The objective of this research is to investigate both mathematically and numerically the effects of vascular geometry upon the cellular dynamics in the endothelium and its consequence in the localisation of atherosclerosis. It is widely accepted that the formation of atherosclerotic plaques preferentially occurs at specific locations in the vasculature, such as arterial branches and bends. It has also been observed that, at the sites of plaque formation, the physiological functions of the vascular endothelium are impaired due to a defect in the production mechanisms of or diminished activities of endothelial nitric oxide (NO). From these observations, a correlation between the vascular geometry, which is effected via local haemodynamic forces, and local bioavailability of endothelial NO has been postulated. The research areas that have been involved in the investigation of atherosclerosis's localisation in the past, haemodynamics, medicine, calcium dynamics, NO kinetics and endothelial cell biology, have been studied individually, and there appears to be no integrated model to date that allows investigation of coupled haemodynamic and cellular mechanism applied in physiologically realistic model geometries. An integrated numerical model that includes these mechanisms will be developed in this research, which will lead to a further, more comprehensive understanding of the pathogenesis of atherosclerosis.

Atherosclerosis is a complex inflammatory disease in which oxidative stress is a major protagonist in the development and progression of the atherosclerotic plaque. All biochemical analysis studies of plaque over the past fifteen years have been carried out on whole plaque with no attempt to localise sites of differing biochemical conditions. This study set out to identify in oxidation levels and inflammatory markers in relation to spatial localisation within the plaque. Advanced plaque samples removed during endarectomy were obtained from the Christchurch Hospital Department of surgery and were dissected into 3-5 mm sections along the longitudinal axis prior to analysis. Samples were analysed for vitamin E, neopterin, total cholesterol and markers of oxidative damage to protein and lipids. Neopterin is a marker of inflammation as it is released by activated macrophages yet it has never been measured in plaques. Initial analysis showed that the acid precipitation method for removing protein from samples prior to HPLC neopterin analysis was causing a significant loss in neopterin. A new acetonitrile based protein removal procedure was developed. Markers of oxidative stress and inflammation where shown to vary across the length of an atherosclerotic plaque. This variation allows for localized incidences of high and low radical flux and microenvironments of depleted antioxidants or areas in which the prooxidative actions of molecular components are favoured. Significant correlations were rarely seen in more then one plaque and trends found in the combined data set generally did not hold true in individual plaques. This reflects upon the complexity of the disease, especially at this advanced stage in which the biochemical morphology of individual plaques is extremely diverse. Separation of the plaques into pre-, post-, and bifurcation areas did produce some trends. These can be related to shear stress variations in the blood flow; further investigations into the biochemical differences between these areas may provide a better understanding of the growth and development of the atherosclerotic plaque.

Studies of aortic plaque reveal the presence of tissue macrophages filled with cholesteryl esters. To study lipoprotein metabolism of in vivo, maturated human macrophages, I isolated cells from human peritoneal effluent. Population analysis using cytochemistry showed substantial numbers of acid-esterase positive monocytic cells, lymphocytes, leukocytes and erythrocytes. Substantial variation in cell populations existed among patients. Human peritoneal cells degraded low density lipoproteins (LDL) and acetylated LDL (AcLDL) by high affinity, receptor-mediated processes. AcLDL degradation saturated at 15 ug protein/ml and LDL degradation saturated at 11 ug protein/ml. Positive correlation of the percentages of monocytic cells with the degradation values (LDL, r =.710; AcLDL, r =.725) and a degradation assay using cells isolated by Lymphoprep showed that the monocytic cells substantially contributed to the degradation of LDL. AcLDL degradation was calcium independent and inhibited by fucoidin. LDL degradation was calcium dependent and very low density lipoprotein and apoE-containing high density lipoprotein (HDL) competed with LDL for receptor uptake; apoE-free HDL, AcLDL and fucoidin did not reduce LDL degradation. Both receptors were pronase-sensitive and degradation was dependent upon lysosomal activity. ACAT activity analysis showed that pre-incubation of cells with LDL or AcLDL stimulated ACAT activity. ACAT activity was greatest for cells preincubated using AcLDL and fresh medium was necessary to maintain the ACAT activity values beyond 24 hrs. LDL-stimulated ACAT activity declined as time was increased above 24 hrs. Flow cytometry analysis of a total cell population and the Lymphoprep-isolated cells revealed a heterogenous T cell population, the presence of monocyte/macrophages, suggested that some of the cells present were activated and confirmed cytochemistry analysis demonstrating that Lymphoprep concentrated the mononuclear cells. Human peritoneal macrophages formed foam cells when incubated in the presence of AcLDL or LDL for 72 hrs. The formation of foam cells in the presence of LDL was dependent upon cell exposure time to the medium. Foam cell formation in the presence of LDL was accompanied by dense vacuolization and in the demonstrated absence of the oxidation of LDL the oil red O stainable material collected outside the vacuoles.

