Impact of Isolation Methods on Extracellular Vesicle Functionality In Vitro and In Vivo.

This study compares the impact of two isolation methods, ultracentrifugation (UC) and size exclusion chromatography (SEC), on small extracellular vesicles (sEVs) from primary human cardiac mesenchymal-derived progenitor cells (CPCs). sEV_UC and sEV_SEC exhibit similar size, marker expression, and miRNA cargo, but sEV_UC contains notably higher total protein levels. In vitro assays show that sEV_UC, despite an equal particle count, induces more robust ERK phosphorylation, cytoprotection, and proliferation in iPS-derived cardiomyocytes (iPS-CMs) compared to sEV_SEC. sEV_UC also contains elevated periostin (POSTN) protein levels, resulting in enhanced focal adhesion kinase (FAK) phosphorylation in iPS-CMs. Importantly, this effect persists with treatment with soluble free-sEV protein fraction from SEC (Prote_SEC), indicating that free proteins like POSTN in sEV_UC enhance FAK phosphorylation. In vivo, sEV contamination with soluble proteins doesn't affect cardiac targeting or FAK phosphorylation, underscoring the intrinsic tissue targeting properties of sEV. These findings emphasize the need for standardized sEV isolation methods, as the choice of method can impact experimental outcomes, particularly in vitro.

Circulating GDF11 exacerbates myocardial injury in mice and associates with increased infarct size in humans.

AIMS: The heart rejuvenating effects of circulating growth differentiation factor 11 (GDF11), a transforming growth factor-ß superfamily member that shares 90% homology with myostatin (MSTN), remains controversial. Here, we aimed to probe the role of GDF11 in acute myocardial infarction (MI), a frequent cause of heart failure and premature death during ageing. METHODS AND RESULTS: In contrast to endogenous Mstn, myocardial Gdf11 declined during the course of ageing and was particularly reduced following ischaemia/reperfusion (I/R) injury, suggesting a therapeutic potential of GDF11 signalling in MI. Unexpectedly, boosting systemic Gdf11 by recombinant GDF11 delivery (0.1 mg/kg body weight over 30 days) prior to myocardial I/R augmented myocardial infarct size in C57BL/6 mice irrespective of their age, predominantly by accelerating pro-apoptotic signalling. While intrinsic cardioprotective signalling pathways remained unaffected by high circulating GDF11, targeted transcriptomics and immunomapping studies focusing on GDF11-associated downstream targets revealed attenuated Nkx2-5 expression confined to CD105-expressing cells, with pro-apoptotic activity, as assessed by caspase-3 levels, being particularly pronounced in adjacent cells, suggesting an indirect effect. By harnessing a highly specific and validated liquid chromatography-tandem mass spectrometry-based assay, we show that in prospectively recruited patients with MI circulating GDF11 but not MSTN levels incline with age. Moreover, GDF11 levels were particularly elevated in those at high risk for adverse outcomes following the acute event, with circulating GDF11 emerging as an independent predictor of myocardial infarct size, as estimated by standardized peak creatine kinase-MB levels. CONCLUSION: Our data challenge the initially reported heart rejuvenating effects of circulating GDF11 and suggest that high levels of systemic GDF11 exacerbate myocardial injury in mice and humans alike. Persistently high GDF11 levels during ageing may contribute to the age-dependent loss of cardioprotective mechanisms and thus poor outcomes of elderly patients following acute MI.

Plasma-derived extracellular vesicles released after endurance exercise exert cardioprotective activity through the activation of antioxidant pathways.

