title: TNF-α mediates choroidal neovascularization by upregulating VEGF expression in RPE through ROS-dependent β-catenin activation.
abstract: OBJECTIVE
  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-α), 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-α antibody or isotype immunoglobulin G (IgG) control, the following measurements were made: 1) TNF-α 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-α 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 β-actin, and quantitative real-time PCR of VEGF mRNA were measured in human RPE cells treated with TNF-α and pretreatment with the nuclear factor kappa B inhibitor, Bay 11-7082 or control. Western blots of β-catenin, VEGF, and p22phox and coimmunoprecipitation of β-catenin and T-cell transcriptional factor were performed in human RPE cells treated with TNF-α following pretreatment with β-catenin transcriptional inhibitors, XAV939 or JW67, or transfection with p22phox siRNA and compared to appropriate controls.
  
  
  RESULTS
  Compared to the non-lasered control, TNF-α 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-α 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-α 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-α treatment increased VEGF expression (p<0.001) and the formation of a transcriptional complex of β-catenin and T-cell transcriptional factor; both were prevented by pretreatment with apocynin or knockdown of p22phox. Inhibition of β-catenin by XAV939, but not the nuclear factor kappa B inhibitor, Bay 11-7082, prevented TNF-α-induced VEGF upregulation.
  
  
  CONCLUSIONS
  Our results support the thinking that TNF-α contributes to CNV by upregulating VEGF production in RPE cells through ROS-dependent activation of β-catenin signaling. These results provide mechanisms of crosstalk between inflammatory mediator, TNF-α, and ROS in RPE cells.
authors.affiliation: The John Moran Eye Center, University of Utah, Salt Lake City, UT.
authors.forename: Haibo
authors.initials: H
authors.lastname: Wang
authors.affiliation: The John Moran Eye Center, University of Utah, Salt Lake City, UT; Department of Ophthalmology, The Fourth Affiliated Hospital of China Medical University, Shenyang, China.
authors.forename: Xiaokun
authors.initials: X
authors.lastname: Han
authors.affiliation: Cell Biology and Physiology, University of North Carolina at Chapel Hill, Chapel Hill, NC.
authors.forename: Erika S
authors.initials: ES
authors.lastname: Wittchen
authors.affiliation: The John Moran Eye Center, University of Utah, Salt Lake City, UT.
authors.forename: M Elizabeth
authors.initials: ME
authors.lastname: Hartnett
mesh_terms: D000818:Animals; D057134:Antibodies, Neutralizing; D015153:Blotting, Western; D002478:Cells, Cultured; D020256:Choroidal Neovascularization / Q000209:etiology* / Q000378:metabolism / Q000473:pathology; D004195:Disease Models, Animal; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005786:Gene Expression Regulation / Q000502:physiology; D007150:Immunohistochemistry; D058449:Intravitreal Injections; D051379:Mice; D008810:Mice, Inbred C57BL; D019255:NADPH Oxidases / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics; D017382:Reactive Oxygen Species / Q000037:antagonists & inhibitors / Q000378:metabolism*; D060888:Real-Time Polymerase Chain Reaction; D055213:Retinal Pigment Epithelium / Q000378:metabolism*; D014162:Transfection; D014409:Tumor Necrosis Factor-alpha / Q000502:physiology*; D015854:Up-Regulation; D042461:Vascular Endothelial Growth Factor A / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism; D051176:beta Catenin / Q000235:genetics / Q000378:metabolism*
chemical_list: D057134:Antibodies, Neutralizing; D004791:Enzyme Inhibitors; D012333:RNA, Messenger; D017382:Reactive Oxygen Species; D014409:Tumor Necrosis Factor-alpha; D042461:Vascular Endothelial Growth Factor A; D051176:beta Catenin; C467480:vascular endothelial growth factor A, mouse; D019255:NADPH Oxidases