VEGF antagonists decrease barrier function of retinal pigment epithelium in vitro: possible participation of intracellular glutathione

Abstract

Purpose: To investigate the influence of VEGF antagonists on the barrier function of the retinal pigment epithelium and underlying mechanisms.

Methods: Porcine RPE cells were cultured on six-well membrane inserts. The cells were exposed to bevacizumab (62.5 microg/mL) or ranibizumab (25 microg/mL) for 24 hours (short term) or 9 days (long term). Transepithelial flux of FITC-dextran and intracellular levels of reduced glutathione (GSH) at normal and low-glucose conditions were investigated at different points in time. The influence of the addition of triamcinolone acetonide (TA) was investigated. The effect of GSH depletion on RPE permeability was examined using L-buthionine sulfoximine (BSO), a gamma-glutamylcysteine synthethase inhibitor.

Results: After short-term exposure, VEGF antagonists increased the transepithelial flux of FITC-dextran significantly on day 2. Bevacizumab, but not ranibizumab, increased permeability up to 9 days. Under long-term exposure, both drugs enhanced permeability for 7 days; bevacizumab had the stronger effect. The addition of TA inhibited this increase. At the ninth day of short- and long-term exposure, bevacizumab-exposed cells, but not ranibizumab-exposed cells, exhibited a significantly lower GSH level. In the low-glucose condition, both drugs accelerated the decrease of intracellular GSH for the first 48 hours. GSH depletion increased the permeability of retinal pigment epithelium. TA had no effect on BSO-induced GSH depletion.

Conclusions: The results suggest that bevacizumab and ranibizumab may decrease RPE barrier function, with bevacizumab exhibiting a prolonged and more profound effect. Combination with TA is thought to be beneficial because of its protective effect on stabilizing RPE junctional integrity.