Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.4.24.3 (collagenase)
 18,340 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Interstitial collagenase (matrix metalloproteinase-1 [MMP-1]) plays an important role in extracellular matrix turnover. Myocardial MMP-1 may contribute to tissue remodelling in the heart. Little is known about collagenase and its regulation in the myocardium. To understand better the nature of this neutral proteinase in the rat myocardium, myocardial collagenase was purified to homogeneity. The purification procedure included a gel-filtration step on Sephacryl S-200 columns and substrate affinity chromatography on type I collagen-Sepharose. Under reducing conditions, collagenase was shown by SDS-PAGE to consist of a single polypeptide chain with a molecular mass of 54 kDa. Purified interstitial collagenase demonstrated a single lytic band on zymography. This band was inhibited by 1,10-phenanthroline (a metal chelator), which indicates that the 54 kDa protein is an MMP. Using a polyclonal antibody to proMMP-1, purified collagenase was characterized by immunoblot analysis. A single band of purified interstitial collagenase was observed on Western blot analysis. This indicated that the purified proenzyme was collagenase. Sequence analysis on cyanogen bromide-digested fragments of latent MMP-1 suggested that the active site sequence of rat myocardial MMP-1 is similar to that of the rat osteoblast collagenase, human skin fibroblast collagenase and Serratia proteinase. The substrate specificity of the purified collagenase was measured against fluorescent-labelled type I collagen. It was observed that after activation, purified collagenase was capable of degrading type I collagen in a time-dependent manner. The half-time for collagen degradation was estimated to be less than 30 s. These results suggest that collagenase is present in the normal adult rat myocardium and that collagen turnover may be regulated by this neutral metalloproteinase. A simple two-step purification protocol is demonstrated for interstitial collagenase. This procedure can be used for routine MMP-1 preparation from tissue sources.
Can J Cardiol 1996 Feb
PMID:Myocardial collagenase: purification and structural characterization. 860 38

Percutaneous transluminal coronary angioplasty is associated with intimal hyperplasia and extracellular matrix deposition of collagen, leading to restenosis in a significant number of cases. The purpose of the present study was to determine the effects of balloon angioplasty on extracellular matrix collagen content and collagenase activity in a porcine coronary artery restenosis model 6 weeks following balloon injury. We tested the hypothesis that in balloon-injured arteries the neointimal extracellular matrix was characterized by increased collagen content and decreased metalloproteinase activity relative to non-injured arteries. Male miniswine maintained on a high cholesterol diet underwent cardiac catheterization and double balloon injury to the right and left circumflex coronary arteries. The coronary arteries were either pressure-perfusion-fixed and prepared for histological examination, or dissected free of adventitia for further collagen and matrix metalloproteinase studies. Collagen synthesis in balloon-injured coronary arteries was compared to non-injured arteries using Northern blot analysis and histochemical stains. Comparative studies on differences between balloon-injured and non-balloon-injured arterial matrix metalloproteinase activity were done using zymography. Balloon angioplasty arterial injury resulted in a significant increase in type I collagen mRNA expression, with increased collagen deposition in the extracellular matrix. In contrast, matrix metalloproteinase activity was markedly decreased. The results suggest that the increased neointimal extracellular matrix observed late in the injury response may be due to not only increased collagen synthesis, but also reduced degradation. The failure to achieve a balance between the synthesis and degradation of extracellular matrix collagen could serve as an important mechanism responsible for restenosis.
J Mol Cell Cardiol 1996 Apr
PMID:Extracellular matrix collagen synthesis and degradation following coronary balloon angioplasty. 873 98

Incubation of cultured neonatal rat cardiomyocytes in hypoxic conditions, mimicking the deprivation of O2 which occurs during in situ myocardial ischemia, leads to a progressive change in cardiomyocytes cytoskeletal components. Confocal scanning laser immunofluorescence microscopy (CSLIM) reveals that the typical striated costameric distribution of vinculin gradually disappears to be replaced by circular, vinculin-containing sarcolemmal rosettes. There is little change in distribution of vinculin in the focal adhesions or in the intercalated disks. This cytoskeletal alteration, like that observed in virally transformed fibroblasts and phorbol ester-treated skeletal myoblasts, is inhibited by genistein, a tyrosine kinase inhibitor. Increased exposure to hypoxic conditions also produces an increase in a 92-kDa collagenase which is immunolocalized only to cardiomyocytes. As with the rosette formation, genistein also inhibits the increased expression of the 92-kDa collagenase. We suggest that this cytoskeletal change with attendant release of 92 kDa collagenase may represent a defensive mechanism on the part of the cardiomyocyte to reduce damage by reducing the cellular coupling to the extracellular collagenous matrix, thereby lessening the stresses imposed by contractile forces.
J Mol Cell Cardiol 1995 Dec
PMID:Hypoxia-induced alterations in cytoskeleton coincide with collagenase expression in cultured neonatal rat cardiomyocytes. 882 74

