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The technical difficulty of separating extracellular vesicles (EVs) from plasma proteins in human blood presents a significant hurdle in EV research, particularly during nano ultra-high-performance liquid chromatography-tandem mass spectrometric (UHPLC-MS/MS) analysis, where detecting "vesicular" proteins among abundant plasma proteins is challenging. Standardisation is a pressing issue in EV research, prompting collaborative global efforts to address it. While the MISEV guidelines offer valuable recommendations, unanswered questions remain, particularly regarding sample storage. We compared size exclusion chromatography (SEC) columns with pore sizes of 35 nm and 70 nm to identify fractions with minimal contaminating proteins and the highest concentration of small EVs (sEVs). Following column selection, we explored potential differences in the quality and quantity of sEVs isolated from platelet-free plasma (PFP) after long-term storage at -80 °C (>2.5 years) compared to freshly drawn blood. Our methodologically rigorous study indicates that prolonged storage, under correct storage and processing conditions, does not compromise sEV quality. Both columns effectively isolated vesicles, with the 70 nm column exhibiting a higher abundance of "vesicular" proteins. We propose a relatively rapid and moderately efficient protocol for obtaining a comparatively pure sEV fraction from plasma, facilitating sEV processing in clinical trials.
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Background and Objectives: Progressive supranuclear palsy (PSP) is a neurodegenerative disease, a tauopathy, which results in a wide clinical spectrum of neurological symptoms. The diagnosis is mostly based on clinical signs and neuroimaging; however, possible biomarkers for screening have been under investigation, and the role of the gut microbiome is unknown. The aim of our study was to identify potential blood biomarkers and observe variations in the gut microbiome within a PSP discordant monozygotic twin pair. Materials and Methods: Anthropometric measurements, neuropsychological tests, and the neurological state were evaluated. Blood was collected for metabolic profiling and for the detection of neurodegenerative and vascular biomarkers. Both the gut microbiome and brain MRI results were thoroughly examined. Results: We found a relevant difference between alpha-synuclein levels and moderate difference in the levels of MMP-2, MB, Apo-A1, Apo-CIII, and Apo-H. With respect to the ratios, a small difference was observed for ApoA1/SAA and ApoB/ApoA1. Using a microbiome analysis, we also discovered a relative dysbiosis, and the MRI results revealed midbrain and frontoparietal cortical atrophy along with a reduction in overall brain volumes and an increase in white matter lesions in the affected twin. Conclusions: We observed significant differences between the unaffected and affected twins in some risk factors and blood biomarkers, along with disparities in the gut microbiome. Additionally, we detected abnormalities in brain MRI results and alterations in cognitive functions.
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Enfermedades Neurodegenerativas , Parálisis Supranuclear Progresiva , Humanos , Parálisis Supranuclear Progresiva/diagnóstico por imagen , Parálisis Supranuclear Progresiva/patología , Imagen por Resonancia Magnética/métodos , Factores de Riesgo , BiomarcadoresRESUMEN
Tumors are intricate ecosystems where cancer cells and non-malignant stromal cells, including cancer-associated fibroblasts (CAFs), engage in complex communication. In this study, we investigated the interaction between poorly (HLE) and well-differentiated (HuH7) hepatoma cells and LX2 fibroblasts. We explored various communication channels, including soluble factors, metabolites, extracellular vesicles (EVs), and miRNAs. Co-culture with HLE cells induced LX2 to produce higher levels of laminin ß1, type IV collagen, and CD44, with pronounced syndecan-1 shedding. Conversely, in HuH7/LX2 co-culture, fibronectin, thrombospondin-1, type IV collagen, and cell surface syndecan-1 were dominant matrix components. Integrins α6ß4 and α6ß1 were upregulated in HLE, while α5ß1 and αVß1 were increased in HuH7. HLE-stimulated LX2 produced excess MMP-2 and 9, whereas HuH7-stimulated LX2 produced excess MMP-1. LX2 activated MAPK and Wnt signaling in hepatoma cells, and conversely, hepatoma-derived EVs upregulated MAPK and Wnt in LX2 cells. LX2-derived EVs induced over tenfold upregulation of SPOCK1/testican-1 in hepatoma EV cargo. We also identified liver cancer-specific miRNAs in hepatoma EVs, with potential implications for early diagnosis. In summary, our study reveals tumor type-dependent communication between hepatoma cells and fibroblasts, shedding light on potential implications for tumor progression. However, the clinical relevance of liver cancer-specific miRNAs requires further investigation.
