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In healthy blood vessels, vascular smooth muscle cells (VSMCs) exist in a contractile, quiescent state but upon vascular insult can switch phenotype to activate proliferation, migration and remodelling of the extracellular matrix. Phenotypically switched VSMCs contribute most cells within neointimal lesions, characteristic of atherosclerosis and in-stent restenosis, diseases that underlie heart attack and stroke. Using multicolour "Confetti" VSMC-specific lineage tracing in animal models of vascular disease, our lab and others have shown that the extensive VSMC contribution to these lesions results from the clonal expansion of few cells. To understand how oligoclonal VSMC lesion contribution arises and to identify the signals activating VSMC proliferation in vivo, I used quantified VSMC clonal development over time in two models of vascular disease. Following acute vascular injury, the number and sizes of patches of clonally expanded VSMCs steadily increased before reaching a plateau, suggesting rare activation of VSMC proliferation in few cells, rather than clonal competition following widespread VSMC activation. Interestingly, only a subset of medial patches gave rise to neointimal patches, suggesting that VSMC lesion invasion represents a second selective event underlying mature lesion oligoclonality. Infrequent activation of VSMC proliferation in atherosclerosis was evidenced by the absence of plaques with high numbers of colours at any stage of plaque development. Tamoxifen-inducible, VSMC-specific deletion of contractile master regulator Myocd in adult mice had modest phenotypic effects on baseline VSMC contractile marker expression, and on injury-induced VSMC transcriptional response and clonality. In both vascular disease models, VSMC activation was greatly enriched in vascular regions displaying elastic lamina alterations, medial acellularity and immune cell recruitment, implicating these as potential proliferation-inducing cues. However, not all VSMCs in these regions formed patches, suggesting that VSMCs must be primed to respond proliferatively. Consistent with the hypothesis that VSMCs marked by stem cell antigen-1 (SCA1) may represent such a primed population, profiling of chromatin accessibility in SCA1+ VSMCs revealed substantial opening of chromatin at genes showing increased expression in injury-activated compared to healthy VSMCs. Manipulation of RUNX1 and CEBP, transcription factors whose motifs were enriched at activation-specific open chromatin regions, could allow for control of VSMC priming and proliferation.

Atherosclerosis is the most common cause of ischaemic heart disease and stroke which represent a global health concern responsible for significant levels of morbidity and mortality worldwide. It is a chronic inflammatory disease characterised by plaque build-up within arterial walls initiated by low density lipoprotein cholesterol. Elevated plasma LDL is a major risk factor for atherosclerosis and its immunogenic oxidation creates neo-epitopes that drive inflammatory immune responses underlying the pathogenesis of atherosclerosis. The germinal centre is found within secondary lymphoid organs and is responsible for the production of high affinity antibody-producing plasma cells and long-lived memory B cells against antigens such as epitopes of oxidised LDL. Class-switched plasma cells and anti-oxLDL IgG antibodies have been detected within atherosclerotic plaques and sera of human and mice suggesting that the germinal centre response is pathogenically dysregulated in atherosclerosis. The tamoxifen-inducible AID-CreERT2-Rosa-EYFP lineage tracing mouse model was used and crossed with the atherosclerosis-prone LDLr-deficient mouse model. This model inducibly and specifically labels germinal centre B cells and their progeny using tamoxifen allowing for tracking of atherosclerosis-specific B cell clones comprising germinal centre, memory, and plasma cells. LDLr-/- and LDLr+/- ('WT') mice were fed chow or high fat diet for up to 8 weeks and upon tamoxifen dosing (timing varied throughout studies) via intra-peritoneal injection or oral gavage, AID-expressing cells (germinal centre B cells) were fluorescently labelled with EYFP. Cells were analysed using flow cytometry and FlowJo software. All graphs and statistics were created using the GraphPad Prism 7.0. In this thesis, I sought to characterise and understand the germinal centre B cell response in atherosclerosis using a lineage tracing mouse model. Firstly, the model was validated as EYFP cells were only detected after tamoxifen administration and were only present in germinal centre-derived cell populations. Furthermore, labelling efficiency was independent of time, diet, or genotype. The germinal centre response was characterised in both WT and LDLr-/- mice demonstrating an time-dependent increase in germinal centre responses. Germinal centre reactions were exacerbated, greater in magnitude and degree of class-switching, by increased plasma cholesterol levels consequent of both HFD and knock out of the LDL receptor. The combination of HFD and LDLr-/- genotype, replicating atherosclerotic conditions, synergistically exacerbated germinal centre responses biased towards pathogenic cellular output due to elevated levels of class switching. Due to the permanence of EYFP labelling, it was possible to track EYFP labelled cells over time and throughout the course of atherosclerosis. Tracking studies revealed that hyperlipidaemic conditions induced by HFD resulted in longer-lived EYFP clones which had increased propensity to undergo class-switching. Furthermore, there was a greater output of EYFP labelled memory and plasma cells. To investigate the mechanism by which HFD induced greater germinal centre responses, interventions were conducted to block the impact of cholesterol and inflammation separately. Use of a cholesterol inhibitor drug and cholesterol free diet revealed that dietary cholesterol is key in driving the exacerbated germinal centre responses observed at early stages i.e., 4 weeks of atherosclerosis. Use of a cytokine inhibitor to dampen inflammation limited germinal centre responses at later stages of atherosclerosis i.e., 8 weeks, suggesting that inflammation plays an important role in germinal centre responses at this later timepoint. Thus, the model proposed is that dietary cholesterol is critical early on in atherosclerosis as disrupted lipid homeostasis results in autoimmune B cell responses with germinal centres primarily reacting to accumulated oxidised LDL while at later stages, the inflammation associated with atherosclerosis fuels exacerbated and pathogenic germinal centre responses. To examine the role of IgG2c and its receptor FcgRIV in atherosclerosis, the impact of IgG2c antibody complexes was tested in vitro. IgG2c significantly enhanced TNF secretion, a marker of inflammation, in FcgRIV-expressing myeloid-derived monocytes. Thus, IgG2c has the potential to exacerbate inflammatory responses from plaque macrophages and dendritic cells. In conclusion, the AID lineage tracing mouse model has been validated for use in the atherosclerosis setting. It has been characterised showing that germinal centre responses are exacerbated during the course of atherosclerosis and are skewed towards production of class- switched longer-lived B cell clones. Dietary cholesterol is the main driver of this pathogenic response in tandem with the inflammatory conditions caused by atherosclerosis. Germinal centre-derived IgG2c antibodies could play an important role in exacerbating inflammation within the plaque through plaque macrophages and dendritic cells. This thesis presents a model whereby atherogenic dyslipidaemia, as a result of elevated serum LDL levels, breaks down B cell tolerance. This results in autoimmune pathology characterised by exacerbated germinal centre responses skewed towards pathogenic antibody isotype clone production in response to LDL-induced inflammation. This provides an opportunity to target B cell-related atherosclerosis-specific responses therapeutically.

Myocardial infarction, a leading cause of mortality and morbidity worldwide, is often the result of coronary plaque rupture. Previous histological work has demonstrated that ruptured plaques are associated with higher stress, with in vivo intravascular studies linking higher plaque structural stress (PSS) with the presentation of acute coronary syndrome. Endothelial shear stress (ESS) on the other hand has been implicated in early plaque development and plaque growth, suggesting that both PSS and ESS can influence future plaque behaviour. The work presented in this thesis first demonstrates that PSS changes with pharmacotherapy, in particular, high-intensity statin (HIS) treatment prevented the rise in PSS in most advanced lesions (those with plaque burden [PB] > 60%). The change in PSS was both dependent on drug therapy and baseline disease severity, and the protective effect of HIS seemed to be mediated by remodelling plaque microstructure and lumen surface. Second, it shows that plaque geometric parameters, especially the longitudinal variation measured by heterogeneity index of lumen roughness, curvature, and irregularity, can identify plaques that subsequently developed adverse clinical events. Lumen roughness heterogeneity showed a weak correlation with PSS and was an independent predictor of adverse clinical events. Finally, the role of combined PSS and ESS in plaque risk prediction was assessed through a subgroup analysis of the PROSPECT (A Prospective Natural-History Study of Coronary Atherosclerosis) study, with results suggesting that incorporation of ESS, ESS gradient, and PSS heterogeneity can improve the capability of VH-IVUS to identify plaques that lead to such events.

Introduction: Semicarbazide-sensitive amine oxidase (SSAO) is both a soluble and membrane bound transmembrane protein expressed in the vasculature. In endothelial cells, SSAO is involved in leukocyte rolling, adhesion, and transmigration into inflammatory sites which may imply a pathological role in the development of atherosclerosis. In vascular smooth muscle cells (VSMCs - the main stromal cells located in the middle layer of the arteries), SSAO has been highlighted as important factor in the oxidation of low-density cholesterol (LDL) and inducer of cell death. Lysyl oxidase (LOX) is another vascular enzyme which contributes to VSMCs extra cellular matrix (ECM) formation and stability. In these cells LOX has been associated with chemotaxis, which may imply a pathological role in the development of atherosclerosis. There is insufficient evidence regarding the interaction of these enzymes in VSMCs, their toxicological consequence, and the mechanism through which they contribute to LDL oxidation and the induction of chemotaxis. This thesis aims to investigate SSAO and LOX catalytic activity in rat aortic VSMCs, and their contributory role in oxidative stress and chemotaxis (important hallmarks in atherosclerosis), through ROS formation and vascular remodelling. Methods: To address this, an in vitro assay was developed and optimised for assessing SSAO and LOX activity in rat cultured aortic VSMCs through H2O2 generation. Toxicity of SSAO substrates and inhibitors was assessed colourimetrically. Cells at passage 3, 5 & 8 and confluency of ~80-90% were treated with the substrates benzylamine and cadaverine, and with LOX and SSAO irreversible suicide inhibitors βAPN and MDL72527. Western blot analysis was utilised to assess the membrane bound forms of the enzymes, vascular adhesion protein 1 (VAP-1) and Pro-LOX, the contractile markers smooth muscle 22α (SM22α) and alpha actin 2 (ACTA2) which indicate VSMCs physiological - contractile phenotype, and the expression of platelet derived growth factor receptor β (PDGFRβ), a receptor implicated in the mechanism of VSMCs chemotaxis. A quantitative polymerase chain reaction (qPCR) was applied to study mRNA levels of the gene that encodes for SSAO, amine oxidase copper containing 3 (AOC3) and LOX (LOX) in the presence of their respective irreversible inhibitors, and after silencing LOX gene. ROS and total glutathione (GSH) content were evaluated with ROS and the recycling GSH assay. Results: SSAO activity was maximal at 6h from addition of reaction mixture. There was a passage dependent increase in SSAO affinity for its substrate benzylamine (**p < 0.01) as shown by a 3-fold reduction in the Km as passage increased from 3 (Km = 0.2084) to 8 (Km = 0.07267). Methylhydrazine was identified as the most potent reversible, and MDL72527 as the most potent irreversible inhibitor. Aminoacetone and methylamine induced VSMCs death at concentrations of 50 & 1000µM, and higher SSAO affinity was detected for aminoacetone comparing to methylamine (Km = 12µM vs 65µM). The cytotoxicity was reversed with MDL72527 which completely abolished cell death. Enhanced cytotoxicity was detected after simultaneous addition of aldehydes and H2O2. VAP-1, Pro-LOX and PDGFRβ were expressed in cultured VSMCs under control conditions with levels maintained between passages. At basal level, LOX activity decreased with passage and its affinity and protein expression were maintained between passages. βAPN abolished LOX activity (**p < 0.01 for 3 vs 8 and *p < 0.05 for 5 vs 8) and had no effect on Pro-LOX expression and LOX mRNA levels; and MDL72527 had no effect on LOX activity, protein, and mRNA expression. At basal level, SSAO activity also decreased with passage and its protein expression was maintained between passages. MDL72527 abolished SSAO activity (****p < 0.0001 for 3 vs 8 and *p < 0.05 for 5 vs 8), VAP-1 expression at passage 5 (**p < 0.01) and passage 8 (****p < 0.0001), and AOC3 mRNA levels at passage 8 (*p < 0.05). βAPN inhibited SSAO activity (****p < 0.0001 for 3 vs 5 and 3 vs 8 and *p < 0.05 for 5 vs 8), VAP-1 expression at passage 3 (*p < 0.05), and AOC3 mRNA levels at passage 3 (*p < 0.05). Benzylamine did not alter VAP-1 and LOX expression and cadaverine reduced VAP-1 at passage 3 (**p < 0.01), passage 5 (*p < 0.05), and passage 8 (**p < 0.01). Loss of contractile markers was observed in non-treated cells and after treatment with MDL72527, cadaverine and benzylamine. PDGFRβ expression was reduced at passage 8 after LOX (*p < 0.05) and passage 3 (*p < 0.05), 5 (*p < 0.05), and 8 (**p < 0.01) after SSAO inhibition. PDGFRβ expression was also reduced at passage 3 (*p < 0.05) after benzylamine and cadaverine treatment. Silencing LOX gene at passage 3 reduced AOC3 mRNA (####p < 0.0001 for SiRNA6 and ###p < 0.001 for SiRNA8) and VAP-1 protein (#p < 0.05 for SiRNA8). Highest ROS production was detected with aminoacetone and benzylamine treatment. MDL72527 abolished ROS in benzylamine (****p < 0.0001), methylamine (****p < 0.0001), and aminoacetone (****p < 0.0001), and βAPN in benzylamine (*p < 0.05) treated cells. Total GSH levels were reduced after benzylamine, methylamine and aminoacetone treatment (****p < 0.0001); however, failed to be restored after addition of MDL72527 and βAPN. Conclusion: A robust method was optimised and validated to effectively characterise SSAO and LOX activity in vitro. A cytotoxic consequence of SSAO catalytic activity was observed in cultured VSMCs and a contributory role of SSAO and LOX was detected in VSMCs chemotaxis, through changes in PDGFRβ expression. A synergistic relationship between SSAO and LOX was identified in early passage VSMCs. In late passage VSMCs, SSAO expressed predominant activity over LOX, and was demonstrated as the leading enzyme in ROS formation. Overall, these novel findings suggest a passage dependent interplay between SSAO and LOX in rat aortic primary VSMCs and associate their activity with VSMCs phenotyping modulation and ROS formation.

Atherosclerosis-related cardiovascular diseases (CVDs) are a major burden on human health. Many studies have reported the central role of dietary nutrients in modulating the inflammatory and oxidative processes involved in the development of atherosclerosis. Selenium (Se) through its incorporation into selenoproteins is an important antioxidant in the body. When dietary Se is suboptimal (i.e. in most European countries) it may be insufficient to maximize cellular selenoenzyme activities leading to lowered protection against oxidative damage and increased risk of oxidative-damage associated diseases including CVD. This study examined the potential for another dietary nutrient, vitamin D, to enhance Se status and to potentially provide protection against oxidative stress and stress-related cellular damage. Vitamin D is a known modulator of inflammation in addition to its classical effects on calcium and bone homeostasis. In the present study, results showed that vitamin D increased selenoenzyme activity within monocyte cells when Se levels were suboptimal. In addition, vitamin D also augmented the reduction in cytokine-stimulated adhesion of monocytes to endothelial cells (ECs), an in vitro model of atherosclerosis. Investigation of the mechanism involved showed there is a reciprocal interaction of vitamin D with Se that acts through regulation of the enzymes 25-hydroxyvitamin D 1-alpha-hydroxylase (CYP27B1) and cystathionine--synthase (CBS). CYP27B1 converts circulating levels of vitamin D form to biologically active vitamin D whereas CBS is required to incorporate organic forms of dietary Se into selenoproteins. Results from this study provide the first evidence to suggest that the impact of suboptimal Se status may be partly alleviated by maintaining adequate vitamin D levels and conversely, Se may act to enhance localized levels and action of active vitamin D. These interactions may have important consequences both for health and for diseases such as atherosclerotic CVDs.

