Reverse Zymography Research Articles

Matrix metalloproteinases and tissue inhibitors of metalloproteinases in gingival crevicular fluid during orthodontic tooth movement

Orthodontic tooth movement requires extensive re-modelling of the periodontium. Matrix metalloproteinases (MMPs) degrade the extracellular matrix during re-modelling, while their activity is regulated by the tissue inhibitors of metalloproteinases (TIMPs). The aim of this study was to investigate differences in MMP and TIMP levels in the gingival crevicular fluid (GCF) at the resorption and apposition sides of orthodontically moved teeth, and to compare these with control teeth. GCF samples were collected from eight orthodontic patients wearing fixed appliances with superelastic nickel-titanium coil springs. The samples were analysed by gelatin zymography, which allows detection of both active and latent MMPs, and reverse zymography for analysis of TIMPs. Western blotting was performed to confirm the identity of MMPs. The data were analysed using either the one-way analysis of variance or the Kruskal-Wallis test. In general, higher levels of MMPs and TIMPs were found at both the resorption and apposition sides compared with the control teeth. Remarkably, partially active MMP-1 was found in GCF from both the resorption and the apposition side but was barely present at the control teeth. TIMP-1 was strongly increased at the apposition side. Gelatinases were mainly present at the resorption side, while gelatinolytic fragments were exclusively detected at the apposition side. MMP-9, which is known to be involved in bone degradation, and a 48 kDa gelatinase were increased at the resorption side. The small increase in TIMP-1 at the resorption side might stimulate bone resorption, whereas the large increase at the apposition side reduces bone resorption. The analysis of MMPs and TIMPs may contribute to the improvement of orthodontic treatment regimens.

Purification and characterization of two novel cysteine protease inhibitors, Eel-CPI-2 and Eel-CPI-3, in the skin mucus of the Japanese eel Anguilla japonica

We have discovered multiple acidic cysteine protease inhibitors, in addition to the known Eel-CPI-1, in the skin mucus extract of the Japanese eel Anguilla japonica by using the two-dimensional gel system of gelatin reverse zymography. Two of the acidic inhibitors, which we have named Eel-CPI-2 and Eel-CPI-3, were purified to homogeneity by anion exchange chromatography on a column of DEAE-Sepharose CL-6B, followed by fast protein liquid chromatography on Superdex 75 10/300 GL and HiTrap Q HP columns. The amino acid compositions of Eel-CPI-2 and Eel-CPI-3 were found to be almost identical and closely similar to that of the eel galectin AJL1. The molecular masses of Eel-CPI-2 and Eel-CPI-3 were elucidated to be 16,089.080 and 16,089.137 Da, respectively, by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The apparent dissociation constant of Eel-CPI-2 and Eel-CPI-3 for cysteine protease papain was determined to be 1.79 × 10−7 and 1.05 × 10−7 M, respectively, by a quartz crystal microbalance technique.

Inhibition of Collagenolytic Cathepsins by β-Lactoglobulin in Milk and Its Suppressive Effect on Bone Resorption

It is well known that the collagenolytic cathepsins play an important role in the degradation of bone matrix. Therefore, the purpose of this study was to clarify the prevention effect of bone resorption by milk components. Using double-layer reverse zymography, we found a 20 kDa protein in milk which inhibited cysteine proteases. This inhibitory protein was identified as beta-lactoglobulin B. The inhibitory activity of beta-lactoglobulin B against cathepsin K was stronger than that of beta-lactoglobulin A. Beta-lactoglobulin B specifically inhibited papain type cysteine proteases such as cathepsins K and L, but not serine proteases, aspartic proteases or metallo proteases. Beta-lactoglobulin B inhibited cathepsin K non-competitively and the Ki value was 10(-5) M. The formation of osteoclastic pits in the culture system was effectively inhibited by 10(-4)-10(-5) M beta-lactoglobulin B in vitro. Furthermore, we demonstrated that beta-lactoglobulin B inhibited degradation of type I-collagen by collagenolytic cathepsins. Using the everted-sac method in rat small intestines, it was found that beta-lactoglobulin was more easily absorbed from the intestines of young rats (5 wk-old) than from those of older rats (more than 20 wk-old). The digested products of beta-lactoglobulin B with lysyl-endopeptidase showed a similar inhibitory activity against cathepsin K to the intact beta-lactoglobulin B did. Therefore, peroral intake of beta-lactoglobulin in milk and its digested peptides are expected to help protect osteoclastic bone resorption via inhibition of collagenolytic cathepsins K and L.

