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Probiotic-derived extracellular vesicles as food-based nanocarriers: Mechanisms, functional applications, and future perspectives in food systems.

Probiotic-derived extracellular vesicles (PDEVs) are a promising type of postbiotic nanoparticle derived by fermentation of probiotics, and have gained growing interest as a potential application in food science and nutrition. These are lipid bilayer vesicles of nanoscale, which are naturally released by probiotic cells and contain a wide variety of bioactive molecules, such as proteins, nucleic acids, and metabolites. Moreover, PDEVs are highly stable, biocompatible, and can be easily engineered to have surfaces with high functionality, which makes them good candidates in functional engineering. In contrast to traditional live probiotics, PDEVs overcome the difficulties of preserving microbial viability during processing and storage, thus providing superior safety, stability, and predictable biological performance. This is a systematic review of the various functions of PDEVs in food systems. We conclude on the processes through which PDEVs control intestinal barrier integrity, alter gut microbiota composition, and alter host immune responses, and their potential to enhance gut health when added to functional foods. In addition to their health-promoting effects, PDEVs have shown significant potential as natural antimicrobial agents to preserve food and as effective nanocarriers of hydrophobic bioactive compounds, including fucoxanthin, to improve their stability, bioavailability, and targeted delivery. Moreover, PDEVs can be used as new regulators of microbial fermentation. However, it should be noted that a lot of the evidence that is available is still preliminary and the effectiveness of these applications in real food-processing and storage conditions has not been fully proven. Although they have potential, there are a number of challenges that still hinder the widespread use of PDEVs in the food industry. These involve the creation of scalable and cost-effective production processes, batch-to-batch consistency, vesicle stability in a variety of food matrices, and regulatory and safety considerations. Other emerging engineering approaches, such as surface functionalization and cargo loading, are also discussed in this review and could further increase the specificity, functionality, and application versatility of PDEVs in food systems. Moving forward, the incorporation of PDEVs into the next generation functional foods, novel food preservation methods, and customized nutrition plans should be prioritized in future studies. Further developments in these fields can make PDEVs useful platforms at the interface of food microbiology, nanotechnology, and human health.

Probiotics

Multicompartment Quantitative Proteomics Revealing Potential Mechanisms Underlying the Treatment Effects of Mesenchymal-Derived Extracellular Vesicles in a Monkey Model of Cortical Injury.

Previous studies have demonstrated that mesenchymal stromal cell-derived extracellular vesicles (MSC-EVs) enhance functional recovery after cortical injury in rhesus monkeys by reducing chronic microglial inflammation, neuronal damage, and myelination deficits. However, the signaling pathways underlying these therapeutic effects remain largely unexplored. In this study, employing a reliable quantitative proteomics platform UHR-IonStar, we identified the protein cargo of MSC-EVs infused intravenously to rhesus monkeys 24 h and 2 weeks post-injury to the motor cortex. We then analyzed global protein expression changes across cerebrospinal fluid (CSF), plasma, and brain tissue of MSC-EV-treated versus vehicle-treated female, aged rhesus monkeys. A total of 1241/431/4124 monkey proteins were reliably quantified in CSF/plasma/tissue samples, respectively. Longitudinal analysis of CSF and plasma samples highlighted a shift from MSC-EV modulation of inflammatory and metabolic proteins in plasma at early recovery (2 weeks), toward modulation of plasticity-related proteins in CSF and brain tissue at later stages (4 weeks). Further protein-protein interaction analysis identified potential MSC-EV targets related to complementary signaling, proteolysis, and aminoglycan stability, which aligned with our previous findings. This comprehensive, multicompartment monkey proteomics study advances understanding of MSC-EV contents and treatment effects, paving the way for novel treatment of cortical injury.

Animals

The extracellular vesicle transcriptome provides tissue-specific functional genomic annotation relevant to disease susceptibility in obesity.

