Summary
Background
Plasma and other body fluids contain membranous extracellular vesicles (EVs), which are considered to derive from activated or apoptotic cells. EVs participate in physiological and ...pathological processes and have potential applications in diagnostics or therapeutics. Knowledge on EVs is, however, limited, mainly due to their sub‐micrometer size and to intrinsic limitations in methods applied for their characterization.
Objectives
Our aim was to provide a comprehensive description of EVs from plasma of healthy subjects.
Methods
Cryo‐transmission electron microscopy combined with receptor‐specific gold labeling was used to reveal the morphology, size and phenotype of EVs. An original approach based on sedimentation on electron microscopy grids was developed for enumerating EVs. A correlation was performed between conventional flow cytometry and electron microscopy results.
Results
We show that platelet‐free plasma samples contain spherical EVs, 30 nm to 1 μm in diameter, tubular EVs, 1–5 μm long, and membrane fragments, 1–8 μm large. We show that only a minority of EVs expose the procoagulant lipid phosphatidylserine, in contrast to the classical theory of EV formation. In addition, the concentrations of the main EV sub‐populations are determined after sedimentation on EM grids. Finally, we show that conventional flow cytometry, the main method of EV characterization, detects only about 1% of them.
Conclusion
This study brings novel insights on EVs from normal plasma and provides a reference for further studies of EVs in disease situations.
Summary
Background
Plasma contains cell‐derived extracellular vesicles (EVs), which participate in physiopathological processes and have potential applications as disease biomarker. However, the ...enumeration of EVs faces major problems, due to their sub‐micrometer size and to intrinsic limitations in methods of characterization, mainly flow cytometry (FCM).
Objectives
Our objective is to enumerate EVs in plasma, by taking as the prototype the population of phosphatidylserine (PS)‐exposing EVs, which constitute one of the major EV populations and are responsible for thrombotic disorders.
Methods
The concentration of PS‐exposing EVs in platelet‐free plasma (PFP) of healthy subjects was measured by FCM using either light scattering or fluorescence as the trigger and fluorescent Annexin‐5 (Anx5) as the specific label. In addition, PS‐exposing EVs were enumerated by electron microscopy (EM) after labeling with Anx5 gold nanoparticles and sedimentation on EM grids.
Results
We show that about 50× more Anx5‐positive EVs are detected by FCM when detection is triggered on fluorescence as compared with light scattering. By fluorescence triggering, concentrations of 22 000–30 000 Anx5‐positive EVs per μL PFP were determined, using two different flow cytometers. The limit of detection of the fluorescence triggering method was estimated at about 1000–2500 Anx5 molecules. Results from EM suggest that EVs down to 100–150 nm diameter are detected by fluorescence triggering.
Conclusion
This study presents a simple method for enumerating EVs. We believe that this method is applicable in a general context and will improve our understanding of the roles of EVs in pathophysiological situations, which will open avenues for the development of EV‐based diagnosis assays.
Abstract Annexins are soluble proteins that bind to biological membranes containing negatively charged phospholipids, principally phosphatidylserine, in a Ca2+ -dependent manner. Annexin-A5 (AnxA5), ...the smallest member of the annexin family, presents unique properties of membrane binding and self-assembly into ordered two-dimensional (2D) arrays on membrane surfaces. We have previously reported that AnxA5 plays a central role in the machinery of membrane repair by enabling rapid resealing of plasma membrane disruption in murine perivascular cells. AnxA5 promotes membrane repair via the formation of a protective 2D bandage at membrane damaged site. Here, we review current knowledge on cell membrane repair and present recent findings on the role of AnxA5 in membrane resealing of human trophoblasts.
