ORAL PRESENTATIONS (Non-competition)
Ultrastructural evaluation of clot properties and gene expression of clotting factor XIII in whole blood of asthma patients
A Phulukdaree1, M N Phasha1, J Bester1, Pretorius E2
Department of Physiology, Faculty of Health Sciences, University of Pretoria1
Department of Physiology, Faculty of Health Sciences, Stellenbosch University2
Asthma is a highly prevalent chronic inflammatory disease of the airways, of which the molecular regulatory mechanisms and relationship to coagulopathies is largely unknown. Coagulation plays an important role in the pathogenesis of asthma and the traditional coagulation factors have an impact on the perpetuation of inflammation in asthma.
The objective of this study was to analyse the viscoelastic property of the clot by using thromboelastography(TEG), the expression of coagulation factor FXIII by qPCR and and ultrastructural analysis of fibrin threads by using scanning electron microscopy(SEM) in allergic asthma patients. Whole blood was collected from 30 patients with allergic asthma and 30 healthy individuals. Plasma was used for SEM and TEG, and whole blood was used for qPCR. Analysis of TEG indicated a significantly higher reaction time (11.64±0.55 min vs. 8.16±0.50 min) maximal amplitude (36.00±1.55 mm vs. 31.45±0.88 mm) and α-angle (41.80o±2.588o vs. 63.58o±1.74o) in asthma patients than controls (p<0.0001, p<0.05, pFibrin and factor XIII are essential parts of haemostatic system. The data indicate that the clot strength and firmness is altered in asthma patients compared to controls, from which we can conclude that the coagulation profile is altered in asthma patients. Analysis of clotting Factor XIII expression demonstrated that the production of coagulation Factor XIII is upregulated in the blood of asthma patients. Moreover, the ultrastructural study of fibrin network also shows an irregular web of fibres with matted appearance. The mechanism by which overexpressed Factor XIII plays a role in altering the cross-linking of fibrin fibres in asthma patients needs to be elucidated by further molecular investigations.
Lipid profile of obese albino rat after treatment with diets from developed recipes with local dietary fibre foodstuffs in Nigeria
Dr Ann I Asouzu1
Ignatius Ajuru University of Education1
Introduction: Eating habit and type of food can pre-dispose one to health risks such as obesity (high body fat and overweight) prevalent among women. Some of the obesity induced foods can increase lipid profile (like cholesterol content, LDL, HDL, Triglycerides (TGL) and VLDL) in the body. Consequently, high lipids levels are likely to escalate issues of blocked arteries, colon cancer, cardiovascular diseases, hypertension, stroke and high mortality rate. Obesity can be treated with diets, hence the need for developing recipes from whole grains, cereals, legumes, roots and tubers, fruits and vegetables. This background prompted an investigation into lipid profile of obese albino rat after treatment with diets from developed recipes with local dietary fibre foodstuffs in Nigeria.
Methods: Random sampling technique was adopted in the selection of 90 subjects (i.e. 20 Nutritionists, 40 Home Economists,10 Agriculturists, 6 Food Analyst, 14 Dieticians and 4 Veterinary Doctors) in Enugu State. Furthermore, the study adopted research and development carried-out in seven phases via: determining the fibre content of the chosen local foodstuffs using the AOAC standard, developing recipes like casiavitas, herb rice, bread fruit and corn, kernel, jack bean risotta, cowpea and maize twist, amarantus delicacy, nestroe, local fruit mix and whole wheat bread. Purchasing of fifty female adult albino rats from Veterinary Department of UNN, taking Body Mass Index Measurements and Lipid Profile Recordings (BMILPR) of the rats before, during and after feeding with the diets, division of the rats into 10 groups of 5 rats each based on body weight, induced obesity on rats with condensed milk and sugar, and lastly treating the rats with the developed dishes. Data obtained was analyzed using mean and standard deviation.
Results: The study revealed that all the diets developed from local recipes contributed significantly to weight loss and reduction in cholesterol and other lipid profiles of albino rats. However, local fruit mix has the highest HDL and TGL reduction (from 40.97 after inducement to 32.29 after treatment) and (from 97.83 to 25.81) respectively. The study recommends that local dietary fibre rich dishes should be used for women with obesity and cholesterol challenges.
Effects of atrazine on the cardiac structure and expression of endothelial Nitric Oxide Synthase (eNOS) in Xenopus juvenile and adult frogs.
Jaclyn Asouzu Johnson1
Felix Mbajiorgu1, Amadi Ihunwo1, Luke Chimuka1
University of the Witwatersrand1
Atrazine is an affordable pesticide. Atrazine’s properties enable it to gain access and contaminate water sources and the environment following agricultural and urban application. Although atrazine has been reported to decrease atria contraction and promote angiogenesis in rats, however little is known about the effects of atrazine on the anuran cardiac tissue. The frog heart uniquely synthesizes endothelial nitric oxide synthase (eNOS) only in the endocardial endothelium; making the Xenopus heart ideal as a model to reveal endothelial cardiac toxicity.
This study investigates the effects of atrazine on the connective tissue profile and expression of eNOS in the heart of Xenopuslaevis juvenile and adult frog. One hundred and twenty laboratory tadpoles aged 10 days bred in at the University of Witwatersrand, Central Animal Services and 60 wild caught (270 days old) male African clawed frogs (Xenopusleavis) procured from the African Xenopus Facility (Knysner, Western Cape, South Africa) were used. They were divided into 4 groups of 15 each and exposed to 0 µg/L (control), 0.01 µg/L, 200 µg/L and 500 µg/L atrazine water concentrations respectively. The atrazine concentration in the exposure tanks was measured weekly. Atrazine concentrations and were verified to be within ±5% for 0.01 µg L-1, and 7 % for both 200 and 400 µg L-1 of target concentrations. Thereafter, all animals were euthanized with benzocaine and the hearts were removed, fixed in 10% buffered formalin, and processed using Mallory- Heidehan rapid one step staining, eNOS immunofluorescence and immunohistochemical staining. Histological examination revealed dilated cardiomyopathy in 0.01µg/L and 200 µg/L groups, and hypertrophied cardiomyopathy effects in 500 µg/L group. eNOS expressions in the endocardial endothelium were reduced in the in 200µg/L and 500 µg/L groups; which are suggestive of decreased cardiac contractility.
Free radical scavenging ability of improved thiosemicarbazone-triazole hybrids and their influence on genes related to diabetes mellitus
Dr Ademola Ayeleso
North West University
Thiosemicarbazones are biologically active compounds which are obtained by the condensation of thiosemicarbazide or a substituted thiosemicarbazide with a suitable aldehyde or ketone. 1,2,3-Triazole is a heterocyclic compound which belong to a class of azole. An azole is a five-membered aromatic ring which contains at least one nitrogen atom and another heteroatom such as nitrogen, oxygen, or sulphur in the ring. Both compounds are known to exhibit health promoting effects. Oxidative stress, an imbalance between the production of free radicals and body antioxidant system has been implicated in the pathogenesis of diabetes. Free radicals attack important macromolecules leading to cell damage. Antioxidants are intimately involved in the prevention of damage caused by free radicals. The anti-diabetic effect of the improved hybrid compounds (2a-h) of thiosemicarbozone and triazole was carried out on glucose transport genes (Glut-4, Mef2a, Pgc-1 and Nrf-1).
The antioxidant capacity (FRAP, TEAC and DPPH) of the hybrids was also carried out. From the results, 2b and 2h showed more pronounced effects in the upregulation of the diabetes associated genes. The addition of methoxy group as an improvement to the previously synthesised compounds could be a reason for the expression of the genes, thereby enhancing glucose transport. All the hybrid compounds showed free radical scavenging abilities. In conclusion, the development of hybrid compounds (2b and 2h) from thiosemicarbozone and triazole could be useful as potential drugs for the management of diabetes mellitus.
The effect of Infra Slow Frequency Neurofeedback on QEEG, HRV, Temperature, EMG, respiration, skin conductance and blood pressure
Peet du Toit1
Department of Physiology, Faculty of Health Sciences, University of Pretoria1
Introduction: Infra Slow Frequency (ISF) Neurofeedback has been shown to influence certain autonomic functions through observations seen during training sessions.
The aim of the study was to determine if it has a significantly measurable change by observing the effect on HRV, temperature, EMG, respiration, skin conductance, BP and the effect on brain functionality by comparison of pre-and post QEEG measurements.
Methods: 20 Male and female subjects with general complaints of anxiety, sleep disturbances and mental performance shortcomings were assessed by means of a QEEG and general questionnaire. Each then completed an average of 12 ISF neurofeedback training sessions of 30 minutes each during which we tracked HRV, peripheral temperature, EMG, respiration and skin conductance as a measurement of physiological response. Blood pressure was measured pre- and post-session. Physiological changes that occurred within sessions as well as changes that occurred over the duration of the study were compared. A post QEEG was performed to determine what changes in brain activity occurred. The questionnaire was also completed again to see which general complaints had improved.
