Extracellular Vesicles Concentration Research Articles

Proteomic Insights Into Gingival Crevicular Extracellular Vesicles in Periodontitis and Gestational Diabetes: An Exploratory Study.

To characterize the gingival crevicular fluid (GCF) and plasma extracellular vesicles (EVs) and explore their proteomic cargo in healthy pregnant women compared to those with gestational diabetes mellitus (GDM) and periodontitis. One-hundred and four pregnant women were recruited at 24-30 gestation weeks. GDM was diagnosed by an oral glucose tolerance test. GCF and plasma samples were obtained to isolate EVs and characterized by nanoparticle tracking, immunoassays, electron microscopy and mass spectrometry. Of the recruits,17.3% women were healthy, 50% had periodontitis and 32.7% had both GDM and periodontitis. Probing depth, clinical attachment loss and bleeding on probing were more severe in GDM and periodontitis pregnancies (p < 0.0001). Additionally, this group showed an increase concentration of total, small and large GCF-EVs (p = 0.0015, p = 0.0011 and p = 0.0008, respectively), with decreased expression of CD9, CD81 and CD81/CD63 ratio (p = 0.0461, p = 0.0164 and p = 0.0005, respectively). No differences were observed in plasmatic EVs concentration or markers expression. Proteomic analysis of GCF-EVs showed peptides of both host and bacterial origin. Gene ontology analysis revealed that proteins of GCF-EVs participate in immune inflammatory responses, glucose metabolism and insulin response mechanisms. GCF-EVs were increased in both GDM and periodontitis, and their proteomic cargo suggest their involvement in immune inflammatory response, glucose metabolism and insulin pathways during pregnancy.

Extracellular Vesicles as Biomarkers of Pregnancy Complications

Extracellular vesicles (EVs) are double-membrane vesicles that facilitate intercellular communication and play a pivotal role in both physiological and pathological processes. A substantial body of evidence suggests that EVs play a role in the pathogenesis of various pregnancy complications. Because EVs can be detected in the peripheral blood, they are potential biomarkers for the early diagnosis of pregnancy complications and foetal developmental disorders. The majority of studies have demonstrated a correlation between alterations in the concentration of EVs and changes in their contents and the occurrence of pregnancy complications. Despite the current limitations in establishing a clear link between these findings and the pathogenesis of the disease, as well as the lack of sufficient evidence to support their use in clinical practice, it is noteworthy to highlight the potential role of specific miRNAs carried by EVs in the development of pregnancy complications. These include miR-210 and miR-136-5p for pre-eclampsia and gestational diabetes mellitus, miR-155, miR-26b-5p, miR-181a-5p, miR-495 and miR-374c for pre-eclampsia and preterm birth. The following miRNAs have been identified as potential biomarkers for preterm birth and gestational diabetes mellitus: miR-197-3p and miR-520h, miR-1323, miR-342-3p, miR-132-3p, miR-182-3p, miR-517-3p, miR-222-3p, miR-16-5p and miR-126-3p. Additionally, miR-127-3p has been linked to foetal growth restriction and preterm birth. Nevertheless, it would be premature to propose that EVs can be employed as biomarkers for pregnancy complications. Further research and the accumulation of results obtained using the methods proposed in the MISEV2023 guidelines will enable a definitive conclusion to be reached.

The proteomic landscape of extracellular vesicles derived from human intervertebral disc cells.

