Macrophage-derived extracellular vesicles alter cardiac recovery and metabolism in a rat heart model of donation after circulatory death.

Conditions to which the cardiac graft is exposed during transplantation with donation after circulatory death (DCD) can trigger the recruitment of macrophages that are either unpolarized (M0) or pro-inflammatory (M1) as well as the release of extracellular vesicles (EV). We aimed to characterize the effects of M0 and M1 macrophage-derived EV administration on post-ischaemic functional recovery and glucose metabolism using an isolated rat heart model of DCD. Isolated rat hearts were subjected to 20 min aerobic perfusion, followed by 27 min global, warm ischaemia or continued aerobic perfusion and 60 min reperfusion with or without intravascular administration of EV. Four experimental groups were compared: (1) no ischaemia, no EV; (2) ischaemia, no EV; (3) ischaemia with M0-macrophage-dervied EV; (4) ischaemia with M1-macrophage-derived EV. Post-ischaemic ventricular and metabolic recovery were evaluated. During reperfusion, ventricular function was decreased in untreated ischaemic and M1-EV hearts, but not in M0-EV hearts, compared to non-ischaemic hearts (p < 0.05). In parallel with the reduced functional recovery in M1-EV versus M0-EV ischaemic hearts, rates of glycolysis from exogenous glucose and oxidative metabolism tended to be lower, while rates of glycogenolysis and lactate release tended to be higher. EV from M0- and M1-macrophages differentially affect post-ischaemic cardiac recovery, potentially by altering glucose metabolism in a rat model of DCD. Targeted EV therapy may be a useful approach for modulating cardiac energy metabolism and optimizing graft quality in the setting of DCD.

Extracellular vesicles selective capture by peptide-functionalized hollow fiber membranes.

Recently, membrane devices and processes have been applied for the separation and concentration of subcellular components such as extracellular vesicles (EVs), which play a diagnostic and therapeutic role in many pathological conditions. However, the separation and isolation of specific EV populations from other components found in biological fluids is still challenging. Here, we developed a peptide-functionalized hollow fiber (HF) membrane module to achieve the separation and enrichment of highly pure EVs derived from the culture media of human cardiac progenitor cells. The strategy is based on the functionalization of PSf HF membrane module with BPt, a peptide sequence able to bind nanovesicles characterized by highly curved membranes. HF membranes were modified by a nanometric coating with a copoly azide polymer to limit non-specific interactions and to enable the conjugation with peptide ligand by click chemistry reaction. The BPt-functionalized module was integrated into a TFF process to facilitate the design, rationalization, and optimization of EV isolation. This integration combined size-based transport of species with specific membrane sensing ligands. The TFF integrated BPt-functionalized membrane module demonstrated the ability to selectively capture EVs with diameter < 200 nm into the lumen of fibers while effectively removing contaminants such as albumin. The captured and released EVs contain the common markers including CD63, CD81, CD9 and syntenin-1. Moreover, they maintained a round shape morphology and structural integrity highlighting that this approach enables EVs concentration and purification with low shear stress. Additionally, it achieved the removal of contaminants such as albumin with high reliability and reproducibility, reaching a removal of 93%.

Injury minimization after myocardial infarction: focus on extracellular vesicles.

Despite improvements in clinical outcomes following acute myocardial infarction, mortality remains high, especially in patients with severely reduced left ventricular ejection fraction (LVEF <30%), emphasizing the need for effective cardioprotective strategies adjunctive to recanalization. Traditional cell therapy has shown equivocal success, shifting the focus to innovative cardioactive biologicals and cell mimetic therapies, particularly extracellular vesicles (EVs). EVs, as carriers of non-coding RNAs and other essential biomolecules, influence neighbouring and remote cell function in a paracrine manner. Compared to cell therapy, EVs possess several clinically advantageous traits, including stability, ease of storage (enabling off-the-shelf clinical readiness), and decreased immunogenicity. Allogeneic EVs from mesenchymal and/or cardiac stromal progenitor cells demonstrate safety and potential efficacy in preclinical settings. This review delves into the translational potential of EV-based therapeutic approaches, specifically highlighting findings from large-animal studies, and offers a synopsis of ongoing early-stage clinical trials in this domain.

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.

Particle profiling of EV-lipoprotein mixtures by AFM nanomechanical imaging.

