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PubMed2006

Cyclooxygenase-2 expression correlates with membrane-type-1 matrix metalloproteinase expression in colorectal cancer tissue.

PURPOSE: Elevated expression of cyclooxygenase-2 has been found in colorectal cancer. One of the mechanisms through which cyclooxygenase-2 affects tumorigenesis is through its overexpression, which leads to increased invasiveness of cancer cells. A crucial step in this pathway is thought to be the induction of membrane-type-1 matrix metalloproteinase, which activates matrix metalloproteinase-2. However, to date there have been few clinicopathologic studies concerning cyclooxygenase-2-mediated invasiveness in human colorectal cancer tissues. METHODS: We performed immunohistochemical analysis of the respective antigens on colorectal cancer specimens obtained by surgical resections from 96 patients with colorectal cancer. RESULTS: Cyclooxygenase-2 and membrane-type-1 matrix metalloproteinase expression was positive exclusively in cancer cells in 88 cases (92 percent) and 23 cases (24 percent), respectively. All 23 cases expressing membrane-type-1 matrix metalloproteinase also expressed cyclooxygenase-2. Matrix metalloproteinase-2 expression was positive in cancer cells in 20 cases (21 percent) and stromal cells in 52 cases (54 percent). Expression of matrix metalloproteinase-2 in cancer cells correlated with lymphatic invasion and local recurrence. Statistically, a significant correlation was found between cyclooxygenase-2 and membrane-type-1 matrix metalloproteinase expression, and membrane-type-1 matrix metalloproteinase and matrix metalloproteinase-2 expression in cancer cells. There was no association between cyclooxygenase-2 expression and matrix metalloproteinase-2 expression. However, immunostaining of serial sections revealed that in the majority of cases examined, nearly 100 percent of cancer cells expressing matrix metalloproteinase-2 also coexpressed cyclooxygenase-2. CONCLUSIONS: This study indicates strong association between both cyclooxygenase-2 and membrane-type-1 matrix metalloproteinase expression, and membrane-type-1 matrix metalloproteinase and matrix metalloproteinase-2 in colorectal cancer. These results support our thesis of a direct correlation between cyclooxygenase-2 and membrane-type-1 matrix metalloproteinase expression--with consequent association between cyclooxygenase-2 and matrix metalloproteinase-2 activation, and tumor invasiveness andrecurrence in certain cases of colorectal cancer.

Adult↗
PubMed1984

Importance of geometry of the extracellular matrix in endochondral bone differentiation.

Subcutaneous implantation of coarse powders (74-420 micron) of demineralized diaphyseal bone matrix resulted in the local differentiation of endochondral bone. However, implantation of matrix with particle size of 44-74 micron (Fine matrix) did not induce bone. We have recently reported that the dissociative extraction of coarse matrix with 4 M guanidine HCl resulted in a complete loss of the ability of matrix to induce endochondral bone; the total loss of biological activity could be restored by reconstitution of extracted soluble components with inactive residue. To determine the possible biochemical potential of fine matrix to induce bone, the matrix was extracted in 4 M guanidine HCl and the extract was reconstituted with biologically inactive 4 M guanidine HCl-treated coarse bone matrix residue. There was a complete restoration of the biological activity by the extract of fine matrix upon reconstitution with extracted coarse matrix. Polyacrylamide gel electrophoresis of the extract of fine matrix revealed similar protein profiles as seen for the extract of coarse matrix. Gel filtration of the 4 M guanidine HCl extract of fine powder on Sepharose CL-6B and the subsequent reconstitution of various column fractions with inactive coarse residue showed that fractions with proteins of 20,000-50,000 mol wt induced new bone formation. These observations demonstrate that although fine bone matrix contains, osteoinductive proteins, matrix geometry (size) is a critical factor in triggering the biochemical cascade of endochondral bone differentiation. Mixing of coarse matrix with Fine results in partial response and it was confined to areas in contact with coarse particles. The results imply a role for geometry of extracellular bone matrix in anchorage-dependent proliferation and differentiation of cells.

Alkaline Phosphatase↗
PubMed1984

The role of intermolecular disulfide bonding in deposition of GP140 in the extracellular matrix.

