A new cell surface, detergent-insoluble glycoprotein matrix of human and hamster fibroblasts. The role of disulfide bonds in stabilization of the matrix.
Explore the source record for details and available documents.
Animals↗
SEARCH · Search PubMed
Results for “Matrix”
Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.
Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.
Clinical evaluation of two thyrotropin radioimmunoassay kits: human serum matrix calibrators and bovine serum matrix calibrators.
Explore the source record for details and available documents.
Animals↗
Matrix metalloproteinases and tissue inhibitor of matrix metalloproteinases expression by tubular epithelia and interstitial fibroblasts in the normal kidney and in fibrosis.
Explore the source record for details and available documents.
Epithelium↗
[Action of radiation and the hydrodynamic effect on the matrix properties of DNA during extracellular synthesis of RNA and thiophosphate protection of matrix properties of T2 DNA during irradiation].
Explore the source record for details and available documents.
Animals↗
Changes in cell, matrix compartment, and fibrillar collagen volumes between growth-plate zones.
To define the contributions of changes in cell, matrix compartment, and fibrillar collagen volumes to longitudinal bone growth, we measured the differences in cell, pericellular/territorial matrix and interterritorial matrix volumes, and fibrillar collagen concentrations between the upper proliferative and lower hypertrophic zones of the proximal tibial physes of six miniature pigs. The mean numerical density of cells decreased from 110,000 cells/mm3 in the upper proliferative zone to 59,900 cells/mm3 in the lower hypertrophic zone. The mean cell volume increased nearly 5-fold (from 1,174 to 5,530 microm3), and the total matrix volume per cell increased 46% (from 8,040 to 11,760 microm3/cell) between the upper proliferative and lower hypertrophic zones. Both the pericellular/territorial matrix volume per cell and the interterritorial matrix volume per cell increased between the upper proliferative and lower hypertrophic zones; the pericellular/territorial matrix volume per cell increased 61% (from 4,580 to 7,390 microm3/cell), whereas the interterritorial matrix volume per cell increased 26% (from 3,460 to 4,370 microm3/cell). The total increase in mean cell volume of 4,356 microm3 exceeded the total increase in mean matrix volume per cell of 3,720 microm3; the total mean pericellular/territorial matrix volume per cell increased more than the total mean interterritorial matrix volume per cell (2,810 compared with 910 microm3/cell). Fibrillar collagen concentration was greater in the interterritorial matrix than in the pericellular/territorial matrix in both zones and increased in both matrix compartments between the upper proliferative and lower hypertrophic zones. The amount of fibrillar collagen per cell also increased in both matrix compartments between the upper proliferative and lower hypertrophic zones (from 1,720 to 3,100 microm3/cell in the pericellular/territorial matrix and from 1,490 to 2,230 microm3/cell in the interterritorial matrix; thus, the total amount of fibrillar collagen per cell increased from 3,210 to 5,530 microm3/cell). Growth rate was inversely related to the cell numerical density in the upper proliferative and lower hypertrophic zones and was directly related to interterritorial matrix volume per cell in the upper proliferative zone and to pericellular/territorial matrix volume per cell in the lower hypertrophic zone. These results show that cell enlargement contributes more to longitudinal bone growth than does increased matrix volume, that increased pericellular/territorial matrix volume makes a greater contribution to growth than does increased interterritorial matrix volume, and that the total amount of fibrillar collagen per cell increases between the upper proliferative and lower hypertrophic zones. The differences between the two matrix compartments in increase in volume, fibrillar collagen concentration, and amount of fibrillar collagen per cell strongly suggest that they differ not only in matrix organization but in rate of matrix accumulation and assembly and that these differences give the two compartments different roles in skeletal growth.
Animals↗
Differential effects of IGF-1 and TGF beta-2 on the assembly of proteoglycans in pericellular and territorial matrix by cultured bovine articular chondrocytes.
