Extracellular Vesicles Subtypes Research Articles

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

IntroductionExtracellular 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 subpopulations 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.MethodsEV 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.ResultsHigh numbers of total EV were present in BM (median value [25–75 percentile]: 14.8 x109/l [8.5–19.3]). Non-significantly lower numbers of total EV were measured in PB (9.2 x109/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 / 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%).ConculsionTaken 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: Overview and Clinical Implications

The release of extracellular vesicles (EVs) is a phenomenon shared by all cell types as a means of communication. Characterization and classification of EVs is challenging and still a matter of debate. However, a general consensus on EVs nomenclatura has been made, on the basis of their size, formation and release mechanisms. EVs has been classified into exosomes, secreted via exocytosis from the late endosome multivesicular bodies, whereas microvesicles (MVs) bud from the plasma membrane and apoptotic bodies are released by cells undergoing apoptosis. Initially regarded as cellular debris, EVs have gained considerable interest in basic sciences and medical research, both as biomarkers and mediators of biological functions. Indeed, EVs carry regulatory molecules including lipid, proteins and different RNA species through the extracellular spaces and deliver these cargos to target cells to modify cellular activity, thereby contributing to both physiological and pathological responses. Because EVs bear markers derived from their parent cells and can be detected in most body fluids, characterization of EVs of different cellular origin is an underestimated source of biological information on cellular activation during disease evolution, and will probably serve as valuable diagnostic and prognostic biomarker in the future. However, the current methods used for the EVs isolation and analysis have several limitations and lack standardization, leading to uncertainties regarding the subtypes of EVs studied and how to interpret the data.After a rapid overview of the current knowledge on the mechanisms of formation, subcellular origin and composition of the different types of EVs, this presentation will focus on microvesicles (MVs). We will first address how their structure/function diversity determines their multifaceted biological functions in coagulation, inflammation, angiogenesis and endothelial dysfunction (ref 1, 2, 3). Secondly, we will summarize the current debate on the different methodologies available for their analysis and quantification in body fluids. A specific attention will be devoted to standardization of sample processing and MVs analysis, and also to recommendations from scientific societies for EVs translational applications as emerging biomarkers measurable in liquid biopsies (ref 4, 5, 6). Finally, focusing on cardiovascular diseases and cancer as emerging field where MVs detection have promising impact to improve patient management, we will illustrate how combination of increasing fundamental knowledge, technological progress and standardization will push MVs towards reliable biomarkers ready for the clinics.1- Ridger VC, Boulanger CM, Angelillo-Scherrer A, Badimon L, Blanc-Brude O, Bochaton-Piallat ML, Boilard E, Buzas EI, Caporali A, Dignat-George F, Evans PC, Lacroix R, Lutgens E, Ketelhuth DFJ, Nieuwland R, Toti F, Tunon J, Weber C, Hoefer IE. Microvesicles in vascular homeostasis and diseases. Position Paper of the European Society of Cardiology (ESC) Working Group on Atherosclerosis and Vascular Biology. Thromb Haemost. 2017;28;117(7):1296-1316.2- Todorova D, Simoncini S, Lacroix R, Sabatier F, Dignat-George F. Extracellular Vesicles in Angiogenesis. Circ Res. 2017;12;120(10):1658-1673.3- Lacroix R, Dubois C, Leroyer AS, Sabatier F, Dignat-George F. Revisited role of microparticles in arterial and venous thrombosis. J Thromb Haemost. 2013;11 Suppl 1:24-35.4- Coumans FAW, Brisson AR, Buzas EI, Dignat-George F, Drees EEE, El-Andaloussi S, Emanueli C, Gasecka A, Hendrix A, Hill AF, Lacroix R, Lee Y, van Leeuwen TG, Mackman N, Mäger I, Nolan JP, van der Pol E, Pegtel DM, Sahoo S, Siljander PRM, Sturk G, de Wever O, Nieuwland R. Methodological Guidelines to Study Extracellular Vesicles. Circ Res. 2017;12;120(10):1632-1648.5-Lacroix R, Judicone C, Mooberry M, Boucekine M, Key NS, Dignat-George F; The ISTH SSC Workshop. Standardization of pre-analytical variables in plasma microparticle determination: results of the International Society on Thrombosis and Haemostasis SSC Collaborative workshop. J Thromb Haemost. 2013 Apr 2.6- Cointe S, Judicone C, Robert S, Mooberry MJ, Poncelet P, Wauben M, Nieuwland R, Key NS, Dignat-George F, Lacroix R. Standardization of microparticle enumeration across different flow cytometry platforms: results of a multicenter collaborative workshop. J Thromb Haemost. 2017;15(1):187-193. DisclosuresNo relevant conflicts of interest to declare.