Cardiovascular diseases (CVD) can cause various conditions, including an increase in reactive oxygen species (ROS) levels that can decrease nitric oxide (NO) availability and promote vasoconstriction, leading to arterial hypertension. Physical exercise (PE) has been found to be protective against CVD by helping to maintain redox homeostasis through a decrease in ROS levels, achieved by increased expression of antioxidant enzymes (AOEs) and modulation of heat shock proteins (HSPs). Extracellular vesicles (EVs) circulating in the body are a major source of regulatory signals, including proteins and nucleic acids. Interestingly, the cardioprotective role of EVs released after PE has not been fully described. The aim of this study was to investigate the role of circulating EVs, obtained through Size Exclusion Chromatography (SEC) of plasma samples from healthy young males (age: 26.95 ± 3.07; estimated maximum oxygen consumption rate (VO2max): 51.22 ± 4.85 (mL/kg/min)) at basal level (Pre_EVs) and immediately after a single bout of endurance exercise (30' treadmill, 70% heart rate (HR) -Post_EVs). Gene ontology (GO) analysis of proteomic data from isolated EVs, revealed enrichment in proteins endowed with catalytic activity in Post_EVs, compare to Pre_EVs, with MAP2K1 being the most significantly upregulated protein. Enzymatic assays on EVs derived from Pre and Post samples showed increment in Glutathione Reductase (GR) and Catalase (CAT) activity in Post_EVs. At functional level, Post_EVs, but not Pre_EVs, enhanced the activity of antioxidant enzymes (AOEs) and reduced oxidative damage accumulation in treated human iPS-derived cardiomyocytes (hCM) at basal level and under stress conditions (Hydrogen Peroxide (H2O2) treatment), resulting in a global cardioprotective effect. In conclusion, our data demonstrated, for the first time, that a single 30-min endurance exercise is able to alter the cargo of circulating EVs, resulting in cardioprotective effect through antioxidant activity.

Erratum: Inflammatory extracellular vesicles prompt heart dysfunction via TLR4-dependent NF-κB activation: Erratum.

[This corrects the article DOI: 10.7150/thno.39072.].

Stress-induced premature senescence is associated with a prolonged QT interval and recapitulates features of cardiac aging.

Rationale: Aging in the heart is a gradual process, involving continuous changes in cardiovascular cells, including cardiomyocytes (CMs), namely cellular senescence. These changes finally lead to adverse organ remodeling and resulting in heart failure. This study exploits CMs from human induced pluripotent stem cells (iCMs) as a tool to model and characterize mechanisms involved in aging. Methods and Results: Human somatic cells were reprogrammed into human induced pluripotent stem cells and subsequently differentiated in iCMs. A senescent-like phenotype (SenCMs) was induced by short exposure (3 hours) to doxorubicin (Dox) at the sub-lethal concentration of 0.2 µM. Dox treatment induced expression of cyclin-dependent kinase inhibitors p21 and p16, and increased positivity to senescence-associated beta-galactosidase when compared to untreated iCMs. SenCMs showed increased oxidative stress, alteration in mitochondrial morphology and depolarized mitochondrial membrane potential, which resulted in decreased ATP production. Functionally, when compared to iCMs, SenCMs showed, prolonged multicellular QTc and single cell APD, with increased APD variability and delayed afterdepolarizations (DADs) incidence, two well-known arrhythmogenic indexes. These effects were largely ascribable to augmented late sodium current (INaL) and reduced delayed rectifier potassium current (Ikr). Moreover sarcoplasmic reticulum (SR) Ca2+ content was reduced because of downregulated SERCA2 and increased RyR2-mediated Ca2+ leak. Electrical and intracellular Ca2+ alterations were mostly justified by increased CaMKII activity in SenCMs. Finally, SenCMs phenotype was furtherly confirmed by analyzing physiological aging in CMs isolated from old mice in comparison to young ones. Conclusions: Overall, we showed that SenCMs recapitulate the phenotype of aged primary CMs in terms of senescence markers, electrical and Ca2+ handling properties and metabolic features. Thus, Dox-induced SenCMs can be considered a novel in vitro platform to study aging mechanisms and to envision cardiac specific anti-aging approach in humans.

De novo DNA methylation induced by circulating extracellular vesicles from acute coronary syndrome patients.

