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Granzyme B in abdominal aortic aneurysm and aortic dissection Chamberlain, Ciara M. 2008

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GRANZYME B IN ABDOMINAL AORTIC ANEURYSM AND AORTIC DISSE CTION by  ClARA M. CHAMBERLAIN  B.Sc (Hon), Simon Fraser University, 2006  A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES (Pathology)  THE UN1VERSITY OF BRITISH COLUMBIA (Vancouver)  June 2008 © Ciara M. Chamberlain, 2008  Abstract  An aneurysm is a permanent focal dilatation of an artery, often associated with atherosclerosis and with a weakening of the vessel wall. An arterial dissection is when a tear in the inner layer of the blood vessel causes blood to flow between the layers and force the blood vessel apart. Aneurysms or dissections can result in rupturing of the vessel, leading to excessive hemorrhaging and death if not immediately repaired. Granzyme B (GrB) is a protein that is expressed and secreted by cytotoxic immune cells. GrB has been identif ied as a key player in atherosclerosis both in the induction of apoptosis in target cells and the degradation of extracellular matrix proteins. We generated GrB/apolipoprotem E (apoE) double knockout (GrB/apoE-DKO) mice, and have shown that advanced atherosclerosi s is significantly reduced in GrB/apoE-DKO mice as compared to apoE-KO mice. Evalua tion of elastin staining indicated a loss of elastic tissue and medial thinning in the aortas of apoE-K O mice that was restored when GrB was absent in the GrB/apoE-DKO mice.  Since medial thinning renders the aorta wall more susceptible to aneurysms or dissections, we hypothesize that the absence of GrB will reduce the incidence of aneurysm format ion and dissection in GrB/apoE-DKO mice as compared to apoE-KO mice during angiotensin II (angli) infusion.  To induce aortic dissections, 3-month-old C57 (wildtype), apoE-KO, and GrB/apoE-DK O mice were implanted with an osmotic mini pump which released a dose of 1 000ng!kglmin of 11  angli (or saline as a control) for 28 days. The mice were euthanized at 28 days, and the aortas were removed and evaluated for gross morphology and, after cross sectional staining, for histopathological lesions.  A significant reduction in aortic dissections and aneurysms was observed in GrB/apoE-DKO mice as compared to apoE-KO mice treated with angli. Further, GrB deficiency corresponds to a significant increase in survival at the 28 day time-point. Aneurysms and dissections were not observed in the saline-treated groups or the angli-treated wild-type controls. In conclusion, our studies suggest that GrB contributes to the loss of vessel wall integrity and to the occurrence of aortic aneurysm and dissection.  111  Table of Contents  Abstract  ii  Table of Contents  iv  List of Tables  vii  List of Figures  viii  Abbreviations Acknowledgements Dedication  xii  Introduction  2  1.1 Cardiovascular Disease  2  1.1.1  Atherosclerosis  2  1.1.2  Abdominal Aortic Aneurysm  4  1.1.3  Aortic Dissection  6  1.2 Animal Models  7  1.2.1  Apolipoprotein E-knockout Mouse  7  1.2.2  Angiotensin TI-induced Abdominal Aortic Aneurysms  8  1.3 GranzymeB  14  1.3.1  GrB-mediated Apoptosis and Signalling  15  1.3.2  Extracellular Granzyme B  19  1.4 Granzyme B in Atheromatous Diseases 1.4.1  Granzyme B in Atherosclerosis and Allograft Vasculopathy  1.5 Hypothesis 1.5.1  21 21 25  Specific Aims  26  Methodology  28  2.1 Animals  28  iv  2.2 ApoE-KO Model of Angiotensin-Il induced AAA  .  2.3 Tissue Fixation, Excision and Processing  29 31  2.3.1  AorticRoots  31  2.3.2  Abdominal and Thoracic Aortic Sections  32  2.3.3  Histological Stains  32  2.4 Atherosclerosis Histological Quantification esuIts  35 37  3.1 Implantation of Osmotic Mini Pump  37  3.2 Survival Following Angiotensin II Infusion  38  3.3 Angiotensin II Associated Pathology is Decreased in GrB/apoE-DKO Mice  39  3.4 AT1 Receptor in Transgenic Mice  44  3.5 Thoracic and Abdominal Aortic Lumen Area  44  3.6 Angiotensin II Causes Medial Thickening  46  3.7 Aortic Root Atherosclerosis  47  3.8 GrB/apoE-DKO Mice and Fibrillin-1 Expression  49 50  4.1 Osmotic Mini Pump Model  50  4.2 GrB Deficiency Protects against Sudden Death and Aortic Dissection  50  4.3 Increased Atherosclerosis and Onset of Abdominal Aortic Aneurysm are Not Related 51 4.4 Aortic Structure and Composition  52  4.5 Inconclusive Apoptosis and CD3 Data  53 54  5.1 Proposed Mechanism  54  5.2 Concluding Remarks  58  V  Future Directions  .  59  Bibliography  61  Appendix  70  vi  List of Tables  Table 1.1. Table 3.1.  Mouse Models of Abdominal Aortic eurysm Summary of Animals Used in this Study  vii  9 38  List of Figures  Figure 1.1. Figure 1.2  Timeline of Angiotensin IT-induced Abdominal Aortic Aneurysm Mechanisms Involving Granzyme B-Mediated Caspase-Dependent and Independent Cell Death  13  17  Figure 1.3.  Putative Mechanism by which Granzyme B Contributes to the Pathogenesis of Atheromatous Disease 25  Figure 2.1.  Examples of ImageProPlus® Tracing  36  Figure 3.1.  35  Figure 3.2.  Mouse Implanted with ALZET® 1004 Pump Kaplan-Meier Survival Curve  Figure 3.3.  Gross Pathology of Aortas  40  Figure 3.4.  41  Figure 3.5.  H&E Staining of Representative Abdominal Aortas Gross Pathological Outcomes  42  Figure 3.6.  Movat’s Pentachrome Staining of Abdominal Aortas  43  Figure 3.7.  AT1 Staining of Aortic Tissue  44  Figure 3.8.  Lumen Area of Thoracic and Abdominal Aortas  45  Figure 3.9.  Medial Thickness of Thoracic and Abdominal Aortas  46  Figure 3.10.  47  Figure 3.11.  Aortic Roots Stained by H&E, Movat’s and ORO Measurements of Aortic Root Lumen and Plaques  48  Figure 3.12.  Fibrillin- 1 Staining in Abdominal Aorta  49  Figure 5.1.  Angiotensin II Causes Abdominal Aortic Aneurysm and Aortic Dissection Through the AT1 Receptor 55 Potential Therapeutic Targets for Inhibition of Abdominal Aortic  Figure 5.2.  Aneurysm  39  57  viii  Abbreviations  A/B/C AAA Abd ACE ADP AIF Angli APAF 1 ApoE AT I ATP AV Bid CAD Casp CTL Cyt c  Abdominal aortic aneurysm Abdominal Angiotensin converting enzyme Adenosine diphosphate Apoptosis-inducing factor Angiotensin II Apoptotic protease-activating factor 1 Apolipoprotein E Angiotensin II type 1 receptor Adenosine triphosphate Allograft vasculopathy BCL-2 interacting domain Caspase-activated deoxynuclease Caspase Cytotoxic T lymphocyte Cytochrome c  D/E/F DFF dATP DKO EC ECM EndoG eNOS  DNA fragmentation factor Deoxyadenosine triphosphate Double-knockout Endothelial cell Extracellular matrix Endonuclease G Endothelial nitric oxide synthase  GJH/ I GrA GrB H&E HIV ICAD ICAM-1 IFN-y  Granzyme A (GrA) Granzyme B (GrB) Hematoxylin and eosin Human immunodeficiency virus Caspase-activated deoxynuclease inhibitor Intercellular adhesion molecule-I Interferon-gamma  J/KJL KO LDL  Knockout Low density lipoprotem ix  Lox-i LRP  Lectin-like oxidized lipoprotein receptor-i Lipoprotein Receptor-related Protein-i  M/N/O M6P Mac MCP- 1 M-CSF MMP MRI NADPH NK OCT  Mannose-6-phosphate Macrophage Macrophage chemoattractive protein -1 Monocyte colony stimulating factor Matrix metalloproteinase Magnetic resonance imaging Nicotinamide adenine dinucleotide phosphate Natural killer cell Optimal cutting temperature  PIQ/R PA Parp PDGFR Perf P1-9 PLD RAA ROCK II ROS  Phosphatidic acid Poly (ADP-ribose) polymerase Platelet derived growth factor receptor Perform Protease inhibitor-9 Phospholipase D Renin-angiotensin-aldosterone system (RAA) Rho-associated coil coiled-containing protein kinase II Reactive oxygen species  S/T/U SEM Smac SMC TGF-13 TIMP- 1 Tregs TUNEL Tx UV  Standard error of the mean Second mitochondria-derived activator of caspases Smooth muscle cell Transforming growth factor Tissue inhibitor of matrix metalloprotease- 1 CD4+/CD25+ regulatory T-cells Terminal deoxynucleotidyl transferase dUTP nick end labelling Thoracic Ultra violet light  V/W/X/Y/Z VCAM VSMC WHO  Vascular cell adhesion molecule Vascular smooth muscle cell World Health Organization  x  Acknowledgements  Thank you kindly to Dr. David Granville for taking me on as a graduate student, and for supporting me in every aspect of my graduate life.  I couldn’t have asked for a better  supervisor or a better environment in which to learn! Thank-you to all of the Granvillites for their support, training and humour- I have thoroughly enjoyed these last two years! Thank you to Dr. David Walker for being a tremendous graduate advisor, teacher, and chair. Thank you to Dr. Bruce McManus and Dr. Pascal Bernatchez for their support and advice as my committee members. Thank you to Dr. Mike Allard for his tremendous open-door policy and pathological insight. Thank you to Claire Smits and Lubos Bohunek for training me on the animal procedures and helping me trouble shoot. Thank you to Hongyan Zhao and Amrit Samra for their excellent histology skills. Much appreciation goes to Dr. Alan Daugherty for sending his SOP for the AAA model, and for answering technical questions we initially encountered.  Thank you to the Michael Smith Foundation for Health Research, the Canadian Institute for Health Research and the University of British Columbia for fellowships and awards, and to the Heart and Stroke Foundation and CIHR for funding our laboratory.  xi  Dedication  This degree could not have been completed without the love and unending support of my parents, Robert and Sharon Chamberlain. Thank you so much, Mom and Dad!  xii  CHAPTER 1  Introduction  1.1  Cardiovascular Disease  In 2004, 30% of all global deaths were attributed to cardiovascular disease making it the . 1 leading cause of death worldwide and accounting for more than 17 million deaths every year Mortality rates between countries vary primarily on prevalence of cardiovascular risk factors and the World Health Organization (WHO) estimates show that obesity, smoking and sedentary lifestyles are continually rising in developing countries. Most Western countries currently face high and increasing rates of cardiovascular disease. As populations age in middle- and low-income countries over the next 25 years, the proportion of deaths due to non-communicable diseases will rise significantly. Globally, deaths each year from cardiovascular diseases will rise to 23.4 million by 20302.  1.1.1  Atherosclerosis  Atherosclerosis is characterized by the thickening of the arterial wall, usually at sites in the arterial tree where laminar flow is disrupted.  This inflammatory vascular disease is  characterized by the excessive accumulation of lipids and modified lipids in the intima, medial damage, and the thickening and structural re-organization of the vessel wall. In 1973, Ross hypothesized that atherosclerosis resulted from injury of the endothelium, leading to a  Chapter 1: Introduction . Physical forces, or the exposure to 3 reparative response that resulted in intimal thickening elevated levels of circulating low density lipoprotein (LDL) or free radicals caused by . 4 smoking, hypertension, or diabetes mellitus can cause endothelial dysfunction  These  factors alter endothelial function by increasing the release of pro-inflammatory cytokines and vasoactive substances, and interfering with normal anti-thrombotic properties and permeable barrier functions, and increasing expression of cell-surface adhesion molecules.  Atherosclerosis begins as a fatty streak consisting of atherogenic lipoproteins entering the intima and becoming modified. The increase of cell-surface adhesion molecules causes the recruitment and intravasation of leukocytes, monocytes and T-cells.  Pro-inflammatory  cytokines expressed within the developing lesion provide chemotactic stimulus to the adherent leukocytes, increasing their migration into the intima. Monocyte colony stimulating factor (M-CSF), which is also produced in the plaque, augments the expression of . Macrophages phagocytose this 5 macrophage scavenger receptors to uptake modified lipids modified lipid in an unregulated manner, causing the formation of foam cells, which make up the fatty streak. Leukocytes, as well as resident vascular wall cells, secrete cytokines and growth factors that promote the migration and proliferation of smooth muscle cells (SMC). Vascular SMC (VSMC) also release factors that degrade elastin and collagen in response to inflammatory stimulation, which allows the cells to migrate through the elastic lamina and collagenous matrix. VSMC proliferate and migrate from the media to the developing plaque in the intima, and contribute to the fatty streak development into an intermediate lesion by excessive extracellular matrix (ECM) secretion. aggregation of lipoproteins.  