Antiproliferative and cytotoxic properties of bevacizumab on different ocular cells

Abstract

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 59-bromo-29-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.

Intravitreal bevacizumab in pigmented rabbit eyes: histological analysis 90 days after injection

“…In vitro studies have shown that doses higher than 1.25 mg bevacizumab can be deleterious to RPE cells (13)(14) . Bevacizumab quickly reaches the subretinal space after an intravitreal application, therefore, the photoreceptor layer and the RPE could be at risk (12,21) .…”

“…It has been shown to be toxic, in vitro, to retinal pigment epithelium (RPE) cells at doses higher than 1.25 mg (13)(14) .…”

Intravitreous anti-VEGF for diabetic retinopathy: hopes and fears for a new therapeutic strategy

“…Other authors have not found the intravitreous injection of bevacizumab (which blocks all circulating VEGF isoforms) to have any direct toxic effects on retinal ganglion cells [54,55]. Moreover, bevacizumab seems to have neutral effects on several types of retinal cell (including ganglion cells) in culture [56][57][58]. However, it should be noted that although no evidence of retinal toxicity can be observed by light microscopy, mitochondrial disruption in the inner segments of photoreceptors (detected by electron microscopy) and a high rate of apoptosis have been reported in rabbit eyes following the intravitreal administration of bevacizumab [59].…”

Impaired Long-Term Expansion and Self-Renewal Potential of Pediatric Acute Myeloid Leukemia–Initiating Cells By PTK787/ZK 222584

“…The used concentration of PTK/ZK in this study is based on our previous results. The used concentration of Bevacizumab in this study is based on literature (14,26). Dimethyl sulfoxide (DMSO) was used as a control.…”