Production Of Matrix Vesicles Research Articles

Unilateral anterior crossbite induces aberrant mineral deposition in degenerative temporomandibular cartilage in rats

To investigate whether mechanical stress induces mineral deposits that contribute to matrix degradation at the onset of osteoarthritis (OA) in temporomandibular joint (TMJ) cartilage. Female Spraguee-Dawley rats were subjected to an unilateral anterior crossbite (UAC) procedure. Histology, electron microscopy, and energy dispersive spectrometer (EDS) were used to examine cartilage matrix structures and composition of mineral deposit in the affected TMJ cartilage. Protein and/or RNA expression of phenotypic markers and mineralization modulators and matrix degradation was analyzed by immunohistochemistry and/or real-time PCR. Synthetic basic calcium phosphate (BCP) and calcium pyrophosphate dehydrate (CPPD) crystals were used to stimulate ATDC5 cells for their impact on cell differentiation and gene expression. Fragmented and disorganized collagen fibers, expanded fibrous spaces, and enhancement of matrix vesicle production and mineral deposition were observed in matrices surrounding hypertrophic chondrocytes in cartilage as early as 2-weeks post-UAC and exacerbated with time. The mineral deposits in TMJ cartilage at 12- and 20-weeks post-UAC had Ca/P ratios of 1.42 and 1.44, which are similar to the ratios for BCP. The expression of mineralization inhibitors, NPP1, ANK, CD73, and Matrix gla protein (MGP) was decreased from 2 to 8 weeks post-UAC, so were the chondrogenic markers, Col-2, Col-X and aggrecan. In contrast, the expression of tissue-nonspecific alkaline phosphatase (TNAP) and MMP13 was increased 4-weeks post-UAC. Treating ADTC5 cells with BCP crystals increased MMPs and ADAMTS5 expression, but reduced matrix production in a time-dependent manner. UAC induces deposition of BCP-like minerals in osteoarthritic cartilage, which can stimulate matrix degradation by promoting the expression of cartilage-degrading enzymes to facilitate OA progression.

Differential miRNA Expression in Cells and Matrix Vesicles in Vascular Smooth Muscle Cells from Rats with Kidney Disease.

Vascular calcification is a complex process and has been associated with aging, diabetes, chronic kidney disease (CKD). Although there have been several studies that examine the role of miRNAs (miRs) in bone osteogenesis, little is known about the role of miRs in vascular calcification and their role in the pathogenesis of vascular abnormalities. Matrix vesicles (MV) are known to play in important role in initiating vascular smooth muscle cell (VSMC) calcification. In the present study, we performed miRNA microarray analysis to identify the dysregulated miRs between MV and VSMC derived from CKD rats to understand the role of post-transcriptional regulatory networks governed by these miRNAs in vascular calcification and to uncover the differential miRNA content of MV. The percentage of miRNA to total RNA was increased in MV compared to VSMC. Comparison of expression profiles of miRNA by microarray demonstrated 33 miRs to be differentially expressed with the majority (~ 57%) of them down-regulated. Target genes controlled by differentially expressed miRNAs were identified utilizing two different complementary computational approaches Miranda and Targetscan to understand the functions and pathways that may be affected due to the production of MV from calcifying VSMC thereby contributing to the regulation of genes by miRs. We found several processes including vascular smooth muscle contraction, response to hypoxia and regulation of muscle cell differentiation to be enriched. Signaling pathways identified included MAP-kinase and wnt signaling that have previously been shown to be important in vascular calcification. In conclusion, our results demonstrate that miRs are concentrated in MV from calcifying VSMC, and that important functions and pathways are affected by the miRs dysregulation between calcifying VSMC and the MV they produce. This suggests that miRs may play a very important regulatory role in vascular calcification in CKD by controlling an extensive network of post-transcriptional targets.

