Objective:  Obesity has become the leading cause of mortality worldwide; however, the fundamental pathophysiology underlying this association remains unclear. The discovery of adipokines, i.e., cytokines produced by adipose cells (adipocytes), revealed that adipose tissue is a highly endocrine organ, thus opening new lines of investigation. The prototypical adipokine leptin increases in obesity, and leptin receptors are found in vascular cells. However, results are contradictory regarding the role of leptin in vascular and endothelial functions. Leptin has been shown to elicit vasodilatation, but has also been linked with atherosclerotic and thrombotic disease. The main aim of the present thesis was to study the association of circulating levels of leptin with markers of endothelial function, and to analyze the effects of leptin infusion in vivo  on vasomotor function and endogenous fibrinolysis. Material:  Four associative studies and two interventional studies were conducted. The former included DISARM (studies 1 and 2), the PIVUS study (study 3), and the Scottish post-infarction study (study 4). The DISARM studies and study 4, respectively, recruited 20 men and 83 men and women with stable ischemic heart disease. Study 3 included a random sample of 1016 subjects (54% women, 70 years old) living in the community of Uppsala, Sweden. For the interventional studies (studies 5 and 6), 10 healthy men were recruited for each study. Methods:  In all studies, endothelial function was estimated based on forearm blood flow (FBF) as measured by strain-gauge venous occlusion plethysmography, at rest or during infusion of vasodilators. In study 3, additional measurement techniques were used, such as brachial ultrasound flow-mediated dilation (FMD) and the aortic augmentation index (AoAIx) by tonometry in the radial artery. Fibrinolytic status was estimated based on basal and stimulated levels of tissue plasminogen activator antigen (t-PA), and by assessment of the endothelial release of t-PA (net t-PA release). Plasma leptin levels were measured by radioimmunoassay. In the associative studies, endothelial function and fibrinolytic status were related to circulating plasma leptin levels. In the experimental studies, exogenous leptin was administered in the brachial artery and endothelial function was assessed by strain-gauge plethysmography Results:  In elderly men and women, leptin was independently associated with decreased endothelial-dependent and -independent vasodilatation, reflecting disturbed endothelial function in resistance vessels. This association was attenuated after adjustment for BMI, and when analyzed among subjects with high plasma leptin levels. FMD (a measure of endothelial function in conduit vessels) was not associated with leptin. Exogenous leptin infusion did not alter vasomotor tone, but the endothelium-dependent and -independent vasodilatation was impaired during concomitant infusion of leptin and vasodilators. Infused leptin in the forearm did not affect blood pressure or pulse rate. Chronic hyperleptinemia, but not acutely induced hyperleptinemia, was associated with release of endothelial tissue plasminogen activator (net t-PA). Conclusions:  In humans, leptin was associated with impaired vasodilatation. However, this relationship was blunted after adjustment for BMI, suggesting that leptin could be the mediator between obesity and impaired vascular function. Furthermore, the observed lack of association in hyperleptinemic subjects may reflect a state of leptin resistance. The experimental result showing attenuated vascular reactivity following leptin infusion is in accordance with the results of the associative studies. The augmented net t-PA release in patients with chronic hyperleptinemia may indicate a state of “vascular activation,” which was not observed in healthy endothelium during a short period of leptin infusion. This thesis addresses several controversial issues regarding the action of leptin on vascular tissue in humans. The final results indicate that the in vivo action of leptin on vascularity is complex and mediated by several mechanisms. Our findings suggest that leptin is an important mediator between obesity and endothelial dysfunction, and should stimulate further investigation of this matter. / Manuel Cruz Gonzalez

