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Role of Transcription Factor C-Jun in Acute Inflammation and Intimal Thickening in Bypassed Vein Grafts: Insights Using Dnazymes

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Role of Transcription Factor C-Jun in Acute Inflammation and Intimal Thickening in Bypassed Vein Grafts: Insights Using Dnazymes Role of transcription factor c-Jun in acute inflammation and intimal thickening in bypassed vein grafts: insights using DNAzymes Alla Waldman, MD Centre for Vascular Research Department of Pathology, The University of New South Wales Submitted for the degree of Doctor of Philosophy (PhD) Thesis Outline 2 TABLE OF CONTENTS ACKNOWLEDGMENT 3 ABSTRACT 4 PUBLICATIONS, PRESENTATIONS, AWARDS 12 ABBREVIATIONS 13 THESIS OUTLINE Chapters 1-3: INTRODUCTION 16 Chapters 4-6: RESULTS AND METHODS 90 Chapter 7: CONCLUSIONS AND FUTURE DIRECTIONS 163 REFERENCES 170 Thesis Outline 3 Acknowledgement I would like to thank my supervisor Professor Levon Khachigian for his patience, guidance, encouragement and continuous support during my PhD. I was very privileged to be part of his research group and to work with many very talented and successful scientists. I am very grateful to Professor Michael Perry, my co-supervisor for his teaching and support. His help in setting up microcirculation studies was invaluable and is greatly appreciated! I would like to thank my colleagues in Levon's lab, in particular Roger Fahmy for his extraordinary teaching, help and support; Dr Ravinay Bhindi for his valuable advice on my animal projects and willingness to help and Dr Valerie Midgley for her help in teaching, her friendship and good humour we shared so many times during the last 3 years. I would like to thank my amazing family, my parents Alex and Raissa Valdman and my brother Michael for always being there for me, for giving me the best opportunities in life to become who I am today. To my husband Joseph and my son Boris, I would have never made it without their unconditional support of my career, your love and friendship. And for my little son Antoine who was born in April 2006-you made it harder for me to finish my thesis on time but you are the most amazing thing in my life and we are blessed to have you! To my best friend Dr Vicky Sundakov thank you for your love, understanding and amazing friendship! Thesis Outline 4 Abstract Atherosclerosis is a key pathological process underlying the development and progression of three major diseases of the vascular system- coronary artery disease, cerebro-vascular and peripheral vascular disease. Chronic vascular wall inflammation is considered as a principal cause in the initiation and progression of atherosclerosis. Intimal thickening that develops in arteries and veins as an adaptive response to an injury has many similarities with atherosclerosis, but at the same time represents a unique pathological entity. This Thesis explores the utility of applying a novel DNAzyme-based approach that targets "master-regulator" transcription factors c-Jun and Egr-1 to in viva and in vitro models of acute inflammation and intimal thickening. Studies included in this Thesis reveal that transcription factor c-Jun plays a key regulatory role in controlling leucocyte movement during an acute inflammation induced by IL-113 through regulation of the expression of adhesion molecules ICAM, VCAM-1, E-selectin and VE-cadherin. Similarly, by applying EDS, a DNAzyme that targets transcription factor Egr-1 to the rat model of mesenteric microcirculation I demonstrate that Egr-1 controls leucocyte movement during an acute inflammation as evidenced by almost complete inhibition of leucocyte flux, adhesion and extravasation by EDS. The rabbit model of bypass grafting shows that Dz13 (a DNAzyme targeting transcription factor c-Jun) significantly reduces intimal Thesis Outline 5 thickening in bypassed vein grafts of chow-fed animals at 28 days in viva and in culture-grown human saphenous veins in vitro. Taken together these findings suggest that a DNAzyme based approach of targeting transcription factor c-Jun has the potential to be used as a modulator of the acute inflammatory response and of intimal thickening formation. Further work needs to be done before this technology is ready for clinical use in humans. Thesis Outline 6 Chapter 1: Atherosclerosis and Inflammation 1.1 Overview of atherosclerosis 16 1.1.2 Introduction 16 1.1.3 Definition of atherosclerosis 16 1.1.4 Definition and causes of endothelial dysfunction 17 1.1.5 Stages of atherosclerotic lesion development 18 1.1.6 Role of SMC in lesion formation in atherosclerosis and 19 intimal thickening 1.2 Stages and mechanisms of leucocytes recruitment during inflammation 24 1.2.1 Introduction 24 1.2.2 Selectins 25 1.2 .3 Adhesion Molecules /CAM-] and VCAM-1 30 1.2 .3 .1 Overview of structure and function 30 1.2 .3 .2 Integrins as ligands for adhesion molecules 33 1.2.3.3 Chemokine-integrin signalling and its role in 35 leucocyte adhesion and transmigration 1.2.4 Control of leucocyte transmigration 37 1.3 Role of chemokines and adhesion