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PubMed2000

Interplay of matrix metalloproteinases, tissue inhibitors of metalloproteinases and their regulators in cardiac matrix remodeling.

Myocardial fibrosis due to maladaptive extracellular matrix remodeling contributes to dysfunction of the failing heart. Further elucidation of the mechanism by which myocardial fibrosis and dilatation can be prevented or even reversed remains of great interest as a potential means to limit myocardial remodeling and dysfunction. Matrix metalloproteinases (MMPs) are the driving force behind extracellular matrix degradation during remodeling and are increased in the failing human heart. MMPs are regulated by a variety of growth factors, cytokines, and matrix fragments such as matrikines. In the present report, we discuss the regulation of MMPs, the role of MMPs in the development of cardiac fibrosis, and the modulation of MMP activity using gene transfer and knockout technologies. We also present recent findings from our laboratory on the regulation of the extracellular MMP inducer (EMMPRIN), MMPs, and transforming growth factor-beta(1) in the failing human heart before and after left ventricular assist device support, as well as the possibility of preventing ventricular fibrosis using different anti-MMP strategies. Several studies suggest that such modulation of MMP activity can alter ventricular remodeling, myocardial dysfunction, and the progression of heart failure. It is therefore suggested that the interplay of MMPs and their regulators is important in the development of the heart failure phenotype, and myocardial fibrosis in heart failure may be modified by modulating MMP activity.

Animals↗

PubMed1997

Gene expression of matrix metalloproteinase-1 (interstitial collagenase) and matrix metalloproteinase-3 (stromelysin-1) in basal cell carcinoma by in situ hybridization using chondroitin ABC lyase.

The gene expression of matrix metalloproteinase-1 and -3 was examined in basal cell carcinomas by in situ hybridization using digoxigenin-labelled riboprobes. Nodulo-ulcerative basal cell carcinomas demonstrated the gene expression for both metalloproteinases but superficial basal cell carcinomas did not present any transcripts for them. Transcripts for matrix metalloproteinase-1 (interstitial collagenase) were demonstrated densely in stromal cells among tumour masses, and those for matrix metalloproteinase-3 (stromelysin-1) were detected only in more advanced cases. Neither were expressed in tumour cells. The two metalloproteinases were produced by stromal cells according to the tumour invasion process, in which various growth factors, cytokines and inflammatory factors, which could regulate gene expressions of matrix metalloproteinases, were involved. It was also found that hybridization signals were enhanced by treatment with chondroitin ABC lyase, which digested abundant glycosaminoglycans in basal cell carcinoma. The procedure for the digestion is simple, and appears to be of value for in situ hybridization studies on tissues containing large amounts of glycosaminoglycans.

Basal Cell Carcinoma↗

PubMed2000

Long-term prevention of renal insufficiency, excess matrix gene expression, and glomerular mesangial matrix expansion by treatment with monoclonal antitransforming growth factor-beta antibody in db/db diabetic mice.

Emerging evidence suggests that transforming growth factor-beta (TGF-beta) is an important mediator of diabetic nephropathy. We showed previously that short-term treatment with a neutralizing monoclonal anti-TGF-beta antibody (alphaT) in streptozotocin-diabetic mice prevents early changes of renal hypertrophy and increased matrix mRNA. To establish that overactivity of the renal TGF-beta system mediates the functional and structural changes of the more advanced stages of nephropathy, we tested whether chronic administration of alphaT prevents renal insufficiency and glomerulosclerosis in the db/db mouse, a model of type 2 diabetes that develops overt nephropathy. Diabetic db/db mice and nondiabetic db/m littermates were treated intraperitoneally with alphaT or control IgG, 300 microgram three times per week for 8 wk. Treatment with alphaT, but not with IgG, significantly decreased the plasma TGF-beta1 concentration without decreasing the plasma glucose concentration. The IgG-treated db/db mice developed albuminuria, renal insufficiency, and glomerular mesangial matrix expansion associated with increased renal mRNAs encoding alpha1(IV) collagen and fibronectin. On the other hand, treatment with alphaT completely prevented the increase in plasma creatinine concentration, the decrease in urinary creatinine clearance, and the expansion of mesangial matrix in db/db mice. The increase in renal matrix mRNAs was substantially attenuated, but the excretion of urinary albumin factored for creatinine clearance was not significantly affected by alphaT treatment. We conclude that chronic inhibition of the biologic actions of TGF-beta with a neutralizing monoclonal antibody in db/db mice prevents the glomerulosclerosis and renal insufficiency resulting from type 2 diabetes.

