The Effect of Glucose Concentration on Expression of VEGF and HGF in Cultured Human RPE Cells

ABSTRACT

PURPOSE: To evaluate the expression of vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF) in response to high glucose concentration in human retinal pigement epithelial (RPE) cells. METHODS: After confluent adult human RPE cells (ARPE-19) were cultured in RPMI 1640 media containing three different concentration of glucose (5.5, 11 and 22 mmol/L) for 3, 7 and 14 days, Western blot analysis was performed. Cell pellets were lysed in lysis buffer and cell lysates were centrifused to collect supernatant fractions for quantifying protein concentration. The proteins were subjected to electrophoresis and transferred to nitrocellulose membranes. After the blots were incubated with primary and secondary antibody to VEGF or HGF consecutively, enhanced chemiluminescence and autoradiography were performed. To evaluate the relationship between protein and mRNA expression, RNA from glucose-treated RPE cells was obtained by using RNAzolB. After cDNA was obtained through reverse transcription of RNA, PCR of cDNA was performed by using primers. RESULTS: We found that incubation with different concentrations of glucose increased the protein expression of VEGF and HGF in concentration-dependent manner in RPE. At 14 days, level of VEGF protein expression was higher in 22 mmol/L glucose group than in other two groups (5.5 mmol/L or 11 mmol/L), but no differences of mRNA expression of VEGF were observed in each group. At 3 days, level of HGF protein expression was higher in 22 mmol/L glucose group than in other groups, but mRNA expression of HGF was not detected in all groups. CONCLUSIONS: These results suggest that RPE cells may participate in angiogenesis in progression of diabetic retinopathy through production of VEGF and HGF.