Redefining Tissue Crosstalk via Shotgun Proteomic Analyses of Plasma Extracellular Vesicles.

Protein signaling between tissues, or tissue cross-talk is becoming recognized as a fundamental biological process that is incompletely understood. Shotgun proteomic analyses of tissues and plasma to explore this concept are regularly challenged by high dynamic range of protein abundance, which limits the identification of lower abundance proteins. In this viewpoint article, it is highlighted how a focus on proteins contained within extracellular vesicles (EVs) not only partially addresses this issue, but can also reveal an underappreciated complexity of the circulating proteome in various physiological and pathological contexts. Furthermore, how quantitative proteomics can inform EV mediated crosstalk is highlighted and the importance of high coverage, sensitive proteomic analyses of EVs to identify both the optimal methods to isolate EV subtypes of interest and proteins that characterize them is stressed.

Minimal information for studies of extracellular vesicles 2018 (MISEV2018): a position statement of the International Society for Extracellular Vesicles and update of the MISEV2014 guidelines

ABSTRACTThe last decade has seen a sharp increase in the number of scientific publications describing physiological and pathological functions of extracellular vesicles (EVs), a collective term covering various subtypes of cell-released, membranous structures, called exosomes, microvesicles, microparticles, ectosomes, oncosomes, apoptotic bodies, and many other names. However, specific issues arise when working with these entities, whose size and amount often make them difficult to obtain as relatively pure preparations, and to characterize properly. The International Society for Extracellular Vesicles (ISEV) proposed Minimal Information for Studies of Extracellular Vesicles (“MISEV”) guidelines for the field in 2014. We now update these “MISEV2014” guidelines based on evolution of the collective knowledge in the last four years. An important point to consider is that ascribing a specific function to EVs in general, or to subtypes of EVs, requires reporting of specific information beyond mere description of function in a crude, potentially contaminated, and heterogeneous preparation. For example, claims that exosomes are endowed with exquisite and specific activities remain difficult to support experimentally, given our still limited knowledge of their specific molecular machineries of biogenesis and release, as compared with other biophysically similar EVs. The MISEV2018 guidelines include tables and outlines of suggested protocols and steps to follow to document specific EV-associated functional activities. Finally, a checklist is provided with summaries of key points.

Exosomes in perspective: a potential surrogate for stem cell therapy.

Exosomes as a unique subtype of small extracellular vesicles (sEVs) have attracted increasing interest in recent years in the fields of mesenchymal stromal cell (MSC) research. Studies have confirmed that exosomes derived from MSCs preserve immunosuppressive phenotype and can mimic therapeutic benefits of their parent cells. This review briefly summarizes most recent findings on the potential of exosomes as an alternative of therapeutic MSCs, focusing on the role of MSCs and their secreted exosomes in regulation of immune cells, preclinical and clinical evidence of therapeutic outcomes of MSC exosomes, and the biodistribution and pharmacokinetic profile of systemically administered exosomes. It is appreciated that exosomes from MSCs of different sources have variable contents including inflammatory mediators, tropic factors, signaling molecules, and nucleic acids (DNA, mRNA, microRNA and long non-coding RNA). Diverse functions of exosomes derived from different sources are expected. More importantly, exosomes isolated in vitro may not mirror that from in vivo, where donor MSCs are exposed to specific disease or injury-related conditions. Simulating in vivo microenvironment by pretreatment of MSCs with relevant chemical mediators may lead to their secretion of therapeutically more efficient exosomes/sEVs. However, we know very little about the key molecules involved and the differences between exosomes released under different conditions. These issues would be of tremendous interest to preclinical research that pursues exosome biology-underlain therapeutic mechanisms of MSCs. Further studies are expected to demonstrate the superiority of MSC-derived exsomes/sEVs as a pharmaceutical entity with regard to efficacy, safety, and practicability.

