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.
Alla Waldman, Roger G Fahmy and Levon M. Khachigian
"DNAzymes targeting c-Jun inhibit inflammation and downregulate endothelial ICAM-
1, VCAM-1 and E-selectin expression"
Abstract presented orally at American Heart Association meeting in Dallas, Texas in
November 2005.
Alla Waldman, Roger G. Fahmy and Levon M. Khachigian
"Role of Transcription Factor c-Jun in Inflammation; DNAzyme Based Approach"
Poster presented at the CSANZ 53 Annual Scientific Meeting in Perth Australia, August
2005.
Travel award from Cardiac Society of Australia and New Zealand to attend and present the poster at the 53 CSANZ Annual Scientific Meeting in Perth, August 2005. Thesis Outline 13
Abbreviations
AMI acute myocardial infarct AP-1 activating protein-I APC activated protein C AS-ODN antisense oligodeoxynucleotides 13 beta CAD coronary artery disease CAIA collagen antibody-induced arthritis COPD chronic obstructive pulmonary disease CR consensus repeat CVD cardio-vascular disease DDT ditholthreitol DNA deoxyribonucleic acid DOTAP 1,2-dioleoyl-3-trimethylammonium propane EAE experimental autoimmune encephalomyelitis EC endothelial cells Egr-1 early-growth response-I FBS foetal bovine serum FGF-2 fibroblast growth factor-2 GlyCAM-1 glycosylation dependent cell adhesion molecule-I HEY high endothelial venules HMEC-I human microvascular endothelial cells- I HUVEC human umbilical vascular endothelial cells h hours IBD inflammatory bowel disease ICAM-1 intracellular adhesion molecule-I IGF insulin-like growth factor IH initimal hyperplasia Thesis Outline 14
IL-lP interleukin 1 beta IL-lra interleukin 1 receptor antagonist IP3 inositol 1,4,5, triphosphate IV intravenous IVM intravital microscopy JAB-1 jun activation domain-binding protein 1 JAM junctional adhesion molecule JNK c-Jun N-terminal kinase LDL low density lipoprotein LDLR low density lipoprotein receptor LFA-1 leucocyte function-associated antigen- I LNA locked nucleic acid M molar MAC-1 CD1lb/CD18 beta-2 integrin MadCAM mucosal address in cell adhesion molecule- I MAPK mitogen activating protein kinase MCP-1 macrophage chemotactic protein- I mg milligram min minute µl microliters ml millilitres MMP matrix metalloproteinase mRNA messenger RNA NI neointima NIH neointimal hyperplasia NF-kappaB nuclear factor kappa B NO nitric oxide eNOS endothelial nitric oxide iNOS inducible nitric oxide OxLDL oxidised LDL Thesis Outline 15
PBS phosphate buffered saline PDGF platelet-derived growth factor PECAM-1 platelet endothelial adhesion molecule-I PI3 phosphatidylinositol 3-kinase PIV parainfluenza virus PMN polymorphonuclear leucocyte PMSF phenulmethylsulfonyl fluoride PND peripheral node aggressins PSGL-1 P-selectin glycoprotein ligand- I RA rheumatoid arthritis RNA ribonucleic acid ROS reactive oxygen species rpm revolutions per minute RSV respiratory syncytial virus RT room temperature SDF-1 stromal cell-derived factor-I siRNAs small interfering RNAs SMC smooth muscle cell SOD-I superoxide dismutase-1 TBS tris-buffered saline THP-1 human acute monocytic leukaemia cell line TEM transendothelial migration TGF-f3 transforming growth factor-beta TIMP tissue inhibitor of matrix metalloproteinase TM thrombomodulin TNF-a tumour necrosis factor alpha VCAM-1 vascular cell adhesion molecule-I VE-cadherin vascular endothelial cadherin VLA-4 very late antigen-4 UAP unstable angina pectoris Chapter 1: Atherosclerosis and Inflammation 16
Chapter 1: Atherosclerosis and Inflammation
1.1 Overview of atherosclerosis
1.1.2 Introduction
Cardiovascular mortality has been the leading cause of death in Australia and other industrialized countries for over 40 years. Moreover, in developing countries the burden of cardiovascular morbidity and mortality is rapidly increasing making this condition one of the most important health issue for the 21 century. The rise in the rates of cardiovascular disease (CVD) is linked to the problems faced by modern societies such as obesity, sedentary life style, smoking, hypertension and diabetes
(Yusuf et al. 2001). In 2004 CVD accounted for 36% of all Australian deaths. The financial burden of CVD in 2002 was $7.8 billion or 12% of all Australian health costs for the same year, making it the most expensive medical condition for our country (Australian Bureau of Statistics www .abs.gov .au/ A USST A TS
Cardiovascular Disease in Australia: A Snapshot, 2004-05 (Cat. No. 4821.0.55.001)).
1.1.3 Definition of atherosclerosis
Cardiovascular disease is principally related to atherosclerosis - a chronic inflammatory, fibro-proliferative condition of medium and large size vessels (Ross
1999). Four major pathological processes are known to contribute to the plaque development: 1) proliferation of smooth muscle cells (SMC), macrophages and lymphocytes; 2) connective tissue matrix accumulation of elastic fibre, collagen and proteoglycans; 3) build up of free and modified cholesterol within the matrix and the Chapter 1: Atherosclerosis and Inflammation 17
associated cells, and 4) cell death (apoptosis) leading to the formation of a necrotic core (Ross 1999). In order to understand the contribution of specific cell types to the atherosclerotic process it is important to consider the overall structure of the affected blood vessels. Anatomically a normal artery consists of three major layers- the intima, the innermost layer of the vessel, made of monolayers of endothelial cells
(EC) and supporting extracellular connective tissue, the media or the middle layer, consisting of smooth muscle cells (SMC) and the outer layer also known as adventitia, composed of connective tissue, some fibroblasts and SMC. The following chapter will concentrate on the roles played by EC and SMC in the development of atherosclerosis.
1.1.4 Definition and causes of endothelial dysfunction
The endothelial layer or endothelium is an innermost layer of the blood vessel with a diverse range of metabolic functions involved in regulation of vessel permeability, thromboginecity, inflammation and vascular tone. The "response to injury" hypothesis proposed more than 30 years ago by Ross and Glomset, regards endothelial dysfunction as the main cause for the development of atherosclerosis
(Ross and Glomset 1976). Endothelial dysfunction arises from a complex interplay between traditional risk factors for CVD (hypertension, smoking, diabetes, elevated native and oxidised LDL, raised triglycerides and homocystine levels, genetic predisposition) and environmental toxins. Characteristically, endothelial dysfunction is associated with a decreased nitric oxide production, local oxidation of circulating Chapter 1: Atherosclerosis and Inflammation 18
lipoproteins and recruitment of circulating blood cells and lipids into the vessel wall
(Davignon and Ganz 2004). The entrance of monocytes and T-lymphocytes into the intima of blood vessel is largely controlled by adhesion molecules expressed on the surface of activated EC. This expression is tightly regulated by locally secreted cytokines, chemokines and growth factors. Once in the intimal layer, monocytes transform into macrophages, ingest lipids and become foam cells that together with lymphocytes and SMC form the earliest lesion known as "fatty streaks" (Ross 1993).
"Fatty streaks" are ubiquitous in humans and already apparent during the first decade of life. Depending on the balance of pro-atherogenic versus anti-atherogenic factors the "fatty streaks" either disappear or progress to more advanced lesions called fibro fatty atheroma and eventually to fibrous plaque. The progression from early to more advanced lesions is marked by accumulation of extracellular lipids, SMC proliferation, deposition of fibrous tissue, focal necrosis and eventually formation of a fibrous cap that overlies a core of lipids and necrotic tissue (Ross 1993).
1.1.5 Stages of atherosclerotic lesion development
Histopathological classification proposed by Stary et al in 1995 describes 6 stages of atherosclerotic lesion development (Stary et al. 1995). Type I, II and III lesions represent early stages of atherosclerosis. They do not cause luminal obstruction and are found almost exclusively in children and young adults. Type I lesion corresponds to the earliest change in intima and contains macrophages and foam cells; type II lesions are made of layers of macrophage foam cells and lipid-laden smooth muscle Chapter 1: Atherosclerosis and Inflammation 19
cells. They represent "fatty streaks", the first grossly visible lesions. Type III lesions are viewed as a bridge between early and advanced atherosclerotic plaques. They contain small amount of extracellular lipids in addition to all components of type II lesions. Advanced lesions are collectively known as type IV, V and VI. In type IV lesions there is an extensive accumulation of extracellular lipids forming a lipid core without a significant increase in fibrous tissue. Typically, type IV lesions produce an eccentric thickening of the artery without lumen narrowing. Type V lesions in addition to the features of type IV lesions, display a prominent fibrous tissue formation, increased vascularity, increased levels of calcium and are made of different cellular layers reflecting the ongoing cycle of damage and repair. Type VI lesions have the morphology of either type IV or type V lesions accompanied by one of the following: surface disruption, haematoma and thrombosis. Plaque disruption with associated acute thrombosis that manifests as unstable angina pectoris, acute myocardial infarction and other acute syndromes, is typically associated with type V and VI lesions (Fig 1.1).
1.1.6 Role of SMC in lesion formation in atherosclerosis and intimal thickening
Another component of the vascular wall essential for the development of atherosclerosis is the smooth muscle cell (SMC). SMCs play an essential role in the development of atherosclerosis and make a significant contribution to the volume of Figure 1.1: Participation of inflammation in all stages of atherosclerosis
A. When the endothelial monolayer becomes inflamed, it expresses adhesion
molecules that bind cognate ligands on leucocytes. Selectins are primarily
responsible for the rolling while integrins mediate firmer adhesion.
Proinflammatory cytokines expressed within atheroma provide a chemotactic
stimulus to the adherent leucocytes directing their migration into the intima.
Inflammatory mediators can augment the expression of macrophage
scavenger receptors leading to uptake of modified lipoprotein particles and the
formation of lipid-laden macrophages.
B. T lymphocytes join macrophages during lesion evolution. Together with
resident vascular wall cells they secrete cytokines and growth factors that
promote the migration and proliferation of SMC. Medial SMC secrete
enzymes that can degrade elastin and collagen in response to inflammatory
stimulation. Degradation of the extracellular matrix allows penetration of
SMC through the elastic lamina and the matrix of the growing plaque.
C. Inflammatory mediators can augment the expression of collagenases by foam
cells within the lesion. These alterations thin and weaken the fibrous cap of
the plaque making it susceptible to rupture. Cross-talk between T
lymphocytes and macrophages heightens the expression of tissue factor a
potent procoagulant. Thus when the fibrous cap ruptures tissue factor triggers
thrombus formation which as a principal cause of most acute complications of
atherosclerosis (Adapted from Peter Libby, Paul M.Ridker and Attilio Maseri
"Inflammation and Atherosclerosis" Circulation 2002 (105): 1135-1143). Chapter 1: Atherosclerosis and Inflammation 20
A
B
C Chapter 1: Atherosclerosis and Inflammation 21
atherosclerotic plaque. They are the major source of growth factors and cytokines such as platelet derived growth factors (PDGF), transforming growth factor-f3 (TGF f3), fibroblast growth factor 2 (FGF-2), tumour necrosis factor alpha (TNF-a) and interleukin 1 beta (IL-1 f3) that act as potent mitogens for SMC and other cells within the vascular wall (Casscells 1991). One of the characteristic features of SMCs is their ability to multiply and migrate in response to mechanical injury, shear stress, growth factors and cytokines (Irvine et al. 2000). This proliferative response of SMCs is a hallmark of a number of pathological and physiological conditions including atherosclerosis (Lindner et al. 1992), restenosis (Irvine et al. 2000), wound healing and repair (Ross et al. 1986), and growth and development (Betsholtz et al. 2001).
Among SMC mitogens PDGF, basic fibroblast growth factor (bFGF) and TGF-f3 are considered to play major roles. PDGF is produced by activated platelets and macrophages and stimulates SMC proliferation and migration in atherosclerosis
(Ross 1995) and arterial injury models (Reidy et al. 1992). The importance of PDGF signalling in SMC in the context of atherosclerosis is underscored by the studies that used antibodies against PDGFa- and f3- receptors in Apo E-/- deficient mice. In a study by Sano et al a 67% reduction in atherosclerotic lesion size and 80% reduction in SMC infiltration of the neointima was reported following the blockade of PDGF-f3 receptors (Sano et al. 2001). TGF-f3 promotes SMCs differentiation in cell culture
(Hautmann et al. 1997), however, the exact role played by TGF-f3 in atherosclerosis in vivo is not absolutely clear. Levels of TGF-f3 rapidly increase within 6-24 h in experimental balloon injury models (Majesky et al. 1991). Interestingly, the Chapter 1: Atherosclerosis and Inflammation 22
overexpression of TGF-13 increases neointima formation, matrix deposition and smooth muscle cell proliferation in several balloon injury models (Schulick et al.
1998; Smith et al. 1999). On the other hand, analysis of human plaques suggested that
TGF-13 signalling may also have a protective effect in the context of atherosclerotic lesion formation (McCaffrey et al. 1999).This observation is supported by studies in
ApoE-/- mice indicating that TGF-13 is critically important for SMC matrix production and the development of a stable fibrotic plaque (Mallat et al. 2001;
Lutgens et al. 2002). FGF-2 is a potent mitogen for vascular SMCs and endothelial cells. Raised levels of FGF-2 are found through all stages of the plaque development
(Hughes et al. 1993). It is released immediately upon injury to the SMC and returns to normal level within few hours. Futhermore, FGF receptors are upregulated in SMC following an injury. In vivo, FGF-2 significantly increases SMC proliferation following balloon catheter denudation in the rat (Lindner et al. 1991 ). Interestingly, injection of FGF-2 neutralizing antibody dramatically decreased SMC proliferation but did not alter the development of intimal thickening at 8 days (Lindner and Reidy
1991). It has also been suggested that in an injured artery FGF-2 promotes endothelial repair (Lindner et al. 1990) and stimulates early but not chronic SMC proliferation.
The proangiogenic ability of FGF-2 means it could be involved in the neovascularization of advanced plaque (Hughes et al. 1993).
SM Cs of the arterial wall are biologically heterogenous. In the normal artery medial
SMCs display a contractile phenotype, characterized by the presence of spindle shape Chapter 1: Atherosclerosis and Inflammation 23
cells that respond to agents controlling vascular tone (Ross 1995) . On the other hand, the synthetic phenotype found in developing and diseased arteries, exhibits an epithelioid shape, enhanced proliferative, migratory and proteolytic activity and increased sensitivity to apoptotic stimuli compared to the contractile phenotype (Hao et al. 2003). Moreover, synthetic SMCs demonstrate an altered ability to metabolise low-density lipoproteins thus contributing to atherogenesis. This phenotype is a hallmark of SMC in atherosclerosis, restenosis and possibly intimal thickening of vein grafts (Orlandi et al. 2006). The switch from contractile to synthetic type occurs in response to growth factors and cytokines produced by inflammatory cells recruited into the vessel wall. As a result of this change SM Cs acquire the ability to synthesize a large number of active substances that promote matrix degradation and SMCs migration towards the intima. The established dogma in the field of atherosclerosis stating that all intimal SMC are derived from migrated and phenotypically altered medial SMC (Ross and Glomset 1976), has recently been challenged by a number of studies that documented the presence of bone marrow- derived cells capable of differentiating into the vascular SMCs to assist vascular repair in animal models of vascular injury (Remy-Martin et al. 1999; Simper et al. 2002) and transplant atherosclerosis(Shimizu et al. 2001; Sata et al. 2002). Studies of the origin of SMCs in humans yielded inconsistent results but generally confirmed the presence of blood derived cells within the developing atherosclerotic lesion that express some early markers of SMCs differentiation (Caplice et al. 2003). Chapter 1: Atherosclerosis and Inflammation 24
Although huge progress has been made in our understanding of molecular mechanisms involved in the regulation of cell trafficking and SMC proliferation, many questions remain unanswered. In particular, molecular mechanisms that directly control SMCs phenotype switching have not been fully delineated. Better understanding of precise contribution of SMCs to atherosclerotic lesion formation and neointima development will enable the creation of more specific therapeutic strategies to target atherogenesis. Interactions between EC and leucocytes form a foundation for the development of atherosclerosis. The next section will discuss the main stages of leucocytes recruitment and principal molecular mechanisms that control them.
1.2 Stages and mechanisms of leucocyte recruitment during inflammation
1.2.1. Introduction
A hallmark of an inflammatory disease process, including atherosclerosis, is the recruitment of leucocytes to the site of inflammation. This process involves the emigration of leucocytes from blood into the surrounding tissues where they release toxic mediators, enzymes and oxygen free radicals, which cause tissue damage.
Leucocyte migration occurs in several distinct steps often referred to as a multi-step paradigm (Butcher 1991; Springer 1994) involving tethering and rolling, arrest, firm adhesion and transendothelial migration or diapedesis. The exact nature of the inflammatory response and the type of leucocyte recruited is regulated by locally Chapter 1: Atherosclerosis and Inflammation 25
produced cytokines and chemokines. Each step of leucocyte emigration is characterised by binding and detachment of adhesion molecules expressed on leucocytes and endothelial cells. Endothelial cell adhesion molecules and their respective leucocyte ligands belong to a number of different families, including selectins, the immunoglobulin gene superfamily and integrins.
1.2.2 Selectins
Selectins are type I transmembrane glycoproteins that mediate leucocyte and platelets rolling and tethering along vascular endothelium (Vestweber and Blanks 1999). Three major selectins have been identified so far: P-, E- and L-selectin. All three share a similar structure, which consists of an N-terminal calcium-dependant lectin domain, an epidermal growth factor-like domain, a single transmembrane domain, linked to a cytoplasmic tail and a variable number of consensus repeat (CR) domains (Fig 1.2).
The major structural difference between selectins is the number of CR domains. In humans P-selectin is the longest with 9 CR domains, followed by E selectin (6 domains) and L-selectin with only 2 CR domains(Vestweber and Blanks 1999). The gene cluster encoding for all three selectins is located on chromosome !(Watson et al.
1990). E-selectin is expressed on activated vascular EC but is absent under baseline conditions, except in skin micro vessels (Keelan et al. 1994). L selectin is constitutively expressed by all granulocytes and monocytes and the majority of lymphocytes (Brady et al. 1992; Fuhlbrigge et al. 1996). Figure 1.2: Selectins structure
(a) Selectins are composed of an N-terminal lectin domain, an epidermal growth factor (EGF) domain, two (L-selectin), six (E-selectin) or nine (P-selectin) consensus repeats with homology to complement regulatory (CR) proteins, a transmembrane domain (black arrow) and a cytoplasmic domain.
(b) Amino acid sequence identity within each domain, among different species
(human, mouse and cow) (top row) and among different selectins in the same species
(bottom row) (Adapted from K.Ley "The role of selectins in inflammation and disease" Trends Mol Med. 2003 Jun: 9(6) 263-8). Chapter 1: Atherosclerosis and Inflammation 26
(a)
Lec·in dam ,in ECF dcmai 2-9 C do ai ., C:r' c:pla. -r11:~ :Jor ,:1 n ,.• ----, ..I .- ·~. _., ·~...... · -~---- .\ ..•_ ___...-- . )
PI ,1 :,rT1,::i
(b) App o:-: r ,ale cu 11no acid 1de1 li1y
A c g same 0'% 40'"o Sf.II8C tin ~ IW8fHl spoP.C1~:·· (P =· 90%} ; I_L :- 95%)
Ai \CJ 1: · diff,;-;re II -~ C, .,2% J ' Noe r one seler.:l1ns i sa_ e species Chapter 1: Atherosclerosis and Inflammation 27
Stimulation of leucocytes by chemoattractants induces L-selectin proteolytic cleavage and shedding. E-selectin undergoes de novo synthesis following stimulation by mediators such as TNF-a and IL-1 with a peak of expression detected at 4 h following the challenge (Bevilacqua et al. 1987). P-selectin is stored in a-granules of platelets and Weibel-Palade bodies of endothelial cells. Rapid release of P-selectin from these structures upon stimulation ensures almost immediate surface expression of this gene (McEver et al. 1989). P-selectin can also by synthesized de novo in human endothelial cells following challenge with IL-4, oncostatin and IL-13 but not
TNFa or IL-l(Pan and McEver 1995; Yao et al. 1996; Woltmann et al. 2000).Both transmembrane and cytoplasmic domains determine localisation of selectin to a particular compartment: E-selectin to the plasma membrane, L-selectin to the tips of microfolds on leucocytes and P-selectin to secretory granules. Selectins bind to their ligands via the N-terminal lectin domain.
The most important and best-characterised selectin-binding ligand is P-selectin glycoprotein ligand 1 (PSGL-1). PSGL-1 is localised to the tips of the microvilli on resting leucocytes (Moore et al. 1995; Laszik et al. 1996). PSGL-1 is not only responsible for more than 90% of P-selectin binding but also serves as the most important ligand for the binding of L-selectin under inflammatory conditions
(Sperandio et al. 2003; Rosen 2004; Sperandio 2006). In acute inflammation in mice
L-selectin was found to be responsible for leucocyte-to-leucocyte interactions known as secondary tethering (Sperandio et al. 2003). On the other hand, in animal models Chapter 1: Atherosclerosis and Inflammation 28
of chronic inflammation, a significant interaction between 1-selectin and its endothelial ligand known as primary tethering has been observed. Moreover, the inflammatory infiltrates found in chronic inflammation exhibit the molecular structure of L-selectin ligands constitutively expressed on high endothelial venules
(HEV) of secondary lymphoid organs (Bistrup et al. 2004). L-selectin's major physiological role involves the regulation of leucocytes homing to lymphoid organs.
In HEVs L-selectin interacts with its specific ligands known as peripheral node aggressins (PNAd) that include glycosylation dependent cell adhesion molecule-1
(GlyCAM-1), CD34, podocalyxin and endomucin (Bargatze et al. 1995; Suzuki et al.
1996). In addition, PSGL-1 plays a role in E-selectin binding but is not considered to be it's major ligand (Norman et al. 2000; Katayama et al. 2003). In addition to PSGL-
1, E-selectin can also bind to glycosylated CD44 and E-selectin ligand 1 (ESLl)
(Hidalgo et al. 2007).
Recently, new insights into the specific function of each selectin have been gained through the mouse models of single, double and triple selectin knockouts. P-selectin appears to be the most versatile of the three selectins as it is able to support leucocyte recruitment and rolling in the absence of both E and L-selectins (Robinson et al.
1999). Even though isolated E-selectin deficiency has no measurable effect on
leucocyte recruitment induced by TNF-a in vivo (Robinson et al. 1999), E-selectin
blockade with specific monoclonal antibodies significantly increases leucocyte
rolling velocity indicating that E-selectin supports leucocyte rolling at slow velocity. Chapter 1: Atherosclerosis and Inflammation 29
Increased leucocyte rolling velocity caused by the removal of E-selectin leads to a moderate reduction in neutrophil arrest indicating the importance of contact time between endothelium and rolling cells in the arrest efficiency (Jung et al. 1998;
Forlow et al. 2000). In addition, E-selectin -/- mice demonstrate a prominent decrease in the number of stable leucocyte adhesions induced by systemic TNF-a administration (Milstone et al. 1998). L-selectin-/- mice display defects in leucocyte capture and rolling resulting in a significant reduction in neutrophil recruitment in several models of inflammation particularly at the late stages of TNF-a induced peritonitis (Jung et al. 1998).
All three selectins can also function as signalling molecules with L-selectin signalling pathways being the most extensively studied. In neutrophils cross-linking of L selectin is associated with elevation of intercellular calcium (Laudanna et al. 1994), upregulation of surface expressed MAC-1, increase in oxidative burst and p 38 MAP kinase and ERK 1/2 phosphorylation (Crockett-Tora bi et al. 1995; Smolen et al.
2000). Moreover, L-selectin activation potentiates neutrophil response to interleukin-
8 (IL-8) (Tsang et al. 1997).
P and E-selectin signalling pathways are less well understood. Increase in intracellular calcium in response to P- and E-selectin cross-linking has been demonstrated in vitro experiments suggesting these selectins can transduce signals across the plasma membrane. In addition, E-selectin- induced signalling involves Chapter 1: Atherosclerosis and Inflammation 30
actin polymerisation and tyrosine phosphorylation of E-selectin. Apart from functioning as signalling molecules, selectins can also transmit signals in adherent cells via interactions with their selectin ligand. For example, in leucocytes adherent to activated platelets there is a P-selectin -dependent increase in superoxide generation and beta 2 integrin activation (Nagata et al. 1993; Evangelista et al. 1996).
Taken together, P, E and L-selectins have both distinct and overlapping functions and are the main molecules involved in regulation of initial stages of leucocyte recruitment - leucocyte rolling and capture. As our body of knowledge regarding molecular mechanisms underlying function of selectins continues to expand, it becomes exceedingly clear that selectins participate in complex interactions with other adhesion molecules such as integrins. Overall, engagement of selectins leads to the initiation of signalling events that prime rolling neutrophils to progress to the next stage of the transmigration cascade - namely, adhesion and extravascular migration.
1.2.3 Adhesion molecules ICAM-1 and VCAM-1
1.2.3.1 Overview of structure and function
Leucocyte adhesion and transmigration are mediated by members of the integrin and immunoglobulin superfamilies. Members of the immunoglobulin superfamily belong to type I transmembrane proteins with a series of repeating extracellular IgG-like domains, a transmembrane region and a short cytoplasmic tail (Stad and Buurman
1994). They include intracellular adhesion molecules (ICAM-1, ICAM-2, ICAM-3), Chapter 1: Atherosclerosis and Inflammation 31
vascular cell adhesion molecule (VCAM-1) and platelet endothelial adhesion molecule-1 (PECAM-1). ICAM-1 and 2 and VCAM-1 serve as principal ligands for leucocyte integrins (reviewed in section 1.2.3.2). PECAM-1 binds primarily to
PECAM-1 expressed by leucocytes.
ICAM-1 expression on endothelial cells can be detected under basal conditions with significant variability between different organs and vascular beds (Stad and Buurman
1994). It increases substantially upon stimulation with pro-inflammatory cytokines or endotoxin peaking at 5 h following endotoxin challenge and correlating with the time of maximum leucocyte adherence to the endothelium (Panes et al. 1995). A constitutive level of VCAM-1 expression is even lower than that of ICAM-1 but increases dramatically following cytokine stimulation (Henninger et al. 1997).
