Japanese encephalitis (JE) caused by Japanese encephalitis virus (JEV) infection remains one of the major types of viral encephalitis. The mechanisms underlying JEV infection and pathogenesis have not been fully elucidated, resulting in the absence of specific therapies for JE. Neuronal apoptosis is a well-established outcome of JEV infection; however, its secondary contributions to JE pathogenesis remain largely uncharacterized. Here, we demonstrate that apoptotic bodies (ApoBDs) derived from JEV-infected neurons package infectious JEV particles, facilitating viral transmission between neurons, as well as between neurons and microglia. Further investigations revealed that microglia predominantly internalize the ApoBDs via phagocytosis and dynamin 2-dependent endocytosis. Moreover, lipids, rather than nucleic acids or proteins, are the key pro-inflammatory constituents of ApoBDs. Subsequent studies demonstrated that ApoBDs activate microglia through the TLR2/TLR4-NF‑κB signalling. In vivo experiments showed that JEV-loaded ApoBDs elicit significantly more severe neuroinflammation and pathological lesions compared to ApoBD-free virions. These findings underscore the non-negligible contribution of neuron-derived ApoBDs to JEV pathogenesis, providing valuable insights for the development of novel therapeutic strategies against JE.
Ovarian cancer remains the most lethal gynaecological malignancy, largely due to late-stage diagnosis and the limited sensitivity of CA-125 for borderline and low-grade tumours. Dysregulated lipid metabolism may provide complementary diagnostic information. This study aimed to develop lipid-based biomarkers to improve the differential diagnosis of adnexal masses. We profiled total plasma and small extracellular vesicle (sEV) lipids in women with benign, borderline and malignant ovarian tumours, using healthy women as controls. Solid-phase extraction demonstrated superior reproducibility, lipid-class specificity and sEV lipid enrichment compared with liquid-liquid extraction. Combined with CA-125, both plasma and sEV lipid biomarkers outperformed CA-125 alone for distinguishing borderline and low-grade serous ovarian cancer from benign tumours (accuracy up to 0.89 vs. 0.67). In patients with normal CA-125 levels, sEV lipids provided the greatest diagnostic advantage, distinguishing invasive and borderline tumours from benign adnexal masses (AUROC up to 0.82). Generative artificial intelligence was used to simulate biomarker performance in synthetic cohorts for differential diagnosis (n = 10,000) and screening (n = 100,000). For differential diagnosis, sEV lipids combined with CA-125 outperformed plasma lipids for identifying endometrioid and mucinous ovarian cancer from benign adnexal masses. For screening, sEV lipids alone achieved accuracies of 0.91 and 0.94 for benign conditions and high-grade serous ovarian cancer, respectively. Simulations restricted to patients with normal CA-125 levels confirmed that sEV lipids provided the highest discriminative performance for low-grade serous ovarian cancer (AUROC up to 0.74), demonstrating their potential where CA-125 alone fails. To the best of our knowledge, this is the first study to directly compare matched plasma and sEV lipid profiles across ovarian cancer histotypes, demonstrating a diagnostic advantage for sEV lipids-particularly for borderline, low-grade serous and mucinous tumours, supporting their integration alongside CA-125 in future differential diagnosis and screening strategies.
Despite advances in percutaneous coronary intervention, ischemia-reperfusion (IR) injury remains a major cause of morbidity and mortality. Adiponectin confers broad cardioprotective effects, motivating the development of adiponectin receptor agonists. Here, we investigated the cardioprotective efficacy and mechanisms of ALY688, a synthetic adiponectin receptor agonist peptide, in myocardial IR injury. In a clinically translatable rat IR model, intravenous administration of ALY688 during ischemia together with subcutaneous dosing that continued for 28 days reduced troponin-I levels, cardiomyocyte death, and infarct size, while preserving cardiac function. ALY688 restored autophagic flux, mitigated reactive oxygen species accumulation, and suppressed apoptosis in both IR hearts and hypoxia-reoxygenation (HR)-treated cardiomyocytes. Proteomic profiling revealed that Rab8a, downregulated by IR, was maintained with ALY688 treatment. Notably, ALY688 increased extracellular vesicle (EV) abundance in myocardium and plasma, and EVs from treated animals displayed distinct proteomic signatures enriched in glycolytic and oxidative stress-related proteins. These EVs conferred protection against HR-induced injury in H9c2 and human iPSC-derived cardiomyocytes. CRISPR-mediated Rab8a knockout impaired ALY688-induced EV biogenesis and attenuated the cytoprotective effects of these EVs. Collectively, these findings identify ALY688 as a promising therapeutic that mitigates IR injury via both direct myocardial protection and Rab8a-dependent EV-mediated cardioprotective signalling.
