Journal of extracellular vesicles
SCOPUS (SonsInc.)SCIE-ISI
2001-3078
Cơ quản chủ quản: John Wiley & Sons Inc. , WILEY
Lĩnh vực:
Cell BiologyHistology
Các bài báo tiêu biểu
Minimal information for studies of extracellular vesicles 2018 (MISEV2018): a position statement of the International Society for Extracellular Vesicles and update of the MISEV2014 guidelinesABSTRACT The last decade has seen a sharp increase in the number of scientific publications describing physiological and pathological functions of extracellular vesicles (EVs), a collective term covering various subtypes of cell‐released, membranous structures, called exosomes, microvesicles, microparticles, ectosomes, oncosomes, apoptotic bodies, and many other names. However, specific issues arise when working with these entities, whose size and amount often make them difficult to obtain as relatively pure preparations, and to characterize properly. The International Society for Extracellular Vesicles (ISEV) proposed Minimal Information for Studies of Extracellular Vesicles (“MISEV”) guidelines for the field in 2014. We now update these “MISEV2014” guidelines based on evolution of the collective knowledge in the last four years. An important point to consider is that ascribing a specific function to EVs in general, or to subtypes of EVs, requires reporting of specific information beyond mere description of function in a crude, potentially contaminated, and heterogeneous preparation. For example, claims that exosomes are endowed with exquisite and specific activities remain difficult to support experimentally, given our still limited knowledge of their specific molecular machineries of biogenesis and release, as compared with other biophysically similar EVs. The MISEV2018 guidelines include tables and outlines of suggested protocols and steps to follow to document specific EV‐associated functional activities. Finally, a checklist is provided with summaries of key points.
Tập 7 Số 1 - 2018
Optimized exosome isolation protocol for cell culture supernatant and human plasmaExtracellular vesicles represent a rich source of novel biomarkers in the diagnosis and prognosis of disease. However, there is currently limited information elucidating the most efficient methods for obtaining high yields of pure exosomes, a subset of extracellular vesicles, from cell culture supernatant and complex biological fluids such as plasma. To this end, we comprehensively characterize a variety of exosome isolation protocols for their efficiency, yield and purity of isolated exosomes. Repeated ultracentrifugation steps can reduce the quality of exosome preparations leading to lower exosome yield. We show that concentration of cell culture conditioned media using ultrafiltration devices results in increased vesicle isolation when compared to traditional ultracentrifugation protocols. However, our data on using conditioned media isolated from the Non‐Small‐Cell Lung Cancer (NSCLC) SK‐MES‐1 cell line demonstrates that the choice of concentrating device can greatly impact the yield of isolated exosomes. We find that centrifuge‐based concentrating methods are more appropriate than pressure‐driven concentrating devices and allow the rapid isolation of exosomes from both NSCLC cell culture conditioned media and complex biological fluids. In fact to date, no protocol detailing exosome isolation utilizing current commercial methods from both cells and patient samples has been described. Utilizing tunable resistive pulse sensing and protein analysis, we provide a comparative analysis of 4 exosome isolation techniques, indicating their efficacy and preparation purity. Our results demonstrate that current precipitation protocols for the isolation of exosomes from cell culture conditioned media and plasma provide the least pure preparations of exosomes, whereas size exclusion isolation is comparable to density gradient purification of exosomes. We have identified current shortcomings in common extracellular vesicle isolation methods and provide a potential standardized method that is effective, reproducible and can be utilized for various starting materials. We believe this method will have extensive application in the growing field of extracellular vesicle research.
Tập 4 Số 1 - 2015
Neutral sphingomyelinases control extracellular vesicles budding from the plasma membraneABSTRACT Extracellular vesicles (EVs) are membrane particles secreted from cells into all body fluids. Several EV populations exist differing in size and cellular origin. Using differential centrifugation EVs pelleting at 14,000 g (“microvesicles” (MV)) and 100,000 g (“exosomes”) are distinguishable by protein markers. Neutral sphingomyelinase (nSMase) inhibition has been shown to inhibit exosome release from cells and has since been used to study their functional implications. How nSMases (also known as SMPD2 and SMPD3) affect the basal secretion of MVs is unclear. Here we investigated how SMPD2/3 impact both EV populations. SMPD2/3 inhibition by GW4869 or RNAi decreases secretion of exosomes, but also increases secretion of MVs from the plasma membrane. Both populations differ significantly in metabolite composition and Wnt proteins are specifically loaded onto MVs under these conditions. Taken together, our data reveal a novel regulatory function of SMPD2/3 in vesicle budding from the plasma membrane and clearly suggest that – despite the different vesicle biogenesis – the routes of vesicular export are adaptable.