We characterized circulating extracellular vesicles (EVs) in obese and lean humans, identifying transcriptional cargo differentially expressed in obesity (277 unique genes; false discovery rate < 10%). Since circulating EVs may have broad origin, we compared this obesity EV transcriptome with expression from human visceral-adipose-tissue-derived EVs from freshly collected and cultured biopsies from the same obese individuals, observing high concordance. Using a comprehensive set of adipose-specific epigenomic and chromatin conformation assays, we found that the differentially expressed transcripts from the EVs were those regulated in adipose by body mass index-associated SNPs (p < 5 &#xd7; 10-8) from a large-scale genome-wide association study (GWAS). Using a phenome-wide association study of the regulatory SNPs for the EV-derived transcripts, we identified a substantial enrichment for inflammatory phenotypes, including type 2 diabetes. Collectively, these findings represent the convergence of the GWAS (genetics), epigenomics (transcript regulation), and EV (liquid biopsy) fields, enabling powerful future genomic studies of complex diseases.

Humans

Enzyme-Metabolite Network Analysis of Endometrial Cancer-Derived Extracellular Vesicles Through Integrated Proteomics and Metabolomics.

Endometrial cancer (EC) is the most common gynecological malignancy in high-income countries. Extracellular vesicles (EVs) are key mediators of intercellular communication and metabolic reprogramming, but their molecular cargo in EC remains poorly characterized. EVs were isolated from four EC cell lines representing Type I and Type II subtypes (AN3CA, ISHIKAWA, HEC1A, and KLE). Untargeted metabolomics was performed by HILIC-LC-MS/MS, proteomics by data-independent acquisition (DIA) mass spectrometry, and multi-omics integration using MetaboAnalyst and OmicsNet. Metabolomic profiling identified 1463 annotated features and revealed significant differences among EC cell lines (PERMANOVA, p = 0.002). Twenty-eight differentially abundant metabolites, including lactic acid, succinic acid, and uric acid, were identified. Proteomic analysis quantified 8513 proteins with subtype-specific expression patterns. Integrated analysis revealed seven significantly enriched pathways, including glycolysis/gluconeogenesis, central carbon metabolism in cancer, and the pentose phosphate pathway. Increased LDHA abundance in metastatic AN3CA-derived EVs was confirmed by Western blot (p = 0.047). EC-derived EVs display subtype- and metastatic-status-specific metabolo-proteomic signatures, with glycolysis, TCA cycle remodeling, and central carbon metabolism as convergent pathway signatures of molecular reprogramming. These findings establish a multi-omics framework for characterizing EV cargo in EC and identify candidate enzyme-metabolite nodes for future biomarker validation in patient-derived specimens.

Female

Bacterial extracellular vesicles exhibit distinct functional potential across biogeographic provinces of the South Pacific Ocean.

Bacterial extracellular vesicles (BEVs) are nanoscale membranous structures released by diverse types of bacteria, and are capable of transporting and delivering biological compounds between cells. Experimental investigation of BEVs in laboratory model systems indicates that these nanoparticles may play a number of roles in the ecophysiology of marine bacterial communities, but their functional potential in the environment remains unclear. Here we describe the proteomic composition of BEV populations over more than 5000 nautical miles of surface waters in the South Pacific, linking BEV cargoes to the bacterial communities producing them. The presence of marine BEVs was consistently observed across a range of biogeochemical conditions, with an overall abundance comparable to that of bacterial cells (up to 108 BEVs L-1). The protein cargo of marine BEVs, however, differed significantly among ocean regions. The BEV populations were enriched in carbohydrate transporters under phytoplankton bloom conditions, and contained iron uptake-related proteins in nutrient-limited waters. These data suggest that BEVs could enable cells to perform key extracellular functions in the marine environment. Our observations highlight the ubiquity of marine BEVs and biogeographic patterns in their ecological potential across oceanic scales.

Extracellular Vesicles

CircExor enables interpretable prediction of circRNA localization into extracellular vesicles.