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.
J Mol Cell Cardiol 1996 Jul
PMID:Post-transcriptional regulation of extracellular matrix metalloproteinase in human heart end-stage failure secondary to ischemic cardiomyopathy. 884 29

Heparin has been shown to stimulate angiogenesis in the border zones surrounding infarcted myocardium. Matrix metalloproteinases (MMP), which are involved in extracellular matrix (ECM) organization, have also been shown to be activated. Cholesterol is required for receptor signaling in the plasma membrane, but a role of MMPs for cholesterol in ECM remodeling has not yet been shown. To examine whether heparin and cholesterol induce MMP and tissue inhibitor of metalloproteinase (TIMP) in human heart fibroblast (HHF) cells, confluent HHF cells were treated with cholesterol (100 microM) or heparin (20 microM). MMP activity was measured using zymography and TIMP was measured by Western blot analysis. The number of HHF cells, measured by a hemocytometer, increased after heparin or cholesterol treatment. Gelatinase A (MMP-2) activity increased in heparin treated cells, and the TIMP-1 level increased in cholesterol-treated cells. Based on Northern blot analysis, we observed that both MMP-1 and MMP-2 were induced at the gene transcription level by heparin and that TIMP-1 was induced by cholesterol. To examine whether the effects of heparin and cholesterol were due to Ca2+ mobilization, we carried out Ca2+ transient assays using FURA-2/AM as a fluorescence probe in HHF cells. Heparin induced a slow rise in the Ca2+ transient with a slow decay, and cholesterol induced a rapid rise with a slow reversal to the baseline calcium level. This suggested that the effect of heparin on Ca2+ release from HHF may be secondary to the receptor binding on the cell membrane but that cholesterol may have a direct effect. Protein kinase inhibitor and Ca2+-channel blocker have been shown to inhibit MMP expression. To examine whether the effect of heparin on MMP expression is mediated through the collagenase promoter activity, we carried out gel-shift assays using a 21-oligonucleotide analogue to the MMP-1 promoter sequence. Results suggested that the increase in MMP promoter activity by heparin is due to a specific transcription factor binding to MMP-1 promoter sequence. The effect of cholesterol on fibroblast cell proliferation is due in part to the tissue inhibitor. This study demonstrated the role of heparin and cholesterol in ECM remodeling and has implications for angiogenesis and athersclerosis, respectively.
J Mol Cell Cardiol 1997 Jan
PMID:Differential regulation of extracellular matrix metalloproteinase and tissue inhibitor by heparin and cholesterol in fibroblast cells. 904 53