The Role of Tissue Inhibitor of Metalloproteinase-3 (TIMP-3) in Hematopoiesis

Matrix metalloproteinase (MMP) activity is regulated by tissue inhibitor of metalloproteinases (TIMPs). We found that while TIMP-1 and -2 expressions were unaffected, and TIMP-4 was not expressed, TIMP-3 mRNA expression decreased 10-fold within the bone marrow (BM) during G-CSF induced mobilization. In addition, through reverse zymography, the level of biologically active TIMP-3 protein was also shown to decrease during mobilization. Down-regulation of TIMP-3 may contribute to the accumulation of active MMPs within the BM, allowing for the release of hematopoieticstem/progenitor cells (HSPC) from the BM matrix. By qRT-PCR we have shown 10-fold greater TIMP-3 expression in endosteal mRNA compared to central BM mRNA in mouse femur (p=0.008). To assess which bone associated cell populations expressed the majority of TIMP-3, pooled bones were crushed, collagenase treated and FACS sorted. Mesenchymal progenitors (CD45-Lin-Sca1+) expressed the highest level of TIMP-3 followed by endothelial cells (CD45-Lin-CD31+) and mature osteoblasts (CD45-Lin-Sca1-CD51+). Erythroid progenitors (CD45+Ter119+Kit+), megakaryocyte progenitors (CD45+CD41+Kit+) and megakaryocytes (CD45+CD41+Kit−) from BM were also found to express TIMP-3, but at a level at least 10-fold lower than those of non-hematopoietic stromal cells. All other BM hematopoietic cell types tested were negative for TIMP-3 expression. Immunohistofluorescence on bone sections validated TIMP-3 expression in megakaryocytes, endothelial cells and osteoblasts. Expression of TIMP-3 in mouse platelets was confirmed by reverse zymography. To investigate TIMP-3 function we over-expressed huTIMP-3 in mice via retroviral transduction with MND-X-IRES-eGFP (MXIE) retroviral vector. BM cells retrovirally transduced with MXIE-huTIMP-3 or empty MXIE control was transplanted into lethally irradiated congenic mice. Engraftment and transduction levels were determined by GFP expression. At 3-months post-transplant there were no significant differences in body weight, total blood, spleen or BM cell counts between the two groups. qRT-PCR data showed that over-expressing huTIMP-3 did not alter the expression level of endogenous mTIMP-3. Flow cytometry analysis showed that in mice transduced with MXIE-huTIMP-3, the frequency of GFP+ B cells (CD11b-B220+) was reduced by 50% in the blood from 23.88±12.00% to 11.94±7.85% (p=0.0315) and by 64% in the BM from 25.06±13.78% to 9.02±7.67% (p=0.0188) when compared to MXIE controls. Conversely, the frequency of GFP+ huTIMP-3 expressing myeloid cells (CD11b+) was significantly increased in the blood from 55.69±17.13% to77.91±6.31% (p=0.0005), BM from 58.67±16.32% to 77.32±12.02% (p=0.0244) and spleen from 14.07±3.75% to 28.82±6.85% (p=0.0002). Unexpectedly, the frequency of untransduced GFP- myeloid and B cells were similar between the two groups. Although huTIMP-3 over-expression did not significantly alter the number of GFP+ HSPC (Linage-Sca1+Kit+, LSK) per femur (MXIE 0.03±0.03%, MXIE-huTIMP-3 0.01±0.01%, p=0.1139), LSK turnover in huTIMP-3 over-expressing cells was increased in vivo from 4.36±2.83% to 13.31±5.61% (p=0.0159) as determined by BrdU incorporation following 3 days of BrdU administration. Similarly, a trend was also observed in vitro after 12days of culture, LSK sorted from MXIE-huTIMP-3 mice proliferate faster than MXIE controls from 2.55^6cells/ml±1.05 to 9.6^6cells/ml±0.54 (p=0.1). In summary, huTIMP-3 over-expression in mice increased HSPC proliferation in vivo and in vitro. And whilst the huTIMP-3 over-expression in mice was not at a sufficient level to observe a global effect on total BM haematopoiesis, our data suggests that forced huTIMP-3 over-expression in vivo skews differentiation towards myelopoiesis at the detriment of lymphopoiesis.