PECAM-1 on the other hand, is constitutively expressed on unstimulated endothelial cells. Targeted deletion of ICAM-1 in mice produces impaired immune responses, decreased contact hypersensitivity, leucocytosis and improved resistance against septic shock. Two models of ICAM-1 deficiency have been described: the so - called
Baylor mouse characteristically expresses low level of alternatively spliced isoforms of ICAM-1, while the Harvard mouse is devoid of any ICAM-1 expression. In both models ICAM-1 deficiency protected mice from a wide range of acute and chronic inflammatory conditions such as experimental colitis, glomerulonephritis, SLE associated vasculitis, ischaemia-reperfusion injury, atherosclerosis and transplant vasculopathy, indicating the importance of this molecule in the pathobiology of Chapter 1: Atherosclerosis and Inflammation 32
inflammation (Sarman et al. 1995; Bullard et al. 1997). Furthermore, studies using endothelial cells from ICAM-1 deficient mice have demonstrated the essential role of this molecule in firm adhesion of monocytes and T-cell emigration across endothelial monolayers (Reiss and Engelhardt 1999). Cellular events occurring as a result of
ICAM-1 gene upregulation are mediated via integrin- ICAM-1 interaction known as inside-out signalling. It involves activation of Rho kinase and proteins regulating actin cytoskeleton such as FAK and contractin. In addition, the short cytoplasmic tail of ICAM interacts with ezrin, a member of ERM (ezrin-radixin-moesin) protein family involved in formation of lamellipodia (Durieu-Trautmann et al. 1994; Heiska et al. 1998). Recently, Barriero et al showed that endothelial cells form special
"docking structures" to "catch" leucocytes and they found co-localization of ezrin,
ICAM-1 and VCAM-1 in these structures (Barreiro et al. 2002).
VCAM-1 was originally identified as a cytokine responsive molecule that facilitates adhesion of lymphocyte, monocytes and eosinophils to activated endothelium. Initial attempts to generate VCAM-1 null mice were unsuccessful since absence of VCAM-
1 resulted in embryonic lethality (Gurtner et al. 1995). The problem was eventually overcome by generating mice that expressed a mutant form of VCAM-1 at markedly reduced levels. Breeding these mice into either ApoE -/- or LDLR -/- mice background helped to gain insight into the role of VCAM-1 in formation of early atherosclerotic lesions (Cybulsky et al. 2001) (reviewed in section 1.3). The role of
VCAM-1 in regulation of lymphocyte and monocyte adhesion to endothelium has Chapter 1: Atherosclerosis and Inflammation 33
been demonstrated with the use of an adenovirus carrying VCAM-1. These experiments showed that VCAM-1 alone was able to support lymphocyte adhesion but not transmigration however, in the monocyte it was sufficient to support rolling, adhesion and extravasation (Gerszten et al. 1998).
Both ICAM-1 and VCAM-1 are involved in the regulation of reactive oxygen species
(ROS) production by endothelial cells. ROS controls the integrity of endothelium and an increased production of ROS is associated with enhanced endothelial permeability
(Wang and Doerschuk 2000). Furthermore, delivery of oxygen radical scavengers that inhibit ROS production reduces leucocyte migration across endothelium (van
Wetering et al. 2002). Overall, cross-linking of ICAM-1 and VCAM-1 through integrin-mediated adhesion of leucocytes activates intracellular signalling in endothelial cells leading to the opening of inter-endothelial cellular junctions (van
Buul and Hordijk 2004).
1.2.3.2 lntegrins as ligands for adhesion molecules
Integrins serve as ligands for binding adhesion molecules and are essential for
leucocyte arrest under conditions of flow. Structurally integrins are heterodimeric transmembrane proteins made of common f3 subunits non-covalently linked with
various a subunits. They are constitutively expressed on leucocytes, endothelial cells
and other cells and mediate cell-cell and cell-matrix interactions (Luscinskas and
Lawler 1994). Upon activation by chemokines and chemoattractants integrins rapidly Chapter 1: Atherosclerosis and Inflammation 34
increase their affinity to cell adhesion molecules (CAM) facilitating CAM binding and signal transduction (Takagi et al. 2001). Out of 20 integrins that have been identified three groups are relevant to leucocyte emigration: 13-1, 13-2 and 13-7 integrin subfamilies. Integrins of the 13-2 subfamily contain one of four different alpha chains linked to a common beta chain. Two integrins from this family deserve special attention: leucocyte function-associated antigen-I or LFA-1 (aLl32 or CDI la/CD18) and Mac-1 ( aMl32 or CD 11 b/CD 18) as they are considered to be the major players in mediating leucocytes adhesion. LFA-1 is expressed on most leucocytes. It causes firm adhesion of leucocytes to endothelium via interaction with ICAM-1 and ICAM-
2. LFA-1 is not stored within leucocytes and undergoes a conformational change upon activation believed to be responsible for its interaction with ICAM-1 (Marlin and Springer 1987). On the other hand, MAC-1, expressed by monocytes and granulocytes, is stored in granules. It rapidly mobilizes to the cell surface upon activation and similarly to LFA-1, binds to ICAM-1 expressed by endothelial cells
(Panes and Granger 1998). In addition, 132 integrins regulate leucocyte rolling velocity in TNF-a-induced inflammation since deletion of CD18, Mac-1 or LFA-1 increases rolling velocities in this setting (Kunkel et al. 2000; Dunne et al. 2002). The functional importance of beta-2 integrins is underscored by the identification of its deficiency in humans. The condition is called leukocyte adhesion deficiency syndrome and is characterized by recurrent infections, impaired wound healing and pus formation as a result of heterogeneous mutation of the CD 18 gene (Anderson et al. 1985). Furthermore, animal studies in 132 knockout mice and with 132 blocking Chapter 1: Atherosclerosis and Inflammation 35
antibodies showed that leucocyte trafficking is critically dependant on this integrin
(Borjesson et al. 2003). Members of ~-1 and ~-7 integrin subfamilies contribute to the recruitment of different leucocyte populations. Beta-I integrin a4~ 1 known as very late antigen-4 or VLA-4 participates in adhesion of lymphocytes, monocytes, eosinophils and natural killer cells to activated endothelium through binding to its counter-receptor, VCAM-1 (Elices et al. 1990). ~-7 integrin a4~7 is expressed on lymphocytes found in gut and gut-associated lymphoid tissue. This integrin can bind to both mucosal address in cell adhesion molecule-I (MAdCAM-1) on high endothelial venules of lymphoid tissue and, to VCAM-1 under inflammatory conditions (Tsuzuki et al. 1996). Integrins expressed on leucocytes are normally in the state of low affinity for their ligands and require activation to mediate firm adhesion. lntegrin activation occurs through the family of chemotactic cytokines known as chemokines.
1.2.3.3 Chemokine-integrin signalling and its role in leucocyte adhesion and transmigration
Chemokines are low molecular weight chemotactic cytokines (8 to 14kDa) with similar structure that bind and signal through seven-transmembrane-spanning G protein-coupled receptors expressed on leucocytes and other cell types (Murdoch and
Finn 2000). Chemokines are classified into 4 groups: C-C, C-X-C, C-XrC and C based on the orientation of cysteine residues. Activated endothelial cells can produce and bind a number of chemokines involved in leucocyte arrest (Weber et al. 1999). Chapter 1: Atherosclerosis and Inflammation 37
increase levels of inositol 1,4,5, triphosphate (IP3) which leads to elevation of intracellular calcium, activation of Rho and mitogen-activated protein kinases and eventual rearrangement of the actin and tubulin cytoskeleton (Servant et al. 1999;
Vicente-Manzanares et al. 1999). This complex signalling cascade is translated by the leucocyte into a cell shape change and forward movement. In addition, chemokines can directly capture rolling leucocytes thus complementing the function of selectins.
Indeed, cross-linking of L-selectin induces CXCR4 expression on lymphocytes leading to an increase in SDF-1-dependant lymphocyte adhesion and transendothelial migration (Ding et al. 2003). It is important to appreciate the difference between leukocyte adhesion in vitro and in vivo. While in vitro leukocytes adhere to activated
EC almost immediately, in vivo they engage in significant rolling before undergoing arrest and firm adhesion (Kunkel et al. 2000). As shown by the elegant study of
DiVietro et al the efficiency of leucocyte arrest depends on chemokine density. In their experiments, an increase in concentration of immobilized chemokine significantly reduced the distance travelled by leukocytes and time to their arrest suggesting that signals received by rolling leukocytes through chemokines have to reach a threshold of activation before they could trigger leucocytes adhesion
(DiVietro et al. 2001)
1.2.4 Control of leucocyte transmigration
The exit of leucocytes across the endothelium into the perivascular space often referred to as transmigration, completes the last stage of leucocyte recruitment in Chapter 1: Atherosclerosis and Inflammation 36
The expression of particular chemokine receptors varies between different subsets of leucocytes, tissue types and inflammatory conditions. This diversity is necessary to provide additional control to leucocyte homing under different inflammatory conditions. No chemokine so far has been shown to be an indispensable regulator of leucocyte arrest, however, some such as IL-8 and stromal cell-derived factor-I (SDF-
1), are considered to play a central role in controlling leucocyte activation. 11-8, one of the principal pro-inflammatory cytokines belongs to the CXC class of chemokines and is involved in neutrophil activation. It binds to the cells expressing CXCRI and
CXCR2 receptors (Rainger et al. 1997). Mice lacking the CXCR2 receptor show severe impairment of leucocyte recruitment and grossly abnormal hematopoiesis
(Cacalano et al. 1994). Despite that, TNF-a applied to cremaster muscle of CXCR2 null mice produces normal leucocyte arrest suggesting that other neutrophil activators play a role under these conditions. SDF-1 or CXCL12 is another chemokine involved in leucocyte adhesion and transendothelial migration (TEM). It is found on the majority of human tissues (Bleul et al. 1996). Similar to IL-8, SDF-1 was shown to be expressed by vascular endothelium in viva (Middleton et al. 1997; Peled et al. 1999).
SDF-1 interacts with CXCR4 on lymphocytes causing rapid redistribution of this receptor to the leading edge of the migrating cell (Vicente-Manzanares et al. 1999).
Multiple signalling pathways induced in transmigrating leucocytes by chemokine integrin interactions have been described (Hughes and Pfaff 1998) but will not be reviewed here in detail. In brief, chemokines binding to their receptors induce a cascade of signalling events via activation of phosphatidylinositol 3 (PI3)-kinase to Chapter 1: Atherosclerosis and Inflammation 38
response to an inflammatory stimulus. This process occurs in two steps; first by the migration between endothelial cells followed by crossing of the basement membrane.
Leucocytes enter the sites of inflammation predominantly between endothelial cells
(ECs) borders (Muller 2001) although direct passing of leucocytes through ECs has been described (Feng et al. 1998).
The principal molecules that control leucocyte transmigration are platelet endothelial cell adhesion molecule-I (PECAM-1 ), vascular endothelial cadherin (VE-cadherin),
CD99, junctional adhesion molecule (JAM) A, B, and C (also known as JAM-I, 2 and 3). In contrast to the heterophilic interactions responsible for rolling and adhesion, most molecular interactions occurring during transmigration are homophilic between a particular adhesion molecule expressed on both EC and leucocyte (Fig 1.3).
PECAM-1 is a member of the immunoglobulin gene superfamily that is found on the surface of leucocytes, platelets and at the border of EC under basal conditions
(Newman et al. 1990). Blocking PECAM-1 with a domain-specific monoclonal antibody practically abolishes diapedesis of leucocytes in vitro (Muller et al. 1993) and in vivo (Liao et al. 1997). However, the PECAM-1 knockout mouse shows normal recruitment of leucocytes in the setting of TNF-a induced inflammation suggesting the involvement of other molecules in this process (Duncan et al. 1999).
Moreover, when IL-1 ~ is injected into PECAM-1 deficient mice, transmigrating Figure 1.3: Overview of distinct steps of leucocyte transendohelial migration
(I) The first stage represents the rolling of leucocytes that involves
adhesion molecules such as selectins.
(II) The second step is characterized by firm adhesion, mediated via
immobilized chemoattractants on the endothelium and adhesion
molecules such as integrins on the leucocytes and CAM on
leucocytes.
(III) The third step is diapedesis of the leucocytes through the endothelial
cell-cell junctions in which homophilic binding molecules such as
PECAM-1, CD-99, JAMs and VE-cadherin play major roles
(Adapted from van Buul, J. D. and P. L. Hordijk "Signaling in
leukocyte transendothelial migration." Arterioscler Thromb Vase
Biol. 2004 24(5): 824-33). Chapter 1: Atherosclerosis and Inflammation 39
Shear stress
J Rolling II Adhesion and Spreading/ 111 Transmigration activation
-~--(-,, -~ --~- Endothelium -- 1•,._,..._-= /, _j' /\ Cherne-attractants \ --.
II 0L~2 L.t- A ) Ill L-selectin 01, ~ 2 '.' "'". I Leukocytes : PSGL-1 PECAM-1 a xB2 10 !,O. ~.1 Sialyl Lewis X 0 ~P1< LA-l) a5 P1/1 ,LA5 t LFA-1 CD99 P-, E-selectin I I ICAM-1,-2 JAM-1 CD99 Endothelium: Glycam-1 CD34 VCA "1-1 PECAM-1 MadCAM -1 Fibronectin VE-Cadhcrin Chapter 1: Atherosclerosis and Inflammation 40
leucocytes are blocked at the level of the subendothelial basement membrane. This data suggests that the relative importance of PECAM-1 involvement in regulating leucocyte transmigration is dependant on the nature of the inflammatory stimulus.
One of the principal functions of PECAM is to regulate vascular permeability. This is evident from chronic inflammatory disease models such as collagen-induced arthritis
(CIA) and in experimental autoimmune encephalomyelitis (EAE) in which PECAM-
1 deficient mice display an exaggerated and prolonged increase in vascular permeability due to a delay in the restoration of vascular integrity following pro inflammatory challenge (Williams et al. 1996; Graesser et al. 2002). Recently,
Mamdouh et al described a novel membrane compartment in the EC that contains about a third of the total cellular PECAM. PECAM-1 from this compartment is continuously recycled and distributed evenly along the endothelial cell border.
However, during an inflammatory response the authors found a significant re distribution of PECAM-1 with its preferential localisation to the endothelial border in direct contact with a transmigrating monocyte. They have hypothesized that PECAM-
1 re-distribution during inflammation may enhance diapedesis and provide a "free pool" of endothelial PECAM-1 that interacts with PECAM-1 expressed by transmigrating leucocytes (Mamdouh et al. 2003).
Another molecule involved in regulation of vascular permeability is VE-cadherin.
VE-cadherin is a transmembrane protein that belongs to the cadherin superfamily. It localises to adherens junctions between EC. The VE-cadherin molecule consists of a Chapter 1: Atherosclerosis and Inflammation 41
conserved cytoplasmic domain and an extracellular domain that links VE-cadherin to the actin cytoskeleton via ~ and y catenin (Ali et al. 1997). Studies using monoclonal blocking VE-cadherin antibodies demonstrated an important role for this molecule in controlling the permeability of adherens junctions (Gotsch et al. 1997) since the deletion of VE-cadherin from lateral junctions greatly enhanced leucocyte transendothelial migration (TEM). In addition, re-organization of the actin cytoskeleton and an increase in monolayer permeability was seen with VE-cadherin antibodies applied to HUVEC monolayers (Hordijk et al. 1999). It has been suggested that neutrophils adherent to endothelium release elastase stored in their granules that disrupts VE-cadherin-catenin complexes at the level of cellular junctions thus widening the gap between endothelial cells (Allport et al. 1997). However, neutrophils from MMP-9- or elastase-deficient mice do not show any defect in transendothelial migration under flow in vitro, raising doubt over the role of elastase in the process of transmigration (All port et al. 2002). By using recombinant VE cadherin protein linked to GFP Luscinskas et al have demonstrated that leucocyte transendothelial migration occurs via opening of pre-existing and de novo gaps, the latter formed only when leucocyte comes in physical contact with the endothelial cell junction. In addition, they showed that different subsets of leucocytes utilize different
mechanisms during their migration with monocytes preferentially initiating formation
of de novo gaps as compared to PMN leucocytes which use the existing gaps
(Luscinskas et al. 2002). Chapter 1: Atherosclerosis and Inflammation 42
CD99 is a heavily O-glycosylated transmembrane glycoprotein that is expressed on all leucocytes, red blood cells and inter-endothelial contacts (Muller 2001), which is involved in regulation of VLA-4 dependent adhesion of T-lymphocytes to endothelial cells under physiological shear stress (Bernard et al. 2000).
Recent in vivo studies suggest that CD99 controls leucocyte transmigration at a later stage compared to PECAM-1. Arrest of leucocytes with the use of anti-CD99 antibodies occurs within inter-endothelial junctions just before transmigration is complete (Schenkel et al. 2002).
Junctional adhesion molecule (JAM) belongs to the lg gene superfamily and comprises 3 structurally related proteins: JAM A, B and C (formerly classified as
JAM-I, 2 and 3). JAM localizes to the apical regions of cell-to cell junctions of both endothelial and epithelial cells and regulates electrical resistance across the monolayers of these cells (Martin-Padura et al. 1998). JAM A is present on all leucocytes, platelets, EC, and epithelial cells and is found on the apical region of inter-endothelial junctions. Homophilic interaction between endothelial and leucocyte-bound JAM-A is believed to be important for leucocyte transmigration. In addition, JAM-A regulates transendothelial migration (TEM) of leucocytes through heterophilic interaction with 132 integrin-LFA-1 (Ostermann et al. 2002). Some reports suggest a decrease in inflammation with the use of anti-JAM-A antibodies
(Martin-Padura et al. 1998) while others did not confirm these findings (Lechner et al.
2000). JAM-B is expressed on vascular and lymphatic endothelium and is implicated Chapter 1: Atherosclerosis and Inflammation 43
in lymphocyte homing (Palmeri et al. 2000; Johnson-Leger et al. 2002). It interncts with /31 integrin receptor-VLA-4 (Cunningham et al. 2002). JAM-C found on EC, platelets, T-lymphocytes and NK-cells acts as a regulator of TEM of leucocytes through its interaction with Mac-1 integrin present on leucocytes (Santoso et al.
2002). Interestingly, JAM-C can also serve as a counter- receptor that mediates JAM
B adhesion to T cells (Arrate et al. 2001). The physiological significance of JAMB
JAM C interaction remains to be discovered.
In summary, there is a great degree of redundancy in adhesion molecules involved in late stages of leucocyte migration into the perivascular space. None of the molecules involved in this process have been shown to be indispensable for leucocyte transmigration in vivo. As our knowledge of molecular interactions continues to accumulate it becomes clear that there is a significant overlap between all three classical stages of leucocyte trafficking. What has been perceived initially as exclusive interactions between particular adhesion molecules and their ligands often extends to more complex interactions as illustrated by integrins, which apart from binding their classical ligands-ICAM-1 and VCAM-1 also engage other receptors including selectins and the JAM families of proteins.
1.3 Role of chemokines and adhesion molecules in atherosclerosis
Selectins and adhesion molecules ICAM-1 and VCAM-1 play an important role in the pathobiology of atherosclerosis. Multiple atherogenic stimuli such as modified Chapter 1: Atherosclerosis and Inflammation 44
LDLs, cytokines, reactive oxygen species (ROS) and shear stress augment the expression of adhesion molecules in cell culture and in vivo (Glagov et al. 1988;
Allen et al. 1998; Chappell et al. 1998). Selectins support monocyte recruitment to vascular endothelium via interactions with PSGL-1 ligand expressed on their surface.
P-selectin and its ligand PSGL-1 play the key regulatory role in this process. P selectin knockout mice show a reduction in the size of atherosclerotic lesions (Dong et al. 2000) and reduced neointima formation after arterial injury (Manka et al. 2001).
Moreover, in Apo-E deficient mice blocking P-selectin significantly reduced monocyte rolling and adhesion to the arterial endothelium (Ramos et al. 1999).
Impressively, even a single dose of PSGL-1 blocking antibody significantly altered neointima (NI) formation in the rat carotid artery in vivo (Phillips et al. 2003). An isolated deficiency of E-selectin seems to play a lesser role in atherosclerotic lesion development compared to P-selectin -/- mice (Collins et al. 2000), but recently was found to be important in NI formation in a rat balloon injury model. In this model E selectin antibodies applied prior to balloon injury significantly attenuated intimal hyperplasia (Gotoh et al. 2004). L-selectin has not been shown to play a significant role in animal models of atherosclerosis at this stage.
Atherosclerotic plaque displays a strong staining for P-selectin, ICAM-1 and VCAM-
1. VCAM-1 and P-selectin expression is detected on endothelium of the ascending aorta and lesion-prone areas following just one week of atherogenic diet in rabbits and before the appearance of intimal macrophages (Li et al. 1993; Sakai et al. 1997). Chapter 1: Atherosclerosis and Inflammation 45
In contrast, ICAM-1 and E-selectin expression is more diffuse and extends to the areas generally protected from atherosclerosis (liyama et al. 1999). Multiple animal studies have linked chemokine MCP-1 and it's receptor CCR2 with the development of early atherosclerotic lesions (Boring et al. 1998; Gosling et al. 1999). Another chemokine abundantly expressed in atherosclerotic plaque is IL-8. Genetically modified mice with the deletion of the receptor for IL-8 showed profoundly reduced lesion size and macrophage accumulation while on an atherogenic diet (Boisvert et al.
1998).
Support for the clinical relevance of adhesion molecules in the development and progression of atherosclerosis comes from population based and controlled randomised studies that found a strong relationship between the level of soluble adhesion molecules at baseline and the rate of future coronary events such as acute myocardial infarct (AMI) or unstable angina pectoris (UAP). For example, elevated level of sICAM-1 was a strong predictor for future coronary events in an asymptomatic cohort of patients (Ridker et al. 2000) and both sICAM-1 and sVCAM-
1 were associated with a higher event rate in patients with pre-existing coronary artery disease (CAD) (Haim et al. 2002; Tanne et al. 2002). In acute coronary syndromes sICAM-1, sVCAM-1, sP selectin and sE-selectin remained elevated for at least 6 months following an event (Mulvihill et al. 2001) and raised concentration of
sVCAM-1 was predictive of the future risk of major adverse cardio-vascular events
(Mulvihill et al. 2001). Chapter 1: Atherosclerosis and Inflammation 46
1.4 Role of IL-1 in inflammation and atherosclerosis
IL-1 is a key regulatory cytokine involved in initiation and propagation of inflammation in general. It plays a major role in many autoimmune and inflammatory conditions such as rheumatoid arthritis (RA), asthma, chronic obstructive pulmonary disease (COPD), inflammatory bowel disease (IBD) (Cominelli and Pizarro 1996), atherosclerosis and diseases of the central nervous system including multiple sclerosis, stroke and Alzheimer's disease (Hallegua and Weisman 2002). Levels of
IL-1 (3 are increased in patients with RA and IBD and have been shown to correlate with the disease activity in RA (Eastgate et al. 1988), COPD (Dentener et al. 2001) and ulcerative colitis (Casini-Raggi et al. 1995).
IL-1 is made of four proteins: IL-la, IL-1(3, IL-lreceptor antagonist (IL-lra) and IL-
18 (known as IFNy-inducing factor) (Dinarello 1997). Both IL-1 a and IL-1 (3 bind to
IL-1 receptor type I (IL-lRl) triggering intracellular signal transduction via p38 mitogen-activated protein kinase (MAPK)-activated phosphorylation cascade that leads to the nuclear translocation of NF-KB and activation of AP-1 transcription factors resulting in synthesis of growth factors, cytokines and vasoactive substances
(Suzuki et al. 1989). The importance of IL-1 signalling through the IL-Rl axis has recently been demonstrated in experiments with IL-lRl/Apo E deficient mice.
Animals with deletion of both alleles for IL-lRl (ApoE+1· / IL-lRl ·1-) showed a 5-fold reduction in the size of atherosclerotic lesions when placed on a high fat diet (Chi et al. 2004). Apart from binding to IL-lRl interleukin-I can also bind to the so-called Chapter 1: Atherosclerosis and Inflammation 47
type II IL-1 receptor, however this binding does not result in cellular activation
(Colotta et al. 1993). In fact, IL-lra is considered to be an endogenous inhibitor of IL-
1 signalling since atherogenesis is reduced in IL-lra transgenic mice on a high fat diet
(Devlin et al. 2002) and administration of IL-lra to ApoE-/- mice is known to decrease fatty streak formation (Elhage et al. 1998).
IL-1 was one of the first cytokines considered to be instrumental in the propagation of vessel wall inflammation in atherosclerosis owing to its proinflammatory effect on endothelial cells (Jirik et al. 1989; Sironi et al. 1989; Garcia et al. 2000), smooth muscle cells (Clinton and Libby 1992; Braun et al. 1995) and macrophages (Sica et al. 1990). In tum, stimulated vascular endothelial, smooth muscle cells, platelets, and different types of leucocytes including monocytes, lymphocytes and activated macrophages can secrete IL-l(Moyer et al. 1991; Galea et al. 1996) thus contributing to the maintenance of the inflammatory milieu by paracrine and autocrine augmentation of cytokine and adhesion molecules expression (Osborn et al. 1989;
Bochner et al. 1991). IL-1 facilitates early lesion formation by augmenting leucocyte adhesion to endothelial cells (Bevilacqua et al. 1985) and mediating leucocyte transmigration (Moser et al. 1989). It stimulates the expression of vascular adhesion molecules and chemokines that in tum attract cells of monocyte lineage, promotes their differentiation and enhances diapedesis (Suzuki et al., 1989). It has been shown that IL-1 is involved in all stages of plaque development including the earliest phase of foam cell accumulation (Galis et al. 1995). In the advanced plaque, IL-1 Chapter 1: Atherosclerosis and Inflammation 48
upregulates production of matrix metalloproteinases and thus contributes to plaque destabilisation and rupture (Galis et al. 1995; Libby et al. 1995; Elhage et al. 1998) so
it's not surprising that certain IL-1~ gene polymorphism is associated with
myocardial infarction in Chlamydia pneumonia seropositive patients (Momiyama et al. 2001) and that pericardial fluid of patients with unstable angina pectoris contains
high levels of IL-1~ (Oyama et al. 2001).
