New Zealand albino rabbits exhibited calcified aortic plaques and maximum average serum cholesterol levels of 1200 mg percent after twenty-three weeks on an atherogenic diet (250 to 500 mg percent cholesterol in ten percent corn oil; 200,00 I.U. vitamin D3 per month). One month following termination of the atherogenic diet, rabbits were treated with disodium edetate (Na2EDTA, 50 mg/kg body weight) via the marginal ear vein, on alternating days for a total of twenty infusions each. Aortae were examined for tissue calcium both quantitatively (direct microcomplexometric analysis) and histologically six weeks after completion of EDTA chelation therapy.

PURPOSE: WIN55,212-2, a potent synthetic agonist of cannabinoid receptor type 1 (CB1) and cannabinoid receptor type 2 (CB2), reduces atherosclerosis in apolipoprotein E (ApoE) null mice. Although pharmacologic evidence suggests the anti-atherosclerotic effects of WIN55,212-2 are mediated CB2, this remains to be confirmed by genetic studies. Therefore, in this study, we investigated the effects of WIN55,212-2 on development of atherosclerosis in low-density lipoprotein receptor (Ldlr) null mice with and without homozygous deletion of the CB2 gene. METHODS: After 6 weeks on an atherogenic diet, groups of CB2 and CB2 Ldlr-null mice received a daily intraperitoneal injection of WIN55,212-2 or vehicle. After two weeks, plasma lipid levels and atherosclerosis in the aortic root were quantified. RESULTS: Plasma cholesterol and triglyceride levels did not differ between CB2 and CB2 mice and WIN55,212-2 had no effect on total cholesterol levels in either genotype. However, triglyceride levels in both CB2 and CB2 mice were significantly lowered by WIN55,212-2. The size of aortic root lesions did not differ significantly between CB2 and CB2 mice with or without WIN55,212-2 treatment. However, WIN55,212-2 treatment significantly lowered lesional macrophage accumulation in CB2 mice, and lesional smooth muscle content in both CB2 and CB2 mice. Lesional apoptosis was also greater in CB2 mice compared to CB2mice, and only reduced by WIN55,212-2 in CB2 mice. Collagen content and elastin fiber fragmentation were unaffected by genotype or WIN55,212-2. CONCLUSIONS: WIN55,212-2 treatment does not alter lesion size in Ldlr null-mice, but does modify lesion cellularity CB2-dependent and CB2-independent mechanisms.

Over the past few years the number of deaths caused due to cardiovascular diseases has been increasing and is of major concern. In the United States, 75% of cardiovascular-related deaths have been attributed to atherosclerosis. Western diets containing large quantities of peroxidized lipids are considered atherogenic. Heated oil in the form of fried food brings high levels of peroxidized fat and its decomposition products in the diet. Peroxidized lipids are known to increase the susceptibility of serum lipoproteins to undergo oxidation, thereby contributing to the progression of atherosclerosis. The intestinal cells are responsible for the absorption of dietary fatty acid peroxides (FAOOH) which has been reported to enhance anti-atherosclerotic effects by inducing apolipoprotein A1 (apoA1) gene and protein levels. Therefore, there is a void in the knowledge of when to expect “harmful” or “beneficial” effects of dietary lipid peroxides. The formation of toxic products like aldehydes from the decomposition of FAOOH is well documented. On the other hand, carboxylic acids particularly azelaic acid, formed as an end product of FAOOH decomposition has been reported to have anti-atherosclerotic effects. Hence, we hypothesize that intestinal cells may decompose FAOOH to aldehydes, which might get converted to carboxylic acids that can be transported across the intestine. Linoleic acid is the most abundant polyunsaturated fatty acid (PUFA) present in the diet. So, we will use peroxidized linoleic acid (13- HPODE) and incubate with intestine derived cells or Caco-2 cells as an in-vitro model for determining its decomposition to aldehydes and carboxylic acids. We propose that the decomposition products of FAOOH in the presence of intestinal cells might be iv responsible for causing an increase in apoA1 levels, which might suggest that lipid peroxidation derived products might actually be beneficial for reducing the progression of atherosclerosis as compared to the absorption of intact FAOOH.

