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PubMed1988

The degradation of type I collagen and human plasma fibronectin by the trypsin-like enzyme and extracellular membrane vesicles of Bacteroides gingivalis W50.

A soluble trypsin-like enzyme (STE) was purified from a cell- and particle-free culture supernatant of this bacterium by a combination of ultra-centrifugation, ammonium-sulphate precipitation and gel-filtration chromatography on Sephacryl S-200. Trypsin-like activity in the culture supernatant was associated with a 58 kDa peptide and also with a higher molecular-weight complex. The STE and extracellular vesicle (ECV) fraction of B. gingivalis W50 rapidly degraded human plasma fibronectin in the presence and the absence of 10 mM dithiothreitol (DTT). The STE yielded a range of lower molecular-weight fibronectin digestion products. Under conditions where little activity was expressed by mammalian trypsin, both STE and ECV depolymerized a denatured and a native type I collagen substrate. Quantitative and qualitative differences were observed in the patterns of digestion products generated by both STE and ECV fraction following incubation with and without 10mM DTT. Inclusion of DTT appeared to reduce the degradative effect of both ECV and STE towards the type I collagen and plasma fibronectin substrates.

Bacterial Outer Membrane Proteins↗

PubMed1987

Changes in the distribution of extracellular matrix vesicles during healing of rat tibial bone (computerized morphometry and electron microscopy).

A study of the distribution of extracellular matrix vesicles during the first 3 weeks of healing of adult rat tibial bone was performed by transmission electron microscopy in combination with computerized morphometry. Bone injury comprised removal of the marrow followed by regeneration of the tissue via a phase of primary mineralization. A total number of 39,498 vesicles were traced on electron micrographs and sorted according to their diameters, distance from the calcified front and types. The different vesicular types were defined as follows: (a) vesicles with electron lucent contents, i.e. "empty", (b) vesicles with amorphous electron opaque contents, i.e. "amorphic", (c) vesicles containing crystalline depositions, i.e. "crystal", and (d) vesicles containing crystalline structures with ruptured membranes i.e. "rupture". The vesicles were studied on the days 3, 6, 14 and 21 after bone injury. Most of the vesicles were concentrated between diameters of 0.07 and 0.17 micron. Most of the vesicles were found in a distance less than 3 microns from the calcified front. The sequence of changes of distances from the calcified front and of the vesicular diameters were recorded as follows: "rupture", "crystal", "amorphic" and "empty", the "rupture" type being the closet to the front and of the largest diameter in each day. 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 Ca and Pi forming amorphous calcium phosphate that is then converted to hydroxyapatite. This is followed by rupture of the vesicular membrane.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1996

Chondrocyte cultures express matrix metalloproteinase mRNA and immunoreactive protein; stromelysin-1 and 72 kDa gelatinase are localized in extracellular matrix vesicles.

Previous studies have shown that costochondral cartilage cell cultures produce extracellular matrix vesicles which contain metalloproteinase activity. In the present study, we examined whether two matrix metalloproteinases (MMPs) known to be present in cartilage, stromelysin-1 and 72 kDa gelatinase, are expressed by fourth passage resting zone and growth zone costochondral chondrocytes and whether they are specifically incorporated into matrix vesicles produced by the cells. We also examined whether the cells synthesize tissue inhibitor of metalloproteinase-1 and -2 (TIMP-1 and TIMP-2). Oligonucleotide primers for stromelysin-1, 72 kDa gelatinase, tissue inhibitor of metalloproteinases-1 and -2 (TIMP-1 and TIMP-2), and GAPDH were synthesized and optimized for use in the reverse transcription-polymerase chain reaction (RT-PCR). It was found that both resting zone and growth zone chondrocytes produced mRNA for both MMPs and the two TIMPs. Further, immunostaining of cell layers with antibodies to 72 kDa gelatinase and stromelysin-1 showed that both cell types produced these MMPs in culture. Substrate gel electrophoresis and Western analysis were used to characterize MMP activity in matrix vesicles, media vesicles, or plasma membranes as well as in conditioned media produced by the chondrocyte cultures. It was found that matrix vesicles but not plasma membranes or media vesicles were selectively enriched in stromelysin-1. Also, 72 kDa gelatinase was found in matrix vesicles, but to a lesser extent than seen in media vesicles. The relative activity of each enzyme detected was cell maturation-dependent. No MMP activity was detected in conditioned media produced by either cell type. The results of this study show that MMPs are expressed by resting zone and growth zone chondrocytes in culture and differentially distributed among three different membrane compartments. This suggests that, in addition to the well-known activators and inhibitors of MMP activity in the matrix, differential membrane distribution may enable more precise control over the site, rate, and extent of matrix degradation by the cell.