Extracellular vesicles (EVs) function as biomarkers and are crucial in cell communication and regulation, with therapeutic potential for intervertebral disc (IVD)-related low back pain (LBP). EV cargo is often affected by tissue health, which may affect the therapeutic potential. There is currently limited knowledge of how the cargo of IVD cell-derived EVs varies with tissue health and how differences in proteomic profile affect the predicted biological functions. Our study purified EVs from human IVD cell conditioned media by size-exclusion chromatography. Nanoparticle tracking analysis was conducted to measure EV size and concentration. Transmission electron microscopy and Western blot were performed to examine EV structure and markers. Tandem mass tag-mass spectrometry was conducted to determine protein cargo. Most EVs were exosomes and intermediate microvesicles with an increasing amount linked to disease progression. Of the proteins detected, 88.6% were shared across the non-degenerate, mildly-degenerate, and degenerate samples. GO and KEGG analyses revealed that cargo from the mildly-degenerate samples was the most distinct, with the proteins in high abundance strongly associated with extracellular matrix (ECM) organization and structure. Shared proteins, highly expressed in the non-degenerate and degenerate samples, showed strong associations with cell adhesion, ECM-receptor interaction, and vesicle-mediated transport, respectively. Our findings indicate that EVs from IVD cells from tissue with different degrees of degeneration share a majority of the cargo proteins. However, the level of expression differs with degeneration grade. Cargo from the mildly-degenerate samples exhibits the most differences. A better understanding of changes in EV cargo in the degenerative process may provide novel information related to molecular mechanisms underlying IVD degeneration and suggest new potential treatment modalities for IVD-related LBP.

The Diagnostic Potential of Extracellular Vesicles Derived From the Blood Plasma of Glioblastoma Patients.

Biomarkers for patients suffering from glioblastoma (GBM) are scarce. Extracellular vesicles (EV) are a promising candidate for a potential biomarker. Therefore, EV concentration could be a potential biomarker of tumor burden, volume, and prognosis. Large EV (lEV) and small EV (sEV) were isolated from 36 GBM patients' blood plasma by differential centrifugation. Nanoparticle tracking was used to measure EV concentration. Quantitative analysis of tumor volume was performed by evaluating T2/FLAIR relaxation times. The mean size of lEV was 173.3 nm ± 18.2 nm, while sEV measured 148.3 ± 9.0 nm. Patients with higher lEV concentrations showed a trend towards longer overall survival (36.1 vs. 16.5 months, p=0.08). Regarding inflammatory markers, higher leukocyte count was positively correlated with higher sEV concentration (r2=0.3887, DF 21, p=0.0015). No significant relationship was found between lEV or sEV concentration and tumor volume. Overall EV concentration in the peripheral blood is not a predictor of tumor volume. sEV concentration is associated with a potential pro-inflammatory metabolism.

Vascular Stress Markers Following Inhalation of Heated Tobacco Products: A Study on Extracellular Vesicles.

The advent of heated tobacco products (HTPs) has introduced new variables in the study of nicotine delivery systems and their health implications. Amidst concerns over cardiovascular effects, this study aims to elucidate the acute impact of HTP inhalation on extracellular vesicles (EV) levels in young, healthy individuals. In this controlled, acute exposure study, 23 young, healthy volunteers were subjected to HTP inhalation. EV levels of endothelial and platelet origin were quantified through flow cytometry before and after exposure. Data analysis was performed using multiple measures ANOVA to assess changes in EV concentrations. Our findings reveal a significant increase in EVs of endothelial and platelet origin following short-term HTP inhalation with nicotine. Notably, no significant change was observed in leukocyte- and neutrophil-derived EVs. This increase in EVs suggests acute vascular stress, with peak levels observed 4h post-exposure. The rise in endothelial and platelet-derived EVs aligns with documented responses to acute vascular injury, paralleling the effects seen with traditional cigarette and e-cigarette use. Despite HTPs being marketed as safer alternatives, our results indicate that nicotine-containing HTPs may still pose significant vascular risks. These findings contribute to the growing body of evidence cautioning against the perceived safety of HTPs and reinforce the importance of regulatory oversight and public health initiatives targeting nicotine delivery technologies. Trial Registrations: ClinicalTrials.gov ID: NCT04824495, registered 2021-01-07.

Monitoring Cell Activation and Extracellular Vesicle Generation in Platelet Concentrates for Transfusion.

