Human essential hypertension is a substantial public health problem with greater than 25% of the adult population affected worldwide. It is a complex disorder and a major risk factor for various cardiovascular diseases such as heart disease, renal disease and stroke. The stroke-prone spontaneously hypertensive rat (SHRSP) is a commonly used model of human essential hypertension. Previous studies identified a quantitative trait locus (QTL) for blood pressure regulation on rat chromosome 2. To further enhance the investigation of this QTL, rat chromosome 2 congenic strains were generated by transferring regions of chromosome 2 from the normotensive WKY onto the genetic background of the SHRSP. Congenic strains exhibited significantly lower blood pressure than the SHRSP parental strain indicating that genes within the congenic region are important for blood pressure regulation. Renal microarray analysis of congenic 2c* and parental strains led to the identification of glutathione s-transferase mu type 1 (Gstm1) as a positional and possible functional candidate gene for hypertension in the SHRSP. Expression of Gstm1 was significantly lower in the SHRSP kidney than in 2c* and WKY and renal oxidative stress was increased in SHRSP. Glutathione s-transferase mu type 1 is part of a large family of antioxidant enzymes and may play a role in hypertension by modulating levels of oxidative stress. This study has utilised small RNAs to examine the role of Gstm1 in cellular oxidative stress and also investigated microRNA (miRNA) expression in the rat congenic strains. RNA interference (RNAi) was utilised to knock-down expression of Gstm1 in a rat kidney tubular epithelial cell line (NRK-52E). Three different short interfering RNA (siRNA) sequences designed to target Gstm1 were evaluated. Each sequence significantly reduced expression of Gstm1 and was confirmed at both mRNA and protein level. Off-target effects on other Gstm isoforms and the interferon response gene, oligoadenylate synthetase 1 were prevented by reducing the concentration of siRNA used. To take knock-down of Gstm1 expression into an in vivo setting, local delivery to the kidney via the renal artery was assessed but was found to cause significant kidney damage. Instead, kidney targeted vectors that can be delivered systemically were evaluated in the SHRSP. Immunohistochemistry confirmed specific targeting of kidney tubules. Plasmid vectors were then generated that express Gstm1 specific short-hairpin RNA (shRNA) molecules based on the sequences that successfully knocked down Gstm1 expression in vitro. Transfection of these plasmids into NRK-52E cells was poor and knock-down of Gstm1 could not be confirmed. The role of Gstm1 in protection against cellular oxidative stress was evaluated in NRK-52E cells by measuring markers of oxidative stress following knock-down of Gstm1 expression. Total GST activity was not reduced in cells transfected with Gstm1 specific siRNA however activity was increased following over-expression of Gstm1. No change in the levels of reduced glutathione was observed in cells following knock-down of Gstm1. Oxidative stress was determined by measuring 8-isoprostane (a marker of lipid peroxidation), 8-hydroxy-2-deoxyguanosine (8-OH-dG) (a marker of oxidative DNA damage) and by the comet assay (DNA damage). No significant difference in the levels of 8-isoprostane or 8-OH-dG was observed in cells treated with Gstm1 specific siRNA compared to control siRNA. However a small but significant increase in ‘comet’ tail length was observed in cells with reduced Gstm1 expression indicating greater DNA damage in these cells. Two miRNAs that map to the chromosome 2 congenic regions were investigated (miR-137 and miR-9-1). The expression of each miRNA was investigated in kidney from SHRSP, WKY and congenic rat strains at 5, 16 and 21 weeks of age. Expression of miR-9-1 was unchanged in the SHRSP but was significantly reduced in 16 week 2c* and WKY compared to 5 week. Expression of miR-137 was unchanged in both WKY and 2k but was significantly increased in 21 week old salt-loaded SHRSP compared to 21 week old SHRSP without salt. Successful over-expression of each miRNA was demonstrated in NRK-52E cells but inhibition of each miRNA could not be confirmed. MicroRNA target prediction methods were employed to identify potential gene targets for each miRNA. A list of predicted targets for each miRNA was generated and combined with gene expression datasets generated from 5 week, 16 week, 21 week and 21 week plus salt kidney microarray analysis carried out previously. Using Ingenuity Pathway Analysis software, predicted target genes were identified according to patterns of expression. Analysis of predicted miR-137 targets failed to find any genes that followed the correct pattern of expression (down-regulated in SHRSP but unchanged in both WKY and 2k). Of the targets predicted for miR-9-1, seven were identified that were up-regulated in both WKY and 2c* but not SHRSP. The expression levels of each of the seven genes were assessed in kidney and NRK-52E cells over-expressing miR-9-1 however none of the predicted targets could be validated. In summary, a wide range of techniques have been employed in an attempt to investigate the function of Gstm1 and the possible role of miRNA that map to the congenic regions implicated in blood pressure regulation. Although a specific role for Gstm1 in protection against oxidative stress has yet to be fully determined, the integration of targeted vectors and modulation of Gstm1 expression will allow the role of Gstm1 to be investigated more fully in vivo.

