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Characterising the in Vitro and in Vivo Function of the Rhog Effector

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Characterising the in Vitro and in Vivo Function of the Rhog Effector Characterising the in vitro and in vivo function of the RhoG effector DOCK4 during angiogenesis and ischemia. Leander Stewart BSc Submitted in accordance with the requirements for the degree of Doctor of Philosophy The University of Leeds Leeds Institute of Cancer and Pathology School of Medicine Sept 2019 ii The candidate confirms that the work submitted is her own and that appropriate credit has been given where reference has been made to the work of others. This copy has been supplied on the understanding that it is copyright material and that no quotation from the thesis may be published without proper acknowledgement. The right of Leander Stewart to be identified as Author of this work has been asserted by her in accordance with the Copyright, Designs and Patents Act 1988. © 2019 The University of Leeds and Leander Stewart iii Acknowledgements I would like to express gratitude to my supervisors Dr Georgia Mavria and Prof Stephen Wheatcroft for their continued support and guidance throughout my PhD. I would also like to thank the British Heart Foundation for the funding for my PhD studies. I offer my deepest gratitude towards Dr Fiona Errington-Mais, for her patience, sage counsel, and encouragement throughout the past 4 years. The support and mentorship of multiple research fellows, PhD students, and laboratory staff across many research groups has enabled me to accomplish the research found within this thesis; including the research groups of Prof Stephen Wheatcroft and Prof Richard Bayliss. My most sincere appreciation to Dr Adam Odell, who greatly aided in developing my initial research skills throughout my first year of my PhD, and Dr Chiara Galloni, for her guidance in research but also for her friendship. To Dr Nadira Yuldasheva, for carrying out all of the HLI surgical procedures, described within this thesis, and also for her invaluable teaching and advice. And to Dr Natalie North, who also mentored me throughout my in vivo research training. To the staff of the FBS protein production facility, Dr Brian Jackson and Laura Wilkinson-Hewitt, for not only providing me with instrumental guidance but also friendship and support throughout the most challenging time period of my PhD. I also wish to direct my deepest gratitude to my family for their patience, understanding, and emotional support throughout the past 4 years. Lastly to my son, Micah Stewart, who has provided me with the love and motivation to continue to persevere in my pursuit of personal and academic success. iv Abstract The RAC1 specific GEF, DOCK4, has been identified as an essential component in the Rho GTPase signalling pathway, imperative for correct vascular patterning and lumenisation during sprouting angiogenesis in vitro. As RAC1 has been previously implicated in the signalling events involved in vascular regrowth within a hypoxic environment, it was hypothesized that DOCK4 may be an important effector in the response to vascular injury and oxygen deprivation. To test this hypothesis, a DOCK4 depleted endothelial co-culture assay was carried out in both hypoxic and normoxic conditions. DOCK4 driven activation of RAC1 has been demonstrated under VEGF signalling, however FGF2 signalling pathways have also been strongly implicated in vascular response to blood vessel injury and hypoxia. Therefore, co-culture assays were carried out to assess sprouting angiogenesis with DOCK4 knockdown in response to FGF2 supplementation. Further, a heterozygous DOCK4 depleted murine model in ischemia studies using a model of HLI was employed together with LDI monitoring of vascular response and regrowth, comparing the response of heterozygous Dock4 KO mice and their WT littermate controls. DOCK4 interacts with the CDC42 GEF DOCK9 but the molecular basis of the interaction is unknown, as is the role of GEF heterodimerization in cell signalling. This study aims to further understand the function of DOCK4 within a pathological sprouting angiogenesis while also investigating the mechanism of interaction between DOCK4 and DOCK9. The two pro-angiogenic growth factors VEGFA and FGF2 drive different phenotypical growth responses during sprouting angiogenesis in vitro. DOCK4 was demonstrated as being an important component of FGF2 stimulated angiogenesis under hypoxia, indicating DOCK4 as important for mechanisms involved in the angiogenic response to ischemia. The specific v site of DOCK9 which interacts with the SH3 domain of DOCK4 was not elucidated during this study, however it was determined that DOCK9 proline rich regions identified as PRR 2, 3, 4, and 9 were unlikely to be involved in the interaction. The small molecule inhibitor QL-47 was demonstrated to be a potent anti-angiogenic compound with VEGFA stimulated ECs being particularly sensitive to QL-47. However, it is highly unlikely that the anti-angiogenic effects are due to disruption of the DOCK4-DOCK9 interaction, as the p.C628 cysteine residue was found to not be involved in DOCK4 SH3 domain interaction. Understanding how Rho GTPases are regulated and mechanisms underpinning their activity will progress the understanding of events that drive blood vessel growth while gaining insight into dysregulation during angiogenic pathologies. vi Table of Contents Acknowledgements ................................................................................................................. iii Abstract ............................................................................................................................. iv Thesis abbreviations ............................................................................................................. xiv Preface .................................................................................................................. xviii 1. Introduction ......................................................................................................................... 1 1.1 The mammalian vascular system ............................................................................. 1 1.2 Blood vessels ........................................................................................................... 3 1.3 Mechanisms of blood vessel growth ..................................................................... 11 1.4 The Rho family of small GTPases ........................................................................... 30 1.1 Rho GTPase signalling in blood vessel lumen formation ....................................... 45 1.5 Hypothesis ................................................................................................................... 61 1.6 Aims .................................................................................................................... 61 2 Materials and methods ....................................................................................................... 62 2.1 Primary cells and cell lines ..................................................................................... 62 2.2 Coating of tissue culture plates ............................................................................. 62 2.3 Cell culture conditions ........................................................................................... 63 2.4 Plasmids ................................................................................................................. 63 2.5 PCR clean-up .......................................................................................................... 67 2.6 Cloning ................................................................................................................... 67 2.7 Bacterial transformation and plasmid preparation ............................................... 67 vii 2.8 Production of pOPINF-DOCK4 SH3, DOCK9 PH-PCIP, DOCK9 PCIP-DHR1, and pOPIN3SC-DOCK9 plasmids in E.coli. ..................................................................................... 68 2.9 Restriction Enzyme Digestion ................................................................................ 69 2.10 Transformation of competent cells for protein expression ................................... 70 2.11 Bacterial culture lysis ............................................................................................. 71 2.12 Transfection of plasmid DNA ................................................................................. 71 2.13 Affinity chromatography ........................................................................................ 71 2.14 Size exclusion chromatography ............................................................................. 72 2.15 Lentiviral shRNA particle generation ..................................................................... 73 2.16 Lentiviral transduction ........................................................................................... 74 2.17 Cell lysis for Co-IP ................................................................................................... 74 2.18 Co-immunoprecipitation (Co-IP) assay ................................................................ 74 2.19 SDS polyacrylamide gel electrophoresis ................................................................ 75 2.20 Western blotting .................................................................................................... 75 2.21 Colorimetric quantification of Co-IP proteins ........................................................ 75 2.22 Stripping and blocking...........................................................................................
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