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Zhen-Yang Zhao, Yan Chen, Jian Wang, Jiyang Cai; Exosomal Export In RPE Cells With Lysosome Inhibition. Invest. Ophthalmol. Vis. Sci. 2012;53(14):1147.
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Exosomes are 40-100nm sized vesicles that are released by fusion of multivesicular bodies (MVBs) with the limiting cell plasma membrane. The MVBs are involved in delivery of cargo proteins, including those aggregated and misfolded ones, to lysosome for degradation and detoxification. We hypothesized that retinal pigment epithelial (RPE) cells with lysosome inhibition will have disrupted sorting machinery of MVBs and will release aberrant cargos via exosomes. The purpose of this study is to determine whether lysosome inhibition in cultured human fetal RPE cells changed protein and RNA profiles of exosomes.
Cultured fetal RPE cells were treated with non-lethal dose of chloroquine (CQ) to inhibit lysosome. Exosomes were harvested by differential ultracentrifugation and their protein contents were measured by Western blot analyses. Exosomal microRNAs were isolated by an ultrafiltration method and analyzed by Exiqon microarrays. The array data were processed in Partek Genomics Suite.
Treatment with 20 μM CQ, which did not affect cell viability but inhibited lysosome degradation, led to increased release of lipidated microtubule-associated protein 1 light chain 3 (LC3-II), a marker of autophagosome in isolated exosomes. The amount of CD9 and Hsc70 was not affected by CQ, indicating lysosome inhibition only increased the contents but not the overall rate of exocytosis from the RPE. The majority of RNA species in RPE exosomes are short non-coding RNAs. Exosomes from CQ-treated RPE cells had higher amount of a number of microRNAs.
Lysosome inhibition altered the exosomal RNA and protein contents released by the RPE. Such mechanisms have implications in the functional interactions between RPE, Bruch’s membrane and choroidal endothelial cells. Further characterization of RPE exosomes might shed light on the mechanisms of extracellular deposit in drusen formation and RPE/choroid degeneration in age-related macular degeneration.
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