Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.4.24.35 (matrix metalloproteinase 9)
 2,207 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Human heart matrix metalloproteinases (MMP) are present in the latent form and activated in the failing heart. To examine whether the MMP activation was due to gene and/or post-translational modification, we analysed tissue from 10 explanted hearts due to coronary heart disease (CHD) and five normal left atrial tissue from donor hearts. Based on in situ immunolabeling MMP-1, tissue inhibitor of metalloproteinase (TIMP-1) and collagen were co-localized in the interstitial tissue. Based on sandwich ELISA, TIMP-1 and MMP-1 levels were 37 +/- 8 ng/mg and 9 +/- 2 ng/mg in normal tissue (P < 0.01) and 12 +/- 5 ng/mg and 75 +/- 11 ng/mg in the infarcted tissue (P < 0.01), respectively. These levels suggest repression of TIMP-1 during myocardial infarction. Northern blot analysis indicated that the mRNAs for both MMP-1 and TIMP-1 were increased three-to four-fold in the infarcted tissue as compared to the normal tissue, suggesting upregulation of MMP and TIMP gene transcription following infarction. Based on in situ tissue overlay zymography, the generalized activation of MMP was observed in the interstitium of the infarcted heart. Zymographic and immunoblot analysis demonstrated the presence of one band at 66 kDa (MMP-2) in the normal tissue and several bands at 92 (MMP-9), 66 (MMP-2) and 54 kDa (MMP-1) in the infarcted heart. Incubation of the zymographic gel with metal chelator (phenanthroline) abolished bands at 92 kDa and 54 kDa but phenanthroline did not abolish the lytic band at 66 kDa. The 66 kDa band was completely abolished in the presence of phenanthroline and phenyl methyl sulfonyl fluoride (PMSF). 2D-zymographic analysis suggested that the lytic band at 66 kDa was a mixture of two neutral proteinases with different isoelectric point. Plasminogen/gelatin zymographic analysis of infarcted tissue extract indicated that the band at 66 kDa was plasmin generated due to increased expression of tissue plasminogen activator (tPA) activity. In relation to increased expression of gelatinase in the infarcted tissue, our data suggest that gelatinase B (92 kDa) is induced in diseased heart. The results suggest that tPA converts plasminogen to plasmin which, in turn, activates MMPs and inactivates TIMP-1 post-translationally following ischemic cardiomyopathy.
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PMID:Post-transcriptional regulation of extracellular matrix metalloproteinase in human heart end-stage failure secondary to ischemic cardiomyopathy. 884 29

Polymorphonuclear neutrophil (PMN) migration across basement membrane is thought to be dependent on the degradation of membrane constituents. PMN gelatinase B, a metalloproteinase able to degrade type IV collagen, may be involved in this phenomenon. PMN gelatinase B is released in the extracellular medium as a latent proform and then activated, mainly by PMN elastase. We investigated the role of gelatinase B in PMN migration across a Matrigel basement membrane matrix coated onto a filter, in a Boyden chamber. The effects of gelatinase and elastase inhibitors on PMN migration in this system were tested. Chemokinesis of PMN was tested in the same Boyden chamber across a filter free of basement membrane. The agarose method was used to test the same inhibitors for effects on PMN chemotaxis. In both systems, FMLP 10(-7)M was used as a chemoattractant. Addition of 10(-8)M TIMP-1 (the preferential gelatinase B inhibitor) inhibited trans-basement membrane PMN migration by 52 +/- 6% (P<0.05), without affecting PMN chemokinesis, chemotaxis, or degranulation. Also, (Ala)(2) Pro Val chloromethyl ketone (AAPVCK) 100 micron, a specific elastase inhibitor, inhibited trans-basement membrane PMN migration by 51 +/- 8% (P<0.05), without affecting PMN chemokinesis, chemotaxis, or degranulation. The AAPVCK-TIMP combination led to a decrease in migration across Matrigel basement membrane (46 +/- 2%, P,0.05)similar to that seen with TIMP alone. AAPVCK was responsible for inhibition of gelatinase B activation, leading to a decrease in activated gelatinase from 14% to 2% of total gelatinase release (P<0.05). All these results strongly suggest that gelatinase B is a major factor of PMN migration across basement membrane and that elastase may contribute to this process by activating pro-gelatinase B.
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PMID:Role of gelatinase B and elastase in human polymorphonuclear neutrophil migration across basement membrane. 884 80

