During atherosclerosis and restenosis, there is an extensive amount of collagen synthesis and degradation. Changes in the types of collagen present can have profound effects on vascular smooth muscle cell (SMC) proliferation and migration. Type VIII collagen, which is normally present at low levels within the mature vascular system, is greatly increased during atherogenesis. The central theme of this thesis is to determine the role of type VIII collagen in the pathogenesis of atherosclerosis and restenosis. In the first study, we demonstrated the importance of type VIII collagen in SMC migration and proliferation. SMCs from type VIII collagen-deficient mice display increased adhesion and decreased spreading, migration, and proliferation compared to SMCs from wild-type mice. Treatment of SMCs from type VIII collagen-deficient mice with exogenous type VIII collagen can rescue the defects. In the second study, we determined that type VIII collagen exerts its effects through regulation of MMP-2 expression. Type VIII collagen-deficient SMCs have decreased levels of MMP-2 and are impaired in chemotaxis toward PDGF-BB and in their ability to contract thick collagen gels. We found that decreasing endogenous MMP-2 levels in normal SMCs or adding exogenous collagen to type VIII collagen-deficient SMCs is sufficient to recapitulate the type VIII collagen-deficient or wild-type SMC phenotype, respectively. In the third study, we investigated the contribution of type VIII collagen to intimal hyperplasia after mechanical injury in the mouse. We found that type VIII collagen-deficient mice display a 35% reduction in intimal hyperplasia and attenuated vessel remodeling after femoral artery wire injury, establishing a role for type VIII collagen in restenosis. The results of the work presented in this thesis demonstrate that production of type VIII collagen confers an SMC phenotype with a greater propencity for proliferation and migration. These effects are in part mediated through regulation of MMP-2 expression and activation. We conclude that the increases in type VIII collagen production that occur during atherosclerosis and restenosis contribute to the capacity of SMCs to alter the existing extracellular matrix in a manner which permits enhanced migration.

Objectives: 1) Evaluate the risk of atherosclerosis in rheumatic disease compared to healthy controls; 2) Assess the lipid profile of children with systemic lupus erythematosus (SLE) at presentation before treatment with corticosteroids; 3) Compare the lipid profiles of children with juvenile dermatomyositis (JDM), systemic juvenile idiopathic arthritis (SJIA), and SLE; 4) Evaluate the extent of early atherosclerosis in children with JDM, SJIA, and SLE; 5) Investigate the progression of early markers of atherosclerosis in children with SLE. Methods. The methods include a systematic review, a cross sectional study of serum lipid levels of a cohort of children with SLE, an analysis of the first time point of a prospective study of cardiovascular disease risk factors and vascular function measures of a cohort of children with JDM, and SJIA, and SLE and a longitudinal study of vascular function measures of a prospective study of a cohort of children with SLE. Results. Our systematic review demonstrated that carotid intima media thickness (CIMT), a surrogate marker of early atherosclerosis, was significantly increased in rheumatic disease populations. We found that newly diagnosed children with SLE before corticosteroid treatment exhibited a pattern of dyslipoproteinemia of increased triglycerides and depressed HDL-cholesterol. When we measured the lipid profiles in children with the rheumatic diseases of JDM, SJIA, and SLE, one third of children had at least one abnormal lipid value. The most common abnormalities were found for total cholesterol and triglyceride levels and most often in children with JDM. One quarter of all patients were found to have insulin resistance. Lastly, when we considered the effects of treatment in children with SLE, we found that improvement in CIMT was possible and it correlated with a higher cumulative dose of prednisone over the study period. Conclusions. Early markers of atherosclerosis in pediatric rheumatic disease are important for determining the risk of these children in developing heart disease as young adults. Chronic inflammation plays a significant role and should be considered an important predictor of premature atherosclerosis.

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Objectives: 1) Evaluate the risk of atherosclerosis in rheumatic disease compared to healthy controls; 2) Assess the lipid profile of children with systemic lupus erythematosus (SLE) at presentation before treatment with corticosteroids; 3) Compare the lipid profiles of children with juvenile dermatomyositis (JDM), systemic juvenile idiopathic arthritis (SJIA), and SLE; 4) Evaluate the extent of early atherosclerosis in children with JDM, SJIA, and SLE; 5) Investigate the progression of early markers of atherosclerosis in children with SLE. Methods. The methods include a systematic review, a cross sectional study of serum lipid levels of a cohort of children with SLE, an analysis of the first time point of a prospective study of cardiovascular disease risk factors and vascular function measures of a cohort of children with JDM, and SJIA, and SLE and a longitudinal study of vascular function measures of a prospective study of a cohort of children with SLE. Results. Our systematic review demonstrated that carotid intima media thickness (CIMT), a surrogate marker of early atherosclerosis, was significantly increased in rheumatic disease populations. We found that newly diagnosed children with SLE before corticosteroid treatment exhibited a pattern of dyslipoproteinemia of increased triglycerides and depressed HDL-cholesterol. When we measured the lipid profiles in children with the rheumatic diseases of JDM, SJIA, and SLE, one third of children had at least one abnormal lipid value. The most common abnormalities were found for total cholesterol and triglyceride levels and most often in children with JDM. One quarter of all patients were found to have insulin resistance. Lastly, when we considered the effects of treatment in children with SLE, we found that improvement in CIMT was possible and it correlated with a higher cumulative dose of prednisone over the study period. Conclusions. Early markers of atherosclerosis in pediatric rheumatic disease are important for determining the risk of these children in developing heart disease as young adults. Chronic inflammation plays a significant role and should be considered an important predictor of premature atherosclerosis.