The widely overlapping physicochemical properties of lipoproteins (LPs) and extracellular vesicles (EVs) represents one of the main obstacles for the isolation and characterization of these pervasive biogenic lipid nanoparticles. We herein present the application of an atomic force microscopy (AFM)-based quantitative morphometry assay to the rapid nanomechanical screening of mixed LPs and EVs samples. The method can determine the diameter and the mechanical stiffness of hundreds of individual nanometric objects within few hours. The obtained diameters are in quantitative accord with those measured via cryo-electron microscopy (cryo-EM); the assignment of specific nanomechanical readout to each object enables the simultaneous discrimination of co-isolated EVs and LPs even if they have overlapping size distributions. EVs and all classes of LPs are shown to be characterised by specific combinations of diameter and stiffness, thus making it possible to estimate their relative abundance in EV/LP mixed samples in terms of stoichiometric ratio, surface area and volume. As a side finding, we show how the mechanical behaviour of specific LP classes is correlated to distinctive structural features revealed by cryo-EM. The described approach is label-free, single-step and relatively quick to perform. Importantly, it can be used to analyse samples which prove very challenging to assess with several established techniques due to ensemble-averaging, low sensibility to small particles, or both, thus providing a very useful tool for quickly assessing the purity of EV/LP isolates including plasma- and serum-derived preparations.

Cardiomyocyte-targeting exosomes from sulforaphane-treated fibroblasts affords cardioprotection in infarcted rats.

BACKGROUND: Exosomes (EXOs), tiny extracellular vesicles that facilitate cell-cell communication, are being explored as a heart failure treatment, although the features of the cell source restrict their efficacy. Fibroblasts the most prevalent non-myocyte heart cells, release poor cardioprotective EXOs. A noninvasive method for manufacturing fibroblast-derived exosomes (F-EXOs) that target cardiomyocytes and slow cardiac remodeling is expected. As a cardioprotective isothiocyanate, sulforaphane (SFN)-induced F-EXOs (SFN-F-EXOs) should recapitulate its anti-remodeling properties. METHODS: Exosomes from low-dose SFN (3 µM/7 days)-treated NIH/3T3 murine cells were examined for number, size, and protein composition. Fluorescence microscopy, RT-qPCR, and western blot assessed cell size, oxidative stress, AcH4 levels, hypertrophic gene expression, and caspase-3 activation in angiotensin II (AngII)-stressed HL-1 murine cardiomyocytes 12 h-treated with various EXOs. The uptake of fluorescently-labeled EXOs was also measured in cardiomyocytes. The cardiac function of infarcted male Wistar rats intramyocardially injected with different EXOs (1·1012) was examined by echocardiography. Left ventricular infarct size, hypertrophy, and capillary density were measured. RESULTS: Sustained treatment of NIH/3T3 with non-toxic SFN concentration significantly enhances the release of CD81 + EXOs rich in TSG101 (Tumor susceptibility gene 101) and Hsp70 (Heat Shock Protein 70), and containing maspin, an endogenous histone deacetylase 1 inhibitor. SFN-F-EXOs counteract angiotensin II (AngII)-induced hypertrophy and apoptosis in murine HL-1 cardiomyocytes enhancing SERCA2a (sarcoplasmic/endoplasmic reticulum Ca2+ ATPase 2a) levels more effectively than F-EXOs. In stressed cardiomyocytes, SFN-F-EXOs boost AcH4 levels by 30% (p < 0.05) and significantly reduce oxidative stress more than F-EXOs. Fluorescence microscopy showed that mouse cardiomyocytes take in SFN-F-EXOs ~ threefold more than F-EXOs. Compared to vehicle-injected infarcted hearts, SFN-F-EXOs reduce hypertrophy, scar size, and improve contractility. CONCLUSIONS: Long-term low-dose SFN treatment of fibroblasts enhances the release of anti-remodeling cardiomyocyte-targeted F-EXOs, which effectively prevent the onset of HF. The proposed method opens a new avenue for large-scale production of cardioprotective exosomes for clinical application using allogeneic fibroblasts.

Methodologies for Scalable Production of High-Quality Purified Small Extracellular Vesicles from Conditioned Medium.

The development of an extracellular vesicles (EV)-based therapeutic product requires the implementation of reproducible and scalable, purification protocols for clinical-grade EV. Commonly used isolation methods including ultracentrifugation, density gradient centrifugation, size exclusion chromatography, and polymer-based precipitation, faced limitations such as yield efficiency, EV purity, and sample volume. We developed a GMP-compatible method for the scalable production, concentration, and isolation of EV through a strategy involving, tangential flow filtration (TFF). We applied this purification method for the isolation of EV from conditioned medium (CM) of cardiac stromal cells, namely cardiac progenitor cells (CPC) which has been shown to possess potential therapeutical application in heart failure. Conditioned medium collection and EV isolation using TFF demonstrated consistent particle recovery (~1013 particle/mL) enrichment of small/medium-EV subfraction (range size 120-140 nm). EV preparations achieved a 97% reduction of major protein-complex contaminant and showed unaltered biological activity. The protocol describes methods to assess EV identity and purity as well as procedures to perform downstream applications including functional potency assay and quality control tests. The large-scale manufacturing of GMP-grade EV represents a versatile protocol that can be easily applied to different cell sources for wide range of therapeutic areas.