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Cardiovascular diseases are a major cause of death worldwide. Aneurysmal rupture in cerebral arteries or loss of endothelial integrity in the course of atherosclerosis or therapeutic angioplasty lead to exposure of vascular smooth muscle cells (SMC) to blood components such as sphingolipids. Sphingosylphosphorylcholine (SPC) and sphingosine 1-phosphate (S1P) are two naturally occurring sphingolipids, which are vasoprotective in the healthy endothelium-lined vessel, but may promote vascular disease by causing functional changes of SMC. Vascular inflammation is an important factor in various pathologies. SPC can activate pro-inflammatory signalling pathways in rat cerebral artery. Here these observations are extended by showing that SPC elicits monocyte chemoattractant protein-1 production in rat cerebral artery SMC ex vivo. Thus, in addition to being a vasoconstrictor, SPC may promote the development of life-threatening prolonged cerebral vasospasm following subarachnoid haemorrhage by supporting vascular inflammation. It is also demonstrated that SPC prevents tumour necrosis factor-a (TNF)-stimulated adhesion of macrophages to rat aortic SMC in vitro by interfering with adhesive properties of SMC, but not macrophages. While this effect appeared to be mediated by the S1P receptor S1P2, S1P itself did not reduce macrophage adhesion. The anti-adhesive action of SPC also depended on lipid rafts. However, SPC did neither prevent TNF-induced nuclear factor kB activation nor cell adhesion molecule expression in SMC. SPC-induced cyclooxygenase 2 expression in aortic SMC was dispensable for its anti-adhesive effect. In contrast, the inhibitory effect of SPC on TNFinduced expression of inducible nitric oxide synthase is probably involved in its anti-adhesive effect because it was mimicked by respective pharmacological blockade. The results also demonstrate that nitric oxide promotes leukocyte adhesion to vascular SMC, while it has the opposite effect on endothelial cells. These findings may help understand cardiovascular diseases and define novel treatment approaches.

Background: Atherosclerosis is a systemic inflammatory disease characterized by the formation of atheromatous plaques within arteries. These plaques can be classified as unstable or stable based on their morphology and cellular infiltrate. Anatomical location of plaques and age of atheroma defines the symptoms of disease. However, there is little in the literature to support this. The study aimed to compare the cellular composition, morphology, lipid biochemistry and 18F- flurodeoxyglucose (18F-FDG) positive emission tomography (PET) uptake between plaques from patients with recently symptomatic carotid disease and patients with symptomatic peripheral arterial disease undergoing intervention. Patients and Method Patients with symptomatic carotid (≥60%) or femoral stenosis undergoing intervention were recruited. All patients underwent 18F-FDG PET scanning prior to operation. The numbers of plaque macrophage and T cells were determined by immunohistochemistry (IHC). Double IHC defined the proportion of classically (M1) activated macrophages (iNOS, MHC II and SOCS-3 positive) or alternatively (M2) activated (dectin-1, CD163; SOCS-1). Plaque composition was quantified by a new morphological definition based on percentage area of fibrooconnective tissue, lipid, calcification and cellular infiltrate. The proportion of fatty acids within plaque lipids was estimated by liquid chromatography. Results 34 patients with symptomatic carotid disease and 34 with symptomatic femoral disease were recruited. 18F-FDG PET imaging was carried out successfully in 29 carotid and 29 femoral artery disease patients. 32 carotid and 25 femoral plaques were obtained. Significant differences were noted between carotid and femoral plaques with respect to the number of macrophages (p<0.001), T cells (p<0.001) and proportion of classical (p<0.001) and alternatively (p<0.001) activated macrophages and morphological analysis with evidence of more inflammation in carotid plaques. Lipid analysis revealed higher triglyceride n-6 PUFAs in carotid compared to femoral plaques (p=0.01). FDG uptake between carotid and femoral plaques was not significantly different and did not correlate with immunohistochemical, plaque morphometry or lipid analysis parameters. FDG uptake correlated with degree of symptomatic carotid stenosis (Spearman‟s coefficient=0.482;p=0.008) and symptomatic ABPI (Spearman‟s coefficient=-0.414;p=0.025). FDG uptake was higher in the symptomatic carotid compared to the contralateral asymptomatic carotid (p=0.016). Conclusion This study has shown substantial difference between morphological and cellular compositions of carotid and femoral plaques. Carotid plaques from recently symptomatic patients exhibited significantly greater percentage areas of lipid deposition, lymphocytic and monocyte/macrophage infiltrate and reduced cap thickness, in line with their more vulnerable nature. Moreover, there were a greater proportion of classically activated macrophages that are associated with plaque vulnerability. In contrast, percentage areas of fibroconnective tissue were higher in the femoral plaques. 18F-FDG PET imaging, although capable of identifying plaque inflammation, may not be adequately sensitive to differentiate between vulnerable and stable complex plaques.

