Matrix metalloproteinases and tissue inhibitors of matrix metalloproteinases in sputum from patients with bronchial asthma.

To examine a possibility that matrix metalloproteinases (MMPs) participate in the pathogenesis of asthma and/or the development of asthma attack, we measured the concentrations of MMP-2, MMP-9, and their respective tissue inhibitors of metalloproteinases (TIMP)-2 and TIMP-1, in induced sputa collected from 28 patients with moderate to severe bronchial asthma. Specimens were collected during both the attack and the remission from 15 age- and sex-matched healthy control subjects. The concentration of MMP-9 was significantly (p < 0.05) higher in the patients, even during the remission, as compared to that in healthy controls. The concentrations of MMP-9 (p < 0.05) and its specific inhibitor TIMP-1 (p < 0.01), and MMP-2 (p < 0.01) in these patients during the attack were significantly higher than those in healthy controls. In these patients, the MMP-9 concentration was significantly higher (p < 0.05) during the attack than during the remission. These results suggest that MMPs and TIMPs may be involved in the pathogenesis of bronchial asthma, and that the increased MMP-9 might be involved in the development of attack in patients with chronic asthma.     

Serum matrix metalloproteinases and tissue inhibitors of metalloproteinases in patients with early rheumatoid arthritis

OBJECTIVE: To analyze serum concentrations of matrix metalloproteinases (MMP) MMP-1, MMP-3, MMP-9, MMP-13, tissue inhibitors of MMP (TIMP) TIMP-1 and TIMP-2, and MMP/TIMP ratios in patients with early rheumatoid arthritis (RA) before and after 6 months of treatment with methotrexate (MTX). METHODS: The study group consisted of 30 patients with RA, not treated with disease modifying antirheumatic drugs or corticosteroids, with disease duration < 3 years. Twenty patients with osteoarthritis (OA) served as a control group. Analysis of serum concentrations of MMP and TIMP was based on a quantitative sandwich ELISA. RESULTS: Serum concentrations of MMP-1, MMP-3, MMP-9, and MMP-13 were higher in untreated patients with early RA than in OA patients (p < 0.001 in all cases). Serum levels of TIMP-1 and TIMP-2 dominated in the serum of RA patients compared with controls (p < 0.01 and p < 0.05, respectively). Ratios of MMP to TIMP were significantly higher in patients with early RA versus controls. Six months' treatment with MTX downregulated serum concentrations of MMP-1 (p < 0.001), MMP-3 (p < 0.001), MMP-9 (p < 0.001), MMP-13 (p < 0.01), and TIMP-1 (p < 0.05) in patients with RA. These changes were accompanied by significantly reduced ratios of MMP to TIMP. MTX treatment decreased markers of RA activity such as the number of painful and swollen joints, erythrocyte sedimentation rate, Disease Activity Score, and C-reactive protein. CONCLUSION: Patients with early RA are characterized by high serum concentrations of tissue-degrading metalloproteinases. Therapy with MTX resulted in clinical improvement and reduced serum MMP levels in patients with RA, confirming effectiveness of MTX in patients in early stages of the disease.     

Matrix metalloproteinases and their tissue inhibitors in endocrinology.

Matrix metalloproteinases (MMPs) and their natural inhibitors (TIMPs) have been widely implicated in tissue resorption, degradation, and fibrosis in a range of normal and abnormal processes, but recently defined additional actions suggest much broader and independent roles for different members of these gene families. This review examines the involvement of hormones in regulation of MMPs and TIMPs and focuses on known and potential roles of particular interest to endocrinologists.     

