ABSTRACT
Neural stem cell (NSC) transplantation induces recovery in animal models of central nervous system (CNS) diseases. Although the replacement of lost endogenous cells was originally proposed as the primary healing mechanism of NSC grafts, it is now clear that transplanted NSCs operate via multiple mechanisms, including the horizontal exchange of therapeutic cargoes to host cells via extracellular vesicles (EVs). EVs are membrane particles trafficking nucleic acids, proteins, metabolites and metabolic enzymes, lipids, and entire organelles. However, the function and the contribution of these cargoes to the broad therapeutic effects of NSCs are yet to be fully understood. Mitochondrial dysfunction is an established feature of several inflammatory and degenerative CNS disorders, most of which are potentially treatable with exogenous stem cell therapeutics. Herein, we investigated the hypothesis that NSCs release and traffic functional mitochondria via EVs to restore mitochondrial function in target cells. Untargeted proteomics revealed a significant enrichment of mitochondrial proteins spontaneously released by NSCs in EVs. Morphological and functional analyses confirmed the presence of ultrastructurally intact mitochondria within EVs with conserved membrane potential and respiration. We found that the transfer of these mitochondria from EVs to mtDNA-deficient L929 Rho0 cells rescued mitochondrial function and increased Rho0 cell survival. Furthermore, the incorporation of mitochondria from EVs into inflammatory mononuclear phagocytes restored normal mitochondrial dynamics and cellular metabolism and reduced the expression of pro-inflammatory markers in target cells. When transplanted in an animal model of multiple sclerosis, exogenous NSCs actively transferred mitochondria to mononuclear phagocytes and induced a significant amelioration of clinical deficits. Our data provide the first evidence that NSCs deliver functional mitochondria to target cells via EVs, paving the way for the development of novel (a)cellular approaches aimed at restoring mitochondrial dysfunction not only in multiple sclerosis, but also in degenerative neurological diseases.
Subject(s)
Extracellular Vesicles/metabolism , Mitochondria/metabolism , Neural Stem Cells/metabolism , Animals , Biological Transport , Cells, Cultured , Female , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Mesenchymal Stem Cells/metabolism , Mesenchymal Stem Cells/physiology , Mice , Mice, Inbred C57BL , Mice, Transgenic , Neural Stem Cells/ultrastructureABSTRACT
Moieties that compete with multivalent interactions or act as cofactors are common in living systems, but their effect on multivalent binding remains poorly understood. We derive a theoretical model that shows how the superselectivity of multivalent interactions is modulated by the presence of cofactors or competitors. We find that the role of these participating moieties can be fully captured by a simple rescaling of the affinity constant of the individual ligand-receptor bonds. Theoretical predictions are supported by experimental data of the membrane repair protein annexin A5 binding to anionic lipid membranes in the presence of Ca2+ cofactors and of the extracellular matrix polysaccharide hyaluronan (HA) binding to CD44 cell surface receptors in the presence of HA oligosaccharide competitors. The obtained findings should facilitate understanding of multivalent recognition in biological systems and open new routes for fine-tuning the selectivity of multivalent nanoprobes in medicinal chemistry.
Subject(s)
Hyaluronic Acid , Receptors, Cell Surface , Annexin A5 , Hyaluronic Acid/chemistry , Ligands , Lipids , Oligosaccharides , Receptors, Cell Surface/metabolismABSTRACT
OBJECTIVE: The lymphatic system is a circulatory system that unidirectionally drains the interstitial tissue fluid back to blood circulation. Although lymph is utilized by leukocytes for immune surveillance, it remains inaccessible to platelets and erythrocytes. Activated cells release submicron extracellular vesicles (EV) that transport molecules from the donor cell. In rheumatoid arthritis, EV accumulate in the joint where they can interact with numerous cellular lineages. However, whether EV can exit the inflamed tissue to recirculate is unknown. Here, we investigated whether vascular leakage that occurs during inflammation could favor EV access to the lymphatic system. Approach and Results: Using an in vivo model of autoimmune inflammatory arthritis, we show that there is an influx of platelet EV, but not EV from erythrocytes or leukocytes, in joint-draining lymph. In contrast to blood platelet EV, lymph platelet EV lacked mitochondrial organelles and failed to promote coagulation. Platelet EV influx in lymph was consistent with joint vascular leakage and implicated the fibrinogen receptor α2bß3 and platelet-derived serotonin. CONCLUSIONS: These findings show that platelets can disseminate their EV in fluid that is inaccessible to platelets and beyond the joint in this disease.
