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J. G. Hu, R. A. Radu, J. Makshanoff, G. H. Travis, D. Bok; Photooxidation Products Activate Complement Attack in Cultured Human Retinal Pigment Epithelium. Invest. Ophthalmol. Vis. Sci. 2010;51(13):1417.
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Recent studies have suggested that inappropriate complement attack is involved in the pathogenesis of age-related macular degeneration (AMD). We examined the response of cultured human retinal pigment epithelium complement activation by photooxidation products.
Cultured human fetal RPE cells were grown to confluence in porous culture wells for 2 months, and were exposed to rod outer segments (ROS) from BALB/C and Albino abca4-/- mice Thereafter, the expression of complement regulatory proteins and anti-oxidative stress genes was measured by qRT-PCR, and the localization and content of the negative regulator, CD46 was imaged by immunohistochemistry. To further test whether photooxidation products such as A2E activate complement attack on RPE, we loaded the cells with A2E and irradiated them with blue light in the presence of 10% human serum as a source of complement. We examined the formation of the C5b-9 membrane attack complex using immunocytochemistry and immunoassay and measured the formation of C3b and C5b in the medium by western analysis.
qRT-PCR showed that the expression of mRNA for complement regulatory proteins, CFH, CD46, CD55, and CD59 was increased by 1.67-, 2.85-, 2.28-, and 2.03-fold respectively after RPE cells were fed ROS from albino abca4 -/- mice compared with BALB/c mice. In addition, expression of the anti-oxidative stress genes SOD1, Metallothionein 1A, Catalase 1,HO and GSTM was increased by 1.9-, 1.35-, 1.22-, 1,29, and 1.32 respectively. Immunostaining of CD46 was elevated in RPE cells exposed to ROS of abca4 -/- mice. The membrane attack complex was detected using anti-C5b-9 antibodies after hRPE cells were exposed to A2E and blue light in the presence of 10% human serum. Western analysis of culture medium containing human serum showed the presence of C3b and C5b.
Our gene expression and morphological data suggest that photooxidation products such as A2E can activate complement attack of cultured human RPE. These results support the notion that A2E and complement activation are involved in AMD pathogenesis.
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