Journal of Extracellular Vesicles最新文献

Synaptic formation impairment is closely correlated with cognitive impairment in Alzheimer's disease (AD), yet the underlying mechanisms remain incompletely understood. Emerging evidence indicates that extracellular vesicles (EVs), critical mediators of intercellular communication, are implicated in the progression of AD. However, the specific mechanisms through which neuron-derived EVs contribute to synaptic formation impairment in AD remain unexplored. In this study, we characterized EVs derived from primary neurons of APP/PS1 transgenic mice (APPNEVs) and investigated their impact on synapse formation. Transmission electron microscopy, nanoparticle flow cytometry, and immunoblotting confirmed that APPNEVs and WT neuron-derived EVs (WTNEVs) had similar morphology, size, and canonical small EVs markers. We further revealed that APPNEVs significantly impaired neuronal synapse formation by downregulating synaptic proteins PSD95 and Synaptophysin (SYP), reducing total synapse number, and shifting synapse morphology toward immature states. Proteomic profiling via mass spectrometry identified APOE as a key upregulated protein in APPNEVs. Pharmacological inhibition of APOE with EZ-482 effectively prevented APPNEV-induced synaptic formation impairment, APPNEV-mediated downregulation of synaptic proteins, and the APPNEV-induced decrease in synaptic maturity. Mechanistically, APPNEVs suppressed Rac1-N-WASP-Arp2/3-mediated filament actin polymerization, a critical pathway for synaptic spine formation, which was prevented by APOE inhibition. In vivo stereotactic injection of APPNEVs into the hippocampus of WT mice further validated their detrimental effects on synaptic integrity, which were prevented by EZ-482 treatment. Collectively, these findings demonstrate that APPNEVs mediate synaptic damage via carrying APOE, providing novel insights into EV-mediated neurodegeneration in AD and highlighting APOE as a potential therapeutic target for preserving synaptic formation.

Ischaemic cardiovascular diseases, particularly myocardial infarction (MI), remain the leading causes of morbidity and mortality worldwide. Targeting extracellular vesicles (EVs) from the gut microbiota by diet may provide opportunities to improve cardiovascular health. Barley leaf (BL) has a long history of use in Traditional Chinese medicine and has been found to beneficially influence the gut microbial composition. Herein, we used a murine model of MI to explore the mechanistic role of gut bacteria-derived EVs in the cardioprotective effects of BL. Dietary supplementation of BL remarkably improved cardiac function and ameliorated adverse remodelling in experimental MI. The cardioprotective effects of BL were linked to enhanced gut epithelial barrier and suppressed transfer of bacterial-derived lipopolysaccharide. Moreover, BL alleviated MI-induced gut microbial dysbiosis, with an enrichment of Lachnospiraceae. Gut microbiota depletion by antibiotic treatment abolished the cardioprotective effects of BL. Furthermore, mice receiving microbiota from BL-fed mice had better cardiac outcomes after MI compared to mice receiving microbiota from mice without BL supplementation. Notably, we identified that BL increased the abundance of Lachnospiraceae_NK4A136_group, a commensal member of the family Lachnospiraceae. Supplementing antibiotic-treated mice with live but not heat-inactivated Lachnospiraceae ameliorated myocardial injury and cardiac remodelling in MI mice. We isolated EVs from Lachnospiraceae and demonstrated that Lachnospiraceae-derived EVs (L-EVs) achieved desirable biosafety, stability and colonic retention effects following oral administration. Mechanistically, estrogen-like metabolites from L-EVs modulated the estrogen receptor alpha (ERα)-solute carrier family 6 member 14 (Slc6a14)-Hippo signalling pathway to promote intestinal stem cell function and ultimately protected against MI-induced adverse remodelling. Our study thus provides novel insights into the role of the microbiota-gut-heart axis in the pathophysiology of MI and underscores the great potential of gut bacteria-derived EVs to reduce pathological outcomes after MI through improving gut health.