Balance between matrix metalloproteinases (MMP) and tissue inhibitors of metalloproteinases (TIMP) in the cervical mucus plug estimated by determination of free non-complexed TIMP

BackgroundThe cervical mucus plug (CMP) is a semi-solid structure with antibacterial properties positioned in the cervical canal during pregnancy. The CMP contains high concentrations of matrix metalloproteinase 8 and 9 (MMP-8, MMP-9) and tissue inhibitor of metalloproteinase 1 (TIMP-1). This indicates a potential to degrade extracellular matrix components depending on the balance between free non-complexed inhibitors and active enzymes.MethodsThirty-two CMPs collected during active labor at term were analyzed. Twelve CMPs were separated into a cellular and an extracellular/fluid phase and analyzed by gelatin and reverse zymography to reveal MMP and TIMP location. Twenty samples were homogenized, extracted and studied by the TIMP activity assay based on gelatin zymography. Enzyme-linked immunosorbent assay (ELISA) was used to determine TIMP-1, MMP-8 and MMP-9 protein concentrations, and gelatin and reverse zymography used to identify gelatinases and TIMPs, respectively. The Western blotting technique was applied for semi-quantification of alpha2-macroglobulin. An ELISA activity assay was used to detect MMP-8 and MMP-9 activity.ResultsProMMP-2, proMMP-9, TIMP-1 and TIMP-2 were almost exclusively located in the fluid phase compared to the cellular phase of the CMP. All the extracted samples contained MMP-8, MMP-9, TIMP-1, TIMP-2 and alpha2-macroglobulin. Free non-complexed TIMP was detected in all the samples analyzed by the TIMP activity assay and was associated with TIMP-1 protein (R = 0.71, p < 0.001) and with the TIMP/MMP molar ratio (1.7 (1.1–2.5) (mean (95% confidence interval)) (R = 0.65, p = 0.002). The ELISA activity assay showed no activity from MMP-8 or MMP-9.ConclusionDue to their extracellular location, potential proteolytic activity from neutrophil-derived MMPs in the CMP could exert a biological impact on cervical dilatation and fetal membrane rupture at term. The functional TIMP activity assay, revealing excess non-complexed TIMP, and a molar inhibitor/enzyme ratio above unity, indicate that refined MMP control prevents CMP-originated proteolytic activity in the surrounding tissue.

Collagenolytic Fragments and Active Gelatinase Complexes in Periodontitis

Periodontal tissues remodel rapidly, which enables quick adaptation to mechanical changes. Matrix metalloproteinases (MMPs) are involved in these remodeling processes under control of tissue inhibitor of metalloproteinases (TIMPs). In periodontitis, overactivity of MMPs results in pathologic tissue degradation. The aim of this study was to analyze MMPs and TIMPs in healthy and diseased gingiva, periodontal ligament (PDL), and gingival crevicular fluid (GCF). Samples of gingiva, PDL, and GCF were obtained from healthy controls (gingiva: n = 18; PDL: n = 15; GCF: n = 8) and subjects with periodontitis (gingiva: n = 11; PDL: n = 18; GCF: n = 12). MMPs and TIMPs were analyzed by gelatin-, collagen-, and reverse zymography and by Western blotting. Total MMP activity was analyzed using a fluorogenic substrate. TIMP-1 and -2, active and pro-MMP-2 and -9, and active MMP-1 and -8 were present in all samples. Large amounts of active MMP-2 complexes and collagenolytic fragments were also found. Their levels were higher in PDL and GCF from subjects with periodontitis. In general, TIMP levels were lower in diseased periodontal tissues. Especially diseased GCF contained more MMPs. Surprisingly, some MMPs were more abundant in healthy gingiva and PDL than in diseased tissue. Unexpected variations in MMP and TIMP levels in gingiva, PDL, and GCF may result from differences in subject characteristics and disease activity. The levels of active MMP-2 complexes and collagenolytic fragments are higher in the periodontium of subjects with periodontitis and might contribute significantly to periodontal destruction.

Matrix Metalloproteinases

Matrix metalloproteinases are a class of enzymes that play an important role in the remodeling of the extracellular matrix in development and cancer metastasis. This unit describes a set of methods--cell-mediated dissolution of type-1 collagen fibrils, direct and reverse zymography, enzyme capture based on alpha2-macroglobulin and TIMP-1 and -2, and demonstration of cryptic thiol groups in metalloproteinase precursors--that are used to characterize the functions of matrix metalloproteinases and their inhibitors.

Differential Effect of 17-ß-Estradiol on Smooth Muscle Cell and Aortic Explant MMP2