Human WI-38 fibroblasts in cultures synthesized at least three molecular forms of the major, extracellular matrix glycoprotein (GP), GP140: (a) cytoplasmic GP140 (1.2 ng of GP140/micrograms of cell protein) was detergent-soluble, underglycosylated, and possessed detectable levels of intermolecular disulfide bonding; (b) matrix GP140 (3.6 ng of GP140/micrograms of cell protein) was detergent-insoluble, more highly glycosylated and polymerized by intermolecular disulfide bonding, and co-distributed in the extracellular matrix with fibronectin; and (c) released GP140 (2 ng of GP140/micrograms of cell protein per 24 h) was recovered in the conditioned culture media and lacked intermolecular disulfide bonding. Cytoplasmic GP140 was the immediate biosynthetic precursor of the matrix form of GP140. In addition, various human adult and fetal tissues contained a form of GP140 that resembled the fibroblast matrix GP140 in the degree of intermolecular disulfide bonding, relative molecular mass, and immunological reactivity. Analysis of the sequence of events in assembly of GP140 and fibronectin in the extracellular matrix detected the following: (a) fibronectin was first to appear in the extracellular matrix; (b) GP140 accumulated in the cytoplasm, then deposited in the extracellular matrix and co-aligned with the established fibronectin; and (c) maturation of the extracellular matrix proceeded by continued intermolecular disulfide bonding. To evaluate possible roles for intermolecular disulfide bonding in cell interactions, a unique assay system was utilized based on the ability of labeled cells to incorporate radioactive matrix components into a biotinylated exogenous matrix. Precipitation of the biotinylated matrix from extracts of the cultures using avidin indicated: (a) disulfide bonding of radioactive GP140 and fibronectin into the exogenous biotinylated matrix required cell contact with the matrix. The newly deposited GP140 and fibronectin derived from the cells and not from GP140 and fibronectin present in the conditioned culture media. (b) Pro-alpha 1 and Pro-alpha 2 procollagens, present in the culture media, bound to the exogenous matrix in a noncovalent manner and were independent of cell contact. (c) SV40 transformed cells (WI-38 VA13) synthesized released form GP140 but did not deposit GP140 into the biotinylated matrix.

Cell Line↗
PubMed2007

Platelet-rich plasma inhibits demineralized bone matrix-induced bone formation in nude mice.

BACKGROUND: It is unclear whether platelet-rich plasma is a clinically effective adjunct to osteoinductive agents such as demineralized bone matrix. It contains platelet-derived growth factor (PDGF), which decreases osteoinduction by human demineralized bone matrix in nude-mouse muscle, suggesting that platelet-rich plasma may also have a negative impact. This study tested the hypothesis that platelet-rich plasma reduces demineralized bone matrix-induced bone formation and that this effect varies with donor-dependent differences in platelet-rich plasma and demineralized bone matrix. METHODS: Human platelet-rich plasma was prepared from blood from six men (average age [and standard error of the mean], 29.2 +/- 2.4 years). Platelet numbers were determined, and growth factors were quantified before and after platelet activation. Human demineralized bone matrix from two donors (demineralized bone matrix-1 and demineralized bone matrix-2) was mixed with activated platelet-rich plasma and was implanted bilaterally in the gastrocnemius muscle in eighty male nude mice (eight implants per variable). Fifty-six days after implantation, the hindlimb calf muscles were harvested for histological analysis. Osteoinduction was evaluated with use of a qualitative score and morphometric measurements of ossicle size, new bone formation, and residual demineralized bone matrix. RESULTS: Compared with platelet-poor plasma, platelet-rich plasma preparations exhibited a fourfold increase in the platelet count, a fifteenfold increase in the amount of transforming growth factor-beta, a sixfold increase in the amount of PDGF-BB, a fivefold increase in the amount of PDGF-AA, and a twofold increase in the amount of PDGF-AB. Demineralized bone matrix-1 was more osteoinductive than demineralized bone matrix-2, as determined on the basis of a greater ossicle area. The effect of platelet-rich plasma was either neutral or inhibitory depending on the demineralized bone matrix batch. When used with demineralized bone matrix-1, platelet-rich plasma did not alter the qualitative score or overall ossicle size, but it decreased the new bone area. When used with demineralized bone matrix-2, platelet-rich plasma reduced the qualitative score, ossicle area, and new bone area and increased the amount of residual demineralized bone matrix. The effects on osteoinduction also varied with the donor of the platelet-rich plasma. CONCLUSIONS: Platelet-rich plasma decreased the osteoinductivity of demineralized bone matrix implanted in immunocom-promised mice, and the activities of both demineralized bone matrix and platelet-rich plasma were donor-dependent.

Adult↗
PubMed2004

Expression of hair keratins in the adult nail unit: an immunohistochemical analysis of the onychogenesis in the proximal nail fold, matrix and nail bed.

