Microvesicles
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Neonatal systemic inflammatory response and multiple organ dysfunction syndrome are the main postnatal insults influencing mortality and morbidity. Critically ill newborns with high predicted mortality are supported by extracorporeal membrane oxygenation (ECMO). Biomarkers of inflammatory response and endothelial injury can be used for early diagnosis and treatment of critical neonatal situations. The aim of our study was to explore plasma proteins and endothelial microvesicles as markers of inflammation and endothelial activation in newborns on ECMO and to compare them with healthy neonates. Thirteen newborns on ECMO and 13 healthy newborns were included in the study. Plasma soluble biomarkers were measured using multiplex immunoassay based on Luminex® xMAP multianalyte profiling platform. The total microvesicle count and plasma level of surface antigen-specific microvesicles were determined by flow cytometry. The plasma concentration of cell-derived microvesicles was measured using annexin-V labeling, and the endothelial origin of microvesicles was determined using lineage-specific antigen labeling of endothelial cell/microvesicle markers (endoglin/CD105, PECAM1/CD31, VEGFR2/CD309, and MadCAM1). Inflammatory markers (procalcitonin, IL-1β, IL-6, and IL-22) were increased in the ECMO group (P < 0.01). The assessment of endothelial markers showed higher concentrations of endocan and angiopoietin-2 (P < 0.01) in the ECMO group while VEGF in the ECMO group was significantly lower (P < 0.01). In the ECMO group, the concentration of annexin-V-positive microvesicles (total microvesicles) and endothelial microvesicles expressing mucosal vascular addressin cell adhesion molecule 1 (MadCAM1) was increased (P = 0.05). In summary, we found increased concentrations of soluble inflammatory and endothelial markers in the plasma of critically ill newborns with multiple organ dysfunction. Increased plasma concentrations of microvesicles may reflect the activation or damage of blood cells and vasculature including endothelial cells. The measurement of cell membrane-derived microvesicles may be added to the panel of established inflammatory markers in order to increase the sensitivity and specificity of the diagnostic process in critically ill newborns.
- MeSH
- biologické markery krev MeSH
- buněčná membrána metabolismus MeSH
- endotel metabolismus MeSH
- endoteliální buňky MeSH
- kritický stav MeSH
- lidé MeSH
- mikropartikule metabolismus MeSH
- mimotělní membránová oxygenace * MeSH
- novorozenec MeSH
- průtoková cytometrie MeSH
- zánět krev MeSH
- Check Tag
- lidé MeSH
- mužské pohlaví MeSH
- novorozenec MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
Human body reacts to physical, chemical and biological insults with a complex inflammatory reaction. Crucial components and executors of this response are endothelial cells, platelets, white blood cells, plasmatic coagulation system, and complement. Endothelial injury and inflammation are associated with elevated blood levels of cell membrane-derived microvesicles. Increased concentrations of microvesicles were found in several inflammatory reactions and diseases including acute coronary syndromes, stroke, vasculitis, venous thromboembolism, multiple sclerosis, rheumatoid arthritis, systemic lupus erythematosus, anti-phospholipid antibody syndrome, inflammatory bowel disease, thrombotic thrombocytopenic purpura, viral myocarditis, sepsis, disseminated intravascular coagulation, polytrauma, and burns. Microvesicles can modulate a variety of cellular processes, thereby having an impact on pathogenesis of diseases associated with inflammation. Microvesicles are important mediators and potential biomarkers of systemic inflammation. Measurement of inflammatory cell-derived microvesicles may be utilized in diagnostic algorithms and used for detection and determination of severity in diseases associated with inflammatory responses, as well as for prediction of their outcome. This review focuses on the mechanisms of release of microvesicles in diseases associated with systemic inflammation and their potential role in the regulation of cellular and humoral interactions.
This review focuses on the formation, composition and function of endothelial microvesicles (MV), often called microparticles (MP). MV release is a controlled event and is considered a hallmark of cellular activation or alteration. MV may affect the function of target cells through surface interaction and receptor activation, cellular fusion and the delivery of intravesicular cargo. Endothelial MV are released as a consequence of endothelial activation during inflammation and have been described to affect hemostasis, various aspects of inflammatory reaction, vessel formation, apoptosis and cell survival, endothelial cell differentiation and function. Recent data suggest the potential use of MV in diagnostics, assessment of severity and prediction of outcomes in inflammatory diseases and their utilization as targets, mediators and vectors in therapy.
- MeSH
- apoptóza MeSH
- biologické markery metabolismus MeSH
- endoteliální buňky cytologie metabolismus MeSH
- exozómy metabolismus MeSH
- extracelulární vezikuly metabolismus MeSH
- lidé MeSH
- mikropartikule metabolismus MeSH
- patologická angiogeneze MeSH
- trombóza patologie MeSH
- zánět patologie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- přehledy MeSH
BACKGROUND AIMS: Microvesicles (MV) shed from the plasma membrane of eukaryotic cells, including human embryonic stem cells (hESC), contain proteins, lipids and RNA and serve as mediators of cell-to-cell communication. However, they may also contain immunogenic membrane domains and infectious particles acquired from xenogenic components of the culture milieu. Therefore, MV represent a potential risk for clinical application of cell therapy. METHODS: We tested the ability of hESC and their most commonly used feeder cells, mouse embryonic fibroblasts (MEF), to produce MV. We found that hESC are potent producers of MV, whereas mitotically inactivated MEF do not produce any detectable MV. We therefore employed a combined proteomic approach to identify the molecules that constitute the major components of MV from hESC maintained in a standard culture setting with xenogenic feeder cells. RESULTS: In purified MV fractions, we identified a total of 22 proteins, including five unique protein species that are known to be highly expressed in invasive cancers and participate in cellular activation, metastasis and inhibition of apoptosis. Moreover, we found that hESC-derived MV contained the immunogenic agents apolipoprotein and transferrin, a source of Neu5Gc, as well as mouse retroviral Gag protein. CONCLUSIONS: These findings indicate that MV represent a mechanism by which hESC communicate; however, they also serve as potential carriers of immunogenic and pathogenic compounds acquired from environment. Our results highlight a potential danger regarding the use of hESC that have previously been exposed to animal proteins and cells.