The present investigation tested the hypothesis that intrinsic gender-related differences exist in rat aortic smooth muscle cell matrix metalloproteinase 2 (MMP2). This investigation comprised 3 sets of experiments. Experiment I: Adult male and female rat aortic smooth muscle cells (RASMCs) at passages 4-8 were stimulated in serum-free media for 48 h with interleukin(IL)1beta at doses encountered in human abdominal aortic aneurysms (2 ng/mL). Messenger RNA was extracted from the RASMCs, and gene expression of MMP2 and tissue inhibitor of metalloproteinase 2 (TIMP2), a major MMP2 inhibitor, was measured by real-time polymerase chain reaction. MMP2 protein levels in conditioned media were measured by Western blotting, and MMP2 and TIMP2 activity quantified by standard and reverse gelatin zymography. Experiment II: Male and female RASMCs were incubated for 48 h in Dulbecco's modified Eagler's medium containing IL-1beta and 17-beta-estradiol at doses from 1x10(-10) to 1x10(-6) molar. MMP2 activity in the conditioned media was then determined. Experiment III: Male rats underwent sustained 17-beta-estradiol exposure for 21 d using extended-release, subcutaneously implanted pellets prior to sacrifice and aortic explantation. Aortas from males, females, and estradiol-treated males were stimulated with IL-1beta for 48-h, and MMP2 activity in the conditioned media was determined. Experiment I: MMP2 gene expression was 3-fold higher in male compared with female IL-1beta stimulated RASMCs (P<0.0001). MMP2:TIMP2 gene expression ratio was 7.5-fold greater in male versus female RASMCs. MMP2 protein levels were 3-fold higher (2.68 versus 0.96 o.d./mg total protein, P=0.003) in male versus female RASMCs. Gelatinolytic activity was more than 6-fold higher (15,010 versus 2,472 o.d./mg total protein, P=0.002) in male versus female RASMCs. Experiment II: MMP2 activity in male and female RASMCs was not altered by a wide range of 17-beta-estradiol concentrations. Experiment III: When pretreated with 17-beta-estradiol, MMP2 activity in the media of male rat whole-aortic explants decreased 2-fold (P=0.002). This post-17-beta-estradiol treatment male level was not different than baseline female aortic explant MMP2 levels. MMP2 is higher in male RASMCs compared to female RASMCs. Exogenous 17-beta-estradiol did not alter MMP2 activity in vitro, but in vivo 17-beta-estradiol exposure greatly decreased male aortic MMP2 production to levels seen in the female aorta. Gender differences in MMP2 are speculated to be associated with phenotypic differences in human abdominal aortic aneurysm formation.

Type I collagen induces tissue factor expression and matrix metalloproteinase 9 production in human primary monocytes through a redox-sensitive pathway.

Tissue factor (TF), the main trigger of coagulation cascade, is a major component of the atherosclerotic plaque. Matrix metalloproteinases (MMPs) are recognized as key mediators of extracellular matrix remodeling during inflammation. It was recently emphasized that both TF and MMP-9 were overexpressed in atherosclerotic plaques, suggesting a role of both molecules in plaque instability and thrombogenicity. The present study was designed to determine whether human monocytes could co-express TF and MMP-9 when the cells interact with type I collagen, a major component of the extracellular matrix and atherosclerotic plaque. Human monocytes were isolated by elutriation and incubated in collagen I-coated plates. Tissue factor and MMP-9 expression were examined using real-time reverse transcription-polymerase chain reaction, flow cytometry, western blot and zymography. The activation of nuclear factor-kappa B (NF-kappaB) and the role of reactive oxygen species (ROS) in TF and MMP-9 production was studied using gel shift experiments, antioxidants pyrrolidine dithiocarbamate (PDTC) and N-acetyl-cysteine (NAC), and apocynin (a specific inhibitor of the NADPH oxidase). Type I collagen induced TF expression and increased MMP-9 production. In addition, the pro-inflammatory tumor necrosis factor-alpha (TNF-alpha), produced in response to collagen I, increased MMP-9 production. PDTC and NAC inhibited NF-kappaB activation during monocyte interaction with collagen I. Finally, both antioxidants and apocynin decreased the expression of TF, TNF-alpha, and MMP-9. These results indicate a new mechanism in the monocyte expression of TF and MMP-9 in response to collagen I involving a ROS-dependent pathway linked to the activation of the NADPH oxidase.

Regulation of cell invasion and signalling pathways in the pituitary adenoma cell line, HP-75, by reversion-inducing cysteine-rich protein with kazal motifs (RECK)

Degradation and remodelling of the extracellular matrix has been investigated, with the main focus on the balance between matrix metalloproteinases (MMP) and tissue inhibitor of metalloproteinases (TIMP). Recent reports disclose the presence of a novel MMP-inhibiting cell membrane-anchored glycoprotein designated 'reversion-inducing cysteine-rich protein with Kazal motifs' (RECK). Our main aim in this study was to elucidate the role of RECK in cell invasion of pituitary adenomas and its contribution to signal transduction. The function of RECK in cell invasion was investigated by comparing data obtained from full-length RECK clone transfection and gene silencing with RECK mRNA-targeting siRNA. RECK expression was confirmed using real-time RT-PCR and Western blotting. Levels of matrix metalloproteinases (MMP-2 and -9) and TIMP-1 were measured by zymography and reverse zymography, respectively. Cell invasion was examined with a 3-D invasion assay. The signal cascade was investigated by cDNA microarray analysis. As expected, expression of RECK was elevated upon cDNA transfection, and diminished using siRNA. We observed elevation of MMP-2 and -9 expression and consequent 3-D cell invasion in cells under-expressing RECK. However, TIMP expression was not affected by RECK. Analysis with cDNA microarray revealed that RECK additionally upregulates growth hormone-releasing hormone receptor (GHRHR) and latrophilin 2 at the transcriptional level. Our findings collectively suggest that RECK regulates the cell signalling pathway, playing a critical neuroendocrinological role in the pituitary adenoma cell line.

