Abstract
Purpose: :
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.
Methods: :
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.
Results: :
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.
Conclusions: :
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.
Keywords: retinal pigment epithelium • cell adhesions/cell junctions • oxidation/oxidative or free radical damage