Immunohistochemical study on tissue inhibitors of metalloproteinases in normal and pathological human livers

The localization of tissue inhibitor of metalloproteinases (TIMP) in normal and pathological livers was examined by immunohistochemistry using monoclonal antibodies at the light microscopic level. In normal liver, immunoreactive TIMP was detected in smooth muscle cells and endothelial cells of blood vessels, fibroblasts, bile duct cells and Kupffer cells, indicating that TIMP is likely to be a general element of the liver. Immunoreactivity was observed in newly-formed blood vessels, proliferating bile ductules, and fibroblasts in the expanded portal area and fibrous septa of chronic active hepatitis and cirrhosis. TIMP was strongly stained in the capsule of hepatocellular carcinoma. The intensity of the immunoreaction in the capsule was generally greater than that in cirrhotic liver apart from the tumor mass. In three of five cases with hepatocellular carcinoma, endothelial walls in contact with tumor cells were positive.     

Immunohistochemical study on tissue inhibitors of metalloproteinases in normal and pathological human livers.

The localization of tissue inhibitor of metalloproteinases (TIMP) in normal and pathological livers was examined by immunohistochemistry using monoclonal antibodies at the light microscopic level. In normal liver, immunoreactive TIMP was detected in smooth muscle cells and endothelial cells of blood vessels, fibroblasts, bile duct cells and Kupffer cells, indicating that TIMP is likely to be a general element of the liver. Immunoreactivity was observed in newly-formed blood vessels, proliferating bile ductules, and fibroblasts in the expanded portal area and fibrous septa of chronic active hepatitis and cirrhosis. TIMP was strongly stained in the capsule of hepatocellular carcinoma. The intensity of the immunoreaction in the capsule was generally greater than that in cirrhotic liver apart from the tumor mass. In three of five cases with hepatocellular carcinoma, endothelial walls in contact with tumor cells were positive.     

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.     

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.     

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.     

Role of fibroblast growth factor-2 in the expression of matrix metalloproteinases and tissue inhibitors of metalloproteinases in human intestinal myofibroblasts

BACKGROUND AND AIMS: The coordinated expression of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) plays a crucial role in tissue remodeling. We investigated the effects of fibroblast growth factor (FGF)-2 on the secretion of MMPs and TIMPs in human intestinal subepithelial myofibroblasts (SEMFs). METHODS: The secretion of MMP-s and TIMPs was determined by ELISA or Western blotting. The mRNA expression of MMPs and TIMPs was assessed by Northern blotting. The activating protein (AP)-1-DNA binding activity was evaluated by electrophoretic gel mobility shift assays (EMSA). RESULTS: Unstimulated intestinal SEMFs constitutively secreted MMP-2 and TIMP-2. FGF-2 stimulated MMP-1, MMP-3 and TIMP-1 secretion, but did not affect MMP-2 or TIMP-2 secretion. FGF-2 induced AP-1-DNA binding activity, and the c-Jun/AP-1 inhibitor curcumin attenuated the FGF-2-induced MMP-1, -3 and TIMP-1 mRNA expression. Mitogen-activated protein (MAP) kinase inhibitors (U0126 and PD098059) also blocked the MMP-1, -3 and TIMP-1 secretion. Furthermore, FGF-2 dose-dependently induced FGF-2 mRNA expression in these cells. CONCLUSIONS: FGF-2 may be one of important regulatory factors for extracellular matrix turnover via a modulation of MMP and TIMP secretion from SEMFs.     

Extracellular matrix remodelling in human aortic valve disease: the role of matrix metalloproteinases and their tissue inhibitors.

