Doctoral Degrees (Medical Physiology)
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- ItemBasic semen parameters assisted by Computer-Aided sperm analysis (CASA) and their correlations with advanced semen parameters in normozoospermic men with different abstinence periods(Stellenbosch : Stellenbosch University, 2018-03) Ayad, Bashir Mohamed; Du Plessis, Stefan S.; Van der Horst, Gerhard; Stellenbonsch university. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences : Medical PhysiologyIntroduction: Affordable basic semen analysis remains a fundamental procedure to be performed routinely during the diagnosis of male infertility. Advanced semen analyses, provide valuable clinical insights in treatment related decision-making, but these are highly expensive and lack universal standardization. The World Health Organization (WHO) guidelines for semen analysis have been adopted by most human andrology and fertility laboratories around the world for more than thirty years. According to the most recent prescribed guidelines of the WHO, subjects must remain abstinent for a minimum period of two days, but not longer than seven days before collecting a sample for a standard semen analysis. Several studies have sought to determine the optimal period for ejaculatory abstinence. However, the results are often found to be contradictory. The aims of this study are two-fold: Aim I: To investigate the effect of short (4 hours) and long (4 days) abstinence periods on sperm quality based on functional and biochemical parameters in a population of normozoospermic men, in addition to the prediction of various basic and advanced semen parameters of the second (4 hours) ejaculate from a set of basic parameters obtained from the first (4 days) ejaculate. Aim II: Establishing a correlation between basic semen parameters assisted by Computer-aided sperm analysis (CASA) and a set of advanced semen analysis tests. To determine cut-off values for advanced semen parameters from various basic parameters based on WHO defined reference values. Methods: Semen samples were collected from one hundred potentially fertile, normozoospermic men (20 to 30 years) who abstained for a period of exactly 4 days and 4 hours prior to collection of the first and second ejaculates respectively. Semen samples were analysed according to the WHO guidelines. Sperm concentration, total sperm count (T.S.C.), total and progressive motility and kinematic/velocity parameters were analysed by CASA. Sperm viability was performed by dye exclusion and morphology via SpermBlueTM staining techniques using Computer Aided Sperm Morphology Analysis (CASMA). Sperm acrosome status was evealuated by fluorescence microscopy. Sperm DNA fragmentation and intracellular superoxide (O2−•) levels were assessed by flow cytometry. Seminal antioxidant status [superoxide dismutase (SOD), catalase (CAT), thiobarbituric acid reactive substances (TBARS)] were measured by means of spectrophotometry. Statistical comparisons between short and long abstinence periods were performed using paired Student’s t-tests on GraphPad Prism™ software, while the prediction of various basic and advanced semen parameters of the second ejaculate from a set of basic semen parameters of the first ejaculate was performed using linear regression models. Correlations were performed using Spearman rank correlation coefficients, while receiver operating characteristic (ROC) curves were used to determine cut-off values. Statistical significance was set at p<0.05. Results I: A significant increase in total and progressive motility as well as in the velocity parameters were observed after short (4 hours) abstinence compared to long (4 days) abstinence periods. DNA fragmentation and intracellular O2−• levels were not significantly different between short and long abstinence periods. Despite the observed decrease in semen volume, sperm concentration and T.S.C. after the short abstinence period, all mean values of the conventional semen parameters still remained above the lower reference limits as recommended by the WHO 5th edition. We were also able to make predictions of various basic (semen volume, sperm concentration, total motility, progressive motility, viability and normal morphology) and advanced (DNA fragmentation, seminal plasma CAT activity and TBARS) parameters of the second ejaculate from a set of basic semen parameters obtained from the first ejaculate with relative certainty. Results II: The proportions of total and progressively motile as well as rapid spermatozoa were positively correlated with CAT activity (p<0.05). A significant negative correlation was observed between VCL, VSL, VAP and both intracellular O2−• and TBARS levels. ALH was significantly and negatively correlated with intracellular O2−• levels and DNA fragmentation, while its correlation with SOD activity was positive (p < 0.05). A negative correlation was also found between the percentage of viable spermatozoa and both O2−• levels and DNA fragmentation, whereas the percentage of normal morphology was negatively correlated with O2−• levels and positively with CAT activity (p < 0.05). The optimal intracellular O2−• cut-off value to differentiate between asthenozoospermic and normozoospermic men was calculated to be 227 median DHE fluorescence intensity [MFI] (p < 0.01). At this cut-off value, the test was 80% sensitive and 86% specific. Sperm viability was associated with a seminal plasma TBARS cut-off value of 9.86 Umol/L (p = 0.02) with sensitivity and specificity of 81% and 80% respectively. Conclusion: Our data challenges the generally accepted guidelines regarding the prescribed prolonged abstinence periods since the results show that 4 hours of sexual abstinence yielded significantly better samples from a sperm functional point of view. The results obtained from this study further support the validity of some CASA parameters as sensitive indicators of changes in sperm oxidative status and DNA integrity. This study also enabled defining the cut-off values and prediction of certain advanced variables from the basic semen analysis.
- ItemCharacterisation of high fat, high sugar diet-induced epigenetic changes in skeletal muscle of wistar rats and metabolic effects of an aspalathin-rich rooibos extract(Stellenbosch : Stellenbosch University, 2021-12) Myataza, Asive; Carmen, Pheiffer; Tarryn, Willmer; Shantal, Windvogel; Rabia, Johnson; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences: Medical Physiology.Background Animal models are widely used to elucidate the pathophysiological mechanisms that underlie obesity and to test the efficacy of anti-obesity therapeutics. However, these models have been largely biased towards males, partly due to the complexity of hormonal fluctuations in females. The primary aim of this study was to elucidate DNA methylation profiles and gene regulatory networks that are altered in the skeletal muscle during the development of obesity in female and male Wistar rats, and to explore whether Afriplex-GRT™ could prevent aberrant DNA methylation patterns and the progression of metabolic disease. Methods Different animal models were employed where female and/or male Wistar rats were fed a standard or a high fat, high sugar (HFHS) diet for 12, 3 or 9 months. The effect of rooibos was investigated in the latter model, where 60 mg/kg of bodyweight Afriplex-GRTTM was co-administered with the diets. Parameters measured included food and water intake, bodyweight, and glucose, insulin, lipid and cytokine concentrations. Skeletal muscle was harvested for histology, gene expression measured using RT2 Profiler™ PCR arrays and Taqman® assays and global and gene-specific DNA methylation were quantified using pyrosequencing. To further explore the effects of DNA methylation, high glucose and fatty acids on skeletal muscle, C2C12 myocytes were differentiated with 7 μM 5-azacytidine, a global DNA methylation inhibitor, 33 mM glucose and 0.5 mM palmitate. Mitochondrial activity and oxidative stress were measured using appropriate assays. Myoblast differentiation and the expression of myoblast determination protein 1 (MyoD), myosin heavy chain 1 (Myh1), insulin growth factor 2 (Igf2), sterol regulatory element binding transcription factor 1 (Srebf1) and DNA methyltransferase 1 (Dnmt1) were assessed. Results The HFHS diet induced visceral adiposity and hypertriglyceridaemia in both male and female rats, while hyperinsulinaemia and significant bodyweight gain was observed in male rats, and systemic inflammation in females only. These changes were accompanied by increased expression of Igf2 and decreased expression of Srebfb1 and Dnmt1 in skeletal muscle of male, but not female rats. No differences in DNA methylation patterns were observed. Treatment with Afriplex-GRT™ did not ameliorate HFHS diet-induced metabolic dysregulation. In C2C12 cells, treatment with 5-azacytidine induced myoblast differentiation and MyoD and Myh1 expression, while palmitate inhibited differentiation and decreased the expression of MyoD, Myh1, Igf2 and Srebf1, which was restored by 5-azacytidine. High glucose increased Igf2 expression but did not affect Srebf1 expression, while a combination of high glucose and 5- azacytidine decreased Srebf1 and Dnmt1 expression. Conclusion The HFHS diet induced different metabolic responses and gene expression patterns in females and males. In general, females exhibited a dampened metabolic response, which we presume may be due to the intrinsic protective effects of female reproductive hormones. Afriplex-GRTTM did not prevent HFHS diet-induced weight gain, which contrasts previous findings where treatment with 60 mg/kg Afriplex-GRTTM decreased bodyweight in obese male rats, suggesting that Afriplex-GRTTM may be better targeted as a therapeutic than a preventative nutraceutical. This study provides novel information on how molecular differences in skeletal muscle may contribute to sex differences in response to HFHS feeding and may have important implications for the identification of therapeutic targets.
