Development of a Novel Mouse Model for Human Pulmonary Arterial Hypertension (PAH)

Permanent Link: http://ufdc.ufl.edu/UFE0021418/00001

Material Information

Title: Development of a Novel Mouse Model for Human Pulmonary Arterial Hypertension (PAH)
Physical Description: 1 online resource (136 p.)
Language: english
Creator: Hong, Kwonho
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2007


Subjects / Keywords: arterial, hypertension, model, mouse, pulmonary
Molecular Cell Biology (IDP) -- Dissertations, Academic -- UF
Genre: Medical Sciences thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation


Abstract: Pulmonary Arterial Hypertension (PAH) is a devastating vascular disorder predisposed by mutation of bone morphogenetic protein receptor type II (BMPR2). Given that the heterozygous BMPR2 mutation incompletely manifests the disease condition of PAH and the endothelial dysfunction has been implicated in PAH individuals, we hypothesized that deletion of Bmpr2 in the pulmonary endothelial cells (ECs) is sufficient to predispose to PAH. To test this hypothesis, a novel PAH model has been established by using the Cre/loxP system. To the end, first we characterized the dynamic pattern of Alk1 expression via in vivo dissection of Alk1 promoter and intron regions. Based on in silico DNA analysis, we isolated a 9.2 kb of Alk1 fragment which precisely recapitulates the endogenous pattern of Alk1 expression. Transgenic lines carrying the 9.2 kb of Alk1 promoter/enhancer-lacZ gene demonstrated a predominant Alk1 expression in the ECs of large arteries and some capillary beds during development. At adult stage, the level of Alk1 in the systemic ECs was greatly diminished, whereas the Alk1 level in the pulmonary ECs was persistant throughout the stage. Next, using the 9.2 kb of Alk1 promoter/enhancer we have developed a novel Cre deleter line, referred as Tg(Alk1-cre)-L1, in which an unique pattern of the Cre-mediated DNA excision in the pulmonary ECs was detected. In the Tg(Alk1-cre)-L1 line, a uniformly distributed Cre activity in the pulmonary ECs was detected from E15.5 on. The homogeneous pattern of Cre activity in the pulmonary ECs was persistant, while mosaic or no Cre activity was detected in the systemic ECs during adult stage. Finally, to investigate cellular impact of the impaired Bmpr2 signaling pathway in pulmonary ECs, the Tg(Alk1-cre)-L1 was bred with mice containing conditional Bmpr2 allele. The Alk1-cre(+);Bmpr2f/f mice were viable and seemingly indistinguishable from control littermates. In the hemodynamic and morphometric analyses, subsets of Alk1-cre(+);Bmpr2+/f and Alk1-cre(+);Bmpr2f/f mice revealed an elevation of right ventricular systolic pressure (RVSP) with increases in the number of muscularized distal arteries and thickness of the medial layer. It has been also demonstrated that the frequency of PAH in the groups was increased in an age-dependent manner. Furthermore, some of hypertensive mice exhibited signs of endothelial dysfunctions such as perivascular infiltration of leukocytes and in situ thrombosis. Taken together, we clearly demonstrated that the deletion of Bmpr2 gene in the pulmonary ECs by itself can predispose to PAH with abnormal vascular remodeling. Because our mouse model is the first genetic model which corresponds to pathophysiological features of human PAH, we believe that mouse lines we developed are useful genetic resources for further studies on elucidating the underlying mechanism of PAH.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Kwonho Hong.
Thesis: Thesis (Ph.D.)--University of Florida, 2007.
Local: Adviser: Oh, Suk P.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2007
System ID: UFE0021418:00001

Permanent Link: http://ufdc.ufl.edu/UFE0021418/00001

Material Information

Title: Development of a Novel Mouse Model for Human Pulmonary Arterial Hypertension (PAH)
Physical Description: 1 online resource (136 p.)
Language: english
Creator: Hong, Kwonho
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2007


Subjects / Keywords: arterial, hypertension, model, mouse, pulmonary
Molecular Cell Biology (IDP) -- Dissertations, Academic -- UF
Genre: Medical Sciences thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation


Abstract: Pulmonary Arterial Hypertension (PAH) is a devastating vascular disorder predisposed by mutation of bone morphogenetic protein receptor type II (BMPR2). Given that the heterozygous BMPR2 mutation incompletely manifests the disease condition of PAH and the endothelial dysfunction has been implicated in PAH individuals, we hypothesized that deletion of Bmpr2 in the pulmonary endothelial cells (ECs) is sufficient to predispose to PAH. To test this hypothesis, a novel PAH model has been established by using the Cre/loxP system. To the end, first we characterized the dynamic pattern of Alk1 expression via in vivo dissection of Alk1 promoter and intron regions. Based on in silico DNA analysis, we isolated a 9.2 kb of Alk1 fragment which precisely recapitulates the endogenous pattern of Alk1 expression. Transgenic lines carrying the 9.2 kb of Alk1 promoter/enhancer-lacZ gene demonstrated a predominant Alk1 expression in the ECs of large arteries and some capillary beds during development. At adult stage, the level of Alk1 in the systemic ECs was greatly diminished, whereas the Alk1 level in the pulmonary ECs was persistant throughout the stage. Next, using the 9.2 kb of Alk1 promoter/enhancer we have developed a novel Cre deleter line, referred as Tg(Alk1-cre)-L1, in which an unique pattern of the Cre-mediated DNA excision in the pulmonary ECs was detected. In the Tg(Alk1-cre)-L1 line, a uniformly distributed Cre activity in the pulmonary ECs was detected from E15.5 on. The homogeneous pattern of Cre activity in the pulmonary ECs was persistant, while mosaic or no Cre activity was detected in the systemic ECs during adult stage. Finally, to investigate cellular impact of the impaired Bmpr2 signaling pathway in pulmonary ECs, the Tg(Alk1-cre)-L1 was bred with mice containing conditional Bmpr2 allele. The Alk1-cre(+);Bmpr2f/f mice were viable and seemingly indistinguishable from control littermates. In the hemodynamic and morphometric analyses, subsets of Alk1-cre(+);Bmpr2+/f and Alk1-cre(+);Bmpr2f/f mice revealed an elevation of right ventricular systolic pressure (RVSP) with increases in the number of muscularized distal arteries and thickness of the medial layer. It has been also demonstrated that the frequency of PAH in the groups was increased in an age-dependent manner. Furthermore, some of hypertensive mice exhibited signs of endothelial dysfunctions such as perivascular infiltration of leukocytes and in situ thrombosis. Taken together, we clearly demonstrated that the deletion of Bmpr2 gene in the pulmonary ECs by itself can predispose to PAH with abnormal vascular remodeling. Because our mouse model is the first genetic model which corresponds to pathophysiological features of human PAH, we believe that mouse lines we developed are useful genetic resources for further studies on elucidating the underlying mechanism of PAH.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Kwonho Hong.
Thesis: Thesis (Ph.D.)--University of Florida, 2007.
Local: Adviser: Oh, Suk P.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2007
System ID: UFE0021418:00001

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2 2007 Kwon-Ho Hong


3 To my parents, Seoung-Wook Hong and Soon-Im Chung. To my family, Jung-Ah Um and Jae-Seoung Hong.


4 ACKNOWLEDGMENTS I wish to acknowledge all of those who have he lped me to complete my graduate studies. Most of all, I would truly like to thank my mentor, Dr. S. Pa ul Oh, for all of his support throughout my graduate career. He is a gr eat mentor and researcher. His continuing encouragement, guidance and enthusiasm toward science are the reason for my successful graduate studies. Next, I would like to tha nk my committee members, Dr. Steve Sugrue, Dr. Barry Byrne, Dr. Naohiro Terada, and Dr. Brian Harfe, for their support during my studies. I would also like to thank our collaborators, Dr. Hedeyuki Be ppu and Dr. Kenneth Bloch at Massachusetts General Hospital a nd Dr. En Li at Novartis, fo r providing us the Bmpr2-floxed mouse line. I am indebted to all of past and present me mbers of Dr. Oh laboratory for their supports during my studies. In particular, I wish to tha nk Dr. Tsugio Seki for his help and support in the Alk1 and Alk5 project. I would al so like to thank Dr. Young-Jae Lee for his help and advice in the ActRIIB project. I would like to acknowledge Dr. Sung-Ok Park for her support in the Tg( Alk1-cre ) project. I would like to thank Dae-Song Jang for his help with lung inflation and histology in the Bmpr2 project. I would like to express my gratitude to Donald Lander and HaLong Nguyen for their help with English style and grammar of disserta tion. Also, I wish to acknowledge the rest of lab members, Eun-Jung Choi, Chul Han, Dr. Hae-Won Han, Eun-Ji Lee, Naime Fliess and Melissa Chen. I would also like to give my special tha nks to my parents, Seoung-Wook Hong and SoonIm Chung for their constant support during my st udy. Finally I would like to thank my beautiful wife, Jung-Ah Um, and wonderful son, Jae-Seoung Hong, for being main part of my happiness.


5 TABLE OF CONTENTS page ACKNOWLEDGMENTS...............................................................................................................4LIST OF TABLES................................................................................................................. ..........8LIST OF FIGURES................................................................................................................ .........9ABSTRACT....................................................................................................................... ............11CHAPTER 1 INTRODUCTION..................................................................................................................13Physiology of Pulmonary Circulation....................................................................................13Classification of PAH.......................................................................................................... ...14Pathological Characteristics of PAH......................................................................................15Remodeling in Intimal Layer...........................................................................................15Remodeling in Medial Layer...........................................................................................17Remodeling in Advantitia Layer.....................................................................................17Treatment of PAH............................................................................................................... ....18Basic Treatment...............................................................................................................18Prostacyclin Therapy.......................................................................................................18Endothelin-1 Receptor Antagonists.................................................................................19Nitric Oxide (NO) Therapy.............................................................................................19Investigating Therapies...................................................................................................20Genetics of PAH................................................................................................................ .....20Transforming Growth Factor(TGF) Signal Transduction.......................................21Bone Morphogenetic Protein Receptor Type II (BMPR2)..............................................22Activin Receptor-Like Kinase-1 (ALK-1)......................................................................25Endoglin....................................................................................................................... ...26Animal Models for PAH.........................................................................................................27Experimental Models.......................................................................................................27Physiological models................................................................................................27Monocrotaline (MCT) model...................................................................................28Genetic Models................................................................................................................29BMPR2 signaling pathway.......................................................................................29Serotonin transporter (5HTT)...................................................................................31Endothelial nitric oxi de synthase (eNOS)................................................................32Atrial natriuretic peptide (ANP)...............................................................................33Vasoactive intestinal peptide (VIP)..........................................................................34S100A4/Mts1...........................................................................................................34Unanswered Questions in PAH..............................................................................................35


6 2 MATERIALS AND METHODS...........................................................................................41Sequence Comparison Analyses.............................................................................................41In Silico Analysis of Alk1 Promoter.......................................................................................41Generation of Transgenic Mouse Lines..................................................................................42Mouse Breeding................................................................................................................. .....43X-Gal Staining................................................................................................................. .......44PCR Analysis................................................................................................................... .......45Wound Healing Study............................................................................................................ .46Histology and Immuno histochemistry....................................................................................46Hemodynamic Analysis..........................................................................................................47Morphometric Analysis..........................................................................................................48Western Blotting............................................................................................................... ......49Statistics..................................................................................................................... .............503 IDENTIFICATION OF ARTERIAL ECS-SPECIFIC ALK1 PROMOTER.........................52Note........................................................................................................................... ..............52Introduction................................................................................................................... ..........52Results........................................................................................................................ .............55Generation of Transgenic Constructs and In Silico Analysis of the Alk1 Promoter.......55Expression Pattern of LacZ in Tg( Alk1-lacZ ) Mice........................................................56Identification of Regulatory Elem ents in the Homologous Regions...............................57Alk1 Is Expressed in the Primitive Blood Vessels During Early Chorioallantoic Placenta Development.................................................................................................58Alk1 Is Differentially Expressed in Umb ilical Arterial Endothelium, and Required for the Distinctive Umbilical Artery and Vein............................................................58Alk1 Is Expressed in the Arteries and Capilla ries in the Labyrinth Layer of Placenta...60Alk1-Deficiency Results in Impairment of Umbilical and Placental Blood Vessel Formation.....................................................................................................................61Discussion..................................................................................................................... ..........614 DEVELOPMENT OF A NOVEL TRANSG ENIC LINE EXPRESSING CRE RECOMBINASE IN E NDOTHELIAL CELLS....................................................................76Notes.......................................................................................................................... .............76Introduction................................................................................................................... ..........76Results and Discussion......................................................................................................... ..80Expression Pattern of Alk1 During Lung Development.................................................80Generation of Tg( Alk1-Cre) Line....................................................................................83


7 5 DEVELOPMENT OF AN ANIMAL MODEL OF HUMAN PULMONARY ARTERIAL HYPERTENSION (PAH)..................................................................................92Note........................................................................................................................... ..............92Introduction................................................................................................................... ..........92Results........................................................................................................................ .............94Bmpr2 Deletion in Pulmonary ECs by a Novel Cre Transgenic Mouse Line.................94Bmpr2 Deletion in Pulmonary ECs Can Induce Elevation of RVSP and RV Hypertrophy.................................................................................................................95Increased Number and Medial Wall Th ickness of Alpha-SMA-Positive Distal Arteries in the Mutant Mice with Elevated RVSP.......................................................96Pathohistological Features of PAH in the Mutant Mice with Elevated RVSP................96Discussion..................................................................................................................... ..........976 CONCLUSION AND PERSPECTIVES..............................................................................107The 9.2 kb of Alk1 Promoter As a Driver of Spatiotemporal Pattern of Alk1 Expression...107A Novel Tg( Alk1-Cre )-L1 Line As a Pulmonary EC-Dominant Cre Deleter......................109Alk1-Cre(+);Bmpr2f/f Mice As an Unique Model System of PAH......................................110Perspectives................................................................................................................... .......111APPENDIX A..................................................................................................................... .........114Publications................................................................................................................... ........114BIOGRAPHICAL SKETCH.......................................................................................................136


8 LIST OF TABLES Tables page 1-1. Diagnostic classification of pulmonary hypertension (PH)...................................................381-2. Functional classification of pu lmonary arterial hypertension (PAH)....................................392-1. Primers used for PCR reactions........................................................................................... ..513-1. List of transcription factor s of which TFBSs were found in the Alk1 regulatory fragment and their respective EC-specific target genes.....................................................65


9 LIST OF FIGURES Figures page 1-1. Signal transduction of TGF...............................................................................................403-1. Transgenic constructs and the dot plot analysis betw een human and mouse ALK1 genomic sequences.............................................................................................................663-2. Artery-specific lacZ expressi on in the blood vessels of Tg( Alk1-lacZ ) embryos.................673-3. Postnatal transgene expressions in Tg( Alk1-lacZ ) mice........................................................683-4. Potential transcriptional factor bindi ng sites identified in the mouse Alk1 pXh4.5-in2 fragment by the rVISTA analysis......................................................................................693-5. Whole-mount X-gal staining of Tg( Alk1-lacZ ) embryos at days E7.75E9.0 during early chorioallantoic development.....................................................................................703-6. Expression patterns of Alk1, Flk1, and Alk5 in E15.5 umbilical vessels.............................713-7. Impaired formation of two distinct umbilical vessels in Alk1-null embryos........................723-8. Comparison of Alk1 and Flk1 expressi on patterns in the definitive placentae.....................733-9. Longitudinal sectio ns of X-gal-stained Alk1+/lacZ placenta....................................................743-10. H&E staining of histological sections of E9.5 placentas.....................................................754-1. Dynamics of Alk1 expression during mouse lung development...........................................864-2. Ontogeny of Alk1 in the E9.5 11.5 lungs............................................................................874-3. Ontogeny of Alk1 in the E13.5 and E15.5 lungs...................................................................884-4. The pAlk1-cre construct.........................................................................................................894-5. The pattern of Cre activity in the lung of Tg( Alk1-cre )-L1 line............................................904-6. Cre activity in the Tg( Alk1-cre )-L1 line during postnatal stages..........................................915-1. Tg( Alk1-cre )-L1 mice express the Cre recombinase in the pulmonary vascular endothelial cells.............................................................................................................. .1025-2. Endothelial Bmpr2 deletion resulted in elevation of RVSP and RV hypertrophy in gene dosage and age dependent manners.................................................................................1035-3. Mice in the PH group exhibited increased nu mber of muscularized distal arteries and thickening of medial layers..............................................................................................104


10 5-4. Previously identified PAH-associated prot ein expressions were elevated in SMC layers of PH mouse lungs...........................................................................................................1055-5. Vascular lesions of the PH lungs mimic some pathological feat ures of human PAH.........106


11 Abstract of Dissertation Pres ented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy DEVELOPMENT OF A NOVEL MOUS E MODEL FOR HUMAN PULMONARY ARTERIAL HYPERTENSION (PAH) By Kwon-Ho Hong August 2007 Chair: S. Paul Oh Major: Medical SciencesMolecular Cell Biology Pulmonary Arterial Hypertension (PAH) is a de vastating vascular disorder predisposed by mutation of bone morphogeneti c protein receptor type II ( BMPR2 ). Given that the heterozygous BMPR2 mutation incompletely manifests the di sease condition of PA H and the endothelial dysfunction has been implicated in PAH indi viduals, we hypothesized that deletion of Bmpr2 in the pulmonary endothelial cells (ECs ) is sufficient to predispose to PAH. To test this hypothesis, a novel PAH model has been established by using the Cre/loxP system. To the end, first we characterized th e dynamic pattern of Alk1 expression via in vivo dissection of Alk1 promoter and intron regions. Based on in silico DNA analysis, we isolated a 9.2 kb of Alk1 fragment which precisely recapitul ates the endogenous pattern of Alk1 expression. Transgenic li nes carrying the 9.2 kb of Alk1 promoter/enhancerlacZ gene demonstrated a predominant Alk1 expression in the ECs of large arteries and some capillary beds during development. At adult stage, the leve l of Alk1 in the systemic ECs was greatly diminished, whereas the Alk1 level in the pulm onary ECs was persistant throughout the stage. Next, using the 9.2 kb of Alk1 promoter/enhancer we have developed a novel Cre deleter line, referred as Tg( Alk1-cre )-L1, in which an unique pattern of the Cre-mediated DNA excision in the pulmonary ECs was detected. In the Tg( Alk1-cre )-L1 line, a uniformly distributed Cre


12 activity in the pulmonary ECs was detected from E15.5 on. The homogeneous pattern of Cre activity in the pulmonary ECs was persistant, whil e mosaic or no Cre activity was detected in the systemic ECs during adult stage. Finally, to investigate cellular impact of the impaired Bmpr2 signaling pathway in pulmonary ECs, the Tg( Alk1-cre )-L1 was bred with mice containing conditional Bmpr2 allele. The Alk1-cre(+);Bmpr2f/f mice were viable and seemingly indistinguishable from control littermates. In the hemodynamic and morphometric analyses, subsets of Alk1-cre(+);Bmpr2+/f and Alk1-cre(+);Bmpr2f/f mice revealed an elevation of ri ght ventricular sy stolic pressure (RVSP) with increases in the number of musculari zed distal arteries and thickness of the medial layer. It has been also demonstr ated that the frequency of PAH in the groups was increased in an age-dependent manner. Furthermore, some of hyp ertensive mice exhibited signs of endothelial dysfunctions such as perivascular infiltration of leukocytes and in situ thrombosis. Taken together, we clearly dem onstrated that the deletion of Bmpr2 gene in the pulmonary ECs by itself can predispose to PAH with abnor mal vascular remodeling. Because our mouse model is the first genetic model which correspon ds to pathophysiologica l features of human PAH, we believe that mouse lines we developed are useful genetic resour ces for further studies on elucidating the underlying mechanism of PAH.


13 CHAPTER 1 INTRODUCTION Physiology of Pulmonary Circulation The lung is a vital and highly vascularized organ whose main functions are to provide oxygen (O2) to peripheral tissues/org ans via the bloodstream and remove carbon dioxide (CO2) from the blood. To fulfill this purpose efficiently, the nature of pulmonary circulation is high flow and low resistance. Pulmonary circulation is very sensitive to any in fluences affecting the blood flow. This is because the lung is the only organ which accommodates for the whole cardiac output with low blood pressure. The right ventricle, a driving force for pulmonary circulation, is a thin-walled tissue that is poorly prepared for a rapid change in loading condition. The average right ventricular systolic pressure (RVSP) is about 25 mmHg and the average right ventricular diastolic pressure is 0-1 mmHg. The average systolic pressure of the pulmonary artery is the same as the average RVSP. On the other hand, the average dias tolic pressure of the pulmonary artery is about 8 mmHg. Anatomically, pulmonary vessels differ from sy stemic vessels in that, for a given caliber, the medial layer of pulmonary arteries is much thinner than that of systemic arteries, which reflects the lower resistance in pulmonary circul ation. Another difference compared to systemic blood vessels is that arterioles, which generate re sistance in systemic circ ulation, are not present in the pulmonary circulation. Th e arterioles are replaced by st ructures called precapillary arteries. The lack of arterioles results in transm ission of the pressure of the pulmonary artery to alveolar capillaries and pulsatile blood flow. Perturbations in the homeostasis of cardio-pu lmonary vascular physiology result in fatal conditions such as pulmonary arterial hyperten sion (PAH). PAH is defined as more than 25 mmHg of mean pulmonary artery pressure (mPAP) at rest, or 30 mmHg with exercise (1). PAH


14 is the third most common cardiov ascular disease in persons mo re than 50 years old, after coronary disease and hypertension (2). Although PAH has been clinically recognized since the late 1800s (3), there are some limitations in studying the underlying mech anism of PAH. One of the biggest limitations is that most of the human lung samples available for studies are from lung biopsies or samples from lung transplants of indivi duals in the end-stages of PAH. Therefore, there is a gap of knowledge in the pathophysiologi cal progress of PAH. Furthermore, because a blood vessel is a multi-layered and dynamic organ, ther e is a paucity of knowledge as to the role of each layer in the initiation or procession of PAH. Therefore, the animal model system is considered as an alternative and plausibl e approach to studying PAH at different pathophysiological stages of the disease. The physiology of a mous e, particularly its pulmonary circulation, is similar to that of a human. For example, the mean systo lic PAP of a mouse is about 24 mmHg and the mean diastolic PAP is 8.5 mmHg (4). Thus, the long-term goal of this study is to develop a novel mouse model for st udying the underlying mechanisms of human PAH. Classification of PAH The current system of classi fication for pulmonary hypertensi on (PH) has been established as the result of three World Health Orga nization (WHO) conferences. The most recent classification of PH followed the results of the 3rd World Conference in Venice in 2003 (5). According to this conference, PH is generally classified as PAH, PH with left heart disease, PH associated with lung diseases and/or hypoxemia, PH due to chronic thromboitic and/or embolic disease (CTEPH), and miscellaneous (Table 1-1). PAH is subclassified into: 1) idiopathic PAH (IPAH, formerly known as primary pulmonary hypertension, or PPH), 2) familial PAH (FPAH), 3) in association with other pathological conditions (APAH), 4) in associ ation with significan t venous or capillary involvement, and 5) persistent PH of the newborn. IPAH is PAH due to an unknown cause.


