Browsing by Author "Van Vuuren, Derick"
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- ItemCardiomyocyte differentiation : experience and observations from 2 laboratories(South African Heart Association, 2017) Patten, Victoria; Chabaesele, I.; Sishi, Balindiwe; Van Vuuren, DerickThe undifferentiated clonal cell line, H9c2, derived from left ventricular rat heart tissue, has been extensively used in cardiovascular research. In the present study, 2 independent laboratories aimed to investigate the cells’ capacity to differentiate into distinct cardiac-like cells. Undifferentiated H9c2 cells were supplemented daily for a period of 6 - 12 days, with varying concentrations of retinoic acid (RA) (10nM, 30nM and 1μM), in standard cell culture medium containing either 1% foetal bovine, or horse serum, in order to stimulate differentiation of the cells into a more cardiac-specific phenotype. Light microscopy confirmed some degree of morphological change associated with differentiation, and a significant increase in oxidative phosphorylation following RA treatment was observed. However, Western blot probing for the cardiac-specific markers Cardiac Troponin T (cTnT) and Myosin Light Chain-2v (MLC2v) indicated little to no differentiation, although immunocytochemistry indicated the presence of cTnT expression. Thus, it was found that the differentiation protocol induced differentiation in some, but not all cells, thereby generating a heterogeneous cell population. Our findings suggest that the H9c2 cell line may display some degree of resistance to differentiation. This should be kept in mind when considering to use this model for cardiovascular research.
- ItemFenofibrate protects endothelial cells against the harmful effects of TNF-alpha(South African Heart Association, 2017) Westcott, Corli; Genis, Amanda; Mthethwa, Mashudu; Graham, Roxanne; Van Vuuren, Derick; Huisamen, Barbara; Strijdom, HansIntroduction: Fenofibrate exerts pleiotropic effects on endothelial cells (ECs) by, amongst others, increasing nitric oxide (NO) production. We aimed to investigate fenofi brate’s putative beneficial actions in healthy or TNF-alpha-induced dysfunctional ECs. Methods: Fenofi brate-induced pro-vasodilatory responses were assessed in aortic rings (50 - 125μM; 30min) with and without L-NMMA (100μM). Rat cardiac microvascular ECs were treated with fenofibrate (30 and 50μM; 1h). In the pre-treatment experiments, fenofibrate (50μM) was administered one hour before TNFalpha treatment (20ng/ml; 24h). NO-production (DAF-2/DA or Griess assay), mitochondrial ROS-production (MitoSox™), cell viability (propidium iodide staining), and changes in the expression/phosphorylation of critical endothelial proteins were measured by Western blotting. Results: Fenofibrate increased NO-production ˜2-fold in healthy ECs (p<0.05 vs. vehicle). A ˜23% pro-vasodilatory response was induced in aortic rings, which was reversed by L-NMMA (p<0.05 vs. fenofibrate). Fenofibrate pretreatment ameliorated TNF-alpha-induced endothelial dysfunction by reversing the loss of NO, improving oxidative stress, restoring cell viability and preventing caspase-3 activation. Protective effects were underpinned by ˜47% and ˜49% up-regulation of activated eNOS and AMP-kinase, respectively (p<0.05 vs. TNFalpha). Conclusions: Fenofibrate protects TNF-alpha-induced dysfunctional ECs via up-regulated eNOS-NO, reduced oxidative stress and improved cell viability. These novel findings warrant further investigations to explore the potential use of fenofibrate as an anti-endothelial dysfunction therapeutic agent.
