Profiling Of Extracellular Vesicles Research Articles

The role of extracellular vesicles in kidney disease progression.

Extracellular vesicles (EVs) are nanosized membranous particles released by nearly all cell types, playing a crucial role in mediating cell-to-cell communication. The molecular profile of EVs often reflects that of their originating cells, rendering them valuable for therapeutic and diagnostic purposes. The kidney comprises various cell types, and urinary EVs are predominantly produced from tubular, glomerular, and urinary bladder cells. Within the nephron, EVs produced from the upper segments, such as glomerular tufts and proximal tubules, can be taken up by their downstream counterparts, thereby altering the physiology of recipient cells. Recent studies have demonstrated that this proximal-distal intra-nephron crosstalk via EVs is crucial for normal kidney physiology. Additionally, EVs from interstitial cells (e.g., fibroblasts and macrophages) have been demonstrated to mediate the exacerbation of kidney damage. This review provides up-to-date findings on the function of renal EVs during the progression of renal diseases. Furthermore, we discussed future directions to use the clinical potential of renal EVs as an early biomarker for renal disorders.

MiRNAs and Neutrophil-Related Membrane Proteins from Plasma-Derived Extracellular Vesicles for Early Prediction of Organ Dysfunction and Prognosis in Septic Patients.

The pathogenesis of sepsis-induced organ dysfunction remains elusive, and the mortality remains alarmingly high. We sought to investigate the profile of extracellular vesicles (EVs)-mediated communication between plasma and polymorphonuclear neutrophils (PMNs) in sepsis, and to elucidate whether miRNAs and PMN-related membrane proteins from plasma-derived EVs (plasma-EVs) are associated with sepsis-induced organ dysfunction and prognosis. PMN-derived EVs (PMN-EVs) were isolated from the blood samples of healthy controls (N=3) and patients with septic shock (N=3) after ICU admission. We performed miRNA sequencing of the isolated EVs, followed by bioinformatic analysis. A miRNA model for comparing PMN-EVs and plasma-EVs was successfully established in the training cohort. Furthermore, miRNAs and PMN-related membrane proteins from the plasma-EV model were confirmed in the validation cohort. A logistic regression model, receiver operating characteristic (ROC) curves, and Kaplan-Meier analyses were performed to evaluate the efficiency of diagnostic and/or prognostic performance. Further, in vivo and in vitro experiments were conducted to explore the involvement of plasma-EVs in PMNs autophagy. Fifty-five miRNAs from PMN-EVs differed significantly between the healthy controls and patients with septic shock. Furthermore, the plasma-EV model (six miRNAs and eight PMN-related membrane proteins) was confirmed in the validation cohort, demonstrating that miR-34a-5p, miR-503-5p, miR-4772-3p, ITGAM, MPO, and MMP9 serve as sepsis biomarkers for distinguishing lung, liver, and kidney dysfunction. Kaplan-Meier survival analysis showed that miR-34a-5p, miR-4772-3p, ITGAM, and MMP9 were potential prognostic predictors. Finally, we found that plasma-EVs from sepsis patients exert an inhibitory effect on PMNs autophagy, which can be reversed by EV inhibitors such as GW4869 and enoxaparin. These findings suggest that miRNAs and PMN-related membrane proteins from plasma-EVs could be valuable diagnostic tools for identifying sepsis-induced organ dysfunction and predicting prognosis, enabling proactive management of sepsis by physicians and improving the prognosis of sepsis patients.

Global Lipidomics Reveals the Lipid Composition Heterogeneity of Extracellular Vesicles from Drug-Resistant Leishmania

