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PubMed2004

Increased matrix metalloproteinase-2 and membrane type 1 matrix metalloproteinase activity and expression in heterotopically transplanted murine tracheas.

BACKGROUND: Bronchiolitis obliterans syndrome (BOS) is the most common long-term cause of morbidity and mortality after heart-lung or lung transplantation. One pathologic feature of BOS is infiltration of fibroblasts and connective tissue products into the airway lumen, which form a fibrous, collagen-rich occlusion. Heterotopically transplanted allogeneic murine tracheal stenosis resemble BOS in the development of obliterans airway disease. Matrix metalloproteinases (MMPs) are key enzymes involved in tissue remodeling and, clinically, have several roles in pulmonary diseases. Among the MMP family, type IV collagenases, MMP-2 and MMP-9, have high gelatinolytic activity and are thought to play a role in several pulmonary diseases. Membrane type 1 MMP (MT1-MMP) activates the zymogen of MMP-2 (proMMP-2, 72 kd), and activated MMP-2 (active MMP-2, 62 kd) degrades type IV collagen and plays an important role in clinical pulmonary disease. In this study, we examine the expression of MMP-2, its activator MT1-MMP and MMP-9 in BOS using murine trachea transplantation models. METHODS: Rats were divided into 5 experimental groups (n = 10 in each group). Group I was a control group with intact tracheas. Animals with tracheal grafts underwent heterotopically syngeneic (Groups II and III) or allogeneic (Groups IV and V) transplantation. The recipient rats were killed 7 days (Groups II and IV) or 28 days (Groups III and V) after transplantation. The harvested tracheal grafts were examined histologically. MMP activity was assessed using gelatin zymography analysis, and MMP-2 and MT1-MMP gene expression was examined by quantitative real-time polymerase chain reaction analysis. Distribution of gelatinolytic activity was studied using in situ zymography. RESULTS: There was little histologic change in the intact trachea (Group I) and in all isografts (Groups II and III). Fibrotic tissues in Group V significantly occluded the tracheal lumen, and there was severe lymphocyte infiltration in Group IV. According to gelatin zymography, proMMP-9 was faint at 7 days, but activated MMP-9 was not present in all groups. The MMP-2 gelatinolytic bands were predominant; the activation in Group V was significantly greater than that in Group IV, and in Group III it was significantly greater than that in Group II. Gene expression of both MMP-2 and MT1-MMP were significantly higher in Group V than in the other groups (p < 0.01), and MMP-2 was clearly activated. Gelatinolytic activity was localized in the fibrotic tissues or lymphocytes of thickening lumen after destruction of the epithelium by stenosis. CONCLUSIONS: These results demonstrate that MMP-2, together with its activator MT1-MMP, may have an important role in the development of BOS, which is associated with destruction of the tracheal epithelium, leading to fibrosis.

Animals↗
PubMed2004

In vitro inhibition of the activation of Pro-matrix Metalloproteinase 1 (Pro-MMP-1) and Pro-matrix metalloproteinase 9 (Pro-MMP-9) by rice and soybean Bowman-Birk inhibitors.

The in vitro inhibitory activity of the rice Bowman-Birk inhibitor (rBBI) or soybean Bowman-Birk inhibitor (sBBI) against trypsin-catalyzed activation of pro-matrix metalloproteinase 1 or 9 (pro-MMP-1 or pro-MMP-9), respectively, was investigated using electrophoresis with silver staining, heparin-enhanced zymography, biotinylated gelatin, Biotrak assay, and fluorescence quenched substrate hydrolysis. rBBI at concentrations of 0.08-0.352 mg/mL dose-dependently inhibited the in vitro activation of 45 microg/mL pro-MMP-1 by trypsin. Heparin-enhanced zymography analysis of pro-MMP-1, trypsin-activated MMP-1, and a mixture of pro-MMP-1-trypsin-rBBI showed clear zones associated with trypsin-activated MMP-1 and the absence of clear zones in lanes containing pro-MMP-1 or a mixture of pro-MMP-1, trypsin, and rBBI. The results of the Biotrak assay also indicated that rBBI dose-dependently suppressed the activation of pro-MMP-1 by trypsin. sBBI dose-dependently inhibited the activation of 100 microg/mL of pro-MMP-9 by trypsin. Biotinylated gelatin assays demonstrated that pro-MMP-9 or pro-MMP-9 in the presence of trypsin and BBI did not hydrolyze gelatin, whereas p-aminophenylmercury acetate (APMA)-activated MMP-9 and trypsin-activated MMP-9 caused significant hydrolysis of gelatin. Quenched fluorescence substrate hydrolysis for total MMP activity showed that pro-MMP-1 or pro-MMP-9 did not hydrolyze the substrate Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH2; active MMP-1 or MMP-9 hydrolyzed the substrate, but lower substrate hydrolysis was obtained when pro-MMP-1 or pro-MMP-9 was incubated with trypsin in the presence of increasing concentrations of rBBI. The results are discussed in light of the role of MMP-1 and MMP-9 in the process of angiogenesis and the potential of rBBI or sBBI as a functional food ingredient.

