Éva Pállinger

Biological properties of extracellular vesicles and their physiological functions.

In the past decade, extracellular vesicles (EVs) have been recognized as potent vehicles of intercellular communication, both in prokaryotes and eukaryotes. This is due to their capacity to transfer proteins, lipids and nucleic acids, thereby influencing various physiological and pathological functions of both recipient and parent cells. While intensive investigation has targeted the role of EVs in different pathological processes, for example, in cancer and autoimmune diseases, the EV-mediated maintenance of homeostasis and the regulation of physiological functions have remained less explored. Here, we provide a comprehensive overview of the current understanding of the physiological roles of EVs, which has been written by crowd-sourcing, drawing on the unique EV expertise of academia-based scientists, clinicians and industry based in 27 European countries, the United States and Australia. This review is intended to be of relevance to both researchers already working on EV biology and to newcomers who will encounter this universal cell biological system. Therefore, here we address the molecular contents and functions of EVs in various tissues and body fluids from cell systems to organs. We also review the physiological mechanisms of EVs in bacteria, lower eukaryotes and plants to highlight the functional uniformity of this emerging communication system.

Detection and isolation of cell-derived microparticles are compromised by protein complexes resulting from shared biophysical parameters

Numerous diseases, recently reported to associate with elevated microvesicle/microparticle (MP) counts, have also long been known to be characterized by accelerated immune complex (IC) formation. The goal of this study was to investigate the potential overlap between parameters of protein complexes (eg, ICs or avidin-biotin complexes) and MPs, which might perturb detection and/or isolation of MPs. In this work, after comprehensive characterization of MPs by electron microscopy, atomic force microscopy, dynamic light-scattering analysis, and flow cytometry, for the first time, we drive attention to the fact that protein complexes, especially insoluble ICs, overlap in biophysical properties (size, light scattering, and sedimentation) with MPs. This, in turn, affects MP quantification by flow cytometry and purification by differential centrifugation, especially in diseases in which IC formation is common, including not only autoimmune diseases, but also hematologic disorders, infections, and cancer. These data may necessitate reevaluation of certain published data on patient-derived MPs and contribute to correct the clinical laboratory assessment of the presence and biologic functions of MPs in health and disease.

Highlights of a new type of intercellular communication: microvesicle-based information transfer

Abstract.Microvesicles (MVs) are membrane-covered cell fragments released by most cell types during apoptosis or activation. They are increasingly considered to play a pivotal role in information transfer between cells. Their presence and role have been proven in several physiological and pathological processes, such as immune modulation in inflammation and pregnancy, or blood coagulation and cancer. MVs represent a newly recognized system of intercellular communications. They not only may serve as prognostic markers in different diseases, but could also hold the potential to be new therapeutic targets or drug delivery systems.The present overview aims to highlight some aspects of this new means of cellular communication: “microvesicular communication”.

The miR-290-295 cluster promotes pluripotency maintenance by regulating cell cycle phase distribution in mouse embryonic stem cells

The mmu-miR-290-295 cluster codes for a family of microRNAs (miRNAs) that are expressed de novo during early embryogenesis and are specific for mouse embryonic stem cells (ESC) and embryonic carcinoma cells (ECC). Detailed sequence analysis and alignment studies of miR-290-295 precursors demonstrated that the cluster has evolved by repeated duplication events of the ancient miR-290 precursor. We show that under serum starvation, overexpression of miR-290-295 miRNAs withhold ES cells from early differentiation, ensures their high proliferation rate and capacity for forming alkaline phosphate positive colonies. Transcriptome analysis revealed that differentiation related marker genes are underexpressed upon high miR-290-295 level. Importantly, miR-290-295 overexpression prevents ES cells from accumulation in G1 phase at low serum level, and seems to regulate cell cycle in different phases. Our data underline that miR-290-295 miRNAs contribute to the natural absence of G1 checkpoint in embryonic stem cells. We define the cell cycle regulators Wee1 and Fbxl5 as potential direct targets of miR-290-295 miRNAs in vitro. Our results suggest that miR-290-295 miRNAs exhibit their effect predominantly through the regulation of cell cycle phase distribution.