This ECM increases the retention and  As the plaque continues to grow, additional lymphocyte  3  Chapter 1: Introduction recruitment follows, and VSMC form a fibrotic cap under the endothelial layer. The fibrous cap eventually becomes thin and weak by a combination of an inhibition of collagen synthesis from VSMC and the expression of collagenases by foam cells. Eventually, a lesion can develop that is vulnerable to rupture, exposing thrombogenic material in the form of necrotic foam cells. The plaque may also grow without rupture, and may eventually obstruct blood flow. The formation of a thrombus which may block blood flow or the obstruction of a ). 6 vessel from plaque formation can lead to ischemia of distal tissue (reviewed in  1.1.2  Abdominal Aortic Aneurysm  An aneurysm is a permanent focal dilatation of an artery and is associated with a weakening of the wall of the vessel. Aneurysm formation can result in rupturing of the vessel, leading to excessive hemorrhaging and death if not surgically repaired. Abdominal Aortic Aneurysm (AAA) is an increasingly common clinical condition with fatal implications, and comprises 80% of all aortic aneurysms. The incidence of AAA in men and women over 60 years of age •5%7• Dilation of the aorta may occur as a consequence of aging, is 4% to 8%, and 0.5% to I  as well as hypertension, atherosclerosis, infection, inflammation, trauma, congenital anomalies, and medial degeneration. Other risk factors for AAA include male gender and ). 8 cigarette smoking (reviewed in  One-third of all AAAs will eventually rupture if left untreated. Currently, the only effective treatment intervention is surgical repair. However, operative mortality for open elective and ruptured AAA repair is 5.6% and 457%9• The risk for rupture of an AAA is directly related to the diameter of the aneurysm’°’  and therefore using a therapeutic strategy to slow the  4  Chapter 1: Introduction rate of expansion of AAAs would be most desirable. However, no therapeutic strategy has been demonstrated to improve clinical outcome for patients with expanding AAA . 7 The exact causes of aneurysms are still under examination. It is currently thought that the primary event in aneurysm development involves proteolytic cleavage of the ECM proteins . 4 ’ 2 collagen and elastin leading to mechanical weakness in the vessel wall’  In tissues  acquired from humans at the end stage of the disease, degradation of medial elastic fibres, thinning of the media, adventitial hypertrophy with an accumulation of T and B lymphocytes, atherosclerosis and thrombi are all observed’ . Proteases such as matrix metalloproteinases 8 ’ 5 (MMP)-2, -7, -9, -12, cathepsins, plasminogen activators, and elastases are all hypothesised . Recently, our laboratory has 4 to contribute to the degradation of fibrillar ECM proteins’ discovered that Granzyme B (GrB) may play an important role in vessel wall thinning during atherosclerosis (Cruz et al, unpublished data). The thinning of vessel walls, resulting in a mechanical weakness, would predispose these vessels to aneurysms. In vitro, we have also shown that GrB cleaves elastin and recently fibrillin- 1 (Boivin et a!, unpublished data).  The important function of fibrillin- 1 is emphasized in cases of deficiency and mutations. Fibrillin- 1 is a 3 50-kDa glycoprotein that forms the structural framework of microfibrils. Microfibril bundles form a template for the deposition of the elastin precursor, tropoelastin. . 9 A mature elastin fiber contains an inner elastin core and a periphery of microfibrils’ Fibrillin- 1 null mice die perinatally from ruptured aortic aneurysm and impaired lung . Mutations in the fibrillin- 1 gene often result in Marfan syndrome, an autosomal 20 function dominant hereditary disorder of connective tissue with major cardiovascular, skeletal and ocular defects. Premature death is often caused by acute aortic dissection, following elastic  5  Chapter 1: Introduction fiber degeneration and progressive dilation of the ascending aorta.  Fibrillin-1 mutations  leading to Marfan syndrome can be a result of altered or reduced secretion of microfibril proteins, improper assembly of microfibrils, or increased susceptibility of microfibrils to . Fibrillin- 1 can also regulate the bioavailability of transforming growth 21 proteolytic damage . 22 factor-13 (TGF-)  TGF- activity has been shown to be enhanced in a mouse Marfan  , and was decreased when aneurysms were reduced in Marfan syndrome 23 syndrome model mice given angil blocker, losartan . 24  1.1.3  Aortic Dissection  An aortic dissection begins with a tear in the aortic intima and inner layer of the aortic media 26 This causes the formation of a false ’ 25 allowing blood to enter and split the aortic media lumen through the media, which is separated from the true lumen by an intimal flap. Medial necrosis or degeneration of aortic media, such as what is seen in aneurysms, is thought to be 8 Mechanical forces contributing to aortic dissection include dissection 2 ’ a prerequisite for 27 flexion forces of the vessel at fixed sites, the radial impact of the pressure pulse, and the shear stress of the blood . Hypertension adds to a mechanical strain on the aortic wall and to 28 the shearing forces exerting a longitudinal stress along the aortic wall. A combination of these factors results in an intimal tear and the propagation of dissection into the aortic media. This is particularly devastating in patients with previous medial degeneration and weakening, such as those with congenital connective tissue diseases.  There are 5 to 30 cases of aortic dissection per million per year. Men are twice as likely to develop an aortic dissection than women, and the peak incidence of aortic dissections are in  6  Chapter 1: Introduction . Not surprisingly, the incidence of aortic dissection is 29 the sixth and seventh decades of life . 26 related to the prevalence of risk factors for aortic dissection in the population that is studied Risk factors for aortic dissection include age, male gender, family history, hypertension, smoking, dyslipidemia, and drug use. Aortic dissections can also be caused by a deceleration ). Connective tissue disorders, such as 30 trauma, such as a fall or car accident (reviewed in Marfan syndrome, also make an individual predisposed to developing an aortic dissection.  1.2  Animal Models  1.2.1  Apolipoprotein E-knockout Mouse  Apolipoprotein E (apoE) is an important mediator in the transport and hepatic metabolic clearance of circulating cholesterol. The absence or dysfunction of apoE leads to elevated . ApoE-deficient mice 31 levels of LDL, triglycerides and total cholesterol in the circulation (apoE-KO) were first produced in 199232. These mice are hyperlipidemic and spontaneously develop atherosclerosis and, with increasing age, develop extensive cutaneous xanthomatosis . When apoE-KO mice are fed a high fat diet, they develop fatty streaks and 3336 and hair 1oss fibroproliferative lesions sooner than on a regular chow diet. The cellular composition of the atherosclerotic lesions in apoE-KO mice is similar to that observed in humans, consisting of macrophages, T cells and smooth muscle cells, as well as the presence of oxidized . Lesions in apoE-KO mice, as in humans, predominantly develop at vascular 37 lipoproteins branch points. The lesions also progress from the foam cell stage to the fibroproliferative stage with well-defined fibrous caps and necrotic lipid cores. In contrast to humans, minimal evidence of plaque rupture has been observed in the apoE-KO model or any other mouse atherosclerosis 38• Atherosclerotic plaque is commonly measured on the aortic ’ model of 31  7  Chapter 1: Introduction root, where the heaviest plaque is seen due to the unique anatomical location and arched architecture, proximity to the heart, contractile pulsatility, and high blood pressure in the aortic arch 38 ’ 37  ApoE-KO mice are commonly utilized to study atherosclerosis and aneurysm pathogenesis. When apoE-KO mice are infused with angiotensin II (angll) for a prolonged period, they exhibit modest increase in atherosclerotic lesions in the aortic root and descending aorta, and . It is through this model that we will begin to 39 develop large AAA, and aortic dissections elucidate the role of GrB in abdominal aortic aneurysms and aortic dissections.  1.2.2  Angiotensin 11-induced Abdominal Aortic Aneurysms  Many animal models of aneurysm formation have been reported in the literature. They are from 40) A good animal model is one that displays key ’ summarized in table 1 (modified 8 aspects of the disease observed from human samples. The main characteristics looked for in animal AAA models include medial degeneration, inflammation, thrombus formation, ’ 40• The apoE-KO angli infusion model 8 association with atherosclerotic plaques, and rupture has all these characteristics. It is for these reasons that we chose this model for our studies.  8  Chapter 1: Introduction  Table 1.1. Models of Abdominal Aortic Aneurysm Characteristics of AAA  Mechanism  Model of AAA  Reference  Elastase infusion  Destroys elastic Iamellae and adventitial tissue.  Medial degeneration, inflammatory response.  Anidjar l990’  Calcium chloride  Application on abdominal aorta causes localized aneurysm.  Disruption of media, inflammatory response, dilation at site of application.  Chiou 200142  LDL-KO, high fat diet  The total plasma cholesterol rose from 246 to> 1,500 mg/dl.  Suprarenal aneurysms, medial degeneration.  Ishibashi 1 99443 Tangirala l995  ApoE-KO, high fat diet  Total plasma cholesterol rose from 132 mgldl to 1821 mg/dl.  Medial degeneration, association with atherosclerotic plaques.  Plump 199232  Angll infusion into LDL receptorKO and apoE-KO  Increased oxidative stress through NADPH oxidase leading to increase inflanmmtion.  Medial degeneration, inflammatory response, medial thrombus, associated with atherosclerotic plaques, rupture.  Daugherty 2000  Blotchy mouse  Abnormal Copper absorption, defective Lox gene, causes abnormal elastin/collagen crosslinking. By 6 months, 100% of mice have thoracic or abdominal aneurysm. Abnormal elastin and collagen crosslinking, death at full term foetus from thoracic aneurysm rupture. Inactivation ofLRPl causes PDGFR overexpression and abnormal activation of PDGFR signalling, resulting in disruption of the elastic layer, SMC proliferation. Mice over-express Angli Rupture of thoracic and abdominal aorta aneurysm within in 10 days of  Medial degeneration, prominent elastin degradation by I month.  Brophy 1988  Medial degeneration, highly fragmented elastic fibers and discontinuity in the smooth muscle cell layers. Medial degeneration, association with atherosclerotic plaques.  Maki 2002  Medial degeneration, inflammation and rupture, independent of blood pressure.  Nishijo l998  Medial degradation in thoracic and abdominal segments. Fragmentation of elastic membranes, thinning of the aortic wall, and rupture of elastic membranes. Medial degradation in thoracic and abdominal segments.  Silence 200l°  Inflammatory response and rupture of vessel.  Shimizu 2004  Lox-I deficiency  SMC-speciflc LRP/LDL receptor-DKO  Tsukuba hypertensive mice, increased salt diet  ‘  Boucher 2003  salt intake.  ApoE/MMP-3DKO, high fat diet  MMP-3 deficiency protected against aortic aneurysms, perhaps by the decrease in MMP-9 activity,  TIMP-I deficiency  No inhibition of MIvIPs, AAA formed.  Allografted aorta  Allografts in IFN-y receptordeficient hosts developed severe AAA formation associated with markedly increased levels of MMP-9 and MMP-12.  Table 1.1.  Silence 2002  51  52  Summary of select mouse models of abdominal aortic aneurysm and their  Abbreviations. AAA, abdominal aortic aneurysm; Angil, angiotensin II; KO, E; IFN-y, interferon-gamma; LDL, low density lipoprotein; Lox, lectin-like oxidized lipoprotein receptor-i LRP, lipoprotein receptor-related protein-i; MMP, matrix metaiioproteinase; PDGFR, platelet derived growth factor receptor; SMC, smooth muscle cell; TIMP- 1, tissue inhibitor of matrix metalioprotease- 1. characteristics.  