BACKGROUND AND AIMS: DNA methylation is associated with gene silencing, but its clinical role in cardiovascular diseases (CVDs) remains to be elucidated. We hypothesized that extracellular vesicles (EVs) may carry epigenetic changes, showing themselves as a potentially valuable non-invasive diagnostic liquid biopsy. We isolated and characterized circulating EVs of acute coronary syndrome (ACS) patients and assessed their role on DNA methylation in epigenetic modifications. METHODS: EVs were recovered from plasma of 19 ACS patients and 50 healthy subjects (HS). Flow cytometry, qRT-PCR, and Western blot (WB) were performed to evaluate both intra-vesicular and intra-cellular signals. ShinyGO, PANTHER, and STRING tools were used to perform GO and PPI network analyses. RESULTS: ACS-derived EVs showed increased levels of DNA methyltransferases (DNMTs) (p<0.001) and Ten-eleven translocation (TET) genes reduction. Specifically, de novo methylation transcripts, as DNMT3A and DNMT3B, were significantly increased in plasma ACS-EVs. DNA methylation analysis on PBMCs from healthy donors treated with HS- and ACS-derived EVs showed an important role of DNMTs carried by EVs. PPI network analysis evidenced that ACS-EVs induced changes in PBMC methylome. In the most enriched subnetwork, the hub gene SRC was connected to NOTCH1, FOXO3, CDC42, IKBKG, RXRA, DGKG, BAIAP2 genes that were showed to have many molecular effects on various cell types into onset of several CVDs. Modulation in gene expression after ACS-EVs treatment was confirmed for SRC, NOTCH1, FOXO3, RXRA, DGKG and BAIAP2 (p<0.05). CONCLUSIONS: Our data showed an important role for ACS-derived EVs in gene expression modulation through de novo DNA methylation signals, and modulating signalling pathways in target cells.

Risk stratification of patients with SARS-CoV-2 by tissue factor expression in circulating extracellular vesicles.

Inflammatory response following SARS-CoV-2 infection results in substantial increase of amounts of intravascular pro-coagulant extracellular vesicles (EVs) expressing tissue factor (CD142) on their surface. CD142-EV turned out to be useful as diagnostic biomarker in COVID-19 patients. Here we aimed at studying the prognostic capacity of CD142-EV in SARS-CoV-2 infection. Expression of CD142-EV was evaluated in 261 subjects admitted to hospital for pneumonia and with a positive molecular test for SARS-CoV-2. The study population consisted of a discovery cohort of selected patients (n = 60) and an independent validation cohort including unselected consecutive enrolled patients (n = 201). CD142-EV levels were correlated with post-hospitalization course of the disease and compared to the clinically available 4C Mortality Score as referral. CD142-EV showed a reliable performance to predict patient prognosis in the discovery cohort (AUC = 0.906) with an accuracy of 81.7%, that was confirmed in the validation cohort (AUC = 0.736). Kaplan-Meier curves highlighted a high discrimination power in unselected subjects with CD142-EV being able to stratify the majority of patients according to their prognosis. We obtained a comparable accuracy, being not inferior in terms of prediction of patients' prognosis and risk of mortality, with 4C Mortality Score. The expression of surface vesicular CD142 and its reliability as prognostic marker was technically validated using different immunocapture strategies and assays. The detection of CD142 on EV surface gains considerable interest as risk stratification tool to support clinical decision making in COVID-19.

Microvesicles released from activated CD4+ T cells alter microvascular endothelial cell function.

