VEGF-R2/Caveolin-1 Pathway of Undifferentiated ARPE-19 Retina Cells: A Potential Target as Anti-VEGF-A Therapy in Wet AMD by Resvega, an Omega-3/Polyphenol Combination

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  Figure 1. RSV/ω3 fatty acids combination disrupts VEGF-A secretion and inhibits phosphorylation of its receptor. (A) Determination of RSG and RSV effects on human retinal ARPE-19 cells. After treatment of ARPE-19 cells with increasing of RSG (0; 0.6; 3; 6; and 12 µM) or RSV (20 µM) concentrations at 37 °C for 24 h, the percentage of cell viability was determined by crystal violet assay. Results are expressed as a percentage of control (mean ± standard deviation of three independent experiments with n = 10). (B) ARPE-19 retina cells were treated for 24 h without serum with RSG (12 µM) or RSV (20 µM). VEGF-A secretion was measured in cell medium by ELISA. The data are mean ± S.D. of four independent experiments with n = 10. p values were determined by one-way ANOVA followed by Tukey’s multiple comparison test. * = p < 0.05; ** = p < 0.01; *** = p < 0.001. (C) ARPE-19 retina cells were treated without (Co) or with H2O2 (200 µM) for 24 h in combination or not with RSG (12 µM) or RSV (20 µM). As in (A) VEGF-A secretion was measured in cell medium by ELISA. The data are mean ± S.D. of four independent experiments with n = 10. p values were determined by one-way ANOVA followed by Tukey’s multiple…

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  Figure 2. Disruption of VEGF-R2/Cav-1 dissociation into lipid rafts by RSV/ω3 fatty acids combination. (A) ARPE-19 retina cells were treated for 24 h by increasing concentrations of RSG (0.6, 3, 6, 12 µM) or with RSV (20 µM) for 24 h. A representative blot of Cav-1 and phospho-Cav-1 (p-Cav-1) is shown from three independent experiments. β-actin was used as a loading control. Densitometry quantification of western blotting is expressed as the mean fold induction ± SEM of three independent experiments. p values were determined by a one-way ANOVA followed by Tukey’s multiple comparison test. ** p < 0.01, *** p < 0.001 and n.s.: not significant. (B) ARPE-19 cells were left untreated (Co) or treated with 0.6 µM RSG for 24 h before fluorescence microscopy analysis of caveolin-1 (Cav-1) and VEGF-R2 coexpression. A representative image of three independent experiments is shown. (C) Lipid composition of ARPE-19 cell lysate fractions after RSG (12 µM) or RSV (20 µM) treatment for 24 h. Cholesterol (black bars) and sphingomyelin (white bars) were expressed in µg/mL of a fraction. Data are expressed as the mean fold induction ± SEM of three independent experiments. (D) The representative blot of the…

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  Figure 3. RSG affects the Raf-MAP kinases pathway in retinal cells. (A) Immunoblot analysis of c-Raf, phospho c-Raf (p-Raf), MEK, phospho MEK (p-MEK), ERK 1/2, phospho-ERK 1/2 (p ERK 1/2) in RSG-treated ARPE-19 cells with increasing concentration (0.6, 3, 6, 12 µM) or with RSV (20 µM) for 24 h. β-actin was used as a loading control. (B) Densitometry quantification of western blotting. Data are expressed as the mean fold induction ± SEM of three independent experiments. P values were determined by a one-way ANOVA followed by Tukey’s multiple comparison test. * p < 0.05, ** p < 0.01 and *** p < 0.001, n.s.: not significant.

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  Figure 4. RSG down-regulates nuclear transcriptional factors c-Jun and c-Fos and their phosphorylated forms. (A) RT-qPCR mRNA expression analysis of c-jun and c-fos. Data represent three independent experiments. Data are expressed as the mean fold induction ± SEM of three independent experiments. p values were determined by a one-way ANOVA followed by Tukey’s multiple comparison test. * p < 0.05, ** p < 0.01, *** p < 0.001 and n.s.: not significant. (B) Immunoblot analysis of c-Jun, phospho Ser63 c-Jun (pSer63 c-Jun), phospho Ser73 c-Jun (pSer73 c-Jun), c-Fos, phospho-c-Fos (p-c-Fos) in RSG-treated ARPE-19 cells with increasing concentration (0.6, 3, 6, 12 µM) or with RSV (20 µM) for 24 h. β-actin was used as the loading control. (C) Densitometry quantification of western blotting. Data are expressed as the mean fold induction ± SEM of three independent experiments. p values were determined by a one-way ANOVA followed by Tukey’s multiple comparison test. * p < 0.05, ** p < 0.01 and *** p < 0.001.

