Mesenchymal Stem Cells (MSCs) are effective therapeutic agents enhancing the repair of injured tissues mostly through their paracrine activity. Increasing evidences show that besides the secretion of soluble molecules, the release of extracellular vesicles (EVs) represents an alternative mechanism adopted by MSCs. Since macrophages are essential contributors toward the resolution of inflammation, which has emerged as a finely orchestrated process, the aim of the present study was to carry out a detailed characterization of EVs released by human adipose derived-MSCs to investigate their involvement as modulators of MSC anti-inflammatory effects inducing macrophage polarization. The EV-isolation method was based on repeated ultracentrifugations of the medium conditioned by MSC exposed to normoxic or hypoxic conditions (EV^Normo and EV^Hypo). Both types of EVs were efficiently internalized by responding bone marrow-derived macrophages, eliciting their switch from a M1 to a M2 phenotype. In vivo, following cardiotoxin-induced skeletal muscle damage, EV^Normo and EV^Hypo interacted with macrophages recruited during the initial inflammatory response. In injured and EV-treated muscles, a downregulation of IL6 and the early marker of innate and classical activation Nos2 were concurrent to a significant upregulation of Arg1 and Ym1, late markers of alternative activation, as well as an increased percentage of infiltrating CD206^pos cells. These effects, accompanied by an accelerated expression of the myogenic markers Pax7, MyoD, and eMyhc, were even greater following EV^Hypo administration. Collectively, these data indicate that MSC-EVs possess effective anti-inflammatory properties, making them potential therapeutic agents more handy and safe than MSCs. © Stem Cells Translational Medicine 2017

Combined administration of anthracyclines (e.g. doxorubicin; Dox) and trastuzumab (Trz), a humanized anti-human epidermal growth factor receptor 2 (HER2; ErbB2), is an effective treatment for HER2-positive breast cancer. However, both agents are associated with cardiac toxicity. Human cardiac-resident mesenchymal progenitor cells (CPCs) secrete extracellular vesicles including nanosized exosomes which protect against myocardial ischaemia. Here, we investigated the effects of these exosomes using a novel model of Dox/Trz-mediated cardiotoxicity. CPCs were derived from cardiac atrial appendage specimens from patients who underwent heart surgery for heart valve disease and/or ischaemic heart disease, and exosomes were purified from CPC conditioned media. Proteomics analyses revealed that CPC exosomes contained multiple proteins involved in redox processes. Dox/Trz induced a significant increase in reactive oxygen species (ROS) in rat cardiomyocytes, which was prevented by CPC exosomes. In vivo, rats received six doses of Dox (Days 1-11), followed by six doses of Trz (Days 19-28). Three doses of either exosomes or exosome suspension vehicle were injected intravenously on Days 5, 11, and 19 in the treatment and control groups, respectively. Dox/Trz induced myocardial fibrosis, CD68+ inflammatory cell infiltrates, inducible nitric oxide synthase expression, and left ventricular dysfunction. CPC exosomes prevented these effects. These vesicles were highly enriched in miR-146a-5p compared with human dermal fibroblast exosomes. Dox upregulated Traf6 and Mpo, two known miR-146a-5p target genes (which encode signalling mediators of inflammatory and cell death axes) in myocytes. CPC exosomes suppressed miR-146a-5p target genes Traf6, Smad4, Irak1, Nox4, and Mpo in Dox-treated cells. Specific silencing of miR-146a-5p abrogated exosome-mediated suppression of those genes leading to an increase in Dox-induced cell death. Human CPC exosomes attenuate Dox-/Trz-induced oxidative stress in cardiomyocytes. Systemic administration of these vesicles prevents Dox/Trz cardiotoxicity in vivo. miR-146a-5p mediates some of the benefits of exosomes in this setting.

Human amniotic fluid stem cells (hAFS) have shown a distinct secretory profile and significant regenerative potential in several preclinical models of disease. Nevertheless, little is known about the detailed characterization of their secretome. Herein we show for the first time that hAFS actively release extracellular vesicles (EV) endowed with significant paracrine potential and regenerative effect. c-KIT⁺ hAFS were isolated from leftover samples of amniotic fluid from prenatal screening and stimulated to enhance EV release (24 hours 20% O₂ versus 1% O₂ preconditioning). The capacity of the c-KIT⁺ hAFS-derived EV (hAFS-EV) to induce proliferation, survival, immunomodulation, and angiogenesis were investigated in vitro and in vivo. The hAFS-EV regenerative potential was also assessed in a model of skeletal muscle atrophy (HSA-Cre, Smn^F7/F7 mice), in which mouse AFS transplantation was previously shown to enhance muscle strength and survival. hAFS secreted EV ranged from 50 up to 1,000 nm in size. In vitro analysis defined their role as biological mediators of regenerative, paracrine effects while their modulatory role in decreasing skeletal muscle inflammation in vivo was shown for the first time. Hypoxic preconditioning significantly induced the enrichment of exosomes endowed with regenerative microRNAs within the hAFS-EV. In conclusion, this is the first study showing that c-KIT⁺ hAFS dynamically release EV endowed with remarkable paracrine potential, thus representing an appealing tool for future regenerative therapy. Stem Cells Translational Medicine 2017;6:1340–1355

Coronavirus-2 (SARS-CoV-2) infection causes an acute respiratory syndrome accompanied by multi-organ damage that implicates a prothrombotic state leading to widespread microvascular clots. The causes of such coagulation abnormalities are unknown. The receptor tissue factor, also known as CD142, is often associated with cell-released extracellular vesicles (EV). In this study, we aimed to characterize surface antigens profile of circulating EV in COVID-19 patients and their potential implication as procoagulant agents. We analyzed serum-derived EV from 67 participants who underwent nasopharyngeal swabs molecular test for suspected SARS-CoV-2 infection (34 positives and 33 negatives) and from 16 healthy controls (HC), as referral. A sub-analysis was performed on subjects who developed pneumonia (n = 28). Serum-derived EV were characterized for their surface antigen profile and tested for their procoagulant activity. A validation experiment was performed pre-treating EV with anti-CD142 antibody or with recombinant FVIIa. Serum TNF-α levels were measured by ELISA. Profiling of EV antigens revealed a surface marker signature that defines circulating EV in COVID-19. A combination of seven surface molecules (CD49e, CD209, CD86, CD133/1, CD69, CD142, and CD20) clustered COVID (+) versus COVID (-) patients and HC. CD142 showed the highest discriminating performance at both multivariate models and ROC curve analysis. Noteworthy, we found that CD142 exposed onto surface of EV was biologically active. CD142 activity was higher in COVID (+) patients and correlated with TNF-α serum levels. In SARS-CoV-2 infection the systemic inflammatory response results in cell-release of substantial amounts of procoagulant EV that may act as clotting initiation agents, contributing to disease severity. Cardiocentro Ticino Institute, Ente ospedaliero Cantonale, Lugano-Switzerland.