Cardiovascular disease is now the leading cause of death worldwide. Serum amyloid A (SAA), a positive acute phase reactant, along with C-reactive protein is used clinically as a marker of cardiovascular disease risk. However, recent data has shed light on a possible causal role of SAA in the development of atherosclerosis, the most pervasive form of cardiovascular disease. Several inflammatory diseases such as diabetes and obesity are known to confer increased risk of developing cardiovascular disease. Individuals with these diseases all have modest but persistent elevation of SAA. To determine if SAA caused the development of atherosclerosis, apoe-/-chow fed mice were injected with either an adenoviral vector expressing human SAA1 (ad-hSAA1), a null adenoviral vector (ad-Null) or saline. Human SAA levels rapidly increased, albeit briefly then returned to baseline within 14 days in mice that received ad-hSAA1. After 16 weeks, mice that received ad-hSAA1 had significantly increased atherosclerosis compared to controls on the aortic intimal surface (p<0.0001), aortic sinus (p<0.05) and the brachiocephalic artery (p<0.05). According to the “response to retention” hypothesis; lipoprotein retention by vascular wall proteoglycans is a key initiating event in the development of atherosclerosis. We previously reported that SAA-stimulated vascular smooth muscle cells expressed biglycan with increased glycosaminoglycan chain length and increased binding affinity for low density lipoprotein. To further test the role of biglycan on the development of atherosclerosis we generated biglycan transgenic mice. These mice were crossed to the ldlr-/- mouse on a C57BL/6 background and fed a pro-atherogenic western diet for 12 weeks. There was a significant increase in atherosclerotic lesion area on the aortic intimal surface (p<0.05) and the aortic sinus (p<0.006), as well as a significant correlation between vascular biglycan content and aortic sinus atherosclerotic lesion area (p<0.0001). These data demonstrate that transiently increased SAA resulted in increased atherosclerosis compared to control mice, possibly via increased vascular biglycan content. In support of this we found that biglycan transgenic mice had significantly increased atherosclerosis compared to wildtype controls, likely through increased lipid retention in the vascular wall.

Cardiovascular disease (CVD) is a progressive disease that presents signs, such as abnormal thickening or stiffening of arteries, early in its preclinical stage, and screening tools such as carotid intima media thickness (CIMT) measurement and pulse wave velocity (PWV) assessment have the potential to identify individuals prior to the clinical manifestation of CVD. The purpose of this study was to determine the prevalence of preclinical atherosclerosis, as indicated by high CIMT and PWV values, in an adult population aged 40-70 years and free of diagnosed CVD using these screening tools. Secondarily, this study aimed to compare established CVD risk factors and other health parameters between those with elevated or normal arterial health values. Sixty subjects made 2 visits to the Ball State University Human Performance Laboratory. The first visit included basic anthropometric measurements as well as assessment of CIMT and PWV. After a one week objective physical activity assessment, subjects returned to the HPL for assessment of blood lipids and body composition via dual energy x-ray absorptiometry scan. Prevalence of preclinical atherosclerosis was calculated from the total sample as well as within both genders, and an independent samples t-test was conducted in order to identify significant differences in health characteristics between those in the normal and high groups. Abnormal CIMT or PWV values were present in 43% of study subjects; 30% and 18% of the test sample met the criteria for elevated CIMT and PWV, respectively. Significant differences existed between normal and high CIMT and PWV study groups for physical activity, body composition, and blood lipid profile variables. Comparisons within each gender revealed differences in health profile elements. Both the CIMT and PWV measurement techniques may be valuable additions for community CVD screenings, as certain health profile abnormalities may impact each marker of arterial health differently. Additional research is needed in order to determine the cost-effectiveness of these screening tools as a preventive health method. / School of Physical Education, Sport, and Exercise Science

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.