knockout; ApoE, apolipoprotein  9  Chapter 1: Introduction The renin-angiotensin-aldosterone (RAA) pathway is a hormone system that regulates longterm blood pressure and extracellular volume in the body. Renin, which is primarily released by the kidneys, stimulates the formation of angiotensin in blood and tissues, which in turn stimulates the release of aldosterone from the adrenal cortex. When renin is released into the blood, it acts upon a circulating substrate, angiotensinogen, which undergoes proteolytic cleavage to form the decapeptide angiotensin I. Angiotensin converting enzyme (ACE), cleaves off two amino acids to form the octapeptide, angli. Angil has several very important functions, all to increase blood pressure. This is done by numerous mechanisms, including constricting resistance vessels, causing the release of aldosterone and vasopressin to increase fluid retention from the kidneys, and stimulate thirst centres within the brain. AngIl has also been shown to stimulate cardiac and vascular hypertrophy. Therapeutic manipulation of the RAA pathway is often used to treat hypertension and heart failure. ACE inhibitors, angil  receptor blockers, and aldosterone receptor blockers are all used clinically to decrease arterial pressure, blood volume, and to inhibit and reverse cardiac and vascular hypertrophy ). 53 (reviewed in  ACE inhibitors have repeatedly been shown to decrease the atherogenic process in ), thus implying a role of angll in the development 45 humans and in animals (reviewed in of atherosclerosis. When renin and angiotensinogen are over-expressed in mice fed a . The increase in atherosclerosis and 54 high fat diet, atherosclerosis is markedly increased aneurysms is attributed to the activation of the angli type 1 receptor (AT 1) (reviewed ). AT 1 is expressed in VSMC, heart, lung, brain, liver, kidney, and adrenal glands. It 55 in is not only involved in salt and water homeostasis and vasoconstriction, but also induces  10  Chapter 1: Introduction reactive oxygen species (ROS) production, cellular hypertrophy and hyperplasia, and . 56 apoptosis  AngIl also enhances gene expression of rae 1 (which is required for nicotinamide adenine . The 5761 dinucleotide phosphate (NADPH) oxidase activation), p22phox, and NOX-1 excessive ROS production in the vessel wall is a result of the linkage of the AT 1-receptor . Excessive ROS production leads to a state 57 to activation of an NADH-NADPH oxidase of oxidant stress, including activation of redox-sensitive genes, such as vascular cell adhesion molecule (VCAM) -1, intercellular adhesion molecule (ICAM)- 1 and macrophage  chemoattractive  protein  (MCP)- 1 62-64•  These  . 55 proinflammatory and have been implicated in atherogenesis  molecules  are  Oxidative stress also  stimulates growth and migration of VSMC, which are key elements in atherosclerotic plaque development . Another devastating consequence of excessive ROS production is 65 . 66 the inactivation of nitric oxide scavenging, contributing to endothelial dysfunction Endothelial dysfunction is a hallmark of atherosclerosis that involves the impairment of . AT1-receptor 67 endothelium-dependent vasodilatation, and contributes to hypertension activation also results in increased oxidation of LDL, uptake of oxLDL by macrophages and foam cell formation, all of which are found in atherosclerotic plaques 6870•  Alan Daugherty’s laboratory was the first to describe the formation of AAA and an increase in atherosclerotic lesions following a 1 month infusion of angil in apoE-KO . No changes in body weight, serum cholesterol concentrations, or distribution of 45 mice serum cholesterol were observed. Blood pressure in anesthetized animals did not change,  11  Chapter 1: Introduction and a follow up study using non-sedated animals was performed and showed only a slight increase in blood pressure. However, blood pressure alone was not sufficient to cause AAA, as increasing the blood pressure to the same value as observed with angli infusion with epinephrine, or reducing the blood pressure to control levels with hydralazine, did not result in AAA . Hypercholesterolemia, which is seen in apoE-KO mice, increases 71 AT1 expression, which would augment the action of angli, contributing to endothelial dysfunction, increased vascular production of ROS, and therefore would promote atherosclerosis. The atherosclerotic lesions formed from angli infusion are overtly similar to those formed during hypercholesterolemia, with infiltration of macrophages and T lymphocytes. The observed aneurysms exhibit many aspects of the human disease including medial degeneration, inflammation, thrombus, and rupture (reviewed in ). 72  One difference between the mouse AAA and human AAA is the location’ . Human 6 AAAs typically form in the infrarenal region, whereas AAA in mice form in the suprarenal region . This is not only the location observed in angli-induced AAA, but 40 also the location of AAA in LDL-receptor-KO, eNOS-KO and aged apoE-KO ’ 45 39 mice The reason for the difference is still unknown. The most popular hypothesis is that hemodynamics caused by altered mechanistic properties of the artery (regional differences in collagen to elastin ratio) may be responsible, or caused by differences associated with being quadrapedal . 76  To date, there have been no studies looking  extensively at the composition of the ECM throughout the mouse aorta.  In a time course study extending this model to 56 days’ , Daugherty’s group determined that 6 atherosclerotic plaques were not contributing to the initiation of angli induced AAA, as the  12  Chapter 1: Introduction lesions were detected after the formation of AAA. The same study’ 6 revealed the sequence of events for this AAA model, which is summarized in Figure 1.1.  ,c)  ‘p Figure 1.1. Timeline of Angiotensin Il-induced Abdominal Aortic Aneurysm. A time course study was performed, and histological observations were collected at various time points. Between 1 and 4 days, macrophages accumulated in the media and adventitia, and disruption of elastin fibers was observed. Between days 7 and 10, medial hematomas were observed, and were associated with macrophages. 10% of mice also died at this stage from aortic rupture. Between 10 and 14 days, the aneurysm tissue matured, as new ECM was deposited. There were also many immune cells present in the aneurysmal tissue. Not until after day 28 were atherosclerotic lesions found associated with aneurysmal location. Adapted from’ . Abbreviations: Angil, angiotensin II; apoE-KO, apolipoprotein E-knockout 6 mouse; ECM, extracellular matrix.  The initial event was an accumulation of macrophages around areas of elastin degradation in the media. This observation is also seen in the early formation of atherosclerotic plaques in apoE-KO mice , and elastin fragmentation is markedly reduced in our own laboratory’s 38 study of GrB/apoE-DKO mice at 6 months of age (Cruz eta!, unpublished data). It has yet to  13  Chapter 1: Introduction be determined in atherosclerosis and aneurysm formation if the elastin fragments act as a chemotactic gradient to recruit monocytes, or if monocyte recruitment leads to elastin degradation. In angli-induced AAA models, evidence suggests that MMPs are involved in the cleavage of elastin. When doxycycline, a broad MMP inhibitor, was administered, the incidence and severity of angli-induced AAA formation 77 in LDL-receptor-KO mice was reduced. It has also been shown (although with some controversy ) that MMP-2 and MMP 78 9 synthesis is promoted by ang11 . Medial dissection then follows during days 4-10 of 7981 angli infusion, with luminal dilation and thrombus formation. Thrombi are contained by adventitial tissue, although in 10% of cases, rupture of the aorta occurs. The thrombi lead to inflammation, indicated by the infiltration of T and B lymphocytes, as well as macrophages. By 28 days, new ECM is deposited in the areas that contain thrombi. Disordered elastin fibers, in the areas where medial elastin fiber breaks are observed, are also present by 28 . 45 days  1.3  Granzyme B  GrB, originally named cytotoxic T-lymphocyte (CTL)-associated gene transcript- 182, plays an important role in the immune system.  It is involved in anti-viral and anti-tumour  functions, and is associated with autoimmunity, transplant rejection, graft-versus-host disease, and thymocyte development . The granzyme family is a highly conserved set of serine 83 proteases. In humans there are five (A, B, H, K and M) arranged in clusters on three separate chromosomes. In mice, there are ten granzymes (A-G, K, M-N) . Granzyme A (GrA) is also 84 involved in immune-mediated killing, and is expressed by both innate and adaptive immune cytotoxic cells while little is known about Granzymes H, K and . 85 M  14  Chapter 1: Introduction  GrB is well known for its contribution to CTL-mediated target cell apoptosis. GrB-deficient mice possess a normal phenotype, with the exception of a slightly reduced CTL-mediated target cell apoptosis, anti-viral responses and tumour cell clearance ’ 86  87,  thus indicating a  redundancy in mechanisms that can be utilized by immune cells to eliminate unwanted cells. GrB-deficient recipient mice exhibit reduced allograft vasculopathy (AV) , and its 88 deficiency in mice leads to increased susceptibility to allergen-induced asthma . In humans, 89 GrB polymorphisms and their implications have begun to be examined 91 with one allele ’ 90 appearing to be correlated with an increased Epstein Barr-virus-associated hemophagocytic risk 92 In samples taken from patients with advanced atherosclerosis ’ lymphohistiocytosis 90 and transplant vascular disease, the presence of GrB was associated with increasing disease severity and cell death. The latter studies observed a marked increase in GrB in macrophages, T cells and the occasional SMC in advanced plaques. Extracellular GrB staining was also absent in advanced disease while no GrB was observed in healthy arteries . 93  1.3.1  GrB-mediated Apoptosis and Signalling  In addition to CTL and natural killer cells (NK), GrB is also expressed in mast cells, CD4+/CD25+ regulatory T-cells (Tregs), blood basophils, SMC and keratinocytes . GrB 9397 is sequestered intracellularly in cytotoxic granules, along with perform  cathepsin C and other  cytotoxic molecules. GrB is produced as a zymogen, and is proteolytically activated by cathepsin C in cytotoxic granules, isolating it from the rest of the cell and thereby preventing effector cell apoptosis ’ 98  .  Upon stimulation of the aforementioned cells by foreign antigen,  GrB and perform are exocytosed into the immunological synapse between the effector and  15  Chapter 1: Introduction target cell. The mechanism by which GrB enters the target cell is an area of significant research and debate. Early reports suggested that GrB entered the cytoplasm of target cells through perform-generated channels in the plasma membrane’°°.  However, more recent  investigations suggest that GrB is endocytosed in a mannose-6-phosphate (M6P) receptor dependent manner with perform triggering the release of GrB from the intracellular vesicles into the cytoplasm of the target cell ’ 101  I 02  In addition to caspase-dependent apoptotic  pathways, GrB can trigger several other apoptotic pathways in target cells including some which can kill cells when caspases are inhibite&° . The classical GrB/perforin pathway is 3 shown in Figure 1.2.  16  Chapter 1: Introduction  Mern bra ne ROCKII  -  I Lamins PARP  MITO  I•CAD  —  CAD/FF  Figure 1.2. Mechanisms Involving Granzyme B-Mediated Caspase-Dependent and Independent Cell Death. Consequences of GrB release from effector cell and internalization into cytoplasm of target cell. Activation of cell death through caspase independent and dependent apoptosis is shown. Abbreviations: ALF, Apoptosis-inducing factor; Apaf- 1, apoptotic protease-activating factor 1; Bid, BCL-2 interacting domain; CAD, caspase-activated deoxynuclease; Casp, caspase; CTL, Cyt c, cytochrome c; cytotoxic T lymphocyte; DFF, DNA fragmentation factor; EndoG, endonuclease G; GrB, granzyme B; gtBID, truncated Bid; I-CAD, caspase-activated deoxynuclease inhibitor; M6P, mannose-6phosphate; MITO, mitochondrion; NK, natural killer cell; PARP, poly (ADP-ribose) polymerase; perf perform; P1-9, Protease inhibitor-9; ROCK II, rho-associated coil coiled containing protein kinase II; Smac, second mitochondria-derived activator of caspases.  