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S. M. Farrell, C. O'Driscoll, C. J. O'Brien, T. G. Cotter; Fibroblast Growth Factor Promotes Retinal Cell Survival via Reactive Oxygen Species. Invest. Ophthalmol. Vis. Sci. 2009;50(13):3626.
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Fibroblastic growth factor (FGF) has been shown to increase survival of photoreceptors in various animal models. Previously we have demonstrated the activation of AKT, ERK and PKA survival pathways following in vitro bFGF administration. In addition we have demonstrated the protective efficacy of bFGF in the light induced mouse retina. However, the exact mechanism by which bFGF protects photoreceptors needs to be further elucidated. Reactive Oxygen Species (ROS) such as hydrogen peroxide have traditionally been thought of as toxic by-products of cell metabolism however more recently they have been recognised to be important cell messengers and associated with cell protection. ROS are capable of deactivating phosphatases, allowing various enzymes to remain in the active phosphorylated form. We hypothesised that bFGF promotes activity of survival pathways via production of reactive oxygen species in the retina.
The 661W mouse photoreceptor derived cell lines and rat retinal ganglion cell derived RGC-5 cell lines were used.For flow cytometric analysis, cells were preincubated with dihydrorhodamine probe 30 mins prior to FGF administration. This probe is oxidised by Reactive oxygen species to rhodamine which is fluorescent. bFGF was administered to achieve concentration of 50ng/ml. Cells were analysed on a BD Facscalibur flow cytometer at FL-1 530nm. 10,000 events were recorded per sample.To assess the effect of antioxidants on reducing the accumulation of activated AKT in response to bFGF, 661W cells were treated with a reactive oxygen species scavanger 15 mins prior to bFGF administration. Total protein was obtained by cell lysis 60mins following FGF administration.
Flow cytometry demonstrated a significant increase in intracellular reactive oxygen species levels within 15 mins of bFGF administration in 661W and RGC-5 cell lines.Levels of pAKT were shown to be elevated following administration of bFGF. Cells treated with CR-6 prior to bFGF administration showed decreased levels of pAKT compared to cells treated with bFGF alone.
Our data suggests that bFGF causes increased activity of the pro-survival AKT pathway by increasing intracellular levels of reactive oxygen species.
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