Although increased deposition of collagen proteins has been described in cardiomyopathy, little is known of the temporal relationship between events in collagen gene transcription and the occurrence of cardiac fibrosis, the removal of collagen by matrix metalloproteinases (MMPs), or of the regulation of these events by angiotensin AT1 receptors in this disease. We sought to study steady-state collagen mRNA abundance and the deposition of specific collagen subtypes in right and left ventricular muscle of Syrian cardiomyopathic (CMP) hamsters at different stages of cardiomyopathy. Using zymography, we also investigated the gelatinolytic activities of different MMPs to gain some information about collagen removal in experimental hearts. Finally, we investigated the effect of AT1 receptor blockade (losartan) on collagen remodeling. We observed that the mRNA levels of types I and III collagens were significantly increased in all four experimental groups (35, 65, 120, and 200 day) in left ventricular tissue when compared to control (F1-beta strain) values. The mRNA levels of these collagen species in experimental right ventricular tissue samples were only elevated significantly in the 35 and 200 day experimental groups when compared to controls. Fibrillar collagen deposition was elevated in left and right ventricular CMP samples after a lag period from the occurrence of corresponding increases in mRNA abundance. Although 2-week losartan treatment of 65, 120 and 200 day experimental groups had no significant effect on left ventricular fibrillar collagen concentration or collagen mRNA abundance when compared to vehicle-infused CMP hamsters, AT1 receptor blockade was associated with complete regression of cardiac hypertrophy. Both MMP-1 (54 kDa band) and MMP-2 (58 and 62 kDa bands) activities were increased in left ventricular CMP tissues at 65, 120 and 200 days when compared to F1-beta controls. Losartan treatment was associated with significant attenuation of MMP activities in cardiomyopathic samples at 65 and 120 days. Thus, elevation of mRNA abundance of fibrillar collagen genes occurs at very early stages in this model of cardiomyopathy, and corresponding collagen proteins were subsequently deposited in the cardiac interstitium at later stages. As collagen concentration was significantly increased in later stages of cardiomyopathy studied herein (120 and 200 day groups), our data support the hypothesis that collagen synthesis exceeds the capacity of collagen removal during the progression of cardiomyopathy. Nevertheless, cardiac collagen remodeling may be facilitated by elevated MMP activity in cardiomyopathic stages in this experimental model, and we suggested that attenuation of MMP activity in the presence of losartan may be a cardioprotective mechanism of this agent.
J Mol Cell Cardiol 1997 Jul
PMID:Cardiac collagen remodeling in the cardiomyopathic Syrian hamster and the effect of losartan. 923 38

Vinculin is a cytoskeletal protein that is believed to be an essential component in the linkage of cytoskeletal actin filaments to the plasma membrane. To investigate the precise function of vinculin in the development of cardiac myofibrils, antisense oligodeoxynucleotides complementary to vinculin mRNA were used to perturb the expression of the protein during myofibril assembly and arrangement in mouse cardiac myocytes. Fetal (day 18-20 post-conception) mouse cardiac myocytes were isolated by collagenase digestion, separated by Percoll density gradient centrifugation, and plated on aligned collagen gels. By 72 h of culture, mouse myocytes displayed an elongated in vivo-like phenotype in parallel with the aligned fibrils of the collagen gels with polarized arrays of myofibrils. Two different antisense oligonucleotides (20-mer) altered the formation of the tissue-like phenotype of myocytes. These antisense oligonucleotides suppressed vinculin protein expression at 43.5+/-26.8% and 48.7+/-20.9% when compared to myocytes that were not treated. Examination of these myocytes by confocal scanning laser and transmission electron microscopy revealed a disruption of the aligned in vivo-like phenotype, assembly of thick and thin filaments, and formulation of Z-bands. Random sequence 20-mer oligonucleotides used as controls had little detectable effect on vinculin protein expression (94.2+/-14.8%), cell shape, normal alignment or assembly of myofibrils. These results indicate that vinculin is a critical cytoskeletal component, that functions in the determination of cell shape and the arrangement and organization of developing myofibrils.
J Mol Cell Cardiol 1997 Aug
PMID:Vinculin is an essential component for normal myofibrillar arrangement in fetal mouse cardiac myocytes. 928 37

Delayed reperfusion has a beneficial effect on prognosis, independent of infarct size. One potential mechanism to explain this observation may be an effect on infarct healing. In this study, the impact of delayed reperfusion on two aspects of the healing process was examined, the activity of matrix metalloproteinase (MMP) enzymes and the expression of fibronectin (FN) mRNA. The rat model of coronary artery ligation was used and rats were randomly assigned to delayed reperfusion (150 min following coronary ligation) or permanent ligation. Animals were subsequently killed 1, 2, 3 and 7 days following infarction. Infarct tissue was harvested for MMP activity (zymography), FN mRNA (RNase protection analysis) and protein (immunofluorescence microscopy and Western analysis), and collagen content (hydroxyproline concentration). Infarction produced marked activation of MMP-1, -2, and -9. Reperfusion significantly attenuated the activity of these enzymes (approximately 50% reduction in MMP-1, P=0.03 and ;60% reduction in MMP-2 at 7 days, P=0.001; approximately 55% reduction in MMP-9 at 24 h and 84% reduction at 48 h, P=0.01 and 0.002, respectively). Delayed reperfusion also produced a trend toward a greater increase in FN mRNA 24 h following infarction and immunofluorescent staining suggested the presence of more FN protein at this point. These data demonstrate that delayed reperfusion alters matrix metalloproteinase activity and fibronectin mRNA expression in the infarct zone. The impact of these changes on infarct healing and their association with the improved prognosis of a patent infarct vessel following infarction will require further study.
J Mol Cell Cardiol 1997 Sep
PMID:Delayed reperfusion alters matrix metalloproteinase activity and fibronectin mRNA expression in the infarct zone of the ligated rat heart. 929 68