Background: Blood lipids, particularly the cholesterol in low-density lipoproteins (LDL-C), are causally related to atherosclerotic cardiovascular disease. Moreover, adiposity, diabetes, and kidney disease-key cardiovascular risk factors-are all linked to perturbations in blood lipids. However, evidence around blood lipids has mostly relied on simple assays of these biomarkers and primarily comes from studies in high-income countries. Methods: In 1998-2004, the Mexico City Prospective Study (MCPS) recruited 159,755 adults aged >35 years, who have been followed for cause-specific mortality until January 1st 2018. All participants had body measures taken and glycosylated haemoglobin (HbA1c) measured. Among 40,349 of these participants, NMR quantified plasma concentrations of about 100 biomarkers involving lipoprotein particles, their lipidic compositions (cholesterol, triglycerides, and phospholipids), apolipoproteins, and other metabolic measures including creatinine. Linear regression analyses examined the cross-sectional associations of adiposity, of diabetes, and of kidney function with NMR biomarkers in individuals aged 35-84 years at recruitment. Cox regression analyses yielded hazard ratios (HRs) for the associations of NMR biomarkers with premature mortality (before age 75) due to vascular occlusive causes (ischaemic heart disease, ischaemic stroke, or peripheral arterial disease). All analyses excluded the few participants (< 1%) who were using lipid-lowering medications at recruitment. To minimise reverse causation, prospective analyses further excluded those with chronic diseases and deaths in the first 10 years of follow-up. Results: At recruitment, of the 36,527 participants aged 35-84 years included in the main cross-sectional analyses, about three-quarters had a body-mass index of 25 kg/m2 or above, 17% had diabetes, and 1.5% had an estimated glomerular filtration rate (eGFR) below 60 mL/kg/1.73 m2 (consistent with chronic kidney disease). The NMR metabolic biomarkers showed a complex pattern of correlation that reflected their biochemical characteristics and their pathophysiological relationships. These biomarkers were also correlated with a number of socio-demographic and lifestyle characteristics. General, abdominal, and gluteo-femoral adiposity were cross-sectionally associated with an NMR metabolic profile characterised by higher levels of ApoB, of very-low density lipoprotein (VLDL) particles (and their lipids), of fatty acids, of cholines, of branched-chain amino acids, and of the inflammation biomarker Glyc-A. Mutual adjustment of general and abdominal adiposity showed that their associations with NMR biomarkers were largely independent. However, the associations of gluteo-femoral adiposity were reversed after adjustment for general and abdominal adiposity. Similarly, diabetes and HbA1c were associated with a metabolic profile that consisted in higher levels of VLDL, of ApoB, of triglycerides in all lipoproteins, of fatty acids, of branched-chain amino acids, and of Glyc-A. Finally, low kidney function (i.e. an eGFR <60 mL/min1.73m2) was associated with a metabolic profile involving higher levels of ApoB, of the ApoB-containing lipoproteins VLDL and LDL, of the cholesterol, triglycerides, and phospholipids in these lipoproteins, of several fatty acids, and of Glyc-A irrespective of diabetes status. The NMR metabolic profiles of adiposity, diabetes, and kidney function tended to be qualitatively similar in men and women and at younger and older age. In the 27,518 participants without diabetes or other prior chronic disease at recruitment, there were 117 vascular occlusive deaths more than 10 years later and before age 75 years. Biomarkers related to ApoB-carrying lipoproteins (VLDL and LDL), and their lipids, ApoB, fatty acids, total cholines, phosphatidylcholine, sphingomyelin, and glucose were associated with risk of vascular occlusive death. Each 1-SD higher baseline level of ApoB and of glucose were associated with 26% (HR of 1.26 [95% CI 1.04, 1.52]) and 42% (HRs of 1.42 [95% CI 1.17, 1.73]) higher risk of vascular occlusive mortality, respectively. Similarly, the most informative linear combination of the NMR biomarkers (the first 'principal component'), which largely reflected ApoB containing particles and their composition, was significantly associated with risk of vascular occlusive death (HR 1.27, 95% CI 1.04, 1.56). Conclusion: Lipid-lowering therapy is substantially underused in Mexico. Metabolic profiling in this Hispanic population with high levels of obesity demonstrates how adiposity, diabetes, and kidney disease associate with lipid and lipoprotein profiles which are linked to vascular occlusive disease. Future research will extend and combine these metabolomics data with genetic data to explore the causal nature of NMR biomarkers to cause-specific mortality risk in this population.

Atherosclerosis is a progressive and chronic condition that belongs to the group of Cardiovascular Diseases (CVDs) which represents the first cause of death worldwide. Although recent advances in strategies for studying the mechanisms underlying the disease progression have led to better understanding of the pathological phases, there is still a lot that needs to be done in order to have a complete knowledge of the molecular factors implicated in the pathophysiology of atherosclerosis. In particular, considering the complexity of atherosclerosis, understanding the processes involved in the advancement from early to advanced plaque phenotype, constitutes an essential step towards monitoring the disease progression. Noncoding RNAs, including small noncoding RNAs (miRNAs) and long noncoding RNAs (lncRNAs), have been characterised as important molecules involved in the regulation of physiological cellular processes and the pathology of diseases. MiR-143/145 is a conserved microRNA cluster involved in the maintenance of vascular smooth muscle cells (vSMCs), whose dysregulation can affect the vSMC phenotype. This is one of the key cellular components involved in the pathophysiology of vascular disease, and specifically in atherosclerosis. Studies have shown that the dysregulation of miR-143/145 affects vSMC function during vascular injury and repair, leading to vSMC phenotypical transition towards a pro-pathological state. CARMN is a conserved lncRNA host gene for miR-143/145, whose role was not explored in vSMC phenotypic transition. In this project, CARMN axis role, including CARMN and miR-143/145, was explored in dysregulated vSMC in vitro and in vivo during atherosclerosis. Key findings of this study have shown the downregulation of CARMN and miR-143/145 during the advancement from early to advanced plaque, in both human and mouse atherosclerosis. Functional studies performed in vSMC, identified dysregulated pathways, - following CARMN axis depletion, which were mostly involved in the regulation of vSMC proliferation, migration, inflammation, and lipid homeostasis. In addition, the expression of miR-143/145 was affected following CARMN depletion. In this study, CARMN downregulation was identified as primary regulator of vSMC proliferation independently from the regulation of miR-143/145, whereas the dysregulation of miR-143/145 expression was found involved in the modulation of vSMC migration and de-differentiation towards a macrophage-like phenotype. Herein, CARMN axis role was also explored in an atherosclerotic mouse model of the disease. In particular, the genetic depletion of CARMN, and associated miR-143/145, induced a more advanced plaque phenotype characterised by increased plaque volume and area versus wild type controls. In addition, the knock-out of CARMN enhanced the content of pro-inflammatory cells, increased cell proliferation of cells within the plaque and reduced the collagen content compared to wild type mice. Collectively, this study identified CARMN loss as a detrimental key event occurring in early plaques, which primes vSMC to a dysregulated phenotype in vitro, and leading to more advanced lesions in vivo.

Coronary heart disease is responsible for around 2 million deaths across Europe each year. This is largely caused by atherosclerosis, a chronic inflammatory disease of the vasculature characterized by development of plaque in the arterial wall. Advanced plaques may become unstable, carrying a risk of rupture or erosion and manifest as acute myocardial infarction. In patients with type 1 and type 2 myocardial infarction, mortality rates remain high; up to 15% and 25%, respectively. Although risk stratification tools like GRACE 2.0 score can robustly predict outcome in these patients, new therapies targeting the unstable plaque are still needed to address this global epidemic. Long noncoding RNAs (lncRNAs) are an emergent class of molecules with diverse functional roles, widely expressed in human physiology and disease. Although some lncRNAs have already been identified in cardiovascular disease, their potential as novel targets in the prevention of atherosclerosis remains unknown. The aim of this project was to discover important lncRNAs in unstable plaque and gain insight into their functional relevance. Analysis of RNA sequencing previously performed on stable and unstable atherosclerotic plaque identified a panel of 47 differentially regulated lncRNAs. I focused on LINC01272, a lncRNA upregulated in unstable plaque previously detected in inflammatory bowel disease, and ultimately renamed it PELATON (plaque enriched lncRNA in atherosclerotic and inflammatory bowel macrophage regulation). PELATON is highly monocyte- and macrophage-enriched across vascular cell types, and almost entirely nuclear by cellular fractionation (90%-98%). In situ hybridization confirmed enrichment of PELATON in areas of plaque inflammation, co-localising with macrophages around the shoulders and necrotic core of human plaque sections. Consistent with its nuclear localization, and despite containing a predicted open reading frame, PELATON did not demonstrate any protein-coding potential in vitro. Functionally, knockdown of PELATON significantly reduced phagocytosis, lipid uptake and reactive oxygen species production in high- content analysis, with a significant reduction in phagocytosis independently validated. Furthermore, CD36, a key mediator of phagocytic oxLDL (oxidized low-density lipoprotein) uptake was significantly reduced with PELATON knockdown. Taken together, these data demonstrate that a novel lncRNA (PELATON) is important to macrophage function in atherosclerotic plaque. Future work should focus on manipulation of PELATON, to potentially reduce plaque instability, and prevent cardiovascular events.

Background: Stroke patients are at high risk of dementia. While cardiovascular comorbidities, such as hyperlipidaemia and atherosclerosis, have been linked to dementia in the general population, it is unclear whether the associations persist in stroke patients, who usually suffer more cardiovascular comorbidities. Statins have been recommended for secondary prevention in stroke patients, but it is unclear whether they have any cognitive effects separate from reducing risk of vascular events. Aim: To triangulate the potential roles of major blood lipids, atherosclerosis and statins in the development of dementia after stroke. Methods: First, I conducted a systematic review to identify the gaps in previous studies on the association of the three factors with post-stroke dementia. In response to the limitations of the previous studies, I conducted three cohort studies using the Clinical Practice Research Datalink (CPRD) for blood lipids, atherosclerosis and statin use, respectively. Given the limitations in the CPRD data, I further used UK Biobank to explore whether the CPRD results could be replicated using similar analytic strategies. Results: My systematic review suggested that hyperlipidaemia was not associated with post-stroke dementia, atherosclerosis was associated with increased risk of post-stroke dementia and post-stroke statin use may reduce the risk. Only one small-scale trial was available, and all other studies were observational with key methodological limitations. My CPRD study on major blood lipids showed that low-density lipoprotein (LDL) cholesterol was associated with increased risk of post-stroke dementia (adjusted hazard ratio [aHR] 1.29, 95% confidence interval [CI] 1.14-1.47, per one log-mmol/L increase), with a linear increasing trend in the risk across quintiles and different guideline targets of LDL cholesterol (all P-trend <0.001). Conversely, triglycerides were associated with decreased risk of dementia (aHR 0.79, 95% CI 0.70-0.90, per one log-mmol/L increase), with a linear decreasing trend in the risk across quintiles of triglycerides (P-trend <0.001). No significant association was found for high-density lipoprotein (HDL) cholesterol. My CPRD study on atherosclerosis suggested that stroke patients with prior atherosclerotic cardiovascular disease (ASCVD) were more likely to develop subsequent dementia (aHR 1.18, 95% CI 1.12-1.25). After full adjustment for potential baseline confounding, however, the risk of post-stroke dementia is attenuated (aHR 1.07, 95% CI 1.00-1.13), with no linear relationship between the age of ASCVD onset or duration of pre-stroke ASCVD and post-stroke dementia (all P-trend >0.05). My CPRD study on statin use found that statin initiation within the first three months after stroke was associated with lower risk of dementia (aHR 0.70, 95% CI 0.64-0.75). After accounting for non-persistence, the risk was further decreased (aHR 0.55, 95% CI 0.50-0.62). My UK Biobank study supported the CPRD findings. The observed associations tended to be stronger than from the CPRD, while mostly not statistically significant due to low incidence of post-stroke dementia in the younger volunteer population in UK Biobank. Conclusions: Blood lipids may affect the risk of post-stroke dementia in different ways, with higher risk associated with LDL cholesterol, lower risk associated with triglycerides, and no association with HDL cholesterol. Stroke patients with prior ASCVD were more likely to develop subsequent dementia but the direct impact of pre-stroke atherosclerosis on the occurrence of post-stroke dementia may not be substantial. Statins have the potential to reduce the risk of dementia over and above future vascular events. Further trials and more real-world studies are needed to confirm the potential benefits of statins, to determine the optimal target of LDL cholesterol and to clarify the roles of triglycerides in the prevention of dementia.

Atherosclerosis is a widespread disease that often leads to serious conditions such as myocardial infarction and stroke. Chronic inflammation plays an overarching role in its pathophysiology, driving initiation and progression of atherosclerosis in humans and animal models. IL-1β is a potent inflammatory cytokine implicated in atherogenesis and other vascular diseases. However, control of its activation and release has been predominantly studied in macrophages, with scant data on whether vascular smooth muscle cells (VSMCs) can release IL-1β. I investigated the expression, activation and release of IL-1β from primary human VSMCs. VSMCs expressed caspase-1, -4, and -8 under basal conditions, but not caspase-5. Stimulation of VSMCs with IL-1α and/or LPS caused rapid processing of IL-1β to the mature form, which could be detected by Western and with a mature IL-1β-specific ELISA. Although IL-1β production and cleavage was dependent on NF-κβ activation, neither caspase or NLRP3 inflammasome inhibitors prevented generation of mature IL-1β, suggesting caspase- and NLRP3-independent processing. Instead, production of mature IL-1β was blocked by the serin protease inhibitor dichloroisocoumarin, with cathepsin G the most likely enzyme responsible for pro-IL-1β activation. Interestingly, mature IL-1β was never detected in the conditioned media and instead accumulated to high levels inside VSMCs. VSMCs expressed the pore-forming protein Gasdermin D, and the active form of Gasdermin D permeabilised VSMCs, yet mature IL-1β was still not released. Finally, mature IL-1β extracted from VSMCs has 13-fold lower activity than equal amounts of mature IL-1β from Thp1 cells, however the existence of a binding partner or inhibitor of IL-1β is unlikely the cause of this phenomenon. In conclusion, the work presented in this thesis suggests that VSMCs have a novel mechanism that specifically prevents the release of active IL-1β, and that perturbation of this could potentially contribute to vascular inflammation and atherogenesis.

This thesis explores the use of susceptibility-weighted imaging (SWI) and quantitative susceptibility mapping (QSM), to characterize carotid artery plaques with and without the use of ultrasmall superparamagnetic iron oxide (USPIO) nanoparticle contrast agents. The overall hypothesis is that QSM can serve to differentiate carotid artery plaque features of different susceptibility and provide a positive contrast mechanism for imaging the uptake of USPIOs. Chapter 1 describes the pathophysiology of carotid atherosclerosis. Vulnerable plaques, i.e. those at risk of rupture, can be characterized by the presence of a lipid rich necrotic core (LRNC), intraplaque haemorrhage (IPH), and inflammation. In addition, plaques may develop calcifications that may be protective of rupture. The chapter describes the established multi-contrast imaging protocols used for characterizing plaques. Furthermore, the use of USPIO-contrast agents to image inflammation is described. Chapter 2 describes the physical principles of MR image generation including the sensitivity to magnetic susceptibility. The principles of T2*w imaging, and susceptibility weighted imaging (SWI) are explained. Chapter 3 reviews the principles and post-processing steps involved in commonly used algorithms for QSM in terms of the underlying physical and mathematical principles which are then demonstrated in the form of numerical simulations. Chapter 4 presents the application of SWI to a group of patients who underwent USPIO enhanced MRI on a 1.5T MRI system. Images were acquired prior to infusion and 48 hours post infusion. SWI and gradient echo phase images were used to depict the field inhomogeneities generated by diamagnetic and paramagnetic materials within the plaques, calcification and USPIO-uptake. These results were then compared to a conventional carotid multi-contrast protocol, which includes R2*-mapping and T2*w imaging, and, where available, CT and histology. In chapter 5 QSM is performed in the carotid artery wall of a cohort of normal volunteers on a 1.5T MRI system. Unlike the brain, the neck contains fat which can cause severe errors in the field estimate, which propagate into the susceptibility map. Therefore, QSM was combined with water-fat separation for application in the neck to correct for these artifacts. This correctly estimated a high fat-fraction in fatty tissue in the neck and allowed for a detailed depiction of the anatomy of healthy volunteers. The susceptibility value measured in fatty tissue agreed with literature values. Chapter 6 applies QSM with water-fat separation to a subset of the patient group on a 1.5T MRI system. On pre-contrast scans QSM successfully identified calcification as diamagnetic tissue and the water-fat separation identified a lipid core. On the post-contrast susceptibility maps, USPIO-uptake was identified as hyperintense signal. This allows QSM to provide quantitative contrast in carotid imaging that can identify multiple features simultaneously and to simplify the imaging of USPIO-contrast. The results were confirmed using the multi-contrast carotid MRI protocol and, where available, histology and CT. Chapter 7 discusses the limitations of the current studies and the potential future improvements of the current methodology in terms of MR acquisition, post-processing algorithms and MR protocols. Future studies could serve to further evaluate the potential of QSM in carotid imaging and use it as a novel tool to quantify USPIO uptake in atherosclerotic carotid arteries.

