Inflammatory extracellular vesicles prompt heart dysfunction via TRL4-dependent NF-κB activation.

Background: After myocardial infarction, necrotic cardiomyocytes release damage-associated proteins that stimulate innate immune pathways and macrophage tissue infiltration, which drives inflammation and myocardial remodeling. Circulating inflammatory extracellular vesicles play a crucial role in the acute and chronic phases of ischemia, in terms of inflammatory progression. In this study, we hypothesize that the paracrine effect mediated by these vesicles induces direct cytotoxicity in cardiomyocytes. Thus, we examined whether reducing the generation of inflammatory vesicles within the first few hours after the ischemic event ameliorates cardiac outcome at short and long time points. Methods: Myocardial infarction was induced in rats that were previously injected intraperitoneally with a chemical inhibitor of extracellular-vesicle biogenesis. Heart global function was assessed by echocardiography performed at 7, 14 and 28 days after MI. Cardiac outcome was also evaluated by hemodynamic analysis at sacrifice. Cytotoxic effects of circulating EV were evaluated ex-vivo in a Langendorff, system by measuring the level of cardiac troponin I (cTnI) in the perfusate. Mechanisms undergoing cytotoxic effects of EV derived from pro-inflammatory macrophages (M1) were studied in-vitro in primary rat neonatal cardiomyocytes. Results: Inflammatory response following myocardial infarction dramatically increased the number of circulating extracellular vesicles carrying alarmins such as IL-1α, IL-1β and Rantes. Reducing the boost in inflammatory vesicles during the acute phase of ischemia resulted in preserved left ventricular ejection fraction in vivo. Hemodynamic analysis confirmed functional recovery by displaying higher velocity of left ventricular relaxation and improved contractility. When added to the perfusate of isolated hearts, post-infarction circulating vesicles induced significantly more cell death in adult cardiomyocytes, as assessed by cTnI release, comparing to circulating vesicles isolated from healthy (non-infarcted) rats. In vitro inflammatory extracellular vesicles induce cell death by driving nuclear translocation of NF-κB into nuclei of cardiomyocytes. Conclusion: Our data suggest that targeting circulating extracellular vesicles during the acute phase of myocardial infarction may offer an effective therapeutic approach to preserve function of ischemic heart

Extracellular vesicles from human cardiac progenitor cells inhibit cardiomyocyte apoptosis and improve cardiac function after myocardial infarction.

AIMS: Recent evidence suggests that cardiac progenitor cells (CPCs) may improve cardiac function after injury. The underlying mechanisms are indirect, but their mediators remain unidentified. Exosomes and other secreted membrane vesicles, hereafter collectively referred to as extracellular vesicles (EVs), act as paracrine signalling mediators. Here, we report that EVs secreted by human CPCs are crucial cardioprotective agents. METHODS AND RESULTS: CPCs were derived from atrial appendage explants from patients who underwent heart valve surgery. CPC-conditioned medium (CM) inhibited apoptosis in mouse HL-1 cardiomyocytic cells, while enhancing tube formation in human umbilical vein endothelial cells. These effects were abrogated by depleting CM of EVs. They were reproduced by EVs secreted by CPCs, but not by those secreted by human dermal fibroblasts. Transmission electron microscopy and nanoparticle tracking analysis showed most EVs to be 30-90 nm in diameter, the size of exosomes, although smaller and larger vesicles were also present. MicroRNAs most highly enriched in EVs secreted by CPCs compared with fibroblasts included miR-210, miR-132, and miR-146a-3p. miR-210 down-regulated its known targets, ephrin A3 and PTP1b, inhibiting apoptosis in cardiomyocytic cells. miR-132 down-regulated its target, RasGAP-p120, enhancing tube formation in endothelial cells. Infarcted hearts injected with EVs from CPCs, but not from fibroblasts, exhibited less cardiomyocyte apoptosis, enhanced angiogenesis, and improved LV ejection fraction (0.8 ± 6.8 vs. -21.3 ± 4.5%; P < 0.05) compared with those injected with control medium. CONCLUSION: EVs are the active component of the paracrine secretion by human CPCs. As a cell-free approach, EVs could circumvent many of the limitations of cell transplantation

Exosomal Expression of CXCR4 Targets Cardioprotective Vesicles to Myocardial Infarction and Improves Outcome after Systemic Administration.