Animals↗

PubMed2002

Molecular mechanism involved in matrix dependent upregulation of matrix metalloproteinases in monocyte/macrophage.

Production of macrophage specific matrix metalloproteinases (MMPs) by monocyte/macrophage (mo/mphi) maintained in vitro on matrix protein substrata has been examined to study the mechanism of matrix protein dependent upregulation of macrophage specific activity. Using specific blocking reagents we have found that interaction of peripheral blood mononuclear cells (PBMC) with extracellular matrix components is crucial for its differentiation to macrophages. Multiwell zymography has shown that production of MMPs was significantly inhibited in cells maintained on fibronectin (FN) pretreated with antibodies to alpha(5), beta(1) integrins and synthetic peptide RGDS. Further, quantification by ELISA showed a significant inhibition in MMP production in cells pretreated with these blocking reagents. Genistein, a non-specific inhibitor of tyrosine kinases, significantly reduced production of MMPs in cells maintained on FN and collagen type IV (COL IV). Immunoblotting analysis has shown that tyrosine phosphorylation occurs in 30 min and two proteins of approximately 115 and approximately 72 kDa are being phosphorylated upon PBMC-FN interaction. These results indicate that integrin mediated downstream signalling involving tyrosine phosphorylation is required for mediating intracellular events associated with differentiation of monocytes to macrophages.

Cell Differentiation↗

PubMed2002

Matrix metalloproteinase activity, bone matrix turnover, and tumor cell proliferation in prostate cancer bone metastasis.

BACKGROUND: The metastasis of prostate cancer to bone is associated with a substantial increase in bone matrix turnover. Matrix metalloproteinases (MMPs) play roles in both normal bone remodeling and invasion and metastasis of prostate cancer. This study was designed to determine the role of MMP activity in prostate cancer that has metastasized to bone. METHODS: Single human fetal bone fragments were implanted subcutaneously in immunodeficient mice. Four weeks later, PC3 human prostate cancer cells were injected directly into some of the implants, and daily treatment was begun with batimastat (a broad-spectrum MMP inhibitor). There were six mice (i.e., six implants) in each of four experimental arms: bone alone with and without batimastat and bone injected with PC3 cells with and without batimastat. Bone implants were harvested after 14 days of treatment and analyzed for MMP expression, bone histomorphometry, osteoclast counts, blood vessel density, and tumor cell proliferation and apoptosis. Complementary data were obtained from bone biopsy samples from patients and a bone organ coculture system. All statistical tests were two-sided. RESULTS: MMPs were detected in tumor and stromal cells of clinical specimens and experimental bone implants. In vivo, MMP inhibition reduced the number of osteoclasts per millimeter in PC3-injected implants-from 8.2 (95% confidence interval [CI] = 7.9 to 8.5) to 3.0 (95% CI = 2.3 to 3.7) (P =.006). In addition, it prevented degradation of marrow trabeculae within the bone implants (cross-sectional area of implant occupied by mineralized trabeculae: untreated implant = 29.1% [95% CI = 27.1% to 31.1%], PC3-injected implant = 14.0% [95% CI = 10.9% to 17.1%] [P =.005 versus untreated], and batimastat-treated PC3-injected implant = 27.2% [95% CI = 22.4% to 32.0%] [P =.03 versus PC3 injected alone]). MMP inhibition reduced proliferating tumor cells from 20.8% (95% CI = 19.9% to 21.7%) to 7.4% (95% CI = 5.2% to 9.6%) (P =.006), without affecting angiogenesis or apoptosis. In vitro, MMP inhibition had no toxic effect on PC3 cells but prevented calcium release from bone fragments cocultured with PC3 cells. CONCLUSIONS: MMP activity appears to play an important role in bone matrix turnover when prostate cancer cells are present in bone. Bone matrix turnover and metastatic tumor growth appear to be involved in a mutually supportive cycle that is disrupted by MMP inhibition.

