proximal consequences of plasminogen activation. The “plasminogen system” is synonymous with the “fibrinolytic system,” a nomenclature that emphasizes the primary substrate of plasmin, fibrin, and function, and the outcome of fibrin degradation, fibrinolysis. However, evidence documents vital roles of components of the plasminogen system in physiological and pathological processes which cannot be attributed to and clearly extend beyond its role in clot dissolution. In particular, components of the plasminogen system can influence cell invasion into tissue, which is dependent on the capacity of plasmin to degrade various extracellular matrix (ECM) proteins, including fibronectins, thrombospondins, von Willebrand factor, and laminin, and to activate certain matrix metalloproteases, enzymes that degrade still other ECM constituents. Many components of the plasminogen system also interact with cells to generate intracellular signals that affect cell survival, proliferation, adhesion, and migration. Some of these roles are directly relevant to endothelial cell (EC) and smooth muscle cell (SMC) biology and it is in this context that we consider the interaction of the plasminogen system with these vascular cells. Why should a book that focuses on pulmonary vascular disease have a chapter dedicated to the interaction of the plasminogen system with the vessel wall? The answer is straightforward; there is compelling evidence linking the plasminogen system to pathological processes in the lung. Keywords Fibrinolysis • Thrombosis • Cell adhesion • Migration • Endothelial cell • Smooth muscle cell • Fibrin • Fibronectin • Plasmin
R. Das (*) Department of Molecular Cardiology, Cleveland Clinic Foundation, 9500 Euclid Avenue NB-50, Cleveland, OH 44195, USA e-mail:
[email protected] 1 Introduction In this chapter, we use the term “plasminogen system” to designate the pathway that controls the conversion of plasminogen to plasmin and the proximal consequences of plasminogen activation. The “plasminogen system” is synonymous with the “fibrinolytic system,” a nomenclature that emphasizes the primary substrate of plasmin, fibrin, and function, and the outcome of fibrin degradation, fibrinolysis. However, evidence from enumerable in vitro studies and numerous studies conducted in mice deficient in various components documents vital roles of components of the plasminogen system in physiological and pathological processes which cannot be attributed to and clearly extend beyond its role in clot dissolution. In particular, components of the plasminogen system can influence cell invasion into tissue, which is dependent on the capacity of plasmin to degrade various extracellular matrix (ECM) proteins, including fibronectins, thrombospondins, von Willebrand factor, and laminin, and to activate certain matrix metalloproteases, enzymes that degrade still other ECM constituents. Many components of the plasminogen system also interact with cells to generate intracellular signals that affect cell survival, proliferation, adhesion, and migration. Some of these roles are directly relevant to endothelial cell (EC) and smooth muscle cell (SMC) biology and it is in this context that we consider the interaction of the plasminogen system with these vascular cells. Why should a book that focuses on pulmonary physiology have a chapter dedicated to the interaction of the plasminogen system with the vessel wall? The answer is straightforward; there is compelling evidence linking the plasminogen system to pathological processes in the lung. In bleomycin-induced pulmonary fibrosis, deficiencies of plasminogen, urokinase plasminogen activator (uPA), uPA receptor (u-PAR), and tissue plasminogen activator (tPA) in mice, all components of the plasminogen system, alter the pathogenic response substantially [1, 2]. Similarly, the response to lung infections and endotoxin-induced lung injury is markedly altered in mice deficient in certain components of the plasminogen system, and expression patterns
J.X.-J. Yuan et al. (eds.), Textbook of Pulmonary Vascular Disease, DOI 10.1007/978-0-387-87429-6_27, © Springer Science+Business Media, LLC 2011
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Fig. 1 A model depicting various interactions of the plasminogen (Plg) system with the vessel wall. Endothelial cells and smooth muscle cells are the major cellular components of the vascular wall. Plg binding to a Plg receptor (Plg-R) either on the endothelial cell surface or on the fibrin surface enhances its activation to plasmin (Plm). Plm activates growth factors (GFs) and matrix metalloproteases (MMPs) and degrades extracellular matrix (ECM) components, which controls vascular remodeling by enhancing cell migration on the ECM. Plm also degrades fibrin to generate fibrin degradation products and promotes fibrinolysis to maintain vascular patency. Plg activation on cell or fibrin surfaces is mediated by Plg activators, either urokinase Plg activator (uPA) or tissue Plg
activator (tPA). uPA or tPA secreted from endothelial cells or smooth muscle cells binds to uPAR or tPA receptors, respectively, to activate Plg to Plm. Both tPA and uPA, either bound to their receptors or free, are inhibited by Plg activator inhibitor (PAI). uPA binding to uPAR generates intracellular signals in association with integrins. This process can lead to Plm-independent events such as cell adhesion, migration, differentiation, and proliferation. The uPA–PAI-1–uPAR complex formed on the cell surface is internalized and free uPAR is recycled back to the cell surface. Plg binding to Plg-R also generates intracellular signals and contributes cellular functions such as proliferation, migration, and adhesion. Unbound Plm is rapidly inhibited by a2-antiplasmin (A-P) in the blood
of these components change in wild-type mice in such models [3]. In ovalbumin-induced bronchial airway inflammation models of asthma, deficiency of plasminogen or inhibitors of plasminogen functions prevents the recruitment of inflammatory cells into lung tissue and spares tissue damage [4]. In such models, deficiencies of plasminogen activator inhibitor (PAI)-1 also alter the development of lung tissue damage [2, 5]. Such results correlate closely with evidence of activation and changes in the plasminogen system in human pulmonary diseases [6].
Interactions of these components with cells regulate many of their functions, including plasminogen activation and plasmin inhibition [7, 8]. Furthermore, the receptors for these components on ECs and other cell types are subject to modulation, thus controlling the proteolytic potential at the cell surface [14, 18]. EC also have the capacity to synthesize and secrete both plasminogen activators and PAI-1 in response to specific stimuli. These secreted components can interact with the cells in an autocrine and paracrine fashion. Many of the components of the plasminogen system, including plasmin(ogen), a2-antiplasmin, and tPA also bind to the surface of a fibrin clot [19]. Thus, there are many parallels between the regulation of the plasminogen system on the cell surface and that on the fibrin surface. We will briefly describe the molecular properties of the individual proteins of the plasminogen system, consider how these proteins interact with cells via their receptors, and then describe specific functions that relate to their interaction with cells within the vessel wall.
2 Components of the Plasminogen System For the purposes of the chapter, the plasminogen system is confined to the molecules and events depicted in Fig. 1. In vertebrates, there are two plasminogen activators, tPA and uPA, which activate the inactive plasminogen zymogen to the active serine protease plasmin. The activity of plasmin is regulated by PAIs, primarily PAI-1, and by plasmin inhibitors, primarily a2-antiplasmin [7, 8]. All the aforementioned components bind directly to cells or influence the interaction of other members of the system with cell surfaces, including ECs [9–14] and SMCs [15–17].
3 Plasminogen and Plasmin Plasminogen is a 92-kDa single-chain glycoprotein of 791 amino acids. Hepatocytes synthesize plasminogen [20] and
27 Interaction of the Plasminogen System with the Vessel Wall
give rise to the high concentrations of plasminogen (approximately 2 mM) in blood, but plasminogen messenger RNA has been detected in other cells as well [21]. The plasminogen molecule can be divided into three regions on the basis of function: (1) N-terminal region: As synthesized, plasminogen has glutamic acid at its N-terminus and is referred to as Glu-plasminogen. The N-terminal region contains several plasmin-sensitive bonds between residues 60 and 80, and their cleavage gives rise to molecules that are referred to collectively as Lys-plasminogen. Proteolytic deletion of the N-terminal region influences the overall conformation of plasminogen, which in turn influences interactions with cells [22]. (2) Kringle region: Plasminogen contains five disulfide-looped kringle domains, each of 80–90 amino acids [23]. Three of the five kringles, KI, KIV, and KV, have lysine-binding sites (LBS). These three kringles display preferences for C-terminal lysine residues, although their specificities are not identical [22]. The LBS mediate plasminogen binding to cell surfaces [24], fibrin [25], and a2-antiplasmin [26]. Therefore, lysine analogues (e.g., e-aminocaproic acid, EACA) [27], tranexamic acid (TXA) [28], and peptides with C-terminal lysines [29] or antibody directed against C-terminal sequences of these binding partners block plasminogen interactions [30]. (3) Protease domain: This domain contains the catalytic triad His603, Asp646, and Ser741A, which is typical of serine proteases. The single-chain plasminogen, which is enzymatically inactive, is converted to two-chain plasmin by cleavage of a single peptide bond (Arg561–Val562) by tPA or uPA. Of the two chains of plasmin, the heavy chain, containing the kringles, and the light chain, containing the protease domain, remain disulfide-linked to each other [31].
3.1 Plasminogen Receptors and Their Modulation Many different cell types have been shown to bind plasminogen in a specific and saturable manner, indicating that they express a discrete numbers of binding sites. These binding sites are heterogeneous in character and are collectively referred to as plasminogen receptors (Plg-Rs). All circulating blood cells, with the exception of erythrocytes [32], express Plg-Rs; the number of Plg-Rs on these cells is great and varies from 104 to more than 106 [7, 33]. Tissue fixed cells also bind plasminogen with high capacity [34]. In general, transformed cells bind plasminogen with higher capacity than their nontransformed counterparts [35–38]. Thus, the almost ubiquitous distribution and the high density of expression are defining characteristics of Plg-Rs. Even though Plg-Rs are heterogeneous, they bind plasminogen with similar affinities; Kd values are generally in the range 0.1–2 mM [33, 35].
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Despite relatively low affinities, the high concentration of plasminogen in local microenvironments ensures that significant occupancy of Plg-Rs occurs on cells. In addition, some much higher affinity Plg-Rs have been identified [9] and some receptors which are selective for plasmin as opposed to plasminogen have been reported [39, 40]. The similarities in the affinities of most Plg-Rs reflect interactions mediated by the LBS of plasminogen. Binding of plasminogen to Plg-Rs via its LBS has several functional consequences which are inherent to LBS engagement [24]: (1) plasminogen activation is enhanced in cell surfaces as compared with solution [32, 41]; (2) plasmin bound to cell surfaces is protected from rapid inactivation by a2-antiplasmin [42, 43]; (3) the transition from Glu-plasminogen to Lys-plasminogen plays an important role in binding and activation at cell surfaces [44, 45]; and (4) plasmin bound to Plg-Rs is catalytically more efficient than free plasmin [46]. Thus, Plg-Rs endow the cell surface with the broad and sustained proteolytic activity of plasmin, which can be utilized for a wide variety of the biological functions of the enzyme, including fibrinolysis, degradation of the ECM, regulation of growth factor activity [47, 48], and generation of intracellular signaling [49, 50]. Many different Plg-Rs have been identified. These can be divided into two groups: those with and those without C-terminal lysines. A BLAST search of the human genome identifies hundreds of proteins with lysine at their C-terminus, and as such they have the potential to interact with the LBS of plasminogen. However, not all these proteins function as Plg-Rs. The residues adjacent to the C-terminal lysine, the availability of the C-terminal lysine at the cell surface, and processing of the C-terminal lysine by proteolytic enzymes, such as the basic carboxypeptidases, which remove C-terminal lysines, can suppress or modulate the ability of proteins with C-terminal lysines to function as Plg-Rs [51]. By the same token, proteolysis to generate a new C-terminal lysine can create a new Plg-R [18, 52]. Cleavage is believed to account for the capacity of p37 (or p36) of the annexin 2 heterotetramer to bind plasminogen [53]. Some of the proteins with C-terminal lysines which have been shown to function as Plg-Rs are listed in Table 1. Of note, many of the known Plg-Rs lack signal sequences and transmembrane domains. Thus, to function as Plg-Rs, they must reach the cell surface by a nonconventional secretory pathway. The Plg-Rs without a C-terminal lysine are also multiple and heterogeneous (Table 1). These include some molecules which bind to the LBS of plasminogen as indicated by the inhibition of binding by EACA or TXA [54–58]. These Plg-Rs contain an internal residue(s) that mimics a C-terminal lysine. Notable in this category are the integrins [56–58]. A few Plg-Rs interact with plasminogen or plasmin and the interactions are not EACAor TXA-sensitive. Heymann nephritis autoantigen, gp330, and a5b1 fall into this category [59, 60]. In the nonprotein
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Table 1 Presence of plasminogen receptors (Plg-Rs) on various cell types and their mechanism of binding Plg-Rs Cell types C-terminal lysine a-Enolase Cytokeratin-8 p11 TATA-binding proteininteracting protein Histone H2B Annexin 2 Actin avb3 aMb2 aIIbb3 Amphoterin
Monocytes, neutrophils, lymphoid cells, myoblasts, neurons, transformed cells Hepatocellular and breast carcinoma Endothelial cells, fibroblasts Monocytoid cells
Present
Neutrophils, monocytoid lineage endothelial cells Endothelial cells, monocyte lineage Endothelial cells, carcinoma, catecholaminergic cells, neuronal cells, fibroblasts Endothelial cells Neutrophils Platelets, rheumatoid arthritis synovial fibroblasts Neuronal cells
Present Absent, but generated by proteolysis Absent, but exposed C-terminal lysine on the cell surface Absent Absent Absent Absent
category are gangliosides [61], which may help to account for the extraordinarily high plasminogen binding capacity of some cells [61]. This interaction is LBS-mediated. Cells may utilize plasminogen in their physiological and pathological responses by modulating their cell-surface expression of Plg-Rs [18]. Processes associated with modulation of Plg-R expression include transformation [24, 36, 37], cell adhesion [62, 63], agonist stimulation [14, 62–65], differentiation [30, 66–68], and apoptosis [69]. Breast tumor with high invasive phenotypes binds more plasminogen than cells lines with lower invasive properties [37]. Thus, the high metastatic potential correlates with high plasminogen binding capacity. Cytokeratin 8 and enolase have been identified as Plg-Rs which are expressed at high levels on tumor cells [38, 70]. With respect to cell adhesion, when cells detach they can display increased capacity to bind plasminogen; when they adhere to substrates, their plasminogen binding capacity is again reduced [62]. These observations indicate that cells are capable of both upregulating and downregulating Plg-Rs; i.e., the modulations can be reversible. Thrombin-stimulated platelets bind fivefold more plasminogen than resting platelets [57], indicating that cells can respond to agonists by enhancing the plasminogen binding capacity. Cells of the monocytoid lineage can also modulate their expression of Plg-Rs in response to maturation and differentiation. When monocytoid cells are differentiated to macrophages, either in vitro or in vivo, their plasminogen binding capacity increases three- to four-fold [30, 66]. Cell-surface expression of four most prominent Plg-Rs on these cells, a-enolase, Histone H2B annexin 2 and p11 (the annexin 2 heterotetramer), leads to the increase in plasminogen binding to the differentiating monocytes. Both in vitro and in vivo studies in mice indicate that histone H2B accounts for approximately 50% of the plasminogen binding capacity of macrophages [30]. The higher plasmin activity on the surface of these cells may facilitate migration of these cells across the ECM to sites of inflammation. Plasminogen binding capacity was
Present Present Present
also observed to increase enormously when premature fat cells differentiate into mature adipocytes [68]. Synovial fibroblasts from patients with rheumatoid arthritis have a higher plasminogen binding capacity than normal synovial fibroblasts [71]. The higher plasmin activity may contribute to the destruction of joint cartilage and bone in patients with rheumatoid arthritis [72].
3.2 Interaction of Plasminogen with ECs The endothelium presents a continuous interface to blood, a rich source of plasminogen. In vitro studies indicate that plasminogen influences EC migration and invasion [58, 73, 74], and studies conducted in plasminogen-deficient mice indicate that plasminogen plays an influential role in EC responses, including angiogenesis [75] and wound healing [76]. Plasminogen also influences complex vascular disease responses, such as atherosclerosis [77] and restenosis [78]. Hence, the interaction of plasminogen with ECs has been analyzed in numerous studies. Most studies of plasminogen binding to ECs have been performed with human umbilical vein ECs (HUVEC). Plasminogen binds to the HUVEC surface via its LBS. Dissociation constants have been estimated to range from 0.3 to 2.0 mM [9, 10, 14]. Glu-plasminogen binds to the HUVEC and is rapidly converted to Lys-plasminogen and to plasmin on the cell surface [45, 53]. The number of plasminogen binding sites ranges from 1.4 × 106to 12.4 × 106. This extraordinarily high number makes it likely that binding is mediated by multiple Plg-Rs. Indeed, several different Plg-Rs have been detected on HUVECs (Table 1). The first Plg-R described on ECs was annexin 2 [53]. Annexin 2 was originally reported to express distinct binding sites for tPA and plasminogen/ plasmin. Annexin 2 does not contain a C-terminal lysine, and it was suggested that it had to be cleaved to become a
27 Interaction of the Plasminogen System with the Vessel Wall
p lasminogen binding protein [53]. However, other studies have suggested that annexin 2 only binds plasminogen when it is associated with its heterotetrameric partner p11 [79]. p11 is synthesized with a C-terminal lysine. The expression of annexin 2 alone or as a heterotetramer in complex with p11 may be regulated by phosphorylation [80]. A second notable Plg-R on ECs is integrin avb3 [58]. There is a substantial body of data implicating avb3 in angiogenesis [81]. Although neither subunit has a C-terminal lysine, the binding of this integrin to plasminogen is LBS-mediated [58]. Interaction of avb3 with plasminogen promotes plasmin-mediated adhesion and migration of bovine aortic ECs [58]. avb3 also binds the antiangiogenic plasminogen fragment angiostatin via its RGD sequence [58]. Plasminogen binding to the EC surface via actin in a kringle-dependent manner has also been reported [82]. We have also detected histone H2B on the surface of HUVECs (unpublished). Our antibody to the C-terminal lysine region of H2B partially inhibits plasminogen binding to HUVECs [30] (and unpublished observations). Modulation of Plg-Rs and plasmin generation on the EC surface has been demonstrated in various studies. Thrombin or other ligands of the protease-activated receptors enhance expression of both annexin 2 and p11 on the HUVEC surface [65]. The upregulation of these Plg-Rs was associated with increased plasminogen binding and enhanced tPA-mediated plasmin generation. Vascular endothelial growth factor (VEGF) stimulation of HUVECs leads to the enhanced fibrinolytic activity by increasing plasmin generation as a result of increased expression of tPA and some matrix metalloproteases by the cells [83]. Our unpublished results also demonstrated an increase in the levels of Plg-Rs when HUVECs are stimulated with VEGF.
3.3 Role of Plasminogen/Plg-Rs in EC Function Upon blood vessel injury, local coagulation culminates in fibrin deposition to prevent excessive blood loss. Fibrinolysis is initiated to restore blood vessel patency and blood flow. The fibrin surface provides binding sites for plasminogen and tPA and their colocalization enhances tPA-mediated plasminogen activation by approximately 500-fold [84]. The EC surface has the capacity to bring plasminogen close to tPA and uPA and also enhances plasminogen activation. Annexin 2 binding of plasminogen enhances its activation by tPA by approximately tenfold [53, 85]. The biological significance of this effect was demonstrated in vivo. Treatment of carotid arteries with recombinant annexin 2 reduced vessel thrombosis induced by ferric chloride injury of the vessel wall in rats [86]. Ling et al. [87] further showed that deficiency of annexin 2 in mice reduces both microvascular fibrin homeostasis and
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macrovascular clearance of thrombi. The contribution of other Plg-Rs on the EC to fibrinolysis has yet to be examined. Since the properties of Plg-Rs with regard to plasminogen binding and activation appear to be similar, one could predict that the contributions of individual Plg-Rs to fibrinolysis would correlate with their expression levels within particular vascular beds. Another major role of plasminogen in EC-dependent responses relates to angiogenesis. Three key steps of angiogenesis are proteolytic degradation of basement membrane, EC migration, and EC proliferation. Plasminogen binding to ECs can play a critical role in each of the three steps. Plasmin can directly degrade ECM proteins, can activate various matrix metalloproteases which are required for cell migration across collagenous matrices, and can also activate latent angiogenic growth factors [47, 48, 88, 89]. Basic fibroblast growth factor (bFGF)-induced formation of capillary tubes from ECs was suppressed in a gelatin sponge assay in mice by inhibitors of plasminogen binding to cells (TXA and EACA) [73]. ECs from plasminogen-/- mice have reduced capillary sprouting in culture and from aortic rings on collagen beds compared with their wild-type counterparts [74, 90]. In a widely used corneal implant model to evaluate angiogenesis in vivo, Oh et al. [75] demonstrated that both bFGF- and VEGF-induced corneal blood vessel formation was significantly reduced in plasminogen-/- mice compared with plasminogen+/+ mice. Angiogenesis was also impaired in annexin 2 null mice [87]. Angiostatin (kringles 1–3) binding to integrin aVb3 inhibited bFGF- and plasmin-induced EC migration [58]. Tissue factor not only binds plasminogen but also angiostatin K 1-3, angiostatin K 1-5 and angiostatin K4 in an LBS-dependent manner, and binding of soluble tissue factor to angiostatin abolished the inhibitory effects of angiostatin on the bFGF-mediated EC proliferation [91]. Kringle 5 of plasminogen promotes caspase activity and induces apoptosis on ECs [92] and, therefore, also functions as an inhibitor of angiogenesis. EC-derived microparticles are found at elevated levels in patients with thrombotic disorders [93] and they can serve as a surface for plasmin generation and also can express uPA and uPAR [94]. Functionally, EC-derived microparticles affected EC tube formation from progenitor cells. Thus, the interaction of components of the plasminogen system with EC-derived microparticles has the potential to influence inflammation, angiogenesis, and atherosclerosis.
4 tPA and tPA Receptors tPA is synthesized primarily by ECs, but not all ECs secrete tPA [95, 96]. In the pulmonary circulation, vessels with diameters of 7–30 mm produce tPA, but capillaries or larger vessels
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do not [96]. The release of tPA from ECs is regulated. Rapid release in response to G-protein-coupled-receptor activation and rises in intracellular Ca2+ levels depend upon secretion of a preexisting pool of tPA, which resides in small storage granules [97]. Release over the longer term also can be induced as well and depends upon new protein synthesis [98]. Released tPA is a 70-kDa single-chain, fully active serine protease [99]. The single-chain form can be cleaved to a two-chain form by plasmin without alteration of proteolytic activity [99]. The N-terminal of tPA is composed of a finger domain, an epidermal growth factor (EGF) domain, and two kringle domains. The serine protease domain resides in the C-terminal region of tPA and is composed of a catalytic triad of His322, Asp371, and Ser478. The enzymatic activity of tPA is tightly regulated by inhibitors of the serpin family, particularly PAI-1. Hence, the ratio of tPA to PAI-1 establishes the capacity of tPA to function as a plasminogen activator within local microenvironments [100]. tPA binds preferentially to fibrin rather than fibrinogen, and this interaction, together with the binding of plasminogen itself to fibrin, targets its plasminogen activator activity to fibrin clots [101]. The finger and the second kringle domain of tPA are involved in its binding to fibrin. tPA also binds to the surfaces of cells. Blood cells which bind tPA include platelets [102], monocytes, and neutrophils [103]. These interactions also depend on the finger and kringle domains of tPA. At least some of these sites are the same C-termini of proteins that also bind plasminogen; [104] and, hence, in competitive situations, the higher plasminogen concentration favors its occupancy of these binding sites.
4.1 Interaction of tPA with ECs A portion of the tPA released from the EC is retained on the cell surface, providing evidence for the presence tPA receptors on such cells [105]. Various groups have examined the interaction of tPA with HUVECs, but the results of these studies have not been entirely consistent. Cobbling these results together, there appear to be three classes of tPA binding sites with respect to affinity. The highest-affinity binding sites for tPA are PAI-1-dependent [12, 106]. PAI-1 is synthesized by EC cells and accumulates in the ECM, where it binds to vitronectin [107]. PAI-1 forms a tight complex with secreted tPA on the matrix; but a portion of PAI-1 associates with the EC surface and contributes to tPA binding to the cells [105]. It is this interaction, which is a consequence of the formation of an enzyme–inhibitor complex, that has been interpreted as a high-affinity receptor for tPA. Annexin 2 on the EC surface contribute to tPA binding as an intermediate-affinity interaction. tPA binds to the N-terminal core 1 domain of annexin 2, whereas plasminogen binds to the core
R. Das and E.F. Plow
4 domain [108]. Unlike plasminogen, tPA binding to annexin 2 is insensitive to lysine analogs but is affected by homocysteine [12, 109]. The Kd value of tPA binding to annexin 2 is 18 nM and accounts for up to 800,000 binding sites on HUVECs [110]. The p11 subunit of the annexin 2 tetramer, which is expressed by ECs, binds to tPA with a Kd of 0.45 mM [104] and may contribute to the low-affinity interaction of tPA with ECs. Actin [55] and tubulin [111] also bind to tPA,; these interactions are inhibited by C-terminal lysine analogs. The mechanism(s) for cell-surface expression of these cytoskeletal proteins is unknown. It is also likely that many of the identified Plg-Rs can be expressed by ECs and also bind tPA.
4.2 Roles of tPA/tPA Receptors in EC Function tPA binds both fibrin and cell surfaces. Binding of tPA to fibrin enhances plasmin generation by 500-fold [112], and binding to the EC surface induces a similar enhancement in plasminogen activation by tPA. Binding of plasminogen and tPA to isolated receptors, such as annexin 2, leads to a significant increase in plasminogen activation [113], although not quite as efficient as on cell surfaces. The functions of tPA overlap those of plasminogen (see earlier). Plasminogenindependent functions of tPA have been described in the brain [114] and activation of extracellular-signal-regulated kinase (ERK) has been attributed to the interaction of tPA with brain.
5 Urokinase Plasminogen Activator uPA is a 54-kDa serine protease. The zymogen is synthesized as a single-chain form (sc-uPA). Upon proteolytic cleavage sc-uPA is converted to a two chain form (tc-uPA), which has plasminogen activator activity dependent on a catalytic triad (Asp225, His204, and Ser356) within its C-terminal domain [115]. tc-uPA can be further cleaved by plasmin, which results in the removal of its growth factor domain and kringle domain from the N-terminal region to generate a two-chain low molecular weight form of uPA (33 kDa). This form retains plasminogen activator activity but no longer binds to the urokinase receptor, uPAR, on cell surfaces [42, 116].
5.1 uPA Receptor A specific cellular receptor for uPA was first demonstrated on monocytoid cells by Vassalli et al. in 1985 [116]. Significant contributions to our early understanding of uPAR were made
27 Interaction of the Plasminogen System with the Vessel Wall
by the Blasi [117] and the Dano [118] groups. Initial studies of the uPAR-deficient mouse failed to identify a significant role of the receptor in fibrinolysis as well as a number of other models [119, 120], but subsequent studies have implicated uPAR in inflammatory cell recruitment in certain models, including models of pulmonary infections [3, 121–123]. uPAR is present on many different cell types, including ECs. It is anchored to the plasma membrane by a glycosyl phosphatidylinositol (GPI) linkage, which permits substantial mobility of uPAR within the membrane [124]. Consequently, uPAR localization on the cell surface can be altered as cells respond to stimuli. On migrating cells, uPAR concentrates at the leading edge but is distributed uniformly on the membrane of nonmigrating cells [125]. uPAR is a glycosylated protein of 50–60 kDa composed of three distinct structural domains (D1, D2, and D3) and two short intradomain linker regions [124]. Most studies have implicated the importance of the D1 and D2 domains in uPA binding [126]. uPAR binds to all uPA forms which contain an intact growth factor domain. It has high affinity for uPA, sc-uPA, and the N-terminal fragment (ATF) that contains the growth factor domain [117]. The affinities of the uPA forms for uPAR range from less than 0.1 nM to more than 2 nM [117]. Affinity of uPAR for uPA can be modulated as cells respond to stimulation [127, 128]. The number of uPAR molecules expressed on cells ranges from 50,000 to 200,000 per cell on monocytoid cells, ECs and fibroblasts, and uPAR expression levels and affinity are subject to modulation depending on stimulation of the cells [125, 128]. uPA can cleave uPAR in the linker region between D1 and D2, releasing D2/D3 domain (soluble uPAR), which has chemotactic activity [129]. Besides uPA, uPAR also binds vitronectin, which is found in serum and the ECM and which also complexes with the uPA inhibitor PAI-1 [129]. Moreover, membrane-anchored uPAR can be released from cells by cleavage of its GPI anchor [124]. In paraxomal hemoglobinurea, the enzyme that attaches the GPI anchor to proteins is mutated and uPAR and other GPI-linked proteins are absent from cell surfaces [130]. It has been suggested that the absence of uPAR may contribute to the prothrombotic phenotype in paraxomal hemoglobinurea patients [130]. PAI-1 inactivates uPA whether it is or is not bound to uPAR [131]. Formation of PAI-1 or PAI-2 complexes with uPA bound to uPAR on the cell surfaces leads to internalization of uPAR/uPA/PAI complexes and drives uPAR cycling back to the cell surface. This process is enhanced by several adaptor molecules such as LDL-receptor-related protein, glycoprotein 130, very low density lipoprotein, urokinasereceptor-associated protein, and Endo 180 [132, 133]. The mannose 6-phosphate receptor, another uPAR-binding protein, specifically binds to the D2/D3 fragment of uPAR and targets it to lysosomes for degradation [134]. In the acidic environment of endosomes, sc-uPA and PAI-1 dissociate from the uPAR-containing complex and are degraded,
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whereas uPAR is recycled back to the cell surface in a functionally active form. In addition to its function as a uPA receptor, uPAR also affects cell adhesion, migration, differentiation, and proliferation via intracellular signaling. These signaling-dependent functions of uPAR depend on its association with other cellular proteins, including integrins, G-protein-coupled receptors [135], and caveolin [136]. The highest affinity of uPAR binding to integrins is with a5b1 and a3b3 and uPAR can promote adhesion of cells to vitronectin, fibronectin, and collagen by regulating these integrins and can even alter the specificities of these integrins for their ligands [137]. uPARdependent intracellular signaling can be induced by binding of uPA to uPAR or by clustering of uPAR. The signaling pathways/events induced by uPAR include the mitogen-activated protein kinase (MAPK) and focal adhesion kinase pathways and the downstream effectors of these pathways, including cytoskeletal rearrangements. uPAR was also found to coprecipitate with the members of Janus kinase/signal transducer and activator of transcription pathway, phosphatidylinositol 3-kinase/Akt pathway, the Src family kinases, and protein kinase C [129]. In addition to uPAR, some studies have suggested the presence of additional mechanisms for uPA to interact with cell surfaces [138]. The identity of these binding sites has not been fully resolved. It has been shown that gangliosides also provide a relatively low affinity binding site for uPA on cells [61].
5.2 uPAR on ECs uPA interacts with uPAR on ECs with high affinity (Kd 95% for IonWorks Quattro
• Drug EC50 influenced by adherence to plate surfaces • No high-resistance membrane seal • No simultaneous ligand addition and recording • No intracellular perfusion • No voltage clamp between reads
2, 4, or 8 wells in parallel; 1 cell/ hole/well (Port-a-Patch)
• Borosilicate chip surface • Primary cell use possible (single-well version) • Internal and external perfusion • Continuous voltage vs. time plots • Ligand addition during recordings • Cumulative/multiple compound additions
• System performance information limited
96-well plates; 16 channels sequentially; 1 cell/hole/well
• • • • •
• Requires a proprietary add-in to solutions to achieve higher seal rate • Though meant for 96x, does not contain 96 amplifiers
1 cell/hole/well
• Constant laminar flow • Positive pressure on electrode is independent of suction in surrounding aperture (mimics manual patch clamp)
IonWorks HT and Quattro (Molecular Devices) 384 wells ; 48 Planar 384-well Perforated WC; channels PatchPlate ~100 MW (HT) sequentially; 1 or 30–50 MW cell/hole/well (Quattro) (HT) or 64 seals cells/holes/well (Quattro)
Patchliner (Nanion Technologies) Planar WC; gigaohm seals NPC-16 chip
SyncroPatch96 (Nanion Technologies) Planar chip WC; gigaohm seals
CytoPatch (Cytocentrics) WC Planar, electrode tip shape surrounded by aperture in borosilicate glass surface IonFlux HT (Fluxion Biosciences) Microfluidic WC; megaohm well plate seals
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Borosilicate chip surface Ligand addition during recordings Internal and external perfusion Continuous voltage vs. time plots Cumulative/multiple compound additions
• Laminar solution flow • Ligand addition during recordings • Cumulative/multiple compound additions • Continuous voltage vs. time plots • Tested with voltage- and ligandgated channels • Separate cell and ligand wells Dynaflow HT (Cellectricon / The Automation Partnership / Astra Zeneca) Microfluidic WC; megaohm 96 channels/plate • Ligand addition during recordings well plate seals • Cumulative/multiple compound additions • Silica microfluidic chip minimizes compound adherence and consequent EC50 shifting • Validated for human ERG and GABAA 96- or 384-well; 16 or 64 (HT) amplifier arrays; 20 cell ensemble recordings
For each, one can control channel activity by altering voltage pulses or the solutions’ (intracellular and extracellular) ionic composition. In the inside-out configuration, the outer face of the membrane patch is sealed within the pipette while the previously inner membrane is exposed to the perfusing solution. This configuration is achieved by rapidly pulling up the
• Not commercially available; used only for in-house screening
• Drug EC50 influenced by adherence to polymer plate surfaces? • No high-resistance membrane seal
• No high-resistance membrane seal
pipette from the attached cell, without breaking the gigaohm seal. The perforated patch configuration allows for the diffusion of small molecules, but not of cytoplasmic proteins and larger molecules. Partial permeability of the membrane (without total rupture) is achieved by the addition of an antibiotic ionophore (e.g., nystatin, amphotericin B) to the
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Fig. 3 Basics of patch clamp electrophysiology. (a) A cell in a gigaohm cell-attached configuration. Basic electronics are depicted: ground (reference) electrode, borosilicate glass microelectrode, AgCl electrode connected to a downstream amplifier which controls voltage output (voltage-clamp mode) or current output (current-clamp mode).
(b) Different patch clamp configurations achievable using conventional, automated, or planar technology; outside-out and inside-out configurations can only be achieved with conventional patch clamp technology. Steps involved in achieving each configuration are outlined in the text
pipette solution and the latter’s subsequent diffusion toward and embedding in the membrane, where it creates small transmembrane openings. Perhaps one of the most widely used configurations involves accessing the entire cytosolic space: the whole-cell configuration. Following stabilization of the gigaohm seal, additional suction or a strong and fast voltage surge (“zap”) applied via the pipette ruptures the membrane patch, giving the user access to the intracellular medium. In this mode, the user can control ion flow across the plasma membrane by modifying the ionic composition in both the pipette and the bath solutions. Macroscopic currents recorded from cells represent the cumulative activity of all the ion channels contained within the cell membrane. The outside-out configuration is generated from the whole-cell configuration by gently pulling up the pipette, thereby stretching the membrane outside the pipette until it reseals itself, forming a miniature cell. It is important to note at this point that only the cell-attached and whole-cell configurations (ruptured and perforated patch) can be achieved using either planar chips or automated patch clamp systems. Each patch clamp configuration has its experimental applications, as evidenced by the sheer volume of articles published in which the patch clamp technique was the tool of choice. Singlechannel current recordings from cell-attached patches provide vital information about the gating and kinetics of individual channel proteins, and allow scientists to distinguish between currents flowing through related channels (e.g. K+ ions flowing through maxi KCa vs. KV channel proteins) on the basis of their unitary conductance. Unfortunately, successful singlechannel recordings require that at least (and preferably only)
one channel protein be present within the membrane patch, which is not always possible depending on the overall protein density of certain channels. The ability to simultaneously record the activity of a whole population of the same channel on the overall plasma membrane is the greatest advantage provided by the whole-cell configuration. Most particularly, the whole-cell mode allows one to record currents generated by ion channels with either small individual conductances or low densities and currents which may be difficult to resolve in cellattached patches. As a disadvantage, the large pipette volume relative to the small cell acts as a “sink” into which normally intracellular metabolic factors can dissolve, potentially leading to channel rundown during the course of an experiment. The prime advantage of the inside-out and outside-out excised-patch configurations is the ability to precisely control the cellular environment. In both scenarios, the operator controls the ionic compositions of solutions on either side of the membrane; this total control of the membrane environment allows for a better understanding of the channels’ permeation processes (pore selectivity and conductance), gating properties (channel opening and closure), and regulation (e.g., by intracellular signaling systems).
3.3 Troubleshooting and Limitations The relative inability to control the cellular environment without modifying the cell’s physiological properties is perhaps the most serious limitation of the patch clamp technique. Because we can only speculate as to the exact composition of the
34 Conventional Patch Clamp Techniques and High-Throughput Patch Clamp Recordings on a Chip
intracellular and extracellular media, the ionic composition of the solutions used in patch clamp experiments becomes a determining factor in isolating and identifying currents. Currents can be identified using ion-selective solutions. For example, in studying voltage-dependent Ca+ currents, K+ may be replaced by Cs+ (or tetraethylammonium) in both the pipette and the bath solution to minimize K+ channel activity. Alternatively, Ba2+ can be used as a replacement for Ca2+ since these channels are more highly permeable to Ba2+. Pharmacological tools are also indispensable for isolating specific currents. For example, tetrodotoxin can be used to selectively block Na+ channels or 4-aminopyridine can be used to block KV channels. The ability to clamp Em also allows for the selective regulation of channel activation. For example, rapidly inactivating Na+ currents are elicited by membrane depolarizations from very negative potentials (–80 to –60 mV), whereas their activation is minimized by using a holding potential of –40 mV since many of the channels are inactivated at this relatively negative Em. Similarly, L-type and T-type Ca2+ channels can be differentiated from each other by using different holding potentials (–70 and -100 mV, respectively). However, the ability to regulate channel activity by modifying the holding potential does not alleviate the need to minimize the activity of other “contaminating” currents. Therefore, ion replacement and pharmacological approaches are still essential in most experiments. Electrophysiologists agree that optimal cellular environment and cell quality, although critical, are not the only limiting factors in completing an experiment. Successful patch clamping is highly dependent on the technical ability of the user, effective cancellation of external electrical noise, attenuation of vibration within the system, and pipette reliability; these factors apply to both manual and automated systems unfortunately. Conventional patch clamp electrophysiology relies on the use of glass microelectrodes maneuvered onto a cell to form a highresistance seal on its membrane. More concisely, a cell is sandwiched between a 150-mm-thick cover glass and a pipette attached to a mechanical micromanipulator positioned inches away (a light year away as far as a cell is concerned!) to achieve the gigaohm seal. The number of things that can go wrong is amazingly high. (1) Moving the pipette toward the cell membrane and forming a seal requires an operator with a high level of training and a steady hand. Because the cell is relatively flat on the cover glass (especially plated and cultured cells), a few extra micrometers of vertical displacement toward the cell may transform a potential gigaohm seal into cell impalement or shattering of the pipette tip. (2) The fabrication of glass microelectrodes is not entirely reliable despite the vast improvements in pipette puller technology. Consequently, the size and geometry of the pipettes can vary from one glass capillary to the next and between pulls on the same day. Correcting for this variation is a time-consuming and tedious process that must be constantly addressed. (3) Most conventional patch rigs are housed within a Faraday cage to insulate them against random external
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electrical noise. The electrode being held high above (and connected to) the cell acts as an effective antenna for electrical noise. Therefore, all components connecting to electrical supply systems must be effectively grounded, especially to allow for the resolution of small currents. (4) Vibration in the system can reduce the success and longevity of an experiment; conventional patch clamp setups are mounted on air tables that absorb floor vibrations and allow maintenance of the seal.
4 T he Advent of Automated Technologies: From Pipettes to Planar Chips 4.1 S tepping Stones to the Development of Planar Electrodes Four significant advances in technology have provided the stepping stones leading to the development of planar electrodes: computation, MEMS, cell lines, and synergy with other high-throughput screening technologies [20]. High-speed data acquisition in parallel across 16–48 channels requires a data throughput of 800 kB/s, with a storage capacity of 1–5 GB each day. Also, analog-to-digital signal conversion with the small cells that are typically used for ion channel screening requires a 500 kHz sampling rate to capture the short capacitative transient during a voltage step in 16 data channels (approximately 30 kHz per channel). This is required to successfully detect the transition from the gigaohm seal to the whole-cell access configuration. Today’s computers can handle the level of multitasking, speed, and storage space required to automate high-throughput data handling and analysis. The development of new micromanufacturing technologies with submicron resolution represents the second significant advance. Advances in microfabrication techniques (reactive ion beam milling, etching, laser ablation) and substrate technologies (silicon, fused silica or quartz, SiO2, glass, plastics, doping and deposition methods) have enabled the production of a 1-mm hole on a planar substrate. Chemical modifications have also improved the “odds” of successful gigaohm seal formation; one example is the high-temperature oxidation of silicon (a semiconductor) to SiO2 (high-resistance dielectric) which is suitable for performing high-quality electrophysiological measurements across the aperture. The use of planar electrodes enables “miniaturizing” and parallelizing the patch clamp technique in an automationready manner. The increased availability of engineered transgenic cell lines has propelled the development of highthroughput devices. Otherwise electrically quiescent cells have been immortalized as perpetual cell lines which may then be transgenically modified to stably express target ion
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channel genes, providing a continuous supply of isolated and uniform cells for automated processing. The emergence of prepackaged automation component technologies (robotic fluidic handling systems, high-precision robots, standardized multiwell plate formats, automation software) under a high-throughput-screening paradigm has facilitated the growth of planar electrode voltage-clamp technology. Their commercial availability has significantly reduced the development time for instrumentation, and made available interfacing technologies and commercial products, as well as the financial burden of developing the instrumentation necessary to interface with the newly developed biochips.
4.2 “ Automatic” Cell Positioning: The First Step to Automation Automated electrophysiology technologies rely on the quality of cells that are isolated and delivered to the electrode in suspension. Cells must be relatively uniform in size and the suspension must be free of debris that could occlude the aperture during cell positioning, a step that is most easily accomplished by negative pressure. However, two exceptions are worth mentioning. Apatchi-1™ moved the pipette to a cell that is already settled onto a surface; therefore, negative pressure is not required to locate the cell onto the electrode. This device is no longer being manufactured. Cytocentrics’s CytoPatch™ places the recording aperture within a larger hole that draws the membrane down onto the recording aperture, thereby mimicking the effect of applying positive pressure with the recording aperture.
4.3 A utomating the System: Glass Pipette Interfaces Throughout the last 25 years, there have been many efforts to provide automation to voltage-clamp experiments. One direction of automation started with fast solution exchangers and picospritzers, and culminated in a high-throughput device from Cellectricon (Dynaflow) which is able to expose a manually voltage clamped cell to very high speed solution changes by moving the cell (clamped on a standard glass pipette) near and across an array of up to 48 microchannels each outflowing solution from a different compound well. Another direction started with specialized chambers to always bring a cell of interest into a predetermined position to simplify the hunt, and had its pinnacle with the development of the Apatchi-1™ system from Sophion [21], a system using computer vision to completely automate the hunting
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for an appropriate cell, patch pipette handling, gigaohm seal formation, and rupture of the membrane into the whole-cell configuration. In one particular embodiment of this approach, a group in Japan automated the hunt by placing the cells into the glass pipette instead of painstakingly chasing one down in the solution outside the pipette, a technique later repeated by Flyion for its Flyscreen product. The early field innovators include the Autopatch (Xention), Flyscreen (Flyion), RoboPatch (Wyeth), Robocyte (ALA Scientific), and OpusXpress (Molecular Devices, formerly Axon Instruments); the last three are for Xenopus laevis oocytes or similar cells, whereas the others were specialized for mammalian cells but were either poorly adopted or never achieved the necessary success rates for drug screening. Table 1 provides a brief description of the individual platforms. Other publications and reviews discuss the operation of these systems and should be referred to for more information [22–26]. Each of these apparatus (as well as the high-throughput devices discussed in Sect. 4.4) contain the six essential elements for any electrophysiology apparatus: an intracellular chamber, an intracellular electrode, an extracellular chamber, an extracellular electrode, a hole between the two chambers that can form a high-resistance seal with a cell, and a conductive medium in each chamber and within the hole. The most important finding with all of these systems is the following: all are equivalent to manual patch clamp recordings in terms of data quality, including true gigaohm seal formation, small access resistance, and stability of recordings. Yet another direction in the automation of electrophysiology started among academia with the making a micron-sized hole in a relatively flat section of a thin plastic tube [16], and culminated with a micromanufactured hole in a planar substrate. It was the planar substrate that finally removed the barrier to the modern higher-density and higher-throughput automation devices that are presently the new standard in electrophysiology.
4.4 Planar Array-Based Approaches Early attempts to produce planar electrodes for voltage clamping failed because of crude and unreliable manufacturing techniques (e.g., poke a fragile glass electrode into plastic, molding around delicate micron-sized features); however, the evolution of modern MEMS produced new methods, such as reactive ion drilling and laser drilling facilitated etching, and finally led to the production of a micronsized hole. “Planar array” refers to the use of multiwell configurations in either a plate-based or a chip-based format to record multiple channels in parallel. For the most part, the systems have integrated robotics which handle cells, solutions, and compounds. Table 2 outlines the major systems
34 Conventional Patch Clamp Techniques and High-Throughput Patch Clamp Recordings on a Chip
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Fig. 4 Chamber configurations for automated and planar patch clamp technologies. (a) PatchPlate used in IonWorks HT. (b) PatchPlate population chip used in IonWorks Quattro. (c) QPlate used in QPatch. (d) SealChip used in PatchXpress. (e) NPC-16 chip used in Patchliner
NPC-16. (f) CytoPatch chip used in CytoPatch. (g) Microfluidic bath used in the IonFlux HT system. (h) Microfluidic bath used in the Dynaflow HT system
which are currently available (as of July 2009) and/or in development. Schematic representations of system assemblies (including bath, perfusion, cell trapping, and electrodes) are provided for each in Fig. 4. The IonWorks HT system from Molecular Devices was the first screening system to come into use [27]. It uses a disposable PatchPlate™ electrode (Fig. 4a) which receives the cells and then experimental compounds. Although the consumable contains 384 wells (recording locations), the recording head has only 48 amplifiers and hence the instrument was designed to sample the clamped cells sequentially rather than simultaneously, imposing discontinuity in the voltage clamp of the cells. Forty-eight currents (perforated patch only) can be measured simultaneously. The technology for the IonWorks Quattro system is identical, except that the amplifier is able to drive a larger current to each chamber. This is because whereas the IonWorks HT consumable has one cell in one hole in each well, the Quattro system has 64 cells in 64 holes (in an 8 × 8 array) for each well (Fig. 4b). Each 384-well plate is capable of patching 150–200 cells for up to 6 min. Neither the HT version nor the Quattro version is able to achieve gigaohm seals;
however the 50–200-MW seals (quasi-voltage-clamped) that are achieved appear to be sufficient to study the timeindependent, or steady-state, currents and the throughput has been unmatched by other devices on the market at the time of this writing. Because of the slow compound delivery and interrupted voltage-clamp methods implemented, this system is not ideally suited to recording fast transient currents or ligand-gated currents. The second screening system released was the Patch Xpress (Molecular Devices), using AVIVA Bioscience’s SealChip16, a 16-well disposable that is capable of patching more than 12 cells (one cell per well) simultaneously for more than 15 min (Fig. 4d). Unlike the IonWorks systems, true gigaohm seals can be achieved (the first to be able to do so) [28, 29]. The PatchXpress became the golden standard in the field for automated electrophysiology because of its success rates with gigaohm seals, the longevity of seals matching that of glass pipettes, and its versatility, allowing the user to program virtually any experimental protocol. At the time of this writing, it is still unmatched in its versatility. The PatchXpress is ideal for all kinds of ion channels, and has very efficient data analysis methods;
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however, its throughput, compared with the throughputs of upcoming systems, is too slow for true high-throughput screening. The third system on the market to automate voltage clamping was a single-cell, low-throughput device from Nanion, Port-a-Patch, engineered for academic users, but lacking any fluidics automation. Nanion later attempted a higher-throughput device, the NPC-16 Patchliner, which was capable of microfluidic exchange of both the intracellular and the extracellular solutions [30] (Fig. 4e). Although the consumable had 16 recording chambers, the instrument only had two to eight parallel recording heads designed to perform an entire uninterrupted experiment on a well before moving to the next well on the consumable. The throughput for this device was insufficient to compete successfully on the market and has remained unpopular. Nanion recently developed the SyncroPatch 96, a large-scale version of the Patchliner which uses a 96-chamber consumable and has 96 simultaneous recording heads. Its fluidics system is designed to handle 384-well plates. This system is designed with one hole per and can achieve gigaohm seals with longevity comparable to that of the gigaohm seals of the PatchXpress, and throughput comparable to that of the IonWorks Quattro. Nanion has validated its application for measuring both ligand-gated and voltage-gated channels. The CytoPatch (Cytocentrics) was the fourth system to be released on the market, with a slightly different design from previous systems. Although it is a planar-based array, an electrode tip shape is micromachinned inside a larger aperture (Fig. 4f). The latter enables easier “capture” and positioning of cells via applying negative pressure to the larger aperture to position the cells while simultaneously applying positive pressure to the “electrode tip” to keep it clean and also to clean the cell surface prior to contact with the tip for the formation of the gigaohm seal, similar to what is done in conventional patch clamping [31]. Although this device has been launched, it is unclear how many instruments have been sold. No validation data are available at this time. The QPatch (Sophion) was the fifth system released (Fig. 4c). The first release had a 16-chamber consumable that could accommodate up to 48 chambers on the same platform. This was provided with 16 simultaneous headstages for a continuous clamp. All the fluidics are retained on the consumable and hence it is limited to using only small volumes, with all compound additions and washouts being left on the consumable [21]. This device is similar to the PatchXpress in many functions; however, it lacks the versatility and the fast washout method, requiring the use of the compound-delivery robotics to provide not only the drug additions but also the washouts. In spite of this, the availability of multiple-compound delivery nozzles allows the QPatch to be used with most ligandgated channels (which are usually more challenging because of the need for a fast washout). The initial release of QPatch
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has now undergone two upgrades, each costing almost as much as the original instrument. The first upgrade increased the number of consumable chambers and the number of headstages to 48, which made its throughput higher than that of the PatchXpress, but still lower than that of the IonWorks systems. The second upgrade increased the current driving capacity of the amplifiers, and there are multiple holes in each well, similar to the IonWorks Quattro. Although the presence of multiple holes prevents the formation of gigaohm seals, it increases the number of useable chambers to nearly 100%. Between 2007 and 2009, two new systems have been announced for primary and secondary drug screening [32]. Fluxion Biosciences introduced the IonFlux, a 16× precursor to the upcoming IonFlux HT which looks like a traditional plate reader, but can, according to the company, deliver between 8,000 and 10,000 assays per day with a 96× consumable. The consumable has a standard 384-well microplate footprint into which cells, compounds, and reagents are loaded. (Fig. 4g). A microfluidic channel network is built into the well plate bottom, allowing for real-time compound addition and cumulative dose–response curves. Electrodes are integrated into the bath as well, and the whole assembly is linked up with 64 amplifiers. Although the system appears to be capable of only loose megaohm seals (20-cell ensemble recordings), the full-time voltage clamp and ability to wash out in the same fluidics channels makes it effective for both voltage- and ligand-gated channels. In partnership with The Automation Partnership (TAP) and AstraZeneca, Cellectricon recently announced the Dynaflow®HT. The system contains cell and compound storage, as well as robotic liquid and plate handling, all of which allow the cells to be prepared in one place (Fig. 4h). The cells are then sucked from 96-well plates into a microfluidic chip. Here the cell membrane forms a seal with the chip where the current changes are monitored. Originally, the system achieved over 7,500 data points per day. With the introduction of a new chip late in 2009, the Dynaflow HT system generates 18,000 data points per day. As with the IonFlux HT system and many other high-throughput planar devices, the system has been developed with multiple cell lines with both voltage- and ligand-gated ion channels.
4.5 W hat About Channels Not on the Plasma Membrane? A number of ion channels are located on the membranes of intracellular organelles such as the sarcoplasmic/endoplasmic reticulum, the mitochondrion, synaptic vessels, and lysosomes. None of the patch clamping technologies described up to this point can possibly measure currents from such channels because the membranes are inaccessible. Therefore, these
34 Conventional Patch Clamp Techniques and High-Throughput Patch Clamp Recordings on a Chip
intracellular channels remain a drug target inaccessible to the automation techniques described so far owing to technological limitations. Furthermore, an important subset of ion channels are electroneutral exchangers, such as Na+–H+ exchanger, or generate a very small current that is difficult to measure under standard voltage-clamp conditions, such as the Na+–Ca2+ exchanger or Na+–K+-ATPase. These also remain inaccessible to planar electrodes because of small currents that may be hidden in the electrical noise from vibratory sources. The SURFE2R (surface electrogenic event reader) developed by IonGate has overcome these limitations (http:// www.iongate.de/index.php?lang=en&cont=4_0). Its technology does not use whole cells, but instead reads the charge displacement from membrane preparations isolated from cell culture. The membranes are coupled via an alkaline thiol and lipid bilayer to gold-surface electrodes, and the latter detect changes in the electrical signal that are produced by a halfreaction step of an exchanger without the need to use reporter molecules. The SURFE2R system is as efficient at measuring the electrical activity of electrogenic pumps such as the Na+–Ca2+ exchanger and the Na+–K+-ATPase pump as it is at measuring the electrical activity of electroneutral channels such as the Na+–H+ exchanger. Its application has been expanded through the use of specialized techniques to include the measurement of ion channels.
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However, this is difficult to do using macroscopic currents (I ) recorded in the whole-cell configuration because I is a function of the single-channel current (i), the number of channels (N ), and the probability of the channel being in the open state (Po) (Eq. 1), none of which can be determined for individual channels in whole-cell patches.
I = iNPo
(1)
Single-channel recordings are typically obtained from cellattached patches, although they can also be obtained from inside-out or outside-out membrane patches. In the simplest scenario, the small membrane patch surrounded by the walls of the glass pipette contains a single channel protein. Nature rarely cooperates this readily, so a more common occurrence is the measurement of multiple step-like channel openings within a membrane patch (Fig. 5a). From these openings, we can measure or calculate the following channel parameters: conductance/amplitude, open probability, and gating.
5 Characterizing Ion Channel Currents Permeation and gating mechanisms control the flux of ions through channel pores. The channel’s pore region contains amino acid sequences which determine the size, geometry, and charge within the channel pore, thereby acting as a filter to select which ions can be conducted and controlling the net rate of ion flux through the channel pore. Closure and opening of the channel (i.e., gating) occurs randomly between the open and closed states. The section that follows provides a brief description of how patch clamping applies to the study of the permeability and gating characteristics of ion channels.
5.1 Unitary Currents Ion channels open and close in a stochastic fashion. However, the probability of finding the channel closed or open is not a fixed number but can be modified/modulated by an external stimulus, such as voltage (i.e., voltage-gated channels), stretch (i.e., mechanosensitive channels), or an agonist (i.e., ligand-gated channels). Patch clamping can provide information on a channel’s open probability and conductance.
Fig. 5 Single-channel analysis. (a) The cell-attached configuration. Although many channel proteins are likely present within the patched area, a single channel has been represented here. The right panel depicts a sample single-channel current recording from a human pulmonary artery smooth muscle cell recorded at +50 mV. The three open channel levels (Ox – representing at least three different channels within the patch) and the closed level (C) are indicated. (b) Amplitude histogram with peaks for open levels 1–3 indicated. An I–V curve from which conductance is derived is shown on the right. (c) Open probability (Popen) is shown as a box plot of Popen as a function of time. Popen can be plotted as a function of patch potential (Epatch) to illustrate the voltage dependence of single-channel gating (right panel)
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5.1.1 C hannel Conductance, Selectivity, and Current Amplitude Each channel opening enables the passive flow of ions through the protein along their electrochemical gradient. The ease of flow of current across the membrane, or conductance (measured in siemens), cannot be directly measured, but it can be approximated from the amplitudes of the channel openings at different voltages. The amplitude, the differential amplitude between the maximum open level of one channel (O1) and the closed level (C), is generally determined from a raw-data-based histogram which reports the number of openings as a function of current amplitude (Fig. 5b, left). Fitting the histogram peaks with Gaussian distributions provides the mean amplitude of each open level (O1 – O3). In the example shown, the amplitudes of the openings were multiples of each other, suggesting that the channel proteins responsible were of the same type. When amplitude values are then plotted as a function of the applied patch potential [Epatch, = –(Eapplied)-|Em|, where Em is –40 mV], a linear regression fit of the data provides the calculated slope (g ), which approximates the conductance of the channel (Fig. 5b, right). The current–voltage relationship can also provide some indication as to the selectivity of the channel. For each concentration gradient, the reversal potential EX ss defined by the Nernst equation (Eq. 2):
EX =
[X ]o RT log , zF [X ]i
(2)
where R is the gas constant, T is the absolute temperature (Kelvins), z is the valence of the ion, F is the Faraday constant, and [X]o/[X]i is the ratio of extracellular to intracellular concentrations of the ion. EX can be determined by measuring the voltage at which the unitary current changes direction (i.e., amplitude goes from positive or negative,or vice versa, on the current–voltage curve).
5.1.2 Channel Open Probability Assuming that single-channel conductance is independent of Em, the measured current can also be described by Eq. 3:
i(t ) = NγPo (E, t )(E − EX ),
(3)
where i(t) is the time-dependent ionic current measured, N is the number of channels in the patch, g is the single-channel conductance, Po(E,t) is the probability that a channel is in the open state, E is the patch potential, and EX is the reversal potential of the current, determined using a double-pulse
protocol or the Nernst equation. As suggested by the above equation, the open probability of a channel protein varies with time and membrane potential. For each patch, the total single channel open probability (NPo) is calculated as follows (Eq. 4):
NPo =
∑ (O n), n
T
(4)
where n is the number of channels in the patch (1, 2, 3…), On is the time spent at the open level for each channel (i.e., 1–n), and T is the total recording time. The fraction of channels in the open state varies between 0 (closed) and 1 (open) depending on the nature of the channel and the stimulus. Figure 5c shows a representative NPo histogram. 5.1.3 Channel Gating Unitary recordings provide information regarding the behavior of the channel protein(s). A dwell level refers to the time a channel spends in the open or closed states. Like amplitude measurements, the durations of the channel transitions from the closed to open states can be measured and sorted into histogram bins from which we can estimate the number of closed and open states and their respective time constants. Typically, dwell times are plotted as a function of the number of observations. Alternatively, dwell times can be plotted as a function of time, as for a time course, to monitor the behavior of a channel over time (e.g., during drug application or in response to an external stimulus). Dwell-time analysis should only be performed in patches with a single open level to better approximate channel behavior, simply because channel closure may be masked by opening of another channel simultaneously. In some cases, the openings are “flickery,” as shown by the left-side openings in Fig. 5d. Dwell-time analysis of these recordings should be done using burst analysis. The latter applies to recordings where consecutive channel openings are separated by a dwell time less than the interburst (definite channel closure) interval. Only the duration of events (openings or closings), not their amplitude, is measured in this mode; therefore, burst analysis cannot be performed on data with more than amplitude level.
5.2 Macroscopic Currents As mentioned earlier, the whole-cell configuration provides a certain degree of experimental flexibility since the user can simultaneously control both intracellular and extracellular ion content and voltage. It allows for the determination of the
34 Conventional Patch Clamp Techniques and High-Throughput Patch Clamp Recordings on a Chip
function of a channel type as a whole family, Therefore, it can be used to successfully characterize a channel protein type in terms of both its permeability and its gating characteristics. 5.2.1 Passive Cell Membrane Properties
I x = g ∆E ,
Since many membrane channels are voltage-dependent, the membrane resistance likewise varies with changes in Em. Experimentally, cell membrane capacitance (Cm) is determined by software based on Eq. 6:
Likening the whole-cell configuration to an electrical circuit allows electrophysiologists to determine passive cell membrane properties, i.e., membrane capacitance, membrane resistance, and cell surface area. The pipette, with its two charged surfaces, is a dielectric substance and therefore acts as a capacitor in the circuit. Pipette capacitance (Cp, measured in Farads) is complicated in character, but its contribution to the overall circuit is usually minimized electronically by injecting a current transient designed to precharge the glass surface to the desired potential. The pipette pore presents a resistance to current flow that may be easily measured before seal formation (Rp, measured in ohms). During wholecell access, however, this resistance is increased by further resistance to current flow due to the contents or geometry of the cell itself (“series resistance,” or “access resistance”), i.e., resistance to filling the entire cytosolic space with the desired amount of charge, or potential due to interaction of charges with proteins or due to limited flux through long cell processes or narrow cell geometry. Once the cytosolic space of the cell has been voltageclamped, the cell membrane also presents its own capacitance. Unlike the pipette, the cell membrane is of relatively uniform thickness and uniform dielectric content. Therefore, in most cells the specific membrane capacitance (Cm), which is normalized by the area of the plasma membrane, is approximately 1 mF/cm2 [2]; cell capacitance is a good indicator of cell size (i.e., the surface area of a cell). The cell membrane itself is a very good dielectric, presenting a resistance of several gigaohms, in effect stopping the flow of charge across the membrane. However, the membrane resistance (Rm) is strongly influenced by the presence of ion conductances through membrane ion channels. Ion channels are selectively permeable to specific cations and have a gating mechanism that may be controlled by voltage or other methods. Ion channels produce a conductance (g, inverse of R) that is dependent on the transmembrane electrical potential energy (DE, measured in volts), and defined by Ohm’s law, which can be alternatively expressed as Eq. 5: (5)
where Ix is the conductance through a channel. The overall membrane resistance is the inverse of the sum of all the conductances present on the membrane. Overall membrane resistance is therefore a good indicator of the amount of current carried through all the open channels on the membrane.
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Cm =
∫I
tr
∆Vcomm
,
(6)
where DVcomm is the amplitude of a small hyperpolarization applied to induce a current transient (Itr). Similarly, membrane input resistance (Rm) can be calculated from Eq. 7:
Rm =
Rtotal × Rseal , Rseal − Rtotal
(7)
where Rseal and Rtotal are the resistances determined, respectively, from the steady-state currents of Itr in response to DVcomm (-5 mV) before and after break-in. The resting membrane potential (RMP) is also a very important property of quiescent cells. Although other techniques may also be used, RMP can be measured in the current-clamp mode once whole-cell access has been established. In many excitable cells, RMP ranges between –85 and –65 mV, which is close to the reversal potential for potassium ions (EK). Coincidentally, as discussed in other chapters, the activity of K+-permeable channels is the main determinant of RMP in many excitable cells, including pulmonary artery smooth muscle cells.
5.2.2 Channel Permeation and Ion Selectivity Channel selectivity can be studied by using the relative permeability (Px/Py) of a channel to different permeant ions. Because the driving force for ions in a cell membrane is dependent on voltage, a simplified voltage form of the Goldman–Hodgkin–Katz equation (Eq. 8) can be used to approximate Px/Py:
Erev =
Px [X ]o RT log . zF Py [Y ]i
(8)
With use of either the outside-out or the whole-cell patch clamp configurations to fix the ionic gradients, this ratio can be easily determined and the reversal potential of a current (Erev) can be measured, providing that ions X and Y are the only permeating ions on the external and internal sides of the membrane. Under whole-cell conditions, the reversal potential (therefore ion selectivity) can be established in two different ways. First, rapid current–voltage (I–V) relationships can be established by using ramp protocols under whole-cell conditions. In this scenario, the reversal potential
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is determined in a fashion similar to that using single-channel analysis. Second, a double-pulse protocol can be utilized whereby cells are depolarized to a single potential, then hyperpolarized to a range of potentials in which the voltage dependence and Erev of the current overlap. A current–voltage relationship generated by plotting the amplitude of the tail current (deactivating current induced by the hyperpolarizing step) as a function of its voltage can clearly reveal Erev and the selectivity of a particular channel.
5.2.3 Channel Gating Properties The stochastic behavior of a channel dictates that it oscillate between its open and closed states, with a jump of an energy barrier required for each state transition (Fig. 6). In some cases, the channels may also be found in intermediate inactivated or subconductance states. Transitions between these conformational states can be dependent on time, voltage, or ligand binding; the speed of these transitions is given by rate constants (v, v¢, w, w¢, x, and z). Establishing a channel’s voltage dependence is typically achieved by a simple voltage step protocol by which the cell membrane is depolarized to a range of potentials before being repolarized to a common negative potential near the cell’s resting Em. Plotting current amplitude as a function of the test potential establishes the current–voltage (I–V) relationship. Analysis of the shape of the currents also provides some information as to the activation (time-to-peak, tact) and inactivation (peak-tosteady state, tinact) kinetics, at least for time-dependent currents, which can be used to derive the rate constants for the channel state transitions. One problem associated with using simple I–V protocols to study the gating properties of a channel is that one cannot discriminate between the combined effect of the gating mechanisms and electrochemical driving force for the ion on channel gating. The simplest way to bypass this issue is to use a double-pulse protocol. An initial voltage step is applied to activate the channels, followed by a step down to a negative potential to allow time-dependent channel closure. Plotting the relative amplitude of the tail current as a function of the step voltage provides an indication of the voltage sensitivity and activation thresholds of the current in question. Relative availability of the channels following current inactivation can also be determined using a similar doublepulse protocol. The slope of the mathematical fit (Boltzmann function) to the activation (as given in Eq. 9) and inactivation C
C
v w
O
z
v w x
I
O y
C
v w
O
v w
O
Fig. 6 Putative state transitions of channels between open and closed states
(use V–V½ in lieu) curves provides an indication of how voltage controls the gating mechanisms, information that is particularly useful when one is studying the functional effect of a drug on channel activity. In this equation, V½ represents the half-maximal activation or inactivation potential and k is the slope factor, which is a good indicator of the steepness of the voltage dependence of channel opening and closing. Many channel-modulating compounds shift or alter V½ and/ or k values to produce their physiological effects. One example is 4-aminopyridine, which shifts the steady-state activation curve of KV currents to more positive potentials, steepens the voltage dependence (k) of activation, and shifts channel availability to more positive potentials in isolated rabbit coronary smooth muscle cells [33]. −1
V −V Y = 1 + exp ½ k
(9)
Finally, one property of inactivating currents remains to be investigated: the recovery from inactivation (I → C transition). The protocol to study recovery also employs a doublepulse strategy, with the determining factor being the delay between two pulses (the latter two being of similar amplitude and duration). A sample protocol is as follows: the membrane is depolarized from the holding potential to a positive membrane potential for 20 s (P1, to allow for full channel inactivation), repolarized to the holding level (approximately RMP), and depolarized again (P2) to the same potential as P1 for a shorter time. The pulses are repeated with the time delay between P1 and P2 varying for each episode of the stimulus train. Percentage recovery (P2/P1 × 100) is then plotted as a function of the time delay between P1 and P2. A recovery time constant (treact) derived from a mathematical fit of the resulting data points describes the recovery kinetics of the current. Repeating this protocol using different holding potentials can be useful in interpreting the voltage dependence of the transition between the inactivated and closed states, i.e., the change in treact may be dependent on voltage.
6 Summary and Future Directions The discovery of channelopathies and the increasing knowledge of the molecular composition of ion channels has placed a heavy burden on research scientists to understand the basic principles regulating ion channel function. As basic scientists learn more, the onus is being put on the pharmaceutical industry to develop compounds which target ion channels and transporters. Ion channels and transporters represent 6% of the overall research drug targets today, a number which has not changed dramatically since the beginning of this millennium. Currently, compound development for ion channel research is at a
34 Conventional Patch Clamp Techniques and High-Throughput Patch Clamp Recordings on a Chip
s tandstill, in part because of the relatively slow development of reliable, efficient, and cost-effective high-throughput devices to replace conventional patch clamping systems. The main goal of this chapter was to provide a simple, yet comprehensive description of the patch clamp technique and its evolution from the motor-driven pipette of conventional patch clamping rigs to the planar chip technology which has rendered manual patching somewhat obsolete. A secondary goal was to elaborate upon the information provided from current recordings and how it can be interpreted on a physiological basis. Advanced molecular biology techniques, as described in other chapters, have provided much information about the molecular composition of ion channels. However, patch clamp electrophysiology remains the tool par excellence for characterizing ion channels and for ascribing biological properties to channel subunits and differentiating between their contributions to the overall physiological state. Ongoing improvements in the high-throughput screening process and technology will no doubt bring to light much more clearly the relative importance of ion channel dysfunction in disease development and treatment. Acknowledgement The authors wish to thank António Guia at Aviva Biosciences for his critique and thoughtful insights regarding automated and high-throughput voltage-clamp devices.
References 1. Höber R (1905) Über den Einfluss der Salze auf den Ruhestrom des Froschmuskels. Pflugers Arch Eur J Physiol 106:599–635 2. Hille B (1992) Ionic channels of excitable membranes, 2nd edn. Sinauer Associates, Sunderland 3. Hodgkin AL, Huxley AF (1952) Propagation of electrical signals along giant nerve fibers. Proc R Soc Lond B Biol Sci 140:177–183 4. Hodgkin AL, Huxley AF (1952) A quantitative description of membrane current and its application to conduction and excitation in nerve. J Physiol 117:500–544 5. Hodgkin AL, Huxley AF (1952) Movement of sodium and potassium ions during nervous activity. Cold Spring Harb Symp Quant Biol 17:43–52 6. Noble D, Tsien RW (1968) The kinetics and rectifier properties of the slow potassium current in cardiac Purkinje fibres. J Physiol 195:185–214 7. Bean RC, Shepherd WC, Chan H, Eichner J (1969) Discrete conductance fluctuations in lipid bilayer protein membranes. J Gen Physiol 53:741–757 8. Neher E, Sakmann B (1976) Single-channel currents recorded from membrane of denervated frog muscle fibers. Nature 260:799–802 9. Rae JL, Levis RA (2003) Fabrication of patch pipets. Curr Protoc Neurosci Unit 6.3 10. Hamill OP, Marty A, Neher E, Sakmann B, Sigworth FJ (1981) Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches. Pflugers Arch 391:85–100 11. Drews J (2000) Drug discovery: a historical perspective. Science 287:1960–1964
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12. Overington JP, Al-Lazikani B, Hopkins AL (2006) How many drug targets are there? Nat Rev Drug Discov 5:993–996 13. Zheng CJ, Han LY, Yap CW, Ji ZL, Cao ZW, Chen YZ (2006) Therapeutic targets: progress of their exploration and investigation of their characteristics. Pharmacol Rev 58:259–279 14. Hopkins AL, Groom CR (2002) The druggable genome. Nat Rev Drug Discov 1:727–730 15. Venter JC, Adams MD, Myers EW et al (2001) The sequence of the human genome. Science 291:1304–1351 16. Kostyuk PG, Krishtal OA, Pidoplichko VI (1975) Effect of internal fluoride and phosphate on membrane currents during intracellular dialysis of nerve cells. Nature 257:691–693 17. Purves RD (1981) Microelectrode methods for intracellular recording and ionophoresis. Academic, London 18. Sakmann B, Neher E (1995) Single-channel recording, 2nd edn. Plenum, New York 19. Coetzee WA, Amarillo Y, Chiu J et al (1999) Molecular diversity of K+ channels. Ann N Y Acad Sci 868:233–285 20. Guia A, Xu J (2005) Planar electrodes: the future of voltage clamp. In: Yuan JX-J (ed) Ion channels in the pulmonary vasculature. Taylor & Francis, Boca Raton, pp 635–651 21. Asmild M, Oswald N, Krzywkowski KM et al (2003) Upscaling and automation of electrophysiology: toward high throughput screening in ion channel drug discovery. Receptors Channels 9:49–58 22. Dunlop J, Bowlby M, Peri R, Vasilyev D, Arias R (2008) High-throughput electrophysiology: an emerging paradigm for ion-channel screening and physiology. Nat Rev Drug Discov 7:358–368 23. Lepple-Wienhues A, Ferlinz K, Seeger A, Schafer A (2003) Flip the tip: an automated, high quality, cost-effective patch clamp screen. Receptors Channels 9:13–17 24. Schnizler K, Kuster M, Methfessel C, Fejtl M (2003) The roboocyte: automated cDNA/mRNA injection and subsequent TEVC recording on Xenopus oocytes in 96-well microtiter plates. Receptors Channels 9:41–48 25. Vasilyev D, Merrill T, Iwanow A, Dunlop J, Bowlby M (2006) A novel method for patch-clamp automation. Pflugers Arch 452:240–247 26. Vasilyev DV, Merrill TL, Bowlby MR (2005) Development of a novel automated ion channel recording method using "inside-out" whole-cell membranes. J Biomol Screen 10:806–813 27. Schroeder K, Neagle B, Trezise DJ, Worley J (2003) Ionworks HT: a new high-throughput electrophysiology measurement platform. J Biomol Screen 8:50–64 28. Tao H, Santa Ana D, Guia A et al (2004) Automated tight seal electrophysiology for assessing the potential hERG liability of pharmaceutical compounds. Assay Drug Dev Technol 2:497–506 29. Xu J, Guia A, Rothwarf D et al (2003) A benchmark study with SealChip™ planar patch-clamp technology. Assay Drug Dev Technol 1:675–684 30. Farre C, Stoelzle S, Haarmann C, George M, Brüggemann A, Fertig N (2007) Automated ion channel screening: patch clamping made easy. Expert Opin Ther Targets 11:557–565 31. Stett A, Burkhardt C, Weber U, van Stiphout P, Knott T (2003) CYTOCENTERING: a novel technique enabling automated cellby-cell patch clamping with the CYTOPATCH chip. Receptors Channels 9:59–66 32. Pearson S (2009) Investigating and focusing on ion channels as drug targets. Genetic Eng Biotechnol News 1–3 33. Remillard CV, Leblanc N (1996) Mechanism of inhibition of delayed rectifier K+ current by 4- aminopyridine in rabbit coronary myocytes. J Physiol 491:383–400
Chapter 35
Measurement of Pulmonary Vascular Structure and Pulmonary Blood Distribution by Multidetector-Row Computed Tomography and Magnetic Resonance Imaging Techniques Sara K. Alford and Eric A. Hoffman
Abstract Pulmonary-based X-ray computed tomography (CT) and magnetic resonance (MR) imaging techniques have been developed for structural and functional assessment of the lungs. CT methods, taking advantage of newer multidetectorrow CT (MDCT) technology, provide a means of quantitatively studying regional pulmonary perfusion (via iodine-based contrast agents) in conjunction with the acquisition of highly detailed information regarding the lung parenchyma and vasculature structure. Additional scanning protocols also allow for regional assessment of ventilation via the use of xenon gas, which is radiodense. Simultaneous to advances in imaging and imaging protocols, software has emerged which provides reliable quantitative tools for automated assessment of structure and function. Texture measures provide information at levels beyond the ability to resolve anatomic structures. CT-based pulmonary vascular structure and assessment for acute and chronic pulmonary embolism (PE) is now common clinical practice, and new functional perfusion techniques are being applied in research studies focusing on pathological changes and the causes of COPD and pulmonary hypertension (PH). Advances in technology have also dramatically improved the image quality and diversity of pulse sequences used for the lung in MR imaging, providing for another powerful imaging technique for the assessment of lung function. This diverse array of MR pulse sequences can be performed back to back to obtain a comprehensive assessment of the lung vasculature with MR angiography (MRA), perfusion imaging, and assessment of the right side of the heart. New functional measures including hyperpolarized noble gas imaging provide new measures of lung function, including ventilation and regional partial oxygen measurements.
S.K. Alford (*) Department of Radiology, University of Iowa, 200 Hawkins Dr., Iowa City, IA 52242, USA e-mail:
[email protected] Keywords X-ray computed tomography • Magnetic resonance imaging • Lung structure • Pulmonary embolism • Angiography
1 Introduction Pulmonary-based X-ray computed tomography (CT) and magnetic resonance (MR) imaging techniques have been developed for structural and functional assessment of the lungs. CT methods, taking advantage of newer multidetector-row CT (MDCT) technology, provide a means of quantitatively studying regional pulmonary perfusion (via iodine-based contrast agents) in conjunction with the acquisition of highly detailed information regarding the lung parenchyma and vasculature structure. Additional scanning protocols also allow for regional assessment of ventilation via the use of xenon gas, which is radiodense. Simultaneous to advances in imaging and imaging protocols, software has emerged which provides reliable quantitative tools for automated assessment of structure and function. Texture measures provide information at levels beyond the ability to resolve anatomic structures. CT-based pulmonary vascular structure and assessment for acute and chronic pulmonary embolism (PE) is now common clinical practice, and new functional perfusion techniques are being applied in research studies focusing on pathological changes and the causes of COPD and pulmonary hypertension (PH). Advances in technology have also dramatically improved the image quality and diversity of pulse sequences used for the lung in MR imaging, providing for another powerful imaging technique for the assessment of lung function. This diverse array of MR pulse sequences can be performed back to back to obtain a comprehensive assessment of the lung vasculature with MR angiography (MRA), perfusion imaging, and assessment of the right side of the heart. New functional measures including hyperpolarized noble gas imaging provide new measures of lung function, including ventilation and regional partial oxygen measurements.
J.X.-J. Yuan et al. (eds.), Textbook of Pulmonary Vascular Disease, DOI 10.1007/978-0-387-87429-6_35, © Springer Science+Business Media, LLC 2011
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2 CT Imaging Techniques 2.1 Development of CT Scanners CT serves as the modality of choice for lung imaging owing to its widespread availability, resolution, high signal-to-noise ratio (SNR) for lung tissue, and fast image-acquisition speed. CT technology has rapidly advanced since its introduction into the medical field in the early 1970s. An early, experimental scanner, known as the Dynamic Spatial Reconstructor, built at the Mayo Clinic housed 14 X-ray sources aimed at a hemicylindrical fluorescent screen, in turn monitored by 14 television cameras. The 20 t gantry rotated at 15 rpm and provided enough projection data to reconstruct up to 240 slices of the body every 1/60 s [1–3]. Many of the early quantitative imaging analysis tools evolved from the efforts to evaluate data obtained with this CT scanner. Data demonstrated the utility of CT imaging in providing regional lung density measures [4–6], lung volumes [7], as well as anatomic detail of the airway and vascular trees [8–11]. Progress regarding volumetric imaging of the lung was delayed largely owing to the clinical availability of volumetric scanning modalities as well as computational resources to evaluate the large data sets derived from volumetric imaging. Spiral CT was introduced by Kalender et al. [12], allowing for volumetric imaging of the lung in a breath-hold. Although single-slice spiral CT scanners with one X-ray source/detector pair were rapidly adopted to acquire volumetric images of the lung, breathhold times were often too long for patients to sustain a breath-hold and slice thicknesses were large relative to the anatomic structure of interest. Thin-slice, high-resolution, step-and-shoot modes, gathering slices widely spaced from the lung apex to the base, were introduced and remained the method of choice for assessing peripheral lung anatomy [13, 14] and functional data were not available clinically. Fourslice MDCT systems became available in 1998, followed by 16-slice and 64-slice systems in 2001 and 2004, respectively. At the time of this writing, scanners are available with 320 slices covering approximately 12 cm of the apical–basal lung axis. MDCT scanners use cone-beam X-ray geometry and multiple detector rows to collect more slices per gantry rotation, thereby allowing for increased z coverage. Their increased speed and ability to image thin slices of isotropic (voxels with the same x, y, and z dimensions) resolution provide significant benefits compared with the earlier generation of single-detector or limited multidetector-row scanners. With isotropic voxels and a voxel dimension on the order of 1 mm or less, one is able to reslice the volumetric image according to anatomic structure of interest. Once 16-slice scanners had been developed, full lung volumes in a breath-hold (10–20 s compared with 40 s for the four-slice scanner) and contrast-enhanced images during multiple phases of the cardiac cycle were now
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feasible. As technology improved, the gantry rotation speed also increased, leading to improvements in technical scanner performance. As detector arrays advanced, 64 slices could be acquired in one gantry rotation in as little as 0.33 s. With MDCT systems, the scanner can also be used in an axial mode where the detector and X-ray source do not spiral through the patient, but rather remain in the same axial position and provide data for lung in the detector plane. This allows for imaging of a slab of lung slices over time to provide functional imaging such as of perfusion. As detector arrays widen, more lung can be covered by the slab using the axial mode. One limitation may be the use of a very broad X-ray beam needed to cover the lung volume without spiraling. The effect of a cone-beam X-ray source on the image reconstruction is currently being explored and new reconstruction algorithms are being developed [15–19].
2.2 Dual-Energy CT A new generation of CT scanner has been introduced by Siemens Medical Systems (Somatom Definition and Somatom Definition Flash) that includes two X-ray sources and two detectors mounted on a rotating gantry and angularly offset approximately 90° from one another [20]. The first dual-source CT (DSCT) scanner system specifications were similar to those of the 64-slice scanner, but could either be used as a dual source, providing twice the coverage for the time, or for dual-energy CT (DECT) imaging, where X-ray sources are set to different energies (kilovolts). DSCT utilizes both sources, thereby increasing temporal resolution, which allows for less motion artifact, which is important for cardiac and lung imaging applications. DECT imaging acquires data from two X-ray guns set to a different energies such as 80 and 140 kV. At the two energy levels, there is a density shift in regions with iodinated or xenon gas contrast agents, but little to no concomitant shift is observed in normal body tissues, allowing for sensitive discrimination between tissue types and contrast agent [21, 22]. The iodine contrast component can be separated out by subtracting lung tissue from the two images and the automatic removal of bone. This iodine map provides a perfused blood volume or microcirculation map. Although the enhancement is also dependent on external factors such as contrast volume, flow rate, and administration site, these maps can serve as an index of regional lung perfusion. In addition, a “diagnostic scan” can be determined from an averaging of the data collected by the two X-ray sources. Alternatively, the iodine signal can be subtracted from the reconstructed image to provide for the ability to assess a virtual unenhanced image for regional lung density analysis. The second-generation DSCT scanner (Siemens Definition Flash) rotates at 0.28 s, provides 128 slices
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per rotation, and spirals through the entire thorax in as little as 0.6 s with a table feed of up to 43 cm/s. Added filters placed in front of each X-ray source provide improved separation of the delivered photon energy spectra from the X-ray tubes set at 80 and 140 kV, providing increased dual-energy-based characterizations.
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voxel value of the 2D maximum intensity projection image. Misrepresentation (an over- or underrepresentation) occurs more frequently with this technique, especially if there are overlapping structures or high-intensity regions in the vasculature such as calcification.
2.4 CT Angiography 2.3 Quantitative Assessment of Volumetric Chest MDCT Images The structure and integrity of the pulmonary vasculature can be assessed with a noncontrast spiral lung volume scan obtained typically at full inspiration. This lung volume provides the best contrast between the air-filled spaces and the parenchyma, airways, and most pathological lung processes. CT contrast is based on the attenuation of different tissues, approximately related to tissue densities. Each voxel reflects the tissue density or X-ray attenuation coefficient by its Hounsfield unit (HU) value. The HU scale is defined as -1,000 HU for air, 0 HU for water, and 1,000 HU for bone. For a noncontrast volumetric lung scan, if one considers the lung as composed of two materials, air at -1,000 HU and “tissue” (including blood volume) at approximately 55 HU, then the density in HU can be converted to the amount of air and “tissue” content per voxel [23]. Volumes are determined on the basis of the product of the segmented in-plane cross-sectional area by the slice thickness, summed over all the slices spanning the segmentation. By combining these two measures, one can determine the absolute volumes of air or tissue in the lung from the region volume multiplied by the fraction of air or tissue. With the recognition that many peripheral pulmonary processes either increase or decrease regional lung density, there is a growing focus on quantifying the distribution of lung attenuation values within CT images. Lung parenchyma voxels can be categorized on the basis of their attenuation or HU values and according to their place in the lung attenuation histogram. This has been applied to emphysema, with density masks of −950 and −910 HU used to determined the percentage of severe and moderate emphysema present in the lung parenchyma [24]. Postprocessing techniques have been developed to aid with management and assessment of large volumetric CT data sets. The near isotropic resolution of the MDCT data allows for 2D and 3D visualization. Three-dimensional volume rendering images of the lung, lobes, airway, or vasculature tree segmentations can be used to visualize the anatomy in three dimensions. Multiplanar reconstructions in the coronal, sagittal, or oblique views are used to evaluate the pulmonary vasculature, specifically for defining the extent of embolization or stenosis. In maximum intensity projection images, the highest HU value attenuation along a user-selected ray becomes the
As a result of the increased image-acquisition speed, improved spatial and temporal resolution, and greater scan volume with newer MDCT scanners, CT angiography (CTA) has become an excellent clinical assessment technique for the pulmonary vasculature and has become the first-line imaging test for the clinical assessment of a suspected acute PE. CTA protocols obtain high-resolution images of the entire chest by acquiring thin high-resolution slices during enhancement from an iodinated contrast medium delivered intravenously with a contrast injector. The timing of the peak contrast enhancement with imaging is crucial and therefore a bolus-triggering software program or a test bolus is used to determine the scan delay time. If images are acquired too early, proper enhancement may not have occurred, resulting in a filling defect. This lessens the contrast between the blood and clot, leading to more difficulty in distinguishing the two. When abnormal hemodynamics are present, such as in the case of low cardiac output or right-sided heart dysfunction, the bolus time delay can vary significantly. Short scanning times help minimize the respiratory motion artifact and ensure high image quality. With scanners acquiring images in as short a time as 0.6 s, dyspneic patients will no longer have trouble with breathholding. In fact, it is possible to acquire full chest scans with no breath-hold at all. For quantitative studies, it will remain a requirement that images be acquired at standardized lung volumes. With the use of slower scanners, respiratory motion results in global parenchymal blurring and can be detected by its presence in all vessels in a region of lung.
2.5 Four-Dimensional X-Ray CT Perfusion Measurements Dynamic perfusion imaging with 4D MDCT scanners allows for quantitative in vivo measurements of regional pulmonary blood flow (PBF) and mean transit time (MTT) [25, 26]. A series of temporal, ECG-gated, breath-hold images are acquired in axial scanning mode (nonspiral scanning mode with fixed z coverage) during the delivery of a central bolus of iodinated contrast, thereby tracking the first pass of iodinated contrast through the pulmonary circulation. A catheter is placed in the superior vena cava and a power injector is used to deliver a fast, sharp bolus. Images are acquired during
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a single breath-hold, allowing for the acquisition of data at a constant lung volume, typically functional residual capacity. ECG-gating minimizes cardiogenic motion by scanning at the same point in each cardiac cycle. As the bolus passes through the pulmonary vasculature and lung parenchyma, a change in tissue radiodensity is reflected by a change in the measured CT voxel HU value. HU values directly increase with increasing concentration of iodinated contrast agent, thereby reflecting the concentration of iodinated contrast in the region at that time point. Lung parenchyma voxels are segmented and filtered to remove airways and vessels. Time– attenuation curves for the pulmonary artery and regions of lung parenchyma are used to calculate regional MTT and PBF using indicator-dilution theory. Wolfkiel et al. measured myocardial blood flow by electron-beam CT, using a singlecompartment model of indicator transport [27]. The basic concept of their model is that regional flow is proportional to maximum enhancement or the total accumulated contrast in the lung region of interest. Therefore, the peak height of the time–attenuation curve is a measure of the flow within that tissue region of interest. The indicator must satisfy several criteria to allow the application of first-pass indicator-dilution theory: (1) contrast indicator should not affect flow by physiologic or volume effects, (2) the indicator must mix with the blood uniformly, and (3) the indicator must remain in the intravascular space. Nonionic iodinated contrast agents used in CT are a suitable indicator. Wu et al., using the DSR scanner, applied this model in pigs to measure PBF and showed a good correlation with microsphere-based flow measurements [28]. The model assumes that tissue accumulation of indicator must be nearly complete before the onset of indicator washout. Wolfkiel and Rich tested this assumption by comparing time–density curves of the pulmonary outflow tract, lung parenchyma, and aortic outflow tract, and determined with a central intravenous, sharp bolus injection the washout was minimal and the assumption was valid [29]. Time–attenuation curves are obtained for regional lung parenchyma and pulmonary artery by plotting the mean HU value for a region of interest with respect to time. Flow per volume is obtained from the ratio of the peak HU value in the parenchymal region of interest to the area under the arterial input curve. In CT imaging, this equation can be rewritten in terms of HU value changes in the lung parenchyma and pulmonary artery. MTT measurements can be obtained by applying the residue-detection model for a bolus injection of contrast. Deconvolution techniques can recover the regional residue function. MTT can be calculated as the ratio of the area under the residue curve to the maximum height of the residue curve. Figure 1 demonstrates a typical time–intensity curve obtained from the region of the pulmonary artery as well as a dependent and nondependent parenchymal lung region. Quantitative measures of MTT and PBF are acquired for each predefined sized region of interest throughout the lung. Also
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shown in Fig. 1 is the ability to detect, without use of contrast agent, detailed anatomic structure of the pulmonary arterial tree. Recently, image processing software has been shown to have the ability to label the arteries and veins separately from the detected pulmonary vascular tree structure [30]. Partial volume effects observed in perfusion images suggest that the time–attenuation curves obtained from voxels of lung parenchyma do not represent flow solely at the capillary level, but may also represent flow from the small arteries. Won et al. demonstrated the ability to use dynamic CT imaging to extract the signal within a single voxel related to microvascular blood flow, thus demonstrating the ability to estimate perfusion parameters at or near the level of gas exchange [26]. This was done by assessing the time–attenuation curve in the feeding pulmonary artery and in the peripheral parenchymal regions to then extract a transfer function, which expresses the mathematical function serving to convert the arterial curve into the parenchymal curve. Deconvolution of the pulmonary artery and regional parenchymal time–attenuation curves consistently yields a bimodal transfer function consisting of a sharp, narrow peak and a second, overlapping more dispersed curve component. It was concluded that the first mode reflected the partialvolume small arteries in the voxel, and the second mode reflected the partial-volume microvascular bed. By separating out the first sharp, narrow curve component from the second, more dispersed component, and reconvolving the arterial input curve with only the second component of the transfer function, they obtained a parenchymal curve reflecting the microvascular perfusion. Therefore, the CT-based approach has the ability to determine the microvascular and arterial curves within peripheral regions of interest and correct for the partial-volume effect of the small arteries within the voxel.
3 MR Imaging Techniques MR imaging relies on the manipulation of gradient fields to create contrast based on the precession of hydrogen atoms when placed in an external magnetic field. These techniques do not use ionizing radiation; therefore, unlike CT, longitudinal studies to follow disease and studies in children are feasible without dose concerns. Traditionally, the lung has been a difficult organ to image with MR imaging. The lung is compose mainly of air, meaning it has a low proton density, resulting in low signal. In addition, the lung has an extremely heterogeneous magnetic susceptibility owing to multiple air–soft tissue interfaces which produce large local gradients that dephase the signal, resulting in very short T2* values [31, 32]. The signal is further reduced owing to respiratory motion, cardiac motion, pulsatility of PBF, and molecular diffusion. Fast imaging sequences are necessary to capture the signal before it decays by using ultrashort echo times (TE) [33]. A fast gradient echo
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Fig. 1 Upper left: Computed tomography (CT)-based regional pulmonary blood flow (PBF, mean normalized) measurements for a 46-year-old male smoker with pack-year history of 31 years. His spirometry test results were normal but CT imaging studies demonstrated early signs of emphysema. CT perfusion imaging studies demonstrated increased heterogeneity in regional PBF and mean transit time (MTT) measurements in subjects with early signs of emphysema compared with never smokers or smokers with normal spirometry and
imaging studies. Upper right: An example of time–intensity curves for the pulmonary artery (top graph) and a dependent (purple) and nondependent (orange) parenchymal region used to calculate PBF and MTT. Lower left: Detailed vascular anatomy is detectable in a nonenhanced CT scan of the lung obtained at a total lung capacity breath-hold. Lower right: New algorithms are being developed with the ability to separate arteries from veins. (Reprinted from [30] with permission)
sequence with ultrashort TE utilizing high-speed, high-strength gradient systems can be used to acquire structural and functional information about the lung. High-performance gradient systems with amplitudes in excess of 20 mT/m and slew rates over 120 mT/m/ms have allowed for ultrafast MR sequences, including ultrafast MRA and significant reduction in the imaging time. Fast imaging sequences typically utilize a fractional echo sampling scheme, high-bandwidth data acquisition, and a truncated RF pulse to minimize the TE.
allowing for the reduction of minimum TE and repetition time (TR). A short TR allows for shorter breath-holds, whereas a short TE minimizes the background signal and susceptibility artifacts. Gadolinium contrast is injected intravenously with a power injector at rates between 2 and 5 mL/s, followed by injection of a saline chaser, causing the blood to appear bright in images. The development of parallel imaging has further improved the ability of 3D MRA to capture the pulmonary vasculature, especially in dyspneic patients who breath-hold poorly. Parallel imaging uses the geometry of numerous surface array coils and sensitivity maps to create k-space information from undersampled regions. The trade-off for faster data-acquisition speeds (temporal resolution) or higher spatial resolution is a reduction of the SNR. By combining parallel imaging with nonCartesian k-space filling pulse sequences such as simultaneous acquisition of spatial harmonics (SMASH) and sensitivity encoding (SENSE), one can further improve image quality. Depending on the clinical indication or research question, parameters can be adjusted to obtain either a set of highspatial-resolution, monophasic images or a multiphasic image set with high temporal resolution, referred to as dynamic time-resolved contrast-enhanced 3D MRA. To obtain higher
3.1 MR Angiography Contrast-enhanced 3D MRA was introduced by Prince et al. [34] and is now the most commonly used MR technique to image the pulmonary vasculature for the assessment of congenital anomalies, PH, or acquired conditions such as PE, specifically in the subset of patients unable to undergo CT (pregnant women or patients who would need following up). MRA images are obtained during a breath-hold using T1-weighted gradient echo MR imaging utilizing a high-performance gradient system,
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temporal resolution with the same image time (ensuring breath-hold), spatial resolution is sacrificed. A high-spatial resolution enables the evaluation of the peripheral pulmonary vasculature including the subsegmental branches, whereas high temporal resolution allows for discrimination of arterial and venous vessels through separation into various phases (arterial phase, parenchymal phase, venous phase). Regional perfusion can be assessed and quantified with contrast-enhanced images obtained from time-resolved angiographic imaging. Dynamic contrast-enhanced MR imaging of pulmonary perfusion acquires a series of images over time, tracking the first pass of a bolus of contrast through the pulmonary circulation. Images are acquired for a single plane (2D) or of the entire lung (3D) using T1-weighted ultrafast imaging sequences with short TR and TE during a low-dose bolus of gadopentetate dimeglumine (Gd-DTPA) contrast agent injected via an intravenous line in the antecubital vein in the arm. This technique uses ultrashort TE that then allow for higher temporal resolution, thereby allowing collection of dynamic information for the direct visualization of regional perfusion. Images are obtained approximately every second over 20–25 s at a slice thickness of 12–20 mm. Quantitative perfusion measurements are obtained on the basis of indicatordilution principles for first-pass bolus studies [35–37]. From signal intensity–time curves, PBF, MTT, and pulmonary blood volume (PBV) measurements are obtained using indicatordilution theory, deconvolution analysis, and the centralvolume principle on a voxel-by-voxel basis. For measurements to be accurate, the measured signal intensity change must be linearly correlated with the local concentration of Gd-DTPA [38]. It is therefore important to use a lower dose of contrast to not cause saturation. The regional PBV is calculated directly from the area of the MR signal intensity–time curve for a region of interest, normalized to the integrated arterial input function from the pulmonary artery. Regionally, PBF is calculated using a relationship between the concentration–time curve for the region of interest and the pulmonary artery. The residue function is determined by deconvolution. The initial height of the deconvolved time–concentration curves, or the residue function evaluated at time zero, equals the PBF. Using the central-volume principle, one determines MTT by PBV divided by PBF. Perfusion maps of the lung can be displayed to demonstrate perfusion deficits.