BACKGROUND: Recently, the expression profiles of the members of the complex hair keratin family have been determined in the human anagen hair follicle. In contrast, the details of hair keratin expression in the human nail unit are poorly known. OBJECTIVES: In order to fill this gap, we have performed an immunohistochemical study of the adult human nail unit by means of specific antibodies against nine hair keratins of both types (hHa2, hHb2, hHa5, hHb5, hHa1, hHb1, hHb6, hHa4 and hHa8) as well as three epithelial keratins (K5, K17 and K10). METHODS: Formalin-fixed paraffin sections of adult nails were examined using monoclonal and polyclonal keratin antibodies, respectively. Longitudinal as well as transverse sections were investigated. RESULTS: Our study revealed two types of epithelial tissue compartments in the nail unit. The first comprised the eponychium and hyponychium and the nail bed, which expressed only epithelial keratins. While keratins K5, K17 (basal) and K10 (suprabasal) were found in the orthokeratinizing eponychium and hyponychium, throughout, the nail bed epithelium expressed only K5 and K17. The second type comprised the apical and ventral matrix which exhibited a mixed pattern of epithelial and hair keratin expression. Thus, K5 and K17 were expressed in the entire multilayered basal cell compartment of the apical and ventral matrix; however, in the latter, K5 and K17 also occurred in the lowermost layers of the overlying keratogenous zone. The hair matrix keratin hHb5, but not its type II partner hHa5, was seen in the entire keratogenous zone of the apical and ventral matrix, but was also located in the uppermost cell layers of the basal compartment of the ventral matrix, where it overlapped with K5 and K17. Similar to their sequential expression in the hair follicle cortex, hair keratins hHa1, hHb1, hHb6 and hHa4 were consecutively expressed in the keratogenous zone of both the ventral and, albeit less distinctly, apical matrix, with hHa1 initiating in the lowermost cell layers. The expression of hHa8 in only single cortex cells of the hair follicle was also preserved in cells of the keratogenous zone. In the region of the so-called dorsal matrix, we observed two histologically and histochemically distinct types of epithelia: (i) a dominant type, histologically similar to the eponychium and an associated K5, K17 and K10 keratin pattern which clearly extended into the apical matrix, and (ii) a minor type, histologically resembling the postulated dorsal matrix without a granular layer and a cuticle, and exhibiting extended K5 expression as well as hair keratin expression in superficial cells. CONCLUSIONS: The coexpression of hHb5 with K5 and K17 in the uppermost cell layers of the basal compartment and the lowermost layers of the keratogenous zone of the ventral matrix prompts us to designate this region the prekeratogenous zone of the ventral matrix. The two alternating types of histology and keratin expression in the dorsal matrix identify this region as a transitional zone between the eponychium and the apical matrix. Finally, our data clearly show that the ventral matrix is the main source of the nail plate. In addition, the mixed scenario of hair and epithelial keratins, including demonstrable amounts of K10, in superficial cells of the apical matrix, lends support to the notion that the dorsal portion of the nail is generated by the apical matrix.

Adult↗
PubMed1993

Apolipoprotein E modulates low density lipoprotein retention by lipoprotein lipase anchored to the subendothelial matrix.

Lipoprotein lipase (lipase), a key enzyme in lipoprotein triglyceride metabolism, has been shown to markedly increase low density lipoprotein (LDL) retention by subendothelial matrix. In the present study we assessed the role that lipoprotein and matrix components play in retention of LDL by lipase anchored to the subendothelial matrix. Lipase addition to subendothelial matrix increased LDL retention by 66-fold. Scatchard analysis of LDL binding to lipase-containing matrix yielded an association constant of 12 nM. Exogenous addition of the matrix components, heparan sulfate and dermatan sulfate (i.e. chondroitin sulfate B), reduced LDL retention by greater than 90%. These glycosaminoglycans (GAGs) also reduced lipolytic activity associated with the matrix, suggesting that lipase was released from its binding sites on the matrix. In contrast, other matrix components (collagen, fibronectin, vitronectin, and chondroitin sulfate A) neither affected LDL release nor matrix lipolytic activity. Thus, heparan sulfate and dermatan sulfate function to anchor lipase to the subendothelial cell matrix. The effects of apolipoprotein E (apoE) and apoA-I were also examined. Preincubation of the subendothelial matrix with apoE, followed by washing, did not affect subsequent lipase binding to the matrix nor its ability to retain LDL. However, the direct addition of apoE alone or in combination with phospholipid liposomes decreased lipase-mediated LDL retention in a concentration-dependent fashion. Addition of apoA-I had no effect. Thus, in these studies apoE functions to displace LDL bound to lipase, but not lipase anchored to the matrix. To further examine the physiologic implications of this process, we assessed the ability of human apoE-rich and apoE-poor high density lipoproteins (HDL) to displace LDL from matrix-anchored lipase. ApoE-rich HDL reduced LDL retention dramatically (86% at 2.5 micrograms/ml). In contrast, apoE-poor HDL, at the highest concentration evaluated (400 micrograms/ml), decreased LDL retention by only 32%. Overall, these data suggest apoE and specifically apoE-containing HDL reduce the lipase-mediated retention of LDL by subendothelial matrix. This observation, in part could explain the protective effects of apoE and apoE-containing HDL against atherosclerosis.

Animals↗
PubMedDate not supplied

Matrix metalloproteinase production in regenerating axolotl spinal cord.

