Antiproliferative and cytotoxic properties of bevacizumab on different ocular cells

Aim: To evaluate the antiproliferative and cytotoxic properties of bevacizumab, a monoclonal antibody against vascular endothelial growth factor ( VEGF), on human retinal pigment epithelium ( ARPE19) cells, rat retinal ganglion cells ( RGC5), and pig choroidal endothelial cells ( CEC). Methods: Monolayer cultures of ARPE19, RGC5, and CEC were used. Bevacizumab ( 0.008 - 2.5 mg/ml), diluted in culture medium, was added to cells that were growing on cell culture dishes. Cellular proliferative activity was monitored by 5'-bromo-2'-deoxyuridine ( BrdU) incorporation into cellular DNA and the morphology assessed microscopically. For cytotoxicity assays ARPE19, RGC5, and CEC cells were grown to confluence and then cultured in a serum depleted medium to ensure a static milieu. The MTT test was performed after 1 day. The Live/Dead viability/cytotoxicity assay was performed and analysed by fluorescence microscopy after 6, 12, 18, 24, 30, 36, and 48 hours of incubation. Expression of VEGF, VEGF receptors ( VEGFR1 and VEGFR2) and von Willebrand factor was analysed by immunohistochemistry. Results: No cytotoxicity of bevacizumab on RGC5, CEC, and ARPE19 cells could be observed after 1 day. However, after 2 days at a bevacizumab concentration of 2.5 mg/ml a moderate decrease in ARPE19 cell numbers and cell viability was observed. Bevacizumab caused a dose dependent suppression of DNA synthesis in CEC as a result of a moderate antiproliferative activity ( maximum reduction 36.8%). No relevant antiproliferative effect of bevacizumab on RGC5 and ARPE19 cells could be observed when used at a concentration of 0.8 mg/ml or lower. CEC and ARPE 19 cells stained positively for VEGF, VEGFR1, and VEGFR2. More than 95% of the CEC were positive for von Willebrand factor. Conclusions: These experimental findings support the safety of intravitreal bevacizumab when used at the currently applied concentration of about 0.25 mg/ml. Bevacizumab exerts a moderate growth inhibition on CEC when used in concentrations of at least 0.025 mg/ml. However, at higher doses ( 2.5 mg/ml) bevacizumab may be harmful to the retinal pigment epithelium.