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

Basic Research in Cardiology (2021) 116:40 https://doi.org/10.1007/s00395-021-00881-9 ORIGINAL CONTRIBUTION 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 Gregorius1 · Chen Wang1 · Oumaima Stambouli2 · Tanja Hussner1 · Yachao Qi1 · Tobias Tertel2 · Verena Börger2 · Ayan Mohamud Yusuf1 · Nina Hagemann1 · Dongpei Yin1 · Robin Dittrich2 · Yanis Mouloud2 · Fabian D. Mairinger3 · Fouzi El Magraoui4 · Aurel Popa‑Wagner5 · Christoph Kleinschnitz1 · Thorsten R. Doeppner6 · Matthias Gunzer4,7 · Helmut E. Meyer4,8 · Bernd Giebel2 · Dirk M. Hermann1 Received: 17 December 2020 / Accepted: 18 May 2021 © The Author(s) 2021 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 MSCsEVs 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% O 2) 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. Keywords Endothelial migration · Microvascular network characteristics · Microvascular remodeling · Neuronal survival · Polymorphonuclear neutrophil · Tube formation Introduction Jonas Gregorius and Chen Wang have equally contributed to this work. * Bernd Giebel * Dirk M. Hermann Extended author information available on the last page of the article Small extracellular vesicles (sEVs), such as exosomes (50–150 nm), play important roles in intercellular communication [37]. In response to injury, sEVs can promote restorative processes [39]. sEVs prepared from mesenchymal stromal cell (MSC) supernatants have been shown to promote neurological recovery and brain remodeling after focal cerebral ischemia in rats and mice [7, 30, 35, 36]. sEVs possess important characteristics which make them 13 Vol.:(0123456789) 40 Page 2 of 19 attractive as therapeutics. In contrast to cell therapies, sEVs are not self-replicating and they lack endogenous tumor formation potentials [19]. sEVs can hardly sense environmental conditions, and thus, 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. Hence, their handling is much easier than that of cells. Due to these promising features, sEVs are rapidly approaching clinical trials in human patients [19]. In a head-to-head study in mice, we have previously demonstrated that sEV preparations obtained from supernatants of MSCs cultured under regular, that is, ‘normoxic’, conditions (21% O 2) equally effectively reduced motor-coordination deficits and increased long-term neuronal survival as their parental MSCs, when they were intravenously administered 24 h after intraluminal middle cerebral artery occlusion (MCAO) [7]. sEV-induced neuroprotection went along with sustained neurogenesis [7]. Neurogenesis and angiogenesis are tightly linked in the ischemic brain [14] and both processes closely accompany successful brain remodeling [12]. Since the cerebral microvasculature, namely endothelial cells, are exposed to intravenously delivered sEVs as firstline targets, the induction of angiogenesis may be instrumental for the capacity of MSC-sEVs to protect ischemic brain tissue. Indeed, evidence of endothelial proliferation was previously found in the ischemic brain after MSC-sEV delivery in mice [7]. The effects of MSC-sEVs on microvessels strongly depend on tissues and pathophysiological states. While MSC-sEV preparations may promote or inhibit angiogenesis in cancer tissues depending on the precise MSC source and tumor microenvironment [6, 18, 41], MSC-sEVs were reported to increase the proliferation and tube formation of cultured human umbilical vein endothelial cells (HUVECs) in an hypoxia-inducible factor-1α (HIF-1α) dependent way [9]. The effects of MSC-sEVs on cerebral microvascular angiogenesis were so far not systematically examined. To evaluate the effects of MSC-sEVs on cerebral angiogenesis, we herein exposed human microvascular endothelial cells (hCMEC/D3) to sEV preparations obtained from MSCs of two randomly selected healthy human donors, which had been cultured under regular ‘normoxic’ conditions (21% O2) or hypoxic conditions (1% O 2), or to sEVs obtained from MSC culture media, which contain platelet lysates. In additional studies, mice that underwent intraluminal MCAO likewise received sEV preparations from ‘normoxic’ MSCs, from hypoxic MSCs or from cell culture media. In some subgroups, polymorphonuclear neutrophil leukocytes (PMN), which have previously been shown to mediate acute neuroprotective effects of MSC-sEVs [30], were depleted. We report that sEVs obtained from hypoxic, but not ‘normoxic’ MSCs or culture media induce angiogenesis as indicated by endothelial proliferation, transwell migration or tube 13 Basic Research in Cardiology (2021) 116:40 formation assays in vitro and microvascular network characteristic analysis in vivo. Interestingly, sEVs from hypoxic MSCs regulated a distinct set of hitherto unrecognized miRNAs in hCMEC/D3 cells that have been linked to angiogenesis. Liquid chromatography/tandem mass spectrometry (LC/MS–MS) revealed previousl (...truncated)