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S. C. Finnemann, C.-C. Yu, C. Scelfo; Relation of RPE Phagocytosis and Oxidative Stress: Molecular Mechanisms. Invest. Ophthalmol. Vis. Sci. 2009;50(13):3523.
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Cumulative oxidative damage of the retinal pigment epithelium (RPE) layer of the retina causes formation of pro-oxidant lipofuscin and contributes to RPE dysfunction and atrophy in dry AMD. We previously showed that oxidative processes secondary to loss of the diurnal rhythm of photoreceptor outer segment phagocytosis cause age-related loss of photoreceptor function (but not viability) and RPE lipofuscin accumulation in β5 integrin knockout mice. Moreover, we demonstrated that dietary supplementation with antioxidants is sufficient to prevent vision loss with age in these mice. Our parallel experiments exploring RPE cells in culture further suggest that minor, by themselves innocuous, delay in lysosomal digestion of engulfed outer segment fragments and inefficient mitochondrial metabolism synergistically impair the RPE’s phagocytic activity towards outer segments. This study aims to identify specific molecules functionally related to phagocytosis and to energy metabolism that are modified by physiologically relevant oxidative stress in the RPE in culture and in the eye.
We raised age- and strain-matched cohorts of wild-type and β5 knockout mice, divided cohorts into groups receiving anti-oxidant supplementation and groups receiving placebo. We collected neural retina and eyecup samples from mice sacrificed at different ages. We compared quantity and quality of oxidative modifications of proteins known to be involved in the diurnal phagocytic activity of the RPE with age among tissue samples. In complementary experiments, we performed the same measurements on samples obtained from RPE cells in culture comparing cells fed or not with isolated photoreceptor outer segment fragments with either intact or impaired mitochondrial ATP synthesis.
Our experiments demonstrated that specific components of the RPE phagocytic machinery itself acquire oxidative modifications as a function of age and in response to chronic oxidative stress. RPE cells of β5 integrin knockout mice fed with antioxidant-enriched diet that is effective in preventing vision loss with age possessed a phagocytic machinery with significantly lower levels of modifications compared to RPE cells of β5 integrin knockout mice on placebo diet.
Chronic, low levels of oxidative stress and gradual decline in cellular functions with age synergize to impair RPE activities that are likely directly relevant for the development of age-related human retinal disease including AMD. Our results are the first to demonstrate that (and via which protein targets) oxidative stress with age impairs the molecular machinery for RPE phagocytosis, a critical function of RPE cells in support of photoreceptor functions.
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