MMP/TIMP expression in spontaneously hypertensive heart failure rats: the effect of ACE- and MMP-inhibition.
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OBJECTIVE: Determine the effect of a matrix metalloproteinase inhibitor (MMPi) and angiotensin converting enzyme inhibitor (ACEi) on collagen, MMP, tissue inhibitors of MMPs (TIMPs) expression in the spontaneously hypertensive heart failure (SHHF) rat. METHODS: Six groups were tested: normotensive 9- and 13-month-old Wistar-Furth (WF) rats, 9-month-old SHHFs (compensatory hypertrophy), 13-month-old SHHFs with HF, and 13-month-old SHHFs orally administered with either an MMPi (PD166793, 5 mgkg(-1)day(-1)) or ACEi (quinapril, 10 mgkg(-1)day(-1)) for 4 months. Collagen volume fraction was assessed histomorphometrically. Left ventricular (LV) mRNA [MMP-1,-2,-3,-7,-9,-11,-13,-14; TIMP-1,-2,-3,-4; and collagen alpha1(I) and alpha1(III)] and protein (MMP-2 and MMP-9 zymographic activity; Western blot analysis of MMP-13, and TIMP-1,-2,-4) levels could be quantified. RESULTS: Collagen mRNA levels were elevated in SHHFs compared to age-matched controls, but collagen volume fraction was elevated only in 13-month-old SHHFs (approximately 2x). Only MMP-2 mRNA levels increased significantly with HF. However, MMP-2 and MMP-9 zymographic activity, and MMP-13 protein levels increased. TIMP-1 and TIMP-2 mRNA and protein levels increased, and TIMP-4 protein levels decreased in SHHFs vs. controls. Both drug treatments reduced LV dilation; preserved systolic function; and normalized MMP/TIMP expression. Both drug treatments also reduced collagen volume fraction, but only quinapril reduced collagen mRNA levels and LV hypertrophy. CONCLUSIONS: The divergent effect of MMPi and ACEi on collagen mRNA levels and hypertrophy indicate that drug efficacy is mediated by different pathways in the SHHF rat. (+info)
Evolution of matrix metalloprotease and tissue inhibitor expression during heart failure progression in the infarcted rat.
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OBJECTIVE: Characterize the timecourse of matrix metalloproteinase (MMP-1, -2, -3, -7, -9, -11, -12, -13, and -14) and endogenous tissue inhibitors of MMPs (TIMP-1, -2, -3, and -4) upregulation during left ventricular (LV) remodeling following myocardial infarction (MI) in rats. METHODS: The descending left coronary artery of male rats (Rattus norvegicus) was ligated to produce a MI. LV function and dilation were assessed from 1 day to 16 weeks post-MI. Protein and mRNA extraction was done on LV samples containing scar and myocardium together. Gelatinase activity was measured by zymography. Westerns were run on the MMPs known to cleave fibrillar collagen in the rat (MMP-8, -13, and -14) as well as TIMP-1, -2, and -4. RESULTS: Average infarct size was 38.6+/-1.1%, and produced LV dysfunction and progressive LV dilation. Thoracic ascites, a marker of congestive heart failure (HF), was not present until 12 weeks post-MI. Upregulation of MMP-2, -8, -9, -13, and -14 and TIMP-1 and TIMP-2 was detected at different timepoints during HF progression. Increased MMP protein levels occurred sometimes without a corresponding elevation in mRNA levels, and increased TIMP mRNA levels without increased protein levels. MMP-13 active form was elevated during the first 2 weeks post-MI while TIMP-1 and TIMP-2 protein levels were not significantly elevated until 2 weeks post-MI. MMP-8 and MMP-14 protein levels increased later during heart failure progression. CONCLUSION: MMP/TIMP upregulation evolves over time following infarction in the rat LV. Some MMPs were significantly elevated during the first week post-MI (MMP-13, -2, and -9) and another was not until 16 weeks post-MI (MMP-14). The dissociation between LV MMP/TIMP mRNA and protein levels shows that post-translation processing occurs in the rat heart. (+info)
Hydrolysis of triple-helical collagen peptide models by matrix metalloproteinases.
