7-oxa-13-prostynoic acid and polyphloretin phosphate as non-specific antagonists of the stimulatory effects of different agents on adenylate cyclase from various tissues.

7-oxa-13-prostynoic acid (OPA) and polyphloretin phosphate (PPP) are believed to act as specific antagonists of prostaglandin action. In order to estimate their specificity, the inhibitory effects of these drugs were tested on the activity of adenylate cyclase from several tissues which were stimulated by prostaglandins and several other compounds. In adenylate cyclase preparation from L-fibroblasts both OPA (0.15-1.5 MM) and PPP (0.01-1.0 MG/ML) antagonized not only the stimulatory effects of PGE but also the stimulatory effects of sodium fluoride and increased enzyme activity due to the previous treatment of cell cultures by cholera toxin. Both OPA and PPP produced a dose dependent depression of adenylate cyclase activity to zero values both under basal conditions and after stimulation by sodium fluoride and various hormones in all preparations studied, including rat liver, heart, brain, epididymal adipose tissue, small intestine, renal cortex and renal medulla. The present results indicate that both prostaglandin antagonists may, in higher concentrations, act as nonspecific inhibitors of the catalytic unit of adenylate cyclase rather than specific antagonists of the prostaglandin effects on adenylate cyclase.     

7-Oxa-13-prostynoic acid and polyphloretin phosphate as non-specific antagonists of the stimulatory effects of different agents on adenylate cyclase from various tissues

7-Oxa-13-prostynoic acid (OPA) and polyphloretin phosphate (PPP) are believed to act as specific antagonists of prostaglandin action. In order to estimate their specificity, the inhibitory effects of these drugs were tested on the activity of adenylate cyclase from several tissues which were stimulated by prostaglandins and several other compounds.

In adenylate cyclaae preparation from L-fibroblasts both OPA (0.15–1.5 mM) and PPP (0.01–1.0 mg/ml) antagonized not only the stimulatory effects of PGE₁ but also the stimulatory effects of sodium fluoride and increased enzyme activity due to the previous treatment of cell cultures by cholera toxin. Both OPA and PPP produced a dose dependent depression of adenylate cyclase activity to zero values both under basal conditions and after stimulation by sodium fluoride and various hormones in all preparations studied, including rat liver, heart, brain, epididymal adipose tissue, small intestine, renal cortex and renal medulla.

The present results indicate that both prostaglandin antagonists may, in higher concentrations, act as nonspecific inhibitors of the catalytic unit of adenylate cyclase rather than specific antagonists of the prostaglandin effects on adenylate cyclase.     

A deficit in collagenase activity contributes to impaired migration of aged microvascular endothelial cells

Angiogenesis is impaired in aging. Delayed neovascularization is due, in part, to slowed endothelial cell migration. Migration requires an optimal level of adhesion to matrix proteins, a process mediated by matrix-degrading metalloproteases (MMPs) such as MMP1. To determine whether impaired angiogenesis in aging is associated with altered synthesis and activity of MMP1, we examined the expression of collagenase and tissue inhibitor of metalloprotease 1 (TIMP1) by immunostain of angiogenic sponge implants from young and aged mice. To characterize the relevance of MMP activity during the movement of aged endothelial cells, the secretion of MMP1 and TIMP1 by late-passage human microvascular endothelial cells (hmEC aged in vitro) and their non-aged (young) counterparts was quantified. The migration of aged human microvascular endothelial cells and the effect of inhibition of TIMP1 on the migration of aged hmEC or collagen I was also measured. Relative to young mice, granulation tissue from aged mice showed less expression of collagenase and increased expression of TIMP1. In vitro, aged hmEC were deficient in MMP1 secretion (55 +/- 13% relative to young cells) and activity but showed increased expression of TIMP1 (280 +/- 109% relative to young cells). Aged hmEC migrated significantly less distance than did young hmEC over a 5-day period (59 +/- 8% relative to young cells). In the presence of a blocking antibody to TIMP1, aged hmEC showed a significant increase in the distance migrated on collagen I over a 5 day period (142 +/- 11% relative to untreated aged hmEC). We propose that deficient MMP1 activity contributes to impaired cellular movement in aged microvascular endothelial cells and that perturbations that enhance collagenase activity increase their migratory ability and angiogenic potential.     