Results: In some subjects, a significant change could be seen in pre- and post QEEG results. In others, the changes were to a lesser extent but still notable. Measurable physiological changes were seen in all parameters measured.
Conclusion: ISF Neurofeedback training has a measurable effect on certain physiological parameters as was recorded in the study. There was also a notable improvement in brain function as was reported by subjects and measured by the QEEG.
Altered Relationship between Aortic Forward and Backward Wave Pressures Following Habitual Physical Exercise in Spontaneously Hypertensive Rats
Dr AdamuJibril Bamaiyi1
Gavin R Norton1, Grace Tade1, Glenda Norman1, Angela J Woodiwiss11
Cardiovascular Pathophysiology and Genomics Research Unit, School of Physiology Faculty of Health Sciences, University of the Witwatersrand1
Background: Although physical exercise training results in an enhanced cardiac systolic function in hypertension, whether through Newton’s 3rd Law of Motion, the increased forward wave pressure (Pf) produced by this effect translates into an increased aortic backward (reflected) wave pressure (Pb) and thus further increases in cardiac afterload is unknown.
Methods: We assessed the impact of 3 months of habitual exercise (spontaneous running on wheels) on aortic pressures determined over an incremental range of mean arterial pressures (MAP) produced by administration of the vasoconstrictor, phenylephrine, in Spontaneously Hypertensive rats (SHR). Aortic Pf, Pb, pulse pressure (PP) and the reflected wave magnitude (RM=Pb/Pf), a Pf-independent measure of reflected wave function, were determined from aortic pressure (catheter) and flow (Doppler in the outflow tract) measurements under isoflurane anaesthesia. Aortic Pf and Pb were determined using wave separation analysis.
Results: Exercise training did not modify tail cuff blood pressures in SHR. Phenylephrine administration (1 mg/kg) resulted in striking increases in Pf, Pb, PP and RM over a range of mean arterial pressure (MAP) and total peripheral resistance (TPR) values (p<0.0001). As compared to sedentary SHR at 2-3 months of age (n=9), sedentary SHR at 5-7 months of age (n=8) demonstrated an increase in the slope of the relationship (β-coefficient) between MAP (index of arteriolar function) and RM (SHR at 5 months of age=0.35±0.03 vs SHR at 2-3 months of age=0.23±0.02, p<0.005). This effect was largely driven by a decreased Pf for a given MAP in older SHR, whilst Pb for a given MAP remained unchanged. Habitual exercise in SHR (n=10) however returned the MAP-RM relationship to values similar to young SHR (0.11±0.04 vs non-exercised SHR, p<0.0005), an effect produced largely by an increased slope of the MAP-Pf relationship, whilst the MAP-Pb relationship remained unchanged. All data were reproduced with TPR rather than MAP employed as an index of arteriolar function.
Conclusion. Independent of effects on arteriolar function, chronic physical exercise in SHR decreases wave reflection and thus with an exercise-induced increase in aortic Pf (by increasing cardiac systolic function) prevents parallel increases in Pb and hence cardiac afterload.
Individual effects of IL-1 β, IL-6 AND IL-8 on erythrocytes and platelets
Dr Janette Bester1
University of Pretoria1, University of Stellenbosch2
Introduction: Interleukins plays a major role in inflammation (1). Over activation of the hemostatic system is known in systemic inflammation (2, 3). IL-1 β, IL-6 AND IL-8 are upregulated in most inflammatory conditions however it is not known how the individual Interleukins act upon the structure of the components of the hemostatic system, and how the viscoelastic clot parameters are changed.
Methods: low physiological levels, representative of chronic inflammation of IL-1 β, IL-6 AND IL-8 was added to control whole blood and Scanning electron microscopy (SEM) was used to study the ultrastructural changes of erythrocytes when exposed to each individual interleukin. Thromboelastography was used to measure the viscoelastic properties of clot formation when exposed to each individual interleukin.
Results: The results showed that all three interleukins caused the viscoelastic properties to display a hypercoagulability trend and the ultrastructural analysis showed abnormal platelets such as hyper activation and spreading. With SEM analysis erythrocyte structure was remarkably affected only in the presence of IL-8, where the morphological changes resembled that typically seen in eryptosis.
Conclusion: It is suggested that erythrocytes and platelets are particularly sensitive to Interleukin presence, and that they are excellent health indicators.
1. Levi M, Poll T. Coagulation in patients with severe sepsis. SeminThrombHemost. 2015;41(1):9-15.
2. Choi G, Schultz MJ, Levi M, van der Poll T. The relationship between inflammation and the coagulation system.
Swiss Med Wkly. 2006;136(9-10):139-44.
3. Karabudak O, Ulusoy RE, Erikci AA, Solmazgul E, Dogan B, Harmanyeri Y. Inflammation and hypercoagulable state in adult psoriatic men. ActaDermVenereol. 2008;88(4):337-40.
Aerobic and anaerobic energy supply during a 2000 m rowing ergometer time trial and relationship to performance in elite rowers
Clark, J.R.1 & du Toit, P.J.1
Department of Physiology, University of Pretoria
Introduction: Exercise training programmes prescribed to athletes are typically based on the physiological demands of the sport, including the nature and extent of energy supply during the activity.
The aims of this study were to describe the relative aerobic and anaerobic energy supply during a 2000 m rowing ergometer time trial, and to examine the correlations between physiological responses and performance.
Methods: Twenty-five elite male rowers (age: [mean ± standard deviation] 21.0 ± 3.6 y, maximum oxygen (O2) uptake [VO2max]: 4.64 ± 0.54 L∙min-1) volunteered as participants. Following surface anthropometry measurements, participants performed a self-paced maximum effort 2000 m time trial under standard conditions, with post-exercise blood lactate concentration determined from arterialised earlobe capillary samples. On a separate laboratory visit participants performed an incremental exercise test spanning ~35% to 85% of mean time trial power output to determine the oxygen uptake (VO2)–power output relationship, peak power output and VO2max. All exercise tests were performed on an air-braked rowing ergometer with pulmonary gas exchange monitored continuously. Time trial accumulated O2 uptake and accumulated O2 deficit were calculated to determine aerobic and anaerobic energy supply, respectively.
Results: Aerobic and anaerobic energy supply respectively represented 80-82% and 18-20% of total energy cost. Performance time correlated (Spearman’s rho) strongly to peak VO2 during the time trial (rho = -0.92; p < 0.001), peak power output (rho = -0.96; p < 0.001), VO2max (rho = -0.92; p < 0.001), and moderately to body mass (rho = -0.52; p = 0.008) and stature (rho = -0.55; p < 0.004). Peak blood lactate concentration following the time trial was not significantly related to measures of anaerobic energy supply.
Conclusion: While aerobic energy supply dominates energy provision it is accompanied by extensive anaerobic energy supply during a maximum effort 2000 m rowing ergometer time trial. Improved understanding of aerobic and anaerobic energy supply during simulated rowing performance trials has practical utility for exercise scientists and coaches in terms of rower identification and management, and the planning, regulating and monitoring of rowing training programmes.
The relationship between emotional wellbeing and health-related physical fitness in young rugby union players
Dr Nicoleen Coetzee1
Megan Payne1, Peet du Toit1, Michael Kleynhans1
University of Pretoria1
Rugby Union is a popular sport both internationally and nationally. The sport has seen a rise in high-intensity activity and is more physically demanding on players since turning professional in 1995. Over the past decade, professional rugby players have made frequent appearances in the media due to lapses in emotional, physical, and social wellbeing, suggesting rugby union players may experience compromised health.
Research has largely ignored rugby players’ health when health is defined beyond injury.
The present study aimed to provide insight into the health of young rugby union players by investigating two components of health, namely emotional and physical wellbeing and the relationship between these facets. Two hundred and thirty-eight (238) young male rugby union players, situated at the Investec Rugby Academy participated in the study. Emotional wellbeing (PW) was measured by the State Trait Personality Inventory (form Y) (STPI-Y) and the Sport Competitive Anxiety Test (adult form) (SCAT-A). Physical wellbeing was defined as health-related physical fitness (HRPF). HRPF was assessed by measurements of the rugby players' body composition, physical fitness, and heart health. Pearson's correlation co-efficient was calculated between the variables used to measure HRPF and PW.
The results showed the rugby players experience average levels of competitive anxiety, above average levels of anger, anxiety, and depression and a relatively high level of curiosity. The rugby players had a high level of HRPF, with the exception of body composition (particularly BMI), cardiovascular endurance, muscle strength and endurance, and heart health (when measured according to the CSI), which indicate a moderate risk to the players’ health. Furthermore, the results show significant correlations between variables of HRPF and PW in the rugby players.
The findings that indicate young rugby union players do not experience optimal PW and HRPF, as well as the significant correlations found between the variables of the rugby players PW and HRPF, suggest these players may be in a state of overtraining or experiencing overtraining syndrome.
