Differential MMP-2 activity induced by mechanical compression and inflammatory factors in human synoviocytes

The anterior cruciate ligament, posterior cruciate ligament, cartilage and meniscus in human knee joint have poor healing ability. Accumulation of MMPs in the joint fluids due to knee injury has been considered as the main reason. Our previous experiments showed that synovium may be the major regulator of MMPs in joint cavity after injury. In this paper, we used human synoviocytes harvested from synovium to determine whether mechanical injury and inflammatory factors will induce MMP-2 production in synoviocytes. With zymography, we found that mechanical compression increased the MMP-2 production by 23% under 6% compressions, 61% under 12% compression and 109% under 14% compressions. In addition, TNF-alpha can also elevate the activity of MMP-2 in a dose dependent manner, while IL-1alpha does not. However, mixture of these two factors dramatically increased MMP-2 production by 201%. In addition, mechanical injury had a strong synergistic effect on MMP-2 production with TNF-alpha, IL-1alpha and their mixture, increasing by 207%, 354% and 468% individually. The generic MMP activity assayrevealed that mechanical compression increased the generic activity. APMA treatment increased the generic activity of MMPs induced by compres-     

Matrix metalloproteinase expression and activity following prostaglandin F(2 alpha)-induced luteolysis

Luteal tissue contains matrix metalloproteinases (MMPs) that cleave specific components of the extracellular matrix (ECM) and are inhibited by tissue inhibitors of metalloproteinases (TIMPs). We previously reported a decrease in luteal TIMP-1 within 15 min of prostaglandin F(2 alpha) (PGF(2 alpha))-induced luteolysis. An increase in the MMP:TIMP ratio may promote ECM degradation and apoptosis, as observed in other tissues that undergo involution. The objectives of these experiments were to determine whether 1) PGF(2 alpha) affects expression of mRNA encoding fibrillar collagenases (MMP-1 and -13), gelatinases A and B (MMP-2 and -9), membrane type (mt)-1 MMP (MMP-14), stromelysin (MMP-3), and matrilysin (MMP-7), and 2) PGF(2 alpha) increases MMP activity during PGF(2 alpha)-induced luteolysis in sheep. Corpora lutea (n = 3-10/time point) were collected at 0, 15, and 30 min and 1, 2, 4, 6, 12, 24, and 48 h after PGF(2 alpha) administration. Northern blot analysis confirmed the presence of all MMPs except MMP-9. Expression of mRNA for the above MMPs (except MMP-2) increased significantly (P < 0.05) by 30 min, and all MMPs increased significantly (P < 0.05) by 6 h after PGF(2 alpha) administration. Expression of MMP-14 mRNA increased significantly (P < 0.05) by 15 min post-PGF(2 alpha) and remained elevated through 48 h. MMP activity in luteal homogenates (following proenzyme activation and inactivation of inhibitors) was increased significantly (P < 0.05) by 15 min and remained elevated through 48 h post-PGF(2 alpha). MMP activity was localized (in situ zymography) to the pericellular area of various cell types in the 0-h group and was markedly increased by 30 min post-PGF(2 alpha). MMP mRNA expression and activity were significantly increased following PGF(2 alpha) treatment. Increased MMP activity may promote ECM degradation during luteolysis.     

Transforming growth factor-beta1 increases airway wound repair via MMP-2 upregulation: a new pathway for epithelial wound repair?

In vivo, transforming growth factor (TGF)-beta1 and matrix metalloproteinases (MMPs) present at the site of airway injury are thought to contribute to epithelial wound repair. As TGF-beta1 can modulate MMP expression and MMPs play an important role in wound repair, we hypothesized that TGF-beta1 may enhance airway epithelial repair via MMPs secreted by epithelial cells. We evaluated the in vitro influence of TGF-beta1 on wound repair in human airway epithelial cells cultured under conditions allowing differentiation. The results showed that TGF-beta1 accelerated in vitro airway wound repair, whereas MMP inhibitors prevented this acceleration. In parallel, we examined the effect of TGF-beta1 on the expression of MMP-2 and MMP-9. TGF-beta1 induced a dramatic increase of MMP-2 expression with an increased steady-state level of MMP-2 mRNA, contrasting with a slight increase in MMP-9 expression. To confirm the role of MMP-2, we subsequently evaluated the effect of MMP-2 on in vitro airway wound repair and demonstrated that the addition of MMP-2 reproduced the acceleration of wound repair induced by TGF-beta1. These results strongly suggest that TGF-beta1 increases in vitro airway wound repair via MMP-2 upregulation. It also raises the issue of a different in vivo biological role of MMP-2 and MMP-9 depending on the cytokine microenvironment.     

