Journal of Extracellular Vesicles最新文献

Intervertebral disc degeneration (IVDD) is a common age-related disorder associated with inflammation, pain and impaired mobility. In this study, we developed a therapeutic system using silk fibroin (SF) hydrogel loaded with mRNA-engineered extracellular vesicles derived from murine bone marrow mesenchymal stem cells (BMSCs-EVs) to modulate macrophage polarization and alleviate IVDD. BMSCs were isolated from 6-week-old C57BL/6 mice, and an acute IVDD model was established via 18G needle puncture of the coccygeal discs (Co7-Co10). RAW 264.7 murine macrophages were used for in vitro assays, with M1 polarization induced by LPS and IFN-γ. The SF/EVs complex was characterized by SEM, FTIR and rheology, confirming its structural suitability for EV delivery. Functionally, SF hydrogel not only served as a biocompatible carrier but also enabled sustained release of EVs, enhancing their anti-inflammatory effects. In vitro, SF/EVs inhibited M1 polarization and promoted M2 marker expression. In vivo implantation improved disc histology and reduced inflammatory macrophage infiltration. High-throughput RNA sequencing identified S100B as a key functional cargo within EVs. Lentivirus-mediated overexpression and knockdown experiments confirmed that EV-derived S100B suppresses M1 polarization and mitigates IVDD progression. In summary, SF hydrogel loaded with S100B-enriched BMSCs-EVs offers a promising strategy to reshape the inflammatory microenvironment and promote disc regeneration in IVDD.

Giardia lamblia, a eukaryotic intestinal parasite, produces small extracellular vesicles (sEVs) as a conserved evolutionary mechanism. This study investigates the functional role of sEVs in modulating drug response traits among G. lamblia parasites. Here, we showed that sEVs derived from metronidazole (MTZ)–resistant clones modify the expression of enzymes involved in MTZ metabolism and the production of reactive oxygen species (ROS) in recipient wild type parasites. The transfer efficiency and phenotypic impact vary depending on the genetic background of the isolates, highlighting a genotype-specific mechanism. Our findings reveal that sEVs act as mediators of phenotypic adaptation in G. lamblia, enhancing parasite survival under drug-induced stress. This study highlights the significance of sEVs in drug-sensitive dynamics and lays the groundwork for investigating therapeutic interventions that target EV-mediated sensitivity in giardiasis.

Mesenchymal stem cell (MSC) transplantation is considered one of the most promising regenerative strategies for treating degenerative musculoskeletal diseases, yet its underlying therapeutic mechanisms remain incompletely understood. In this study, we demonstrate that transplanted MSCs regulate apoptosis and DNA damage repair (DDR) in senescent nucleus pulposus cells (NPCs) by releasing apoptotic extracellular vesicles (ApoEVs), thereby delaying the process of intervertebral disc degeneration (IVDD). Mechanistically, we found that NPCs in degenerated discs exhibit abnormal subcellular localization of the deubiquitinase ubiquitin specific peptidase 5 (USP5), with excessive cytoplasmic retention leading to aberrant ubiquitination and degradation of the E2F transcription factor 1 (E2F1). Following transplantation into the degenerative disc microenvironment, MSCs undergo extensive apoptosis in the short-term and release ApoEVs enriched in highly acetylated USP5. These vesicles promote nuclear translocation of USP5 in NPCs, which stabilizes E2F1 by preventing its ubiquitin-mediated degradation. This cascade reduces DNA damage and apoptosis in NPCs and enhances their functional activity. Overall, our findings reveal a previously unrecognized mechanism by which apoptotic donor MSCs exert therapeutic effects through intercellular communication, specifically by modulating recipient NPCs apoptosis and DDR pathways. This study underscores the critical role of donor cell apoptosis in the therapeutic efficacy of stem cell transplantation and provides new insights for optimizing regenerative medicine strategies.