Both hypertrophic (HCM) and dilated cardiomyopathy (DCM), though differing in their aetiologies, share features of impaired resting energetics. The aim of this thesis was to determine if cardiac high energy phosphate metabolism, measured as the phosphocreatine (PCr)/ATP ratio using 31Phosphorus magnetic resonance spectroscopy (31P MRS), is further impaired during exercise in these pathologies. This would provide a possible explanation for the high incidence of exercise related death in HCM and DCM as well as the blunted inotropic response to exercise in DCM. Furthermore, this thesis investigates the role of stress perfusion and stress tissue oxygenation in HCM (as these are hypothesized to exacerbate the primary defect in energetics) and exercise training in DCM (which is hypothesized to improve function though the mechanisms are uncertain). This work developed a novel protocol for measuring 31P MRS in a clinically acceptable time frame. The traditional acquisition is at least 20 minutes (as much as 40 minutes in subjects with lower pulse rates). This is a particularly long time to allow for exercise in the magnet particularly in the symptomatic DCM cohort. Hence this work meticulously developed a shorter 8 minute protocol. Its validity, reproducibility and application to exercise were confirmed. The post processing of the MRS data was further improved for calculating blood contamination and tested with both simulated and patient data, including normal, hypertrophied and thinned myocardium. Applying this new method, this thesis is the first to report a further decrease in exercise energetics in HCM. The relationship between perfusion, tissue de-oxygenation and energetic compromise during exercise was then explored in HCM. Athletes, with physiological hypertrophy, were used as an additional control group in these experiments. These results demonstrated a strikingly blunted oxygenation response of the HCM heart to stress even in the pre-hypertrophy HCM mutation carriers. However, as a group, the data did not show a correlation between the blunted oxygenation response and the percentage change in PCr/ATP during exercise. None-the-less, these results can potentially be useful for distinguishing between hypertrophy in the athletes and pathological hypertrophy in HCM and for distinguishing HCM mutation carriers’ pre hypertrophy and the normal heart. In the DCM cohort, this thesis explored the impact of exercise training on cardiac metabolism and function. The results showed no change in cardiac energetics and left ventricular ejection fraction during 8 minutes of exercise. In addition, an eight week home exercise programme did not alter resting or exercise cardiac PCr/ATP, but improved cardiac function during rest and exercise, and increased exercise tolerance and quality of life scores. In conclusion, this thesis reports further insights into cardiac exercise energetics in HCM and DCM and its relationship to perfusion and oxygenation in HCM and to exercise training in DCM. Therapies that decrease the energy cost of cardiac work during exercise may prove beneficial targets to explore further in these conditions.

The management of the asymptomatic patients with severe aortic stenosis (AS) is challenging; clinicians have to balance the risks of early surgery against the risk that irreversible myocardial damage may occur with a conservative management strategy. It has become increasingly apparent that prognosis in asymptomatic AS depends not only on the degree of valvular stenosis, but also on the myocardial response to pressure overload and understanding the mechanisms of myocardial decompensation may help to guide management in the future. The degree of myocardial fibrosis, microvascular dysfunction, hypertrophy and left ventricular (LV) geometry may all play important roles. However, current guidelines for management of asymptomatic AS limit assessment of the myocardium to the measurement of ejection fraction with echocardiography. More advanced techniques may provide greater information that could be clinically useful. This thesis seeks to further our understanding of the mechanisms of the myocardial response to AS, using Cardiac Magnetic Resonance (CMR) in patients with moderate and severe AS. Myocardial perfusion in AS is examined in chapter 3. The results show that CMR first pass perfusion can be carried out safely and is well tolerated by AS patients. Microvascular dysfunction in these patients was associated with age, exercise time and markers of diastolic dysfunction. Myocardial strain is examined in chapter 4, utilizing a new software tool to look at strain throughout the left ventricle, and also to explore the relationship between strain and myocardial fibrosis. The results show that there are significant variations in circumferential strain measurements, depending on slice position in the LV, and also that there was no relationship found between strain and the degree of LV fibrosis. In chapter 5, the potential of CMR T1 mapping to identify fibrosis is examined using a new shortened non-contrast sequence (ShMOLLI - Shortened Modified Look-Locker Inversion) developed in our unit. CMR T1 values were validated against histological quantification of myocardial fibrosis in a large group of moderate and asymptomatic AS. A good correlation was found between ShMOLLI derived T1 values, with T1 values increasing with the severity of AS. The clinical value of measuring myocardial perfusion and LV global strain is examined in chapter 6 by linking these to prognosis. Measurement of circumferential strain could predict prognosis in asymptomatic AS, but myocardial perfusion showed poor ability to predict events. In conclusion, this thesis offers further insights into the changes that occur in the myocardium of patients with asymptomatic moderate and severe AS, using established and new CMR techniques. The clinical value of measuring these CMR parameters to aid risk stratification is shown, and the future potential for monitoring new therapies in these patients is discussed in the final chapter.

