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Haibo Wang, Manabu McCloskey, Erika Wittchen, M Elizabeth Hartnett; Activation of Rap1 prevents tumor necrosis factor alpha-induced ROS generation in RPE. Invest. Ophthalmol. Vis. Sci. 2013;54(15):1803.
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Both inflammation and oxidative stress are associated with the pathogenesis and progression of AMD. Macrophage-derived TNFα has been found in CNV membranes surgically removed from patients with AMD, and in vascular endothelial cells, TNFα induces NADPH oxidase to generate reactive oxygen species (ROS), which are proposed as involved in the development of neovascular AMD. We recently showed that activation of a GTPase, Rap1, increased RPE barrier integrity and reduced CNV size in a laser injury model. We now test the hypothesis that TNFα increases ROS generation in RPE by activating NADPH oxidase, and that activation of Rap1 prevents TNFα-induced ROS generation in RPE.
Using H2DCFDA, TNFα-induced ROS generation was measured in: RPE transfected with p22phox siRNA or control siRNA; in RPE infected with an adenoviral vector expressing GTPase-activating protein (Ad-RapGAP) to inhibit endogenous Rap1 activity or incubated with an Epac-specific cAMP analogue, 8-pCPT-2_OMe-cAMP (8-CPT) to activate endogenous Rap1; or co-infected with Ad-RapGAP and adenoviral vector expressing active Rap1a (Ad-63E) or Rap1b (Ad-G12V). Activation of NADPH oxidase by TNFα was determined by co-immunoprecipitation of membrane bound subunit p22phox and cytosolic subunit p47phox in RPE infected with the Ad-RapGAP or treated with 8CPT. Adenoviral vector expressing GFP (Ad-GFP) was used as control. In a laser-induced CNV model, ROS generation was measured in fresh-frozen sections of RPE-choroid from mice injected with 8-CPT or control PBS using dihydroethidium fluorescence staining. Statistics were performed using ANOVA.
TNFα treatment induced ROS generation in RPE, and this induction was prevented in RPE transfected with p22phox siRNA. Compared to control Ad-GFP, TNFα-induced ROS generation was further enhanced in RPE infected with the Ad-RapGAP, but blocked in RPE either co-infected with Ad-RapGAP and Ad-63E or incubated with 8CPT. Compared to RPE infected with control Ad-GFP, TNFα-induced co-immunoprecipitation of p47phox and p22phox was increased in RPE infected with Ad-RapGAP, and decreased in RPE treated with 8-CPT. Compared to PBS, laser injury-induced ROS generation in the RPE/choroid was decreased by 8CPT treatment.
Activated Rap1 prevented TNFα-induced ROS generation in RPE and laser induced ROS generation. The molecular mechanism may involve inhibition of NADPH oxidase activation.
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