GrB has also been shown to directly cleave rho-associated coiled-coil containing protein kinase II (ROCK II) in the absence of active caspases to induce apoptotic cell membrane blebbing in a caspase-independent manner . Target cells induced to undergo apoptosis in 95 response to GrB in vitro and in vivo exhibit signs of mitochondrial inner membrane transmembrane potential collapse and mitochondrial cytochrome c (cyt c) release. These  17  Chapter 1: Introduction events were insensitive to caspase inhibition, showing that mitochondrial disruption was GrB-dependent and caspase-independent’° . The release of cyt c from the mitochondrion is 4 believed to be initiated directly through the cleavage of Bid by GrB to generate a truncated form (gtBid) that instigates cyt c release’° . 5108  Cyt c, along with deoxyadenosine  triphosphate (dATP), apoptotic protease-activating factor 1 (Apaf- 1), and procaspase-9, initiates apoptosome formation to facilitate the autocatalytic activation of procaspase-9’° . 4 Caspase-9 in turn activates the executioner caspases which cleave specific structural, functional and reparative proteins, resulting in the manifestation of apoptotic cell death’° . 9  GrB exhibits a broad range of substrate specificity, but preferentially cleaves after aspartate residues, and is capable of processing caspases -3, -7, -8,-lO; cleaving intracellular substrates (DNA fragmentation factor 45 (DFF45), caspase-activated deoxynuclease inhibitor (ICAD), ROCK II, and nuclear lamins); and proteolyzing certain proteoglycans (aggrecan) and extracellular proteins (fibronectin, vitronectin, SMC ECM  )95. 110-115  Protease inhibitor-9 (P1-9) is the only known endogenous cellular, irreversible inhibitor of ’ 116 GrB  It is often believed that CTL granule release is strictly unidirectional into the  target cell. However, in vitro evidence indicates that non-target cells in the proximity of the target cell may also undergo apoptosis in a granule-dependent mechanism’ 18 Further, in vivo work has shown that local serum concentrations of GrB are highly elevated in areas where a CTL response is occurring’ . Thus, bystander cells and proteins in the proximity of an 19 immune response are susceptible to GrB-mediated apoptosis and cleavage, respectively. It has been suggested that immune cells express P1-9, or its murine homologue Spi-6, to evade  18  Chapter 1: Introduction accidental cell death” . In the normal vasculature, endothelial cells and smooth muscle cells 6 express P1-9 suggesting that they may be protected against GrB insult under these ’ 17 conditions’  120  However, P1-9 expression in endothelial cells may be reduced in the  presence of elevated oxidative stress such as that which occurs during ischemia and reperftision associated with transplantation, or atherosclerosis, rendering these cells susceptible to GrB-induced apoptosis. In support, oxidative stress reduces cellular levels of other anti-apoptotic factors, such as c-FLIP, in endothelial cells thereby inducing increased susceptibility to FasL-mediated apoptosis . 121  Further, decreased P1-9 protein levels are  observed in native human atherosclerotic 1 lesions 7 ’  1.3.2  Extracellular Granzyme B  Although it has long been believed that GrA and GrB release is strictly unidirectional towards the target cell, measurements of serum concentrations of GrA and GrB are markedly elevated in conditions of chronic inflammation. For instance, GrA and GrB are present at low levels in the plasma of healthy patients while rising 150-300 fold in the synovial fluid of patients with the chronic inflammatory condition rheumatoid arthritis (Reviewed in ). 122 Patients with ongoing CTL responses, such as infections by Ebstein-Barr virus or human immune-deficiency virus (HIV) type 1,  have elevated plasma  GrA and GrB levels” . 9  Patients with high levels of circulating GrB had more ischemic lesions on cerebral magnetic resonance imaging (MRI), and elevated plasma GrB was an indicator of plaque instability and cerebrovascular events . GrB has been detected in the blood vessels, surrounding cells 123 and ECM of two inflammatory vascular disorders, atherosclerosis and transplant vascular disease, and the level of GrB correlated with disease severity . 93  19  Preliminary results  Chapter 1: Introduction measuring GrB in plasma from patients with chronic obstructive pulmonary disease show an elevated level of GrB’ . In Kawasaki disease, a disease involving vasculitis of medium24 sized vessels and death from the coronary artery aneurysms and dissections, serum GrB levels are elevated . GrB is also found to be released extracellularly in vitro when 125 keratinocytes are grown to confluence, and that the extracellular GrB retains its activity . 97  Hence, persistent leakage of GrB from the effector cell results in a rise in serum levels. This extracellular rise may be eliciting matrix remodelling in chronic inflammatory states such as atherosclerosis, diabetes, and AV.  Recently, GrB has been implicated in extracellular  proteolytic activities. Immunohistochemical studies have shown the expression of P1-9 by non-immune cells in the testis, and the absence of apoptosis of these and surrounding cells. These findings led to the suggestion that GrB may be affecting matrix remodelling in this . These observations suggest that GrB could have some perform-independent effects 26 organ’ in certain cells/tissues. In support of the latter concept, it has been demonstrated in vitro that GrB can induce smooth muscle cell apoptosis in the absence of perform which may involve the cleaving of extracellular matrix proteins, such as fibronectin, which may lead to cell death through anoikis ’ 88  114, 115  Another example is using ultraviolet light (UV) to activate  GrB production in keratinocytes.  UVB activation of keratinoctyes resulted in cellular  production of GrB’ , and knocking-down GrB resulted in inhibition of cell detachment and 27 cell death, and reduced cell migration through fibronectin and vitronectin matrix upon UVA . 28 exposure’  20  Chapter 1: Introduction Recently our laboratory has found that GrB cleaves fibrillin-1 in vitro (Boivin et al, unpublished data). This is a completely novel finding, as nothing else is known about the interaction of fibrillin- 1 and GrB. This is very intriguing, as it may help elucidate a mechanism for the reduced atherosclerosis observed in our laboratory’s study of GrB/apoE DKO mice (Cruz et al, unpublished data). It may also lead to a possible role of GrB in aneurysm diseases, particularly Marfan syndrome, where defects in fibrillin-1 are the causative agent of the disease. A recent paper revealed that in a mouse model of Marfan syndrome (using heterozygous fibrillin- 1 mutant mice) an inhibitor of AT 1, losartan, prevented the formation of aortic aneurysms. This is very relevant to our current study, as we investigate the potential cleavage of fibrillin- 1 by GrB in the context of AAA and aortic dissections induced by angli acting through the AT1 receptor . 24  1.4  Granzyme B in Atheromatous Diseases  1.4.1  Granzyme B in Atherosclerosis and Allograft Vasculopathy  Atherosclerotic lesions exhibit increased endothelial cell turnover (reviewed in’ ). 29 Endothelial injury and activation leads to increased permeability to lipids, pro-inflammatory cytokine release, expression of adhesion molecules and other pro-atherogenic events. Although the precise cause of apoptosis in atherosclerosis and AV is not understood and likely involves a multitude of factors, there is evidence to suggest that GrB may play a key role in such vasculopathies as discussed below.  AV is an accelerated immune-driven form of arteriosclerosis that affects the vessels of vascularized solid organ allografts and is the primary cause of chronic transplant failure in  21  Chapter 1: Introduction patients that survive greater than one year after transplantation’ . 30  Initial studies  demonstrated that GrB was abundantly expressed in advanced atherosclerosis and AV lesions of human patients . When examining diseased atherosclerotic vessels, GrB was found in the 93 superficial intima, deep intima, and the media indicating a significant increase in GrB protein in all regions of vessels with advanced atherosclerosis. In AV, a significant GrB increase was observed only in the deep intima. In the intima of both diseases, GrB was observed  around and within lipid-rich regions where it localized to terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL)-positive foam cells of atherosclerotic plaques. This suggests that GrB is lipophillic and contributing to cell death in these lesions. There was also an accumulation of GrB in and around smooth muscle cells, macrophages, and T cells, and infiltrating leukocytes in AV showed expression of GrB. In addition, extracellular GrB staining was observed . 93  To further understand the role of GrB in AV, a mouse  heterotrophic heart transplant model of AV was utilized. In these studies, hearts from 1 29J donor mice where heterotopically transplanted into 1 29J (syngeneic control), C57 wild-type (allogeneic control), C57/GrB-KO, or C57/perforin-KO mice. Hearts transplanted into GrB KO mice or perforin-KO mice exhibited significantly smaller lesions and luminal narrowing compared to that of wild-type recipients. Reduced endothelial apoptosis was also observed ’ 88 114  The role of GrB in native atherosclerosis requires further elucidation. Recent research suggests that GrB/perforin may be affecting the stability of atherosclerotic plaques, as patients with unstable angina have a population of perform-expressing CD4+ T cells that are absent in patients with stable angina. In addition, perform-expressing CD4+ T-cells have  22  Chapter 1: Introduction been isolated from the peripheral blood of patients with atherosclerosis, and it has been shown that these cells do in fact induce endothelial cell apoptosis through the perform . 31 pathway’  All of this evidence allows the speculation that GrB contributes to smooth  muscle cell apoptosis in the mtima of both atherosclerosis and AV lesions’ . However, it 29 should be noted that the role of smooth muscle cell and macrophage apoptosis in atherosclerosis progression might depend on the stage of lesion affected. In early, stable lesions, increased smooth muscle cell apoptosis may be a beneficial way to reduce intimal hyperplasia.  Conversely, in advanced, unstable lesions, increased smooth muscle cell  apoptosis decreases the cellularity of affected lesions and reduces synthesis of extracellular matrix components. The latter events result in necrotic core formation and thinning of the fibrous cap, respectively, which ultimately contributes to plaque destabilization and . A mentioned previously, patients with elevated plasma GrB levels had unstable 29 rupture’ plaques, and increased cerebrovascular events’ 23  As discussed, the GrB/perforin pathway plays a key role in the pathogenesis of AV ’ 88  132  Damage to the endothelium resulting from immune attack may be an initiating event in ’ 33 AV’  134  Endothelial injury activates CTL which may result in endothelial cell apoptosis  through FasL and granule-mediated pathways’ 35138• Additionally, there is extensive medial damage in AV that is characterized by medial thinning and an increase in smooth muscle cell . This medial damage is known to be largely mediated by CTLs and leads to 39 apoptosis’ impaired vasoconstriction . T lymphocytes may play a key role in the pathogenesis of AV 140 through the induction of apoptosis via the Fas/FasL andlor GrB/perforin pathways. However, it has been demonstrated that CTL kill endothelial cells predominantly through the  23  Chapter 1: Introduction GrB/perforin pathway’ . 37  Thus, it is likely that CTL/GrB-mediated apoptosis may be  responsible for AV-associated medial thinning.  As previously discussed, GrB can induce smooth muscle cell anoikis in the absence of perform by cleaving several extracellular proteins’ ’ 14  115  Cleavage of extracellular proteins  by GrB may contribute to extracellular matrix alterations and vascular remodelling events that occur in AV. It has been shown that disruption of smooth muscle cell attachment to extracellular matrices with integrin antibodies induces smooth muscle cell apoptosis in vivo in a balloon injury model of vascular disease’ . The proposed mechanism for which GrB 41 may be initiating downstream effects leading to the remodelling of the extracellular matrix and the recruitment of immune cells to the lesion is shown in Figure 1.3.  