Response to anti-VEGF-A treatment in co-cultured endothelial and retinal pigment epithelial cells

摘要

Purpose: Anti-VEGF molecules Ranibizumab (RA) and Aflibercept (AF) are widely employed to treat neovascular maculopathies. Blockage of secreted VEGF-A, however, may be associated to an increased expression and secretion of other VEGF family members. The aim of this study is to investigate the impact of RA and AF in an in vitro model of interacting RPE and endothelial cells. Methods: Human RPE (ARPE-19) and endothelial (HECV) cells were co-cultured in DMEM (1 g/L glucose) and 10% FBS. Cells were treated with clinical doses of either RA (0.125 mg/ml) or AF (0.5 mg/ml) for 24 (T1) and 72 (T2) hours, then drugs were removed and cells were co-cultured for further 48 hours (T3). At every experimental timepoint we evaluated: cell viability (via fluorimetric assay); mRNA expression (via RealTime-PCR) and secretion of VEGF-A, -B, -C and PlGf (via ELISA); influence of conditioned media on endothelial cell migration and proliferation (Scratch assay).br / Results: Cell viability was not affected by exposure to RA or AF. At T3 viability of HECV remained unaffected, whilst viability of ARPE-19 cells was significantly increased either with RA or AF compared to control cells. mRNA expression of VEGFs was differently regulated in ARPE-19 and HECV: i.e. VEGF-A (T1) is decreased by RA in RPE, but not in HECV; VEGF-A (T2) is increased by AF only in HECV; HIF-1?± (T3) is decreased by both RA and AF in RPE, but not in HECV. VEGF-A was not detectable in surpernatants of cells cultured with RA or AF, and remained very low even at T3. Levels of VEGF-B were not affected by RA nor AF. Levels of VEGF-C is slightly increased at T1, and returned to controlsa?? level at T2. Levels of PlGF was very low in cells grown with AF at every time points. Scratch assay showed that wound healing was prevented by media collected from AF (T1), RA and AF (T2), and AF (T3).br / Conclusions: These findings suggest that at clinical dose RA and AF do not exert a cytotoxic effect on RPE and endothelial cells. However, significant functional changes have been observed in both cell lines as revealed by RT-PCR and ELISA. Therefore the results suggest that the concentrations of different VEGFs in the media contribute to establishing an anti-angiogenic environment, which can be maintained even after drug removal. The strongest inhibition of endothelial cells migration was reached with media collected from RA (T2).