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Signalling Interactions Between Platelets and Lymphatic Endothelial SIGNALLING INTERACTIONS BETWEEN PLATELETS AND LYMPHATIC ENDOTHELIAL CELLS, LINKED TO LYMPHANGIOGENESIS By STACEY ANNE LANGAN A thesis submitted to the University of Birmingham for the degree of DOCTOR OF PHILOSOPHY Centre for Cardiovascular Sciences School of Clinical & Experimental Medicine College of Medical & Dental Sciences University of Birmingham October 2014 University of Birmingham Research Archive e-theses repository This unpublished thesis/dissertation is copyright of the author and/or third parties. The intellectual property rights of the author or third parties in respect of this work are as defined by The Copyright Designs and Patents Act 1988 or as modified by any successor legislation. Any use made of information contained in this thesis/dissertation must be in accordance with that legislation and must be properly acknowledged. Further distribution or reproduction in any format is prohibited without the permission of the copyright holder. ABSTRACT The platelet receptor CLEC-2 is the only known endogenous ligand for the transmembrane receptor podoplanin, which is expressed on lymphatic endothelial cells (LEC) as well as a number of other cell types. Both CLEC-2 and podoplanin are required for normal lymphangiogenesis as mouse embryos lacking either protein develop a phenotype in which blood is detected in the lymphatic vessels. This thesis examines the role of the podoplanin- CLEC-2 interaction in the migratory and tube-forming capabilities of LEC. Addition of platelets or antibody-mediated podoplanin crosslinking both inhibited migration of LEC in transfilter migration assays in the presence, but not absence, of vascular endothelial growth factor (VEGF)-C. Similarly, platelets and podoplanin crosslinking reduced stability of LEC networks formed in co-cultures with fibroblasts. We also found that siRNA- mediated knockdown of podoplanin negated the pro-migratory effects of VEGF-C and VEGF- A. Furthermore, we obtained evidence that podoplanin signalling may involve RhoA and Rho-kinase, and that the effect of podoplanin might be linked to its phosphorylation by protein kinase A downstream of VEGF receptor signalling. These data suggest that the interaction of podoplanin and CLEC-2 prevents connection between blood and lymphatic vessels through reductions in LEC migration and stability of cell-cell interactions. ACKNOWLEDGEMENTS The work described in this thesis was carried out in the School of Clinical and Experimental Medicine at the University of Birmingham and was funded by the Medical Research Council and British Heart Foundation. Firstly, I would like to thank my supervisors, Professor Gerard Nash and Professor Steve Watson, for their support and guidance throughout the course of my studies. In particular, thanks to Professor Nash for proof-reading end-of-year reports, conference abstracts and drafts of this thesis. Secondly, I would like to thank Dr Leyre Navarro-Núñez for advice on experimental design, originally teaching me how to perform some of the techniques used in this thesis, providing reagents and being a constant source of support and advice. I would also like to thank Dr Mike Tomlinson and Jasmeet Reyat for advice on tetraspanins and Dr Helen McGettrick for providing reagents. Thanks also to members of the Nash/Rainger and Watson groups that I have worked with over the last three years. In particular, thanks to Clare, Hafsa, Bonita, Jasmeet, Emma and Phil for making our office a great place to work. Finally, my thanks go to my family and friends. Thanks to my parents, Pat and Kevin, and my brother, Jamie, for your support and understanding. Thanks to Lou and Laura, for endless cups of tea and making me feel at home for the last 18 months. Lastly, Adam, thank you for your unfaltering support and your belief that I could achieve this. CONTENTS Chapter One: General Introduction……………………………………………………………………………………0 1.1 Overview…………………………………………………………………………………………………………………………1 1.2 The lymphatic system and lymphangiogenesis…………………………………………..…………………..1 1.2.1 Functions of the lymphatic system…………………………………………………………………………….1 1.2.2 Structure of the lymphatic system……………………………………………………………………………..2 1.2.3 Embryonic lymphatic development……………………………………………………………………………3 1.2.4 Lymphangiogenesis in adults……………………………………………………………………………………..7 1.3 Endothelial cells………………………………………………………………………………………………………………9 1.3.1 Functions of endothelial cells…………………………………………………………………………………….9 1.3.2 Differences between vascular and lymphatic endothelial cells………………………………...11 1.3.3 Cell migration…………………………………………………………………………………………………………...12 1.3.3.1 Focal adhesions…………………………………………………………………………………………..