Activin A Suppresses Osteoblast Mineralization Capacity by Altering Extracellular Matrix (ECM) Composition and Impairing Matrix Vesicle (MV) Production

During bone formation, osteoblasts deposit an extracellular matrix (ECM) that is mineralized via a process involving production and secretion of highly specialized matrix vesicles (MVs). Activin A, a transforming growth factor-β (TGF-β) superfamily member, was previously shown to have inhibitory effects in human bone formation models through unclear mechanisms. We investigated these mechanisms elicited by activin A during in vitro osteogenic differentiation of human mesenchymal stem cells (hMSC). Activin A inhibition of ECM mineralization coincided with a strong decline in alkaline phosphatase (ALP(1)) activity in extracellular compartments, ECM and matrix vesicles. SILAC-based quantitative proteomics disclosed intricate protein composition alterations in the activin A ECM, including changed expression of collagen XII, osteonectin and several cytoskeleton-binding proteins. Moreover, in activin A osteoblasts matrix vesicle production was deficient containing very low expression of annexin proteins. ECM enhanced human mesenchymal stem cell osteogenic development and mineralization. This osteogenic enhancement was significantly decreased when human mesenchymal stem cells were cultured on ECM produced under activin A treatment. These findings demonstrate that activin A targets the ECM maturation phase of osteoblast differentiation resulting ultimately in the inhibition of mineralization. ECM proteins modulated by activin A are not only determinant for bone mineralization but also possess osteoinductive properties that are relevant for bone tissue regeneration.

Apoptosis regulates human vascular calcification in vitro: evidence for initiation of vascular calcification by apoptotic bodies.

The mechanisms involved in the initiation of vascular calcification are not known, but matrix vesicles, the nucleation sites for calcium crystal formation in bone, are likely candidates, because similar structures have been found in calcified arteries. The regulation of matrix vesicle production is poorly understood but is thought to be associated with apoptotic cell death. In the present study, we investigated the role of apoptosis in vascular calcification. We report that apoptosis occurs in a human vascular calcification model in which postconfluent vascular smooth muscle cell (VSMC) cultures form nodules spontaneously and calcify after approximately 28 days. Apoptosis occurred before the onset of calcification in VSMC nodules and was detected by several methods, including nuclear morphology, the TUNEL technique, and external display of phosphatidyl serine. Inhibition of apoptosis with the caspase inhibitor ZVAD.fmk reduced calcification in nodules by approximately 40%, as measured by the cresolphthalein method and alizarin red staining. In addition, when apoptosis was stimulated in nodular cultures with anti-Fas IgM, there was a 10-fold increase in calcification. Furthermore, incubation of VSMC-derived apoptotic bodies with (45)Ca demonstrated that, like matrix vesicles, they can concentrate calcium. These observations provide evidence that apoptosis precedes VSMC calcification and that apoptotic bodies derived from VSMCs may act as nucleating structures for calcium crystal formation.

Pulsed electromagnetic field stimulation of MG63 osteoblast-like cells affects differentiation and local factor production.

Pulsed electromagnetic field stimulation has been used to promote the healing of chronic nonunions and fractures with delayed healing, but relatively little is known about its effects on osteogenic cells or the mechanisms involved. The purpose of this study was to examine the response of osteoblast-like cells to a pulsed electromagnetic field signal used clinically and to determine if the signal modulates the production of autocrine factors associated with differentiation. Confluent cultures of MG63 human osteoblast-like cells were placed between Helmholtz coils and exposed to a pulsed electromagnetic signal consisting of a burst of 20 pulses repeating at 15 Hz for 8 hours per day for 1, 2, or 4 days. Controls were cultured under identical conditions, but no signal was applied. Treated and control cultures were alternated between two comparable incubators and, therefore, between active coils; measurement of the temperature of the incubators and the culture medium indicated that application of the signal did not generate heat above the level found in the control incubator or culture medium. The pulsed electromagnetic signal caused a reduction in cell proliferation on the basis of cell number and [3H]thymidine incorporation. Cellular alkaline phosphatase-specific activity increased in the cultures exposed to the signal, with maximum effects at day 1. In contrast, enzyme activity in the cell-layer lysates, which included alkaline phosphatase-enriched extracellular matrix vesicles, continued to increase with the time of exposure to the signal. After 1 and 2 days of exposure, collagen synthesis and osteocalcin production were greater than in the control cultures. Prostaglandin E2 in the treated cultures was significantly reduced at 1 and 2 days, whereas transforming growth factor-beta1 was increased; at 4 days of treatment, however, the levels of both local factors were similar to those in the controls. The results indicate enhanced differentiation as the net effect of pulsed electromagnetic fields on osteoblasts, as evidenced by decreased proliferation and increased alkaline phosphatase-specific activity, osteocalcin synthesis, and collagen production. Pulsed electromagnetic field stimulation appears to promote the production of matrix vesicles on the basis of higher levels of alkaline phosphatase at 4 days in the cell layers than in the isolated cells, commensurate with osteogenic differentiation in response to transforming growth factor-beta1. The results indicate that osteoblasts are sensitive to pulsed electromagnetic field stimulation, which alters cell activity through changes in local factor production.