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Carcinoma Hepatocelular , Neoplasias Hepáticas , Humanos , Carcinoma Hepatocelular/genética , Sindecano-1 , Colágeno Tipo IV , Ecosistema , Neoplasias Hepáticas/genética , Fibroblastos , Comunicación , ProteoglicanosRESUMEN
Extracellular vesicles (EVs) are membrane-enclosed subcellular structures released by all cell types. EVs have important roles in both cellular homeostasis and intercellular communication. Recent progress in the field revealed substantial heterogeneity of EVs even within the size-based EV categories. Here we addressed the question whether the exportin-1 (XPO1)-mediated nuclear export of RNAs contributed to the EV heterogeneity. Size-based populations were separated from the conditioned media of three cell lines (U937, THP-1 and 5/4E8) in steady-state condition. The effects of activation and leptomycin B treatment (to inhibit the XPO1-mediated nuclear export of RNAs) were also tested in the case of the two monocytic cell lines. Agilent Pico and Small chips were used to characterize RNAs, fragment analysis was performed, and EV-associated miRNAs were tested by Taqman assays. As expected, we found the highest small RNA/total RNA ratio and the lowest rRNA/total RNA proportion in small EVs (~ 50-150 nm). Profiles of the small RNAs within different size-based EV categories significantly differed based on the activation status of the EV releasing cells. Leptomycin B had a differential inhibition on the tested small RNAs in EVs, even within the same EV size category. A similar heterogeneity of the EV miRNA content was observed upon cellular activation and nuclear export inhibition. Here we complement the already existing knowledge on EV heterogeneity by providing evidence that the RNA cargo varies depending on the EV size-based category, the releasing cell type, the functional status of the releasing cells and the exportin-1-mediated nuclear export of RNAs.
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Vesículas Extracelulares , MicroARNs , Animales , Humanos , Ratones , Transporte Activo de Núcleo Celular , Comunicación Celular , Vesículas Extracelulares/metabolismo , Carioferinas/genética , Carioferinas/metabolismo , MicroARNs/genética , MicroARNs/metabolismo , Proteína Exportina 1RESUMEN
Nowadays, extracellular vesicles (EVs) raise a great interest as they are implicated in intercellular communication between cancer and stromal cells. Our aim was to understand how vesicular NME1 and NME2 released by breast cancer cells influence the tumour microenvironment. As a model, we used human invasive breast carcinoma cells overexpressing NME1 or NME2, and first analysed in detail the presence of both isoforms in EV subtypes by capillary Western immunoassay (WES) and immunoelectron microscopy. Data obtained by both methods showed that NME1 was present in medium-sized EVs or microvesicles, whereas NME2 was abundant in both microvesicles and small-sized EVs or exosomes. Next, human skin-derived fibroblasts were treated with NME1 or NME2 containing EVs, and subsequently mRNA expression changes in fibroblasts were examined. RNAseq results showed that the expression of fatty acid and cholesterol metabolism-related genes was decreased significantly in response to NME1 or NME2 containing EV treatment. We found that FASN (fatty acid synthase) and ACSS2 (acyl-coenzyme A synthetase short-chain family member 2), related to fatty acid synthesis and oxidation, were underexpressed in NME1/2-EV-treated fibroblasts. Our data show an emerging link between NME-containing EVs and regulation of tumour metabolism.
