Extracellular vesicles are a heterogeneous group of cell-derived membranous structures comprising exosomes and microvesicles, which originate from the endosomal system or which are shed from the plasma membrane, respectively They are present in biological fluids and are involved in multiple physiological and pathological processes Extracellular vesicles are now considered as an additional mechanism for intercellular communication, allowing cells to exchange proteins, lipids and genetic material Knowledge of the cellular processes that govern extracellular vesicle biology is essential to shed light on the physiological and pathological functions of these vesicles as well as on clinical applications involving their use and/or analysis However, in this expanding field, much remains unknown regarding the origin, biogenesis, secretion, targeting and fate of these vesicles

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Shedding light on the cell biology of extracellular vesicles.

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.

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Biological properties of extracellular vesicles and their physiological functions

Extracellular vesicles (EVs) have become the focus of rising interest because of their numerous functions in physiology and pathology. Cells release heterogeneous vesicles of different sizes and intracellular origins, including small EVs formed inside endosomal compartments (i.e., exosomes) and EVs of various sizes budding from the plasma membrane. Specific markers for the analysis and isolation of different EV populations are missing, imposing important limitations to understanding EV functions. Here, EVs from human dendritic cells were first separated by their sedimentation speed, and then either by their behavior upon upward floatation into iodixanol gradients or by immuno-isolation. Extensive quantitative proteomic analysis allowing comparison of the isolated populations showed that several classically used exosome markers, like major histocompatibility complex, flotillin, and heat-shock 70-kDa proteins, are similarly present in all EVs. We identified proteins specifically enriched in small EVs, and define a set of five protein categories displaying different relative abundance in distinct EV populations. We demonstrate the presence of exosomal and nonexosomal subpopulations within small EVs, and propose their differential separation by immuno-isolation using either CD63, CD81, or CD9. Our work thus provides guidelines to define subtypes of EVs for future functional studies.

https://www.pnas.org/content/pnas/113/8/E968.full.pdf

Proteomic comparison defines novel markers to characterize heterogeneous populations of extracellular vesicle subtypes.

The ability of exosomes to transfer cargo from donor to acceptor cells, thereby triggering phenotypic changes in the latter, has generated substantial interest in the scientific community. However, the extent to which exosomes differ from other extracellular vesicles in terms of their biogenesis and functions remains ill-defined. Here, we discuss the current knowledge on the specificities of exosomes and other types of extracellular vesicles, and their roles as important agents of cell-to-cell communication.

Specificities of secretion and uptake of exosomes and other extracellular vesicles for cell-to-cell communication.

The emergence of publications on extracellular RNA (exRNA) and extracellular vesicles (EV) has highlighted the potential of these molecules and vehicles as biomarkers of disease and therapeutic targets. These findings have created a paradigm shift, most prominently in the field of oncology, prompting expanded interest in the field and dedication of funds for EV research. At the same time, understanding of EV subtypes, biogenesis, cargo and mechanisms of shuttling remains incomplete. The techniques that can be harnessed to address the many gaps in our current knowledge were the subject of a special workshop of the International Society for Extracellular Vesicles (ISEV) in New York City in October 2012. As part of the “ISEV Research Seminar: Analysis and Function of RNA in Extracellular Vesicles (evRNA)”, 6 round-table discussions were held to provide an evidence-based framework for isolation and analysis of EV, purification and analysis of associated RNA molecules, and molecular engineering of EV for therapeutic intervention. This article arises from the discussion of EV isolation and analysis at that meeting. The conclusions of the round table are supplemented with a review of published materials and our experience. Controversies and outstanding questions are identified that may inform future research and funding priorities. While we emphasize the need for standardization of specimen handling, appropriate normative controls, and isolation and analysis techniques to facilitate comparison of results, we also recognize that continual development and evaluation of techniques will be necessary as new knowledge is amassed. On many points, consensus has not yet been achieved and must be built through the reporting of well-controlled experiments. Keywords: extracellular vesicle; exosome; microvesicle; standardization; isolation (Published: 27 May 2013) Citation: Journal of Extracellular Vesicles 2013, 2 : 20360 - http://dx.doi.org/10.3402/jev.v2i0.20360

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Standardization of sample collection, isolation and analysis methods in extracellular vesicle research

