Anita N. Böing

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 discussed.

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.

Recent developments in the nomenclature, presence, isolation, detection and clinical impact of extracellular vesicles.

The research field of extracellular vesicles (EVs), such as microparticles and exosomes, is growing exponentially. The goal of this review is to provide an overview of recent developments relevant to the readers of the Journal of Thrombosis and Haemostasis. We will discuss nomenclature, the presence of EVs in fluids, methods of isolation and detection, and emerging clinical implications. Although research on EVs has been performed within the ISTH for over a decade, most of the recent research on EVs has been brought together by the International Society on Extracellular Vesicles (ISEV). To achieve an overview of recent developments, the information provided in this review comes not only from publications, but also from latest meetings of the ISEV (April 2015, Washington, DC, USA), the International Society on Advancement of Cytometry (June 2015, Glasgow, UK), and the ISTH (June 2015, Toronto, Canada).

Microparticles and exosomes: impact on normal and complicated pregnancy.

Eukaryotic cells release vesicles into their environment by membrane shedding (ectosomes or microparticles) and secretion (exosomes). Microparticles and exosomes occur commonly in vitro and in vivo. The occurrence, composition and function(s) of these vesicles change during disease (progression). During the last decade, the scientific and clinical interest increased tremendously. Evidence is accumulating that microparticles and exosomes may be of pathophysiological relevance in autoimmune, cardiovascular and thromboembolic diseases, as well as inflammatory and infectious disorders. In this review, we will summarize the discovery, biology, structure and function of microparticles and exosomes, and discuss their (patho‐) physiological role during normal and complicated pregnancy.

Reproducible extracellular vesicle size and concentration determination with tunable resistive pulse sensing

Introduction The size of extracellular vesicles (EVs) can be determined with a tunable resistive pulse sensor (TRPS). Because the sensing pore diameter varies from pore to pore, the minimum detectable diameter also varies. The aim of this study is to determine and improve the reproducibility of TRPS measurements. Methods Experiments were performed with the qNano system (Izon) using beads and a standard urine vesicle sample. With a combination of voltage and stretch that yields a high blockade height, we investigate whether the minimum detected diameter is more reproducible when we configure the instrument targeting (a) fixed stretch and voltage, or (b) fixed blockade height. Results Daily measurements with a fixed stretch and voltage (n=102) on a standard urine sample show a minimum detected vesicle diameter of 128±19 nm [mean±standard deviation; coefficient of variation (CV) 14.8%]. The vesicle concentration was 2.4·109±3.8·109 vesicles/mL (range 1.4·108–1.8·1010). When we compared setting a fixed stretch and voltage to setting a fixed blockade height on 3 different pores, we found a minimum detected vesicle diameter of 118 nm (CV 15.5%, stretch), and 123 nm (CV 4.5%, blockade height). The detected vesicle concentration was 3.2–8.2·108 vesicles/mL with fixed stretch and 6.4–7.8·108 vesicles/mL with fixed blockade height. Summary/conclusion Pore-to-pore variability is the cause of the variation in minimum detected size when setting a fixed stretch and voltage. The reproducibility of the minimum detectable diameter is much improved by setting a fixed blockade height.

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 determined.

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.

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 endothelium.

Extracellular vesicles, tissue factor, cancer and thrombosis – discussion themes of the ISEV 2014 Educational Day

Although the association between cancer and venous thromboembolism (VTE) has long been known, the mechanisms are poorly understood. Circulating tissue factor–bearing extracellular vesicles have been proposed as a possible explanation for the increased risk of VTE observed in some types of cancer. The International Society for Extracellular Vesicles (ISEV) and International Society on Thrombosis and Haemostasis (ISTH) held a joint Educational Day in April 2014 to discuss the latest developments in this field. This review discusses the themes of that event and the ISEV 2014 meeting that followed.

Circulating Platelet-derived and Placenta-derived Microparticles Expose Flt-1 in Preeclampsia

Background. Flt-1 is secreted by various cells and elevated concentrations are present in preeclampsia affecting vascular function. Microparticles from these cells may expose Flt-1. We evaluated whether Flt-1 is microparticle-associated in preeclampsia, and established the origin of Flt-1-exposing microparticles. Methods. The concentration of Flt-1 was measured in samples from preeclamptic patients, pregnant and nonpregnant women by enzyme-linked immunosorbent assay. Microparticles were analyzed by flow cytometry. Western blot determined the different forms of Flt-1. Results. Noncell bound Flt-1 was elevated in preeclampsia compared to controls. A fraction (5%) was associated with microparticles in preeclampsia. Flt-1-exposing microparticles were increased in preeclampsia compared to normotensive pregnancy (p = 0.02). Full-length Flt-1, was identified in microparticles of platelet and placental origin. Conclusion. Full-length Flt-1 is associated with platelet and placenta-derived microparticles. Possibly, the presentation of Flt-1 on the membrane of a microparticle might alter its function, particularly if it acts in synergism with other exposed vasoactive molecules.