Journal of extracellular biology最新文献

Extracellular vesicles (EVs) have gained increasing attention in recent years due to their pivotal role in both health and disease. Emerging from both eukaryotic and prokaryotic cells, EVs serve as essential mediators of intercellular communication, exceeding the simplistic interactions observed with individual molecules. In this comprehensive review, we will focus on both Bacterial Extracellular Vesicles (BEV) and on Host derived Extracellular Vesicles (HEV) and highlight mechanistic principles, as well as their transformation into diagnostic and therapeutic tools. We will start with a short introduction into the biogenesis and principal properties of BEV and HEV. We will then focus on the composition of BEV and introduce OMICs-based studies that helped to unravel their complex constitution. As both BEV and HEV interact with different epithelial and endothelial barriers and shape their properties, we will highlight mechanistic principles for both EV types. Starting from the intestinal system, where we will look at BEV and how these BEV overcome the intestinal barrier to change distant organs and the patient's immune system. We will then visit other endothelial and epithelial sites of the human body and summarize how HEV shapes these barriers and how HEV can overcome these barriers. We will then turn towards diagnostic and therapeutic approaches. As both BEV and HEV are currently suggested as diagnostic markers and are being investigated as potential therapeutic agents. Lastly, we will discuss current challenges and provide an outlook on the future in the field. This review seeks to raise awareness for both bacterial and host-derived EVs, highlighting that they present two sides of the same coin.

Alterations to the community structure and function of the microbiome are associated with changes to host physiology, including immune responses. However, the contribution of microbe-derived RNAs carried by outer membrane vesicles (OMVs) to host immune responses remains unclear. This study investigated the role of OMVs and OMV-associated small RNA (sRNA) species from pathogenic and commensal Bacteroides fragilis (ETBF and NTBF, respectively) in eliciting different immune responses from intestinal epithelial cells. To distinguish the differences in the sRNA profiles of the two strains and their OMVs, RNA-seq, qRT-PCR, and northern blotting were conducted to identify enrichment of discrete sRNA species in OMVs, which were also differentially expressed between the two strains. Specifically, coding and non-coding RNAs were enriched in OMVs from NTBF and ETBF, with BF9343_RS22680 and BF9343_RS17870 being significantly enriched in ETBF OMVs compared to NTBF. To understand the effects of OMVs on pattern recognition receptors, reporter cells of Toll-like receptor (TLR) activation were treated with OMVs, demonstrating activation of TLRs 2, 3, and 7. Treatment of Caco-2 and HT29-MTX cells with OMVs demonstrated increased expression of IL-8. Surprisingly, we discovered that degradation of RNase-accessible RNAs within ETBF OMVs, but not NTBF OMVs, resulted in vesicles with enhanced capacity to stimulate IL-8 expression, indicating that these extravesicular RNAs exert an immunosuppressive effect. This suggests a dual role for OMV-associated RNAs in modulating host immune responses, with implications for both bacterial pathogenesis and therapeutic applications.

Small extracellular vesicles (sEVs), ranging from 30 to 200 nm in diameter, are a subset of extracellular vesicles that play an essential role in intercellular communication by transporting bioactive molecules between cells. Due to their diverse cargo and origins, sEVs form a highly heterogeneous population. Previous studies have suggested that recipient cells uptake specific sEVs to ensure accurate signalling, but direct experimental evidence on how cells effectively communicate in such a complex and noisy environment has been lacking. In this study, we provide direct experimental evidence by developing a method to retrieve and analyze internalized sEVs from the intracellular membrane compartments of recipient cells. Using this sEV retrieval method, we isolated sEV subpopulations from two types of breast cancer cells after incubation with sEVs derived from mixed cell cultures. We then assessed their functional roles by evaluating the phenotypic responses of cells treated with these retrieved sEV subpopulations. Our results reveal apparent differences in their functional impacts, indicating that cells employ a function-based mechanism to selectively uptake sEVs. This finding advances our understanding of how cells enhance communication efficiency via sEVs. Further investigation into this phenomenon could offer deeper insights into intercellular communication and its role in health and disease.

