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V. V. Lyzogubov, R. G. Tytarenko, P. Jha, P. S. Bora, N. S. Bora; Suppression of Complement Factor H Increases Deposition of Membrane Attack Complex and Vessel Growth in Mouse Model of CNV. Invest. Ophthalmol. Vis. Sci. 2009;50(13):3752.
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We have previously reported that complement activation and membrane attack complex (MAC) formation via the alternative pathway plays a crucial role in the development of laser-induced CNV in mice. This study was undertaken to investigate the role of complement factor H (CFH) the key regulator of the alternative pathway in laser induced CNV.
CNV was induced in C57BL/6 mice by photocoagulation using Argon laser. Animals received CFH siRNA, control siRNA and vehicle separately via the subretinal route using lipid based delivery system. Samples were collected on days 1, 3, 5 and 7 post-laser photocoagulation. Suppression of CFH was confirmed by RT-PCR and Western blotting. For CNV size evaluation animals were perfused with FITC-dextran, images of injured sites were captured using confocal microscopy of flat mounted RPE-choroid-sclera. Additionally, RPE-choroid-sclera flat mounts were stained for MAC using immunohistochemistry. Size of CNV complex and intensity of MAC positive immunofluorescence was determined with ImageJ program.
Subretinal injection of CFH siRNA decreased CFH mRNA and protein expression in RPE-choroid. Quantitative immunohistochemical analysis demonstrated that suppression of CFH leads to increased MAC deposition (89-250%) on RPE cells. Compared to the animals receiving control siRNA and vehicle alone, a sharp increase in the size of CNV complex (292% at day 3, 75% at day 5 and 81% at day 7) was observed in the animals in which factor H was suppressed by factor H siRNA.
Factor H inhibition leads to increased MAC deposition on RPE cells and predisposes to early and more intensive vessel growth in mouse after laser photocoagulation. Our results further suggest that the inhibition of the alternative pathway of complement activation may be used as a therapeutic tool in the treatment of wet AMD in future.
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