3.2 Cardiac Imaging for Right Ventricular Assessment Cardiovascular MR imaging techniques have been developed for quantitative measurements of right ventricular anatomy, shape and function, velocity profiles, stress and strain measurements, coronary artery anatomy, and myocardium viability. The
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assessment of the right ventricular anatomy and function is necessary along with pulmonary vasculature measurements to fully understand pulmonary vasculature diseases, specifically for pulmonary arterial hypertension. Protocols combining MRA, lung perfusion imaging, and the assessment of rightsided heart function with cardiovascular MR imaging allow MR protocols to provide a comprehensive evaluation of the lung and heart. Delayed contrast enhancement imaging is widely used to assess myocardium viability following myocardial infarctions, but different delayed enhancement patterns have also been documented in subjects with PH. In addition to the first-pass kinetics and demonstration of pulmonary vasculature anatomy from MRA, images taken 10–15 min following contrast administration demonstrate a pattern of delayed enhancement which allows for the assessment of myocardial viability. In delayed-enhancement imaging, there is hyperenhancement of nonviable tissue, reflecting irreversibly damaged regions of myocardium necrosis and fibrosis [39, 40]. MR quantitative volumetric assessment of the ventricles has become the gold standard and work has been done to determine normative values for cardiac parameters based on age and sex [41]. Many pulse sequences, including spin echo (“black blood”) and steady-state free precession (“bright blood”), have been developed for the visualization of the heart chambers and anatomy. Static image sets are acquired at enddiastole and end-systole while the subject is holding his/her breath at end expiration. A dynamic cine image set can capture multiple phases of the cardiac cycle. Although 2D static images can provide data on the anatomy, a 3D contiguous stack of short-axis or transverse images with at least two phases of the cardiac cycle is necessary for volumetric measurements. Manual or semiautomatic segmentation of the endocardial and epicardial contours is performed and cardiac parameters including ventricular volumes, stroke volume, ejection fraction and myocardial mass can be calculated. Right ventricular volume for end systole (ESV) and right ventricular volume for end diastole (EDV) are determined on the basis of Simpson’s rule. Stroke volumes can be determined by subtracting ESV from EDV. Ejection fraction is the stroke volume divided by the EDV. Ventricular mass is the myocardial volume multiplied by the specific weight of muscle (1.05 g/cm3). Phase-contrast MR imaging is capable of determining velocity and blood flow in the pulmonary vasculature and has been used to differentiate pulmonary disease such as pulmonary arterial hypertension [42]. Images are obtained in a plane perpendicular to the dominant flow and voxels represent the signal phase, which is velocity-encoded. Volumetric flow measurements are obtained by multiplying the spatial mean velocity (cm/s) of blood flow with the cross-sectional area of the vessel (cm2). Measurements of the velocity and flow of the pulmonary artery can be used to determine stroke volume, cardiac output, ejection fraction, and regurgitation fraction. Many new techniques and applications of cardiovascular MR
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imaging are currently in development. Myocardial tagging sequences track the motion of the heart by using RF pulses to form grid lines across images which deform as the heart contracts. Three-dimensional global and local deformation such as strain patterns can be determined for the right ventricle [43]. Real-time MR imaging is being used to perform MR-guided endovascular catheterization procedures. Catheter placement is visualized by susceptibility artifacts and right ventricle and pulmonary artery pressures can be determined and correlated with ventricular volume measurements to construct right ventricular pressure–volume curves [44, 45].
3.3 Arterial Spin Labeling Arterial spin labeling (ASL) is an MR perfusion method for quantitatively measuring regional PBF by using arterial blood as an endogenous tracer. It is completely noninvasive, does not require any contrast injection, and is repeatable. The protons in the water of pulmonary arterial blood are magnetically tagged, and the quantity of tracer in the lung parenchyma can be determined on the basis of measurable magnetization effects of the water present in the lung. ASL magnetizes the blood by applying an inversion RF pulse proximal to the imaging slice, thereby tagging the blood. MR imaging for ASL is typically performed using a 1.5-T scanner with a phased-array coil and ECG gating to minimize cardiac motion. Breath-holding at functional residual capacity ensures consistent lung volume, minimizes respiratory motion, and optimizes the volume of blood relative to the air in the lung and thus improves the SNR. ASL techniques can be divided into two categories: steady-state and pulsed sequences. Steady-state ASL was introduced by Detre et al. [46, 47] and applied to the lungs of human subjects by Roberts et al. [41, 48, 49]. The steady-state ASL technique measures perfusion on the basis of a detected change of steady-state magnetization due to the labeling of arterial water supplying an organ. Pulsed ASL sequences were introduced by Edelman et al. [50] and now several specialized pulsed ASL sequences have been developed, including signal targeting alternating RF (STAR) [51–53], flow-sensitive alternating inversion recovery (FAIR), and more recently, FAIR with an extra RF pulse (FAIRER) [54]. These techniques provide a perfusionweighted image generated by the subtraction of a tagged and nontagged image. The pulsed ASL technique labels the arterial blood by inverting the protons proximal to the imaging slab by applying a selective inversion recovery RF pulse. For the lung, spins are typically magnetized in the right ventricle or main pulmonary artery. After a transit time delay, a tagged image is acquired. This delay allows for spins to distribute throughout the pulmonary circulation. A control image (no tagging) is also obtained. These two images are then pair-
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wise subtracted, yielding a perfusion-weighted image. The signal intensity of each voxel in the perfusion-weighted image is proportional to the volume of arterial blood delivered to the voxel during the time delay, which in turn is proportional to the local perfusion. Quantification of regional blood flow can be determined from ASL images by the application of mathematical models that take into account other physiological and MR factors. The Bloch equation has been modified to include flow effects and kinetic models have been developed for the various pulse sequences [55]. Perfusion-weighted images have near-complete background suppression since the signal from stationary protons present in both images will cancel out during the subtraction. This subtraction is extremely sensitive to motion. Another consideration is that although ASL provides perfusion-weighted images, it provides a snapshot of blood flow located in many vessels, not just the capillary bed. All flowing blood entering the image slice from outside the tagged region will contribute to the signal intensity of the perfusion-weighted images; therefore, changes in signal intensity could reflect alterations in local arterial blood flow, venous blood flow, or both.
3.4 Hyperpolarized 3He MR Imaging The use of a hyperpolarized gas such as 3He with MR imaging was introduced in the mid 1990s, and hyperpolarized 3He MR imaging provides a variety of techniques capable of providing functional and morphological information on the lung. 3He is an inert, nontoxic noble gas, not absorbed in the body after inhalation. To enhance the signal by 4–5 orders of magnitude, the gas can be hyperpolarized by optical pumping techniques (spin-exchange method or metastability-exchange method). Hyperpolarization is a process in which atoms are brought to a higher energy level by the introduction of laser light at appropriate wavelengths. Depending on method, 30–60% polarization can be achieved. The hyperpolarized gas is inhaled and diffuses rapidly into the air spaces of the lung, acting as a contrast agent for the ventilated airways and alveolar spaces. To image with hyperpolarized 3He, the MR scanner must have broadband RF transmit/receive hardware and have a coil tuned to the Larmor frequency of 3He, not 1H. 3He is currently an investigational new drug and has not been approved by the Food and Drug Administration for general clinical use. No adverse effects have been reported to date, but continuous pulse oximetry is performed to monitor oxygen saturation levels to ensure oxygenation does not fall with the breathing of anoxic or hypoxic gas mixtures during scanning. Four techniques for hyperpolarized 3He MR imaging have been developed: static-ventilation, dynamic-ventilation, diffusion-weighting, and oxygen-sensitive sequences. For a static representation of lung ventilation, the lung is imaged
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during a 10–12-s breath-hold following a single inhalation of 3 He gas. A normal lung will have a homogenous gas distribution, demonstrating ventilation (or delivery of the gas) throughout the lung, whereas a diseased lung will have multiple signal voids or ventilation defects. Dynamic imaging sequences with high temporal resolution can be performed while a subject continuously breathes following a single breath of gas. A series of images are obtained, demonstrating the dynamic distribution of ventilation, allowing for the calculation of transit times of the gas to the trachea, bronchi, airways, and alveolar spaces. On the basis of normal transit times and prolonged washout times, impaired ventilation patterns and air trapping can be detected. Diffusion-weighting imaging provides information about the pulmonary microstructure by measuring the Brownian motion or the random microscopic molecular movement of the gas. To perform this, bipolar gradients are applied to dephase and rephrase spins prior to signal acquisition, and therefore only diffusing spins will contribute to the signal. From a diffusion-weighted image set, the apparent diffusion coefficient (ADC) is calculated regionally, providing ADC maps. Diffusion, assessed by the ADC, is related to the dimensions of the air spaces. Typically, the gas is highly restricted by the walls and alveoli in the lung. But, if the gas is less restricted, it can diffuse over greater lengths. In emphysema, there is destruction of lung parenchyma, leading to an increase in air spaces. The gas is now less restricted, which is reflected by higher ADC values. Oxygen-sensitive sequences can determine the regional alveolar oxygen pressures (pAO2), which reflect the regional pulmonary perfusion, V/Q ratio, and oxygen uptake in the lung. This technique is based on the linear correlation between the partial pressure of oxygen and the loss of hyperpolarization of 3 He gas. Depolarization rates of the hyperpolarized 3He gas are significantly affected by the RF pulses and the paramagnetic properties of oxygen [56]. The dipolar interaction of oxygen will lead to a gradual decrease in the magnetization of polarized 3He. This effect, once separated from the affect of RF pulses, can be used to determine regional measurements of partial pressure of oxygen (pAO2) and depletion rates. Different pulse-sequence techniques have been developed, including a double-acquisition method [57, 58], a single-breath-hold acquisition [59, 60], and a 3D acquisition scheme [61]. The doubleacquisition technique allowed for determination of pAO2 by distinguishing RF from O2 effects with two identical sequences performed with different timing delays. A limitation of this technique was the necessity for identical breath-holds (i.e., equal lung volumes) during the two inhalations of hyperpolarized 3He gas for reliable analysis. The single-breath-hold acquisition scheme uses a pulse sequence with a changing interscan time to acquire information on the flip angle and oxygen effect during one breath-hold. A very short interscan time is used for the acquisition of the first two images, which are used to
S.K. Alford and E.A. Hoffman
Fig. 2 (a) A series of hyperpolarized 3He images over time from one slice of a 3D single-scan, pO 2 imaging sequence demonstrate loss of signal which is both time- and oxygen-concentration-dependent. (b) From this longitudinal series, a color map with partial pressures of oxygen (pAO2) can be determined. (Adapted and reprinted with permission from [55])
d etermine the effect from the flip angle. The remaining images of the series are acquired with a longer interscan time and are used to calculate the molecular oxygen relaxation rate. In healthy subjects, the pAO2 distributions are very homogeneous. An example of this is shown in Fig. 2. The relaxation time, T1, is a function of time because pulmonary gas exchange leads to a fall in alveolar pO2 during a breath-hold. For a breath-hold of up to 35 s, the oxygen uptake is perfusion-limited, not diffusion-limited, and therefore the drop in pO2 is assumed to be a linear process; therefore, the loss of hyperpolarized gas polarization over time is proportional to the concentration of alveolar pO2. A method to derive regional V/Q ratios from pAO2 measurements has been developed from mass-balance relationships for alveolar CO2 and O2 gas exchange [62]. In addition to pAO2 MR measurements, this method requires venous blood obtained by a pulmonary artery catheter for calculations.
4 CT- and MR-Based Blood Pool Agents Blood pool agents provide contrast in the vasculature over a longer time so visualization of structure can occur without reinjection of contrast. These agents could provide the ability to use imaging for the assessment of treatment, such as tissue plasminogen activator therapy in acute PE, without the nephrotoxic effect of contrast agents. The ideal blood pool agent should have the following properties: a long half-life, remain exclusively in the intravascular space, low immuno-
35 Measurement of Pulmonary Vascular Structure and Pulmonary Blood Distribution by Multidetector-Row
Fig. 3 Three-dimensional (3D) cardiac reconstructions extracted from an multidetector-row CT (MDCT) (Siemens Sensation 64) image data set acquired from an anesthetized supine sheep 3 h after intravenous infusion of liposomal contrast agent [63–66]. (Image data from an unpublished study by the authors in collaboration with Ananth Annapragada, Ketan Ghaghada, and Edwin van Beek)
genicity, low toxicity, be excretable, and have a high number of reporter groups. Two methods have been developed for blood pool agents. The first group consists of in vivo or ex vivo labeling of blood cells or plasma components. The second category includes synthetic macromolecules, colloids, or liposomes carrying a diagnostic label. Liposome-based blood pool agents are currently being developed for CT and MR imaging [63–66]. The liposomes contain contrast agent, iodinated contrast for CT and Gd-DTPA for MR imaging, in the central core of the liposome. This way, the contrast is protected from breakdown and the stealth coating of the liposome, coupled with its 100-nm size, prolongs its residence time and keeps it from being cleared by the kidney. Instead of renal clearance of the contrast agent, the blood pool agent is taken up and cleared by the reticuloendothelial system. A first application for these agents is envisioned to be in a so-called triple rule out whereby a patient with chest pain can be given a single dose of the liposomal contrast agent and evaluated for pulmonary emboli, peripheral sources of clot, aortic aneurysm, and coronary artery disease coupled with a dynamic study of myocardial function. An example image from a sheep study is provided in Fig. 3, which shows a cardiac image acquired from a sheep into which the liposomal agent was injected and which was imaged 3 h after injection via a Siemens Sensation 64-slice MDCT scanner.
5 Diagnosing Pulmonary Vascular Disease with CT or MR Imaging CT and MR imaging are emerging as complementary imaging modalities for the assessment of the lung. They both have strength in the assessment of the pulmonary anatomy, including
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the structure of the pulmonary vasculature and assessment of abnormalities suggestive of pulmonary vasculature disease (pulmonary emboli, PH, vascultitis, and other anomalies). In addition, imaging studies have proven helpful for a variety of less common conditions, congenital and acquired, such as pulmonary sequestration, pulmonary arteriovenous malformations, and complex congenital heart defects such as partial anomalous pulmonary venous return. Although qualitative methods may provide some information such as in the case of acute PE, quantitative methods to evaluate regional pulmonary perfusion can be used to more precisely assess the pulmonary function and may help with the diagnosis of certain disease states, such as pulmonary arterial hypertension.
5.1 Pulmonary Embolism CTA has been the first line for assessment of subjects suspected of having acute PE, and MRA has provided another excellent imaging modality in the case when CTA is not possible (pregnant woman, contrast allergy, etc.). In 1992, Remy et al. introduced pulmonary CTA with the assessment of 42 patients with central artery emboli [67]. They demonstrated 100% sensitivity and 96% specificity with 5-mm collimation. With the improved speed and thinner submillimeter collimation of newer scanners, CTA of the pulmonary arteries is capable of imaging the pulmonary arteries at the segmental and subsegmental levels, thereby detecting peripheral, small pulmonary emboli. Studies have demonstrated that the clinical validity of using CTA to rule out PE is similar to that of using conventional pulmonary angiography [67–70]. The high negative predictive value of a normal CTA study and its association with beneficial patient outcome has been demonstrated [71]. The value of CTA for acute PE assessment is not solely due to its ability to visualize the pulmonary arteries down to the subsegmental and fifth generation, but also in its ability to provide clinicians with alternative diagnostic information. CT offers a complete assessment of the lung parenchyma and mediastinal structures, which has led to alternative diagnoses in a large subset of patients initially studied for suspected PE. Utilizing mediastinal (soft tissue) window leveling, one can assess acute PE by the presence of a pulmonary arterial filling defect. Regions of hypoenhancement may be central, marginal, or complete. To confirm the defect is arterial, one can switch to lung windows to demonstrate an accompanying bronchus or follow the course of the vessel back centrally. Lung parenchyma findings include wedgeshaped, peripheral or pleural-based consolidation, representing an area of hemorrhage or possible infarction. Dual-source MDCT imaging has been applied to the assessment of pulmonary emboli [71]. Endoluminal clots can be detected with an average of images from the two-tube X-ray sources as
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S.K. Alford and E.A. Hoffman
Fig. 4 A 60-year-old male with a smoking history of 30 pack-years with small-sized peripheral clots present in the right upper lobes with a corresponding perfusion defect. On the contralateral side, there is a small perfusion defect without clot present on transverse images. Image parameters: tube A, 140 kV; tube B, 80 kV;
effective milliampere seconds, 265/46; 120 mL of 35% contrast agent at an injection rate of 4 mL/s. Dose–length product, 281 mGy cm. (Courtesy of Martine Remy-Jardin and Jacques Remy, Department of Thoracic Imaging, University Center of Lille, Lille, France)
efficiently as with single-source CTA, with the perfusion map providing additional information regarding the lung parenchyma. Typical triangular-shaped wedge defects, as seen in Fig. 4, can be demonstrated and are helpful in distinguishing acute PE from a nonspecific lung consolidation.
right side of the heart. Although both MDCT and MR imaging are used to assess the heart, cardiovascular MR imaging is the preferred method for assessment of the right side of the heart. Follow-up of subjects with PH provides information on quantitative ventricular mass, ventricular volume, and ejection fraction, which can be tracked over time to determine disease progression. Evaluation of septal bowing and the curvature from short-axis cine images can be determined. Short-axis curvature measures have been found to have a clear linear relationship to invasive systolic pulmonary artery pressure [73]. Delayed-enhancement imaging provides information on the functionality of the myocardium, with infarcted tissue demonstrated as bright hyperenhanced regions of myocardium.
5.2 Pulmonary Hypertension In the case of PH, CTA or MR imaging studies are required to make a diagnosis, to identify the type of PH, to evaluate the functional and hemodynamic impairment, and to monitor changes in disease in response to therapy. MR imaging is increasingly used for the evaluation of PH owing to its ability to provide a good assessment of the pathological and functional changes in both the heart and pulmonary circulation. Imaging can discriminate between the two main contributing factors: a primary lung parenchymal disease and a primary cardiac process. Imaging signs indicative of PH include central pulmonary artery dilatation, tapering of the peripheral pulmonary arteries, and enlargement of the right side of the heart. Measurement of the diameter of the main pulmonary artery to assess for dilatation is done with either CT or MR imaging. The enlargement of the lobar arteries to a diameter greater than that of the accompanying bronchus is another useful sign of pulmonary arterial hypertension. To help discriminate the type of PH, the assessment of the diameter and distribution of the pulmonary veins and venous flow can aid in the diagnosis of postcapillary PH. Enlarged pulmonary veins indicate chronic pulmonary venous hypertension secondary to left-sided heart disease. Perfusion imaging has been applied to PH in a few small studies. Contrast-enhanced MR perfusion imaging demonstrated long time to peak enhancement times in a group of PH subjects compared with normal subjects [72]. Right-sided heart disease, including ventricular hypertrophy and enlargement, is a common and expected secondary finding of PH due to the increased workload placed on the
5.3 Hypoxic Pulmonary Vasoconstriction The normal response of the lung to hypoxia is hypoxic pulmonary vasoconstriction (HPV) resulting in the shunting of blood toward a better-ventilated region of lung for improved oxygenation. CT-based perfusion methods have been used for the characterization of acute lung injury and HPV. There are suggestions in the literature that, in response to inflammatory processes, HPV is blocked [74, 75]. Easley et al. [76] used MDCT to demonstrate in a sheep model the regional failure of HPV with an endotoxin injury (known to inhibit HPV) compared with lung lavage injury where PBF redistributed consistent with a preserved HPV. Hoffman et al. showed the coexistence of HPV failure and normal HPV response in a sheep with both pneumonia and an endobronchial valve that blocked ventilation to an unaffected lung region (Fig. 5) [61]. MR perfusion imaging techniques have been applied to highaltitude pulmonary edema (HAPE) and provided insights into its pathogenesis. Dehnert et al. demonstrated with dynamic contrast-enhanced MR imaging that the inhomogeneity of HPV increases with hypoxia in all subjects, but more so in HAPE-susceptible individuals [77]. Hopkins et al. [78] used ASL MR imaging to spatially quantify PBF
35 Measurement of Pulmonary Vascular Structure and Pulmonary Blood Distribution by Multidetector-Row
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Fig. 5 Before (top row) and after (bottom row) endobronchial valve placement. The left column provides the view of one CT section at the level of the diaphragm dome. Note the significant dependent pneumonia (a). The center column demonstrates ventilation overlaid in color as assessed by xenon-CT. Note that there is little ventilation in either the baseline or the post valve in the region of the dependent pneumonia. By design, there is a large region where ventilation was eliminated by the valve placement (b). The right column shows a color overlay of the perfusion measurements. Note that in region (c) coinciding with region (b) from the central column (no ventilation due to valve placement) there is a regional loss of perfusion, indicating an intact hypoxic pulmonary vasoconstrictor response. At the
same time, in the region of pneumonia (d), where there is little or no ventilation, blood flow is enhanced after valve placement. Presumably, blood flow shunted away from the valve-based hypoxic pulmonary vasoconstriction is shunted straight toward the region of inflammation, presumably because the inflammation has served to blunt hypoxic pulmonary vasoconstriction in this region, leaving this region as the path of least resistance for the blood flow diverted from the region affected by hypoxic pulmonary vasoconstriction (HPV). Thus, this image demonstrates that lung has the ability to locally modulate HPV on the basis of local inflammation. (Adapted from [61] with permission. Official journal of the American Thoracic Society copyright American Thoracic Society)
heterogeneity in HAPE-susceptible individuals, HAPEresistant individuals, and individuals without altitude exposure. Hopkins et al. found that HAPE-susceptible individuals have increased PBF heterogeneity in acute hypoxia, consistent with uneven HPV. It has been hypothesized that uneven HPV in HAPE-susceptible individuals may lead to overperfused regions of lung with increased capillary pressures, resulting in hydrostatic edema to those regions.
the vascular bed related to the severity of the parenchymal destruction has been demonstrated in COPD subjects [79], changes in the early COPD have just recently been explored. Increased heterogeneity of perfusion measures such as the PBF and MTT has recently been demonstrated in smokers with normal PFTs but with very early signs of emphysema, possibly indicating the predilection of these subjects to the disease [80, 81]. Data in animals demonstrate that, in the presence of inflammation, HPV is blocked [61], thus preserving the normal cascade of events involved in the inflammatory process, including the delivery of bone-marrow-derived progenitor cells to repair collateral damage. Hoffman et al. have hypothesized that the failure of inherent mechanisms to block the normal HPV response in the presence of inflammation may lead to a failure of the normal mechanisms serving to limit the inflammatory response and reparative process, thus leading to the emphysema [61]. We are taught that the normal response to regional hypoxia in the lungs is to shunt blood toward better-ventilated regions. However, when local hypoxia
5.4 Emphysema Recent evidence suggests that vascular changes may be important to the pathogenesis and progression of emphysema and may relate to an abnormal HPV response in the presence of inflammation. Dynamic perfusion measurements with CT and MR imaging can provide valuable regional information about vasculature changes related to COPD. Although a reduction of
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is induced by smoking-related inflammatory processes which flood alveoli such as in peripheral bronchiolitis (micro nodules) and/or local interstitial edema (ground glass), it becomes counterproductive to shut down blood flow to that region needing flow to maintain the cascade of defense and reparative mechanisms such as the enhanced delivery of bone-marrowderived mesenchymal stem cells. In support of the above hypothesis is the observation by Remy-Jardin et al. [62] that there is an increased propensity for the lung to develop emphysema in regions where prior inflammatory processes (illdefined ground-glass opacities/micronodules) have been observed. In an individual unable to block HPV in the presence of inflammation, increased vascular resistance to inflamed lung regions is compounded by gravitational effects which further limit flow to the lung apices, thus providing an explanation as to why smoking-related emphysema tends to preferentially occur at the apices.
6 Conclusion CT and MR imaging techniques for the lung have seen great advancements over a short time and can now be used to provide structural and functional information regarding the pulmonary vasculature. Imaging techniques for the assessment of PE have become first-line choices for physicians. New imaging techniques including perfusion imaging capable of regional blood flow assessment are being developed along with new scanner technologies, including DECT, which holds promise for new methods to better explore pulmonary vascular diseases. With the advancements in imaging to assess both structure and function, and to correlate regional structural changes in the lungs to regional functional alterations, and to quite possibly image functional changes as a precursor to structural alterations, there is a growing effort to utilize imaging-based phenotypes to understand the pathological processes leading to disease, to identify underlying genotypes associated with disease susceptibility, and to utilize quantitative imaging tools for drug and interventional device discovery, safety testing, and outcome assessment.
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524 69. Teigen CL, Maus TP, PFd S, Johnson CM, Stanson AW, Welch TJ (1993) Pulmonary embolism: diagnosis with electron-beam CT. Radiology 188:839–845 70. van Rossum AB, Pattynama PM, Ton ER et al (1996) Pulmonary embolism: validation of spiral CT angiography in 149 patients. Radiology 201:467–470 71. Thieme SF, Becker CR, Hacker M, Nikolaou K, Reiser MF, Johnson TRC (2008) Dual energy CT for the assessment of lung perfusion - correlation to scintigraphy. Eur J Radiol 68:369–374 72. Ohno Y, Hatabu H, Murase K et al (2007) Primary pulmonary hypertension: 3D dynamic perfusion MRI for quantitative analysis of regional pulmonary perfusion. Am J Roentgenol 188:48–56 73. Roeleveld RJ, Marcus JT, Faes TJC et al (2005) Interventricular septal configuration at MR imaging and pulmonary arterial pressure in pulmonary hypertension. Radiology 234:710–717 74. Hlastala MP, Lamm WJ, Karp A, Polissar NL, Starr IR, Glenny RW (2004) Spatial distribution of hypoxic pulmonary vasoconstriction in the supine pig. J Appl Physiol 96:1589–1599 75. Lamm WJ, Starr IR, Neradilek B, Polissar NL, Glenny RW, Hlastala MP (2004) Hypoxic pulmonary vasoconstriction is heterogeneously distributed in the prone dog. Respir Physiol Neurobiol 144: 281–294
S.K. Alford and E.A. Hoffman 76. Easley RB, Fuld MK, Fernandez-Bustamante A, Hoffman EA, Simon BA (2006) Mechanism of hypoxemia in acute lung injury evaluated by multidetector-row CT. Acad Radiol 13:916–921 77. Dehnert C, Risse F, Ley S et al (2006) Magnetic resonance imaging of uneven pulmonary perfusion in hypoxia in humans. Am J Respir Crit Care Med 174:1132–1138 78. Hopkins SR, Levin DL (2006) Heterogeneous pulmonary blood flow in response to hypoxia: a risk factor for high altitude pulmonary edema? Respir Physiol Neurobiol 151:217–228 79. Ley-Zaporozhan J, Ley S, Eberhardt R et al (2007) Assessment of the relationship between lung parenchymal destruction and impaired pulmonary perfusion on a lobar level in patients with emphysema. Eur J Radiol 63:76–83 80. Alford S, van Beek E, Hudson M, Baumhauer H, McLennan G, Hoffman E (2008) Characterization of regional alterations in pulmonary perfusion via MDCT in nonsmokers and smokers. Eur Radiol 18:330–331 81. Alford S, Van Beek E, McLennan G, Hoffman E (2006) CT-based blood flow measurements in lung parenchyma of normal controls, normal smokers and smokers with emphysema [abstr]. Paper presented at: Radiological Society of North America 92nd Annual Meeting, Chicago, IL
Chapter 36
Quantification of DNA, RNA, and Protein Expression Fiona Murray, Jason X.-J. Yuan, and Paul A. Insel
Abstract Advances in molecular biology and biochemistry have provided tools with which to explore and increase our understanding of mediators that regulate development and pathological changes in the pulmonary circulation. Combinations of techniques have allowed researchers to start to unravel complex biological pathways that have direct relevance to pulmonary research, such as those that contribute to cellular proliferation and vascular permeability. The aim of this chapter is to describe some of the established protocols to quantify DNA, RNA, and protein expression, discuss advances in these techniques, and illustrate how they can be applied. Expression levels of cell signaling components can be detected at the level of DNA, RNA, and protein. Southern blotting (also termed “Southern hybridization” and named for Edwin Southern, who developed the technique) allows for the detection of a specific DNA sequence in a preparation of DNA, typically from cellular nuclei. This method was modified and extended later for the detection of RNA and protein, named Northern and Western blots, respectively. All three of these protocols are based on similar principles and utilize similar methods. Alternatively, measurement of gene expression [using messenger RNA (mRNA) as a starting material for the generation of complementary DNA (cDNA)] can also be performed via reverse-transcription (RT) polymerase chain reaction (PCR), followed by agarose gel electrophoresis. A modification of this technique, real-time PCR (also termed “quantitative PCR”), allows for quantitative analysis of gene copy number. A number of other high-throughput techniques exist for the profiling of gene and protein expression, such as by use of DNA and protein microarrays; however, these will be discussed in more detail in subsequent sections. Many variations exist for the protocols outlined below and most buffers can be made using a variety of recipes or can be purchased from a number of suppliers. However, the principles remain the same; F. Murray (*) Departments of Medicine and Pharmacology, University of California, San Diego, BSB 3073, 9500 Gilman Drive, La Jolla, San Diego, CA 92093-0636, USA e-mail:
[email protected] understanding the stages in each process and their purpose is key. This chapter outlines the methods that have proven to be versatile, reliable, and sensitive. Keywords Molecular biology • Methodology • Western blot • Northern blot • Southern blot • RT-PCR • mRNA expression
1 Introduction Advances in molecular biology and biochemistry have provided tools with which to explore and increase our understanding of mediators that regulate development and pathological changes in the pulmonary circulation. Combinations of techniques have allowed researchers to start to unravel complex biological pathways that have direct relevance to pulmonary research, such as those that contribute to cellular proliferation and vascular permeability. The aim of this chapter is to describe some of the established protocols to quantify DNA, RNA, and protein expression, discuss advances in these techniques, and illustrate how they can be applied. Expression levels of cell signaling components can be detected at the level of DNA, RNA, and protein. Southern blotting (also termed “Southern hybridization” and named after Edwin Southern, who developed the technique) allows for the detection of a specific DNA sequence in a preparation of DNA, typically from cellular nuclei [1]. This method was modified and extended by Stark and Towbin for the detection of RNA and protein, named northern and western blots, respectively [2–4]. All three of these protocols are based on similar principles and utilize similar methods, composed of five main steps: (1) sample preparation, (2) size-based separation by gel electrophoresis, (3) transfer to a solid support, (4) hybridization of a probe, and (5) detection (Fig. 1). Alternatively, measurement of gene expression [using messenger RNA (mRNA) as a starting material for the generation of complementary DNA (cDNA)] can also be performed via reverse-transcription (RT) polymerase chain reaction
J.X.-J. Yuan et al. (eds.), Textbook of Pulmonary Vascular Disease, DOI 10.1007/978-0-387-87429-6_36, © Springer Science+Business Media, LLC 2011
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sequence variation. Such polymorphisms are sometimes used to track the association of a DNA sequence variation with a genetic disease and to measure recombination rates that allow the generation of maps that measure the distance between RFLPs. Examples of the use of Southern blots include (1) the assessment of patients for mutations in bone morphogenetic protein receptor type II, which can contribute to the development of primary pulmonary hypertension, (2) the identification of hypoxia-induced oxidative base modifications in the vascular endothelial growth factor hypoxia-response element, and (3) characterization of the decreased ratio of mitochondrial DNA to nuclear DNA in the left and the right ventricle after 21 days of chronic hypoxia [14–16].
Fig. 1 Flow diagram depicting the five main steps common to Southern, northern, and western blotting, sample preparation, size-based separation by gel electrophoresis, transfer to a solid support, hybridization of the probe, and its detection
(PCR), followed by agarose gel electrophoresis [5, 6]. A modification of this technique, real-time PCR (also termed “quantitative PCR”), allows for quantitative analysis of gene copy number [7, 8]. A number of other high-throughput techniques exist for the profiling of gene and protein expression, such as by use of DNA and protein microarrays; however, these will be discussed in more detail in subsequent sections. Many variations exist for the protocols outlined below and most buffers can be made using a variety of recipes or can be purchased from a number of suppliers. However, the principles remain the same; understanding the stages in each process and their purpose is key. Below we outline the methods that have been or are used in our laboratory and have proven to be versatile, reliable, and sensitive. More detailed or alternative methods have been published [8–13].
2 DNA Quantification 2.1 Southern Blotting Southern blotting is most commonly used to detect specific DNA sequences in the genome, typically in DNA prepared from tissue or cells. Southern blotting can identify if a specific gene is present, the number of copies, the degree of similarity between the chromosomal gene and the probe, and if abnormalities exist in the structure of the gene. Mutations that result in DNA deletion, insertion, or rearrangement are readily detectable by Southern blotting and hence it is routinely used in the initially genotyping of transgenic animals. The procedure for Southern blotting can be used to detect restriction fragment length polymorphisms (RFLPs), which are indicative of DNA
2.1.1 DNA Isolation Approximately 200–250 mg of DNA can be purified from blood (10 mL), cultured pulmonary artery cells (approximately 5 × 107), and lung tissue biopsies (up to 1 cm3) using a variety of buffers to extract the DNA. Many such buffers are available from commercial suppliers and avoid the use of phenol–chloroform extraction, which was a classically used approach to prepare DNA. Extraction works best if tissues are homogenized or ground to a fine powder under liquid nitrogen before the extraction process. Tissue or cells are suspended in TE buffer [10 mM tris(hydroxymethyl) aminomethane–hydrogen chloride (Tris–HCl) pH 8.0, 1 mM ethylenediaminetetraacetic acid (EDTA)] then mixed with 10 mL/mL DNA extraction buffer [10 mM Tris–HCl (pH 8.0), 1 mM EDTA, 20 mg/mL RNAse, 0.5% sodium dodecyl sulfate (SDS)] and incubated for 1 h at 37°C. Proteinase K is added to give a final concentration of 100 mg/mL and the samples are incubated for a further 3 h at 50°C. An equal volume of phenol equilibrated with 0.5 M Tris–HCl (pH 8.0) is then added and the two phases are mixed for 10 min. The viscous phase is removed using a wide-pore pipette and the phenol extraction is repeated. Two preferred approaches can then be used to recover DNA: either dialyze the aqueous phase four times at 4°C against 4 L of 50 mM Tris–HCl (pH 8.0), 10 mM EDTA (pH 8.0) or add 0.2 vol of 10 mM ammonium acetate followed by 2 vol of ethanol, after which the DNA is removed using a Pasteur pipette, washed with 70% ethanol, and then resuspended in TE buffer (pH 8.0) at approximately 5 × 106 cells/ml. The DNA is left to dissolve overnight at 4°C; the concentration and purity are then measured using a spectrophotometer (to quantify A260/A280). Ideally this ratio should be 1.75–2.0; if A260/A280 is less than 1.75, protein contamination is present and the sample should be repurified. Commercial kits such as the QIAamp DNA mini kit (Qiagen) are available for rapid purification of DNA, which is increasingly the preferred choice of many researchers.
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2.1.2 Gel Electrophoresis and Transfer Once purified, the DNA (approximately 10 mg) is digested with restriction endonuclease enzymes [e.g., HindIII, EcoRI together with its appropriate 1× enzyme buffer and 1× acetylated bovine serum albumin (BSA) (New England Biolabs)]. At the end of digestion, gel-loading buffer [10× sample buffer: (65% (w/v) sucrose, 10 mM Tris–HCl (pH 7.5), 10 mM EDTA, and 0.3% (w/v) bromophenol blue] is added and the DNA is separated by electrophoresis alongside an appropriate DNA marker on a 0.7% agarose gel cast in 0.5× stock TBE buffer containing 0.5 mg/mL ethidium bromide [10× stock TBE buffer: 0.9 M Tris base, 0.9 M boric acid, 0.25 M EDTA, (pH 8.3)]. After adequate separation has been achieved, the DNA is visualized using UV light, and then the gel is directly soaked in an alkaline buffer (1.5 M NaCl, 0.5 M NaOH) to denature the double-stranded fragments for 45 min. This is followed by incubation with neutralization buffer (1.5 M NaCl, 1 M Tris–HCl, pH 7.4) for 30 min before transfer onto a membrane of choice. Nylon-based (e.g., Amersham Hybond N, N+, GE Healthcare), nitrocellulose, or poly(vinylidene fluoride) (PVDF) membranes can be employed. Nylon membranes are stronger, easier to recycle, and bind small DNA fragments more efficiently; however, they tend to give higher background values. Capillary transfer, which runs overnight, was used in Southern’s original method and still remains popular, at least in part owing to its low cost. For capillary transfer, the gel is laid on Whatman 3MM paper, placed in a reservoir of transfer buffer (10× stock standard saline citrate (SSC): 1.5 M NaCl, 0.15 M sodium citrate, pH 7.0) and covered with the membrane. To aid in removing air bubbles and ensuring complete contact with the Whatman 3MM paper, a stack of paper towels and a weight are used to hold the stack down. Transfer buffer moves from a reservoir below the gel into the dry stack of paper towels above the membrane, thereby carrying the nucleic acid with it until it is trapped on the membrane. The transfer process can be shortened to 30–60 min by using vacuum blotting or electrophoresis (according to the manufacturer’s instructions). The transferred DNA is fixed to the membrane by baking at high temperature (approximately 80°C for 0.5–2 h for both nitrocellulose and nylon membranes) or through cross-linking by exposure to UV radiation (nylon membranes).
2.1.3 Hybridization of Probe and Detection For prehybridization the membrane is incubated for 1–2 h at 68°C in hybridization buffer (6× stock SSC, 5× stock Denhardt’s reagent, 0.5% SDS, 100 mg/mL denatured, fragmented salmon sperm DNA) to block nonspecific attachment of the probe to the membrane. One then adds a radiolabeled probe specific for the gene of interest, either a DNA probe (cDNA clone, genomic
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fragment, oligonucleotide) or an RNA probe for the required period. Preparation of radiolabeled DNA and RNA probes is not within the scope of this chapter; however, other publications provide detailed methods [9]. Radioactive probes, most commonly with 32P, 33P, and 35S, which all produce b particles, can readily be detected on X-ray film. Washing the membrane to remove unbound probe is performed in several steps of increasing degrees of stringency depending on the application and degree of sensitivity required: typically, several washing steps are carried out in buffers starting with high salt to low salt concentrations (2× stock SSC to 0.1× stock SSC, 0.5% SDS at 65°C) and stringent conditions. Hybridization is detected by visualization on X-ray film by autoradiography, which allows for the identification of the sizes and the number of fragments of genes that bind to the probe. A major advance in Southern blotting is that nonradioactive probes, such as fluorescent or chromogenic dyes, are now available that allow detection of hybridization by the development of color on the membrane. These nonradioactive probes, such as digoxogenin, biotin, and fluorescein-11dUTP, are sensitive yet much safer, easier to dispose of, and less expensive than radioactive probes [13].
3 RNA Quantification The variety of methods that exist for the quantification of the transcript are northern blotting, in situ hybridization, RNase protection assays, RT-PCR, and cDNA arrays, all of which require the isolation of high-quality RNA.
3.1 RNA Isolation RNA can be readily purified from lungs and isolated pulmonary cells; however, since RNA is unstable compared with DNA, a number of steps need to be employed to ensure that its integrity is maintained throughout the procedure. It is important to inhibit the activity of RNases, avoid introduction of RNases from other sources, and work quickly: RNase-free glassware and plastics should be used or alternatively autoclaved, rinsed with chloroform, or treated with diethyl pyocarbonate (DEPC) before use, and addition of RNase inhibitors is recommended during cell/tissue lysis procedures. Furthermore, when tissue or cells are harvested for extraction of RNA at a later date, samples must be snap-frozen in liquid nitrogen or treated in a way to stabilize the RNA, e.g., with the addition of RNAlater (Qiagen). A number of methods exist for isolating RNA from cells and tissues, such as acid–phenol extraction coupled with alcohol precipitation, and guanidine isothiocyanate extraction
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coupled with cesium chloride ultracentifugation [17–19]. Commercially available RNA extraction kits and reagents use a combination of the above-mentioned extraction techniques or patented technology and are generally thought to be safer, yet effective, for extracting high-quality RNA from both tissue and cells; TRIzol® (a monophasic solution of phenol and guanidine isothiocyanate, Invitrogen) and Qiagen RNeasy kits are both routinely used in our laboratory. The choice of kit depends on downstream application and price; it is recommended to incorporate a DNase treatment step (available from each supplier) to remove genomic contamination. For isolation of RNA from pulmonary artery branches and lungs it is recommended to grind them to a fine powder in liquid nitrogen with a mortar and pestle and place them in lysis buffer, followed by further homogenization and then either to pass the lysate through a 25-gauge needle five times or to pipet samples onto a QIAshredder (Qiagen, Valencia, CA, USA) column (to disrupt the RNA. The expected yield of RNA using these kits is roughly 2 mg/mg of lung tissue and 5 mg per 1 × 106 cultured pulmonary artery smooth muscle cells (PASMCs). Purified RNA is dissolved in RNase free water and its concentration and purity are determined using a spectrophotometer (quantified A260/A280). RNA absorbs UV radiation at 260 nm with an extinction coefficient of 40 mg/mL when using a cuvette with a path length of 1 cm. The ratio A260/A280 for pure RNA is roughly 2 (range 1.75–2.0 is usually deemed an appropriate value to use the RNA for subsequent studies) but this ratio decreases in the presence of protein (which is preferentially detected at 280 nm). RNA integrity should also be confirmed by assaying 2–4 mg of the RNA sample by gel electrophoresis on a denaturing 0.7% agarose gel cast in 0.5× TBE containing 0.5 mg/mL ethidium bromide (see above), especially when the RNA is to be used for applications such as microarrays. Good-quality RNA should reveal (as identified with ethidium bromide) discrete 18S and 28S ribosomal RNA (rRNA) bands with the 28S rRNA band approximately twice as intense as the 18S rRNA band. If desired, poly(A) + RNA (mRNA, 1–2% of total RNA) can be purified from total cellular RNA by chromatography on oligo(dT)-cellulose, since for gene expression this is the RNA required [20, 21]. RNA selection is not always necessary, although for some uses, e.g., as a template to construct cDNA libraries, mRNA is required. Next we discuss the downstream applications of northern blotting and RT-PCR.
3.2 Northern Blotting Northern blotting employs a similar approach to that of Southern blotting but is designed to detect RNA rather than DNA. Northern blotting relies on the hybridization of a
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labeled probe to a target mRNA and is valuable for examining expression of specific genes in development, disease in response to various stimuli, and for detecting alternatively spliced gene variants. Northern blotting is still commonly performed and, for example, has been used to demonstrate the upregulation of genes such as angiopoietin-1 and vasoactive intestinal peptide receptor in patients with pulmonary hypertension [22, 23].
3.2.1 Gel Electrophoresis and Transfer Denaturation of RNA (by glyoxal and dimethyl sulfoxide, methylmercuric hydroxide, or formaldehyde) allows for its tight binding to nitrocellulose membranes and the hybridization of radioactive probes [24–26]: below we describe a method for the electrophoresis of RNA through gels containing formaldehyde. Samples are prepared by mixing 20 mg of purified RNA (4.5 mL), formaldehyde gel-running buffer [2.0 mL, 5× stick formaldehyde gel-running buffer: 0.1 M 3-morpholinopropanesulfonic acid sodium salt (pH 7.0), 40 mM sodium acetate, 5 mM EDTA (pH 8.0)], formaldehyde (3.5 mL), and formamide (10.0 mL), followed by incubation at 65°C for 15 min. The samples are mixed with loading buffer [10× stock sample buffer: 65% (w/v) sucrose, 10 mM Tris–HCl (pH 7.5), 10 mM EDTA, and 0.3% (w/v) bromophenol blue] and together with a molecular weight marker are run on a denaturing 1% agarose gel (the percentage of agarose depends on the degree of separation required) cast in 1× stock formaldehyde gel-running buffer. After adequate separation, the gel is removed, cut, and stained (in the marker lanes that contain size standards) with ethidium bromide (0.5 mg/mL) and photographed so that the size of the bands on the membrane after hybridization can be estimated. Before transfer the formaldehyde must be removed from the gel by washing with DEPC-treated water, after which the RNA is transferred (by capillary elution, vacuum transfer or electroblotting) in 20× stock SSC (3 M NaCl, 0.3 M sodium citrate, pH 7.0) to a solid support (either nitrocellulose or charged nylon membrane) similar to that outlined earlier for Southern blotting, and then the membrane is dried and baked at 80°C (or by UV cross-linking).
3.2.2 Hybridization of Probe and Detection Membranes are incubated for 1–2 h at 68°C in hybridization buffer (6× stock SSC, 2× Denhardt’s reagent, 0.1% SDS) and then with the radiolabeled probe for 16–24 h. After the membrane has been washed (0.2× SSC: 30 mM NaCl, 3 mM sodium citrate, pH 7.0 containing 0.1% w/v SDS, twice at 50–60°C for 10 min, depending on the probe), the bands on the gel are visualized by autoradiography (exposed for
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24–48 h at –70°C). The membrane can be incubated for 10 min at 96°C in “stripping buffer” (10 mM Tris–HCl, pH 7.5 containing 1 mM EDTA, and 1% SDS) and then washed three times for 10 min to allow for hybridization of a new probe. Stripping and reprobing is, however, only recommended on the stronger Nylon+ membranes and although this can be done repeatedly, we recommend no more than five cycles. Quantification of mRNA expression is measured by band intensity via densitometery. The gel is either scanned or photographed and the pixels in each band are measured using image quantification. We use ImageJ, free software available from NIH (http://rsb.info.nih.gov/ij/). A shape is drawn round the largest band and then copied to each band that requires quantification. It is important to determine the background around each band so as to normalize the intensity and always to run samples on the same gel and use different exposure times to confirm that the signal is not saturated. As controls for equal RNA loading in each lane on the gels, one uses a “housekeeping gene” such as glycerol 3-phosphate dehydrogenase (GAPDH) or b-actin and corrects results for differences in loading. As will be discussed below, no single housekeeping gene is ideal for such studies. Advances in northern blotting methods have included the introduction of nonradioactive probes such as those labeled with digoxogenin and biotin [13]. However, the greatest advance in detecting changes in gene expression has been the introduction of RT-PCR and microarray approaches, which have advantages compared with northern blots for studies of RNA.
3.3 Reverse-Transcription PCR PCR allows the amplification of specific DNA sequences in vitro by primer extension of complementary strands of DNA [5, 6]. PCR is generally less expensive, easier, quicker, and more sensitive than other methods currently used to assess gene expression. The general method of PCR involves thermostable DNA (taq) polymerase-catalyzed amplification by multiple rounds of melting, annealing primers, and extension. Template cDNA mixed with a buffer that allows for maximal polymerase activity, primers specific for the gene of interest, and all four deoxynucleotides (dNTPs) are necessary for the reaction: it is recommended that a “master mix” be used to increase the accuracy of the procedure. Specific PCR conditions should be optimized for each application. Therefore, as the general method is outlined below, each parameter that influences the specificity of the PCR will be discussed. We have used PCR to show the expression pattern of ion channels and phosphodiesterases (PDEs) in PASMCs and changes with hypoxia or the development of pulmonary hypertension [27–29]. To use PCR to measure gene expression, isolated RNA must be converted into cDNA. RT converts almost exactly
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the same proportion of RNA into cDNA (e.g., addition of 1 mg RNA in a 20-mL RT reaction gives cDNA with a final concentration of 50 ng/mL), using dNTP (10 mM each) building blocks, enzymes such as Moloney murine leukemia virus or avian myeloblastosis virus, primers for the reaction, and RNase H to eliminate residual RNA [30]. An identical reaction without addition of reverse transcriptase should always be included to verify the absence of genomic DNA (“no-RT control”). Primers routinely used to aid in the synthesis cDNA are generic sequences (2 pmol), random hexamers (50 ng) or oligo(dT) (200 ng). Oligo(dT) binds to the poly(A) tail of mRNA and thus only synthesizes cDNA from mRNA, whereas random hexamers nonspecifically prime cDNA synthesis from nonpolyadenylated transfer RNA and rRNA. We use random hexamers since we generally normalize our data to 28S RNA (see later for the choice of housekeeping gene). Commercial kits are available for RT, and provide all the reagents required for the reaction: we routinely use both Iscript (Bio-Rad Laboratories) and Superscript III (Invitrogen).
3.3.1 Primer Design PCR amplifications are performed using gene-specific forward and reverse primers. Primer length, percent G–C content, melting temperature, and 3¢ end sequence needs are optimized for successful PCR [31, 32]. We generally design primers on the basis of the product of interest’s mRNA sequence (http:// www.ncbi.nlm.nih.gov/sites/entrez?db = nuccore&itool = toolbar). Primer specificity can be confirmed via NCBI BLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi). Many Web sites exist to aid in the design of primers (e.g., Primer3, http://frodo.wi. mit.edu). A number of general rules should be followed to prevent mispriming or the generation of primer dimers: • Primers should be 18–25 bases in length. • Base composition should be 50–60% (G + C). • Melting temperature (Tm) is typically 50–70°C (same Tm for both primers). • Primers should not be self-complementary (form hairpins). • The 3¢ end should end in a G or a C, or CG or GC (as this increases the efficiency of priming); however, the 3¢ end of each primer should not be complementary (leading to the formation of primer dimers) or contain runs of three or more Cs or Gs (which promote mispriming at G- or C-rich sequences). • Primers should span exons so genomic contamination can be easily detected (since the product size will be much larger). • The optimal primer concentration for PCR is approximately 0.2 mM. As an important positive control we validate all our primers using species-specific reference RNA (Stratagene). If no
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product appears in PCRs that contain cDNA from our cells of interest but the positive control yields a PCR product, the lack of product from the cells of interest is thus not the result of poor reaction conditions.
3.3.2 PCR Reagents and Buffers All reagents should be in excess in the PCR, thereby allowing the cDNA to be limiting. PCR is performed in a standard buffer (10 mM Tris–HCl, pH 8.0, 50 mM KCl, 1.5 mM MgCl2, 0.01% Tween 20): increasing the MgCl2 concentration can increase the efficiency of the reaction, and addition of dimethyl sulfoxide (2%v/v) helps prevent the formation of secondary structures. A dNTP mixture (final concentration, 0.2 mM) is essential to provide the nucleotides for efficient extension from the primers. The main component of the PCR is the enzyme Taq DNA polymerase, which can be obtained from numerous commercial sources (1 unit of Taq per reaction is usually sufficient). Modifications to Taq have allowed it to be kept inactive at room temperature, such as Platinum® Taq (Invitrogen), which is only active after it has been denatured at 94°C, thus preventing nonspecific primer annealing at room temperature.
3.3.3 PCR Cycles PCRs are performed in programmable thermal cyclers that have the ability to quickly ramp up and down in temperature. Typical PCR protocols begin with a 95°C denaturation for 10 min to fully melt the template, exposing single-stranded template DNA for primer annealing, followed by 35–40 cycles of the following three steps (example given for primers with a Tm of 60°C for a 250-bp product): • Denaturation: 95°C for 15 s (denatures the newly synthesized products) • Annealing: 55°C for 30 s (binding of the primers to their target site) • Extension: 72°C for 1 min (approximately 1 min per kilobase DNA synthesis of primer) A final extension of 72°C for 5 min is not essential but is usually included to allow complete synthesis of any partial products that may have formed in the later stages of PCR. Since temperatures of 95°C can lead to evaporation, this should be prevented by overlaying the reaction with oil or performing the PCR in a thermal cycler with a heated lid. The annealing temperature is dependent on the Tm of the primers and is the most variable parameter that requires adjustment: the annealing temperature can be easily optimized using a gradient thermal cycler. In general, the annealing temperature is about 3–5°C below the Tm.
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3.3.4 Quantification of PCR Products To analyze the PCR products 10× stock sample buffer [65% (w/v) sucrose, 10 mM Tris–HCl (pH 7.5), 10 mM EDTA, and 0.3% (w/v) bromophenol blue] is added to give a final concentration of 1× and then this mix is electrophoresed together with appropriate molecular weight standards on a nondenaturing gel (1% agarose gel cast in 0.5× TBE containing 0.5 mg/mL ethidium bromide) and visualized using UV light. The first time primers are used, it is important to excise and sequence each product and verify its identity; we use the QIAquick gel extraction kit (Qiagen) for this procedure. Gene expression is quantified by measuring the band intensity as described earlier for northern blot. For semiquantification of the PCR products, GAPDH, b-actin, rRNAs, or other housekeeping genes are used as an internal control. The inclusion of a loading or internal control (i.e., an RNA that does not change with treatment of cells) is required for all techniques that measure expression. The choice of a housekeeping gene is somewhat controversial [33, 34]. For example, expression of GAPDH mRNA can be upregulated in proliferating cells, including aggressive cancers [35]. Furthermore, the use of 18S or 28S to normalize the results (which can only be used if cDNA synthesis was primed using random hexamers) may not always represent the overall cellular mRNA population since it is a rRNA [36]. We believe that it is best to assess several different loading controls until one has been validated for use with a particular target cell or tissue. In our experience, 28S rRNA is optimal for normalizing RNA expression in human PASMCs and lung tissue [36]. It is important to note that PCR is at best semiquantitative since one measures generated product at an end point (e.g., after 40 cycles) when the product formation will be at a plateau. Quantification of the PCR product at the end of each cycle would be required to enhance sensitivity, a process that would be extremely time consuming. A key advance in PCR has been the development of real-time PCR, which is more sensitive and accurate for quantification of mRNA and is discussed in more detail next [7, 8].
3.4 Real-Time PCR The general principles of real-time PCR are similar to those of traditional PCR but fluorophore probes that bind to double-stranded DNA are used for the detection and quantification of the PCR product. These fluorescent probes include nonspecific DNA intercalating dyes (SYBR Green I) or sequence-specific fluorescent oligonucleotide probes
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(TaqMan, molecular beacons, and Scorpion probes that use Förster resonance energy transfer [8, 37–42]). Fluorescence intensity is measured after each cycle; the intensity increases proportional to the increase in the amount of PCR product. Fluorescence versus cycle number is then plotted for each sample and a threshold fluorescence is set within the linear amplification range (Fig. 2). The cycle number at which the threshold fluorescence crosses the amplification plot is known as the Ct or threshold cycle. The greater the amount of template, the lower the number of cycles required to reach the Ct value. Because real-time PCR offers a 107-fold dynamic range, a variety of ratios of target and normalizer can be assayed with equal sensitivity and that eliminates post-PCR processing. Real-time PCR is the preferred technique in our laboratory to measure gene expression. We have used it to demonstrate the relative expression of PDEs and caveolins in PASMCs and how the expression of PDE isoforms is altered in PASMCs isolated from patients with pulmonary hypertension [43, 44].
3.4.1 Choice of Probe The first step in real-time PCR is to decide which dye/probe is best for a particular experiment. The four different probes that are currently available for real-time PCR are TaqMan, molecular beacons, Scorpion probes, and SYBR Green I, each of which allows the detection of PCR products via the generation of a fluorescent signal. SYBR Green I (an intercalating dye that fluoresces upon binding to double-stranded DNA) is the cheapest, most widely used, and most flexible
Fig. 2 Example of amplification plots obtained from a real-time PCR experiment showing 28S and Epac-1 in human pulmonary artery smooth muscle cells (PASMCs). The threshold cycle (Ct) is inversely proportional to the initial copy number. DCt is the difference between the Ct of 28S and the Ct of Epac-1 for both samples
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probe. SYBR Green fluoresces when it binds to doublestranded DNA, which facilitates its use with any gene-specific primer. However, it also can bind to nonspecific products and primer dimers, although melt curves allow the detection of the latter [45]. TaqMan, molecular beacons, or Scorpion gene-specific probes can be designed, and are oligonucleotides (20–30 bases long with a Tm 10°C higher than for the primers) with a reporter dye such as 5¢ FAM (5-carboxyfluorescein) attached to the 5¢ end and a quencher dye such as 3¢ MGB (minor groove binder) attached to the 3¢ end. TaqMan probes specifically anneal between the forward and reverse primers and prior to replication of the template. The 5' exonuclease activity of the polymerase cleaves the probe, releasing the reporter molecule from its close proximity to the quencher, thereby increasing fluorescence intensity [46]. Use of any these probes can allow for multiplexing, for example, so the housekeeping gene is amplified in the same tube as the gene of interest, as long as each probe is designed with a spectrally unique fluorophore [39, 47, 48]. TaqMan probes require very little optimization and have recently been used to define the anatomic distribution of G-protein-coupled receptors (GPCRs) [49].
3.4.2 General Protocol The general protocol for real-time PCR is similar to that for PCR, although a few modifications are required to optimize the efficiency of the reaction. It is important to keep the PCR products optimally sized between 100 and 150 bp, since it is believed longer products create a stronger signal. It has been suggested that products can be as small as 50 bp; however, this can make sequencing of products more difficult. Owing to the increased sensitivity of the real-time reaction, it is usually best to use lower concentrations of both cDNA and primers: a general guide is 8 ng and 100 nM per reaction, respectively; however optimization is usually required. One needs to determine the efficiency of each reaction so as to allow comparison of gene expression within a tissue or cell (see below). We currently use the Bio-Rad DNA engine Opticon 2 thermocycler that allows for reactions to be performed in both 96-well and tube format, and qPCR MasterMix for SYBR Green I [dNTPs, HotGoldStar DNA polymerase, MgCl2 (5 mM final concentration), uracil-N-glycosylase, SYBR Green I] from Eurogentec: many real-time thermocylers and SYBR Green master mixes are currently commercially available. As with PCR, controls that lack the template and RT are required for each experiment and multiple replicates should always be run. If the Ct between replicates differs by more than 0.5, the values should be excluded. Below we outline an example of the real-time PCR protocol using qPCR Mastermix for SYBR
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Green I for primers with a Tm of 60°C that detect a product of 120 bp: 1. Uracil-N-glycosylase step: 50°C for 2 min (cleaves contaminating template) 2. Initial denaturation: 95°C for 10 min (activates Taq and denatures uracil-N-glycosylase) 3. Denaturation: 95°C for 15 s (denatures newly synthesized products) 4. Annealing: 55°C for 30 s (binding of primers to their target site) 5. Extension: 72°C for 1 min (approximately 1 min per kilobase, DNA synthesis off the primer) 6. Plate-read, and repeat steps 3–5, 39 times 7. Melt curve 60–95°C, reading every 0.2°C, hold 0.1 s The last step in any real-time PCR that uses SYBR Green should be the inclusion of a melt curve, which provides information on specificity. The temperature is increased from 60 to 95°C and the fluorescence is continuously measured. At a specific temperature the product breaks, thereby resulting in a drop of fluorescence as the SYBR Green dissociates from the double-stranded DNA: the dissociation temperature depends on the length and composition of the product. A good melt curve should yield a single peak, as shown in Fig. 3. If primer dimers are formed, a small peak around a lower temperature (e.g., 70°C) is observed.
3.4.3 Reaction Efficiency The efficiency of the reaction needs to be calculated for each target [50]. To calculate the efficiency, we use reference
Fig. 3 The first derivative of raw fluorescence plotted against increasing temperature in the melt curve. The single melt peaks at 78°C (28S) and 81.5°C (Epac-1) indicate that a single PCR product from PASMCs has been amplified in each well
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cDNA (Stratagene) as the template, since some primers may show poor efficiency in a tissue of interest if the gene is poorly expressed. The easiest way to determine efficiency is to construct a standard curve by diluting the template (four or five points on the standard curve can accurately determine the efficiency) [51]. We use the dilutions 1:8, 1:64, and 1:512 since each should be exactly three Ct different if the reaction is 100% efficient (23, 26, and 29 respectively). Plotting the logarithm of the cDNA concentration against the Ct number will determine the linearity and sensitivity of the reaction. The efficiency, which should be 90–100% (slope between −3.1 and −3.6, where −3.32 represents most efficient), can be calculated by the following equation:
efficiency = 10 (−1 / slope ) − 1.
(1)
The linearity of the PCR assay is determined by R2 (the correlation coefficient, where 1 represents a perfect fit). If R2 is less than 0.98, this indicates that no linear relation exists between Ct and the concentration of the DNA and suggests the reaction is not efficient.
3.4.4 Normalization and Quantification Methods The main problems associated with real-time PCR are the uncontrolled variables and the interpretation of the results: normalization can correct for some of these variables. As outlined already, data should be normalized to a housekeeping gene whose expression is abundant and remains constant between samples. The same problems exist for real-time PCR as with PCR for determining the best housekeeping gene; however, we believe that the best option is to use multiple housekeeping genes for normalization. Absolute quantification measures the exact number of copies of the gene, whereas relative quantification defines the level of expression of the gene of interest relative to that of another gene, typically that of a housekeeping gene [52, 53]. Absolute quantification requires a standard curve constructed with known concentrations of the target (using a standard such as cDNA plasmids). Unknown concentrations are determined by interpolating values from the standard curves. In our laboratory, for example, we have used this type of quantification to investigate the stoichiometry of GPCR signaling pathways [54]. The most widely used quantification technique is relative or comparative quantification, in which one quantitates target genes without the need for a standard curve. Gene expression is calculated relative to a calibrator, which can be, for example, control patient samples, untreated cells, or the lowest expressed gene in the sample studied. Usually the DDCt method is used, which compares the normalized Ct level between the gene in the sample versus the calibrator. Initially,
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the DCt for the target gene is calculated for both the sample and the calibrator by
∆Ct = Ct target gene − Ct housekeeping gene .
profile gene expression and microRNA expression in single cells [55, 56].
(2)
The difference between the DCt of the sample and that of the calibrator is then calculated, giving the DDCt value: Relative quantity to the calibrator = 2-DDCt, where
4 Protein Quantification
Tools for analyzing mRNA expression are now widely available. However, the quantification of protein levels is ∆∆Ct = (Ct target gene − Ct housekeeping gene )calibrator necessary for optimal understanding of physiological and − (Ct target gene − Ct housekeeping gene )sample . (3) pathological states, including in the pulmonary circulation: mRNA expression does not always correlate with protein As long as the target and normalizer have similar dynamic expression and therefore one can draw erroneous concluranges, the comparative Ct method (DDCt method) is the most sions if only RNA expression is assayed [57]. practical method. Figure 4 shows the expression (whereby One of the most widely used techniques to identify prowhen one uses DCt, a lower Ct yields higher expression) of a tein expression is immunoblotting, commonly termed “westnumber of PDEs in PASMCs isolated from control subjects ern blotting.” Western blotting not only detects an individual and patients with pulmonary hypertension (n = 3). These data protein in a complex mixture, but it also provides informashow the relative expression of these PDEs, since primer effi- tion on its weight, abundance and can provide accurate ciency is the same for each product, in control PASMCs to be quantification [4, 9, 58]. Western blotting involves the sepaPDE3A > PDE5A > PDE4B > PDE4A > PDE4C > PDE2A > P ration of proteins (on the basis of molecular mass) on SDS– DE1C > PDE1A > PDE4D > PDE3B. However, this pattern polyacrylamide gels, then transfer to a membrane on which changes in PASMC samples from patients with pulmonary the protein can be detected using specific primary antibodies hypertension: PDE5A > PDE1C > PDE1A > PDE3A > PDE4B followed by labeled secondary antibodies. Western blotting > PDE4A > PDE4C > PDE3B > PDE2A > PDE4D. Thus mRNA is highly dependent on the quality of primary antibody that is expression of PDE1A, PDE1C, PDE3B, and PDE5A signifi- used and its specificity for the protein of interest. Although cantly increases with pulmonary hypertension. many antibodies are obtainable from commercial sources, if Advances in real-time PCR have allowed this technology a particular protein is novel, one will need to generate an to be assessable to most laboratories. More recently, improved antibody, which can sometimes be difficult and costly in performance of real-time PCR has allowed it to be adapted to terms of both time and money. Western blotting has been
Fig. 4 Messenger RNA expression of multiple phosphodiesterase (PDE) isoforms in control and pulmonary arterial hypertension PASMCs expressed as Ct normalized to 28S rRNA (mean ± standard error of the mean)
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used in many published works to identify proteins that are expressed in the pulmonary circulation and/or whose expression changes with disease or stimuli [29, 43, 44, 59].
4.1 Protein Isolation Before solutions of proteins can be separated by gel electrophoresis, tissues and cells need to be lysed. A number of lysis buffers and conditions can be used; these differ in ionic strength, pH, and nature/concentration of the detergent depending on the protein of interest. Use of detergents is essential for studies of membrane-bound proteins, but different types of detergents (e.g., ionic, nonionic, zwitterionic) can have widely differing effects in terms of yield, denaturation, and impact on charge of solubilized proteins and thus on migration patterns when the protein solutions are subsequently electrophoresed [9]. It is important to keep the sample on ice at all times and add protease and phosphatase inhibitors (if one is studying proteins that are phosphorylated) to the lysis buffer to minimize protein degradation. Unless specialized methods of extraction are required, owing to its broad utility we typically use the detergent NP40 in our lysis buffer [50 mM Tris–HCl (pH 8.0), 150 mM NaCl, 0.02% sodium azide, 100 mg/mL phenylmethylsulfonylfluoride (a protease inhibitor), 1 mg/mL aprotinin (a protease inhibitor), and 1% NP-40]. For PASMCs, we typically add 100 mL lysis buffer per well of a six-well plate and incubate on ice for 10 min, after which the cells are scraped and sonicated three times for 15 s; it is advisable to wash the cells with ice-cold phosphate-buffered saline to remove residual serum. Lung tissue should be ground under liquid nitrogen before the addition of 200 mL lysis buffer per 100 mg tissue (in general 2 vol of buffer for each volume of tissue), after which the sample should be homogenized using a handheld rotor homogenizer. Lysates are centrifuged at 12,000 rpm for 2 min at 4°C to remove debris and the supernatant is assayed for protein content so as to ensure equal loading of samples onto gels. One of the most commonly used methods for protein quantification is the Bradford assay (available commercially from Bio-Rad) [60]. The protein content of the sample is estimated using a standard curve constructed with known concentrations of BSA. Samples are prepared by heating them at 70°C for 10 min in sample buffer (50 mM Tris–HCl (pH 6.8), 2% SDS, 0.1% bromophenol blue, 10% glycerol, 2.5% b-mercaptoethanol): in general we recommend loading 10–15 mg protein per well. Both the high temperature and the reducing agent (b-mercaptoethanol) destroy tertiary protein folding and quaternary protein structures by breaking disulfide bonds and preventing their reformation. SDS denatures secondary structures and non-disulfide-linked tertiary structures, thus unfolding and linearizing the proteins.
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4.2 Gel Electrophoresis and Transfer Proteins are typically separated by SDS–polyacrylamide gel electrophoresis using a discontinuous buffer system in a vertical gel system (e.g., Invitrogen). Precast gels are commercially available (NuPage® Novex® Bis-Tris mini gels, Invitrogen) or gels can be poured individually. The concentration of acrylamide can vary depending on the size of the target protein; typically separation gels are 8–15%: a higher percentage of acrylamide is used to separate proteins with lower molecular weight. We prepare separating gels with a final concentration of 10 or 12% acrylamide [components of separating gel: 10/12% acrylamide, 0.375 M Tris-Base (pH 8.8), 0.1% (w/v) SDS, 0.05% (w/v) ammonium persulfate, 0.05% (v/v) N,N,N¢,N¢,-tetramethylethylenediamine (TEMED), and distilled H2O]. After setting of the separation gel, a 6% acrylamide stacking gel [6% acrylamide, 0.125 M Tris base (pH 6.7), 0.1% (w/v) SDS, 0.05% (w/v) ammonium persulfate, 0.1% (v/v) TEMED, and distilled H2O] is poured on top and a tenwell comb or a 12-well comb is inserted. After complete polymerization has occurred, the combs are removed, the gel is mounted in electrophoresis running buffer [25 mM Tris, 250 mM glycine (pH 8.3), 0.1% SDS], and the samples are loaded together with molecular weight standards (colored Novex sharp prestain protein standards are useful as they also determine transfer efficiency; Invitrogen). Gels are then run at a constant voltage (200 V) until the dye front is at the bottom. Transfer from the gel to a solid support (typically either nitrocellulose or PVDF) takes advantage of the negative charge of the proteins (which has been imparted by the SDS). For the conventional apparatus, the gel and membrane are sandwiched between pieces of Whatmann 3MM paper and sponges that had been presoaked in transfer buffer (39 mM glycine, 48 mM Tris-base, 0.037% SDS, 20% methanol, pH 8.3) with the membrane on the anodic side. The chamber is filled with transfer buffer and the gel is run at 100 V for 60 min. We currently use the Iblot dry blotting system (Invitrogen), which allows transfer in as little as 8 min without the need to prepare buffers. After transfer, the membrane can be stained (for protein) with Ponceau S to ensure good transfer, although we use the colored marker as a guide.
4.3 Hybridization of Probe and Detection To optimize the sensitivity of western blotting, it is important to block nonspecific binding. Typically, 5% nonfat dry milk or BSA in TBST [0.05% Tween 20 (a nonionic detergent) in phosphate-buffered saline or TBS (Tris-buffered saline)] is used depending on the compatibility of the detection system. The membrane is typically blocked for 2–3 h at room temperature, after which the primary antibody [at the optimal
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concentration (generally the one recommended by the manufacturer) in 2% milk or BSA in TBST) is incubated with the membrane overnight at 4°C. The membrane is then washed three times for 10 min with TBST, incubated with the secondary antibody (according to the manufacturer’s instructions) for 1 h at room temperature, and then the washes are repeated. All stages of the protocol are carried out on an orbital shaker to ensure equal coverage and washing of the membrane. Most secondary antibodies are conjugated with horseradish peroxidase, which, if exposed to hydrogen peroxide and an appropriate chemiluminescent agent (luminol), produces fluorescence in proportion to the amount of bound protein. Enhanced chemiluminescence (ECL) detection is a sensitive method that avoids use of radioactivity. Equal volumes of ECL detection reagents (2.5 mM luminol, 1.1 mM p-coumaric acid, 0.1 M Tris base, pH 8.5, and 0.02% hydrogen peroxide, 0.1M Tris base, pH 8.5) are mixed to give a final volume of 125 mL/cm2 on the membrane and are applied to the membrane for 1 min: we also use ECL Advance (Amersham), which provides increased sensitivity. To visualize immunoreactive bands, the membrane is then exposed to X-ray film for 10 s to 10 min depending on the intensity of the signal. Furthermore, we and many other laboratories use the UVP Bioimaging system (CCD camera), which captures images of the developed membrane without the need for film, developer, or fixer and which allows one to document the migration pattern of proteins on the membrane and save this information on a computer for subsequent analysis and presentation of the results. Membranes can be stripped to remove the initially used primary antibodies and then reprobed with other antibodies by incubating the blot with gentle agitation in a buffer [100 mM b-mercaptoethanol, 2% SDS, 62.5 mM Tris–HCl, pH 6.7, or RestoreTM stripping buffer (Thermo Scientific)] at 37°C for 1 h. After removal of the stripping buffer and washing of the membrane (three times for 10 min in the wash buffer), the membrane is reblocked and probed with a new antibody as outlined above. Protein quantification is measured by band intensity via densitometry, using ImageJ. Samples to be compared should always be run on the same gel and several different exposure times in rapid succession should be used to ensure the signal is not saturated so as to prevent misinterpretation of results. Protein-loading controls such as GAPDH and b-actin should be used to normalize the data to ensure equal loading of samples. If the target protein and the loading control have different molecular weights, the membrane can be cut and probed for both antibodies simultaneously; otherwise the membrane should be stripped and reprobed. Figure 5 shows the protein expression by immunoblotting of PDE5A, GAPDH, and caveolin-1 in whole cell lysates from lungs isolated from control and monocrotaline-treated rats. These data show that protein expression of PDE5A increases, whereas that of caveolin-1
535 PDE5A 100kDa
α β
Cav-1 21-24 kDa
GAPDH 36 kDa Control 2
MCT-treated
*
1 0
PDE5A
CAV-1 α
CAV-1 β
−1 −2 *
−3
n=5 −4
*
Fig. 5 Example of an image obtained by western blotting that depicts increased expression of PDE5A protein and decreased expression of caveolin-1 protein in whole lung from monocrotaline-treated rats compared with the control. Glycerol-3-phosphate dehydrogenase was used to normalize the data for equal protein loading and ImageJ was used to quantify the changes. Cav-1a and cav-1b refer to the two bands of caveolin 1 shown on the western blot
decreases, with monocrotaline treatment. These changes associated with monocrotaline are verified by showing equal protein loading via GAPDH and by measuring the band intensity using ImageJ. The technique described above provides a semiquantitative analysis of protein expression; however, for more accurate quantification, one needs to load a series of diluted protein standards (i.e., purified protein that is commercially available or obtained by cloning, expression, and purification). A dilution range of up to 50-fold is recommended; however, this depends on the dynamic range, and gels should be run in duplicate to provide greater accuracy. To calculate the concentration of the unknown, the band intensity (using the image analysis program) of each diluted purified protein is measured to generate a standard curve to define the protein content of unknowns. Our laboratory has used this method along with radioligand binding to determine the stoichiometry of GPCR signaling in rat myocytes [61]. Advances in the detection and quantification of proteins with disease or stimuli have included the development of many commercially available products that reduce nonspecific binding, increase the sensitivity, and reduce the time of the procedure. One recent approach is the use of secondary antibodies that are directly conjugated with a nearinfrared (NIR) fluorophore such as IRDyeTM800 or Alexa
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Fluor 680, which can even be used simultaneously for multiplexed analysis of two proteins (e.g., to detect total and phosphorylated protein at the same time). This technique has been shown to give a wider range of linear detection and unlike chemiluminescence the signal is measured in a static state [62]. Furthermore, since the NIR fluorescent blots are stable, they can be rescanned months later without any loss of the signal. One disadvantage is the need to buy a new detection system, such as the Odyssey infrared imaging system from LI-COR Biosciences, which is currently quite expensive.
5 Summary We have described techniques, experimental procedures, and some of the applications for the study of DNA, RNA, and protein, in particular as relevant to pulmonary research and medicine. The availability of new types of instrumentation as well as kits and standard reagents from commercial suppliers has helped simplify a number of the methods. In effect, the availability of these instruments and reagents helps create “best practices” that will help minimize interlaboratory differences in results. The application of these techniques should not only allow researchers to gain more insight into the correlation of gene expression with physiological function, but will hopefully also aid in the generation of new therapeutic targets as well as improvements in diagnostic techniques. Ultimately, their application and use may help guide the management of patients who are at high risk of developing or who present to physicians with diseases such as pulmonary hypertension. Acknowledgements This work was supported by grants from the National Heart, Lung and Blood Institute of the National Institutes of Health, the Lymphoma and Leukemia Society, and the Ellison Medical Foundation.
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537 pathophysiology of idiopathic pulmonary arterial hypertension. FASEB J 21:2970–2979 45. Morrison TB, Weis JJ, Wittwer CT (1998) Quantification of lowcopy transcripts by continuous SYBR Green I monitoring during amplification. Biotechniques 24:954–958, 960, 962 46. Hiyoshi M, Hosoi S (1994) Assay of DNA denaturation by polymerase chain reaction-driven fluorescent label incorporation and fluorescence resonance energy transfer. Anal Biochem 221:306–311 47. Rickert AM, Lehrach H, Sperling S (2004) Multiplexed real-time PCR using universal reporters. Clin Chem 50:1680–1683 48. Vrettou C, Traeger-Synodinos J, Tzetis M, Palmer G, Sofocleous C, Kanavakis E (2004) Real-time PCR for single-cell genotyping in sickle cell and thalassemia syndromes as a rapid, accurate, reliable, and widely applicable protocol for preimplantation genetic diagnosis. Hum Mutat 23:513–521 49. Regard JB, Sato IT, Coughlin SR (2008) Anatomical profiling of G protein-coupled receptor expression. Cell 135:561–571 50. Schmittgen TD, Livak KJ (2008) Analyzing real-time PCR data by the comparative C(T) method. Nat Protoc 3:1101–1108 51. Pfaffl MW (2001) A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res 29:e45 52. Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2-DDCT) method. Methods 25:402–408 53. Sellars MJ, Vuocolo T, Leeton LA, Coman GJ, Degnan BM, Preston NP (2007) Real-time RT-PCR quantification of Kuruma shrimp transcripts: a comparison of relative and absolute quantification procedures. J Biotechnol 129:391–399 54. Fuhs SR, Bundey RA, Ling Z, Insel PA (2007) Stoichiometry of GPCR-Gq/11 signaling components in MDCK cells: Are there “spare receptors”? FASEB J 21:568.17 55. Edvardsson L, Olofsson T (2009) Real-time PCR analysis for blood cell lineage specific markers. Methods Mol Biol 496:313–322 56. Schmittgen TD, Lee EJ, Jiang J et al (2008) Real-time PCR quantification of precursor and mature microRNA. Methods 44:31–38 57. Richter W, Jin SL, Conti M (2005) Splice variants of the cyclic nucleotide phosphodiesterase PDE4D are differentially expressed and regulated in rat tissue. Biochem J 388:803–811 58. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685 59. Rabinovitch M (2008) Molecular pathogenesis of pulmonary arterial hypertension. J Clin Invest 118:2372–2379 60. Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254 61. Post SR, Hilal-Dandan R, Urasawa K, Brunton LL, Insel PA (1995) Quantification of signalling components and amplification in the beta-adrenergic-receptor-adenylate cyclase pathway in isolated adult rat ventricular myocytes. Biochem J 311:75–80 62. Picariello L, Carbonell Sala S, Martineti V et al (2006) A comparison of methods for the analysis of low abundance proteins in desmoid tumor cells. Anal Biochem 354:205–212
Chapter 37
Gene Cloning, Transfection, and Mutagenesis Ellen C. Breen and Jason X.-J. Yuan
Abstract Mutagenesis is a change or alteration in the DNA sequences of a gene. Mutagenic events may occur spontaneously within the genome of an organism and many times do not lead to functional consequences or an altered phenotype. However, at times a change in the coding sequence of a gene manifests itself as a dysfunctional phenotypic trait or predisposes an individual to a particular disease. The human genome may contain variations or mutations in an individual or in a related group of individuals that predispose them to a particular disease. Identifying and understanding these mutations (or polymorphisms) in gene structure can aid in the understanding, diagnosis, and treatment of patients. Quite the opposite to the mutations that spontaneously occur and may be difficult to identify, purposefully introducing mutations into a candidate gene that is then expressed in a cell or mouse model systems can readily reveal important information. This chapter provides an overview of the strategy to clone a gene, express it in a cultured cell system, and elucidate the function of the expressed gene product using mutagenesis methods. The value of this type of experimental approach will be highlighted by reviewing two pulmonary genes that have been analyzed using in vitro mutagenesis methods, the potassium channel Kv1.5 and the bone morphogenetic protein receptor II. Keywords Gene mutation • Polymorphism • Heritable pulmonary arterial hypertension • KCNA5 • BMPR2 • Genotype and phenotype
1 Introduction Mutagenesis is a change or alteration in the DNA sequences of a gene. Mutagenic events may occur spontaneously within the genome of an organism and many times do not lead to E.C. Breen (*) Division of Physiology, Department of Medicine, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0623, USA e-mail:
[email protected] functional consequences or an altered phenotype. However, at times a change in the coding sequence of a gene manifests itself as a dysfunctional phenotypic trait or predisposes an individual to a particular disease. A classic example is the gene mutation first described in 1917 by Emmel found in those with sickle cell anemia [1]. In this rare genetic disease, the recessive gene for hemoglobin S is inherited. Expression of hemoglobin S leads to fragile “sickle-cell”-shaped red blood cells that are unable to adequately transport oxygen to organs throughout the body. Although autosomal recessive diseases in general are rare in the overall population, the human genome may contain variations or mutations in an individual or in a related group of individuals that predispose them to a particular disease. Identifying and understanding these mutations (or polymorphisms) in gene structure can aid in the understanding, diagnosis, and treatment of patients. Quite the opposite to the mutations that spontaneously occur and may be difficult to identify, purposefully introducing mutations into a candidate gene that is then expressed in a cell or mouse model systems can readily reveal important information. For instance, specifically designed mutations within the coding region of a gene can lead to the elucidation of how that particular expressed gene product functions within a cell to produce a biological effect. Systematic mutational analysis of untranslated gene regulatory regions (cis-acting DNA elements located in promoters and introns) is often used as a tool to identify the interaction between DNA binding sites and the transcription factors that recognize these specific DNA elements and determine when and how a gene is transcribed. Finally, the re-creation of gene mutations (polymorphisms) that appears at a higher frequency in human populations with lung diseases, such as primary pulmonary hypertension, chronic obstructive pulmonary disease, and asthma, can allow investigators to determine if these mutations may contribute to the early onset or progression of disease symptoms. This chapter will provide an overview of the strategy to clone a gene, express it in a cultured cell system, and elucidate the function of the expressed gene product using mutagenesis methods. The value of this type of experimental approach will
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be highlighted by reviewing two pulmonary genes that have been analyzed using in vitro mutagenesis methods, the potassium channel Kv1.5 and the bone morphogenetic protein (BMP) receptor II (BMPRII) [2–24]. The first gene to be discussed is one of the oxygen-sensitive potassium channels, Kv1.5, expressed in a subset of pulmonary artery smooth muscle cells (PASMCs) located in the precapillary resistance vessels [25, 26]. The importance of the second gene product, BMPRII, in pulmonary biology was brought under scrutiny owing to the identification of a high percentage of polymorphisms or mutations in patients who exhibit pulmonary arterial hypertension (PAH) [10, 11, 27–33]. Review of the analysis of these two genes using mutagenesis approaches will provide examples of how these methods can be applied to further our understanding of pathogenic mechanisms of lung vascular diseases and lay a foundation for rationale-based drug design to treat pulmonary hypertension.
2 Gene Cloning To study the function of a gene, the first requirement is to know the composition and order of the base pairs that make up the DNA gene sequence. A sensible strategy to clone and sequence a gene of interest will depend on how much information is already known about the particular gene, genes within the same family, or the same gene in related species.
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2.2 PCR Cloning With the advent of recombinant DNA technology and the thermostable DNA polymerase (Taq enzyme), PCR cloning is probably the easiest and fastest approach for cloning a gene sequence today [35]. The PCR is a reaction in which a region of DNA can be amplified (or exponentially synthesized) between two known primer regions (approximately 20–30 bp in length) of DNA [38]. If regions of DNA are highly conserved among species or within similar gene products in the same species, these conserved regions may be used as the initial primers to amplify or generate the intervening unknown DNA sequence from the species of interest. Usually the amplified DNA fragment produced is not long enough to contain all the sequence information to encode the gene product. This partial DNA fragment can then be used to screen a DNA library [36, 37] or the complete gene can be synthesized using one-sided anchored PCR methods or rapid amplification of cDNA ends [39]. The later method relies on knowing enough of the DNA sequence information to design one sequence-specific primer and utilizes a generic primer (such as the poly A sequence) to accomplish the exponential amplification of the unknown intervening sequence through cycles of DNA synthesis with the thermostable DNA polymerase (Taq).
2.3 Library Screening 2.1 Computer Resources The NIH GenBank provides a tremendous resource to start this process. Many genes have already been cloned or partially cloned and are available from several commercial vendors (http://www.ncbi.nlm.nih.gov/Genbank34). In addition BLAST (which stands for “basic local alignment search tool”) is a computer algorithm that allows comparison of gene sequences across species or with an unknown DNA sequence and identifies regions that are similar (http://www. ncbi.nlm.nih.gov/blast). The main advantage of having some or all of the base pair information about a gene of interest is that this can be used to “read” the DNA sequence, the coding region, which will be used to design specific mutations for functional analysis. If only part of the sequence information is known or similar sequences have been cloned in other species or genes within the same family of proteins, conserved and/or known regions may be used to design primers to screen for the gene of interest using PCR cloning [35]. Alternatively, partial DNA fragments may be used as hybridization probes to screen a genomic DNA or complementary DNA (cDNA) library and identify, isolate, and amplify the full-length gene sequence [36, 37].
A second method to isolate the full-length gene sequence, if a partial region of the gene is known, is to screen a DNA library. DNA libraries are collections of DNA fragments from a particular tissue or species that in total contain all the sequence information from the organs or species from which they were derived. The DNA may be genomic, containing all the intron and exon information, or a cDNA library can be assembled from cDNA that is reverse-transcribed from isolated messenger RNA (mRNA). Many types of genomic and cDNA libraries from a variety of organs and species are commercially available for screening a given gene within a particular species. The collection of DNA fragments that make up a library are “housed” in vectors (plasmids, cosmids, and phage) which are vehicles for amplifying the collection of DNA sequences in a bacterial strain that is then screened for the correct DNA containing clone. Once identified, this gene-containing bacterial colony can be grown in larger quantities to further amplify, isolate, and purify the DNA clone of interest. If a partial DNA fragment or short oligonucleotide is available, this DNA sequence information can be used to screen for full-length sequences by filter hybridization [36, 40]. If no DNA sequence information is known, then an alternative screening method must be designed, and
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this experimental strategy is referred to as “expression cloning.” This could be the use of an antibody to screen for the expressed protein or possibly a functional assay, for instance, an enzyme activity or unique biological response [41]. By whichever method is chosen to identify, isolate, and purify the gene of interest, the final cloned DNA is sequenced to determine the precise composition and order of base pairs that will be transcribed and translated into the final gene product.
3 Gene Expression in Cell Systems 3.1 Expression Vectors and Cell Systems Once the coding region of the gene is known, it is subcloned into an expression vector, which will allow the gene product to be produced within a cell. The key features of expression plasmids are (1) a strong (or efficient) promoter that will allow expression of the gene using the endogenous factors present in the cell system chosen, (2) a translational start site to allow expression of both the cloned gene and a bacterial selectable marker gene (i.e., antibiotic resistance gene), and (3) a poly A sequence at the 3¢ end to allow for efficient transcription and stabilization of the transcribed mRNAs. In addition, often an intervening sequence (intron) that is spliced out in the final mRNA is included for the purpose of enhancing expression efficiency. These are the basic elements that will allow the gene to be expressed within a cell. In addition, a mammalian selectable marker gene is very often included. This allows the separation of cultured cells, which express the cloned cells from a mixed population of cells that are both positive (expressing) and negative (nonexpressing) for the introduced gene. Included in the design of the vector may be the further option to remove this selectable gene sequence at a later stage. Thus, the next consideration is what type of cell system will work best for studying the gene of interest. Several heterologous cell systems have been routinely used for gene expression, including HEK 293, COS-7 cells, CHO cells, and Xenopus oocytes. In these heterologous cell systems, the gene of interest is often not endogenously expressed or is expressed at low levels, and this feature allows the newly incorporated gene to be more easily studied. However, often these more generic cell systems do not contain all the accompanying cellular factors to allow true gene expression under basal or experimental conditions. For example, heterologous expression of the Kv1.5a subunit in HEK 293 cells and mouse L cells differs in the voltage-dependent activation and slow inactivation kinetic profile monitored by in vivo patch clamp experiments. In this case an associated b subunit, endogenous Kvb2.1, is expressed in HEK 293 cells
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but is not expressed in mouse L cells [42]. Thus, in the interpretation of data from these types of heterologous expression experiments, one must consider the type of cell system. Cell lines derived from more differentiated organs or even primary cells from patients carrying a disease-related polymorphism may provide an environment that much more closely represents the conditions that occur in vivo in an intact organism. In the case of primary pulmonary hypertension, PASMCs and endothelial cells are often studied. The disadvantage of using a primary cell culture is that these cells may be limited in the number of times they can be passaged before they start to lose their unique phenotype or proliferative capacity. Thus, both cell lines and primary cell cultures are valuable systems and deciding which one is most appropriate will depend on the type of mutagenesis method utilized and the experimental question being addressed.
3.2 Methods of Gene Delivery 3.2.1 Transient Transfection Methods Once the gene or interest has been cloned into an expression vector, there are several methods for expressing that gene within a cultured cell [43]. The choice of gene delivery will depend on the cell type, the proliferative or quiescent state of the cell culture, the length of the gene, the required efficiency (number of cells that take up the gene), and the expression levels required for end analysis. The process of introducing an expression plasmid into cells in culture is referred to as “transfection.” In cell culture studies several basic methods that employ agents or chemicals that encourage plasmid DNA to be taken up by the cell are routinely used, including diethylaminoethyl (DEAE)-dextran, calcium phosphate, and lipofectin. In the DEAE-dextran and calcium phosphate methods, the DNA forms a precipitate on the surface of the cell that is engulfed by a form of “endocytosis” [44, 45]. A requirement for these chemical-based transfection methods is that the cells in culture must be actively dividing or proliferating to incorporate the plasmid DNA. Calcium phosphate and DEAE-dextran routinely provide a transfection efficiency of 50% or greater. For liposomes to mediate transfection of foreign DNA into the cell, plasmid DNA is mixed with cationic and neutral lipids,which forms a compact structure through electrostatic interaction with the nucleic acid backbone. The cationic “charged” DNA–lipid complexes are able to cross the negatively charged, hydrophobic cell membrane and enter the cells [46]. For cell types that divide more slowly (i.e., fully differentiated or primary culture cells) or for transfection into quiescent cells, alternative methods must be considered. One of these alternative transfection methods is electroporation. In this method a current of
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specific amplitude and duration, depending on the cell type, is applied to a cell in contact with the plasmid DNA and temporarily creates pores or openings in the cell membrane that allow DNA uptake. Once the plasmid DNA has been incorporated into the cell, by any of these described methods, it is generally only transiently expressed over the next several days (72 h), and only a small percentage cells (on the order of 1 in 104 depending on the cell type) actually incorporate the foreign DNA into the cell chromosomes. For many experimental designs, this type of “transient” expression pattern is perfectly adequate. However, if long-term consistent expression is desired, cells that integrate the transgene and maintain expression may be selected in a process that establishes a “stably” expressing cell line.
3.2.2 Selection of Stable Transfected Cells There are two basic ways to create populations of cells that stably (or continuously) express a transfected gene through incorporation into the cell’s genome. The first is to use a dominant selectable marker. This is a gene that is usually expressed along with the subcloned gene in the expression vector. After transfection, the cells that expresses both the gene-coding region and the selectable marker are cultured in a medium that contains a gene-specific agent that only allows transfected cells to survive. There are several types of selectable marker genes, such as the commonly used neomycin resistance (NeoR) or hygromycin resistance (Hyg or hph) genes. Cells then cultured with aminoglycoside antibiotics, gentamicin (G418), or hygromycin, which interferes with eukaryotic cell protein translation, do not survive unless they also express the corresponding resistance gene, NeoR or Hyg. After several rounds of selection and propagation, a cell line which expresses high levels of the transfected gene is obtained. One potential drawback of this type of approach is that if the expressed gene has a negative effect on cell growth or proliferation, it will be impossible to select a stable population. Furthermore, this process usually takes several weeks, during which time primary cell types may change or lose the expression of some of their unique differentiated genes before a stable, high-expressing population of cells is selected. The other possibility is to select the genes shortly after the initial transfection procedure. This can be done by expressing a selectable “tag.” For instance, fluorescent molecules may be expressed as part of a fusion product included in the cloned gene product or cells may be cotransfected with a second plasmid encoding a fluorescent reporter molecule. Another option is to coexpress a marker gene or a gene “label” in tandem on the same plasmid using an internal ribosomal entry site element. This allows two genes to be translated from the same vector. Thus, if the second gene is a fluorescent protein, cells can be readily identified and isola-
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tion of the cells expressing the newly incorporated DNA is possible by fluorescence-activated cell sorting. This also eliminates the possibility that the introduced gene will be silenced by methylation modification over the extended time it would take to purify the transfected population with a dominant selectable marker gene.
3.2.3 Viral Vectors In addition to transfection of expression plasmids, viral vectors provide a highly efficient way to express foreign DNA in cultured cells. Viral vectors are particularly useful for transfecting primary cultures, which are quiescent or slowly proliferating, and are therefore not easily transfected (i.e., with calcium phosphate, DEAE-dextran, or cationic lipid reagents). The production of virus vectors is a bit more involved as it requires viral proteins and target genes to be expressed and assembled in specialized packaging cell lines from which the recombinant virus is subsequently isolated and purified. Once again, the choice of viral vector will depend on whether the cells are actively dividing, whether “transient” or “stable” transfection is required, and the length of the coding sequence expressing the gene of interest. Three viral vectors, among those commonly used in cell culture systems, are retroviruses, adenoviruses, and adeno-associated viruses (AAVs). As the name implies, retroviruses are RNA viruses [47]. Retroviral particles require actively dividing cells for the viral complex to enter into the host nucleus and stable incorporation into the host cell genome. Once infected with a retrovirus expressing recombinant DNA, the gene product may then be studied in transiently expressing cells or selected for stable expression. A limitation to consider when working with a recombinant retrovirus is that very large coding regions (more than 8–11 kb) are too large to be packaged into the recombinant retroviral particle [48]. An adenovirus is a DNA virus that also provides a high level of expression in mammalian cells. Recombinant adenoviruses have been engineered to express genes of interest along with fluorescent or selective genes and readily infect both dividing and nondiving cells. However, this expression is transient and the introduced DNA does not stably integrate into the cells genome. Commercially, adenovirus vectors can incorporate coding sequences up to about 8 kb. The last virus discussed here is the AAV, which contains viral elements similar to those of the recombinant adenovirus except it uses an additional helper virus sequence to provide the viral genes used in the production and assembly of the recombinant AAV. In addition, recombinant AAVs will infect nondividing cells, a feature that may be useful for expression in primary or fully differentiated cells. Recombinant AAVs provide high-level long-term expression and roughly 5–10% of the transduced cells will stably integrate the foreign gene into the cell’s
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chromosomes. A limitation of the AAV system is it will not accommodate large coding regions (more than 3 kb) [49]. All these recombinant-type viral tools have been designed to be replication-incompetent by removing several of the viral encoded proteins (i.e., E1 and E3). This also provides an increased capacity for including the nonviral coding sequence (the target gene). Since these recombinant viruses can infect a cell once and produce the gene product without repeatedly infecting many cells, they are safer to work with than wildtype viruses. Although this design provides a degree of biosafety when working with these viral vectors, it is necessary to implement proper safety procedures when working in the laboratory.
3.2.4 Heterologous Overexpression Overexpression of the introduced DNA is then measured using standard methods to quantify gene expression (Fig. 1). These methods include reverse-transcription PCR to measure mRNA levels and western blot analysis and enzymelinked immunosorbent assays for assessment of the final protein level. Fluorescence microscopy or standard immunohistochemistry may also be used to detect the subcellular location, particularly if the expressed gene is a fluorescent fusion product or contains a generic tag (i.e., a FLAG), given that the resulting chimeric protein does not interfere with the protein or lipid interactions of the native protein. Finally, the cellular function is ready to be tested.