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BACKGROUND: tert-butylhydroquinone (tBHQ) is an antioxidant commonly used as a food additive. Studies suggest that tBHQ could modulate immune responses to influenza and SARS-CoV-2 infection. In our transcriptomic analysis we explored the molecular mechanisms behind tBHQ's modulatory properties and the relationships to respiratory viral infections. METHODS: tBHQ was administered per os to BALB/c mice (1.5% [w/w]) for 20 days. Splenic T cells were isolated with magnetic separation and subjected to transcriptomic analysis. Gene-set enrichment analysis and g:Profiler was conducted to provide a functional interpretation of significantly changed genes. Further analysis for AHR/NRF2 binding sites was performed with GeneHancer. RESULTS: In CD4+ cells, we found significantly altered expression of 269 genes by tBHQ. Of them, many had relevance in influenza infection such as genes responsible for virus entry (Anxa1/2, Cd14), interferon signaling (Dusp10, Tnfsf13), or prostaglandin synthesis (Ptgs1/2). In SARS-CoV-2 infections, interferon signaling (Ifitm1), proteolytic enzymes (CtsB), and also cell-surface proteins (Cd14, Cd151) were among the prominent alterations after tBHQ exposure. Of these genes, many had one or more binding sites for AHR and NRF2, two major xenosensors triggered by tBHQ. CONCLUSIONS: Our results strongly suggest that a common food additive, tBHQ, can modulate virus-dependent processes in both influenza and SARS-CoV-2 infections.
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The release of extracellular vesicles (EVs) is increased under cellular stress and cardiomyocyte damaging conditions. However, whether the cardiomyocyte-derived EVs eventually reach the systemic circulation and whether their number in the bloodstream reflects cardiac injury, remains unknown. Wild type C57B/6 and conditional transgenic mice expressing green fluorescent protein (GFP) by cardiomyocytes were studied in lipopolysaccharide (LPS)-induced systemic inflammatory response syndrome (SIRS). EVs were separated both from platelet-free plasma and from the conditioned medium of isolated cardiomyocytes of the left ventricular wall. Size distribution and concentration of the released particles were determined by Nanoparticle Tracking Analysis. The presence of GFP + cardiomyocyte-derived circulating EVs was monitored by flow cytometry and cardiac function was assessed by echocardiography. In LPS-treated mice, systemic inflammation and the consequent cardiomyopathy were verified by elevated plasma levels of TNFα, GDF-15, and cardiac troponin I, and by a decrease in the ejection fraction. Furthermore, we demonstrated elevated levels of circulating small- and medium-sized EVs in the LPS-injected mice. Importantly, we detected GFP+ cardiomyocyte-derived EVs in the circulation of control mice, and the number of these circulating GFP+ vesicles increased significantly upon intraperitoneal LPS administration (P = 0.029). The cardiomyocyte-derived GFP+ EVs were also positive for intravesicular troponin I (cTnI) and muscle-associated glycogen phosphorylase (PYGM). This is the first direct demonstration that cardiomyocyte-derived EVs are present in the circulation and that the increased number of cardiac-derived EVs in the blood reflects cardiac injury in LPS-induced systemic inflammation (SIRS).
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Movimiento Celular , Vesículas Extracelulares/metabolismo , Miocardio/patología , Miocitos Cardíacos/patología , Síndrome de Respuesta Inflamatoria Sistémica/patología , Animales , Movimiento Celular/efectos de los fármacos , Clusterina/metabolismo , Vesículas Extracelulares/efectos de los fármacos , Glucógeno Fosforilasa/metabolismo , Proteínas Fluorescentes Verdes/metabolismo , Integrasas/metabolismo , Lipopolisacáridos , Masculino , Ratones Endogámicos C57BL , Ratones Transgénicos , Miocardio/metabolismo , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/metabolismo , Especificidad de Órganos/efectos de los fármacos , Fenotipo , Síndrome de Respuesta Inflamatoria Sistémica/sangre , Síndrome de Respuesta Inflamatoria Sistémica/fisiopatología , Tamoxifeno/farmacología , Troponina I/metabolismoRESUMEN
Mast cells have been shown to release extracellular vesicles (EVs) in vitro. However, EV-mediated mast cell communication in vivo remains unexplored. Primary mast cells from GFP-transgenic and wild type mice, were grown in the presence or absence of lipopolysaccharide (LPS), and the secreted EVs were separated from the conditioned media. Mast cell-derived EVs were next cultured with LPS-naïve mast cells, and the induction of TNF-α expression was monitored. In addition, primary mast cells were seeded in diffusion chambers that were implanted into the peritoneal cavities of mice. Diffusion chambers enabled the release of GFP+ mast cell-derived EVs in vivo into the peritoneal cavity. Peritoneal lavage cells were assessed for the uptake of GFP+ EVs and for TNF-α production. In vitro, LPS-stimulated mast cell-derived EVs were efficiently taken up by non-stimulated mast cells, and induced TNF-α expression in a TLR4, JNK and P38 MAPK dependent manner. In vivo, using implanted diffusion chambers, we confirmed the release and transmission of mast cell-derived EVs to other mast cells with subsequent induction of TNF-α expression. These data show an EV-mediated spreading of pro-inflammatory response between mast cells, and provide the first in vivo evidence for the biological role of mast cell-derived EVs.
