Differential effects of bevacizumab, ranibizumab and aflibercept on cell viability, phagocytosis and mitochondrial bioenergetics of retinal pigment epithelial cell

Abstract

Purpose: To evaluate the short- and long-term effects of most clinically used anti-vascular endothelial growth factor agents, including bevacizumab, ranibizumab or aflibercept, on cell viability, phagocytosis, mitochondrial bioenergetics and the oxidant acrolein-induced oxidative stress of human adult retinal pigment epithelial (ARPE)-19 cells.

Methods: In cultured ARPE-19 cells, cell viability was measured by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay, phagocytotic activity and intracellular reactive oxygen species (ROS) level were determined by flow cytometry, mitochondrial bioenergetics was assessed using a Seahorse XF24 Extracellular Flux Analyzer, and protein expression was measured by Western blotting.

Results: Long-term exposure to all three agents had no effect on cell viability; but rescued the ARPE-19 cells from acrolein-induced decrease in cell viability. Bevacizumab, but not ranibizumab or aflibercept, suppressed the phagocytotic activity of ARPE-19 cells and exerted significantly less protection against acrolein-induced inhibition of phagocytosis. Both ranibizumab and aflibercept increased basal respiratory rate and maximal mitochondrial respiratory capacity after 1-hr exposure; but returned to baseline following 24- or 72-hr exposure. In contrast, both responses were reduced on short-term exposure, but augmented after long-term exposure to bevacizumab. Long-term pretreatment with all three agents reversed acrolein-induced impairment of mitochondrial bioenergetics, overproduction of ROS and phosphorylation of the mitogen-activated protein kinases in ARPE-19 cells.

Conclusion: Bevacizumab might affect mitochondrial bioenergetics differently from that by ranibizumab and aflibercept. Ranibizumab and aflibercept at their therapeutic dose protect against acrolein-induced oxidative cytotoxicity in human ARPE-19 cells via an increase in mitochondrial bioenergetics. An early protective action on mitochondrial bioenergetic capacity might be used to predict possible long-term antioxidative effects of the agents in the eye.

Keywords: aflibercept; bevacizumab; mitochondrial bioenergetics; oxidative stress; phagocytosis; ranibizumab; retinal pigment epithelial cell.