The signal transduction pathway induced by IL-18 is similar the one induced by IL-
i a and IL-1 ~ and this translates into similar biological functions. IL-18 upregulates the expression of ICAM-1 by monocytes (Dinarello 1999) and VCAM-1 by endothelial cells (Vidal-Vanaclocha et al. 2000) and is localised to macrophages in human carotid atherosclerotic pl<;tque (Mallat et al. 2001). Recently, an elevated
plasma level of IL-18 was found to be a strong predictor of cardiovascular death in
patients with stable and unstable angina (Blankenberg et al. 2002). Few strategies
have been used to block IL-1 activity in an attempt to modify inflammation in chronic
conditions such as RA. Currently the only drug used in clinical practice to inhibits IL
i signalling is a recombinant IL-1 RA protein called anakinra approved for use in the
United States in patients with severe destructive RA.
1.5 Intravital microscopy as a tool for studying inflammation in vivo
lntravital microscopy (IVM) is a technique of studying translucent tissues of
experimental animals and has been used for research purpose since the 19th century Chapter 1: Atherosclerosis and Inflammation 49
(Mempel et al. 2004). IVM allows direct observation of leucocyte-endothelial interactions in vivo thus offering an invaluable tool for studying cellular-endothelial interaction during inflammation and ischaemia/reperfusion. Careful preparation of the tissue of interest is essential for the success of this technique in order to avoid unnecessary trauma that can precipitate local and systemic inflammatory responses.
To achieve reliable and reproducible results tissue is kept warm with a buffer balanced for pH, gas content and electrolytes. Other variables such as contamination from exteriorisation of the tissue following surgery and the effect of anaesthetics have to be controlled as well. IVM allows a real time in vivo observation and recording of leucocyte behaviour, systemic or local delivery of drugs and with the use of fluorescent labelling techniques a direct visualisation of different leucocytes subsets.
Brightfield microscopy is generally performed on transparent tissues. However, with the emergence of fluorescence microscopy and low-light detection technology, visualisation of solid tissues by epi-illumination has become possible as well
(Mempel et al. 2004). Using brightfield transillumination leucocytes appear as colourless spheres of uniform size. Mesenteric venules have been extensively used for studying Ieucocytes trafficking, adhesion and transmigration in different clinical settings. It has become a standard model for assessing effects of inflammation and ischemia/reperfusion or their inhibitors on leucocyte/endothelial interactions in vivo.
Many circulatory beds have been utilized for IVM studies the most common being submucosal mesenteric venules of different species (Zweifach 1973), hamster cheek Chapter 1: Atherosclerosis and Inflammation 50
pouch (Raud et al. 1989), cremaster muscle in rats and mice (Gavins and Chatterjee
2004) and implanted ear chambers (Asano et al. 1988). Chapter 2: Transcription factors c-Jun and Egr-1 51
Chapter 2: Transcription factors c-Jun and Egr-1
2.1 Biological/unction of AP-1 and c-Jun
Activating protein 1 (AP-1) is a key transcriptional regulator of cell proliferation, survival, death and immune response (Shaulian and Karin 2001). AP-1 is a collective term for a group of functionally and structurally related genes that include the Jun protein family (c-Jun, JunB and JunD), the Fos protein family (c-Fos, FosB, Fral and
Fra2), Maf and the A TF sub-families (Shaulian and Karin 2002). They share a conserved basic DNA-binding domain collectively called bZIP which is coupled with a leucine zipper region responsible for the dimerization required for DNA binding by the basic domain (Hess et al. 2004). AP-1 binds to consensus sequence 5' -
TGAG/CTCA-3' called TREs (TPA-responsive elements) on the promoter of several genes containing AP-1 binding sites. AP-1 transcription factors are induced by a variety of physiological and pathological stimuli including serum, cytokines (e.g. IL-
1, TNFa) growth factors, T cell activators, UV radiation and oncogenes such as v-Src or Ha-Ras (Shaulian and Karin 2001).
c-Jun is one of the best-characterized components of AP-1. It is a 39-kDa protein that consists of a C-terminal basic region, a leucine zipper (bZIP) DNA binding domain and an N-terminal transcriptional activation domain. Basal levels of c-Jun expression are low but increase dramatically following stimulation with growth factors, cytokines and UV radiation through phosphorylation of c-Jun by the JNK group of
mitogen activated protein kinases (MAPKs) on serines 63 and 73. This leads to an Chapter 2: Transcription factors c-Jun and Egr-1 52
increased stability and enhanced transcriptional activity of c-Jun (Smeal et al. 1994).
Similar to other members of the AP-1 family c-Jun can either enhance or inhibit cell proliferation and differentiation (Hess et al. 2004). For example, c-Jun plays a well established role in angiogenesis through regulation of VEGF gene expression in endothelial cells (Alfranca et al. 2002) but at the same time has a pro-apoptotic action via the extrinsic death receptor pathway involving FasL and TNFa both of which contain AP-1 binding sites (Eichhorst et al. 2000). c-Jun is indispensable for normal embryogenesis since c-Jun double knockout mice are not viable (Mechta-Grigoriou et al. 2001). Primary fibroblasts derived from both heterozygous and homozygous mutant c-Jun mice exhibit greatly reduced growth rates in culture. In contrast to the primary fibroblasts cultured from a wild-type mice, they do not respond to the addition of mitogens (Johnson et al. 1993). In addition, fibroblasts from mice embryos with homozygous c-Jun deficiency are resistant to apoptosis induced by UV radiation (Shaulian et al. 2000). Recently, the importance of c-Jun in regulation of angiogenesis and solid tumour growth has been demonstrated by our laboratory with the use of c-Jun specific DNAzymes (Zhang et al. 2004).
2.2.1 Genes activated by c-Jun in response to vascular injury/ inflammation
Data continues to accumulate regarding the importance of c-Jun in vascular pathobiology. AP-1 binding sites have been identified in a variety of genes involved in inflammation, thrombosis and atherogenesis such as ICAM-1, VCAM-1, MCP-1, Chapter 2: Transcription factors c-Jun and Egr-1 53
endothelin-1, tissue factor and PAI-1 (Lee et al. 1991; Parry and Mackman 1995;
Shyy et al. 1995; Olman et al. 1999). Recently, a key role of c-Jun in the regulation of
SMC growth and proliferation was demonstrated by our laboratory with the use of a
c-Jun specific DNAzyme (Dz13). In these experiments Dz13 but not its scrambled
counterpart-Dz13scr inhibited SMC proliferation in vitro and attenuated neointima
(NI) formation in a rat model carotid artery injury (Khachigian et al. 2002). The
importance of the c-Jun/AP-1 complex in atherogenesis is reinforced by an observation that native and oxidized LDLs induce expression of ICAM-1 in human
vascular EC and rat mesangial cells through binding to the c-Jun/ AP-1 site in the
ICAM-1 gene promoter region (Wang et al. 2001; Wu et al. 2003). Moreover, c
Jun/AP-1 is required for NP-kappa B induction of VCAM-1 expression by TNFa in
HMEC as shown by an elegant study of Ahmad et al that demonstrated a significant down-regulation of VCAM-1 expression by c-Jun and c-Fos inhibitors (Ahmad et al.
1998). An up-regulation of ICAM-1 expression by c-Jun has also been documented
with other pro-inflammatory / atherogenic stimuli such as insulin-like growth factor
(IGF)-1 (Che et al. 2002). Both c-Jun and c-Fos were found to play an important role
in regulating the expression of ICAM-1 and MCP-1 in human vascular endothelial
cells stimulated with PMA (Wang et al. 1999).
The role of c-Jun in transcriptional activation of selectins is less clear. There is no
direct link between P or L-selectin and c-Jun/ AP-1 in the published literature.
However, c-Jun was shown to be important in the regulation of E-selectin expression
in an ischemia/reperfusion model of murine testis (Lysiak et al. 2003). Recently, a Chapter 2: Transcription factors c-J un and Egr-1 54
group from Italy showed that Jun activation domain-binding protein I (JAB-I) that
coactivates c-Jun, interacts with the 132 subunit of LFA-1, an ICAM-1 ligand
expressed by activated leucocytes, thus linking c-Jun to signal transduction via
integrins for the first time (Bianchi et al. 2000).
2.2 Egr-1 and atherosclerosis
2.2.1 Egr-1 in regulation ofpro-inflammatory and pro-thrombotic events relevant to inflammation and vascular injury
The early growth response (Egr-1) is a product of an immediate early gene and belongs to the superfamily of the zinc finger transcription factors involved in regulation of cell growth and differentiation (Milbrandt 1988; Gashler and Sukhatme
1995). Egr-1 is an 80-82kD DNA-binding protein that interacts with the GC-rich consensus region to initiate transcription of a diverse set of genes. Egr-1 expression increases rapidly from a low or undetectable basal level upon stimulation by different agents. Among the potent inducers of Egr-1 expression are shear stress (Khachigian et al. 1997), hypoxia (Yan et al. 1999) and a variety of cellular stressors such as growth factors (Silverman et al. 1997), oxidized and native LDL (Stoyanova et al.
2001), pro-inflammatory cytokines (IL-1, TNFa), hormones and angiotensin II (Day et al. 1999). The role of Egr-1 in vascular pathobiology has been extensively studied for over a decade. Egr-1 is considered to be an important player in the development
and progression of atherosclerosis based on its role in the regulation of tissue factor
(TF) (Cui et al. 1996; Pawlinski et al. 2003) and plasminogen activator inhibitor-I Chapter 2: Transcription factors c-Jun and Egr-1 55
(PAI-1) expression (Liao et al. 2006), SMC mitogens (PDGFs, bFGF, TGFP)
(Midgley and Khachigian 2004), adhesion molecules (ICAM-1, CD-44, VCAM-1)
(Harja et al. 2004), chemokines (MCP-1) (Yan et al. 2000), and oxidative stress via superoxide dismutase 1 (SODl) (Mine et al. 1999). The level of Egr-1 is increased almost 5-fold in human carotid atherosclerotic plaque with a corresponding elevation of Egr-1-inducible genes (McCaffrey et al. 2000). In addition, Egr-1 staining of atherosclerotic lesions in LDLR-/- mice increases exponentially with the progression of atheromatous lesions (McCaffrey et al. 2000). In human atherosclerotic lesions
Egr-1 was found to localize preferentially to macrophages, a-SM-actin-positive cells and endothelial cells all of which are known to be involved in the development of atherosclerosis (Du et al. 2000; McCaffrey et al. 2000). In the atherosclerosis prone
ApoE knockout mice the loss of Egr-1 (Egr-1-/-/ApoE-/-) protected these animals from the development of extensive lesions in the aorta at 14 and 24 weeks of age
(Harja et al. 2004). Recently, Egr-1 has been linked to chronic vascular inflammation through the induction of TF expression in mouse macrophages infected with
Chlamydia pneumoniae (Bea et al. 2003 ). Importantly, Egr-1 expression in atherosclerotic lesions can be altered with drugs commonly used for treatment of atherosclerosis such as statins. For example, simvastatin inhibited Egr-1 expression in lesions of Apo-E deficient mice by more than 60% (Bea et al. 2003).
The key role of Egr-1 in regulating intimal hyperplasia following vascular injury has been demonstrated in viva with a variety of gene silencing approaches. Chapter 2: Transcription factors c-Jun and Egr-1 56
For example, when the catalytic DNAzyme (EDS) that targets rat Egr-1 was delivered to the rat common carotid artery it suppressed the development of intimal thickening following both balloon angioplasty (Santiago et al. 1999) and permanent carotid artery ligation (Lowe et al. 2002). Targeting Egr-1 with DNAzyme in a porcine model of vascular injury reduced in-stent restenosis in pig coronary arteries at 30 days (Lowe et al. 2001). Another technique for silencing Egr-1 is the cis -element oligonucleotide decoy bearing an Egr-1 binding site. This agent has successfully inhibited intimal hyperplasia after balloon injury to carotid arteries of hypercholesterolemic rabbits (Ohtani et al. 2004).
In addition to its prominent role in atherosclerosis and the development of intimal thickening, Egr-1 has been singled out as a "master switch" regulator of tissue factor and inflammatory mediators in several mouse models including LPS-induced endotoxemia (Pawlinski et al. 2003) and ischaemia-reperfusion induced injury to the lungs (Yan et al. 2000). In both models Egr-1 knock-out mice demonstrated a significant downregulation of tissue factor and PAI-1 (Yan et al. 2000; Pawlinski et al. 2003 ), pro-inflammatory cytokines and chemokines such as IL-1 j), MIP-2,
RANTES, IP-10 (Yan et al. 2000) and MCP-1 and IL-6 (Pawlinski et al. 2003) and the suppression of the expression of adhesion molecules such as ICAM-1 (Yan et al.
2000). Other vascular conditions that utilize the Egr-1 axis signalling include cardiac hypertrophy (Saadane et al. 2000) and cardiac allograft vasculopathy (Okada et al.
2002). Chapter 2: Transcription factors c-Jun and Egr-1 57
2.3 Targeting c-Jun and Egr-1 with novel molecular approaches
2.3.1 DNAzymes
Deoxyribozymes are catalytic oligodeoxynucleotides composed entirely of DNA and capable of cleaving single stranded RNA between unpaired purine and paired pyrimidine junctions. Typically, a DNAzyme molecule consists of a cation dependant catalytic core of 15 nucleotides flanked by two arms of 6 to 12 nucleotides (Fig 2.1).
The sequence of the nucleotides of flanking arms confers DNAzyme specificity for a given mRNA (Khachigian 2002). DNAzymes were originally created by the screening of randomly selected DNA molecules for their ability to cleave RN As in vitro (Breaker and Joyce 1994). In 1997 Santoro and Joyce produced a "10-23" catalytic DNAzyme domain. It was obtained from the 23 rd clone following the 10th round of amplification (Santoro and Joyce 1997). Further advances in DNAzyme design were introduced by creating a 3' inversion on the 3' terminus that significantly improved resistance of the DNAzyme to degradation by serum or cellular exonucleases (Dass et al. 2002). As a result, up to 50% of modified DNAzymes remain catalytically active after 72 hours of incubation in DMEM with 10% FBS
(Dass et al. 2002). More recently, locked nucleic acids (LNA) have emerged as attractive monomers for modifying DNAzymes. The advantages of LNAs include improved thermal stability and solubility, enhanced resistance to 3 '-exonuclytic Figure 2.1: Structure of catalytic DNAzyme
Cleavage of human c-Jun mRNA between unpaired purine and paired pyrimidine junctions is indicated by a black arrow.
Dz13 molecule consists of a catalytic core made of 15 nucleotides (dash dot arrow)
that is flanked by 9 nucleotides on each side (square dot arrow) that bind to
nucleotide sequence of c-Jun mRNA based on Watson-Crick base-pairing rules. Chapter 2: Transcription factors c-Jun and Egr-1 58
hum c-Jun RNA 1302 1310 11312 1320 I I t I I ••• 5'-CAA CGC CUCG UUC CUC CCG UC-3' ..
GTT GCG GAG AAG GAG GGC-5' 3'-J. GA GG ...... C C -·-·-·~ A T A A CAT CG
Dz13 Chapter 2: Transcription factors c-Jun and Egr-1 59
degradation and easy synthesis and delivery into cells with cationic transfection agents (Petersen and Wengel 2003). DNAzymes offer a novel therapeutic approach to manipulating gene expression in viva. Their potency and efficacy has been demonstrated in a variety of animal models. For example, DNAzyme targeting transcription factor Egr-1 has been successfully used to inhibit cardiac ischaemia/reperfusion injury following the ligation of the left anterior descending artery in rats (Bhindi et al. 2006) and porcine intracoronary stent implantation (Lowe et al. 2001). Another DNAzyme, Dz13 targeting transcription factor c-Jun, has recently been shown to modulate inflammation (Fahmy et al. 2006) and inhibit tumour growth and corneal neovascularization in viva (Zhang et al. 2004). Many other successful in viva applications of DNAzymes have been reported including targeting transforming growth factor j31 (TGF-j31) in a rat model of experimental glomerulonephritis (lsaka et al. 2004), blocking vascular endothelial growth factor receptor 2 to inhibit angiogenesis and tumour growth (Zhang et al. 2002) and down regulating PAI-1 expression to improve myocardial regeneration in rats following ischaemic injury (Xiang et al. 2005). DNAzyme technology when compared to other gene silencing strategies, offers a great specificity, low immunogenicity, enhanced serum stability and relatively low cost of synthesis. The main current limitations of
DNAzymes are the limited efficacy of transfection vectors, their cellular toxicity as well as the inability to deliver DNAzymes systemically. Chapter 2: Transcription factors c-Jun and Egr-1 60
2.3.2 siRNAs
Small interfering RNAs (siRNA) were developed through recognition of the naturally occurring phenomenon called RNA interference (RNAi) in which double-stranded
RNA (dsRNA) molecules suppress expression of a target protein by stimulating the specific degradation of the complimentary target mRNA (Zamore 2001; Hannon
2002). Initial introduction of long dsRNAs into mammalian cells resulted in activation of a non-specific interferon response, however this problem has been bypassed by introduction of short 21-23mer base pairs of siRNAs. When administered, siRNAs are incorporated into RNA-induced silencing complexes
(RISC) that destroy target mRNA homologous to the integral siRNA. The final result is a rapid degradation of the target mRNA with a decrease in protein expression
(Hammond et al. 2000). RNAi contributes to the silencing of repetitive genetic elements or so-called "jumping genes" (Sijen and Plasterk 2003) and is considered to be a part of the nucleic-acid-based immune system since RN Ai protects human cells from viral infections by destroying viral transcripts (Gitlin and Andino 2003 ).
Therefore, RNAi represents an excellent strategy for modulating gene expression as it takes advantage of physiological gene-silencing machinery. siRNAs have been widely used as part of a functional genomic approach for in vitro screening of multiple signal transduction pathways particularly in viral and cancer cells (Billy et al. 2001; Jacque et al. 2002; Wilda et al. 2002). At the same time there has been an enormous growth in the application of siRNA technology in vivo. siRNA technology though in its infancy, is vastly superior to antisense oligonucleotide based technology Chapter 2: Transcription factors c-Jun and Egr-1 61
due to its exceptional specificity to a given mRNA, high resistance to ribonucleases and nonimmunogenic nature (Bertrand et al. 2002). Currently siRNAs are best suited to local delivery. Few studies have used systemic administration of siRNAs as it requires high volume pump injection of the drug which often induces acute cardiac failure in mice due to volume overload (Song et al. 2003). Systemically delivered siRNAs accumulate predominantly in the liver, spleen and kidneys with other organs showing only low to moderate uptake (Braasch et al. 2004). The best-suited route for local administration of siRNA at present is delivering them to the airway epithelium by inhalation. Intranasal administration of siRNAs to mice that targets two viruses involved in the pathogenesis of croup, pneumonia and bronchiolitis, namely RSV
(respiratory syncytial virus) and PIV (parainfluenza virus), virtually aborted viral replication and protected the lungs of the animals from damage (Bitko et al. 2005).
Currently two clinical trials of siRNA targeting either VEGF or VEGF receptor are proceeding in patients with an age-related macular degeneration (AMD). Preliminary data from Sirna Therapeutics from a phase I trial indicates clinical safety and excellent tolerability of siRNAs (ARVO, 1-5 May 2005, Fort Lauderdale, FL, USA).
In September 2006, a different company, Acuity Pharmaceuticals has also reported positive results of their phase 2 clinical trial of the drug known as bevasiranib sodium
(previously called Cand5), another si-RNA targeting VEGF. Further clinical trials of siRNA-based compounds are coming up in the near future and will be targeting a broad spectrum of clinical conditions from viral infections and cancer to ocular and metabolic disorders. Chapter 2: Transcription factors c-Jun and Egr-1 62
2.3.3 Antisense oligodeoxynucleotides
Antisense oligodeoxynucleotides (AS-ODN) are usually made of 15-25 nucleotide bases. They hybridise to a target mRNA by Watson-Crick base pairing rules and block translation to a protein by either inhibiting ribosome movement along mRNA or by inducing cleavage of mRNA by endogenous RNaseH (Kurreck 2003). RNaseH destroys the RNA but leaves the ODN intact thereby allowing the same molecule to hybridise with yet another mRNA target. Unmodified AS-ODNs are rapidly destroyed in biological fluids by nucleases, a problem that has been significantly overcome through a vast number of chemical modifications, designed to increase the stability of AS-ODN (Herdewijn 2000).
One of the most promising new developments in the field of gene-based therapies has been the introduction of locked nucleic acid (LNA), a ribonucleotide containing a methylene bridge that connects the 2' -oxygen of the ribose with 4' -carbon (Orum and
Wengel 2001). DNA LNA oligonucleotides possess an enhanced stability against nucleolytic degradation and very high target affinity (Kurreck et al. 2002). An improved cellular uptake has also been reported for LNA and probably accounts for their superior antisense potency. Several successful applications of LNAs in vivo have been reported in the literature including targeting of HIV -1 RNA (Darfeuille et al. 2004), silencing a large subunit of RNA polymerase II (Fluiter et al. 2003) and rat delta opioid receptor (Wahlestedt et al. 2000) as well as developing better molecular diagnostic drugs for in vivo tumour imaging (Schmidt et al. 2004). One commercially Chapter 2: Transcription factors c-Jun and Egr-1 63
available antisense drug Vitravene has been used since 1998 for the treatment of
CMV retinitis in patients with AIDS.
2.4 Transfection agents for the delivery of DNA therapeutics
One of the main challenges in the field of gene-based therapeutics is the development of the efficient delivery systems since the cellular uptake of naked DNA molecules is an extremely inefficient process owing to a combination of several factors including
DNA charge, size and stability. For example, the interaction between the negatively charged phosphate backbone of DNA and the negatively charged cell surface results in electrostatic repulsion that prevents DNA entering a cell. In addition, chemically unmodified molecules have poor stability in vivo due to their susceptibility to degradation by endo- and exonucleases. Thus, tremendous efforts have been put into developing strategies for the delivery of DNA into the cells over the last few decades.
DNA delivery methods can be divided into three general groups: electrical techniques, mechanical transfection and vector-assisted delivery systems (Patil et al.
2005). We will focus on describing non-viral vector-assisted delivery systems in particular liposomal systems that have been used by us throughout animal and in vitro experiments presented in this Thesis. An ideal DNA delivery vector should possess a
high transfection efficiency combined with a high degree of target cell specificity,
low immunogenecity, chemical stability and biodegradability. In addition, it should
be simple to formulate and produce. Vector-assisted DNA delivery systems can be Chapter 2: Transcription factors c-Jun and Egr-1 64
divided into 2 groups: viral and non-viral delivery systems. Viral delivery systems
have been developed to utilize the innate ability of viruses to transfer DNA molecules into the cells with extreme efficiency. For therapeutic purposes, a viral genome gets assembled with a transgene of interest that is released from the virus once it enters a cell. The gene then enters the nucleus, integrates into the host genome and becomes expressed (Kamiya et al. 2001). Many attenuated non-pathogenic viruses have been used for the delivery of DNA molecules (Mah et al. 2002). Currently viruses are used in more than 70% of human clinical gene therapy trials worldwide (Walther and Stein
2000). The main advantage is their very high level of transfection efficiency (45-
95%) even when it comes to the transfecting primary human endothelial and smooth muscle cells that are notoriously difficult to transfect (Garton et al. 2002). Despite this, there are several concerns over the viral delivery systems. The major ones are related to their toxicity and the potential of the viruses to integrate into the human genome. High doses of the viral material have been detected in seminal vesicles, testes and lymph nodes of mice (Timme et al. 1998) and recently were also found in seminal fluid of a male patient who underwent gene therapy, raising concerns over possible "contamination" of the germ line by viruses upon loss of their replication deficiency (Tenenbaum et al. 2003). Other concerns include a possibility of reduced potency after a long-term storage of viral preparations (Nyberg-Hoffman and
Aguilar-Cordova 1999), high production cost (particularly for retroviruses)
(McTaggart and Al-Rubeai 2002) and a potential for the loss of transgene expression
upon cell maturation (Bachrach et al. 2002). For these reasons nonviral delivery
systems are emerging as strong alternatives to viral vectors. They can be divided into Chapter 2: Transcription factors c-Jun and Egr-1 65
2 major groups: polymeric delivery systems and liposomal delivery systems. We will
now pay attention to the liposomal delivery systems.
Liposomes are vesicles made of an aqueous compartment enclosed in a phospholipid
bi-layer that have recently emerged as one of the most versatile tools for the delivery
of DNA therapeutics (Godbey and Mikos 2001). Liposomes have a number of
advantages over the viral vectors, as they are easy to synthesize and modify, appear to
be non-immunogenic and do not interfere with the human genome. Their major
problems are related to cellular toxicity, lower transfection efficiency in comparison to the viral vectors and inability to reach tissues beyond the vasculature unless
injected directly into the tissue (Tousignant et al. 2000). Liposomes work as a DNA
drug delivery system by entrapping DNA inside the aqueous core or complexing
DNA with the phospholipid lamellae. This complex gets delivered inside a cell by
either fusion with the plasma membrane of the cell or via endocytosis. Cationic
liposomal formulations are made from a mixture of cationic and zwitterionic lipid
(Feigner et al. 1994). The cationic lipids such as DOTAP serve as DNA complexing
agents while commonly used zwitterionic lipids such as DOPE or cholesterol help in
membrane perturbation and fusion. The best-known and the most frequently used
cationic transfection agents include DOTAP/DOPE, DOTAP/Cholesterol FuGENE6,
Lipofectamine, Effectine and Transfectam. DOT AP/DOPE is currently used in a
small number of human trials looking at novel anticancer treatments (Merdan et al.
2002). Chapter 2: Transcription factors c-Jun and Egr-1 66
One of the critical issues associated with the use of these agents is related to the characterisation of their mechanism of action in different biological environments.
