Abstract
Systemic transplantation of oxygen−glucose deprivation (OGD)-preconditioned primary microglia enhances neurological recovery in rodent stroke models, albeit the underlying mechanisms have not been sufficiently addressed. Herein, we analyzed whether or not extracellular vesicles (EVs) derived from such microglia are the biological mediators of these observations and which signaling pathways are involved in the process. Exposing bEnd.3 endothelial cells (ECs) and primary cortical neurons to OGD, the impact of EVs from OGD-preconditioned microglia on angiogenesis and neuronal apoptosis by the tube formation assay and TUNEL staining was assessed. Under these conditions, EV treatment stimulated both angiogenesis and tube formation in ECs and repressed neuronal cell injury. Characterizing microglia EVs by means of Western blot analysis and other techniques revealed these EVs to be rich in TGF-β1. The latter turned out to be a key compound for the therapeutic potential of microglia EVs, affecting the Smad2/3 pathway in both ECs and neurons. EV infusion in stroke mice confirmed the aforementioned in vitro results, demonstrating an activation of the TGF-β/Smad2/3 signaling pathway within the ischemic brain. Furthermore, enriched TGF-β1 in EVs secreted from OGD-preconditioned microglia stimulated M2 polarization of residing microglia within the ischemic cerebral environment, which may contribute to a regulation of an early inflammatory response in postischemic hemispheres. These observations are not only interesting from the mechanistic point of view but have an immediate therapeutic implication as well, since stroke mice treated with such EVs displayed a better functional recovery in the behavioral test analyses. Hence, the present findings suggest a new way of action of EVs derived from OGD-preconditioned microglia by regulating the TGF-β/Smad2/3 pathway in order to promote tissue regeneration and neurological recovery in stroke mice.
Highlights
Cerebral ischemia induces energy depletion, excitotoxicity, and cellular demise [1], resulting in high levels of cytokines and chemokines at the early stage of the disease
Isolation and characterization of extracellular vesicles (EVs) from oxygen−glucose deprivation (OGD)-preconditioned microglia and TGF-β1 knockdown microglia Verifying our hypothesis that EVs from preconditioned microglia are the true biological mediators of these cells, EVs were first characterized according to the ISEV guidelines [27]
Some endothelial cells were incubated with EVs enriched with TGF-β1 (i.e., EVs from preconditioned microglia), whereas other endothelial cells were treated with EVs plus a TGF-β1 receptor inhibitor
Summary
Cerebral ischemia induces energy depletion, excitotoxicity, and cellular demise [1], resulting in high levels of cytokines and chemokines at the early stage of the disease. The release of such factors further aggravates secondary brain injury due to enhanced excitotoxicity, cytolysis, oxidative stress, and thromboinflammation [2,3,4]. M2 microglia alter the cellular microenvironment of the ischemic brain by clearing cell debris and by releasing immunomodulatory factors such as galectin-3 and IL-10 [9,10,11,12] Such activated M2 microglia contribute to axonal growth and cerebral repair for which VEGF, BDNF, IGF-1, and MMP-9 play a pivotal role. The transition of the microglial phenotype, depends on the cellular activation state during the course of ischemic brain injury
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