Background: Cardiovascular disease (CVD) is still the leading cause of death in the developed as well as developing country. Atherosclerosis and neointima formation considered as chronic inflammatory diseases, in which many cells including inflammatory smooth muscle cell (iSMC) were involved. ISMC differentiation from vascular adventitia stem/progenitor cells (AdSPCs) has been recently recognised as a critical determinant in cardiovascular diseases. One study have recently reported an important role for microRNA-214 (miR-214) in mature vascular SMC functions and injury-induced neointima formation. However, little is known about the functional involvements of miR-214 in iSMC differentiation from AdSPCs and its contribution to adverse arterial remodelling. Purpose: In this PhD study, I aimed to study the functional importance of miR-214 and its target gene in iSMC differentiation from AdSPCs and neointima SMC hyperplasia. Results: miR-214 expression was significantly increased during SMC differentiation from AdSPCs in response to TGFβ1. miR-214 gain/loss-offunction assays showed that miR-214 plays an important role in SMC differentiation from AdSPCs. By co-incubating AdSPCs with TGFβ1 and TNFα, AdSPCs were induced to differentiate toward iSMCs as evident by a decreased expression level of SMC-specific genes, but an increased level of inflammatory genes in iSMCs. Importantly, such phenotype could be reverted by miR-214 over-expression. I observed that sonic hedgehogglioma- associated oncogene 1 (Shh-GLI1) signal was closely modulated during iSMC differentiation, and demonstrated that Suppressor of Fused (SuFu) is the functional target of miR-214 controlling iSMC generation from AdSPCs. Further mechanistic studies revealed that increased amount of the GLI1 protein was translocalized into nuclei in miR-214 over-expressing or 6 SuFu knockdown cells, and the consensus sequence (GACCACCCA) of GLI1 within gene promoters of smooth muscle alpha-actin (SMαA) and serum response factor (SRF) was required for their regulation by miR-214 and SuFu. Additionally, Sufu also functions as a positive regulator for inflammatory gene regulations in iSMCs. In vivo, I demonstrated that locally enforced expression of miR-214 in the injured vessels significantly reduced SuFu expression level, iSMC generation, and inhibited neointima SMC hyperplasia after injury. Importantly, I observed that perivascularly transplanted AdSPCs significant contribute to neointima SMC hyperplasia by differentiating to iSMCs, and miR-214 over-expression could reverse such an effect. Finally, a decreased expression level of miR-214, but increased expression level of Sufu was observed in diseased human arteries. Conclusions/Implications: This thesis reports an unexplored role for miR- 214 in iSMC differentiation from AdSPCs and controlling neointima iSMC hyperplasia. These findings provide new insights into the therapeutic effect of miR-214 in vascular diseases.

Deregulation of vascular smooth muscle cell (VSMC) phenotypic switching is implicated in vascular disease development and VSMCs produce the majority of cells in late-stage atherosclerotic plaques. However, VSMC accumulation in mouse models of vascular disease arises from clonal expansion of very few medial cells. The source of these cells and mechanism of their selection are unknown. Hypothesising that VSMCs which expand in disease would be distinctive within the healthy population, single cell RNA sequencing (scRNA-seq) was combined with mouse VSMC-specific lineage tracing to profile VSMC transcriptomes from healthy aorta and two in vivo disease models (CL – carotid ligation and HFD – high-fat diet-induced atherosclerosis). This identified VSMCs expressing stem cell antigen-1 (Sca1), which lacked expression of conventional VSMC markers, but were also distinct from adventitial and endothelial cell profiles. Induction of Sca1 was evident in phenotypically switched VSMCs in vitro and in both in vivo disease models. Notably, Sca1+ VSMCs were a rare subset in the healthy aorta, but more prevalent in the two disease datasets and expressed an activated, responsive gene signature in all three environments. This gene signature contains known regulators of plaque progression; suggestive that they may be candidates for those cells selectively proliferating in disease. Focusing on VSMCs from CL, the scRNA-seq dataset lacked discrete clusters of gene expression. Instead a transition in expression profiles could be seen, allowing for cells to be mapped onto a linear trajectory, using an unbiased approach. This trajectory showed contractile marker downregulation over pseudotime, corresponding to increased expression of Sca1 and proliferation marker Ki67. Moreover, Ki67+Sca1+ VSMCs were identified post-CL via flow cytometry, indicating that Sca1+ VSMCs indeed expand in disease. Investigation of candidate genes with differential expression across this pseudotime revealed enrichment for VSMC activation and disease-relevant gene ontology pathways. Further disease relevance of these candidates was highlighted using immunostaining of mouse and human plaque sections and comparison to other scRNA-seq datasets. Finally, a lineage traced tissue explant model was developed to study clonal VSMC proliferation in vitro. Traditional dissociated primary cell culture causes spontaneous VSMC phenotypic switching and general proliferation, which is not representative of the in vivo VSMC response. The explant model presented here maintains cell-cell and cell-extracellular matrix contact, resulting in limited proliferation of VSMCs and formation of monoclonal patches of VSMCs, comparable to those seen in vivo. Additionally, the model replicated published observations of differences in proliferation of VSMCs derived from two distinct aortic regions and increased proliferation with growth factor treatment. Lentiviral transduction of the explanted VSMCs allowed for genetic manipulation, providing a platform to test the influence of individual genes on VSMC clonal expansion. In summary, this work identified a rare Sca1+ VSMC population which may represent a source of clonally expanding VSMCs. A comprehensive resource of transcriptional data from healthy and disease associated VSMCs has been generated and interrogation revealed commonality in activated Sca1+ VSMC expression profiles. Further investigation demonstrated a trajectory of gene expression, implicating many disease-relevant genes in VSMC activation. When combined with the in vitro lineage labelled model of VSMC proliferation, this provides a basis for identification and screening of genes involved in clonal expansion, with the ultimate aim to isolate or selectively target specific detrimental VSMC subpopulations.

Inflammation plays a key role in the disease processes of both atherosclerosis and myocardial infarction (MI). Regulatory T (Treg) cells are a subset of the adaptive immune system involved in immune tolerance. Animal models show that increasing Treg cell numbers in atherosclerosis can limit disease progression. Several small clinical studies have used low dose interleukin-2 (ld-IL-2) to increase Treg cells in patients with various autoimmune but not cardiovascular diseases. Therefore, a phase 1/2a, randomised, placebo-controlled, double-blind, dose-escalation, two-part clinical trial was performed to test ld-IL-2 (aldesleukin) given once daily subcutaneously, for five consecutive days. In Part A, 25 patients with stable ischaemic heart disease were treated in 5 dose groups (0.3, 0.6, 1.2, 2.4 and 3 x106 IU/day); whilst in Part B, 16 patients with acute coronary syndrome were treated in two dose groups (1.5 and 2.5 x106 IU/day). Results showed that ld-IL-2 was safe and well tolerated across the dose range. The 1.5 x106 IU/day dose increased Treg cell levels by the pre-specified 75% and did not increase effector T cells numbers. There were additional novel findings in other immune cells and cytokine markers. B cell subsets in animal models worsen post-MI inflammation and remodelling. Antibody mediated depletion of B cells in mice limited myocardial injury and improved cardiac function. Rituximab is a monoclonal antibody targeted against human B cells and has been used in the treatment of certain autoimmune diseases and cancers. However, its use in cardiovascular disease is untested and is currently contraindicated. Therefore, a phase 1/2a, open-label, dose-escalation, single-arm, clinical trial was set up to test rituximab in patients with ST-elevation MI. In total, 24 patients were treated in 4 escalating dose groups (200, 500, 700 and 1000 mg). Rituximab was administrated intravenously as a single dose within 48hours of symptoms onset. Rituximab was well tolerated at all doses. Rituximab caused rapid and significant depletion of B cells within 30 minutes of starting the infusion which suggests its use in acute MI is biologically plausible. At 6 months B cell repopulation occurred in a dose-dependent manner. In addition, several new insights into the pharmacodynamics of rituximab were gained. These results have paved the way for future phase 2b trials, one of which is underway.

Atherosclerosis and plaque rupture are widely known as multifactorial problems. To isolate the significance of wall shear stress in these problems, the work of this thesis explores the hypothesis that the frequency composition of the wall shear stress signal is associated with the different plaque compositions and disease characteristics in animal models of atherosclerosis. In this thesis, a lattice Boltzmann simulation tool was developed to test the hypothesis with the basic functionality of an existing code being enhanced for blood flow simulation and wall shear stress calculation. The wall shear stress signals computed from the simulation tool were analysed in terms of the frequency composition to recover the harmonic amplitude and phase information. This information was then used in comparing the different animal models. Compared to the healthy, non-diseased vessel, disease models are known to result in a decrease in the time-averaged wall shear stress from the reduction in blood flow rate and local complex flow patterns. Further to this, the simulation of these models showed a decrease in the first harmonic amplitude along the length of the vessel. This is a key result of this thesis as the decreased first harmonic amplitude is associated with an increase in the expression of adhesion molecules and proinflammatory factors in endothelial cells. The uniformity in wall shear stress in regions of different plaque type, however, suggests the dominance of circumferential stretch effects over wall shear stress effects in the disease process. Blood flow simulations in the mouse, rabbit and human vessels were also performed to deduce scaling relationships of the zeroth and first harmonic amplitudes between mammals. The body mass exponent of the first harmonic amplitude was found to be higher than that of the zeroth harmonic amplitude. This suggests an increased significance of the first harmonic component in the wall shear stress signal relative to the zeroth harmonic amplitude in larger mammals. The absence of plaque rupture in the atherosclerotic minipig, however, also suggests the dominance of genomic effects over wall shear stress effects in the disease process. A key issue in atherosclerosis research is the absence of plaque rupture in the mouse model. The apparent dominance of circumferential stretch and genomic differences shown here suggests that the wall shear stress alone cannot explain the lack of plaque rupture in atherosclerotic mice. How these differences affect plaque composition would be key in understanding the absence of plaque rupture in mouse models and how studies in mice can be applied to benefit human treatments.

Degenerative abdominal aortic aneurysms (AAA) are characterised by aortic smooth muscle apoptosis, elastin degradation and a chronic inflammatory cell infiltrate. The inciting antigen is yet unknown. AAAs share many features with autoimmune conditions whilst overlapping several risk factors with atherosclerosis. The majority of AAAs of an operable size are lined with intraluminal thrombus (ILT), which is in constant contact with the aortic wall and flowing arterial blood. Flow mediated dilatation (FMD) is a surrogate marker for endothelial dysfunction and a predictor of future cardiovascular events. Patients with AAAs have a deteriorating FMD, which improves after surgery, thus implicating the aneurysm-thrombus complex as a potential driver of the systemic endothelial dysfunction. The immunological contribution of ILT to the pathology is under investigated. This thesis aims to comprehensively define the immune landscape of the aortic wall and intraluminal thrombus through deep phenotyping, thus providing new insights into the mechanisms of degeneration in advanced stage aneurysms. Furthermore, it aims to explore a mechanistic link between ILT and systemic endothelial dysfunction. Patients were prospectively recruited on to the Oxford Abdominal Aortic Aneurysm study. Peripheral blood, aortic wall and ILT specimens were collected intraoperatively and optimised for deep phenotyping with Time-of-flight mass cytometry (CyTOF). Cell sorting the aortic complex tissues provided high quality single cell suspensions for discovery analyses using different approaches. The aortic wall was replete with T and B cells demonstrating new evidence of T and B cell crosstalk. In particular, T follicular helper cells were identified, thus providing evidence for class-switching to memory B cells in the aortic wall. The aortic and ILT lymphocytes contained the cytotoxic granule Granzyme B. This is a novel alternate mechanism of tissue destruction in late stage disease and its finding in the peripheral blood suggests a role as a potential biomarker. The myeloid cells had significant overlap in their markers of expression and the ILT also contained active monocyte derived dendritic cells, which is another novel finding. Both the T cells and myeloid cells had no dominant effector phenotype and rather represented a complex continuum aiming to achieve homeostatic balance. Finally, the ILT secretome was found to be cytotoxic to aortic endothelial cells and induced changes in gene expression consistent with endothelial dysfunction. This thesis defines in detail the immune landscape of the aortic aneurysm-thrombus complex. It highlights the involvement of both innate and adaptive immune systems, as well as pro- and anti-inflammatory pathways involved in chronic non-resolving inflammation. The finding that ILT induces gene changes of endothelial dysfunction provides a novel mechanistic link for the changes in FMD of patients with AAA. These observations highlight potential targets for translational therapeutics.

37 million people are currently living with HIV worldwide. With the development of Antiretroviral Therapy (ART) AIDS mortality has reduced, with cardiovascular disease (CVD) now the leading cause of HIV deaths. HIV associated CVD has been linked to chronic immune activation through the association of inflammatory biomarkers, clinical events and asymptomatic atherosclerosis; even from a young age. Microparticles (MPs), markers of cellular activation and injury, are also elevated in adults with HIV, and demonstrate the capacity to contribute to inflammation, endothelial dysfunction and coagulation. Monocytes play a key role in atherosclerosis initiation and display an activated, pro-inflammatory phenotype in paediatric HIV. Monocytic MPs (MMPS) are elevated in adults with HIV, thus the aim of this thesis was to quantify MMPS in HIV infected children and investigate their effects on monocytic function. A novel negative isolation method was first optimised to enable the study of human monocyte function 'ex vivo'. This isolation procedure allowed the negative enrichment of all three circulating monocyte subsets (Classical, Non-classical and Intermediate), with high purity and minimal activation. Extracted monocytes displayed minimal alterations in marker expression while retaining their phagocytic, migratory and cytokine secretion function 'ex vivo'. Using a monocytic cell line (THP-1), MP release under different stimulatory and apoptotic conditions was investigated. Under stimulatory conditions with cytokines and a calcium ionophore (A23187), MPs were released in a concentration-dependent manner, with endotoxin stimulation resulting in the highest quantity. Furthermore, MPs displayed a similar trend in surface marker expression in comparison to their parent cell. Next, we investigated the effects of THP-1 derived MMPS on human monocyte function employing the isolation method established. MMPS evoked the release of pro-inflammatory cytokines in addition to enhancing CD11b expression, adherence and transendothelial migration. This work demonstrates that MMPS activates monocytes and enhances endothelial migration, presenting a mechanism that contributes to atherosclerosis pathogenesis. Finally, MMPS and MPs from an endothelial, platelet and T cell origin were enumerated in children with HIV infection pre/post ART initiation and healthy sex-age-matched controls. Plasma samples were collected during the CHAPAS-3 CV sub-study, from which we analysed 16 children receiving ART (≥2 years), 11 treatment-naïve children and 15 controls. Circulating MPs from an endothelial, platelet and T cell origin found in children with HIV infection normalised to levels found in healthy controls, following ART initiation and experience. MMPS were elevated within the treatment-naïve cohort and remained elevated despite treatment intervention; this elevation was also observed in the treatment-experienced cohort. The persistence of elevated MMPS indicates consistent monocyte activation despite successful viral suppression, supporting the activated phenotype reported in literature. This data combined with the functional implications of MMPS on monocytic function described in this thesis elucidates a mechanism that contributes to elevated vascular inflammation reported within this population.