AIMS: Aortic valve diseases are characterized by pathological remodelling of valvular tissue but the cellular and molecular effectors involved in these processes are not well known. The role of matrix metalloproteinase (MMP)-2, MMP-9, MMP-3, MMP-7, and tissue inhibitor of matrix metalloproteinase (TIMP)-1 and TIMP-2 are investigated here. METHODS AND RESULTS: Histological analysis of pathological valves [aortic stenosis (AS) (n=49), aortic regurgitation (AR) (n=23)] and control valves (n=8) was performed. The main tissue abnormalities (calcification, inflammatory cells, and capillaries) observed in AS were less severe or absent in AR. However, both groups of pathological valves displayed similar histological signs of extracellular matrix (ECM) remodelling. Biochemical analysis of MMPs and TIMPs (gelatin and casein zymography and ELISA) was performed on valve extracts. MMP-2 activity was not significantly different in control and pathological valves. Increases in MMP-9 and MMP-3 in AS demonstrated an inflammatory state. Finally, there was a four- to seven-fold increase of TIMP-1 in pathological valves. TIMP-1, TIMP-2, and MMP-2 were synthesized by the valvular interstitial cells in primary culture. CONCLUSION: This study demonstrates the involvement of the MMP/TIMP system in ECM remodelling of both AS and AR. These findings provide evidence of inflammatory injury more severe in AS than in AR and involvement of mesenchymal cell response.     

Ovarian tissue remodeling: role of matrix metalloproteinases and their inhibitors

Follicular formation, growth or atresia, and ovulation as well as luteal formation and subsequent regression are dependent upon cyclical remodeling of the extracellular matrix (ECM). The proteinaceous and nonproteinaceous components of the ECM provide the tissue specific, extracellular architecture to which cells attach. Furthermore, the ECM modulates cellular activities through cellular surface receptors and serves as a reservoir for specific growth factors, cytokines, and binding proteins. The ability of the ECM to direct the proliferation, differentiation and function of cells implicates ECM remodeling in normal ovarian function. Specific components of the ECM are cleaved by matrix metalloproteinases (MMPs) whose activities are specifically inhibited by tissue inhibitors of metalloproteinases (TIMPs). MMPs are zinc- and calcium-dependent enzymes that collectively degrade proteinaceous components of the ECM. Controlled turnover of ECM by MMPs and TIMPs may be essential for creating and (or) preserving microenvironments conducive to follicular and luteal function and is likely dependent upon the ratio of enzyme to inhibitor. To date, most studies have focused upon correlating ovarian expression of MMPs and TIMPs with various stages of the reproductive cycle. From these studies, many potential key regulators of ovarian ECM remodeling have been identified. This review presents evidence for the involvement of MMPs and TIMPs in ECM remodeling associated with follicular and luteal function.     

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.     

Application of real-time RT-PCR to quantifying gene expression of matrix metalloproteinases and tissue inhibitors of metalloproteinases in human abdominal aortic aneurysm

BACKGROUND: The relative expression levels of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs), key regulators in remodeling of extracellular matrix, are considered to play a pivotal role in the development of abdominal aortic aneurysm (AAA). However, few data exist regarding quantitative assessment of their expression in clinical settings. METHODS: In 22 patients with AAA who underwent graft replacement, tissue samples of the AAA and non-dilated aorta were obtained. Using a real-time RT-PCR method that enabled quantitative measurement of mRNA levels in small tissue samples, we determined gene expression levels of MMPs and TIMPs relative to that of glutaraldehyde 3-phosphate dehydrogenase in each sample. RESULTS: The expression levels of the MMP-1 and -3 genes were significantly augmented in AAA compared with non-dilated regions (4.48 +/- 2.01 versus 0.26 +/- 0.12, P < 0.01 and 1.89 +/- 1.00 versus 5.01 +/- 0.97, P < 0.05, respectively). Although genes for TIMP-1, -2 and -3 tended to be upregulated in AAA, relative expression levels of MMP-1 to TIMP-1, MMP-1 to TIMP-2, MMP-1 to TIMP-3, and MMP-3 to TIMP-2 were still higher in AAA than in non-dilated regions (1.12 +/- 0.63 versus 0.10 +/- 0.03, 4.13 +/- 1.12 versus 0.43 +/- 0.11, 1.61 +/- 0.59 versus 0.14 +/- 0.03, and 7.81 +/- 1.60 versus 2.56 +/- 0.76, respectively, P < 0.05). CONCLUSION: These results demonstrate that the present real-time RT-PCR method is reliable for the determination of mRNA levels in small samples of vascular tissue and that disproportional expression of both MMP-1 and MMP-3 relative to TIMPs relates pathologically to the evolution of AAA.     

