University of Louisville ThinkIR: The University of Louisville's Institutional Repository Electronic Theses and Dissertations 5-2014 Epigenetic modifications associated with aortic remodeling in hyperhomocysteinemia. Nithya Narayanan University of Louisville Follow this and additional works at: https://ir.library.louisville.edu/etd Part of the Biophysics Commons, and the Physiology Commons Recommended Citation Narayanan, Nithya, "Epigenetic modifications associated with aortic remodeling in hyperhomocysteinemia." (2014). Electronic Theses and Dissertations. Paper 1042. https://doi.org/10.18297/etd/1042 This Doctoral Dissertation is brought to you for free and open access by ThinkIR: The University of Louisville's Institutional Repository. It has been accepted for inclusion in Electronic Theses and Dissertations by an authorized administrator of ThinkIR: The University of Louisville's Institutional Repository. 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EPIGENETIC MODIFICATIONS ASSOCIATED WITH AORTIC REMODELING IN HYPERHOMOCYSTEINEMIA By Nithya Narayanan B.Tech., Anna University, 2009 M.S., University of Texas at Dallas, 2011 M.S., University of Louisville, 2013 A Dissertation Submitted to the Faculty of School of Medicine of the University of Louisville In Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy Department of Physiology and Biophysics University of Louisville Louisville, Kentucky May 2014 EPIGENETIC MODIFICATIONS ASSOCIATED WITH AORTIC REMODELING IN HYPERHOMOCYSTEINEMIA By Nithya Narayanan B.Tech., Anna University, 2009 M.S., University of Texas at Dallas, 2011 M.S., University of Louisville, 2013 A Dissertation Approved on April 16, 2014 by the following Dissertation Committee __________________________________ Suresh C.Tyagi, Ph.D., Mentor __________________________________ Utpal Sen, PhD., C-Mentor __________________________________ Andrew M. Roberts, Ph.D. __________________________________ Irving G. Joshua, Ph.D. __________________________________ William B. Wead, Ph.D. __________________________________ Adrienne P. Bratcher, Ph.D. ii DEDICATION This dissertation is dedicated to my parents Mrs. Radha Narayanan & Mr. M. Narayanan and my grandparents Mrs. Swarna Krishnamurthi & Mr. R. Krishnamurthi for believing in me and encouraging me throughout my life. iii ACKNOWLEDGEMENTS I would like to thank my mentor, Dr. Suresh C. Tyagi for his constant support and encouragement throughout my graduate program. His vision and passion for science have been the inspiration for this dissertation. I am grateful to Dr. Utpal Sen, for his valuable suggestions to encapsulate my dissertation. I would like to express my deepest gratitude to Dr. Andrew M. Roberts for his valuable inputs and guidance for drafting this dissertation. I would like to thank Dr. William B. Wead for his help and advice during my PhD program. I would like to thank Dr. Irving G. Joshua and Dr. Adrienne P. Bratcher for their guidance as committee members and also for the opportunity to conduct myobath studies in their laboratory. I would like to thank Dr. David Lominadze for his valuable suggestions during my graduate program. My sincere thanks to all the professors at the Department of Physiology for providing me a profound insight into the fundamentals of physiology through one of the best classroom experiences in my academic life. I would like to thank the administrative staff at the Department of Physiology for helping me during my graduate program. I am grateful to my lab members for providing me a supportive work atmosphere to pursue my research work. Without the support and advice of Dr. Sathnur B. Pushpakumar and Dr. Srikanth Givvimani, conducting these promising experiments would have been impossible. I would like to extend my heartfelt thanks to them, for guiding me through iv my PhD research. My special thanks to Dr. Sourav Kundu for helping me with the experiments and for creating a memorable work atmosphere. I am extremely grateful to our lab manager, Naira Metreveli, for providing me the necessary facilities and kind words when I needed. I am thankful to Dr. Neetu Tyagi and Mrs. Reeta Tyagi for guiding me when I started working in this lab. I would like to thank all the summer research students for being a memorable part of my lab life in UofL. I am thankful to Dexter James for helping me with the myobath studies. I would like to thank all my friends and family for their unconditional support. I am extremely grateful to my younger brother, Pramodh and my aunt, Jayanthi for being the beacons of love and inspiration in my life. I am very thankful to my uncle, Kalyan for his care and encouragement. I would like to thank Kannan’s and Naren’s families for