Atherosclerosis is the leading cause of death in the Western world. Although mitochondrial DNA (mtDNA) damage has been implicated in atherosclerosis, it is unclear whether the damage is sufficient to impair mitochondrial respiration, and mitochondrial dysfunction has not been demonstrated. Treatment of vascular smooth muscle cells (VSMCs) with an atherogenic lipid, oxidised low-density lipoprotein (OxLDL), dose dependently decreased basal and maximal respiration and fat-feeding of apolipoprotein E deficient (ApoE-/-) mice reduced mitochondrial DNA copy number relative to nuclear DNA in aortas. Mitochondrial respiration of ApoE-/- mouse aortas, assessed through a 24-well Seahorse extracellular flux analyser, was not affected prior to the development of atherosclerotic plaques. Developed human carotid atherosclerotic plaques were dissected into defined regions including healthy media, shoulder region, fibrous cap and core and their respiration was investigated. The respiratory reserve capacity (RRC) of the shoulder region was similar to the media. However, the cap RRC was significantly reduced compared to healthy media. In contrast, the extracellular acidification rates (ECAR) of the media, shoulder, cap and core regions were similar. In addition, mtDNA copy number was significantly reduced in tissues derived from human plaques compared to healthy arteries and expression of complexes I and II of the electron transfer chain (ETC) were significantly reduced in plaque VSMCs. OxLDL induced mitophagy in human VSMCs and plaque VSMCs demonstrated increased levels of mitophagy without compensatory upregulation of proteins involved in mitochondrial biogenesis. Understanding the role of mitochondrial metabolism and signalling is important for our understanding of disease progression and may lead to future therapeutic targets.

Stroke remains the most common cause of long-term disability with approximately two-thirds of ischaemic strokes resulting from underlying carotid atherosclerotic disease. The clinical management of carotid artery disease relies primarily on the evaluation of the severity of carotid artery luminal stenosis. However, there is growing evidence that morphological and functional information about carotid plaques may also be crucial for the assessment of the severity of atherosclerosis. High-resolution magnetic resonance (MR) imaging is an emerging imaging technique, which has shown great promise in providing detailed information about plaque pathobiology besides luminal stenosis. Using a special contrast medium called ultrasmall superparamagnetic particles of iron oxide (USPIO), MR imaging can also help in the assessment of carotid plaque inflammation. In this dissertation, I explore the different MR imaging modalities for the assessment of morphological, biomechanical and functional characteristics of the atherosclerotic plaques in different patient cohorts, to assess the efficacy of these techniques in identifying high-risk atherosclerotic plaques. It is important to identify these individuals with vulnerable plaques and are the risk of future cerebrovascular events so that they receive optimal medical therapy and timely surgical intervention. Overall the results of this dissertation provide strong support for the use of functional MR imaging techniques i.e. USPIO-enhanced and Dynamic contrast-enhanced MR imaging for detailed morphological and functional assessment of plaques. This may help refine risk stratification especially in subgroups of patients with asymptomatic carotid disease. This may assist in in improving our standing of the pathogenesis of cardiovascular disease in different patient cohorts and help to determine disease severity and prognosis, as well as providing a biomarker to assess the efficacy of established or novel therapeutic interventions.

The total number of annual deaths caused by cardiovascular diseases (CVDs), aortic aneurysms (AAs) and aortic dissections (ADs) is more than from any other cause. Most CVDs are a result of atherosclerosis, in which a fibrous cap covered lipid-rich plaque is formed in the arteries. The fibrous cap can rupture, blocking the artery and limiting the flow of blood to vital organs. AAs and ADs manifest as localised dilatations of the aorta, while simultaneously weakening the aortic wall. In atherosclerosis, AAs and ADs disruptions of the fibrous network and incorporation of microstructural defects make the artery vulnerable to rupture. This dissertation is aimed at describing the relationship between microstructure and mechanical, including fracture behaviour of arterial tissue. Unnotched and notched mechanical tests were performed on tissue samples from aneurysm-affected aortas. Mechanical parameters of ultimate material strength and extreme extensibility were measured, showing arterial layer and direction dependent differences in both unnotched and notched test. Similar differences were observed in fracture parameters of J-integral and crack tip curvature at failure. Interestingly, aneurysmal tissues were found to be notch-insensitive as the notched samples did not fail at a low stretch level. Histopathological analysis was performed on the mechanically tested tissues to investigate the influence of collagen, elastin, macrophage and glycosaminoglycan (GAG) contents, as well as collagen fibre dispersion on the mechanical and fracture behaviours. Both collagen and GAGs were associated with tissue strength, while higher GAG deposition was found to result in larger local collagen fibre dispersion in media and adventitia layers, but not in the intima. Stress-stretch behaviour of healthy, aneurysmal and atherosclerotic tissues were characterised with the modified Mooney-Rivlin constitutive material model. Curve fitting was used to compute the corresponding values of the model constants for each tissue type. Fitted material constants were found to differ amongst distinct types of tissue, while histological analyses showed that material constants were associated with waviness and dispersion of collagen fibres. To study the stretch driven microstructural reorganisations of the fibrous network, tensile tests with unnotched and notched tissue strips from porcine carotid arteries were performed while imaging with multiphoton microscopy. In general, fibres rotated towards the direction of stretch, where in notched samples, fibres rearranged themselves to redistribute loads away from the notch tip. Finally, a complete microstructural component based finite element model of the arterial tissue was developed, which consisted of the collagen fibres and the other structural entities such as elastin and GAGs. The collagen fibres were modelled as discrete entities within the model, where all the other structural entities were modelled in the form of ground matrix. The developed model was used to study the influence of distinct microstructural parameters on the mechanical behaviour of arterial tissues.

Introduction. Calcific aortic valve disease (CAVD) and atherosclerosis are important public health problems. Our ability to allocate patients to treatment strategies optimally and monitor progression is limited. Hybrid positron emission and computed tomography (PET/CT) is able to demonstrate both anatomy (CT) and molecular processes (PET) in vivo. The 18F-sodium fluoride (18F-fluoride) isotope has shown promise in early cardiovascular studies. The purpose of this thesis was to elucidate the mechanisms of cardiovascular 18Ffluoride uptake and define how 18F-fluoride PET/CT might play a role in the assessment of CAVD and atherosclerosis. Methods. Three cohorts were recruited constituting patients with: CAVD, coronary atherosclerosis and carotid atherosclerosis. Using in vitro, ex vivo and in vivo techniques, a model of the mechanism of 18F-fluoride uptake and its pharmacology was elaborated. In observational clinical studies, 18F-fluoride and 18F-FDG uptake in aortic valvular, coronary and carotid arterial tissue was assessed. Techniques for uptake quantification were evaluated for accuracy and reliability. Tissue uptake was related to established clinical and image-based variables as well as prospectively gathered clinical outcome data. Results. In the CAVD cohort, 121 volunteers were recruited. 18F-fluoride PET/CT correlated with tissue markers of active calcification and predicted the genesis of new areas of calcification within the aortic valve. 18F-fluoride uptake was associated with disease progression and clinical events. In the coronary cohort, 80 volunteers were recruited. In patients with acute myocardial infarction the highest coronary 18F-fluoride uptake was seen in the culprit plaque (median maximum tissue-to-background ratio: culprit 1·66 [IQR 1·40-2·25] versus highest non-culprit 1·24 [1·06-1·38], p < 0·0001). In patients with stable angina, plaques with focal 18F-fluoride uptake were associated with more high-risk features on intravascular ultrasound than those without uptake. In the carotid cohort, 38 volunteers were recruited (26 +12, separate studies). 18F-fluoride plaque uptake was associated with neurovascular symptoms (log10 mean standardized uptake value 0.29±0.10 versus 0.23±0.11, P=0.001) as well as image and tissue derived features of high-risk plaque. 18F-fluoride selectively highlighted areas of pathologically high risk nascent microcalcification and was proven to have pharmacological properties highly favourable for PET imaging. Conclusions. 18F-fluoride PET/CT is a valuable tool for exploring pathobiology in CAVD and atherosclerosis may represent an attractive method for assessing response to novel therapies.

Insulin resistance (IR) and dysfunctional adipose tissue (AT) secretome are believed to mediate the mechanistic links between metabolic and vascular disease. However, the mechanistic details of the interactions between vascular IR, AT-derived products and vascular disease phenotypes in humans are poorly understood. In the first part of my thesis, I attempted to characterise downstream vascular insulin signalling in patients with atherosclerosis and how this could be modified to regulate vascular insulin sensitivity. In the second part of this work, I explored the role of AT-derived Wnt5a (a novel, AT-secreted glycoprotein with inadequately characterised vascular roles) as a link between metabolic and vascular disease via direct regulation of vascular redox state and endothelial function. For these purposes, I used the Oxford cohort for Fat, Vessels and Fat (OxHVF), a uniquely phenotyped cohort of >1,000 patients undergoing cardiac surgery) to perform observational association studies and functional ex vivo bioassays on intact vascular tissues, which were also complemented by appropriate in vitro mechanistic experiments. I firstly demonstrated that vascular IR is a universal feature of human atherosclerosis irrespectively of the presence of systemic IR, characterised by disproportionally increased Erk1&2 activation as opposed to Akt activation in response to insulin. This can be reversed by pre-treatment with a dipeptidyl peptidase 4 (DD4) inhibitor, thus identifying vascular insulin-sensitising roles for this class of drugs. In the second part of my thesis, I revealed that systemic Wnt5a bioavailability is increased in obesity, diabetes and vascular disease, while Wnt5a (partially originating from AT) can induce vascular NADPH-oxidase activation via a novel USP17 interaction through paracrine and endocrine ways, also accompanied by endothelial dysfunction, eNOS uncoupling and redox-sensitive VSMC phenotype changes. My findings provide novel links between obesity, insulin resistance and vascular disease mechanisms and pose significant therapeutic implications.

Cardiovascular disease is a severe health problem, especially in the Western world. Its primary cause is atherosclerosis, which is characterized by the arterial wall thickening. Modern therapeutic strategies have restricted efficacy and the mortality still remains high. Current research has supported the idea of targeting dysregulated endothelial cell (EC) metabolism as a novel therapeutic strategy. In the scope of this PhD research work, we aim to further explore the possibilities for an improved treatment of this life threatening disease. EC glycolytic flux is up-regulated during angiogenesis and it is controlled by 6-phosphofructo2-kinase/fructose-2,6-bisphosphatase (PFKFB3), which is hence an innovative target for atherosclerosis therapy. PFKFB3 is a homodimeric bifunctional enzyme that has a very high kinase to phosphatase activity ratio. Its activity is controlled by the N-terminus autoregulatory domain in the kinase region. The main task of this research work was to explore the alternatives for PFKFB3 modulation apart from potentially problematic ATP kinase inhibition. Virtual screening was performed on the targeted allosteric binding site and here we present the synthesis and biological evaluation of the selected libraries of PFKFB3 allosteric phosphatase modulators deriving from two design strategies. In vitro activity measurement and binding assays were performed on the isolated recombinant enzyme. A phosphatase activity measurement method was developed inhouse using LC-MS and the binding assay was performed using microscale thermophoresis. Three peptides (HM-20-22) were found to be able to bind PFKFB3 with a micromolar affinity. HM-21 and 22 were able modulate the PFKFB3 phosphatase activity and interestingly showed the same overall effect on the enzyme kinetics as in the case of a very potent ATP competitive inhibitor. The outcome of the research work presented here suggests that it is possible to use an alternative approach in blocking PFKFB3 activity without inhibiting the kinase. This intriguing discovery could have a significant impact on further research about this important metabolic target and the possibility of its use in atherosclerosis therapy.

Several types of cells actively involved in atherosclerosis undergo a metabolic reprogramming that comprises an accelerated glycolytic flux to the detriment of mitochondrial oxidative phosphorylation. This phenomenon, known as the Warburg effect, allows cells to produce ample energy and biomass with negligible oxygen consumption, resulting in uncontrolled high-speed proliferation. This phenotypic dysfunction underlies both inflammation and angiogenesis, two processes that are foundation of the pathological behaviour of the plaque and promote its destabilisation. The preponderant kinase domain of inducible PFKFB3 enzyme catalyses the synthesis of F2,6P, which simultaneously promotes glycolysis and inhibits gluconeogenesis. PFKFB3 has been found to be one of the most abundant and overly expressed isoenzymes in the Warburg effect, suggesting its key role in the pathology. From a clinical perspective, PFKFB3 represents an emerging biological target, and its inhibition would be an innovative therapeutic strategy for the treatment of atherosclerotic lesions. Our study revolves around the identification of new PFKFB3 kinase inhibitors. The rational design was carried out with the aid of computational methods, based on the 3D-structures of both PFKFB3 protein and known active ligands. In particular, two classes of inhibitors were used as reference ligands for the development of two distinct virtual screening workflows that led to the identification of two generations of candidate inhibitors. The first hierarchical workflow consisted of a pharmacophore-based library filtration, followed by molecular docking and molecular dynamic simulations, whereas the second consisted of a ligandoptimisation process based on the design of a focussed synthesisable library of analogues that were submitted to molecular docking along with a concomitant similarity search for scaffold hopping. The first- and the second-generation candidate compounds, which were predicted to have good in silico affinity towards PFKFB3, were synthesised and biologically evaluated through kinase assay, providing further insights about these selected drug-like molecules and new structure-activity relationship information.

Background: Cardiovascular disease is the leading cause of morbidity and mortality worldwide. Obesity-related systemic inflammation and derangements in lipoproteins metabolism, quality and functionality are important risk factors for cardiovascular diseases in obese individuals and diabetes. It is important to assess qualitative changes in lipoproteins and changes in high density lipoprotein (HDL) functionality in obese patients to realise the exact nature of derangements in lipoproteins metabolism. Standard lipid profile test used routinely in clinical practice fails to detect these important derangements. In obesity and diabetes HDL`s capacity to promote cholesterol efflux and reverse cholesterol transport is compromised. Many other aspects of HDL functionality are also impaired. It is important to assess and understand the impact of weight loss and pharmacological interventions on systemic inflammation, lipoproteins metabolism and quality as well as HDL`s functions. Aim: Investigate improvement in HDL metabolism and functionality, lipoprotein (a) (Lp(a)), oxidised phospholipid on apolipoproteins B (ox-PL-apoB), glycaemia, systemic and vascular inflammation markers following bariatric surgery-induced weight loss and extended-release niacin/laropiprant (ERN/LRPT) and atorvastatin (high and low dose) treatments. Method: This thesis is composed of three clinical studies. In the first study, the effect of bariatric surgery-induced weight loss on systemic inflammation, HDL functions, lipoproteins, Lp(a) and ox-PL-apoB was studied. Patients before and after bariatric surgery were assessed. The second study was a randomised controlled double-blind cross-over trial of ERN/LRPT of 38 hyperlipidemic statin-treated patients, the effect of ERN/LRPT on HDL functions, Lp(a) and ox-PL-apoB were assessed. In the third study, the effect of high and low dose atorvastatin on cholesterol efflux capacity (CEC) and serum amyloid A were studied in 119 diabetic patients in a randomised double-blind study. Patients were randomised to receive low dose atorvastatin 10mg (N=59) or high dose atorvastatin 80mg (N=60) daily, the effect of atorvastatin after 12 months of randomisation were assessed. Results: Cholesterol efflux capacity, HDL-C, Lp(a) and serum paraoxonase1 activity increased significantly after bariatric surgery-induced weight loss. Serum triglyceride, myeloperoxidase mass, myeloperoxidase peroxidation activity, serum amyloid A, anti-apoA-I antibodies, Ox-PL-apoB, tumor necrosis factor alpha, interleukin 6, high-sensitivity C-reactive protein, resistin, monocyte-chemotactic protein 1, Inter cellular adhesion molecule 1, E-selectin, weight, body mass index, waist circumference, HbA1c, insulin and homeostasis model assessment of insulin resistance decreased significantly following surgery. However total cholesterol (TC) and low density lipoprotein cholesterol (LDL-C) did not change significantly. ERN/LRPT increased cholesterol efflux capacity and HDL-C significantly compared to placebo. Furthermore, serum triglyceride, TC, LDL-C, Lp(a), Serum amyloid A, apoB and cholesteryl ester transfer protein activity were reduced significantly following treatment. Comparing high to low dose atorvastatin, at one year following treatment, triglyceride, TC, LDL-C and apoB were significantly lower in high dose group than low dose group. However CEC and HDL-C did not change significantly. Conclusions: HDL functionality markers and its capacity to promote cholesterol efflux improved significantly following bariatric surgery-induced weight loss. HDL-C increased and CEC capacity improved modestly after ERN/LRPT treatment. Glucose metabolism and systemic and vascular inflammation biomarkers all improved significantly after surgery. High dose atorvastatin lowered atherogenic lipoproteins more than the low dose. However, atorvastatin had no effect on HDL-C, CEC or markers of HDL functionality.