Subject(s)
Arthritis, Rheumatoid/physiopathology , Blood Platelets/physiology , Extracellular Vesicles/physiology , Lymph/physiology , Animals , Blood Platelets/metabolism , Capillary Permeability , Disease Models, Animal , Mice, Inbred C57BL , Serotonin/metabolismABSTRACT
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.
Subject(s)
Blood Specimen Collection/methods , Blood Specimen Collection/standards , Extracellular Vesicles/metabolism , Biomarkers/blood , Cardiovascular Diseases/blood , Cardiovascular Diseases/diagnosis , Exosomes/metabolism , Flow Cytometry/methods , HumansABSTRACT
Adeno-associated virus (AAV) is a safe and effective vector for gene therapy for retinal disorders. Gene therapy for hearing disorders is not as advanced, in part because gene delivery to sensory hair cells of the inner ear is inefficient. Although AAV transduces the inner hair cells of the mouse cochlea, outer hair cells remain refractory to transduction. Here, we demonstrate that a vector, exosome-associated AAV (exo-AAV), is a potent carrier of transgenes to all inner ear hair cells. Exo-AAV1-GFP is more efficient than conventional AAV1-GFP, both in mouse cochlear explants in vitro and with direct cochlear injection in vivo. Exo-AAV shows no toxicity in vivo, as assayed by tests of auditory and vestibular function. Finally, exo-AAV1 gene therapy partially rescues hearing in a mouse model of hereditary deafness (lipoma HMGIC fusion partner-like 5/tetraspan membrane protein of hair cell stereocilia [Lhfpl5/Tmhs-/-]). Exo-AAV is a powerful gene delivery system for hair cell research and may be useful for gene therapy for deafness.
Subject(s)
Dependovirus/genetics , Exosomes/metabolism , Gene Transfer Techniques , Genetic Vectors/genetics , Hair Cells, Auditory, Inner/metabolism , Hearing/genetics , Animals , Cells, Cultured , Dependovirus/classification , Evoked Potentials, Auditory, Brain Stem/genetics , Female , Gene Expression , Genes, Reporter , Genetic Therapy , Genetic Vectors/administration & dosage , Male , Mice , Mice, Knockout , Phenotype , Transduction, Genetic , TransgenesABSTRACT
Defect in membrane repair contributes to the development of limb girdle muscular dystrophy type 2B (LGMD2B) and Miyoshi myopathy. In healthy skeletal muscle, unraveling membrane repair mechanisms requires to establish an exhaustive list of the components of the resealing machinery. Here we show that human myotubes rendered deficient for Annexin-A5 (AnxA5) suffer from a severe defect in membrane resealing. This defect is rescued by the addition of recombinant AnxA5 while an AnxA5 mutant, which is unable to form 2D protein arrays, has no effect. Using correlative light and electron microscopy, we show that AnxA5 binds to the edges of the torn membrane, as early as a few seconds after sarcolemma injury, where it probably self-assembles into 2D arrays. In addition, we observed that membrane resealing is associated with the presence of a cluster of lipid vesicles at the wounded site. AnxA5 is present at the surface of these vesicles and may thus participate in plugging the cell membrane disruption. Finally, we show that AnxA5 behaves similarly in myotubes from a muscle cell line established from a patient suffering from LGMD2B, a myopathy due to dysferlin mutations, which indicates that trafficking of AnxA5 during sarcolemma damage is independent of the presence of dysferlin.
