PURPOSE Extracellular vesicles (EVs) can mediate long-distance communication in polarized RPE monolayers. Specifically, EVs from oxidatively stressed donor cells (stress EVs) rapidly reduced barrier function (transepithelial resistance, TER) in naïve recipient monolayers, when compared to control EVs. This effect on TER was dependent on dynamin-mediated EV uptake, which occurred rapidly with EVs from oxidatively stressed donor cells. Here, we further determined molecular mechanisms involved in uptake of EVs by naïve RPE cells. METHODS RPE cells were grown as monolayers in media supplemented with 1% FBS followed by transfer to FBS-free media. Cultures were used to collect control or stress EVs upon treatment with H2O2, others served as naïve recipient cells. In recipient monolayers, TER was used to monitor EV-uptake-based activity, live-cell imaging confirmed uptake. EV surface proteins were quantified by protein chemistry. RESULTS Clathrin-independent, lipid raft-mediated internalization was excluded as an uptake mechanism. Known ligand-receptor interactions involved in clathrin-dependent endocytosis include integrins and proteoglycans. Desialylated glycans and integrin-receptors on recipient cells were necessary for EV uptake and subsequent reduction of TER in recipient cells. Protein quantifications confirmed elevated levels of ligands and neuraminidase on stress EVs. However, control EVs could confer activity in the TER assay if exogenous neuraminidase or additional ligand was provided. CONCLUSIONS In summary, while EVs from both stressed cells and control contain cargo to communicate stress messages to naive RPE cells, stress EVs contain surface ligands that confer rapid uptake by recipient cells. We propose that EVs potentially contribute to RPE dysfunction in aging and disease.

中文翻译：

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应激性视网膜色素上皮细胞单层中细胞外小泡摄取的机制。

目的细胞外囊泡（EVs）可以在极化RPE单层中介导长距离通讯。具体而言，与对照电动车相比，来自氧化应激供体细胞的电动车（应力电动车）在幼稚的受体单层中迅速降低了屏障功能（跨上皮电阻，TER）。这种对TER的作用取决于动力蛋白介导的EV摄取，这种作用在氧化应激供体细胞的EV中迅速发生。在这里，我们进一步确定了天真的RPE细胞摄取EV所涉及的分子机制。方法RPE细胞在添加1％FBS的培养基中单层生长，然后转移至无FBS的培养基中。用H2O2处理后，可将培养物用于收集对照或应激EV，其他则用作幼稚的受体细胞。在受体单层中，TER被用来监测基于EV摄取的活动，活细胞成像证实摄取。EV表面蛋白通过蛋白质化学定量。结果排除了网格蛋白非依赖性，脂筏介导的内在化为摄取机制。与网格蛋白依赖性内吞有关的已知配体-受体相互作用包括整联蛋白和蛋白聚糖。受体细胞上的去唾液酸化聚糖和整联蛋白受体对于EV摄取和随后受体细胞中TER的降低是必需的。蛋白质定量证实了应激电动汽车上配体和神经氨酸酶水平的升高。但是，如果提供了外源性神经氨酸酶或其他配体，则对照EV可以在TER分析中赋予活性。结论总而言之，虽然来自受压单元和控制单元的电动汽车都装有货物，可将压力信息传达给未成熟的RPE单元，压力电动汽车包含表面配体，可让受体细胞快速吸收。我们建议，电动汽车可能会导致衰老和疾病中的RPE功能障碍。