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PubMed1976

Matrix vesicle heterogeneity: possible morphogenetic functions for matrix vesicles.

Extracellular membranous matrix vesicles were localized and described using electronmicroscopy during chondrogenesis, osteogenesis, and dentinogenesis. Evidence indicates that matrix vesicles in each of these specific tissue types function to concentrate and transport ions and enzymes which serve as nucleation sites for the mineralization of hydroxylapatite. We have examined different developmental stages of Meckel's cartilage, alveolar bone and epithelial-mesenchymal interactions associated with tooth formation in newborn mice. These ultrastructural studies indicate matrix vesicle heterogeneity. Whereas most matrix vesicles contain alkaline phosphatase activity during cartilage, bone and dentine mineralization, in earlier developmental stages matrix vesicles contain acid phosphatase activities and little, if any, alkaline phosphatase. Tissue type, specific developmental stage, and ultrastructural criteria indicate various "classes" of matrix vesicles. During epithelial-mesenchymal interactions in tooth development, mesenchymal cells (preodontoblasts) appear to be the source of matrix vesicles as indicated by the complementarity between H-2 histocompatibility alloantigen specificity on the cell surface and that of the matrix vesicle outer surface; matrix vesicles are limited by a trilaminar membrane derived from the mesenchymal cells. Some of the vesicles located adjacent to dividing inner enamel epithelial cells contain RNA's as determined by electron microscopic autoradiography in situ, as well as by direct biochemical assays. We postulate that matrix vesicles have many different and important biological functions, one of which may be to mediate developmental information from mesenchyme to epithelia during "instructive" stages of tooth development.

Animals↗

PubMed1991

Haemin-restriction influences haemin-binding, haemagglutination and protease activity of cells and extracellular membrane vesicles of Porphyromonas gingivalis W50.

Porphyromonas gingivalis strain W50 was grown in a chemostat either under haemin limitation or haemin excess at pH 7.3. Cells and the extracellular vesicle (ECV) and extracellular protein (EP) fractions were separated, quantified, and assayed for haemagglutination, protease activity and haemin binding. Under haemin-limitation, despite a reduction in cell yield, there was a 2.5-fold increase in the gravimetric yield of extracellular vesicles. Cells and vesicles from haemin-limited cultures, haemagglutinated sheep red blood cells to higher titres than their haemin-excess counterparts. Growth in haemin-excess conditions resulted in increased haemin-binding capacities of ECV, cells and EDTA-extracted outer membrane. Cells grown under haemin-excess showed a 2-fold elevation in specific activity towards the substrate N-alpha-benzoyl-L-arginine-p-nitroanilide (L-BAPNA) compared to haemin-limited cells. The specific activities against L-BAPNA for haemin-limited ECV were 3-fold greater than their haemin-excess counterparts. These vesicle activities represented 25% and 3% of the total culture protease activity under haemin limited and haemin excess conditions respectively.

Animals↗

PubMed2002

Extracellular membrane vesicles from tumor cells promote angiogenesis via sphingomyelin.

Actively growing tumor cells shed membrane vesicles into the extracellular milieu both in vivo and in vitro. Extracellular membrane vesicles from tumor cells contain most surface antigens and proteases present on these cells. They facilitate the escape of tumors from immune surveillance and promote tumor cell invasion. Here, we demonstrate that tumor membrane vesicles stimulate an additional important activity for tumor growth and metastasis by promoting endothelial cell migration, invasion, and tube formation, and inducing in vivo neovascularization. Our data show that tumor vesicles are one of the multiple effectors involved in tumor-induced angiogenesis. Heat-treated vesicles and lipid extracts from the vesicles also induce endothelial cell migration and in vivo angiogenesis. We identify sphingomyelin as the active component for vesicle-induced endothelial cell migration, tube formation, and neovascularization. Together with previously reported results, our data demonstrate that shed tumor vesicles play multiple roles in tumor growth and metastasis by promoting angiogenesis, tumor invasion, and immune escape.

Cell Division↗

PubMed1992

Isolation and purification of extracellular matrix vesicles from blood vessels.

Extracellular membrane-bound vesicles (called matrix vesicles) which occur in abundance in atherosclerotic blood vessels are believed to be associated with lipid accumulation and calcification. A technique has been developed to isolate them from experimental aneurysms in sheep in which they are known to be plentiful. The matrix vesicles were isolated by differential centrifugation following extraction by hypotonic salt solution. Most of the vesicles were pelleted at 30,000g and fell within the size range of matrix vesicles in situ in the aneurysmal wall. Preliminary characterization of the enzymatic activities indicates that many of these vesicles are formed from cell membranes rather than being derived from lysosomes, mitochondria or endoplasmic reticulum. Morphologically they are similar to matrix vesicles of other mineralizing tissues.

