Journal of extracellular biology最新文献

Extracellular vesicles (EV) which play critical roles in intercellular communication, have garnered interest as biomarkers with researchers studying brain-related disease processes due to their ability to be isolated from various biofluids. Astrocytes, a type of glial cell, play a critical role in neuronal regulation and function. As such, EV enriched from astrocytes can be used to interrogate cargo and identify mechanisms by which astrocytes communicate with other cells of the central nervous system or shed light on pathophysiological conditions. This manuscript compared five EV isolation methods (differential ultracentrifugation [dUC], precipitation, precipitation + purification, silicon carbon resin and size exclusion chromatography [SEC]) using small volumes of human plasma and serum with a focus on immunocapture of astrocyte-enriched EV (AEEV), with the excitatory amino acid transporter 1, or GLAST. Methods were evaluated on yield, purity, recovery and downstream application to include immunoassays for tetraspanin, immune and astrocyte markers. Results revealed that whilst precipitation-based methods such as ExoQuick yielded higher EV concentrations, size exclusion (SmartSEC, qEV) provided greater purity, emphasizing a trade-off between yield and purity. This study provides a comprehensive resource for researchers in selecting EV isolation methods tailored to small biobanked clinical samples, with the goal of advancing biomarker discovery in Neuroscience.

Proteasomes are essential for protein degradation and maintaining cellular balance, yet their roles in extracellular fluids are not well understood. Our study investigates the freely circulating proteasome in blood, to uncover its unique molecular characteristics, compared to its intracellular counterparts. Using a transgenic mouse model, mass spectrometry, and biochemical tools, we show that the predominant proteasome in serum is the free uncapped 20S particle, which seems to assemble intracellularly before entering the bloodstream. This serum proteasome is composed of constitutive and immuno subunits and exhibits all three catalytic activities. Moreover, the complex displays distinct post-translational modifications, indicating specialization for extracellular roles, as demonstrated by its enhanced caspase-like activity. We also found that physiological stress significantly upregulates serum 20S proteasome levels, paralleling human data. This research highlights the specialized characteristics of circulating proteasomes, offering new insights into protein turnover in the blood with significant implications for understanding proteostasis beyond the intracellular environment.

Amyloid β (Aβ) has emerged as a pathophysiological driver in age-related macular degeneration (AMD), emphasizing its significance in the aetiology of this prevalent sight-threatening condition. The multifaceted nature of AMD pathophysiology, presumably involving diverse retinal cascades, corresponds with the complexity of Aβ-induced retinopathy. Therefore, targeting a broad array of pathogenic processes holds promise for therapeutic intervention in AMD-associated retinal pathology. This study investigates the potential of exosomes derived from adipose tissue mesenchymal stem cells (AT-MSC-Exosomes) in alleviating Aβ-induced retinotoxicity. Through intravitreal injections in wild-type rats and RPE-like cell culture experiments, we examined the protective effects of AT-MSC-Exosomes against Aβ42 retinotoxicity. Our findings reveal that pre-treatment with AT-MSC-Exosomes enabled nearly-intact retinal function in vivo and maintained retinal cell viability in vitro, evidenced by longitudinal electroretinography (ERG) and XTT proliferation assays, respectively. Fluorescent labelling demonstrated increased migration of AT-MSC-Exosomes towards retinal cells under conditions of amyloid-related toxicity. Proteomic analysis indicated a decrease in the retinal levels of heat-shock proteins activated by pathogenic Aβ fibrils following AT-MSC-Exosome treatment. Similarly, immunostaining highlighted the modulation of α-crystallin expression in retinal astrocytes by AT-MSC-Exosomes. These results suggest the potential therapeutic relevance of AT-MSC-Exosomes in Aβ-related retinal pathology, offering a promising avenue for future AMD treatment strategies.

Mesenchymal stromal cell-derived small extracellular vesicles (MSC-sEVs) are pivotal for the curative effects of mesenchymal stromal cells, but their translation into clinical products is hindered by the technical challenges of scaled production and purification. Ultrafiltration, a pressure-driven membrane separation method, is well known as an efficient, scalable, and cost-effective approach for bioseparation. However, there has been little study so far that comprehensively evaluates the potential application of ultrafiltration for scaled sEV isolation and purification. In this study, the feasibility and effectiveness of ultrafiltration for MSC-sEV isolation and purification are studied, and the effects of key process design and operational parameters, including the membrane pore size, transmembrane pressure (TMP), stirring speed (shear rate), feed concentration, are quantified using a stirred cell setup. Results revealed that 500 kDa molecular weight cut-off (MWCO) polyethersulfone membrane demonstrated superior suitability for MSC-sEV separation, yielding higher purity and productivity compared to 100 and 300 kDa MWCO membranes of the same material. The MSC-sEV productivity and purity could also be improved by applying a moderate stirring speed and lower operational pressure, respectively. Isovolumetric diafiltration was incorporated to enhance the purity of MSC-sEVs, successfully removing about 99% of protein contaminants by six diafiltration volumes (DVs). Subsequently, a fed-batch ultra-diafiltration (UF/DF) process with optimised filtration parameters was developed and compared with the currently most used ultracentrifugation (UC) method, showing exceptional effectiveness and performance in the isolation of MSC-sEVs: it increased the recovery of MSC-sEV from 20.59% to 60.88% (about three folds increase) and nearly doubled the purity, while also reducing processing time from over 4 h to 3.5 h, with a potential further reduction to less than 2.5 h through automation. The study concludes that ultrafiltration could be a promising method for both lab-scale preparation and industrial-scale manufacture of MSC-sEVs, offering advantages of high recovery, scalability, fast, and cost-effectiveness.