Background: The rise of drug-resistant Leishmania strains presents a significant challenge in the treatment of Leishmaniasis, a neglected tropical disease. Extracellular vesicles (EVs) produced by these parasites have gained attention for their role in drug resistance and host–pathogen interactions. Methods: This study developed and applied a novel lipidomics workflow to explore the lipid profiles of EVs from three types of drug-resistant Leishmania infatum strains compared to a wild-type strain. EVs were isolated through ultracentrifugation, and their lipid content was extracted using a modified Matyash protocol. LC-MS analysis was performed, and data processing in MS-DIAL enabled lipid identification and quantification. Statistical analysis in MetaboAnalyst revealed strain-specific lipid alterations, highlighting potential links between lipid composition and drug resistance mechanisms. Results: Our results show distinct alterations in lipid composition associated with drug resistance. Specifically, drug-resistant strains exhibited reduced levels of phosphatidylcholine (PC) and phosphatidylglycerol (PG), particularly in the amphotericin B-resistant strain LiAmB1000.1. Sterol and glycerolipid species, including cholesteryl ester (CE) and triacylglycerol (TG) were also found to be diminished in LiAmB1000.1. These changes suggest significant lipid remodeling under drug pressure, potentially altering the biophysical properties of EV membranes and their capacity for molecule transfer. Furthermore, the lipidomic profiles of EVs from the other resistant strains, LiSb2000.1 and LiMF200.5, also displayed unique alterations, underscoring strain-specific adaptations to different drug resistance mechanisms. Conclusions: These significant alterations in lipid composition suggest potential lipid-based mechanisms underlying drug resistance in Leishmania, providing new avenues for therapeutic intervention.

Modeling the Impact of Extracellular Vesicle Cargoes in the Diagnosis of Coronary Artery Disease

Objectives: We aimed to assess the relationship among circulating extracellular vesicles (EVs), hypoxia-related proteins, and the conventional risk factors of life-threatening coronary artery disease (CAD) to find more precise novel biomarkers. Methods: Patients were categorized based on coronary CT angiography. Patients with a Segment Involvement Score > 5 were identified as CAD patients. Individuals with a Segment Involvement Score < 5 were considered control subjects. The characterization of EVs and analysis of the plasma concentration of growth differentiation factor-15 were performed using multicolor or bead-based flow cytometry. The plasma protein levels of glycogen phosphorylase, muscle form, clusterin, and carboxypeptidase N subunit 1 were determined using an enzyme-linked immunosorbent assay. Multiple logistic regression was used to determine the association of the biomarkers with the CAD outcome after accounting for established risk factors. The analysis was built in three steps: first, we included the basic clinical and laboratory variables (Model 1), then we integrated the plasma protein values (Model 2), and finally, we complemented it with the circulating EV pattern (Model 3). To assess the discrimination value of the models, an area under (AUC) the receiver operating curve was calculated and compared across the three models. Results: The area under the curve (AUC) values were 0.68, 0.77, and 0.84 in Models 1, 2, and 3, respectively. The variables with the greatest impact on the AUC values were hemoglobin (0.2 (0.16–0.26)) in Model 1, carboxypeptidase N subunit 1 (0.12 (0.09–0.14)) in Model 2, and circulating CD41+/CD61+ EVs (0.31 (0.15–0.5)) in Model 3. A correlation analysis showed a significant impact of circulating CD41+/CD61+ platelet-derived EVs (p = 0.03, r = −0.4176) in Model 3. Conclusions: Based on our results, the circulating EV profile can be used as a supportive biomarker, along with the conventional laboratory markers of CAD, and it enables a more sensitive, non-invasive diagnostic analysis of CAD.

Proteomics of circulating extracellular vesicles reveals diverse clinical presentations of COVID-19 but fails to identify viral peptides.

Extracellular vesicles (EVs) released by virus-infected cells have the potential to encapsulate viral peptides, a characteristic that could facilitate vaccine development. Furthermore, plasma-derived EVs may elucidate pathological changes occurring in distal tissues during viral infections. We hypothesized that molecular characterization of EVs isolated from COVID-19 patients would reveal peptides suitable for vaccine development. Blood samples were collected from three cohorts: severe COVID-19 patients (G1), mild/asymptomatic cases (G2), and SARS-CoV-2-negative healthcare workers (G3). Samples were obtained at two time points: during the initial phase of the pandemic in early 2020 (m0) and eight months later (m8). Clinical data analysis revealed elevated inflammatory markers in G1. Notably, non-vaccinated individuals in G1 exhibited increased levels of neutralizing antibodies at m8, suggesting prolonged exposure to viral antigens. Proteomic profiling of EVs was performed using three distinct methods: immunocapture (targeting CD9), ganglioside-capture (utilizing Siglec-1) and size-exclusion chromatography (SEC). Contrary to our hypothesis, this analysis failed to identify viral peptides. These findings were subsequently validated through Western blot analysis targeting the RBD of the SARS-CoV-2 Spike protein's and comparative studies using samples from experimentally infected Syrian hamsters. Furthermore, analysis of the EV cargo revealed a diverse molecular profile, including components involved in the regulation of viral replication, systemic inflammation, antigen presentation, and stress responses. These findings underscore the potential significance of EVs in the pathogenesis and progression of COVID-19.