ET(A)-receptor blockade prevents matrix metalloproteinase activation late postmyocardial infarction in the rat

Endothelin (ET) A (ET(A)) receptors activate matrix metalloproteinases (MMP). Since endothelin-1 (ET) is increased in myocardium late postmyocardial infarction (MI), we hypothesized that stimulation of ET(A) receptors contributes to activation of myocardial MMPs late post-MI. Three days post-MI, rats were randomized to treatment with the ET(A)-selective receptor antagonist sitaxsentan (n = 12) or a control group (n = 12). Six weeks later, there were rightward shifts of the left ventricular (LV) end-diastolic and end-systolic pressure-volume relationships, as measured ex vivo by the isovolumic Langendorff technique. Both shifts were markedly attenuated by sitaxsentan. In LV myocardium remote from the infarct, the activities of MMP-1, MMP-2, and MMP-9 were increased in the post-MI group, and the increases were prevented by sitaxsentan treatment. Expression of tissue inhibitor of MMP-1 was decreased post-MI, and the decrease was prevented by sitaxsentan treatment. Chronic post-MI remodeling is associated with activation of MMPs in myocardium remote from the infarct. Inhibition of ET(A) receptors prevents MMP activation and LV dilation, suggesting that ET, acting via the ET(A) receptor, contributes to chronic post-MI remodeling by its effects on MMP activity.     

Lipoprotein receptor-mediated induction of matrix metalloproteinase by tissue plasminogen activator

Although thrombolysis with tissue plasminogen activator (tPA) is a stroke therapy approved by the US Food and Drug Administration, its efficacy may be limited by neurotoxic side effects. Recently, proteolytic damage involving matrix metalloproteinases (MMPs) have been implicated. In experimental embolic stroke models, MMP inhibitors decreased cerebral hemorrhage and injury after treatment with tPA. MMPs comprise a family of zinc endopeptidases that can modify several components of the extracellular matrix. In particular, the gelatinases MMP-2 and MMP-9 can degrade neurovascular matrix integrity. MMP-9 promotes neuronal death by disrupting cell-matrix interactions, and MMP-9 knockout mice have reduced blood-brain barrier leakage and infarction after cerebral ischemia. Hence it is possible that tPA upregulates MMPs in the brain, and that subsequent matrix degradation causes brain injury. Here we show that tPA upregulates MMP-9 in cell culture and in vivo. MMP-9 levels were lower in tPA knockouts compared with wild-type mice after focal cerebral ischemia. In human cerebral microvascular endothelial cells, MMP-9 was upregulated when recombinant tPA was added. RNA interference (RNAi) suggested that this response was mediated by the low-density lipoprotein receptor-related protein (LRP), which avidly binds tPA and possesses signaling properties. Targeting the tPA-LRP signaling pathway in brain may offer new approaches for decreasing neurotoxicity and improving stroke therapy.     