Podocyte injury and endothelial cell dysfunction are the hallmarks of glomerular disease. How these two events are connected remains largely unknown. This study aimed to delineate the role of extracellular vesicles (EVs) in mediating podocyte-endothelial communication in glomerular disease. Podocyte-derived EVs were characterized by nanoparticle tracking analysis and electron microscopy. Proteomic analyses were used to characterize proteins in the EVs. The involvement of integrin αvβ1, focal adhesion kinase (FAK), endoplasmic reticulum (ER) stress-related proteins was investigated using siRNA inhibition, neutralizing antibodies, small molecule inhibitors in vitro and in vivo. We found that podocyte injury was associated with an increased secretion of EVs, in which integrin αvβ1 was upregulated and enriched. Podocyte-derived EVs were recruited by endothelium via secreting fibronectin. Integrin αvβ1 from podocyte EVs was then transferred to endothelial cells, in which it activated the FAK and triggered ER stress, ultimately leading to endothelial cell apoptosis. In mouse models of glomerular disease, intravenous injection of the EVs from injured podocytes exacerbated endothelial ER stress and apoptosis, while inhibition of integrin β1 signaling blocked this effect. Similarly, inhibition of EV secretion by dimethyl amiloride preserved endothelial integrity and ameliorated glomerulosclerosis in vivo. These studies indicate that podocyte injury causes endothelial dysfunction by releasing integrin αvβ1-enriched EVs, which trigger FAK-mediated ER stress and apoptosis. Therefore, targeted blockade of EV secretion or integrin αvβ1 signaling may hold promise for protecting against endothelial dysfunction in glomerular disease.

Early detection of oesophageal squamous cell carcinoma (ESCC) is critical for improving survival, yet current screening is hampered by the lack of effective, non-invasive methods. Here, we developed and prospectively validated an extracellular vesicle (EV) protein-based blood test for the preclinical detection of ESCC. We first engineered BarFlare, a high-sensitivity platform for serum EV protein analysis, and identified a novel biomarker panel that includes EV-associated squamous cell carcinoma antigen (SCC) and matrix metalloproteinase-13 (MMP13). These biomarkers were integrated with clinical factors into an interpretable multi-criteria decision-making classification fusion (MCF) machine-learning framework. The MCF model was trained and validated in prospective, multicentre diagnostic cohorts (n = 1018), and its preclinical detection capability was assessed in a prospective, population-based longitudinal cohort. The MCF framework accurately distinguished patients with ESCC from healthy controls in a test set (AUC, 0.987) and two external validation cohorts (AUCs, 0.926 and 0.960), including those with early-stage disease (AUCs, 0.901-0.980). Critically, in the longitudinal cohort, the framework identified individuals who would later develop ESCC from their baseline blood samples with a median lead time of 34.9 months (range, 0.4-72.5) before clinical diagnosis (AUC, 0.864; sensitivity, 73.3%; specificity, 82.2%). The risk score of the model correlated with time to diagnosis, and its dynamic increase significantly outperformed that of traditional serum SCC for preclinical risk stratification. Our validated, blood-based EV protein signature not only accurately detects prevalent ESCC but also identifies high-risk individuals years before clinical presentation, providing a powerful, non-invasive tool that supports risk-stratified screening and creates a critical window for early, potentially curative intervention. Trial Registration: Chinese Clinical Trial Registry identifiers: ChiCTR2200066733 and ChiCTR2200065610.

Small extracellular vesicles (sEVs) have emerged as next-generation multifunctional nanotherapeutics due to their parental-cell traits and role in intercellular communication. Among them, immune cell–derived sEVs are uniquely positioned to couple innate immunomodulatory activities with therapeutic payload delivery, making them highly attractive for cancer therapy. In particular, M1 macrophage-derived sEVs (M1-sEVs) preserve the tumour-suppressive functions of their parent cells, including tumour microenvironment (TME) reprogramming, immune activation, and inhibition of cancer progression. However, the mechanisms by which these activities are coordinated within the TME, and whether they act independently or synergistically, remain poorly understood. Clarifying these mechanisms is crucial for harnessing their intrinsic bioactivity in combination with their natural capacity as drug delivery nanocarriers to optimize therapeutic efficacy. Here, we demonstrate that M1-sEVs exhibit intrinsic stability and circulation longevity via ‘do not eat me’ ligands, as well as tumour-homing ability revealed by proteomic profiling, enabling efficient uptake and deep infiltration in breast cancer models. Functionally, M1-sEVs deliver antiproliferative microRNAs that suppress tumour metabolism, growth, and progression by inhibiting self-renewal, adhesion, migration, motility, and invasion. Importantly, by integrating this endogenous bioactivity with exogenous doxorubicin loading, we achieved synergistic efficacy: a 3-fold reduction in IC₅₀ in vitro (0.46 µM vs. 1.45 µM for free drug) and 70.18% tumour growth inhibition in vivo. These findings highlight M1-sEVs as dual-action nanotherapeutics that combine innate immune-regulatory and tumour-inhibitory functions with efficient drug delivery, advancing their development as powerful platforms for cancer therapy.