…13 1.3.3.2 Integrins………………………………………………………………………………………………………..15 1.3.3.3 RhoA signalling………………………………………………………………………………………………17 1.4 Vascular endothelial growth factors………………………………………………………………………………22 1.4.1 Expression of vascular endothelial growth factor receptors by endothelial cells………22 1.4.2 Signalling downstream of vascular endothelial growth factor receptors…………………..25 1.4.3 Role of VEGF-C and VEGFR3 in lymphangiogenesis……………………………………………………27 1.5 Platelets in lymphatic development………………………………………………………………………………..29 1.5.1 Platelet development………………………………………………………………………………………………..30 1.5.2 Functions of platelets………………………………………………………………………………………………..31 1.5.3 CLEC-2 and its signalling pathway………………………………………………………………………………35 1.5.4 Physiological roles of CLEC-2……………………………………………………………………………………..40 1.5.5 Platelets in lymphatic development and maintenance….……………………………………………42 1.6 Podoplanin…………………………………………………………………………………………………………………….…46 1.6.1 Structure of podoplanin……………………………………………………………………………………………..46 1.6.2 Functions of podoplanin…………………………………………………………………………………………….47 1.6.2.1 Podoplanin in lymphatic development…………………………………………………………..50 1.6.3 Signalling downstream of podoplanin………………………………………………………………………..52 1.7 Hypothesis and aims……………………………………………………………………………………………………….59 Chapter Two: Materials and Methods………………………………………………………………………………..61 2.1 Ethics statement………………………………………………………………………………………………………………62 2.2 Source of antibodies………………………………………………………………………………………………………..62 2.3 Cell culture………………………………………………………………………………………………………………………62 2.3.1 Source of cells…………………………………………………………………………………………………………….62 2.3.2 Lymphatic endothelial cells…………………………………………………………………………………………63 2.3.3 Vascular endothelial cells……………………………………………………………………………………………63 2.3.4 Human dermal fibroblasts…………………………………………………………………………………………..65 2.3.5 Detachment of cells from flasks………………………………………………………………………………….65 2.3.6 Freezing and thawing of LEC and HDF…………………………………………………………………………66 2.4 siRNA knockdown of podoplanin………………………………………………………………………………………66 2.4.1 Source of duplexes………………………………………………………………………………………………………66 2.4.2 Transfection of LEC with siRNA……………………………………………………………………………………68 2.5 Flow cytometry…………………………………………………………………………………………………………………68 2.5.1 Staining of LEC for podoplanin, VEGFR2, VEGFR3 or CD31………………………………………….68 2.6 Preparation of washed platelets and platelet microvesicles…………………………………………….71 2.6.1 Preparation of washed human platelets using prostacyclin…………………………………………71 2.6.2 Preparation of washed human platelets using theophylline…………………………………………71 2.6.3 Preparation of platelet releasate………………………………………………………………………………….72 2.6.4 Preparation of washed mouse platelets……………………………………………………………………….72 2.6.5 Preparation of human and mouse platelet microvesicles…………………………………………….73 2.6.5.1 Staining of microvesicles for CD41 and CLEC-2……………………………………………………….73 2.7 Transfilter migration assays………………………………………………………………………………………………74 2.7.1 Selection of VEGF-C concentration and assay timepoint………………………………………………74 2.7.2 Determination of platelet number and addition time…………………………………………………76 2.7.3 Determination of crosslinking strategy……………………………………………………………………….77 2.7.4 Transfilter assays with addition of inhibitors, growth factors or antibodies………………..78 2.8 Scratch wound assay………………………………………………………………………………………………………..83 2.8.1 Scratch wound assay with addition of platelets…………………………………………………………..83 2.8.2 Scratch wound assay with podoplanin crosslinking……………………………………………………..84 2.9 Formation of endothelial “tubes” in co-cultures with human dermal fibroblasts……………..86 2.9.1 Addition of platelets…………………………………………………………………………………………………….86 2.9.2 Addition of crosslinking antibodies………………………………………………………………………………87 2.9.3 Post-staining of co-cultures………………………………………………………………………………………….87 2.9.4 Analysis of co-culture experiments………………………………………………………………………………88 2.10 RhoA activation assay……………………………………………………………………………………………………..91 2.10.1 Timecourse of response to VEGF-C…………………………………………………………………………….91 2.10.2 Effect of crosslinking of podoplanin……………………………………………………………………………93 2.11 Co-immunoprecipitation and Western blotting to analyse binding partners of podoplanin……………………………………………………………………………………………………………………………..93 2.11.1 Surface biotinylation of LEC and co-immunoprecipitation…………………………………………93 2.11.2 Western blot: Odyssey exposure………………………………………………………………………………..94 2.12 Statistical analysis……………………………………………………………………………………………………………96 Chapter Three: The effect of the podoplanin-CLEC-2 interaction on lymphatic endothelial cell migration…………………………………………………………………………………………………………………………..97 3.1 Introduction……………………………………………………………………………………………………………………..98 3.2 Results………………………………………………………………………………………………………………………………99 3.2.1 The effect of platelets on migration of LEC, HMEC-1 and HUVEC………………………………..99 3.2.2 The effect of siRNA transfection on surface expression of podoplanin……………………..105 3.2.3 The effect of podoplanin
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