Differences in uptake and intracellular fate between Bevacizumab and Aflibercept after repetitive long-term treatment in the RPE

In this study, we have investigated the effects of anti-VEGF treatment (bevacizumab, aflibercept) after long-term, repeated treatment on uptake, storage and subcellular localization. Methods: Experiments were conducted in primary porcine RPE cells in first passage and in ARPE-19 cell line. Cells were treated with 250 µg/ml bevacizumab, aflibercept, or, as a non-VEGF inhibiting antibody, rituximab once a week for 1 day, 7 days, 4 weeks and 12 weeks. Cell survival was evaluated with MTT assay. Uptake and localization of compounds was investigated with immunofluorescence microscopy. Selective intracellular protein s were stained with specific respective primary antibodies; actin cytoskeleton was stained with phalloidin. For quantitative analysis, intracellular signals were normalized to light intensity and exposure time. Intracellular association with lysosomes (Lamp2) and exosomes (CD63) was also quantified. In addition, subcellular fractions (nucleus, plasma, membrane, cytoskeleton) were generated and analyzed in Western blot. Results: Weekly treatment up to 12 weeks displayed no toxic effects on RPE cells in any substance tested. Intracellular signal of bevacizumab and aflibercept was strongest afte r 1 day, decreased after 1 and 4 weeks, but increased again after 12 weeks. The signal of intracellular bevacizumab was significantly stronger than of aflibercept. In addition, in primary RPE, aflibercept was significantly more associated with Lamp2, indicating degradation of a...
Source: Ophthalmic Research - Category: Opthalmology Source Type: research