Platelets play a crucial role in blood transfusions, and understanding the changes that occur during their storage is important for maintaining the quality of preparations. In this study, we examined key alternating factors, with a particular focus on platelet activation and the release of extracellular vesicles. Additionally, we compared two detection methods-imaging flow cytometry (IFC) and nanoparticle tracking analysis (NTA)-for their effectiveness in detecting particles. Platelet concentrates were prepared by pooling buffy coats from five blood group-compatible donors in an additive solution. Samples were analysed after one, three, and seven days of storage for residual white blood cells (WBCs), glucose levels, platelet activation, and extracellular vesicle concentrations. Over the storage period, the total platelet concentration decreased slightly, while the residual WBC count remained stable. Glucose levels declined, whereas platelet activation and extracellular vesicle concentration increased, with a positive correlation between the two. The particle size remained relatively unchanged throughout the storage period. Ultimately, despite controlled processing and storage conditions, platelet activation, and the release of extracellular vesicles still occurred, which may have implications for transfusion recipients. Although an optimised method is still needed, IFC has proved to be specific and potentially appropriate for detecting extracellular vesicles in transfusion preparations.

Extracellular vesicles characterization in patients with hypertrophic cardiomyopathy

Abstract Background Hypertrophic cardiomyopathy (HCM) is a genetic disorder, diagnosed according to the presence of phenotypical traits of the myocardium. A specific biomarker which can associate with the severity of HCM is lacking. Extracellular Vesicles (EVs), nanoparticles released by cells into biological fluids, hold promise as accessible diagnostic tools, as their abundance and molecular composition can reflect the severity of cardiac diseases (Rizzuto 2024). We aim at characterising plasma-derived EVs isolated from 28 HCM patients and 18 healthy volunteers (CTR). Methods The whole cohort underwent transthoracic echocardiography, whereas magnetic resonance and exome sequencing was performed on HCM patients. EVs were isolated via ultracentrifugation from plasma of both groups. Quantitative and qualitative assessments of EVs were performed by nanoparticle tracking analysis, transmission electron microscopy, Western blot, targeted (Olink) and untargeted (nLC-MS/MS) proteomics and FACS analysis. Plasma-derived EV subpopulations were characterised according to their cellular origin. Data are expressed as median and interquartile range (25th, 75th). Results Most patients were male (68% HCM and 66% CTR) with a median age of 58 (49-69) years (HCM) and 47 (44-52) (CTR). Among HCM patients, missense mutations in the MYH7 gene were the most identified. The median maximum wall thickness (WTMax) in HCM was 16 mm (15-19) vs 8 mm (7-9) in CTR. The isolated EVs expressed tetraspanins (CD9, CD63 and CD81), Alix and β-integrin and showed an intact phospholipid bilayer of 100 nm in size. EV concentration was reduced in HCM vs CTRL, respectively 2.8*109 EV/ml/cell count (2*109-4*109) and 4.6*10⁹ EV/ml/cell count (3*109-6*109). Among 15 plasma-derived EV subpopulations studied, those released from platelets (CD41a) and activated platelets (CD62P and CD40L) were increased in HCM patients vs CTR by 1.7 fold. Negative associations were found between E/e’, parameter of diastolic function, and cardiomyocyte-derived EVs (r= -0.2658), and between left ventricle ejection fraction and platelet-derived EVs (r=-0.2189); a positive correlation was found between WTMax and EVs derived from activated endothelial cells (r=0.2330). The proteomic analysis of EVs shows an enrichment in proteins related to platelets adhesion and inflammation in HCM patients. Conclusions HCM patients present a peculiar phenotypic pattern of EVs that may associate with diagnostic clinical features of HCM.

Salivary Extracellular Vesicles Separation: Analysis of Ultracentrifugation-Based Protocols.

The clinical potential of extracellular vesicles (EVs) is widely acknowledged, yet the standardization and reproducibility of its separation remain challenging. This study compares three protocols: ultracentrifugation (UC), UC with purification step (UC + PS), and a combined protocol using polymer-based precipitation and UC (PBP + UC). Salivary samples were collected from healthy donors. EVs were separated (UC, UC + PS, and PBP + UC) and characterized using transmission electron microscopy, nanoparticle tracking analysis, EV purity, RNA concentration, and Western blotting. miRNA expression was evaluated by quantitative RT-PCR. Statistical analyses comparing groups were performed using ANOVA. All methods successfully separated CD9+ and CD63+ EVs from saliva. The UC + PS and PBP + UC protocols yielded the highest concentrations of EVs, enriched in < 200 nm vesicles. EV purity and RNA recovery were comparable among all methods. Expression of miR-16, miR-27a, and miR-99a was successfully detected using all methods. The UC + PS and PBP + UC protocols demonstrate comparable efficiency in separating salivary EVs. However, the combined PBP + UC protocol, with its simplified processing capability, offers a significant advantage, particularly in the initial phase of EV separation. This finding suggests its potential application in clinical settings where time-sensitive simple processing is critical. Further validation is needed to confirm its effectiveness for transcriptomic and proteomic analyses.