Certain circular RNAs (circRNAs) are selectively enriched in extracellular vesicles (EVs), in which they contribute to intercellular communication and represent promising biomarkers, yet the sequence determinants of their sorting remain unclear. Existing computational predictors are optimized mainly for linear RNAs and rarely address circRNA localization into EVs. Here we introduce circExor, the first framework specifically designed for circRNA EV localization. We curate a dedicated benchmark data set of 2102 circRNAs and implement a variable-length end-to-end concatenation strategy together with k-mer frequency encoding to accommodate circular topology, long sequence length, and length heterogeneity. Using a tree-based classifier, circExor achieves superior performance compared with RNAlocate-v3 and ExoGRU, reaching an AUROC of 0.743 on the internal test set and an average AUROC of 0.680 on the held-out test set. SHAP-based analysis, sequence perturbation analysis, motif mapping, and cell-based experimental validation support the predicted EV tendency and identify YBX1, HNRNPK, HNRNPL, and NOVA2 as candidate RBPs potentially associated with circRNA sorting. CircExor therefore provides a predictive and interpretable framework that links in silico modeling to mechanistic hypotheses, and supports biomarker discovery and candidate prioritization for downstream studies of EV-associated circRNAs.

Journal Article

Parkin Induces Ubiquitination and Large Extracellular Vesicle Release of HMGB1 to Activate Antitumor Immunity.

UNLABELLED: Parkin (PRKN) is a mitochondria-associated E3 ubiquitin ligase that mediates mitophagy and organelle quality control. More recently, PRKN has been implicated in stimulating antitumor immunity and reprogramming the tumor immune microenvironment. In this study, we showed that PRKN ubiquitinates the alarmin molecule, high-mobility group box-1 (HMGB1) on Lys146 (K146) using predominantly K48 linkages. By molecular modeling, the in-between-ring domain of PRKN (Gln326-Leu358) made extensive contacts with the amino-terminus A-box of HMGB1 (Met1-Ser42), forming a mitochondria-associated PRKN-HMGB1 complex that juxtaposes K146 to ubiquitin active site residues Gly76 and Arg74. Instead of proteasomal degradation, PRKN ubiquitination of K146 enabled the loading of HMGB1 but not HMGB1 K146A mutant, onto autophagy- and mitochondria-derived large extracellular vesicles (LEV). In turn, released PRKN-HMGB1-LEV stimulated a potent IFN and cytokine response in recipient cells, expanding CD8+ T-cell subsets with effector (CD69+/KLRG1+), self-renewal (TCF1+/PD-1+), and cytotoxic (KLRG1+/GrzB+) properties. Conditional expression of PRKN induced HMGB1 release, activated intratumoral CD8+ T cells, and suppressed syngeneic tumor growth in vivo in a response that was abolished by HMGB1 silencing. These data identify that PRKN-LEV-regulated release of HMGB1 reprograms antitumor immunity via stimulation of IFN signaling and expansion of specialized CD8+ T-cell subsets. SIGNIFICANCE: Parkin ubiquitinates the alarmin molecule HMGB1 to enable its regulated release in large extracellular vesicles that activate interferon signaling, expand specialized CD8+ T-cell subsets, and promote antitumor immunity.

HMGB1 Protein

Proteomic Analysis of Extracellular Vesicles Reveals Vitronectin and Laminin Subunit Alpha-3 as Candidate Biomarkers for Gastric Cancer.

