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The prevalence of diabetes mellitus is increasing dramatically worldwide. Approximately three out of four diabetic patients will die of cerebro- and cardiovascular disease (CVD). Atherosclerosis, a chronic inflammatory disease of the medium-large arteries, is the major underlying cause of most CVDs. Despite the established progressive relationship between diabetes and CVD, the molecular mechanisms by which diabetes promotes atherosclerosis are not well understood. This has impeded the development of strategies to block or slow atherogenesis in diabetic patients. The objective of my thesis is to investigate the molecular alterations by which diabetes accelerates the development of atherosclerosis using comprehensive metabolomics techniques. We first investigated the development and progression of atherosclerosis at the molecular level in apolipoprotein E-deficient mice. We identified specific changes in plasma-borne metabolites that are associated with the pathogenesis and progression of atherosclerosis. In addition, glycerophospholipid and sphingolipid metabolism were found to be the most significantly altered pathways. Using comprehensive metabolomics techniques, we were able to differentiate atherosclerotic plasma metabolome from healthy control and delineate different stages of atherosclerotic progression. Next, we characterized multiple mouse models of hyperglycemia-induced accelerated atherosclerosis. We showed that the vascular effects of glucosamine supplementation are comparable to streptozotocin-induced and genetically-induced (Ins2Akita) hyperglycemia in terms of lesional glucosamine, endoplasmic reticulum (ER) stress levels and atherosclerotic burden. In addition, we showed that a chemical chaperone (4-phenylbutyric acid) reduces ER stress levels and attenuates accelerated atherogenesis in each of these models. Together these findings support the mechanism involving glucosamine-induced ER stress in hyperglycemia-induced accelerated atherosclerosis. Lastly, metabolomics techniques were used to investigate the molecular alterations by which hyperglycemia promotes the accelerated development of atherosclerosis in several disease models. The three mouse models induced both unique and common changes in the plasma metabolome. Identification of the commonly altered metabolite features revealed alterations in glycerophospholipid and sphingolipid metabolisms, and key atherosclerosis-associated processes including inflammation and oxidative stress. Together, we showed that comprehensive metabolomics techniques can be used to identify specific alterations in the metabolome that are associated with a particular disease genotype and phenotype. These data highlight the important roles of the glycerophospholipid and sphingolipid metabolisms in the pathogenesis of atherosclerosis and diabetic atherosclerosis. The clear difference in the level of several metabolites supports the use of plasma lipid profiling as a diagnostic tool of atherogenesis. / Thesis / Doctor of Science (PhD)

Prevention of macrophage apoptosis in advanced atherosclerotic lesions can help stop atherosclerosis progression to vulnerable plaques. High density lipoprotein (HDL) can protect macrophages from apoptosis that has been induced by a variety of agents. We hypothesize that this is the consequence of the sphingolipid, sphingosine-1-phosphate (S1P), specifically carried by HDL, and transferred to S1P receptor 1 (S1P1) on the cells via the HDL receptor, scavenger receptor class B type 1 (SR-B1). Apoptosis was induced in murine peritoneal macrophages from wild type and different knockout mice with, tunicamycin, thapsigargin, staurosporine, or UV irradiation. Apoptosis was measured by terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL) or with cleaved caspase-3 (CC3) staining. Treatment of cells with HDL or S1P protected them against apoptosis induced by a variety of stimuli. In contrast, pre-treatment of HDL with S1P lyase, which irreversibly cleaves S1P, eliminated the ability of HDL to protect macrophages. Inhibition of SR-B1’s lipid transport activity reduced HDL dependant protection against apoptosis. Furthermore, HDL dependent protection against apoptosis induced by tunicamycin was prevented when the S1P receptor S1P1 was knocked out. However, this protection was not prevented when apoptosis was induced by staurosporine. These results suggest that the HDL mediated protection of macrophages against apoptosis is multi-faceted and one approach may involve SR-B1 mediated delivery of S1P from HDL to the S1P1. Understanding the mechanisms by which HDL elicits atheroprotective signalling in macrophages will provide insight into new targets for therapeutic intervention in atherosclerotic disease. / Thesis / Master of Science in Medical Sciences (MSMS) / Atherosclerosis, is a disease where in the artery walls thicken due to cholesterol build-up, is the major underlying cause for cardiovascular diseases, which is currently a leading cause of death in many populations. We believe that HDL, the “good” cholesterol and S1P, a small molecule carried by HDL, can help prevent the progress of atherosclerosis by preventing macrophages, cells that absorb the cholesterol, from dying. We attempt to prove this by providing S1P or HDL to macrophages that are made to undergo cell death. Results show that both HDL and S1P can protect cells against cell death induced by many factors. However, HDL can protect against certain cell death inducing stimuli without the need for S1P and more research is required to fully understand HDL’s protective role in atherosclerosis. Understanding how HDL elicits atheroprotective signalling in macrophages will help in finding new drugs and therapies to reduce atherosclerosis-based deaths across the world.