- ItemCompounds specific to Aspalathus linearis protects the diabetic heart against oxidative stress: a mechanistic study(Stellenbosch : Stellenbosch University, 2016-12) Dludla, Phiwayinkosi Vusi; Johnson, Rabia; Huisamen, Barbara; Essop, M. Faadiel; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences: Medical Physiology.ENGLISH ABSTRACT : In diabetics, hyperglycemia, hyperlipidemia and inflammation potentiates the development of cardiovascular diseases (CVDs). These conditions provoke excessive generation of oxidative stress that has been implicated in the pathogenesis of diabetic cardiomyopathy (DCM). In the diabetic state, excessive generation of free radicals can cause oxidative damage to DNA and alter protein and lipids, leading to the activation of various death-induced signaling pathways. Activation of these death pathways result in structural and functional modifications to the myocardium. Current diabetic drug therapies do not protect the diabetic heart at risk from developing cardiovascular complications. Thus, in search for new therapeutics, we aim to unravel the molecular mechanisms associated with the protective effect of two major bioactive compounds from Aspalathus linearis, phenyl pyruvic acid-2-O-β-D-glucoside (PPAG) and aspalathin against hyperglycemia-induced oxidative stress and apoptosis in H9c2 cardiomyocytes. The study showed that PPAG and aspalathin were able to decrease mitochondrial membrane depolarization and prevent hyperglycemia-induced myocardial apoptosis by increasing the Bcl2/Bax ratio. We revealed that while both compounds were able to reduce hyperglycemia-induced apoptosis, only aspalathin could ameliorate lipid toxicity and oxidative stress-associated with insulin resistance. An important feature of the failing heart is the observed shift in mitochondrial substrate preference that precedes the onset of oxidative damage. The current study revealed that aspalathin improved glucose metabolism by decreasing fatty acid uptake and subsequent β-oxidation. This was achieved through decreasing the expression of adenosine monophosphate-activated protein kinase threonine 172 (pAMPK (Thr172)) and carnitine palmitoyltransferase 1 (Cpt1), while increasing that of acetyl-CoA carboxylase (Acc) and glucose transporter 4 (Glut4). Additionally, it is known that cardiomyocytes have a very low antioxidant capacity and a shift in mitochondrial substrate preference can result in accelerated oxidative damage. In this study, we showed that aspalathin ameliorated oxidative stress by increasing the antioxidant capacity of the cells through activation of the antioxidant response pathway, nuclear factor erythroid 2 (NF-E2)-related factor 2 (Nrf2) and its downstream target genes. Moreover, we showed that aspalathin was able to reverse lipid toxicity by increasing the expression of Adiponectin, C1Q and collagen domain containing (Adipoq) and concomitantly decreasing cluster of differentiation 36 (Cd36) and Cpt1 mRNA expression. We further observed that Adipoq negatively regulated sterol regulatory element binding transcription factor 1 (Srebf1), stearoyl-Coenzyme A desaturase 1 (Scd1) and solute carrier family 27 (fatty acid transporter), member 3/5 (Slc27a3/5). This led to reduced lipid accumulation in H9c2 cardiomyocytes, with an associated decrease in total cholesterol, triglycerides and low-density lipoprotein in leptin resistant db/db mice. This was accompanied by decreased mRNA expression of inflammation markers in H9c2 cells, including interleukin 3 and 6 (IL3 and IL6), tumor necrosis factor receptor superfamily, member 1b and 13 (Tnfrsf1b and Tnfsf13), Janus kinase 2 (Jak2) and mitogen-activated protein kinase 3 (Mapk3). Together our results infer that aspalathin can slow down the progression of DCM, and thus protect the myocardium against causal factors associated with the development and progression of CVD.
- ItemContribution of highly active anti-retroviral therapy to the development of non-alcoholic fatty liver disease with concomitant cardiovascular dysfunction in an obese rat model(Stellenbosch : Stellenbosch University, 2018-03) Kamau, Festus Maina; Ruduwaan, Salie; Waweru, Peter Maina; Strijdom, Hans; Stellenbosch University. Faculty of Medical and Health Sciences. Dept. of Biomedical Sciences : Medical Physiology.Introduction: HIV/AIDS mortality is declining due to successful highly active anti-retroviral therapy (HAART). However, obesity, non-alcoholic fatty liver disease (NAFLD) and cardiovascular disease (CVD) in treated HIV-infected populations are rising. Impaired peroxisome proliferator-activated receptor (PPARα/γ) activity is partly implicated. Aims: To assess the contribution of a protease inhibitor (Lopinavir/Ritonavir (LPV/r) and nucleoside reverse transcriptase inhibitor (Azidothymidine (AZT)/lamivudine (3TC)) HAART regimen in the development of NAFLD and CVD in rats with high caloric diet (HCD)-induced obesity, and to investigate dual PPARα/γ stimulation in limiting HAART-induced NAFLD and CVD. Methods: Wistar rats were randomised into rat chow and HCD groups (n=88/group; 16 weeks). From week 10, each group was further sub-divided into: vehicle/control, HAART, HAART+PPARα/γ agonist (Saroglitazar) and Saroglitazar only (n=22/group) administered via oral gavage. Endpoints: Daily food/water consumption, weekly total body mass (TBM), random+fasting blood glucose measurements (n=8/group); hearts exposed to either 20min global ischaemia/10min reperfusion for Western blot (WB) analysis (n=6/group) or 35min regional ischaemia/60min reperfusion (n=8/group) for haemodynamic and infarct size (IS) determinations. Liver samples were histologically assessed (n=12/group) and analysed by WB. Intraperitoneal (IP) fat was weighed. Fasting serum lipids, insulin and oxidative stress markers were measured (n=8/group). WB analyses of important signalling proteins in pre/post-cardiac ischaemia, liver and aorta tissues. Thoracic aorta (n=8/group) segments were subjected to isometric tension studies. Results: The HCD resulted in obesity (increased: TBM, %IP fat (HCD control 6.50±0.40% vs. lean control 3.60±0.3%; p<0.0001), liver mass, insulin and triglycerides (TGs). Additionally, HCD induced insulin resistance (IR). HAART+Saroglitazar led to reduced %IP fat in lean and HCD groups. HAART-induced IR, elevated cardiac mass and insulin in obese rats were limited by Saroglitazar co-treatment. HCD+HAART-induced oxidative stress (elevated conjugated dienes), was limited in combined HAART+Saroglitazar. Liver histology: HAART induced moderate hepatic steatosis in ~67% of obese rats and moderate inflammation in ~25% of cases. Combined HAART+Saroglitazar limited these changes and upregulated adenosine monophosphate-activated protein kinase (AMPK) and protein kinase B (PKB/Akt) activity, which were downregulated by HAART in HCD. Heart/aorta studies: Untreated obese rats had smaller %IS compared to lean control rats (19.1±1.6 vs. 26.1±1.6, respectively; p<0.05). IS for HCD+HAART animals were smaller (despite poor cardiac performance) compared to untreated obese rats. Post-ischaemia activity of extracellular-signal-regulated kinase (Erk1/2), PKB/Akt, AMPK and endothelial nitric oxide synthase (eNOS) was downregulated, whereas expression of p22-phox and caspase-3 was accentuated. HAART+Saroglitazar upregulated (1.5-fold) the expression of Erk1/2, PKB/Akt, AMPK and eNOS, and downregulated caspase-3 and p22-phox. Obese+HAART rats demonstrated poor aortic relaxation accompanied by downregulated eNOS and PKB/Akt, and upregulated p22-phox. However, Saroglitazar+HAART in obese animals improved aortic relaxation by ~30%, accompanied by upregulation of eNOS, and PKB/Akt, and downregulated p22-phox. Discussion and conclusion: HAART-induced NAFLD and CVD in obesity were limited by PPARα/γ agonist co-administration. HAART treatment for six weeks was not a cardiovascular risk factor per se, but it potentiated HCD-induced cardiovascular effects. Monitoring cardiovascular risk factors in obese+HAART patients is crucial, and our findings suggest that there is therapeutic potential in co-treatment with PPARα/γ agonists. The metabolic, functional and signalling disturbances in the liver, heart and aorta tissues in obese+HAART rats are interlinked, and partially limited by co-treatment with a dual PPARα/γ agonist.
- ItemThe dietary ionic effects on sex ratios in animal models(Stellenbosch : Stellenbosch University, 2016-03) Linge, Augustine Peter Kavoo; Du Plessis, S. S.; Kimwele, C.; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences. Medical Physiology.ENGLISH ABSTRACT: X-linked disorders are more expressed in male offspring and prevention of these hereditary diseases is the only recourse to date. Influencing conception towards female offspring can circumvent this problem; however sex ratio adjustment remains highly contentious. Treatment of genetic disorders through sex ratio adjustments has been examined and adopted as acceptable, easier, cheaper, safer and legal. Historically, society has been rife with allegations that diet does influence sex ratios though this has not been proven fully. The dietary chemical compositions have been claimed to act as modulators that affect the electrical charges or potential of the membranes of the oocytes and cause allosteric modification or electrotactism through a process referred to as galvanotropism and cause selective attraction towards either of the male gametes and subsequently influence the gender of the conceptus. This study was performed in an attempt to address the various questions, allegations and speculations that have been rife in many societies concerning interplay between diet, fertilization and sex ratios so as to verify the validity of these social claims by taking them to the laboratory for experimental verification. Swiss Webster mice study In a double-blind fashion, nine double groups were set up comprising of 144 families of Swiss (Webster) mice, each receiving different ionic formulations in their drinking water: 1) water and 2) glucose as controls; high serum concentrations of single elements of 3) sodium, 4) potassium, 5) calcium and 6) magnesium; combined double elements 7) sodium + potassium and 8) calcium + magnesium; and finally a cocktail of the four elements 9) sodium + potassium + calcium + magnesium. Tests included the perinatal mortality rate; the relationship between high chemical composition diet and serum levels; the effects of the study chemicals on weight gains of the study models; the effects of birth order on sex ratios and the effects of seasonal variations on sex ratios. There were 1528 deliveries with 13,040 (6,348 females and 6,692 males) pups at 8.5 pups on average per litter. a) Glucose, sodium, potassium and sodium + potassium supplementation influenced the sex ratios towards male progeny (p<0.001). Calcium (p<0.014), magnesium (p<0.008) and calcium + magnesium (p<0.001) supplementation influenced sex ratios towards the female progeny. The water (p>0.61) and cocktail solutions (p>0.0609) had no influence. b) The perinatal mortality rate was 32/1000 and was female biased among the magnesium (p<0.005) and combined calcium + magnesium (p<0.044) groups only. c) Normal serum levels were observed in the control groups (p>0.165), while significant elevated serum levels were observed among the experimental groups (p<0.0001). d) The total mean weight gains were 11.12g and 10.55g among the females and males respectively. The weight trends were used to track the general wellbeing of the animal models. e) The mean litter size was 8.5 per delivery in all the groups and generations, while no influence due to birth order were detected. f) Seasons affect the litter size, in particular the rainy season, but not the gender ratios (p>0.061). Cat fish study Parallel double blind studies looking at the dietary chemical ionic effects on the oocyte membrane electrical potential were done utilising a cat fish model (n=108). The study sought to find out effects of the following solutions on the oocyte electrical charges: 1) plain electrolyte solution, 2) glucose solution 3) sodium solution 4) potassium solution 5) calcium solution 6) magnesium solution 7) sodium + potassium solution 8) calcium + magnesium solution and the 9) cocktail solution of the four elements combined. The results revealed that oocytes retrieved from the two control groups had baseline oval polar attraction significantly more towards the positive than the negative pole (p<0.0003). There was however more significant oocyte polar attraction towards the positive electrode among the oocytes retrieved from the sodium, potassium and the combined sodium + potassium solutions (p<0.0001). Oocytes retrieved from calcium, magnesium and combined calcium + magnesium solutions had significant affinity towards the negative electrode and minimal affinity towards the positive pole (p<0.0001). Oocytes harvested from the solutions constituted with all the salts demonstrated dual attraction with more attraction to the positive electrode than the negative electrode but of no statistical significance (p>0.0530). These study findings do confirm the social allegations that a positive relationship does exist between dietary components and sex ratios. The chemicals acted as the dietary modulators that ultimately influenced the electrical cellular gametal charges and subsequently the resulting progeny. Our platform of comfort is unlike artificial sex ratio adjustment methods; the natural sex ratio adjustment methods that include the dietary method under scrutiny in this study are practiced always at the comfort of many people’s homes and are difficult to quantify or have legislation on. However, this study shows that their long term effects conform to the Fishers principle of evolution towards 1:1 sex ratios and therefore do not have significant social gender skewing on a long term basis. The study clearly explains the molecular basis upon which ions of single valency attracts the Y-bearing sperm leading to a male conceptus and how cations of double valency attracts the X-bearing sperm leading to a female conceptus despite being positively charged. The study further reaffirms the natural feminine supremacy by demonstrating that it is the ova and by extension the woman who determines the sex of the conceptus. The study ultimately confirms that the dietary ionic effects on sex ratios can be used for prevention of X-linked disorders.