Animals↗

PubMed1983

Correlative morphometric and biochemical analysis of purified extracellular matrix vesicles from rat alveolar bone.

Extracellular matrix vesicles were isolated from rat alveolar bone and purified by either gel filtration or discontinuous sucrose density gradient. Morphometric evaluation of electron-micrographs of pellets of purified and nonpurified vesicle fractions was correlated with the activity of vesicular enzymes. A high correlation was found between the percentage of area occupied by vesicles with electron-dense content (electron-dense vesicle fractional area) and the enzymatic activity. Highest enzymatic specific activities and electron-dense vesicle fractional area were recorded in the "light" vesicle-enriched fraction obtained after equilibrium density centrifugation. These parameters revealed lowest values in the "heavy" vesicle-enriched fraction resolved by the same methods. The combined electron-dense and electron-lucent fractional area (vesicular fractional area) was similar in the different purified fractions. It is therefore suggested that the fraction obtained by gel filtration contains both "light" and "heavy" vesicles. Morphometric study is proposed as an additional criterion for the degree of purification of matrix vesicle preparations.

Acid Phosphatase↗

PubMed1987

Trypsin-like enzyme activity of the extracellular membrane vesicles of Bacteroides gingivalis W50.

Trypsin-like enzyme activity in spent culture media from 3-d-old batch cultures of Bacteroides gingivalis W50 was measured by using the hydrolysis of N alpha-benzoyl-L-arginine-p-nitroanilide. The cell-free culture medium was fractionated by differential centrifugation at 10,000 g and 75,000 g, yielding two particulate fractions and a soluble supernatant fraction. About 80% of the total recoverable activity was associated with the particulate fractions, the remainder being in the supernatant. Electron microscopy of ruthenium-red/osmium stained ultrathin sections of the pellet fractions showed them to be composed of vesicular particles (extracellular vesicles), between 50 and 250 nm in diameter. Enzyme activity in all three fractions was enhanced by dithiothreitol. Gel-permeation chromatography of the soluble fraction yielded one peak of activity which contained 64 kDa and 58 kDa polypeptides. Enzyme activity from the vesicular fractions could be solubilized by sonication, giving a similar chromatographic profile to the supernatant fraction. The main peak of activity was composed of 64 kDa and 58 kDa polypeptides. In addition, there was a higher molecular mass enzyme activity peak composed of the 64 kDa and 58 kDa components along with 111 kDa, 93 kDa and 70 kDa polypeptides. We conclude that the trypsin-like enzyme of B. gingivalis is released as a soluble protein and is also associated with extracellular vesicles, in which it may exist as a soluble component and also as a protein complex.

Bacteroides↗

PubMed2025

Rapid screening and identification of genes involved in bacterial extracellular membrane vesicle production using a curvature-sensing peptide.

Bacteria secrete extracellular membrane vesicles (EMVs). Physiological functions and biotechnological applications of these lipid nanoparticles have been attracting significant attention. However, the details of the molecular basis of EMV biogenesis have not yet been fully elucidated. In our previous work, an N-terminus-substituted FAAV peptide labeled with nitrobenzoxadiazole (NBD; nFAAV5-NBD) was developed. This peptide can sense the curvature of a lipid bilayer and selectively bind to EMVs even in the presence of cells. Here, we applied nFAAV5-NBD to a genome-wide screening of hyper- and hypo-vesiculation transposon mutants of a Gram-negative bacterium, Shewanella vesiculosa HM13, to identify the genes involved in EMV production. We analyzed the transposon insertion sites in hyper- and hypo-vesiculation mutants and identified 16 and six genes, respectively, with a transposon inserted within or near them. Targeted gene-disrupted mutants of the identified genes showed that the lack of putative dipeptidyl carboxypeptidase, glutamate synthase β-subunit, LapG protease, metallohydrolase, RNA polymerase sigma-54 factor, inactive transglutaminase, PepSY domain-containing protein, and Rhs-family protein caused EMV overproduction. On the other hand, disruption of the genes encoding putative phosphoenolpyruvate synthase, d-hexose-6-phosphate epimerase, NAD-specific glutamate dehydrogenase, and sensory box histidine kinase/response regulator decreased EMV production. This study demonstrates the utility of a novel screening method using a curvature-sensing peptide for mutants with altered EMV productivity and provides information on the genes related to EMV production.IMPORTANCEConventional methods for isolation and quantification of extracellular membrane vesicles (EMVs) are generally time-consuming. nFAAV5-NBD can detect EMVs in the culture without separating EMVs from cells. In situ detection of EMVs using this peptide facilitated screening of the genes related to EMV production. We succeeded in identifying various genes associated with EMV production of Shewanella vesiculosa HM13, which would contribute to the elucidation of bacterial EMV formation mechanisms. Additionally, the hyper-vesiculating mutants obtained in this study would be valuable for EMV applications, such as secreting useful substances as EMV cargoes and producing artificially functionalized EMVs.