Animals↗

PubMed1998

Matrix metalloproteinase-2 production and its binding to the matrix are increased in abdominal aortic aneurysms.

Degradation of the elastic media is a hallmark of abdominal aortic aneurysms (AAAs). We examined the expression of 2 elastolytic matrix metalloproteinases (MMPs), MMP-2 and MMP-9, in AAA aortic tissues compared with those from atherosclerotic occlusive disease (AOD) and nondiseased control tissues. Quantitative competitive reverse transcription-polymerase chain reaction and gelatin zymography showed increased MMP-9 mRNA and protein in both AAA and AOD tissues compared with those in control tissue, but there was no significant difference between AAA and AOD. In contrast, MMP-2 mRNA and protein levels were significantly higher in AAA than in AOD or control tissues. Sequential extraction of the MMPs from the aortic tissue with a physiological salt solution, 2% dimethylsulfoxide (DMSO), and 10 mol/L urea showed that large amounts of MMP-2 and MMP-9 were bound to the matrix. The most conspicuous finding was that the levels of MMP-2 were significantly elevated in the DMSO fraction in AAA tissues compared with AOD and control tissues. In addition, a large portion of MMP-2 found in the DMSO and urea fractions was in the active 62-kDa form, indicating that the precursor of MMP-2 in AAA is largely activated locally and binds to the tissue matrix tightly. By immunolocalization, MMP-9 was found to be primarily produced by macrophages and MMP-2 by mesenchymal cells. The production of MMP-2 was prominent when mesenchymal cells were surrounded by inflammatory cells, suggesting paracrine modulation of MMP-2 expression in AAAs. These observations emphasize that MMP-2 participates in the progression of AAAs by degrading aortic tissue matrix components.

Aged↗

PubMed2001

Impact of mutations of cartilage matrix genes on matrix structure, gene activity and chondrogenesis.

OBJECTIVE: Chondrocytes in the growth plate at different stages of differentiation synthesize characteristic extracellular matrix (ECM) components. Mutations in some ECM genes result in chondrodysplasia in humans and mice. We aimed to evaluate the impact of loss- and gain-of-function mutations of ECM genes on matrix structure, gene expression and formation of the growth plate. DESIGN: We review information on the impact of deficiencies in proteoglycans, and types X and II collagens on skeletal development. Additionally, we compare the impact of a glycine904 to cysteine (G904C) mutation in the triple helical coding domain of mouse Col2a1 with two previously reported Col2a1 mutations (exon7 deletion (Del1) and G85C). The G904C Col2a1 gene was introduced as a transgene into mice. Transgenic newborn mice were examined for skeletal development. The histology of the epiphyseal cartilage and the growth plate, and the ultrastructure of chondrocytes and collagen fibrillar morphology in the ECM were studied in 18.5-day transgenic and wild-type fetuses. The distribution of the mRNAs for Col2a1, Col11a1, Col9a1, Matn1, Agc and Ihh in the growth plate of 18.5-day G904C/G904C and wild type fetuses were compared by in situ hybridization. RESULTS: Heterozygous transgenic mice harbouring five copies of the G904C Col2a1 transgene developed skeletal abnormalities and dwarfism. Homozygous G904C/G904C mice died at birth, showing cleft palate, disrupted zonation of chondrocytes and reduction of the zone of hypertrophic chondrocytes. Fewer collagen fibrils were found in ECM of the cartilage. Rough endoplasmic reticulum of the chondrocytes of G904C/+ and G904C/G904C mice was distended. In G904C/G904C mutant mice, Agc gene activity was extended to the hypertrophic zone. Expression of the other genes studied was unchanged. Calcified materials that were not found normally in the maturing and only at low abundance in the hypertrophic zones of the wild type growth plate, were present in these zones in G904C/G904C mice. Despite phenotypic similarities for the G904C and Del1 mice, reduced expression of types I, II, IX, X collagens and aggrecan were reported for the latter mutation. Changes in gene activity and matrix organization in the growth plate also accompanied deficiencies in aggrecan, perlecan and collagen II. CONCLUSIONS: The data suggest that a single amino acid alteration in collagen II could lead to skeletal abnormalities through multiple secondary effects on the synthesis and assembly of ECM components. The functional impact of mutations of ECM genes reveals that chondrodysplasia is caused not just by the formation of abnormal matrix molecules, but that the alteration of one ECM component may lead to a cascade of disruption of other gene activities in chondrocytes which collectively contribute to the pathological changes in the architecture of the growth plate.

