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R. A. Radu, J. Hu, J. H. Peng, M. Lloyd, G. H. Travis, D. Bok; Role of A2E-lipofuscin in Complement Activation in the abca4 Knockout Mouse. Invest. Ophthalmol. Vis. Sci. 2008;49(13):2667.
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The accumulation of toxic lipofuscin fluorophores such as A2E, a vitamin A derivative, in the retinal pigment epithelium (RPE) is involved in the etiology of macular and retinal degenerations. Previously we showed that oxidation products of A2E (A2E-oxiranes) are increased in mice exposed to a high light intensity. Moreover, albino abca4 -/- mice showed high levels of A2E-oxiranes when compared to age-matched pigmented KO mice. Recently it has been shown that the gene for complement factor H (CFH) is a strong susceptibility locus for AMD. In the current study, we investigate the role of lipofuscin fluorophores in activating the complement system.
Age-matched abca4 +/+ (WT) and abca4 -/- (KO) mice on a BALB/c background were considered for this study. Serum, liver, retina and eyecup containing RPE were collected at designated time-points. Protein extracts from different tissues were tested for various complement components (CFH, CRP, C3, C3a and iC3b) by immunoblot analyses. Eyes were also fixed and processed for immunocytochemistry. Retina sections were analyzed by light and electron microscopy. Retinoids and lipofuscin fluorophores extracted from eyecups were analyzed by high-performance liquid chromatography.
In contrast to age-matched WT mice, three-month-old KO mice showed increased C3 fragment staining predominantly within the basal infolding of the RPE. The C3 breakdown products, C3a and iC3b, were highly increased in the KO RPE cells. Significant reduction in the outer segments and the thickness of the outer nuclear layers was seen by light microscopy 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.
(i) A2E levels and autofluorescence in the eye are significantly higher in the KOmice and increase steadily with age. (ii) Retinal degeneration in albino KO mice was present in seven-month old mice and showed progression with age. (iii) Increased C3 immunoreactivity and high C3a and iC3b levels in KO mice indicate an inflammatory response in the RPE as early as three month of age. Ongoing studies are addressing whether specific molecular species of vitamin A derivatives preferentially trigger the activation of the complement system and if chronic inflammation contributes to progression of the RPE dysfunction with subsequent visual loss.
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