Introduction Coronary artery disease (CAD) is the most common cause of death in Europe and North America and early detection of atherosclerosis is a clinical priority. Diagnosis of CAD remains conventional angiography, although recent technology has introduced non-invasive imaging of coronary arteries using computed tomographic coronary angiography (CTCA), which enables the detection and quantification of coronary artery calcification (CAC). CAC forms within the arterial wall and is usually found in or adjacent to atherosclerotic plaques and is consequently known as sub-clinical atherosclerosis.  The conventional cardiovascular (CV) risk factors used to quantify the estimated 10-year coronary event risk comprise dyslipidaemia, hypertension, diabetes mellitus, obesity, smoking and family history of CAD. Nevertheless, their relationship with significant (&gt;50%) stenosis, their interaction with the CAC score and their predictive ability for CAC presence and extent has not been fully determined in symptomatic patients.   Methods   For Papers 1-4 we took patients from the Euro-CCAD cohort, an international study established in 2009 in Umeå, Sweden. The study data gave us the CAC score and the CV risk factor profile in 6309 patients, together with angiography results for a reduced cohort of 5515 patients. In Papers 1 and 2 we assessed the risk factors for significant stenosis, including CAC as a risk factor. Paper 2 carried out this analysis by geographical region: Europe vs USA and northern vs southern Europe. Paper 3 investigated the CV risk factors for CAC presence, stratified by age and gender, while Paper 4 assessed the CV risk factors for CAC extent, stratified by gender.  In paper 5 we carried out a systematic review and meta-analysis of all studies of the risk factor predictors of CAC presence, extent and progression in symptomatic patients. From a total of 884 studies, we identified 10 which fitted our inclusion criteria, providing us with a total of 15,769 symptomatic patients. All 10 were entered in the systematic review and 7 were also eligible for the meta-analysis.   Results Paper 1:           Among risk factors alone, the most powerful predictors of significant coronary stenosis were male gender followed by diabetes, smoking, hypercholesterolaemia, hypertension, family history of CAD and age; only obesity was not predictive. When including the log transformed CAC score as a risk factor, this proved the most powerful predictor of &gt;50% stenosis, but hypercholesterolaemia and hypertension lost their predictive ability. The conventional risk factors alone were 70% accurate in predicting significant stenosis, the log transformed CAC score alone was 82% accurate but the combination was 84% accurate and improved both sensitivity and specificity.  Paper 2:           Despite some striking differences in profiles between Europe and the USA, the most important risk factors for &gt;50% stenosis in both groups were male gender followed by diabetes. When the log CAC score was included as a risk factor, it became by far the most important predictor of &gt;50% stenosis in both continents, followed by male gender. In the northern vs southern Europe comparison the result was similar, with the log CAC score being the most important predictor of &gt;50% stenosis in both regions, followed by male gender.  Paper 3:           Independent predictors of CAC presence in males and females were age, dyslipidaemia, hypertension, diabetes and smoking, with the addition of family history of CAD in males; obesity was not predictive in either gender. The most important predictors of CAC presence in males were dyslipidaemia and diabetes, while among females the most important predictors of CAC presence were diabetes followed by smoking. When analysed by age groups, in both males and females aged &lt;70 years, diabetes, hypertension and dyslipidaemia were predictive, with diabetes being the strongest; in females aged &lt;70 years, smoking was also predictive. Among those aged ≥70 years, the results are completely different, with only dyslipidaemia being predictive in males but smoking and diabetes were predictive in females.  Paper 4:           In the total cohort, age, male gender, diabetes, obesity, family history of CAD and number of risk factors predicted an increasing CAC score, with the most important being male gender and diabetes. In males, hypertension and dyslipidaemia were also predictive, although diabetes was the most important predictor. Diabetes was similarly the most important risk factor in females, followed by age and number of risk factors. Among patients with CAC, hypertension, dyslipidaemia and diabetes predicted CAC extent in both males and females, with diabetes being the strongest predictor in males followed by dyslipidaemia, while diabetes was also the strongest predictor in females, followed by hypertension. Quantile regression confirmed the consistent predictive ability of diabetes.  