Low-density lipoprotein mimics blood plasma-derived exosomes and microvesicles during isolation and detection

Circulating extracellular vesicles have emerged as potential new biomarkers in a wide variety of diseases. Despite the increasing interest, their isolation and purification from body fluids remains challenging. Here we studied human pre-prandial and 4 hours postprandial platelet-free blood plasma samples as well as human platelet concentrates. Using flow cytometry, we found that the majority of circulating particles within the size range of extracellular vesicles lacked common vesicular markers. We identified most of these particles as lipoproteins (predominantly low-density lipoprotein, LDL) which mimicked the characteristics of extracellular vesicles and also co-purified with them. Based on biophysical properties of LDL this finding was highly unexpected. Current state-of-the-art extracellular vesicle isolation and purification methods did not result in lipoprotein-free vesicle preparations from blood plasma or from platelet concentrates. Furthermore, transmission electron microscopy showed an association of LDL with isolated vesicles upon in vitro mixing. This is the first study to show co-purification and in vitro association of LDL with extracellular vesicles and its interference with vesicle analysis. Our data point to the importance of careful study design and data interpretation in studies using blood-derived extracellular vesicles with special focus on potentially co-purified LDL.

Improved Characterization of EV Preparations Based on Protein to Lipid Ratio and Lipid Properties

In recent years the study of extracellular vesicles has gathered much scientific and clinical interest. As the field is expanding, it is becoming clear that better methods for characterization and quantification of extracellular vesicles as well as better standards to compare studies are warranted. The goal of the present work was to find improved parameters to characterize extracellular vesicle preparations. Here we introduce a simple 96 well plate-based total lipid assay for determination of lipid content and protein to lipid ratios of extracellular vesicle preparations from various myeloid and lymphoid cell lines as well as blood plasma. These preparations included apoptotic bodies, microvesicles/microparticles, and exosomes isolated by size-based fractionation. We also investigated lipid bilayer order of extracellular vesicle subpopulations using Di-4-ANEPPDHQ lipid probe, and lipid composition using affinity reagents to clustered cholesterol (monoclonal anti-cholesterol antibody) and ganglioside GM1 (cholera toxin subunit B). We have consistently found different protein to lipid ratios characteristic for the investigated extracellular vesicle subpopulations which were substantially altered in the case of vesicular damage or protein contamination. Spectral ratiometric imaging and flow cytometric analysis also revealed marked differences between the various vesicle populations in their lipid order and their clustered membrane cholesterol and GM1 content. Our study introduces for the first time a simple and readily available lipid assay to complement the widely used protein assays in order to better characterize extracellular vesicle preparations. Besides differentiating extracellular vesicle subpopulations, the novel parameters introduced in this work (protein to lipid ratio, lipid bilayer order, and lipid composition), may prove useful for quality control of extracellular vesicle related basic and clinical studies.

T Lymphocytes are Targets for Platelet- and Trophoblast-Derived Microvesicles During Pregnancy

Microvesicles (MVs) can derive from several cell types and their membranes contain cell surface elements. Their role is increasingly recognized in cell-to-cell communication, as they act as both paracrine and remote messengers, occurring in circulating form as well as in plasma. Successful pregnancy requires a series of interactions between the maternal immune system and the implanted fetus, such that the semi-allograft will not be rejected. These interactions occur at the materno-placental interface and/or at a systemic level. In the present study we identified for the first time the in vivo plasma pattern of the MVs of third-trimester, healthy pregnant women, their cellular origin, and their target cells using flow cytometry and confocal laser microscopy. We searched for the cellular target molecules of thrombocyte-derived MVs with the help of neutralizing antibodies. We examined the in vitro effects of MVs on STAT3 phosphorylation of primary lymphocytes and Jurkat cells. We found that both placental trophoblast-derived and maternal thrombocyte-derived MVs bind to circulating peripheral T lymphocytes, but not to B lymphocytes or NK cells. We were able to show that the P-selectin (CD62P)-PSGL-1 (CD162) interaction is one mechanism binding platelet-derived MVs to T cells. We were also able to demonstrate that MV-lymphocyte interactions induce STAT3 phosphorylation in T cells. Our findings indicate that both thrombocyte- and trophoblast-derived MVs may play an important role in the immunomodulation of pregnancy. We suggest that the transfer of different signals via MVs represents a novel form of communication between the placenta and the maternal immune system, and that MVs contribute to the establishment of stable immune tolerance to the semi-allograft fetus.