Alzheimer's disease (AD) is an age-related neurodegenerative pathology. Brain-derived extracellular vesicles (EVs) have been demonstrated to be implicated in AD pathogenesis by facilitating the propagation of Tau, amyloid-β and inflammatory cytokines. However, the impact of peripheral EVs (pEVs) in AD pathogenesis remains poorly investigated. The objective of our study was to compare the passage of pEVs from adults, cognitively healthy elderly, and AD patients through the blood-brain barrier (BBB), to evaluate their uptake in the brain and to assess their impact on the microglia activity using in vitro and in vivo models. To this end, pEVs were enriched, characterized, and fluorescently labelled. The passage of pEVs through the endothelial bEnd.3 cells was studied in a Transwell device with either neuronal or microglia cells seeded at the bottom of the well. Following the internalization of pEVs from AD patients, microglia adopted an amoeboid morphology and released a heightened level of pro-inflammatory cytokine IL-6. To further assess their in vivo transport across the BBB, pEVs were injected into the blood circulation of 2-days post-fertilization Tg(flk1:EGFP) zebrafish. The biodistribution of pEVs was monitored at 1 and 24 h post-injection using confocal microscopy. We demonstrated that pEVs traverse the BBB by transcytosis and subsequently diffuse progressively into the brain. pEVs were then internalized by neuronal and radial glial cells as seen in Tg(huc:EGFP) and Tg(gfap:EGFP) zebrafish, respectively. Additional experiments were performed with the intrahippocampal injection of pEVs in the mouse, indicating their spreading throughout the brain and their uptake by neuronal and glial cells. These findings contribute to novel insights into the fate of pEVs following their passage through the BBB in vitro and in vivo, and demonstrate for the first time that pEVs from AD patients affect microglia activity. This suggests a potential mechanism through which peripheral tissue cues may contribute to AD pathogenesis.

The extracellular vesicle release in red blood cell concentrates reflects progressive accumulation of storage lesions and could represent a new measure to be implemented routinely in blood centres in addition to haemolysis. Nevertheless, there is currently no standardized isolation protocol. In a previous publication, we developed a reproducible ultracentrifugation-based protocol (20,000 × g protocol) that allows to classify red blood cell concentrates into three cohorts according to their vesiculation level. Since this protocol was not adapted to meet routine requirements, the goal of this study was to develop an easier method based on low-speed centrifugation (2,000 × g protocol) and limited red blood cell concentrate volumes to match with a non-destructive sampling from the quality control sampling tubing. Despite the presence of contaminants, mainly in the form of albumin and lipoproteins, the material isolated with the 2,000 × g protocol contained red blood cell-derived vesicular structures. It was reproducible, could predict the number of extracellular vesicles obtained with the 20,000 × g protocol and better discriminated between the three vesiculation cohorts than haemolysis at the legal expiry date of 6 weeks. However, by decreasing red blood cell concentrate volumes to fit with the volume in the quality control tubing, particle yield was highly reduced. Therefore, centrifugation time and relative centrifugal force were adapted (1,000 × g protocol), allowing for the recovery of a similar particle number and composition between small and large volumes sampled from the main unit, in different vesiculation cohorts over time. A similar observation was made with the 1,000 × g protocol between small volumes sampled from the quality control tubing and the mother-bag. In conclusion, our study paves the way for the use of the 2,000 × g protocol (adapted to a 1,000 × g protocol with the quality control sampling tubing) for particle measurement in blood centres.

