Philosophiae Doctor - PhD / In this century most major medical advances have resulted in part from research on animals and non-human primates such as the African green monkey and therefore often serve as a critical link between basic research and human clinical application. Due to its close evolutionary relationship to humans, the African green monkey is known to be an excellent and most sought after models for studies of human cardiovascular disease (CVD). While the human genome project and some others related to model organisms are very well advanced or even complete, little sequence information has been acquired for the African green monkey. Given the importance of this species in biomedical research generally and CVD specifically, and the fundamental significance of sequence data, it is critical that this paucity of genome information concerning this specific animal model be addressed in order to better define the molecular basis and to further understand the mechanism of cholesterol metabolism in this species which will also contribute immensely to primatology. There is a growing interest in the role of genetic polymorphisms in predicting susceptibility to disease and responsiveness to drug interventions. Since plasma lipid abnormalities are risk factors for coronary atherosclerosis, determination of these plasma lipid concentrations, especially for genes involved in lipid transport and metabolism may be influenced by genetic variations. In this study, the African green monkey was used as a model to evaluate the effect of niacin on plasma lipids and reverse cholesterol transport by examine gene expression and the influence of several polymorphisms found in genes that are involved in cholesterol metabolism in humans. A survey of genetic variation spanning ten prioritised “candidate” genes was conducted, all of which are known to produce proteins that play key roles in the reverse cholesterol pathway (RCT), and in the homeostatic regulation of blood lipid profiles related to cardiovascular health and disease. everse transcription polymerase chain reaction (RT-PCR) was used to evaluate mRNA expression of those “candidate” genes. Twenty two coincident singlenucleotide polymorphisms (cSNPs), reported to play a vital role in RCT, were genotyped within these genes. This study’s findings implicate a subset of six of the twenty two genetic variants, spanning five “candidate” genes. To assess possible involvement of these prioritised “candidate” genes and their polymorphisms, biochemical analyses of known risk factors of coronary artery disease such as HDL-C and LDL-C were conducted. Eight healthy African green monkeys were entered in this study of which four were treated with niacin at an escalating dosage. Their mean lipid-lowering response following drug therapy was analysed, compared to those with the same genotype in a control group. Niacin treatment was associated with a considerable reduction in LDL-Cholesterol, up-regulation of HDL synthesis, and increase of apo A-1 levels. Gene expression had minimal effect on niacin treatment, except CYP7A1 which was down-regulated at the same time when considerable change in HDL-C, LDL-C and apoA-1 levels was observed. The presence of CYP7A1:Asn233Ser polymorphism may have played a critical role in metabolising niacin and influencing the up-regulation of HDL-C synthesis in the African green monkey. Although cholesterol lowering alone may explain the anti-atherosclerotic effect of niacin on HDL-C, in this study, gene expression data also shed some light in supporting the hypothesis that genetic variants may influence the expression of genes involved in RCT, which may also have played a role in the anti-atherosclerotic effect of the drug.