Cell therapy has been evaluated to enhance heart function after injury. Delivered cells mostly act via paracrine mechanisms, including secreted growth factors, cytokines, and vesicles, such as exosomes (Exo). Intramyocardial injection of cardiac-resident progenitor cells (CPC)-derived Exo reduced scarring and improved cardiac function after myocardial infarction in rats. Here, we explore a clinically relevant approach to enhance the homing process to cardiomyocytes (CM), which is crucial for therapeutic efficacy upon systemic delivery of Exo. By overexpressing exosomal CXCR4, we increased the efficacy of plasmatic injection of cardioprotective Exo-CPC by increasing their bioavailability to ischemic hearts. Intravenous injection of Exo <sup>CXCR4</sup> significantly reduced infarct size and improved left ventricle ejection fraction at 4 weeks compared to Exo <sup>CTRL</sup> (p < 0.01). Hemodynamic measurements showed that Exo <sup>CXCR4</sup> improved dp/dt min, as compared to Exo <sup>CTRL</sup> and PBS group. In vitro, Exo <sup>CXCR4</sup> was more bioactive than Exo <sup>CTRL</sup> in preventing CM death. This in vitro effect was independent from SDF-1α, as shown by using AMD3100 as specific CXCR4 antagonist. We showed, for the first time, that systemic administration of Exo derived from CXCR4-overexpressing CPC improves heart function in a rat model of ischemia reperfusion injury These data represent a substantial step toward clinical application of Exo-based therapeutics in cardiovascular disease

Cooking pots, tableware, and the changing sounds of sociability in Italy, 1300–1700

This article considers how the sounds produced by the preparation and consumption of meals in Italy changed between around 1300 and 1700. It argues that by focusing on sound, and by using ecological approaches, we can rediscover obscured connections between different categories of material objects. By examining material and textual evidence for three categories of objects associated with cooking and dining – metalwork, ceramics, and glass – the article traces changes in the material cultures of kitchen and table, and the clear impact these changes had on domestic soundscapes. It considers these sound-producing objects as agents of social interaction, exploring the social relationships they constructed, and the role sound played in those relationships. The article then focuses on the practices of cooking and dining, and the way they shaped the sound of objects. Finally, the article situates objects and social practices within the spatial context of the home, tracing an increasing impetus to manage and control specific types of sound in relation to gender. In the discourse on hospitality, noise came to signify a badly-managed, and therefore morally dubious, household, while silence testified to decorous and authoritative domestic management

Studio pilota sull'impiego di cellule CD133+ immunoselezionate per il trattamento di vasculopatie periferiche.

Background: l’ischemia critica degli arti inferiori (CLI) costituisce uno stato avanzato di arteriopatia obliterante periferica caratterizzata dalla presenza di dolore a riposo associato a lesioni ulcerative la cui prognosi è severa: ≈50% dei pazienti va incontro ad amputazione ad 1 anno dalla diagnosi. Per tali motivi esiste la necessità di nuove terapie per i pazienti “no-option”. In modelli animali l’iniezione intramuscolare di cellule progenitrici endoteliali (EPC) CD133+ circolanti di origine midollare favorisce i processi di vasculogenesi e neo-angiogenesi. Obiettivo del nostro studio era dimostrare la fattibilità di mobilizzare, raccogliere, selezionare EPC da sangue periferico dopo stimolazione con fattori di crescita ed iniettarle per via intramuscolare in pazienti affetti da CLI severa. Metodi: i soggetti arruolati erano pazienti “no-option” considerati candidati all’amputazione dell’arto. Le EPC sono state mobilizzate con GCSF e le CD133+ selezionate utilizzando il sistema “Miltenyi Biotec Clinimacs”. Quantificazione e caratterizzazione delle CD133+ sono state condotte mediante citometria a flusso (FACS). È stato inoltre valutato il numero di colonie di Hill ottenute da cellule mononucleate (MNC) degli stessi pazienti pre-mobilizzazione e al momento della raccolta leucaferetica e confrontato con quello ottenuto da donatori sani. Le CD133+ sono state iniettate nei muscoli dell’arto inferiore interessato dalle lesioni (50/60 iniezioni), in regime di anestesia locale. Un’aliquota di cellule è stata infine utilizzata per saggi di clonogenicità. I pazienti sono stati sottoposti ad ankle-brachial index (ABI) test ed ecocontrastografia in condizioni basali e dopo 3, 6, 9, e 12 mesi dal trapianto per valutare lo stato di perfusione dei tessuti. Il confronto è stato quantizzato mediante calcolo dell’area sotto la curva (AUC). Risultati: abbiamo arruolato un totale di 8 pazienti (6 uomini e 2 donne). La procedura è stata ben tollerata e non si sono osservati eventi avversi. L’analisi FACS ha dimostrato una variabilità di mobilizzazione interindividuale come atteso. Al follow-up a 12 mesi si è registrato un netto miglioramento della circolazione periferica rispetto al basale in 6 pazienti su 8 come dimostrato dalla misurazione ABI e della AUC. Nei 6 responders dopo 3 mesi il dolore era scomparso e la ferita rimarginata. I 2 pazienti non responsivi all’infusione di CD133+ erano entrambi diabetici. Il numero di colonie di Hill era significativamente inferiore nei pazienti rispetto ai controlli sani, ma dopo stimolazione con G-CSF i due valori diventavano paragonabili; nei 2 pazienti in cui la terapia non ha prodotto giovamento, il numero di colonie post G-CSF è rimasto invariato. Conclusioni: i risultati dello studio pilota condotto, indicano che le procedure di mobilizzazione, raccolta leucaferetica ed iniezione intramuscolare di CD133+ in pazienti affetti da CLI sono fattibili e sicure. I nostri dati sembrano inoltre indicare una buona efficacia della terapia. L’analisi della conta delle colonie di Hill indica che la scarsa mobilizzazione di cellule con proprietà clonogeniche potrebbe spiegare la mancanza di risposta nei pazienti diabetici

