Evaluation of cytotoxicity of bevacizumab on VEGF-enriched corneal endothelial cells

To evaluate the cytotoxicity of varying doses of Bevacizumab on corneal endothelial cells in the presence of a range of concentrations of vascular endothelial growth factor (VEGF). Bevacizumab, a drug widely used in the treatment of neovascular glaucoma neutralizes all isoforms of VEGF and ameliorates neovascularization after intracameral administration. However, the safety of intracameral administration of Bevacizumab and dose-dependent toxicity on corneal endothelial cells has not been established. Bovine corneal endothelial (BCE) cells were treated with VEGF (50 ng/ml) and/or Bevacizumab (0.1-2 mg/ml) for 72 h. Cell proliferation was measured with the water soluble tetrazolium salts (WST-1) assay. Morphological changes were recorded by bright-field microscopy of cells. Cytotoxicity in response to Bevacizumab was evaluated by trypan blue exclusion, as well as annexin V/propidium iodide (PI) staining. Bevacizumab was not cytotoxic at the concentrations tested and the percentage of Bevacizumab-treated cells staining positively for both PI and Annexin V was less than 1%. The anti-proliferative effects of Bevacizumab on BCE cells were dose-dependent; a dose of 1.5 mg/ml or 2 mg/ml produced a 33% (p=0.005) or 47% (p=0.001) decrease in cell proliferation compared to controls. Similar results were obtained in cells treated with a combination of Bevacizumab and VEGF. VEGF (50 ng/ml) had no significant effect on cell proliferation compared to controls. Morphology of cells was unchanged after treatment with Bevacizumab and/or VEGF compared to controls. Bevacizumab was safe and not toxic to BCE cells at concentrations commonly used in clinical practice.