Animals↗

PubMed1989

The distribution of trypsin-like enzyme activity in cultures of a virulent and an avirulent strain of Bacteroides gingivalis W50.

The distribution of trypsin-like enzyme activity was studied in 48- and 72-h batch cultures of Bacteroides gingivalis W50 and an avirulent variant (W50/BE1) of the parent strain. Activity was measured at pH 7.4 in cells, the extracellular vesicle (ECV) and soluble extracellular protein (EP) fractions recovered by ammonium sulphate precipitation from spent growth medium. Both organisms produced cell surface and extracellular vesicles, but whilst strain W50 produced more ECV, W50/BE1 yielded more of the EP fraction by weight. Whole cultures of W50 displayed a three-fold greater trypsin activity than those of W50/BE1. However, 90% of the total enzyme activity of W50 cultures was associated with the particulate fraction (cells and ECV totalled), whereas this fraction accounted for only 10-30% of the total for W50/BE1. Unlike W50/BE1, the specific activities of W50 cells and ECV rose in 72-h cultures. Conversely, cultures of W50/BE1 displayed an increase in the yield and specific activity of the EP fraction.

Bacteroides↗

PubMed1987

Extracellular matrix vesicle distribution in primary mineralization two weeks after injury to rat tibial bone (ultrastructural tissue morphometry).

Primary mineralization on the 14th day of bone healing served as a model to study the distribution of extracellular matrix vesicles by means of transmission electron microscopy combined with computerized morphometry. Vesicles were traced on electron micrographs and classified according to diameter, distance from the calcified front, and type. The different types were determined as follows: electron-lucent vesicles ("empty"), vesicles with amorphous contents ("amorphic"), vesicles containing crystalline depositions ("crystal"), and vesicles with crystals and ruptured membranes ("rupture"). The majority of the vesicles measured between 0.02 and 0.07 micron and were located at a distance of less than 3 micron from the calcified front. They were distributed according to "empty", "amorphic", "crystal" and "rupture" type in concentrations of 10%, 31%, 51% and 8%, respectively. The diameters of the "rupture" vesicles were significantly larger than those of the "empty" and "amorphic" types. The sequence of their location, starting at the calcified front, ran as follows: "rupture", "crystal", "amorphic" and "empty", with the "rupture" type proximate to the front. According to the working hypothesis on calcification via extracellular matrix vesicles, it is thought that the "empty" vesicles are secreted by the cell and that subsequently amorphous Ca and Pi accumulate intravesicularly to form a hydroxyapatite crystal which, in turn, brings about rupture of the vesicle's membrane. The results of the present study support this theory and, additionally, show that the maturation process is accompanied by an increase of the vesicular diameter and by its approximation to the calcifying front.

Animals↗

PubMed1975

Calcification of rachitic rat cartilage in vitro by extracellular matrix vesicles.

Growth plate cartilage from rachitic rats was studied to assess the role in calcification of extracellular matrix vesicles, which are thought to participate in the initial stage of mineralization of connective tissue. The concentration of matrix vesicles and their distribution within the longitudinal septa was found to be normal in rats made rachitic by feeding by a diet low in vitamin D and phosphate for 3 weeks after weaning. Rachitic cartilage matrix did not contain circumvesicular clusters of apatite as does normal cartilage; however, occasional vesicles did enclose one or a few apatite needles. When slices of rachitic cartilage were incubated at 37 C in a metastable calcium phosphate solution ([Ca++] times [PO SEE ARTICLE] equals 3.5 mM identical to 2), apatite formation was initiated in association with matrix vesicles. Under these conditions, mineralization was prominent in the upper hypertrophic cartilage, where matrix vesicles became encrusted with apatite after only 2 to 3 hours of incubation. Vesicular apatite accumulation was inhibited by preheating the cartilage to 60 C for 30 minutes. Measurements of 45Ca uptake by rachitic cartilage slices from metastable calcium phosphates solution also indicated inhibition of calcification by heat. Light microscopic autoradiographs showed 45Ca localization primarily in the matrix of longitudinal septa and substantiated the inhibition site of mineralization in healing rachitic cartilage. The presence of apatite within rachitic vesicles prior to heating and the inhibition of vesicle calcification by heat suggests an active, enzymatically and mediated mechanism of vesicular calcification.

Animals↗

PubMed1988

The relationship between extracellular matrix vesicles and the calcifying front on the 21st day after injury to rat tibial bone.