- MeSH
- antigeny heterofilní imunologie MeSH
- antigeny nádorové imunologie metabolismus MeSH
- apolipoproteiny imunologie metabolismus MeSH
- buněčné linie MeSH
- elektronová mikroskopie MeSH
- embryonální kmenové buňky cytologie imunologie metabolismus MeSH
- fibroblasty cytologie imunologie metabolismus MeSH
- genové produkty gag imunologie metabolismus MeSH
- kokultivační techniky MeSH
- lidé MeSH
- mikropartikule imunologie metabolismus MeSH
- myši MeSH
- proteiny regulující apoptózu imunologie metabolismus MeSH
- proteomika MeSH
- riskování MeSH
- skot MeSH
- tandemová hmotnostní spektrometrie MeSH
- tkáňová terapie - dějiny škodlivé účinky MeSH
- transferin imunologie metabolismus MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- skot MeSH
- zvířata MeSH
- Publikační typ
- práce podpořená grantem MeSH
Flow cytometry is a powerful method, which is widely used for high-throughput quantitative and qualitative analysis of cells. However, its straightforward applicability for extracellular vesicles (EVs) and mainly exosomes is hampered by several challenges, reflecting mostly the small size of these vesicles (exosomes: ~80-200 nm, microvesicles: ~200-1,000 nm), their polydispersity, and low refractive index. The current best and most widely used protocol for beads-free flow cytometry of exosomes uses ultracentrifugation (UC) coupled with floatation in sucrose gradient for their isolation, labeling with lipophilic dye PKH67 and antibodies, and an optimized version of commercial high-end cytometer for analysis. However, this approach requires an experienced flow cytometer operator capable of manual hardware adjustments and calibration of the cytometer. Here, we provide a novel and fast approach for quantification and characterization of both exosomes and microvesicles isolated from cell culture media as well as from more complex human samples (ascites of ovarian cancer patients) suitable for multiuser labs by using a flow cytometer especially designed for small particles, which can be used without adjustments prior to data acquisition. EVs can be fluorescently labeled with protein-(Carboxyfluoresceinsuccinimidyl ester, CFSE) and/or lipid- (FM) specific dyes, without the necessity of removing the unbound fluorescent dye by UC, which further facilitates and speeds up the characterization of microvesicles and exosomes using flow cytometry. In addition, double labeling with protein- and lipid-specific dyes enables separation of EVs from common contaminants of EV preparations, such as protein aggregates or micelles formed by unbound lipophilic styryl dyes, thus not leading to overestimation of EV numbers. Moreover, our protocol is compatible with antibody labeling using fluorescently conjugated primary antibodies. The presented methodology opens the possibility for routine quantification and characterization of EVs from various sources. Finally, it has the potential to bring a desired level of control into routine experiments and non-specialized labs, thanks to its simple bead-based standardization.
- Publikační typ
- časopisecké články MeSH
In the ciliate Tetrahymena thermophila, lysosome-related organelles called mucocysts accumulate at the cell periphery where they secrete their contents in response to extracellular events, a phenomenon called regulated exocytosis. The molecular bases underlying regulated exocytosis have been extensively described in animals but it is not clear whether similar mechanisms exist in ciliates or their sister lineage, the Apicomplexan parasites, which together belong to the ecologically and medically important superphylum Alveolata. Beginning with a T. thermophila mutant in mucocyst exocytosis, we used a forward genetic approach to uncover MDL1 (Mucocyst Discharge with a LamG domain), a novel gene that is essential for regulated exocytosis of mucocysts. Mdl1p is a 40 kDa membrane glycoprotein that localizes to mucocysts, and specifically to a tip domain that contacts the plasma membrane when the mucocyst is docked. This sub-localization of Mdl1p, which occurs prior to docking, underscores a functional asymmetry in mucocysts that is strikingly similar to that of highly polarized secretory organelles in other Alveolates. A mis-sense mutation in the LamG domain results in mucocysts that dock but only undergo inefficient exocytosis. In contrast, complete knockout of MDL1 largely prevents mucocyst docking itself. Mdl1p is physically associated with 9 other proteins, all of them novel and largely restricted to Alveolates, and sedimentation analysis supports the idea that they form a large complex. Analysis of three other members of this putative complex, called MDD (for Mucocyst Docking and Discharge), shows that they also localize to mucocysts. Negative staining of purified MDD complexes revealed distinct particles with a central channel. Our results uncover a novel macromolecular complex whose subunits are conserved within alveolates but not in other lineages, that is essential for regulated exocytosis in T. thermophila.
- MeSH
- exocytóza genetika MeSH
- lyzozomy metabolismus MeSH
- organely metabolismus MeSH
- sekreční vezikuly genetika metabolismus MeSH
- Tetrahymena thermophila * genetika MeSH
- Tetrahymena * MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, N.I.H., Extramural MeSH