Diabetic kidney disease (DKD) is a health burden that lacks specific and early diagnostic biomarkers. For their discovery, we sequenced urinary extracellular vesicle miRNAs in a type 1 diabetes cohort of males with and without DKD. The results were replicated by sequencing or qPCR in two independent cohorts and six published datasets, including type 1 and 2 diabetes, and both sexes. We also validated stable reference gene candidate miRNAs. Chronic kidney disease, hypertensive nephropathy, IgA nephropathy, polycystic kidney disease, kidney stones, prostate cancer and non-diabetic cohorts served as additional controls. MiRNAs changed due to urine collection type or centrifugation before storage were excluded. We analyzed differentially expressed miRNAs and their correlations with clinical measurements, receiver operating characteristic curves and target mRNAs, proteins and pathways, incorporating single-cell data and circulating proteins of type 1 and 2 diabetes cohorts. By studying the uEV miRNAs (N = 490 individuals total) and plasma proteins (N = 4335), we pinpointed 6 stable miRNAs, 11 differentially expressed miRNAs, 9 target proteins and 16 DKD-associated pathways. Differentially expressed miRNAs overlapped between diabetes subtypes and sexes, with strongest evidence for miR-192-5p, miR-146a-5p, miR-486-5p and miR-574-5p. The miRNAs alone or combined with clinical measurements classified individuals with the fastest kidney function decline (sensitivity 0.75–1.00, specificity 0.83–1.00) even in the normoalbuminuria group. The differentially expressed miRNAs did not cluster the control cohorts except for the chronic kidney disease cohort, which showed some clustering based on proteinuria status. Altogether, the miRNAs showed potential to identify early kidney function decline and may target key kidney cells, mRNAs, proteins and pathogenic mechanisms in DKD.

Amniotic fluid (AF) is a valuable source of extracellular vesicles (EVs) derived from the fetoplacental unit. Preclinical and clinical studies have highlighted promising applications of AF-EVs and their role in cellular communication, yet our understanding of AF-EV physiology remains limited. This study aimed to examine the physiological importance of AF-EVs in fetal development from the second trimester to term gestation. We obtained AF samples from routine second-trimester amniocentesis and prelabour Caesarean section at term. We isolated EVs using a combination of differential centrifugation, filtration and ultracentrifugation and characterised them using nanoparticle tracking analysis, cryo-electron microscopy and Western blotting. The differential EV proteome was analysed using label-free proteomics. We assessed the second trimester and term AF-EV properties through an enrichment analysis. The EV size and protein enrichment difference revealed a gestational-age-dependent variation in the predominant EV subtype. Second-trimester-derived EVs were enriched in ectosomes, while term EVs contained a significant proportion of exosomes. We identified several morphologies of AF-EVs, including unilamellar, multilamellar, multicompartmental and granular-centred EVs, across gestational ages. Proteomics analysis of AF-EVs identified 4137 proteins with high confidence, of which 1090 exhibited significant differential enrichment between the two groups. Second-trimester-enriched AF-EV proteins represented molecule assembly processes, metabolism and organogenesis. At term, AF-EV proteins corresponded to impending newborn functions such as immunity and digestion. In conclusion, we provide compelling evidence that EV biogenesis and secretion in the fetoplacental unit undergo significant alterations across gestational ages, revealing a complex and dynamic physiology and intercellular communication that adapts to the needs of the developing fetus.

Human milk is rich in extracellular vesicles (EV) that may contribute to shaping neonatal immunity. Here, we evaluated whether freezing, and the addition of sodium citrate (SC), affect the characteristics of human milk EVs and their miRNAs. Freezing may compromise the milk EV population and their miRNA profile by creating artificial vesicles due to cell lysis. Furthermore, SC can be added to clear the EV fraction of micelles, that is, protein aggregates that co-isolate with milk EVs, and may affect certain downstream analyses. To investigate potential differences between milk EV and their miRNA cargo when isolated from fresh and frozen samples, mature milk samples were collected from 10 women and subjected to four different treatments: fresh and frozen; fresh^SC and frozen^SC. Ultracentrifugation was used for EV isolation, and subsequently characterized by Nanoparticle tracking analysis, flow cytometry, Western blot and electron microscopy. While freezing without SC has no impact on the evaluated EV parameters, freezing with SC significantly altered particle mean size as measured by NTA and protein levels as studied by MACSPlex flow cytometry. Importantly, neither freezing nor SC had an impact on the EV miRNA cargo, measured by qPCR. These findings also suggest that EV isolates from frozen samples, in comparison to freshly isolated ones, can produce valid results concerning morphology, size, surface markers and the EV miRNA profile.

Extracellular vesicles (EVs) are present in body fluids and act as disease biomarkers. Emerging evidence has proven that EVs are released not only from mammalian cells but also from bacteria. Ovarian cancer has a dismal prognosis because of difficulties in early detection. This study aimed to identify bacterial EV (BEV) proteins associated with ovarian cancer. Fifteen patients with ovarian cancer or non-cancer were recruited, and EVs were isolated from the ascites. BEVs were recovered from seven in vitro-cultured strains of bacteria present in the vaginal microbiota, and LC-MS/MS analysis was performed. The detected peptide data were annotated to both human and bacterial references, and the human data showed that the profiles of cancer EVs were distinct from those of patients with non-cancer. As analysed by bacterial proteins, P15636_Protease1 was found as the BEV-associated protein highly expressed in patients with cancer. To distinguish patients with cancer, the area under the curve was 0.88 (95% CI, 0.64–1.00). In addition, homology analysis showed that P15636_Protease1 is a unique protein only detected in bacteria. In this study, ovarian cancer-specific bacterial proteins were identified on EVs, and BEVs in bodily fluids are promising in the discovery of disease biomarkers.