Phenotyping of circulating extracellular vesicles (EVs) in obesity identifies large EVs as functional conveyors of Macrophage Migration Inhibitory Factor

ObjectiveObesity-associated metabolic dysfunctions are linked to dysregulated production of adipokines. Accumulating evidence suggests a role for fat-derived extracellular vesicles (EVs) in obesity-metabolic disturbances. Since EVs convey numerous proteins we aimed to evaluate their contribution in adipokine secretion. MethodsPlasma collected from metabolic syndrome patients were used to isolate EV subtypes, namely microvesicles (MVs) and exosomes (EXOs). Numerous soluble factor concentrations were measured successively on total, MV- and EXO-depleted plasma by multiplexed immunoassays. ResultsCirculating MVs and EXOs were significantly increased with BMI, supporting a role of EVs as metabolic relays in obesity. Obesity was associated with dysregulated soluble factor production. Sequential depletion of plasma MVs and EXOs did not modify plasma levels of these molecules, with the exception of Macrophage Migration Inhibitory Factor (MIF). Half of plasma MIF circulated within MVs, and this MV secretory pathway was conserved over different MIF-producing cells. Although MV-associated MIF triggered rapid ERK1/2 activation in macrophages, these functional MV-MIF effects specifically relied on MIF tautomerase activity. ConclusionOur results emphasize the importance of reconsidering MIF-metabolic actions with regard to its MV-associated form and opening new EV-based strategies for therapeutic MIF approaches.

Extracellular Vesicles: How Drug and Pathology Interfere With Their Biogenesis and Function.

Extracellular vesicles (EV) are at the center of an intense activity of investigation, both for their possible employment as biomarkers of ongoing pathologic processes and for their broad range of biological activities. EV can promote tissue repair in very different pathologic settings, including hindlimb and myocardial ischemia. Importantly, the exact mode of action of EV is still partly understood, since they may act by modulating growth factors and cytokines, signaling pathways, and by transferring non-coding RNAs to target cells. However, the term EV identifies cell derived, enveloped particles very heterogeneous in size, composition, and biogenesis. Therefore, part of the controversies on the biological effects exerted by EV is a consequence of differences in methods of separation that result in the enrichment of different entities. Since technical challenges still hamper the highly specific sorting of different EV subpopulations, up to now only few investigators have tried to verify differences in the biological effects of specific EV subtypes. This review summarizes the current state of the art on the comprehension of mechanisms involved in EV biogenesis and release, which is a prerequisite for understanding and investigating the impact that pathology and drug therapy may exert on the secretion and composition of EV. Finally, we described both the mechanism involved in the modulation of EV secretion by drugs commonly used in patients affected by heart failure, and how pathophysiological mechanisms involved in heart disease modify EV secretion.

New Insights into the Role of Exosomes in the Heart After Myocardial Infarction.

Intercellular communications play a pivotal role in several cardiac pathophysiological processes. One subtype of extracellular vesicles, so-called exosomes, became known as important intercellular communication mediators in the heart. Exosomes are lipid bilayer biological nanovesicles loaded with diverse proteins, lipids, and mRNAs/microRNAs. All major cardiac cell types can modulate recipient cellular function via the release of exosomes. After myocardial infarction (MI), exosomes, especially those secreted by different cardiac stem cells, have been shown to confer cardioprotective effects, activate regenerative signals, and participate into cardiac repair. In this review, we rapidly recall the biology of exosomes at the beginning. Then we summarize the exosomes secreted by different myocardial cells and their function in cardiac intercellular communication. At last, we discuss the role of these vesicles in cardiac repair after MI.