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Fig. 1 Characterization of expression and function of the human Kv1.5 potassium channel heterologously expressed in HEK293 cells. The construct [85] includes a human Kv1.5 encoding sequence (nucleotides –22 to +1,895) subcloned into XbaI and KpnI sites of the pBK-CMV expression vector. (a) DNA products of the regular (upper) and singlecell (lower) reverse-transcription PCR performed with messenger RNA isolated from native HEK293 cells (WT) and from HEK293 cells transfected with the human Kv1.5 expression construct (Kv1.5). The Kv1.5 transcription level in Kv1.5-transfected cells is much higher than in wild-type cells. (b) Western blots of total cell protein samples prepared from native HEK293 cells (WT) and from HEK293 cells transfected with human Kv1.5 expression construct (Kv). The amount of Kv1.5 protein product heterologously expressed in Kv1.5 transfected cells is very high compared with nontransfected cells. (c) Whole-cell currents from HEK293 cells transfected with empty pBK-CMV vector (-hKv1.5) and from HEK293 cells transfected with humans Kv1.5 expression construct (+hKv1.5). The current amplitude from Kv1.5-transfected cells is almost 25 times larher than in nontransfected cells. (d) Reversible inhibition by 4-aminopyridine (4-AP) of Kv1.5 channels heterologously expressed in HEK293 cells. The significant and reversible current decrease in Kv1.5-transfected cells treated with 4-AP reflects reversible inhibition of expressed ion channels by the voltage-dependent potassium channel blocker and shows the specificity of this channel. (Reproduced from [102] with permission)
4 Design of Recombinant Mutant Clones Over the last 10–20 years, several very clever and creative methods have been invented, and modified as molecular biology methods have evolved, to incorporate a mutation into cloned DNA and then select and amplify the mutated DNA clones for further analysis. Mutations may be randomly incorporated using chemicals or nucleases and then screened for changes in phenotype or function. However, there are several advantages of being able to precisely incorporate specific mutations in precise locations within a DNA sequence. Large changes in gene structure may be accomplished using restriction enzymes to remove or insert regions from a DNA clone that is then religated and transformed into bacteria. Using a variety of techniques, one can also incorporate multiple mutations over much more defined regions using a method such as linker scanner mutagenesis or scanner saturation mutations [50, 51]. Such methods are often used to elucidate the functional regions within 5¢ promoter regions or scan for peptide binding sites in expressed proteins. PCR either with incorporated restriction enzyme sites
or with gene splicing using overlap extensions can be used to introduce or exchange regions of DNA from another gene or to reintroduce DNA regions which contain specific deletions or insertions, or clusters of mutations. In addition to gross changes in gene structure (deletions, duplication, rearrangement, and multiple mutations over a region), precise mutations directed to a single or a few base pairs are also possible through site-directed mutagenesis methods [52, 53]. Practically, any type of change can be made with precision within the DNA sequences. A directed mutagenesis approach is very useful for studying proteins localized in the membrane (i.e., receptors and channels) since these proteins are not easily secreted and isolated for analysis. In addition, more information may be gained from studying their function in the context of native membranes (plasma membranes or endoplasmic reticulum) in which channel proteins or receptors interact with associated proteins and/or lipids. The next section will describe the design of methods to incorporate gross and site-directed single mutations (Fig. 2).
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Fig. 2 Designs for mutagenic clones: large deletion, chimera, and site-directed mutant. (a) Gene deletions may be created by restriction digestion of the cloned gene using sites that flank the region or subunit to be removed. The clone may be digested with one enzyme that cuts twice, two enzymes with compatible ends, or unique enzymes that are then filled in to form blunt ends. The resulting DNA fragments are separated by electrophoresis. The remaining cloned DNA is religated with T4 ligase and used to transform competent bacteria. (b) Overlapping PCR chimeric proteins may be created by interchanging subunits from similar or related genes (e.g., Kv1.2. Kv1.5, and Kv1.2). The two target genes (gene 1 and gene 2) are first amplified separately by PCR using four different primers (P1, P2, P3, and P4). P2 is designed to be homologous to gene 2 and P3 is homologous to gene 1. The outside primers P1 and P4 include restriction enzyme sites to allow the joined fragments to be reintroduced into the full-length gene clone. The two newly synthesized double-stranded DNA PCR
products are denatured to separate the DNA stands and allowed to anneal. This allows the homologous end regions to bind. Then Taq polymerase is used to extend the attached linear DNA. The resulting chimeric DNA is amplified using end primers (P1 and P4) and subcloned back into the original gene clone. (c) Site-directed mutants can be created using primers that contain the precise change in one or two base pairs. The target gene clone is denatured and the primers are allowed to anneal to the target sequence. The new strand is synthesized using a high-fidelity DNA polymerase (i.e., Pfu) in a linear reaction. The original DNA template contains methylated and hemimethylated sites, whereas the new DNA synthesized off the parent template is unmethylated. Incubation with the restriction enzyme DpnI selectively digests the parental methylated DNA. The mutated gene remains but this DNA also contains several “nicks” or gaps in the sequence. This new mutated DNA is transformed into a bacterial strain that allows the “nicked” DNA to be repaired
4.1 Large Deletions
that recognizes and cleaves sites near the ends of the DNA fragment, which will be removed. The resulting restriction digest fragment is separated by agarose electrophoresis from the remaining plasmid DNA (the plasmid backbone and remainder of the cloned gene), which is isolated from the agarose gel, religated with T4 DNA ligase, and transfected into the appropriate bacterial strain for amplification, isolation,
If the gene has already been cloned into a plasmid, a straightforward approach to creating a large deletion is to use the endogenous restriction enzyme sites that are encoded in the cloned DNA sequence. The plasmid, containing the gene of interest, is isolated and digested with a restriction enzyme
37 Gene Cloning, Transfection, and Mutagenesis
and eventual delivery into a mammalian cell system. If the identical restriction sites are not conveniently located at the ends of the region to be deleted, then two unique restriction enzymes with compatible ends may be used or the “sticky” ends can be filled in with a DNA polymerase (i.e., Taq or Klenow) to create blunt ends that can be ligated. Alternatively, directed mutagenesis using the PCR may be utilized to incorporate a particular restriction enzyme within the gene sequence.
4.2 C onstruction of Chimeras with Overlapping PCR Another useful mutation to define the subunit specificity of a protein is to replace one functional region with another and create a hybrid protein [54]. For instance, in Kv1.5 it has been shown that the T1 domain in the N-terminal region dictates the kinetics of channel inactivation. Furthermore, this property is conserved between channels as a chimeric protein containing the N-terminal of Kv1.1 or Kv1.3 and a shortened form of Kv1.5 were able to reproduce the inactivation kinetics found in full-length Kv1.5 [21]. One way to create a chimera is to employ a method based on sequential PCRs using overlapping PCR. In this scheme two genes or regions of DNA to be joined are first amplified in separate reactions that incorporate ends suitable for joining and reinserting the newly created DNA into the appropriate expression vector. One primer for each of the two reactions is designed to include end-restriction sites used for reinsertion of the joined DNA fragments into the expression vector. The “overlapping” component of this technique is to design internal primers that are homologous to the other target gene. In other words, primer 2 used to synthesize target gene 1 would be homologous to the end of target gene 2 and primer 3 would be homologous to the end of target gene 1. The resulting PCR-amplified target genes are mixed, denatured, and reannealed so the homologous primer can bind. Single-stranded connected DNA fragments are then extended with Taq polymerase to form double-stranded products that are subsequently amplified by PCR using the external primer (primers 1 and 4). This new chimeric region is now ready to be recloned into an expression vector for functional analysis.
4.3 Site-Directed Mutagenesis Kunkel et al. were the first to describe an elegant method to create single base pair changes in DNA [53, 55]. In this design a new mutated strand is synthesized using a mis-
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matched “mutated” primer and a single-stranded M13 DNA template containing several dUTP residues in place of TTP. This special uracil-containing template is isolated from M13 phages that have been amplified in Escherichia coli deficient in the enzyme dUTPase. This –dut –ung E. coli strain (E. coli CJ236 or equivalent dut − ung − F¢ strain) is also deficient in the glycolase that would normally catalyze the removal of uracil incorporated into DNA, uracil N-glyosylase (ung). The new mutated strand that complements this UTP-containing template is synthesized in a reaction containing the normal proportion of each dNTP and results in a new strand without uracil resides. The resulting double-stranded DNA product is then reintroduced into a wild-type bacterial strain (E. coli +dut, +ung) which will selectively amplify the mutated DNA [53, 55]. An obvious improvement on this method is to synthesize the new mutated DNA again using a mismatched primer but in this case the PCR is used to amplify the mutated DNA. This design results in incorporation of the precise mutation with a very high efficiency [52]. The resulting DNA fragment is then recloned into the original expression vector using either restriction enzyme sites (endogenous or incorporated into the PCR primer design) or by overlapping PCR (as described earlier). One caution with PCR-based methods is that there is an inherent mutation rate in Taq polymerase. Several high-fidelity thermostable polymerases are currently commercially available that synthesize DNA with a lower mutation rate and thus reduce the probability of creating unwanted mutations in the cloned gene. Yet another variation of using mismatched primers, but eliminating the PCR amplification step, has been developed to introduce site-directed changes in a gene sequence and to further reduce the risk of incorporating random spurious mutation (developed by Stratagene, “Quick Exchange” [56]). In this design, primers which incorporate the precise change in DNA sequence are annealed to a plasmid template. New double-stranded products are produced by extending the primers with the high-fidelity DNA polymerase (PfuTurbo) in a linear fashion from each primed end. This step differs from the repeated cycles of DNA polymerase synthesis used to exponentially amplify the DNA in a standard PCR. This DNA synthesis reaction results in a new mutated plasmid containing the precise mutation. However, staggered DNA nicks are also present throughout this newly synthesized strand. The reaction product containing the new “nicked” DNA and original template are then incubated with DpnI endoculease, which selectively recognizes methylated and hemimethylated sites and digests the parental DNA. The nicked plasmid containing the desired mutation is then transformed into a bacterial strain able to repair the “nicked” DNA strand and amplify the new construct.
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5 The Kv1.5 Potassium Channel and BMPRII: Key Genes Implicated in Pulmonary Hypertension This section will describe examples of two genes that are important for pulmonary vascular function. Analysis of these two pulmonary genes, using experimental mutagenesis approaches, has been valuable in both revealing their biological function in the lung as well as understanding how dysfunction of these expressed pulmonary proteins may contribute to pulmonary hypertension. The first gene to be discussed is an oxygen-sensitive K+ channel expressed by PASMCs located in the resistance (or precapillary) vessels. In this situation mutagenesis approaches have provided a wealth of knowledge on how this related Kv1.5 channel functions to maintain vasomotor tone and contributes to the hypoxic pulmonary vasoconstriction (HPV) response to acute hypoxia. In addition to understanding the conformation and function of Kv1.5 and related Kv channel a subunits, this same mutagenesis approach has allowed a rational approach to the design of drugs for the treatment of pulmonary hypertension [57–59]. The second pulmonary gene discussed in the following sections is BMPRII. This tyrosine kinase receptor is also implicated in regulating PASMC proliferation, and heterozygous germ line mutations have been identified and implicated in predisposing patients to familial and idiopathic pulmonary hypertension [10, 11, 27–30, 33, 60–65]. In this case, mutagenesis has been used as a very important tool for analyzing the mutation already found in patients that are predisposed to or exhibiting familial or idiopathic pulmonary hypertension.
5.1 The Oxygen-Sensitive Potassium Channel, Kv1.5 Kv1.5 belongs to a family of related potassium channels (for reviews, see [25, 26, 66]). Voltage-sensitive Kv channels conduct potassium currents at or near the diffusion-limited rates and this property contributes to the maintenance of membrane potential (Em) in PASMCs and consequently resting vasomotor tone. In response to a decrease in the level of environmental O2, hypoxia, the pulmonary resistance vessels very rapidly constrict in a response that is thought to divert blood flow away from poorly ventilated alveoli. The benefit of this adaptive response may be to limit ventilation perfusion inequalities and maintain systemic oxygen levels [67]. PASMCs express a variety potassium channels, including voltage-gated K+ channels (Kv), inward rectifier K+ channels (KIR), calciumactivated K+ channels (KCa), and tandem-pore two-domain K+ channels (K2P) [68]. However, certain members of the Kv family of delayed rectifier (IK,DR) and noninactivating (IK,N) channels (Kv1.2, Kv1.5, Kv2.1, Kv3.1b, and Kv9.3) are selectively expressed in resistance vessel PASMCs [69, 70],
E.C. Breen and J.X.-J. Yuan
and in particular Kv2.1 and Kv1.5 are thought to be major contributors to the oxygen-sensitive change in the voltage sensitivity and activation kinetics that regulate K+ current across the PASMC membrane [26, 71, 72]. In response to an acute hypoxic exposure, the resistance vessel PASMCs are thought to generate less reactive oxygen species (ROS) [25]. This decrease in the amount of ROS leads to an inhibition of the Kv1.5-gated K+ current. Kv channels close, preventing the tonic efflux of K+ ions and the cell depolarizes as positive charge accumulates within the cell. This new membrane potential favors Ca2+ channel opening and Ca2+ ions move into the PASMC down a steep concentration gradient. This rise in the level of intracellular calcium signals myocytes to contract around the vessel, or vasoconstriction. The entire process is referred to as “hypoxic pulmonary vasoconstriction” (HPV) [67, 69, 73, 74]. The inhibition of ROS in response to hypoxia is unique to pulmonary arteries and although the oxygen sensor in this system is not yet definitively known, the release of ROS (from mitochondrial or NADPH oxidoreductase) is thought to signal channel activity inhibition and downregulate Kv1.5 expression through a hypoxia-inducible factor 1a dependent pathway [25]. Interestingly, upon exposure to chronic hypoxia (greater than 3 weeks), a blunted HPV response is observed upon return to hypoxia that is accompanied by decreased expression of Kv 2.1, Kv 1.5, and Kv 9.3 [75, 76]. However, the HPV can be rescued in chronically exposed mice by overexpression of adenovirally delivered Kv1.5 [77]. However, unlike exposure to chronic hypoxia, in which pulmonary artery remodeling and HPV are eventually restored to normal upon return to a normoxic environment, pulmonary hypertension is associated with a persistent decrease in Kv1.5 expression and K+ current that lead to sustained vessel tension [78, 79]. In addition, the role of Kv1.5 channels in regulating PASMC proliferation and apoptosis could contribute to the aberrant vascular remodeling and the formation of plexiform lesions in the precapillary arteries, two hallmarks of idiopathic PAH (IPAH) and familial PAH (FPAH) [66, 80, 81]. Recently, a potential dysfunction role of Kv1.5 in PAH has been supported by the finding of several single nucleotide polymorphisms in the Kv1.5 gene in PASMCs from patients with IPAH [82]. A great amount of information has been gained through the functional analysis of cloned Kv1.5 channels designed with a strategically positioned mutation. Most of the studies used the patch clamp technique first described by Neher and Sakmann [83] to record the current through single K+ channels and plot the kinetics of channel activation and inactivation in response to a voltage change. The Kv1.5 channel was first identified and cloned from rabbit vascular smooth muscle cells [84] and the human gene was isolated and cloned from the heart in 1993 [85]. As illustrated in Fig. 3, Kv1.5 is composed of six subunits or domains the span the plasma member (S1–S6) along with both C-terminal and N-terminal tail
37 Gene Cloning, Transfection, and Mutagenesis
Fig. 3 Structure of KCNA5 and characteristics of Kv1.5 channel currents. (a) KCNA5 gene organization showing the start codon (dotted line with arrow), the heteromerization domain (Het dom), and six transmembrane (TM) segments (TM1–TM6). Noncoding flanking 5¢- and 3¢-untranslated regions (UTR) are also indicated by hatched boxes. Right, pictogram of human chromosome 12 highlighting the p13 region containing KCNA5 (see the arrow at top). (b) Kv channel subunit protein folding motif and regulatory subunit binding; the pore region (P) is shown between TM5 and TM6. (c) Cross-sectional view of the arrangement of a and bsubunits into a tetrameric Kv channel with an ion-selective pore. (d) Representative single-channel Kv1.5 currents (left) at different test potentials (TP) and the current–voltage (I–V) relationship curve (middle) recorded
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from a cell-attached membrane patch of a human pulmonary artery smooth muscle cell (PASMC) transiently transfected with KCNA5. The fluorescence microscopy image (right) depicts human a PASMC transfected with KCNA5, identifiable by the green fluorescence. e Representative single-channel Kv1.5 currents at +100 mV from a COS-7 cell, a rat PASMC, a HEK293 cell, and a human coronary artery smooth muscle cell (CASMC) transfected with KCNA5 (left). The summarized I–V curve is constructed from single-channel Kv1.5 currents recorded from multiple cell types. The images identify KCNA5-transfected COS-7 cells, rat PASMCs, HEK-293 cells, and rat mesenteric artery smooth muscle cells (rMASMC) by their green fluorescence (right and bottom). (Reproduced from [82] with permission)
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regions that confer distinct biological properties. The last two helices (S5 and S6) are connected by a linker region. Four Kv a subunits assemble to form homomeric or heteromeric channels and are associated with b subunits, which bind to the N-terminal region of the channel and modulate channel function. Much of the early work on the organization of Kv channels has come from study the drosophila prototype, Shaker, and the Kv 1.2 channel crystallographic structure was solved in 2005 [86]. However, in the small resistance pulmonary
arteries, Kv1.5 has a major role in the rapid HPV response to acute hypoxia and altered Kv1.5 function and expression are implicated in the pathogenesis of PAH [26, 70, 87]. Mutagenesis studies using cloned Kv1.5 have been utilized to elucidate the subunit domains that sense a change in voltage to activate the channel as well as the functional domains responsible for “gating” the selective flux of K+ ions across the PASMC membrane (Table 1). For instance, creation of a chimeric construct in which Kv1.5 and Kv2.1 are
Table 1 Characterization of Kv1.5 functional domains through expression of cloned recombinant mutants Cell system and Mutations Method of mutagenesis functional assay Finding
References
N-terminal mutations Kv1.5/Kv1.2
[71]
Overlapping PCR to express both a subunits in tandem
Mouse L cells Patch clamp
Kv1.5DN209
N-terminal deletion using restriction enzyme sites
HEK 293 Patch clamp
Kv1.5DN19 Kv1.5DN91 Kv1.5DN119 Kv1.1 N/Kv1.5 Kv1.3 N/Kv1.5 KV1.5AAQL Kv1.5 N/Kv1.2 Kv1.2 N/Kv1.5
Bal31 5¢ nuclease digestion and PCR subcloning; subcloning using restriction enzymes and PCR; and primer exchange
HEK293 Xenopus laevis oocytes Patch clamp
Site-directed primer exchange
Mouse Ltk– cells Patch clamp
Established the importance of the first proline in the Pro-X-Pro “hinge” motif for channel gating
[13]
PCR site-directed mutagenesis
Mouse Ltk– cells Patch clamp
Residues near the Pro-X-Pro “hinge” sequence affect the stability of the activation pore
[16]
Site-directed primer exchange
HEK293 Patch clamp
[17]
Site-directed primer exchange
HEK293 Ltk– cells Patch clamp
Excess H+ or Zn2+ can bind to H463 in the pore turret and stabilizes a channel conformation that is in an inactivated state Identified a group of amino acid residues (including but not solely H463) in the pore turret that modulate slow inactivation
S6 mutations PVPV (WT) AVAV AVPV AVPP PVAV PVPP T505I T505V V512A V512M S515E Y519F Y519N H463Q H463G (H463Q, R487V) Small, neutral H463G H463A Large, charged H463R H463K H463E Cysteine substitutions T462C⇒P468C
Kv1.2 and Kv1.5 associated to form functional O2-sensitive heteromeric K+ channel The truncated from of Kv1.5 displays accelerated inactivation from closed states relative to full-length Kv1.5 Established interactions with the Kv1.5T1 subunit imparts U-type slow-inactivation channel kinetics
[20]
[21]
[18]
(continued)
37 Gene Cloning, Transfection, and Mutagenesis
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Table 1 (continued) Mutations S4 and S6 mutations S4 domain R410N S6 domain P511G
Method of mutagenesis Coexpressed as monomers or in tandem within a subunit
(V407I, I410L, S414T) Site-directed primer exchange W472F R487V H463G C-terminal membrane localization Kv1.5∆C10 Kv1.5∆C13 Kv1.5∆C18 Kv1.5∆C28 Kv1.4/Kv1.5 Kv1.4∆C37 +1.5 Kv1.5∆C28 + 1.4 Interaction with PDZ domain FL-Kv1.5-A
FL-Kv1.5-A FL-Kv1.5Gly- ∆SAP97
Src adaptor Kv1.5-DPro Kv1.4-Kv1.5 Kv1.4-Pro
Cell system and functional assay Ltk– cells
Xenopus oocytes Voltage clamp fluorimetry
Finding
References
Coupling between voltage sensing (S4 domain) and gating (S6 domain) occurs by intrasubunit interactions Dynamic monitoring of voltage sensing by S4 coupled to channel opening and closing of the K+-selective filter
[14]
[12, 90, 91, 93, 94]
Mutations using endogenous restriction enzymes and PCR site-directed mutagenesis
GFP-tagged constructs monitored by confocal microscopy and patch clamp recording
Surface expression governed by C-terminal motif VXXSL
[23]
Mutations generated using PCR to replace the terminal TDL residues with AAA and tag the construct with the FLAG epitope Kv1.5 N-terminal deletion and glycine. Series of SAP97 deletions
Cardiac myocytes COS-7 Xenopus oocytes
SAP97 and Kv1.5 C-terminal functional interactions
[101]
Cos-7 CHO
Caveolin 3 and SAP97 in PDZ2 and PDZ3 recruit Kv1.5 and regulate K currents.
[15]
Deletions and insertions of proline-rich motif RPLPPLP
HEK293 Patch clamp
Kv1.5 subunits function as an Src homology domain (SH3)dependent adaptor allowing phosphorylation of associated a subunits that lack an SH3 domain
[95]
expressed in tandem in heterologous cells has determined that both of these Kv channels confer oxygen sensitivity, although the voltage-gating properties of these Kv proteins expressed separately differ from those of heteromeric channels. In L cells expressing Kv1.2/Kv1.5 the whole-cell current is reversibly inhibited by hypoxia in the voltage range of the PASMC resting Em [71]. As stated previously, Kv1.5 is a rapidly activating delayed rectifier channel which is inactivated by a slower pore-dependent mechanism termed “C-type inactivation” that involves the constriction of the outer mouth of the channel pore [6]. Realization that the N-terminal tail region of the channels plays a role in this slow inactivation process came from the comparison of a truncated N-terminal Kv1.5 isoform, found to occur naturally in cardiac myocytes, with full-length Kv1.5. The truncated Kv1.5 isoform (Kv1.5D209) displays an accelerated inactivation from
closed states in patch clamp experiments. The voltage–inactivation relationship for Kv1.5D209 is U shaped, in which more complete inactivation occurs at intermediate changes in membrane potential and relief of inactivation takes place at very positive potentials. Systematic deletion and sitedirected mutagenesis of the N-terminal region narrowed down a specific T1 domain within the N terminus that imparts U-type inactivation. This region is conserved among a subset of Kv channels, including Kv2.1, Shaker, and Kv3.1 [21]. Thus, creation of chimeras that include the N-terminal domain of Kv2.1 plus transmembrane segments and the C-terminal region of Kv1.5 result in a U-shaped inactivation–voltage relationship. The opposite effect was found when the Kv1.5 N-terminal tail was combined with Kv2.1 transmembrane domains, and the U-type inactivation profile was attenuated compared with a native Kv2.1 channel.
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However, for voltage-dependent activation of the channel to occur there must be a mechanism to sense a change in membrane potential. In Kv channels this is thought to occur predominantly through the S4 domain, which contains several charged residues (arginines) located in the membrane electric field which could potentially change their position in response to a field change [88]. In Kv1.5, precise mutations support S4 as the voltage-sensing domain, whereas S6 controls activation–inactivation kinetics by controlling the equilibrium between an open and a closed pore region [16]. Furthermore, these voltage-sensing and gating functions are coupled through intrasubunit interactions (not intersubunit interaction within individual Kv channels) [14]. A conserved Pro-X-Pro sequence is located in the region between helices S5 and S6 that forms a “hinge” allowing the gate to close and open. To understand how the S6 transmembrane region modulates channel activation and deactivation, site-directed mutagenesis was used to change residues in and around this Pro-X-Pro “hinge” motif. The first proline residue in the ProX-Pro “hinge” motif was found to be vital for channel gating and replacing a nearby proline partially restores open and closing of the channel although it does so with altered activation kinetics [13]. Additional studies have systematically substituted cysteine residues in the “turret” of the pore with neutral or charged amino acids to identify residues that modulate the conductance of K+ through the permeation pore. Specific functional domains or a group of particular amino acid residues (including but not exclusively H463) in the pore turret regulate the slow inactivation properties of the Kv1.5 channels [17, 18]. A very interesting study was recently reported by Vaid et al. in which a fluorophore, attached to particular amino acids in the S3–S4 linker, was used to dynamically monitor the macroscopic voltage-sensitive movements of the channel during the time course of activation and inactivation [12]. By recording both the voltage-dependent K+ current and the change in fluorescence, they could decipher macroscopic movement of the S4 voltage-sensing domain and conformational rearrangements that couple opening of the intracellular pore gate (Fig. 4). In this study, S4 movement was reflected as rapid fluorescence quenching associated with activation of the voltage sensors upon depolarization. In the case of Kv1.5, this is followed by a later dequenching of the fluorescent signal. This later dequenching phase is not present when the same technique is used to monitor Shaker channels [89]. To further understand this late dequenching phase of the voltage-dependent fluorescent signal, previously well characterized site-directed mutations were again studied. The triple ILT mutation (V407I,/I410L/S414T [90]) was used as a way to dissociate channel opening from voltage sensing and led to the suggestion that Kv1.5 fluorescence dequenching reflects the channel opening late in the activation pathway. Immobilization of the outer pore by sitedirected mutation (W472F, H463G or R487V [91–94]) or
E.C. Breen and J.X.-J. Yuan
raising the extracellular K+ concentration revealed that this dequenching is likely associated with a K+ selectivity filter in the outer pore as opposed to the intracellular pore gate [12]. For the Kv channels to sense and gate K+ currents, they must be translocated (or be expressed) on the cell membrane. This cellular trafficking was first attributed to a motif (VXXSL) in the C-terminal domain [23]. Within the membrane, Kv1.5 a subunits interact with additional proteins and lipids that modulate the amplitude and duration of the K+ current. Specifically the last three amino acid residues (TDL) of the N-terminal tail have been shown to interact with SAP97 located in the PDZ membrane complex [5]. More recently it has been shown that both N-terminal and C-terminal regions interact with components of PDZ domains and influence the magnitude of the K+ current across the channel [19]. In particular, caveolin 3 and SAP97 are thought to play a role in recruiting Kv1.5 to the membrane and stabilize its location in a tripartite structure [15]. Kv1.5 has also been shown to interact with Src tyrosine protein kinase and serve as an adaptor to initiate the phosphorylation of heteromeric Kv channels that do not contain a similar SH3 domain [95]. Thus, mutagenesis approaches have been extensively used as a tool to elucidate the complex structure–function relationships within the a subunit of Kv1.5 and also its interactions with other Kv a subunits, associated proteins, and lipids that control the flux of K+ through the membrane.
5.2 BMP Receptor Polymorphisms Associated with Pulmonary Hypertension In 2000, two separate laboratories reported results suggesting that familial primary pulmonary hypertension was caused by mutations in BMPRII [27, 28]. The study by Deng et al. [28] used positional cloning to identify five mutations that predicted premature termination and two missense mutations in the BMPRII gene from a screen of individuals from 19 families [28]. In a separate publication form the International PPH Consortium, two frame-shift, two nonsense, and three missense mutations were found in a panel of eight kindreds studied [27]. Over the years, several studies of large cohorts of patients around the world have uncovered a strong link between BMPRII mutations and a predisposition to FPAH or IPAH. It is estimated that heterozygous germline mutations are present in more than 50% of patients with FPAH and in approximately 20% of those with sporadic IPAH [29, 60, 61]. In addition, these patients often have disease symptoms an average of 10 years earlier than noncarriers and exhibit more severely compromised hemodynamic dysfunction [64]. BMPRII is member of the transforming growth factor b (TGF-b) receptor superfamily that includes several type II
37 Gene Cloning, Transfection, and Mutagenesis
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Fig. 4 Characteristics of the tetramethylrhodamine maleimide (TMRM) fluorescence report from Kv1.A397C channels. Representative ionic current (a, d) and fluorescence (b, e) traces recorded from oocytes expressing Shaker A359C (a, b) or Kv1.5 A397C (d, e) channels labeled with TMRM. Voltage-clamp pulses were applied from −80 to +60 mV from a holding potential of −80 mV (only highlighted traces are shown for clarity). (c, f) Mean G–V and F–V relations for Shaker A359C (c) and Kv1.5 A397C (f). V1/2 and k values for the G–V relation of Shaker A359C channels were −16.2 ± 1.3 and 16.8 ± 1.1 mV, respectively, and the corresponding values for the F–V relation were −40.9 ± 1.9
and 17.0 ± 1.7 mV, respectively. V1/2 and k values for the G–V relation of Kv1.5 A397C were 7.3 ± 1.6 and 16.4 ± 1.2 mV, respectively, and the corresponding values for the F–Vpeak relation were 1.9 ± 1.9 and 19.0 ± 1.3 mV, respectively. The F–Vpeak relation was not significantly shifted from the G–V relation. The voltage dependence of the dequenching component of fluorescence from Kv1.5 A397C (calculated as the peak minus the end fluorescence amplitude, F–Vdecay) is also shown in (f); V1/2 and k values were 31.0 ± 2.4 and 15.5 ± 1.9 mV, respectively (Copyright Vaid et al. [103]. Originally published in the Journal of General Physiology. doi:10.1085/jpg.200809978)
receptors [BMPRII, and activin receptors (ActR-IIA and ActR-IIB)] and type I receptors [ActR-I (ALK2), BMPR-IA (ALK3) and BMPR-IB (ALK6)] that function as heterodimers in both vascular endothelial cells and PASMCs. In the small pulmonary arteries, selective binding of BMP ligands is thought to maintain the balance of PASMC and endothelial cell turnover. In contrast, in human PASMCs from patients with PAH carrying a BMPRII mutation, uncontrolled PASMC proliferation contributes to the aberrant pulmonary artery remodeling that occludes vessels and leads to persistent elevated pulmonary artery pressures. Thus, several in vitro experiment have been designed to recreate the missense and frame-shift mutations that have been identified in FPAH and IPAH to better understand how these polymor-
phisms could predispose an individual to a disease phenotype (Table 2). The first BMPRII mutants to be analyzed in vitro recreated some of the common polymorphisms found in patients with FPAH [27, 60]. In vitro expression of a kinase domain missense mutation (D485G) and the frame-shift mutation [1,860 fs(+10)] in HeLa cells and the mammary epithelial cell line NMuMG (a cell type known to be competent for TGF-b signaling) showed decreased basal and BMP4induced transcriptional activation of Smad binding element reporter constructs [60]. This early study was followed the analysis of a more extensive number of mutant BMPRII by Rudarakanchana et al. [1]. A series of site-directed mutations were created in the ligand binding, kinase domain and
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Table 2 Functional analysis of bone morphogenetic protein (BMP) receptor II (BMPRII) mutants Mutation Method of mutagenesis Cell system Functional consequence
References
Mammary-gland epithelium cell (NMuMG) line that is constitutively competent for TGF-b signaling HeLa cells, primary PASMCs and NMuMG cells
Mutants revealed decreased transcriptional activation of a Smad binding element transcriptional reporter
[60]
Revealed different effects of mutants expressed in heterologous cells and NMuMG on Smad-dependent transcription, MAPK activity, and cell proliferation
[1]
Recombinant adenovirus
Human PASMCs
Suggest that the kinase domain is essential for Smad signaling and that the C-terminal tail plays a role in BMP7-induced apoptosis
[8]
Recombinant adenovirus
PASMCs from BMPRIILox/Lox mice and delivered AdvCre to delete the BMPRII gene
Short and long isoforms of BMRPII were both found to restore BMP-mediated growth inhibition and osteogenic differentiation of PASMCs in response to BMP4 and BMP7
[7]
D485G1,860 fs(+10)
Restriction digest Site-directed primer exchange
Ligand binding C(60)Y C(117)Y C(118)W C(123)R/S Kinase domain C(347)Y C(420)R C(483)R D(485)G R(491)W/Q Cytoplasmic tail K(512)T N(519)K S(532)X R(899)X Kinase domain C347Y Truncated C-terminus I860X Deleted C-terminus ∆T AdvLF – long AdvSF – ∆T
Site-directed primer exchange
Fig. 5 Functional domain structure of the human bone morphogenetic receptor II (BMPRII). BMPRII consists of a signal peptide (SP) followed by the ligand binding domain, transmembrane domain (TM), kinase domain, and a unique C-terminal cytoplasmic tail
C-terminal cytoplasm tail of BMPRII that were expressed in HeLa cells, PASMCs, and NMuMG cells (Fig. 5). Cysteine substitutions in the ligand binding and kinase domain were found to prevent trafficking of the BMPRII receptor to the cell surface and consequently basal and BMP4-induced transcription activation of a BMP/Smad reporter construct. Interestingly non-cysteine substitutions in the kinase domain localized to the cell membrane but transcriptional activation of the BMP/Smad reporter construct was suppressed. Finally, cytoplasmic terminal mutants translocated to the cell surface
but in this case retained BMP signaling. When any of the above-mentioned mutants were transfected into NMuMG epithelial cells, activation of p38MAPK signaling and increased proliferation occurred even in the absence of a BMP4 ligand. This study was the first to indicate that BMPRII can signal through both the Smad and the mitogen-activated protein kinase pathways, but even more intriguing was that in the absence of a BMP ligand, the cues to prevent proliferation were lost. Subsequent in vitro mutagenesis studies focused on the cytoplasmic C-terminal domain, which is unique to BMPRII. Using a PASMC cell system in which the BMPRII gene was conditionally deleted in vitro, Yu et al. demonstrated that the ability of BMP2 and BMP4 to inhibit PASMC growth was lost. However, in these same BMPRII-deficient cells BMP6 and BMP7 signaling was augmented. The BMPRIIdeficient PASMCs could transduce BMP6 and BMP7 signals through an altered set of receptors, in this case the activinIIA receptor and a subset of type I receptors that do not normally interact with BMPRII [3]. In a subsequent study, reintroduction of the BMPRII gene as either a complete long
37 Gene Cloning, Transfection, and Mutagenesis
form or short form (not including the C-terminal cytoplasmic tail) was able to restore BMP4- and BMP7-mediated growth inhibition of platelet-derived-growth-factor-activated SMCs and restore osteogenic differentiation [7]. In a study by Lagna et al. [8], mutations in the serine/threonine kinase mutation (C347Y), a truncated COOH tail domain (I860X), or complete deletion of the C-terminal domain revealed a PASMC population that was resistant to apoptosis [8]. So although it was first thought that BMPRII mutations, which in most cases abrogate BMPRII expression, were due to haplotype insufficiency [60], it appears that some mutations actually lead to a gain of function [3, 8]. However, both PASMCs and vascular endothelial cells express BMPRII receptors as well as additional type II and type I receptors that compete for BMP ligand binding and these precise cellspecifc ligand binding mechanisms are just beginning to be elucidated [4, 96]. However, irrespective of whether mutations in human PASMCs result in altered signaling owing to haplotype insufficiency or to a new dominant phenotype that works through an alternative set of BMP receptors and ligands, it is still unknown why these mutations are silent for many years until the symptoms associated with PAH begin to surface in young adults. A “second hit” hypothesis suggests that additional genetic or environmental factors are necessary to provide the conditions in which BMPRII mutated cells begin to express an abnormal phenotype. These secondary cues may be inflammatory stimuli and/or possibly altered interactions with a modifying factor, such as RACK1 or Src tyrosine kinase [9, 97, 98]. In vivo, mice with a heterozygous deletion of BMPRII require an additional inflammatory stress to develop PAH [99]. However, heterozygous or homozygous BMPRII gene deletion targeted to endothelial cells is sufficient to predispose a subset of mice to PAH [100]. Thus, current research is focused on understanding the cell-specific additional genetic and environmental factors that trigger the onset of PAH. Further understanding these pathways through mutant phenotypes that disrupt the balance of PASMCs and endothelial cells will hopefully allow for better design of treatment strategies that target the abnormal cellular processes unique to each susceptible individual.
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Chapter 38
Approaches for Manipulation of Gene Expression Ying Yu and Jason X.-J. Yuan
Abstract Disease phenotypes are typically a result of interactions between both genetic variation and environmental conditions. More than 25,000 genes that make up the human genetic blueprint have been decoded. This extensive gene database along with the advent of DNA microarray technology and bioinformatics enable researchers to generate gene expression profiles in any given cell or tissue of interest. It is therefore possible to identify altered gene expression that occurs in a particular disease condition. These molecular breakthroughs open up new avenues for accurate diagnoses and genetic counseling, as well as opportunities for gene or protein therapy. Manipulating the expression of specific genes into their respective final protein products is an important tool for understanding the function of a specific gene that may have potential as a gene therapy. Traditional gene manipulation has focused on introducing a target gene into the cells and tissues through recombinant DNA and gene transfer techniques to express the gene of interest. To downregulate or inhibit the expression of a gene, antisense DNA and RNA interference (RNAi) fragments, which block RNA processing or translation of specific messenger RNAs (mRNAs), are methods that provide powerful and specific tools for studying gene regulation. This chapter discusses some of the most widely used molecular biology tools for downregulating gene expression: antisense-oligonucleotidemediated gene silencing and RNAi-mediated gene silencing. Keywords Gene expression profile • Gene manipulation • Gene silencing • Small interference RNA • Molecular biology • Methodology
1 Introduction Disease phenotypes are typically a result of interactions between both genetic variation and environmental conditions [1]. The completion of the Human Genome Project in 2003 is a historic achievement and a fundamental milestone in life science research. More than 25,000 genes that make up the human genetic blueprint have been decoded. This extensive gene database along with the advent of DNA microarray technology and bioinformatics enable researchers to generate gene expression profiles in any given cell or tissue of interest. It is therefore possible to identify altered gene expression that occurs in a particular disease condition. These molecular breakthroughs open up new avenues for accurate diagnoses and genetic counseling, as well as opportunities for gene or protein therapy [2]. Manipulating the expression of specific genes into their respective final protein products is an important tool for understanding the function of a specific gene that may have potential as a gene therapy. Traditional gene manipulation has focused on introducing a target gene into the cells and tissues through recombinant DNA and gene transfer techniques to express the gene of interest. To downregulate or inhibit the expression of a gene, antisense DNA and RNA interference (RNAi) fragments, which block RNA processing or translation of specific messenger RNAs (mRNAs), are methods that provide powerful and specific tools for studying gene regulation. In this chapter, we will discuss some of the most widely used molecular biology tools for downregulating gene expression: (1) antisense-oligonucleotide-mediated gene silencing; and (2) RNAi-mediated gene silencing.
2 Antisense-Olignucleotide-Mediated Gene Silencing 2.1 Concept and Mechanism Y. Yu (*) Department of Medicine, University of California, San Diego, 9500 Gilman Drive, MC 0725, La Jolla, CA 92093, USA email:
[email protected] The concept underlying antisense-oligonucleotide-mediated gene silencing is relatively straightforward. Based on
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Watson–Crick base-pairing interactions, an exogenous DNA sequence is employed which specifically hybridizes to the complementary RNA of the target gene and sterically inhibits the mRNA translation. As the transcribed RNA molecule is the sense strand of a given gene, any DNA sequence that hybridizes to that RNA molecule must be on the antisense strand. Therefore, the antisense oligonucleotide would, via the formation of an mRNA–DNA duplex, specifically prevent the translation of that mRNA into protein [3]. There are two major mechanisms by which antisense oligonucleotides may inhibit gene translation: (1) Direct antisense oligonucleotide DNA–RNA hybridization. In this case, the DNA–RNA duplex exerts a direct steric effect and physically blocks the RNA splicing and translational machinery. These types of antisense oligonucleotides are efficient only when targeted to the 5¢ or AUG initiation codon region. (2) Antisense targeted RNase H cleavage. RNase H is a ubiquitous enzyme required for DNA replication [4]. The RNase H enzyme recognizes the DNA–RNA hybrid and cleaves the RNA strand, resulting in fragmentation of the original full-length mRNA into two fragments, one lacking a 5¢-cap structure and one lacking a 3¢-poly(A) tail. Such fragments are usually rapidly degraded by exonuclease activity, and are thus no longer available for subsequent translation and protein synthesis [5]. Compared with antisense oligonucleotides designed as a steric blocker, RNase H-dependent oligonucleotides can inhibit protein expression when targeted to virtually any coding region of the mRNA [6].
2.2 Design and Synthesis Antisense oligonucleotides are usually designed to contain ten to 30 nucleotides complementary to a target mRNA by standard Watson–Crick base-pairing rules. However, if the target site in the mRNA is picked randomly, only a few complementary oligonucleotides can effectively inhibit targeted mRNA expression. The criteria for selection of antisense oligonucleotides are as followed: (1) specific target recognition by Watson–Crick base pairing; (2) good structural mimicry to the natural DNA–RNA; and (3) activation of RNase H to promote the target mRNA cleavage. To date, computational approaches have been developed to predict biological active antisense oligonucleotides for a precise target sequence. A computational approach significantly increases the probability for selecting an efficacious antisense oligonucleotide.
2.3 Limitations and Prospects in Therapeutic Application The study of the use of short antisense oligonucleotides to block translation of specific mRNAs was first reported by
Y. Yu and J.X.-J. Yuan
Zamecnik and Stephenson in 1978. It was demonstrated that antisense oligonucleotides complementary to the 3¢- and 5¢-terminal repeat sequences of Rous sarcoma virus inhibit viral replication and the expression of viral proteins in chicken embryo fibroblasts [7, 8]. Since then, antisense oligonucleotides have continued to be developed to selectively inhibit expression of a wide variety of genes at the translational level, and potentially treat a broad range of diseases, including human types of cancer, diabetes, asthma, and pulmonary vascular diseases. In 1998, the first antisense drug, Vitravene, an inhibitor of cytomegalovirus replication, was approved by the Food and Drug Administration for the treatment of retinitis in AIDS patients [9]. However, to date, most antisense therapies have not produced significant clinical results [10, 11]. A distinct disadvantage of the antisense approach is the instability of the single-stranded antisense oligonucleotides. Several modifications are being used to increase nuclease resistance (or antisense stability) as well as improving membrane permeability and binding affinity. One of the most frequently used modifications is the incorporation of a phosphorothioate group [12]. A phosphorothioate modification of antisense oligonucleotides is accomplished by replacing the nonbridging oxygen on the phosphate linkage with a sulfur atom. This type of modification significantly increases the stability of antisense oligonucleotides as a result of enhanced resistance to enzymatic hydrolysis. However, some phosphorothioate-modified antisense oligonucleotides may also fail to activate RNase H, which is considered to be an important step in the antisense mechanism of action [13]. Overall, the optimal use of antisense oligonucleotides for inhibiting target gene expression requires the resolution of problems relating to effective design, enhanced biological activity, and efficient target delivery. These issues are currently being actively addressed and will hopefully continue to shed light on ways to increase therapeutic efficacy and specificity [3].
3 RNAi-Mediated Gene Silencing 3.1 Concept and Mechanism RNAi represents an innovative new strategy for using small RNA molecules to manipulate target gene expression [14]. The concept for the RNAi-dependent gene regulation process was originally derived from the discovery of doublestranded RNA (dsRNA)-induced gene silencing in Caenorhabditis elegans [15]. Studies of the dsRNA-mediated gene silencing mechanism demonstrated it was initiated by an ATP-dependent, progressive cleavage of the long dsRNA template into short double-stranded fragments of 21–23 base pairs by the RNase Dicer [16–18]. These short
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double-stranded fragments are called small interfering RNA (siRNA) and are incorporated into a protein complex called RNA-induced silencing complex (RISC). ATP-dependent unwinding of the siRNA duplex generates an active RISC with single antisense strands. Once unwound, the singlestranded antisense guides RISC to the target mRNA that has a complementary sequence. Argonaute, the catalytic component of the RISC, endonucleolytically cleaves the target mRNA in the cytoplasm of cells and, thereby, prevents it from being used as a translation template [19–21] (Fig.1). Further studies have shown that RNAi is not only an important regulator of gene expression in many eukaryotic cell types, but also plays important roles in defending cells against parasitic genes, viruses, and transposons [22].
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RNAi was not initially considered a useful experimental or therapeutic tool in mammalian cells owing to highly toxic effects of long dsRNA and dsRNA-triggered interferon responses [23]. To eliminate nonspecific side effects of long dsRNA and exclude long dsRNA from the RNAi process, Elbashir et al. initiated the use of siRNAs that were 21-mer duplexes with 5¢ phosphates and two-base 3¢ overhangs on each strand. These shorter siRNAs were demonstrated to selectively and efficiently degrade targeted mRNAs upon introduction into several mammalian cell lines [24]. Recently, siRNA has been shown not only to be a valuable experimental tool in a variety of biological systems, but is also a potential therapeutic method for several diseases [25].
3.2 siRNA Design and Synthesis 3.2.1 siRNA Design
Fig. 1 RNA interference (RNAi) pathway. Long double-stranded RNA (dsRNA) molecules, which share sequence-specific homology with a target messenger RNA (mRNA), are cleaved as 21–23-nucleotide small interfering RNA duplexes (siRNAs) by an RNase III like enzyme known as Dicer. The siRNAs are recruited into a multiprotein siRNA–protein complex called RNA-induced silencing complex (RISC). Once activated by ATP, an activated RISC is generated with a single antisense strand of siRNA, which serves as a probe to target mRNA that has a complementary sequence. When a complementary sequence is found, silencing is mediated via endonucleolytic cleavage
Exogenous siRNA typically consists of a 19–23 base-paired duplex with two-nucleotide 3¢ overhangs. In principle, any siRNA containing the appropriate antisense sequence can be directed against the target mRNA that has a complementary sequence. However, only a few randomly selected siRNAs show effective silencing activity and lead to detectable altered phenotypes in functional studies [26, 27]. Specific criteria for designing the most efficient siRNA have been established [24, 28–31]. Ideally, the target mRNA sequence should begin with an AA dinucleotide as AA(N19)TT. N19 represent any 19 nucleotides of the target mRNA. The 3¢-UU overhangs, or their DNA counterparts, 3¢-TT, are often included to stabilize the siRNAs against RNases and are generally preferred over other sequences [24]. Additional important criteria for selecting the target gene sequence are a low G/C content, lack of inverted repeats, low internal stability at the sense strand 3¢ terminus, and sense strand base preferences (positions 3, 10, 13, and 19) [31]. Ui-Tei et al. further developed the siRNA selection rules to include (1) A/U at the 5¢ end of the antisense strand; (2) G/C at the 5¢ end of the sense strand; (3) at least five A/U residues in the 5¢ terminal one third of the antisense strand; and (4) the absence of any GC stretch of more than nine nucleotides in length [32]. Current criteria for siRNA design have also been embedded in the development of computer algorithms to predict effective siRNA sequences. Several siRNA design tools and search engines are available on the Internet to help select the highly efficient siRNAs for silencing experiments [27]. An investigator can simply input the target mRNA sequence in any of the siRNA design Web sites and generate lists of candidate siRNAs. As a practical strategy for obtaining the most effective siRNA, several siRNAs need to be designed according to the empirical rules and tested in cellular systems.
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3.2.2 siRNA Synthesis Several methods are used for preparing or synthesizing siRNAs. These methods include chemical synthesis, in vitro transcription, generation of a mixture of siRNAs by using long dsRNA and recombinant RNase III or Dicer [33], endogenous expression as short hairpin RNAs (shRNAs) from expression plasmids or viral vectors, and endogenous expression from PCR-derived siRNA expression cassettes. Moreover, functional validated siRNA or shRNA libraries are also commercially available. Although chemically synthesized siRNAs provide a promising tool for studying siRNA functions, this approach is limited by its high cost. To reduce the cost of siRNA selection, many investigators generate siRNAs using a less expensive preparation method such as in vitro transcription [34, 35]. siRNA Synthesis via In Vitro Transcription. The key to this method is to use RNA polymerase (e.g. T7) to generate individual strands of the siRNA. Templates for the transcription reactions are the sense and antisense strands of DNA oligonucleotides encoding the desired siRNA strands followed by a T7 promoter region, respectively. The RNA polymerase binds the T7 promoter and initiates RNA synthesis. After transcription, the reactions are combined to anneal the two siRNA strands. Then, the siRNA preparation is then treated with DNase (to remove the DNA template), RNase (to polish the ends of the dsRNA), and purified. In vitro transcription methods provide a rapid, convenient, and highly effective way to produce sequence-specific siRNAs. Furthermore, several commercially available kits, such as the silencer® siRNA construct kit (Ambion) and the BLOCKITTM RNAi TOPO transcription kit (Invitrogen), are available to aid investigators in the production of sequence-specific siRNAs. DNA-Based Vectors for Expression of shRNAs. Although the in vitro transcribed siRNA method provides investigators with a simple method to screen and identify effective siRNAs, two concerns have been raised regarding its further application: (1) a single transient transfection of siRNA into cells may not provide a sufficient window for functional gene inhibition to study proteins with a long half-life; (2) the transcribed siRNAs in functional genomics studies are easily affected by the variable transfection efficiencies in difficult-to-transfect cell lines [36, 37]. These issues have been addressed by the development of DNA-based plasmid vectors or viral vectors that express siRNA oligonucleotides [38–40]. This method is based on siRNAs that can be transcribed under the control of RNA polymerase III (pol III) promoters. Presently, U6 and H1 pol III promoters have been preferentially exploited for producing siRNAs [41]. Two types of pol III promoter based siRNA expression system are widely used. In the shRNA expression system, the vector contains a single pol III promoter followed by a segment of DNA
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that encodes a short hairpin structure with a stem of 19–29 base pairs and a short loop of four to ten nucleotides. The transcribed shRNA is then processed into siRNAs by Dicer within the transfected cell. In the tandem siRNA expression system, the vector contains dual pol III promoters arranged in a convergent manner that drive the expression of two short complementary RNA strands from a single 19-base-pair target region. Then two RNA strands can anneal and form siRNA duplexes with overhangs of four U residues at each 3¢ end. Since the target sequences that work well as synthesized siRNAs also work when incorporated as stem–loop hairpins into vectors [42], investigators usually construct the siRNA expression vectors on the basis of their target sequence selection from the inhibitory effects of the in vitro transcribed siRNA candidates. Vector-based siRNA expression systems not only allow for the creation of stable cell lines depleted in a specific target gene [43], but also permit coexpression of reporter genes such as green fluorescence protein and luciferase, which facilitates tracking and/or selection/enrichment of transfected/transduced cells. Viral vectors have been used extensively in gene therapy to deliver DNA to target cells. Viruses evolved to specialize in gene transduction can also be used to ferry siRNA into cells. The use of viral vectors, such as lentiviruses [44, 45] and adenovirus [46, 47], allows the expression of siRNAs in difficultto-transfect cell lines and primary cell cultures [48].
3.3 siRNA Delivery The major bottleneck of siRNA application, as for most antisense or nucleic acid based strategies, is their poor cellular uptake associated with negatively charged siRNA and the hydrophobic nature of the cellular membrane [49, 50]. How to realize sufficient siRNA delivery is a major challenge for its application [51]. siRNA delivery systems are required to facilitate siRNA diffusion across the cell membrane, escape from endosome and/or lysosome degradation or errant siRNA compartmentalization, and target siRNA bioactivity to the appropriate intracellular site of action [51]. Several delivery systems have been developed, including: (1) Cationic liposomal delivery system: Commercially available cationic liposomes such as Lipofectamine (Invitrogen), RNAifect (Qiagen), and DharmaFECT (Dharmacon) are more commonly used for delivery of synthesized siRNAs and siRNA expression plasmid vectors into cultured cells. However, the large size and the toxicity of cationic lipid particles significantly affect their utilization in vivo [52]. (2) Viral delivery systems: There is no doubt that viral vector-based shRNAs are highly efficient in both in vitro and in vivo studies. However, the use of viruses in vivo has significant safety
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concerns and limitations, including toxic immune responses and inadvertent gene expression changes following random integration into the host genome [53, 54]. (3) Cationic cell-penetrating peptides (CPPs) delivery system: CPPs are a class of small cationic peptides derived from protein transduction domains and membrane transduction sequences. These molecules are composed of about ten to 30 amino acids and have the ability to engage the anionic cell surface through electrostatic interactions and rapidly induce their own cellular internalization through various forms of endocytosis [55–57]. The most well characterized CPPs include TAT peptide (the HIV-1 TAT protein), penetratin, transportan, polyarginine, and MPG (a short amphi pathic peptide) [58–62]. These cationic peptides are powerful carriers for cellular uptake for a variety of macromolecules, including proteins, peptides, and oligonucleotides, making them attractive candidates for siRNA delivery in vitro and in vivo [57]. For the CPP-based siRNA delivery, however, most of the siRNA internalized to the cell is entrapped in endosomes [56]. Strategies for endosomal escape of siRNAs must therefore be included when designing and synthesizing CPPs. It has been reported that histidine polymers are able to absorb protons in the acidic environment of the endosome, leading to osmotic swelling and membrane disruption, which promote the escape of plasmid DNA from the endosomes [63]. We propose that the CPP fused to five histidine residues and one cysteine residue at both the C-terminus and the N-terminus (C-5H-CPP-5H-C) is an effective peptide model for siRNA delivery. Two cysteine residues at the C- and N-terminal ends of the delivery peptide are used to enhance stability of peptide–siRNA complexes through the formation of interpeptide disulfide bonds. Selection of the most effective CPP is another challenge for CPP-based siRNA delivery. High transduction efficacy and low cellular cytotoxicity are major characters for effective CPPs. Some novel CPPs have been generated as chimeras of natural CPPs, such as YM2 peptide “PVRRPRRRRRR” and YM3 peptide “THRLPRRRRRR” with improved cell penetrating activity over wild-type TAT peptide “YGRKKRRQRRRR” [64]. In addition to further improvement in the penetration ability of CPPs, the most challenging task will be searching for effective CPPs for tissue- or cell-specific delivery. (4) Nanoparticle delivery systems: The cationic polymer such as polyethyleneimine have been used to successfully deliver siRNA to target cells. Safety evaluation of this potential approach is essential for its in vivo application [65]. (5) Cholesterol-conjugated siRNA delivery systems: Cholesterol-conjugated siRNA was successfully applied for tissue-specific siRNA delivery based on conjugation of lipophilic groups that can enhance cell uptake and enhance the pharmacokinetics and tissue biodistribution of oligonucleotides [66]. This approach may have high clinical significance, particularly as it relates to the development of new siRNA therapeutic
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strategies. (6) Specific siRNA delivery systems: Antibodymediated siRNA delivery system [67] and cell-type-specific delivery system [68, 69]. It is believed that siRNA delivery strategies will soon be further improved and developed for any given clinical condition [51].
3.4 Limitations and Prospects in Therapeutic Application siRNA has been widely adopted by investigators as a standard tool to investigate gene function in molecular and cell biology studies. siRNA approaches also have many very promising potential therapeutic applications to combat disorders with identified molecular targets [70]. However, a number of problems and hurdles remain for siRNA-based therapeutics, such as the instability of siRNA, efficient siRNA delivery, and siRNA-mediated unspecific immune response. As naked siRNAs are easily degraded in human plasma with a half-life of minutes [71], the instability of siRNA was an important obstacle for its application in humans. Fortunately, recent chemical modifications of siRNA have made significant progress to overcome this issue of instability [72, 73]. Currently, the biggest hurdle in developing RNAibased therapies is the in vivo barriers issue. This includes (1) rapid clearance by the kidneys, (2) degradation by serum and tissue nuclease, (3) uptake by phagocytes of the reticuloendothelial system leading to sequestration in liver and spleen, (4) failure to cross the capillary endothelium, (5) slow diffusion/ binding in extracellular matrix, (6) inefficient endocytosis by tissue cells, and (7) inefficient release from endosomes [50]. As we described above, following rapid advancement in understanding the details of the RNAi biological pathway, novel siRNA delivery systems are being developed and traditional delivery systems will continue to improve. It is expected that more exciting discoveries and applications of siRNAmediated therapy will emerge soon. Human clinical trials of RNAi-based drugs by several pharmaceutical companies are currently under way. Intravitreal administration of siRNA targeting vascular endothelial growth factor (VEGF) for treating age-related macular degeneration (Sirna Therapeutics) and intranasal administration of siRNA for treating respiratory syncytial virus infection (Alnylam Pharmaceutics) are just two examples [74]. As siRNA biology is complex, it is essential to validate the mechanisms underlying the observed biological effects before attributing them to RNAi-mediated therapeutic effects. siRNAs have been shown to activate immune response in a sequence- and concentration-dependent manner, leading to nonspecific gene silencing [75, 76]. An example of this type of concern comes from a recent study of siRNAs targeting VEGF or its receptor to inhibit retinal angiogenesis in an animal model of macular degeneration [77].
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A comprehensive analysis revealed that the observed reduction of angiogenesis by “naked” VEGF siRNA was not due to RNAi-mediated inhibition of VEGF expression, but rather was caused by sequence-independent stimulation of Toll-like receptor 3 and downstream interferon-g and IL-12 mediated suppression of neovascularization [77]. Thus, the possible interplay of siRNA with various members of the Toll-like receptor family of cell-surface proteins must be considered in the design of experiments [50]. Although it remains an open question concerning the safety, tolerability, tissue biodistribution, and biological effectiveness of RNAi therapy, it is almost certain that RNAi-based therapy will have a profound effect in the coming decades.
4 The Application of siRNA to Elucidate the Role of the Canonical Transient Receptor Potential 6 Channel in Idiopathic Pulmonary Arterial Hypertension 4.1 Background Idiopathic pulmonary arterial hypertension (IPAH) is a progressive and ultimately fatal lung disorder characterized by increased resting pulmonary arterial pressure [78–80]. Sustained contraction and excessive proliferation of pulmonary arterial smooth muscle cells (PASMCs), mediated by persistent Ca2+ entry through Ca2+ channels, may play critical roles in the development of IPAH [81]. Recently, we demonstrated that canonical transient receptor potential 6 (TRPC6), an essential component of the receptor-operated Ca2+ channels, is significantly upregulated in PASMCs isolated from IPAH patients (IPAH-PASMCs) [82]. As upregulation of TRPC6 channel expression is an important initial step in the elevation of cytosolic free Ca2+ concentration required for mitogen-mediated PASMC proliferation [83], we hypothesized that upregulation of canonical TRPC6 channels in IPAH-PASMCs may serve as a critical Ca2+ entry pathway, raise intracellular Ca2+ concentration, and stimulate PASMC proliferation. To evaluate the potential therapeutic effects of TRPC6 inhibition on the abnormal PASMC proliferation in IPAH patients, we generated siRNA to specifically target TRPC6.
4.2 Synthesis and Selection of Efficient TRPC6 siRNA Using In Vitro Transcription Four sets of sense and antisense oligonucleotide primers were designed using Ambion’s siRNA Target Finder
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(http://www.ambion.com/techlib/misc/siRNA_finder.html). By using the silencer siRNA construction kit (Ambion) with these four sets of primers, we synthesized four siRNAs which targeted the following 21 nucleotides of the human TRPC6 mRNA sequence (GenBank accession no. NM_004621). The target sequences corresponding to siRNAs were as follows: siRNA1 (AAGTGCAATGACT-GCAACCAG); siRNA2 (AAGGTCTTTATGCAATTGCTG); siRNA3 (AAGTTCCTTGTGGTCCTTGCT) and siRNA4 (AAGCC TTCACAACAGTTGAAG). A scrambled 21-nucleotide control oligonucleotide (AAGCGCGCTTTG-TAGGATTCG) was also generated which bore no significant homology to any mammalian gene sequence. To identify the most effective siRNA for the inhibition of TRPC6 gene expression, COS-7 (African green monkey SV40-transfected kidney fibroblast cell line) cells were cotransfected with 2 mg of TRPC6-expressing plasmid and 100 nM siRNA or 100 nM scrambled control using Lipofectamine (Invitrogen). Protein levels were evaluated by western blot analysis after 72 h. As shown in Fig. 2a and b, two siRNA candidates (siRNA1 and siRNA2) suppressed TRPC6 protein expression levels, with siRNA2 being more effective [82].
4.3 Generation of Recombinant Adenovirus Expressing TRPC6 siRNA On the basis of the previously presented selection data, we firstly constructed a DNA-based vector (pSilencer 2.0-U6-TRPC6) that expressed TRPC6 siRNA targeting AAGGTCTTT ATGCAATTGCTG (nucleotides 2212–2232 of TRPC6 mRNA, NM_004621). Efficient delivery of siRNA or siRNA expression vector into cells is a critical step for RNAi-based gene silencing experiments. As IPAH-PASMCs are primary cultured cells, traditional transfection reagents may be inefficient for siRNA delivery. Recombinant adenovirus carrying siRNA targeting human TRPC6 was generated using an Invitrogen ViraPower adenoviral expression kit. The siRNA expression DNA cassettes, including RNA pol III promoter U6, a hairpin siRNA target sequence and terminator, were subcloned into pENTR1A entry vector. Scrambled control vector was also constructed as a negative control after efficient recombination of the entry vector into the promoterless pAd/PL-DEST Gateway® vectors, followed by viral production and transduction. The adenoviral particles for TRPC6 siRNA (Ad-siRNA-TRPC6) and the scramble control siRNA (Ad-siRNA-scramble) were plaque-purified and amplified in 293A cells. The titer of the adenoviral stock was measured by a spectrophotometer. Reverse transcription PCR and western blot data showed that adenoviral TRPC6 siRNA effectively knocked down TRPC6 expression in IPAHPASMCs [82] (Fig. 2c, d).
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Fig. 2 Downregulation of canonical transient receptor potential 6 (TRPC6) with siRNA inhibits 1-oleoyl-2-acetyl-sn-glycerol (OAG)induced Ca2+ influx and cell proliferation in pulmonary arterial smooth muscle cells isolated from idiopathic pulmonary arterial hypertension patients (IPAH-PASMCs). (a) COS-7 cells were cotransfected with pCMS-EGFP-TRPC6 (human TRPC6 expression vector) and one of four TRPC6 siRNA candidates (each siRNA at 100 nM) or scrambled siRNA. Western blot analysis demonstrates the effect of siRNA candidates and scrambled siRNA on TRPC6 protein level in COS-7 cells. The control lane indicates cells transfected with an empty vector. b-Actin controls are shown below. (b) A bar graph showing the normalized (to b-actin) TRPC6 protein expression in the presence of the different siRNA candidates. Among them, siRNA2 significantly decreased TRPC6 protein expression. (c, d) On the basis of the selection data, recombinant adenovirus expressing siRNA2 targeting human TRPC6 (TRPC6 siRNA) was generated. As a control, recombinant adenovirus expressing siRNA targeting human glyceraldehyde
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3-phosphate (GAPDH; GAPDH siRNA) or without targeting (scrambled siRNA) were used. mRNA (c) and protein (d) expression levels of TRPC6, canonical transient receptor potential 4, and GADPH are shown in IPAH-PASMCs treated with scrambled siRNA, GADPH siRNA, or TRPC6 siRNA. (e) Representative records of cytosolic free Ca2+ concentration changes in response to OAG in IPAH-PASMCs treated with scrambled siRNA or TRPC6 siRNA (upper panel). Summarized data of the resting cytosolic free Ca2+ concentration (left) and OAG-induced cytosolic free Ca2+ concentration increases (right) in IPAH-PASMCs treated with scrambled siRNA or TRPC6 siRNA. **P 2,000, but local conditions are very important. If a constant ratio of blood density to viscosity is assumed, turbulence is encouraged by increasing the velocity of flow or the diameter of the vessel. Regions of the arterial tree with arches, extensive branching, or partial obstruction are more likely to experience disturbed flow.
2.1.2 Shear Values in the Vessels of the Pulmonary and Systemic Vasculature The vasculature, both pulmonary and systemic, consists of large conduit vessels (macrocirculation) that provide convective transport over large distances and smaller vessels (microcirculation) that permit diffusive exchange between blood and tissue. Shear stress in these vessels depends on the flow rate and vessel diameter. Vascular shear stress of large conduit arteries typically varies between 5 and 20 dyn/cm2; however, significant instantaneous values range from near zero up to 40 dyn/cm2 during states of increased cardiac output. Table 1 shows typical wall shear stress values for vessels of various size in the human
54 Shear Stress, Cell Signaling, and Pulmonary Vascular Remodeling Table 1 Shear stress in selected human blood vessels (data from [3, 4]) Vessel Shear stressa(dyn/cm22) Aorta Large arteriole Small arteriole Capillary Small vein Large vein Pulmonary artery a Approximate normal values
5 8 10 24 11 5 2
system circulatory system [3, 4]. Because the lung has a highly compliant, high-capacity vascular bed, the particle flow is relatively slow (although bulk flow is equal to the flow of the systemic circulation) and the wall shear stress is correspondingly lower compared with that in systemic vessels.
3 Physiological Response to Shear It is now well established that hemodynamic forces such as shear play an important role in physiological responses of endothelial cells. Shear resulting from blood flow leads to immediate responses in the form of altered ion channel activity and activation of signaling molecules and subsequent later responses manifested by changes in structure, metabolism, and gene expression [1, 5–8]. Overall these changes occur as a result of complex interactions between the mechanical component of shear and the cytoskeletal and biochemical components of the cell. In vivo studies have demonstrated that arteries adapt to chronic changes in blood flow by altering their structure to accommodate the new condition [1, 8]. However, in vivo studies are not suitable for accurately monitoring changes in cellular phenotype or gene expression. Thus, the use of endothelial cells in culture or isolated intact vessels in vitro has filled the gap. These models can be subjected to controlled shear stresses, and monitored for endothelial responses and properties, including release of mediators, gene expression, and protein production [6, 7, 9, 10]. Most of the published studies utilized cells cultured under static conditions and subjected to the onset of shear. This relatively unphysiologic preparation can give information regarding the cellular machinery that is involved in the response to shear but might not accurately reflect the changes that occur with altered shear in vivo. A more physiologic preparation is isolated cells that have been flow adapted in vitro and then subjected to altered shear. This section will present three aspects of mechanotransduction. Firstly, candidate flow sensors will be discussed from the perspective of molecules at the luminal cell surface that by virtue of being in direct contact with the flowing blood are activated directly by physical changes (conformational
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change) or indirectly by ligand–receptor interactions. Secondly, the physiological changes with immediate and long-term shear will be presented with emphasis on ion channel activation and the endothelial gene expression involved in adaptation to shear. The third aspect will be the signaling pathway associated with change of shear.
3.1 Flow Sensors and Mechanosensing Although the identity of the endothelial cell component that is responsible for sensing shear has not been precisely identified, a number of candidate cell membrane shear sensors have been suggested for endothelial cells. A cell membrane protein would be the ideal candidate, but an internal sensor based on a change in the internal stresses also is possible. This latter condition has been termed “cellular tensegrity” [11]. Tensegrity as defined by Buckminster Fuller [12] is the mechanism for stabilization of shape of a structure by continuous tension or “tensional integrity.” The cellular tensegrity model proposes that the whole cell is a prestressed structure where tensional forces are borne by cytoskeletal microfilaments and intermediate filaments, and these forces are balanced by interconnected structural elements (extracellular matrix adhesion and microtubules) that resist compression. The tensional prestress (pre-existing tensile stress) that stabilizes the whole cell is generated by the contractile machinery of the cell (actomyosin apparatus). Changes in shear can then be sensed by changes in the internal cell “tension.” The alternative sensing mechanism is that the change in the local environment of a cell membrane protein results in a “biochemical” response.
3.1.1 Ion Channels A change in membrane potential has been demonstrated as one of the earliest responses to a change in flow. Patch clamp studies have shown that an initial K+ current is activated with onset of flow and is followed by a Cl- current. This presumably reflects a change in activity of endothelial plasma membrane ion channels. Two types of ion channels have been reported to be flow-sensitive: (1) an inwardly rectifying K+ channel that with activation hyperpolarizes the cell membrane, whereas channel inactivation results in membrane depolarization [13], and (2) an outwardly rectifying Cl- channel that when activated depolarizes the endothelial membrane [14, 15]. These channels are activated independently of each other and exhibit different sensitivities to applied shear stress [16]. Inwardly rectified K+ channels (KIR) represent a relatively large family of proteins that are divided into six types. The KATP channel is included in this family and is classified as KIR type 6. KATP channels (specifically KIR6.2) are expressed at low levels in
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isolated, cultured endothelial cells, but are induced during long-term (48–72-h) exposure to shear [17]. Induction of these channels during flow adaptation indicates that some other cellular protein serves as a flow sensor to mediate KATP channel gene expression. The KATP channels appear to be maintained in an active state by flow and are inactivated (decreased open probability) by loss of shear [18]. Another inwardly rectified K+ channel, KIR2.1, has also been reported to be flow-sensitive but its presence and physiological role in endothelial cells have not been confirmed [19]. Other ion channels that may be involved in the response to flow include members of the transient receptor potential (TRP) superfamily. TRP channels in the endothelium belong to the subfamily TRPC, including TRPC1 and TRPC6, which display physiological responses in a pure bilayer [20, 21]. The TRPC channels are important in regulating Ca2+ entry into cells. At least one of these channels, TRPC1, is located in caveolae, thought to be a site of mechanosensing (see later). TRPC4 has been reported to be activated by cell swelling, which represents in part a mechanical stimulus [22, 23].
3.1.2 Integrins Integrins are transmembrane receptors that link cytoskeletal proteins with the proteins in the extracellular matrix so as to facilitate cell–matrix communication. Integrins connect to the cytoskeleton through focal adhesions that contain multiple actin-associated proteins, including talin, vinculin, paxillin, and zylin [24]. The cytoskeleton responds mechanically to forces transferred via the extracellular matrix and channeled through integrins by rearranging its interlinked actin microfilaments, microtubules, and intermediate filaments. Focal adhesions undergo constant remodeling on the abluminal side of endothelial cells and integrins are suggested to exert their influence through the activation of focal adhesion kinase. Integrins have two subunits, a and b. One of the major integrins in vascular endothelium is avb3, which interacts with the matrix protein fibronectin. Shear-induced signaling has been shown to decrease in the presence of anti-avb3 antibodies [25]. 3.1.3 Caveolae Caveolae are lipid-rich invaginations on the cell surface that serve as a platform for clustering of many cell-membraneassociated proteins. They participate in endocytosis and also are reported to serve as mechanotransduction sites within the plasma membrane. A role for caveolae in flow sensing in the pulmonary endothelium has been proposed [26–29]. Chronic exposure to shear stress altered caveolin expression and distribution, increased the density of caveolae, and led to enhanced mechanosensitivity to subsequent changes in hemodynamic forces by endothelial cells in culture [26]. Depletion of caveolae prevented the endothelial
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response to stop of flow [28, 29]. Caveolae associate with b1 integrins during flow and have been reported to be functionally linked in their flow-sensing properties [26, 30]. Cells with disrupted caveolar structural domains show attenuated integrin-dependent caveolin-1 phosphorylation and activation of Src (a tyrosine kinase) in response to alterations in shear.
3.1.4 Cytoskeleton The cytoskeleton and other structural components can transmit shear-induced deformation of the cell surface throughout the cell via changes in cytoskeletal tension. The proteins involved include focal adhesion sites, integrins, cellular junctions, and extracellular matrix [5]. The cytoskeleton is composed of three major types of protein filaments: microtubules, microfilaments, and intermediate filaments. These form a cytoskeletal network that provides a scaffold for organizing several signaling molecules whose activation appears to be mediated through the cytoskeleton. The tensional forces that are generated within contractile microfilaments and transmitted throughout the cell are balanced by internal microtubules and by adhesions to extracellular matrix and to other cells [5]. This allows cells to shift compressive forces between microtubules and extracellular matrix adhesions. Thus, extracellular matrix bears most of the shear stress in cells spread on adhesive substrates, whereas microtubules bear the mechanical forces in cells with fewer anchoring points.
3.1.5 Mechanosensory Complexes In addition to individual mechanosensors, a multicomponent complex has been reported to mediate shear response in endothelium. This complex is composed of the platelet endothelial cell adhesion molecule (PECAM), vascular endothelial growth factor receptor 2, and vascular endothelial cadherin [31]. Together these receptors confer shear responsiveness to endothelial cells. This is further supported by observations in vivo where PECAM null lungs do not show the endothelial response to disturbed flow, i.e., activation of nuclear factor kB (NF-kB) and inflammatory genes [31].
3.2 Immediate Response to Shear Responses to either acute or chronic alterations in blood flow in the vasculature are endothelium-dependent [32]. The earliest change that is detected in endothelial cells in culture upon onset or cessation of flow, respectively, is activation [13] or deactivation of ion channels [18]. This is followed by
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other signaling events, such as increases in cytosolic Ca2+ concentration, activation of intracellular kinases, and generation of nitric oxide (NO) and reactive oxygen species (ROS). Cells that are initially cultured under static conditions and then exposed to flow undergo adaptive changes that result in a flow-adapted state. The vascular endothelium in vivo is assumed to be flow-adapted and the demonstration of physiological responses to flow cessation in vitro requires prior adaptation of cells to flow.
3.3 Adaptation to Flow and Development of a Stable Phenotype Endothelial cells in conducting vessels of the arterial vascular tree exhibit an ellipsoidal shape and are aligned with the direction of blood flow. On the other hand, the shape of individual cells near vessel branch points fails to show any orientation, consistent with local temporal and spatial fluctuations in flow [8, 33]. Exposure of endothelial cells in vitro to unidirectional laminar shear induces a time- and forcedependent change in cell shape and alignment that appears to reflect the in vivo condition. The changes are reversible upon cessation of flow [8]. These changes in cell shape and alignment are seen with endothelial cell cultures that have been derived from large conducting vessels, but to date have not been reported with endothelial cells derived from the microcirculation. The shape changes in endothelial cells require reorganization of the cytoskeleton [1]. Phenotypic changes may extend to the level of the individual endothelial cells that compose the continuous vascular lining. Prolonged exposure of endothelial cells to flow in vitro also causes changes in gene expression. These changes represent the response of shear-sensitive elements that trigger a biochemical cascade. This cascade activates protein kinases that in turn lead to activation of cytosolic transcription factors and regulation of gene transcription in the nucleus. Persistent changes in blood flow, such as those resulting from diseaserelated or experimentally induced vascular narrowing or shunts, also can cause structural remodeling involving cell proliferation and apoptosis and hypertrophy of extracellular matrix. These longer-term structural modifications are discussed in section 5.
3.3.1 Shear-Dependent Endothelial Cell Gene Regulation Flow-dependent regulation of endothelial cell gene expression has been demonstrated in vitro by measuring messenger RNA and protein levels as a function of time after the application of continuous laminar shear stress [7, 34, 35]. With
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use of gene array methods, shear stress was found to significantly affect the expression of about 3,000 endothelial cell genes, of which 232 genes were affected by the frequency of shear and about 3,000 genes were affected by shear magnitude. Of the 232 genes sensitive to differences in frequency, 185 were also sensitive to mean shear [36]. Several patterns of response of genes to shear have been described: 1. Rapid induction under shear followed by rapid return to the baseline [37, 38]. An example of this pattern is shown by c-fos, a proto-oncogene that encodes a DNA binding protein of the activator protein (AP-1) family. c-fos has low transcript levels in cells cultured under static conditions, but rapid induction to a high level of expression (20-fold over the basal level) in response to shear followed by a rapid return to the baseline [37, 38]. 2. Initially high transcript levels under basal conditions that increase further upon exposure to shear. Examples include platelet-derived growth factor (PDGF)-B chain, endothelial nitric oxide synthase (eNOS), c-jun, PDGF-A, monocyte chemotactic protein-1 (MCP-1), transforming growth factor- b (TGF-b), basic fibroblast growth factor, tissue plasminogen activator and intercellular adhesion molecule (ICAM)-1 [39–42]. 3. A third response is biphasic with a relatively slight increase associated with onset of flow followed by a decrease in expression to levels below basal. This response has been described for endothelin-1 [43]. 4. A fourth response requires a cofactor for induction. Human umbilical vascular endothelial cells exposed to shear exhibit relatively little change in vascular cell adhesion molecule (VCAM)-1 messenger RNA expression, but show significant induction if they are pre-exposed to endotoxin or various cytokines [41].
3.3.2 Shear Stress Response Element The observation that gene transcription in endothelial cells can be modulated by laminar shear stress led to the search for conserved cis elements in the promoter sequences of shear-inducible genes. Several such cis elements have now been identified, indicating that there may be various sequences that regulate transcription upon shear. The most common transcriptional regulatory unit is a six-base-pair sequence, GAGACC, that was identified on the basis of the effect of shear on the promoter of PDGF-B [44]. Several endothelial genes unrelated to PDGF have been reported to be responsive to laminar shear stress and encode the shear stress response element (SSRE) or its complementary sequence (GGTCTC) in their promoter regions. Examples of genes that have an SSRE in their respective promoters include eNOS, MCP-1, and TGF-b [44].
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3.4 Signaling Associated with Altered Shear Experimental models to study the effects of altered shear have evaluated either the onset of shear in cells cultured under static conditions or the cessation of shear in flowadapted cells. Cellular responses with these two approaches have many similarities, leading to the generalization that cells respond to a change from the steady-state condition. The model of abrupt cessation of shear in flow-adapted cells mimics the expected response to acute arterial obstruction such as that associated with an embolus. The in vivo counterpart of the increased laminar shear model in cells cultured under static conditions is less clear; a more physiological in vitro model would be the study of increased laminar flow in cells that have been flow-adapted (shear-adapted), but those studies have not been done. The effects of disturbed flow (oscillatory or turbulent) have been interpreted as an indication of the changes in a vessel with partial luminal occlusion, such as the change associated with atherosclerosis, or with vasoconstriction. Here again, flow-adapted cells may represent a better basal (control) condition. Although the procedure to “flow adapt” endothelial cells in vitro is in principal straightforward, it is time-consuming and requires close attention to maintain sterility [18, 29, 45]. Nevertheless, use of this procedure can be expected to result in a significant increase in physiological relevance. A schematic of a commercially available chamber that has been used for this purpose is shown in Fig. 2. The physiological changes with altered shear range from virtually instantaneous electrochemical responses to more delayed changes in gene expression and cell phenotype.
3.4.1 Onset of Flow Candidate flow sensors as mentioned already can directly or indirectly sense flow and generate a biochemical signaling
Fig. 3 Time course of endothelial response to onset of laminar shear stress in cells cultured under static conditions. See the text for details
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cascade in response. The timescale for shear-induced responses by endothelial cells can be grouped into three categories (Fig. 3). Immediate responses generated subsequent to the acute sensing of shear are rapid, on the order of seconds, and may be initiated by changes in ion permeability due to the activation of flow-sensitive K+ and Cl− channels [13, 14]; these changes play an important role in regulating overall endothelial responsiveness to flow. Subsequent responses include an increase in intracellular free Ca2+ concentration, increased adenylate cyclase activity, inositol trisphosphate generation [1, 5, 13, 46], and the phosphorylation of the serine/threonine kinase Akt [15]. Intermediate
Fig. 2 A chamber that is used for adapting endothelial cells in vitro to flow. The artificial capillary system (FiberCell Systems) is made up of semipermeable polypropylene hollow fibers approximately 200 mm in diameter into which cells are seeded. The fibers are encased in a cartridge, with the ends forming the inlet and outlet ports to allow for perfusion. Before the seeding, the fibers are coated with a fibronectin matrix. After the seeding, the chamber is perfused via the side ports to provide substrate while allowing the cells to attach. After 24 h, perfusion is re-routed to the main ports and cells are subjected to laminar shear stress for an additional 24–72 h to obtain a flow-adapted state. Experimentally, re-routing the perfusate to the side ports simulates ischemia (loss of shear) while maintaining delivery of O2 and substrates. This chamber can be used to obtain cells that show a programmed response to ischemia [17, 28, 29, 45]. (Adapted from [45] with permission)
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responses that develop over minutes include activation of intracellular signaling molecules, release of mediators such as ROS and TGF-b [42], and the redistribution of cytoskeletal elements. Late responses (hours) are cellular adaptations and involve regulation of protein synthesis, changes in the cell shape, and alignment of cells in the direction of flow [6, 47].
3.4.2 Cessation of Flow Endothelial cells also show an immediate response to an abrupt decrease in shear. On the basis of a limited number of studies, there is a threshold for this effect, with indications that the shear stress must be decreased by more than 80% from the steady-state value for a response to be elicited [48]. Decreased shear can occur with various diseases owing to vascular obstruction associated with embolism/thrombosis, tumor growth, or (rarely in the lung) atherosclerosis. From a research standpoint, the lung permits study of the effects of the mechanical component of flow (i.e., decreased shear stress) without the attendant tissue anoxia that accompanies stop of blood flow in the vascular beds of other organs. Thus, ischemia/reperfusion in the lung can be differentiated from anoxia/reoxygenation. Decreased shear (i.e., ischemia) in the pulmonary vasculature has been studied using both the intact lung (isolated perfused rat or mouse lung) and in vitro flow-adapted pulmonary endothelial cells. In these systems, the control condition is the continuously perfused lung or cells; ischemia is achieved by stopping the perfusate flow. The use of realtime high-resolution digital imaging of the subpleural microvasculature in the intact lung by either confocal or epifluorescence microscopy in the presence of fluorophores has enabled the measurement of the rapid changes that occur in endothelial cells with the abrupt stop of flow. These events include changes in plasma membrane polarity, in the generation of ROS and NO, and increase in the intracellular Ca2+ concentration [18, 49–52]. The use of cells and lungs from gene-targeted mice has allowed the determination of the role of various mediators and pathways in the lung response. These responses are described in greater detail in the next section.
4 Cellular Response to Altered Shear 4.1 Membrane Depolarization Membrane-potential-sensitive dyes, such as bisoxonol and di-8-ANEPPS, localize in the membrane lipid bilayer and show increased fluorescence when the membrane is in a depolarized state. On the basis of changes in fluorescence, cell membrane potential decreases within several seconds after stop of flow [50]. Pretreatment of the lungs with a KATP
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channel agonist to maintain the open state blocked membrane depolarization, indicating that inactivation (closure) of these channels could be responsible for the change in membrane potential with ischemia. Lungs from KATP (KIR6.2) null mice showed a greatly diminished response to the abrupt decrease in shear [51]. Patch clamp studies with isolated flow-adapted endothelial cells confirmed the presence of a K+ current that was responsive to modulators of KATP channel activity and that decreased with abrupt flow cessation [18]. These studies led to the conclusion that inactivation of a cell membrane KATP channel resulting in membrane depolarization is one of the initial events following cessation of flow. The change in cell membrane potential with ischemia is not seen in cells from caveolin null mice (that lack caveolae), indicating that these organelles may serve as a flow sensor that is coupled to the subsequent inactivation of KATP channels.
4.2 Generation of ROS Various fluorophores, including dihydrochlorofluorescein, hydroethidine, and Amplex red, are readily oxidized, resulting in increased cellular fluorescence. These fluorophores have been used with intact organ and cell systems to image the cellular production of ROS. In the presence of these dyes, there is a rapid increase in endothelial cell fluorescence in the intact lung in the immediate period (minutes) subsequent to stop of flow [49–51]. A similar finding has been observed with endothelial cells in vitro provided they have been flowadapted prior to the experiment. Thus, ROS formation appears to be an early event associated with altered shear stress.
4.2.1 Role of NADPH Oxidase in ROS Generation Investigation into the pathway for endothelial generation of ROS with ischemia revealed that diphenyleneiodonium (a flavoprotein inhibitor that inhibits NADPH oxidase) effectively prevented ischemia-induced ROS generation. It is now known that endothelial cells possess a plasma membrane NADPH oxidase enzyme system that is similar to that described for phagocytic cells and is called “NOX2” [53, 54]. The generation of a functional NADPH oxidase necessitates the translocation of three cytosolic protein components (p47phox, p67phox, p21rac1) to the plasma membrane, where they associate with the cytochrome b-558 heterodimer (gp91phox and p22phox) [54, 55]; this assembly is required for ROS generation. Mice with “knockout” of gp91phox, the flavoprotein of the multicomponent NADPH oxidase enzyme complex, fail to generate ROS with lung ischemia, providing a strong indication that NOX2 is the enzyme responsible [49, 52]. NOX2 is a transmembrane protein that transfers electrons
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across the plasma membrane to generate superoxide anion (O2•−) in the extracellular (intravascular) space. O2•− in turn dismutes to H2O2, either spontaneously or through catalysis by extracellular superoxide dismutase (SOD). With the in vitro flow-adapted endothelial cell system, generation of ROS was demonstrated by the SOD-dependent reduction of cytochrome c added to the medium. These results indicate that the ROS produced with ischemia is O2•−, as expected for NADPH oxidase. Further, reduction of extracellular cytochrome c indicates that O2•− is generated on the extracellular side of the plasma membrane with ischemia, again consistent with the known activity of NOX2. The activation of NOX2 with altered shear is linked to cell membrane depolarization as shown by the absence of ROS generation in the presence of agents that prevent the closure of KATP channels [52]. A linkage between membrane potential and activation of NADPH oxidase has also been demonstrated for phagocytic cells [56, 57] although the mechanism is still undefined.
4.3 Intracellular Ca2+ Cessation of flow in the pulmonary endothelium in situ or in flow-adapted endothelial cells in vitro results in a rapid rise in the intracellular Ca2+ concentration [50, 58–60]. This effect is also dependent on the change in membrane potential (depolarization) of the lung endothelium in flow-adapted bovine pulmonary artery endothelial cells. The concentration of intracellular Ca2+ (as monitored by Fura-2) was estimated at approximately 135 nM under control conditions and began to increase within 30 s after flow cessation, reaching a plateau within 10 min at approximately double the basal concentration. The increase was inhibited by removal of Ca2+ from the perfusate, indicating that at least some of the increase in the internal Ca2+ concentration came by translocation from the extracellular medium across the cell membrane. It is generally accepted that voltage-gated Ca2+ channels mediate Ca2+ influx in response to cell membrane depolarization [61]. Ca2+ influx in endothelial cells in response to ischemia was not altered by inhibitors of L-type Ca2+ channels, which led to a search for T-type channels in these cells. These channels have indeed been detected in freshly isolated microvascular endothelial cells, but they are poorly expressed in endothelial cells cultured under static conditions [62]. However, the expression of the two subunits of this channel, the a1G and b3 proteins, is significantly increased during flow adaptation, suggesting that these channels are present and function in cells in vivo [60]. Ca2+ influx is blocked if cells are pretreated with the semispecific T-type channel blocker mibefradil. T-type Ca2+ channels open after a relatively small depolarization (10–20 mV),
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unlike L-type Ca2+ channels which require a significantly larger depolarization [63]. Calculations based on fluorescence imaging and using the Nernst equation show that membrane potential in the in situ lung decreases by approximately 15–20 mV with ischemia, compatible with the window for T-type channel activation [58, 60]. In addition to influx of Ca2+ through the cell membrane channels, there is some release of Ca2+ from its intracellular stores into the cell cytoplasm [58].
4.4 NO Generation Increased NO production has been detected in the pulmonary endothelium with cessation of flow in the intact lung as well as in flow-adapted endothelial cells in vitro. NO generation was observed through use of the fluorophore diaminofluorescein, and temporally appeared to follow the increase in intracellular Ca2+ concentration. Inhibition by Nw-nitro-l-arginine methyl ester indicates that NO generation with ischemia is due to activation of nitric oxide synthase. Further, inhibition by chelation of Ca2+ or treatment with a calmodulin inhibitor suggests that eNOS is the isoform responsible for the major fraction of NO production [28].
4.5 Phosphorylation Signaling Cascades Mitogen-activated protein kinases appear to be important mediators of mechanosignal transduction. Ischemia in the flow-adapted endothelial cell model leads to an increase in phosphorylation of extracellular-signal-regulated kinase (ERK) 1 and 2 during the first 10 min of stop of flow and reaches a plateau at 20–30 min. ERK phosphorylation (activation) is suppressed when ROS generation is inhibited. On the other hand, inhibitors of ERK phosphorylation suppressed NO generation but had no effect on ROS generation. These results indicate that ERK phosphorylation is downstream of (dependent on) ROS but upstream of eNOS [28].
4.6 Activation of Transcription Factors The initial responses to ischemia, viz., increased levels of ROS, Ca2+, and NO, are compatible with cell signaling and led to a search for activation of transcription factors under these conditions. Analysis of nuclear extracts from flow-adapted endothelial cells by electrophoretic mobility shift assay at
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1 h of ischemia showed increased NF-kB components (p65 and p50), indicating activation of this transcription factor. AP-1 also showed activation at 1 h of ischemia, with increases of the c-Jun/c-fos subunits of AP-1 [45]. Activation of NF-kB as well as AP-1 was abolished by pretreatment of cells with inhibitors of ROS generation, indicating that their activation is linked to oxidative events.
4.7 Cell Proliferation ROS generation is known to mediate Ras-induced cell cycle progression and mammalian cell proliferation [64]. Further, activation of transcription factors, NF-kB and AP-1, reportedly is coupled to increased cell division. On the basis of these observations, the proliferative response of flow-adapted endothelial cells subjected to ischemia was evaluated. Pulmonary artery endothelial cells demonstrated twice as much 3H-thymidine incorporation as control cells at 24 h after stop of flow, indicating an increase in DNA synthesis. Further, pulmonary microvascular endothelial cells studied by flow cytometry showed a 2.5-fold increase in the cellular proliferation index with ischemia, whereas an increase in the number of cells in the S and G2/M phases was observed with cell cycle analysis [29, 45]. Cell proliferation is blocked by inhibition of ROS generation, thus linking the ROS produced with ischemia to DNA synthesis, activation of transcription factors, and cell division [65, 66].
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of eNOS and increased synthesis of NO. The latter represents another mechanism for the restoration of blood flow, that is, via NO-mediated vasodilation. Of course, production of ROS and NO can result in cellular manifestations of oxidative/nitrosative stress such as lipid peroxidation and protein nitration under conditions where the rate of oxidant generation exceeds the oxidant scavenging ability of the cells. The signaling cascade in response to acute ischemia is shown in Fig. 4.
5 Manifestations of Disease The role of hemodynamic factors in diseases of the systemic circulation has long been established with the association between regions of disturbed flow and atherosclerotic lesions in vivo [67, 68]. Studies with endothelial cells in vitro have also shown that changing shear forces or hemodynamics alters endothelial cell structure, function, and gene expression and activates endothelial cell intracellular signaling pathways as described in previous sections [1, 9]. The changes accompanying altered shear in the pulmonary circulation have been less intensively studied, but clearly alteration of lung structure/function is seen with increased shear such as that accompanying left-to-right blood shunt or the decreased shear associated with thromboembolic disease. This section will present the pathophysiological alteration in the lung associated with altered shear. An important, unresolved issue
4.8 Summary of the Endothelial Signaling Cascade with Altered Shear The studies described in the previous sections are consistent with a defined endothelial cell signaling cascade that is initiated by the abrupt cessation of flow. A comparable cascade may be initiated by increased shear, although the precise details in this latter circumstance are not as well described. After sensing in the cell membrane, the initial response is cell membrane depolarization due to inactivation of KATP channels; this results in activation of NADPH oxidase and generation of ROS. The consequences of increased ROS production include mitogen-activated protein kinase (ERK) activation, activation of transcription factors (NF-kB, AP-1), and endothelial cell proliferation. Cell proliferation with ischemia may be an attempt to generate new capillaries to restore impeded blood flow. Cell membrane depolarization also leads to opening of T-type voltage-dependent Ca2+ channels in endothelium, with consequent Ca2+ influx and elevated intracellular Ca2+ concentration, which in turn leads to activation
Fig. 4 Endothelial cell response to abrupt loss of shear stress (simulated ischemia). See the text for details. (Modified from [87] with permission from Springer)
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regarding the contribution of altered flow to maintaining and advancing vascular disease is the relative contribution of altered shear versus the continuing contributions of the underlying disease process. In other words, it is important to differentiate the lung disease caused by altered shear from the manifestations of the underlying disease process.
5.1 Disturbed Flow and Cell Proliferation The normal structure and function of the vasculature is dependent upon the normal shear stresses produced by laminar blood flow at physiological levels. Laminar flow maintains vascular homeostasis and is considered to be antiapoptotic antiatherosclerotic, and antiproliferative [5, 6, 8]. Under normal laminar flow, the production of NO, TGF-b, PDGF, and other factors by the endothelium which regulate smooth muscle cell growth is maintained in a basal state [1]. This balance is altered in the presence of disturbed (turbulent or oscillatory) shear. Disturbed flow promotes endothelial dysfunction in the form of endothelial cell damage, increased macromolecular permeability, lipoprotein accumulation, and leukocyte adhesion [33, 69], and leads to structural modifications of the vessel wall. The response includes abnormal proliferation of both endothelial and smooth muscle cells and increased synthesis of matrix proteins. These changes associated with shear can amplify lung alterations owing to progression of the underlying disease process or can incite new disease processes. The production of ROS through activation of vascular NADPH oxidase appears to play a central role in cell proliferation [70] as demonstrated experimentally with smooth muscle proliferation induced by the NOX2 activator angiotensin II [71, 72].
5.2 Inflammatory Responses to Shear Disturbed flow also has been associated with induction of vascular wall inflammation. A physiological level of laminar shear stress is considered to be anti-inflammatory and maintains mediators of inflammation in a basal state [73]. This anti-inflammatory role of laminar shear stress may be mediated partly by NO since NO synthesis limits the production of adhesion molecules, cytokines, and chemokines. Endothelial cells exposed to disturbed flow in vitro over prolonged periods develop a proinflammatory phenotype with increased expression of adhesion molecules (ICAM, VCAM, P-selectin, and E-selectin), chemokines (monocyte chemotactic protein) and cytokines (tumor necrosis factor, interleukins) [35, 74, 75]. Like the mechanisms for cell proliferation, the effect on inflammations occurs at least in part through endothelial
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ROS production mediated via activation of vascular NADPH oxidase [35, 75].
5.3 Rarefaction of Pulmonary Vasculature and Increased Pulmonary Vascular Resistance Increased or disturbed shear can result in an increased pulmonary vascular resistance that is attributed, in part, to remodeling of the walls of resistance vessels. Thus, intimal, medial, and adventitial hypertrophy leads to reduction of the diameter of the vascular lumen and increased vascular resistance. The results of vasculature remodeling in the lung are a reduction of the total number of pulmonary arterioles with consequent altered perfusion of the pulmonary alveoli and abnormal ventilation–perfusion relationships [76]. This effect of shear in the pulmonary vasculature contrasts with its effects in the systemic vasculature, where vascular disease promotes angiogenesis. In a rat model of pulmonary hypertension, extensive new vessel formation was observed in the peribronchial region and on the pleural surface arising from systemic (bronchial and intercostal) vessels, but there was no evidence of new vessel formation in the alveolar walls [77]. A similar response has been seen with decreased shear associated with experimental pulmonary artery ligation [78]. These results imply that unlike in the systemic vasculature, angiogenesis does not occur in the adult pulmonary circulation. The mechanisms for the differential responses of the pulmonary and systemic vascular beds are not clear.
