KRUPPEL-LIKE TRANSCRIPTION FACTORS: MASTER REGULATORS OF VASCULAR ENDOTHELIUM by PANJAMAPORN SANGWUNG Submitted in partial fulfillment of the requirements For the degree of Doctor of Philosophy Dissertation Advisor: Mukesh K. Jain, MD. Department of Physiology and Biophysics CASE WESTERN RESERVE UNIVERSITY August, 2017 CASE WESTERN RESERVE UNIVERSITY SCHOOL OF GRADUATE STUDIES We hereby approve the thesis/dissertation of Panjamaporn Sangwung candidate for the degree of Doctor of Philosophy*. Committee Chair George R. Dubyak, Ph.D. Committee Member Thomas N. Nosek, Ph.D. Committee Member Julian E. Stelzer, Ph.D. Committee Member Jonathan D. Smith, Ph.D. Dissertation Advisor Mukesh K. Jain, M.D. Date of Defense July 6th, 2017 *We also certify that written approval has been obtained for any proprietary material contained therein. DEDICATION This dissertation is dedicated to my family, especially my parents (Boonma and Usanee Saengwang) and my grandparents (Anan and Aonkaew Intana) for their constant source of support and love. TABLE OF CONTENTS LIST OF TABLES………………………………………………………….…………..v LIST OF FIGURES.………………..…………………………..………………………vi ACKNOWLEDGEMENTS………………………… … ……..………….……….….viii LIST OF ABBREVIATIONS………………… …………..…….…………………….xii ABSTRACT……………………………………………….……...……….……..……. 1 CHAPTER 1. SIGNIFICANCE AND INTRODUCTION……………..……………. 3 1.1 Significance ……………………………………………………………….……. 3 1.2 Structure and functional importance of vascular endothelium …..…… 5 Basic structure of blood vessels……………………………………...……... 5 Functional Importance of vascular endothelium…………………………… 7 1.3 Dysfunction of endothelial cells and diseases ………………………..… 14 1.4 Regulation of endothelial function ……………………………………….... 16 Biochemical stimuli……………………………………..………………….… 16 Biomechanical stimuli……………………………………………..…….…… 17 1.5 Kruppel-like factor (KLF) ………………………………………….……..….. 18 Structure of KLFs……………………………………………………………….. 18 Identification and characterization of endothelial KLFs………………....….. 19 KLF2 – Identification and characterization………………..……………..... 19 Role of endothelial KLF2…………………………………….………….…... 21 Regulation of endothelial KLF2………………………………………….…. 25 KLF4 – Identification and Characterization……………………………….. 30 i Role of endothelial KLF4………………………….…………………….…... 31 Regulation of endothelial KLF4…………………………………….….…… 32 KLF6 – Identification, characterization, and functions………………....... 33 1.6 Endothelial hemoglobin …………………………………..……………….... 35 Functional importance of hemoglobin …………………………………..….... 35 Hemoglobin and disorders…………………………………………………..… 36 Structure of hemoglobin ……………………………………………….……..... 37 Regulation of hemoglobin………………………………………………….…... 37 Hemoglobin in non-erythroid cells…………………………………………..… 38 CHAPTER 2. KLF2 AND KLF4 CONTROL ENDOTHELIAL IDENTITY AND VASCULAR INTEGRITY…..…………………………..………………………....... 41 2.1 Abstract………..…………………………………………………….……….….. 41 2.2 Introduction………..…………………………………………………..……...… 42 2.3 Materials and methods…………………………………………….………...... 44 2.4 Results…….……………………………………………………...…..………..... 52 Endothelial-specific Klf2 and Klf4 deletion leads to rapid death of adult Mice..………………………………………………………………………...... 52 Endothelial-specific deletion of Klf2 and Klf4 leads to vascular leak and systemic coagulopathy……………………………………………........ 59 Endothelial-specific Klf2 and Klf4 deletion results in profound alterations in the EC transcriptome…………………………….…...……… 67 2.5 Discussion and conclusion...………………………………………...……… 69 ii 2.6 Author contributions……...…………………………………………...……… 73 2.7 Acknowledgments…………...………………………………………...………. 73 CHAPTER 3. REGULATION OF ENDOTHELIAL HEMOGLOBIN ALPHA EXPRESSION BY KRUPPEL-LIKE FACTORS……………..…………………... 74 3.1 Abstract………..………………………………………………...………...……. 74 3.2 Introduction………..………………………………………………...……..…… 75 3.3 Materials and methods………………………………………………..….…… 77 3.4 Results…….…………………………………………………………………..…. 85 KLF2 and KLF4 induce HBA expression in the endothelium…………………… 85 The transactivation of the human hemoglobin alpha gene promoter in ECs by KLF2/KLF4………………………………………………………... 93 Direct binding of KLF4 to the endogenous hemoglobin alpha promoter in the EC………………………………………………….............. 98 3.5 Discussion…….…………………………………………………..…………… 100 3.6 Conclusion…….………………………...…………………………..………… 102 3.7 Acknowledgment……………………...…………..………………..………… 102 CHAPTER 4. DISCUSSION AND SUMMARY STATEMENTS OF THE THESIS PROJECTS …………………………………………………………. 103 4.1 Discussion …….………………………...…………………………..……….... 103 4.2 Summary statements of the thesis projects…..…..…………....…..….... 106 iii CHAPTER 5. FUTURE DIRECTIONS…….………………......................…...... 107 BIBLIOGRAPHY.…………………....…………………………...……………….... 109 iv LIST OF TABLES Table 2.1 Primer sequences for qPCR (TaqMan) analysis………………….….. 