Effects of bevacizumab on apoptosis, Na-K⁺-adenosine triphosphatase and zonula occludens 1 expression on cultured corneal endothelial cells

Background: This laboratory study was undertaken to investigate the influence of bevacizumab on apoptosis, Na⁺-K⁺-adenosine triphosphatase (Na⁺-K⁺-ATPase) and zonula occludens 1 (ZO-1) expression on cultured human corneal endothelial cells (HCECs). Methods: Annexin V binding combined with propidium iodide (PI) costaining was used to distinguish viable, early and late apoptotic cells. Immunolocalization of ZO-1 and Na⁺-K⁺-ATPase was performed to analyze intercellular cell integrity after exposure to 5.0 mg/ml bevacizumab for 24 h. Results: No significant induction of apoptosis or necrosis was seen in HCECs after exposure to 5.0 mg/ml bevacizumab (p = 0.689, p = 0.516, respectively). The mean number of annexin-V-FITC- and PI-positive cells did not change significantly. Additionally, no significant changes in expression were detectable, neither for ZO-1 nor for Na⁺-K⁺-ATPase in comparison with the control. For ZO-1, 70.0% of the cells stained intensely, 24.7% stained moderately, and 5.3% stained weakly in the control group. After exposure to 5.0 mg bevacizumab, only minor changes were observable: 68.8% stained intensely, 25.4% moderately and 5.8% weakly (p = 0.524). For Na⁺-K⁺-ATPase, 19.3% of the cells stained intensely, 59.4% moderately, and 21.3% weakly in the control group. After exposure to 5.0 mg bevacizumab, again only minor changes were observable in the expression pattern: 18.2% stained intensely, 60.3% moderately and 21.5% weakly. The changes were not significant compared with the control (p = 0.492). Conclusions: Bevacizumab, at concentrations used clinically, did not induce apoptosis or necrosis in HCECs in vitro. Additionally, no alteration of ZO-1 or Na⁺/K⁺-ATPase expression was detected after exposure to 5.0 mg/ml bevacizumab for 24 h.