Small extracellular vesicles obtained from hypoxic mesenchymal stromal cells have unique characteristics that promote cerebral angiogenesis, brain remodeling and neurological recovery after focal cerebral ischemia in mice

Jonas Gregorius,Verena Börger,Chen Wang,Yanis Mouloud,Dongpei Yin,Thorsten R Doeppner,Helmut E Meyer,Yachao Qi,Tanja Hussner,Robin Dittrich,Nina Hagemann,Christoph Kleinschnitz,Fouzi El Magraoui ,Matthias Gunzer,Ayan Mohamud Yusuf,Fabian Mairinger ,Oumaima Stambouli,Aurel Popa‐Wagner ,Bernd Giebel,Tobias Tertel,Dirk M Hermann

doi:10.1007/s00395-021-00881-9

Abstract

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.

Highlights

Small extracellular vesicles, such as exosomes (50–150 nm), play important roles in intercellular communication [37]
While small extracellular vesicles (sEVs) obtained from mesenchymal stromal cell (MSC) cell culture media (sEVplatelet; Fig. 1A, D) or sEVs from ‘normoxic’ MSCs (sEVnormoxic, source 41.5; Fig. 1B, D) did not influence endothelial proliferation at any dose examined, sEVs obtained from hypoxic MSCs (sEVhypoxic, again source 41.5) dose-dependently increased the proliferation of hCMEC/ D3 cells (Fig. 1C, D)
If the angiogenic effect of sEVs obtained from hypoxic MSCs translates into enhanced post-stroke angiogenesis and brain remodeling in vivo, mice were exposed to transient intraluminal middle cerebral artery occlusion (MCAO) followed by delivery of vehicle, sEVplatelet, sEVnormoxic or sEVhypoxic at 24, 72 and 120 h post-ischemia

Summary

Introduction

Small extracellular vesicles (sEVs), such as exosomes (50–150 nm), play important roles in intercellular communication [37]. SEVs can hardly sense environmental conditions, and their biological activity can be predicted more reliably than that of cells [19]. Due to their small size, sEV products can be sterilized by filtration. Their handling is much easier than that of cells. Due to these promising features, sEVs are rapidly approaching clinical trials in human patients [19]

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Results

Conclusion