Extracellular vesicle (EV)-enriched secretomes are emerging as a new and innovative therapeutic option in the field of regenerative medicine. The clinical use of EV-enriched secretome-based products requires manufacturing processes and quality control (QC) testing that comply with current good manufacturing practice (GMP). The goal of this work was to develop a robust and reproducible large-scale GMP-compliant process for the production of an EV-enriched secretome derived from cardiovascular progenitor cells (CPC), including the vesiculation of CPC, purification and concentration of the product; and sterilising filtration. QC strategies for in-process and release testing of an investigational medicinal product (IMP) were developed to guarantee quantity, safety, purity and identity. The IMP showed biological activity and was non-immunogenic in vitro, and showed no signs of toxicity or tumour development in vivo. The IMP was approved for use in a single-centre Phase I clinical trial by the French National Agency for Medicines and Health (ANSM) for the treatment of heart failure. The IMP is stored between –65°C and –85°C and can be easily diluted by the hospital pharmacy for infusion to the patient. This work represents a major advance for the use of CPC derived EV-enriched secretomes as a biological drug for cardiac clinical applications.

Trial Registration: ClinicalTrials.gov identifier: NCT05774509

In spite of the numerous clinical trials conducted on mesenchymal stromal cells and their extracellular vesicles (MSC-EVs) across a wide range of diseases, the field faces challenges in reaching a consensus on crucial parameters such as source, marker definition, and culture conditions, adding to heterogeneous efficiencies. Nevertheless, there is widespread acceptance of the pro-inflammatory activation of MSCs with IFN-γ and TNF-α to enhance immune modulation. Our study highlights the impact of activation duration on MSC-EV-mediated immune modulation of macrophages. Extended activation periods (24 h) revealed elevated levels of IFN-γ-induced CD317 on the MSC surface. CD317 is known to tether enveloped viral particles to the cell membrane, impeding viral diffusion and spread. We demonstrated the accumulation of EVs on the MSC cell surface following activation or lentiviral CD317 overexpression. In contrast, CD317 knockdown eliminated the enrichment of EVs on the cell surface, significantly enhancing the MSC-EV-mediated immune modulation of macrophages. Considering the pivotal role of IFN-γ in MSC immune modulation and its inevitable contact in patients, our findings propose CD317 as a potential modulator of MSC-based therapy efficacy in clinical applications.

Small extracellular vesicles (sEV) are a class of natural vesicles rich in heterogeneous cargos with the great advantage of non-invasive detection; these vesicles exhibit complex intercellular crosstalk and mediate important biological functions. However, the potential value of plasma sEV in clinical prognosis prediction of triple-negative breast cancer (TNBC) and their biological functions have not been well elucidated. In this study, we isolated sEV from non-metastatic and metastatic TNBC plasma samples. We found that the expression of reticulin 4 (RTN4) in metastatic patients was significantly higher than that in non-metastatic patients. At the same time, clinical data showed that RTN4 was associated with poor prognosis and advanced-stage TNBC patients. Subsequently, in vivo and in vitro assays showed that compared to RTN4^Low sEV, RTN4^high sEV significantly promoted tumour cell migration, invasion, epithelial-mesenchymal transition (EMT) and lung metastasis, and upregulated the expression of PD-L1 in tumour tissues and inhibited CD8⁺T cell infiltration. Regarding mechanism research, we found that RTN4 within sEV drives tumour EMT and PD-L1 expression by activating the NF-κB signalling pathway. Further, through the combined treatment experiment of anti-PD-1 and anti-RTN4, it was found that the combination of the two drugs was significantly superior to monotherapy in inhibiting tumour metastasis, EMT, and promoting CD8⁺T cell infiltration. Our results highlight the molecular mechanism of sEV protein RTN4 in tumour progression and immune system regulation, indicating that RTN4 targeting and anti-PD-1 combined therapy have clinical potential. sEV protein RTN4 is a potential new prognostic marker for non-invasive detection of TNBC and a new target for TNBC treatment.

Despite extensive proof for the tumour-supporting function of cancer-derived small extracellular vesicles (sEVs), attributions of pathological effects to specific sEV subpopulations are poorly described. In this study, we aimed to characterise a distinct sEV species under the control of Syntenin, a key regulator of endosomal sEV biogenesis, regarding its proteomic cargo and pro-tumourigenic functions. Using mass spectrometry (MS), we detected 178 down- and 236 up-regulated proteins on sEVs from breast cancer cells upon Syntenin knockout (KO). Pathway enrichment analysis suggested that Syntenin depletion was particularly associated with adhesion-related processes. Accordingly, sEVs from Syntenin-deficient 4T1 and MCF-7 breast cancer cells showed a reduced expression of several focal adhesion and cell–cell junction proteins. Syntenin silencing reduced the Fibronectin-binding capacity of sEVs from both cell lines, which was mediated by sEV-associated Integrin alpha-V/beta-3 (α_Vβ₃). Compared to sEVs from wildtype cells, Syntenin KO sEVs showed decreased tropism towards the Fibronectin-rich liver microenvironment in vivo, provided less adhesive support for 4T1 cells and thereby failed to induce cancer cell migration, which appeared to be independent of EV uptake. In summary, this study revealed that Syntenin has a large-scale effect on the proteomic cargo of sEVs and regulates their adhesive, organotropic and pro-migratory properties in breast cancer.