Adeno-associated virus (AAV) vectors are showing promise in gene therapy trials and have proven to be extremely efficient biological tools in basic neuroscience research. One major limitation to ...their widespread use in the neuroscience laboratory is the cost, labor, skill and time-intense purification process of AAV. We have recently shown that AAV can associate with exosomes (exo-AAV) when the vector is isolated from conditioned media of producer cells, and the exo-AAV is more resistant to neutralizing anti-AAV antibodies compared with standard AAV. Here, we demonstrate that simple pelleting of exo-AAV from media via ultracentrifugation results in high-titer vector preparations capable of efficient transduction of central nervous system (CNS) cells after systemic injection in mice. We observed that exo-AAV is more efficient at gene delivery to the brain at low vector doses relative to conventional AAV, even when derived from a serotype that does not normally efficiently cross the blood-brain barrier. Similar cell types were transduced by exo-AAV and conventionally purified vector. Importantly, no cellular toxicity was noted in exo-AAV-transduced cells. We demonstrated the utility and robustness of exo-AAV-mediated gene delivery by detecting direct GFP fluorescence after systemic injection, allowing three-dimensional reconstruction of transduced Purkinje cells in the cerebellum using ex vivo serial two-photon tomography. The ease of isolation combined with the high efficiency of transgene expression in the CNS, may enable the widespread use of exo-AAV as a neuroscience research tool. Furthermore, the ability of exo-AAV to evade neutralizing antibodies while still transducing CNS after peripheral delivery is clinically relevant.
Cancer cells are exposed to major compressive and shearing forces during invasion and metastasis, leading to extensive plasma membrane damage. To survive this mechanical stress, they need to repair ...membrane injury efficiently. Targeting the membrane repair machinery is thus potentially a new way to prevent invasion and metastasis. We show here that annexin-A2 (ANXA2) is required for membrane repair in invasive breast and pancreatic cancer cells. Mechanistically, we show by fluorescence and electron microscopy that cells fail to reseal shear-stress damaged membrane when ANXA2 is silenced or the protein is inhibited with neutralizing antibody. Silencing of ANXA2 has no effect on proliferation in vitro, and may even accelerate migration in wound healing assays, but reduces tumor cell dissemination in both mice and zebrafish. We expect that inhibiting membrane repair will be particularly effective in aggressive, poor prognosis tumors because they rely on the membrane repair machinery to survive membrane damage during tumor invasion and metastasis. This could be achieved either with anti-ANXA2 antibodies, which have been shown to inhibit metastasis of breast and pancreatic cancer cells, or with small molecule drugs.
Supported lipid bilayers (SLBs) are popular models of cell membranes with potential bio-technological applications. A qualitative understanding of the process of SLB formation after exposure of small ...lipid vesicles to a hydrophilic support is now emerging. Recent studies have revealed a stunning variety of effects that can take place during this self-organization process. The ensemble of results in our group has revealed unprecedented insight into intermediates of the SLB-formation process and has helped to identify a number of parameters that are determinant for the lipid deposition on solid supports. The pathway of lipid deposition can be tuned by electrostatic interactions and by the presence of calcium. We emphasize the importance of the solid support in the SLB-formation process. Our results suggest that the molecular-level interaction between lipids and the solid support needs to be considered explicitly, to understand the rupture of vesicles and the formation of SLBs as well as to predict the properties of the resulting SLB. The impact of the SLB-formation process on the quality and the physical properties of the resulting SLB as well as implications for other types of surface-confined lipid bilayers are discussed.
Supported lipid bilayers (SLBs) are popular models of cell membranes with potential biotechnological applications and an understanding of the mechanisms of SLB formation is now emerging. Here we ...characterize, by combining atomic force microscopy, quartz crystal microbalance with dissipation monitoring, and ellipsometry, the formation of SLBs on mica from sonicated unilamellar vesicles using mixtures of zwitterionic, negatively and positively charged lipids. The results are compared with those we reported previously on silica. As on silica, electrostatic interactions were found to determine the pathway of lipid deposition. However, fundamental differences in the stability of surface-bound vesicles and the mobility of SLB patches were observed, and point out the determining role of the solid support in the SLB-formation process. The presence of calcium was found to have a much more pronounced influence on the lipid deposition process on mica than on silica. Our results indicate a specific calcium-mediated interaction between dioleoylphosphatidylserine molecules and mica. In addition, we show that the use of PLL-
g-PEG modified tips considerably improves the AFM imaging of surface-bound vesicles and bilayer patches and evaluate the effects of the AFM tip on the apparent size and shape of these soft structures.