5.4 Pulmonary Hypertension Pulmonary hypertension has multiple causes with a range of underlying mechanisms resulting in areas of the vasculature that are subjected to either excessive or decreased shear [79, 80]. Thus, increased shear results from increased blood flow, disturbed (turbulent) shear results from partial intravascular obstruction, and decreased shear is associated with vascular occlusion. Clinical examples include left-to-right shunt (increased shear), vasculitis (intravascular obstruction), and thromboembolic disease (occlusion). Pulmonary vasoconstriction would be expected to result in increased shear, but turbulent shear or decreased shear could also result depending on the pulmonary blood flow. In most cases of pulmonary hypertension (e.g., caused by lung damage), areas of increased shear, turbulent shear, and decreased shear coexist within the lung circulation. Persistent changes in pulmonary shear stress result in structural alterations of the vessel wall, indicated by changes
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in wall diameter and thickness, with eventual modification of vessel diameter. This vascular remodeling is characterized by thickening of all three layers of the blood vessel wall, viz., the adventitia, the media, and the intima, due to hypertrophy and/or proliferation (hyperplasia) of smooth muscle, endothelial, and fibroblastic cells as well as increased deposition of extracellular matrix components such as collagen, elastin, and fibronectin. The effects of altered shear stress on vascular remodeling have been demonstrated in animal models of increased pulmonary blood flow due to experimental left-toright shunts [81, 82]. Another manifestation of altered shear is the formation of neointimal lesions that are hyperplastic areas composed of smooth muscle cells and extracellular matrix [83]. These lesions are seen with severe pulmonary endothelial injury. Experimentally, the pulmonary vascular injury associated with the monocrotaline model of pulmonary hypertension is characterized by the development of the neointimal pattern. The intimal changes can include fibrotic or cellular thickening and acute and organizing thrombi, all of which can result from or lead to disordered flow. Finally, a hallmark of obstructive intimal remodeling is the pathological structure called the “plexiform lesion” that is associated with severe pulmonary artery hypertension [84]. This lesion is manifested by granulation tissue that forms to repair transmural destruction of the vessel wall. The lesions generally develop at the branch point of a muscular artery, compatible with an important role for shear stress in their pathogenesis. These lesions are most commonly observed in primary pulmonary hypertension, but can occur in other forms of pulmonary hypertension as well.
5.5 Atherosclerosis Shear stress appears to play a contributing role in development of atherosclerotic lesions since atheroma (plaque) are rarely seen in straight sections of the arterial tree, where flow is laminar, but develop in regions associated with disturbed or oscillatory flows near arterial bifurcations, branch ostia, and curvatures. Arteries such as the coronary, carotid, cerebral, iliac, and splenic arteries are selectively involved in comparison with other branches. Although pulmonary artery atherosclerosis is rare, there are reports of macrophage-rich lesions in patients with severe pulmonary hypertension [85]. Inflammation is thought to play an important role in the pathogenesis and progression of atherosclerosis [86]. As plaque forms, monocytes present in the plaque proliferate, oxidize LDL, and generate multiple cytokines that act as chemoattractants for other inflammatory cells [86]. These cells induce neointimal lesions with increased extracellular matrix production, thereby contributing to the progression of atherosclerotic lesions.
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6 Conclusions Shear stress plays an important role in lung (and other vascular) disease and disease processes. Laminar shear is sensed by the endothelium and perhaps other sections of the vascular wall and helps to maintain those cells in a quiescent state, consistent with their functions in pulmonary perfusion and gas exchange. Disturbed or abruptly decreased flow can be associated with inflammation and alterations of the vascular wall, including neointimal and plexiform lesions. Thus, the pattern of shear represents a fundamental mechanism that is crucial to the health and viability of the lung as the organ of gas exchange.
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S. Chatterjee and A.B. Fisher 38. Nishida K, Harrison DG, Navas JP et al (1992) Molecular cloning and characterization of the constitutive bovine aortic endothelial cell nitric oxide synthase. J Clin Invest 90:2092–2096 39. Hsieh HJ, Li NQ, Frangos JA (1991) Shear stress increases endothelial platelet-derived growth factor mRNA levels. Am J Physiol 260:H642–H646 40. Shyy YJ, Hsieh HJ, Usami S et al (1994) Fluid shear stress induces a biphasic response of human monocyte chemotactic protein 1 gene expression in vascular endothelium. Proc Natl Acad Sci USA 91:4678–4682 41. Nagel T, Resnick N, Atkinson WJ et al (1994) Shear stress selectively upregulates intercellular adhesion molecule-1 expression in cultured human vascular endothelial cells. J Clin Invest 94:885–891 42. Ohno M, Cooke JP, Dzau VJ et al (1995) Fluid shear stress induces endothelial transforming growth factor beta-1 transcription and production. Modulation by potassium channel blockade. J Clin Invest 95:1363–1369 43. Malek AM, Gibbons GH, Dzau VJ et al (1993) Fluid shear stress differentially modulates expression of genes encoding basic fibroblast growth factor and platelet-derived growth factor B chain in vascular endothelium. J Clin Invest 92:2013–2021 44. Resnick N, Yahav H, Khachigian LM et al (1997) Endothelial gene regulation by laminar shear stress. Adv Exp Med Biol 430:155–164 45. Wei Z, Costa K, Al-Mehdi AB et al (1999) Simulated ischemia in flow-adapted endothelial cells leads to generation of reactive oxygen species and cell signaling. Circ Res 85:682–689 46. Cooke JP, Rossitch E Jr, Andon NA et al (1991) Flow activates an endothelial potassium channel to release an endogenous nitrovasodilator. J Clin Invest 88:1663–1671 47. Davies PF, Tripathi SC (1993) Mechanical stress mechanisms and the cell. An endothelial paradigm. Circ Res 72:239–245 48. Al-Mehdi AB, Zhao G, Fisher AB (1998) ATP-independent membrane depolarization with ischemia in the oxygen-ventilated isolated rat lung. Am J Respir Cell Mol Biol 18:653–661 49. Al-Mehdi AB, Zhao G, Dodia C et al (1998) Endothelial NADPH oxidase as the source of oxidants in lungs exposed to ischemia or high K+. Circ Res 83:730–737 50. Song C, Al-Mehdi AB, Fisher AB (2001) An immediate endothelial cell signaling response to lung ischemia. Am J Physiol Lung Cell Mol Physiol 281:L993–L1000 51. Zhang Q, Matsuzaki I, Chatterjee S et al (2005) Activation of endothelial NADPH oxidase during normoxic lung ischemia is KATP channel dependent. Am J Physiol Lung Cell Mol Physiol 289:L954–L961 52. Zhang Q, Chatterjee S, Wei Z et al (2008) Rac and PI3 kinase mediate endothelial cell-reactive oxygen species generation during normoxic lung ischemia. Antioxid Redox Signal 10:679–689 53. Jones SA, O'Donnell VB, Wood JD et al (1996) Expression of phagocyte NADPH oxidase components in human endothelial cells. Am J Physiol 271:H1626–H1634 54. Babior BM (2000) The NADPH oxidase of endothelial cells. IUBMB Life 50:267–269 55. DeLeo FR, Quinn MT (1996) Assembly of the phagocyte NADPH oxidase: molecular interaction of oxidase proteins. J Leukoc Biol 60:677–691 56. Korchak HM, Weissmann G (1978) Changes in membrane potential of human granulocytes antecede the metabolic responses to surface stimulation. Proc Natl Acad Sci U S A 75:3818–3822 57. Cameron AR, Nelson J, Forman HJ (1983) Depolarization and increased conductance precede superoxide release by concanavalin A-stimulated rat alveolar macrophages. Proc Natl Acad Sci USA 80:3726–3728 58. Tozawa K, al-Mehdi AB, Muzykantov AB et al (1999) In situ imaging of intracellular calcium with ischemia in lung subpleural microvascular endothelial cells. Antioxid Redox Signal 1:145–154
54 Shear Stress, Cell Signaling, and Pulmonary Vascular Remodeling 59. Manevich Y, Al-Mehdi A, Muzykantov V et al (2001) Oxidative burst and NO generation as initial response to ischemia in flowadapted endothelial cells. Am J Physiol Heart Circ Physiol 280:H2126–H2135 60. Wei Z, Manevich Y, Al-Mehdi AB et al (2004) Ca2+ flux through voltage-gated channels with flow cessation in pulmonary microvascular endothelial cells. Microcirculation 11:517–526 61. Catterall WA (2000) Structure and regulation of voltage-gated Ca2+ channels. Annu Rev Cell Dev Biol 16:521–555 62. Wu S, Haynes J Jr, Taylor JT et al (2003) Cav3.1 a1G T-type Ca2+ channels mediate vaso-occlusion of sickled erythrocytes in lung microcirculation. Circ Res 93:346–353 63. Perez-Reyes E (1999) Three for T: molecular analysis of the low voltage-activated calcium channel family. Cell Mol Life Sci 56:660–669 64. Boonstra J, Post JA (2004) Molecular events associated with reactive oxygen species and cell cycle progression in mammalian cells. Gene 337:1–13 65. Milovanova T, Manevich Y, Haddad A et al (2004) Endothelial cell proliferation associated with abrupt reduction in shear stress is dependent on reactive oxygen species. Antioxid Redox Signal 6:245–258 66. Milovanova T, Chatterjee S, Manevich Y et al (2006) Lung endothelial cell proliferation with decreased shear stress is mediated by reactive oxygen species. Am J Physiol Cell Physiol 290:C66–C76 67. Kohler TR, Jawien A (1992) Flow affects development of intimal hyperplasia after arterial injury in rats. Arterioscler Thromb 12: 963–971 68. Kimura BJ, Russo RJ, Bhargava V et al (1996) Atheroma morphology and distribution in proximal left anterior descending coronary artery: in vivo observations. J Am Coll Cardiol 27:825–831 69. Bussolari SR, Dewey CF Jr, Gimbrone MA Jr (1982) Apparatus for subjecting living cells to fluid shear stress. Rev Sci Instrum 53:1851–1854 70. Weber DS, Rocic P, Mellis AM et al (2005) Angiotensin II-induced hypertrophy is potentiated in mice overexpressing p22phox in vascular smooth muscle. Am J Physiol Heart Circ Physiol 288:H37–H42 71. Ushio-Fukai M, Zafari AM, Fukui T et al (1996) p22phox is a critical component of the superoxide-generating NADH/NADPH oxidase system and regulates angiotensin II-induced hypertrophy in vascular smooth muscle cells. J Biol Chem 271:23317–23321 72. Griendling KK, Sorescu D, Lassegue B et al (2000) Modulation of protein kinase activity and gene expression by reactive oxygen species and their role in vascular physiology and pathophysiology. Arterioscl Thromb Vasc Biol 20:2175–2183
799 73. Sampath R, Kukielka GL, Smith CW et al (1995) Shear stressmediated changes in the expression of leukocyte adhesion receptors on human umbilical vein endothelial cells in vitro. Ann Biomed Eng 23:247–256 74. Chappell DC, Varner SE, Nerem RM et al (1998) Oscillatory shear stress stimulates adhesion molecule expression in cultured human endothelium. Circ Res 82:532–539 75. Hwang J, Saha A, Boo YC et al (2003) Oscillatory shear stress stimulates endothelial production of O2- from p47phox-dependent NAD(P)H oxidases, leading to monocyte adhesion. J Biol Chem 278:47291–47298 76. Partovian C, Adnot S, Raffestin B et al (2000) Adenovirus-mediated lung vascular endothelial growth factor overexpression protects against hypoxic pulmonary hypertension in rats. Am J Respir Cell Mol Biol 23:762–771 77. Schraufnagel DE (1990) Monocrotaline-induced angiogenesis. Differences in the bronchial and pulmonary vasculature. Am J Pathol 137:1083–1090 78. Mitzner W, Lee W, Georgakopoulos D et al (2000) Angiogenesis in the mouse lung. Am J Pathol 157:93–101 79. Voelkel NF, Tuder RM (1995) Cellular and molecular mechanisms in the pathogenesis of severe pulmonary hypertension. Eur Respir J 8:2129–2138 80. Rubin LJ (1997) Primary pulmonary hypertension. N Engl J Med 336:111–117 81. Bousamra M 2nd, Rossi R, Jacobs E et al (2000) Systemic lobar shunting induces advanced pulmonary vasculopathy. J Thorac Cardiovasc Surg 120:88–98 82. Friedli B, Kent G, Kidd BS (1975) The effect of increased pulmonary blood flow on the pulmonary vascular bed in pigs. Pediatr Res 9:547–553 83. Tanaka Y, Schuster DP, Davis EC et al (1996) The role of vascular injury and hemodynamics in rat pulmonary artery remodeling. J Clin Invest 98:434–442 84. Yi ES, Kim H, Ahn H et al (2000) Distribution of obstructive intimal lesions and their cellular phenotypes in chronic pulmonary hypertension. A morphometric and immunohistochemical study. Am J Respir Crit Care Med 162:1577–1586 85. Botney MD (1999) Role of hemodynamics in pulmonary vascular remodeling: implications for primary pulmonary hypertension. Am J Respir Crit Care Med 159:361–364 86. Ross R (1999) Atherosclerosis-an inflammatory disease.[see comment]. N Engl J Med 340:115–126 87. Chatterjee S, Chapman KE, Fisher AB (2008) Lung ischemia: a model for endothelial mechanotransduction. Cell Biochem Biophys 52:125–138
Chapter 55
Pulmonary Hypertension and the Extracellular Matrix Marlene Rabinovitch
Abstract The extracellular matrix (ECM) plays a pivotal role in regulating cell shape and cell signaling, in maintaining cell–cell communication, and in controlling cell differentiation and dedifferentiation. The stiffness of a vessel is directly related to the proportion of the various ECM components. It is not surprising then that even haploinsufficiency of a single component of the ECM such as elastin or fibrillin can lead to profound developmental abnormalities and propensity to diseases of blood vessels. For example, haploinsufficiency of elastin (Williams syndrome) is associated with pulmonary and systemic stenoses and haploinsufficiency of fibrillin (Marfan syndrome) leads to aneurismal dilatation of the aorta. A variety of growth factors and cytokines regulate the production of the various components of the ECM and are in fact bound by different components of the ECM. Turnover of the ECM and release of growth factors is controlled by the balance between proteolytic enzymes such as serine elastases and matrix metalloproteinases (MMPs) and their endogenous inhibitors. So it is not surprising that abnormalities in the regulation of the ECM would contribute in a fundamental way to the pathobiology of pulmonary vascular disease leading to pulmonary arterial hypertension (PAH). Keywords Elastase • Elastin • Fibrillin • Vascular wall stiffness • Growth factors • Pulmonary vascular remodeling
1 Introduction The extracellular matrix (ECM) plays a pivotal role in regulating cell shape and cell signaling, in maintaining cell– cell communication, and in controlling cell differentiation and dedifferentiation. The stiffness of a vessel is directly related to the proportion of the various ECM components. It M. Rabinovitch (*) Department of Pediatrics, Wall Center for Pulmonary Vascular Diseases, Stanford University School of Medicine, CCSR-Room 2245B, 269 Campus Drive, Stanford, CA 94305-5162, USA e-mail:
[email protected] is not surprising then that even haploinsufficiency of a single component of the ECM such as elastin or fibrillin can lead to profound developmental abnormalities and propensity to diseases of blood vessels. For example, haploinsufficiency of elastin (Williams syndrome) is associated with pulmonary and systemic stenoses and haploinsufficiency of fibrillin (Marfan syndrome) leads to aneurismal dilatation of the aorta. A variety of growth factors and cytokines regulate the production of the various components of the ECM and are in fact bound by different components of the ECM. Turnover of the ECM and release of growth factors is controlled by the balance between proteolytic enzymes such as serine elastases and matrix metalloproteinases (MMPs) and their endogenous inhibitors. So it is not surprising that abnormalities in the regulation of the ECM would contribute in a fundamental way to the pathogenic mechanisms of pulmonary vascular remodeling leading to pulmonary arterial hypertension (PAH).
2 Pulmonary Hypertension and Elastase Activity Ultrastructural assessment of pulmonary arteries (PA) in lung biopsy tissue from patients with congenital heart defects and PAH [1] first suggested that changes in the ECM could contribute to the pathobiology of PAH. In association with alterations in endothelial structure and function, we noted fragmentation of elastic laminae (Fig. 1). These early features, apparent by electron microscopy, preceded the later development of occlusive lesions. Gaps in the internal elastic laminae were also reported in patients with PAH and neointimal lesions [2]. Fragmentation of elastin occurred in experimental rats in which PAH was induced in association with endothelial injury [3] following injection of the toxin monocrotaline [4]. Consistent with the ultrastructural changes reflecting degradation of PA elastin was heightened activity of a serine elastase in the PA wall [4]. The increased activity of this endogenous vascular elastase (EVE) preceded the development of PAH and the accompanying vascular lesions. Those vascular changes consisted of abnormal muscularization and
J.X.-J. Yuan et al. (eds.), Textbook of Pulmonary Vascular Disease, DOI 10.1007/978-0-387-87429-6_55, © Springer Science+Business Media, LLC 2011
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Fig. 1 (a) A section of pulmonary artery 92 mm in diameter in a patient with normal pulmonary arterial pressure shows an intact elastic lamina. (b) In a section from a pulmonary artery 108 mm in diameter in a patient with increased pulmonary blood flow and pressure, microfibrillar material is present in the subendothelium but there is no true internal elastic lamina. The endothelial cells (EC) and smooth muscle cells (SMC) are separated by only a thick basement membrane (bm). Bars 1 mm. (Modified from [1] with permission)
loss of peripheral alveolar duct and wall PAs and medial hypertrophy of the more proximal intra-acinar PAs at the level of the terminal and respiratory bronchioli [5]. Moreover, inhibition of EVE resulted in attenuation of the PAH as well as the associated vascular changes in a variety of experimental rodent models [5]. More recently in a mouse that overexpresses S100A4 and that develops neointimal lesions after injection of the murine gamma herpes virus 68 (MHV-68) [6], we showed that serine elastase activity is increased after infection of the virus and with reactivation. In an effort to understand how EVE activity was regulated in the vessel wall, we cultured PA smooth muscle cells (SMC). We were able to show that EVE could be induced by serum and endothelial factors, to which SMC would be exposed under conditions of endothelial injury [7]. It appeared that these factors signaled through the mitogen-activated protein kinase pathway, causing induction and nuclear translocation of phosphorylated extracellular signal-regulated kinase 1 and 2 (pERK1/2) that was inhibited by NO. We showed that pERK1/2 nuclear translocation was necessary for the activity of the transcription factor for elastase, AML1 [8]. Then we documented that EVE could release SMC mitogens such as basic fibroblast growth factor that are normally stored in the ECM in an inactive form [9]. In addition, we showed that elastin peptides [10], produced in response to EVE, could mediate both SMC proliferation and fibronectindependent SMC motility [11] (Fig. 2). As well, elastin peptides are chemoattractants for inflammatory cells [12], and can induce proliferation and migration of SMC [13, 14]. Further studies revealed that EVE, either directly or through activation of MMPs [15], could, by degrading collagen and exposing cryptic RGD sites, cause heightened signaling through avb3 integrins [16]. This signaling was responsible for production and secretion of tenascin-C, a glycoprotein that binds to and clusters avb3 integrins [16]. In so doing,
M. Rabinovitch
tenascin-C alters the actin cytoskeleton and this causes clustering and activation of growth factor receptors [16]. These receptors transduce both pro-proliferative and cell survival signals in PA SMCs. So when we suppressed EVE or MMP activity in proliferating PA SMCs, we not only prevented their proliferation but also induced apoptosis [16]. We then documented that we could induce regression of hypertrophied PAs in the intact animal by inhibiting EVE and by inducing PA SMC apoptosis [17] (Fig. 3). Interestingly, inhibition of EVE also reversed the loss of small precapillary vessels (alveolar duct and wall arteries), suggesting that this therapy may also regenerate the lost microcirculation associated with PAH. Although heightened MMP activity is associated with the development of PAH, we could not reverse experimentally induced PAH in the monocrotaline-treated rat with MMP inhibition, although the length of survival was improved [18]. Others have shown that inhibition of MMPs may actually worsen hypoxia-induced PAH [19]. In fact, we have shown that MMPs are activated in SMCs downstream of bone morphogenetic protein receptor II (BMPRII) signaling [20, 21] and are necessary for normal migratory and proliferative behavior of SMCs and pericytes in development, and potentially in response to injury. An extensive body of work has shown that metalloproteinase activity is required for the regression of chronic-hypoxia-induced PAH [22].
3 Elafin, an EVE Inhibitor To further address the importance of EVE in the pathology of cardiovascular disease, we made a mouse that overexpressed human elafin, a molecule that inhibits leukocyte elastase but that we had shown was also an endogenous inhibitor of EVE [23]. Elafin was initially isolated from bronchial secretions [24] and from psoriatic skin [25] as a high-affinity serine elastase inhibitor (ki for neutrophil elastase 4.2 ×10−9 M). It is similar functionally, structurally, and by chromosomal location to secreted leukocyte protease inhibitor [26] but with limited targets of inhibitory activity. These include neutrophil elastase, pancreatic elastase, and proteinase 3 [25]. Elafin is produced as a 12-kDa pro-form called trappin and in this form consists of a signal peptide, a transglutaminase domain that binds to a variety of ECM components and an elastase inhibitory domain. Upon cleavage of the signal peptide and the transglutaminase domain, the 6-kDa elafin is released. In designing the transgenic mouse, we overexpressed elafin under the regulation of the preproendothelin promoter. Thus, it was induced under conditions of endothelial injury when endothelin transcription is activated. The elafinoverexpressing mouse was not only protected against PAH [27], but also against a variety of other cardiovascular diseases
55 Pulmonary Hypertension and the Extracellular Matrix
Fig. 2 (a) We speculate, on the basis of our studies in cultured cells and in experimental animals, as to how a stimulus could induce activity of an elastolytic enzyme and how this might stimulate the remodeling process. In response to an injurious stimulus, the first casualty is the endothelial cell. As a result of structural and functional alterations in endothelial cells, some of the barrier function would be lost, allowing a leak into the subendothelium of a serum factor normally excluded from this region. The serum factor could induce activity of an endogenous vascular elastase (EVE). This enzyme released from precursor or mature smooth muscle cells (SMCs) would activate growth factors normally stored in the extracellular matrix in an inactive form, which are known to induce smooth muscle hypertrophy and proliferation and increases in connective tissue (CT) protein (e.g., collagen and elastin) synthesis. The growth factors could also result in the differentiation of precursor cells to mature smooth muscle
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related to the muscularization of normally nonmuscular small peripheral arteries. Elastase activity could also contribute to the loss of small arteries. (b) As a result of elastase activity, a proteolytic cascade could be amplified through the activation of matrix metalloproteinases. We have shown that proteolysis of the matrix leads to the induction of tenascin-C, which in clustering to b3 integrins results in the clustering and activation of growth factor receptors which send important survival as well as growth signals to the cells. Continued elastase activity would cause migration of smooth muscle cells in several ways. The elastin peptides or degradation products of elastin can stimulate fibronectin, a glycoprotein that is pivotal in altering smooth muscle cell shape and in switching smooth muscle cells to the motile phenotype. Inhibition of elastase activity could lead to smooth muscle cell apoptosis and regression of pulmonary vascular disease (PVD). (Reproduced from [11] with permission)
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Fig. 3 Cellular mechanism responsible for reversal of pulmonary artery muscularity. Elastase inhibition arrests tenascin-C accumulation and proliferation and induces apoptosis and loss of extracellular matrix (such as elastin). (a–p) Days refer to time after injection of monocrotaline: (a, e, j, m) day 21; (b, f, j, n) day 28; (c, g, k, o) day 28; (d, h, l, p) day 28. (a–d) Saline-perfused pulmonary arteries stained with Movat pentachrome stain. (e–h) Pulmonary arteries after tenascin-C
immunohistochemistry. Arrows indicate positive brown peroxidase staining. (i–l) In situ terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assays identifying apoptosis. Arrows indicate TUNEL-positive vascular cells. (m–p) Proliferating vascular cells, shown by immunohistochemistry for proliferating cell nuclear antigen (PCNA); dark nuclei are PCNA-positive cells. Scale bars 5 mm. (Reproduced from [17] with permission)
such as myocardial infarction [28], myocarditis [23], and carotid artery stenosis [29]. Gene transfer of elafin attenuated vein graft atherosclerosis [30] and infusion of elafin in rabbits attenuated both posttransplant coronary artery disease and rejection [31].
occlusion. Haploinsufficiency of elastin causes PAH and replacement of the null allele with the human tropoelastin gene to generate 35% of normal tropoelastin [32] further increases the severity of PAH and vascular stiffness. Structural changes in the distal PA vasculature are not, however, very severe [33] (Fig. 4). A variety of mice have been created with loss of elastin assembly genes such as fibrillin-1 and fibulin-5, and deletions of some of these genes (fibulin-4) cause lethality in embryonic or early postnatal life, so the presence of PAH has not been assessed. In the newborn calf that is made hypoxic, there is evidence for marked stimulation of tropoelastin synthesis in the media and adventitia of the PAs [34] (Fig. 5). We observed that in rats treated with monocrotaline, there is a twofold increase in net production of both elastin and collagen,
4 Elastin, Elastin Assembly Genes, and Pulmonary Hypertension Intact well-assembled elastin plays a critical role in vascular resilience and in preventing stiffening of the vasculature and in controlling SMC proliferation. Deletion of elastin results in neonatal lethality owing to proliferation of SMCs and vascular
55 Pulmonary Hypertension and the Extracellular Matrix Fig. 4 (a) Right ventricular (RV) pressure was measured using a microcatheter introduced through the internal jugular vein; six animals per genotype. Bars represent mean systolic (solid bars) and diastolic (shaded bars) pressures ± the standard error. *P 12 mmHga MPAP mean pulmonary artery pressure, PVR pulmonary vascular resistance, TPG transpulmonary gradient [MPAP minus pulmonary artery occlusion pressure (PAOP)] a This parameter replaces the PAOP (also known as pulmonary capillary wedge pressure) criteria proposed by the ERS Task Force [5]
M.J. Krowka
2 Etiology and Pathogenetic Mechanisms It is hypothesized that in genetically susceptible patients, the existence of portal hypertension allows circulating factors to bypass normal hepatic metabolism and adversely affect the pulmonary vascular bed [7–9]. Specifically, obstruction to arterial flow is caused by proliferation of pulmonary arterial endothelium and smooth muscle, in situ thrombosis, and vasoconstriction (Fig. 2). The presence of classic plexogenic arteriopathy has been well documented [10, 11]. Abnormal platelet aggregations have been documented at the small vessel and capillary level in POPH patients who have died of right-sided heart failure during LT attempts [12]. Consistent with this “bypass” hypothesis is the increased frequency of POPH seen after portocaval shunt surgery [13, 14]. Interestingly, the microscopic pulmonary pathologic changes seen in POPH are identical to those seen in idiopathic pulmonary artery hypertension [15]. Furthermore, a deficiency in endothelial prostacyclin synthase and increase in circulating endothelin-1 levels have been reported in POPH [16, 17]. Increased measured gradients of endothelin-1 between the pulmonary artery and pulmonary veins, but not between the hepatic vein and inferior vena cava have been documented (K.L. Swanson, Mayo Clinic, personal communication).
3 Prevalence and Clinical Manifestations
Fig. 1 Pulmonary hemodynamics associated with portal hypertension can be categorized into three main groups on the basis of measured mean pulmonary artery pressure (MPAP), cardiac output (CO), and pulmonary arterial occlusion pressure (PAOP). Pulmonary vascular resistance (PVR) is a calculated value [(MPAP-PAOP)/CO]
Fig. 2 Autopsy specimen from patient with mild portopulmonary hypertension (POPH) (MPAP 34 mmHg; PVR 160 dyn s cm–5, CO 9.4 L/min following treatment with sildenafil 20 mg three times daily for 3 months). Sudden cardiac arrest 3 days after liver transplantion. Distinction between in situ clot and embolic clot may be difficult (patient had no radiographic findings to suggest pulmonary emboli). Proliferation of endothelium and smooth muscle noted with plexogenic change caused vascular obstruction to flow
Although earlier autopsy studies suggested a 0.25–0.73% frequency of pulmonary artery hypertension complicating portal hypertension or cirrhosis, the clinical diagnosis of POPH in the era of pulmonary artery hypertension therapeutic advances and LT is more prevalent [18–20]. Data from the REVEAL registry (Registry to Evaluate Early and LongTerm Pulmonary Artery Hypertension Management),
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p rovided by pulmonary hypertension clinics throughout the USA since 2006 (n = 3,250), suggest that the diagnosis of POPH comprises nearly 5% of all data submissions [21]. A recent French study has described POPH as the fourth leading cause of pulmonary artery hypertension seen in the 17-member hospital consortium and with a frequency of 10.4% of 674 patients seen in the time span from 2002 to 2003 [22]. Two of the largest LT centers in the USA reported a 5.3–8.5% frequency of POPH in LT candidates [23, 24]. POPH can complicate any hepatic disorder that results in portal hypertension and usually manifests 4–7 years following a given hepatic diagnosis [25, 26]. Not infrequently, patients referred to pulmonary hypertension clinics with a diagnosis of idiopathic pulmonary hypertension are subsequently found to have POPH, as the only presenting clue may be a marked reduction in the serum platelet counts (without changes in the levels of serum bilirubin or liver enzymes) due to splenomegaly caused by portal hypertension. An increased frequency of POPH is suggested in autoimmune hepatic problems and the female gender [27]. Rarely, POPH occurs in the pediatric age group [28]. No relationship exists between the severity of POPH and the severity of liver disease as characterized by the Child–Pugh–Turcotte scores (ascites, encephalopathy, bilirubin, albumin and prothrombin time), model for end-stage liver disease scores (bilirubin, creatinine, and international normalized ratio) or the degree of the hepatic vein–portal gradient [4, 5, 26, 27]. As obstruction to pulmonary arterial flow worsens, progressive exertional dyspnea is usually the first patient complaint. Late
symptoms may include chest pain and syncope, caused by right-sided heart strain and reduced cardiac output, respectively [4]. The physical examination may reveal an increased intensity and wide splitting of the second heart sound, elevated jugular pressures, a systolic murmur of tricuspid regurgitation, and normal breath sounds. A hyperdynamic precordium is usually present and a right ventricular heave may be palpable. Cyanosis is not a usual sign in POPH [4]. Abnormalities in the posterolateral chest radiograph include cardiomegaly and enlarged central pulmonary arteries, but these findings are associated with at least moderate to severe pulmonary artery hypertension [4]. Further changes in chest radiographs may demonstrate other complications of advanced liver disease that occur independent of the POPH and reflect the dual nature of the concomitant pulmonary and hepatic problems (Fig. 3). Electrocardiogram changes suggest right ventricular hypertrophy, right atrial enlargement, a rightward QRS axis, and strain on the right ventricle as manifested by T-wave inversions in the precordial V1 to V4 leads (a sign of moderate to severe POPH). Pulmonary function testing is not particularly helpful and frequently reveals a nonspecific decrease in the single breath diffusing capacity for carbon monoxide; lung volumes and expiratory airflows are normal. Arterial blood gases may show a slight abnormality in oxygenation and marked reduction in carbon dioxide with a respiratory alkalosis pattern, but again these are nonspecific findings [4, 5].
Fig. 3 Chest radiograph demonstrating cardiomegaly and enlargement of central pulmonary arteries; patient receiving intravenous administration of prostacyclin. Four years later the patient presented with metastatic hepatocellular carcinoma and pericardial effusion in the setting of stable POPH
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4 Screening and Diagnostic Criteria Specific diagnostic criteria for the diagnosis of POPH have been formulated via a Pulmonary-Hepatic Vascular Disorder Task Force convened in 2004 [5]. Increased mean pulmonary artery pressure (MPAP) and pulmonary vascular resistance (PVR), accompanied by a normal pulmonary artery occlusion pressure (PAOP), form the key elements of the criteria (Table 1). The latter criterion (normal PAOP) has recently been modified and replaced by an increased transpulmonary gradient by some investigators (see later) [23]. With diagnostic criteria now recognized, practice guidelines have been proposed by the American Association for the Study of Liver Disease that recommend screening transthoracic echocardiography for all LT candidates [6]. The goal of such screening is to noninvasively identify patients with any form of pulmonary hypertension and select those that should undergo diagnostic right-sided heart catheterization. As to the clinical relevance for such screening, the presence of moderate to severe POPH (MPAP greater than 35 mmHg) in LT candidates has been associated with several intraoperative deaths and an approximate 35% frequency of posttransplant hospitalization mortality. This degree of mortality was documented in both a 2000 literature review (n = 48 cases of POPH with attempted LT) and a 2004 report from a multicenter, prospective data collection effort of LT candidates (n = 66) with POPH [29, 30]. Thus, screening and documentation of POPH has significant clinical management implications. Two key features can be elicited from echocardiography to guide the clinician toward further study: an estimate of the pulmonary artery systolic pressure and cardiac chamber size
Fig. 4 Frequency of pulmonary hemodynamic patterns documented by right-sided heart catheterization (RHC) that followed screening transthoracic echocardiography; RHC was accomplished if right ventricular systolic pressure was more than 50 mmHg. (Reproduced from [23] with permission)
M.J. Krowka
and function [31–33]. Patients with advanced liver disease commonly have a hyperdynamic circulatory state that causes high flow in the pulmonary vascular bed. Cardiac and/or renal dysfunction may complicate liver diseases, leading to excess central volume. Less commonly, obstruction to pulmonary artery flow due to endothelial/smooth muscle proliferation and/or in situ thrombosis that characterizes POPH may occur. Importantly, any or all of these mechanisms may result in increased pulmonary artery systolic pressure in a given patient that can be estimated by the right ventricular systolic pressure (as long as the pulmonic valve is normal) using the modified Bernoulli equation. In addition, measurement of the left atrial size may help discern volume status and left-sided heart function [32]. Finally, qualitative assessment of right ventricular size and function can be made, as well as documentation of abnormal shape of the left ventricle (“D” shape) in the setting of severe pulmonary artery hypertension [33]. In the largest prospective screening and diagnostic study reported to date from 1,235 patients seen at the Mayo Clinic Liver Transplant Program, 101 (8.2%) underwent right-sided heart catheterization because the right ventricular systolic pressure was found to be greater than 50 mmHg [23] (Fig. 4). Of all patients screened, 66 of 1,235 (5.3%) had both increased MPAP and increased PVR, thus fulfilling the POPH diagnostic criteria (Table 2). Selected pulmonary hemodynamic relationships observed in POPH patients are shown graphically in Fig. 5. A high flow state with normal PVR was documented in 35 patients (35%) with right ventricular systolic pressure greater than 50 mmHg. Clinically, normal PVR patients would not need pulmonary vasomodulating therapy, nor would they
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Table 2 Pulmonary hemodynamic patterns associated with advanced liver disease (mean right-sided heart catheterization data; n = 101) MPAP (mmHg) CO (L/min) PVR (dyn s cm–5) PAOP (mmHg) TPG (mmHg) 31 ± 9 8.6 ± 2.6* 142 ± 58 16 ± 6* High flow (decreased PVR) (n = 35) 28 ± 8 8.2 ± 2.3 154 ± 60 12 ± 2 Normal volume (n = 20) 34 ± 10 9.1 ± 3.0 125 ± 52 21 ± 4 Increased volume (n = 15) POPH (increased PVR) (n = 66) 49 ± 11* 6.1 ± 2.0 533 ± 247* 12 ± 6 Normal volume (n = 50) 48 ± 11 5.9 ± 2.0 571 ± 257 10 ± 3 Increased volume (n = 16) 53 ± 9 6.8 ± 2.0 407 ± 171 21 ± 5 CO cardiac output, *p 37 12 patients 0 patients p = 0.036 (Fisher’s exact) From Smith et al. [185] with permission from Elsevier
noted. This pharmacokinetic study laid the groundwork for an ongoing randomized, placebo-controlled, double blind trial of intravenously administered citrulline in children at risk for postoperative PH after congenital cardiac surgery. Further research with both orally administered and intravenously administered citrulline is needed.
9.3.5 Sildenafil Therapy Sildenafil is a PDE5 inhibitor that blocks degradation of cGMP. Use of both orally administered sildenafil (clinically available) and intravenously administered sildenafil (clinically not available) has been reported in the postoperative period in small series of patients. In ten infants with severe postoperative PH also being treated with iNO, orally administered sildenafil lowered PAP without significant effects on systemic pressure or oxygenation [187]. Larger doses of orally administered sildenafil up to 2 mg/kg did not offer any added benefit. However, absorption of an oral medication in the immediate postoperative period may be erratic and limit effectiveness. In a randomized-sequence open-label study in 15 patients at risk for postoperative PH, intravenously administered sildenafil was equally effective as iNO at reducing PAP but was also associated with a drop in systemic arterial pressure [188]. More concerning was an increase in the A–a gradient associated with use of sildenafil, presumably due to reversal of hypoxic pulmonary vasoconstriction and increased intrapulmonary shunting. Intravenously administered sildenafil is presently not commercially available, which limits further investigation. Despite biologic plausibility as a therapy for PH, sildenafil has not been adequately studied in pediatric patients with perioperative PH.
9.3.6 Aerosolized Prostacyclin * Vanderbilt University has filed for patents and licensed citrulline as a therapeutic agent with Asklepion Pharmaceuticals. Frederick Barr has had grant support from Asklepion Pharmaceuticals for the study of intravenously administered citrulline.
Iloprost, a commercially available prostacyclin analog, is of potential interest as a selective pulmonary vasodilator but, like the previously discussed therapeutic agents, has not been
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adequately studied in children with postoperative PH. Two small case series reported functional responses to inhaled iloprost with a drop in mean PAP without systemic side effects. No randomized controlled trials have been reported in this population. Further investigation is warranted.
9.4 Management of PH in the Setting of Repaired CHD There are no evidence-based therapies for children with severe disease following surgical repair of their cardiac defects. Generally, positive anecdotal results have been reported using prostacyclin analogs [171, 189, 190], endothelin receptor blockade [22, 191–193], and phosphodiesterase inhibition [194] in pediatric patient groups, which contrasts with the universally dismal prognosis in the absence of therapy. The premature infant with PH and left-to-right shunt lesions deserves special mention. Beyond standard anticongestive therapies, the benefits of operative or transcatheter repair of structural heart disease must be weighed against the inherent risks. Attention to coexisting comorbidites, such as intraventricular hemorrhage, necrotizing enterocolitis, chronic aspiration, and airway abnormalities must also be considered. Lim and Matherne reported on the closure of an atrial septal defect in such a child with mechanical ventilatory dependence using transcatheter techniques [195]. Although it is not recommended that all patients with atriallevel shunts with BPD undergo closure, as many defects can and will close spontaneously, this therapeutic strategy represents an important shift in perspective toward less invasive early intervention in an at-risk patient population.
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disease and elevations in PAP can progress from mild to severe in a relatively short time frame. Despite its limitations, Doppler echocardiography is the most readily available tool for noninvasive and serial screening. A framework for prospective identification and ongoing surveillance of infants with persistent pulmonary disease has been developed and implemented at Vanderbilt Children’s Hospital (Fig. 5). We propose that infants who remain ventilated or who have a fractional inspired oxygen requirement greater than 0.3 be prospectively screened at 30 days of life and at least monthly thereafter for echocardiographic evidence of PH (Fig. 5, group A). More frequent assessments are recommended for those in whom mild PH is evident (Fig. 5, group B) and for those with moderate to severe PH or for whom therapy has been initiated (Fig. 5, group C). This approach is predicated on the knowledge that disease severity can progress or regress over time and that a previously normal echocardiogram may not remain normal in the face of ongoing lung or heart disease. Therefore, disease assessment must bridge the transition from NICU or pediatric intensive care unit (PICU) to outpatient management. A multidisciplinary clinic format involving pediatric pulmonology and pediatric cardiology departments may offer the optimal approach to long-term follow-up and management. There are no well-publicized guidelines for the appropriate medical workup of an infant or child newly diagnosed with later-onset PH. We suggest that this evaluation should include an assessment of gastroesophageal reflux and chronic aspiration, and the adequacy of ventilation and oxygenation. Poor nutrition is frequently coexistent; its correction is critical to lung growth and recovery. Although the ideal oxygen saturation target for this population is unknown, it is clearly important to avoid recurrent hypoxemic events. For infants with BPD and a diagnosis of PH, it may be necessary to adjust upward the oxygen saturation targets currently thought
10 Proposed Guidelines for Surveillance and Management It is unfortunate that the diagnosis of PH in children is often not made until advanced stages of the disease. This is largely because of lack of guidelines for proactive surveillance and limitations of the current techniques for assessment of PH in the pediatric population. An unproven tenet is that identification of infants and children at earlier stages of PH will result in more effective interventions and improved outcomes. For this to occur, guidelines for prospective identification of patients at risk and algorithms for longitudinal surveillance and management must be developed. The dynamic nature of PH in the pediatric patient must be considered when designing such algorithms; PH can develop at nearly any time in the course of underlying lung or heart
Fig. 5 A framework for prospective identification and ongoing surveillance by echocardiogram of infants with BPD at risk for later-onset PH
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Fig. 6 Suggested algorithm for medical evaluation of the infant and young child with PH and chronic lung disease. (Modified with permission from McLaughlin and McGoon [199])
to be beneficial in the first weeks of life to avoid the development of BPD. On an individual basis, consideration should be given to a metabolic workup for inborn errors of metabolism, storage diseases and thyroid dysfunction, hematologic diseases such as SCD, coagulation defects including genetic thrombophilias, such as factor V Leiden mutations, and infectious diseases, including HIV (Fig. 6).
11 Gaps in Knowledge Much work must be done before we can translate pharmacologic interventions into evidenced-based therapies for infants and children with PH and structural lung and cardiac disease. This goal will require not just additional basic laboratory research but also a focused effort from the clinical community. We urge the pediatric cardiology community to define and adopt standardized echocardiographic end points for age-based classifications of mild, moderate, and severe PH. This will facilitate prospective monitoring of disease progression and response to therapy. Also needed are age-appropriate and clinically relevant end points for pediatric clinical trials. The 6-min walk test is not useful for infants and most young children with PH. Attention should be given to developing therapies for RV dysfunction in children. We also applaud efforts to develop new imaging modalities for PH, including MRI assessment of lung volume, lung vascular area, and right-sided heart geometry. The validity and predictive power of biomarkers,
such as plasma BNP and troponin, for infants and children with PH and myocardial dysfunction must be determined. Criteria should be developed for referral of patients with progressive PH to an interventional pediatric cardiologist for cardiac catheterization and vasodilator testing. The optimal multidisciplinary approach involving the pediatric pulmonologist, pediatric cardiologist, dietician, and others in the outpatient management of patients with chronic lung or cardiac disorders who are at risk of or who have documented PH must be studied and implemented across centers. More data are needed on the natural history and outcomes of children, including preterm infants, who develop PH beyond the first weeks of life. A wide range of treatment regimens including combination therapies are being used clinically, despite the lack of appropriately powered and randomized efficacy and safety trials. A mechanism beyond the occasional case series is needed for recording the responses to these therapies and the outcomes of these patients. Enrollment of infants and children with PH that persists or develops beyond the first weeks of life in to a pediatric PH registry, such as the TOPP Registry, could potentially meet this need. Although it is true that such registries are observational and descriptive in nature, the systematic accrual of data could help clarify patient phenotypes, genetic and clinical risk factors, and practice patterns. A registry could provide much needed information about the timing and antecedents of diagnosis, the types of therapies employed and whether early detection and treatment confers a benefit [196]. Designed correctly, a registry would provide an accessible cohort to generate and test hypotheses, as demonstrated
75 Pediatric Pulmonary Hypertension: An Integrated View from Pediatric Subspecialists
by the tremendous strides made in the genetic understanding of heritable PAH with simultaneous discoveries of BMPR2 mutations made possible by the Vanderbilt and Columbia PAH registries [197, 198]. A database registry will ultimately enable the conduct of clinical trials by better defining prevalence and phenotypes and assembling random treatment strategies into a comprehensible pattern. Formation of a pediatric PH clinical trials network would be an important step toward addressing the significant obstacles to the conduct of randomized clinical trials in this patient population. These obstacles include limited patient numbers in any one center, disease heterogeneity, lack of standardized definitions for disease onset, severity and progression, multiple treatment paradigms, limited funding, and ethical considerations. Much work remains to achieve similar success for neonates and children with PH associated with structural changes in the lungs as has been realized for infants with reactive pulmonary vascular disease and PPHN. Effective treatment will most likely require long-term administration of a combination of vasodilators and innovative therapies that block progression of the vascular remodeling process and promote regression of established vascular changes. Potential targets for pharmacologic intervention include extracellular matrix components, vasoregulatory and antiproliferative proteins, angiogenic proteins, and intracellular signal transduction cascades. Laboratory and clinical studies are needed to define the roles of genetic polymorphisms, inflammation, and oxidants, vasodilator/vasoconstrictor imbalance, ion channel dysfunction, angiogenesis and differentiation, thrombosis, and hemodynamics in the pathogenesis of neonatal and pediatric pulmonary hypertensive diseases. The spectrum of pediatric PH is broad, crossing disciplines, age groups, and the boundaries between acute and chronic care. A successful institutional pediatric PH program must acknowledge these realities and be both longitudinal and comprehensive in scope. Collaboration between cardiologists, neonatologists, pulmonologists, and intensivists is needed as the diseases producing PH in children span these subspecialty borders and require multidisciplinary expertise. A comprehensive pediatric PH program would also include a multidisciplinary team of investigators with expertise in basic and clinical trials research, epidemiology, pharmacology, genetics, and informatics. The goals and scope of such a program would include basic scientific discovery, translation of discovery into interventional clinical trials, epidemiologic-outcomes-based research, longitudinal clinical care, and data collection for purposes of quality improvement and dissemination of potentially better practices. Core principles would include disease prevention and stabilization, communication, and continuity of care, with special attention to these tenets during vulnerable windows between NICU or PICU discharge and outpatient management. These
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collaborative efforts will likely stimulate advances in the broader field of PH. As pediatric and adult subspecialists and investigators collectively approach the disease from different directions and their unique perspectives, we may then begin to fill the gaps in our knowledge and in the care we provide our patients. Acknowledgements Supported by award numbers R01 HL075511 (J.L.A.), R01 HL068572 (C.D.F), and 5RO1HL073317 (F.E.B.) from the National Heart, Lung, and Blood Institute (NHLBI). The content is solely the responsibility of the authors and does not necessarily represent the official views of the NHLBI or the National Institutes of Health. The authors acknowledge Robin Roller for her editorial assistance and to Neeru Kaushik for her assistance with the MRI image.
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Chapter 76
Persistent Pulmonary Hypertension of the Newborn: Mechanisms and Treatment Steven H. Abman, Robin H. Steinhorn, and Judy L. Aschner
Abstract Some infants fail to achieve or sustain the normal decrease in pulmonary vascular resistance (PVR) at birth, leading to severe respiratory distress and hypoxemia, which is referred to as persistent pulmonary hypertension of the newborn (PPHN). PPHN is a major clinical problem, affecting up to 10% of full-term and preterm infants in neonatal intensive care, and contributing significantly to high morbidity and mortality. Newborns with PPHN are at risk for severe asphyxia and its complications, including death, chronic lung disease, and neurodevelopmental sequelae, and other problems. This chapter will review the pathophysiology of PPHN, and clinical strategies that utilize a physiologic approach to the treatment of newborns with severe PPHN. Keywords Persistent pulmonary hypertension in the newborn • Preterm infants • Development defect
1 Introduction Some infants fail to achieve or sustain the normal decrease in pulmonary vascular resistance (PVR) at birth (described in Chap. 9), leading to severe respiratory distress and hypoxemia, which is referred to as persistent pulmonary hypertension of the newborn (PPHN). PPHN is a major clinical problem, affecting up to 10% of full-term and preterm infants in neonatal intensive care, and contributing significantly to high morbidity and mortality [1, 2]. Newborns with PPHN are at risk for severe asphyxia and its complications, including death, chronic lung disease, and neurodevelopmental sequelae, and other problems. This chapter will review the pathophysiology of PPHN, and clinical strategies that utilize a physiologic approach to the treatment of newborns with severe PPHN.
S.H. Abman (*) Department of Pediatrics, The Children’s Hospital, 1717 East Arizona Avenue, Denver, CO 80210, USA e-mail:
[email protected] 2 Pathophysiology of Experimental PPHN Mechanisms that lead to the failure of PVR to fall at birth have been pursued in various animal models to better understand the pathogenesis and pathophysiology of PPHN. Such models have included exposure to acute or chronic hypoxia after birth, chronic hypoxia in utero, placement of meconium into the airways of neonatal animals, sepsis, and other treatment. Although each model demonstrates interesting physiologic changes that may be especially relevant to particular clinical settings, most studies have examined only brief changes in the pulmonary circulation, and mechanisms underlying altered lung vascular structure and function of PPHN remain poorly understood. Clinical observations that neonates with severe PPHN who die during the first few days after birth already have pathologic signs of chronic pulmonary vascular disease suggest that intrauterine events may play an important role in this syndrome [3]. Adverse intrauterine stimuli during late gestation, such as abnormal hemodynamics, changes in substrate or hormone delivery to the lung, hypoxia, and inflammation, may potentially alter lung vascular function and structure, contributing to abnormalities of postnatal adaptation. Several investigators have examined the effects of chronic intrauterine stresses, such as hypoxia and hypertension, in animal models in the attempt to mimic the clinical problem of PPHN. Whether chronic hypoxia alone can cause PPHN is controversial. A past report suggested that maternal hypoxia in rats increases pulmonary vascular smooth muscle thickening in newborns, but this observation has not been reproduced in maternal rats or guinea pigs with more extensive studies [4]. Pulmonary hypertension induced by early closure of the ductus arteriosus in fetal lambs alters lung vascular reactivity and structure, causing the failure of postnatal adaptation at delivery, and providing an experimental model of PPHN [5–7]. Over days, pulmonary artery pressure and PVR progressively increase, but flow remains low and PaO2 is unchanged [7]. Marked right ventricular hypertrophy and structural remodeling of small pulmonary arteries develops after 8 days of hypertension. After delivery, these lambs have persistent elevation of PVR despite mechanical ventilation
J.X.-J. Yuan et al. (eds.), Textbook of Pulmonary Vascular Disease, DOI 10.1007/978-0-387-87429-6_76, © Springer Science+Business Media, LLC 2011
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with high oxygen concentrations. Studies with this model have shown that chronic hypertension without high flow can alter fetal lung vascular structure and function. This model is further characterized by endothelial cell dysfunction and altered smooth muscle cell reactivity and growth, including findings of impaired NO production and activity and downregulation of lung endothelial NO synthase messenger RNA and protein expression [8–12]. Fetal pulmonary hypertension is also associated with decreased cyclic GMP (cGMP) concentrations, associated with decreased soluble guanylate cyclase and upregulated cGMP-specific phosphodiesterase type 5 (PDE5) activities, suggesting further impairments in downstream signaling [13–15] (Fig. 1). Thus, multiple alterations in the NO-cGMP cascade appear to play an essential role in the pathogenesis and pathophysiology of experimental PPHN, contributing to altered structure and function of the developing lung circulation, and leading to failure of postnatal cardiorespiratory adaptation. Recent evidence indicates that excessive production of reactive oxygen species such as superoxide in the pulmonary vasculature may further contribute to the disruption in NO-cGMP signaling in this model [12, 16]. Upregulation of endothelin-1 (ET-1) may also contribute to the pathophysiology of PPHN. Circulating levels of ET-1, a potent vasoconstrictor and co-mitogen for vascular smooth muscle cell hyperplasia, are increased in human newborns with severe PPHN [17]. In the experimental model of PPHN due to compression of the ductus arteriosus in fetal sheep, lung ET-1 messenger RNA and protein content is markedly increased, and the balance of endothelin receptors is altered, favoring vasoconstriction [18, 19]. Chronic inhibition of the endothelin A receptor attenuates the severity of pulmonary hypertension, decreases pulmonary artery wall thickening, and improves the fall in PVR at birth in this model [20]. Thus, experimental studies have shown the important role of
Fig. 1 Abnormalities of the NO-cyclic GMP cascade in the pathophysiology of persistent pulmonary hypertension of the newborn (PPHN). (Reprinted with permission from Abman [65])
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the NO-cGMP cascade and the ET-1 system in the regulation of vascular tone and reactivity of the fetal and transitional pulmonary circulation. There is mounting evidence that oxidant stress plays an important role in the pathogenesis of PPHN. Increases in the levels of reactive oxygen species such as superoxide and hydrogen peroxide (H2O2) in the smooth muscle and adventitia of pulmonary arteries have been demonstrated in the ductal ligation model of persistent pulmonary hypertension [16, 21]. There appear to be multiple sources for elevated production of reactive oxygen species, including mitochondrial dysfunction, increased expression and activity of NADPH oxidase, and uncoupled nitric oxide synthase activity [16, 22–24]. Once present in the lung, elevated concentrations of reactive oxygen species promote vasoconstriction directly, and through multiple mechanisms, including increased endothelin levels [25], and oxidization of free fatty acids to create vasoconstrictor metabolites such as isoprostanes [26]. Superoxide anions rapidly combine with NO, inactivating it and in the process forming peroxynitrite, a potent oxidant with the potential to produce vasoconstriction and cytotoxicity. Increased levels of reactive oxygen species in the pulmonary vasculature of the ductal ligation model promote dysfunction of NO-cGMP signaling at multiple steps in the pathway, including blunted nitric oxide synthase expression, uncoupled endothelial nitric oxide synthase activity (thus further promoting reactive oxygen species production), and increased activity and expression of cGMPspecific phosphodiesterases [12, 27]. Superoxide dismutases (SOD) catalyze the conversion of superoxide anions to H2O2 and O2. Owing to the efficiency of the reaction between NO and superoxide, the local concentration of SOD is a key determinant of the biological half-life of endogenous NO [28]. Decreased SOD activity has been observed in models of PPHN, although characterization of alterations for specific isoforms is ongoing. Further evidence for the critical role of SOD is the recent observation that administration of a single intratracheal dose of recombinant human SOD (at birth or at 4 h of age) in neonatal lambs with PPHN produced a sustained increase in oxygenation over a 24-h period, reduced production of isoprostanes and peroxynitrite, and restored normal endothelial nitric oxide synthase expression and function. Finally, in addition to the vasoactive mediators described above, it has become clear that alterations of growth factors, such as vascular endothelial growth factor and platelet-derived growth factor (PDGF), are likely to play key roles in the modulation of vascular maturation, growth, and structure. For example, inhibition of PDGF-B attenuates smooth muscle hyperplasia in experimental pulmonary hypertension in fetal lambs, suggesting a potential role in the pathogenesis of PPHN [29]. Additional new data suggest that maternal exposure to selective serotonin reuptake inhibitors (SSRIs) during late gestation is associated with a sixfold increase in the prevalence of PPHN [30], although it is not clear how many infants
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developed severe disease. Newborn rats exposed in utero to fluoxetine develop pulmonary vascular remodeling, abnormal oxygenation, and higher mortality when compared with vehicle-treated controls [31]. However, these findings showed only mild changes in right ventricular hypertrophy and pulmonary vascular remodeling in the neonatal rat pups, with minimal changes in vasoreactivity after maternal SSRI exposure. Whether these effects were related to a direct impact on the fetal lung circulation or were secondary to altered maternal or umbilical–placental function remains unknown. As SSRIs have been reported to reduce pulmonary vascular remodeling in adult models of pulmonary hypertension, these findings also serve to highlight the unique vulnerability of fetal pulmonary vascular development.
3 Clinical Aspects of PPHN The first reports of PPHN described term newborns with profound hypoxemia who lacked radiographic evidence of parenchymal lung disease and echocardiographic evidence of structural cardiac disease (Fig. 2). In these patients, hypoxemia was caused by marked elevations of PVR leading to right-to-left extrapulmonary shunting of blood across the
Fig. 2 Chest X-rays illustrating common disorders associated with PPHN
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patent ductus arteriosus (PDA) or foramen ovale (PFO) during the early postnatal period. Owing to the persistence of high PVR and blood flow through these “fetal shunts,” the term “persistent fetal circulation” was originally used to describe this group of patients. Consequently, it was recognized that this physiologic pattern can complicate the clinical course of neonates with diverse causes of hypoxemic respiratory failure. As a result, the term “PPHN” has been considered as a syndrome, and is currently applied more broadly to include neonates that have a similar physiology in association with different cardiopulmonary disorders, such as meconium aspiration, sepsis, pneumonia, asphyxia, congenital diaphragmatic hernia (CDH), and respiratory distress syndrome (RDS). Striking differences exist between these conditions, and the mechanisms that contribute to high PVR can vary between these diseases. However, these disorders are included in the syndrome of PPHN owing to common pathophysiologic features, including sustained elevation of PVR leading to hypoxemia due to right-to-left extrapulmonary shunting of blood flow across the ductus arteriosus or PFO. In many clinical settings, hypoxemic respiratory failure in term newborns is often presumed to be associated with PPHN-type physiology; however, hypoxemic term newborns can lack echocardiographic findings of extrapulmonary shunting across the PDA or PFO. Thus, “PPHN” should be reserved to
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describe neonates in whom extrapulmonary shunting contributes to hypoxemia and impaired cardiopulmonary function. Recent estimates suggest an incidence for PPHN of 1.9 per 1,000 live births, or an estimated 7,400 cases per year [32]. Diseases associated with PPHN are often classified within one of three categories: (1) maladaptation – vessels are presumably of normal structural but have abnormal vasoreactivity; (2) excessive muscularization – increased smooth muscle cell thickness and increased distal extension of muscle to vessels which are usually nonmuscular; and (3) underdevelopment – lung hypoplasia associated with decreased pulmonary artery number. This designation is imprecise, however, and high PVR in most patients likely involves overlapping changes among these categories. For example, neonates with CDH are primarily classified as having vascular “underdevelopment” due to lung hypoplasia, yet lung histologic examination of fatal cases typically shows marked muscularization of pulmonary arteries, and clinically, these patients can respond to vasodilator therapy. Similarly, neonates with meconium aspiration often have clinical evidence of altered vasoreactivity, but excessive muscularization is often found at autopsy. As described above, autopsy studies of fatal PPHN demonstrate severe hypertensive structural remodeling even in newborns who die shortly after birth, suggesting that many cases of severe disease are associated with chronic intrauterine stress. However, the exact intrauterine events that alter pulmonary vascular reactivity and structure are poorly understood. Epidemiology studies have demonstrated strong associations between PPHN and maternal smoking and ingestion of cold remedies that include aspirin or other nonsteroidal anti-inflammatory products [33, 34]. Since these agents can induce partial constriction of the ductus arteriosus, it is possible that pulmonary hypertension due to antenatal ductal narrowing contributes to PPHN (see later). Other perinatal stresses, including placenta previa and abruption, and asymmetric growth restriction, are associated with PPHN; however, most neonates who are exposed to these prenatal stresses do not develop PPHN. Circulating levels of l-arginine, the substrate for NO, are decreased in some newborns with PPHN, suggesting that impaired NO production may contribute to the pathophysiology of PPHN. It is possible that genetic factors increase susceptibility for pulmonary hypertension. A recent study reported strong links between PPHN and polymorphisms of the carbamoyl phosphate synthase gene [35]. However, the importance of this finding is uncertain, and further work is needed in this area. Studies of adults with idiopathic primary pulmonary hypertension have identified abnormalities of bone morphogenetic protein receptor genes; whether polymorphisms of genes for the bone morphogenetic protein or transforming growth factor b receptors, other critical growth factors, vasoactive substances, or other products increase the risk for some newborns to develop PPHN is unknown.
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4 Clinical Presentation and Evaluation Clinically, PPHN is most often recognized in term or nearterm neonates, but clearly can occur in premature neonates as well (Table 1). PPHN typically presents as respiratory distress and cyanosis within 6–12 h of birth. Although PPHN is often associated with indicators of perinatal distress, such as asphyxia, low Apgar scores, and meconium staining, idiopathic PPHN can present without signs of acute perinatal distress. Laboratory findings can include low levels of glucose, hypocalcemia, hypothermia, polycythemia, or thrombocytopenia. Radiographic findings are variable, depending upon the primary disease associated with PPHN. Classically, Table 1 Disorders associated with neonatal pulmonary hypertension Pulmonary
Meconium aspiration syndrome Respiratory distress syndromea Lung hypoplasia – primary Congenital diaphragmatic hernia Pneumonia/sepsis Idiopathic Transient tachypnea of the newborn Alveolar-capillary dysplasia Associated abnormalities in lung development: – Congenital lobar emphysema (rare association) – Cystic adenomatoid malformation (rare association) – Idiopathic, with impaired distal alveolarization – Pulmonary interstitial glycogenosis – Surfactant B and C deficiency – ABCA 3 deficiency – Others
Cardiovascular
Myocardial dysfunction (asphyxia, infection, stress) Structural cardiac disease: – Mitral stenosis, cor triatriatum – Endocardial fibroelastosis – Pompe’s disease – Aortic atresia, coarctation of the aorta, interrupted aortic arch – Transposition of the great vessels – Ebsteins’s anomaly, tricuspid atresia Hepatic arteriovenous malformations Cerebral arteriovenous malformations Total anomalous pulmonary venous return Pulmonary vein stenosis (isolated) Pulmonary atresia
Neuromuscular disease Metabolic disease Polycythemia Thrombocytopenia Maternal drug use or smoking (especially selective serotonin reuptake inhibitors) a Term and preterm newborns
Associations with other diseases
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the chest X-ray in idiopathic PPHN shows an oligemic lung, normally or slightly hyperinflated, and lacking parenchymal infiltrates. In general, the degree of hypoxemia is disproportionate to the severity of radiographic evidence of lung disease. Not all term newborns with hypoxemic respiratory failure have PPHN-type physiology [36]. Hypoxemia in the newborn can be due to extrapulmonary shunt, as described earlier, in which high pulmonary artery pressure at systemic levels leads to right-to-left shunting of blood flow across the PDA or PFO. However, in many infants intrapulmonary shunt or ventilation–perfusion mismatch is the predominant abnormality, in which hypoxemia results from the lack of mixing of blood with aerated lung regions owing to parenchymal lung disease, without the shunting of blood flow across the PDA and PFO. In the latter setting, hypoxemia is related to the amount of pulmonary arterial blood that perfuses nonaerated lung regions. Although PVR is often elevated in hypoxemic newborns without PPHN, high PVR does not contribute significantly to hypoxemia in these cases. Several factors can contribute to high pulmonary artery pressure in neonates with PPHN-type physiology. Pulmonary hypertension can be due to vasoconstriction or structural vascular lesions that directly increase PVR. Changes in lung volume in neonates with parenchymal lung disease can also be an important determinant of PVR. PVR increases at low lung volumes owing to dense parenchymal infiltrate and poor lung recruitment, or at high lung volumes owing to hyperinflation associated with overdistension or gas trapping. Cardiac disease is also associated with PPHN. High pulmonary venous pressure due to left ventricular dysfunction (e.g., asphyxia or sepsis) can also elevate pulmonary artery pressure, causing right-to-left shunting, with little vasoconstriction. In this setting, enhancing cardiac performance and systemic hemodynamics may lower pulmonary artery pressure more effectively than promoting pulmonary vasodilation. Thus, understanding the cardiopulmonary interactions is key to improving outcome in PPHN. PPHN is characterized by hypoxemia that is poorly responsive to supplemental oxygen. In the presence of right-to-left shunting across the PDA, “differential cyanosis” is often present, which is difficult to detect by physical examination, and is defined by a difference in PaO2 between right radial artery and descending aorta values of more than 10 Torr, or an O2 saturation gradient of more than 5%. However, postductal desaturation can be found in ductus-dependent cardiac diseases, including hypoplastic left-sided heart syndrome, coarctation of the aorta, and interrupted aortic arch. The response to supplemental oxygen can help to distinguish PPHN from primary lung or cardiac disease. Although supplemental oxygen traditionally increases PaO2 more readily in lung disease than in cyanotic heart disease or PPHN, this may not be obvious with more advanced parenchymal lung disease. Marked
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improvement in SaO2 (increase to 100%) with supplemental oxygen suggests the presence of V/Q mismatch due to lung disease or highly reactive PPHN. Most patients with PPHN have at least a transient improvement in oxygenation in response to interventions such as high levels of inspired oxygen and/or mechanical ventilation. Acute respiratory alkalosis induced by hyperventilation to achieve PaCO2 < 30 Torr and pH >7.50 may increase PaO2 to more than 50 Torr in PPHN, but rarely in cyanotic heart disease. The echocardiogram plays an important diagnostic role and is an essential tool for managing newborns with PPHN. The initial echocardiographic evaluation rules out structural heart disease causing hypoxemia or ductal shunting (e.g., coarctation of the aorta and total anomalous pulmonary venous return). Further, as stated already, not all term newborns with hypoxemia have PPHN physiology. Although high pulmonary artery pressure is commonly found in association with neonatal lung disease, the diagnosis of PPHN is uncertain without evidence of bidirectional or predominantly right-to-left shunting across the PFO or PDA. Echocardiographic signs suggestive of pulmonary hypertension (e.g., increased right ventricular systolic time intervals and septal flattening) are less helpful. In addition to demonstrating the presence of PPHN physiology, the echocardiogram is critical for the evaluation of left ventricular function and diagnosis of anatomic heart disease, including such “PPHN mimics” as coarctation of the aorta, total anomalous pulmonary venous return, and hypoplastic left-sided heart syndrome,. Studies should carefully assess the predominant direction of shunting at the PFO as well as the PDA. Although right-to-left shunting at the PDA and PFO is typical for PPHN, predominant right-to-left shunting at the PDA but left-to-right shunting at the PFO may help to identify the important role of left ventricular dysfunction in the underlying pathophysiology. In the presence of severe left ventricular dysfunction with pulmonary hypertension, pulmonary vasodilation alone may be ineffective in improving oxygenation. In this setting, efforts to reduce PVR should be accompanied by targeted therapies to increase cardiac performance and decrease left ventricular afterload. In the setting of impaired left ventricular performance, cardiotonic therapies that increase systemic vascular resistance may further worsen left ventricular function and increase pulmonary artery pressure. Thus, careful echocardiographic assessment provides invaluable information about the underlying pathophysiology and will help guide the course of treatment.
5 Treatment of PPHN In general, management of the newborn with PPHN includes the treatment and avoidance of hypothermia, hypoglycemia,
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hypocalcemia, anemia, and hypovolemia; correction of metabolic acidosis; diagnostic studies for sepsis; and serial monitoring of arterial blood pressure, pulse oximetry (pre- and postductal), and transcutaneous PCO2. Therapy includes optimization of systemic hemodynamics with volume and cardiotonic therapy (dobutamine, dopamine, and milrinone), to enhance cardiac output and systemic O2 transport. Failure to respond to medical management, as evidenced by failure to sustain improvement in oxygenation with good hemodynamic function, often leads to treatment with extracorporeal membrane oxygenation (ECMO) [37]. Although ECMO can be a life-saving therapy, it is costly, labor-intensive, and can have severe side effects, such as intracranial hemorrhage. Since arteriovenous ECMO usually involves ligation of the carotid artery, the potential for acute and long-term CNS injuries continues to be a major concern. The goal of mechanical ventilation is to improve oxygenation, achieve “optimal” lung volume to minimize the adverse effects of high or low lung volumes on PVR, and minimize the risk for lung injury (“volutrauma”). Mechanical ventilation using inappropriate settings can produce acute lung injury (ventilator-induced lung injury, VILI), causing pulmonary edema, decreased lung compliance, and promoting lung inflammation owing to increased cytokine production and lung neutrophil accumulation. The development of VILI is an important determinant of the clinical course and the eventual outcome of newborns with hypoxemic respiratory failure, and postnatal lung injury worsens the degree of pulmonary hypertension [38]. On the other hand, failure to achieve adequate lung volumes (functional residual capacity) contributes to hypoxemia and high PVR in newborns with PPHN. Some newborns with parenchymal lung disease with PPHN physiology improve oxygenation and decrease right-to-left extrapulmonary shunting with aggressive lung recruitment during high-frequency oscillatory ventilation (HFOV) [39] or with an “open lung approach” of higher positive end-expiratory pressure with low tidal volumes, as more commonly utilized in older patients with acute RDS [40]. Marked controversy and variability exists between centers regarding the use of hyperventilation to achieve alkalosis to improve oxygenation. Past studies have clearly shown that acute hyperventilation can improve PaO2 in neonates with PPHN, providing a diagnostic test and potential therapeutic strategy. However, there are many issues with the use of hypocarbic alkalosis for prolonged therapy. Depending upon the ventilator strategy and underlying lung disease, hyperventilation is likely to increase VILI, and the ability to sustain decreased PVR during prolonged hyperventilation is unproven. Experimental studies suggest that the response to alkalosis is transient, and that alkalosis may paradoxically worsen pulmonary vascular tone, reactivity, and permeability edema [41, 42]. In addition, prolonged hyperventilation
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reduces cerebral blood flow and oxygen delivery to the brain, potentially worsening neurodevelopmental outcome. Additional therapies, including infusions of sodium bicarbonate, surfactant therapy, and the use of intravenous vasodilators, are also highly variable between centers. Although surfactant improved oxygenation and reduced ECMO in some lung diseases, such as meconium aspiration and RDS, a multicenter trial showed benefit in infants with relatively mild disease, and failed to show a reduction in ECMO utilization in the subset of newborns with idiopathic PPHN [43]. The use of intravenous vasodilator drug therapy, with agents such as tolazoline, magnesium sulfate, prostacyclin, and sodium nitroprusside, is also controversial owing to the nonselective effects of these agents on the systemic circulation. Systemic hypotension may worsen right-to-left shunting, impair oxygen delivery, and worsen gas exchange in patients with parenchymal lung disease. In addition, the initial response to such agents as tolazoline is often transient, and severe adverse effects such as gastrointestinal hemorrhage have been reported. Endotracheal administration of vasodilators, including tolazoline, sodium nitroprusside, and prostacyclin, may cause selective pulmonary vasodilation and minimize systemic hypotension. However, these data are largely limited to animal studies, and evidence is needed to confirm the safety and efficacy of this approach in humans. Inhaled nitric oxide (iNO) therapy at low doses (5–20 ppm) improves oxygenation and decreases the need for ECMO therapy in patients with diverse causes of PPHN [44–49] (Fig. 3). Multicenter clinical trials support the use of iNO in near-term (more than 34 weeks’ gestation) and term newborns, although the use of iNO in infants of less than 34 weeks’ gestation remains largely investigational. Studies support the use of iNO in infants who have hypoxemic respiratory failure with evidence of PPHN, who require mechanical ventilation and high inspired oxygen concentrations. The most common criterion employed has been the oxygenation index (OI; mean airway pressure times FiO2 times 100 divided by PaO2). Although clinical trials commonly allowed for
Fig. 3 Summary of multicenter randomized trials of inhaled NO in term newborns with hypoxemia and PPHN, showing reduction of extracorporeal membrane oxygenation utilization
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enrollment with an OI of more than 25, the mean OI at entry to the study in multicenter trials was approximately 40. It is unclear whether infants with less severe hypoxemia benefit from iNO therapy [50]. The first studies of iNO treatment in term newborns reported initial doses that ranged from 20 to 80 ppm [46, 51]. In the former report, rapid improvement in PaO2 was achieved at low doses (20 ppm) for 4 h, and this response was sustained with prolonged therapy at 6 ppm. Subsequent multicenter studies confirmed the efficacy of this dosing strategy [44, 48], and showed that increasing the dose beyond 20 ppm in nonresponders did not improve outcomes [48]. The available evidence, therefore, supports the use of doses of iNO beginning at 20 ppm in term newborns with PPHN, since this strategy decreased ECMO utilization without increasing adverse effects. Although brief exposures to higher doses (40–80 ppm) appear to be safe, sustained treatment with 80 ppm NO increases the risk of methemoglobinemia [45]. In our practice, we discontinue use of iNO if the FiO2 is less than 0.60 and the PaO2 is more than 60 without evidence of “rebound” pulmonary hypertension or an increase in FiO2 of more than 15% after iNO withdrawal. Weaning can generally be accomplished in 4–5 days, and prolonged need for iNO therapy without resolution of disease should lead to a more extensive evaluation to determine whether previously unsuspected anatomic lung or cardiovascular disease is present (e.g.,, pulmonary venous stenosis, alveolar capillary dysplasia, and severe lung hypoplasia) [52]. In newborns with severe lung disease, HFOV is frequently used to optimize lung inflation and minimize lung injury. The combination of HFOV with iNO often enhances the improvement in oxygenation in newborns with severe PPHN complicated by diffuse parencyhmal lung disease and underinflation (e.g., RDS and pneumonia). A randomized, multicenter trial of infants with severe PPHN demonstrated that treatment with iNO in combination with HFOV was successful in many patients who failed to respond to HFOV or iNO alone [47]. For patients with PPHN complicated by severe lung disease, response rates for the combination of HFOV and iNO were better than those for HFOV alone or iNO with conventional ventilation. In contrast, for patients without significant parenchymal lung disease, both iNO and HFOV in combination with iNO were more effective than HFOV alone. This response to combined treatment with HFOV and iNO likely reflects both improvement in intrapulmonary shunting in patients with severe lung disease and PPHN (using a strategy designed to recruit and sustain lung volume, rather than to hyperventilate the lung) and augmented NO delivery to its site of action. Although iNO may be an effective treatment for PPHN, it should be considered only as part of an overall clinical strategy that simultaneously addresses the role of parenchymal lung disease, cardiac performance, and systemic hemodynamics.
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Although clinical improvement during iNO therapy occurs with many disorders associated with PPHN, not all neonates with acute hypoxemic respiratory failure and pulmonary hypertension respond to iNO therapy. Several mechanisms may explain the clinical variability in responsiveness to iNO therapy (Table 2). As noted above, an inability to deliver NO to the pulmonary circulation due to poor lung inflation is a major cause of poor responsiveness. In addition, poor NO responsiveness may be related to myocardial dysfunction or systemic hypotension, severe pulmonary vascular structural disease, and unsuspected or missed anatomic cardiovascular lesions (such as total anomalous pulmonary venous return, coarctation of the aorta, alveolar capillary dysplasia, pulmonary interstitial glycogenosis, and surfactant protein deficiency). Since iNO is usually delivered with high concentrations of oxygen, there is also the potential for enhanced production of reactive oxygen and reactive nitrogen metabolites, both of which may contribute to vasoconstriction and/or inadequate responses to iNO. Although hyperoxic ventilation is standard therapy for PPHN, it may be toxic to the developing lung through formation of reactive oxygen species, such as superoxide anions [53]. As noted previously, superoxide rapidly combines with and inactivates NO and in the process forms peroxynitrite; both are potent oxidants with the potential to produce vasoconstriction, cytotoxicity, and vascular smooth muscle cell proliferation. Studies in newborn lambs with pharmacologic pulmonary hypertension have indicated that iNO responsiveness is significantly blunted after even brief (30-min) periods of ventilation with 100% O2, and that oxidant stress alters NO responsiveness in part through increasing expression and activity of cGMPspecific phosphodiesterases [27, 54]. These and other studies indicate that another important mechanism of poor responsiveness to iNO may be altered smooth muscle cell responsiveness, and there are a number of emerging therapies that take advantage of our increased understanding of the cellular effects of iNO. As the response to iNO is mediated primarily by activation of soluble guanylate cyclase and cGMP-dependent protein kinase, it is logical to pursue other mechanisms that might enhance cGMP accumulation, such as inhibition of cGMP-metabolizing Table 2 Mechanisms underlying poor responses to inhaled NO Primary disease and pathophysiology Poor lung inflation Anatomic cardiac disease Left ventricular dysfunction Structural vascular disease (pulmonary venous obstruction, severe remodeling) Biochemical: increased PDE5 activity, increased O2− and ET-1 production, others
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PDE5 activity [55, 56]. Recently approved by the FDA for the treatment of male erectile dysfunction, sildenafil is a potent and highly specific PDE5 inhibitor that has been used in several preclinical studies in animal models. For instance, in lambs with experimental pulmonary hypertension, both enterically administered and aerosolized sildenafil dilate the pulmonary vasculature and augment the pulmonary vascular response to iNO [57, 58]. Intravenously administered sildenafil was found to be a selective pulmonary vasodilator with efficacy equivalent to iNO in a piglet model of meconium aspiration, although hypotension and worsening oxygenation resulted when sildenafil was used in combination with iNO [59, 60]. Data have only recently begun to emerge on the use of sildenafil alone or in combination with iNO in clinical populations, and investigations in newborn infants have been limited because sildenafil is currently available only in enteric administration form. A recent report demonstrated that enterically administered sildenafil improved oxygenation and survival in human infants with PPHN compared with a placebo [61]. Preliminary findings from a pilot study of intravenously administered sildenafil in newborns with pulmonary hypertension indicate that the infusion was generally well tolerated, with improvements in oxygenation noted in the cohorts that received higher infusion doses [62]. Similar to cGMP, cyclic AMP (cAMP) also stimulates vasodilatation, and amplification of the cAMP signaling pathway may enhance pulmonary vasodilation. Prostacyclin is a second central vasodilator that is upregulated after birth in response to ventilation of the lung. Prostacyclin stimulates adenylate cyclase to increase intracellular cAMP levels, which, similar to increased intracellular cGMP levels, lead to vasorelaxation through a decrease in intracellular calcium concentrations. Emerging data indicate that prostacyclin stimulates membrane-bound adenylate cyclase, increases cAMP levels, and inhibits pulmonary artery smooth muscle cell proliferation in vitro. Rebound pulmonary hypertension following withdrawal of iNO has been mitigated by intravenously administered prostacyclin in children with pulmonary hypertension associated with congenital heart disease. However, as noted above, the use of systemic infusions of prostacyclin may be limited by systemic hypotension. Inhaled prostacyclin has been shown to have vasodilator effects limited to the pulmonary circulation. The actions of inhaled prostacyclin and iNO appear to be additive in humans and even synergistic in some studies. Reports of inhaled prostacyclin use in neonates with PPHN are limited, but case reports indicate it may enhance oxygenation in infants that are poorly responsive to iNO [63]. Another potential approach taking advantage of cAMP signaling is inhibition of phosphodiesterase type 3 (PDE3), which metabolizes cAMP. Milrinone, a PDE3 inhibitor, has been shown to decrease pulmonary artery pressure and resistance, and to act additively with iNO in animal studies. A recent report indi-
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cates that the intravenous administration of milrinone to neonates with severe PPHN and poor iNO responsiveness was associated with improvements in oxygenation without compromising hemodynamic status [64]. Finally, although newer therapies, including HFOV and iNO, have led to a dramatic reduction in the need for ECMO therapy, ECMO remains an effective and potentially lifesaving rescue modality for severe PPHN. Current patterns of ECMO use demonstrate persistent use in neonates with CDH and patients with severe hemodynamic instability, with less need for ECMO in meconium aspiration, RDS, idiopathic PPHN, and other disorders. Ongoing laboratory and clinical studies are currently exploring newer agents that may soon have therapeutic roles in PPHN such as SOD, soluble guanylate cyclase activators and stimulators, Rho kinase inhibitors and others (Table 3).
6 Summary PPHN is a clinical syndrome that is associated with diverse cardiopulmonary diseases, with pathophysiologic mechanisms including pulmonary vascular, cardiac, and lung disease. Experimental work on basic mechanisms of vascular regulation of the developing lung circulation and models of perinatal pulmonary hypertension has improved our therapeutic approaches to neonates with PPHN. iNO has been shown to be an effective pulmonary vasodilator for infants with PPHN, but successful clinical strategies require meticulous care of associated lung and cardiac disease. More work is needed to expand our therapeutic repertoire to further improve the outcome of the sick newborn with severe hypoxemia, especially in patients with lung hypoplasia and advanced structural vascular disease.
Table 3 New mechanisms and emerging therapies for persistent pulmonary hypertension of the newborn Mechanism Specific therapy Increased superoxide generation High PDE5 activity Impaired/oxidized sGC
rhSOD PDE5 inhibitors (sildenafil) sGC activators/stimulators (BAY 58-2667, BAY 41-2272) rhVEGF ET receptor antagonists (bosentan) Prostacyclin analogs
Impaired VEGF signaling Increased ET-1 Altered prostacyclin production High Rho kinase activity Rho kinase inhibitors (fasudil) ET endothelin; rhSOD recombinant human superoxide dismutase; rhVEGF recombinant human vascular endothelial growth factor; sGC soluble gunylate cyclase; VEGF vascular endothelial growth factor Reprinted with permission from Abman [65]
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References 1. Levin DL, Heymann MA, Kitterman JA, Gregory GA, Phibbs RH, Rudolph AM (1976) Persistent pulmonary hypertension of the newborn. J Pediatr 89:626–633 2. Kinsella JP, Abman SH (1995) Recent developments in the pathophysiology and treatment of persistent pulmonary hypertension of the newborn. J Pediatr 126:853–864 3. Geggel R, Reid LM (1984) The structural basis for PPHN. Clin Perinatol 11:525–549 4. Murphy J, Aronovitz M, Reid L (1986) Effects of chronic in utero hypoxia on the pulmonary vasculature of the newborn guinea pig. Pediatr Res 20:292–295 5. Levin DL, Hyman AI, Heymann MA, Rudolph AM (1978) Fetal hypertension and the development of increased pulmonary vascular smooth muscle: a possible mechanism for persistent pulmonary hypertension of the newborn infant. J Pediatr 92:265–269 6. Morin FC III, Eagan EA (1989) The effect of closing the ductus arteriosus on the pulmonary circulation of the fetal sheep. J Dev Physiol 11:245–250 7. Abman SH, Accurso FJ (1989) Acute effects of partial compression of ductus arteriosus on fetal pulmonary circulation. Am J Physiol Heart Circ Physiol 26:H626–H634 8. Storme L, Rairigh RL, Parker TA, Kinsella JP, Abman SH (1999) Acute intrauterine pulmonary hypertension impairs endothelium dependent vasodilation in the ovine fetus. Pediatr Res 45: 575–581 9. Villamor E, LeCras TD, Horan MP, Halbower AC, Tuder RM, Abman SH (1997) Chronic intrauterine pulmonary hypertension impairs endothelial nitric oxide synthase in the ovine fetus. Am J Physiol Lung Cell Mol Physiol 272:L1013–L1020 10. Shaul PW, Yuhanna IS, German Z, Chen Z, Steinhorn RH, Morin FC III (1997) Pulmonary endothelial NO synthase gene expression is decreased in fetal lambs with pulmonary hypertension. Am J Physiol Lung Cell Mol Physiol 272:L1005–L1012 11. McQueston JA, Kinsella JP, Ivy DD, McMurtry IF, Abman SH (1995) Chronic pulmonary hypertension in utero impairs endothelium-dependent vasodilation. Am J Physiol Heart Circ Physiol 268:H288–H294 12. Farrow KN, Lakshminrusimha S, Reda WJ et al (2008) Superoxide dismutase restores eNOS expression and function in resistance pulmonary arteries from neonatal lambs with persistent pulmonary hypertension. Am J Physiol Lung Cell Mol Physiol 295: L979–L987 13. Hanson KA, Abman SH, Clarke WR (1996) Elevation of pulmonary PDE5-specific activity in an experimental fetal ovine perinatal pulmonary hypertension model. Pediatr Res 39:334A 14. Steinhorn RH, Russell JA, Morin FC III (1995) Disruption of cyclic GMP production in pulmonary arteries isolated from fetal lambs with pulmonary hypertension. Am J Physiol Heart Circ Physiol 268:H1483–H1489 15. Tzao C, Nickerson PA, Russell JA, Gugino SF, Steinhorn RH (2001) Pulmonary hypertension alters soluble guanylate cyclase activity and expression in pulmonary arteries isolated from fetal lambs. Pediatr Pulmonol 31:97–105 16. Brennan LA, Steinhorn RH, Wedgwood S et al (2003) Increased superoxide generation is associated with pulmonary hypertension in fetal lambs. A role for NADPH oxidase. Circ Res 92:683–691 17. Rosenberg AA, Kennaugh J, Koppenhafer SL, Loomis M, Chatfield BA, Abman SH (1993) Elevated immunoreactive endothelin-1 levels in newborn infants with persistent pulmonary hypertension. J Pediatr 123:109–114 18. Ivy DD, Le Cras TD, Horan MP, Abman SH (1998) Increased lung preproET-1 and decreased ETB-receptor gene expression in fetal pulmonary hypertension. Am J Physiol 274:L535–L541
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19. Ivy DD, Ziegler JW, Dubus MF, Fox JJ, Kinsella JP, Abman SH (1996) Chronic intrauterine pulmonary hypertension alters endothelin receptor activity in the ovine fetal lung. Pediatr Res 39:435–442 20. Ivy DD, Parker TA, Ziegler JW, Galan HL, Kinsella JP, Tuder RM, Abman SH (1997) Prolonged endothelin A receptor blockade attenuates pulmonary hypertension in the ovine fetus. J Clin Invest 99:1179–1186 21. Fike CD, Slaughter JC, Kaplowitz MR, Zhang Y, Aschner JL (2008) Reactive oxygen species from NADPH oxidase contribute to altered pulmonary vascular responses in piglets with chronic hypoxiainduced pulmonary hypertension. Am J Physiol Lung Cell Mol Physiol 295:L881–L888 22. Konduri GG, Ou J, Shi Y, Pritchard KA Jr (2003) Decreased association of HSP90 impairs endothelial nitric oxide synthase in fetal lambs with persistent pulmonary hypertension. Am J Physiol Heart Circ Physiol 285:H204–H211 23. Wedgwood S, Steinhorn RH, Bunderson M et al (2005) Increased hydrogen peroxide downregulates soluble guanylate cyclase in the lungs of lambs with persistent pulmonary hypertension of the newborn. Am J Physiol Lung Cell Mol Physiol 289:L660–L666 24. Konduri GG, Bakhutashvili I, Eis A, Pritchard KA (2007) Oxidant stress from uncoupled nitric oxide synthase impairs vasodilation in fetal lambs with persistent pulmonary hypertension. Am J Physiol Heart Circ Physiol 292:H1812–H1820 25. Wedgwood S, Black SM (2003) Role of reactive oxygen species in vascular remodeling associated with pulmonary hypertension. Antioxid Redox Signal 5:759–769 26. Lakshminrusimha S, Russell JA, Wedgwood S et al (2006) Superoxide dismutase improves oxygenation and reduces oxidation in neonatal pulmonary hypertension. Am J Respir Crit Care Med 174:1370–1377 27. Farrow KN, Groh BS, Schumacker PT et al (2008) Hyperoxia increases phosphodiesterase 5 expression and activity in ovine fetal pulmonary artery smooth muscle cells. Circ Res 102:226–233 28. Faraci F, Didion S (2004) Vascular protection: Superoxide dismutase isoforms in the vessel wall. Arterioscler Thromb Vasc Biol 24:1367–1373 29. Balasubramaniam V, Le Cras TD, Ivy DD, Kinsella J, Grover TR, Abman SH (2003) Role of platelet-derived growth factor in the pathogenesis of perinatal pulmonary hypertension. Am J Physiol Lung Cell Mol Physiol 284:L826–L833 30. Chambers CD, Hernandez-Diaz S, Van Marter LJ et al (2006) Selective serotonin-reuptake inhibitors and risk of persistent pulmonary hypertension of the newborn. N Engl J Med 354:579–587 31. Fornaro E, Li D, Pan J, Belik J (2007) Prenatal exposure to fluoxetine induces fetal pulmonary hypertension in the rat. Am J Respir Crit Care Med 176:1035–1040 32. Walsh-Sukys MC, Tyson JE, Wright LL et al (2000) Persistent pulmonary hypertension of the newborn in the era before nitric oxide: practice variation and outcomes. Pediatrics 105:14–20 33. Van Marter LJ, Leviton A, Allred EN (1996) PPHN and smoking and aspirin and nonsteroidal antiinflammatory drug consumption during pregnancy. Pediatrics 97:658–663 34. Alano MA, Ngougmna E, Ostrea EM Jr, Konduri GG (2001) Analysis of nonsteroidal antiinflammatory drugs in meconium and its relation to persistent pulmonary hypertension of the newborn. Pediatrics 107:519–523 35. Pearson DL, Dawling S, Walsh WF et al (2001) Neonatal pulmonary hypertension–urea-cycle intermediates, nitric oxide production, and carbamoyl-phosphate synthetase function. N Engl J Med 344:1832–1838 36. Abman SH, Kinsella JP (1995) Inhaled nitric oxide for persistent pulmonary hypertension of the newborn: the physiology matters. Pediatrics 96:1153–1155
1118 37. UK Collaborative ECMO Trial Group (1996) UK collaborative randomised trial of neonatal extracorporeal membrane oxygenation. Lancet 348:75–82 38. Patterson K, Kapur SP, Chandra RS (1988) PPHN: pulmonary pathologic effects. In: Rosenberg HS, Berstein J (eds) Cardiovascular diseases, perspectives in pediatric pathology, vol 12. Karger, Basel, pp 139–154 39. Kinsella JP, Abman SH (2000) Clinical approach to inhaled NO therapy in the newborn. J Pediatr 136:717–726 40. Acute Respiratory Distress Syndrome Network (2000) Ventilation with lower tidal volumes as compared with traditional tidal volumes for acute lung injury and the ARDS. N Engl J Med 342:1301–1308 41. Gordon JB, Martinez FR, Keller PA, Tod ML, Madden JA (1993) Differing effects of acute and prolonged alkalosis on hypoxic pulmonary vasoconstriction. Am Rev Resp Dis 148:1651–1656 42. Laffey JG, Engelberts D, Kavanaugh BP (2000) Injurious effects of hypocapnic alkalosis in the isolated lung. Am J Resp Crit Care Med 162:399–405 43. Lotze A, Mitchell BR, Bulas DI, Zola EM, Shalwitz RA, Gunkel JH (1998) Multicenter study of surfactant (beractant) use in the treatment of term infants with severe respiratory failure. Survanta in Term Infants Study Group. J Pediatr 132:40–47 44. Clark RH, Kueser TJ, Walker MW et al (2000) Low dose nitric oxide therapy for persistent pulmonary hypertension of the newborn. N Engl J Med 342:469–474 45. Davidson D, Barefield ES, Kattwinkel J et al (1998) Inhaled nitric oxide for the early treatment of persistent pulmonary hypertension of the term newborn: a randomized, double-masked, placebo-controlled, dose-response, multicenter study. Pediatrics 101:325–334 46. Kinsella JP, Shaffer E, Neish SR, Abman SH (1992) Low-dose inhalational nitric oxide in persistent pulmonary hypertension of the newborn. Lancet 340:819–820 47. Kinsella JP, Truog WE, Walsh WF et al (1997) Randomized, multicenter trial of inhaled nitric oxide and high-frequency oscillatory ventilation in severe, persistent pulmonary hypertension of the newborn. J Pediatr 131:55–62 48. Neonatal Inhaled Nitric Oxide Study Group (1997) Inhaled nitric oxide in full-term and nearly full-term infants with hypoxic respiratory failure. New Engl J Med 336:597–604 49. Roberts JD, Fineman J, Morin FC III et al (1997) Inhaled nitric oxide and persistent pulmonary hypertension of the newborn. New Engl J Med 336:605–610 50. Konduri GG, Solimani A, Sokol GM et al (2004) A randomized trial of early versus standard inhaled nitric oxide therapy in term and near-term newborn infants with hypoxic respiratory failure. Pediatrics 113:559–564 51. Roberts JD, Polaner DM, Lang P, Zapol WM (1992) Inhaled nitric oxide in persistent pulmonary hypertension of the newborn. Lancet 340:818–819
S.H. Abman et al. 52. Goldman AP, Tasker RC, Haworth SG, Sigston PE, Macrae DJ (1996) Four patterns of response to inhaled nitric oxide for persistent pulmonary hypertension of the newborn. Pediatrics 98:706–713 53. Lakshminrusimha S, Russell JA, Steinhorn RH, Ryan RM, Gugino SF, Morin FC III, Swartz DD, Kumar VH (2006) Pulmonary arterial contractility in neonatal lambs increases with 100% oxygen resuscitation. Pediatr Res 59:137–141 54. Lakshminrusimha S, Russell JA, Steinhorn RH et al (2007) Pulmonary hemodynamics in neonatal lambs resuscitated with 21%, 50%, and 100% oxygen. Pediatr Res 62:313–318 55. Ziegler JW, Ivy DD, Wiggins JW, Kinsella JP, Clarke WR, Abman SH (1998) Effects of dipyridamole and inhaled nitric oxide in pediatric patients with pulmonary hypertension. Am J Resp Crit Care Med 158:1388–1395 56. Atz AM, Wessel DL (1999) Sildenafil ameliorates effects of inhaled nitric oxide withdrawal. Anesthesiology 91:307–310 57. Ichinose F, Erana-Garcia J, Hromi J, Raveh Y, Jones R, Krim L, Clark MWH, Winkler JD, Bloch KD, Zapol WM (2001) Nebulized sildenafil is a selective pulmonary vasodilator in lambs with acute pulmonary hypertension. Crit Care Med 29:1000–1005 58. Weimann J, Ullrich R, Hromi J, Fujino Y, Clark MWH, Bloch KD, Zapol WM (2000) Sildenafil is a pulmonary vasodilator in awake lambs with acute pulmonary hypertension. Anesthesiology 92:1702–1712 59. Shekerdemian LS, Ravn HB, Penny DJ (2002) Intravenous sildenafil lowers pulmonary vascular resistance in a model of neonatal pulmonary hypertension. Am J Resp Crit Care Med 165:1098–1102 60. Shekerdemian LS, Ravn HB, Penny DJ (2004) Interaction between inhaled nitric oxide and intravenous sildenafil in a porcine model of meconium aspiration syndrome. Pediatr Res 55:413–418 61. Baquero H, Soliz A, Neira F, Venegas ME, Sola A (2006) Oral sildenafil in infants with persistent pulmonary hypertension of the newborn: a pilot randomized blinded study. Pediatrics 117: 1077–1083 62. Steinhorn RH, Kinsella JP, Butrous G, Dilleen M, Oakes M, Wessel DL (2007) Open-label, multicentre, pharmacokinetic study of IV sildenafil in the treatment of neonates with persistent pulmonary hypertension of the newborn (PPHN). Circulation 116:II-614 63. Kelly LK, Porta NF, Goodman DM, Carroll CL, Steinhorn RH (2002) Inhaled prostacyclin for term infants with persistent pulmonary hypertension refractory to inhaled nitric oxide. J Pediatr 141: 830–832 64. McNamara PJ, Laique F, Muang-In S, Whyte HE (2006) Milrinone improves oxygenation in neonates with severe persistent pulmonary hypertension of the newborn. J Crit Care 21:217–222 65. Abman SH (2007) Recent advances in the pathogenesis and treatment of persistent pulmonary hypertension of the newborn. Neonatology 91(4):283–290
Chapter 77
The Pulmonary Circulation in Congenital Heart Disease Thomas J. Kulik and Mary P. Mullen
Abstract The right-to-left shunting in patients with a large ventricular septal defect (VSD) is caused by increased pulmonary vascular resistance (PVR) secondary to pulmonary vascular disease. The pulmonary circulation hugely impacts the clinical phenotype of many congenital heart lesions. For example, PVR is a major determinant of pulmonary blood flow (PBF; hence left ventricular output and therefore the likelihood of congestive heart failure) in infants with a large interventricular or great vessel communication (e.g., VSD; patent ductus arteriosus). In babies with a single cardiac ventricle, the magnitude of PVR often is a major determinant of both ventricular volume load and systemic oxygen delivery. On the other hand, over the longer run, many cardiac malformations can cause PVR to increase, often resulting in serious disability for the patient so afflicted. This is especially true for single-ventricle patients outside the neonatal period, for whom operative palliation with a cavopulmonary circulation (e.g., surgically directing the systemic venous return directly to the lungs, without an inter mediary pumping chamber) requires a healthy (i.e., low PVR) pulmonary circulation. But patients with two ventricles can also suffer disability and early demise from increased PVR related to a cardiac defect. So, depending upon the circumstances, with heart malformations the pulmonary vascular bed may prove problematic owing to too little (actually, normal) or too much PVR. The primary focus of this chapter is on the latter, increased PVR secondary to increased pulmonary artery (PA) pressure and/or flow caused by cardiac structural malfor mations. We use pulmonary vascular disease (PVD) to denote this increased PVR, which is accounted for by both pathological remodeling of the pulmonary circulation and active vasoconstriction of small PAs, in varying relative degrees. Keywords Eisenmenger syndrome • Septal defect of the heart • Patent ductus arteriosus • Right-to-left shunt • Pulmonary vasoconstriction • Pulmonary blood flow T.J. Kulik (*) Department of Cardiology, Children’s Hospital Boston, 300 Longwood Avenue, Boston, MA 02115, USA e-mail:
[email protected] 1 Introduction In 1958 the great Paul Wood related, in the Croonian Lecture at the Royal College of Physicians, how investigators rather haltingly came to discern the physiology and pathology of Eisenmenger syndrome [1]. It was not until the late 1940s to early 1950s that people tumbled to the now-obvious notion that right-to-left shunting in patients with a large ventricular septal defect (VSD) is caused by increased pulmonary vascular resistance (PVR) secondary to pulmonary vascular disease; previous hypotheses attributed the observed cyanosis to reduced oxygenation within the lungs or decreased cardiac output. Although Wood, and others, got much of the story right even 50 years ago, our understanding of how structural malformations of the heart impact the pulmonary circulation, and vice versa, has advanced enormously since then, and this knowledge critically informs practitioners of cardiology and cardiac surgery, especially in pediatrics. Indeed, the “lesser” circulation hugely impacts the clinical phenotype of many congenital heart lesions. For example, PVR is a major determinant of pulmonary blood flow (PBF; hence left ventricular output and therefore the likelihood of congestive heart failure) in infants with a large interventricular or great vessel communication (e.g., VSD; patent ductus arteriosus). In babies with a single cardiac ventricle, the magnitude of PVR often is a major determinant of both ventricular volume load and systemic oxygen delivery. The fact that PVR is normally much lower than systemic resistance can actually be disadvantageous in this setting, as higher than normal PVR (up to a point) enhances systemic oxygen delivery in unoperated infants with a single ventricle [2]. On the other hand, over the longer run, many cardiac malformations can cause PVR to increase, often resulting in serious disability for the patient so afflicted. This is especially true for single-ventricle patients outside the neonatal period, for whom operative palliation with a cavopulmonary circulation (e.g., surgically directing the systemic venous return directly to the lungs, without an intermediary pumping chamber) requires a healthy (i.e., low PVR) pulmonary circulation. But patients with two ventricles can also suffer disability and early demise from increased PVR related to a cardiac defect.