15 IPAH is a rare disease with an in cidence of 2-3 cases per million per year, and there are twice as many female as male patients (6). The mean age of diagnosis is 37 years (7). Shortness of breath (dyspnea) and fatigue are the mo st common symptoms of PAH, followed by chest pain, fainting spells (syncope), and heart failure. Although it is dependent on the study, around 10% of PAH individuals are estimated to be FPAH. FPAH is a form of inherited PAH due to mutations of bone morphogenetic protein recepto r type II (BMPR2) or activin re ceptor-like kinase 1 (ALK-1). The symptoms are similar to those of IPAH. Ot her pathological conditions such as connective tissue diseases (CTD), congenital heart diseases, HIV infection, and some drugs may also lead to the development of PAH. Other occlusive vasc ulopathy, such as pulmonary occlusive venopathy and pulmonary microvasculopathy, can also cause PAH. The prognosis of survivor and clinical trials of PAH subjects are generally estimated by WHO functional class [formally known as New York Heart Association (NYHA) functional class] (Table 1-2) exercise endurance (distance walked in 6 minutes), and hemodynamics (8). Pathological Characteristics of PAH Pathologically, PAH is characterized by obstruc tion of the small pulm onary arteries in association with medial hyperplasia, neointim a formation, plexiform lesions, and occasionally thrombotic lesions. Remodeling in Intimal Layer The intimal layer (approximately 1-2 m of thickness) consists of a base membrane and a single-layered lining of endothelial cells (ECs) localized between th e internal elastic laminae and vascular lumen. Pulmonary ECs seem to be a very quiescent cell-type with a doubling time of more than 0.5-1yr. Although the contribution of ECs to PAH is poorly understood, excessive proliferation of ECs is one of th e key features of PAH. One rema rkable pathological feature of PAH is neointima formation. The term reflects the formation of cell layers with an extracellular


16 matrix (ECM) between the endothelium and internal elastic laminae in small and large arteries (9). The major cell-type composing the neointima is myofibroblast, which may test positive with some smooth muscle cell (SMC) markers such as -smooth muscle actin ( SMA) and vimentin. The myofibroblast does not express EC-marke r genes such as CD31 and the von Willebrand factor (vWF) (9). The origin of th e myofibroblast is currently unknown. In in vitro analysis, it has been shown that the myofibroblast isolated neointima exhibited a different response to growth factors compared to cell isolated from vascular SMCs (10). The data suggests that the myofibroblast might be originated from not only SMCs but different cell-types such as transdifferentiation of EC or advantitia cells. Although the rule of monolayer of endothelium is generally maintained in the PAH lung, studies have shown that tumors or tumorletlike clusters of ECs contributed by monoclonal endothelial proliferation in plex iogenic-lesions can form (11;12) Plexiform lesion is a hallmark of a severe form of PAH showing intimal prolif eration with the formation of multiple capillarylike channels in a pulmonary artery (13). The celltype responsible for plexiform lesions is still under debate, though it has been su ggested that ECs, responding to cytokine, growth factors, or vascular injury, underlie the initi ation of the lesions. Cell-types in the plex iform lesion include ECs, myofibroblast, and interm ingled connective tissues (11). Interestingly, most pulmonary vessels with plexiogenic lesions show perivascular infiltration of the leukocytes (11;14). Among the cell-types consisting of blood vessel, EC is a dominant celltype in the plexiform lesion (11). Using the conventional hematoxilin-eosin (H&E) staining, it has been shown that plexiform lesions have been detected in only 10-20% of total pulmonary artery samples (15). The plexiform lesion can be found in any vessel of the lung. One typical ex ample of a plexiform lesion is in the supernumerary ar tery. If there is a plexiform lesion in the vessel, a concentric


17 laminar intimal fibrosis (CLIF) is found at the close to branching point, and followed by a plexiform lesion. After the plexiform lesion, an angiomatoid (aneurismal dilation of vessel) lesion is found (13). Although plexiform lesions can occur without CLIF, CLIF is associated with plexiform lesions. A recent study suggested that ECs with a mutation of BMPR2 seem to increase the susceptibility to apoptosis which re sults in fragile ECs of the precapillary arteries. However, after repeated apoptosis, the rema ining apoptosis-resistant ECs seem to undergo massive proliferation and eventually develop plexiform lesions (16). Remodeling in Medial Layer Vascular SMCs are major components of the medial layer. Beside the SMCs, elastic laminae are stacked between SMCs. Like ECs, SMCs are also replicatively quiescent and relatively unresponsive to mitogen (17). Hyperplasia in the medial layer is a common feature in all the different forms of PAH. Medial thickeni ng in PAH reflects the role of SMCs in the control of vascular tone. In prec apillary vessels, cells inside internal elastic laminae seem to differentiate into SMCs (18). Depending on the location of the blood vessel, the source of SMC differentiation varies. In distal ve ssels lacking elastic laminae, peri cyte and interstitial fibroblast from surrounding lung parenchyma appear to contribu te to the process of muscularization (19). Remodeling in Advantitia Layer PAH also causes thickening of advetitia laye r by increasing the numbe r of fibroblasts. A recent study using a hypoxia model has been show n that CD68-positive circulating mesenchymal precursor of a monocyte/macrophage lineage contributes to the thickening of the advantitia layer (20). Many of the cells produce type I collagen and are SMA-positive cells. It seems that the induction of many proinflammatory cytoki nes and chemokines such as monocyte chemoattractant protein (MCP)-1, macrophage inflammatory prot ein (MIP)-2, interleukin (IL)1 IL-6 are involved in the remodeli ng of the advantitia layer (21).


18 Treatment of PAH Basic Treatment To prevent the worsening of a diseases prog ress, some lifestyle modifications such as low-level aerobic exercises, avoiding exposur e to hypoxic conditions like high altitudes, and restriction from a high-salt di et are recommended (5;22). The hemodynamic changes during pregnancy, labor, and the postpar tum period are devastating to PAH patients (23). Diuretic therapy can be used to reduce the right ventricl e preload in PAH indivi dual with heart failure. Increases in intracellular calcium and calcium-m ediated signals have an effect on muscle contraction (24;25). A study has shown that a hi gh dose of calcium channel blockers have a beneficial effect on the survival rate of some PAH patients (26). Prostacyclin Therapy Prostacyclin (Prostaglandin I2) and thromboxane A2 (TxA2) are major metabolites of arachidonic acid in blood vessels Prostacyclin is a potent va sodilator thro ugh production of cyclic AMP (cAMP) and inhibits the grow th of SMCs (27). On the other hand, TxA2 is a potent vasoconstrictor. It has been shown that PAH patients showed a decr eased urinary level of prostacyclin metabolites (6-keto-prostacyclin F2 ), whereas the urinary level of TxA2 metabolites (TxB2) has increased (28). The data suggests th at the balance between vasodilator and vasoconstrictor is severely im paired in PAH individuals. The intravenous infusion of e poprostenol (prostacyclin) was firs t used to treat PAH in the early 1980s (29). Studies showed that continuous intravenous epoprostenol clearly has beneficial effects to WHO functional class III or IV (30; 31). Epoprostenol treatment improved exercise tolerance, hemodynamics, and overall survival rates (31). The intr avenous treatment of epoprostenol has some side effects such as jaw pain, headache, diarrhea, and nausea (32;33). As


19 an alterative method to intravenous delivery of pr ostacyclin, subcutaneous (treprostinil), oral (beraprost), or inhaled (ilopros t) delivery of protacyclin an alogues are being used (34-36). Endothelin-1 Receptor Antagonists Endothelin-1 (ET-1) is mainly released by ECs and exerts its effect in vascular SMC in a paracrine manner. ET-1 works as a vasocons trictor and has a mitogenic effect on SMC proliferation (37). Its va scular effect is mainly mediated through ETA and ETB, which are G protein-coupled receptors (38). ETB is predominantly expressed in ECs, whereas ETA is mainly expressed in SMCs (39;40). It has been shown th at individuals with PAH exhibit high levels of circulating ET-1 (41;42). Bose ntan, a US Food and Drug Admi nistration (FDA) approved drug, is a nonselective ET receptor anta gonist which can work on both ETA and ETB. Studies demonstrate that the treatment of bosentan on WHO functional class III patient significantly ameliorate hemodynamics and 6MWT (43;44). Because ETA is the ET-1 receptor expressed in SMCs, selective ETA receptor blockers such as sita xsentan and ambrisentan are being investigated for the tr eatment of PAH (45;46). Nitric Oxide (NO) Therapy NO produced by ECs functions as a potent vasodilator through the stimul ation of soluble guanylate cyclase and increased pr oduction of intracellular cyclic GMP (cGMP) in SMCs (47). The cellular effects of an increased level of cGMP include decreased sensitivity to myosin and the inhibition of calcium released from the sarc oplasmic reticulum (47). The level of cGMP is regulated by phosphodiesterase type 5 (PDE5) (48). Studies have demonstrated that changes in NO pathways were found in lungs with PAH (49; 50). It has also been shown that PDE5 is upregulated in the hypoxia-mediate d animal model for PAH (51;52). Inhaled NO gas relaxes pulmonary arteries, which decreases vascul ar resistance, PA pressure, and RV afterload (53;54). A recent stu dy demonstrated that the oral treatment of


20 cidenafil citrate (ViagraTM), an inhibitor of PDE5, improved exercise capacity, WHO functional class, and hemodynamics in PAH patients (55). Investigating Therapies Simvastatin (ZocorTM) is a lipid-lowering agent derived from Aspergillus terreus It has been shown that simvastatin has a proapoptotic e ffect on neointimal SMCs in an experimental PAH model (56). Oral treatment of simvastatin (20-80mg/day) improves exercise endurance and hemodynamics without adverse effects (57). Studies have also shown that a platelet-derived growth fact or (PDGF) receptor antagonist, imatinib mesylate (STI571, GleevecTM) reverses the experimental PAH model and improves exercise capacity, hemodynamics, and cardiac indices (58;59). Genetics of PAH Progress has been made in elucidating the cellular impact of a mutation in PAH genes during the development of PAH, since the BMPR2 gene was first identified as a PAH gene (60;61). Studies show that a mutation of the BMPR2 gene is responsible for as much as 70% of all FPAH cases and up to 26% of IPAH cases (62). BMPR2 is a member of type II receptors in the transforming growth factor(TGF) signaling pathway. Recent studies have also demonstrated that other TGFreceptors, activin receptor-like kinase-1 (ALK-1) and endoglin (Eng), are also related to PAH (63-68). Mutations in ALK-1 and ENG are also linked to another inherited vascular disorder called heredita ry hemorrhagic telangiectasia (HHT) (69-72). Interestingly, loss of BMPR2 expression is also found in human prostate cancer cells, suggesting that the BMPR2 signal plays an important role in the regulation of cell proliferation (73).


21 Transforming Growth Factor(TGF) Signal Transduction TGFis a large cytokine family that contri butes to diverse cellu lar processes. TGFwas originally named after its ability to cause a phe notypic transformation of cu ltured epithelial cells. However, the cellular effects of each member s eem to be varied or even opposite, depending upon the cell-type and its developmental stage. Some of these cellula r effects include cell proliferation, migration, apoptosis, pa ttern formation, and immunosupression. TGFfamily members include TGFs, bone morphogenic proteins (BMPs) /growth and differentiation factors (GDFs), and activins/inhibin. Each subgroup is categorized by sequence homology and the utilization of cytoplasmic medi ator proteins, SMADs (74-76). TGFwas initially identified by a finding that a pool of growth fact ors secreted from a transformed mouse fibroblast was able to induce the formation of foci in the soft agar assay (77). Later, Roberts et al. found that TGFwas one of the active factors in that pool of growth factors (78;79). Since then, the TGFsuperfamily has expanded. In mammals, more than 30 ligands have been identified as members of the TGFsuperfamily. TGFsignal transduction is initiated by the bind ing of a ligand to a heteromeric complex of transmembrane serine/threonine type II and type I receptors. Once ligands bind to a type II receptor, the ligand-type II receptor complex recr uits and trans-phosphorylates type I receptor, which, in turn, activates cytoplasmic SMAD prot eins. Although most lig ands are functionally different, they utilize a limited number of r eceptors and SMADs. For more than 30 known TGFfamily ligands, only five type II receptors a nd seven type I receptors have been identified, indicating that each receptor mediates multiple signals. One of the key features of TGFsignaling pathways is that there is a consider able amount of redundancie s in the interaction between ligands and type II receptors, as well as type II and type I recep tors (80). Biochemical


22 studies have determined that activin type II receptors (ACVR2A and ACVR2B) utilize ALK2 (ACVR1), ALK4 (ACVR1B), and ALK7 (ACVRL 1C) depending on their interacting ligands including activins (INHBA, IN HBB, INHBC, and INHBE), N odal, BMP7, and GDF11. Activin receptor-like kinase 5 (ALK5) (T RI; TGFBR1) has been shown to interact with the TGFtype II receptor (T RII; TGFBR2) and to mediate TGFsubfamily signals. Recent studies have implicated ALK5 in mediating other TGFfamily signals, such as GDF8 and GDF11 (81). Eight SMAD proteins have been identified in mammals so far. The activated type I receptor phosphorylates receptor-regulated SMADs (R-SMADs). R-SMADs then form a complex with a common partner, SMAD4 (Co-SMAD) then enter into the nucleus. Once there, the SMAD complex interacts with diverse transcri ptional coactivators or corepressors, according to their genetic makeup and ce llular contexts. Depending on wh ich R-SMAD is utilized, TGFsignaling can be generally separated into two pathways: SMAD 2/3 for TGFand SMAD 1/5/8 for BMP. Bone Morphogenetic Protein Receptor Type II (BMPR2) Human BMPR2 consisting of a total of 13 e xons is localized on chromosome 2q33 (82-84). BMPR2 is comprised of 4 major domains: th e extracellular domain (ED), transmembrane domain (TD), kinase domain (KD) and the long cytoplasmic domain (CD). The exons 1-3 encode the ED, exon 4 encodes the TD, exons 511 encode the serine/thr eonine KD, and exons 12 and 13 encode the CD. It has been shown that each domain is highly conserved among species (85). The large C-terminar CD is pr esent only in the BMPR2 among receptors in TGFsuperfamily. Studies have suggested that human BMPR2 is produced by alternative splicing, in which a short form lacks exon 12 encoding a part of CD (84;86). The short form is only 30 amino acids (AA) long compared to 527 AA in the long form after kinase domain (85;87).


23 Currently, the function of the long CD is larg ely unknown. Interestingly, a mutation in exon 12 is commonly observed in PAH patients, suggesting th at the domain has a specific function; for example, crosstalk with other signaling pathways (88-90). Ramos et al. showed that BMPR2 is predominantly expressed in ECs, SMCs, type II pneumocyte, and epithelial cells (91). Studies also demonstrated that BMPR2 is localized in lipid rafts where BMPR2 in teracts with caveolin1 and -1 (91;92). Atkinson et al. demonstrated that BMPR2 mRNA and pr oteins are markedly reduced in the lung of a PAH individual with a heterozygous mu tation of the BMPR2 gene, as compared to normal individuals (14). A lesse r reduction of BMPR2 was also found in individuals with secondary PH. The data suggests that reduction of the BMPR2 leve l primarily contributes to the development of PAH in patie nts with a BMPR2 mutation. Mutation of BMPR2 can be found in any of th e exons except exon 13 (90). The types of mutations that can be found in BMPR2 incl ude missense, nonsense, frameshift, deletion, duplication, abnormal splicing, and single nucleotide polymorphism (SNP) (88-90). More than 50% of BMPR2 mutations are predicted to l ead to protein truncation through a premature termination codon and that there is an age vari ability for the initial onset of PAH (93). One of the key genetic features of PAH is that a mutation of any of the PAH genes incompletely manifestates PAH. Studies have re vealed that a BMPR2 mutation develops PAH in around 20-30% of individuals with heterozygous BMPR2 mutations, suggesting that other factors might be involved in the development of PAH (62;94). Studies al so showed that Bmpr2 null or hypomorphic mice are embryonic lethal due to defects in mesoderm formation and outflow tract formation of the heart (95;96).


24 So far, more than 20 BMP/GDF ligands have been identified in humans (97). In biochemical studies, some of the BMPs/GDFs such as BMP2, 4, 6, 7, 9, 15, and GDF-5 can bind to BMPR2 and utilize ALK-1, -2, -3, -5, or -6 as a type I receptor (9 8-101). Depending on the utilization of a type I receptor, signals th rough BMPR2 are mediated by either SMAD2/3 or SMAD1/5/8. BMP signaling through BMPR2 has b een implicated for various biological processes such as cardiac development, bone and cartilage formation, craniofacial development, and reproduction (98;102104). Cellular effects of the BMPR2 si gnal in the vasculature seem to be heterogeneous rather than homogeneous, depending on the cell-type. For example, BMPR2 signal by BMP2, 4, or 7 seems to cause apoptosis in the human pulmonary artery SMC by downregulating Bcl-2 and in a caspases (caspase 3, 8, or 9)-dependent manner (105;106). Studies have shown that the reducti on of the BMPR2 resulting from knockdown or overexpression of the mutant form of BMPR2 can rescue from BMP7-med iated apoptosis in SMCs (106). Even in the pulmonary artery SMC, the cellular effect of BMPR2 seems to be dependent upon the BMP protein. Frank et al. showed that BMP4 has an effects of proproliferation and promotion of cell migration of pulmonary artery SMCs, wher eas BMP2 has an opposite effect (107). A more complicated model of BMPR2 signalin g in SMC was recently reported (108). The authors showed that in the absence of Bmpr2, BMP2/4 can utilize ActRIIa and Alk2 or Alk3, and BMP7 can utilize ActRIIa and Alk2. The re dundancy can partially explain why a BMPR2 mutation incompletely develops PAH. On the other hand, a BMPR2 mutation in the pulmonary artery ECs initially causes an increase in apoptos is (16). The study suggested that after a certain amount of apoptosis has occurred, the survivi ng ECs proliferate in an uncontrolled manner, which in effect causes increased cellularity in the intimal la yer. Although a dependence on celltype for the biphasic function of BMPR2 seems to exist, cell-cell intera ction between ECs and


25 SMCs can also contribute to the development of PAH. A recent study has shown that SMCs exhibit a marked increase in cell proliferation in the presence of a conditioned medium from ECs from PAH individuals (109). The authors have dem onstrated that one of the factors contributing to SMCs proliferation is an increased level of serotonin (5HT) due to elevated level of tryptophan hydroxylase (TPH), a required enzyme for 5HT production pathway, in ECs associated with PAH. Activin Receptor-Like Kinase-1 (ALK-1) ALK-1 is encoded by 10 exons and is locali zed on chromosome 12q11-q14 (110;111). It has been shown that a mutation of ALK-1 is responsible for HHT2 (72). Although some geographical regions may show a higher rate of in cidence for HHT than ot hers (110), the disease affects more than 1 in 10,000 individuals worldwide (112). It is char acterized by recurrent epistaxis, mucocutaneous telangiectasia, ga strointestinal hemorrh age, and arteriovenous malformation (AVM) in the pulmonary, cereb ral, or hepatic va sculature (112). Homozygous deletion of Alk1 causes embryoni c lethality by embryonic day (E) 10.5 with severe defects in vascular development (113;114). Recently, Srinivasan et al. has shown that heterozygous Alk1 mice display the human HHT phenotype in a number of features showing age-dependent disease penetrance, vascular lesions in the specific organs and pathohistological similarity of the lesions (115). Interestingly, blood vessel malformation in the female uterus was also evident in aged Alk1+/LacZ mice with 129/Sv background (Unpublished observation). The observation is reminiscent of a vascular defect in Eng+/mice with 129/Ola background (116). Seki et al. showed that arterial ECs show a r obust Alk1 expression over venous ECs during embryonic and early postnatal stages, but a grea tly diminished level of Alk1during adult life, except in pulmonary ECs (117).


26 Recently, studies have shown that some HHT patients with heterozygous mutation of ALK-1 exhibit pathophysiological conditions of PAH (63;65;66;68). These patients exhibit an increase in mPAP and plexiform lesions in the pulmonary vasculatur e in addition to HHT phenotypes. The result is paradoxical because the pulmonary pathophysiologys of PAH and HHT are considered to be in opposition. Fo r example, HHT patient s carrying pulmonary arteriovenous malformation (PAVM) suffer severe dilation in the pulmonary vessels, which may lead to a decrease in pulmonary resistance. On the other hand, PAH patients exhibit obliteration of small pulmonary arteries, whic h result in markedly increased pulmonary vascular resistance (63). The molecular mechanism by which hete rozygous mutation of ALK-1 develops such opposite vascular phenotypes remains unknown. Endoglin Eng, which is encoded by 14 exons, is a type III receptor in TGFsignaling pathways. Eng is localized on chromosome 9q33-q34. It ha s been shown that a mutation of Eng is responsible for HHT1 (70;71). A recent study demonstrated that a mutation of Eng can cause HHT phenotypes as well as PAH (68). The study showed that a substitution of a nucleotide is identified within the putative splicing site of intron 12 of Eng, 22 bp upstream of exon 13. The substitution seems to cause a loss of exon 13 without disr uption of the readi ng frame of the Eng. Another study showed that an HHT individu al with a mutation in Eng developed PAH after taking an appetite suppr essant, dexfenfluramine (67). Although the underlying mechanism of PAH in a person with an Eng mutation is co mpletely unknown, the studies suggest that the mutation of Eng influences other TGFsignaling pathways, which then display PAH phenotypes, rather than Eng mutation alone causing PAH.