Results from the clinic support, encouragement and advice section showed an overwhelming patient perception of a very poor service from their health professionals as far as exercise was concerned. Hospital b showed consistently higher scores than the other three clinics but still only half of the respondents gave a positive impression of exercise advice and support. Over 2/3 of all respondents reported a preference for an exercise advisor at their clinic. In order to obtain a balanced picture, it was necessary to check the perceptions of the health professionals against those of the patients as research has shown that patients may believe they have not been given information from their doctors and nurses even when they have been, due to the stressful situation of hospital visits. Study 3 was designed to produce information about health professionals' real views and behaviours towards exercise both for themselves and for their patients. The methodology employed was that of focus groups which were taped, transcribed and analysed using qualitative and quantitative methods. It was clear from the results that the patients' perceptions of an inferior exercise education programme from the clinics was accurate. The health professionals freely admitted that exercise had been put last on the agenda, largely because the health professionals themselves did not understand or have knowledge of the possible protective value that exercise could have for their patients' cardiovascular, stress-reactivity and psychological well-being systems. There is a clear need for a large scale randomised control trial with long term follow up on the physiological and psychological benefits of exercise for people with insulin dependent diabetes.

The impending global pandemic of obesity, type 2 diabetes and vascular disease suggests an urgent need for both prevention strategies and effective treatment. Of all the common ethnic groups South Asians, who make up a fifth of the world’s population, have the highest prevalence of both diabetes and vascular disease. The high rates of diabetes, in particular, occur with lower average adiposity levels, suggesting that South Asians are more susceptible to the effects of obesity. Differences in insulin sensitivity and diabetes prevalence between South Asians and Europeans cannot be fully explained by differences in adiposity alone. The aim of this thesis was to investigate whether differences in oxidative capacity and capacity for fatty acid utilisation in South Asians might contribute, using a range of whole-body and skeletal muscle measures. Twenty South Asian men and 20 age and BMI-matched white European men underwent exercise and metabolic testing and muscle biopsy to determine expression of oxidative and lipid metabolism genes and of insulin signalling proteins. In fully adjusted analyses, South Asians, compared to Europeans, exhibited significantly reduced insulin sensitivity; lower VO2max and reduced fat oxidation during submaximal exercise at the same exercise intensities. South Asians exhibited significantly higher skeletal muscle gene expression of CPT1A and FASN and significantly lower skeletal muscle protein expression of PI3K and PKB Ser473 phosphorylation. Fat oxidation during submaximal exercise and VO2max both correlated significantly with insulin sensitivity index and PKB Ser473 phosphorylation, with VO2max or fat oxidation during exercise explaining 10–13% of the variance in insulin sensitivity index, independent of age, adiposity and physical activity. These data suggest that reduced oxidative capacity and capacity for fatty acid utilisation at the whole body level are key features of the insulin resistant phenotype observed in South Asians, but that this is not the consequence of reduced skeletal muscle expression of oxidative and lipid metabolism genes.