In urodele amphibian spinal cord regeneration, the ependymal cells lining the central canal remodel the lesion site to favor axonal regrowth. We profiled the production of matrix metalloproteinases by injury-reactive mesenchymal ependymal cells in vivo and in vitro and found that matrix metalloproteinases are involved in this remodeling process in the axolotl (Ambystoma mexicanum). The production of cell-associated matrix metalloproteinases in vivo was shown to be identical to that in our cultured ependymal cell model system. Activated and zymogen forms of matrix metalloproteinases were identified using zymography, chemical inhibitors of matrix metalloproteinases, and cleavage of propeptides by organomercurials. The principal cellular proteinases consisted of matrix metalloproteinase-2 (gelatinase A) and matrix metalloproteinase-1 (type I collagenase), which display characteristic shifts in molecular weight following proenzyme processing by organomercurials. In addition, ependymal cell conditioned medium contained secreted forms of the enzyme undetectable in situ. Matrix metalloproteinase-9 (gelatinase B) as well as matrix metalloproteinase-2 and matrix metalloproteinase-1 were secreted and casein substrate zymography showed the presence of a small amount of a very high molecular weight matrix metalloproteinase-3 (prostromelysin) secreted into the culture medium. Matrix metalloproteinases were still present at 4 weeks post-lesioning when the ependymal cells have just re-epithelialized, but decreased near the completion of regeneration (8 weeks post-lesioning). Zymography showed no detectable matrix metalloproteinases in unlesioned cord but the presence of tissue inhibitor of metalloproteinase-1 in intact cord was seen by Western blotting. This study shows that matrix metalloproteinases are associated with urodele spinal cord regeneration and validates the use of our ependymal cell tissue culture model system to evaluate ependymal cell behavior during spinal cord regeneration.

Ambystoma mexicanum↗
PubMed2004

Effects of mutations in an HIV-1 gag gene containing a 107-codon tandem repeat in the matrix region on assembly and processing of the protein product.

It has been demonstrated previously that a human immunodeficiency virus (HIV) type 1 Gag mutant (MA2) with a tandem repeat of 107-matrix codons in the matrix domain could direct virus particle assembly and budding [Wang et al. (2000c): J Med Virol 61:423-432]. Since the regions involved functionally in HIV Gag assembly and transport have been mapped to the matrix domain, it was interesting to test the effects of the duplicated matrix-coding sequence on Gag assembly, transport, and virus processing of some assembly-defective HIV matrix mutants. In this study, a number of HIV matrix mutations were introduced into either the proximal or distal copy of the duplicated matrix-coding sequence. Assembly, release, processing, and subcellular localization of the Gag mutants were analyzed by transient expression in 293T cells. The result indicates that the budding defect of HIV matrix mutants could be moderately or significantly reversed when the additional 107-matrix codons were present; however, these matrix double mutations affected significantly the virus particle processing. Mislocalized matrix mutants could also be redistributed to a certain degree in the presence of the duplicated matrix copy. Although the subcellular distribution patterns of the matrix mutants did not correlate completely with the budding efficiency, the data suggest that the budding defect caused by the matrix mutations could be masked to some extent by the duplicated matrix coding sequence.

Cell Culture Techniques↗
PubMed2002

Identification of matrix metalloproteinase-9 in amniotic fluid and amniochorion in spontaneous labor and after experimental intrauterine infection or interleukin-1 beta infusion in pregnant rhesus monkeys.

OBJECTIVE: The purpose of this study was to examine the roles of intrauterine infection, inflammation, and spontaneous labor on the expression of matrix metalloproteinase-9 in fetal membranes and amniotic fluid of late pregnant rhesus monkeys. STUDY DESIGN: Pregnant rhesus monkeys with timed gestations were chronically catheterized to allow serial sampling of amniotic fluid before and during experimentally induced intrauterine inflammation. Six animals received group B streptococci into the chorionic-decidual space, and 4 animals received intra-amniotic interleukin-1 beta infusions (10 microg). Three additional animals were serially sampled by amniocentesis through late pregnancy until spontaneous term labor. Amniotic fluid samples were examined by zymography for matrix metalloproteinase-9 and -2 and Western immunoblot for matrix metalloproteinase-9 and -2 and tissue inhibitors of metalloproteinase-1 and -2. Fetal membranes were obtained at cesarean delivery during labor (before rupture), formalin fixed, and embedded in paraffin for immunocytochemistry of matrix metalloproteinase-9 and in situ hybridization of matrix metalloproteinase-9 messenger RNA. Tissues from 2 additional animals were collected as age-matched non-labor controls. RESULTS: In amniotic fluid, the 92-kd latent matrix metalloproteinase-9 was detectable in late pregnancy and increased dramatically, followed by the appearance of the 83-kd active form before spontaneous term delivery. Amniotic fluid matrix metalloproteinase-2 levels (both latent and active forms) remained relatively constant throughout pregnancy and in labor. Both bacteria and interleukin-1 beta rapidly increased the signal of latent matrix metalloproteinase-9 without a consistent increase in the active form before the onset of labor. Chorionic-decidual inoculation of group B streptococci was followed by the expression of latent matrix metalloproteinase-9 before the appearance of group B streptococci in amniotic fluid or the onset of labor. Matrix metalloproteinase-2 increased to a new steady-state level or remained unchanged after group B streptococci inoculation or interleukin-1 beta infusion, respectively. Amniotic fluid tissue inhibitors of metalloproteinase-1 declined and tissue inhibitors of metalloproteinase-2 remained unchanged during early group B streptococci infection, after interleukin-1 beta infusion and on the day of spontaneous term labor. However, both tissue inhibitors of metalloproteinase-1 and -2 levels increased after preterm labor that was induced by group B streptococci. Immunocytochemistry localized matrix metalloproteinase-9 protein to amnion and chorion epithelial and mesenchymal cells and decidual stromal cells. Granular matrix metalloproteinase-9 staining was observed in the connective tissue layer of inflamed fetal membranes. In situ hybridization for messenger RNA confirmed the production of matrix metalloproteinase-9 by amnion and chorion. CONCLUSION: Bacterial- and interleukin-1 beta-induced preterm labor and spontaneous term labor are preceded and accompanied by progressive increases in amniotic fluid matrix metalloproteinase-9 (92 kd) in rhesus monkeys. Amniotic fluid matrix metalloproteinase-9 may serve as a clinical marker for the onset of both preterm and term labor.