The effect of photoshopping on heterosexual males' perception of females
Dr Nicoleen Coetzee1
Bendoline Holtzhausen1, Peet du Toit2, Michael Kleynhans2, Evangeline Nortje2
Department of Psychology1 and Department of Physiology2, University of Pretoria
Attractiveness is a phenomenon that is highly prioritised in society and has a variety of implications on the health, mental health, occupational, judicial and economic spheres. The media is known to have an impact on how certain norms, expectations and beliefs in society are formed, including, but not limited to, the creation of beauty ideals. Despite this importance, little research has been done on how men have come to perceive the attractiveness of women due to daily exposure to media and photoshopped images of women.
This study therefore attempts to address this gap in research by studying the impact that media and the use of photoshopping might have on males' perception of female attractiveness. A mixed-method approach was followed. The quantitative data entailed a comparison of heart rate, breathing rate, heart rate variability and viewing time for 24 participants between non-photoshopped and photoshopped images. The qualitative section comprised eye tracking data and a questionnaire on the topic of media, photoshopping, features of attractiveness and what impact these three constructs might have on each other for 25 participants.
The results indicate on a physiological level there are limited and sporadic changes in how men view, and respond to, photoshopped images of women. Most participants report that especially on a psychological level, media and photoshopping has in impact on their perceptions of attractiveness.
Chronic depressive symptoms desensitize the renin system to protect against volume overloading hypertension in Blacks: the SABPA study
Arnoldeen C de Vos1
Leone Malan1, Marike Cockeran2, Jacobus D Scheepers1,Nicolaas T Malan1
Hypertension in Africa Research Team (HART), School for Physiology, Nutrition, and Consumer Science, North-West University, Potchefstroom1, Medicine Usage in South Africa (MUSA), North-West University, Potchefstroom2<
Background: Raised renin levels in White albeit low-renin levels in Black populations have been associated with volume-loading hypertension. Depressive symptoms, frequently co-occurring with vascular dysregulation, might reflect a disturbed renin-angiotensin-aldosterone-system (RAAS). We aimed to investigate prospective changes in depression symptoms, RAAS (renin, aldosterone), blood pressure, and estimated glomerular filtration rate (eGFR) in a bi-ethnic sex cohort.
Methods: Socio-economically similar participants (43.7 ± 9 years) participated in both phases of this study. Exclusion criteria comprised hypertension medication users, clinically diagnosed diabetics, or human immunodeficiency virus infected individuals [N=195: 68 Blacks (33 males, 35 females); 127 Whites (54 males, 73 females)]. Depressive symptoms (PHQ-9), 24h blood pressure measurements and fasting blood samples were obtained.
Results: Blacks had higher prevalence of depressive symptoms and diastolic hypertension (DBP-HTN), higher eGFR but lower aldosterone and renin levels (p ≤ 0.01). Multivariate regression and receiver operating characteristics analyses showed an inverse association between chronic depression and renin levels [Adjusted R2 0.38; β -0.27 (-0.5, -0.1), p = 0.009] with AUC=0.61 (0.47-0.74) and sensitivity/specificity of 63.6/61.0% in Blacks only. Chronic depressive symptoms in this group were also associated with chronic DBP-HTN [AUC = 0.58 (0.45-0.72); sensitivity/specificity 72.4/46.2%]. No associations existed between eGFR, the RAAS and depressive symptoms in the bi-ethnic cohort.
Conclusion: We propose that chronic depressive symptoms in Blacks activated central neural control centres and desensitization of the RAAS. Subsequent lower renin activity may protect against volume-loading hypertension. These findings emphasize the potential impact of depression on low renin-hypertension in Blacks in terms of prevention, diagnosis, and treatment.
Boiling practices in Bapedi traditional medicine: Crude extract antimicrobial potency vs boiling intervals
Marthienus J Potgieter2, Emmanuel Tshikalange3
Department of Physiology and Environmental Health, University of Limpopo1
Department of Biodiversity, UL2, Department of Plant and Soil Sciences, UP3
Introduction: Traditional medicine as the sum total of the knowledge, skills, and practices based on theories, beliefs, and experiences in the context of local culture and environment are used to maintain health through prevention, diagnosis or treatment of illnesses. It demands a far deeper understanding of their practices, than merely which plants to use for what ailments. It is frequently noted that ethnic groups such as the Bapedi, agree on the plant-ailment interface. However, when preparing these concoctions some variations have been observed in the boiling intervals used. This study investigated the merit of different boiling times on the efficacy of crude extracts.
Methods: Three plant species [Aloe marlothii, Catharanthusroseus and Ziziphusmucronata] were identified from ethnobotanical studies. Subterranean parts were collected, properly identified, dried and powdered. Powdered parts were subjected to boiling intervals ranging from just adding it to boiling water [T0] to 20 minutes [T20] of boiling. Concoctions were filtered through No.1 Whatman paper and the filtrate subjected to freeze drying. The freeze-dried yield was used to prepare the stock solutions for the antimicrobial assays. Pathogens subjected to the minimum inhibitory concentration assays included Neisseria gonorrhoea, Proteus vulgaris and Staphylococcus aureus. Ciprofloxacin was used as a positive control.
Results: In general, A. marlothii exhibited improved antimicrobial activity as boiling time progressed. After 20 minutes of boiling the MIC was 0.78mg/ml for all pathogens tested. Ziziphusmucronata showed little change in activity against N. gonorrhoea and P. vulgaris as boiling time increased. However, activity against S. aureus changed considerably, from 6.25 mg/ml to 0.19 mg/ml [T0–T15] and then to 12.5 mg/ml [T20]. Anti-microbial activity of C. roseus was the least affected by boiling time. In all cases the MIC detected at T0 remained unchanged until T20.
Conclusion: Predominantly our findings are in support of either an unchanged or improved anti-microbial state as boiling time is increased. This highlights the notion that extended boiling times seems to be essential for some plant species, but not for others.
A practical response to increasing class sizes: the use of artificial urine as an approach to standardizing a renal physiology practical session for online assessment
Kennedy H. Erlwanger1
Vernice R. Peterson1, Glenda Norman1, Samantha Kerr1
School of Physiology, University of the Witwatersrand1
Introduction: In response to the increasing numbers of BSc students taking Physiology as a second year major, we developed and adopted new strategies. For renal physiology, the students have a practical on kidney function. Previously, the students used their own urine for the laboratory session. Due to the students’ uncontrolled fluid intake and variations in hydration status prior to the practical sessions, the results obtained lacked normal values to compare to pathophysiological states thus making assessment of their laboratory reports difficult. Hence, preparation for the labs and marking the written reports was time consuming.
Methods: Artificial urine was constituted using water, food colourant and various additives to mimic different physiological and pathological states. The students (n=196 male and female ) used commercial urine analysis reagent strips to analyze the artificial urine and recorded the volumes and specific gravity of what represented serial collections of urine following various ‘physiological interventions’.
Additionally, a model demonstrating the filtered load and excreted quantities of various substances filtered by the kidneys was displayed. The students analyzed the data collected during the lab session and then answered questions related to data and physiological scenarios from the laboratory session using the e-learning platform SAKAI.
Results: The preparation and marking time for the laboratory sessions was cut down significantly and produced consistent results between groups. The use of an electronic system gave the students immediate feedback on their performance. In addition the electronic nature of the assignment allows for an item analysis for future refinement.
Conclusion: The use of artificial urine should be considered as a standardized teaching tool for physiology laboratory sessions on renal function.
Carotid Intima-Media Thickness, but Not Plaque Score Associate with Stroke in Black South Africans
Pitchou Zonga Gazwa1
Eitzaz Sadiq2, Gavin R Norton1, Philanathi Mabena1
Angela J Woodiwiss, Girish Modi, Olebogeng HI Majane
Schools of Physiology, Faculty of Health Sciences, University of the Witwatersrand, Johannesburg1
School of Medicine, Faculty of Health Sciences, University of the Witwatersrand, Johannesburg2
Background: Ischemic stroke caused by arterial occlusion accounts for most cases of stroke and the majority of cases of ischemic stroke occur as a consequence of excess atherosclerosis which may be indexed by changes in the carotid artery. However, whether carotid intima-media thickness (IMT) reflects a compensatory (medial hypertrophy) or pathological (atherosclerotic) change in groups of African descent is uncertain.
Methods: Carotid IMT and the presence of atherosclerotic plaque (B mode ultrasound) were determined in 137 patients of African descent with ischemic stroke admitted to a neurology unit in Gauteng and in 195 age, sex- and ethnicity-matched healthy participants selected from a community study (random selection).
Results: With adjustments for conventional risk factors related to stroke (hypertension and high density lipoprotein concentrations), carotid IMT (mm) was increased in patients with stroke (0.74±0.01) as compared to the control group (0.68±0.01, p<0.0001). The multivariate adjusted odds of having a carotid IMT ≥0.9 (mm) was also independently associated with stroke (Odds ratio=2.74, confidence intervals=1.31-5.89, p=0.008). However, only 25.6% of patients with stroke had carotid plaque as compared to 17.4% of controls (p=0.073).
Conclusion: In a group of African ancestry, carotid IMT, but not the presence of carotid plaque is independently associated stroke. Therefore, in groups of African ancestry carotid IMT, although not closely associated with carotid plaque, is likely to reflect a pathological rather than compensatory change in large vessels.