OBJECTIVES: Knowledge of matrix assembly is necessary to understand the pathogenesis of disease processes and to find solutions for repair of articular cartilage lesions. The influence of growth factors on matrix assembly is largely unknown. We investigated whether, and to what degree, insulin-like growth factor (IGF-1) and transforming growth factor beta-2 (TGF beta-2) influence proteoglycan synthesis and accumulation in the cell-associated matrix compartment (consisting of pericellular and territorial matrix) compared to the further-removed matrix compartment (consisting of the interterritorial matrix). DESIGN: Bovine articular chondrocytes were cultured in alginate beads for day 13. The effect of addition of 25 ng/ml IGF-1 or 25 ng/ml TGF beta-2 during the last 7 days in culture was determined. Cell-associated and further-removed matrix compartments were separated by centrifugation after sodium citrate/EDTA treatment. The amount of DNA, the total amount of proteoglycans and the amount of newly synthesized proteoglycans were analyzed biochemically. Morphometric analysis on electron micrographs was used to calculate the volumes of the cell-associated and further-removed matrix components. RESULTS: It was demonstrated in control beads that 25 +/- 8% of the proteoglycans were laid down in the cell-associated matrix compartment compared with 75 +/- 8% in the further-removed matrix compartment. The cell-associated matrix compartment in intact beads could be recognized in electron microscopy by a delineation of dense amorph material. Morphometric evaluation showed a relative volume of the cell-associated matrix compartment of 5.2 +/- 0.6% compared with 91.3 +/- 0.8% of the further-removed matrix compartment and 3.5 +/- 0.3% of the area occupied by cells. Combination of biochemical and morphometric results showed that the concentration of proteoglycans in the cell-associated matrix compartment was 3.63 +/- 0.32 mg/ml. By adding IGF-1 or TGF beta-2, the amount of both total accumulated proteoglycans and newly synthesized [35S]proteoglycans at day 13 in culture increased. In addition to an overall rise in proteoglycan content, IGF-1 significantly increased (24%) the percentage of proteoglycans laid down in the cell-associated matrix compartment while not changing the relative volume of this compartment (5.2 +/- 0.8%). This leads to a 82% (P < 0.05) increase in the proteoglycan concentration in the cell-associated matrix compartment compared to control beads. In contrast, TGF beta-2 significantly decreased (24%) the relative amount of proteoglycans in the cell-associated matrix compartment which was paralleled by a reduction of the relative volume from 5.2 +/- 0.6 to 3.6 +/- 1.4%. This leads to a significant increase of 87% of the proteoglycan concentration in the cell-associated matrix compartment. CONCLUSIONS: This study demonstrates that both IGF-1 and TGF beta-2 significantly but differently influence proteoglycan synthesis and accumulation in the cell-associated matrix compartment of cultured bovine chondrocytes in alginate. Both growth factors increase the concentration of proteoglycans in the cell-associated matrix compartment. However, addition of TGF beta-2 to bovine articular chondrocytes cultured in alginate beads for 13 days results in a significant reduction of the relative volume of the pericellular matrix compartment compared to controls, indicating differences in assembly of the matrix.
Animals↗
Matrix metalloproteinases and aggrecanase: their role in disorders of the human intervertebral disc.
STUDY DESIGN: A comprehensive immunohistochemical study of matrix metalloproteinase activity in discs from patients with different disc diseases. OBJECTIVES: To identify individual matrix metalloproteinase enzymes that could contribute to the degeneration of the matrix of the intervertebral disc, to identify the cells that produce matrix metalloproteinases (for example, the endogenous disc cells or invading cells associated with vascularisation), and to determine if "aggrecanase" contributes to degradation of proteoglycans in disc disorders. SUMMARY OF BACKGROUND DATA: Matrix disorganization and loss of substance are the most common findings in degenerate discs, and proteinase enzyme activity is one means of causing these changes. METHODS: Forty-nine discs from 46 patients with degenerative disc disease, posterior anular tears, spondylolisthesis, or disc herniation were studied immunohistochemically to determine the presence of matrix metalloproteinases 1, 2, 3, 7, 8, 9 and 13, tissue metalloproteinases 1 and 2, and proteoglycan degradation products generated by either matrix metalloproteinases or aggrecanase activity. In addition, in situ zymography was used to confirm matrix metalloproteinase activity. RESULTS: The most extensive staining was seen for matrix metalloproteinases 1, 2, 3, and 9, with 91%, 71%, 65%, and 72% of samples having some immunopositivity for the respective antibodies. In contrast, staining for matrix metalloproteinases 7 and 8 was much less (38% for both). Tissue inhibitor of metalloproteinases 1 and 2 were expressed in 34% and 79% of specimens, respectively. Matrix metalloproteinases were found particularly in cell clusters and blood vessels of degenerate discs, with staining correlating positively with macroscopic degenerative grade. For all of the enzymes, there was most staining in the herniation specimens and least in the autopsy samples. The opposite was true of staining for the matrix metalloproteinases inhibitor, tissue inhibitor of metalloproteinases 2, with most found in the autopsy specimens. Enzyme activity was confirmed by in situ zymography and staining for matrix metalloproteinase degradation products of proteoglycans. In addition, there was staining with antibodies demonstrating aggrecanase degradation products. CONCLUSIONS: Matrix metalloproteinase activity is more prevalent in herniated discs than in other disc disorders studied, although matrix metalloproteinases may have been more common earlier in the disease progression. Matrix metalloproteinases can be produced by invading blood vessels and associated cells, as well as by indigenous disc cells. Aggrecanase activity, although present in some samples, was not as obvious as that of matrix metalloproteinases. In addition to altered matrix metalloproteinase production, there appears to be a change in the balance between enzymes and endogenous inhibitors, tissue inhibitors of metalloproteinases. This study highlights specific matrix metalloproteinases that might be most efficient to target in developing therapeutics for minimizing degradation of the extracellular matrix of the disc.