Label-Free Prostate Cancer Detection by Characterization of Extracellular Vesicles Using Raman Spectroscopy.

Mammalian cells release extracellular vesicles (EVs) into their microenvironment that travel the entire body along the stream of bodily fluids. EVs contain a wide range of biomolecules. The transported cargo varies depending on the EV origin. Knowledge of the origin and chemical composition of EVs can potentially be used as a biomarker to detect, stage, and monitor diseases. In this paper, we demonstrate the potential of EVs as a prostate cancer biomarker. A Raman optical tweezer was employed to obtain Raman signatures from four types of EV samples, which were red blood cell- and platelet-derived EVs of healthy donors and the prostate cancer cell lines- (PC3 and LNCaP) derived EVs. EVs’ Raman spectra could be clearly separated/classified into distinct groups using principal component analysis (PCA) which permits the discrimination of the investigated EV subtypes. These findings may provide new methodology to detect and monitor early stage cancer.

Arrestin-Domain Containing Protein 1 (Arrdc1) Regulates the Protein Cargo and Release of Extracellular Vesicles.

Extracellular vesicles (EVs) are lipid-bilayered vesicles that are released by multiple cell types and contain nucleic acids and proteins. Very little is known about how the cargo is packaged into EVs. Ubiquitination of proteins is a key posttranslational modification that regulates protein stability and trafficking to subcellular compartments including EVs. Recently, arrestin-domain containing protein 1 (Arrdc1), an adaptor for the Nedd4 family of ubiquitin ligases, has been implicated in the release of ectosomes, a subtype of EV that buds from the plasma membrane. However, it is currently unknown whether Arrdc1 can regulate the release of exosomes, a class of EVs that are derived endocytically. Furthermore, it is unclear whether Arrdc1 can regulate the sorting of protein cargo into the EVs. Exosomes and ectosomes are isolated from mouse embryonic fibroblasts isolated from wild type and Arrdc1-deficient (Arrdc1-/- ) mice. Nanoparticle tracking analysis-based EV quantitation shows that Arrdc1 regulates the release of both exosomes and ectosomes. Proteomic analysis highlights the change in protein cargo in EVs upon deletion of Arrdc1. Functional enrichment analysis reveals the enrichment of mitochondrial proteins in ectosomes, while proteins implicated in apoptotic cleavage of cell adhesion proteins and formation of cornified envelope are significantly depleted in exosomes upon knockout of Arrdc1.

Extracellular vesicles and their nucleic acids for biomarker discovery

Extracellular vesicles (EVs) are a heterogenous population of vesicles originate from cells. EVs are found in different biofluids and carry different macromolecules, including proteins, lipids, and nucleic acids, providing a snap shot of the parental cells at the time of release. EVs have the ability to transfer molecular cargoes to other cells and can initiate different physiological and pathological processes. Mounting lines of evidence demonstrated that EVs' cargo and machinery is affected in disease states, positioning EVs as potential sources for the discovery of novel biomarkers. In this review, we demonstrate a conceptual overview of the EV field with particular focus on their nucleic acid cargoes. Current knowledge of EV subtypes, nucleic acid cargo and pathophysiological roles are outlined, with emphasis placed on advantages against competing analytes. We review the utility of EVs and their nucleic acid cargoes as biomarkers and critically assess the newly available advances in the field of EV biomarkers and high throughput technologies. Challenges to achieving the diagnostic potential of EVs, including sample handling, EV isolation, methodological considerations, and bioassay reproducibility are discussed. Future implementation of 'omics-based technologies and integration of systems biology approaches for the development of EV-based biomarkers and personalized medicine are also considered.