Lower high-sensitivity cardiac Troponin T cut points in Blacks to predict hypertension: the SABPA Study
Malan L,1 Delport R,2 , Cockeran M,3 REIMANN M4
Hypertension in Africa Research Team (HART), North-West University, Potchefstroom1
Department of Chemical Pathology, School of Medicine, Faculty of Health Sciences, University of Pretoria2
Medicine Usage in South Africa (MUSA), North-West University, Potchefstroom3
Autonomic and Neuroendocrinological Laboratory Dresden, University Hospital Carl Gustav Carus, TechnischeUniversität Dresden, Germany4
Introduction: High sensitivity cardiac Troponin T (hs-cTnT) is a validated measure of myocardial damage or cardiac stress which in turn may reflect silent myocardial ischemia (SMI) and hypertension. Our aim was to determine: 1) cardiac stress, indicative of more SMI events and duration; 2) whether hs-cTnT predicts systolic blood pressure (SBP) ambulatory hypertension; and 3) if hs-cTnT cut-points are similar within a bi-ethnic cohort in risk prediction of SBP hypertension.
Methods: A bi-ethnic sex cohort (n=404) with similar socio-economic status (blacks n=198, whites n=206, aged 20-65) participated where 24h ambulatory blood pressure, -ECG and overnight fasting cardiometabolic variables were measured.
Results: Blacks showed susceptibility with elevated glycated haemoglobin (HbA1C), hs-cTnT, more SMI events, -heart rate and –hypertension prevalence. A lower Receiver Operating Characteristic (ROC) hs-cTnT cut-point of 4.16 pg/ml predicting SBP hypertension [AUC 0.67 (95% CI: 0.59; 0.74), sensitivity/specificity:64%/68%] was observed in blacks in comparison to the higher 5.58 pg/ml cut-point in whites [AUC 0.66 (95% CI: 0.57; 0.75), sensitivity/specificity: 61%/71%]. Multivariate linear regression analysis showed positive associations between SMI events and hs-cTnT in black males [Adj. R2 = 0.27; β 0.43 (0.25; 0.61); p <0.001]. In black females, positive associations existed between SMI event maximum duration and hs-cTnT. [Adj. R2 = 0.17, β 0.43 (0.17; 0.69), p <0.01].
Conclusion: We propose hs-cTnT cut-points of 4.16 pg/ml in blacks and 5.58 pg/ml in whites to predict SBP hypertension. The lower cut-points in blacks may be due to an increased cardiovascular susceptibility in black men and metabolic susceptibility in black women.
Key words: high sensitivity Troponin T, Hypertension, Ethnicity, Sex, Silent Ischemia
Disease Severity Impacts the Relationships of Apelin concentrations with Arterial Function in Patients with Rheumatoid Arthritis
Chanel Robinson1, Patrick Dessein1, Aletta Millen1
University of Witwatersrand
Background: Patients with (RA) experience inflammation induced impaired arterial and endothelial function. Apelin can improve arterial function by enhancing the expression of endothelial nitric oxide synthase. We examined the potential effects of apelin on arterial function in RA.
Methods: Associations of apelin concentrations with SphygmoCor determined arterial stiffness (pulse wave velocity), wave reflection (augmentation index, reflected wave pressure and reflection magnitude) and pressure pulsatility (central systolic pressure (CSP), central pulse pressure (CPP), peripheral pulse pressure (PPP), pulse pressure amplification (PPA) and forward wave pressure (Pf)) were identified in comprehensively adjusted multivariate regression models among 170 RA patients. Left ventricular stroke volume and ejection fraction were determined by echocardiography.
Results: Apelin concentrations were not independently associated with arterial function measures (p>0.05). Joint deformity counts impacted the apelin-CSP, apelin-CPP, apelin-PPamp and apelin-Pf relations (interaction p = 0.004, 0.01, 0.01 and s study. A loss of apelin protective effects on arterial function may contribute to the link between heightened cardiovascular risk and RA severity.
Impact of Aortic Rather Than Brachial Pulsatile Haemodynamics on Variations in End-Organ Measures Across the Full Adult Blood Pressure Range
Gavin R Norton1, Imraan Ballim1, Carlos D Libhaber1
Cardiovascular Pathophysiology and Genomics Research Unit, School of Physiology, Faculty of Health Sciences, University of the Witwatersrand1
Background: The extent to which the adverse effects of blood pressure (BP) are mediated by pulsatile haemodynamic changes across the normotensive as compared to the hypertensive adult brachial BP range is unknown.
Methods: In 1307 community participants the contribution of pulsatile haemodynamics (applanation tonometry and SphygmoCor software) to variations in left ventricular mass index (LVMI)(echocardiography) (n=920), carotid intima-media thickness (IMT)(n=712) and estimated glomerular filtration rate (eGFR)(n=1164) was assessed.
Results: In normotensives (50.5%) independent of steady-state pressure (mean arterial pressure), significant relations between aortic backward wave pressure (Pb) and LVMI (partial r=0.16, phy (Odds ratios [95% CI], 1.125 [1.059 to 1.195] vs 1.054 [1.027 to 1.082] and 1.042 [1.020 to 1.066, p- and SBP-LVMI relations.
Conclusions: Beyond brachial BP, pulsatile haemodynamics rather than steady-state pressures account for end-organ effects more consistently across the normotensive than the hypertensive BP range. Hence, targeting aortic pulsatile haemodynamic changes may best limit BP-related cardiovascular risk within the normotensive BP range.
Myocardial stress and inflammation in Africans – a cycle of events: The SABPA prospective study
Esmé Jansen van Vuren1
Leoné MalaN1,Roland von Känel1,2, Leandi Lammertyn1 , Cockeran M3, Nicolaas T Malan1
Hypertension in Africa Research Team (HART), School for Physiology, Nutrition, and Consumer Science, North-West University, Potchefstroom1
Department of Neurology, Inselspital, Bern University Hospital, and University of Bern Switzerland2
Medicine Usage in South Africa (MUSA), North-West University, Potchefstroom3
A pro-inflammatory profile has been related to the development of cardiac remodelling.
The aim of this study was to determine whether inflammation may lead to increased myocardial stress and predict the development of cardiac remodelling in Africans over a three year period. This prospective study was conducted in 298 Black and White teachers (aged 20-65 years) from South Africa. Fasting blood samples were obtained to measure Troponin T (cTnT), NT-proBNP, C-reactive protein (CRP), Interleukin (IL)-6 and tumor-necrosis factor (TNF)-α. 24-hour ambulatory blood pressure and 2-lead ECG were also determined.
A resting 12-lead ECG determined left ventricular hypertrophy (LVH). Over the three-year period, Blacks showed greater increases in IL-6 (p<0.001) and BP (p<0.001) values than Whites, whereas Whites had a greater increase in NT-proBNP (p=0.01) levels than Blacks. CRP (OR=0.83, 95% CI 0.72 to 0.97, p=0.02) predicted myocyte injury in Black men. Also in Black men, NT-proBNP was associated with CRP (β=0.36; 95% CI 0.14 to 0.58; p≤0.01) and MAP (β=0.33; 95% CI 0.11 to 0.55; p≤0.01). In White men, TNF-α (OR=1.75, 95% CI 1.05 to 2.91, p=0.03) and NT-proBNP (OR=1.06, 95% CI 1.01 to 1.10, p=0.01) predicted myocyte injury.
The findings suggest that myocyte injury may facilitate an immune response leading to an increase in myocardial stress that may further exacerbate cardiac injury. Ultimately the risk for subclinical cardiac remodelling and end organ damage increases in Africans.
Keywords: cardiac remodelling, myocyte injury, inflammation, Africans, myocardial stress
Characterizing the role(s) of the transketolase protein family in metabolic tissues
Gaurang Deshpande1, Veronique Human1, M. Faadiel Essop1
The transketolase (TKT) family of enzymes catalyze key steps of the non-oxidative pentose phosphate pathway, linking glycolysis with biosynthetic reactions and NADPH production. Hyperglycemia blunts TKT activity, while its upregulation reverses damaging intracellular mechanisms. TKT proteins therefore emerge as putative clinical biomarkers and/or therapeutic targets. The transketolase family comprises of TKT, transketolase-like 1 (TKTL1) and transketolase-like 2 (TKTL2) proteins. The contribution of TKTL1 and/or -2 to TKT activity is controversial, e.g. some studies found TKTL1-specific enzymatic activities in addition to the classic TKT reactions. This study therefore aimed to gain greater insights into the TKT protein family, focusing on functional roles. It was hypothesized that TKT family proteins display distinct expression patterns and enzyme activities in metabolic tissues that may be altered by hyperglycemia.
We aimed to: 1) perform bioinformatics modeling to construct TKT protein homology models and predict enzyme activity; 2) evaluate TKT family expression in leukocytes from diabetic persons (vs. pre-diabetic and matched controls), and 3) assess heart, liver and muscle tissue expression of TKT proteins in human, rat and mouse samples.
