Antipermeability and antiproliferative effects of standard and frozen bevacizumab on choroidal endothelial cells.

Abstract

Bevacizumab is an antiangiogenic compound developed to target tumour vessels. Its off-label use in ophthalmology requires in vitro testing on ocular cells. To quantify the antipermeability and antiproliferative effects of bevacizumab on cultured choroidal endothelial cells (CECs). It was examined whether deep-freezing of bevacizumab attenuates its antiangiogenic activity. Porcine CECs were cultured in permeable insert systems. Permeability of the cell monolayers was quantified by a fluorescent isothiocyanate-dextran assay after treatment with vascular endothelial growth factor (VEGF; 20-100 ng/ml) alone and in combination with bevacizumab (0.1-1 mg/ml). Proliferation of the CECs was tested using a "wound scratch" assay. The experiments were repeated with bevacizumab after freezing at -20 degrees C for 5 days. Bevacizumab significantly reduced VEGF-induced permeability in a dose-dependant manner. A molar ratio of 2.6:1 of bevacizumab to VEGF was required for complete blocking of VEGF-induced rise in permeability. CEC proliferation was significantly blocked by bevacizumab (0.5 mg/ml). Thawed bevacizumab after deep freezing showed a moderate, but not statistically significant loss in activity. Bevacizumab significantly reduces VEGF-induced permeability and proliferation of CECs. Freezing and thawing of bevacizumab will affect its biological activity.