Toshio Nakanishi H. Scott Baldwin Jeffrey R. Fineman Hiroyuki Yamagishi Editors Molecular Mechanism of Congenital Heart Disease and Pulmonary Hypertension Molecular Mechanism of Congenital Heart Disease and Pulmonary Hypertension Toshio Nakanishi • H. Scott Baldwin Jeffrey R. Fineman • Hiroyuki Yamagishi Editors Molecular Mechanism of Congenital Heart Disease and Pulmonary Hypertension Editors Toshio Nakanishi Department of Pediatric Cardiology Tokyo Women's Medical University Tokyo Japan Jeffrey R. Fineman UCSF Benioff Children's Hospital University of California San Francisco, CA USA H. Scott Baldwin Department of Pediatrics and Cell and Developmental Biology Vanderbilt University Medical Center Nashville, TN USA Hiroyuki Yamagishi Division of Pediatric Cardiology Department of Pediatrics Keio University School of Medicine Tokyo Japan This book is an open access publication. 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The registered company address is: 152 Beach Road, #21-01/04 Gateway East, Singapore 189721, Singapore v Preface How does the advance in science happen? Is it a sudden phenomenon like an unex- pected illumination or a result of collective addition of many small facts? This book is based on the 8th Takao International Symposium on Molecular Mechanism of Cardiopulmonary Disease—New Insight into the Development of Pulmonary Circulation and Ductus Arteriosus—in October 2017 in Matsue, Japan. The Takao International Symposium was first held in 1978 by Dr. Atsuyoshi Takao who was a professor and the chief of Pediatric Cardiology at the Heart Institute of Japan in Tokyo Women’s Medical University from 1972 to 1990 (passed away on August 8, 2006, at the age of 81). In the 1970s, Dr. Takao wanted to facilitate basic science in pediatric cardiology by holding international conferences, putting researchers together in a small place, and letting them discuss the etiology of con- genital heart diseases (CHD). Since the success of the first symposium, it has been held every 4–5 years and after he passed away, twice in recent 5 years. The proceed- ing books were published after each symposium in 1980, 1984, 1990, 1995, 2000, 2005, 2015, and this time in 2020. It is amazing that so much advance in science has been accomplished since the first Takao symposium, with path-breaking changes occurring in fields ranging from experimental teratology to molecular biology. At this time of the 8th Takao symposium, we, organizers, thought that it might be a good time to change the field of discussion somewhat from cardiac morphogenesis to lung development. Pulmonary circulation consists of airway, lung parenchyma, and pulmonary vessels, and each component can have diseases closely related with CHD. Development of the lung in the embryonic stage may have a common path- way of heart development. Researchers in the field of cardiac morphogenesis may be interested to talk with researchers in the field of pulmonary circulation. Also, between the heart and lung, there is a ductus arteriosus, which has unique character- istics, although it is so close to the pulmonary artery. Thus, the 8th Takao sympo- sium focused on new insights into the development of pulmonary circulation as well as basic science of pulmonary hypertension (PH) and ductus arteriosus. The aim of the meeting was to mix old friends studying cardiac morphogenesis with new friends studying pulmonary circulation and ductus arteriosus. Although PH in the field of pediatric cardiology shares common features of adult PH, it is associated with diverse diseases with onset at any age. The etiology of pediatric PH is quite different to that of adults, including idiopathic pulmonary arte- rial hypertension, PH associated with CHD, and/or developmental lung diseases. vi Recently, medical and surgical treatment of CHD has been well established, and the associated PH and/or pulmonary arterial disorders have become important causes of morbidity and mortality in children. The management of such children remains challenging as treatments are basically dependent on evidence-based studies in adult and/or the clinical expertise of pediatric cardiologists. Moreover, there is still a lack of data on treatment strategies, effectiveness, pharmacokinetics as well as basic science of pulmonary vascular development and disorders. The ductus arteriosus is an essential fetal structure, which provides a fetal arterial shunt between the main pulmonary artery and the descending aorta. Normally, the ductus arteriosus begins to close immediately after birth, but