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Sijia Cao, Jiangyuan Gao, Aikun Wang, Jing Z Cui, Joanne A Matsubara; Drusen component amyloid beta promotes membrane attack complex formation on ARPE-19. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):3774. doi: https://doi.org/.
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Drusen are hallmark deposits associated with age-related macular degeneration (AMD) and amyloid beta (Aβ) and membrane attack complex (MAC) have been both reported in drusen. However, the relationship between Aβ and complement activation on RPE cells is not yet known. In this study we test the hypothesis that Aβ promotes MAC formation on RPE cells by stimulating RPE cells with Aβ in the presence of normal human serum (NHS) as a source of complement proteins. To further understand the effect of Aβ on the regulation of complement cascade, several complement regulators were also measured.
ARPE-19 cells were stimulated with fibrillar Aβ in the presence of NHS (25%) or heat-inactivated (HI)-NHS for 24 hours. Positive controls included stimulation with zymosan and NHS. Negative controls included Aβ or serum-free medium alone. Next, ARPE-19 cells were fixed and immunostained for MAC (1:50, Clone aE11, Dako) and the nuclei were counterstained by DAPI. The MAC formation was quantified as the percentage of MAC immunoreactive cells over the total number of cells per 40× field. The gene and protein expressions of complement regulators were measured by quantitative PCR and western blot (WB) in cells treated with Aβ or controls.
The level of MAC immunoreactive cells was higher in the ARPE-19 treated with NHS and Aβ compared to its corresponding negative control (mean ± standard error of mean (SEM), 14.9±1.2% versus 0.4±0.2%, n=6, p < 0.01, Student’s t test). The complement regulator CD55 was downregulated by Aβ by 20 folds (p < 0.05, Student’s t test), while CD46, CD59, CFH and CFI did not change. The protein level of CD55, as measured by WB, showed a decreased trend after 6-hour treatment with Aβ.
The drusen component, Aβ, induced MAC formation on ARPE-19 cells, and it may promote this process by downregulating the expression of the complement regulator CD55. It is known that the Y402H at-risk variant in the CFH gene is associated with greater MAC deposition in choroid compared to the protective variant. Thus, a concomitant exposure to drusen components, such as Aβ, may further exacerbate MAC formation leading to RPE dysfunction in outer retina.
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