Biotinylation↗
PubMed2001

Role of matrix metalloproteinases 1, 2, and 9 and tissue inhibitor of matrix metalloproteinase-1 in chronic venous insufficiency.

PURPOSE: Increased transforming growth factor-beta(1) (TGF-beta(1)) activity is associated with chronic venous insufficiency (CVI) disease progression and dermal skin pathology. Because TGF-beta(1) stimulates collagen synthesis and alters the levels of matrix metalloproteinases (MMPs) and their inhibitors (TIMPs), we investigated the hypothesis that increased TGF-beta(1) activity is associated with differences in messenger RNA and protein levels of MMPs and TIMP-1 in patients with CVI. METHODS: One hundred ten biopsies of the lower calf and lower thigh in 73 patients were snap frozen in liquid nitrogen and stratified into six groups according to the clinical etiologic anatomic distribution pathophysiology disease classification. One set of lower-calf and lower-thigh biopsies were analyzed for MMP-1 and TIMP-1 gene expression with quantitative reverse transcription and competitive polymerase chain reaction. A second set of biopsies was analyzed for the active and latent forms of MMP-1, MMP-2, and MMP-9 as well as for TIMP-1 by western blotting, gelatin zymography, and tissue localization by immunohistochemistry (IHC). RESULTS: Compared with the control, MMP-1 messenger RNA was increased in class-4 and class-6 patients (P < or =.01), whereas TIMP-1 was increased in class-6 patients only (P < or =.05). However, there were no differences in total protein between MMP-1 and TIMP-1. Active MMP-2 protein increased in class-4 and class-5 patients compared with active MMP-1 and TIMP-1 (P < or =.01). Western blotting did not identify the active component of MMP-9. Similarly, only the latent form of MMP-9 was observed by gelatin zymography, whereas both the latent and active forms of MMP-2 were observed. IHC demonstrated MMP-1 and MMP-2 in dermal fibroblasts and in perivascular leukocytes. TIMP-1 was observed in basal-layer keratinocytes of the epidermis only. MMP-9 was not detected by IHC. CONCLUSION: MMP synthesis is regulated at both the transcriptional and post-transcriptional levels in CVI. Our data suggest that post-translational modifications are key to functional regulation. Dermal fibroblasts and migrating leukocytes are probable cellular sources of MMPs. Increased active MMP-2 levels in class-4 and class-5 patients indicate tissue remodeling caused by pre-ulcer and postulcer environmental stimuli. These data suggest that alterations in MMP-2 activity, in conjunction with TGF-beta(1)-mediated events, cause an imbalance in tissue remodeling leading to a pro-ulcer-forming environment.

Blotting, Western↗
PubMed2001

Claudin promotes activation of pro-matrix metalloproteinase-2 mediated by membrane-type matrix metalloproteinases.

Genes associated with regulation of membrane-type matrix metalloproteinase-1 (MT1-MMP)-mediated pro-MMP-2 processing were screened in 293T cells by a newly developed expression cloning method. One of the gene products, which promoted processing of pro-MMP-2 by MT1-MMP was claudin-5, a major component of endothelial tight junctions. Expression of claudin-5 not only replaced TIMP-2 in pro-MMP-2 activation by MT1-MMP but also promoted activation of pro-MMP-2 mediated by all MT-MMPs and MT1-MMP mutants lacking the transmembrane domain (DeltaMT1-MMP). A carboxyl-terminal deletion mutant of pro-MMP-2 (proDeltaMMP-2) was processed to an intermediate form by MT1-MMP in 293T cells and was further converted to an activated form by introduction of claudin-5. In contrast to the stimulatory effect of TIMP-2 on pro-MMP-2 activation by MT1-MMP, activation of pro-MMP-2 by DeltaMT1-MMP in the presence of claudin-5 and proDeltaMMP-2 processing by MT1-MMP were both inversely repressed by expression of exogenous TIMP-2. These results suggest that TIMP-2 is not involved in cluadin-5-induced pro-MMP-2 activation by MT-MMPs. Stimulation of MT-MMP-mediated pro-MMP-2 activation was also observed with other claudin family members, claudin-1, claudin-2, and claudin-3. Amino acid substitutions or deletions in ectodomain of claudin-1 abolished stimulatory effect. Direct interaction of claudin-1 with MT1-MMP and MMP-2 was demonstrated by immunoprecipitation analysis. MT1-MMP was co-localized with claudin-1 not only at cell-cell borders, but also at other parts of the cells. TIMP-2 enhanced cell surface localization of MMP-2 mediated by MT1-MMP, and claudin-1 also stimulated it. These results suggest that claudin recruits all MT-MMPs and pro-MMP-2 on the cell surface to achieve elevated focal concentrations and, consequently, enhances activation of pro-MMP-2.