24,25-(OH)2D3 regulation of matrix vesicle protein kinase C occurs both during biosynthesis and in the extracellular matrix.

Plasma membranes and matrix vesicles isolated from rat costochondral resting zone chondrocyte cultures contain predominantly protein kinase C alpha (PKCalpha) and PKCzeta, respectively, and the level of PKC specific activity in these membrane fractions is regulated by 24,25-(OH)2D3 [14]. In the present study, we examined whether the effect of 24,25-(OH)2D3 on membrane PKC is via genomic mechanisms during biogenesis and through a nongenomic mechanism after the matrix vesicles are resident in the matrix. There was a dose-dependent decrease in matrix vesicle PKC specific activity and a significant increase in plasma membrane enzyme activity in cultures treated for 90 minutes with 10(-9)-10(-7) M 24,25-(OH)2D3. However, at 12 hours, matrix vesicle PKC was stimulated, but no effect was seen in the plasma membranes, suggesting that the effect seen at 90 minutes was due to a direct action of the hormone on PKC activity in the membrane, and that the effect seen at 12 hours was due to new matrix vesicle production with altered PKC content. Neither actinomycin D nor cycloheximide inhibited matrix vesicle PKC at 30, 60, or 90 minutes, but by 12 hours, these inhibitors blocked the effect of the hormone. 24,25-(OH)2D3-dependent plasma membrane PKC was sensitive to both actinomycin D and cycloheximide at early time points, but by 12 hours, no effect of the inhibitors was seen. Monensin did not alter basal plasma membrane PKC activity or the 24, 25-(OH)2D3-dependent increase, suggesting that this increase was due to translocation of cytosolic PKC rather than new membrane synthesis. Monensin did not affect matrix vesicle PKC at early time points, but it decreased 24,25-(OH)2D3-dependent enzyme activity at later times, indicating that new matrix vesicle production was blocked. At least part of the effect of 24,25-(OH)2D3 on PKC involved phospholipase A2 (PA2). Quinacrine (a PA2 inhibitor) alone had no effect on matrix vesicle PKC, but in cultures treated for 12 hours with quinacrine and 24,25-(OH)2D3, a synergistic increase in matrix vesicle PKC was observed. Quinacrine caused a time-dependent decrease in matrix vesicle PKC and a dose- and time-dependent increase in plasma membrane PKC when incubated directly with the membranes, supporting the hypothesis that PA2 plays a role in the nongenomic regulation of PKC by 24,25-(OH)2D3. Experiments using anti-isoform specific antibodies showed that 24,25-(OH)2D3 modulated the distribution of PKCalpha, beta, and zeta between the plasma membrane and matrix vesicle compartments via translocation and new PKC synthesis. Thus, the data support the hypothesis that 24, 25-(OH)2D3 regulates matrix vesicles through two pathways: a genomic one at the stage of biosynthesis and packaging, and a second nongenomic mechanism acting directly upon matrix vesicles in the matrix. These data also indicate that matrix vesicle regulation consists of complex events with several different points of regulation.