BACKGROUND: Microvesicles are vesicles shed by plasma membranes following cell activation and apoptosis. The role of lymphocyte-derived microvesicles in endothelial function remains poorly understood. METHODS: CD4+ T cells isolated from peripheral blood of healthy human donors were stimulated using anti-CD3/anti-CD28-coated beads. Proteomic profiling of microvesicles was performed using linear discriminant analysis (LDA) from activated T cells (MV.Act) and nonactivated T cells (MV.NAct). In addition, data processing analysis was performed using MaxQUANT workflow. Differentially expressed proteins found in MV.Act or MV.NAct samples with identification frequency = 100%, which were selected by both LDA (p < .01) and MaxQUANT (p < .01) workflows, were defined as "high-confidence" differentially expressed proteins. Functional effects of MV.Act on human primary microvascular endothelial cells were analysed. RESULTS: T cells released large amounts of microvesicles upon stimulation. Proteomic profiling of microvesicles using LDA identified 2279 proteins (n = 2110 and n = 851 proteins in MV.Act and MV.NAct, respectively). Protein-protein interaction network models reconstructed from both differentially expressed proteins (n = 594; LDA p ≤ .01) and "high-confidence" differentially expressed proteins (n = 98; p ≤ .01) revealed that MV.Act were enriched with proteins related to immune responses, protein translation, cytoskeleton organisation and TNFα-induced apoptosis. For instance, MV.Act were highly enriched with IFN-γ, a key proinflammatory pathway related to effector CD4+ T cells. Endothelial cell incubation with MV.Act induced superoxide generation, apoptosis, endothelial wound healing impairment and endothelial monolayer barrier disruption. CONCLUSIONS: T cell receptor-mediated activation of CD4+ T cells stimulates the release of microvesicles enriched with proteins involved in immune responses, inflammation and apoptosis. T cell-derived microvesicles alter microvascular endothelial function and barrier permeability, potentially promoting tissue inflammation.

Supervised and unsupervised learning to define the cardiovascular risk of patients according to an extracellular vesicle molecular signature.

Cardiovascular (CV) disease represents the most common cause of death in developed countries. Risk assessment is highly relevant to intervene at individual level and implement prevention strategies. Circulating extracellular vesicles (EVs) are involved in the development and progression of CV diseases and are considered promising biomarkers. We aimed at identifying an EV signature to improve the stratification of patients according to CV risk and likelihood to develop fatal CV events. EVs were characterized by nanoparticle tracking analysis and flow cytometry for a standardized panel of 37 surface antigens in a cross-sectional multicenter cohort (n = 486). CV profile was defined by presence of different indicators (age, sex, body mass index, hypertension, hyperlipidemia, diabetes, coronary artery disease, cardiac heart failure, chronic kidney disease, smoking habit, organ damage) and according to the 10-year risk of fatal CV events estimated using SCORE charts of European Society of Cardiology. By combining expression levels of EV antigens using unsupervised learning, patients were classified into 3 clusters: Cluster-I (n = 288), Cluster-II (n = 83), Cluster-III (n = 30). A separate analysis was conducted on patients displaying acute CV events (n = 82). Prevalence of hypertension, diabetes, chronic heart failure, and organ damage (defined as left ventricular hypertrophy and/or microalbuminuria) increased progressively from Cluster-I to Cluster-III. Several EV antigens, including markers for platelets (CD41b-CD42a-CD62P), leukocytes (CD1c-CD2-CD3-CD4-CD8-CD14-CD19-CD20-CD25-CD40-CD45-CD69-CD86), and endothelium (CD31-CD105) were independently associated with CV risk indicators and correlated to age, blood pressure, glucometabolic profile, renal function, and SCORE risk. EV profiling, obtained from minimally invasive blood sampling, allows accurate patient stratification according to CV risk profile.

Extracellular Vesicle Surface Markers as a Diagnostic Tool in Transient Ischemic Attacks.

