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      Impairing autophagy in retinal pigment epithelium leads to inflammasome activation and enhanced macrophage-mediated angiogenesis

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          Abstract

          Age-related decreases in autophagy contribute to the progression of age-related macular degeneration (AMD). We have now studied the interaction between autophagy impaired in retinal pigment epithelium (RPE) and the responses of macrophages. We find that dying RPE cells can activate the macrophage inflammasome and promote angiogenesis. In vitro, inhibiting rotenone-induced autophagy in RPE cells elicits caspase-3 mediated cell death. Co-culture of damaged RPE with macrophages leads to the secretion of IL-1β, IL-6 and nitrite oxide. Exogenous IL-6 protects the dysfunctional RPE but IL-1β causes enhanced cell death. Furthermore, IL-1β toxicity is more pronounced in dysfunctional RPE cells showing reduced IRAK3 gene expression. Co-culture of macrophages with damaged RPE also elicits elevated levels of pro-angiogenic proteins that promote ex vivo choroidal vessel sprouting. In vivo, impaired autophagy in the eye promotes photoreceptor and RPE degeneration and recruitment of inflammasome-activated macrophages. The degenerative tissue environment drives an enhanced pro-angiogenic response, demonstrated by increased size of laser-induced choroidal neovascularization (CNV) lesions. The contribution of macrophages was confirmed by depletion of CCR2 + monocytes, which attenuates CNV in the presence of RPE degeneration. Our results suggest that the interplay between perturbed RPE homeostasis and activated macrophages influences key features of AMD development.

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          Most cited references42

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          Drusen proteome analysis: an approach to the etiology of age-related macular degeneration.

          Drusen are extracellular deposits that accumulate below the retinal pigment epithelium on Bruch's membrane and are risk factors for developing age-related macular degeneration (AMD). The progression of AMD might be slowed or halted if the formation of drusen could be modulated. To work toward a molecular understanding of drusen formation, we have developed a method for isolating microgram quantities of drusen and Bruch's membrane for proteome analysis. Liquid chromatography tandem MS analyses of drusen preparations from 18 normal donors and five AMD donors identified 129 proteins. Immunocytochemical studies have thus far localized approximately 16% of these proteins in drusen. Tissue metalloproteinase inhibitor 3, clusterin, vitronectin, and serum albumin were the most common proteins observed in normal donor drusen whereas crystallin was detected more frequently in AMD donor drusen. Up to 65% of the proteins identified were found in drusen from both AMD and normal donors. However, oxidative protein modifications were also observed, including apparent crosslinked species of tissue metalloproteinase inhibitor 3 and vitronectin, and carboxyethyl pyrrole protein adducts. Carboxyethyl pyrrole adducts are uniquely generated from the oxidation of docosahexaenoate-containing lipids. By Western analysis they were found to be more abundant in AMD than in normal Bruch's membrane and were found associated with drusen proteins. Carboxymethyl lysine, another oxidative modification, was also detected in drusen. These data strongly support the hypothesis that oxidative injury contributes to the pathogenesis of AMD and suggest that oxidative protein modifications may have a critical role in drusen formation.
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            Oxidative damage-induced inflammation initiates age-related macular degeneration.

            Oxidative damage and inflammation are postulated to be involved in age-related macular degeneration (AMD). However, the molecular signal(s) linking oxidation to inflammation in this late-onset disease is unknown. Here we describe AMD-like lesions in mice after immunization with mouse serum albumin adducted with carboxyethylpyrrole, a unique oxidation fragment of docosahexaenoic acid that has previously been found adducting proteins in drusen from AMD donor eye tissues and in plasma samples from individuals with AMD. Immunized mice develop antibodies to this hapten, fix complement component-3 in Bruch's membrane, accumulate drusen below the retinal pigment epithelium during aging, and develop lesions in the retinal pigment epithelium mimicking geographic atrophy, the blinding end-stage condition characteristic of the dry form of AMD. We hypothesize that these mice are sensitized to the generation of carboxyethylpyrrole adducts in the outer retina, where docosahexaenoic acid is abundant and conditions for oxidative damage are permissive. This new model provides a platform for dissecting the molecular pathology of oxidative damage in the outer retina and the immune response contributing to AMD.
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              Advances in measuring cellular bioenergetics using extracellular flux.

              Cell-based assays have become a favored format for drug discovery because living cells have relevant biological complexity and can be highly multiplexed to screen for drugs and their mechanisms. In response to a changing extracellular environment, disease and/or drug exposure, cells remodel bioenergetic pathways in a matter of minutes to drive phenotypic changes associated with these perturbations. By measuring the extracellular flux (XF), that is the changes in oxygen and proton concentrations in the media surrounding cells, one can simultaneously determine their relative state of aerobic and glycolytic metabolism, respectively. In addition, XF is time-resolved and non-invasive, making it an attractive format for studying drug effects in vitro.
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                Author and article information

                Journal
                Sci Rep
                Sci Rep
                Scientific Reports
                Nature Publishing Group
                2045-2322
                05 February 2016
                2016
                : 6
                : 20639
                Affiliations
                [1 ]School of Clinical Sciences, University of Bristol , Bristol, UK
                [2 ]School of Cellular and Molecular Medicine, University of Bristol , Bristol, UK
                [3 ]Department of Internal Medicine II, University Hospital Regensburg , Regensburg, Germany
                [4 ]Institute of Ophthalmology, University College London , London, UK
                [5 ]National Institute for Health Research (NIHR) Biomedical Research Centre , London, UK
                Author notes
                Article
                srep20639
                10.1038/srep20639
                4742917
                26847702
                84249d13-8fde-4312-9fbc-7ec2c3c082f3
                Copyright © 2016, Macmillan Publishers Limited

                This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/

                History
                : 28 October 2015
                : 06 January 2016
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