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

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

A dynamic clamping approach using in silico IK1 current for discrimination of chamber-specific hiPSC-derived cardiomyocytes.

Human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes (CM) constitute a mixed population of ventricular-, atrial-, nodal-like cells, limiting the reliability for studying chamber-specific disease mechanisms. Previous studies characterised CM phenotype based on action potential (AP) morphology, but the classification criteria were still undefined. Our aim was to use in silico models to develop an automated approach for discriminating the electrophysiological differences between hiPSC-CM. We propose the dynamic clamp (DC) technique with the injection of a specific IK1 current as a tool for deriving nine electrical biomarkers and blindly classifying differentiated CM. An unsupervised learning algorithm was applied to discriminate CM phenotypes and principal component analysis was used to visualise cell clustering. Pharmacological validation was performed by specific ion channel blocker and receptor agonist. The proposed approach improves the translational relevance of the hiPSC-CM model for studying mechanisms underlying inherited or acquired atrial arrhythmias in human CM, and for screening anti-arrhythmic agents.

Plasma Extracellular Vesicles as Liquid Biopsy to Unravel the Molecular Mechanisms of Cardiac Reverse Remodeling Following Resynchronization Therapy?

Cardiac resynchronization therapy (CRT) has become a valuable addition to the treatment options for heart failure, in particular for patients with disturbances in electrical conduction that lead to regionally different contraction patterns (dyssynchrony). Dyssynchronous hearts show extensive molecular and cellular remodeling, which has primarily been investigated in experimental animals. Evidence showing that at least several miRNAs play a role in this remodeling is increasing. A comparison of results from measurements in plasma and myocardial tissue suggests that plasma levels of miRNAs may reflect the expression of these miRNAs in the heart. Because many miRNAs released in the plasma are included in extracellular vesicles (EVs), which protect them from degradation, measurement of myocardium-derived miRNAs in peripheral blood EVs may open new avenues to investigate and monitor (reverse) remodeling in dyssynchronous and resynchronized hearts of patients.

Methods for the identification and characterization of extracellular vesicles in cardiovascular studies: from exosomes to microvesicles.

Extracellular vesicles (EVs) are nanosized vesicles with a lipid bilayer that are released from cells of the cardiovascular system, and are considered important mediators of intercellular and extracellular communications. Two types of EVs of particular interest are exosomes and microvesicles, which have been identified in all tissue and body fluids and carry a variety of molecules including RNAs, proteins, and lipids. EVs have potential for use in the diagnosis and prognosis of cardiovascular diseases and as new therapeutic agents, particularly in the setting of myocardial infarction and heart failure. Despite their promise, technical challenges related to their small size make it challenging to accurately identify and characterize them, and to study EV-mediated processes. Here, we aim to provide the reader with an overview of the techniques and technologies available for the separation and characterization of EVs from different sources. Methods for determining the protein, RNA, and lipid content of EVs are discussed. The aim of this document is to provide guidance on critical methodological issues and highlight key points for consideration for the investigation of EVs in cardiovascular studies.

Identification of a serum and urine extracellular vesicle signature predicting renal outcome after kidney transplant.

BACKGROUND: A long-standing effort is dedicated towards the identification of biomarkers allowing the prediction of graft outcome after kidney transplant. Extracellular vesicles (EVs) circulating in body fluids represent an attractive candidate, as their cargo mirrors the originating cell and its pathophysiological status. The aim of the study was to investigate EV surface antigens as potential predictors of renal outcome after kidney transplant. METHODS: We characterized 37 surface antigens by flow cytometry, in serum and urine EVs from 58 patients who were evaluated before, and at 10-14 days, 3 months and 1 year after transplant, for a total of 426 analyzed samples. The outcome was defined according to estimated glomerular filtration rate (eGFR) at 1 year. RESULTS: Endothelial cells and platelets markers (CD31, CD41b, CD42a and CD62P) in serum EVs were higher at baseline in patients with persistent kidney dysfunction at 1 year, and progressively decreased after kidney transplant. Conversely, mesenchymal progenitor cell marker (CD1c, CD105, CD133, SSEEA-4) in urine EVs progressively increased after transplant in patients displaying renal recovery at follow-up. These markers correlated with eGFR, creatinine and proteinuria, associated with patient outcome at univariate analysis and were able to predict patient outcome at receiver operating characteristics curves analysis. A specific EV molecular signature obtained by supervised learning correctly classified patients according to 1-year renal outcome. CONCLUSIONS: An EV-based signature, reflecting the cardiovascular profile of the recipient, and the repairing/regenerative features of the graft, could be introduced as a non-invasive tool for a tailored management of follow-up of patients undergoing kidney transplant.

