Tnf-Alpha Mediates Choroidal Neovascularization By Upregulating Vegf Expression In Rpe Through Ros-Dependent Beta-Catenin Activation

Purpose: Inflammation, oxidative stress, and angiogenesis have been proposed to interact in age-related macular degeneration. It has been postulated that external stimuli that cause oxidative stress can increase production of vascular endothelial growth factor (VEGF) in retinal pigment epithelial (RPE) cells. In this study, we tested the hypothesis that the inflammatory cytokine, tumor necrosis factor alpha (TNF-alpha), contributed to choroidal neovascularization (CNV) by upregulating VEGF in RPE through intracellular reactive oxygen species (ROS)-dependent signaling and sought to understand the mechanisms involved.Methods: In a murine laser-induced CNV model, 7 days after laser treatment and intravitreal neutralizing mouse TNF-alpha antibody or isotype immunoglobulin G (IgG) control, the following measurements were made: 1) TNF-alpha protein and VEGF protein in RPE/choroids with western blot, 2) CNV volume in RPE/choroidal flatmounts, and 3) semiquantification of oxidized phospholipids stained with E06 antibody within CNV with immunohistochemistry (IHC). In cultured human RPE cells treated with TNF-alpha or PBS control, 1) ROS generation was measured using the 2',7'-dichlorodihydrofluorescein diacetate (DCFDA) fluorescence assay, and 2) NOX4 protein and VEGF protein or mRNA were measured with western blot or quantitative real-time PCR in cells pretreated with apocynin or nicotinamide adenine dinucleotide phosphate-oxidase (NADPH) inhibitor, VAS 2870, or transfected with p22phox siRNA, and each was compared to its appropriate control. Western blots of phosphorylated p65 (p-p65), total p65 and beta-actin, and quantitative real-time PCR of VEGF mRNA were measured in human RPE cells treated with TNF-alpha and pretreatment with the nuclear factor kappa B inhibitor, Bay 11-7082 or control. Western blots of beta-catenin, VEGF, and p22phox and coimmunoprecipitation of beta-catenin and T-cell transcriptional factor were performed in human RPE cells treated with TNF-alpha following pretreatment with beta-catenin transcriptional inhibitors, XAV939 or JW67, or transfection with p22phox siRNA and compared to appropriate controls.Results: Compared to the non-lasered control, TNF-alpha and VEGF protein were increased in the RPE/choroids in a murine laser-induced CNV model (p<0.05). An intravitreal neutralizing antibody to mouse TNF-alpha reduced CNV volume, and VEGF protein in the RPE/choroids (p<0.01) and oxidized phospholipids within CNV compared to IgG control (p<0.05). In cultured RPE cells and compared to controls, TNF-alpha induced ROS generation and increased activation of NOX4, an isoform of NADPH oxidase; both were prevented by pretreatment with the apocynin or VAS2870 or knockdown of p22phox, a subunit of NADPH oxidase. TNF-alpha treatment increased VEGF expression (p<0.001) and the formation of a transcriptional complex of beta-catenin and T-cell transcriptional factor; both were prevented by pretreatment with apocynin or knockdown of p22phox. Inhibition of beta-catenin by XAV939, but not the nuclear factor kappa B inhibitor, Bay 11-7082, prevented TNF-alpha-induced VEGF upregulation.Conclusions: Our results support the thinking that TNF-alpha contributes to CNV by upregulating VEGF production in RPE cells through ROS-dependent activation of beta-catenin signaling. These results provide mechanisms of crosstalk between inflammatory mediator, TNF-alpha, and ROS in RPE cells.