Minimal effects of VEGF and anti-VEGF drugs on the permeability or selectivity of RPE tight junctions.
OBJECTIVE
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.
Peng
Shaomin
S
Department of Surgery, Yale University, New Haven, Connecticut 06520-8062, USA.
Adelman
Ron A
RA
Rizzolo
Lawrence J
LJ
D020533:Angiogenesis Inhibitors / Q000494:pharmacology*; D000911:Antibodies, Monoclonal / Q000494:pharmacology; D061067:Antibodies, Monoclonal, Humanized; D000068258:Bevacizumab; D001813:Blood-Retinal Barrier / Q000187:drug effects; D002199:Capillary Permeability / Q000187:drug effects*; D002478:Cells, Cultured; D057167:Claudins / Q000235:genetics; D017097:Electric Impedance; D004797:Enzyme-Linked Immunosorbent Assay; D019084:Fluorescent Antibody Technique, Indirect; D015870:Gene Expression; D006801:Humans; D015151:Immunoblotting; D011092:Polyethylene Glycols / Q000378:metabolism; D011188:Potassium / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D000069579:Ranibizumab; D055213:Retinal Pigment Epithelium / Q000187:drug effects* / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012964:Sodium / Q000378:metabolism; D019108:Tight Junctions / Q000187:drug effects* / Q000378:metabolism; D042461:Vascular Endothelial Growth Factor A / Q000378:metabolism / Q000494:pharmacology*
D020533:Angiogenesis Inhibitors; D000911:Antibodies, Monoclonal; D061067:Antibodies, Monoclonal, Humanized; D057167:Claudins; D012333:RNA, Messenger; C467484:VEGFA protein, human; D042461:Vascular Endothelial Growth Factor A; D000068258:Bevacizumab; D011092:Polyethylene Glycols; C028210:monomethoxypolyethylene glycol; D012964:Sodium; D011188:Potassium; D000069579:Ranibizumab