in some cases, it remains patent after birth and results in severe complications including PH, right ventricular dysfunction, and respiratory failure. On the other hand, patent ductus arteriosus after birth is required for patients with some complex CHD in which the systemic or pulmonary circulation is dependent on the blood flow through the duc- tus arteriosus. Understanding of the precise molecular mechanism underlying duc- tus closure is, therefore, important in the field of pediatric cardiology. The final step of ductus closure is as a result of vascular remodeling events driven by the cascade of signaling pathways that have not been fully elucidated. In this proceedings book, many interesting results in the field of “Basic Science of Lungs and Pulmonary Circulation,” “Clinical/Translational Science of Pulmonary Hypertension,” “Basic and Clinical Science of Ductus Arteriosus and Vessels,” as well as “Development and Regeneration of Cardiovascular System” are presented. These interactive chapters must contribute to an enthusiastic and exciting advance in science. We hope to shed light on the basic mechanisms of PH by studying basic sci- ence in the development of the lung, pulmonary vessels, ductus arteriosus, and heart. Finally, we would like to note that the Takao symposium and its publications have been supported by the Akemi-chan Fund from The Sankei press since its inception. The Akemi-chan Fund was set up over 50 years ago to save the life of Akemi-chan who was a Japanese girl with ventricular septal defect. Since that time, the fund has been supported by the donation from many persons from all over Japan. Also, we would like to appreciate the support from the Japanese Society of Pediatric Cardiology and Cardiac Surgery, Japan Research Promotion Society for Cardiovascular Diseases, and the Cardiovascular Development Forum in Japan. The purpose of this Forum is to study the etiology and management of CHD, from both basic and clinical aspects, as well as to exchange the results among the members in order to contribute to the development of medicine which totally overlaps with the concept propounded by Dr. Takao. We do hope that this book would shed light on the direction in which we should proceed in the research field in the next 5 years, to ultimately save the lives of those who suffer from congenital and acquired heart and lung diseases. Tokyo, Japan Toshio Nakanishi Tokyo, Japan Hiroyuki Yamagishi Preface vii Contents Part I Basic Science of Pulmonary Development and Pulmonary Arterial Disease 1 Perspective for Part I . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 Hiroyuki Yamagishi 2 The Alveolar Stem Cell Niche of the Mammalian Lung . . . . . . . . . . . . 7 Brigid L. M. Hogan 3 Lung Development and Notch Signaling . . . . . . . . . . . . . . . . . . . . . . . . 13 Mitsuru Morimoto 4 Specialized Smooth Muscle Cell Progenitors in Pulmonary Hypertension . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 Fatima Zahra Saddouk, Aglaia Ntokou, and Daniel M. Greif 5 Diverse Pharmacology of Prostacyclin Mimetics: Implications for Pulmonary Hypertension . . . . . . . . . . . . . . . . . . . . . . . 31 Lucie H. Clapp, Jeries H. J. Abu-Hanna, and Jigisha A. Patel 6 Endothelial-to-Mesenchymal Transition in Pulmonary Hypertension . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63 Benoît Ranchoux, Virginie F. Tanguay, and Frédéric Perros 7 Extracellular Vesicles, MicroRNAs, and Pulmonary Hypertension . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71 Tianji Chen and J. Usha Raj 8 Roles of Tbx4 in the Lung Mesenchyme for Airway and Vascular Development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79 Keiko Uchida, Yu Yoshida, Kazuki Kodo, and Hiroyuki Yamagishi 9 A lacZ Reporter Transgenic Mouse Line Revealing the Development of Pulmonary Artery . . . . . . . . . . . . . . . . . . . . . . . . . . 83 Reina Ishizaki, Keiko Uchida, Akimichi Shibata, Takatoshi Tsuchihashi, Jun Maeda, Katsuhiko Mikoshiba, and Hiroyuki Yamagishi viii 10 Roles of Stem Cell Antigen-1 in the Pulmonary Endothelium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 87 Jun Maeda, Keiko Uchida, Kazuki Kodo, and Hiroyuki Yamagishi 11 Morphological Characterization of Pulmonary Microvascular Disease in Bronchopulmonary Dysplasia Caused by Hyperoxia in Newborn Mice . . . . . . . . . . . . . . . . . . . . . . . . . 91 Hidehiko Nakanishi, Shunichi Morikawa, Shuji Kitahara, Asuka Yoshii, Atsushi Uchiyama, Satoshi Kusuda, and Taichi Ezaki 12 Involvement of CXCR4 and Stem Cells in a Rat Model of Pulmonary Arterial Hypertension . . . . . . . . . . . . . . . . . . . . . . . . . . . 