Rivaroxaban, in combination with low-dose aspirin, is associated with a reduction in proinflammatory and prothrombotic circulating vesicle signatures in patients with cardiovascular disease

BackgroundDespite secondary prevention with aspirin, patients with stable cardiovascular disease (CVD) remain at elevated long-term risk of major adverse cardiovascular events. The Cardiovascular Outcomes in People Using Anticoagulant Strategies (COMPASS) double-blind, randomized clinical trial demonstrated that aspirin plus low-dose rivaroxaban (COMPASS regime) significantly decreased the incidence of major adverse cardiovascular events by 24% compared with aspirin alone. However, the mechanisms underlying these potential synergistic/nonantithrombotic effects remain elusive. Extracellular vesicles (EVs) are crucial messengers regulating a myriad of biological/pathological processes and are highly implicated in CVD. ObjectivesWe hypothesized that circulating EV profiles reflect the cardioprotective properties of the COMPASS regime. MethodsA cohort of stable CVD patients (N = 40) who participated in the COMPASS trial and were previously randomized to receive aspirin were prospectively recruited and assigned a revised regimen of open-label aspirin plus rivaroxaban. Blood samples were obtained at baseline (aspirin only) and 6-month follow-up. Plasma EV concentration, size, and origin were analyzed by nanoparticle tracking analysis and flow cytometry. EVs were enriched by ultracentrifugation for proteomic analysis. ResultsThe COMPASS regime fundamentally altered small (<200 nm) and large (200-1000 nm) EV concentration and size compared with aspirin alone. Crucially, levels of platelet-derived and myeloperoxidase-positive EVs became significantly decreased at follow-up. Comparative proteomic characterization further revealed a significant decrease in highly proinflammatory protein expression at follow-up. ConclusionThe observed changes in EV subpopulations, together with the differential protein expression profiles, suggest amelioration of an underlying proinflammatory and prothrombotic state upon dual therapy, which may be of clinical relevance toward understanding the fundamental mechanism underlying the reported superior cardiovascular outcomes associated with this antithrombotic regimen.

Extracellular Vesicle Characteristics in Local Fluid and Plasma Measured by Nanoparticle Tracking Analysis Can Help Differentiate High-Grade Serous Carcinoma from Benign Ovarian Pathology

Background: High-grade serous carcinoma (HGSC) is the most lethal of gynecological cancers in developed countries. It usually presents late with non-specific symptoms and most cases are diagnosed at an advanced stage, with 5-year overall survival being around 40%. Biomarkers for screening and early diagnosis of this aggressive disease are, thus, a research priority. Extracellular vesicles (EVs) that reflect the cell of origin and that can be isolated from local fluid and plasma by minimally invasive liquid biopsy are such promising biomarkers. Besides EV concentration and molecular profile, which have been the main focus of research for many years, recent studies have also called attention to EV size distribution. The aim of our study was to evaluate the potential of EV concentration and size distribution in local fluid and plasma as diagnostic biomarkers for HGSC. Methods: Paired pretreatment ascites and plasma samples from 37 patients with advanced HGSC and paired pretreatment free peritoneal fluid (FPF) and plasma samples from 40 controls with benign ovarian pathology (BOP) were analyzed using nanoparticle tracking analysis (NTA). Results: We observed a significant difference in EV concentration in local fluid, but not in plasma, between HGSC patients and the control group. We also found a significant difference in EV size distribution in both local fluid and plasma between HGSC patients and the control group. The receiver operating characteristics (ROC) curve analysis of EV characteristics showed excellent diagnostic performance for the mode, D10, and D50 in local fluid and acceptable diagnostic performance for EV concentration and mean EV size in local fluid, as well as for the mode and D10 value in plasma. Conclusions: The results of our study show that EV concentration in local fluid and more importantly EV size distribution in both local fluid and plasma are significantly changed in the presence of HGSC. Future research of size-dependent molecular profiling of EVs could help identify novel diagnostic biomarkers for HGSC.