糜酶转基因小鼠心脏组织中基质金属蛋白酶9的活化

为研究糜酶在心脏中的功能 ,用明胶酶谱法和放免法检测了糜酶转基因小鼠心脏组织中基质金属蛋白酶及糜酶的活力 .糜酶转基因小鼠心脏组织中糜酶样活力较转基因阴性小鼠升高了约80 % ;而其心脏匀浆液凝胶酶谱分析结果显示在 92 k D处明胶酶活力也升高约 30 % ;经 Western印迹鉴定为基质金属蛋白酶 9,而在蛋白水平上与转基因阴性小鼠无显著差异 .结果提示 ,糜酶转基因小鼠心脏组织中糜酶活力的升高可活化基质金属蛋白酶 9,从而影响心脏胶原代谢 .     

Characteristics of a 95-kDa matrix metalloproteinase produced by mammary carcinoma cells

A Mr 95,000 matrix metalloproteinase (MMP) produced by rat mammary carcinoma cells has been isolated and characterized. The MMP was secreted in a proteolytically inactive form that was free from bound tissue inhibitor of metalloproteinases. The enzyme was highly glycosylated as evident from an apparent drop of Mr from 95,000 to 83,000 after treatment with N-glycanase. Rotary shadowing electron micrographs of purified proenzyme preparations revealed a uniform set of ellipsoidal molecules. Treatment of the proenzyme with 1% SDS resulted in generation of catalytic activity and exposed a cryptic unpaired Cys residue. The latent proenzyme may be activated in at least three additional ways: either spontaneously upon storage, by treatment with organomercurials, or by limited proteolysis by trypsin. Each mode of activation yielded a distinct pattern of cleavage of the enzyme. The activated enzyme cleaved gelatin (denatured type I collagen) and native type IV and V collagen at 30-37 degrees C. Noncollagenous proteins including alpha 1-proteinase inhibitor, casein, and fibrinogen also were cleaved. The rat mammary carcinoma cell line that produces the Mr 95,000 MMP is composed of two distinct (epithelial- and myoepithelial-like) cell types. The enzyme is expressed constitutively by the epithelial cells. This suggests that expression of the Mr 95,000 MMP is regulated differently from that of interstitial collagenase, which is produced by the epithelial cells only in response to specific inductive factor(s) from the myoepithelial-like cells. Monoclonal antibodies raised against the purified latent Mr 95,000 form of the enzyme bind specifically to the Mr 95,000 MMP and have been used to localize the enzyme to the Golgi region and cytoplasmic granules of the epithelial cells.     

Expression of matrix metalloproteinase activity in idiopathic dilated cardiomyopathy: A marker of cardiac dilatation

BACKGROUND: Idiopathic dilated cardiomyopathy (DCM), ventricular systolic dysfunction and chamber dilatation are accompanied by architectural remodeling, wall thinning and cardiac myocyte slippage. Recent work has demonstrated an association between collagen degradation and an increased expression of matrix metalloproteinases (MMPs). Accordingly, we have sought to correlate (a) collagen degradation with MMP elevations and, (b) assay the neutralizing potential of a known inhibitor of MMP, tetracycline on MMPs in DCM. METHODS: Assessment of LV volume and shape by 2-D echocardiography was performed. Light microscopic assessment of histopathology in picrosirius red stained biopsy samples of 11 DCM patients and six post-transplant patients was performed. Zymographic estimation of MMP activity and influence of tetracycline on MMP activity was assessed. RESULTS: Small amount of interstitial collagen was noted in the control group, whereas in the DCM hearts, chamber dilatation was associated with areas of scanty myocyte necrosis, islands of excess collagen, and focal areas of absent or scanty collagen with intact myocytes. In cardiomyopathic tissue, collagenase activity was markedly elevated at 63% compared with 8% in post-transplant tissue. Tetracycline at a concentration of 285+/-10 microM (IC50) inhibited collagenase activity by 50% in cardiomyopathic tissue. CONCLUSIONS: Areas of focal interstitial collagen accumulation were accompanied by collagen fiber lysis and increased collagenase activity in dilated cardiomyopathy. This enhanced collagenolytic activity found in endomyocardial biopsy tissue was inhibited by tetracycline. The non-antibiotic property of tetracycline may be of potential value in the prevention of ventricular dilatation in idiopathic dilated cardiomyopathy.     