It was the aim of the present study to characterize the hemodynamic, biochemical and morphologic effect of angiotensin II receptor blockade on hypoxia-induced right ventricular hypertrophy in rats. Isolated right ventricular hypertrophy was induced in female Sprague-Dawley rats by intermittent hypoxia (IH; 10% O2, 8 h/day, 5 days/week, 20 days of exposition, n=15). After completion of IH, left- (LV) and right-ventricular (RV) hemodynamic parameters were measured under room air conditions in the intact, thiopental-anesthetized animals with special Millar ultraminiature tipcatheter-manometers. Cardiac output was determined using the thermodilution method. Cell volume (CV) of isolated cardiomyocytes was measured with a Coulter Channellyzer after collagenase cell isolation. The specific activities of the myocardial pentose phosphate pathway enzymes glucose-6-phosphate-dehydrogenase (G-6-PD) and 6-phosphogluconate-dehydrogenase (6-PGD) were determined using a spectrophotometric assay. IH caused a rise in right ventricular systolic pressure (RVSP) from 38.1+/-0.83 to 58.1+/-1.42 mmHg and an increase in the RV weight/body weight ratio (RVW/BW) from 0.884+/-0. 053 to 1.166+/-0.049 mg/g. The activities of G-6-PD and 6-PGD were significantly increased after IH in the RV, but not in the LV. CV was increased from 24 248+/-1193 to 29 541+/-1765 micrometer 3, myocardial cell length was unchanged. IH had no influence on the LV parameters or cardiac output. Co-infusion of the angiotensin II receptor antagonist losartan (LO; 12 mg/kg/d i.p., n=14) during the IH period reduced the rises in RVSP (49.4+/-2.06 mmHg), RVW/BW (0. 99+/-0.072 mg/g), G-6-PD and 6-PGD significantly, but not completely. The increase in CV, however, was prevented (24 524+/-2370 micrometer 3) entirely. We conclude from these data that the IH-induced RV-hypertrophy was primarily of the concentric type. LO attenuated the hypoxia-induced isolated RV hypertrophy and significantly reduced the metabolic response of the RV. The LO effect was most potent with regard to the increase in cardiomyocyte volume.
J Mol Cell Cardiol 1997 Nov
PMID:Effects of angiotensin II receptor blockade on hypoxia-induced right ventricular hypertrophy in rats. 940 68

Calcium tolerant pig and rabbit cardiomyocytes were isolated using retrograde aortic perfusion of nominally calcium-free collagenase. Preconditioning protocols used 1 or 3x10-min episodes of ischemic pelleting or pre-incubation with 100 micro M adenosine, followed by a 15-min post-incubation and 180-240-min ischemic pelleting. Control cells were incubated and washed in parallel with the experimental groups. Injury was assessed by determination of cell morphology, trypan blue permeability following osmotic swelling, lactate and HPLC analysis of adenine nucleotides. Preconditioned pig cardiomyocytes had a reduced rate of ischemic contracture, but protection occurred without conservation of ATP. Preconditioned rabbit cardiomyocytes were protected without significant changes in rates of ischemic contracture or ATP depletion. Incubation of ischemic cells with the protein phosphatase inhibitor, fostriecin, at PP2A-selective concentrations (0.1-10 micro M), mimicked preconditioning in both rabbit and pig cardiomyocytes. In rabbits, the KATP channel blocker, 5-hydroxydecanoate (5-HD), did not block preconditioning or fostriecin protection. In the pig, 5-HD blocked both preconditioning and fostriecin protection, with return of the rates of ischemic contracture to control. However, 5-HD was an effective blocker of protection only in early ischemia. Fostriecin mimicked preconditioning in the rabbit and the early responses of the preconditioned pig. Preconditioning appears associated with protein phosphorylation in both the rabbit and the pig, but major pathways leading to protection may differ in the two species.
J Mol Cell Cardiol 1997 Nov
PMID:Comparison of in vitro preconditioning responses of isolated pig and rabbit cardiomyocytes: effects of a protein phosphatase inhibitor, fostriecin. 940 76


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