We have previously uncovered the impact of oncogenic and differentiation processes on extracellular vesicles (EVs) in cancer. This is of interested in the context of glioma stem cells (GSC) that are ...responsible for recurrent nature of glioblastoma multiforme (GBM), while retaining the potential to undergo differentiation and self renewal. GSCs reside in vascular niches where they interact with endothelial cells through a number of mediators including bioactive cargo of EVs. GSCs can be classified as proneural (PN) or mesenchymal (MES) subtypes on the basis of their gene expression profiles and distinct biological characteristics. In the present study we investigated how GSC diversity and differentiation programmes influence their EV-mediated communication potentials. Indeed, molecular subtypes of GBMs and GSCs differ with respect to their expression of EV-related genes (vesiculome) and GSCs with PN or MES phenotypes produce EVs with markedly different characteristics, marker profiles, proteomes and endothelial stimulating activities. For example, while EVs of PN GSC are largely devoid of exosomal markers their counterparts from MES GSCs express ample CD9, CD63 and CD81 tetraspanins. In both GSC subtypes serum-induced differentiation results in profound, but distinct changes of cellular phenotypes including the enhanced EV production, reconfiguration of their proteomes and the related functional pathways. Notably, the EV uptake was a function of both subtype and differentiation state of donor cells. Thus, while, EVs produced by differentiated MES GSCs were internalized less efficiently than those from undifferentiated cells they exhibited an increased stimulatory potential for human brain endothelial cells. Such stimulating activity was also observed for EVs derived from differentiated PN GSCs, despite their even weaker uptake by endothelial cells. These findings suggest that the role of EVs as biological mediators and biomarkers in GBM may depend on the molecular subtype and functional state of donor cancer cells, including cancer stem cells.
Abbreviations: CryoTEM: cryo-transmission electron microscopy; DIFF: differentiated GSCs; EGF: epidermal growth factor; DUC: differential ultracentrifugation; EV: extracellular vesicle; FGF: fibroblast growth factor; GBM: glioblastoma multiforme; GFAP: glial fibrillary acidic protein; GO: gene ontology; GSC: glioma stem cells; HBEC-5i: human brain endothelial cells; MES: mesenchymal cells; MTS - 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt; PMT1: proneural-to-mesenchyman transition cell line 1; PN: proneural cells; TEM: transmission electron microscopy; WB: western blotting
Owing to the relationship between extracellular vesicles (EVs) and physiological and pathological conditions, the interest in EVs is exponentially growing. EVs hold high hopes for novel diagnostic ...and translational discoveries. This review provides an expert-based update of recent advances in the methods to study EVs and summarizes currently accepted considerations and recommendations from sample collection to isolation, detection, and characterization of EVs. Common misconceptions and methodological pitfalls are highlighted. Although EVs are found in all body fluids, in this review, we will focus on EVs from human blood, not only our most complex but also the most interesting body fluid for cardiovascular research.
Abstract The high biocompatibility and versatile nature of liposomes made these particles keystone components in many hot-topic research areas. For transfection and cell labelling purposes, synthetic ...cationic lipids are often added, but in most studies, little attention has been paid to their cytotoxic effects. In the present work, cationic magnetoliposomes (MLs), i.e. iron oxide cores enwrapped by a phospholipid bilayer (dimyristoylphosphatidylcholine or sphingomyelin) doped with cationic lipids (1,2-distearoyl-3-trimethylammonium propane), serve as a model to examine cationic lipid toxicity. Mechanisms of cytotoxic effects were found to be either dependent or independent of actual particle internalisation according to data obtained in the absence or presence of several endocytosis inhibitors. The former seem to be caused by the generation of reactive oxygen species (ROS) leading to a Ca2+ influx at high ROS levels. The latter are due to a destabilisation of the cell plasma membrane upon transfer of the cationic lipid from the ML bilayer into the plasma membrane. However, these adverse effects can be diminished by the use of a ROS scavenger, a Ca2+ -channel blocker or by modulating the liposome size, lipid bilayer constitution or by stabilising the membrane by anchoring it on a solid core. Careful attention must be paid in terms of assessing cell viability as the effects are highly time dependent and the data suggest the incompatibility of using the well-known MTT assay when high levels of ROS species are generated.
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