Subject(s)
Annexin A5/metabolism , Cell Membrane/metabolism , Muscle Fibers, Skeletal/metabolism , Muscle Fibers, Skeletal/pathology , Wound Healing , Adult , Annexin A5/ultrastructure , Cell Line , Dysferlin , Extracellular Space/metabolism , Humans , Lasers , Lipid Bilayers/metabolism , Membrane Proteins/deficiency , Membrane Proteins/metabolism , Muscle Fibers, Skeletal/ultrastructure , Muscle Proteins/deficiency , Muscle Proteins/metabolism , Muscular Dystrophies, Limb-Girdle/metabolism , Muscular Dystrophies, Limb-Girdle/pathology , Mutation/genetics , Myoblasts/metabolism , Myoblasts/pathology , Recombinant Proteins/metabolism , Sarcolemma/pathology , Subcellular Fractions/metabolismABSTRACT
Cells release membrane vesicles in their surrounding medium either constitutively or in response to activating signals. Two main types of extracellular vesicles (EVs) are commonly distinguished based on their mechanism of formation, membrane composition and size. According to the current model, EVs shed from the plasma membrane, often called microvesicles, expose phosphatidylserine (PS) and range in size from 100 nm to 1 µm, while EVs originating from endosomal multi-vesicular bodies, called exosomes, contain tetraspanin proteins, including CD63, and range in size from 50 to 100 nm. Heijnen et al. [1] have shown that activated platelets release EVs corresponding to these two types of vesicles, using negative staining electron microscopy (EM) and immuno-gold labeling. Here, we apply cryo-EM and immuno-gold labeling to provide a quantitative analysis of EVs released by platelets activated by thrombin, TRAP and CRP-XL, as well as EVs from serum. We show that EVs activated by these three agonists present a similar size distribution, the majority of them forming a broad peak extending from 50 nm to 1 µm, about 50% of them ranging from 50 to 400 nm. We show also that 60% of the EVs from TRAP or CRP-XL activation expose CD41, a majority of them exposing also PS. To explain the presence of large EVs CD41-negative or PS-negative, several alternative mechanisms of EV formation are proposed. We find also that the majority of EVs in activated platelet samples expose CD63, and distinguish two populations of CD63-positive EVs, namely large EVs with low labeling density and small EVs with high labeling density.
Subject(s)
Blood Platelets/metabolism , Cell-Derived Microparticles/metabolism , Cryoelectron Microscopy/methods , Exosomes/metabolism , Immunohistochemistry/methods , Staining and Labeling/methods , Biomarkers/metabolism , Blood Platelets/cytology , Blood Platelets/drug effects , Carrier Proteins/pharmacology , Cell-Derived Microparticles/chemistry , Cell-Derived Microparticles/classification , Exosomes/chemistry , Exosomes/classification , Humans , Particle Size , Peptides/pharmacology , Phosphatidylserines/metabolism , Platelet Activation/drug effects , Platelet Activation/physiology , Receptors, Thrombin/chemistry , Tetraspanin 30/metabolism , Tetraspanins/metabolism , Thrombin/pharmacologyABSTRACT
Annexin-A5 (AnxA5) is the smallest member of the annexins, a group of soluble proteins that bind to membranes containing negatively-charged phospholipids, principally phosphatidylserine, in a Ca(2+)-dependent manner. AnxA5 presents unique properties of binding and self-assembling on membrane surfaces, forming highly ordered two-dimensional (2D) arrays. We showed previously that AnxA5 plays a central role in the machinery of cell membrane repair of murine perivascular cells, promoting the resealing of membrane damages via the formation of 2D protein arrays at membrane disrupted sites and preventing the extension of membrane ruptures. As the placenta is one of the richest source of AnxA5 in humans, we investigated whether AnxA5 was involved in membrane repair in this organ. We addressed this question at the level of human trophoblasts, either mononucleated cytotrophoblasts or multinucleated syncytiotrophoblasts, in choriocarcinoma cells and primary trophoblasts. Using established procedure of laser irradiation and fluorescence microscopy, we observed that both human cytotrophoblasts and syncytiotrophoblasts repair efficiently a µm²-size disruption. Compared to wild-type cells, AnxA5-deficient trophoblasts exhibit severe defect of membrane repair. Through specifically binding to the disrupted site as early as a few seconds after membrane wounding, AnxA5 promotes membrane resealing of injured human trophoblasts. In addition, we observed that a large membrane area containing the disrupted site was released in the extracellular milieu. We propose mechanisms ensuring membrane resealing and subsequent lesion removal in human trophoblasts. This article is part of a Special Issue entitled: 13th European Symposium on Calcium.
