Journal of Extracellular Vesicles最新文献

Up-regulation of Centrosomal Protein 55 (CEP55) in cancer cells increases malignancy, and the protein can be transferred via exosomes. However, the mechanism of how CEP55 is delivered to exosomes is unknown. In this study, we addressed this issue and analysed trafficking of EGFP-CEP55 from early to late endosomes by using high-resolution microscopy. Our data show that endogenous as well as EGFP-CEP55 appeared as dot-like structures in cancer cells. However, we did not find an internalization of CEP55 into early Rab5- and late Rab7-positive endosomes but only into secretory late CD63-positive endosomes. In addition, an association of the CEP55 dots with the endoplasmic reticulum and with ALG-2-interacting protein X (Alix) dots was detected. Moreover, mutation of the CEP55-Alix interaction site strongly reduced the formation of CEP55 dots as well as CEP55 localization in extracellular vesicles. In summary, our data indicate that delivery of CEP55 into exosomes does not occur by the canonical early-to-late endosome pathway but by Alix-mediated recruitment to secretory late secretory CD63 endosomes.

Histones are conserved nuclear proteins that function as part of the nucleosome in the regulation of chromatin structure and gene expression. Interestingly, extracellular histones populate biofluids from healthy individuals, and when elevated, may contribute to various acute and chronic diseases. It is generally assumed that most extracellular histones exist as nucleosomes, as components of extracellular chromatin. We analysed cell culture models under normal and stressed conditions to identify pathways of histone secretion. We report that core and linker histones localize to extracellular vesicles (EVs) and are secreted via the multivesicular body/exosome pathway. Upregulation of EV histone secretion occurs in response to cellular stress, with enhanced vesicle secretion and a shift towards a population of smaller EVs. Most histones were membrane associated with the outer surface of EVs. Degradation of EV-DNA did not impact significantly on EV-histone association. Individual histones  and histone octamers bound strongly to liposomes and EVs, but nucleosomes did not, showing histones do not require DNA for EV binding. Histones colocalized to tetraspanin positive EVs but using genetic or pharmacological intervention, we found that all known pathways of exosome biogenesis acted positively on histone secretion. Inhibition of autophagy and lysosomal degradation had a strong positive effect on EV histone release. Unexpectedly, EV-associated histones lacked the extensive post-translational modification of their nuclear counterparts, suggesting loss of PTMs may be involved in their trafficking or secretion. Our data does not support a significant role for EV-histones existing as nucleosomes. We show for the first time that histones are secreted from cells as membrane proteins via EVs/exosomes. This fundamental discovery provides support for further investigation of the biological activity of exosome associated histones and their role in disease.

Stochiometric issues, encompassing both the quantity and heterogeneity of extracellular vesicles (EVs) derived from tumour or other tissues in blood, pose important challenges across various stages of biomarker discovery and detection, affecting the integrity of data, introducing losses and artifacts during blood processing, EV purification and analysis. These challenges shape the diagnostic utility of EVs especially within the framework of established and emerging methodologies. By addressing these challenges, we aim to delineate crucial parameters and requirements for tumour-specific EV detection, or more precisely, for tumour identification via EV based assays. Our endeavour involves a comprehensive examination of the layers that mask or confound the traceability of EV markers such as nucleic acids and proteins, and focus on ‘low prevalence—low concentration’ scenario. Finally, we evaluate the advantages versus limitations of single-particle analysers over more conventional bulk assays, suggesting that the combined use of both to capture and interpret the EV signals, in particular the EV surface displayed proteins, may ultimately provide quantitative information on their absolute abundance and distribution.