Profibrotic influence of high glucose concentration on cardiac fibroblast functions: effects of losartan and vitamin E

Long-standing diabetes can result in the development of cardiomyopathy, which can be accompanied by myocardial fibrosis. Although exposure of cultured kidney and skin fibroblasts to high glucose (HG) concentration is known to increase collagen synthesis, little is known about cardiac fibroblasts (CFs). Therefore, we determined the influence of HG conditions on CF functions and the effects of losartan and vitamin E in these responses. We cultured rat CFs in either normal glucose (NG; 5.5 mM) or HG (25 mM) media and assessed changes in protein and collagen synthesis, matrix metalloproteinase (MMP) activity, and levels of mRNA for ANG II type 1 (AT(1)) receptors. Results indicate that HG-level CFs synthesized more protein and collagen, and these effects were not due to changes in osmotic pressure. The addition of ANG II stimulated protein and collagen synthesis in NG-concentration but not HG-concentration CFs. Interestingly, losartan pretreatment blocked the HG- or ANG II-induced increases in both protein and collagen synthesis. HG or ANG II decreased total MMP activity. Decreases in MMP activity were blocked by losartan. AT(1) mRNA levels were upregulated with HG concentration. Vitamin E pretreatment blocked the effects of HG on total protein synthesis and stimulated MMP activity. Results suggest that HG levels may promote fibrosis by increasing CF protein and collagen synthesis and decreasing MMP activity. HG levels may cause these effects via the upregulation of AT(1) receptors, which can be blocked by losartan. However, vitamin E can alter HG concentration-induced changes in CF functions independently of AT(1) mRNA levels.     

Leptin regulates MMP-2, TIMP-1 and collagen synthesis via p38 MAPK in HL-1 murine cardiomyocytes

A clear association between obesity and heart failure exists and a significant role for leptin, the product of the obese gene, has been suggested. One aspect of myocardial remodeling which characterizes heart failure is a disruption in the balance of extracellular matrix synthesis and degradation. Here we investigated the effects of leptin on matrix metalloproteinase (MMP) activity, tissue inhibitor of metalloproteinase (TIMP) expression, as well as collagen synthesis in HL-1 cardiac muscle cells. Gelatin zymographic analysis of MMP activity in conditioned media showed that leptin enhanced MMP-2 activity in a dose- and time-dependent manner. Leptin is known to stimulate phosphorylation of p38 MAPK in cardiac cells and utilization of the p38 MAPK inhibitor, SB203580, demonstrated that this kinase also plays a role in regulating several extracellular matrix components, such that inhibition of p38 MAPK signaling prevented the leptin-induced increase in MMP-2 activation. We also observed that leptin enhanced collagen synthesis determined by both proline incorporation and picrosirius red staining of conditioned media. Pro-collagen type-I and pro-collagen type-III expression, measured by real-time PCR and Western blotting were also increased by leptin, effects which were again attenuated by SB203580. In summary, these results demonstrate the potential for leptin to play a role in mediating myocardial ECM remodeling and that the p38 MAPK pathway plays an important role in mediating these effects.     