Subject(s)
Annexin A5/metabolism , Cell Membrane/metabolism , Trophoblasts/metabolism , Annexin A5/genetics , Cell Line, Tumor , Cell Membrane/pathology , Female , Humans , Pregnancy , Trophoblasts/pathologyABSTRACT
Plasma contains cell-derived extracellular vesicles (EVs) which participate in various physiopathological processes and have potential biomedical applications. Despite intense research activity, knowledge on EVs is limited mainly due to the difficulty of isolating and characterizing sub-micrometer particles like EVs. We have recently reported that a simple flow cytometry (FCM) approach based on triggering the detection on a fluorescence signal enabled the detection of 50× more Annexin-A5 binding EVs (Anx5+ EVs) in plasma than the conventional FCM approach based on light scattering triggering. Here, we present the application of the fluorescence triggering approach to the enumeration and phenotyping of EVs from platelet free plasma (PFP), focusing on CD41+ and CD235a+ EVs, as well as their sub-populations which bind or do not bind Anx5. Higher EV concentrations were detected by fluorescence triggering as compared to light scattering triggering, namely 40× for Anx5+ EVs, 75× for CD41+ EVs, and 15× for CD235a+ EVs. We found that about 30% of Anx5+ EVs were of platelet origin while only 3% of them were of erythrocyte origin. In addition, a majority of EVs from platelet and erythrocyte origin do not expose PS, in contrast to the classical theory of EV formation. Furthermore, the same PFP samples were analyzed fresh and after freeze-thawing, showing that freeze-thawing processes induce an increase, of about 35%, in the amount of Anx5+ EVs, while the other EV phenotypes remain unchanged. The method of EV detection and phenotyping by fluorescence triggering is simple, sensitive and reliable. We foresee that its application to EV studies will improve our understanding on the formation mechanisms and functions of EVs in health and disease and help the development of EV-based biomarkers.
Subject(s)
Extracellular Vesicles/chemistry , Flow Cytometry/methods , Annexin A5/chemistry , Fluorescence , Fluorescent Dyes/chemistry , Humans , Kinetics , Limit of Detection , Phenotype , Platelet Membrane Glycoprotein IIb/chemistry , Staining and LabelingABSTRACT
Skin injury induces the cell surface exposure of phosphatidylserine (PS) on damaged and dying cells to activate coagulation and repair processes. Annexins can bind to PS and may modulate the healing response. Here, we determine the relevance of annexins for skin wound healing using AnxA1- and AnxA5-deficient mice and recombinant annexins with distinct PS binding properties. Wound inflammation, closure and the formation of granulation tissue were not altered in AnxA1- or AnxA5-deficient mice or after increasing AnxA5 serum concentrations (100 nM) in wild-type mice. Increased serum concentrations (1 µM) of AnxA5 induced massive bleeding, but wound hemostasis was not delayed by AnxA1. Both annexins interact with PS, but only AnxA5 can form 2-dimensional (2D) arrays on the cell surface. The injection of an AnxA5 mutant that binds to PS but lacks the ability of 2D array formation failed to induce bleeding. 2D lattice-forming AnxA4, with high affinity to PS also caused bleeding, while hemostasis was not affected by AnxA8 with low affinity or the AnxA8 mutant with medium affinity for PS and the lack of 2D formation. Increased concentrations of AnxA4 and AnxA5 also delayed coagulation pathway activation in vitro. This effect was attenuated for the AnxA5 mutant as well as for AnxA1 and AnxA8. In conclusion, endogenous AnxA1 and AnxA5 are dispensable for wound hemostasis and repair, but pharmacologically excessive concentrations of AnxA4 and AnxA5 inhibit hemostasis in skin wounds.