INTRODUCTION: The focus of this MD thesis has been relaxin, a member of the insulin family, which is a protein composed of two disulphide linked chains of approximately 6000 Daltons. Relaxin has been traditionally recognised as a hormone of parturition, though more recently it has been postulated that relaxin may be involved in cardiovascular regulation. We used concentrations similar to those found in the plasma in physiological (non-pregnant, pregnancy) and pathophysiological (chronic heart failure) states. Firstly, we characterised the effects of relaxin in small human resistance arteries ex vivo using wire myography obtained from gluteal biopsies taken from patients with coronary heart disease (CHD) and normal left ventricular systolic function. We also studied the same effects in larger calibre arteries (internal mammary) and veins (saphenous) using standard organ bath techniques. The effect of relaxin in veins has not previously been described. Internal mammary arteries and saphenous veins were obtained from patients undergoing coronary artery bypass surgery. Small pulmonary arteries were obtained from patients undergoing thoracotomy for bronchial carcinoma. In addition, we wished to determine if a transcardiac or transpulmonary gradient of relaxin could be measured to suggest either pulmonary or cardiac secretion or clearance of the hormone. Relaxin secretion in heart failure has previously been described. Lastly, we wished to determine whether an increased relaxin plasma concentration in patients with chronic heart failure (CHF), is of prognostic importance. METHODS AND RESULTS i)comparative potency of relaxin compared to other vasodilators: Small resistance arteries were obtained from biopsies taken from patients with CHD. Each set of vessels was preconstricted with noradrenaline. Thereafter, cumulative concentration response (relaxation) curves (CRCs) were constructed with known vasodilators 25 atrial natriuretic peptide (ANP), epoprostenol, substance P and relaxin (n=8). Relaxin was found to be a more potent vasodilator than ANP and equipotent to epoprostenol. ii) mechanism of vasorelaxation: CRCs to relaxin (as above) were constructed to identify the importance of the endothelium – following the removal of the endothelium by the established method of intraluminal rubbing with a human hair. We found that relaxin is endothelium dependent. iii) interaction of relaxin with nitric oxide and other possible mechanisms of vasodilation and importance of ACE inhibitor treatment: We identified the importance of the effect of ACE inhibitor treatment on the action of relaxin in human resistance arteries. Relaxin’s vasodilatory action was significantly reduced in those patients on ACE inhibitors (n=28) compared with those patients not on ACE inhibitors (n=30). In patients treated with an ACE inhibitor, we found that manipulation of prostanoids is important. Indomethacin, (a cyclooxygenase inhibitor) (n=8) blocked relaxin’s vasodilatory action. Manipulation of the cAMP second messenger system, with milrinone, (a cAMP phosphodiesterase inhibitor) (n=6) is also important as relaxin’s vasodilatory action was enhanced. Manipulation of cyclic GMP second messenger system is also important. ODQ, (a guanylate cyclase inhibitor) (n=10) reduced relaxin’s action while zaprinast, (a cGMP phosphodiesterase inhibitor) (n=7) enhanced relaxin’s action. Manipulation of nitric oxide with L-NAME (n=8) and L-NOARG (n=10), nitric oxide synthase (NOS) inhibitors and EDHF with apamin and charybdotoxin (potassium channel blockers) (n=7) had a curious effect causing the opposite action to that expected, by enhancing relaxin’s vasodilatory action. In patients not treated with an ACE inhibitor, we found that manipulation of nitric oxide with L-NAME (n=8) and LNOARG (n=8), is important, as both reduced relaxin’s vasodilatory action. Manipulating the cGMP second messenger system with ODQ (n=8) greatly reduced relaxin’s action. but zaprinast (n=9) did not. Manipulation of EDHF with apamin and charybdotoxin (n=8) had no effect on relaxin’s action. Manipulation of prostanoids with indomethacin (n=10) reduced relaxin’s action but manipulation of cAMP with milrinone (n=8), had no effect. 