Many bacteria produce extracellular vesicles (EVs) that play critical roles in various biological processes and hold significant potential for biomedical applications. However, the mechanisms underlying EV biogenesis remain incompletely understood. Using transmission electron microscopy, we demonstrate that X-ray irradiation induces outward blebbing of the inner membrane in Pseudomonas aeruginosa PAO1 (P. aeruginosa PAO1), leading to the formation of outer-inner membrane vesicles (OIMVs) through outer membrane pinching-off. The endolysin Lys, which is negatively regulated by PrtR and positively regulated by PrtN, is essential for OIMV production. Lys translocates into the periplasmic space, where it disrupts the peptidoglycan layer, causing morphological changes from rod-shaped to round cells and facilitating OIMV release. Furthermore, deletion of YciB, a protein crucial for inner membrane integrity, significantly increases OIMV production. In a murine model of acute pneumonia, OIMV immunisation significantly improves pulmonary bacterial clearance, reduces lung injury and enhances survival rates. Our findings reveal inner membrane blebbing as a novel mechanism of OIMV biogenesis in P. aeruginosa PAO1 under X-ray irradiation and highlight the potential of OIMVs as promising vaccine candidates against P. aeruginosa infections.

Cytolysin A (ClyA) is a pore-forming protein from a strongly silenced gene in non-pathogenic Escherichia coli, including typical commensal isolates in the intestinal microbiome of healthy mammalian hosts. Upon overproduction, ClyA-expressing bacteria display a cytolytic phenotype. However, it remains unclear whether sublytic amounts of native ClyA play a role in commensal E. coli-host interactions in vivo. Here, we show that sublytic amounts of ClyA are released via outer membrane vesicles (OMVs) and affect host cells in a remarkable manner. OMVs isolated from ClyA⁺ E. coli were internalised into cultured colon cancer cells. The OMV-associated ClyA caused reduced levels of cancer-activating proteins such as H3K27me3, CXCR4, STAT3 and MDM2 via the EZH2/H3K27me3/microRNA 622/CXCR4 signalling axis. Our results demonstrate that sublytic amounts of ClyA in OMVs from non-pathogenic E. coli can influence the stability of the EZH2 protein, reducing its activity in epigenetic regulation, causing elevated level of the tumour suppressor protein p53.

The immune evasion strategies and lifelong latency of herpes simplex virus type 2 (HSV-2) present significant challenges for effective treatment. Recent studies have demonstrated that the commensal microbiota plays an important role in regulating immunity against viral infections. We previously reported that Lactobacillus rhamnosus GG (LGG) activates the expression of type I interferons (IFN-I) to inhibit HSV-2 infection. However, the specific molecular mechanisms remain unclear. Bacterial extracellular vesicles (EVs) are small lipid bilayer-bound particles secreted by bacteria, which can serve as intercellular communication vehicles between the host and pathogens, functioning as immunomodulatory vectors defending against viral infections. In this study, we confirmed that LGG-EVs activate the nucleotide-binding oligomerisation domain-containing protein 2 (NOD2)-IFN-I signalling pathway, inducing the expression of interferon-stimulated genes (ISGs) to combat HSV-2 infection both in vivo and in vitro. Furthermore, we explored the specific components within LGG-EVs and identified the presence of muramyl dipeptide (MDP). We demonstrated that MDP-enriched LGG-EVs effectively inhibit HSV-2 infection via activation of the NOD2-IFN-I pathway. These findings suggest that LGG-EVs could serve as a novel therapeutic strategy for HSV-2 and provide a mechanistic foundation for future antiviral research.