- ItemEffect of an obesogenic diet on the epigenetic profile of livers from Wistar rats and treatment with an aspalathin-rich rooibos extract(Stellenbosch : Stellenbosch University, 2021-12) Abrahams, Yoonus; Pheiffer, Carmen; Johnson, Rabia; Samodien, Mugammad Ebrahim; Windvogel, Shantal; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences: Medical Physiology.Background There is a scarcity of knowledge about obesity and metabolic syndrome development and progression in females, who are often under-represented in preclinical research. The aims of this study were thus to investigate the effects of high fat, high sugar (HFHS) feeding on gene expression and DNA methylation patterns in the livers of female and male Wistar rats and to determine whether an Aspalathin-rich rooibos extract, Afriplex® GRT, could prevent HFHS diet-induced metabolic dysregulation. Methods This study employed different animal models. Female and/or male Wistar rats were fed a HFHS diet for 12, 24 or 36 weeks. The effectiveness of Afriplex® GRT was investigated by daily supplementation with 60 mg/kg of bodyweight co-administered with diets for 36 weeks. Food and water intake, bodyweight, fasting blood glucose concentrations and glucose tolerance were measured regularly. After termination, organ weights, liver histology, fasting serum insulin, high- and low-density lipoprotein and cholesterol concentrations were measured. Gene expression was assessed using RT2 Profiler Rat Fatty Liver PCR arrays and TaqMan® gene expression assays. DNA methylation was quantified by pyrosequencing. The effects of DNA methylation were further investigated in the HepG2/C3A hepatocarcinoma cell line. Cells were treated with 2.5 μM of the DNA methyl transferase inhibitor, 5-aza-2’-deoxycytidine for 24 hours to induce DNA hypomethylation. Thereafter, steatosis and insulin resistance were induced by exposing cells to a 1:1 mixture of 0.5 mM palmitic and oleic acids for 24 hours. Cell viability, mitochondrial function, mitochondrial membrane potential, lipid accumulation, reactive oxygen species accumulation and gene expression were assessed. Results The HFHS diet increased visceral adiposity and hypertriglyceridaemia in both female and male rats, while hyperinsulinaemia and sustained obesity were observed in males only. Males fed the HFHS diet for 12 weeks showed reduced peroxisome proliferator-activated receptor gamma coactivator-1 alpha (Pgc1a) expression and hypermethylation of the Pgc1a promoter, but this was not observed in males fed the same diet for 36 weeks. Females fed HFHS showed a sex-specific reduction in peroxisome proliferator-activated receptor gamma (Pparg) expression. Afriplex® GRT did not prevent obesity or metabolic dysregulation. In vitro analysis demonstrated that DNA hypomethylation reduced lipid accumulation but did not affect insulin sensitivity. Steatosis and insulin resistance in the presence of DNA hypomethylation was associated with increased expression of uncoupling protein 2 (UCP2) and CCAAT/enhancer-binding protein beta (C/EBPß). Conclusion The HFHS diet elicited different responses in female and male Wistar rats. Male rats exhibited obesity, hyperinsulinaemia, glucose intolerance and altered methylation of Pgc1a. However, females exhibited a dampened metabolic response, potentially mediated through sex-specific downregulation of hepatic Pparg. Prophylactic daily supplementation with 60 mg/kg Afriplex® GRT did not prevent diet-induced obesity or metabolic dysregulation. For the first time, we show that DNA hypomethylation in HepG2 hepatocarcinoma cells decreases lipid accumulation in vitro and show that hypermethylation of a conserved CpG site in the promoter of Pgc1a is associated with decreased expression in the livers of male Wistar rats. We observed sex-specific hepatic differences between females and males which may have important implications for the development of therapeutic targets. In future, this may allow for the development of effective treatments free of sex-biased adverse effects and which confer protection against metabolic disease, reduce mortality and improve the quality of life of affected persons globally.
- ItemThe effect of creatine supplementation on myocardial metabolism and function in sedentary and exercised rats(Stellenbosch : University of Stellenbosch, 2010-12) Webster, Ingrid; Du Toit, E. F.; Huisamen, Barbara; University of Stellenbosch. Faculty of Health Sciences. Dept. of Biomedical Sciences. Medical Physiology.ENGLISH ABSTRACT: Background: There has been a dramatic increase in the use of dietary creatine supplementation among sports men and women, and by clinicians as a therapeutic agent in muscular and neurological diseases. The effects of creatine have been studied extensively in skeletal muscle, but knowledge of its myocardial effects is limited. Objectives: To investigate the effects of dietary creatine supplementation with and without exercise on 1) basal cardiac function, 2) susceptibility to ischaemia/reperfusion injury and 3) myocardial protein expression and phosphorylation and 4) mitochondrial oxidative function. Methods: Male Wistar rats were randomly divided into control or creatine supplemented groups. Half of each group was exercise trained by swimming for a period of 8 weeks, 5 days per week. At the end of the 8 weeks the open field test was performed and blood corticosterone levels were measured by RIA to determine whether the swim training protocol had any effects on stress levels of the rats. Afterwards hearts were excised and either freeze-clamped for biochemical and molecular analysis or perfused on the isolated heart perfusion system to assess function and tolerance to ischaemia and reperfusion. Five series of experiments were performed: (i) Mechanical function was documented before and after 20 minutes global ischaemia using the work heart model, (ii) A H2O filled balloon connected to a pressure transducer was inserted into the left ventricle to measure LVDP and ischaemic contracture in the Langendorff model, (iii) The left coronary artery was ligated for 35 minutes and infarct size determined after 30 minutes of reperfusion by conventional TTC staining methods. (iv) Mitochondrial oxidative capacity was quantified. (v) High pressure liquid chromatography (HPLC) and Western Blot analysis were performed on blood and heart tissue for determination of high energy phosphates and protein expression and phosphorylation. Results: Neither the behavioural studies nor the corticosterone levels showed any evidence of stress in the groups investigated. Hearts from creatine supplemented sedentary (33.5 ± 4.5%), creatine supplemented exercised rats (18.22 ± 6.2%) as well as control exercised rats (26.1 ± 5.9%) had poorer aortic output recoveries than the sedentary control group (55.9 ± 4.35% p < 0.01) and there was also greater ischaemic contracture in the creatine supplemented exercised group compared to the sedentary control group (10.4 ± 4.23 mmHg vs 31.63 ± 4.74 mmHg). There were no differences in either infarct size or in mitochondrial oxygen consumption between the groups. HPLC analysis revealed elevated phosphocreatine content (44.51 ±14.65 vs 8.19 ±4.93 nmol/gram wet weight, p < 0.05) as well as elevated ATP levels (781.1 ±58.82 vs 482.1 ±75.86 nmol/gram wet weight, p<0.05) in blood from creatine supplemented vs control sedentary rats. These high energy phosphate elevations were not evident in heart tissue and creatine tranporter expression was not altered by creatine supplementation. GLUT4 and phosphorylated AMPK and PKB/Akt were all significantly higher in the creatine supplemented exercised hearts compared to the control sedentary hearts. Conclusion: This study suggests that creatine supplementation has no effects on basal cardiac function but reduces myocardial tolerance to ischaemia in hearts from exercise trained animals by increasing the ischaemic contracture and decreasing reperfusion aortic output. Exercise training alone also significantly decreased aortic output recovery. However, the exact mechanisms for these adverse myocardial effects are unknown and need further investigation.
