Mesenchymal stem cells (MSCs) play important roles in tissue repair and regeneration, such as the induction of angiogenesis, particularly under hypoxic conditions. However, the molecular mechanisms underlying hypoxic MSC activation remain largely unknown. MSC-derived extracellular vesicles (EVs) are vital mediators of cell-to-cell communication and can be directly utilized as therapeutic agents for tissue repair and regeneration. Here, we explored the effects of EVs from human hypoxic olfactory mucosa MSCs (OM-MSCs) on angiogenesis and its underlying mechanism. EVs were isolated from normoxic (N) OM-MSCs (N-EVs) and hypoxic (H) OM-MSCs (H-EVs) using differential centrifugation and identified by transmission electron microscopy and flow cytometry. In vitro and in vivo, both types of OM-MSC-EVs promoted the proliferation, migration, and angiogenic activities of human brain microvascular endothelial cells (HBMECs). In addition, angiogenesis-stimulatory activity in the H-EV group was significantly enhanced compared to the N-EV group. MicroRNA profiling revealed a higher abundance of miR-612 in H-EVs than in N-EVs, while miR-612 inactivation abolished the N-EV treatment benefit. To explore the roles of miR-612, overexpression and knock-down experiments were performed using a mimic and inhibitor or agomir and antagomir of miR-612. The miR-612 target genes were confirmed using the luciferase reporter assay. Gain- and loss-of-function studies allowed the validation of miR-612 (enriched in hypoxic OM-MSC-EVs) as a functional messenger that stimulates angiogenesis and represses the expression of TP53 by targeting its 3′-untranslated region. Further functional assays showed that hypoxic OM-MSC-EVs promote paracrine Hypoxia-inducible factor 1-alpha (HIF-1α)-Vascular endothelial growth factor (VEGF) signaling in HBMECs via the exosomal miR-612-TP53-HIF-1α-VEGF axis. These findings suggest that hypoxic OM-MSC-EVs may represent a promising strategy for ischemic disease by promoting angiogenesis via miR-612 transfer. Graphical Abstract

Nasal olfactory mucosa mesenchymal stem cells (OM‐MSCs) have the ability to promote regeneration in the nervous system in vivo. Moreover, with view to the potential for clinical application, OM‐MSCs have the advantage of being easily accessible from patients and transplantable in an autologous manner, thus eliminating immune rejection and contentious ethical issues. So far, most studies have been focused on the role of OM‐MSCs in central nervous system replacement. However, the secreted proteomics of OM‐MSCs have not been reported yet. Here, proteins secreted by OM‐MSCs cultured in serum‐free conditions were separated on SDS‐PAGE and identified by LC‐MS/MS. As a result, a total of 274 secreted proteins were identified. These molecules are known to be important in neurotrophy, angiogenesis, cell growth, differentiation, and apoptosis, and inflammation which were highly correlated with the repair of central nervous system. The proteomic profiling of the OM‐MSCs secretome might provide new insights into their nature in the neural recovery. However, proteomic analysis for clinical biomarkers of OM‐MSCs needs to be further studied.

Na_v1. 9 voltage-gated sodium channel is preferentially expressed in peripheral nociceptive neurons. Recent progresses have proved its role in pain sensation, but our understanding of Na_v1.9, in general, has lagged behind because of limitations in heterologous expression in mammal cells. In this work, functional expression of human Na_v1.9 (hNa_v1.9) was achieved by fusing GFP to the C-terminal of hNa_v1.9 in ND7/23 cells, which has been proved to be a reliable method to the electrophysiological and pharmacological studies of hNa_v1.9. By using the hNa_v1.9 expression system, we investigated the electrophysiological properties of four mutations of hNa_v1.9 (K419N, A582T, A842P, and F1689L), whose electrophysiological functions have not been determined yet. The four mutations significantly caused positive shift of the steady-state fast inactivation and therefore increased hNa_v1.9 activity, consistent with the phenotype of painful peripheral neuropathy. Meanwhile, the effects of inflammatory mediators on hNa_v1.9 were also investigated. Impressively, histamine was found for the first time to enhance hNa_v1.9 activity, indicating its vital role in hNa_v1.9 modulating inflammatory pain. Taken together, our research provided a useful platform for hNa_v1.9 studies and new insight into mechanism of hNa_v1.9 linking to pain.

Mesenchymal stromal cells (MSCs) have been used for the treatment of neuronal injury and neurodegenerative diseases. Their underlying mechanism may involve increased secretion of paracrine factors, which promotes tissue repair. Presently, exosomes have been regarded as important components of paracrine secretion and paracrine factors. MSC exosomes represent a promising opportunity to develop novel cell-free therapy approaches. In this study, exosomes from nasal olfactory mucosa MSCs (OM-MSCs) were extracted and purified using ultracentrifugation, resulting in exosome diameters of 40-130 nm. Similar to other exosomes, OM-MSC exosomes were CD63- and CD81-positive and calnexin-negative. Functionally, OM-MSC exosomes promoted human brain microvascular endothelial cell (HBMEC) proliferation and migration. The present study analyzed the OM-MSC exosome paracrine proteome. A total of 304 exosome-associated proteins were identified by LC-MS/MS, including plasminogen activator inhibitor 1 (SERPINE 1), insulin-like growth factor binding protein family members (IGFBP 4 and 5), epidermal growth factor receptor (EGFR), neurogenic locus notch homolog protein 2 (NOTCH 2), apolipoprotein E (APOE), and heat shock protein HSP90-beta (HSP90AB1). These molecules are known to be important in neurotrophic, angiogenesis, cell growth, differentiation, apoptosis, and inflammation and are highly correlated with the mechanism of tissue repair and neural restoration. These observations may provide a basis for further evaluation of OM-MSC exosome potential as a novel therapeutic modality.