Atherosclerosis causes hundreds of thousands of deaths in the US alone every year. Fibrous cap rupture is one of the leading causes of these fatalities. Thin cap atheromas are commonly regarded as vulnerable plaque, however the effect of calcium upon a thin fibrous cap with lipid pool is poorly understood. Some studies have shown that calcium adds to stability of the lesion, while others have proven otherwise. An article by Li et al. 2007 suggests location is the key factor. By varying the percentage of calcium and lipid within a defined region, the stress on the cap was estimated using an idealized finite element arterial model. Also the thickness of the fibrous cap was varied to determine whether the stress was solely a function of lipid percentage or a combination. Plaque, arterial wall, lipid, and calcium were modeled using linear elastic, isotropic, and incompressible material properties. The first test varied the thin cap thickness from 65 to 500 microns and tested the calcified lipid model at varying lipid/calcium percentages. The lipid/Calcium pool increased/decreased 10% each test. As the cap thickness becomes thinner than 100 microns, the stress level increases rapidly. The second test compared a model with lipid pool and calcium behind the lipid with a thin cap of 65 microns to a model with lipid pool of the same size and thin cap of 65 microns but only fibrous tissue surrounding (no calcium). The lipid pool increased from 10 to 90% lipid. The result of this test found that at higher lipid percentages, the calcium increased the stress on the cap. By understanding the material properties of plaque and the structure of the lesion, future developments may be able to evaluate rupture risk. This idealized study illustrates the ability of computation models to provide insight into clinical situations.

No description available.

<p>Atherosclerosis is a multi-factorial disease and is the major cause of death in the western world. Numerous risk factors, including hyperlipidemia, obesity, diabetes, smoking, hypertension, and family history increase the risk of atherosclerosis and death from cardiovascular disease (CVD). Clinical and epidemiological studies have now shown that hyperhomocysteinemia (HHcy) is an independent risk factor for CVD. Further, we and others have demonstrated that HHcy accelerates atherosclerosis in apolipoprotein Edeficient ( apoff1-) mice. Although several studies have reported that homocysteineinduced endoplasmic reticulum (ER) stress causes growth arrest and programmed cell death (PCD) in cultured vascular endothelial cells, the cellular factors responsible for this effect and their relevance to atherosclerosis have not been completely elucidated.</p><p>Previously, we have demonstrated that homocysteine induces the expression of Tcell death associated gene 51 (TDAG51), a member of the pleckstrin homology-related domain family, in cultured human vascular endothelial cells. Transient overexpression of TDAG51 elicited significant changes in cell morphology, decreased cell adhesion and promoted detachment-mediated PCD. In support of these in vitro findings, TDAG51 expression was increased and correlated with PCD in the atherosclerotic lesions from apoff1-mice fed hyperhomocysteinemic diets, compared to mice fed control diet. To investigate the in vivo significance of TDAG51 on atherosclerotic lesion development and progression, knockout mice deficient in both TDAG51 and apoE genes were generated. Our findings show that TDAG51-1-/apoff1-double knockout (DKO) mice fed control chow diet have significantly reduced atherosclerotic lesion size, compared to ageand sex-matched apoff1-control mice. Atheroprotective function of TDAG51 deficiency may be explained in part by the observation that there is a significant upregulation of peroxisome proliferator-activated receptor y (PPAR-y) in TDAG51-deficient (TDAG51 _1_) cells including mouse embryonic fibroblasts (MEFs), compared to control wildtype MEFs. Given that PPAR-y has both atheroprotective and anti-inflammatory properties, TDAG51 may represent a unique negative regulator of PPAR-y and its downstream gene targets. Taken together, my findings demonstrate that TDAG51 is a novel cellular mediator involved in the development and progression of atherosclerosis.</p><p>In addition to its anti-atherogenic properties, I have demonstrated that TDAG5 l _1_ MEFs have increased migratory properties following monolayer disruption or in response to chemotaxis on fibronectin-coated Boyden chambers, compared to wildtype control MEFs. Although TDAG51-induced cell migration could potentially affect atherosclerotic lesion development, our recent observations suggest that TDAG51 may also have a role in wound healing. Our studies have shown that dorsal skin wounds within TDAG5 l _ 1_ mice healed slowly, compared to those in control mice through a mechanism involving impaired myofibroblast differentiation. Since the underlying mechanisms of wound healing and fibrosis are similar, it is conceivable that TDAG51 may have role in fibrosis.</p><p>In summary, this thesis provides novel evidence that TDAG5 l is involved in the pathogenesis of atherosclerosis and wound healing. Furthermore, TDAG51 may represent a novel therapeutic target for attenuating atherosclerotic lesion development, thereby reducing the risk of cardiovascular disease and its complications.</p> / Thesis / Doctor of Philosophy (PhD)

