Possible influence of hypoxia on the expression of G-protein signaling 5 regulators in human retinal pigment epithelial cells and its mechanism

Abstract

Objective To observe the possible influence of hypoxia on the expression of regulators of G-protein signaling 5 (RGS5) in human retinal pigment epithelial (RPE) cells. Methods Experimental study. To establish a hypoxia model, human RPE cells were cultured with 200 μmol/L CoCl2. RPE cells were sorted for showing the expressions of RGS5 and vascular endothelial growth factor (VEGF) in the following time frames: 0, 1, 3, 6, 12 and 24 hours. RPE cells were grouped according to treatment protocols: the hypoxia and VEGF inhibitor Bevacizumab groups at 12 and 24 hour time points, treated with concentrations of Bevacizumab at 25, 100 and 250 μ,g/ml. The expression of RGS5 and VEGF and their mRNA was examined by immunofluorescence, Western blot and RT-PCR. All results were statistically evaluated using a student's t test. Results RGS5 protein was mainly found in the cytoplasm of cultured RPE cells in normoxic conditions. The expression of RGS5 protein and mRNA increased in hypoxia conditions. Expression for both of them peaked at 24 hours after hypoxia (0.932±0.104, t=3.106, P=0.011; 0.742±0.083, t=2.852, P=0.017). The highest levels of VEGF protein and mRNA expression occurred at 12 hours (1.022±0.141, t=4.144, P=0.002;0.491±0.063, t=5.707, P＜0.01), and levels were still higher than normal at 24 hours (0.942±0.125,t=3.306, P=0.008; 0.425±0.080, t=3.239, P=0.011). The expression of RGS5 protein and mRNA was inhibited by Bevacizumab in the 100 μg/ml (t=2.953, P=0.014; t=3.009, P=0.013) and 250 μg/ml (t=2.913, P=0.015; t=4.243, P=0.002) groups, following the inhibition of VEGF (t=2.794, P=0.019; t=2.823, P=0.018; t=3.396, P=0.007; t=9.584, P＜0.01). Conclusion The expressions of VEGF increase earlier than RGS5 in hypoxia conditions, and Bevacizumab can effectively inhibit the expression levels of RGS5 and VEGF in human RPE cells. This may suggest RGS5 could be located downstream from the VEGF signaling pathway, and play an important role in regulating RPE-related hypoxic-ischemic ophthalmopathy. Key words: Choroidal neovascularization; Vascular endothelial growth factor A; Regulon, G-protein signaling 5 ; Pigment epithelial,eye