Effects of hypoxia and high-concentration glucose and high-concentration insulin on VEGF expression in Miller cells

Objective Retinal neovascularization is a main cause of blindness in diabetic retinopathy. Our previous study has proved the overexpression of VEGF in Maller cells in high glucose and insulin environment,but its mechanism is still unclear. This study was to investigate the change of VEGF released by cultured rabbit retinal MUller cells at high concentration glucose and high concentration insulin under hypoxia environment in vitro. Methods Rabbit＇ s retina was cultured by tissue suspension culture method to establish the primary culture system to obtain and identify the cultured Mailer cells by GFAP stain. Three gas incubator and low-flow sustained ventilation was used to control hypoxia environment. The cultured Mailer cells were digested by 0.25% trypsin and suspensed in H-DMEM containing 10% fetal bovine serum and then were treated under the hypoxia and further under the 50 mmol/L of glucose,4 μmol/（ min·L） insulin or 50 mmol/L glucose ＋ 4 μmol/（ min·L） insulin condition respectively for 1 day,2 days and 3 days. The AO/EB stain was used to detect apoptotic Mailer cells in different time points. Immunocytochemical stain was adopted to observe the effects of hypoxia, high concentration of glucose and high concentration of insulin on expression of VEGF in the Mailer cells. Results 95% positive Maller cells for GFAP were obtained. The apoptotic Mailer cells were 20% and 32% in the 3rd and 4th days under the hypoxia. The expression of VEGF in MUller cells was significantly increased in 50 mmol/L glucose and 4 μmol/（ min·L） insulin,50 mmol/L glucose ＋ 4 μmol/ （ min·L） insulin group under the normoxia condition （ P 〈 0. 05 ,P 〈 0. 01 ）. The statistically significant difference in VEGF expression was found between only hypoxic group and normal in the 2nd and 3rd day（ P 〈 0. 05 ）. Conclusion High concentration of glucose or high concentration of insulin can stimulate VEGF expression more obviously under the hypoxia than normoxia condition. Key words: Muller cells;  VEGF;  hypoxia;  diabetic retinopathy;  high glucose;  high insulin