Acute ischemic events associated with atherosclerosis are most often caused by rupture or erosion of unstable plaques. Clinical studies have demonstrated that in-vivo multi- contrast MRI can characterize plaque morphology and composition to evaluate the vulnerability of atherosclerotic plaques. The standard protocol for carotid imaging uses the Double-Inversion-Recovery (DIR) Fast-Spin-Echo (FSE) pulse sequence to acquire black-blood 2D high-resolution cross-sectional T\W, PDW and T2W images. With the addition of bright-blood Time-of-Flight images, it was demonstrated that in-vivo multi- contrast MRI could discriminate the major plaque components: lipid-rich necrotic core, intra-plaque haemorrhage, fibrous tissue and calcification. Given the nature and the large amount of multi-contrast MRI data, clinical studies of atherosclerosis would benefit from the availability of reliable and accurate automated techniques for image registration, segmentation and plaque classification. Recent multi-contrast MRI studies presented automatic plaque characterization methods that showed promising results under ex-vivo and in-vivo conditions. This thesis investigates some weaknesses in the current image acquisition and analysis techniques, which can affect the results of in- vivo MRI plaque characterization, and then proposes novel methods to advance the understanding of atherosclerosis in the carotid arteries. An automated multi -contrast registration algorithm that corrects for misalignments between carotid images caused by patient motion using sub-pixel accuracy and different similarity metrics was developed and validated. This project also used an alternative in-vivo carotid imaging approach based on the DIR Multi-Echo-Spin-Echo (Multi-SE) pulse sequence that acquired a series of black-blood 2D high-resolution cross-sectional images at different echo times. Quantitative T2 maps and synthetic multi-contrast images of carotid arteries were calculated from the Multi-SE images. T2 maps were automatically segmented and classified to provide in-vivo T2 measurements of the main plaque components, while Multi-SE synthetic images were compared with FSE images to demonstrate that the FSE acquisition strategy causes a significant loss of vessel edge sharpness.

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Inflammation plays a crucial role in atherosclerotic plaque generation/progression. Dendritic cells (DCs). cellular immune-response components linking innate and adaptive immune systems, have been found in atherosclerotic plaques. In this study, Des were examined as a possible therapeutic tool to modulate the inflammatory immune response underlying plaque formation. Apolipoprotein (apo) B-100 derived antigens are believed to modulate humoral immune responses to achieve atheroprotection, but their role in cellular immunity remains unclear. Therefore, one objective was to characterise the immunomodulatory effect of apoB-100-derived peptides (P2, P45, P210) on immature DCs (iDCs) and naive T lymphocytes ill vitro. iDCs were generated from bone-marrow progenitor-cells of male apoE-'- mice. Peptide up-take and processing was studied by confocal microscopy after 6h, 2411 and 48h. Peptide P45 was found in the endolysosomal compartments, co-localising with MHC-I and :MHC-II antigen-presenting complexes. The phenotypic and differentiation characteristics of P2, P45 and P21O-Joaded DCs were studied by flow cytometry, and cytokine and matrix metalloproleinase production by PCR/ELISA after 48h. Proliferation and differentiation of T lymphocytes driven by peptide-loaded DCs was also studied. Peptide-loaded DCs displayed a tolerogenie phenotype similar to that of unloaded, iDCs, and inhibited CD4+ proliferation induced by mature DCs when co-cultured. My results suggest that the protective effect of the peptides could be mediated by DCs presenting them to T cells. A second objective was to examine the effect of vaccination with tolerogenic DCs (toIDCs), generated in vitro through incubation with IL-10 and TGF-β for 6 days, on atherosclerotic progression in apoE-/- mice. This showed that immunisation with tolDCs increased the number of CD8+CD25'FoxP3+ T regulatory cells as well as secretion of IL-l0 within the spleen of immunised mice. IL- l0 levels were also elevated in the serum, while cholesterol levels were reduced, although plaque size remained unchanged. These results provide new insights for treatment and prevention of atherosclerosis through vaccination.

C-Myb is a DNA-binding transcription factor that functions in apoptosis, proliferation and differentiation. The role of c-Myb in vascular injury has been investigated in vitro and in vivo. Knock-down of c-Myb leads to a reduction in proliferation and an increase in apoptosis in vascular smooth muscle cells (VSMCs). Reduction of c-Myb activity has also been shown to reduce the incidence of neointimal formation in vivo by reducing VSMC proliferation. In contrast, over-expression of c-Myb in vivo leads to increased survival rates in certain cell types. Despite these observations, c-Myb expression has not yet been investigated in atherosclerosis. The balance between apoptosis and proliferation is pivotal in the pathogenesis of the disease. The aim of this thesis w as to investigate c-Myb expression in atherosclerotic lesions in addition to elucidating possible mechanisms by which c-Myb functions in atherosclerosis.