The effect of Ddr1 deletion on the expression of genes involved in atherosclerotic vascular remodeling and on the development of atherosclerotic calcification Pamela J. Ahmad, PhD Institute of Medical Science, 2008 During atherosclerosis, collagen molecules, which are abundant in the healthy vessel, are extensively degraded, re-synthesized or newly synthesized, and remodeled to induce profound changes in VSMCs as they colonize and expand atherosclerotic lesions. The central theme of this thesis was to investigate the effect of genetic deletion of a collagen receptor, DDR1, on VSMC processes during atherosclerosis. In the first study, we demonstrated a role for DDR1 as an important regulator of gene expression in synthetic VSMCs. We have profiled the expression of vascular collagen matrix molecules, MMPs and TIMPs in synthetic VSMCs and we have demonstrated that deletion of Ddr1 is sufficient to accelerate ECM remodeling in synthetic VSMCs, which may influence cell migration during atherosclerosis. Moreover, we have extended our knowledge of DDR1 function in synthetic VSMCs, by demonstrating that DDR1 limits VSMC proliferation in a complex matrix microenvironment representative of the ECM produced in the vessel wall during vascular disease. In the second study, we investigated the role of DDR1 in atherosclerotic calcification, a feature of advanced atherosclerotic disease. Here, we demonstrated that intimal calcification in Ldlr-/- mice fed a high-fat/ high-cholesterol diet may be mediated through the initiation of a chondrogenic transcriptional regulatory program and that deletion of Ddr1 significantly attenuated the frequency and extent of atherosclerotic mineralization in vivo, as well as the ability of vascular smooth muscle cells to calcify in vitro, suggesting an important role for DDR1 in VSMCs as a positive regulator of this pathological process. In our third study, we provided evidence of a biochemical association between MMP-2 and DDR1b in VSMCs, which involves a direct interaction between MMP-2 and the extracellular region of the DDR1 receptor. In addition, we reported an association between endogenous MMP-2 and Stat1 in VSMCs, providing a platform for future research to investigate the functional consequences of these novel interactions.

During atherosclerosis and restenosis, there is an extensive amount of collagen synthesis and degradation. Changes in the types of collagen present can have profound effects on vascular smooth muscle cell (SMC) proliferation and migration. Type VIII collagen, which is normally present at low levels within the mature vascular system, is greatly increased during atherogenesis. The central theme of this thesis is to determine the role of type VIII collagen in the pathogenesis of atherosclerosis and restenosis. In the first study, we demonstrated the importance of type VIII collagen in SMC migration and proliferation. SMCs from type VIII collagen-deficient mice display increased adhesion and decreased spreading, migration, and proliferation compared to SMCs from wild-type mice. Treatment of SMCs from type VIII collagen-deficient mice with exogenous type VIII collagen can rescue the defects. In the second study, we determined that type VIII collagen exerts its effects through regulation of MMP-2 expression. Type VIII collagen-deficient SMCs have decreased levels of MMP-2 and are impaired in chemotaxis toward PDGF-BB and in their ability to contract thick collagen gels. We found that decreasing endogenous MMP-2 levels in normal SMCs or adding exogenous collagen to type VIII collagen-deficient SMCs is sufficient to recapitulate the type VIII collagen-deficient or wild-type SMC phenotype, respectively. In the third study, we investigated the contribution of type VIII collagen to intimal hyperplasia after mechanical injury in the mouse. We found that type VIII collagen-deficient mice display a 35% reduction in intimal hyperplasia and attenuated vessel remodeling after femoral artery wire injury, establishing a role for type VIII collagen in restenosis. The results of the work presented in this thesis demonstrate that production of type VIII collagen confers an SMC phenotype with a greater propencity for proliferation and migration. These effects are in part mediated through regulation of MMP-2 expression and activation. We conclude that the increases in type VIII collagen production that occur during atherosclerosis and restenosis contribute to the capacity of SMCs to alter the existing extracellular matrix in a manner which permits enhanced migration.