Pharmacological tools to selectively modulate extracellular vesicle (EV) secretion are scarce. Here, we identify the ALK5 (TGF-β receptor I) inhibitor SD-208 as a potent suppressor of small EV (sEV) secretion that acts independently of its canonical anti-fibrotic activity. SD-208 not only reversed myofibroblast activation but also markedly inhibited sEV secretion. Strikingly, this inhibitory effect persisted in non-activated cardiac fibroblasts and non-fibrotic HEK293 cells, demonstrating that SD-208 regulates EV secretion through mechanisms uncoupled from TGF-β/Smad signalling. Mechanistic analyses revealed that SD-208 disrupts vesicle trafficking rather than EV biogenesis. Reduced secretion of CD63+ EVs was accompanied by intracellular accumulation of CD63+ structures and their selective diversion into LAMP1+ lysosomes. Proteomic profiling of SD-208-treated and control HEK293 cells and cardiac fibroblasts revealed dysregulation of vesicle trafficking pathways, enrichment of ubiquitin ligase complexes, and enhanced endosome-to-lysosome transport. Together, these findings demonstrate that SD-208 diverts CD63+ multivesicular bodies (MVBs) from a secretory fate toward lysosomal degradation. This work identifies SD-208 as a small-molecule tool to interrogate the secretory-versus-degradative fate of MVBs and uncovers a new regulatory link between lysosomal pathways and EV trafficking. Beyond its established role as an anti-fibrotic agent, SD-208 provides mechanistic and therapeutic opportunities for the control of EV secretion in diseases such as fibrosis, cardiac remodelling, hypertrophic cardiomyopathy, and cancer.
Neutrophils effectively eliminate Candida albicans from human blood, but a subset of fungal cells escapes clearance and remains extracellular and viable. Here we show that this evasion is independent of known immune-escape traits of C. albicans. Instead, neutrophil-derived extracellular vesicles (EVs) enriched in antimicrobial proteins and neutrophil surface markers (CD66b, CD45, CD63, and complement receptors CR1, CR3 and CR4) promote this state. Isolated EVs bound to C. albicans preferentially in a complement-dependent manner, and this binding was partially inhibited by anti-CD11b, supporting CR3 involvement. Despite their antimicrobial cargo, EVs did not impair fungal growth. Instead, EV coating reduced neutrophil phagocytosis in purified-cell and whole-blood settings. These findings reveal a dual role for neutrophil-derived EVs at the host-pathogen interface: although enriched for innate effector molecules with potential antifungal activity, their deposition on C. albicans does not impair growth but is associated with reduced phagocytosis and maintenance of an extracellular population.
Helminths modulate host immunity, and in this respect, the porcine whipworm Trichuris suis has been explored clinically as an immunotherapy for inflammatory diseases. T. suis secretes a complex mixture of excretory/secretory products (ESP), including extracellular vesicles (EVs), nanosized membranous particles with bioactive cargoes of lipids, proteins and nucleic acids that are implicated in intercellular communication within organisms. Here, we characterise the EV protein and miRNA cargo of T. suis to identify potential roles in host-parasite communication. In contrast to EVs from clade III/V helminth species characterised to date, we show that T. suis EVs did not suppress LPS-induced TNF production by human macrophages under the conditions tested, in contrast to non-vesicular ESP fractions which did. By generating the first extracellular miRNA complement for T. suis and quantifying EV-associated versus free miRNAs across larval and adult stages, we identify selective enrichment of two miRNAs in adult EVs with homology to human miR-22-3p and miR-29-3p, canonical anti-fibrotic and tumour-suppressor miRNAs. Functional assays in human colon cancer epithelial cells reveal that these helminth-derived orthologues downregulate conserved oncogenic targets, indicating evolutionary conservation of their regulatory capacity. Our findings uncover a previously unrecognised axis of whipworm biology in which selective EV-mediated export of anti-fibrotic miRNAs has the potential to modulate host tissue remodeling independently of classical immunomodulation, with implications for understanding helminth-driven epithelial homeostasis and cancer risk.