Conformational selection and collagenolysis in Type III collagen

Matrix metalloproteases (MMPs) cleave native collagen at a single site despite the fact that collagen contains more than one scissile bond that can, in principle, be cleaved. For peptide bond hydrolysis to occur at one specific site, MMPs must (1) localize to a region near the unique scissile bond, (2) bind residues at the catalytic site that form the scissile bond, and (3) hydrolyze the corresponding peptide bond. Prior studies suggest that for some types of collagen, binding of noncatalytic MMP domains to amino acid sequences in the vicinity of the true cleavage site facilitates the localization of collagenases. In the present study, our goal was to determine whether binding to the catalytic site also plays a role in determining MMP specificity. To investigate this, we computed the conformational free energy landscape of Type III collagen at each potential cleavage site. The free energy profiles suggest that although all potential cleavage sites sample unfolded states at relatively low temperatures, the true cleavage site samples structures that are complementary to the catalytic site. By contrast, potential cleavage sites that are not cleaved sample states that are relatively incompatible with the MMP active site. Furthermore, our findings point to a specific role for arginine residues in modulating the structural stability of collagen near the collagenase cleavage site. These data imply that locally unfolded potential cleavage sites in Type III collagen sample distinct unfolded ensembles, and that the region about the true collagenase cleavage site samples states that are most complementary to the MMP active site. Proteins 2010. © 2009 Wiley‐Liss, Inc.     

Developmental changes of calcium transients and contractility during the cultivation of rat neonatal cardiomyocytes

In the normal myocardium matrix metalloproteinases (MMP) are present in the latent form. To examine whether MMP are activated following infarction or idiopathic dilated cardiomyopathy (DCM), we extracted and measured MMP activity in tissue derived from 7 explanted, failing human hearts due to either previous myocardial infarction (MI) or DCM. MMP activity in infarcted left ventricle (LV), noninfarcted IV and right ventricle (RV) from MI patients, as well as tissue from either ventricle of DCM patients, were compared to the activity of donor heart tissue. SDS-PAGE and dye-binding assays were used to determine total protein concentration, while collagenase activity was measured by SDS-PAGE type substrate gels embedded with type I gelatin (zymography). Accuracy of the zymographic technique was shown for tissue samples as small as 0.05 mg and was comparable to results obtained by a spectrophotometric method.. After normalization for total protein concentration, we found 3 ± 1 % collagenase activity in normal atrial tissue which could be activated to 80–90% by trypsin or plasmin, indicating that collagenase is normally inactive or in a latent form in human heart. In endo- and epimyocardium of infarcted LV on the other hand, collagenase activity was 85–95% and 10–20%, respectively, while 5–10% and 3–5%, respectively, in noninfarcted LV In DCM, collagenolytic activity in the endo and epimyocardium was 75 ± 5 and 35 ± 5% in the LV and 35 ± 7 and 20 ± 5% in the RV, respectively. Thus, in dilated failing human hearts secondary to previous MI or DCM, MMP activity is increased. This is particularly the case within the endomyocardium of the infarcted and noninfarcted portions of either ventricle with MI and in both ventricles in DCM. This suggests that an activation of collagenase throughout the myocardium may contribute to its remodeling that includes ventricular dilatation and wall thinning.This work was supported in part by NIH grant GM-48595 and by a Grant-In-Aid from the American Heart Association, Missouri Affiliate (92-10517).     

Catalytic activities and substrate specificity of the human membrane type 4 matrix metalloproteinase catalytic domain.

Membrane type (MT) matrix metalloproteinases (MMPs) are recently recognized members of the family of Zn(2+)- and Ca(2+)-dependent MMPs. To investigate the proteolytic capabilities of human MT4-MMP (i.e. MMP-17), we have cloned DNA encoding its catalytic domain (CD) from a breast carcinoma cDNA library. Human membrane type 4 MMP CD (MT4-MMPCD) protein, expressed as inclusion bodies in Escherichia coli, was purified to homogeneity and refolded in the presence of Zn(2+) and Ca(2+). While MT4-MMPCD cleaved synthetic MMP substrates Ac-PLG-[2-mercapto-4-methylpentanoyl]-LG-OEt and Mca-PLGL-Dpa-AR-NH(2) with modest efficiency, it catalyzed with much higher efficiency the hydrolysis of a pro-tumor necrosis factor-alpha converting enzyme synthetic substrate, Mca-PLAQAV-Dpa-RSSSR-NH(2). Catalytic efficiency with the pro-tumor necrosis factor-alpha converting enzyme substrate was maximal at pH 7.4 and was modulated by three ionizable enzyme groups (pK(a3) = 6.2, pK(a2) = 8.3, and pK(a1) = 10.6). MT4-MMPCD cleaved gelatin but was inactive toward type I collagen, type IV collagen, fibronectin, and laminin. Like all known MT-MMPs, MT4-MMPCD was also able to activate 72-kDa progelatinase A to its 68-kDa form. EDTA, 1,10-phenanthroline, reference hydroxamic acid MMP inhibitors, tissue inhibitor of metalloproteinases-1, and tissue inhibitor of metalloproteinases-2 all potently blocked MT4-MMPCD enzymatic activity. MT4-MMP is, therefore, a competent Zn(2+)-dependent MMP with unique specificity among synthetic substrates and the capability to both degrade gelatin and activate progelatinase A.     

