Harrington Elizabeth O, Braza Julie, Shil Aparna, Chichger Havovi
Vascular Research Laboratory, Providence Veterans Affairs Medical Center, Providence, RI, USA.
Department of Medicine, Alpert Medical School of Brown University, Providence, RI, USA.
Pulm Circ. 2020 Sep 21;10(3):2045894020951759. doi: 10.1177/2045894020951759. eCollection 2020 Jul-Sep.
The novel endosome protein, p18, and the early endosome GTPase, Rab4, play a significant role in protecting the pulmonary vasculature against permeability associated with acute respiratory distress syndrome. Recently, endothelial-derived extracellular vesicles have been identified to play a key role in the endothelial permeability associated with acute respiratory distress syndrome. Therefore, we investigated the effect of these microparticles, released from endothelial cells overexpressing p18 and Rab4, on pulmonary endothelial barrier function. Endothelial-derived extracellular vesicles isolated from lung microvascular endothelial cells which overexpressed cDNA for wild-type p18 protected a naïve monolayer against lipopolysaccharide-induced permeability. In contrast, endothelial-derived extracellular vesicles from cells overexpressing the non-endosomal binding p18 mutant (p18) exerted no protective effect on the endothelial monolayer. Cells overexpressing either dominant active or inactive Rab4 released endothelial-derived extracellular vesicles which had no effect on lipopolysaccharide-induced permeability. miRNA analysis and permeability studies of endothelial-derived extracellular vesicle isolated from wild-type p18-overexpressing cells demonstrates that let-7i-5p, miR-96-5p, and miR-137-3p are endothelial-derived extracellular vesicle cargo which exert protective effects on the pulmonary endothelium. Finally, we observed down-regulation of p18 protein expression in both the lung and endothelium in an in vivo and in vitro model of acute respiratory distress syndrome. These results demonstrate that endothelial-derived extracellular vesicle released from cells overexpressing p18, but not Rab4, contain miRNA cargo which likely promote a barrier-protective effect on the pulmonary endothelium in settings of acute respiratory distress syndrome. Findings indicate the importance of p18 in the pulmonary vasculature and demonstrate that targeting this protein may provide a novel therapeutic strategy to reduce endothelial permeability associated with acute respiratory distress syndrome.
新型内体蛋白p18和早期内体GTP酶Rab4在保护肺血管系统免受与急性呼吸窘迫综合征相关的通透性影响方面发挥着重要作用。最近，已确定内皮细胞衍生的细胞外囊泡在与急性呼吸窘迫综合征相关的内皮通透性中起关键作用。因此，我们研究了这些从过表达p18和Rab4的内皮细胞释放的微粒对肺内皮屏障功能的影响。从过表达野生型p18 cDNA的肺微血管内皮细胞中分离出的内皮细胞衍生的细胞外囊泡可保护未处理的单层细胞免受脂多糖诱导的通透性影响。相比之下，来自过表达非内体结合p18突变体（p18）的细胞的内皮细胞衍生的细胞外囊泡对内皮单层没有保护作用。过表达显性活性或无活性Rab4的细胞释放的内皮细胞衍生的细胞外囊泡对脂多糖诱导的通透性没有影响。对从过表达野生型p18的细胞中分离出的内皮细胞衍生的细胞外囊泡进行的miRNA分析和通透性研究表明，let-7i-5p、miR-96-5p和miR-137-3p是内皮细胞衍生的细胞外囊泡货物，它们对肺内皮具有保护作用。最后，我们在急性呼吸窘迫综合征的体内和体外模型中观察到肺和内皮中p18蛋白表达的下调。这些结果表明，从过表达p18而非Rab4的细胞中释放的内皮细胞衍生的细胞外囊泡含有可能在急性呼吸窘迫综合征情况下对肺内皮产生屏障保护作用的miRNA货物。研究结果表明p18在肺血管系统中的重要性，并证明靶向该蛋白可能提供一种新的治疗策略，以降低与急性呼吸窘迫综合征相关的内皮通透性。