Biomarker research in cardiovascular disease (CVD) is increasingly challenging with very few biomarkers being adopted into clinical practice. CVD risk assessment scores are in place to guide primary and secondary prevention however, they require considerable improvement. At present, there is no blood test that can predict who is at higher risk of first or recurrent cardiovascular events. Since CVD is a complex inflammatory disease with several underlying biological pathways, one biomarker is unlikely to define an individual's cardiovascular risk. However, it is possible to improve the current risk assessment scores using a multimarker approach. To date no one has investigated the many known and potentially novel markers in individuals at various levels of CVD risk. Therefore, in this present work, 344 participants at various levels of CVD risk (according to the European Society of Cardiology risk score), were recruited to explore potential biomarkers for risk stratification. Proteins belonging to the tumour necrosis factor alpha (TNFα) inflammatory pathway, which has previously been shown to play a major role in CVD initiation and complications, as well as novel proteins associated with atherosclerosis, plaque rupture and thrombosis were explored using ELISA, multiplex proximity extension assays (PEA) developed by Olink Proteomics®, MSD® MULTI-SPOT Assay System and quantitative real-time PCR. Results reveal a complex panel of markers that were able to identify individuals at very high risk of CVD. Furthermore, more specific panels of markers were discovered that were able to further stratify patients at very high risk of CVD according to their cardiac history and co-morbidities. The present investigation overall demonstrates that a combination of proteins from several inflammatory pathways are necessary to evaluate an individual's risk of first or recurrent cardiovascular events as well as the risk of developing CVD-associated co-morbidities and treatment response. The blood biomarker panels discovered in this present work can be used to establish a unique proteomic disease signature for each individual recruited to the study. This will aid future long term prospective longitudinal studies in evaluating the clinical utility of such panels alone or in combination with current risk prediction tools.

Atherosclerosis is an inflammatory disorder characterized by the formation of plaque inside an artery wall. Despite the significance of atherosclerotic cardiovascular disease to healthcare, the pathophysiology of atherosclerosis is not fully understood. To allow us to examine the dynamical process of atherogenesis, a theoretical approach has the potential to increase knowledge of the interactions involved. A computational model of atherosclerosis has been built to study the process of atheroma formation and to suggest therapeutic hypotheses. Previously, computational models of disease pathways have aided in combinatorial drug discovery, and have led to the generation of therapeutic hypotheses. The model has been developed to conform to Systems Biology Markup Language (SBML) and Systems Biology Graphical Notation (SBGN) open standards. Collating parameters from multiple sources, the curated model displays atherosclerosis-like behaviour such as lipoprotein oxidation, cellular build-up, extra-cellular matrix formation and reverse cholesterol transport. Publicly available genomic data has been utilised to evaluate the changes in pathway dynamics across population subgroups. Data taken from the 1000 Genome Project, a worldwide effort to create an expansive catalogue of human variation, has been used to predict a tertiary protein structure for all proteins contained within the mathematical model, and the variation in structure for a collection of mutations is studied. A combination of molecular dynamics methods and electrostatic potential analysis are then used to estimate how the binding rates of these proteins are affected by individual mutations. These updated binding rates are subsequently used to reparameterise the mathematical model. With population data available from the 1000 Genomes Project, these new parameters can be used to study population specific dynamics of atherosclerosis, and subsequently suggest new therapeutic responses.

A carotid atherosclerotic plaque represents a nidus of inflammation mere centimetres below the blood-brain barrier. This inflammation, along with associated regions of microcalcification, are histopathological features of atheroma at risk of rupture (so-called “vulnerable plaques”) that trigger thromboembolic stroke. While conventional clinical imaging simply measures the degree of vessel stenosis, it is a crude measure that reveals little of the metabolic processes affecting plaque vulnerability. Our research demonstrates the utility of positron emission tomography (PET) using 18F-fluorodeoxyglucose (FDG) and 18F-sodium fluoride (NaF), measuring inflammation and microcalcification respectively, to identify culprit carotid atheroma in vivo, and establish how these processes influence plaque vulnerability. Furthermore, for stroke care it is the downstream thromboembolic effects upon the brain that are key. While proinflammatory conditions may increase the risk of stroke, the relationship between atheroma inflammation and the peri-infarct inflammatory response following a stroke remains poorly defined. Our work demonstrates how inflammatory activity in symptomatic carotid atheroma, measured using PET, influences both chronic small vessel disease and the evolution of lesion volume in the post-stroke period. Using metabolic imaging we can both identify vulnerable atheroma in vivo and demonstrate how these processes affect infarct evolution. We show that whilst inflammation is a generalised process, microcalcification is a focal process that may represent a point of maximum vulnerability. These results also reveal the complexity of the atheroma-brain interaction that may simultaneously trigger events while also influencing stroke evolution in the early recovery period. This has important implications for understanding pathophysiology of both atherosclerosis and stroke evolution, advancing drug-discovery, and potential clinical applications to minimise the impact from this devastating disease.

Atherosclerosis is characterised by the progressive growth of a plaque, where a fibrous cap covers a lipid-rich core. Rupture of this fibrous cap can lead to thrombosis and a heart attack or stroke. This dissertation considered the role of plaque microstructure in the mechanics and rupture risk of atherosclerotic plaques. Fibre structures in human atherosclerotic plaques were characterised through scanning electron microscopy, histology, and image processing. Local primary fibre orientation and the fibre dispersion were calculated. Plaque shoulder regions, where rupture is most frequent, had higher fibre dispersion and were more misaligned with the lumen wall. Comparative FE models were made from the plaque geometries and fibre structures found by image processing: one with evenly dispersed fibres (isotropic) and one with a preferred fibre orientation (anisotropic). The isotropic and anisotropic FE models predicted significantly different stresses. Stresses were often highest in the shoulder regions. Anisotropic stresses were calculated relative to fibres: axial stresses were highest, shear stresses were intermediate, and transverse stresses were low. Since the tissue is strongest in the fibre direction (axially), axial and shear failure modes should be considered. Material tests (uniaxial tension, trouser tear, and notched specimen) were used to characterise material properties in healthy porcine arteries. Fibre structures were evaluated by histology, multiphoton microscopy, and image processing. Samples were stiffer circumferentially and toughest against radial tears. Fractures progressed between fibres, rather than by breaking them. Material properties and fracture mechanisms were explained by fibre structures. In summary, atherosclerosis altered the fibre structure of the arteries, in ways that were mechanically significant and that explained clinical observations.

FOXO3a is a pro-apoptotic transcription factor which shows increased activation in vascular smooth muscle cells (VSMCs) of advanced atherosclerotic plaques, specifically within the intimal layer. Since VSMC apoptosis plays a crucial role in the pathophysiology of atherosclerosis, we investigated the mechanisms underlying FOXO3a-mediated cell death in this particular cell type. We aimed to characterise a novel VSMC system (FOXO3aA3ERTM) and use these cells to validate MMP-13 and TIMP3 as new FOXO3a target genes. Also, we sought to determine the mechanisms of FOXO3aA3ERTM-mediated VSMC apoptosis, particularly regarding MMP-13 and TIMP3, potential MMP-13 substrates in the extracellular matrix and the precise apoptotic signalling involved. Furthermore, we aimed to investigate whether VSMC-specific activation of FOXO3aA3ERTM in mouse affects vascular remodelling during injury and whether this is reliant on MMP-13. Lastly, we aimed to address if endogenous FOXO3a upregulates MMP-13 in mouse and human VSMCs. Our laboratory has created a transgenic rat VSMC line which stably expresses an inducible FOXO3a mutant allele known as FOXO3aA3ERTM and previous microarray experiments identified matrix metalloproteinase 13 (MMP-13) as a potential novel FOXO3a target gene. Initially, we described several key features of the FOXO3aA3ERTM VSMCs used throughout this thesis, and subsequently demonstrated that MMP-13 is a bona fide target whose expression is rapidly upregulated upon FOXO3a activation, leading to markedly higher levels of protein, cleavage and proteolytic capacity. This induction of MMP-13 was responsible for the vast majority of FOXO3a-mediated apoptosis which was accompanied by prominent degradation of fibronectin, a glycoprotein found in the extracellular matrix. However, we could not identify a terminal apoptotic pathway. FOXO3a also downregulated the endogenous MMP inhibitor TIMP3, the recombinant protein of which reduced both MMP-13 proteolysis and FOXO3a-mediated apoptosis. Activation of FOXO3aA3ERTM in the VSMCs of medium and large arteries in mice resulted in heightened expression of MMP-13 in the vessel wall, which contributed to enhanced neointimal formation during carotid ligation. Finally, endogenous FOXO3a activation leads to increased MMP-13 expression in human VSMCs, but not mouse. Overall, we have shown that FOXO3a promotes VSMC apoptosis through MMP-13 both in vitro and in vivo, a novel pathway that has important implications for the pathogenesis and treatment of vascular disease.

Vascular smooth muscle cell (VSMC) accumulation is a hallmark of atherosclerosis and vascular injury. However, fundamental aspects of proliferation and the phenotypic changes within individual VSMCs, which underlie vascular disease remain unresolved. In particular, it is not known if all VSMCs proliferate and display plasticity, or whether individual cells can switch to multiple phenotypes. To assess whether proliferation and plasticity in disease is a general characteristic of VSMCs or a feature of a subset of cells, multi-colour lineage labelling is used to demonstrate that VSMCs in injury-induced neointimal lesions and in atherosclerotic plaques are oligo-clonal, derived from few expanding cells, within mice. Lineage tracing also revealed that the progeny of individual VSMCs contribute to both alpha Smooth muscle actin (aSma)-positive fibrous cap and Mac-3-expressing macrophage-like plaque core cells. Co-staining for phenotypic markers further identified a double-positive aSma+ Mac3+ cell population, which is specific to VSMC-derived plaque cells. In contrast, VSMC-derived cells generating the neointima after vascular injury generally retained expression of VSMC markers and upregulation of Mac3 was less pronounced. Monochromatic regions in atherosclerotic plaques and injury-induced neointima did not contain VSMC-derived cells expressing a different fluorescent reporter protein, suggesting that proliferation-independent VSMC migration does not make a major contribution to VSMC accumulation in vascular disease. Similarly, VSMC proliferation was examined in an Angiotensin II perfusion model of aortic aneurysm in mice, oligo-clonal proliferation was observed in remodelling regions of the vasculature, however phenotypic changes were observed in a large proportion of VSMCs, suggesting that the majority of VSMCs have some potential to modulate their phenotype. To understand the mechanisms behind the inherent VSMC heterogeneity and observed functionality, the single cell transcriptomic techniques Smart-seq2 and the Chromium 10X system were optimized for use on VSMCs. The work within this thesis suggests that extensive proliferation of a low proportion of highly plastic VSMCs results in the observed VSMC accumulation after injury, and the atherosclerotic and aortic aneurysm models of cardiovascular disease.

Ischaemic heart disease remains the single leading cause of death throughout the world. Rupture of an advanced atheromatous coronary plaque precipitates the majority of these clinical events, resulting in thrombosis and myocardial infarction. Post-mortem studies have identified thin-cap fibroatheroma (TCFA) as the plaque subtype most prone to rupture with prospective virtual-histology intravascular ultrasound (VH-IVUS) studies linking VH-TCFA to future adverse clinical events. VH-TCFA are however common along the coronary tree with the majority remaining clinically silent, suggesting that factors other than plaque phenotype play an important role in determining rupture and future plaque behaviour. Rupture is thought to occur when the structural stress within the plaque exceeds the material strength of the overlying fibrous cap. Previous histological work has demonstrated that ruptured plaques are associated with higher stress compared to non-ruptured controls, with in vivo VH-IVUS studies linking higher plaque structural stress (PSS) with the presentation of acute coronary syndrome. Wall shear stress (WSS) on the other hand has been implicated in early plaque development and plaque growth suggesting that both PSS and WSS can influence future plaque behaviour. The work presented in this thesis is associated with a number of novel findings. First, it is the only work to demonstrate that in vivo PSS is higher in coronary atherosclerotic plaques with rupture vs. no rupture across a range of plaque subtypes and irrespective of whether analysis of the entire plaque or of regions close to the minimal luminal area is performed. Second, it shows that the pattern and extent of plaque progression and regression defined as an increase and decrease in plaque area, respectively, are associated with specific biomechanical environments at baseline, in the only study that examines the role of both PSS and WSS in this process. More specifically, high PSS is associated with changes consistent with increased vulnerability both in areas of progression and regression. On the other hand, lower WSS at baseline is associated with greater increases in plaque area and burden in areas that progress and with smaller decreases in areas that regress largely due to changes in fibrous tissue. Although the role of WSS in determining future plaque behaviour has been previously examined, this is the first time that this is assessed specifically in areas of progression and regression, particularly important in view of the dynamic nature of atherosclerotic plaques. More importantly, the work presented in this thesis demonstrates that the interplay of these biomechanical forces is associated with specific patterns of plaque progression and regression despite the fact that PSS and WSS are independent of each other. This has never been previously demonstrated and further suggests that incorporation of biomechanical analysis can play role in the identification of plaques that lead to future clinical events. Finally, the ability of PSS to identify plaques that lead to adverse clinical events was assessed through a propensity core matched analysis of the PROSPECT (A Prospective Natural-History Study of Coronary Atherosclerosis) study. The analysis presented here is the largest, most extensive and thus most significant work to ever examine this with results suggesting that incorporation of PSS and associated parameters can improve the capability of VH-IVUS to identify plaques that lead to such events. In summary, the results of this thesis suggest that coronary PSS plays an important role in the pathophysiology of plaque rupture, and that its incorporation in routine plaque assessment may improve our current ability to identifying coronary plaques that lead to future adverse clinical events. The interplay between PSS and WSS may also affect future plaque behaviour and in particular progression and regression. Prospective studies are now required to fully evaluate the role of these biomechanical forces in plaque development, and whether their incorporation in plaque evaluation can be of clinical significance.