Matrix metalloproteinases and tissue inhibitors of metalloproteinases in colonic adenomas-adenocarcinomas

Colonic adenocarcinomas evolve through a multistep process from tubular adenomas to invasive adenocarcinomas. Matrix metalloproteinases (MMPs) have been implicated in proteolysis of basement membrane for initiation of metastatic cascade. METHODS: By immunocytochemical staining, hyperplastic polyps, tubular adenomas, tubovillous adenomas, villous adenomas to adenocarcinomas were systematically examined for the presence of MMP-2 (gelatinase A) and MMP-9 (gelatinase B) and tissue inhibitor of MMP (TIMP)-1 and TIMP-2, respectively. RESULTS: MMP-2 and MMP-9, and TIMP-1 and TIMP-2 were immunolocalized in scattered stromal cells, whereas epithelial cells of normal mucosa and hyperplastic polyps were weakly stained. From tubular adenomas to villous adenomas, immunolocalization of gelatinases and TIMPs showed increasing gradually, and in situ carcinomas showed a definite positive, immunolocalization of gelatinases and TIMPs. CONCLUSION: Increasing immunolocalization of gelatinases and TIMPs from tubular adenomas to adenocarcinomas coincides with a multistep process of colonic tumorigenesis.Presented in part at the 20th International Congress of International Academy of Pathology, Hong Kong, China, October 9 to 14, 1994.     

Matrix metalloproteinases and tissue inhibitors of metalloproteinases in synovial fluids from patients with rheumatoid arthritis or osteoarthritis

OBJECTIVE: Matrix metalloproteinases (MMPs) are expressed in joint tissues of patients with rheumatoid arthritis (RA) and osteoarthritis (OA). The objective of this study was to define the steady state levels of seven different MMPs and two tissue inhibitors of metalloproteinases (TIMPs) as well as the potential metalloproteinase activity in the synovial fluid (SF) to provide more insight into the role of MMPs in cartilage destruction in RA and OA. METHODS: Levels of MMP-1, MMP-2, MMP-3, MMP-7, MMP-8, MMP-9, MMP-13, TIMP-1, and TIMP-2 in SF aspirated from knee joints of 97 patients with RA and 103 patients with OA were measured by the corresponding one step sandwich enzyme immunoassays. Proteolytic activity of MMPs in these SFs was examined in an assay using [(3)H]carboxymethylated transferrin substrate in the presence of inhibitors of serine and cysteine proteinases after activation with p-aminophenylmercuric acetate (APMA). Destruction of RA knee joints was radiographically evaluated. RESULTS: Levels of MMP-1, MMP-2, MMP-3, MMP-8, and MMP-9 were significantly higher in RA SF than in OA SF. MMP-7 and MMP-13 were detectable in more than 45% of RA SFs and in less than 20% of OA SFs, respectively. Among the MMPs examined, MMP-3 levels were extremely high compared with those of other MMPs. Direct correlations were seen between the levels of MMP-1 and MMP-3 and between those of MMP-8 and MMP-9 in RA SF. Although the levels of MMP-1 and MMP-3 increased even in the early stage of RA, those of MMP-8 and MMP-9 were low in the early stage and increased with the progression of RA. Molar ratios of the total amounts of the MMPs to those of the TIMPs were 5.2-fold higher in patients with RA than in OA, which was significant. APMA-activated metalloproteinase activity in SF showed a similar result, and a direct correlation was seen between the molar ratios and the activity in RA SF. CONCLUSIONS: Our results show that high levels of MMP-1, MMP-2, MMP-3, MMP-8, MMP-9, and TIMP-1 are present in RA SF and suggest that once these MMPs are fully activated, they have an imbalance against TIMPs, which may contribute to the cartilage destruction in RA.     

Matrix metalloproteinases and tissue inhibitors of metalloproteinases: structure,function, and biochemistry

Matrix metalloproteinases (MMPs), also designated matrixins, hydrolyze components of the extracellular matrix. These proteinases play a central role in many biological processes, such as embryogenesis, normal tissue remodeling, wound healing, and angiogenesis, and in diseases such as atheroma, arthritis, cancer, and tissue ulceration. Currently 23 MMP genes have been identified in humans, and most are multidomain proteins. This review describes the members of the matrixin family and discusses substrate specificity, domain structure and function, the activation of proMMPs, the regulation of matrixin activity by tissue inhibitors of metalloproteinases, and their pathophysiological implication.     