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So, depending upon the circumstances, with heart malfor mations the pulmonary vascular bed may prove problematic owing to too little (actually, normal) or too much PVR. The primary focus of this chapter is on the latter, increased PVR secondary to increased pulmonary artery (PA) pressure and/ or flow caused by cardiac structural malformations. We use pulmonary vascular disease (PVD) to denote this increased PVR, which is accounted for by both pathological remo deling of the pulmonary circulation and active vasoconstriction of small PAs, in varying relative degrees. Our use of this term in this way, which rolls together both pathological remodeling and active vasoconstriction, requires some explanation, as “pulmonary vascular disease” (PVD) usually refers specifically to pathological anatomic changes in the vascular bed. We decline to cleave vasoconstriction from structural vascular changes when conceptualizing the problem of increased PVR for two reasons. Firstly, for any population of patients so afflicted, many or most will have both vasoconstriction and remodeling. Moreover, even if we measure the effect of a pulmonary vasodilator on an indi vidual’s pulmonary hemodynamics, we will still have only a partial understanding of how PVR is partitioned into active and anatomic components for that patient: our ability to dilate the lung’s circulation is imperfect, and we can never be certain how much residual smooth muscle contractile tone remains with a dilator. To dichotomize active and fixed pathological anatomic changes therefore implies a level of understanding actually lacking, and leeches away an important nuance from thinking about this problem. One could argue that it is useful to (as best as possible) separate (irreversible) anatomic changes from (reversible) vasoconstriction because fixed changes are not amenable to resolution with known therapy, with the important clinical implications that follow. Although there is utility in that, when one considers that chronic vasodilator therapy can substantially reduce PVR which does not respond acutely to dilators, the line between “fixed” and reversible causes of increased PVR blurs, whether because “irreversible” anatomic changes are not so irreversible after all, or because release of vasoconstriction can sometimes take days or weeks rather than seconds or minutes. Secondly, though our understanding of the pathobiology of PVD is pitifully limited, it seems likely that active vasoconstriction both directly reduces vascular luminal area and causes pathological remodeling (and, conversely, that vasoconstriction in the remodeled circulation has a disproportionate effect on PVR). It therefore seems important to consider these two factors as being intimately associated, realizing that they are in some ways distinct. We will also briefly discuss abnormalities of the pulmonary circulation that occur in conjunction with cardiac malformations with right-sided obstruction. These cardiac lesions tend to be associated with decreased PA pressure and flow (but not invariably so) and any PA disease usually resides in the large (main, lobar, or proximal segmental) PAs.
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2 The Pathology and Physiology of PVD The term “PVR” is used here as is customary: PVR = mean PA pressure – mean left atrial pressure/PBF. PVR is commonly expressed in dyne . sec/cm5, or Wood units (mmHg/l min; 1 Wood unit = 80 dyne . sec/cm5 usually indexed to body surface area in pediatrics. Normal PVR is 1–3 units . m2 after the first approximately 2 months of life [3]. (NB: Some older papers report total pulmonary resistance, which is PA pressure divided by PBF.). Precisely measuring PVR, and interpreting changes in this variable – e.g., after administration of a pharmacological agent – in patients with cardiac shunting lesions requires care and can be difficult. With unrestrictive interventricular or great vessel communications (i.e., the systolic pressures in the pulmonary and systemic circulations are the same), mean PA pressure may change little with change in PVR, and an alteration in resistance may be largely manifest in a change in PBF [4]. However, alteration in systemic vascular resistance, independent from alteration in PVR, will also change PBF in these circumstances, complicating interpretation of observed hemodynamics. Also, because PVR falls with an increase in flow due to passive mechanical effects [5], it may be difficult to parse any alteration in vascular tone from these passive changes. Finally, accurately measuring PBF with certain lesions (e.g., patent ductus arteriosus) using clinically available methods may be impossible. Increased PVR can result not only from structural changes (decreased luminal area of arteries ± veins, ± a diminished number of vessels) but also from active vaso constriction of pulmonary resistance vessels. We know this because if patients with cardiac malformations and increased PVR are given vasodilators, some show an acute decrease in both mean PA pressure and PVR [6–11]. This is particularly true for infants, but even older children and adults with markedly increased PVR are often “reactive” [6, 12–14]. A variety of agents can vasodilate the lung under these circumstances (increased FiO2, inhaled nitric oxide, acetylcholine, tolazoline hydrochloride, isoproterenol), and multiple vasodilators in combination have been shown to be more effective than either alone (e.g., high FiO2plus acetylcholine [6], and high FiO2 plus inhaled nitric oxide) [15, 16]. Since these agents differ in their mechanism of action (opening K+ channels; increasing the level of intracellular cyclic GMP directly or via endothelial release of nitric oxide; a-adrenergic blockade; increasing the level of intracellular cyclic AMP), these observations afford little clue as to the mechanism of the smooth muscle activation with increased pressure and flow. This reactivity, when present, is variable in magnitude, and because it is probably impossible to eliminate all active tone from pulmonary vessels in vivo, one cannot know precisely how PVR is partitioned between structural vascular changes and vasoconstriction.
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3 The Microarchitecture of PVD
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The progressive changes in pulmonary vascular structure that occur with defects involving an unrestrictive communication between the systemic and pulmonary circulations (e.g., large VSD; patent ductus arteriosus) were described (Fig. 1). Heath–Edwards (H-E) grade 1 changes consist of medial hypertrophy, extension of smooth muscle into nor mally nonmuscular arteries, and adventitial thickening and fibrosis. Grade 2 changes include increased medial hypertrophy and cellular intimal proliferation, sometimes sufficient to occlude the vessel, in arteries of less than 300-mm diameter. Grade 3 changes include extension of cellular intimal proliferation into arteries of 300–500-mm diameter, and intimal fibrosis in vessels smaller than 300 mm, resulting in widespread occlusion of these small arteries. Longitudinal fasciculi of smooth muscle develop in the
hypertrophied media of medium- and large-diameter arteries. In late stage 3, generalized dilation lesions are seen. Though not emphasized, splitting “or elastosis” of the internal elastic lamina was also described. The arteries in grade 4 show thinning of the media and generalized dilation, as well as local areas of dilation (“dilation lesions”). Dilation lesions are of three types: “plexiform,” “angiomatoid,” and veinlike branches of hypertrophied (usually occluded) muscular PAs. Thrombi are sometimes seen in these lesions. In grade 4, plexiform lesions are present, but the other types of dilation lesions are found in grade 5, where medial fibrosis is also found. The rarely seen grade 6 indicates the presence of the preceding plus necrotizing arteritis. Little attention was given to the pulmonary veins, except to say that with VSDs the veins are normal, but that intimal fibrosis is seen with atrial septal defects (ASDs). The virtue of this description is that it clearly identifies multiple fundamental morphological characteristics of PVD caused by increased pressure and flow: medial hypertrophy, intimal hyperplasia, intimal and medial fibrosis, dilation of small PAs, and dilation and plexiform lesions. Heath and Edwards also suggested that these abnormalities occur in a stereotyped sequence (although they did not actually show that in their paper). Its most important limitations are that no quantification of the pathological changes is provided, and that other important characteristics of the remodeled circulation were not uncovered (the reduction in the number of small PAs) or described in much detail (distal extension of smooth muscle).
Fig. 1 Heath–Edwards classification of pulmonary vascular pathological changes observed in patients with congenital cardiac malformations with intracardiac shunting. (a) Grade 1 – medial hypertrophy (magnification ×150). (b) Grade 2 – cellular intimal proliferation (×250). (c) Grade 3
– intimal fibrosis (×150). (d) Grade 4 – dilation and intimal fibrosis (×150). (e) Grade 5 – plexiform lesion (×95). (f) Grade 6 – necrotizing arteritis (×250). See the text for details. (Reproduced from [158] with permission)
Donald Heath and Jesse Edwards gave the first systematic description of pulmonary vascular remodeling with left-toright shunting lesions in 1958 [17]. Subsequent work by multiple investigators has provided additional important insights, and a better means of quantifying pathological changes in small pulmonary blood vessels.
3.1 Observations of Heath and Edwards
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3.2 Morphometric Insights A second method of assessing the pulmonary vascular bed was subsequently developed which has provided additional information regarding the earliest changes in growth and development of the lung associated with pulmonary arteriopathy. The morphometric approach quantifies the thickness of the arterial muscular coat, the degree of abnormal distal extension of smooth muscle, and the density of small PAs relative to the number of alveoli. Although these studies initially used whole lung injected with a barium–gelatin mixture, modified morphometric analysis has also been applied to uninjected postmortem lungs and biopsy material [18–21]. Morphometric structural findings are graded as follows: grade A – appearance of smooth muscle more peripherally than normal (into normally nonmuscular arteries) with or without modest medial hypertrophy (medial thickness less than or equal to 1.5 times the normal thickness); grade B – distal extension of muscle plus medial thickness 1.5 – 2.0 times normal thickness (mild), or greater than 2 times normal thickness (severe); grade C – grade B plus a decreased number of peripheral arteries relative to alveoli. Grade C “mild” indicates a less than 50% reduction in the number of peripheral arteries, and “severe” denotes a reduction greater than 50%. Grade A changes are seen with increased PBF but normal PA pressure (these are normal vessels from the standpoint of H-E criteria). Grade B mild changes usually occur with increased PA pressure (there is always PA hypertension with severe grade B). Any of the three morphometric changes can be observed with any of the H-E 1–3 grades, although grades A and B mild are rarely seen with any H-E abnormality, and H-E 3 is almost always accompanied by grade C findings [20, 22]. Pulmonary venous abnormalities with increased PBF ± pressure are quite variable: often the veins are normal, but increased medial thickness is sometimes found, as well as arterialization (development of an external elastic lamina), and occasionally intimal fibrosis in older patients [20, 21, 23, 24] (Fig. 2). Other observations made using morphometric techniques are relevant to the architecture of pulmonary vascular remodeling. For example, Haworth carefully examined lungs from patients who died with a variety of different types of heart lesions and described the pattern of early intimal pathology: intimal proliferation and fibrosis reduces the cross-sectional area of the vascular bed both by reducing the lumen of very small PAs and by completely occluding small branch vessels, reducing the number of small PAs [24]. By establishing that abnormal extension of smooth muscle and a reduction of small artery density are as much a part of PVD as medial hypertrophy and intimal changes, morphometric analysis has contributed importantly to our understanding of the architecture of pulmonary vascular remodeling. Also, by quantifying early changes caused by increased flow and pressure (distal extension and mild
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hypertrophy) this analysis has enhanced our understanding of the evolution of pathological remodeling. There are some puzzles related to data derived from this approach, however, which require further exploration. For example, some [20] but not all [24] reports indicate that grade C changes are almost always seen in the context of increased PVR. The external diameter of intra-acinar arteries (i.e., arteries accompanying the most distal respiratory unit – respiratory bronchiole, alveolar duct, and alveolus) with increased pressure and flow appears to be quite variable [20, 21, 24]. Not all studies of increased pressure and flow lesions have observed a reduced number of peripheral arteries [25], and there are other differences in morphometric findings between different types of defects with similar hemodynamic profiles that lack clear explanation [24].
3.3 Angiographic Assessment of Pathological Remodeling Investigators have performed angiography of small PAs in patients with congenital heart disease and compared angiographic findings with hemodynamics and lung biopsy. Nihill and McNamara [26] found dilation of small vessels with increased PBF (but normal pressure), but that with increased PVR there is also abrupt tapering of small PAs (about 1–2 mm in diameter), a reduction in the number of supernumerary arteries, and an incomplete, patchy, and reticular (as compared with ground glass) capillary blush. They also found these pathological changes, which they attributed to obstruction of small vessels, to be randomly distributed within the lung. Little correlation was found between H-E biopsy findings (which ranged from normal to grade 5) and PA pressure and PVR, but that with a patchy, incomplete capillary blush and PVR of 6 units . m2 or more, H-E grade 3 or greater changes were almost always present on biopsy. Rabinovitch et al. [27] quantified the rate of taper in small axial PAs (1.5–2.5 mm in diameter), and found that the rate of taper increased as did PVR, which correlated with morphometric findings on biopsy. Capillary blush was reduced in patients with morphometric abnormality on biopsy, especially with the most severe pathological remodeling.
4 When Is PVD Reversible? The issue of “reversibility,” i.e., reduction or normalization of PVR with elimination of the associated cardiac structural lesion, is of great clinical and biological interest. Perhaps the best place to start a discussion of reversibility of PVD is Eisenmenger syndrome, the ultimate incarnation of
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Fig. 2 Comparison of lung biopsy at time of corrective surgery for conditions associated with pulmonary artery (PA) hypertension (VSD, ventricular septal defect; DTGA, d-transposition of the great arteries; CAVC, complete atrioventricular canal. “Other” lesion include: left-sided regurgitation or obstruction; atrial septal defect; truncus arteriosus; patent ductus arteriosus, aortopulmonary window). (a) Mean PA pressure measured 1 year after surgery. (b) Mean pulmonary vascular resistance measured 1 year after surgery. Vertical lines separate normal from abnormally elevated values. Horizontal lines separate biopsy grades. Age at repair is shown. Patients who underwent repair within the first 8 months of life, regardless of biopsy grade, had normal or nearnormal PA pressure and normal resistance 1 year after surgery. (Reproduced from [22] with permission from Lippincott Williams and Wilkins)
irreversibility. Paul Wood’s definition of Eisenmenger syndrome is probably as good as any of those definitions which have followed: pulmonary hypertension at the systemic level, due to high PVR (more than 800 dyne · sec/cm5) with reversed (i.e., right-to-left) or bidirectional shunt [1]. These patients have severely remodeled pulmonary vasculature (which includes the entire range of H-E grades) and, except in childhood, seldom respond to vasodilators with a decline in PVR, especially if the shunting is purely from right to left [1, 12– 14]. Surgical therapy for such patients is precluded: closure of ventricular or great vessel shunts attended by pure rightto-left shunting lacks a theoretical rationale (since, lacking the “pop off,” right ventricular pressure will increase to suprasystemic level, which is poorly tolerated). Clinical
experience confirms persistently elevated or increased PVR following repair, and poor outcome [1]. Adults with increased PA pressure and PVR of more than 800 dyne · sec/cm5, but substantial left-to-right shunting, are also at risk for progressive PVD even after surgical correction, although the response of the pulmonary vascular bed to elimination of the shunt can be much more favorable under those circumstances, as is discussed later. For patients with only modestly elevated PVR, especially those under about 2 years of age, elimination of the shunt almost always results in normal PVR. In fact, the possible responses of the pulmonary vascular bed to repair of a shunting lesion exist on a continuum, ranging from complete resolution of the PVD to no change or even an increase in resistance.
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What is the anatomic/physiological basis of alleviation of PVD, and what factors influence the likelihood of resolution of PVD following elimination of the cardiac lesion? As previously noted, there are two components of increased PVR in this setting (active vasoconstriction and structural remodeling of the pulmonary vascular bed) and both, depending upon the circumstances, may be ameliorated after repair of the cardiac defect. In some settings, vasoconstriction probably accounts for much or all of the increase in PVR related to the cardiac defect. As detailed in Sect. 5.1, relief of vasoconstriction is a very large component of the fall in PVR following relief of pulmonary venous hypertension. Dalen et al. [28] studied five adults having had mitral valve replacement and found that their average PVR fell from 1,234 dyne · sec/cm5 (preoperative) to about 675 dyne · sec/cm5 by the second postoperative day, and by the fourth day it was down to about 400 dyne · sec/ cm5 in three patients in whom this measurement was made. Atz et al. [29] investigated the effect of inhaled nitric oxide on 15 children with mitral stenosis, most of whom were studied immediately after surgical or catheter intervention on the valve. They found that inhaled nitric oxide acutely decreased mean PVR from 5.8 ± 0.7 to 2.9 ± 0.4 units · m2 (Fig. 3). Increased PVR in cardiac malformations with increased PA pressure and flow is also often due to vasoconstriction. Neutze et al. [10] studied the effect of isoproterenol infusion on pulmonary hemodynamics in patients with VSD. In 22 patients with moderately increased PVR (5.0–5.9 units · m2), ranging in age from 3 to 84 months (mean 28), with isoproterenol infusion PVR decreased in all patients, falling from 6.2 to 3.2 units · m2; cardiac catheterization at an average of 5.5 years after closure of the defect showed PVR to be about the same as the preoperative isoproterenol value. Thirty-six patients had severe PVD, with PVR of 8 units or more (range, 8–43 units · m2), 2–392 months old, yet nearly half decreased
Fig. 3 Atz et al. gave 15 patients with congenital mitral stenosis and pulmonary hypertension nitric oxide by inhalation for 15 min. Most of the patients were studied immediately after cardiopulmonary bypass or interventional catheterization. Mean PA pressure fell in all patients. (Reprinted from [29] with permission from Elsevier)
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their PVR to 7 units · m2 or less with isoproterenol infusion, and in four patients it fell to less than 3 units · m2. Vogel et al. made observations consistent with these [8]. Lock et al. [4] found that 20% of patients with VSD and increased mean PA pressure (44 mmHg or more) and PVR (pulmonary-to-systemic vascular resistance ratio of 0.3 or more ) decreased their pulmonary-to-systemic vascular resistance ratio to the normal level (0.2 or less) with 100% inspired oxygen. There is not much room left for a fixed reduction in the cross-sectional area of the pulmonary vascular bed to account for increased PVR when acute interventions can reduce PVR to the normal level, or nearly so. When one considers that only a single vasodilator was used in the above-mentioned studies, and that at least some residual vascular tone therefore likely remained, it emphasizes the importance of vasoconstriction in increased PVR with congenital defects, even in patients beyond the first year of life. However, as described above, there are patients (who tend to be older and with higher PVR) who are not “responders,” with a severely pathologically remodeled vascular bed, for whom structural changes presumably account for the vast majority of the increased resistance. To what extent the microvasculature undergoes “reverse” architectural remodeling is poorly defined. It has generally been assumed that medial hypertrophy, and perhaps intimal proliferation, can regress after removal of increased pressure and/or flow [30], whereas intimal and medial fibrosis and plexiform lesions cannot. There is actually very little information directly pertaining to remodeling of the pulmonary circulation following repair, and the authors are aware of only two papers that directly address this issue. Investigators compared lung biopsies taken before and 2.5–10 years after placement of a PA band, to reduce PA pressure, in patients with a variety of different types of congenital shunting lesions [31]. A reduction of pressure was correlated with regression of medial hypertrophy, intimal proliferation, and, possibly, mild intimal fibrosis. However, there were a limited number of patients, the histological findings were variable (especially regarding intimal changes), and few hemodynamic data were provided. Damman et al. examined lung biopsies taken before and several years after PA banding, and also concluded that medial hypertrophy and intimal “thickening” had regressed, but their data are too scanty to shed much light on the question of regression of intimal lesions [32]. Inferential data suggesting that remodeling can occur also come from studies of chronic infusion of prostacyclin, as is discussed in Sect. 7.3.2 . The authors are aware of no data regarding whether the number of small PAs is altered with favorable remodeling. It is unfortunate that we do not have a better understanding of precisely how the microcirculation undergoes reverse remodeling in the human. That said, it seems unlikely that histological observations could supplant assessment of the most physiologically (and clinically) relevant variable, i.e.,
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resistance to blood flow across the lungs. And even if biopsy did provide a perfect measure of postrepair remodeling and hemodynamics, the natural reluctance to subject patients to this procedure would greatly limit its utility. Our under standing of reversibility is largely informed by observations of pulmonary hemodynamics before and some time after repair, not by direct inspection of the microarchitecture of the vascular bed. A considerable body of literature has accumulated regarding this question, and although our understanding of reversibility is still imperfect, we have learned that four variables have a major impact on the likelihood that a given patient will have reversal of PVD after repair:
3. The PVR at the time of operation. Normal PVR stays normal after operation, whereas – generally speaking – severely increased PVR stays high or increases (beyond the first year of life or so). For patients whose PVR is moderately increased, there is considerable variability in the response to repair. 4. As yet undefined factors inherent in the individual. Reports of large series of patients studied before and following closure of cardiac shunting lesions show that there is considerable individual variability in the pulmonary circulatory response to shunt elimination (Fig. 4). The biological basis for this is unclear.
1. The type of cardiac lesion, mostly because the nature of the lesion determines the PA pressure and flow characteristics. As detailed later, PVD caused by lesions which increase PA pressure but not flow (e.g., mitral stenosis) is much more likely to resolve upon repair than that associated with increased PA pressure and flow. There are a few lesions [e.g., d-transposition of the great arteries (d-TGA)] asso ciated with a particularly high risk of or precocious deve lopment of PVD, for reasons that are unclear (Table 1). 2. The age of the patient at time of repair. For lesions that cause PVD in the first few years of life (e.g., VSD), earlier repair confers a greater likelihood of resolution of PVD. To what extent this is a simple matter of prevention of pathological remodeling, versus reversal of same, is unknown, although the fact that advanced (and irreversible) H-E changes tend to occur only in older patients suggests that prevention rather than resolution predominates.
These considerations are further discussed in the context of specific heart lesions next.
5 Cardiac Structural Lesions Causing PVD Consideration of the general types of structural lesions associated with PVD (and their impact on PA pressure and flow) is important partly because of the obvious clinical implications, and because it also affords clues to the biological processes involved in the vascular remodeling. As elaborated upon in the following subsections: (1) increased PA pressure (alone) is quick to cause active vasoconstriction and medial hypertrophy, but slow to provoke irreversible pathological remodeling of the pulmonary circulation; (2) increased PBF (alone) is very slow to cause significant vascular
Table 1 Summary of the pulmonary circulatory and clinical characteristics of some representative congenital cardiac malformations Physiological Pathological Time course Hemodynamics Lesion characteristics characteristics Risk of PVD of PVD Reversibility of PVD Increased PA pressure
MS
Increased PBF
ASD
Increased PA pressure and PBF
VSD
H-E 1–3; Active pulmonary plexiform vasoconstriction lesions rare accounts for a large fraction of the increased PVR Active vasoconstriction H-E 1–6 and structural changes Active vasoconstriction H-E 1–6, morphometric and structural A–C changes increase PVR; fixed structural changes with very high PVR Active vasoconstriction H-E 1–6, morphometric and structural A–C changes
Unclear
Unclear, but can occur in early childhood
Even very high PVR in adults is usually reversible
~10–15%
Seldom before ~20 years of age
See the text
Infancy on
Moderately increased PVR at least partially reversible, even in adults Almost always reversible for the first 1–2 years of age; likelihood of reversal falls with age and increasing PVR Reversible, but requires correction or palliation at very early age
Unclear, See the text d-TGA/VSD Complex likely high malformations with increased PA pressure and flow ASD atrial defect, d-TGA d-transposition of the great arteries, H-E Heath–Edwards, MS mitral stenosis, PA pulmonary artery, PBF pulmonary blood flow, PVD pulmonary vascular disease, PVR pulmonary vascular resistance, VSD ventricular septal defect
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Fig. 4 Grosse-Brockhoff et al. reported the effect of surgical repair of VSD in 65 patients operated on at various ages. The first postoperative catheterization was 6–8 weeks after the operation. In general, the likelihood of normal mean PA pressure after closure of the defect was greatest for patients operated on at younger age, and for those with the lowest preoperative pressure. (Reproduced from [72] with permission)
remodeling, and does so in only a relatively small minority of people; (3) increased pressure and flow together are considerably more pernicious in causing pathological remodeling, especially high-grade lesions, than either increased pressure or flow alone; and (4) other factors besides PA pressure and flow importantly influence the propensity to develop PVD. Both the clinician and the biologist need to take these observations into account when they set about to do their good work (Table 1).
5.1 Increased PA Pressure Increased pressure in the pulmonary circulation is alone sufficient to cause pathological remodeling. Cardiac lesions associated with increased pulmonary venous pressure but normal, or decreased PBF (e.g., mitral stenosis, cor triatriatum, or obstructed pulmonary venous connection), cause increased PA pressure out of proportion to pulmonary venous pressure, and pathological pulmonary vascular remodeling, even in early childhood [33]. Mitral stenosis is the most common structural lesion causing pulmonary venous hypertension, where medial hypertrophy and intimal fibrosis are observed in both arteries and veins, although plexiform lesions only rarely develop [33–38]. With mitral stenosis, PVR is highly variable, ranging from normal (even with left atrial pressure greater than 30 mmHg) to more than 1,000 dyne · sec/cm5 [28, 33, 39, 40]. As noted above, the increased PVR is usually mostly due to active pulmonary vasoconstriction, as revealed by an acute fall in PA pressure and PVR with vasodilators, and the very rapid fall in PA pressure and resistance after surgery to relieve the
stenosis [28, 29]. In fact, adult patients with mitral stenosis, even those with greatly elevated PVR, typically show a large reduction in PVR, often to normal levels, following relief of the left atrial hypertension [28, 40, 41]. This is of some interest, since intimal fibrosis, which is commonly thought to infer irreversible vascular disease, is commonly observed with mitral stenosis [37, 38]. Much less information is available for pediatric patients, although substantial reduction of PVR following reduction of pulmonary venous hypertension also appears to be true for infants and children, at least in the case of isolated elevation in pulmonary venous pressure (i.e., without the presence of other cardiac defects) [33, 42]. Cardiac malformations which increase pulmonary venous pressure in utero cause remodeling of the pulmonary vascular bed in the fetus and newborn. Hypoplastic left-sided heart syndrome with highly restrictive connection between the left and right atria is associated with pulmonary venous hypertension in utero, and the same is likely true for at least some patients with total anomalous pulmonary venous connection with obstruction. Marked medial hypertrophy and “arterialization” of the pulmonary veins, and dilation of pulmonary lymphatics are noted even in neonates. Abnormalities of the small PAs are less notable in the newborn, but develop early in the neonatal period [43–45].
5.2 Increased PBF Small increases in PBF (and without elevation in pressure), as occur with a small ASD, VSD, or patent ductus arteriosus, pose no appreciable risk for the pulmonary circulation [46].
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On the other hand, a large increase in PBF, even when PA pressure is initially normal, will cause PVD in a significant percentage of people. With a sufficiently large ASD – the most common lesion associated with this physiological state – marked elevation of PA pressure and PVR and advanced H-E changes may develop over time [47]. The risk of developing PVD with an ASD is relatively low. Data vary widely in the percentage of adults with ASD and PVD, probably partly because of variability in case ascertainment and partly because “PVD” is variably defined in published studies, but 10–15% seems a reasonable estimate [48–51]. What is quite clear is that the risk of PVD in the first two decades of life with ASD is very low [47, 52], in contradistinction to lesions causing increased pressure and flow (e.g., unrestrictive VSDs), where irreversible PVD in the first several years of life is not uncommon. Having said that, severe PVD in the first few months or years of life with ASD is well described [19, 53], and comes up periodically for practitioners who treat pulmonary hypertension. It is unclear whether this precocious pathological pulmonary vascular remodeling is caused by the increased PBF, or if the presence of the cardiac defect is only coincidental in these unusual cases. There are several reports of pulmonary hemodynamics in adults studied months to years after repair of ASD [54–57]. Preoperatively, these patients had at most moderate PA hypertension (mean PA pressure 60 mmHg or lower). Patients with normal or near-normal mean PA pressure preoperatively (lower than about 30 mmHg) had a fall in pressure after ASD closure and PA pressures were within the normal range; those with significant PA hypertension before repair also had a reduction in PA pressure, although PA pressure often remained abnormally high. With both normal and elevated preoperative PA pressure, postoperative PVR fell after repair in two of the studies [55, 56], but was unchanged or increased in two others [54, 57]. Lueker at al. [57] reported total pulmonary resistance rather than PVR. Some postoperative ASD patients, even with normal resting PA pressure, show an increase rather than the normal decline [58] in PVR with exercise [56, 57], suggesting that the pulmonary vascular bed remains abnormal despite closure of the defect.
5.3 Increased PA Pressure and Flow In cardiac lesions with an unrestrictive communication between the systemic and the pulmonary circulations, there is increased PA pressure and flow (the latter as long as PVR is less than systemic vascular resistance). Examples include, but are not limited to, isolated, unrestrictive VSD, large patent ductus arteriosus, double-outlet right ventricle without pulmonary stenosis, single ventricle malformations without significant obstruction of PBF, and excessively large surgi-
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cally placed systemic-to-pulmonary shunts. The risk of development and rate of progression of PVD is highly variable and poorly defined, although it is known that several factors clearly impact the likelihood of and the rate of progression of PVD: the type of malformation, the size of the communication between the systemic and pulmonary circuits, the propensity of the individual to develop PVD, and environmental factors (e.g., alveolar hypoxia). The best characterized lesion of this type is the isolated VSD. The microvascular remodeling associated with this lesion begins in early infancy, and consists of distal extension of smooth muscle, seen with increased PBF but normal pressure [20]. Medial hypertrophy is also a consistent finding in infants with increased PA pressure, and a reduction in the number of small PAs is often observed [20, 22, 23, 59]. Intimal proliferation (H-E grade 2) is very unusual in early infancy (but Newfeld et al. [60] found it common) but begins to appear near the end of the first year of life in patients with increased pressure and PVR [21, 22]. Two important points are worth noting: (1) Progressive PVD occurs over time. Wood [1] and others thought that adult patients with irreversible PVD from unrestrictive communication between the systemic and pulmonary circulations had never had the normal postnatal decline in PVR, and thus PVR had been markedly elevated since birth. It is now clear that most patients with an unrestrictive VSD who develop elevated PVR have a substantial fall in PVR in infancy, followed later by an increase in resistance [61, 62]. (2) Remodeling in the first year of life is generally limited to distal extension of smooth muscle, medial hypertrophy, and a reduction of the number of small arteries; medial hypertrophy, at least, is reversible with alleviation of the increased PA pressure. Higher grade H-E changes are usually not observed until the third year of life [21, 63], which is consistent with clinical observations documenting a reliable fall in PVR with VSD closure in the first 2 years of life, but less reliably so at older ages (described below). Precise data regarding the risk of developing PVD with VSD are limited. Babies with large a VSD often develop congestive heart failure and either succumb to or have surgical closure of the defect, hence the “natural history” of pulmonary vascular remodeling is often either interrupted by death or modified by therapy. Keith et al. reported experience at the Hospital for Sick Children in Toronto with about 1,500 patients with isolated VSD [62, 64]. Two hundred patients were catheterized in the first year of life, and again “some years later.” No infant with a highly restrictive defect (normal PVR and PBF less than twice the systemic value) (n = 43) developed elevated PVR over the course of observation. There were 146 infants with PBF greater than twice the systemic value, with or without an increase in PVR; 21 (14%) of them had elevated PVR (more than 20% of the systemic value) at follow-up cardiac catheterization, which was an
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increase in PVR in all but three of these patients. This is likely an underestimate of the risk of developing PVD, since some of the patients had an operation to close the VSD during the period of observation. (They also described 11 neonates with low flow/high PVR who at follow-up evaluation had normal PVR; they attributed this to a delay in the normal postnatal fall in PVR.) The Joint Study on the Natural History of Congenital Heart Defects made similar observations. Five hundred and thirty-seven patients with an isolated VSD (21 years old or younger, half of whom were 2–11 years old) were observed over a time period of 4–8 years [46]. None of these patients had surgical closure or palliation of the defect. In patients with normal PVR, PVR rarely increased over the period of observation, but as resistance at initial observation increased, so did the risk of progression (Table 2). Hence, although we do not know the precise relationship between PA pressure, PBF, and PVR as a function of time, it is clear (for isolated VSD; see later for other increased pressure/flow lesions) that for patients with increased PBF but normal PA pressure the risk of developing PVD – at least over a period of several years – is exceedingly small, but that increased PVR tends to increase with time, at least in patients outside the early neonatal period. Reversibility has been extensively investigated following repair of VSD. Closure of a VSD in the first 2 years of life, even when PVR is markedly elevated, reliably (but with a few exceptions) results in normal or near-normal PVR at follow-up, and the likelihood of favorable pulmonary vascular remodeling is probably even better if operation is performed in the first year of life [22, 63, 65–67]. If PVR is increased, the status of the pulmonary circulation is much less favorable at older ages of operation. Although an occasional older child or adult with substantially elevated PVR has a marked fall after repair [65, 67–69], in general patients much beyond the first few years of life or so with increased PVR preoperatively continue to have increased resistance after surgery (although the PA pressure often falls as a result of the reduced volume of PBF), and some show a progressive rise despite closure. Friedli et al. [70] reported their experience with VSD closure in 57 children (average age 4.8 years, range 11 months to 17 years, at operation). PA pressure fell
Table 2 Summary of observations from The Joint Study on the Natural History of Congenital Heart Defects regarding risk of progression of PVD in patients with VSDs PVR-to-SVR ratio at initial evaluation 0.7 485 29 18 32 N (patients) 1.5 38 89 100 Percentage of patients with PVR-to-SVR ratio more than 0.2 at follow-up (4–8 years) Rarely did normal PVR increase. Substantially increased PVR was unusual, but tended to increase with time. See the text for details
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in nearly all patients after closure, although it often remained elevated; seven of ten patients with a preoperative pulmonary-to-systemic resistance ratio of more than 1:3 had a progressive increase in PVR following VSD closure, as compared with two of eight with a pulmonary-to-systemic resistance ratio of less than 1:3. Their experience was somewhat less favorable than that of many others, but persistent elevation of PA pressure (although it may fall after closure) and persistent elevation of PVR are the rule rather than the exception when higher-resistance VSDs are closed beyond the first about 3–4 years of life; [54, 65, 67–69, 71, 72] for the exception, see Ikawa and et al. [73] (Fig. 4). Further evidence of residual abnormality of the pulmonary circulation after VSD repair comes from studies showing in many patients PVR increases with exercise [57, 71, 74, 75] and/or they have an exaggerated increase in PVR as a response to subambient oxygen [57]. Maron et al. found an abnormal increase in PA pressure with exercise even in postoperative patients with normal resting PA pressure, and found that the magnitude of increase in pressure with exercise correlated positively with the age at operation [76]. The clinical consequences of persistent elevation of PVR after VSD closure appear to be related to the magnitude of residual PA pressure elevation, although available data are mostly inferential. Moller et al. [77] observed a large reduction in 30–35-year postoperative survival in patients operated on with preoperative PVR of more than 7 units, many of whose deaths were related to PVD; the negative effect on survival was considerably less marked for lesser elevation of PVR at operation. The report of Cartmill et al. [68] is consistent with this. It is important to note that although VSD is the best characterized of the increased pressure/flow cardiac lesions relative to the pulmonary circulation, it is not perfectly representative of other simple left-to-right post-atrial-shunting lesions. For example, large patent ductus arteriosus may be associated with early development of PVD, probably because the pulmonary vascular bed is exposed to increased pressure and flow in both systole and diastole. And, as discussed next, a few more complex pressure/flow lesions are associated with a greater propensity to early development of PVD than isolated VSDs.
5.4 Complex Lesions with Increased PA Pressure and Flow Variables other than PA pressure and flow importantly impact the risk of, and the pace of developing PVD. For example, patients with unrepaired d-TGA have increased PBF but normal PA pressure after the early neonatal period, yet are at risk of developing markedly increased PVR and advanced H-E changes in the first year or two of life, a tempo far more precocious than that observed with ASD. Patients with d-TGA and VSD and/or patent ductus arteriosus are even
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more notorious for developing early PVD, and are at high risk for developing grade 4 H-E lesions in the first year of life [24, 78–82]. The reason for this propensity is unclear, and a number of possible factors have been adduced to explain this unusual tendency for pathological remodeling: polycythemia (common in these patients), increased bronchial flow, observed microthrombi in small PAs, and preferential ejection of blood into the right PA (hence increased flow to the corresponding lung) [30]. It is unclear which, if any, of these factors are involved. Two other observations also suggest that the pulmonary vascular bed in d-TGA is unusually prone to increased PVR. Progressive and severe PVD following repair in patients without PVD, although uncommon, is well described [79, 83–86], whereas it is hardly ever seen with other cardiac lesions repaired in neonates. And acute and labile pulmonary vasoconstriction in newborns with d-TGA (resulting in severe hypoxemia) is not uncommonly seen [87, 88], even though such labile PVR is rarely apparent with other neonatal congenital heart defects. (However, it is possible that the increased propensity for pulmonary vasoconstriction for d-TGA is more apparent than real, since arterial oxygen saturation in neonates with d-TGA is probably more dependent on PBF than for most other cyanotic lesions.) Other malformations, e.g., truncus arteriosus [25], are associated with an increased tendency to develop PVD. The notion is well entrenched that patients with trisomy 21 are prone to early development of fixed pulmonary vascular changes, especially in the context of complete atrioventricular canal defects. Multiple papers have appeared on this subject, yet convincing confirmatory evidence is lacking [89]. Alveolar hypoxia, even at the modestly elevated altitude of Denver, Colorado (5,280 ft), augments PVR with congenital cardiac defects [8, 90, 91].
5.5 Right-Sided Obstructive Lesions This is a complex group of lesions relative to the pulmonary circulation. Some (e.g., severe tetralogy of Fallot) may have decreased PBF in utero and certainly ex utero, until surgical palliation or repair is undertaken, which may be at several months of age. Others (e.g., pulmonary atresia with an intact ventricular septum) may have decreased flow in utero (since the only source of PBF is the ductus arteriosus, which is usually restrictive in this setting), but surgical palliation in the early neonatal period often results in a normal or increased volume of PBF. Patients with pulmonary atresia and VSD (aka tetralogy of Fallot and pulmonary atresia) are complex cases: depending upon the anatomy of the central PAs, and their blood supply from the aorta, the microvascular bed in any given region of the lung may be exposed to low, normal, or increased pressure and flow. Much less
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attention has been directed to the pulmonary microvascular bed with right-sided obstructive lesions than those with leftto-right shunts, probably because increased PVR due to small-vessel abnormalities is much less likely to impact the clinical phenotype of these patients (possibly excepting those with pulmonary atresia and VSD) than those with unrestrictive communication between the systemic and pulmonary circulations. Tetralogy of Fallot is the archetypical cardiac lesion causing decreased PBF. One morphometric study investigated injected lungs from six children with this lesion, aged 1.5–4.5 years, two having had previous surgical palliation. The intra-acinar arteries and veins were increased in number, and the PAs were somewhat thicker than normal, with distal extension of muscle into normally nonmuscular arteries. Other studies have not found medial hypertrophy in this setting [92]. Any abnormality of resistance PAs in tetralogy of Fallot must seldom be of physiological consequence, however, as increased PVR after repair is rarely seen unless the patient had a previously-placed shunt (and therefore exposure to increased pressure and flow) [93]. Haworth and Reid [94] used morphometric techniques to examine the pulmonary circulation in six neonates with pulmonary atresia with an intact ventricular septum and two patients with pulmonary atresia with VSD. In all cases, the only source of PBF was via a ductus arteriosus, which they found to be restrictive (3 mm or less in diameter) in all but one case. Hence, these patients presumably had decreased PBF in utero relative to the normal fetus. One patient had had an aortopulmonary shunt placed surgically 25 days prior to death. Their findings were variable, but in most cases the pre- and intra-acinar arteries were smaller than normal, had medial muscle, and were fewer in number than normal. With pulmonary atresia and VSD there are two basic anatomic arrangements for blood supply to the lungs (albeit with some overlap): (1) a patent ductus arteriosus supplies central PAs, which distribute to all or nearly all segments of the lungs, and (2) the blood supply to the lungs derives solely from aortopulmonary collateral vessels, arising from the aorta (most often the descending aorta), or occasionally its branches. In some cases, the ductus arteriosus may provide the sole supply to a large percentage of lung, the remainder being supplied by aortopulmonary collateral vessels. The aortopulmonary collateral vessels, most likely remnants of the primitive arterial supply to the lungs which normally regress when the intrapulmonary PAs become connected to the sixth aortic arches, may be one or multiple in number, and are extraordinarily variable in their size and distribution. They may be so large as to transmit high pressure and flow to the distal vascular bed, or small or stenotic in their proximal course, and therefore flow- and pressure-restrictive. In most cases small central PAs are present, and are connected to the aortopulmonary collateral vessels, but their distribution to
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the lung segments is variable and may be quite deficient; parts of the lung may be connected only to aortopulmonary collateral vessels. In addition, multiple stenoses are often present in the central PAs and collateral vessels, which substantially increase resistance to flow through the pulmonary circuit [95, 96] (Fig. 5). Haworth and McCartney [95] reported somewhat variable morphometric findings in seven specimens from patients with pulmonary atresia and VSD. The percentage medial thickness was normal or reduced in small arteries in five cases, without abnormal distal extension of smooth muscle, but in three cases there was medial hypertrophy and distal extension of muscle. In a few segments there was mild eccentric intimal “change” or fibrosis. The diameter of the peripheral arteries was normal or small, and quite variable. The arterial number was normal. The authors interpreted these findings as being compatible with a reduction in pulmonary blood flow. The size of the aortopulmonary collateral vessels supplying the examined segments was not clear from their report, and it seems likely that their observations were of areas supplied by pressure- and flow-restrictive collateral vessels. Although published data are sparse, anecdotal observations suggest that a significant fraction of patients who have “repair” of this lesion (i.e., connecting many or all of the aortopulmonary collateral vessels, and any central PA, to a conduit connected to the right ventricle and closing the VSD) are left with increased PVR. In some cases this seems to be mostly related to increased PVR at the level of the small, resistance PAs; in others, abnormalities in the central PAs (small size, focal stenosis, lack of distribution to all segments of the lung) are operative. Peripheral PA stenosis can occur as an isolated lesion, or in conjunction with other congenital cardiac malformations, especially valvar pulmonary stenosis and tetralogy of
Fig. 5 Descending aortogram of an infant with pulmonary atresia and VSD. Multiple aortopulmonary collateral vessels arising from the descending aorta supply multiple segments of the right and the left lungs, and are connected to a large central left pulmonary artery
T.J. Kulik and M.P. Mullen
Fallot. Isolated PA stenosis can affect the main or proximal branch PAs or distal lobar or segmental PA segments; narrowing at the takeoff of lobar or segmental branches is common. The stenotic areas may be focal and discrete, or long segments of the PA may be diffusely narrow [97, 98]. Peripheral PA stenosis is most commonly diagnosed in infancy and childhood, but is well described in adults [99]. The histological findings are variable, and include fibrous intimal hyperplasia, medial hypertrophy, or hypoplasia, but diffusely hypoplastic PAs may be histologically normal [99, 100]. This condition is strongly associated with a variety of congenital syndromes, most commonly Williams syndrome, Noonan syndrome, Alagille syndrome, and congenital rubella [99]. Right ventricular pressure ranges from normal (with mild or unilateral stenosis) to suprasystemic. Interestingly, at least in patients with Williams syndrome, there is a tendency for PA stenoses to resolve with time, sometimes remarkably so [101].
6 The Biology of PVD As noted already, there are multiple distinct, but biologically related components of PVD caused by congenital heart lesions, which are variably present: (1) active constriction of pulmonary resistance arteries, (2) extension of smooth muscle distally, into normally nonmuscular PAs, (3) medial hypertrophy (arteries and veins), (4) a reduction in the number of small PAs, (5) cellular intimal proliferation, (6) intimal and medial fibrosis, (7) plexiform and other complex lesions, and (8) miscellaneous abnormalities, such as intraluminal thrombi, adventitial thickening, and atheromatous lesions in large, elastic PAs. For none of these features do we have a mechanistic understanding of their genesis. Although factors distinct from PA pressure and flow clearly impact the development of PVD secondary to cardiac malformations, the reasonably stereotyped response of the human pulmonary vascular bed to hemodynamic abnormalities suggests that increased wall stress and endothelial shear stress must somehow play a key role. Indeed, it has been long appreciated that hemodynamic forces help shape systemic arterial architecture [102], and experimental studies have suggested that increased wall stress and endothelial shear stress are important modulators of pulmonary vascular remodeling (see later). The following is a very brief outline of a few ways in which mechanical factors may play a role of the pathogenesis of PVD, considering first vasoconstriction, and then pathological remodeling. It is beyond the scope of this review to discuss the massive accumulation of other information related to the general issue of pathological pulmonary vascular remodeling (for recent reviews, see [103–105]), although cellular and molecular mechanisms unrelated to hemodynamic
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forces must also be important components of pulmonary vascular remodeling.
6.1 Mechanical Forces and Vasoconstriction Vasoconstriction is a common feature of PVD with congenital malformations, and likely increases PVR both by decreasing the cross-sectional area of resistance vessels and via stimulating structural changes in the microcirculation. The latter could occur in two ways. First, constriction of small PAs increases intraluminal pressure (and therefore a stimulus for remodeling) in the arteries proximal to them. Second, smooth muscle contractile activation itself shares intracellular biochemical pathways with the control of growth and phenotype [106]. (Indeed, most endogenous vasoconstrictors, e.g., endothelin and epinephrine, also stimulate growth of vascular smooth muscle [107, 108].) At least under certain circumstances, contractile activation may therefore stimulate smooth muscle hypertrophy and/or hyperplasia, and connective tissue synthesis. There are at least three mechanisms by which increased PA pressure could stimulate smooth muscle contraction: 1. Stretch-inducted (myogenic) contraction. There is a large body of work investigating myogenic tone in systemic arteries, and stretch-induced contraction of small PAs has been shown in vitro [109, 110] and in vivo [111]. Some have speculated that stretch-induced contraction serves to normalize wall tension in blood vessels, by reducing the vessel radius in response to increased intraluminal pressure [112]; if so, myogenic tone could be relatively high in the hypertensive pulmonary circulation. However, it is not clear if or how this property of smooth muscle operates to activate contraction in the steady state in vivo. Both the length of and the load born by smooth muscle cells in the vascular wall are a function of multiple factors and would not necessarily be abnormally high with increased intraluminal pressure. 2. Reflex contraction of resistance PAs caused by distention of the central PAs. Distention of the main- or large-branch PAs causes PVR to increase substantially [113–115]. This effect has been demonstrated in immature and mature animals [116], and human infants [117]. If the distended region of the PA is infiltrated with lidocaine, or is dissected through to the media of the artery to disrupt any nerves traversing the region, the vasoconstrictor reflex is abolished [114]. Although 6-hydroxydopamine (which destroys adrenergic nerve terminals) abolishes the hypertensive response [114], a-adrenergic blocking agents do not affect the response [113, 114], suggesting that nonadrenergic, noncholinergic transmission may be involved. This reflex could serve as a positive-feedback loop for
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increasing PVR since increased intraluminal pressure distends the large PAs. Unfortunately, this “reflex” is incompletely characterized and what if any role it plays in PVD is unknown, although it is tempting to adduce both myogenic tone and this reflex-mediated vasoconstriction – actually, their sudden reduction – to explain the rapid fall in PVR after relief of mitral stenosis. 3. Endothelial dysfunction. Pulmonary endothelial elaboration of vasoconstrictors, or reduction of endothelial production of vasodilators, could increase vascular tone. Abnormalities of pulmonary endothelial cells, including their morphology, and an increased density of microfilament bundles and rough endoplasmic reticulum, have been observed with increased PA pressure and flow in humans [118]. Although these ultrastructural abnormalities tell us little about the behavior of these cells, subsequent observations suggest deficient endothelium-mediated pulmonary vasodilation with increased pressure/flow lesions in humans [119], and in an animal model of increased PA pressure and flow [120].
6.2 Mechanical Forces and Vascular Remodeling In vitro studies of cultured systemic [121] and PA smooth muscle cells, and segments of PAs, have shown that mechanical stretch can serve as a growth stimulus and increase matrix protein synthesis [122–126]. In vivo experiments are also consistent with the notion that increased PA pressure and flow stimulate a variety of cellular responses associated with remodeling. For example, Reddy et al. developed a neonatal lamb model of increased PA pressure and flow (using a surgically placed aortopulmonary shunt) which has a pulmonary vascular phenotype consistent with that seen in the human (medial hypertrophy and distal extension of smooth muscle, although an increased number of small PAs is also observed) [127]. This model has been used to investigate the effect of altered PA hemodynamics on the expression of a number of factors which may play a role in vascular remodeling. The levels of endothelin-1, transforming growth factor b1, vascular endothelial growth factor, and fibroblast growth factor 2 are increased (relative to controls) in PAs and/or lung tissue from shunted lambs, as are alterations in expression of the cognate receptors consistent with growth promotion [128– 131]. Experiments using other in vivo models have also shown that PA pressure and/or flow critically modulate the microarchitecture of the pulmonary vascular bed [132–134]. Shear stress modulates endothelial cell phenotype, and expression of a variety of factors which affect the biological processes of adjacent cells and are thought to be operative in vascular remodeling. The notion has evolved that temporally
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and spatially uniform shear stress of the appropriate magnitude promotes endothelial expression of substances which negatively modulate growth and matrix protein synthesis (e.g., nitric oxide and prostacyclin); conversely, disturbed flow or low shear stress decreases synthesis of these negative growth regulators, and increases endothelial expression of smooth muscle mitogens, endothelin-1, and other substances operative in pathological remodeling [135–137]. Most of the work in this arena has focused on the systemic vascular bed, but surely many of the same biological processes apply to the pulmonary circulation. There is still much to be learned before a comprehensive and detailed theory of pulmonary vascular modeling related to hemodynamic disturbance can evolve. The search for such a theory is confounded by the complexity of the phenomenon, by our relatively crude understanding of how mechanical forces affect biological processes in vivo, and the lack of specific antagonists for (or genetically engineered models of) stretch- or shear-stress-mediated cellular perturbations. Insights into the pathogenesis of other forms of PVD will doubtlessly apply to uncovering the biological processes of that related to cardiac malformations. In particular, it will be interesting if gene mutations associated with idiopathic pulmonary hypertension (e.g., BMPR2 and ALK-1 mutations) are in any way linked with cardiac-malformationrelated PA hypertension; available data at this time suggest no association with BMPR2 mutations [138], or are too limited to be definitive [139].
7 Clinical Management 7.1 Prevention The best cure for PVD related to congenital heart lesions is prevention, by correction of the defect early enough to prevent permanent pulmonary vascular remodeling. As outlined already, the “window” of opportunity for prevention is reasonably wide, and to some extent lesion-specific. Although the time of operation is, to some extent, center-specific, in general ASDs are repaired by age 4–5 years, VSDs within the first year of life, complete atrioventricular canal defects and large patent ductus arteriosi by 6 months of age, and d-TGA/VSD is either repaired or is palliated using a pulmonary arterial band in the first few weeks of life. For neonates with a single cardiac ventricle the aim is to absolutely minimize pathological remodeling, and surgery to secure normal PA pressure (if needed) is usually undertaken in the first few weeks. Patients of any type who show evidence of higher than expected PVR may require operation sooner, and other factors (most often, congestive heart failure) may also dictate earlier surgery.
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7.2 Deciding When Surgical Intervention Is Appropriate For the unusual patient with a severely remodeled pulmonary circulation, things are more complicated and less satisfactory, starting with the decision of whether repair is appropriate. (We refer here to patients with increased PA pressure/flow lesions, not those with left-sided inflow obstruction; as noted previously, for the latter even markedly increased PVR in adults generally responds well to relief of pulmonary venous hypertension.) Few patients less than 1–2 years old have severely elevated PVR (pulmonary-to- systemic vascular resistance ratio of more than 0.75) and, even for those who do, given the considerable capacity of the young pulmonary circulation to “reverse” remodel, surgical repair is usually offered unless PVR is suprasystemic. On the other hand, older patients with severely elevated PVR are at risk for having persistently increased PA pressure, or even a progressive increase in PVR after repair. Significantly increased PVR also increases the risk of mortality immediately after operation in patients of all ages [10, 16] owing to labile and at times severe acute pulmonary vasoconstriction. This variably increased PVR is apparently caused by one or more endogenous vasoconstrictors, or perhaps lack of an endogenous vasodilator(s), in combination with the pathologically remodeled vascular bed [140]. More than increased smooth muscle cell mass may be involved in this hyperreactivity; e.g., abnormal endothelial function may play a role [9, 119, 141], but see [142]. At increased risk are patients with an unrestrictive ventricular or great vessel communication, truncus arteriosus having increased PVR before operation, and pulmonary venous hypertension (e.g., total anomalous pulmonary connection with obstruction or mitral stenosis) [22, 143, 144]. For patients with high, but not systemic-level PVR, we do not have a sufficiently sensitive and specific method for determining whom will respond favorably to operation (i.e., will have a substantial and lasting fall in PA pressure). Wood [1], 50 years ago, suggested that surgical repair is indicated for those whose PVR does not exceed 10 units, and whose pulmonary-to-systemic flow ratio is at least 2:1. Borderline (for repair) cases have PVR of 10–12 units, and a pulmonary-to-systemic flow ratio of 1.75–2. A more modern approach to determining operative suitability is to consider resting PVR along with the reactivity of the pulmonary circulation (enhanced inspired oxygen, tolazoline, isoproterenol, and inhaled nitric oxide have been used). Vogel et al. found that patients with VSD, even those having PVR exceeding 1,000 dyne · sec/cm5, who were “reactive” (i.e., responded to a 1 mg/kg bolus of tolazoline with a reduction of PVR to 450 dyne · sec/cm5 or less) had a substantial fall in PVR postoperatively, whereas nonreactors did
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not. But the patients of Vogel et al. were studied in Denver (alveolar hypoxia adds a reactive component to the PVR not present at sea level [91]) and nearly all of the reactive patients were 5 years old or younger. Neutze et al. [10] studied patients with VSD and found that for those with PVR of less than 8 units (average age, 34 months) reactive patients (i.e., those whose decreased PVR to less than 7 units with isoproterenol infusion) had normal or modestly increased PVR after surgical closure (n = 15). Four of the unreactive patients had surgical repair, at least three of whom died or had evidence of progressive PVD (one was lost to follow-up). Balzer et al. [16] collected data on patients with a variety of cardiac malformations (median age 28 months), including a small percentage evaluated for heart transplantation, who had vasodilator testing with enhanced FiO2 and inspired nitric oxide (n = 124). They found that if a combination of FiO2 = 1.0 and inhaled nitric oxide were used to evaluate reactivity, PVR of less than 5.3 units . m2 and a pulmonary-to-systemic resistance ratio of less than 0.27 (with vasodilators) was 97% sensitive and 90% specific for identifying patients with a good outcome (alive and without right-sided heart failure) after operation. On the other hand, Lock et al. found no relationship between pulmonary vascular response to 100% inspired oxygen and outcome after repair of VSD [4]. Lung biopsy (usually as an adjunct to hemodynamic data) has been advocated as a means of determining suitability for surgical repair. Indeed, it has been shown that preoperative biopsy (analyzed by H-E criteria) may be predictive of persistent PA hypertension in patients older than 2 years of age following repair of interventricular communications [22]. Unfortunately, there are only limited follow-up data correlating preoperative biopsy with eventual outcome, and the available information suggests that biopsy findings are neither highly sensitive nor specific for determining the presence of irreversible vascular disease. For example, Braunlin et al. [145] provided long-term follow-up data on 57 patients who had had preoperative lung biopsy with VSD. Although patients with H-E grades 1 and 2 changes generally did well, four of 47 patients died of PVD, and it is unclear how many (if any) of the survivors had increased PVR. There were only five patients with grade 3 changes, with limited follow-up information (though one clearly died of PVD). Of four patients with grade 4 changes, at least three died of PA hypertension, suggesting that advanced lesions reliably portend a poor outcome, but their numbers were few, and exceptions have been reported [146]. Other reports have shown considerable overlap between patients with severe pathological changes who do well following repair and those who do not [21, 147]. Despite multiple papers advocating the use of this modality over the last three decades, the authors are aware of no centers that routinely employ biopsy for decision making in difficult cases.
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7.3 Therapy There are multiple therapeutic modalities available for patients with PVD for whom surgery is not suitable. Unfortunately, none are nearly as effective as one would like. Most of these modalities are supportive rather than designed to decrease PVR (e.g., diuretics, erythrophoresis, and balloon atrial septostomy) and will not be approached here, nor will lung transplantation. Only therapies whose primary intended effect is to decrease PVR are discussed. 7.3.1 PA Banding Experimental work in rats suggests that the pathologically remodeled pulmonary circulation can undergo “reverse” remodeling (regression of hypertrophy and distal muscularization of distal PAs) if the PA pressure is reduced [133]. This suggests that if PA pressure can be reduced via placing a restrictive band on the main PA, so that pressure distal to the band is decreased, favorable remodeling might follow. Castaneda et al. [67] banded 13 infants (average age 3.5 months) with a large VSD (and, although not stated, probably significantly increased PBF), and found that PVR fell from an average of 1,950 to 700 dyne · sec cm5 when the patients were studied about 3–5 years later. Also, as discussed already, there are two other published reports suggesting that PA banding can reverse some pathological remodeling [32, 35]. However, this approach is rarely feasible for patients without an unrestrictive interventricular communication, since right ventricular pressure would be acutely increased by the band, which is poorly tolerated. And with a large VSD, PA banding will cause unacceptable hypoxemia if the combination of PVR and resistance to flow across the band critically reduces PBF. We do not know which patients are most likely to tolerate and usefully decrease their PVR with PA banding, but certainly only a very small number stand to derive benefit (leaving aside infants with little or no increase in PVR, banded in anticipation of surgery at an older age). 7.3.2 Pharmacological Therapy 1. Enhanced inspired oxygen. Alveolar hypoxia causes acute pulmonary vasoconstriction, and chronic hypoxia causes increased PVR and medial hypertrophy. The effect of hypoxia on the pulmonary circulation with increased pressure/flow is likely even greater than on the otherwise normal lung, given the experience in Denver with shunting lesions and mitral stenosis [90, 91]. Increasing the fraction of inspired oxygen might therefore be rational therapy, especially for patients with cardiac malformations with hypoventilation or lung disease. The authors are
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unaware of any published data on the effect of chronic oxygen therapy on PVR in patients with congenital shunting lesions, although there is one report describing its feasibility in children [13]. 2. Inhaled nitric oxide. Virtually all known vasoactive agents, notably excepting hypoxia and acidosis, have the same directional (constriction vs. dilation) affect on the pulmonary and systemic circulations. Nitric oxide, which increases the levels of intracellular cyclic GMP, relaxes both pulmonary and systemic vessels, but when given by inhalation selectively dilates the pulmonary circulation since it is mostly inactivated by blood hemoglobin before it can affect systemic vascular smooth muscle. Inhaled nitric oxide has thus proven valuable in treating postoperative pulmonary hypertensive “crises” (the acute and labile increase in PVR after cardiac surgery described earlier) [148], since systemic vasodilation in this setting is usually counterproductive. This drug has largely supplanted most other agents and maneuvers (e.g., tolazoline hydrochloride, hyperventilation) previously used for acute pulmonary vasoconstriction, although it is not invariably effective therapy. 3. Calcium channel blockers. These drugs were the first effective therapy for idiopathic pulmonary vasoconstriction (albeit for only a small minority of adult patients), and thus might prove effective with PVD due to congenital heart malformations. However, these drugs are ineffective in those patients with idiopathic PA hypertension who are not acutely responsive to vasodilators, a quality which describes most patients (beyond childhood) with severely increased PVR due to increased PA pressure and flow. Published experience with long-term use of nifedipine does not permit an accurate assessment of its effect on PVR in patients with congenital cardiac lesions [149]. 4. Prostacyclin. Two studies found chronic (3 months or more) infusion of prostacyclin in children and adults with PVD secondary to congenital cardiac shunting lesions (repaired and unrepaired) to decrease PVR, improve oxygen delivery, and increase exercise capacity [150, 151]. No effect on survival was demonstrated in these studies (which were without placebo controls). The fall in PVR was substantial (about 50%, although PVR remained very elevated), and the fact that it occurred in patients who seldom show an acute response to vasodilators suggests that prostacyclin may have effected partial remodeling of the pulmonary circulation (although other explanations are possible). There is anecdotal experience that chronic infusion of prostacyclin decreased PVR sufficiently in some patients to permit closure of defects considered inoperable before therapy [152, 153]. 5. Bosentan. There are several studies of this endothelin receptor (A and B) blocker in patients with Eisenmenger
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syndrome, two of which include hemodynamic data. One study of adult patients found 16 weeks of therapy with orally administered bosentan very modestly decreased both PA pressure and PVR [154], whereas another found a very small reduction in PVR but no change in PA pressure after 12 months of therapy [155]. Both showed an increase in systemic oxygen delivery, exercise capacity, and functional class. It is not known if bosentan affects survival in this setting. 6. Sildenafil. A report of this phosphodiesterase type V inhibitor in seven adults with Eisenmenger syndrome found that after 6 months mean PA pressure and PVR were substantially decreased and systemic oxygen delivery was increased; New York Heart Association functional class was improved, but exercise capacity was not significantly different from the baseline [156]. Multiple other studies have shown symptomatic improvement and increase in exercise capacity with sildenafil in Eisenmenger syndrome patients [157].
8 Summary Generally speaking, congenital cardiac malformations are associated with an abnormal pulmonary circulation in one of two ways: (1) Defects which increase PA pressure and/or PBF cause PVD to develop over time. The evolution of PVD generally occurs over months and years, although a few defects are associated with abnormal pulmonary hemodynamics in utero and PVD in those cases can be manifest even in the neonate. (2) Congenital abnormalities of the large PAs are a feature of certain congenital heart malformations (e.g., pulmonary atresia and VSD). Regarding malformations which increase PA pressure and flow, although there is considerable variability in the pulmonary vascular phenotype expressed even within a population of apparently similar patients, three observations stand out: (1) both active vasoconstriction and pathological remodeling operate to increase PVR, (2) increased PA pressure and increased PBF, in isolation, are considerably less likely to cause PVD than when both occur together, and (3) excepting a few high-risk malformations (e.g., d-TGA and VSD), permanent or progressive pathological changes seldom occur when malformations are corrected in the first 1–2 years of life. Our mechanistic understanding of the biological processes involved in pulmonary vasoconstriction and vascular remodeling is primitive; although many pieces of the puzzle have been accumulated, nobody knows how many are missing, nor how to fit the accumulated pieces together. Because any successful mechanistic explication of PVD in this setting must account for the effect of physical forces on pulmonary
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vascular tone and remodeling, it is heartening that multiple in vitro and in vivo models have been developed which have and will yield yet more information pertinent to the biological effects of increased PA flow and pressure. New genetic discoveries will also doubtlessly play an increasing role in uncovering the mechanisms involved in PVD, as well as a better understanding of vascular biological processes in general. Therapy for PVD related to congenital heart defects is primarily prevention, which has been much enhanced by advances in the diagnosis and repair of congenital cardiac malformations. Early experience indicating that some drugs may ameliorate PVD previously thought to be irreversible gives hope that reverse remodeling – at least to some degree – may be possible even in older patients.
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Chapter 78
Pulmonary Hypertension Secondary to Congenital Systemic-to-Pulmonary (Left-to-Right) Shunts Antonio A. Lopes
Abstract Pulmonary arterial hypertension associated with congenital left-to-right shunts remains a matter of concern not only in underserved areas, but also in developed countries, in both un-operated on and operated on patients. The risk of developing advanced pulmonary vascular disease generally but not invariably depends on the size and location of the defect. Patients with restrictive ventricular septal defects (VSDs) are unlikely to acquire pulmonary arterial hypertension (the prevalence is only 3%). On the other hand, the likelihood of severe pulmonary hypertension and development of Eisenmenger syndrome is considerable in patients with nonrestrictive defects (greater than 1.5 cm in diameter); in this case, 50% will be affected. This is in contrast with subjects with ASDs, for whom the risk of acquiring pulmonary hypertension is not higher than 10% with a late onset (90% during the adulthood). However, pulmonary artery pressure and vascular resistance tend to be more frequently elevated in patients with sinus venosus defects than in those with secundum defects. Despite the general concept that pulmonary hypertension is not a matter of concern if patients undergo repair of the cardiac shunts during the first year of life, certain anomalies are known to be associated with early elevation of pulmonary vascular resistance. There is general agreement that this is the case for truncus arteriosus, atrioventricular septal defects, and transposition of the great arteries with VSD. For unknown reasons, some patients with simple defects such as VSD or patent ductus arteriosus have no history of pulmonary congestion or failure to thrive during the first months of life, suggesting early-onset pulmonary arterial hypertension. These patients cannot be safely assigned for repair of the defects on the basis of noninvasive evaluation only. Rather, complete evaluation including direct measurement of pulmonary vascular resistance is necessary to make a decision about the therapeutic strategies. This chapter explores how the appropriate recognition of a state of A.A. Lopes (*) Department of Pediatric Cardiology and Adult Congenital Heart Disease, Heart Institute (InCor), University of São Paulo School of Medicine, São Paulo-SP 05403-000, Brazil e-mail:
[email protected] increased pulmonary blood flow or a state of increased pulmonary vascular resistance allows for decisions to be made between the therapeutic options that are applicable to patients with left-to-right shunts associated with pulmonary hypertension (correction of the defect, pulmonary artery banding, medical treatment, or combinations). Although there is not much evidence to support such decisions, expertise that has been accumulated in tertiary reference centers with high standards of medical assistance allows for proper selection of the therapeutic strategies. The issue of adults with ASD is particularly focused on. The pathogenic mechanisms of pulmonary vascular disease in left-to-right shunts and the correlations of pathological features with the outcomes are also addressed. Keywords Left-to-right shunt • Ventricular septal defect • Atrial septal defect • Eisenmenger syndrome
1 Introduction “In developed countries, pulmonary arterial hypertension is not a matter of concern in patients with congenital cardiac defects associated with left-to-right shunting, since the defects are repaired in early infancy, thus eliminating the hemodynamic stimuli for development of severe pulmonary vasculopathy.” Although this statement emphasizes the rationale for early repair of congenital cardiac shunts, it is not totally correct. Three decades ago, there was a general concept that at least 30% of patients with left-to-right shunts were at risk for development of pulmonary vascular disease [1]. In 1993, the Second Natural History Study of Congenital Heart Defects (NHS-2) showed that more than 50% of patients with a large, nonrestrictive ventricular septal defect (VSD) developed Eisenmenger syndrome [2]. Of note, 13.8% of patients with successfully repaired VSDs (no residual defects) had persistent pulmonary hypertension. More recently, the Euro Heart Survey on Adult Congenital Heart Disease showed that 12% of 377 patients with repaired atrial septal defects (ASDs) and 13% of 275 patients with repaired
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VSDs had residual pulmonary hypertension. For subjects with open defects, the prevalence of pulmonary hypertension was 34 and 28% respectively. The overall prevalence of Eisenmenger syndrome in patients followed up for 5 years in this survey was 7% (133 of 1,877 adults) [3]. On the other hand, of 201 patients with Eisenmenger syndrome followed up from 1976 to 1992 in a single tertiary center in India, the prevalence of ASDs, VSDs, and patent ductus arteriosus was 29.8, 33.3, and 14.2%, respectively [4]. Thus, pulmonary arterial hypertension associated with congenital left-to-right shunts remains a matter of concern not only in underserved areas, but also in developed countries, in both unoperated on and operated on patients. The risk of developing advanced pulmonary vascular disease generally but not invariably depends on the size and location of the defect. Patients with restrictive VSDs are unlikely to acquire pulmonary arterial hypertension (the prevalence is only 3%). On the other hand, the likelihood of severe pulmonary hypertension and development of Eisenmenger syndrome is considerable in patients with nonrestrictive defects (greater than 1.5 cm in diameter); in this case, 50% will be affected [5]. This is in contrast with subjects with ASDs, for whom the risk of acquiring pulmonary hypertension is not higher than 10% with a late onset (90% during the adulthood) [6]. However, pulmonary artery pressure and vascular resistance tend to be more frequently elevated in patients with sinus venosus defects than in those with secundum defects [7]. Despite the general concept that pulmonary hypertension is not a matter of concern if patients undergo repair of the cardiac shunts during the first year of life, certain anomalies are known to be associated with early elevation of pulmonary vascular resistance. There is general agreement that this is the case for truncus arteriosus, atrioventricular septal defects, and transposition of the great arteries with VSD. For unknown reasons, some patients with simple defects such as VSD or patent ductus arteriosus have no history of pulmonary congestion or failure to thrive during the first months of life, suggesting early-onset pulmonary arterial hypertension. These patients cannot be safely assigned for repair of the defects on the basis of noninvasive evaluation only. Rather, complete evaluation including direct measurement of pulmonary vascular resistance is necessary to make a decision about the therapeutic strategies. This chapter explores how the appropriate recognition of a state of increased pulmonary blood flow or a state of increased pulmonary vascular resistance allows for decisions to be made between the therapeutic options that are applicable to patients with left-to-right shunts associated with pulmonary hypertension (correction of the defect, pulmonary artery banding, medical treatment, or combinations). Although there is not much evidence to support such decisions, expertise that has been accumulated in tertiary reference centers with high standards of medical assistance allows
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for proper selection of the therapeutic strategies. The issue of adults with ASD is particularly focused on. The pathogenic mechanisms of pulmonary vascular disease in left-to-right shunts and the correlations of pathological features with the outcomes are also addressed.
2 Pathogenic Mechanisms For many years it was reasoned that patients with congenital heart defects and left-to-right shunting had pulmonary vascular disease as a consequence of increased mechanical forces acting on the wall of the pulmonary vessels. Until recently, except for Down syndrome, which seems to be associated with early development of pulmonary vasculopathy, no other pathogenic factors were proposed. In recent years, there has been increasing interest in genetic susceptibility, in view of its importance in the pathogenesis of idiopathic and familial pulmonary arterial hypertension. With or without a genetic factor, it seems reasonable to assume that the shear stress and the circumferential stretch associated with increased pulmonary flow and pressure are important initiating/triggering events. Vascular cells are equipped with receptors that allow them to convert mechanical inputs into biochemical events. Many pathways, including the mitogen-activated protein kinase cascade can be activated by flow or stretch leading to activation of transcription factors with subsequent gene expression [8, 9]. Endothelial cells use multiple sensing mechanisms to detect changes in mechanical forces, and the cytoskeleton plays a pivotal role in mechanotransduction [10]. In pulmonary hypertension associated with congenital heart defects, transmission election microscopy has been used to demonstrate increased density of endothelial microfilament bundles corresponding to the cytoskeleton [11]. Shear- and stretch-induced cytoskeletal reorganization in endothelial cells is followed by formation of the so called focal adhesion complexes where a number of substrates are phosphorylated by several kinases. Downstream of such signaling cascades, multiple transcription factors are activated, including AP-1, NF-kB, Sp-1 and Egr-1. The actions of these factors on their responsive elements result in the transcription of several genes [12]. Of note, nitric oxide production can be induced in pulmonary vascular endothelial cells by circumferential stretch. The phosphatidylinositol 3-hydroxy kinase pathway and endothelial nitric oxide synthase (eNOS) phosphorylation seem to be involved [13]. The results of studies on the expression of nitric oxide synthase genes (eNOS and the inducible isoform, iNOS) in pulmonary hypertension associated with increased pulmonary blood flow/pulmonary artery pressure are not totally uniform. The differences may be partly due to differences in
78 Pulmonary Hypertension Secondary to Congenital Systemic-to-Pulmonary (Left-to-Right) Shunts
the models (humans, animals, isolated-perfused lungs, etc.). Black et al. [14] used aortopulmonary vascular grafts in fetal lambs to demonstrate a 2.4:1 and 2.08:1 increase, respectively, in lung eNOS messenger RNA (mRNA) and protein in comparison with age-matched controls. On the other hand, Berger et al. [15] used immunohistochemistry to analyze both isoforms in lung tissue of patients with congenital heart disease, and showed differential (increased) expression of iNOS (but not eNOS) in patients with increased pulmonary blood flow and pulmonary artery pressure compared with those with increased flow but normal pressure, those with congestive pulmonary vasculopathy and controls. It is not known if overexpression of nitric oxide synthase genes and production of nitric oxide induced by increased shear stress or circumferential stretch are effective in counteracting the actions of the vasoconstrictors present in the scenario. Interestingly, using the same model of in utero aortopulmonary shunts in lams, Steinhorn et al. [16] were able to demonstrate impaired endothelium-dependent relaxation of pulmonary arteries which was significantly improved by pretreatment with superoxide dismutase and catalase, but not l-arginine. Flow- and pressure-related damage to the pulmonary vascular endothelium has been shown to induce proteolytic activities within the vascular wall leading to cellular proliferation and deposition of extracellular matrix. The loss of endothelial integrity is associated with entry of circulating factors to the subendothelial space. The presence of these factors in the subendothelium likely accounts for stimulation of smooth muscle cells to produce proteases such as endovascular elastase [17]. Pericellular proteolysis leads to release of peptide growth factors from their proteoglycan storage sites within the matrix. In addition, extracellular matrix degradation results in tight interactions between matrix proteins (collagens) and the smooth muscle cell membrane via integrin superfamily receptors, leading to further synthesis of matrix glycoproteins such as fibronectin and tenascin. Subsequent interactions of the cell with the modified matrix (collagen, tenascin) via integrins result in cytoskeleton organization, intracellular signaling with the formation of focal adhesion complexes, and clustering of growth factor receptors (e.g., epidermal growth factor) which become sensitive to agonists [17]. Colocalization of tenascin and epidermal growth factor with smooth muscle cells of hypertensive pulmonary arteries has been demonstrated [18]. The end results of these processes include changes in smooth muscle cell phenotype to a synthetic pattern, cellular proliferation, and migration to the intima which is facilitated by the presence of fibronectin in the matrix [19]. There has been evidence of increased production of endothelin in lungs of patients with congenital heart disease [20]. However, in humans, it is not known whether endothelin and its receptors (ETA and ETB, which mediate vasoconstriction and vasodilatation, respectively) play a substantial
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role in the early stages of pulmonary vascular remodeling associated with congenital cardiac shunts. Anyway, because endothelin (particularly endothelin-1) is a potent vasoconstrictor and proliferation-stimulating agent, there has been general agreement that it contributes to the progression of the disease. Controversy remains about the stimulus that triggers the expression of endothelin and its receptors in lungs of patients with cardiac shunts (increased flow, increased flow and pressure, increased diastolic pressure in the pulmonary circulation). In fetal lambs with aortopulmonary shunts (presumably increased pulmonary flow and pressure), increased levels of endothelin-converting enzyme-1 and ETA receptor mRNA and protein have been observed in lung tissue. However, ETB receptor mRNA and protein have been shown to be decreased [21]. Lutz et al. demonstrated increased density of ETA receptors in lung arteries and parenchyma of patients with increased pulmonary vascular resistance in comparison with those with increased flow but low resistance, whereas the density of ETB receptors was low and not related to hemodynamics [22]. Vincent et al. observed increased circulating endothelin levels in children with congenital heart defects associated with a pulmonary to systemic flow ratio higher than 1.5 [23]. On the other hand, Ishikawa et al. demonstrated increased plasma levels of endothelin-1 in patients with VSDs (increased pulmonary flow and pressure) in contrast to those with high flow and low pressure associated with ASDs. Interestingly, in patients with severe pulmonary congestion due to pulmonary venous stenosis, plasma levels of endothelin-1 were even higher compared with those in patients with VSDs [24]. Thus, it can be hypothesized that increased diastolic pressure in the pulmonary circulation is the principal stimulus for induction of endothelin. Further studies are required to refine the mechanisms that trigger the expression of endothelin and its receptors in patients with congenital cardiac shunts, and for a better understanding of their behavior on a long-term basis following repair of the anomalies. Several other mechanisms described for pulmonary arterial hypertension in general likely play a role in the development of pulmonary vasculopathy associated with congenital heart defects. For example, abnormalities in the behavior of ion channels (particularly Kv channels) which have been extensively studied in idiopathic pulmonary arterial hypertension are probably present in congenital heart disease. Limsuwan et al. reported that the activity of K+ channels was reduced in pulmonary vascular smooth muscle cells exposed to serum from patients with pulmonary arterial hypertension associated with congenital cardiac defects [25]. In the specific setting of peptide growth factors, increased expression of vascular endothelial growth factor which has been demonstrated in patients with congenital heart disease [26] associated with defective transforming growth factor b (TGF-b) signaling may account for loss of the equilibrium
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between cellular growth and apoptosis in favor of proliferation. Mutations in the gene that encodes the bone morphogenetic protein receptor type II (BMPR-II), a member of the TGF-b superfamily of receptors, have been identified in approximately 60% of patients with familial pulmonary arterial hypertension and in 25% of individuals with the sporadic form of the disease [27]. The BMPR-II mutations are known to alter the pattern of phosphorylation of cytoplasmic signaling proteins within the smooth muscle cells, leading to signal transduction, gene transcription, and cellular proliferation, thus explaining the tendency for cellular growth observed in patients with pulmonary arterial hypertension [28]. Interestingly, BMPR-II mutations have been reported in pulmonary hypertension associated with congenital heart disease [29]. Even more exciting are the observations that members of the bone morphogenetic protein/ TGF-b pathway may be involved in developmental anomalies of the heart that are known to be associated with pulmonary arterial hypertension such as atrioventricular septal and conotruncal defects [30–32]. This leads to the fascinating hypothesis that abnormalities of a single gene family might account for both the congenital cardiac anomaly and the tendency for development of advanced pulmonary vasculopathy, with flow and pressure acting as triggering factors.
3 Pathology The pulmonary vascular lesions that accompany the congenital cardiac anomalies with left-to-right shunting were classified by Donald Heath and Jesse E. Edwards in 1958 [33]. The lesions were classified in six grades according to the severity. This classification remains useful and has been considered as the most important qualitative approach to pulmonary vasculopathy in congenital heart disease. It con-
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stitutes the basis for definition of reversibility of vascular lesions following repair of the septal defects. Grade I corresponds to medial hypertrophy of the pulmonary arteries, whereas in grade II lesions intimal proliferation is present, with progressive accumulation of cellular elements and fibroelastic tissue in the vascular lumen leading to complete occlusion (grade III) (Fig. 1). Grades IV–VI correspond to vascular dilatation sometimes associated with angiomatoid structures or plexiform lesions (thin-walled lesions with capillary-like proliferation inside) and, finally, necrotizing arteritis with reactive inflammatory exudate throughout the vascular layers [34]. Subsequently, Rabinovitch et al. proposed a morphometric (quantitative) approach to the pulmonary vascular abnormalities that occur in congenital heart disease, which consisted of measuring the amount of smooth muscle in the pulmonary arteries and determining the number of small intraacinar arteries relative to the number of alveoli [35]. The first observed change is an abnormal extension of smooth muscle into peripheral, normally nonmuscular arteries (grade A). In grade B, medial thickness is present as a result of hypertrophy and hyperplasia of preexisting smooth muscle cells and increase in the amount of extracellular matrix, and is determined relative to the arterial diameter. Grade C corresponds to a decrease in arterial concentration likely due to impaired growth, and is established on the basis of the alveolar to arterial ratio. This ratio is around or above 20:1 in neonates, but normally decreases to less than 10:1 by the age of 5 or 6 years owing to accelerated growth of intraacinar arteries relative to the alveoli. In children with congenital cardiac shunts and pulmonary hypertension, the alveolar to arterial ratio remains elevated. There has been general agreement that Heath–Edwards grade III–VI lesions are associated with irreversible disease and persistent pulmonary hypertension following surgical
Fig. 1 a Muscular pulmonary artery with marked medial hypertrophy and absence of intimal proliferation (Heath–Edwards grade I). b Small pulmonary artery totally occluded by intimal proliferation and fibrosis (Heath–Edwards grade III). (Courtesy of Vera D. Aiello, Heart Institute, São Paulo, Brazil)
78 Pulmonary Hypertension Secondary to Congenital Systemic-to-Pulmonary (Left-to-Right) Shunts
repair of the cardiac defects. This is particularly so when a morphometric grade C is detected. However, Rabinovitch et al. observed that when patients are subjected to surgical treatment early in life (before 9 months of age), pulmonary vascular resistance decreases 1 year postoperatively to normal levels regardless of the severity of pulmonary vascular lesions in biopsy specimens [36]. Furthermore, all of the drugs that are currently available for treatment of pulmonary arterial hypertension (namely, prostanoids, endothelin receptor antagonists, and phosphodiesterase inhibitors) probably have antiproliferative properties as suggested in experimental conditions. Therefore, the criteria for reversibility of pulmonary vascular abnormalities in congenital heart disease will probably change in the years to come with the emerging possibilities of combining surgical and medical (drug) interventions. There has been much discussion about the usefulness of lung biopsies to establish the severity of pulmonary vasculopathy and predict outcomes, in particular in view of the possibility of challenging the whole pulmonary circulation with vasodilators during cardiac catheterization. However, it should be considered that as new therapies become available, more information is needed about their actions on pulmonary vascular remodeling. New biological markers in lung tissue will probably be validated on the basis of the correlations between their expression and long-term outcomes. Thus, in many institutions, intraoperative lung biopsies are still performed in selected patients suspected of having moderate to severe disease and likely to require long-term postoperative treatment. Obviously, biopsies should be performed only in tertiary centers with expert pathologists. Moreover, lung biopsy specimens (preferably obtained from the upper lobes) must be adequate for analysis. The results must include not only qualitative information, but also morphometric data, a detailed description of intraacinar arteries (dilated thin-walled vessels may be suggestive of severely occlusive proximal lesions not seen in the material), and reference to the presence or absence of perivascular inflammatory elements.
4 Clinical Assessment of Pulmonary Hemodynamics and Prediction of Outcomes The decision to repair large, nonrestrictive septal defects, frequently referred to as “operability,” is based on the likelihood of persistent pulmonary hypertension following the procedure, since this may be associated with right-sided cardiac failure, low cardiac output, and, eventually, fatal outcome. This is particularly important in patients who require openheart operations, since cardiopulmonary bypass is often fol-
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lowed by a rise in pulmonary vascular resistance. Theoretically, moderate to severe pulmonary vascular lesions known to be associated with postoperative pulmonary hypertension, in particular in the presence of vasoconstrictor stimuli, should be detected by direct examination of the pulmonary microvasculature. However, lung biopsies are not routinely performed preoperatively. On the other hand, postoperative pulmonary hypertensive crises may occur even in patients with mildly elevated pulmonary vascular resistance, and are difficult to predict in these instances. Therefore, the propensity to pulmonary vasoconstriction per se does not define a given patient as inoperable. Rather, it reinforces the need for specific perioperative therapeutic interventions. The definition of inoperability is based on the likelihood of moderate to severe pulmonary vasculopathy (obstructive lesions) which is established after complete preoperative noninvasive and invasive evaluation. Direct examination of lung biopsy specimens may be necessary in selected cases. There are no doubts about the importance of differentiating between “predominantly increased pulmonary blood flow” (either spontaneously or during vasodilator administration) and “predominantly increased pulmonary vascular resistance” (fixed) since, as mentioned, patients in the second category are at a higher risk for persistent pulmonary hypertension following repair of the cardiac defects. However, the application of this concept in practice requires caution in view of questions that remain unanswered. First, there is no clear-cut value of pulmonary vascular resistance that defines which levels are “considerably high.” Second, the magnitude of vasodilator-induced drop in pulmonary vascular resistance that defines a “significant response” in terms of predicting favorable outcomes has been a matter of debate. Also, there has been considerable discussion about the intensity of the vasodilator stimulus that should be used in vasoreactivity tests. Last, neither the baseline level of pulmonary vascular resistance nor its change by vasodilators can accurately predict the occurrence of postoperative pulmonary hypertensive crises which may be associated with fatal outcomes in some cases. Despite these difficulties, attempts to correlate preoperative hemodynamics with outcomes deserve consideration. Examples of such attempts are shown in Table 1 [37–41]. In patients with congenital cardiac shunts associated with pulmonary hypertension, pulmonary vasoreactivity has been tested using a number of vasodilator stimuli and protocols. Inhaled oxygen and inhaled nitric oxide, either alone or in combination, have been the most frequently used vasodilators. Nitric oxide has been used in concentrations ranging from 5 to 80 ppm. Maximal pulmonary vasodilatation can be achieved by using 80 ppm nitric oxide in the presence of high oxygen concentrations (90–100%). However, controversy remains about the use of such a strong stimulus to define operability.
11
Turanlahti et al. [40] Not pre-established >10% drop in PVR/SVR in 11 patients >20% drop in PVR/SVR in 9 patients ³20% drop in PVR/SVR or PVR/SVR 10% drop in PVR/SVR in 11 operated on patients
Balzer et al. [41]
124
• ~100% O2 • ~100% O2 + 10–80 ppm NO (median 60 ppm NO)
Not specified PVR/SVR < 0.27 (optimal balance in sensitivity and specificity in retrospect) RA room air (21–23% oxygen), NO inhaled nitric oxide, PVR pulmonary vascular resistance, PVR/SVR pulmonary to systemic vascular resistance ratio
• RA + 20, 40, 80 ppm NO • 90–100% O2 + 20, 40, 80 ppm NO
100% O2 RA + 80 ppm NO 91% O2 + 80 ppm NO 100% O2 RA + 5 ppm NO RA + 40 ppm NO
7 (immediate) and 4 (late) of 74 operated on patients
2 of 11 operated on patients
1 of 15 operated on patients 3 of 15 operated on patients (all of them with a ³20% drop in PVR preoperatively) 6 of 11 operated on patients
15
Yasuda et al. [39]
• • • • • •
Not mentioned
19
Atz et al. [38]
Fatal outcome 1 of 7 operated on patients None of 10 operated on patients
Positive response + PVR/ SVR £0.3 Positive response + PVR 10% drop in PVR and PVR/SVR ³20% drop in PVR
13
Berner et al. [37]
• RA + 35 ppm NO
Postoperative pulmonary hypertensive crises
Table 1 Correlation between pulmonary vascular reactivity and outcome in patients with left-to-right shunts Patients in the study References with unrepaired shunts Vasodilatation protocols Positive response Criteria for operability
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Some specialists would not consider an open-heart operation for patients who respond to strong stimuli only. On the other hand, the use of oxygen at high concentrations in patients with large septal defects may lead to error in the calculation of pulmonary blood flow. In these instances, even mild variations in oxygen saturation in the pulmonary artery lead to considerable inaccuracy in the estimation of pulmonary flow by the Fick method. Furthermore, it is not possible to measure oxygen consumption in patients receiving about 100% O2. The magnitude of the drop in pulmonary vascular resistance during vasodilator administration has been used to define operability. Ideally, the response should be linked to the intensity of the vasodilator stimulus used in the test. For example, using 80 ppm nitric oxide with about 100% oxygen, Balzer et al. [41] reported a 97% sensitivity and 90% accuracy in predicting favorable outcomes when a final pulmonary to systemic vascular resistance ratio of less than 0.33 was used as the criterion for operability. In their study, the specificity was only 8% when a 20% decrease in pulmonary to systemic resistance ratio was used as the criterion. Other authors prefer to adopt a 20% or greater drop in pulmonary vascular resistance as the responsiveness criterion, but to define operability only in the presence of pulmonary vascular resistance levels below 6 units·m2 during vasodilator administration. On the basis of the observations by the authors listed in Table 1 and others, very stringent hemodynamic criteria for operability can be proposed. The majority of specialists agree that patients with cardiac shunts associated with pulmonary hypertension are expected to have a favorable postoperative outcome if they have a baseline pulmonary vascular resistance index of less than 6 units·m2 and a pulmonary to systemic vascular resistance ratio of less than 0.3. For patients above these levels, a 20% or greater drop in both parameters during inhalation of low to intermediate concentrations of nitric oxide (e.g., 20–40 ppm), with final levels of less than 6 units·m2 and less than 0.3, respectively, would be predictive of favorable outcome as well. Pulmonary vasodilator tests are generally performed if baseline pulmonary vascular resistance is in the range of 6.0–9.0 units·m2, which corresponds to a pulmonary to systemic vascular resistance ratio of 0.3–0.5. Although patients above these limits can be considered for surgical treatment if the mentioned criteria are met, the risk of postoperative complications and unfavorable outcome is considerable. These policies are obviously stringent, and would probably exclude patients who might benefit from repair of their defects. However, for adoption of less stringent criteria, randomized controlled studies are required. Finally, it is noticeable that even stringent criteria cannot totally eliminate the risk of postoperative pulmonary hypertensive crises. In this way, some intensivists strongly argue in favor of having a pulmonary arterial line for a better postoperative management of patients who are considered “at risk.”
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Also, many argue in favor of the “prophylactic” use of nitric oxide postoperatively in these patients, although there are no controlled studies to support such a recommendation. Thus, prediction of outcomes in patients with congenital cardiac shunts associated with pulmonary hypertension remains a challenge. For the majority of patients with large defects causing significant left-to-right shunting who are assisted early in life, repair can be indicated on the basis of noninvasive evaluation. However, for patients who do not have a clear history of congestive heart failure, a careful analysis of results of noninvasive and sometimes invasive evaluation becomes mandatory. In these instances, the decision to operate and prediction of outcomes cannot be based purely on hemodynamic data. Rather, the presence of associated syndromes (especially Down syndrome), the patient age, the type of the anomaly, the direction of flow across the defect (left-to-right or bidirectional), and the peripheral oxygen saturation recorded sequentially over several days must be taken into account for decision making.
5 Treatment Modalities Ideally, patients with simple defects such as VSDs should undergo complete repair, particularly if they are seen early in life. However, as discussed, persistent pulmonary hypertension following repair is still a problem in some of them. Patients with persistent pulmonary hypertension are generally treated with the drugs that are available for management of pulmonary arterial hypertension, namely, prostanoids, endothelin receptor antagonists, and phosphodiesterase inhibitors. In fact, in many clinical studies designed to investigate the efficacy of these drugs, patients with residual pulmonary hypertension following repair of congenital cardiac defects have been enrolled [42–44]. Alternatively, patients who are considered “at risk” for persistent pulmonary hypertension can be offered pulmonary artery banding initially. If this is performed in young children, one expects that removal of the hemodynamic stimuli (mainly increased pressure) will be followed by improvement of pulmonary vascular abnormalities [45]. If so, complete repair will become possible months later, with a lower risk of poor outcomes due to heightened pulmonary artery pressure and vascular resistance. Favorable effects of hemodynamic unloading were demonstrated by Wagenvoort et al. [46] with reversal of pulmonary vasculopathy following pulmonary artery banding in patients with congenital cardiac shunts. In experimental pulmonary hypertension, hemodynamic unloading is associated with decreased number of proliferating cells in the wall of pulmonary arteries and decreased medial thickness [47]. However, there are unsolved problems with the use of
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pulmonary artery banding in humans. There have been no controlled studies demonstrating the efficacy of this procedure in specific patient subgroups (different ages and different defects). The magnitude of the reduction in pulmonary artery pressure that would be necessary to induce reversal of vascular remodeling is not known. In some instances, distal pulmonary artery pressure does not decrease substantially, unless a pharmacological intervention is proposed as part of the treatment. Telemetrically adjustable pulmonary artery banding devices are now available [48], and may be advantageous in such instances. Ineffectiveness of banding may be associated with extreme thickness of the media of small pulmonary arteries [49]. As a general rule, pulmonary artery banding may be beneficial in selected cases, if a substantial reduction in distal pulmonary artery pressure can be achieved (the magnitude and appropriate duration have not been defined yet) in the absence of hypoxemia. There are, in fact, several possible therapeutic approaches to congenital cardiac shunts associated with pulmonary hypertension. For the majority of patients seen within the
first months of life with large defects, complete repair is the treatment of choice. At the other side of the range, there is a smaller group of children, even young ones, with no history of pulmonary congestion or failure to thrive who present with peripheral oxygen desaturation and heightened pulmonary vascular resistance unresponsive or poorly responsive to vasodilators. In these instances, medical treatment should be always considered first. In many cases, it is the only treatment acceptable. Between these extremes, there are several situations where patients could possibly benefit from combined medical and surgical approaches. Unfortunately again, there are no controlled studies to support such recommendations. However, expertise coming from reference centers with high standards of assistance can be used a general guide for decision making, at least in the less complex situations. On the basis of the authors listed in Table 1 and others [50–52], Fig. 2 was constructed as an attempt to summarize the strategies that can be used in the management of patients with VSD and moderate to severe pulmonary hypertension. Accordingly, only patients in categories A and B can be
Fig. 2 Possible therapeutic strategies for patients with ventricular septal defects (VSDs) at different levels of pulmonary vascular resistance (PVR), pulmonary to systemic vascular resistance ratio (PVR/SVR), and response to vasodilators. Patients with the profiles in a and b are likely to become free of medication (assuming that in b pulmonary artery banding will be followed by VSD closure). Patients with the profiles in c and d are unlikely to benefit from complete repair, and will probably need medication for
treatment of pulmonary arterial hypertension on a long-term basis. Even defined as operable, some patients “are likely” to require special care postoperatively (e). This is particularly so for subjects with marked pulmonary vasoreactivity shown in preoperative tests. The therapeutic proposals shown are based on the opinion of experts, but are not supported by evidence. ASD atrial septal defect, PA pulmonary artery. (The artwork is courtesy of Valéria de Melo Moreira, Heart Institute, São Paulo, Brazil)
78 Pulmonary Hypertension Secondary to Congenital Systemic-to-Pulmonary (Left-to-Right) Shunts
expected to become free of medication on a long-term basis (assuming that patients in category B will have the VSD corrected sometime after pulmonary artery banding). Patients in categories C and D are likely to need drugs for treatment of pulmonary arterial hypertension for their whole life. Again, the situations depicted in Fig. 2 are illustrative and should not be used as evidence-based guidelines.