26 iv)relaxin and small human pulmonary arteries: We determined, using wire myography, that relaxin is not a vasodilator of small pulmonary resistance arteries (n=5). v)relaxin and large calibre vessels: We determined, using the organ bath technique, that relaxin is not a vasodilator of larger calibre arteries i.e. internal mammary arteries removed from patients during coronary artery bypass surgery (n=5).Relaxin is not a venodilator studying saphenous veins removed from patients during coronary artery bypass surgery (n=5). vi)transmyocardial and transpulmonary gradient of relaxin: Plasma relaxin concentrations were measured using a validated assay. Samples were taken from patients undergoing CABG surgery, from the aorta, coronary sinus, pulmonary artery and pulmonary vein. We found that in 20 patients with normal left ventricular function that there was no transpulmonary gradient but there was a transcardiac gradient suggesting net cardiac extraction of relaxin. vii)prognostic value of relaxin in patients with chronic heart failure: Relaxin was compared with N-terminal pro brain natriuretic peptide to determine whether relaxin is of prognostic importance. Plasma concentrations of the hormones were measured in 87 patients admitted with CHF. These patients were followed up for a year during which time hospitalisations due to CHF and death were recorded. While NT-proBNP was found to be a powerful and independent predictor of outcome in these patients, relaxin was not. CONCLUSION. In addition to its established role in pregnancy, relaxin has many other actions. In particular, its antihypertensive, antithrombotic and vasodilatory properties suggest that relaxin may have a central role in cardiovascular regulation.

Patients with diabetic renal disease develop elevated urinary albumin excretion rates [AER] and hypertension. Preliminary data from several groups suggest that diabetic patients handle transferrin, a protein similar in size and weight, in a different fashion from albumin. In the first part of this thesis I report the results of clinical studies of urinary transferrin excretion [TER] in diabetes mellitus. More than 80% type 1 [insulin dependent] diabetic patients have increased TER but less than 40% have increased AER. TER may be provoked by exercise in uncomplicated type 1 diabetes and the rise is proportionally far greater than that for AER. Newly diagnosed type 2 [non-insulin dependent] diabetic subjects have increased TER which falls with improved glycaemic control. Interventional studies with lisinopril, an angiotensin converting enzyme [ACE] inhibitor, in microalbuminuric and macroalbuminuric diabetic subjects show a reduction in TER independent of reduction in blood pressure. Data are presented suggesting a role for altered renal tubular function in TER in diabetes. The second part of the thesis examines the role of the system in the hypertension of diabetic renal disease. Patients with elevated AER have increased resting plasma renin activity. Those with uncomplicated diabetes show an exaggerated blood pressure reponse to exercise. ACE inhibition reduces blood pressure in hypertensive patients and AER in both hypertensive and normotensive patients.

The adrenal steroid dehydroepiandrosterone (DHEA) and its sulphate ester, DHEAS are the most abundant circulating steroid hormones in humans. Uncongugated DHEA predominately exerts its effects via its downstream conversion to active sex steroids in peripheral target tissues. In contrast the conversion of DHEAS to androgens first requires cleavage of the sulfate group, catalysed by the microsomal enzyme steroid sulfatase (STS). Conversely, DHEA is converted to inactive DHEAS by the activity of the cytosolic enzyme DHEA sulphotransferase (SULT2A1). However, in addition, evidence is growing that DHEA and DHEAS can have specific, direct effects. In this thesis, I have demonstrated that abrogation of DHEA metabolism can result in the manifestation of pathophysiological conditions. SULT2A1 requires 3'-phosphoadenosine-5'-phosphosulfate (PAPS) for catalytic activity. I have identified compound heterozygous mutations in the gene encoding human PAPS synthase 2 (PAPSS2) in a girl with androgen excess and confirmed the inactivating nature of the mutations via in vitro activity analysis. These observations indicate that PAPSS2 deficiency is a novel monogenic adrenocortical cause of androgen excess. In addition, I have demonstrated that DHEA can have specific direct effects, attenuates human adipogenesis, while enhancing glucose uptake in mature adipocytes. These findings highlight DHEA metabolism, particularly by SULT2A1, as important mechanisms regulating DHEA activity.

