Abstract
Purpose :
Complement activation is strongly correlated with the onset of age-related macular degeneration (AMD), yet its activator remains controversial. During the visual cycle, abnormal buildup of all-trans-retinaldehyde (atRAL) leads to the formation of retinal pigment epithelium(RPE) lipofuscin, Moreover, atRAL is potent in the generation of reactive oxygen species (ROS) that may have the ability to activate complement. Accordingly, in this study we investigated the role of atRAL in complement activation and its underlying mechanisms.
Methods :
Complement activation was assessed by C5b9 deposition in RPE by immunoflurorescence staining and Western blots in aged Abca4-/-Rdh8-/- DKO mice as well as young mice after 10K lux light exposure. In primary porcine RPE cells, the expression levels of complement regulatory proteins, including CD46, CD55, CD59 and complement factor H (CFH), were measured by q-RT-PCR and Western blots after 15 μM atRAL treatment. Complement activation was examined by C5b9 immunofluroresce staining in atRAL laden cells incubated with normal human serum (NHS) for another hour. C3a and C5a levels were measured by ELISA in culture supernatant. Intracellular ROS levels in atRAL-treated cells were quantified using 2′,7′-dichlorofluorescin-diacetate (DCFA) staining by flow cytometry. ROS scavenger N-acetyl-L-cysteine (NAC) was used to inhibit ROS.
Results :
Compared with age-matched wild-type mice, the deposition of C3 and C5b9 was increased in Abca4-/-Rdh8-/- DKO mice. The level of C5b9 in the RPE was also increased after exposure of young Abca4-/-Rdh8-/- DKO mice to 10K lux light for 2 hours. After treating primary porcine RPE cells with 15 μM atRAL for 24 hours, the expression of CD46, CD55, CD59 and CFH was significantly suppressed at the transcriptional level, and a decrease in protein expression of CD59 and CFH was also detected. Furthermore, the staining of C5b9 complex was significantly increased after NHS incubation, suggesting that atRAL treatment activates complement pathway. The suppression of ROS generated from atRAL by NAC restored the expression of complement regulatory proteins, and thereby inhibited complement activation.
Conclusions :
Aberrant accumulation of atRAL activates complement pathway in the RPE by stimulating intracellular ROS to downregulate complement regulatory proteins.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.