Extracellular vesicles from microglial cells activated by abnormal heparan sulfate oligosaccharides from Sanfilippo patients impair neuronal dendritic arborization

BackgroundIn mucopolysaccharidosis type III (MPS III, also known as Sanfilippo syndrome), a pediatric neurodegenerative disorder, accumulation of abnormal glycosaminoglycans (GAGs) induces severe neuroinflammation by triggering the microglial pro-inflammatory cytokines production via a TLR4-dependent pathway. But the extent of the microglia contribution to the MPS III neuropathology remains unclear. Extracellular vesicles (EVs) mediate intercellular communication and are known to participate in the pathogenesis of adult neurodegenerative diseases. However, characterization of the molecular profiles of EVs released by MPS III microglia and their effects on neuronal functions have not been described.MethodsHere, we isolated EVs secreted by the microglial cells after treatment with GAGs purified from urines of Sanfilippo patients (sfGAGs-EVs) or from age-matched healthy subjects (nGAGs-EVs) to explore the EVs’ proteins and small RNA profiles using LC–MS/MS and RNA sequencing. We next performed a functional assay by immunofluorescence following nGAGs- or sfGAGs-EVs uptake by WT primary cortical neurons and analyzed their extensions metrics after staining of βIII-tubulin and MAP2 by confocal microscopy.ResultsFunctional enrichment analysis for both proteomics and RNA sequencing data from sfGAGs-EVs revealed a specific content involved in neuroinflammation and neurodevelopment pathways. Treatment of cortical neurons with sfGAGs-EVs induced a disease-associated phenotype demonstrated by a lower total neurite surface area, an impaired somatodendritic compartment, and a higher number of immature dendritic spines.ConclusionsThis study shows, for the first time, that GAGs from patients with Sanfilippo syndrome can induce microglial secretion of EVs that deliver a specific molecular message to recipient naive neurons, while promoting the neuroinflammation, and depriving neurons of neurodevelopmental factors. This work provides a framework for further studies of biomarkers to evaluate efficiency of emerging therapies.Graphical

High-throughput, multiplexed quantification, and sorting of single EVs at single-molecule level.

We have developed a platform for the high-throughput, multiplexed, and ultra-sensitive profiling of individual extracellular vesicles (EVs) directly in plasma, which we call BDEVS - Agarose B ead-based D igital Single Molecule-Single EV S orting. Unlike conventional approaches, BDEVS achieves single molecule sensitivity and moderate multiplexing (demonstrated 3-plex) without sacrificing the throughput (processing ten thousand of EVs per minute) necessary to resolve EVs directly in human plasma. Our platform integrates rolling circle amplification (RCA) of EV surface proteins, which are cleaved from single EVs, and amplified within agarose droplets, followed by flow cytometry-based readout and sorting, overcoming steric hindrance, non-specific binding, and the lack of quantitation of multiple proteins on EVs that have plagued earlier approaches. We evaluated the analytical capabilities of BDEVS through head-to-head comparison with gold-standard technologies, and demonstrated a ∼100x improvement in the limit of detection of EV subpopulations. We demonstrate the high throughput (∼100k beads / minute) profiling of individual EVs for key immune markers PD-L1, CD155, and the melanoma tumor marker TYRP-1, and showed that BDEVS can precisely quantify and sort EVs, offering unprecedented resolution for analyzing tumor-immune interactions and detecting rare EV subpopulations in complex clinical specimens. We demonstrate BDEVS's potential as a transformative tool for EV-based diagnostics and therapeutic monitoring in the context of cancer immunology by analyzing plasma samples from patients with melanoma, where EV heterogeneity plays a critical role in disease progression and response to therapy.

