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V. S. Brar, R. K. Sharma, R. Keshavamurthy, K. V. Chalam; Vascular Endothelial Growth Factor Protects Against Oxidative Stress in Differentiated Retinal Ganglion Cells. Invest. Ophthalmol. Vis. Sci. 2009;50(13):379.
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This experiment is aimed at evaluating potential VEGF protection against oxidative stress in a differentiated model of retinal ganglion cells and whether treatment with bevacizumab can abrogate this effect. Vascular endothelial growth factor (VEGF) has a neuroprotective function in the central nervous system neurons in conditions of hypoxia and oxidative stress. Anti-VEGF therapy is an established treatment in retinal neovascular disorders and exudative age related macular degeneration (ARMD). Oxidative stress has been implicated in the pathogenesis of ARMD. Thus, to gain a better understanding whether anti-VEGF treatment might have deleterious consequence on retinal neurons, we investigated the effect of VEGF on oxidative stress induced injury to a model of differentiated retinal ganglion cells, RGC-5.
Rat retinal ganglion cells (RGC-5) were differentiated using the tyrosine kinase inhibitor staurosporine. Evidence of differentiation was obtained through evaluation of changes in morphology and expression of the neuronal marker class III β-tubulin by immunocytochemistry. Differentiated RGC-5 cells were treated with H2O2 for 24 hrs in the presence of increasing doses of VEGF. Cytotoxicity was assessed by quantifying cell numbers using the WST-1 assay. Specificity of oxidative stress was assessed by conducting the experiments in the presence of the glutathione reductase inhibitor BSO. Cytotoxic effects of bevacizumab were assessed by incubating differentiated RGC-5 with increasing concentrations of bevacizumab for 24h and assessing cell numbers using WST-1 and CV assays. Effect of increasing concentrations of bevacizumab on the protective effect of VEGF on H2O2 induced cell death was also assessed.
24h treatment with staurosporine produced cell processes which were increased in number and elongated compared with untreated RGC-5. These processes were shown to be neuronal in nature through the expression of class III β-tubulin by immunocytochemistry. VEGF (200 ng/mL) protected differentiated RGC-5 against oxidative stress produced by H2O2 (800 µM). This effect was eliminated by the addition of BSO and was blocked by co-treatment with bevacizumab (2.0 mg/mL). Bevacizumab was not found to be cytotoxic at all doses tested (0.1, 1.0, & 2.0 mg/mL).
VEGF protects retinal ganglion cells against oxidative stress induced cytotoxicity. This neuroprotection may rely on the glutathione reductase pathway and is blocked by bevacizumab, in a model of differentiated retinal ganglion cells.
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