Co-isolation of extracellular vesicles and high-density lipoproteins using density gradient ultracentrifugation

Extracellular vesicles (EVs) facilitate intercellular communication by carrying bioactive molecules such as proteins, messenger RNA, and micro (mi)RNAs. Recently, high-density lipoproteins (HDL) isolated from human plasma were also reported to transport miRNA to other cells. HDL, when isolated from human plasma, ranges in density between 1.063 and 1.21 g/mL, which grossly overlap with the reported density of EVs. Consequently, HDL and EV will be co-isolated when using density gradient ultracentrifugation. Thus, more stringent isolation/separation procedures of EV and HDL are essential to know their relative contribution to the pool of circulating bioactive molecules

Reproducibility of extracellular vesicle research

Publisher Copyright: © 2022Cells release membrane-delimited particles into the environment. These particles are called “extracellular vesicles” (EVs), and EVs are present in fluids contacting cells, including body fluids and conditioned culture media. Because EVs change and contribute to health and disease, EVs have become a hot topic. From the thousands of papers now published on EVs annually, one easily gets the impression that EVs provide biomarkers for all diseases, and that EVs are carriers of all relevant biomolecules and are omnipotent therapeutics. At the same time, EVs are heterogeneous, elusive and difficult to study due to their physical properties and the complex composition of their environment. This overview addresses the current challenges encountered when working with EVs, and how we envision that most of these challenges will be overcome in the near future. Right now, an infrastructure is being developed to improve the reproducibility of EV measurement results. This infrastructure comprises expert task forces of the International Society of Extracellular Vesicles (ISEV) developing guidelines and recommendations, instrument calibration, standardized and transparent reporting, and education. Altogether, these developments will support the credibility of EV research by introducing robust reproducibility, which is a prerequisite for understanding their biological significance and biomarker potential.Peer reviewe

Clinical requirements for extracellular vesicle assays

The scientific and clinical interest in extracellular vesicles (EV) has grown exponentially during the past 15 years. As most research indicates that EVs can be utilised in diagnostics, prognostics and therapeutics, we may be on the brink of establishing the clinical utility of EV measurement, but how can we make this a reality? If we are to introduce EVs as biomarkers into clinical laboratories it will be necessary to offer fully validated, International Organization for Standardization (ISO) standard 15189 assays. ISO 15189 defines the quality management system requirements particular to medical laboratories and is used internationally to determine accreditation. In order for a clinical laboratory to offer an accredited test for EVs, this assay must have been subjected to a thorough assay validation process. This process requires the generation of data related to defined performance characteristics, to ensure that an assay is performing in accordance with the needs of its clinical users. Each of the defined performance characteristics will be discussed in this review, along with the issues that specifically affect EV analysis. Accreditation is increasingly important for all clinical laboratories and the standards required to achieve this are becoming more and more stringent. Therefore, as companies seek to develop the best assays to detect EVs and their molecular contents for clinical utility, and as we move rapidly towards our goal of offering EV analysis in the diagnosis and monitoring of disease, it is timely to highlight the requirements for the clinical accreditation of such assays. It is essential to consider these parameters to ensure that we develop the highest quality assays possible and ultimately the best outcomes for patients

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

P2Y12 antagonist ticagrelor inhibits the release of procoagulant extracellular vesicles from activated platelets

Background: Activated platelets release platelet extracellular vesicles (PEVs). Adenosine diphosphate(ADP) receptors P2Y1 and P2Y12 both play a role in platelet activation, The present hypothesis hereinis that the inhibition of these receptors may affect the release of PEVs.Methods: Platelet-rich plasma from 10 healthy subjects was incubated with saline, P2Y1 antagonistMRS2179 (100 μM), P2Y12 antagonist ticagrelor (1 μM), and a combination of both antagonists.Platelets were activated by ADP (10 μM) under stirring conditions at 37°C. Platelet reactivity wasassessed by impedance aggregometry. Concentrations of PEVs– (positive for CD61 but negative forP-selectin and phosphatidylserine) and PEVs+ (positive for all) were determined by a state-of-the-artflow cytometer. Procoagulant activity of PEVs was measured by a fibrin generation test.Results: ADP-induced aggregation (57 ± 13 area under curve {AUC] units) was inhibited 73%by the P2Y1 antagonist, 86% by the P2Y12 antagonist, and 95% when combined (p < 0.001 for all).The release of PEVs– (2.9 E ± 0.8 × 108/mL) was inhibited 48% in the presence of both antagonists(p = 0.015), whereas antagonists alone were ineffective. The release of PEVs+ (2.4 ± 1.6 × 107/mL)was unaffected by the P2Y1 antagonist, but was 62% inhibited by the P2Y12 antagonist (p = 0.035),and 72% by both antagonists (p = 0.022). PEVs promoted coagulation in presence of tissue factor.Conclusions: Inhibition of P2Y1 and P2Y12 receptors reduces platelet aggregation and affects therelease of distinct subpopulations of PEVs. Ticagrelor decreases the release of procoagulant PEVs fromactivated platelets, which may contribute to the observed clinical benefits in patients treated with ticagrelor

Human bone marrow contains high levels of extracellular vesicles with a tissue-specific subtype distribution

