EXTENDED REPORT

University Eye Clinic, Department I, Eberhard-Karls University, University of Tuebingen, Tuebingen, Germany

Correspondence to:
Correspondence to:
Dr Martin Spitzer
University Eye Clinic, Department I, Eberhard-Karls University, Tuebingen, Schleichstrasse 12, 72076 Tuebingen, Germany; martin.spitzer{at}med.uni-tuebingen.de

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.

Abbreviations: AMD, age related macular degeneration; BrdU, 5'-bromo-2'-deoxyuridine; CEC, choroidal endothelial cells; CNV, choroidal neovascularisation; HUVEC, human umbilical vein endothelial cells; MTT, 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazoliumbromide; RGC, retinal ganglion cells; RPE, retinal pigment epithelium; VEGF, vascular endothelial growth factor

Keywords: bevacizumab; Avastin; cytotoxicity; ocular cells

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