Rap1GAP Promotes Invasion via Induction of Matrix Metalloproteinase 9 Secretion, Which Is Associated with Poor Survival in Low N-Stage Squamous Cell Carcinoma

The objective of the current study was to investigate the effects of Rap1GAP on invasion and progression of head and neck squamous cell carcinoma (SCC) and the role of matrix metalloproteinase (MMP) 9 and MMP2 in this process. Rap1GAP functions by switching off Rap1, the Ras-like protein that has been associated with carcinogenesis. Previous findings suggest that Rap1GAP acts as a tumor suppressor protein in SCC by delaying the G(1)-S transition of the cell cycle. However, cells transfected with Rap1GAP exhibit a more invasive phenotype than corresponding vector-transfected control cells. MMP2 and MMP9 are enzymes that mediate SCC invasion via degradation of the extracellular matrix. Using SCC cells transfected with empty vector or Rap1GAP, cell invasion and MMP secretion were determined by Matrigel assays and gelatin zymography, respectively. Rap1GAP up-regulated transcription and secretion of MMP2 and MMP9, as assayed by quantitative reverse transcription-PCR and zymography. Furthermore, chemical and RNA interference blockade of MMP2/MMP9 inhibited invasion by Rap1GAP-transfected cells. Immunohistochemical staining of a human oropharyngeal SCC tissue microarray showed that Rap1GAP and MMP9 expression and staining intensity are correlated (P < 0.0001) and that, in early N-stage lesions of SCC, high MMP9 is prognostic of poor disease-specific survival (P < 0.05). Furthermore, Rap1GAP staining is correlated with MMP2 (P < 0.03). MMP2 in combination with N stage has a prognostic effect on time to indication of surgery at primary site. MMP2 intensity is also positively correlated with T stage (P < 0.015). In conclusion, Rap1GAP inhibits tumor growth but induces MMP2- and MMP9-mediated SCC invasion and tumor progression, suggesting a role for this protein as a biomarker for early N-stage, aggressive SCCs.

Tissue inhibitor of metalloproteinase-1 exacerbated renal interstitial fibrosis through enhancing inflammation

Tissue inhibitor of metalloproteinase-1 (TIMP-1) is associated with renal fibrosis. Furthermore, it is a multi-functional protein, and whether it has other roles in renal fibrosis is unknown. As several inflammatory mediators are substrates of matrix metalloproteinases (MMPs), TIMP-1 might affect renal fibrosis via inflamatory pathways. Plasmids containing the sense and antisense human TIMP-1 sequence were stably transfected into the human kidney proximal tubular epithelial cell line (HKC), MMP-2 and MMP-9 siRNA were transiently transfected into HKC and the transfected cells were stimulated with phorbol 12-myristate 13-acetate (PMA). In vivo, we established unilateral ureteral obstruction (UUO) models by using homozygote human TIMP-1 transgenic mice. The expression of intercellular adhesion molecule-1 (ICAM-1) in transfected cells and F4/80-positive cells in the renal interstitium were examined by indirect immunofluorescence. Protein levels in the cells and UUO models were examined by western blot, and the activities of the gelatinases and TIMP-1 were examined by gelatin zymography and reverse zymography, respectively. After stimulation with PMA, the activities of the gelatinases were decreased, ICAM-1 was upregulated, and soluble ICAM-1 in the supernatant was decreased, in HKC transfected with sense TIMP-1, and ICAM-1 was increased in HKC transfected with MMP-9 siRNA. At 14 days after UUO, it was found that compared with wild-type mice, in transgenic mice, with upregulation of TIMP-1, activities of gelatinases were downregulated, ICAM-1, transforming growth factor-beta1 (TGF-beta1), collagens I and III were upregulated, and the extent of renal fibrosis and infiltration of macrophages was more severe. Overexpression of TIMP-1 could promote renal interstitial fibrosis through the inflammatory pathway, which might be partly induced by upregulating ICAM-1.