Suppression of fibroblast metalloproteinases by ajulemic acid, a nonpsychoactive cannabinoid acid

Production of matrix metalloproteinases (MMP) in joint tissue of patients with inflammatory arthritis facilitates cartilage degradation and bone erosion, and leads to joint deformities and crippling. Thus, MMPs are important targets for agents designed to treat inflammatory arthritis. Oral administration of ajulemic acid (AjA), a synthetic, nonpsychoactive cannabinoid acid, prevents joint tissue injury in rats with adjuvant arthritis. AjA binds to and activates PPARgamma directly. Therefore, we investigated the influence of AjA on MMP production in human fibroblast-like synovial cells (FLS), and examined the role of PPARgamma in the mechanism of action of AjA. FLS, treated or not with a PPARgamma antagonist, were treated with AjA then stimulated with TNFalpha or IL-1alpha. Release of MMPs-1, 3, and 9 was measured by ELISA. The influence of AjA on MMP-3 release from stimulated PPARgamma positive (PPAR+/-) and PPARgamma null (PPAR-/-) mouse embryonic fibroblasts (MEF) was also examined. Addition of AjA to FLS suppressed production of MMPs whether or not PPARgamma activation was blocked. Secretion of MMP-3 was also suppressed by AjA in both TNFalpha- and IL-1alpha-stimulated PPARgamma+/- and PPARgamma-/- MEF. Suppression of MMP secretion from FLS by AjA appears to be PPARgamma independent. Prevention by AjA of joint tissue injury and crippling in the rat adjuvant arthritis model may be explained in large part by inhibition of MMPs. These results suggest that AjA may be useful for treatment of patients with rheumatoid arthritis and osteoarthritis.     

Purified human chondroitin-4-sulfate reduced MMP/TIMP imbalance induced by iron plus ascorbate in human fibroblast cultures

Imbalance between matrix metalloproteinases (MMPs) and tissue inhibitor of matrix metalloproteinases (TIMPs) is an important control point in tissue remodelling. Several findings have reported a marked MMP/TIMP imbalance in a variety of in vitro models in which oxidative stress was induced. Since previous studies showed that commercial hyaluronan and chondroitin-4-sulphate are able to limit lipid peroxidation during oxidative stress, we investigated the antioxidant capacity of purified human plasma chondroitin-4-sulfate in reducing MMP and TIMP imbalance in a model of ROS-induced oxidative injury in fibroblast cultures. Purified human plasma chondroitin-4-sulfate was added to the fibroblast cultures exposed to FeSO4 plus ascorbate. We assayed cell death, MMP and TIMP mRNA expression and protein activities, DNA damage, membrane lipid peroxidation, and aconitase depletion. FeSO4 plus ascorbate produced severe death of cells and increased MMP-1, MMP-2 and MMP-9 expression and protein activities. It also caused DNA strand breaks, enhanced lipid peroxidation and decreased aconitase. TIMP-1 and TIMP-2 protein levels and mRNA expression remain unaltered. Purified human plasma C4S, at three different doses, restored the MMP/TIMP homeostasis, increased cell survival, reduced DNA damage, inhibited lipid peroxidation and limited impairment of aconitase. These results further support the hypothesis that these biomolecules possess antioxidant activity and by reducing ROS production C4S may limit cell injury produced by MMP/TIMP imbalance.     

Preconditioning decreases ischemia/reperfusion-induced release and activation of matrix metalloproteinase-2

Release and activation of matrix metalloproteinases (MMPs) significantly contribute to myocardial stunning injury immediately after ischemia and reperfusion, however, their role in preconditioning remains unknown. We therefore examined the effects of preconditioning and subsequent ischemia/reperfusion on MMP activity in isolated rat hearts. Hearts were subjected to a preconditioning protocol (three consecutive 5-min periods of global ischemia interspersed with 5 min of reperfusion) followed by 30 min ischemia and 5 min reperfusion. To measure MMP release, coronary effluent was collected: (a) during aerobic perfusion, (b) in reperfusion following each preconditioning ischemia, and (c) during the final reperfusion following test ischemia. MMP-2 activities could be detected by gelatin zymography in the ventricles and coronary effluent samples from the perfused hearts. The levels of MMP-2 activity in the effluent were markedly increased in effluent following test ischemia from control hearts without preconditioning. This was accompanied by a decrease in corresponding tissue MMP activities. Preconditioning significantly decreased the MMP-2 activity in the coronary effluent following test ischemia/reperfusion and preserved the MMP-2 protein content and activity in the myocardium. Our results demonstrate that classic preconditioning inhibits ischemia/reperfusion induced release and activation of MMP-2. These results suggest that preconditioning may exert part of its cardioprotective effects through the reduction of MMP-2 release.     

