Dynamic release of neuronal extracellular vesicles containing miR-21a-5p is induced by hypoxia

Extracellular Vesicles

Hypoxia induces changes in the secretion of extracellular vesicles (EVs) in several non-neuronal cells and pathological conditions. EVs are packed with biomolecules, such as microRNA(miR)-21-5p, which respond to hypoxia. However, the true EV association of miR-21-5p, and its functional or biomarker relevance, are inadequately characterised. Neurons are extremely sensitive cells, and it is not known whether the secretion of neuronal EVs and miR-21-5p are altered upon hypoxia. Here, we characterised the temporal EV secretion profile and cell viability of neurons under hypoxia. Hypoxia induced a rapid increase of miR-21a-5p secretion in the EVs, which preceded the elevation of hypoxia-induced tissue or cellular miR-21a-5p. Prolonged hypoxia induced cell death and the release of morphologically distinct EVs. The EVs protected miR-21a-5p from enzymatic degradation but a remarkable fraction of miR-21a-5p remained fragile and non-EV associated. The increase in miR-21a-5p secretion may have biomarker potential, as high blood levels of miR-21-5p in stroke patients were associated with significant disability at hospital discharge. Our data provides an understanding of the dynamic regulation of EV secretion from neurons under hypoxia and provides a candidate for the prediction of recovery from ischemic stroke.

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Recent Publications

Cigarette smoke (CS) represents one of the most relevant environmental risk factors for several chronic pathologies. Tissue damage caused by CS exposure is mediated, at least in part, by oxidative stress induced by its toxic and pro-oxidant components. Evidence demonstrates that extracellular vesicles (EVs) released by various cell types exposed to CS extract (CSE) are characterized by altered biochemical cargo and gained pathological properties. In the present study, we evaluated the content of oxidized proteins and phospholipid fatty acid profiles of EVs released by human bronchial epithelial BEAS-2B cells treated with CSE. This specific molecular characterization has hitherto not been performed. After confirmation that CSE reduces viability of BEAS-2B cells and elevates intracellular ROS levels, in a dose-dependent manner, we demonstrated that 24 h exposure at 1% CSE, a concentration that only slight modifies cell viability but increases ROS levels, was able to increase carbonylated protein levels in cells and released EVs. The release of oxidatively modified proteins via EVs might represent a mechanism used by cells to remove toxic proteins in order to avoid their intracellular overloading. Moreover, 1% CSE induced only few changes in the fatty acid asset in BEAS-2B cell membrane phospholipids, whereas several rearrangements were observed in EVs released by CSE-treated cells. The impact of changes in acyl chain composition of CSE-EVs accounted for the increased saturation levels of phospholipids, a membrane parameter that might influence EV stability, uptake and, at least in part, EV-mediated biological effects. The present in vitro study adds new information concerning the biochemical composition of CSE-related EVs, useful to predict their biological effects on target cells. Furthermore, the information regarding the presence of oxidized proteins and the specific membrane features of CSE-related EVs can be useful to define the utilization of circulating EVs as marker for diagnosing of CS-induced lung damage and/or CS-related diseases.

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