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J. Tyrrell, P. Yang, G. J. Jaffe; Effect of Alternative Pathway Complement Activation on Cell Mortality and Survival Factor Expression in Cultured Human RPE Cells. Invest. Ophthalmol. Vis. Sci. 2008;49(13):488.
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Retinal pigment epithelium (RPE) cell death is a characteristic of late neovascular and non-neovascular AMD. Drusen in eyes of patients with AMD contain complement pathway molecules, such as C5 and C5b-9, suggesting that complement may play an important role in AMD pathology. Herein we determined the effect of complement activation on RPE cell mortality, and survival factor and anti-apoptotic molecule expression.
Cultured human RPE were exposed to zymosan A (1mg/ml or 4mg/ml) and normal human serum complement (NHS) or heat-inactivated normal human serum in gelatin veronal buffer (GVB). Following exposure, media complement activation was assayed with a SC5b-9 ELISA kit (Quidel). To measure the effect of complement activation on cell permeability/lysis, cells were pre-loaded with calcein-AM and then exposed to zymosan and NHS. To quantify calcein-AM released from RPE, supernatant fluorescence was measured and the percent specific calcein release was calculated. RNA was harvested from complement-exposed cells and analyzed by RT real-time PCR for gene expression levels of several survival factors as well as pro-apoptotic molecules.
There were high levels of SC5b-9 in samples containing zymosan and NHS, confirming alternative complement pathway activation. Following exposure to zymosan and NHS, there was no significant difference in RPE cell permeability compared to controls. RNA analysis showed increased expression of survival factors Traf1, Traf2, Ciap1, Ciap2, C-flip and Bcl-xl compared to the control groups. There was no significant change in expression of pro-apoptotic factors Bax, Bak1, and Bim compared to control groups.
RPE may not be sensitive to complement mediated cell death. Activated complement may actually protect RPE from apoptosis through up regulation of survival factors.
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