- ItemIschaemic postconditioning : from bench to bedside …(Clinics Cardiv Publishing, 2008-12) Van Vuuren, Derick; Lochner, AmandaThe increase in the incidence of ischaemic heart disease and acute myocardial infarction (AMI) in both high- and lowincome countries necessitates the development of myocardial salvaging/protection interventions, to be applied alongside standard reperfusion therapies. Although the phenomenon of ischaemic preconditioning (IPC) is associated with the desired protective capacity, the necessity of its application before sustained ischaemia limits its clinical potential. The recently described phenomenon of postconditioning (postC), or short cycles of reperfusion/ischaemia applied at the onset of reperfusion, falls within the clinically relevant time period of reperfusion, but can it elicit reliable and potent cardioprotection? The answer to this problem is intimately related to the question whether postC can be translated from a laboratory technique to a clinical therapy. In this brief overview of postconditioning, the experimental set-ups and postC algorithms utilised, and their associated outcomes in all animal models studied (dog, rabbit, mouse, rat and pig) are discussed. The therapeutic potential of postC is also addressed by discussing reported preliminary studies on the efficacy and feasibility of postC (both ischaemic and pharmacological) in humans.
- ItemThe role of Protein Phosphatase 2A (PP2A) in myocardial ischaemia/reperfusion injury(Stellenbosch : Stellenbosch University, 2014-04) Van Vuuren, Derick; Lochner, Amanda; Engelbrecht, Anna-Mart; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences, Division of Medical Physiology.ENGLISH ABSTRACT: Ischaemic heart disease is a major contributor to global morbidity and mortality rates. Manoeuvres such as ischaemic preconditioning confer cardioprotection against ischaemia/reperfusion (I/R) injury by activating several intracellular signalling pathways. These pathways have been defined solely in terms of the kinases involved, despite the realization in recent years that protein phosphatase activity also contributes significantly to the attributes of the propagated signal. Protein phosphatase 2A (PP2A) is a heteromultimeric enzyme involved in an array of phosphatase reactions. We hypothesized that PP2A is an important participant in the myocardial response to I/R by regulating intracellular signalling. This project aimed to (i) characterize PP2A during myocardial I/R; (ii) determine the importance of its contribution to the cellular response to I/R; and (iii) investigate its role in the signalling pathways mediated by PKB/Akt, GSK-3β, ERK p42/p44 and p38 MAPK. Two models were used to characterize PP2A during I/R: (i) H9c2 cells exposed to simulated ischaemia (SI) buffer in conjunction with hypoxia (0.5% O2) for a maximum of 2 hours, followed by reoxygenation in standard growth medium for up to 30 minutes; and (ii) isolated working rat hearts exposed to a maximum of 20 minutes global ischaemia and 10 minutes reperfusion. In both models samples were collected at several time points during I/R for Western blotting analysis. PP2A-C (the catalytic subunit) accumulated in the nucleus during early ischaemia, but later redistributed to the cytosol. At the end of ischaemia there was an elevation of PP2A-C relative to PP2A-A in the unfractionated whole cell preparation concomitant with an increase in the inhibitory phosphorylation of PP2A-C. The impact of PP2A activity was evaluated by either inhibiting PP2A using okadaic acid (OA, 10 nM) or activating it by administering FTY720 (1 μM) in an isolated working rat heart model exposed to either 35 minutes of regional ischaemia (RI) with infarct size (IFS) as primary end-point, or 20 minutes global ischaemia (GI) with functional recovery as end-point. The results showed that the pre-ischaemic administration of OA or FTY720 reduced or exacerbated IFS respectively, indicating that PP2A activation during I/R favours cell death. OA and FTY720 were also employed to assess the contribution of PP2A to intracellular signalling in an isolated working rat heart exposed to I/R. Samples were collected at several timepoints and analyzed using Western Blotting. Pre-ischaemic administration of OA enhanced the phosphorylation of PKB/Akt, ERK p42/p44 and GSK-3β at the onset of reperfusion, while FTY720 given before ischaemia reduced the phosphorylation of GSK-3β, p38 MAPK and PKB/Akt at the end of ischaemia and onset of reperfusion. In summary, PP2A is part of an early nuclear-based response to ischaemia, while long-term ischaemia induces an increase in PP2A-C. A portion of this PP2A-C is stored in an inactive form, while an active portion acts as a regulator of the pro-survival signalling components PKB/Akt, GSK- 3β and ERK p42/p44 at the end of ischaemia and the onset of reperfusion. PP2A is therefore an important component of the myocardial response to I/R by regulating pro-survival signalling.