Shewanella↗

PubMed1995

Immunomodulatory effects of extracellular secretory vesicles isolated from bovine semen.

We demonstrate that extracellular secretory vesicles isolated from bovine seminal plasma have immunomodulatory properties. They inhibit mitogen induced proliferation of bovine and human peripheral blood lymphocytes in a dose dependent fashion. They also inhibit phagocytosis of latex particles by bovine neutrophils. Phagocytosis of opsonised Staphylococcus aureus however was not affected. Furthermore phorbol ester and chemotactic peptide induced superoxide production was decreased especially when a suboptimal dose of stimulants was used. We suggest that extracellular secretory vesicles may preserve sperm survival in the female reproductive tract.

Animals↗

PubMed2026

Extracellular vesicles-mediated delivery of SpCas9 RNPs for therapeutic gene editing in Spinocerebellar Ataxia Type 3.

Spinocerebellar Ataxia Type 3 (SCA3) is a neurodegenerative dominantly-inherited disorder caused by an overexpansion of a CAG tract within the ATXN3 gene, conferring toxic properties to the ataxin-3 protein. Genome editing with CRISPR-Cas9 enzymes is a promising strategy to inactivate mutant ATXN3 alleles, however, in vivo delivery remains challenging. Extracellular vesicles (EVs) are promising delivery vehicles for Cas9 and single guide RNA (sgRNA) ribonucleoproteins that minimize genomic exposure to highly active endonucleases. In this study, we designed SpCas9 with a palmitoylation motif that enables SpCas9 and sgRNA enrichment into EVs. Introduction of a photocleavable linker - PhoCl - allowed the photo-inducible release of SpCas9 from the palmitoylation motif in EVs, increasing target engagement to ATXN3 in vitro. EVs loaded with SpCas9 ribonucleoproteins resulted in ATXN3 knockout in SCA3 patient-derived iPSCs and two SCA3 animal models. These findings highlight an innovative route for transient delivery of gene editing tools. This approach provides a promising therapeutic platform for the treatment of genetic diseases, including SCA3.

Humans↗

PubMed2026

Mast Cells Selectively Deliver Extracellular Vesicle-Encapsulated mRNA to Colorectal Cancer Cells.

Mast cells (MCs), a type of granulocytic immune cell, exert contrasting effects on tumorigenesis. The anti- or pro-tumorigenic activity of MCs depends on the cancer type, tumor microenvironment, and MC localization within the tumor. Consequently, their role remains controversial and poorly understood across multiple cancer types, including colorectal cancer (CRC). Most proposed mechanisms underlying MC activity in CRC have focused on MC secretion of biological factors. In this study, we demonstrated that MCs transfer extracellular vesicles containing mRNAs and proteins to CRC cells. This process occurs through a tightly regulated mechanism that requires direct cell-cell contact, calcium signaling, and integrin-mediated interactions. Such requirements resemble aspects of immunological synapses observed between lymphocytes and cancer cells. The novel mode of intercellular communication between MCs and cancer cells described here may help refine our understanding of MC functions in cancer biology.

Mast Cells↗

PubMed2004

Exosomes: endosomal-derived vesicles shipping extracellular messages.

Exosomes are membrane vesicles released into the extracellular environment upon exocytic fusion of multivesicular endosomes with the cell surface. They have a particular composition reflecting their origin in endosomes as intraluminal vesicles. In vitro and in vivo studies support the contribution of exosomes to an acellular mode of communication, leading to intercellular transfer of molecules. Exosomes may have regulatory functions in the immune system and their application in cancer immunotherapy is promising. The mechanisms involved in exosome secretion and interaction with target cells are as yet unclear. A better understanding of these mechanisms is also essential to determine the link between exosomes and retroviruses.

