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Haibo Wang, Erika Wittchen, M. Elizabeth Hartnett; The small GTPase Rap1 regulates intracellular ROS generation in RPE. Invest. Ophthalmol. Vis. Sci. 2012;53(14):4277.
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We have previously shown that NADPH oxidase-mediated generation of reactive oxygen species (ROS) is involved in pathologic steps of neovascular AMD. Activation of NADPH oxidase involves assembly of the cytosolic (p47phox, p67phox, and Rac1) and membrane-associated components (NOX and p22phox). We recently showed that activation of a GTPase, Rap1, reduced CNV size in a laser injury model. We now tested the hypothesis that inhibition of Rap1 increases ROS generation in RPE cells by interfering with NADPH oxidase subunit assembly and activation.
Using CM-H2DCFDA, intracellular ROS generation was measured in RPE cells infected with an adenoviral vector expressing GTPase-activating proteins (Ad-RapGAP) to inhibit Rap1 activity or control vector expressed with GFP (Ad-GFP). Protein interaction of Rap1 and NADPH oxidase subunit, p22Phox, in RPE cells was determined by co-immunoprecipitation of Rap1 and p22phox in RPE cells infected with the Ad-RapGAP or control Ad-GFP. To activate Rap1, RPE were exposed to an Epac-specific cAMP analogue, 8-pCPT-2_OMe-cAMP (8-CPT, Tocris Bioscience). Statistics were performed using ANOVA.
Compared with control Ad-GFP, ROS generation was increased in RPE cells infected with the Ad-RapGAP (P=0.003) and decreased in RPE cells incubated with 8-CPT (P=3.3464E-15). Compared to RPE infected with control Ad-GFP without 8-CPT treatment, infection of RPE with Ad-RapGAP decreased endogenous Rap1 bound to p22phox (P=0.000036), while incubation of RPE with 8-CPT restored the binding affinity of endogenous Rap1 to p22phox.
Activated Rap1 prevented ROS generation in RPE cells. The molecular mechanism may involve inhibition of NADPH oxidase activation by interfering with NADPH oxidase subunit assembly.
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