ABCA1 is believed to affect macrophage inflammatory responses, but the mechanism by which ABCA1 may impact cytokine secretion in macrophages has yet to be fully defined. We observed that the induction of ABCA1 expression in three different cell lines, namely BHK, RAW 264.7 macrophages, and primary bone marrow derived macrophages (BMDMs), results in a significant increase in phosphorylated CREB, a known protein kinase A (PKA) substrate. In RAW macrophages, induction of ABCA1 expression by the LXR-agonist T0901317 is correlated with a decrease in LPS-stimulated secretion of proinflammatory cytokines IL-6 and TNF-α. Additionally, the secretion of anti-inflammatory cytokine IL-10 was increased upon ABCA1 induction. A similar trend was observed in BMDMS: ABCA1-expressing BMDMs released less TNF-α and more IL-10 compared to ABCA1-knockout BMDMs. We speculated that the inflammation modulating effects of ABCA1 in macrophages could be a result of PKA activation. Indeed, we found that the LXR-induced ABCA1 phenotype can be mimicked by cAMP in macrophages. 8-bromo-cAMP, a PKA activator, dose-dependently suppressed inflammatory cytokine secretion while promoting IL-10 release in the absence of ABCA1 expression. Finally, we found that the T0901317-induced ABCA1 expression is correlated with higher expression levels of MKP-1, a downstream target of PKA known to suppress inflammatory responses. Together, our results suggest that ABCA1 expression may activate PKA and CREB and that such activation may contribute to the inflammatory modulating effects of ABCA1.

Mechanical forces are important regulators of cell function in many tissues including, for example, bone and components of the cardiovascular system. The endothelial lining of blood vessels has been shown to respond in an atheroprotective manner to unidirectional, laminar flow-induced shear stress and in an atherogenic manner to oscillating and low levels of shear. We have developed a cone and plate shear apparatus to simulate fluid shear stress on endothelial cells in vitro. The significant feature of this apparatus is that, unlike other in vitro flow systems, it accurately produces varying levels of shear stress, consistent with those created in vivo during the cardiac cycle. Flow characteristics of this system were verified by computational fluid dynamics (CFD) and laser Doppler velocimetry (LDV). Cellular responses were monitored by cell morphology and protein expression. These responses are consistent with in vivo responses as well as previous work using other in vitro flow systems.

Each year in the United States over a million people experience a myocardial infarction. The majority of these attacks are caused by coronary artery plaque cap rupture with subsequent thrombus formation. Because rupture is a mechanical event and the tendency of a plaque to rupture is due in part to increases in the mechanical stresses in the fibrous cap, mechanical analyses are important to understanding plaque stability. Histology is the only method capable of identifying plaque features that are associated with vulnerability. Therefore, minimally distorted histologic sections should serve as a basis for constructing the models used in mechanical analyses. Further, because substantial longitudinal variations in geometry and mechanical properties often exist, models should be three-dimensional (3-D). Finally, given the complex geometries of atherosclerotic plaques and the fact that they are composed of different materials, the finite element (FE) method should be used to determine the distribution of stress under physiological loading. Until now, a critical need has existed to determine the distribution of stress in 3-D FE models of human coronary atherosclerotic plaques based on minimally distorted histologic sections. In this research study, a method to measure and correct for distortions caused by acrylic histologic processing was first created. The devised strain-based method yields a limited set of parameters needed for a first order correction. Thus, corrections can be easily implemented using FE methods. Next, a methodology to create 3-D finite FE models of human coronary atherosclerotic plaques based on stable acrylic histologic sections was developed. Models of plaques, ranging in disease severity, were generated using the developed methodology. Lastly, the distributions of stress in these models were obtained and the effects of some plaque features on stresses were determined. Results from this study confirm that morphological description of a plaque is not sufficient to predict plaque rupture. The findings suggest that in many cases the 3-D stress field within a plaque must be known in order to assess plaque stability. Finally, the results show that patient specific models must be developed if the 3-D stress field within a plaque is to be determined.

Cardiovascular Disease (CVD) continues to be the leading cause of death in western countries according to the statistics update by the American Heart Association. Atherosclerosis is estimated to be responsible for a large portion of CVD and affects 60 million people in the United States. Accurate diagnosis is crucial for proper treatment planning. Currently, the clinical standard screening technique for diagnosing atherosclerosis is x-ray angiography, which reveals the residual lumen size. X-ray angiographic images possess good resolution and contrast, however, lumen size is not always a proper criterion given the positive remodeling nature of atherosclerotic plaques. In the past decade, it has been shown that most plaques responsible for a fatal or nonfatal myocardial infarction are less than 70% stenosed. Clinical data support the idea that plaques producing non-flow-limiting stenoses account for more cases of plaque rupture and thrombosis than plaques producing a more severe stenosis. Due to this fact, plaque itself must be imaged in order to assess its vulnerability. A wealth of literature suggests that multicontrast MRI has the potential of characterizing plaque constituents, and thus is a promising technique for plaque imaging. Because of the technical difficulties associated with in-vivo plaque imaging and the fact that our research was aimed at developing new methodologies, our approaches was to image excised coronary arteries under simulated in-vivo conditions in a tissue culture chamber. It is shown by this research that automatic plaque characterization techniques developed under ex-vivo conditions still apply for in-vivo studies. Based on this finding, an automatic plaque characterization technique using multicontrast MRI was developed. Furthermore, "shared k-space" reconstruction techniques were interrogated to assess their feasibility in accelerating multicontrast MRI acquisition. Results show that these techniques are promising in accelerating multicontrast MRI acquisitions.