Induction of macrophage matrix metalloproteinase biosynthesis by surfactant protein D

Recent studies strongly suggest that surfactant protein D (SP-D) plays important roles in pulmonary host defense and the regulation of immune and inflammatory reactions in the lung. Although SP-D can bind to alveolar macrophages and can elicit their chemotaxis, relatively little is known about the direct cellular consequences of SP-D on the function of these cells. Because matrix metalloproteinases (MMPs) are synthesized in increased amounts in response to various proinflammatory stimuli, we investigated the capacity of SP-D to modulate the production of MMPs by freshly isolated human alveolar macrophages. Unexpectedly we found that recombinant rat SP-D dodecamers selectively induce the biosynthesis of collagenase-1 (MMP-1), stromelysin (MMP-3), and macrophage elastase (MMP-12) without significantly increasing the production of tumor necrosis factor alpha and interleukin-1beta. SP-D did not alter the production of these MMPs by fibroblasts. Phosphatidylinositol, a surfactant-associated ligand that interacts with the carboxyl-terminal neck and carbohydrate recognition domains of SP-D, inhibited the SP-D-dependent increase in MMP biosynthesis. A trimeric, recombinant protein consisting of only the neck and carbohydrate recognition domain did not augment metalloproteinase production, suggesting that the stimulatory effect on MMP production depends on an appropriate spatial presentation of trimeric lectin domains. Although SP-D dodecamers can selectively augment metalloproteinase activity in vitro, this effect may be competitively inhibited by tissue inhibitors of metalloproteinases or surfactant-associated ligands in vivo.     

Induction of multiple matrix metalloproteinases in human dermal and synovial fibroblasts by Staphylococcus aureus: implications in the pathogenesis of septic arthritis and other soft tissue infections

Infections of body tissue by Staphylococcus aureus are quickly followed by degradation of connective tissue. Patients with rheumatoid arthritis are more prone to S. aureus-mediated septic arthritis. Various types of collagen form the major structural matrix of different connective tissues of the body. These different collagens are degraded by specific matrix metalloproteinases (MMPs) produced by fibroblasts, other connective tissue cells, and inflammatory cells that are induced by interleukin-1 (IL-1) and tumor necrosis factor (TNF). To determine the host's contribution in the joint destruction of S. aureus-mediated septic arthritis, we analyzed the MMP expression profile in human dermal and synovial fibroblasts upon exposure to culture supernatant and whole cell lysates of S. aureus. Human dermal and synovial fibroblasts treated with cell lysate and filtered culture supernatants had significantly enhanced expression of MMP-1, MMP-2, MMP-3, MMP-7, MMP-10, and MMP-11 compared with the untreated controls (p < 0.05). In the S. aureus culture supernatant, the MMP induction activity was identified to be within the molecular-weight range of 30 to >50 kDa. The MMP expression profile was similar in fibroblasts exposed to a combination of IL-1/TNF. mRNA levels of several genes of the mitogen-activated protein kinase (MAPK) signal transduction pathway were significantly elevated in fibroblasts treated with S. aureus cell lysate and culture supernatant. Also, tyrosine phosphorylation was significantly higher in fibroblasts treated with S. aureus components. Tyrosine phosphorylation and MAPK gene expression patterns were similar in fibroblasts treated with a combination of IL-1/TNF and S. aureus. Mutants lacking staphylococcal accessory regulator (Sar) and accessory gene regulator (Agr), which cause significantly less severe septic arthritis in murine models, were able to induce expression of several MMP mRNA comparable with that of their isogenic parent strain but induced notably higher levels of tissue inhibitors of metalloproteinases (TIMPs). To our knowledge, this is the first report of induction of multiple MMP/TIMP expression from human dermal and synovial fibroblasts upon S. aureus treatment. We propose that host-derived MMPs contribute to the progressive joint destruction observed in S. aureus-mediated septic arthritis.     

