As transmitters of biological information, extracellular vesicles (EVs) are crucial for the maintenance of physiological homeostasis, but also contribute to pathological conditions, such as thrombotic disorders. The ability of EVs to support thrombin generation has been linked to their exposure of phosphatidylserine, an anionic phospholipid that is normally restricted to the inner leaflet of the plasma membrane but exposed on the outer leaflet during EV biogenesis. Here, we investigated whether EVs of different cellular origin and from different settings, namely platelets and red blood cells from blood bank units and a monocyte-like cell line (THP-1), differ regarding their potential to support factor XII-dependent thrombin generation. EVs were isolated from blood products or THP-1 cell culture supernatants using differential centrifugation and characterized by a combination of flow cytometry, nanoparticle tracking analysis, and Western blotting. Soluble factors co-enriched during the isolation of EVs were depleted from blood-cell derived EV fractions using size exclusion chromatography, while proteins bound to the surface of EVs were degraded by mild protease treatment. We found that platelet-derived and red blood cell-derived EVs supported factor XII-dependent thrombin generation to comparable extents, while monocytic EVs failed to support thrombin generation when added to EV-depleted human plasma. We excluded a major contribution of co-enriched soluble proteins or of proteins bound to the EV surface to the thrombogenicity of blood cell-derived EVs. Our data suggest that the enhanced potential of blood cell-derived EVs to support thrombin generation is rather due to enhanced exposure of phosphatidylserine on the surface of blood cell-derived EVs. Extending these investigations to EVs from other cell types, such as mesenchymal stromal cells, will be crucial for their future therapeutic applications.
Growing interest in extracellular vesicles (EVs) has prompted the advancements of protocols for improved EV characterization. As a high-throughput, multi-parameter, and single particle technique, flow cytometry is widely used for EV characterization. The comparison of data on EV concentration, however, is hindered by the lack of standardization between different protocols and instruments. Here, we quantified EV counts of platelet-derived EVs, using two flow cytometers (Gallios and CytoFLEX LX) and nanoparticle tracking analysis (NTA). Phosphatidylserine-exposing EVs were identified by labelling with lactadherin (LA). Calibration with silica-based fluorescent beads showed detection limits of 300 nm and 150 nm for Gallios and CytoFLEX LX, respectively. Accordingly, CytoFLEX LX yielded 40-fold higher EV counts and 13-fold higher counts of LA+CD41+ EVs compared to Gallios. NTA in fluorescence mode (F-NTA) demonstrated that only 9.5% of all vesicles detected in scatter mode exposed phosphatidylserine, resulting in good agreement of LA+ EVs for CytoFLEX LX and F-NTA. Since certain functional characteristics, such as the exposure of pro-coagulant phosphatidylserine, are not equally displayed across the entire EV size range, our study highlights the necessity of indicating the size range of EVs detected with a given approach along with the EV concentration to support the comparability between different studies.
Inflammatory bowel disease increases the odds of developing colitis-associated cancer. We hypothesized that Western-style diet (WD) aggravates azoxymethane (AOM)/dextran sulfate sodium salt (DSS)-induced colitis-associated tumorigenesis and that switching to the standard AIN93G diet will ameliorate disease symptoms even after cancer initiation. Female BALB/c mice received either WD (WD group) or standard AIN93G diet (AIN group) for the whole experimental period. After five weeks, the mice received 12.5 mg/kg AOM intraperitoneally, followed by three DSS cycles. In one group of mice, the WD was switched to AIN93G the day before starting the first DSS cycle (WD/AIN group). Feeding the WD during the whole experimental period aggravated colitis symptoms, shortened the colon (p < 0.05), changed microbiota composition and increased tumor promotion. On molecular level, the WD reduced proliferation (p < 0.05) and increased expression of the vitamin D catabolizing enzyme Cyp24a1 (p < 0.001). The switch to the AIN93G diet ameliorated this effect, reflected by longer colons, fewer (p < 0.05) and smaller (p < 0.01) aberrant colonic crypt foci, comparable with the AIN group. Our results show that switching to a healthy diet, even after cancer initiation is able to revert the deleterious effect of the WD and could be an effective preventive strategy to reduce colitis symptoms and prevent tumorigenesis.
The emerging role of extracellular vesicles (EVs) as biomarkers and their envisioned therapeutic use require advanced techniques for their detailed characterization. In this context, we investigated gas-phase electrophoresis on a nano electrospray gas-phase electrophoretic mobility molecular analyzer (nES GEMMA, aka nES differential mobility analyzer, nES DMA) as an alternative to standard analytical techniques. In gas-phase electrophoresis, single-charged, surface-dry, native, polydisperse, and aerosolized analytes, e.g., proteins or bio-nanoparticles, are separated according to their electrophoretic mobility diameter, i.e., globular size. Subsequently, monodisperse particles are counted after a nucleation step in a supersaturated atmosphere as they pass a focused laser beam. Hence, particle number concentrations are obtained in accordance with recommendations of the European Commission for nanoparticle characterization (2011/696/EU from October 18th, 2011). Smaller sample constituents (e.g., co-purified proteins) can be detected next to larger ones (e.g., vesicles). Focusing on platelet-derived EVs, we compared different vesicle isolation techniques. In all cases, nanoparticle tracking analysis (NTA) confirmed the presence of vesicles. However, nES GEMMA often revealed a significant co-purification of proteins from the sample matrix, precluding gas-phase electrophoresis of less-diluted samples containing higher vesicle concentrations. Therefore, mainly peaks in the protein size range were detected. Mass spectrometry revealed that these main contaminants belonged to the group of globulins and coagulation-related components. An additional size exclusion chromatography (SEC) step enabled the depletion of co-purified, proteinaceous matrix components, while a label-free quantitative proteomics approach revealed no significant differences in the detected EV core proteome. Hence, the future in-depth analysis of EVs via gas-phase electrophoresis appears feasible. Graphical abstract
Extracellular vesicles (EVs) are in the scientific spotlight due to their potential application in the medical field, ranging from medical diagnosis to therapy. These applications rely on EV stability during isolation and purification—ideally, these steps should not impact vesicle integrity. In this context, we investigated EV stability and particle numbers via nano electrospray gas-phase electrophoretic mobility molecular analysis (nES GEMMA) and nanoparticle tracking analysis (NTA). In nES GEMMA, native, surface-dry analytes are separated in the gas-phase according to the particle size. Besides information on size and particle heterogeneity, particle number concentrations are obtained in accordance with recommendations of the European Commission for nanoparticle characterization (2011/696/EU, 18 October 2011). Likewise, and in contrast to NTA, nES GEMMA enables detection of co-purified proteins. On the other hand, NTA, yielding data on hydrodynamic size distributions, is able to relate particle concentrations, omitting electrolyte exchange (and resulting EV loss), which is prerequisite for nES GEMMA. Focusing on EVs of different origin, we compared vesicles concentrations and stability, especially after electrolyte exchange and size exclusion chromatography (SEC). Co-isolated proteins were detected in most samples, and the vesicle amount varied in dependence on the EV source. We found that depletion of co-purified proteins was achievable via SEC, but was associated with a loss of EVs and—most importantly—with decreased vesicle stability, as detected via a reduced nES GEMMA measurement repeatability. Ultimately, we propose the repeatability of nES GEMMA to yield information on EV stability, and, as a result, we propose that nES GEMMA can yield additional valuable information in EV research.
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