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Final Copy 2020 09 29 Dicks This electronic thesis or dissertation has been downloaded from Explore Bristol Research, http://research-information.bristol.ac.uk Author: Dickson, Emily J Title: The polarised secretion of the human umbilical vein endothelial eell General rights Access to the thesis is subject to the Creative Commons Attribution - NonCommercial-No Derivatives 4.0 International Public License. A copy of this may be found at https://creativecommons.org/licenses/by-nc-nd/4.0/legalcode This license sets out your rights and the restrictions that apply to your access to the thesis so it is important you read this before proceeding. Take down policy Some pages of this thesis may have been removed for copyright restrictions prior to having it been deposited in Explore Bristol Research. However, if you have discovered material within the thesis that you consider to be unlawful e.g. breaches of copyright (either yours or that of a third party) or any other law, including but not limited to those relating to patent, trademark, confidentiality, data protection, obscenity, defamation, libel, then please contact [email protected] and include the following information in your message: •Your contact details •Bibliographic details for the item, including a URL •An outline nature of the complaint Your claim will be investigated and, where appropriate, the item in question will be removed from public view as soon as possible. The Polarised Secretion of the Human Umbilical Vein Endothelial Cell Emily Dickson School of Biochemistry February 2020 Word Count: Thirty-two thousand, eight hundred and nineteen i Abstract Endothelial cells (ECs) are polarised, allowing them to secrete different proteins into both the circulating fluid, and the underlying matrix. However, there is little understanding of how this polarised secretion occurs. Using mass spectrometry, I have examined the role liprin-α1 plays in the secretion of proteins, which had previously been identified to be involved in the secretion of fibronectin from the basolateral surface. I have shown that liprin-α1 affects the polarised secretion of a subset of proteins, particularly those involved in the assembly of the extracellular matrix (ECM) such as collagen-α1 and α2. Previous studies have suggested that atorvastatin treatment has a positive pleiotropic effect upon ECs, reducing the risk of atherosclerosis. I have shown that atorvastatin treatment affected the secretion of certain proteins from both the apical and basolateral surface of the ECs. One major group of proteins affected were proteins involved in the ECM. Dysregulation of the ECM has previously been shown to be involved in the pathogenesis of atherosclerosis. Additionally, atorvastatin treatment increased the apical/basal secretion ratio of some proteins also known to be involved in the pathogenesis of atherosclerosis, including biglycan. However, atorvastatin also increased the apical secretion of protein- glutamine gamma-glutamyltransferase 2, which is known to decrease rupture of atherosclerotic plaques. Whilst my study provides a slight insight into the polarised secretion from ECs and the role atorvastatin treatment may have in patients with atherosclerosis, further research is needed to validate these results. ii Dedication and Acknowledgements Firstly, I would like to thank my supervisor Professor Harry Mellor for his assistance, guidance and patience throughout my project. I would also like to express my gratitude to Dr Ananthalakshmy Sundararaman and Dr Haoche Wei for their support throughout my project, for teaching me new techniques, and providing suggestions and insights to allow me to develop as an individual scientist. I would like to especially extend my thanks to Haoche for generously spending his time assisting me with my initial experiments, and Ananthalakshmy for her assistance during imaging. I would also like to acknowledge the Wolfson Bioimaging Facility at the University of Bristol for access to the microscopes used throughout my project. I would like to pay special regards to my family for supporting me throughout my entire study, and whom without none of this would be possible. They have given an enormous amount of time and dedication towards my education, and their encouragement has enabled to finish my study. I would like to thank my sister Anna for providing criticisms and feedback on my draft versions. I would like to thank my friends for their constant support, and the University of Bristol Swimming and Water Polo club for making my time at University unforgettable. iii Authors declaration I declare that the work in this dissertation was carried out in accordance with the requirements of the University's Regulations and Code of Practice for Research Degree Programmes and that it has not been submitted for any other academic award. Except where indicated by specific reference in the text, the work is the candidate's own work. Work done in collaboration with, or with the assistance of, others, is indicated as such. Any views expressed in the dissertation are those of the author. SIGNED: ............................................................. DATE:.......................... iv Page of Contents Abstract ..................................................................................................................... ii Dedication and Acknowledgements ...................................................................... iii Authors declaration ................................................................................................ iv Page of Contents ...................................................................................................... v List of Tables ......................................................................................................... viii List of Figures ......................................................................................................... ix Chapter 1 Endothelial cells ..................................................................................... 1 1.1 Endothelial Cell Functions ................................................................................. 2 1.1.1 Transport ..................................................................................................... 2 1.1.1.1 Glucose................................................................................................. 2 1.1.1.2 Amino acids .......................................................................................... 3 1.1.1.3 Albumin ................................................................................................ 4 1.1.2 Secretion..................................................................................................... 6 1.1.2.1 Extracellular matrix proteins ................................................................. 6 1.1.2.2 Coagulation factors ............................................................................... 8 1.1.2.3 Nitric oxide .......................................................................................... 10 1.1.2.4 Inflammatory mediators ...................................................................... 10 1.2 The Vascular System ...................................................................................... 12 1.2.1 The cardiovascular system ....................................................................... 12 1.2.1.1 Arteries ............................................................................................... 12 1.2.1.2 Microvasculature ................................................................................ 15 1.2.1.3 Veins .................................................................................................. 17 1.2.2 Lymphatic system ..................................................................................... 19 1.3 Endothelial cell subtypes................................................................................. 22 1.3.1 Specific differences between endothelial cells .......................................... 22 1.3.1.1 Continuous endothelium in the blood-brain-barrier ............................. 22 1.3.1.2 Discontinuous endothelium in the liver ............................................... 23 1.3.1.3 Fenestration endothelium in the kidney .............................................. 24 1.3.2 Mechanisms of endothelial cell heterogeneity .......................................... 25 1.4 Secretory Pathway .......................................................................................... 27 1.4.1 Epithelial secretion .................................................................................... 27 v 1.4.2 Extracellular vesicles ................................................................................ 31 1.4.2.1 Exosomes ........................................................................................... 31 1.4.2.2 Microvesicles ...................................................................................... 33 1.4.3 Endothelial cell secretion .......................................................................... 34 1.4.4 Polarised EC secretion ............................................................................. 37 1.5 Statins ............................................................................................................. 40 1.6 Aims of Project ................................................................................................ 41 Chapter 2 Materials and Methods ........................................................................
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