Extracellular vesicles derived from adipose mesenchymal stem cells (AMSCs) reduce autophagic flux via microRNA-25-3p transfer after ischemic stroke

Causal therapy of ischemic stroke remains available to only a minority of patients despite the success of systemic thrombolysis and thrombectomy. Consequently, new adjuvant therapeutic options are urgently needed. MSC transplantation leads to improved neurological recovery in preclinical stroke models. However, MSCs do not directly act on ischemic brain tissue, but rather mediate their effects via the secretion of EVs. The latter are a heterogeneous group of corpuscular structures with a diameter of 30-1000 nm, which contain non-coding RNA, DNA, microRNA, and proteins. The application of EVs seems to be a more effective and low-risk treatment than stem cell transplantation in experimental stroke models. Hence, MSC-EVs provide an adjunctive approach to the treatment of ischemic stroke. Although the mechanisms by which MSC-EVs act remains elusive, recent evidence suggests that MSC-EVs may be responsible for neuroprotection in pathological stroke conditions. Herein, it was first confirmed that the application of AMSC-EVs is not inferior to the use of AMSCs in an in vitro hypoxia model. Incubation of neurons with AMSCs or AMSC-EVs increases the resistance to hypoxic cell injury. Interestingly, the inhibition of EV secretion from AMSCs by GW4869 leads to a loss of the therapeutic effect of AMSCs. This observation again underscores the importance of EVs as a biologically active agent in stem cell treatment of stroke. For the neuroprotective effect of AMSC-EVs under experimental stroke conditions, I for the first time illustrated the interaction of intravesicular microRNA-25-3p with the p53-BNIP3 signaling pathway. The regulation of this pathway by EVs is accompanied by inhibition of the autophagic flux, which in turn leads to reduced neuronal death after OGD. Indeed, the observations described above can be reversed by the use of anti-oligonucleotides and thus reduce the therapeutic effect of EVs. The functional significance of the p53-BNIP3 signaling pathway and the modulation of the autophagic flux via intravesicular transfer of microRNA-25-3p were further investigated in a mouse stroke model. It was shown for the first time in vivo that the application of AMSC-EVs via this mechanism leads to a reduction of cerebral tissue damage and better functional recovery of the animals after stroke. The present work makes an important contribution to the analysis of the mechanisms of EV-based therapy of ischemic stroke. Further preclinical work in this field is needed before EVs can be used as adjuvant therapy in stroke patients. In conclusion, the research results presented herein provide a novel insight into the mechanisms by which AMSC-EVs induce therapeutic actions under experimental stroke settings. This study also provides evidence that native AMSC-EVs yield enhanced neurological recovery and neuroprotection by inhibiting ischemia-induced autophagy. The autophagy inhibition, in turn, is mainly responsible for EVs transferring microRNA-25-3p from AMSCs to their recipient cells. The latter results in interference with the autophagy regulation signaling pathway, p53- BNIP3. These new observations on EVs derived from AMSC may lead to the development of new therapeutic targets and strategies to treat ischemic stroke.2022-03-1