Glucocorticoid (GC) excess is characterised by central obesity, hypertension, proximal myopathy, insulin resistance and in some cases overt type 2 diabetes (T2D). However, the precise molecular mechanisms responsible for these observation have not been defined in detail. We have shown that GCs reduce the insulin sensitivity of skeletal muscle by impacting upon the insulin signalling cascade at several critical points: IRS1, PI3K and AS160. Furthermore, we have described a novel role of GC, and GCs with insulin, in the regulation of intramyocellular lipid metabolism, which may underpin GC-induced insulin resistance in this tissue. We have also highlighted the importance of 11\(\beta\)-hydroxysteroid dehydrogenase type 1 (11\(\beta\)-HSD1), which controls local GC availability, as a critical regulator of skeletal muscle insulin sensitivity, and have provided new insight into the insulin sensitizing actions of selective 11\(\beta\)-HSD1 inhibitors. In summary, these data highlight the importance of GCs, and pre-receptor GC metabolism in the regulation of lipid metabolic pathways and response to insulin stimulation in skeletal muscle.

Invariant natural killer T (iNKT) cells constitute an important regulatory arm of the immune system. Defects in the number and activities of iNKT cells have been linked to the development of autoimmune diseases. The glycoprotein CD1d plays an integral part in the recognition and presentation of lipid antigens such as α-galactosylceramide (α-GalCer) to iNKT cells, producing a variety of anti-inflammatory (T\(_H\)2) cytokines, such as interleukin-4 (IL-4), and pro-inflammatory (T\(_H\)1) cytokines, such as interferon-γ (IFN-γ). A decreased number of iNKT cells and defects in their capacity to produce T\(_H\)2 cytokines is associated with autoimmune diseases, such as type 1 diabetes (T1D). α-GalCer stimulates both T\(_H\)1 and T\(_H\)2 responses. Some analogues of α-GalCer preferentially induce the production of T\(_H\)2 cytokines, highlighting the possibility that such compounds could have therapeutic potential with regards to T\(_H\)1 cell-mediated autoimmune diseases, such as T1D and SLE. A library of α-GalCer analogues was synthesised and their ability to modulate immune responses analysed. Altering the length of the phytosphingosine chain in α-GalCer analogues was shown to drastically affect the T\(_H\)1:T\(_H\)2 response, with truncated phytosphingosine chains of 9 carbons skewing the response towards a predominantly T\(_H\)2 response. Substituting the galactose sugar head for glucose (α-GlcCer) or L-fucose (α-L-FucCer) also elicited differences in the immunological profile of α-GalCer analogues, with lymphocytic proliferation being greatest in the galactose analogue, followed by L-fucose, followed by a glucose analogue. These differences in activity were also mirrored in the cytokine responses of the analogues, suggesting the C4’ hydroxyl group plays a key part in antigen recognition and activity. Analogues incorporating 2 double bonds in the \(N\)-acyl chain exhibited T\(_H\)2 cytokine profiles on a par with α-GalCer, yet dramatically decreased T\(_H\)1 responses were observed. They also considerably delayed the clinical presentation of glucosuria in NOD mice. These results have provided important insights into the nature of antigen binding with CD1d, recognition of the antigen by iNKT cell receptors, and how such factors play a role in skewing the immune response, thus highlighting areas where structural diversity could be introduced in order to exploit immunomodulating potential, and find a possible prophylactic therapy for the prevention and treatment of autoimmune diseases, such as T1D.