Characterization of Mca-Lys-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH2, a fluorogenic substrate with increased specificity constants for collagenases and tumor necrosis factor converting enzyme

Matrix metalloproteinases (MMPs) and the related tumor necrosis factor converting enzyme (TACE) are involved in tissue remodeling, cell migration, and processing of signaling molecules, such as cytokines and adhesion molecules. Fluorescence-quenched peptide substrates have been widely used to quantitate the actual enzymatic activity of MMPs. However, the various MMPs have very different specific activities toward these substrates. This restricts their value for the determination of composite proteolytic activity of mixtures of metalloproteinases in biological fluids. The N-terminal elongation of the most widely used MMP substrate (FS-1) with a Lys to the sequence Mca-Lys-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH(2) (FS-6) yields a fluorogenic peptide with improved substrate properties. As compared to FS-1, the specificity constant (kcat/Km) of FS-6 for collagenases (MMP-1, MMP-8, MMP-13) and MT1-MMP (MMP-14) is increased two- to ninefold and threefold, respectively, while those for gelatinases and matrilysin remain equally high. Using high-performance liquid chromatography-fluorescence detection, MMP activity can be quantitated in the picomolar range. FS-6 shows up to twofold higher specificity constants (kcat/Km of 0.8x10(6)M(-1)s(-1)) for TACE, as compared to standard substrates Mca-PLAQAV-Dpa-RSSSAR-NH(2) and Dabcyl-LAQAVRSSSAR-EDANS. FS-6 is fully water soluble and thus allows measurement of metalloproteinase activity in tissue culture conditions, e.g., on the surface of viable cells in situ.     

Role of matrix metalloproteinases in inflammatory bowel disease

Recent evidence demonstrates that the increased expression and activity of matrix metalloproteinases (MMPs) may contribute to intestinal tissue injury and inflammation in inflammatory bowel disease, and that MMP inhibition might be a new therapeutic approach to controlling inflammatory response. In addition, MMPs may play a crucial role in physiological and pathophysiological reactions such as leukocyte accumulation into inflamed tissue, cytokine production from inflammatory and epithelial cells, T lymphocyte homing to the intestine, wound healing and proliferation of epithelial cells, and intestinal innate immunity. This review focuses on recent progress in elucidating the biological and pathological roles of MMPs in inflammatory bowel disease.     

Hepatoprotective Effect of MMP-19 Deficiency in a Mouse Model of Chronic Liver Fibrosis

Liver fibrosis is characterized by the deposition and increased turnover of extracellular matrix. This process is controlled by matrix metalloproteinases (MMPs), whose expression and activity dynamically change during injury progression. MMP-19, one of the most widely expressed MMPs, is highly expressed in liver; however, its contribution to liver pathology is unknown. The aim of this study was to elucidate the role of MMP-19 during the development and resolution of fibrosis by comparing the response of MMP-19-deficient (MMP19KO) and wild-type mice upon chronic liver CCl₄-intoxication. We show that loss of MMP-19 was beneficial during liver injury, as plasma ALT and AST levels, deposition of fibrillar collagen, and phosphorylation of SMAD3, a TGF-ß1 signaling molecule, were all significantly lower in MMP19KO mice. The ameliorated course of the disease in MMP19KO mice likely results from a slower rate of basement membrane destruction and ECM remodeling as the knockout mice maintained significantly higher levels of type IV collagen and lower expression and activation of MMP-2 after 4 weeks of CCl₄-intoxication. Hastened liver regeneration in MMP19KO mice was associated with slightly higher IGF-1 mRNA expression, slightly increased phosphorylation of Akt kinase, decreased TGF-ß1 mRNA levels and significantly reduced SMAD3 phosphorylation. In addition, primary hepatocytes isolated from MMP19KO mice showed impaired responsiveness towards TGF-ß1 stimulation, resulting in lower expression of Snail1 and vimentin mRNA. Thus, MMP-19-deficiency improves the development of hepatic fibrosis through the diminished replacement of physiological extracellular matrix with fibrotic deposits in the beginning of the injury, leading to subsequent changes in TGF-ß and IGF-1 signaling pathways.     