Amnion↗
PubMed1998

Experimental posterolateral lumbar spinal fusion with a demineralized bone matrix gel.

STUDY DESIGN: A controlled rabbit model of lumbar posterolateral intertransverse process arthrodesis was used to evaluate a bone graft substitute. OBJECTIVE: To determine the efficacy of demineralized bone matrix gel as an autograft extender, using different ratios of demineralized bone matrix to autograft and to determine the efficacy of demineralized bone matrix as an autogenous bone graft enhancer by adding it to the usual quantity of autograft. SUMMARY OF BACKGROUND DATA: Autogenous bone is considered the most effective bone graft material for posterolateral lumbar arthrodesis, yet nonunions occur in up to 30% of patients. In addition, donor site complications may occur in 25-30% of patients. This has prompted the search for and investigation of bone graft extenders, enhancers, and substitutes. Commercially available demineralized bone matrix gel is one possible graft extender and enhancer, which, unlike mineralized allografts, has osteoinductive properties. Although the gel is in common use, the efficacy of demineralized bone matrix when used for posterolateral spine arthrodesis has not been examined in prospective clinical studies. Furthermore, no known animal studies have tested demineralized bone matrix gel in a posterolateral arthrodesis model. METHODS: Forty-seven New Zealand white rabbits underwent bilateral posterolateral spine arthrodesis at L5-L6 using autogenous iliac crest bone graft alone or in combination with demineralized bone matrix. Four groups were formed on the basis of the ratio of autograft to demineralized bone matrix: autograft alone (3 mL), 100:0 group; autograft (3 mL) and demineralized bone matrix (1.5 mL), 100:50 group; autograft (1.5 mL) and demineralized bone matrix (1.5 mL), 50:50 group; and autograft (0.75 mL) and demineralized bone matrix (2.25 mL), 25:75 group. Rabbits were killed 6 weeks after surgery. Inspection, manual palpation, radiographic film, and histologic evaluation were used to assess fusion. RESULTS: All groups had similar fusion rates (66-73%) based on manual palpation. Rabbits implanted with demineralized bone matrix had more mature fusion masses, evidenced by the greater trabecular bone formation seen on radiographic film and histologic study. CONCLUSIONS: Demineralized bone matrix was effective as a graft extender when used in up to a 3:1 ratio with autograft in a rabbit posterolateral spine fusion model. When less than the standard volume of autograft was used, the addition of demineralized bone matrix gel lead to fusion success rates comparable to those of the standard amount of autograft alone. However, demineralized bone matrix did not increase the frequency of successful fusion when added to the standard amount of autograft.

Animals↗
PubMed1991

The remodeling of synaptic extracellular matrix and its dynamic relationship with nerve terminals at living frog neuromuscular junctions.

The question of whether the synaptic extracellular matrix undergoes remodeling and how this remodeling is related to nerve terminal plasticity was examined in living neuromuscular junctions of adult frogs. Sartorius muscles were double stained with a fluorescent nerve terminal dye 4-(4-diethylamino-styryl)-N-methylpyridinium iodide (4-Di-2-Asp) and rhodamine-tagged peanut agglutinin (PNA) which recognizes synaptic extracellular matrix. Both nerve terminals and synaptic extracellular matrix in 200 identified normal junctions were visualized in vivo two or three times over a period of 2.6-6 months. The majority of neuromuscular junctions (NMJs) showed remodeling of both nerve terminals and synaptic extracellular matrix. Only 2.5% showed no changes in either synaptic element. The most commonly seen remodeling involved correlated changes in both nerve terminals and synaptic extracellular matrix. In this large group, while some junctions (20%) showed overall proportionate changes in all branches, most junctions (68%) showed disproportionate extension and/or retraction of some but not all individual branches. Another group of NMJs (9.5%) showed mismatched changes in the nerve terminal and synaptic extracellular matrix. In this group, some NMJs showed a decrease in the nerve terminal length without a corresponding reduction in synaptic extracellular matrix length. In other junctions that displayed extension of branches, the PNA-stained matrix was longer than the distal tip of the nerve terminal. Morphometric analysis indicated an average increase of 15.6% in total nerve terminal length and 13.6% in total synaptic extracellular matrix length. Although almost all NMJs displayed remodeling in at least one branch, about 50% of the 2201 individual branches examined did not show changes. The average change was 8.9% growth in the length of individual nerve terminal branches and 8.3% growth in the length of individual branches of synaptic extracellular matrix. There was no significant difference in the morphometry between the repeatedly observed junctions and the previously unobserved control junctions. Furthermore, junctions in which the synaptic extracellular matrix was longer than the nerve terminal also were seen in control as well as in experimental muscles. Cases where the nerve terminals were longer than the synaptic extracellular matrix were never observed in newly arising junctional branches. The present study has shown extensive remodeling in not only the nerve terminal but also the synaptic extracellular matrix in adult living frog NMJs. Results suggest that nerve terminals retract before the synaptic extracellular matrix. A hypothesis that extension of synaptic extracellular matrix precedes nerve terminal growth during synaptic remodeling is proposed.