27 Animal Models for PAH Experimental Models Physiological models A hypoxia-caused mismatch in the ratio between ventilation (V) and perfusion (Q), is a potent factor in the development of PAH. Th e hypoxia-induced vasoc onstriction is a unique feature of pulmonary physiology. Thus, if the l ung is exposed to a hypoxic condition which then causes low partial pressure of arterial oxygen (PaO2), pulmonary vascular resistance (PVR) is increased to match the V/Q ratio. Although acute hypoxia might be beneficial to enhance the oxygenation of peripheral blood vessels, chroni c hypoxia causes el evation in mPAP. According to the Poiseuilles equation, PVR is inversely propor tional to the fourth power of the radius of the pulmonary arteries. Therefore, small changes in the radius of a pulmona ry artery can cause a drastic increase in pulmonary ar terial pressure. Such chronic hypoxia is followed by vascular remodeling (118). Medial thickness is a remarkab le feature of hypoxia-induced PH. Persons who dwell at high altitudes show an increase in hemodynamics, the number of red blood cells (polycythemia), right ventricu lar hypertrophy, and vascular remodeling (119). Although the molecular mechanism underlying hypoxia-mediated vascular remodeling is not clearly defined, the oxygen sensor(s)-mediated change of ion channe ls has been implicated as being involved in the vascular remodeling and constriction of SM Cs (120;121). It has be en shown that hypoxia causes change in the production of mitochondrial r eactive oxygen species (ROS), which then results in an increase in cytoplasmic calcium ([Ca2+]cyt) concentration via increased influx and the release of Ca2+ (122;123). The increased [Ca2+]cyt results in phosphorylation of the myosin light chain, which causes an increase in va scular tone. Hypoxia is also followed by depolarization of the membrane by inhibition of voltage-gated K+ channel (Kv) activity (120). Recently, Takahashi et al. showed that sustained exposure to hypoxic conditions causes a


28 reduction in the BMPR2 level in the experimental model (124). It seems that a loss of BMPR2 in SMC causes a reduction of Kv expression, which results in depolarization of the membrane (125). A recent study demonstrated that sustained hypoxia causes perivascular accumulation of CD45, CD11b, CD14, and CD68-positive cells, termed fi brocytes, in pulmonary artery advantitia. The study suggests that circulating mesenchymal precursor cells play an important role in remodeling of pulmonary arteries with a hypoxic condition (20). In contrast to V/Q mismatch with hypoxia, an over-perfusion of pulmonary circulation due to congenital heart diseases can cause PAH. Studies have shown that an over-circulationinduced experimental model is sufficient to develop PAH with abnormal vascular remodeling, increases in ET1, inducible NOS (iNOS), and vascular endothelial growth factor (VEGF) expressions (126;127). The studies also showed that PAH by induction of over-circulation in a porcine model could be prevented by admi nistration of bosentan and sidenafil. Monocrotaline (MCT) model Monocrotalline (MCT) is a pyrroliz idine alkaloid plant toxin de rived from the seed of the Crotalaria spectabilis Like the hypoxia-induced PAH animal model, administration of MCT to a rodent is a widely investigating model syst em in the PAH field. Because the pyrrolizidine alkaloid needs to be bioactivated to a pyrroli c derivative [MCT pyrrole (MCTP)] by cytochrome P450 in the liver, it often causes hepatic damage as well (128). It has been shown that treatment of MCTP causes megalocytosis of ECs and SMCs, characterized by markedly enlarged cells with enlarged nuclei and organelles ( 129). Studies have indicated that the endothelial golgi is a main target of the MCTP by disruption of the Caveolin -1/raft function due to blockage in trafficking of the raft scaffoldi ng proteins (129;130). An in vitro study demonstrated that treatment of MCTP to cultured pulmonary arterial ECs causes apoptosis with an increase in permeability (131). The administration of MCT or MCTP causes, initially, an increase in cardiac output with


29 the muscularization of small arteries, followed by a gradual increase in vascular resistance. After three weeks of MCT treatment, cardiac output is dropped and pulm onary arterial pressure is further increased. Accumulating data suggests that MCT or MC TP injures both ECs and SMCs, which results in a dysfunction in both cell-types. A recent study also suggests that administration of MCT causes marked downregulation of Bmpr2, Alk1, Alk6, and Smad4, 5, 6, and 8 expressions (132). The study suggests that MCT cause s not only the dysfunction of vascular cells, but also downregulation in th e expression of PAH genes. Genetic Models BMPR2 signaling pathway The significance of the heteroz ygous Bmpr2 mouse is still considered co ntroversial as far as being a model system for human PAH is concerned. Beppu et al. has observed a mild elevation of pulmonary arterial pressure with m ild muscularization of sm all pulmonary arteries in heterozygous Bmpr2 null mice (133). The Bmpr2+/mice do not exhibit vascular lesions such as neointima formation or plexioform, except in the muscularization of precapillary arteries. However, once Bmpr2+/mice were challenged with a hypoxic stimulus, there was no difference observed in the hemodynamics between Bmpr2+/mice and the wild-type mice. Paradoxically, a hypoxic condition in a Bmpr2+/mouse causes less muscularization of the pulmonary arteries (133). On the other hand, with the same mouse, other independ ent groups failed to observe an elevation of pulmonary arterial pressure (134; 135). They did demonstrate that an adenovirusmediated pulmonary overexpression of 5-lipoxygenas e (5LO), a mediator of inflammation, and a chronic infusion of serotonin (5HT) develops PAH in Bmpr2+/mice. The result suggests that subjects carrying mutations in the BMPR2 gene ar e predisposed to the de velopment of PAH, but not sufficient for development of PAH-associated vascular le sions. The studies raised the possibility that an additional ge netic and/or environmental sec ond hit is necessary to reach


30 clinical manifestation of PAH in subj ects with heterozygous BMPR2 mutation. The Bmpr2+/mice and human studies suggested that a mechanis tic model of PAH, called haploinsufficiency of the BMPR2 gene in the onset of PAH, does not s eem to be the precise mechanism for the development of PAH (89;90;93). A study highlighted the role of the Bmpr2 gene in SMCs by showing an elevation of RV pressure in transgenic mice by overexpressing a dominant-negative form of Bmpr2 in SMCs (136). In the study, the authors employed a met hod called inducible transgenesis in that a truncated Bmpr2, by insertion of a base pair at a kinase domain, was designed to be overexpressed by a SMC-specific SM22promoter under the regulati on of tetracycline. The transgenic mice exhibited elevated RV pressure with muscularization of pulmonary arteries. However the mouse line does not show any of the ot her vascular injuries observed in the severe form of PAH such as neointima formation a nd plexiogenic lesions. Th e data suggests that impairment of the Bmpr2 signal in SMC is suffici ent to cause elevation of pulmonary arterial pressure, but not to further other pathological conditions. A recent study demonstrated that Bmp4 plays a role in the development of hypoxia-related PAH (107). Frank et al. showed that mice in a hypoxic condition experience selective upregulation of Bmp4 with reduction of phosphor ylation of Smad1/5/8 and Id1, a downstream gene of the Smad1/5/8, expression. However, the hypoxia-mediated hypertension was less severe in Bmp4 heterozygous mice ( Bmp4+/lacZ) compared to wild-type mice. Further analysis suggested that Bmp4 derived from ECs plays an important ro le in the migration and proliferation of SMCs in the hypoxia-induced PAH model. The study showed that Bmp4 is one of the factors involved in the development of PAH under certain conditions.


31 Serotonin transporter (5HTT) Serotonin (5-hydroxytryptamine, 5HT) is know n to function as a vasodilator in the systemic vessel, and vasoconstrictor in pulmona ry blood vessels (137;138). 5HT is also known as a potent mitogen to SMCs (139). It has been implicated that 5HT is capable of causing PAH after an outbreak of the disease in individuals who used aminorex fumarate, an appetite suppressant that inhibits 5HT re uptake by platelet (138). Plasma levels of 5HT are normally low because most of the 5HT in the body is metaboliz ed by the liver and stored by platelets (140). 5HT is synthesized from L-tryptophan, an esse ntial amino acid (109). Over 95% of 5HT is produced from enterochromaffin cells in the gut, wh ereas the remaining amount is synthesized in the other cell-types in the brain and lung (141). The synthesis is regulated by an enzyme called typtophan hydroxylase (TPH). There are two diffe rent isotypes of PTH, PTH1 and PTH2, involved in the synthesis of 5HT (109). Eddahibi et al. showed that TPH1 is more widely expressed in different organs su ch as intestine, brain and lung whereas TPH2 is predominantly expressed in the brain. 5HT mediates its signal through at least five classes of 5HT receptors which belong to G protein-coupled receptor (GP CR) and serotonin transporter (5HTT). Because aminorex fumarate is a substrate for 5HTT, the outbreak of PAH after taking the drug has opened a new avenue of investig ating the involvement of 5HT in the development of PAH. It also has been shown that a polymorphism in the human 5HTT promoter alters the level of gene expression (142). Generally, the polymorphism is co mposed of two alleles: a 44-bp insertion or deletion, designated the L and S alleles. In PASMCs with the L/L genotype, 5-HTT expression is 1.5-fold higher than that in LS or SS cells under basal c onditions, and this difference is even more marked when 5-HTT expression is increased by external stimuli (143;144). Interestingly, the ECs from an IPAH individual showed high le vels of TPH1 mRNA. On the other hand, 5HTT is upregulated in the SMCs from an IPAH i ndividual (109). In recent studies, however, two


32 independent groups failed to find any correla tion between 5HTT polymorphism and BMPR2 in the age at diagnosis or survival interval of PAH patients (145;146). It has been shown, however, that 5HTT is upregulated in diffe rent forms of PAH in the remodeled SMCs (147). Furthermore, Guignabert et al. demonstrated that an early increase d level of 5HTT after MCT treatment was observed, and an administration of fluoxetine, an inhibitor of 5HTT, prevented and reversed MCT-induced PAH (148). It has been shown that a null mutation of 5HTT results in a less severe PAH with sustained hypoxia (143). Recent studies have demonstated that an overexpression of 5HTT causes elevated RV pressure with muscular ization of pulmonary ar teries (149;150). In the same line of evidence, Long et al. observed that Bmpr2+/mice exhibited an increase in susceptibility upon the administration of 5HT (135). However, the authors found that the synergistic effect was mediated by both 5HT2 and 5HT1 receptors. Although some discrepancies exist, previous in vitro and in vivo studies s uggest that 5HT, through 5HTT, plays an important role in the remodeling of pulmonary vasculature. Endothelial nitric oxide synthase (eNOS) It has been shown endothelial nitric oxide synthase (eNOS) is strongly expr essed in the pulmonary ECs and the epithelium of normal lungs (49). In the same study, it was also shown that the expression of eNOS (NOS3) is se verely impaired in a PAH individual. Giaid et al. demonstrated that the staining in tensity of eNOS was negatively co rrelated with the severity of pathological condition. Interestingly, the study showed that there was an inverse correlation between the level of eNOS expression and total vascular resistance of pulmonary vessels in PAH patients with plexiform lesion. Steudel et al. showed that deletion of eNOS causes pulmonary arterial pressure to elevate, with a decrease in vasodilatory response to acetylcholine (151). However the authors failed to find any morphometric changes in eNOS-/mice, suggesting that eNOS modulated vascular tone ra ther than vascular remodeling. Another study demonstrated that


33 the muscularization of pulmonary arteries was more severe than normal in both female and male eNOS-/mice during embryonic stages and at birth, but the abnor mal muscularization was persisted only in the male eNOS-/mice, suggesting a gender bias in eNOS-/mice (152). Paradoxically mice with hypoxia-induced PH displaye d an increased level of the eNOS protein without a concomitant increase in NO bioactivity (153;154). Recent studies highlighted that a co-f actor for eNOS, tetrahydrobiopterin (BH4), plays an important role in the pulmonary vessel homeostas is (155;156). The studie s demonstrated that a hyperphenylalaninemic mutant mouse (hph-1) e xhibits a 90% reduction in GTP-cyclohydrolase 1 (GTP-CH1), which catalyzes the rate-limiting step for the de novo production of BH4. BH4 functions as an essential co-f actor for several enzymes, incl uding phenylalanine-4-hydroxylase (PAH), tyrosine-3-hydroxylase (T H), tryptophan-5-hydroxylase (TPH), all three forms of nitric oxide synthase (NOS1, NOS2, and NOS3), and glyceryl-eth er mono-oxygenase (157). Interestingly, the elevation of RV pressure a nd muscularization of pulm onary arteries with a reduction of NOS activity were evident in hph-1 mice compared wildtype mice. The hypertensive phenotype in hph-1 mice can be rescued by crossing them with a transgenic line selectively overexpressing GTP-CH1 in ECs. The data suggests that an impairment in BH4 production of the ECs can cause PAH, presumably through the eNOS pathway. Atrial natriuretic peptide (ANP) ANP, a member of natriuretic peptides, is a cardiac hormone with potent diuretic, vascular relaxant, and antiproliferative properties. In systemic circul ation, it modulates intravascular volume and the hypertensive res ponse to salt loading (158). Like other natriuretic peptides, ANP acts on a membrane-bound receptor that activates particulate gua nylate cyclase and promotes cGMP accumulation (159).


34 In pulmonary circulation, ANP seem s to play a role as a physiological modulator in the regulation of vascular resistance and vascular remodeling. Klinger et al. showed that ANPdeficient mice have moderately elevated RV systolic pressures (22 2 vs. 15 1 mmHg) and thickened RV walls when compared to heterozygous or wild-type mice. After 3 weeks of hypoxia, the mean RV systolic pressures for homozygous muta nts were higher (29 3) than heterozygous or wild-type mice (23 1 and 22 2 mmH g, respectively). There was a greater degree of vascular remodeling of the distal pulmonary vessels in homozygous mice than in heterozygous or wild-type mice under both hypoxic and normoxic conditions (158). Vasoactive intestinal peptide (VIP) Studies demonstrated that VIP plays an importa nt role as a vasodila tor, an inhibitor of SMC proliferation, an anti-inflammatory and an anti-apoptotic agent in pulmonary vessels (160;161). A recent study demonstrated that deletion of the VIP gene leads to a mild elevation of RV pressure with right ventricular (RV) hypertrophy and pulmonary vascular remodeling (162). Immunohistochemical analysis showed that a thickening of the medial layer resulted from an increase in SMCs and collagen deposition. However the study failed to show the massive proliferation of ECs which is typically seen in the severe form of the disease. Another feature of VIP-/mice is that a perivascular infiltration of inflammatory cell was observed surrounding affected vessels. S100A4/Mts1 The S100 family members are known to be involved in multiple cellular processes including cell motility, interce llular adhesion, extracellular signal transduction, and cell proliferation and differentiation (163). S100A4/Mt s1, a member of the S100 family of calciumbinding proteins, has been im plicated as being induced in malignant metastatic breast cancer and in the stimulation of angiogenesis (163;164). Ambartsumian et al. demonstrated that the


35 S100A4/Mts1 protein is capable of enhancing the endothelial cell motility in vitro and stimulating the corneal neovascularization in vivo (163). Lawrie et al. showed that S100A4/Mts1 can facilitate both the prolifer ation and migration of the human pulmonary artery SMC through receptor for advanced glycation end products (RAGE). Furthermore, it has been shown that a subset of transgenic mice (~5%) overexpres sing S100A4/Mts1 coul d develop plexogenic arteriopathy with intimal hyperpla sia which led to the occlusion of the vessel lumen (165). The study also showed that the expression of S100A 4/Mts1 was induced in the medial layer of vessels with plexiogenic lesi ons and neointima formation. Unanswered Questions in PAH Although much progress has been made in the P AH field, a number of questions are yet to be answered. First, one debating issue in the etiology of PAH still exists regarding the vascular components underlying the initiation and process of the pathological condition of PAH. Because all blood vessel components exhibit abnormal remode ling in a PAH individual, there is a paucity of information about the cell-type initiating the abnormal vascular remode ling. Studies show that a subset of PAH individuals possess endothelial dysfunctions including perivascular infiltration of leukocytes, an increase in inflammatory cytokines, and in situ thrombosis (3;14;166-168). The data has raised the possibility that dysfunction in the endothelium may lead to an imbalance in vasodilators vs. vasoconstrictors, which then triggers the aforementioned abnormal vascular remodeling. Nevertheless, no part of the study has clearly demonstrated th at the cellular impact of the mutation of PAH genes are in a speci fic cell-type of pulmonary blood vessels. Secondly, an emerging body of evidence has implicat ed that an addition al factor (modifier) might be necessary to reach the pathophysio logical condition, along with a heterozygous mutation of BMPR2 gene. In this vein, another issue needing to be answered in the BMPR2 signaling pathway is which downstream mediat or impaired by BMPR2 mutation causes PAH.


36 SMAD is known as a canonical mediator of the BMPR2 signal. A study shows that phosphorylation of Smad1/5 was reduced in the pulmona ry arterial SMCs of PAH individuals with BMPR2 mutation (169). The study suggests th at not only BMPR2 expression is reduced, but additionally, the activation of the SMAD path way is severely impaired by a mutation of BMPR2 in PAH individuals. On the other ha nd, because the BMP signaling pathway has a crosstalk with other pathways, there is a mounting evidence that the mitogen-activated protein kinases (MAPKs), including p38MAPK, p42/44MAPK (ERK1/2), and c-Jun-N-terminal kinase/stress-activated protein kinase (JNK/SAPK), are regulated by BMPs and TGFs in certain cell-types (92;170). Recent studies have shown that exogenous BMP ligands affect phosphorylation of p38MAPK and p42/44MAPK, and have a proliferation effect on SMCs (108;169). The studies suggest that thos e MAPK pathways somehow interact with SMAD pathways. However, no data has demonstrated whic h pathway(s), ie. SMAD-dependent or SMADindependent, is responsible for PAH due to BMPR2 mutation. Thirdly, because the plexiogenic lesion seems to be due to monoclonal proliferation of ECs, as shown in the previous studies (12;171), the possibility of a loss of heterozygosity (LOH) of BMPR2 gene in those cells has been suggested. The idea is that the monoclonal cell growth within such lesions is considered to be akin to neoplas ia. Because homozygous null of Bmpr2 results in early embryonic lethality, it has been su ggested that a mutation of one allele of the BMPR2 gene is transmitted through the germ line, whereas a mutation of the other allele is brought about by somatic mutation. Although there wa s a limitation in the number of samples, Machado et al. failed to find out the LOH of the BMPR2 gene in the plexiform lesion of a PAH lung (172). Nevertheless, to test the observati on made in humans, the conditionally homozygous deletion of the BMPR2 gene in an animal would answer the issue.


37 Lastly, the current mechanism for the hyperpla sia in the vascular wall is based on the finding of a resistance to apoptos is and proproliferation in SMCs of the MCT model and in PAH patients (16;173;174). Besides the hypothe tic model based on proliferation vs. apoptosis, recent studies have shown that circul ating EC progenitor cells or b one marrow-derived cells (BMDCs) can also contribute to, to some degree, vascular remodeling (175-178). Although the physiological effects of transplanted BMDCs in the experimental model are controversial or various, the studies do demonstrat e that the BMDCs or circulating EC progenitor cells can be incorporated into remodeling medial and in timal layers. Based on the above findings, one important question that can be raised is which cell-type, ie. preexisting SMCs or ECs vs. circulating progenitor cells or BMDCs, is th e dominant player in the abnormal vascular remodeling. The studies suggest that both may occur in the process of PAH individuals. Nevertheless, none of the studies have demonstrated the issue.


38 Table 1-1. Diagnostic classificati on of pulmonary hypertension (PH) Pulmonary arterial hypertension Idiopathic Familial Associated with Collagen vascular disease Congenital left-to-right shunt Portal hypertension Infection with human immunodeficiency virus Drugs and toxins Other conditions Associated with substantial ve nous or capillary involvement Pulmonary veno-occlusive disease Pulmonary capillary hemangiomatosis Persistent pulmonary hypertension of the newborn Pulmonary hypertension with left heart disease Left-sided atrial or ventricular heart disease Left-sided valvular heart disease Pulmonary hypertension associated w ith lung disease or hypoxemia or both Chronic obstructive pulmonary disease Interstitial lung disease Sleep-disordered breathing Alveolar hypoventilation disorders Chronic exposure to high altitude Developmental abnormalities Pulmonary hypertension due to chronic th rombotic or embolic disease or both Thromboembolic obstruction of proximal pulmonary arteries Thromboembolic obstruction of distal pulmonary arteries Nonthrombotic pulmonary embolism (tum or, parasites, foreign material) Miscellaneous Sarcoidosis, pulmonary Langerhans-cel l histocytosis, lynphangiomatosis, and compression of pulmonary vessels (adenopat hy, tumor, and fibrosing mediastinitis) This classification is adapted from Humbert et al ., 2004 (5)


39 Table 1-2. Functional cla ssification of pulmonary ar terial hypertension (PAH) Class Description Class I Pulmonary arterial hypertension witout a resulting limita tion of physical activity. Ordinary physical activity do es not cause under dyspnea or fatigue, chest pain, or near syncope Class II Pulmonary arterial hypertension resulti ng in a slight limita tion of physical activity. The patient is comfortable at rest, but ordinary physical activity causes under dyspnea or fatigue, ch est pain, or near-syncope Class III Pulmonary arterial hypertension resulti ng in a marked limitation of physical activity. The patient is comfortable at rest, but less than ordinary activity causes under dyspnea or fatigue, ch est pain, or near-syncope Class IV Pulmonary arterial hypertension resulting in an ability to ca rry out any physical activity without symtoms. Dyspnea, fati gue, or both may be present even at rest, and discomfort is incr eased by any physical activity. This classification is adapted from Humbert et al. 2004 (5)


40 Figure 1-1. Signal transduction of TGFThere are more than 30 known ligands in the TGFsuperfamily. TGFfamily members include TGFs, bone morphogenic proteins (BMPs)/growth and differentiation factor s (GDFs), and activins/inhibin. The ligand starts signaling by binding to and phos phorylating the serine/threonine kinase receptors, and then the signal is prop agated through phosphorylation of the cytoplasmic SMAD proteins. There are seven type I, five type II and two type III receptors in the signaling pathway. The type II receptor includes TGFtype II receptor (T RII), activin type II receptors (ActRIIA and ActRIIB), bone morphogenetic protein type II receptor (BMPR2), and Mllerian inhibiting substance type II receptor (MISR2). The type I recep tors include activin receptor-like kinase (ALK) 1-7. The type III receptors include -glycan and endoglin. Eight SMAD proteins have been identified in mammal s so far. The activated type I receptor phosphorylates receptor-regulated SMADs (R-SMADs). The R-SMADs then form a complex with a common partner SMAD 4 (C o-SMAD), and enter into the nucleus. The inhibitory (I)-SMADs, SMAD 6 and SMAD 7, negatively modulate TGFsignaling by inhibiting the in teraction between R-SMADs and type I receptor or SMAD 4 and by targeted degradation of receptors.