6 The Special Issue of Atrial Septal Defects ASDs are prevalent in adults with congenital heart disease, corresponding to more than 40% of all the anomalies detected in adulthood [3]. In normal subjects, the pulmonary artery systolic to diastolic pressure ratio is about 2:1. In patients with uncomplicated ASDs (left-to-right shunting), this ratio increases, with heightened pulmonary artery systolic pressure due to increased right ventricular ejection volume, and normal or even decreased pulmonary artery diastolic pressure due to the septal defect itself. Pulmonary vascular disease is a relatively rare complication in this disease. It occurs in 5–10% of patients with unrepaired defects [6], but a prevalence of 35% has been reported in adults [3]. As pulmonary vasculopathy develops, the pulmonary artery systolic to diastolic pressure ratio decreases owing to increased diastolic pressure. In a recent report, pulmonary arterial hypertension was identified as the best independent predictor of functional limitation in adults with ASDs, with an odds ratio of 25.2 [53]. Importantly, pulmonary arterial hypertension may be detected in about 12% of adults with repaired defects [3]. This leads to a considerable change in the natural history of the disease, with need for long-term vasodilator therapy and eventually atrial septostomy. There has been general agreement that adults with ASDs should be offered corrective procedures, in particular since long-term functional limitations and hemodynamic abnormalities seem to be more prevalent among patients with an open defect compared with those with a closed defect [53]. Unfavorable outcomes in patients above the age of 40 years with unrepaired defects are associated with a mean pulmonary artery pressure of more than 35 mmHg and peripheral oxygen saturation of 80% or less [54]. On the other hand, anatomic closure of ASDs in adulthood is generally performed in patients with pulmonary artery systolic pressure of less than 70 mmHg and a pulmonary to systemic blood flow ratio of more than 1.7 [55]. Even in the era of new drugs for treatment of pulmonary arterial hypertension, there is no reason for closing defects in patients with right-to-left shunting or a pulmonary vascular resistance of more than 14 Wood units·m2 [56]. Temporary balloon occlusion of the ASD during cardiac catheterization may be of help to assess the impact of the acute shunt elimination on right atrial and pulmonary artery pres-
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sures. The procedure may also be helpful in patients with left-sided diseases, to analyze left atrial pressure and decide which subjects will tolerate a corrective procedure. There are alternatives for the management of patients with an ASD and elevated pulmonary artery pressure and vascular resistance. Although there are no controlled studies to support recommendations of long-term therapy with vasodilators, there have been some reports suggesting that these drugs may be beneficial [57, 58]. In particular, there have been reports of patients treated with intravenous administration of epoprostenol or oral administration of bosentan with successful closure of the defect subsequently [59, 60]. Alternatively, fenestrated devices can be used for partial closure of the defects in patients with elevated pulmonary artery pressure [61], since in late follow-up, many patients undergoing percutaneous closure with conventional devices do not have complete normalization of pulmonary hemodynamics [62]. All these single or combined procedures require controlled studies for them to be recommended. Only case series have been reported until now. Anyway, in view of the poor outcomes in patients with closed defects and moderate to severe residual pulmonary hypertension, with survival curves similar to those for patients with idiopathic pulmonary arterial hypertension, there has been increasing interest in the use of vasodilators before making a decision about corrective procedures.
7 Drug Therapy There are different potential objectives of drug therapy in congenital cardiac shunts associated with pulmonary hypertension, depending on the clinical and hemodynamic conditions. An obvious objective of the treatment is to improve peripheral oxygen saturation, the physical capacity, and the quality of life in severely hypoxemic patients with advanced Eisenmenger syndrome. In terms of clinical studies, there has been considerable effort to achieve this goal. For example, prostanoids [63] and the endothelin receptor antagonist bosentan [64–66] have proved beneficial up to 1 year of treatment. However, the efficacy of bosentan seems to decline over the second year of therapy [44, 67], particularly in children. A second goal to be achieved is the management of elevated pulmonary artery pressure postoperatively. Pulmonary hypertensive crises may occur even after successful repair of the cardiac anomalies in patients for whom operability is defined on the basis of complete noninvasive and invasive evaluation. Increased pulmonary vasoreactivity following cardiopulmonary bypass is a well-known phenomenon likely related to decreased nitric oxide production and/or its inactivation by free radicals produced during the ischemic process
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[68, 69]. Furthermore, increased plasma levels of endothelin-1 have been demonstrated after cardiopulmonary bypass in patients with congenital heart defects and pulmonary hypertension [70]. Substances that increase nitric oxide production such as its precursor l-arginine might theoretically improve the postoperative pulmonary endothelial dysfunction that occurs in these patients [71]. Alternatively, nitric oxide can be administered by inhalation to counteract the effects of endogenous vasoconstrictors [72–74], with daily measurement of the methemoglobin level. One randomized study demonstrated favorable effects of inhaled nitric oxide after congenital heart surgery [75], although its use against a background of hyperoxic alkalosis does not seem to be associated with any benefits [76]. Also, there seems to be no benefit from doses exceeding 10–20 ppm, and effects have been demonstrated with 2 ppm nitric oxide [77]. Preliminary data suggest that prostanoids, endothelin receptor antagonists, and phosphodiesterase inhibitors may be useful to control postoperative changes in pulmonary hemodynamics. Aerosolized iloprost seems to be as effective as nitric oxide in selectively lowering pulmonary vascular resistance [52]. In experimental models of pulmonary hypertension secondary to increased pulmonary blood flow, endothelin receptor antagonists seem to effectively block the increase in pulmonary vascular resistance that occurs after bypass [78, 79]. Intravenously administered sildenafil appears to be a potent pulmonary vasodilator when used in children with congenital heart disease in the catheterization laboratory, as well as postoperatively [80]. In addition, sildenafil has proved useful to facilitate weaning from inhaled nitric oxide and prevent rebound pulmonary hypertension [81, 82]. Rebound pulmonary hypertension is an undesired condition that leads to hemodynamic instability and hypoxemia, and requires reinstitution of inhaled nitric oxide therapy with prolonged ventilation and delayed tracheal extubation. Despite the usefulness of all these pharmacological agents, there have been no controlled studies demonstrating benefits with their prophylactic use in children who are considered “at risk” for postoperative pulmonary hypertensive crises. The third issue that needs to be addressed is the advantage (if any) of giving vasodilators preoperatively (and eventually postoperatively) to patients who are considered operable, but are at risk for persistent pulmonary hypertension after hospital discharge. The typical scenario is that of a 2-year-old child with preoperative pulmonary vascular resistance of 9–10 Wood units·m2 decreasing to less than 5 Wood units·m2 during nitric oxide inhalation, discharged from hospital after successful repair of an atrioventricular septal defect. Survival curves for patients who are seen with residual pulmonary hypertension after repair of congenital cardiac shunts are considerably worse than those for unoperated on subjects [83]. Thus, decreasing pulmonary artery pressure by means of vasodilator therapy initiated
A.A. Lopes
before or immediately after operation could be tremendously beneficial. Unfortunately, there are no controlled studies to support such recommendations, and decisions are based on careful analysis of individual data. However, on the basis of the benefits that have been demonstrated with the use of prostanoids, endothelin receptor antagonists, and phosphodiesterase inhibitors in children with pulmonary hypertension in general [84–87], it seems reasonable to suppose that these drugs may be of help in the management of pulmonary hypertension associated with congenital cardiac shunts before and after operation. The recent advances in the treatment of pulmonary arterial hypertension lead to a new dilemma, which is the last point to be addressed in this session. Can the new drugs reverse advanced pulmonary vascular lesions associated with congenital cardiac shunts so that inoperable patients become operable? Although there are potential merits of the “treatand-repair” approach using the recent therapies, the discussion will probably remain open for a long time [88]. This is not the case for hypoxemic subjects with advanced Eisenmenger syndrome. Thus, if pulmonary vasodilatation actually occurs, surgical palliation will become necessary to protect pulmonary vessels (those not completely damaged) from increased blood flow. Adjustable pulmonary artery banding could be an option in such instances. Alternatively, a fenestrated patch could be used for partial closure of an ASD or a VSD (Fig. 3). To test these hypotheses, specific studies are needed taking into consideration the drugs (single or combined), the patient age, the nature of the cardiac anomaly, and the severity of pulmonary vasculopathy. On the basis of current knowledge and survival curves, there is no rationale for closing septal defects in patients with advanced pulmonary vascular disease.
8 Summary Early recognition of congenital cardiac shunts and advances in postoperative care have allowed for successful correction of the anomalies with decreased risk of residual pulmonary hypertension associated with progressive pulmonary vasculopathy. In the vast majority of cases, assignment for corrective procedures is based on noninvasive evaluation, with no need for cardiac catheterization. In some cases, however, a noninvasive diagnostic approach does not suffice. These include patients who seek medical care later in life (e.g., by the age of 2 years or above) and those, even younger, with specific defects such as truncus arteriosus, atrioventricular septal defects, transposition of the great arteries, and large, unrestrictive VSDs. In these instances, despite recent advances with imaging, cardiac catheterization is needed. Direct measurement of pulmonary vascular resistance and
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these disorders toward much poorer outcomes. With the availability of new drugs for treatment of pulmonary arterial hypertension, the criteria for operability tend to change toward a “treat-and-repair” paradigm. But controlled studies are required to determine which therapies will prevent immediate postoperative pulmonary hypertensive crises often associated with fatal outcomes, and long-term residual pulmonary hypertension. Until then, patients should be carefully evaluated, and decisions should be made on an individual basis using multiple data. A full picture is better!
References
Fig. 3 Definition of operability in patients with congenital cardiac shunts associated with pulmonary hypertension. In patient 1, the decision to catheterize was based on the absence of a clear history of failure to thrive, the presence of bidirectional flow across the VSD, and periods of peripheral oxygen desaturation. Operability was defined on the basis of the patient’s age, the presence of pulmonary congestion on the chest X-ray, and favorable hemodynamic findings. A pulmonary vasoreactivity test was considered unnecessary. In patient 2, cardiac catheterization was indicated on the basis of age, the absence of pulmonary congestion, bidirectional shunting, and periods of peripheral oxygen desaturation. The more than 20% drop in PVR index (PVRi) and PVR/SVR during nitric oxide (NO) inhalation indicated that vasoconstriction was present. However, since the final values of PVRi and PVR/SVR were above 6 units m2 and 0.3, respectively, complete VSD closure was not indicated. A fenestrated patch was used for partial closure of the defect after 45 days of vasodilator therapy with sildenafil. The patient had an uneventful postoperative course. MR mitral regurgitation, PAP pulmonary artery pressure, Qp/Qs pulmonary to systemic blood flow ratio, SatO2 peripheral oxygen saturation
determination of pulmonary vasoreactivity using appropriate vasodilatation protocols are crucial for making decisions about the therapeutic strategies. The survival curves for unoperated on patients with congenital cardiac shunts and pulmonary hypertension are considerably better than those for patients with idiopathic pulmonary arterial hypertension, suggesting that inappropriate closure of septal defects may shift the natural history of
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Chapter 79
Surgical Evaluation of Congenital-Heart-Disease-Associated Pulmonary Hypertension Clive J. Lewis and Andrew A. Klein
Abstract Congenital heart disease (CHD) is the most common birth defect, with an incidence of approximately 0.8% of live births. Improvements in surgery have altered the impact of CHD enormously, not only improving mortality in many conditions but also allowing some infants to survive into adulthood when previously they would have died within the first few days after birth. This increased survival has led to a huge increase in the number of survivors of surgery for CHD in childhood now reaching adulthood. This group of “grown-up CHD” (GUCH) patients have distinct needs outside both pediatric and adult cardiology environments and are best served by specialists in this field who are trained to understand the long-term complications these patients face. Pulmonary arterial hypertension (PAH) associated with CHD (CHD-PAH) is relatively common, occurring in 5–10% of patients. It may complicate both simple and more complex lesions and may occur early or late in the natural or operative history of the condition. It results in significant morbidity and early mortality of patients compared with those without evidence of PAH. This chapter discusses the evaluation of CHD patients with dynamic PAH where surgical correction of lesions may be safely undertaken. Surgical or interventional correction is desirable, particularly early in the natural history of CHD before advanced changes in the pulmonary vasculature are established, provided there is evidence to support reversibility of PAH afterward, although this may be difficult to predict in some difficult or marginal patients. Special consideration is given to established, moderate to severe PAH, in particular patients with Eisenmenger syndrome. Discussion of operability most often arises in older children and adults with large shunts and therefore this chapter concentrates on this group of patients. Consideration is also given to advanced therapies, including transplantation, and the importance of anesthetic and intensive care management in patients with CHD-PAH.
C.J. Lewis (*) The Transplant Unit, Papworth Hospital NHS Foundation Trust, Papworth Everard, CB3 8RE, Cambridge, UK e-mail:
[email protected] Keywords Surgical treatment • Operability • Inoperable patients • Interventional correction
1 Introduction Congenital heart disease (CHD) is the most common birth defect, with an incidence of approximately 0.8% of live births [1]. Advances over the last five decades have led to increased recognition of congenital defects both in utero and in early life, particularly through improved imaging but also through earlier screening and improved recognition. This has led to improved outcomes through better medical and surgical treatment but also a better understanding about the timing of intervention to reduce longer-term complications. Improvements in surgery have altered the impact of CHD enormously, not only improving mortality in many conditions but also allowing some infants to survive into adulthood when previously they would have died within the first few days after birth. This increased survival has led to a huge increase in the number of survivors of surgery for CHD in childhood now reaching adulthood. This group of “grown-up CHD” (GUCH) patients have distinct needs outside both pediatric and adult cardiology environments and are best served by specialists in this field who are trained to understand the long-term complications these patients face. Pulmonary arterial hypertension (PAH) associated with CHD (CHD-PAH) is relatively common, occurring in 5–10% of patients [2]. It may complicate both simple and more complex lesions and may occur early or late in the natural or operative history of the condition. It results in significant morbidity and early mortality of patients compared with those without evidence of PAH [3]. The majority of patients with CHD-PAH are those who have large intracardiac or extracardiac shunt lesions which have not been repaired or where they have been repaired late. In CHD-PAH, pulmonary vascular resistance (PVR) starts to increase at a variable time point following birth and there are many factors which influence the development of PAH, including the anatomical lesion, length of life, degree of
J.X.-J. Yuan et al. (eds.), Textbook of Pulmonary Vascular Disease, DOI 10.1007/978-0-387-87429-6_79, © Springer Science+Business Media, LLC 2011
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shunting, previous intervention, and genetics. Early in the natural history of CHD, changes in the pulmonary vasculature giving rise to increased PVR may be reversible and therefore lesion correction would be the optimal management. Once changes in the vascular bed are established, correction of the shunt lesion is high risk and may worsen the natural history. The earlier detection and treatment of CHD and a better understanding about the timing of intervention will hopefully translate into a significant reduction of the incidence of CHD-PAH. Landzberg described CHD-PAH in five different scenarios: [4] (1) dynamic (relating to high shunt flow and responding to closure of shunt); (2) late postoperative PAH (therapeutic options include pulmonary vasodilator therapy); (3) immediate postoperative (reactive); (4) unusual forms of CHD where hemodynamics depend on low PVR such as the Glenn and Fontan shunts in tricuspid atresia or single ventricle circulations; (5) The physiologic characteristics of Eisenmenger syndrome includes left-to-right shunting, which results in significant elevation of pulmonary artery pressure (PAP) and PVR to systemic or near-systemic levels, resulting in bidirectional or reversed right-to-left shunting. Both simple and more complex lesions such as atrioventricular septal defects, truncus arteriosus, and single ventricle circulations can give rise to Eisenmenger physiology. Symptoms may not appear until the second decade of life, especially if the presence of CHD has not been recognized early in life. The outlook is poor, with progressive right ventricular (RV) failure and premature death due to sudden arrhythmia, heart failure, and hemoptysis. Thus, early recognition of CHD, especially in patients with large, unrestrictive shunts, is imperative to prevent the sequelae of progressive PAH. Earlier diagnosis, recognition, and treatment of CHD has already resulted in a significant decrease in patients presenting later in life with Eisenmenger physiology and it is hoped that this trend will continue [5]. However, even in developed countries, late presentation of missed shunts means that a number of patients continue to present with previously undiagnosed CHD and established changes in the pulmonary vascular bed. In developing countries, owing to a lack of resources and health-care infrastructures, early diagnosis and treatment of even simple CHD is not available, which still leads to large numbers of children and adults with untreated CHD, many of whom will have significant PAH. This chapter discusses the evaluation of CHD patients with dynamic PAH where surgical correction of lesions may be safely undertaken. Surgical or interventional correction is desirable, particularly early in the natural history of CHD before advanced changes in the pulmonary vasculature are established, provided there is evidence to support reversibility of PAH afterward, although this may be difficult to predict in some difficult or marginal patients in whom it is not clear that here will be reversal of PAH. Special consideration is given to established, moderate to severe PAH, in particular patients
C.J. Lewis and A.A. Klein
with Eisenmenger physiology previously thought to be very high risk or inoperable, where recent advances in the management of PAH, particularly with pulmonary vasodilators, result in reduction of PAP and/or PVR to levels which may allow safe correction of the congenital lesion. Discussion of operability most often arises in older children and adults with large shunts and therefore this chapter concentrates on this group of patients. Consideration is also given to advanced therapies, including transplantation, and the importance of anesthetic and intensive care management in patients with CHD-PAH.
2 Investigations To Evaluate Operability of CHD-PAH Appropriate selection of patients is essential for good outcomes after surgery or intervention, and full clinical assessment and invasive and noninvasive investigations are necessary to aid selection. Of particular importance are anatomical definition, shunt definition, and exclusion of confounding precipitants of PAH. Evaluation of patients with CHD-PAH should therefore be carried out in centers with particular expertise in CHD to safely characterize patients who may be suitable for corrective procedures after extensive assessment. Generally, CHD-PAH is reversible provided that correction occurs prior to changes in the pulmonary vascular bed becoming established and “fixed.” Improved understanding of the natural history of lesions and when PAH tends to become irreversible has therefore changed clinical practice, with increased vigilance and surveillance for progressive increase in PAH, and this has led to earlier intervention. However, it is difficult to determine exactly when PAH has become irreversible and there are no clearly established parameters. Indeed, recent experience with the use of novel oral pulmonary vasodilators in CHD-PAH has shown that even patients assumed to have established “fixed” PAH can have dramatic falls in PAP, which has the potential to allow late, high-risk surgical correction of the underlying lesion. Several factors help determine whether PAH is established, including pulmonary blood flow (Qp), PVR, and PAH reversibility by pulmonary vasodilatation. Poor postoperative outcome is likely when there is high pulmonary blood flow and PVR, little evidence of reversibility, and poor RV function, in terms of both immediate postoperative problems with right-sided heart failure and pulmonary hypertensive crises but also longer-term residual significant PAH.
2.1 Clinical Assessment Full comprehensive clinical examination and noninvasive investigation (oxygen saturations, electrocardiogram, chest X-ray) provide important information. Favorable findings
79 Surgical Evaluation of Congenital-Heart-Disease-Associated Pulmonary Hypertension
suggesting operability include those indicating ongoing leftto-right shunting: shunt murmur with absence of loud single S2 and pulmonary regurgitation on clinical examination; normal saturations exceeding 95%; no desaturation on the 6-min walk test; absence of right-sided heart strain and RV hypertrophy on the electrocardiogram and prominent lung vascularity on the chest X-ray [6] (Table 1). Consideration should also be given to exclude conditions with can increase PVR, such as upper airway obstruction (tracheomalacia, enlarged adenoids), hypoventilation (sleep apnea, neuromuscular disease), restrictive lung disease (chest wall abnormalities, scoliosis, interstitial lung disease), small airway obstruction (asthma, bronchiolitis), and parenchymal lung disease (consolidation/collapse, pulmonary edema).
2.2 Open-Lung Biopsy Open-lung biopsy has been used to gauge the potential for PAH reversibility using histopathological grading. Generally, PVR reactivity to vasodilatation provides a more physiologic method of assessing reversibility, so open-lung biopsy should only be considered in selected cases where the PVR reversibility studies are not conclusive. Open-lung biopsy is high risk in CHD-PAH and should only be considered in appropriate centers. The extent of structural change in the pulmonary vascular bed correlates with pulmonary hemodynamics including postoperative residual pulmonary hypertension and can therefore be useful to select patients for surgical correction but by itself is not reliable in predicting operability [7, 8]. Three progressively severe stages are recognized and incorporate the Heath and Edwards classification with pulmonary hemodynamics. Grade A describes smooth muscle cell migration to the subendothelium of arterioles. It is associated
with increased pulmonary blood flow and raised pulse pressure, but with normal mean PAP. Grade B describes proximal artery medial hypertrophy with increased intercellular connective tissue and is associated with an increase in PAP. In grade C there are fewer distal pulmonary arterioles, with a resultant increase in PVR. Vascular remodeling of the pulmonary vascular bed may be seen on lung biopsy following pulmonary vasodilator therapy in patients with very high PAP.
2.3 Cardiac Imaging Imaging has evolved greatly in the last few decades; 2D and 3D echocardiography, cardiac computed tomography (CT), and magnetic resonance imaging (MRI) with 3D reconstruction have revolutionized the way CHD is assessed. In many cases, imaging methods have replaced conventional cardiac catheterization and angiography. Echocardiography and MRI can be used not only for anatomical definition but can also allow calculation of the ratio of pulmonary to systemic blood flow (Qp/Qs), an important concept to help understand whether congenital lesions can be corrected in the presence of PAH. Echocardiography is very frequently used in the assessment of CHD-PAH and gives a number of important pieces of information to aid clinical evaluation. 2D echocardiography allows clear definition of cardiac anatomy, including understanding the nature of a shunt. The size of any communication may be ascertained as well as the pressure gradient across a shunt, which will determine whether a lesion is restrictive or unrestrictive; the latter is more likely to be associated with PAH. PAP can be estimated from assessment of tricuspid valve regurgitation velocity, which gives RV systolic pressure, calculated from systolic PAP = 4V2 + RAP, where RAP is right atrial pressure and V is the maximum tricuspid
Table 1 Clinical assessment of hemodynamics in left-to-right shunts Modality Clues suggesting operability History
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Clues suggesting inoperability
Feeding difficulty, failure to thrive, tachypnea, and frequent respiratory infections in infancy
Absence of symptoms or “improvement” in status as suggested by improved feeding, weight gain, and resolution of tachypnea Visible cyanosis, quiet precordium, loud single S2, Physical examination Increased precordial activity, labored respiratory efforts absence of flow murmurs, early diastolic murmur of (subcostal and intercostal recession), split second heart pulmonary regurgitation sound (S2), mid-diastolic “flow” murmurs Oxygen saturation a Normal (>95%) Reduced ( 2, small gradient across lesion (less than 25 mmHg), bidirectional color flow across a lesion, and more severe tricuspid regurgitation. Cardiac CT and MRI are especially useful for assessment of patients who have poor echocardiographic windows. Both techniques are able to define cardiac anatomy, including shunt flow, sizes, and location. CT is especially useful to look for complications of PAH which may have a bearing on suitability for surgery, including pulmonary embolism/infarction and thrombosis in situ. MRI, through cine imaging, allows myocardial motion, valves, and blood flow to be imaged. Both cine imaging and 3D magnetic resonance angiography allow collateral arteries to be visualized, which is essential when planning intervention. Phase velocity mapping allows functional questions to be answered such as the severity of a
Fig. 1 Typical transthoracic echocardiographic findings in pulmonary arterial hypertension associated with congenital heart disease (CHD-PAH). a A 2D apical long-axis image showing grossly dilated right atrium and ventricle with flattening of the interventricular septum. Failure of tricuspid valve apposition results in severe
regurgitation as shown in color Doppler echocardiography (b) and in continuous-wave Doppler echocardiography (c). (d) Tricuspid annular plane systolic excursion is used to assess right ventricular function. (Echocardiographic images courtesy of Dr. Rosemary Rusk, Papworth hospital, Cambridge, UK)
79 Surgical Evaluation of Congenital-Heart-Disease-Associated Pulmonary Hypertension
stenotic/regurgitant lesion and measurement of shunt or collateral flow. MRI is especially useful for assessment of RV and left ventricular size, function, and mass measurement. In many cases, the images and measurements obtained complement those obtained by other noninvasive investigations and avoid the need for invasive cardiac catheterization. However, cardiac MRI in CHD patients is complex and requires expertise, planning, and clinical supervision from appropriately specialized practitioners owing to the variability of anatomy and clinical conditions. Special consideration should be given to assessment of the right ventricle as preserved postoperative RV function is critical for the success of correcting CHD lesions. Patients with idiopathic PAH (iPAH) have a much worse prognosis than patients with CHD-PAH (discussed in Sect. 9.1), which in part may be due to the length of time the right ventricle has been exposed to progressive but slow increases in PAP, leading to adaptive RV hypertrophy and preservation of RV function. Assessment of RV function is difficult; however, recent improvements in our understanding of RV function have improved imaging diagnostic ability. Cardiac MRI is the “gold standard” test for RV function, allowing measurement of RV size and ejection fraction. However, better echocardiography including 3D techniques to measure RV volumes and ejection fraction and tissue Doppler echocardiography assessment has also improved quantification of RV function.
2.4 Invasive Hemodynamic Assessment Cardiac catheterization provides a direct invasive measurement of pulmonary hemodynamics, the ability to determine the potential reversibility of PAP and PVR using pulmonary vasodilators (such as high-flow oxygen, nitric oxide, calcium antagonists, prostacyclin analogues, and phosphodiesterase inhibitors) and the ability to test therapeutic potentials (temporary balloon occlusion of shunt). Since the severity and the significance of a shunt are used to make decisions on surgical or interventional closure of lesions, it is important to document accurate values; thus, catheter-based studies of PAP and PVR are the accepted “gold standard.” Important confounding factors in cardiac catheterization include accuracy of data during acquisition, distal or branch PA stenosis, pulmonary vein stenosis, and multiple sources of pulmonary blood flow. In addition, there are a number of different calculations used to assess hemodynamics, including the Fick principle and thermodilution, which each have limitations and assumptions about oxygen consumption and mixing of blood in shunts. Using the Fick principle, one may estimate the blood flow to shunt relationship as Qp/Qs = (aortic saturation–mixed venous saturation)/(pulmonary vein saturation–PA saturation). Sources of error have to be con-
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sidered when considering the validity of the Fick result, including blood gas analysis in anemia, cyanotic conditions, hepatic derangement, and sampling and measurement errors. In addition, PAP and PVR are often labile and change in response to stress stimuli or exercise, and cardiac catheterization provides hemodynamic assessment only at one time point. Exercise performed during invasive monitoring can therefore be useful and may be performed by a patient using a bicycle pedal ergometer on the catheterization table. Further consideration should also to be given to the degree of shunting. Shunts with high flow and therefore greatly increased volume of blood passing through the lung may recruit upper and middle lobe vascular beds, which could result in an underestimate of PVR and PAP. Following shunt closure, subsequent to decreased flow, the previously recruited vascular beds no longer contribute to the PVR, resulting in a significantly lower than expected fall in PAP postoperatively. Pulmonary blood flow in CHD may be made up from multiple sources, including the native RVOT, surgically created shunts (e.g., Blalock–Taussig shunt), and aortopulmonary (AP) or other venous/systemic collaterals, and each may have different flow and oxygen saturation characteristics. In these cases, it is important to isolate each source of blood flow and to measure pulmonary blood flow and PVR in isolated lung segments to assess reversibility. This can be achieved using a balloon to temporarily occlude shunts/collaterals and by using a balloon PA catheter manipulated into different lung segments to obtain the pulmonary capillary wedge pressure (PCWP) and hence calculate PVR (mean PAP–mean PCWP/ cardiac output), which can then be used with the calculated PVR in each segment to accurately determine overall PVR. The following have been used as thresholds beyond which surgical or interventional outcomes are significantly worse: Qp/Qs > 1.5–2, PVR > 15 Wood units (WU), PVR indexed 6–8 WU/m2, and pulmonary to systemic resistance ratio (Rp/ Rs) £ 1/3 [9]. Other factors to be considered include the type of congenital lesion, reversibility of PAH/PVR on vasodilator testing, and temporary shunt occlusion; this is discussed further in Sect. 3.
2.5 PVR Reversibility Studies PVR studies for potential reversibility depend on pulmonary bed vasodilatation using a variety of agents [2]. Calcium antagonists such as nifedipine have been used in the past, although ideally a selective pulmonary vasodilator should be used since systemic vasodilatation and consequent fall in blood pressure will also result in a fall in PAP. Assessment of pulmonary vasoreactivity can be performed with short-acting pulmonary vasodilators, including 100% inspired oxygen, nitric oxide, nebulized or intravenously
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administered prostacyclin analogues, adenosine, and phosphodiesterase inhibitors such as sildenafil. There is no consensus as to which agent or specific protocol provides the optimal information. A good vasoreactive response could be considered as a fall in PAP of more than 10 mmHg or to a level below 40 mmHg, without a significant fall in systemic blood pressure or cardiac output [10]. However, there is limited guidance on vasoreactivity criteria in the literature when considering surgical correction of CHD-PAH. One study demonstrated that with the use of a combination of oxygen and inhaled nitric oxide, a greater number of appropriate candidates for corrective cardiac surgery or transplantation could be identified during preoperative vasoreactivity testing when the ratio of pulmonary and systemic vascular resistance (Rp/Rs) was less than 0.33 [11]. Data relating vasoreactivity to long-term outcome after surgery are lacking. However, Post et al. reported correlation between the baseline response to nitric oxide and the mid-term outcome to 5 years in adult patients with CHD-PAH [12].
2.6 Temporary Shunt Occlusion Balloon test occlusion of shunts may provide additional information to aid clinical decision making. If the shunt is temporarily occluded, this allows assessment of PAP and PVR under test conditions representing the immediate postintervention state, allowing better prediction of postintervention results. A rise in right-sided heart filling pressures, PCWP, PAP, RV systolic pressure or a fall in cardiac index would suggest an adverse outcome for proceeding with shunt correction. Temporary shunt occlusion is easily facilitated using a balloon for a patent ductus arteriosus (PDA) or an atrial septal defect (ASD) but is difficult in ventricular septal defects (VSDs). Several case series have reported the utility of this test to aid decisions regarding the potential for permanent shunt closure. In one series, responders to balloon test occlusion were defined as having a fall of at least 25% in PAP or at least a 50% fall in the PAP to systemic arterial pressure ratio [6]. Fourteen of 18 responders were shown to have sustained reductions in PAP (even in the presence of high PVR and low Qp/Qs), with a progressive rise in nonresponders. The utility of balloon test occlusion to determine suitability for percutaneous transcatheter PDA device closure in patients with severe PAH has been demonstrated by Yan et al. [13]. Twenty of 29 patients who underwent successful PDA closure demonstrated a mean fall in PAP of 37 mmHg with saturations maintained above 95% following temporary PDA occlusion. The criteria used to determine that the remaining patients were unsuitable for PDA closure included worsening of symptoms, increase in PAP (mean 10 mmHg), and saturations below 86% or below 95% with supplemental oxygen.
C.J. Lewis and A.A. Klein
3 Consideration When Undertaking Repair of CHD Lesions Associated with Left-to-Right Shunting All lesions associated with significant left-to-to right shunting lead to increased pulmonary blood flow and may lead to pathological and physiological changes in the pulmonary vascular bed, which produce progressive pulmonary hypertension and increasing PVR. Large defects, unrestrictive flow dynamics, and unrepaired defects are most likely to lead to the development of PAH, but the response to increased pulmonary blood flow is not predictable, resulting in significant difficulty in deciding whether to close a lesion. Patients with more complex lesions such as truncus arteriosus and atrioventricular septal defects are at high risk for developing PAH early. VSDs are common and approximately 50% of large, unrestrictive VSDs will develop Eisenmenger physiology if they are not closed within the first 18 months of life. Overall, for all types of VSDs there is a moderate risk (approximately 20%) for a patient developing PAH, with a similar risk for a large PDA. Simple shunts such as ASDs are often associated with mild to moderate elevation of PAP, in part due to increased pulmonary blood flow, and these may be safely closed. However, assessment of PAP and PVR is important prior to closure and during long-term follow-up since PAP may remain elevated or even rise progressively. In some forms of CHD, primary repair is best carried out when an infant has grown and so initial banding of the PA is performed, for example, in a large VSD. This allows protection of the pulmonary vascular bed and prevents the increase in PVR which would otherwise ensue, so that correction of the defect and debanding can be delayed. Intraoperative assessment of the PA band is important so that a sufficient gradient is generated to reduce pulmonary blood flow and prevent the development of PAH while maintaining adequate oxygenation. Frequent postoperative evaluation is also important to ensure that there is sufficient blood flow as the infant grows and that PAH does not develop. Cardiac catheterization is required to assess PAP and PVR if there is clinical suspicion that PAH may have developed (low gradient on echocardiography across the PA band). PA banding in severe PAH with Eisenmenger physiology has been shown in a small number of patients to allow regression of the histological findings of PAH and reduction in PAP, allowing surgical correction [14]. Two of four patients with severe PAH who were considered high risk for conventional surgery benefited from PA banding, with subsequent reduction in PVR allowing later surgical correction and a good medium-term outcome; the remaining patients failed to respond. Other evidence suggests that PVR only improves to a small extent and only in very young patients after PA banding. There are no selection criteria to determine which patients will respond to banding and therefore this strategy
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cannot be recommended to improve the chances of operability in established CHD-PAH. Patients with variants of tetralogy of Fallot may need additional pulmonary blood supply owing to the RVOT obstruction and subsequent cyanosis and “spelling.” Various systemic–pulmonary shunts have been developed to increase pulmonary blood flow: the Blalock–Taussig shunt (modified – subclavian to PA); Waterston and Pott’s shunts (AP connections, now rarely encountered). The AP shunts are particularly associated with the development of PAH due to overshunting at systemic pressure. In the modern era, overshunting is unusual owing to appropriate selection of shunt size, but may still result in pulmonary vascular disease or distortion of the branch PA and it is important to exclude these prior to undertaking surgical repair. Pulmonary atresia is often associated with single or multifocal AP collateral arteries, which must be carefully assessed when considering operative repair. Collaterals may subject the pulmonary vasculature to systemic-level pressure and increasing PVR but may also contain stenoses protecting the lung. Different lung segments may have differing arterial supply, including dual supply (from collaterals and native pulmonary blood flow), which makes assessing PVR in these patients more complex. Strategies for early or late surgical repair of this condition may allow prevention of PAH developing, e.g., unifocalization of collaterals, formation of a central PA, or interventional coil embolization of collateral arteries. There is a lack of evidence and guidance when deciding on the operability of patients where there is late presentation, moderate to severe PAH, and the suspicion of “established” or “fixed” changes in the pulmonary vascular bed. Factors which have been shown to affect outcome after surgical correction of CHD are explored in Table 2 and include age at repair, preoperative PAP to systemic pressure ratio, and Rp/Rs ratio, although there is no predictable relationship or cutoff value [15, 16]. However, when considering surgical correction, if there is significant systemic cyanosis (i.e., representing
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right-to-left shunting at atrial level), the severity of PAH and its irreversibility is likely to prevent successful surgery. Established CHD-PAH has previously been viewed as irreversible and therefore these patients have been considered unsuitable for surgical or interventional treatment owing to the high perioperative risk and poor short- and medium-term outcome, including residual PAH and RV failure. Recent advances in the management of CHD-PAH include pulmonary vasodilator therapy (discussed in Sect. 5), intervention to reduce pulmonary blood flow, and atrial septostomy. In severe PAH, shunting at atrial level can partially relieve high right atrial pressure and improve symptoms at the expense of systemic cyanosis due to right-to-left shunting. Indeed, the clinical benefit of creating a right-to-left shunt to “off-load” the right side of the heart by performing atrial septostomy is recognized in selected severe PAH patients who are very symptomatic [17]. Atrial septostomy is usually performed by interventional catheterization using a blade but has significant morbidity associated with it. Up to nearly a third of Eisenmenger patients have evidence of pulmonary vasoreactivity in response to inhaled nitric oxide, meriting the suggestion that responders could be targeted with specific treatments to reduce PAP [18]. The use of pulmonary vasodilators, in particular, has shown promise in reducing PVR and PAH in congenital patients with the additional potential benefit of the proposed vascular remodeling effects of endothelin antagonists [19]. With the expected increase in the number of CHD patients on these therapies, we are likely to see a number of patients, who with appropriate follow-up and reinvestigation or rechallenging, may meet conventional criteria which would allow consideration for surgical correction in the future in patients previously thought inoperable. This may allow not only improvement in the significant morbidity associated with CHD-PAH, but may also potentially improve mortality. Very symptomatic patients who are deemed too high risk for corrective intervention and who fail a trial of pulmonary vasodilator therapy should be referred for consideration of lung or combined heart and lung transplantation.
Table 2 Criteria suggesting adverse outcome following shunt closure Hemodynamics Vasodilator testing Temporary shunt occlusion Rise in filling pressures SPAP >70 mmHg Decrease in SPAP (PCWP, PAP, RVSP) 40 mmHg Decrease in Pp/Ps 2/3 Fall in systemic Fall in cardiac index Qp/Qs >1.5–2 blood pressure or Saturations 7.45 Hyperoxia, increase FiO2 to 1.0 Reduce mean intrathoracic pressure to 6–10 mmHg by altering ventilator parameters Nebulized prostacyclin analogue (iloprost 20 mg over 15 min) Phosphodiesterase inhibitor loading dose/infusion (enoximone, milrinone) Intra-aortic counterpulsation device (intra-aortic balloon pump) Intravenous administration of sildenafil Intravenous infusion of dipyridamole (0.5 mg/kg/h) Open pericardium/sternum Nitric oxide 10–50 ppm in inspiratory limb of ventilator circuit Cardiopulmonary bypass
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reduction in pulmonary hemodynamics which may allow shunt closure, although this remains an unproven strategy. Carefully selected CHD-PAH patients with severe symptoms may benefit from combined heart and lung transplantation or lung transplantation with correction of CHD.
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18. Budts W, Van Pelt N, Gillyns H et al (2001) Residual pulmonary vasoreactivity to inhaled nitric oxide in patients with severe obstructive pulmonary hypertension and Eisenmenger syndrome. Heart 86:553–558 19. Galiè N, Beghetti M, Gatzoulis MA et al (2006) Bosentan Randomized Trial of Endothelin Antagonist Therapy-5 (BREATHE-5) Investigators. Bosentan therapy in patients with Eisenmenger syndrome: a multicenter, double-blind, randomized, placebo-controlled study. Circulation 114:48–54 20. Uzun O, Wong JK, Bhole V et al (2006) Resolution of protein-losing enteropathy and normalization of mesenteric Doppler flow with sildenafil after Fontan. Ann Thorac Surg 82:e39–e40 21. Takahashi K, Mori Y, Yamamura H et al (2003) Effect of beraprost sodium on pulmonary vascular resistance in candidates for a Fontan procedure: a preliminary study. Pediatr Int 45:671–675 22. Jones DK, Higenbottam TW, Wallwork J (1987) Treatment of primary pulmonary hypertension intravenous epoprostenol (prostacyclin). Br Heart J 57:270–278 23. Steiropoulos P, Trakada G, Bouros D (2008) Current pharmacological treatment of pulmonary arterial hypertension. Curr Clin Pharmacol 3:11–19 24. Fernandes SM, Newburger JW, Lang P et al (2003) Usefulness of epoprostenol therapy in the severely ill adolescent/adult with Eisenmenger physiology. Am J Cardiol 91:632–635 25. Channick RN, Simonneau G, Sitbon O et al (2001) Effects of the dual endothelin-receptor antagonist bosentan in patients with pulmonary hypertension: a randomised placebo-controlled study. Lancet 358:1119–1123 26. Galiè N, Rubin LJ, Hoeper M et al (2008) Treatment of patients with mildly symptomatic pulmonary arterial hypertension with bosentan (EARLY study): a double-blind, randomized controlled trial. Lancet 371:2061–2062 27. Schulze-Neick I, Gilbert N, Ewert R et al (2005) Adult patients with congenital heart disease and pulmonary arterial hypertension: first open prospective multicenter study of bosentan therapy. Am Heart J 150:716 28. Galiè N, Ghofrani HA, Torbicki A et al (2005) Sildenafil Use in Pulmonary Arterial Hypertension (SUPER) Study Group. Sildenafil citrate therapy for pulmonary arterial hypertension. N Engl J Med 353:2148–2157 29. Imanaka K, Kotsuka Y, Takamoto S et al (1998) Atrial septal defect and severe pulmonary hypertension in an adult who needed nitric oxide inhalation after repair. Kyobu Geka 51:403–405 30. Lim ZS, Salmon AP, Vettukattil JJ et al (2007) Sildenafil therapy for pulmonary arterial hypertension associated with atrial septal defects. Int J Cardiol 118:178–182 31. Kovacikova L, Zahorec M, Nosal M (2007) Sildenafil as a pulmonary vasodilator after repair of congenital heart disease. Bratisl Lek Listy 108:453–454 32. Ikonomidis JS, Hilton EJ, Payne K et al (2007) Selective endothelinA receptor inhibition after cardiac surgery: a safety and feasibility study. Ann Thorac Surg 83:2153–2160 33. Hopkins WE (2005) The remarkable right ventricle of patients with Eisenmenger syndrome. Coron Artery Dis 16:19–25 34. Thistlethwaite PA, Kaneko K, Madani MM et al (2008) Technique and outcomes of pulmonary endarterectomy surgery. Ann Thorac Cardiovasc Surg 14:274–282 35. Frigiola A, Tsang V, Nordmeyer J et al (2008) Current approaches to pulmonary regurgitation. Eur J Cardiothorac Surg 34: 576–580 36. Dimopoulos K, Peset A, Garzoulis MA (2008) Evaluating operability in adults with congenital heart disease and the role of pretreatment with targeted pulmonary arterial hypertension therapy. Int J Cardiol 129:163–171 37. Beghetti M (2006) Pulmonary arterial hypertension related to congenital heart disease. Elsevier, Munich
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Chapter 80
Pulmonary Veno-occlusive Disease Peter F. Clardy and Jess Mandel
Abstract Pulmonary veno-occlusive disease (PVOD) is a rare and highly lethal disorder of the pulmonary vasculature. In contrast to the insights into pathophysiology and management that have characterized the diagnosis and treatment of idiopathic pulmonary arterial hypertension (IPAH; formerly known as primary pulmonary hypertension) over the past decade, the pathophysiologic mechanisms underlying PVOD are incompletely understood, the clinical diagnosis is notoriously difficult, and therapy is largely unsatisfactory. Historically, what is now called PVOD has been variably termed “isolated pulmonary venous sclerosis,” “obstructive disease of the pulmonary veins,” or “the venous form of primary pulmonary hypertension.” Over the past decade, “pulmonary obstructive venopathy” has been proposed as a more accurate alternative description, but this phrase has not been adopted into wide clinical use. It is important to note that PVOD is completely distinct from stenosis of one or more of the four main pulmonary veins. Stenosis of these large pulmonary veins is primarily a result of congenital malformation, but may also develop as a complication following cardiothoracic surgery. In addition, large-vessel pulmonary venous stenosis has been increasingly reported as a complication following radio-frequency ablation for atrial fibrillation or other dysrhythmias. Keywords Stenosis • Pulmonary vein • Venous obstruction • Venous sclerosis
1 Introduction Pulmonary veno-occlusive disease (PVOD) is a rare and highly lethal disorder of the pulmonary vasculature [1, 2]. In contrast to the insights into pathophysiology and management that have characterized the diagnosis and treatment of
P.F. Clardy (*) Department of Medicine, Beth Israel Deaconess Medical Center, 330 Brookline Avenue, KsB-23, Boston, MA 02215, USA e-mail:
[email protected] idiopathic pulmonary arterial hypertension (IPAH; formerly known as primary pulmonary hypertension) over the past decade, the pathophysiologic mechanisms underlying PVOD are incompletely understood, the clinical diagnosis is notoriously difficult, and therapy is largely unsatisfactory. The initial clinicopathologic description of PVOD was documented by Julius Höra at the University of Munich in 1934 [3]. The initial patient presented with progressive dyspnea, edema, and cyanosis, resulting in death over the course of approximately 1 year. The presumptive clinical diagnosis was mitral stenosis, but at autopsy, diffuse obstruction of the pulmonary venules with loose fibrous tissue was noted, in the absence of mitral valve disease or other causes of left atrial hypertension. Höra postulated that an infection, perhaps streptococcal, was responsible for the pathologic findings, but no organisms could be demonstrated on stains or cultures. Historically, what is now called PVOD has been variably termed “isolated pulmonary venous sclerosis,” “obstructive disease of the pulmonary veins,” or “the venous form of primary pulmonary hypertension.” The term “pulmonary venoocclusive disease” (PVOD) was popularized in the 1960s by Heath, Brown, and others [4–7]. Over the past decade, “pulmonary obstructive venopathy” has been proposed as a more accurate alternative description, but this phrase has not been adopted into wide clinical use [8]. It is important to note that PVOD is completely distinct from stenosis of one or more of the four main pulmonary veins. Stenosis of these large pulmonary veins is primarily a result of congenital malformation, but may also develop as a complication following cardiothoracic surgery [9, 10]. In addition, large-vessel pulmonary venous stenosis has been increasingly reported as a complication following radio-frequency ablation for atrial fibrillation or other dysrhythmias [11–13].
2 Epidemiology Although PVOD is clearly a rare disease, the true incidence and prevalence of PVOD are challenging to precisely measure for two reasons: the fact that mild cases may not come
J.X.-J. Yuan et al. (eds.), Textbook of Pulmonary Vascular Disease, DOI 10.1007/978-0-387-87429-6_80, © Springer Science+Business Media, LLC 2011
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to medical attention, and the suspicion that many cases of PVOD are misclassified as IPAH given the difficulty in distinguishing the two syndromes without tissue biopsy [14]. Despite these challenges, an estimate of the annual incidence of PVOD can be extrapolated from analysis of several series of patients with IPAH from which the fractions of patients fulfilling criteria for PVOD were reported. Pooling of seven such series published between 1970 and 1991 (comprising a total of 465 patients) suggests that the incidence of PVOD is approximately 10% of that of true IPAH [15–22]. Using an annual incidence of IPAH of one to two cases per million persons in the general population, this predicts an annual incidence of PVOD of around 0.1–0.2 cases per million persons in the general population [23, 24]. However, this may represent an underestimate of the true burden of disease, since a number of patients with PVOD are probably misclassified as having either interstitial lung disease or heart failure because of similarities in the radiographic appearance of all of these disorders. No well-designed studies have examined the magnitude of this likely misclassification phenomenon. Unlike IPAH, which is more common in women, there does not appear to be a clear gender imbalance among patients with PVOD [2, 25, 26]. The age at diagnosis has ranged from within 9 days of birth to the seventh decade of life.
3 Pathology The pathologic characterization of pulmonary vasculopathy remains challenging, even for experienced pathologists. To minimize errors in interpretation, lung specimens should be fixed in a distended state, and at least five blocks from each lobe should be sectioned and examined. In addition to hematoxylin and eosin staining, examination using Movat, Masson, Verhoeff–van Gieson, and Perls iron stains is recommended. Immunohistochemical markers for smooth muscle and endothelial antigens (e.g., factor VIII, CD31, CD34) may also be useful [8].
Fig. 1 Pulmonary veno-occlusive disease. Elastic stain distinguishes a small artery (a) from an involved small vein (b) (×20). (Reprinted with permission from Mandel and Taichman [154]. Copyright Elsevier 2006)
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The dominant pathologic abnormality observed in PVOD is the extensive and diffuse obliteration of small pulmonary veins or venules by fibrous tissue, which may be either loose and edematous, or dense, sclerotic, and acellular [2, 8, 15, 27, 28]. Of note, lesions within the venous system tend to be eccentric or trabeculated, similar to the changes that are observed in arterial structures that have undergone recanalization following thrombotic occlusion [29]. The media of the venules may undergo arterialization with an increase in the number of elastic fibers, and these fibers become calcified in some cases (Fig. 1). In addition, a foreign body giant cell response to these calcified fibers has been reported (Fig. 1) [8]. Pulmonary arteriolar changes frequently accompany venous changes, although it is not clear if they occur concomitantly or develop later as a consequence of venous obstruction. These arteriolar changes contribute to the misclassification of PVOD as IPAH. In approximately 50% of patients with PVOD, pulmonary arterioles demonstrate marked medial hypertrophy, which is easily confused with IPAH unless the venous structures are carefully surveyed. Plexiform lesions are less commonly encountered than is the case in IPAH, and arteritis or venulitis is distinctly uncommon [14, 30, 31]. Pleural and parenchymal lymphatic channels can become markedly dilated, presumably because of the increased volume of fluid moving from pulmonary capillaries to the interstitial space as a result of pulmonary venous hypertension [32]. In PVOD, capillaries become engorged and tortuous, occasionally leading to a misdiagnosis of pulmonary capillary hemangiomatosis (PCH; also called pulmonary microvasculopathy) [33]. Both of these disorders are also associated with prominent lymphadenopathy, and a study comparing lymph node disease in patients with PVOD and PCH showed similar nonspecific changes in lymph node architecture, including vascular transformation of the sinuses, intrasinusal hemorrhage with erythrophagocytosis, and lymphoid follicular hyperplasia [34]. These changes are very rare in
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patients with IPAH and suggest, along with other lines of evidence, that PVOD and PCH may exist along a continuum of disorders [35]. Areas of microscopic pulmonary hemorrhage and hemosiderosis are rare in IPAH but are frequently observed in PVOD, and presumably develop as a consequence of capillary endothelial damage and extravasation of erythrocytes due to pulmonary capillary hypertension [14]. Determining whether observed areas of pulmonary hemorrhage are due to PVOD or reflect tissue disruption by biopsy procedures can be challenging. Patients with PVOD commonly demonstrate hemosiderin within alveolar macrophages or the interstitium, and in some cases this feature is sufficiently prominent that the diagnosis of idiopathic pulmonary hemosiderosis or a vasculitis associated with alveolar hemorrhage (e.g., Wegener’s granulomatosis) is considered (Fig. 2) [2, 36]. In contrast to many other causes of pulmonary hypertension, pulmonary parenchymal abnormalities are commonly observed in PVOD [14]. Interstitial edema is commonly seen, and is particularly prominent within lobular septa. Collagen deposition in these regions, and occasionally also involving alveolar walls, may appear similar to changes observed in long-standing mitral stenosis [37]. Lymphocytes and monocytes are commonly seen within the interstitium, and may be sufficiently numerous to erroneously suggest usual interstitial pneumonitis as the primary diagnosis [28]. Because of a number of shared features, it has been proposed that IPAH, PVOD, and PCH may represent a continual spectrum of manifestations of a single disease process termed “pulmonary vascular occlusive disease” [8, 35, 38]. Advocates of this paradigm emphasize that obliterative pulmonary
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vascular lesions frequently are not limited to only the arterial, capillary, or venous structures, but can involve a number of different vascular areas within the same patient. Further evidence that these disorders may represent a single disease process includes the presence of bone morphogenic protein receptor type 2 (BMPR2) abnormalities in both IPAH and PVOD (see later). Although such a conceptualization has not yet found general acceptance, the third and fourth World Health Organization Conferences on Pulmonary Hypertension (Venice 2003, Dana Point 2008) classified PVOD and PCH together in the subcategory of “pulmonary arterial hypertension associated with significant venous or capillary involvement” [9].
4 Etiology The cause of PVOD remains unknown, and no well-designed and adequately powered case-control or cohort studies have been performed specifically to explore the potential causes of this condition. The situation is further complicated by the hypothesis that PVOD represents a final common clinicopathologic pathway triggered in a susceptible host by a number of discrete causes. Despite these uncertainties, a variety of risk factors have been proposed, based upon case reports and small case series.
5 Etiology 5.1 Genetic Susceptibility
Fig. 2 Pulmonary veno-occlusive disease. Hematoxylin and eosin staining shows obstructive intimal fibrosis of small vein and extensive hemosiderin-laden intra-alveolar macrophages (×20). (Reprinted with permission from Mandel and Taichman [154]. Copyright Elsevier 2006)
Reports of PVOD developing in siblings have led for many years to suspicions that a genetic predisposition may underlie development of the disease in some instances. In most cases involving siblings, PVOD developed before the third decade of life, and nonaffected siblings have also been detailed in a number of these families [7, 21, 28, 39, 40]. No definite genetic abnormality has been demonstrated in these sibling pairs, and the development of disease in these cases could also be the result of common environmental exposures to inciting agents. Since the identification of mutations of the BMPR2 gene as a major cause of familial IPAH in 2000, there has been speculation that abnormalities at this site may also be involved in the pathogenesis of PVOD [14, 41–43]. A total of six distinct BMPR2 mutations have been described in patients with PVOD [14]. In one compelling case report, a BMPR2 mutation resulting in a truncated and nonfunctional protein was described in a patient who developed PVOD at age 36 [44]. The patient’s mother had died of pulmonary
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hypertension several decades earlier, but lung tissue had not been examined to determine if she had suffered from PVOD or IPAH. The authors reported that at the time of the publication, the index patient had been stable for 5 years while receiving continuous intravenous administration of epoprostenol, a course that is somewhat atypical in PVOD. The description of BMPR2 mutations in patients with PVOD suggests that IPAH and PVOD may share a common genetic basis, and the pathologic phenotype that develops may be due to modifier genes or differing environmental exposures. It has been suggested that loss of a functional BMPR2 allele might contribute to the development of PVOD by diminishing the normal antiproliferative effects of bone morphogenic protein on pulmonary vascular endothelial and smooth muscle cells, resulting in unchecked proliferation in response to other ligands of the transforming growth factor b family [45]. A common BMPR2 defect might also help explain the overlap in pathologic features found in IPAH and PVOD, including the development of scattered venous lesions found in some IPAH patients with predominantly precapillary disease, and the presence of precapillary lesions and abnormal precapillary resistance in many patients with PVOD [46, 47]. Expanded genotyping of patients with PVOD should help clarify the genetic overlap of these two diseases, but at present this is informed only by occasional reports in isolated patients.
5.2 Infectious Agents Since Höra’s initial description of PVOD, infection has been postulated to play an etiologic role in the initiation or progression of disease [3, 48]. Höra believed that streptococcal infection played a role in his patient’s illness; since that time, nonbacterial pathogens have been more commonly hypothesized to play a role in the development of PVOD. In some cases, an antecedent influenza-like illness or serologic evidence of recent infection with agents such as measles and Toxoplasma gondii has been noted to coincide with the diagnosis of PVOD, and other patients have had manifestations of Epstein–Barr or cytomegalovirus infection, such as lymphadenopathy, fever, and erythrophagocytosis [4, 5, 49–51]. Several cases of PVOD have been reported among patients infected with the human immunodeficiency virus (HIV) [52–54]. However, despite these reports, it is difficult to reconcile the time courses of acute infectious illnesses and PVOD, given that it requires months to years to develop right ventricular hypertrophy and the symptom complex associated with PVOD. It is more likely that the significance of an acute illness near the time of PVOD diagnosis is that it may bring an undiagnosed patient to medical attention, and stimulate a diagnostic evaluation that ultimately demonstrates the presence of PVOD.
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In contrast to acute infection, HIV and other lentiviruses could pursue a chronologic course of infection that is more congruent with the time course required for the development of observed cardiovascular adaptations to PVOD. Despite this, viral inclusions or DNA has not been definitively documented in the pulmonary vascular lesions of affected patients.
5.3 Toxic Exposures An association between exposure to certain drugs or chemicals and the development of PVOD has been suggested by several case reports. As an example, the disease developed in a 14-yearold boy with a 2-year history of ingesting and sniffing a powdered cleaning product containing silica, soda ash, dodecyl benzyl sulfonate, and trichloro-s-triazinetriome [55]. Exposure to drugs delivered with therapeutic intent is also linked to the development of PVOD. As awareness developed that patients could manifest hepatic veno-occlusive disease following antineoplastic chemotherapy, reports began to emerge linking PVOD to these agents as well. Many of the patients with PVOD diagnosed after the treatment of malignancy received radiation and a variety of different antineoplastic drugs over several years, making identification of a specific culprit agent problematic; nonetheless, the most commonly implicated compounds are bleomycin, mitomycin, carmustine, and gemcitabine [56–62]. Anecdotal reports suggest that PVOD may be more common following either allogeneic or autologous bone marrow or stem cell transplantation than following conventional cytoreductive chemotherapy; however, there are insufficient data to definitively test this assertion [63–66]. The mechanism by which certain drugs might cause pulmonary venous remodeling is unknown. It is possible that these chemotherapeutic agents are metabolized to toxic intermediates in pulmonary capillaries and then cause damage to pulmonary venous structures. Cocaine, amphetamines, and anorectic agents are associated with pulmonary arterial hypertension, but have not been definitively linked to PVOD [23, 24]. Similarly, “bush teas” containing pyrrolizidine alkaloids have been responsible for case clusters of hepatic veno-occlusive disease but have not been associated with the development of PVOD [67, 68].
5.4 Thrombophilia It has long been hypothesized that thrombophilia may play a role in the pathogenesis of PVOD, both because venules and small veins in PVOD have an appearance similar to recanalized, thrombotically occluded arteries, and because
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lung specimens occasionally have evidence of fresh thrombi in affected vessels. Although coagulation and rheologic parameters have not been assessed in PVOD patients by state-of-the-art techniques, several reports from the 1960s described increased platelet adhesiveness in patients with the disorder, in one case to a degree four standard deviations above the mean for normals [4, 69]. In addition, several cases of PVOD have occurred in patients with a risk factor for hypercoagulability, such as pregnancy or oral contraceptive use [70, 71]. Although cases of PVOD have indeed occurred in the setting of documented thrombophilia, such comorbidities are unusual [72]. The thrombophilia hypothesis also fails to explain the fact that extrapulmonary venous or arterial thrombi, which would be expected to develop commonly in hypercoagulable states, are rare in patients with PVOD. Finally, careful examination of lung specimens fails to demonstrate acute pulmonary venous thrombi in the majority of patients with PVOD [32].
5.5 Autoimmune Disorders Venulitis, either primary or occurring secondary to an infectious vasculitis, followed by thrombosis, remodeling, or both, could presumably explain the pathologic changes seen in PVOD, but inflammation is not a typical feature of this disorder. Granulomatous venulitis rarely has been described to accompany sarcoidosis, and a similar vascular lesion has been described in a 21-year-old man with an extensive history of cannabis smoking and the clinical syndrome of PVOD [31, 73]. Although most patients with PVOD do not display manifestations of autoimmunity, a number of individuals have developed PVOD in the context of positive antinuclear antibodies, alopecia, myopathy, rheumatoid arthritis, Felty’s syndrome, systemic lupus erythematosus, mixed connective tissue disease, or the scleroderma spectrum of conditions (including features such as calcinosis, Raynaud’s phenomenon, esophageal dysmotility, sclerodactyly, and telangiectasias) [74–86]. However, the fact that only a minority of cases display associated autoimmune findings argues against autoimmunity playing a fundamental role in the development of PVOD.
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many patients are initially diagnosed with an acute respiratory infection. In general, PVOD is subsequently diagnosed only after patients fail to improve significantly after treatment with antimicrobial agents [5, 51, 87]. Progressive pulmonary hypertension may result in right upper quadrant pain (secondary to hepatic congestion), pedal edema, and/or exertional syncope. Orthopnea is more common and severer in patients with PVOD than in those with IPAH or other causes of pulmonary arterial hypertension [36]. Unusual clinical presentations of PVOD include symptomatic diffuse alveolar hemorrhage and sudden cardiac death [88–90]. Chronic subacute alveolar hemorrhage appears common on the basis of lung histopathologic findings and bronchoalveolar lavage (BAL) results, but massive and life-threatening hemoptysis rarely develops [88, 91]. Physical examination tends to reveal nonspecific findings of pulmonary hypertension, with or without overt right ventricular failure. Classic findings include increased intensity of the pulmonic component of the second heart sound, a right ventricular heave or lift, a right-sided third or fourth heart sound, elevated jugular venous pressure, hepatomegaly, and/ or peripheral edema. Murmurs of tricuspid regurgitation and, less commonly, pulmonic insufficiency may be appreciated in some patients. Murmurs or gallops present in this setting generally are augmented with inspiration, as increased venous return to the thorax increases turbulent flow through right-sided valvular defects [92]. Digital clubbing is sometimes present, and basilar rales, which are unusual in IPAH, are appreciated in some patients (Fig. 3) [51]. Pleural effusions are relatively common in patients with PVOD, in contrast to their infrequent association with purely precapillary causes of pulmonary hypertension [93, 94]. The likely pathophysiologic explanation for this observation is that PVOD and other postcapillary causes of pulmonary
6 Clinical Manifestations The majority of patients with PVOD present with nonspecific symptoms of pulmonary hypertension, including dyspnea on exertion and fatigue [14, 26]. Patients may develop a chronic cough (either productive or nonproductive), and
Fig. 3 Prominent clubbing of the digits in a patient with pulmonary veno-occlusive disease. (Reprinted with permission from Mandel and Taichman [154]. Copyright Elsevier 2006)
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hypertension produce elevated pulmonary capillary and visceral pleural hydrostatic pressures, leading to transudation of fluid into the pleural space. In contrast, the predominant high-resistance areas of the pulmonary circulation are precapillary in IPAH, and thus pleural effusions do not generally accumulate until elevated hydrostatic pressure develops in the central systemic veins, leading to elevations in hydrostatic pressure within the parietal pleural capillaries.
6.1 Radiographic Features Chronic pulmonary capillary hypertension can produce transudation of fluid into the pulmonary interstitium, with consequent engorgement of pulmonary lymphatics. This results in the radiographic appearance of Kerley B lines on plain chest radiographs. Other possible findings on chest radiographs include enlargement of central pulmonary arteries, peribronchial cuffing, and scattered parenchymal opacities (Fig. 4) [4, 95]. However, these findings are not pathognomonic of PVOD, and the absence of any or all of these findings does not eliminate the possibility that PVOD is present [26, 80, 96, 97]. Computed tomography images may display smooth thickening of the septa, diffuse or mosaic ground-glass opacities, multiple well-defined or poorly defined small noncalcified nodules, pleural effusions, or areas of alveolar consolidation which may be gravitationally dependent or centrilobular in their distribution (Fig. 5) [93, 98–106]. The pathologic correlate of ground-glass attenuation seen in these patients is not
Fig. 4 A posteroanterior chest radiograph from a 28-year-old woman with pulmonary veno-occlusive disease demonstrated at autopsy shows increased interstitial markings diffusely and Kerley B lines that are most prominent in her lower lateral right chest. (Reprinted with permission from Mandel and Taichman [154]. Copyright Elsevier 2006)
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entirely clear, although alveolar hemorrhage, focal areas of hydrostatic edema corresponding to regions with markedly obstructed venous outflow, nonspecific interstitial inflammation, and alveolar septal thickening with associated epithelial hyperplasia have each been proposed to explain the finding. Importantly, the central pulmonary veins and the left atrium are not enlarged, in contrast to patients with mitral stenosis, cor triatriatum, left atrial myxoma, or large pulmonary vein stenosis secondary to electrophysiologic ablation, prior cardiothoracic surgery, or congenital malformation. Prominent mediastinal lymphadenopathy has been reported in a number of cases [1, 9, 14, 34, 101, 102, 107, 108]. Radionuclide ventilation–perfusion images display normal ventilation but commonly show multiple focal areas of hypoperfusion, sometimes approximating a segmental pattern [109]. This finding may be misinterpreted as supportive of the diagnosis of chronic thromboembolic pulmonary hypertension [26, 36, 87, 88, 97, 98, 110–113].
6.2 Cardiac Catheterization It is notoriously difficult to obtain a satisfactory pulmonary artery occlusion (wedge) pressure in patients with PVOD. Flushing of the catheter when the distal port of the catheter is in the wedged position and the balloon is inflated results in a disproportionate rise in recorded pressure, which then falls extremely slowly to the baseline. These phenomena are the result of impaired runoff of infused saline due to downstream
Fig. 5 Computed tomographic image through the lung bases shows numerous thickened septal lines (double arrow) and patchy foci of ground-glass attenuation (thin arrow). The arteries are enlarged relative to bronchi (thick arrow), whereas pulmonary veins appear of normal caliber (curved arrow). (Used with permission from Mandel et al. [2]. Copyright American Thoracic Society)
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obstruction and diminished cross-sectional area of the pulmonary venules and small veins [4, 88, 114]. To document successful catheter wedging in this situation, it may be helpful to demonstrate that blood gas analysis of a sample slowly drawn from the distal catheter port while the balloon is inflated results in values similar to those for a specimen drawn simultaneously from an arterial source. If a pulmonary artery occlusion pressure can be successfully measured, one should record values in several different locations to ensure that spurious values have not been obtained as a result of local phenomena. In patients with PVOD, the pulmonary artery occlusion pressure is generally normal or decreased, despite the fact that postcapillary obstruction does in fact produce pulmonary capillary hypertension [14, 51, 115]. The cause of this seeming paradox is that the pulmonary artery occlusion pressure is determined by left atrial pressures, not by hydrostatic pressures within the pulmonary capillaries, as the term “pulmonary capillary wedge pressure” is sometimes taken to imply. Rather, with the balloon inflated and the catheter in the wedged position, a static column of blood is created that extends from the catheter tip, through the pulmonary capillaries, venules, and veins to the left atrium, the latter of which determines the recorded pressure, and is generally normal in PVOD. Extensive stenosis of the small pulmonary veins in PVOD tends to dampen this pressure tracing to some degree, but normal left atrial pressures remain the fundamental determinant of the pulmonary artery occlusion pressure [116, 117]. Short-acting pulmonary arterial vasodilators are routinely administered to patients with pulmonary arterial hypertension at the time of cardiac catheterization to determine if pulmonary vasoreactivity is present [24]. However, in patients with PVOD, such medications may lead to the development of acute life-threatening pulmonary edema [14, 51, 108, 118]. Presumably, pulmonary edema develops in this setting because of pulmonary arterial vasodilation without concomitant pulmonary venodilation, producing a rapid increase in transcapillary hydrostatic forces and transudation of fluid into the pulmonary interstitium and alveoli.
6.3 Other Studies Patients with PVOD generally display normal spirometry. The single-breath diffusing capacity for carbon monoxide is usually reduced, and a mild to moderate restrictive ventilatory defect is observed in some cases [51, 97, 98]. Laboratory parameters are usually unremarkable, although isolated cases have displayed otherwise unexplained features such as microangiopathic hemolytic anemia [60], heavy proteinuria [5], or elevated serum IgG or IgM concentrations [76]. Bronchoscopy may reveal intense hyperemia and longitudinal vascular engorgement of the lobar and segmental
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bronchi in a series of patients with PVOD [111]. This finding is thought to result from engorgement of the bronchial arterial system, which in segmental and more distal bronchi normally drains into the pulmonary circulation, but which has poor distal runoff in PVOD. In this series, hyperemia was not seen in the trachea or main bronchi, where systemic bronchial veins are well developed.
7 Diagnosis The classic triad of severe pulmonary hypertension, radiographic evidence of pulmonary edema, and a normal pulmonary artery occlusion pressure is frequently sufficient to warrant a clinical diagnosis of PVOD [117]. However, many patients with PVOD do not have all three components of this triad, and thus its absence cannot reliably exclude the diagnosis. Delays in diagnosis are almost universally encountered by patients with PVOD, with many patients believed to suffer from heart failure because of radiographic evidence of pulmonary edema and pleural effusions, or chronic thromboembolic pulmonary hypertension because of nonresolving radionuclide perfusion defects. In cases of PVOD where interstitial changes are radiographically or histologically prominent, alternative diagnoses of diffuse parenchymal lung diseases such as sarcoidosis, pneumoconioses, cystic fibrosis, and idiopathic pulmonary fibrosis may be considered [119]. Evidence of pulmonary hemorrhage may be noted in BAL fluid in patients with PVOD, but this finding is not specific and may also be noted in patients with IPAH or other forms of pulmonary vascular disease. However, the demonstration of occult pulmonary hemorrhage by BAL in the proper clinical setting may support the diagnosis if lung tissue has not been obtained [91]. Transbronchial biopsy almost never yields enough tissue for a firm pathologic diagnosis, and carries significant bleeding risk when performed in the setting of pulmonary hypertension. Definitive diagnosis requires surgical lung biopsy, and this procedure should be contemplated to confirm the clinical suspicion of PVOD. Although the need for lung biopsy has been questioned because therapy for PVOD is so unsatisfactory, establishing the diagnosis has significant implications for prognosis, medical therapy, and timing of lung transplantation, and therefore biopsy should be considered in patients in whom the diagnosis is suspected and surgical risk is not prohibitive.
8 Prognosis and Treatment The prognosis of PVOD is poor, with most patients dying within 2 years of diagnosis. However, cases have been reported in which individuals have survived for 5 years or
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more, generally when therapy with orally administered calcium channel antagonists or intravenous epoprostenol therapy has been well tolerated [44, 120, 121]. Because PVOD is a rare disease, large treatment trials cannot be easily performed, and the degree to which any of the current therapies influence survival and quality of life is speculative [122]. With the possible exceptions of lung transplantation and intravenous administration of epoprostenol (if tolerated), the positive impact of current therapies does not seem profound.
8.1 Conventional Supportive Therapy Patients with PVOD should receive conventional supportive therapy similar to that prescribed for patients with IPAH, unless contraindications are present [123]. Of note, none of these therapies have been documented in highquality randomized trials to be efficacious in patients with pulmonary hypertension of any cause; they are employed because of theoretical consideration and/or case reports, case series, or retrospective analyses that have suggested possible benefit. Warfarin generally is titrated to an International Normalized Ratio of approximately 2.0, based upon the suggestion of several nonrandomized studies in IPAH that survival may be modestly improved [124, 125]. Episodic small-volume hemoptysis generally does not require discontinuation of anticoagulation, but the medication is usually stopped if more than 50 mL of blood is expectorated over a 24-h period, if significant extrapulmonary hemorrhage occurs, or if syncope or other risk factors for head trauma develop. Long-term oxygen therapy should be initiated if an oxygen saturation of 89% or less, or an arterial oxygen partial pressure of 59 mmHg or less, is documented. These recommendations are based upon clinical trials of oxygen therapy in patients with chronic obstructive pulmonary disease rather than primary pulmonary vascular disease, and not all patients with pulmonary hypertension show improvement in pulmonary hemodynamics after oxygen therapy is begun [126–128]. Transtracheal oxygen therapy permits delivery of oxygen at higher flow rates than via nasal cannulae and may be considered when epistaxis is problematic, although local bleeding complications can also occur [129]. Diuretics should be used as necessary to maintain euvolemia. Both dehydration and hypervolemia should be avoided, and patients should monitor their weight daily. On the basis of the improvement in cardiac output described in patients with IPAH treated with digoxin, the drug is routinely prescribed to patients with PVOD unless contraindications are present [130].
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8.2 Calcium Channel Antagonists and Epoprostenol A minority of patients with IPAH will display acute pulmonary vasoreactivity in response to vasodilator medications such as adenosine, epoprostenol, inhaled nitric oxide, and calcium channel antagonists [123, 124]. Pulmonary vasoreactivity was defined in older literature as a 20–25% decrease in mean pulmonary artery pressure and pulmonary vascular resistance, or more recently as a reduction in mean pulmonary artery pressure of more than 10 mmHg to a value of 40 mmHg or below, with a normal or high cardiac output [131]. Vasoreactive patients tend to have an excellent prognosis when treated with calcium channel antagonists. In IPAH patients without an acute hemodynamic response and who thus cannot be treated with calcium channel antagonists, therapy with continuous intravenous administration of epoprostenol has been associated with improved survival [132]. Therapy with other prostanoids (e.g., treprostinil, iloprost), endothelin-1 receptor antagonists (e.g., bosentan, ambrisentan), or phosphodiesterase-5 inhibitors (e.g., sildenafil) likewise is associated with improved outcomes [133, 134]. The generalizability of such data to PVOD is unclear. Theoretically, a reduction in pulmonary arterial resistance without a concomitant reduction in pulmonary venous resistance could cause pulmonary edema, whereas a parallel decrease in both arterial and venous resistance should be well tolerated and advantageous. The clinical experience with vasodilator/remodeling medications in patients with PVOD has been mixed. Modest improvements in hemodynamics and exercise tolerance have been reported in a small number of patients with nifedipine, hydralazine, and prazosin, but in general these benefits have not been well maintained over time [121, 135]. Epoprostenol has been reported to have salutary effects on pulmonary hemodynamics and to decrease vasomotor tone in pulmonary venules in some patients, but has produced fulminant pulmonary edema and death in others [44, 51, 108, 120, 136, 137]. Unfortunately, pulmonary edema may result from chronic vasodilator therapy, even if the initial diagnostic challenge was associated with beneficial changes in the patient’s hemodynamic profile [14]. There is limited experience with inhaled nitric oxide or iloprost in this condition, although initial descriptions of the use of these agents in this context suggested that improvement in cardiac output resulted without the development of pulmonary edema [138]. Because the therapeutic options in PVOD are so limited, a cautious trial of epoprostenol is generally indicated. If the use of epoprostenol is tolerated, epoprostenol is generally initially given to patients at a dosage of 2–4 ng/kg/min, then this is uptitrated by 1–2 ng/kg/min every 4 weeks as permitted by side effects. Whether endothelin-1 antagonists exert a beneficial or detrimental effect has not been firmly established.
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8.3 Lung Transplantation Either single-lung and double-lung transplantation can be performed to treat PVOD [139]. Heart–lung transplantation is rarely required, as impaired right ventricular function usually improves following the transplantation of normal lungs [139, 140]. Rare recurrence of PVOD after transplantation has been reported, although the risk of recurrence is difficult to estimate because the number of transplants performed for this diagnosis has been relatively small [99, 141, 142].
8.4 Immunosuppressive Agents Immunosuppressive medications such as glucocorticoids and azathioprine have occasionally have been employed in the treatment of PVOD, although protocols have been neither standardized nor randomized, making it difficult to form unequivocal conclusions about their effectiveness. Nonetheless, only rare responses to therapy have been reported, and only a minority of these cases have shown sustained improvements [76, 84, 143, 144]. Most patients do not have prominent autoimmune features or biochemical indices suggestive of an inflammatory process, and thus would not be expected to respond to such interventions [2, 119]. In general, immunosuppressive agents do not have a role in the treatment of PVOD, although a 4-week trial of 0.75– 1.0 mg/kg of prednisone may be considered in patients with associated nonspecific interstitial pulmonary inflammation, or an autoimmune feature such as arthritis, alopecia, or an elevated erythrocyte sedimentation rate. If improvement is seen in symptoms, radiographs, diffusing capacity, and/or alveolar–arterial oxygen gradient, the dosage is then slowly tapered to 20–40 mg/day.
8.5 Experimental Therapies Although endothelin-1 receptor antagonists such as bosentan and phosphodiesterase-5 inhibitors such as sildenafil are finding roles in the treatment of pulmonary arterial hypertension, there is little published experience with these medications in the treatment of PVOD [134, 145–147]. Both classes of medications could prove either beneficial or deleterious in PVOD for similar reasons as vasodilators, and at present no recommendation regarding their use can be made. A number of experimental therapies have been investigated for hepatic veno-occlusive disease, such as defibrotide, recombinant tissue plasminogen activator, and antithrombin-III concentrate [148, 149]. No data are available regarding the potential utility of these treatments in
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PVOD, and given the likely differences in pathogenesis of the two conditions, none of these agents are recommended to treat patients with PVOD pending specific data supporting their efficacy. The beneficial therapeutic effect of the platelet-derived growth factor receptor inhibitor, imatinib (Gleevec), in patients with IPAH has been described in several case studies [150–152]. Use of this agent was reported in a single patient with presumed PVOD, with rapid improvement in clinical status and radiographic abnormalities [153]. The value of this approach in other patients remains speculative, and the absence of both tissue biopsy prior to therapy and hemodynamic evaluation following the initiation of therapy are limitations of this report. However, given the otherwise poor prognosis and lack of response to treatment seen in most patients with PVOD, these findings are suggestive that platelet derived growth factor receptor inhibition may play a role in the future management of PVOD.
9 Conclusion Despite more than seven decades since its initial description, PVOD remains a highly lethal and poorly understood syndrome, and fundamental questions regarding the cause and optimal treatment remain unanswered. The diagnosis of PVOD requires a high degree of clinical suspicion and in most cases necessitates surgical lung biopsy to definitively establish the presence of this disorder. Because the prognosis and management of PVOD are sufficiently different from those of precapillary causes of pulmonary hypertension, the risk associated with surgical biopsy is generally justified. In particular, the benefits and risks associated with medical therapy using prostanoids or endothelin-1 receptor antagonists are different for patients with PVOD than for those with IPAH. Although these treatments may be of benefit to selected patients with PVOD, significant harm or death may occur if PVOD goes unrecognized or is misdiagnosed as IPAH. Additional research is required to more precisely delineate the epidemiologic factors, cause, risk factors and optimal therapy for this syndrome.
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Chapter 81
Left Ventricular Diastolic Heart Function and Pulmonary Hypertension Stuart Rich and Mardi Gomberg-Maitland
Abstract Pulmonary venous hypertension is now one of the most common causes of pulmonary hypertension (PH) diagnosed by both pulmonologists and cardiologists. Patients with pulmonary venous hypertension will typically have elevated pulmonary venous pressure (as reflected in the pulmonary capillary wedge pressure), most frequently as a reflection of increased left ventricular end-diastolic pressure. Although mitral stenosis was the most common cause of this entity decades ago, left ventricular diastolic dysfunction is the most common cause of pulmonary venous hypertension seen in the Western world today. The mechanisms for the hypertension in mitral stenosis and in left ventricular diastolic abnormalities are thought to be similar. A chronic elevation in the diastolic filling pressure of the left side of the heart causes a backward transmission of the pressure to the pulmonary venous system which appears to trigger vasoconstriction in the pulmonary arterial bed. The current accepted designation is PH secondary to heart failure with a preserved ejection fraction. Keywords Pulmonary venous hypertension • Pulmonary venous pressure • Mitral stenosis • Diastolic function
1 Introduction Pulmonary venous hypertension is now one of the most common causes of pulmonary hypertension (PH) diagnosed by both pulmonologists and cardiologists. Patients with pulmonary venous hypertension will typically have elevated pulmonary venous pressure (as reflected in the pulmonary capillary wedge pressure), most frequently as a reflection of increased left ventricular end-diastolic pressure [1]. Although mitral stenosis was the most common cause of this entity
S. Rich (*) Section of Cardiology, University of Chicago Medical Center, 5841 South Maryland Avenue, Chicago, IL 60637, USA e-mail:
[email protected] decades ago, left ventricular diastolic dysfunction is the most common cause of pulmonary venous hypertension seen in the Western world today. The mechanisms for the hypertension in mitral stenosis and in left ventricular diastolic abnormalities are thought to be similar. A chronic elevation in the diastolic filling pressure of the left side of the heart causes a backward transmission of the pressure to the pulmonary venous system which appears to trigger vasoconstriction in the pulmonary arterial bed [2]. The current accepted designation is PH secondary to heart failure with a preserved ejection fraction (HFpEF) [3].
2 Pathology Histologically, abnormal thickening of the veins and formation of a neointima is seen [4]. The latter can be quite extensive. As a secondary feature there is medial hypertrophy and with time thickening of the neointima on the arterial side of the pulmonary circulation [5]. Reversibility of these changes with treatment of the underlying cause of the venous hypertension may be possible. The variability in the response of the pulmonary arterial circulation to the elevated venous pressure indicates that there may be genetic factors that control individual response and potential reversibility of the disease. For example, insulin resistance, frequently observed in association with impaired left ventricular diastolic function, is independently linked to the development of pulmonary arterial hypertension (PAH) [6].
3 Mechanism of PH in HFpEF The development of PH in HFpEF has largely focused on the role of left ventricular diastolic dysfunction with a passive effect leading to pulmonary venous hypertension [7]. As with mitral stenosis, in many patients the passive contribution of pulmonary venous hypertension may not by itself account for the increased pulmonary artery systolic pressure.
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The greater severity of PH in HFpEF may be caused by an additional precapillary component of PAH [8]. In patients with long-standing pulmonary congestion, precapillary PH may be mediated by a reactive increase in pulmonary arterial tone or development of a congestive arteriopathy characterized by pulmonary arteriolar remodeling, medial hyperplasia, and intimal fibrosis [9]. The same abnormality is seen in patients with mitral stenosis and systolic heart failure.
4 Left Ventricular Compression Versus Left Ventricular Underfilling The classic echocardiographic appearance of PH with HFpEF is one of a markedly dilated right ventricle and a small left ventricle. It has been debated whether the mechanism of the small left ventricle is compression which is contributing to the increased left ventricular end-diastolic pressure, or underfilling as a consequence of the failure of the right ventricle. Bernheim described this interdependence between the right and left ventricles, hypothesizing that the geometric changes from progressive left ventricular enlargement and hypertrophy could directly compress the right ventricle and thereby reduce right ventricular filling. This “Bernheim effect” could lead to elevated central venous pressure and peripheral edema in cases of right ventricular failure [10]. The “reverse” Bernheim phenomenon (i.e., enlargement of the right ventricle leading to compression and underfilling of the left ventricle) has also been described in both animals and humans with PH and right ventricular failure [11]. The mechanisms of interdependence are better understood by use of new imaging techniques. On the one hand, direct ventricular interaction could limit left ventricular diastolic filling. Several echocardiographic studies have shown a high incidence of decreased early left ventricular filling in PH and right ventricular enlargement [12, 13]. The left ventricle appears distorted and compressed in the setting of severe right ventricular pressure overload, and the interventricular septum functions more as part of the right ventricle than as part of the left ventricle. Typically there is reversal of the E/A transmitral pattern, a lower stroke volume index, and a lower cardiac index in PAH versus control patients. Using magnetic resonance imaging, patients with idiopathic PAH had a reduction in left ventricular peak filling rate and left ventricular stroke volume as compared with control patients [14]. Investigators also found that leftward curvature of the interventricular septum correlated with left ventricular filling rate and concluded that direct ventricular interaction impairs ventricular filling in PH. On the other hand, underfilling of the left side of the heart is also a rational explanation for the observed physiologic characteristics [15]. The high pulmonary vascular resistance and low cardiac output observed in severe PH may lead to
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underfilling of the left side of the heart. A study in patients with chronic thromboembolic PH (CTEPH) pre- and postoperatively observed this phenomenon [15]. Preoperatively, the interventricular septum was not particularly hypertrophied, and its thickness matched that of the posterior wall of the left ventricle. Also, although the left ventricle in CTEPH appeared compressed within the pericardium by the right ventricle, pericardiectomy did not lead to changes in left ventricular shape or diastolic pressure/volume relationships. After successful thromboendarterectomy, transmitral Doppler E velocity increased significantly and the E/A ratio normalized. The normal preoperative thickness of the interventricular septum and posterior wall of the left ventricle also did not change after surgery. The finding of an abnormal transmitral filling pattern (E/A) together with a normal mitral Em velocity before thromboendarterectomy supports the concept that the abnormal relaxation pattern of transmitral left ventricular filling in CTEPH appears to be due to a low left ventricular preload and underfilling rather than right ventricular hypertrophy, enlargement, and left ventricular compression. Postoperatively, the dramatic increases in transmitral E and pulmonary venous flow velocities after thromboendarterectomy are consistent with a marked improvement in left ventricular preload. The rapid normalization of the E/A ratio after thromboendarterectomy and the presence of normal preoperative interventricular septal thickness by echocardiography would also argue against any significant lingering effects from septal hypertrophy. Finally, the coexistence of an E/A transmitral filling pattern and an essentially normal mitral annular Em velocity before thromboendarterectomy is perhaps the most persuasive evidence of left ventricular underfilling with relatively preserved diastolic function.
5 Prevalence of PH in HFpEF The prevalence of PH in HFpEF has not been evaluated in prospective studies and is unknown. Severe PH from HFpEF was described in early isolated case reports of elderly hypertensive patients with HFpEF [16]. In two recent series of patients with HFpEF, PH was diagnosed by echocardiography in 44% in the clinic [17] and in 25% of patients hospitalized for symptomatic heart failure [18]. Of note, the latter study also identified elevated pulmonary artery systolic pressure as an independent predictor of mortality in HFpEF (Fig. 1).
6 Evaluation The clinical profile of left ventricular diastolic dysfunction is characteristically observed in an older patient with hypertension, diabetes, coronary artery disease, and/or obesity (Fig. 2).
81 Left Ventricular Diastolic Heart Function and Pulmonary Hypertension
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Table 1 The diagnosis of pulmonary hypertension from heart failure with preserved ejection fraction Clinical assessment Features Symptoms
Fig. 1 Kaplan–Meier survival curves in patients with heart failure with a preserved ejection fraction with pulmonary artery systolic pressure above and below the median. Patients were stratified on the basis of the pulmonary artery systolic pressure determined by Doppler echocardiography and their being followed for 3 years. Those with severer pulmonary hypertension had worse survival. (Reprinted from Lam et al. [3], with permission from Elsevier)
Fig. 2 The differential diagnoses to the syndrome of heart failure with normal ejection fraction. Conditions that can cause or contribute to dyspnea in patients with normal left ventricular systolic function are illustrated. They can be characterized as cardiac and noncardiac in origin. Many patients will have more than one cause. (Reprinted from Maeder and Kaye [38], with permission from Elsevier)
The clinical signs and symptoms that are characteristic of pulmonary venous hypertension include dyspnea with effort, and eventually right ventricular failure with edema, similar to category 1 PAH [19]. However, important and distinctive symptoms are orthopnea and paroxysmal nocturnal dyspnea, which is not a feature of PAH. Atrial arrhythmias also seem to be more common in patients with pulmonary venous hypertension than has been noted in PAH.
Orthopnea Paroxysmal nocturnal dyspnea Signs Elevated jugular venous pressure Edema Electrocardiogram RVH may be absent LVH may be present Chest X-ray Pulmonary vascular congestion Pulmonary edema Pleural effusions Chest CT scan Mosaic perfusion pattern Ground-glass opacities Echocardiogram RV enlargement Elevated PA pressure by Doppler echocardiography Normal LV ejection fraction Cardiac Normal LV systolic function catheterization Elevated PCWP Normal PCWP and normal cardiac output (consider challenge with exercise or short acting pulmonary vasodilator) Normal PCWP and reduced cardiac output (consider challenge with exercise or inotropic agents) RVH right ventricular hypertrophy, LVH right ventricular hypertrophy, RV right ventricular, LV left ventricular, PA pulmonary arterial, PCWP pulmonary capillary wedge pressure
The clinical evaluation of these patients will often reveal findings that support pulmonary venous hypertension [20] (Table 1). The electrocardiogram may show left ventricular hypertrophy rather than right ventricular hypertrophy. The chest X-ray will often show pulmonary vascular congestion, pleural effusions, and on occasion pulmonary edema. A highresolution chest CT scan can be particularly helpful because it will often reveal ground-glass opacities consistent with chronic pulmonary edema, and a mosaic perfusion pattern. These constellations of findings should alert the clinician that the patient likely has pulmonary venous hypertension and not PAH. The use of Doppler echocardiography has become a popular screening tool for the diagnosis of PH and in some clinical trials has been the only measure of the severity of the PH in response to treatment. However, in spite of the common perception that Doppler echocardiography can accurately measure pulmonary pressures, the data suggest that it is very imprecise [21]. In addition, because Doppler echocardiography cannot determine pulmonary capillary wedge pressure and cardiac output, two critical measurements in making an accurate diagnosis, the use of Doppler echocardiography alone to diagnose and initiate treatment of patients should be discouraged. Cardiac catheterization is essential as demonstration of an elevated pulmonary capillary wedge pressure secures the diagnosis. Clinicians need to be reminded that an accurate assessment of left ventricular end-diastolic pressure or
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pulmonary capillary wedge pressure can only be made at end expiration; using a digitally derived mean pulmonary capillary wedge pressure will generally yield erroneous information and usually a misclassification of the patient as having normal filling pressures [22]. Establishing the pulmonary capillary wedge pressure in a patient with PH is paramount, as it has critical implications. As most of the treatments for PAH increase cardiac output, when given to patients with left ventricular diastolic dysfunction they run the risk of causing acute or chronic pulmonary edema and worsening hypoxemia. The potential for clinical worsening and pulmonary edema from the PAH therapies is so great that, in our opinion, a direct measurement of the left ventricular end-diastolic pressure should be performed in any patient in whom an adequate pulmonary capillary wedge pressure cannot be obtained, or is obtained but its validity is in doubt. Two hemodynamic profiles have been described that are common in these patients [19]. Some patients will have an elevation in pulmonary arterial pressure with only a minimal increase in the transpulmonary gradient (mean PA pressure minus pulmonary capillary wedge pressure), as a reflection of the passive increase in pulmonary arterial pressure necessary to overcome the increase downstream resistance. Indeed, a preserved right ventricle must generate high systolic pressures to ensure adequate forward blood flow in these patients, and thus moderate degrees of PH are not only characteristic, but also favorable. However, a subset of these patients will have reactive pulmonary vasoconstriction resulting in marked elevations in pulmonary arterial pressure beyond that which is necessary to maintain cardiac output. These patients are frequently distinguished by a marked elevation in pulmonary arterial diastolic pressure. It is believed that these patients have a permissive genotype such that, when exposed to high pulmonary venous resistance, they develop reactive PH with severer arterial changes, including neointimal formation, than the other subgroup [23]. There are also patients with left ventricular diastolic dysfunction who have normal left ventricular end-diastolic pressure at rest [24] (Fig. 3). When this occurs in the setting of reactive PH, it is very difficult to know if these patients actually have PAH or pulmonary venous hypertension. Using an inotropic challenge at the time of diagnostic cardiac catheterization may be helpful. If the response shows a significant increase in cardiac output without an accompanying increase in pulmonary capillary wedge pressure, the patient likely has PAH. On the other hand, if the increase in cardiac output is accompanied by an increase in pulmonary capillary wedge pressure, the patient likely has pulmonary venous hypertension. Many physicians will use the information from acute vasodilator testing to provide insight into the effects of therapies other than calcium channel blockers. Because of the many different effects these medications can have, it is important to note their impact on the interaction of pre- and
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Fig. 3 End-diastolic pressure–volume relationships from patients with left ventricular diastolic dysfunction and from normal patients. The steep curve in those with diastolic dysfunction illustrates how it is possible to have normal left ventricular end-diastolic pressure at rest. It also shows how increasing left ventricular filling, either by exercise (as done here) or with inotropic drugs, raises the left ventricular end-diastolic pressure to abnormal levels. (Adapted with permission [24])
postcapillary pressures, transpulmonary blood flow, and gas exchange in every patient who undergoes vasoreactivity testing. Determining the effects of vasodilators on pulmonary capillary wedge pressure is essential. Since it can be difficult to distinguish patients with idiopathic PAH from those with left ventricular diastolic dysfunction, one needs to be particularly careful when evaluating pulmonary venous and/or left ventricular end-diastolic pressures during drug testing.
7 Treatment The current dilemma relates to how best to treat these patients. Again, lessons learned from treating patients with mitral stenosis can be instructive [25]. Patients who present with pulmonary edema should satisfactorily respond to the use of diuretics as a temporizing measure. However, the only definitive treatment of patients with mitral stenosis and PH is mitral valve repair or replacement [26, 27]. Several clinical studies have shown that removing the mitral valve gradient, either surgically or percutaneously, will result in an immediate fall in the pulmonary arterial pressure. The magnitude of the fall, however, can be quite variable, with some patients achieving normal hemodynamics within 24 h, and others taking many months to improve [28]. The magnitude and rate of improvement may be related to the severity of the vascular disease dictated both by the duration of the PH and genetic factors that either induce severer vascular disease or that impede regeneration and remodeling following removal of the mitral valve obstruction [29]. It is likely that these same issues are relevant to PH in patients with left ventricular diastolic dysfunction.
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To date, there are no proven therapies in HFpEF [30]. Treatment recommendations as outlined in heart failure guidelines are empiric and have not changed over time. Vasodilator therapy aimed at PH in HFpEF is therefore an appealing consideration but has been tempered by concern that increases in right-sided heart output with pulmonary vasodilators may result in further increases in left atrial pressure in patients with left-sided heart disease and hear failure. Since the major hemodynamic effect of these therapies is to raise cardiac output, it will predictably cause a worsening of pulmonary edema if the pulmonary venous obstruction is not being relieved [31, 32]. In fact these therapies are contraindicated in patients with pulmonary venous hypertension, and there are multiple reports of rapid deterioration and death when pulmonary vasodilators were used in the presence of pulmonary venous hypertension [33]. Indeed, the use of epoprostenol was associated with increased mortality in systolic heart failure, although the mechanism for increased mortality was unclear [34]. Similarly, trials of endothelin receptor antagonists in systolic heart failure failed to show clinical benefit [35]. Recent small trials using phosphodiesterase-5 inhibitors in systolic heart failure have shown beneficial effects, including improvement in exercise capacity and quality of life [36]. In fact, evidence exists that phosphodiesterase-5 inhibition may not only improve pulmonary tone and right-sided heart function but that it also exerts pleiotropic effects on left ventricular structure, and ventricular function [37]. Our approach to treating patients with PH in HFpEF has been to use medical measures to lower left ventricular filling pressures (such as nitrates, diuretics, and aggressive treatment of systemic hypertension). When successful, we have found that the pulmonary arterial pressure will also fall, and the cardiac output will increase.