Animals↗
PubMed1998

Articular cartilage: tissue design and chondrocyte-matrix interactions.

The unique biologic and mechanical properties of articular cartilage depend on the design of the tissue and the interactions between the chondrocytes and the matrix that maintain the tissue. Chondrocytes form the macromolecular framework of the tissue matrix from three classes of molecules: collagens, proteoglycans, and noncollagenous proteins. Type II, IX, and XI collagens form a fibrillar meshwork that gives the tissue as form and tensile stiffness and strength. Type VI collagen forms part of the matrix immediately surrounding the chondrocytes and may help the chondrocytes to attach to the macromolecular framework of the matrix. Large aggregating proteoglycans (aggrecans) give the tissue its stiffness to compression and its resilience and contribute to its durability. Small proteoglycans, including decorin, biglycan, and fibromodulin, bind to other matrix macromolecules and thereby help to stabilize the matrix. They may also influence the function of the chondrocytes and bind growth factors. Anchorin CII, a noncollagenous protein, appears to help to anchor chondrocytes to the matrix. Cartilage oligomeric protein may have value as a marker of turnover and degeneration of cartilage, and other noncollagenous proteins, including tenascin and fibronectin, can influence interactions between the chondrocytes and the matrix. The matrix protects the cells from injury due to normal use of the joint, determines the types and concentrations of molecules that reach the tells and helps to maintain the chondrocyte phenotype. Throughout life, the tissue undergoes continual internal remodeling as the cells replace matrix macromolecules lost through degradation. The available evidence indicates that normal matrix turnover depends on the ability of chondrocytes to detect alterations in the macromolecular composition and organization of the matrix, including the presence of degraded molecules, and to respond by synthesizing appropriate types and amounts of new molecules. In addition, the matrix acts as a signal transducer for the cells. Loading of the tissue due to use of the joint creates mechanical, electrical, and physicochemical signals that help to direct the synthetic and degradative activity of chondrocytes. A prolonged severe decrease in the use of the joint leads to alterations in the composition of the matrix and eventually to loss of tissue structure and mechanical properties, whereas use of the joint stimulates the synthetic activity of chondrocytes and possibly the internal tissue remodeling Aging leads to alterations in the composition of the matrix and the activity of the chondrocytes, including the ability of the cells to respond to a variety of stimuli such as growth factors. These alterations may increase the probability of degeneration of the cartilage.

Animals↗
PubMed2000

Defective extracellular matrix reorganization by chronic wound fibroblasts is associated with alterations in TIMP-1, TIMP-2, and MMP-2 activity.

Chronic leg wounds are characterized by defective remodeling of the extracellular matrix, failure of reepithelialization, and prolonged inflammation. The hypothesis that this defective extracellular matrix remodeling is associated with phenotypic differences in the activity of the matrix metalloproteinases and tissue inhibitors of metalloproteinases was studied in chronic wound and patient-matched normal fibroblasts in three-dimensional collagen lattice systems. Chronic wound fibroblasts exhibited no differences in morphology or proliferation (p > 0.1) compared with patient-matched uninvolved dermal fibroblasts. The ability of chronic wound fibroblasts to reorganize extracellular matrix was significantly impaired, however, in comparison to the uninvolved dermal fibroblasts (p < 0.01). This difference in extracellular matrix reorganization was not related to differences in proliferation within the collagen lattices (p > 0.05) or attachment to type I collagen (p > 0.1). Marked differences were evident in matrix metalloproteinase-2 activity between chronic wound and patient-matched normal fibroblasts. Whereas levels of pro-matrix metalloproteinase-2 were similar between the two fibroblast populations (p > 0.1), the chronic wound fibroblasts exhibited significantly decreased levels of the 62 kDa active form of matrix metalloproteinase-2 (p < 0.01). Reverse zymography and enzyme-linked immunosorbent assay demonstrated that the decreased matrix metalloproteinase-2 activity was associated with increased production of tissue inhibitors of metalloproteinase-1 and -2 by the chronic wound fibroblasts (p < 0.05). Increased production of tissue inhibitors of metalloproteinases in chronic wound fibroblasts was also reflected in decreased levels of matrix metalloproteinase-1 (p < 0.005). These data suggest that the impaired ability of chronic wound fibroblasts to reorganize extracellular matrix in vitro is related to decreased levels of active matrix metalloproteinase-2 and matrix metalloproteinase-1 resulting from increased production of tissue inhibitors of metalloproteinase-1 and -2 by chronic wound fibroblasts. These findings provide a mechanism to explain the impaired cellular responses and extracellular matrix reorganization observed in chronic leg wounds in vivo.