Abstract 4056: Immune regulation by miR-1227 LO

Abstract Immunotherapy offers great promise for the treatment of prostate cancer; however prostate cancer often acquires mechanisms to dampen the tumoricidal immune response. Understanding how prostate cancer dampens the immune system will facilitate the generation of more effective immunotherapies. Here we investigate how large oncosomes (LO)-mediated transfer of miR-1227 to immune cells modulates the immune response. Extracellular vesicle (EV)-encapsulated miRNAs are being increasingly recognized for playing a key role in cancer progression and immune modulation. miR-1227 is a poorly characterized miRNA that is enriched in EV secreted by prostate cancer cells in comparison to non-malignant prostate epithelial cells. miR-1227 expression is increased in migratory glioblastoma cells in comparison to non-migratory glioblastoma cells, and in radiation resistant esophageal cells and colorectal cancer stromal cells indicating that miR-1227 is upregulated in different cancer types. However, the role of miR-1227 in cancer is poorly understood. We demonstrate here that EV obtained from prostate cancer cells overexpressing miR-1227, induce the migration of prostate cancer cells and fibroblasts. A comparative analysis between different EV subtypes indicates that miR-1227 is enriched in LO, a class of EV that are secreted by highly invasive amoeboid-migrating cells. We also show that LO carry more RNA than the more widely studied exosomes indicating that LO may be a more robust source of EV-encapsulated miRNA. RNA sequencing from miR-1227 stably expressing prostate cancer cells and 5 different in silico miRNA target prediction methods were used to identify putative miR-1227 targets. IPA pathway analysis from these predicted targets highlighted several functions related to the immune system, including T cell homeostasis, T cell development, and development of Th17 cells. A more in-depth analysis of the 51 genes involved in T cell homeostasis showed enrichment of genes associated with Th17 and Th1 T cell differentiation. Alterations in Th17 and Th1 differentiation modulates T cell cytokine secretion, which are important mediators of cancer growth and progression. Transient transfection of miR-1227 in immune cells altered the cytokine expression profile, resulting in the upregulation of several cytokines that promote cancer growth including CCL5 and IL6. We tested whether LO can be taken up by activated T cells to directly modulate cytokine secretion. To determine whether miR-1227 can be functionally transferred to recipient cells via LO we isolated LO from prostate cancer cells stably expressing miR-1227 using ultracentrifugation followed by density gradient purification. The isolated LO were used to treat immune cells to show that LO-encapsulated miR-1227 alters cytokine profiles. These results indicate that LO-encapsulated miR-1227 may be involved in cancer immune modulation through targeting specific genes involved in T-cell differentiation. Citation Format: Andrew R. Chin, Dolores Di Vizio, Valentina R. Minciacchi, Mariana Sobreiro, Cristiana Spinelli. Immune regulation by miR-1227 LO [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4056.

The ESCRT-III Protein CHMP1A Mediates Secretion of Sonic Hedgehog on a Distinctive Subtype of Extracellular Vesicles

SUMMARYEndosomal sorting complex required for transport (ESCRT) complex proteins regulate biogenesis and release of extracellular vesicles (EVs), which enable cell-to-cell communication in the nervous system essential for development and adult function. We recently showed human loss-of-function (LOF) mutations in ESCRT-III member CHMP1A cause autosomal recessive microcephaly with pontocerebellar hypoplasia, but its mechanism was unclear. Here, we show Chmp1a is required for progenitor proliferation in mouse cortex and cerebellum and progenitor maintenance in human cerebral organoids. In Chmp1a null mice, this defect is associated with impaired sonic hedgehog (Shh) secretion and intraluminal vesicle (ILV) formation in multivesicular bodies (MVBs). Furthermore, we show CHMP1A is important for release of an EV subtype that contains AXL, RAB18, and TMED10 (ART) and SHH. Our findings show CHMP1A loss impairs secretion of SHH on ART-EVs, providing molecular mechanistic insights into the role of ESCRT proteins and EVs in the brain.