ABSTRACT Extracellular vesicles (EVs) offer compelling opportunities for diagnostics and therapeutics, yet translation toward routine clinical use and commercial readiness remains limited. In diagnostics, EV‐based assays may reach practice as laboratory‐developed tests, while in therapeutics unproven “exosome” offerings have prompted safety communications, together underscoring the need for fit‐for‐purpose regulatory science. Here, we organize implementation challenges into six recurring bottlenecks: Identity; Purity; Potency; Measurement comparability; Manufacturing control; and Safety. We summarize how international standardization efforts led by the International Society for Extracellular Vesicles (ISEV), including MISEV2023 and task‐force outputs, strengthen reproducibility and comparability, while highlighting remaining gaps that arise when descriptive guidance must be converted into operational specifications and submission‐ready evidence packages. Using Japan as a case example, we illustrate how regulatory‐authority discussions, professional‐society statements, and emerging implementation‐oriented guidance can clarify review questions, distinguish therapeutic EV products from conditioned medium, and support trust‐preserving translation. Finally, we propose a non‐binding, deliverables‐based roadmap across near‐, mid‐, and long‐term horizons to enable clinical and commercial readiness. The roadmap should be adapted to product context, risk profile, and jurisdiction, and it emphasizes precompetitive collaboration across academia, industry, and government to build shared infrastructure for measurement, standardization, reproducibility, and quality evaluation, thereby translating scientific consensus into operational development and review frameworks.
ABSTRACT Tumour‐derived small extracellular vesicles (sEV) are increasingly recognized as key mediators of cancer progression and metastasis by facilitating intercellular communication. In this study, we identify nidogen 1 (NID1), a basement membrane glycoprotein, as significantly enriched in sEV from metastatic hepatocellular carcinoma (HCC) cells. We demonstrate that the sEV‐mediated delivery of NID1 enhances oncogenic behaviours, including proliferation, invasion and metastasis. Knockout mice of NID1 exhibited a marked delay in liver tumour formation induced by N‐diethylnitrosamine (DEN) and carbon tetrachloride (CCL4), or hydrodynamic injection delivery of oncogenes, underscoring the role of NID1 in hepatocarcinogenesis. Importantly, we find that a stiffer extracellular matrix, a characteristic of tumour microenvironments, stimulates increased NID1 level in sEV. Using NID1 mutants, we identify the C‐terminal domain as critical for its oncogenic functions, including promoting tumour growth and metastasis. Proteomic profiling reveals that transglutaminase 3 (TGM3) interacts with NID1 via its C‐terminus. Subsequent analyses indicate that sEV‐NID1 elevates TGM3 levels, leading to dysregulation of integrins and activation of the YAP/TAZ signalling pathway, thereby contributing to tumour progression. To explore therapeutic potential, we developed a novel monoclonal anti‐NID1 antibody (anti‐NID1 mAb) and evaluated its efficacy in multiple HCC mouse models. The antibody markedly suppressed liver tumour growth and metastasis of human and murine HCC cells. The therapeutic effect was further enhanced when combined with sorafenib, a standard treatment for HCC. Our findings delineate a novel sEV‐NID1‐TGM3 regulatory axis that promotes hepatocarcinogenesis and metastasis and suggest that targeting sEV‐delivered NID1 with our homemade monoclonal antibody offers a promising avenue for therapeutic intervention in HCC.