Introduction Extracellular vesicles (EV) are shed from a broad variety of cells and play an important role in activation of coagulation, cell to cell interaction and transport of membrane components. They are usually measured as circulating EV in peripheral blood (PB) and other body fluids. However, little is known about the distribution, presence and impact of EV and their sub-populations in bone marrow (BM). In our study, we focused on the analysis of different EV subtypes in human BM as compared to EV subsets in PB. Methods EV in BM and PB from 12 healthy stem cell donors were measured by flow-cytometry using Annexin V and cell-specific antibodies for hematopoietic stem cells, leucocytes, platelets, red blood cells, and endothelial cells. Additionally, concentrations of tissue factor-bearing EV were evaluated. Results High numbers of total EV were present in BM (median value [25-75 percentile]: 14.8 x10(9)/l [8.5-19.3]). Non-significantly lower numbers of total EV were measured in PB (9.2 x10(9)/l [3.8-14.5]). However, distribuation of EV subtypes showed substantial differences between BM and PB: In PB, distribution of EV fractions was similar as previously described. Most EV originated from platelets (93.9%), and only few EV were derived from leucocytes (4.5%), erythrocytes (1.8%), endothelial cells (1.0%), and hematopoietic stem cells (0.7%). In contrast, major fractions of BM-EV were derived from red blood cells or erythropoietic cells (43.2%), followed by megacaryocytes I platelets (27.6%), and by leucocytes as well as their progenitor cells (25,7%);only low EV proportions originated from endothelial cells and hematopoietic stem cells (2.0% and 1.5%, respectively). Similar fractions of tissue factor- bearing EV were found in BM and PB (1.3% and 0.9%). Conculsion Taken together, we describe EV numbers and their subtype distribution in the BM compartment for the first time. The tissue specific EV distribution reflects BM cell composition and favours the idea of a BM-PB barrier existing not only for cells, but also for EV

Extracellular vesicles from amniotic fluid, milk, saliva, and urine expose complexes of tissue factor and activated factor VII

Background Tissue factor (TF) is expressed in the adventitia of the vessel wall and on extracellular vesicles (EVs) in body fluids. TF and activated coagulation factor (F) VII(a) together form the so-called extrinsic tenase complex, which initiates coagulation. Aim We investigated whether EVs in amniotic fluid, milk, saliva, and urine expose functional extrinsic tenase complexes that can trigger coagulation. Methods Milk, saliva, and urine were collected from healthy breastfeeding women (n = 6), and amniotic fluid was collected from healthy women undergoing routine amniocentesis (n = 7). EVs were isolated from body fluids by size exclusion chromatography (SEC) and clotting experiments were performed in the presence and absence of antibodies against TF and FVIIa in normal plasma and in FVII-deficient plasma. The ability of body fluids to generate FXa also was determined. Results Amniotic fluid, milk, saliva, and urine triggered clotting of normal plasma and of FVII-deficient plasma, which was almost completely inhibited by an anti-FVII antibody and to a lesser extent by an anti-TF antibody. Fractionation of body fluids by SEC showed that only the fractions containing EVs triggered clotting in normal plasma and FVII-deficient plasma and generated FXa, which again was almost completely inhibited by an anti-FVII antibody and partially by an anti-TF antibody. Conclusion Here we show that EVs from amniotic fluid, milk, saliva, and urine expose complexes of TF and FVIIa (i.e., extrinsic tenase complexes) that directly activate FX. Based on our present findings we propose that these EVs from normal body fluids provide hemostatic protection

Synovial microparticles from arthritic patients modulate chemokine and cytokine release by synoviocytes

Synovial fluid from patients with various arthritides contains procoagulant, cell-derived microparticles. Here we studied whether synovial microparticles modulate the release of chemokines and cytokines by fibroblast-like synoviocytes (FLS). Microparticles, isolated from the synovial fluid of rheumatoid arthritis (RA) and arthritis control (AC) patients (n = 8 and n = 3, respectively), were identified and quantified by flow cytometry. Simultaneously, arthroscopically guided synovial biopsies were taken from the same knee joint as the synovial fluid. FLS were isolated, cultured, and incubated for 24 hours in the absence or presence of autologous microparticles. Subsequently, cell-free culture supernatants were collected and concentrations of monocyte chemoattractant protein-1 (MCP-1), IL-6, IL-8, granulocyte/macrophage colony-stimulating factor (GM-CSF), vascular endothelial growth factor (VEGF) and intracellular adhesion molecule-1 (ICAM-1) were determined. Results were consistent with previous observations: synovial fluid from all RA as well as AC patients contained microparticles of monocytic and granulocytic origin. Incubation with autologous microparticles increased the levels of MCP-1, IL-8 and RANTES in 6 of 11 cultures of FLS, and IL-6, ICAM-1 and VEGF in 10 cultures. Total numbers of microparticles were correlated with the IL-8 (r = 0.91, P < 0.0001) and MCP-1 concentrations (r = 0.81, P < 0.0001), as did the numbers of granulocyte-derived microparticles (r = 0.89, P < 0.0001 and r = 0.93, P < 0.0001, respectively). In contrast, GM-CSF levels were decreased. These results demonstrate that microparticles might modulate the release of chemokines and cytokines by FLS and might therefore have a function in synovial inflammation and angiogenesis

Essentials of extracellular vesicles: posters on basic and clinical aspects of extracellular vesicles

The past decade has witnessed an exponential development in the field of extracellular vesicles. Sporadic observations have reached a critical level and the scientific community increasingly recognizes the potential biomedical significance of these subcellular structures present in all body fluids as significant components of the cellular secretome. The Educational Committee of the International Society for Extracellular Vesicles prepared two posters ("Basic aspects of extracellular vesicles" and "Clinical aspects of extracellular vesicles") to provide essential pieces of information on extracellular vesicles at glance for anyone not familiar with the field