Solar Ultraviolet Irradiation Induces Decorin Degradation in Human Skin Likely via Neutrophil Elastase

Exposure of human skin to solar ultraviolet (UV) irradiation induces matrix metalloproteinase-1 (MMP-1) activity, which degrades type I collagen fibrils. Type I collagen is the most abundant protein in skin and constitutes the majority of skin connective tissue (dermis). Degradation of collagen fibrils impairs the structure and function of skin that characterize skin aging. Decorin is the predominant proteoglycan in human dermis. In model systems, decorin binds to and protects type I collagen fibrils from proteolytic degradation by enzymes such as MMP-1. Little is known regarding alterations of decorin in response to UV irradiation. We found that solar-simulated UV irradiation of human skin in vivo stimulated substantial decorin degradation, with kinetics similar to infiltration of polymorphonuclear (PMN) cells. Proteases that were released from isolated PMN cells degraded decorin in vitro. A highly selective inhibitor of neutrophil elastase blocked decorin breakdown by proteases released from PMN cells. Furthermore, purified neutrophil elastase cleaved decorin in vitro and generated fragments with similar molecular weights as those resulting from protease activity released from PMN cells, and as observed in UV-irradiated human skin. Cleavage of decorin by neutrophil elastase significantly augmented fragmentation of type I collagen fibrils by MMP-1. Taken together, these data indicate that PMN cell proteases, especially neutrophil elastase, degrade decorin, and this degradation renders collagen fibrils more susceptible to MMP-1 cleavage. These data identify decorin degradation and neutrophil elastase as potential therapeutic targets for mitigating sun exposure-induced collagen fibril degradation in human skin.     

Enhanced matrix metalloproteinase activity in skeletal muscles of rats with congestive heart failure

Patients with congestive heart failure (CHF) are prone to increased skeletal muscle fatigue. Elevated circulatory concentrations of tumor necrosis factor (TNF)-alpha and monocyte chemoattractant protein-1, which may stimulate matrix metalloproteinase (MMP) activity and, thereby, contribute to skeletal muscle dysfunction, are frequently found in CHF. However, whether skeletal muscle MMP activity is altered in CHF is unknown. Hence, we have used a gelatinase assay to assess the activity of MMP and tissue inhibitors of MMP in single skeletal muscles of rats with CHF 6 wk after induction of myocardial infarction. Sham-operated (Sham) rats were used as controls. We also measured the gene expression and protein contents of MMP-2 and MMP-9 in skeletal muscles of these rats. Plasma MMP activity was nearly seven times higher (P < 0.05) in CHF than in Sham rats. Concomitantly, the MMP activity within single slow- and fast-twitch skeletal muscles of CHF rats increased two- to fourfold compared with Sham animals, whereas tissue inhibitor of MMP activity did not differ (P > 0.05). Preformed MMP-2 and MMP-9 were probably activated in CHF, because neither their gene expression nor protein levels were altered (P > 0.05). Serum concentrations of TNF-alpha and monocyte chemoattractant protein-1 remained unchanged (P > 0.05) between CHF and Sham rats during the 6-wk observation period. We conclude that development of CHF in rats enhances MMP activity, which in turn may distort the normal contractile function of skeletal muscle, thereby contributing to increased skeletal muscle fatigue.     