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The matrix metalloproteinase (MMP) family has been implicated in the process of a variety of diseases such as arthritis, atherosclerosis, and tumor cell metastasis. To study the mechanisms of MMP action on collagenous substrates, we have constructed homotrimeric triple-helical peptide (THP) models of the collagenase cleavage sites in types I and II collagen. The THPs incorporate either the alpha1(I)772-786 or the alpha1(II)772-783 sequence. The alpha1(I)772-786 and alpha1(II)772-783 THPs were hydrolyzed by MMP-1 at the Gly-Ile and Gly-Leu bonds, respectively, analogous to the bonds cleaved in corresponding native collagens. Thus, the THPs contained all necessary information to direct MMP-1 binding and proteolysis. Subsequent investigations using the alpha1(I)772-786 THP showed hydrolysis by MMP-2, MMP-13, and a COOH-terminal domain-deleted MMP-1 (MMP-1(Delta(243-450))) but not by MMP-3 or a COOH-terminal domain-deleted MMP-3 (MMP-3(Delta(248-460))). Kinetic analyses showed a k(cat)/K(m) value of 1,808 s(-1) m(-1) for MMP-1 hydrolysis of alpha1(I)772-786 THP, approximately 10-fold lower than for type I collagen. The effect is caused primarily by relative K(m) values. MMP-2 and MMP-13 cleaved the THP more rapidly than MMP-1, but MMP-2 cleavage occurred at distinct multiple sites. Comparison of MMP-1 and MMP-1(Delta(243-450)) hydrolysis of alpha1(I)772-786 THP showed that both can cleave a triple-helical substrate with a slightly higher K(m) value for MMP-1(Delta(243-450)). We propose that the COOH-terminal domain of MMPs is necessary for orienting whole, native collagen molecules but may not be necessary for binding to and cleaving a THP. This proposal is consistent with the large distance between the MMP-1 catalytic and COOH-terminal domains observed by three-dimensional structural analysis and supports previous suggestions that the features of the catalytic domain contribute significantly toward enzyme specificity. (+info)
Involvement of matrix metalloproteinases 2 and 9, tissue inhibitor of metalloproteinases and apoptosis in tissue remodelling in the sheep placenta.
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Placental growth and development is crucial for successful pregnancy. The aim of this study was to characterize the activity and localization of the matrix metalloproteinase 2 (MMP-2) and MMP-9, which are capable of degrading basement membrane collagen (predominantly collagen type IV), and their endogenous tissue inhibitor of matrix metalloproteinases (TIMPs), in amniotic fluid and in the developing ovine placenta. Cell deletion by apoptosis during placental development was also examined. Zymography with gelatin as substrate indicated that MMP-2 (72 kDa gelatinase A; predominantly latent form) was present in increasing amounts in amniotic fluid from day 70 of gestation to labour (days 140-145), and MMP-9 (92 kDa gelatinase B; predominantly latent form) was detectable from day 125 to labour; there was no increase in MMP-2 or -9 in labour. A broad range of TIMPs was detected in amniotic fluid; the molecular masses corresponded to TIMP-1, -2 and -3. Immunohistochemical techniques localized MMP-2, MMP-9 and TIMP-3 in the sheep placenta, predominantly in the trophoblast layer in uninucleate, but not binucleate, cells. However, MMP-2 and -9 activated proteins in placental homogenates were low throughout pregnancy. Apoptosis was identified by morphological criteria and also by TdT-mediated dUTP nick end labelling. Apoptosis was present in discrete regions in the placenta, predominantly in trophoblast cells near the tips and the basal regions of the fetomaternal interdigitations. During pregnancy the sheep placenta becomes more complex and the area of the fetomaternal interface increases. MMP-2 and -9 are likely to be involved in breaking down basement membranes to allow cell migration during this process. It is suggested that digestion of supporting extracellular matrix may trigger apoptosis and in some way increase the branching pattern in the villi. (+info)
Membrane type 4 matrix metalloproteinase (MMP17) has tumor necrosis factor-alpha convertase activity but does not activate pro-MMP2.
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Membrane type 4 matrix metalloproteinase (MT4-MMP) shows the least sequence homology to the other MT-MMPs, suggesting a distinct function for this protein. We have isolated a complete cDNA corresponding to the mouse homologue which includes the signal peptide and a complete pro-domain, features that were lacking from the human form originally isolated. Mouse MT4-MMP (mMT4-MMP) expressed in COS-7 cells is located at the cell surface but does not show ability to activate pro-MMP2. The pro-catalytic domain was expressed in Escherichia coli as insoluble inclusions and active enzyme recovered after refolding. Activity of the isolated catalytic domain against synthetic peptides commonly used for MMP enzyme assays could be inhibited by TIMP1, -2, and -3. The recombinant mMT4-MMP catalytic domain was also unable to activate pro-MMP2 and was very poor at hydrolyzing components of the extracellular matrix with the exception of fibrinogen and fibrin. mMT4-MMP was able to hydrolyze efficiently a peptide consisting of the pro-tumor necrosis factor alpha (TNFalpha) cleavage site, a glutathione S-transferase-pro-TNFalpha fusion protein, and was found to shed pro-TNFalpha when co-transfected in COS-7 cells. MT4-MMP was detected by Western blot in monocyte/macrophage cell lines which in combination with its fibrinolytic and TNFalpha-converting activity suggests a role in inflammation. (+info)
Identification and characterization of human endometase (Matrix metalloproteinase-26) from endometrial tumor.
