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C-Jun N-Terminal Kinase Primes Endothelial Cells at Atheroprone Sites for Apoptosis

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C-Jun N-Terminal Kinase Primes Endothelial Cells at Atheroprone Sites for Apoptosis c-Jun N-terminal kinase primes endothelial cells at atheroprone sites for apoptosis Hera Ashraf Chaudhury Cardiovascular Science Unit, Imperial College London PhD ~ 1 ~ ACKNOWLEDGEMENTS Firstly, I would like to thank my supervisor Dr Paul Evans for his invaluable time, support and guidance throughout the course of my PhD. I would also like to thank him for his help and understanding during the writing and submission of this thesis. I would also like to thank everyone in the Cardiovascular Medicine Unit for providing an excellent and enjoyable environment for a novice scientist to achieve this level of credibility. I would particularly like to express my gratitude to Dr Graeme M Birdsey and Dr Joseph J. Boyle for their technical advice and especially to Mr Mustafa Zakkar for all his contribution; it has been a real pleasure to work with you. An extra special thanks to Danuta, Jay, Karine, Gunhild, Le, Shahir, Simon and Kim who made the bad days easier to deal with - thanks for all your support. I also wish to acknowledge Adam Platt (UCB Celltech, Cambridge, UK) for kindly providing the small molecule inhibitors of JNK and p38, Dr Jayne Dennis (Microarray Centre, Hammersmith Hospital, UK) for her invaluable assistance with the microarray set-up, Professor Roger Davis, (University of Massachusetts) for providing JNK1 knockout mice and Dr Andrew Clark, (Imperial College London) for supplying MKP-1 knockout mice. Finally, I would like to thank my parents, my siblings; Haseeb and Nayab and the entire family for all their love and support during the course of the PhD, especially my mother for all the late night drives to the lab to set-up extremely important experiments and Faisal for his understanding during the thesis writing stages. ~ 2 ~ DECLARATION This thesis comprises results produced by myself or with the help of members from the laboratory. It also contains illustrations from other laboratories which are stated in the figure legends. This work has been carried out at the Cardiovascular Science Unit, Imperial College London. ~ 3 ~ ABSTRACT Atherosclerosis can be initiated by pro-inflammatory activation of endothelial cells (EC) which leads to the recruitment of leukocytes to the vessel wall, and also by endothelial apoptosis which elevates the permeability of arteries to lipoproteins. The greater curvature of the aorta is exposed to high shear and is protected from EC apoptosis, inflammation and atherosclerosis, whereas the lesser curvature is exposed to low shear and is susceptible to atherosclerosis. Pro-inflammatory mediators (e.g. TNFα, LPS) trigger phosphorylation of c- Jun N-terminal kinase (JNK) and p38 MAP kinases to positively regulate these process and influence atherosclerosis. I examined the effects of JNK activation on EC physiology at atherosusceptible sites. En face staining revealed that phosphorylation of JNK in EC occurs constitutively at the susceptible site of the murine aortic arch and can be enhanced by LPS treatment. In contrast, JNK activation was suppressed at the protected site by mitogen-activated protein kinase phosphatase-1 (MKP-1), a negative regulator of JNK and p38 MAP kinases. To study the function of JNK and p38 in vascular endothelium, I identified the transcriptional programs that they regulate by applying specific pharmacological inhibitors to cultured EC and assessing the transcriptome using microarrays. Functional annotation revealed that JNK and p38 positively regulate the expression of numerous pro-inflammatory and pro-apoptotic molecules. Subsequent gene silencing studies demonstrated that JNK1 positively regulates pro-apoptotic molecule expression in EC. I validated my findings in vivo by analyzing EC in aortae of wild-type, JNK1-/- and MKP-1-/- mice. I observed that EC at an atherosusceptible site express pro-apoptotic proteins and are primed for apoptosis and proliferation in response to LPS through a JNK1-dependent mechanism, whereas EC at a protected site expressed lower levels of pro-apoptotic molecules and was protected from injury by MKP-1. These findings indicate that the spatial variation of JNK1 activity delineates the spatial distribution of apoptosis and turnover of EC in arteries, and may influence the punctuate development of atherosclerotic lesions. ~ 4 ~ TABLE OF CONTENTS C-JUN N-TERMINAL KINASE PRIMES ENDOTHELIAL CELLS AT ATHEROPRONE SITES FOR APOPTOSIS ....................................................................... 1 ACKNOWLEDGEMENTS .................................................................................................... 2 DECLARATION...................................................................................................................... 3 ABSTRACT .............................................................................................................................. 4 TABLE OF CONTENTS ........................................................................................................ 5 LIST OF FIGURES ............................................................................................................... 10 LIST OF TABLES ................................................................................................................. 13 CHAPTER 1: INTRODUCTION ......................................................................................... 14 INFLAMMATION............................................................................................................... 15 Activation of endothelium during inflammation ............................................................... 15 Tumor necrosis factor (TNFα).......................................................................................... 18 Lipopolysaccharide (LPS) ................................................................................................ 20 Inflammation-related signaling pathways ........................................................................ 22 NF-κB ........................................................................................................................... 22 Mitogen activated protein kinases (MAPK) ................................................................. 24 p38 ............................................................................................................................. 24 JNK ........................................................................................................................... 26 CELL DEATH ..................................................................................................................... 28 Apoptosis .......................................................................................................................... 31 Caspases ........................................................................................................................... 32 Pro-apoptotic signaling .................................................................................................... 33 Extrinsic pathway.......................................................................................................... 35 Intrinsic pathway ........................................................................................................... 36 Role of JNK in apoptosis .................................................................................................. 37 ATHEROSCLEROSIS ........................................................................................................ 40 ~ 5 ~ Inflammation in atherosclerosis ....................................................................................... 42 Apoptosis in atherosclerosis ............................................................................................. 43 EC Apoptosis in atherosclerosis ....................................................................................... 45 JNK signaling in atherosclerosis ...................................................................................... 46 SHEAR STRESS ................................................................................................................ 47 Shear stress regulates atherosclerosis ............................................................................. 50 Effects of shear stress on EC activation and inflammation .............................................. 51 Effects of shear stress on EC apoptosis ............................................................................ 54 HYPOTHESIS AND AIMS ......................................................................................................... 57 CHAPTER 2: MATERIALS AND METHODS ................................................................. 58 REAGENTS ......................................................................................................................... 59 Tissue culture .................................................................................................................... 59 Cytokines and Pharmacological inhibitors ...................................................................... 59 Antibodies ......................................................................................................................... 59 CELL CULTURE ................................................................................................................. 63 Harvesting and culture of primary human umbilical vein endothelial cells (HUVEC) ... 63 Harvesting and culture of porcine aortic endothelial cells (PAEC) ................................ 63 Maintenance of primary cells ..........................................................................................
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