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BACKGROUND: Macrophages in atherosclerotic plaques are believed to play a central role in plaque instability. However, the cell-specific molecular mechanisms involved are incompletely characterized. To address this, we performed genome-wide gene expression analyses using microarrays on RNA extracted from macrophage-rich regions of stable and unstable human atheromatous plaques. METHODS AND RESULTS: Plaques derived from carotid endarterectomy specimens (n=118) were designated as stable or unstable according to clinical, radiological and histopathological criteria. Macrophage-rich regions were excised from unstable and stable plaques by laser microdissection. Transcriptional profiling of extracted RNA was carried out using Affymetrix U133plus2 microarrays. The expression profiles were characteristic of activated macrophages. Expression profiles from stable and unstable samples clustered into distinct groups. Nine hundred and fourteen genes differentially expressed between stable and unstable sample groups were identified at a false discovery rate of 10%. The findings were confirmed by real-time PCR for eleven of the twelve genes with the highest fold expression difference (p<0.05). The analysis of the gene expression of the twelve highest fold expression genes by their principal components demonstrated a distribution of the samples along an axis of stability-instability in gene expression space. More specifically, components of the PPAR/Adipocytokine signalling pathway were significantly differentially expressed (p=5.4x10-7) between stable and unstable plaques with greater expression in unstable plaques. Key components of the pathway, fatty acid binding protein 4 (FABP4) and leptin, showed nine-fold (p=0.0086) and five-fold (p=0.0012) greater expression in unstable plaques respectively. The immunohistochemistry of the protein products for FABP4 and Leptin confirmed their co-localisation with the macrophages in the plaque. CONCLUSION: Differences in gene expression signatures are present between macrophages isolated from stable and unstable human atheromatous plaques. Components of the PPAR/adipocytokine signalling pathway, specifically FABP4 and Leptin, are expressed at higher levels in macrophages from unstable plaques. These findings suggest that down-regulation of PPAR/adipocytokine signalling within plaques may have therapeutic potential.

In the early stage of atherosclerosis, monocytes in the circulation become adherent and migrate into the intima of the blood vessel wall in response to stimuli, such as injury and inflammation, and inflammatory factors and metalloproteinases promote this process (Reaven and Witztum 1996). THP-1 monocytic cells have been used as a model to investigate the process of atherosclerosis as they grow in suspension, but become adherent, form processes and produce metalloproteinases and cytokines after phorbol 12-myristate 13-acetate (PMA) treatment. PMA treatment of THP-1 cells results in elevation of steady state levels of MMP9 at 9h, but ADAMTS4 was significantly induced at later time points, which suggested that other factors may be involved in it. The aim of this thesis was to investigate the factors involved in ADAMTS4 and MMP9 regulation.

Chronic exposure of humans to arsenic contaminated drinking water promotes the development of atherosclerosis and is a problem in many countries worldwide. The pathogenic mechanisms are ill-defined, although arsenite-induced endothelial dysfunction has been hypothesised. Ageing is a major risk factor for atherosclerosis and aged (senescent) endothelial cells have been observed in human and experimental atherosclerosis, including in people with arsenicosis. Senescence is a cellular state of irreversible growth arrest, the accumulation of such cells could contribute to the development of age-related diseases. The aim of this thesis was to determine whether arsenite induced premature senescence in human vascular endothelial cells in vitro and whether the mechanisms involved reactive oxygen species-mediated cellular damage. Since mitochondria are a major source of cellular oxidants, studies to determine whether mitochondria were the primary targets for arsenic toxicity were carried out. At sub-cytotoxic concentrations, using a cell culture model, arsenite caused stressinduced premature senescence in HUVECs which was quantitatively similar to induction of senescence by low levels of serial peroxide. Microarray-based transcriptomics revealed that antioxidant responses, including Nrf2-oxidative stress response pathway and metallothioneins, were upregulated in HUVECs treated with subcytotoxic arsenite concentrations supporting arsenite-induced oxidative stress as a possible mechanism. This was accompanied by an increase in nuclear DNA damage observed using the Comet assay. Although no statistically significant effects on mitochondrial biogenesis or mtDNA damage were seen using qPCR, analysis by extracellular flux analyzer revealed a decline in mitochondrial function, specifically the mitochondrial reserve capacity. These findings cast serious doubt that mitochondria are the primary targets of arsenite in endothelial cells, with the oxidative stress occurring through cytoplasmic sources and leading to cellular damage. This forms a possible mechanism for the observed premature senescence in endothelial cells due to arsenite which is linked with the process of atherosclerosis in humans.

