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A. S. Lewin, A. Dinculescu, R. Miller, J. Pang, Z. Shan, M. K. Raizada, W. W. Hauswirth, Q. Li; Down-Regulation of p22phox in Retinal Pigment Epithelial Cells Inhibits Choroidal Neovascularization in Mice. Invest. Ophthalmol. Vis. Sci. 2008;49(13):1128.
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Choroidal neovascularization (CNV) arises in variety of chorioretinal diseases, including age-related macular degeneration (AMD). Oxidative stress has been thought to play an important role in the development of CNV. NADPH oxidase has been shown to be one of the major intracellular sources of reactive oxygen species (ROS) in the vascular system. We investigated the expression of p22phox, an integral subunit in the NADPH oxidase complex in the mouse eye, and tested the feasibility of directly targeting this pathway using siRNA against p22phox in an experimental animal model of CNV.
Expression of p22phox in the eye was examined by immunocytochemistry, in situ hybridization, and western blot analysis. A synthetic cDNA coding for siRNA against p22phox gene was cloned into an AAV vector under the control of H1 promoter, along with a GFP reporter gene under the control of the chicken -actin/CMV enhancer promoter (CBA). A scrambled siRNA was cloned into the same vector as the control. The vector was injected either intravitrealy or subretinally into the right eyes of adult C57BL/6 mice. Laser photocoagulation was performed 4-6 weeks after injection (serotype 2 of AAV) or 24 hrs before injection (serotype 5 of AAV) to focally rupture Bruch’s membrane in both eyes (4-5 sites each eye). The development of CNV at each laser burn was evaluated 14 days later by angiographic choroidal flat-mount image analysis, as well as by histology of transverse retinal sections.
p22phox was expressed in the RPE and other retinal neurons in mouse eye. Eyes receiving subretinal injection of AAV2-siRNA-p22phox showed significant reduction of CNV area. Eyes receiving either subretinal injection of control siRNA vector or intravitreal injection of AAV2-siRNA-p22phox were essentially indistinguishable from control eyes. Subretinal treatment with AAV5-siRNA-p22phox, which has much faster onset of expression than AAV2, 24 hrs after laser was as effective as AAV2 administered 6 weeks prior to laser.
These results demonstrate that RPE cells express p22phox, an essential component of NADPH oxidase, and that inhibition of p22phox by siRNA delivered via AAV vectors effectively blocks CNV development in the laser induced CNV murine model. This inhibition provides evidence for involvement of oxidative stress in CNV development. Targeting this pathway presents a new opportunity for therapeutic intervention against the CNV seen in patients with the exudative form of AMD.
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