Regulated production of mineralization-competent matrix vesicles in hypertrophic chondrocytes.

Matrix vesicles have a critical role in the initiation of mineral deposition in skeletal tissues, but the ways in which they exert this key function remain poorly understood. This issue is made even more intriguing by the fact that matrix vesicles are also present in nonmineralizing tissues. Thus, we tested the novel hypothesis that matrix vesicles produced and released by mineralizing cells are structurally and functionally different from those released by nonmineralizing cells. To test this hypothesis, we made use of cultures of chick embryonic hypertrophic chondrocytes in which mineralization was triggered by treatment with vitamin C and phosphate. Ultrastructural analysis revealed that both control nonmineralizing and vitamin C/phosphatetreated mineralizing chondrocytes produced and released matrix vesicles that exhibited similar round shape, smooth contour, and average size. However, unlike control vesicles, those produced by mineralizing chondrocytes had very strong alkaline phosphatase activity and contained annexin V, a membrane-associated protein known to mediate Ca2+ influx into matrix vesicles. Strikingly, these vesicles also formed numerous apatite-like crystals upon incubation with synthetic cartilage lymph, while control vesicles failed to do so. Northern blot and immunohistochemical analyses showed that the production and release of annexin V-rich matrix vesicles by mineralizing chondrocytes were accompanied by a marked increase in annexin V expression and, interestingly, were followed by increased expression of type I collagen. Studies on embryonic cartilages demonstrated a similar sequence of phenotypic changes during the mineralization process in vivo. Thus, chondrocytes located in the hypertrophic zone of chick embryo tibial growth plate were characterized by strong annexin V expression, and those located at the chondro-osseous mineralizing border exhibited expression of both annexin V and type I collagen. These findings reveal that hypertrophic chondrocytes can qualitatively modulate their production of matrix vesicles and only when induced to initiate mineralization, will release mineralization-competent matrix vesicles rich in annexin V and alkaline phosphatase. The occurrence of type I collagen in concert with cartilage matrix calcification suggests that the protein may facilitate crystal growth after rupture of the matrix vesicle membrane; it may also offer a smooth transition from mineralized type II/type X collagen-rich cartilage matrix to type I collagen-rich bone matrix.

Effects of hydroxyapatite implants on primary mineralization during rat tibial healing: biochemical and morphometric analyses.

The effect of 40- to 60-mesh hydroxyapatite (HA) granules (Calcitek, Inc., Carlsbad, CA) on the process of primary mineralization during bone healing was examined following insertion of the HA granules into rat tibial bone after marrow ablation. Response to HA was assessed by monitoring morphometric and biochemical changes in matrix vesicles, which are extracellular organelles associated with initial calcification. Following insertion of HA, matrix vesicle-enriched membranes (MVEMs) were isolated from the tissue adjacent to the implant and from the endosteum of the contralateral limb at 3, 6, 14, and 21 days and from a nonimplanted control group (t = 0). MVEM alkaline phosphatase- and phospholipase A2-specific activities were increased on days 6 (peak) and 14; phosphatidylserine content was also elevated on days 6 and 14 (peak). Comparable changes were seen in the contralateral limb but at lesser magnitudes. Morphological changes were observed as well. The number of matrix vesicles/micron2 matrix increased on days 6 (peak) and 14. The mean diameter of the matrix vesicles was elevated on days 6 (peak), 14, and 21. Mean distance from the calcifying front increased on day 6 but was decreased on days 14 and 21. These results indicated that HA behaves like bone-bonding implants in that there is a stimulation of matrix vesicle enzymes, increased phosphatidylserine content, and increase numbers of matrix vesicles. However, the increases occur only after 6 days postimplantation, indicating a delay in response when compared to normal healing. This delay is confirmed by the morphometric measurements. HA causes a reduction in the response associated with marrow ablation. In addition, the effects of HA are comparable locally and systemically but with different intensity. These observations suggest that osteogenic cells are able to compensate for the inhibitory effects of HA and primary calcification involves normal matrix vesicle production and maturation, if somewhat delayed and reduced in magnitude. The ability to support primary mineral formation may contribute to the successful bonding of HA with surrounding osseous tissue.