Indices of extracellular matrix turnover in human masseter muscles as markers of craniofacial form--a preliminary study

Environmental remodelling of the craniofacial musculature is obligatory for successful outcomes following interventions such as functional appliance therapy or orthognathic surgery. Genetically driven remodelling of the craniofacial musculature is also seen in individuals with altered facial form. The processes that are involved in the remodelling of intramuscular connective tissue need to be activated in such situations. Such processes require activity of matrix metalloproteinases (MMPs) and the tissue inhibitors of matrix metalloproteinases (TIMPs), which are responsible for extracellular matrix (ECM) turnover. The aim of this study was, therefore, to establish the expression of MMP-2 and MMP-9 and their inhibitors, TIMP-1 and TIMP-2, in the masseter muscle of humans with both normal and increased vertical facial form and to assess whether this expression had any value as a predictor of facial form. Biopsies were taken from 20 subjects (10 with vertical facial deformity and 10 with normal vertical facial form to act as a control group). The sample group consisted of 15 females and 5 males and the average age of the donors +/- standard deviation (SD) was 26.04 +/- 6.16 years (range: 17.67-31.25 years). Biopsy samples were then subjected to zymography and reverse zymography to assess MMP and TIMP expression, respectively. Lateral skull cephalograms were analysed for each subject using Spearman's rho correlation coefficients and Mann-Whitney U-tests. TIMP-1 activity was consistently expressed in human masseter muscle. MMP-2, MMP-9, and TIMP-2 activity, when detected, was at a low level. These data indicate that in most individuals, an excess of TIMP-1, compared with MMP-2 and MMP-9, limits ECM turnover in human masseter muscle. There was a demonstrable variation in proteinase expression between different individuals. These preliminary findings, however, do not confirm that indices of ECM turnover are a reflection of an individual's vertical facial form.

Protease-Activated Receptor-2 Augments Experimental Crescentic Glomerulonephritis

Protease-activated receptor-2 (PAR-2) is a cellular receptor expressed prominently on epithelial, mesangial, and endothelial cells in the kidney and on macrophages. PAR-2 is activated by serine proteases such as trypsin, tryptase, and coagulation factors VIIa and Xa. It induces pleiotropic effects including vasodilatation, increasing plasminogen activator inhibitor (PAI-1) expression, mesangial cell proliferation, and cytokine production by macrophages. The role of PAR-2 in renal inflammation was studied in antiglomerular basement membrane antibody-induced crescentic glomerulonephritis (CGN) using PAR-2-deficient (PAR-2(-/-)) mice and wild-type littermate controls. PAR-2(-/-) mice had reduced crescent formation, proteinuria, and serum creatinine compared with wild-type mice 21 days after initiation of CGN. Glomerular accumulation of CD4(+) T cells and macrophages and the number of proliferating cells in glomeruli were similar in both groups. Glomerular fibrin deposition was significantly reduced in PAR-2(-/-) mice, and this was associated with reduced renal plasminogen activator inhibitor expression and increased renal matrix-metalloprotinase-9 activity. These results demonstrate a proinflammatory role for PAR-2 in CGN that is independent of effects on glomerular leukocyte recruitment and mesangial cell proliferation. PAR-2-mediated augmentation of renal plasminogen activator inhibitor expression and inhibition of matrix-metalloprotinase-9 activity may contribute to increased glomerular fibrin accumulation and glomerular injury in CGN.

Modulation of interleukin‐6 and matrix metalloproteinase 2 expression in human fibroblast‐like synoviocytes by functional ionotropic glutamate receptors

Patients with rheumatoid arthritis (RA) have increased concentrations of the amino acid glutamate in synovial fluid. This study was undertaken to determine whether glutamate receptors are expressed in the synovial joint, and to determine whether activation of glutamate receptors on human synoviocytes contributes to RA disease pathology. Glutamate receptor expression was examined in tissue samples from rat knee joints and in human fibroblast-like synoviocytes (FLS). FLS from 5 RA patients and 1 normal control were used to determine whether a range of glutamate receptor antagonists influenced expression of the proinflammatory cytokine interleukin-6 (IL-6), enzymes involved in matrix degradation and cytokine processing (matrix metalloproteinase 2 [MMP-2] and MMP-9), and the inhibitors of these enzymes (tissue inhibitor of metalloproteinases 1 [TIMP-1] and TIMP-2). IL-6 concentrations were determined by enzyme-linked immunosorbent assay, MMP activity was measured by gelatin zymography, and TIMP activity was determined by reverse zymography. Fluorescence imaging of intracellular calcium concentrations in live RA FLS stimulated with specific antagonists was used to reveal functional activation of glutamate receptors that modulated IL-6 or MMP-2. Ionotropic and metabotropic glutamate receptor subunit mRNA were expressed in the patella, fat pad, and meniscus of the rat knee and in human articular cartilage. Inhibition of N-methyl-D-aspartate (NMDA) receptors in RA FLS increased proMMP-2 release, whereas non-NMDA ionotropic glutamate receptor antagonists reduced IL-6 production by these cells. Stimulation with glutamate, NMDA, or kainate (KA) increased intracellular calcium concentrations in RA FLS, demonstrating functional activation of specific ionotropic glutamate receptors. Our findings indicate that activation of NMDA and KA glutamate receptors on human synoviocytes may contribute to joint destruction by increasing IL-6 expression.