BACKGROUND/AIMS: Clinically useful noninvasive biomarkers for gastric cancer remain limited. Extracellular vesicles (EVs) carry a molecular cargo reflective of their cells of origin and have emerged as promising candidates for blood-based cancer biomarkers. We aimed to identify EV-associated protein biomarkers for gastric cancer via a proteomic approach. METHODS: Proteomic profiling of EVs was performed using one normal gastric cell line (Hs738st/int) and two gastric cancer cell lines (AGS and NCI-N87). Selected proteins were validated in blood-derived EVs isolated from plasma samples of 10 healthy controls and 36 patients with gastric cancer. RESULTS: Proteomic analysis identified 224 differentially expressed proteins whose expression was consistently altered in gastric cancer cell line-derived EVs. Among these, vitronectin (VTN) and laminin subunit alpha-3 (LAMA3) were selected based on their consistent upregulation. EV-associated LAMA3 levels were significantly higher in patients with gastric cancer than in healthy controls (p=0.003), with significant elevations observed from stage II onward (p=0.041, p=0.017, and p=0.004 for stages II, III, and IV, respectively). EV-associated VTN levels were not significantly different overall (p=0.089); however, stage-specific analysis demonstrated significant increases in VTN levels in patients with stage III (p=0.036) and stage IV (p=0.005) gastric cancer. Both EV-associated VTN and LAMA3 levels showed significant positive correlations with the cancer stage (&#x3c1;=0.564 and &#x3c1;=0.611, respectively; both p<0.001). CONCLUSIONS: The levels of EV-associated VTN and LAMA3 appear to be more closely associated with disease progression than with early-stage detection of gastric cancer. These findings suggest that EV-based proteomic biomarkers may have clinical utility for monitoring tumor progression in patients with clinically advanced gastric cancer.

Humans

Extracellular Vesicle- MDM2 -DNA as a Potential Liquid Biopsy Biomarker for Disease Identification in Retroperitoneal Liposarcoma.

OBJECTIVE: We aimed to assess the levels of MDM2 -DNA within extracellular vesicles (EVs) isolated from the serum of retroperitoneal liposarcoma (RLS) patients versus healthy donors, as well as within the same patients at the time of surgery versus postoperative surveillance visits. To determine whether EV- MDM2 may serve as a possible first-ever biomarker of liposarcoma recurrence. BACKGROUND: A hallmark of well-differentiated and dedifferentiated (WD/DD) retroperitoneal liposarcoma is elevated MDM2 due to genome amplification, with recurrence rates of >50% even after complete resection. Imaging technologies frequently cannot resolve recurrent WD/DD-RLS versus postoperative scarring. Early detection of recurrent lesions, for which biomarkers are lacking, would guide surveillance and treatment decisions. METHODS: WD/DD-RLS serum samples were collected both at the time of surgery and during follow-up visits from 42 patients, along with sera from healthy donors (n=14). EVs were isolated, DNA purified, and MDM2 -DNA levels determined through q-PCR analysis. Nonparametric tests were employed to compare EV- MDM2 DNA levels from patients versus the control group, as well as the time of surgery versus postsurgery conditions. RESULTS: EV -MDM2 levels were significantly higher in WD/DD-RLS than controls ( P =0.00085). Moreover, EV- MDM2 levels were remarkably decreased in WD/DD-RLS patients after resection ( P =0.00036), reaching values comparable to control group ( P =0.124). During postoperative surveillance, significant increases of EV- MDM2 were observed in some patients, correlating with computed tomography scan evidence of recurrent or persistent postresection disease. CONCLUSIONS: Serum EV- MDM2 may serve as a potential biomarker of early recurrent or postoperatively persistent WD/DD-RLS, a disease currently lacking such determinants.

Humans

FACS-Proteomics strategy toward extracellular vesicles single-phenotype characterization in biological fluids: exploring the role of leukocyte-derived EVs in multiple sclerosis.

