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Valeriy V. Lyzogubov, Ruslana G. Tytarenko, Purushottam Jha, Xiaobo Wu, Grant Kolar, John P. Atkinson, Puran S. Bora, Nalini S. Bora; Role Of CD46 In Laser Induced Choroidal Neovascularization. Invest. Ophthalmol. Vis. Sci. 2011;52(14):2326.
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Several studies have established an important role for the complement system in the development of choroidal neovascularization (CNV) associated with exudative age-related macular degeneration (AMD). CD46, a widely expressed transmembrane glycoprotein that serves as complement inhibitor, is present on many cell types in the human eye. However, in the mouse, CD46 it is only known to be expressed on the inner acrosomal membrane of spermatozoa while a related protein with a similar functional repertoire takes its place in other tissues.
RT-PCR, Western blot analysis and immunohistochemistry (IHC) were used to detect mouse CD46. We compared the development of CNV in mice with homozygous deficiency of CD46 (CD46-/-) and C57BL/6 mice, the wild type (WT) control. CNV was induced by photocoagulation using Argon laser. Animals were perfused with FITC-dextran and then flat mounts of RPE-choroid were examined under confocal microscope at days 2, 3 and 7. The FITC-positive signal (CNV size) was measured using ImageJ program. In addition, RPE-choroid tissue was stained using IHC for membrane attack complex (MAC) and vascular endothelial growth factor (VEGF). MAC and VEGF were also assessed by Western blot.
CD46 mRNA and protein were primarily detected in the posterior segment, including RPE and choroid. Confocal analyses of RPE-choroid flat mounts revealed that CD46-/- mice were more susceptible to laser-induced CNV. In these mice, 20% of laser spots were positive for CNV at day 2 post-treatment but were not detected in WT. At day 3, 43% of the laser spots were positive for CNV in CD46-/- mice while only 11% were CNV positive in controls. Fully developed CNV complex was noted in both CD46-/- and WT mice at day 7; however, CNV was more severe in CD46-/- mice and Western blot and IHC of paraffin sections demonstrated increased MAC and VEGF.
Our data provide 1) the initial evidence for CD46 expression in the mouse eye and 2) support an important role in the development of laser-induced CNV.
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