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Calponin and Cytoskeleton Dynamics in Macrophage Functions and the Ap Thogenesis of Atherosclerosis Rong Liu Wayne State University

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Calponin and Cytoskeleton Dynamics in Macrophage Functions and the Ap Thogenesis of Atherosclerosis Rong Liu Wayne State University Wayne State University Wayne State University Dissertations 1-1-2016 Calponin And Cytoskeleton Dynamics In Macrophage Functions And The aP thogenesis Of Atherosclerosis Rong Liu Wayne State University, Follow this and additional works at: http://digitalcommons.wayne.edu/oa_dissertations Part of the Immunology and Infectious Disease Commons, Medicine and Health Sciences Commons, and the Physiology Commons Recommended Citation Liu, Rong, "Calponin And Cytoskeleton Dynamics In Macrophage Functions And The aP thogenesis Of Atherosclerosis" (2016). Wayne State University Dissertations. 1650. http://digitalcommons.wayne.edu/oa_dissertations/1650 This Open Access Dissertation is brought to you for free and open access by DigitalCommons@WayneState. It has been accepted for inclusion in Wayne State University Dissertations by an authorized administrator of DigitalCommons@WayneState. CALPONIN AND CYTOSKELETON DYNAMICS IN MACROPHAGE FUNCTIONS AND THE PATHOGENESIS OF ATHEROSCLEROSIS by RONG LIU DISSERTATION Submitted to the Graduate School of Wayne State University, Detroit, Michigan in partial fulfilment of the requirements for the degree of DOCTOR OF PHILOSOPHY 2016 MAJOR: PHYSIOLOGY Approved By: Advisor Date © COPYRIGHT BY RONG LIU 2016 All Rights ReserveD DEDICATION To my departed loving father for your guidance and encouragement during my youth and career development; to my dear mother for your love and support. ii ACKNOWLEDGEMENTS I am grateful to my dissertation advisor, Dr. J.-P. Jin. Your training and insights have been invaluable to me. Thank you for your tireless teaching and guidance. I sincerely thank the members of my dissertation committee, Dr. Steven Cala, Dr. Robert Lasley, Dr. Takeshi Sakamoto, Dr. Assia Shisheva and Dr. Jianjun Wang, for their support and advice. I want to thank all the Jin lab members for their help and friendship, particularly Dr. Hanzhong Feng who is my first instructor of research skills. The following Jin lab members and collaborators have also helped my dissertation research in significant ways: Dr. M. Moazzem Hossain, Dr. Xuequn Chen, and Dr. Alan Daugherty at the University of Kentucky. I want to further thank the Physiology Ph.D. Program and the Department of Physiology and our graduate officers, Dr. Douglas Yingst and Dr. Steven Cala. The Physiology Department at Wayne State University is my first home in the United States. Lastly, my many thanks go to Ms. Christine Cupps. I am grateful to your patience and all the help in the past four years which has been the best time of my life. iii TABLE OF CONTENTS Dedication ........................................................................................................................ ii Acknowledgements ......................................................................................................... iii List of Tables .................................................................................................................. vii List of Figures ................................................................................................................. viii List of Abbreviations ......................................................................................................... x Chapter 1 Introduction ...................................................................................................... 1 The cardiovascular system and coronary heart disease ............................................. 1 Atherosclerosis is an inflammatory disease ................................................................ 2 Role of macrophage in the development of atherosclerosis ....................................... 3 Calponin and isoforms ................................................................................................ 5 Tissue distribution of calponin isoforms ...................................................................... 6 Structure-function relationship of calponin .................................................................. 9 Role of calponin 2 in the regulation of actin cytoskeleton in non- muscle cells............................................................................................................... 12 Calponin 2 in the regulation of macrophage function ................................................ 13 Aims of dissertation research .................................................................................... 15 Chapter 2 – Cloning of cDNA Encoding Mouse ApoE, Expression of Recombinant Protein in Bacterial Culture, and Approach to Developing Hybridoma Monoclonal Antibodies .......................................... 18 Abstract ..................................................................................................................... 18 Introduction ............................................................................................................... 19 Materials and Methods .............................................................................................. 20 Results ...................................................................................................................... 24 Discussion ................................................................................................................. 28 Chapter 3 – Deletion of Calponin 2 in Macrophages Alters Cytoskeleton- based Functions and Attenuates the Development of iv Atherosclerosis ........................................................................................... 32 Abstract ..................................................................................................................... 32 Introduction ............................................................................................................... 33 Materials and Methods .............................................................................................. 35 Results ...................................................................................................................... 43 Discussion ................................................................................................................. 54 Summary ................................................................................................................... 59 Chapter 4 – Mechanical Regulation of Cytoskeleton Function in Blood Vessels: The Role of a Calponin-related Protein, SM22 ............................ 60 Abstract ..................................................................................................................... 60 Introduction ............................................................................................................... 61 Materials and Methods .............................................................................................. 63 Results ...................................................................................................................... 70 Discussion ................................................................................................................. 78 Chapter 5 – Physiological Contractility of Cardiomyocytes in the Wall of Mouse and Rat Azygos Vein .................................................................. 83 Abstract ..................................................................................................................... 83 Introduction ............................................................................................................... 84 Materials and Methods .............................................................................................. 86 Results ...................................................................................................................... 91 Discussion ................................................................................................................. 97 Chapter 6 Conclusions ................................................................................................. 102 Appendix A IACUC Protocol Approval Letter ............................................................... 105 Appendix B NCBI database for calponin isoforms analyzed for Figure 2 ..................... 106 Appendix C NCBI database for calponin and SM22 isoforms analyzed for Figure 28 ................................................................................................................. 107 v Appendix D Copyright License Agreement for Chapter 3 ............................................ 108 Appendix E Copyright License Agreement for Chapter 5 ............................................. 109 References ................................................................................................................... 110 Abstract ........................................................................................................................ 135 Autobiographical Statement ......................................................................................... 137 vi LIST OF TABLES Table 1: Calponin isoform genes and their tissue-specific expression ............................. 6 Table 2: Primers used in qPCR ...................................................................................... 70 vii LIST OF FIGURES Figure 1: The initial inflammatory response in atherosclerosis ........................................ 3 Figure 2: Evolutionary lineage of vertebrate calponin isoforms ........................................ 8 Figure 3: Linear structure and comparison of calponin isoforms .................................... 10 Figure 4: Faster migration
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