The relationship between extracellular matrix vesicles and the calcifying fronts was examined by studying vesicular diameters and types. Transmission electron microscopy combined with computerized morphometry three weeks after injury to the tibial bone in rats was used. The different vesicle types were defined as: (1) vesicles with electron lucent contents referred to as empty; (2) vesicles with amorphous electron opaque contents, called amorphic; (3) vesicles containing crystalline depositions, called crystalline; and (4) vesicles containing crystalline structures with ruptured membranes, referred to as ruptured. The diameters of most vesicles ranged between 0.07 and 0.17 micron. More than 95% of the vesicles were located less than 2 micron from the calcified front. The vesicles were distributed among the categories as follows: empty, 9.6%; amorphic, 19.3%; crystal, 39.2%; and ruptured, 31.9%, respectively. The diameters of the crystalline and ruptured vesicles were significantly larger than those of the empty and amorphic types. The ruptured type had the largest diameters. The sequence of distances from the calcified front was recorded as follows: ruptured, crystalline, amorphic, and empty, with the ruptured and crystalline types being the closest to the front. This study supports the accepted theory on matrix vesicle mineralization. The cell is responsible for secretion of empty vesicles that accumulate amorphous Ca and Pi to form a hydroxyapatite crystal. This is followed by rupture of the vesicular membrane. The propagation of the process is accompanied by an increase in the vesicular diameter and its approximation to the calcifying front.

Animals↗

PubMed2007

Analysis of the extracellular matrix vesicle proteome in mineralizing osteoblasts.

Many key processes central to bone formation and homeostasis require the involvement of osteoblasts, cells responsible for accumulation and mineralization of the extracellular matrix (ECM). During this complex and only partially understood process, osteoblasts generate and secrete matrix vesicles (MVs) into the ECM to initiate mineralization. Although they are considered an important component of mineralization process, MVs still remain a mystery. To better understand their function and biogenesis, a proteomic analysis of MVs has been conducted. MVs were harvested by two sample preparation approaches and mass spectrometry was utilized for protein identification. A total of 133 proteins were identified in common from the two MV preparations, among which were previously known proteins, such as annexins and peptidases, along with many novel proteins including a variety of enzymes, osteoblast-specific factors, ion channels, and signal transduction molecules, such as 14-3-3 family members and Rab-related proteins. To compare the proteome of MV with that of the ECM we conducted a large-scale proteomic analysis of collagenase digested mineralizing osteoblast matrix. This analysis resulted in the identification of 1,327 unique proteins. A comparison of the proteins identified from the two MV preparations with the ECM analysis revealed 83 unique, non-redundant proteins identified in all three samples. This investigation represents the first systematic proteomic analysis of MVs and provides insights into both the function and origin of these important mineralization-regulating vesicles.

Animals↗

PubMed1991

Expression of a negative regulator of human erythropoiesis by fluidized lymphocyte plasma membranes.

Regulation of hematopoiesis by paracrine molecules occurs in vitro. In some cases, hematopoietic paracrine factors have been localized to the plasma membrane of accessory cells. We have purified a unique integral membrane glycoprotein from normal human B cells that functions in vitro as a paracrine factor whose activity is directed toward erythroid progenitor cells. This factor is also spontaneously exfoliated from the cell surface as a component of extracellular vesicles. Analysis of the lipid and protein compositions and membrane lipid order of these extracellular vesicles reveals them to be biochemically distinct and more fluid than their parent membranes. Evidence in nonhematopoietic culture systems indicates that cell membrane function may be altered by modifying membrane fluidity. In an effort to accelerate growth factor release, plasma membranes of B cells were fluidized by incubation with an emulsion of Liposin II, phosphatidylcholine, and phosphatidylethanolamine. Fluidity assessed by steady-state fluorescence polarization was reduced in lipid-treated cells. Exfoliation was 3-4-fold higher from lipid-treated cells relative to untreated cells. Unexpectedly, a negative signal for burst-forming unit-erythroid (BFU-E) proliferation was expressed in membranes, in shed extracellular vesicles, and in supernatants of medium conditioned by the fluidized cells. Purification of the inhibitor is under way. The data are consistent with the hypothesis that accessory cell plasma membranes may positively or negatively regulate erythroid differentiation, depending upon the exchange of cholesterol and phospholipids between plasma membrane and ambient lipid pools.

B-Lymphocytes↗

PubMed1989

Changes in extracellular matrix vesicles during healing of rat tibial bone: a morphometric and biochemical study.