Animals↗
PubMed2002

A unique substrate binding mode discriminates membrane type-1 matrix metalloproteinase from other matrix metalloproteinases.

In our study, we characterized the substrate recognition properties of membrane type-1 matrix metalloproteinase (MT1-MMP; also known as MMP-14), a key enzyme in tumor cell invasion and metastasis. A panel of optimal peptide substrates for MT1-MMP was identified using substrate phage display. The substrates can be segregated into four groups based on their degree of selectivity for MT1-MMP. Substrates with poor selectivity for MT1-MMP are comprised predominately of the Pro-X-X- downward arrow-X(Hy) motif that is recognized by a number of MMPs. Highly selective substrates lack the characteristic Pro at the P(3) position; instead they contain an Arg at the P(4) position. This P(4) Arg is essential for efficient hydrolysis and for selectivity for MT1-MMP. Molecular modeling indicates that the selective substrates adopt a linear conformation that extends along the entire catalytic pocket of MT1-MMP, whereas non-selective substrates are kinked at the conserved P(3) Pro residue. Importantly, the selective substrates can be made non-selective by insertion of a proline kink at P(3), without significantly reducing overall k(cat)/K(m) values. Altogether the study provides a structural basis for selective and non-selective substrate recognition by MT1-MMP. The findings in this report are likely to explain several aspects of MT1-MMP biology.

Catalytic Domain↗
PubMed2004

Increased intrahepatic cyclooxygenase 2, matrix metalloproteinase 2, and matrix metalloproteinase 9 expression is associated with progressive liver disease in chronic hepatitis C virus infection: role of viral core and NS5A proteins.

BACKGROUND: Cyclooxygenase 2 (COX-2) and matrix metalloproteinases (MMPs) have been implicated in tissue injury and fibrogenesis in animal models but little is known regarding their role in hepatitis C virus (HCV) related liver disease in humans. AIMS: To characterise the intrahepatic expression pattern of COX-2 and MMPs in chronic HCV infection and determine whether HCV core and NS5A proteins could promote their expression in cultured hepatocyte derived cell lines. PATIENTS: Thirty two anti-HCV+ and 10 anti-HCV- patients were studied. METHODS: Western blot, reverse transcription-polymerase chain reaction (RT-PCR), enzyme immunoassay, and immunohistochemistry were used to assess the expression pattern of COX-2 and MMPs in liver biopsy samples from all patients. COX-2 gene expression and MMP-9 protein levels were also determined by immunoblot, RT-PCR, and luciferase assays in core and NS5A transfected hepatocyte derived cells. RESULTS: The intrahepatic expression level of COX-2, MMP-2, and MMP-9 was significantly higher in HCV+ than in HCV- patients, increasing with the fibrotic stage of liver disease. We further demonstrated that COX-2 mRNA, protein, and activity were induced in resting and activated core and NS5A transfectants. Both viral proteins induced transcriptional activity of the COX-2 gene promoter whereas core, but not NS5A, exerted an inducer effect on MMP-9 protein levels in cultured hepatocyte derived cells. CONCLUSIONS: Intrahepatic COX-2, MMP-2, and MMP-9 overexpression is associated with progressive hepatic fibrosis in chronic HCV infection, suggesting their pathogenic role in fibrogenesis. HCV core and NS5A proteins were able to upregulate COX-2 and MMP-9 gene expression in hepatocyte derived cells, providing a potential mechanism for hepatic fibrosis during chronic HCV infection.

Adult↗
PubMed2005

High serum levels of matrix metalloproteinase-9 and matrix metalloproteinase-1 are associated with rapid progression in patients with metastatic melanoma.