95 Tingting Zhang, Nanako Kawaguchi, Emiko Hayama, Yoshiyuki Furutani, and Toshio Nakanishi 13 Ca 2+ Signal Through Inositol Trisphosphate Receptors for Cardiovascular Development and Pathophysiology of Pulmonary Arterial Hypertension . . . . . . . . . . . . . . . . . . . . . . . . . . . 97 Akimichi Shibata, Keiko Uchida, Katsuhiko Mikoshiba, and Hiroyuki Yamagishi Part II Abnormal Pulmonary Circulation in the Developing Lung and Heart 14 Perspective for Part II . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 103 Toshio Nakanishi 15 Pathophysiology of Pulmonary Circulation in Congenital Heart Disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 109 Megumi Hashimoto, Masahiro Kaneko, Naohisa Nishida, Hina Serizawa, Kohei Kawata, Rika Sekiya, and Hideaki Senzaki 16 Development of Novel Therapies for Pulmonary Hypertension by Clinical Application of Basic Research . . . . . . . . . . . . . . . . . . . . . . . 125 Kimio Satoh and Hiroaki Shimokawa 17 Using Patient-Specific Induced Pluripotent Stem Cells to Understand and Treat Pulmonary Arterial Hypertension . . . . . . . . . . 131 Mingxia Gu 18 Modeling Pulmonary Arterial Hypertension Using Induced Pluripotent Stem Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 139 Amer A. Rana, Fedir N. Kiskin, and C.-Hong Chang 19 Dysfunction and Restoration of Endothelial Cell Communications in Pulmonary Arterial Hypertension: Therapeutic Implications . . . . 147 Christophe Guignabert Contents ix 20 Inflammatory Cytokines in the Pathogenesis of Pulmonary Arterial Hypertension . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 157 Yoshikazu Nakaoka, Tadakatsu Inagaki, and Mikiyasu Shirai 21 Genotypes and Phenotypes of Chinese Pediatric Patients with Idiopathic and Heritable Pulmonary Arterial Hypertension: Experiences from a Single Center . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 163 Hong-Sheng Zhang, Qian Liu, Chun-Mei Piao, Yan Zhu, Qiang-Qiang Li, Jie Du, and Hong Gu 22 Fundamental Insight into Pulmonary Vascular Disease: Perspectives from Pediatric PAH in Japan . . . . . . . . . . . . . . . . . . . . . . . 173 Yoshihide Mitani and Hirofumi Sawada 23 Risk Stratification in Paediatric Pulmonary Arterial Hypertension . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 177 Shahin Moledina 24 The Adaptive Right Ventricle in Eisenmenger Syndrome: Potential Therapeutic Targets for Pulmonary Hypertension? . . . . . . . 183 Rebecca Johnson Kameny, Sanjeev A. Datar, Jason Boehme, and Jeffrey R. Fineman 25 Impaired Right Coronary Vasodilator Function in Pulmonary Hypertensive Rats Assessed by In Vivo Synchrotron Microangiography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 193 Tadakatsu Inagaki, Hirotsugu Tsuchimochi, James T. Pearson, Daryl O. Schwenke, Keiji Umetani, Mikiyasu Shirai, and Yoshikazu Nakaoka 26 Relationship Between Mutations in ENG and ALK1 Genes and the Affected Organs in Hereditary Hemorrhagic Telangiectasia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 197 Toru Iwasa, Osamu Yamada, Hiroko Morisaki, Takayuki Morisaki, Ken-ichi Kurosaki, and Isao Shiraishi 27 A Genetic Analysis for Patients with Pulmonary Arterial Hypertension . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 201 Yu Yoshida, Keiko Uchida, Kazuki Kodo, Yoshiyuki Furutani, Toshio Nakanishi, and Hiroyuki Yamagishi 28 Evaluation and Visualization of the Right Ventricle Using Three-Dimensional Echocardiography . . . . . . . . . . . . . . . . . . . . . . . . . . 205 Yui Ito, Yusaku Kamiya, Azuma Ikari, and Katsuaki Toyoshima 29 Pulmonary Hypertension Associated with Postoperative Tetralogy of Fallot . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 209 Jun Yasuhara and Hiroyuki Yamagishi Contents x 30 Microscopic Lung Airway Abnormality and Pulmonary Vascular Disease Associated with Congenital Systemic-to- Pulmonary Shunt . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 213 Hirofumi Sawada, Yoshihide Mitani, Hiroyuki Ohashi, Shoichiro Otsuki, Noriko Yodoya, Hidetoshi Hayakawa, Hironori Oshita, Jane C. Kabwe, Takeshi Konuma, Kyoko Imanaka-Yoshida, Hideto Shimpo, Kazuo Maruyama, and Masahiro Hirayama 31 Respiratory Syncytial Virus Infection in Infants with Heart and Lung Diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 215 Hiroyuki Yamagishi Part III Ductus Arteriosus: Bridge Over Troubled Vessels 32 Perspective for Part III . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 223 Toshio Nakanishi 33 The Ductus Arteriosus, a Vascular Outsider, in Relation to the Pulmonary Circulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 227 Adriana C. Gittenberger-de Groot, Arno A. W. Roest, Regina Bökenkamp, Monique R. M. Jongbloed, Margot M. Bartelings, Marco C. DeRuiter, and Robert E. Poelmann 34 Molecular, Genetic, and Pharmacological Modulation of the Ductus Arteriosus: K ATP Channels as Novel Drug Targets . . . . . . . 235 Elaine L. Shelton and Jerod S. Denton 35 New Mediators in the Biology of the Ductus Arteriosus: Lessons from the Chicken Embryo . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 243 Saskia van der Sterren, Riazudin Mohammed, and Eduardo Villamor 36 Constriction of the Ductus Arteriosus with K ATP Channel Inhibitors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 251 Kazuo Momma, Katsuaki Toyoshima, Emiko Hayama, and Toshio Nakanishi 37 New Insights on How to Treat Patent Ductus Arteriosus . . . . . . . . . . . 259 Utako Yokoyama, Rika Aoki, Shujiro Fujita, Shiho Iwasaki, Kazuo Seki, Asou Toshihide, Munetaka Masuda, Susumu Minamisawa, and Yoshihiro Ishikawa 38 Antenatal Administration of Betamethasone Contributes to Intimal Thickening of the Ductus Arteriosus . . . . . . . . . . . . . . . . . . . 265 Takahiro Kemmotsu, Utako Yokoyama, Junichi Saito, Satoko Ito, Azusa Uozumi, Shiho Iwasaki, Shigeru Nishimaki, Shuichi Ito, Munetaka Masuda, Toshihide Asou, and Yoshihiro Ishikawa Contents xi 39 Prostaglandin E-EP4-Mediated Fibulin-1 Up-regulation Plays a Role in Intimal Thickening of the Ductus Arteriosus . . . . . . . . . . . . . 267 Satoko Ito, Utako Yokoyama, Junichi Saito, Munetaka Masuda, Toshihide Asou, and Yoshihiro Ishikawa 40 Transcriptional Profiles in the Chicken Ductus Arteriosus During Hatching . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 269 Satoko Shinjo, Toru Akaike, Eriko Ohmori, Ichige Kajimura, Nobuhito Goda, and Susumu Minamisawa 41 Inhibition of Cyclooxygenase Contracts Chicken Ductus Arteriosus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 273 Toru Akaike and Susumu Minamisawa 42 Prostaglandin E 2 Receptor EP4 Inhibition Constricts the Rat Ductus Arteriosus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 275 Toshiki Sakuma, Toru Akaike, and Susumu Minamisawa 43 Dilatation of the Ductus Arteriosus by Diazoxide in Fetal and Neonatal Rats . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 277 Katsuaki Toyoshima, Kazuo Momma, Tetsuko Ishii, and Toshio Nakanishi 44 The Effect of Long-term Administration of Prostaglandin E1 on Morphological Changes in Ductus Arteriosus . . . . . . . . . . . . . . . . . 281 Ryuma Iwaki, Hironori Matsuhisa, Susumu Minamisawa, Toru Akaike, Masato Hoshino, Naoto Yagi, Kiyozo Morita, Gen Shinohara, Yukihiro Kaneko, Syuichi Yoshitake, Masashi Takahashi, Takuro Tsukube, and Yoshihiro Oshima 45 Significance of SGK1 as a Protein Kinase Transcriptionally Regulated by ALK1 Signaling in Vascular Endothelial Cells . . . . . . . . 285 Osamu Nakagawa, Yusuke Watanabe, Yukihiro Harada, Toru Tanaka, and Teruhisa Kawamura 46 Fabrication of Implantable Human Arterial Graft by Periodic Hydrostatic Pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 289 Junichi Saito, Utako Yokoyama, Toshio Takayama, Hiroaki Ito, Tomomi Tadokoro, Yoshinobu Sugo, Kentaro Kurasawa, Miyuki Ogawa, Etsuko Miyagi, Hideki Taniguchi, Makoto Kaneko, and Yoshihiro Ishikawa 47 Optimum Preparation of Candida albicans Cell Wall Extra (CAWE) for the Mouse Model of Kawasaki Disease . . . . . . . . . . . . . . . 293 Yukako Yoshikane, Tamaki Cho, Mitsuhisa Koga, Seiji Haraoka, and Atsushi Ogawa Contents xii Part IV Development and Regeneration of the Cardiovascular System 48 Perspective for Part IV . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 297 H. Scott Baldwin 49 Advances in the Second Heart Field . . . . . . . . . . . . . . . . . . . . . . . . . . . . 301 Robert G. Kelly 50 Novel Cardiac Progenitors for All Components of the Heart Except for the Right Ventricle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 309 Hiroki Kokubo, Masayuki Fujii, Akane Sakaguchi, Masao Yoshizumi, and Yumiko Saga 51 Regional and TBX5- Dependent Gene Expression in the Atria: Implications for Pulmonary Vein Development and Atrial Fibrillation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 315 Jeffrey D. Steimle, Brigitte Laforest, Rangarajan D. Nadadur, Michael T. Broman, and Ivan P. Moskowitz 52 The Endocardium as a Master Regulator of Ventricular Trabeculation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 331 Xianghu Qu and H. Scott Baldwin 53 The Role of Alternative mRNA Splicing in Heart Development . . . . . 