>Magister Scientiae - MSc / Being overweight or obese are major factors contributing to the increased morbidity and mortality due to non-communicable diseases such as type 2 diabetes, cardiovascular disease and cancer. The treatment of obesity with pharmaceutical drugs is plagued by side effects. Plants and their phytochemicals possess a number of beneficial health effects including anti-oxidant,anti-mutagenic, anti-inflammatory, anti-obesity and anti-cancer effects, mediated by activation of the adenosine monophosphate protein kinase (AMPK).AMPK controls many metabolic processes including glucose uptake and utilisation, and adipogenesis, and is often referred to as the master regulator establishing cellular homeostasis.Cyclopia maculata, commonly known as honeybush, is an indigenous South Africa plant possessing anti-oxidant, anti-inflammatory and anti-cancer properties. Recently, others in our laboratory have shown that a hot water extract of fermented C. maculata inhibits adipocyte differentiation in 3T3-L1 pre-adipocytes, with some evidence of weight regulatory properties in a Wistar rat model of diet-induced obesity. In the rat study, 21 day old weanlings were fed a high fat, high sugar cafeteria diet for 3 months with (n=10) or without (n=10) C. maculata supplementation. This group of rats was referred to as the lean group (n=20). Another group of rats were fed a cafeteria diet for 4 months to induce obesity (obese group, n=20) and thereafter treated as described for the lean rats. The aim of this MSc study was to determine whether C. maculata induces AMPK activation.Proteins were extracted from the liver and muscle tissue of lean and obese Wistar rats using an optimized extraction method with a commercial lysis buffer and the TissueLyser.Treatment with the C. maculata extract had no effect on the protein yield in lean and obese rats. Interestingly, the protein yield in the liver of obese rats was significantly higher than that observed in lean rats. Although C. maculata treatment slightly increased AMPK activation (calculated as the ratio of phosphorylated AMPK to total AMPK) in the liver of lean and obese rats, the difference was not statistical significant. Conversely, C.maculata treatment decreased AMPK activity in muscle of lean and obese rats, with statistical significance observed in the lean group only (2.3-fold, p<0.05). Differences in AMPK activation between the groups were also noted, a 1.3-fold decreased activity observed in obese groups compared to their lean counterparts, although this was not statistically significant. Expression of PPARα, a downstream protein target affected by AMPK activation was reduced in the liver of lean and obese rats after C. maculata treatment. Moreover, PPARα expression was significantly higher in obese compared to lean rats (2.7-fold, p<0.001). PPARα is a transcription factor mediating fat metabolism (β-oxidation) and its expression is induced by circulating free fatty acids, which are increased in obese compared to lean rats. The expression of PPARα in muscle was too low for Western blot analysis and quantification.Cyclopia maculata treatment did not affect hepatic expression of UCP2, another protein important in establishing energy homeostasis. The expression of UCP2 was 2.9-fold higher in the liver of obese rats compared to their lean counterparts, although the difference was not statistically significant. The opposite results were observed in the muscle where C. maculata treatment decreased UCP2 expression in lean rats (2.8-fold,p<0.0001), and UCP2 expression was decreased 1.4-fold in obese rats compared to lean rats, although the difference was not statistically significant.ELISA results for AMPK activation revealed that C. maculata treatment increased AMPK activity, although not statistically significant. Histological analysis of retroperitoneal fat showed that C. maculata did not affect adipocyte size and number, although a slight decrease in adipocyte size was observed after treatment .This study has demonstrated that treatment of the cafeteria diet fed Wistar rats with 300 mg/kg of a hot water extract of fermented C. maculata does activate AMPK. This study revealed important differences between lean and obese rats. In particular, increased hepatic protein content, PPARα and UCP2 expression was observed in obese rats compared to the lean group. This suggests an adaptive response to the increased circulating free fatty acids during obesity and an increase in β-oxidation in these animals.

An infarct is an area which has lost its blood supply due to obstruction, thrombosis or embolism. It is the third leading cause of death in the Western world, following non-cerebral cardiovascular disease and cancer. This research study focused on determining the infarct prevalence according to age, sex and brain areas most affected by infarcts. The prevalence of different infarct types was also determined. Brain MRI statistics were obtained from a Private Radiology practice in Pretoria for a 13-month period. A total of 1844 brain MRI examinations were evaluated, of which 299 patients presented with infarcts. Their age and sex were noted and their individual reports were obtained to record the anatomical structures and brain lobes that were infarcted. The infarct types were also noted. Diffusion-weighted images were used to measure new infarcts, while FLAIR images were used to measure old infarcts. Results showed an overall incidence of 16.10% and vascular structures accounted for 26.63% of these. Most infarcts were new (56.80%) and mainly affected patients aged 70–79 years (31.36%). Normal cerebral infarcts (72.49%) and embolic infarcts (14.50%) were the most common. The parietal lobe (34.91%) and right middle cerebral artery (11.54%) presented with the most infarcts. The right hemisphere (34.91%) presented with slight infarct predominance, but this was not significant when compared to the left (31.95%) hemisphere (Chi square p>0.05). No significant difference was found concerning the overall male to female ratio (Chi square p>0.05). Females aged 18–39 years of age presented with three times more infarcts than their male counterparts. This may possibly be due to their use of oral contraceptives and pregnancy, which increases the risk of thrombosis and embolism. Females over 80 years also presented with higher infarct prevalence, which is expected, since men die at earlier ages due to other co-morbidities such as cancer. / Dissertation (MSc)--University of Pretoria, 2009. / Anatomy / MSc / Unrestricted