ABSTRACT Bacterial outer membrane vesicles (OMVs) are nano‐sized, spherical structures released by Gram‐negative bacteria that play diverse roles in bacterial physiology, including communication, nutrient acquisition and host interactions. These vesicles bud from the bacterial outer membrane and contain lipopolysaccharides, periplasmic proteins, nucleotides and other biomolecules. The vesicle nucleating peptide (VNp) is a short peptide tag that, when fused to the amino terminus of a protein of interest, promotes the formation of bespoke recombinant extracellular vesicles (EVs) in Escherichia coli, enabling efficient production and simplified purification of recombinant proteins. Here, we characterise VNp‐induced extracellular vesicles (VNp‐EVs) and compare their composition and organisation with OMVs produced from E. coli expressing a periplasmic targeting fusion. While both vesicle types possess a single outer membrane‐derived lipid bilayer, recombinant protein is highly enriched within the VNp‐EVs compared to OMVs containing the periplasm targeting ssDsbA‐fusion protein. VNp‐fusions and the periplasm‐targeted recombinant protein localise to distinct vesicle populations, with VNp‐fusions showing markedly higher luminal concentrations and relative vesicular abundance, compared to the vesicles containing a periplasmic targeted fusion protein. OmpX co‐expression further enriched the VNp‐fusion content of vesicles, further enhancing yield. The VNp‐vesicle lumen is an oxidising environment, thus supports formation of inter‐ and intra‐molecular disulfide bonds within encapsulated proteins. Overall, VNp‐EVs represent a distinct class of recombinant EVs that offer a simple and efficient route for producing and purifying concentrated, correctly folded recombinant proteins, expanding the utility of bacterial vesicle systems for biotechnological applications.
ABSTRACT The biogenesis of small extracellular vesicles (sEVs) is only partially understood. Our recent findings provide evidence that a newly described sEV secretion pathway, the amphiectosome release followed by the sEV discharge by the ‘torn bag mechanism’ are present in all tested cell lines and mouse organs. Surprisingly, in in situ fixed steady‐state cells, transmission electron microscopy did not reveal sEV release via exocytosis of multivesicular endosomes (MVEs). In the current study, we extended our previous analysis to additional mouse organs and confirmed the presence of secreted amphiectosomes in all of them. Furthermore, we investigated which parameters influence the activation of the distinct sEV release mechanisms in HEK cells. Our results show that under stress conditions (such as Ca2+ ionophore‐induced membrane stress or metabolic stress, induced by serum starvation), exocytosis of MVEs is activated, while this process is absent in steady‐state conditions. By silencing ATG5 (a key regulator of autophagy) and RAB27a (an essential small GTPase for MVE exocytosis), we selectively modulated these two mechanisms, respectively. Amphiectosome release depended on both autophagy and ATG5, while exocytosis of MVE was autophagy‐independent but RAB27a‐dependent. Our findings suggest that sEV release via the ‘torn bag mechanism’ is a general and essential secretion pathway in non‐stressed, steady‐state mammalian cells, while stress conditions induce the sEV release via MVE exocytosis.
ABSTRACT Plasma proteins are increasingly recognized as key regulators of extracellular vesicle (EV) behaviour in circulation. However, von Willebrand factor (vWF)—the largest multimeric glycoprotein in blood and the only blood protein activated by shear—has received little attention in this context. Under elevated shear stress, vWF transitions from a compact globular form to an extended adhesive conformation. Although elevated plasma vWF levels are associated with hypercoagulation in conditions such as malaria, COVID‐19, and cancer, its interactions with EVs or cells under physiological flow have not been systematically investigated. Here, we show that shear‐activated vWF functions as a size‐selective molecular filter that preferentially captures objects smaller than ∼4 µm, including platelets and tumour‐derived EVs, while excluding larger cells. Although intact tumour cells do not directly bind vWF under physiological flow, the coordinated binding of EVs and platelets to extended vWF promotes platelet aggregation, which subsequently traps circulating tumour cells and fosters metastatic dissemination. Our findings reveal a previously unrecognized role of vWF as a shear‐dependent EV‐binding protein that brings EVs and platelets together to enhance coagulation and metastasis. These EV–vWF–platelet aggregates may represent promising biomarkers or therapeutic targets for the prevention of hypercoagulation in cancer and other thrombo‐inflammatory diseases.