Subject(s)
Annexin A1/deficiency , Annexin A4/pharmacology , Annexin A5/pharmacology , Hemorrhage/drug therapy , Hemostasis/drug effects , Wound Healing/physiology , Animals , Annexin A1/genetics , Annexin A5/deficiency , Annexin A5/genetics , Flow Cytometry , Humans , Mice , Mice, Inbred C57BL , Mice, Knockout , Phosphatidylserines/metabolism , Prothrombin Time , Rats , Recombinant Proteins/pharmacology , Skin/injuriesABSTRACT
PURPOSE OF REVIEW: Platelet microparticles are small extracellular vesicles abundant in blood. The present review will introduce the mechanisms underlying the generation of microparticles, and will describe the diverse microparticle subtypes identified to date. The most appropriate methodologies used to distinguish microparticle subtypes will be also presented. RECENT FINDINGS: Both the megakaryocytes and platelets can generate microparticles. Circulating microparticles originating from megakaryocytes are distinguished from those derived from activated platelets by the presence of CD62P, LAMP-1, and immunoreceptor-based activation motif receptors. Close examination of platelet activation has shed light on a novel mechanism leading to microparticle production. Under physiologic flow, microparticles bud off from long membrane strands formed by activated platelets. Furthermore, mounting evidence supports the notion of microparticle heterogeneity. Platelet microparticles are commonly characterized by the expression of surface platelet antigens and phosphatidylserine. In fact, only a fraction of platelet microparticles harbor phosphatidylserine, and a distinct subset contains respiratory-competent mitochondria. During disease, the microparticle surface may undergo posttranslational modifications such as citrullination, further supporting the concept of microparticle diversity. SUMMARY: An appreciation of the microparticle heterogeneity will support their development as potential biomarkers and may reveal functions unique to each microparticle subtype in health and disease.
Subject(s)
Blood Platelets/cytology , Cell-Derived Microparticles , Cell-Derived Microparticles/metabolism , Flow Cytometry , Humans , Megakaryocytes/cytology , Mitochondria/physiology , Phosphatidylserines/metabolism , Platelet ActivationABSTRACT
The BclA protein is a major component of the outermost layer of spores of a number of bacterial species and Clostridium difficile carries three bclA genes. Using insertional mutagenesis each gene was characterized and spores devoid of these proteins had surface aberrations, reduced hydrophobicity and germinated faster than wild-type spores. Therefore the BclA proteins were likely major components of the spore surface and when absent impaired the protective shield effect of this outermost layer. Analysis of infection and colonization in mice and hamsters revealed that the 50% infectious dose (ID50 ) of spores was significantly higher (2-logs) in the bclA1(-) mutant compared to the isogenic wild-type control, but that levels of toxins (A and B) were indistinguishable from animals dosed with wild-type spores. bclA1(-) spores germinated faster than wild-type spores yet mice were less susceptible to infection suggesting that BclA1 must play a key role in the initial (i.e. pre-spore germination) stages of infection. We also show that the ID50 was higher in mice infected with R20291, a 'hypervirulent' 027 strain, that carries a truncated BclA1 protein.