Hydrogen sulfide (H2S), is a gasotransmitter with several key roles in metabolism and vascular function. The effects of H2S are dependent on concentration and target organ. For example, increased H2S concentrations impair liver metabolic function but protect against vascular dysfunction and atherosclerosis. Thiosulfate sulfurtransferase (TST), a nuclear encoded mitochondrial matrix enzyme, is proposed to be a component of the sulfide oxidising unit (SOU) which metabolises H2S. Preliminary data has shown that Tst deletion in mice (Tst-/-) increases circulating H2S levels measured in whole blood. Therefore, it was hypothesised that Tst-/- mice would exhibit worsened metabolic function in the liver but also protection of vascular function under conditions of vascular stress e.g. atherosclerosis. Liver metabolism was assessed by extensive metabolic phenotyping of Tst-/-mice fed control diet and in conditions of metabolic dysfunction induced by a high fat diet (HFD). Tst deletion altered glucose metabolism in mice; gluconeogenesis was increased in liver from Tst-/-mice fed control diet. Glucose intolerance in HFD-fed Tst-/-mice was also more severe than HFDfed C57BL/6 controls. In vitro metabolic investigations in primary hepatocytes isolated from Tst-/-mice demonstrated that mitochondrial ATP-linked and leak respiration were increased compared to controls. The effect of Tst deletion on vascular function was investigated in Tst- /-mice fed control or HFD using myography. Tst deletion did not alter vessel function when mice were maintained on a normal diet. HFD feeding (20 weeks) reduced maximal vessel constriction in the presence of endothelial nitric oxide synthase and cyclooxygenase inhibitors in C57BL/6 aorta. However, in Tst-/-mice fed HFD there was no reduction in maximal constriction suggesting a protective action of Tst deletion. The effects of Tst deletion on atherosclerotic lesions was investigated by generating double knock-out (DKO) mice by deletion of the Tst gene in ApoE-/- mice and (ApoE-/-Tst-/-). Atherosclerotic lesion formation was accelerated by feeding mice a western diet. Within the brachiocephalic branch lesion volume and total vessel volume were reduced in DKO mice fed western diet for 12 weeks, indicating that Tst deletion reduced lesion formation. Plasma cholesterol was reduced in DKO mice compared to ApoE-/- controls and a trend towards reduced systolic blood pressure was also noted. Overall this work supported the hypothesis that Tst deletion engenders metabolic dysfunction but vascular protection. The findings are consistent with the reported effects of increased H2S signalling. Overall inhibition of TST represents a novel target for treatment of atherosclerosis, with the caveat that glycaemia may be worsened due to hepatic metabolic dysfunction.

PET imaging, using the bone tracer Na18F, allows the non-invasive location of atherosclerotic plaques that are at risk of rupture. However, the spatial resolution of PET is only 4-5 mm, limiting the mechanistic information this technique can provide. In this thesis, the use of fluorescence and Raman imaging to elucidate the mechanism of micro-calcification within atherosclerotic plaques has been investigated. A number of fluorescent probes to detect fluoride and calcium have been synthesised. One of the fluoride probes has been shown to be selective for fluoride however, the concentration of fluoride required to activate the probe is order of magnitudes higher than the amount of Na18F used for PET imaging making it problematic to use for future studies. On the other hand, a calcium probe has been shown to: selectively bind to hydroxyapatite (HAP); permit visualisation and quantification of HAP in both vascular and bone cell models; and effectively stain cultured aortic sections and whole mouse aorta for OPT imaging. Building on these preliminary data, fluorescence imaging and immunohistochemistry (IHC) imaging of both healthy and atherosclerotic tissue that were previously subjected to PET imaging, were successfully carried out showing the ability of the probe to detect HAP in human vascular tissue. IHC staining for Osteoprotegerin (OPG) and Osteopontin (OPN), two bone proteins recently detected in vascular tissue, showed the co-localization of OPG with the probe. Conversely, the OPN was shown to localize in areas surrounding high OPG and probe signal. To determine the exact composition of vascular calcification, Raman spectroscopy was also used. It is believed that the biosynthetic pathway to HAP passes through a series of transitional states; each of these has different structural characteristics which can be studied using Raman spectroscopy. In particular, HAP has a strong characteristic Raman peak at 960 cm-1. An increase in HAP concentration has been detected by Raman in both calcified cell models and aortic sections. When human vascular tissue was analysed, an additional peak at 973 cm-1 was present suggesting the presence of whitlockite (WTK) in this tissue as well as HAP.

BACKGROUND: Cardiovascular disease, principally manifest as myocardial infarction or stroke, is the dominant cause of death worldwide and despite therapeutic advances, the global burden of these conditions continues to increase. In order to address this ongoing disease burden, there is a clear need to more effectively target the use of existing and novel diagnostic investigations and medical therapies. Emerging cardiovascular biomarkers include the biochemical, such as high-sensitivity cardiac troponin, and the radiological, such as computed tomography coronary angiography (CTCA) and 18Ffluoride positron emission tomography (PET). Cardiac troponins can now be reliably quantified in clinically stable or asymptomatic populations and provide information about myocardial pathophysiology, whilst CTCA can non-invasively quantify atherosclerotic burden and 18F-fluoride PET imaging offers insight into plaque vulnerability. Improved targeting of diagnostic investigations requires more reliable estimation of pre-test probability of coronary disease whilst optimizing the use of pharmacological or interventional treatments requires more accurate prognostic stratification. Achieving both objectives in an equitable manner across all population groups will depend upon updated clinical guidelines containing improved risk models and enhanced management pathways. The objective of this thesis was to investigate the potential clinical benefit of novel approaches to the diagnostic and prognostic assessment of coronary heart disease. EVALUATION OF THE 2016 NATIONAL INSTITUTE FOR HEALTH AND CARE EXCELLENCE (NICE) GUIDANCE ON THE ASSESSMENT OF SUSPECTED STABLE ANGINA. A post-hoc analysis was undertaken of the Scottish COmputed Tomography of the HEART (SCOT-HEART) trial of 4,146 participants with suspected angina randomised to assessment with computed tomography coronary angiography or standard care. Patients were dichotomised according to guideline definitions into groups representing possible angina and non-anginal presentations. The primary (diagnostic) endpoint was diagnostic certainty of angina at 6 weeks and the prognostic endpoint comprised fatal and non-fatal myocardial infarction. In 3,770 eligible participants, CTCA increased diagnostic certainty more in those with possible angina (relative risk [RR] 2.22 (95% CI 1.91-2.60), p < 0.001) than those with non-anginal symptoms (RR 1.30 (1.11-1.53), p=0.002; pinteraction < 0.001). In the possible angina cohort, CTCA did not change rates of invasive angiography (p=0.481) but markedly reduced rates of normal coronary angiography (hazard ratio [HR] 0.32 (0.19-0.52), p < 0.001). In the non-anginal cohort, rates of invasive angiography increased (HR 1.82 (1.13-2.92), p=0.014) without reducing rates of normal coronary angiography (HR 0.78 (0.30-2.05), p=0.622). At 3.2 years of follow-up, fatal or nonfatal MI was reduced in patients with possible angina (3.2% to 1.9%; HR 0.58 (0.34- 0.99), p=0.045) but not in those with non-anginal symptoms (HR 0.65 (0.25-1.69), p=0.379). Overall the updated NICE guidance on patient assessment maximises the benefits of CTCA with respect to diagnostic certainty, the use of invasive coronary angiography, and reductions in fatal and non-fatal myocardial infarction. Patients with non-anginal chest pain derive minimal benefit from CTCA, which instead increases rates of invasive investigation. EXTERNAL VALIDATION OF THE PROSPECTIVE MULTICENTER IMAGING STUDY FOR EVALUATION OF CHEST PAIN (PROMISE) TOOL FOR DETERMINING MINIMAL-RISK OF CORONARY ARTERY DISEASE. The PROspective Multicenter Imaging Study for Evaluation of chest pain (PROMISE) minimal-risk tool was recently developed to identify patients with suspected stable angina at very low risk of coronary artery disease and clinical events. The external validity of this tool was investigated within the context of the Scottish Computed Tomography of the HEART multicenter randomised controlled trial of patients with suspected stable angina due to coronary artery disease. Model discrimination and calibration was determined amongst 1,764 patients in whom complete CCTA data were available and compared with the European Society of Cardiology guideline-endorsed Coronary Artery Disease Consortium (CADC) risk score. The PROMISE minimal-risk tool improved discrimination compared with the CADC model (c-statistic 0.785 vs 0.730, p < 0.001) and was improved further following re-estimation of covariate coefficients (c-statistic 0.805, p < 0.001). Model calibration was initially poor (c2 197.6, Hosmer-Lemeshow [HL] p < 0.001), with significant overestimation of probability of minimal risk, but improved significantly following revision of the PROMISE minimal-risk intercept and covariate coefficients (c2 5.6, HL p=0.692). HIGH-SENSITIVITY CARDIAC TROPONIN I IN THE DIAGNOSIS OF STABLE CORONARY ARTERY DISEASE In a pre-specified sub-study of the Scottish COmputed Tomography of the Heart trial, plasma cardiac troponin was measured using a high-sensitivity single molecule counting assay in 943 adults with suspected stable angina who had undergone coronary computed tomography angiography. Rates of obstructive coronary artery disease were compared with the pre-test probability determined by the European Society of Cardiology Coronary Artery Disease Consortium risk model with and without cardiac troponin concentrations. External validation was undertaken in an independent study population from Denmark comprising 487 patients with suspected stable angina. Higher cardiac troponin concentrations were associated with obstructive coronary artery disease with a 5-fold increase across quintiles (9 to 48%, p < 0.001) independent of known cardiovascular risk factors (odds ratio [OR] 1.35 [95% confidence interval (CI) 1.25-1.46] per doubling of troponin). Cardiac troponin concentrations improved the discrimination of the ESC model for identifying obstructive coronary artery disease (c-statistic 0.785 to 0.800, p=0.003) and improved classification into ESCrecommended categories of clinical risk (net reclassification improvement 0.143 [95% CI, 0.093-0.193]). The revised model achieved similar improvements in discrimination and net reclassification when applied in the external validation cohort. HIGH-SENSITIVITY CARDIAC TROPONIN I IN CARDIOVASCULAR RISK STRATIFICATION OF PATIENTS WITH CHRONIC OBSTRUCTIVE PULMONARY DISEASE AND HEIGHTENED CARDIOVASCULAR RISK. The association between plasma high-sensitivity cardiac troponin I concentration and cardiovascular events in patients with chronic obstructive pulmonary disease and heightened cardiovascular risk was examined within the context of a double-blind randomised controlled trial of inhaled corticosteroids and bronchodilators (1 placebo arm and 3 different treatment arms). Plasma cardiac troponin I concentrations were measured with a high-sensitivity assay in a subgroup of 1,599 patients. The cardiovascular endpoint was a composite of cardiovascular death, myocardial infarction, stroke, unstable angina and transient ischaemic attack during follow-up of 1.5 years. Baseline plasma cardiac troponin I concentrations were above the lower limit of detection (1.0 ng/L) in 1,559 (97.5%) patients and were unaffected by inhaled therapies at 3 months (p > 0.05 for all). Compared with the lowest tertile (cardiac troponin I ≤3.0 ng/L), patients in the highest tertile (≥ 5.5 ng/L) were at greater risk of cardiovascular events (hazard ratio 3.0, 95% confidence interval 1.5 to 6.2, p=0.002) and cardiovascular death (hazard ratio 9.6, 95% confidence interval 2.6 to 35.6, p < 0.001) after adjustment for cardiovascular risk factors. There were no differences in COPD exacerbations between tertiles even after adjustment (p > 0.05)., REPRODUCIBILITY OF CORONARY 18F-FLUORIDE PET-CT IMAGING The inter-observer and scan-rescan reproducibility of coronary 18F-fluoride PET-CT imaging was investigated in 20 patients with clinically stable but high risk multi-vessel coronary artery disease who underwent repeated 18F-fluoride PET-CT scans 11.5±4.5 days apart. Scan analysis using the currently accepted approach of normalisation to a referent coronary segment (TBRREFERENT) identified 10 (50%) patients with evidence of focal coronary 18F-fluoride uptake and demonstrated moderate agreement across observers on a per-patient level (k = 0.56). This was similar to the level of agreement achieved with visual assessment alone (k = 0.64). Reproducibility was improved by semi-quantitative reporting combining visual assessment with a threshold uptake value for determining the presence of tracer uptake (k = 0.84). Using the optimised approach achieved excellent agreement on overall segmental uptake counts (intra-class correlation = 0.97). CONCLUSION: Cardiovascular diagnostic and prognostic assessments represent a complex endeavour and established tools for risk prediction can demonstrate suboptimal predictive accuracy when evaluated in patient cohorts that are independent of the population used for model derivation.

Myocardial infarction (MI) occurs following acute thrombotic occlusion of a coronary artery, and triggers a robust inflammatory response. Within hours, neutrophils are recruited to the infarcted myocardium followed by the infiltration of pro-inflammatory Ly6Chi monocytes. Transition from the pro-inflammatory macrophage phenotype (M1) to an anti-inflammatory, pro-resolution phenotype (M2-like) is critical to successful infarct healing. Interventions that polarize macrophages towards an anti-inflammatory 'M2-like' phenotype improve infarct healing in the experimental MI mouse model and reduce subsequent adverse remodelling of the myocardium, but the endogenous mechanisms that regulate repair are not well understood. Furthermore, differences in the resolution of inflammation in C57BL/6 and BALB/c mice, which are two of the commonly used wild-type mouse strains in experimental MI have not been characterised. We previously found that low peripheral blood eosinophil count is associated with increased short-term risk of mortality in low-intermediate risk patients with ischaemic heart disease. This suggests that eosinophils may have a role in the successful remodelling and repair of the heart following myocardial infarction. Eosinophils express a number of immuno-modulating cytokines and lipid mediators implicated in the resolution of inflammation. Increasingly prominent is interleukin-4 (IL-4), a cytokine that has been found to maintain the anti-inflammatory M2-like phenotype in macrophages. We therefore hypothesised that IL-4Rα signalling and recruitment of eosinophils to the myocardium following infarction are key in regulating the subsequent inflammatory response and scar tissue formation during infarct repair and cardiac remodelling. Experimental MI was induced by permanent left anterior descending artery ligation in isofluorane anaesthetized 12-15 week-old male wild-type (WT) BALB/c, WT C57BL/6, IL4Rα-/-, IL-4Rαflox/-, IL-4Rαflox/-LysMCre mice and eosinophil-deficient ΔdblGATA mice. Cardiac function was characterised by high-resolution ultrasound and immune cell infiltration by flow cytometry of single cell infarct and remote zone tissue digests. Blood eosinophil count and 6-month all-cause mortality were assessed in 732 consecutive patients undergoing primary percutaneous coronary intervention for ST-segment elevation myocardial infarction (STEMI). The rate of mortality due to cardiac rupture was significantly higher in C57BL/6 mice in comparison with BALB/c mice at Day 7 post-MI. This was associated with a higher proportion of pro-inflammatory Ly-6Chi macrophages infiltrating the infarct zone tissue of C57BL/6 mice following MI. An accompanying reduction in the number of splenic Ly-6Chi monocytes post-MI, suggestive of splenic monocyte mobilisation, was seen in C57BL/6 mice but not found in BALB/c mice. Furthermore, C57BL/6 mice had a delayed transition in macrophage polarisation towards an anti-inflammatory phenotype. Disruption of IL4Rα signalling, in mice null for the IL4Rα gene, resulted in increased F4/80+ macrophage and pro-inflammatory Ly6Chi macrophage infiltration of the infarct zone and reduced expression of the anti-inflammatory macrophage marker CD206, compared to wild-type controls. Furthermore, expression of GATA3 and ST2, both associated with the immunosuppressive function of (CD4+ Foxp3+) regulatory T cells, was reduced in infarct zone regulatory T cells from IL4Rα-/- mice. These findings were associated with defective wound healing with impaired angiogenesis, increased scar size, disarrayed infarct zone collagen deposition, accompanied by modified expression of plod2 that encodes the collagen cross-linking enzyme lysyl hydroxylase 2. Resulting in greater left ventricular dilatation and loss of cardiac function, as well as a higher 7- day mortality due to cardiac rupture in IL4Rα-/- mice. This indicates that successful infarct repair requires the engagement of IL-4Rα signalling to facilitate the accumulation of anti-inflammatory macrophages and highly immunosuppressive ST2+ regulatory T cells in the heart following MI. Resident cardiac macrophages from naïve hearts of IL-4Rαflox/-LysMCre mice failed to undergo LysMCre-mediated deletion of the IL-4Rα gene, potentially because low or absent expression of Lyz2 (encoding lysozyme M). In both ST-elevation MI (STEMI) patients and mice after acute MI, there was a decline in peripheral blood eosinophil count, with activated eosinophils being recruited to the infarct zone and paracardial adipose tissue of mice. The transcription factors GATA-1 plays a role in the differentiation of eosinophils from eosinophil progenitor cells. Deletion of GATA-1 results in loss of the eosinophil lineage and has been exploited to develop the eosinophil-deficient ΔdblGATA mouse. ΔdblGATA mice were used to address the role of eosinophils in cardiac remodelling following MI. ΔdblGATA mice had increased left ventricular dilatation and reduced ejection fraction after induction of MI, relative to wild-type mice. ΔdblGATA mice had increased scar size with disarrayed infarct zone collagen deposition, accompanied by modified expression of the genes plod2 and lox, which are associated with collagen cross-linking. The proportion of CD206+ anti-inflammatory macrophages was less in the infarct zone of ΔdblGATA mice, but was restored by adoptive transfer of eosinophils from WT mice. Furthermore, adverse cardiac remodelling in eosinophil-deficient ΔdblGATA mice was rescued by provision of IL-4 complex following MI. In conclusion, an enhanced inflammatory response following MI underlies the increased risk of cardiac rupture seen with WT C57BL/6 mice in comparison to WT BALB/c mice. WT BALB/c mice are protected from cardiac rupture, which was associated with an absence of splenic monocyte mobilisation following ischaemic injury. The resolution of inflammation was found to be dependent on IL4Rα signalling which is crucial for cardiac repair and remodelling, through modulating inflammatory cell recruitment and phenotype, as well as scar formation. Eosinophils are recruited to the heart post-MI and are essential for regulating cardiac repair and remodelling, likely through provision of IL-4. Therefore, we were able to show that IL-4Rα signalling and recruitment of eosinophils to the myocardium following infarction are both key in regulating the subsequent inflammatory response and scar tissue formation during infarct healing and cardiac remodelling.