ABSTRACT Extracellular vesicles (EVs) mediate intercellular communication within the tumour microenvironment by carrying cargoes from paracrine parent cells. EVs have attracted great research interest for their ability to carry nucleic acids into recipient cells and modulate cellular functions. However, previous studies have largely focused on RNA sequence information rather than RNA structure features. Here, we observed that EVs derived from colorectal cancer cells are enriched with endogenous double‑stranded RNA (dsRNA), a danger‑associated molecular pattern (DAMP) that leads to the activation of dsRNA‑sensing pathways in recipient cells. Crucially, we investigated the specific crosstalk between tumour‐derived EVs and circulating platelets. As anucleate cells, platelets are uniquely suited models for isolating the effects of exogenous nucleic acids. Our analysis reveals that endogenous dsRNA from tumour EVs activates the platelet OAS–RNASEL innate immune ribonuclease cascade and the RNASEL/ABCE1/PELO axis, resulting in the decay of ribosomal protein mRNAs. This study, spanning from clinical observation to mechanistic validation, uncovers a novel pathway of tumour–platelet communication. We identify EV‐enriched endogenous dsRNA as a functional mediator that enables tumour cells to directly reprogram platelet transcriptomes, revealing a new dimension of tumour–immune modulation.
ABSTRACT Non‐Hodgkin lymphoma (NHL), predominantly B cell lymphomas (B‐NHL), is currently treated with chemotherapy combined with rituximab (RTX), an anti‐CD20 monoclonal antibody. Despite substantial therapeutic advances, treatment resistance and disease relapse continue to affect a significant fraction of patients. The mechanisms by which the tumour microenvironment and other factors influence RTX efficacy are not fully elucidated. Herein, we hypothesized that CD20+ extracellular vesicles (EVs) shed by B cell lymphomas are recognized by RTX forming immune complexes that modulate natural killer (NK) cell activity via Fcγ receptor (FcγR) interactions. EVs isolated from lymph node explants and plasma samples of B‐NHL patients contained abundant CD20+ vesicles, which were particularly enriched in advanced disease. RTX specifically bound CD20 on these EVs, generating EV‐RTX immune complexes. Functional studies employing EVs from a B‐NHL cell line and from patient‐derived samples demonstrated that, whereas EVs suppressed NK cell activation and cytotoxicity, EV‐RTX immune complexes reversed this inhibitory effect and enhanced NK cell effector functions. Indeed, EV‐RTX immune complexes specifically triggered FcγRIIIa‐dependent NK cell activation, evidenced by increased Syk phosphorylation, CD69 expression, and enhanced lytic activity against target cells. Our findings uncover a previously unrecognized mechanism by which RTX, through the formation of immune complexes with CD20+ EVs, promotes NK cell activation and may enhance therapeutic efficacy. More broadly, these results demonstrate that antibody binding can endow EVs with novel immunomodulatory properties, revealing a potential mechanism by which therapeutic antibodies reshape EV function and influence anti‐tumour immunity.
ABSTRACT Temporomandibular joint osteoarthritis (TMJ‐OA) is highly prevalent with an insidious onset. Severe inflammation and significant degenerative changes are often associated with the condition, and current clinical treatments remain inadequate. In this study, we focus on pyroptosis and engineered the camouflage protein (GSDMD‐C) attached to the membrane surface of small extracellular vesicles (sEVs)–sEV‐p. sEV‐p has two key effects: firstly, the inherent immunomodulatory and nutritional support properties of sEVs promote the recovery of cellular function under pathological conditions; secondly, camouflage protein bind to the activated caspase‐1, reducing the cleavage of endogenous GSDMD. The therapeutic effects of sEV‐p were evaluated through in vitro experiments and treatment of TMJ‐OA models in mice and Bama pigs. We further elucidated the mechanisms by single‐cell RNA sequencing analysis. Results show that sEV‐p alleviates the abnormal activation of inflammatory factors induced by pyroptosis, accompanied by a reduction in the proportion of inflammatory cells and a mitigation of acute inflammatory responses.