46 Table 2.2 Differentially expressed genes (q<0.05) in the Hallmark coagulation pathway (EC-DKO vs CRE) at day 6 post-tamoxifen……… 72 Table 3.1 Primer sequences for qPCR (TaqMan) analysis……………….…….. 79 Table 3.2 The HBA promoter mutagenic oligonucleotide primers (5’-3’)………. 83 Table 3.3 Identified peptides of HBA protein in the MEJ of KLF4 overexpressing HCAECs using LC-MS/MS………………..…….……….. 92 v LIST OF FIGURES Figure 1.1 Basic structure of normal artery…………………………………………. 5 Figure 1.2 Schematic representation of both tight and adherens junctions in ECs…………………………………………………...…............... 9 Figure 1.3 The TM/protein C/protein S anticoagulation system….……………… 11 Figure 1.4 NO production by eNOS………………………………………….…….. 13 Figure 1.5 A schematic representation of common structure and functional domains for Krüppel-like factors (KLFs)………………………... 18 Figure 1.6 Human hemoglobin genes located on chromosome 11and 16 ….… 38 Figure 1.7 Possible mechanism of NO control at the MEJ………………….…… 40 Figure 2.1 Expression of Klf2, Klf4, and their targets in primary cardiac microvascular EC……………………………….………………………….…. 54 Figure 2.2 Endothelial-specific Klf2 and Klf4 deletion leads to rapid death of adult mice……………….……………………….….……………….. 55 Figure 2.3 Electrocardiogram (EKG) recording………….……………….……….. 56 Figure 2.4 Echocardiographic analysis at day 6 after tamoxifen administration……………………….……………..…………………….……. 57 Figure 2.5 Gross post-mortem examination in EC-Klf2-KO and EC-Klf4-KO mice…………………………………..……………..…………… 58 Figure 2.6 Endothelial-specific deletion of Klf2 and Klf4 leads to vascular leak and systemic coagulopathy………………..….…...………… 62 Figure 2.7 Endothelial-specific deletion of Klf2 and Klf4 results in vascular leak…………………..……………………..…………..…….……… 64 vi Figure 2.8 Representative electron microscopic (EM) images of brain indicate degeneration of EC and extravascular erythrocytes in the EC-DKO mice at day 6 post-tamoxifen……………………..………….. 65 Figure 2.9 Expression of F3, Serpine1, and F2rl3 mRNA in primary cardiac microvascular EC at day 6 post-tamoxifen……………...………… 66 Figure 2.10 Endothelial-specific Klf2 and Klf4 deletion results in profound alterations in the EC transcriptome……………………………………….… 68 Figure 3.1 Hemoglobin alpha mRNA expression in the EC by KLF2/KLF4…… 87 Figure 3.2 Expression of KLF2 or KLF4 mRNA in ECs………………………….. 88 Figure 3.3 KLF2/KLF4-mediated hemoglobin alpha protein expression in the EC cultured alone on a petri dish…………………………................ 89 Figure 3.4 Hemoglobin alpha protein expression in the EC by KLF2/KLF4…... 90 Figure 3.5 KLF2/KLF4-mediated hemoglobin alpha protein expression in the EC and the MEJ obtained from the co-cultured model…………….. 91 Figure 3.6 KLF2/KLF4 mediate a transactivation of the HBA promoter………... 95 Figure 3.7 KLF2/KLF4 fail to mediate a transactivation of the mutant HBA promoter………………………………………………….……………… 96 Figure 3.8 Critical KLF-binding sites on the HBA promoter……………………... 97 Figure 3.9 Direct binding of KLF4 on the HBA promoter in the EC…………...... 99 vii ACKNOWLEDGEMENTS I would like to express my wholehearted thanks to all those who helped me to make my PhD study and dissertation work a success. This dissertation would not have been possible without help of many people. First and foremost, I would like to express my grateful thanks to my mentor, Dr. Mukesh Jain, for giving me the opportunity to join the lab and for giving me mental and technical advice on my professional and personal life. Dr.Jain is one of the best mentors I have ever had in my life. Dr.Jain provided leadership development, useful guidance, tutelage, consultation, and feedback to me. He also provided me with opportunity to explore research, full material supports, and expert. He connected me to resources to ensure progress of the projects. He motivated, supported, and challenged me to push myself beyond the limits to achieve the goals and success in my profession. Dr.Jain is an outstanding physician scientist, a great speaker, and creative writer. I have learned a tremendous amount from him. I am pleased to acknowledge and thank my dissertation committees (Drs. George Dubyak, Jonathan Smith, Thomas Nosek, and Julian Stelzer). I met my dissertation committees at least once a year. My committees helped me directing my research and troubleshooting problems. Dr. Dubyak is one of the best teachers I have ever met. He has tremendous amount of knowledge in vascular biology and cell signaling. He was always available for me when needed. Dr. Dubyak provided guidance to access to internal and external resources, and viii served as troubleshooters. He helped me plan a graduation timeline and made sure that things moved along. Dr. Smith encouraged me to think critically by challenging me with logical questions, and to become better problem solvers. He has massive amount