Cell Adhesion↗
PubMed1983

Association of actin with the nuclear matrix from bovine lymphocytes.

Nuclear matrix prepared from bovine lymphocytes contained a significant amount of actin. Both nuclear matrix actin and rabbit muscle actin showed the same electrophoretic mobility on SDS-gel. The matrix-associated actin could be separated into three isoproteins which may correspond to alpha-, beta- and gamma-actin. The most acidic spot of these isoproteins co-migrated with rabbit muscle actin (alpha-actin) on two-dimensional electrophoresis. The amino-acid composition of the nuclear matrix actin was closely related to that of rabbit muscle and to that of porcine brain actin. Moreover, the actin filaments, treated with 0.75 M guanidine hydrochloride, changed from the polymerized form of the nuclear matrix actin into a monomeric form (G-actin), which had strong inhibitor activity against pancreatic DNase I. From this inhibition, the actin content of the nuclear matrix was estimated to be about 12% of total matrix protein. When the nuclear matrix was digested with trypsin, the bulk of matrix protein was hydrolyzed, but about 80% of the actin remained associated with sphere structures (trypsin-treated nuclear matrix) precipitable by low speed centrifugation. SDS-gel analysis revealed that actin was one of the major components of the trypsin-treated nuclear matrix, which had a similar size and structure as the untreated nuclear matrix. The fibrogranular structure and residual nucleoli of the original nuclear matrix were well preserved against trypsin digestion; however, the peripheral lamina was removed. These results indicate that the matrix-associated actin is localized predominantly in the matrix interior, where it presumably interacts closely with the fibrogranular structure and/or the residual nucleoli.

Actins↗
PubMed2000

Reduction-oxidation (redox) state regulation of matrix metalloproteinase activity in human fetal membranes.

OBJECTIVE: The mechanisms underlying membrane rupture at term and preterm are obscure. Collagenolytic activity of matrix metalloproteinases in amniochorionic membranes increases during spontaneous term and preterm labor associated with intra-amniotic infection. We sought to test the hypothesis that reduction-oxidation homeostasis, which is altered in inflammatory states, directly regulates amniochorionic matrix metalloproteinases. STUDY DESIGN: Membranes were collected from 7 patients undergoing elective cesarean delivery at term, rinsed thoroughly, and immediately incubated in phosphate-buffered sodium chloride solution at 37 degrees C for 24 hours. Matrix metalloproteinase activity in the culture medium was assayed by substrate-gel electrophoresis and normalized against the dry weight of the tissue incubated. Superoxide anions were generated in the presence of membranes by a xanthine (2 mmol/L) and xanthine oxidase (20 mU/mL) mixture and monitored by reduction of ferri-cytochrome c to ferro-cytochrome c. Incubations were performed in the presence of xanthine alone, a xanthine-xanthine oxidase mixture, superoxide dismutase (500 U/mL), a xanthine-xanthine oxidase-superoxide dismutase mixture, nitro-L-arginine (a nitric oxide synthase inhibitor, 1 mmol/L), xanthine-xanthine oxidase-nitro-L-arginine, S-nitroso-N -acetylpenicillamine (a nitric oxide donor, 10 mmol/L), xanthine-xanthine oxidase-S-nitroso-N -acetylpenicillamine, N -acetylcysteine (a thiol-containing antioxidant, 0.1, 1, or 10 mmol/L), lipopolysaccharide (100 ng/mL), or lipopolysaccharide-N -acetylcysteine. Intracellular generation of superoxide anions was monitored by the reduction of nitroblue tetrazolium to formazan. RESULTS: Basal matrix metalloproteinase 9 and matrix metalloproteinase 2 levels were detected in all samples. Superoxide anions significantly increased matrix metalloproteinase 9 activity but did not increase matrix metalloproteinase 2 activity, which effect was reversed by the addition of superoxide dismutase. N-acetylcysteine reduced basal activity of both matrix metalloproteinase 9 and matrix metalloproteinase 2 to 20%. Importantly, N-acetylcysteine completely inhibited intracellular formazan formation in cultured membranes both in the absence and in the presence of lipopolysaccharide. Neither nitric oxide synthase inhibition nor the nitric oxide donor S-nitroso-N -acetylpenicillamine had any effect on fetal membrane matrix metalloproteinase activity. CONCLUSION: Matrix metalloproteinase activity in human fetal membranes is reduction-oxidation (redox)-regulated. Matrix metalloproteinase 9 activity in human fetal membranes is directly increased by superoxide anion, a byproduct of macrophages and neutrophils. Neither nitric oxide donors nor nitric oxide synthase inhibitors significantly affect matrix metalloproteinase activity in human fetal membranes. The glutathione precursor N-acetylcysteine dramatically inhibits amniochorionic matrix metalloproteinase activity in addition to inhibiting intrinsic superoxide generation within the tissue. Thus thiol-reducing agents, such as N-acetylcysteine, may be beneficial in preventing preterm premature rupture of the membranes.

Acetylcysteine↗
PubMedDate not supplied

Identification of matrix metalloproteinases in canine cutaneous mast cell tumors.

