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Diabetes is one of the fastest growing diseases today, affecting over a million people in the UK. Numerous medical complications, such as heart disease, are regularly associated with diabetes. Despite advances in methods of diagnosis and treatment there is still a need for greater understanding of these diseases. This will include research directed towards the influence of specific treatments and reasons for the high incidence of diabetes and heart disease in 'at risk' populations. Changes in elemental status are associated as the cause or effect of various diseased states. Elemental imbalance in diabetics can result in impaired glucose tolerance and insulin resistance and in sufferers of heart disease elemental changes impair heart rate and elasticity of blood vessels. In the UK 10,000 patients with Ischaemic Heart Disease undergo coronary artery bypass grafting (CABG) surgery each year. Elemental analysis has been carried out on blood samples collected from a group of patients admitted to hospital for bypass surgery. Proton Induced X-ray Emission (PIXE) and Instrumental Neutron Activation Analysis (INAA) have been applied as complementary analytical tools for determining elemental concentrations. Differences have been examined between CABG patients with and without diabetes. Both experimental methods have been used to investigate elemental levels in whole blood, erythrocytes and plasma. Elemental concentration varied according to the blood constituent and reflected short and long-term influences on elemental homeostasis. Plasma was found to concentrate Na, Mg and Ca the highest using both experimental techniques. All blood samples were collected and prepared at St. George's Hospital, Tooting in the UK. An additional study was conducted to investigate the influence of the bypass operation on the patient's elemental status. Whole blood was obtained at pre (1h before operation), post (1-2 hours after operation) and recovery (24 hours after completion of the operation) stages of bypass surgery. Differences between the three phases were observed, individual variations have been plotted so rates of change can be seen and evaluated with the particular medical history. Concentrations of Na, Mg, Al, P, S, Cl, K, Ca and Fe in whole blood were determined. The two measurement techniques found different concentrations however results showed a general trend that post operative concentrations were elevated compared to pre operative values. Analysis of blood drawn during the recovery phase, 24 hours after the surgery, found that concentration were typically approaching pre operative levels. Both PIXE and INAA found concentrations of Na, Mg and Al peaked post operation and then decreased in the recovery phase, towards values measured pre surgery. Various factors may be responsible for the elemental changes occurring during surgery including, hormone production, routine administration of intra-operative fluids and contact of blood with non- endothelial surfaces. Hierarchical cluster analysis has been used to confirm differences between elemental levels in pre, post and recovery stages of bypass surgery. The dendograms produced indicate significant distinction between the three stages. The explosive impact of diabetes in the UK resident Asian population is discussed and the influence of diabetogenic agents introduced. Examination of research literature revealed that betel nut has been implicated as a causative agent in several medical conditions. Samples of Betel nut and six associated chewing materials widely used in Asian communities has been collected and prepared for analysis. Instrumental neutron activation analysis has been used to determine the concentration of Na, Mg, Al, Cl, Ca, V, Mn, Cu and Br in the samples by means of short-lived radionuclides.

Pre-clinical studies suggest that granulocyte-colony stimulating factor (GCSF) holds promise for the treatment of ischaemic heart disease (IHD). However, its safety and efficacy in this setting, and in particular in patients with chronic refractory 'no-option' IHD, is unclear. Therefore, a clinical study was initiated in 20 such 'no-option' patients, with the aim of assessing the safety and efficacy of both G-CSF administration, and also, that of an intracoronary infusion of G-CSF mobilised CD133+ cells. The study involved initial baseline cardiac ischaemia assessment (symptom based questionnaire, exercise stress test (EST), nuclear Sestamibi (MIBI) and dobutamine stress echocardiographic (DSE) imaging). Stable 'no-option' IHD patients then received open-label G-CSF commencing at 10μg/kg s/c for five days, with an EST on days four and six (to facilitate myocardial cytokine generation and stem cell trafficking). After three months, cardiac ischaemia assessment and the same regimen of G-CSF and ESTs were repeated, but in addition, leukapheresis and then a randomised double-blinded intracoronary infusion of CD133+ or unselected cells were performed. Final cardiac ischaemia assessment was three months thereafter. Eighteen male and two female subjects (mean age 62.4) were enrolled. Eight events occurred that fulfilled pre-specified 'adverse event' criteria: four ischaemic (troponin positive) episodes, two episodes of transient thrombocytopaenia (one profound), one episode of gout and one unscheduled hospitalisation for exhaustion. Troponin was positive on 17 further occasions (all CK-MB negative), however, at these instances angina severity was identical to baseline. Importantly, no adverse event(s) resulted in any detectable long-term adverse sequelae for any subject. From baseline to final follow-up, the administration of two cycles of G-CSF was associated with statistically significant improvements in a range of subjective outcomes, including anginal symptoms, quality of life and EST performance (all p < 0.005). However, the objective MIBI and DSE scans showed only trends towards improvement (all p > 0.1). Compared to unselected cells, an intracoronary infusion of CD133+ cells did not improve either subjective or objective outcomes. In conclusion, administering G-CSF to patients with refractory 'no-option' IHD warrants careful monitoring, but may be performed with safety. A larger, randomised double-blind placebo-controlled trial of G-CSF in these patients appears warranted.