Aggrecans↗

PubMed1995

Leukemic cells (HL-60) produce a novel extracellular matrix-degrading proteinase that is not inhibited by tissue inhibitors of matrix metalloproteinases (TIMPs).

In addition to the known 94-kd gelatinase (matrix metalloproteinase 9, MMP-9), HL-60 leukemia cells release a hither-to undescribed 45-kd metalloproteinase into the culture medium. This enzyme cleaves the synthetic substrate Pro-Gln-Gly-Ile-Ala-Gly-Gln-Arg, which represents the cleavage site for collagenases in collagen type I not between isoleucine and alanine--the typical cleavage site for collagenases--but between alanine and glycine. The enzymatic activity was purified through a combination of zinc-chelate-Sepharose column chromatography, precipitation with Fractogel TSK-AF Red and gelatin-Sepharose, and subsequent sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Microsequence analysis of the NH2-terminus of the purified 45-kd proteinase revealed the sequence Asp-Ile-Ser-Lys-Tyr-Thr-Thr-Thr-, which could not be found in other proteins when searched in several protein data bases. Incubation of the enzyme immobilized on nitrocellulose membranes with polyclonal antibodies to collagenase and stromelysin or gelatinases revealed no cross-reactivity. The proteolytic activity was not increased by treatment with trypsin, 8M urea, acid, or organomercurials. The proteinase, which was inhibited by chemical inhibitors of metalloproteinases, such as phenanthrolene or EDTA, is able to degrade several matrix constituents, such as collagen type IV, fibronectin, gelatin, and proteoglycans. In contrast to all known MMPs, the proteolytic activity of the 45-kd enzyme was not abolished upon incubation with recombinant tissue inhibitors of matrix metalloproteinases (TIMP) 1 or 2. Thus, the novel enzyme may influence extracellular matrix (ECM) turnover in vivo because its activity is not influenced by specific inhibitors of MMPs.

Amino Acid Sequence↗

PubMedDate not supplied

Mesangial cell-matrix interactions: modulation of matrix expression in mesangial cells by extracellular matrices.

Aberrant extracellular matrices (ECMs) observed in mesangial areas during the progression of glomerulonephropathy could be produced by mesangial cells (MCs), interacting with altered ECM components, under the influence of various mediators. We studied the interaction between MCs and ECMs, especially focusing on matrix expression of MCs in contact with ECMs. We analyzed mRNA expression and protein synthesis of ECM components by cultured rat MCs which were plated on different types of ECMs, basement membrane-type matrix (BMM), type IV collagen and type I collagen. The level of mRNA for ECM components (alpha 1 type I collagen, alpha 1 type IV collagen, fibronectin and laminin B2) increased in MCs on the two types of collagen compared with BMM. The relative increase of alpha 1 type I collagen mRNA expression in MCs on type I collagen was greater than that of alpha 1 type IV collagen expression. At the protein level, type I collagen production by MCs on coated type I collagen showed a 2.2-fold increase relative to that on BMM. MCs on type IV collagen produced it at an intermediate level. The phenomena were not associated with differences of proliferative activity, but were accompanied by minor morphological differences. These results indicate that ECMs can modulate the phenotype of MCs in terms of matrix expression and suggest that MC-matrix interaction may in part contribute to abnormal ECM accumulation in diseased glomeruli.

Animals↗

PubMed1998

Remodeling of collagen matrix by human tumor cells requires activation and cell surface association of matrix metalloproteinase-2.