Presence of matrix metalloproteinases has been associated with tumor invasion and metastasis in human neoplasia. The presence of matrix metalloproteinase 2 and matrix metalloproteinase 9 was determined in canine mast cell tumor tissue and normal stromal tissue from 24 dogs with spontaneously occurring cutaneous mast cell tumors. Seventeen of the mast cell tumors were of histologic grade 2, and 7 were of histologic grade 3. Gelatin zymography and computer assisted densitometry image analysis were used to quantify matrix metalloproteinase concentration. Bands from canine tissues migrated in the same location as human proenzyme and active enzyme matrix metalloproteinase 2 and matrix metalloproteinase 9 standards. A semiquantitative value for each patient sample was obtained by comparing the optical assessment density of each unknown band to the optical density of the human standard. The presence of matrix metalloproteinase 2 and matrix metalloproteinase 9 in histologic grade 2 mast cell tumors and histologic grade 3 mast cell tumors was compared, as was presence of matrix metalloproteinases in tumor and stromal tissue. There was dramatically more proenzyme matrix metalloproteinase 9 activity in histologic grade 3 mast cell tumors when compared to grade 2 tumors (P = .03). There was also dramatically more active enzyme matrix metalloproteinase 2 and active enzyme matrix metalloproteinase 9 activity in tumor tissue compared to stromal tissue (P = .02, P < .0001). This study demonstrates that the proenzyme and active enzyme forms of matrix metalloproteinase 2 and matrix metalloproteinase 9 are present in canine mast cell tumors. This appears to be related to the degree of histologic malignancy, although histologic grade 1 tumors were not evaluated.

Animals↗
PubMed2003

New insights into the roles of matrix metalloproteinases in colorectal cancer development and progression.

This review outlines new concepts that are emerging for the functions of matrix metalloproteinases in colorectal cancer development and progression. The two main concepts that will be discussed are the role of matrix metalloproteinases in the early stages of colorectal tumour development and the functional mechanisms by which matrix metalloproteinases contribute to colorectal tumour invasion and metastasis. The matrix metalloproteinases are a group of enzymes, which have been best characterized for their ability to degrade extracellular matrix proteins and thus they have been extensively studied in tumour invasion. It is now becoming recognized that the matrix metalloproteinases have key roles in a variety of biological processes that are distinct from their well-defined role in matrix degradation. This group of enzymes has been shown to interact with a broad range of non-matrix proteins including growth factors and their receptors, mediators of apoptosis, and cell adhesion molecules. The elucidation of novel biological roles for the matrix metalloproteinases also challenges the current predominant concept of matrix metalloproteinases as enzymes only involved in matrix degradation. Recent studies have shown that several matrix metalloproteinases, especially matrilysin (MMP-7), interact with the specific molecular genetic and signalling pathways involved in colorectal cancer development. In particular, matrilysin is activated at an early stage of colorectal tumourigenesis by the beta-catenin signalling pathway. Furthermore, studies are now elucidating specific mechanisms by which individual matrix metalloproteinases, especially membrane-type matrix metalloproteinases, interact with specific cell adhesion molecules and cytoskeletal proteins and thus contribute dynamically to colorectal tumour invasion.

Cell Adhesion↗
PubMed2005

Matrix metalloproteinases synthesized in autosomal dominant polycystic kidney disease play a role in development of a concurrent abdominal aortic aneurysm.

Abdominal aortic aneurysm is well known to be associated with autosomal dominant polycystic kidney disease. Kidney tubules of autosomal dominant polycystic kidney disease synthesize and secrete high levels of matrix metalloproteinase 2, 3, and 9, especially matrix metalloproteinase 2, and serum matrix metalloproteinase 1 and plasma matrix metalloproteinase 9 concentrations in the disease are significantly higher than those in healthy controls. On the other hand, matrix metalloproteinases play a crucial role in the pathogenesis of abdominal aortic aneurysm. Inflammatory cell expression of matrix metalloproteinase 9 plays a critical role in an experimental model of aortic aneurysm disease. Macrophage-derived matrix metalloproteinase 9 and mesenchymal cell matrix metalloproteinase 2 are both required and work in concert to produce abdominal aortic aneurysm. The plasma matrix metalloproteinase 9 levels are significantly higher in the patients with abdominal aortic aneurysm than in the patients with aortoiliac occlusive disease or the healthy patients. Remarkably elevated matrix metalloproteinase 2 mRNA and protein levels in abdominal aortic aneurysm tissues as compared with normal and atherosclerotic aortic tissues are detected, and matrix metalloproteinase 2 proteolytic activity is several-fold higher in abdominal aortic aneurysms than in other pathological or normal states. Patients with abdominal aortic aneurysm elevate matrix metalloproteinase 2 levels in the vasculature remote from the aorta, supporting both the systemic nature of aneurysmal disease and a primary role of matrix metalloproteinase 2 in aneurysm formation. The authors propose a novel hypothesis that matrix metalloproteinases, synthesized and secreted by kidney tubules of autosomal dominant polycystic kidney disease, play a critical role in development of a concurrent abdominal aortic aneurysm.

Animals↗