Yang Dexin, Li Zongyang, Gao Guoqing, Li Xiaofeng, Liao Zijun, Wang Yachao, Li Weiping, Zhang Yuan, Liu Wenlan
Department of Neurosurgery, Shenzhen Key Laboratory of Neurosurgery, Shenzhen Second People's Hospital/The First Affiliated Hospital of Shenzhen University, Shenzhen 518035, China.
The Central Laboratory, Shenzhen Second People's Hospital/The First Affiliated Hospital of Shenzhen University, Shenzhen 518035, China.
ACS Omega. 2021 Aug 18;6(34):22410-22421. doi: 10.1021/acsomega.1c03248. eCollection 2021 Aug 31.
Endothelial cell damage is an important pathological basis for the deterioration of acute ischemia stroke. Our previous studies have been exploring the mechanism of blood-brain barrier (BBB) endothelial cell injury in the early stage of cerebral ischemia. Exosomes act as an important intercellular player in neurovascular communication. However, the characteristic of exosomes derived from BBB endothelial cells in early ischemic stroke is poorly understood. We exposed cultured brain microvascular endothelial cells (bEnd.3) to 3 h oxygen glucose deprivation (OGD) to mimic early cerebral ischemia in vitro and compared miRome and surface protein contents of exosomes derived from bEnd.3 cells by miRNA sequencing and the proximity barcoding assay (PBA). A total of 346 differentially miRNA (159 upregulated and 187 downregulated) were identified via miRNA-Seq in bEnd.3 cells after exposure to OGD for 3 h. Moreover, Gene Ontology (GO) and KEGG pathway analyses showed that cell proliferation- and angiogenesis-associated miRNAs were significantly affected. The abnormal changes in top eight miRNAs were further verified by a quantitative polymerase chain reaction (qPCR). PBA experiments showed that the numbers of exosomes carrying the following proteins increased significantly under ischemia, including bFGF, CD146, EPHA2, ABCB5, and ITGB2. These proteins were related to angiogenesis, cell proliferation, and cell inflammation. The network analysis combining PBA data with miRNA-Seq data showed that 79 miRNAs were related to 24 membrane proteins and predicted that there were surface proteins associated with a variety of miRNA molecules, such as ITGA9, XIAP, ADAM1, ITGA2, ITGA3, PDPN, and ITGB1. Meanwhile, there were miRNAs related to various surface proteins including miR-410-3p, miR-378b, and miR-1960. Taken together, our data demonstrated for the first time the changes of exosomal miRNAs and surface protein profiles derived from ischemic microvascular endothelial cells, which may provide new therapeutic targets for BBB protection in ischemic stroke.
内皮细胞损伤是急性缺血性卒中病情恶化的重要病理基础。我们之前的研究一直在探索脑缺血早期血脑屏障（BBB）内皮细胞损伤的机制。外泌体在神经血管通讯中是重要的细胞间参与者。然而，早期缺血性卒中时源自BBB内皮细胞的外泌体特征仍知之甚少。我们将培养的脑微血管内皮细胞（bEnd.3）暴露于3小时氧糖剥夺（OGD）以在体外模拟早期脑缺血，并通过miRNA测序和邻近条形码分析（PBA）比较源自bEnd.3细胞的外泌体的miRome和表面蛋白含量。通过miRNA测序在暴露于OGD 3小时后的bEnd.3细胞中总共鉴定出346个差异miRNA（159个上调和187个下调）。此外，基因本体（GO）和KEGG通路分析表明，与细胞增殖和血管生成相关的miRNA受到显著影响。前八个miRNA的异常变化通过定量聚合酶链反应（qPCR）进一步验证。PBA实验表明，在缺血状态下携带以下蛋白质的外泌体数量显著增加，包括碱性成纤维细胞生长因子（bFGF）、CD146、EPH受体A2（EPHA2）、ATP结合盒转运蛋白B5（ABCB5）和整合素β2（ITGB2）。这些蛋白质与血管生成、细胞增殖和细胞炎症有关。将PBA数据与miRNA测序数据相结合的网络分析表明，79个miRNA与24种膜蛋白相关，并预测存在与多种miRNA分子相关的表面蛋白，如整合素α9（ITGA9）、X连锁凋亡抑制蛋白（XIAP）、解聚素和金属蛋白酶1（ADAMl）、整合素α2（ITGA2）、整合素α3（ITGA3）、血小板源性蛋白（PDPN）和整合素β1（ITGB1）。同时，存在与各种表面蛋白相关的miRNA，包括miR-410-3p、miR-378b和miR-1960。综上所述，我们的数据首次证明了缺血性微血管内皮细胞来源的外泌体miRNA和表面蛋白谱的变化，这可能为缺血性卒中的血脑屏障保护提供新的治疗靶点。