Epithelial sodium channels (ENaC) are of immense importance, controlling Na+ transport across epithelia and thus playing a critical role in all aspects of fluid clearance as well as numerous other functions. Although extensive studies have been carried out to invistegate the regulation mechanism of ENaC, many questions still remain unclear. Therefore, we employed various techniques including electrophysiology and molecular biology approaches to investigate the mechanisms underlying the regulation of ENaCs by lipid metabolites, oxygen and mechanical stress. We have identified profound regulation mechanisms of ENaC in distal renal epithelial cells and vascular endothelial cells by lipid metabolites, heme and mechanical forces. Our results revealed a novel O\(_2\) sensitive regulation pathway of ENaC channels, in which hemeoxygenase acts as the O\(_2\) sensor and the substrate and product of which either inhibits or stimulates ENaC activity. This finding may eventually lead to novel clinic strategies in dealing with diseases e.g. renal failure, kidney reperfusion injury, hypertension, pulmonary edema and pre-clampsia. In addition, we, at the first time, have revealed that ENaC is functionally expressed in a variety of endothelial cells and is able to serve as mechano-transducing sensors in the vascular endothelial cells which play important roles in vascular physiology and pathology progresses.

Cachexia is a prognostically important development in patients with heart failure. The most commonly used definition of cardiac cachexia is loss of a percentage of body weight over time. Muscle wasting has been assumed to be the major contributor to this weight loss, and cytokine activation is postulated to be central to the pathogenesis. We hypothesised that elevated circulating cytokines in cachectic heart failure patients would be associated with muscle inflammation, injury and impaired ability to repair. The aim of this doctoral work was to characterise the nature of cachexia in patients with heart failure (HF) and stable coronary artery disease (CAD), to quantify the loss of muscle mass, and test the hypothesis that muscle wasting is mediated by the activation of tissue cytokines and cell cycle inhibitors. We studied five subject groups. Three were groups of patients with stable coronary artery disease: 1) HF-cachexia - patients with HF, reduced left ventricular systolic function and cachexia, n=10; 2) HF-no cachexia - those with HF, reduced systolic function but no cachexia, n=20; and 3) CAD - those with CAD, no symptoms of HF and preserved systolic function, n=10. The other subject groups were: 4) IDCM - patients with idiopathic dilated cardiomyopathy, n=7; and 5) HC - healthy controls, n=9. Subjects were characterised by New York Heart Association (NYHA) classification, left ventricular ejection fraction (LVEF), peak oxygen consumption (VO2), weight history and body composition analysis. Circulating levels of tumour necrosis factor-α (TNF-α), interleukin-6 (IL-6), C-reactive protein (CRP), leptin, adiponectin, and Btype natriuretic peptide (BNP) were measured. Skeletal muscle biopsies were analysed for the expression of messenger ribonucleic acid (mRNA) for TNF-α, IL-6, interleukin-1β (IL-1β), interleukin-18 (IL-18) and the cell cycle inhibitors (cyclin dependent kinase (CDK) inhibitors) p21, p27 and p57. We found that the HF-cachexia group had significantly lower body mass index (BMI) and percentage body fat than all the other subject groups. In contrast, there was no significant reduction in fat free mass index (FFMI). In addition, the HF-cachexia group had higher rates of fat oxidation than all other groups. While the HF-cachexia group had elevated circulating levels of TNF-α and IL-6, there was no increased expression of cytokines or CDK inhibitors in the skeletal muscle. Circulating adiponectin and BNP levels were elevated in the HF-cachexia group. There was a positive association between adiponectin and BNP, and a negative relationship of each with BMI and percentage body fat. In addition, adiponectin positively correlated with rate of fat oxidation and TNF-α concentration. A possible causal relationship between adiponectin and increased rate of fat oxidation was further investigated in an additional study of young healthy male subjects performing an exercise program specifically designed to maximise fat metabolism (n=11). Despite inducing significantly increased rates of fat oxidation and adiponectin concentrations no relationship was observed between them. In conclusion, cachexia in patients with heart failure and stable coronary artery disease predominantly involves the loss of adipose tissue, with no evidence of muscle wasting or inflammation. The presence of increased circulating levels of adiponectin and BNP, their association with each other, and the relationship of each with body composition, energy metabolism and TNF-α suggests these peptides may play an important role in the pathogenesis of cardiac cachexia.