Culture and differentiation of chondrocytes entrapped in alginate gels

We studied the response to culture conditions and the differentiative ability in suspension culture in alginate gels of resting chondrocytes from the preosseous cartilage of adult pig scapula. It was found that the maximum rate of chondrocyte duplication is reached at the fourth day in culture whereas the rate of proteoglycan synthesis and alkaline phosphatase expression do not gain a maximum value before the seventh day. During the culture time, the chondrocytes undergo differentiation as it is demonstrated by the alkaline phosphatase specific activity increase and by morphological criteria (hypertrophy, increase of the number of mitochondria per cell, increased endoplasmic reticulum, matrix vesicle production). The alginate gels can be easily dissolved to obtain cell populations in which the variation of cytosolic calcium concentration following a proliferative stimulus can be conveniently observed using the conventional procedure of Fura 2.

Regulation of matrix vesicle phospholipid metabolism is cell maturation-dependent

We have developed a chondrocyte culture model for assessing the regulation of matrix vesicles at two different stages of chondrogenic maturation. These chondrocytes, resting zone (RC) and growth zone (GC), retain their phenotypic markers in culture, including production of matrix vesicles with distinctive lipid compositions and enzyme activities. Isolated matrix vesicles incubated in vitro with 1,25-(OH)2D3 (1,25) or 24,25-(OH)2D3 (24,25) respond differentially. 1,25 stimulates phospholipase A2 (PA2) in GC vesicles, but not on those from RC. 24,25 inhibits PA2 in RC vesicles, but has no effect on GC. PA2 activity is required for fatty acid turnover and is the rate-limiting step in prostaglandin production. Plasma membrane phospholipids are more susceptible to the release of arachidonic acid by PA2 than are matrix vesicle phospholipids. Matrix vesicles are distinct from the plasma membrane in terms of lipid composition and arachidonic acid incorporation. 1,25 and 24,25 stimulate arachidonic acid turnover in their target cells, but by different mechanisms. 1,25 has no effect on arachidonic acid turnover in RC; however, 24,25 inhibits turnover in RC and GC. 1,25 and 24,25 also affect isolated matrix vesicle membrane fluidity. These results suggest that vitamin D metabolites modulate PA2 activity, change the composition of membrane phospholipids by altering fatty acid composition, and affect calcium transport. The effects are mediated by altering membrane fluidity and is dependent on the stage of cell differentiation.

Epithelial Cell Lines That Induce Bone Formation In Vivo Produce Alkaline Phosphatase-Enriched Matrix Vesicles in Culture

Hypertrophic chondrocytes and osteoblasts produce alkaline phosphatase (ALPase)-enriched matrix vesicles in vivo and in vitro and, along with certain epithelial cell lines and osteoblast precursors, induce bone when implanted in mesenchymal tissues. This study examined whether ALPase-enriched matrix vesicle production in vitro was a general property of cells that induce bone in vivo. Epithelial cell lines FL, WISH, and OK 16; connective tissue cell lines HEPM 1 and HEPM 2; neonatal rat muscle cells; rat costochondral chondrocytes; and human fibroblasts were implanted intramuscularly into nude mice. The FL and WISH cells produced tumors and induced large islands of bone with focal areas of cartilage immediately adjacent to the tumors. The chondrocytes formed cartilage nodules but did not induce bone, indicating that the ability of the cells to form a solid mass was not an a priori requirement for bone formation. No other cell type produced tumors or nodules or induced bone formation, although connective tissue cells have been shown to induce chondrogenesis in vitro and osteogenesis in vivo. Only matrix vesicles from normal chondrocytes, FL, WISH, and OK16 cultures exhibited enriched ALPase-specific activity. Matrix vesicles from FL and WISH cultures exhibited ALPase specific activities similar to those isolated from osteoblast or chondrocyte cultures. These data suggest that the ability to produce ALPase-enriched matrix vesicles in culture may be associated with the ability of cells to induce bone or cartilage in vivo.