Although heart attack is caused by occlusion of a major coronary artery, some patients have occlusion without heart attack because these patients have sufficient collateral circulation to provide an alternate pathway for blood supply to the myocardium at ischemic risk. The growth of new capillary vessels (angiogenesis) and enlargement of preexisting vessels play an important role in the collateral development. We evaluated the hypothesis that extracellular matrix metalloproteinase (MMP) expression is altered in coronary collateral arteries (0.5-1 mm o.d.) isolated from canine hearts 2-4 months after surgical placement of an ameroid occluder around the proximal left circumflex artery (n = 4), during the development of collateral vessels and restructuring new vessels. Histologic studies (hematoxylin and eosin, trichrome, and van Gieson stains) indicated cellular proliferation and increased collagen and elastin content in collateral vessels compared with comparable-sized unoccluded arterial segments of the left anterior descending (LAD) artery. In situ MMP activity of collateral vessels, measured using denatured collagen in the gel matrix, indicated an increase in total MMP activity in the intima of collateral vessels compared with normal LAD vessels. To further identify the type of MMP, tissue homogenates were prepared from collateral and LAD vessels and analyzed by SDS-PAGE zymography. The results suggest induction of gelatinase A and gelatinase B expression in collateral vessels compared with normal LAD tissue, when identical amounts of total protein were loaded onto each lane in the gel. Based on plasminogen-casein zymography, we observed the tissue plasminogen activator level to be increased in collateral vessels. On the basis of immunoblot and mRNA (Northern blot) analyses, we determined that the MMP-1 level was induced in collateral vessels 2 and 4 months after ameroid occlusion. In contrast with MMP-1, the level of TIMP-1 (tissue inhibitor of metelloproteinases) was decreased significantly (p < 0.001) in collateral compared with LAD vessels, suggesting a role for arterial TIMP in anti-angiogenic activity. Collectively, these results suggest that chronic occlusion of a major coronary artery induces upregulation of vascular remodeling mechanisms subserving collateral development. Increased MMP-2 activity in collaterals may be associated with decreased levels of tissue inhibitor of metalloproteinases and fibrous tissue remodeling following angiogenic and (or) adaptive responses of the myocardium to chronic ischemia.
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PMID:Temporal expression of extracellular matrix metalloproteinases and tissue plasminogen activator in the development of collateral vessels in the canine model of coronary occlusion. 896 Mar 89

Matrix metalloproteinases are a growing family of neutral pH optima, zinc atom-dependent endopeptidases that collectively degrade all components of the extracellular matrix. This family of related proteases is further defined by their inhibition of protease activity by a class of low-molecular-weight endogenous inhibitors known as tissue inhibitors of metalloproteinases or TIMPs. Reverse zymography is an electrophoretic technique used to identify TIMP inhibitory activity within acrylamide gels. Previous methods have generally used biochemically complex sources of proteolytic activity (such as cell culture conditioned media) copolymerized with a proteinase substrate in the gel to identify the zones of inhibited proteolysis. We describe a novel system for reverse zymography using purified recombinant human gelatinase A or gelatinase B in place of conditioned media. These reverse zymograms using recombinant gelatinase have sensitivities for TIMPs that are favorable in comparison to immunoblotting techniques but have the benefit of visualizing multiple inhibitors simultaneously. We have developed and characterized these methods for the evaluation of inhibitors and have shown them to be highly sensitive, convenient, and reproducible. Both systems detect TIMPs 1, 2, and 3 simultaneously, but with differential sensitivities for TIMPs 1 and 2. Using gelatinase A the system can detect as little as 1 pg of rTIMP-2, but the limit of detection for rTIMP-1 is 40 pg. Gelatinase B shows less differential activity in that the limits of detection are 60 and 40 pg for TIMP-2 and TIMP-1, respectively. We demonstrate how these varied sensitivities of the gelatinases for the TIMPs can contribute to potential pitfalls in systems using uncharacterized reagents (i.e., conditioned media).
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PMID:Quantitative reverse zymography: analysis of picogram amounts of metalloproteinase inhibitors using gelatinase A and B reverse zymograms. 902 22