Detection of brain metastases from blood using Brain nanoMET sensor: Extracellular vesicles as a dynamic marker for metastatic brain tumors

Brain metastases account for a significant number of cancer-related deaths with poor prognosis and limited treatment options. Current diagnostic methods have limitations in resolution, sensitivity, inability to differentiate between primary and metastatic brain tumors, and invasiveness. Liquid biopsy is a promising non-invasive alternative; however, current approaches have shown limited efficacy for diagnosing brain metastases due to biomarker instability and low levels of detectable tumor-specific biomarkers. This study introduces an innovative liquid biopsy technique using extracellular vesicles (EVs) as a biomarker for brain metastases, employing the Brain nanoMET sensor. The sensor was fabricated through an ultrashort femtosecond laser ablation process and provides excellent surface-enhanced Raman Scattering functionality. We developed an in vitro model of metastatic tumors to understand the tumor microenvironment and secretomes influencing brain metastases from breast and lung cancers. Molecular profiling of EVs derived from brain-seeking metastatic tumors revealed unique, brain-specific signatures, which were also validated in the peripheral circulation of brain metastasis patients. Compared to primary brain tumor EVs, we also observed an upregulation of PD-L1 marker in the metastatic EVs. A machine learning model trained on these EV molecular profiles achieved 97% sensitivity in differentiating metastatic brain cancer from primary brain cancer, with 94% accuracy in predicting the primary tissue of origin for breast metastasis and 100% accuracy for lung metastasis. The results from this pilot validation suggest that this technique holds significant potential for improving metastasis diagnosis and targeted treatment strategies for brain metastases, addressing a critical unmet need in neuro-oncology.

Squeezable Hydrogel Microparticles for Single Extracellular Vesicle Protein Profiling.

Extracellular vesicles (EVs) are promising for molecular diagnostics, but current analyses are limited by the rarity and compositional heterogeneity of EV protein expression. Therefore, single EV profiling methods require high sensitivity, multiplexing, and throughput to address these issues. Here a single EV analysis technique that utilizes squeezable methacrylated hyaluronic acid hydrogel microparticles (MHPs) is described as a scaffold to immobilize EVs and perform an integrated rolling circle amplification (RCA) assay for an ultra-sensitive and multiplex analysis of single EV proteins. EVs are prepared into MHPs in a high-throughput manner with droplet microfluidics and optimally labeled with antibody-oligonucleotide conjugates in MHPs without steric limitations. By designing MHPs with high compressibility, single EV protein signals are amplified as RCA products that can be aligned on the same plane by physically squeezing MHPs and visualized with low magnification. This method provides a simple and scalable single EV imaging analysis pipeline for identifying multiplex marker expression patterns from single EVs. For validation, the single EV heterogeneity of highly expressed cancer cell markers is profiled across different cancer cell lines. These findings exemplify squeezable MHPs as a robust platform with high sensitivity, multiplexing, and scalability for resolving single EV heterogeneity and advancing molecular assay technologies.

Proteomic profiling of extracellular vesicles in takotsubo syndrome

Abstract Background/Purpose Takotsubo syndrome (TS) is an acute form of heart failure characterized by transient regional wall motion abnormalities triggered by a stressful event. The mechanisms underlying its development and recovery are poorly understood, resulting in a lack of disease-specific treatments and diagnostic markers. Extracellular vesicles (EVs) play a significant role in cellular communication and have been shown to be important in various diseases. Their involvement in cardiac conditions like TS is an emerging area of research. The primary objective of this study is to isolate and characterize EVs, as well as profile their proteomic profile, from the heart tissue of rats induced with TS. Methods Rats underwent TS induction through the infusion of 1 mg/kg isoprenaline over 15 minutes (TS24h), or were given saline (control). High-resolution echocardiography verified apical ballooning at 6 hours. After 24 hours, heart tissues from apical and basal segments were collected and processed. EV isolation involved tissue slicing, enzymatic digestion, and differential centrifugation steps, including a final iodixanol cushion separation for large and small EVs (Figure 1). Tandem mass tags and mass spectrometry were utilized for global proteomics and any differentially expressed proteins (DEPs) were identified. Analytical techniques such as volcano plots, heatmaps, enrichment analysis, and protein clustering/networks were employed. The strongest clusters (lowest False Discovery Rate and highest intensity) were selected, and their Gene Ontology (GO) terms/pathways were identified. Results Pure, cup-shaped, vesicles ranging from 50-800nm were successfully isolated (figure 1). EVs from apex of control and TS24h hearts had lower protein concentrations compared to their corresponding basal segments. Global proteomic analysis revealed a distinct protein profile in EVs from the apical segment of TS hearts at 24 hours. A total of 256 (TS24h-apex vs control-apex) and 561 proteins (TS24h-apex vs TS24h-base) were differentially expressed. No DEPs were observed when comparing the basal segments of TS24h and control hearts. Functional enrichment analysis of the DEPs showed an abundant change of biological processes related to metabolic lipid processes, inflammatory response, complement activation, reorganization of extracellular matrix. A downregulation was seen for biological processes related to mitochondrial ATP synthesis coupled electron transport and muscle contraction. Conclusion This study demonstrates a distinct EV protein profile in the apical segments of TS hearts, with marked changes in proteins related to metabolic lipid processes, inflammation, and mitochondrial activity. This extensive catalogue of novel proteins sets the stage for future research aimed at identifying potential therapeutic targets and diagnostic biomarkers, and enabling proof-of-concept studies in TS.