FLIM reveals alternative EV-mediated cellular up-take pathways of paclitaxel

In response to physiological and artificial stimuli, cells generate nano-scale extracellular vesicles (EVs) by encapsulating biomolecules in plasma membrane-derived phospholipid envelopes. These vesicles are released to bodily fluids, hence acting as powerful endogenous mediators in intercellular signaling. EVs provide a compelling alternative for biomarker discovery and targeted drug delivery, but their kinetics and dynamics while interacting with living cells are poorly understood. Here we introduce a novel method, fluorescence lifetime imaging microscopy (FLIM) to investigate these interaction attributes. By FLIM, we show distinct cellular uptake mechanisms of different EV subtypes, exosomes and microvesicles, loaded with anti-cancer agent, paclitaxel. We demonstrate differences in intracellular behavior and drug release profiles of paclitaxel-containing EVs. Exosomes seem to deliver the drug mostly by endocytosis while microvesicles enter the cells by both endocytosis and fusion with cell membrane. This research offers a new real-time method to investigate EV kinetics with living cells, and it is a potential advancement to complement the existing techniques. The findings of this study improve the current knowledge in exploiting EVs as next-generation targeted drug delivery systems.

Placental Exosomes During Gestation: Liquid Biopsies Carrying Signals for the Regulation of Human Parturition.

Parturition is defined as the action or process of giving birth to offspring. Normal term human parturition ensues following the maturation of fetal organ systems typically between 37 and 40 weeks of gestation. Our conventional understanding of how parturition initiation is signaled revolves around feto-maternal immune and endocrine changes occurring in the intrauterine cavity. These changes in turn correlate with the sequence of fetal growth and development. These important physiological changes also result in homeostatic imbalances which result in heightened inflammatory signaling. This disrupts the maintenance of pregnancy, thus leading to laborrelated changes. However, the precise mechanisms of the signaling cascades that lead to the initiation of parturition remain unclear, although exosomes may be a mediator of this process. Exosomes are a subtype of extracellular vesicles characterised by their endocytic origin. This involves the trafficking of intraluminal vesicles into multivesicular bodies (MVB) and then exocytosis via the plasmatic membranes. Exosomes are highly stable nanovesicles that are released by a wide range of cells and organs including the human placenta and fetal membranes. Interestingly, exosomes from placental origin have been uncovered in maternal circulation across gestation. In addition, their concentration is higher in pregnancies with complications such as gestational diabetes and preeclampsia. In normal gestation, the concentration of placental exosomes in maternal circulation correlates with placental weight at third trimester. The role of placental exosomes across gestation has not been fully elucidated, although recent studies suggest that placental exosomes are involved in maternal-fetal inmmuno-tolerance, maternal systemic inflammation and nutrient transport. The content of exosomes is of particular importance, encompassing a large range of molecules such as mRNA, miRNAs, DNA, lipids, cell-surface receptors, and protein mediators. These can in turn interact with either adjacent or distal cells to reprogram their phenotype and regulate their function. Many of the pro-parturition proinflammatory mediators reach maternal compartments from the fetal side via circulation, but major impediments remain, such as degradation at various levels and limited halflife in circulation. Recent findings suggest that a more effective mode of communication and signal transport is through exosomes, where signals are protected and will not succumb to degradation. Thus, understanding how exosomes regulate key events throughout pregnancy and parturition will provide an opportunity to understand the mechanisms involved in the maternal and fetal metabolic adaptations during normal and pathological pregnancies. Subsequently, this will assist in identifying those pregnancies at risk of developing complications. This may also allow more appropriate modifications of their clinical management. This review will hence examine the current body of data to summarise our understanding of how signaling pathways lead to the beginning of parturition. In addition, we propose that extracellular vesicles, namely exosomes, may be an integral component of these signaling events by transporting specific signals to prepare the maternal physiology to initiate parturition. Understanding these signals and their mechanisms in normal term pregnancies can provide insight into pathological activation of these signals, which can cause spontaneous preterm parturition. Hence, this review expands on our knowledge of exosomes as professional carriers of fetal signals to instigate human parturition.