8 Outcome A recent series on PH from HFpEF listed independent predictors of death, including age, stroke, chronic obstructive pulmonary disease, cancer, diabetes, low glomerular filtration rate, and hyponatremia, findings that mirror those of other large epidemiology studies of patients with HFpEF [3]. Taken together, these studies emphasize that cardiac and noncardiac comorbidities, along with hear failure, appear to play an important role in the increased morbidity and mortality in patients with HFpEF. Because patients with HFpEF often have important comorbid conditions, and because these comorbidities strongly influence outcomes, clinicians should aggressively identify and treat conditions such as hypertension, coronary artery disease, atrial fibrillation, diabetes, chronic kidney disease, and cerebrovascular disease in these patients rather than waiting for new PH- and HFpEF-specific treatments to emerge.
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As the frequency of diagnosis of PH secondary to HFpEF is increasing, we propose the following guidelines: • One should always attempt to treat the underlying disease first. • The use of conventional therapies (diuretics, oxygen) should be tried initially to correct related clinical problems. Exercise testing may be helpful to uncover exerciseinduced hypoxemia which may benefit from treatment. • The acute testing of vasodilators with hemodynamic guidance is recommended prior to initiating chronic use to evaluate the potential for beneficial or adverse effects. • Patients who respond favorably to pulmonary vasodilators should manifest clear improvement in their symptoms related to the PH and this should be corroborated with clinical testing (echocardiography, exercise testing, and catheterization). • Patients who fail to improve, or who demonstrate worsening clinical findings (tachycardia, hypoxemia, hypotension, or worsening edema), should have the vasodilator therapy promptly discontinued. Beneficial effects should be objectively apparent within 4–6 weeks.
References 1. Redfield MM (2004) Understanding "diastolic" heart failure. N Engl J Med 350:1930–1931 2. Wood P (1958) Pulmonary hypertension with special reference to the vasoconstrictive factor. Br Heart J 20:557 3. Lam CSP, Roger VL, Rodeheffer RJ, Borlaug BA, Enders FT, Redfield MR (2009) Pulmonary hypertension in heart failure with preserved ejection fraction. J Am Coll Cardiol 53:1119–1126 4. Wagenvoort C, Wagenvoort N (1977) Pathology of pulmonary hypertension, 2nd edn. Wiley, New York 5. Pietra GG Capron F, Stewart S, Leone O et al (2004) Pathologic assessment of vasculopathies in pulmonary hypertension. J Am Coll Cardiol 43:25S–32S 6. Hansmann G, Wagner RA, Schellong S et al (2007) Pulmonary arterial hypertension is linked to insulin resistance and reversed by peroxisome proliferator-activated receptor-g activation. Circulation 115:1275–1284 7. Kessler KM, Willens HJ, Mallon SM (1993) Diastolic left ventricular dysfunction leading to severe reversible pulmonary hypertension. Am Heart J 126:234–235 8. Willens HJ, Kessler KM (1993) Severe pulmonary hypertension associated with diastolic left ventricular dysfunction. Chest 103:1877–1883 9. LaBourene JI CJ, Johnson DJ, Mehra A, Keeley FW, Rabinovitch M (1990) Alterations in elastin and collagen related to the mechanism of progressive pulmonary venous obstruction in a piglet model. A hemodynamic, ultrastructural, and biochemical study. Circ Res 66:438–456 10. Santamore WP, Dell’Italia LJ (1998) Ventricular interdependence: significant left ventricular contributions to right ventricular systolic function. Prog Cardiovasc Dis 40:289–308 11. Little WC, Badke FR, O’Rourke RA (1984) Effect of right ventricular pressure on the end-diastolic left ventricular pressure volume relationship before and after chronic right ventricular pressure overload in dogs without pericardia. Circ Res 54:719–730
1188 12. Boussuges B, Pinet C, Molenat F et al (2000) Left atrial and ventricular filling in chronic obstructive pulmonary disease. An echocardiographic and Doppler study. Am J Respir Crit Care Med 162:670–675 13. Schena M, Clini E, Errera D, Quadri A (1996) Echo-Doppler evaluation of left ventricular impairment in chronic cor pulmonale. Chest 109:1446–1451 14. Benza R, Biederman R, Murali S, Gupta H (2008) Role of cardiac magnetic resonance imaging in the management of patients with pulmonary arterial hypertension. J Am Coll Cardiol 52:1683–1692 15. Gurudevan SV, Malouf PJ, Auger WR, Waltman TJ, Madani M, Raisinghani AB, DeMaria AN, Blanchard DG (2007) Abnormal left ventricular diastolic filling in chronic thromboembolic pulmonary hypertension: true diastolic dysfunction or left ventricular underfilling? J Am Coll Cardiol 49:1334–1339 16. Shapiro BP, McGoon MD, Redfield MM (2007) Unexplained pulmonary hypertension in elderly patients. Chest 131:94–100 17. Klapholz M, Maurer M, Lowe AM et al (2004) Hospitalization for heart failure in the presence of a normal left ventricular ejection fraction: Results of the New York Heart Failure Registry. J Am Coll Cardiol 43:1432–1438 18. Kjaergaard J, Akkan D, Iversen KK et al (2007) Prognostic importance of pulmonary hypertension in patients with heart failure. Am J Cardiol 99:1146–1150 19. Rich S, Rabinovitch M (2008) The diagnosis and treatment of secondary (non-category 1) pulmonary hypertension. Circulation 118:2190–2199 20. Angeja BG, Grossman W (2003) Evaluation and management of diastolic heart failure. Circulation 107:659–663 21. Fisher MR, Forfia PR, Chamera E et al (2009) Accuracy of Doppler echocardiography in the hemodynamic assessment of pulmonary hypertension. Am J Respir Crit Care Med 179:615–621 22. Halpern SD, Taichman DB (2009) Hemodynamic assessment of pulmonary hypertension. Am J Respir Crit Care Med 179:615–621 23. Du L, Sullivan C, Chu D et al (2003) Signaling molecules in nonfamilial pulmonary hypertension. New Engl J Med 348:500–509 24. Westermann D, Kasner M, Steendijk P et al (2008) Role of left ventricular stiffness in heart failure with normal ejection fraction. Circulation 117:2051–2060 25. Mahoney P, Loh E, Blitz L, Herrmann H (2001) Hemodynamic effects of inhaled nitric oxide in women with mitral stenosis and pulmonary hypertension. Am J Cardiol 87:188–192 26. Braunwald E, Braunwald N, Ross J et al (1965) Effects of mitral valve replacement on pulmonary vascular dynamics of patients with pulmonary hypertension. N Engl J Med 273:509–514
S. Rich and M. Gomberg-Maitland 27. Zener JC, Hancock EW, Shumway NE, Harrison DC (1972) Regression of extreme pulmonary hypertension after mitral valve surgery. Am J Cardiol 30:820–826 28. Levine M, Weinstein J, Diver D, Berman AD, Wyman RM (1989) Progressive improvement in pulmonary vascular resistance following percutaneous mitral valvuloplasty. Circulation 79:1061–1067 29. Fawzy ME, Hassan W, Stefadouros M, Moursi M (2004) El Shaer F, Chaudhary MA. Prevalence and fate of severe pulmonary hypertension in 559 consecutive patients with severe rheumatic mitral stenosis undergoing mitral balloon valvotomy. J Heart Valve Dis 13:942–947 30. Senni M, Redfield MM (2001) Heart failure with preserved systolic function. A different natural history? J Am Coll Cardiol 38: 1277–1282 31. Palmer S, Robinson L, Wang A, Gossage J, Bashore T, Tapson V (1998) Massive pulmonary edema and death after prostacyclin infusion in a patient with pulmonary veno-occlusive disease. Chest 113:237–240 32. Humbert M, Maître S, Capron F, Rain B, Musset D, Simonneau G (1998) Pulmonary edema complicating continuous intravenous prostacyclin in pulmonary capillary hemangiomatosis. Am J Respir Crit Care Med 157:1681–1685 33. Preston I, Klinger J, Houtchen J, Nelson D, Mehta S, Hill N (2002) Pulmonary edema caused by inhaled nitric oxide therapy in two patients with pulmonary hypertension associated with CREST syndrome. Chest 121:656–659 34. Califf RM, Adams KF, McKenna WJ et al (1997) A randomized controlled trial of epoprostenol therapy for severe congestive heart failure: The Flolan International Randomized Survival Trial (FIRST). Am Heart J 134:44–54 35. Anand I, McMurray J, Cohn JN et al, for the EARTH Investigators (2004) Long-term effects of darusentan on left-ventricular remodelling and clinical outcomes in the Endothelin A Receptor Antagonist Trial in Heart Failure (EARTH): randomised, double-blind, placebo controlled trial. Lancet 364:347–354 36. Guazzi M, Tumminello G, Di Marco F, Fiorentini C, Guazzi MD (2004) The effects of phosphodiesterase-5 inhibition with sildenafil on pulmonary hemodynamics and diffusion capacity, exercise ventilatory efficiency, and oxygen uptake kinetics in chronic heart failure. J Am Coll Cardiol 44:2339–2348 37. Takimoto E, Champion HC, Li M et al (2005) Chronic inhibition of cyclic GMP phosphodiesterase-5A prevents and reverses cardiac hypertrophy. Nat Med 11:214–222 38. Maeder MT, Kaye DM (2009) Heart failure with normal ejection fraction. J Am Coll Cardiol 53:905–918
Chapter 82
Pulmonary Hypertension Associated with Chronic Obstructive Pulmonary Diseases Norbert F. Voelkel, Catherine Grossman, and Herman J. Bogaard
Abstract Chronic obstructive lung diseases and so-called cor pulmonale have traditionally been connected by the link of pulmonary hypertension (PH). The concept that right ventricular hypertrophy is attributed to the afterload of the right ventricle, i.e., attributable to PH, has not been challenged and clinicians continue to attempt to explain cor pulmonale as a consequence of chronic PH. Both pulmonary parenchyma disease and primary vascular disease can lead to cor pulmonale. Among the obstructive lung diseases, chronic obstructive pulmonary disease (COPD)/ emphysema has been recognized as the most common cause of PH in countries where smoking, but not schistosomiasis, is endemic. Until recently, clinical investigators conceptually associated PH in the setting of COPD/emphysema with chronic hypoxia. Pulmonary angiograms documented the extent of vascular involvement and vascular loss in patients with emphysema. Chronic oxygen supplementation therapy was introduced into clinical management of COPD patients on the basis of the finding that oxygen treatment increased survival and that improved survival was associated with a small reduction in mean pulmonary artery pressure at rest. This chapter discusses the pathogenic mechanisms, diagnostic and therapeutic approaches for pulmonary hypertension associated with chronic obstructive pulmonary diseases. Keywords Chronic hypoxia and hypoxemia • Emphysema • Cor pulmonale • Pulmonary parenchyma disease
N.F. Voelkel (*) Division Pulmonary and Critical Care Medicine, Department of Internal Medicine, Virginia Commonwealth University, 1101 E. Marshall Street, Richmond, VA, USA e-mail:
[email protected] 1 Chronic Obstructive Pulmonary Disease, Pulmonary Hypertension, and Cor Pulmonale: The History Chronic obstructive lung diseases and so-called cor pulmonale have traditionally been connected by the link of pulmonary hypertension (PH). The first clinical description of cor pulmonale goes back to White in 1931 [1]. The concept that right ventricular hypertrophy is attributed to the afterload of the right ventricle, i.e., attributable to PH, has not been challenged and clinicians continue to attempt to explain cor pulmonale as a consequence of chronic PH. An expert committee of the WHO defined “cor pulmonale” in 1963 [2] as stress or strain of the right ventricle as a consequence of diseases that affect the structure or function of the lung and thus effect a pressure increase in the lung circulation. According to this definition, both pulmonary parenchyma disease and primary vascular disease can lead to cor pulmonale. Among the obstructive lung diseases, chronic obstructive pulmonary disease (COPD)/emphysema has been recognized as the most common cause of PH in countries where smoking, but not schistosomiasis, is endemic. Until recently, clinical investigators conceptually associated PH in the setting of COPD/emphysema with chronic hypoxia [3] although substantial pathological vascular abnormalities, difficult to ascribe to hypoxia, had already been described by Liebow in 1958 (see later) [4]. At the same time, pulmonary angiograms documented the extent of vascular involvement and vascular loss in patients with emphysema [5]. Chronic oxygen supplementation therapy was introduced into clinical management of COPD patients on the basis of the finding that oxygen treatment increased survival and that improved survival was associated with a small reduction in mean pulmonary artery pressure at rest [6]. Although it is unclear if improved survival of these patients was attributable to a reduction of the pulmonary artery pressure, the pharmaceutical industry continues to be interested in the development of treatments for PH in COPD patients. MacNee [7, 8] reviewed PH, COPD, and cor pulmonale and there are also more recent reviews of this topic [9–11].
J.X.-J. Yuan et al. (eds.), Textbook of Pulmonary Vascular Disease, DOI 10.1007/978-0-387-87429-6_82, © Springer Science+Business Media, LLC 2011
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The German literature distinguishes between “manifest PH” (at rest), which is most likely also “fixed,” because of remodeling of the vasculature or vessel loss, and “latent PH,” which emerges with exercise. Both Burrows et al. [12] and Weitzenblum et al. [13] have emphasized the importance of exercise testing in the evaluation of PH in COPD patients. Figure 1 depicts the pulmonary vessel loss which was assessed by a postmortem angiogram of a patient with emphysema. A lung function parameter which relates to lung vessel loss in COPD is the diffusing capacity of carbon monoxide (DLCO). Early on, investigators of PH in COPD distinguished between a “hypoxic form of cor pulmonale” and a “vascular form of cor pulmonale.” Alveolar hypoventilation leading to hypoxia, hypercapnia, and pulmonary arteriolar vasoconstriction explained the hypoxic form, whereas vascular occlusions explained the vascular form [14]. The “vascular occlusions” concept is apparently undergoing a renaissance as pulmonary embolic events are now more frequently being appreciated as a cause of COPD exacerbations (see later). Although the term “cor pulmonale” was coined to distinguish right ventricular impairment caused by lung disease, new data may point to an involvement of the left ventricle as well in patients with COPD (see later). This begins to put into question the strict use of the term “cor pulmonale” in the setting of COPD. In fact, in some COPD patients there may be a combination of precapillary and postcapillary PH, and
Fig. 1 Pulmonary arteriogram of a segment of artery of a normal lung and segmental arteriogram of a lung from a patient with severe emphysema. A artery, V vein. (Reproduced with permission [5])
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this aspect may change our treatment and practice guidelines. A “classic” paper which examines the basic pulmonary hemodynamics is entitled “Alveolar pressure, pulmonary venous pressure, and the vascular waterfall,” by Permut et al. [15]; these key words all need to be considered when investigating and treating PH in COPD.
2 The Clinical Presentation of PH in COPD PH in COPD patients may not only characterize a particular COPD patient phenotype, but importantly PH in COPD patients is a survival-determining factor [16]. Figure 2 shows that mean pulmonary artery pressure and age predict survival of COPD patients. Patients with COPD/emphysema are short of breath, initially with strenuous work or exercise and, as the disease progresses, at rest. Understandably the clinician focuses his/her attention on the airflow limitation, bronchoconstriction, and symptoms associated with bronchitis. Since exercise limitation and dyspnea are also cardinal symptoms of severe PH, the clinical investigation of PH in COPD patients is usually delayed until signs of heart failure appear. Nevertheless, many attempts have been made over the years to define the lung function, and blood gas variables, which characterize a COPD patient who is likely to develop PH. The approach has generally been to collect data from a reasonably large cohort of COPD patients and to correlate the forced expiratory volume in 1 s (FEV1), SO2, PaO2, PCO2, or DLCO data with the pulmonary artery pressure. Figures 3 and 4 provide examples of such plots. These correlations (plots) are based on the assumption that the pulmonary artery pressure
Fig. 2 Survival of chronic obstructive pulmonary disease (COPD) patients with pulmonary hypertension related to the mean pulmonary artery pressure and related to the age of the patient. (Reproduced with permission of the American College of Chest Physicians [16])
82 Pulmonary Hypertension Associated with Chronic Obstructive Pulmonary Diseases
Fig. 3 Relationship between the mean pulmonary artery pressure and the PaO2 in patients with severe pulmonary hypertension and COPD. (a) Patients have no other causes of pulmonary hypertension, reflecting a rare subgroup of COPD patients with severe
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pulmonary hypertension. These patients were hypoxemic and had a median survival of 26 months. (b) Patients show a relationship between pulmonary artery pressure and PaO2. (Reproduced with permission [23])
Fig. 4 Relationship between mean pulmonary artery pressure, diffusing capacity of carbon monoxide (a) PaO2 (b) and oxygen saturation (c and d) (Reproduced with permission [50])
at rest should relate to known pathobiological concepts; that is pulmonary artery pressure relates to hypoxia, loss of lung vessels, and so on. The histology and morphology of the lung tissue and of lung vessels is not considered because lung tissue is usually not available for analysis. On the other hand, the degree of hyperinflation or inspiratory capacity, which affect the venous return and the nocturnal oxygen desaturation, are also part of the equation [17, 18]. Astutely,
the early clinical COPD investigators noticed and distinguished the edematous, blue and bloated and the thin (often cachectic) pink and puffing phenotype, and autopsy studies demonstrated a greater degree of right ventricular hypertrophy in the blue and bloated patients [18]. An untested hypothesis is that the so-called blue bloater phenotype of COPD disappeared after the introduction of oxygen therapy for COPD. Another untested hypothesis is that PH is severer or
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occurs more frequently in COPD patients living at altitude. Kessler et al. investigated the natural history of PH in a series of 131 COPD patients and found that the pulmonary artery pressure at rest and that during exercise are independent predictors of the subsequent development of PH and that the progression of PH in patients with mild or moderate hypoxemia is slow [19].
3 Diagnosis of PH in COPD The diagnosis of PH in patients with COPD/emphysema is established like in all other forms of PH using noninvasive and invasive methods. The diagnosis of PH should be considered in patients with obstructive airway diseases (including cystic fibrosis, bronchiectasis, and obliterating bronchiolitis) as well as in patients with alveolar hypoventilation and sleep apnea syndromes. Both electrocardiogram and chest radiographic findings are unreliable when it comes to establishing a diagnosis of PH in COPD patients. Unfortunately, echocardiographic examination is also fraught with problems and the chance of obtaining tricuspid valve regurgitation signals of sufficient quality is slim [20–22]. The frustration of the experienced echocardiographer to interpret signals and patterns in patients with hyperinflated lungs together with the widespread therapeutic nihilism are often factors which prevent the vigorous pursuit of the diagnosis of PH in clinical practice, and the gold-standard method of hemodynamic assessment is not contemplated. Weitzenblum’s group in Strassbourg, France, has the greatest experience in the hemodynamic study of COPD patients and recently reported right-sided heart catheter data of 998 COPD patients [23]. These data are extremely helpful although they may not reflect the entire spectrum of PH in COPD patients since only patients living at or near sea level were included. Nevertheless, these data show that severe PH (defined as a resting mean pulmonary artery pressure of more than 40 mmHg) is rare in COPD patients. Likely severe PH defines a subgroup of COPD patients as also recently described by Thabut et al. [24]. This group described a “cluster of patients” characterized by moderate airway obstruction but profound hypoxemia without hypercapnea. In fact, these patients were hypocapnic and thus resembled patients with idiopathic PAH. If the diagnosis of PH matters in the overall treatment plan of a patient, then a right-sided heart catheterization with exercise should be performed. Figure 5 illustrates the variable pulmonary artery pressure response of COPD patients during exercise and the fact that exercise-induced dyspnea in some COPD patients can indeed be attributed (also) to their PH. The factors that account for the variability of exercise-induced PH and the degree of the pulmonary artery pressure response in an individual patient remain unclear. This variability is
Fig. 5 The most important components of the cardiovascular system response and how they interact when a COPD patient who has a hyperinflated lung exercises
likely due to a number of components, including (but not limited to) hyperinflation (auto-positive end-expiratory pressure), loss of lung vessels, and even an element of left ventricular dysfunction. Although technically demanding, measurement of the pulmonary capillary wedge pressure during exercise may unmask a left-sided heart failure component.
4 Pathophysiology of PH in COPD Hypoxic pulmonary arteriolar vasoconstriction, chronic pulmonary vascular remodeling, and in situ thrombosis are determinants of the pulmonary artery pressure. Polycythemia can be another factor, yet COPD patients are more likely to be anemic. A still unexplained aspect of the pathophysiological characteristics of the PH is the observation that oxygen supplementation blunts the exercise-induced rise in the pulmonary artery pressure as originally reported by Burrows et al. [12]. Oxygen could blunt the dyspnea of the patient, affecting a lower respiratory rate, which in turn may cause less air trapping; however, oxygen can also reduce the cardiac output and flow-dependent PH as well as hypoxic vasoconstriction. Respiratory and metabolic acidosis can worsen hypoxic vasoconstriction, which may be prevented by oxygen treatment (Fig. 5).
5 Pathobiology of PH Both pulmonary vascular remodeling and lung vessel loss are cellular processes controlled by proteins and genes. As already mentioned, the three principal histological manifestations of the lung vessel abnormalities in COPD/emphysema are muscularization and fibrosis of arterioles, capillary loss, and intravascular abnormalities related to thrombosis [11]. Until recently, hypoxic vasoconstriction had been consi dered the major driver of pulmonary vascular remodeling in
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COPD/emphysema although convincing cellular concepts were lacking. “Vasoconstriction causes pulmonary vascular smooth muscle hypertrophy” has been a popular creed [3, 16]. However, it is now clear that pulmonary muscularization of arterioles can develop as a consequence of inflammation and independently of hypoxia [25], and indeed Santos et al. [26] have described pulmonary vascular remodeling in the lungs from patients with mild COPD without hypoxia. In addition, exposure of experimental animals to cigarette smoke causes pulmonary vascular remodeling [27]. Capillary loss can now be explained by endothelial cell apoptosis as a consequence of chronic oxidant stress or secondary to the destruction of the matrix on which the endothelial cells sit (anoikis). The third manifestation – in situ thrombosis – is the least understood aspect of pulmonary vascular disease in COPD/ emphysema, although a hypercoagulable state is perhaps best explained with the concept of endothelial cell dysfunction [28, 29]. We believe that an entirely rational and productive approach to the pathobiology of lung vessels in COPD/ emphysema is the investigation of endothelial cell dysfunction. Endothelial cell dysfunction and endothelial cell “disease” could be important organizing principles of the COPD/ emphysema vascular disease aspect. There is both functional as well as histological evidence for endothelial cell dysfunction in COPD. It has been shown that NO-dependent vasodilation of small arteries is impaired [29] and histologically that the expression of the prostacyclin synthase [30] and endothelial nitric oxide synthase [31] proteins is lost in small pulmonary arteries in the lungs from patients with COPD/ emphysema (Fig. 6). The levels of vascular endothelial growth factor and vascular endothelial growth factor receptor 2 proteins are reduced in the tissue from end-stage COPD patients [32]. Taken together, these findings mean that the case for endothelial cell disease in the COPD lung is quite strong. It has been shown that pulmonary microvascular endothelial cell apoptosis stimulates vascular smooth muscle growth via transforming growth factor b [33]. Thus, during the last few years, the concept of “vasoconstriction” has been complemented by the concepts of “inflammation” and “endothelial cell dysfunction.” It is apparent that these new disease models call for new treatment principles rather than for pulmonary vasodilators. To end on a historical note regarding loss of capillaries (in emphysema): “Whether the process is initiated by air trapping or the result of atrophy otherwise induced, the effect is that the rich vascular beds vanish with the tissues they once supplied. The loss of vascularity and alveoli in emphysema noted so many years ago is still not certainly explained.” And: “In advanced emphysema, whether or not there is congestive heart failure, the bronchial veins become markedly expanded and can be traced along the bronchi…” and “it may be said that such changes as appear to be induced by very high levels of pressure in the pulmonary arteries occur very uncommonly in pulmonary emphysema” [4].
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Fig. 6 Mean pulmonary artery pressure measurements in COPD patients at rest and during exercise demonstrating the variable pulmonary artery pressure response to exercise. (Reproduced with permission from Macmillan Publishing Group [3])
How can we fit these histopathological data with recent polymorphism studies which conclude that a serotonin transporter (5-HTT) polymorphism and an interleukin-6 gene polymorphism are associated with the development of PH in COPD [34, 35]? How can we fit these morphological derangements of the lung with data that indicate that lungs from COPD patients have acquired a decrease in the expression of histone deacetylase 2 expression [36]? Could this be the explanation for the altered emphysema lung gene expression which is characterized by a “no-growth” signature [37]? Are acroleinated proteins (Fig. 6) decorating endothelial cells and vascular smooth muscle cells of the emphysema lung signals of permanent molecular and protein “scars,” which, together with suppressed growth signals, preclude vascular repair of the lung?
6 Cor Pulmonale: COPD, Associated PH, and “COPD-Associated Heart Disease” As readers may have noticed by now, it is our intention to steer away from PH as an outcome-defining aspect of the COPD/emphysema syndrome and the question rather becomes: What are the underlying causes of the high cardiovascular mortality in patients with COPD/emphysema? We offer in the form of postulates three interrelated causes. We postulate that COPD is an endothelial disease, that in
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COPD/emphysema, as a consequence of the endothelial cell disease, there is a significant in situ thrombosis component, and lastly, that “cor pulmonale” is not strictly a right ventricular (by increased afterload determined) problem, but is also a problem of the left ventricle. This topic was recently reviewed by Lee-Chiong and Matthay [38]. The question of a potential left-sided heart involvement was addressed first 40 years ago and has still not been entirely resolved. Some studies have shown that left ventricular function remains normal in COPD patients and others suggest left ventricular dysfunction [39, 40]. Even studies of the contractility of the right ventricle, using end-systolic pressure–volume analysis, indicated that the right ventricular contractility was preserved [41, 42]. Rutten et al. [43] examined patients with mild/moderate degrees of COPD and confirmed the diagnosis of chronic heart failure using cardiac MRI in 20% of these COPD patients. COPD patients with heart failure have high levels of brain natriuretic peptide, a greater BMI, and a slightly lower cardiac index when compared with patients without heart failure. Paudel et al. [44] performed echocardiography in 60 patients with COPD and found evidence for left ventricular systolic dysfunction in 26% of these patients. A comprehensive literature search [45] of cross-sectional or prospective studies which had used CT scanning or pulmonary angiography (550 patients) found an overall prevalence of pulmonary embolism of about 20%. Thus, taken together, and entirely from a clinical view point, both pulmonary embolism and left-sided heart involvement are significant
Fig. 7 Immunohistology of lungs from patients with COPD/ emphysema. (a) Tissue stained with an antibody directed against prostacyclin synthase; loss of prostacyclin synthase expression in small-vessel endothelial cells (left) when compared with control lungs. (b) Loss of endothelial nitric oxide synthase expression in a COPD/ emphysema lung (left) when compared with a normal lung. (c) Vessels and a long-term smoking history. The antibody is directed against acroleinated proteins. Note that both vascular endothelial cells and smooth muscle cells test positive for acroleinated proteins
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problems in patients with COPD; unfortunately the prevalence of severe PH in patients with COPD is hard to come by and the “heart and lungs” in COPD – close friends in real life – are still separated in clinical practice [46] (Fig. 7). We propose that a common denominator of COPD, pulmonary embolism, and chronic heart failure is the vasculature, and more precisely the endothelium. The Emphysema and Cancer Action Project study report demonstrated an impairment of flow-mediated vasodilation in COPD patients which remarkably was related to the CT-scan-determined degree of emphysema but not to the forced expiratory volume [45]! Lusuardi et al. reached a similar conclusion of impaired endothelial cell (EC) function in a smaller cohort of 44 COPD patients: here, however, flow-mediated vasodilation was inversely related to the FEV1 to forced vital capacity ratio [46]. Lack of vasodilator production by the endothelium, a prothrombotic state, and capillary loss are likely important components of the COPD syndrome (Fig. 8).
7 Therapy of PH in COPD On the basis of the concepts and postulates we have outlined, the treatment plan for patients with COPD and PH may not fundamentally differ from the treatment plan we may now contemplate for all patients with severely disabling COPD. Chronic oxygen treatment is likely indicated for all patients with resting hypoxia. Should all patients with
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endothelial disease may be central in particular when COPD/ PH is associated with other morbidities such as diabetes, hyperlipidemia, obesity, and high blood pressure. Perhaps we can postulate that it will be productive – reducing both morbidity and mortality – to consider that a microangiopathy, that involves several organs, not only the lung, and chronic systemic inflammation become more defined treatment targets for investigating the effects of statins in COPD patients with PH as a start [53].
References Fig. 8 The relationship between COPD, pulmonary hypertension, and chronic heart disease
exercise-induced hypoxemia be treated with supplemental oxygen? The pulmonary artery pressure per se may no longer become a treatment target in contrast to the endothelial cell dysfunction that we need to understand better and need to treat. At this juncture we cannot provide treatment recommendations, we can only ask questions. Should COPD patients who do not have contraindications be anticoagulated? Should COPD patients be treated with “statins” if for no other reason than that a retrospective data base analysis found that statins, angiotensin-converting-enzyme inhibitors, or angiotensin receptor blockers appear to reduce the mortality of patients with COPD [47]. What about the heretical thought to treat these patients with cardioselective b-adrenergic receptor blockers? A reasonably large cohort of COPD patients was followed in the Netherlands for more than 15 years and the analysis of the data shows a reduction of mortality in COPD patients undergoing vascular surgery [48]. This finding is also of interest in the context of another recent report which suggests that the treatment of COPD patients with inhaled b-adrenergic agonists was associated with increased cardiovascular risk [49]. Holverda et al. compared the exercise characteristics of COPD patients with and without PH and demonstrated a significantly reduced peak workload in COPD patients with PH [50]. Sildenafil acutely decreased the mean pulmonary artery pressure during submaximal exercise [51], but after 12 weeks of therapy did not affect the cardiac stroke volume or exercise capacity [52], illustrating again that the cardiac output rather than the pulmonary artery pressure determines exercise limitation.
8 Summary and Perspective We have provided a contextual view of the problem of PH associated with COPD/emphysema. The “big picture” of the patient with COPD plus PH is, in our opinion, that the
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1196 19. Kessler R, Faller M, Weitzenblum E et al (2001) “Natural history” of pulmonary hypertension in a series of 131 patients with chronic obstructive lung disease. Am J Respir Crit Care Med 164:219–224 20. Torbicki A, Skwarski K, Hawrylkiewicz I, Pasierski T, Miskiewicz Z, Zielinski J (1989) Attempts at measuring pulmonary arterial pressure by means of Doppler echocardiography in patients with chronic lung disease. Eur Respir J 2:856–860 21. Laaban JP, Diebold B, Zelinski R, Lafay M, Raffoul H, Rochemaure J (1989) Noninvasive estimation of systolic pulmonary artery pressure using Doppler echocardiography in patients with chronic obstructive pulmonary disease. Chest 96:1258–1262 22. Tramarin R, Torbicki A, Marchandise B, Laaban JP, Morpurgo M (1991) Doppler echocardiographic evaluation of pulmonary artery pressure in chronic obstructive pulmonary disease. A European multicentre study. Working Group on Noninvasive Evaluation of Pulmonary Artery Pressure. European Office of the World Health Organization, Copenhagen. Eur Heart J 12:103–111 23. Chaouat A, Bugnet AS, Kadaoui N et al (2005) Severe pulmonary hypertension and chronic obstructive pulmonary disease. Am J Respir Crit Care Med 172:189–194 24. Thabut G, Dauriat G, Stern JB et al (2005) Pulmonary hemodynamics in advanced COPD candidates for lung volume reduction surgery or lung transplantation. Chest 127:1531–1536 25. Daley E, Emson C, Guignabert C et al (2008) Pulmonary arterial remodeling induced by a Th2 immune response. J Exp Med 205:361–372 26. Santos S, Peinado VI, Ramirez J et al (2002) Characterization of pulmonary vascular remodelling in smokers and patients with mild COPD. Eur Respir J 19:632–638 27. Wright JL, Churg A (1991) Effect of long-term cigarette smoke exposure on pulmonary vascular structure and function in the guinea pig. Exp Lung Res 17:997–1009 28. Peinado VI, Barbera JA, Ramirez J et al (1998) Endothelial dysfunction in pulmonary arteries of patients with mild COPD. Am J Physiol 274:L908–L913 29. Dinh-Xuan AT, Higenbottam TW, Clelland CA et al (1991) Impairment of endothelium-dependent pulmonary-artery relaxation in chronic obstructive lung disease. N Engl J Med 324:1539–1547 30. Nana-Sinkam SP, Lee JD, Sotto-Santiago S et al (2007) Prostacyclin prevents pulmonary endothelial cell apoptosis induced by cigarette smoke. Am J Respir Crit Care Med 175:676–685 31. Barbera JA, Peinado VI, Santos S, Ramirez J, Roca J, RodriguezRoisin R (2001) Reduced expression of endothelial nitric oxide synthase in pulmonary arteries of smokers. Am J Respir Crit Care Med 164:709–713 32. Kasahara Y, Tuder RM, Cool CD, Lynch DA, Flores SC, Voelkel NF (2001) Endothelial cell death and decreased expression of vascular endothelial growth factor and vascular endothelial growth factor receptor 2 in emphysema. Am J Respir Crit Care Med 163:737–744 33. Sakao S, Taraseviciene-Stewart L, Wood K, Cool CD, Voelkel NF (2006) Apoptosis of pulmonary microvascular endothelial cells stimulates vascular smooth muscle cell growth. Am J Physiol Lung Cell Mol Physiol 291:L362–L368 34. Eddahibi S, Chaouat A, Morrell N et al (2003) Polymorphism of the serotonin transporter gene and pulmonary hypertension in chronic obstructive pulmonary disease. Circulation 108:1839–1844 35. Eddahibi S, Chaouat A, Tu L et al (2006) Interleukin-6 gene polymorphism confers susceptibility to pulmonary hypertension in chronic obstructive pulmonary disease. Proc Am Thorac Soc 3:475–476
N.F. Voelkel et al. 36. Barnes PJ (2009) Role of HDAC2 in the pathophysiology of COPD. Annu Rev Physiol 71:451–464 37. Golpon HA, Coldren CD, Zamora MR et al (2004) Emphysema lung tissue gene expression profiling. Am J Respir Cell Mol Biol 31:595–600 38. Lee-Chiong T, Matthay RA (2003) Pulmonary hypertension and cor pulmonale in COPD. Semin Respir Crit Care Med 24:263–272 39. Murphy ML, Adamson J, Hutcheson F (1974) Left ventricular hypertrophy in patients with chronic bronchitis and emphysema. Ann Intern Med 81:307–313 40. Fluck DC, Chandrasekar RG, Gardner FV (1966) Left ventricular hypertrophy in chronic bronchitis. Br Heart J 28:92–97 41. Burghuber OC, Salzer-Muhar U, Gotz M (1988) Right ventricular contractility is preserved in patients with cystic fibrosis and pulmonary artery hypertension. Scand J Gastroenterol Suppl 143:93–98 42. MacNee W, Wathen CG, Hannan WJ, Flenley DC, Muir AL (1983) Effects of pirbuterol and sodium nitroprusside on pulmonary haemodynamics in hypoxic cor pulmonale. Br Med J (Clin Res Ed) 287:1169–1172 43. Rutten FH, Vonken EJ, Cramer MJ et al (2008) Cardiovascular magnetic resonance imaging to identify left-sided chronic heart failure in stable patients with chronic obstructive pulmonary disease. Am Heart J 156:506–512 44. Paudel B, Dhungel S, Paudel K, Pandru K, Paudel R (2008) When left ventricular failure complicates chronic obstructive pulmonary disease: hypoxia plays the major role. Kathmandu Univ Med J 6:37–40 45. Rizkallah J, Man SF, Sin DD (2009) Prevalence of pulmonary embolism in acute exacerbations of chronic obstructive pulmonary disease: a systematic review and meta-analysis. Chest 135(3):786–793 46. Lusuardi M, Garuti G, Massobrio M, Spagnolatti L, Bendinelli S (2008) Heart and lungs in COPD. Close friends in real life– separate in daily medical practice? Monaldi Arch Chest Dis 69:11–17 47. Mancini GB, Etminan M, Zhang B, Levesque LE, FitzGerald JM, Brophy JM (2006) Reduction of morbidity and mortality by statins, angiotensin-converting enzyme inhibitors, and angiotensin receptor blockers in patients with chronic obstructive pulmonary disease. J Am Coll Cardiol 47:2554–2560 48. van Gestel YRBM, Hoeks SE, Sin DD et al (2008) Impact of cardioselective beta-blockers on mortality in patients with chronic obstructive pulmonary disease and atherosclerosis. Am J Respir Crit Care Med 178:695–700 49. Macie C, Wooldrage K, Manfreda J, Anthonisen N (2008) Cardiovascular morbidity and the use of inhaled bronchodilators. Int J Chron Obstruct Pulm Dis 3:163–169 50. Holverda S, Bogaard HJ, Groepenhoff H, Postmus PE, Boonstra A, Vonk-Noordegraaf A (2008) Cardiopulmonary exercise test characteristics in patients with chronic obstructive pulmonary disease and associated pulmonary hypertension. Respiration 76:160–167 51. Holverda S, Rietema H, Bogaard HJ et al (2008) Acute effects of sildenafil on exercise pulmonary hemodynamics and capacity in patients with COPD. Pulm Pharmacol Ther 21:558–564 52. Rietema H, Holverda S, Bogaard HJ et al (2008) Sildenafil treatment in COPD does not affect stroke volume or exercise capacity. Eur Respir J 31:759–764 53. Lee TM, Chen CC, Shen HN, Chang NC (2009) Effects of pravastatin on functional capacity in patients with chronic obstructive pulmonary disease and pulmonary hypertension. Clin Sci (Lond) 116:497–505
Chapter 83
Pulmonary Hypertension Associated with Interstitial Lung Disease Mary E. Strek and Julian Solway
Abstract Interstitial lung diseases (ILDs) are a distinct type of chronic respiratory disorder that can result in pulmonary hypertension. There are numerous causes of ILD but all are characterized by dyspnea and abnormal lung function, with arterial oxygen desaturation occurring as the disease advances. Patients with idiopathic pulmonary fibrosis (IPF), a relentlessly progressive form of ILD, are particularly likely to develop pulmonary hypertension. Both chronic hypoxemia with subsequent pulmonary vasoconstriction and obliteration of the pulmonary vascular bed as a result of interstitial fibrosis have traditionally been considered the pathways by which pulmonary hypertension develops in ILD. Not all patients with pulmonary hypertension from ILD have a severe restrictive ventilatory deficit or pulmonary fibrosis, however, suggesting that other pathologic mechanisms may contribute to the development of pulmonary vascular disease and cor pulmonale in patients with ILD. Sarcoidosis, pulmonary Langerhans cell histiocytosis (PLCH), and lymphangioleiomyomatosis (LAM) are types of ILD that may directly involve the pulmonary vasculature, in addition to causing interstitial fibrosis. Collagen vascular diseases such as scleroderma may cause pulmonary hypertension as a result of progressive ILD or by direct pulmonary vascular involvement. In patients with chronic lung disease, worsening dyspnea and hypoxemia are often mistaken for progressive ILD rather than recognized as heralding the onset of pulmonary hypertension. Although physical findings, echocardiography results, and serum brain natriuretic peptide (BNP) levels can suggest the presence of pulmonary hypertension, right-sided heart catheterization remains the diagnostic gold standard. In this chapter, we review the current classification schemes of both pulmonary hypertension and ILD. The epidemiology of pulmonary hypertension in specific types of ILD is discussed. New information about the origin, pathogenesis, and pathologic findings in ILD-related pulmonary hypertension is described. Clinical manifestations and means of diagnosis M.E. Strek (*) Department of Medicine, University of Chicago, Chicago, IL 60637, USA e-mail:
[email protected] are reviewed. We end the chapter with a focus on current treatment options and prognosis. Keywords Interstitial lung disease • Sarcoidosis • Langerhans cell histiocytosis • Lymphangioleiomyomatosis • Idiopathic pulmonary fibrosis
1 Introduction Chronic lung disease is a major cause of pulmonary hypertension and subsequent right-sided heart failure or cor pulmonale. Interstitial lung diseases (ILDs) are a distinct type of chronic respiratory disorder that can result in pulmonary hypertension. There are numerous causes of ILD but all are characterized by dyspnea and abnormal lung function, with arterial oxygen desaturation occurring as the disease advances. Patients with idiopathic pulmonary fibrosis (IPF), a relentlessly progressive form of ILD, are particularly likely to develop pulmonary hypertension. Both chronic hypoxemia with subsequent pulmonary artery vasoconstriction and obliteration of the pulmonary vascular bed as a result of interstitial fibrosis have traditionally been considered the pathways by which pulmonary hypertension develops in ILD. Not all patients with pulmonary hypertension from ILD have a severe restrictive ventilatory deficit or pulmonary fibrosis, however, suggesting that other pathologic mechanisms may contribute to the development of pulmonary vascular disease and cor pulmonale in patients with ILD. Sarcoidosis, pulmonary Langerhans cell histiocytosis (PLCH), and lymphangioleiomyomatosis (LAM) are types of ILD that may directly involve the pulmonary vasculature, in addition to causing interstitial fibrosis. Collagen vascular diseases such as scleroderma may cause pulmonary hypertension as a result of progressive ILD or by direct pulmonary vascular involvement (see Chap. 70). In patients with chronic lung disease, worsening dyspnea and hypoxemia are often mistaken for progressive ILD rather than recognized as heralding the onset of pulmonary hypertension. Although physical findings, echocardiography
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results, and serum brain natriuretic peptide (BNP) levels can suggest the presence of pulmonary hypertension, right-sided heart catheterization remains the diagnostic gold standard. Supplemental oxygen for arterial oxygen desaturation and treatment of the underlying ILD have been the primary modes of therapy, but improved understanding of the pathogenic mechanisms of pulmonary hypertension in patients with ILD suggests there is a role for pulmonary artery vasodilators, as well as therapies that prevent inflammation and remodeling of the pulmonary vasculature. Pulmonary hypertension as a result of ILD, especially of IPF, portends a poor prognosis. In this chapter, we review the current classification schemes of both pulmonary hypertension and ILD. The epidemiology of pulmonary hypertension in specific types of ILD is discussed. New information about the origin, pathogenesis, and pathologic findings in ILD-related pulmonary hypertension is described. Clinical manifestations and means of diagnosis are reviewed. We end the chapter with a focus on current treatment options and prognosis.
2 Background Our understanding of pulmonary hypertension and its causes has improved dramatically in recent years leading to the development of effective therapies [1, 2]. Pulmonary hypertension is defined as a sustained mean pulmonary artery pressure (mPAP) greater than 25 mmHg at rest or greater than 30 mmHg with exercise [2]. The World Health Organization has classified pulmonary hypertension into five different categories with diseases grouped together on the basis of similar pathophysiologic changes, clinical presentation, and potential therapy [1]. Pulmonary arterial hypertension may be idiopathic, which was previously referred to as “primary” pulmonary hypertension. Since idiopathic pulmonary arterial hypertension shares similar features with pulmonary hypertension associated with collagen vascular disease, portal hypertension, congenital shunts, and drugs and toxins they are grouped together in the Dana Point 2008 “Updated Clinical Classification of Pulmonary Hypertension” (Table 1). Other major causes include left-sided heart disease, chronic lung disease, chronic thromboembolic disease, and a group of miscellaneous causes. Pulmonary hypertension associated with lung diseases and/or hypoxemia can result from a variety of chronic pulmonary disorders including chronic obstructive pulmonary disease, obstructive sleep apnea, and ILD [3]. The ILDs are a heterogeneous group of diseases that primarily affect the pulmonary interstitium [4]. These diseases result in varying degrees of inflammation and fibrosis of the lung parenchyma. They can be caused by occupational exposure to asbestos or silica dusts, a hypersensitivity response to environmental exposures, or collagen vascular diseases [5–7]. In many
M.E. Strek and J. Solway Table 1 Updated clinical classification of pulmonary hypertension (Dana Point, 2008) Pulmonary arterial hypertension Idiopathic, heritable, collagen vascular disease, congenital heart diseases, portal hypertension, drugs and toxins Pulmonary hypertension with left-sided heart disease Pulmonary hypertension associated with lung diseases and/or hypoxia Pulmonary hypertension due to chronic thrombotic and/or embolic disease Multifactorial Mechanisms Sarcoidosis, pulmonary Langerhans cell histiocytosis, lymphangioleiomyomatosis
cases, despite a thorough evaluation including surgical lung biopsy, the type or cause of the ILD remains unidentified. Recent advances in our understanding of the idiopathic ILDs has led to an international consensus statement on idiopathic interstitial pneumonias that suggests these be grouped according to clinical, radiographic, and pathologic patterns with a multidisciplinary approach to diagnosis [8]. The most common idiopathic ILD is IPF, which is characterized pathologically by usual interstitial pneumonia (UIP) with architectural destruction, fibroblastic foci, and honeycomb fibrosis. In addition to this specific pathologic criteria, characteristic high-resolution chest computed tomography (CT) scan findings in UIP obviate surgical lung biopsy in many cases [4, 9]. Nonspecific interstitial pneumonia (NSIP) is a pathologic pattern that is most often seen with collagen vascular disease or a hypersensitivity response to an environmental antigen, but it too can be idiopathic [10]. Patients with NSIP tend to be younger and have a better prognosis than those with IPF. There are numerous poorly understood inflammatory causes of ILD including sarcoidosis, which is a multisystem granulomatous disorder that most commonly involves the lungs [11]. PLCH results from proliferation of a specific population of dendritic cells in the small airways and lung parenchyma and is predominantly seen in cigarette smokers [12]. LAM is caused by mutations in tuberous sclerosis genes with smooth muscle proliferation in many compartments of the lung and a remarkable restriction to the female gender [13]. Sarcoidosis, PLCH, and LAM may directly involve the pulmonary vasculature, in addition to causing interstitial fibrosis and are grouped separately under the “miscellaneous” category in the classification scheme noted above (Table 1). Collagen vascular diseases, especially scleroderma, may directly involve the pulmonary arterial bed, resulting in a clinical picture very similar to that of idiopathic pulmonary arterial hypertension or result in pulmonary hypertension from ILD and hypoxemia [7, 14]. Some of these patients have ILD but disproportionately severe pulmonary hypertension, suggesting the connective tissue disease affects both the pulmonary interstitial and the vascular compartments [15, 16]. This is discussed in more detail in Chap. 70.
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The best studied populations with pulmonary hypertension complicating ILD are patients with IPF, scleroderma, and sarcoidosis [17, 18].
3 Epidemiology of Pulmonary Hypertension in ILD Pulmonary hypertension is a common complication of ILDs (Table 2). The apparent incidence and prevalence vary depending on the type and severity of ILD as well as the method used for diagnosis of the pulmonary hypertension. A retrospective analysis examined the incidence of pulmonary hypertension in 163 consecutively enrolled patients with either idiopathic or collagen vascular disease associated interstitial pneumonias [19]. Among the 78 cases of idiopathic interstitial pneumonia, 43 patients had IPF. Pulmonary hypertension defined by systolic pulmonary artery pressure (sPAP) of 40 mmHg or more on echocardiography was seen in 28% of the patients with idiopathic interstitial pneumonia and in 21% of the patients with collagen vascular disease associated ILD. In scleroderma ILD, pulmonary hypertension was noted in approximately 20% of the patients who underwent screening echocardiography in two separate studies [15, 16]. In IPF, the most serious form of ILD, sPAP greater than 35 mmHg was noted in 74 of 88 patients at presentation to the Mayo Clinic, after excluding 48 patients with IPF who also had left ventricular or valvular heart disease and those in whom pulmonary pressures could not be estimated [20]. A much lower number was noted in 78 unselected patients with IPF who were prospectively followed for up to 14 years [21]. In this study, right-sided heart catheterization data in 61 patients revealed 8.1% with mPAP greater than 25 mmHg. Among patients with advanced IPF, 32% of patients u ndergoing lung Table 2 Common causes of interstitial lung disease (ILD) and percentage with pulmonary hypertension Percentage with pulmonary hypertensiona Causes of ILD Screeningb Before transplant Idiopathic pulmonary fibrosis 8–84 32–46 Collagen vascular disease 20 Unknown related ILD Hypersensitivity Rare Unknown pneumonitis Asbestosis 6 Unknown Sarcoidosis 6 74 Pulmonary Langerhans 12 92 cell histiocytosis Lymphangioleiomyomatosis Rare Uncommon a Most of the data are from retrospective case series of selected populations. b These figures are mostly based on echocardiographic rather than rightsided heart catheterization determination of pulmonary artery pressures. IPF, idiopathic pulmonary fibrosis.
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transplant evaluation had elevated mPAP greater than 25 mmHg measured by right-sided heart catheterization [22]. In a larger series of patients with IPF undergoing right-sided heart catheterization for lung transplantation evaluation, 46.1% had mPAP of 25 mmHg or greater, with 9% having values of more than 40 mmHg [23]. In a small study of 16 men with asbestosis, 15 of whom were cigarette smokers, right-sided heart catheterization showed that only one patient had pulmonary hypertension using the currently accepted definition [24]. In a study of 48 ex-miners with suspected coal workers’ pneumoconiosis, 63% of whom were cigarette smokers, 21 patients had mPAP of 20 mmHg or more [25]. The presence of emphysema or progressive massive fibrosis was not higher in this group compared with those with mPAP lower than 20 mmHg. The prevalence of pulmonary hypertension diagnosed by echocardiography in patients with both diffuse parenchymal lung disease with fibrosis (ILD) and cigarette-smoking-related emphysema was 47% at diagnosis and 55% during follow-up [26]. Compared to patients with IPF, patients with combined pulmonary fibrosis and emphysema have an increased incidence of pulmonary hypertension, normal lung volumes and higher mortality. The incidence of pulmonary hypertension defined as sPAP of 40 mmHg or greater in a Japanese population of patients with sarcoidosis was 6% in over 200 patients screened by echocardiography [27]. A retrospective study of 363 patients with advanced sarcoidosis listed for lung transplantation from 1995 to 2002 showed that 73.8% of the patients had mPAP of 25 mmHg or greater measured by right-sided heart catheterization [28]. In a study comparing pulmonary hemodynamics measured by right-sided heart catheterization in patients with severe chronic respiratory disease listed for lung transplantation, patients with PLCH had more severe pulmonary hypertension compared with patients with IPF or COPD [29]. At a center with 123 patients with PLCH, 17 underwent echocardiography for suspected pulmonary hypertension or symptoms [30]. Fifteen patients had sPAP greater than 35 mmHg, with nine having sPAP greater than 50 mmHg. Of 39 patients who underwent lung transplantation for PLCH, pulmonary hypertension was observed in 92% of cases, with moderate to severe elevations (mPAP og 35 mmHg or greater) in 72.5% of cases [31]. LAM is characterized by proliferation of smooth muscle cells around bronchovascular structures; however, clinically significant pulmonary hypertension is not well described [3, 13].
4 Etiology and Pathogenic Mechanisms The mechanism by which the various types of ILD result in inflammation and fibrosis of the lung parenchyma is poorly understood [4, 8]. In patients with ILD, ventilation–perfusion heterogeneity from disordered lung architecture results in
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hypoxemia. That this and not diffusion limitation is the main contributor to the gas-exchange abnormalities has been confirmed in studies of patients with ILD using multiple inert gas techniques [32, 33]. During exercise, however, structural vascular changes may cause diffusion limitation and prevent redistribution of blood away from poorly ventilated lung units, causing worsening ventilation–perfusion inequality and lower PaO2. It is likely that in some patients with ILD, decreased respiratory muscle tone with sleep may result in nocturnal hypoxemia [34]. By whatever mechanism, chronic or sustained hypoxemia results in pulmonary artery vasoconstriction, which may persist in adults as a physiologic mechanism to preserve ventilation–perfusion homogeneity in the lung [35]. Chronic pulmonary artery vasoconstriction results in pulmonary hypertension. Acidosis from CO2 retention, which may occur in patients with severe pulmonary fibrosis, has an additive effect on increasing pulmonary pressures [35]. In animal models, chronic hypoxia results in “muscularization” of small pulmonary arteries that usually do not have a smooth muscle layer [36]. This creates pulmonary vascular resistance in vascular segments that were previously lowresistance units, thereby increasing pulmonary artery pressure. Compromise of the pulmonary vascular bed from inflammation and fibrosis of the lung parenchyma also contributes to increased pulmonary artery pressures and in some patients there is a correlation between the severity of the ILD and the presence of pulmonary hypertension [9, 21, 22]. Hypoxic pulmonary vasoconstriction and architectural destruction of the pulmonary capillary bed, however, are likely not the whole story. In some patients, pulmonary hypertension is more severe than the ILD and cannot be explained by the extent of pulmonary fibrosis or the degree of hypoxemia [37, 38]. In a study of patients with advanced IPF undergoing lung transplant evaluation, neither total lung capacity nor forced vital capacity (FVC) differed between those with pulmonary hypertension and those without pulmonary hypertension [22]. In addition, supplemental oxygen does not typically reverse the pulmonary hypertension once it is present. These observations have prompted numerous studies, some of which are discussed below, that suggest the following may contribute to pulmonary vascular disease in ILD independent of chronic hypoxemia: (1) inflammation and fibrosis of the pulmonary vascular bed with altered growth factor and cytokine expression, and (2) activation of the coagulation cascade which may contribute to a thrombotic angiopathy. In animal models, chronic hypoxia has been shown to produce an inflammatory response within the pulmonary vasculature with recruitment of inflammatory cells and mediators [36]. Gene expression patterns in patients with IPF who had estimated right ventricular systolic pressure greater than 30 mmHg on echocardiography revealed 19.8% of the genes known to be associated with the vascular endothelium were underexpressed, including vascular endothelial growth factor
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(VEGF) and platelet endothelial cell adhesion molecule, whereas genes that regulate inflammation such as phospholipase A2 were overexpressed [39]. Analysis of messenger RNA expression in lung tissue from patients with UIP or NSIP showed a decrease in the level of VEGF in the alveolar wall compared with normal lung tissue [40]. Vascular density was evaluated in open lung biopsies in patients with IPF, scleroderma-related ILD, and normal lung tissue. Reduced vessel density was noted in both scleroderma-related ILD (3.9%) and IPF (4.5%) compared with controls (20.4%) [41]. Expression of the angiostatic protein pigment epitheliumderived factor was increased in the fibroblastic foci from lungs of patients with IPF, whereas the VEGF level was decreased [42]. Ebina et al. confirmed that vascular density was reduced in areas of dense fibrosis in lung tissue from patients with UIP compared with normal controls [43]. This study suggested that vascular remodeling was heterogeneous since vascular density was significantly higher in areas of low-grade fibrosis than in control lungs. Plasma concentrations of the angiogenic cytokine interleukin-8 (IL-8) and endothelin-1 levels were increased in patients with idiopathic ILD compared with healthy volunteers [44]. Increased VEGF levels correlated with increasing chest CT scan fibrosis score and decreased FVC. Lung tissue from patients with IPF demonstrate higher levels of IL-8 than controls [45]. In lung tissue from patients with IPF, immunostains for endothelin-1 were prominent compared with minimal staining in control lungs [46]. In patients who also had pulmonary hypertension, staining was present in pulmonary vascular endothelial cells. Animal models of bleomycin lung injury, a form of ILD, suggest that thrombin expression may be increased, with thrombin inhibition shown to decrease lung collagen deposition [47]. Increased tissue factor expression and fibrin deposition were seen in lung tissue from patients with IPF and scleroderma-related ILD compared with control lung tissue [48]. Immunofluorescence staining for thrombomodulin, which exerts a protective effect against blood coagulation, was not seen in capillary endothelial cells from lung tissue in patients with UIP or fibrotic NSIP [40]. In a study of 19 patients thought to have IPF, immune-based microvascular injury was noted with patchy areas of capillary injury and elevated factor VIII levels in most patients, with antiphospholipid antibodies noted in 18 patients [49]. Although none of the patients were diagnosed with a collagen vascular disorder, many had serologic findings suggestive of collagen vascular disease and nine had NSIP on lung biopsy, a pathologic finding often seen in association with connective tissue disease, suggesting the antiphospholipid antibodies and pulmonary vascular disease were related to an autoimmune process in these patients. Sarcoidosis and PLCH result in pulmonary hypertension by a number of mechanisms, hence their separate classification from other chronic respiratory disorders that are associated with pulmonary hypertension. Severe, chronic
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ILD from sarcoidosis and PCLH can result in pulmonary fibrosis and hypoxemia. Additionally, there is evidence that sarcoidosis and PLCH can directly and primarily involve the pulmonary vasculature. In 40 autopsy cases of sarcoidosis, pulmonary vascular involvement was noted in all patients, even those who were clinically thought to lack lung involvement, and was noted at all levels from large elastic pulmonary arteries to venules [50]. In addition, compression of large blood vessels by fibrogranulomatous involvement of proximal lymph nodes was seen. In this study, the degree of pulmonary vascular involvement on pathological changes correlated with the degree of pulmonary parenchymal involvement. In a study of 22 patients with pulmonary hypertension from sarcoidosis, 15 patients had pulmonary fibrosis, whereas two had no radiographic evidence of pulmonary sarcoidosis [51]. Granulomatous vascular involvement with occlusive venopathy was noted in the five patients undergoing lung transplantation [51]. Short-term vasoresponsiveness to pulmonary vasodilators was noted in the majority of patients in a small study of moderate to severe pulmonary hypertension from sarcoidosis [52]. Both diastolic and systolic cardiac dysfunction can cause pulmonary hypertension from elevated left-sided heart pressures in patients with cardiac sarcoidosis [53]. Direct pulmonary vascular involvement of the muscular arteries and veins has been noted in PLCH [29, 31]. Left-sided heart disease may contribute to pulmonary hypertension in some patients, especially those with sarcoidosis. Elevated pulmonary capillary wedge pressures were seen in 16.1% of 48 patients with both IPF and pulmonary hypertension [54]. In 22 patients with IPF and mild to moderate pulmonary hypertension, enhanced images obtained by tissue Doppler echocardiography showed impaired right ventricular function, reversal of left ventricular diastolic filling to late diastole, and lower peak myocardial velocities in early systole compared with healthy controls, suggestive of impaired left ventricular diastolic function [55]. In many patients, pulmonary hypertension worsens over time and right-sided heart failure develops. Rapid clinical deterioration from right-sided heart failure has been noted in some patients once pulmonary hypertension is present. Right ventricular pressure overload can cause decreased left ventricular function through ventricular interdependence, decreasing cardiac output, and mixed venous oxygen saturation, resulting in severe hypoxemia and worsened pulmonary hypertension [56]. A fall in cardiac output in a patient with ILD has greater physiologic consequences than for a patient without chronic lung disease since the associated decrease in mixed venous oxygen saturation may cause a dramatic increase in arterial oxygen desaturation owing to ventilation–perfusion mismatch in the affected lung. In summary, hypoxic pulmonary vasoconstriction, destruction of the vascular bed from pulmonary fibrosis, vascular inflam-
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mation and remodeling, alteration of the coagulation cascade, and left-sided heart disease all may play a role in the development of pulmonary hypertension in patients with ILD.
5 Pathology Pulmonary vascular remodeling, with dilation of elastic pulmonary arteries, medial hypertrophy of elastic and muscular arteries, and right ventricular hypertrophy, is described in all forms of pulmonary hypertension [57]. Lung biopsies from patients with ILD frequently reveal pulmonary artery hypertrophy (Fig. 1). Pulmonary artery
Fig. 1 Lung biopsy in a patient with idiopathic pulmonary fibrosis and pulmonary hypertension. a Background of usual interstitial pneumonia with moderate interstitial fibrosis and a fibroblastic focus (thick arrow). Smaller branch of the pulmonary artery with mild to moderate medial hypertrophy (thin arrow) (hematoxylin–eosin stain, original ×100). b Background of microscopic honeycombing. Pulmonary artery (arrow) with marked medial hypertrophy and intimal fibrosis which significantly narrows the lumen (hematoxylin–eosin stain, original ×100). Photographs courtesy of Dr. Aliya Husain.
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medial and intimal thickening can be a nonspecific finding in ILD in the absence of clinically evident pulmonary hypertension [57]. In IPF, increased numbers of fibroblasts and myofibroblasts and increased extracellular matrix deposition result in advential thickening around pulmonary vessels [17]. Lung explants from 26 patients with IPF undergoing lung transplantation were examined for vascular abnormalities both in areas of dense fibrosis and in relatively preserved lung [58]. In the dense fibrotic areas, more than two thirds of the muscular pulmonary arteries had moderate or greater intimal fibrosis and medial hypertrophy. A similar number of pulmonary arterioles had moderate muscularization. The majority had moderate luminal narrowing by intimal fibrosis of the small pulmonary veins and venules. In architecturally preserved lung zones, occlusion of the pulmonary venules occurred in lung tissue from 17 of 26 patients, with associated findings suggestive of an occlusive venopathy. Plexiform lesions were not seen. In situ thrombosis has been described in small muscular pulmonary arteries consistent with the prothrombotic environment described above [17]. Sarcoidosis and PLCH both may directly involve pulmonary vessels. Granulomas were seen throughout the pulmonary vasculature from large pulmonary arteries to venules, with venous involvement more prominent than arterial involvement in one autopsy study of patients with sarcoidosis [50]. Granulomas were seen more prominently in the veins, with occlusive intimal fibrosis and recanalization resulting in an occlusive venopathy in a study of explanted lungs from patients undergoing lung transplantation for sarcoidosis [51]. No plexiform or thrombotic lesions were noted. In a study of 12 patients with severe pulmonary hypertension from PLCH undergoing lung transplantation, a proliferative arteriopathy with intimal fibrosis and medial hypertrophy with arterial obliteration was noted in 60% of the cases and intimal fibrosis and medial hypertrophy and obliteration of septal pulmonary veins were noted in 75% of patients [29]. In addition, venular obliteration, hemosiderosis, and capillary dilatation similar to veno-occlusive disease were present in one third of the patients. Thus, in striking contrast to the pathologic findings in pulmonary arterial hypertension that is idiopathic, when it is caused by ILD, plexiform lesions are not seen, whereas perivascular fibrosis and an occlusive venopathy may be noted.
6 Clinical Manifestation The development of pulmonary hypertension in patients with ILD is typically not recognized in the early stages, with clinical manifestations of pulmonary hypertension mistakenly attributed
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to the underlying ILD. Dyspnea, especially with exertion, is the first and most common symptom of both ILD and pulmonary hypertension. Patients with pulmonary hypertension may also report fatigue and exercise limitation as well as chest pain and palpitations. Complaints of weight gain, increased abdominal girth, or pedal edema suggest volume retention from right-sided heart dysfunction. Findings on physical examination include an elevated jugular venous pulsation from increased right atrial pressure, a right ventricular heave at the left lower sternal border, fixed splitting or a loud pulmonic component of the second heart sound, a right ventricular fourth heart sound, a holosystolic tricuspid regurgitation or early diastolic pulmonic regurgitation murmur, ascites, and lower-extremity edema (Table 3). Many of these findings do not occur until later in the course of the disease. Syncope is concerning for the development of severe pulmonary hypertension. New or worsening arterial oxygen desaturation, especially with exercise, should increase the suspicion of pulmonary hypertension in patients with ILD. The arterial oxygen saturation may be disproportionately reduced compared with the severity of the reduction in lung function. In patients with IPF undergoing lung transplant evaluation, those who had mPAP greater than 25 mmHg had a lower mean arterial oxygen saturation and decreased exercise capacity as measured on a standardized 6-min walk test, compared with those who did not have pulmonary hypertension, with similar total lung capacity and FVC in both groups [22]. In a retrospective study of patients with IPF and pulmonary hypertension from a registry of patients listed for lung transplant, need for supplemental oxygen was as an independent variable associated with pulmonary hypertension, whereas both increased age and carbon dioxide tension were risk factors for severe pulmonary hypertension [23]. Formal evaluation of exercise ability with the 6-min walk test may reveal a decrease in the previously measured walk distance, increased symptoms at the end of the walk, or new or worsened oxygen desaturation in patients who have developed pulmonary hypertension [59].
Table 3 Clinical manifestations of pulmonary hypertension Symptoms Dyspnea, especially with exertion Exercise limitation Syncope Physical examination Decreased arterial oxygen saturation Increased jugular venous pulsation Loud pulmonic component of the second heart sound Pedal edema Laboratory findings Increased serum brain natriuretic peptide level Enlarged pulmonary artery on chest radiograph Decreased diffusing capacity
83 Pulmonary Hypertension Associated with Interstitial Lung Disease
These findings were also noted in patients with pulmonary hypertension from sarcoidosis [28, 60]. Chest radiography can suggest the diagnosis of pulmonary hypertension but is not particularly sensitive. A chest radiograph finding of the right interlobar pulmonary artery diameter greater than 15 mm on the frontal view suggests pulmonary artery enlargement [37]. Attenuation of peripheral pulmonary vasculature and right ventricular enlargement may also be seen. Electrocardiography may show right-axis deviation and right ventricular hypertrophy. Pulmonary function tests may reveal a disproportionate reduction in the diffusing capacity in patients with ILD or sarcoidosis who develop pulmonary hypertension [54, 61]. Nathan et al. noted a modest association between mean diffusing capacity less than 30% of predicted and a twofold higher prevalence of pulmonary hypertension [54]. There was a trend toward higher prevalence and greater severity of pulmonary hypertension in those with mean FVC more than 70% of that predicted compared with those with FVC less than 40% of predicted again, suggesting that reduction in lung volumes may not correlate with the presence of pulmonary hypertension. In the study by Shorr et al., a low forced expiratory volume in 1 s was a risk factor for severe pulmonary hypertension, perhaps owing to concomitant airflow obstruction [23].
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IPF [62]. Zisman et al. then validated this screening method in a follow-up study of 60 patients with IPF [63]. They found that the estimated mPAP using this equation was within 5 mmHg of the mPAP measured by right-sided heart catheterization 72% of the time [63]. An estimated mPAP greater than 21 mmHg was associated with a sensitivity of 95% and a negative predictive value of 96% for mPAP greater than 25 mmHg measured by right-sided heart catheterization. Percent predicted diffusing capacity, but not FVC, correlated with the presence of pulmonary hypertension [62]. Chest CT scans findings that suggest the presence of pulmonary hypertension include increased size of the main pulmonary artery trunk compared with the aorta, increased pulmonary artery size compared with the accompanying airway, right ventricular enlargement, and leftward shift of the ventricular septum (Fig. 2). In a study of patients with advanced lung disease of many types, the sensitivity of the diameter of the main pulmonary artery 29 mm or greater for
7 Diagnosis Pulmonary hypertension in patients with ILD requires a high index of suspicion and is suggested by increasing dyspnea, worsening hypoxemia, and a disproportionate reduction in the diffusing capacity. Other causes such as left-sided heart disease, valvular heart disease, obstructive sleep apnea, pulmonary embolism, and portal hypertension must be excluded [1]. Pulmonary capillary hemangiomatosis and pulmonary venoocclusive disease can be mistaken for ILD-associated pulmonary hypertension. Nocturnal oximetry should be performed in those without evidence for hypoxemia at rest or with exercise. Chest radiographs, chest CT scans, echocardiograms and serum BNP levels all can suggest the diagnosis but the gold standard remains the measurement of pulmonary artery pressures by right-sided heart catheterization. None of these other diagnostic tests have demonstrated sufficient accuracy to preclude invasive and direct measurement of pulmonary pressures in the majority of patients in whom pulmonary hypertension is suspected to document the presence, assess the severity, and demonstrate improvement with treatment. Although individual measures of lung volumes cannot be used to screen for pulmonary hypertension, Zisman et al. used the ratio of percent predicted FVC to percent predicted diffusing capacity and room air resting pulse oximetry to develop an equation that predicted mPAP in patients with
Fig. 2 Chest computed tomography (CT) scan findings in a patient with IPF and pulmonary hypertension. a The chest CT scan shows a typical UIP pattern with peripheral reticular interstitial changes, traction bronchiectasis, and honeycombing (arrow). The main pulmonary artery trunk is larger than the aorta. b The same patient with a section through the lung bases. Peripheral pulmonary artery branches are larger than the accompanying airway (circle)
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pulmonary hypertension was 65.9% and increased to 85.7% when combined with the ratio of the diameter of the lobar artery to the diameter of the lobar bronchiole greater than 1 [64]. A recent study of 65 patients with advanced IPF, refuted the claim that pulmonary artery size on chest CT scan could predict the presence of pulmonary hypertension [65]. No significant correlation between main pulmonary artery diameter or the ratio of the main pulmonary artery diameter to aorta diameter and mPAP as measured by right-sided heart catheterization was found. In addition, the degree of pulmonary fibrosis on chest CT scan also did not correlate with pulmonary hypertension in this study. Transthoracic Doppler echocardiography is a noninvasive method to screen patients for pulmonary hypertension. Right ventricular size, wall thickness, and function can also be assessed [66]. If pulmonary outflow tract obstruction is not present, estimated right ventricular systolic pressure measured by echocardiography has been shown to correlate with sPAP measured invasively [67]. Transthoracic Doppler echocardiography detects regurgitation of blood backward across the tricuspid valve during right ventricular systole. The tricuspid regurgitant jet velocity is used to estimate the right ventricular pressure after the addition of an estimate of central venous pressure. Using a modification of the Bernoulli equation, the change in pressure equals 4 times the tricuspid regurgitant jet velocity squared [68]. A value of more than 3.0 m/s correlates reasonably well with elevated right-sided heart pressures measured by catheterization. Significant discordance is not uncommon and the absence of a tricuspid regurgitant jet in 20–30% of cases does not rule out pulmonary hypertension. In a cohort study of 374 lung transplant candidates with advanced lung disease, with a prevalence of pulmonary hypertension of 25%, estimation of the sPAP was possible in 166 patients (44%) [69]. The correlation between sPAP measured by cardiac catheterization and that estimated by echocardiography was good (r = 0.69, P 1,100 dyn s cm−5 Rup > 60%
+a PVR > 1,100 dyn s cm−5 Rup 20 mmHg) A field in the state of Minas Gerais, Brazil Systolic pulmonary artery pressure >40 mmHg on echocardiography; the results were confirmed with invasive haemodynamics measurements Systolic pulmonary artery pressure ranging from 40 to 126 mmHg with Doppler echocardiography
References [69] [151]
[150, 152]
[134]
[153] [142]
[141]
present in just five of the nine patients. These patients underwent contrast-enhanced multidetector-row computed tomography and an increased diameter of the pulmonary artery trunk, tapering of peripheral pulmonary arteries, and cardiac enlargement were observed (Figs. 6, 7). Lapa et al. [142] studied 65 patients followed up in the gastroenterology department for hepatosplenic manifestation of S. mansoni with transthoracic Doppler echocardiography. They found 12 patients with pulmonary artery systolic pressure above 40 mmHg. Thirteen of these patients underwent right-sided heart catheterization, which confirmed significant pulmonary hypertension in five patients (7.7%) (three had been classified as having precapillary pulmonary hypertension and two had postcapillary pulmonary hypertension). On the other hand, in a prospective study of sixty-five patients hospitalized with S. japonicum infection with hepatosplenic disease, only one patient had pulmonary hypertension [136]. The relatively uncommon appearance of severe S. japonicum infections may reflect the difference in the antigenicity between species. It is therefore difficult to estimate the real prevalence of pulmonary vascular diseases, particularly when we have to consider many factors, such as the range of clinical presentation, pathological involvement, difference in the antigenicity, and progress of the pathological changes, as well as the role of coinfection, current treatment, and control and multiple exposures. All these factors can contribute to the real prevalence. It is suggested from our unpublished current experimental observation that the changes in the pulmonary vasculature after Schistosoma infection are far more common but may not always be associated with significant increases in the total vascular resistance and may appear as benign pulmonary hypertension and clinical pulmonary hypertension. This, however, needs further investigation.
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Ghazwan Butrous and Angela P. Bandiera
8 The Clinical Presentations The early symptoms of acute schistosomiasis are usually nonspecific symptoms such as nocturnal fever, cough, dyspnea, myalgia, headache, and abdominal tenderness with eosinophilia, and lymphadenopathy. In some patients, chest radiography usually shows patchy or diffuse pulmonary infiltrates. These signs and symptoms appear 3–6 weeks after infection and they are referred to as Katayama syndrome [35, 143]. This most commonly affects older people who are heavily exposed to schistosomiasis for the first time. The condition is usually thought to be due to proinflammatory cytokines which may be related to Th-1 responses [35]. Diagnosis requires a high index of suspicion and, therefore, cannot be made by screening urine or feces for eggs. Serological testing may be required [144]. Chronic pulmonary disease is more common in endemic areas but there are no typical clinical distinguishing features and it may manifest itself as asthma, dyspnea on exertion, anemia, fatigue, weakness, cough, giddiness and fainting, palpitation, thoracic pain, pericardial pain, and hemoptysis. The electrocardiogram may show right ventricular hypertrophy and strain. Finally, the main branches of the pulmonary artery in some patients may become dilated and may develop large aneurysms (Figs. 6, 7). Eventually the right side of the heart hypertrophies and fails (cor pulmonale) [25, 145–148]. Chest radiography may show an enlarged pulmonary arterial trunk, right ventricular hypertrophy, and evidence of pleural effusion (Fig. 8). These clinical and radiological findings are
Fig. 7 The tomographic scan of a 51-year-old female with a pulmonary artery systolic pressure of 71 mmHg showing enlargement of the main pulmonary arteries
Fig. 8 Chest radiograph in a patient with World Health Organization class IV pulmonary hypertension secondary to schistosomiasis from Brazil showing severe right ventricular dilatation and enlargement of the pulmonary trunk with evidence of heart failure
Fig. 6 A 58-year-old woman who had hepatosplenic schistosomal disease and pulmonary artery systolic pressure of 68 mmHg. A multidetector-row tomographic scan of the thorax showing dilated pulmonary arteries and showing abrupt reduction in the diameter of the peripheral pulmonary vasculature
similar to those associated with other causes of pulmonary hypertension [149]. In most reported studies, the majority of the published data indicate that the majority of patients with mild pulmonary hypertension do not develop severe right-sided heart failure [53, 150]. One series from two endemic area in Brazil showed that in 141 patients with hepatosplenic schistosomiasis, pulmonary hypertension occurred in 13% and cor pulmonale in 2.1% [53]. In our experience, particularly in the patients
91 Pulmonary Hypertension Due to Schistosomiasis
referred to specialized pulmonary hypertension centers in endemic areas, 62% of these patients are in New York Heart Association functional class III or IV with an average right ventricular systolic pressure of 83.48 ± 24.61 mmHg.
9 M anagement of Pulmonary Hypertension Secondary to Schistosomiasis There has been no controlled clinical trial on the use of pulmonary-hypertension-specific drugs in patients with schistosomiasis-associated pulmonary hypertension. In our experience a phosphodiesterase-5 inhibitor (sildenafil) was used in an open-label study in 85 patients (unpublished data), resulting in improvement in exercise capacity as assessed with the 6-min walk test. There were a few cases with successful treatment with the endothelin receptor antagonist bosentan. Affordable safe and efficacious medications are needed to help this group of patients, and thus double-blind clinical trials are needed. Antischistosomal therapy usually has no effect, but it is given to prevent further progression of disease. However, Bouree et al. reported a remission after treatment with praziquantel in patients with S. haematobium pulmonary hypertension [64]. Pulmonary embolisms of dead worms may follow treatment for schistosomiasis, with an abrupt increase in pulmonary pressure and development of acute cor pulmonale [53].
10 The Issues and Problems We can only speculate on the number of patients affected by schistosomiasis-associated pulmonary vascular disease as the real prevalence is not yet known because more than 200 million people are infected worldwide. We can nevertheless consider that schistosomiasis is the major cause of pulmonary hypertension worldwide, and it may even be the leading cause of pulmonary hypertension in endemic areas. A concerted effort is therefore needed in various parts of the word owing to the diversity of the parasite itself and the many factors that are associated with its pathophysiological effects as described in this chapter. The apparent role of inflammation, as discussed in this chapter, can be useful as a good model to assess and understand more thoroughly the role of inflammation; not only in schistosomiasis-associated pulmonary vascular disease but also in general. The clinical profile, pattern, and natural history of the development of pulmonary hypertension need further evaluation. Recent advances in the treatment of pulmonary hypertension have not been implemented in the management of patients with pulmonary hypertension secondary to schistosomiasis. Therefore, the need to develop an inexpensive and affordable treatment warrants further attention.
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Chapter 92
Pulmonary Hypertension Due to Capillary Hemangiomatosis Mourad Toporsian, David H. Roberts, and S. Ananth Karumanchi
Abstract The pulmonary circulation is a high-compliance, low-resistance vascular bed with enormous volume reserve capacity. These characteristics allow it to accommodate the entire cardiac output while being subjected to a low mean pulmonary artery pressure (mPAP) of 25 mmHg. Elevations in mPAP beyond 25 mmHg at rest or 30 mmHg during exercise are signs of pulmonary hypertension (PH), a devastating disorder that can affect individuals of all ages worldwide. Given the diverse pathophysiological manifestations, causes, and clinical management strategies of PH, it has been divided into five categories on the basis of the modified World Health Organization (WHO) Evian classification. Pulmonary arterial hypertension (PAH), or WHO group 1, consists of all forms of PH which are of unknown cause and involve extensive arteriopathy or loss/remodeling of the pulmonary arterial vasculature. This chapter will focus on an extremely rare form of PAH referred to as pulmonary capillary hemangiomatosis (PCH). Unlike other forms of PAH where changes in the pulmonary vasculature are limited solely to the arteries, PCH is a very intriguing disease of uncontrolled endothelial cell growth that although specific to the lung involves proliferation of capillary-sized blood vessels. Keywords Pulmonary arteriopathy • Rare disease • Endo thelial cell growth • Precapillary arterioles • Capillary-sized blood vessel
1 Introduction The pulmonary circulation is a high-compliance, lowresistance vascular bed with enormous volume reserve capacity. These characteristics allow it to accommodate the entire cardiac output while being subjected to a low mean pulmonary artery pressure (mPAP) of 25 mmHg. Elevations M. Toporsian (*) Beth Israel Deaconess Medical Center, 330 Brookline Avenue, 02215 Boston, MA, USA e-mail:
[email protected] in mPAP beyond 25 mmHg at rest or 30 mmHg during exercise are signs of pulmonary hypertension (PH), a devastating disorder that can affect individuals of all ages worldwide. Given the diverse pathophysiological manifestations, causes, and clinical management strategies of PH, it has been divided into five categories on the basis of the modified World Health Organization (WHO) Evian classification (reviewed in detail in Chap. 65). Pulmonary arterial hypertension (PAH), or WHO group 1, consists of all forms of PH which are of unknown cause and involve extensive arteriopathy or loss/remodeling of the pulmonary arterial vasculature. This chapter will focus on an extremely rare form of PAH referred to as pulmonary capillary hemangiomatosis (PCH). Unlike other forms of PAH where changes in the pulmonary vasculature are limited solely to the arteries, PCH is a very intriguing disease of uncontrolled endothelial cell growth that although specific to the lung involves proliferation of capillary-sized blood vessels.
2 Historical Perspective Three decades ago, PCH was first described by Wagenvoort et al. in a 71-year-old woman with progressive dyspnea, hemoptysis, and hemorrhagic pleural effusions [1]. They described this new clinical entity as an angiomatous growth with distinctive atypical proliferation of capillary-like channels in the lung tissue. PCH is now characterized as a diffuse proliferation of capillaries that can form glomeruloid nodules and project into pulmonary veins, arteries, interstitium, and, albeit less commonly, the airways [2, 3]. Similar to what occurs in other types of PAH, intimal thickening and medial hypertrophy of small pulmonary arteries are present, contributing to elevated pulmonary vascular resistance and mPAP. Since its initial description, PCH has been sporadically described in the literature, with fewer than 50 cases reported to date. Given the lack of awareness and difficulty in diagnosis, most cases have been discovered after death, suggesting that the prevalence of PCH may be underestimated and the mechanisms underlying its pathogenesis understudied.
J.X.-J. Yuan et al. (eds.), Textbook of Pulmonary Vascular Disease, DOI 10.1007/978-0-387-87429-6_92, © Springer Science+Business Media, LLC 2011
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3 Clinical Features and Pathology PCH typically affects young adults (age range 6–71 years), has a very poor prognosis, and often leads to death. Patients usually present with dyspnea, cough, fatigue, weight loss, and abnormal pulmonary function tests, and have a median survival of 3 years from initial clinical discovery. Although the cause of this disease remains unknown, a hereditary form was reported by Langleben et al. [4], suggesting a genetic susceptibility. However, PCH has not been recognized as a disease of congenital onset, and it is unclear if this hereditary form of the disease is the same as the one seen in adults [5]. PCH often manifests itself as PH and hemoptysis leading to right ventricular hypertrophy and failure [6, 7]. To date, only one case without PH has been reported [8]. In fact, PCH is classified as PAH associated with significant venous or capillary involvement [9]. Consistent with the prevalence of PAH being more common in females than in males, oral contraceptive use has been proposed as an important risk factor for this condition [10]. It is believed that the mechanical obstruction of blood flow in small venules and veins by invading capillaries leads to increased upstream pulmonary artery pressure and contributes to the observed PH. Patients exhibit normal or low pulmonary capillary wedge pressures (PCWP) [6], as this parameter assesses the pressure in the largest pulmonary veins and the left atrium [11], structures that are not affected in PCH. This criterion is helpful in the clinical differential diagnosis as a normal PCWP eliminates the possibility of other abnormalities such as obstructed or stenotic larger pulmonary veins, left atrial myxoma, mitral stenosis, or left ventricular failure. PCH has been described as a slowly progressive neoplasm or angioproliferative defect of the lung. Transbronchial biopsy of the lung may often reveal nonspecific hemosiderosis. Lung biopsies using video-assisted thoracic surgery are needed to make the diagnosis. The most distinctive histologic feature of PCH is proliferation of capillary channels
Fig. 1 (a) Lung biopsy specimen (hematoxylin and eosin stain) from a 44-year-old patient with pulmonary capillary hemangiomatosis (PCH) showing capillary proliferation within the alveolar septae and interstitium. (b) CD31 staining of the lung tissue obtained (from the
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within the alveolar walls. Immunohistochemical studies showing CD31 (platelet/endothelial cell adhesion molecule 1) and factor VIII related antigen staining of PCH/PM = pulmonary microvasculopathy lungs have confirmed that endothelial cells are the cells of origin that invade the pulmonary vasculature, interstitium, and airways (Fig. 1a, b). Highresolution CT scans typically show a diffuse ground-glass pattern and septal thickening (Fig. 1c). The proliferation of at least two layers of thin-walled alveolar capillary blood vessels with infiltration of these structures is diagnostic [2]. Invasion of the alveolar septa and bronchioles by proliferating endothelial cells disrupts gas exchange by reducing the surface area and the diffusion capacity of the lung. Muscularization and concomitant narrowing of pulmonary arteries coupled with the obstruction of veins compromise arterial supply and venous drainage, respectively. These changes result in PH and are accompanied by recurrent pulmonary hemorrhage, scar formation, and thrombosis leading to vascular obliteration. Moreover, there is compensatory redistribution of ventilation/perfusion from abnormal to less affected areas of the lung. Restrictive remodeling as a consequence of endothelial cell invasion of the pulmonary vasculature and interstitium stiffens the lung, compromising inspiratory capacity and increasing lung collapse during expiration. Although PCH is a non-hypoxia-related form of PH, chronic hypoxia and metabolic acidosis, and their associated sequelae, are inevitably a consequence.
3.1 Pulmonary Veno-occlusive Disease The other form of PAH that is associated with significant venous involvement is pulmonary veno-occlusive disease (PVOD). The hemodynamics of PVOD and PCH are the consequence of a widespread vascular obstructive process. However, unlike in PCH, this obstruction originates in the
same subject described in a) confirms capillary proliferation. (c) Representative high-resolution CT scan image (from the subject with PCH described in a) showing ground-glass opacities and thickening of interlobular septa
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pulmonary venules and small veins in PVOD. The consequential increase in capillary hydrostatic pressure may exceed the osmotic pressure of blood, leading to pulmonary edema as well as engorgement and dilatation of the subpleural and interlobular septal lymphatic channels. Histologically, PVOD is characterized by intimal fibrosis that occludes the pulmonary veins. These venous lesions are accompanied by looplike dilatations of the capillary bed, leading to edema and dilated lymphatic spaces in the interlobular septa [12]. Secondary remodeling of the lung parenchyma and of the pulmonary arteries are caused by increased intravascular pressure due to obstructed venous flow. In contrast, these secondary changes are due to well-circumscribed proliferative capillary lesions in PCH. PVOD and PCH have with very similar signs and symptoms, but the presence of radiologic septal lines primarily in venous structures is indicative of PVOD [13].
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5.2 Obesity A link between obesity and PCH has been previously proposed, yet a direct causal relationship remains controversial. Recently, an autopsy study of 76 patients revealed histologic features of PH, particularly venous hypertension, and PCH in 72% of obese subjects. PCH was observed in 50% of morbidly obese subjects [19]. The authors posit that the pulmonary capillary proliferation in the obese is most likely related to chronic hypoxia and left ventricular dysfunction. Interestingly, however, obesity is also associated with increased levels of inflammatory cytokines and immune activation, suggesting that persistent and chronic inflammation may indeed play a role in PCH.
6 Management 4 Differential Diagnosis Although PCH specifically involves the invasion of the pulmonary vasculature and parenchyma by proliferating capillaries, this disorder is usually misdiagnosed as the more common microvasculopathies such as PVOD (see Chap. 80) or idiopathic PAH. PCH is also to be differentiated from cavernous hemangiomatosis or pulmonary angiomatosis, which typically involve vessels of larger caliber [14]. Other disorders considered as part of the differential diagnosis include usual interstitial pneumonitis, recurrent pulmonary thromboembolism, and idiopathic pulmonary hemosiderosis [15]. Definitive diagnosis of PCH usually requires histological examination of the lung tissue (often at autopsy), and unsuspected incidental findings of PCH have been found after death in patients without clinical disease.
5 Risk Factors 5.1 Infection and Immunity An immune-mediated cause has been suggested owing to the sporadic occurrence of PCH. Moreover, although most cases are idiopathic, PCH has also been described in patients with autoimmune diseases such as systemic lupus erythematosus [16], scleroderma, Takayasu arteritis, and Kartagener syndrome [15, 17]. Recently, a case report showing the occurrence of de novo PCH in a 45-year-old woman 3 months after bilateral lung transplantation suggests that persistent infection or inflammation may be an inciting factor in the dysregulated angiogenesis [18].
Given the paucity of clinical cases, difficult diagnosis, and unknown cause of PCH, treatments are on an experimental basis and available as case reports. By far, the best outcomes are currently afforded by pneumonectomy [6], heart-lung transplantation [20], or bilateral lung transplantation [21], which may be curative. To this end, pharmacological interventions are considered supportive until surgery. In some cases, immunosuppression with a-interferon [17, 22] has shown some promise whereas, in others, antiangiogenic therapy such as doxycycline has been attempted [23]. Although corticosteroids have been used in this disorder, they have not been found to be of significant benefit. Interestingly, although it is widely accepted that vasodilator therapy with prostacyclin or calcium channel blockers can improve pulmonary hypertensive arteriopathy, selective pulmonary vasodilators such as these medications can cause life-threatening pulmonary edema in patients with pulmonary postcapillary vasculopathies such as PCH and PVOD, and are therefore contraindicated [17, 24, 25]. There are no data available regarding a potential use of several recently approved antiangiogenic agents, such as bevacizumab [anti-vascular endothelial growth factor (VEGF) antibody] and rapamycin (mammalian target of rapamycin inhibitor), in PCH therapy.
7 Mechanisms of Disease 7.1 Hypoxia Adaptation to hypoxia by capillary proliferation has been proposed as a possible mechanism in the development of PCH [3]. This concept is supported by our understanding of
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the mechanisms involved in other diseases characterized by the proliferation of normal capillaries, including childhood hemangiomas, angiofibromas and psoriasis. Potent hypoxiainducible factors, such as VEGF, basic fibroblast growth factor (bFGF), and platelet-derived growth factor (PDGF), have been shown to play prominent roles in the onset and progression of these diseases [26]. Although these factors have been implicated in the pathogenesis of PCH to differing degrees, it remains to be clarified if concomitant aberrant changes in endothelial oxygen sensing and responses contribute to the onset of PCH. Other proposed mechanisms for the pathogenesis of PCH speculate that the lesion is a hamartoma [27] or a low-grade vascular neoplasm [28]. However, the typical histological findings of diffuse vascular proliferation throughout the lung do not lend support to these hypotheses.
7.2 A ngiogenic Factors (VEGF, bFGF, and PDGF) VEGF is an endothelial-cell-specific mitogen. It promotes angiogenesis via its interactions with two high-affinity receptor tyrosine kinases, VEGF receptors 1 (fms-like tyrosine kinase 1, Flt-1) and 2 (KDR in humans, Flk-1 in mice) [29]. Recent studies have demonstrated alternatively spliced variants of Flt-1, resulting in the synthesis and secretion of an endogenous soluble form, termed sFlt-1, that can bind VEGF and inhibit its angiogenic properties. sFlt-1 can also bind placental growth factor, another member of the VEGF family [30]. Angiogenesis is necessary for normal alveolarization during postnatal lung growth and development [31]. Treatment of newborn rats with a single dose of the VEGF receptor antagonist SU5416 has been shown to reduce lung vascular density, alveolarization, weight, and cause PAH [32]. These effects persist well into adulthood and suggest that the inhibition of lung vascular development during a critical period of postnatal lung growth impairs alveolarization. These findings suggest an intimate cross talk between pulmonary epithelial and vascular endothelial cells, which is at least partly mediated by VEGF. The juxtaposition of the capillary endothelium with alveolar epithelial cells suggests the possibility that PCH may be an epithelial defect related to excessive VEGF secretion. This would be consistent with the lung-specific manifestations of PCH. bFGF belongs to a family of heparin-binding polypeptides and shows multiple functions, including cell proliferation, differentiation, survival, and motility. In particular, it is one of the most potent angiogenic factors, providing a basis for its potential role in PCH. In a special case report by Ginns et al. [23], an elevated urinary bFGF level was observed in a 20-year-old man with PCH. This patient also displayed atypical endotheliomatosis, suggesting the potential efficacy of a matrix metalloprotease (MMP) inhibitor such as doxycycline.
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Interestingly, the use of doxycycline led to a complete remission and normalization of abnormally high bFGF levels. Whether the elevated bFGF levels were specific to the observed atypical endotheliomatosis remains unknown. PDGF is a heterodimeric molecule that is composed of two similar polypeptides (A and B). It plays a central role in angiogenesis and acts as a potent mitogen for pulmonary artery smooth muscle cells via its tyrosine kinase receptor (PDGFR). PDGF and its downstream signaling has been shown to be upregulated in hypoxia- and monocrotalineinduced PH, as well as in lung homogenates of patients with PAH [33–35]. The PDGFR-b antagonist imatinib mesylate, currently used to treat chronic myelogenous leukemia, reverses PAH in these animal models of disease and has also recently shown promise as a new treatment in humans with PAH. Recent tissue microarray analyses have demonstrated increased levels of PDGF-B and PDGFR-b in lungs of patients with PCH [12]. This suggests a common link between the vasculopathy seen in PCH patients and that seen in patients with other forms of PAH. PDGF also plays an important role in the recruitment of pericytes into newly formed blood vessels, regulatory cells involved in the promotion of angiogenesis. The recent finding of prominent pericytic components in the nodules of PCH patients suggests a potential causal role for PDGF-induced excessive angiogenesis in PCH as well as its potential value as a cellular target to induce the regression of capillary ingrowths.
7.3 Matrix Proteases The extracellular matrix (ECM) is a reservoir of growth factors. These factors are released by matrix protease-mediated ECM degradation, which, in turn, promotes cellular invasiveness through the ability of these factors to enhance the expression and activity of matrix-degrading MMP-2 and MMP-9 [36]. Both these MMPs play a pivotal role in endothelial cell invasion and angiogenesis. As mentioned previously, the MMP inhibitor doxycycline has been used successfully in one case of PCH, suggesting that a better understanding of specific MMPs involved in this disease may pave the way to effective therapy [23]. Recent findings in lungs of patients with PCH suggest more biochemical similarities between this disease and other forms of PH. For instance, increased numbers of mast cells, as measured by increased levels of CD117 staining [12], have been documented in lungs of PCH patients. Tryptase is a proangiogenic component of mast cell granules that can induce endothelial cells to form tubelike structures, degrade the ECM, and permits capillary invasion into the pulmonary interstitium. Expression of this factor is increased in tumors and it is involved in tumor progression via its effects on angiogenesis. Its potential role in PCH remains to be elucidated.
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7.4 Endothelial Nitric Oxide Synthase Reduced nitric oxide (NO) bioavailability in the pulmonary vasculature is generally observed in animal models of PAH and in patients with this disease. It is believed that reduced endothelial NO synthase (eNOS) expression and/or its abnormal activation are contributing factors. A report by Kradin et al. [37] demonstrated that patients with PCH have reduced eNOS levels in abnormal capillary vessels. This observation was exclusive to PCH patients displaying pulmonary arterial hypertensive arteriopathy, suggesting that decreased eNOS expression may be a causal factor. Although NO is a very potent vasodilator and likely involved in the regulation of pulmonary vascular tone, its specific role in the prevention of PAH remains to be elucidated. eNOS null mice do not spontaneously develop PH, although their exposure to hypoxia results in an accelerated and severe form of the disease [38]. It is likely that additional factors acting in concert are necessary in PAH. Increased oxidative stress has been shown in numerous animal models of PH and in humans with this disease. Interestingly, the failure of eNOS to couple oxygen to l-arginine metabolism results in an increased amount of eNOS-derived O2•−, and reduced NO release [39]. The enzyme is thus rendered uncoupled and this condition has quickly emerged as an important mechanism in numerous vasculopathies, including PAH [40–43]. Further studies are needed to evaluate a role for eNOS uncoupling in the pathogenesis of PH patients due to a variety of causes, including PCH [44].