Atherosclerosis is a chronic inflammatory disease characterized by plaque or clot build-up in the arterial vessel wall, which leads to blood vessel occlusion and consequently heart attack and stroke. Diabetes is a metabolic disorder characterized by elevated sugar (hyperglycemia), which is a known risk factor for the development and exacerbation of atherosclerosis. Recent studies identified fibrinolytic factors (e.g. plasminogen activator inhibitor 1 (PAI-1), thrombin-activatable fibrinolysis inhibitor (TAFI)) that are linked with worsening of atherosclerosis and diabetes. In addition, since activated TAFI (TAFIa), possesses both antifibrinolytic and anti-inflammatory properties, it is a molecule of interest within the context of atherosclerosis progression. Therefore, we hypothesize that fibrinolysis is influenced by atherosclerosis and diabetes. To test this, we used mice that are prone to developing atherosclerosis (ApoE-/-) and hyperglycemia (Ins2+/Akita) from which the heart and plasma samples were collected at 15- or 25-weeks of age. Overall, no differences in plasma clot lysis times were observed between male (hyperglycemic) and female (normal glycemic) ApoE-/-:Ins2+/Akita mice. Quantitation of plaque volume showed a significant increase at 25-weeks compared with 15-weeks, consistent with previous reports. Closer examination revealed that in 15-week-old mice, plaque volume and PAI-1 levels displayed a trend with lysis time, but not in 25-week-old mice. The lysis times showed no difference with hyperglycemia or age. When TAFIa was inhibited, 15-week-old mice had longer lysis times compared with 25-week-old mice independent of hyperglycemia. In addition, elevation of cholesterol and triglyceride levels from hyperglycemia were only observed at 15-weeks. Total PAI-1 levels appeared to decrease with age. TAFI zymogen levels did not change with hyperglycemia or age. Fragment 1.2 levels, which indicate coagulation activation/thrombin generation increase with age but were not correlated with hyperglycemia. Overall, hyperglycemia does not appear to impact fibrinolysis directly, but rather indirectly through atherosclerosis in ApoE-/-:Ins2+/Akita mice. / Thesis / Master of Science (MSc) / Atherosclerosis is a disease of the large arteries that leads to unwanted blood clot formation that restrict blood flow to the heart and the brain causing heart attack and stroke, respectively. Diabetes mellitus is a metabolic disorder characterized by elevated blood sugar levels (hyperglycemia). Hyperglycemia directly alters protein and cellular structure, and indirectly influences cardiovascular complications (e.g. exacerbation of atherosclerosis progression). Although fibrinolysis, the process of removing clots, has also been linked with hyperglycemia, the mechanism remains unclear. To study the effect of hyperglycemia- mediated atherosclerosis exacerbation on fibrinolysis, we used mice that are prone to atherosclerosis with and without hyperglycemia and quantified: (a) atherosclerosis (lesion size, lipids), (b) blood sugar, and (c) fibrinolysis (lysis times, coagulation/fibrinolytic factors). We found strong correlation between lesion size and lysis time but not with other fibrinolytic markers. Hyperglycemia also did not affect fibrinolysis directly. Therefore, hyperglycemia may indirectly influence fibrinolysis through atherosclerosis.