Animals↗

PubMed1979

Ultrastructural and biochemical study of extracellular matrix vesicles in normal alveolar bone of rats.

The occurrence of vesicles in the extracellular matrix of alveolar bone of normal young rats was demonstrated by both ultrastructural and enzymatic studies. Transmission electron microscopy revealed abundant vesicles in the matrix. The presence of hydroxyapatite crystals, both within the vesicles and in the matrix, was affiliated with rupture of the vesicular membrane. Calcifying nodules were scarce. High levels of both specific and total activities of alkaline- and pyrophosphatases were found in the fraction of isolated vesicles. This fraction also showed activities of different ATPases and acid phosphatase.

Acid Phosphatase↗

PubMed1989

Production of cell-bound and vesicle-associated trypsin-like protease, alkaline phosphatase and N-acetyl-beta-glucosaminidase by Bacteroides gingivalis strain W50.

Bacteroides gingivalis strain W50 was grown in batch and continuous culture on complex medium with haemin. In batch culture, cell-bound levels of trypsin-like protease (EC 3.4.21.4), alkaline phosphatase (EC 3.1.3.1) and N-acetyl-beta-glucosaminidase (EC 3.2.1.30) increased during the exponential phase of growth. These enzyme activities were also detected in extracellular vesicles and in extracellular soluble forms in the supernatant fluid, but in lower amounts per unit biomass compared to cell-bound levels. In continuous culture, at high relative growth rates (0.7-0.9 murel), the highest proportions of enzyme activities were cell-bound. In contrast, at low relative growth rates (0.1-0.2 murel), highest enzyme levels were detected in the extracellular vesicle fraction. Levels of extracellular soluble enzymes were always low compared to cell-bound or extracellular vesicle levels, but were highest at low relative growth rates. All three enzymes appeared to be relatively stable in their soluble forms. Vesicle production appeared to be associated with actively growing cells but was influenced by growth rate. The results are consistent with the hypothesis that cell-bound 'periplasmic' enzymes are encapsulated into vesicles which are subsequently released by the cells. Therefore, levels of total extracellular enzyme (extracellular vesicle plus extracellular soluble) may depend on the rate of vesicle formation superimposed on the rates of production of 'periplasmic' enzymes in the cell.

Acetylglucosaminidase↗

PubMed1991

Extracellular vesicle-associated and soluble trypsin-like enzyme fractions of Porphyromonas gingivalis W50.

Soluble vesicle-associated trypsin-like enzyme fractions (VSF) were prepared by sonication from extracellular vesicles (ECV) from strains W50 and W50/BE1. High-(H), intermediate-(I) and low-(L) molecular-weight VSF enzyme subfractions were identified by non-dissociative gel filtration chromatography with Mr 160, 95 and 60 kDa respectively. The chromatographic profiles of W50 VSF from 48-h and 72-h cultures were identical. W50/BE1 VSF displayed a higher ratio of the 160 to 60 kDa components. This ratio was reduced in VSF from 72-h cultures. Extracellular soluble protein (EP) trypsin profiles were similar to their respective VSF, but the 60 kDa component predominated. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) revealed a loss of soluble extracellular polypeptides with culture age. A polyclonal antiserum to EP subfraction L reacted in immunoblots with a 50 kDa peptide of subfraction L of W50. Whole EP and its subfraction H displayed a 50 kDa immunoreactive peptide but no peptides of higher molecular weight. This antiserum reacted with a similar sized peptide, and with lower-molecular-weight components in whole ECV. Gelatin substrate zymography of whole EP following non-reducing SDS-PAGE revealed a major 80 kDa protease that increased with culture age. Minor protease bands of 70 and 50 kDa were also detected.

Bacterial Outer Membrane Proteins↗

PubMed1990

Stability of soluble and extracellular vesicle-associated trypsin-like protease (TLP) activity of Bacteroides gingivalis W50.

Comparison was made of the specific activities of whole extracellular soluble protein (EP) and extracellular vesicle (ECV)-associated trypsin-like protease (TLP) activity from batch cultures of Bacteroides gingivalis W50. Rapid loss of activity occurred when these fractions were maintained at 37 degrees C in the presence of DTT. Residual levels of activity were detected after incubation of ECV and EP for up to 8 days under non-reducing conditions. Rates of activity loss in EP and ECV were similar. Mixtures of EP and ECV, in the same proportions as found in the culture supernatant showed neither depression nor elevation of total activity from the expected compound activities of the two separate fractions.