Our homology models predict that despite amino acid deletions between TKT family members, TKTL1 and -2 contain key conserved residues to allow homo-dimerization and co-factor and substrate binding (as found for TKT).
The expression data show that leukocyte TKTL1 mRNA increased by 72% and 73% in pre-diabetic and diabetic individuals, respectively, vs. non-diabetic controls, while TKT expression was not significantly altered. Human tissue protein analysis showed distinct expression patterns, i.e. a 66 kDa TKT band was expressed in liver, but lacking in heart and muscle. TKTL1 was detected as a 50 kDa monomer and 100 kDa dimer in liver and skeletal muscle, with an additional smaller 30 kDa band – also the main fragment in heart tissue. A 66 kDa TKTL2 band was expressed in human liver, with a smaller 45 kDa band in muscle. Such bands were absent in heart samples. These protein expression data were mostly mirrored in rat and mouse tissues.
Our data reveal that TKT, TKTL1 and TKTL2 exhibit distinct tissue-specific expression and dimerization patterns in metabolic tissues, suggesting unique tissue-specific metabolic functions.
Voluntary ingestion of a high-fat high-carbohydrate diet: a model for prediabetes
Prof Musa Mabandla1, Dr Andile Khathi1
University of KwaZulu-Natal1
Increased dependency on diets that are high in fats and carbohydrates coincided with the increased prevalence of type 2 diabetes. Type 2 diabetes is normally preceded by pre-diabetes, a condition that is not well established in animal models for diabetes. In this study, we evaluated the effects of a high-fat high-carbohydrate diet in the development of prediabetes.
Animals were randomly assigned to the following diets; standard rat chow (ND+H2O), modern high-fat high-carbohydrate (HFHC+Fructose), high-fructose (ND+Fructose) and a high-fat high-carbohydrate (HFHC+H2O). Animals had access to food and fluids ad libitum during experimentation. We measured blood glucose, total cholesterol and triglyceride concentration, OGT response and the HOMA-IR index. Body weight gain, food intake, fluid intake and caloric intake were also monitored. Plasma insulin, ghrelin, leptin, glycosylated haemoglobin (HbA1c) and gastric ghrelin concentrations were also measured.
When compared to the other groups, the OGT response in correspondence with AUCglucose values in HFHC+Frucose group resulted in increased blood glucose concentration. This was accompanied by an increase in glucose, triglyceride and HbA1c concentrations as well as caloric intake despite lower body weight gain. There was also an increase in plasma insulin and ghrelin concentrations while plasma leptin concentration was decreased. The HOMA-IR value was above 2.9 in the HFHC+Fructose group suggesting insulin resistance.
Voluntary ingestion of a high-fat high-carbohydrate diet with fructose leads to sustained high blood glucose concentration despite increased plasma insulin that is associated with increased HbA1c and ghrelin concentration suggesting reduced insulin sensitivity. This suggests that we were able to create an animal model for pre-diabetes that develops from voluntary ingestion of a high-fat high-carbohydrate diet with fructose thus opening avenues for a better understanding of this condition.
Keywords: high-fat high-carbohydrate diet, OGT, HbA1c, insulin, ghrelin, leptin, HOMA2-IR, pre-diabetes
The effect of the potent inflammogen, lipopolysaccharide (LPS) on blood coagulation
Dr Janette Bester1, Prof Douglas B Kell2, Prof Resia Pretorius1
Department of Physiology, University of Pretoria1; School of Chemistry, The Manchester Institute of Biotechnology, and Centre for Synthetic Biology of Fine and Speciality Chemicals, The University of Manchester2
One of the hallmarks of inflammation is a pathological coagulation system, which translates to hypercoagulation. It is well known that a variety of inflammatory diseases are accompanied by hypercoagulability, and a number of more-or-less longer-term signalling pathways and a plethora of inflammatory molecules have been shown to be involved. One such molecule is lipopolysaccharide (LPS), which is located on the outer membrane of gram negative bacteria. LPS is a potent circulating inflammogen and is present during inflammation.
Here we show that the addition of tiny concentrations (0.2 ng.L-1) of bacterial lipopolysaccharide (LPS) from E. coli (E. coli O111:B4 (Sigma, L2630)) to and platelet-poor plasma (PPP) of healthy donors leads to marked changes in the nature of the fibrin fibres so formed, as observed by ultrastructural and fluorescence microscopy. Our results show that when LPS is added to PPP, it causes matted fibrin formation and this clot structure resemble those seen in a number of inflammatory diseases, consistent with an involvement of LPS in their aetiology.
We also present results that show that PPP with added LPS causes hypercoagulability and changes in viscoelasticity, as seen with thromboelastography. We could also demonstrate that these changes in fibre structure are mirrored when the experiment is done simply with purified fibrinogen and thrombin (± 0.2 ng.L-1 LPS).
We also found, with super-resolution microscopy, that fibrin exposed to LPS was stainable with the amyloid-selective stain Thioflavin T (ThT). Hense we suggest that LPS causes the fibrin(ogen) protein molecules to become amyloid in nature. This is the first report of the process of amyloidogenesis in fibrin(ogen).
This ratio of concentrations of LPS:fibrinogen in vivo represents a molecular amplification by the LPS of more than 108-fold, a number that is probably unparalleled in biology. The observation of a direct effect of such highly substoichiometric amounts of LPS on both fibrinogen and coagulation opens up this process to further mechanistic analysis.
Local neoplastic invasion and metastasis formation are diminished due to aberrant actin stress fiber formation and extracellular milieu modifications in response to novel microtubule disruptors
Laurence Lafanechère2, Renaud Prudent3, Michael Pepper4
Department of Physiology, University of Pretoria1
Institut Albert Bonniot, INSERM U1209/ UMR CNRS 5309, Université Grenoble Alpes, Grenoble, France2
Cellipse, Minatec Bat 52A 411b, 7 Parvis Louis Néel, Grenoble, France3
Department of Immunology, Institute for Cellular and Molecular Medicine, and SAMRC Extramural Unit for Stem Cell Research and Therapy, University of Pretoria4
Introduction: Candidate anti-cancer drugs should not only target neoplastic cell cytotoxicity, but should also impede local invasion, neovascularization and the formation of distant metastasis. Pharmacokinetic and cytotoxicity profiles of 2-methoxyestradiol were improved via in silico-design of sulphamoylated analogues. This T0 study aimed at investigating two analogues, namely 2-ethyl-3-O-sulphamoyl-estra-1,3,5(10)15-tetraene-3-ol-17one (ESE-15-one) and 2-ethyl-3-O-sulphamoyl-estra-1,3,5(10)16-tetraene (ESE-16), for their potential as novel anti-cancer agents.
Methods: The effect of the compounds was assessed on cell motility and local invasion on cervical (HeLa) and breast adenocarcinoma (MDA-MB-231) cells via wound healing assays and MatrigelTM invasion chambers. Human umbilical vein endothelial cells (HUVEC) were used to determine the effect of ESE-15-one and ESE-16 on neovascularization. Signalling pathways were elucidated using reverse phase protein arrays and Western blots. Fluorescence microscopy evaluated the response of the actin- and microtubule cytoskeleton to compound exposure. These in vitro results were translated into in vivo setting using chick chorioallantoic membrane (CAM) assays.
Results: The compounds demonstrate anti-invasion effects with decreased distant metastasis in both the in vitro and in ovo assays. Nanomolar drug doses disrupted microtubule dynamics, together with formation of thick actin stress filaments. The Rho family of small GTPases was implicated, with sustained cofilin phosphorylation causing the adynamic phenomena of the actin filaments. Time-dependent phosphorylation of Erk and JNK linked these changes to death- and autophagic processes. Furthermore, as intended in the initial design of the drug, preferential binding to carbonic anhydrase could explain the augmented expression of tissue inhibitor of metalloproteinase 2 (TIMP2) and the tumour suppressor phosphatase and tensin homolog (PTEN). Angiogenic pathways were also implicated, as demonstrated by the exposure-dependent upregulation of the vascular endothelial growth factor receptor 2 (VEGF-R2), which is also linked to stress fibre formation via the Rho-GTPase pathway.
Conclusion: The novel compounds retarded cell migration at concentrations insufficient to induce cell death. Disrupted microtubule dynamics altered the membrane fluidics causing aberrant actin reorganization and the accumulation of P-ezrin/radixin/moesine which co-localizes with Fas on the membrane to induce apoptosis. Thus, these molecules display anti-invasion and anti-metastatic effects using pathways traditionally associated with the positive regulation of these properties. Future studies will investigate whether these effects translate into murine models.