339 Douglas C. Bittel, Nataliya Kibiryeva, Naoya Kenmochi, Prakash Patil, Tamayo Uechi, Brenda Rongish, Mike Filla, Jennifer Marshall, Michael Artman, Rajasingh Johnson, and James E. O’Brien Jr 54 Progress in the Generation of Multiple Lineage Human-iPSC-Derived 3D-Engineered Cardiac Tissues for Cardiac Repair . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 353 Fei Ye, Shuji Setozaki, William J. Kowalski, Marc Dwenger, Fangping Yuan, Joseph P. Tinney, Takeichiro Nakane, Hidetoshi Masumoto, and Bradley B. Keller 55 Quantification of Contractility in Stem Cell-Derived Cardiomyocytes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 363 Konrad Brockmeier, Moritz Haustein, Markus Khalil, and Tobias Hannes 56 A Neurotrophic Factor Receptor GFRA2, a Specific Surface Antigen for Cardiac Progenitor Cells, Regulates the Process of Myocardial Compaction . . . . . . . . . . . . . . . . . . . . . . . . . . 369 Hidekazu Ishida, Shigeru Miyagawa, Keiichi Ozono, Ken Suzuki, Yoshiki Sawa, and Kenta Yashiro 57 Cardiac Cell Specification by Defined Factors . . . . . . . . . . . . . . . . . . . . 373 Yuika Morita and Jun Takeuchi Contents xiii 58 A Temporo-Spatial Regulation of Sema3c Is Essential for Interaction of Progenitor Cells during Cardiac Outflow Tract Development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 377 Kazuki Kodo, Shinsuke Shibata, Sachiko Miyagawa-Tomita, Sang-Ging Ong, Hiroshi Takahashi, Tsutomu Kume, Hideyuki Okano, Rumiko Matsuoka, and Hiroyuki Yamagishi 59 Spatiotemporally Restricted Developmental Alterations in the Anterior and Secondary Heart Fields Cause Distinct Conotruncal Heart Defects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 381 Mayu Narematsu and Yuji Nakajima 60 Significance of Transcription Factors in the Mechanisms of Great Artery Malformations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 385 Yusuke Watanabe and Osamu Nakagawa 61 The Different c-kit Expression in Human Induced Pluripotent Stem (iPS) Cells Between With Feeder Cells and Without Feeder Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 389 Nanako Kawaguchi, Emiko Hayama, Yoshiyuki Furutani, and Toshio Nakanishi 62 Establishment of Induced Pluripotent Stem Cells from Immortalized B Cell Lines and Their Differentiation into Cardiomyocytes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 391 Emiko Hayama, Yoshiyuki Furutani, Nanako Kawaguchi, Eiko Oomichi, Mitsuyo Shimada, Kei Inai, and Toshio Nakanishi 63 Establishment of an In Vitro LQT3 model Using Induced Pluripotent Stem Cells from LQT3 Patient-Derived Cardiomyocytes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 393 Yoshiyuki Furutani, Emiko Hayama, Nanako Kawaguchi, Yasuhiro Katsube, Eiko Oomichi, Mitsuyo Shimada, Kei Inai, and Toshio Nakanishi 64 Genetic Assessments for Clinical Courses of Left Ventricle Noncompaction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 395 Yoshitsugu Nogimori, Akio Kato, Kazuhisa Sato, Yosuke Kitagawa, Takuya Wakamiya, Shin Ono, Ki-Sung Kim, Sadamitsu Yanagi, and Hideaki Ueda 65 Elucidating the Pathogenesis of Congenital Heart Disease in the Era of Next-Generation Sequencing . . . . . . . . . . . . . . . . . . . . . . . 397 Yu Nakagama and Ryo Inuzuka Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 399 Contents Part I Basic Science of Pulmonary Development and Pulmonary Arterial Disease 3 © The Editor(s) (if applicable) and The Author(s) 2020 T. Nakanishi et al. (eds.), Molecular Mechanism of Congenital Heart Disease and Pulmonary Hypertension , https://doi.org/10.1007/978-981-15-1185-1_1 H. Yamagishi ( * ) Division of Pediatric Cardiology, Department of Pediatrics, Keio University School of Medicine, Tokyo, Japan e-mail: hyamag@keio.jp 1 Perspective for Part I Hiroyuki Yamagishi The lungs are the primary organs of the respiratory system in vertebrates, other than fish, that function as an efficient gas exchanger. This respiratory system is physi- cally and physiologically connected to the cardiovascular system to extract oxygen from the atmosphere and transfer it into the bloodstream and to release carbon diox- ide from the bloodstream into the atmosphere. The normal pulmonary circulation, in contrast to the systemic circulation, has low pressure because of a low-resistant vascular bed that enables highly efficient gas exchange between the millions of alveoli and capillaries. Pulmonary hypertension (PH) is a rare pulmonary arterial disease with a high mortality and morbidity although