Interplay of matrix metalloproteinases, tissue inhibitors of metalloproteinases and their regulators in cardiac matrix remodeling

Myocardial fibrosis due to maladaptive extracellular matrix remodeling contributes to dysfunction of the failing heart. Further elucidation of the mechanism by which myocardial fibrosis and dilatation can be prevented or even reversed remains of great interest as a potential means to limit myocardial remodeling and dysfunction. Matrix metalloproteinases (MMPs) are the driving force behind extracellular matrix degradation during remodeling and are increased in the failing human heart. MMPs are regulated by a variety of growth factors, cytokines, and matrix fragments such as matrikines. In the present report, we discuss the regulation of MMPs, the role of MMPs in the development of cardiac fibrosis, and the modulation of MMP activity using gene transfer and knockout technologies. We also present recent findings from our laboratory on the regulation of the extracellular MMP inducer (EMMPRIN), MMPs, and transforming growth factor-beta(1) in the failing human heart before and after left ventricular assist device support, as well as the possibility of preventing ventricular fibrosis using different anti-MMP strategies. Several studies suggest that such modulation of MMP activity can alter ventricular remodeling, myocardial dysfunction, and the progression of heart failure. It is therefore suggested that the interplay of MMPs and their regulators is important in the development of the heart failure phenotype, and myocardial fibrosis in heart failure may be modified by modulating MMP activity.     

Occurrence of matrix metalloproteinases and tissue inhibitors of metalloproteinases in tuberculous pleuritis

OBJECTIVE: Matrix metalloproteinases (MMP) and tissue inhibitors of metalloproteinases (TIMP) have been found in high concentrations in pleural effusions. Because MMP and TIMP may play a part in the causation of the fibrosis seen in tuberculous (TB) pleuritis their occurrence was examined. DESIGN: Pleural effusion fluid and plasma concentrations of MMP-1, MMP-2, MMP-3, MMP-8, MMP-9, TIMP-1 and TIMP-2 were determined by ELISA in 21 patients with TB pleuritis. To adjust for the total protein content, respective ratios were calculated. Activities of MMP-2 and MMP-9 were measured by gelatine zymography and the MMP-9/MMP-2 ratios calculated. Pleural effusions and plasma of 15 patients with congestive heat failure (CHF) and plasma of 15 healthy persons (CON) served as controls. RESULTS: Immunoreactive pleural fluid concentrations of MMP-1, MMP-2, MMP-8, and MMP-9 were higher in TB compared to CHF, but plasma concentrations were not different between the groups. TB pleural fluid concentrations of MMP-1, MMP-2, TIMP-1, and TIMP-2 were higher compared to TB plasma. MMP-3 was found in trace amounts only. The MMP-9/total protein ratios in pleural fluid were higher in TB compared to CHF (0.4492+/-0.1633 vs 0.0364+/-0.0145, P<0.005) but the TIMP-1 ratios were lower (139.0+/-28.7 vs 517.8+/-183.7, P<0.0005). In TB pleural fluid vs TB plasma, the respective MMP-1, MMP-2, TIMP-1, and TIMP-2 ratios were increased (0.46+/-0.10 vs 0.17+/-0.02; 25.2+/-2.8 vs 4.2+/-0.9; 139.0+/-28.7 vs 27.8+/-8.2; 0.67+/-0.13 vs 0.18+/-0.04, P<0.0005 each). Gelatine zymography demonstrated MMP-2 and MMP-9 bands of different brightness in TB effusions but in CHF effusions the MMP-9 band was barely visible. The MMP-9/MMP-2 effusion ratios were therefore higher in TB compared to CHF (0.46+/-0.15 vs 0.05+/-0.04, P<0.0005). CONCLUSION: Compartmentalized MMP-1, MMP-2, TIMP-1, and TIMP-2 and, compared to CHF, a surplus of MMP-1, MMP-2, MMP-8, and MMP-9 in the pleural space obviously contribute to the fibrotic reactions in TB pleuritis.     