Expression of catalytically active matrix metalloproteinase‐1 in dermal fibroblasts induces collagen fragmentation and functional alterations that resemble aged human skin

Increased expression of matrix metalloproteinase‐1 (MMP‐1) and reduced production of type I collagen by dermal fibroblasts are prominent features of aged human skin. We have proposed that MMP‐1‐mediated collagen fibril fragmentation is a key driver of age‐related decline of skin function. To investigate this hypothesis, we constructed, characterized, and expressed constitutively active MMP‐1 mutant (MMP‐1 V94G) in adult human skin in organ culture and fibroblasts in three‐dimensional collagen lattice cultures. Expression of MMP‐1 V94G in young skin in organ culture caused fragmentation and ultrastructural alterations of collagen fibrils similar to those observed in aged human skin in vivo. Expression of MMP‐1 V94G in dermal fibroblasts cultured in three‐dimensional collagen lattices caused substantial collagen fragmentation, which was markedly reduced by MMP‐1 siRNA‐mediated knockdown or MMP inhibitor MMI270. Importantly, fibroblasts cultured in MMP‐1 V94G‐fragmented collagen lattices displayed many alterations observed in fibroblasts in aged human skin, including reduced cytoplasmic area, disassembled actin cytoskeleton, impaired TGF‐β pathway, and reduced collagen production. These results support the concept that MMP‐1‐mediated fragmentation of dermal collagen fibrils alters the morphology and function of dermal fibroblasts and provide a foundation for understanding specific mechanisms that link collagen fibril fragmentation to age‐related decline of fibroblast function.     

Skeletal muscle collagen content in humans after high-force eccentric contractions.

The purpose of this study was to investigate the effects of high-force eccentric muscle contractions on collagen remodeling and on circulating levels of matrix metalloproteinases (MMP) and tissue inhibitors of metalloproteinases (TIMP) in humans. Nine volunteers [5 men and 4 women, mean age 23 (SD 4) yr] each performed a bout of 100 maximum voluntary eccentric contractions of the knee extensors. Muscle biopsies were taken before exercise and on days 4 and 22 afterward. Image analysis of stained tissue sections was used to quantify endomysial collagen staining intensity. Maximum voluntary contractile isometric force was recorded preexercise and on days 1, 2, 3, 4, 8, 11, and 14 postexercise. Venipuncture blood samples were also drawn on these days for measurement of serum creatine kinase activity and concentrations of MMP-9, TIMP-1, TIMP-2, and the MMP-2/TIMP-2 complex. Maximum voluntary contractile force declined by 39 +/- 23% (mean +/- SD) on day 2 postexercise and recovered thereafter. Serum creatine kinase activity peaked on day 4 postexercise (P < 0.01). Collagen type IV staining intensity increased significantly on day 22 postexercise to 126 +/- 29% (mean +/- SD) of preexercise values (P < 0.05). Serum MMP-9 levels increased on day 8 postexercise (P < 0.01), and serum TIMP-1 was also significantly elevated on days 1, 2, 3, 4, and 14 postexercise (P < 0.05). These results suggest that a single bout of eccentric muscle contractions results in remodeling of endomysial type IV collagen, possibly via the MMP pathway.     

