Carotid atherosclerosis, one of the leading causes of ischemic stroke worldwide, can induce severe narrowing or even occlusion of the vessel, restricting blood flow to the brain and resulting in perfusion deficits. The plaque that has a high probability of undergoing rapid progression or future ruptures is defined as “vulnerable plaque”. Identifying vulnerable plaque is of great importance in clinical carotid atherosclerosis imaging. To date, a multi-contrast magnitude-based MR approach with blood suppression technique has been widely used to detect vulnerable plaque features. However, due to the limitations of magnitude-based methods, developing new MR techniques that have better sensitivity to hemorrhage and calcification is of great interest. Quantitative Susceptibility Mapping (QSM) is a technique that utilizes the MR phase information and has been widely used for quantifying the tissue susceptibility in the brain. The susceptibility contrast is extremely sensitive to hemorrhage and calcium which makes QSM a potential tool for carotid plaque imaging to identify intraplaque hemorrhage (IPH) and calcification. However, existing QSM methods have not been successfully implemented in the neck due to several challenges. The presence of air/tissue interface, plaque that has high susceptibility, and fat surrounding the carotid arteries can cause severe phase aliasing and other problems that will induce errors in the resultant susceptibility maps. To overcome these challenges and thus, develop a robust method for carotid QSM, a protocol that includes both data acquisition strategy and post-processing methods is proposed. For data acquisition, four echoes including two water/fat in-phase echoes and two water/fat out-of-phase echoes were collected. For data post-processing, temporal domain algorithm Catalytic Multiecho Phase Unwrapping Scheme (CAMPUS) was used to unwrap the phase images and local QSM was proposed. This protocol is able to properly unwrap the phase images even with the presence of high susceptibility plaque and eliminate the water/fat chemical shift effect in QSM reconstructions which will generate reliable susceptibility maps. From our results, the proposed QSM protocol has demonstrated the ability to generate reliable susceptibility maps and excellent sensitivity to IPH and calcification. Combining QSM with existing magnitude-based methods will lead to a major improvement in the diagnosis of carotid atherosclerosis. / Thesis / Master of Applied Science (MASc)

The underlying pathological process of coronary artery disease (CAD) is the development of coronary artery atherosclerotic occlusions and associated myocardial infarction. Both increased chronic inflammation and plasma low-density lipoprotein (LDL) cholesterol levels promote atherosclerosis. Inhibiting proprotein convertase subtilisin/kexin type 9 (PCSK9) is widely known for its role in enhancing LDL receptor (LDLR)-mediated cholesterol lowering when the LDLR-apolipoprotein E (APOE) axis is intact and protecting against atherosclerosis progression by reducing plasma cholesterol levels. In this thesis, we sought to test the effects of PCSK9 inhibition mediated cholesterol lowering on pre-existing CAD as well as the plasma cholesterol independent effects of PCSK9 inhibition on CAD by utilizing different mouse models. One year old scavenger receptor class B type I (Sr-b1) knockout (KO) mice which have an intact LDLR-APOE axis, develop coronary artery atherosclerosis and myocardial fibrosis induced by a high fat, high cholesterol and cholate containing (HFCC) diet. Weekly anti-PCSK9 antibody treatment initiated one week before switching to an HFCC diet increased hepatic LDLR protein levels, and reduced plasma cholesterol levels and the progression of atherosclerosis in both the aortic sinus and coronary arteries in one year old Sr-b1 KO mice (maintained on an HFCC diet for 7 weeks). Weekly anti-PCSK9 antibody treatment initiated 7 weeks after switching to an HFCC diet also increased hepatic LDLR protein levels and reduced plasma cholesterol levels in one year old Sr-b1 KO mice (maintained on an HFCC diet for 12 weeks). More importantly, anti-PCSK9 antibody treatment during the last 5 weeks of the 12-week HFCC diet feeding period also slowed down the growth of pre-existing atherosclerosis in both the aortic sinus and coronary arteries and reduced myocardial fibrosis and damage. Mice deficient in both Sr-b1 and ApoE (Sr-b1/ApoE double KO (dKO) mice) spontaneously and rapidly develop features reminiscent of human CAD. Whole body Pcsk9 genetic KO in both female and male Sr-b1/ApoE dKO mice did not affect plasma cholesterol levels despite increased hepatic LDLR protein levels, presumably due to the lack of APOE. However, genetic Pcsk9 inactivation significantly attenuated atherosclerosis in both the aortic sinus and coronary arteries, myocardial fibrosis and damage, left ventricle (LV) dysfunction and cardiac enlargement in both female and male Sr-b1/ApoE dKO mice. Restoring circulating PCSK9 by a recombinant adeno associated virus 8 (AAV8)-mediated hepatic expression of a Pcsk9 cDNA in Pcsk9/Sr-b1/ApoE triple KO mice reversed the plasma cholesterol independent protective effects of genetic PCSK9 KO on aortic sinus and coronary artery atherosclerosis and myocardial fibrosis and damage in both females and males. Treatment of Sr-b1/ApoE dKO mice with an anti-PCSK9 antibody which disrupts the interaction between the LDLR and PCSK9 protected against aortic sinus and coronary artery atherosclerosis in males but not in females and did not protect either males or females against myocardial fibrosis and damage, LV dysfunction or cardiac enlargement. My thesis demonstrates that anti-PCSK9 antibody mediated plasma cholesterol lowering delays the continued development of pre-existing CAD. My thesis also demonstrates that liver-derived, circulating PCSK9 promotes CAD in a plasma cholesterol independent manner in Sr-b1/ApoE dKO mice and these effects appear to be largely independent of the PCSK9-LDLR interaction, particularly in females. / Thesis / Doctor of Philosophy (PhD)