- ItemThe effect of Cyclopia maculata extract on β-cell function, protection against oxidative stress and cell survival(Stellenbosch : Stellenbosch University, 2014-12) Chellan, Nireshni; Muller, Christo John Frederick; Strijdom, Hans; Joubert, Elizabeth; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences. Medical Physiology.ENGLISH ABSTRACT: Insights into the role of oxidative stress and pancreatic β-cell dysfunction in the pathogenesis of type 2 diabetes (T2D) reveals an opportunity for the development of novel therapeutics that directly protect and preserve β-cells. The protective role of dietary antioxidants, such as plant polyphenols, against oxidative stress induced diseases, including T2D, is increasingly under scrutiny. Polyphenol-rich extracts of Cyclopia spp, containing mangiferin, may provide novel therapeutics. An aqueous extract of unfermented Cyclopia maculata, containing more than 6 % mangiferin, was assessed for its protective effect in pancreatic β-cells in vitro, ex vivo and in vivo under conditions characteristic of T2D. The effect of mangiferin was also evaluated in vitro and ex vivo, with N-acetyl cysteine (NAC) as an antioxidant control. In this study, we established in vitro toxicity models in RIN-5F insulinoma cells based on conditions β-cells are exposed to in T2D; i.e. lipotoxicity, inflammation and oxidative stress conditions. To achieve this, cells were exposed to the following stressors: palmitic acid (PA), a pro-inflammatory cytokine combination and streptozotocin (STZ), respectively. Thereafter, the ability of the C. maculata extract, mangiferin and NAC to protect RIN-5F cells from the effects of these stressors was assessed by measuring β-cell viability, function and oxidative stress. Cell viability was assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, adenosine triphosphate and annexin-V and propidium iodide assays. Cell function was evaluated by measuring glucose stimulated insulin secretion, cell proliferation and cellular calcium. To assess oxidative stress in the RIN-5F cells, diaminofluorescein-FM and dihydroethidium fluorescence, and superoxide dismutase enzyme activity were measured. The in vitro findings were then verified in isolated pancreatic rat islets using methods and models established in the RIN-5F experiments. The protective effect of the extract, NAC and metformin was assessed in STZ induced diabetic Wistar rats, using two treatment regimes, i.e. by treating rats with established diabetes and by pretreating rats prior to induction of diabetes by STZ. Glucose metabolism, oxidative stress and pancreatic morphology were assessed by performing an oral glucose tolerance test, measuring serum insulin, triglycerides, nitrites, catalase and glutathione. Hepatic thiobarbituric acid reactive substances and nitrotyrosine were also assessed. Immunohistochemical labelling of pancreata with insulin, glucagon and MIB-5 was used for morphological assessment. The extract improved β-cell viability, function and attenuated oxidative stress, most apparently in STZ and PA induced toxicity models comparable with NAC both in vitro and in isolated islets. Mangiferin was not as effective, showing only marginal improvement in RIN-5F cell and islet function, and oxidative stress. Pretreatment of STZ induced diabetic Wistar rats with extract was as effective as, if not better than, metformin in improving glucose tolerance, hypertriglyceridaemia and pancreatic islet morphology related to improved β-cell function. This study demonstrated that the aqueous extract of unfermented C. maculata was able to protect pancreatic β-cells from STZ and PA induced toxicity in vitro and ex vivo. In vivo, pretreatment with the extract improved glucose metabolism and pancreatic islet morphology in STZ induced diabetic Wistar rats.
- ItemEffect of different substrates on functional performance and kinase activation during reperfusion after ischaemia in hearts from obese insulin resistant rats(Stellenbosch : Stellenbosch University, 2017-03) Fan, WenJun; Huisamen, Barbara; Lochner, Amanda; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences: Medical PhysiologyENGLISH ABSTRACT : Obesity is an important risk factor for the development of insulin resistance, the metabolic syndrome and diabetes and has also been implicated as one of the major risk factors for coronary heart disease. Ischaemic heart disease impacts on both cardiac metabolism and function. During early reperfusion after ischaemia, several protein kinases are specifically activated, including PI3K/PKB, MAPKs (ERK, JNK and p38 MAPK), and the tyrosine kinases. Activation of PKB and ERK, the so-called reperfusion injury salvage kinase (RISK) pathway, is associated with a reduction in infarct size and/or improvement in functional recovery. PKB is an enzyme central also to insulin signalling and glucose uptake. Activation of the JNK signaling pathway has been suggested to be a prerequisite for PKB activation; however, its role in ischaemia/reperfusion (I/R) injury remains controversial. We hypothesize that obesity-induced insulin resistance will affect infarct size, functional recovery and interactions between PKB, JNK, ERK, p38MAPK and PTEN activation during reperfusion after exposure to ischaemia. The aim of the study was therefore to assess the effects of hyperphagia-induced obesity and insulin resistance in rats on the response of the heart to I/R injury, with particular attention to the intracellular signalling pathways during early reperfusion. To further elucidate the role of JNK, we used SP600125, a specific inhibitor of JNK. Methods: Insulin resistance was induced by feeding rats a high caloric diet for 16 weeks (DIO). Hearts from DIO and age-matched controls (C) were perfused in the working mode (preload 15cm H2O; afterload 100cm H2O) and subjected to (i) 15 min global ischaemia followed by different reperfusion times for evaluation of functional recovery and freeze-clamping of tissues for Western blot or (ii) 35 min regional ischaemia followed by 2 hours reperfusion for infarct size determination (IS), using tetrazolium staining. Substrates were glucose (G) (10mM), glucose (10mM) plus BSA (3%) (G+B), and glucose (10mM) plus fatty acid (1.2mM palmitate / 3% BSA) (G+FA). The JNK inhibitor, SP600125, was administered either before ischaemia or during reperfusion after ischaemia. Infarct size, functional recovery as well as expression and activation of PKB, ERK, JNK, p38MAPK and PTEN were used as endpoints. Results: (I) In the presence of glucose alone as substrate, the hearts from DIO rats exhibited an improved tolerance to ischaemia/reperfusion (I/R) injury as reflected by an increase in functional recovery (after exposure to 15 min global ischaemia) as well as a reduction in infarct size (after 35 min regional ischaemia) compared with the age-matched controls. This was associated with early activation of PKB and JNKp54/p46 at 10 min reperfusion, with down regulation of activation of these kinases after 30 min reperfusion. (II) Contrary to expectations, the combination of a high concentration of fatty acids and glucose as substrates (G+FA) afforded significantly more protection against I/R injury in hearts from both DIO and control rats, when compared with the respective groups perfused with glucose alone as substrate. This improved protection in both groups was associated with increased activation of the PKB pathway. Interestingly, perfusion with glucose and a high concentration of fatty acid maintained PKB activation throughout the reperfusion phase, in contrast to the transient activation seen with glucose alone as substrate. (III) SP600125 (10 uM), administrated either before ischaemia or during early reperfusion after ischaemia, almost completely inhibited the JNK pathway and exacerbated myocardial I/R injury, particularly in hearts from DIO rats. Conclusion: Our study demonstrates, in contrast to several other studies, that dietary-induced obesity and high perfusate fatty acid concentrations, increase the tolerance of the ex vivo myocardium to I/R injury. It was also found that, contrary to expectations, a high concentration of circulating fatty acid was not detrimental to hearts of normal rats during I/R, indicating the beneficial actions of fatty acids on the outcome of I/R injury. This protection was shown to be associated with activation of PKB and JNK during early reperfusion. Administration of the selective JNK inhibitor, SP600125, before or after myocardial ischaemia indicates that JNK and its downstream signalling pathways are critical in mediating protection against I/R in our study. SP-induced effects were also associated with lower activation of PKB. Our results suggest that the cross-talk between the JNK and PKB pathways in the post-ischaemic myocardium may be a major contributing factor to the outcome of I/R injury The data presented here, although seemingly dichotomous, actually solidify the hypothesis that JNK signalling specifically and simultaneously modulates pro- and antiapoptotic effector mechanisms within cardiomyocytes. They also reflect an extraordinary complexity of the heart‘s metabolic, functional, and structural changes in obesity. In addition, the results obtained showed that moderate hyperphagia-induced obesity does not have a harmful effect on the ischaemic-reperfused heart and in fact, reduced the sensitivity of the heart to I/R damage. This was further substantiated by the beneficial effects of fatty acids in the perfusate. Taken together, our results are potentially of clinical significance, and confirm the importance of events during early reperfusion as possible therapeutic targets.
