Abstract 1226 Poster Session IV, Tuesday, 5/4 (poster 257)

Retinopathy of Prematurity (ROP) is a neovascularizing disease that causes blindness in premature infants. Vascular endothelial growth factor (VEGF) is an endothelial cell specific mitogen that has been implicated in the increased vasculogenesis that is seen in ROP. VEGF gene expression increases in response to hypoxia. HIF-1 is a heterodimeric hypoxia inducible transcription factor that causes transcriptional activation of several hypoxia inducible genes including VEGF. Our objective was to demonstrate the presence of HIF-1 and elucidate the kinetics of HIF-1 and VEGF induction in retinal cells. Human retinal pigment epithelial cells were incubated in 1% oxygen for 0.5-30 hours. Total RNA, nuclear protein and whole cell extract was analysed by gel shift, northern blot and western analysis for HIF-1 alpha and VEGF. VEGF levels were measured by ELISA in the media. HIF-1 DNA binding by gel shift analysis was present at 30 minutes and continued to increase until 30 hours of hypoxia. HIF-1 alpha mRNA expression was unchanged by hypoxia. HIF-1 alpha protein levels increased from 3.5 fold to 19 fold after 30 hours of hypoxia. VEGF mRNA expression increased from 1.5 fold at 1 hour-2 hours to 2.8 fold after 30 hours of hypoxia and gradually decreased to normoxic levels on reexposure of the cells to room air. VEGF protein levels in the media by ELISA began increasing after 30 minutes of exposure to hypoxia and continued to increase upto 3.6 fold after 30 hours of hypoxia. No decrease in VEGF protein concentration was noted on reexposure of the cells to normoxia.

We conclude that in human retinal pigment epithelial cells HIF-1 DNA binding, VEGF mRNA and protein increases within 30 minutes-2 hours of hypoxia. Reexposure of cells to a normoxic environment leads to a gradual decrease in VEGF mRNA but not to a decrease in VEGF protein levels. Further experiments are being done to determine the mechanism by which hypoxia causes an increase in VEGF mRNA and protein secretion in retinal cells.