Bacteroides↗

PubMed1982

Occurrence of actin-like protein in extracellular matrix vesicles.

Preliminary indications of the occurrence of actin and myosin in crude matrix vesicle preparations have been reported previously. In the present study extracellular matrix vesicles from rat alveolar bone were isolated. They were further purified by a sucrose density gradient. SDS-polyacrylamide gel electrophoresis of the purified vesicles revealed the presence of a polypeptide with a molecular weight of 43 K daltons and with electrophoretic mobility identical to that of blood platelet actin. The limited proteolysis of both 43 K dalton vesicular polypeptide and actin by Staphylococcus aureus-V8-protease revealed three fragments with identical electrophoretic mobility. In addition, the vesicular preparations inhibited the activity of DNase I, a property typical of actin monomers. Filamentous material extracted from matrix vesicles showed ultrastructuraL features of F-actin. Reaction of this material with heavy meromyosin resulted in arrowhead formation, which is characteristic of acto-heavy meromyosin. The occurrence of actin in extracellular matrix vesicles may account for their budding from the osteoblastic plasma membrane, their possible motility in the matrix, and maintenance of the spherical shape.

Actins↗

PubMed1988

The distribution of extracellular matrix vesicles in healing of rat tibial bone three days after intramedullary injury.

The distribution of extracellular matrix vesicles on the third day of bone healing was studied by morphometric analysis of transmission electron micrographs. Detection and grouping of the vesicles was performed according to type, diameter, and distance from the calcified front. The different types were selected as follows: vesicles with electron-lucent contents ("empty"), vesicles with amorphous electron-opaque contents ("amorphic"), vesicles containing crystalline depositions ("crystal"), and vesicles containing crystalline structures with ruptured membranes ("rupture"). The majority of vesicles were between 0.07 micron and 0.12 micron in diameter and were located at less than 3 micron from the calcified front. The distribution of the "empty", "amorphic", "crystal", and "rupture" vesicles was 23.2%, 74%, 2.5%, and 0.3% respectively. Their sequence of arrangement according to diameter was as follows: "empty", "amorphic", "crystal", and "rupture", the empty vesicles constituting the smallest and the "rupture" the largest type. Distances from the calcified front were similar for the "empty", "amorphic", and "crystal" vesicles, while the "rupture" type was located nearest to the front. The present observations support the widely acknowledged hypothesis on the role of extracellular matrix vesicles in mineralization. It is thought that the secretion of "empty" vesicles from the cell is followed by intravascular accumulation of amorphous Ca and Pi to form a hydroxyapatite crystal that, in turn, ruptures the vesicle's membrane.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1987

Ultrastructural tissue morphometry of the distribution of extracellular matrix vesicles in remodeling rat tibial bone six days after injury.

A study of the distribution of extracellular matrix vesicles on the 6th day of bone healing was performed by methods of transmission electron microscopy combined with computerized morphometry. The vesicles were detected on the electron micrographs and grouped according to their diameters, distance from the calcified front and type. The different types were selected as follows: vesicles with electron-lucent contents, i.e., 'empty'; vesicles with amorphous electron-opaque contents, i.e., 'amorphous'; vesicles containing crystalline depositions, i.e., 'crystal', and vesicles containing crystalline structures with ruptured membranes, i.e., 'rupture'. Most of the vesicles were concentrated between diameters of 0.02 and 0.22 microns. Most of the vesicles were found within a distance of less than 3 micron from the calcified front. The vesicles were distributed according to their types: 'empty', 'amorphous', 'crystal' and 'rupture' in 14, 39, 34 and 13%, respectively. The diameters of the 'crystal' and 'rupture' vesicles were significantly larger than those of the 'empty' and 'amorphous' types. The sequence of distances from the calcified front was recorded as follows: 'rupture', 'crystal', 'amorphous' and 'empty', the 'rupture' type being the closest to the front. The results of the present study confirm the accepted hypothesis on calcification via extracellular matrix vesicles. It is thought that the cell secretes 'empty' vesicles that accumulate amorphous Ca and P to form a hydroxy-apatite crystal. This is followed by rupture of the vesicular membrane. The propagation of the process is accompanied by increase in the vesicular diameter and its approximation to the calcifying front.

Animals↗