Atherosclerosis is a major cause of cardiovascular disease, which is among the leading causes of death globally. Elevated plasma concentration of low density lipoprotein (LDL) cholesterol is a risk factor for atherosclerosis, while a high plasma level of high density lipoprotein (HDL) cholesterol is considered protective. Uptake of HDL cholesterol by hepatocytes during reverse cholesterol transport, and athero-protective signaling induced by HDL in other cells are mediated by the scavenger receptor class B, type 1 (SR-B1). SR-B1 deficiency in mice that are susceptible to atherosclerosis results in exacerbation of atherosclerosis, and in mice with mutations in apolipoprotein E (apoE), renders mice uniquely susceptible to occlusive coronary artery (CA) atherosclerosis and myocardial infarction. In this thesis, the impact of a lack of SR-B1 on the development of atherosclerosis is characterized in otherwise wild type mice, and in mice that also lack the LDL receptor (LDLR). We demonstrate that after prolonged feeding of a high fat, high cholesterol cholate-containing diet, SR-B1 knockout (KO) mice develop similar levels of diet-induced atherosclerosis to LDLR KO mice and apoE KO mice in traditionally susceptible arteries, and significantly more atherosclerosis in arteries that are typically resistant to plaque development, such as the CAs. SR-B1/LDLR double KO mice develop extensive occlusive CA atherosclerosis accompanied by myocardial infarction, and exhibit reduced iv survival compared to LDLR KO control mice when fed a variety of atherogenic diets. In both SR-B1 single KO and SR-BI/LDLR dKO mice, CA atherosclerosis is accompanied by splenomegaly, elevated numbers of circulating leukocytes and increased expression of VCAM-1 in CA endothelium. Interestingly, removal of the spleen has no effect on circulating leukocyte numbers or atherosclerosis in SR-B1/LDLR dKO mice, suggesting the enlarged spleens in SR-B1 deficient mice do not influence atherosclerosis in these animals. We conclude that SR-B1 is important for the protection against atherosclerosis in mice, particularly in CAs. This is likely through roles in multiple cell types including hepatocytes, endothelial cells and bone marrow-derived cells. Future studies should focus on evaluating the impact of cell-specific SR-B1 activity in different cell types on murine atherosclerotic CA disease. / Thesis / Doctor of Philosophy (PhD)

The prevalence of diabetes is increasing rapidly around the world. People with diabetes are 2–4 times more likely to die from cerebro and cardio-vascular causes than people with no history of diabetes, even after controlling for other risk factors. Atherosclerosis, the underlying cause of most cardiovascular disease (CVD), is accelerated in people with diabetes, but several clinical trials have questioned the efficacy of glucose lowering therapies. A better understanding of the molecular pathways by which diabetes accelerates atherosclerosis will expand the scope of current targets and strategies for more effective therapies. In this thesis we investigate a novel mechanism and establish and characterize new hyperglycemic mouse models for the study of diabetic atherosclerosis. Firstly, we investigate the effects of hyperglycemia on the vasa vasorum, the microvascular network that surrounds and supplies large vessels, and correlate those effects to the development of atherosclerosis. In normoglycemic ApoE-/- mice, the vasa vasorum expands as atherosclerotic lesions grow. However, in hyperglycemic ApoE-/- mice there is no significant neovascularization of the vasa vasorum despite the enhanced atherosclerotic development. We hypothesize that the ability of hyperglycemia to disrupt vasa vasorum neovascularization may promote the development and progression of atherosclerosis in diabetes. Secondly, we establish, characterize and manipulate a new model of hyperglycemia-induced atherosclerosis: the ApoE-/-:Ins2+/Akita mouse. We describe sex-specific differences of the ApoE-/-:Ins2+/Akita mouse model. Male ApoE-/-:Ins2+/Akita mice develop chronic hyperglycemia and accelerated atherosclerosis. Castration slows atherosclerosis in ApoE-/-:Ins2+/Akita mice but enhances it in normoglycemic controls. Female ApoE-/-:Ins2+/Akita mice are only transiently hyperglycemic but still present with accelerated atherosclerosis. Ovariectomized ApoE-/-:Ins2+/Akita mice are chronically hyperglycemic and show indications of advanced atherosclerosis. Lastly, we investigate the effects of a western-type diet on the hyperglycemic ApoE-/-:Ins2+/Akita mice. We demonstrate the pernicious phenotype of the mice leading to a significantly shortened lifespan correlated with massive atherosclerosis that extends to the aortic sinus, ascending and descending aorta, brachiocephalic artery and coronary arteries. In conclusion we provide insights for a new mechanism by which hyperglycemia may accelerate atherosclerosis and possible role of the vasa vasorum in the progression of atherosclerosis in hyperglycemic mice. We also establish new mouse models that illuminate the action of sex hormones on pancreatic beta-cell function and the vasculature. These models will provide a test bed to further study sex hormone effects, as well as the diabetic pathways that promote atherosclerosis. / Thesis / Doctor of Philosophy (PhD)