41 CHAPTER 2 MATERIALS AND METHODS Sequence Comparison Analyses Mouse and human ALK1 genomic DNA sequences were obtained from the UCSC Genome browser (http://genome.ucsc.edu ). To visualize the mouse and human ALK1 genomic sequence homology, dot-plot sequence comparisons were performed by using the Dot Matcher program available at European Molecular Biol ogy Open Software Suite (http://p8090bioinfo.pbi.nrc.ca.lp.hscl.ufl.edu/EMBOSS/ ), and DNA sequence homology alignment was conducted using the Blast 2 Sequences program (http://www.ncbi.nlm.nih.gov.l p.hscl.ufl.edu/gorf/bl2.html ). Blast 2 Sequences was also used to search for common regulatory elements between Alk1 and endotheliumor artery-specific genes. The following genes were included: Tek (Tie-2) Nos3 (eNOS) and Cdh5 (VE-cadherin) for endothelium-specific genes; and Efnb2 (Ephrin-B2), Bmx, Nrp (Neuropilin-1), Dll4, Notch1, Notch3, and Notch4 for artery-specific genes. Full-length gene sequences as well as 10 kb 5' regulatory regions from each gene were used as queries, a nd comparisons against 9.2 kb pXh4.5in2 sequence were performed with default settings. In Silico Analysis of Alk1 Promoter To identify potential transcripti onal factor bindi ng sites (TFBSs) in the pXh4.5-in2 sequence, the rVISTA 2.0 program provided by Lawrence Livermore National Laboratory was utilized (http://rvista.dcode.org/ ). The program matches a TFBS consensus matrix from the TRANSFAC 7.3 database (http://www.biobase.de ) to query sequences and tests whether matched consensus matrixes are conserved between two query sequences. Some tr anscriptional factors (TFs) have multiple consensus matrixes with different names in the database. Therefore, the number of potential TFBS consensus matrixes is usually greater than the number of potential


42 TFs. In addition, the number of TFBSs is usua lly greater than the number of TFBS consensus matrixes because each TFBS consensus matrix can be matched to multiple locations in the query sequences. For the Alk1 regulatory element analysis, the same set of sequences used for the dotplot analysis were inputted into the rVISTA 2.0 program. Potential TFBSs that were conserved between the mouse and human sequences were then obt ained. The program located 471 conserved potential TFBSs in the 9.2 kb m ouse sequence, where each TFBS was matched to one of the 112 TFBS consensus matrixes. Among these112 matrixes, 16 TFBS consensus matrixes for the 12 TFs were known to regulate EC-specific genes. Generation of Transgenic Mouse Lines The pXh4.5-SIBN and pBam9-SIB constructs were generated from 4.5 kb of Xho I and 9.2 kb of Bam HI fragments of Alk1 which contain 2.7 kb and 8 kb of the promoter regions, respectively, as well as exon 1 through a 5' region of intron 2. For the third construct, pXh4.5in2-SIB, the 3' end of the 4.5 kb of Xho I fragment was extended to 3' region to include the rest of intron 2, exon 3, and a 5' region of intron 3, for a total length of 9.2 kb. Each Alk1 genomic fragment was connected to either the SIBN or SIB cassette, wh ich contained SV40 splicing donor/acceptor signals (SD/SA), internal ri bosomal entry sequence (IRES), -galactosidase gene, poly A signal, and with or wit hout a neomycin resistant gene cassette driven by the PGK promoter. The DNA constructs were microinjected into the male pronuclei of fertilized eggs from the FVB strain using the established pro cedures. The founder line s were screened by genomic Southern blot or PCR analyses. Several founder lines from pXh4.5-SIBN, pBam9-SIB and pXh4.5-in2-SIB were further examined for their transgene expression by whole-mount X-gal staining of F1, F2, or F3 embryos. Conse quently, four pXh4.5-SIBN, one pBam9-SIB, and two


43 pXh4.5-in2-SIB mouse lines were establishe d. The mouse lines from the pXh4.5-in2-SIB construct will be referred as Tg( Alk1-lacZ ) hereafter. For the generation of Tg( Alk1-cre ) lines, the 9.2 kb of Alk-1 promoter/enhancer element was used. The 9.2 kb of Alk1 fragment was connected to a Cre transgene. For proper splicing and initiation for transcription, the Cre cassette also contained SD/SA, IRES and Cre genes. Two independent founder lines, Tg( Alk1-cre )-E and Tg( Alk1-cre )-L1, were obtained from the pAlk1Cre construct. To test the ability of the Cre-mediated DNA excision, the Tg( Alk1-cre ) F1 mouse was crossed with a Cre tester line, R26R, and subsequently Xgal staining and histology were carried out. Mouse Breeding All procedures performed on animals were reviewed and approved by the University of Florida Institutional Animal Care and Use Committee. The mice used for the procedures were wild-type C57BL/6J and FVB mice, which we re purchased from Harlan (Indianapolis, Ind). The establishment of Alk1+/lacZ, Alk5+/lacZ, and Flk+/lacZ reporter lines were described previously (81;117;179). The pregnant female mice were sacrificed at specific gestational days. To examine the expression pattern of Alk1 in embryos, male Tg( Alk1-lacZ ) mice were crossed with either the C57BL/6J or FVB wild-type mice. Once a vagi nal plug was found, the stage of the embryo was considered as at embryonic days (E) 0.5. The pre gnant female mice were dissected at various embryonic stages such as E7.75, 8.5, 9.5, 10.5, 11.5, 12.5, 13.5, 15.5, and 18.5 embryos. The harvested embryos were then subjected to X-gal staining. To examine the expression pattern of Alk1 at postnatal stages, various organs such as lung, heart, intestine, eyes, brain, kidney, and skin were isolated and used for X-gal staining.


44 For examination of the expression pattern of Alk1, Flk1, or Alk5 gene in the placenta, female Tg( Alk1-lacZ ), Alk1+/lacZ, Alk5+/lacZ, or Flk1+/lacZ mice were intercrossed with wild-type C57BL/6J male mice. The embryos and uteri/p lacentas were used for examining expression patterns of LacZ in the maternal and fetal blood vessels in the placenta and umbilical vessels. To generate Alk1-null embryos, male and female Alk1+/lacZ mice were intercrossed. At E9.5 and 10.5, placentas with embryos were harvested, fi xed and stained with an X-gal solution. To test the Cre-mediated DNA excision, a male Tg( Alk1-cre ) F1 mouse was crossed with a female R26R mice. At E9.5, 10.5, 11.5, 13.5 and 15.5 em bryos were harvested, fixed and stained with an X-gal solution. To examine the Cre-me diated DNA excision in a germ cell, male or female Tg( Alk1-cre );R26R bigenic mice were crossed with wild-type C57BL/6J mice. At various embryonic stages, embryos were harveste d and subjected to X-gal staining. Conditional allele of Bmpr2 was generated in a previous study (180). Mice carrying Bmpr2 conditional allele were bred with the Tg( Alk1-cre )-L1 line. After multiple rounds of the breeding process, the following mouse groups containing different genetic combination were generated: Alk1-cre(+);Bmpr2f/f, Alk1-cre(+);Bmpr2+/f,and Alk1-cre(-);Bmpr2f/f. To visualize the Cremediated Bmpr2 deletion, an R26R transgene was introduced into a few of the mice in each group, and a PCR analysis was carried out with a primer set flanking two loxP sequences. X-Gal Staining For examination of the embryonic expression pattern of Alk1 in the Tg( Alk1-lacZ ) line, the pregnant female mice were euthanized by cervica l dislocation. Mouse uteri with embryos were removed, placed in PBS, and opened to expose th e embryos. The residual uterine wall and yolk sac were carefully removed. If the embryos were older than E12.5, chest and abdominal walls of the embryos were opened, and internal organs we re isolated and processed together with the embryos. The isolated embryos were subjecte d to fixation with a fixative solution (1%


45 formaldehyde, 0.2% glut araldehyde, 2 mM MgCl2, 5 mM EGTA, and 0.02% NP-40) for 10 minutes at room temperature. After fixation, th e embryos were washed with PBS for 5 minutes. The washing was repeated three times on a rocker Next the embryos were put into an X-gal solution [5 mM K3Fe(CN)6, 5 mM K4Fe(CN)6, 2 mM MgCl2, 0.01% NaDeoxycholate, and 0.5 mg/ml X-gal]. The embryos were incubate d in the X-gal solu tion overnight at 37 The next morning, the stained embryos were washed with PBS, fixed with 4% paraformaldehyde for 2hrs, washed again with PBS and kept at 4 for future use. To examine the LacZ expression in organs at postnatal stages, each organ was remo ved, sliced to 1-2 mm thickness and stained with the same X-gal staining procedure as the embryos. To examine the -galactosidase activities in th e extraembryonic proper of Tg( Alk1-lacZ ), Alk1+/lacZ, Alk5+/lacZ, or Flk1+/lacZ embryos, the uterine wall and yolk sac were opened, and the placenta with the embryo attached were fixed fo r 15 minutes in the X-gal fixative solution. For E12.5 15.5 stages, placentas were sagittally diss ected to 1-2 mm thickness for better penetration of X-gal solution. After washing with PBS, the same X-gal staining procedure that was employed on the embryos was applied. The stained samples were either embedded in paraffin for histology or cleared with organi c solvent (Bezyl alcohol :Benzyl benzoate = 1:1) for whole mount imaging. PCR Analysis Tail biopsies were carried out at three week s post-delivery. A small piece of the yolk sac was used for the genotyping of embryonic stages. Th e clipped tail or yolk sac was lysed in lysis buffer [50 mM Tris (pH 8.0), 0.5% TritonX-100] with proteinase K (1 mg/ml) for overnight at 55 The next morning the lysed tail samples were centrifuged at 12000 rpm for 10 minutes to precipitate tissue debris. Th e PCR reaction mixture consisted of the following: 2.5 l of 10X


46 buffer, 3 l of MgCl2 (25 mM), 0.5 l of dNTPs (25 pM), 0.5 l of each primer, 1 l of genomic DNA, and 15 l of H2O. Mineral oil was overlaid to preven t the PCR mixture from evaporation. A hot start PCR reaction was prepared under th e following directions: the mixture was stayed at 94 for 10 minutes then the temperature was lowered to 72 Once the temperature reached 72 2 l of a Taq polymerase was added. After adding the Taq polymerase, the PCR cycle was run in the following way: 35 cycles at 94 for 45 seconds, 1 cycle at 60 for 45 seconds, and 1 cycle at 72 for 1 minute. The primers used for genot yping are summarized in the Table 2-1. Wound Healing Study Four-month-old mice were anesthetized with an intraperitoneal (I.P. ) injection consisting of 0.015 mL of 2.5% Avertin per gram of body weight. The back of the mouse was shaved and swabbed with Betadine. Three 4mm-diameter full-thickness excisional wounds were placed on the mid-dorsum using a biopsy punch. The wounds were not sutured and were treated with Betadine. The wounded areas we re completely recovered after 14 days, in most cases. The mice were euthanized at six different time points (3, 5, 8, 10, 12, and 14 days after wounding), and full dorsal-side skins were rem oved and stained with X-gal, followed by fixation and clearing. Histology and Immunohistochemistry The pieces of tissue and embryo samples were dehydrated with increasing concentrations of ethanol (70 % 95 % 100 %) for 15 minutes each and incubated in an organic solvent for another 15 minutes. After incubation in the orga nic solvent, the samples were incubated in paraffin for at least 2 hrs. The embedded embryo s, placentas, and adult tissue samples were cut into 6-7 m of thicknesses. For morphometric analysis after hemodynamic study, the left lobe of lung was transversely cut to 5 m thickness. Tissue secti ons were deparaffinized in CitrosolV for at least 15 minutes and sequentially rehydr ated with an ethanol series (100 % 95 % 70 %, 15


47 minutes/each) and water. Some of the sections were subjected to hematoxylin and eosin (H&E) staining. To determine the X-gal posi tive cells, the sections were c ounterstained with nuclear fast red (NFR). For immunohistochemistry, the st andard ABC method was used w ith a Vector staining kit (Vector laboratories, Inc., CA). Briefl y, sections were hydrated and endogenous peroxidase activity was blocked by treat ing them with 3% (v/v) hydrogen peroxide (H2O2) at room temperature for 10 minutes. After two times of wa shes with PBS, the sections were incubated with blocking serums corresponding to their s econdary antibodies species for 1 hour at room temperature, followed by an incubation of primar y antibodies. Biotinated secondary antibodies were incubated for thirty minutes after bei ng washed with PBS. After secondary antibody incubation, sections were treate d with peroxidase-conjugated avid in/biotin complex for thirty minutes followed by two PBS washes. Color development was carried out with a DAB+ substrate chromogenic solution (Vector laboratories, Inc., CA). The antibodies used for immunohistochemistry are the following: SMA (clone: 1A4; Sigma, 1:800), Pecam1 (clone: Mec13.3; PharMingen, 1:200), phosphor-Smad1/5/8 (Cat. # 9511; Cell Signaling Tech., 1:300), CD68 (clone: M-20; Santa Cruz Biothech., 1:40 0), cytokeratin (Cat. #: Z0622, DAKO, 1:600), Ki-67 (clone: M-19 Santa Cruz Biothech., 1:400) von Willebrand factor (Cat #: A0082; DAKO, 1:400), fibrin(ogen) (Cat. #: A0080; DAKO, 1:400), Tenascin-C (Cat. #: AB19013; Chemicon, 1:200), cleaved caspase-3 (clone: 5A1; Cell Sign aling Tech., 1:400), and Serotonin transporter (clone: C-20; Santa Cruz Biotech., 1:200) antibodies. Hemodynamic Analysis Indirect systemic pressure was recorded by using the tail-cuff method. A pneumatic pulse sensor was distally placed on the tail to an occlusion cuff controlled by a Programmed Electro-


48 Sphygmomanometer (PE-300, Narco Bio-Systems, TX). To evaluate pulmonary pressure, right ventricular systolic pressure (RVSP) was measured by right h eart catheterization through the right jugular vein. Briefly, each mouse was anes thetized with Ketamine (100 mg/kg)/Xylazine (15 mg/kg) and placed in a supine position. A 2-3 cm incision was made to expose the right jugular vein. A Mikro-Tip pressu re transducer (SPR-835, Millar Instrument, TX) was inserted into the right external jugular ve in and advanced into the right ventricle. All electrical outputs from the tail cuff, the pulse sensor and trans ducer were recorded and analyzed by a Powerlab 8/30 data acquisition system and the asso ciated Chart software (ADinstrument, CO). After the hemodynamic study, the mouse was euthanized, and the organs, including the heart and lungs, were isolated for further analyses. The outflow tract and atria were removed prior to the weighing of the right and left ventricle plus the septum. Morphometric Analysis After the hemodynamic analysis, the left lung was then inflated with 20-25 cmH2O of gravity for 20 minutes followed by flow with 4% paraformaldehyde for another 20 minutes. The trachea of the inflated lung was tied up with sutu re silk and fixed with 4% paraformaldehyde overnight. The next morning, the lung sample was washed two times with PBS for 10 minutes each, then transversely sliced into 0.5 cm sec tions. To determine the muscularized peripheral vessels and wall thic kness, immunostaining with SMA was performed. In each section, SMApositive vessels were categorized by their locations such as vessels at the level of terminal bronchioli, respiratory bronchioli, alveolar ducts, or alveolar sac. To determine muscularization of pulmonary vessels, peripheral blood vessels ranging from 30-70 m of diameter were counted in at least four fields at X20 magnification. The counted vess els were categorized as fully muscularized (75-100% of me dial layer is covered by SMA staining), partially muscularized


49 (1-74% of medial layer is covered by SMA staining), or non-muscular vessels at the level of alveolar duct and alveolar sac. The percentage of pulmonary vessels in each category was calculated by dividing the number of vessels in the category by the total number of counted vessels in the same field. To calculate the per centage of wall thickness (W T), circular and fully muscularized vessels with ranges of 30-70 m of diameter were selected. A thickness (WT1) between the outer boundary a nd the inner boundary of the SMA-positive medial layer was measured. Another thickness (WT2), the diam etrically opposite point of the vessel, was measured. At the same time, the external diameter (ED) was also measured. The percentage medial thickness for these vessels were calculated as [(WT1 + WT2) X 100]/ED. Western Blotting One of the right lung lobes were used for Western blot analysis. Once removed from a 80 freezer, the lung lobe was placd into a Chem icon lysis buffer [50 mM Tris (pH 6.8), 1 mM EDTA and 2% SDS], disrupted with a pestle un til the lung tissue was able to move freely, sonicated three times for 5 seconds and spun at 13000 rpm for 15 minutes. The concentration of protein was determined using the Biorad DC Assa y kit. Forty micrograms of total proteins were fractionated by 12% of SDS-PAGE, transferred to PVDF membranes and incubated with the primary antibody for BMPR2 (Cat #: 612292; BD Transduction Lab., 1:500). After incubation with horseradish peroxidase-linked anti-mouse Ig G, a chemiluminescent detection reagent (ECL PlusTM, Amersham Pharmacia Biotech Inc, NJ) was used to detect the protein. The antibodies used for the other Western blotting analysiss are the following; phospho-ERK (clone: E-4; Santa Cruz Biothech., 1:800), total ER K (p44/42) (Cat #: 9102; Cell Signaling Tech., 1:800), Smad 1 (clone: T-20; Santa Cruz Biothech., 1:400), Al k1 (clone: D-20; Santa Cruz Biothech., 1:400),


50 eNOS (Cat #: 610296; BD Transduction La b., 1:800), GAPDH (Cat. #: ab8245; abcam, 1:10,000), and -actin (clone:A5441; Sigma, 1:10,000). Statistics Data were described as mean SEM. The difference between groups was determined by the Students t-Test or Two-wa y ANOVA. A value of p< 0.05 was considered as a statistically significant.




52 CHAPTER 3 IDENTIFICATION OF ARTERIAL ECS-SPECIFIC ALK1 PROMOTER Note The work presented in this chapter was publishe d in Isolation of a Regulatory Region of Activin Receptor-Like Kinase 1 Gene Sufficient for Arterial Endothelium-Specific Expression by Seki T, Hong KH, Yun J, Kim SJ, Oh SP, 2004 Circulation Research;94:e72., and Activin receptor-like kinase 1 (ALK1) is essential for placental vascular development in mice by Hong KH, Seki T, and Oh SP, 2007 Labor atory Investigation; 87(7):670-679. Introduction Blood vessels consist of a netw ork of arteries, capillaries, and veins. Recent discoveries of arteryor vein-specific genes, such as ephrinB2 and its receptor EphB4, and some molecules involved in the Notch-delta signa ling pathway have contributed to a significant advancement in understanding the mechanisms by which arteries and veins gain their distinct identities during vascular development (181). In c ontrast to the long-standing belief that the acquisition of arterial and venous identities is largely determined by different physiological parameters (eg, blood flow, blood pressure, and shear st ress), genetic studies in mice and fish suggest th at arterial and venous ECs acquire their distinct molecular identities be fore the establishment of blood flow (182;183). Studies have shown that the aforementioned arteryor vein-specific genes play crucial roles in angiogenesis and segregation of the two blood vessel ty pes (182;184;185). It remains unclear, however, whether those genes are dir ectly involved in the morphogenesis of vessel typespecific architecture during development. ALK1 is a TGFtype I receptor in vascular ECs ( 113;186). Haploinsufficiency of ALK1 in a human causes HHT2, also known as Osler-Web er-Rendu syndrome, which is characterized by recurrent epistaxis, localized mucocutaneous telangiectas es, and arteriovenous malformations


53 (AVM) in the lungs, liver, and brain (187;188) Mutations on the gene include nonsense, missense, frameshift, and interference of splicing, all of which seem to result in loss-of-function mutations (188). Clinical and gene tic data have demonstrated th at 60% of HHT cases are caused by such exonic mutations; however, types of mutati on in the remaining 40% of the cases are still inexplicable (189;190). Although there has been si gnificant progress in understanding the genetics of HHT, an etiological role of non-exonic mutations of ALK1 in HHT still remains to be studied. Pathologically, the telangiectasia is divided into three stag es. In the first stage, the cutaneous vascular bed is obvious with the dilation of post capillary venule followed by a thickening of vessel walls resulting from accumula tion of excessive pericytes. In the second stage, dilated arterioles are obs erved with intervening capillary beds. In the end stages, the venule becomes gradually dilated and spreaded throughout the skin in a tortuous pattern. The capillary network is no longer observed and is re placed by direct connections between arterioles and venules. The arteriole and venule portions of each arteriovenous connection appear to maintain characteristics of their origins (191). Our lab and another group have reported that Alk1 plays an important role in vascular development and remodeling (113;114;117). Ho mozygous Alk1 mutant mice show embryonic lethality around E10.5 with severe vascular defects characterized by hyperdilation of large vessels, and failure in recruitment of VSMCs sh eathing ECs tube (113). Consistent with this result, a mutation in the zebrafish Alk1 gene, vbg resulted in an increased number of ECs within the affected vessel, suggesting th e role of Alk1 in vascular rem odeling (192). It has also been shown that Alk1 heterozygous mice display HHT -like phenotypes in a number of characteristics including age-dependent pene trance, lesions in specific anatomical locations, histological


