P31: Tight junction integrity and blood retinal barrier function: effects of endostatin and VEGF

The disruption of tight junction protein expression and the subsequent breakdown of the blood retinal barrier (BRB) occurs in a number of retinal diseases including diabetic retinopathy, sickle cell anaemia and cystoid macula edema. The endothelial cells of the retinal microvasculature are joined by highly specialized tight junctional complexes that confer highly selective properties, and together with other capillary cells maintain the integrity of the inner blood retinal barrier (iBRB). The vasopermeabilizing agent VEGF has been shown to open endothelial tight junctions and alter barrier function, but how this is modulated by other vasoactive agents remains unelucidated. The purpose of this study was to evaluate the effects of endostatin, an endothelial-specific angiogenic inhibitor that inhibits ocular angiogenesis, and VEGF on tight junctions and barrier function in vitro. Results showed endostatin caused a four-fold increase in occludin levels and post-translational modifications. Treatment with VEGF165 (10 ng/mL) for 24 h significantly increased permeability in retinal microvascular endothelial cell (RMEC) monolayers by four-fold, with P = 0.05, as determined by FITC-Dextran-70 and FITC-Dextran-4 flux. For determination of effects of endostatin on VEGF-induced permeability, RMECs were first treated with VEGF165 (10 ng/mL) for 2 h, followed by addition of endostatin (20 ng/mL), with cells incubated for an additional 22 h. When RMECs were exposed to endostatin there was a decrease in VEGF-induced permeability (P = 0.05). Concomitantly, endostatin-treated RMECs demonstrated a marked up-regulation of occludin protein and mRNA. All experiments were performed in triplicate and statistical analyses of results was performed with Student's t-test. In conclusion, using an in vitro model of the BRB, it is evident that this endostatin can promote integrity of the retinal endothelial barrier possibly by inducing expression and phosphorylation of the tight junction protein occludin.