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The Role of Cyclin-Dependent Kinase 8 in Vascular Disease Desiree Leach University of South Carolina

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The Role of Cyclin-Dependent Kinase 8 in Vascular Disease Desiree Leach University of South Carolina University of South Carolina Scholar Commons Theses and Dissertations Fall 2017 The Role Of Cyclin-Dependent Kinase 8 In Vascular Disease Desiree Leach University of South Carolina Follow this and additional works at: https://scholarcommons.sc.edu/etd Part of the Biomedical and Dental Materials Commons Recommended Citation Leach, D.(2017). The Role Of Cyclin-Dependent Kinase 8 In Vascular Disease. (Doctoral dissertation). Retrieved from https://scholarcommons.sc.edu/etd/4523 This Open Access Dissertation is brought to you by Scholar Commons. It has been accepted for inclusion in Theses and Dissertations by an authorized administrator of Scholar Commons. For more information, please contact [email protected]. THE ROLE OF CYCLIN -DEPENDENT KINASE 8 IN VASCULAR DISEASE by Desiree Leach Bachelor of Science Coastal Carolina University, 2010 Submitted in Partial Fulfillment of the Requirements For the Degree of Doctor of Philosophy in Biomedical Science School of Medicine University of South Carolina 2017 Accepted by: Taixing Cui, Major Professor Wayne Carver, Committee Member Joseph Janicki, Committee Member Igor Roninson, Committee Member Udai Singh, Committee Member Cheryl L Addy, Vice Provost and Dean of the Graduate School © Copyright by Desiree Leach, 2017 All Rights Reserved. ii ACKNOWLEDGEMENTS I would like to thank my mentor, Dr. Taixing Cui, for all his guidance and support. Thank you for supplying me with the knowledge and techniques to grow as a scientist. I would also like to thank my committee members: Dr. Wayne Carver, Dr. Igor Roninson, Dr. Udai Singh and Dr. Joseph Janicki. I appreciate all of your constructive criticism and your efforts to help me grow as a scientist. I would also like to thank my lab members: Bryan Mathis, Qu Chen, Wei Wei Wu, Qi Lei and Chris Bowen. I appreciate all of you for sharing your knowledge, resources, and devoting time out of your schedule to help me in any way. I would also like to thank the faculty and administrative staff: Dr. Edie Goldsmith, Ansley Roberts, Larialmy Allen, Judy Lawrence, Debra Poston, and Heather Willingham. Thank you for making sure I stayed on track throughout the program. Lastly, I would like to thank my family and friends for all their love, prayers and support. If it weren’t for them, I wouldn’t be in this position today. Thank you for your unwavering faith in me and for keeping my morale high. iii ABSTRACT In response to injury, mature vascular smooth muscle cells (SMCs) undergo dedifferentiation, also known as phenotype modulation or switch. This process is characterized by a downregulation or loss of expression of contractile genes and concomitant with an increase in cell proliferation, migration, and extracellular matrix production, thereby leading to vascular lesion formation. However, the underlying molecular mechanism is not fully understood. The objective of my study was to determine whether cyclin-dependent kinase 8 (CDK8), a transcription-regulating kinase, plays a mediator role in vascular SMC dedifferentiation and lesion formation. Our results from immunochemical staining and Western blot revealed that CDK8 expression was upregulated in vascular SMCs in mouse injured arteries and human arteries with atherosclerosis. In cultured rat aortic SMCs (RASMCs), the dedifferentiated phenotype exhibiting the downregulation of SMC contractile genes such as smooth muscle 22 alpha (SM22α), calponin-1 (CNN1), and alpha smooth muscle actin (αSMA) as well as increased proliferation, migration, and cytokine production was dramatically suppressed and reversed into a more differentiated state by CDK8 inactivation via highly selective CDK8 inhibitor, Senexin A, and shRNA knockdown approaches. Peri-vascular delivery of CDK8 inhibitor, Senexin A attenuated ligation-induced neointima (NI) formation in mouse carotid arteries. At the molecular level, we uncovered that CDK8 facilitated the activation of AKT to inhibit GSK3β which phosphorylates c-MYC for degradation by iv proteasomes, thereby promoting vascular SMC dedifferentiation. In addition, activated CDK8 formed signaling complex with AKT thereby activating AKT in vascular SMCs. In conclusion, our results demonstrate that CDK8 is a critical mediator of vascular SMC dedifferentiation at least partly by facilitating the AKT-mediated inhibition of GSK3β to stabilize c-MYC, thereby contributing to NI formation. Targeting CDK8 may be a novel therapeutic approach for the treatment of occlusive vascular disease due to the abnormal growth of vascular SMCs. v TABLE OF CONTENTS ACKNOWLEDGEMENTS ........................................................................................................ iii