DEFINING THE ROLE OF HYPOXIA AND HIFs IN VASCULAR SMOOTH MUSCLE CELLS by ANNA CLARK HENRY BORTON Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy Department of Pathology CASE WESTERN RESERVE UNIVERSITY August, 2018 CASE WESTERN RESERVE UNIVERSITY SCHOOL OF GRADUATE STUDIES We hereby approve the thesis/dissertation of Anna Clark Henry Borton candidate for the PhD Degree*. Committee Chair Nicholas P. Ziats, Ph.D. Committee Members Diana L. Ramirez-Bergeron, Ph.D. Aaron Proweller, M.D., Ph.D. George R. Dubyak, Ph.D. Clive R. Hamlin, Ph.D. Date of Defense June 25, 2018 *We also certify that written approval has been obtained for any proprietary material contained therein. 2 Table of Contents List of Tables……………………………………………………………………. 7 List of Figures…………………………………………………………………… 8 Acknowledgements…………………………………………………………….. 10 List of Abbreviations……………………………………………………………. 12 Abstract………………………………………………………………………….. 16 Chapter 1: Introduction…………………………………………………........ 18 Vascular Smooth Muscle ……………………………………………… 18 Structure of the vascular wall.....……………………………… 18 Diverse developmental origins of VSMCs……………………. 20 Vascular smooth muscle contractile function………………... 22 Phenotypic plasticity in VSMCs……………………………….. 27 Hypoxia-Inducible Factors……………………………………………... 29 Oxygen dependent regulation of HIFs………………………... 30 Alternative HIF stabilization pathways………………………... 32 HIF function in the vasculature………………………………... 33 HIF in pulmonary VSMCs……………………………………… 33 HIF in VSMCs of the systemic vasculature………………….. 35 Peripheral Vascular Disease…………………………………………... 37 Epidemiology and disease burden……………………………. 38 Clinical presentation and diagnosis…………………………… 38 Pathogenesis and vascular compensation…………………... 40 Therapeutic interventions……………………………………… 44 3 HIF in VSMCs of the peripheral vasculature………………………… 45 A murine model of PVD: HLI…………………………………... 45 HIFs in HLI model………………………………………………. 46 HIF as a therapeutic……………………………………………. 48 Summary and Hypothesis……………………………………………… 48 Chapter 2: Aryl Hydrocarbon Receptor Nuclear Translocator in Vascular Smooth Muscle Cells is Required for Optimal Peripheral Perfusion Recovery…………………………………………………………… 50 Authors…………………………………………………………………… 50 Summary………………………………………………………………… 51 Introduction……………………………………………………………… 53 Methods………………………………………………………………….. 56 Results…………………………………………………………………… 66 Generation of smooth muscle specific Arnt knockout mouse model…………………………………………………………….. 66 ArntSMKO mice show impaired blood flow recovery in HLI model…………………………………………………………….. 68 Limb ischemia stimulates collateral remodeling in ArntSMKO and Arntlox/lox mice………………………………………………. 70 Early capillary angiogenesis does not depend on VSMC Arnt in HLI……………………………………………………….. 73 ArntSMKO mice show increased hypoxia and damage in gastrocnemius in response to ischemia……………………… 81 4 Altered vascular smooth muscle morphology in ArntSMKO mice………………………………………………………………. 84 Loss of ARNT alters VSMC phenotype………………………. 87 Discussion……………………………………………………………….. 91 Chapter 3 Loss of Vascular Smooth Muscle Cell Aryl Hydrocarbon Receptor Nuclear Translocator Impairs Vasoconstriction…………….. 97 Authors…………………………………………………………………… 97 Summary………………………………………………………………… 98 Introduction……………………………………………………………… 99 Methods………………………………………………………………….. 101 Results…………………………………………………………………… 105 Transcriptional expression of contractile elements are reduced in VSMCs lacking ARNT…………………………….. 105 Large vessel structure and tunica media thickness is not affected in ArntSMKO…………………………………………….. 107 Loss of Arnt in VSMCs impairs aortic vasoconstriction…….. 107 Loss of Arnt in VSMCs does not affect aortic vasorelaxation 109 Discussion……………………………………………………………….. 112 Conclusions……………………………………………………………… 115 Chapter 4: Discussion and Closing …………………………………......... 116 Discussion……………………………………………………………….. 116 Molecular dysregulation………………………………………... 116 5 Implications for other vascular pathologies………………….. 123 Potential for translation to human disease…………………… 129 Closing………………………….………………………………………... 135 Future Directions………………………………………………………... 136 References……………………………………………………………………... 139 6 List of Tables Supplemental Table 1: qPCR primers………………………………………... 64 7 List of Figures Figure 1.1. Layers of the vascular wall……………………………………….. 19 Figure 1.2. VSMCs and ECs of the vascular wall………………………..…. 21 Figure 1.3. VSMC myofilament components………………………………… 23 Figure 1.4. Regulation of VSMC contraction and relaxation……………….. 25 Figure 1.5. Canonical HIF regulation…………………………………………. 31 Figure 1.6. Vascular responses to flow limitation…………………………… 43 Figure 1.7. Hind limb ischemia: a murine model of PAD………………....... 47 Figure 2.1. Characterization of ArntSMKO mice……………………………….. 