Short Pulse of Clinical Concentration of Bevacizumab Modulates Human Retinal Pigment Epithelial Functionality

PURPOSE. Cross-talk between Notch signaling and vascular endothelial growth factor (VEGF) is a major driver of angiogenesis. Here we investigated the temporal effect of bevacizumab (BEV) on Notch signaling and the functional features of cultured primary retinal pigment epithelial (PRPE) cells. METHODS. Human (cadaver) PRPE cells were treated with clinical concentrations of BEV (0.25 mg/mL). Notch signaling pathway receptors, ligands, and downstream target genes were analyzed with quantitative real-time polymerase chain reaction (qRT-PCR). Cell proliferation along with phagocytosis and transmembrane potential was analyzed by fluorescent activated cell sorter (FACS) and immunofluorescence. RESULTS. Bevacizumab-treated PRPE cultures revealed a significant temporal downregulation of notch4 (P < 0.05) and Delta-like-4 (P < 0.005) gene (16% reduced) and protein (29.7% reduced) expression only at the 2-hour exposure, though secreted VEGF levels were significantly blocked (P < 0.005) at all the time points (2, 4, 6 hours). Further, a significant downregulation (P < 0.005) in cell cycle (reduced by 34.1%) and a concurrent (P < 0.005) upregulation of F-actin staining (increased by 2.5-fold) could be detected. Bevacizumab-treated PRPE cells revealed an elevated transmembrane potential (by 63%) and significant decrease (P < 0.01) in phagocytosis (by 19.25%) in comparison to untreated controls. CONCLUSIONS. There is temporal interaction between BEV and the Notch signaling pathway, specifically with Notch4 and Delta-like-ligand-4 in PRPE cultures. This transient decrease in Notch signaling can impact the functionality of RPE cells. These findings can help to provide a better understanding of the effect of long-term usage of anti-VEGF agents in the treatment of retinal degenerative and vitreoretinopathy diseases.