54 similarities between the lesions, and even the recapitulation of the secondary phenotype of cardiac pathology (115). Using Alk1-lacZ "knock-in" mice ( Alk1+/lacZ), we have previously observed a dynamic spatiotemporal expression pattern of Alk1 : Alk1 is expressed predomin antly in arterial ECs throughout the developmental and postnatal grow th stages, and its expression is diminished in the adult stage; Alk1 expression is induced in reconstructing and rem odeling arteries during angiogenesis prompted by either wound healing or tumorigene sis (117). The data from these expressions together with the VSMC defect in Alk1 -deficient embryos suggest that ALK1 may be directly involved in the morphogenesis of arterial development and remodeling. As proposed in the earlier study, HHT has been generally considered as a disease that is initiated in the vein (112). However, our demonstration, which shows predominant Alk1 expression in arterial ECs, suggests that the artery is the primary vessel affe cted, and the remodeling of the vein could be a secondary effect caused by he modynamic changes (117). The placenta is a vital organ for exchanging gases, nutrients, waste products, and various cytokines as a maternal and fe tal interface (193;194) Morphogenesis of the chorioallantoic placenta begins with formation of the chorion from extra-embryonic ectoderm and the allantois from mesoderm at the posterior end of the em bryo during gastrulation. Subsequently, allantois reaches to the chrionic plate, and feto-place ntal blood vessels are established by E8.5 (195). Extensive branching morphogenesis by the allant oic mesoderm and the chorionic trophoblasts (syncytiotrophoblasts) take s place to structure the labyrinth (Lab) layer at which the maternalfetal exchange occurs. Both mouse and human placentas are similar because both belong to the hemochorial placenta, in that the lining of the fe tal trophoblast cells are in direct contact with the maternal blood. Trophoblasts from the ectoplacen tal cone contribute to the spongiotrophoblast


55 (Sp) layer, in which maternal arterial endotheli al cells are eroded away and replaced by fetal trophoblast cells. This replacement step is importan t because it makes the maternal arteries low resistant vessels (194). The main goal of this chapter is to characteri ze the nature of the spa tiotemporal pattern of Alk1 expression using the convent ional transgenic approach, and to further analyze both embryonic and extraembryonic Alk1 expr ession in the transgenic line. Results Generation of Transgenic Constructs and In Silico Analysis of the Alk1 Promoter To investigate the cis -acting elements required for the spatiotemporal expression of the Alk1 gene (Figure 3-1A), three different transgen ic constructs containi ng various regions/lengths of putative regulatory sequences connected to the lacZ gene were generated (Figure 3-1B). The first construct, pXh4.5-SIBN, contained a 2.7 kb promoter, exon 1, and a part of 5' region of intron 2. Five independent founder lines were established from this construct, and the transmission of the transgene to F1 offspring was confirmed via genomic Southern blot analyses. None of the embryos from these lines, however displayed the vascular-specific expression of the lacZ gene. To investigate whet her the essential regulatory elements resided in upstream sequences, the 2.7 kb promoter region was extended to a tota l length of 8 kb in the second construct, pBam9-SIB. As in the first case, a mouse line from this construct did not exhibit a vascular-specific expression pattern of the transgene either. These results prompted us to se arch for the potential regulatory elements by comparing DNA sequences between the human and mouse ALK1 gene loci. Intriguingly, dot plot and blast analyses revealed the presence of highly conserved sequences at the 3' region of intron 2 (Figure 3-1C). The span of a 1.8 kb conserved region in the 3' region of intron 2 consisted of at least five homologous regions ranging from 37 to 145 bp in length, and each sequence cluster showed more


56 than 80% identity (data not shown). Based on these results, a third construct, pXh4.5-in2-SIB, which was similar to the first construct, pXh4.5-SIBN, but with the extension of the 3' region to include the remaining intron 2, exon 3, and approximately 300 bp of 5' region of intron 3, for a total length of 9.2 kb were generated (Figure 3-1B). Two founder lines from this construct showed artery-specific expression of the lacZ gene (Figure 3-2A), which recapitulated the pattern seen in Alk1+/LacZ embryos (Figure 3-2B). To examine detailed regulatory activities of the 9.2 kb Alk1 sequences, a pattern of X-gal stainings in various stages of embryos, adult organs, as well as in wounded skins, were prepared and compared to the results to those of the Alk1+/lacZ mice. Expression Pattern of LacZ in Tg( Alk1-lacZ ) Mice Two transgenic mouse lines from the pXh4.5-in2-SIB construct, Tg( Alk1-lacZ ) C2 and K1 showed identical expression patterns with slightly different intensity [thus, will be collectively referred as Tg( Alk1-lacZ ) hereafter]. In embryos, the transgene expression pattern of Tg( Alk1lacZ ) was virtually indistinguishable from Alk1+/LacZ except for the intensity levels (Figure 3-2). Strong expressions in the capillaries of perineural tissues in E10.5 to 13.5 embryos were consistently observed (Figure 3-2C and 2D). The transgene expression in Tg( Alk1-lacZ ) was detected in developing arterial ECs, but neither in VSMCs nor in venous ECs (Figure 3-2E and 2F). The artery-specific expression was also clearly shown in internal thorac ic arteries (Figure 32G and 2H), mesenteric arteries (Figure 3-2I), and desce nding aorta and intercostal arteries (Figure 3-2J and 2K). Strong expression in the pulmonary vessels (Figure 3-2L and 2M), and the lack of staining in the liver (data not shown) were also c onsistent with those in Alk1+/LacZ. The X-gal staining patterns of Tg( Alk1-lacZ ) mice during postnatal life were, to a large extent, identical to the ones of Alk1+/LacZ mice, in which the intense Alk1 expression in arterial vessels during the postnatal gr owth phase was greatly diminished in most of the tissues except for


57 the lungs at the adult stage. Consistent with these observations, Tg( Alk1-lacZ ) mice showed intense vascular staining, such as in the brain an d the iris (Figure 3-3A and 3B), during the early postnatal growth phase In the adult stage, however, positive staining was barely detectable in the capillary-like small vessels throughout the body, except for a moderate amount of expression in the lungs (Figure 3-3C through 3E). In the skin, the Tg( Alk1-lacZ ) mice showed intense staining of arteries at the newborn stage (Figure 3-3F) as observed in Alk1+/LacZ. Unlike Alk1+/lacZ mice, however, the expression was greatly reduced in the 2-week-old mice. Next, a transgene expression during induced angiogenesis in adult mice using a skin wound healing model was also examined. Although overall X-gal staining intensity in the skin was lower in Tg( Alk1-lacZ ) mice when compared with the Alk1+/lacZ mice, the expression pattern was comparable. During wound healing in the adult stage, the transgene expression was induced in arterial vessels in a similar fashion as the Alk1+/lacZ mice, although the staining was significantly lighter and limited in both duration and area (Figure 3-3G through 3J). Although the vascular staining in the wounded area of the Alk1+/lacZ mice was present from days 3 to 12 after wounding, staining of the transgenic mice was observed only from days 3 to 8. Also, the staining was restricted to the vessels adjacent to the wounds and did not extend to distant arteries that fed the wound lesion in the transgenic mice. Identification of Regulatory El ements in the Homologous Regions Our transgenic studies indicate that the conserved intronic regions may contain the enhancer element(s) for the artery-specific Alk1 expression. To investigate whether the 9.2 kb regulatory fragment of the Alk1 gene contains common regul atory elements, DNA sequence homology alignments between the 9.2 kb regulatory sequence and regu latory regions of other known endotheliumor artery-specific genes were perf ormed. Extensive homology searches, however, did not reveal any significant continuous homology region.


58 To identify potential transcripti onal factor bindi ng sites (TFBSs) in the 9.2 kb regulatory fragment, comprehensive in silico analysis with the rVISTA 2.0 program was performed. In the analysis, 16 conserved TFBSs for 12 transcriptions factor s known to regulate EC-specific genes within the 9.2 kb regulatory fragment were found (Table 3-1, Figure 3-4). The in vivo relevance of the conserved intronic regions and TFBSs remains to be investigated. Alk1 Is Expressed in the Primitive Blood Vesse ls During Early Chorioallantoic Placenta Development Utilizing Alk1+/lacZ as well as Tg( Alk1-lacZ ) lines, the expression pattern of Alk1 during early chorioallantoic placental development wa s investigated. The first detectable Alk1 expression was observed in the allantoic bud at around E7.75 (Figure 3-5A). At the subsequent stages when the allantoic mesoderm forms pr imitive umbilical vessels and reaches to the chorionic plate, Alk1 expression was detected in these primitive vascular beds formed in the allatoic mesoderms as well as in the embryonic blood vessels (Figure 3-5B). Most Alk1-positive cells in these extra-embryonic tissues were positive for Pecam1, a mature endothelial cellspecific marker (Figure 3-5C and 5D). Alk1 expr ession was apparent in patent blood vessels formed in the chorio-allantoic connection of E9.0 embryos (Figure 3-5E and 5F). Alk1 Is Differentially Expressed in Umbilical Arterial Endothelium, and Required for the Distinctive Umbilical Artery and Vein To further investigate whether the arterial endothelium-specific expression pattern that we have observed in the embryo prope r is conserved in extraembryonic vessels, we examined the Xgal staining pattern in the umbilical vessels of E15.5 Alk1+/lacZ and Tg( Alk1-lacZ ) mice and compared it with that of Flk1+/lacZ and Alk5+/lacZ mice. X-gal-positive cells were found mostly in the umbilical arterial endothelium of Alk1+/lacZ mice (Figure 3-6A and 6B). No X-gal positive cells were found in the vascular smooth muscle or adventitial layers, a nd the venous endothelial cells had a very weak X-gal positive staining. Tg( Alk1-lacZ ) mice exhibited the same expression


59 pattern with the Alk1+/lacZ mice, except that the X-gal st aining intensity in the venous endothelium was stronger than that of Alk1+/lacZ mice (Figure 3-6C and 6D). In contrast, X-galpositive cells were found in both the arterial and ve nous endothelium as well as in the capillaries in the advantitial layer of the umbilical artery in Flk1+/lacZ mice (Figure 3-6E and 6F). Interestingly, the X-gal staining intensity in the venous endothelium was stronger than that of the arterial endothelium (Figure 3-6E). In addi tion, X-gal-positive staini ng in the microvessels surrounding medial layer of ar teries were detected in Flk1+/lacZ (Figure 3-6E), but not in Alk1+/lacZ mice (Figure 3-6C). These results demonstrate heterogeneity of expression of endothelialspecific genes in a vessel type-dependent manner. The Alk5 expression was predominantly detected in the medial layers but not in the e ndothelium of both umbili cal arteries and veins (Figure 3-6G and 6H). The non-overlappi ng expression patterns of these TGFtype I receptors are consistent with our previous study in embryonic vessels, suggesting that TGFmight be involved in vascular morphogenesis utilizing two distinct type I recepto rs: ALK1 in ECs and ALK5 in vascular SMCs. The vessel type-specific expression pattern s as well as the function of Alk1 were investigated with Alk1+/lacZ and Alk1lacZ/lacZ embryos at E9.5 and 10.5 stages. The differential expression pattern was not obvious at the E9.5 st age (Figure 3-7A), but became noticeable by the E10.5 stage (Figure 3-7C). Umbilical arterial and venous lumens were clearly separated in Alk1+/lacZ mice along the entire length of umbilical vessels (Figure 3-7A and 7C), whereas only one large lumen was found in the Alk1 -null embryos at both E9.5 (four out of four embryos) and E10.5 (three out of three embryos), especially ar ound the middle of umbilical vessels (Figure 37B and 7D).


60 Alk1 Is Expressed in the Arteries and Capillaries in the Labyrinth Layer of Placenta Next, the Alk1 expression in the midand late gestational stages of placentas was examined. Expression patterns in both Tg( Alk1-lacZ ) and Alk1+/lacZ placentas were essentially identical, although the overall stai ning intensity appeared to be higher in the Tg line (Figure 38A 8F). A murine placenta is a multiple laye red organ consisting of an endometrium-derived deciduas (Dec), a junctional zone containing tr ophoblast giant cells, a spogiotrophoblast (Sp), and a labyrinth (Lab) where phys iological exchanges occur ( 193). The Lab layer contains multiple cellular barriers: a single layer of m ono-nucleated cytotrophoblasts, two layers of multinucleated syncytiotrophoblasts, and fetal capillary endothelial cells (194). Predominant arterial staining was again manifested, and fetal arteries projecting to the Lab layer of the definitive placenta were distinctively highlighted by the X-gal staining. On the ot her hand, X-gal staining was not detected in the Sp as well as Dec la yers of placentas in the Alk1 reporter lines. Dissimilar from Alk1 reporter lines, X-gal-positive fetal arteries projecting into the Lab layer were unobservable in the Flk1+/lacZ mice (Figure 3-8G 8I). In addition, Flk1 expression was detected from maternal vessels in the decidua and uterine walls (Figure 3-8G). Histological sections of X-gal stained Alk1+/lacZ placenta confirmed the restricted Alk1 expressions in the umbilical vess els and the Lab layer (Figure 3-9A 9D). No detectable X-gal staining was observed from fetal and maternal vessels in the Sp and Dec layers. To determine the cell type expressing Alk1 in the Lab layer, we stained E12.5 placenta s ections with anti-Pecam1 or cytokeratin (CK) antibodies. We used E12.5 placentas because the villu s density in the Lab layer is less compact than that of E15.5, and thus different cell layers can be identified more easily. The X-gal-positive cells in the Lab layer appeared to be Pecam1-positive, but mostly nonoverlapping with CK-positive cells, showing that Alk1 is expressed in endothelial cells but not in syncytiotrophoblasts.


61 Alk1-Deficiency Results in Impairment of Um bilical and Placental Blood Vessel Formation To investigate the role of Alk1 in placental development, Alk1-null placentas at E9.5 stage were examined. The branching morphogenesis of the chorionic ectoderm and invasion of allantoic mesoderm into the Lab layer of Alk1lacZ/lacZ placentas were largely unaffected (Figure 310A and 10B). In Alk1-null placentas, however, the chorionic vessels were severely dilated and showed signs of fusions, similar to the vasc ular abnormality observed in Alk1-null embryos (113). Discussion In this chapter, we have demonstrated, us ing transgenic mice, that the 9.2 kb fragment of the Alk1 gene contains essential regulatory elements for its spatiotemporal expression and that heterogeneous Alk1 expression is conserved in extra-embryonic vasc ulature and is restricted to fetal ECs. Also implicated, an intriguing possibility is that the cons erved region in intron 2 may contain the enhancer elements for the artery-specifi c gene expression. This result suggests that intron 2 contains a potential re gulatory element(s) which dir ects its arterial EC-specific expression. Supporting this notion, a homol ogy study between mouse and human ALK1 revealed that at least five homologous regions ranging from 37 to 145 bps exist at the 3 end of intron 2. Each homologous region shows more than 80% homol ogy between the two species. The data also demonstrated that 16 conserved transcriptional f actor binding sites (TFBSs) for 11 transcription factors known to regulate EC-specific genes within the homologous regions of 3 end of intron 2. The in silico data suggests a potential role of the intronic sequence in the spatiotemporal regulation of Alk1 expression durin g vascular development. Thus, the analysis of role of the homologous intronic sequences in the regulati on of Alk1 expression may provide novel genetic insights in arteriogenesis. This study also suggests that the expr ession pattern observed in the


62 Alk1+/lacZ mice was not a consequence of Alk1 haplo insufficiency. The presented 9.2 kb regulatory fragment, as the first regulatory elements specific for arterial endothelial cells, would provide invaluable information regarding regulatory mechanisms of Alk1 as well as other arteryspecific genes. Furthermore, this regulatory fragment can be used to activate or silence a gene in developing arteries to study its function in arteriogenesis and in the remodeling of arterial vessels. We also showed that Alk1-deficiency re sults in severe dilation and fusion of chorioallantoic and umbilical vessels, sugges ting a vasodilatory phe notype of the Alk1-null embryos. The variance of expr ession patterns between Tg( Alk1-lacZ ) and Alk1+/lacZ mice in the skin wounds may be due to either the strain differences, or a lack of additional cis -acting elements involved in the Alk1 expression during wound-induced angiogenesis. Using lacZ reporter lines we also demonstrat ed heterogeneous Alk1 e xpression patterns in umbilical and fetal placental vessels and that Alk1-deficiency leads to persistent umbilical vessels and extensive dilation of chorioallantoi c vessels. Alk1 expression was first detected in the allantoic bud and then in the allenochorionic primitive vessels when a llantois reaches to the chorion. Alk1-null embryos formed a large persis tent vessel instead of a separated umbilical artery and vein. At present it is not clear whether the two umb ilical vessels might have been formed and then laterally fused, or that separa tion had not occurred during development of the umbilical vessels of the mutant embryos. At later stages, Alk1 was shown to be expresse d predominantly in the arterial endothelium of umbilical vessels. This result is consistent with our previo us findings in embryonic vessels (117), indicating that oxygen concentration is no t the underlying basis of the vessel type-specific differential Alk1 expression. Recent studies suggest that arterial and venous endothelial cells have distinct molecular identitie s prior to patent vessel formation. Ephrin-B2 was expressed in


63 both arterial ECs and SMCs, wher eas EphB4 (a putative receptor for Ephrin-B2) was expressed almost exclusively in venous ECs prior to the onset of circulation ( 196;197). More recently, several other artery-specific genes have been repo rted in vertebrate embryos, including a Notch ligand Delta (Dll4) (198;199) and a Notch-Delta downstr eam transcription factor (Gridlock) (200). Studies have clearly shown that these genes are involved eith er in early lineage determination between arterial and venous ECs or in segregating two ve ssel identities at the capillary level. However, it is unclear whether th ese arteryor vein-speci fic genes are involved in creating a structural and functi onal distinction between arterial or venous vasculature. Unlike Ephrins and Delta ligands, which are membrane-bou nd and thus require cell-to-cell contact, Alk1 interacts with TGFligands as paracrine or endoc rine signals. It has been demonstrated that Alk1-deficiency leads to impa ired differentiation or recruitment of SMCs and severe dilation and fusion of embryonic and extra-embryonic vessels (113;114). With the correlation between the thickness of smooth muscle layers and the level of Alk1 expression in the artery and vein, we speculate that ALK1 may play an important role in arteriogenesis and in the remodeling of arteries. Temporal deletion of the Alk1 gene at later stages of development would provide a direct answ er to this hypothesis. Alk1-null embryos exhibited severe dilation of blood vessels with an elevated VEGF mRNA label (113). A question was ra ised as to whether such vascul ar defects were a response to a hypoxic condition. In the study, we presented evid ence that the process of establishing the labrynthine layer was unaffected in Alk1-null placenta, yet chorionic vessels were severely dilated to the same degree to the embryonic vessels. This result suggests that the phenomenon of blood vessel dilation is primarily due to Alk1-defici ency per se rather th an a secondary response to hypoxia.


64 ALK1 shares numerous common characterist ics with ENG. Both ALK1 and ENG are plasma membrane proteins mediating TGFfamily signals, are involved in the same genetic disease, and are expressed in vascular endothe lial cells. Recent reports have suggested that elevated sENG in conjunction with sFLT1 of pl acental origin might be a major pathogenetic marker and cause of preeclampsia (201;202). Pr eeclampsia is characterized by hypertension and proteinuria in the third trimester of pregnancy and occurs in 5% of all pregnancies (203;204). Deficiency or incompletion of replacing mate rnal arterial endothelium with trophoblasts increases vascular resistance and maternal bl ood pressure, and thus may contribute to the pathogenesis of preeclampsia. Unlike Flk1 (VEG F receptor 2) whose expression is limited to endothelial cells, a strong fetal Flt expr ession was observed in spongiotrophoblasts and syncytiotrophoblasts in addition to ECs (205). Eng was also shown to be highly expressed in the syncytiotrophoblasts (206). Since both ALK1 an d ENG interact as a si gnaling partner and are involved in the same genetic disease, there is an emerging question whether ALK1 is also involved in preeclampsia (207). In the study, a difference in the expression patter n of Alk1 from those of Eng or Flt1 in the placenta has been found: Alk1 expression was restri cted to fetal endothelial cells while Eng and Flt1 were shown to be expressed in the pl acental trophoblast cells (205;206). This difference suggests that Alk1 is most likely not involve d in preeclampsia at the level of placenta.


65 Table 3-1. List of tran scription factors of whic h TFBSs were found in the Alk1 regulatory fragment and their respective EC-specific target genes Binding sites TFs In promoter In enhancer SP1 VEGFR2, VE-cadherin, ICAM2, eNOS SP3 VE-cadherin AP2 VEGFR2 CREB VEGFR1 NFB VEGFR2, PECAM, ICAM2 GATA vWF VEGFR2 GATA1 ICAM2 GATA2 PECAM, endothelin1, ICAM2, eNOS ETS ICAM2 Tie2, VEGFR2 ETS1 VEGFR1, VEGFR2, VE-cadherin, vWF ETS2 VEGFR1, VEGFR2 ELF1 Tie2 SP1 indicates specific protein 1; SP3, specific pr otein 3; AP2, adaptor protein 2; CREB, cyclicAMP response element binding protein; NFB, nuclear factorB; ELF1, E74-like factor 1; VEGFR, vascular endothelial gr owth factor receptor; VEcadherin, vascular endothelial cadherin; ICAM2, intercellular adhesion molecule ; eNOS, endothelial nitric oxide synthase; PECAM, platelet/endothelial cell adhesion molecule; and vWF, von Willebrand factor.


66 Figure 3-1. Transgenic constructs and the dot plot analysis between human and mouse ALK1 genomic sequences. A) Full mouse Alk1 gene structure. Restriction enzyme sites used for generating the transgenic constructs are indicated by X ( Xho I) and B ( Bam HI). Boxes represent exons, and exon numbers are indicated above. B) Schematic representations of 3 transgen ic constructs. Detailed constr uct information is available in the Materials and Methods. C) Dot pl ot analysis between partial genomic sequences of human (y-axis) and mouse (x-axis) ALK1 gene. Exon 1 of the human ALK1 gene is split in 2 exons (1 and 2) in the mouse Alk1 gene. Continuous sequence homologies are highlighted by gray shaded ar eas. Note that the 3' regions of mouse intron-2 and human intron-1 are highly conserved (gray bracket).