The Use of Avian Epiphyseal Chondrocytes for in Vitro Studies of Skeletal Metabolism

Chondrocytes from the postembryonic avian epiphyseal growth plate are easily obtained and can be successfully cultured for a considerable length of time. Under the proper culture conditions, the chondrocytes will become confluent and achieve a hypertrophic morphology followed by mineralization of the extracellular matrix. Although this can be accomplished with a serum-free medium, the process is accelerated in the presence of serum. Minimal growth factor requirements for the serum-free medium include fibroblast growth factor and insulin-like growth factor. Transforming growth factor-beta and parathyroid hormone are also mitogenic for chondrocytes. Chondrocytes may have more critical requirements for amino acids because further supplementation of a conventional medium such as Dulbecco’s modified Eagle’s medium results in improved performance of the cultured cells. Ascorbic acid in the medium is very important for matrix vesicle production and mineralization of cartilage nodules.

Matrix Vesicles in Bone Tumors

Bone tumors were categorized into alkaline phosphatase (ALPase)-positive (2 ossifying fibromas, 1 benign osteoblastoma and 16 osteosarcomas) and negative (2 chondromas, 2 chondrosarcomas, 3 non-ossifying fibromas, 2 malignant fibrous histiocytomas and 6 giant cell tumors of bone) groups. Production and distribution of matrix vesicles (MVs) in the tumor tissues were examined to clarify their role in neoplastic bone formation. Four distinct types of MV were isolated primarily in ALPase positive bone tumors: empty, amorphous, crystalline and ruptured MVs. They were formed by budding off from the cytoplasmic projections of the osteoblastic tumor cells. The significance of differences in the production rate of MVs between ALPase-positive and negative bone tumors was investigated in view of the predominantly high production of MVs in ALPase-positive bone tumors. Many more mature MVs (crystalline and ruptured) were observed in the osteoblastic lesions of osteosarcoma than in the fibroblastic and MFH-like lesions, suggesting an intimate relationship with maturation and differentiation of the osteoblastic tumor cells. The above findings indicate that production of MVs is one of the diagnostic parameters for osteoblast-derived bone tumors, as well as ALPase activity, and that vesicle-induced mineralization is a major mineralization mechanism in neoplastic bone formation.

Matrix vesicle enzyme activity in endosteal bone following implantation of bonding and non.‐bonding implant materials

The effect of bone bonding (KGy-Cera) and non-bone bonding (KGy-213) implant materials on primary mineralization was examined in endosteal bone repair following marrow ablation. Comparisons were made to determine implant effect on concentration and biochemical parameters of matrix vesicles, as contrasted to vesicles in normal bone healing. Matrix vesicle number was determined by high-resolution computerized morphometric analysis, and implant effect on the specific activity of alkaline phosphatase and phospholipase A2 was measured. Bone responses differed according to the composition of the implant material. The bone bonding implant in this study stimulated matrix vesicle formation, alkaline phosphatase specific activity, and, to a lesser extent, phospholipase A2 activity. The effect of the non-bonding implant on healing bone was of suppression of enzyme specific activities and reduced matrix vesicle production. The results indicate that the bone bonding implant material (KGy-Cera) promotes the initiation of primary mineralization, whereas failure of the KGy-213 to bond may be related to toxic materials that leach from the implant and inhibit the normal sequence of events in the mineralization cascade. The results also demonstrate that the implant materials alter the healing process distal to the injury site. Changes observed in the contralateral control limb mimic the changes observed in the injured limb, but at lower magnitude.