Pre-clinical studies suggest that granulocyte-colony stimulating factor (GCSF) holds promise for the treatment of ischaemic heart disease (IHD). However, its safety and efficacy in this setting, and in particular in patients with chronic refractory 'no-option' IHD, is unclear. Therefore, a clinical study was initiated in 20 such 'no-option' patients, with the aim of assessing the safety and efficacy of both G-CSF administration, and also, that of an intracoronary infusion of G-CSF mobilised CD133+ cells. The study involved initial baseline cardiac ischaemia assessment (symptom based questionnaire, exercise stress test (EST), nuclear Sestamibi (MIBI) and dobutamine stress echocardiographic (DSE) imaging). Stable 'no-option' IHD patients then received open-label G-CSF commencing at 10μg/kg s/c for five days, with an EST on days four and six (to facilitate myocardial cytokine generation and stem cell trafficking). After three months, cardiac ischaemia assessment and the same regimen of G-CSF and ESTs were repeated, but in addition, leukapheresis and then a randomised double-blinded intracoronary infusion of CD133+ or unselected cells were performed. Final cardiac ischaemia assessment was three months thereafter. Eighteen male and two female subjects (mean age 62.4) were enrolled. Eight events occurred that fulfilled pre-specified 'adverse event' criteria: four ischaemic (troponin positive) episodes, two episodes of transient thrombocytopaenia (one profound), one episode of gout and one unscheduled hospitalisation for exhaustion. Troponin was positive on 17 further occasions (all CK-MB negative), however, at these instances angina severity was identical to baseline. Importantly, no adverse event(s) resulted in any detectable long-term adverse sequelae for any subject. From baseline to final follow-up, the administration of two cycles of G-CSF was associated with statistically significant improvements in a range of subjective outcomes, including anginal symptoms, quality of life and EST performance (all p < 0.005). However, the objective MIBI and DSE scans showed only trends towards improvement (all p > 0.1). Compared to unselected cells, an intracoronary infusion of CD133+ cells did not improve either subjective or objective outcomes. In conclusion, administering G-CSF to patients with refractory 'no-option' IHD warrants careful monitoring, but may be performed with safety. A larger, randomised double-blind placebo-controlled trial of G-CSF in these patients appears warranted.

Background: Excess adiposity, diabetes mellitus, and ischaemic heart disease are common important causes of morbidity and premature mortality in postmenopausal women in the UK. A large amount of data exists on known risk factors for these conditions, and for risk factors men and women share there is little evidence to suggest sex-based differences. It has been suggested that factors unique to women (such as parity and breastfeeding) may also influence risk. The nature of the relationship between childbearing and these conditions remains to be clarified. In this thesis I explore the association between women’s childbearing histories and their adiposity, and risk of diabetes or ischaemic heart disease after the menopause, to provide evidence on the character, repeatability and public health relevance of the associations. Aim: To explore the hypothesis that childbearing (specifically parity and breastfeeding) is associated with women’s body weight and risk of excess adiposity, and also with women’s risk of diabetes mellitus, and ischaemic heart disease after the menopause. Methods: Data are analysed from a large population-based cohort of middle-aged UK women recruited in 1996 to 2001 (the Million Women Study) with complete childbearing information, and who had baseline anthropometry, and were followed for incident diabetes or ischaemic heart disease through repeat survey questionnaires, hospital admission records, and central registry databases. Results: In a large ethnically homogeneous population of postmenopausal UK women increasing parity was associated with an increase in BMI, however this increase was offset in women who breastfed. The associations between parity, breastfeeding and BMI were of a similar order of magnitude to established risk factors known to be associated with BMI, for example smoking, and physical activity. The associations between childbearing and women’s risk of diabetes mellitus after the menopause appear to be largely due to the effects of childbearing on maternal BMI. There is only limited evidence to suggest a direct effect of childbearing on women’s risk of diabetes after the menopause. There is statistically significant evidence of an association between childbearing and women’s risk of ischaemic heart disease after the menopause. Parity was associated with a modest increase in risk whereas breastfeeding was associated with a small decrease in risk, however the effects were small in comparison to known important risk factors. Conclusions In a large population of UK women childbearing was found to have a persistent influence on women’s mean BMI after the menopause, and through this postmenopausal risk of diabetes mellitus. Childbearing was also found to be mod-estly associated with women’s risk of ischaemic heart disease after the menopause.