Macrophage foam cells are the major culprits in atherosclerotic lesions, having a prominent role in both lesion initiation and progression. With atherosclerosis being the main factor underlying cardiovascular complications, there is a long-standing interest on finding ways to reverse lipid buildup in plaques. Studies have shown that promoting reverse cholesterol transport (RCT) from macrophage foam cells is anti-atherogenic because it alleviates the cholesterol burden of the plaques. The goal of this thesis was to gain insight into the mechanisms that govern cholesterol efflux from macrophage foam cells. The first part of this study looked at the ability of different oxysterols to promote cholesterol efflux in unloaded as compared to lipid-loaded macrophages, and our major finding here is that epoxycholesterol decreases efflux in lipid-loaded macrophages. It appears that epoxycholesterol does so by impairing the release cholesterol from its cellular storage site, the lipid droplet (LD), where it accumulates in the form of cholesteryl esters (CE). These results highlighted the importance of cholesterol release from LDs for efflux; indeed, this process is increasingly being recognized as the rate-limiting step for RCT in vivo. Subsequent experiments aimed at elucidating the mechanisms that govern LD CE hydrolysis in macrophage foam cells lead to the discovery of a novel pathway involved in cholesterol efflux. Macrophage CE hydrolysis is classically defined as being entirely dependent on neutral CE hydrolases. In the second part of this study, we demonstrate that in addition to the canonical CE hydrolases, which mediate neutral lipid hydrolysis, lysosomal acid lipase (LAL) also participates in the hydrolysis of cytoplasmic CE. Autophagy is specifically triggered in macrophages by atherogenic lipoproteins and delivers LD CE to LAL in lysosomes, thus generating free cholesterol for efflux. This autophagy-mediated cholesterol efflux is a process that is primarily dependant on the ABCA1 transporter and, importantly, is important for whole-body RCT. Overall, the studies presented in this thesis support that macrophage LD CE hydrolysis is rate-limiting for cholesterol efflux and shed light on the mechanisms of cholesterol mobilization for efflux in macrophage foam cells.

Angiotensin II (Ang II) is an important modulator for development of atherosclerosis from early stage foam cell formation to advanced stage plaque rupture. Recently, the importance of locally generated Ang II, especially in macrophages, has become more evident. Generation of Ang II by several enzymes other than ACE and renin has been shown mainly in vitro. Cathepsin G is one these enzymes which is expressed in neutrophils and macrophages. Macrophages are one of the primary and crucial cells in atherosclerotic lesions which become lipid-laden foam cells through lipoprotein uptake. We hypothesized that activation of nuclear factors in foam cells increases Ang II by modulation of the renin angiotensin system (RAS) genes and cathepsin G. We also hypothesized that cathepsin G, through its Ang II generating activity and its other catalytic functions, promotes atherosclerosis. The present study assessed the Ang I and II levels and expression of the RAS genes in THP-1 cells, a human acute monocytic leukemia cell line, and in peritoneal and bone marrow-derived macrophages after exposure to acetylated LDL (ac-LDL). I also evaluated how RAS blockade would affect foam cell formation in THP-1 cells. In parallel, I assessed the role of cathepsin G in Ang II generation and in the progression of atherosclerosis in cathepsin G heterozygous knockout mice on an Apoe-/- background (Ctsg+/-Apoe-/- mice). Ac-LDL treatment increased Ang I and Ang II levels in cell lysates and media from THP-1 cells but not in peritoneal or bone marrow-derived macrophages from wild type C57BL/6 mice. In ac-LDL-treated THP-1 cells, ACE and cathepsin G mRNA levels and activities were elevated. Angiotensinogen mRNA is increased but not the angiotensinogen protein concentration. Renin mRNA level and activity were not altered by ac-LDL treatment. Blocking RAS by an AT1 receptor blocker, ACE inhibitors or a renin inhibitor decreased cholesteryl ester content of THP-1 cells after exposure to ac-LDL. To confirm that the Ang II effect on foam cell formation was not unique to ac-LDL, we treated the THP-1 macrophages with a renin inhibitor or an AT1 receptor inhibitor after exposure to oxidized LDL (ox-LDL). RAS blockade in ox-LDL-treated cells also abolished cholesteryl ester formation. To see how Ang II plays a role in foam cell formation we assessed the effect of RAS inhibitors on SR-A, the principal receptor for mediating ac-LDL entry into the cells and on acyl-CoA:cholesterol acyl transferase (ACAT-1), the enzyme responsible for intracellular cholesterol esterification. RAS blockade in both ac-LDL- and ox-LDL-treated cells decreased SR-A and ACAT-1 protein levels. Cathepsin G partial deficiency on an Apoe-/- background did not change Ang II levels in peritoneal or bone marrow-derived macrophage cell lysates or media. This deficiency also did not affect immunoreactive angiotensin peptide levels in atherosclerotic lesions. After 8 weeks on a high fat diet Ctsg+/-Apoe-/- mice were similar to Ctsg+/+Apoe-/- mice in terms of lesion size and serum cholesterol levels but the Ctsg+/+Apoe-/- mice had more advanced lesions with more collagen and smooth muscle cells and fewer macrophages. Moreover, Ctsg+/+Apoe-/- mice had more apoptotic cells than their Ctsg+/-Apoe-/- littermates. Overall, our findings indicate that Ang II is increased in foam cells and this endogenous Ang II is involved in cholesteryl ester formation, possibly by regulating the levels of ACAT-1 and SR-A. We did not find any role for cathepsin G in generation of Ang II in mice but cathepsin G does, nevertheless, promote the progression of atherosclerotic lesions to a more advanced stage.