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BACKGROUND The paradigm that atherosclerosis is just luminal stenosis has now been superseded by work illustrating that it is a complex interplay of inflammatory processes with vessel wall remodelling. Non-invasive cardiovascular magnetic resonance (CMR) techniques can play an important role in providing quantitative volumetric assessment of atherosclerotic plaque burden, identifying early atherosclerotic changes and determining the composition of plaque constituents. We aimed to demonstrate how bulk motion artefacts can potentially lead to deleterious quality image, how novel methods of bulk motion correction can provide better image quality, to use a new CMR approach to characterise plaque, and apply the technique in a novel "at risk" population through imaging of the carotid arteries. METHODS AND RESULTS We used a novel method of detecting swallowing and established that swallowing during image acquisition can have a profound adverse effect on image quality. We showed that application of an accept or reject algorithm which deletes data acquired during swallowing, improves image quality. Respiratory movement of the carotid arteries has a similar detrimental effect on image quality but to a lesser extent and is variable between individuals. We demonstrated that the use of an ultra-short echo time (UTE) sequence can detect areas of calcification within plaque in ex vivo carotid artery specimens and correlated this with computerised tomography and histological data. Finally, we showed there was a positive effect on early atherosclerosis in patients with Behçet"s Disease treated with interferon-alpha. We showed that this may be related to stimulation of homeostatic IgM antibodies with improved clearance of biologically active lipids which have undergone oxidation and improvement in endothelial function. CONCLUSIONS Through sequence development, we have shown how bulk motion artefact can be overcome to improve image quality of carotid artery wall imaging, and how we can further characterise plaque constituents. Furthermore, we demonstrate how CMR of the vessel wall can be applied in clinical trials to assess early atherosclerotic plaque burden, with novel results of the improvement in atherosclerosis using interferon, which has not previously been described.

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No description available.

Endothelial inflammation leading to vascular dysfunction is a major contributor to the development of atherosclerosis. The release of adenosine at sites of inflammation represents an endogenous mechanism for limiting excessive inflammation and tissue damage. The majority of the anti-inflammatory effects of adenosine are mediated by signalling through the A2AAR and activation of the A2AAR has been shown to be protective in numerous models of inflammatory disease. Little is known about the molecular mechanisms behind these effects. However, in vitro studies using cultured endothelial cells indicate that signalling through the A2AAR can suppress activation of the NF kappa B and JAK/STAT proinflammatory signalling pathways. NF kappa B appears to be primed for activation in atherosclerosis-prone regions of the aorta indicating that suppression of NF kappa B signalling may protect against the development of atherosclerosis. In this study, the role of the A2AAR in regulating NF kappa B and JAK/STAT signalling pathway activation in the aorta was studied using A2AAR-deficient mice subjected to an LPS-induced model of septic shock. In response to LPS treatment, these mice displayed markedly elevated plasma levels of the pro-inflammatory cytokines TNF-alpha, IL-6, IL-1 beta and GMCSF compared to wild-type mice. Consistent with this finding, heightened activation of the NF kappa B and JAK/STAT pathways was detected in aortic protein samples from A2AAR-deficient mice as demonstrated by increased levels of the phosphorylated forms of I kappa B alpha and STAT1. However, expression of the NF kappa B and STAT1-regulated genes ICAM-1, VCAM-1 and TAP-1 was unaffected indicating the involvement of compensatory negative feedback mechanisms. These findings confirm a role for the A2AAR in regulation of pro-inflammatory signalling in the aorta. Further analysis of mechanisms which mediate this response may reveal new targets for therapeutic intervention to suppress inflammation in inflammatory disorders such as atherosclerosis. While the wide range of anti-inflammatory and tissue-protective responses elicited by the A2AAR have been well documented, the molecular regulation of the A2AAR has been less well studied. The presence of several serine and threonine residues in the extended C-terminal tail of the A2AAR suggests that it may be regulated by phosphorylation events occurring in this region. Indeed, the canine A2AAR is phosphorylated in response to PKC activation. Interestingly, several proteins have recently been identified as being able to interact with the C-terminal tail of the A2AAR. However, how these interactions are regulated is not known. One of the aims of this study was to characterise phosphorylation of the human A2AAR and to determine whether this could provide a means for regulating the binding of C-terminal interacting proteins. This was examined using human umbilical vein endothelial cells infected with recombinant adenovirus encoding the human A2AAR. It was found that phosphorylation of the human A2AAR could be induced in HUVECs by treatment with PMA or by stimulation of endogenous histamine H1 receptors. This was due to activation of PKC, as phosphorylation was inhibited by the PKC inhibitor GF109203X and by depletion of PKC following chronic treatment with PMA. Treatment of cells with the calcium-chelating agent BAPTA/AM did not inhibit PMA-induced phosphorylation indicating that a calcium-insensitive isoform of PKC was responsible. Meanwhile an siRNA-mediated gene silencing approach confirmed that PKC epsilon was not responsible indicating the involvement of either PKC delta or PKC theta. Previously reported interactions between the A2AAR and TRAX and 14-3-3 tau were confirmed in vitro by GST pull-down assay. Binding of 14-3-3 tau to the A2AAR appeared to be unaffected by treatment of HUVECs with PMA. However, A2AAR complex formation with TRAX was significantly reduced in samples from PMA-stimulated cells. These findings indicate that PKC-mediated phosphorylation may represent a means of regulating which proteins can interact with the C-terminal tail of the A2AAR. This may allow the A2AAR to initiate distinct signalling pathways depending on the cellular context in order to achieve the appropriate response.