Background and Purpose: Extracellular vesicles (EVs) are promising biomarkers for cerebral ischemic diseases, but not systematically tested in patients with transient ischemic attacks (TIAs). We aimed at (1) investigating the profile of EV-surface antigens in patients with symptoms suspicious for TIA; (2) developing and validating a predictive model for TIA diagnosis based on a specific EV-surface antigen profile. Methods: We analyzed 40 subjects with symptoms suspicious for TIA and 20 healthy controls from a training cohort. An independent cohort of 28 subjects served as external validation. Patients were stratified according to likelihood of having a real ischemic event using the Precise Diagnostic Score, defined as: unlikely (score 0­1), possible-probable (score 2­3), or very likely (score 4­8). Serum vesicles were quantified by nanoparticle tracking analysis and EV-surface antigen profile characterized by multiplex flow cytometry. Results: EV concentration increased in patients with very likely or possible-probable TIA (P<0.05) compared with controls. Nanoparticle concentration was directly correlated with the Precise Diagnostic score (R=0.712; P<0.001). After EV immuno-capturing, CD8, CD2, CD62P, melanoma-associated chondroitin sulfate proteoglycan, CD42a, CD44, CD326, CD142, CD31, and CD14 were identified as discriminants between groups. Receiver operating characteristic curve analysis confirmed a reliable diagnostic performance for each of these markers taken individually and for a compound marker derived from their linear combinations (area under the curve, 0.851). Finally, a random forest model combining the expression levels of selected markers achieved an accuracy of 96% and 78.9% for discriminating patients with a very likely TIA, in the training and external validation cohort, respectively. Conclusions: The EV-surface antigen profile appears to be different in patients with transient symptoms adjudicated to be very likely caused by brain ischemia compared with patients whose symptoms were less likely to due to brain ischemia. We propose an algorithm based on an EV-surface-antigen specific signature that might aid in the recognition of TIA.

Characterization of Circulating Extracellular Vesicle Surface Antigens in Patients With Primary Aldosteronism.

[Figure: see text].

Circulating extracellular vesicles are endowed with enhanced procoagulant activity in SARS-CoV-2 infection.

BACKGROUND: 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. METHODS: 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. FINDINGS: 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. INTERPRETATION: 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. FUNDING: Cardiocentro Ticino Institute, Ente ospedaliero Cantonale, Lugano-Switzerland.

Structural and Electrophysiological Changes in a Model of Cardiotoxicity Induced by Anthracycline Combined With Trastuzumab.

BACKGROUND: Combined treatment with anthracyclines (e.g., doxorubicin; Dox) and trastuzumab (Trz), a humanized anti-human epidermal growth factor receptor 2 (HER2; ErbB2) antibody, in patients with HER2-positive cancer is limited by cardiotoxicity, as manifested by contractile dysfunction and arrhythmia. The respective roles of the two agents in the cardiotoxicity of the combined therapy are incompletely understood. OBJECTIVE: To assess cardiac performance, T-tubule organization, electrophysiological changes and intracellular Ca2+ handling in cardiac myocytes (CMs) using an in vivo rat model of Dox/Trz-related cardiotoxicity. METHODS AND RESULTS: Adult rats received 6 doses of either Dox or Trz, or the two agents sequentially. Dox-mediated left ventricular (LV) dysfunction was aggravated by Trz administration. Dox treatment, but not Trz, induced T-tubule disarray. Moreover, Dox, but not Trz monotherapy, induced prolonged action potential duration (APD), increased incidence of delayed afterdepolarizations (DADs) and beat-to-beat variability of repolarization (BVR), and slower Ca2+ transient decay. Although APD, DADs, BVR and Ca2+ transient decay recovered over time after the cessation of Dox treatment, subsequent Trz administration exacerbated these abnormalities. Trz, but not Dox, reduced Ca2+ transient amplitude and SR Ca2+ content, although only Dox treatment was associated with SERCA downregulation. Finally, Dox treatment increased Ca2+ spark frequency, resting Ca2+ waves, sarcoplasmic reticulum (SR) Ca2+ leak, and long-lasting Ca2+ release events (so-called Ca2+ "embers"), partially reproduced by Trz treatment. CONCLUSION: These results suggest that in vivo Dox but not Trz administration causes T-tubule disarray and pronounced changes in electrical activity of CMs. While adaptive changes may account for normal AP shape and reduced DADs late after Dox administration, subsequent Trz administration interferes with such adaptive changes. Intracellular Ca2+ handling was differently affected by Dox and Trz treatment, leading to SR instability in both cases. These findings illustrate the specific roles of Dox and Trz, and their interactions in cardiotoxicity and arrhythmogenicity.

An exosomal-carried short periostin isoform induces cardiomyocyte proliferation.

