ENDOSTATIN REDUCES VEGF–MEDIATED VASOPERMEABILITY IN RETINAL MICROVASCULAR ENDOTHELIAL CELLS

Abstract

Abstract: : Purpose:The endothelial cells of the retinal microvasculature are joined by highly specialised 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 vasopermeabilising 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. It has been reported that the endogenous anti–angiogenic peptide endostatin may reduce retinal vasopermeability and the current study has examined the effect of endostatin on retinal microvascular endothelial cell (RMEC) tight junctions and barrier function in vitro. Methods:Monolayers of RMECs were grown on Transwell^® Costar^® inserts and exposed to 10ng/ml VEGF₁₆₅ for 24 hours. After this period, barrier function was determined by FD–70 (FITC–Dextran–70) and FD–4 flux and Trans–Endothelial Electrical Resistance (TEER) measurements. In some experiments, RMECs were first treated with VEGF₁₆₅ (10 ng/ml) for two hours, followed by addition of endostatin (20ng/ml), with cells incubated for an additional 22h, and VEGF–induced permeability determined. The protein and mRNA expression of the tight junction component occludin was also determined using Western analysis and RT–PCR. Phosphorylation states of occludin were determined using phospho–specific antibodies by immunoprecipitation, whilst cellular localisation of occludin was determined by Confocal Laser Scanning Microscopy (CLSM). Experiments were performed in triplicate and statistical analyses of results was performed with Student T test. Results:VEGF₁₆₅ significantly increased permeability in RMEC monolayers (Four–fold increase in FD–70/FD–4 flux, with P ≤0.05). When RMECs were exposed to endostatin there was a time–dependent decrease in VEGF–induced permeability (P ≤0.05) and this was independent of exposure route. Concomitantly, endostatin–treated RMECs demonstrated a marked up–regulation of occludin protein and mRNA. Endostatin caused increased phosphorylation of occludin on serine residues, and endostatin treatment did not affect occludin localisation, as determined by CLSM. Conclusions:This is the first report of the effects of physiological concentrations of Endostatin on the permeability of an in vitro model of the blood retinal barrier. Endostatin may serve to counteract VEGF–induced retinal capillary permeability through modulating the expression of occludin at the BRB.

Keywords: retinal degenerations: cell biology • vascular cells • diabetic retinopathy