Hyperpolarization via the Dynamic Nuclear Polarization (DNP) technique has revolutionized our ability to study metabolic changes in real time. The aim of this thesis was to build upon previous work centered around the use of DNP within the isolated perfused rat heart, a well established model system for the study of cardiac metabolism, to enhance the information that can be obtained through the combination of DNP with perfused organs. Initially this was done by using the widely studied DNP probe, [1-¹³C]pyruvate, to generate images of metabolism within the isolated perfused rat heart. The developed technique was then successfully demonstrated in two models of myocardial infarction. The thesis then proceeds to develop an understanding of how the supra-physiological concentrations of [1-¹³C]pyruvate commonly used in DNP experiments can affect metabolism in the isolated perfused rat heart, and the way in which the myocardium responds to those changes if it is not adequately supplied with substrates ordinarily present in vivo, namely fatty acids. New methods of providing the heart with these required substrates were developed, without significant interference to the biochemical information acquired from DNP experiments. As [1-¹³C]pyruvate only provides information on a small subset of carbohydrate metabolism, the next chapter develops new compounds to be used with DNP, which would allow the exploration of short chain fatty acid metabolism (butyrate) as well as ketone body metabolism (β-hydroxybutyrate and acetoacetate), and other aspects of carbohydrate metabolism (lactate and alanine). These compounds were developed and then tested for their potential usefulness in the isolated perfused heart. Finally, as the isolated perfused rat heart lacks the diversity of genetic disease models available in the mouse, the final chapter expanded the use of DNP to the isolated perfused mouse heart with all the size challenges that this entails, and makes the transition from the heart to the liver, in order to provide an alternative metabolic viewpoint on the biochemistry of disease models. This thesis thereby permits studies involving isolated perfused organs to be carried out whilst exploiting all the tools that DNP has to offer and consequentially, allows for a vast array of physiologically derived information to help us better understand metabolic diseases.

The studies presented in this thesis were undertaken to investigate the cellular and molecular mechanisms responsible for sympathetic hyperactivity that is observed in the Spontaneous Hypertensive Rat (SHR) and whether these abnormalities arise even before the onset of hypertension. Moreover, selected molecular candidates related to oxidative state in cardiac autonomic signalling have been explored for their potential therapeutic effects. <b>Chapter One</b> is an overview of (i) the relevance of autonomic dysfunction in cardiovascular disease in both human and animal models, (ii) the physiological basis of cardiac sympathetic neurotransmission, (iii) the neuromodulators of peripheral cardiac sympathetic-vagal balance discussed along with how they may be involved in cardiac adrenergic control of neurotransmission and NO-cGMP signalling. This develops the formulation of the specific aims of the thesis. <b>Chapter Two</b> outlines a detailed rationale for the experimental approach taken to (i) characterise protein expression in the pre-hypertensive animal model with immunohistochemistry and Western blotting, (ii) manipulate selected gene expression to amplify NO-cGMP signalling in vivo and in vitro via viral gene transfer, (iii) investigate calcium handling in cardiac sympathetic stellate neurons with calcium imaging , (iv) measure cardiac noradrenergic neurotransmission from double atria using radioactive-labelled [³H]-noradrenaline. <b>Chapter Three</b> demonstrated abnormal NO-cGMP signalling in pre-hypertensive SHRs. Endogenous nNOS protein residing in both cardiac parasympathetic and sympathetic neurons was significantly lower in the pre-hypertensive SHR compared to aged-matched WKYs. This was associated with lower cGMP levels. An enhanced depolarization evoked [Ca²⁺]i transient was observed in cardiac stellate neurons from pre-hypertensive SHR when compared with the WKY, an effect that was reversed by nNOS or sGC inhibition. <b>Chapter Four</b> investigated the role of nNOS and brain natriuretic peptide (BNP) in cGMP signalling pathways. Gene transfer of nNOS via adenoviral vector in SHR cardiac sympathetic neurons increased cGMP concentration and normalised neuronal calcium handling during depolarization. BNP significantly reduces [3H]- noradrenaline release. Overexpression of PDE2 which facilitates the breakdown of cGMP caused an increase in [³H]- noradrenaline release in response to field stimulation and also prevented the inhibitory action of BNP. <b>Chapter Five</b> examined the role of the nNOS adaptor protein, CAPON in NO-cGMP signalling. Endogenous CAPON protein is present in cardiac sympathetic neurons in the WKY, and is significantly reduced in pre-hypertensive SHR cardiac neurons. Artificial up-regulation of cardiac sympathetic CAPON via targeted gene transfer directly attenuated neuronal Ca²⁺ transients, resulting in decreased noradrenaline release in the SHR. <b>Chapter Six</b> is a concluding discussion summarising the main findings from this thesis, placing them in a physiological context and discussing avenues for further research.

