Pia Siljander

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.

Vesiclepedia: A Compendium for Extracellular Vesicles with Continuous Community Annotation

Vesiclepedia is a community-annotated compendium of molecular data on extracellular vesicles.

Use of Synthetic Peptides to Locate Novel Integrin α2β1-binding Motifs in Human Collagen III

A set of 57 synthetic peptides encompassing the entire triplehelical domain of human collagen III was used to locate binding sites for the collagen-binding integrin α2β1. The capacity of the peptides to support Mg2+-dependent binding of several integrin preparations was examined. Wild-type integrins (recombinant α2 I-domain, α2β1 purified from platelet membranes, and recombinant soluble α2β1 expressed as an α2-Fos/β1-Jun heterodimer) bound well to only three peptides, two containing GXX′GER motifs (GROGER and GMOGER, where O is hydroxyproline) and one containing two adjacent GXX′GEN motifs (GLKGEN and GLOGEN). Two mutant α2 I-domains were tested: the inactive T221A mutant, which recognized no peptides, and the constitutively active E318W mutant, which bound a larger subset of peptides. Adhesion of activated human platelets to GER-containing peptides was greater than that of resting platelets, and HT1080 cells bound well to more of the peptides compared with platelets. Binding of cells and recombinant proteins was abolished by anti-α2 monoclonal antibody 6F1 and by chelation of Mg2+. We describe two novel high affinity integrin-binding motifs in human collagen III (GROGER and GLOGEN) and a third motif (GLKGEN) that displays intermediate activity. Each motif was verified using shorter synthetic peptides.

EVpedia: a community web portal for extracellular vesicles research

MOTIVATION Extracellular vesicles (EVs) are spherical bilayered proteolipids, harboring various bioactive molecules. Due to the complexity of the vesicular nomenclatures and components, online searches for EV-related publications and vesicular components are currently challenging. RESULTS We present an improved version of EVpedia, a public database for EVs research. This community web portal contains a database of publications and vesicular components, identification of orthologous vesicular components, bioinformatic tools and a personalized function. EVpedia includes 6879 publications, 172 080 vesicular components from 263 high-throughput datasets, and has been accessed more than 65 000 times from more than 750 cities. In addition, about 350 members from 73 international research groups have participated in developing EVpedia. This free web-based database might serve as a useful resource to stimulate the emerging field of EV research. AVAILABILITY AND IMPLEMENTATION The web site was implemented in PHP, Java, MySQL and Apache, and is freely available at http://evpedia.info.

Platelet Adhesion Enhances the Glycoprotein VI–Dependent Procoagulant Response Involvement of p38 MAP Kinase and Calpain

Abstract— In the final stages of activation, platelets express coagulation-promoting activity by 2 simultaneous processes: exposure of aminophospholipids, eg, phosphatidylserine (PS), at the platelet surface, and formation of membrane blebs, which may be shed as microvesicles. Contact with collagen triggers both processes via platelet glycoprotein VI (GPVI). Here, we studied the capacity of 2 GPVI ligands, collagen-related peptide (CRP) and the snake venom protein convulxin (CVX), to elicit the procoagulant platelet response. In platelets in suspension, either ligand induced full aggregation and high Ca2+ signals but little microvesiculation or PS exposure. However, most of the platelets adhering to immobilized CRP or CVX had exposed PS and formed membrane blebs after a prolonged increase in cytosolic [Ca2+]i. Platelets adhering to fibrinogen responded similarly but only when exposed to soluble CRP or CVX. By scanning electron microscopic analysis, the bleb-forming platelets were detected as either round, spongelike structures with associated microparticles or as arrays of vesicular cell fragments. The phosphorylation of p38 mitogen-activated protein kinase (MAPK) elicited by CRP and CVX was enhanced in fibrinogen-adherent platelets compared with that in platelets in suspension. The p38 inhibitor SB203580 and the calpain protease inhibitor calpeptin reduced only the procoagulant bleb formation, having no effect on PS exposure. Inhibition of p38 also downregulated calpain activity. We conclude that the procoagulant response evoked by GPVI stimulation is potentiated by platelet adhesion. The sequential activation of p38 MAPK and calpain appears to regulate procoagulant membrane blebbing but not PS exposure.

Evidence-Based Clinical Use of Nanoscale Extracellular Vesicles in Nanomedicine

Recent research has demonstrated that all body fluids assessed contain substantial amounts of vesicles that range in size from 30 to 1000 nm and that are surrounded by phospholipid membranes containing different membrane microdomains such as lipid rafts and caveolae. The most prominent representatives of these so-called extracellular vesicles (EVs) are nanosized exosomes (70-150 nm), which are derivatives of the endosomal system, and microvesicles (100-1000 nm), which are produced by outward budding of the plasma membrane. Nanosized EVs are released by almost all cell types and mediate targeted intercellular communication under physiological and pathophysiological conditions. Containing cell-type-specific signatures, EVs have been proposed as biomarkers in a variety of diseases. Furthermore, according to their physical functions, EVs of selected cell types have been used as therapeutic agents in immune therapy, vaccination trials, regenerative medicine, and drug delivery. Undoubtedly, the rapidly emerging field of basic and applied EV research will significantly influence the biomedicinal landscape in the future. In this Perspective, we, a network of European scientists from clinical, academic, and industry settings collaborating through the H2020 European Cooperation in Science and Technology (COST) program European Network on Microvesicles and Exosomes in Health and Disease (ME-HAD), demonstrate the high potential of nanosized EVs for both diagnostic and therapeutic (i.e., theranostic) areas of nanomedicine.