Tập 6 Số 1 - 2017
Size and concentration analyses of extracellular vesicles by nanoparticle tracking analysis: a variation studyABSTRACT Current methods for characterisation of extracellular vesicles (EVs) need further standardisation in order to obtain an acceptable level of data comparability. Size and concentration of EVs can be determined by nanoparticle tracking analysis (NTA). However, both the heterogeneity of EVs and the choice of instrument settings may cause an appreciable analytical variation. Intra‐assay (within‐day, n = 6) and inter‐assay (day‐to‐day, n = 6) variations (coefficient of variation, % CV) of different preparations of EVs and artificial vesicles or beads were determined using two NanoSight NS500 instruments, located at different laboratories. All analyses were performed by the same operator. The effect of applying identical software settings or instrument‐optimised settings for each sample type and instrument was also evaluated. Finally, the impact of different operators and the use of two different software versions were investigated. The intra‐assay CVs were 1–12% for both EVs and artificial samples, measured on the same instrument. The overall day‐to‐day variation was similar for both instruments, ranging from 2% to 25%. However, significantly different results were observed between the two instruments using identical software settings. The effect of applying instrument‐optimised settings reduced the mismatch between the instruments, resulting in little to no significant divergences. The impact of using different operators and software versions when analysing silica microspheres and microvesicles from monocytes using instrument‐optimised settings on the same instrument did not contribute to significant variation compared to the overall day‐to‐day variation of one operator. Performance differences between two similar NTA instruments may display significant divergences in size and concentration measurements when analysing EVs, depending on applied instrument settings and technical conditions. The importance of developing a streamlined and standardised execution of analysis, as well as monitoring longitudinal variation parameters on both biological and synthetic samples, should be highlighted.
Tập 6 Số 1 - 2017
Effect of storage on physical and functional properties of extracellular vesicles derived from neutrophilic granulocytesAim To carry out a systematic study on the effect of different storage conditions on the number as well as the physical and functional properties of antibacterial extracellular vesicles (EVs) derived from human neutrophilic granulocytes. Methods Production of EVs with antibacterial properties was initiated by opsonized Zymosan A particles. The number of released fluorescent EVs was determined by flow cytometry following careful calibration. Physical properties and size of EVs were investigated by flow cytometry, dynamic light scattering and electron microscopy. Functional properties of EVs were tested by bacterial survival assay. Results Storage at +20°C or +4°C resulted in a significant decrease of EV number and antibacterial effect after 1 day. Storage at −20°C did not influence the EV number up to 28 days, but induced a shift in EV size and almost complete loss of antibacterial function by 28 days. Storage at −80°C had no significant effect either on EV number or size and allowed partial preservation of the antibacterial function up to 28 days. Snap‐freezing did not improve the results, whereas the widely used cryoprotectants induced EV lysis. Conclusion Storage significantly alters both the physical and functional properties of EVs even if the number of EVs stays constant. If storage is needed, EVs should be kept at −80°C, preferably not longer than 7 days. For functional tests, freshly prepared EVs are recommended.
Tập 3 Số 1 - 2014
Evaluation of circulating small extracellular vesicles derived miRNAs as biomarkers of early colon cancer: a comparison with plasma total miRNAsABSTRACT Early diagnosis of colon cancer (CC) is clinically important, as it can significantly improve patients' survival rate and quality of life. Although the potential role for small extracellular vesicles (sEVs) in early detection of many diseases has been repeatedly mentioned, systematic screening of plasma sEVs derived early CC specific biomarkers has not yet been reported. In this work, plasma sEVs enriched fractions were derived from 15 early‐stage (TisN0M0) CC patients and 10 normal controls (NC). RNA sequencing identified a total number of 95 sEVs enriched fraction derived miRNAs with differential expression between CC and NC, most of which (60/95) was in well accordance with tissue results in the Cancer Genome Atlas (TCGA) dataset. Among those miRNAs, we selected let‐7b‐3p, miR‐139‐3p, miR‐145‐3p, and miR‐150‐3p for further validation in an independent cohort consisting of 134 participants (58 CC and 76 NC). In the validation cohort, the AUC of 4 individual miRNAs ranged from 0.680 to 0.792. A logistic model combining two miRNAs (i.e. let‐7b‐3p and miR‐145‐3p) achieved an AUC of 0.901. Adding the 3rd miRNA into this model can further increase the AUC to 0.927. Side by side comparison revealed that sEVs miRNA profile outperformed cell‐free plasma miRNA in the diagnosis of early CC. In conclusion, we suggested that circulating sEVs enriched fractions have a distinct miRNA profile in CC patients, and sEVs derived miRNA could be used as a promising biomarker to detect CC at an early stage.
