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H. Mahaffy, S. McFarlane, D.A. Simpson, W.J. Curry, A.W. Stitt; Advanced Glycation Endproducts Accumulate in RPE Lysosomal Compartments and Induce Widespread Alteration in Gene Expression: Possible Role in Age-related Dysfunction . Invest. Ophthalmol. Vis. Sci. 2003;44(13):2274.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Advanced glycation endproducts (AGEs) are known to accumulate during aging and have been localised to Bruch’s membrane. We have studied trafficking of AGE-modified proteins to intracellular compartments in ARPE-19 cells. Parallel microarray analysis has been conducted to determine AGE-mediated dysregulation of mRNA expression in these cells. Methods: Lysosomal-like compartments were identified in ARPE-19 monolayers using a low-pH lysotracker fluorophore. Localisation of immunoreactive AGEs was also studied using confocal microscopy. Since it is known that ARPE-19 cells exposed to rod outer segments (ROS) show phagocytosis and lysosomal degradative activation, these cells were treated with either AGE-modified ROS (AGE-ROS) or non-modified control ROS. This provided an in vitro model whereby AGEs could be appropriately exposed to RPE. To measure changes in gene expression RNA was extracted, reverse transcribed and hybridized to a cDNA microarray containing 12,000 expressed human sequences (Agilent human 1). Fluorescent labelling was performed with a 3DNA Submicro Kit (Genisphere) and spot intensities measured using ScanAlyze. Fold changes below 2.0 were disregarded. Results: Immunoreactive AGEs were detected within ARPE-19 intracellular compartments, in a perinuclear pattern. AGE-immunoreactivity showed strong co-localisation with low pH organelles highlighted by lysotracker fluorophore. This indicated that AGEs were trafficked to lysosome-like organelles in these cells. Exposure of ARPE-19 to AGE-ROS induced a range of mRNA expression changes that were subsequently confirmed by real time RT-PCR. While some unknown mRNAs were altered, interesting candidates upregulated in AGE-ROS-exposed RPE included Cytochrome c oxidase subunit VIIa-related protein (~3.5 fold), superoxide dismutase (~3.3 fold), cytochrome c (~2.9 fold), cathepsin A (~3.0 fold), Connexin 43 (~3.1 fold). Conclusions: This study has demonstrated that AGEs are trafficked to the RPE lysosomal compartment. Binding and uptake of AGE-modified ROS material can lead to widespread alteration in RPE gene expression with possible implications for age-related dysfunction.
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