Session Title: AMD I
Session Date/Time: Thursday 17/09/2015 | 08:30-10:30
Paper Time: 10:06
First Author: : M.Nicolo ITALY
Co Author(s): : A. Puddu R. Sanguineti G. Viviani C. Traverso
PURPOSE:Members of the vascular endothelial growth factor (VEGF) family, such as VEGF-A and Placental Growth Factor (PlGF), play a critical role in angiogenesis. Synergism between VEGF-A and PlGF contributes to both physiological angiogenic processes and pathological plasma extravasation. Ranibizumab (RBZ), an anti-VEGF-A molecule, and Aflibercept (AFL), a recombinant decoy receptor that binds both VEGF-A and PlGF, are widely employed to treat age-related macular degeneration (AMD). The aim of this study is to investigate whether treatments with RBZ or AFL activate different responses in an in vitro models of RPE and endothelial cells in co-culture.
Department of Internal Medicine and Department of Neuroscience, Ophthalmology, Genetics and Maternal Science - University of Genova, Italy
Human RPE (ARPE-19) and endothelial (HECV) cells were co-cultured in DMEM low glucose and 10% FBS. Cells were treated with clinical doses of either RBZ (0.125 mg/ml) or AF (0.5 mg/ml) for 24 (T1) and 72 (T2) hours. At every experimental timepoint we evaluated: cell viability (via fluorimetric assay); mRNA expression and protein secretion of VEGF-A and PlGF (via RealTime-PCR and ELISA).
Cell viability was not affected by exposure to RBZ or AFL. Treatment with RBZ maintained mRNA levels of VEGF-A and PlGF comparable to those observed in control cells. As expected, treatments with RBZ prevented detection of VEGF-A in surpernatants of cells at every time points. Although culture with RBZ did not affect secretion of PlGF at T1, RBZ significantly decreased it at T2. Treatment with AFL strongly increased VEGF-A gene expression at T1 in both cell lines, and at T2 only in HECV. Moreover culture with AFL enhanced mRNA levels of PlGF in ARPE-19 cells at T1. As expected, treatments with AFL prevented detection of VEGF-A and PlGF in surpernatants of cells at both time points.
The main interesting finding of this study is that prolonged exposure to RBZ decreases the release of PlGF in comparison to control, suggesting that blockage of VEGF-A may contribute to reduce PlGF amount. The decreased amount of PlGF from cells treated for 72h with RBZ is not related to lowered mRNA levels, suggesting that other mechanisms are involved in this process. Furthermore these results suggest that mRNA expression of VEGFs was differently regulated by clinical dose of RBZ or AFL, as revealed by RT-PCR: while the blockage of VEGF-A did not induce a compensatory response in these cells, the concomitant blockage of PlGF caused by AFL up-regulated mRNA levels of VEGF-A in both cell types. Since it has been proposed that PlGF contributes to stimulate angiogenesis by displacing VEGF-A from the binding to VEGFR-1, thereby increasing the fraction of VEGF-A available to activate VEGFR-2, our findings related to AFL may be due to the loss of this “braking signal”. In conclusion these results highlight that blockage of VEGF-A due to the chronic exposure to RBZ leads to progressive reduction of PlGF, on the contrary, drastic depletion of PlGF caused by AFL may lead to sustained activation of endothelial cells.