High-throughput capture and in situ protein analysis of extracellular vesicles by chemical probe-based array.

Extracellular vesicles (EVs) are small particles with phospholipid bilayers that carry a diverse range of cargoes including nucleic acids, proteins and metabolites. EVs have important roles in various cellular processes and are increasingly recognized for their ubiquitous role in cell-cell communications and potential applications in therapeutics and diagnostics. Although many methods have been developed for the characterization and measurement of EVs, analyzing them from biofluids remains a challenge with regard to throughput and sensitivity. Recently, we introduced an approach to facilitate high-throughput analysis of EVs from trace amounts of sample. In this method, an amphiphile-dendrimer supramolecular probe (ADSP) is coated onto a nitrocellulose membrane for array-based capture and to enable an in situ immunoblotting assay. Here, we describe the protocol for our array-based method of EV profiling. We describe an enhanced version of the method that incorporates an automated printing workstation, ensuring high throughput and reproducibility. We further demonstrate the use of our array to profile specific glycosylations on the EV surface using click chemistry of an azide group introduced by metabolic labeling. In this protocol, the synthesis of ADSP and the fabrication of ADSP nitrocellulose membrane array can be completed on the same day. EVs are efficiently captured from biological or clinical samples through a 30-min incubation, followed by an immunoblotting assay within a 3-h window, thus providing a high-throughput platform for EV isolation and in situ targeted analysis of EV proteins and their modifications.

Protein profile of extracellular vesicles derived from adult Parascaris spp.

BackgroundParascaris spp. represent a significant threat to equine health worldwide, particularly in foals. The long-term survival of parasites in the host necessitates persistent modulation of the host immune response. Intercellular communication achieved through the exchange of molecules via extracellular vesicles (EVs) released from the parasite could be a crucial factor in this regard. This study aimed to isolate and characterize EVs released by adult male and female Parascaris worms and conduct a proteomic analysis to identify sex-specific proteins and potential immunomodulatory factors.MethodsLive adult Parascaris worms were collected, and EVs were isolated from spent culture media using differential ultracentrifugation. Nanoparticle tracking analysis and transmission electron microscopy confirmed the size, concentration, and morphology of the isolated EVs. Proteins within the isolated EVs were analyzed using mass spectrometry-based proteomics (LC–MS/MS).ResultsProteomic analysis revealed a total of 113 proteins in Parascaris EVs, with several proteins showing homology to known helminth exosome proteins and exhibiting immunomodulatory functions. Sex-specific differences in EV protein composition were observed, with a distinct abundance of C-type lectins in female EVs, suggesting potential sex-specific roles or regulation. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses revealed metabolic pathways shared between male and female Parascaris EVs, as well as differences in signal transduction, and cell growth and death pathways, indicating sex-specific variations.ConclusionsThese findings imply that Parascaris EVs and their protein cargo are complex. This data potentially opens avenues for discovering innovative approaches to managing and understanding helminth infection.Graphical

Associations of age and sex with characteristics of extracellular vesicles and protein-enriched fractions of blood plasma.