24  Chapter 1: Introduction  a  2. Non—specific Extracellular releeGrB  (  Matrix degradation Matrixremodelling Thini,:.f  ECM  Anur:yn!  firi cap  em otaxis  Autoa ntigens  Fragments Inflammation  Unidire a l 1. 1 intercellular re 1€ 5 C  _——Capoptosis SMC apoptosis  ‘  Vascular dysfunction  Caspases Bid  I —,  gtBid  —  Media  MITO  Intima  +  +  MedIal Thinnir,  Acellu l;rit  Peduced C,ntractilIt  Reduced ECM Prducti:n Thinnin;offIrcu cap  Susceptibilit. t aneursrns  Necrticcrc frn,atin Plaque in:tabiIit  Figure 1.3. Putative Mechanism by which Granzyme B Contributes to the Pathogenesis of Atheromatous Disease. (1) Classical/Intracellular Pathway: GrB is released from cytotoxic immune cells towards the target cells (EC/SMC). Perform facilitates the uptake of GrB into SMC. Once inside the cell GrB executes the apoptotic machinery. Pathological consequences of EC/SMC apoptosis are indicated. (2) Non-specfic/Extracellular GrB Activity: GrB release is not completely unidirectional. In conditions of chronic inflammation, GrB gradually accumulates on lipid-rich regions of plaques and hydrophobic extracellular matrix proteins. Chronic degradation of extracellular matrix proteins may lead to the production of matrix chemotactic fragments that promote the recruitment of immune cells. Abbreviations: CTL, cytotoxic T lymphocyte; EC, endothelial cell; GrB, granzyme B; MITO, mitochondrion; NK, natural killer cell; SMC, smooth muscle cell.  1.5  Hypothesis  Our laboratory studies the mechanisms by which immunological and non-immunological factors contribute to vascular injury, dysfunction and atherogenesis. To study the role of GrB in native atherosclerosis in settings of hyperlipidemia, our laboratory is currently utilizing the well-characterized apoE-KO model of atherosclerosis. We have recently discovered that the  25  Chapter 1: Introduction absence of GrB in GrB/apoE-DKO mice significantly reduced the size and frequency of atherosclerotic plaques compared to that of apoE-KO mice. In addition we have recently shown that in the hyperlipidemic environment of the apoE-KO mice, GrB accumulates on elastin fibers and may contribute to elastm degradation. We have also obtained preliminary data that GrB can cleave fibrillin- 1 in vitro, and that elastin and fibrillin- 1 staining, along with medial thinning are reduced in GrB/apoE-DKO compared to apoE-KO mice. This suggests that GrB is involved in the degradation of elastin and fibrillin- 1 microfibrils in apoE-KO mice.  As such, we hypothesize that the absence of GrB will reduce the incidence of aneurysm formation and dissection.  GrB contributes to the development of aortic aneurysms through  the cleavage of ECM proteins andlor induction of medial smooth muscle cell apoptosis. In particular, that GrB cleavage of fibrillin-1 leads to the degradation of elastic components of the aortic wall, leading to the development of aortic aneurysms. The goal of this research project is to investigate the role of OrB in aneurysms.  Specifically, we wish to further  examine the protective effect of the GrB/apoE-DKO phenotype on medial thinning using a well-established model of AAA and aortic dissections.  1.5.1  Specific Aims  The specific aims of this study are: 1. To determine if OrB deficiency in apoE-KO mice is protective against the development of aneurysms.  26  Chapter 1: Introduction 2. To determine whether GrB deficiency affects the 28-day survival rates of angil treated apoE-KO mice. 3. To determine the mechanism by which GrB contributes to vessel wall instability, and aortic aneurysms and dissections.  27  CHAPTER 2  Methodology  2.1  Animals  Protocols in this study were approved by the Animal Experimentation Committee of the University of British Columbia (see Appendix II for certificate) and involved 50 male mice. C57B116 mice, apoE-KO mice (C57B1/6 background) and GrB-KO mice (C57B1!6 background) were obtained from Jackson Laboratories (Bar Harbor, ME). The GrB/apoE DKO mice were generated by crossing the apoE-KO and GrB-KO mouse strains. Genotyping of the mice was performed using primers and Polymerase Chain Reaction (PCR) protocols designed from Jackson Laboratories (GrB primers: 5’-TGAAG ATCCT CCTGC TACTG C-3’ and 5’-TCCTG AGAAA GACCT CTGCC-3’;  apoE primers: 5’-GCCTA  GCCGA GGGAG AGCCG-3 t and 5’-TGTGA CTTGG GAGCT CTGCA GC-3’) (University of British Columbia NAPS Facility, Vancouver, BC). The pups were weaned at 3 weeks of age and then maintained on a 12-hour day and night cycle with food and water provided ad libitum in barrier conditions at the Genetic Engineered Models (GEM) facility (The James Hogg iCAPTURE Centre for Cardiovascular and Pulmonary Research, UBC/St. Paul’s Hospital, Vancouver, BC). At 3 months of age, the mice were randomly assigned to receive either 28 days of angil infusion, or saline infusion from a subcutaneous 1004 model ALZET® mini osmotic pump.  Chapter 2: Methodology  2.2  ApoE-KO Model of Angiotensin-Il-induced AAA  To induce aneurysms, we used the well-characterized angli osmotic minipump method . 45 ALZET® osmotic pumps (DURECT Corporation, Cupertino, CA) are miniature, implantable pumps commonly used for research in mice and rats. These infusion pumps continuously deliver drugs and other test agents at controlled rates from one day to four weeks without the need for external connections or frequent handling. ALZET® pumps operate by osmotic displacement. An empty reservoir within the core of the pump is filled with the drug solution to be delivered. Due to the presence of a high concentration of salt in a chamber surrounding the reservoir, water enters the pump through its outer surface. This entry of water increases the volume in the salt chamber, causing compression of the flexible reservoir and delivery of the drug solution into the animal.  Mice were weighed immediately before calculating doses, and the mean weight of mice per group was used. Since mice on a regular diet gain O.5g of weight per week, the drug dose was calculated based on the projected mid weight of the experiment (so mice were slightly overdosed for the first two weeks and under-dosed for the final two weeks). The dose was calculated based on the pump fill volume, mean pumping rate, and the midpoint weight of the mice to achieve a value of 1.44mg/kg/day, (equivalent to l000ng/minlkg). To ensure accurate filling, pumps were weighed before and after filling, and a value of 1mg equal to 1iL was used when assessing fill volume. Angil was obtained from Sigma in 5mg aliquots, and stored at -20°C until use. Fresh angil was diluted in saline for each experiment. All pumps and solutions were worked with under sterile conditions. After filling of the pump with angli or vehicle control, they were stored in sterile tissue culture tubes, containing  29  Chapter 2: Methodology enough saline to completely cover the pump. All test tubes were placed in the incubator at 37 °C for 24 hours prior to implantation. Normally the pumps should incubate for 48 hours before they will begin to release angli at the proper flow rates and doses. However, this 24 hour incubation allowed the pumps to partially prime. They should not begin releasing angil at full dose for an additional 24 hours after implantation. This will allow the mice 1 day to recover from the surgery of implantation, prior to the potential stress of angil infusion.  On day 1, mice were brought into the GEM micro-procedure room.  Animals were  anesthetised with gaseous anaesthetic at a flow rate of 1 .5L per minute of oxygen with 2.5% of isoflurane delivered via a Baines system using a calibrated tabletop anaesthetic machine, administered from a rodent nose cone. Depth of anaesthesia was monitored by toe pinch response and breathing. Eyes were protected using ocular lubricant. The back of the mouse was shaved, and the legs were taped down. The back of the mouse was then sterilized with 70% ethanol, followed by iodine. The mouse was then moved to the surgical table, so that the dissecting microscope could be utilised.  Under sterile conditions, a lateral incision was made below the scapula, and the skin was blunt dissected from the subcutaneous layer, making a hole large enough to fit the minipump. The minipump was inserted with the flow regulator angled towards the head. Incisions were closed with 2-4 dissolving discontinuous sutures. Isoflurane was then shut off, allowing the mouse to breathe pure oxygen, and a dose of buprenorphine was administered for pain relief. After 1 hour, when the mice had recovered from anaesthesia, they were moved back to the animal holding room. The mice were monitored daily for the remainder of the experiment.  30  Chapter 2: Methodology At day 28, tissues from the surviving mice were collected. Blood was collected by cardiac puncture following CO 2 euthanization. The mouse was placed on ice, the chest is opened, the right atrium cut, and a needle is placed in the left ventricle. Saline, and then 4% para formaldehyde, was perfused at a constant pressure of 1 OOmmHg using a pressurized tubing system until no blood is observed exiting the incision in the right atria. The heart, aorta to the iliac bifurcation, and kidneys were dissected from the mouse and photographed. At this point, a gross description of the aorta is made. In the case of the mice that were found deceased, tissue perfusion was not an option. collected without perfusion. lost.  In these cases, the aorta and heart were  Due to autolysis, some tissue was difficult to dissect and was  The tissue was stored in fresh 4% para-formaldehyde overnight, and photographed  again before being sectioned and embedded.  2.3  Tissue Fixation, Excision and Processing  2.3.1  Aortic Roots  The upper half of the heart (containing the atria and aortic arch) was frozen in optimum cutting temperature embedding medium (OCT)  (Cryomatrix, Shandan)  for serial  cryosectioning covering 1 0tm of the root. From each heart 10-20 sections were obtained. Sections were stained with Oil red 0 (ORO), Hematoxylin and eosin (H&E) and Movat’s Pentachrome (Movat’s) stains to examine the presence of atherosclerotic lesions present in the aortic root (see below for protocol).  The slides were then viewed under a light  microscope. ImageProPlus® (MediaCybernetics, Silver Spring, MD) was used to trace and quantify the atherosclerotic lesions, as well as the lumen area and valve area of each sample.  31  Chapter 2: Methodology Atherosclerotic lesions were expressed as the cross-section area of the lesions, as well as the ratio of lesion to valve cross sectional area, and lesion to lumen area.  2.3.2  Abdominal and Thoracic Aortic Sections  Sections were isolated from the descending aorta immediately above the diaphragm, and the thoracoabdominal aorta immediately above the renal arteries. These sections were embedded and frozen in OCT, and serially cryosectioned into lOj.tm specimens.  2.3.3  Histological Stains  Hematoxylin & Eosin Tissue sections were washed in two, 5 mm changes of water to remove OCT. Slides were then immersed in hematoxylin for 5 mm, removed, washed in dH O for 1 mm, differentiated 2 in 1% acid alcohol rapidly (5-10 sec), washed in dH O for 1 mm, and then immersed in 2 lithium chloride for 30 sec. The tissue was then washed in dH O for I mm and immersed in 2 70% isopropyl alcohol for 30 sec before staining with eosin. Slides were immersed in 1% eosin in 80% alcohol for 30 sec, drained, air-dried over night, and immersed in xylene for automatic coverslipping.  All slides were examined under light microscopy and  photomicrographs obtained on a  SpotTM  digital camera. The exposure was automatically  calculated subsequent to white balancing.  Movat’ s Pentachrome Tissue sections were washed in two, 5 mm changes of water to remove OCT.  