Primary mineral formation in woven bone has been associated with the production of extracellular matrix vesicles. Previous studies have demonstrated an increase in phospholipid: Ca:Pi complexes (CPLX) immediately prior to hydroxyapatite formation. Since matrix vesicles are enriched in phosphatidylserine and PS is the major phospholipid in CPLX, the present study examined whether the morphologic appearance of matrix vesicles and initial formation of crystals within them could be correlated to changes in their phospholipid composition and metabolism. Ablation of the tibial marrow in rats was used as the model since this procedure induces endosteal repair with primary mineralization. The morphologic appearance of the matrix vesicles was assessed by morphometric analysis at the electron microscopic level. Matrix vesicles were divided into 4 categories: empty, amorphic, crystal, and rupture. There was time dependent decrease in the number of empty and amorphic matrix vesicles which correlated with an increase in crystal and rupture type. Distance from the calcification front decreased as more rupture-type vesicles were noted. In a parallel set of experiments, matrix vesicle-enriched membranes (MVEM) were isolated from homogenates of endosteal tissue removed from the treated tibia as well as from the contralateral control. There was an increase at 6 days in MVEM alkaline phosphatase and phospholipase A2 specific activities in both limbs, the magnitude of response being significantly greater in the treated legs. The phospholipid composition of the MVEM changed with time. SPH was highest at day 3, PS was detectable only in day 6 and 14 samples, and PC exhibited a time dependent decrease.(ABSTRACT TRUNCATED AT 250 WORDS)

Alkaline Phosphatase↗

PubMed1988

Structurally distinct plasma membrane regions give rise to extracellular membrane vesicles in normal and transformed lymphocytes.

Shedding of extracellular membranes from the cell surface may be one of the means through which cells communicate with one another. In an attempt to elucidate whether cell surface exfoliation is a directed or random process, we investigated the membrane lipid and protein composition and membrane lipid order of shed extracellular membranes and of plasma membranes from which they arose in normal circulating lymphocytes and in the B-lymphoblastoid cell lines Raji, WI HF2 729 and the T-lymphoblastoid cell line Jurkat. Extracellular membranes derived from transformed cell lines were more rigid as assessed by steady state polarization of 1,6-diphenylhexatriene (DPH) and were highly enriched in cholesterol when compared with the corresponding plasma membrane. The extracellular membranes from normal lymphocytes, on the other hand, were more fluid and contained more polyunsaturated acyl chains than did the plasma membranes from these cells. Our results suggest that extracellular membranes are shed from specialized regions of the lymphocyte plasma membrane and that membrane exfoliation is likely to be a directed event.

Animals↗

PubMed1981

The occurrence of hydroxyapatite crystals in extracellular matrix vesicles after surgical manipulation of the rabbit knee joint.

Free autologous grafts of synovial tissue were transplanted into experimental defects produced in the articular cartilage of rabbit knee joints. The grafted tissue underwent transformation into fibrocartilage. Extracellular matrix vesicles associated with calcified areas were present at the grafted sites. Hydroxyapatite crystals were found within these vesicles and in their vicinity. No calcification occurred in articular cartilage from sham operated joints in which defects were produced but no grafts made and in normal controls. These tissues showed abundant matrix vesicles devoid of crystalline mineral. A careful study of normal synovial tissue did not reveal matrix vesicles and calcifications. The present observations suggest that matrix vesicles in normal articular cartilage exist in a latent form. Vesicle mineralization following surgical manipulations of the joint is probably a manifestation of the metabolic stage of the tissue.

Animals↗

PubMed1979

The relationship between extracellular matrix vesicles and calcospherities in primary mineralization of neoplastic bone tissue. TEM and SEM studies on osteosarcoma.

Primary mineralization in neoplastic tissue was studied in osteosarcoma, correlating observations obtained by SEM to those found with TEM. The process is characterized by extracellular matrix vesicles, distributed in the matrix between the forming neoplastic cells and the calcifying fronts. The occurrence of osmiophilic material and solitary hydroxyapatite crystals within the vesicles is followed by accumulation of apatite crystals, disappearance of the vesicular membrane and formation of calcospherites and calcified fronts. The process described here in neoplastic tissue is essentially similar to primary calcification in normal calcified tissues.

Apatites↗

PubMed1981

Transmission electron microscopy of reparative dentin in rat molar pulps. Primary mineralization via extracellular matrix vesicles.

A reparative dentin bridge was induced in rat molar teeth by pulp exposure and capping with calcium hydroxide. Transmission electron-microscopic examination after 10 days revealed the presence of odontoblastic cells and collagenous matrix with focal calcifications. The calcifying fronts were composed of hydroxyapatite crystals. Numerous extracellular matrix vesicles were scattered between the forming cells and the calcifying fronts. Some of the vesicles contained electron-dense material and in others, apatite crystals were detected. Matrix vesicles could not be identified in normal, mature calcifying dentin matrix. In view of the present observations and studies on surgical manipulations in articular cartilage, it is concluded that matrix vesicle calcification may result from alterations in the metabolic state of mesenchymal tissues. These changes can be induced surgically or chemically.