In order to achieve the best transfection efficiency for a given agent one has to
understand its biophysical and chemical properties. FuGENE6 has been on the market since 1997 through Roche Applied Science and represents one of the best currently available cationic agents. FuGENE6 is well characterized and compares favourably to other vectors such as DOT AP/DOPE, DOT A/Cholesterol. It has low cellular toxicity in vitro, high transfection efficacy and good stability. Faneca et al compared FuGENE6 with DOTAP/DOPE and DOT AP/Cholesterol and FuGENE6 exhibited the highest level of transfection efficiency across different cell types
(Faneca et al. 2002). The chemical structure of FuGENE6 is under a patent from
Roche and has not been disclosed. Before the transfection, DNA is mixed with
FuGENE6 in a serum-free medium and left to form complexes for at least 15 minutes. Once added to the cells it can be removed after several hours or left until
assay time with some cells showing better transfection efficiency with longer
exposure while others remain unaffected (Faneca et al. 2002). Importantly, no
additional cellular cytotoxicity has been observed when FuGENE6 was left until the
time of assay. The FuGENE6: DNA complex is stable for up to 2 hours (Jacobsen et
al. 2004). The major disadvantage of FuGENE6 compared to the DOT AP/DOPE
formulation is the fact that FUGENE6 is only approved for in vitro applications and
had not been approved for use in human clinical trials. Chapter 3: Coronary artery bypass surgery 67
Chapter 3: Coronary artery bypass surgery
3.1 Introduction
Coronary artery disease (CAD) is one of the major health problems faced by modem societies. The overall mortality from CAD has been steadily declining in the last three decades, however the absolute number of people affected by this condition continues to grow due to the increased longevity and improved medical and surgical treatment of this disease. Coronary revascularization comprising percutaneous intervention and coronary artery bypass grafting (CABG) is the mainstay of treatment for CAD and is known to improve patient symptoms and overall survival. Despite a growing number of percutaneous coronary interventions performed each year, CABG remains an important and frequently performed procedure. The main conduits used for CABG are saphenous veins due to their availability, length and vessel size. Although the last two decades have seen a significant increase in the use of arterial conduits such as mammary and radial arteries, saphenous veins remain a principal vessel for bypass surgery. The major limitation of saphenous veins as conduits for bypass surgery is their long-term survival. A 50% of graft failure rate by 10 years from the time of surgery was reported in the mid nineties (Fitzgibbon et al. 1996). The survival of vein grafts has improved significantly over the last decade owing to improved surgical technique and more aggressive management of traditional risk factors with more recent figures showing between 60 to 70% patency rate at 10 years of follow up
(Goldman et al. 2004). Despite this improvement, the rate of bypass graft failure remains unacceptably high thus representing a significant medical and economic Chapter 3: Coronary artery bypass surgery 68
problem since re-operation is associated with a higher risk of complications and
additional cost.
3.2 Mechanisms of bypass graft failure
Vein grafts placed into the arterial circulation invariably develop a degree of intimal hyperplasia (IH) that is considered to be a foundation for the later development of accelerated graft atherosclerosis. IH formation is currently viewed as a complex multifactorial process in which endothelial injury, smooth muscle cell (SMCs) proliferation, increased shear stress and hypercholesterolemia play a major role.
Endothelial injury originates from surgical manipulation of vein grafts, hypoxia and the hemodynamic shear stress of a high-pressure arterial circulation. Loss of endothelial integrity leads to enhanced platelet aggregation, fibrin deposition, and activation of the extrinsic coagulation cascade and leucocytes adhesion. In response to the above-mentioned factors SM Cs of the medial layer undergo a switch from a quiescent contractile state to a synthetic state that results in their migration to the intimal layer where they form a major cellular component of the neointima. This response of SMCs is evident from day 4 of injury and reaches its peak at 2 weeks.
Further thickening of the neointima is produced by accumulation of extracellular matrix proteins-elastin, collagens, glycoproteins and proteoglycans. Eventually, vein
grafts develop atherosclerosis that leads to late (more that 5 years after grafting) graft occlusion. Vein graft atherosclerosis often termed "accelerated atherosclerosis" is
different from "spontaneous atherosclerosis" of native arterial circulation (Ip et al. Chapter 3: Coronary artery bypass surgery 69
1990). Morphological lesions of accelerated atherosclerosis are more diffuse and concentric, with increased cellularity and a variable degree of macrophage infiltration and lipid accumulation (Davies et al. 1999). Consistent with this observation is the fact that venous tissue has an increased avidity for lipid uptake, superior to that of arterial tissue of the same species (Fuchs et al. 1972). In a rabbit model of bypass grafting Davies et al showed that hypercholesterolemia at the time of graft implantation leads to the increased duration of endothelial cell recovery, prolonged
subendothelial oedema, greater accumulation of macrophages in endothelial and subendothelial spaces and infiltration of subendothelial spaces with foam cells from one week onwards (Davies et al. 1999).
3.3 Animal models of bypass grafting
3.3.1 Main advantages and disadvantages of commonly used models
A number of animal models have been used to investigate the pathophysiologic
mechanisms of graft failure and to study potential interventions designed to reduce
graft failure rate. The best studied models include rat models of iliolumbar vein to
iliac artery (McGeachie et al. 1981) and epigastric vein to femoral artery interposition
grafting (Westerband et al. 2001), a mouse model of jugular vein to carotid artery
end-to-end anastomosis (Zou et al. 1998), a canine femoral artery-internal jugular
vein model (Ulus et al. 2000), a porcine model of carotid artery-saphenous vein
interposition grafting (Dashwood et al. 1998) and a rabbit model of external jugular
vein to common carotid artery grafting (ltoh et al. 1994). Chapter 3: Coronary artery bypass surgery 70
The main advantage of a mouse model of bypass grafting is the availability of transgenic mice that can be used to study the genetic basis of vein graft atherosclerosis (Zou et al. 1998). The main disadvantage is a technical challenge of creating arterio-venous anastomosis in a small animal. A rat model of bypass grafting
has proved to be useful in investigating early histological changes of arterialised vein grafts but with the exception of a few studies it is not widely used for studying therapeutic interventions to reduce graft failure. On the other hand, canine and porcine models of bypass grafting have turned out to be an invaluable tool for advancing our knowledge on the mechanisms of graft failure. An important advantage of these models is related to their closer similarity in general anatomy and vascular histology to that described in man. Furthermore, an application of the special therapeutic strategies such as external stenting can be best performed in large animals
(Violaris et al. 1993 ). One of the main disadvantages is the high cost of larger animals and special requirements for housing, intra-operative anaesthesia and postoperative care. A limited number of studies has employed the technique of
saphenous-coronary bypass grafting in dogs and pigs with or without the use of a
bypass pump. This methodology closely resembles the one used in humans. These
studies were predominantly designed to perfect the technique of bypass grafting and
to investigate novel surgical methods such as robot assisted laparoscopic grafting
(Hoagland et al. 2004). A rabbit model of bypass grafting is one of the most
commonly used animal models. It has a number of advantages over other models
such as no special anaesthetic requirements compared to the canine and porcine Chapter 3: Coronary artery bypass surgery 71
models, a relatively small cost, easy availability and an option to study the effect of cholesterol on the development of intimal thickening. However, the histology of rabbit jugular vein is very different to the human saphenous vein so the results obtained in a rabbit model may not be applicable to the bypassed human saphenous vein.
3.3.2 Morphologicalfeatures of vein grafts from hypercholesterolemic versus normocholesterolemic animals
The rabbit model of arterio-venous grafting is the most widely used animal model of bypass grafting. It involves engrafting of the external jugular vein to the ipsilateral common carotid artery. The surgery is carried out under a general anaesthetic in a sterile environment; the placement of microsurgical arterio-venous anastomosis is accomplished with the use of an operating microscope or loupes and can be done in either end-to-side or end-to-end fashion, the former being a preferred method.
Considerable operator skill is required to achieve a reliable and reproducible result.
Neointima formation is observed within the first 2 weeks post-implantation. A commonly used end point in this model is NI development 4 weeks following bypass grafting but longer time points of 3 and 6 months have been used in some studies
(Ehsan et al. 200 I). Pre and postoperative hypercholesterolemia significantly alters the development of neointima as has been shown by the pioneering work of M.
Klyachkin and collegues. They found an accelerated NI development in hypercholesterolemic animals compared to controls. In their experiments cholesterol- Chapter 3: Coronary artery bypass surgery 72
fed rabbits developed a twofold increase in NI after 2 weeks on the diet and a three fold difference at 4 weeks compared to controls. Hypercholesterolemia not only produced quantitative changes in neointima formation but was also associated with significant qualitative differences: NI of cholesterol fed animals contained lipid-laden
SMC and macrophages whereas NI of control animals consisted of layers of SMC with collagen and a small amount of elastin (Klyachkin et al. 1993). The same authors demonstrated for the first time that hypercholesterolemia was associated with an impaired relaxation and enhanced contractility of vein grafts that contributed to endothelial dysfunction. Similarly, Davies and colleagues showed that hypercholesterolemia was associated with a prolonged endothelial recovery after vein implantation, increase in subendothelial oedema and debris deposition, early macrophage accumulation and the presence of foam cells in subendothelium and vein wall from day 7 onwards (Davies et al. 1999).
3.4 Gene based approach and molecular targets for the treatment of vein graft intimal thickening
There is currently no effective pharmacological treatment for vein graft failure; at the same time surgery provides a unique opportunity for an ex-vivo delivery of gene therapy. Gene based approaches to the treatment of graft failure have been studied for over a decade. Basic research has identified specific targets for gene therapy in vein graft failure that include coagulation pathways, endothelial dysfunction, cellular proliferation, matrix metalloproteinase activity and ROS production. Genetic based Chapter 3: Coronary artery bypass surgery 73
interventions that have been used in experimental animal models to reduce the rate of vein graft thickening are summarized in Table 3.1.
In broad terms there are two main strategies for gene-based therapies. The first approach involves replacement/augmentation of an inadequately functioning gene and this is most frequently achieved with adenoviral vector transfer. The second is based on the suppression of gene function in order to inhibit a particular pathological process and usually relies on nucleic acid based technology. Adenoviral gene transfer has been widely used for the delivery of gene therapy to vein grafts in cell culture and in vivo. Host cells that successfully express the target gene are called transduced.
Gene transfer efficacy is somewhat reduced in vein grafts compared to arteries but nevertheless clinically relevant levels of transduction are well documented. Most investigators report high initial levels of transduction in vivo with adenovirus that rapidly declines after 7 to 10 days (Channon et al. 1997).
Another gene-based approach that has been employed for the treatment of experimental graft failure is antisense or decoy oligodeoxynucleotides (ODN) delivered with the help of either transfection agents or mechanical pressure (Kusch et al. 2006). The main attraction of the nucleic acid based gene therapy is that small synthetic oligodeoxynucleotides (typically 1/1000 the size of an entire gene) can be easily delivered to the cells without requiring a viral vector. Chapter 3: Coronary artery bypass surgery 74
Table 3.1 Genetic interventions to reduce the rate of vein graft failure in animal models
Gene /cell type Type of gene Model Animal Effect on References targeted therapy used NI formation
Neuronal NOS Adenovirus Jugular vein- Rabbit >50% (West et al. carotid artery 2001)
Inducible NOS Adenovirus Jugular-carotid Pig 36% (Kibbe et artery al. 2001)
Endothelial Hemaggl utinating Jugular vein- Rabbit, 30% (Ohta et al. NO synthase virus of Japan carotid artery cholesterol- 2002) fed
TIMP-2 Adenovirus Vena cava - Mouse >50% (Hu et al. common carotid 2001) artery
c-Jun/c-Fos Antisense ODN Femoral artery- Rat 30% (Fulton et epigastric vein al. 1998; graft Suggs et al. 1999) PCNA Antisense ODN Jugular vein- Rabbit 26% (Fulton et carotid artery al. 1998)
TF Recombinant TF Jugular vein Rabbit 21% (Huynh et pathway inhibitor carotid artery al. 2001)
bFGF Antisense ODN Jugular vein Rabbit 34% (Yamashita carotid artery et al. 2003) Chapter 3: Coronary artery bypass surgery 75
Table 3.1 continued
TIMP-3 Adenovirus Saphenous vein Pig 58% (George et carotid artery al. 2000)
ICAM-1 !CAM-deletion by Vena cava - ICAM-1 30-50% (Zou et al. genetic carotid artery double 2000) manipulation knock-out mice CC-chemokine Recombinant Vena cava - Apo-E 65% (Ali et al. adenovirus common carotid knock-out 2005) artery mice
SMC Virulence- Jugular vein - Rabbit >50% (Curi et al. attenuated herpes carotid artery 2003) simples virus
p53 P53 double Vena cava - Genetically Increase in (Mayr et knockout mice common carotid modified NI by al. 2002) artery mice >50%
Retinoblastoma Adenovirus Jugular vein- Rabbit 22% (Schwartz protein (delta carotid aretry et al. 1999) Rb)
SOD, TIMP-1 Adenovirus Jugular vein- Rabbit 50% (Turunen and CC- carotid artery et al. 2006) chemokine
MCP-1 Adenovirus Vena cava- Mouse 51% (Schepers carotid artery et al. 2006)
C-type Adenovirus Jugular vein Rabbit >50% (Ohno et natriuretic carotid artery al. 2002) peptide Chapter 3: Coronary artery bypass surgery 76
Transfection agents used to facilitate delivery of ODN to the cells are not strictly
required for the delivery of ODN to vein grafts. Mann et al showed that a non
distending pressure of 39.9 kPa applied to the human saphenous vein wall for 10
minutes resulted in a greater than 80% uptake of ODN by venous endothelial cells
(Mann et al. 1999). The main limitations of adenoviral delivery systems are the
potential for introduction of the foreign genetic material into human cells,
immunogenecity and limited long-term efficacy whereas antisense-based technology
is limited by the problems related to the effective delivery of genetic material to the
intact vascular cells and concerns over the safety of transfection agents.
3.4.1 Endothelial injury, ischaemialreperfusion and oxidative stress
All vein grafts undergo a period of warm ischaemia followed by reperfusion, which
leads to local generation of reactive oxygen species (ROS) within the vessel wall.
ROS injure endothelial and SMC through both direct cytotoxicity and indirectly via triggering a secondary inflammatory response with accumulation of neutrophils and
monocytes (West et al. 2001). These cells further amplify cytokine production, which
result in local cell activation, migration and proliferation (Eslami et al. 2001).
Naturally occurring cytoprotective proteins such as heat shock protein-70 and
scavenging enzymes with antioxidant properties such as catalase, superoxide
dismutase and heme oxygenase-1 have been examined for their utility in the models
of arterial injury and cardiac reperfusion with some encouraging outcomes
(Jayakumar et al. 2001; Li et al. 2001). Superoxide dismutase (SOD) attenuates the Chapter 3: Coronary artery bypass surgery 77
damage caused by the release of free oxygen radicals. Adeno-viral mediated
overexpression of SOD was associated with a significant reduction in neointima formation in a rabbit model of bypass grafting (Turunen et al. 2006).
Endothelial injury alters normal vascular tone leading to a decreased production of a
potent vasodilator, nitric oxide (NO). As a result, experimental vein grafts
demonstrate significant impairment in endothelium-dependent relaxation (Komori et al. 1991; Ishii et al. 1993). In addition to its regulatory role in maintaining vascular tone, nitric oxide suppresses leucocyte-endothelial interaction (Kubes et al. 1991 ),
platelet adhesion (Azuma et al. 1986) and vascular smooth muscle cell proliferation
(Garg and Hassid 1989). It also acts as a potent anti-inflammatory agent by inhibiting
cytokine production and expression of adhesion molecules ICAM-1 and VCAM-1 by
vascular endothelium. Furthermore, NO protects SMC from the effects of vasoactive
substances released by platelets. Delivery of endothelial nitric oxide (eNOS) has been
achieved in cell culture and in vivo with the use of adenovirus. In a porcine model,
adenoviral transfection of inducible nitric oxide synthase (iNOS) into the vein grafts
led to decreased cell proliferation at 3 and 7 days after bypass and a 30% reduction in
NIH at 21 days (Kibbe et al. 2001). Similarly, a positive effect on graft remodelling
including a reduction in SMC intimal hyperplasia and superoxide production was
observed at 28 days following adenoviral nitric oxide synthase transfer into the rabbit
vein grafts (West et al. 2001). Chapter 3: Coronary artery bypass surgery 78
3.4.2 Role of coagulation cascade
Tissue factor (TF) is one of the principal molecules upregulated by endothelial injury.
Activation of TF leads to up-regulation of the extrinsic coagulation pathway through factor VII, IX, X and the generation of thrombin and fibrin. A TF-mediated thrombin production is important in the pathogenesis of intimal hyperplasia in several arterial injury models (Jang et al. 1995; Oltrona et al. 1997). Thrombin, fibrin and activated factor X (Xa) are involved in the stimulation of vascular SMC proliferation and migration (McNamara et al. 1993). Furthermore, both thrombin and fibrinogen can participate in platelet activation and promote leucocyte chemotaxis (Speidel et al.
1996). Thrombosis is a common endpoint of all failed grafts. Early thrombosis often has an underlying anatomical basis and is seen most frequently in the small-calibre conduits placed into a low blood flow environment. The thrombosis results from a complex interplay between pro and antithrombotic factors. Amongst naturally occurring anticoagulant mechanisms are synthesis of heparin sulfate proteoglycans, the protein C/thrombomodulin and the hydrolysis of adenosine nucleotides involved in platelet activation. Both protein C and thrombomoudlin (TM) are the major contributors to vascular thromboresistance. Once bound to TM thrombin in rendered incapable of enzymatically cleaving fibrinogen to form fibrin but acquires the ability to activate protein C. Activated protein C (APC) participates in proteolytic hydrolysis
of factors Va and VIiia thus inhibiting further generation of thrombin. Targeting of
the TM/ protein C pathway with gene-based therapies has been successful in the
treatment of arterial thrombosis (Waugh et al. 1999; Waugh et al. 1999) but produced Chapter 3: Coronary artery bypass surgery 79
mixed results in models of bypassed vein grafts. For example, restoring TM
expression in rabbit vein grafts with adenovirus vector-mediated gene transfer
significantly reduced thrombin activity but did not translate into attenuation of
neointima development at 28 days (Kim et al. 2002). On the other hand, ex-vivo
treatment of veins with direct thrombin inhibitors reduced intimal hyperplasia and
resulted in better preservation of Iuminal diameter in a rat model of bypass grafting
(Mureebe et al. 2004).
3.4.3 Inflammation and adhesion molecules
Peri-operative vein manipulation combined with stretching of the vein, ischemia
reperfusion injury and the deformation caused by exposure to a pulsatile high
pressure of arterial circulation leads to the activation of endothelium which promotes
an influx of circulating white cells into the vein wall. Endothelial activation is
characterized by an upregulation of ICAM-1, VCAM-1 and monocyte
chemoattractant protein (MCP-1) and is evident even during the early stages of vein
implantation (Stark et al. 1997). It has been known for a while that MCP-1 and its
receptor CCR-2 play a key role in vascular inflammation and monocyte chemotaxis
in the context of arterial injury and atherosclerosis. More recently, the pivotal role of
the MCP-1-CCR-2 pathway in controlling intimal thickening has been demonstrated
with the use of a CCR-2 receptor inhibitor that produced an impressive 51 %
reduction of IH in vein grafts of hypercholesterolemia-prone ApoE3Leiden mice
(Schepers et al. 2006). In line with the abovementioned data is the observation that Chapter 3: Coronary artery bypass surgery 80
MCP-1 production and monocyte adhesion are increased more than three fold following saphenous vein graft manipulation and brief exposure to the arterial circulation compared to controls (Eslami et al. 2001 ). Apart from MCP-1 pathway, movement of monocytes/macrophages inside the vessel wall also relays on ICAM-
11p2 integrin and VCAM-1/ VLA-4 mediated mechanisms (Crook et al. 2002). Once inside the wall macrophages become engorged with lipids and transform into lipid laden macrophages or "foam"cells. "Foam cells" secrete a wealth of growth factors that promote neointimal proliferation. Similar to monocyte/macrophage entry into the vessel wall, movement of neutrophils through the vessel wall requires interactions between cellular adhesion molecules expressed on the circulating white cells and venular endothelium. Neutrophils that enter the vessel wall become capable of secreting PDGF-like substance that leads to SMC proliferation and intimal hyperplasia (Shuhaiber et al. 2002).
A cause-effect relationship between ICAM-1 and intima hyperplasia (IH) is evident from ICAM-1-deficient mice that display 30-50% reduction in IH following bypass grafting (Zou et al. 2000). At the histological level the loss of ICAM-1 produced a significant reduction in leucocyte adhesion to the endothelium of vein grafts in
ICAM-1 -/- mice compared to the wild-type controls. Recently, ICAM-1 siRNAs have been found to be successful in reducing ICAM-1 expression in cultured venous endothelial cells in vitro thus opening a door for applying siRNAs to the treatment of human vein graft failure in the future (Walker et al. 2005). Chapter 3: Coronary artery bypass surgery 81
3.4.4 Cytokines, SMC mitogens and regulators of cell proliferation
A number of cytokines and growth factors have been implicated in the development of NI hyperplasia. These include basic fibroblast growth factor (bFGF), platelet derived growth factor (PDGF), interleuikin-1, transforming growth factor-13 (TGF-13) and tumor necrosis factor-a. PDGFs, bFGF and TGF-13 are very strong SMCs mitogens involved in SMCs growth, proliferation and migration (Hoch et al. 1995).
Their synthesis is significantly increased by hypoxia, shear stress and mechanical injury.
SMC proliferation is a hallmark of NI formation following an arterial injury and vein grafting therefore arresting the growth of SMC via manipulation of the proteins involved in the cell cycle, has been explored over the last decade in both experimental and clinical settings. Inhibition of genes that control cell cycle progression such as cyclin-dependent kinases, proliferating cell nuclear antigen (PCNA) and protooncogenes c-myc and c-myb has been achieved with antisense molecules
(Morishita et al. 1994; Fulton et al. 1997). Simultaneous inhibition of several transcription factors produced a better outcome compared to a single target. For example, rabbit vein grafts treated simultaneously with antisense ODN to PCNA and cell division cycle-2 kinase demonstrated a better remodelling with a striking attenuation of neointimal and medial hypertrophy at 6 months in hypercholesterolemic rabbits (Ehsan et al. 2001). This effect was accompanied by an improved endothelial function, namely better vasomotor responses and decreased expression of adhesion molecules and monocyte binding. Transcription factor E2F is Chapter 3: Coronary artery bypass surgery 82
known to up regulate a large number of cell-cycle genes and in the late nineties it was singled out as an ideal target for gene therapy of vein graft failure (DeGregori et al.
1995). The blockade of E2F with double-stranded decoy ODN that bears the consensus E2F binding site markedly inhibited NIH in both rat carotid artery injury and rabbit vein graft models (Morishita et al. 1995; Ehsan et al. 2001). E2F decoy application (Edifoligide) to human veins at the time of coronary artery bypass surgery progressed to phase 3 clinical trials (PREVENT IV) however, no angiographic difference in vein graft failure was observed between Edifoligide treated and control veins at the 12 month follow up, an outcome that precluded further investigation of this novel approach (Alexander et al. 2005).
3.4.5 Matrix remodelling
The process of SMC migration requires the matrix surrounding the cell to be broken down. Matrix metalloproteinases (MMPs) are the family of structurally related endopeptidases capable of degrading extracellular proteins including elastin and collagen (Okada et al. 1993; Katsuda and Okada 1994). MMPs are secreted by resident vascular cells as well as inflammatory cells recruited into the vein graft wall.
Increased proteolytic activity of MMPs is well documented in response to endothelial injury and is associated with NI formation (Jenkins et al. 1998). MMP activity is controlled by tissue inhibitors of MMPs (TIMPs). TIMPs consist of four major isoforms-TIMP 1, 2, 3 and 4. The balance between MMPs and TIMPs determines a local proteolytic milieu (Kranzhofer et al. 1999). Since matrix accumulation is one of Chapter 3: Coronary artery bypass surgery 83
the key features of a vein graft lesion, therapeutic strategies designed to inhibit its production have been extensively explored. An adenovirus-mediated overexpression of TIMP-1, 2 and 3 reduced NIH and improved vein graft remodelling in a mouse and porcine models in vivo and in human veins in vitro (George et al. 1998; George et al.
2000; Hu et al. 2001).
Other molecules involved in maintaining balance between matrix accumulation and matrix degradation are plasmin and plasminogen activator inhibitor-I. They have been successfully targeted with an adenoviral approach in animal models of intimal thickening (Kairuz et al. 2005).
3.4.6 Adventitia and perivascular fibroblast
Adventitia is the outermost layer of a blood vessel that contains bundles of SMC, collagen and elastin fibres. The vasa vasorum of adventitia are responsible for providing nutrients and oxygen to the wall of large vessels (Kachlik et al. 2003). This is particularly true for the veins where vasa vasorum penetrates much closer to the intima compared to the arteries so that damage to the adventitia and the related structures can significantly compromise oxygen supply and promote graft occlusion.
In arteries occlusion of vasa vasorum correlates with atherosclerosis and neointima formation (Barker et al. 1993). Similarly, in vein grafts the damage to vasa vasorum is associated with the development of neointimal hyperplasia and impaired wound healing around saphenous vein grafts (O'Brien et al. 1997). Chapter 3: Coronary artery bypass surgery 84
The role of adventitial fibroblasts in neointima development has received a considerable attention in recent times. Using a porcine model of saphenous vein grafting Shi et al have shown that fibroblasts translocate through the media of newly grafted veins and differentiate into myofibroblasts, acquiring alpha-smooth muscle actin (Shi et al. 1997). Furthermore, intima of old human grafts retrieved at the time of repeat bypass graft surgery, exhibit a profile of cystoskeletal proteins similar to that of myofibroblasts in a porcine graft model thus suggesting the involvement of these cells in NI formation (Motwani and Topol 1998).
3.5 Therapeutic interventions for reducing graft failure rate in humans
As discussed earlier, bypass graft failure is a multifactorial process resulting from a complex interplay between local and systemic factors. Vein harvesting for surgery typically disrupts endothelial venous lining thus compromising cell function. In addition, mechanical distension and hypoxia contribute to an early endothelial dysfunction therefore a "no touch technique" of handling tissues during harvesting has been adopted to minimize damage and preserve endothelial integrity and function
(Tsui et al. 2001). The "no touch technique" which involves minimal handling of the veins during surgery together with preservation of the perivascular tissue surrounding the vein, has translated into much higher patency rate at the18 month follow-up compared to the standard vein harvesting protocol (Souza et al. 2001).
Cardiopulmonary bypass and endothelial dysfunction typically lead to the activation Chapter 3: Coronary artery bypass surgery 85
of the coagulation cascade by TF with deposition of fibrin and thrombosis that
contributes to the early graft failure. Anticoagulation with heparin in the peri
operative period and long-term antiplatelet therapy with aspirin reduces the risk of
acute and subacute graft thrombosis (Goldman et al. 1994). Smoking is an important
modifiable risk factor for the progression of atherosclerosis, not surprisingly, patients
who quit smoking following CABG have a greater mid and long-term survival benefits compared to the ones who continue to smoke (van Domburg et al. 2000). In addition, they are less likely to require repeated CABG and have half the rate of subsequent myocardial infarction (Cavender et al. 1992; Voors et al. 1996). Blood
pressure control reduces the rate of progression of atherosclerosis following CABG as well as the risk of stroke and myocardial infarction (Domanski et al. 2000).