ABSTRACT .......................................................................................................................... iv LIST OF FIGURES ............................................................................................................... viii LIST OF ABBREVIATIONS ..................................................................................................... xi CHAPTER 1. THE HISTORY OF VASCULAR DISEASE AND CYCLIN -DEPENDENT KINASE 8 FUNCTION ..............................................................................................................................1 1.1 VASCULAR DISEASE ..............................................................................................1 1.2 CELLULAR MECHANISMS OF VASCULAR DISEASE ................................................2 1.3 MOLECULAR MECHANISMS REGULATING SMOOTH MUSCLE CELL PLASTICITY ...6 1.4 CYCLIN -DEPENDENT KINASE 8 ...........................................................................12 CHAPTER 2. CYCLIN -DEPENDENT KINASE 8 IS A POSITIVE REGULATOR IN VASCULAR SMOOTH MUSCLE CELL DEDIFFERENTIATION ....................................................................22 2.1 INTRODUCTION ....................................................................................................22 2.2 MATERIAL AND METHODS ...................................................................................25 2.3 RESULTS ..............................................................................................................29 2.4 DISCUSSION .........................................................................................................31 CHAPTER 3. CYCLIN -DEPENDENT KINASE 8 IS A POSITIVE REGULATOR IN VASCULAR SMOOTH MUSCLE CELL DEDIFFERENTIATION VIA AKT/GSK3 Β-MEDIATED ENHANCEMENT OF C -MYC STABILIZATION ........................................................................40 3.1 INTRODUCTION ....................................................................................................40 3.2 MATERIAL AND METHODS ...................................................................................42 vi 3.3 RESULTS ..............................................................................................................46 3.4 DISCUSSION .........................................................................................................51 CHAPTER 4. CYCLIN -DEPENDENT KINASE 8-MEDIATED VASCULAR SMOOTH MUSCLE CELL DEDIFFERENTIATION CONTRIBUTES TO NEOINTIMAL FORMATION .....................................67 4.1 INTRODUCTION ....................................................................................................67 4.2 MATERIAL AND METHODS ...................................................................................68 4.3 RESULTS ..............................................................................................................71 4.4 DISCUSSION .........................................................................................................72 CHAPTER 5. SUMMARY AND CONCLUSIONS .......................................................................78 REFERENCES .......................................................................................................................80 APPENDIX A. PATIENT MEDICAL HISTORY .........................................................................95 vii LIST OF FIGURES Figure 2.1. CDK8 is expressed in vasculature of normal male adult mice ........................33 Figure 2.2. CDK8 expression is upregulated after carotid ligation injury in mice ............33 Figure 2.3. CDK8 protein expression is upregulated after carotid ligation in mice ..........34 Figure 2.4. CDK8 is expressed in human coronary arteries with atherosclerotic lesions. 35 Figure 2.5. CDK8 protein expression is upregulated in RASMC cell culture ...................35 Figure 2.6. CDK8 inhibition reduced RASMC proliferation ............................................36 Figure 2.7. CDK8 inhibition reduced DNA synthesis .......................................................36 Figure 2.8. CDK8 inhibition prevented RASMC cell cycle progression ..........................37 Figure 2.9. CDK8 inhibition promoted RASMC differentiation .......................................37 Figure 2.10. Senexin A reduction on DNA synthesis is CDK8 specific ...........................38 Figure 2.11. CDK8 inhibition attenuated FBS-induced migration of RASMCs ...............38 Figure 2.12. Senexin A treatment increased extracellular matrix secretion ......................39 Figure 3.1. Senexin A treatment downregulated c-MYC protein expression but had little to no effect on STAT1 phosphorylation ............................................................................53 Figure 3.2. CDK8 inhibition by lentiviral approach reduced c-MYC protein expression .53 Figure 3.3. CDK8 inhibition did not affect
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