Atherosclerotic cardiovascular disease is the leading cause of death of adults in North America. Diabetes is a classical risk factor for atherosclerotic cardiovascular disease. Plasminogen activator inhibitor-1 (PAI-1) is the major physiological inhibitor of fibrinolysis. Elevated levels of PAI-1, oxidized low-density lipoprotein (oxLDL) and glycated LDL (glyLDL) were detected in patients with diabetes. Increased oxidative stress is associated with diabetic cardiovascular complications. Previous studies in our laboratory demonstrated that oxLDL or glyLDL increased the production of PAI-1 or reactive oxygen species (ROS) in vascular endothelial cells (EC). This study was undertaken to investigate transmembrane signaling mechanisms involved in oxLDL or glyLDL-induced upregulation of PAI-1 in cultured vascular EC. Further, we examined the mechanism for oxLDL or glyLDL-induced oxidative stress in EC. The results of the present studies demonstrated novel transmembrane signaling pathway for oxLDL-induced PAI-1 production in vascular EC. We demonstrated that lectin-like oxLDL receptor-1, H-Ras, a small G-protein and Raf-1/ERK-1/2 mediate oxLDL-induced PAI-1 expression in cultured EC. GlyLDL may activate EC via a distinct transmembrane signaling pathway. The results of the present study demonstrated that receptor for advanced glycation end products, NADPH oxidase and H-Ras/Raf-1 are implicated in the upregulation of heat shock factor-1 or PAI-1 in vascular EC under diabetes-associated metabolic stress. We investigated the effects of oxLDL or glyLDL on mitochondrial function in EC. Treatment with oxLDL or glyLDL significantly impaired the activities of electron transport chain (ETC) enzymes and also increased mitochondria-associated ROS in EC. The findings suggest that oxLDL or glyLDL attenuated activity of ETC and increased ROS generation in EC, which potentially contributes to oxidative stress in vasculature. In conclusion, diabetes-associated lipoproteins may upregulate stress response mediators and PAI-1 production via distinct transmembrane signaling pathways. OxLDL or glyLDL may increase ROS production via NOX activation and the impairment of mitochondrial ETC enzyme activity in EC. The understanding and identification of the regulatory mechanisms involved in diabetes-associated lipoprotein-induced signaling may help pharmacological design for the management of diabetic cardiovascular complications.

An important part of repair processes activated by vascular injury is the recruitment of vascular smooth muscle cells (SMC) from the existing contractile coat. Phenotypic modulation of SMCs enables these cells to proliferate and migrate into the vessel intima. Despite its importance in vessel repair, this plasticity of SMCs can also promote both the pathogenesis of atherosclerosis as well as neointimal formation following revascularization- induced injury. Vascular growth factors are major contributors to the migratory and proliferative responses to injury. IGF-1 is one such growth factor that elicits a response via its receptor, the IGF-1R, a classical tyrosine kinase receptor. However, it has been suggested that the IGF-1R may also be coupled to a heterotrimeric G protein and can thus initiate cellular responses via this alternate pathway. The objective of this study was to investigate the structural aspects of IGR-1R coupling to a heterotrimeric G protein in SMCs, as well as the contribution of this pathway to the cellular responses. In a porcine primary SMC culture model, IGF-1R co-precipitated with both the α- and β-subunits of a G protein, with the latter demonstrating activation dependent precipitation. The specific Gα class activated by IGF-1R was Gαi, in a manner that was independent of the activity of the tyrosine kinase. Both Gαi1 and Gαi2 directly interacted with the receptor. Gβγ mediated the activation of MAPK and its inhibition was sufficient to attenuate both the proliferation and migration of SMCs in vitro. In contrast, the contribution of Gαi was related to regulation of protein translation and histone modification. The data supports the conclusion that IGF-1 regulates the phenotype of vascular SMCs at least partially via a non-classical G protein-coupled receptor. Investigation into the individual subunits of the G protein complex led to the elucidation of a model in which both components play an integral role in the IGF-1 response, independent of the receptor tyrosine kinase activity. In one case, an interplay of specific Gαi-subunits leads to modulation of the VSMC translational and transcriptional responses, while in the other, release of the Gβγ-subunit activated the MAPK response in a manner that significantly contributes to both the migration and proliferation of SMCs.