Granulocyte-Derived Extracellular Vesicles Activate Monocytes and Are Associated With Mortality in Intensive Care Unit Patients.

To understand how extracellular vesicle (EV) subtypes differentially activate monocytes, a series of in vitro studies were performed. We found that plasma-EVs biased monocytes toward an M1 profile. Culturing monocytes with granulocyte-, monocyte-, and endothelial-EVs induced several pro-inflammatory cytokines. By contrast, platelet-EVs induced TGF-β and GM-CSF, and red blood cell (RBC)-EVs did not activate monocytes in vitro. The scavenger receptor CD36 was important for binding of RBC-EVs to monocytes, while blockade of CD36, CD163, CD206, TLR1, TLR2, and TLR4 did not affect binding of plasma-EVs to monocytes in vitro. To identify mortality risk factors, multiple soluble factors and EV subtypes were measured in patients’ plasma at intensive care unit admission. Of 43 coagulation factors and cytokines measured, two were significantly associated with mortality, tissue plasminogen activator and cystatin C. Of 14 cellular markers quantified on EVs, 4 were early predictors of mortality, including the granulocyte marker CD66b. In conclusion, granulocyte-EVs have potent pro-inflammatory effects on monocytes in vitro. Furthermore, correlation of early granulocyte-EV levels with mortality in critically ill patients provides a potential target for intervention in management of the pro-inflammatory cascade associated with critical illness.

Granulocyte-derived extracellular vesicles activate monocytes and are associated with mortality in ICU patients

Abstract To understand how extracellular vesicle (EV) subtypes differentially activate monocytes, a series of in vitro studies were performed. We found that plasma-EVs biased monocytes toward an M1 profile. Culturing monocytes with granulocyte-, monocyte- and endothelial-EVs induced several pro-inflammatory cytokines. In contrast, platelet-EVs induced TGF-β and GM-CSF, and red blood cell (RBC)-EVs did not activate monocytes in vitro. The scavenger receptor CD36 was important for binding of RBC-EVs to monocytes, while blockade of CD36, CD163, CD206, TLR1, TLR2, and TLR4 did not affect binding of plasma-EVs to monocytes in vitro. To identify mortality risk factors, multiple soluble factors and EV subtypes were measured in patients’ plasma at intensive care unit (ICU) admission. Of 43 coagulation factors and cytokines measured, two were significantly associated with mortality, TPA and cystatin C. Of 14 cellular markers quantified on EVs, 4 were early predictors of mortality, including the granulocyte marker CD66b. In conclusion, granulocyte-EVs have potent pro-inflammatory effects on monocytes in vitro. Furthermore, correlation of early granulocyte EV levels with mortality in critically ill patients provides a potential target for intervention in management of the pro-inflammatory cascade associated with critical illness.

Low cardiorespiratory fitness is associated with higher extracellular vesicle counts in obese adults.