PURPOSE: Matrix metalloproteinases (MMP) are proteolytic enzymes that play an important role in various aspects of cancer progression. In the present work, we have studied the prognostic significance of serum levels of gelatinase B (MMP-9), collagenase-1 (MMP-1), and collagenase-3 (MMP-13) in patients with advanced melanoma. EXPERIMENTAL DESIGN: Total pretreatment serum levels of MMP-9 in 71 patients and MMP-1 and MMP-13 in 48 patients were determined by an assay system based on ELISA. Total MMP levels were also assessed in eight healthy controls. The active and latent forms of MMPs were defined by using Western blot analysis and gelatin zymography. RESULTS: Patients with high serum levels of MMP-9 (> or = 376.6 ng/mL; n = 19) had significantly poorer overall survival (OS) than patients with lower serum MMP-9 levels (n = 52; median OS, 29.1 versus 45.2 months; P = 0.033). High MMP-9 levels were also associated with visceral or bone metastasis (P = 0.027), elevated serum alkaline phosphatase level (P = 0.0009), and presence of liver metastases (P = 0.032). Serum levels of MMP-1 and MMP-13 did not correlate with OS. MMP-1 and MMP-9 were found mainly in latent forms in serum, whereas the majority of MMP-13 in serum was active (48 kDa) form. MMP-13 was found more often in active form in patients (mean, 99% of the total MMP-13 level) than in controls (mean, 84% of the total MMP-13 level; P < 0.0001). After initiating the therapy, patients with elevated levels of MMP-1 (> or = 29.8 ng/mL, n = 10) progressed more rapidly than patients with lower levels (median, 1.9 versus 3.5 months; P = 0.023). Serum levels of MMP-9 and MMP-13 did not correlate with the time to progression (TTP). In multivariate analysis with age and gender, MMP-9 or MMP-1 turned out to be independent prognostic factors for OS [P = 0.039; hazard ratio (HR), 1.8; 95% confidence interval (95% CI), 1.03-3.3] or TTP (P = 0.023; HR, 2.7; 95% CI, 1.15-6.4), respectively. CONCLUSIONS: Our findings provide evidence that MMP-1, MMP-9, and MMP-13 play important roles at different phases of metastatic melanoma spread and that serum MMP-9, in particular, could have clinical value in identifying patients at high risk for melanoma progression.

Adult↗
PubMed2004

Comprehensive analysis of matrix metalloproteinase and tissue inhibitor expression in pancreatic cancer: increased expression of matrix metalloproteinase-7 predicts poor survival.

PURPOSE: To enable the design of improved inhibitors of matrix metalloproteinases (MMPs) for the treatment of pancreatic cancer, the expression profiles of a range of MMPs and tissue inhibitors of MMPs (TIMPs) were determined. EXPERIMENTAL DESIGN: Nine MMPs (MMPs 1-3, 7-9, 11, 12, and 14) and three TIMPs (TIMPs 1-3) were examined in up to 75 pancreatic ductal adenocarcinomas and 10 normal pancreata by immunohistochemistry. Eighteen additional pancreatic ductal adenocarcinomas and an additional eight normal pancreata were also analyzed by real-time reverse transcription-PCR and additionally for MMP-15. RESULTS: There was increased expression by immunohistochemistry for MMPs 7, 8, 9, and 11 and TIMP-3 in pancreatic cancer compared with normal pancreas (P < 0.0001, 0.04, 0.0009, 0.005, and 0.0001, respectively). Real-time reverse transcription-PCR showed a significant increase in mRNA levels for MMP-11 in tumor tissue compared with normal pancreatic tissue (P = 0.0005) and also significantly reduced levels of MMP-15 (P = 0.0026). Univariate analysis revealed that survival was reduced by lymph node involvement (P = 0.0007) and increased expression of MMP-7 (P = 0.005) and (for the first time) MMP-11 (P = 0.02) but not reduced by tumor grade, tumor diameter, positive resection margins, adjuvant treatment, or expression of the remaining MMPs and TIMPs. On multivariate analysis, only MMP-7 predicted shortened survival (P < 0.05); however, increased MMP-11 expression was strongly associated with lymph node involvement (P = 0.0073). CONCLUSIONS: We propose that the principle specificity for effective inhibitors of MMPs in pancreatic cancer should be for MMP-7 with secondary specificity against MMP-11. Moreover, these studies indicate that MMP-7 expression is a powerful independent prognostic indicator and potentially of considerable clinical value.

Adult↗
PubMed2004

Loss of matrix metalloproteinase-9 or matrix metalloproteinase-12 protects apolipoprotein E-deficient mice against atherosclerotic media destruction but differentially affects plaque growth.