Mechanical stretch induces MMP-2 release and activation in lung endothelium: role of EMMPRIN

High-volume mechanical ventilation leads to ventilator-induced lung injury. This type of lung injury is accompanied by an increased release and activation of matrix metalloproteinases (MMPs). To investigate the mechanism leading to the increased MMP release, we systematically studied the effect of mechanical stretch on human microvascular endothelial cells isolated from the lung. We exposed cells grown on collagen 1 BioFlex plates to sinusoidal cyclic stretch at 0.5 Hz using the Flexercell system with 17-18% elongation of cells. After 4 days of cell stretching, conditioned media and cell lysate were collected and analyzed by gelatin, casein, and reverse zymograms as well as Western blotting. RT-PCR of mRNA extracted from stretched cells was performed. Our results show that 1) cyclic stretch led to increased release and activation of MMP-2 and MMP-1; 2) the activation of MMP-2 was accompanied by an increase in membrane type-1 MMP (MT1-MMP) and inhibited by a hydroxamic acid-derived inhibitor of MMPs (Prinomastat, AG3340); and 3) the MMP-2 release and activation were preceded by an increase in production of extracellular MMP inducer (EMMPRIN). These results suggest that cyclic mechanical stretch leads to MMP-2 activation through an MT1-MMP mechanism. EMMPRIN may play an important role in the release and activation of MMPs during lung injury.     

Matrix metalloproteinases and collagen ultrastructure in moderate myocardial ischemia and reperfusion in vivo

Severe ischemic injury or infarction of myocardium may cause activation of matrix metalloproteinases (MMPs) and damage the interstitial matrix. However, it is unknown whether MMP activation and matrix damage occur after moderate ischemia and reperfusion that result in myocardial stunning without infarction, and if so whether such changes contribute to postischemic myocardial expansion and contractile dysfunction. To address these questions, open-chest anesthetized pigs underwent 90 min of regional ischemia (subendocardial blood flow 0.4 +/- 0.1 ml. g(-1). min(-1)) and 90 min of reperfusion. After ischemia plus reperfusion, histological and ultrastructural examination revealed no myocardial infarction or inflammatory cell infiltration. Myocardial MMP-9 content increased threefold with a fourfold increase in the active form (P < 0.001). Myocardial collagenase content doubled (P < 0.01) but remained in latent form. MMP-2 and tissue inhibitors of metalloproteinases were unaffected. Despite increases in MMPs, collagen ultrastructure (assessed by cell maceration scanning electron microscopy) was unaltered. Intracoronary administration of the MMP inhibitor GM-2487 did not prevent or attenuate myocardial expansion (assessed by regional diastolic dimensions at near-zero left ventricular pressure) or contractile dysfunction. We conclude that although moderate ischemia and reperfusion alter myocardial MMP content and activity, these effects do not result in damage to interstitial collagen, nor do they contribute to myocardial expansion or contractile dysfunction.     

MT1-MMP-dependent neovessel formation within the confines of the three-dimensional extracellular matrix

During angiogenesis, endothelial cells initiate a tissue-invasive program within an interstitial matrix comprised largely of type I collagen. Extracellular matrix-degradative enzymes, including the matrix metalloproteinases (MMPs) MMP-2 and MMP-9, are thought to play key roles in angiogenesis by binding to docking sites on the cell surface after activation by plasmin- and/or membrane-type (MT) 1-MMP-dependent processes. To identify proteinases critical to neovessel formation, an ex vivo model of angiogenesis has been established wherein tissue explants from gene-targeted mice are embedded within a three-dimensional, type I collagen matrix. Unexpectedly, neither MMP-2, MMP-9, their cognate cell-surface receptors (i.e., beta3 integrin and CD44), nor plasminogen are essential for collagenolytic activity, endothelial cell invasion, or neovessel formation. Instead, the membrane-anchored MMP, MT1-MMP, confers endothelial cells with the ability to express invasive and tubulogenic activity in a collagen-rich milieu, in vitro or in vivo, where it plays an indispensable role in driving neovessel formation.