Ischaemic heart disease is the most common cause of mortality in the western hemisphere and it is rapidly becoming the leading cause of death globally. Moreover, therapeutic interventions by cardiologists and cardiac surgeons frequently subject the heart to acute I/R injury, which in itself can cause mortality. Recent investigations of adult stem cells have primarily focused on their regenerative potential for chronic ischaemic heart disease. In this thesis, I have investigated the hypothesis that adult bone marrow derived stem cells are cardioprotective in acute regional myocardial I/R injury. In a rat model of left anterior descending coronary artery (LAD) reversible occlusion and reperfusion, I demonstrate that an intravenous bolus of adult bone marrow derived (1) bone marrow mononuclear (BMNNC) and (2) mesenchymal stem cells (MSC) upon reperfusion can attenuate infarct size. This effect is comparable to ischaemic preconditioning (IPC), which is the gold standard for cardioprotection. Next, I demonstrated the mechanisms for adult stem cell cardioprotection are principally anti-apoptotic and depend upon stem cell secreted factors to (1) activate phosphatidylinositide 3-kinase (PI3)/Akt cell survival kinase-signaling pathway (2) inhibit glycogen synthase kinase-3β (3) inhibit p38MAPK (4) inhibit nuclear translocation of p65NF-κB. 7 Proteomic analysis of myocardium subjected to I/R and treated with either BMMNC or BMMNC derived supernatant (BMS) upon reperfusion demonstrated higher expression of a whole host of pro-survival proteins. These were notably (1) 14-3-3-ε protein (2) anti-oxidant peroxiredoxin-6 (3) heat shock protein (HSP) αB-crystallin, HSP72, HSP tumour necrosis factor receptor-1 associated protein, and HSP ischaemia responsive protein-94 (4) glycolytic protein glyceraldehyde-3-phosphate dehydrogenase (5) mitochondrial aconitase and mitochondrial voltage-dependent anionselective channel protein-1. Thereafter, I investigated the mobilization of endogenous bone marrow stem cells and trafficking to the ischaemic myocardium by stromal cell derived factor-1 (SDF-1) /chemokine, receptor type 4 (CXCR4) signaling. I demonstrate high up-regulated expression of CXCR4 and CD26 in BMMNC following IPC, which might have a role in IPC-mediated cardioprotection. Finally, and in concordance with this finding I demonstrate that both IPC and an exogenous MSC bolus upon reperfusion can synergize to abolish acute myocardial I/R injury.

Cardiovascular disease (CVD) is a complex combination of multiple conditions. The majority of deaths within CVD include heart attacks and strokes caused by atherosclerotic disease. The pathophysiological process for atherosclerotic disease occurs within the endothelial lining of the vessels of the body. This prolonged process occurs when cholesterol deposits form irregularity in luminal flow resulting in decreased blood flow and ischaemia. This unstable cholesterol plaque can rupture resulting in clot formation and artery occlusion. Within this thesis I aim to show background to the relevant pathophysiology of ischaemic heart disease (IHD) with the main emphasis on acute myocardial infarction (AMI), the history of its therapy to current therapy. I will discuss the theorised role of stem cell therapy within animal models and previous clinical trials within regenerative medicine and AMI. I will describe and discuss the method and the results of the REGENERATE-AMI trial (Clintrial.gov: NCT00765453), which will include the safety and efficiency of the therapy, and the possible cytokine mechanism by which this therapy may exert it effect. Additionally I will describe the potential for assessing myocardial oedema using 3-slice T2-STIR short axis stack imaging post AMI compared to the conventional 10-slice T2-STIR technique to assess its feasibility and clinical similarity to assess its use as a tool in translational research.