Matrix metalloproteinases and their inhibitors in gastric cancer

Background—The matrix metalloproteinases (MMPs)and tissue inhibitors of matrix metalloproteinases (TIMPs) are stronglyimplicated in tumour invasion and metastasis.
Aims—To investigate the presence of individualMMPs and TIMPs in gastric cancer.
Methods—The presence of MMP-1, MMP-2, MMP-3,MMP-9, TIMP-1, and TIMP-2 was identified in a group of gastric cancers(n=74) by immunohistochemistry using monoclonal antibodies. Theseantibodies were effective on formalin fixed, paraffin wax embedded sections.
Results—A large proportion (94%) of gastriccancers contained MMP-2; MMP-1 and MMP-9 were also detected in 73% and70% of tumours respectively. MMP-3 was only present in 27% oftumours. MMP-1 and MMP-9 were found predominantly in intestinal typetumours. TIMP-1 and TIMP-2 were identified in 41% and 57% of tumoursrespectively. Immunoreactivity for individual MMPs or TIMPs was notidentified in normal stomach.
Conclusions—This study shows the presenceof matrix metalloproteinases, particularly MMP-2, and TIMPs in stomachcancer. Antibodies which are effective in formalin fixed, paraffin waxembedded sections are useful for the identification of MMPs and TIMPsin diagnostic specimens.

Keywords:immunohistochemistry; matrix metalloproteinase; neoplasm; stomach

     

Matrix metalloproteinases and the thyroid.

Matrix metalloproteinases (MMPs) are proteolytic enzymes that degrade components of the extracellular matrix (ECM) and basement membrane. They play a critical role in many physiological and pathological processes, such as tumor metastasis. The original concept-that MMP activity during metastasis is restricted solely to invasion of the basement membrane and destruction of ECM components-has been modified to encompass multiple aspects of tumor progression: tumor establishment, growth, angiogenesis, intravasation, extravasation, and almost all metastatic steps. Moreover, the role of tissue inhibitors of matrix metalloproteinases (TIMPs), originally believed to exhibit anti-invasion properties solely by virtue of their inhibition of MMPs, has been extended to include their multiple biological effects, such as growth promotion. In thyroid neoplasia as well, MMPs, in particular MMP-2, seem to be associated with metastatic potential. It would seem that similar and divergent patterns regulate MMP and TIMP gene expression in benign and malignant human thyrocytes, in many instances in agreement with the concept of MMPs playing the role of stimulating, and TIMPs inhibiting cell invasion.     

Effect of repeated infliximab therapy on serum matrix metalloproteinases and tissue inhibitors of metalloproteinases in patients with rheumatoid arthritis

OBJECTIVE: Matrix metalloproteinases (MMP) are involved in the articular tissue destruction processes in the pathogenesis of rheumatoid arthritis (RA). We investigated the effects of multiple infusions of infliximab, a chimeric anti-tumor necrosis factor-a (anti-TNF-a) antibody, on concentrations of serum MMP and tissue inhibitors of metalloproteinases (TIMP) in patients with active RA. METHODS: Patients with RA were scheduled to receive 9 infusions of infliximab (3 mg/kg) at Weeks 0, 2, 6, and every 8 weeks thereafter. The therapy was combined with methotrexate (MTX) (7.5-20 mg/week). Serum concentrations of interstitial collagenase (MMP-1), stromelysin-1 (MMP-3), gelatinase B (MMP-9), TIMP-1, and TIMP-2 were measured by ELISA prior to infusion at Weeks 0, 2, 6, 14, 38, and 62. RESULTS: The initial infusion of infliximab downregulated serum levels of MMP-1 (p < 0.001), MMP-3 (p < 0.001), MMP-9 (p < 0.001), TIMP-1 (p < 0.01), and TIMP-2 (p < 0.05). The second drug administration caused even more remarkable reduction of measured MMP (p < 0.001 in all cases) but not of TIMP levels. These changes were accompanied by decreased ratios of measured MMP to TIMP. Further infliximab therapy also significantly suppressed serum MMP levels, but was less effective. Before the first infliximab infusion serum concentrations of MMP and TIMP correlated with markers of RA activity such as the Disease Activity Score and C-reactive protein levels. After further drug administrations such associations, although less significant, were also noted. CONCLUSION: Anti-TNF-a antibody therapy combined with MTX resulted in rapid clinical improvement and reduced serum MMP concentrations in patients with RA. Further infusions of infliximab maintained the decrease of MMP, although to a lesser extent than the first and second doses.     