Small extracellular vesicles (sEVs) derived from cytotoxic T lymphocytes (CTLs) are emerging as potential mediators of antitumor immunity; however, their subcellular origins and functional properties remain incompletely defined. In this study, we investigated the intracellular routes and cytotoxic potential of CTL-derived exosomes. Using correlative light and electron microscopy, we discovered that CTL-derived exosomes originate from both classical multivesicular bodies (MVBs) and the recently identified multi core granules (MCGs). Through total internal reflection fluorescence microscopy, we demonstrated that, in contrast to MVB-derived exosomes, MCG-derived exosomes are released at the immunological synapse in a stimulus-dependent manner. To enable functional characterization, we developed a scalable primary cell culture method for the isolation of high-purity exosomes. Super-resolution microscopy revealed significant heterogeneity in exosome size and tetraspanin composition. Notably, MCG-derived exosomes exhibited fivefold higher cytotoxic activity than MVB-derived exosomes, inducing apoptosis in tumor cells via a caspase 3-dependent mechanism. These findings reveal that CTLs exploit distinct secretory pathways to release heterogeneous exosome populations with differential cytotoxic capacities, offering new insights into CTL-mediated immune responses and providing a basis for the development of novel exosome-based immunotherapies.

Platelet-activating factor (PAF) is a potent phospholipid mediator with therapeutic potential in neuroregeneration, but its therapeutic application is hindered by rapid degradation and systemic proinflammatory effects. Here, we present an engineered extracellular vesicle (EV)-based delivery strategy that stabilizes and targets PAF to sites of hippocampal injury, restoring neuronal structure and cognitive function. EVs derived from EP4 antagonist-primed mesenchymal stem cells (GWEVs) exhibit enhanced secretion and selective enrichment of bioactive lipids, particularly PAF, which promotes neuroregeneration, attenuates gliosis and rescues spatial memory. Mechanistic studies reveal that PAF's therapeutic activity depends not on classical PTAFR engagement but on neuronal metabolism via PAF-acetylhydrolase (PAFAH), particularly the PAFAH1B1 subunit. The hydrolysis-resistant analogue MPAF fails to confer benefit, underscoring the requirement for enzymatic processing. To address translational needs, we developed a bioorthogonal click-labelling platform that enables real-time SPECT imaging of EV biodistribution while preserving function. GWEVs preferentially accumulate in injured hippocampi, confirming targeted delivery. This study defines a previously unrecognized lipid metabolism-dependent repair mechanism and demonstrates the feasibility of leveraging EVs for CNS-targeted delivery of labile lipid therapeutics. These findings offer a platform for advancing regenerative strategies in neurodegenerative diseases and traumatic brain injury.

Endometriosis (EM) is a chronic inflammatory disease that affects ∼10% of women during reproductive age. It is characterised by ectopic (ECT) growth of endometrial-like tissue mainly in the pelvic cavity. Small extracellular vesicles (sEVs) mediate cellular interactions, but their function remains poorly understood in the pathogenesis of EM. 3D endometrial epithelial organoids (EEOs) from ECT lesions and eutopic (EUT) endometrium from EM patients and controls were established to investigate sEVs. Multiplex bead-based flow cytometry revealed CD133/1 and EpCAM as dominant markers on EEO-sEVs, with ECT EEO-sEVs showing upregulation of CD44, CD29 and downregulation of EpCAM compared to EUT EEO-sEVs. Peritoneal fluid (PF)-sEVs displayed high and correlated CD133/1 and EpCAM expression, indicating a major contribution from endometrial epithelial (EE) cells, alongside sEVs of lymphocyte and endothelial origin. Functionally, both ECT EEO-sEVs and PF-sEVs from EM patients significantly suppressed macrophage phagocytosis, as assessed by pH-sensitive fluorescent bioparticles. The effect was reversed by CD47 blockade. The coexpression of CD47 with CD133/1 and EpCAM on PF-sEVs indicates the involvement of EE cell-derived sEVs in CD47/SIRP-α mediated suppression. This study provides the first thorough characterisation of EE-derived sEVs utilising EEO models in EM and demonstrates their potential immunomodulatory role in the peritoneal microenvironment via CD47/SIRP-α signalling.