Slides were  oxidized by saturation in aqueous picric acid for 10 mm at room temperature, washed in  32  Chapter 2: Methodology running water until colourless, rinsed in dH O and then rapidly immersed in 3% acetic acid. 2 Slides were then immediately immersed in Alcian Blue (Ig Alcian Blue, 3mL glacial acetic acid in lOOmL dH O) for 30 mm, rinsed in 3% acetic acid, and washed in warm running tap 2 water for 10 mm. After rinsing in 2 dH O , slides were immersed in Verhoeff’s stain for 45 mm, then rinsed and soaked in warm tap water for 5 mm. Tissue sections were then washed in 2 dH O , immersed in Biebrich Scarlet-Acid Fuchsin (0.8g Biebrich Scarlet, 0.6g Acid Fuchsin, l.6g Phosphotungstic acid in l4OmL dH O) for 10 mm, rinsed in 2 2 dH O , and the colour differentiated in 5% phosphotungstic acid for 2 mm.  Finally, the slides were rinsed  with dH O and de-hydrated in 3 changes of 100% ethanol before staining in alcoholic saffron 2 (6g saffron in lOOmL ethanol) for 10 mm at 60°C (modified from’ ) The tissue sections 42 were set to air dry over night, and then immersed in xylene before coverslipping using an automatic machine.  AT1 and Fibrillin-l Staining Tissue sections were washed in two, 5 mm changes of water to remove OCT. Heat-based antigen retrieval was performed by boiling slides for 15 mm  in citrate buffer (pH 6.0)  followed by 30 mm of cooling in order to unmask antigenic epitopes that are modified by formalin-fixation. Slides were then washed in Phosphate Buffered Saline (PBS) twice, each time for 5 minutes. Slides were then quenched in 3% hydrogen peroxide, and washed in 3 changes of PBS. Slides were blocked with 10% normal goat serum in PBS for 30 mm at room temperature. Blocking serum was removed, and 10% normal goat serum with either rabbit anti-fibrillin- 1 (Dako; Carpinteria, CA, USA), or rabbit anti-AT 1 (Santa Cruz; Santa Cruz, CA, USA) overnight in a humidified chamber at 4°C.  33  The primary antibody was  Chapter 2: Methodology removed, and the slides washed two times in PBS for 5 mm  in each wash at room  temperature before incubation in a 1:350 dilution of biotin goat anti-rabbit in 5% normal goat serum for 30 minutes in chamber. The secondary antibody was then removed, the slides washed in PBS (pH 7.4) three times for 5 mm  in each wash.  Prepared ABC reagent  (VECTASTA1N® ABC (Horseradish Peroxidase) kit, Burlingame, CA) was added to each section, and incubated for 30 mm  at room temperature. Slides were then washed in two  changes of PBS with 0.1% tween (PBST) for 5 mm each wash, and then one time in PBS for 5 mm. To visualize staining Nova-red solution was incubated for 5-6 mm, and following incubation slides were immediately washed in water. Slides were then counterstained with hematoxylin for 1 mm and washed in water. The tissue sections were set to air dry over night, and then immersed in xylene before coverslipping using an automatic machine.  ApopTag Peroxidase In Situ Apoptosis Detection Staining ApopTag® staining on OCT-embedded sections was utilized to assess DNA fragmentation and was carried out as per the manufacturer’s instructions (Chemicon Intematural, Inc.). Slides were washed in water to remove OCT in the same manner as described above for immunohistochemistry. Slides were then washed twice in PBS for 5 mm in each wash and incubated with 20ig/mL proteinase K for 20 mm at room temperature to permeabilize the tissue and to digest DNA-associated proteins. Slides were washed three times in PBS (for 5 mm in each wash and residual peroxidase activity in tissues quenched with the addition of 3% H 0 for 15 mm at room temperature. After washing three times in PBS, tissue sections 2 were incubated in equilibration buffer for at least 10 sec and then with TdT enzyme in the presence of digoxigenin UTP for lh at 37°C.  34  TdT enzyme was then inactivated by  Chapter 2: Methodology incubation in Stop Buffer for 10 mm. After washing the slides three times in PBS, anti digoxigenin antibody conjugated to HRP was added for 30 mm at room temperature to detect the DNA strand breaks that have been labelled with biotinylated UTP by TdT. Slides were then washed three times in PBS and staining was visualized by incubating slides in DAB for 5-10 mi  Hematoxylin was used as a nuclear counterstain before coverslipping and  microscopic examination of the stained sections.  Photomicrographs were obtained as  described above for immunohistochemistry.  2.4  Atherosclerosis Histological Quantification.  Serial 10 jim sections of the aortic roots isolated as described were stained with H&E, Movat’s pentachrome, or ORO. ImageProPlus® was used to quantify the lesion area per lumen cross section in ten to twenty sections from each mouse that survived to the 28 day endpoint, which were then averaged to provide mean lesion area per mouse (Figure 2.1). To calculate aortic lumen area and medial thickness, the internal and external elastic lamina from Movat’s pentachrome stained sections were traced with ImageProPlus®.  The  maximum lumen area was calculated using the internal elastic lamina perimeter. The medial thickness was calculated by subtracting the maximum area calculated from the external elastic lamina from the maximum area calculated from the internal elastic area, and then dividing by internal elastic lamina perimeter value.  35  Chapter 2: Methodology  Figure 2.1. Examples of ImageProPlus® Tracing. Movat’s pentachrome stain of aortic roots (A,B) and abdominal aorta (C,D). ImageProPlus® tracing is indicated by green line in each panel. A) Tracing of an aortic root lumen. B) Tracing of one plaque. C) Tracing of the internal elastic lamina. D) Tracing of the external elastic lamina.  36  •CHAPTER 3  Resuits  3.1  Implantation of Osmotic Mini Pump  We successfully optimized the implantation of ALZET® 1004 minipumps into our mice (Figure 3.1). There was a 0% mortality rate from surgical complications. One mouse was euthanized early due to observed weight loss attributed to a mal-occluded tooth, and this animal was discarded from all of our data analysis. Table 3.1 details the ages of mice at time of implant, start and change in weight, and number surviving to 28 days. Mice were slightly older in the angll infusion GrB/apoE-DKO group, due to availability of litters.  I “-  J I ...  Figure 3.1. Mouse Implanted with AEZET® 1004 Pump. Three month old mice were implanted with ALZET® 1004 model pumps containing either 1 OOi.tL of saline or angiotensin II. The pumps remained in the animal for the remainder of the 28 day experiment. There were no complications or deaths attributed to the pump implantation procedure.  Chapter 3: Results Table 3.1. Summary of Animals Used in this Study Genotype  Treatment  n at 0 days  n at 28 days  Average age (days)  Starting weight (g)  Weight change (g)  apoE-KO  saline  8  8  109.8  27.7  1.3  apoE-KO  angll  16  9  104.3  28.2  1.0  GrB/apoE DKO  saline  11  11  112.7  29.8  1.2  GrB/apoE DKO  angll  15  13  136.1  29.1  -0.3  Table 3.1. A total of 50 male mice were used in this study. 24 apoE-KO mice received either saline or angEl, and 26 GrB/apoE-DKO mice received either saline or angil. The starting ages and weights are listed for all mice. The weight change value is calculated from only the mice that survived to the 28-day time point. Abbreviations. angil, angiotensin II; apoE-KO, apolipoprotein E-knockout; GrB/apoE-DKO, gralizyme B/apoE-double knockout mouse.  3.2  Survival Following Angiotensin II Infusion  Figure 3.2 shows a Kaplan-Meier curve detailing the survival over the 28 day experiment. No mortality was observed in either group infused with saline, and GrB/apoE-DKO mice had a significant increase in survival (83%) versus apoE-KO mice (56%) to 28-days with chronic angil infusion.  38  Chapter 3: Results  100 a)  A ‘.  80  -k-  60  -‘—  U)  a) a) (3  apoE-KO, saline apoE-KO, angil apoE/GrB-DKO, saline apoE/GrB-DKO, angil  40  I-  0  20 0 0  I  I  I  I  7  14  21  28  Days Figure 3.2. Kaplan-Meier Survival Curve. Lines represent percentage of mice alive on each day. No death was observed in either control group (n = 8 for apoE-KO, n= 11 for GrB/apoE-DKO. In contrast, 86.67% of GrB/apoE-DKO (n 15) and 56.25% of apoE-KO mice (n = 16) infused with angil survived to 28 days. Curves were significantly different, as measured by the Log-rank (Mantel-Cox) Test (p 0.0037). Abbreviations: angil, angiotensin II; apoE-KO, apolipoprotein E-knockout; GrB/apoE-DKO, granzyme BIapoE double knockout mouse.  3.3  Angiotensin II Associated Pathology is Decreased in GrB/apoE-DKO Mice  Figure 3.3 shows characteristic gross pathology assessed at 28 days. Aortic dissections were defined by blood accumulation between the media and outer aortic wall, and abdominal aneurysms were defined by the presence of a small, medial thrombus. Mice that were found dead without any preceding signs of suffering were defined as sudden death. Figure 3.4 shows representative H&E images of a healthy aorta, a small AAA, and a large aortic dissection.  39  Chapter 3: Results Saline Control  Abdominal Aortic Aneurysm  Aortic Dissection  Figure 3.3. Gross Pathology of Aortas. At day 28, tissues from the surviving mice were collected. Blood was collected by cardiac puncture following CO 2 euthanization. The mouse was placed on ice, the chest was opened, the right atrium was cut, and a needle was placed in the left ventricle. Saline, and then 4% para-formaldehyde, were perfused at a constant pressure of lOOmmHg until no blood is observed exiting the incision in the right atria. The heart, aorta to the iliac bifurcation, and kidneys were dissected from the mouse and photographed. A healthy aorta is shown in the left panel, which is representative of what was observed in all saline infused animals. An aorta with an abdominal aortic aneurysm is shown in the middle panel (indicated by red arrow), which is representative of 30% of the GrB/apoE-DKO mice. An aorta with an aortic dissection is shown in the far right panel (dissection length indicated by 2 red arrows), which is representative of what was observed in the majority of surviving apoE-KO mice. Abbreviations: apoE-KO, apolipoprotein E knockout; GrB/apoE-DKO, granzyme B/apoE-double knockout mouse.  40  Chapter 3: Results  Figure 3.4. H&E Staining of Representative Abdominal Aortas. A. B normal, healthy blood vessel. B. A vessel with a small medial thrombus, indicative of a small aneurysm. C. A vessel with a large amount of blood in the media, indicative of a large dissecting aneurysm. Scale bar = 1 OOOtm.  The differences in pathological outcome are shown graphically in Figure 3.5. No pathology was observed with either saline group. The surviving mice in the apoE-KO angil group developed aortic dissections in contrast to the small AAA observed in 30% of the GrB/apoE DKO surviving group. Our pathological assessments were confirmed by H&E and Movat’s staining of cross sections from thoracic and abdominal aortas (Figure 3.6).  41  Chapter 3: Results >1  0  Sudden Death AAA Aortic Dissection No Pathology  0. C, C 0. U, C)  U,  C a, U h.  a,  0.  Figure 3.5. Gross Pathological Outcomes. Bar graph showing the outcome of all mice used in experiment. Sudden death was defined as mice that expired without any signs of suffering. On necropsy, it appeared that death was caused by a rupture of the aorta, in all but one of the apoE-KO mice and the single GrB/apoE-DKO mouse that died early. No pathology was observed in the saline groups, and aortic dissections were seen only in the angli infused apoE-KO group. Abbreviations: AAA, abdominal aortic aneurysm; angli, angiotensin II; apoE-KO, apolipoprotein E-knockout; GrB/apoE-DKO, granzyme BIapoE double knockout mouse.  42  Chapter 3: Results B.  A.  Figure 3.6. Movat’s Pentachrome Staining of Abdominal Aortas. A. apoE-KO, angli; B. apoE-KO, saline; C. GrB/apoE-DKO, saline; D. GrB/apoE-DKO, angli. No pathology was observed with saline infusion in either the apoE-KO or the GrB/apoE-DKO groups. Following 28-days of angli infusion, most apoE-KO surviving mice displayed a dissecting aneurysm. GrB/apoE-DKO mice, however, displayed no pathology, or small AAAs. Scale bar = l000j.tm. Abbreviations: AAA, abdominal aortic aneurysm; angli, angiotensin II; apoE-KO, apolipoprotein E-knockout; GrB/apoE-DKO, granzyme B/apoE-double knockout mouse.  43  Chapter 3: Results  3.4  AT1 Receptor in Transgenic Mice  Immunohistochemistry revealed the presence of the AT1 receptor in both the apoE-KO and GrB/apoE-DKO vessels. A wildtype mouse (C57) was used as a positive control (Figure 3.7). This result verified that the AT1 receptor was not inadvertently affected during the generation of the transgenic strains.  