The incidence of obesity, type 2 diabetes and cardiovascular disease (CVD) is increasing at alarming rates worldwide. Obesity is associated with a chronic nutrient surplus that contributes to chronic low-grade inflammation, ectopic lipid deposition and insulin resistance. Insulin resistance is an important factor contributing to the development of both type 2 diabetes and CVD. Therefore, therapies that can address multiple aspects of cardio-metabolic diseases could have significant clinical utility to reduce morbidity and mortality associated with these conditions. Several distinct glucose lowering therapies have been developed, targeting unique molecular targets. Interestingly, three district glucose lowering therapies, metformin, canagliflozin and salsalate have been shown to potently activate the central energy regulating enzyme, AMP activated protein kinase (AMPK). Activation of AMPK has been shown to be important for regulating fatty acid and cholesterol synthesis, fatty acid oxidation, glucose homeostasis, inflammation and whole-body energy expenditure. Therefore, the objective of this thesis was to examine the effects of metformin, canagliflozin and salsalate, on obesity, atherosclerosis, hepatic lipid metabolism, and macrophage inflammatory signalling and to delineate the mechanism(s) by which these changes occur. In this thesis we show that metformin reduces obesity through a circulating hormone GDF15, and that AMPK is not required for metformin induced GDF15 secretion. Additionally, we show that canagliflozin reduces hepatic cholesterol synthesis and macrophage IL1-1β secretion through mechanisms requiring AMPKβ1. Lastly, we show that salsalate reduces atherosclerosis in a manner dependent on macrophage AMPK β1 and this is associated with reduced macrophage proliferation in vitro and in vivo. These insights into the mechanisms by which these glucose lowering therapies elicit beneficial effects on obesity and atherosclerosis further our understanding of the potential use of these agents for treatments beyond improved glycemic control. Furthermore, this evidence can direct future drug development or drug combinations to more effectively treat multiple aspects of these common chronic diseases that affect over a billion people worldwide. / Thesis / Doctor of Philosophy (Medical Science)

No / Endothelln-converting enzyme (ECE-1) is a critical enzyme in the production of the potent vasoconstrictor peptide endothelin (ET-1). It has previously been shown that the levels of both ET-1 and ECE-1 are raised in atherosclerosis, but the possible relevance of the isoforms of ECE-1 in these changes has not yet been investigated. The aim of this study was to examine the expression of the ECE-1a and ECE-1c isoforms in human atherosclerotic pathologies. Immunohistochemical analysis was carried out on sections from atherosclerotic and non-atherosclerotic vascular tissue using a combination of ECE-1 isoform-specific antibodies, anti-¿-actin antibodies to identify smooth muscle cells (SMC) and anti-CD68 antibodies to identify macrophages. ECE-1 isoform expression was also examined in cultured SMC and in macrophages isolated from human blood. Results indicated differences in isoform expression in atherosclerotic lesions, with distinct patterns of staining for ECE-1 a and ECE-1 c. ECE-1 c immunoreactivity was seen in macrophages, and also correlated with actin staining. ECE-1a was also localized to macrophages and SMC. Results of this study suggest that these local changes influence the expression patterns of the ECE-1 isoforms within individual cell types. Correlation of these isoform expression patterns with the stage of atherosclerosis could provide novel indicators of disease progression.

published_or_final_version / abstract / Pharmacology / Doctoral / Doctor of Philosophy

Increased physical activity and exercise have been associated with reduction in risk for cardiovascular disease. It has been postulated that increased physical activity along with diet modification and smoking cessation can lead to atherosclerotic regression. The purpose of this review of research was to examine the effects of lifestyle modifications on atherosclerotic progression/regression. Dietary modifications, increasing physical activity levels, and a combination of the two were examined for their effectiveness in altering the progression of atherosclerosis. Twenty- eight studies were examined to determine what lifestyle modifications have proven effective in reducing factors of cardiac disease and atherosclerosis. The findings were consistent in showing that alterations in diet and increased physical activity levels were successful in altering the progression of atherosclerosis. Altering dietary intake of fat amount and type can provide protective effects against plaque formation while physical activity provides a reduction in pro-atherogenic factors. The combination of diet and exercise was associated with initial reductions in atherosclerosis as well as long term effects including a reduction in the degree of vascular stenosis and a decrease in progression or an increase in regression of atherosclerosis when compared to those who did not participate in the interventions. The interventions reviewed in this study included only non-pharmacological forms of management. Dietary restrictions, such as 10% total fat calorie intake, may be difficult for individuals to maintain and commit to long term. The effects of lipid and cholesterol management through the use of medications with diet and exercise modifications should be reviewed.

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