Lack of Association of Carotid-Femoral Pulse Wave Velocity with Occlusive Arterial Events in a South African population
Tshegofatso H Motau1
Gavin R Norton1, Nomvuyo Manyatsi1, Andrea Kolkenbeck-Ruh1
School of Physiology, Cardiovascular Pathophysiology and Genomics Research Unit (CPGRU), Faculty of Health Sciences, University of the Witwatersrand, Johannesburg1
Introduction: Stiffness of the aorta, as indexed by carotid-femoral pulse wave velocity (PWV), is thought to play a major role in the pathogenesis of large artery disease. However, aortic stiffness increases mainly later in life and hence the role in populations whom experience large artery events at a largely young-to-middle age, is uncertain.Methods: We evaluated the association of PWV with the occlusive arterial events, stroke (ischaemic) (n=55) and critical limb ischaemia (CLI) (n=61) in patients recruited from the Charlotte-Maxeke Johannesburg Academic Hospital. Comparisons were made with 116 age-, sex- and ethnicity-matched healthy participants (controls) recruited from SOWETO. Aortic PWV was determined using carotid and femoral applanation tonometry and SphygmoCor software, and associations between PWV and occlusive arterial events compared with the association of carotid artery atherosclerosis, as determined from intima-media thickness (IMT) measurements (using B-mode ultrasound, SonoSite) with occlusive arterial events.Results: The median age (interquartile range) of patients and controls was 57.4 (49.2-67.9) and 59.0 (49.0-66.5) years, respectively. Whilst as compared to controls (0.719 ± 0.13 mm), unadjusted carotid IMT was increased in patients with occlusive arterial events (0.834 ± 0.18 mm, p=0.0003), a difference which remained after adjustments for conventional risk factors (0.723 ± 0.02 mm versus 0.824 ± 0.01 mm, p=0.0011), no differences in PWV were noted between the control (8.40 ± 0.74 m/sec versus 9.59 ± 0.80 m/sec, p=0.3009) and case groups.Conclusions: In contrast to carotid IMT which associates independently with occlusive arterial events at a relatively young age, aortic PWV does not. Hence, aortic stiffness is unlikely to be a major pathophysiological mechanism responsible for occlusive arterial events in young-to-middle age individuals of African ancestry.
Pharmacokinetics and pharmacodynamics of novel oestradiol analogues
Mercier, A. E1; Cromarty, D2; Rheeder, M3; Naidoo, V4; Arbi, S5; Bester, M. J5; Olorunju, S.A.S6; Joubert, A.M1
Department of Physiology, School of Medicine, Faculty of Health Sciences, University of Pretoria1
Department of Pharmacology, School of Medicine, Faculty of Health Sciences, University of Pretoria2
Biomedical Research Centre, Laboratory Animal Technology, Faculty of Veterinary Sciences, University of Pretoria; Department of Pharmacology, Biomedical Research Centre, Faculty of Veterinary Sciences, University of Pretoria4
Department of Anatomy, School of Medicine, Faculty of Health Sciences, University of Pretoria5
Biostatistics Unit, Medical Research Council, Pretoria6
Introduction: 2-Methoxyestradiol (2ME) is an endogenous 17β-oestradiol metabolite that exerts antiproliferative-, antiangiogenic- and antitumor activity on cancer cells both in vitro and in vivo. However, the use of 2ME as potential anticancer agent is limited due to its poor oral bioavailability and short half-life. Three sulphamoylated 2ME analogues were synthesized following in silico design. The basis of this research was focused on improving treatment systems to enable the anticancer drug to only affect the cancer cell and at low dosages with less frequent treatment intervals.
Methods: Screening limits of each analogue were set to 5 µg/ml using a LC-MS/MS method by analysing serial dilutions of each compound in serum. Therapeutically relevant oral bioavailability was assessed using a murine oral absorption model. In vivo oral absorption studies were conducted where 150 mg/kg of individual compounds were administered orally to CD-1 mice; blood was collected two hours post-dosing and was analysed for the presence of the dosed compound and potential metabolites via LC-MS/MS. Liver samples were collected to evaluate the compounds’ cytotoxic effect via immunohistochemistry determination of HIF-1α, CAIX and PCNA protein binding levels. Hypotheses regarding the mechanism of action of these analogues was assessed ex vivo using whole blood and serum of CD-1 mice models via the Rapid Equilibrium Dialysis (RED).
Results and conclusion: Results indicated successful screening of these analogues via LC-MS/MS in serum (Retention time between 3.76 and 4.85 min). In vivo absorption and retention of these compounds was achieved at 3.9 min. for ESE-15-one, 3.76 min. for ESE-15-ol and 5.24 min. for ESE-16. The RED system showed specific binding of the oestradiol analogues to red blood cells, providing a validation of results discovered in silico and in vitro showing the potential of the novel drug binding to carbonic anhydrase IX delaying early metabolism. There was an increase with the expression of PCNA, HIF-1α and CAIX with ESE-15-one (P<0.001) when compared to the other experimental groups. The results from this study indicate that there is merit to further investigate the mode of action of ESE-15-one, ESE-16 and ESE -15-ol in vivo.
Disruption of both actin- and microtubule dynamics sensitizes triple negative breast cancer cells to radiation
Annie Joubert1, Roy Lakier1, Laurence Lafanechère1
University of Pretoria1
Introduction: Both microtubules and actin filaments, components of the cellular cytoskeleton, are highly dynamic and rigorously controlled. Neoplastic dysregulation of these structures have been implicated in tumour growth, local invasion and distant metastasis formation. In this study a novel 2 methoxyestradiol analogue 2-ethyl-3-O-sulphamoyl-estra-1,3,5(10)16-tetraene (ESE-16), was investigated to ascertain the mechanism whereby it presensitizes breast cancer cells to radiation therapy. This was contrasted to the action of a novel LIM kinase inhibitor, 9-benzoyloxy-5,11-dimethyl-2H,6H-pyrido[4,3-b]carbazol-1-one (Pyr1), which stabilizes microtubules and abrogates actin filaments.
Methods: Triple negative BT-20 breast cancer cells were exposed to low doses ESE-16 or Pyr1 24-hours prior to 8 Gy radiation. Induction of apoptosis and signalling pathways were elucidated with flow cytometric quantification of reactive oxygen species (ROS), mitochondrial transmembrane potential, externalization of phosphatidylserine and cell cycle analysis. Deoxyribonucleic acid (DNA) damage was quantified with micronuclei scoring. Signalling of DNA repair mechanisms were evaluated by determining ataxia-telangiectasia mutated (p-ATM) and Ku70 expression by Western blot, and phosphorylation of histone H2AX (γH2AX) via flow cytometry. Light microscopy was used to investigate the morphological changes induced on the combination-treated cells. Additionally, the bystander effect of these drug treatments were investigated via flow cytometric quantification of hydroethidine and Mitocapture™.
Results: Both compounds displayed a radiosensitizing effect on BT-20 cells, by increasing ROS production, decreasing the mitochondrial transmembrane potential and inducing apoptosis more so than in the individual treatment controls. Light microscopy revealed an increase in apoptotic body formation with a decrease in cell density. Although DNA damage was not significantly increased on exposure to radiation, DNA repair mechanisms were retarded in the cells presensitized with the drugs. Amplification of the apoptotic signalling via cell-to-cell communication was demonstrated on analysis of the bystander effect.
Discussion: Disruption of cytoskeletal dynamics resulting from drug pre-exposure rendered the chromosomes more susceptible to radiation damage and impaired trafficking of DNA repair proteins to the nucleus. Future studies will ascertain the precise role of actin skeleton dynamics modulation in the radiosensitizing effect of Pyr1 as a potential clinical target.
Do Increases in Aortic Forward or Backward Wave Pressures Account for Adrenergic-Induced Increases in Pulse Pressure in Rats?
Angela J Woodiwiss1, Grace Tade1, Gavin R Norton1<
Cardiovascular Pathophysiology and Genomics Research Unit, School of Physiology Faculty of Health Sciences, University of the Witwatersrand, Johannesburg1
Background: Ejection of blood from the left ventricle generates aortic forward wave pressures (Pf), which through Newton’s 3rd Law of Motion reflect from distal arterial sites to generate aortic backward wave pressures (Pb). Pf and Pb constitute the wave components of pulse pressure (PP). Although Pb is of a lower magnitude than Pf, Pb may contribute more than Pf to variations in PP, and hence to cardiovascular load. However, conflicting results have rendered uncertainty in this field.
Methods: We compared the impact of the administration of adrenaline (6.6mg/kg, n=16), an α-and β-adrenergic receptor agonist with marked inotropic and vasoconstrictor properties to that of phenylephrine (54 mg/kg, n=13), an α-adrenergic receptor agonist with mainly vasoconstrictor properties on aortic Pf, Pb and PP in Sprague Dawley rats. Aortic Pf, Pb, PP and the reflected wave magnitude (RM=Pb/Pf), a Pf-independent measure of reflected wave function, were determined from aortic pressure (catheter) and flow (Doppler in the outflow tract) measurements under isoflurane anaesthesia. Aortic Pf and Pb were determined using wave separation analysis.