Acetaminophen (APAP) overdose can cause severe liver injury and life-threatening conditions that may lead to multiple organ failure without proper treatment. N-acetylcysteine (NAC) is the accepted and prescribed treatment for detoxification in cases of APAP overdose. Nonetheless, in acute liver failure (ALF), particularly when the ingestion is substantial, NAC may not fully restore liver function. NAC administration in ALF has limitations and potential adverse effects, including nausea, vomiting, diarrhoea, flatus, gastroesophageal reflux, and anaphylactoid reactions. Mesenchymal stromal cell (MSC)-based therapies using paracrine activity show promise for treating ALF, with preclinical studies demonstrating improvement. Recently, MSC-derived extracellular vesicles (EVs) have emerged as a new therapeutic option for liver injury. MSC-derived EVs can contain various therapeutic cargos depending on the cell of origin, participate in physiological processes, and respond to abnormalities. However, most therapeutic EVs lack a distinct orientation upon entering the body, resulting in a lack of targeting specificity. Therefore, enhancing the precision of natural EV delivery systems is urgently needed. Thus, we developed an advanced targeting technique to deliver modified EVs within the body. Our strategy aims to employ bioorthogonal click chemistry to attach a targeting molecule to the surface of small extracellular vesicles (sEVs), creating exogenous chimeric antigen receptor-modified sEVs (CAR-sEVs) for the treatment. First, we engineered azido-modified sEVs (N₃-sEVs) through metabolic glycoengineering by treating MSCs with the azide-containing monosaccharide N-azidoacetyl-mannosamine (Ac4ManNAz). Next, we conjugated N₃-sEVs with a dibenzocyclooctyne (DBCO)-tagged single-chain variable fragment (DBCO-scFv) that targets the asialoglycoprotein receptor (ASGR1), thus producing CAR-sEVs for precise liver targeting. The efficacy of CAR-sEV therapy in ALF models by targeting ASGR1 was validated. MSC-derived CAR-sEVs reduced serum liver enzymes, mitigated liver damage, and promoted hepatocyte proliferation in APAP-induced injury. Overall, CAR-sEVs exhibited enhanced hepatocyte specificity and efficacy in ameliorating liver injury, highlighting the significant advancements achievable with cell-free targeted therapy.

Alzheimer´s disease (AD) is the most frequent neurodegenerative disorder in the world and is characterised by the loss of memory and other cognitive functions. Metabolic changes associated with AD are important players in the development of the disease. However, the mechanism underlying these changes is still unknown. Extracellular vesicles (EVs) are nano-sized particles that play an important role in regulating pathophysiological processes and are a non-invasive manner to obtain information of the cell that is secreting them. The analysis of brain-derived EVs (bdEVs) will provide new insights in the metabolic processes associated with AD. To characterize bdEVs in AD, we optimised a method to isolate them from tissue of different brain regions, obtaining the highest enrichment in isolations from the temporal cortex. We performed unbiased untargeted metabolomics analysis on post-mortem human temporal cortex tissue and bdEVs from the same region of AD patients and healthy controls. Both, univariate and multivariate statistical analysis were used to determine the metabolites that influence the separation between AD patients and controls. Interestingly, a clear separation between control and AD groups was obtained with bdEVs, which allowed to select 12 relevant features by a validated PLS-DA model. Furthermore, comparison of tissue and bdEVs identified 68 common features. The pathway enrichment analysis of the common metabolites showed that the alanine, aspartate and glutamate pathway and the arginine, phenylalanine, tyrosine pathway were the most significant ones in the separation between the AD patients and controls. The phenylalanine, tyrosine and tryptophan pathway, still had a very high influence in the separation between groups, albeit not significant. Notably, some metabolites were identified for the first time in bdEVs. For example, the N-acetyl aspartic acid (NAA) metabolite present in bdEVs was suitable to differentiate AD patients from healthy controls. Furthermore, the analysis of the hippocampus, midbrain, temporal and entorhinal cortex and their respective bdEVs indicated that the metabolic profiles of different brain areas were distinct and showed some correlation between the metabolome of the tissue and its respective bdEVs. Thus, our study highlights the potential of bdEVs to understand the metabolic fingerprint associated with AD and their potential use as diagnostic and therapeutic targets.

Extracellular vesicles (EVs) from brain-seeking breast cancer cells (Br-EVs) breach the blood-brain barrier (BBB) via transcytosis and promote brain metastasis. Here, we defined the mechanisms by which Br-EVs modulate brain endothelial cell (BEC) dynamics to facilitate their BBB transcytosis. BEC treated with Br-EVs show significant downregulation of Rab11fip2, known to promote vesicle recycling to the plasma membrane and significant upregulation of Rab11fip3 and Rab11fip5, which support structural stability of the endosomal compartment and facilitate vesicle recycling and transcytosis, respectively. Using machine learning and quantitative global proteomic, we identified novel Br-EV-induced changes in BECs morphology, motility, and proteome that correlate with decreased BEC cytoplasm and cytoskeletal organization and dynamics. These results define early steps leading to breast-to-brain metastasis and identify molecules that could serve as targets for therapeutic strategies for brain metastasis.