We assessed the functional significance of tumor cell-associated matrix metalloproteinase (MMP)-2 in extracellular matrix remodeling compared with that of the soluble enzyme by evaluating the contraction of three-dimensional collagen lattices by human glioma U251.3 and fibrosarcoma HT-1080 cell lines. In this model, the constitutive synthesis and activation of the MMP-2 proenzyme were modulated by stable transfections of tumor cells with cDNA encoding membrane type 1-MMP (MT1-MMP). The efficiency of transfected cells in contracting collagen lattices was shown to be dependent on the MT1-MMP-mediated activation of MMP-2 accompanied by cell surface association of activated MMP-2, on the cell-matrix interactions controlled by collagen-specific integrins, and on the integrity of actin and microtubule cytoskeletons. Each one of these mechanisms was essential but was not sufficient by itself in accomplishing gel contraction by MT1-MMP-transfected cells. Both MMP-2 activation and gel contraction by transfected glioma cells were inhibited by tissue inhibitor of metalloproteinase (TIMP)-2 and the recombinant COOH-terminal domain of MMP-2. However, the kinetics and mechanisms of their inhibitory effects were different, because TIMP-2 and the COOH-terminal domain of MMP-2 preferentially inhibited the MT1-MMP-dependent and autocatalytic steps of MMP-2 activation, respectively. By contrast, TIMP-1, an efficient inhibitor of soluble MMP-2 activity, failed to affect gel contraction. In addition, soluble MMP-2 activated by either organomercurials or cells was not able to induce the contraction of collagen lattices when added to transfected cells. Therefore, soluble activated MMP-2, sensitive to TIMP-1 inhibition, does not mediate collagen gel contraction by tumor cells, whereas the activity of cell surface-associated MMP-2 plays a critical role in remodeling of the extracellular matrix in vitro. These mechanisms of functional and spatial regulation of MMP-2 may also be applicable to different aspects of tissue reorganization in vivo, including cell migration and invasion, angiogenesis, and wound healing.

Collagen↗

PubMed1998

Matrix metalloproteinase-mediated extracellular matrix protein degradation in human pulmonary emphysema.

The aim of this study was to investigate the extracellular degrading proteolytic cascade proteins referred to as matrix metalloproteinase-1 (MMP-1), MMP-2, MMP-9, membrane-type matrix metalloproteinase-1 (MT1-MMP), tissue inhibitors of matrix metalloproteinase-1 (TIMP-1), TIMP-2, neutrophil elastase, and alpha1-antitrypsin in human pulmonary emphysema. Localization of MMP-1, MMP-2, MMP-8, MMP-9, MT1-MMP, TIMP-1, and TIMP-2 was verified by immunohistochemical analysis. The results of our study indicated that the immunoreactivity of MMP-1, MMP-8, MMP-9, and TIMP-1 was absent, whereas MT1-MMP and MMP-2 were mainly observed in pneumocytes, fibroblasts, and alveolar macrophages. Although MT1-MMP and MMP-2 were observed both in emphysematous and normal lung tissue, these immunoreactivities were intense in the emphysematous samples. The presence of MMP-1, MMP-2, MMP-9, TIMP-1, and TIMP-2 was confirmed at mRNA level by reverse transcription-PCR analysis and enzyme immunoassay (EIA). However, the only statistical difference that was observed was in MMP-2 and MMP-9 (MMP-2: emphysematous samples, 19.1+/-2.1 versus control samples, 5.2+/-0.60 microg/g protein, p < 0.05; MMP-9: emphysematous samples, 18.4+/-5.6 versus control samples, 8.1+/-2.7 microg/g protein, p < 0.05). Results of the neutrophil elastase as analyzed by EIA, and alpha1-antitrypsin levels as detected by laser nephelometric immunoassay, indicated no statistical difference between the emphysematous and control groups. In addition to the presence of mRNA levels, the level of MT1-MMP according to immunoblot analysis increased in the emphysematous samples. Gelatin zymographic analysis confirmed the presence of both pro and active forms of MMP-2, and the increased ratio of the active form of MMP-2 in emphysematous samples (25.9%+/-2.0% versus 11.2%+/-3.3%, p < 0.05), indicated in situ activation of MMP-2 by MT1-MMP. Elastin zymographic analysis showed elastolytic activity by MMP-2 and MMP-9 but not the reported band of macrophage metalloelastase (MMP-12). The data suggest that the MT1-MMP/MMP-2/TIMP-2 system plays a significant role in the MMP-mediated extracellular matrix degradation and tissue remodeling of emphysematous lungs, and thus may contribute to the weakening of lung parenchyma and lead to the formation of emphysema.