7.5 Osler–Rendu–Weber Syndrome Osler–Rendu–Weber syndrome, or hereditary hemorrhagic telangiectasia (HHT), is an autosomal dominant vascular dysplasia characterized by dilated blood vessels, telangiectases, and arteriovenous malformations [45]. Haploinsufficiency in endoglin (ENG) and activin-like kinase 1 receptor (ACVLR1) genes is the underlying cause of HHT type 1 [46] and type 2 [47], respectively. Recently, mutations in the ACVRL1 gene have been reported in patients presenting with PAH [48–50]. The ACVLR1 gene encodes the activin-like kinase 1 (ALK1) receptor, a type I receptor of the transforming growth factor b (TGF-b) superfamily that is mainly expressed in the vascular endothelium and particularly abundant in pulmonary blood vessels. Functionally, ALK1 acts via Smad1/Smad5/Smad8 [51] and regulates endothelial cell proliferation and migration in vitro [52], and angiogenesis in vivo [53, 54], in response to TGF-b1. More recently, bone morphogenetic protein (BMP) 9 and 10 have been identified as high-affinity ligands for ALK1 which in conjunction with BMP receptor 2 (BMPR2) can regulate BMP signaling in endothelial cells [55]. These findings underscore
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the importance of ALK1 in BMP-mediated endothelial cell function and further accentuate its potential involvement in PAH associated with abnormal BMPR2 signaling. However, it is still not well understood how loss of BMPR2 signaling leads to vascular remodeling exclusively in the lung. BMPR2 mutation has also been described for a patient suffering from PVOD, suggesting a close pathogenetic link between idiopathic PAH and PVOD [56]. A recent case report showed PCH arising in a patient with HHT [57]. Immunohistochemical staining for eNOS was also lower in the affected capillary endothelial cells of PCH compared with normal capillary endothelium. We have demonstrated that human and mouse endoglin-deficient (HHT mouse) endothelial cells and tissues have reduced eNOS levels [58]. Endoglin is an endothelial cell-surface glycoprotein and coreceptor for various ligands of the TGF-b superfamily. It resides in caveolae, where it associates with eNOS and promotes its normal activation. Endoglin-deficient endothelial cells display uncoupled eNOS activity [58]. Endoglin is known to regulate angiogenesis [59–61], local vascular tone, and TGF-b and BMP receptor signaling via ALK1, suggesting a critical role for endoglin in endothelial cell function [55] that is potentially impaired not only in HHT but also in PAH and PCH.
8 Conclusion As in other forms of hemangiomatosis, dysregulated angiogenesis significantly contributes to PCH pathogenesis. Aberrant adaptation to hypoxia and chronic infection/inflammation leading to overexpression of angiogenic factors such as VEGF, TGF-b, and bFGF signaling are likely key mechanisms in PCH pathogenesis. A common signaling defect may be an underlying cause of PAH and HHT, providing valuable clues to our understanding of PCH, a rare but devastating disorder. Future work on identifying new genes responsible for this disorder in the hereditary form may shed clues to the pathogenesis of this disease. It is imperative to create new animal models that mimic the human condition to develop new treatments for this devastating disorder.
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Chapter 93
Molecular Basis of Right Ventricular Hypertrophy and Failure in Pulmonary Vascular Disease Yuichiro J. Suzuki
Abstract Right ventricular hypertrophy and right heart failure occur in patients with pulmonary hypertension. The mechanisms of left ventricular hypertrophy are well understood, however, information on right ventricular hypertrophy is limited. Studies of embryonic and adult hearts provided evidence that the molecular signaling mechanisms are different for right and left ventricles. Thus, the development of therapeutic strategies specific for the right heart is needed. This chapter summarizes current understanding of cell growth signaling mechanisms in the right ventricle. Keywords Pulmonary hypertension • Right heart failure • Right ventricle • Right ventricular hypertrophy • Signal transduction
1 Introduction to Right Ventricular Hypertrophy and Failure in Pulmonary Vascular Disease In response to pulmonary vascular diseases in which the pulmonary vascular resistance increases, the right ventricle is subjected to increased load or volume, which ultimately has lethal consequences. The term “cor pulmonale,” which was originally introduced by Paul D. White in 1931 [1], has been used to describe these conditions. “Cor pulmonale” is now defined as an alteration in the structure and function of the right ventricle, resulting from diseases affecting the function and/or structure of the lung, except when these pulmonary alterations are the result of diseases that primarily affect the left side of the heart [2]. Acute cor pulmonale, which can occur in response to pulmonary embolism, results in dilation of the right ventricle, whereas chronic cor pulmonale, which is associated with chronic obstructive pulmonary disease and
Y.J. Suzuki (*) Department of Pharmacology, Georgetown University Medical Center, 3900 Reservoir Road NW, 20057 Washington, DC,USA e-mail:
[email protected] idiopathic pulmonary arterial hypertension, exhibits right ventricular hypertrophy. As right ventricular failure is the major cause of death among patients with pulmonary hypertension, understanding the molecular basis of how right ventricular failure develops is highly important. Knowledge of the right ventricle, however, is limited. Many of the molecular mechanisms of cardiac hypertrophy and failure have been studied in the left ventricle, and it is not clear how many of the mechanisms of left and right ventricular hypertrophy and failure are common. Presently, no strategies specific for the right side of the heart are available to treat right-sided heart failure, and it has become apparent that agents to treat left-sided heart failure are not necessarily effective in right-sided heart failure. Although the development of therapeutic strategies to treat right-sided heart failure is needed, information on the mechanism of right cardiac hypertrophy and failure is surprisingly lacking. In 2005, National Heart, Lung, and Blood Institute convened a meeting of the Working Group on Cellular and Molecular Mechanisms of Right Heart Failure to foster understanding of the mechanism and the development of new treatments for right-sided heart failure (http://www.nhlbi.nih.gov/meetings/ workshops/right-heart.htm; see also [3]). There is a strong need for the medical community to learn about known mechanisms of right ventricular signaling to foster basic and clinical research on right ventricle to identify molecular targets to develop agents for treating right-sided heart failure. Table 1 lists the learning objectives of this chapter. By definition, left and right ventricles are quite different, as the left ventricle serves the systemic circulation, whereas the right ventricle serves the pulmonary circulation, with markedly lower blood pressure (Table 2). Further, as described in the latter part of this chapter, developmentally, the right and left ventricles are derived from different precursors. This chapter first covers known molecular mechanisms of cardiac hypertrophy and failure, in general, and then provides information on the differences in right and left ventricles. This chapter will focus on the molecular and cellular basis of the right side of the heart. For information on right ventricular anatomy and physiology, the reader should refer to some recent review articles [3–8].
J.X.-J. Yuan et al. (eds.), Textbook of Pulmonary Vascular Disease, DOI 10.1007/978-0-387-87429-6_93, © Springer Science+Business Media, LLC 2011
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Table 1 Learning objectives 1. 2. 3. 4. 5. 6.
Know the definition of “ventricular hypertrophy and failure” Know the importance of “ventricular hypertrophy and failure” in pulmonary vascular disease Appreciate that the biology of right side of the adult heart is not well understood Know the molecular mechanisms of right ventricular development Know the structural and functional differences between adult right and left ventricles Know the molecular mechanisms of right ventricular hypertrophy and failure
Table 2 Right ventricle versus left ventricle 1. 2. 3. 4. 5.
The right ventricle pumps blood into the low-pressure pulmonary vasculature The mechanical afterload in the right ventricle is significantly smaller The free wall of the right ventricle is much thinner The action potential duration is shorter in the right ventricle There is a higher density of transient outward potassium current in the right ventricle
2 Molecular Basis of Cardiac Hypertrophy and Failure In response to increased vascular resistance, the heart ventricle (right or left) initially adapts by changing its structure. Increased systemic vascular resistance influences the left ventricle, and increased pulmonary vascular resistance influences the right ventricle. Chronic pressure overload by such diseases such as pulmonary and systemic hypertension often initially results in adaptation via cardiac hypertrophy. The term “hypertrophy” is defined by an enlarged organ or part of the body or overgrowth of an organ or part of the body owing to the increased size of the constituent cells. Exercise training can cause physiological hypertrophy, which is a temporary increase in the size of an organ or part to provide for a natural increase of function. Pressure overload initially leads to concentric cardiac hypertrophy by increasing the ventricular wall thickness. This type of hypertrophy occurs owing to increased width of cardiac muscle by increasing sarcomere units in parallel, perhaps designed to increase the force of contraction to overcome increased vascular resistance. There has been debate as to whether this type of hypertrophy is a truly adaptive event or a pathologic event. Clinically observed concentric hypertrophy is somewhat different from exercisetraining-induced physiologic hypertrophy. In concentrically hypertrophied cardiac muscle cells, only the width is increased, whereas physiologic hypertrophy is associated with increases in both the width and the length of myocytes. Moreover, physiologic hypertrophy is reversible according to the need for the muscle contraction force. For example,
Fig. 1 Cardiac hypertrophy and dilation
stopping exercise training results in a return of the ventricular thickness to that before the onset of training. In contrast, clinical concentric hypertrophy in response to pressure overload is often not readily reversible and can result in the development of a dilated heart (Fig. 1). This process is often referred to as “transition from cardiac hypertrophy to heart failure.” Although the general mechanisms of the development of concentric cardiac hypertrophy have been well studied, less is known about how hypertrophied hearts transition to failed hearts. At the cell level, cardiac myocytes can undergo (1) physiologic hypertrophy, (2) concentric hypertrophy, and (3) eccentric hypertrophy (Fig. 2). Concentric hypertrophy occurs by the parallel addition of sarcomeres; thus, it requires synthesis of contractile proteins such as myosin, actin, tropomyosin, and troponin. Among a number of transcription factors that have been shown to regulate synthesis of these proteins as well as others, GATA-4 transcription factor regulates gene transcription of important hypertrophic genes such as myosin heavy chains (MHCs), myosin light chains, troponin C, and angiotensin type 1a receptor as well as fetal genes that are reexpressed during adult clinical hypertrophy such as atrial natriuretic factor (ANF) [9].
3 Development of the Right Side of the Heart Although the mechanisms of left ventricular hypertrophy are well understood, it is not clear if the information obtained from studies of the left ventricle directly applies to the right ventricle [3]. In the adult heart, many observations both in humans and in experimental models suggest that the properties of ventricular hypertrophy in the left and right ventricles in response to pressure overload are remarkably similar; however, whether some differences in genetic programming
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Fig. 2 Morphology of ventricular muscle cells in cardiac hypertrophy and failure. Phenotypically distinct changes in the morphology of myocytes occur. The expression of embryonic genes is increased in eccentric and
concentric hypertrophy, but not in physiologic hypertrophy, in response to exercise training. (Reprinted with permission from Hunter and Chien [72]. Copyright Massachusetts Medical Society. All rights reserved)
and/or signal transduction events occur in the left and right ventricle is not clear. Since no specific treatment strategies for the management of right ventricular hypertrophy and failure have been established, understanding the differences between molecular mechanisms for left and right ventricular hypertrophy and failure may provide clues for developing treatment specific for the right side of the heart. As described below, the molecular mechanisms of the development of left and right ventricles are quite different; thus, it is conceivable that adult left and right ventricles may exhibit differential molecular signaling events during the course of clinical cardiac hypertrophy and failure. In this section, known differences in molecular mechanisms of left and right ventricular formations are described. Considering similarities in molecular responses in adult left and right ventricles, it is remarkable to learn that cells from left and right ventricles originate from differential precursors. Cells in the first heart field (also called the primary heart field) contribute to the formation of left ventricular myocardium, whereas the second heart field (also referred to as the anterior heart field) contributes to the right ventricular myocardium [10, 11]. The first heart field derives from cells in the anterior lateral plate mesoderm that forms a crescent shape at approximately embryonic day 7.5 in the mouse embryo (corresponding to week 2 of human gestation) [12]. Second heart field cells, which lie dorsal to the straight heart tube, migrate into the anterior and posterior ends of the tube to form the right ventricle, conotruncus, and part of the atria (Fig. 3). Precursors of the left ventricle are sparsely populated by the second heart field and are largely derived from the first heart field [12]. The existence of differential molecular mechanisms for the formation of left and right ventricles is well demonstrated particularly through studies of basic helix–loop–helix (bHLH) domain containing transcription factors eHand (Hand1) and dHand (Hand2). “dHand” stands for “deciduum membrane,
heart, autonomic nervous system, and other neural-crestderived tissues”; and “eHand” stands for “extraembryonic membrane, heart, autonomic nervous system, and other neural-crest-derived tissues.” During mouse cardiogenesis, dHand is expressed specifically in the right ventricle, whereas eHand is expressed in the left ventricle [13]. Gene deletion of dHand in mouse embryos resulted in the inhibition of right ventricular formation without affecting the left ventricle [13]. More recent work on Gata4 knockout mice further delineated the molecular pathways governing chamber-specific regulation by showing that these mice also develop right ventricular hypoplasia [14]. GATA-4 has been shown to regulate dHand gene transcription [15]. These results demonstrate the molecular mechanism for the chamber-specific formation of right and left ventricles. In the adult heart, however, the chamber-specific expression of dHand and eHand is lost and both transcription factors can be detected in the myocardium of both chambers [16]. The free wall thickness and force development of right and left ventricles are similar in the fetus. After birth and in infancy, however, right ventricular hypertrophy regresses and the typical postnatal heart with a crescent-shaped right ventricle and an elliptic left ventricle develops. The crescent shape allows the right ventricle to have a lower volume-to-surface area ratio and thus a highly complaint chamber.
4 Electrophysiological and Contractile Properties of the Right Side of the Adult Heart At the cellular level, both adult right and left ventricles develop to have similar excitation–contraction mechanisms for muscle contraction, although slight differences have been
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Fig. 3 Mammalian heart development. Oblique views of whole embryos and frontal views of cardiac precursors during human cardiac development are shown. At fay 15, first heart field (FHF) cells form a crescent shape in the anterior embryo with second heart field (SHF) cells medial and anterior to the FHF. At day 21, SHF cells lie dorsal to the straight heart tube and begin to migrate (arrows) into the anterior and posterior ends of the tube to form the right ventricle (RV), conotruncus (CT), and part of the atria (A). At day 28, following rightward looping of the heart tube, cardiac neural crest (CNC)
cells also migrate (arrow) into the outflow tract from the neural folds to septate the outflow tract and pattern the bilaterally symmetric aortic arch arteries (III, IV, and VI). At day 50, septation of the ventricles, atria, and atrioventricular valves (AVV) results in the fur-chambered heart. V ventricle, LV left ventricle, LA left atrium, RA right atrium, AS aortic sac, Ao aorta, PA pulmonary artery, RSCA right subclavian artery, LSCA left subclavian artery, RCA right carotid artery, LCA left carotid artery, DA ductus arteriosus. (Reproduced from [12] with permission from Elsevier)
observed, as described in this section. Cardiac muscle contraction is initiated by depolarization of the sarcolemmal L-type Ca2+ channel, which in turn transports Ca2+ from extracellular to intracellular space. This transported Ca2+ serves as a second messenger, which in turn targets another Ca2+ channel located on the membrane of an intracellular organelle called the sarcoplasmic reticulum. This Ca2+-release channel is called a ryanodine receptor, which opens in response to binding of Ca2+ on the cytosolic side, triggering a mechanism called Ca2+-induced Ca2+ release, which amplifies the amount of Ca2+ in the cytosolic space. Increased cytosolic Ca2+ concentration triggers binding of Ca2+ to troponin C and results in actin–myosin interactions and muscle contraction. During the relaxation phase, Ca2+ is extruded out of the cytosolic space to the sarcoplasmic reticulum via Ca2+ATPase (SERCA2 or also called ATP-dependent Ca2+ pump) and to the extracellular space via the Na+–Ca2+ exchanger. In adult rodents, the action potential duration at a fixed heart rate has been reported to be shorter in the right ventricle than in the left ventricle [17–19]. Whole-cell voltage-clamp recordings revealed that the transient outward K+ current, Ito, density was significantly higher in the mouse right ventricle than in the left ventricle [20]. Similarly, Ito density was found to be higher in the canine right ventricle than in the left ventricle [21, 22]. In rats, right ventricular myocytes contracted more rapidly than those from the left ventricle [23].
Contractile activity differences between right and left ventricles were studied by Kondo et al. [24]. They monitored intracellular Ca2+ transients and contractile function in freshly isolated myocytes from mouse right and left ventricles. They found that the Ca2+-induced Ca2+-release amplitude is smaller in the right ventricle than in the left ventricle. Radiolabeled [45] Ca2+ uptake activities in isolated sarcoplasmic reticulum vesicles from right and left ventricles showed no differences. The messenger RNA levels of Ca2+-handling proteins such as the sarcoplasmic reticulum Ca2+-ATPase, phospholamban, ryanodine receptor, and Na+–Ca2+ exchanger did not show any differences between right and left ventricles in mice. In contrast, Sathish et al. [25] found, in rats, that ATPdependent Ca2+ uptake of the sarcoplasmic reticulum is nearly fourfold lower in the right ventricle than in the left ventricle. Similarly, depolarization-induced Ca2+ transients in the right ventricle decayed more slowly than those in the left ventricle. This may be attributed to the lesser interactions of the Ca2+-ATPase with phospholamban. Lower Ca2+ uptake by the sarcoplasmic reticulum and a lower Ca2+-ATPase phosphoenzyme formation were noted in the right ventricle compared with the left ventricle. Less than 50% of the sarcoplasmic reticulum Ca2+ pump in the right ventricle is catalytically active, despite the expression level of this protein being only about 15% lower in the right ventricle than in the left
93 Molecular Basis of Right Ventricular Hypertrophy and Failure in Pulmonary Vascular Disease
ventricle. Coimmunoprecipitation experiments revealed that the level of phospholamban-bound Ca2+-ATPase molecules in the sarcoplasmic reticulum of the right ventricle is greater than that in the sarcoplasmic reticulum of the left ventricle. In isolated rat cardiac myocytes, the Ca2+-induced Ca2+release kinetics is significantly prolonged in right ventricular myocytes compared with myocytes from the left ventricle. The slow decay of cytosolic Ca2+ concentration in the right ventricle and the consequent decrease in the rate of right ventricular relaxation may promote synchrony of right and left ventricles, and the reduced sarcoplasmic reticulum Ca2+ pump activity in the right ventricle may reflect a higher diastolic reserve which may maintain right ventricular function in the condition of increased pulmonary vascular resistance [25].
5 M echanisms of Adult Right Ventricular Hypertrophy and Failure in Pulmonary Vascular Disease Despite the right and left ventricles being developed from different precursors, the protein expression patterns of adult right and left ventricles are very similar. Our laboratory performed two-dimensional gel electrophoresis studies to compare the protein expression patterns between right and left ventricles from adult rats. We found that the protein expression patterns in right and left ventricles are remarkably similar and our Coomassie-stained gels did not exhibit any detectable differences (unpublished results). Although a large body of results on the mechanisms of left ventricular hypertrophy exists in the literature, results on right ventricular hypertrophy are substantially less developed. Lack of studies of right cardiac hypertrophy and failure is apparent by examining the number of published studies. For example, as of December 2010, a literature search on PubMed using the keyword “ventricular hypertrophy” resulted in 28,989 hits and “left ventricular hypertrophy” gave 21,132 hits, indicating that approximately half of the studies of ventricular hypertrophy might have been on the left side. However, in contrast, the keyword “right ventricular hypertrophy” resulted in only 4,786 hits, suggesting that studies on cardiac hypertrophy have focused on the left side of the heart and/or the experiments did not consider differences between right and left sidedness. These searches as well as literature searches with similar keywords clearly indicate the lack of published studies concerning the right side of the heart, especially those pertaining to the mechanisms of cardiac hypertrophy and heart failure. The available results from studies of the mechanisms of right ventricular hypertrophy largely resemble what is known for left ventricular hypertrophy. Studies of rodent models of pulmonary hypertension have shown changes in gene expression in the right side of
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the heart. Many studies have demonstrated that ANF is upregulated in the hypertrophied right ventricle [26, 27]. ANF is a well-known marker for ventricular hypertrophy and has been shown to be upregulated in left ventricular hypertrophy. Therefore, these findings in the right ventricle are consistent with what has been shown in the left side of the heart. Some studies have also demonstrated the altered expression of MHC in the hypertrophied right ventricle [26, 28, 29]. Since GATA-4 transcription factor plays important roles in the regulation of ANF and MHC gene expression as well as other hypertrophic markers [9], our laboratory examined if GATA-4 is activated in the hypertrophied right ventricle in response to pulmonary hypertension. Rats were subjected to chronic hypoxia at 10% O2, which caused pulmonary hypertension and right ventricular hypertrophy [30]. In these rats, GATA-4 DNA binding activity was found to be upregulated in the right ventricle, but not in the left [31]. Although the activation of GATA-4 in the right ventricle in response to pulmonary hypertension was expected and is consistent with other forms of cardiac hypertrophy [32], the mechanism of the activation appears to be unique in the right ventricular hypertrophy induced by pulmonary hypertension. Although much of GATA-4 activation has been attributed to the involvement of posttranslational modifications such as nuclear factor of activated T cells/GATA-4 interactions [33] and GATA-4 phosphorylation [34, 35], GATA-4 activation in the right ventricle in response to pulmonary hypertension is due to increased gene transcription of Gata4 [31]. Our laboratory cloned the Gata4 promoter and identified an important regulatory element in the proximal region, namely, the CCAAT box, which can bind to transcription factors such as CBF/NF-Y [36]. The activation of Gata4 gene transcription in the right ventricle in response to pulmonary hypertension involves the activation of CCAATbinding factor (CBF)/nuclear factor-Y (NF-Y) binding to the CCAAT box of the Gata4 promoter through a novel signaling mechanism involving protein carbonylation of annexin A1 [37]. Figure 4 depicts a putative mechanism for the GATA-4 activation via the promotion of annexin A1 carbonylation and degradation, which in turn liberates CBF/ NF-Y for binding to the Gata4 promoter and gene activation in the right ventricle. As discussed in various chapters in this book, pulmonary hypertension and pulmonary vascular remodeling are mediated by various factors. Notably, endothelin-1 and serotonin play important roles in both vasoconstriction as well as pulmonary vascular thickening. Since these molecules also mediate cardiac hypertrophy and failure, the roles of mediators of pulmonary hypertension such as endothelin-1 and serotonin in the development of cardiac hypertrophy and failure should be considered. Endothelin-1, a peptide composed of 21 amino acids, is a potent vasoconstrictor originally identified in vascular
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Fig. 4 Proposed mechanism for GATA-4 activation in the right ventricle in response to pulmonary hypertension. Pulmonary hypertension exerts pressure overload on the right ventricle and also produces growth factors such as endothelin-1 and serotonin. These stimuli generate reactive oxygen species in the right ventricle, which in turn carbonylate annexin A1, which is bound to CBF/NF-Y transcription factor. Carbonylated annexin A1 is degraded by the proteasomes, resulting in liberated CBF/NF-Y that can bind to the CCAAT box within the Gata4 promoter. CBF/NF-Y binding enhances gene transcription of Gata4 and increased levels of GATA-4 transcription factor, which in turn promote gene expression of various hypertrophic regulators
endothelial cells [38]. The plasma endothelin-1 level was found to be elevated in patients with idiopathic pulmonary arterial hypertension [39, 40]. Histology studies of lung tissues from patients with pulmonary hypertension demonstrated the production of excess endothelin-1 and increased expression of preproendothelin-1, the precursor for endothelin-1 [40]. Similarly, increased endothelin-1 expression was observed in lungs of fawn-hooded rats with increased susceptibility to developing pulmonary hypertension [41]. In secondary pulmonary hypertension, plasma levels of endothelin-1 correlated with the severity of the disease [42, 43]. In various animal models of secondary pulmonary hypertension, endothelin receptor antagonists have been shown to block the progression of the disease [44–51]. Human studies showed that the endothelin receptor antagonist bosentan increased exercise capacity and improved hemodynamics in patients with pulmonary hypertension [52, 53]. Bosentan (Tracleer®) has been approved by the FDA for the treatment of pulmonary arterial hypertension. Soon after the discovery of endothelin-1, this vasoactive peptide was found to be a potent promoter of cardiac muscle cell hypertrophy [54–57]. Several years later, endothelin-1 was also found to play a role in the functional deterioration of left ventricles during the transition from compensatory hypertrophy to congestive heart failure in salt-sensitive hypertensive rats [58]. Thus, endothelin-1, the level of which is elevated in pulmonary hypertension patients, plays a role
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in the development of right ventricular hypertrophy and/or in the transition from compensatory right ventricular hypertrophy to right-sided heart failure. However, studies of the roles of endothelin-1 in right ventricular hypertrophy and failure have not yet been reported. Serotonin (5-hydroxytryptamine) is a potent vasoconstrictor and a mitogen of pulmonary artery smooth muscle cells. Evidence for the role of serotonin in the development of pulmonary hypertension was first recognized in fawnhooded rats, in which a genetic deficit in serotonin platelet storage and high plasma levels of serotonin are associated with the susceptibility to developing pulmonary hypertension [59]. Further studies showed that a continuous intravenous infusion of serotonin during the exposure of rats to hypoxia potentiated the development of pulmonary hypertension [60]. The role of serotonin in pulmonary hypertension is further supported by results showing that serotonin-transporter-deficient mice develop less hypoxic pulmonary hypertension [61]. In mice, overexpression of serotonin transporter enhanced the hypoxia-induced increase in pulmonary vascular remodeling, right ventricular pressure, and right ventricular hypertrophy [62]. In patients with idiopathic pulmonary arterial hypertension, high levels of plasma serotonin were observed [63]. Eddahibi et al. [64] reported that pulmonary artery smooth muscle cells from patients with pulmonary hypertension grow faster than cells from control subjects in response to serotonin. On the basis of these results, possible therapeutic strategies for pulmonary hypertension by targeting serotonin pathways have been proposed [65, 66]. Although the existence of serotonin metabolic enzymes and serotonin receptors in the heart has been known for some time, the roles of serotonin in cardiac disease only recently emerged. Nebigil et al. [67] found that serotonin 2B receptor mutant mice have dilated cardiomyopathy without a compensatory hypertrophic response. In contrast, overexpression of this receptor results in cardiac hypertrophy [68], suggesting that serotonin signaling through serotonin 2B receptor may play a role in the development of concentric cardiac hypertrophy. Bianchi et al. [69] also showed that serotonin can promote cardiac myocyte hypertrophy through serotonin transporter and monoamine oxidase A. Brattelid et al. reported that serotonin 2A receptor may be involved in cardiac hypertrophy, whereas serotonin 4A receptor may contribute to the transition to heart failure [70]. Although serotonin levels have been shown to be increased in patients with pulmonary hypertension [63], the roles of serotonin in right ventricular hypertrophy and failure have not been investigated. Recently, our laboratory found that serotonin promotes protein oxidation specifically in the right ventricle, which might elicit right ventricular hypertrophy and/or rightsided heart failure [71].
93 Molecular Basis of Right Ventricular Hypertrophy and Failure in Pulmonary Vascular Disease
6 Summary Pulmonary hypertension results in right ventricular hypertrophy and right-sided heart failure. Although the mechanisms of left ventricular hypertrophy have been well studied, information on right ventricular hypertrophy is limited. Recent results from embryonic and adult hearts provided evidence that the molecular signaling mechanisms are different for the right and left ventricles, indicating the importance of developing therapeutic agents specific for the right side of the heart. This chapter summarized current understanding of growth signaling mechanisms in the right ventricle.
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Y.J. Suzuki 53. Rubin LJ, Badesch DB, Barst RJ et al (2002) Bosentan therapy for pulmonary arterial hypertension. N Engl J Med 346:896–903 54. Suzuki T, Hoshi H, Mitsui Y (1990) Endothelin stimulates hypertrophy and contractility of neonatal rat cardiac myocytes in a serumfree medium. FEBS Lett 268:149–151 55. Ito H, Hirata Y, Hiroe M et al (1991) Endothelin-1 induces hypertrophy with enhanced expression of muscle-specific genes in cultured neonatal rat cardiomyocytes. Circ Res 69:209–215 56. Sugden PH, Fuller SJ, Mynett JR, Hatchett RJ 4th, Bogoyevitch MA, Sugden MC (1993) Stimulation of adult rat ventricular myocyte protein synthesis and phosphoinositide hydrolysis by the endothelins. Biochim Biophys Acta 1175:327–332 57. Yamazaki T, Komuro I, Kudoh S et al (1996) Endothelin-1 is involved in mechanical stress-induced cardiomyocyte hypertrophy. J Biol Chem 271:3221–3228 58. Iwanaga Y, Kihara Y, Hasegawa K et al (1998) Cardiac endothelin-1 plays a critical role in the functional deterioration of left ventricles during the transition from compensatory hypertrophy to congestive heart failure in salt-sensitive hypertensive rats. Circulation 98:2065–2073 59. Sato K, Webb S, Tucker A et al (1992) Factors influencing the idiopathic development of pulmonary hypertension in the fawn-hooded rat. Am Rev Respir Dis 145:793–797 60. Eddahibi S, Raffestin B, Pham I et al (1997) Treatment with 5-HT potentiates development of pulmonary hypertension in chronically hypoxic rats. Am J Physiol 272:H1173–H1181 61. Eddahibi S, Hanoun N, Lanfumey L et al (2000) Attenuated hypoxic pulmonary hypertension in mice lacking the 5-hydroxytryptamine transporter gene. J Clin Invest 105:1555–1562 62. MacLean MR, Deuchar GA, Hicks MN et al (2004) Overexpression of the 5-hydroxytryptamine transporter gene: effect on pulmonary hemodynamics and hypoxia-induced pulmonary hypertension. Circulation 109:2150–2155 63. Hervé P, Launay JM, Scrobohaci ML et al (1995) Increased plasma serotonin in primary pulmonary hypertension. Am J Med 99:249–254 64. Eddahibi S, Humbert M, Fadel E et al (2001) Serotonin transporter overexpression is responsible for pulmonary artery smooth muscle hyperplasia in primary pulmonary hypertension. J Clin Invest 198:1141–1150 65. Eddahibi S, Adnot S (2006) The serotonin pathway in pulmonary hypertension. Arch Mal Coeur Vaiss 99:621–625 66. MacLean MR (2007) Pulmonary hypertension and the serotonin hypothesis: where are we now? Int J Clin Pract 61:27–31 67. Nebigil CG, Hickel P, Messaddeq N et al (2001) Ablation of serotonin 5-HT(2B) receptors in mice leads to abnormal cardiac structure and function. Circulation 103:2973–2979 68. Nebigil CG, Jaffré F, Messaddeq N et al (2003) Overexpression of the serotonin 5-HT2B receptor in heart leads to abnormal mito chondrial function and cardiac hypertrophy. Circulation 107: 3223–3229 69. Bianchi P, Pimentel DR, Murphy MP, Colucci WS, Parini A (2005) A new hypertrophic mechanism of serotonin in cardiac myocytes: receptor-independent ROS generation. FASEB J 19:641–643 70. Brattelid T, Qvigstad E, Birkeland JA et al (2007) Serotonin responsiveness through 5-HT2A and 5-HT4 receptors is differentially regulated in hypertrophic and failing rat cardiac ventricle. J Mol Cell Cardiol 43:767–779 71. Liu L, Marcocci L, Wong CM, Park AM, Suzuki YJ (2008) Serotonin-mediated protein carbonylation in the right heart. Free Radic Biol Med 45:847–854 72. Hunter JJ, Chien KR (1999) Signaling pathways for cardiac hypertrophy and failure. N Engl J Med 341:1276–1283
Chapter 94
Right Ventricular Dysfunction in Pulmonary Hypertension Francois Haddad, Mehdi Skhiri, and Evangelos Michelakis
Abstract We now know that there are many key differences between the right ventricle (RV) and the left ventricle (LV), ranging from embryological origin, structure, function (metabolism and perfusion), neurohormonal activation and response to increased afterload. Although the role of the RV in the low-pressure normal pulmonary circulation may not be critical, its critical role may become apparent in the diseased pulmonary circulation (e.g. pulmonary hypertension, PHT) or in high-altitude environments. Similarly, although in utero the RV is hypertrophied, within a few weeks after birth, its metabolism and structure is switching to the adult phenotype. Thus, the RV may be designed to be more “plastic.” However, its dynamic responses to physiologic or nonphysiologic triggers increase the risk of maladaptation. We now know that a maladaptive, failed RV is the most important factor in the morbidity and mortality in PHT, regardless of its specific cause. Right ventricular dysfunction is also a very strong predictor of outcome in patients with heart failure due to left ventricular dysfunction. Yet, at this time, few studies have focused on the RV, without extrapolating concepts form the LV. Thus, the concept of RV-specific therapies remains embryonic. In 2006, the NIH formed a task force focusing on increasing awareness and promoting RV studies specifically. Here we review the basic principles of right ventricular dysfunction, clinical diagnosis, as well as therapy, introducing the concept of RV-specific therapies. However, the biggest question in this field, i.e. what is the difference between the adapted RV (for example, the RV in a patient with severe PHT due to congenital heart disease that offers the patient decades-long survival) and the maladaptive RV [for example, the RV in a patient with idiopathic pulmonary arterial hypertension (PAH) of similar magnitude, which fails relatively quickly and allows only a few years of survival], remains unknown. It is hoped that the next edition of this textbook will provide some answers to this critical question.
F. Haddad (*) Division of Cardiovascular Medicine, Stanford School of Medicine, 770 Welch Road, Suite 400, Palo Alto, CA 94304-5715, USA e-mail:
[email protected] Keywords Right ventricular hypertrophy • Afterload • Maladaptation • Right heart dysfunction and failure
1 Introduction “…And I ask, as the lungs are so close at hand, and in continual motion, and the vessel that supplies them is of such dimensions, what is the use or meaning of this pulse of the right ventricle? And why was nature reduced to the necessity of adding another ventricle for the sole purpose of nourishing the lungs?” William Harvey Exercitatio Anatomica de Motu Cordis et Sanguinis in Animalibus, 1628 The answers to Harvey’s questions remain unanswered. Since the pulmonary circulation has a very low resistance compared with the systemic circulation, it was fair to question why another heart chamber was dedicated to “pushing blood through,” even in the form of a rhetorical question. This is particularly intriguing if one realizes that the right ventricle (RV)-ejected blood is not “nourishing the lung” as Harvey implied. The lungs consume a minimal amount of oxygen and they are “nourished” by the bronchial circulation. As opposed to every other organ in the body, the lungs are oxygen suppliers, not consumers. This is a fundamental difference between the left and the right circulation. Thus, a critical concept of left ventricular failure, i.e. the neurormonal hypothesis, which explains the cardiovascular remodeling in response to neurohormones secreted by hypoperfused target organs, may not apply fully to right ventricular failure (RVF). We now know that there are many key differences between the RV and the left ventricle (LV), ranging from embryological origin, structure, function (metabolism and perfusion), neurohormonal activation and response to increased afterload [1–3]. Going back to Harvey’s rhetorical question, one can speculate that although the role of the RV in the low-pressure normal pulmonary circulation may not be critical, its critical role may become apparent in the diseased pulmonary circulation (e.g. pulmonary hypertension, PHT)
J.X.-J. Yuan et al. (eds.), Textbook of Pulmonary Vascular Disease, DOI 10.1007/978-0-387-87429-6_94, © Springer Science+Business Media, LLC 2011
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or in high-altitude environments. Similarly, although in utero the RV is hypertrophied, within a few weeks after birth, its metabolism and structure is switching to the adult phenotype. Thus, the RV may be designed to be more “plastic.” However, its dynamic responses to physiologic or nonphysiologic triggers increase the risk of maladaptation. We now know that a maladaptive, failed RV is the most important factor in the morbidity and mortality in PHT, regardless of its specific cause [4–9]. Right ventricular dysfunction is also a very strong predictor of outcome in patients with heart failure due to left ventricular dysfunction [5, 6, 9–14]. Yet, at this time, few studies have focused on the RV, without extrapolating concepts form the LV. Thus, the concept of RV-specific therapies remains embryonic. In 2006, the NIH formed a task force focusing on increasing awareness and promoting RV studies specifically [15]. Here we review the basic principles of right ventricular dysfunction, clinical diagnosis, as well as therapy, introducing the concept of RV-specific therapies. However, the biggest question in this field, i.e. what is the difference between the adapted RV (for example, the RV in a patient with severe PHT due to congenital heart disease that offers the patient decades-long survival) and the maladaptive RV [for example, the RV in a patient with idiopathic pulmonary arterial hypertension (PAH) of similar magnitude, which fails relatively quickly and allows only a few years of survival], remains unknown. It is hoped that the next edition of this textbook will provide some answers to this critical question.
2 Definition of Right Ventricular Failure RVF is defined as a complex clinical syndrome that can result from any structural or functional cardiac disorder that impairs the ability of the right side of the heart to fill or eject appropriately [16, 17]. The cardinal clinical manifestations of RVF are (a) fluid retention, manifested as peripheral edema or ascites, (b) decreased systolic reserve or low cardiac output syndrome, which may present as exercise intolerance, fatigue or altered mentation, and (c) atrial or ventricular arrhythmias [16–18]. Functionally, the affected RV may be in the subpulmonary (usual) or systemic position (in congenitally corrected levotranspositions of great vessels or dextrotranspositions of great vessels following an atrial-switch repair). Patients may present with a clinical picture of biventricular failure or predominantly right-sided heart failure. The causes of predominant right-sided heart failure include pulmonary embolism, right ventricular myocardial infarction, PAH, chronic pulmonary disease, postcardiotomy RVF, tricuspid valve disease, and selected forms of congenital heart disease, i.e. tetralogy of Fallot, pulmonary stenosis, and systemic RV (Table 1). When RVF is considered as a progressive disorder, patients with asymptomatic ventricular dysfunction are considered to
F. Haddad et al. Table 1 Causes and mechanisms of right-sided heart failure (RHF) Mechanism of right ventricular dysfunction Specific cause Pressure overload
LHF(most common cause) Acute pulmonary embolism (common) PH RVOT obstruction Double chambered Systemic right ventricle (TGA) Volume overload Tricuspid regurgitation Pulmonary regurgitation Atrial septal defect Total or partial anomalous pulmonary return, Carcinoid syndrome (stenotic component possible) Ischemia and infarction Right ventricular myocardial ischemia or infarction Intrinsic myocardial Cardiomyopathy or infiltrative process process Arrhythmogenic right ventricular dysplasia Inflow limitation Tricuspid stenosis Superior vena cava stenosis Complex congenital Ebstein's anomaly malformation Tetralogy of Fallot Double-outlet right ventricle with mitral atresia Hypoplasic right ventricle Pericardial disease Constrictive pericarditis Source: Adapted from Haddad et al. [20] with permission
Fig. 1 The theoretical progression of right ventricular failure (RVF) from normal physiological function to compensated right ventricular remodeling and to overt RVF. As the right ventricle fails, it dilates and the septum shifts to the left because of ventricular interdependence. This stage is often associated with decreased reserve and exercise tolerance of the right ventricle. (Adapted with permission from Champion et al. [23])
be in the early stages of RVF (Fig. 1) [16]. Analogous for the staging proposed for left-sided heart failure, patients may progress from being at risk of RVF (stage A), to asymptomatic right ventricular dysfunction (stage B), to RVF (stage C), and finally refractory RVF (stage D) [16]. Patients with more advanced stages of RVF often have a greater degree of dilatation to hypertrophy (greater volume to mass ratio). Finally, it is also practical to divide RVF as to whether it is acute or chronic.
3 Pathophysiology of Right Ventricular Dysfunction and Failure Following initial myocardial stress or injury, several factors may contribute to progression to RVF, including the timing of myocardial stress, the type of stressor, myocardial ischemia,
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as well as possible neurohormonal and immunologic activation [14, 15, 19, 20]. In general, the RV adapts better to volume rather than pressure overload and to chronic rather than acute stressors. Pediatric patients also appear to have a more favorable course or adaptation than adult patients for similar levels of elevation in pulmonary pressure. Although it was previously thought that right ventricular volume overload is very well tolerated by the RV, recent data from MessikaZeitoun et al. have demonstrated that the presence of a flail tricuspid valve is associated with decreased survival and a higher incidence of symptomatic heart failure and atrial fibrillation [21]. This points toward a more aggressive strategy for surgical repair of flail tricuspid valves, a trend similar to that currently followed for mitral regurgitation. With the exception of Eisenmenger’s syndrome, the RV is unable to sustain long-term severe pressure overload. Early on, right ventricular hypertrophy is mainly an adaptive response (compensated state). As the disease progresses, the RV dilates and RVF eventually occurs (maladaptative right ventricular remodeling; Fig. 1) [22]. The compensatory phase is significantly shorter in the RV than in the LV and explains largely the increased mortality observed in patients with PAH compared with patients with systemic hypertension. Similarly, cardiovascular collapse is not uncommon in patients with massive pulmonary embolism but is rare in patients with a hypertensive urgency. Although this increased vulnerability may be in part due to the lower mass of the RV, several other mechanisms may be involved. An intriguing hypothesis involves the loss of the “protective” molecular, metabolic, and structural phenotype of the fetal RV. When the transition to the adult phenotype occurs, the RV becomes more vulnerable to failure in the presence of increased afterload. This is in contrast to what occurs in patients who develop Eisenmenger’s syndrome in the first year of life who may maintain the fetal phenotype and better adaptation to pressure overload. Several mechanisms are now recognized to contribute to maladaptative right ventricular remodeling, including (1) a switch in contractile protein isoforms from a-myosin heavy chain (a-MHC) to b-myosin heavy chain (b-MHC), (2) mitochondrial and metabolic remodeling, (3) electrical remodeling, including alterations in enzymes and ion channels involved in myocyte excitation– contraction coupling, (4) matrix remodeling with increased fibrosis, and (5) neurohormonal and cytokine activation (Fig. 2) [22–24]. As in left-sided heart disease, a decrease in the amount of a-MHC and an increase in the amount of b-MHC, a feature of the “stressed myocardium,” are also observed in the failing adult RV [25]. In the normal adult human RV, the a-MHC isoform makes up approximately 23–34% of total myosin heavy chain, and b-MHC makes up the remainder [22]. In the pressure-overloaded RV, the change in contractile protein isoforms leads to a decrease in cardiomycyte function. In the LV, microRNA-208 has been identified as one of the key
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Fig. 2 Postulated molecular mechanisms of RVF
regulators of this contractile protein switch, but whether the same or a similar molecular trigger also occurs in the RV remains unknown [26]. Attention has also been drawn to the role of mitochondrial and metabolic remodeling in PAH and right ventricular hypertrophy [24, 27–30]. In the hypertrophied RV, there is a switch to glycolysis from fatty acid oxidation, i.e. the primary fuel source becomes glucose (glycolysis taking place in the cytoplasm) as opposed to fatty acid oxidation (taking place in mitochondria). At first, this appears paradoxical since oxidative phosphorylation in the mitochondria produces much more ATP than does glycolysis. However, we now know from lessons in cancer that there are “secondary gains” from such a switch [31]. For example, this metabolic switch is associated with mitochondrial hyperpolarization, a state in which mitochondria-dependent apoptosis is suppressed. This metabolic switch explains in part the resistance to apoptosis in cancer. It is possible that in the presence of increased afterload, the myocardium suppresses mitochondria-dependent apoptosis, at the expense of suboptimal contractility, at least acutely. As in cancer, long-term response leads to increased uptake of glucose in the cytoplasm and upregulation of the glycolysis, catching up to the energetic efficiency of the mitochondrial oxidative phosphorylation. Thus, as right ventricular hypertrophy increases, the right ventricular mitochondria become more hyperpolarized [28] and 18F-fluorodeoxyglucose (FDG) uptake (measured by PET in vivo) increases [32] in an impressive similarity to what occurs in cancer. Since this metabolic remodeling can be detected and quantified in vivo by FDG-PET and is restricted to the RV in PAH, metabolic targeting therapies might be RV-specific and feasible. Among the neurohormones involved in RVF, evidence is stronger for angiotensin II, catecholamines, and natriuretic
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peptides [33–35]. However, the role of these neurohormones has not been as extensively studied in RVF and direct extrapolation from the left ventricular literature may not be wise. Cytokine activation may also play an important role in patients with RVF. In patients with selected forms of congenital heart disease and right ventricular dysfunction, elevated levels of tumor necrosis factor and endotoxin were associated with more symptomatic disease (lower functional class or more edema) [36, 37]. A recent study demonstrated that genes may be differentially regulated in the pressure-overloaded RV as compared with the pressure-overloaded LV, again supporting the view that the two ventricles and their response to disease might have significant differences. The differentially expressed genes were involved in the Wnt signaling pathway, apoptosis, migration of actin polymerization, and processing of the ubiquitin system [38]. As discussed earlier, the differential expression of genes in the right side of the heart and the left side of the heart is not surprising in view of the different embryological origin of the RV and the LV and their different physiological environments [2, 3]. A more detailed review of the molecular mechanisms of RVF can be found in this textbook and in a recent review by Bogaard et al. [22]. Ventricular interdependence also plays an essential part in the pathophysiology of RVF. Although always present, ventricular interdependence is most apparent with changes in loading conditions such as those seen with volume loading, respiration, or sudden postural changes [39]. Ventricular interdependence helps maintain hemodynamics in early stages of RVF. Experimental studies have shown that in the absence of a dilated RV, left ventricular systolic contraction contributes 20–40% of right ventricular systolic pressure generation [39, 40]. Diastolic ventricular interdependence contributes to the development of left ventricular systolic dysfunction in patients with RVF. Right ventricular enlargement or increased afterload may shift the interventricular septum and increase pericardial constraint on the LV; both of these changes can alter left ventricular geometry and decrease left ventricular preload and contractility (Fig. 1) [39, 41]. Compression of the left main coronary artery by a dilated main pulmonary artery, which is occasionally observed in PAH, may contribute to left ventricular dysfunction [42]. Tricuspid regurgitation and ongoing ischemia may also contribute to the progression of RVF. Although the RV often fails in the presence of pressure overload, it often, but not always, retains an amazing ability for reverse remodeling once the stressor has been removed. Right ventricular volumes and function can return to normal following lung transplantation for severe PAH or after pulmonary artery thrombarterectomy for chronic thromboembolic disease or pulmonary valve replacement for tetralogy of Fallot [43]. Identifying which
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RVs will be able to recover function after the afterload decreases will be an important priority of noninvasive imaging in the near future.
4 Evaluation of Patients with Right Ventricular Failure A complete history, physical examination, and selective clinical studies help establish the cause and severity of right ventricular dysfunction as well as the extent of end-organ damage (renal or liver dysfunction) or the presence of associated conditions. Signs of right ventricular hypertrophy or right ventricular dilatation may occasionally be detected in routine electrocardiography (ECG) or chest X-ray studies. Although echocardiography plays a key role in the diagnosis of right-sided heart disease, magnetic resonance imaging (MRI) is emerging as the gold standard for evaluating right ventricular size and function. It is also becoming clearer that in order to better understand right ventricular physiology, one has to adequately characterize right ventricular contractility and afterload and preload of the RV. In this section, we will first discuss key physiological concepts in the evaluation of the RV as well as specific modalities in the assessment of the right-sided heart disease.
5 Key Physiological Concepts An ideal index of contractility should be independent of afterload and preload, sensitive to change in inotropy, independent of heart size and mass, easy and safe to apply, and feasible in the clinical setting [4, 44]. Ventricular elastance is considered by many investigators as the most reliable index of contractility (Table 2, Fig. 3). Ventricular elastance is measured using pressure–volume loop tracing at different loading conditions; it is the slope of the end-systolic pressure–volume relationship (Fig. 3) [23, 45, 46]. Because conductance catheterization is invasive and time-consuming, it is predominantly used as a research tool for the assessment of ventricular function [47]. More recently, a simplified method of deriving ventricular elastance has been derived using single-beat elastance measures. This method uses the maximal pressure of isovolumic beat (as the second point in the curve) and may be derived using MRI and single pressure measurements [48, 49]. This method could potentially simplify the measure of ventricular elastance and facilitate its clinical use [48]. Recent work has shown that single-beat elastance methods may be useful in assessing ventricular adaptation to PHT [2, 50].
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Table 2 Selected parameters in the assessment of right ventricular function Functional parameters Normal value Load dependence RVEF (%)
61 ± 7% (47–76%) [77] >45% >32% [75]
Clinical utility
+++
Clinical validation, wide acceptance Prognostic value in cardiopulmonary disorders [41] RVFAC (%) +++ Good correlation with RVEF Prognostic value in coronary artery disease [75] TAPSE (mm) > 18 [75] +++ Simple measure not limited by endocardial border recognition: good correlation with RVEF RVMPI 0.28 ± 0.04 [69] ++ Global nongeometric index of systolic and diastolic function, prognostic value PH, CHD [70–72] dP/dt max (mmHg/s) 100–250 [41] ++ Not a reliable index of contractility [76] More useful in assessing directional change when preload accounted for IVA (m/s2) 1.4 ± 0.5 [74] + Promising new noninvasive index of contractility, studies in CHD [47, 74] Ees (mmHg/ml) 1.30 ± 0.84 [46] + Most reliable index of contractility [41] Ees/Ea (measure of ventriculoat- ~1.5 + Useful to study the mechanisms of disease. Value in erial coupling) right-sided heart disease currently under investigation Pulmonary arterial input Ratio of pulsatile Not applicableValue in right-sided heart disease currently under impedance pulmonary pressure measure of arterial investigation to pulsatile flow load RVEF right ventricular ejection fraction, RVFAC, right ventricular fractional area change, TAPSE, tricuspid annular plane systolic excursion, Sm tissue Doppler echocardiography maximal systolic velocity at the tricuspid annulus, RVMPI right ventricular myocardial performance index, dP/ dt max maximal positive first derivative of pressure, IVA, isovolumic acceleration, Ees maximal right ventricular elastance, Ea arterial elastance, CHD, congenital heart disease Fig. 3 Right ventricular pressure–volume (PV) relationship in patients with pulmonary vascular disease. (a) The placement of the conductance catheter in the right ventricle to obtain PV data. (b) The end-systolic PV loop relationship and the end-diastolic PV relationship. Ea refers to arterial elastance and is a measure of arterial afterload; Ea is calculated as the slope of the line that links end-systolic pressure and end-diastolic volume. (c) Representative tracing of PV loops in borderline and late pulmonary arterial hypertension. (d) The key features of summary impedance spectra. PVR pulmonary vascular resistance, PWV pulse wave velocity. (Adapted with permission from Champion et al. [23])
It is now also becoming clear that better understand right ventricular physiology, it is important to consider the RV and the pulmonary artery as a unit [23]. Pulmonary vascular impedance is a way of expressing opposition to flow from the RV to the pulmonary artery (Fig. 3) [51]. The factors that determine the flow from the RV include the resistance of the pulmonary bed (pulmonary vascular resistance), the ability of the arterial vessels to accommodate the ejected blood
bolus with a small rise in pressure at the ventricular outlet (i.e. the pulmonary arterial compliance), the inertance of the blood that has to be accelerated during ejection, pulse wave reflections, and pulmonary vascular recruitment [51]. Considering these properties of the pulmonary circulation may help improve prediction of cardiovascular outcome in cardiovascular disease. A simple calculation of pulmonary compliance as the ratio of stroke volume to pulmonary pulse
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pressure has been suggested to have independent and strong prognostic significance in PAH [52, 53]. Future studies will help establish the clinical value of more complex modeling of pulmonary flow and RV-pulmonary artery coupling on the basis of invasively derived pulmonary impedance.
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6.1 Electrocardiography
Although the symptoms of RVF are similar regardless of the cause, e.g. fluid retention or decreased exercise tolerance, careful history-taking and physical examination can usually provide important hints to the cause of RVF. For example, a classic mitral valve murmur (whether systolic or diastolic) will suggest mitral valve disease as a cause of secondary PHT; or a murmur suggesting an atrial septal defect may suggest congenital heart disease. Signs that are important to keep in mind that suggest RVF regardless of the cause include jugular venous distension, right-sided S3 gallop, the presence of right ventricular heave, and the classic murmur of tricuspid regurgitation (holosystolic systolic murmur that increases with inspiration).
ECG is a specific but less sensitive means of diagnosing right venricular hypertrophy and for this reason, although simple, it is not a good screening test (Fig. 4a). Nonetheless, if right venricular hypertrophy is detected, PHT is likely present [54]. Lehtonen et al. compared four sets of ECG criteria for diagnosing right ventricular hypertrophy in patients whose right ventricular thickness was measured at autopsy. Hearts were considered normal if the total weight was less than 250 g, the RV weighed less than 65 g, and the combined LV plus septum weight was less than 190 g (ratio of weight of LV + weight of septum/weight of RV 2.3–3.3). By combining aspects of the four sets of ECG criteria, they achieved a diagnostic sensitivity of 63% and a specificity of 96%. These combined criteria included right-axis deviation greater than 110°, R-wave equal to or greater than S-wave (in V1 or V2), R-wave equal to or less than S-wave in lead V6, a calculated value AR/PL of 0.7 (where A is the maximum R-wave amplitude in lead V1 or lead V2, R is the maximal S-wave amplitude in lead I or lead V6, and PL is the minimal S-wave amplitude in lead I or lead V6). This set of criteria was quite specific, despite the
Fig. 4 Use of echocardiography in assessing right ventricular hypertrophy. A chest X-ray and right ventricular hypertrophy in a patient with pulmonary arterial hypertension and right ventricular hypertrophy. Note the signs of right ventricular hypertrophy on the electrocardiogram and the signs of right ventricular enlargement and pulmonary arterial dilatation on the chest X-ray, as discussed in the text. Representative echocardiographic tracings of tricuspid annular systolic excursion (a, b) and Doppler signals
of the right ventricular outflow and tricuspid regurgitation (c, d). Decreased acceleration time and the presence of pulmonary notch are associated with severer pulmonary vascular disease. Right ventricular myocardial performance index is calculated as the ratio of the isovolumic acceleration and the relaxation time divided by the ejection time. ET ejection time, AT acceleration time, Vmax maximal tricuspid pressure gradient at peak tricuspid regurgitation signal (modal frequency), RV right ventricle
6 History and Physical Examination
94 Right Ventricular Dysfunction in Pulmonary Hypertension
presence of left ventricular hypertrophy and myocardial infarction in the study population. A limitation of this study is that the autopsy was restricted to patients dying from respiratory failure and perhaps these criteria might not be applicable in other causes of PHT. For example, the chest size is enlarged in patients with chronic airway disease and influences the voltage and the axis in ECG. The ECG criteria for right ventricular hypertrophy become less specific in the presence of posterior myocardial infarction, left posterior hemiblock, and Wolff–Parkinson–White syndrome. These conditions, all rare in PHT patients, may cause right-axis deviation and/or a predominant RV in lead V1, similar to the findings in right ventricular hypertrophy. Lastly, patients with right ventricular hypertrophy typically have right ventricular enlargement (known as P-pulmonale), i.e. asymmetrical peaked P-waves greater than 0.25 mV in lead 2.
6.2 Chest X-ray Enlargement of the RV is an important clue to the presence of severe PHT and is suggested by the filling in of the retrosternal space on the lateral view of the chest X-ray (Fig. 4a). Additional signs may suggest a specific cause. For example, “pruning” of the peripheral pulmonary arteries is a classic sign, which reflects vascular obliteration of the small arteries in the periphery of the lungs in PAH. Important signs of secondary PHT can be also documented with a chest X-ray; for example, the presence of septal thickening or pleural effusions will suggest venous hypertension to left ventricular dysfunction, venoocclusive disease, or pulmonary capillary angiomatosis.
6.3 Laboratory Evaluation Obtaining baseline renal and liver function tests and albumin and uric acid levels as well as B-type natriuretic peptide levels may be of particular interest in determining the prognosis of right-sided heart disease [55–61]. Data are also emerging on the prognostic value of renal dysfunction and hyponatremia in patients with PAH [60, 62]. In the last 5 years, studies have also identified interleukin-1 receptor like protein soluble isoform (sST2) as a novel biomarker in heart failure; elevation in the sST2 level predicted mortality in heart failure [63–65]. In mouse models, IL-33/ST2L signaling antagonizes cardiomyocyte hypertrophy induced by transverse aortic constriction and agonists (angiotensin II and phenylepinephrine), while improving systolic function [22, 64]. The value of sST2 is currently being investigated in patients with right-sided heart failure. Other laboratory studies should be individualized depending of the suspected cause of RVF.
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For example, serological tests for HIV and connective tissue disease (e.g. antinuclear antibody) are often routinely performed in patients with PAH. Finally, in patients with edema and severe hypoalbuminemia, protein-losing enteropathy should be excluded with the assay of stool a1-antitrypsin.
6.4 Echocardiography Echocardiography plays a key role in the diagnosis of rightsided heart disease. Signs of right-sided heart disease on an echocardiogram can include right ventricular enlargement, right ventricular systolic dysfunction, tricuspid regurgitation, and PHT, congenital heart defects, valvular heart disease, or left-sided heart disease. Common indices of right ventricular function include right ventricular fractional area change (RVFAC), tricuspid annular plane systolic excursion (TAPSE), and the right ventricular myocardial performance index (RVMPI). RVFAC represents the ratio of systolic area change to diastolic right ventricular area. It is measured in the four-chamber view and can be systematically incorporated in the basic echocardiographic study. In end-stage pulmonary disease, a reasonable correlation has been reported between RVFAC and right ventricular ejection fraction [66]. TAPSE is another useful quantitative measurement of right ventricular systolic performance. TAPSE reflects the longitudinal systolic excursion of the lateral tricuspid valve annulus toward the apex (Fig. 4b). It is usually measured using M-mode imaging in the four-chamber view [67]. Studies showed moderate correlation between TAPSE and right ventricular ejection fraction measured by radionuclide angiography [68]. Finally, RVMPI, which is the ratio of isovolumic time intervals to ventricular ejection time, has been described as a nongeometric index of global ventricular function (Fig. 4) [69]. RVMPI appears to be relatively independent of preload, afterload, and heart rate and has been useful in assessing patients with congenital heart disease and PHT [70–72]. In recent years, a novel noninvasive index of contractility based on the myocardial isovolumic acceleration assessed by tissue Doppler echocardiography has been described (Fig. 5) [47, 73, 74]. In close-chested animal models, isovolumic acceleration was found to reflect right ventricular myocardial contractile function and was less affected by preload and afterload within a physiologic range when compared with either dP/dt max or elastance. Its value was validated clinically in congenital heart disease, i.e. after repair of tetralogy of Fallot and in transposition of great arteries [47, 74]. Further validation of this new index is being actively pursued in PHT and heart failure. Table 2 summarizes common indices used in the study of right ventricular function [41, 69–72, 75–77].
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c ardiac output, shunt fraction, and pulmonary vasoreactivity [79]. Left-sided heart catheterization is often performed to precisely measure left ventricular end-diastolic pressure.
6.7 Exercise Testing
Fig. 5 Tissue Doppler echocardiography spectral curve at the level of the proximal right ventricular myocardial proximal wall. The isovolumic myocardial acceleration is calculated as the difference between the baseline and the peak velocity (stars in the right panel) during isovolumic contraction divided by the time interval. (Reproduced from Vogel et al. [74] with permission from Elsevier)
6.5 Magnetic Resonance Imaging MRI is becoming the goal standard for evaluating right-sided heart structure and function. Except when contraindicated, MRI is now the modality of choice for determining right ventricular ejection fraction, a position previously held by radionuclide angiography. Pulmonary angiography may also be performed using MRI. Performing MRI is particularly useful in patients with complex congenital heart defects (e.g. Ebstein’s anomaly, hypoplasic RV), in patients in whom precise quantification of valvular regurgitation is important, in assessment of the pulmonary vasculature or arteriovenous malformation, for planning of complex surgery or procedures, or for research purposes [20]. Recent studies using MRI have also demonstrated the prognostic value of right ventricular end-diastolic volumes and pulmonary compliance assessed by MRI in PAH [52, 78]. Although currently the utilization of MRI is limited by availability and cost, it is possible that the ability of the MRI to offer a “single stop shop” comprehensive assessment of the right ventricular cardiopulmonary unit might in the future prove to be an essential part of the evaluation of RVF (see Chaps. 35, 100).
6.6 Right-Sided Heart Catheterization Right-sided heart catheterization is an important part of the evaluation of right-sided heart disease. Indications for rightsided heart catheterization include assessment of pulmonary vascular resistance or impedance, pulmonary pressures,
Exercise testing is also very useful in objectively assessing clinical deterioration in patients with PAH or congenital heart disease. Caution is, however, advised in performing maximal exercise testing in patients with severe pulmonary vascular disease and RVF (contraindicated in the recent American Heart Association consensus on congenital heart disease) [80]. Other studies should be individualized depending of the suspected cause of RVF. In patients with PAH, a ventilation– perfusion scan, pulmonary function tests, and overnight oximetry are often performed. Lung or heart biopsy is rarely indicated in patients with isolated right-sided heart disease. Genetic counseling should be pursued in patients with congenital heart disease or arrhythmogenic right-sided heart dysplasia. Depending on the cause and severity of RVF, patients are usually followed at differing intervals (usually 3 months to 1 year). The recent guidelines for congenital heart disease and PAH offer individualized timing for follow-up depending on the conditions [80].
7 Managing Right Ventricular Failure Management of RVF should always take into account the cause and setting in which RVF occurs. Figures 6 and 7 summarize the management of acute and chronic RVF. In contrast to patients with chronic ischemic or nonischemic cardiomyopathy, patients with RVF often have significantly abnormal afterload (e.g. PHT) or valvular heart disease (acquired or congenital pulmonary or tricuspid disease). It is therefore not surprising that therapy for RVF should primarily target its cause. It is useful to divide the RVF syndrome into four clinical categories, i.e. biventricular failure, systemic RVF, predominant subpulmonary RVF, and hypoplasic RV syndrome [80]. The management of patients with a hypoplasic RV is beyond the scope of this chapter and the reader is referred to the recent consensus statement of Warnes et al. [80]. As in left-sided heart failure, specific treatment goals include optimization of preload, afterload, and contractility and maintenance of sinus rhythm and atrioventricular synchrony, to increase cardiac output and decrease venous pressure (i.e. right atrial pressure). The management of patients with RVF remains more empiric than the management of patients with left-sided heart failure [16, 17]. Clinical trials of patients with
94 Right Ventricular Dysfunction in Pulmonary Hypertension
Fig. 6 Management of chronic RVF. ACC, American College of Cardiology, AHA American Heart Association, ACEI angiotensin converting enzyme inhibitor, ARB angiotensin receptor blocker, CTEPH
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chronic thromboembolic pulmonary hypertension, ERB, endothelin receptor blockers, PDE5, phosphodiesterase 5, TOF, tetralogy of Fallot, TV, tricuspid valve
Fig. 7 Management of acute RVF. PE pulmonary embolism, PH pulmonary hypertension, PR, pulmonary regurgitation, TR, tricuspid regurgitation, endocarditis, RVAD, right ventricular assist device, ECMO extracorporal membrane oxygenation, TV, tricuspid valve, A-V, atrioventricular
RVF have also not been powered for mortality end points. Among patients with RVF, the evidence is best established for patients with PAH [16, 80, 81]. In PAH it is, however, more difficult to distinguish whether the beneficial effects of therapy are due to changes in pulmonary vasculature or RV-specific effects; we therefore often consider the effects PAH therapy
in the context of the RV-pulmonary artery unit. In patients with congenital heart disease, the effects of therapy have not been consistently studied across functional class severity (New York Heart Association functional class). Because the prevalence of right-sided heart failure is relatively small compared with the prevalence of left-sided heart failure, finding
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appropriate surrogate end points has been an important focus of research [79]. Surrogate end points being considered include exercise capacity, clinical worsening, ventricular remodeling, and measures of vascular impedance in PHT.
8 Chronic Right Ventricular Failure In patients with biventricular failure, the management is based on the recommendations of the American College of Cardiology [16]. In addition to the standard therapies, new therapies are emerging that may be particularly important for patients with biventricular failure. Recent studies have shown that the use of sildenafil may improve pulmonary hemodynamics and exercise capacity in patients with chronic systolic heart failure [82, 83]. Whether sildenafil will improve hemodynamics and outcome of patients with heart failure and PHT or RVF is the subject of ongoing investigation. Table 3 summarizes the results of selected clinical studies on sildenafil in patients with PAH, left-sided heart failure, and chronic thromboembolic disease [84–87]. In patients with predominately RVF, the management is closely related to the cause of RVF (Fig. 6). Patients with PAH benefit from prostanoid therapy, endothelin receptor blockers,
sildenafil, and anticoagulation. Although both endothelin receptor blockers and sildenafil have proven to improve the 6-min walk distance, recent studies suggest that sildenafil may also have positive inotropic effects on the RV [88]. Whether these differences will translate into beneficial long-term effects still remains to be proven. Digoxin has not been shown to acutely improve hemodynamics in patients with right-sided heart failure and PAH; chronic benefits have not been clearly demonstrated (Table 4) [89, 93]. Inhibition of the angiotensin system or beta blockade may be considered in selected patients with systemic RVs although their benefit remains uncertain and controversial [80, 94]. Tables 5 and 6 summarize the clinical evidence on the use of angiotensin system inhibition or beta blockade in patients with right-sided heart failure [95–100]. Resynchronization therapy or right-sided heart pacing may become useful in selected patients with RVF, if further validated. It has currently been tested mainly in patients with congenital heart disease (Table 7) [101–104]. A multicenter trial is currently planned to assess the benefits of resynchronization therapy in patients with PAH. Timely repair of congenital lesions is also essential to avoid progressive right ventricular dysfunction in congenital heart disease. Tricuspid valvuloplasty, either surgical or percutaneous, or cardiac support devices may also have a role in managing chronic RVF in the future.
Table 3 Selected studies on phosphodiesterase type 5 inhibition in patients with right ventricular failure (RVF) or PH Study Study population Characteristics n Design Main findingsa Galiè et al. [84]
PAH
NYHA class II/III MPAP 49–56 mmHg CI 2.2–2.5 L/min/m2 6MWT 339–347 m
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RCT Orally administered sildenafil vs. placebo 12 weeks
Beneficial effect: increase in 6MWT by 14%. Dose-dependent increase in CI, and decrease in MPAP, PVR, and RAP
Lewis et al. [85]
LHF with PH
NYHA class II/III MPAP 33 mmHg PCWP 19 mmHg Stroke volume 44 mL
34
RCTOrally administered sildenafil vs. placebo 12 weeks
Beneficial effect: increase in peak VO2 by 14%, decrease in exercise stroke volume by 60%, decrease in PVR by 18% at rest and 28% with exercise
Lepore et al. [86] LHF with PH
NYHA class III or IV MPAP 37 mmHg PCWP 22 mmHg CI 2.1 L/min/m2
11
RCT Orally administered sildenafil vs. iNO vs. combination
Additive beneficial effect: decrease in PVR by 50%, decrease in SVR by 24%, increase in CI by 30%, decrease in MPAP 14% (NS)
Ghofrani et al. [87]
CTEPH
NYHA class NA MPAP 52 mmHg CI 2.0 L/min/m2 6MWT 312 m
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Beneficial effect: increase in 6MWT Prospective study by 17%, increase in CI by 20%, Orally administered decrease in PVR by 30% decrease sildenafil for 6 months in MPAP by 15%
Stocker et al. [123]
CHD
Infants at risk of PH Detrimental effect: decrease in PaO2 16 RT after cardiac surgery by 29.9 mmHg when intravenously iNO and intravenously administered sildenafil given first, Ventilated infants administered sildenafil leading to an early termination MPAP 68 mmHg of the study CI 3.9 L/min/m2 a Unless otherwise specified, the results refer to statistically significant findings (p 26 years RVEF 47% Left ventricular fractional shortening of 33% TTPG 43 mmHg
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Retrospective study ACE-I from 6 to 126 months
Beneficial effect: increase in exercise time by 18%, decrease in regurgitant volume by 63.5%, increase in RVEF by 6% ‡ No difference in exercise duration, peak VO2, or cardiac index
No difference in peak VO2 or exercise duration
No difference in TTPG or exercise duration but subgroup of patients who were better responders a Unless otherwise specified, the results refer to statistically significant findings (p 18 years by 6% ‡. No change in peak VO2 Carvedilol for 12 months NYHA class II and III RVEF 34% No difference in peak VO2, RVEF, Norozi CHD Tetralogy of Fallot 33 RCT et al. [131] ventricular volumes, NYHA class NYHA class I and II Bisoprolol vs. placebo for 6 months Adults LVEF 57% CI 3.8 L/min/m2 a Unless otherwise specified, the results refer to statistically significant findings (p 25 mmHg 50% Thabut et al. [157] Severe COPD patients undergoing evaluation for lung transplant or lung MPAP > 35 mmHg 13.5% volume reduction surgery Scharf et al. [158] Patients with severe emphysema FEV1 = 27% MPAP > 20 mmHg 90.8% Chaouat et al. [14] COPD patients with chronic respiratory FEV1 = 33% MPAP > 40 mmHg 2.7% failure All studies are based on right-sided heart catheterization PH pulmonary hypertension, FEV1, forced expiratory volume in 1 s, VC, vital capacity, MPAP mean pulmonary artery pressure
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retrospective study of 88 patients with IPF referred to the Mayo Clinic, 84% had a Doppler-estimated PAP greater than 35 mmHg [16]. However, pretransplant RHC data in patients with severe IPF suggest that fewer patients (30–46%) have PH, defined as a mean PAP greater than 25 mmHg [17, 18]. A retrospective cross-sectional study of 619 patients with scleroderma found PH and interstitial lung disease in 18% of patients, based on echocardiography and pulmonary function test performed within 6 months of one another [10]. The prevalence of cor pulmonale in patients with sarcoidosis varies from estimates as high as 73.8%, based on RHC in patients listed for lung transplant, to as low as 5.7%, in a cross-sectional study based on Doppler echocardiography [19, 20].
M. Gomberg-Maitland et al.
The histological manifestations of cor pulmonale vary depending on the underlying lung disease, and a thorough histological examination of both the pulmonary vasculature
and the parenchyma at the time of biopsy or autopsy is recommended. The vascular disease is primarily manifest in small arteries and arterioles. The microscopic findings involve both the tunica media and the intima of small muscular arteries and arterioles, which are located adjacent to small bronchioles and within alveolar septa, respectively. One characteristic feature of cor pulmonale is the muscularization of small arterioles. Normally, arterioles within alveolar septa are nonmuscular. This feature (muscularization of small pulmonary arteries) is recapitulated in animal models of cor pulmonale created by exposing animals to chronic hypoxia [23]. The finding of smooth muscle fibers surrounding arterioles within alveolar septa indicates the presence of distal extension of muscularization (Fig. 4a). This tissue changes are prominent in emphysema, and are often the only vascular abnormality seen [24, 25]. On the other hand, IPF manifests both distal extension of arteriolar muscularization and medial hypertrophy of small arteries [26, 27]. Medial hypertrophy consists of increased numbers of smooth muscle fibers and nuclei in the tunica media, resulting in an increased thickness of the arterial wall (Fig. 4b, c). Sarcoidosis with PH manifests itself as direct infiltration of nonnecrotizing granulomas in the pulmonary vasculature [28, 29]. The walls of small to medium-sized arteries are invaded by collections of histiocytes, often with prominent foreign-body giant cells (Fig. 4d). Asteroid bodies, cytoplasmic inclusions within the granulomas in sarcoidosis, are characteristic but not diagnostic [30]. Finally, the histologic finding that characterizes the lung disease associated with connective tissue disease is intimal fibrosis of pulmonary artery branches [28, 31]. In this case, the tunica intima becomes concentrically thickened by collagen and proliferating endothelial cells (Fig. 4e). In summary, emphysema is characterized by arteriolar muscularization, IPF by medial hypertrophy, sarcoidosis by direct invasion of vessels by granulomas, and connective tissue disease by intimal fibrosis. Findings that differentiate PAH from cor pulmonale include the involvement of large vessels in PAH, which often have intimal plaques and the
Fig. 3 The heart in cor pulmonale. (a) Cross-section of the heart from a patient with cor pulmonale showing right ventricular hypertrophy and dilation and a D-shaped left ventricle (chamber on the right of the pho-
tograph). (b) Histology of right ventricular myocardium in cor pulmonale showing myofiber hypertrophy with enlarged “boxcar” nuclei (inset), hematoxylin and eosin (H&E) stain, high power with magnified insert
1.4 Histopathology 1.4.1 The RV The pulmonary vascular remodeling in cor pulmonale increases RV afterload, leading to RVH and RV dilation [21, 22]. The RV wall thickness increases and may even double in cor pulmonale. The trabeculae become more prominent, and may bridge the ventricular lumen (Fig. 3a). Histologically, myocardial hypertrophy is evidenced by an increase in nuclear size and change in shape to rectangular, or “boxcar” nuclei (Fig. 3b). With increasing RV pressure and blood flow, the interventricular septum may bow into the left ventricle, with the compression creating a D-shaped left ventricle. This finding reflects the severity of the PH and is not specific for cor pulmonale.
1.4.2 The Lung
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Fig. 4 Lung histology in cor pulmonale. (a) Arteriolar muscularization in emphysema, H&E stain, high power. (b) Medial hypertrophy, H&E stain, high power. (c) Medial hypertrophy, trichrome stain, high power. (d) Vessel involved by granulomas in sarcoidosis, H&E
stain, low power. (e) Intimal fibrosis in connective tissue disease associated lung disease, H&E stain, high power. (f) Plexiform lesion in idiopathic pulmonary arterial hypertension, H&E stain, high power
presence of plexiform lesions in PAH [32, 33]. Plexiform lesions consist of capillary-like, angioproliferative vascular channels that form within the lumen of small muscular arteries, and often appear at branch points (Fig. 4f). The luminal diameter of these channels may vary, giving them a disordered appearance. Fibrin thrombi are often present within the lumina of plexiform lesions. These lesions are not seen in cor pulmonale and are mentioned here only to contrast them with the histologic findings of cor pulmonale.
of catecholamines may contribute to systemic hypertension and perhaps to cor pulmonale. Alveolar hypoxia is the major mechanism involved in the pathogenesis of PH in cor pulmonale [11]. Acutely, hypoxia causes pulmonary vasoconstriction (hypoxic pulmonary vasoconstriction, HPV) [35], whereas chronic hypoxia leads to pulmonary vascular remodeling by promoting cell proliferation [36, 37] in addition to activating constrictor pathways, such as endothelin synthesis and activation of Rho kinase [38]. Although endothelin-1 is detectable in both the peripheral blood and the lavage of COPD patients, the levels are only modestly elevated compared with those of controls and are not statistically greater in patients with cor pulmonale than in COPD controls [39]. In contrast to its vasodilatory effect on the systemic circulation, acute hypoxia causes vasoconstriction of precapillary pulmonary resistance vessels (HPV) through contraction of pulmonary smooth muscles. HPV is a homeostatic mechanism to optimize ventilation– perfusion matching by shunting the blood flow away from poorly ventilated lungs [35]. HPV is initiated by inhibition of voltage-gated potassium (Kv) channels, notably Kv1.5, in pulmonary artery smooth muscle cells (PASMCs) [40]. In the PASMC, hypoxia inhibits outward potassium currents, thereby causing membrane depolarization and entry of calcium through the L-type voltage-gated calcium channels. In addition, hypoxia causes calcium sensitization, which sustains HPV [41], and also promotes release of calcium stores
1.5 Pathophysiology Cor pulmonale results from a variety of active and passive mechanisms that alter pulmonary hemodynamics and ultimately RV function (Fig. 5).
1.5.1 Role of Catecholamines, Hypoxia, Hypercarbia, and Acidosis In normal individuals, altitude increases systemic blood pressure and plasma arterial noradrenaline and adrenaline concentrations (3.7-fold and 2.4-fold, respectively) [34]. Thus, chronic hypoxia activates the sympathetic nervous system in healthy humans; this elevation in the concentrations
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Fig. 5 Confirmed and suspected mechanisms leading to pulmonary hypertension in chronic obstructive pulmonary diseases
from the sarcoplasmic reticulum via store-operated channels [42]. Hypoxia also stimulates Rho kinase, which in turn inhibits myosin light chain phosphatase, thereby increasing phosphorylation of the light chain and augmenting contraction [35, 40]. HPV is transient and resolves rapidly. Although HPV persists in COPD patients [43], there is a general tendency for HPV to decrease in patients (and animals) exposed to chronic hypoxia. Indeed, HPV is largely absent in species that are native to highlands, such as the yak. Thus, HPV likely plays only a minor role in the pathogenesis of chronic cor pulmonale and is generally beneficial in these patients (as manifest by a worsening shunt and hypoxemia when HPV is suppressed in COPD using a calcium channel blocker [44]). However, excessive HPV may contribute to the acute worsening of PH in patients with COPD during acute exacerbations, exercise, and sleep [11].
1.5.2 The Basic Science of Cor Pulmonale Chronic hypoxia results in pulmonary vascular remodeling characterized by muscular medial hypertrophy of the small pulmonary arteries, neomuscularization of the pulmonary arterioles, and a variable degree of intimal fibrosis [45]. The mechanism underlying chronic-hypoxia-induced pulmonary vascular remodeling is unclear. Stenmark et al. have documented excessive proliferation in PASMCs of the calf in response to hypoxia, but remind us that the affects of hypoxia touch each layer of the pulmonary artery [46, 47]. They noted that there is smooth muscle heterogeneity within the pulmonary artery wall and that only certain populations of PASMCs proliferate in response to hypoxia (PASMCs that
stain negative for the cytoskeletal protein metavinculin) [47]. We have found a similar diversity in the expression of O2-sensitive Kv channels in the pulmonary artery, noting that Kv1.5 channel expression is enriched in resistance PASMCs, the site of maximal HPV [48, 49]. In vitro studies on human pulmonary artery cells and in vivo rat models, as well as human studies, have suggested that chronic hypoxia produces an imbalance of endotheliumderived vasoactive and vasoproliferative mediators that favors constriction and proliferation. Hypoxia increases the synthesis of vasoconstrictive endothelin and angiotensin II, whereas it decreases the production of the potent vasodilators nitric oxide and prostacyclin [50–54]. In addition, hypoxia induces the synthesis of vascular endothelial growth factor (VEGF) and platelet-derived growth factor [51]. Several other vasoactive and proliferative mediators, such as serotonin and serine elastases, have also been observed to be released by hypoxia [55, 56]. These vasoactive and vasoproliferative mediators interact in a highly complex fashion, leading to increased pulmonary vascular tone and proliferation of pulmonary vascular cells [46].
1.5.3 T he Mitochondria: Hypoxia-Inducible Factor 1a: Kv Pathway in PH Abnormalities in a mitochondria–hypoxia-inducible factor 1a (HIF-1a)–Kv pathway, recently discovered in an experimental model of PAH, may also contribute to excess smooth muscle cell proliferation and hypertrophy in cor pulmonale. From animal models we know that the PASMCs in cor pulmonale are calcium-overloaded and prone to constriction
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and proliferation. The fawn-hooded rat is a mutant strain which offers useful information about the basic mechanism of cor pulmonale [57]. These rats have mild PH and polycythemia and are hypoxia-sensitive (i.e. they develop PH in response to modest hypoxia or even spontaneously in normoxia as they age) [36, 57–61]. In the fawn-hooded rat, the ionic remodeling appears to relate to normoxic activation of HIF-1a, which promotes a metabolic shift and causes downregulation of Kv1.5 (one of the Kv channels involved in HPV). The fawn-hooded rats are reminiscent of patients from the Chuvash region of Russia, in whom homozygous germline mutations (598 C->T) of the von Hippel–Lindau gene lead to HIF-1a upregulation during normoxia, resulting in high erythropoietin levels, polycythemia, and PH [62]. In chronic hypoxia (in rats) there is also evidence that hypoxia activates the master transcription factor HIF-1a and that this (in part) explains the loss of Kv1.5 and development of PH. The decrease in expression of specific Kv channels (notably Kv1.5) in chronic hypoxia has been confirmed by several groups [61, 63], causes PASMC depolarization [64], and favors PASMC proliferation and vascoconstriction [57]. Further supporting the pathological role of HIF-1a in cor pulmonale, mice that are haploinsufficient for HIF-1a are resistant to the development of hypoxic PH and do not downregulate their PASMC Kv current in response to hypoxia [65, 66]. Thus, there is some basic science to suggest that abnormalities in mitochondrial metabolism and redox signaling in PASMCs whether inherited, as in the fawn-hooded rat, or acquired, as occurs with exposure to chronic hypoxia, leads to normoxic activation of HIF-1a and ultimately Kv channel downregulation, which contributes to PH [57]. This mitochondria–HIF-1a–Kv pathway may promote proliferating apoptosis-resistant PASMCs that contribute to cor pulmonale. This pathway offers several appealing therapeutic targets to treat cor pulmonale. Although most of the evidence for this mechanism comes from rodent studies,
similar mitochondrial abnormalities, resulting in impaired respiration and enhanced glycolysis, occur in PASMCs [57] and endothelial cells [67] of humans with PAH. There are no human cor pulmonale data assessing this pathway. In rodents, experimental cor pulmonale can be regressed by restoring oxidative mitochondrial metabolism (using the pyruvate dehydrogenase kinase inhibitor dichloroacetate [37, 57]) or even by Kv1.5 gene therapy [36]. Dichloroacetate appears to be beneficial by reducing proliferation and enhancing apoptosis in PASMCs. Hypercarbia and acidosis also play a role in the development of cor pulmonale, although the impact of these factors is less than that of hypoxia. Hypercarbia induces hyperventilation, which in turn produces changes in lung mechanics, leading to increased PAP. It has also been suggested that hypercarbia augments HPV [68]. Likewise, acidemia acts synergistically with hypoxia to enhance pulmonary vasoconstriction [69].
Fig. 6 Airway pressure to pulmonary blood flow relationships relevant to cor pulmonale. The relationship of flow and pressure in the pulmonary circulation obtained after increasing the flow through the left pulmonary artery by balloon occlusion of the right pulmonary artery. (a) The influence of alveolar pressure on pulmonary blood flow and pressure in cor pulmonale is assessed by increasing mouth pressure.
(b) The relationship between flow and pressure of the pulmonary circulation in normal subjects with increased mouth pressure is similar to the relation in patients with chronic obstructive pulmonary disease. (Adapted with permission from MacNee [11]. The official journal of the American Thoracic Society, copyright American Thoracic Society)
1.5.4 Abnormal Lung Mechanics In addition to the structural disease in the pulmonary vasculature, elevated alveolar pressure in patients with COPD may contribute to the increased PVR [70]. A linear relationship exists between arterial pressure and flow in the pulmonary circulation, when the intraalveolar pressure is normal. However, in patients with obstructive lung disease, when the alveolar pressure increases owing to increased airway resistance, the PAP increases out of proportion to flow, owing to compression of the pulmonary vasculature by the overdistended alveoli. Heath and coworkers [71] elegantly demonstrated this by increasing the alveolar pressure by increasing the gas pressure in the mouth (Fig. 6). As alveolar pressures are increased, the pressure in the pulmonary circulation initially increases steeply and then changes in a curvilinear
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pattern as opposed to a normal linear pattern. Airway resistance affects pulmonary hemodynamics, particularly when there is increased ventilatory pressure, as occurs during COPD exacerbations [11].
1.5.5 Inflammation The role of inflammation as a mediator of cor pulmonale in patients with chronic lung disease is controversial [72]. The number of inflammatory cells, especially CD8 lymphocytes, in the pulmonary vascular wall has been shown to correlate with the enlargement of the intimal layer [73]. Systemic levels of inflammatory cytokines, such as C-reactive protein and interleukin 6, correlate with mean PAP in patients with COPD [74, 75]. Recent studies suggest that oxidants in cigarette smoke cause inflammation, releasing endothelium-derived vasoconstrictive and vasoproliferative mediators such as endothelin and VEGF [76]. Oxidants in cigarette smoke also interfere with nitric oxide mediated vasodilatation by decreasing nitric oxide bioactivity. Pulmonary vascular remodeling has been reported in smokers even when their lung function is normal [77].
1.5.6 Miscellaneous Additional mechanisms that contribute to cor pulmonale include increased blood viscosity from polycythemia, emphysematous reduction of the pulmonary capillary bed, and the anatomical distortion of the pulmonary vasculature by parenchymal changes seen in interstitial lung diseases. Polycythemia, secondary to hypoxia, increases blood viscosity, which in turn augments PVR on the basis of Poiseuille’s equation. Reducing blood volume and thereby blood viscosity decreases PAP and PVR in COPD patients with polycythemia [78]. Theoretically, destruction of the pulmonary vascular bed in emphysema can result in increased PVR. However, the report of Hicken et al. contradicts this thinking, noting a poor correlation between the presence of RVH, a
Fig. 7 The response of the right and left ventricles to an acute increase in afterload. (Adapted with permission from MacNee [11]. The official journal of the American Thoracic Society, copyright American Thoracic Society)
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sign of significant cor pulmonale, and the total alveolar surface area, a reflection of the size of the pulmonary vascular bed [79]. Thus, the role of emphysematous reduction of the pulmonary capillary bed in the pathogenesis of cor pulmonale is unclear. Nonetheless, loss of the capillary vascular bed may contribute to the worsening of PH in these patients during exercise, which may expose a reduced reserve capacity to accommodate increased pulmonary blood flow [5].
1.6 RV Dysfunction The RV is a thin-walled, crescentic, compliant chamber formed by a concave free wall opposite to a convex interventricular septum. The RV both contracts against the septum in systole and also shortens in the long axis. Although the RV pumps the same stroke volume as the left ventricle, its stroke work is significantly less than that of the left ventricle since the PVR is significantly lower than the systemic vascular resistance. In view of its geometric configuration, the RV has a lower volume to surface area ratio than the left ventricle; thus, it acts as a volume pump rather than a pressure pump [80]. When the RV afterload increases acutely, as in acute pulmonary embolism, the RV cannot generate a mean PAP greater than 40 mmHg, and in such circumstances, the RV stroke volume decreases linearly with the increase in the afterload [11] (Fig. 7). In addition, the shortening of the RV in the long axis is diminished in PH, as reflected by a diminished tricuspid annular plane systolic excursion (TAPSE) on M-mode echocardiography. On the other hand, sustained pressure overload, secondary to chronic PH, leads to RVH and RV enlargement. In the presence of RVH, the perfusion pressure gradient between the right coronary artery, which supplies the RV both during systole and diastole, and the RV cavity decreases. The increased demand due to the hypertrophy coupled with the decreased supply results in a relative RV ischemia, leading to RV diastolic and systolic dysfunction, and eventually right-sided heart failure [81].
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PH associated with chronic lung disease is generally mild to moderate, not the systemic levels of PAP seen in patients with PAH [13]. In addition, PH in cor pulmonale tends to progress slowly over years, allowing the RV to adapt [15]. Thus, it is unclear whether the concept of sustained pressure overload should be used at all to describe the pathophysiological features of cor pulmonale [5]. Patients with moderate to severe COPD, but normal arterial PO2, usually have normal RV function during periods when their lung disease is stable [82]. Likewise, RV contractility, assessed by the end systolic pressure–volume relation, is generally preserved in patients with cor pulmonale when their underlying lung disease is stable [83, 84]. In patients with chronic lung disease, RV dysfunction and right-sided heart failure primarily occur during acute exacerbations of the lung disease, with sleep, during exercise, and when there is an acute increase in PAP (due to HPV, acidosis, or altered lung mechanics) [85–87]. Severe PH in a patient with chronic lung disease should elicit a search for other causes of PH, making cor pulmonale a diagnosis of exclusion.
1.7 Clinical Presentation Dyspnea is the most common symptom of cor pulmonale, but clinical recognition is difficult as most patients with chronic lung disease often have dyspnea. The presence of other symptoms such as chest pain, light-headedness, syncope, and worsening dyspnea may indicate the presence of cor pulmonale [88]. Typical physical examination findings suggestive of PH and RVH such as an elevated jugular venous distension, a palpable RV heave, a loud pulmonic component of the second heart sound, a holosystolic murmur secondary to tricuspid regurgitation, and an early diastolic murmur, due to pulmonary regurgitation, may be masked by the hyperinflation of the chest [7]. However, cor pulmonale patients tend to have elevated jugular venous pressure, ascites, and edema when they develop decompensated right-sided heart failure. It is vital to recognize that edema in patients with cor pulmonale is not always synonymous with right-sided heart failure. Some patients may develop edema even in the absence of right-sided heart failure. Although the exact mechanism for this is unclear, hypercapnia and hypoxia have been suggested to cause renal vasoconstriction, leading to salt and water retention [89].
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2.1 Electrocardiography The most common electrocardiographic signs of cor pulmonale include an enlarged P wave in lead II (P-pulmonale), rightward P-wave axis deviation, and S1Q3T3 pattern (measuring a dominant S wave in lead I, a Q wave in III, and an inverted T wave in lead III) (Fig. 8). Other electrocardiographic signs include evidence of RVH, rightward QRS axis deviation, and right bundle branch block [90]. There are several criteria for RVH, but generally tall R waves in the anteriorly and rightward directed leads (aVR, V1, V2) and deep S waves with small R waves in the leftward-directed leads (I, aVL, and lateral precordial leads) suggest RVH. Patients with cor pulmonale associated with COPD have been reported to have low QRS voltage because of hyperinflated lungs that can decrease the amplitude of EKG deflections and often have delayed R-wave progression, rather than R-wave predominance in the precordial leads. The amplitude of the R wave in V1 and the R/S ratio in V2 correlate with PAP in patients with cor pulmonale [91]. Unfortunately, EKG findings are not very sensitive for diagnosing cor pulmonale. In a study of 68 patients with COPD, electrocardiography had a specificity of 86% but a sensitivity of only 51% for diagnosing cor pulmonale compared with RHC. The sensitivity was particularly low (38%) in patients with mild PH [92]. Thus, EKG changes suggestive of right-sided heart involvement in patients with chronic lung disease if present can identify cor pulmonale; however, a normal EKG does not exclude it.
2.2 Chest Radiography Chest radiography is inferior to electrocardiography for the diagnosis of cor pulmonale. The classic radiographic signs of cor pulmonale on chest X-ray (Fig. 9) include enlargement of the main pulmonary artery and its branches, with marked tapering of the vessels in the lung periphery, known as “pruning.” In more advanced stages, when the RV is enlarged, one can see filling in of the retrosternal space on the lateral view. In patients with COPD, the presence of cor pulmonale has been related to a right descending pulmonary artery diameter greater than18 mm [93]. Unfortunately a normal chest X-ray does not rule out the presence of mild cor pulmonale. The value of the chest radiograph is that it is relatively inexpensive, easily performed, and can help to exclude other underlying lung disease and pulmonary venous hypertension.
2 Diagnostic Evaluation Because the clinical signs of cor pulmonale are relatively insensitive, noninvasive testing is a useful adjunct to enable early diagnosis and to quantify PH, RVH, and RV volume/ function.
2.3 Echocardiography Transthoracic echocardiography (TTE) is the most commonly used noninvasive screening test for quantifying cor pulmonale.
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Fig. 8 The electrocardiogram in cor pulmonale. A typical electrocardiogram from a patient with cor pulmonale demonstrating right-axis deviation, right ventricular strain pattern, low voltage in the limb leads, and delayed R-wave transition
Fig. 9 The chest X-ray in cor pulmonale. Typical chest X-ray seen from a patient with sarcoidosis-associated cor pulmonale. Note the prominent pulmonary arteries, pruning of vessels at the periphery, and right ventricular enlargement
TTE can detect elevated PAP, evaluate RV function, and rule out other causes of PH such as left-sided heart disease, shunting, and congenital heart disease (Fig. 10). Pulmonary artery systolic pressure (PASP) is equivalent to the RV systolic pressure (RVSP) in the absence of pulmonary outflow
obstruction. RVSP can be estimated by the measurement of the peak velocity of the tricuspid regurgitant jet, on continuous-wave Doppler echocardiography, using the simplified Bernoulli equation. The RVSP is calculated as 4V2, where V is the peak velocity of the tricuspid regurgitant flow with contin-
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Fig. 10 The echocardiogram in cor pulmonale. Echocardiogram seen in the apical four chambers and short-axis views from a patient with severe pulmonary hypertension. Left: Apical four-chamber view. Right: Parasternal short axis view
Fig. 11 Assessment of pulmonary artery pressure in cor pulmonale using continuous-wave Doppler echocardiography of tricuspid regurgitation
uous-wave Doppler echocardiography. The formula requires estimation of the mean right atrial pressure (RAP). PASP = RVSP = 4V2 + RAP [94] (Figs. 11, 12). The RAP can be estimated by observing the dimension and collapse of the inferior vena cava with a maneuver such as inspiration [95] (Table 4). If the inferior vena cava collapses more than 50% with inspiration, the RAP is usually less than 10 mmHg [96]. Doppler-estimated PASP correlates well with cathetermeasured PASP in patients with cardiac disease [97, 98]; however, this method has two major limitations in patients with chronic lung disease. First, it can be difficult to visualize tricuspid regurgitant jet in patients with chronic lung disease owing to hyperinflation of the lungs and a poor acoustic window, with the rate of an adequate examination ranging from 24 to 76%. This is particularly true in patients with obstructive lung disease and in those with a residual volume greater than 150% of that predicted. Second, Doppler-estimated PASP is frequently inaccurate in patients with chronic lung disease. In a cohort study of 374 lung transplant candidates, only 48% of patients had less than 10 mmHg discrepancy
between Doppler-estimated and catheter-measured PASP (Fig. 13). In the same study, the accuracy increased only modestly when the estimated RAP was replaced with cathetermeasured RAP. Fifty-two percent of pressure estimations were found to be inaccurate (more than 10 mmHg difference compared with the measured pressure), and 48% of patients were misclassified as having PH by echocardiography [12]. The sensitivity, specificity, and positive and negative predictive values of systolic PAP estimation for diagnosis of PH were 85, 55, 52, and 87%, respectively [12]. Morphologic and functional evaluation of the RV is important in the TTE screening of patients suspected to have cor pulmonale. When evaluating the morphology of the chambers of the right side of the heart, it is important to detect RVH, RV and right atrium volume, and the presence of a pericardial effusion. RVH is determined by measuring the thickness of the RV free wall at end-diastole. A value greater than 5 mm is abnormal and is considered to be a sign of increased afterload [99]; this, however, is not true for acute RV pressure overload, where these morphologic changes to the RV wall may not have had time to develop. RV volume is difficult to determine quantitatively by echocardiography, as the RV is a complex cavity that changes in orientation in the presence of different loading conditions and its relationship to the left ventricle. There have been several attempts at volume estimations by single plane dimensions (Fig. 14) and areas of geometric figures have shown poor correlation with volumes determined by angiography [100]. The use of real-time, threedimensional echo has improved the noninvasive echocardiographic assessment of RV volume and function [101]. RV function can be noninvasively evaluated in patients with cor pulmonale using several Doppler echocardiographic measurements. These measurements either assess the motion of the tricuspid annulus or measure time intervals in regard to contraction and relaxation times of the RV [102, 103]. The RV Tei index, defined as the ratio of the sum of the isovolumetric contraction time and the isovolumetric relaxation time to the ejection time, is a measure of global RV function. In general, Tei index increases with PH; a cutoff value of 0.47
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Fig. 12 Estimation of pulmonary artery pressure in cor pulmonale using Bernoulli’s equation. Estimation of right ventricular pressure by using the velocity of the tricuspid regurgitant jet. The velocity of the jet is 4.26 m/s. Using Bernoulli’s equation, the estimated right ventricular systolic pressure is 4 × (4.04)2 = 72 mmHg + the right atrial pressure (estimated from physical examination or assessment of inferior vena cava size and motion)
Table 4 Determination of the right atrial pressure using the inferior vena cava (IVC) size and collapsibility Estimation of mean right atrial pressure Right atrial pressure IVC diameter IVC collapsibility with (mmHg) (mm) respiration (%) 5 10 15 20
60%
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