BACKGROUND: The isolation and proteomics characterization of extracellular vesicles (EVs) from body fluids is challenging due to their vast heterogeneity. We have recently demonstrated that Fluorescence-activated Cell Sorting (FACS) efficiently isolates the whole EV circulating compartment directly from untouched body fluids enabling a comprehensive EV proteomics analysis. RESULTS: Here, we characterized, for the first time, a single-phenotype EV subset by sorting leukocyte-derived EVs (Leuko EVs) from peripheral blood and tears of healthy volunteers.&#xa0;Using an optimized and patented staining protocol of the whole EV compartment we identified and excluded non-EV particles, debris and damaged EVs. We further isolated, using an anti-CD45 antibody, Leuko EVs (CD45+&#x2009;EVs), reaching a high level of purity (>&#x2009;90%). Purified Leuko EVs were characterized using atomic force microscopy, nanoparticle tracking, and shotgun proteomics analysis revealing a similar coded protein cargo in both biological fluids. Subsequently, the same workflow was applied to tears from Relapsing-Remitting Multiple Sclerosis (RRMS) patients, revealing a Leuko EVs protein cargo enrichment that reflects the neuroinflammatory condition characteristics of RRMS. This enrichment was evidenced by the activation of upstream regulators TGFB1 and NFE2L2, which are associated with inflammatory responses. Additionally, the analysis identified markers indicative of endothelial cell proliferation and the development of enhanced vascular networks, with AGNPT2 and VEGF emerging as activated upstream regulators. These findings indicate the complex interplay between inflammation and angiogenesis in RRMS. CONCLUSIONS: In conclusion, our combined FACS-Proteomics strategy offers a promising approach for biomarker discovery, analysing cell-specific EV phenotypes directly from untouched body fluids, advancing the clinical value of tears EVs and improving the understanding of EV-mediated processes in vivo. Data are available via ProteomeXchange with the identifier PXD049036 and in EV-TRACK knowledgebase with ID: EV240150.

Humans

Downregulated lysyl oxidase in plasma extracellular vesicles: a biomarker linked to brain metastasis risk in lung adenocarcinoma.

BACKGROUND: Brain metastasis (BrM) is a leading cause of mortality in patients with lung adenocarcinoma (LUAD). Extracellular vesicles (EVs), which carry bioactive molecules, play a critical role in tumor microenvironment remodeling and exhibit metastatic organotropism, holding promise as liquid biopsy biomarkers. This study aims to identify plasma EV-derived proteins associated with LUAD-BrM. METHODS: A multi-omics framework was applied. Plasma EVs from 59 stage IV LUAD patients (30 BrM vs 29 non-BrM) were profiled using data-independent acquisition mass spectrometry proteomics. Candidate proteins were screened via bioinformatics and machine learning (LASSO/RF/SVM). Initial validation included tissue proteomics (n&#x2009;=&#x2009;13), single-cell transcriptomics (TISCH2), and Western blot analysis of a subset of the discovery samples. Functional experiments were conducted in vitro. The lead candidate was ultimately validated in an independent plasma cohort (n&#x2009;=&#x2009;158) through ELISA. RESULTS: Proteomic analysis implicated collagen-containing extracellular matrix (ECM) pathways. Lysyl oxidase (LOX), a key ECM cross-linking enzyme, was identified as a lead candidate. LOX and its family member LOXL1 were consistently downregulated in BrM tissues and plasma EVs. Single-cell analysis revealed decreased LOX expression specifically in BrM-associated fibroblasts, which showed suppressed ECM-related pathways. In vitro experiments supported a PI3K/AKT-LOX-ECM regulatory axis. Plasma EV-derived LOX demonstrated strong diagnostic performance in the independent cohort, with an AUC of 0.786 (95% CI 0.713iated fi. CONCLUSIONS: Our study establishes plasma EV-derived LOX as a promising non-invasive biomarker for LUAD-BrM through a comprehensive multi-omics validation strategy. We propose a model wherein downregulation of LOX, potentially driven by PI3K/AKT signaling in tumor-associated fibroblasts, contributes to ECM degradation and may promote brain-tropic metastasis. This finding offers new insights for risk stratification and timely intervention in LUAD patients.

Humans

Small extracellular vesicles are the key players in ochratoxin A-induced kidney toxicity.