Aged↗

PubMed1996

Secretion of matrix metalloproteinase-2 (72 kD gelatinase/type IV collagenase = gelatinase A) by malignant human glioma cell lines: implications for the growth and cellular invasion of the extracellular matrix.

Human glioma cells (T98G and A172 cell lines) were cultured on various extracellular matrix (ECM) components including type I, IV and V collagens, fibronectin, laminin, and reconstituted basement membrane (Matrigel), and the role of matrix metalloproteinases (MMPs) in their growth and invasion was examined. T98G glioma cells grew well on these ECM components and invaded the reconstituted basement membrane. In contrast, A172 glioma cells showed growth inhibition on collagen types IV and V and Matrigel without invasion of the Matrigel. Gelatin zymography and enzyme immunoassays demonstrated that T98G glioma cells, but not A172 cells, secrete a large amount of matrix metalloproteinase-2 (MMP-2, 72 kD gelatinase/type IV collagenase = gelatinase A), and this was confirmed by immunoblotting and immunohistochemistry. Of the two different tissue inhibitors of metalloproteinases (TIMP-1 and TIMP-2), T98G cells produced only TIMP-1 during culture on Matrigel, whereas A172 cells secreted both. Although both human recombinant TIMP-1 and TIMP-2 stimulated T98G cell growth slightly on Matrigel, the in vitro invasiveness was significantly reduced by only recombinant TIMP-2. These results suggest that MMP-2 plays an important role in the ECM invasion of T98G human glioma cells in vitro.

Brain Neoplasms↗

PubMed2000

Collagen and elastin degradation by matrix metalloproteinases and tissue inhibitors of matrix metalloproteinase in aortic dissection.

The degradation of collagen fibrils and elastic fibers in 21 cases of acute aortic dissection (AD) was ultrastructurally and immunohistochemically investigated; and the expression of the catabolic matrix metalloproteinases (MMPs)-1, -2, -3, and -9 and their inhibitors, the tissue inhibitors of matrix metalloproteinase (TIMPs)-1 and -2, was studied. The features of the entry site of the dissection (ES; 21 ascending aortas) were compared with those of fully remote sites (RS; 19 nondissected abdominal aortas) and the ascending aortas from 10 control cases. By electron microscopy, the medial layer at the ES and adjacent intact aortic wall demonstrated spirally thickened collagen fibrils with a typical banding pattern that were almost always colocalized with elastic lamellae, which often exhibited attenuation, fragmentation, or disruption. In addition, the basement membrane surrounding the smooth muscle cells (SMCs) comprising the media was frequently thinned or lost at the ES. These findings were rarely seen at the RS or in the aortas of controls. Immunohistochemically, the expression of MMP-1 was significantly in the cytoplasm of SMCs of both the intima and media at the ES and adjacent intact wall, and significant expression of MMP-2 and -9 was found in SMCs of the intima compared with the RS and controls. Significant expression of TIMP-1 and -2 was demonstrated in the cytoplasm of SMCs at the ES and adjacent intact wall compared with that at the RS and the control specimens. These findings suggest that the degradation of proteins associated with fibrosis and the occurrence of AD are not merely coincident, but rather that AD is induced by alterations of the extracellular matrix caused by changes of SMCs at a segment of the ascending aorta made vulnerable through hemodynamic stress, especially that caused by hypertension.

Adult↗

PubMed1998

Matrix metalloproteinase expression increases after cerebral focal ischemia in rats: inhibition of matrix metalloproteinase-9 reduces infarct size.