molecules in atherosclerosis 43 1.4 Role of IL-1 in inflammation and atherosclerosis 46 1.5 lntavital microscopy as tool for studying inflammation in vivo 48 Thesis Outline 7 Chapter 2: Transcription factors c-Jun and Egr-1 2.1 Biological function of AP-1 and c-Jun 51 2.1 .1 Genes activated by c-Jun in response to vascular 52 injury/inflammation 2.2 Egr-1 and atherosclerosis 54 2.2.1 Role of Egr-1 in regulation of pro-inflammatory and 54 pro-thrombotic events relevant to inflammation 2.3 Targeting c-Jun and Egr-1 with novel molecular approaches 57 2.3.1 DNAzymes 57 2.3.2 siRNAs 60 2.3.3 Antisense oligodeoxynucleotides 62 2.4 Transfection agents for the delivery of DNA therapeutics 63 Chapter 3: Coronary artery bypass surgery 3.1 Introduction 67 3.2 Mechanisms of bypass graft failure 68 3.3 Animal models of bypass grafting 69 3.3.1 Main advantages and disadvantages of 69 commonly used models 3.3.2 Morphological features of vein grafts from 71 hypercholesterolemic versus normocholesterolemic animals Thesis Outline 8 3.4 Gene based approach and molecular targets for the treatment 72 of vein graft intimal thickening 3.4.1 Endothelial injury, ischaemialreperfusion and oxidative stress 76 3.4.2 Role of coagulation cascade 78 3.4.3 Inflammation and adhesion molecules 79 3.4.4 Cytokines, SMC mitogens and regulators of cell proliferation 81 3.4.5 Matrix remodelling 82 3.4.6 Adventitia and perivascular fibroblasts 83 3.5 Therapeutic interventions for reducing graft failure rate in humans 84 3.6 Conclusion 87 3.7 Hypothesis and approaches 88 Chapter 4: Materials and Methods 4.1 Introduction 90 4.2 Cell culture, DNAzyme and siRNA synthesis, and transfection 90 for in vitro experiments 4.3 DNAzymes targeting transcription factors c-Jun and Egr-1 92 in models of acute inflammation and intimal thickening in viva 4.3.1 Rat model of mesenteric microcirculation 92 4.3.1.1 Surgical procedure and intravital microscopy 92 4.3.1 .2 Transfection with DNAzymes and 94 delivery of FITC-labelled Dzl 3scr 4.3.1.3 Tissue harvesting 96 Thesis Outline 9 4.3.2 Rabbit model of bypass grafting 96 4.3.2.1 Surgical procedure and ex-viva transfection 96 with DNAzyme 4.3.2.2 Harvesting of bypassed vein grafts 98 4.3.2.3 Histomorphometric analysis of vein grafts 99 4.4 lmmunohistochemistry 100 4.5 Human saphenous vein culture, transfection with DNAzymes and 103 histomorphometric analysis 4.6 Western blot 104 4.7 Co-culture model of inflammation 107 Chapter 5: DNAzymes targeting transcription factors c-Jun and Egr-1 as novel anti-inflammatory agents in model systems 5.1 Introduction and Aim 109 5.2 Results 110 5.2.1 Dzf 3 and EDS abolish inflammation in rat mesenteric 110 venules 5.2.2 FITC labelled Dzl 3scr transfection in viva 112 5.2.3 Leucocyte rolling velocity in viva 113 5.2.4 Dzf 3 and c-Jun siRNA inhibit adhesion ofmonocytes to the 119 Thesis Outline 10 endothelial cells in a co-ulture model of inflammation in vitro 5 .2 .5 Dzl 3 inhibits the expression of c-Jun and multiple 124 proinflammatory genes in JL-1 /3 stimulated endothelial cells in vitro 5.2.6 Dzl3 inhibits expression of c-Jun and other genes 124 in the rat mesenteric venules in vivo 5.3 Discussion 131 Chapter 6: DNAzyme targeting c-Jun inhibits intimal thickening in a rabbit model of bypass surgery and in human saphenous veins in vitro 6.1 Introduction and Aim 140 6.2 Results 142 6.2.1 Dzl3 but not Dzl3scr inhibits neointima development 142 in human saphenous veins in vitro 6.2.2 Dzl3 but not Dzl3scr inhibits neointimaformation 145 in cholesterol-fed rabbits at 21 days post surgery 6.2.3 Dzl3 but not Dzl3scr inhibits the development of 145 neointima in bypassed vein grafts of normal chow-fed rabbits 6.2.4. lmmunohistochemistry for SMC in 4 week-old rabbit 148 vein grafts Thesis Outline 11 6.2.5 Dzl 3 suppresses MMP-2 expression in 4 week-old 148 rabbit vein grafts by immunohistochemistry 6.2.6 Expression of RAM-I I in 4 week-old rabbit vein 148 grafts by immunohistochemistry 6.3 Study limitations 154 6.4 Discussion 155 6.5 Overview of technical difficulties faced by the candidate 161 during the candidature Chapter 7: Conclusions and Future Directions 7 .1 Overview of the major findings 163 7.2 Future directions 163 7.2.1 Explore the potential role for the local application of 163 Dzl 3 in inflammatory conditions such as rheumatoid arthritis 7.2 .2 Determine the applicability of Dzl 3 for the treatment of 166 neointimal hyperplasia through more animal and in vitro experiments 7.2.3 Explore the utility of targeting of transcription factor Egr-1 by 168 DNAzymes in the setting of neointimal formation 7.2 .4 Explore the potential for the use of c-Jun and Egr-1 siRNA 168 as anti-inflammatory and anti-proliferative agents References 170 Thesis Outline 12 Publications, presentations and awards arising from this Thesis Fahmy RG, Waldman A, Zhang A, Mitchell A, Tedla N, Cai H, Geczy C, Chesterman C, Perry M and Khachigian L (2006) "Suppression of vascular permeability and inflammation by targeting of the transcription factor c-Jun." Nat Biotechnol 24(7): 856-63.
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