Effects of Bevacizumab on Apoptosis, Na⁺-K⁺-Adenosine Triphosphatase and Zonula Occludens 1 Expression on Cultured Corneal Endothelial Cells

Abstract

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.

Preclinical aspects of anti-VEGF agents for the treatment of wet AMD: ranibizumab and bevacizumab

“…29 In vitro, bevacizumab was shown to be non-toxic or not to alter cell viability of rat neurosensory retinal (R28), 30 retinal ganglion, 31 and medulla-derived PC12 cells 32 or human RPE, 30,33 microvascular endothelial (HMVECad), 30 corneal keratinocyte, 34 corneal fibroblast, 34 and corneal endothelial cells. 34,35 In addition, bevacizumab was demonstrated to reduce VEGF-induced permeability and proliferation of cultured porcine choroidal endothelial cells, 18 inhibit VEGF-stimulated proliferation of monkey choroidal endothelial cells, 33 and inhibit VEGF-induced migration of HUVEC. 36 In contrast to these studies, bevacizumab was shown to have a stimulatory effect on cell proliferation in the rat retinal ganglion cell line RGC-5, which was suggested to be due to a nonspecific effect via increasing the protein contents of the cell growth environment.…”

Bevacizumab and Rapamycin Can Decrease Corneal Opacity and Apoptotic Keratocyte Number following Photorefractive Keratectomy

“…36 However, Yoeruek et al reported that bevacizumab at concentrations used clinically did not induce apoptosis or necrosis in human corneal endothelial cells in vitro. 37 Additionally, bevacizumab reduced the formation of nitric oxide (NO) in the action mechanism of VEGF, suppressed vascularization, and then reduced vascular permeability. 38,39 Bevacizumab also has an antiproliferative effect on keratocytes as well as fibroblasts, which can improve chances of successful filtration operation.…”