Extracellular vesicles (EVs) affect the function of cells in living animals. Yet, cell type-specific EV abundances and their distribution in biological fluids are technically challenging to study. Thus, we aimed to develop an in vivo EV reporter system to monitor cell-type-specific EVs, with a focus on adipocyte-derived EVs. While our previously generated EV reporter construct had insufficient sensitivity, we successfully created a sensitive EV reporter using an adeno-associated virus (AAV) vector with Cre-activated expression of human CD63 fused to NanoLuc (CD63-NanoLuc). Moreover, we designed a control AAV construct for monitoring constitutive secretion of NanoLuc (sec-NanoLuc). AAV administration to mice induced adipocyte-specific expression of both reporters. NanoLuc activity was detected in plasma. While sec-NanoLuc was predominantly in plasma and urine, CD63-NanoLuc was highest in adipose tissues (ATs). We challenged mice with a 2-week high-fat diet (HFD), which had minimal effects on body weight and adipogenic markers. Still, the HFD-fed CD63-NanoLuc mice, but not sec-NanoLuc mice, showed significantly higher NanoLuc activity in ATs, lungs, kidneys and urine. Thus, our CD63-NanoLuc and sec-NanoLuc constructs revealed an early effect of HFD on the abundance and distribution of adipocyte-derived EVs and provide a sensitive system for monitoring cell-type-specific EVs in health and disease.

Neuroinflammaging, a moderate, chronic, and sterile inflammation in the hippocampus, contributes to age-related cognitive decline. Neuroinflammaging comprises the activation of the nucleotide-binding domain, leucine-rich repeat family, and pyrin domain-containing 3 (NLRP3) inflammasomes, and the cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) pathway that triggers type 1 interferon (IFN-1) signalling. Studies have shown that extracellular vesicles from human induced pluripotent stem cell-derived neural stem cells (hiPSC-NSC-EVs) contain therapeutic miRNAs that can alleviate neuroinflammation. Therefore, this study examined the effects of late middle-aged (18-month-old) male and female C57BL6/J mice receiving two intranasal doses of hiPSC-NSC-EVs on neuroinflammaging in the hippocampus at 20.5 months of age. Compared with animals receiving vehicle treatment, the hippocampus of animals receiving hiPSC-NSC-EVs exhibited reductions in astrocyte hypertrophy, microglial clusters, and oxidative stress, along with elevated expression of antioxidant proteins and genes that maintain mitochondrial respiratory chain integrity. Moreover, hiPSC-NSC-EVs therapy decreased the levels of various proteins involved in the activation of the NLRP3 inflammasome, p38/mitogen-activated protein kinase, cGAS-STING-IFN-1, and Janus kinase and signal transducer and activator of transcription signalling pathways. Furthermore, in vitro assays using genetically engineered RAW cells and hiPSC-NSC-EVs, with or without targeted depletion of specific miRNAs, demonstrated that miRNA-30e-3p and miRNA-181a-5p, both present in hiPSC-NSC-EVs, can significantly inhibit the activation of the NLRP3 inflammasome and the STING pathway, respectively. Additionally, single-cell RNA sequencing conducted 7 days post-treatment revealed that hiPSC-NSC-EVs induce widespread transcriptomic changes in microglia, including increased expression of numerous genes that enhance oxidative phosphorylation and reduced expression of abundant genes that drive multiple proinflammatory signalling pathways. These changes mediated by hiPSC-NSC-EVs were also associated with improved cognitive and memory function. Thus, intranasal hiPSC-NSC-EVs therapy in late middle age can effectively diminish proinflammatory microglial transcriptome and signalling cascades that drive neuroinflammaging in the hippocampus, contributing to better brain function in old age.