- ItemThe effect of green rooibos extract on rat hearts in a pre-diabetic model : an evaluation of the function and mechanisms involved(Stellenbosch : Stellenbosch University, 2019-03) Smit, Sybrand Engelbrecht; Huisamen, Barbara; Marais, Erna; Johnson, Rabia; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences: Medical Physiology.ENGLISH ABSTRACT: Background— Cardiovascular diseases (CVD) remains the leading cause of death globally, with a rising prevalence of individual risk factors such as obesity and insulin resistance. Furthermore, diabetic patients are particularly at risk for developing ischemic heart disease and strokes. In patient suffering an ischemic event, most of the myocardial damage incurred happens once blood flow is restored – a phenomena known as reperfusion injury. Mitochondrial ROS production is implicated as one of the major contributors to cell death in the reperfused heart and the importance of mitophagic processes (mitochondrial housekeeping) could be a potential therapeutic target to prevent ischemia/reperfusion injury (I/R-I). Aspalathus linearis (commonly known as rooibos) is an indigenous South African plant grown exclusively in the Western Cape fynbos region. Rooibos is rich in bioactive phenolic compounds, including aspalathin, a C-linked dihydrochalcone glucoside unique to rooibos, known for its hypoglycemic and strong antioxidant potential. Methods— The present study made use of 300 Wistar rats randomly allocated into controls and rats receiving a 16-week high-fat, high-caloric diet (HCD). After 10 weeks on the respective diets, half of each group received 60 mg/kg/day Afriplex green rooibos extract (GRT), containing 12% aspalathin, for 6 weeks. The primary aim was to investigate its therapeutic potential in treating 20 min global ischemia/reperfusion injury (I/R-I) in rats with increased susceptibility for CVD, as well as determine the mitochondrial oxidative phosphorylation (OxPhos) during four stages of I/R-I. The secondary aim was to elucidate GRT’s effect on cardiac signaling and mitophagy with regards to I/R-I. Results— HCD over 16 weeks resulted in daily increased food intake (22.30±1.27 g vs 18.00±0.54 g = ↑24%; p<0.001), decreased daily water intake (19.55±0.58 mL vs 29.74±0.74 mL = ↓34%; p<0.001), leading to increased body weight (403.9±5.1 vs373.6±4.8 g = ↑8%; p<0.001) and increased intraperitoneal fat (23.69±0.89 vs 13.98±0.43 g = ↑69%; p<0.001) compared to age-matched controls. Furthermore, HCD increased fasting blood glucose (5.75±0.16 vs 5.21±0.16 mM = ↑10%; p<0.05), fasting blood insulin (6.20±0.57 vs 4.31±0.54 ng/mL = ↑44%; p<0.05) and insulin resistance through raised HOMA-IR (3.52±0.35 vs 2.30±0.28 = ↑53%; p<0.05) compared to age-matched controls. GRT supplementation for 6 weeks had no significant effect on biometric parameters in either controls or HCD. Pre-ischemia, HCD rats presented with worse functional heart parameters, such as diastolic and systolic pressure, aortic output (AO), cardiac output (CO) and total work (WT) (10.69±0.14 vs 12.6±0.19 mW = ↓15%; p<0.001) compared to controls, while GRT supplementation was able to significantly improve these parameters in both controls (13.91±0.23 mW = ↑10%; p<0.001) and HCD (11.98±0.20 mW = ↑12%; p<0.001). GRT administration also lowered the heart rate during stabilization in both control and HCD by an average 10 bpms (p<0.05). Post-ischemia, HCD hearts were weaker than controls and had a lower WT recovery (55.51±2.28 vs 64.8±2.04% = ↓14%; p<0.01), while GRT restored WT recovery in HCD (63.35±2.49% = ↑14%; p<0.05). HCD rats also had a greater infarct size compared to controls (34.99±11.56 vs 16.42±8.71% = ↑113%; p<0.01) following a 35 min regional ischemia protocol. GRT supplementation led to a remarkable reduction in infarct size (13.22±6.66% = ↓62%; p<0.001), while conferring no added protection to controls (14.58±5.43%). Regarding cardiac and mitochondrial signaling, prior to ischemia HCD heart had a decreased dependence on insulin-dependent AMPK and increased inflammation via upregulated p38, whereas GRT treatment presented with decreased insulindependent PKB and AS160 signaling (together with increased FA OxPhos compared to carbohydrates, however showed more mitochondrial uncoupling, decreasing basal metabolic rate and thereby potentially less ROS production), inhibited GSK3β and conferred an anti-inflammatory effect by significantly reducing p38 activation. HCD also had higher mitophagy rates through Parkin and LC3 signaling. After 20 min ischemia, in HCD, ATM and AMPK were upregulated and insulin-dependent PKB downregulated with GSK3β. Mitophagy signals, such as PINK1 and p62 were elevated, but autophagic flux remained low during ischemia in all groups. GRT supplementation resulted in an opposite profile with AMPK downregulated and showing inhibition of ERK1/2. In early reperfusion, all protective signaling were downregulated in HCD including AMPK, PKB, AS160, GSK3b and ATM. Mitophagy was also activated through Parkin, p62 and LC3 while having increased OxPhos potential in FA compared to carbohydrates. GRT supplementation reduced oxidative stress and inflammation through downregulation of JNK1/2 and p38, and initiated mitophagy through an AMPKdependent mechanism and Parkin. GRT supplementation also inhibited mitochondrial OxPhos after reperfusion culminating in a potentially decreased ROS production. Conclusion— This study showed the cardioprotective effect of Afriplex GRT supplementation by improving functional heart recovery and reducing infarct size post-ischemia in rats with elevated risk for CVD. Another novel find is the reduction in heart rate induced by GRT treatment through inhibition of pacemaking cells. This could have potential therapeutic application in patients suffering from ischemic heart disease. GRT ilicits a cardiotonic effect in I/R-I through anti-inflammatory mechanisms pre- and post-ischemia. In early reperfusion, GRT treatment resulted in decreased oxidative stress, inhibition of mitochondrial OxPhos and enabled AMPKdependent mitophagy. GRT shows promise as a strong antioxidant and anti-inflammatory agent in managing adverse outcomes in patients at risk for CVD.
- ItemThe effects of a green rooibos extract on the reproductive function of obesity-induced insulin resistant or hypertensive male wistar rats(Stellenbosch : Stellenbosch University, 2019-12) Manirafasha, Claudine; Du Plessis, S. S.; Huisamen, Barbara; Aboua, Yapo Guillaume; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences: Medical Physiology.ENGLISH ABSTRACT: Diet-induced obesity (DIO) due to a high caloric diet (HCD) predisposes an individual to the development of diabetes and cardiovascular diseases, with high prevalence in young populations. Existing evidence supports the sentiment that insulin resistance and hypertension (HT) affect male reproduction. A greater understanding of the influence of insulin resistance and/or HT on male reproduction is required in order to prevent or treat male infertility. Due to the limitations of orthodox drugs, there is currently a strong movement towards and support for studies on phytomedicine. Rooibos (Aspalathus linearis) has been used in several studies and is known to have natural antioxidant effects and anti-obesogenic, antidiabetic, anti-hypertensive and anti-infertility activities. Currently, the company Afriplex (Pty) Ltd is producing an aspalathin-rich laboratory standardized extract prepared from green rooibos called Afriplex GRTTM (GRT). However, there is very little knowledge regarding the use of GRT in obesity-related insulin resistance and/or HT, and specifically, its effects on male reproductive health. Aim This study aimed to explore the effect of GRT on the reproductive function in obesity-induced insulin resistant and hypertensive male Wistar rats. Methods A prospective randomized control and experimental animal study design was used. Two different diets were used to induce obesity-related insulin resistance with or without HT in male Wistar rats. Subsequently, the possible protective properties of GRT on the male reproductive system were evaluated. Animals (weighing 120 ±10 g, approximately 7 weeks old) were randomly assigned to seven groups with seven rats each. All rats had unrestricted access to their respective diets and water for 16 weeks. At baseline (week 0–10), we had three groups: 1) lean control (LC) – animals that received standard rat chow; 2) obese (OB) – animals that received a diet to induce obesity associated with insulin resistance; and 3) obese with hypertension (OBHT) – animals were placed on a slightly modified DIO and additionally developed HT. From weeks 11 to 16, one LC, OB and OBHT group were each treated with GRT (prepared and supplied by Afriplex (Pty) Ltd) at 60 mg/kg/day as a dietary supplement in the form of jelly blocks. An additional group of OBHT animals was treated during the same period for 6 weeks with Captopril, an angiotensin-converting-enzyme (ACE) inhibitor (positive control for HT) at 60 mg/kg per day. Food and water intake were monitored on a daily basis. An oral glucose tolerance test was performed during the 10th week after the onset of the respective diets and during the 16th week, after which the animals were sacrificed. Blood pressure measurements were taken once per week throughout the experimental period. After the 16-week period, animals were killed and blood, testis and epididymal tissue were harvested for further analysis. Body weight, intra-peritoneal fat, non-fasting glucose levels, IL-1β, IL-6, IL-12, IL-18 and TNFα, oxidative stress (OS) markers (superoxide dismutase and catalase activity, malondialdehyde), testosterone and estradiol, sperm concentration, viability, morphology, total motility, progressive motility and various velocity parameters were measured. Results and conclusion Both diets successfully induced insulin resistance with or without hypertension and demonstrated detrimental effects on male reproductive function as evidenced by OS and hormone dysregulation. Treatment with GRT reversed OS and balanced the androgens. This study provided insight into the pharmacological effects of GRT in the treatment of pathophysiological changes that occur in DIO associated with insulin resistance or HT. These findings will hopefully inspire further research into the clinical setting related to the GRT and could possibly lead to the development of new drugs from this compound.
- ItemThe effects of early life trauma on the neurochemistry and behaviour of the adult rat(Stellenbosch : University of Stellenbosch, 2006-12) Uys, Joachim De Klerk; Daniels, M. W. U.; Stein, D. J.; University of Stellenbosch. Faculty of Health Sciences. Dept. of Biomedical Sciences. Medical Physiology.Early life trauma leads to behavioural abnormalities later in life. These include mood and anxiety disorders such as depression and posttraumatic stress disorder (PTSD). This association may be due in part to the effects of trauma on brain development. Data from basic and clinical experiments suggest that alterations in the hippocampus may be fundamental to the development of these disorders. Here we used an animal model of early life trauma to investigate its effects on the behaviour and neurochemistry of the adult rat. Adolescent rats were subjected to time-dependent sensitization stress consisting of a triple stressor (2 hours restraint, 20 min swim stress and exposure to ether vapour) on post-natal day (PND) 28, a single re-stress on PND 35 (20 min swim stress), and a second re-stress in adulthood (PND 60, 20 min swim stress). The rationale was that the frequency of exposure to situational reminders contributes to the maintenance over time of fear-related behavioural disturbances. The effects of trauma on the hypothalamus-pituitary-adrenal-axis, hippocampal and plasma neurotrophin levels, behaviour and phosphoinositide-3 kinase (PI-3 kinase) signaling proteins were initially investigated. In addition, proteomic technologies such as protein arrays and 2D-SDS PAGE combined with liquid chromatography tandem mass spectrometry (LC-MS/MS) were employed to study trauma-induced effects on the hippocampus. Traumatized animals showed a decrease in glucocorticoid receptors in the dentate gyrus of the hippocampus and an increase in basal corticosterone levels 24 hours after adulthood re-stress. These effects were reversed by pretreatment with the serotonin selective reuptake inhibitor, escitalopram. A decrease in the neurotrophins, BDNF and NT-3 were evident 8 days, but not 24 hours after adulthood re-stress. This decrease was not accompanied by decreases in plasma neurotrophin or PI-3 kinase, protein kinase B (PKB), phosphatase and tensin homologue (PTEN), phospho-forkhead and phospho-AFX protein levels. In addition, traumatized animals showed increased rearing in both the elevated plus maze and open field. Proteomic analysis of trauma-induced changes in the hippocampus show increases in Ca2+ homeostasis / signaling proteins such as S-100B, phospho-JNK and calcineurin. Apoptotic initiator proteins, including caspase 9, -10 and -12 were increased and there was evidence of cytoskeletal protein dysregulation. Furthermore, cell cycle regulators and energy metabolism proteins were decreased. These effects indicate to a cellular state of cell cycle arrest after increased calcium influx to avoid apoptosis. Our data suggest that adolescent trauma with adulthood re-stress may affect numerous systems at different levels. These include neuroendocrine-, protein systems and behaviour, and confirmed that a systems biology approach is needed for a better understanding of the neurobiology of mental disorders.