Leukocyte Extracellular Vesicles Predict Progression of Systolic Dysfunction in Heart Failure with Mildly Reduced Ejection Fraction (LYCHEE) - A Prospective, Multicentre Cohort Study.

Risk stratification in heart failure with mildly-reduced ejection fraction (HFmrEF) remains challenging. We evaluated the predictive value of advanced glycation end products (AGEs) and plasma concentrations of extracellular vesicles (EVs) for the systolic and diastolic dysfunction progression in HFmrEF patients. Skin AGE accumulation was measured using AGE Reader. Plasma EV concentrations were measured using flow cytometry. Among 74 patients enrolled, 13 (18%) had systolic dysfunction progression and 5 (7%) had diastolic dysfunction progression during 6.5months follow-up. Leukocyte EVs concentrations were higher in patients with systolic dysfunction progression (p = 0.002) and predicted the progression with 75.0% sensitivity and 58.3% specificity, independent of other clinical variables (OR 4.72, 95% CI 0.99-22.31). Skin AGE levels and concentrations of other EV subtypes were not associated with systolic or diastolic dysfunction progression. Increased leukocyte EVs concentrations are associated with 4.7-fold higher odds of systolic dysfunction progression in HFmrEF patients.

Extracellular vesicles from cancer cell lines of different origins drive the phenotype of normal oral fibroblasts in a CAF-like direction.

Normal oral fibroblasts (NOFs) are located in the connective tissue of the oral mucosa. The NOFs play an important role in wound healing, tumor progression, and metastasis. They are subjected to influence by external and internal stimuli, among them extracellular vesicles (EVs), that are considered as important players in cell to cell communication, especially in carcinogenesis and metastatic processes. During tumorigenesis, stromal NOFs may undergo activation into cancer-associated fibroblasts (CAFs) that modify their phenotype to provide pro-oncogenic signals that in turn facilitate tumor initiation, progression, and metastasis. The aim of the study was to reveal the effect of EVs derived from local (oral squamous cell carcinoma - OSCC) and distant (pancreatic adenocarcinoma - PDAC; malignant melanoma brain metastasis - MBM) cancer origin on NOFs and their possible change into a CAF-like direction. The effect of each of the cancer EV types on NOFs proliferation, viability, and migration was tested. Also, changes in gene expression of the well-established CAF biomarkers ACTA2, FAP, PDGFR, and two putative CAF biomarkers, the Ca2+- activated ion channels ANO1 and KCNMA, were studied. Obtained results indicate that NOFs receive and process signals transmitted by EVs originating from both OSCC, PDAC, and MBM. The fibroblast response was dependent on EV origin and concentration, and duration of EV exposure. The present results indicate that the molecular cargo of the EVs direct NOFs towards a pro-tumorigenic phenotype.

Platinum-perovskite nanocomposite-based Exosensor for specific detection of prostate cancer in clinical settings.

Exosomes, extracellular vesicles (EVs) with an average size of 50-150nm, transfer various biomolecules and exchange signaling molecules between cells in a paracrine manner. Molecular investigations have revealed that EVs can reflect real-time metabolic changes in normal- and cancer-origin cells and thus harbor valid diagnostic biomarkers. Despite these advantages, the detection of low concentrations of cancer cell EVs in biological fluids is still a great challenge. Here, a new electrochemical Exosensor based on platinum-perovskite is developed for the direct detection of EVs using a biotinylated monoclonal CD63 antibody as a capture element. The label-free method exhibited higher sensitivity with a lower limit of quantification of 2000 EVs/µL with a dynamic linear range (LDR) of 2000 to 14,000 EVs/μL compared with other available methods. To enhance the selectivity of detection, EVs were simultaneously sandwiched between secondary antibodies of PSA (prostate-specific antigen), as an FDA-approved prostate cancer biomarker. Data indicated that this Exosensor can distinguish normal and cancer EVs in samples from healthy individuals and prostate cancer patients. Taken together, this technology offers a unique approach to label-free quantification of EVs and cancer detection in the early stages.

