VEGF–Induced Human Retinal Endothelial Cell Permeability and Curcumin

Abstract

Purpose: : This study evaluates the specific molecular action of curcumin on VEGF induced Human Retinal Endothelial Cell (HREC) permeability. Curcumin (diferuloylmethane), a small–molecular weight compound isolated from turmeric (Curcuma longa Linn) has been shown to have anti–inflammatory, antioxidant and anti–proliferative effects.

Methods: : HRECs were isolated and cultured within 20 hours of postmortem from human donors. HRECs were exposed to varying concentrations of VEGF (2.5, 5, 10, 50 and 100 ng/ml) and the permeability changes were measured as Transendothelial Electrical Resistance (TER) by Electrical Cell–substrate Impedance Sensing system (ECIS). The effect of different doses of curcumin (1, 5 and 10µM) on VEGF induced TER changes were then measured. The expression of PKC is an index of the permeability induced by VEGF. Total PKC activity was measured by γ³²P incorporation. Effect of 10µM curcumin on VEGF induced PKC ßII expression was analysed by western blot.

Results: : VEGF in a dose–dependent manner decreased TER that reflects the increase in HREC permeability. 10ng/ml VEGF showed a 27% decrease in the TER when compared to cells with no treatment. On addition of curcumin this decrease in the TER was reverted. Different concentrations of curcumin 1, 5 and 10µM suitably decreased this VEGF induced permeability by 3, 17 and 26.3% respectively. Total PKC activity was increased to 3.4 fold on exposure of HRECs to 10ng/ml VEGF and this effect was significantly attenuated by 10µM curcumin from 21 ± 2.75 pmoles of phosphate incorporation (VEGF control) to 9.1± 0.1 pmoles (p < 0.01). To measure the specific isoform involved, western blot analysis was carried out. VEGF induced the overexpression of PKC ß II in the membrane fraction of HRECs and this was significantly inhibited by 10µM curcumin (26.8% vs Vehicle control). However, no significant effect was seen with PKC α expression in the cytosol and membrane fractions of HRECs.

Conclusions: : The VEGF induced HREC permeability was significantly inhibited by curcumin and the mechanism of its action seems to be through inhibition of VEGF–mediated PKC ß II expression/activity.

Keywords: retinal culture • pump/barrier function • second messengers: pharmacology/physiology