- ItemEffects of insulin and leptin on human spermatozoa function and their cross-talk with nitric oxide and cytokines(Stellenbosch : University of Stellenbosch, 2009-12) Lampiao, Fanuel; Du Plessis, S. S.; Strijdom, Hans; University of Stellenbosch. Faculty of Health Sciences. Dept. of Biomedical Sciences. Medical Physiology.ENGLISH ABSTRACT: In recent years there has been an increase in obesity and diabetes mellitus (DM). These conditions have for a long time been associated with infertility. Obesity is characterized by high levels of circulating leptin and cytokines as well as insulin resistance. Type I DM is associated with low or no insulin whereas, Type II DM is characterised by insulin resistance. As the prevalence of obesity and DM continues to rise, it is likely that the incidence of infertility associated with these pathological conditions will likewise increase. The effects of insulin and leptin on male reproductive function have been reported on the endocrine and spermatogenesis level, but their effects on cellular level of human ejaculated spermatozoa are yet to be elucidated. This study presents data on the role of insulin and leptin on human ejaculated spermatozoa and their interaction with cytokines and nitric oxide. In the first part of the study, we established the suitable concentrations of glucose, insulin and leptin that could be administered to human spermatozoa in vitro. Glucose concentration of 5.6 mM was chosen as the suitable concentration to be administered to human spermatozoa because it has previously been reported in the literature; furthermore, it is within the range of the physiological glucose levels found in the blood of fasting humans. Insulin and leptin concentrations of 10 μIU and 10 nmol were chosen respectively because they gave much improved sperm function and this was within the range of insulin and leptin levels previously measured in human ejaculated spermatozoa. This was followed by investigating the signalling pathway of insulin and its beneficial effects on human spermatozoa function. Endogenous insulin secretion from human ejaculated spermatozoa was blocked by nifedipine and its receptor tyrosine phosphorylation effects were inhibited by erbstatin while phosphatidylinositol 3-kinase (PI3K) phosphorylation activity was inhibited by wortmannin. Exogenous insulin administration significantly increased human sperm motility parameters as well as the sperm ability to acrosome react. The inhibition of endogenous insulin release from spermatozoa as well as the inhibition of the insulin receptor substrate (IRS) tyrosine phosphorylation significantly decreased motility parameters and the ability of spermatozoa to acrosome react. The study also investigated the effects of insulin and leptin on human sperm motility, viability, acrosome reaction and nitric oxide (NO) production. Both insulin and leptin significantly increased sperm motility parameters, acrosome reaction and NO production. The NO production induced by insulin and leptin was via PI3K signalling as evidenced by a reduction in NO levels when PI3K activity was inhibited by wortmannin. To investigate whether insulin and leptin could improve motility parameters of asthernozoospermic and teratozoospermic spermatozoa, the spermatozoa were separated into two fractions by means of a double density gradient technique. The gradient system was able to separate spermatozoa into high morphologically abnormal and less motile spermatozoa similar to that of asthernozoospermic and teratozoospermic patients as well as a more motile fraction. Insulin and leptin significantly increased the motility parameters of spermatozoa from the immature and less motile fraction. The fourth part of the study was aimed at investigating the effects of the cytokines, tumor necrosis factor alpha (TNF-α) and interleukin-6 (IL-6), on human sperm motility, viability, acrosome reaction and NO production. The study shows that TNF-α and IL-6 significantly reduced motility parameters and acrosome reaction in a dose4 and time-dependent manner. These cytokines were also shown to significantly increase NO production from human spermatozoa. The decreased motility parameters induced by these cytokines could be attributed to their ability to induce excessive NO production. It is not yet clear how they inhibit spermatozoa to undergo the acrosome reaction. The fifth part of the study was to investigate the expression and localization of glucose transporter 8 (GLUT8) in human spermatozoa. This study shows that GLUT8 is constitutively expressed and located in the midpiece region of the human spermatozoa. The study also showed that stimulating spermatozoa with insulin led to an increase in GLUT8 expression as well as translocation to the acrosomal region. In the last part of the study we wanted to investigate why the increase in NO generation by spermatozoa due to insulin and leptin stimulation is accompanied with increased sperm function whereas NO increased due to TNF-α and IL-6 stimulation is accompanied with decreased sperm function. We observed that TNF-α and IL-6 not only increased NO production but also ROS production. This study speculates that the decrease in sperm motility and acrosome reaction when TNF-α and IL-6 were administered was due to the concomitant high increase in NO and ROS they induced. In conclusion, this study has established in vitro beneficial effects of insulin and leptin in normozoospermic and asthernozoospermic human sperm function. These hormones influence sperm function via the PI3K signalling pathway in two ways. Firstly, by increasing GLUT8 expression and translocation thereby possibly increasing glucose uptake and metabolism and secondly, by increasing NO production. The study has also established that TNF-α and IL-6 have detrimental effects on human spermatozoa in a dose and time dependent manner. These effects are mediated via their ability to stimulate both NO and ROS production in human spermatozoa. This study reports that GLUT8 is expressed in the midpiece region of human spermatozoa and that insulin stimulation upgrades its expression and leads to its translocation to the acrosomal region.
- ItemEpacs exchange protein directly activated by cAMP role in obesity-induced cardiovascular diseases(Stellenbosch : Stellenbosch University, 2020-03) Ndlovu, Zibele; Marais, Erna; Strijdom, Hans; Stellenbosch University. Faculty of Medical Sciences. Dept. of Biomedical Sciences: Medical Physiology.ENGLISH ABSTRACT: Introduction and aims: The strong association between obesity and cardiovascular diseases (CVD) stresses the necessity of elucidating the underlying molecular mechanisms linking these pathologies. Evidence suggests that Epac (exchange protein directly activated by cAMP) could be a new therapeutic target for obesity and CVD. This study aimed to elucidate the role of Epac in (i) myocardial I/R injury of ex vivo hearts from normal weight; and diet-induced obese rats; (ii) vascular reactivity of ex vivo aortas from diet-induced obese rats and (iii) free fatty acid (FFA)-induced endothelial dysfunction in rat aortic endothelial cells (RAECs). Methods: Male Wistar rats of normal weight (250 to 350 g) or receiving a high-calorie diet (HC) for 16 weeks or age-matched controls (CD), were used. Mechanical function of isolated perfused working rat hearts were evaluated. Regional ischaemia (35 minutes) was followed by 60 minutes reperfusion and infarct size determination. Hearts were perfused with a selective Epac1 agonist (8-CPT-2’-O-Me-cAMP, CPT, 2 μM) or antagonist (ESI-09, 5 μM) for 10 minutes before (pre-treatment) or after (posttreatment) regional ischaemia and also post-treated with MEK-ERK inhibitor (PD98059, 10 μM) or PKB inhibitor (A6730, 2.5 μM). Thoracic aortas were isolated from rats fed either the HC or CD diet. Aortas were pre-incubated for 15 min with CPT, ESI-09 or nitric oxide synthase (NOS) inhibitor (L-NAME, 100 μM). Vascular reactivity was evaluated by phenylephrine-precontraction followed by acetylcholine-induced relaxation. Cardiac and aortic signalling proteins were detected with Western blotting. Cultured RAECs were supplemented with FFAs (Palmitic acid and Oleic acid) to induce endothelial injury. The effects of FFAs, CPT and ESI-09 on intracellular levels of NO, superoxide (ROS) and cell viability were determined with fluorescence-based assays. Results: After 16 weeks the HC animals presented with significantly increased body weight and visceral fat. In hearts, HC diet had no deleterious effects on post-ischaemic cardiac function and infarct size. Post-treatment with CPT (2 μM) reduced infarct size and improved cardiac recovery. This protection was abolished by post-treatment with A6730 in HC hearts, but not with PD98059. Furthermore, CPT increased CaMKII activation but reduced eNOS phosphorylation. Epac inhibitor (ESI-09, 5 μM) was detrimental to cardiac function. This was associated with: decreased phosphorylated ERK1/2 and GSK-3b; increased phosphorylated AMPK, ULK-1 and PARP cleavage. HC diet did not adversely affect vascular reactivity. CPT significantly improved vasorelaxation in both diets which were abolished by ESI-09. CPT induced vasorelaxation in a dose-dependent manner in both diets via NOS. ESI-09 reduced PKB phosphorylation in CD aortas and increased AMPK phosphorylation in both diets. In normal RAECs CPT improved NO production and reduced intracellular ROS. CPT could not reverse the detrimental effect of FFAs on RAECs. Conclusion: Post-ischaemic Epac activation is cardioprotective against I/R injury in ex vivo isolated rat hearts from HC, partially via the PKB pathway. Epac activation improved vasorelaxation via NOS in ex vivo aortas from both diets. In addition, Epac activation increased NO production and reduced ROS generation in normal RAECs. Therefore Epac may be a potential therapeutic target in the protection against CVD.