Determination of matrix metalloproteinase activity using biotinylated gelatin

Matrix metalloproteinases (MMPs) and, specifically, MMP-2 (gelatinase A) and MMP-9 (gelatinase B) are strongly associated with malignant progression and matrix remodeling. These enzymes are a subject of intensive studies involving screening of comprehensive chemical libraries of synthetic inhibitors. There is no simple method available for measurement of activity of gelatinases and related MMPs. Here, we report a simple, inexpensive, and highly sensitive assay for MMP activity. The assay performed in a 96-well microtiter plate format employs biotin-labeled gelatin (denatured collagen type I) as a substrate. Following the substrate cleavage, only the proteolytic fragments bearing biotin moieties are captured by streptavidin coated on the plastic surface and the captured fragments with at least two biotin molecules should be revealed by streptavidin conjugated with horseradish peroxidase. The frequency of lysine residues is low in collagen type I relative to the MMP cleavage sequences (PXGX). Accordingly, the majority of the cleavage products must be devoid of biotin or possess only one biotin group. Both of these types of fragments cannot be recognized by the horseradish peroxidase-streptavidin conjugate. Therefore, higher gelatinolytic activity is associated with lower signal in the assay. This 2-h assay allows identification of gelatinolytic activity of MMP-2 in concentrations as low as 0.16 ng/ml. The sensitivity of this ELISA-like assay is comparable to that of gelatin zymography, a method widely used to detect gelatinases. However, in contrast to zymography, the assay directly measures the enzymatic activity of MMP samples. The gelatinolytic activity assay permits efficient analyses and screening of the MMP inhibitor panels and allows quantitation of gelatinolytic activity of various MMPs in solution as well as on cell surfaces.     

Expression of matrix metalloproteinase activity in idiopathic dilated cardiomyopathy: A marker of cardiac dilatation

Background: Idiopathic dilated cardiomyopathy (DCM), ventricular systolic dysfunction and chamber dilatation are accompanied by architectural remodeling, wall thinning and cardiac myocyte slippage. Recent work has demonstrated an association between collagen degradation and an increased expression of matrix metalloproteinases (MMPs). Accordingly, we have sought to correlate (a) collagen degradation with MMP elevations and, (b) assay the neutralizing potential of a known inhibitor of MMP, tetracycline on MMPs in DCM. Methods: Assessment of LV volume and shape by 2-D echocardiography was performed. Light microscopic assessment of histopathology in picrosirius red stained biopsy samples of 11 DCM patients and six post-transplant patients was performed. Zymographic estimation of MMP activity and influence of tetracycline on MMP activity was assessed. Results: Small amount of interstitial collagen was noted in the control group, whereas in the DCM hearts, chamber dilatation was associated with areas of scanty myocyte necrosis, islands of excess collagen, and focal areas of absent or scanty collagen with intact myocytes. In cardiomyopathic tissue, collagenase activity was markedly elevated at 63% compared with 8% in post-transplant tissue. Tetracycline at a concentration of 285 ± 10 μM (IC50) inhibited collagenase activity by 50% in cardiomyopathic tissue. Conclusions: Areas of focal interstitial collagen accumulation were accompanied by collagen fiber lysis and increased collagenase activity in dilated cardiomyopathy. This enhanced collagenolytic activity found in endomyocardial biopsy tissue was inhibited by tetracycline. The non-antibiotic property of tetracycline may be of potential value in the prevention of ventricular dilatation in idiopathic dilated cardiomyopathy. (Mol Cell Biochem 264: 183–191, 2004)     

Red blood cells increase secretion of matrix metalloproteinases from human lung fibroblasts in vitro

Tissue remodeling is an important process in many inflammatory and fibrotic lung disorders. RBC may in these conditions interact with extracellular matrix (ECM). Fibroblasts can produce and secrete matrix components, matrix-degrading enzymes (MMPs) and tissue inhibitors of metalloproteinases (TIMPs). Imbalance in matrix synthesis/degradation may result in rearrangement of tissue architecture and lead to diseases such as emphysema or fibrosis. Neutrophil elastase (NE), a protease released by neutrophils, is known to activate MMP. We hypothesized that RBC can stimulate secretion of MMPs from human lung fibroblasts and that NE can augment this effect. Human fetal lung fibroblasts were cultured in floating collagen gels with or without RBC. After 4 days, the culture medium was analyzed with gelatin zymography, Western blot, and ELISA for MMP-1, -2, -3 and TIMP-1, -2. RBC augmented NE-induced fibroblast-mediated collagen gel contraction compared with NE alone (18.4+/-1.6%, 23.7+/-1.4% of initial gel area, respectively). A pan-MMP inhibitor (GM-6001) completely abolished the stimulating effect of NE. Gelatin zymography showed that RBC stimulated MMP-2 activity and that NE enhanced conversion to the active form. Addition of GM-6001 completely inhibited MMP-2 activity in controls, whereas it only partially altered RBC-induced MMP activity. Western blot confirmed the presence of MMP-1 and MMP-3 in fibroblasts stimulated with RBC, and ELISA confirmed increased concentrations of pro-MMP-1. We conclude that stimulation of MMP secretion by fibroblasts may explain the ability of RBC to augment fibroblast-mediated collagen gel contraction. This might be a potential mechanism by which hemorrhage in inflammatory conditions leads to ECM remodeling.     