Adolescent↗
Immunolocalization of matrix metalloproteinases in partial-thickness defects in pig articular cartilage. A preliminary report.
BACKGROUND: Partial-thickness defects in mature articular cartilage do not heal spontaneously. Attempts at repair often result in limited integration between the repair tissue and the surrounding cartilage, with formation of chondrocyte clusters adjacent to a zone of cartilage necrosis. In wound repair, spatially and temporally controlled expression of matrix metalloproteinases and their inhibitors have been implicated in proteolytic degradation of damaged extracellular matrix components, but the sequence of events following damage to cartilage is unknown. To determine this sequence, we studied the distribution of matrix metalloproteinases and their inhibitors during early in vivo repair of partial-thickness defects in pig articular cartilage. METHODS: With use of a model that elicits the ingrowth of mesenchymal cells into partial-thickness defects, partial-thickness defects were created in knee joint cartilage. The distributions of matrix metalloproteinase-1, 2, 3, 9, 13, and 14; tissue inhibitors of metalloproteinase-1 and 2; and the neoepitope DIPEN341 specifically generated following matrix metalloproteinase cleavage of aggrecan were determined by immunolocalization of repair tissue and surrounding cartilage excised from immature pigs during the first eight weeks of repair and from adult minipigs at eight days and three weeks. RESULTS: Synthesis of matrix metalloproteinase-13 was usually confined to hypertrophic chondrocytes in immature cartilage and to the radial zone in adult cartilage. Following injury, strong induction of matrix metalloproteinase-13 synthesis was observed in chondrocyte clusters surrounding lesions in all of the animals. The migration of macrophages into defects was prominent at two and eight days, with synthesis and deposition of matrix metalloproteinase-9 onto damaged cartilage matrix and newly synthesized matrix in the defect. The DIPEN341 neoepitope was localized to damaged cartilage matrix at eight days and six weeks, indicating partial degradation of aggrecan. Focal synthesis of matrix metalloproteinase-1, 3, and 14 and of tissue inhibitor of metalloproteinase-1 occurred at later times, suggesting continuous remodeling of the increasingly compact repair tissue. CONCLUSIONS: The expression of matrix metalloproteinase-13 by normal hypertrophic chondrocytes and the induction of synthesis in chondrocyte clusters adjacent to the zone of cartilage necrosis suggest that this enzyme participates in the pericellular proteolysis required for lacunar expansion. The localization of matrix metalloproteinase-9 to damaged cartilage matrix suggested matrix proteolysis, which was confirmed with DIPEN341 localization. Reduced matrix metachromasia persisted and was colocalized with DIPEN341 at six weeks. However, under the conditions investigated, there was only limited proteolytic degradation in the zone of cartilage necrosis. This may render the zone mechanically weakened, thereby contributing to subsequent instability of the region, and may form a barrier to integration of repair tissue with viable cartilage. CLINICAL RELEVANCE: Osteoarthritis initially involves the superficial layers of cartilage. The development of procedures to promote the healing or repair of early defects will have major advantages in terms of disease alleviation as well as economic importance. Identification of the enzymes involved in the early repair of partial-thickness defects in articular cartilage is clinically relevant because proteolysis of damaged matrix has to take place in order for repair tissue to integrate with surrounding healthy cartilage.
Aggrecans↗
Regulation of matrix metalloproteinases in a model of colonic wound healing in a rabbit.