Atherosclerosis is a chronic inflammatory disease of the arteries that represents the root cause of the majority of heart attacks and strokes. The accumulation of lipid droplets (LDs) in macrophages and their subsequent transformation into foam cells is one of the key steps in the development of atherosclerotic lesions. It has been traditionally thought that this process is largely dependent on the accumulation of oxidised low-density lipoprotein (OxLDL) via uptake by macrophage scavenger receptors. However, as DNA signatures from a wide array of bacterial species have been identified in human atherosclerotic lesions, this project aimed to investigate whether and by which mechanisms these organisms may modulate macrophage foam cell formation, with a particular emphasis on the potential roles played by Toll-like receptor (TLR) signalling. The present findings establish that exposure of murine or human macrophages to diverse heat-killed atheroma-associated bacteria, or stimulants of any TLR, leads to the accumulation of cholesterol ester and foam cell formation. This process is dependent on TLR signalling, particularly TLR2 and TLR4, but independent of lipoprotein oxidation or scavenger receptor uptake, and although it is dose-dependently potentiated by LDL, it can also occur in the absence of LDL supplementation. TLR-dependent lipid accumulation is not due to enhanced pinocytosis or LDL receptor-dependent LDL uptake. Instead, the results indicate both reduced cholesterol efflux to HDL and increased de novo lipid synthesis, via TLR-dependent modulation of key proteins of the two pathways, the ABCA1/G1 cholesterol efflux proteins, and 3-hydroxy-3-methylglutatyl CoA reductase (HMGCR), respectively. Taken together, the results point towards a novel mechanism of foam cell formation that begins in circulating monocytes exposed to bacteria or their products before they are recruited to developing lesions. Novel therapies for the prevention of atherosclerosis could be developed to target the mechanisms of crosstalk between the TLR and lipid regulatory pathways identified by the current project.

In order to provide more information on platelet activation that would be of interest to all clinicians involved in the care of the patient with atherosclerosis, this thesis sets out to provide data to either support or refute the following hypotheses. (1). Platelet activation in patients with stable coronary heart disease increases with the angiographic severity of disease. (2). Patients presenting with the acute manifestations of atherosclerosis have a greater degree of platelet activation than patients with stable disease. (3). Peripheral artery intervention results in the release of the cytokine, sCD40L, by platelets. (4). Platelet microparticle levels are higher in patients with Type 2 Diabetes who develop symptomatic macrovascular disease.;Using both flow cytometric and Enzyme Linked Immunosorbent Assay (ELISA) based measurements of platelet activation, this thesis confirms that platelet activation can be related to the clinical severity of atherosclerotic disease. In addition, the development of symptomatic atherosclerotic disease is associated with increased platelet microparticle levels. Peripheral artery angioplasty has also been shown to increase sCD40L release. However, there is no association between platelet activation status and the angiographic severity of coronary heart disease.

The primary aim of this thesis was to quantify the collagenase concentrations in carotid plaques and to relate them to markers to plaque instability.;Recent studies have shown that strain therapy decreases cardiovascular risk, even in patients with normal cholesterol levels. A further aim of this thesis was to observe the effects of statins on clinical and biochemical indicators of plaque instability.;Atherosclerotic plaques were collected from 159 patients undergoing carotid endarterectomy. The presence and timing of carotid territory symptoms was ascertained. Pre-operative embolisation was recorded by transcranial Doppler. Each plaque was assessed for histological features of instability. Plaque MMP and cytokine concentrations was quantified using ELISA.;Significantly higher concentrations of active MMP-8 were observed in the plaques of symptomatic patients (p=0.0002), emboli-positive patients (p=0.0037) and in those plaques demonstrating histological evidence of rupture (p=0.0036). No differences were seen in the levels of MMP-1 and MMP-13. Immunohistochemistry, in situ by hybridisation and colocalisation studies confirmed the presence of MMP-8 protein and mRNA within the plaque, which colocalised with macrophages. These data suggest that the active form of MMP-8 may be partly responsible for degradation of the collagen cap of atherosclerotic plaques. This enzyme represents an attractive target for drug therapy aimed at stabilising vulnerable plaques.;Patients on statins were less likely to have suffered symptoms in the month prior to surgery (p=0.0049) and less likely to have cerebral embolisation detected (p=0.0459). Carotid plaques retrieved from statin-taking patients, revealed significantly lower concentrations of MMP-9 (p=0.0018) and IL-6 (p=0.0005). These data suggest that statins may stabilise plaques by lowering MMP and cytokine levels, resulting in decreased embolisation and symptoms.