Although a small number of cardiomyocytes may reenter the cell cycle after injury, the adult mammalian heart is incapable of a robust cardiomyocyte proliferation. Periostin, a secreted extracellular matrix protein, has been implicated as a regulator of cardiomyocyte proliferation; however, this role remains controversial. Alternative splicing of the human periostin gene results in 6 isoforms lacking sequences between exons 17 and 21, in addition to full-length periostin. We previously showed that exosomes (Exo) secreted by human cardiac explant-derived progenitor cells (CPC) carried periostin. Here, we aimed to investigate their cell cycle activity. Methods: CPC were derived as the cellular outgrowth of ex vivo cultured cardiac atrial explants. Exo were purified from CPC conditioned medium using size exclusion chromatography. Exosomal periostin was analyzed by Western blotting using a pair of antibodies (one raised against aa 537-836, and one raised against amino acids mapping at exon 17 of human periostin), by ELISA, and by cryo-EM with immune-gold labeling. Cell cycle activity was assessed in neonatal rat cardiomyocytes, in human induced pluripotent stem cell (iPS)-derived cardiomyocytes, and in adult rat cardiomyocytes after myocardial infarction. The role of periostin in cell cycle activity was investigated by transfecting donor CPC with a siRNA against this protein. Results: Periostin expression in CPC-secreted exosomes was detected using the antibody raised against aa 537-836 of the human protein, but not with the exon 17-specific antibody, consistent with an isoform lacking exon 17. Periostin was visualized on vesicle surfaces by cryo-EM and immune-gold labeling. CPC-derived exosomes induced cell proliferation in neonatal rat cardiomyocytes both in vitro and in vivo, in human iPS-derived cardiomyocytes, and in adult rat cardiomyocytes after myocardial infarction. Exo promoted phosphorylation of focal adhesion kinase (FAK), actin polymerization, and nuclear translocation of Yes-associated protein (YAP) in cardiomyocytes. Knocking down of periostin or YAP, or blocking FAK phosphorylation with PF-573228 nullified Exo-induced proliferation. A truncated human periostin peptide (aa 22-669), but not recombinant human full-length periostin, mimicked the pro-proliferative activity of exosomes. Conclusions: Our results show, for the first time, that CPC-secreted exosomes promote cardiomyocyte cell cycle-reentry via a short periostin isoform expressed on their surfaces, whereas recombinant full-length periostin does not. These findings highlight isoform-specific roles of periostin in cardiomyocyte proliferation.

Immune profiling of plasma-derived extracellular vesicles identifies Parkinson disease.

OBJECTIVE: To develop a diagnostic model based on plasma-derived extracellular vesicle (EV) subpopulations in Parkinson disease (PD) and atypical parkinsonism (AP), we applied an innovative flow cytometric multiplex bead-based platform. METHODS: Plasma-derived EVs were isolated from PD, matched healthy controls, multiple system atrophy (MSA), and AP with tauopathies (AP-Tau). The expression levels of 37 EV surface markers were measured by flow cytometry and correlated with clinical scales. A diagnostic model based on EV surface markers expression was built via supervised machine learning algorithms and validated in an external cohort. RESULTS: Distinctive pools of EV surface markers related to inflammatory and immune cells stratified patients according to the clinical diagnosis. PD and MSA displayed a greater pool of overexpressed immune markers, suggesting a different immune dysregulation in PD and MSA vs AP-Tau. The receiver operating characteristic curve analysis of a compound EV marker showed optimal diagnostic performance for PD (area under the curve [AUC] 0.908; sensitivity 96.3%, specificity 78.9%) and MSA (AUC 0.974; sensitivity 100%, specificity 94.7%) and good accuracy for AP-Tau (AUC 0.718; sensitivity 77.8%, specificity 89.5%). A diagnostic model based on EV marker expression correctly classified 88.9% of patients with reliable diagnostic performance after internal and external validations. CONCLUSIONS: Immune profiling of plasmatic EVs represents a crucial step toward the identification of biomarkers of disease for PD and AP.

Exosomes: Beyond stem cells for cardiac protection and repair.