BACKGROUND: Despite growing evidence of ochratoxin A (OTA)-induced kidney toxicity, the underlying mechanisms remain elusive. Emerging evidence suggests that small extracellular vesicles (sEVs) act as mediators of intercellular communication to recipient cells during various physiological and pathological conditions. Given the distinctive properties of sEVs, it is hypothesized that OTA-induced sEVs might mediate the OTA-induced kidney pathogenesis. METHODS: To explore the involvement of sEVs in OTA-induced kidney toxicity, sEVs were isolated and characterized from OTA-exposed rat kidney epithelial cells (NRK52E). Later, these sEVs were used to treat NRK52E cells and Wistar rats to assess the impact of OTA-induced sEVs on kidney toxicity. Label-free proteomics was also performed on OTA-induced sEVs, and key proteins were identified and validated. The biodistribution of sEVs in rats was also assessed using live imaging. The role of validated protein/s in kidney toxicity was further confirmed via a gene silencing and overexpression study. RESULTS: OTA exposure increased sEV secretion into conditioned media of NRK52E cells and into the urine of Wistar rats. Interestingly, we found that OTA-induced sEVs cause similar kidney toxicity in vitro and in vivo systems as OTA exposure, and blocking of sEV secretion markedly alleviated OTA-mediated kidney toxicity. Proteomics analysis identified annexin A2 and fibrinogen-&#x263; as common proteins detected in sEVs derived from OTA-exposed NRK52E cells or rat urine. However, immunoblotting validated that annexin A2 was the only sEV-associated protein, expressed significantly in both NRK52E and rat urine following OTA exposure. Notably, silencing of annexin A2 attenuated the ability of OTA-induced sEVs to cause kidney toxicity, whereas overexpression exacerbates it. CONCLUSIONS: Our findings identify the annexin A2-enriched sEVs as key mediators of OTA-induced kidney toxicity. Annexin A2, along with other kidney injury markers, offers a promising non-invasive translational biomarker for early detection and monitoring of OTA-induced kidney toxicity.

Ochratoxins

Extracellular Vesicles From Glioblastoma Cells Reflect 2D vs. 3D Culture Adaptation and Resistance to Temozolomide.

Glioblastoma (GBM) is an aggressive brain tumor marked by extensive heterogeneity, resistance to therapy, and dismal prognosis. Extracellular vesicles (EVs) have emerged as key players in GBM biology, mediating intercellular communication and therapy adaptation. However, the exact functions and molecular impact of EVs in GBM remain incompletely understood. In this study, we performed a comparative proteomic analysis of U87MG GBM cells grown in two-dimensional (2D) monolayers and three-dimensional (3D) spheroids following temozolomide (TMZ) treatment, alongside characterization of EVs derived from both culture systems. 3D-spheroids secreted more EVs of smaller size and exhibited a more TMZ-resistant, stem-like proteome under TMZ-induced genotoxic stress. In contrast, 2D cell cultures demonstrated greater proteome remodeling, with EVs enriched in protein families involved in DNA repair, oxidative stress adaptation, and methylation processes. Notably, several methyltransferases were decreased intracellularly but selectively retained in EVs, suggesting active sorting to influence the tumor microenvironment or modulate epigenetic states in recipient cells. EVs also carried adhesion molecules and signaling proteins linked to migration, invasion, and Wnt pathway activation, as well as metabolic enzymes connecting serine metabolism and redox control to TMZ resistance. Mapping EV and cellular proteomes onto The Cancer Genome Atlas (TCGA) dataset identified prognostic protein families associated with either poor or favorable patient outcomes. Our data demonstrate that EV cargo composition mirrors TMZ-induced phenotypic adaptation and reveals molecular mechanisms underlying therapeutic resistance. These EV-associated signatures may serve as clinically actionable biomarkers for patient stratification and offer potential targets to overcome chemoresistance in GBM.

Humans

MicroRNAs signatures in small extracellular vesicles for psychological resilience in young adults using machine learning.

AIMS: Psychological resilience refers to an individual's capacity to adapt to adverse events. MicroRNAs (miRNAs) play a crucial role in regulating post-transcriptional processes, while small extracellular vesicles (sEVs) act as transport vehicles. This study aimed to employ genome-wide profiling to identify and validate differences in the expression of resilience-associated sEV-miRNAs between low resilience (LR) and high resilience (HR) in young adults. METHODS: Eighty participants were divided into LR or HR based on the Connor - Davidson Resilience Scale (CD-RISC). The expression levels of the target sEV-miRNAs in LR and HR were compared and analyzed. RESULTS: Expression analyses demonstrated significant differences in let-7b, miR-151b, miR-335, and miR-193a between LR and HR (p&#x2009;<&#x2009;0.01), with let-7b showing the highest discriminative ability. The AUC values for each sEV-miRNA ranged from 0.74 to 0.94, based on logistic regression and three machine learning models: random forest, support vector machine, and eXtreme gradient boosting. Based on leave-one-out cross-validation in different models, the combined four sEV-miRNAs demonstrated strong performance for detecting LR (AUC&#x2009;=&#x2009;0.87-0.90). Sex-specific differences were also observed, with female participants showing more pronounced resilience signatures in targeted sEV-miRNAs. CONCLUSIONS: These findings suggest that sEV-miRNAs hold potential as biomarkers for psychological resilience in young adults.