Abstract MP35: Angiotensin II-induced vascular response is enhanced by Extracellular Vesicles

Intro: The exact effects of extracellular vesicles (EVs) on renal hemodynamics and blood pressure remain to be determined. In this study, we examined the effect of EVs from hypertensive mice and humans on Angiotensin II (ANG II)-induced vascular response in an ex vivo model of isolated and perfused renal afferent arterioles (Af-Arts) and also assessed their effect on renal blood flow (RBF) and systolic blood pressure (SBP) in normotensive mice in vivo. Method: Circulating EVs were collected from mice with ANG II-induced hypertension (n=5) and hypertensive humans (n=2). Differential centrifugation (DCF) (5000G x 30min, followed by 17.000G for 30 minutes) was applied to generate an EV pellet (p20) which was subsequently run through size exclusion chromatography (SEC). EV size and concentration was determined by nanoparticle tracking analysis and spectral flow cytometry. Basic EV characterization was performed with cryo electron microscopy and western blotting of EV and non EV proteins. Following a 30-minute perfusion with EVs (p20 EV pellet (10 10 /ml) or fraction 1-3 or 4-6 of SEC (doses 1-9 7/ml )), dose-response curves to ANG II (10 -12 to 10 -6 mol/l) were measured in Af-Arts of C57BL/6 mice. Similar doses of EVs were infused into the penile vein of C57BL/6 mice and RBF and SBP were measured. As EVs might deliver AT1R we explored the final common pathway in vasoconstriction in vessels treated with EVs from hypertensive animals vs controls by performing immunoblotting of myosin light chain II S19 phosphorylation. Results: EVs deriving from ANG II-treated mice and hypertensive humans, but not those from normotensive control mice and humans, significantly enhanced the vasoconstrictor response to ANG II in Af-Arts of C57BL/6 mice. The same vasoconstrictor response was observed when Af-Arts were perfused with combined EV preps from DCF and SEC (fraction 1-3 vs fraction 1-6) from hypertensive animals. In vivo injection of EVs from ANG II-treated hypertensive mice reduced RBF and increased SBP in C57BL/6 mice compared to EVs from normotensive control mice. Immunoblotting of myosin light chain II S19 phosphorylation was inconclusive. Conclusion: These data suggest that EVs from hypertensive animals and humans directly affect ANG II response, renal hemodynamic and blood pressure regulation. Further work is needed to identify the EV effector candidate for enhanced ANG II response and possible effect on ART1 activation.

Analysis of plasma-derived small extracellular vesicle characteristics and microRNA cargo following exercise-induced skeletal muscle damage in men.

Extracellular vesicle (EV) cargo is known to change in response to stimuli such as muscle damage. This study aimed to assess particle size, concentration and microRNA (miR) content within small EV-enriched separations prepared from human blood taken before and after unaccustomed eccentric-biased exercise-induced muscle damage. Nine male volunteers underwent plyometric jumping and downhill running, with blood samples taken at baseline, 2, and 24 h post-exercise. EVs were separated using size exclusion chromatography (SEC) and their characteristics evaluated by nanoparticle tracking. No changes in EV size or concentration were seen following the muscle-damaging exercise. Small RNA sequencing identified 240 miRs to be consistently present within the EVs. RT-qPCR analysis was performed: specifically, for known muscle-enriched/important miRs, including miR-1, -206, -133a, -133b, -31, -208b, -451a, -486 and - 499 and the immune-important miR-21, -146a and - 155. Notably, none of the immune-important miRs within the EVs tested changed in response to the muscle damage. Of the muscle-associated miRs tested, only the levels of miR-31-5p were seen to change with decreased levels at 24 h compared to baseline and 2 h, indicating involvement in the damage response. These findings shed light on the dynamic role of EV miRs in response to exercise-induced muscle damage.

Malondialdehyde-specific natural IgM inhibit NETosis triggered by culprit site-derived extracellular vesicles from myocardial infarction patients.

