Single-step isolation of extracellular vesicles by size-exclusion chromatography

Background: Isolation of extracellular vesicles from plasma is a challenge due to the presence of proteins and lipoproteins. Isolation of vesicles using differential centrifugation or density-gradient ultracentrifugation results in co-isolation of contaminants such as protein aggregates and incomplete separation of vesicles from lipoproteins, respectively. Aim: To develop a single-step protocol to isolate vesicles from human body fluids. Methods: Platelet-free supernatant, derived from platelet concentrates, was loaded on a sepharose CL-2B column to perform size-exclusion chromatography (SEC; n=3). Fractions were collected and analysed by nanoparticle tracking analysis, resistive pulse sensing, flow cytometry and transmission electron microscopy. The concentrations of high-density lipoprotein cholesterol (HDL) and protein were measured in each fraction. Results: Fractions 9–12 contained the highest concentrations of particles larger than 70 nm and platelet-derived vesicles (46%±6 and 61%±2 of totals present in all collected fractions, respectively), but less than 5% of HDL and less than 1% of protein (4.8%±1 and 0.65%±0.3, respectively). HDL was present mainly in fractions 18–20 (32%±2 of total), and protein in fractions 19–21 (36%±2 of total). Compared to the starting material, recovery of platelet-derived vesicles was 43%±23 in fractions 9–12, with an 8-fold and 70-fold enrichment compared to HDL and protein. Conclusions: SEC efficiently isolates extracellular vesicles with a diameter larger than 70 nm from platelet-free supernatant of platelet concentrates. Application SEC will improve studies on the dimensional, structural and functional properties of extracellular vesicles

Microparticles in vascular disorders: How tissue factor-exposing vesicles contribute to pathology and physiology

Coagulation is initiated by tissue factor (TF). Coagulant TF is constitutively expressed by extravascular cells, but there is increasing evidence that TF can also be present within the blood, in particular during pathological conditions. Such TF is exposed on circulating cell-derived vesicles, and its presence has been associated with development of disseminated intravascular coagulation and venous thrombosis. For example, the presence of TF-exposing vesicles in the blood of cancer patients may be associated with their high risk of developing venous thromboembolism. Remarkably, high levels of coagulant TF-exposing vesicles are present in other body fluids such as saliva and urine of healthy persons, suggesting that these vesicles play a physiological role. We postulate that the presence of TF-exposing vesicles in body fluids as saliva and urine provides an additional source of coagulant TF that promotes coagulation, thereby reducing blood loss and contributing to host defence by reducing the risk of microorganisms entering the "milieu interieur". (C) 2012 Elsevier Ltd. All rights reserve

Platelet microparticles contain active caspase 3

During storage, platelets undergo processes resembling apoptosis, including microparticle release, aminophospholipid exposure, and procaspase 3 processing. Recently, we showed that microparticles from endothelial cells contain caspase 3, one of the executioner enzymes of apoptosis. In this study we determined whether platelet-derived microparticles (PMP) contain caspase 3 in vitro (stored platelet concentrate) and ex vivo (plasma from healthy humans). In addition, we studied the underlying mechanism of caspase 3 formation in PMP, and the ability of such PMP to induce apoptosis in human macrophages (THP-1 cells). The presence of caspase 3 (antigen) was studied by Western blot and flowcytometry, and activity was determined by Ac-DEVD-pNA and ROCK I cleavage. In vitro, PMP numbers increased during storage. From day one onwards, PMP contained procaspase 3, whereas caspase 3 (antigen and activity) was detectable after 5-7 days of storage. PMP contained caspase 9 but not caspase 8, and the time course of caspase 9 formation paralleled procaspase 3 disappearance and caspase 3 appearance. In addition, PMP in human plasma also contained detectable quantities of caspase 3. Incubation of THP-1 cells with PMP induced apoptosis. Taken together, PMP contain caspase 3 in vitro and ex vivo. Our data implicate that procaspase 3 is likely to be processed by caspase 9 in PMP during storage. PMP induce apoptosis of human macrophages, but whether this induction is due to the transfer of caspase 3 remains to be determine

Platelet extracellular vesicles as biomarkers for arterial thrombosis

Arterial thrombosis is a major and global cause of human death and disability. Considering the socioeconomic costs of arterial thrombosis, identification of biomarkers to predict and detect arterial thrombosis at an early stage is an important public health goal. Platelet extracellular vesicles (PEV) are a new candidate biomarker of arterial thrombosis. PEV can be measured in biorepositories, thereby offering the possibility to validate PEV in multicenter clinical trials. PEV analysis has been hitherto hampered by lack of standardized methodology, but substantial technological improvements of PEV detection techniques have been achieved recently. However, before PEV emerge from research tools to clinical applications, a number of issues should be clarified. To facilitate validation of PEV as biomarkers of thrombosis, we discuss (i) whether PEV are useful as biomarkers of thrombosis, (ii) why previous conclusions on PEV concentrations, composition and functions require re-evaluation, and (iii) which questions have to be answered before PEV become clinically usefu