Low cardiorespiratory fitness (CRF) is associated with cardiovascular disease (CVD) independent of obesity. Extracellular vesicles (EVs) are a novel target of CVD, however, it remains unknown if obese individuals with very poor fitness (VPF) have elevated EVs versus people with poor fitness (PF). Thus, we tested whether VPF was associated with greater EV subtypes in obese adults. Subjects with VPF (n = 13, VO 2peak: 15.4 ± 0.6 mL/kg/min, BMI: 34.1 ± 1.7 kg/m2) and PF (n = 13, VO 2peak: 25.9 ± 3.0 mL/kg/min, BMI: 32.1 ± 1.2 kg/m2) were compared in this cross‐sectional study. After an overnight fast, AnnexinV (AV) +/− platelet (CD31+/CD41+), leukocyte (CD45+/CD41−), and endothelial EVs (CD105+, CD31+/CD41−) were analyzed from fresh platelet poor plasma via imaging flow cytometry. Body fat, blood pressure (BP), and glucose tolerance (OGTT) were also tested. Body weight, BP, and circulating glucose were similar between groups, although VPF subjects were older than PF (64.0 ± 2.1 vs. 49.8 ± 4.2 year; P < 0.05). People with VPF, compared with PF, had higher total AV− EVs (P = 0.04), AV − platelet EVs (CD31+/CD41+; P = 0.006), and AV − endothelial EVs (CD31+/CD41−; P = 0.005) independent of age and body fat. Higher AV− platelet and endothelial EVs were associated with lower VO 2peak (r = −0.56, P = 0.006 and r = −0.55, P = 0.005, respectively). Endothelial‐derived AV−/CD31+/CD41−EVs were also related to pulse pressure (r = 0.45, P = 0.03), whereas AV−/CD105 was linked to postprandial glucose (r = 0.41, P = 0.04). VPF is associated with higher AnnexinV− total, endothelial, and platelet EVs in obese adults, suggesting that subtle differences in fitness may reduce type 2 diabetes and CVD risk through an EV‐related mechanism.

Extracellular vesicles as mediators of immunopathology in infectious diseases.

In the last decades, extracellular vesicles have emerged as important elements in cell-cell communication and as key players in disease pathogenesis via transmission of their cargo between different cells. Various works have described different subpopulations of these membrane structures, based on their cell of origin, biogenesis, size, biophysical properties and cargo. In addition to their pathophysiological role in the development and progression of different diseases including infectious diseases, neurodegenerative disorders and cancer, extracellular vesicles are now recognized for their potential as novel therapeutic targets and intelligent drug delivery system. Here, we have reviewed the most recent data on different subtypes of extracellular vesicles, focusing on microvesicles and exosomes and their subpopulations, their involvement in immune-mediated pathogenesis of various infectious diseases and their role as potential therapeutic targets.

Extracellular Vesicle Subtypes Released From Activated or Apoptotic T-Lymphocytes Carry a Specific and Stimulus-Dependent Protein Cargo.

Extracellular vesicles (EVs) are released from nearly all mammalian cells and different EV populations have been described. Microvesicles represent large EVs (LEVs) released from the cellular surface, while exosomes are small EVs (SEVs) released from an intracellular compartment. As it is likely that different stimuli promote the release of distinct EV populations, we analyzed EVs from human lymphocytes considering the respective release stimuli (activation Vs. apoptosis induction). We could clearly separate two EV populations, namely SEVs (average diameter <200 nm) and LEVs (diameter range between 200 and 1000 nm). Morphology and size were analyzed by electron microscopy and nanoparticle tracking analysis. Apoptosis induction caused a massive release of LEVs, while activated T-cells released SEVs and LEVs in considerably lower amounts. The release of SEVs from apoptotic T-cells was comparable with LEV release from activated ones. LEVs contained signaling proteins and proteins of the actin-myosin cytoskeleton. SEVs carried cytoplasmic/endosomal proteins like the 70-kDa heat shock protein 70 (HSP70) or tumor susceptibility 101 (TSG101), microtubule-associated proteins, and ubiquitinated proteins. The protein expression profile of SEVs and LEVs changed substantially after the induction of apoptosis. After apoptosis induction, HSP70 and TSG101 (often used as exosome markers) were highly expressed within LEVs. Interestingly, in contrast to HSP70 and TSG101, gelsolin and eps15 homology domain-containing protein 3 (EHD3) turned out to be specific for SEVs irrespective of the stimulus causing the EV release. Finally, we detected several subunits of the proteasome (PSMB9, PSMB10) as well as the danger signal HMGB1 exclusively within apoptotic cell-released LEVs. Thus, we were able to identify new marker proteins that can be useful to discriminate between distinct LEV subpopulations. The mass spectrometry proteomics data are available via ProteomeXchange with identifier PXD009074.