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Vesículas Extracelulares/metabolismo , Sistema de Señalización de MAP Quinasas , Mastocitos/metabolismo , Animales , Células Cultivadas , Vesículas Extracelulares/genética , Regulación de la Expresión Génica/efectos de los fármacos , Inflamación/inducido químicamente , Inflamación/genética , Inflamación/metabolismo , Lipopolisacáridos/toxicidad , Ratones , Ratones Transgénicos , Factor de Necrosis Tumoral alfa/biosíntesis , Factor de Necrosis Tumoral alfa/genéticaRESUMEN
Extracellular vesicles (EV) are membrane-surrounded vesicles that represent a novel way of intercellular communication by carrying biologically important molecules in a concentrated and protected form. The intestinal epithelium is continuously renewed by a small proliferating intestinal stem cell (ISC) population, residing at the bottom of the intestinal crypts in a specific microenvironment, the stem cell niche. By using 3D mouse and human intestinal organoids, we show that intestinal fibroblast-derived EVs are involved in forming the ISC niche by transmitting Wnt and epidermal growth factor (EGF) activity. With a mouse model that expresses EGFP in the Lgr5+ ISCs, we prove that loss in ISC number in the absence of EGF is prevented by fibroblast-derived EVs. Furthermore, we demonstrate that intestinal fibroblast-derived EVs carry EGF family members, such as amphiregulin. Mechanistically, blocking EV-bound amphiregulin inhibited the EV-induced survival of organoids. In contrast, EVs have no role in transporting R-Spondin, a critical niche factor amplifying Wnt signaling. Collectively, we prove the important role of fibroblast-derived EVs as a novel transmission mechanism of factors in the normal ISC niche.
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Vesículas Extracelulares/metabolismo , Mucosa Intestinal/fisiopatología , Intestinos/fisiopatología , Nicho de Células Madre/genética , Anciano , Humanos , Masculino , Persona de Mediana EdadRESUMEN
Stem cell-based therapies raise hope for cell replacement and provide opportunity for cardiac regenerative medicine and tumor therapy. Extracellular vesicles are a membrane-enclosed intercellular delivery system with the potential to improve the therapeutic efficacy of the treatment of a variety of disorders. As the incidence of breast cancer continues to rise, radiotherapy has emerged as a leading treatment modality. Radiotherapy also increases the risk of coronary heart disease and cardiac mortality. In a chest-irradiated mouse model of cardiac injury, we investigated the effects of local irradiation. We found an increased lethality after 16 Gy irradiation. Importantly, radio-detoxified LPS (RD-LPS) treatment prolonged the survival significantly. By flow cytometry, we demonstrated that upon administration of RD-LPS, the number of bone marrow-derived endothelial progenitor cells increased in the bone marrow and, in particular, in the circulation. Furthermore, mass spectrometry analysis showed that RD-LPS altered the proteomic composition of bone marrow cell-derived small extracellular vesicles (sEVs). RD-LPS treatment increased interferon-induced transmembrane protein-3 (IFITM3) expression markedly both in bone marrow cells and in bone marrow cell-derived small extracellular vesicles. This is the first study to demonstrate that radio-detoxified LPS treatment induces an increase of circulating endothelial progenitor cells (EPCs) in parallel with a reduced radiotherapy-related mortality. While the total number of bone marrow-derived extracellular vesicles was significantly increased 24 h after treatment in the RD-LPS groups, the number of endothelial progenitor cells was reduced in animals injected with GW4896 (a chemical inhibitor of exosome biogenesis) as compared with controls. In contrast to these in vivo results, in vitro experiments did not support the effect of sEVs on EPCs. Our data raise the intriguing possibility that IFITM3 may serve as a marker of the radio-detoxified LPS treatment.
