Abstract
Purpose: :
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.
Methods: :
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.
Results: :
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).
Conclusions: :
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.
Keywords: drug toxicity/drug effects • ganglion cells • oxidation/oxidative or free radical damage