67 Figure 3-2. Artery-specific lacZ e xpression in the blood vessels of Tg( Alk1-lacZ ) embryos. A) and B) Whole-mount X-galstained E11.5 Tg(Alk1-lacZ) and Alk1+/LacZ embryos, showing the identical staining pattern but with much higher intensity in Tg(Alk1lacZ) C) and D) Strong LacZ -positive capillary-like vessels in the developing neural tube; C) the section of E11.5 embryo and D) a dorsal view of E12.5 neural tube. E) and F) Sections of X-galstained E11.5 Tg(Alk1-lacZ) embryos counterstained with nuclear fast red (NFR) or immunostained with antiSMA. Note that the X-gal positive cells were found in a single laye r of endothelium in the artery (a, arrowheads), but not in the vein (v), and were not concomitant with SMCs. G) and H), Blood vessels on the thoracic wall of newborn pups before and after X-gal staining, showing that only arteries and conn ecting small arteries were X-gal positive. I) Whole-mount X-galstained mesenteric ve ssels of E18.5 fetuses. Only mesenteric artery (a) but not ve in (v) showed the transgene expr ession. J) and K) X-galstained aorta and intercostal blood vessels before a nd after clearing with the organic solvents. Similar sizes of intercostal arteries (a) a nd veins (v) were running side-by-side J), whereas only arteries and dorsal aorta (ao) were X-gal positive. X-galpositive staining in the rib skeleton (*) is from the endogenous -gal activity. L) Intense X-gal staining in the lungs an d aorta (ao) of E15.5 Tg(Alk1-lacZ) embryos. M) NFR counterstained section of E15.5 lungs s howed X-galpositive arteries (a) and capillaries (arrowheads). Note that bronchi al epithelial cells were X-gal negative. Scale bars in C, E, F, and M = 50 m.


68 Figure 3-3. Postnatal tran sgene expressions in Tg( Alk1-lacZ ) mice. A) D) X-galstained blood vessels in the brain and the iris. A) and B) Intense tran sgene expression in newborn brain and 2-week-old iris were greatly dimini shed in the adult stage. C) and D) Only speckled staining in small branches (arrowh eads) was observed. E) Adult lung section counterstained with NFR. Alve olar capillaries expressed the transgene in the adult stage. F) Intense staining in the arteries of newborn skin. G) J) X-gal staining in the skin during days 3, 5, 8, and 10 after w ounding. LacZ expression was induced during wound healing (arrowheads), but in a limited extent. Number and intensity of X-gal positive vessels peaked at day 5 after wounding and diminished completely by day 10. Asterisks (*) in G) through J) indicate the wounds.


69 Figure 3-4. Potential transcrip tional factor binding sites identified in th e mouse Alk1 pXh4.5-in2 fragment by the rVISTA analysis. Top, 16 TFBS matrixes, which represent TFBSs for 12 different transcription factors. Some transcription factors have multiple TFBS matrixes. For example, SP1 has 4 different TFBS matrixes (SP1, SP1_Q2, SP1_Q4, and SP1_Q6). Positions of each conserved TF BS are indicated as a bar within the matrix. Bottom, Mouse Alk1 gene structure (the top lin e), and the sequence homology between mAlk1 and hALK1 sequences. Color legends in the boxed area indicate the nature of the highly homologous regions. Repe titive sequences are indicated by green boxes on the bottom line. Only the transcrip tional factors known to have active roles in regulating EC-sp ecific genes are shown.


70 Figure 3-5. Whole-mount X-gal staining of Tg( Alk1-lacZ ) embryos at days E7.75E9.0 during early chorioallantoic development. A) The first detectable Alk1 expression was found in the allantoic bud of E7.75 embryos. al, allantois. B) D) X-gal staining was detected in the developing blood vessels of the embryo proper as well as in the primitive chorioallantoic vessels of E8.5 em bryos. C) Transverse sections of X-gal stained E8.5 embryos at the level of the alla ntois were counterstai ned with NFR or D) immunostained with Pecam1 antibodies, showin g that the majority of X-gal-positive cells are Pecam1-positive. E) G) Longit udinal section of E9.0 embryos shows X-gal staining in embryonic and extra-embryonic ECs. F) and G) Higher magnification views of allantoic mesoderm, showing deve lopment of patent blood vessels positive for X-gal and Pecam1. Arrows in D) and G) indicate some representative cells positive for both X-gal and Pecam1.


71 Figure 3-6. Expression patterns of Alk1, Flk1, and Alk5 in E15.5 umbilical vessels. Transverse sections of whole-mount X-galstained umbilical vessels of Alk1+/lacZ; A) and B), Tg( Alk1-lacZ ); C) and D), Flk1+/lacZ; E) and F), and Alk5+/lacZ; G) and H) embryos were counterstained with NFR in A), C) E), and G) or immunostained with SMA antibody in B), D), F), and H). A) D) Note that X-gal-positive cells were mostly detected in the endothelium of the umbilical artery (UA) of Alk1+/lacZ and Tg( Alk1lacZ ) embryos. E) and F) X-gal-staining intens ity was stronger in the umbilical vein (UV) than in the UA, and capillaries in th e medial and adventitial areas were also Xgal-positive in Flk1+/lacZ mice. G) and H) X-gal-positive cells were localized in smooth muscle layers, but not in endothelium of the UA and UV in Alk5+/lacZ. A highmagnification view of the UA is shown as an inset in G).


72 Figure 3-7. Impaired formation of two distinct umbilical vessels in Alk1-null embryos. A) and C) Histological analysis of umbilical vessels of Alk1+/lacZ and B) and D) Alk1lacZ/lacZ embryos at E9.5 and E10.5. Transverse se ctions of whole-m ount X-gal-stained umbilical vessels were counterstained with NFR. Two distinct blood vessels were apparent in Alk1+/lacZ, whereas only one large persis tent vessel was present in Alk1lacZ/lacZ embryos. C) It is interesting to note that the UA-predominant Alk1 expression became evident in E10.5 embryos.


73 Figure 3-8. Comparison of Alk1 a nd Flk1 expression patterns in the definitive placentae. A) I) Whole mount X-gal-stai ned placentae of E15.5 Alk1+/lacZ, Tg( Alk1-lacZ ), and Flk1+/lacZ embryos were cleared with organi c solvents. B), E) and H) Higher magnification of longitudinal vi ews of A), D) and G) C), F) and I) Horizontal views of X-gal-stained placenta from fetus side. No te that Alk1 expression is limited to the Lab layer and strong in fetal arteries projec ting into the Lab layer. Ar, fetal arteries; Dec, decidua basalis; Emb, embryoni c side; Lab, labyrinth layer; Sp, spongiotrophoblast layer; Ut, uterine wall; Ve, fetal veins.


74 Figure 3-9. Longitudinal se ctions of X-gal-stained Alk1+/lacZ placenta. A) D) Histological sections of E15.5 placenta shows that X-galpositive cells are localized mostly in fetal blood vessels at umbilical arteries and capil laries in the Lab layer, whereas no X-galpositive cells are detected in the Sp and Dec layers or in the uterine wall. E) and F) Immunostaining (shown as brown color) of the Lab areas of X-gal-stained E12.5 placenta with Pecam1) or CK) antibodies, showing that EC, but not syncytiotrophoblasts, are X-gal positive.


75 Figure 3-10. H&E staining of histological sections of E9.5 placentas from A) and C) Alk1+/lacZ mice, and B) and D) Alk1lacZ/lacZ mice. Asterisks and arrows in C) and D) indicate chorioallantoic blood vessels and areas of allantoic mesodermal invasion into developing Lab, respectively. C h, chorionic ectodermal cells.


76 CHAPTER 4 DEVELOPMENT OF A NOVEL TRANSGENIC LINE EXPRESSING CRE RECOMBINASE IN ENDOTHELIAL CELLS Notes The work presented in this chapte r will be published in part with In vitro dissection of TGFsignal transduction pathways reveals ALK5and TGF R2-independent role of ALK1 for pathogenesis of hereditary hemorrhagic telangiec tasia 2 (HHT2). by Park SO, Lee YJ, Seki T, Hong KH, Flies N, Jiang Z, Park A, Wu X, Ka rlsson, S, Kaartinen V, Moses HL, Raizada MK, Roman BL, and Oh SP, 2007 (submitted) and Genetic ablation of the Bmpr2 gene in pulmonary endothelium is sufficient to predispose pulmonary arterial hypertension. by Hong KH, Lee YJ, Beppu H, Bloch K, Li E, Raizada MK, and Oh SP (in preparation). Introduction Using a mouse as a model system for studying human genetic diseases has been considered to be advantageous for multiple reasons. First, mice have a relatively short lifecycle. Second, its genome has been completely sequenced and gene delivery methods using microinjection, electroporation, chemicals, viruses, and naked DNA to different developmental stages of mice gamete have been well established with high e fficacy. Third, embryonic stem (ES) cells showing germ-line transmission are available from various mouse strains including inbred strains (129 and C57BL/6) or hybrid strain s between 129 and C57BL/6. Forth, mouse ES cells have a high efficiency of homologous recombination compared to other species. Lastly, mouse physiology is comparable to human physiology (http://www.informatics.jax.org/mg ihome/other/mouse_facts3.shtml ). Much progress in the manipul ation of the mouse genome has been made since the Cre / lox P system was firstly used (208). A lthough some lim itations in the Cre/lox P system still exist, one of the biggest advanc es contributed by the Cre / lox P system is that it allows us to bypass


77 embryonic lethality due to the lack of a gene, and st udy the role of a gene of interest in a specific cell-type or tissue. On the other hand, one of the critical limitations of the Cre/lox P system is that once genomic DNA is recombined by the Cre recombinase in a cell type, the progenitor cells, which might have an ability to differentiate into other cell types, also contain the recombined allele. Even though the main feature of the Cre/lox P system is beneficial for some lineage tracing or fate-mapping studies during development, there is a possibility that the loss of a gene in unwanted cell types or tissues may then cause phenotypes in those cell types or tissues. To overcome this drawback of the Cre/lox P system, it is required that a better gene manipulation system need to be found or multiple mouse lines showing different patterns of Cre-mediated DNA excisions need to be developed. The cardiovascular system, in mammals, includes the heart, blood and blood vessels. These three components are anatomically and f unctionally closely related to maintain the homeostasis of our body system. Because of the close relationship, one of the difficulties in studying the etiology of a particular cardiovascular disease lies in wh ich type of cell, in a certain component, is primarily responsible for the di sease. For example, an endothelium, which displays functional and morphol ogical heterogeneity depending on its location or subcellular environments, is a continuum tissue from the h eart to peripheral organs. Developmentally, the origin of ECs seems to be vari ous. Initially, it has been proposed that only the mesodermal cells that migrated from the primitive streak into th e yolk sac produces ECs and hematopoietic stem cell (HSC) progenitors in situ in mammals (209). However, subsequent studies have shown that such progenitors also arose in traembryonically, specifically in th e para-aortic mesoderm serving as the precursor to the aorta-gonad-mesonephros (AGM) region (210). The placenta has also been implicated as an organ presenting both EC and hematopoietic precursor cells (211).


78 Although the exact time of specificat ion of the mesodermal cells to ECs remains to be answered, it is generally accepted that the vasculogenesis, a process of blood vessel formation occurring by a de novo production of ECs, begins first in the yolk sac, followed by the AGM region, and then the placenta, in mammalian embryos (212). At th e stages of ECs specification, EC precursors and cardiac progenitor cells seem to share some common markers such as fetal liver kinase-1 [ Flk1 or vascular endothelial growth fact or receptor-2 (VEGFR-2)] and LIM-homeobox transcription factor islet-1 ( Isl1), and can be trans-differen tiated between each other. Mice lacking Flk1 exhibited embryonic lethality between E8.5 and 9.5 with defects in the development of haematopoietic cells and ECs. Yolk-sac blood islands were absent at E7.5 days, organized blood vessels were not observed in the embryo or yolk sac at any stage, and haematopoietic progenitors were severely reduced (179;213). In a r ecent study, it has been shown that Flk1positive cells have an ability to be differentiated to cardiomyocytes, SMCs, and ECs as well (214). Another fate-mapping study demonstrated that the Isl1 delineating the second cardiogenic progenitor field can mark cell populations that contribute to myocardi al cells, subsets of endocardium, and aortic endothelium (215;216). So far, multiple transgenic (Tg) and knockin (KI) lines expressing the Cre in ECs have been developed. Those mice include Tg( Tie2-cre ), Tg( Tie1-cre ), Tg( Flk1-cre ), Flk1+/cre, and Tg( Vecad-cre ) Among them, the most commonly used mouse line is the Tg( Tie2-cre ) line (217). A recent study demonstrated that Tie2-cre-mediated -galactosidase activity was found in the endocardium, the mesenchyme of the atrioventricular (AV) cushions as well as ECs (218). Indeed, the onset of Cre-mediated recombin ation in the ECs and the mesenchyme of AV cushions of the Tg( Tie2-cre ) ; R26R mice was readily detected at E9.5 (217). During early heart valve formation, a subset of ECs which specifies the cushion-forming regions delaminates and


79 invades into the cardiac jelly, a ma ss of connective tissue, where they subsequently proliferate and complete their differentiation into mesenchymal cells (219;220). Although the Tg( Tie2-cre ) line is widely using to delete a gene of interest in the ECs, it possibly deletes a gene in the AV cushion, which then causes defect s in the cardiac valve. Supp orting this possibility, recent studies showed that conditional abolition of some genes using the Tg( Tie2-cre ) line lead to defects in the valve formation as well as mi nor or detrimental phenotypes in the blood vessel (221-223). The studies suggest that the Tg( Tie2-cre ) line is a useful model for studying the function of a gene in the entire ECs or for fate-mapping studies, but is not suitable for studying functions of genes in the EC s of specific vascular beds. It has been shown that the Tg( Flk1-cre ) line exhibits strong Cre activity in the hematopoietic cells as well as ECs of developi ng blood vessels. Aside from that, Cre activity is very weakly detected in the pulmonary vascul ature (224). Furthermore, Cre-mediated lineage tracing study using Flk1+/cre mice have shown that both vascul ar endothelium and cardiac muscle arise from Flk1-positive mesodermal pr ogenitors during development (225). Like the Tg( Tie2-cre ) line, the Tg( Tie1-cre ) line also exhibits the Cre-mediated DNA excision in the mesenchymal cells in the cardiac cushion (226;227). Song et al. showed that the Tie1-cre-mediated -galactosidase activity in the Tg( Tie1-cre ) ; R26R bigenic mice was readily detected at E9.5 with whole-mount staining (227). Histological an alysis revealed that the Tie1Cre-mediated -galactosidase activity was detected in the endocardial cells, mesenchymal cells in the AV cushion and ECs, indicating that th e Tie1-Cre-mediated DNA r ecombination occurred prior to endothelial-mesenchymal tran sition (EMT) during AV cushion formation. As described above, the current Cre deleter line s used in endothelial biology have multiple advantages and disadvantages simu ltaneously. Therefore, the goal of this chapter is to establish a


80 stable transgenic line expressing Cre in ar terial and pulmonary ECs using the 9.2 kb of Alk1 promoter/enhancer. Because the Tg( Alk1-cre ) line developed in this study will be used in the development of a mouse model for PAH, the pr imary focus of this chapter lies in the development and screening of the Tg( Alk1-cre ) line(s) which exhibits an activity of Cremediated DNA excision in the lung ECs dominan tly during late gestation and adult stages. Results and Discussion Expression Pattern of Alk1 During Lung Development To examine the detailed expression pattern of endogenous Alk1 in the lung, lungs from various stages of the Tg( Alk1-lacZ ) embryos were subjected to X-gal staining and histological analysis. Mouse lung development has been propose d to be divided into five stages based on epithelial processes: 1) embryonic (or lung bud) stage, 2) (pseudo)gl andular stage, 3) canalicular stage, 4) saccular stage, and 5) alveolar stage (228;229). Th e lung bud stage (E8.0 9.0) occurs as the lung primordium is budded out from the foregut endoderm and subsequently envaginated into surrounding splanchnic mesenchyme. Th e endodermal bud branches and differentiates within the surrounding mesoderm, which ultimat ely gives rise to the airways, blood vessels, and alveoli of the mature lung together. The glandul ar stage (E9.0 16.0) is characterized by rapid and further branching of epithelial cells. The primary phase of vessel formation occurs during the canalicular stage (E16.0~) as blood vessels extend more peripherally following the airway branches. During maturation stages, the saccul ar and the alveolar stages (E17~), are characterized by formation of thic k-walled saccules that are capab le of respiratory function and eventually become the de licate septae of mature alveoli (230). As shown in Figure 4-2A, the Alk1-positive cells were readily detected in the primordial lung bud at E9.5. Using Tg( Tie2lacZ ) line, it has been shown that already at E 9.5 10.0, ECs expressing Tie2 was part of the splanchnic plexus surrounding the developing esophagus and airways (231). In the study, Parera


81 et al clearly showed that ECs e xpressing other markers such as Flk1, Pecam1 and Fli1 were present surrounding lung epithelial cells. As shown in Figure 4-1A, the branching of prim ordial lung to both left and right sides was obvious at E10.5. The whole mount X-gal staining of the lung revealed that a continuum of Alk1-positive cells was present at the proxima l pulmonary vessels, whereas some patchy or speckled Alk1-positive cells were seen at the distal part of the lung (Figure 4-1A). The Alk1positive cells in the forming of the proximal or branching pulmonary vessels were readily identified in the X-gal stained sections (Figure 4-2B). In the analysis, the forming afferent vessel, in which only the proximal part of the vessel contained erythrocyte-filled lu men, was continuous from the 6th aortic arch (Figure 4-2B). Although the Al k1-positive cells were weakly stained with anti-Pecam1, most of the X-gal stained cells were overlapped with the ma rker (Figure 4-2C). At E11.5, the Alk1-positive cells were further extended into the distal lung and formed efferent vessels which would give rise to the pulmonary vein (Figur e 4-1B). Histological analysis showed that most of the Alk1-positive cells we re overlapped with Pecam1 staining, and distal vessels were evident of formation of erythroc yte-containing lacunae (F igure 4-2F). It is interesting to note that the SMA-positive cells started to sheathe the proximal part of the airway but not the vessels (Figure 4-2E). The data suggests that although the ECs expressing Pecam1 and Alk1 form the architecture of primitive lung bl ood vessels, the vessels are not mature yet. In the E12.5 and 13.5 lungs, Alk1-positive cells started to delineate lines of connection from the pulmonary artery to the pulmonary vein ECs (Figure 4-1C and 1D). A cross section of an E13.5 lung showed that Alk1-positive cells ex tended further to the distal lung and surrounded the epithelial cells (Figure 4-3A).


82 In the E15.5 lung, the immunohistochemistry cl early indicated that the Alk1-positive cells were limited in the ECs (Figure 4-3B and 3C). The SMA staining clearl y marked SMCs surrounding epithelial cells and Alk1 -positive ECs (Figure 4-3D). The pattern of X-gal staining in the lung was comparable with immunostaining with Pecam1 in the wildtype lung (Figure 4-3E and 3F). In the present study, the expression patte rn of Alk1 during lung development was examined. As discussed in Chapter 3, Alk1 is continuously expressed in pulmonary ECs, whereas other systemic vessels exhibit reduced or no Alk1 expression du ring adult stages. The data presented in this chapter suggest that pulmonary ECs star t to express Alk1 from E9.5 and continue to express Alk1 throughou t the rest of the developmental stages and adult life. Using vascular casting and electron microscopy, it has b een proposed that pulmonary vessel formation is established by two distinct processes: the cen tral (proximal) vessels formation is occurred by angiogenesis and peripheral (dis tal) vessels by vasculogenesis (232). The study suggested that the connection between the central and peripheral vascular lume n is established around E13.014.0 where the pulmonary circulation starts. However, Schachtner et al. showed that the connection between proximal vessels containing lumen and distal vessels lacking lumen was already formed by E10.5, indicating that the distal vessel is also formed in angiogenesis (230). Consistent with the finding, a recent study also demonstrated that using the Tg( Tie2-lacZ ) line, the proximal and distal vessels are connected to each other during the lung bud stage (231). Although in the presented study we can not complete ly rule out the possibility that Alk1 is expressed in the angioblasts, e ndothelial progenitor ce lls, Alk1-positive cells were present with a patchy and discontinuous cluster in the distal lung. The data, alt hough it is substantial, suggests that Alk1 plays a role in the vasculogenesis du ring lung development. Another important finding


83 made known through this study is that like um bilical venous ECs, pulmonary venous ECs also express Alk1 from at least E12.5. Generation of Tg( Alk1-Cre) Line To develop the mouse line expressing the Cr e recombinase in the arterial ECs, we constructed a p Alk1-cre vector which contained the 9.2 kb of Alk1 promoter, SV40 splice donor and acceptor (SDSA), the internal ribosome entr y site (IRES), and the Cre recombinase gene (Figure 4-4). By using the standard DNA injectio n technique, two founder lines were obtained (data not shown). The founders we re screen by PCR analysis. The founder lines were mated with C57BL/6J mice to generate F1 offspring. To determine the tissue-specificity of the Cre-mediated DNA excision (which will be referred to as Cre activity hereafter), the F1 Tg( Alk1-cre ) lines were intercrossed with a Cre tester line, R26R mice containing stop codon flanked by loxP sequences and a gene encoding -galactosidase in the ROSA26 locus (233). Then, to determine whether the Cre activity could be s een in germ cells, some of the Alk1-cre(+); R26R bigenic mice were crossed with wild type C57BL/6J mice. Interestingly, one of the Tg( Alk1-cre ) line, called Tg( Alk1-cre )-E, exhibited an ectopic ons et of Cre activity in the germ cells. From the mating scheme, E10.5 embryos genotyped Alk1-cre(+); R26R, Alk1-cre(-); R26R, Alk1-cre(+) and wild type were obtained. After X-ga l staining and PCR genotyping, like Alk1-cre(+); R26R embryo, embryos genotyped Alk1-cre(-); R26R displayed Cre activity throughout their entire body, suggesting that the Cre-mediated DNA excision ha d occurred during germ cell formation in the Tg( Alk1-cre )-E line (data not shown). In contrast to the Tg( Alk1-cre )-E line, the other line called Tg( Alk1-cre )-L1 exhibited an onset of Cre activity during the la te embryonic stages. In this lin e, the patchy X-gal staining was observed in E13.5 lung as shown in Figure 4-5A. A histological se ctioning of the lung displayed


84 discontinuous X-gal staining in the pulmonary vessels. Immunostaining with anti-Pecam1 showed incomplete colocalization of the immuno staining on the X-gal stained ECs (Figure 4-5B and 5C). At E15.5, however, the Tg( Alk1-cre )-L1 showed a uniform X-gal staining in the pulmonary ECs (Figure 4-5D). As shown in Figure 4-5E and 5F, the X-gal staining was completely overlapped with Pecam1 immunostain ing. Interestingly, Cre activity in the Tg( Alk1cre )-L1 line was dominant in the pulmonary vess el whereas mosaic or no activities were observed in the systemic vessels. Newborn pups from the Tg( Alk1-cre ) and R26R cross were obtained. As shown in Figure 4-6A, whole-mount X-gal staining was seen in the dorsal aorta and branchi ng intercostal arteries in the Alk1-cre(+); R26R bigenic mouse, but not in the Alk1-cre(-); R26R monogenic mouse (Figure 4-6B). In the histological analys is, however, a mosaic Cre activity judged by discontinuous X-gal staining was de tected in the cross section of the dorsal aorta (Figure 4-6C). Next, the pattern of X-gal staining during postn atal life was carefully examined. As shown in Figure 4-6D, the staining intensity and patt ern of X-gal staining wa s robustly and evenly detected in most of the ECs at 2 week -old lungs. The Cre activity in the Tg( Alk1-cre )-L1 line was detected in most likely all of the pulmonary ECs including arteries, cap illaries and veins. In this study, we presented a novel Tg( Alk1-cre )-L1 line which exhibited uniform and persistent Cre activity in the pulmonary ECs fr om E15.5 on. The immediate goal of this chapter was to develop a novel Tg( Alk1-cre ) line for the development of an animal model of human PAH in Chapter 5. As discussed in Chapter 3, because it has been shown that Tg( Alk1-lacZ ) line displays the absence of LacZ e xpression in the endocardiac ECs, there are clear advantages of using the Tg( Alk1-cre )-L1 line over already established Cre lines driven by other endothelial cell-specific promoters, such as Tie2-Cre Tie1-Cre Flk1-Cre and Flk1+/cre KI lines. To


85 overcome any early developmental defects in cardiogenesis and pulmonary blood vessel formation, the Tg( Alk1-cre )-L1 line is useful because of pul monary EC-dominant Cre activity. Moreover the deletion of Bmpr 2 by crossing with the Tg( Tie2-cre ) line can cause severe cardiac defects (personal communication). The presented Tg( Alk1-cre )-L1 line will be a unique mouse line for studying the underlying mechanis m of Bmpr2-related PAH in ECs.


