The Antiproliferative Effect of Bevacizumab on Human Tenon Fibroblasts Is Not Mediated by Vascular Endothelial Growth Factor Inhibition

PURPOSE. Vascular endothelial growth factor-signaling in human tenon fibroblasts (hTFs) has recently become a target for antifibrotic treatment in glaucoma filtration surgery The anti-VEGF antibody bevacizumab (BVC) has been shown to increase filtration bleb size. Given the relatively high concentration of BVC needed to obtain an effect, we investigated whether BVC acts through VEGF inhibition or via non-antigen-dependent ways. METHODS. Human tenon fibroblast primary cultures were obtained from strabismus surgery subjects. Under low (0.2%) and high (10%) serum conditions, cells were incubated with BVC, ranibizumab (RNB), aflibercept (AFB), or rituximab (RTX) at different concentrations. Total number of cells and number of dead or proliferating (5-bromo-2-deoxy-uridinepositive) cells were assessed after 24 hours. Concentrations of VEGF-A in cell culture media was measured with ELISA. Intracellular IgG was detected with immunostaining and Western blot analysis. RESULTS. In quiescent hTF culture (0.2% serum) the addition of 5 mg/mL BVC induced widespread cell death. Under proliferative conditions (10% serum), BVC reduced the number of proliferating cells. No such effect was observed with 2.5 mg/mL BVC or with 10 mg/mL AFB or 2.5 mg/mL RNB, although they were equally effective in binding free VEGF-A in the culture media. Instead, the CD20 antibody RTX, which did not bind VEGF, induced hTF death and inhibited proliferation in a BVC-comparable fashion. Bevacizumab, AFB, and RTX were detected intracellularly in a concentration-dependent manner. CONCLUSIONS. The cell death-inducing and antiproliferative effect of 5 mg/mL BVC appeared not to depend on VEGF inhibition. Our data question a direct role of VEGF for hTF survival and proliferation.