Inducible Pluripotent Stem Cell-Derived Small Extracellular Vesicles Rejuvenate Senescent Blood-Brain Barrier to Protect against Ischemic Stroke in Aged Mice

Blood-brain barrier (BBB) breakdown after ischemic stroke exacerbates brain injury and BBB senescence can cause severe neurological deficits in aged ischemic stroke population. Recent evidence reveals that inducible pluripotent stem cell-derived small extracellular vesicles (iPSC-sEVs) possess phenomenal antisenescence capability. However, whether iPSC-sEVs can rejuvenate BBB senescence to improve stroke outcomes in aged mice remains unknown. Here, we showed that long-term treatment with iPSC-sEVs alleviated aging-induced BBB senescence in aged mice. In aged stroke mice, iPSC-sEVs significantly mitigated BBB integrity damage, reduced the following infiltration of peripheral leukocytes, and decreased the release of pro-inflammatory factors from the leukocytes, which ultimately inhibited neuronal death and improved neurofunctional recovery. Mechanism studies showed that iPSC-sEVs could activate the endothelial nitric oxide synthase (eNOS) and up-regulate sirtuin 1 (Sirt1) in senescent endothelial cells. Blocking the activation of eNOS abolished iPSC-sEV-mediated rejuvenation of BBB senescence and the protection of BBB integrity. Proteomics results demonstrated that iPSC-sEVs were enriched with bioactive factors including AKT serine/threonine kinase 1 (AKT1) and calmodulin (CALM) to activate the eNOS-Sirt1 axis. Further investigation showed that AKT1 and CALM inhibitors blocked iPSC-sEV-afforded activation of the eNOS-Sirt1 axis in senescent endothelial cells. Taken together, iPSC-sEVs can protect against ischemic stroke in aged mice by rejuvenating BBB senescence, partially, through delivering AKT1 and CALM to activate eNOS-Sirt1 axis, which indicates that iPSC-sEVs treatment is an effective alternative to treat ischemic stroke in the aged population.