Abstract
Purpose :
Complement activation has been increasingly implicated in the pathogenesis of AMD. Apolipoprotein E (ApoE) and complement activation products such as membrane attack complex (MAC) have been detected in Bruch’s membrane (BrM) and drusen from eyes with AMD. We previously reported complement activation induced time-dependent ApoE accumulation in human RPE cells. Herein, we test the hypothesis that the interaction between ApoE and MAC contribute to increased cell-associated ApoE. We also evaluated the relationship between complement activation and ApoE expression in human eyes and mouse models.
Methods :
Cultured human RPE cells were primed with a complement-fixing antibody and then treated with either 6% C1q-depleted human serum (C1q-Dep) to elicit cell surface MAC formation or heat inactivated C1q-Dep to block complement activation. Repetitive complement attack was induced every other day for 1 week (three attacks). RPE cells were incubated for 15 minutes with 0.02N ammonium hydroxide 48 hrs after the third complement attack. Western blot was performed on RPE cell-conditioned media, total cellular lysate proteins and extracellular matrix (ECM). ApoE and MAC co-localization were assessed on cultured human RPE cells and human eyes. C3d and ApoE were examined by immunofluorescent microscopy in ApoB 100 transgenic mice and CD55/CD59 double deficient (KO) mice.
Results :
After a single complement attack, when compared to controls, decreased cell-associated ApoE was accompanied by increased release of ApoE into conditioned media at days 1, 2 and 3 post complement challenge. Cell-associated S58 priming antibody was decreased on RPE cells in a pattern similar to that observed with ApoE protein. ApoE and MAC were frequently co-localized in complement-treated cells, but not in control cells. ApoE was co-distributed with MAC in sub-RPE deposits and drusen from human eyes. Repeated complement attack dramatically enhanced RPE ApoE accumulation and caused ApoE ECM deposition. C3d and ApoE were detected in BrM of ApoB 100 transgenic mice as well as CD55/CD59 KO mice.
Conclusions :
Complement-mediated RPE cell-associated ApoE accumulation and subsequent release may be one of mechanisms that accounts for ApoE in drusen. This information will enhance our understanding of the role that complement activation plays to mediate drusen formation and composition, and may elucidate potential therapeutic targets.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.