helping me during my stay in the United States. I would like to thank all my teachers and professors for their guidance. I would like to thank the Almighty for bringing the ray of hope during my dark times. Without His blessings, nothing would have been there in my life. Last but not least, I am indebted to my wonderful husband, Nitin, for always being there for me and making my life more meaningful. v ABSTRACT EPIGENETIC MODIFICATIONS ASSOCIATED WITH AORTIC REMODELING IN HYPERHOMOCYSTEINEMIA Nithya Narayanan April 16, 2014 Background: Hyperhomocysteinemia (HHcy) is prevalent in hypertensive patients and is an independent risk factor for aortic pathologies. HHcy is known to cause an imbalance between matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) leading to accumulation of collagen in the aorta resulting in stiffness and development of hypertension. Although the exact mechanism of extracellular matrix remodeling (ECM) is unclear, emerging evidence implicates epigenetic regulation involving DNA methylation. The purpose of the study was to investigate whether inhibition of DNA methylation reduces high blood pressure by regulating aortic ECM remodeling in HHcy. Methods: In the first set of experiments, we studied the effect of HHcy in causing epigenetic changes leading to aortic remodeling resulting in hypertension. Cystathionine beta synthase heterozygous knockout (CBS+/-) mice of C57BL/6J background were used vi as HHcy model and their Wild type (WT) littermates were used as controls. Hcy level in plasma was measured by HPLC-UV. Blood pressure was measured using tail-cuff method. The mRNA expression levels of ECM proteins and epigenetic modifications enzymes in the aorta were measured by RT-PCR. Global methylation level was measured by ELISA. In the second set of experiments, we studied effects of an epigenetic DNA methyltransferase inhibitor, 5-Aza-2’-deoxycytidine (Aza) in HHcy. For in vivo studies, we used 8-12 weeks old WT and CBS+/- mice and administered them with Aza for four weeks. During the treatment period, blood pressure was measured using tail-cuff method. Aorta wall thickness, lumen diameter and resistive index were measured before and after treatment using ultrasound. Aortic responses to vasoconstrictor and vasodilators were measured using tissue myobath. Collagen accumulation in the aortic walls was measured by Mason Trichrome and Picrosirius red staining. ECM proteases, Hcy metabolism enzymes and DNMT1 expression were measured using immunostaining. Global methylation levels were assessed using ELISA. For cell culture studies, mouse aortic smooth muscle cells were treated with Aza and Hcy for 48 hrs. MMP9 activity was measured using gelatin zymography and DNMT1, 3a and 3b expression was measured using immunoblotting. Results: In CBS+/- mice, increased plasma Hcy levels were associated with an increase in blood pressure. There was an increase in the mRNA levels of collagen and decrease in elastin in HHcy mice. Global hypermethylation was accompanied by an increased expression of methylation proteins in HHcy. Aza treatment normalized plasma SAM, SAH and Hcy levels and blood pressure in HHcy mice. Thoracic and abdominal aorta vii ultrasound revealed reduction in resistive index and wall-to-lumen ratio. Vascular response to vasoactive agents improved following Aza treatment in HHcy. Histology showed a marked reduction in collagen deposition in the aorta. Aza treatment decreased the expression of DNMT1, 3b, MMP9, TIMP1 and S-adenosyl homocysteine hydrolase (SAHH) and upregulated methylene tetrahydrofolate reductase (MTHFR) Conclusion: We conclude that reduction of DNA methylation in HHcy reduces adverse aortic remodeling to mitigate hypertension. viii TABLE OF CONTENTS ACKNOWLEDGEMENTS………………………………………………………………iv ABSTRACT……………………………………………………………………………...vi TABLE OF CONTENTS…………………………………………………………………ix LIST OF TABLES………………………………………………………………………..xi LIST OF FIGURES……………………………………………...………………………xii CHAPTER I: INTRODUCTION………………………………………………………….1 CHAPTER II: BACKGROUND Aortic diseases…………………………………………………………………….4 Homocysteine……………………………………………………………………..9 Hyperhomocysteinemia and vascular dysfunction…….……………….………. 16 Extracellular matrix remodeling…………………………………………….…...17 Epigenetic Modifications………………………………………….……………..25 CHAPTER III: HYPOTHESIS AND SPECIFIC AIMS…………………………….…..31 CHAPTER IV: AORTIC REMODELING IN HYPERHOMOCYSTEINEMIA……….34 ix CHAPTER V: EFFECT OF 5-AZA-2’DEOXYCYTIDINE IN AORTIC REMODELING IN HYPERHOMOCYSTEINEMIA…………………………………..52 CHAPTER VI: SUMMARY, CONCLUSION AND FUTURE DIRECTIONS……….94 REFERENCES……………………………………………………………………..……97 LIST OF ABBREVIATIONS…………………………………………………………..116 CURRICULUM VITAE………………………………………………………………120