ABSTRACT Efficient production of iron‐engineered extracellular vesicles (EVs) is of interest for their use as delivery systems in biotherapy. In this study, we investigated the role of iron in EV biogenesis and evaluated a scalable fluidic strategy for their mass production using turbulence stimulation in bioreactors. Murine mesenchymal stem cells were loaded with iron via ferric quinate or iron oxide nanoparticles (IONPs) and subsequently subjected to turbulence stimulation or serum starvation to induce EV secretion. Using elemental spectroscopy and cryogenic electron microscopy, we found that prolonged incubation with ferric quinate combined with turbulence stimulation resulted in the highest EV yield—60,000 EVs per cell in 3 h—and enabled efficient iron transfer from cells to EVs. In contrast, IONP treatment did not increase EV yield compared to control and showed high variability in iron transfer, as probed by EV magnetophoretic mobility. TEM and XEDS analyses confirmed ferritin‐bound iron encapsulation within EVs, whereas IONPs were poorly internalised. These results highlight the promising role of ionic iron in EV formation and content, demonstrating how iron source, concentration, and incubation duration influence both EV biogenesis and iron engineering.
ABSTRACT Cerebrospinal fluid (CSF) is drained into the systemic lymphatics via paravertebral lymph nodes. Superficial and deep cervical lymph nodes collect CSF in mice, but the exact and quantified routes are unknown. Recently, we simultaneously visualized cervical, sacral and iliac lymph nodes via serial imaging on the intrathecal [64Cu]Cu‐albumin positron emission tomography. Paravertebral lymph nodes might act as sentinels to monitor the CSF, brain, and spinal cord. We used 64Cu‐labeled Escherichia coli extracellular vesicles, outer membrane vesicles (OMVs), as lymph node seekers for intrathecal administration and quantified the differential amounts of various paravertebral lymph nodes along the axis of the brain and spinal cord in mice. The quantified results revealed 77.3% in superficial and deep cervical lymph nodes, 11.4% in abdominal/pelvic lymph nodes and 11.3% in sacral lymph nodes. Click‐labeled [64Cu]Cu‐OMVs were drained to reach and stop at the lymph nodes on serial quantification. The cervical lymph nodes drained most of the OMV‐laden CSF, which is proportional to the surface areas of the brain (70%) and spinal cord in mice. We propose that all paravertebral lymph nodes monitor the segmental regions of the brain and spinal cord as immediate sentinel lymph nodes against the central nervous system.
ABSTRACT Metabolic dysfunction‐associated steatotic liver disease (MASLD) and metabolic dysfunction‐associated steatohepatitis are leading causes of chronic liver disease, yet the contribution of neutrophils to early hepatocellular lipid accumulation remains poorly understood. Here, we investigated the role of neutrophils in hepatic lipid deposition during early MASLD development. Neutrophils acquired extracellular fatty acids (FAs) via FATP2 and CD36 and stored them as triglycerides (TGs). These lipid‐laden neutrophils (LNs) did not utilize FAs for energy production or lipid mediator synthesis but instead transferred lipid cargo to hepatocytes through extracellular vesicles (EVs). Neutrophil‐derived EVs were enriched with TGs and lipid metabolism‐regulating microRNAs, augmenting TG accumulation in hepatocytes. Peripheral neutrophils isolated from high‐fat diet‐fed mice exhibited a lipid‐laden phenotype, and adoptive transfer of LNs and EVs derived from LNs increased hepatic fat accumulation in recipient mice. In patients with MASLD, circulating neutrophils showed lipid droplet accumulation with increased TGs and enrichment of lipid‐associated miRNAs while plasma EVs were also enriched with TGs and lipid‐associated miRNAs. Single‐cell RNA sequencing of peripheral immune cells identified a neutrophil subpopulation characterized by enhanced lipid‐handling and EV‐related gene expression. These findings identify neutrophil‐mediated lipid transfer via EVs as a mechanistic link between innate immune activation and hepatic lipid accumulation during early MASLD.