86 Figure 4-1. Dynamics of Alk1 expression durin g mouse lung development. The whole mount view of X-gal stained Tg( Alk1-lacZ ) lungs at E10.5 13.5. A) At E10.5, lung branching to left and right lobes were r eadily detected (marke d white dot line) and continuous distributions of Alk1-positive cells were evident in the afferent vessels (arrow head). On the other hand, a patchy distribution of Alk1-positive cells was detected in the distal lung. B) At E 11.5, the primitive lung lobes were obvious. Alk1 expression is further extended to the di stal lung. C) and D) At E12.5 and 13.5, Alk1 expression was detected in the large vessels such as aorta (Ao) and branching left and right common carotid arteries (LCA and RCA, respectively) a nd right and left subclavian arteries (RSA and LSA, respect ively). At the stages, Alk1 expression is detected in the pulmonary arteries and ve ins (arrow). Eso; esophagus, RL; right lobe, LL; left lobe, CL; cranial lobe, ML; medial lobe, CDL; caudal lobe, SL; sacral lobe.


87 Figure 4-2. Ontogeny of Alk1 in the E9.5 11.5 l ungs. A) The isolated Alk1-positive cells were readily detected in the primordial lung bud at E9.5 (arrow head). B) The branching of primordial lung to both left and right side s was obvious at E10.5. A continuous line of Alk1-positive cells were detect ed at the proximal part of lung (arrow head), whereas patchy Alk1-positive cells were detected at distal lung at E10.5. C) Most of Alk1positive cells were colocaliz ed with Pecam1 immunostaining. D) At E11.5, the Alk1expressing cells were further extended to distal lung. E) Im munostaining with SMA defines bronchial SMCs. Note that no SMA-positive cells were detected in the vessels. F) Immunostaining with Pecam1 was completely overlapped with X-gal staining. Tr; trachea, Eso; esophagus.


88 Figure 4-3. Ontogeny of Alk1 in the E13.5 and E15.5 lungs. A) and B) The Alk1-positive cells were further extended to di stal lung during E13.5 15.5. C) A high magnification of view of E15.5 revealed the Alk1expressi on in the ECs. D) Immunohistochemistry with SMA further confirmed that the Alk1 e xpression were limited in the ECs. E) and F) The pattern of immunostaining with Pecam1 was indistinguishable with the pattern of X-gal staining at E15.5.


89 Figure 4-4. The pAlk1-cre construct. The 9.2 kb of Alk1 promoter/enhancer is connected to Cre cassette. For proper splicing and transc ription, the Cre cassette contains SV40 splicing donor/acceptor signals (SD/SA), internal ri bosomal entry sequence (IRES), Cre gene and poly A signal.


90 Figure 4-5. The pattern of Cre activity in the lung of Tg( Alk1-cre )-L1 line. A) At E13.5, a patchy Cre activity was detected in ECs. B) and C) Immunostaining with Pecam1 demonstrated colocalization of X-gal staine d ECs. Note that a subset of pulmonary ECs exhibits Cre activity. D) F) At E 15.5, most of pulmonary ECs displayed Cre activity confirmed by Pecam1 immunostaining.


91 Figure 4-6. Cre activity in the Tg( Alk1-cre )-L1 line during postnatal stages. A) Whole mount Xgal staining showed Cre activities in th e aorta (red arrowheads) and branching intercostal arteries in the Alk1-cre(+) ;R26R bigenic mice, but not in the Alk1-Cre() ;R26R monogenic mice B). C) Histology reveal ed a mosaic pattern of Cre activity in the aortic ECs. D) In the 2wks lung, a uniform pattern of Cre activity in the pulmonary ECs was detected. D) A high ma gnification view of one of pulmonary arteries. Black arrowheads represent ECs containing a recombined allele via Cre activity.


92 CHAPTER 5 DEVELOPMENT OF AN ANIMAL MODEL OF HUMAN PULMONARY ARTERIAL HYPERTENSION (PAH) Note The work presented in this chapter will be published in the Gen etic ablation of the Bmpr2 gene in pulmonary endothelium is sufficient to predispose pulmonary arterial hypertension. by Hong KH, Lee YJ, Beppu H, Bloch K, Li E, Raizada MK, and Oh SP (in preparation) Introduction Pulmonary arterial hypertension (PAH) is a rare lung disorder (2-3 per million per year), in which pulmonary arterial mean pressure rises far above normal levels (25 mmHg at rest and 30 mmHg during exercise) (234-236). Pat hological features of PAH include intimal fibrosis, medial hypertrophy, adventitial pro liferation, plexiform lesions, and the obliteration of small arteries in the lung. Such alterations in precapillary pulmonar y arteries cause increased pulmonary vascular resistance, which leads to right ventricular hypertrophy and eventua lly to heart failure. Prognosis of PAH is still poor, despite recent advances in therapeutics showing a beneficial effect on the survival rate of some PAH patients (8). The pathogenesis of PAH is largely unknown, but there are ample evidence implicating the involvement of diverse vascular effector s. In general, pulmonary hypertension can be promoted by hormones, growth factors, neurotransmitters, and environmental stresses that induce pulmonary vascular constriction, cell prolif eration, or vascular re modeling (1;6). Genetic studies have shown that impairment of bone morphogenic protein receptor type II (BMPR2) signaling plays a critical role in pathogenesis of PAH. Most cases of PAH are sporadic, but about 10% of them are inherited in an autosomal-do minant manner. Heterozygous mutations of the BMPR2 gene were found in 70% of familial PAH cases (60;90;93;237). Furthermore, 26% of apparently sporadic PAH cases also contain germline BMPR2 mutations (62). The characteristics


93 of BMPR2 mutations indicate haploinsufficiency as th e molecular mechanism of disease (14). In addition to the BMPR2 gene, recent studies have shown that PAH also develops in hereditary hemorrhagic telangiectasia (HHT) patients with ALK1 (Activin receptor Like Kinase-1) or ENG (ENDOGLIN) mutations (63;65;66). Studies have shown that abut 20% of people harboring a heterozygous BMPR2 mutation exhibit PAH phenotype, sugges ting that the heterozygous BMPR2 mutations are necessary, but by themselves insufficient, to account for the clinical manifestation of PAH (238). Environmental or genetic second hits may play a pivotal role in triggering the disease. Mononuclear cell infiltrations were often observe d in the PAH vascular lesions, implicating inflammation as a potential environmental factor triggering PAH (14). Inflammation inflicted by viral transduction of 5-lipoxygenase (5LO), an inflammation mediator in Bmpr2 hetrozygous mice ( Bmpr2+/-) mice displayed PAH phenotype (134). Serot onin also increased susceptibility to PAH in Bmpr2+/mice by inhibiting BMP signaling in sm ooth muscle cells (135). In terms of genetic second hit, loss of heterozygosity (LOH) of BMPR2 allele was investigated. A report showed that BMPR2 heterozygosity was retained in plexiform and c oncentric vascular lesions of 7 familial PAH patients harboring BMPR2 mutations (172), demonstrating that BMPR2 LOH is not required for abnormal vasc ular remodeling in PAH. It remains unknown, however, whether or how homozygous BMPR2 mutations impact on morphogenetic changes of pulmonary vessels pertinent to PAH. The cellular origin critical for ini tiation of PAH pathogenesis has not been clearly demonstrated. Hyper-proliferation SMC layer is a key feature of PAH pathogenesis. Plexiform lesions consist of over-pro liferating endothelial cells. Adventitia l proliferation is also involved in PAH pathology. BMPR2 is expressed in multiple cell types in the lung. In blood vessels,


94 BMPR2 expression was observed in SMC and adventitial fibroblasts as well as in ECs (14;239). Transgenic mice in which a dominant-negative fo rm of Bmpr2 was expr essed selectively in SMCs developed pulmonary hypertension (136), implicating perturbed BMPR2 signaling in SMCs for the pathobiology of PAH. It remains unknown, however, whether BMPR2 mutations in pulmonary ECs can also trigger the disease. Here we show using the Cre/loxP system that heterozygous Bmpr2 deletion in pulmonary ECs can predispose to PAH. We also demonstrate that homozygous Bmpr2 deletion in pulmonary ECs is insufficient to cause PAH, bu t increases susceptibility to PAH. This novel animal model will provide valuable resources wi th which to further ou r understanding of the etiology and pathogenesis of PAH. Results Bmpr2 Deletion in Pulmonary ECs by a Novel Cre Transgenic Mouse Line To investigate the role of BMPR2 signaling in pulmonary vascular endothelium, we utilized a Bmpr2 conditional knockout mouse strain (180) and a novel Cre transgenic mouse line, designated as Tg( Alk1-cre )-L1 (Park et al ., unpublished). The Tg( Alk1-cre )-L1 line was established during the screening of multiple transgenic founder lines in which the Cre recombinase is driven by a 9.2 kb Alk1 genomic fragment (240). Unlike other Tg( Alk1-cre ) and known endothelium-specific Cre lines, Cre-medi ated DNA excision (Cre activity, hereafter) was not detected in the endocardial cells and their progeny in Tg( Alk1-cre )-L1 mice. On the other hand, the Cre activity was observed in pulmonary ECs from its emergence during embryogenesis till adulthood. We have monitored the Cre activity of neonatal and adult Tg( Alk1-cre )-L1 mice by crossing them with R26R mice in which cons titutive lacZ expressi on is designed to be activated by Cre. As shown in Figure 5-1A and 1B, the Cre activity was detected in vast areas of the lung of 5 month-old Tg( Alk1-cre )-L1;R26R mice. Histological sections showed that


95 pulmonary ECs were positive, whereas the bronchial epithelia and smooth muscle layers were negative for the Cre activity. We crossed Tg( Alk1-cre )-L1 mice with Bmpr2f/f females to generate Bmpr2+/f;Tg( Alk1 -cre)-L1 male mice, that were subsequently crossed with Bmpr2f/f;R26R females. The Cre(+); Bmpr2f/f mice were viable over 8 months. Genomic PCR analysis on multiple organs/tissues of tw o month-old Cre(+); Bmpr2f/f mice revealed that the Cre-mediated Bmpr2 gene excision was detected only in the lung samp le, indicating a lung-specific Cre expression in the L1 line (Figure 5-1C). Western blot anal ysis on whole lung extracts with anti-BMPR2 antibody revealed a reduced amount of BMPR2 protein in Cre(+); Bmpr2+/f and Cre(+); Bmpr2f/f lungs compared to Cre(-); Bmpr2f/f controls (Figure 5-1D). Bmpr2 Deletion in Pulmonary ECs Can Indu ce Elevation of RVSP and RV Hypertrophy To assess the pulmonary artery pressure, righ t ventricular pressure was measured by right heart catheterization th rough the right jugular vein. At 2-4 month-old ages, mean right ventricular systolic pressure (RVSP) of control [Cre(-); Bmpr2f/f] mice was 23 0.68 mmHg (n=11). The mean RVSP of Cre(+); Bmpr2+/f was not significantly higher than that of control, but one mouse had RVSP of 44 mmHg (Figure 5-2A). The mean RVSP of Cre(+); Bmpr2f/f was significantly higher (30.1 2.1 mmHg; p < 0.05) than that of control (Figure 5-2A). Out of 24 Cre(+); Bmpr2f/f mice examined, 7 of them showed RVSP higher than 30 mmHg. This result indicates that endothelial heterozygous and homozygous Bmpr2 deletion can elevate pulmonary arterial pressure in a gene-dosage dependent manner. To determine whether the frequency or se verity of pulmonary hypertension (PH) is affected by aging, we measured the RV pressure on 6 month-old mice. As shown in Figure 5-2B, the frequency of PH in Cre(+); Bmpr2+/f (3/10) and Cre(+); Bmpr2f/f (6/9) 6 month-old group was increased compared to that in 2-4 monthold corresponding genotype groups. There was no


96 significant difference in the systemic pressure among different genotype groups, nor between PH (i.e., above 30 mmHg RVSP) and non-PH (N-P H; below 30 mmHg RVSP) groups (Figure 52C). The RV/(LV+septum ) ratio, an indicato r of RV hypertrophy, was significantly higher in the PH group compared to the control or nonPH group, confirming the presence of sustained elevation of RV pressure (Figure 5-2D). Increased Number and Medial Wall Thickness of Alpha-SMA-Posit ive Distal Arteries in the Mutant Mice with Elevated RVSP To determine whether the elevation of RVSP and right ventri cular hypertrophy were associated with pulmonary vascular remodeli ng, lung tissue sections were examined by H&E staining and immunohistochemistr y. General aspects of lung development and morphology were indistinguishable among genotype groups and between the PH and non-PH groups. Immunostaining of the lung sections with SMA antibodies revealed increased number of SMA-positive small arteries and thickness of SMC layers of small arteries in the PH group compared to the non-PH group (Figure 5-3A a nd 3B). Quantitative morphometric analysis demonstrated that the percentages of SMA-positive small arteries (30-70 m outer diameter) in the PH group were significantly greater than those in the non-PH group in both Cre(+); Bmpr2+/f and Cre(+); Bmpr2f/f mice (Figure 5-3C). It is note worthy that the percentage of SMA-positive small arteries in the non-PH Cre(+); Bmpr2f/f mice was also significantly greater than that in the non-PH Cre(+); Bmpr2+/f mice (Figure 5-3C). The th ickness of medial layer of SMA-positive small arteries in the PH group was also signifi cantly greater than th at in the non-PH group (Figure 5-3D). Pathohistological Features of PAH in the Mutant Mice with Elevated RVSP Elevated serotonin signaling and tenascin-C (Tn-C) expression have been implicated in the pathogenesis of PAH. Transgenic mice overe xpressing the 5-hydroxytryptamine transporter


97 (5HTT) gene in smooth muscles develop pulmona ry hypertension (150). Tenascin-C (Tn-C) is an extracellular matrix glycoprotein induced in a number of experimental and clinical forms of PAH, including familial PAH, monocrotalin-i nduced PAH, and flow-induced vascular remodeling (241-243). Immunostaini ng with anti-5HTT and Tn-C an tibodies visualized marked elevation of these proteins in the SMC layer of the PH lungs (Figure 5-4C and 4F). We found a high incidence of focal leukocyte in filtrations at the periphery of pulmonary vessels of Cre(+); Bmpr2+/f (38%; 5/13) and Cre(+); Bmpr2f/f (44%; 11/25) mice compared to the control mice (0/11) (Figure 55A and 5B). Among Cre(+); Bmpr2f/f mice, frequency of the infiltration appeared to be highe r in the PH group (8/14) than that in the N-PH (3/11) group. Most of the infiltrate d cells were CD68-positive monocyte/ macrophages (Figure 5-2B inset). Neointima formation in occlusive arteries was al so observed in a limited number of the PH lungs (Figure 5-2C 2E). In situ thrombosis of small muscular ar teries was also observed in about 50% (9/18) of the PH lungs. As shown in Figur e 5-5F, lumens of the affected vessels were partially or completely occluded by fibrin(ogen)-positive thrombi. Discussion We show here that some, but not a ll, mice having the genetic ablation of Bmpr2 in pulmonary ECs exhibited an elevation of RV SP with various histopa thological features reminiscent of human PAH lungs, demonstrating for the first time in vivo that Bmpr2 mutation in endothelium is sufficient to predispose PAH. Homozygous Bmpr2 deletion, by itself, was not sufficient to cause PAH, but increased the susceptibility to PAH. PAH is a heterogeneous disorder, ha ving multiple etiology. A mounting body of evidence, however, has shown that BMP signa ling pathway plays a pivotal role in the pathogenesis of PAH. Genetic studies ha ve demonstrated that heterozygous BMPR2 mutations predispose PAH in sporadic as well as fam ilial cases (60;62;90;93;237). In addition, recent


98 studies revealed that impaired BMPR2 signaling might be a central mechanism for PAH caused by hypoxia and MCT administration, two major expe rimental animal models for PAH (132;243). Since the pathogenesis of PAH involves all vasc ular components, identity of the principal cell type for PAH pathogenesis has been investigated. West et al. have shown by an inducible transgenic approach that overe xpression of a dominant-negative form of Bmpr2 resulted in elevation of pulmonary arterial pressure, suggesting that impairment of the Bmpr2 signal in SMC might be sufficient to cause PAH (136). Despite th e lack of direct evidence, pulmonary ECs have been suspected as a primary cell type in which BMPR2 mutations elicit PAH. Occurrence of plexiform, which consist of pro liferative endothelial cells in severe form of PAH, suggests that pulmonary ECs are the principal source of PAH pathogenesis. Predisposition of PAH by mutations in ALK-1 or ENG genes (63;65;66), which express primarily in endothelial cells (117;244), also support this speculation. The data presented here is the first direct in vivo evidence, however, that endothelial Bmpr2 mutation is sufficient to predispose PAH. Since familial PAH is a dominantly inherited disease and haploinsufficiency is a common mechanism for familial PAH, Bmpr2+/mice received much attention as a genetic model. Although an initial report showed modest elevation of mean pulmonary arterial pressure in Bmpr2+/mice (133), more recent studies using the same mouse strain showed no significant difference of RVSP between Bmpr2+/and control mice (134;135). We showed here dichotomy of PAH phenotype among mi ce harboring heterozygous Bmpr2 deletion in pECs: 1/11 (9%) in 24 month-old group and 3/9 (33%) in 6 month-old group. The reas on for this discrepancy is unclear, but perhaps the differences in a number of experimental conditions used might have contributed to it. These include methods for hemodynamic study ( i.e direct cardiac puncture vs cardiac catheterization), anes thesia conditions, and number age and strain of mice.


