Obtained from the right cell-type, mesenchymal stromal cell (MSC)-derived small extracellular vesicles (sEVs) promote stroke recovery. Within this process, microvascular remodeling plays a central role. Herein, we evaluated the effects of MSC-sEVs on the proliferation, migration, and tube formation of human cerebral microvascular endothelial cells (hCMEC/D3) in vitro and on post-ischemic angiogenesis, brain remodeling and neurological recovery after middle cerebral artery occlusion (MCAO) in mice. In vitro, sEVs obtained from hypoxic (1% O₂), but not ‘normoxic’ (21% O₂) MSCs dose-dependently promoted endothelial proliferation, migration, and tube formation and increased post-ischemic endothelial survival. sEVs from hypoxic MSCs regulated a distinct set of miRNAs in hCMEC/D3 cells previously linked to angiogenesis, three being upregulated (miR-126-3p, miR-140-5p, let-7c-5p) and three downregulated (miR-186-5p, miR-370-3p, miR-409-3p). LC/MS–MS revealed 52 proteins differentially abundant in sEVs from hypoxic and ‘normoxic’ MSCs. 19 proteins were enriched (among them proteins involved in extracellular matrix–receptor interaction, focal adhesion, leukocyte transendothelial migration, protein digestion, and absorption), and 33 proteins reduced (among them proteins associated with metabolic pathways, extracellular matrix–receptor interaction, focal adhesion, and actin cytoskeleton) in hypoxic MSC-sEVs. Post-MCAO, sEVs from hypoxic MSCs increased microvascular length and branching point density in previously ischemic tissue assessed by 3D light sheet microscopy over up to 56 days, reduced delayed neuronal degeneration and brain atrophy, and enhanced neurological recovery. sEV-induced angiogenesis in vivo depended on the presence of polymorphonuclear neutrophils. In neutrophil-depleted mice, MSC-sEVs did not influence microvascular remodeling. sEVs from hypoxic MSCs have distinct angiogenic properties. Hypoxic preconditioning enhances the restorative effects of MSC-sEVs.

间充质基质细胞 (MSC) 衍生的小细胞外囊泡 (sEV) 取自正确的细胞类型，可促进中风恢复。在此过程中，微血管重塑发挥着核心作用。在此，我们评估了MSC-sEVs对体外人脑微血管内皮细胞（hCMEC/D3）增殖、迁移和管形成以及大脑中动脉闭塞后缺血后血管生成、脑重塑和神经功能恢复的影响。 MCAO）在小鼠中。在体外，从低氧（1% O ₂ ）而不是“常氧”（21% O ₂ ）MSC 获得的 sEV 剂量依赖性地促进内皮增殖、迁移和管形成，并增加缺血后内皮存活。来自缺氧 MSC 的 sEV 在先前与血管生成相关的 hCMEC/D3 细胞中调节一组不同的 miRNA，其中 3 个上调（miR-126-3p、miR-140-5p、let-7c-5p）和 3 个下调（miR-186- 5p、miR-370-3p、miR-409-3p)。 LC/MS-MS 揭示了来自低氧和“常氧”MSC 的 sEV 中 52 种蛋白质的丰度存在差异。 19种蛋白质被富集（其中涉及细胞外基质-受体相互作用、粘着斑、白细胞跨内皮迁移、蛋白质消化和吸收的蛋白质），33种蛋白质被减少（其中涉及代谢途径、细胞外基质-受体相互作用、局灶性粘着斑相关的蛋白质）。缺氧 MSC-sEV 中的粘附和肌动蛋白细胞骨架）。 MCAO 后，来自缺氧 MSC 的 sEV 在长达 56 天内增加了先前缺血组织中的微血管长度和分支点密度（通过 3D 光片显微镜评估），减少了迟发性神经元变性和脑萎缩，并增强了神经功能恢复。 sEV 诱导的体内血管生成依赖于多形核中性粒细胞的存在。在中性粒细胞耗尽的小鼠中，MSC-sEVs 不影响微血管重塑。来自缺氧 MSC 的 sEV 具有独特的血管生成特性。低氧预处理增强 MSC-sEV 的恢复效果。