Extracellular vesicles (EVs) are nanosized particles that are released by various cell types and play vital roles in intercellular communication. They carry biological molecules reflecting the physiological and pathological states of their source cells and tissues, showing potential as biomarkers. However, the impact of demographic factors like age and sex on the properties of blood plasma EVs remains underexplored. This study aims to fill this gap by evaluating how these factors influence the particle count and proteomic profiles of plasma EV preparations and corresponding protein fractions. Plasma samples from 120 healthy volunteers were collected and pooled into six groups: young males (age: 27.6 ± 4.0), young females (27.4 ± 3.8), middle-aged males (48.8 ± 3.8), middle-aged females (48.9 ± 3.9), old males (69.3 ± 3.9), and old females (69.4 ± 4.3). EV- and protein-enriched fractions were separated by size-exclusion chromatography (SEC). Fractions were characterized for particle number concentration and protein composition to identify characteristics affected by age and biological sex. Plasma EVs and corresponding protein fractions exhibited distinct characteristics, with differential enrichment of markers related to EVs and other blood components, including lipoproteins. Proteomic profiles of both EVs and protein fractions displayed sex- and age-dependent differences. Differentially abundant proteins displayed functions previously identified in the context of aging and sex differences, highlighting their utility as biomarkers. Age and sex significantly affect the characteristics of plasma EVs and proteins, potentially influencing their efficacy and interpretation as biomarkers in clinical applications. This study lays the groundwork for detailed mechanistic research to understand how EVs mediate age- and sex-related effects in health.

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.

Proteomic analysis of plasma-derived extracellular vesicles: pre- and postprandial comparisons

Extracellular vesicles (EVs) are key in intercellular communication, carrying biomolecules like nucleic acids, lipids, and proteins. This study investigated postprandial characteristics and proteomic profiles of blood-derived EVs in healthy individuals. Twelve participants fasted overnight before baseline assessments. After consuming a controlled isocaloric meal, EVs were isolated for proteomic and flow cytometric analysis. Plasma triacylglyceride levels confirmed fasting completion, while protein concentrations in plasma and EVs were monitored for postprandial stability. Proteomic analysis identified upregulated proteins related to transport mechanisms and epithelial/endothelial functions postprandially, indicating potential roles in physiological responses to nutritional intake. Enrichment analyses revealed vesicle-related pathways and immune system processes. Flow cytometry showed increased expression of CD324 on CD9+CD63+CD81+ large extracellular vesicles postprandially, suggesting an epithelial origin. These findings offer valuable insights into postprandial EV dynamics and their potential physiological significance, highlighting the need for stringent fasting guidelines in EV studies to account for postprandial effects on EV composition and function.

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.

Proteomic Profiles of Maternal Plasma Extracellular Vesicles for Prediction of Preeclampsia.

Preeclampsia is a heterogeneous syndrome of diverse etiologies and molecular pathways leading to distinct clinical subtypes. Herein, we aimed to characterize the extracellular vesicle (EV)-associated and soluble fractions of the maternal plasma proteome in patients with preeclampsia and to assess their value for disease prediction. This case-control study included 24 women with term preeclampsia, 23 women with preterm preeclampsia, and 94 healthy pregnant controls. Blood samples were collected from cases on average 7 weeks before the diagnosis of preeclampsia and were matched to control samples. Soluble and EV fractions were separated from maternal plasma; EVs were confirmed by cryo-EM, NanoSight, and flow cytometry; and 82 proteins were analyzed with bead-based, multiplexed immunoassays. Quantile regression analysis and random forest models were implemented to evaluate protein concentration differences and their predictive accuracy. Preeclampsia subgroups defined by molecular profiles were identified by hierarchical cluster analysis. Significance was set at p < 0.05 or false discovery rate-adjusted q < 0.1. In preterm preeclampsia, PlGF, PTX3, and VEGFR-1 displayed differential abundance in both soluble and EV fractions, whereas angiogenin, CD40L, endoglin, galectin-1, IL-27, CCL19, and TIMP1 were changed only in the soluble fraction (q < 0.1). The direction of changes in the EV fraction was consistent with that in the soluble fraction for nine proteins. In term preeclampsia, CCL3 had increased abundance in both fractions (q < 0.1). The combined EV and soluble fraction proteomic profiles predicted preterm and term preeclampsia with an AUC of 78% (95% CI, 66%-90%) and 68% (95% CI, 56%-80%), respectively. Three clusters of preeclampsia featuring distinct clinical characteristics and placental pathology were identified based on combined protein data. Our findings reveal distinct alterations of the maternal EV-associated and soluble plasma proteome in preterm and term preeclampsia and identify molecular subgroups of patients with distinct clinical and placental histopathologic features.

Untargeted Metabolomic Profiling of Extracellular Vesicles Isolated from Human Seminal Plasma.