- ItemEvaluation of mitochondrial and molecular derangements in cardiac adipose tissue during type 2 diabetes and relationship with cardiovascular risk(Stellenbosch : Stellenbosch University, 2023-06) Nyawo, Thembeka Amanda; Pheiffer, Carmen; Mazibuko-Mbeje, Sithandiwe; Phiwayinkosi, Dludla; Hanel, Sadie Van Gijsen; Hans, Strijdom; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences. Division of Medical Physiology.ENGLISH ABSTRACT: Cardiovascular disease (CVD) affects hundreds of millions of people globally, and 18.6 million deaths were attributed to CVD during 2019 alone. Type 2 diabetes (T2D) and obesity contribute significantly towards the increasing prevalence of CVD. Ageing and adipose tissue dysfunction are important mechanisms in the pathology of these metabolic diseases and their downstream cardiovascular complications. The roles of visceral (VAT) and subcutaneous (SAT) adipose depots in T2D and CVD development have been well documented; however, there is limited evidence on the pathological contribution of cardiac fat (CF) to CVD. The aim of this study was to elucidate the role of CF, in comparison to retroperitoneal (RF) and inguinal (IF) fat depots, representatives of VAT and SAT, respectively, in the development and progression of CVD in an experimental mouse model of obesity and diabetes. The study used male obese, diabetic db/db mice and their lean db/+ counterparts to explore morphological features of CF, gene expression signatures, mitochondrial bioenergetics, and associations with CVD risk factors. Briefly, mice were monitored for 8, 12 and 18 weeks, during which body weight and fasting blood glucose concentrations were measured weekly. Glucose tolerance was assessed using the oral glucose tolerance test one week prior to euthanasia. Blood and tissue samples of the heart, CF, RF, and IF were collected for assessment of biochemical markers, histological examination using Haematoxylin and Eosin staining, and gene expression analysis using quantitative real time PCR. In addition, CF, RF and IF were harvested for the assessment of mitochondrial function in adipose-derived stromal cells (ADSCs). Phenotypic and metabolic parameters deteriorated with disease and age, where db/db mice displayed conditions of hyperglycaemia, hyperinsulinaemia, hyperlipidaemia and glucose intolerance with ageing. In addition, an age-related increase in adiponectin serum levels were observed in db/+ mice, while in db/db mice, adiponectin levels decreased with age. Furthermore, histological analysis showed that adipocyte size in all depots increased over time in both the non-diabetic and diabetic state. Adipose depot-, disease- and age-related changes in gene expression signatures in CF, RF and IF were observed, with RF and IF in db/db mice exhibiting upregulation of genes involved in inflammation and oxidative stress, while CF appears to possess increased expression of genes representing thermogenic capacity. Disease- and age-related differential regulation of circulating CVD risk markers were observed. Dysregulation of markers such as metalloproteinase 9 (MMP9), intercellular adhesion molecule 1 (ICAM1), platelet endothelial cell adhesion molecule 1 (Pecam1) and Thrombomodulin (THBD) suggested vascular remodelling and dysfunction during CVD progression. Moreover, circulating levels of MMP9, ICAM and P-Selectin positively correlated with CF adipocyte size. In heart tissue, signs of histological myocardial changes with microarchitecture disruption and the presence of intramyocardial lipid droplets in db/db mice were observed in with ageing. Moreover, gene expression analysis in heart tissue revealed a disease- and age- related downregulation of brain natriuretic peptide (BNP) levels in db/db mice. Furthermore, ADSCs from CF had higher mitochondrial bioenergetics parameters compared to ADSCs from RF. This may be attributed to the higher uncoupling protein 1 (UCP1) expression in CF which reportedly decreases oxidative phosphorylation through thermogenesis. In conclusion, the positive correlation between CVD risk markers with CF adipocyte size in the diabetic state indicates a relationship with CF, supporting the notion that increased CF adiposity is associated with increased CVD risk. Importantly, CF, unlike RF and IF, displays beige-like adipocytes and thermogenic capacity, which may help mitigate the harmful effects of diabetes and its cardiovascular complications.
- ItemExposure of cardiac microvascular endothelial cells to harmful stimuli : a study of the cellular responses and mechanisms(Stellenbosch : Stellenbosch University, 2014-04) Genis, Amanda; Strijdom, Hans; Huisamen, Barbara; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences. Division of Medical Physiology.ENGLISH ABSTRACT: Exposure to harmful stimuli can render vascular endothelial cells dysfunctional, characterised by reduced nitric oxide (NO) bioavailibility. Endothelial dysfunction (ED) is a reversible precursor of ischaemic heart disease (IHD), and understanding the mechanisms underlying the development of ED could lead to clinical strategies in preventing/treating IHD. Very little is known about the responses of cardiac microvascular endothelial cells (CMECs) to pro-ED stimuli, as most studies are conducted on macrovascular endothelial cells. The current dissertation set out to comprehensively investigate the responses of cultured primary adult rat CMECs to known harmful stimuli, viz. hypoxia and tumor necrosis factor-alpha (TNF-α; proinflammatory cytokine). We were interested to investigate whether this distinct endothelial cell type would develop classical features of ED, and if so, what the underlying mechanisms were. First we aimed to establish a baseline characterization of the CMECs under control conditions. Next, we developed a model of hypoxia-induced cell injury and measured apoptosis/necrosis, intracellular NO and reactive oxygen species (ROS), expression and activation of signalling proteins involved with NObiosynthesis, hypoxia and apoptosis, and differential regulation of proteins. Finally, we characterised CMEC responses to treatment with TNF-α. We assessed apoptosis/necrosis, intracellular NO and ROS levels, NO-biosynthesis pathway proteins and large-scale differential protein regulation. The above measurements were performed by morphological assessment (light and fluorescence microscopy), FACS analysis, western blotting and large-scale proteomic analyses. Data showed that CMECs shared many baseline features with other endothelial cell types, including morphological appearance, LDL-uptake, NO-production, and expression of eNOS protein. In a novel observation, proteomic analysis revealed the expression of 1387 proteins. Another novel finding was the high abundance of structural mitochondrial proteins, suggesting that CMECs require mitochondria for non-respiration purposes as well. High expression of vesicle, glycolytic and RAS signalling proteins were other features of the baseline CMECs. CMECs exposed to hypoxia responded by increased apoptosis/necrosis and expression of the hypoxia-marker, HIF-1α. Interestingly, hypoxic CMECs showed increased eNOS-NO biosynthesis, associated with increased mitochondrial ROS and reduced anti-oxidant systems, suggestive of oxidative stress. In accordance with the literature, several glycolytic proteins were up-regulated. A novel finding was the up-regulation of proteins involved with protein synthesis, not usually described in hypoxic cell studies. The CMECs responded to TNF-α-treatment by exhibiting hallmarks of ED, namely attenuated biosynthesis of PKB/Akt-eNOSderived NO and the development of outspoken response to oxidative stress as indicated by the up-regulation of several anti-oxidant systems. The data showed that TNF-α treatment elicited classical TNF-Receptor 1-mediated signalling characterized by the dual activation of pro-apoptotic pathways (BID and caspase-3) as well as the protective, pro-inflammatory IKB-alpha–NF-KB pathway. In conclusion, this is the first study of its kind to describe a comprehensive characterisation of CMECs under baseline and injury-inducing conditions. On the whole, although it appeared as if the CMECs shared many responses and mechanisms with more frequently researched endothelial cell types, the data also supplied several novel additions to the literature, particularly with the application of proteomics. We believe that this dissertation has provided more insights into endothelial heterogeneity in the vascular system and into the mechanisms adopted by CMECs when exposed to stimuli typically associated with cardiovascular risk.
- ItemGamete proteomic profile of male patients suffering from sexually transmitted infections(Stellenbosch : Stellenbosch University, 2016-03) Flint, Margot Gwyneth; Du Plessis, Stefan; Van der Horst, Gerhard; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences. Medical Physiology.ENGLISH ABSTRACT: The aim of this study was to provide better insight into the effects of Neisseria gonorrhoea, Chlamydia trachomatis, and Trichomonas vaginalis on semen characteristics. In addition, a crucial focus was also to determine the protein profile of spermatozoa isolated from these infected semen samples. Identification of semen samples positive for bacterial colonisation of N. gonorrhoea and T. vaginalis were done through the use of differential isolation mediums. For the detection of samples positive for C. trachomatis, the ImmunoComb® was employed, which is a quantitative indirect enzyme immunoassay. Macro- and microscopic semen and spermatozoa parameters were assessed, including: volume, pH, viscosity, concentration, motility, morphology, viability, acrosome reaction, leukocyte count, and PMN-elastase concentration. To assess prostate and seminal vesicle functioning, the seminal concentrations of citric acid and fructose were photometrically quantified. The quantification of the level of reactive oxygen species (ROS) production was determined by means of flow cytometry and the DNA fragmentation was detected using a commercially available assay. For proteomic analysis, samples were iTRAQ (isobaric tags for relative and absolute quantification) labelled and underwent liquid chromatography-mass spectrometry, followed by data analysis, protein identification, and quantification. Results of the study showed that amongst the three microorganisms, the most prevalent occurrence rate in the population tested was that of T. vaginalis. The effects of the studied sexually transmitted infections (STIs) on the spermatozoa parameters demonstrate the negative impact of the microorganisms on the fertility outcome of the male partner. The study has shown the relationship between leukocyte-derived ROS and DNA fragmentation, which can significantly impair the fertility outcome. Additionally, the findings of decreased concentrations of fructose in the ejaculates positive for N. gonorrhoea, demonstrates the effect of an STI on the glandular functioning of the secretory activity of the seminal vesicles. The observed negative correlation between ROS and DNA fragmentation in samples positive for N. gonorrhoea demonstrates the impact of a pro-oxidant overload on spermatozoa DNA integrity. A considerable number of 178 differentially expressed proteins (DEPs) were identified in the STI positive sample groups. The study subsequently focused on specific proteins according to their role in male fertility. Insight into the role DEPs may play in spermatozoa metabolism and the impact on the motility was provided. Amongst structural related proteins, the down-regulation of outer dense fibre 2, in samples positive for C. trachomatis, was evaluated. Oxidative stress related proteins included the up-regulated superoxide dismutase 1 in the ejaculates positive for N. gonorrhoea and down-regulated peroxiredoxin 5 in the C. trachomatis positive group. From the proteins involved in the response to physiological stress, heat shock proteins was discussed, with the down-regulation of the 70-kDa heat shock protein and the up-regulated HSP 90-kDa-beta member 1, both identified in samples positive for N. gonorrhoea. Among immune response proteins, prolactin-inducible protein (C. trachomatis) was found to be down-regulated, while azurocidin (N. gonorrhoea) and filamin-B (T. vaginalis) were up-regulated. Proteins involved in DNA condensation included down-regulated protamine-2 (C. trachomatis). The identification of proteins, which are differentially expressed between spermatozoa from samples positive for STIs, can provide crucial insight into their possible influence on male fertility and role as potential biomarkers for further research. Research on a proteomic and molecular level could allow for the prevention of the long-term obstacles facing partners experiencing compromised fertility. Despite the ongoing research focusing on the sperm proteome, it can be stated with reasonable certainty, that this is the first study into the proteomic profile and corresponding parameters of spermatozoa isolated from semen samples positive for STIs.