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We report the discovery, cloning, and characterization of a novel human matrix metalloproteinase 26 (MMP-26) (matrixin) gene, endometase, an endometrial tumor-derived metalloproteinase. Among more than three million expressed sequence tags sequenced, the endometase gene was only obtained from human endometrial tumor cDNA library. Endometase mRNA was expressed specifically in human uterus, not in other tissues/cells tested, e.g. testis, heart, brain, lungs, liver, thymus, and melanoma G361. Endometase protein has a signal peptide, a propeptide domain, and a catalytic domain with a unique "cysteine switch" propeptide sequence, PHCGVPDGSD, and a zinc-binding motif, VATHEIGHSLGLQH. Endometase is 43, 41, 41, and 39% identical to human metalloelastase, stromelysin, collagenase-3, and matrilysin, respectively. The zymogen was expressed and isolated from Escherichia coli as inclusion bodies with a molecular mass of 28 kDa. The identity and homogeneity of the recombinant protein was confirmed by protein N-terminal sequencing, silver stain, and immunoblot analyses. The pro-enzyme was partially activated during the folding process. Endometase selectively cleaved type I gelatin and alpha(1)-proteinase inhibitor; however, it did not digest collagens, laminin, elastin, beta-casein, plasminogen, soybean trypsin inhibitor, or Bowman-Birk inhibitor. It hydrolyzed peptide substrates of matrixins and tumor necrosis factor-alpha converting enzyme. Endometase may selectively cleave extracellular matrix proteins, inactivate serpins, and process cytokines. (+info)
Angiopoietin-1 induces endothelial cell sprouting through the activation of focal adhesion kinase and plasmin secretion.
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Angiopoietin-1 (Ang1) is a strong inducer of endothelial cell sprouting, which is a first step in both angiogenesis and neovascularization. We examined the mechanisms underlying Ang1-induced cell sprouting using porcine pulmonary artery endothelial cells. Ang1 induced the nondirectional and directional migration of endothelial cells mediated through the Tie2 but not the Tie1 receptor. Ang1 induced tyrosine phosphorylation of p125(FAK), and this phosphorylation was dependent on phosphatidylinositol (PI) 3'-kinase activity. Ang1 induced the secretion of plasmin and matrix metalloproteinase-2 (MMP-2), which is inhibited by PI 3'-kinase inhibitors. Ang1 also induced the secretion of small amounts of proMMP-3 and proMMP-9 but not proMMP-1. Ang1 suppressed the secretion of tissue inhibitor of metalloproteinase-2 (TIMP-2), but not of TIMP-1. Addition of alpha(2)-antiplasmin, a combination of TIMP-1 and TIMP-2, or PI 3'-kinase inhibitors inhibited Ang1-induced sprouting activity. Therefore, Ang1-induced sprouting activity in endothelial cells may be accomplished by cytoskeletal changes and secretion of proteinases and may be largely mediated through intracellular PI 3'-kinase activation. (+info)
The Ras-mitogen-activated protein kinase pathway is critical for the activation of matrix metalloproteinase secretion and the invasiveness in v-crk-transformed 3Y1.
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To search for the intracellular signaling pathway critical for the secretion of matrix metalloproteinases (MMP), we studied the effects of dominant negative Ras (S17N Ras) and dominant negative MEK1 (MEK1AA) expression in v-crk-transformed 3Y1. Expression of either S17N Ras or MEK1AA dramatically suppressed the augmented secretion of MMP-2 and MMP-9 in v-crk-transfected 3Y1. Similarly, a Ras farnesyltransferase inhibitor, manumycin A, and a MEK1 inhibitor, U0126, suppressed MMP secretion in a dose-dependent manner, whereas a PI3 kinase inhibitor, wortmannin, could not. In addition, the suppression of MMP secretion by S17N Ras showed good correlation with the inhibition of in vitro invasiveness of the cells. In contrast, expression of dominant negative C3G did not suppress MMP secretion, although it substantially blocked the c-Jun N-terminal kinase activation. Taken together, the Ras-MEK1 pathway, but not the C3G-JNK pathway, seems to play a key role in the activation of MMP secretion and, hence, the invasiveness of v-crk-transformed cells. (+info)