Improved circulating microparticle analysis in acid-citrate dextrose (ACD) anticoagulant tube

INTRODUCTION Recently extracellular vesicles (exosomes, microparticles also referred to as microvesicles and apoptotic bodies) have attracted substantial interest as potential biomarkers and therapeutic vehicles. However, analysis of microparticles in biological fluids is confounded by many factors such as the activation of cells in the blood collection tube that leads to in vitro vesiculation. In this study we aimed at identifying an anticoagulant that prevents in vitro vesiculation in blood plasma samples. MATERIALS AND METHODS We compared the levels of platelet microparticles and non-platelet-derived microparticles in platelet-free plasma samples of healthy donors. Platelet-free plasma samples were isolated using different anticoagulant tubes, and were analyzed by flow cytometry and Zymuphen assay. The extent of in vitro vesiculation was compared in citrate and acid-citrate-dextrose (ACD) tubes. RESULTS Agitation and storage of blood samples at 37 °C for 1 hour induced a strong release of both platelet microparticles and non-platelet-derived microparticles. Strikingly, in vitro vesiculation related to blood sample handling and storage was prevented in samples in ACD tubes. Importantly, microparticle levels elevated in vivo remained detectable in ACD tubes. CONCLUSIONS We propose the general use of the ACD tube instead of other conventional anticoagulant tubes for the assessment of plasma microparticles since it gives a more realistic picture of the in vivo levels of circulating microparticles and does not interfere with downstream protein or RNA analyses.

The role of membrane vesicles in tumorigenesis

Membrane vesicles are membrane-covered cell fragments generated by all cell types. They comprise a recently recognized new system of intercellular communication, believed to play a pivotal role in information transfer between cells, as they display a large number of biomolecules enclosed within the membrane as well as in the membrane proper. The phenotype of the donor cell is reflected in the vesicular protein content, which also allows the identification of the original cell. Membrane vesicles have been implicated in several physiological and pathological processes, most notably in tumorigenesis. Tumor-derived vesicles may serve as prognostic markers, they were detected in blood plasma and in other body fluids. Their size varies between 30 and 1000 nm. All of them reflect the special potential of tumor cells for survival and for the expansion of the tumor, independently from cell-to-cell contact. Tumor-derived vesicles have the potential to facilitate the escape of tumor cells from immune surveillance through their protein and RNA content, at the same time they are involved in the establishment of a beneficial environment for newly formed and migrating tumor cells, influencing angiogenesis and the reorganization of the extracellular matrix. Elucidating the properties of tumor-derived vesicles should increase our understanding in tumor biology and open new perspectives in cancer treatment. Tumor-derived vesicles are involved in tumorigenesis at multiple level and drugs themselves can be expulsed from tumor cells via vesicles. Consequently, interfering with the formation, release and propagation of these vesicles can be a novel and alternative issue in cancer therapy. The present review is an overview of the roles of membrane vesicles in tumorigenesis showing also the potential to consider them as new targets in tumor therapy.

Lymphocyte phenotype analysis and chromosome aberration frequency of workers occupationally exposed to styrene, benzene, polycyclic aromatic hydrocarbons or mixed solvents

The aim of our study was to investigate the immunotoxicity of benzene, styrene and polycyclic aromatic hydrocarbon exposure, to establish the correlation between immunological and genotoxicological parameters, and to assess the possible effect of confounding factors such as age and smoking. The immune status of the donors was characterized by measuring the surface antigens of peripheral lymphocytes. The studied antigens were the following: CD3, CD4, CD8, CD14, CD19, CD25, CD38, CD45, CD45RO, CD54, CD56, CD62L, CD71 and HLA-DR. In our studies, we compared the immunological and genotoxicological parameters (chromosome aberration, sister chromatid exchange frequency, unscheduled DNA synthesis) of the different groups with healthy controls. Analysis revealed changes in the expression of surface antigens on peripheral lymphocytes in correlation with exposure. Confounding factors, such as smoking, increased the proportion of CD4 positive T lymphocytes and influenced the surface expression of several antigens. In our investigation the occurrence of chromosome aberrations negatively correlated with CD25 (IL-2R) expression in both CD4 and CD8 positive T cells. The presented data suggest that solvents such as benzene, styrene and PAHs activate peripheral lymphocytes, and cause changes in the incidence of CD25+/CD4+ T lymphocytes that may represent a distinct subset of immune-regulatory T cells.