Abstract
Purpose:
SIRT1, a NAD+ dependent histone deacetylase, has been shown to act as a key regulator of angiogenesis. Hypoxia is a critical pathological factor in new vessel growth in wet AMD. The current study was to investigate the role of SIRT1 on a mimic hypoxic condition in human fetal retinal pigment epithelial (fRPE) cells.
Methods:
Human fRPE cells (passage2-4) were incubated in DMED supplemented with 10% fetal bovine serum at 37°C with 5% CO2. Addition of cobalt chloride (CoCl2) to DMEM was used to mimic hypoxic condition. Cell viability was accessed after exposing fRPE cells to different concentrations of CoCl2 (0, 50, 100, 200, 500, 1000uM) for 24 hours using PrestoBlue™ reagent. The fRPE cells were transfected using specific SIRT1 siRNA (10nM) following by CoCl2 (100uM) treatment for 24 hours or pretreating the cells with resveratrol (SIRT1 activator) (50uM) for 16 hours before CoCl2 treatment. At the end point, the HIF-1a protein accumulation in the cell lysate was analyzed by western blotting and ELISA were performed to detect the secreted VEGF protein in the supernatant.
Results:
HIF-1a protein expression was increased in a dose-dependent manner in CoCl2-treated fRPE cells which reached a maximum at 100uM CoCl2. Loss of SIRT1 by specific SIRT1 siRNA transfection even further augmented this increase of 1.5-fold (p<0.05). Treatment of rRPE cells with resveratrol inhibited HIF-1a accumulation in hypoxia significantly (p<0.01) and this inhibition effect attenuated by knocking down of SIRT1. Furthermore, VEGF secreted in the supernatant was up-regulated by 1.5-fold after incubating fRPE cells with CoCl2 and even further increased with additionally adding SIRT1 siRNA. While using resveratrol down-regulated the VEGF secretion by 2-fold in the supernatant in hypoxia. This down-regulation effect was also reversed by SIRT1 siRNA.
Conclusions:
SIRT 1 and its activator are able to inhibit HIF-1a/VEGF pathway under a mimic hypoxia condition. The results suggest that target SIRT1 could be a therapeutic potential for the treatment of wet AMD.
Keywords: 453 choroid: neovascularization •
701 retinal pigment epithelium