Ischaemic heart disease is a major contributor to premature death and heart failure in the Westernised world. Ischaemic injury within the heart may be beneficially modulated by the nucleoside adenosine. Derived from catabolism of ATP, adenosine was initially known as a potent bradycardic and hypotensive agent. However, more recently the protective function of adenosine has been investigated. The protective effects of adenosine are mediated via activation of adenosine receptors: A1, A2A, A2B, and A3 receptors. Activation of these potentially protective (or retaliatory) adenosine receptors hinges upon accumulation of adenosine during ischaemia-reperfusion. This Thesis examines the role and mechanisms of adenosine mediated cardioprotection in young and aged hearts, exploring endogenous and exogenous adenosine receptor activation, genetic manipulation of A1 receptors and adenosine deaminase and pharmacological manipulation of adenosine metabolism. The effects of age on ischaemic responses and adenosine handling and protection are also assessed. The core approach to assess each of the above issues involved the Langendorff isolated mouse heart preparation. Experiments within Chapter 3 focuses on the contractile effects of adenosine receptors in normoxic hearts. This study indicates A2A receptors have no direct effect on contractility, while adenosine exerts positive inotropy independently of coronary flow and perfusion pressure (i.e. Independent of the Gregg phenomenon). In addition, investigations in genetically modified hearts hint at positive inotropy in response to A1 receptors. Since the latter is only evidenced in modified lines, it is possible A1-mediated inotropy may be abnormal or supraphysiological. In Chapter 4 the impact of genetic 'deletion' of A1ARs and/or adenosine deaminase (ADA) on intrinsic tolerance to ischaemia were studied. Data demonstrate that genetic deletion of A1 receptors significantly limits the ability of the mouse myocardium to withstand injury during ischaemic insult. Thus, providing strong support for a role of A1ARs in determining intrinsic tolerance to ischaemia-reperfusion. ADA KO mice confirm protection afforded by endogenous adenosine and the notion of adenosine metabolism modification as a protective strategy. Interestingly, effects of A1AR KO differ from A1AR overexpression or A1AR agonism in that the latter decrease contractile diastolic dysfunction while A1AR KO selectively increase systolic dysfunction and increase oncosis without altering diastolic injury. This challenges current dogma regarding the action of A1 adenosine receptors on ischaemic injury. In Chapter 5 the effects of adenosine metabolism inhibition (via adenosine deaminase (ADA) and adenosine kinase (AK) inhibitors) were studied. Inhibition of adenosine deaminase with the drug EHNA, and adenosine phosphorylation with iodotubercidin significantly protected mouse hearts from ischaemia-reperfusion, reducing contractile dysfunction and cardiac enzyme efflux. However, inhibitors failed to improve the outcome of the aged myocardium. 8-SPT and 5-HD reduced the protective effects of EHNA and iodotubercidin demonstrating thus; cardioprotection via ADA and AK appears to rely on adenosine receptor activation and involves a mitoK ATP dependent mechanism. Since aging is of considerable importance with regard to outcomes of ischaemic heart disease, experiments in Chapter 6 focused on effects of aging (and gender) on cardiovascular function and injury during ischaemia-reperfusion. In assessing post ischaemic outcomes in hearts from young adult (2-4 months), mature adult (8 months), middle aged (12 months), aged (18 months) and senescent (24-28 months) C57/BL/6J mice, data reveal a substantial age-related decline in ischaemic tolerance (which appears selective for myocardial vs. vascular injury). The decline in ischaemic tolerance is expressed primarily within the initial 12 months in both males and females with relatively little further decline with continued aging. There is evidence of a modest improvement in tolerance in senescence vs. aged hearts possibly reflecting selection of a protected phenotype in senescent populations. In addition, mature and middle-aged female hearts showed improved tolerance to ischaemia-reperfusion compared to males, supporting a role for hormonal changes. Effects of aging and purine metabolism were studied in Chapter 7. Data suggest impaired tolerance to ischaemia-reperfusion in older hearts may stem in part from shifts in myocardial purine catabolism. Data reveal reduced accumulation of salvageable and cardioprotective adenosine and enhanced accumulation of poorly salvaged (and potentially injurious) hypoxanthine and xanthine. These changes may potentially predispose the aged myocardium to ischaemic injury and radical generation via the xanthine oxidase reaction. The final data Chapter of this Thesis describes preliminary data regarding aging, signalling and adenosine mediated protection. It was found that protein kinase C (PKC) and A1 receptors mediate protection in young hearts and also that A1 receptors appear to mediate protection via a PKC LindependentLi signalling cascade. In addition, experiments in aged hearts (attempting to elucidate mechanisms behind impaired adenosinergic protection with age) suggest a PKC-independent A1-mediated protection path may be preserved with aging, since A1 receptors continue to offer some protection while PKC activation does not. It is possible the failure of exogenous adenosine to offer protection in aged hearts may result from a lesion at or downstream of PKC.