Hyperlipidemia prior to and following bypass grafting is a significant predictor of future conduit failure. It is strongly associated with the development of accelerated
graft atherosclerosis in epidemiological studies. Large randomised clinical trials demonstrated that aggressive peri-and postoperative therapy with statins significantly
improves long-term vein graft survival (Sacks et al. 1996; 1998; Knatterud et al.
2000). Statins are inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A reductase
and are currently the most potent cholesterol-lowering drugs available for clinical
use. Their main mechanism of action involves inhibition of the conversion of 3-
hydroxy-3-methylglutaryl coenzyme A into mevalonate, which is a rate-limiting step
in cholesterol synthesis. In addition, statins inhibit synthesis of other intermediates Chapter 3: Coronary artery bypass surgery 86
involved in cholesterol pathways such as geranylgeranyl pyrophosphate and farnesyl pyrophosphate that prevent the activation of membrane associated pathways known as Rho and Ras (Corsini et al. 1999). This mechanism of action is believed to be responsible for the numerous pleiotropic effects of statins, which are independent of their cholesterol-lowering properties (Waldman and Kritharides 2003). Among the most clinically relevant pleiotropic effects of statins are their anti-inflammatory, anti proliferative and anti-thrombotic actions. Statins promote re-endothelialization of injured endothelium (Walter et al. 2002), alleviate endothelial dysfunction through an improved production of NO (Tamai et al. 1997), reduce platelet aggregation
(Notarbartolo et al. 1995) and tissue factor activity (Cortellaro et al. 2002) and induce an activation of local fibrinolysis via induction of tPA (Essig et al. 1998; Mussoni et al. 2000). Statins also affect clot formation through the inhibition of plasminogen activator inhibitor 1 (PAl-1), the effect that is partially driven by their LDL cholesterol lowering properties (Bourcier and Libby 2000). The inhibition of TF expression by statins is well documented in vitro and this action is believed to be independent of their cholesterol lowering effect (Colli et al. 1997). In vivo, a significant reduction of TF protein level and activity has been found in the carotid enderectomy specimens extracted from patients who received atorvastatin (one of the statins) for 4-6 months prior to surgery (Cortellaro et al. 2002). A downregulation of the markers of inflammation by statins is well established in clinical trials. They decrease plasma levels of C-reactive protein and IL-6 independent of their LDL- Chapter 3: Coronary artery bypass surgery 87
lowering action (Ridker et al. 1999) and reduce both cytokine and adhesion molecule expression (A viram et al. 1992; Lefer et al. 1999).
3.6 Conclusion
Despite significant progress in understanding and treating vein graft failure, it continues to be a major limitation of bypass surgery. The pathogenesis of graft failure involves a complex interplay between intrinsic and extrinsic factors. On the intrinsic level, numerous cytokines and growth factors are involved in the propagation of signalling pathways that culminate in cell cycle progression and SMC proliferation.
On the extrinsic level, traditional risk factors for cardiovascular disease make a significant contribution to the progression of vein graft atherosclerosis in a similar way in which they influence the progression of atherosclerosis in the native arteries.
Clinical measures aimed at controlling modifiable risk factors for the progression of vein graft atherosclerosis such as lowering cholesterol, high sugars and blood pressure, inhibiting platelets activity and stopping smoking prolong vein graft survival but are not capable of averting vein graft failure completely due to the intrinsic nature of the response induced in the veins placed in a foreign environment of arterial circulation. So far, clinical trials of novel genetic therapies to combat vein graft failure in humans have not been successful, but despite these setbacks both scientists and clinicians are confident that it is only a matter of time until genetic targets and delivery systems are refined to produce clinically meaningful results. It is almost certain that a combination of therapies including pre-operative, intra-operative Chapter 3: Coronary artery bypass surgery 88
and post-operative measures will be required for the best chance of improving vein graft survival. In the future, intra-operative delivery of gene therapy targeting multiple pathways involved in the pathogenesis of IH in combination with aggressive therapies aimed at modifying risk factors would offer the best chance of prolonging vein graft survival thereby providing an improved quality of life for the patients.
3.7 Hypothesis and Approaches
Despite the enormous progress made in the last decade towards a better understanding of the molecular mechanisms involved in inflammation, much more work is required to advance our knowledge of this complex process. The aim of this thesis is to investigate the specific role of transcription factor c-Jun in two types of pathological processes: an acute inflammatory response secondary to a cytokine challenge and intimal thickening of arterialised vein grafts. Both conditions are relevant to human pathobiology and share many common molecular pathways.
The role played by c-Jun in regulation of inflammation is not fully understood because of two main reasons outlined below. Firstly, mice with the homozygous deficiency for c-Jun do not survive making it impossible to create an ideal in viva model to study the effect of c-Jun knock out in different biological systems.
Secondly, gene-silencing techniques available to study the role of this transcription factor in human pathobiology, have been rather imperfect until recently as they lacked a high level of specificity for c-Jun. In 2002 our laboratory generated a novel Chapter 3: Coronary artery bypass surgery 89
DNAzyme called Dz13 that possesses an extreme target specificity for c-Jun without affecting other members of the AP-1 family (Khachigian et al. 2002). Since discovery, Dz13 has been applied to different in vivo models such as arterial balloon injury in rats with excellent results (Khachigian et al. 2002). Previously published literature described c-Jun as one of the principal regulators of ICAM-1, VCAM-1 and
E-selectin expression in endothelial and other cell types (Wang et al. 2001; Holzberg et al. 2003). In addition, data from our own laboratory indicates that c-Jun is involved in regulation of SMC proliferation and migration, a pathological pathway important for the development of intimal thickening (Miano et al. 1993; Khachigian et al.
2002). Based on all of the above, we hypothesised that blocking c-Jun expression by
Dz13 will translate into a significant reduction in leucocyte recruitment to the site of inflammation through inhibition of the expression of multiple adhesion molecules and an attenuation in the development of intimal thickening in arterialised vein grafts most likely via two independent pathways: inhibition of the initial stages of the inflammatory response and SMC proliferation/migration. To test our hypothesis further we selected the two well-known in vivo models: firstly, a model of rat mesenteric microcirculation was used to study the effect of acute inflammation on leucocyte recruitment to mesenteric microvasculature as it allowed direct topical delivery of both DNAzyme and IL-1(3; secondly, a rabbit interposition arterio-venous
grafting was employed to study the changes in the intimal thickening following topical delivery of Oz 13. The next three chapters will present the methodology and
main results of our in vitro and in vivo experiments. Chapter 4: Materials and Methods 90
Chapter 4: Materials and Methods
4.1 Introduction
This chapter details the methodology of in vivo and in vitro experiments that form a foundation of this Thesis that focused on investigating the role of DNAzyme in acute
inflammation and intimal thickening in vivo. For this purpose we chose two in vivo
models: the mesenteric microcirculation in the rat and interposition bypass grafting
in the rabbit. In addition, we performed a number of in vitro experiments designed to
gain insights into some of the mechanisms of DNAzyme action in the context of
acute inflammation.
4.2 Cell culture, DNAzyme and siRNA synthesis, and transfection
for in vitro experiments
The immortalized human microvascular endothelial cell line, HMEC-1 (American
Type Culture Collection, Rockville, MD) was grown in MCDB 131 medium
(Invitrogen, Gaithersburg, MD) containing 10% feta) bovine serum (FBS) lOng/ml
epidermal growth factor, 2mm L-glutamine, lµg/ml hydrocortisone, and 5 U/ml
penicillin/streptomycin (all from Invitrogen). THP-1 cells, a human acute monocytic
leukaemia cell line (a kind gift from the Inflammatory Diseases Research Unit of the
University of NSW) were grown in RPMI 1640 (Gibco, USA) supplemented with
10% of fetal bovine serum. Both serum and FBS were heat-treated and filtered as per Chapter 4: Materials and Methods 91
standard protocol to remove any trace of LPS to prevent non-specific activation of
THP-1 cells.
DNAzymes were synthesized with an inverted thymidine at the 3' position (Tri Link
Biotechnologies, San Diego CA) and purified by high-pressure liquid
chromatography (HPLC). Dz13, a c-Jun targeting DNAzyme with 9+9 nucleotide
arms and its scrambled counterpart Dz13scr had the following sequences: 5' -
CGGGAGGAAggctagctacaacgaGAGGCGTTG-Ti-3' and 5'
GCGACGTGAggctagctacaacgaGTGGAGGAG-Ti-3' respectively. Nucleotides in
lower case represent the 10-23 catalytic domains; Ti refers to a 3 '-3' -linked inverted
thymidine. Jun siRNA was synthesized with the sequence, 5-
r(CAGCUUCCUGCCUUUGUAA)dTT-3'. The scrambled counterpart, Jun
siRNAscr, had the following sequence-5' -r(GAUUACUAGCCGUCUUCCU)dTT-
3'.
The agent used for transfection with DNAzymes was commercially available
FuGENE6 from Roche Diagnostics (Castle Hill, Sydney, Australia) and for siRNA
RNAiFect from Qaigen that was prepared according to the manufacturer's
instructions. We used a 3: 1 ratio between FuGENE6 and DNAzymes based on
previous experience in our laboratory. DNAzymes were resuspended in sterile H2o
at the indicated concentrations. Control cells were mock transfected with vehicle
alone. Cells were transfected 6 hours after they had been placed in serum-free Chapter 4: Materials and Methods 92
medium to initiate growth arrest. The transfected cells were incubated for a further
18 h in serum-free medium and then transfected a second time with the same
DNAzyme, siRNA or vehicle alone (so called a double transfection protocol). IL-1 ~ was added to HMEC-1 cells 24 hours after their placement into serum-free medium in a standard concentration of 20 ng/ml.
4.3 DNAzymes targeting transcription factors c-Jun and Egr-1 in models of acute inflammation and intimal thickening in vivo
4.3.1 Rat model of mesenteric microcirculation
60 male Sprague-Dawley rats (230-300 g) were obtained from the Animal Resources
Centre, Perth, Australia and housed in the biological resource centre (brc) of UNSW.
Rats were fed a normal chow diet and water ad libitum. The experimental procedure used in this study was reviewed and approved by the animal ethics committee of
University of NSW (approval number 02/139).
4.3.1.1 Surgical procedure and intravital microscopy
Rats were anaesthetized lightly with Halothane (4% in room air) followed by
intraperitoneal injection of sodium thiobarbital (lnactin, 100 mg/kg of body weight).
Tracheostomy was performed for airway management throughout the experiment.
An arterial line was inserted into the right femoral artery for blood pressure
monitoring and intravenous saline administration. Following midline abdominal Chapter 4: Materials and Methods 93
incision, the small bowel and its mesentery were exteriorised and placed on a temperature-controlled Plexiglass chamber for observation of mesenteric circulation using intravital microscopy. Special care was taken in handling the tissue during exteriorisation. The small bowel was covered with moist gauze to prevent it from drying. Both mesentery and small bowel were continuously superfused with modified Krebs-Henseleit solution at 37°C made of 131. lmM NaCl, 4.7mM KCl, l.2mM MgSO4, 20mM NaHCO3 and 2.0 mM CaC12 and adjusted to pH of 7.4. The mesenteric microcirculation was observed through an orthostatic microscope
(Olympus) with a 20-x long working distance objective and a 10-x eyepiece. A high resolution video camera (JVC) mounted on the microscope-projected images to a colour monitor. All images were recorded on Super-VHS (Maxwell, Japan) tapes for offline analysis. Single unbranched mesenteric venules of 25-50 µm in diameter and
> 100 µm in length were studied. Venular diameter was measured on line using a video calliper. Centreline red blood velocity was also measured on line with an optical Doppler velocimeter. Leucocyte rolling velocity was determined by measuring the time required for a leucocyte to travel a distance of 100µm along the length of the venule and was expressed as µms-'. The number of rolling, adherent and extravasated leucocytes was determined off-line by analysis of videotaped images.
Rolling leucocyte flux was determined as the number of cells crossing a fixed reference point in the vessel per minute. The same reference point was used throughout the experiment as leucocytes may roll for only a section of the vessel before re-entering the blood flow or becoming firmly adherent. Adherent leucocytes Chapter 4: Materials and Methods 94
were expressed as the number of stationary cells per 100 µm of vessel. To be
considered as adherent a leucocyte had to be stationary for at least 30 seconds or
longer. Leucocyte extravasation was expressed as number of white blood cells per
microscopic visual field. Venules were allowed to stabilise for 20 minutes following
surgery at which point leucocyte flux, adhesion and extravasation were assessed on line. If at that point leucocyte flux was> 35 cell/min, adhesion was> 3 cells/l00µm and extravasation was> 10 cells per field the venules were excluded from experiment due to the high level of background inflammation.
4.3.1.2 Transfection with DNAzymes and delivery of FITC-labelled
Dzl3scr
To assess the effect of DNAzymes targeting transcription factors c-Jun and Egr-1 in
acute inflammation, animals were divided into 6 groups: Control, vehicle alone,
Dz13, EDS, Dz13scr and ED5scr. 1Llf3 was added to the superfusion solution at the
concentration of 20ng/ml. DNAzymes were delivered in 100 µI of solution
containing 35 µg of DNAzyme, 40µ1 of FuGENE6, and 5µ1 of MgCl in sterile PBS.
This solution was applied topically for 10 minutes during which time superfusion
was stopped to maximize absorption of DNAzymes. The control group received 100
µI of PBS solution for 10 minutes while the vehicle group was given a solution
containing PBS, FuGENE6 and MgCl 2 but no DNAzymes. After 10 minutes,
superfusion with 1Llf3 was commenced and continued for one hour. The recordings
were made at the start of 1Llf3 treatment and at 60 minutes. Chapter 4: Materials and Methods 95
To identify the best time point for assessing the effect of IL-1 j3 on inflammation we
performed a number of pilot experiments. During pilot experiments the recordings
were made every 15 minutes for the total duration of 2 hours. These experiments
demonstrated a progressive increase in the parameters of inflammation once the total
duration of the experiment exceeded 90 minutes. They also showed that IL-113
induced changes were apparent between 30 to 60 minutes from the start of
superfusion hence we chose 60 minutes of superfusion as a cut-off point for the analysis. A ten minute incubation time point with DNAzymes was selected for two
main reasons: the fact that out pilot experiments demonstrated a non-specific
increase in the parameters of inflammation once the time exceeded 10 minutes and
the knowledge that due to the unique structure of the mesentery there is a rapid
absorption of topically applied drugs. Some experiments were done in a blind fashion to improve the validity of the data routinely analysed by the same
investigator (myself). In these experiments I was given two tubes containing either
Dz13 or Dz13scr and marked A and B that were prepared by an independent
researcher not involved in this project. Drugs A and B were administered to 6 rats (3
per group) according to the abovementioned protocol. Blind analysis of the recording
from these experiments correctly identified Dz13 and Dz13scr preparations. Chapter 4: Materials and Methods 96
FITC labelled DZ13scr was administered to 2 rats to identify the uptake of the
DNAzyme by venular endothelial cells. Experiments were conducted as per standard
protocol with special care taken to avoid light exposure as much as possible.
4.3.1.3 Tissue harvesting
To collect the specimens of rat mesenteric venules, the hepatic artery was cannulated
with a blunted 22G needle at the end of the experiments, and the vasculature was flushed with warm PBS (37°C). The inferior vena cava was divided to allow blood and PBS to escape. Next, the mesentery was fixed with 10% formalin in PBS via infusion through hepatic artery. The mesenteric venule under investigation together
with the surrounding tissue was excised, mounted on a piece of cork and secured to it
with four pins. It was placed immediately into 10% formalin overnight at 4° C and the tissue was processed according to the protocol outlined in section 4.4.
4.3.2 Rabbit model of bypass grafting
56 NZ white rabbits weighing 3-3.5 kg were used for this study. The first set of
experiments was performed on 32 rabbits fed a 1% cholesterol diet (prepared by
adding commercially available cholesterol supplement to a normal rabbit chow) for 4
weeks prior to the surgery and 3 weeks following the operation. In this group
animals were sacrificed for vein collection 21 days post-operatively. The remaining
24 rabbits were fed a normal chow throughout and were sacrificed 28 days post
operatively. Chapter 4: Materials and Methods 97
4.3.2.1 Surgical procedure and ex-vivo transfection with DNAzyme
Rabbits were anaesthetised with ketamine (Parnell laboratories, Australia) (60
mg/kg) and xylazine (Ilium Xylazil-20, Australia) (5 mg/kg) given as an
intramuscular injection. Anaesthesia was maintained with inhaled isoflurane (Laser animal health) administered at the flow rate of 2 1/min in 50% oxygen. All surgery was performed in the animal operating theatre with aseptic technique and was approved by University of NSW Animal Ethics Committee (approval number 04/09).
Rabbits were placed in a supine position and a cannula was inserted into the superficial ear vein for fluids, antibiotics and heparin administration. Prior to the skin incision animals received cephazolin (Mayne Pharma, Australia) in a dose of
300mg/kg of body weight intravenously and 30 mls of normal saline. A midline incision was used to expose the right external jugular vein. A 2-3 cm section of the
vein was excised with all side branches ligated and placed in a Petri dish containing
15 ml of normal saline with 1000 i.u of heparin (David Bull Laboratories, Australia).
Heparin in a dose of 330 i.u per kg of body weight was also administered
intravenously. Animals were randomly allocated into the 3 treatment groups: control,
Dz13 and Dz13scr. For an ex-vivo transfection with DNAzymes the vein was placed
into a sterile eppindorff tube and incubated at 37°C for 30 minutes. Total volume of
the transfection solution was 200µ1 made of 200µg of either Dz13 or Dz13scr, 40µ1
of FuGENE6 and 5 µI of MgCl2 in sterile PBS. Next, the right common carotid artery
was exposed and proximal and distal hemostatic clips applied. A small longitudinal
arteriotomy was performed and flushed with heparinized saline. The jugular vein was Chapter 4: Materials and Methods 98
reversed and the distal end of the vein was anastomosed to the proximal carotid artery in an end-to-side manner using 8.0 double-ended prolene suture (catalogue number 8730H, Johnson&Johnson). Distal anastomosis was accomplished in a similar way. The anastomosis was created with the help of Zeiss surgical loops (3.5 x
400) (Carl Zeiss, Germany). Finally, the right common carotid artery between the anastomosis was ligated with 4.0 silk suture (Silkam, ref 0762202) and divided to create unidirectional blood flow. The patency of anastomosis was confirmed by the presence of palpable pulsatile blood flow through the vein on the completion of surgery. The neck incision was closed in two layers with 4.0 ethylon suture
(catalogue number 1670 Johnson & Johnson) and 50ml of normal saline given IV prior to the removal of the cannula. Each rabbit received 2mg/kg of Rimadyl (Pfizer
Animal Health) intramuscularly, at the end of surgery for analgesia. Rabbits were placed on their side under a warming light and given 21/min of oxygen via a mask until full recovery from the anaesthetic. Rabbits were checked daily for the first 5 days for general well being and wound condition.
4.3.2.2 Harvesting of bypassed vein grafts
Vein specimens for morphometric analysis were collected from all animals. Two time points were chosen for vein harvesting: 21 days for animals on the cholesterol supplemented diet and 28 days for the ones on a normal chow diet. Chapter 4: Materials and Methods 99
At the time of vein collection animals were anaesthetised in exactly the same way as for surgery. The vein was exposed via a midline incision and carefully dissected from surrounding tissues. Then the proximal and distal carotid segments were cannulated. The vein was perfusion fixed with 10% formalin in phosphate-buffered saline. The mid-portion of the vein was removed and placed into 10% formalin. The remaining venous tissue went into RNAase later solution (Ambion ,Inc USA) overnight and was stored at -80°C for future experiments. In 6 animals contralateral
(left external jugular) vein was collected and placed into RNAase later solution as described above. Animals were sacrificed by intracardiac injection of 5mls of
Phenobarbital (David Bull Laboratories, Australia).
4.3.2.3 Histomorphometric analysis of vein grafts
For morphometric analysis veins were sectioned every 500 microns from the
midpoint and processed with a modified Masson's trichrome and Verhoeff's elastin
stain. Four slides were made form each level for immunohistochemistry. Veins were
photographed at x40 magnification and the images were transferred to the computer.
In most cases the cross-sectional image of the vein did not fit into the visual field so
2 or more images of the same vein were taken and later reconstructed to a full image
using Adobe Photoshop Elements Program version 3. Veins were analysed for the
development of neointima (NI) with the help of image J program downloaded from
the following website (http://rsb.info.nih.gov/ij). The degree of newly developed
intimal hyperplasia (IH) was expressed as the percentage of the area occupied by NI Chapter 4: Materials and Methods 100
to the total vessel area. Intimal hyperplasia was measured as an area bound by the
new intima on the inside and internal elastic lamina on the outside; a total vessel area
was measured as an area bound by the new intima on the inside and external elastic
lamina on the outside. Each vein specimen was analysed for the development of IH on at least 4 (average between all samples was 4.8) different anatomical levels at 500 microns intervals and an average of these measurement was taken as a percentage of
IH for a given specimen. In the cholesterol-fed animals, due to the high rate of thrombosis, which occurred in both Dz13-treated and Dz13scr groups, IH was analysed through taking photographs of the vein grafts and determining the weight on NI to the weight of the whole vessel.
4.4 Immunohistochemistry
IHC was performed on formalin fixed, paraffin embedded tissue according to the following protocol: slides were de-paraffinised and dehydrated in an automated
Leica autostain XL machine and placed in RO-H2o. For the antigen retrieval slides
were boiled in 500mls of citrate solution for 10 mins (9 ml of 0.1 M citric acid
monohydrate (Sigma), 41 ml 0.lM sodium citrate dihydrate (Sigma) and RO-H2o to
make up 500ml, adjusted to pH 6.0). Next, slides were cooled off to room
temperature (RT) and equilibrated with lxTBS for 15 mins. The tissue was circled
with Pap-pen (Sigma) and lOmls of buffer containing 9 mls of methanol (APS
Finechem, NSW, Australia) and 1ml of 30% H20 2 was added to completely cover the
slides for 30 mins. The buffer was washed off with Ix tris-buffered saline (TBS Chapter 4: Materials and Methods 101
made of 0.15 M NaCl/0.1 M Tris/HCI buffer, pH 7.4) twice for 5 mins. Next, a
blocking serum was applied for 30 mins ( 1 part serum: 5 parts of 2% BSA in TBS).
The selection of blocking serum was based on the secondary antibodies used e.g. if
secondary antibody was goat- then we used a goat serum. Blocking serum was left
on for 30 mins. Serum was flicked off the slides (no washing) and 50 µI of primary
antibodies diluted in 2%BSA in TBS was applied. The following primary antibodies
and dilutions were used on rat tissue: goat anti-rat ICAM-1 (R&D Systems, Inc)
1:50, rabbit polyclonal antibodies to VCAM-1, JAM-1, E-selectin, and c-Jun (Santa
Cruz Biotechnology, Inc) in dilution of 1:80 for c-Jun and 1:50 for all others, goat
polyclonal antibodies to PECAM-1 (Santa Cruz Biotechnology, Inc) in 1:50 dilution.
The VE-cadherin antibody was a rabbit polyclonal from Alexis Biochemicals and
was used in a dilution 1: 100. For the rabbit veins we used mouse monoclonal anti
MMP-2 and MMP-9 primary antibodies from Calbiochem in 1:50 dilution.
The following day slides were washed twice in lxTBS for 5 mins. For the rat tissue
fifty µI of either goat anti-rabbit or rabbit anti-goat antibodies (Dako) based on
primary antibodies used in a dilution of 1:200 in 2% BSA-TBS, were added and left
for 30 mins at RT. For detection of MMP-2 and MMP-9 on rabbit tissue we used 50
µl of biotinylated anti-mouse antibody (Vector laboratories). Following the
incubation with an appropriate secondary antibody the slides were washed with
lxTBS for 5 min twice and 50µ1 of streptavidin (Dako) in 2% BSA in TBS was Chapter 4: Materials and Methods 102
applied to each slide and left for lh. Following application of streptavidin slides were
washed for the last time with 1xTBS for 5 mins.
In the rat tissue the antigen was visualised with 3-amino, 9 ethyl-carbazole (AEC) chromogen. To prepare AEC solution we used 2375µ1 of acetate buffer (74mls of
0.2M Acetic Acid (Sigma) and 176mls of 0.2M Sodium Acetate (Sigma) in H20 to make up to IL), 125µ1 of AEC substrate prepared by dissolving 1 tablet of 3-amino,
9 ethyl-carbazole (Sigma) in 2.5mls of NN-dimethyl formamide and 1.25µ1 of 30%
H20 2) that was applied to each slide with a transfer pipette. Once the colour change was observed AEC was washed off with sterile water (Baxter). All slides apart from the ones stained for c-Jun were counterstained manually with haematoxylin for
10sec, washed under running RO-H20 until the water turned clear and dipped in
Scott's Blue 5 times and washed again. Excess water was gently wiped off and a drop of faramount mounting aqueous medium (Dako) was applied to each slide; it was allowed to dry overnight.
In rabbit veins MMP-2 and MMP-9 stain was visualized with a commercially
available Vector VIP substrate kit for peroxidase as per manufacturer's protocol
(Vector Laboratories, Inc, USA). Staining of the rabbit tissue for macrophages and
SMC was performed using Bond X™ automated immunostainer (Vision BioSystems,
Melbourne, Australia). Primary antibodies used were mouse monoclonal anti-rabbit
macrophage antibody RAM-11 (M063) and mouse monoclonal anti-smooth muscle Chapter 4: Materials and Methods 103
antibody (M0635) both from Dako in 1:50 dilution. The secondary antibody used to detect primary antibody binding was a goat anti-mouse biotinylated (Dako) diluted to 1:200. Staining was done using Intense R detection system, which is a LSAB system and contains 3% of hydrogen peroxide, HRP-conjugated streptavidin,
3 '3 '-diaminobenzidine (DAB) and haematoxylin. Staining was assessed using an
Olympus BXS 1 microscope and photographs were taken with an Olympus DP70 digital camera.
4.5 Human saphenous vein culture, transfection with DNAzymes and histomorphometric analysis
The experiments were designed and performed in collaboration with Dr Paul Bannon from the cardiothoracic surgical unit of Royal Prince Alfred hospital, Sydney. A segment of long saphenous vein approximately 8-10 cm in length was obtained during bypass surgery. The vein was placed in sterile saline and kept at room temperature; it was promptly transported from the hospital to the University of NSW where the vein was cut using a sterile scalpel blade into small segments approximatelyl.0 cm in length and placed into RPMI-1640 (Gibco, USA) medium supplemented with 20% fetal bovine serum.
