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Diabetes mellitus dramatically increases the risk for atherosclerotic cardiovascular disease. It has been established that chronic hyperglycemia promotes an increase in glucose flux through the hexosamine biosynthesis pathway (HBP). Central to this pathway is glutamine:fructose-6-phosphate amidotransferase (GFAT), the rate-limiting enzyme controlling the conversion of glucose to glucosamine. We have shown that glucosamine is a potent inducer of endoplasmic reticulum (ER) stress, which is characterized by the accumulation of misfolded proteins in the ER. Chronic ER stress can initiate a multifaceted response that results in lipid accumulation, inflammation and apoptosis: the hallmark features of atherosclerosis. We hypothesized that conditions of chronic hyperglycemia, associated with diabetes mellitus, can accelerate the development of atherosclerosis by a mechanism that involves increased HBP flux resulting in glucosamine-induced ER stress and the subsequent activation of pro-atherogenic pathways. In support of the hypothesis we found that glucosamine-supplemented apoE-/- mice had elevated levels of ER stress and atherosclerosis. Mechanistically, our data showed that glucosamine induced ER stress by interfering with the lipid-linked oligosaccharide biosynthesis pathway and protein N-glycosylation. These findings support a model by which conditions of hyperglycemia promote vascular complications through a glucosamine-intermediate. / Thesis / Doctor of Philosophy (PhD) / Diabetes mellitus dramatically increases the risk for heart attacks and strokes. High blood glucose is utilized in cells through its conversion into metabolites, such as glucosamine. We hypothesized that conditions of high blood glucose can led to an increase in intracellular glucosamine which can initiate pathways involved in accelerating atherosclerosis. Our results show that this is possible in both human cells and mice.

BACKGROUND: Despite aggressive dietary modification, lipid lowering medications, and other medical therapy, vascular proliferative diseases continue to account for 50% of all mortality in the United States. It is a significant medical and socioeconomic problem contributing to the mortality of multiple diseases including myocardial infarction (MI), stroke, renal failure, and peripheral vascular disease. With a growing number of children becoming obese and an increase in the number of patients with co-morbidities such as metabolic syndrome and Type 2 diabetes mellitus, epidemiological studies project the morbidity and mortality of these diseases to increase. Among these vascular proliferative diseases are primary atherosclerosis, vascular restenosis, and allograft vasculopathy, all of which are the result of chronic inflammation believed to stem from initial endothelial injury. Once activated by any number of potential injurious agents, endothelial cells (ECs) secrete cytokines that act on multiple cell types. Stimulation of resident vascular smooth muscle cells (VSMCs) results in a phenotypic switch from a normally contractile state to a proliferative state. Following this phenotypic shift, VSMCs migrate from the media to the intima of the artery where they begin secretion of both pro- and anti-inflammatory cytokines. Vascular proliferative disease ensues as a result of the autocrine and paracrine signaling of these cytokines between many different cell types including ECs, VSMCs, macrophages, and T-cells. As a result of the integral role pro- and anti-inflammatory cytokines play in the development of vascular proliferative diseases, they have become the subject of intense study in the field of cardiovascular research. Interleukin-19 (IL-19) is a newly described member of the IL-10 sub-family of anti-inflammatory cytokines. Discovered in 2000, it was originally only thought to be basally expressed in monocytes and lymphocytes, however in 2005 our lab discovered that while uninjured arteries have no detectable IL-19, arteries of patients with vascular proliferative diseases have notable IL-19 expression. Since its discovery in multiple cell types of injured arteries, our lab has subsequently shown that IL-19 inhibits proliferation, migration, spreading, production of reactive oxygen species (ROSs), and expression of pro-inflammatory genes in VSMCs, while in ECs IL-19 has been shown to promote angiogenesis, proliferation, migration, and spreading. AIMS and HYPOTHESIS: The first aim of the current study is to show that IL-19 is expressed in atherosclerotic plaque, and to test that IL-19 can reduce experimental atherosclerosis in susceptible mice. The second aim of the study is to show that IL-19 can regulate development of intimal hyperplasia in a murine model of restenosis. For both aims, we sought to identify potential intracellular signaling mechanisms of IL-19 which produce the observed effect. These aims directed our overall hypothesis that the anti-inflammatory properties of IL-19 can attenuate the vascular response to injury in various animal models of vascular proliferative disease. METHODS and RESULTS: The first aim of this dissertation showed that LDLR-/- mice fed an atherogenic diet and injected with either 1.0ng/g/day or 10.0ng/g/day rmIL-19 had significantly less plaque area in the aortic arch compared with controls (p<0.0001). Weight gain and serum lipid levels were not significantly different. IL-19 could halt, but not reverse expansion of existing plaque. Gene expression in splenocytes from IL-19 treated mice demonstrated immune cell Th2 polarization, with decreased expression of T-bet, IFNgamma, IL-1β and IL-12β, and increased expression of GATA3 messenger ribonucleic acid (mRNA). A greater percentage of lymphocytes were Th2 polarized in IL-19 treated mice. Cellular characterization of plaque by immunohistochemistry demonstrated IL-19 treated mice have significantly less macrophage infiltrate compared with controls (p<0.001). Intravital microscopy revealed significantly less leukocyte adhesion in wild-type mice injected with IL-19 and fed an atherogenic diet compared with controls. Treatment of cultured EC, VSMC, and bone marrow-derived macrophages (BMDM) with IL-19 resulted in a significant decrease in chemokine mRNA, and in the mRNA-stability protein HuR. In the second aim of this dissertation we showed that IL-19 attenuates vascular restenosis in response to carotid artery ligation. Carotid artery ligation of hyper-responsive friend leukemia virus B (FVB) wild-type mice injected with 10ng/g/day rIL-19 had significantly lower neointima/media ratio (I/M) compared with phosphate buffered saline (PBS) controls (p=0.006). Conversely, carotid artery of IL-19-/- mice demonstrated significantly higher I/M ratio compared with wild-type mice (p=0.04). Importantly, the increased I/M ratio in the knockout mice could be rescued by injection of 10ng/g/day IL-19 (p=0.04). VSMC explanted from IL-19-/- mice proliferated significantly more rapidly compared with wild-type (p=0.04). Surprisingly, in this model, IL-19 does not modulate adoptive immunity. Rather, addition of IL-19 to cultured wild-type VSMC did not significantly decrease VSMC proliferation, but could rescue proliferation in IL-19-/- VSMC to wild-type levels (p=0.02). IL-19-/- VSMC expressed significantly greater levels of inflammatory mRNA including IL-1β, TNFα, and MCP-1 in response to TNFα stimulation (p<0.01 for all). No polarization of adaptive immunity was noted in these mice. CONCLUSIONS: These data are the first to report that IL-19 is a potent inhibitor of experimental atherosclerosis via diverse mechanisms including immune cell polarization, decrease in macrophage adhesion, and decrease in gene expression. In addition, these data are also the first to show that IL-19 plays a previously unrecognized protective role in vascular restenosis. Together, these data suggest IL-19 is both anti-atherogenic and anti-restenotic and may identify IL-19 as a novel therapeutic to limit vascular inflammation. / Physiology

Atherosclerosis, considered a chronic inflammatory disease, is the underlying mechanism for several cardiovascular diseases. Hyperlipidemia is the number one risk factor for atherogenesis. Caspase-1 is an inflammatory caspase, which can be activated by the metabolic stresses through pathogen associated molecular patterns (PAMPs)-recognition receptors, (PRR) recognition and inflammasome assembly. Activated caspase-1 can initiate inflammation in multiple ways. Thus, regulating inflammasome components expression is essential to control caspase-1 activation and its subsequent inflammatory processes. I hypothesized that the readiness of inflammasome component expression for caspase-1 activation in tissues is an index for inflammation privilege. Endothelial cells (EC) which are the innermost layer of the vessel and are the critical gatekeeper for monocyte migration. The first step of atherogenesis is activation of ECs, which allows monocyte adhesion and migration into the sub-endothelial layer. I also hypothesized that caspase-1 can sense hyperlipidemia and regulate EC activation and inflammation during early atherogenesis. I first determined the expression profiles of inflammasome components, pro-inflammatory caspases and PRRs is different among tissues, and cardiovascular tissues express relative less PRRs via a database-mining method. According to the readiness of inflammasome components, tissues could be classified into three tiers. The first tier consists of tissues with constitutively expressed inflammasomes. The second tier of tissues includes potentially inducible expression of one inflammasome component. The third tier of tissues has inducible expression of at least two inflammasome components. This three-tier model can be applied to determine the inflammation privilege of tissues in response to pro-inflammatory stimuli. I also demonstrated that hyperlipidemia induced caspase-1 expression and activation in aorta along with the atherogenesis in apolipoprotein E (ApoE)-/- mice with high fat (HF) diet, experimentally. We then generated the ApoE-/-/Casp-1-/- double knockout mice, and found that the ApoE-/-/Casp-1-/- mice contained significantly less atherosclerotic lesion in aortic sinus and less cytokine and chemokine expression in aortic tissues compared with ApoE-/- mice. ApoE-/-/Casp-1-/- mice also had less CD11b+/F4/80- neutrophil and CD11b+/F4/80+ monocyte recruitments into aorta compared with ApoE-/- mice. However, the percentage of monocyte subsets in peryphery blood remained at the same level in between ApoE-/- mice and ApoE-/-/Casp-1-/- mice. I then proposed that perhaps the caspase-1 activation in vascular cells, in ECs played the essential role of controling monocyte migraion. My in vitro data demonstrated that oxidized low density lipoprotein (ox-LDL) and its componnents could induced caspase-1 activation in human aortic ECs (HAECs) through ROS pathway which then led to EC activation and pyroptotic cell death. Deficiency of caspase-1 in aortic EC attenuated hyperlipidemia induced EC activation and inflammtion. Mechanically, I found that caspase-1 deficiency accumulated an anti-atherogenic protein, Sirt-1 in the aorta. Collectively, our data suggested that caspase-1/inflammasome in ECs can sense hyperlipidemia, become activated, drive EC activation, and promote monocyte recruitment and early atherosclerosis. / Pharmacology