Systemic inflammatory networks and local signaling cascades trigger culprit pathogenic mechanisms relating clinical cardiovascular disease (CVD) risk factors to atherosclerotic plaque progression and rupture. Imaging vascular inflammation affords a valuable marker of atherosclerotic disease activity to reveal important mechanistic insights for CVD research, to quantify early anti-inflammatory effects of new atherosclerosis drugs, and, ultimately, to help improve CVD risk prediction. While carotid, aortic, and peripheral arterial inflammation can be measured by 18F-fluorodeoxyglucose (FDG) PET-computed tomography (CT), as a glucose analog, high 18F-FDG signal spillover owing to physiological myocardial muscle metabolism prevents reliable coronary interpretation. Lack of cell specificity, and the influence of hypoxia on 18F-FDG uptake within macrophages and other plaque cells, are further limitations that drive the search for an alternative PET tracer for imaging inflammation in atherosclerosis. Up-regulation of the G-protein coupled receptor somatostatin receptor subtype-2 (SST2) occurs on the cell surface of activated macrophages. The central hypothesis tested in this thesis is that vascular SST2 PET imaging using 68Ga-DOTATATE might offer a more accurate marker of macrophage inflammation than 18F-FDG, with superior coronary imaging and therefore better power to discriminate high-risk vs. low-risk atherosclerotic lesions. Comprehensive molecular, histological and clinical evaluation of this experimental CVD imaging biomarker was undertaken, including a prospective clinical study with head-to-head comparison to 18F-FDG in coronary, carotid, and aortic vasculature. In vitro data showed that (i) target SSTR2 gene expression occurred in “pro-inflammatory” M1 macrophages, (ii) specific 68Ga-DOTATATE ligand binding to SST2 receptors occurred in CD68-positive macrophage-rich carotid plaque regions, and (iii) and carotid SSTR2 mRNA was highly correlated with both the pan-macrophage marker CD68 and in vivo 68Ga-DOTATATE PET signals. In clinical imaging, increased 68Ga-DOTATATE inflammatory signals correctly identified culprit vs. non-culprit arteries in patients with acute coronary syndrome and transient ischemic attack/stroke. 68Ga-DOTATATE also demonstrated good diagnostic accuracy for high-risk coronary CT features, and strong correlations with clinical CVD risk factors and 18F-FDG-defined vascular inflammation. While 18F-FDG also differentiated culprit vs. non-culprit carotid and high-risk coronary arteries, myocardial 18F-FDG overspill rendered coronary scans uninterpretable in most patients. In contrast, 68Ga-DOTATATE allowed unimpeded coronary interpretation in all patients. Findings of this thesis provide compelling evidence, from gene, to cell, to plaque, to patient, that SST2 PET imaging using 68Ga-DOTATATE provides a quantifiable marker of macrophage-related atherosclerotic inflammation and disease activity. Further studies are needed to establish whether 68Ga-DOTATATE PET can improve CVD risk prediction when added to current clinical methods, or offer a novel imaging platform to rapidly test the anti-inflammatory capacity of emerging atherosclerosis drugs. Broader translational applications of 68Ga-DOTATATE PET include possible use in diagnosis and therapeutic monitoring of vasculitis, endocarditis, myocarditis, and other manifestations of cardiovascular inflammation.

Psoriasis is an immune mediated inflammatory disease which affects 2-3% of the world's population. Over the last decade, psoriasis has been acknowledged as an independent risk factor for atherosclerosis. The precise mechanism or mechanisms of the heightened risk is widely speculated. Endothelial cells and macrophages are central players in the immunopathological development of both diseases. Interleukin-36 cytokines (IL-36) have been heavily implicated in psoriasis immunopathology. Significant upregulation of epidermal IL-36 is a recognised characteristic of psoriatic skin inflammation. IL-36 induces inflammatory responses in dendritic cells, fibroblasts and epithelial cells. While vascular alterations are a hallmark of psoriatic lesions and dermal endothelial cells are well known to play a critical role in dermal inflammation, the effects of IL-36 on endothelial cells have not been defined. We report that endothelial cells including dermal microvascular cells express a functionally active IL-36 receptor. Adhesion molecules VCAM-1 and ICAM-1 are upregulated following IL-36γ stimulation, and this is reversed in the presence of the endogenous IL-36 receptor antagonist. IL-36γ-stimulated endothelial cells secrete the proinflammatory chemokines IL-8, CCL2 and CCL20. Chemotaxis assays showed increased migration of T-cells following IL-36γ stimulation of endothelial cells. Both resident and infiltrating inflammatory myeloid cells contribute to the immunopathology of psoriasis by promoting the IL-23/IL-17 axis. We show that IL-36γ induces the production of psoriasis-associated cytokines from macrophages (IL-23, TNFα) and that this response is enhanced in macrophages from psoriasis patients. This effect is specific for IL-36γ and could not be mimicked by other IL-1 family cytokines such as IL-1α. Furthermore, IL-36γ stimulated macrophages potently activated endothelial cells as illustrated by ICAM-1(CD54) upregulation, and led to increased adherence of monocytes, effects that were markedly more pronounced for psoriatic macrophages. Interestingly, regardless of stimulus, monocytes isolated from psoriasis patients showed increased adherence to both the stimulated and unstimulated endothelium when compared to monocytes from healthy individuals. Collectively, these findings add to the growing evidence for IL-36γ having roles in psoriatic responses, by enhancing endothelium directed leukocyte infiltration into the skin and strengthening the IL-23/IL-17 pathway. Our findings also point to a cellular response which could potentially support cardiovascular comorbidities in psoriasis.

Background: The incidence of coronary heart diseases, including atherosclerosis, increases with ageing. The factors which influence arterial function, and which may be changed with ageing, are multiple but effects of perivascular adipose tissue (PVAT) on large arteries have not previously been considered. A key role for nitric oxide (NO) in mediating the anti-contractile capacity of PVAT has been suggested. Caveolin-1 (Cav-1) modulates the production of NO in vivo by tonic inhibition of eNOS. The influence of aortic PVAT and the contribution of NO to vascular reactivity in ageing C57BL/6 mice, atherosclerotic ApoE knockout mice (ApoE-/-), Cav-1 knockout mice (Cav-1-/-) and atheroprotected ApoECav-1 double knockout mice (ApoE-/-Cav-1-/-) is unknown. Hypothesis: The influence of PVAT on vascular function is modulated by ageing and the development of atherosclerosis via NO bioavailability. Methods: Male mice were used in this study. C57BL/6 mice were obtained at 4 weeks of age and maintained on a normal rodent diet (ND) for 8, 16 or 26 weeks. ApoE-/- and Cav-1-/- mice were bred from in-house colonies and ApoE-/-Cav-1-/- mice were generated by interbreeding ApoE-/- and Cav-1-/- mice. Upon weaning, ApoE-/-, Cav-1-/- and ApoE-/-Cav-1-/- mice were maintained on either a ND or Western-type diet (WD) for 8, 16 or 26 weeks. Vascular reactivity studies on isolated aortic ring preparations were performed in the presence or absence of PVAT. The contribution of NO to the vascular reactivity of aortic PVAT was determined using pharmacological inhibition of NO synthase. Aortic PVAT was assessed for evidence of morphological and/or compositional changes associated with ageing or a WD. Results: NO mediated an anti-contractile effect of aortic PVAT in C57BL/6 mice fed a ND up to 16 weeks. The anti-contractile capacity of aortic PVAT was lost after 26 weeks on a ND and preceded endothelial dysfunction. Loss of the PVAT anti-contractile effect was accompanied by alterations in PVAT morphology and composition. Aortic PVAT from ND-fed ApoE-/- mice was dysfunctional and did not exert an anti-contractile effect. Furthermore, a WD did not alter the influence of PVAT on vascular reactivity in ApoE-/- mice and PVAT morphology and composition was unchanged. NOS inhibition did not alter the contractile responses. The aortic PVAT of ND-fed Cav-1-/- mice did not exert an anti-contractile effect and PVAT composition was unchanged with increasing age. However, after 26 weeks on a WD, aortic PVAT from Cav-1-/- mice potentiated contractions to phenylephrine and white adipocyte hypertrophy was observed. NOS inhibition revealed a pro-contractile effect of aortic PVAT from Cav-1-/- mice. Loss of Cav-1-/- conferred significant protection against the development of atherosclerosis in WD-fed ApoE-/-Cav-1-/- mice despite a proatherogenic lipid profile. Aortic PVAT from ND-fed ApoE-/-Cav-1-/- mice did not exhibit an anti-contractile capacity and PVAT morphology was unchanged with ageing. Additionally, a WD did not influence the effect of PVAT on vascular reactivity in ApoE-/-Cav-1-/- mice although white adipocyte hypertrophy was observed after 26 weeks of high fat feeding. NOS inhibition revealed a pro-contractile effect of aortic PVAT in 8-week ND-fed ApoE-/-Cav-1-/- mice. Conclusions: This work has produced novel insights into the influence of aortic PVAT and NO on vascular reactivity and the morphology of aortic PVAT in ageing C57BL/6 mice, atherosclerotic ApoE-/- mice, Cav-1-/- mice and athero-protected ApoE-/-Cav-1-/- double knockout mice. Ageing to pre-middle age in C57BL/6 mice results in a loss of the anti-contractile effect of PVAT prior to endothelial dysfunction. This is associated with altered NO bioavailability and changes to the morphology and composition of PVAT. This may reveal potential therapeutic targets to restore the anti-contractile capacity of PVAT if comparable age-related PVAT dysfunction is observed in humans. Aortic PVAT of ApoE-/- mice does not exert an anti-contractile effect which may be attributed to decreased basal eNOS activity. A WD does not alter the vascular reactivity of PVAT. In addition, aortic PVAT from Cav-1-/- mice does not exhibit an anti-contractile capacity yet it exerts a pro-contractile effect after 26 weeks on a WD. The aortic PVAT of ApoE-/-Cav-1-/- mice does not modulate vascular reactivity and this is unaltered with feeding of a WD although white adipocyte hypertrophy was observed within the PVAT. The critical role of Cav-1 in the initiation and progression of atherosclerosis is reinforced by the atheroprotected phenotype of the ApoE-/-Cav-1-/- mice even though a severely proatherogenic lipid profile is observed in both the ND and WD-fed mice. Therapeutically targeting LDL transcytosis into the arterial wall could potentially prevent or halt the development of atherosclerosis. Aortic PVAT of ND-fed Cav-1-/- and ApoE-/-Cav-1-/- mice may not be dysfunctional but unable to modulate vascular reactivity due to attenuated vasoconstrictor responses of PVAT-denuded aortic rings as a result of excess NO, although this requires further investigation.

Atherosclerosis is a multifactorial, chronic inflammatory condition characterised by endothelial dysfunction, hyperlipidaemia and the accumulation of fatty deposits within the tunica intima of medium-to-large sized muscular arteries. This disease can prove fatal with patients suffering lethal myocardial infarction or stroke. Recently, two studies investigating the role of G-protein-coupled receptor 55 (GPR55) in atherosclerosis reported conflicting results; one reported a pro-atherogenic role for GPR55 and the other, an anti-atherogenic role for this receptor. Interestingly, another study demonstrated that the activation of GPR55 by lysophosphatidylinositol (LPI) in cultured rat neonatal ventricular cardiomyocytes provokes distinct cellular functions that are dependent on the location of GPR55, leading to suggestions that GPR55 may regulate cardiomyocyte function at two cellular sites and be a potential therapeutic target for cardiac disorders. While it has been demonstrated that GPR55 is important in the maintenance of cardiac function of healthy mice, what is currently unknown is if GPR55 has a role in the cardiovascular remodelling and cardiac function of atherosclerosis prone mice. To address this, the present studies were conducted to investigate 1) the role of GPR55 in atherogenesis, 2) if GPR55 has a role in the cardiac function of mice suffering from atherosclerosis, 3) the signalling pathway by which LPI activates cardiomyocytes, 4) the impact of GPR55 activation on the outcome of myocardial ischaemia/reperfusion (I/R) injury and, 5) the signalling mechanisms by which GPR55 elicits any observed effects on the myocardium in response to such injury. Using C57BL/6 (wildtype; WT), apolipoprotein E knockout (ApoE-/-; mouse model of atherosclerosis), GPR55 knockout (GPR55-/-) and novel ApoE-/-/GPR55-/- mice, this study has established that in the presence of high fat feeding (to accelerate atherosclerosis), GPR55 has a complex role whereby it both regulates risk factors associated with atherosclerosis (i.e. body weight and fat mass) yet promotes the development of fatty streaks within the vasculature, via a lipid independent mechanism. In terms of cardiac function, GPR55 exerted a protective role by maintaining the systolic function of high fat fed ApoE-/- mice, yet negatively affected the contractile reserve of these mice. With regard to infarct size, the present study established that LPI-induced activation of GPR55 (pre-global ischaemia) exacerbates myocardial tissue injury via a Rho-associated protein kinase (ROCK) dependent mechanism. Finally, this study established that LPI signals through the same signalling pathway as it did in the isolated heart, in both mouse and human-induced pluripotent stem cell-derived cardiomyocytes thus suggesting a translational role for GPR55 in the human heart. In conclusion, despite further research being required, the data presented within this thesis provides evidence that GPR55 may have the potential to be targeted for therapeutic gains in atherosclerosis and myocardial I/R injury.