乙型肝炎病毒X蛋白对MMPs及TIMPs的影响

目的全面分析乙型肝炎病毒X蛋白（Hepatitis Bvirus Xprotein，HBx）对基质金属蛋白酶（Matrix Metalloproteinases，MMPs）及组织金属蛋白酶抑制物（Tissue Inhibitors of Metalloproteinases，TIMPs）的影响，探讨其在肝细胞癌的侵袭转移中的可能作用。方法PCR扩增HBVX基因并克隆人真核表达载体pcDNA3．1／HisC，重组载体及空载体分别以Lipofectamine2000转染HepG2细胞并以800μg／mlG418筛选抗性细胞克隆。以Western blot检测抗性细胞HBx表达。抽提细胞总RNA，半定量RT-PCR检测MMPs及TIMPs。收集细胞培养液上清，以明胶酶谱检测MMP2及MMP9活性，反相明胶酶谱检测TIMPs活性。结果构建了HBx重组载体pcDNA3．1-XB。该重组载体及对照空载体转染HepG2细胞后，G418筛选，分别获得抗性细胞克隆HepG2-XB及HepG2-HIS，前者经Westernblot证实可表达HBx。半定量RT—PCR显示HBx可促进MMP2、7、13、14、16、17、19、23、24及TIMP1、4基因的转录，抑制MMP1、3、8、9、10、11、12、15、20及TIMP2、3基因的转录。明胶酶谱检测显示HBx可促进酶原MMP2（Pro—MMP2）及活性MMP9（Active—MMP9）表达，抑制酶原MMP9（Pro—MMP9）表达；反相明胶酶谱显示HBx可促进TIMP1、TIMP4的表达，同时抑制TIMP2及糖基化TIMP3的表达。结论HBx蛋白对MMPs、TIMPs转录表达的影响是多方面的，但这种影响在HBx促进肝癌细胞侵袭转移过程中的确切机制有待进一步阐明。     

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.     

Localization of matrix metalloproteinases and tissue inhibitor of metalloproteinases-2 in normal human and rabbit stomachs.

BACKGROUND/AIMS: Matrix metalloproteinases (MMPs) are endopeptidases that degrade extracellular matrix and are involved in the pathogenesis of gastrointestinal ulcer and cancer along with tissue inhibitors of metalloproteinases (TIMPs). The purpose of this study is to examine their localization and functions in the normal stomach. METHODS: We examined the localization of MMP-1, MMP-2, MMP-9 and TIMP-2 in normal human and rabbit stomachs by light- and electron-microscopic immunohistochemistry and Western blotting, and the enzymatic activities of MMP-2 and MMP-9 by gelatin zymography. RESULTS: Immunohistochemistry revealed their localization in parietal cells, and surface and foveolar epithelial cells. Electron-microscopic immunohistochemistry of parietal cells showed immunolabeling of MMP-2 and TIMP-2 in the cisternae of the rough endoplasmic reticulum, and that of MMP-1 and MMP-9 in tubular structures in their cytoplasm. Western blotting revealed that the densities of MMP-2 and MMP-9 bands were higher for the fundic gland region than for the pyloric gland region. Gelatin zymography revealed that tissue extracts of the fundic gland region exhibited higher enzymatic activity of MMP-2 and MMP-9 than those of the pyloric gland region. CONCLUSION: Normal rabbit and human stomachs contain MMP-1, MMP-2, MMP-9, and TIMP-2 and these are mainly localized in, and synthesized by parietal cells.