NOVEL FUNCTIONS OF ADRENOMEDULLIN, RECEPTOR ACTIVITY-MODIFYING PROTEIN 2, AND GPR182 IN CARDIOVASCULAR DISEASE AND CANCER Daniel Oliver Kechele A dissertation submitted to the faculty of the University of North Carolina at Chapel Hill in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Department of Cell Biology and Physiology (Cell and Molecular Physiology) Chapel Hill 2016 Approved by: Kathleen Caron Keith Burridge Andrew Dudley John Rawls Carol Otey i ©2016 Daniel Oliver Kechele ALL RIGHTS RESERVED ii ABSTRACT Daniel Oliver Kechele: Novel Functions of Adrenomedullin, Receptor Activity-Modifying Protein 2, and Gpr182 in Cardiovascular Disease and Cancer (Under the direction of Kathleen Caron) The multifunctional circulating peptide, adrenomedullin (AM) signaling through its G protein-coupled receptor (GPCR) complex, comprised of the calcitonin receptor-like receptor (CLR) and receptor activity-modifying protein 2 (Ramp2), is required for lymphatic development and embryonic survival, as well as numerous functions in adult cardiovascular physiology. However these functions, especially during adulthood, have not been fully elucidated. Using both gain- and loss-of-function mouse models these studies aimed to further elucidate the roles of AM signaling during cardiovascular homeostasis and tumor progression. Endothelial restoration of Ramp2 is sufficient to rescue the embryonic lethality- associated with global loss of Ramp2. However, the developmental loss of Ramp2 in non- endothelial cells led to spontaneous hypotension, dilated cardiomyopathy, and multi-organ inflammation in adult survivors. Smooth muscle cell- or global-deletion of CLR did not recapitulate these phenotypes, indicating that Ramp2 is likely interacting with other GPCRs, including parathyroid hormone and glucagon receptors, to maintain cardiovascular homeostasis. While AM signaling is essential for lymphatic development and maintenance, it is unclear how AM influences tumor lymphatics. Tumor cells engineered to overexpress or knockdown AM did not alter tumor growth or angiogenesis. High tumor-derived AM caused tumor and lymph node lymphangiogenesis and increased metastasis. While AM is typically considered protective, these data suggest that AM signaling could enhance tumor progression. iii While CLR/Ramp2 is the canonical AM receptor, it was previously thought that the AM receptor was the orphan GPCR, Gpr182, of which almost nothing is known. Using a knock-in reporter mouse model to map Gpr182 expression in vivo, Gpr182 was enriched in the active stem cells in the small intestine. Gpr182 was dispensable during development and homeostasis, but when challenged with irradiation injury or adenoma model, reduced Gpr182 led to intestinal hyperproliferation during regeneration and enhanced adenoma formation, respectively. Together, these studies demonstrated the importance of endothelial AM signaling during development, the complexity of GPCR/RAMP interactions in vivo, the underappreciated functions of AM signaling in tumor lymphatic metastasis, and the role of Gpr182 as a negative regulator of intestinal proliferation; all of which could represent pharmacological-tractable targets to treat a number of cardiovascular and neoplastic diseases. iv ACKNOWLEDGEMENTS I would like to thank all members, past and present, of the Caron lab for intellectual discussions and technical assistance in all my projects. With regards to scientific data presented throughout this dissertation, I would like to thank the University of North Carolina Lineberger Animal Histopathology Core (NIH CA16086) and Kirk McNaughton and Ashley Ezzell of the University of North Carolina Histology Research Core for tissue embedding, sectioning, and staining expertise. I would also like to acknowledge Dr. Robert Bagnell and the University of North Carolina Microscopy Services Laboratory for confocal microscopy assistance. Regarding the data presented in Chapter II, I would like to thank the University of North Carolina Animal Models Core and the University of North Carolina Rodents Advanced Surgical Models Core, specifically Dr. Mauricio Rojas and Dr. Brian Cooley with echocardiography and intra-arterial blood pressure measurements, respectively. I would also like to thank Dr. Lin Xiao and Dr. Andrew Dudley (UNC-CH Cell Biology and Physiology) for endothelial isolation expertise, and Helen Willcockson, Dr. Samantha Hoopes, John Pawlak, and Dr. Wenjing Xu for technical assistance. For preliminary data presented in Chapter IV, I would like to thank Dr. James Dunleavey of the Dudley lab for providing melanoma cell line and tumor and flow cytometry expertise. For study presented in Chapter VI, I would like to thank all members of Dr. Kay Lund’s laboratory (UNC-CH Cell Biology and Physiology) for reagents and discussions. I would like to thank Dr. Linda Samuelson (University of Michigan Molecular and Integrative Physiology) for provided Notch RNA and continued mentorship. Finally, I would like to acknowledge that research conducted in this dissertation was supported by NIH RO1-HL091973, RO1-HD060860 and RO1-DK099156 grants to K.M.C.; NIH F31-CA174194 and the University of North Carolina v Cell and Molecular Physiology Fellner Fellowship to D.O.K.; an American Heart Association 12PRE11710002 to S.E.W-S.; an American Heart Association 15PRE25680001 to C.E.T.; NIH 2-T32-DK07686 to A.T.M; NIH 1-F30-CA200345 and T32-CA071341-17 to M.B.S; and NIH RO1-DK040247-19 to P.K.L. vi TABLE OF CONTENTS LIST OF TABLES .................................................................................................................. x LIST OF FIGURES ............................................................................................................... xi LIST OF ABBREVIATIONS ................................................................................................ xiii PART I Chapter I: Adrenomedullin Function in Vascular Endothelial Cells: Insights from Genetic Mouse Models .................................................................................................... 1 Overview ................................................................................................................. 1 Introduction ............................................................................................................. 1 Development ........................................................................................................... 6 Physiology and Pathology ......................................................................................10 Summary and Future Directions .............................................................................16 Author Contributions...............................................................................................16 Tables ................................................................................................................... 17 Figures ................................................................................................................... 19 Chapter II: Endothelial restoration of receptor activity-modifying protein 2 is sufficeint to rescue lethality, but survivors develop dilated cardiomyopathy ..........................21 Overview ................................................................................................................21 Introduction ............................................................................................................22 Methods .................................................................................................................23 Results ...................................................................................................................25 Discussion ..............................................................................................................32 Perspectives ..........................................................................................................35 Novelty and Significance ........................................................................................36 vii Author Contributions ..............................................................................................37 Tables ....................................................................................................................38 Figures ...................................................................................................................40 Supplemental Methods ...........................................................................................47 Supplemental Tables..............................................................................................54 Supplemental Figures ............................................................................................58 Chapter III: Dosage of adrenomedullin correlates with tumor and lymph node lymphangiogenesis ............................................................................................61 Overview ................................................................................................................61 Introduction ............................................................................................................62 Materials and Methods ...........................................................................................64 Results ...................................................................................................................68 Discussion ..............................................................................................................73