Metabolism of the transport protein albumin is known to be regulated by long-term nutrient status. To assess the short-term response to feeding, studies were performed which measured the rate of albumin synthesis once after an overnight fast and once after one of two feeding regimens consisting of five small hourly meals or one large meal. Albumin synthesis increased by approximately 25% and 31% above the fasting value for the small meals and large meal regimens respectively, thus demonstrating that albumin synthesis is acutely sensitive to nutrient intake. A supplementary animal experiment indicated that this response is part of an overall increase in liver protein synthesis. Elevated plasma fibrinogen concentration has been implicated as a risk factor for the development of cardiovascular disease. As cigarette smokers are known to have a significantly raised plasma fibrinogen concentration, studies were performed to investigate the metabolic mechanism responsible for this by comparing the rate of fibrinogen synthesis in smokers and non-smokers and smokers who had abstained from smoking for 14 days. Smokers had a significantly greater absolute rate of fibrinogen synthesis (mean SD, 21.5 1.9 versus 16.0 1.4 mg/kg/d, p<0.05). Although smokers were found to have moderate leucocytosis, there did not appear to be any increased metabolic activity of lymphocytes. In a separate group of smokers, abstention from smoking for 14 days resulted in a significant reduction in plasma fibrinogen concentration of 19%. The fractional rate of fibrinogen synthesis also decreased significantly by 14% suggesting that a substantial part of the hyperfibrinogenaemia observed in smokers is the product of increased output by the liver.

Basic and clinical research documents the benefits of dietary milled flaxseed (MFX), a rich source of alpha-linolenic acid (ALA) and lignans, in the attenuation of risk factors key to regulating cardiovascular disease (CVD) progression. ALA has antihypertensive properties and the lignan metabolites, enterodiol (END) and enterolactone (ENL), have antioxidative potential. The effectiveness of these bioactives to reduce risk factors of CVD may be dependent upon their plasma concentrations. To study this, we first designed and validated a method using supported liquid extraction and gas chromatography/mass spectrometry to isolate and quantify enterolignans in plasma. Applying this technique, we examined MFX doses of 10-40 g/d administered to healthy, younger adults (18-49 years of age) for 4 weeks. Ten g/d was sufficient to significantly increase circulating ALA (1.5 fold) and enterolignans (5-31 fold). There was no significant dose-dependent response. In another investigation, younger (18-29 years of age) and older (45-69 years of age) healthy adults were studied to determine if age influenced enterolignan metabolism. CVD is associated with advanced age but older people may not be able to obtain lignan metabolites from dietary MFX. Following 4 weeks of MFX consumption, both age groups increased plasma total enterolignans (END + ENL) with no between-group differences. This suggested that older and younger adults metabolize MFX lignans equally. A final study assessed MFX bioactives in plasma of peripheral artery disease patients >40 years of age. Plasma enterolignans increased 10-50 fold and ALA 1-2 fold after only one month of MFX ingestion. Dietary MFX also attenuated total (11%) and LDL (15%) cholesterol in these patients after 1-6 months of administered MFX compared to placebo. The attenuation in cholesterol was due to the high fiber content of flaxseed, and not to ALA and enterolignans, despite their marked increase in circulation. MFX did not interfere with cholesterol-lowering medications but instead decreased cholesterol levels beyond the effects of medications alone. To conclude, dietary supplementation with MFX resulted in an increase in plasma enterolignan and ALA concentrations in healthy younger and older adults and in patients with pre-existing CVD. The cholesterol-lowering benefits of MFX were additional to cholesterol-lowering drugs and likely attributed to MFX fiber. / May 2016

Magister Public Health - MPH / Background: Hypertension (HTN) complications are one of the leading causes of disability and mortality worldwide, with increasing trends noted in Africa. The most neglected causes of uncontrolled HTN and its complications are unhealthy diets, excess alcohol consumption and physical inactivity. Adherence to recommended lifestyle modifications remains low in Zimbabwe. This study seeks to explore the factors influencing adherence to World Health Organisation (WHO) lifestyle modification recommendations in patients with hypertension at Parirenyatwa Hospital, Harare. Methodology: An analytic cross-sectional study design was utilized. 328 hypertensive patients aged at least 18, receiving care at Parirenyatwa Hospital were recruited into the study. A self-administered questionnaire was used to collect information on demographics, knowledge and adherence to WHO recommended lifestyle modifications from participants. Statistical Package for Social Scientists (SPSS) version 20 was used for data analysis. The Spearman test was used to test for linear correlation among variables and the 5-point Likert Scale was utilized to categorize the extent of practice of dietary and physical activity recommendations by WHO.