Inhibition of microparticle release triggers endothelial cell apoptosis and detachment

Endothelial cell cultures contain caspase 3-containing microparticles (EMP), which are reported to form during or after cell detachment. We hypothesize that also adherent endothelial cells release EMP, thus protecting these cells from caspase 3 accumulation, detachment and apoptosis. Human umbilical vein endothelial cells (HUVEC) were incubated with and without inhibitors of microparticle release (Y-27632, calpeptin), both in the absence or presence of additional "external stress", i.e. the apoptotic agent staurosporin (200 nM) or the activating cytokine interleukin (IL)-1alpha (5 ng/ml). Control cultures contained mainly viable adherent cells and minor fractions of apoptotic detached cells and microparticles in the absence of inhibitors. In the presence of inhibitors, caspase 3 accumulated in adherent cells and detachment tended to increase. During incubation with either staurosporin or IL-1alpha in the absence of inhibitors of microparticle release, adherent cells remained viable, and detachment and EMP release increased slightly. In the presence of inhibitors, dramatic changes occurred in staurosporin-treated cultures. Caspase 3 accumulated in adherent cells and >90% of the cells detached within 48 hours. In IL-1alpha-treated cultures no accumulation of caspase 3 was observed in adherent cells, although detachment increased. Scanning electron microscopy studies confirmed the presence of EMP on both adherent and detached cells. Prolonged culture of detached cells indicated a rapid EMP formation as well as some EMP formation at longer culture periods. Inhibition of EMP release causes accumulation of caspase 3 and promotes cell detachment, although the extent depends on the kind of "external stress". Thus, the release of caspase 3-containing microparticles may contribute to endothelial cell surviva

Classification, functions, and clinical relevance of extracellular vesicles

Both eukaryotic and prokaryotic cells release small, phospholipid-enclosed vesicles into their environment. Why do cells release vesicles? Initial studies showed that eukaryotic vesicles are used to remove obsolete cellular molecules. Although this release of vesicles is beneficial to the cell, the vesicles can also be a danger to their environment, for instance in blood, where vesicles can provide a surface supporting coagulation. Evidence is accumulating that vesicles are cargo containers used by eukaryotic cells to exchange biomolecules as transmembrane receptors and genetic information. Because also bacteria communicate to each other via extracellular vesicles, the intercellular communication via extracellular cargo carriers seems to be conserved throughout evolution, and therefore vesicles are likely to be a highly efficient, robust, and economic manner of exchanging information between cells. Furthermore, vesicles protect cells from accumulation of waste or drugs, they contribute to physiology and pathology, and they have a myriad of potential clinical applications, ranging from biomarkers to anticancer therapy. Because vesicles may pass the blood-brain barrier, they can perhaps even be considered naturally occurring liposomes. Unfortunately, pathways of vesicle release and vesicles themselves are also being used by tumors and infectious diseases to facilitate spreading, and to escape from immune surveillance. In this review, the different types, nomenclature, functions, and clinical relevance of vesicles will be discusse

Simvastatin-induced endothelial cell detachment and microparticle release are prenylation dependent

Statins reduce cardiovascular disease risk and affect endothelial function by cholesterol-dependent and independent mechanisms. Recently, circulating (detached) endothelial cells and endothelial microparticles (EMP) have been associated with endothelial functioning in vitro and in vivo. We investigated whether simvastatin affects endothelial detachment and release of EMP. Human umbilical vein endothelial cells (HUVECs) were incubated with clinically relevant concentrations of simvastatin (1.0 and 5.0 microM), with or without mevalonic acid (100 microM) or geranylgeranylpyrophosphate (GGPP; 20 microM) for 24 hours, and analyzed by flowcytometry and Western blot. Simvastatin at 1.0 and 5.0 microM increased cell detachment from 12.5+/-4.1% to 26.0+/-7.6% (p=0.013) and 28.9 +/- 2.2% (p=0.002) as well as EMP release (p=0.098 and p=0.041, respectively). The majority of detached cells was apoptotic, although the fraction of detached cells that showed signs of apoptosis (>70%) was unaffected by simvastatin. Detached cells and EMP contained caspase 3 and caspase 8, but not caspase 9. Restoring either cholesterol biosynthesis and prenylation (mevalonate) or prenylation alone (GGPP) reversed all simvastatin-induced effects on cell detachment and EMP release. Adherent cells showed no signs of simvastatin-induced apoptosis. Simvastatin promotes detachment and EMP release by inhibiting prenylation, presumably via a caspase 8-dependent mechanism. We hypothesize that by facilitating detachment and EMP release, statins improve the overall condition of the remaining vascular endotheliu