We studied inflammatory and oxidative stress-related parameters and cytotoxic response of human umbilical vein endothelial cells (HUVEC) to a 24 h treatment with milled particles simulating debris involved in sandblasting of orthopedic implants (OI). We used different abrasives (corundum—(Al2O3), used corundum retrieved from removed OI (u. Al2O3), and zirconia/silica composite (ZrO2/SiO2)). Morphological changes were observed by scanning electron microscopy (SEM). Concentration of Interleukins IL-6 and IL-1β and Tumor Necrosis Factor α (TNF)-α was assessed by enzyme-linked immunosorbent assay (ELISA). Activity of Cholinesterase (ChE) and Glutathione S-transferase (GST) was measured by spectrophotometry. Reactive oxygen species (ROS), lipid droplets (LD) and apoptosis were measured by flow cytometry (FCM). Detachment of the cells from glass and budding of the cell membrane did not differ in the treated and untreated control cells. Increased concentration of IL-1β and of IL-6 was found after treatment with all tested particle types, indicating inflammatory response of the treated cells. Increased ChE activity was found after treatment with u. Al2O3 and ZrO2/SiO2. Increased GST activity was found after treatment with ZrO2/SiO2. Increased LD quantity but not ROS quantity was found after treatment with u. Al2O3. No cytotoxicity was detected after treatment with u. Al2O3. The tested materials in concentrations added to in vitro cell lines were found non-toxic but bioactive and therefore prone to induce a response of the human body to OI.

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Inflammatory, Oxidative Stress and Small Cellular Particle Response in HUVEC Induced by Debris from Endoprosthesis Processing

This CORR Insights®is a commentary on the article “Does Surgeon Experience Impact the Risk of Complications After Bernese Periacetabular Osteotomy?” by Novais and colleagues available at: DOI: 10.1007/s11999-016-5010-1 . The author certifies that she, or any members of her immediate family, have no funding or commercial associations (consultancies, stock ownership, equity interest, patent/licensing arrangements, etc.) that might pose a conflict of interest in connection with the submitted article. All ICMJE Conflict of Interest Forms for authors and Clinical Orthopaedics and Related Research editors and board members are on file with the publication and can be viewed on request. The opinions expressed are those of the writers and do not reflect the opinion or policy of CORR® or The Association of Bone and Joint Surgeons®.

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CORR Insights®: Does Surgeon Experience Impact the Risk of Complications After Bernese Periacetabular Osteotomy?

The interaction between charged membrane surfaces, separated by a solution containing charged nanoparticles was studied experimentally and theoretically. The nonlocal theory for the nanoparticles was developed where finite size of nanoparticles and spatial distribution of charge within a particle were taken into account. It was shown that for large enough membrane surface charge densities and large enough dimensions of nanoparticles, the force between equally charged membranes may be attractive due to spatially distributed charges within the nanoparticles.

Interaction between charged membrane surfaces mediated by charged nanoparticles

Surfactin molecules with a cone-like structure promote the formation of membrane domains with negative spontaneous curvature and induce membrane invaginations.

Small cellular particles (SCPs) are being considered for their role in cell-to-cell communication. We harvested and characterized SCPs from spruce needle homogenate. SCPs were isolated by differential ultracentrifugation. They were imaged by scanning electron microscope (SEM) and cryogenic transmission electron microscope (cryo TEM), assessed for their number density and hydrodynamic diameter by interferometric light microscopy (ILM) and flow cytometry (FCM), total phenolic content (TPC) by UV-vis spectroscopy, and terpene content by gas chromatography-mass spectrometry (GC-MS). The supernatant after ultracentrifugation at 50,000× g contained bilayer-enclosed vesicles whereas in the isolate we observed small particles of other types and only a few vesicles. The number density of cell-sized particles (CSPs) (larger than 2 μm) and meso-sized particles (MSPs) (cca 400 nm–2 µm) was about four orders of magnitude lower than the number density of SCPs (sized below 500 nm). The average hydrodynamic diameter of SCPs measured in 10,029 SCPs was 161 ± 133 nm. TCP decreased considerably due to 5-day aging. Volatile terpenoid content was found in the pellet after 300× g. The above results indicate that spruce needle homogenate is a source of vesicles to be explored for potential delivery use.

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Veronika Kralj-Iglič

Papers

Inflammatory, Oxidative Stress and Small Cellular Particle Response in HUVEC Induced by Debris from Endoprosthesis Processing

CORR Insights®: Does Surgeon Experience Impact the Risk of Complications After Bernese Periacetabular Osteotomy?

Interaction between charged membrane surfaces mediated by charged nanoparticles

Surfactin molecules with a cone-like structure promote the formation of membrane domains with negative spontaneous curvature and induce membrane invaginations.

Small Cellular Particles from European Spruce Needle Homogenate