Background: Small extracellular vesicles (sEV) secreted by mesenchymal stem cells (MSC) derived from human induced pluripotent stem cells (iPSC, iMSC-sEV) are considered to have great potential in treating ischemic diseases. Angiogenesis play an important role in post-stroke recovery. However, no studies have yet been conducted to systemically examine the effect and the underlying mechanism of iMSC-sEV on angiogenesis under brain ischemia conditions.Methods: Ischemic stroke model was performed in rats induced by middle cerebral artery occlusion (MCAO), and the pro-angiogenic capacity of iMSC-sEV was measured. The in vitro effects of iMSC-sEV on the migration and tube formation of endothelial cells were investigated, respectively. Autophagy and autophagy-related signaling pathway were detected in vivo and in vitro.Results: We found that iMSC-sEV significantly reduced infarct volume, enhanced angiogenesis, and alleviated long-term neurological deficits in rats after stroke. We also demonstrated that iMSC-sEV increased migration and tube formation of endothelial cells in vitro. A further mechanism study revealed that the pro-angiogenic effect of iMSC-sEV was correlated with a reduction in autophagy. Furthermore, iMSC-sEV significantly activated signal transducer and activator of transcription 3 (STAT3), and suppression of STAT3 abolished iMSC-sEV-induced inhibition of autophagy and promotion of angiogenesis in vivo and in vitro.Conclusions: Taken together, our data indicate that iMSC-sEV promote angiogenesis after ischemic stroke, potentially, by inhibiting autophagy, a process that is partially dependent on STAT3 activation.

Regulatory T cells (Tregs) are known to protect against ischemic stroke. However, the low frequency of Tregs restricts their clinical utility. This study investigated whether expanding the number of Tregsin vivowith the IL-2/IL-2 antibody complex (IL-2/IL-2Ab) could improve stroke outcomes and further elaborated the mechanisms of protection in male mice. C57BL/6 mice received IL-2/IL-2Ab or isotype IgG (IsoAb) intraperitoneally for 3 d before (pretreatment) or starting 2 h after (posttreatment) 60 min middle cerebral artery occlusion (MCAO). IL-2/IL-2Ab selectively increased the number of Tregs in the blood, spleen, and lymph nodes. The IL-2/IL-2Ab treatment significantly reduced infarct volume, inhibited neuroinflammation, and improved sensorimotor functions, as manifested by rotarod test and foot fault test, compared with IsoAb-treated stroke mice. Treg depletion was then achieved by diphtheria toxin (DT) injection into transgenic mice expressing the DT receptor under the control of the Foxp3 promoter (DTR mice). The depletion of Tregs completely eliminated IL-2/IL-2Ab-afforded neuroprotection. Interestingly, adoptive transfer of Tregs collected from IL-2/IL-2Ab-treated mice demonstrated more potent neuroprotection than an equal number of Tregs prepared from IsoAb-treated mice, suggesting that IL-2/IL-2Ab not only elevated Treg numbers, but also boosted their functions. Mechanistically, IL-2/IL-2Ab promoted the expression of CD39 and CD73 in expanded Tregs. CD73 deficiency diminished the protective effect of IL-2/IL-2Ab-stimulated Tregs in stroke mice. The results show that IL-2/IL-2Ab expands Tregsin vivoand boosts their immunomodulatory function. The activation of CD39/CD73 signaling in Tregs may participate as a potential mechanism underlying IL-2/IL-2Ab-afforded neuroprotection against ischemic brain injury.SIGNIFICANCE STATEMENTRegulatory T cells (Tregs) are known to protect against ischemic stroke. However, the low frequency of Tregs restricts their clinical utility. This study reported that systemic administration of the IL-2/IL-2 antibody complex (IL-2/IL-2Ab) robustly and selectively expanded the number of Tregs after stroke. IL-2/IL-2Ab pretreatment or posttreatment significantly improved stroke outcomes in a rodent model of ischemic stroke. We further discovered that IL-2/IL-2Ab not only elevated Treg numbers, but also boosted their functions and enhanced the expression of CD39 and CD73. Using CD73-deficient mice, we confirmed the importance of CD73 in the protective effect of IL-2/IL-2Ab-stimulated Tregs in stroke mice. These results shed light on IL-2/IL-2Ab as a clinically feasible immune therapy to boost endogenous Treg responses and ameliorate ischemic brain injury.

Endogenous neurogenesis holds promise for brain repair and long‐term functional recovery after ischaemic stroke. However, the effects of exosomes from human urine‐derived stem cells (USC‐Exos) in neurogenesis remain unclear. This study aimed to investigate whether USC‐Exos enhanced neurogenesis and promoted functional recovery in brain ischaemia. By using an experimental stroke rat model, we found that intravenous injection of USC‐Exos enhanced neurogenesis and alleviated neurological deficits in post‐ischaemic stroke rats. We used neural stem cells (NSCs) subjected to oxygen‐glucose deprivation/reoxygenation (OGD/R) as an in vitro model of ischaemic stroke. The in vitro results suggested that USC‐Exos promoted both proliferation and neuronal differentiation of NSCs after OGD/R. Notably, a further mechanism study revealed that the pro‐neurogenesis effects of USC‐Exos may be partially attributed to histone deacetylase 6 (HDAC6) inhibition via the transfer of exosomal microRNA‐26a (miR‐26a). Taken together, this study indicates that USC‐Exos can be used as a novel promising strategy for brain ischaemia, which highlights the application of USC‐Exos.

Background: Despite recent evidence demonstrating a potent protective effect of adoptively transferred regulatory T cells (Tregs) in ischemic stroke, the mechanism for Treg mobilization and activation in the ischemic brain is, remarkably, unknown. This study determines the role of C‐C chemokine receptor type 5 ( CCR 5) in mediating the docking and activation of transferred Tregs in their protection of early blood‐brain barrier disruption after stroke. Methods and Results: Adoptive transfer of CCR 5 ^−/− Tregs failed to reduce brain infarct or neurological deficits, indicating an indispensable role of CCR 5 in Treg‐afforded protection against cerebral ischemia. Two‐photon live imaging demonstrated that CCR 5 was critical for Treg docking at the injured vessel wall, where they interact with blood‐borne neutrophils/macrophages after cerebral ischemic injury. CCR 5 deficiency on donor Tregs deprived of their early protection against blood‐brain barrier damage. Using flow cytometry, real‐time polymerase chain reaction, and immunostaining, we confirmed that the expression of CCL 5, a CCR 5 ligand, was significantly elevated on the injured endothelium after cerebral ischemia, accompanied by CCR 5 upregulation on circulating Tregs. In a Treg‐endothelial cell coculture, CCR 5 expression was induced on Tregs on their exposure to ischemia‐injured endothelial cells. Furthermore, CCR 5 induction on Tregs enhanced expression of the inhibitory molecule programmed death ligand 1, which in turn inhibited neutrophil‐derived matrix metallopeptidase 9. Conclusions: These results suggest that CCR 5 is a critical molecule for Treg‐mediated blood‐brain barrier protection and a potential target to optimize Treg therapy for stroke.