Human milk extracellular vesicles (EVs) are crucial mother-to-baby messengers that transfer biological signals. These EVs are reported to survive digestion and transport across the intestine. The mechanisms of interaction between human milk EVs and the intestinal mucosa, including epithelial uptake remain unclear. Here, we studied the interaction of human milk EVs with the gut barrier components, including intestinal biofluids, enzymes, mucus and epithelium. Additionally, we probed the endocytic mechanisms mediating the EV intestinal uptake. Finally, using proteomic analysis, we determined the existence and identification of proteins enriched in the EV fraction transported across the intestinal epithelium. We show that human milk EVs are largely stable in the biochemical gut barriers and demonstrate high mucus diffusivity. EVs show a high level of epithelial cell uptake (∼70%) and efficient transport across Caco-2 monolayers. Whilst cell uptake of EVs was mediated by multiple routes, none of the pathway-specific inhibitors inhibited their epithelial translocation. Proteomic analysis of EVs transported across Caco-2 monolayers identified 14 enriched EV proteins that may facilitate intestinal transport. These findings significantly expand our understanding of the interactions between human milk EVs and the gut barriers, including their intestinal uptake.

Current state-of-the-art tools for analysing extracellular vesicles (EVs) offer either highly sensitive but unidimensional bulk measurements of EV components, or high-resolution multiparametric single-particle analyses which lack standardization and appropriate reference materials. This limits the accuracy of the assessment of marker abundance and overall marker distribution amongst individual EVs, and finally, the understanding of true EV heterogeneity. In this study, we aimed to define the standardized operating procedures and reference material for fluorescent characterization of EVs with two commonly used EV analytical platforms—nanoparticle tracking analysis (NTA) and nano-flow cytometry (nFCM). We achieved quantitative fluorescence analyses on ZetaView NTA and NanoAnalyzer nFCM instruments, by utilizing yellow-green FluoSpheres (FS) with assigned ERF (equivalent reference fluorophore) values. This standardization technique allowed for fluorescent EV signal to be expressed in ERF units (indicative of bound fluorescent antibodies per EV), thus enabling measurement of target protein marker abundance on individual EVs, and in the whole EV population. The NTA's and nFCM's limits of detection (LoD) were evaluated at 21 and 9 Alexa Fluor 488 (AF488) molecules, respectively. To complement the limited quantification of markers expressed in a few copies per single EV, in-line bulk fluorescence measurements with a plate reader were performed. This provided absolute marker quantification and more insightful analyses of EV heterogeneity and marker stoichiometry. The standardization method outlined in this work unlocks the full analytical potential of NTA and nFCM, enabling cross-platform data comparison. At the same time, it highlights some of the technical challenges and considerations and thus contributes to the ongoing efforts towards the development of EV analytical tools.

Hallal, S. M., Sida, L. A, Tűzesi, Á., Shivalingam, B., Sim, H.-W., Buckland, M. E, Satgunaseelan, L., & Alexander, K. L (2024). Size matters: Biomolecular compositions of small and large extracellular vesicles in the urine of glioblastoma patients. Journal of Extracellular Biology, 3, e70021. https://doi.org/10.1002/jex2.70021

In the originally-published article, author Ágota Tűzesi's name was incorrectly given as Csilla Ágota Tűzesi. This has been corrected in the online version of the article.

We apologize for this error.

Extracellular vesicles (EVs) are cell-derived small membrane structures that transport various molecules. They have emerged as potential circulating biomarkers for monitoring responses to cancer therapies. This study aimed to comprehensively characterize plasma-carried EVs in hormone receptor-positive (HR⁺) metastatic breast cancer (MBC) patients treated with first-line CDK4/6 inhibitors (iCDK4/6) combined with endocrine therapy. MBC patients were classified into three groups based on their response to therapy: resistant, intermediate or sensitive. In a prospective cohort, we monitored the concentration of circulating EVs, analyzed their lipid signature and correlated these factors with treatment response. To facilitate the translation of EV research to clinical practice, we established a three-step procedure: (1) EVs were isolated from plasma using semi-automatized size exclusion chromatography (SEC); (2) EV concentration, termed vesiclemia, was determined by drop counting via interferometric light microscopy (ILM); and (3) EV lipid composition was analyzed by mass spectrometry. ILM-based vesiclemia values were highly fluctuating upon iCDK4/6 treatment, while early increase associated with accelerated progression. Of note, vesiclemia remained a steady parameter over a 1-year period in age-matched healthy women. Additionally, analysis of the EV cargo unveiled a distinct sphingolipid profile, characterized by increased levels of ceramides and sphingomyelins in resistant patients within the first 2 months of treatment. Based on 16 sphingolipid species, sensitive and resistant patients were correctly classified with an overall accuracy of 82%. This specific sphingolipid pattern was exclusively discernible within EVs, and not in plasma, highlighting the significance of EVs in the early prediction of individual responses to iCDK4/6 and disease progression. Overall, this study provides insights of the longitudinal characterization of plasma-borne EVs in both a healthy group and HR⁺ MBC patients under iCDK4/6 therapies. Combined vesiclemia and EV sphingolipid profile emphasize the promising potential of EVs as non-invasive biomarkers for monitoring early treatment response.