treatment options have improved in the last decades. PH is defined by a mean pulmonary artery pressure (mPAP) greater than or equal to 20 mmHg at rest and is classified into five main subgroups based on the etiology and hemodynamic criteria. Because PH is hetero- geneous at the genetic and molecular levels, a deeper understanding is required for the development of more effective therapies. In Part I, the basic science for the development of the lungs and the pathogenesis of PH is discussed towards a better understanding of the etiology and pathophysiol- ogy of PH. Drs. Hogan and Morimoto overview the morphogenesis and epithelial development of the lung. Each alveolus in lungs is composed of epithelial and mes- enchymal populations. Dr. Hogan (Chap. 2) focuses on the interesting function of Type 2 epithelial cells, as epithelial stem cells for the alveoli, capable of long-term self-renewal and differentiation into Type 1 epithelial cells that are extremely large and thin to be specialized for gas exchange. The relative contribution of the WNT, FGF, EGF, and BMP signaling pathways as well as cytokines produced by immune 4 cells to alveolar homeostasis, repair and regrowth is discussed. Dr. Morimoto (Chap. 3) focuses on Notch signaling as a reciprocal mesenchymal-epithelial inter- action that controls the development of the airway branching structure. Notch- mediated cell fate selection is utilized to organize three epithelial cell types during the bronchial tree is extending. As for the mesenchymal population that gives rise to endothelial cells, pericytes, several different fibroblast subpopulations, and immune cells, Dr. Uchida et al. (Chap. 8) suggest that the expression of a transcription factor Tbx4 in the lung mesenchyme may be involved in the formation of both airways and vessels in the lungs using animal experiments. It is also known that mutations of the TBX4 gene lead to PH in human. Dr. Ishizaki et al. (Chap. 9) demonstrate that pul- monary arterial smooth muscle layers, probably derived from mesenchyme of the mesodermal origin, may elongate gradually from the proximal pulmonary arterial trunk to bilateral peripheral pulmonary arteries by using a transgenic mouse line harboring the LacZ reporter gene in the specific locus. This observation supports the “distal angiogenesis” model for pulmonary arterial development. PH is also known as a progressive pulmonary vascular remodeling disease. Histopathological changes in PH include an increase in smooth muscle cells in the pulmonary vasculature with muscularization of normally non-muscularized distal pulmonary arterioles. Dr. Greif’s team (Chap. 4) identified novel smooth muscle cell progenitors located at the muscular-unmuscular arteriole border that have a unique molecular signature and are the source of the vast majority of pathological distal arteriole smooth muscle cells in PH. Molecular mechanisms underlying induction, proliferation, migration, and differentiation of these specialized progeni- tor cells are discussed. In addition to the above-described fashions to increase smooth muscle cells in the pulmonary vasculature of PH, recent studies have dem- onstrated that a part of smooth muscle cells from intimal and plexiform lesions has an endothelial origin through the process of endothelial-mesenchymal transition (EMT). Dr. Ranchoux et al. (Chap. 6) describe how EMT appears to play a crucial role in PH progression including the underlying molecular mechanisms. As another advanced understanding for pathogenesis of PH, Drs. Chen and Raj (Chap. 7) focus on extracellular vesicles. Subsets of extracellular vesicles are microvesicles, exo- somes, and apoptotic bodies. They are released from a variety of cell types and carry cargo such as proteins and microRNAs that may play an important role in the patho- genesis of PH. Utilizing animal models, three important findings about the pathogenesis of PH are also discussed in Part I. Dr. Nakanishi et al. (Chap. 11) show morphological changes of pulmonary microvasculature in the mouse model of bronchopulmonary dysplasia (BPD). As BPD is often associated with PH, their study may provide insights into secondary PH due to pulmonary diseases. Dr. Zhang et al. (Chap. 12) identify significant increased expression of chemokine receptor type 4 (CXCR4) and some stem cell markers in PH model rats as putative mediators of pulmonary vascular remodeling. Dr. Shibata et al. (Chap. 13) demonstrate that Ca 2+ signal through the inositol trisphosphate receptor (IP 3R) may be involved in the patho- physiology