Subject(s)
Bacterial Proteins/metabolism , Clostridioides difficile/pathogenicity , Enterocolitis, Pseudomembranous/metabolism , Spores, Bacterial/pathogenicity , Animals , Clostridioides difficile/metabolism , Cricetinae , Gene Expression Regulation, Bacterial , Mice , Spores, Bacterial/metabolismABSTRACT
The level of circulating platelet-, erythrocyte-, leucocyte- and endothelial-derived microparticles detected by high-sensitivity flow cytometry was investigated in 37 ß-thalassaemia major patients receiving a regular transfusion regimen. The phospholipid procoagulant potential of the circulating microparticles and the microparticle-dependent tissue factor activity were evaluated. A high level of circulating erythrocyte- and platelet-microparticles was found. In contrast, the number of endothelial microparticles was within the normal range. Platelet microparticles were significantly higher in splenectomized than in non-splenectomized patients, independent of platelet count (P < 0·001). Multivariate analysis indicated that phospholipid-dependent procoagulant activity was influenced by both splenectomy (P = 0·001) and platelet microparticle level (P < 0·001). Erythrocyte microparticles were not related to splenectomy, appear to be devoid of proper procoagulant activity and no relationship between their production and haemolysis, dyserythropoiesis or oxidative stress markers could be established. Intra-microparticle labelling with anti-HbF antibodies showed that they originate only partially (median of 28%) from thalassaemic erythropoiesis. In conclusion, when ß-thalassaemia major patients are intensively transfused, the procoagulant activity associated with thalassaemic erythrocyte microparticles is probably diluted by transfusions. In contrast, platelet microparticles, being both more elevated and more procoagulant, especially after splenectomy, may contribute to the residual thrombotic risk reported in splenectomized multi-transfused ß-thalassaemia major patients.
Subject(s)
Blood Platelets/physiology , Blood Transfusion , Cell-Derived Microparticles/physiology , Thrombophilia/blood , beta-Thalassemia/blood , Adolescent , Adult , Autoantibodies/blood , Autoantibodies/immunology , Blood Platelets/ultrastructure , Cell-Derived Microparticles/classification , Combined Modality Therapy , Diabetes Mellitus/etiology , Erythrocyte Membrane/ultrastructure , Female , Fetal Hemoglobin/immunology , Flow Cytometry , Humans , Hypogonadism/etiology , Iron/blood , Iron Overload/blood , Iron Overload/etiology , Male , Membrane Lipids/blood , Middle Aged , Oxidative Stress , Phosphatidylserines/blood , Risk , Splenectomy , Thrombophilia/etiology , Transfusion Reaction , Young Adult , beta-Thalassemia/complications , beta-Thalassemia/surgery , beta-Thalassemia/therapyABSTRACT
Free-standing lipid bilayers in nano- and micro-pores are interesting membrane models and attractive for biotechnological applications. We describe here the controlled preparation of proteo-lipid mono- and bilayers using the Langmuir-Schaefer transfer or Langmuir-Blodgett technique, respectively on hydrophobic and hydrophilic surfaces. We demonstrate the formation of suspended proteo-lipid layers by Transmission Electron Microscopy (TEM) and in situ Atomic Force Microscopy (AFM) imaging. Using Annexin-A5 as a membrane-associated protein, continuous proteo-lipid mono- and bilayers were formed, which span pore arrays over areas of several square-micrometers. The 2D organization of proteins associated to lipid monolayer is well preserved during the transfer process and the protein association is Ca(2+)-dependent and therefore reversible. The simple formation and reliable transfer of stabilized free-standing lipid films is a first crucial step to create biomimetic membranes for biotechnological applications and membrane protein research.
Subject(s)
Annexin A5/chemistry , Lipid Bilayers/chemistry , Hydrophobic and Hydrophilic Interactions , Microscopy, Atomic Force , Microscopy, Electron, TransmissionABSTRACT
Microvesicles (MVs) are a subtype of extracellular vesicles that can transfer biological information over long distances, affecting normal and pathological processes including skin wound healing. However, the diffusion of MVs into tissues can be impeded by the extracellular matrix (ECM). We investigated the diffusion of dermal wound myofibroblast-derived MVs into the ECM by using hydrogels composed of different ECM molecules such as fibrin, type III collagen and type I collagen that are present during the healing process. Fluorescent MVs mixed with hydrogels were employed to detect MV diffusion using fluorometric methods. Our results showed that MVs specifically bound type I collagen and diffused freely out of fibrin and type III collagen. Further analysis using flow cytometry and specific inhibitors revealed that MVs bind to type I collagen via the α2ß1 integrin. These data demonstrate that MV transport depends on the composition of the wound environment.