Abstract Background and Aims Neutrophil extracellular traps (NETs) trigger atherothrombosis during acute myocardial infarction (AMI), but mechanisms of induction remain unclear. Levels of extracellular vesicles (EV) carrying oxidation-specific epitopes (OSE), which are targeted by specific natural immunoglobulin M (IgM), are increased at the culprit site in AMI. This study investigated EV as inducers of NETosis and assessed the inhibitory effect of natural anti-OSE–IgM in this process. Methods Blood from the culprit and peripheral site of ST-segment elevation myocardial infarction (STEMI) patients (n = 28) was collected, and myocardial function assessed by cardiac magnetic resonance imaging (cMRI) 4 ± 2 days and 195 ± 15 days post-AMI. Extracellular vesicles were isolated from patient plasma and cell culture supernatants for neutrophil stimulation in vitro and in vivo, in the presence of a malondialdehyde (MDA)-specific IgM or an isotype control. NETosis and neutrophil functions were assessed via enzyme-linked immunosorbent assay and fluorescence microscopy. Pharmacological inhibitors were used to map signalling pathways. Neutrophil extracellular trap markers and anti-OSE–IgM were measured by ELISA. Results CD45+ MDA+ EV and NET markers were elevated at the culprit site. Extracellular vesicles induced neutrophil activation and NET formation via TLR4 and PAD4, and mice injected with EV showed increased NETosis. Malondialdehyde-specific IgM levels were inversely associated with citH3 in STEMI patient blood. An MDA-specific IgM inhibited EV-induced NET release in vitro and in vivo. CD45+ MDA+ EV concentrations inversely correlated with left ventricular ejection fraction post-AMI. Conclusions Culprit site–derived EV induce NETosis, while MDA-specific natural IgM inhibit this effect, potentially impacting outcome after AMI.

Circulating extracellular vesicles in Systemic Lupus Erythematosus: physicochemical properties and phenotype

ObjectiveThis study aimed to identify the physicochemical and phenotypic characteristics of circulating Extracellular Vesicles (EVs) in the plasma of patients with SLE, with or without Lupus Nephritis (LN), and their...

Lipidomic and Proteomic Insights from Extracellular Vesicles in Postmortem Dorsolateral Prefrontal Cortex Reveal Substance Use Disorder-Induced Brain Changes.

Substance use disorder (SUD) significantly increases the risk of neurotoxicity, inflammation, oxidative stress, and impaired neuroplasticity. The activation of inflammatory pathways by substances may lead to glial activation and chronic neuroinflammation, potentially mediated by the release of extracellular particles (EPs), such as extracellular condensates (ECs) and extracellular vesicles (EVs). These particles, which reflect the physiological, pathophysiological, and metabolic states of their cells of origin, might carry molecular signatures indicative of SUD. In particular, our study investigated neuroinflammatory signatures in SUD by isolating EVs from the dorsolateral prefrontal cortex (dlPFC) Brodmann's area 9 (BA9) in postmortem subjects. We isolated BA9-derived EVs from postmortem brain tissues of eight individuals (controls: n=4, SUD: n=4). The EVs were analyzed for physical properties (concentration, size, zeta potential, morphology) and subjected to integrative multi-omics analysis to profile the lipidomic and proteomic characteristics. We assessed the interactions and bioactivity of EVs by evaluating their uptake by glial cells. We further assessed the effects of EVs on complement mRNA expression in glial cells as well as their effects on microglial migration. No significant differences in EV concentration, size, zeta potential, or surface markers were observed between SUD and control groups. However, lipidomic analysis revealed significant enrichment of glycerophosphoinositol bisphosphate (PIP2) in SUD EVs. Proteomic analysis indicates downregulation of SERPINB12, ACYP2, CAMK1D, DSC1, and FLNB, and upregulation of C4A, C3, and ALB in SUD EVs. Gene ontology and protein-protein interactome analyses highlight functions such as cell motility, focal adhesion, and acute phase response signaling that is associated with the identified proteins. Both control and SUD EVs increased C3 and C4 mRNA expression in microglia, but only SUD EVs upregulated these genes in astrocytes. SUD EVs also significantly enhanced microglial migration in a wound healing assay.This study successfully isolated EVs from postmortem brains and used a multi-omics approach to identify EV-associated lipids and proteins in SUD. Elevated C3 and C4 in SUD EVs and the distinct effects of EVs on glial cells suggest a crucial role in acute phase response signaling and neuroinflammation.