PURPOSE: Matrix metalloproteinases occur in the colon at an anastomosis but not in the normal colon. Matrix metalloproteinase synthesis can be regulated by cytokines, for example interleukin-1 beta and growth factors, such as transforming growth factor beta and basic fibroblast growth factor. The aim of this study was to investigate the regulation of matrix metalloproteinases at an anastomosis by identifying the cell types that synthesize matrix metalloproteinases, examining factors that might regulate their synthesis, determining whether they occur in an active form, and assessing the effect of suture type on these parameters. METHODS: An anastomosis was formed in the distal colon of rabbits using either polyglactin or polydioxanone and the animals were killed six hours or seven days later. The distribution of matrix metalloproteinases and cytokines and the cell types were assessed by immunohistochemistry. Matrix metalloproteinase-2, matrix metalloproteinase-3, and matrix metalloproteinase-9 were detected also by zymography. RESULTS: Immunohistochemistry showed that matrix metalloproteinases were restricted to the suture line. Although zymography demonstrated that matrix metalloproteinase-2 was present mainly in an active form, matrix metalloproteinase-9 and matrix metalloproteinase-3 were present in the pro-form. The active form of matrix metalloproteinase-3 occurred more often in the polydioxanone-sutured rabbits. With the exception of matrix metalloproteinase-9, the matrix metalloproteinases were synthesized by fibroblasts. Interleukin-1 beta and transforming growth factor beta were more widespread than in the normal colon and were localized adjacent to the matrix metalloproteinases. Basic fibroblast growth factor was also more widespread postoperatively but occurred deeper in the anastomosis than the matrix metalloproteinases. CONCLUSIONS: This study has shown that interleukin-1 beta and transforming growth factor beta may regulate the synthesis of the matrix metalloproteinases by fibroblasts and that minor differences that occur in the matrix metalloproteinase profile are dependent on the suture type.
Anastomosis, Surgical↗
Increased degradation and altered tissue distribution of cartilage oligomeric matrix protein in human rheumatoid and osteoarthritic cartilage.
We investigated the degradation and tissue distribution of cartilage oligomeric matrix protein in normal, osteoarthritic, and rheumatoid arthritic articular cartilage of the human knee. Cartilage was subjected to sequential extractions with buffers containing neutral salt, with EDTA, and finally with guanidine/HCl and then was analyzed by Western blotting with a polyclonal antiserum to human cartilage oligomeric matrix protein. Western blots of the nine neutral salt extracts from normal cartilage revealed mostly intact pentameric molecules of cartilage oligomeric matrix protein, in contrast to the 13 osteoarthritic and five rheumatoid arthritic cartilage samples that demonstrated marked degradation of cartilage oligomeric matrix protein as noted by a predominance of reduction-sensitive bands at approximately 150 kDa and nonreduction-sensitive bands in the 67-94 kDa range. The EDTA and guanidine/HCl extracts from all groups were similar and showed mostly intact molecules of cartilage oligomeric matrix protein, with smaller amounts of degraded cartilage oligomeric matrix protein identical to those resolved by the Western blots of the neutral salt extracts. Western blots of matched pairs of synovial fluid and cartilage extracts demonstrated cartilage oligomeric matrix protein fragments of the same molecular mass. Competitive enzyme-linked immunosorbent assay revealed significantly less cartilage oligomeric matrix protein in rheumatoid articular cartilage than in either normal or osteoarthritic cartilage. In contrast to normal cartilage, where cartilage oligomeric matrix protein was predominantly localized to the interterritorial matrix throughout all zones of the matrix, with increased staining in the deeper cartilaginous zones, the most intense staining in osteoarthritic cartilage was in the superficial zones of fibrillated cartilage, with little to no immunostaining in the midzones and relatively poor staining in the deeper cartilaginous zones. This distribution was the inverse of that for proteoglycans, as demonstrated by toluidine blue staining, where proteoglycans were depleted primarily from the superficial fibrillated cartilage. In mild to moderately affected rheumatoid cartilage, the tissue distribution of cartilage oligomeric matrix protein was similar to the distribution of proteoglycans, with relatively uniform staining of the interterritorial and territorial matrics. In more severely affected rheumatoid cartilage, the superficial zones demonstrated punctate immunostaining for cartilage oligomeric matrix protein in the interterritorial and territorial matrics, and staining was restricted to the territorial matrix in the deep cartilaginous zones. It is evident from this study that (a) noncollagenous proteins such as cartilage oligomeric matrix protein are greatly affected in arthritis, (b) degradation fragments released from the matrix into the synovial fluid reflect the processes occurring within the matrix, and (c) different zones of the articular cartilage are susceptible to degradation of cartilage oligomeric matrix protein in the different disease processes.
Adult↗
Functional heterogeneity of the matrix compartment in the cat caudate nucleus as demonstrated by the cholinergic presynaptic regulation of dopamine release.
