Regulation of retinal pigment epithelium barrier function by Vascular Endothelial Growth Factor (VEGF)

Abstract

Abstract: : Purpose: To study the effect of pathological conditions such as hyperglycemia and hypoxia on the barrier function of the retinal pigment epithelium (RPE) and to analyze the signaling pathways involved in its regulation. For this purpose, experiments were performed on human RPE cell line. Methods: Human RPE cells (ARPE–19) were cultured in vitro under either hypoxic (CoCl2) conditions up to 16 hours or hyperglycemic conditions up to 24 hours. Gene expression of VEGF, VEGF receptors, COX2 and HIF–1 alpha in RPE cells was studied using RT–PCR and immunohistochemistry. RPE cells were grown on filters for 4 weeks and the effect of hyperglycemia, hypoxia and added exogenous VEGF on the function of the tight junctions of RPE cells was evaluated using measurement of transepithelial electrical resistance (TER) and immunohistochemistry for occludin, a marker for tight junction and F–actin. Results: The expression of VEGF and HIF–1 alpha in the RPE cells was increased by both hypoxia and hyperglycemia treatments. The RPE monolayer grown on filters for 4 weeks with a concentration of 10% FBS showed a low TER (50 Ω /cm²) that increased after changing the medium to 2% serum (70–80 Ω /cm²). Adding VEGF in 2% serum induced a biphasic early (10min) and late (6 hours) decrease of (50–60 Ω /cm² and 25–35 Ω /cm² respectively). Accordingly, in the culture conditions, occludin and F–actin immunoreactivity in the cells was reduced 24 hours after administration of VEGF. Conclusions: These data indicate that RPE barrier function may be regulated in pathological conditions through VEGF signaling pathway.

Keywords: retinal pigment epithelium • hypoxia • gene/expression