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  Figure 5. RSG inhibits the translocation of phosphorylated c-Jun forms into the nucleus of ARPE-19 cells. (A) Immunofluorescence analysis of expression and localization of c-Fos, c-Jun, and phospho Ser63 c-Jun, phospho Ser 73 c-Jun into untreated (Co) or treated ARPE-19 retina cells with RSG (12 µM) or with RSV (20 µM) for 24 h. Left panel, representative images of specific proteins expression (green fluorescence) counterstaining with Hoechst 33342 (nuclei, blue). Right panel quantification of a specific protein on merged pictures (300 cells per line) with Image J software. p values were determined by a one-way ANOVA followed by Tukey’s multiple comparison test. ** p < 0.01, *** p < 0.001 and n.s.: not significant. (B) Representative blot of c-Jun, phospho Ser63 c-Jun (pSer63 c-Jun), phospho Ser73 c-Jun (pSer73 c-Jun) protein expression in RSG (12 µM)-treated ARPE-19 cells or with RSV (20 µM) for 24 h. Nuclear fractions (N) and cytosolic (C) fractions are shown from three independent experiments. Densitometry quantification of western blotting. Data are expressed as the mean fold induction ± SEM of three independent experiments. p values were determined by a one-way ANOVA followed by…

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  Figure 6. RSG inhibits vegf-a and vegf-r2 mRNA expressions through a disruption of the AP-1 binding sites. ARPE-19 cells were untreated (Co) or treated with RSG (12 µM) or with RSV (20 µM) for 24 h. (A) Upper panel, analysis on the putative binding sites of AP-1 on the vegf-a gene promoter. Down panel, ChIP analysis of the interaction between AP-1 and the vegf promoter in ARPE-19 cells. Analysis of the putative B.S.3 for AP-1 on the vegf promoter (−4976 −4964) is shown. For the putative B.S.1, B.S.2, B.S.4, and B.S.5, any amplification was obtained. (B) RT-qPCR mRNA expression analysis of vegf-a. Data represent three independent experiments. Data are expressed as the mean fold induction ± SEM of three independent experiments. p values were determined by a one-way ANOVA followed by Tukey’s multiple comparison test. * p < 0.05, ** p < 0.01 and *** p < 0.001. (C) Upper panel, analysis of the putative binding sites of AP-1 on the vegf-r2 gene promoter. Down panel, ChIP analysis of the interaction between AP-1 and the vegf-r2 promoter in ARPE-19 cells. Analysis of the putative B.S.5 and B.S.6 for AP-1 on the vegf-r2 promoter (−3942, −3930, and −3864, −3852, respectively) is shown. For the…

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  Figure 7. Suggested model to counteract VEGF-A secretion in retinal cells with AMD phenotype by nutraceuticals. Contrary to the physiopathological mechanism observed in retinal cells mimicking AMD, nutraceutical with resveratrol/ω3 fatty acids combination promotes (1) VEGF-R2 relocalization into lipid rafts and its binding with Cav-1, a constitutive protein of lipid rafts. This VEGF-R2-Cav-1 association, which acts as a negative regulator of the VEGF-A signaling cascade, prevents (2) the activation of MAP kinase pathway and subsequently decreases (3) phosphorylation of transcriptional nuclear factors such as c-Jun. Reduction of phospho-c-Jun level is associated with a (4) disruption of c-Jun into the nucleus. Furthermore, nutraceuticals also seemed to disrupt and antagonize AP-1 binding sites on the promoters of vegf and vegfr genes and contribute to the decrease in their mRNA and protein levels.