Excess production and secretion of very low-density lipoprotein (VLDL) by the liver into the circulatory system is directly related to atherosclerosis, a chronic cardiovascular disease that threatens the lives of many worldwide and continues to be a leading cause of death in the United States. The rate-limiting step in VLDL secretion is its transport from the site of biogenesis, the hepatic endoplasmic reticulum to the cis-Golgi. This step is mediated by a specialized ER- derived vesicle, the VLDL transport vesicle (VTV). Upon exit of the ER the VTV targets, fuses and delivers VLDL into the lumen of the Golgi. The targeting and fusion of the VTV with the Golgi is facilitated by specific set of soluable N-ethylmaleimide sensitive factor attachment protein receptor (SNARE) proteins that form a SNARE complex, which is required for the VTV-Golgi fusion and thus delivery to the Golgi. Data from our laboratory indicates that the formation of the SNARE complex requires cytosolic factors. Through the purification of liver cytosol, chromatographic steps, detailed mass spectrometry, immunodepletion and western blotting data it was identified that the protein necessary for SNARE complex formation is Hsc-70. Although Hsc-70's identification is significant, the role it plays in SNARE complex formation for VTV -Golgi fusion is a predicament and yet to be unraveled. In this study we performed a series of co-immunoprecipitation reactions to identify its role in SNARE-complex assembly. Using western blot data we confirmed binding of Hsc-70 with Sec22b, the v-SNARE on the VTV. Moreover, we confirmed the interaction of Hsc-70 with t-SNAREs, (syn5, rBet1 and GOS28) on the Golgi membrane. Removal of Hsc-70 from the liver cytosol resulted in significant reduction of SNARE-complex formation. Ultimately, the identification proteins involved in the process of VLDL delivery to the Golgi would offer therapeutic targets to control VLDL secretion into the blood by the liver.