Extracellular vesicles (EVs) from plants, particularly traditional medicinal plants, have garnered therapeutic interest. However, therapeutic utility requires establishing effective extraction and purification protocols for manufacturing. Here, we systemically optimized an enzyme-based EV extraction method and demonstrated the generalizability with different parts from seventeen medicinal plants, fresh, frozen or dried. The enzymatic pulping process (henceforth denoted as Phyto-EVpure) of fresh ginger, Morus alba leaves and Isatis indigotica Fort. roots improved EV yield and purity over grinding. Ginger EVs processed via Phyto-EVpure preserved bioactivities and conferred hepatoprotection in a non-alcoholic fatty liver disease model in vitro and in vivo. Phyto-EVpure efficiently isolated EVs from leaf, rhizome and root stored fresh, frozen or dried. Phyto-EVpure enabled us to demonstrate that EVs from phloem of previously intractable dried vine samples exhibited greater antitumour activities than xylem in vitro. Collectively, we provided a manufacturing-friendly protocol for medicinal plant EV isolation and demonstrated the generalizability of Phyto-EVpure for efficient isolation of EVs from a wide spectrum of traditional medicinal plants under different conditions.

Systemic inflammatory state found in obesity increases the risk of developing numerous diseases. While endurance training seems effective to reduce this inflammation, the underlying mechanisms are not fully understood. Among those, extracellular vesicles (EVs) have been proposed to be actors in the anti-inflammatory intercellular crosstalk induced by exercise training. This study aimed to investigate how endurance training modulates the EV proteome in the context of an inflammatory state in adults with obesity. Thirteen lean sedentary adults and 10 sedentary adults with obesity participated in a 12-week endurance training programme. Skeletal muscle, abdominal subcutaneous adipose tissue and venous blood samples were taken prior to and after the training period. The systemic and adipose tissue inflammatory states were assessed, and plasma EVs were isolated by size exclusion chromatography. EV content was analysed by mass spectrometry. EVs isolated from the medium of myotubes stimulated by electrical pulse stimulation in vitro were quantified, and their content was analysed by western blot. After the endurance training, C-reactive protein (CRP) levels decreased in participants with obesity. In abdominal subcutaneous adipose tissue, the phosphorylation state of nuclear factor-kappa B (NF-κB) was not affected by training, but interleukin (IL)-6 and IL-1β protein levels were reduced after the 12 weeks in both groups. Conversely, interferon gamma (IFNγ) level reduction was exclusively found in the obesity group. Despite no changes in EV abundance, EV proteome was modified by training. Among the modified proteins in participants with obesity, the antioxidant enzyme peroxiredoxin (PRDX) 1 abundance was increased after training. Additionally, the PRDX1 content of EVs isolated from stimulated myotubes was increased compared to control conditions. In conclusion, our results suggest that the anti-inflammatory effects of exercise training are not directly mediated by EV anti-inflammatory proteome changes. However, exercise training increases circulating EV antioxidant content, possibly through contractile activity of skeletal muscle during repeated exercise.

Extracellular vesicles (EVs) are lipid nano-to-micro-sized vesicles increasingly studied for their role in intercellular communication and their potential as minimally invasive molecular indicators in various diseases. However, challenges remain in characterizing specific surface molecules on EVs due to cargo heterogeneity and the lack of convenient quantification methods. In this study, we show the isolation, characterization, detection, and quantification of Trop2-carrying EVs (EV-Trop2) in serum of prostate cancer patients. This work combines the unique advantages of our EV isolation method with ELISA to enable surface-protein-specific EV analysis directly from serum. This is, to our knowledge, the first demonstration to isolate and quantify EV-Trop2 from prostate cancer patient serum to study its expression patterns in relation to prostate cancer status. Analysis of serum samples from three patient groups: high-risk prostate cancer (n = 22), low-risk prostate cancer (n = 23), and cancer-free groups (n = 21), revealed significantly different levels of EV-Trop2 expression between the high-risk and low-risk patient groups (p = 0.0015) and between high-risk patient and cancer-free groups (p < 0.0001). Multivariate modeling further showed that EV-Trop2 contributed to improved classifier metrics across the three sample groups. These findings highlight a strategy for probing EV-associated surface targets and suggest broader applicability of this approach across multiple cancers.