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

由于其在缺血性中风动物模型中的促进恢复作用，从间充质基质细胞（MSCs）中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。尽管动物模型的疗效数据越来越多，但我们对msc - sev的作用机制和作用部位的基本知识仍然很少。正常血脂或饮食性高脂血症是加剧缺血后神经炎症的临床相关血管危险因素，我们计划在脑腔内大脑中动脉闭塞模型中表征msc - sev介导缺血后脑组织恢复的免疫调节机制。免疫反应将通过流式细胞术、组织化学和3D薄层显微镜在血液、大脑微血管和大脑中进行表征。靶细胞将在体外和体内通过基因工程方法通过摄取染料标记的sev或装载CD63-mCherry或CRE重组酶的sev来鉴定。为了揭示msc - ev的作用机制，将对分离的靶细胞进行全局基因表达谱和sEV蛋白质组谱分析。通过抗体介导或药理学研究，我们将从功能上使血小板聚集和/或相关免疫反应失活，剖析msc - sev靶向的血栓炎症事件。

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.