Tập 8 Số 1 - 2019
Comparative analysis of EV isolation procedures for miRNAs detection in serum samplesExtracellular vesicles (EVs) are emerging as potent non‐invasive biomarkers. However, current methodologies are time consuming and difficult to translate to clinical practice. To analyse EV‐encapsulated circulating miRNA, we searched for a quick, easy and economic method to enrich frozen human serum samples for EV. We compared the efficiency of several protocols and commercial kits to isolate EVs. Different methods based on precipitation, columns or filter systems were tested and compared with ultracentrifugation, which is the most classical protocol to isolate EVs. EV samples were assessed for purity and quantity by nanoparticle tracking analysis and western blot or cytometry against major EV protein markers. For biomarker validation, levels of a set of miRNAs were determined in EV fractions and compared with their levels in total serum. EVs isolated with precipitation‐based methods were enriched for a subgroup of miRNAs that corresponded to miRNAs described to be encapsulated into EVs (miR‐126, miR‐30c and miR‐143), while the detection of miR‐21, miR‐16‐5p and miR‐19a was very low compared with total serum. Our results point to precipitation using polyethylene glycol (PEG) as a suitable method for an easy and cheap enrichment of serum EVs for miRNA analyses. The overall performance of PEG was very similar, or better than other commercial precipitating reagents, in both protein and miRNA yield, but in comparison to them PEG is much cheaper. Other methods presented poorer results, mostly when assessing miRNA by qPCR analyses. Using PEG precipitation in a longitudinal study with human samples, we demonstrated that miRNA could be assessed in frozen samples up to 8 years of storage. We report a method based on a cut‐off value of mean of fold EV detection versus serum that provides an estimate of the degree of encapsulation of a given miRNA.
Tập 5 Số 1 - 2016
MiR155‐5p in adventitial fibroblasts‐derived extracellular vesicles inhibits vascular smooth muscle cell proliferation via suppressing angiotensin‐converting enzyme expressionABSTRACT Proliferation of vascular smooth muscle cells (VSMCs) plays crucial roles in vascular remodelling and stiffening in hypertension. Vascular adventitial fibroblasts are a key regulator of vascular wall function and structure. This study is designed to investigate the roles of adventitial fibroblasts‐derived extracellular vesicles (EVs) in VSMC proliferation and vascular remodelling in normotensive Wistar‐Kyoto rat (WKY) and spontaneously hypertensive rat (SHR), an animal model of human essential hypertension. EVs were isolated from aortic adventitial fibroblasts of WKY (WKY‐EVs) and SHR (SHR‐EVs). Compared with WKY‐EVs, miR155‐5p content was reduced, while angiotensin‐converting enzyme (ACE) content was increased in SHR‐EVs. WKY‐EVs inhibited VSMC proliferation of SHR, which was prevented by miR155‐5p inhibitor. SHR‐EVs promoted VSMC proliferation of both strains, which was enhanced by miR155‐5p inhibitor, but abolished by captopril or losartan. Dual luciferase reporter assay showed that ACE was a target gene of miR155‐5p. MiR155‐5p mimic or overexpression inhibited VSMC proliferation and ACE upregulation of SHR. WKY‐EVs reduced ACE mRNA and protein expressions while SHR‐EVs only increased ACE protein level in VSMCs of both strains. However, the SHR‐EVs‐derived from the ACE knockdown‐treated adventitial fibroblasts lost the roles in promoting VSMC proliferation and ACE upregulation. Systemic miR155‐5p overexpression reduced vascular ACE, angiotensin II and proliferating cell nuclear antigen levels, and attenuated hypertension and vascular remodelling in SHR. Repetitive intravenous injection of SHR‐EVs increased blood pressure and vascular ACE contents, and promoted vascular remodelling in both strains, while WKY‐EVs reduced vascular ACE contents and attenuated hypertension and vascular remodelling in SHR. We concluded that WKY‐EVs‐mediated miR155‐5p transfer attenuates VSMC proliferation and vascular remodelling in SHR via suppressing ACE expression, while SHR‐EVs‐mediated ACE transfer promotes VSMC proliferation and vascular remodelling.