- ItemHIV/AIDS and air pollution as emerging cardiovascular risk factors in Cape Town populations : is endothelial function a marker of effect(Stellenbosch : Stellenbosch University, 2020-03) Everson, Frans Pieter; Strijdom, Hans; De Boever, P.; Nawrot, T. S.; Goswami, N.; Stellenbosch University. Faculty of Medical Sciences. Dept of Biomedical Sciences: Medical Physiology.ENGLISH ABSTRACT: Background: HIV and antiretroviral therapy (ART) are associated with cardiovascular disease (CVD). Concomitantly,air pollution is a global health concern and associated with CVD. Although South Africa (SA) has the largest HIV population, ART roll-out programme and also one of the most carbon-intensive economies in world, the effects of these emerging cardiovascular risk factors remain under investigated. Aim: The current study aimed to investigate whether endothelial function (an early marker of cardiovascular risk/disease) is a marker of effect of HIV, ART and air pollution in a study cohort residing in the Cape Town region. Methods: Volunteering participants were recruited from health-care clinics in Worcester and Cape Town. A health questionnaire was completed (demographic, lifestyle, and socioeconomic information), anthropometric measurements taken (BMI and blood pressure) and fasting blood and urine samples collected from each participant for chemical pathology and biomarker analyses. Sub-study 1 followed a repeated measures design (baseline and 18-month follow-up visit) to investigate the effects of HIV (viral load) and ART (pre- vs. post-ART treatment and an 18-month ART treatment period) on markers of endothelial function. Sub-study 2 investigated the effects of personal air pollution exposure (NO2 and BTEX via passive diffusion samplers) in a repeated measures design (baseline and 6-month follow-up visit) on markers of endothelial function. Markers of endothelial function for both sub-studies included: tumor necrosis factor-alpha (TNF-α), high sensitivity C-reactive protein (hsCRP), intercellular adhesion molecule-1 (ICAM-1),vascular cellular adhesion molecule-1 (VCAM-1), e-selectin, p-selectin, vascular endothelial growth factor (VEGF), plasminogen activator inhibitor-1 (PAI-1), retinalmicrovascular calibres (including central retinal arteriolar/venular equivalent (CRAE; CRVE), CRAE/CRVE ratio (AVR)) and flow-mediated dilation (FMD). Results: Sub-study 1: Each interquartile range (IQR) increment increase in viral load (1300 copies mRNA/ml) was associated with CRVE (9.29 μm), AVR (-0.016) and %FMD (-2.13%). Compared to baseline, initiating ART was associated with VCAM-1 (-148 ng/ml), VEGF (40.6%), PAI-1 (14.12 ng/ml) and CRVE (-6.42 μm). An 18-month ART treatment period was associated with TNF-α (-1.22 pg/ml), ICAM-1 (-45%), e-selectin (-5.57 ng/ml) CRVE (–7.00 μm) and % FMD (-9.8%). Sub-study 2: Each IQR increment increase in NO2 (7.0 μg/m³) was associated with VEGF (-18.9%), CRVE (-2.93 μm) and baseline brachial artery diameter (-0.29 μm). Benzene (IQR: 3.3 μg/m³) was associated with p-selectin (-5.8 pg/ml), toluene (IQR: 30.0 μg/m³) was associated with PA1-1 (7.2 ng/ml). Ethyl-benzene (IQR: 3.8 μg/m³) was associated with VCAM-1 (-4.9%) and PA1-1 (9.1 ng/ml). m+p-Xylene and o-Xylene (IQR 3.8 μg/m³ respectively) were associated with VCAM-1 (-1.47% and -4.5%) and PA1 (3.08 ng/ml and 11.7 ng/ml). 3+4MHA (1380 ng/ml) was associated with %FMD (-0.40%). Discussion and Conclusion: The study showed that endothelial function is a marker of effect of HIV, ART and air pollutants (NO2 and BTEX) in the current study population, and that HIV and air pollution contribute to an increased cardiovascular risk profile while ART exhibited varying effects. This study underscores the relevance of these emerging cardiovascular risk factors in South Africa and the greater sub-Saharan Africa region. This study strongly supports the need for further investigation, also in study populations beyond the Western Cape.
- ItemHypoxia and the heart : the role of nitric oxide in cardiac myocytes and endothelial cells(Stellenbosch : University of Stellenbosch, 2007-03) Strijdom, Hans; Lochner, Amanda; Moolman, Johan; University of Stellenbosch. Faculty of Health Sciences. Dept. of Biomedical Sciences. Medical Physiology.Nitric oxide (NO) is a major signaling molecule in the heart with various biological effects. The putative role of NO as a cardioprotective agent against ischaemiareperfusion injury and in ischaemic preconditioning (IP) has made it one of the fastest growing fields in basic cardiovascular research. However, NO may also be associated with harmful effects, especially when released in excessive amounts. Little is known about the relative contributions to NO-production by the cardiac microvascular endothelial cells (CMECs) and the adjacent cardiomyocytes. Furthermore, the respective roles of endothelial NOS (eNOS) and inducible NOS (iNOS) are not well characterized in these cell types, particularly in hypoxia. In order to gain a better understanding of the role of NO in the hypoxic/ischaemic heart, the aims of this study were to: (1) develop an isolated cardiomyocyte model in which hypoxia and early IP can be induced and the role of NO assessed; (2) measure NOproduction in cardiomyocytes and CMECs under baseline and hypoxic conditions; and (3) evaluate the expression, regulation and activation of eNOS and iNOS in cardiomyocytes and CMECs (baseline and hypoxia) and establish the relationship with NO-production under these conditions. Cardiomyocytes isolated from adult rat hearts and commercially purchased rat CMECs were used as cell models.
- ItemIdentification of novel DNA methylation signatures in the development of cardiovascular disease(2021) Jooste, Tracey; Johnson, Rabia; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences: Medical Physiology.ENGLISH ABSTRACT: Cardiovascular diseases (CVDs) remain the leading cause of death worldwide, claiming approximately 17.9 million lives annually. The last few decades have seen an exponential increase in the prevalence of major CVD risk factors, such as obesity, insulin resistance (IR), and type 2 diabetes mellitus (T2DM) in underdeveloped countries, including South Africa. This increase is concomitant to escalating CVD incidence and in part due to increased consumption of diets high in fat and sugar, and sedentary lifestyles. Several mechanisms have been implicated in the pathogenesis of diabetic-induced CVD and recently, increasing evidence suggests that dysregulation of the epigenome may play an important role in the development of T2DM and related cardiac complications. More specifically, aberrant DNA methylation has been extensively investigated and implicated in the development of chronic diseases such as obesity, T2DM and CVD. Nonetheless, it has been suggested that some active dietary compounds may reverse this epigenetic phenomenon which allows for the modification of the transcription of critical genes associated with the progression of CVD. Several plant-polyphenols have been reported to influence gene transcription by altering DNA methylation status. Thus, targeting DNA methylation could provide a promising approach for alternative prevention strategies. This study explored the gene expression networks activated during diet-induced CVD and the ability of a green rooibos extract, Afriplex GRT, to alter this consequence. In addition, the study aimed to evaluate aberrant DNA methylation associated with diet-induced CVD to further elucidate pathophysiology. To this end, cardiomyocytes exposed to high glucose and palmitate (HG + Pal) displayed a diminished antioxidant defence system, mitochondrial dysfunction and increased apoptosis, indicative of cardiac stress. Additionally, the combinatory treatment with HG + Pal induced transcriptional changes associated with inflammation, oxidative stress, altered lipid metabolism and increased contractile dysfunction, ultimately promoting the development of atherosclerosis and hypertrophic cardiomyopathy. Interestingly, post treatment with Afriplex GRT or Aspalathin had no significant effect on the metabolic and molecular derangements induced under HG + Pal stress. Similarly, RNA sequencing conducted on cardiac tissue of Wistar rats that received a high fat, high sugar (HFHS) diet, revealed the downregulation of differentially expressed genes (DEGs) involved in host antioxidant activity and inflammatory response, accompanied with an increase in hypertrophic gene expression possibly affecting cardiac muscle functionality. Supplementation with Afriplex GRT™ yielded no high confidence results for the amelioration of the transcriptomic signatures resulting from HFHS diet feeding. To profile DNA methylation throughout disease progression, cardiac tissue of male Wistar rats maintained on a HFHS diet were subjected to whole genome bisulfite sequencing (WGBS). The latter revealed aberrant DNA methylation of genes linked to the phagosome, platelet activation, toll-like receptor signalling and diabetic cardiomyopathy. Furthermore, hypomethylation within the intergenic and gene body regions of several differentially methylated genes (DMGs) overlapped with DEGs identified in the RNA sequencing analysis. Collectively these results demonstrate the ability of the HFHS diet to act as a pathological stimulus capable of inducing altered gene expression and DNA methylation associated with a heightened proinflammatory and lipid metabolism response that increased the risk of CVD development.
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