Veins were allocated to the four treatment groups: control, Dz13, Dz13scr and vehicle alone. Transfection with either 0.4 µM of DNAzymes or vehicle was done on day 0 and day 7. Veins were grown for 14 days with a daily change of the culture Chapter 4: Materials and Methods 104
medium. On day 14 veins were placed into 10% formalin for fixation and left at 4°C
overnight. Veins were then cut cross-sectionally and stained with a modified
Masson' s trichrome and Verhoeff' s elastin stain.
The measurement of intimal hyperplasia (IH) was done by using image J program downloaded from the following website (http://rsb.info.nih.gov/ij). Neointima (NI) was measured as an area bound by the new intimal thickening on the inside and internal elastic lamina on the outside; the vessel media area was calculated as an area between internal elastic lamina on the inside and external elastic lamina on the outside. IH was expressed as a percentage of NI to the area of the media.
4.6 Western blot
Human microvascular endothelial cells (HMEC-1) were grown in Petri dishes to 80-
90% confluence in MCDB 131 medium containing glutamine and 10% FBS and transfected twice with 0.4 µM of Dz13, Dz13scr or vehicle alone as per the protocol outlined in section 4.2. Following the second transfection the serum free medium containing 20 ng/ml of IL-113 was added to all plates except control. Four hours later cells were washed with icy cold PBS before 500µ1 of 1x RIPA buffer (lS0mM NaCl
(Sigma), S0mM Tris-HCl pH 7.5, 1% deoxycholate (ICN), 0.1 % sodium dodecyl
sulfate (SOS), 1% aprotinin trasylol (Bayer, AG, Germany), 2mM phenylmethyl
sulphonyl fluoride (PMSF) (Sigma), lOµg/ml leupeptin hemisulphate (Sigma)) was
added to each plate and scraped using a Costar cell lifter. The contents were frozen to Chapter 4: Materials and Methods 105
-80°C and then thawed upon ice and fully disrupted by pipetting followed by
centrifuging at 14,000 rpm for 5 mins at 4°C. BCA Protein Assay Reagent Kit
(Pierce, USA) was used to determine protein concentration of each sample as per manufacturer's instructions. The samples were read on a Spectra MAX plus spectrophotometer (Molecular Devices Corporation, USA) at an absorbency of
562nm using SOFf max PRO computer program (Version 2.2.1, 1998, Molecular
Devices Corporation, USA).
Following protein estimation, 5 µg of protein were mixed with 7 .5 µI of 4 x SOS
protein loading sample buffer (500µ1 lM Tris-HCI ph 6.8, 800µL 100% glycerol
(Merck), 900µ1 20% SOS, 400µ1 0.005% bromophenol blue (Sigma), 500µ1 dH 20)) and 2µ1 of 0.5M OTT was added and boiled for 5 mins at 100° C and then
immediately placed on ice before 2µ1 of 0.5M iodoacetamide (Sigma) was added to
each sample. Samples were loaded onto 10% SOS resolving gel (2.5ml of 1.5M Tris
pH 8.8, 2.5ml 40% denaturating acrylamide/bis (Bio-Rad), 100µ1 10% SOS, 5µ1
TEMED (Progen Industries, Qld, Australia), 50µ1 10% ammonium persulphate
(APS) (Sigma), 4.86ml dH20) and a stacking gel ( 1.26ml 0.5M Tris pH 6.8, 500µ1
40% denaturating acrylamide/bis, 50µ1 10% SOS, 5µ1 TEMED, 25µ1 10% APS and
3.16ml dH20) and electrophoresis ran at 100V in 1x SOS running buffer (6g Tris
base, 28.8g glycine (ICN), 2g SOS and dH20 up to lL) until bromophenol blue dye
ran off the gel. Chapter 4: Materials and Methods 106
Transfer buffer was prepared (3g Tris base, 200ml methanol (APS Finechem, NSW,
Australia) and dH20 up to lL) and a Immobilon-P polyvinyldene fluoride nylon
transfer membrane (Millipore, USA) was cut to size, presoaked in 100% ethanol and
thoroughly rinsed with RO- H20. Following electrophoresis, the gel was loaded into
the transfer sandwich in pre-cooled transfer buffer and run at 100V in transfer buffer for lh. The membrane was air-dried and gel was stained for lh in GelCode Blue
stain reagent (Pierce) and rinsed in RO-H20 for lh. The dried membrane was then
washed with 100% ethanol and RO- H20 and placed into 5% skim milk powder in
0.05% Tween 20 (Sigma) in PBS at 4°C overnight.
The following day, the membrane was washed three times for 15 mins with Tween
20-PBS at RT. Primary polyclonal rabbit antibodies to ICAM-1, VCAM-1, E
selectin, JAM-1, p-JNK-1, beta-actin and VE-cadherin (Santa Cruz Biotechnology,
Inc., Alexis Biochemicals) were diluted 1: 1000 with 1% bovine serum albumin
(BSA) (Sigma) in Tween 20-PBS and applied to the membrane for 1 h at RT
following which the membrane was washed again three times with Tween 20-PBS
for 15 mins at RT. Once the washing was complete a secondary goat anti-rabbit IgG
anitibody conjugated with horseradish peroxidase in dilution 1: 1000 in 1%
BSAffween 20-PBS was added. Following 1 h incubation at RT, secondary antibody
was washed 3x with Tweeen 20 in PBS for 15 mins. Chapter 4: Materials and Methods 107
Chemilluminescent visualisation was performed mixing equal parts of each
Renaissance chemiluminescence reagents (NEN Life Sciences, USA), which were
incubated with the membrane for 1 min. The membrane was gently blotted twice on
blotting paper and placed into a clear plastic bag. The membrane was exposed to X
Ray Hyperfilm (Amershem Life Science, Eng UK) for various time points and the film was developed manually to control the intensity of bands as well as background.
4.7 Co-culture model of inflammation
HMEC-1 cells were plated on fibronectin-coated wells of a 24-well plate at 0.5x 106 cells/well and grown in MCDB 131 medium containing glutamine and 10% FBS. 80-
90% confluent cells were double transfected with either 0.05 µM of Dz13 / Dz13scr or 0.2 µM of c-Jun siRNA/siRNAscr and stimulated with IL-113 as described in the
section 4.2. Six hours later serum free medium was removed and monocytes (THP-1
cell line) were added to each well at 0.25x 106 per well. The plate was incubated at
37°C for 30 minutes then washed three times gently with 1 ml of warm sterile PBS
to remove all non-adherent cells. Monocytes adherent to the endothelium were
counted as the number of translucent cells per visual field using phase-contrast
Olympus microscope (X 100) magnification. There was no difference in endothelial
cells density in a 24-well plate by visual assessment.
In the reverse experiment, THP-1 monocytic cells were placed in RPMI-1640 (Gibco
USA) medium with 5% FBS and transfected with 0.05 µM of Dz13 or Dz13scr twice
as outlined in section 4.2. HMEC-1 cells were stimulated with 20 ng/ml of IL-113. Chapter 4: Materials and Methods 108
THP-1 cells were added to HMEC-1 cells at a concentration of 0.25x 106 per well.
Incubation of THP-1 with HMEC-1 and cell counting was identical to the protocol outlined above. Using ATC-labelled Dz13scr THP-1 cells were double transfected with 0.05 µM of Dz13scr as per the protocol outlined in section 4.2. Chapter 5: DNAzymes targeting transcription factors c-Jun and Egr-1 as novel anti inflammatory agents in model systems 109
Chapter 5: DNAzymes targeting transcription factors c-Jun and Egr-1 as novel anti-inflammatory agents in model systems
5.1 Introduction and Aim
Inflammation is a principal pathological process that forms a foundation of many common diseases such as atherosclerosis, sepsis, rheumatoid arthritis and asthma.
These and many other inflammatory conditions are characterized by endothelial activation and recruitment of inflammatory cells to the sites of inflammation. Our understanding of the molecular events that control cell movement during inflammation has grown enormously over the last two decades, however, this knowledge has not yet translated into the availability of better and safer anti inflammatory drugs. Thus, there is an ongoing effort to find more specific therapies to target inflammation and a gene-based approach seems to offer a potential advantage over the standard anti-inflammatory therapies based on steroidal and non steroidal drugs that are often flawed with significant side effects for the patients.
Our interest in the immediate early response transcription factors Egr-1 and c-Jun in inflammation was based on the special role of these molecules in regulating the expression of multiple proinflammatory genes in a wide range of conditions such as atherosclerosis (Wang et al. 2001; Harja et al. 2004), inflammation (Pawlinski et al.
2003) and ischaemia-reperfusion (Yan et al. 2000; Lysiak et al. 2003). DNAzyme- Chapter 5: DNAzymes targeting transcription factors c-Jun and Egr-1 as novel anti inflammatory agents in model systems 110 based technology that targets these transcription factors has been successfully applied to a variety of animal models by our laboratory (Santiago et al. 1999; Lowe et al.
2001; Khachigian et al. 2002; Lowe et al. 2002) but has not yet been tested by us or others in the setting of an acute inflammation. We hypothesized that blocking c-Jun with Dz13 (Khachigian et al. 2002), and Egr-1 with EDS (Santiago et al. 1999) would be beneficial in acute inflammation. To test our hypothesis further we chose a well established model of microcirculation that utilizes rat mesenteric venules. This model is well characterised, has been used extensively to study inflammation in vivo and was ideal for the local delivery of IL-1 ~ and DNAzymes (Matheson and Garrison
2005). We then extended our findings to different in vitro models of inflammation to look for a cause-effect relationship between the inhibition of c-Jun and downregulation of the genes known to be involved in modulating an inflammatory response.
5.2 Results
5.2.1 Dzl3 and EDS abolish inflammation in rat mesenteric venules
Leucocyte flux, adhesion and extravasation are three main parameters that are routinely used to assess pro-inflammatory properties of different agents in models of microcirculation in vivo and were carefully examined in this study.
Leucocyte flux is regulated predominantly by selectins expressed on the surface of activated endothelium and rolling cells; it refers to the movement of cells through a Chapter 5: DNAzymes targeting transcription factors c-Jun and Egr-1 as novel anti inflammatory agents in model systems 111 blood vessel and was calculated as a number of rolling white cells crossing a set point in the vessel per minute.
Leucocyte adhesion requires enhanced expression of adhesion molecules by endothelium with corresponding upregulation of their ligands on the surface of circulating blood cells. Leucocyte adhesion invariably increases in response to inflammation. Adherent leucocytes were counted per 100 µm of vessel and only the cells that remained stationary for 30sec or longer were considered as adherent.
Extravasation is a final stage of a simplified three-step process of leucocyte recruitment by proinflammatory stimuli and refers to a leucocyte that exits from the intravascular to the perivascular space; it is predominantly controlled by families of cadherins and junctional adhesion molecules. Extravasation was counted as a number of perivascular white blood cells per microscopic visual field.
Targeting transcription factors Egr-1 with EDS and c-Jun with Dz13 was very effective in reducing all three parameters of inflammation assessed in this study.
There was no significant difference between leucocyte flux, adhesion and extravasation in the control group at baseline and 60min with corresponding values for each parameter of 21± 4.47 versus 21.1 ±5.47 (Fig 5.1), 1± 0.27 versus 1.4 ± 0.42
(Fig 5.2) and 5 ± 0.68 versus 7 .3 ± 0.9 respectively (Fig 5.3). As expected, IL-1 j3 delivered topically via a continuous superfusion for 60min, produced an acute inflammatory response that led to a dramatic increase in all three parameters of Chapter 5: DNAzymes targeting transcription factors c-Jun and Egr-1 as novel anti inflammatory agents in model systems 112 inflammation. Leucocyte flux increased from 27±4.16 at baseline to 60.8± 7.34 at
60min (Fig 5.1) and both adhesion and extravasation went up from 0.8±0.25 and
4.9±0.4 (Fig 5.2) to 9.9±1.03 and 20±3.28 respectively (Fig 5.3).
Dz 13 and EDS applied to the rat mesentery 10min prior to the start of IL-1 ~ superfusion effectively blocked the inflammatory response induced by this cytokine.
Dz13 but not Dz13scr reduced leucocyte flux following 60min of superfusion with
IL-1~ from 49.4 ± 8.7 to 27.9 ± 6.3 (Fig 5.1), leucocyte adhesion from 9.6 ± 1.34 to
1.6 ± 0.46 (Fig 5.2) and extravasation from 19.9 ± 3.3 to 7.6 ± 1.23 (Fig 5.3).
Topical delivery of EDS was also extremely effective in blocking IL-1~ driven acute inflammation in a rat mesenteric venule. In comparison to ED5scr leucocyte flux was reduced from 39.4 ± 4.16 to 13.7 ± 2.49 at 60min (Fig 5.1). Leucocyte adhesion was virtually completely blocked from 11.4 ± 1.16 cells in ED5scr treated group at 60min to 1.0 ± 0.53 cells in a group that received EDS prior to IL-1~ exposure (Fig 5.2).
Similarly, leucocyte extravasation in EDS treated vessels was substantially lower at
60min as compared to ED5scr treated group with corresponding values of 22.1 ± 2.07 and 8.4 ± 0.78 respectively (Fig 5.3).
5.2.2 FITC labelled DZ13scr transfection in vivo
FITC-labelled DZ13scr was applied to rat mesenteric venules (n=2) for lOmin as
indicated in the protocol described in section 4.3.1.2. A discrete, punctate Chapter 5: DNAzymes targeting transcription factors c-Jun and Egr-1 as novel anti inflammatory agents in model systems 113 fluorescence in the inner layer of the mesenteric venule representing an uptake of
DNAzyme by endothelial cells, was observed in a vessel treated with FITC-Dz13scr
(Fig 5.4 A) while a control venule did not show punctate fluorescence but rather
uniform low-grade green light fluorescence throughout the whole vessel wall (Fig 5.4
B).
5.2.3 Leucocyte rolling velocities in vivo
Leucocyte rolling velocity was measured in control venules, venules treated with
Dz13, Dz13scr and IL-1 Bto ascertain the effect of Dz13 administration on leucocyte
rolling in vivo. A significant drop in leucocyte rolling velocity was observed following administration of IL-1 Bfor 60min from 52 ± 1.45 in the control group to
22 ± 2.6 µm s- 1 for the group that received IL-1 B- However, pre-treatment of
mesenteric venules with Dz13 for lOmin prior to the delivery of IL-lB led to a
significant increase in leucocyte velocity to 95 ± 6.0, which is 50% above the level
seen in the control group at 60min (Fig 5.5). Leucocyte rolling velocity in Dz 13cr
group was 33 ± 5.4 µm s- 1, which was similar to that observed in IL-1 Btreated
venulae. Figure 5.1: Dz13 and EDS inhibit leucocyte rolling following 60min of superfusion of the rat mesentery with IL- lf} in vivo
Microcirculation studies of the rat mesenteric venule were performed as described in section 4.3 .1.1 of "Materials and Methods".
EDS and Dzl3 were delivered in 100 µl of solution containing 35 µg of DNAzyme with the rest being a transfection agent and MgCI. A significant inhibition of flux was seen with both agents as compared to their scrambled counterparts.
*Indicates statistically significant difference in leucocyte flux between Dzl3 vs Dzl3scr and EDS vs ED5scr treated groups by Student's t-test with p <0.05, n=8 per each treatment group.
## Indicates statistically significant difference in leucocyte flux between Il-113 and
EDSscr treated groups by Student's t-test with p <0.05; n=8 per each treatment group. Chapter 5: DNAzymes targeting tran cription factor c-Jun and Egr-1 a novel anti- inflammatory agents in model y tern 114
D Control D IL- 1~
~ Dzl3+1L- I~
[Il] Dzl3scr+ IL- 1~ -:: ....::"' D ED5+ IL- 1~ ~ ~ II ~ ED5 er+ IL- 1 ~ i( :I • C e/J JO .5 0 I. ..:i ~ )) 0 II :I II J IO
60min Figure 5.2: Dz13 and EDS inhibit leucocyte adhesion foil owing 60min of superfusion of the rat mesentery with IL-lP in vivo
Microcirculation studies of the rat mesenteric venule were performed as described in section 4.3 .1.1 of "Materials and Methods".
EDS and Dz13 were delivered in 100 µl of solution containing 35 µg of DNAzyme with the rest being a transfection agent and MgCI. Leucocyte adhesion was completely suppressed by both DNAzymes but not their scrambled counterparts.
*Indicates statistically significant difference between Dz13 vs Dz13scr and EDS vs
ED5scr treated groups by Student's t-test with p <0.05, n=8 per each treatment group. Chapter 5: DNAzymes targeting transcription factors c-Jun and Egr-1 as novel anti inflammatory agents in model systems 115
1-1
12
Control 10 D t: -~ IL- 1~ .::C D i 1/J 1/J II ..> § Dzl3+IL-l~ 0 E i Oz 13scr+ IL- 1~ I. [II] II Q, C 0 wt~7 '@ ,;-...... ED5+ IL-1~ 'vi ~ II ~~j; fJ t: '\,-.$ 'O ('l @·'··' II ED5scr+ IL-1 ~ ... m~'Wffe~-:-.~ J >, CJ 0 • CJ ::l II -l
0 60min Figure 5.3: Dz13 and EDS inhibit leucocyte extravasation following
60min of superfusion of the rat mesentery with IL- tp in vivo
Microcirculation studies of the rat mesenteric venule were performed according to the protocol described in section 4.3.1.1 of "Materials and Methods".
EDS and Dz13 were delivered in 100 µl of solution containing 35 µg of DNAzyme with the rest being a transfection agent and MgCl. Leucocyte adhesion was completely suppressed by both DNAzymes but not their scrambled counterparts.
*Indicates statistically significant difference between Dz13 vs Dz13scr and EDS vs
ED5scr treated groups by Student's t-test with p <0.05, n=8 per each treatment group. Chapter 5: DNAzymes targeting transcription factors c-Jun and Egr-1 as novel anti inflammatory agents in model systems 116
30
,-.. 't) "ii 25 i: D Control -; -{/)~= ::::. 0 IL-1~ -; ~ 20 "ii {/) {/) 5I Dzl3+IL- I~ ;,.~ .c..~ ~ 't) -~ 15 0= ~ "ii ._,II C 0 -;: 10 C':I {/) C':I ;,. C':I ..I. l< ~ ~ ...... II 0 II
=~ .J
0 60min Figure 5.4: FITC-labelled Dz13scr uptake by endothelial cells in vivo
FITC-labelled Dz13scr is seen localised to the endothelial cells of a mesenteric venule
(panel A) as evidenced by a fluorescent signal identified in the innermost layer of the venulae (white arrow), which corresponds to the endothelial layer of the vessel. Panel C displays a magnified view of punctate fluorescence marked with three thin white arrows.
On the other hand, no punctate fluorescence is seen in the control vessel not treated with
FITC-labelled Dz13scr (panel B), which demonstrates a uniform fluorescence through the entire vessel wall. Chapter 5: DNAzymes targeting transcription factors c-Jun and Egr-1 as novel anti inflammatory agents in model systems 117 Figure 5.5: Rolling leucocyte velocity
Rolling leucocyte velocity was measured in control, IL-1(3, Dz13 and Dz13scr groups. It was calculated as the time required for a randomly selected leucocyte to cross 100 microns of the vessel length. Velocity was recorded for 8 leucocytes in each treatment group.
*Indicates statistically significant difference between control and IL-1 (3 treated groups by
Student's t-test with p <0.05.
++ Indicates statistically significant difference between Dz13 and Dz13scr treated groups by Student's t-test with p <0.05. Chapter 5: DNAzymes targeting transcription factors c-Jun and Egr-1 as novel anti inflammatory agents in model systems 118
120 -.------,
100
~ QJ r,:i a so ::::t 0 ~ ·-0 1 60 OJ:) ·-C -0 i. ## QJ 40 ;;.. -~ 0 ~ * =QJ ~ 20
0 -I----'---,.______
D Control IL-1(3
~ Dz 13 + IL-1 f3 Ii Dz13scr +IL-1 f3 Chapter 5: DNAzymes targeting transcription factors c-Jun and Egr-1 as novel anti inflammatory agents in model systems 119
5.2.4 Dz13 and c-Jun siRNA inhibit adhesion of monocytes to the endothelial cells in a co-culture model of inflammation in vitro A co-culture model of inflammation was used to assess the effect of c-Jun inhibition on monocyte adhesion to the cytokine-stimulated endothelial cells in vitro as described in section 4.7 of Chapter 4. c-Jun expression was targeted with two different gene-silencing techniques: firstly, c-Jun specific siRNA (Fig 5.6) and secondly, DNAzyme (Dz13) (Fig 5.7). Both methods were highly successful in blocking adhesion of THP-1 monocytic cells to the IL-1 Bactivated HMEC-1 in vitro.
Pre-treatment of HMEC-1 with IL-1 Bincreased monocyte adhesion from 42 ± 2.6 cells to 136.5 ± 5.7 per well. c-Jun siRNA reduced the number of adherent monocytes to 46 ± 2.6 cells per well as compared to 111.5 ± 4.7 per well in the scrambled group
( 3 wells per each treatment group in 2 independent experiments). Similarly, Dz13 reduced the number of monocytes attached to the stimulated endothelial cells from
342 ± 16.2 for Dz 13scr to 83 ± 14.1 in Dz13 treated group (3 wells per each treatment group in 2 independent experiments) (Fig 5.7 A).
In reverse experiments, which involved stimulation of HMEC-1 with IL-1 Bfollowed by adding monocytes transfected with either Dz13 or Dz13scr, no suppression of monocyte adhesion was seen in cells that received Dz13 versus Dz13scr. In these experiments monocyte adhesion in the control group was 47.7 ± 3.0 and while it was significantly increased in the IL-lB treated group (307±32.3), no difference was seen between Dz13 and Dz13scr groups with corresponding values of 293 ± 15.7 and Chapter 5: DNAzymes targeting transcription factors c-Jun and Egr-1 as novel anti inflammatory agents in model systems 120
302±43 respectively (Fig 5.7 B). Monocyte transfection with FITC-labelled Dz13scr confirmed high level of DNAzyme uptake in the treatment group (Fig 5.8 A) as compared to the control (Fig 5.8 B). Figure 5.6: c-Jun siRNA but not c-Jun siRNAscr inhibits adhesion of
THP-1 cells to the activated HMEC-1 in vitro
HMEC-1 was stimulated with 20ng/ml of IL-lj3 and transfected with c-Jun siRNA or its scramble counterpart before the addition of THP-1 cells as outlined in section 4.2 of the "Materials and Methods". Three wells were counted in each experimental group. The data is representative of 2 independent experiments.
* Indicates statistically significant difference between siRNA and siRNAscr treated groups by Student's t-test, p < 0.05. Chapter 5: DNAzymes targeting transcription factors c-Jun and Egr-1 as novel anti inflammatory agents in model systems 121
160
140
120 'Ii Q,I -u~ 0 C 0 100 E C -Q,I I,, Q,I .c 80 "O ~ I,., 0 I,, Q,I 60 .c E * :: z 40
20
0 eo...... eo.. ~ .... Joo,oi ~ + Joo,oi ~ + :i. N i 0 N :.. ~ 0 [I) :z< :z< ~ ~ [I) [I) 0 ·- ·- -:.. eo.. C C .... ::, ::, I C ~ ~ -0 ~ I I u Joo,oi ~ ~ Figure 5.7: Dz13 but not Dz13scr inhibits adhesion of THP-1 cells to the activated HMEC-1 in vitro
Panel A: HMEC-1 cells were stimulated with 20ng/ml of IL-1 f3 and transfected with
either Dz13 or Dz13scr before the addition of THP-1 cells as outlined in the section
4.2; Transfection of the endothelial cells with Dz13 but not Dz13scr significantly
reduced adhesion of THP-1 cells to their surface. Three wells were counted per each
treatment group and the data is representative of 2 independent experiments.
Panel B: HMEC-1 cells were stimulated with 20ng/ml of IL-lf3 but not transfected
with DNAzymes, instead THP-1 were transfected with either Dz13 or Dz13scr before they were added.to HMEC-1. Transfection of THP-1 with Dz13 did not reduce their
adhesion to the IL-1 f3 stimulated endothelial cells, the effect shown in Panel A. Three
wells were analyzed per treatment group and the data is representative of 2
independent experiments.
* Indicates statistically significant difference between Dz 13 and Dz 13scr treated
groups by Student's t-test, p < 0.05. Chapter 5: DNAzymes targeting transcription factors c-Jun and Egr-1 as novel anti inflammatory agents in model systems 122
450
400 A r,i ....~ CJ;,.. 350 0 =0 300 ....8
=~ 250 i. .c~ "O 200 ~ ~ 0 i. 150 ~ .Q 8 100 z= 50
0
400--~------,
r,i ....~ 350 ,>, CJ O 300 =0 6 ..,. 250
=~ i.. ~ 200 .c "O ~ I.., 0 i. ~ ,i:. 6 z:, 50
0
-....0s.. =0 u Figure 5.8: Monocyte FITC-Dz13scr transfection in vivo
THP-1 cells were transfected with HTC-labelled Dz13scr according to the protocol described in section 4.7 of Chapter 4
Panel A demonstrates FITC uptake by transfected THP-1 cells (white thin arrows).
Panel B shows no fluorescence in control group that did not receive HTC-labelled
Dz13scr ( a non-transfected THP-1 cell is circled in white). Chapter 5: DNAzymes targeting transcription factors c-Jun and Egr-1 as novel anti inflammatory agents in model systems 123
A
FITC-Dz13scr transfected THP-1 cells
B Chapter 5: DNAzymes targeting transcription factors c-Jun and Egr-1 as novel anti inflammatory agents in model systems 124
5.2.5 Dzl.3 inhibits the expression of c-Jun and multiple proinflammatory genes in IL-1/J stimulated endothelial cells in vitro
We found a time-dependent increase in c-Jun protein expression by Western blot that peaked at 4 and 8 hours and returned to the basal level by 24h and a progressive induction of ICAM-1 protein expression over a 24-hour period following stimulation of HMEC with IL-1 f3 (Fig 5.9 A). Importantly, the expression of c-Jun and other genes involved in inflammation such as E-selectin, ICAM-1 and VCAM-1 was abolished or significantly reduced (VE-cadherin) by a prior transfection of HMEC with Dz13 but not Dz13scr (Fig 5.9 B).The expression of other adhesion molecules, namely PECAM-1 and JAM-I was unaffected by Dz13. Moreover, Dz13 had no effect on level of JNK-1, an upstream regulator of c-Jun phosphorylation that enhances transcriptional activity of c-Jun, thus emphasizing the specificity of Dz13 for c-Jun mRNA. Beta actin levels were equal between all samples (Fig 5.10).