Here, we describe the production of recombinant human tissue inhibitor of metalloproteinases-1 (rTIMP-1) and wild-type and mutant human collagenase type I (rMMP-1) proteins in SF9 cells by the baculovirus expression system. Wild-type MMP-1, as well as the MMP-1 mutant lacking the C-terminal hemopexin-like domain [des-(248-450)-MMP-1], exhibit enzymatic activity upon cleavage of the prodomain by treatment with trypsin or 4-aminophenylmercuric acetate. Enzyme activity of both proteins can be inhibited by addition of rTIMP. Deletion of the complete active-site [des-(161-228)-MMP-1] within the catalytic domain, or mutation of a single His residue of the Zn2+ binding domain (His199), generates stable forms of MMP-1 proteins which are unable to digest collagen type I or beta-casein. In addition to co-immunoprecipitation analysis, we have established a rapid and sensitive ELISA assay using immobilized rTIMP to determine the structural requirements of MMP-1 to form complexes with its inhibitor. Only the activated and not the latent forms of wild-type and C-terminal mutant des-(248-450)-MMP-1 proteins are able to form complexes with TIMP. Neither mutation of His199, nor deletion mutants des-(161-228)-MMP-1 and des-(161-228/248-450)-MMP-1, interact with TIMP. This demonstrates that the C-terminal hemopexin domain of MMP-1, in contrast to the corresponding regions of gelatinase A and gelatinase B, does not interact with TIMP-1. In summary, we have shown that the integrity of the catalytic domain of MMP-1 and its ability to bind Zn2+ is absolutely required for complex formation with TIMP-1, which further underlines the importance of this region for proper regulation of enzymatic activity of MMP-1.
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PMID:The catalytic domain of activated collagenase I (MMP-1) is absolutely required for interaction with its specific inhibitor, tissue inhibitor of metalloproteinases-1 (TIMP-1). 906 49

Connective tissues synthesise and secrete a family of matrix metalloproteinases (MMPs) which are capable of degrading most components of the extracellular matrix. Animal studies suggest that the MMPs play a role in bone turnover. Using specific polyclonal antisera, immunohistochemistry was used to determine the patterns of synthesis and distribution of collagenase (MMP-1), stromelysin (MMP-3), gelatinase A (MMP-2) and gelatinase B (MMP-9) and of the tissue inhibitor of metalloproteinases-1 (TIMP-1) within developing human osteophytic bone. The different MMPs and TIMP showed distinct patterns of localisation. Collagenase expression was seen at sites of vascular invasion, in osteoblasts synthesising new matrix and in some osteoclasts at sites of resorption. Chondrocytes demonstrated variable levels of collagenase and stromelysin expression throughout the proliferative and hypertrophic regions, stromelysin showing both cell-associated and strong matrix staining. Intense gelatinase B expression was observed at sites of bone resorption in osteoclasts and mononuclear cells. Gelatinase A was only weakly expressed in the fibrocartilage adjacent to areas of endochondral ossification. There was widespread but variable expression of TIMP-1 throughout the fibrous tissue, cartilage and bone. These results indicate that MMPs play a role in the development of human bone from cartilage and fibrous tissue and are likely to have multiple functions.
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PMID:Distribution of matrix metalloproteinases and their inhibitor, TIMP-1, in developing human osteophytic bone. 927 57

Thickening and reduplication of the tubular basement membrane has been reported as an early event in diabetic nephropathy. In the current study we examined the effects of elevated D-glucose concentrations on human proximal tubular (HPTC) type IV collagen and fibronectin turnover. Incubation of confluent growth arrested HPTC with 25 mM D-glucose led to accumulation of both type IV collagen and fibronectin. This effect was maximal at 48 hours and represented a sevenfold increase for fibronectin (N = 4, P = 0.04), and a threefold increase for type IV collagen (N = 3, P = 0.03) over cells exposed to 5 mM D-glucose controls. This increase was not dependent on new gene transcription for either protein. Tissue inhibitor of metalloproteinases (TIMP 1 + TIMP 2) were induced following addition of 25 mM D-glucose, but not when cells were exposed to 5 mM D-glucose. Twenty-four hours after the addition of 25 mM D-glucose there was an eightfold increase in TIMP 1 (P = 0.009, N = 4), and a tenfold increase in TIMP 2 levels (P = 0.003, N = 4), over the control values for both inhibitors. The increase in both TIMP 1 and TIMP 2 in response to 25 mM D-glucose was abrogated in a dose dependent manner by the aldose reductase inhibitor sorbinil. Gelatin-substrate gel zymography showed increased activity of gelatinase A, but not of gelatinase B in response to the addition of 25 mM D-glucose to HPTC. The induction of gelatinase A was accompanied by increased gelatinase A mRNA expression, which was inhibited both by protein kinase C (PKC) depletion using PMA pre-treatment, and by the addition of a PKC inhibitor. These data demonstrate that the glucose-induced accumulation of type IV collagen and fibronectin is unrelated to increased gene transcription, but may involve alterations in the degradative pathway of these basement membrane constituents. Furthermore, the data demonstrate that glucose may simultaneously activate two intracellular pathways (the polyol pathway and a PKC dependent activation pathway), which are involved in mediating separate, complementary effects on cell function.
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PMID:Exposure of human renal proximal tubular cells to glucose leads to accumulation of type IV collagen and fibronectin by decreased degradation. 932 36