Chemical exposure in humans rarely consists of a single chemical. The everyday exposure is characterized by thousands of chemicals mainly present at low levels. Despite that fact, risk assessment of chemicals is carried out on a chemical-by-chemical basis although there is a consensus that this view is too simplistic. This thesis aims to validate a statistical method to study the impact of mixtures of contaminants and to use that method to investigate the associations between circulating levels of a large number of environmental contaminants and atherosclerosis and the metabolic syndrome in an elderly population. Contaminants measured in the circulation represented various classes, such as persistent organic pollutants, plastic-associated chemicals and metals. There was little co-variation among the contaminants and only two clusters of PCBs could be discerned. Gradient boosted CARTs were used to assess additive and multiplicative associations between atherosclerosis, as measured by the intima-media thickness (IMT) and the echogenicity of the intima-media complex (IM-GSM), and prevalent metabolic syndrome. Systolic blood pressure was the most important predictor of IMT while the influence of the contaminants was marginal. Three phthalate metabolites; MMP, MEHP and MIBP were strongly related to IM-GSM. A synergistic interaction was found for MMP and MIBP, and a small antagonistic interaction was found for MIBP and MEHP. Associations between the contaminants and prevalent metabolic syndrome were modest, but three pesticides; p,p’-DDE, hexachlorbenzene and trans-nonachlor along with PCBs 118 and 209 and mercury were the strongest predictors of prevalent metabolic syndrome. This thesis concludes that many contaminants need to be measured to get a clear picture of the exposure. Boosted CARTs are useful for uncovering interactions. Multiplicative and/or additive effects of certain contaminant mixtures were found for atherosclerosis or the metabolic syndrome.

Atherosclerosis is a chronic inflammatory disease of arterial blood vessels, characterized by deposition of lipoproteins in the arterial wall. Atherosclerotic plaques form preferentially in distinct regions of the vasculature such as branch points, curvatures and bifurcations, suggesting that local hemodynamic forces may contribute to disease susceptibility. Shear stress imparted on endothelial cells (ECs) by the flowing blood has been shown to modulate gene expression and remodelling of the artery. In this thesis, an in vitro model was established to recreate the contrasting environments found in atherosclerosis-prone and atherosclerosis-resistant regions of the vasculature to demonstrate a direct causal-relationship between shear stress and expression of endothelial nitric oxide synthase (eNOS) and p65 in ECs. In vitro assessment of cell shape and expression patterns of these anti- and atherogenic genes demonstrated that shear stress can induce cell morphology and gene expression patterns that are similar to ECs in atherosclerosis-prone and atherosclerosis-resistant regions of the mouse vasculature. Regulation of eNOS transcription by shear stress was demonstrated using a transgenic mouse model and in vitro heterogeneous nuclear RNA (hnRNA) quantification. Similar to ECs in atherosclerosis-prone regions, epithelial cells lining the small intestine lumen express high levels of p65. To investigate the effects of p65 expression levels on constitutive and tumour necrosis factor α (TNFα)-induced nuclear factor-κB (NF-κB) signalling, p65 expression was suppressed in HeLa cells by RNA interference. Lower p65 expression resulted in reduced TNFα-induced expression of NF-κB target genes, including many subunits of inhibitor of nuclear factor κB (IκB), demonstrating modulation of NF-κB priming by p65 expression levels. Suppression of p65 also affected constitutive expression levels of IκB, and resulted in re-setting of the NF-κB/IκB equilibrium. Experiments using inhibitors of canonical NF-κB signalling found that basal expression of NF-κB components is independent of nuclear factor κB kinase β (IKKβ) activity and proteasome-mediated degradation of IκBα. Together, these studies elucidate the mechanism of flow-mediated gene regulation and the effect of resulting changes in p65 expression on NF-κB signalling.