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Células de la Médula Ósea/metabolismo , Células Progenitoras Endoteliales/metabolismo , Vesículas Extracelulares/metabolismo , Rayos gamma , Lipopolisacáridos/farmacología , Lipopolisacáridos/efectos de la radiación , Animales , Células de la Médula Ósea/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Células Progenitoras Endoteliales/efectos de los fármacos , Vesículas Extracelulares/efectos de los fármacos , Vesículas Extracelulares/ultraestructura , Silenciador del Gen , Proteínas de la Membrana/metabolismo , Ratones Endogámicos C57BL , Protectores contra Radiación/farmacologíaRESUMEN
Small extracellular vesicles (EVs) are membrane enclosed structures that are usually released from cells upon exocytosis of multivesicular bodies (MVBs) as a collection of separate, free EVs. In this study, we analysed paraffin embedded sections of archived human colorectal cancer samples. We studied 3D reconstructions of confocal microscopic images complemented by HyVolution and STED imaging. Unexpectedly, we found evidence that large, MVB-like aggregates of ALIX/CD63 positive EV clusters were released en bloc by migrating tumour cells. These structures were often captured with partial or complete extra-cytoplasmic localization at the interface of the plasma membrane of the tumour cell and the stroma. Their diameter ranged between 0.62 and 1.94 µm (mean±S.D.: 1.17 ± 0.34 µm). High-resolution 3D reconstruction showed that these extracellular MVB-like EV clusters were composed of distinguishable internal particles of small EV size (mean±S.D.: 128.96 ± 16.73 nm). In vitro, HT29 colorectal cancer cells also showed the release of similar structures as confirmed by immunohistochemistry and immune electron microscopy. Our results provide evidence for an en bloc transmission of MVB-like EV clusters through the plasma membrane. Immunofluorescent-based detection of the MVB like small EV clusters in archived pathological samples may represent a novel and unique opportunity which enables analysis of EV release in situ in human tissues.
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Extracellular vesicles (EVs) are membrane-surrounded structures that transmit biologically important molecules from the releasing to target cells, thus providing a novel intercellular communication mechanism. Since EVs carry their cargo in a protected form and their secretion is generally increased in tumorigenesis, EVs hold a great potential for early cancer diagnosis. By 3D culturing, we provide evidence that colorectal cancer (CRC) patient-derived organoids, representing a state-of-the-art established and essential approach for studying human CRC, is a suitable model for EV analysis. When testing the effects of major factors promoting CRC progression on EV release in the organoid model, we observed that Apc mutation, leading to uncontrolled Wnt activation and thus to tumorigenesis in the vast majority in CRC patients, critically induces EV release by activating the Wnt pathway. Furthermore, the extracellular matrix component collagen, known to accumulate in tumorigenesis, enhances EV secretion as well. Importantly, we show that fibroblast-derived EVs induce colony formation of CRC organoid cells under hypoxia. In contrast, there was no major effect of tumor cell-derived EVs on the activation of fibroblasts. Collectively, our results with CRC and Apc-mutant adenoma organoids identify Apc mutation and collagen deposition as critical factors for increasing EV release from tumors. Furthermore, we provide evidence that stromal fibroblast-derived EVs contribute to tumorigenesis under unfavorable conditions in CRC.
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Proteína de la Poliposis Adenomatosa del Colon/genética , Neoplasias Colorrectales/patología , Vesículas Extracelulares/patología , Intestinos/patología , Organoides/patología , Animales , Carcinogénesis/genética , Carcinogénesis/metabolismo , Carcinogénesis/patología , Línea Celular Tumoral , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/metabolismo , Progresión de la Enfermedad , Vesículas Extracelulares/genética , Humanos , Ratones Endogámicos C57BL , Mutación , Organoides/metabolismo , Células Tumorales Cultivadas , Vía de Señalización WntRESUMEN
AIMS: The prognosis of patients with pancreatic cancer has remained virtually unchanged with a high mortality rate compared to other types of cancers. An earlier detection would provide a time window of opportunity for treatment and prevention of deaths. In the present study we investigated extracellular vesicle (EV)-associated potential biomarkers for pancreatic cancer by directly assessing EV size-based subpopulations in pancreatic juice samples of patients with chronic pancreatitis or pancreatic cancer. In addition, we also studied blood plasma and pancreatic cancer cell line-derived EVs. METHODS: Comparative proteomic analysis was performed of 102â¯EV preparations from human pancreatic juices, blood, and pancreatic cancer cell lines Capan-1 and MIA PaCa-2. EV preparations were also characterized by electron microscopy, tunable resistive pulse sensing, and flow cytometry. RESULTS: Here we describe the presence of EVs in human pancreatic juice samples. Pancreatic juice EV-associated proteins that we identified as possible candidate markers for pancreatic cancer included mucins, such as MUC1, MUC4, MUC5AC, MUC6 and MUC16, CFTR, and MDR1 proteins. These candidate biomarkers could also be detected by flow cytometry in EVs found in pancreatic juice and those secreted by pancreatic cancer cell lines. CONCLUSIONS: Together our data show that detection and characterization of EVs directly in pancreatic juice is feasible and may prove to be a valuable source of potential biomarkers of pancreatic cancer.
