Hypoxia initiates sirtuin1-mediated vascular endothelial growth factor activation in choroidal endothelial cells through hypoxia inducible factor–2α

PURPOSE Hypoxia is a critical pathological factor in a variety of retinal diseases, including age-related macular degeneration. It upregulates angiogenic growth factors and promotes neovascularization. Hypoxia changes the cellular redox state and activates class III histone deacetylase sirtuin1 (SIRT1). Activated SIRT1 signals hypoxia inducible factor (HIF)-2α, which transactivates vascular endothelial growth factor (VEGF) and erythropoietin. In this study, we investigated the role of hypoxia induced SIRT1 in choroidal neovascularization in relation to age-related macular degeneration. METHODS Choroidal endothelial cells (RF/6A) were maintained in a semiconfluent state and hypoxia was induced by exposing the cells to cobalt chloride for 24 h. Induction of hypoxia was confirmed by flow cytometric analysis and the levels of SIRT1 were noted in a hypoxic condition as well in the cells after blocking SIRT1 activity using sirtinol. The role of SIRT1 in the activation of HIF-2α and nuclear factor-κB (RelA/p65) during hypoxia in the presence or absence of SIRT1 was assessed using immunoblot analysis. VEGF levels were quantified using enzyme-linked immunosorbent assay. RESULTS Hypoxic induction was confirmed using flow cytometric analysis, which showed cell cycle arrest starting at a 200 µM concentration of cobalt chloride. Hypoxic treatment (200 µM concentration of cobalt chloride) increased SIRT1 levels to 7.8%, which reduced to control level after its activity was inhibited (p<0.05). Activated SIRT1 mediates HIF-2α and nuclear factor-κB (RelA/p65) expression to 4.5 fold and fivefold, respectively, compared to control, and the levels were suppressed following sirtinol treatment (4.1% and 39.3% respectively; p=0.01). Hypoxic treatment increased VEGF levels by 94.9±19.6 pg/ml compared to control levels (25.58±3.58 pg/ml). These levels decreased to 10.29±0.2 pg/ml after blocking SIRT1 activity using sirtinol, compared to control (p<0.01). CONCLUSIONS Our study results demonstrate that hypoxia mimetic cobalt chloride induces SIRT1 and augments HIF-2α, which activates and releases VEGF.