Targeting post-ischemic thromboinflammation by mesenchymal stromal cell (MSC)-derived small extracellular vesicles

由于它们在缺血性中风动物模型中的恢复促进作用，从间充质基质细胞（MSC）获得的小细胞外囊泡（sEV）正在迅速接近人类中风患者的临床概念验证研究。尽管在动物模型中的疗效数据越来越多，但我们对MSC-sEV的作用机制和作用位点的基本知识仍然很少。使用表现出血脂正常或饮食诱导的高脂血症（这是一种临床相关的血管风险因素，可加重缺血后神经炎症）的小鼠，我们计划在管腔内大脑中动脉闭塞模型中表征介导MSC-sEV诱导的缺血后脑组织恢复的免疫调节机制。将通过流式细胞术、组织化学和3D光片显微镜表征血液、脑微血管和脑中的免疫应答。靶细胞将在体外和体内通过摄取染料标记的sEV或经由基因工程方法负载有CD 63-mCherry或CRE重组酶的sEV来鉴定。为了阐明MSC-EV的作用机制，将进行分离的靶细胞的全局基因表达谱和sEV蛋白质组谱分析。使用抗体介导或药理学研究，我们将功能性灭活血小板聚集和/或相关免疫反应，剖析MSC-sEV靶向的血栓炎性事件。

As a consequence of their recovery-promoting actions in animal models of ischemic stroke, small extracellular vesicles (sEV) obtained from mesenchymal stromal cells (MSCs) are rapidly approaching clinical proof-of-concept studies in human stroke patients. Despite increasing efficacy data in animal models, our basic knowledge about the mechanisms of action and sites of action of MSC-sEVs remains sparse. Using mice exhibiting normolipidemia or dietary-induced hyperlipidemia, which is a clinically relevant vascular risk factor that exacerbates post-ischemic neuroinflammation, we plan to characterize immunomodulatory mechanisms mediating post-ischemic brain tissue recovery induced by MSC-sEVs in an intraluminal middle cerebral artery occlusion model. Immune responses will be characterized in the blood, cerebral microvessels and brain by flow cytometry, histochemistry and 3D light sheet microscopy. Target cells will be identified in vitro and in vivo via the uptake of dye labelled sEVs or sEVs loaded with CD63-mCherry or CRE recombinase via genetic engineering approaches. For unravelling the mechanisms of action of MSC-EVs, global gene expression profiles of isolated target cells and sEV proteome profiling will be performed. Using antibody-mediated or pharmacological studies, we will functionally deactivate platelet aggregation and/or associated immune responses, dissecting thromboinflammatory events targeted by MSC-sEVs.