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Adenocarcinoma/diagnóstico , Biomarcadores de Tumor/genética , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Vesículas Extracelulares/química , Mucinas/genética , Neoplasias Pancreáticas/diagnóstico , Subfamilia B de Transportador de Casetes de Unión a ATP/genética , Subfamilia B de Transportador de Casetes de Unión a ATP/metabolismo , Adenocarcinoma/genética , Adenocarcinoma/metabolismo , Adenocarcinoma/patología , Biomarcadores de Tumor/metabolismo , Línea Celular Tumoral , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Diagnóstico Diferencial , Vesículas Extracelulares/metabolismo , Expresión Génica , Humanos , Mucinas/metabolismo , Páncreas , Jugo Pancreático/química , Jugo Pancreático/metabolismo , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patología , Pancreatitis Crónica/diagnóstico , Pancreatitis Crónica/genética , Pancreatitis Crónica/metabolismo , Pancreatitis Crónica/patología , Pronóstico , Proteoma/genética , Proteoma/metabolismo , ProteómicaRESUMEN
Intestinal regulatory T cells (Tregs) are fundamental in peripheral tolerance toward commensals and food-borne antigens. Accordingly, gut-draining mesenteric lymph nodes (mLNs) represent a site of efficient peripheral de novo Treg induction when compared to skin-draining peripheral LNs (pLNs), and we had recently shown that LN stromal cells substantially contribute to this process. Here, we aimed to unravel the underlying molecular mechanisms and generated immortalized fibroblastic reticular cell lines (iFRCs) from mLNs and pLNs, allowing unlimited investigation of this rare stromal cell subset. In line with our previous findings, mLN-iFRCs showed a higher Treg-inducing capacity when compared to pLN-iFRCs. RNA-seq analysis focusing on secreted molecules revealed a more tolerogenic phenotype of mLN- as compared to pLN-iFRCs. Remarkably, mLN-iFRCs produced substantial numbers of microvesicles (MVs) that carried elevated levels of TGF-ß when compared to pLN-iFRC-derived MVs, and these novel players of intercellular communication were shown to be responsible for the tolerogenic properties of mLN-iFRCs. Thus, stromal cells originating from mLNs contribute to peripheral tolerance by fostering de novo Treg induction using TGF-ß-carrying MVs. This finding provides novel insights into the subcellular/molecular mechanisms of de novo Treg induction and might serve as promising tool for future therapeutic applications to treat inflammatory disorders.