Previously, using a new in vitro microsuperfusion procedure, we have demonstrated marked differences in the cholinergic presynaptic regulation of the release of [3H]dopamine continuously synthesized from [3H]tyrosine in two close striosomal- and matrix-enriched areas of the cat caudate nucleus. A tetrodotoxin-resistant stimulatory effect of acetylcholine mediated by muscarinic receptors was observed in both compartments. However, in addition, two opposing types of tetrodotoxin-sensitive acetylcholine-evoked regulation of [3H]dopamine release were only seen in the matrix: one facilitatory, involving nicotinic receptors located on as yet unidentified neurons, and the other inhibitory, mediated by muscarinic receptors located on dynorphin-containing neurons. In the present study, using the same approach, a functional heterogeneity was demonstrated in the matrix. Indeed, in various conditions the effects of acetylcholine (50 microM) on the release of [3H]dopamine were different in a matrix-enriched area (matrix 2) distinct from that previously investigated (matrix 1); these areas being characterized by the presence or absence of islands of striatonigral cells, respectively. As in matrix 1, acetylcholine induced a short-lasting stimulation of [3H]dopamine release in matrix 2 but, in contrast to that observed in matrix 1, the acetylcholine-evoked response in matrix 2 was not modified in the presence of tetrodotoxin (1 microM). Experiments made in the presence of the tetrodotoxin and atropine (1 microM) indicated that both muscarinic and nicotinic receptors are located on dopaminergic nerve terminals in matrix 2 while muscarinic receptors are only present in matrix 1. In the absence of tetrodotoxin, the short-lasting stimulation of [3H]dopamine release was transformed into a long-lasting response in the presence of pempidine (50 microM), in matrix 2 but not in matrix 1 while prolonged responses were seen in both matrix areas in the presence of atropine. Finally, the acetylcholine short stimulatory effect on [3H]dopamine release was transformed into a long stimulatory response in the presence of bicuculline (50 microM) but not naloxone (1 microM) in matrix 2 while the reverse was observed in matrix 1. By providing further evidence for a functional heterogeneity of the matrix, our results suggest that depending on the matrix area investigated, dynorphin- or GABA-containing neurons are involved in the indirect cholinergic inhibitory control of dopamine release.
Acetylcholine↗
Functional role of matrix metalloproteinases in ovarian tumor cell plasticity.
OBJECTIVE: We previously demonstrated that aggressive ovarian cancer cells are able to display in vitro vasculogenic mimicry, which is reflected by their ability to form vasculogenic-like networks in 3-dimensional cultures and to express vascular cell-associated markers. The goal of this study was to examine the functional role of specific matrix metalloproteinases in the formation of vasculogenic-like networks and extracellular matrix remodeling in vitro. We also investigated the clinical relevance of matrix metalloproteinase-2 and -9 and membrane type 1-matrix metalloproteinase in human ovarian cancers with evidence of tumor cell-lined vasculature. STUDY DESIGN: Ovarian cancer cells (A2780-PAR, SKOV3, and EG) were seeded onto separate 3-dimensional cultures that contained either Matrigel or type I collagen, in the absence of endothelial cells or fibroblasts. These cultures were treated with either chemically modified tetracycline-3 (general matrix metalloproteinase inhibitor), recombinant tissue inhibitor of metalloproteinase-1 or -2, or function-blocking antibodies to matrix metalloproteinase-2 or -9 or membrane type 1-matrix metalloproteinase. In addition, 78 invasive epithelial ovarian cancers were evaluated for expression of matrix metalloproteinase-2 and -9 and membrane type 1-matrix metalloproteinase and correlated with various clinical parameters. RESULTS: The aggressive ovarian cancer cells (SKOV3 and EG) were able to form in vitro vasculogenic-like networks and contract 3-dimensional collagen I gels, whereas the poorly aggressive A2780-PAR cell line did not. Chemically modified tetracycline-3 completely blocked the network formation. Blocking antibodies to matrix metalloproteinase-2 and membrane type 1-matrix metalloproteinase inhibited the formation of the vasculogenic-like networks and collagen gel contraction, but the antibody to matrix metalloproteinase-9 had no effect on network formation and minimal effect on gel contraction. Treatment of 3-dimensional cultures with tissue inhibitor of metalloproteinase-2 retarded the network formation and only small, partially developed structures were noted that did not form network connections. Tissue inhibitor of metalloproteinase-1 had no appreciable effect on the extent or efficiency of network formation. Human invasive ovarian cancers with evidence of tumor cell-lined vasculature were significantly more likely to have strong epithelial and stromal matrix metalloproteinase-2 and -9 and membrane type 1-matrix metalloproteinase expression (all probability values were <.05). CONCLUSION: Matrix metalloproteinase-2 and membrane type 1-matrix metalloproteinase appear to play a key role in the development of vasculogenic-like networks and matrix remodeling by aggressive ovarian cancer cells. Human ovarian cancers with matrix metalloproteinase overexpression are more likely to have tumor cell-lined vasculature. These results may offer new insights for consideration in ovarian cancer treatment strategies.