5.2.6 Dzl.3 inhibits expression of c-Jun and other genes in rat mesenteric venules in vivo
Extensive immunohistochemistry performed on the venules treated with either Dz13 or Dz13scr showed virtually a complete suppression of c-Jun expression (Fig 5.11) and a significant reduction in expression of ICAM-1 (Fig 5.12), VCAM-1, E-selectin and VE- cadherin while the expression of PECAM-1 and JAM-I (Fig.5.13) was largely unchanged by the treatment with Dz13 (Table 5.1). Figure 5.9: Expression of c-Jun and other genes by Western blot
(A) Western blot analysis of total extracts ofHMEC-1 exposed to 20 ng/ml ofIL-1~ for the times indicated using antibodies to c-Jun and ICAM-1 shows a bi-phasic pattern of induction of c-Jun protein expression (maximum at 4 and 8 hours) and time-dependent progressive increase in ICAM-1 protein expression over a 24 hour period.
(B) Western blot analysis of total extracts of HMEC-1 exposed to 20 ng/ml of IL-1 ~ with extracts harvested 4h after cytokine treatment with indicated antibodies demonstrates that pre-treatment of HMEC-1 with Dz13 but not Dz13scr abolishes the expression of c-Jun, E-~electin, VCAM-1 and ICAM-1, reduces the expression of
VE-cadherin and does not affect the expression of PECAM-1, JAM-I or JNK-1.
These experiments were performed by Mr. Roger Fahmy Chapter 5: DNAzymes targeting transcription factors c-Jun and Egr-1 as novel anti inflammatory agents in model systems 125
0 1 2 4 6 8 24h IL-1beta
37kD•· I I c-Jun
82-1 I ICAM-1
A Coomassie
] 2 3 4
llkD•·I c-Jun 1. Control 150, E-selectin 2. IL-lfl 82, VCAM-1 3. Dz13+IL-lfl
82, ICAM-1 B 4. Dz13scr+ IL-lfl 135, VE-cadherin
Jl-1 I JAM-1 150-1 I PECAM-1
&4.1 I p-JNK-1
J).I I beta-Actin I Coomassie I Figure 5.10: Expression of c-Jun and other genes by Western blot
The expression of different genes by Western blot was normalized to beta-actin. As seen prior to normalization, Dz13 abolished the inducible expression of c-Jun, E selectin, VCAM-1, ICAM-1 and VE-cadherin without affecting the expression of
PECAM-1, JAM-I or JNK-1 confirming that the changes in gene expression seen with Dz13 and Dz13scr were not related to the difference in loading of the samples.
* Indicates statistically significant difference between Dz13 treated and Dz13scr groups by Student's t-test, p <0.05. The data is representative of 2 independent experiments. Chapter 5: DNAzymes targeting transcription factors c-Jun and Egr-1 as novel anti inflammatory agents in model systems 126
1 D No addition ·-..-C 0.9 LJ IL-1beta (.) D IL·1beta+ Dz13 i .0 0.7 Xi 0 :,: .... 0.6 O.> > .... 0.5 ::i i ·-~ ·:, 1 -O.> t ~i ~ 0.4 , I C ' I 01 = I 0.3 ::r:,.,.;= ':! ·-IJJ1 l.l IJ) - - lI I '. O.> 0,2 l :!c .. 1 ' - ,:;i; c. "' ,',, )( J *' II* .I - lI ; IJJ 0.1 :r :!< ::cc, !J ! 0 ~ I~ rLJi Figure 5.11: Immunohistochemical staining of venular endothelial cells for c-Jun
IHC was performed as per the protocol outlined in section 4.4 of Materials and Methods using rabbit polyclonal antibody to c-Jun in a dilution of 1:80 (Santa Cruz Biotechnology,
Inc). c-Jun was visualised with AEC chromogen; no counterstain was used.
Panel A shows the absence of any detectable staining within the vessel treated with Dz13 as indicated by a white arrows pointing to the vessel wall, while Panel C demonstrates abundant staining for c-Jun through the different layers of the vessel wall in the vein treated with Dz13scr (x 200 magnification) appearing as punctate red pigment. Panel B depicts a part of the same vessel wall as panel C taken at higher magnification (x 600) and shows localisation of c-Jun to endothelial (regular black arrow) and SMC layer (open head arrow). Chapter 5: DNAzymes targeting transcription factors c-Jun and Egr-1 as novel anti inflammatory agents in model systems 127
C Figure 5.12: Immunohistochemical staining of venular endothelial cells forICAM-1
IHC was performed as per the protocol outlined in section 4.4 of Materials and Methods using goat anti-rat ICAM-1 primary antibody (R&D Systems, Inc) in a 1:50 dilution.
ICAM-1 was visualised with AEC chromogen; the slides were counterstained with haematoxylin for 10sec.
Panel A shows the absence of any detectable staining within the vessel treated with Dz13 while Panel C demonstrates abundant staining for ICAM-1 within the endothelial layer of the vein treated with Dz13scr seen as red pigment (x200 magnification). Panel B depicts a part of the same vessel wall as Panel C taken at higher magnification (x600) and reveals red pigment stain within the endothelial cells (black arrow). Chapter 5: DNAzymes targeting transcription factors c-Jun and Egr-1 as novel anti inflammatory agents in model systems 128 Figure 5.13: Immunohistochemical staining of venular endothelial cells for JAM-1
IHC was performed as per the protocol outlined in section 4.4 of Materials and Methods using rabbit polyclonal primary antibody (Santa Cruz
Biotechnology, Inc) in a 1:50 dilution. JAM-1 was visualised with AEC chromogen; the slides were counterstained with haematoxylin for 10sec.
Abundant staining for JAM-1 is seen in both Dz13 treated vessel (Panel A) and in Dz13scr treated venule (Panel C) within all three layers of the vein as red pigment (x200 magnification). Panel B depicts a part of the same vessel wall as Panel A taken at higher magnification (x600) and reveals red pigment stain within the endothelial cells (red arrow). Chapter 5: DNAzymes targeting transcription factors c-Jun and Egr-1 as novel anti inflammatory agents in model systems 129
. ~
,,
» f Table 5.1: The intensity of immunohistochemical staining for different antigens in the rat mesenteric venules
Minus sign (-) denotes no detectable staining.
The intensity of positive immunohistochemical staining was divided into thee categories: mild, corresponding to one plus(+), moderate, corresponding to two plus
(++) and strong, described as three plus(+++). Chapter 5: DNAzymes targeting transcription factors c-Jun and Egr-1 as novel anti inflammatory agents in model systems 130
Antigen Dz13 Dz13scr c-Jun - ++
E-selectin - ++
VCAM-1 - ++
ICAM-1 - +++
VE-cadherin - ++
JAM-1 ++ ++
PECAM-1 ++ ++
c-Fos ++/+++ ++/+++ Chapter 5: DNAzymes targeting transcription factors c-Jun and Egr-1 as novel anti inflammatory agents in model systems 131
5.3 Discussion
New strategies targeting the key regulators of inflammation can offer a novel therapeutic approach to the treatment of atherosclerosis and many other conditions linked to acute or chronic inflammation. Choosing the right target for genetic intervention is of paramount importance. The focus of the research for many years has been in identifying so-called "master regulators" of gene expression that can be specifically manipulated by early intervention. One such target is transcription factors that act as upstream regulators of gene expression. In particular, transcription factors c-Jun and Egr-1 are very attractive targets for intervention since they are immediate early response factors and their activation controls the expression of a vast number of downstream genes. Role of Egr-1 in different pathological settings is relatively well defined in comparison to the role of c-Jun due to the availability of Egr-1 knockout mice, Egr-1 decoys, antisense ODN and DNAzymes. Egr-1 has been coined as a
"master switch" involved in regulation of SMC proliferation, ischaemia-reperfusion and atherosclerosis in vivo (Santiago et al. 1999; Yan et al. 1999; Harja et al. 2004).
On the other hand, lack of specific c-Jun inhibitors and inability to generate c-Jun knockout mice hampered our understanding of the specific role played by this transcription factor in regulating complex biological processes.
This study has systematically examined the role of c-Jun in inflammation through in vitro and in vivo experiments. The data indicates that c-Jun plays a regulatory role in controlling leucocyte infiltration in vivo during an acute inflammatory response Chapter 5: DNAzymes targeting transcription factors c-Jun and Egr-1 as novel anti inflammatory agents in model systems 132 induced by 11-113 as evidenced by an almost complete suppression of inflammation following a topical delivery of Dz13 to the rat mesenteric venule. Extensive immunohistochemistry and Western blot analysis revealed some of the molecular mechanisms that link c-Jun to the regulation of an IL-ll3-driven inflammatory response. Similar to previous reports emphasizing the role of c-Jun in controlling the expression of E-selectin and adhesion molecules ICAM-1 and VCAM-1, this study found that Dz13 abolished the expression of c-Jun and resulted in a significant downregulation of E-selectin, ICAM, VCAM-1 and VE-cadherin in endothelial cells both in viva and in vitro. Even though it is generally accepted that VCAM-1 expression is under a predominant transcriptional regulation of NP-kappa B (Neish et al. 1992), more recent studies demonstrated that VCAM-1 promoter contains AP-1/ c-Jun binding sites the integrity of which is essential for VCAM-1 induction by NP kappa B in the context of TNP-a stimulation (Ahmad et al. 1998) . These results suggest that the binding sites for c-Jun identified through in vitro experiments on the promoter region of VCAM-1 (Ahmad et al. 1998) and ICAM-1 (Wang et al. 2001) are functionally significant in regulating the expression of these genes under the condition of IL-113- induced inflammation in viva. It is not known at present whether c-Jun directly regulates VE-cadherin transcription, although c-Jun recognition elements have been described on the rodent VE-cadherin promoter (Acc. No.
8D062710). Chapter 5: DNAzymes targeting transcription factors c-Jun and Egr-1 as novel anti inflammatory agents in model systems 133
The initial stage of the inflammatory response results in increased leucocyte flux to the sites of injury and is controlled by a family of selectins. Particular attention was paid to E-selectin in this study given the fact that this selectin is under partial transcriptional regulation of c-Jun (Lysiak et al. 2003) and is important for leucocyte recruitment (Kunkel and Ley 1996). It has been established that E-selectin supports leucocyte rolling at slow velocity. When leucocyte rolling velocity was examined in four groups of animals: control, IL-lj3, Dz13 and Dz13scr treated groups it became apparent that IL-113 caused a significant drop in rolling velocity while Dz13 administration led to a 50% increase in the rolling velocity compared to the control group. This change in rolling velocity was most likely achieved through inhibition of
E-selectin expression in vivo by Dz13. Administration of Dz13scr on the other hand, did not increase leucocyte velocity which was similar to the leucocyte velocity found in IL-113 treated animals. The role of E-selectin in modulating leucocyte flux is less clear due to a number of conflicting reports with some citing up to 50% reduction in leucocyte rolling flux when E-selectin is blocked (Olofsson et al. 1994; oude Egbrink et al. 2002) while others failed to demonstrate the same association (Labow et al.
1994; Milstone et al. 1998). Interestingly, in one of these studies, despite the lack of measurable difference in leucocyte flux between E+/E+ and E-;E selectin mice the latter displayed a significantly reduced density of leucocytes adhesion to the endothelium (Milstone et al. 1998). In addition, recently published data implicates E selectin involvement in LFA-1 activation leading to amplification of LFA-1/ICAM-1 dependent rolling and accelerated neutrophil recruitment to the sites of inflammation Chapter 5: DNAzymes targeting transcription factors c-Jun and Egr-1 as novel anti inflammatory agents in model systems 134
(Chesnutt et al. 2006). Thus, it is plausible to suggest that one of the mechanisms through which c-Jun-dependent inhibition of E-selectin expression by Dz13 affects leucocyte flux is the interference with LFA-1/ICAM-1 dependent rolling.
Furthermore, loss of E-selectin can contribute to the enhanced leucocyte adhesion by increasing leucocyte rolling velocity and decreasing contact time between rolling cells and endothelium. The role of other selectins in the context of c-Jun inhibition was not addressed by this study. It is also possible that altered rolling and adhesion may be due to c-Jun dependent activation and/or altered expression of pre-existing molecules such as P-selectin, even though no c-Jun binding sites have been identified on the P-selectin promoter so far.
It is not clear how c-Jun controls expression of VE-cadherin. Based on the presence of a putative AP-1 binding site on the promoter of rodent VE-cadherin it seems plausible to suggest that c-Jun may be involved in transcriptional regulation of this gene, however, further studies are needed to address this issue in more detail.
Contrary to the reduction in leucocyte transmigration observed in this study, others have reported that inhibition of VE-cadherin expression in vitro leads to the opening of lateral cellular junctions resulting in enhanced transmigration of neutrophils into the perivascular space (Gotsch et al. 1997; Hordijk et al. 1999). We believe that reduction in leucocyte transmigration by Dzl3 was secondary to the blocking of leucocyte adhesion to endothelium through c-Jun dependent downregulation of the Chapter 5: DNAzymes targeting transcription factors c-Jun and Egr-1 as novel anti inflammatory agents in model systems 135 expression of E-selectin and adhesion molecules ICAM-1 and VCAM-1 which was more profound compared to the inhibition of VE-cadherin.
Topically delivered Dz13scr was localised to the venular endothelial cells. It is well known that endothelial cell c-Jun-dependent gene expression is critical for regulation of inflammatory gene expression such as ICAM-1, VCAM-1 and E-selectin. On the other hand, in this study monocytes that incorporated Dz13 were refractory to any suppressive effect of c-Jun inhibition in these cells indicating that c-Jun pathway is not crucial to the control of adhesive properties of monocytes. Monocyte migration and adhesion involves complex interactions between monocyte surface receptors: f31 and f32 integrins and external ligands leading to the activation of transcription factors and inductions of genes encoding for IL-113, TNF-a and tissue factor (Fan and
Edgington 1991; Rezzonico et al. 2000). NF-KB nuclear transcription factor, that controls the expression of multiple inflammatory genes, has been identified as the main transcription factor involved in monocyte activation (Haskill et al. 1991) with no significant role in this process ascribed to c-Jun. Other signalling pathways upregulated by integrin ligation include various protein kinases such as ERK, FAK
(Shiratsuchi and Basson 2004), PI-3K (Reyes-Reyes et al. 2002), and Src-family kinases (Lin et al. 1995).
The role of Egr-1 in inflammation was examined in the context of rat mesenteric microcirculation studies only. Chapter 5: DNAzymes targeting transcription factors c-Jun and Egr-1 as novel anti inflammatory agents in model systems 136
Administration of EDS resulted in a virtual abrogation of inflammation - the result almost identical to the one observed with Dz13. Egr-1 is known to control a large family of genes involved in coagulation and inflammatory cascades. Of a particular relevance to this work are the findings of other researchers that established a profound downregulation of the expression of ICAM-1, MCP-1 and VCAM-1 in the absence of Egr-1 in murine models of ischaemia/reperfusion (Yan et al. 2000), endotoxemia (Pawlinski et al. 2003) and atherosclerosis (Harja et al. 2004). In addition, ICAM-1 promoter has been shown to have functional Egr-1 DNA-binding motifs, which allow for the direct induction of ICAM-1 by Egr-1 (Maltzman et al.
1996). Interestingly, in the model of LPS induced mouse endotoxemia no difference was seen in the target organ infiltration by inflammatory cells between Egr-1 +rand
Egr-1 -t mice leading authors to conclude that Egr-1 is unlikely to be involved in the regulation of the early inflammatory response (Pawlinski et al. 2003). However, our data suggests that Egr-1 is important in the recruitment of leucocytes to the mesenteric circulation in response to IL-1 p. Further in vivo and in vitro experiments are needed to clarify the role of Egr-1 as a regulator of the acute inflammatory response in different pathological settings.
A major discrepancy regarding our results was related to the following two observations: the finding that c-Jun blockade with Dz13 abolishes an acute
inflammatory response following topical application of IL-lP for 60min in vivo and the Western blot results which showed no significant increase in ICAM-1, VCAM-1 Chapter 5: DNAzymes targeting transcription factors c-Jun and Egr-1 as novel anti inflammatory agents in model systems 137 and E-selectin protein levels in the first few hours of IL-1 (3 stimulation in vitro. This is a consequence of typical differences between in vitro and in vivo model systems.
Acute changes in the mesenteric microcirculation in response to different stimuli in vivo are well documented with changes in leucocyte rolling, adhesion and extravasation observed as early as within the first 15min of a topical application of platelet activating factor (PAP), (Sanz et al. 2002; Sanz et al. 2005). By 60min the same authors reported a 4 fold increase in leucocyte rolling flux with a concomitant two-fold decrease in leucocyte rolling velocity while leucocyte adhesion and extravasation were 13 and 21 times higher respectively at the same time point compared to baseline (Sanz et al. 2002; Sanz et al. 2005). Furthermore, our Westerns were also totally consistent with published literature that most adhesion molecules require de novo synthesis following stimulation with pro-inflammatory cytokines so that significant changes in their protein levels are not seen for the first few hours and best appreciated from 4h onwards. This raises a very important question: can the inhibition of acute inflammatory response seen with topical administration of Dz13 be attributed to the reduction of the expression of the adhesion molecules studied in this work. To answer this question a few other lines of evidence derived from this study as well as the literature review were examined. First of all, this study demonstrated a significant increase in ICAM-1 and E-selectin expression by the venular endothelium at 60min as determined by IHC. This increase occurred much earlier in vivo than the corresponding change in the level of the same adhesion molecules expressions in vitro. To understand these findings better, one should Chapter 5: DNAzymes targeting transcription factors c-Jun and Egr-1 as novel anti inflammatory agents in model systems 138 consider significant differences that exist between in vitro and in viva environments.
While the in vitro model assessed a direct effect of cytokine stimulation on endothelial cells it does not take into account complex interactions between adhesion molecules expressed by activated endothelium and their corresponding leucocyte receptors. It is plausible to suggest that these interactions cause much greater and faster upregulation of the expression of adhesion molecules in viva as opposed to the in vitro setting possibly through a different pattern of adhesion molecules expression in cultured endothelial cells compared to their normal microenvironment in viva.
Indeed, other researchers have also found a rapid and dramatic upregulation in the expression of E-selectin and ICAM-1 in vitro measured directly by flow cytometry.
For example, in a study reported by Ahluwalia et al, the level of ICAM-1 was 470 times higher at 90min compared to the baseline while E-selectin expression increased only by 1.2 times following an intraperitoneal administration of IL-lj3 (Ahluwalia et al. 2004). So despite seemingly "contradictory" findings between the microcirculation study and Western blot data, there is no doubt that Dz13 inhibits c-Jun and adhesion molecules expression known to regulate leucocyte flux, adhesion and extravasation, and temporal differences observed in this study relate to the nature of the models used.
In summary, the use of c-Jun specific DNAzyme in this study allowed the examination of the role of this transcription factor in acute inflammation and showed that inhibition of c-Jun provides a powerful suppression of inflammation in viva and Chapter 5: DNAzymes targeting transcription factors c-Jun and Egr-1 as novel anti inflammatory agents in model systems 139 in vitro. These findings identify c-Jun as another "master regulator" of significant clinical importance and suggest that manipulation of c-Jun expression can provide new opportunities for intervention in the different pathological settings involving inflammation. Chapter 6: DNAzymes targeting c-Jun inhibits intimal thickening in a rabbit model of bypass surgery and in human saphenous veins in vitro 140
Chapter 6: DNAzyme targeting c-Jun inhibits intimal thickening in a rabbit model of bypass surgery and in cultured human saphenous veins in vitro
6.1 Introduction and Aim
Despite significant progress towards much better surgical survival of bypass grafts made in the last decade, the graft failure rate remains unacceptably high (Goldman et al. 2004). Gene based intervention to improve graft rate survival has been a topic of extensive research in recent years. So far no breakthrough in this area has been achieved despite many promising preliminary results obtained from animal studies
(Schachner et al. 2006) and early stages of human trials (Alexander et al. 2005).
The majority of animal studies have targeted a single gene identified by the investigators as an important causative factor in the development of vein graft neointima with a 30 to 50% reduction in NI formation in most cases (a summary of animal studies appears in table 3.1, pages 74-75 Chapter 3). The only human trial of gene based therapy for combating neointima development in bypassed saphenous veins was a trial of double-stranded decoy ODN that blocks transcription factor E2F, which is known to be involved in the regulation of a large number of cell-cycle genes.
Unfortunately, despite promising pre-clinical data, the results of a phase 3 human trial
(PREVENT IV) were disappointing as no difference was seen in the degree on neointimal formation between the treatment and control groups at 12 months
(Alexander et al. 2005). Chapter 6: DNAzymes targeting c-Jun inhibits intimal thickening in a rabbit model of bypass surgery and in human saphenous veins in vitro 141
Veins used for bypass surgery represent an ideal target for the gene-based therapy.
All veins have to be extracted (harvested) prior to implantation into the arterial circulation, which mean no extra steps are required for ex vivo transfection.
Importantly, even with the best surgical handling endothelial injury related to vein harvesting cannot be fully avoided, and can lead to accelerated neointima (NI) formation in arterialised vein grafts. Finally, co-existing problems such as hypercholesterolemia, high blood pressure and diabetes, which are very common in patients undergoing bypass surgery, are known to contribute significantly to a persistent inflammatory state and promote graft failure (Sacks et al. 1996; 1998;
Knatterud et al. 2000). Therefore, new therapies especially the ones that can be delivered locally to the targeted tissue are needed to effectively prolong vein graft survival. Animal models have been extensively used for preclinical evaluation of novel approaches for the treatment of vein graft failure. A rabbit model of bypass grafting is well characterized, reproducible and relatively cheap to perform. In addition, it can be used to study the effect of hypercholesterolemia on NI formation because rabbits fed a cholesterol-rich diet are known to develop extensive atherosclerotic lesions similar to human atheroma (Davies et al. 1999).
The purpose of this project was to evaluate the role of transcription factor c-Jun in the complex process of intimal thickening in arterialised vein grafts of cholesterol-fed and normal chow-fed rabbits by using DNAzyme that targets c-Jun. In addition, the Chapter 6: DNAzymes targeting c-Jun inhibits intimal thickening in a rabbit model of bypass surgery and in human saphenous veins in vitro 142 ability of Dz13 to inhibit intimal thickening in cultured human saphenous veins in vitro was examined.
6.2 Results
6.2.1 Dz13 but not Dz13scr inhibits neointima development in human saphenous veins in vitro
Human saphenous veins were grown in culture medium and double transfected with
Dz13 as outlined in section 4.5 of "Materials and Methods". Intimal hyperplasia was measured as the area bounded by the new intima on the inside and internal elastic lamina on the outside; the vessel media area was calculated as an area between the internal elastic lamina on the inside and external elastic lamina on the outside.
Neointimal thickening is expressed as a ratio of the area occupied by intimal hyperplasia to the area of the media.
NI to media ratio was 11.7 ± 2.1 % in Dz13 treated veins (n=5) as compared to 18.6 ±
2.6 (n=5) and 17.8 ± 0.85 (n=4) for Dz13scr treated and control veins respectively
(Fig 6.1). Representative images of Dz13 and Dz13scr treated saphenous veins are shown in figure 6.2 (Fig 6.2). Figure 6.1: Dz13 but not Dz13scr inhibits neointima formation in cultured human saphenous veins in vitro
Saphenous veins treated with Dz13 (n=S) displayed a significant 37% reduction in NI formation compared to Dz13scr (n=S) treated and control veins (n=4).
*Indicates statistically significant difference in NI formation between Dz13 and Dz13scr treated groups by Student t-test with p value of <0.05. Chapter 6: DNAzymes targeting c-Jun inhibits intimal thickening in a rabbit model of bypass surgery and in human saphenous veins in vitro 143
25 --,------,
...0 20 ,-1 ~ I. ...~ ~ QJ e 15 * 0 ,-1 ...e~ ,-1 10 ... =0 zQJ ~ 5
0 ...... ___
Control Dz13 0.4µM Dz13scr 0.4µM Figure 6.2: Representative images of saphenous veins treated with
DNAzyme
Panel A shows a saphenous vein treated with Dz13scr that displays significantly more NI formation compared to the vein in panel B treated with Dz13.
"M" refers to the vein medial layer.
"Ad" refers to adventitia.
The adventitia is partially cut off in image B. Chapter 6: DNAzymes targeting c-Jun inhibits intimal thickening in a rabbit model of bypass surgery and in human saphenous veins in vitro 144
Internal elastic Neointima lamina
Neointima
Internal elastic lamina Chapter 6: DNAzymes targeting c-Jun inhibits intimal thickening in a rabbit model of bypass surgery and in human saphenous veins in vitro 145
6.2.2 Dz13 but not Dzl3scr inhibits neointimaformation in cholesterol
/ ed rabbits at 21 days post surgery
Veins of cholesterol-fed animals were harvested 21days post-operatively. In total 24
veins were collected but only 13 were available for the analysis of NI. The rest of the
specimens were either completely occluded or thrombosed. Both the control and the
Dz13scr group had a very high intima to the whole vessel area ratio with corresponding values of 69 ± 5.2 and 68 ± 8.5 respectively. This ratio was
significantly reduced to 27± 6.8 by the treatment with Dz13 (Fig 6.3).
6.2.3. Dzl3 but not Dz13scr inhibits the development of neointima in bypassed vein grafts of normal chow-fed rabbits
Vein grafts treated with a topical application of Dz13 for 30 min prior to implantation
into the arterial circulation displayed a prominent reduction in intimal thickening
development. NI thickening was reduced by 41 % from 26.1 ± 4.5 in the Dz 13scr
group to 15.3 ± 2 in Dz13 treated veins by 28 days of follow up (Fig 6.4). No
difference was seen between NI development in control: 25.8 ± 1.7 versus DZ13scr:
26.1 ± 4.5 groups. Figure 6.3: Dz13 but not Dz13scr inhibits neointima formation in arterialised vein grafts of cholesterol fed rabbits
Vein grafts of cholesterol-fed rabbits (n=4) treated with 200 µg of Dz13 demonstrated a significant reduction in NI formation in comparison to the veins treated with 200 µg of
Dz13scr (n=4); n=S for the Control group.
A photograph of a failed vein graft shows a large intraluminal thrombus marked by "T".