99 We showed that endothelial Bmpr2 deletion can cause muscularization of the medial layer of distal arteries. Since no Cre activity was detected in SMCs, the BMPR2 signaling must be intact in SMCs of these mice. Therefore, me dial hypertrophy in the PH group mice is due to indirect effect of defects in endothelial cells. We observed a hi gher incidence of infiltration of CD68-positive mononuclear cells in the perivascular regi ons of Cre(+); Bmpr2+/f and Cre(+); Bmpr2f/f mice compared to Cre(-); Bmpr2f/f controls, and a higher frequency in the PH group than the N-PH group. Th is data suggests that Bmpr2 deletion in pulmonary ECs may cause some forms of endothelial dysfunction, which make s the pulmonary vessels more susceptible to secondary triggers such as in flammation or thrombosis. This result is consistent with pathological findings in human PAH samples, and also consis tent with the report demonstrating that Bmpr2+/develop PAH upon treatment with pro-in flammatory reagents or serotonin (134;135). What remains to be investigated, ho wever, is the nature of the endothelial dysfunction, and the underlying mechanism by which Bmpr2 deletion in pulmonary ECs causes such a condition. Based on the observation that cells in the plexiogenic lesion are monoclonal (12;171), LOH of the BMPR2 gene in those cells has been s uggested as a potential pathogenetic mechanism for PAH. Machado et al. investigated microsatellite in stability in 5 loci including a BMPR2 locus on DNA samples isolated from plexifor m and concentric vascular lesions of 7 familial PAH patient lungs by laser microdissect ion technique, but heterozygosity of the BMPR2 gene appeared to be maintained (172). This result demonstrated that LOH of BMPR2 is not necessary for the development of such vascular lesions. We dem onstrated here that homozygous Bmpr2 deletion in pulmonary ECs by itself is not su fficient to develop PAH. Our data showed, however, that homozygous Bmpr2 deletion significantly increases the susceptibility to PAH,

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100 compared to heterozygous Bmpr2 deletion. We observed a high incidence of infiltration of CD68-positive mononuclear cells in the perivasc ular regions of PAH lungs, suggesting that Bmpr2 deletion in pulmonary ECs may cause some forms of endothelial dysfunction, which makes the pulmonary vessels more susceptible to secondary triggers su ch as inflammation or thrombosis. This result is consistent with pathol ogical findings in human PAH samples, and also consistent with the report demonstrating that Bmpr2+/develop PAH upon treatment with proinflammatory reagents or serotonin (134;135). What remains to be investigated, however, is the nature of the endothelial dysfunction, and the underlying mechanism by which Bmpr2 deletion in pulmonary ECs causes such a condition. It is unclear why some, but not all, mutant mice develop PAH. There was no gender bias for the PAH phenotype in the mutant mice. To examine the possibility that the phenotypic variation resulted from variance of Bmpr2 deletion in pulmonary ECs, we monitored the Cre activity by introducing the R26R allele. No differe nces in the intensity and pattern of X-gal staining were detected between PH and N-PH groups regardless of thei r genotypes, indicating that the phenotypic variation should not be attributed to incomplete excision of Bmpr2 gene in pulmonary ECs. Also, the PAH phenotype had no corre lation with the presence of R26R allele. We showed here that about 30% of Cre(+); Bmpr2+/f mice showed PAH phenotype at 6 months of age. This statistic result is si milar to human studies showing th at about 20% of people harboring BMPR2 mutations develop PAH (238). Since mice used in this present study were on 129Sv, C57BL/6, and FVB mixed backgrou nd, strain-specific genetic modifiers might have played a role for controlling the susceptibility to the second hit. The underlying pathogenetic mechanisms for PAH is poorly understood. One of the major impediments for this task is limited access to biol ogical samples. PAH is a rare disease, and only

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101 available pathological samples are from lung transplantation patients, at the very last stage of the disease. The animal models for PAH will theref ore provide relevant pathological samples from early to late stages of PAH. Once established, thes e animals can also be used to test the efficacy of novel therapeutic drugs. Tg( Alk1-cre )-L1 as well as Cre(+); Bmpr2f/f mouse lines presented here will be useful geneti c resources to further our know ledge about PAH pathogenesis, specifically for the role of BMPR2 si gnaling in pulmonary endothelium. Currently available endothelial Cre lines, including Tg( Tie1-cre ), Tg( Tie2-cre ), Flk1+/cre, and Tg( Vecad-cre ) lines, express the Cre recombinase in the endocardial cells progeny of which constitute atrioventricular cushions (217; 225;226;245). Conditional deletion of several TGF/BMP receptors using these Cre lines appeared to have some form of cardiac malformations, such as cardiac valve or ve ntricular septal defects (221; 227;246). When we deleted the Bmpr2 gene using Tg( Tie2-cre ) mice, most Tg( Tie2-cre ); Bmpr2f/mice died before the weaning age because of the presence of cardiac malformations (unpublished; Beppu et al .). In this regard, the Tg( Alk1-cre )-L1 line is superior to these known endothe lial Cre lines for evaluating the impact of a gene deletion in pulmonary ECs because Cre is not expressed in the endocardial cells and their progeny during cardiogenesis stages (Park et al unpublished). The Tg( Alk1-cre )-L1 line will also be useful to assess the role of other BMPR2 signaling partners (e.g BMPR1A and SMAD1) or of potential downstream medi ators in PAH pathogenesis of PAH.

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102 Figure 5-1. Tg( Alk1-cre )-L1 mice express the Cre recombinase in the pulmonary vascular endothelial cells. A) and B) The cells in which Cr e-mediated recombination has occurred were visualized by stai ning the lungs of 5-month-old Tg( Alk1-cre )-L1;R26R bigenic mice with X-gal for the lacZ e xpression. Sections of whole mount X-gal stained lungs were counter stained with nuclear fast red (NFR; A), or with SMA antibody. Insets are magnified views of the area indicated by arrows. Note that X-gal positive cells resided in vascular ECs, but neither in airway epithelial cells nor smooth muscle cells. C) PCR detection of the Bmpr2 Ex4,5 allele (i.e., deletion of exons 4 and 5 of Bmpr2 by Cre) from genomic DNA isolated from multiple organs/tissues of 2-month-old Cre(-); Bmpr2f/f (left) and Cre(+); Bmpr2f/f (right), demonstrating a lung-specific Cre activity. A primer set amplifying an Alk1 locus (~190 bps) was used as a control for PCR r eaction. D) Western blotting analysis with BMPR2 antibody on whole lung lysate show s reduced levels of BMPR2 protein (~120 kd) in Cre(+); Bmpr2+/f and Cre(+); Bmpr2f/f lungs compared to Cre(-); Bmpr2f/f lungs. GAPDH (~36 kd) was used as a loading control.

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103 Figure 5-2. Endothelial Bmpr2 deletion resulted in elevati on of RVSP and RV hypertrophy in gene dosage and age dependent manners. A) and B) RV pressure of control, Cre(+); Bmpr2+/f and Cre(+); Bmpr2f/f mice was measured at 2-4 and 6 months of age. Each dot represents RVSP of an individual mouse. Mean value (m) of RVSP of each genotype group was given and indicated by horizontal bars. Pulmonary hypertensive (PH) group (i.e. RVSP greater than 30 mmHg; open bar) and none-PH (N-PH; solid bar) group were separated in each genotype in C) and D). C) Systolic systemic pressure was recoded by an indirect tail cuff method. There was no significant difference among any comparisons. D) The RV/(LV+S) ratios in PH groups were significantly greater than that in N-PH groups regardless of their genotypes. Levels of statistical significance are indicated.

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104 Figure 5-3. Mice in the PH group e xhibited increased number of musc ularized distal arteries and thickening of medial layers. A) and B) Representative hist ological sections of control and PH lungs, stained with SMA antibody. Insets in A) a nd B) are magnified views of the areas indicated by arrows Note readily identifiable strong SMA-positive small arteries in the PH group lungs, and th ickening of arterial walls (inset in B). Scale bars in insets represent 25 m. C) and D) Percentages of small arteries positive for SMA staining and medical thickness of di stal arteries were compared among five groups, i.e. control, PH and N-PH groups in each mutant genotype. Mice in PH group in either genotype showed significant in creases in number of SMA-positive small arteries and in thickness of medial layers.

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105 Figure 5-4. Previously identifie d PAH-associated protein expre ssions were elevated in SMC layers of PH mouse lungs. A) F) Immunos taining of control and mutant lungs with antibodies against serotonin transporter (5HTT) and tena scin-C (Tn-C) revealed elevated expression of both 5HTT and Tn-C in SMC layers of PH group samples compared to control and N-PH group samp les. Representative areas containing similar sized vessels were shown. Scale bars represent 25 m.

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106 Figure 5-5. Vascular lesi ons of the PH lungs mimic some pa thological features of human PAH. A) and B) H&E staining of c ontrol and PH lungs, showing focal leukocyte infiltration around pulmonary arteries of PH lungs of Cre(+); Bmpr2f/+ or Cre(+); Bmpr2f/f mice. Immunohistochemical studies revealed what the infiltrated cells were mostly CD68positive mononuclear cells (inset in B). Br Bronchus; PA, pulmonary artery. C) and D) Immunostaining with SMA and von Willebrand factor (vWF) antibodies showed occlusive arteries with neointima forma tion in a PH lung. E) Elastic van Geison staining showed neointima formati on. Most cells in neointima were SMA-positive. eEL, external elastic lamina. F, H&E staining showing a representative in situ thrombotic lesion in a PH lung. Lumen of the affected vessel was occluded by formation of fibrin(ogen)positive thrombus (inset).

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107 CHAPTER 6 CONCLUSION AND PERSPECTIVES In the presented studies, two distinct TGFreceptors linked to i nherited cardiovascular diseases, i.e HHT2 and PAH, were studied. Altho ugh the biological significance of TGFhas been implicated in a variety of biological pr ocesses for decades, multiple debatable and unknown issues regarding the role of the receptors in the vascular biology are yet to be answered. Throughout the studies, most of the work has be en engaged in elucidating the physiologic or pathogenic roles of the receptors in the endothelial biology. In Chapter 3, we sought to characterize th e regulatory mechanism of dynamic pattern of Alk1 expression via in vivo analysis of the Alk1 promoter and intron 2 regions. In Chapter 4, we developed and characterized a novel Tg( Alk1-cre )-L1 line displaying a un ique pattern of Cre activity in the pulmonary ECs. Fi nally, in Chapter 5, using the Tg( Alk1-cre )-L1 line we sought to test a long-awaited and evidence-based hypothe sis in the PAH field by selectively deleting Bmpr2 in the pulmonary ECs. The 9.2 kb of Alk1 Promoter As a Driver of Sp atiotemporal Pattern of Alk1 Expression The in silico approach used to analyze the Alk1 promoter revealed highly homologous regions between mouse and human ALK1 in th e promoter and intron 2 regions. Based on the data, the Alk1 promoter was further dissect ed in order to determine what the key regulator/enhancer for Alk1 expression. Because as noted in Chapter 1, Alk1 exhibits a heterogeneity in the tissue-specificity of expression, the well-established in vivo transgenesis approach was employed to bypass any disagreement that may have been caused by an in vitro culture of the ECs. To our su rprise, the 9.2 kb of pXh4.5-in2-SIB construct, containing whole homologous sequences in intr on 2, precisely recapitulated the endogenous Alk1 expression, whereas the two other cons tructs failed to show an Alk1-sp ecific expression pattern. The X-gal

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108 staining in the Tg( Alk1-lacZ ) (from the pXh4.5-in2-SIB construc t) was predominantly limited in the arterial ECs of large vessels and perineural and pulmonary capillaries during development. As expected, Alk1 expression was greatly dimini shed during the adult stage, but could be induced with wound challenge. However, the in ducibility of Alk1 duri ng wound-healing was not as robust as in Alk1+/lacZ mice. The difference between the lines is most likely caused by a lack of an additional regulatory element(s) which would be necessary for precise induciblity of the Alk1 gene in adult. Consistent with the phenotype displaying excessive dila tion/fusion in embryonic vessels of Alk1lacZ/lacZ, null mutation of Alk1 also caused the malformation of umbilical vessels. The phenotype includes dilation of the vessels at E9.5, followed by fusion of the vessels at E10.5. Of interest, although the Alk1 expr ession was detected in both umb ilical arterial and umbilical venous ECs, the Alk1 expression in the umbilical arterial ECs is still dominant. The data indicates that because partial pressure of oxygen in the umbilical vein is higher than that in the umbilical artery, the level of oxygen does not seem to be a direct mechanism in the regulation of the Alk1 expression. Supporting th is view, a recent microarray analysis showed that no significant changes of oxygen-sensitive genes in the blood outgrowth endo thelial cells (BOECs) isolated from HHT patients were detected (247). HHT is a vascular disease caused by a mutation in the TGFtype receptor (Eng or ALK1) or in a cytoplasmic mediator (SMAD4). Most of the diagnostic screening for this mutation has been carried out with direct sequencing of coding exons. It ha s been shown that 60% of HHT cases are caused by such exonic mutations, however the types of mutations in the rest of the cases are still inexplicable (189). The presented re sults are the first to demonstrate the potential role of intronic sequence in the regulation of Alk1 expression. Thus, the results from the study

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109 may provide genomic sequence information for screening non-exonic mutations of ALK1 in HHT2 patients. A future prospect would be follow-up studi es on further dissecting and defining the homologous regions in intron 2. To directly determine whet her the homologous sequence in intron 2 does function as a tr ue enhancer element governing EC-dominant expression, an in vivo transient transgenic approach would be a bene ficial tool. According to this method, various transgenic constructs containing a minimal Alk1 promoter, intron 2 sequence with or without homologous region along with the lacZ reporter gene would be designed, introduced into fertilized mouse eggs and embryos would be harv ested to visualize the transgenic constructs expression through X-gal staining. The confirmed promoter and homologous sequence would be a useful resource for a viral ve ctor system. Furthermore, the in vivo dissection of the homologous region would provide us sequence in formation for the identification of trans-acting factors in the regulation of dynamic pattern of Alk1 expression. A Novel Tg( Alk1-Cre )-L1 Line As a Pulmonary EC-Dominant Cre Deleter Using the 9.2 kb Alk1 fragment, a novel Tg( Alk1-cre )-L1 line was developed. In the line, uniform Cre activity was persis tently observed in pulmonary ECs from E15.5 on. On the other hand, the ECs of systemic vessels displayed mosaic Cre activity. Consistent with the pattern of Alk1 expression in pulmonary vessels, pulmonary Cre activity in the line was shown in almost all of the pulmonary ECs during embryonic and early postnatal stages. In the Tg( Alk1-lacZ ) line, however, the X-gal staining was mostly limited to small vessels, such as small arteries and capillaries, during the adult stage. As discussed in Chapter 4, once an allele is recombined by Cre in a cell, progenitor cells from th e cell also contain the recombined allele. This is most likely the reason why the homogeneous pattern of X-gal st aining in all pulmonary ECs persisted through the late embryonic and in th e adult stages of the Tg( Alk1-cre )-L1;R26R bigenic mice. More

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110 importantly, unlike any other pan-endot helium-specific Cre lines, the Tg( Alk1-cre )-L1 line exhibited minimal Cre activity in the ECs of the coronary vessel and capilla ries in the heart and no endocardial Cre activity. As discussed in Chapter 1, based on the trace of X-gal staining pattern in the Tg( Alk1-cre );R26R bigenic mice, the turnover ra te for pulmonary EC seems to be very slow in adults. Alk1-Cre(+);Bmpr2f/f Mice As an Unique Model System of PAH In Chapter 5, the pathogenic role of pe rturbed endothelial Bmpr2 signaling in the development of PAH was highlighted. Based on th e pathohistological ev idence of endothelial dysfunction and low disease pe netrance in PAH individuals we hypothesized that the homozygous deletion of Bmpr2 in the pulmonary ECs increases the genetic susceptibility to PAH. Indeed, endothelial Bmpr2 deletion could predispose elevat ion of RVSP, muscularization of small arteries and right ventricular hypertrophy. Although th e disease penetrance was still incomplete in our mouse model, homozygous deletion of Bmpr2 in ECs led to higher frequency of PAH than heterozygous dele tion with an age-dependent manne r. The data suggests that the factors altering gene dos age of Bmpr2 play a pathogenic ro le in the development of PAH. Certainly, none of the mutant mice appeared to display congenital heart defects such as septal defects or valvular pulmonary stenosis. This result was possibly achieved due to the lack of endocardial Cre activity in the Tg( Alk1-cre )-L1 line. Our initial attempt with collaboration was to conditionally delete Bmpr2 in ECs by using the Tg( Tie2-cre ). However, the Tie2cre(+);Bmpr2f/f mice displayed postnatal anomalies incl uding heart defect. The result further indicates that unlike any other EC-specific Cre lines, the Tg( Alk1-cre )-L1 line is a unique line as a pulmonary EC-dominant Cre deleter without causing heart defect. The Tie2-cre(+);Bmpr2f/f mice would be a useful resource for studying other t ypes of PH such as persistent PH caused by congenital heart defects. Interestingly, the s ubset of hypertensive mi ce exhibited signs of

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111 endothelial dysfunction such as perivascular infiltration and in situ thrombosis. Although the subset of PAH individuals also exhibit the signs of endothelial dysfunction, the mechanism underlying such dysfunctions is still open to questi on. It is tempting to speculate that the Bmpr2 signal plays a protective role in the endothelium from injuries such as inflammation responses and mechanical forces such as flow shear stress. Perspectives Although our murine models have helped to explai n some critical issues in the etiology of PAH, a number of mechanistic questions are st ill unanswered. For subsequent examination of inflammatory responses as one of the multiple hi ts in our model system, a chronic infusion of inflammatory cytokine such as IL-1 IL-6 or MCP-1 into Alk1-cre(+);Bmpr2+/f and Alk1cre(+);Bmpr2f/f mice would be a useful approach to reso lving this possibility. Because the mouse model we used in the PAH study harbors mixed genetic backgrounds ( i.e. 129Sv/J, C57BL/6 and FVB), strain-specific modifier gene(s) mi ght play a role in the development of pathophysiological manifestation in the Alk1-cre(+);Bmpr2+/f or Alk1-cre(+);Bmpr2f/f mice. Therefore, PCR-based genome-wide scan for the identification of potential quantitative trait loci (QTL) linked to PAH in the hypertensive mice would be useful approach to test this possibility. As discussed in Chapter 1, PAH is known to exhibit opposite vascular phenotypes of HHT. It has been demonstrated that the subset of HHT individuals with the ALK1 mutation also displayed clinical manifestations of PAH. Ther efore, one of the fundame ntal questions in the genetics of PAH would be how the ALK1 mutation genocopies the BM PR2 deficiency. A recent study suggests that in the presen ce of BMP9, BMPR2 can preferenti ally interact with ALK1 in ECs and inhibit growth factors stimulated-cell migrat ion and proliferation (99). Moreover, Yu et al. demonstrated that loss of the BMPR2 f unction can be compensated by another TGFtype II

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112 receptor, ActRIIa, in SMCs (108). Therefore, to further test the mechanistic role of the interactions between the receptors, some genetic compound mice such as Alk1cre(+);Bmpr2f/f;ActRIIa-/or Alk1-cre(+);Bmpr2f/f;Alk1+/f would provide a direct answer for the questions. Follow-up studies with the mouse strains would help to explain the incomplete disease penetrance in our mouse model and BMPR2 mutation carrier as well. Because the Alk1cre(+);Alk1f/f mice exhibit postnatal lethality with ma lformation in the pulm onary vessels, the Alk1-cre(+);Bmpr2f/f;Alk1+/f mice would at least in part prov ide answers for the interwoven link among the receptors as a disease-causing mechanism. Furthermore, the mouse line would be a useful genetic resource to study a decision -point of the two distinct diseases, i.e. HHT and PAH, caused by ALK1 mutation. Another intriguing question in the PAH field is whether PAH as a consequence of the BMPR2 mutation is mediated by a SMAD-dependent signaling mechanism. To investigate the pathogenic significance of SMAD -dependent signaling as a do wnstream contributor to the development of PAH, the conditional de letion of endothelial Smad1 in compound Alk1cre(+);Smad1f/f mice would be worthy. A recent study s howed that homozygous inactivation of endothelial Smad5 in Tie2-cre(+);Smad5-/f mice did not impair blood vessel formation, suggesting SMAD1 to be a main downstream medi ator of BMPR2 in ECs (248). Indeed, reduced levels of phosphorylated SMAD1 was observed in the pulmonary vessels in PAH individuals with a heterozygous BMPR2 mutation (169). Thus, the Alk1-cre(+);Smad1f/f mice would further provide evidence of the depe ndence of SMAD1 signaling in the development of PAH. The isolated pulmonary EC would be a good resear ch resource to investigate the impact of endothelial BMPR2 mutation in vitro To nullify the Bmpr2 allele, ROSA26 -creER(+);Bmpr2f/f mice would be generated, the pulmonary ECs can be isolated from the mouse strain, and

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113 tamoxifen would be treated to the isolated ECs. Because the CreER efficiently becomes active by tamoxifen, the system would provide sets of experiments to test th e impaired Bmpr2 function in the endothelial biology under various conditions. The isolated ECs would be a useful resource to investigate questions regardi ng the perturbed trafficking of mutant Bmpr2 to membrane, and desensitization of mutant Bmpr 2 to ligands. It has been shown that BMPR2 containing some types of mutation fail to reach the cell membrane (249). Th e data suggests that the failure in targeting BMPR2 to membrane might be one of th e disease-causing mechanisms with or without heterozygous BMPR2 mutation. The functional significance of membrane trafficking of BMPR2 was recently reviewed (250). Morrell et al. showed that SMCs isolated from the pulmonary artery of individuals with IPAH exhibited altered growth responses to the BMP-2, -4, and -7, as compared with SMCs isolated from controls (251 ). The dynamics of BMPR2 on the plasma membrane seem to be a complex system (252). Currently it is unknown how the mutant BMPR2 desensitizes the ligands; thus, it would be worthy to test the possible involvement of the receptor desensitization in ECs containing mutant Bmpr2. In conclusion, using genetically modified mi ce we have demonstrated that the endothelial deletion of Bmpr2 can predispose PAH. The mouse lines, as powerful research resources, have the potential to be used in the isolation of new th erapeutic targets and disc overy of target drug(s) for PAH. The presented data also will meet our common endeavor in the improvement of public health.

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114 APPENDIX A Publications 1. Seki,T., Hong,K.H. Yun,J., Kim,S.J., and Oh,S.P. 2004. Isol ation of a regulatory region of activin receptor-like kinase 1 gene sufficien t for arterial endothelium-specific expression. Circ.Res. 94:e72-e77. 2. Park,S., Lee,Y.J., Lee,H.J., Seki,T., Hong,K.H. Park,J., Beppu,H., Lim,I.K., Yoon,J.W., Li,E. et al 2004. B-cell translocation gene 2 (Btg 2) regulates vert ebral patterning by modulating bone morphogeneti c protein/smad signaling. Mol.Cell Biol. 24:10256-10262. 3. Seki,T.*, Hong,K.H. *, and Oh,S.P. 2006. Nonoverlapping expression patterns of ALK1 and ALK5 reveal distinct roles of ea ch receptor in vascular development. Lab Invest 86:116-129. (* equally contributed) 4. Lee,Y.J., Hong,K.H. Yun,J., and Oh,S.P. 2006. Generation of activin receptor type IIB isoform-specific hypomorphic alleles. Genesis. 44:487-494. 5. Hong,K.H. Seki,T., and Oh,S.P. 2007. Activin recep tor-like kinase 1 is essential for placental vascular development in mice. Lab Invest 87:670-679.

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136 BIOGRAPHICAL SKETCH Kwon-Ho Hong was born in Naju city of Je onnam province, Korea. Then he moved to Seoul when he was 10 years-old, and grew up there. Following graduation from the Gyeongseong high school, he went to the KONKUK University in Seoul where he earned a Bachelor of Science degree in animal science in 1997. He went to the Korean Army in 1991 and served for 28 months. During his college years, he enjoyed an extracurricular activity in a club named The Workshop where his passion for the biological science was built up. In 1997, he joined Prof. Hoon-Taek Lees laboratory at th e same university and studied the biological function of Leptin using transgenic mice. He received a Master of Science degree in 1999. In 2000, he originally started a Ph.D. program in the department of Animal Sciences, University of Florida. After two semesters in the department, he moved to Dr. S. Paul Ohs laboratory in the department of Physiology & Functional Genomic s in 2001. After one year of working in his laboratory as a research assistant, he reentered the Interdisciplinary Program (IDP) in Biomedical Sciences, University of Florida in 2002. He rejoined Dr. S. Pa ul Ohs laboratory in 2003. During Ph.D. degree, the majority of his work was engaged in the studies of the in vivo role of TGFreceptors in the vascular biology.