Isolation and characterization of platelet-derived extracellular vesicles.

Background Platelet-derived extracellular vesicles (EVs) participate, for example, in haemostasis, immunity and development. Most studies of platelet EVs have targeted microparticles, whereas exosomes and EV characterization under various conditions have been less analyzed. Studies have been hampered by the difficulty in obtaining EVs free from contaminating cells and platelet remnants. Therefore, we optimized an EV isolation protocol and compared the quantity and protein content of EVs induced by different agonists. Methods Platelets isolated with iodixanol gradient were activated by thrombin and collagen, lipopolysaccharide (LPS) or Ca2+ ionophore. Microparticles and exosomes were isolated by differential centrifugations. EVs were quantitated by nanoparticle tracking analysis (NTA) and total protein. Size distributions were determined by NTA and electron microscopy. Proteomics was used to characterize the differentially induced EVs. Results The main EV populations were 100–250 nm and over 90% were <500 nm irrespective of the activation. However, activation pathways differentially regulated the quantity and the quality of EVs, which also formed constitutively. Thrombogenic activation was the most potent physiological EV-generator. LPS was a weak inducer of EVs, which had a selective protein content from the thrombogenic EVs. Ca2+ ionophore generated a large population of protein-poor and unselectively packed EVs. By proteomic analysis, EVs were highly heterogeneous after the different activations and between the vesicle subpopulations. Conclusions Although platelets constitutively release EVs, vesiculation can be increased, and the activation pathway determines the number and the cargo of the formed EVs. These activation-dependent variations render the use of protein content in sample normalization invalid. Since most platelet EVs are 100–250 nm, only a fraction has been analyzed by previously used methods, for example, flow cytometry. As the EV subpopulations could not be distinguished and large vesicle populations may be lost by differential centrifugation, novel methods are required for the isolation and the differentiation of all EVs.

Different gDNA content in the subpopulations of prostate cancer extracellular vesicles: Apoptotic bodies, microvesicles, and exosomes

Extracellular vesicles (EVs) are cell‐derived membrane vesicles. EVs contain several RNAs such as mRNA, microRNAs, and ncRNAs, but less is known of their genomic DNA (gDNA) content. It is also unknown whether the DNA cargo is randomly sorted or if it is systematically packed into specific EV subpopulations. The aim of this study was to analyze whether different prostate cancer (PCa) cell‐derived EV subpopulations (apoptotic bodies, microvesicles, and exosomes) carry different gDNA fragments.

Platelet-Derived Microvesicles: Multitalented Participants in Intercellular Communication

Platelets can release a heterogeneous pool of vesicles which include plasma membrane-derived microparticles (PMPs) and multivesicular body-derived exosomes. As both vesicle types are generated upon activation and their distinction is complicated due to an overlap in their molecular properties and sizes, they are best discussed as an entity, the platelet-derived microvesicles (PMVs). PMPs can be formed through several induction pathways, which determine their different molecular profiles and facilitate tailor-made participation in intercellular communication. This dynamic variability may lie behind the multifaceted and sometimes very different observations of the PMPs in physiological and pathological settings. Currently, little is known of platelet-derived exosomes. In all, PMVs not only participate in several homeostatic multicellular processes, such as hemostasis, maintenance of vascular health, and immunity, but they also play a role in thrombotic and inflammatory diseases and cancer progression. In the past few years, the number of original articles and reviews on microvesicles has dramatically increased, but the data simultaneously raise further questions, the answers to which depend on forthcoming analytical improvements. In this article, the differential activation pathways and the molecular and functional properties of PMVs are reviewed in context with their sometimes paradoxical role in health and in disease. Also, the methodological issues of PMV detection and analysis are discussed in the light of recent advances within the field.

Microvesicle- and exosome-mediated drug delivery enhances the cytotoxicity of Paclitaxel in autologous prostate cancer cells.

BACKGROUND Extracellular vesicles (EVs) are naturally occurring membrane particles that mediate intercellular communication by delivering molecular information between cells. In this study, we investigated the effectiveness of two different populations of EVs (microvesicle- and exosome-enriched) as carriers of Paclitaxel to autologous prostate cancer cells. METHODS EVs were isolated from LNCaP- and PC-3 prostate cancer cell cultures using differential centrifugation and characterized by electron microscopy, nanoparticle tracking analysis, and Western blot. The uptake of microvesicles and exosomes by the autologous prostate cancer cells was assessed by flow cytometry and confocal microscopy. The EVs were loaded with Paclitaxel and the effectiveness of EV-mediated drug delivery was assessed with viability assays. The distribution of EVs and EV-delivered Paclitaxel in cells was inspected by confocal microscopy. RESULTS Our main finding was that the loading of Paclitaxel to autologous prostate cancer cell-derived EVs increased its cytotoxic effect. This capacity was independent of the EV population and the cell line tested. Although the EVs without the drug increased cancer cell viability, the net effect of enhanced cytotoxicity remained. Both EV populations delivered Paclitaxel to the recipient cells through endocytosis, leading to the release of the drug from within the cells. The removal of EV surface proteins did not affect exosomes, while the drug delivery mediated by microvesicles was partially inhibited. CONCLUSIONS Cancer cell-derived EVs can be used as effective carriers of Paclitaxel to their parental cells, bringing the drug into the cells through an endocytic pathway and increasing its cytotoxicity. However, due to the increased cell viability, the use of cancer cell-derived EVs must be further investigated before any clinical applications can be designed.