Damage to the endothelium is an important contributor to the initiation and progression of atherosclerosis. GRP78 is an endoplasmic reticulum (ER)-resident molecular chaperone in normal healthy endothelium that functions to assist in the correct folding of newly synthesized proteins and to prevent the aggregation of folding intermediates. In addition, GRP78 is present as a transmembrane protein on the surface of lesion-resident endothelial cells. Surface GRP78 is known to act as a surface signaling receptor in cancer cells and is activated by anti-GRP78 autoantibodies (GRP78a-Abs) isolated from the serum of cancer patients. However, the role of cell surface GRP78 on endothelial cells and the influence of GRP78a-Abs in atherosclerosis is unknown. The objectives of this study were to investigate the effects of GRP78a-Abs on lesion development, examine whether engagement of cell surface GRP78 by GRP78a-Abs modulates endothelial cell function, and determine whether GRP78a-Abs were associated with cardiovascular disease (CVD) in humans. This research showed that ApoE-/- mice with advanced atherosclerotic lesions have elevated serum levels of GRP78a-Abs and ApoE-/- mice immunized against recombinant GRP78 demonstrated a significant increase in GRP78a-Abs titers as well as accelerated lesion growth. Furthermore, this work demonstrated that activation of surface GRP78 on endothelial cells by GRP78a-Abs significantly increases gene expression of adhesion molecules ICAM-1 and VCAM-1 as well as leukocyte adhesion through the NFκB pathway. Additionally, middle-aged to elderly adults at risk for CVD showed a tendency toward elevated circulating GRP78a-Ab levels. Our results suggest that signaling through cell surface GRP78 can activate intracellular pathways that contribute to endothelial cell activation and augment atherosclerotic lesion development. These findings demonstrate a novel role for GRP78a-Abs and surface GRP78 receptor activity in endothelial cell function and the early stages of lesion development, as well as establish an initial framework for future work involving circulating GRP78a-Abs and atherosclerotic disease in humans. Furthermore, this work indicates inhibiting the interaction of GRP78a-Abs with cell surface GRP78 could present a novel therapeutic strategy to modulate lesion growth, thereby reducing the risk for atherosclerosis and cardiovascular disease. / Thesis / Doctor of Philosophy (PhD)

Atherosclerosis is a chronic inflammatory disease affecting large- and medium-sized arteries and is considered the major cause behind cardiovascular diseases (CVD). Elevated low-density lipoprotein (LDL)-cholesterol and low high-density lipoprotein (HDL)-cholesterol are considered major risk factors for the CVD. HDL mediates a variety of atheroprotective actions, many of them involving the interaction with the scavenger receptor class B, type 1 (SR-B1). Despite the efforts placed in raising HDL-cholesterol, no improvement has been achieved in reducing CVD risk, suggesting that other components of the HDL particles may be responsible for HDL-mediated atheroprotection. One of these may be sphingosine-1-phospate (S1P). In this thesis, the role of S1P receptors (S1PRs) in atherosclerosis is explored, with emphasis in macrophage apoptosis. In particular, the importance of the macrophage S1PR1 and its role in apoptosis and atherosclerosis was evaluated. We demonstrated that diabetes exacerbates atherosclerosis development and myocardial infarction in SR-B1 KO/apoE-hypomorphic mice and that treatment with FTY720, a S1PR agonist, protects against diabetes pro-atherogenic effects. We also show that S1PR1 agonists protected macrophages against apoptosis through phosphoinositide 3-kinase (PI3K)/AKT, and that HDL failed to protect S1PR1 deficient macrophages against apoptosis. In vivo, macrophage S1PR1 deficiency translated into increased atherosclerosis, necrotic core formation and numbers of apoptotic cells in the atherosclerotic plaque. BIM deficiency in BM cells was protective against atherosclerosis development and HDL treatment reduced BIM protein levels in cells exposed to ER stressors, suggesting that the pro-apoptotic protein may be an important target for HDL in macrophages. We conclude that signaling through the S1PRs, in particular S1PR1 is important in controlling macrophage apoptosis and atherosclerosis development. Our data suggests that S1PR1 signaling axis and the pro-apoptotic protein BIM play an important role in mediating HDL anti-apoptotic signaling, however further studies are required to clarify the interaction between all of these factors. / Thesis / Doctor of Philosophy (PhD)