This electronic thesis or dissertation has been downloaded from the King’s Research Portal at https://kclpure.kcl.ac.uk/portal/ Vascular Stem Cell Migration in Response to Dkk3 in Vascular Diseases The emergence of a novel chemokine-like protein Bhaloo, Shirin Issa Awarding institution: King's College London The copyright of this thesis rests with the author and no quotation from it or information derived from it may be published without proper acknowledgement. END USER LICENCE AGREEMENT Unless another licence is stated on the immediately following page this work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International licence. https://creativecommons.org/licenses/by-nc-nd/4.0/ You are free to copy, distribute and transmit the work Under the following conditions: Attribution: You must attribute the work in the manner specified by the author (but not in any way that suggests that they endorse you or your use of the work). 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Oct. 2021 Vascular Stem Cell Migration in Response to Dkk3 in Vascular Diseases The emergence of a novel chemokine-like protein By Shirin Issa Bhaloo A thesis submitted to King’s College London for the degree of Doctor of Philosophy Cardiovascular Division Faculty of Life Sciences & Medicine King’s College London May 2017 0 In dedication to my Family 1 Acknowledgements I wish to express my sincere gratitude to Professor Qingbo Xu for giving me the opportunity to pursue a PhD degree in his laboratory at King’s College London. His support, trust, advice, motivation and encouragement throughout my PhD were invaluable and I will always take them with me. I would like to specially thank Dr. Alexandra Le Bras for her daily supervision, for all the knowledge, expertise and time she shared, for the motivation and support she gave and for her precious advice, not only for my student life but also for my daily life. I also wish to convey my appreciation to Dr. Yanhua Hu for her kind help and suggestions, particularly regarding animal work, and for her valuable support and encouragement during my PhD. I would like to thank Ms. Sherrie King for her indispensable help in so many aspects of my PhD journey and for her daily smile and deeply cherished support and friendship. Many thanks to Zhongyi Zhang for sharing his technical expertise and for his positivity and patience. I also wish to thank my colleagues Ms. Wenduo Gu (Wendy), Mr. Yao Xie, Dr. Xuechong Hong (Stella), Dr. Ka Hou Lao (Raymond), Dr. Ana Moraga, Ms. Peiyi Luo, Mr. Zhichao Ni and Dr. Witold Nowak, for all the memorable moments we shared, including so many dinners, adventures and trips, which I hope they continue for a long and fun time. Most importantly, I would like to thank my dear Parents and Sister, for always believing in me, supporting me and giving me so much love and courage. Finally, I wish to express my appreciation to the Marie Curie Initial Training Network Research Fellowship Program and the British Heart Foundation for funding my PhD project. 2 Declaration I, Shirin Issa Bhaloo, confirm that the work presented in this thesis is my own and I have been involved in the design, planning and conduct of all the experiments and the thesis writing. Expert assistance was provided in some aspects of the project by the following colleagues from the Cardiovascular Division of King’s College London. Dr. Yanhua Hu isolated vascular progenitor cells and smooth muscle cells from murine aorta. For the ex vivo experiments, Dr. Hu also isolated murine aorta vessels. The vector pShuttle-FLAG-HA was kindly provided by Dr. Lingfang Zeng. 3 Abstract Stem/Progenitor cells, such as Sca-1+ cells, are abundant in the vascular adventitia and they can differentiate into SMCs and ECs. These cells participate in the vascular wall remodeling observed in atherosclerosis, injury-induced neointimal hyperplasia and vein graft atherosclerosis. The detailed mechanisms of progenitor cell migration towards the intima have not been fully investigated. We hypothesize that Dkk3 plays an important role in resident Sca-1+ vascular progenitor cell migration. In this work, mouse vascular progenitor cells were isolated from the adventitia and sorted for the Sca-1 marker. Using transwell and wound healing assays, we showed that Dkk3 induced adventitia- derived Sca-1+ vascular progenitor cell (Sca-1+ APCs) migration in vitro. Additionally, the aortic ring assay demonstrated that Dkk3 was also able to induce Sca-1+ cell migration ex vivo. Analysis of the signaling pathways revealed that MAPK kinase signalling cascade, PI3K/AKT pathway and Rho- GTPases family of proteins were involved in the regulation of Dkk3-induced Sca-1+ progenitor cell migration. Our experiments also identified similarities in the migratory response of Sca-1+ cells to Dkk3 and Sdf-1α. Sdf-1α receptors are well established and they include CXCR4 and CXCR7. In contrast, the receptor for Dkk3 has not been identified yet. The current study aimed at identifying the receptor(s) involved in Dkk3-driven migration of Sca-1+ cells. Co-IP analysis and Affinity Binding assay revealed that Dkk3 binds to CXCR7. The downregulation of CXCR7 by SiRNA transfection reduced Dkk3-mediated Sca-1+ APC migration and the activation of the downstream signalling pathways. Overexpression of CXCR7, on the contrary, enhanced cell migration. In order to perform an exhaustive analysis of the potential binding partners of Dkk3 and identify additional receptors, we performed a Yeast Two Hybrid experiment. The results showed that integrin α5 (ITGα5) and integrin β1 (ITGβ1) interacted with Dkk3. Co-IP and Affinity Binding assays confirmed that Dkk3 bound to ITGα5 and ITGβ1. Furthermore, downregulation of these integrins by SiRNA transfection supressed the migration of Sca-1+ APCs triggered by Dkk3. Our work shows for the first time that Dkk3 acts as a chemokine-like protein able to induce the migration of resident Sca-1+ vascular progenitor cells. The pioneer identification of Dkk3 receptors and the elucidation of the signalling pathways involved in Sca-1+ APC migration could be of major interest for the development of novel drug-targeted therapies in vascular diseases. 4 Table of Contents Abstract ................................................................................................................................................... 4 Abbreviations ........................................................................................................................................ 10 List of Figures ........................................................................................................................................ 15 List of Tables ......................................................................................................................................... 19 Chapter 1: Introduction .................................................................................................................. 20 1.1 The Vascular System ............................................................................................................. 21 1.1.1 The evolution on the knowledge of the arterial adventitia .......................................... 22 1.2 Vascular progenitor cells ....................................................................................................... 23 1.2.1 Circulating vascular progenitor cells .................................................................................... 23 1.2.2 Resident vascular progenitor cells ....................................................................................... 25 1.2.3 Sca-1+ Vascular Progenitor Cells ................................................................................... 30 1.3 Atherosclerosis ...................................................................................................................... 31 1.3.1 Pathogenesis of Atherosclerosis ................................................................................... 31 1.3.2 Endothelial cells and Smooth muscle cells in atherosclerosis ...................................... 33 1.3.3 The role of vascular progenitor cells in atherosclerosis ............................................... 35 1.3.4 Treatment of atherosclerosis ........................................................................................ 38 1.3.5 Complications ................................................................................................................ 39 1.4 Cytokines in atherosclerosis ................................................................................................. 41 1.4.1 The Chemokine – Chemokine receptor System ............................................................ 44 1.5 Cell migration ........................................................................................................................ 57 1.5.1 Signalling pathways involved in cell migration ............................................................. 61 1.5.2 The role of integrins in cell migration ..........................................................................