Matrix metalloproteinases in blood from patients with LAM

Pulmonary lymphangioleiomyomatosis (LAM) is characterized by the proliferation of abnormal smooth muscle cells (LAM cells) and destruction of alveolar structure. Immunohistochemical studies suggest that excess matrix metalloproteinases (MMPs) synthesized by LAM cells function in the proteolytic mechanisms of this disease. We postulated MMP levels in the blood are elevated in LAM patients. Serum samples were collected from 36 LAM patients and 25 controls, and gelatinolytic activities were semi-quantified by gelatin zymography. The reliability of serum data for MMP-9 was confirmed by the measurement of MMP-9 concentration in plasma by enzyme-linked immunosorbent assay as well as by gelatin zymography. Serum levels of MMP-9 (0.7+/-0.1 AU), but not MMP-2, were significantly elevated in LAM patients compared with controls (0.1+/-0 AU). Plasma and serum levels of MMP-9 significantly correlated. These results suggest the involvement of MMP-9 in LAM.     

Serum matrix metalloproteinases and tissue inhibitors of metalloproteinases in different histological variants of rheumatoid synovitis.

OBJECTIVE: Rheumatoid synovitis is characterized by an invasive and tissue-destructive infiltrate of lymphocytes, macrophages and synoviocytes. Matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) produced by these cells are important in the remodelling of the articular tissues in rheumatoid arthritis (RA). The aim of this study was to explore whether the serum concentrations of MMPs and their inhibitors were correlated with the histological appearance of the disease. METHODS: Tissue and serum samples were obtained from 37 patients with clinically active RA and 30 with osteoarthritis (OA). Morphological analysis allowed the division of RA synovial specimens into two distinct types. In 22 samples only diffuse infiltrates of mononuclear cells without further microanatomical organization were found. In 15 specimens we observed lymphocytic conglomerates with germinal centre-like structures. Serum concentrations of interstitial collagenase (MMP-1), stromelysin-1 (MMP-3), gelatinase B (MMP-9), TIMP-1 and TIMP-2 were measured with an ELISA technique. RESULTS: Unique serum profiles of MMPs and TIMPs were identified in each of the two histological types of RA synovitis. The serum concentrations of MMP-1, MMP-3 and MMP-9 were higher in RA patients than in OA patients used as a control group (P<0.001 for all comparisons). These three MMPs dominated in the serum of RA patients with follicular synovitis compared with those with diffuse synovitis (P<0.05, P<0.01 and P<0.001 respectively). The analysis of the serum concentrations of TIMP-1 and TIMP-2 showed that their levels were also elevated in RA patients compared with OA patients (P<0.001 and P<0.01 respectively). Only TIMP-1 was found in a significantly higher concentration in the serum of RA patients with follicular synovitis than in those with diffuse synovitis (P<0.05). The serum concentrations of MMPs and TIMP-1 clearly identified patients with two different histological types of rheumatoid synovitis and with OA. Additionally, the analysis of clinical data showed that the rheumatoid disease in patients with follicular synovitis seemed to be more active than in those with diffuse synovitis. CONCLUSION: The morphological appearance of rheumatoid synovitis and the serum MMP and TIMP-1 profile were correlated with the clinical activity of the disease, confirming the heterogeneity of RA. These associations also suggest that patients with different histological forms of RA might require different treatment regimens.     

Matrix metalloproteinases and tissue inhibitors of metalloproteinases in hamster aortic atherosclerosis: correlation with in-situ zymography

Atherogenesis requires extracellular matrix (ECM) alterations, a process possibly mediated by matrix-degrading metalloproteinases (MMPs) and their endogenous tissue inhibitors (TIMPs). The objective of this study was to examine the immunohistochemical expression patterns of MMPs-1, -2, -3 and -9 and their tissue inhibitors, TIMPs-1, -2, -3 and -4 during the three major stages of atherosclerotic lesion development in hypercholesterolemic Syrian Golden hamsters. Aortic atherosclerotic lesions (fatty streak, fibro-fatty and advanced) were histologically characterized in treated hamsters at 12, 24, and 49 weeks. The immunochemistry expression of these MMPs and TIMPs were examined in treated aortic sections with lesions and control aortic sections without lesions. MMP activity in control aortas and atherosclerotic lesions was characterized by in-situ zymography. Positive immunoreactivity for MMPs-2, -3, -9 and TIMPs-1, -2,-3, and -4 was observed in both control and atherosclerotic aortic arch segments, while MMP-1 was only observed in atherosclerotic lesions. Using in-situ zymography, we identified casein and gelatin degradation in fatty streak, fibro-fatty and advanced lesions. The immunohistochemical expression of these MMPs and TIMPs were examined in treated aortic sections with lesions and control aortic sections without lesions. In all lesion stages, substrate degradation was inhibited with 1,10-phenanthroline. Degradation of these substrates was not observed in control aortas. In addition, substrate degradation was inhibited with 1,10-phenanthroline. These findings suggested that in control segments, the net proteolytic balance was shifted in favor of MMP inhibition. Alternatively, despite the colocalization of MMPs and TIMPs in the treated segments, net proteolytic balance favored the catalytic MMPs.     