B cell maturation is crucial for effective adaptive immunity. It requires a complex signalling network to mediate antibody diversification through mutagenesis. B cells also rely on queues from other cells within the germinal centre. Recently, a novel class of intercellular signals mediated by extracellular vesicles (EVs) has emerged. Studies have shown that B cell EV-mediated signalling is involved in immune response regulation and tumorigenesis. However, the mechanistic role of B cell EVs is not yet established. We herein study the biological properties and physiological function of B cell EVs during B cell maturation. We use emerging technologies to profile B cell EV surface marker signatures at the single particle level, molecular cargo and physiological roles in B cell maturation. EV ncRNA cargo, characterised by RNA-seq, identified an EV-mediated novel non-coding RNA (ncRNA) regulatory network for B cell maturation. We show that a previously uncharacterised micro-RNA (miR-5099) in combination with a set of long ncRNA are carried within B cell EVs and could contribute to antibody diversification. The physiological role of EVs in B cell maturation is investigated using EV blockade assays and complementation studies using diverse EV sources further confirmed the physiological role and mode of action of EVs in B cell maturation.

Microglial phagocytosis of haematomas is crucial for neural functional recovery following intracerebral haemorrhage (ICH), a process regulated by various factors from within and outside the central nervous system (CNS). Extracellular vesicles (EVs), significant mediators of intercellular communication, have been demonstrated to play a pivotal role in the pathogenesis and progression of CNS diseases. However, the regulatory role of endogenous EVs on the phagocytic capacity of microglia post-ICH remains elusive. Utilising multi-omics analysis of brain tissue-derived EVs proteomics and single-cell RNA sequencing, this study identified that bone marrow-derived macrophages (BMDMs) potentially enhance microglial phagocytosis via EVs following ICH. By blocking BMDMs and reducing ARG1 in BMDM-derived EVs, we demonstrated that BMDMs facilitate erythrophagocytosis by delivering ARG1 to microglia via EVs post-ICH. EVs-carried ARG1 was found to augment phagocytosis by promoting RAC1-dependent cytoskeletal remodelling in microglia. Collectively, this research uncovers an intercellular communication pathway from BMDMs to microglia mediated by EVs post-ICH. This provides a novel paradigm for EV-mediated intercellular communication mechanisms and suggests a promising therapeutic potential for BMDM-derived EVs in the treatment of ICH.

CprA is a short-chain dehydrogenase/reductase (SDR) that contributes to resistance against colistin and antimicrobial peptides. The cprA gene is conserved across Pseudomonas aeruginosa clades and its expression is directly regulated by the two-component system PmrAB. We have shown that cprA expression leads to the production of outer membrane vesicles (OMVs) that block autophagic flux and have a greater capacity to activate the non-canonical inflammasome pathway. In a murine model of sepsis, a P. aeruginosa strain deleted for cprA was less virulent than the wild-type (WT) strain. These results demonstrate the important role of CprA in the pathogenicity of P. aeruginosa. It is worth noting that CprA is also a functional ortholog of hemolysin F (HlyF), which is encoded by virulence plasmids of Escherichia coli. We have shown that other cryptic SDRs encoded by mammalian and plant pathogens, such as Yersinia pestis and Ralstonia solanacearum are functional orthologs of CprA and HlyF. These SDRs also induce the production of OMVs which block autophagic flux. This study uncovers a new family of virulence determinants in Gram-negative bacteria, offering potential for innovative therapeutic interventions and deeper insights into bacterial pathogenesis.

Extracellular vesicles (EVs) can be isolated and purified from cell cultures and biofluids using different methodologies. Here, we explored a novel EV isolation approach by combining superabsorbent polymers (SAP) in a dialysis membrane with size exclusion chromatography (SEC) to achieve high concentration and purity of EVs without the use of ultracentrifugation (UC). Suspension HEK293 cells transfected with CD63 coupled with Thermo Luciferase were used to quantify the EV yield and purity. The 500 mL conditioned medium volume was initially reduced by pressure ultrafiltration, followed by UC, SAP or a centrifugal filter unit (CFU). Using either of these methods, the EVs were concentrated to a final volume of approximately 1 mL, with retained functionality. The yield, quantified by luciferase activity, was highest with UC (70%–80%), followed by SAP (60%–70%) and CFU (50%–60%). Further purification of the EVs was performed by iodixanol density cushion (IDC) or SEC (Sepharose CL-2B or 6B, in either 10 or 20 mL columns). Although the IDC and Sepharose CL-2B (10 mL) achieved the highest yields, the purity was slightly higher (30%) with IDC. In conclusion, combining SAP concentration with CL-2B SEC is an alternative and efficient way to isolate EVs without using UC.