Objective To investigate the expression of heparanase(Hpa) 、vascular endothelial growth factor(VEGF) and RNA polymerase II (Pol II) and to analyse the effect of Hpa on transcription activity of VEGF promoter in glucose-induced human retinal microvascular endothelial cells (HRECs) hypoglucose model.
Methods glucose-induced HRECs hypoglucose model was used in this study. Two experimental groups were investigated: normal control group, hypoglucose group (glucose 30mmol/L, 72h).The expression of heparanase、VEGF and Pol II in HRECs of normal and hypoglucose groups was analyzed with double immunofluorescence. Cells in both groups were used for ChIP with anti-Hpa and anti-Pol II antibodies to identify high-confidence Hpa-binding regions across the entire promoter, and use real-time PCR to demonstrate the interaction between Hpa and the VEGF promoter region.
Results   Double immunofluorescence studies showed that the expression of heparanase and VEGF in cytoplasm and nuclear was intense in hypoglucose HRECs, but faint in normal group; heparanase and Pol II in nucleu was also intense in hypoglucose HRECs, and the distribution of Hpa is consistent with that of Pol II. Real-time PCR-based ChIP results showed the high-confidence Hpa-binding regions was -1165 to -984 (containing  hypoxia response element) inVEGF promoter, and the cells treated with 30 mmol/L glucose showed an increase of Hpa and Pol II in VEGF gene promoter region, compared with the untreated cells (t=-3.244, P=0.032; t=-6.096, P=0.004, respectively).
Conclusions Nuclear heparanase is directly combined to VEGF promoter, and involved in the regulation of VEGF gene transcription in hypoglucose HRECs.