BACKGROUND: Epidemiological and histological evidence implicates proteinases of the matrix metalloproteinase (MMP) family in atherosclerosis and aneurysm formation. We previously indicated a role for urokinase-type plasminogen activator in atherosclerotic media destruction by proteolytic activation of MMPs. However, the role of specific MMPs, such as MMP-9 and MMP-12, in atherosclerosis remains undefined. METHODS AND RESULTS: MMP-9- or MMP-12-deficient mice were crossed in the atherosclerosis-prone apolipoprotein E-deficient background and fed a cholesterol-rich diet. Mice were killed at 15 or 25 weeks of diet to study intermediate and advanced lesions, respectively. Loss of MMP-9 reduced atherosclerotic burden throughout the aorta and impaired macrophage infiltration and collagen deposition, while MMP-12 deficiency did not affect lesion growth. MMP-9 or MMP-12 deficiency conferred significant protection against transmedial elastin degradation and ectasia in the atherosclerotic media. CONCLUSIONS: This study is the first to provide direct genetic evidence for a significant involvement of MMP-9, but not of MMP-12, in atherosclerotic plaque growth. In addition, deficiency of MMP-9 or MMP-12 protected apolipoprotein E-deficient mice against atherosclerotic media destruction and ectasia, mechanisms that implicate the involvement of these MMPs in aneurysm formation.

Animals↗
PubMed2005

Furin-like proprotein convertases are central regulators of the membrane type matrix metalloproteinase-pro-matrix metalloproteinase-2 proteolytic cascade in atherosclerosis.

BACKGROUND: Accumulation of macrophages and their in situ expression of matrix metalloproteinases (MMPs) are important determinants of plaque stability. Activation of membrane-bound MT1-MMP, the major activator of pro-MMP-2, requires intracellular endoproteolytic cleavage of its precursor protein. This type of activation typically requires suitable furin-like proprotein convertases (PCs), specifically furin and PC5. The present study was done to investigate the function of MT1-MMP as well as furin-like PCs in mononuclear inflammatory cells. METHODS AND RESULTS: Macrophage differentiation of human monocytic THP-1 cells was accompanied by increased expression of furin, PC5, and MT1-MMP. Some pro-MMP-2 activation was found in macrophages, but pro-MMP-2 level or activation was not enhanced after stimulation with the proinflammatory mediators tumor necrosis factor-alpha or lipopolysaccharide. However, culturing of macrophages in conditioned medium from serum-starved vascular smooth muscle cells, which constitutively secrete pro-MMP-2, resulted in a strong pro-MMP-2 activation. Inhibition of furin-like PCs with the specific pharmacological inhibitor decanoyl-RVKR-chloromethylketone (dec-CMK) inhibited MT1-MMP activation in macrophages. Dec-CMK or furin-specific small interfering RNA significantly inhibited macrophage MT1-MMP-dependent activation of vascular smooth muscle cell-derived pro-MMP-2. Flow cytometry demonstrated that human circulating monocytes express furin and PC5, and MT1-MMP and immunohistochemistry revealed their colocalization in macrophages in advanced human atherosclerotic lesions. CONCLUSIONS: Furin-like PCs (furin and PC5) play a central role in a MT-MMP-MMP-2 proteolytic cascade, involving provision of macrophage MT1-MMP for the activation of pro-MMP-2 synthesized by other cells. Furin and PC5 are expressed in human peripheral blood mononuclear cells and colocalize with MT1-MMP in macrophages in the atherosclerotic plaque, supporting the hypothesis that they are potential targets in atherosclerosis.

Arteriosclerosis↗
PubMed2003

The triterpenoid CDDO inhibits expression of matrix metalloproteinase-1, matrix metalloproteinase-13 and Bcl-3 in primary human chondrocytes.

A synthetic triterpenoid, 2-cyano-3,12-dioxoolean-1,9-dien-28-oic acid (CDDO), has been reported to have anti-inflammatory properties and to decrease the interleukin-1 (IL-1)-induced expression of matrix metalloproteinase-1 (MMP-1) and MMP-13. We have shown previously that IL-1 induces expression of the inhibitor of NF-kappaB (IkappaB) family member Bcl-3, and that this contributes to MMP-1 expression. To quantify the effects of CDDO on IL-1-induced MMP-1, MMP-13 and Bcl-3 expression, we stimulated the chondrosarcoma cell line SW-1353 and human primary chondrocytes with IL-1, in the presence or absence of CDDO. Harvested RNA was subjected to quantitative real-time reverse-transcriptase polymerase chain reaction. In SW-1353 cells, 300 nM CDDO significantly decreased the induction of MMP-1 and MMP-13 by IL-1. In human primary chondrocytes, 300 nM CDDO inhibited the induction of these genes by IL-1 to an even greater extent. In both cell types, inhibition of MMP-1 required 24 hours of pretreatment with CDDO, whereas MMP-13 could be inhibited when CDDO and IL-1 were added simultaneously to culture. In human primary chondrocytes, IL-1-induced Bcl-3 expression was inhibited when cells were pretreated with CDDO. To determine whether the inhibitory effect of CDDO on MMP worked through inhibition of Bcl-3 gene expression, SW-1353 cells stably transfected with a Bcl-3 expression plasmid were treated with IL-1 and/or CDDO, and MMP gene expression was assayed. Overexpression of Bcl-3 increased MMP-1, but not MMP-13, mRNA levels. Furthermore, overexpressed Bcl-3 could sustain the CDDO-dependent inhibition of IL-1-induced MMP-1 expression. Our data demonstrate that CDDO inhibits IL-1-induced MMP-1 and MMP-13 expression in human chondrocytes. CDDO also inhibits the expression of Bcl-3, an IL-1-responsive gene that preferentially contributes to MMP-1 gene expression.