Sex-dependent differences in the prevalence of diabetes and cardiovascular diseases are well established. The objective of this project is to investigate the molecular mechanisms by which estrogen modulates chronic disease progression. Our lab, and others, have previously implicated endoplasmic reticulum (ER) stress in the development and progression of diabetes and cardiometabolic disease. We hypothesize that estrogens protect pancreatic beta cell health, and slow the progression of cardiometabolic disease, by modulating the unfolded protein response (UPR) in response to ER stress. Two distinct mouse models were used in these studies. The ApoE-/-Ins2+/Akita mouse model of hyperglycemia-induced atherosclerosis, in which females are significantly protected from hyperglycemia and atherosclerosis relative to males, and the TALLYHO/JngJ mouse model, in which females are protected from chronic hyperglycemia relative to males. We found that ovariectomy of female ApoE-/-Ins2+/Akita or TALLYHO/JngJ mice promoted chronic hyperglycemia. Supplementation with exogenous 17-beta estradiol significantly lowered blood glucose levels in ovariectomized ApoE-/-Ins2+/Akita mice and reduced atherosclerotic lesion development in both male and ovariectomized female mice. Pancreatic islets from sham operated ApoE-/-Ins2+/Akita female mice showed a significant increase in the expression of protective UPR factors and a decrease in pro-apoptotic factors, compared to males or ovariectomized females. To determine if alleviating ER stress could moderate hyperglycemia, male and ovariectomized female TALLYHO/JngJ mice were treated with the chemical chaperone 4-phenylbutryic acid (4-PBA). We showed that 4-PBA treatment significantly lowered fasting blood glucose levels and improved glucose tolerance. The results of this thesis suggest that estrogens play a protective role in the maintenance of beta cell health and blood glucose regulation by activating the adaptive UPR. This mechanism may explain the protection observed in premenopausal women and may lead to the development of targeted therapies to treat diabetes and cardiometabolic diseases. / Thesis / Doctor of Philosophy (PhD) / People who suffer from diabetes mellitus have a higher risk of developing heart attack and stroke compared to those who do not have diabetes. Moreover, the risk of heart attack and stroke is higher in men than in women. We still do not understand the underlying reasons for these differences. This thesis project has used unique mouse models that display many of the same sex differences in disease progression that we see in humans to study the pathways and mechanisms that promote diabetes development. Specifically, we examined the protective effects of estrogen towards the development of diabetes and cardiovascular disease and how this hormone affected specific cells and tissues. The results of these studies are important because they will provide more information regarding the effects of menopause and aging on chronic disease progression in women.

Psoriasis is an immune mediated inflammatory disease which affects 2-3% of the world’s population. Over the last decade, psoriasis has been acknowledged as an independent risk factor for atherosclerosis. The precise mechanism or mechanisms of the heightened risk is widely speculated. Endothelial cells and macrophages are central players in the immunopathological development of both diseases. Interleukin-36 cytokines (IL-36) have been heavily implicated in psoriasis immunopathology. Significant upregulation of epidermal IL-36 is a recognised characteristic of psoriatic skin inflammation. IL-36 induces inflammatory responses in dendritic cells, fibroblasts and epithelial cells. While vascular alterations are a hallmark of psoriatic lesions and dermal endothelial cells are well known to play a critical role in dermal inflammation, the effects of IL-36 on endothelial cells have not been defined. We report that endothelial cells including dermal microvascular cells express a functionally active IL-36 receptor. Adhesion molecules VCAM-1 and ICAM-1 are upregulated following IL-36γ stimulation, and this is reversed in the presence of the endogenous IL-36 receptor antagonist. IL-36γ-stimulated endothelial cells secrete the proinflammatory chemokines IL-8, CCL2 and CCL20. Chemotaxis assays showed increased migration of T-cells following IL-36γ stimulation of endothelial cells. Both resident and infiltrating inflammatory myeloid cells contribute to the immunopathology of psoriasis by promoting the IL-23/IL-17 axis. We show that IL-36γ induces the production of psoriasis-associated cytokines from macrophages (IL-23, TNFα) and that this response is enhanced in macrophages from psoriasis patients. This effect is specific for IL-36γ and could not be mimicked by other IL-1 family cytokines such as IL-1α. Furthermore, IL-36γ stimulated macrophages potently activated endothelial cells as illustrated by ICAM-1(CD54) upregulation, and led to increased adherence of monocytes, effects that were markedly more pronounced for psoriatic macrophages. Interestingly, regardless of stimulus, monocytes isolated from psoriasis patients showed increased adherence to both the stimulated and unstimulated endothelium when compared to monocytes from healthy individuals. Collectively, these findings add to the growing evidence for IL-36γ having roles in psoriatic responses, by enhancing endothelium directed leukocyte infiltration into the skin and strengthening the IL-23/IL-17 pathway. Our findings also point to a cellular response which could potentially support cardiovascular comorbidities in psoriasis.

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