*Indicates statistically significant difference in NI formation between Dz13 and Dz13scr treated groups by Student t-test, p value <0.05. Chapter 6: DNAzymes targeting c-Jun inhibits intimal thickening in a rabbit model of bypass surgery and in human saphenous veins in vitro 146
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Control Dz13 Dz13scr Figure 6.4: Dzl3 but not Dzl3scr inhibits neointima formation in arterialised vein grafts of normal chow fed rabbits
Vein grafts of non-cholesterol fed rabbits treated with 200 µg of Dz13 (n=6) showed a significant reduction in NI formation in comparison to the veins treated with 200 µg of
Dz13scr (n=5).
*Indicates statistically significant difference in NI formation between Dz13 and Dz13scr treated groups by ANOV A. Chapter 6: DNAzymes targeting c-Jun inhibits intimal thickening in a rabbit model of bypass surgery and in human saphenous veins in vitro 147
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Control Dz13 Dz13scr Chapter 6: DNAzymes targeting c-Jun inhibits intimal thickening in a rabbit model of bypass surgery and in human saphenous veins in vitro 148
Representative images of Dz13 and Dz13scr treated vessels are depicted in figure 6.5 and demonstrate significantly more NI in the vein treated with Dz13scr (image B) as compared to the vein that was treated with Dz13 (image A). Figure 6.6 shows the structure of new intima at high magnification (x400 times) and demonstrates a more compact appearance of NI in the vein that received Dz13scr.
6.2.4 Immunohistochemistry for SMC in 4 week-old rabbit vein grafts
The density of SMC stain per unit area was the same in Dz13 and Dz13scr-treated groups (Fig 6.7). However, the absolute number of SMC was greater in veins treated with Dz13scr due to the larger area of the NI in this group (Fig 6.5).
6.2.5 MMP-2 and MMP-9 expression in 4 week-old rabbit vein grafts by immunohistochemistry
IHC staining for MMP-2 and MMP-9 expression in vein grafts from animals fed a normal chow diet demonstrated a complete suppression of MMP-2 expression by
Dz13 while Dz13scr had no such effect (Fig 6.8). There was no detectable staining for MMP-9 in vein grafts treated with either Dz13 or Dz13scr.
6.2.6 Expression of RAM- 11 in 4 week-old rabbit vein grafts by immunohistocehmistry
IHC staining for a marker of macrophage expression, RAM-11 was negative in both
Dz13 and Dz13scr treated veins (Fig 6.9). Figure 6.5: Images of vein grafts treated with Dz13 and Dz13scr
Panel A is an example of a vein graft treated with 200 µg of Dz13. New intimal thickening was evenly distributed throughout the vessel.
Panel Bis a vein graft that received 200 µg of Dz13scr; it demonstrates considerably more intimal thickening compared to panel A with associated loss of the intraluminal diameter.
Both vein samples were collected at 28 days. Chapter 6: DNAzymes targeting c-Jun inhibits intimal thickening in a rabbit model of bypass surgery and in human saphenous veins in vitro 149 Figure 6.6: Structure of intimal thickening in arterialised vein grafts of non-cholesterol fed rabbits
Panel A shows the structure of intimal thickening in a vein graft treated with Dz13
(x400).
Panel B depicts the structure of intimal thickening in a vein graft treated with Dz13scr
(x400).
NI of DZ13scr treated veins is more compact and cellular in comparison to Dz13 group.
Both veins were collected at 28 days and treated with 200µg of DNAzyme. Chapter 6: DNAzymes targeting c-Jun inhibits intimal thickening in a rabbit model of bypass surgery and in human saphenous veins in vitro 150
... ,. Internal elastic lamina ,, ,. • •
Internal elastic Jamin Figure 6.7: Immunohistochemical staining of rabbit vein grafts for alpha SMC antigen
IHC was performed as detailed in section 4.4 of Chapter 4.
No difference was seen in the intensity of staining between Dzl3 treated vein grafts
(panel A) and the grafts that received Dz13scr (Panel B). Chapter 6: DNAzymes targeting c-Jun inhibits intimal thickening in a rabbit model of bypass surgery and in human saphenous veins in vitro 151
,,
A
B Figure 6.8: Immunohistochemical staining of chow-fed rabbit vein grafts for MMP-2
IHC was performed as detailed in section 4.4 of Chapter 4.
Treatment with 200 µg of Dz13 (panel A) completely suppressed MMP-2 expression (red staining seen in NI and media) in a 4 week old rabbit vein graft while treatment with
Dz13scr had no such effect (Panel B). Chapter 6: DNAzymes targeting c-Jun inhibits intimal thickening in a rabbit model of bypass surgery and in human saphenous veins in vitro 152
A
Internal elastic lamina Figure 6.9: Immunohistochemical staining of rabbit vein grafts for
RAM-11
IHC was performed as detailed in section 4.4 of Chapter 4.
No staining for RAM-11 was seen in rabbit veins treated with Dz13 (panel A) or Dz13scr
(Panel B) at 4 weeks following the surgery. Chapter 6: DNAzymes targeting c-Jun inhibits intimal thickening in a rabbit model of bypass surgery and in human saphenous veins in vitro 153
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\ .. B t : Chapter 6: DNAzymes targeting c-Jun inhibits intimal thickening in a rabbit model of bypass surgery and in human saphenous veins in vitro 154
6.3 Study limitations This project was initially designed to test the effect of Dz13 on intimal thickening development in bypassed vein grafts of cholesterol-fed rabbits based on the following considerations: the knowledge that morphological changes induced in rabbit vein grafts by a high level of cholesterol closely resemble the changes observed in human vein grafts (Lie et al. 1977; Zwolak et al. 1989) and the evidence that hypercholesterolemic animals exhibit more cellular infiltrate with a higher proportion of inflammatory cell content than their normocholesterolemic counterparts (Davies et al. 1999). A greater degree of inflammation in vein grafts exposed to hypercholesterolemia was considered to be an important feature of this project, which was designed following the success of Dz13 in reducing an acute inflammatory response in a rat and other models of inflammation (Fahmy et al. 2006).
Unfortunately, the validity of data collected from cholesterol-fed rabbits was affected by two major adverse events: a high rate of graft thrombosis and graft stenosis
(absence of palpable blood flow at 3 weeks following the surgery) resulting a graft failure rate of 54% at 3 weeks. The remaining grafts often contained a partial thrombus that made their morphometric analysis unreliable. The analysis of the available specimens, demonstrated that Dz13 produced an anticipated reduction in NI growth as compared to the control and Dz13scr groups, which was encouraging.
However, rather than persevering with hypercholesterolemic rabbits, the decision was made to change the project and perform a second lot of surgery in rabbits placed on a normal chow-diet to avoid hypercholesterolemia that could have been a significant Chapter 6: DNAzymes targeting c-Jun inhibits intimal thickening in a rabbit model of bypass surgery and in human saphenous veins in vitro 155 contributor to the high rate of graft thrombosis and failure. In retrospect, however, it seems that these problems were most likely related to the technical nature of the surgery itself and my lack of experience in creating microsurgical anastomosis. To overcome a problem with thrombosis, the dose of intravenous heparin was increased in the second project from 250u/kg body weight to 330u/kg body weight. Prior to commencing the new set of experiments permission was obtained from the Animal
Ethics Committee of the University of New South Wales to conduct additional experiments designed to refine the microsurgical technique and to ensure that the operator had sufficient surgical skills required for the creation of anastomosis. From a surgical viewpoint the second lot of experiments was much more successful with a higher patency rate of 80% and no thrombosis. Only five grafts out of 24 animals failed; two grafts due to the failed anastomosis and three other grafts due to wound infection in the postoperative period. Another limitation of this project is related to the relatively small number of animals (between 5 and 6) per treatment group. In addition, due to the restraints of time and financial cost of the project, I did not collect vein specimens at different time points. This would be helpful in determining a time course of c-Jun inhibition by Dz13 and documenting the changes in the expression of inflammatory molecules and MMPs as a result of c-Jun silencing.
6.4 Discussion
New molecular approaches that target intimal thickening of bypassed grafts have been a topic of extensive in vitro and in viva research for more than a decade and led Chapter 6: DNAzymes targeting c-Jun inhibits intimal thickening in a rabbit model of bypass surgery and in human saphenous veins in vitro 156 to much better understanding of the basic molecular events involved in the control of vein graft failure. Multiple gene delivery techniques targeting a wide range of key molecular processes involved in pathophysiology of graft failure have been tried in the past but did not yield expected results (reviewed in detail in section 1.7.4 of
Chapter 1). Recently, a much publicised trial of E2F decoy (Prevent IV) was stopped prematurely when no significant difference in graft failure rate following CABG surgery was demonstrated between placebo and the treatment group at 12 months of follow-up (Alexander et al. 2005). Based on our lab's extensive experience with
DNAzymes in general and Dz13 in particular we felt that a DNAzyme-based approach to the treatment of intimal thickening could offer some important advantages over a Decoy approach and was worth exploring further. Compared to decoy ODN, DNAzymes offer many advantages such as single strandiness, higher transfection efficiency and a greater stability due to the inverted T.
Transcription factor c-Jun is an attractive target for interventions designed to improve the long-term survival of venous grafts for a number of reasons: firstly, inhibition of c-Jun leads to the suppression of SMC proliferation and migration in vitro and the reduction of NI formation following a rat carotid artery ligation model in vivo by 60%
(Khachigian et al. 2002). Moreover, c-Jun has been shown to be a key regulator of an acute inflammatory response through controlling the expression of different proinflammatory genes in diverse pathological settings as detailed by elegant studies of Fahmy and colleagues (Fahmy et al. 2006). Finally, the role of c-Jun in the Chapter 6: DNAzymes targeting c-Jun inhibits intimal thickening in a rabbit model of bypass surgery and in human saphenous veins in vitro 157 pathobiology of graft failure is emphasized by the observation that c-Jun is upregulated in failed vein grafts by micro-array analysis (Hilker et al. 2003). In addition, inhibition of c-Jun expression with the use of antisense ODN in a rat model of bypass grafting has been found to be moderately successful (Suggs et al. 1999).
In this study inhibition of c-Jun by DNAzyme at the time of surgery was associated with a significant reduction in the development of intimal thickening at 21 days in hypercholesterolemic rabbits and 28 days in rabbits fed a normal diet. Different time points for the two sets of experiments were chosen based on the fact that vein grafts of cholesterol-fed rabbits develop significantly more neointima than the grafts of the animals placed on a normal chow diet (Davies et al. 1999). Furthermore choosing a shorter postoperative follow-up was beneficial in minimising the cost of animal housing, which is particularly high for cholesterol-fed rabbits who spend an extra 4 weeks on cholesterol supplemented diet prior to undergoing surgery. A modest but statistically significant reduction in NI formation was observed in cultured human saphenous veins in vitro. IHC performed on 4-week-old rabbit vein grafts showed inhibition of MMP-2 but not MMP-9 expression. To my knowledge this is the first successful application of a DNAzyme to a model of bypass grafting in vivo. In rabbits placed on a normal diet Dz13 produced a 41 % reduction of the intimal thickening in bypassed grafts as compared to Dz13scr. This result is consistent with other studies that used a genetic approach to inhibiting NI formation in animal models of bypass grafting (table 3.1 of Chapter 3, page 74). Chapter 6: DNAzymes targeting c-Jun inhibits intimal thickening in a rabbit model of bypass surgery and in human saphenous veins in vitro 158
To investigate some of the mechanisms responsible for the reduction in intimal thickening secondary to inhibition of c-Jun expression by Dz13, IHC staining for rabbit macrophages and alpha-actin SMC was performed in 28-day-old grafts.
Unexpectedly, both Dz13scr and Dz13 treated groups showed no detectable staining for macrophages (RAM-11). According to published literature, vein grafts of non cholesterol fed rabbits have a relatively small number of macrophages in comparison to cholesterol-fed animals where there is an abundance of the cells (Davies and
Hagen 1995; Davies et al. 1999). One would have anticipated a reduction in the number of macrophages with Dz13 treatment based on the anti-inflammatory property of Dz13 demonstrated in microcirculation studies in the rat (Fahmy et al.
2006). The immunohistochemical finding that macrophages were absent in both groups in this study suggests that inflammation was not an underlying pathophysiological mechanism of neointima development in bypassed grafts not exposed to cholesterol feeding. The intensity of stain for alpha-actin SMC antigen per unit area of the vein was similar between Dz13 and Dz13scr groups, however the area was larger in Dz13scr treated vessels as evidenced by a greater degree of new intima formation. This finding taken together with inhibition of intima thickening by Dz13 indicates that the primary mechanism responsible for the observed effect of Dz13 on intimal development is an absolute reduction in a number of SMC in vein grafts exposed to Dz13 but not Dz13scr. The results of this study indicate that SMC Chapter 6: DNAzymes targeting c-Jun inhibits intimal thickening in a rabbit model of bypass surgery and in human saphenous veins in vitro 159 hyperplasia rather than macrophage infiltration is the main pathophysiological mechanism of NI development in veins of the chow-fed rabbit.
The finding that Dz13 inhibited the expression of MMP-2 but not MMP-9 in a 4- week-old vein grafts was interesting and somewhat unexpected as MMP-9 is a c-Jun dependent gene (Shin et al. 2002) and both MMP-9 and MMP-2 had previously been inhibited by Dz13 in squamous cell carcinoma (Zhang et al. 2006) and vascular endothelial cells (Zhang et al. 2004). One of the possible explanations for this unexpected result could be the timing of vein collection. Recently, it has been shown that MMP-9 mRNA level in bypassed rabbit vein grafts increase dramatically in the first few days following the surgery peaking at day 3 and returning to baseline level within 14 days (Berceli et al. 2006). Similar changes were also observed in pro
MMP-9 zymographic activity that increased 300-fold at day 1 but returned to normal levels by day 7. In contrast to the changes seen in MMP-9 activity, levels of active
MMP-2 decreased in the first few days post-implantation but went up progressively with the development of intimal thickening at day 7 and 14. In addition, the expression and activity of MMP-2 was markedly increased under poor flow condition, which is associated with accelerated neointimal growth (Berceli et al.
2006). It is believed that the initial surge in MMP-9 activity acts to facilitate an early entry of monocytes into the vein wall (Cook-Mills and Deem 2005), the hypothesis supported by in vitro migration assay data demonstrating an indispensable role of
MMP-9 in degradation of tight junction proteins and the mediation of cytokine-driven Chapter 6: DNAzymes targeting c-Jun inhibits intimal thickening in a rabbit model of bypass surgery and in human saphenous veins in vitro 160 migration of inflammatory cells through an intact monolayer (Ichiyasu et al. 2004).
MMP-2 upregulation, on the other hand, is essential for SMC migration into the developing neointima (Godin et al. 2000). Therefore, by looking at the expression of
MMP-2 and MMP-9 at 4 weeks only, the effect of Dz13 on early upregulation of
MMP-9 was most likely missed while downregulation of MMP-2 level by Dz13 could still be appreciated in 28 day old vein grafts. This theory would have to be tested further with IHC and Western blot performed on vein grafts collected at different time points including the first few days post surgery.
Even though our initial results from in viva experiments look rather promising they should be interpreted with caution until more animal experiments are conducted with a longer follow-up period of at least 3 to 6 months. Even then, achieving a meaningful reduction in NI formation in animal models does not guarantee the success of the same intervention in humans as has recently been seen in the
PREVENT IV trial (Alexander et al. 2005). The major significance of this work comes from the fact that this is the first successful application of a DNAzyme-based genetic intervention to the targeting of neointimal development in bypassed vein grafts. However, a lot more work has to be done to understand the mechanisms behind this success. To continue this project other reserchers in our lab are planning to perform RT-PCR and Western blot on the vein samples collected at different time points to assess the effect of Dz13 on the levels of c-Jun, ICAM-1, VCAM-1, E selectin, MMP-2 and MMP-9 mRNA and protein expression respectively. In Chapter 6: DNAzymes targeting c-Jun inhibits intimal thickening in a rabbit model of bypass surgery and in human saphenous veins in vitro 161 addition, performing IHC on vein graft samples at specific time points after implantation would help to understand changes in the pattern of expression of different molecules and their response to c-Jun inhibition by Dz13.
6.5 Overview of technical difficulties faced by the candidate during the candidature
The current Thesis is based on two in vivo projects: a rat model of microcirculation and a rabbit model of external jugular vein to common carotid artery grafting. Both models are technically challenging projects requiring a high level of proficiency from a candidate in order to obtain reproducible and reliable results. Even though practical help in setting up microcirculation studies was available from my co-supervisor
A/Professor Michael Perry, it was difficult to get experiments started at the beginning for a number of reasons: the lab that was previously used for microcirculation studies was not in a proper working order as no experiments had been conducted in this lab for over a year. Another difficulty was the absence of technical personnel with practical experience in the relevant microcirculation techniques. As a result, I faced a couple of months delay in starting the project. Once these difficulties were overcome it took several months of practice to generate reliable control data.
The second in vivo project, a rabbit model of interposition grafting, turned out to be even more challenging than the first. This model relies on the creation of
microsurgical anastomosis between jugular vein and carotid artery, which in turn Chapter 6: DNAzymes targeting c-Jun inhibits intimal thickening in a rabbit model of bypass surgery and in human saphenous veins in vitro 162 required considerable experience in microsurgical technique as a flawed technique can profoundly influence the development of intimal thickening. I learnt the procedure by myself relying mostly on published literature for surgical protocols.
Subsequent to this, the first set of experiments conducted in cholesterol-fed animals over an 8-month period produced inconsistent data due to the high rate of graft thrombosis, which in retrospect was predominantly related to lack of experience on my behalf rather than cholesterol feeding. I abandoned cholesterol feeding and spent another 7 months on the same project using normal chow-fed rabbits. These multiple setbacks had obvious impact on my overall productivity during the candidature. Chapter7: Conclusions and future directions 163
Chapter 7: Conclusions and future directions
7.1 Overview of the major findings arising from this Thesis
• The transcription factors c-Jun and Egr-1 are important regulators of
leucocyte recruitment during acute inflammation induced by IL-1 (3 in the rat
mesenteric venules as demonstrated with the use of DNAzymes targeting
these transcription factors.
• c-Jun plays a regulatory role in controlling the expression of adhesion
molecules ICAM-1 and VCAM-1, E-selectin and VE-cadherin in the context
of IL-1(3 induced inflammation.
• c-Jun is critical in modulating adhesion of monocytic cells to the stimulated
endothelial cells in vitro as shown with c- Jun specific DNAzyme and siRNA.
• Targeting c-Jun with DNAzyme reduces neointima development in bypassed
vein grafts of the chow-fed rabbit. The main cellular mechanism responsible
for the observed effect was related to the inhibition of SMC proliferation by
the DNAzyme.
• c-Jun DNAzyme inhibits neointima formation in cultured human saphenous
veins.
• c-Jun DNAzyme suppresses the expression of MMP-2 in vein grafts of the
chow-fed rabbit at 28 days as assessed by immunohistochemistry.
7.2 Future directions Chapter 7: Conclusions and future directions 164
7.2.I Explore the potential role for the local application of
Dzl3 in inflammatory conditions such as rheumatoid arthritis
Based on the findings of this Thesis and other published literature the transcription factor c-Jun appears to be a promising target for a wide range of inflammatory and proliferative conditions. Using different genetic approaches to blocking c-Jun expression, namely c-Jun specific DNAzyme (Dz13) and siRNA, this lab has recently demonstrated that c-Jun is involved in regulation of vascular permeability, leucocyte movement and leucocyte-endothelial interactions during an acute inflammatory response (Fahmy et al. 2006). These findings are of great interest since they represent the first systematic evaluation of anti-inflammatory properties of transcription factor c-Jun. One of the models used by the lab to examine anti-inflammatory properties of c-Jun was a model of the collagen antibody-induced arthritis (CAIP) in mice. In this model, local administration of Dz13 three days after the induction of arthritis resulted in inhibition of neovascularization and neutrophil accumulation into the synovium. In addition, Dz13 blocked bone erosions and reduced the number of multinucleated osteoclast-like cells (Fahmy et al. 2006). CAIP is a well-characterized murine model of rheumatoid arthritis (RA), a chronic autoimmune disorder associated with a widespread involvement of joints and visceral organs. Synovial inflammation in RA is characterised by endothelial activation with enhanced expression of adhesion molecules such as VCAM-1 and ICAM-1 in response to locally produced inflammatory mediators (TNF a and IL-1) and thought to be critical to the influx of leucocytes into a rheumatic joint. TNFa blockade in RA is associated with reduced Chapter 7: Conclusions and future directions 165
levels of VCAM-1 and decrease in T cell trafficking to the synovium (Tak et al.
1996). In addition, ICAM-1 deficient mice or mice treated with ICAM-1 neutralizing antibodies exhibits less severe joint inflammation thought to be secondary to impaired leucocyte trafficking (Kakimoto et al. 1992; Bullard et al. 1996). Joint involvement in RA is associated with bone destruction appearing at early stages of the disease and leading to the long-term deformities, chronic pain and loss of function. The last 10 years has seen an enormous progress in the treatment of this debilitating condition. A number of immunomodulating drugs have come to the clinical use with the potential to preserve joint function and modify the natural course of this disorder (Tremoulet and Albani 2006). Despite this, many sufferers of RA live in constant pain from their condition. Currently, corticosteroids are the principal agents used for intra-articular injections. They provide effective pain relief in a short to medium term and can even prevent long-term joint destruction (Kirwan et al. 2007) however, their use on a regular basis carries a significant risk of adverse effects. For these reasons new therapies delivered into the affected joints are eagerly anticipated.
New genetic compounds such as DNAzymes or siRNA offer a potential advantage over steroids since they seem to have an excellent safety profile (extensively tested in animals only). If these new therapies prove to be useful for the treatment of rheumatoid arthritis, their use could be extended to other inflammatory conditions with joint involvement such as psoriasis and inflammatory bowel disease. The future work in this direction should involve a longer follow-up and the use of other animal Chapter 7: Conclusions and future directions 166
models of arthritis to validate the efficacy of c-Jun inhibition in more than one in viva setting. Extended follow up would also be essential to determine any systemic side effects resulting from intraarticular administration of Dz13. For Dz13 to progress to human clinical trials more work will have to be done on the pharmacokinetics and biodistribution of this compound. It would also be appropriate to determine any potential interaction between locally delivered Dz13 and systemically administered drugs commonly used for the treatment RA such as NSAID, methotrexate and corticosteroids.
7.2.2 Determine the applicability of Dzl3 for the treatment of neointimal hyperplasia through more animal and in vitro experiments
Experiments presented in Chapter 6 demonstrate that Dz13 was effective in blocking
NI formation during a short-term follow-up in the rabbit model of bypass grafting. To add further validity to this initial data the follow-up period should be extended to at least 3 months. The main mechanism involved in inhibition of NI formation in the chow-fed rabbit in this study was the reduction of SMC hyperplasia by Dz13, the effect that has been documented in the past by our lab in different in vitro and in viva models (Khachigian et al. 2002). Cholesterol feeding in the rabbit is associated with increased infiltration of the tissue by macrophages and upregulation of inflammatory markers such as adhesion molecules. It would be important to test the effect of Dz13 on intimal hyperplasia (IH) in cholesterol-fed animals, theoretically, Dz13 should be Chapter 7: Conclusions and future directions 167
effective in inhibiting an inflammatory response associated with cholesterol feeding
based on the data presented in Chapter 5.
Another plausible mechanism of Dz13 action in vein grafts worth exploring further is
the possible protective effect of c-Jun loss on SMC apoptosis. SMC apoptosis following graft implantation into the arterial circulation has been described as one of
the earliest cellular events in neointima formation (Mayr et al. 2000) followed by
infiltration of the graft with leucocytes and monocytes. To address these possibilities
it would be important to determine the expression of c-Jun and inflammatory markers
in vein grafts through different time points by IHC and by quantifying their
respective protein expression by Western blot. In addition, performing an apoptosis
assay on the graft tissue with commercially available apoptosis kits will help to
understand whether loss of c-Jun protectes venous SMC from early apoptosis induced
by mechanical stretch. Extensive IHC staining of vein grafts for SMC, macrophages,
endothelial cells and leucocyte integrin CD18 would help to understand the
mechanisms by which the loss of c-Jun affects the infiltration of a vein graft by
different subsets of cells.
7.2.3 Explore the utility of targeting of transcription factor Egr-1 by
DNAzymes in the setting ofneointimalformation
Based on what is known about Egr-1 and the genes controlled by Egr-1 (reviewed in
depth in section 1.4.2. of Chapter 1) it would be interesting to explore the role of this Chapter 7: Conclusions and future directions 168
transcription factor in neointimal formation. This can be achieved with two approaches - either by targeting Egr-1 with a genetic-based intervention such as
ODN, DNAzyme or siRNA in large animal models or by conducting experiments in
Egr-ldouble knockout mice. The advantage of the former approach is availability of cholesterol-fed animals that display vein graft changes closely resembling the ones observed in humans.
7.2.4 Explore the potential role for the use of c-Jun and Egr-1 siRNA as anti- inflammatory and anti-proliferative agents
This Thesis have demonstrated that c-Jun and Egr-1 play a crucial role in controlling • leucocyte movement during an acute inflammatory response through targeting these transcription factors by their specific DNAzymes. In addition, transcription factor c
Jun was inhibited with other molecular drugs such as c-Jun siRNA in a co-culture assay model in vitro with similar efficacy to the DNAzyme based approach. As discussed in section 2.3.2 siRNAs represent a new, exciting and rapidly developing area in the field of gene-based therapeutics. siRNA molecules offer few important advantages over other molecular approaches including ODN and DNAzymes. Their degree of specificity to a given mRNA is superior to the other commonly used gene based drugs, they are completely non-immunogenic and posses high resistance to
ribonucleases (Bertrand et al. 2002). More importantly, siRNAs do not have to transfer through the nuclear membrane for their activity and therefore require less
sophisticated delivery systems compared to DNAzymes. Furthermore, their small size Chapter 7: Conclusions and future directions 169
offers a good opportunity for delivering a cocktail of several siRNAs targeting multiple genes implicated in the development of a particular disease. For these reasons, it would be important to explore the therapeutic utility of siRNAs targeting c-Jun and Egr-1 in the setting of inflammation and in the development of intimal thickening.
In conclusion, this Thesis details the regulatory role, played by the transcription factors c-Jun and Egr-1 in acute inflammation through the use of a gene-targeting approach with DNAzyme. Furthermore, by applying Dz13 to vein grafts in vivo and in vitro I have demonstrated for the first time that targeting c-Jun with DNAzyme represents a promising approach in inhibiting NI formation in bypassed vein grafts.
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