Results: Both adrenaline and phenylephrine administration resulted in marked increases in all aortic pressures as well as RM (p<0.0001). In addition, adrenaline, but not phenylephrine administration resulted in increases in aortic flow and left ventricular endocardial fractional shortening. After phenylephrine administration, the contribution of aortic Pb to increases in PP (β-coefficient or slope=3.10±0.10) by far exceeded that of the contribution of Pf to increases in PP (slope=1.47±0.18, p<0.0001). Similarly however, after adrenaline administration the contribution of aortic Pb to increases in PP (slope=3.49±0.15) also by far exceeded that of the contribution of Pf to increases in PP (slope=1.27±0.06, p<0.0001). Consequently, in both groups RM was strongly associated with increases in PP (p<0.0001), with no differences noted in the relationships between adrenaline and phenylephrine and with RM showing a markedly stronger contribution to increases in PP than Pf alone.
Conclusion:In contrast to conventional thought, the main effect of adrenergic activation on increases in PP, is through vasoconstrictor-mediated increases aortic backward wave pressures, rather than through inotropic-induced increases in Pf.
Marked Associations Between Concentric Cardiac Remodelling and Circulating Inflammatory Markers Independent of Hemodynamic Loads in a Community Sample
Chanel Robinson1,Glenda Norman1, Carlos D Libhaber1
Cardiovascular Pathophysiology and Genomics Research Unit, School of Physiology Faculty of Health Sciences, University of the Witwatersrand, Johannesburg
Background: An explanation for the ability of concentric left ventricular (LV) remodelling to predict cardiovascular outcomes independent of conventional risk factors and LV mass (LVM), has not been provided. Whether LV remodelling is independently associated with inflammatory changes beyond loading conditions, is unknown.
Methods: In 682 randomly selected participants of a community sample we assessed independent relations between LV relative wall thickness (RWT) (echocardiography) and several circulating pro-inflammatory markers.
Results: Left ventricular RWT was not independently associated with C-reactive protein (CRP) (p=0.64) concentrations. However, independent of confounders and either office, 24-hour or central aortic blood pressure (BP), or of aortic pulse wave velocity, circulating interleukin-6 (IL-6) (partial r=-0.12, pnamic factors that influence LV load, a concentric LV geometry is markedly and independently associated with inflammation, as indexed by circulating TNF-α, IL-6 and galectin-3 concentrations, and this may in-part explain the ability to predict adverse cardiovascular effects.
Evidence for increased dense granule release in platelets from rheumatoid arthritis patients and potential contribution to prothrombosis and synovial pathophysiology
Prashilla Soma1, Etheresia Pretorius1
Department of Physiology, University of Pretoria1
Introduction: Platelets are participants in the immune arsenal contributing to the pathophysiology of rheumatoid arthritis (RA). Mechanisms through which they could be involved in proinflammatory activities were examined via investigation of their internal granulation patterns.
Methods: Ultrastructural analyses of citrated whole blood samples comprising 452 platelets from 29 RA patients and 242 platelets from 14 control subjects was performed using transmission electron microscopy (TEM).
Results: Analyses of cell-specific platelet organelles showed fewer dense (D) granules in RA platelets compared to controls. A Mann-Whitney U test confirmed that there were 50% fewer D granules in RA platelets compared to the control platelet group (p = 0.0042). This finding was corroborated by increased detection of expressed pseudopodia, intercellular aggregates, and platelet masses within RA samples. Significantly large and dense platelet masses were identified in two RA patient samples who had suffered myocardial infarctions. Other smaller platelet aggregates were identified in 4 patients while various platelet-leukocyte interactions were identified in 8 patients’ samples.
Discussion and Conclusion: Dense granule expulsion triggers the formation of pseudopodia, serotonin (5HT) release, and increased platelet intercellular recruitment activities. Our suspicion of D granule release activities has supporting evidence in its contribution to arterial inflammation and thrombosis post-traumatically in atherosclerotic mice (1). D granule release can lead to enhanced vascular permeability and extravasation of inflammatory mediators at synovia (2). Recruitment of leukocytes and endothelial cells are facilitated via P-selectin and CD63 secreted from D granules (3, 4). CD63 also aids fibrinogen binding by platelet integrin αIIbβ3 (5). ATP, ADP and Ca2+ secretions from the D granules may also be employed in priming RA platelets for activating the coagulation cascade (6, 7). An alternate proposition, demonstrated using storage pool disease models, is that during intra-vesicular sorting, excessive utilization of granulophysin (also CD63) in other azurophilic granules reduces its availability for dense granule packaging (8, 9). Depletion of granulophysin by lysosomes due to increased uptake and degradation of platelet aggregates may explain its relatively reduced availability for dense granules in RA platelets (6). Findings suggest D granule release is crucial to the platelet’s role in the regulation of RA inflammatory pathophysiology.
Nesfatin-1 and Visfatin expression is associated with reduced atherosclerotic disease risk in patients with rheumatoid arthritis
Sule Gunther1, Aletta Millen1, Patrick Dessein2
University of Witwatersrand1; VrijeUniversiteit Brussel2
Background: Nesfatin-1 comprises a peptide that is involved in appetite suppression, energy homeostasis and fluid regulation, and was recently documented to participate in a range of cardiometabolic pathways. There is currently a need for the identification of novel biomarkers in the elucidation of CVD risk and its stratification in persons with rheumatoid arthritis (RA). The role of nesfatin-1 in cardiovascular disease risk among RA patients is uncertain.
Objective: We investigated the potential impact of nesfatin-1 on subclinical cardiovascular disease manifestations in patients with RA by determining the associations of nesfatin-1 concentrations with atherosclerosis and circulating levels of matrix metalloproteinase (MMP)-2 that mediates plaque stability and those of MMP-3 and MMP-9 that cause plaque vulnerability.
Methods: Nesfatin-1 concentrations were measured in 213 (104 black; 109 white) RA patients. Relationships of nesfatin-1 concentrations with ultrasound determined carotid intima-media thickness (cIMT) and plaque and MMP levels were identified in confounder adjusted multivariate regression models.
Results: Nesfatin-1 concentrations were inversely associated with c-IMT (β (SE) = -0.022 (0.008), p = 0.00) and directly with MMP-2 levels (β (SE) = 0.117 (0.031), p = 0.00). After adjustment for conventional risk factors and RA characteristics, these associations remained (c-IMT: β (SE) = -0.017 (0.008), p = 0.04; MMP-2: β (SE) = 0.116 (0.033), p = 0.00). Patient characteristics and disease activity did not influence the nesfatin-1-to-cIMT relation or nesfatin1-1-MMP-2 relation.
Ten weeks of training combined with creatine monohydrate supplementation maintains physique and improves running economy compared to placebo in ultraendurance runners
James Robert Clark1
Department of Physiology, Division of Biokinetics and Sport Science, Faculty of Health Sciences, University of Pretoria1
Introduction: Creatine monohydrate (CrM) supplementation has been shown to optimize muscle phosphocreatine (PCr) stores, thereby supporting rapid adenosine triphosphate (ATP) rephosphorylation during periods of high enegy demand. CrM has, therefore, become one of the most widely used permitted performance-enhancing substances in sport. CrM supplementation is considered necessary to optimize maximal anaerobic exercise performance in strength athletes engaged in resistance training. However, its potential benefits to aerobic endurance performance has received much less attention. The aim of this research was to investigate the effects of CrM supplementation in runners adhering to a standardized, individually-tailored endurance training programme.
Methods: Seventeen trained male ultraendurance runners paired on fitness were, in a double-blind fashion, assigned to either a group consuming CrM (n = 9; age: 38 ± 7.8 y; VO2 max: 57.2 ± 5.3 ml·kg-1·min-1) or placebo (n = 8; age: 37 ± 8.2 y; VO2 max: 52.1 ± 5.7 ml·kg-1·min-1). Participants ingested 6 g·d-1 supplement for six days followed by 3 g·d-1 for nine weeks. Groups were compared with respect to change from baseline to 10 wk using ANCOVA with covariate baseline value. Within group effects were assessed using Student’s paired t-test. Effect size was reported as Cohen d. For parameters not normally distributed, ANCOVA for ranks was employed and within group comparisons done using the Wilcoxon-matcehd pairs signed ranks test.
Results: CrM supplementation, relative to placebo, resulted in a retention of 1.0 kg body mass (p = 0.194, Cohen d = 0.7). CrM intake significantly increased (p = 0.042) and, compared to placebo, prevented a loss of mesomorphy (p = 0.037, Cohen d = 0.6). Measurements for running economy (i.e. oxygen uptake and internal energy expenditure) at treadmill speeds of 10 km·h-1 and 12 km·h-1 significantly improved (p ˂ 0.05) with CrM compared to placebo supplementation.
Conclusion: Findings of the study are consistent with literature reporting CrM supplementation to augment physiological adaptations to training over that of placebo ingestion. However, the present study is the first to demonstrate the effect in ultraendurance runners. Thus, CrM supplementation in conjunction with training may maintain physique and improve running economy in endurance athletes.