Adult↗
Function blocking autoantibodies against matrix metalloproteinase-1 in patients with systemic sclerosis.
Systemic sclerosis is characterized by fibrosis and systemic autoimmunity; however, roles of autoantibodies in the development of fibrosis remain unknown in systemic sclerosis. The net accumulation of extracellular matrix is dependent on the balance between the synthesis and degradation of extracellular matrix components, the latter process regulated by matrix metalloproteinases. Matrix metalloproteinase-1 (interstitial collagenase-1) can initiate degradation of collagen types I-III that are major extracellular matrix constituents in affected skin of systemic sclerosis. In this study, we tested the hypothesis that systemic autoimmunity in systemic sclerosis induced anti-matrix metalloproteinase-1 autoantibodies that inhibited matrix metallo-proteinase-1 activity, resulting in collagen accumulation. Enzyme-linked immunosorbent assay using human recombinant matrix metalloproteinase-1 revealed that IgG anti-matrix metalloproteinase-1 autoantibody levels were significantly elevated in sera from patients with systemic sclerosis, but not patients with active systemic lupus erythematosus or dermatomyositis, relative to normal controls. IgG anti-matrix metalloproteinase-1 autoantibody levels were significantly higher in patients with diffuse cutaneous systemic sclerosis than those found in patients with limited cutaneous systemic sclerosis. Furthermore, IgG anti-matrix metalloproteinase-1 antibody levels significantly correlated with the extent of fibrosis in the skin, lung, and renal blood vessels. The presence of IgG anti-matrix metalloproteinase-1 autoantibody in sera from systemic sclerosis patients was confirmed by immunoblotting analysis. Remarkably, IgG anti-matrix metalloproteinase-1 autoantibody in sera from systemic sclerosis patients inhibited matrix metalloproteinase-1 collagenase activity. Collectively, the results of this study suggest that anti-matrix metalloproteinase-1 autoantibody contributes to the development of fibrosis by inhibiting matrix metalloproteinase-1 collagenase activity and reducing the extracellular matrix turnover and suggest that the presence of anti-matrix metalloproteinase-1 autoantibody in systemic sclerosis is the link between systemic autoimmunity and fibrosis.
Adult↗
Dynamics of the matrix metalloproteinases MMP-1 and MMP-8 in acute open human dermal wounds.
Extracellular matrix degradation during dermal wound healing involves multiple levels of regulation by several enzymes of the matrix metalloproteinase family, their activators, and their inhibitors. This study tested the hypothesis that a temporal pattern of interstitial collagenase appearance occurs during normal dermal wound healing, with matrix metalloproteinase-8 originating from neutrophils appearing earlier than the fibroblast-derived matrix metalloproteinase-1. Open (6 mm) full-thickness dermal wounds, which were covered by transparent occlusive dressings, were made in healthy human volunteers (n = 20). Wound fluids from under the dressings were collected daily through day 8, and wound tissue biopsies were obtained on days 0, 2, 4, 14, and 28. Collagenases were extracted from homogenized tissue biopsies for analysis. Samples were analyzed for the presence of matrix metalloproteinase-1 and matrix metalloproteinase-8 by enzyme-linked immunosorbent assays and by collagenase activity assays using purified types I and III collagen as substrates. In addition, tissue inhibitor of metalloproteinases-1 and matrix metalloproteinase-1/tissue inhibitor of metalloproteinases-1 complexes in wound fluids were measured. Results showed a differential temporal pattern of matrix metalloproteinase-1 and matrix metalloproteinase-8 in wound exudates with peak levels of matrix metalloproteinase-8 occurring on day 4 and matrix metalloproteinase-1 peak levels on day 7. Maximal levels in tissue for both enzymes occurred on day 2. At all time points examined, levels of matrix metalloproteinase-8 were statistically higher than matrix metalloproteinase-1 (100-fold to 200-fold). Tissue inhibitor of metalloproteinases-1 levels declined over time, whereas levels of matrix metalloproteinase-1/tissue inhibitor of metalloproteinase-1 complexes increased to a plateau on day 7. This study provides new evidence implicating matrix metalloproteinase-8 as a major collagenase in healing human dermal wounds. It also shows a temporal pattern in the appearance of the matrix metalloproteinases, tissue inhibitor of metalloproteinase-1, and matrix metalloproteinase-1/tissue inhibitor of metalloproteinases-1 complexes, suggesting that a tightly regulated pattern of expression of matrix metalloproteinases and their inhibitors is essential for normal wound healing in humans.