Methylmercury (MeHg) is known predominantly as a neurotoxicant, however emerging experimental and epidemiological evidence has shown associations between MeHg exposure and the potential for increased risks of cardiovascular diseases. This thesis investigated the in vitro cytotoxic effects of MeHg in two cardiomyocyte cell lines, H9C2 rat neonatal cell line and AC16 adult human cell line. We observed significant increases in cell death at concentrations from 1 – 10 µM. ROS production and intracellular calcium concentrations increased dose-dependantly with MeHgCl exposure. Furthermore, while assessing mitochondrial function, a decline in maximal respiration at 1 µM was seen. However, these observations may in turn be a direct consequence of decreased cell numbers following exposures. Additionally, this study highlighted the differences in cellular bioenergetics which may impact how certain cells respond to contaminant stressors. The distribution of MeHg and total Hg in rat heart tissues was also examined and we observed increasing concentrations of MeHg in high and low dosed rat groups as compared to the vehicle controls. No difference was observed in Hg levels between the normal and high fat and sugar diet groups. The urinary isoprostane levels, which are indicative of systemic oxidative stress, showed significant increases in lean rats exposed to the high dose treatment. It was also observed that a high fat and sugar diet in lean and obese rats can contribute to increasing oxidative stress regardless of the level of contaminants they were dosed with. This thesis demonstrated several in vitro effects of MeHg on heart cells as well as determine the distribution of Hg levels in heart tissues and oxidative stress markers from an in vivo study.

Acute myocardial infarction (MI) is the end result of many cardiovascular diseases and is one of the leading causes of death in the western world. Cell therapy, using circulating angiogenic cells (CACs) or CD34+ cells from peripheral blood, is one approach under investigation for restoring blood flow and function to the ischemic heart. However, the numbers of CACs and CD34+ circulating cells are inversely proportional to the severity of cardiovascular disease and age; therefore, there is a need to increase their numbers and/or function for therapy. One possibility is to enhance the therapeutic potential of the cells with the use of a biomaterial. In this study, we used a collagen matrix to culture human CD34+ circulating cells, and evaluated the effect of the matrix on CD34+ cell properties and function. The matrix was able to successfully increase proliferation, migration, CD34+ phenotype and branching in an angiogenesis assay. These functional benefits may be associated with the sonic hedgehog (Shh) pathway. The collagen matrix was previously shown to enhance the function of healthy CACs, but its ability to do the same for CACs from coronary artery disease patients is unknown. In this study, the matrix was shown to enhance the viability, proliferation and angiogenic potential of patient CACs. Furthermore, gene expression for integrins and Shh pathway components in the sub-population of CD34+ cells was similar between patient and healthy donors when isolated from CACs. This work provides insight into the mechanisms for the observed matrix-enhanced function of therapeutic CACs and CD34+ cells from both healthy and CAD patient donors.

Magister Pharmaceuticae - MPharm / Hypertension is by far the most common risk factor for cardiovascular disease (CVD), which has been identified as the leading cause of death worldwide and a major economic burden in developing countries. Over the years, there has been an increased interest in isolating and identifying bioactive compounds from medicinal plant, with the aim of finding alternative sources of therapy to some of the problematic synthetic drugs and to validate the therapeutic use of some traditional plants. The renin angiotensin aldosterone system is a key regulator of blood pressure, on which ACE (Angiotensin Converting Enzyme) inhibitors act and have been at the forefront of therapeutic strategy for treatment and management of hypertension and CVD. But despite the success of ACEI’s, their long term use has been associated with side- effects coupled with its contraindication in pregnancy. The plant of interest, Centella asiatica is a widely known medicinal plant, used in treatment of a variety of conditions including hypertension. There is currently no scientific evidence validating its claimed use in hypertension. This study therefore, investigated the ACE inhibitory effects of Centella asiatica. Crude methanol, ethanol and aqueous extracts of Centella asiatica were assayed for ACE inhibition activity. Methanol and ethanol crude extract(s) was subjected to a bioassay guided fractionation process to isolate and identify the active compounds. A fluorescence based ACE assay was utilized at various stages of the process including HPLC purification stage to screen the fractions and compounds for ACE inhibition activity.