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Vesículas Extracelulares/inmunología , Ganglios Linfáticos/inmunología , Células del Estroma/inmunología , Linfocitos T Reguladores/inmunología , Animales , Línea Celular , Vesículas Extracelulares/genética , Vesículas Extracelulares/ultraestructura , Factores de Transcripción Forkhead/inmunología , Factores de Transcripción Forkhead/metabolismo , Perfilación de la Expresión Génica/métodos , Mesenterio/inmunología , Ratones Endogámicos BALB C , Ratones Noqueados , Microscopía Electrónica de Rastreo , Microscopía Electrónica de Transmisión , Células del Estroma/metabolismo , Células del Estroma/ultraestructura , Linfocitos T Reguladores/metabolismo , Factor de Crecimiento Transformador beta/genética , Factor de Crecimiento Transformador beta/inmunologíaRESUMEN
Recently, biological roles of extracellular vesicles (which include among others exosomes, microvesicles and apoptotic bodies) have attracted substantial attention in various fields of biomedicine. Here we investigated the impact of sustained exposure of cells to the fluoroquinolone antibiotic ciprofloxacin on the released extracellular vesicles. Ciprofloxacin is widely used in humans against bacterial infections as well as in cell cultures against Mycoplasma contamination. However, ciprofloxacin is an inducer of oxidative stress and mitochondrial dysfunction of mammalian cells. Unexpectedly, here we found that ciprofloxacin induced the release of both DNA (mitochondrial and chromosomal sequences) and DNA-binding proteins on the exofacial surfaces of small extracellular vesicles referred to in this paper as exosomes. Furthermore, a label-free optical biosensor analysis revealed DNA-dependent binding of exosomes to fibronectin. DNA release on the surface of exosomes was not affected any further by cellular activation or apoptosis induction. Our results reveal for the first time that prolonged low-dose ciprofloxacin exposure leads to the release of DNA associated with the external surface of exosomes.
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Antibacterianos/farmacología , ADN/metabolismo , Exosomas/metabolismo , Vesículas Extracelulares/metabolismo , Membranas Intracelulares/metabolismo , Apoptosis/efectos de los fármacos , Línea Celular , Supervivencia Celular/efectos de los fármacos , Ciprofloxacina/farmacología , ADN Mitocondrial , Humanos , Células JurkatRESUMEN
There is no available blood marker for the preoperative diagnosis of adrenocortical malignancy. The objective of this study was to investigate the expression of extracellular vesicle-associated microRNAs and their diagnostic potential in plasma samples of patients suffering from adrenocortical tumors. Extracellular vesicles were isolated either by using Total Exosome Isolation Kit or by differential centrifugation/ultracentrifugation. Preoperative plasma extracellular vesicle samples of 6 adrenocortical adenomas (ACA) and 6 histologically verified adrenocortical cancer (ACC) were first screened by Taqman Human Microarray A-cards. Based on the results of screening, two miRNAs were selected and validated by targeted quantitative real-time PCR. The validation cohort included 18 ACAs and 16 ACCs. Beside RNA analysis, extracellular vesicle preparations were also assessed by transmission electron microscopy, flow cytometry and dynamic light scattering. Significant overexpression of hsa-miR-101 and hsa-miR-483-5p in ACC relative to ACA samples has been validated. Receiver operator characteristics of data revealed dCT hsa-miR-483-5p normalized to cel-miR-39 to have the highest diagnostic accuracy (area under curve 0.965), the sensitivity and the specifity were 87.5 and 94.44, respectively. Extracellular vesicle-associated hsa-miR-483-5p thus appears to be a promising minimally invasive biomarker in the preoperative diagnosis of ACC but needs further validation in larger cohorts of patients.
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Neoplasias de la Corteza Suprarrenal/diagnóstico , Neoplasias de la Corteza Suprarrenal/genética , MicroARN Circulante/genética , Vesículas Extracelulares/metabolismo , Neoplasias de la Corteza Suprarrenal/sangre , Adulto , Anciano , Anciano de 80 o más Años , MicroARN Circulante/metabolismo , Dispersión Dinámica de Luz , Vesículas Extracelulares/ultraestructura , Femenino , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Humanos , Masculino , Persona de Mediana Edad , Curva ROC , Reproducibilidad de los ResultadosRESUMEN
Extracellular vesicles are produced in all organisms. The most intensively investigated categories of extracellular vesicles include apoptotic bodies, microvesicles and exosomes. Among a very wide range of areas, their role has been confirmed in intercellular communication, immune response and angiogenesis (in both physiological and pathological conditions). Their alterations suggest the potential use of them as biomarkers. In this paper the authors give an insight into the research of extracellular vesicles in general, and then focus on published findings in hematological malignancies. Quantitative and qualitative changes of microvesicles and exosomes may have value in diagnostics, prognostics and minimal residual disease monitoring of hematological malignancies. The function of extracellular vesicles in downregulation of natural killer cells' activity has been demonstrated in acute myeloid leukemia. In chronic lymphocytic leukemia, microvesicles seem to play a role in drug resistance. Orv. Hetil., 2016, 157(35), 1379-1384.