Tập 9 Số 1 - 2020
An experimental strategy unveiling exosomal microRNAs 486‐5p, 181a‐5p and 30d‐5p from hypoxic tumour cells as circulating indicators of high‐risk rectal cancerABSTRACT Tumour hypoxia contributes to poor treatment outcome in locally advanced rectal cancer (LARC) and circulating extracellular vesicles (EVs) as potential biomarkers of tumour hypoxia and adverse prognosis have not been fully explored. We examined EV miRNAs from hypoxic colorectal cancer cell lines as template for relevant miRNAs in LARC patients participating in a prospective biomarker study (NCT01816607). Five cell lines were cultured under normoxia (21% O2 ) or hypoxia (0.2% O2 ) for 24 h, and exosomes were isolated by differential ultracentrifugation. Using a commercial kit, exosomes were precipitated from 24 patient plasma samples collected at the time of diagnosis. Exosome size distribution and protein cargo were determined by cryo‐electron microscopy, nanoparticle tracking analysis, immunoblotting and flow cytometry. The vesicles harboured strong cell line‐specific miRNA profiles with 35 unique miRNAs differentially expressed between hypoxic and normoxic cells. Six of these miRNAs were considered candidate‐circulating markers of tumour hypoxia in the patients based on the frequency or magnitude of variance in hypoxic versus normoxic cell line experiments and prevalence in patient plasma. Of these, low plasma levels of exosomal miR‐486‐5p and miR‐181a‐5p were associated with organ‐invasive primary tumour (p = 0.029) and lymph node metastases (p = 0.024), respectively, both attributes of adverse LARC prognosis. In line with this, the plasma level of exosomal miR‐30d‐5p was elevated in patients who experienced metastatic progression (p = 0.036). Our strategy confirmed that EVs from colorectal cancer cell lines were exosomes containing the oxygen‐sensitive miRNAs 486‐5p, 181a‐5p and 30d‐5p, which were retrieved as circulating markers of high‐risk LARC.
Tập 8 Số 1 - 2019
Prostate cancer extracellular vesicles mediate intercellular communication with bone marrow cells and promote metastasis in a cholesterol‐dependent mannerAbstract Primary tumours can establish long‐range communication with distant
organs to transform them into fertile soil for circulating tumour cells to
implant and proliferate, a process called pre‐metastatic niche (PMN) formation. Tumour‐derived extracellular vesicles (EV) are potent mediators of PMN formation due to their diverse complement of pro‐malignant molecular cargo and their propensity to target specific cell types (Costa‐Silva et al., 2015; Hoshino et al., 2015; Peinado et al., 2012; Peinado et al., 2017). While significant progress has been made to understand the mechanisms by which pro‐metastatic EVs create tumour‐favouring microenvironments at pre‐metastatic organ sites, comparatively little attention has been paid to the factors intrinsic to recipient cells that may modify the extent to which pro‐metastatic EV signalling is received and transduced. Here, we investigated the role of recipient cell cholesterol homeostasis in prostate cancer (PCa) EV‐mediated signalling and metastasis. Using a bone metastatic model of enzalutamide‐resistant PCa, we first characterized an axis of EV‐mediated communication between PCa cells and bone marrow that is marked by in vitro and in vivo PCa EV uptake by bone marrow myeloid cells, activation of NF‐κB signalling, enhanced osteoclast differentiation, and reduced myeloid thrombospondin‐1 expression. We then employed a targeted, biomimetic approach to reduce myeloid cell cholesterol in vitro and in vivo prior to conditioning with PCa EVs. Reducing myeloid cell cholesterol prevented the uptake of PCa EVs by recipient myeloid cells, abolished NF‐κB activity and osteoclast differentiation, stabilized thrombospondin‐1 expression, and reduced metastatic burden by 77%. These results demonstrate that cholesterol homeostasis in bone marrow myeloid cells regulates pro‐metastatic EV signalling and metastasis by acting as a gatekeeper for EV signal transduction.
Tập 10 Số 2 - 2020