BACKGROUND AND PURPOSE: Matrix metalloproteinases (MMPs) are a family of proteolytic enzymes that degrade the extracellular matrix and are implicated in numerous pathological conditions including atherosclerosis, inflammation, and tumor growth and metastasis. In the brain, the endothelial cell wall, strengthened by tight junctions, defines the blood-brain barrier (BBB). The extracellular matrix molecules constitute the basement membrane underlying the vasculature and play a critical role in maintaining the integrity of the BBB. After focal stroke, there is a breakdown of the BBB with an associated increase in vascular permeability, inflammatory cell influx, and neuronal cell death. The present study was designed to investigate the effects of MMP expression after stroke. METHODS: Focal stroke was produced by permanent middle cerebral artery occlusion (MCAO) in the rat, and MMP protein expression was measured by Western blot and zymogram analysis over a time course ranging from 6 hours to 30 days (n=32). Immunohistochemistry at 1 and 5 days (n=8 and 6, respectively) was also utilized to characterize the expression of several MMPs and related proteins after stroke, including their cellular source. To test the hypothesis that early increased MMP-9 expression is involved in ischemic brain injury, a neutralizing monoclonal antibody directed against MMP-9 was administered intravenously (n=7 per group) 1 hour before MCAO, and infarct size was measured 24 hours later. RESULTS: MMP expression increased progressively over time after stroke. After 12 hours, significant (P<0.05) MMP-9 activity was observed that reached maximum levels by 24 hours (P<0.001), then persisted for 5 days at this level and returned to basal (zero) levels by 15 days. On the basis of morphological criteria, MMP-9 appeared to stain with endothelial cells and neutrophils identified both within and at the periphery of the infarct within 24 hours of focal ischemia. After 5 days, MMP-9 appeared to stain with macrophages present within the infarcted brain. MMP-2 activity was significantly (P<0.001) increased by 24 hours and was maximum after 5 days following MCAO. MMP-2 appeared to stain with macrophages present within the infarcted region. Unlike MMP-9 and MMP-2, tissue inhibitor of metalloproteinase-1 was identified at comparable levels in both control and ischemic tissue after MCAO. MMP-1 and MMP-3 could not be detected in the brain after focal stroke. When an MMP-9-neutralizing monoclonal antibody was administered systemically, animals exhibited significantly reduced infarct size (ie, a 30% reduction compared with non-immune antibody controls; P<0.05). CONCLUSIONS: These results demonstrate that early increased MMP-9 expression in endothelial cells and infiltrating neutrophils is a significant response to cerebral focal ischemia and that selective inhibition of MMP-9 activity can significantly reduce brain injury after stroke.

Animals↗

PubMed2000

Dm1-MMP, a matrix metalloproteinase from Drosophila with a potential role in extracellular matrix remodeling during neural development.

We have cloned and characterized a cDNA encoding Dm1-MMP, the first matrix metalloproteinase (MMP) identified in Drosophila melanogaster. The isolated cDNA encodes a protein of 541 residues that has a domain organization identical to that of most vertebrate MMPs including a signal sequence, a prodomain with the activation locus, a catalytic domain with a zinc-binding site, and a COOH-terminal hemopexin domain. Northern blot analysis of Dm1-MMP expression in embryonic and larval adult tissues revealed a strong expression level in the developing embryo at 10-22 h, declining thereafter and being undetectable in adults. Western blot analysis confirmed the presence of pro- and active forms of Dm1-MMP in vivo during larval development. In situ hybridization experiments demonstrated that Dm1-MMP is expressed in a segmented pattern in cell clusters at the midline during embryonic stage 12-13, when neurons of the central nervous system start to arise. Recombinant Dm1-MMP produced in Escherichia coli exhibits a potent proteolytic activity against synthetic peptides used for analysis of vertebrate MMPs. This activity is inhibited by tissue inhibitors of metalloproteinases and by synthetic MMP inhibitors such as BB-94. Furthermore, Dm1-MMP is able to degrade the extracellular matrix and basement membrane proteins fibronectin and type IV collagen. On the basis of these data, together with the predominant expression of Dm1-MMP in embryonic neural cells, we propose that this enzyme may be involved in the extracellular matrix remodeling taking place during the development of the central nervous system in Drosophila.

Amino Acid Sequence↗

PubMed1997

Membrane-type matrix metalloproteinases 1 and 2 exhibit broad-spectrum proteolytic capacities comparable to many matrix metalloproteinases.