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Biomarcadores de Tumor/sangre , Vesículas Extracelulares/metabolismo , Neoplasias Hematológicas/sangre , Comunicación Celular , Micropartículas Derivadas de Células/metabolismo , Humanos , Tamaño de la PartículaRESUMEN
The biodistribution of extracellular vesicles (EVs) is a fundamental question in the field of circulating biomarkers, which has recently gained attention. Despite the capabilities of nuclear imaging methods, such as single-photon emission computed tomography, radioisotope labeling of EVs and the use of the aforementioned methods for in vivo studies hardly can be found in the literature. In this article, the authors describe a novel method for the radioisotope labeling of erythrocyte-derived EVs using the (99m)Tc-tricarbonyl complex. Moreover, the capability of the developed labeling method for in vivo biodistribution studies is demonstrated in a mouse model. The authors found that the intravenously administered (99m)Tc-labeled EVs mostly accumulated in the liver and spleen. The in vivo stability of the labeled EVs was assessed by the comparison of the obtained biodistribution of EVs with that of the free (99m)Tc-tricarbonyl. According to the authors' data, only a minor fraction of the radioactive label became detached from the EVs.
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Vesículas Extracelulares/metabolismo , Marcaje Isotópico/métodos , Compuestos de Organotecnecio/química , Radiofármacos/farmacocinética , Animales , Eritrocitos/química , Eritrocitos/ultraestructura , Exosomas/metabolismo , Vesículas Extracelulares/química , Humanos , Masculino , Ratones , Ratones Endogámicos BALB C , Compuestos de Organotecnecio/farmacocinética , Radiofármacos/química , Tomografía Computarizada por Tomografía Computarizada de Emisión de Fotón Único/métodos , Distribución TisularRESUMEN
Circulating extracellular vesicles have emerged as potential new biomarkers in a wide variety of diseases. Despite the increasing interest, their isolation and purification from body fluids remains challenging. Here we studied human pre-prandial and 4 hours postprandial platelet-free blood plasma samples as well as human platelet concentrates. Using flow cytometry, we found that the majority of circulating particles within the size range of extracellular vesicles lacked common vesicular markers. We identified most of these particles as lipoproteins (predominantly low-density lipoprotein, LDL) which mimicked the characteristics of extracellular vesicles and also co-purified with them. Based on biophysical properties of LDL this finding was highly unexpected. Current state-of-the-art extracellular vesicle isolation and purification methods did not result in lipoprotein-free vesicle preparations from blood plasma or from platelet concentrates. Furthermore, transmission electron microscopy showed an association of LDL with isolated vesicles upon in vitro mixing. This is the first study to show co-purification and in vitro association of LDL with extracellular vesicles and its interference with vesicle analysis. Our data point to the importance of careful study design and data interpretation in studies using blood-derived extracellular vesicles with special focus on potentially co-purified LDL.
Asunto(s)
Exosomas/química , Vesículas Extracelulares/química , Lipoproteínas LDL/sangre , Adulto , Biomarcadores/sangre , Plaquetas/química , Femenino , Humanos , Masculino , Periodo PosprandialRESUMEN
Extracellular vesicles (including exosomes, microvesicles and apoptotic bodies) are currently attracting rapidly increasing attention from various fields of biology due to their ability to carry complex information and act as autocrine, paracrine and even endocrine intercellular messengers. In the present study we investigated the sensitivity of size-based subpopulations of extracellular vesicles to different concentrations of detergents including sodium dodecyl sulphate, Triton X-100, Tween 20 and deoxycholate. We determined the required detergent concentration that lysed each of the vesicle subpopulations secreted by Jurkat, THP-1, MiaPaCa and U937 human cell lines. We characterized the vesicles by tunable resistive pulse sensing, flow cytometry and transmission electron microscopy. Microvesicles and apoptotic bodies were found to be more sensitive to detergent lysis than exosomes. Furthermore, we found evidence that sodium dodecyl sulphate and Triton X-100 were more effective in vesicle lysis at low concentrations than deoxycholate or Tween 20. Taken together, our data suggest that a combination of differential detergent lysis with tunable resistive pulse sensing or flow cytometry may prove useful for simple and fast differentiation between exosomes and other extracellular vesicle subpopulations as well as between vesicular and non-vesicular structures.