67 Figure 2.2. Bulk perfusion and functional recovery is reduced in ArntSMKO mice following femoral artery ligation…………………………………………. 69 Figure 2.3. VSMC ARNT is not required for ischemia induced collateralization………………………………………………………………….. 71 Supplemental Figure 2.1. Collateral vessel lumen cross sectional area vs limb perfusion…………………………………………………………………… 72 Supplemental Figure 2.2. Proximal HLI model………………………………. 75 Figure 2.4. Histological assessment of capillary number and perfusion status in gastrocnemius muscle (GC)………………………………………… 76 Supplemental Figure 2.3. Capillary density and smooth muscle cell colocalization in gastrocnemius (GC) at day 7………………………………. 78 Supplemental Figure 2.4. Regional assessment of capillary density and perfusion status in gastrocnemius muscle (GC)…………………………….. 79 8 Figure 2.5. Ischemic skeletal muscle regeneration and vessel integrity are impaired in ArntSMKO mice…………………………………………………. 83 Figure 2.6. Smooth muscle morphology and perimural wrapping of small arterioles…………………………………………………………………………. 85 Supplemental Figure 2.5. Additional images of arterioles in skeletal muscle……………………………………………………………………………. 86 Figure 2.7. Transcriptional expression of proliferation and migration regulators in response to hypoxia…………………………………………….. 89 Figure 2.8. Assessment of VSMC phenotype in vitro………………………. 90 Figure 3.1. Contractile gene expression is reduced in ArntSMKO VSMCs…. 106 Figure 3.2. Large vessel structure unchanged in ArntSMKO………………… 108 Figure 3.3. ArntSMKO vessels have impaired vasoconstriction……………… 110 Figure 3.4. Vasorelaxation is intact in ArntSMKO……………………………... 111 Figure 4.1. Effects of hypoxia exposure on human iliac artery VSMCs…... 130 Figure 4.2. Effects of hypoxia exposure on human iliac vein VSMCs…….. 133 Figure 4.3. Iliac vein smooth muscle cells are more susceptible to H2O2 induced apoptosis………………………………………………………………. 134 9 Acknowledgements Numerous people have helped me reach this milestone, and for each of their contributions, I am tremendously grateful. It has been an outstanding privilege to train under Diana Ramirez-Bergeron. Her enthusiasm and love of science are positively infectious, as is the commitment to excellence, scientific rigor, and collaborative spirit she instills in her trainees. Her mentorship inspired me to step outside my academic comfort zone, encouraged me through challenges, and helped me develop skills I will use for the rest of my career. My thesis project, spanned the expertise of the Ramirez and Proweller laboratories. I am incredibly grateful to Aaron Proweller for sharing his scientific guidance, technical expertise, and perspective as a physician scientist. Enormous thanks also to my thesis committee members, Nicholas Ziats, George Dubyak, and Clive Hamlin for all their scientific insight and advice. Science does not happen in a vacuum: I am grateful to have had the opportunity to work with and learn from all the past and present members of the Ramirez and Proweller laboratories. I would also like to thank Mukesh Jain, Xudong Liao, Lalitha Nayak, Yuan Lu, Andrei Maiseyeu, Anne Hamik, Alex Huang, Rongli Zhang, Bryan Benson, Lee Neilson, Iulia Barbur, Stephanie Lapping, Amer Alaiti, and Keiki Sugi for their technical assistance, professional and scientific advice, goodwill, and encouragement. 10 I have been fortunate to have a wonderful community of peers including the MSTP entering classes of 2011 and 2012 and my CVRI graduate student compatriots Nelson, David, and Liyan. I am grateful to the MSTP, our director Cliff Harding, the co-directors, and the administrative team for the opportunity to chase the dream of a career as a physician scientist. Thanks also to the CVRI and the Departments of Pathology and Medicine and their administrators for being my academic home during the PhD. Overwhelming thank yous are also due to my friends and extended family for graciously listening to the minutia of experimental success and failure, and cheering me through the celebratory moments and the frustrations. I am so grateful to my family for fostering curiosity and dedication and for their love, support, and encouragement. Finally, a tremendous thank you to my wonderful husband Peter. He has been there every step of the way as my greatest supporter and the baker of thesis committee appeasing banana bread. 11 List of Abbreviations ABI – Ankle-brachial index ACC – American College of Cardiology Foundation Ach – Acetylcholine AHA – American Heart Association AHR – Aryl hydrocarbon receptor ANG1 – Angiopoietin AngII – Angiotensin II ARNT; HIF-1β – Aryl hydrocarbon receptor nuclear translocator Arntlox/lox – Animals and cells with floxed ARNT gene ArntSMKO – Smooth muscle specific ARNT deletion model AT1 – Angiotensin receptor 1 BSA – Bovine serum albumin Ca – Calcium CaCm – Calcium calmodulin CLI – Critical