Peripheral arterial disease (PAD) defines atherosclerotic disease of the arteries to the legs. PAD begins early in life and remains asymptomatic over long periods. The ankle brachial index (ABI) is an important diagnostic test which can identify asymptomatic individuals and serve as a good marker of the underlying peripheral and systemic atherosclerosis. Recent advances in vascular biology proposed a role of inflammatory and haemostatic mechanisms in atherosclerotic disease. Although inflammatory and haemostatic markers have been associated with coronary atherosclerosis in large scale epidemiological studies their role in PAD development is not well established and for many markers unknown. Also, their relationship with the progression of early asymptomatic disease has not been studied before. The aim of this thesis was to examine 12 markers of inflammation and haemostasis in relation to peripheral atherosclerotic progression and incident PAD. The Edinburgh Artery Study was used for this analysis. This is a population based cohort study of 1,592 men and women recruited in 1987. ABI was measured at baseline and at two follow up examinations which were conducted after 5 and after 12 years. Also, subjects were followed up for cardiovascular events for 17 years. Conventional cardiovascular risk factors, C-reactive protein (CRP), interleukin-6 (IL-6), intercellular adhesion molecule 1 (ICAM-1), vascular adhesion molecule-1 (VCAM-1), E-selectin, fibrinogen, D-dimer, tissue plasminogen activator (t-PA), vonWillebrand factor (vWF), factor VII, fibrinopeptide A (FpA) and prothrombin fragments 1+2 (F1+2) were measured at baseline. Valid ABI measurements were available for 1,582 subjects at baseline, for 1,081 subjects at the 5 year follow up and for 816 subjects at the 12 year follow up. The population showed a progression in atherosclerotic disease assessed by the mean ABI decline over time. The mean change in ABI was -0.04 (0.18) after 5 years and -0.06 (0.19) after 12 years. From inflammatory markers, CRP (p <0.01), IL-6 (p <0.001) and ICAM-1 (p <0.01) were associated with atherosclerotic progression after 12 years, independently of baseline ABI and of conventional cardiovascular risk factors. Also, from haemostatic markers, fibrinogen (p =0.05) and D-dimer (p ≤ 0.05) were significantly associated with atherosclerotic progression independently of baseline ABI and cardiovascular risk factors. Moreover, subjects with higher levels of both D-dimer and IL-6 at baseline had the greatest ABI decline. Also, IL-6 showed the stronger independent effect on atherosclerotic progression and retained statistical significance after adjustments for all inflammatory markers and for fibrinogen and D-dimer. Approximately 26% of the baseline population developed at least one event of major CVD and 14% of the baseline population developed symptomatic PAD after 17 years of follow up. Inflammatory markers, CRP and IL-6 showed modest associations with PAD which lost statistical significance in the multivariable model. On the other hand, these markers were associated with incident major CVD with hazard ratios (95% CI) 1.6 (1.2, 2.3) and 1.8 (1.3, 2.6) respectively (top vs. bottom tertile) in the multivariable model. ICAM-1 showed weak associations with incident CVD, however, was significantly associated with PAD with hazard ratio (95% CI) 1.8 (1.2, 2.7) (top vs. bottom tertile) after adjustments for cardiovascular risk factors and CVD at baseline. Haemostatic markers, fibrinogen and D-dimer were associated with 2.2 (95% CI: 1.5, 3.2) and 1.7 (1.2, 2.6) increase in the risk of PAD development and 1.8 (1.3, 2.3) and 1.6 (1.2, 2.1) increase in the risk of CVD independently of cardiovascular risk factors and history of CVD at baseline, respectively. This analysis showed a major role of inflammatory markers, CRP, IL-6 and ICAM-1 in atherosclerotic development and progression. In addition, fibrinogen and D-dimer, but not other haemostatic factors, were associated with progressive and incident peripheral atherosclerosis. Since D-dimer and fibrinogen are acute phase reactants, these data support the hypothesis that inflammation is more related to atherosclerosis than is hypercoagulation. Most importantly, the majority of the reported associations were not explained by increased levels of cardiovascular risk factors or pre-existing clinical or subclinical arterial disease. Thus these markers are more likely to have a causal than a consequential role in atherosclerotic disease.