B-Cell Lymphoma 3 Protein↗
PubMed2002

[The role of matrix metalloproteinases in extracellular matrix remodelling in chronic obstructive pulmonary disease rat models].

OBJECTIVE: To study the role of metalloproteinases(MMPs) in the airway extracellular matrix (ECM) remodelling of chronic obstructive pulmonary disease (COPD) rat models. METHODS: The COPD rat models which was established by intratracheal instillation of 200 microg lipopolysaccharide once for every two weeks(twice), and exposed to 5% smoke for 0.5 h/d for 4 weeks. The pathological changes were observed, lung function and blood gas changes were also deter mined. The fibroblasts, lymphocytes of bronchial walls and alveolar macrophages were counted. The hydroxyproline of bronchial lung tissue homogenates were deter mined by biochemistry method. The expression of MMP-9,MMP-2 and tissue inhibitor of metalloproteinase-1(TIMP-1) in bronchi and lung tissue was verfied by immunohistochemical analysis and by reverse transcription-polymerase chain reaction analysis. The gelatinolytic activities of MMPs of lung tissue were performed by gelatin zymographic analysis. RESULTS: The pathological changes of bronchi and lung tissue, the changes of lung function and blood gas analysis were similar to those of the COPD patients. The number of fibroblasts, lymphocytes and alveolar macrophages of model group were significantly increased than those of control group (P < 0.001). The hydroxyproline of model group was significantly in creased than that of control group (P < 0.001). By using image analyzer, immunoreactivity of MMP-9, MMP-2,TIMP-1 were markedly increased in epithelial cells of bronchi, fibroblasts, macrophages, endothelial cells and pneumocytes in model group as compared with those of control group. The protein expressions of MMP-9, MMP-2 and TIMP-1 in model group were significantly increased than those in control group(P < 0.0001 or P < 0.01). The mRNA expression of MMP-2, MMP-9 and TIMP-1 in COPD model group (1.11 +/- 0.06,1.04 +/- 0.26 and 0.85 +/- 0.34,respectively) were significantly increased than those in control group (0.30 +/- 0.17,0.36 +/- 0.09 and 0.23 +/- 0.08,respectively) as well(P < 0.001 or P < "0.01). The relative gelatinolytic activities of 72 000 MMP-2, 92 000 MMP-9 in model group (3 263.5 +/- 665.1 and 1 338.4 +/- 241.2, respectively) were also significantly higher than those in the model group(388.6 +/- 60.8 and 116.1 +/- 49.8,respectively, P < 0.001). CONCLUSION: The findings suggested that there were up regulations of MMP-9,MMP-2 and TIMP-1 of lung tissue in COPD model group which may contribute to the pathogenesis of airflow limitation through the airway remodelling and alveolar structure destruction(emphysema). The ECM degradation and deposition were imbalanced and abnormally activated. The evaluation of MMP-9,MMP-2 which are responsible for the inflammation and destruction process, and the evaluation of TIMP-1 which is responsible for the repair and remodelling process of the airways, may play an important role in the airway ECM remodelling in COPD.

Animals↗
PubMed2005

[Comparison of doxycycline, losartan, and their combination on the expression of matrix metalloproteinase, tissue inhibitor of matrix metalloproteinase, and collagen remodeling in the noninfarcted myocardium after acute myocardial infarction in rats].