Cilostazol attenuates MCP‐1 and MMP‐9 expression in vivo in LPS‐administrated balloon‐injured rabbit aorta and in vitro in LPS‐treated monocytic THP‐1 cells

Monocyte chemoattractant protein-1 (MCP-1) and matrix metalloproteinase-9 (MMP-9) are involved in vascular inflammation. We tested the hypothesis, and explored the underlining mechanisms that cilostazol, a phosphodiesterase 3 inhibitor with antiplatelet and antithrombotic properties, inhibits lipopolysaccharide (LPS)-induced MCP-1 and MMP-9 expression. In a rabbit aorta balloon-injury model, administration of LPS increased macrophage infiltration and MCP-1 and MMP-9 expression; cilostazol supplementation prevented this phenomenon and reduced intimal hyperplasia. In contrast, the reverse zymography showed that cilostazol did not affect TIMP-1 expression in serum. In monocytic THP-1 cells, cilostazol and N6,O2'-dibutyryl-cAMP (dioctanoyl-cAMP, a cAMP analog) dose-dependently inhibited LPS-induced MCP-1 protein expression and MMP-9 activation, but did not affect the tissue inhibitor of metalloproteinase-1. Quantitative real-time polymerase chain reaction (PCR) showed that cilostazol inhibited MCP-1 and MMP-9 mRNA expression. Cilostazol significantly inhibited LPS-induced activation of p38, JNK, and nuclear factor-kappaB, and the respective inhibitors of p38 and JNK greatly reduced the level of LPS-induced MCP-1 and MMP-9, suggesting the involvement of the p38 and JNK pathways. In conclusion, cilostazol administered with LPS in vivo reduced neointimal hyperplasia and macrophage infiltration in the balloon-injured rabbit aorta; in vitro, cilostazol inhibits LPS-induced MCP-1 and MMP-9 expression. These data suggest that cilostazol may play an important role in preventing endotoxin- and injured-mediated vascular inflammation.

Migration of culture-expanded human mesenchymal stem cells through bone marrow endothelium is regulated by matrix metalloproteinase-2 and tissue inhibitor of metalloproteinase-3

Mesenchymal stem cells (MSC) are adult stem cells that can be expanded many fold in vitro and have the therapeutic potential to restore the bone marrow microenvironment and support hematopoietic recovery after myeloablative conditioning for hematopoietic stem cell transplantation. Successful homing to the target tissue, such as bone marrow, implies that MSC are able to extravasate after systemic administration. However, the extravasation capacity of MSC and the underlying mechanisms are poorly understood to date. We studied in vitro the capacity of MSC to migrate through bone marrow endothelium. In vitro invasion and transendothelial migration assays were performed. The expression of matrix metalloproteinase (MMP) was analyzed by reverse transcriptase polymerase chain reaction (RT-PCR) and zymography. Migration of cells cultured at high or low confluence was compared and differential gene expression in these conditions was analyzed with microarray and real-time RT-PCR. The functional involvement in MSC migration was assessed using neutralizing anti-MMP-2 antibody, MMP-2 short interfering RNA or recombinant tissue inhibitor of metalloproteinase (TIMP-3). We demonstrated that MSC can invade reconstituted basement membrane and that bone marrow endothelial cells stimulate this process. We also showed that the transendothelial migration of MSC is at least partially regulated by MMP-2. High culture confluence was found to increase production of the natural MMP-inhibitor TIMP-3 and decrease transendothelial migration of MSC. We show that MSC have the potential to migrate through bone marrow endothelium and that this process involves MMP-2. Moreover, the migration of MSC is significantly influenced by the level of culture confluence. Increased culture confluence impairs migration and is related to an upregulation of TIMP-3. The therapeutic use of MSC would benefit from a selection of culture conditions that allow optimal extravasation of these cells.