ABSTRACT Gut microbial dysbiosis has been observed in several diseases. Although causal links and direct effects on host cells remain unclear, bacteria‐derived extracellular vesicles (BEVs) from the gut microbiota may regulate the host immune response. We examined the impact of severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) infection on the gut microbiome and BEVs release, and the effects of released BEVs on cytokine responses in monocyte‐derived cell lines. Fecal samples from 17 patients with coronavirus disease 2019 (COVID‐19) and 20 healthy individuals were collected to isolate bacterial and BEV fractions. Parental BEV‐releasing bacteria were identified from vesicle‐encapsulated bacterial DNA by 16S rRNA gene sequencing. Patients with COVID‐19 exhibited altered gut microbiota composition and the profile of bacterial DNA‐containing BEVs (dcBEVs) release compared to healthy controls. BEVs from patients, but not from healthy individuals, significantly changed cytokine levels in U937 monocyte cells. Following COVID‐19 recovery, dcBEV profiles diverged into two distinct groups: those that retained the capacity to induce cytokines in monocytes and those that lost this functionality. BEVs from single bacterial cultures within families altered after COVID‐19 onset affected the expression of genes in monocytes, primarily immune‐response genes, notably chemokine ligands and G protein‐coupled receptors. SARS‐CoV‐2–induced dysbiosis alters the profile of dcBEVs release, thereby modulating the host immune response and potentially contributing to COVID‐19 pathogenesis.
ABSTRACT Fibrosis is a critical component of ventricular remodelling after myocardial ischemia, and the activation and expansion of cardiac fibroblasts represent key drivers of this process. Our previous work identified S100A9‐dependent macrophage‐to‐myofibroblast transition (MMT) as a newly recognized source of myofibroblasts in the post‐MIR heart. However, the mechanisms by which S100A9 regulates MMT remain incompletely understood. Here, using multiple genetically engineered mouse models, MIR and MI model, and in vitro multicellular co‐culture systems, we investigated the role of S100A9 in regulating fibroblast‐derived migrasome release during MMT. We found that macrophage‐derived S100A9 promotes mitochondrial dysfunction and migrasome release in cardiac fibroblasts through TLR4/PGC1α signalling following MIR. These fibroblast‐derived migrasomes, in turn, activate integrin/Src signalling in macrophages, triggering MMT and accelerating cardiac fibrosis. Importantly, we delineate how macrophage‐derived S100A9 orchestrates crosstalk between fibroblasts and immune cells and identify migrasome‐mediated activation of MMT as a previously unrecognized mechanism driving post‐MIR fibrotic remodelling. Our findings suggest that targeting S100A9‐induced migrasome release and downstream MMT signalling may represent a promising therapeutic strategy to mitigate pathological cardiac fibrosis.
Extracellular vesicles (EVs) in the mammalian oviduct constitute a key maternal regulatory system that maintains redox balance during early embryogenesis, yet their molecular cargo and functional relevance in human embryos remain poorly defined. Here, we show that human Fallopian tube-derived EVs (oEVs) are rapidly internalized by human preimplantation embryos and improve developmental quality in vitro, increasing high-quality Day 3 embryo formation and blastocyst development. Label-free proteomics identified 6505 oEV proteins, with metabolic, antioxidant and stress-response pathways strongly enriched. Cross-reference with four independent datasets revealed a conserved protein subset shared across secretory-phase oviduct fluid, pluripotent stem cell-derived EVs and in-vivo-developed embryos; among these, YWHAZ was prioritized for functional validation because of its abundance in oEVs, its presence across embryo-related datasets, and its known involvement in stress-response pathways. We found that Ywhaz-deficient mouse embryos exhibit elevated oxidative and apoptotic stress, transcriptional signatures of impaired glutathione metabolism, and failure to survive to birth despite normal blastocyst morphology. Recombinant YWHAZ protein alone failed to enter intact embryos, whereas engineered YWHAZ-loaded EVs were efficiently internalized and significantly reduced intracellular ROS and apoptosis, restoring redox status towards in vivo levels without compromising implantation or foetal growth. Taken together, these findings identify YWHAZ protein as a conserved vesicle-delivered regulator of redox homeostasis and demonstrate that EV-mediated molecular delivery can partially rescue the oxidative stress burden characteristic of in vitro embryo culture. This work provides mechanistic insight into the maternal redox support system of the oviduct and establishes a foundation for EV-based engineering of next-generation embryo culture strategies. Fallopian tube extracellular vesicles enhance human IVF embryo development by delivering functional proteins such as YWHAZ to regulate oxidative stress and blastocyst formation. Large Scale Data Proteomic data are available in the PRIDE database under accession number PXD054946 (https://www.ebi.ac.uk/pride/). Ywhaz-KO embryo RNA-sequencing data are available in the GEO database under accession number GSE294735 https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE294735.