Paper 5:           In the systematic review, age was strongly predictive of both CAC presence and extent but not of CAC progression. The results for CAC presence were overwhelmed by data from one study of almost 10,000 patients, which found that white ethnicity, diabetes, hypertension and obesity were predictive of CAC presence but not male gender, dyslipidaemia, family history or smoking. With respect to CAC extent, only male gender and hypertension were clearly predictive, while in the one study of CAC progression, only diabetes and hypertension were predictive. In the meta-analysis, hypertension followed by male gender, diabetes and age were predictive of CAC presence, while for CAC extent mild-moderate CAC was predicted by hypertension alone, whereas severe CAC was predicted by hypertension followed by diabetes.   Conclusion Our investigation of the Euro-CCAD cohort showed that the CAC score is far more predictive of significant stenosis than risk factors alone, followed by male gender and diabetes, and there was little benefit to risk factor assessment over and above the CAC score for &gt;50% stenosis prediction. Regional variations made little difference to this result. Independent predictors of CAC presence were dyslipidaemia and diabetes in males and diabetes followed by smoking in females. The risk factor predictors alter at age 70. The most important risk factor predictors of CAC extent were male gender and diabetes; when analysed by gender, diabetes was the most important in both males and females. Our studies have consistently shown the strong predictive ability of male gender in the total cohort and diabetes in males and females and this is reflected in the meta-analysis, which also found hypertension to be independently predictive. Interestingly, dyslipidaemia does not appear to be a strong risk factor.

Chemokine-induced macrophage recruitment into the vascular wall is an early pathological event in the progression of atherosclerosis. Macrophage activation and chemotaxis during cell recruitment are mediated by chemokine ligation of multiple G- protein coupled receptors. The Regulator of G-Protein Signalling-l (RGS-l) acts to down-regulate the response to sustained chemokine stimulation. Studies in this laboratory have shown Rgsl is up-regulated in atherosclerotic ApoE1- mice in association with atherosclerotic plaque progression and published findings have reported that RGS 1 is highly expressed in leukocytes. However an in vivo role for RGS-l in macrophage function or in atherosclerosis has not been investigated. This thesis aimed to address the hypothesis that RGS 1 has an important role in atherosclerosis and modulates the inflammatory response by controlling chemokine signalling and macrophage chemotaxis to atherosclerotic plaques. To investigate the role of RGS 1 in macrophage function and the development of atherosclerosis, Rgsrl- mice were characterised on the ApoE1- background. Flow cytometric analysis of leukocytes in blood, spleen and bone marrow indicated Rgsrl- ApoE1- mice had no significant differences in the numbers of monocytes or lymphocytes compared to ApoE1- mice. Rgsl was found to be highly expressed in macrophages from ApoE1- mice compared to B-Iymphocytes, where it has a non-redundant role, and other cells involved in plaque formation. Furthermore, Rgsl is up-regulated with monocyte- macrophage activation by innate stimuli. For the first time, RGS 1 'was shown to affect chemokine receptor signalling in macrophages in vitro. RgsrlApoE1- macrophages showed significantly enhanced chemotaxis to CCL2, CCL3 and CCLS and impaired homologous desensitisation to the chemokine CCLS in comparison to ApoE1- cells. To determine the role of RGS-l in leukocyte trafficking and atherosclerosis, a detailed atherosclerosis study was carried out. RgsrlApoE1- mice had significantly less lesion formation in the aortic roots at 9-weeks and in the aorta at 16-weeks on a chow diet in comparison to ApoE1- mice. This was accompanied with decreased macrophage content in the aortic root at 9-weeks. To further investigate aortic leukocyte recruitment, an Angiotensin IT-induced model of acute vascular inflammation was used. At 9 weeks of age, Rgsrl-ApoE1- mice had significantly less aortic CD4S+ leukocytes and cons' myeloid cells recruited to the aorta in comparison to ApoE1- mice. Collectively, these findings identify a new role for RGS-l in macrophage function and support a role for RGS-l in leukocyte recruitment and retention in the initial stages of atherosclerotic plaque formation. These results identify RGS 1 as a novel target for the treatment of acute vascular inflammation and early atherosclerosis.