Adult↗
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.
Animals↗
Matrix metalloproteinase inhibitor BB-3103 unlike the serine proteinase inhibitor aprotinin abrogates epidermal healing of human skin wounds ex vivo.
Several matrix metalloproteinases and serine proteinases are upregulated in migrating keratinocytes during cutaneous wound repair. Single cell culture studies indicate the necessity for matrix metalloproteinases but not for serine proteinases in keratinocyte locomotion. To account for epithelial-mesenchymal interactions, an ex vivo human skin wound model was used to investigate the contribution of matrix metalloproteinases and serine proteinases to wound healing by treatment with broad-spectrum inhibitors of matrix metalloproteinases (BB-3103) or serine proteinases (aprotinin). Human skin explants with circular 3 mm superficial defects were incubated in culture medium without (controls) or with the proteinase inhibitors for 7 d. BB-3103 abrogated epithelialization (p < 0.001), whereas aprotinin-treated wounds and controls were covered with new epithelium. Lack of epithelialization was unlikely due to cytotoxicity because the matrix metalloproteinase inhibitor did neither influence viability of cultured epidermal keratinocytes nor apoptosis in wounds. Involvement of specific matrix metalloproteinases in epithelialization was analyzed by gelatin zymography, western blotting, immunohistochemistry, and in situ hybridization. Wound healing was accompanied by active matrix metalloproteinase-1 and increased active matrix metalloproteinase-2 but irrespectively of active matrix metalloproteinase-9. BB-3103 blocked activation of matrix metalloproteinase-2 and matrix metalloproteinase-9 but not of matrix metalloproteinase-1. Active matrix metalloproteinase-2 localized solely to the dermis, whereas matrix metalloproteinase-9 was consistently found in new epithelium. Membrane-type 1 matrix metalloproteinase was undetectable in wound keratinocytes. BB-3103 and aprotinin reduced tumor necrosis factor-alpha in media but did not appreciably alter amounts of other soluble regulators of matrix metalloproteinases and epithelialization. Our findings demonstrate that keratinocyte migration is associated with active matrix metalloproteinase-2 but occurs independently of serine proteinases and active matrix metalloproteinase-9 in fibrin-deficient skin wound healing.
Adult↗
Activation of latent transforming growth factor beta1 by stromelysin 1 in extracts of growth plate chondrocyte-derived matrix vesicles.
Previous studies have shown that matrix vesicles isolated from cultures of costochondral growth zone chondrocytes and treated with 1alpha,25-dihydroxyvitamin D3 [1alpha,25(OH)2D3] can activate recombinant human latent transforming growth factor beta1 (rhTGF-beta1). It is unknown what enzyme or other factor in the extracellular organelles is responsible for the activation. This study tested the hypothesis that enzymes present in matrix vesicles can activate latent TGF-beta1 and that this is regulated by 1alpha,25(OH)2D3. To do this, we examined the ability of matrix vesicle extracts to activate small latent rhTGF-beta1. In addition, enzymes previously determined to be present in matrix vesicles were screened for their ability to activate small latent rhTGF-beta1. Recombinant human matrix metalloproteinase 2 (rhMMP-2; 72 kDa gelatinase), rhMMP-3 (stromelysin 1), purified human plasminogen, and purified urokinase (plasminogen activator) were each tested at varying concentrations. To assess the role of cell maturation, we used a cell culture model in which chondrocytes are derived from two distinct zones of rat costochondral cartilage, the resting zone and the growth zone. Matrix vesicles were isolated from these cultures and then tested. The results showed that extracts of matrix vesicles produced by both growth zone and resting zone chondrocytes were able to activate small latent rhTGF-beta1. The effects were dose and time dependent, with greater activity being found in extracts of matrix vesicles from the growth zone chondrocyte cultures. Only rhMMP-3 was able to activate small latent rhTGF-beta1, indicating that stromelysin-1, but not MMP-2, plasminogen, or urokinase, was involved. As observed in the extracts, the effect of rhMMP-3 was time and dose dependent. When anti-MMP-3 antibody was added to matrix vesicle extracts from both cell types, activation of small latent rhTGF-beta1 was dose-dependently blocked. Neither 1alpha,25(OH)2D3 nor 24R,25(OH)2D3 had a direct effect on activation