Matrix remodeling in experimental and human heart failure: a possible regulatory role for TIMP-3

In the failing heart, an imbalance in matrix metalloproteinases (MMPs) and their biological regulators, the tissue inhibitors of MMPs (TIMPs), may result in cardiac dilatation from matrix degradation. We hypothesized that a reduction of myocardial TIMP-3 is associated with adverse matrix remodeling in both human and experimental heart failure. Cardiomyopathic hamsters at age 15 wk (normal), 25 wk (compensated stage), and 35 wk (overt failure) were compared with age-matched normal controls. MMP activity (gelatinase bioassay) was increased in cardiomyopathic hearts (P = 0.03) and peaked during the transition to overt heart failure. TIMP-3 content (immunoblot) was decreased compared with normal controls (74 +/- 5% at 25 wk, 69 +/- 10% at 35 wk; P = 0.001) and its reduction was associated with increased MMP activity (r = -0.6; P = 0.004). TIMP-1 increased progressively (P = 0.001), whereas TIMP-2, TIMP-4, and MMP protein levels were unchanged. Myocardial collagen (hydroxyproline content) increased with time during the progression to end-stage cardiac failure (P < 0.0001). Collagen synthesis ([(14)C]proline uptake) was elevated in cardiomyopathy at 15 and 25 wk (P < 0.05). The collagen cross-linking ratio (insoluble:soluble collagen) was reduced (P = 0.003) as the left ventricle dilated. By confocal microscopy restricted to viable myocardium, collagen content was reduced (P = 0.04) with fragmentation (P < 0.0001) and thinning (P = 0.003) of perimysial collagen fibers. Similarly, patients with end-stage congestive heart failure (n = 7) compared with nonfailing controls (n = 2) had elevated gelatinase MMP activity (P = 0.02) associated with isolated reductions in TIMP-3 (55 +/- 5% of normal; P = 0.003). Reductions of TIMP-3 parallel adverse matrix remodeling in the cardiomyopathic hamster and the failing human heart. TIMP-3 may contribute to the regulation of myocardial remodeling and its reduction may promote a transition from compensated to end-stage congestive heart failure.     

Expression and enzymatic activity of human disintegrin and metalloproteinase ADAM19/meltrin beta

The adamalysins are involved in proteolysis, adhesion, fusion, and intracellular signaling. Human ADAM19/adamalysin-19 (A disintegrin and metalloproteinase 19) was identified from primary dendritic cell cDNA libraries. It has a signal sequence, a pro-domain with a "cysteine-switch" residue, a metalloproteinase domain with a zinc-binding site, a disintegrin, a cysteine-rich domain, an epidermal-growth-factor-like domain, a transmembrane domain, and a cytoplasmic domain with putative SH3 ligand binding sites. Its mRNA was expressed in the placenta, heart, bladder, lymph nodes, and leukocytes, colorectal adenocarcinoma SW 480, and other organs/cells. The hADAM19 recombinant protein was expressed in human cells. It formed a complex with and cleaved alpha-2 macroglobulin (alpha2-M). Its proteolytic activity was blocked by 1,10-phenanthroline, EDTA, EGTA, and a synthetic matrix metalloproteinase (MMP) inhibitor and not by the tissue inhibitors of metalloproteinases TIMP-1 and TIMP-2. It did not cleave the MMP substrates tested, e.g., type I collagen and gelatin, casein, and four peptide substrates. Thus, hADAM19 is an active metalloproteinase and may have a specific substrate profile.