The adult human heart has limited regenerative capacity; hence, stem cell therapy has been investigated as a potential approach for cardiac repair. However, a large part of the benefit of the injection of stem and progenitor cells into injured hearts is mediated by secreted factors. Exosomes-nano-sized secreted extracellular vesicles of endosomal origin-have emerged as key signaling organelles in intercellular communication, and are now viewed as the key regenerative constituent of the secretome of stem and progenitor cells. Exosomes released from mesenchymal stem cells, cardiac-derived progenitor cells, embryonic stem cells, induced pluripotent stem cells (iPSCs), and iPSC-derived cardiomyocytes exhibit cardioprotective, immunomodulatory, and reparative abilities. This concise review discusses the therapeutic benefit of exosomes secreted by stem and progenitor cells in preclinical models of ischemic heart disease.

Circulating extracellular vesicles as non-invasive biomarker of rejection in heart transplant.

BACKGROUND: Circulating extracellular vesicles (EVs) are raising considerable interest as a non-invasive diagnostic tool, as they are easily detectable in biologic fluids and contain a specific set of nucleic acids, proteins, and lipids reflecting pathophysiologic conditions. We aimed to investigate differences in plasma-derived EV surface protein profiles as a biomarker to be used in combination with endomyocardial biopsies (EMBs) for the diagnosis of allograft rejection. METHODS: Plasma was collected from 90 patients (53 training cohort, 37 validation cohort) before EMB. EV concentration was assessed by nanoparticle tracking analysis. EV surface antigens were measured using a multiplex flow cytometry assay composed of 37 fluorescently labeled capture bead populations coated with specific antibodies directed against respective EV surface epitopes. RESULTS: The concentration of EVs was significantly increased and their diameter decreased in patients undergoing rejection as compared with negative ones. The trend was highly significant for both antibody-mediated rejection and acute cellular rejection (p < 0.001). Among EV surface markers, CD3, CD2, ROR1, SSEA-4, human leukocyte antigen (HLA)-I, and CD41b were identified as discriminants between controls and acute cellular rejection, whereas HLA-II, CD326, CD19, CD25, CD20, ROR1, SSEA-4, HLA-I, and CD41b discriminated controls from patients with antibody-mediated rejection. Receiver operating characteristics curves confirmed a reliable diagnostic performance for each single marker (area under the curve range, 0.727-0.939). According to differential EV-marker expression, a diagnostic model was built and validated in an external cohort of patients. Our model was able to distinguish patients undergoing rejection from those without rejection. The accuracy at validation in an independent external cohort reached 86.5%. Its application for patient management has the potential to reduce the number of EMBs. Further studies in a higher number of patients are required to validate this approach for clinical purposes. CONCLUSIONS: Circulating EVs are highly promising as a new tool to characterize cardiac allograft rejection and to be complementary to EMB monitoring.

An extracellular vesicle epitope profile is associated with acute myocardial infarction.

The current standard biomarker for myocardial infarction (MI) is high-sensitive troponin. Although powerful in clinical setting, search for new markers is warranted as early diagnosis of MI is associated with improved outcomes. Extracellular vesicles (EVs) attracted considerable interest as new blood biomarkers. A training cohort used for diagnostic modelling included 30 patients with STEMI, 38 with stable angina (SA) and 30 matched-controls. Extracellular vesicle concentration was assessed by nanoparticle tracking analysis. Extracellular vesicle surface-epitopes were measured by flow cytometry. Diagnostic models were developed using machine learning algorithms and validated on an independent cohort of 80 patients. Serum EV concentration from STEMI patients was increased as compared to controls and SA. EV levels of CD62P, CD42a, CD41b, CD31 and CD40 increased in STEMI, and to a lesser extent in SA patients. An aggregate marker including EV concentration and CD62P/CD42a levels achieved non-inferiority to troponin, discriminating STEMI from controls (AUC = 0.969). A random forest model based on EV biomarkers discriminated the two groups with 100% accuracy. EV markers and RF model confirmed high diagnostic performance at validation. In conclusion, patients with acute MI or SA exhibit characteristic EV biomarker profiles. EV biomarkers hold great potential as early markers for the management of patients with MI.

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-1ß 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.