The metabolic response of HepG2 cells to extracellular vesicles derived from Raphanus sativus L. var. caudatus Alef microgreens probed by chemometrics-assisted LC-MS/MS analysis

Extracellular vesicles (EVs) derived from Thai rat-tailed radish (Raphanus sativus L. var. caudatus Alef) microgreens were previously reported as novel bioactive bioparticles against cancer. This study aimed to investigate the metabolic disruption associated with the antiproliferative effect against HepG2 liver cancer cells, a representative of metabolizing cells and tissue. In this study, the neutral red uptake assay was performed to screen for the antiproliferative effect and determine the cytotoxic concentrations of EVs against HepG2 cells. An untargeted approach to cellular metabolomics was conducted using liquid chromatography coupled with the high-resolution mass spectrometry system with multivariate and univariate analyses to determine the metabolic changes of HepG2 liver cancer cells after EV treatment. EVs showed an antiproliferative effect in HepG2 cells with a half-maximal inhibitory concentration (IC50) of 685.5 ± 26.4 and 139.7 ± 4.2 μg/ml at 24 and 48 h, respectively. In the metabolomics study, 163 metabolites were annotated, with 61 significantly altered metabolites. Among these significant metabolites, 18 were related to glycerophospholipid metabolism. Phosphatidylcholine—the important lipid building blocks for cell membranes, lipid mediators for cell proliferation, and immunosuppressive signaling—was mainly decreased by EV treatment. The alteration of cellular phospholipids in cancer was discussed. This finding suggested the possible mechanism of anticancer action of EVs by disrupting phospholipid metabolism and survival signaling in cancer cells. Further studies should be made to confirm EVs' potential as single and combination therapy in vivo to reduce cancer resistance. This may close the gap between in vitro study and clinical setting.

The Concentration of Salivary Extracellular Vesicles Is Related to Obesity.

Saliva is essential for the proper dilution and distribution of taste molecules on the tongue. It harbors extracellular vesicles (EVs), which mediate cell-cell communication. Changes in the composition of salivary EVs may arise under obese conditions and may potentially be involved in taste sensation and dysregulated eating behavior. Therefore, this study addresses the relationship between the size and concentration of salivary EVs and metabolic shifts in obesity or factors of taste sensation. A total of 119 participants in the Obese Taste Bud (OTB) Study were included, who performed a standardized taste test, underwent taste bud density assessment, and were phenotypically characterized for anthropometrics, blood- and saliva adipokine levels, and various metabolic factors. Utilizing size exclusion chromatography followed by ultrafiltration, EVs were extracted from 2 mL of actively secreted saliva. EVs were characterized using nanoparticle tracking analyses, Western blot, and scanning transmission electron microscopy. Finally, group comparisons and bivariate correlation analyses were conducted. Among the total cohort, the median size of salivary EVs was 190.05 nm, and the overall concentration ranged from 1.4 × 107 to 1.76 × 109 per mL of saliva. The size range and concentration of EVs per mL are negatively correlated (p = 0.0002, r = -0.264). Comparing lean participants (mean rank of 45.98) with those presenting obesity (mean rank of 34.46), a significant difference in the salivary EV content was observed (p = 0.029). Body weight, BMI, arm and calf circumferences, as well as the percentage of body fat were all negatively related to the concentration of EVs in all study participants (all p < 0.05, r > -0.2). No associations were found between the EV parameters and taste perception but serum alkaline phosphatase levels were negatively correlated (p = 0.007, r = -0.284) and adiponectin serum levels were positively correlated to the EV concentration (p = 0.036, r = 0.208). The current study provides evidence for the relation between salivary EVs and anthropometric as well as metabolic parameters of obesity. This can provide the basis for further research on the cargo of salivary EVs and how they may influence taste sensation, and may elucidate their potential connection to altered eating habits in obesity.