Matrix metalloproteinases 2, 9, and 13, and tissue inhibitors of metalloproteinases 1 and 2 in experimental lung silicosis.

Exposure to silica induces granulomatous lung inflammation evolving to fibrosis through yet unclear pathogenic mechanisms. We examined the expression of extracellular matrix remodeling molecules: collagenase 3, gelatinases A and B, and TIMP-1 and TIMP-2 in experimental lung silicosis. Rats were instilled with 50 mg of silica and sacrificed after 15 and 60 d. At 60 d a significant increase in lung collagen content was found (170.2 +/- 34.4 versus 88.2 +/- 20.8 microgram/mg in controls, p = 0.01). Gelatin zymography of bronchoalveolar lavage fluid (BALF) from 15 and 60 d revealed bands of progelatinase A and progelatinase B, and lung tissue zymograms showed in addition, the active gelatinase A form at 15 d. By in situ hybridization and immunohistochemistry, early silicotic granulomas exhibited intense staining for all matrix metalloproteinases (MMPs) and TIMPs assayed. Labeling was restricted inside granulomas and surrounding areas. Late silicotic granulomas at 60 d showed lower MMP expression than did early lesions, and in highly fibrotic nodules scarce signal was usually found. TIMP-1 and TIMP-2 showed a moderate reduction in 60-d silicotic nodules. These findings suggest that an imbalance in the expression of MMPs and TIMPs may be implicated in extracellular matrix remodeling and basement membrane disruption during experimental lung silicosis.     

Proinflammatory cytokines regulate tissue inhibitors of metalloproteinases and disintegrin metalloproteinase in cardiac cells.

OBJECTIVE: Tissue inhibitors of metalloproteinases (TIMPs) are downregulated in the failing human heart. The objective of the present study was to test the hypothesis that cytokines might be involved in the regulation of TIMPs in cardiac cells. METHODS: Neonatal Sprague-Dawley rat ventricular cells were exposed to 100 units/ml tumor necrosis factor-alpha and/or 5 ng/ml interleukin-1 beta. The mRNA and protein expression of TIMPs-1-4 and disintegrin metalloproteinase was analyzed using Northern blot, ELISA and/or Western blot, respectively. Proteolytic activity and extracellular matrix degradation and turnover were determined using gelatin zymography and pulse-chase experiments. RESULTS: The TIMP-1 mRNA was upregulated in cardiac cells, while TIMP-1 protein levels were unchanged in myocytes but downregulated in non-myocytes. The TIMP-2 expression did not change with the cytokine treatment. TIMP-3 was downregulated at both the mRNA and protein levels in cardiac cells. TIMP-4 protein was transiently increased and then returned to control level. In contrast, disintegrin metalloproteinase mRNA and protein were significantly upregulated in those cells. The gelatinolytic activity and extracellular matrix protein degradation were significantly increased. CONCLUSIONS: Tumor necrosis factor-alpha and interleukin-1 beta regulate the expression of TIMPs and disintegrin metalloproteinase, which may in turn contribute to the increased matrix degradation in cardiac cells. Since heart failure in humans is characterized by both re-expression of myocardial cytokines and remodeling of the extracellular matrix, those in vitro results suggest a potential role for those cytokines in the regulation of extracellular matrix remodeling and therefore in the transition to the end-stage heart failure phenotype.