Cardiovascular disease remains the commonest cause of death worldwide. The majority of deaths are caused by atherosclerotic plaque rupture with resultant myocardial infarction or stroke, or rupture of abdominal aortic aneurysms. Conventional imaging modalities have consistently failed to identify atherosclerotic plaques or aneurysms with high-risk pathological features that are at highest risk of rupture or progression. The development of modern molecular imaging techniques targeted at these features could lead to the identification of such high-risk plaques and aneurysms in vivo and guide the development of novel treatment strategies. The aim of this thesis was to evaluate whether novel molecular modalities have a role in providing new insights into biological disease processes, and identify high-risk plaques and aneurysms. Using positron emission tomography-computed tomography (PET-CT), 18F-fluorodeoxyglucose and 18F-fluoride were utilised as markers of metabolic inflammation and active calcification. Cellular inflammation was assessed using ultrasmall superparamagnetic particles of iron oxide (USPIO) enhanced magnetic resonance imaging (MRI). In a prospective trial, 80 patients with myocardial infarction (n=40) and stable angina (n=40) underwent 18F-fluoride and 18F-fluorodeoxyglucose PET-CT, and invasive coronary angiography (Chapter 3). Intense 18F-fluoride uptake localised to recently ruptured plaque in patients with acute myocardial infarction. In patients with stable coronary artery disease, 18F-fluoride uptake identified coronary plaques with high-risk features on intravascular ultrasound. 18F-fluoride PET-CT is the first noninvasive imaging method to identify and localise ruptured and high-risk coronary plaques. Aortic vascular uptake of 18F- fluorodeoxyglucose was studied in patients with myocardial infarction and stable angina (Chapter 4). In a separate outcome of 1,003 patients enrolled in the Global Registry of Acute Coronary Events, we further evaluated whether infarct size predicted recurrent coronary events. Patients with myocardial infarction had higher remote atherosclerotic tracer uptake that correlated with the degree of myocardial necrosis, and exceeded that observed in patients with stable coronary disease. The outcome cohort demonstrated that patients with higher degree of myocardial necrosis had the highest risk of early recurrent myocardial infarction. This supports the hypothesis that acute myocardial infarction exacerbates systemic atherosclerotic inflammation and remote plaque destabilization: myocardial infarction begets myocardial infarction. In a prospective imaging cohort, the role inflammation and calcification was assessed in 63 patients with abdominal aortic aneurysms and 19 age and sex matched patients with atherosclerosis (Chapter 5). Compared to non-aneurysmal segments, enhanced inflammation and calcification was observed within the wall of aortic aneurysmal segments. In comparison to matched controls with atherosclerosis, the entire aorta in those with aortic aneurysm appears more highly inflamed, suggesting presence of a global aortopathy rather than a disease confined only to the abdominal region of the aorta. Aortic aneurysms have greater active inflammation and calcification than atherosclerotic controls suggesting a more intense, destructive and transmural pathological process. A subgroup of fifteen patients with aortic aneurysms underwent imaging with both PET-CT with 18F-fluorodeoxyglucose, and T2*- weighted MRI before and 24 h after administration of USPIO (Chapter 6). Whilst there was a moderate correlation between the two tracers, there were distinct differences in the pattern and distribution of uptake suggesting a differential detection of macrophage glycolytic and phagocytic activity respectively. These studies provide novel insights into vascular biological processes involved in the initiation, progression and rupture of atherosclerotic plaques and aortic aneurysms. Future longitudinal studies are needed to establish whether these techniques have a role in improving the clinical management and treatment of patients with coronary artery disease and aortic aneurysms.

Atherosclerotic cardiovascular disease is a leading cause of death worldwide. Dysregulation of vascular redox state plays a crucial role in the atherosclerotic process. Increased production of vascular superoxide (O2·-) and other reactive oxygen species (ROS) leads to endothelial dysfunction, a key early step in atherogenesis. Adipose tissue is a source of vasoactive, hormone-like molecules which are termed adipokines. One of the most important adipokines is adiponectin. Adiponectin has been shown to have antioxidant, anti-atherosclerotic effects in cell culture studies and animal models. However, its role in human cardiovascular disease has not been extensively investigated. More specifically, its effects on the human vascular wall and the mechanisms regulating its synthesis in adipose tissue have not been studied before in humans. The aim of my thesis is to explore the role of adiponectin in human atherosclerosis. This was achieved through use of the Oxford CABG Bioresource: a well-phenotyped cohort and tissue bank of patients undergoing cardiac surgery. By employing a range of in vivo and ex vivo techniques, I demonstrate for the first time in humans that adiponectin has direct antioxidant effects in the vascular wall, by directly suppressing pro-oxidant vascular enzymes and restoring redox balance. These effects persist in type 2 diabetes, presence of which is linked to reduced circulating adiponectin levels. Indeed, a variety of stimuli affect adiponectin synthesis in human adipose tissue, with brain natriuretic peptide being a major driver of adiponectin synthesis. However, different adipose tissue depots demonstrate diverse responses to stimuli affecting adiponectin synthesis, owing to their functional and morphological differences. Of particular interest is the fact that synthesis of adiponectin in perivascular adipose tissue is driven by the oxidative stress status of the underlying vessel. This observation led me to document for the first time in humans the existence of a reciprocal, two-way interaction between perivascular adipose tissue and the vascular wall: high vascular oxidative stress leads to release of factors with the ability to up-regulate adiponectin expression in perivascular adipose tissue, acting as a local paracrine defence mechanism attempting to restore vascular redox state. My thesis provides proof-of-concept for this novel cross-talk between adipose tissue and the vascular wall. This can have significant impact in designing new therapeutic strategies against atherosclerosis.

Monocytes comprise a key component of the innate immune system. Their excessive and/or prolonged recruitment and/or activation can however be harmful and has been associated with the pathogenesis of numerous inflammatory disorders, most notably atherosclerosis. Consequently there is much interest in deciphering the mechanisms by which monocytes migrate into sites of inflammation. In this context, whilst many studies have investigated the roles of adhesion molecules and chemokine receptors, very little attention has been placed on the potential involvement of proteases. To help address this point, and considering cigarette smoke-induced recruitment of monocytes to the lung is impaired in mice deficient in neutrophil elastase (NE-/-), we sought to further investigate the role of NE, a serine protease, in monocyte migration. To achieve this we successfully crossed CX3CR1-eGFP-knock-in mice, which exhibit fluorescently labelled monocytes, with NE-/- mice. By comparing NE-/- and NE+/+ littermates we demonstrated, for the first time, NE expression in murine monocytes. Peritonitis, ear, and cremaster muscle models of inflammation were subsequently employed to assess the involvement of NE in monocyte recruitment. In CCL2-induced peritonitis, a predominantly monocyte-driven inflammatory model, monocyte infiltration was significantly reduced after 16 hours in NE-/- mice compared to NE+/+ controls. Monocyte transmigration was also defective in the ears (though interestingly not the cremaster muscles) of NE-/- mice stimulated with CCL2, with the majority of monocytes seemingly unable to penetrate the venular endothelium. Furthermore, immunostaining revealed transmigrating monocytes were depleted of their intracellular NE stores. Taken together, it appears the release of NE by monocytes promotes their transmigration in response to CCL2, identifying NE as a potential therapeutic target for monocyte-driven pathologies.

Atherosclerosis is the root cause of a wide range of cardiovascular diseases. Although it is a global arterial disease, some of the most severe consequences, heart attack and stroke, are caused by ischemia due to local plaque rupture. The risk of rupture is related to the mechanical properties of the plaque. Magnetic resonance elastography (MRE) images tissue elasticity by inverting, externally excited, harmonic wave displacement into a stiffness map, known as an elastogram. The aim of this thesis is to computationally and experimentally investigate the application of MRE to image the mechanical properties of atherosclerotic plaques. The cardiac cycle, lumen boundary, size and inhomogeneous nature of atherosclerotic plaques pose additional complications compared to more well-established MRE applications. Computational modelling allowed these complications to be assessed in a controlled and simplified environment, prior to experimental studies. Computational simulation of MRE was proposed by combining steady state shear waves, yielded by finite element analysis, with the 2D Helmholtz inversion algorithm. The accuracy and robustness of this technique was ascertained through models of homogeneous tissue. A computational sensitivity study was conducted through idealised atherosclerotic plaques, incorporating the effects of disease variables and mechanical, imaging and inversion parameters on the wave images and elastograms. Subject to parameter optimisation, a change in local plaque shear modulus with composition was established. Amongst other variables, an increase of the lipid pool volume in 10mm3 increments was shown to decrease the predicted shear modulus for stenosis sizes between 50% and 80%. The limitations of the Helmholtz inversion algorithm were demonstrated. A series of arterial phantoms containing plaques of various size and stiffness were developed to test the experimental feasibility of the technique. The lumen was identifiable in the wave images and elastograms. However the experimental wave propagation, noise and resolution left the vessel wall and plaque unresolvable. A computational replica of the phantoms yielded clearer wave images and elastograms, indicating that changes to the experimental procedure could lead to more successful results. The comparison also highlighted certain areas for improvement in the computational work. Imaging protocol for in vivo MRE through the peripheral arteries of healthy volunteers and peripheral artery disease patients was developed. The presence of physiological motion and low signal to noise ratios made the vessel anatomy unidentifiable. The application of MRE to atherosclerotic plaques through simulations, arterial phantoms, healthy volunteers and patients has shown that although there is the potential to identify a change in shear modulus with composition, the addition of realistic experimental complications are severely limiting to the technique. The gradual addition of complications throughout the thesis has allowed their impact to be assessed and in turn has highlighted areas for future research.

Introduction: Rheumatoid arthritis (RA) is a systemic autoimmune condition, characterised by an inflammatory arthritis. It is associated with a 50% increased risk of cardiovascular (CV) mortality. Chronic inflammation is thought to lead to accelerated atherosclerosis in RA. There is some evidence to suggest that patients have a more inflammatory, unstable atherosclerotic plaque phenotype. The impact of advances in RA treatment, on cardiovascular co-morbidity remains unclear. The aims of the current study were to employ non-invasive imaging techniques to test the hypothesis that RA patients have more inflammatory, unstable atherosclerotic plaques compared to unaffected individuals and that treatment of active arthritis would lead to alterations in plaque composition and inflammation. Secondary aims were to evaluate the association of clinical phenotype and potential serological biomarkers of CV risk with plaque presence and phenotype. Methods: A prospective pilot study of patients with active RA and age and sex matched controls was conducted. Subjects underwent clinical and serological evaluation, then carotid artery ultrasound was performed to screen for carotid plaque. Dynamic contrast enhanced magnetic resonance imaging (DCE-MRI) was performed on those with suitable plaque. A subgroup of patients had a carotid artery positron emission tomography (PET) scan. Patients were followed up with repeat clinical, serological and DCE-MRI assessments. The primary outcome evaluated was difference in plaque inflammation measured on DCE-MRI between patients and controls and in patients longitudinally. Secondary outcomes included differences in plaque composition on DCE-MRI, plaque inflammation on PET and the relationship of clinical, serological and imaging findings. Results: 130 patients and 52 controls were recruited and screened for carotid plaque. There was a higher prevalence of plaque on ultrasound in the patient group (53% vs 36%) and plaque was independently associated with high sensitivity c reactive protein (hsCRP). Carotid DCE- MRI data was analysed in 15 patients and 5 controls. There was no significant difference in plaque inflammation on DCE-MRI between the groups. However there was a significantly higher rate of plaque calcification in patients, despite similar plaque burden in both groups (73.3% vs 20%, p=0.038). All 15 patients exhibited features of high-risk plaque. Plaque inflammation was seen in all 13 patients in whom PET imaging was undertaken. No significant improvement in plaque inflammation was detected on DCE-MRI over time, which was in keeping with the lack of clinical improvement found in most cases. ConclusionsIncreased prevalence of atherosclerosis and differences in plaque phenotype were observed in this study and findings would support the hypothesis that patients have a more high-risk plaque phenotype. The high prevalence of calcified lesions in RA is a novel finding which warrants further investigation. The study was underpowered to detect significant changes in plaque inflammation, measured on DCE-MRI, between the groups and in patients over time. However, this study provides valuable data with which to plan a larger study to investigate the effects of anti-inflammatory therapy on atherosclerosis in RA in the future.

Coronary artery disease (CAD) is caused by atherosclerosis, a chronic inflammatory response to modified lipoproteins. A key pathophysiological event is the lipid-induced transformation of macrophages into lipid-laden foam cells and their accumulation in atherosclerotic plaques. Heritable CAD risk is associated with common genetic variants at over 40 genomic loci; the underlying causal mechanisms remain largely unknown and could affect transcriptional regulation in foam cells. Epigenetic and gene expression changes were measured in primary human macrophages before and after exposure to atherogenic, oxidized low-density lipoprotein—with resultant foam cell formation. This unbiased approach involved open chromatin mapping with formaldehyde-assisted isolation of regulatory elements with enhancer and transcription factor mapping using chromatin immuno-precipitation. Foam cell formation was associated with changes in a subset of open chromatin and enhancer sites that were strongly correlated with expression of nearby genes. OxLDL-regulated enhancers were enriched for several transcription factors—including C/EBP-beta— that have no previously documented role in foam cell formation. OxLDL exposure up-regulated C/EBP-beta expression and increased C/EBP-beta binding across the genome, most prominently around genes involved in inflammatory response pathways. Variants at CAD-associated loci were enriched in the subset of oxLDLregulated open chromatin sites. These included rs72664324 in an oxLDL-induced super-enhancer at the PPAP2B locus. OxLDL increased C/EBP-beta binding at rs72664324. C/EBP-beta binding, enhancer activity and oxLDL-induced upregulation of PPAP2B were stronger with the protective A allele of rs72664324. The PPAP2B protein product LPP3 was expressed in foam cells in human atherosclerotic plaques and was upregulated by oxLDL exposure in macrophages, so increasing the degradation of pro-inflammatory mediators. I also found several other CAD risk candidate genes were regulated by oxLDL: Phosphatase and actin regulator 1 (PHACTR1) and macrophage inducible Ca2+ dependent C-type lectin (Mincle). This led us to find a novel expression-quantitative-trait locus for PHACTR1 in macrophages and define new glycolipid ligands for Mincle. Our results demonstrate a genetic mechanism contributing to CAD risk at the PPAP2B locus and highlight the value of integrating gene expression and epigenetic changes to study disease processes involving pathogenic environmental stimuli.

The role of inducible nitric oxide synthase (iNOS) and/or nitric oxide (NO) in atherosclerosis remains elusive. Several researchers argued whether iNOS and/or NO are pathogenic or cardio protective. The pathogenesis of atherosclerosis is complex and includes mechanisms associated with inducible nitric oxide synthase (iNOS). We have demonstrated that the expression and function of iNOS may be selectively down regulated by pro-oxidants such as antimycin A and diethyl maleate (DEM). To further explore the underlying mechanisms associated with these effects we have investigated whether antimycin A and/or DEM modulated the activation of key cellular signalling molecules associated with the induction of iNOS. Expression of p38 mitogen activated kinase (MAPK) and Akt were induced by exposure to lipopolysaccharide (LPS) and interferon-gamma (IFN-γ). Oxidative stress (OS) was induced using antimycin A, DEM and hydrogen peroxide (H2O2). All three OS inducers caused a significant generation of free radicals whereas only antimycin A and DEM generated superoxide radical (O2-). Also nitrite production and iNOS expression may be down regulated, in part; by pro-oxidants generating O2- but not hydroxyl radicals (OH-). Antimycin A and DEM concentration dependently inhibited the phosphorylation of p38 MAPK and Akt and this was restored when the cells were pre-treated with Atorvastatin whereas H2O2 was without any significant effect. Taken together, the data suggest novel actions for both pro-oxidants and atorvastatin which may have important implications in coronary artery disease where suppression of iNOS may be deleterious and maintaining its expression may be cardio-protective.