ABSTRACT
Spores of Clostridium difficile play a key role in the dissemination of this important human pathogen, and until recently little has been known of their functional characteristics. Genes encoding six spore coat proteins (cotA, cotB, cotCB, cotD, cotE, and sodA) were disrupted by ClosTron insertional mutagenesis. Mutation of one gene, cotA, presented a major structural defect in spore assembly, with a clear misassembly of the outermost layers of the spore coat. The CotA protein is most probably subject to posttranslational modification and could play a key role in stabilizing the spore coat. Surprisingly, mutation of the other spore coat genes did not affect the integrity of the spore, although for the cotD, cotE, and sodA mutants, enzyme activity was reduced or abolished. This could imply that these enzymatic proteins are located in the exosporium or alternatively that they are structurally redundant. Of the spore coat proteins predicted to carry enzymatic activity, three were confirmed to be enzymes using both in vivo and in vitro methods, the latter using recombinant expressed proteins. These were a manganese catalase, encoded by cotD, a superoxide dismutase (SOD), encoded by sodA, and a bifunctional enzyme with peroxiredoxin and chitinase activity, encoded by cotE. These enzymes being exposed on the spore surface would play a role in coat polymerization and detoxification of H2O2. Two additional proteins, CotF (a tyrosine-rich protein and potential substrate for SodA) and CotG (a putative manganese catalase) were shown to be located at the spore surface.
Subject(s)
Bacterial Proteins/metabolism , Clostridioides difficile/growth & development , Clostridioides difficile/metabolism , Spores, Bacterial/growth & development , Spores, Bacterial/metabolism , Bacterial Proteins/genetics , Clostridioides difficile/genetics , Enzymes/genetics , Enzymes/metabolism , Gene Knockout Techniques , Mutagenesis, Insertional , Spores, Bacterial/geneticsABSTRACT
Extracellular vesicles (EVs) are nanosized vesicles with a lipid bilayer that are released from cells of the cardiovascular system, and are considered important mediators of intercellular and extracellular communications. Two types of EVs of particular interest are exosomes and microvesicles, which have been identified in all tissue and body fluids and carry a variety of molecules including RNAs, proteins, and lipids. EVs have potential for use in the diagnosis and prognosis of cardiovascular diseases and as new therapeutic agents, particularly in the setting of myocardial infarction and heart failure. Despite their promise, technical challenges related to their small size make it challenging to accurately identify and characterize them, and to study EV-mediated processes. Here, we aim to provide the reader with an overview of the techniques and technologies available for the separation and characterization of EVs from different sources. Methods for determining the protein, RNA, and lipid content of EVs are discussed. The aim of this document is to provide guidance on critical methodological issues and highlight key points for consideration for the investigation of EVs in cardiovascular studies.
Subject(s)
Cardiovascular System , Cell-Derived Microparticles , Exosomes , Extracellular Vesicles , Myocardial Infarction , Humans , Exosomes/metabolism , Extracellular Vesicles/metabolism , Cell-Derived Microparticles/metabolism , RNA/metabolism , Myocardial Infarction/metabolismABSTRACT
A simple synthetic route is developed to achieve gold functionalized radial mesoporous silica nanoparticles (Au-MsNP) synthesized by a one step procedure fully compatible with basic conditions required for the preparation of monodispersed nanospheres. In a second step, Au-MsNP particles have been coated with phospholipid bilayers in order to design an advanced biofunctional platform with the gold metallic nanoparticles previously grown into the pore channels and responsible for a plasmonic activity relevant for biosensing. The size of Au-MsNP is checked by dynamic light scattering while zeta potential measurements reflect their surface charge. The particle morphology is characterized by transmission and scanning electron microscopy and the Si/Au ratios are obtained from energy dispersive X-ray analysis. The textural properties of Au-MsNP, specific surface area and pore size, are determined from N(2) adsorption. The supported bilayers are achieved from vesicles of different phospholipids incubated with Au-MsNP particles. The coating efficiency is investigated by zeta potential and cryo- transmission electron microscopy. The plasmonic activities of bare Au-MsNP particles and coated lipid bilayer Au-MsNP platform are evidenced for two model systems: direct adsorption of bovine serum albumin and molecular recognition events between avidin molecules and biotin receptors integrated in the supported lipid bilayer.