Matrix Vesicles as a Marker of Endochondral Ossification

Endochondral ossification in bone development and repair, and in induced bone formation in mesenchymal tissues, involves recruitment of mesenchymal cells, their differentiation into chondrocytes, and calcification of the cartilagenous matrix. Stimulation of proteoglycan synthesis is used as a biochemical marker of chondrogenesis, however it does not distinguish among chondrogenic phenotypes. Chondrocytes derived from the resting zone and adjacent growth zone cartilage of the costochondral junction of young rats, produce matrix vesicles in culture which are enriched in alkaline phosphatase specific activity with respect to the plasma membrane. Matrix vesicles isolated from cultures of neonatal rat muscle mesenchymal cells are not enriched in this enzyme activity. Alkaline phosphatase in matrix vesicles produced by growth zone chondrocytes is stimulated by 1,25(OH)2D3; enzyme in matrix vesicles produced by resting zone chondrocytes is stimulated by 24,25(OH)2D3; enzyme in matrix vesicles isolated from mesenchymal cell cultures is responsive to neither metabolite. Matrix vesicle phospholipase A2 is stimulated by 1,25(OH)2D3 in growth zone chondrocytes cultures; inhibited by 24,25(OH)2D3 in resting zone chondrocyte cultures; and is unaffected by either metabolite in mesenchymal cell cultures. These observations suggest that matrix vesicle production, as defined by alkaline phosphatase enrichment, and responsiveness of matrix vesicle enzymes to vitamin D metabolites, can be used as markers of phenotypic maturation during chondrogenesis in vivo and in vitro.

Differences in matrix vesicle concentration among growth plate zones.

We studied the proximal tibial growth plates of 15-day-old mice to determine if matrix vesicle concentration varies among growth plate zones or between the pericellular and territorial matrix compartment and the interterritorial matrix compartment. Growth plates were examined by electron microscopy and divided into five zones: reserve zone (RZ), upper proliferative zone (UPZ), lower proliferative zone (LPZ), upper hypertrophic zone (UHZ), and lower hypertrophic zone (LHZ) which included the calcifying zone. We measured the diameter and volume fraction of matrix vesicles and calculated their numerical density and volume per cell and number per cell in the pericellular and territorial matrix and in the interterritorial matrix of each zone. In the pericellular and territorial matrix compartment, the matrix vesicle concentration progressively decreased from the RZ to the LHZ. Changes in matrix vesicle concentration in the interterritorial matrix followed a different pattern. Between the RZ and the UPZ, matrix vesicle numerical density declined slightly and then increased to peak values in the LPZ and UHZ, followed by a decline between the UHZ and the LHZ. These changes in matrix vesicle concentration paralleled previously reported changes in intramitochondrial calcium content, suggesting that matrix vesicle production in growth plate may be related to intracellular calcium concentration. The existence of the maximum concentration of matrix vesicles in the LPZ and UHZ longitudinal septa which do not mineralize followed by a decline in matrix vesicle concentration in the LHZ longitudinal septa which mineralize suggests that a high concentration of matrix vesicles may be needed to prepare the matrix for mineralization or to initiate mineralization and that matrix vesicles are depleted during mineralization.

Effect of trifluoperazine and other drugs on matrix vesicle formation by chicken growth plate chondrocytes in primary cell culture

Growth plate chondrocytes in primary culture release alkaline phosphatase (AP)-rich matrix vesicles (MV) into the culture medium. While these are thought to derive from the plasma membrane by a membrane fusion-dependent process, the mechanism is not fully understood. Recently, a cytosolic protein, synexin, has been shown to promote membrane fusion in a number of systems, and thus may be involved in MV formation. Since the action of synexin is selectively inhibited by trifluoperazine (TFP) and other phenothiazines, we examined the effects of these drugs, and imipramine, on cellular AP production and formation of AP-containing MV by cultured chondrocytes. In addition, we studied the effect on cell division, protein biosynthesis, and incorporation of palmitate into cellular and MV lipids. All of the drugs reduced cellular AP in a concentration-dependent manner; however, at the higher levels, chlorpromazine (CPZ) and TFP caused a transient sharp rise in MV AP activity. At IC 50 levels, the drugs were more inhibitory to cellular AP than to MV AP, appearing to enhance significantly the transfer of available cellular AP into MV. In contrast, the drugs stimulated incorporation of [ 3H]palmitate into cellular lipids, but either had no effect on, or actually inhibited, incorporation of the fatty acid into MV. At these levels, the drugs had little effect on cell division and protein biosynthesis. The inhibitory effect of IC 50 levels of CPZ on palmitate incorporation into MV appears to have resulted from impairment of vesicle formation per se, since at these levels the drug stimulated incorporation of the fatty acid into the cells. The transient stimulatory effect of higher levels of CPZ on MV AP levels, and the enhanced transfer of AP into MV by the drugs generally, may result from the effect of the drugs on membrane structure. Since TFP was not inhibitory to MV formation, it is doubtful that synexin was directly involved.