of PH using IP 3R knockout mice. CXCR4 or IP 3R might be a potential candidate for new therapeutic targets of PH. H. Yamagishi 5 Finally, Dr. Clapp’s group (Chap. 5) discuss about basic science for the treatment of PH. Although there were no treatments available for PH 20 years ago, current therapies come from four drug classes, namely, prostanoid analogs, endothelin receptor antagonists, phosphodiesterase type 5 inhibitors, and soluble guanylate cyclase stimulators, and through focused research, there are more in development. Intravenous prostacyclin, as a prostanoid analog, remains the most efficacious treat- ment for PH, and several prostacyclin analogs are approved for use via different administration routes. They act as vasodilators but potently inhibit platelet aggrega- tion, cell proliferation, and inflammation. Dr. Clapp et al. discuss how prostacyclins might rescue BMPR2 and TASK-1 dysfunction and the importance of prostanoid EP2 receptors as negative modulators of vascular tone, proliferation, and fibrosis. BMPR2 is known as the most important genetic cause of PH, and TASK-1 is a TWIK-related acid-sensitive potassium channel that, they believe, is likely to be a key target for prostacyclins. As basic research for future stem cell therapy of PH, Dr. Maeda et al. (Chap. 10) suggest that stem cell antigen-1 in the pulmonary endothe- lium may potentially make progenitor cell populations stay resident in adult murine lungs. The field of pulmonary vascular biology continues to capitalize on advances in basic science. Part I includes a number of exciting advances in basic science and how these advances are impacting the patient with PH. This knowledge should be required for physicians and scientists to be translated into future clinical practice. Open Access This chapter is licensed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license and indicate if changes were made. The images or other third party material in this chapter are included in the chapter's Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the chapter's Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. 1 Perspective for Part I 7 © The Editor(s) (if applicable) and The Author(s) 2020 T. Nakanishi et al. (eds.), Molecular Mechanism of Congenital Heart Disease and Pulmonary Hypertension , https://doi.org/10.1007/978-981-15-1185-1_2 B. L. M. Hogan ( * ) Department of Cell Biology, Duke University Medical School, Durham, NC, USA e-mail: brigid.hogan@duke.edu 2 The Alveolar Stem Cell Niche of the Mammalian Lung Brigid L. M. Hogan Abstract The alveolar region of the mammalian lung evolved to enable highly efficient gas exchange between the millions of air-filled sacs known as alveoli and blood cir- culating through the pulmonary vessels. Each alveolus is composed of epithelial and mesenchymal populations; Type 1 (AT1) and Type 2 (AT2) epithelial cells line the sacs while the mesenchymal compartment is composed of endothelial cells, pericytes, several different fibroblast subpopulations, and immune cells. Compared with organs such as the intestine and skin, there is normally little cell turnover in the adult lung. However, if the alveolar epithelium is damaged, for example, by toxic agents or viral infections, or if a lung lobe is removed, there is extensive repair or compensatory regrowth (neoalveolarization) of the tissue. It is now well accepted that AT2s function as epithelial stem cells for the alveoli, capable of long-term self-renewal and differentiation into AT1s. However, many important questions remain, in particular, about the functional heterogeneity of AT2s and how the different components of their local environment or niche inter- act to regulate their proliferation and differentiation. Known signaling factors include ligands and antagonists of the WNT, FGF, EGF and bone morphogenetic (BMP) signaling pathways as well as cytokines produced by immune cells. The relative contribution of these factors to alveolar homeostasis, repair and regrowth is discussed. Keywords Lung · Alveolar stem cell niche · Compensatory regrowth · Signaling pathways 8 2.1 Introduction: The Alveolar Type 2 Epithelial Stem Cell Niche Our current understanding of the alveolar stem cell niche of the mammalian lung is schematized in Fig. 2.1 [1–4]. Each alveolus contains two kinds of epithelial cells: Type 2 cells (AT2s), specialized for producing, secreting and recycling surfactant lipids and proteins, and Type 1 cells (AT1s) that are extremely large and thin and specialized for gas exchange [5]. It is now well accepted AT2s