Animals↗

PubMed1981

The occurrence of extracellular matrix vesicles in pulmonary alveolar microlithiasis.

Mineralization in pulmonary alveolar microlithiasis was studied by transmission electron microscopy. The disease is characterized by psammoma like calcifications composed of hydroxyapatite crystals. The calcifications were surrounded by typical forming cells and matrix composed of collagen fibers arranged in a longitudinal pattern. Abundant calcifying extracellular matrix vesicles were found between the cells and the calcified fronts. The type of calcification found in the present lesion is similar to ectopic primary mineralization in other diseases, embryonal ossification and bone wound healing.

Adult↗

PubMed1981

Primary mineralization and extracellular matrix vesicles in rat bone after administration of glass-ceramic implants.

Glass-ceramic implants were administered into cavities prepared in rat femoral shaft. Electron microscope examination revealed formation of collagenous rich matrix in the implant-bone interface. Features typical to primary mineralization as well as bone and implant resorption were present in the interface. Primary mineralization was characterized by the occurrence of active forming cells, extracellular matrix vesicles and calcifying calcospheritic structures. Intensive primary mineralization in association with the implants indicates that glass-ceramic may be stimulative to ossification, allowing favorable tissue-implant relationship.

Animals↗

PubMed2026

Probiogenomic analysis of functional potential and safety of L. plantarum 8p-a3 and DMC-S1 strains: in silico vs in vitro and in vivo data.

The molecular basis of the beneficial effects and the causes of the negative effects of probiotics are not entirely clear. Clarifying these issues is important for understanding the biology and assessing the safety of the microbes. Omics technologies have opened up new resources for obtaining relevant knowledge. Here, for the first time, we present the results of a comparative analysis of the functional potential and safety of two L. plantarum strains: the approved probiotic 8p-a3 and the Drosophila intestinal resident, which exhibit opposite effects on D. melanogaster as the model host organism. Through genomic analysis, extracellular vesicle studies, and in vitro and in vivo assays, we have identified the common and specific characteristics of the strains. The strains proved to be similar in a set of genes that determine benefits to the host organism, as well as in the presence of some risk factors. Significant differences between the strains are related to genes responsible for adhesion, sialic acid metabolism, mucin degradation, antimicrobial peptides, tannin resistance, and immunomodulation. In silico data correlated with in vitro and in vivo data, with the exception of antimicrobial sensitivity. Pronounced differences between the strains were found in terms of the composition and biological effects of their vesicles. In vivo data on the effects of the strains correlate with the corresponding data of their vesicles in the fruit fly model. The results obtained open up new facets in L. plantarum strains relevant for evaluating the functionality and safety of probiotics.IMPORTANCEUsing a probiogenomic approach, common and specific features regarding functionality and safety were identified in the strains (the approved probiotic strain L. plantarum 8p-a3 and the Drosophila intestinal bacterium L. plantarum DMC-S1), which exhibit opposite effects on the model host organism (D. melanogaster). The genomic analysis was supplemented by the analysis of extracellular vesicles of the strains. Comparative analysis of in silico data in combination with in vitro and in vivo studies was performed, and unexpected capabilities of the strains were discovered. Novel factors, essential for evaluating the safety of probiotics, were identified. New facets in the interplay of probiotic bacterium with host organism have been revealed.

Animals↗

PubMed2025

Identification of molecular markers and exploration of the oncogenic role of exomeres in hepatocellular carcinoma.

Extracellular vesicles and particles (EVPs) serve as functional mediators delivering their cargoes to specific destinations. Exomeres (EMs) represent a newly discovered subset of nanoparticles, with limited understanding of their biophysical characteristics and functionalities. Here, we isolated and studied EMs from different normal and cancer cell lines. Proteomic analysis reveals distinctive features of EMs compared to small extracellular vesicles (sEVs) and identifies galactosamine (N-acetyl)-6-sulfatase (GALNS) and mannosidase alpha class 2B member 1 (MAN2B1) to be expressed in EMs, indicating their potential as specific EM molecular markers. Subsequent investigations into tumor-derived EMs demonstrate their oncogenic properties to support cancer growth and metastasis. Furthermore, analysis of murine hepatocellular carcinoma-derived EM reveals their ability to induce cell cycle progression and metabolic alterations. Collectively, our data highlight the distinct nature of EMs as a nanoparticle subpopulation different from sEVs, with cancer-derived EMs significantly contributing to tumor growth and dissemination.

Carcinoma, Hepatocellular↗