Seminal extracellular vesicles (SemEVs) are repositories of biomolecules, including metabolites involved in the regulation of sperm function. The correlation between the metabolite profile of SemEVs and semen parameters, along with their role in regulating sperm function, is an unexplored area. This preliminary study evaluated the metabolomic content of SemEVs. Semen samples were obtained from 18 healthy men, and SemEVs were extracted from seminal plasma using the size exclusion chromatography qEV Gen 2-35 nm column coupled with an automatic fraction collector. The physical characterization of SemEVs was carried out with the ZetaView PMX-430-Z QUATT laser system. EV protein markers were detected using Western blot. In addition, these SemEVs were used for metabolomic profiling and functional bioinformatic analysis. The mean concentration of isolated SemEVs was 1.7 ± 1.1 × 1011/mL of seminal plasma, whereas SemEVs size and zeta potential were 129.5 ± 5.5 nm and -40.03 ± 3.99 mV, respectively. Western blot analysis confirmed the presence of EV specific markers such as CD81, ALIX, and TSG101. A total of 107 metabolites were identified using this untargeted metabolomic approach in SemEVs. Bioinformatics analysis further revealed that metabolites associated with tyrosine metabolism were highly enriched in these SemEVs. Ingenuity Pathway Analysis (IPA) also indicated that these metabolites present in SemEVs were involved in the regulation of the free radical scavenging pathway. Furthermore, our metabolomic results suggest that these SemEV-associated metabolites may play a pivotal role in the maintenance of seminal plasma redox homeostasis.

Quantitative proteomics reveals differential extracellular vesicle cargo from M1 and M2 monocyte-derived human macrophages.

Extracellular vesicles (EVs) mediate intercellular communication by carrying molecular cargo that facilitate diverse physiological processes. Macrophages, playing central roles in immune responses, release EVs that modulate various cellular functions. Given the distinct roles of M1 and M2 macrophage states, understanding the proteomic profiles of their EVs is important for elucidation of EV-mediated signalling and identifying potential biomarkers for diseases involving macrophage polarisation. We employed quantitative proteomics combined with bioinformatics to characterise the proteomic profile of EVs released by M1 and M2 monocyte-derived macrophages. We identified 1,731 proteins in M1/M2 EVs, 132 of which were significantly differentially between M1 and M2. Proteomic data, together with pathway analysis, found that M1/M2 macrophage EV cargo relate to cellular source, and may play roles in shaping immune responses, with M1 EV cargo associated with promotion of pro-inflammatory and antiviral functions, while M2 EV cargo associated with immune regulation and tissue repair. M1 EV cargo was associated with cytokine/chemokine signalling pathways, DNA damage, methylation, and oxidative stress. M2 EV cargo were associated with macrophage alternative-activation signalling pathways, antigen presentation, and lipid metabolism. We also report that macrophage EVs carry metallothioneins, and other related proteins involved in response to metals and oxidative stress.

Extracellular vesicle miRNome during subclinical mastitis in dairy cows

Bovine mastitis is one of the main inflammatory diseases that can affect the udder during lactation. Somatic cell counts and sometimes microbiological tests are routinely adopted during monitoring diagnostics in dairy herds. However, subclinical mastitis is challenging to identify, reducing the possibility of early treatments. The main aim of this study was to investigate the miRNome profile of extracellular vesicles isolated from milk as potential biomarkers of subclinical mastitis. Milk samples were collected from a total of 60 dairy cows during routine monitoring tests. Small RNA sequencing technology was applied to extracellular vesicles of milk samples collected from cows classified according to the somatic cell count to identify differences in the miRNome between mastitic and healthy cows. A total of 1997 miRNAs were differentially expressed between both groups. Among them, 68 miRNAs whose FDRs were < 0.05 were mostly downregulated, with only one upregulated miRNA (i.e., miR-361). Functional analysis revealed that miR-455-3p, miR-503-3p, miR-1301-3p and miR-361-5p are involved in the regulation of several biological processes related to mastitis, including immune system-related processes. This study suggests the involvement of extracellular vesicle-derived miRNAs in the regulation of mastitis. Moreover, these findings provide evidence that miRNAs from milk extracellular vesicles can be used to identify biomarkers of mastitis. However, further studies must be conducted to validate these miRNAs, especially for subclinical diagnosis.