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

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.

Effects of a single intravitreal injection of aflibercept and ranibizumab on glomeruli of monkeys.

PURPOSE: It is known that endothelial cells in the kidney are also strongly VEGF-dependent. Whether intravitreal drugs can be detected within the glomeruli or affect VEGF in glomerular podocytes is not known. Therefore, the aim of this pilot study was to investigate the effects of a single intravitreal injection of aflibercept and ranibizumab on glomeruli of monkeys. METHODS: The kidneys of eight cynomolgus monkeys, which were intravitreally injected either with 2 mg of aflibercept or with 0.5 mg of ranibizumab, were investigated one and seven days after injection. Two animals served as controls. The distribution of aflibercept, ranibizumab and VEGF was evaluated using anti-Fc- or anti-F(ab)-fragment and anti-VEGF antibodies respectively. The ratio of stained area/nuclei was calculated using a semi-quantitative computer assisted method. Glomerular endothelial cell fenestration was quantified in electron microscopy using a systematic uniform random sampling protocol and estimating the ratio of fenestrae per µm. RESULTS: Compared to the controls, the anti-VEGF stained area/nuclei ratio of the ranibizumab-treated animals showed no significant changes whereas the stained areas of the aflibercept-treated monkeys showed a significant decrease post-treatment. Immune reactivity (IR) against aflibercept or ranibizumab was detected in aflibercept- or ranibizumab treated animals respectively. The number of fenestrations of the glomerular endothelial cells has shown no significant differences except one day after aflibercept injection in which the number was increased. CONCLUSION: Surprisingly, both drugs could be detected within the capillaries of the glomeruli. After a single intravitreal injection of aflibercept, VEGF IR in the podocytes was significantly reduced compared to controls. Ranibizumab injection had no significant effect on the glomeruli's VEGF level. Whether this is caused by aflibercept's higher affinity to VEGF or because it is used in a higher stoichiometric concentration compared to ranibizumab remains to be investigated.