A dissertation submitted to the Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, in fulfillment of the requirements for the degree of Master of Science in Medicine Johannesburg, 2005 / ABSTRACT INTRODUCTION The risk of cardiovascular disease in patients with end stage renal disease (ESRD) is far greater than in the general population. Amongst patients with ESRD, the prevalence of coronary artery disease (CAD) and congestive heart failure is approximately 40% compared with 5-12% in the general population. The excess risk is caused by multiple traditional and non-traditional risk factors for ischaemic heart disease present in these patients. There is little information on CAD and its risk factors in black haemodialysis patients as most of these studies were carried out in the white population. This study is therefore aimed at determining the risk factors for atherosclerosis in Black and non-black (White and Indian) South African patients on haemodialysis. METHODS Fifty-eight black patients and twenty-six non-black patients on haemodialysis were recruited. Sixty-three age and sex matched controls (staff, students and kidney donors) were also recruited. Fasting venous blood samples were drawn for measurement of Creactive protein, homocysteine, Lp (a), serum lipids and adiponectin. Carotid intima-media thickness and plaque occurrence was measured by B-mode ultrasonography. Echocardiography was used to determine LVH. vi RESULTS Haemodialysis (HD) patients had significantly lower total cholesterol, LDL cholesterol and triglycerides compared with controls (p<0.001; p= 0.042). Hs-CRP, adiponectin and homocysteine levels were significantly higher in patients compared with controls (p< 0.001). The prevalence of plaques was significantly higher among HD patients (32%) compared with controls (7%) X2 = 60.72 p< 0.001. LVMI was significantly higher among HD patients (194.25± 7.69gm/m2) compared with controls (93.21 ± 3.27 gm/m2) p < 0.001. No significant difference between patients (Black or Asian/White) and controls with respect to CIMT was found. CVD risk factors in black haemodialysis patients and black controls showed a similar pattern to the whole study population combined. Risk factors associated with CIMT on regression analysis were total cholesterol, LDL-cholesterol, age, Hs-CRP, family history of CKD. Risk factors associated with plaque occurrence on logistic regression analysis were age, systolic blood pressure, male gender, smoking, calcium phosphate product and serum phosphate. CONCLUSION HD patients have a high prevalence of traditional and non-traditional risk factors for atherosclerosis and this is independent of race. Traditional risk factors like lipids were much lower in ESRD patients. HD patients showed a high prevalence of atherosclerosis as measured by increased carotid intima-media thickness and plaque occurrence in carotid arteries. Hs-CRP correlated significantly with a surrogate marker of atherosclerosis (CIMT).

Functional biomaterials are being developed as scaffolds to support endogenous cells and to promote the regeneration of ischemic tissue. The aim for this study was to develop a new translational platform for injectable hydrogels using recombinant human collagen (rHC) of two types: type I (TI) and type III (TIII). The collagen solutions were characterized to ensure batch-to-batch consistency and protein integrity. The hydrogel preparation protocol was extensively monitored to ensure ease of use and high-quality production. Post-gelation, rHC TIII have a higher viscosity compared to rHC TI, yet water content was high for both hydrogels. The cross-linking degree is similar for both rHC hydrogels, which are stable well above physiological temperatures, but rHC TI is more susceptible to enzymatic degradation than rHC TIII. Furthermore, the micro-architecture differed with pore size dimensions of rHC TIII being significantly larger than that of rHC TI. Cardiac fibroblasts were cultured on the rHC hydrogels, and cells attached readily to the scaffold environment, which promoted proliferation. The rHC matrices mechanical and biological properties provide structural support, and demonstrate biodegradability and biocompatibility. The intrinsic physical differences between the rHC hydrogels will likely have implications in future studies. In conclusion, the rHC TI and TIII hydrogels are proven to be suitable matrices for continued investigation towards future translational applications.