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R. A. Radu, J. Hu, Q. Yuan, J. Makshanoff, D. Welch, M. Lloyd, G. H. Travis, D. Bok; Complement System Dysregulation and Oxidative Stress in the abca4-/- Mice. Invest. Ophthalmol. Vis. Sci. 2010;51(13):1412.
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Chronic inflammation due to activation of the complement system is an important cause of age-related macular degeneration (AMD). Accumulation of autofluorescent lipofuscin pigments, such A2E, within RPE cells is seen in several forms of macular degeneration including AMD. A2E activates complement in cultured RPE cells following light exposure. Oxidative stress also plays a role in AMD pathogenesis. Previously, we showed that oxidation products of A2E are increased in abca4 -/- mice exposed to high intensity light. In the current study, we investigated the role of lipofuscin pigments and oxidative stress in activating the complement system in abca4 -/- eyes.
Retinas and eyecups containing RPE were collected at designated time-points from age-matched wild-type (WT) and abca4 -/- (KO) mice. Protein extracts were tested for complement activation components and C-reactive protein by immunoblotting. The expression levels for complement-regulatory protein (CRP) and oxidative stress genes were measured by qRT-PCR and immunocytochemistry. Lipid peroxidation levels in mouse RPE were determined by TBARS (for MDA) and ELISA (for 4-HNE). Retina sections were analyzed by light and electron microscopy. Visual retinoids and lipofuscin pigments were quantitated by high-performance liquid chromatography.
Retinal degeneration was seen in seven-mo-old KO mice. A2E and A2E-precursors were significantly elevated in the KO vs. WT eyes at all time points. Consistently, RPE autofluorescence was higher in the KO mice. Bruch’s membrane was significantly thickened in 11-mo-old KO vs. WT mice. The KO mice also showed increased complement C3 immunoreactivity predominantly within the basal infoldings of the RPE. Quantitative Western blotting showed that iC3b was 1.7-fold higher in KO vs. WT RPE cell homogenates. MDA and 4-HNE levels in KO eyecups homogenates were 2.1-, and 3.1-fold respectively higher than in WT. By qRT-PCR, all oxidative-stress genes were upregulated in KO RPE. Surprisingly, expression of all CRP genes was reduced in the KO eyes.
Our data suggest that loss of ABCA4 causes dysregulation of the complement system, chronic inflammation, and increased oxidative stress due to A2E accumulation in the RPE. Moreover, down-regulation of CRP genes, perhaps as a result of chronic insult, renders the RPE more susceptible to further complement attack.
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