Soluble proenzyme forms of the catalytic domains of membrane-type matrix metalloproteinases 1 and 2 (MT1-MMP and MT2-MMP) and a form of MT1-MMP containing the catalytic and hemopexin domains were expressed as soluble recombinant proteins. Purified, activated forms of the MT-MMP were shown to degrade fibronectin, tenascin, nidogen, aggrecan and perlecan. Only MT2-MMP showed activity against laminin. MT1-MMP retaining the hemopexin domain was able to specifically cleave native type-I and type-III collagens into the 3/4-1/4 fragments typical of the specific collagenases. The catalytic domain alone did not retain this activity. The MT-MMP did not degrade interleukin-1beta, but, similarly to many other MMP, could process a pro [tumor necrosis factor (TNF) alpha] fusion protein to release mature TNF. However, the latter was subsequently degraded into smaller fragments. These results demonstrate that, in addition to their ability to activate other MMP, such as progelatinase A/proMMP2 and procollagenase-3/proMMP13, MT-MMP degrade a number of extracellular matrix macromolecules. Their location at the surface of cells implies that they could play a significant role in the modulation of cell-matrix interactions.

Aggrecans↗

PubMed1994

Increased expression of matrix metalloproteinases and matrix degrading activity in vulnerable regions of human atherosclerotic plaques.

Dysregulated extracellular matrix (ECM) metabolism may contribute to vascular remodeling during the development and complication of human atherosclerotic lesions. We investigated the expression of matrix metalloproteinases (MMPs), a family of enzymes that degrade ECM components in human atherosclerotic plaques (n = 30) and in uninvolved arterial specimens (n = 11). We studied members of all three MMP classes (interstitial collagenase, MMP-1; gelatinases, MMP-2 and MMP-9; and stromelysin, MMP-3) and their endogenous inhibitors (TIMPs 1 and 2) by immunocytochemistry, zymography, and immunoprecipitation. Normal arteries stained uniformly for 72-kD gelatinase and TIMPs. In contrast, plaques' shoulders and regions of foam cell accumulation displayed locally increased expression of 92-kD gelatinase, stromelysin, and interstitial collagenase. However, the mere presence of MMP does not establish their catalytic capacity, as the zymogens lack activity, and TIMPs may block activated MMPs. All plaque extracts contained activated forms of gelatinases determined zymographically and by degradation of 3H-collagen type IV. To test directly whether atheromata actually contain active matrix-degrading enzymes in situ, we devised a method which allows the detection and microscopic localization of MMP enzymatic activity directly in tissue sections. In situ zymography revealed gelatinolytic and caseinolytic activity in frozen sections of atherosclerotic but not of uninvolved arterial tissues. The MMP inhibitors, EDTA and 1,10-phenanthroline, as well as recombinant TIMP-1, reduced these activities which colocalized with regions of increased immunoreactive MMP expression, i.e., the shoulders, core, and microvasculature of the plaques. Focal overexpression of activated MMP may promote destabilization and complication of atherosclerotic plaques and provide novel targets for therapeutic intervention.

Arteries↗

PubMed1998

The activation and function of host matrix metalloproteinases in dentin matrix breakdown in caries lesions.

Matrix metalloproteinases (MMPs) are a family of enzymes which, in concert, are capable of degrading collagen. We investigated whether human MMPs could participate in the degradation of dentin organic matrix after demineralization. We performed Western blot analyses using MMP-specific antibodies to identify MMPs in human dental caries lesions. Enzymography and functional activity assays, with 125I-labeled gelatin as substrate or quantitating the degradation of type I collagen, were used to determine the activity of purified and salivary gelatinolytic (MMP-2 and MMP-9) and collagenolytic (MMP-8) enzymes with and without acid-activation in pHs relevant to caries. Respective analyses were done with caries-related bacteria. We performed electron microscope analyses to assess the degradative activity of sterilized salivary host MMPs on demineralized human dentin. Human MMP-2, MMP-8, and MMP-9 were identified in demineralized dentinal lesions. The latent purified forms of these enzymes were activated at low pH (4.5), followed by neutralization, mimicking the conditions during caries progression. Incubation of human saliva at low pH followed by neutralization resulted in a four-fold increase in the gelatinolytic activity. No gelatinolytic or collagenolytic activity was observed in bacterial samples. The activated enzymes in saliva degraded demineralized dentin organic matrix in vitro. These results demonstrate the pH-dependent activation mechanism of MMPs, which may have a distinct role in different physiological and pathological conditions. They further demonstrate that host MMPs, activated by bacterial acids, have a crucial role in the destruction of dentin by caries.

Bacterial Proteins↗