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Coronary artery disease (CAD) is one of the main causes of premature mortality in the Western world, however with early and targeted treatment it is treatable. Calcium scoring multi-slice computed tomography (MSCT) imaging is used to visualize small calcified plaques located in the coronary arteries which represent a possible arterial narrowing, thus limiting or preventing the supply of oxygenated blood to the heart muscle. A clinician digitally quantifies the patient plaque burden to determine appropriate treatment, such as lipid lowering drugs, based on defined risk categories. MSCT is capable of imaging the entire heart within a single breath hold, thus exposing the patient to lower dose compared to coronary angiography, however this results in lower contrast images that may be affected by acquisition and motion artefacts. Clinically significant calcified plaques are located in the main coronary arteries, whereas other clinically insignificant calcified plaques can be found in close proximity but will not affect blood flow through the coronary arteries and thus does not concern the clinician for calcium scoring. This can lead to inter- and intra- observer variability when distinguishing between clinically significant and insignificant plaques, resulting in incorrect plaque burden quantification. Reducing the subjectivity and reproducibility in calcium scoring can lead to greater accuracy in patient treatment prescription as small score shifts will affect atherosclerosis risk categorisation. For a disease where treatment can include drug prescription, the reduction of non-required drug prescription is an important objective. A preliminary automation of the calcium scoring method was developed presenting good correlation between automated and observer generated risk assignment, however, variation in automated scores signified over or under inclusion of non clinically significant or clinically significant plaques respectively. Thus it was theorised that the extraction of the coronary artery tree would facilitate focussed calcified plaque assessment and quantification. To achieve this goal, a fully automated observer-less calcium scoring framework has been presented employing automated heart isolation and coronary artery tracking. Ground truth coronary artery trees are applied to aid vessel tracking, compensating for the poor image clarity and low axial resolution of MSCT calcium scoring images. Validation of this system employs ground truth clinical calcium scores obtained by an observer study using simulated CT images from the XCAT digital phantom. Assessment of atherosclerosis risk categorisation of the resulting scores from the framework presented in this thesis against ground truth shows a 93.8% system accuracy with a 85.4% sensitivity and 96.5% specificity when examined against the minimum cases required to represent agreement between all observers. However, combined observer performance of 0.729 from ROC analysis affirms observer variability in manual calcium scoring. Overall score difference analysis between framework and observer scores show an average of 10% overscore and underscore respectively, thus indicating compensation by the automated framework for observer variability based on the use of vessel tracking to identify actual coronary calcified plaques. Key words: coronary artery disease, segmentation, computer aided diagnosis, automation, active contour, vessel tracking, calcium scoring, observer variability, MSCT

Cell therapy is a promising strategy for treating ischaemic chronic heart failure. However, its therapeutic efficacy has not been fully established. In addition, arrhythmia occurrence is a concern of this treatment. In this work, therapeutic benefits, arrhythmogenicity and underlying mechanisms, which were hypothesised to be modulated by cell-delivery route into the heart, were investigated with the aim of optimising cell therapy. Injection of either skeletal myoblasts or mononuclear bone marrow cells into the rat ischaemic chronically failing heart via either direct intramyocardial or retrograde intracoronary route similarly improved both cardiac function and physical activity over the 84 days analysed. Survival of the grafted cells in the myocardium was extremely poor and trans-differentiation or fusion of the grafted cells into cardiomyocytes or vessels were only rarely identified via either celldelivery route. Therefore, paracrine effects including increased neovascular formation and attenuated fibrosis in the myocardium were considered to play an important role in the therapeutic benefits of cell therapy using either cell-type. Of note, direct intramyocardial injection of either cell-type, but not retrograde intracoronary injection, produced spontaneous ventricular arrhythmias including ventricular tachycardia in the early periods following cell injection. Local heterogeneity in the myocardium induced by clusters of grafted cells, which also involved inflammatory response, was considered to be a cause of the arrhythmias following intramyocardial cell injection. In contrast, in the late periods, injection of skeletal myoblasts via either route, but not mononuclear bone marrow cells, caused latent ventricular tachycardia possibly via regression of connexin43 in the native myocardium. Efficiency of engraftment of mononuclear bone marrow cells in the myocardium following intracoronary injection was very poor in the normal heart, but was enhanced by induction of ischaemia-reperfusion prior to cell injection. Most of the enhanced cell-engraftment was dependent on P-selectin-mediated cellular interaction between donor cells and endothelium.

The endothelial nitric oxide synthase (eNOS) co-factor tetrahydrobiopterin (BH4) has been shown to play a pivotal role in maintaining endothelial function in experimental vascular disease models. In BH4-deficient states, eNOS becomes enzymatically ‘uncoupled’, generating reactive oxygen species instead of nitric oxide, thus promoting endothelial dysfunction. In humans with coronary artery disease (CAD), higher vascular BH4 levels have been shown to be associated with improved endothelial function, and genetic variation in endogenous BH4 synthesis has implicated a causal role. Accordingly, BH4 has been proposed as a potential therapeutic target in vascular disease states. The work in this thesis aims to further elucidate the roles of exogenous and endogenous BH4 in humans. In a randomised, placebo-controlled clinical trial of oral BH4 therapy in patients with CAD, exogenous BH4 had no effect on endothelial function or vascular oxidative stress. Subsequent pharmacokinetic and pharmacodynamic analysis revealed that oral BH4 significantly augmented BH4 levels in plasma and in venous tissue (but not in arterial tissue), but also increased levels of the oxidation product dihydrobiopterin (BH2), which lacks eNOS cofactor activity. Thus, there was a null effect on overall biopterin redox status. To further understand the mechanics of exogenous BH4 oxidation, ex vivo studies of human blood and vascular tissue demonstrated that exogenous BH4 is very rapidly oxidised to BH2; co-administration with an antioxidant had only a modest effect on preventing BH4 oxidation in blood, with no beneficial effect on biopterin redox state in the vasculature. Finally, using a “Mendelian randomisation” approach, I studied the effects of a haplotype of GCH1 (the gene encoding the rate limiting enzyme in BH4 synthesis) on endogenous BH4 bioavailability and vascular function in healthy individuals. In patients with CAD, this haplotype has been associated with decreased BH4 bioavailability and eNOS uncoupling, however in healthy individuals the haplotype exerted no significant effect, likely due to reduced inflammatory stimulation of GCH1.