C57  apoE-KO  GrB/apoE-DKO  Figure 3.7. AT1 Staining of Aortic Tissue. The AT1 receptor was present in the apoE-KO and GrB/apoE-DKO mouse. AT1 stain is indicated by red colour. Scale bar lOO!Im. Abbreviations: AT 1, angiotensin II type 1 receptor; apoE-KO, apolipoprotein E-knockout; C57, C57 wildtype mouse; GrB/apoE-DKO, granzyme B/apoE-double knockout mouse.  3.5  Thoracic and Abdominal Aortic Lumen Area  Figure 3.8 shows the dimensions of the thoracic and abdominal aortas, expressed in area. A one-way ANOVA/ Dunn’s Multiple Comparison Test was performed to compare all values. The only significant difference (p<O.05) was between the abdominal lumen area of apoE-KO mice that received saline or angli for 28 days.  44  Chapter 3: Results  p<O.05  I  I  C4  E Cu  C a)  E -J  Figure 3.8. Lumen Area of Thoracic and Abdominal Aortas. Samples were collected following 28 days of angli or saline infusion. Formalin fixed tissue was embedded in OCT, sectioned on a cryostat, and stained with Movat’s pentachrome. The internal elastic lamina was traced using ImageProPlus, and the maximum diameter was calculated. Angil caused a significant increase between apoE-KO abdominal samples (one-way ANOVA/ Dunn’s Multiple Comparison Test, p<O.O5). Bars represent mean value, error bars represent standard error of the mean (SEM). Abbreviations: Abd, abdominal aorta; angil, angiotensin II; apoE KO, apolipoprotein E-knockout; GrB/apoE-DKO, granzyme B/apoE-double knockout mouse; Tx, thoracic aorta.  45  Chapter 3: Results  3.6  Angiotensin II Causes Medial Thickening  The medial thickness of both the thoracic and abdominal samples was measured. Although not significant, except for the apoE-KO saline versus angli thoracic samples (p<O.O ), there 5 is a trend for medial thickness to increase following angli infusion, as was expected in the literature (Figure 3.9).  (0  .  0  a)  Figure 3.9. Medial Thickness of Thoracic and Abdominal Aortas. Samples were collected following 28 days of angil or saline infusion. Formalin fixed tissue was embedded in OCT, sectioned on a cryostat, and stained with Movat’s pentachrome. The internal and external elastic lamina were traced with ImageProPlus. The medial thickness was calculated by subtracting the maximum area calculated from the external elastic lamina from the maximum area calculated from the internal elastic area, and then dividing by internal elastic lamina perimeter value. Bars represent mean values, error bars represent standard error of the mean (SEM). Significant differences were only achieved between the thoracic values for apoE-KO mice (one-way ANOVA/Dunn’s Multiple Comparison Test, p<O.O5). Although not significant, there is a trend for angil infusion to increase medial thickness. Abbreviations. Abd, abdominal aorta; angli, angiotensin II; apoE-KO, apolipoprotein E knockout; GrB/apoE-DKO, granzyme B/apoE-double knockout mouse; Tx, thoracic aorta. 46  Chapter 3: Results  3.7  Aortic Root Atherosclerosis  No significant differences were observed between the amount of atherosclerosis present in the aortic root between the 4 groups (p>O.O5) (Figure 3.10). Figure 3.11 represents the percentage of plaque in the lumen. No significant difference was observed with lumen or plaque measurements (p>O.05).  apoE-KO  DKO  apoE-KO  DKO  apoE-KO  DKO  saline  angli  H&E  MOVAT’S  ORO  Figure 3.10. Aortic Roots Stained by H&E, Movat’s and ORO. Formalin fixed hearts collected from mice that survived to 28 days were embedded in OCT, and sectioned on the cryostat. Slides were stained with H&E, Movat’s, and ORO. When the aortic root lumen area and plaque area were obtained, no significant difference was observed in calculated percent lumen covered by plaque between either genotype on either treatment. Scale bar = 1mm. Abbreviations: angil, angiotensin II; apoE-KO, apolipoprotein E-knockout; DKO, granzyme B/apoE-double knockout mouse; H&E, Hematoxylin and eosin; ORO, Oil red 0.  47  Chapter 3: Results  .  a)  100  .  C.)  C.) 0  a) E ‘I  80 60 40  0  4-I  a) C.) Ia)  0.  20 0  .  I  U..... I..... ._._._ _. U  “••‘  I  I[111111HI  Figure 3.11. Measurements of Aortic Root Lumen and Plaques. Aortic roots were collected from animals surviving to 28 days. No significant difference was observed between either genotype on either treatment (one-way ANOVA! Dunn’s Multiple Comparison Test, p>O.05). Each experimental group is made from 10-20 sections per mouse, and each group had 7-1 1 mice per group (apoE-KO saline, n=7; GrB/apoE-DKO saline, n=7; apoE-KO angil, n=9; GrB/apoE-DKO, n=1 1). Bars represent percentage of aortic root lumen covered by plaque, error bars represent standard error of mean (SEM). Abbreviations: angli, angiotensin II; apoE-KO, apolipoprotein E-knockout; GrB!apoE-DKO, granzyme B/apoE-double knockout mouse.  48  Chapter 3: Results  3.8  GrB/apoE-DKO Mice and Fibrillin-1 Expression  Staining with antifibrillm- 1 revealed a greater amount of fibrillin- 1 in GrB/apoE-DKO mice versus apoE-KO in both saline and angil treatment groups (Figure 3.12). Interestingly, in areas around the thrombus of an AAA or dissection, very minimal fibrillin- 1 staining was observed (Figure 3.12, bottom right panel).  GrB/apoE-DKO  apoE-KO  saline  .“. -r  .  angli —  Figure 3.12. Fibrihin-1 Staining in Abdominal Aorta. Samples were collected following 28 days of angll or saline infusion. Formalin fixed tissue was embedded in optimal cutting temperature (OCT), sectioned on a cryostat, and stained with anti-fibrillin-l. Decreased fibrillin-l staining, as indicated by red colour, was observed in apoE-KO mice from saline and angll- infusion, versus GrB/apoE-DKO mice. Scale bar = lOOm. Abbreviations: angll, angiotensin II; apoE-KO, apolipoprotein E-knockout; GrB/apoE-DKO, granzyme B/apoE-double knockout mouse.  49  CHAPTER 4  Discussion  4.1  Osmotic Mini Pump Model  The use of ALZET® minipumps was a novel protocol for our animal centre. We had little trouble optimizing the filling of the pump and the implantation into the mice. Excitingly, since this project has begun, other groups in our centre have switched from multiple injection protocols to pump implantation protocols. This technology has reduced the costs associated with multiple injections and technician time, and reduced the stress on animals that was associated with daily handling. It also allows for a constant, chronic dosing as opposed to an oscillating dose associated with injections.  4.2  GrB Deficiency Protects against Sudden Death and Aortic Dissection  Over the 28-day experiment, GrB-deficiency caused a significant increase of survival (83%) over apoE-KO mice that still possessed GrB (53%). GrB/apoE-DKO mice that did develop AAA were very small, early stage AAA that did not dissect. This was in contrast to the AAA observed in the apoE-KO mice that were larger and had dissected. When the apoE-KO mice that died early during the experiment were examined, large blood clots were found in the thoracic cavity. Often, the aorta was also dissected up to the level of the heart. This suggests that death was caused by rupturing of the aorta. With this, we suggest that GrB plays an  Chapter 4: Discussion important, detrimental role in the development of AAA and the progression of dissecting AAA, and that its deficiency is protective. In addition, we observed a lack of fibrillin-l staining in the aortas of apoE-KO mice compared to GrB/apoE-DKO mice.  With this  observation and our laboratory’s recent finding that GrB can cleave fibrillin-l in vitro, we suggest that GrB contributes to AAA formation and progression by cleaving fibrillin-l (please see chapter 5 for a detailed mechanism).  4.3  Increased Atherosclerosis and Onset of Abdominal Aortic Aneurysm are Not Related  No difference in the amount of atherosclerosis in the aortic root was observed between apoE KO and GrB/apoE-DKO mice receiving saline or angli. This result was initially surprising. We had anticipated that both apoE-KO and the GrB/apoE-DKO mice would show an increase in atherosclerotic plaques when infused with angil over 28 day, as is reported in the . However, our mice were on a regular chow diet and were only 3 months of age 45 literature in contrast to the high fat diet in the majority of AAA studies and our own laboratory’s observations. Moreover, other groups have reported great variability in the extent and stage of atherosclerotic lesions in apoE-KO mice younger than 4 months in age fed a regular diet . 38 What is unique about this finding is that the GrB/apoE-DKO mice were protected against developing AAAs, aortic dissections, and aortic ruptures while having no change in atherosclerosis.  This adds to the argument that angli-induced AAA do not require  atherosclerosis, which is an area of controversy in the understanding of this relatively new model (previously discussed in the introduction). This finding has also been reported in apoE/MMP-3-DKO mice, where atherosclerotic lesions increase and aneurysm prevalence  51  Chapter 4: Discussion . Atherosclerotic lesions also decrease in apoE/TIMP- 1 -DKO mice while AAA 50 decreases incidence increases . However, in the majority of mouse aneurysm studies, atherosclerosis 51 severity and AAA incidence and severity both increase/decrease together (reviewed 40 in In ). human populations, 90% of abdominal aortic aneurysms are associated with atherosclerotic plaques, but this may be a result of the shared risk factors for both diseases, and the two may not be correlated.  4.4  Aortic Structure and Composition  As expected, angll infusion did increase medial thickness in both the thoracic sections and abdominal sections of the aorta. Lumen dimensions of abdominal compared to thoracic aorta sections in all groups did not change with saline infusion.  However, apoE-KO mice  exhibited an increase in abdominal aorta lumen expansion following angli infusion.  It  should be noted that only mice without aneurysm or dissection were included in this analysis, and this data shows that apparently “healthy” apoE-KO mice receiving angli infusion exhibited signs of early AAA, in this case a dilated lumen.  When examining abdominal aortic sections stained with Movat’s pentachrome, there appeared to be a consistent increase in collagen staining in the apoE-KO mice compared to the GrB/apoE-DKO mice perfused with angli. In particular, the area of the lumen in contact with intramural thrombus showed increased collagen staining. Increased collagen-i deposition is an indirect marker of increased transforming growth factor (TGF)-f3 . Unfortunately, we were unable to quantify this increase due to staining being 43 signalling’  52  Chapter 4: Discussion done on different days. A collagen specific stain, such as Sirius red would help elucidate this mechanism.  4.5  Inconclusive Apoptosis and CD3 Data  When staining for apoptotic cells, there were no positive nuclei in any of our samples. Since angll causes medial hypertrophy and hyperplasia, we were not surprised that we did not see an increase in apoptotic cells in our experiment.  However, the staining quality was  problematic. With the CD3 stain, once again very few positive nuclei were observed. This may be because we are looking too late to see CD3+ leukocytes in the developed AAA, or because we were using formalin fixed tissues.  When we perform our next group of  experimental mice to collect blood pressure data (see next chapter), we will be collecting fresh frozen aortic tissue and will be staining again for leukocytes and macrophages, as well as repeating the apoptosis stain.  53  CHAPTER 5  Conclusion  5.1  Proposed Mechanism  The role of GrB in the angli-induced AAA and aortic dissections is multifactoral. From the evidence presented in this study, we can conclude that GrB-deficiency has a protective role against aneurysm and dissection formation and rupture. This appeared to be independent of any change in atherosclerotic plaque formation.  The effects of angil are mediated by  signaling through the AT1 receptor, and are depicted in Figure 5.1.  In VSMC, angil  activates phospholipase D (PLD)-mediated phosphatidylcholine hydrolysis (PLD), causing a production of phosphatidic acid (PA). ’ 57 R0S  PA stimulates NADPH oxidase, which produces  ROS causes VSMC hypertrophy and hyperplasia, lipid peroxidation, activates  MMP-2 and -9, and activates an inflammatory response, recruiting monocytes and leukocytes . Vascular ROS also inactivates NO, which results in endothelial cell 57 to the vasculature dysfunction. Endothelial cells are also affected directly through AT1 signaling, which causes an increase in E-selectin and VCAM- 1, which causes increased leukocyte adhesion ’ 63  .  