Humans

TNF&#x3b1;-induced endothelial extracellular vesicles regulate astrocyte function: an integrated transcriptomic and proteomic study.

Endothelial cells and astrocytes are critical structural and functional components of the blood-brain barrier. In many neuroinflammatory diseases, endothelial cells are among the first to respond to inflammatory stimuli and release extracellular vesicles (EVs). However, whether inflammatory stimulation alters EV RNA cargo and subsequently regulates astrocyte function remains unclear. In this study, we performed integrated RNA sequencing and proteomic analyses to investigate the effects of TNF&#x3b1;-stimulated endothelial EVs on astrocytes. RNA profiling revealed significant alterations in EV cargo after TNF&#x3b1; stimulation, including 867 upregulated and 577 downregulated mRNAs, 317 upregulated and 15 downregulated lncRNAs, and 88 upregulated and 62 downregulated miRNAs. The results of functional enrichment analysis suggested that altered EV RNAs may primarily promote inflammatory responses, cell migration, and RNA splicing in astrocytes while reducing their regulatory effects on neuronal projection and calcium homeostasis. Further integrative analysis of EV RNAs and astrocytic proteomics revealed key overlapping targets, including upregulated expression of ICAM1, SOD2, TFPI2, and TNFAIP8, whereas NFKBIA expression was consistently decreased. Network analysis revealed NF-&#x3ba;B as the central regulatory node. Reduced levels of EV-derived NFKBIA mRNA were associated with decreased I&#x3ba;B&#x3b1; protein levels in astrocytes, which promoted NF-&#x3ba;B activation and inflammatory cytokine release. Finally, overexpression of I&#x3ba;B&#x3b1; in astrocytes significantly attenuated TNF&#x3b1; EV-induced IL-1&#x3b2; and IL-6 secretion. Collectively, these findings demonstrate that TNF&#x3b1;-stimulated endothelial EVs coordinately regulate astrocyte function through mRNA, lncRNA, and miRNA cargo and that the I&#x3ba;B&#x3b1;/NF-&#x3ba;B axis may be a key mechanism underlying endothelial EV-mediated inflammatory disruption of the blood-brain barrier.

Astrocytes

Food-derived extracellular vesicles as delivery platforms for medicine-food homology components in metabolic syndrome.

Diet-induced obesity and associated metabolic syndromes have become major global public health challenge, highlighting the urgent need for safe and effective strategies. Recently, food-derived extracellular vesicles (FDEVs) have garnered increasing attention as natural nanocarriers due to their excellent biocompatibility and specific targeted delivery capabilities. FDEVs can efficiently deliver medicine-food homology components (MFHCs) to precisely regulate lipid metabolism, inflammatory responses, and insulin sensitivity, thereby improving obesity and its metabolic abnormalities. This systematic review summarizes recent advances in the use of FDEVs as delivery vehicles for MFHCs to suppress diet-induced obesity and metabolic syndrome, with a particular focus on the underlying molecular mechanisms, including signaling pathway regulation and cellular metabolic remodeling. In addition, the clinical translational potential and industrial application prospects of FDEVs are evaluated, and key challenges related to preparation techniques, safety assessment, and large-scale production are discussed. By integrating current evidence, this review aims to provide theoretical framework and future perspectives for the development of FDEVs as a novel targeted delivery platform and treatment of metabolic diseases.