OBJECTIVE: To compare the effects of matrix metalloproteinase (MMP) inhibitor doxycycline, losartan, and their combination on the expression of MMP-8, 13, tissue inhibitor of MMP-1, 2 (TIMP-1, 2), and collagen remodeling in the noninfarcted myocardium after acute myocardial infarction (AMI) in rats. METHODS: Two hundred and fifty-four AMI rats, induced by left coronary ligation, were randomly assigned to the following groups: (1) AMI controls group (n = 64); (2) doxycycline group (30 mg x kg(-1) x d(-1), n = 63); (3) losartan group (10 mg x kg(-1) x d(-1), n = 62); (4) concomitant doxycycline and losartan group (30 and 10 mg x kg(-1) x d(-1) respectively, n = 65); and (5) Sham-operated rats (n = 30), which were randomly selected to serve as noninfarction controls. Each group was further divided into three subgroups of 1, 2, and 4 weeks that received treatment. After the completion of treatment, the rats were killed. The mRNA and protein expression of MMPs and TIMPs in the noninfarcted myocardium were quantified by RT-PCR and Western blot, respectively. The type I and type III collagen volume fraction (CVF) of the noninfarced myocardium were assessed immunohistochemically. RESULTS: No significant difference existed in myocardial infarction sizes among the 12 subgroups of AMI controls and the three treatment groups (42%-48%, all P > 0.05). Compared with sham operated rats, the mRNA and protein expression of MMP-8 and 13 significantly increased by 39%-183% in all three subgroups of AMI controls (all P < 0.05), except both of their mRNA expressions in 2-week subgroups; the mRNA and protein levels of TIMP-1 increased only in 1-week subgroup of AMI controls by 104% and 67%, respectively (both P < 0.05); the mRNA of TIMP-2 increased in all 1, 2, and 4-week subgroups by 144%-232% (all P < 0.05), but its protein expression lagged and only enhanced in 2 and 4-week subgroups of AMI controls by 231% and 332%, respectively (both P < 0.05). Meanwhile, both type I and type III CVF of noninfarcted myocardium significantly increased in all three subgroups of AMI controls (type I CVF: 3.01%-5.64% vs 1.53%-1.67%, P < 0.01-0.001; type III CVF: 2.19%-4.42% vs 1.46%-1.59%, P < 0.05-0.001), with type I CVF being higher in 4-week than in 1 and 2-week subgroups (5.64% vs 3.01% and 3.02% respectively, all P < 0.05). Compared with AMI controls, all three kinds of treatment significantly reduced the increased mRNA and protein expressions of MMP-8, 13 and TIMP-1, 2 after AMI by 14%-60% (all P < 0.05), as well as type I/III CVF in their 2 and 4-week subgroups (type I CVF: 1.56%-2.38% vs 3.02%-5.64%, P < 0.05-0.001; type III CVF: 1.92%-2.65% vs 4.19%-4.42%, P < 0.05-0.01), except for doxycycline's effect on type III CVF in any of its three subgroups (all P > 0.05). Among the three treatment groups, significant differences existed in the above mentioned indicators only at some subgroup levels (all P < 0.05). CONCLUSIONS: Like losartan, doxycycline can also suppress the enhanced mRNA and protein expression of MMP-8, 13 and TIMP-1, 2, and reduce type I collagen deposition in the noninfarcted myocardium after AMI in rats. However, it has no effect on type III collagen deposition.

Angiotensin II Type 1 Receptor Blockers↗
PubMed1999

The effect of solvent and matrix combinations on the analysis of bacteria by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry.

The ability to rapidly identify the taxonomic class of the wide variety of microorganisms involved in human and animal disease is becoming increasingly important, especially with the increasing development of resistance to the antibiotics which form the main defence against them. A number of groups have recognised the utility of matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry MALDI-TOF in the analysis of these microorganisms. However, no consistent methodology has been developed which is in general use. In particular the use of different solvent extraction systems and mass spectrometric matrices can have significant effects on the quality of the data obtained. We have now studied a number of the commonly used matrices and a range of solvent systems of widely varying polarity in an attempt to devise an optimum analytical strategy for the rapid characterisation of these organisms by MALDI-TOFMS. The E. coli ATCC 9637 organisms were initially washed to remove growth medium contaminants, followed by extraction with one of a range of solvents prior to admixing with a number of different single matrices or binary and ternary combinations of these matrices. The results obtained indicate that a binary combination of 2-(4-hydroxyphenylazo)benzoic acid and 2-mercaptobenzothiazole (1:1) as matrix provides the best data after the proteinaceous material from the organism cell surface was extracted with 17% formic acid, 33% isopropyl alcohol and 50% water, (solvent 2 in this work).

2-Propanol↗
PubMed2000

Continuous flow infrared matrix-assisted laser desorption/ionization with a solvent matrix

Continuous flow matrix-assisted laser desorption/ionization (MALDI) was demonstrated with infrared laser desorption and an ethanol matrix. A capillary was used to deliver an analyte solution dissolved in ethanol to a metal frit embedded in a sample stage. Typical flow rates were 1.7&mgr;L/min. An optical parametric oscillator tuned to 2.8&mgr;m was used for desorption and ionization, and mass analysis was achieved with a 1 m linear time-of-flight mass spectrometer. Flow injection studies were performed with low picomolar quantities of insulin and myoglobin in solutions containing 0.1 to 1.0% glycerol in ethanol. Copyright 2000 John Wiley & Sons, Ltd.