that express surfac- tant protein C (Sftpc) can function as stem cells during homeostasis and repair by self-renewing and giving rise to AT1s. However, the rate at which AT2s proliferate and differentiate depends on local “demand.” At steady state the rate is very low. If lungs are experimentally depleted of only AT2s then their proliferation increases but differentiation is still minimal [6]. However, if AT1s are also damaged or if whole new alveoli need to be generated in response to partial (left lobe) pneumonectomy (PPnx), then AT2s both self-renew and show robust AT1 differentiation [7–9]. How AT2s detect the absence of AT1s is unclear; perhaps they sense denuded basal lam- ina or a reduction in cellular contacts with AT1s or, in the case of PPnx, an increase in mechanical tension [10]. Matrix fibroblast Septum with myofibroblast Airway smooth muscle Bronchiolalveolar duct junction Alveolar macrophage Type2 Type1 Endothelial cell and pericyte TASC Interstitial immune cell Fig. 2.1 Schematic representation of the alveolar region of the mouse lung. The Type 2 stem cell niche (boxed) includes fibroblasts known as TASCs that have long cytoplasmic extensions, AT1 cells, capillary endothelial cells and pericytes as well as alveolar macrophages and interstitial immune cells. Alveolar septae contain myofibroblasts, and other fibroblast cell types (matrix fibro- blasts) are present in the stroma B. L. M. Hogan 9 2.2 Evidence for Heterogeneity in the AT2 Population An important unanswered question is whether all AT2s have the same stem cell potential or whether this is a property of a privileged few. Significantly, two groups have recently shown that at steady state a minority of AT2s express a reporter for Axin2 , a downstream target of canonical Wnt signaling. One group [11] found that only 1% of the AT2s were positive although more cells became active in response to injury. By contrast, a second group [12] reported that 20–30% of AT2s were positive at steady state and constituted a stable subpopulation which they termed alveolar epithelial progenitors (AEPs). In both cases evidence was presented that the Wnt ligand responsible for upregulating Axin2 and promoting AT2 proliferation is pro- duced by niche fibroblasts in close proximity to AT2s, termed MANCs (mesenchy- mal alveolar niche cells). These cells can also produce Wnt antagonists, which reduce AT2 proliferation and promote differentiation, and may, therefore, fine tune the dynamic response of the stem cells during repair [13]. In the light of these studies, one may ask whether AT2 subpopulations have been detected by single-cell transcriptomic analysis. One recent study does show a minor AT2 subpopulation in the steady state lung, defined as “alveolar bipotential progeni- tors” [14]. However, these cells are distinguished by co-expressing markers of AT2s and AT1s but not by Axin2 levels. In support of the single-cell RNA-seq data, a recent report [8] identified by immunohistochemistry a small subpopulation (less than 1%) of AT2s co-expressing Sftpc and Ager (advanced glycosylation and end product-specific receptor, a marker for AT1s). The proportion of these dual-positive cells increases to 20% by 7 days after PPnx when compensatory cell proliferation is active. In addition, when isolated AT2 cells are placed in 3D organoid culture under conditions in which single cells give rise to structures known as “alveolospheres” containing both AT2s and AT1s, all of the cells initially and transiently become dual positive for Sftpc and Ager [8]. This behavior may indicate a reversion of mature AT2s to a more plastic, bipotential state under conditions promoting repair. New genetic tools are clearly needed to test the in vivo significance of the dual-positive cells and their relation, if any, to Axin2+ epithelial cells [11, 12]. 2.3 Signaling Pathways in the Stem Cell Niche Besides WNTs and their antagonists, a number of other signaling factors have been identified as playing a role in the mouse lung alveolar stem cell niche. Pathways include those regulated by Fgfs (largely produced by fibroblast populations), Vegf (secreted by AT2s), Bmps and Egfs. Egf signaling is thought to be mediated both by the ligand itself and by peptides released from the extracellular matrix after injury. This latter mechanism has been proposed for the proliferation of AT2s after PPnx [15]. Specifically, it appears that in response to PPnx pulmonary capillary endothe- lial cells (PCECs) activate the expression of the matrix metalloproteinase MMP14. This degrades