Minimal Effects of VEGF and Anti-VEGF Drugs on the Permeability or Selectivity of RPE Tight Junctions

Abstract

Purpose.: Bevacizumab and ranibizumab are currently used to treat age-related macular degeneration by neutralizing vascular endothelial growth factor (VEGF). In this study, the potential side effects on the outer blood–retinal barrier were examined. Methods.: Human fetal RPE (hfRPE) cells were used because they are highly differentiated in culture. The claudin composition of RPE tight junctions was determined by RT-PCR, immunoblot analysis, and immunofluorescence. ELISA assays monitored the secretion and trafficking of VEGF and a fluid-phase marker, methylpolyethylene glycol (mPEG). Tight junction functions were assessed by the conductance of K⁺ and Na⁺ (derived from the transepithelial electrical resistance, TER) and the flux of NaCl and mPEG. Results.: Claudin-3, claudin-10, and claudin-19 were detected in RPE tight junctions. VEGF was secreted in equal amounts across the apical and basolateral membranes, but the apical membrane was more active in endocytosing and degrading VEGF. Exogenous VEGF and mPEG crossed the RPE monolayer by transcytosis, predominantly in the apical-to-basal direction. RPE tight junctions were selective for K⁺, but did not discriminate between Na⁺ and Cl⁻. VEGF, bevacizumab, and ranibizumab had minimal effects on TER, permeation of mPEG, and selectivity for K⁺, Na⁺, and Cl⁻. They had minimal effects on the expression and distribution of the claudins. Conclusions.: RPE has mechanisms for maintaining low concentrations of VEGF in the subretinal space that include endocytosis and degradation and fluid-phase transcytosis in the apical-to-basal direction. RPE tight junctions are selective for K⁺ over Na⁺ and Cl⁻. Permeability and selectivity of the junctions are not affected by VEGF, bevacizumab, or ranibizumab.