Journal Article↗
PubMed2004

Matrix reactivity of Al and Ga atoms (M) in the presence of silane: generation and characterization of the eta2-coordinated complex M.SiH4, the insertion product HMSiH3, and the MI species MSiH3 in a solid argon matrix.

Matrix isolation experiments give evidence for the formation of the loosely bonded metal-silane complex M.SiH(4) by the spontaneous reaction of Al or Ga atoms (M) with silane in a solid Ar matrix at 12 K; however, Ga(2) appears to insert spontaneously into an Si--H bond to form HGaGaSiH(3), probably with the structure HGa(micro-SiH(3))Ga. In M.SiH(4) the metal atom is eta(2)-coordinated by the silane, resulting in a species with C(2v) symmetry. The complex has a distinctive photochemistry: it can be converted on photolysis at lambda approximately 410 or approximately 254 nm to its tautomer, HMSiH(3), which also has a doublet ground electronic state and from which it can be regenerated with lambda approximately 580 nm radiation. Broadband UV-visible photolysis (lambda=200-800 nm) results in decomposition of HMSiH(3), the univalent species MSiH(3) being the only detectable product. The experimental data collected for several silane isotopomers (SiH(4), SiD(4), and SiD(3)H) and different reagent concentrations, together with the results of sophisticated quantum chemical calculations, are used to explore in detail the properties of the detected species and the reaction pathways compassing their formation.

Journal Article↗
PubMed2002

Tri-alpha-naphthylbenzene as a crystalline or glassy matrix for matrix-assisted laser desorption/ionization: a model system for the study of effects of dispersion of polymer samples at a molecular level.

Tri-alpha-naphthylbenzene (TalphaNB) can exist as either a crystalline or glassy solid at ambient temperatures, making it a unique matrix in matrix-assisted laser desorption/ionization (MALDI) spectroscopy. Electrosprayed TalphaNB is crystalline and has a melting point of 180 +/- 2 degrees C, as measured by differential scanning calorimetry (DSC). A glass of TalphaNB is obtained upon heating above the crystalline melting point with a glass transition temperature of 68 +/- 2 degrees C having no remaining crystallinity. MALDI samples containing mass fraction 1% polystyrene (PS) are run in both the crystalline and amorphous states. In the crystalline state, there is a strong spectrum typical of PS, but upon melting and quenching to the glassy state, the MALDI signal disappears. If the transparent, amorphous sample is treated with 1-butanol, it becomes white, and the MALDI signal returns. DSC shows that the 1-butanol treatment leads to the return of some of the crystallinity. Small angle neutron scattering (SANS) shows that the crystalline state has large aggregations of PS while the amorphous state has molecularly dispersed PS molecules. MALDI gives strong signals only when there are large aggregations of polymer molecules, with individually dispersed molecules producing no signal.

Journal Article↗
PubMed1992

Purification of liver aldehyde dehydrogenase by p-hydroxyacetophenone-sepharose affinity matrix and the coelution of chloramphenicol acetyl transferase from the same matrix with recombinantly expressed aldehyde dehydrogenase.

p-Hydroxyacetophenone was coupled to epoxy-activated Sepharose 6B to generate an affinity chromatographic matrix to purify aldehyde dehydrogenase. Purified beef liver mitochondrial aldehyde dehydrogenase specifically bound to the support and could be eluted with p-hydroxyacetophenone. A post-ammonium sulfate (30-55%) fraction of bovine liver was applied to the affinity gel column and aldehyde dehydrogenase was effectively purified, although not to complete homogeneity, indicating the potential selectivity of the matrix. Both beef liver cytosolic and mitochondrial aldehyde dehydrogenase bound to the column. A post-Cibacron blue Sepharose Cl-6B affinity-fractionated liver mitochondrial aldehyde dehydrogenase was purified to complete homogeneity by p-hydroxyacetophenone-Sepharose, thus eliminating the need for the isoelectric focusing step often employed. p-Hydroxyacetophenone was found to be a competitive inhibitor against propionaldehyde and noncompetitive against NAD. Escherichia coli lysates of recombinantly expressed aldehyde dehydrogenase were purified from E. coli lysates with one major 25-kDa protein contaminant also binding to the column, as detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. The 25-kDa contaminant was found to be chloramphenicol acetyl transferase from sequence analysis and binding studies.

Acetophenones↗