A large number of studies have reported on the association between the obligate intracellular bacterium, Chlamydia pneumoniae and atherosclerosis. These studies suggest that C. pneumoniae may potentially play a role in the atherosclerotic process, as not all the current atherosclerotic risk factors account for the resulting complications, such as angina, myocardial infarction, heart failure and stroke. The research presented in this thesis analysed whether there are any reliable markers of chronic C. pneumoniae vascular infection, including chlamydial sero-prevalence as defined by two commercial serological tests, detection of C. pneumoniae DNA in the peripheral circulation, the presence or absence of risk factors and symptomatic status. The presence of the bacterium in atherosclerotic carotid specimens was diagnosed directly using a C. pneumoniae-specific polymerase chain reaction (PCR) and a genus-specific immunofluorescent (IF) assay. Eighteen of the 54 (33%) carotid artery diseased (CAD) specimens were positive for the presence of C. pneumoniae DNA by PCR detection, whereas the IF assay detected only six positive samples. PCR analysis found that only two of 43 (5%) patients had C. pneumoniae DNA present within their peripheral blood mononuclear cell (PBMC) fraction. Chlamydial antibodies were detected by Focus microimmunofluorescence and/or Medac recombinant enzyme-linked immunosorbert assay (rELISA) in 56% (24/43) of CAD patients tested. Traditional risk factors, symptomatic status, antigen detection and PCR-based detection of C. pneumoniae in PBMCs, all failed to correlate with the presence of a chlamydial vascular infection. In conclusion, the existing non-invasive diagnostic tests (serology and peripheral blood-based PCR detection) are inefficient for diagnosing a vascular Chlamydia infection, suggesting that a different chlamydial antigen should be tested targeted to identify a chronic C. pneumoniae infection in CAD patients. Given the observation that numerous previously published studies have detected C. pneumoniae in atherosclerotic arterial tissue, yet at widely different detection rates (0% to 100%), it was clear that the location and quantity of clinical specimen could directly affect the detection rate. Previous reports have not used a standard and validated procedure for sampling arterial specimens for C. pneumoniae DNA. The inconsistent detection rates of chlamydial DNA in atherosclerotic plaque are a result of low concentration and irregular distribution of the bacterium, as reported in this study. Our research concluded that a minimum of 15 (30ìm-thick) sections should be analysed by PCR to minimize these sampling variables and obtain a 95% chance of detecting all true C. pneumoniae-positive samples. All previous studies may have under estimated the prevalence of C. pneumoniae, as stringent sampling and repeat testing of the bacterium is required to minimise false-negative results. An interesting finding was that C. pneumoniae DNA was present in all 10 atherosclerotic arteries, although extensive sampling of the carotid was crucial for detection. The third area of research examined the question of possible strain differences between C. pneumoniae isolates infecting human atherosclerotic carotid arteries. Whole genome sequencing as well as specific gene typing suggests that there is relatively little genetic variation in human isolates of C. pneumoniae. To date, there has been little genomic analysis of strains from human cardiovascular sites. We analysed the genotypes of C. pneumoniae present in human atherosclerotic carotid plaque and found several polymorphisms in the variable domain-4 (VD4) region of the outer membrane protein-A (ompA) gene and the intergenic region between the ygeD and uridine kinase (ygeD-urk) genes. Our research identified four different genotypes of C. pneumoniae in human atherosclerotic carotid arteries, including an isolate that appears genetically identical to a strain previously detected in koalas. Two genotypes of C. pneumoniae were present in both human carotid specimens and koala PBMC fractions, suggesting that these genotypes of C. pneumoniae may be capable of crossing the host barrier. The study showed that diversity exists in both the ompAVD4 gene and the ygeD-urk intergenic region enabling fine-detailed differentiation between five different genotypes found in respiratory and/or vascular C. pneumoniae isolates. The importance of the diversity of C. pneumoniae isolates in its role in atherogenesis needs to be further studied.