Background: Myocardial infarction (MI), also known as heart attack, is the leading cause of morbidity and mortality among the heart diseases spectrum. It results from an insufficient supply of blood to the heart muscles. According to the United States (U.S.) Centers for Disease Control and Prevention (CDC), about 610 000 people die of heart disease in the U.S. every year. Myocardial infarction contributes 370 000 of these deaths annually. Every 40 seconds, someone in the U.S. experience heart attack. This burden is disproportionately distributed within the U.S. population. The rate of heart disease in Central Appalachia is 249 per 100 000, 42% higher than the national rate. Exploring further within the region, rural areas experience higher heart disease mortality rates; 27% higher than the region’s metro counties. According to 2018 America Health Ranking, the prevalence of heart attack in Tennessee is 5.9%, compared to the 4.9% nationwide, with the majority of the burden seen among adults aged ≥65 years and with a 1:1.8 female to male ratio. Patients with heart disease often have other comorbid conditions such as peripheral arterial disease (PAD), hypertension, diabetes, dyslipidemias, which contribute immensely to this chronic condition. Therefore, the aim of this study is to explore the association between cardiovascular comorbidities such as diabetes mellitus, hypertension and dyslipidemia, and the prevalence of heart attack among patients with PAD in rural Central Appalachia. Methods: We used a cross-sectional data of patients diagnosed with PAD in the Central Appalachian region. A total of 13455 patients with PAD were recruited using ICD 9 and 10 search terms for PAD from the electronic medical records (EMR) system between January 1, 2008, and April 30, 2018. Descriptive statistics of the variables were extracted. The association between the comorbidities, including hypertension, diabetes, dyslipidemia, body mass index(BMI) and the prevalence of MI was determined using a binomial logistic regression model. All analysis was done using IBM SPSS statistics 25. Results: Of the total 13455 patients with PAD, 3045 had MI (37.7% female and 62.3% male) with a mean age of 69±10.5years. While 93% had hypertension, 56% had diabetes. For the lipids, the mean of HDL, Cholesterol, and LDL among participants with a history of MI is 40.99mg/dl±13, 156.32mg/dl±45, 82.08mg/dl±36.35 respectively. The results of binomial logistic regression with stratification based on gender shows that female patients with diabetes had 86% increased odds of MI [OR: 1.858, C.I: 1.308-2.638, p-value=0.001), and for female hypertensives, 4.51 times increased odds of MI was found (C.I: 1.576-12.895, p-value=0.005). The male diabetics and hypertensives showed a similarly increased odds of MI with (OR 1.138, C.I: 0.870-1.489 p-value=0.345) and (OR 3.697C.I: 1.559-8.736, p-value=0.003) respectively. No significant association was found among the various lipid profiles examined. Conclusion: The results showed that female PAD patients with hypertension and diabetes have a significantly increased likelihood of having MI. In contrast, male with PAD also showed increased likelihood (although to a lesser degree) of MI in those with hypertension, but not those with diabetes. These findings underscore the importance of a proactive approach to preventive care and adequate control among PAD patients with diabetes and hypertension in a bid to curbing the morbidity and mortality associated with myocardial infarction among residents in Central Appalachia.

No description available.

Elevated plasma cholesterol is a risk factor for cardiovascular disease. Proprotein convertase subtilisin/kexin type 9 (PCSK9) hinders the uptake of low-density lipoprotein cholesterol (LDL-c) by mediating degradation of LDL receptors (LDLRs) in the liver. Gain-of-function (GOF) mutations in PCSK9 cause familial hypercholesterolemia (FH). In normolipidemic human plasma, 30-40% of PCSK9 is bound to LDL particles, and this association with LDL inhibits PCSK9’s ability to mediate LDLR degradation in cultured cells. To further investigate the physiological relevance of this interaction, we analyzed natural GOF mutations in PCSK9 and assessed their effects in vitro on LDL binding, LDLR binding and LDLR degradation. Our results indicate that several GOF mutations severely inhibit LDL binding compared to wild type (WT) PCSK9, and only modestly affect LDLR affinity and LDLR degradation. These findings shed light on the potential physiological relevance of the PCSK9-LDL interaction, which may have an inhibitory effect on PCSK9 activity in vivo.

Magister Pharmaceuticae - MPharm / Cardiovascular diseases (CVDs) such as ischaemic heart diseases, heart failure and stroke remain a major cause of death globally. Various deep-rooted factors influence CVD development; these include but are not limited to elevated blood lipids, high blood pressure, obesity and diabetes. A considerable number of proteins are involved directly and indirectly in the transport, maintenance and elimination of plasma lipids, including high and low-density lipoprotein cholesterol (HDL-C and LDL-C). There are several mechanisms involved in the removal of LDL particles from systemic circulation. One such mechanism is associated with the gene that encodes proprotein convertase subtilisin/kexin type 9 (PCSK9), which has become an exciting therapeutic target for the reduction of residual risk of CVDs. Currently, statins are the mainstay treatment to reduce LDL-C, and a need exists to further develop more effective LDL-C-lowering drugs that might supplement statins. This study was aimed at contributing to the generation of knowledge regarding the effect of niacin in reducing LDL levels through PCSK9 interaction.