Effect of amino acid levels on matrix vesicle formation by epiphyseal growth plate chondrocytes in primary culture.

The effect of varying the amino acid concentrations of the culture medium on matrix vesicle formation was studied in primary cultures of chicken epiphyseal growth plate chondrocytes grown in Dulbecco's modified Eagle's medium (DME) supplemented with 10% fetal bovine serum (FBS). Decreasing the levels of free amino acids in the culture medium to levels of one-half, one quarter, and one eighth of the values normally present in DME caused a progressive decline in matrix vesicle (MV) formation. Increasing the level in the culture medium of those amino acids that are enriched in extracellular fluid (ECF) of growth plate cartilage significantly increased formation of matrix vesicles (MV), as assayed by the alkaline phosphatase (AP) activities present in high-speed sediments from spent culture media. However, adjusting the levels of all amino acids to match those of the ECF produced the greatest stimulation of MV formation. Of the amino acids that are notably enriched in ECF, glutamate (GLU), alanine (ALA), serine (SER), asparagine (ASN), and taurine (TAU) individually enhanced MV production, whereas proline (PRO), glycine (GLY), and aspartate (ASP) had essentially no effect. The simple combination of ECF levels of ALA and GLU resulted in a stimulation of MV formation equal to that observed when the eight aforementioned amino acids were elevated to ECF levels. Other combinations of ASP and GLY, or of TAU, SER, and ASN showed some stimulation, but at a lower level. Increasing the amino acid concentrations, alone or in combination, also increased the levels of cellular AP, and to a lesser extent cellular protein. While increases in cellular AP were generally correlated with increased formation of AP-rich MV, this was not uniformly true. These results indicate that in addition to hormones and growth factors, nutritional factors such as the levels of amino acids are also critical for normal phenotypic expression, growth, and matrix formation by epiphyseal chondrocytes.

The morphology of matrix vesicles produced in experimental arterial aneurysms of rabbits

The morphology and possible modes of matrix vesicle production in the walls of experimental rabbit aneurysms were investigated at an ultrastructural level. The abundant matrix vesicles were mostly circular or oval, and in 95% the largest dimension measured 25 to 130nm. Many had been formed by the disintegration of whole smooth muscle cells or portions thereof but more frequently they were related to and even embedded in the thickened, dystrophic basement membranes of viable smooth muscle cells. Budding was an infrequent feature. Many matrix vesicles were associated with fragments of plasma membrane in the matrix and areas which were possibly interruptions of the plasma membranes. Phagocytosis was seen, but more often the matrix vesicles underwent calcification, and groups of larger closely packed, electron translucent vesicles (80-500nm) indicated lipid accumulation. Experimental aneurysms provide a rich source of matrix vesicles for future investigations.

Effect of parathyroid hormone (PTH), calcitonin (CT), and prostaglandin E 2(PGE 2) on production of matrix vesicles (MV) by primary cultures of epiphyseal growth plate chondrocytes

Effect of parathyroid hormone (PTH), calcitonin (CT), and prostaglandin E 2(PGE 2) on production of matrix vesicles (MV) by primary cultures of epiphyseal growth plate chondrocytes