Myosins and MyomiR Network in Patients with Obstructive Hypertrophic Cardiomyopathy.

Hypertrophic cardiomyopathy (HCM) is the most common genetic cardiomyopathy. The molecular mechanisms determining HCM phenotypes are incompletely understood. Myocardial biopsies were obtained from a group of patients with obstructive HCM (n = 23) selected for surgical myectomy and from 9 unused donor hearts (controls). A subset of tissue-abundant myectomy samples from HCM (n = 10) and controls (n = 6) was submitted to laser-capture microdissection to isolate cardiomyocytes. We investigated the relationship among clinical phenotype, cardiac myosin proteins (MyHC6, MyHC7, and MyHC7b) measured by optimized label-free mass spectrometry, the relative genes (MYH7, MYH7B and MYLC2), and the MyomiR network (myosin-encoded microRNA (miRs) and long-noncoding RNAs (Mhrt)) measured using RNA sequencing and RT-qPCR. MyHC6 was lower in HCM vs. controls, whilst MyHC7, MyHC7b, and MyLC2 were comparable. MYH7, MYH7B, and MYLC2 were higher in HCM whilst MYH6, miR-208a, miR-208b, miR-499 were comparable in HCM and controls. These results are compatible with defective transcription by active genes in HCM. Mhrt and two miR-499-target genes, SOX6 and PTBP3, were upregulated in HCM. The presence of HCM-associated mutations correlated with PTBP3 in myectomies and with SOX6 in cardiomyocytes. Additionally, iPSC-derived cardiomyocytes, transiently transfected with either miR-208a or miR-499, demonstrated a time-dependent relationship between MyomiRs and myosin genes. The transfection end-stage pattern was at least in part similar to findings in HCM myectomies. These data support uncoupling between myosin protein/genes and a modulatory role for the myosin/MyomiR network in the HCM myocardium, possibly contributing to phenotypic diversity and providing putative therapeutic targets.

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.

Investigating the Paracrine Role of Perinatal Derivatives: Human Amniotic Fluid Stem Cell-Extracellular Vesicles Show Promising Transient Potential for Cardiomyocyte Renewal.

Cardiomyocyte renewal represents an unmet clinical need for cardiac regeneration. Stem cell paracrine therapy has attracted increasing attention to resurge rescue mechanisms within the heart. We previously characterized the paracrine effects that human amniotic fluid-derived stem cells (hAFSC) can exert to provide cardioprotection and enhance cardiac repair in preclinical models of myocardial ischemia and cardiotoxicity. Here, we analyze whether hAFSC secretome formulations, namely, hAFSC conditioned medium (hAFSC-CM) over extracellular vesicles (hAFSC-EVs) separated from it, can induce cardiomyocyte renewal. c-KIT+ hAFSC were obtained by leftover samples of II trimester prenatal amniocentesis (fetal hAFSC) and from clinical waste III trimester amniotic fluid during scheduled C-section procedures (perinatal hAFSC). hAFSC were primed under 1% O2 to enrich hAFSC-CM and EVs with cardioactive factors. Neonatal mouse ventricular cardiomyocytes (mNVCM) were isolated from cardiac tissue of R26pFUCCI2 mice with cell cycle fluorescent tagging by mutually exclusive nuclear signal. mNVCM were stimulated by fetal versus perinatal hAFSC-CM and hAFSC-EVs to identify the most promising formulation for in vivo assessment in a R26pFUCCI2 neonatal mouse model of myocardial infarction (MI) via intraperitoneal delivery. While the perinatal hAFSC secretome did not provide any significant cardiogenic effect, fetal hAFSC-EVs significantly sustained mNVCM transition from S to M phase by 2-fold, while triggering cytokinesis by 4.5-fold over vehicle-treated cells. Treated mNVCM showed disorganized expression of cardiac alpha-actinin, suggesting cytoskeletal re-arrangements prior to cell renewal, with a 40% significant downregulation of Cofilin-2 and a positive trend of polymerized F-Actin. Fetal hAFSC-EVs increased cardiomyocyte cell cycle progression by 1.8-fold in the 4-day-old neonatal left ventricle myocardium short term after MI; however, such effect was lost at the later stage. Fetal hAFSC-EVs were enriched with a short isoform of Agrin, a mediator of neonatal heart regeneration acting by YAP-related signaling; yet in vitro application of YAP inhibitor verteporfin partially affected EV paracrine stimulation on mNVCM. EVs secreted by developmentally juvenile fetal hAFSC can support cardiomyocyte renewal to some extension, via intercellular conveyance of candidates possibly involving Agrin in combination with other factors. These perinatal derivative promising cardiogenic effects need further investigation to define their specific mechanism of action and enhance their potential translation into therapeutic opportunity.

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.