Extracellular vesicles (EVs) contain biological molecules and are secreted by cells into the extracellular milieu. The endothelial sodium channel (EnNaC) plays an important role in modulating endothelial cell stiffness. We hypothesized EVs secreted from human aortic endothelial cells (hAoEC) positively regulate EnNaC in an autocrine dependent manner. A comprehensive lipidomic analysis using targeted mass spectrometry was performed on multiple preparations of EVs isolated from the conditioned media of hAoEC or complete growth media of these cells. Cultured hAoEC challenged with EVs isolated from the conditioned media of these cells resulted in an increase in EnNaC activity when compared to the same concentration of media derived EVs or vehicle alone. EVs isolated from the conditioned media of hAoEC but not human fibroblast cells were enriched in MARCKS Like Protein 1 (MLP1). The pharmacological inhibition of the negative regulator of MLP1, protein kinase C, in cultured hAoEC resulted in an increase in EV size and release compared to vehicle or pharmacological inhibition of protein kinase D. The MLP1 enriched EVs increased the density of actin filaments in cultured hAoEC compared to EVs isolated from human fibroblast cells lacking MLP1. We quantified 141 lipids from glycerolipids, glycerophospholipids, and sphingolipids in conditioned media EVs that represented twice the number found in control media EVs. The concentrations of sphingomyelin, lysophosphatidylcholine and phosphatidylethanolamine were higher in conditioned media EVs. These results provide the first evidence for EnNaC regulation in hAoEC by EVs and provide insight into a possible mechanism involving MLP1, unsaturated lipids, and bioactive lipids.

中文翻译：

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人主动脉内皮细胞中细胞外囊泡增加内皮钠通道活性：MLP1和生物活性脂质的推定作用

细胞外囊泡 (EV) 含有生物分子，由细胞分泌到细胞外环境中。内皮钠通道 (EnNaC) 在调节内皮细胞硬度中起重要作用。我们假设人类主动脉内皮细胞 (hAoEC) 分泌的 EV 以自分泌依赖方式正向调节 EnNaC。使用靶向质谱法对从 hAoEC 的条件培养基或这些细胞的完全生长培养基中分离的多种 EV 制剂进行了全面的脂质组学分析。与相同浓度的培养基衍生的 EV 或单独的载体相比，用从这些细胞的条件培养基中分离的 EV 挑战培养的 hAoEC 会导致 EnNaC 活性增加。从 hAoEC 的条件培养基中分离出来的 EVs 而不是人类成纤维细胞中富含 MARCKS 样蛋白 1 (MLP1)。与蛋白激酶 D 的载体或药理学抑制相比，MLP1 的负调节因子蛋白激酶 C 在培养的 hAoEC 中的药理学抑制导致 EV 大小和释放的增加。富含 MLP1 的 EV 增加了培养的 hAoEC 中肌动蛋白丝的密度与从缺乏 MLP1 的人成纤维细胞中分离的 EV 相比。我们从条件培养基 EV 中的甘油脂、甘油磷脂和鞘脂中量化了 141 种脂质，其数量是对照培养基 EV 中发现的数量的两倍。在条件培养基 EV 中，鞘磷脂、溶血磷脂酰胆碱和磷脂酰乙醇胺的浓度较高。