Extracellular Vesicles

TGF-&#x3b2; Receptor-dependent Tissue Factor Release and Proteomic Profiling of Extracellular Vesicles from Mechanically Compressed Human Bronchial Epithelial Cells.

In asthma, tissue factor (TF) concentrations are elevated in the lung. In our previous studies using mechanically compressed human bronchial epithelial (HBE) cells, which are a well-defined in&#xa0;vitro model of bronchoconstriction during asthma exacerbations, we detected TF within extracellular vesicles (EVs) released from compressed HBE cells. Here, to better characterize the potential role of this mechanism in asthma, we tested the extent to which the transcriptional regulation of epithelial cell-derived TF varied between donors with and without asthma. Using RNA in situ hybridization, we detected epithelial expression of F3, the TF protein-encoding gene, in human airways. Next, to determine the role of TGF-&#x3b2; receptor (TGF-&#x3b2;R) in the regulation of TF, we exposed well-differentiated HBE cells to mechanical compression in the presence or absence of a pharmacological inhibitor of TGF-&#x3b2;R. Furthermore, to identify the protein cargo of EVs released from HBE cells, we used tandem mass tag mass spectrometry. Our findings revealed significantly higher F3 expression in the airways of patients with asthma compared with healthy control subjects. However, we observed no differences in F3 expression or TF release between asthmatic and nonasthmatic HBE cells, both at baseline and after compression. Mechanistically, compression-induced F3 expression in HBE cells depended on TGF-&#x3b2;R. Our proteomic analysis identified 22 differentially released proteins in EVs, with higher concentrations in compressed cells compared with controls. Gene Ontology analysis indicates that these proteins are involved in diverse biological processes, highlighting a potential role for epithelial cell-derived EVs during asthma exacerbations.

Humans

Tumor-associated macrophages display differential protein cargo sorting in extracellular vesicles associated with poor survival in ovarian cancer.

Ovarian cancer (OC) progression and metastasis are promoted by ascites, which constitutes a central part of the tumor microenvironment (TME). In this fluid, tumor-associated macrophages (TAMs) represent a prominent immune cell type. In addition to tumor and other host cells such as TAMs, ascites is highly enriched in soluble factors as well as extracellular vesicles (EVs). How TAMs contribute to the EV compartment of the OC TME remains, however, underexplored. In this work peripheral blood monocytes from healthy donors were differentiated into monocyte-derived macrophages (MDMs) and polarized into classically activated (M1-like), alternatively activated (M2-like) and TAM-like (by ascites incubation). For all subtypes, serum-free conditioned medium was collected for 24&#xa0;h and EVs were isolated and characterized by nano-flow cytometry (nFC), label-free mass spectrometry-based proteomics and electron microscopy, among others. Our results demonstrated distinct traits for EV release and cargo across the different macrophage subtypes. Specifically, TAM-like macrophages exhibited impaired release of small EVs and reduced frequency of tetraspanin-positive particles. These EV subpopulations displayed sizing profiles closer to M1-like than to M2-like samples. Also, the low EV release in TAM-like MDMs was accompanied by altered expression of biogenesis-related markers like flotillin-1 (FLOT1) and a decreased N-glycosylation of CD63 protein, which was validated in patient-derived samples. Remarkably, the EV-associated proteome of TAMs displayed significant enrichment in both pro- and anti-inflammatory molecules with clinical value. Markers significantly enriched in the ascites TAM-EV signature were mostly associated with poor prognosis, whereas M1-like EV-related markers (pro-inflammatory) were mostly associated with longer survival. Our results confirmed previous data for proteins like CD163 and MRC1 to be associated to TAM-EVs, while also describing novel candidates with diagnostic (i.e., COLEC12) and/or prognostic (i.e., MSR1) value in plasma. Taken together, our data support a unique secretory profile of TAMs in OC and provide new EV-associated biomarkers with translational impact. Our results pave the way for a better understanding of the mechanisms behind TAM-EV cargo loading and function, and how these cells participate in the TME landscape.

Humans