Macrophages are also affected directly through AT 1 signaling. Macrophages increase 12/15 lipoxygenase, peroxide production, LDL oxidation, cholesterol synthesis and decrease . 69 cholesterol efflux  Chapter 5: Conclusion The recruited immune cells release GrB, which can cleave the extracellular matrix proteins elastin and fibrillin- 1. The newly formed elastin fragments can act as a chemotactic gradient, recruiting more immune cells. MMP-2 and MMP-9 also have elastolytic properties and are expressed by macrophages’. The newly formed fibriliin-l fragments from GrB cleavage are hypothesized to release TGF. AT1 receptor signalling can also increase TGF-f3 22 ligands and receptors, and the expression of thormbospondin-l, which is an activator TGF-43. TGF-13 further activates MMP-2 and MMP-9, and increases collagen-i synthesis’ . The 43 combination of collagen synthesis and elastin degradation leads to mechanical weakness of the vessel wall, leading to aneurysms and dissections.  *GrBJ —  -  .— —  Uib-l frag  I  Figure 5.1. Figure legend on next page  55  Chapter 5: Conclusion Figure 5.1. Angiotensin II Causes Abdominal Aortic Aneurysm and Aortic Dissection Through the AT1 Receptor. AT 1 receptors are represented by yellow diamonds. In VSMC, angli activates phospholipase D-mediated phosphatidyicholine hydrolysis (PLD) through signaling through AT1 receptor, causing a production of phosphatidic acid (PA). PA stimulates NADPH oxidase, which produces ROS. ROS cause VSMC hypertrophy and hyperplasia, lipid peroxidation, may activate MMP-2 and -9, and activates an inflammatory response, recruiting monocytes and leukocytes to the vasculature. Vascular ROS also inactivates NO, which results in endothelial cell dysfunction. EC are also affected directly through AT 1 signaling, which causes an increase in E-selectin and VCAM- 1, which causes an increase in leukocyte adhesion. Macrophages increase 12/15 lipoxygenase, peroxide production, LDL oxidation, cholesterol synthesis and decrease cholesterol efflux through AT 1 receptor signaling. The recruited immune cells release GrB, can cleave ECM proteins elastin and fibrillin- 1. Elastin fragments can act as a chemotactic gradient, recruiting more immune cells. MMP-2 and MMP-9 also have elastolytic properties. The fibrillin- 1 fragments released by GrB may activate TGF-p. AT 1 receptor signalling can increase TGF ligands and receptors, and the expression of thormbospondin-1, which is an activator TGF-f3. TGF-p further activates MMP-2 and MMP-9, and increases collagen-i synthesis. The combination of collagen synthesis and elastin degradation leads to mechanical weakness of the vessel wall, leading to aneurysms and dissections. Abbreviations. AAA, abdominal aortic aneurysm; angil, angiotensin II; AT 1 a, angiotensin II type I a receptor; EC, endothelial cell; eNOS, endothelial nitric oxide synthase; Fib-i frag, fibrilllin- 1 fragments; GrB, granzyme B; LDL, low density lipoprotein; MMP, matrix metailoproteinase; NADPH, nicotinamide adenine dinucleotide phosphate; NO, nitric oxide; ROS, reactive oxygen species; TGF-j3, transforming growth factor; VCAM- 1, vascular cell adhesion molecule.  No one study has completely delineated the entire pathway, but many studies have looked at  specific molecules. Vitamin E is an antioxidant, and when orally administered to apoE-KO mice during angll infusion, AAA formation was significantly reduced’ . Administration of 45 losartan, an AT 1 blocker, resulted in decreased AAA in apoE-KO mice infused with ang-11 , 77 and also in a Marfan syndrome mouse model . 24 reduction in AAA formation’ . 46  AT1/apoE-DKO mice also exhibit a  eNOS/apoE-DKO mice have a high incidence of  spontaneous AAA, showing a role for NO in the development of AAA . When p47phox, a 71 component of NADPH oxidase, is knocked out of apoE-KO mice, AAA formation is greatly . Figure 5.2 details the specific points the mechanism of AAA formation is 47 decreased’ inhibited.  56  Chapter 5: Conclusion  ection Figure 5.2. Potential Therapeutic Targets for Inhibition of Abdominal Aortic Aneurysm. AAA have been prevented or reduced in mouse models of AAA using the following therapeutic targets. Vitamin E is an antioxidant which scavenges ROS, and when orally administered to apoE-KO mice during angli infusion, AAA formation was significantly reduced. Administration of losartan, an AT 1 blocker, resulted in decreased AAA in apoE-KO mice infused with angil, and also in a Marfan syndrome mouse model. AT1/apoE-DKO mice are also exhibit a reduction in AAA formation. eNOS/apoE-DKO mice have a high incidence of spontaneous AAA, showing a role for NO in the development of AAA. When p47phox, a component of NADPH oxidase, is knocked out of apoE-KO mice, AAA formation is drastically decreased. Abbreviations: AAA, abdominal aortic aneurysm; angli, angiotensin II; apoE-KO, apolipoprotein E-knockout; AT1a, angiotensin II  type 1 a receptor; EC, endothelial cell; eNOS, endothelial nitric oxide synthase; Fib- 1 frag, fibrilllin-l fragments; GrB, granzyme B; LDL, low density lipoprotein; MMP, matrix metalloproteinase; NADPH, nicotinamide adenine dinucleotide phosphate; NO, nitric oxide; PA, phosphatidic acid; PLD phospholipase D-mediated phosphatidylcholine hydrolysis; ROS, reactive oxygen species; TGF-13, transforming growth factor; VCAM-l, vascular cell adhesion molecule.  57  Chapter 5: Conclusion  5.2  Concluding Remarks  Following angil or saline infusion, no change in atherosclerosis is observed between GrB/apoE-DKO and apoE-KO mice. GrB ablation in GrB/apoE-DKO mice results in higher 28 day survival rates, and less severe AAA and aortic dissections than apoE-KO mice following 28 days of angli infusion. Saline infusions had no pathological effects in either group of mice. Atherosclerosis quantification was perfonned on aortic root sections from mice surviving to the 28-day time point. No significant changes were observed between the  two genotypes or following the angil treatment. As expected, angil infusion did increase medial thickness in both the thoracic sections and abdominal sections of the aorta. Lumen dimensions of abdominal compared thoracic aorta sections in all groups of mice did not change with saline infusion. However, apoE-KO mice exhibited an increase in abdominal aorta lumen expansion following angli infusion, thus revealing that even “healthy” apoE-KO mice receiving angil infusion exhibited signs of early AAA. Fibrillin- 1 staining is reduced in apoE compared to GrB/apoE-DKO mice, which supports our hypothesis that GrB is cleaving fibrillin- 1, which would result in decreased mechanical structure as well as potentially deregulating the bio-availability of TGF-3, leading to an increased risk of AAA and atherosclerosis.  In summary, GrB-deficiency protects against the development of AAA, aortic dissections, and ruptures in an angil infusion apoE-KO model of AAA.  GrB may be exhibiting its  detrimental effects by degrading ECM proteins, in particular fibirillin- 1, and leading to mechanical weakness and subsequent AAA or aortic dissection.  GrB has now been  identified as a potential therapeutic target for prevention of AAA and aortic dissection.  58  CHAPTER 6  Future Directions  One additional area to examine in the angli AAA model is blood pressure (BP). Although we do expect to see a modest rise in blood pressure with angll infusion, it should have no effect on our study outcome. Evidence supporting this include the observation that C57 mice develop the same increase in BP when infused with angil as apoE-KO, but do not develop . 45 AAA  Norepinephrine increase in BP does not cause AAA in apoE-KO mice . 148  ApoE/eNOS-DKO mice are not protected against spontaneous AAA development when the BP is normalized with hydralazine ’ 71  Vitamin E has no effect on BP, but protects against  AAA in angli-infused apoE-KO mice . However, we are currently underway completing a 145 small group of mice where we will be measuring changes in BP weekly. We would like to perform our experimental model in a group of GrB-KO mice (mice that contain the apoE gene) to verify that GrB is not having a role in AAA development in a normal cholesterol environment. These mice are currently being bred, and the experiments will be completed in our laboratory in the near future. We would also like to perform our study on perforin-KO mice. Perform is the molecule that allows for GrB entry into the target cell. Without perform, all of GrB effect would be from extracellular sources of GrB, and not from intracellular, apoptotic functions (discussed in’ ). 22  Chapter 6: Future Directions It has been proposed that fibrillin-l regulates the bioavailability of TGF-f3.  Deregulated  TGF-f3 signalling has been implicated in select manifestations of Marfan syndrome. We are currently in collaboration with the University of Hawaii examining nuclear translocation of Smad2, which is a marker of TGF-B signalling. The increase in collagen-i synthesis that we observed in our model of AAA is consistent with an increase in TGF-B signalling. Based on the significance of the results described in this study, we will also use inhibitors of GrB, such as serpins and GrB-siRNA, to look at therapeutic intervention in this model.  60  CHAPTER 7  Bibliography  1. 2. 3.  4. 5. 6. 7. 8. 9. 10.  11.  12. 13. 14.  15.  Mackay, J. & Mensah, G. 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Circulation 103, 448-54 (2001).  69  Appendix  Animal Ethics Certificate -}  TOT tNWERS1TY OF 00171011 COLUMBIA  1AL CARE CERTIFICAtE 1 ANJ AppI denY  her: .°.06-C110  Jncesdgaoar or Course Director: Do::d S Crsnr:1:e Onpurtesrur: Patholorvjo Cabors:rrvXlednme Aniatals:  MireTSOBLOIO Mi:epecobprl do lIre Geeoor.’cte B  brordoorur 0-i  -  1.0cr porform l.Zi:o ApcE  ---  310  Mice Groor ro  SItes Dale:  Edna  Irceokeror 06  April 10,2006  Apprar-alDsre:  April30. 2008  Fundiug Sources: FnrodiugAgenrvr FnordlngTirlo:  dOnna C-roar  FssudtscgAgracoc Tussdiss lisle:  l,flchuel Smirk Fcoordscior OooHeal:Roorao:b Grosoyrocel akprip:deocuuosonors6 diroodsec  FcsucilnoAgrnc r 5 Fnndincllrte:  C.roadroaierrinodOs o606eckb Research CIhdil C-car eeAaedlicdorbeeoeoteeoo aodlcrrsrr  eaa 0 FnrrdiugA c Fnnrling Title:  Caaodcoaclcrorr:rarec of ldrilth Reorao-ctriCii-IRI Geoaro-atuB irru e-redu:eftrvneclipidronio-acce 0  Fundincfrgeuov: Fussdtno Title:  dOosoaadircckeooordo:iooofBrrrhcaamddrdccoo Rclo and oeccda:iorr of Gnscorcme Sat arheo-oma:oao drres.ues  Funding Ageuco-: Fsmdiuglirlc:  Lrvofl:iedicirro LOC 0:uor Cp 9rorrdrrc  FssndtrsnAnsnas-: Psandinllirle:  UBC Focrfrv of S Irdicicor S:ooenp fcredi::c  Fussdiaa4genrr: Fundtrr Tide:  Casradiae ccerotes oiookReseacAliDL Coovose ERnie irs ‘:arrrpls::: :oscrolnr dlreaceurd ossroiared vororalar dvr:i.rcct:no  Funding .Ogessrv: Frnsdinn Tide:  Cosrsdn1rro:ibooesaf$OecdrRerrnda1Cii-j ?to:rnsedehibiroo-Oloei?onrenso lsdrib:rec-d: P.oleinhl.uor Troarpisar: Rreooioo  Fussdtargiogenno: Funding Title:  Moclrmllothlorncdooron for Healti: Resooreb Rehra105irro oo:d rode nOr en -sets B in illrneoroa:oroa oamsst  IundtucAgeoec’: Funding Title:  M:chaelladhFnSioo fnet{o&thRoroooch ranoyeceBisratheromntonndimmtc 0 Regral.sinoorodeoleoi  Fnndioglgesson-: Funding Tine:  Ca ostharr Is r: tears ofHmuhRecehdCiiWi Role and oerolr:ia:: of ratosorne B::: ad:saomu:ousd:seusec  Unfrsrsd.sl dde:  To  Hual,rol:!ed c,o::rrCe Bch-rprriperr-arco-::areddoeordeer  trdcrosaoeoorxoo:hoarores:r & Iriadoro  Tlr AromaS Cone Cnrrror::ree has enonined and eppone-ed tIre -ace niacin-is fee to, aboec esoperoosorreal peojed. TOTs reoddonto a s-all in: one Vole donor the above roan nrapprnai dare (echjcheve: is tassel peoooded here A:rrooni enorew :e ojaroned by lee CCAC and rome o-rroioo agerrciec  rICO :icnolge  A rape- oftisis cnrsifirrote means Ore dispia -ed in fasse nraiaarst inaitity. 1  066cr of Research SenIors aardAimmaiorrsoior. 201. 6290 .3oonenor: Road, Vaooorovor, BC OTT ITT Phone- 001-tM-Ill I For 901-022-0195  70  roche —coperimealal pro-sedonro.  

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