of small latent rhTGF-beta1 by the extracts. However, when intact matrix vesicles were treated with 1alpha,25(OH)2D3, their ability to activate small latent rhTGF-beta1 was increased. Inhibition of phospholipase A2 with quinacrine blocked the 1alpha,25(OH)2D3-dependent effect. These results suggest that the ability of 1alpha,25(OH)2D3-treated matrix vesicles to activate small latent TGF-beta1 is via action of the secosteroid on the matrix vesicle membrane, not on the enzymes responsible for activating latent TGF-beta1. Because matrix vesicles isolated from growth zone chondrocytes have been shown to contain increased phospholipase A2 activity after treatment with 1alpha,25(OH)2D3, it is likely that this secosteroid promotes loss of membrane integrity through phospholipase A2-dependent formation of lysophospholipids, resulting in the release of MMP-3 into the matrix, where latent TGF-beta1 is stored. Taken together, the results of the current study show that matrix vesicles produced by growth plate chondrocytes contain MMP-3, that this enzyme is at least partially responsible for activation of small latent TGF-beta1 in the matrix, and that 1alpha,25(OH)2D3 regulates MMP release from matrix vesicles.
Animals↗
Coexpression of integrin alpha(v)beta3 and matrix metalloproteinase-2 (MMP-2) coincides with MMP-2 activation: correlation with melanoma progression.
Tumor cell invasion and metastasis formation depend on both adhesive and proteolytic mechanisms. Previous studies have shown that expression of matrix metalloproteinase-2 and integrin alphavbeta3 correlate with melanoma progression. Recently, direct binding of matrix metalloproteinase-2 to alpha(v)beta3 was implicated in presenting activated matrix metalloproteinase-2 on the cell surface of invasive cells. In this study we investigated this, using the highly metastatic, alpha(v)beta3-negative melanoma cell lines MV3 and BLM, their beta3-transfected alpha(v)beta3 expressing counterparts, xenografts derived from these cell lines, and fresh human cutaneous melanoma lesions comprising all stages of melanoma progression. Expression and activation status of matrix metalloproteinase-2 were studied by reverse transcription-polymerase chain reaction, immunohistochemistry, western blotting, and zymographic analysis, respectively. Matrix metalloproteinase-2 protein expression in vitro was similar in both alpha(v)beta3-negative and alpha(v)beta3-positive cell lines Remarkable differences, however, exist in the localization of inactive and active matrix metalloproteinase-2. Soluble active matrix metalloproteinase-2 was detectable only in the conditioned medium of alpha(v)beta3-negative cell lines and undetectable in the alpha(v)beta3-positive cell lines. Conversely, active matrix metalloproteinase-2 was present exclusively on the cell surface of the alpha(v)beta3 expressing transfectants. Western blot analysis of other components that are involved in matrix metalloproteinase-2 activation showed that processing of proMT1-matrix metalloproteinase to the activated form was enhanced in beta3 transfectants, whereas secretion of tissue inhibitor of metalloproteinase-2 was decreased. In vivo, the presence of functionally active matrix metalloproteinase-2 was significantly higher in xenografts derived from the alpha(v)beta3 expressing MV3 and BLM cell lines. In human cutaneous melanoma lesions, neither matrix metalloproteinase-2 nor integrin alpha(v)beta3 is detectable in melanoma in situ as determined by immunohistochemistry. In contrast, the number of matrix metalloproteinase-2-positive and alphavbeta3-positive tumor cells was clearly increased in primary melanomas, and melanoma metastases. Double staining experiments and confocal laser microscopy demonstrated that the percentage of cells coexpressing matrix metalloproteinase-2 and alpha(v)beta3 increased in advanced primary melanomas and melanoma metastases. In addition, zymography showed that functionally active matrix metalloproteinase-2 was frequently present in melanoma metastases. In these lesions a high proportion of matrix metalloproteinase-2- and alphavbeta3-double-positive melanoma cells were detectable. Our study demonstrates that the presence of activated matrix metalloproteinase-2 correlates with expression of alpha(v)beta3 in human melanoma cells both in vitro and in vivo, and also in fresh human melanoma lesions. These findings strongly suggest that co-ordinated expression of both factors may be required for melanoma cell invasion and metastasis formation.
Disease Progression↗