Purified exosomes from human cardiac progenitor cells improve cardiac function after myocardial infarction in vivo

Background: Cardiac progenitor cell (CPC) transplantation improves cardiac function after myocardial infarction. Exosomes (Exo) are secreted nano-sized membrane vesicles that act as intercellular carriers of proteins and RNAs. Here, we investigated the role of Exo in the paracrine secretion by human CPCs (Exo-CPC), as compared to those from normal human fibroblasts (Exo-NHDF). Methods: CPC were derived from atrial explants of patients who underwent heart valve surgery. Exo were precipitated with ExoQuickTM. Cytoprotective and proangiogenic assays were performed with mouse cardiomyocytes (CM) and human endothelial cells (HUVEC), respectively. The in vivo effects of Exo- CPC and Exo-NHDF were tested in a rat model of acute myocardial infarction. Results: In vitro, Exo- CPC significantly reduced starvation-induced CM apoptosis by 59% (p<0.05), whereas Exo-NHDF did not. They also stimulated tube formation by HUVEC in Matrigel (total tube length 7912.38±1076 a.u.) compared with Exo-NHDF (total tube length 5095.25±747 a.u.; p<0.05). When injected into infarcted rat hearts, Exo-CPC, but not Exo-NHDF, significantly reduced infarct scar (0.58±0.08 a.u. vs. Exo-NHDF, 0.76±0.01 a.u.; p<0.05) while increasingnew vessel formation (84±13 vs. 34±5 vessels/mm2; p<0.01). Left ventricular ejection fraction decreased from baseline to 7 days in hearts injected with PBS (-21.3±4.5%) or Exo-NHDF (-12±6.3%) but was maintained in those injected with Exo-CPC (+0.8±6.8%; p<0.05 vs. PBS). Conclusions: Exo accounts for proangiogenic and antiapoptotic activities of human CPCs. Exo-CPC injected into infarcted hearts improves cardiac function early after MI. As a cell-free product, Exo-CPC has a potential for circumventing many of the limitations of cell therapy for cardiac repair

γ-Irradiated cord blood MNCs: Different paracrine effects on mature and progenitor endothelial cells

Cell-based therapies have been employed to promote neovascularization mainly through the release of paracrine factors inhibiting apoptosis and supporting migration and proliferation of resident differentiated cells. We tested in vitro pro-angiogenic effects of apoptotic cord blood-derived mononuclear cells (CB-MNCs) and their conditioned medium (CM) on mature endothelial cells (HUVECs) and peripheral blood-derived endothelial progenitor cells (ECFCs).CB-MNCs were γ-irradiated to induce apoptosis and cultured for 72. h to obtain the release of CM. MNCs viability, evaluated by flow cytometry, decreased progressively after γ-irradiation reaching 41% at 72. h. γ-Irradiated MNCs (γMNCs) released increasing amounts of EGF, PDGF-AB and VEGF in their CM over time, as assessed by ELISA. γ-MNCs and their CM enhanced capillary-like network formation (in a dose-dependent and time-persistent manner), proliferation and migration of HUVECs in vitro, while they primed capillary-like network formation (dose-independent and not time-persistent) and induced migration but did not support proliferation of ECFCs. Our data support the hypothesis of paracrine mechanism as prevalent in regenerative medicine and demonstrate the efficacy of MNCs secretome in inducing neovascularization. To our knowledge, this is the first paper highlighting differential pro-angiogenic effects of CM on mature and progenitor endothelial cells, adding a tile in the understanding of mechanisms involved in neovascularization. © 2014 Elsevier Inc