Identification of Cysteine Proteases and Screening of Cysteine Protease Inhibitors in Biological Samples by a Two-Dimensional Gel System of Zymography and Reverse Zymography

We have developed a two-dimensional (2D-) gel system of zymography and reverse zymography for the detection and characterization of proteases and protease inhibitors. Isoelectric focusing (IEF) agarose gels with pH gradients were employed for separation in the first-dimension and sodium dodecyl sulfate (SDS)-polyacrylamide gel copolymerized with gelatin used for the second dimension. Proteases and protease inhibitors separated by IEF gel were applied on the second gel without trichloroacetic acid (TCA) fixation. Protease activity in the 2D-gel was visualized as transparent spots where gelatin substrate was digested after commassie brilliant blue (CBB) staining. Some of the transparent spots from the skin mucus extract of rainbow trout were determined to be a cysteine protease through use of E-64 or CA-074. In the reverse zymography technique, the gel was incubated with papain solution at 37°C for 18 h. Cysteine protease inhibitors from broad bean seeds were detected as clear blue spots after CBB staining. The amino (N-) terminal sequences of four papain inhibitor spots thus detected were demonstrated to be identical to that of favin β chain, a broad bean lectin. Taken together, our system can be considered to be an efficient technique for discovering and characterizing new proteases and protease inhibitors in biological samples. This is the first report describing a 2D-gel system of zymography and reverse zymography.

Matrix metalloproteinase (MMP) induction and inhibition at different doses of recombinant tissue plasminogen activator following experimental stroke

Although recombinant tissue plasminogen activator (rt-PA) is successfully used for thrombolysis in human stroke, it may increase the risk of haemorrhagic complications. It was shown that the matrix metalloproteinase (MMP) system is critically involved in basal lamina degradation after middle cerebral artery occlusion and reperfusion following rt-PA administration. We describe the effects of different doses of rt-PA (saline, 0.9, 9, or 18 mg rt-PA/kg body weight) on the MMPs, their specific inhibitors (TIMPs), and also their inducer protein EMMPRIN following experimental cerebral ischemia (3 hours [h], 24 h reperfusion, suture model) in rats. The amount of MMP-2 and -9 was measured by gelatine zymography, TIMP-1 and -2 by reverse gelatine zymography, and the content of EMMPRIN and the basal lamina component collagen type IV by Western blotting. The amount of both MMPs steadily rose with increasing doses of rt-PA (p<0.05). In contrast, their endogenous inhibitors TIMPs decreased (p<0.001). A balance between the proteases and their inhibitors was achieved at the low dose of 0.9 mg/kg rt-PA in the rats, which significantly coincided with the demonstrated protection of collagen type IV degradation at this dose. The inducer protein EMMPRIN increased in parallel to its substrate MMP-2. Exogenous rt-PA leads to an increase of the MMP-inducing system by EMMPRIN, and a rise of the degrading MMPs follows. However, at low to moderate doses of rt-PA the microvascular basal lamina was protected, probably due to inhibition of MMP-2 and MMP-9 by the upregulation of their inhibitors. This strongly supports use of the lowest effective dosage of rt-PA available.

Inhibitory effect of antisense RNA on the gene expression of membrane-type 1 matrix metalloproteinase and invasiveness of human gastric carcinoma cells

AIM:To investigate the influence on the gene expression of membrane-type 1 matrix metalloproteinase(MT1-MMP)and invasiveness of human gastric carcinoma cell line BGC823 by antisense RNA. METHODS:The eukaryotic expression vector carrying MT1-MMP antisense RNA was con- structed with recombinant technology and then transfected into human gastric cancer cell line BGC823.The changes of MT1-MMP mRNA ex- pression,cell proliferation,activation of gelatin- ase A and cell invasion ability were examined by reverse transcription-polymerase chain reaction (RT-PCR),MTT assay,zymography and Tran- swell invasion assay,respectively. RESULTS:The eukaryotic expression vector (named pasMMP14)containing MT1-MMP an- tisense RNA was successfully constructed and transfected into BGC823 cells.The expression of MT1-MMP mRNA was down-regulated with an inhibitory rate of 36%,in comparison with that in negative control group.At the 48th hour after transfection with pasMMP14,the activity of gelatinase A was dramatically inhibited.Af- ter 72 hours,cell proliferation was significantly decreased as compared with that in pcDNA3.0 group and normal control group(t=2.358,P0.01;t=2.727,P0.01).Transwell invasion assay showed that the invasive property was greatly suppressed in pasMMP14-transfected group as compared with that in pcDNA3.0 group and normal control group(t=5.744,P0.01;t=5.695,P0.01). CONCLUSION:Antisense RNA can evidently inhibit the gene expression of MT1-MMP and invasiveness of gastric cancer cells,suggesting that MT1-MMP gene may be a molecular target of anti-invasion therapy for gastric cancer.