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Sankarathi Balaiya, Lee R. Ferguson, Kakarla V. Chalam; Evaluation of Sirtuin Role in Neuroprotection of Retinal Ganglion Cells in Hypoxia. Invest. Ophthalmol. Vis. Sci. 2012;53(7):4315-4322. doi: 10.1167/iovs.11-9259.
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Hypoxia-induced apoptosis is responsible for reduced retinal ganglion cell (RGC) viability in a variety of chronic ocular disorders. Sirtuin 1 (SIRT1) plays an important role in preserving cell viability during hypoxia. We investigated the role of SIRT1 in sustaining RGC viability in an in vitro model of hypoxia.
Staurosphorine-differentiated RGCs (RGC-5) received varying hypoxic concentrations (100–500 μM) of cobalt chloride (CoCl2) for 24 hours. Hypoxia-induced cell viability was assessed by WST-1 assay. The role of SIRT1 in promoting viability was determined indirectly via sirtinol (SIRT1 inhibitor). Hypoxia-induced apoptosis was evaluated by measuring stress-activated protein kinase/c-jun N-terminal kinase (SAPK/JNK) and caspase 3 activity. Vascular endothelial growth factor (VEGF) was measured to ascertain the influence of SIRT1.
CoCl2 concentrations greater than 100 μM resulted in significantly reduced RGC viability (P = 0.01). CoCl2 treatment increased SIRT1 levels significantly (P < 0.01): 100 (6.5-fold), 200 (6-fold), 300 (3.5-fold), and 400 μM (4.5-fold). Phosphorylated SAPK/JNK increased 36-fold (200 μM CoCl2 concentration), then plateaued at the 300- (25-fold) and 400-μM (27.8-fold) CoCl2 concentrations (P < 0.01). CoCl2 and sirtinol treatment increased Caspase 3 activity (P < 0.05). VEGF release was significantly higher than control at the 100-μM CoCl2 concentrations (P < 0.01). Sirtinol reduced RGC viability, SIRT1 levels, and VEGF release (P < 0.01) while having greater effect on SAPK/JNK phosphorylation.
SIRT1 significantly influences RGC viability. Sirtinol's effect reflects the interaction SIRT1 has with apoptotic signaling proteins. This investigation demonstrated SIRT1 importance in forestalling the effects of hypoxia-induced apoptosis.
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