The effects of obesogenic feeding and a patented green Rooibos extract on in vivo adiposity and ex vivo function of cultured adipose-derived stromal cells
Sybrand Smit2, Mignon van Vuuren2, Barbara Huisamen2
Division of Endocrinology, Department of Medicine, Faculty of Medicine and Health Sciences, Stellenbosch University1
Division of Medical Physiology, Department of Biomedical Sciences, Faculty of Medicine and Health Sciences, Stellenbosch University1
Background: Naïve progenitor cells isolated from adipose tissue (adipose-derived stromal cells: ADSCs) can be differentiated into mature adipocytes in culture and used to study adipocyte biology ex vivo. We investigated the effects of obesogenic feeding on in vivo visceral adiposity and on the ex vivo differentiation of ADSCs from subcutaneous and visceral fat (scADSCs and pvADSCs, respectively), and whether these effects could be counteracted by the patented Afriplexreen Rooibos extract GRT.
Methods: Male Wistar rats were placed on the following diets for 16 weeks: control; high fat diet (HFD); and high fat/high fructose/cholesterol diet (HF-FCD). Within each diet group, a subgroup of animals received Afriplex GRT (60mg/kg body weight) from weeks 10 to 16. The animals (n=8 from each group) were subsequently sacrificed and inguinal subcutaneous (SC) and perirenal visceral (PV) adipose tissue biopsies were harvested. Total body weight (BW) and PV fat pad weight were recorded. ADSCs were isolated from adipose tissue and differentiated into mature adipocytes through treatment with adipocytic induction media for 12 days. Intracellular lipid droplets were stained with Oil Red O (ORO) and quantified with image analysis software.
Results: The in vivo visceral adiposity index (PV weight / total body weight) increased with HFD (P<0.001) and HF-FCD (P<0.001) and was normalised with HFD-GRT. The ex vivo visceral adiposity index (% ORO staining in pvADSCs / % ORO staining in scADSCs from the same animal) was increased with HFD (P<0.05) and HF-FCD (P<0.05) and unexpectedly also in all GRT groups (P<0.01). AM-induced lipid accumulation was suppressed in scADSCs from HFD, HF-FCD and all GRT groups.
Conclusions: Obesogenic feeding resulted in long-term changes in the adipogenic response of ADSCs towards increased visceral adiposity. GRT did not normalise lipid accumulation in scADSCs from HFD or HF-FCD animals, but rather suppressed lipid accumulation in scADSCs independent of diet. Consequently, the dietary use of GRT may be associated with abnormal adipose tissue function.
Eryptosis and spontaneous fibrin formation associated with commonly used synthetic contraceptive hormones
Albe Carina Swanepoel1
Department of Physiology, Faculty of Health Sciences, University of Pretoria1
Introduction: Combined oral contraceptives (COCs), used for both family planning and numerous non-contraceptive benefits, are unquestionably associated with increased occurrence of venous thrombosis (VT). The VT risk associated with these synthetic hormone formulations depend on the estrogen concentration and specific type of progestin. Atypical interactions between erythrocytes and fibrinogen/fibrin form part of the pathophysiology of VT. It is therefore important to investigate the impact of different concentrations of the synthetic estrogenethinylestradiol (EE) as well as specific types of synthetic progestins used in COC formulations on whole blood clot formation.
Methods: EE at two concentrations and different synthetic progestins namely Drospirenone (DRSP), Levonorgestrel (LNG), Norgestrel (NG) and Medroxyprogesterone acetate (MPA) were added to whole blood (WB) from healthy individuals. Viscoelasticity of WB clots were determined using thromboelastography (TEG). Light microscopy (LM) was used to determine axial ratios of erythrocytes while scanning electron microscopy (SEM) was employed to investigate ultrastructural changes to erythrocytes. Analysis of variance (ANOVA) was performed for all viscoelastic and axial ratio data using GraphPad Prism 5 with a p-value of ≤0.05 considered as statistically signiﬁcant.
Results: The viscoelastic profile of WB exposed to synthetic hormones indicated that the highest concentration of EE and all progestins tested increased the reaction rate and kinetics of whole blood clots but did not affect the clot strength or stability.
Erythrocyte axial ratios were increased indicating overall shape changes for the highest concentration of EE and all progestins tested. Closer investigation with SEM confirmed erythrocyte shape changes, indicative of eryptosis. Increased rouleaux formation for specific progestins was also evident. Spontaneous fibrin formation occurred and interaction with erythrocytes was also observed.
Conclusion: The physiological functioning of erythrocytes in circulation is dependent on both their shape and membrane flexibility. Synthetic hormones induced eryptosis and the interaction of these abnormal erythrocytes with spontaneously formed fibrin masses may explain the increased risk of VT associated with these hormones.
Altered Relationship Between Aortic Forward and Backward Wave Pressures Following an Increased Sodium Intake in Dahl Salt-sensitive Rats
Angela J Woodiwiss1, Glenda Norman1, Gavin R Norton1
Cardiovascular Pathophysiology and Genomics Research Unit, School of Physiology Faculty of Health Sciences, University of the Witwatersrand, Johannesburg1
Background: Increases in pulse pressure (PP) are a major cause of cardiovascular damage. There is considerable evidence that these effects are mediated by an enhanced aortic backward (Pb)(reflected) wave pressure, but the factors that determine Pb are uncertain. Although cross sectional community-based studies suggest a relationship between a higher sodium (Na+) intake and increased aortic reflected wave pressures independent of effects on steady-state pressures (mean arterial pressure-MAP), there is no direct evidence to support a cause-effect relationship.
Methods: We assessed the impact of 1 month of 1% NaCl in the drinking water on aortic pressures determined over an incremental range of mean arterial pressures (MAP) produced by administration of the vasoconstrictor, phenylephrine, in Dahl salt-sensitive (DSS) rats. Aortic forward wave pressures (Pf), Pb, PP and the reflected wave magnitude (RM=Pb/Pf), a Pf-independent measure of reflected wave function, were determined from aortic pressure (catheter) and flow (Doppler in the outflow tract) measurements under isoflurane anaesthesia. Aortic Pf and Pb were determined using wave separation analysis.
Results: NaCl added to the drinking water resulted in an increase in tail cuff blood pressures inDSS rats. Phenylephrine administration (1 mg/kg) caused striking increases in Pf, Pb, PP andRM over a range of diastolic blood pressures (DBP) and total peripheral resistance (TPR)values (p<0.0001). As compared to DSS not receiving NaCl in the drinking water (control, n=7),DSS receiving NaCl in the drinking water (n=7) demonstrated increased RM values over anMAP (index of arteriolar function) range of 110 to 150 mm Hg (p<0.05). This effect was largelydriven by a decreased Pf for a given MAP in DSS receiving NaCl in the drinking water, whilst Pbfor a given MAP remained unchanged. All data were reproduced with TPR rather than MAPemployed as an index of arteriolar function.
Conclusion. Independent of effects on arteriolar function, an increased sodium intake in salt-sensitive rats results in increases in aortic wave reflection so that reductions in aortic Pf (bydecreasing cardiac systolic function) do not translate into parallel decreases in Pb (Newton’s 3 rdLaw of Motion) and hence reductions in cardiac afterload.
Association of Blood Pressure Variability Ratio with Glomerular Filtration Rate Independent of Blood Pressure and Pulse Wave Velocity
Angela J Woodiwiss1
Gavin R Norton1, Iddo Z Ben-Dov2, Benjamin Gavish3, Michael Bursztyn2
Cardiovascular Pathophysiology and Genomics Research Unit, School of Physiology University of the Witwatersrand, Johannesburg1
Nephrology and Hypertension Services (IZB-D) Hadassah-Hebrew University Medical Center, Jerusalem, Israel2
Yazmonit Ltd, Eshtaol, Israel3
Background: Blood pressure variability ratio (BPVR)(derived from within subject standard deviation of 24-hour ambulatory blood pressure [BP]) predicts all-cause mortality independent of BP and has a similar prognostic ability to ambulatory arterial stiffness (AASI). Whether BPVR, which like AASI is an index of arterial stiffening (dynamic relationship between changes in stiffness and pressure), offers prognostic information beyond measurements of arterial stiffness at a given pressure, as indexed by pulse wave velocity (PWV), is not known.
Methods: We assessed whether BPVR and AASI were associated with indices of subclinical organ damage (TOD) [estimated glomerular filtration rate (eGFR), left ventricular mass index (LVMI), early-to-late transmitral velocity (E/A), carotid intima-media thickness (IMT)] independent of BP, and whether BPVR-TOD and AASI-TOD relations were independent of PWV (applanation tonometry) in 772 randomly selected participants from an urban, developing community. AASI was derived from 24-hour diastolic BP versus systolic BP standard linear regression.
Results: On bivariate analyses, BPVR, AASI and PWV were correlated with all indices of TOD (p<0.0005). However, after adjustments for potential confounders including age and 24-hour mean BP, BPVR and PWV (p<0.005 to p<0.0001), but not AASI (p>0.25), were independently associated with eGFR, but not other indices of TOD. Importantly, the BPVR-eGFR relation was independent of BP variability (p<0.005) and PWV (p<0.001).
Conclusions: BPVR was negatively associated with eGFR independent of mean BP, BP variability and PWV. Therefore, in the prediction of cardiovascular risk, measurements of arterial stiffening (BPVR and sym-AASI) may provide information beyond the impact of arterial stiffness.