Subject(s)
Biosensing Techniques , Gold/chemistry , Lipid Bilayers/chemistry , Metal Nanoparticles/chemistry , Nanospheres/chemistry , Adsorption , Animals , Cattle , Cryoelectron Microscopy , Gold/metabolism , Lipid Bilayers/metabolism , Microscopy, Electron, Scanning , Microscopy, Electron, Transmission , Particle Size , Receptors, Growth Factor/chemistry , Receptors, Growth Factor/metabolism , Serum Albumin, Bovine/chemistry , Serum Albumin, Bovine/metabolism , Silicon Dioxide/chemistry , Silicon Dioxide/metabolism , Surface PropertiesABSTRACT
Uveal melanoma (UM) is the most common primary intraocular tumor and often spreads to the liver. Intercellular communication though extracellular vesicles (EVs) plays an important role in several oncogenic processes, including metastasis, therapeutic resistance, and immune escape. This study examines how EVs released by UM cells modify stellate and endothelial cells in the tumor microenvironment. The surface markers, and the concentration and size of EVs derived from UM cells or choroidal melanocytes were characterized by high-resolution flow cytometry, electron microscopy, and Western blotting. The selective biodistribution of EVs was studied in mice by fluorescence imaging. The activation/contractility of stellate cells and the tubular organization of endothelial cells after exposure to melanomic EVs were determined by traction force microscopy, collagen gel contraction, or endothelial tube formation assays. We showed that large EVs from UM cells and healthy melanocytes are heterogenous in size, as well as their expression of phosphatidylserine, tetraspanins, and Tsg101. Melanomic EVs mainly accumulated in the liver and lungs of mice. Hepatic stellate cells with internalized melanomic EVs had increased contractility, whereas EV-treated endothelial cells developed more capillary-like networks. Our study demonstrates that the transfer of EVs from UM cells leads to a pro-fibrotic and pro-angiogenic phenotype in hepatic stellate and endothelial cells.
Subject(s)
Extracellular Vesicles , Melanoma , Mice , Animals , Tumor Microenvironment , Endothelial Cells , Tissue Distribution , Extracellular Vesicles/metabolism , Melanoma/metabolismABSTRACT
Platelets are hyperactivated in coronavirus disease 2019 (COVID-19). However, the mechanisms promoting platelet activation by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are not well understood. This may be due to inherent challenges in discriminating the contribution of viral vs host components produced by infected cells. This is particularly true for enveloped viruses and extracellular vesicles (EVs), as they are concomitantly released during infection and share biophysical properties. To study this, we evaluated whether SARS-CoV-2 itself or components derived from SARS-CoV-2-infected human lung epithelial cells could activate isolated platelets from healthy donors. Activation was measured by the surface expression of P-selectin and the activated conformation of integrin αIIbß3, degranulation, aggregation under flow conditions, and the release of EVs. We find that neither SARS-CoV-2 nor purified spike activates platelets. In contrast, tissue factor (TF) produced by infected cells was highly potent at activating platelets. This required trace amounts of plasma containing the coagulation factors FX, FII, and FVII. Robust platelet activation involved thrombin and the activation of protease-activated receptor (PAR)-1 and -4 expressed by platelets. Virions and EVs were identified by electron microscopy. Through size-exclusion chromatography, TF activity was found to be associated with a virus or EVs, which were indistinguishable. Increased TF messenger RNA (mRNA) expression and activity were also found in lungs in a murine model of COVID-19 and plasma of severe COVID-19 patients, respectively. In summary, TF activity from SARS-CoV-2-infected cells activates thrombin, which signals to PARs on platelets. Blockade of molecules in this pathway may interfere with platelet activation and the coagulation characteristic of COVID-19.