Exposure to the chemotherapeutic drug bleomycin leads to pulmonary fibrosis in humans and has been widely used in animal models of the disease. Using C57BL/6 bleomycin-sensitive mice, pulmonary fibrosis was induced by multiple intraperitoneal injections of the drug. An increase in the relative amounts of steady-state alpha1(I) procollagen, alpha1(III) procollagen, and fibronectin mRNA as well as histopathological evidence of fibrosis was observed. The effect of bleomycin on the expression of the enzymes responsible for extracellular matrix degradation, the matrix metalloproteinases (MMPs), and their inhibitors (TIMPs), was selective and showed temporal differences during the development of fibrosis. Of the MMPs tested, bleomycin treatment resulted in the up-regulation of gelatinase A and macrophage metalloelastase gene expression in whole-lung homogenates, whereas gelatinase B, stromelysin-1, and interstitial collagenase gene expression was not significantly changed. Timp2 and Timp3, the murine homologues of the respective TIMP genes, were constitutively expressed, whereas Timp1 was markedly up-regulated during fibrosis. The strong correlation between enhanced extracellular matrix gene expression, differential MMP and TIMP gene expression, and histopathological evidence of fibrosis suggest that dysregulated matrix remodeling is likely to contribute to the pathology of bleomycin-induced pulmonary fibrosis.
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PMID:Differential expression of extracellular matrix remodeling genes in a murine model of bleomycin-induced pulmonary fibrosis. 950 24

During cutaneous wound healing a number of migratory and remodeling events occur that require the action of matrix metalloproteinases (MMPs) and their natural inhibitors (TIMPs). In this study, we analyzed the temporal and spatial expression patterns of these molecules during the healing of murine excisional skin wounds. Our data imply that defined phases of repair rely on distinct repertoires of MMP activity and TIMP counterregulation. Reepithelialization was found to be associated with active production of collagenase, 92-kDa gelatinase, and stromelysins-1 and -2 by distinct subpopulations of keratinocytes at the migrating border. Notably, no TIMP transcripts were expressed in the epidermis, but TIMP-1 expression in the wound colocalized with expression of collagenase, 92-kDa gelatinase, and stromelysin-1, albeit in distinct cells. Concomitant with the formation of an extensive hyperproliferative epithelium, TIMP-1 transcripts accumulated at the mesenchymal/epidermal border of the granulation tissue. During later phases of wound repair, we observed an increase in 72-kDa gelatinase and MT1-MMP expression, whereby the transcripts of these colocalizing MMPs were detected exclusively and at high levels in the granulation tissue. At completion of reepithelialization, the expression levels of the MMPs and TIMP-1 seen in epidermal and dermal compartments declined to near-basal levels, whereas the macrophage-specific metalloelastase (MME) reached maximum expression. In reepithelialized wound tissue, MME transcripts were detected in deep layers of reconstituted dermis and seemed to cluster around vascular structures. Systemic glucocorticoid treatment, which is known to result in impaired wound healing, led to a nearly complete shut-off of MME expression. These observations imply an additional role of macrophage-related proteolysis, independent of its classical roles during earlier, inflammatory phases of cutaneous wound repair.
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PMID:Matrix metalloproteinases (MMPs) and their physiological inhibitors (TIMPs) are differentially expressed during excisional skin wound repair. 966 17

The present study examines the effect of transforming growth factor-beta1 (TGF-beta1) and interleukin-1beta (IL-1beta) on the regulation of gelatinase A, gelatinase B, tissue inhibitor of metalloproteinase-I (TIMP-I) and TIMP-II in human glomerular epithelial cells (GEC). The addition of TGF-beta1 resulted in the increased production and secretion of both gelatinase A (72-kD type IV collagenase) and gelatinase B (92-kD type IV collagenase), in a dose- and time-dependent manner. In contrast, the addition of IL-1beta to GEC resulted in the stimulation of secretion of gelatinase B but not gelatinase A. When the secretion of the regulatory inhibitors was examined, IL-1beta or TGF-beta1 both resulted in an increased secretion of TIMP-I, whereas the secretion of TIMP-II was downregulated. Such results demonstrate an independent and opposite regulation of the enzymes and their inhibitors. Of particular interest was the observation of the differential regulation of gelatinase A and its specific inhibitor TIMP-II (which binds to the latent form of this enzyme) in response to TGF-beta1. These results for the first time indicate that in human GEC, matrix metalloproteinases (MMP), as well as their specific inhibitors, are independently regulated by different cytokines. MMP and their regulatory tissue inhibitors (TIMP) play an important role in tissue remodeling. The results of the present study serve to emphasize both the complex regulation of matrix metabolism in the glomerulus and the potential pathologic role of an imbalance between the proteinases and their inhibitors in various forms of glomerular disease.
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PMID:Differential regulation of matrix metalloproteinases and their inhibitors in human glomerular epithelial cells in vitro. 972 71


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