Tissue Factor Coagulant Activity is Regulated by the Plasma Membrane Microenvironment

Background Tissue factor (TF) can be present in a non-coagulant and coagulant form. Whether the coagulant activity is affected by the plasma membrane microenvironment is unexplored. Objective This article studies the presence and coagulant activity of human TF in plasma membrane micro-domains. Methods Plasma membranes were isolated from human MIA PaCa2 cells, MDA-MB-231 cells and human vascular smooth muscle cells by Percoll gradient ultracentrifugation after cell disruption. Plasma membranes were fractionated by OptiPrep gradient ultracentrifugation, and the presence of TF, flotillin, caveolin, clathrin, protein disulphide isomerase (PDI), TF pathway inhibitor (TFPI) and phosphatidylserine (PS) were determined. Results Plasma membranes contain two detergent-resistant membrane (DRM) compartments differing in density and biochemical composition. High-density DRMs (DRM-H) have a density (ρ) of 1.15 to 1.20 g/mL and contain clathrin, whereas low-density DRMs (DRM-L) have a density between 1.09 and 1.13 g/mL and do not contain clathrin. Both DRMs contain TF, flotillin and caveolin. PDI is detectable in DRM-H, TFPI is not detectable in either DMR-H or DRM-L and PS is detectable in DRM-L. The DRM-H-associated TF (> 95% of the TF antigen) lacks detectable coagulant activity, whereas the DRM-L-associated TF triggers coagulation. This coagulant activity is inhibited by lactadherin and thus PS-dependent, but seemed insensitive to 16F16, an inhibitor of PDI. Conclusion Non-coagulant and coagulant TF are present within different types of DRMs in the plasma membrane, and the composition of these DRMs may affect the TF coagulant activity

Handling and storage of human body fluids for analysis of extracellular vesicles

Because procedures of handling and storage of body fluids affect numbers and composition of extracellular vesicles (EVs), standardization is important to ensure reliable and comparable measurements of EVs in a clinical environment. We aimed to develop standard protocols for handling and storage of human body fluids for EV analysis. Conditions such as centrifugation, single freeze–thaw cycle, effect of time delay between blood collection and plasma preparation and storage were investigated. Plasma is the most commonly studied body fluid in EV research. We mainly focused on EVs originating from platelets and erythrocytes and investigated the behaviour of these 2 types of EVs independently as well as in plasma samples of healthy subjects. EVs in urine and saliva were also studied for comparison. All samples were analysed simultaneously before and after freeze–thawing by resistive pulse sensing, nanoparticle tracking analysis, conventional flow cytometry (FCM) and transmission (scanning) electron microscopy. Our main finding is that the effect of centrifugation markedly depends on the cellular origin of EVs. Whereas erythrocyte EVs remain present as single EVs after centrifugation, platelet EVs form aggregates, which affect their measured concentration in plasma. Single erythrocyte and platelet EVs are present mainly in the range of 100–200 nm, far below the lower limit of what can be measured by conventional FCM. Furthermore, the effects of single freeze–thaw cycle, time delay between blood collection and plasma preparation up to 1 hour and storage up to 1 year are insignificant (p>0.05) on the measured concentration and diameter of EVs from erythrocyte and platelet concentrates and EVs in plasma, urine and saliva. In conclusion, in standard protocols for EV studies, centrifugation to isolate EVs from collected body fluids should be avoided. Freezing and storage of collected body fluids, albeit their insignificant effects, should be performed identically for comparative EV studies and to create reliable biorepositories

Extracellular vesicles in human follicular fluid do not promote coagulation

Body fluids contain extracellular vesicles expressing tissue factor on their surface and serve as an additional trigger for coagulation. During the menstrual cycle ovarian tissue restoration is mandatory and it is unknown whether follicular fluid might provide procoagulant substances. Within an observational study, follicular fluid from women undergoing IVF/intracytoplasmic sperm injection (ICSI) was analysed by fluorescence-activated cell sorting (FACS), electron microscopy, resistive pulse sensing (RPS), nanoparticle-tracking analysis (NTA) and fibrin generation tests (FGT). The presence of extracellular vesicles, especially CD9-positive extracellular vesicles in follicular fluid, was proven. However, clotting tests revealed no procoagulant properties of the detected extracellular vesicle