Expression of Matrix Metalloproteinases and Their Tissue Inhibitors in Human Brain Tumors

In this study, we investigated the expression patterns of 15 matrix metalloproteinases (MMPs) and three tissue inhibitors of metalloproteinase in gliomas, medulloblastomas, and normal brain tissue. By Northern blot analysis we found increased levels of mRNAs encoding for gelatinase A, gelatinase B, two membrane-type MMPs (mt1- and mt2-MMP), and tissue inhibitors of metalloproteinase-1 in glioblastomas and medulloblastomas. We observed a significant increase of mt1-MMP, gelatinase A, gelatinase B, and tissue inhibitors of metalloproteinase-1 in glioblastomas as compared with low-grade astrocytomas, anaplastic astrocytomas, and normal brain. In medulloblastomas, the expression of mt1-MMP, mt2-MMP, and gelatinase A were also increased, but to a lesser extent than that observed in glioblastomas. These data were confirmed at the protein level by immunostaining analysis. Moreover, substrate gel electrophoresis showed that the activated forms of gelatinases A and B were present in glioblastomas and medulloblastomas. These results suggest that increased expression of mt1-MMP/gelatinase A is closely related to the malignant progression observed in gliomas. Furthermore, the present study demonstrates, to our knowledge for the first time, that medulloblastomas express high levels of MMP.     

Expression of Matrix Metalloproteinases 1, 2, 9 and Their Tissue Inhibitor-1 in Cartilage-Forming Osteal Tumors

The expression of MMP-1, -2, -9 and TIMP-1 was studied in 10 benign cartilage-forming osteal tumors (5 osteochondromas and 5 chondromas) and 39 chondrosarcomas (14 central, 4 periosteal, 7 dedifferentiated, and 14 secondary tumors). No expression of MMP and TIMP-1 was detected in benign cartilage-forming osteal tumors. In chondrosarcomas, the expression of MMP-1 was detected in 84.6%, of MMP-2 in 71.8, of MMP-9 in 97.4, and of TIMP-1 in 82.4% cases, the levels of expression of these markers varied from 10 to 60%. The expression of MMP-1 was not associated with patient gender, maximum size and degree of differentiation of the tumor, but was linked with age. The expression of MMP-1 was more often detected in central and dedifferentiated chondrosarcomas; the expression of MMP-1(+) was significantly associated with 3-year relapse-free and 5-year overall survival of the patients. The expression of MMP-1 in the tumor was associated with unfavorable course of the disease. The values of MMP-2 expression in chondrosarcomas did not reflect the main clinical morphological characteristics of the disease and its prognosis. The level of MMP-9 protein expression in chondrosarcomas ≥40% is prognostically unfavorable, while <40% is a favorable factor for 3-year relapse-free survival. The risk of disease relapse within 1 year after the beginning of therapy was maximum in T₃ tumors with expression of MMP-9 protein ≥40%. No relationships between the parameters of TIMP-1 expression in chondrosarcomas and the main clinical morphological characteristics of the disease and its prognosis were detected.     

牙周炎牙龈组织MMP-2、TIMP-1的免疫组化研究

目的　探讨来源于宿主的基质金属蛋白酶MMPs及其抑制因子在牙周炎发病中的作用机制。方法　本研究利用免疫组化方法检测 15例牙周炎患者牙龈组织中和 4例健康牙龈中基质金属蛋白酶 2(MMP 2 )、组织抑制因子 1(TIMP 1)的蛋白表达。结果　 15例成人牙周炎组牙龈组织中MMP 2在上皮内及上皮下的炎性组织中均有阳性表达 ,而且阳性率明显高于正常牙龈 (P <0 .0 5 ) ;TIMP 1在牙周炎以及正常的牙龈组织中表达无明显差异。结论　本研究提示基质金属蛋白酶MMP 2参与牙周组织破坏过程 ,参与牙周炎的病理生理机制     

Temperospatial Expression of Matrix Metalloproteinases 1, 2, 3, and 9 During Early Tooth Development

Odontogenesis involves a complex series of processes including epithelial-mesenchymal interactions, morphogenesis, differentiation, fibrillogenesis, and mineralization. Extracellular (ECM) remodeling plays a critical role in the rapid morphological changes that accompany these events. It is proposed that matrix metalloproteinases (MMPs) participate in the remodeling of tooth-specific matrices that accompanies the developmental events. MMPs are zinc-requiring endopeptidases that are centrally involved in the controlled turnover of ECM components and are key to a varied range of developmental processes. Thus, the aim of this study was to examine the expression of MMPs 1, 2, 3, and 9 within the developing tooth germ of Wistar rats, using immunohistochemical localisation. During the bud stage, MMPs 1, 2, 3, and 9 were expressed within both epithelial and mesenchymal cells. Later on, during the cap stage, differential expression was observed; of note was the expression of MMP 3 within the enamel knot. This study reports the temperospatial expression of MMPs 1, 2, 3, and 9 during early tooth development, and points to them having a key role during this important developmental period.     

Expression and Activities of Matrix Metalloproteinases under Oscillatory Shear in IL-1-Stimulated Synovial Cells

Matrix metalloproteinases (MMPs) are known to play a critical role in tissue disintegration, and an elevated level of MMPs is observed in synovium and synovial fluid of joints with rheumatoid arthritis. During joint movement, synovial tissue receives various mechanical stimuli, but effects of mechanical challenges on regulation of MMPs in rheumatic synovium are poorly understood. Focusing on cellular responses to oscillatory fluid shear in human synovial cells, we determined the expression of MMP-1 and MMP-13 by polymerase chain reaction and immunoblotting as well as proteolytic activities of total MMPs by a fibril degradation assay and zymography. The results revealed that ~0.5 dyn/cm 2 oscillatory shear at 1 Hz not only reduced an mRNA level and a protein level of MMP-1 and MMP-13, but it also decreased collagenase and gelatinase activities of total MMPs. Furthermore, the induction of the MMP expression and activities by interleukin-1 was suppressed by the oscillatory shear. Interestingly, the oscillatory shear upregulated the mRNA expression of TIMP-1 and TIMP-2. Our results support a potential role of oscillatory shear in regulating expression and activities of MMPs in the presence and the absence of proinflammatory cytokine.     

Expression of Matrix Metalloproteinases in Invasive Pulmonary Adenocarcinoma with Bronchioloalveolar Component and Atypical Adenomatous Hyperplasia

To investigate the association between the expression of matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) and the clinicopathological features in lepidic and invasive components of adenocarcinoma of the lung, we performed immunostaining for type IV collagen, various MMPs, and TIMPs in 27 cases of invasive adenocarcinomas and 5 cases of atypical adenomatous hyperplasia of alveolar epithelial cells (AAH). Mean extent of lepidic growth was 61% and the survival was significantly better in cases with 50% or more lepidic component. The preservation of type IV collagen in lepidic areas correlated inversely with lymphatic or vascular invasion (P = 0.02 and 0.002, respectively). Five-year survival was reduced in cases showing destruction of type IV collagen (P = 0.004) or expression of MMP-2 (P = 0.008) in lepidic areas. MMP-2 co-localized with MT-1-MMP (its activating enzyme) and TIMP-2 in neoplastic cells. Reactivity for other MMPs and TIMPs did not correlate with destruction of type IV collagen or prognosis. Type IV collagen was preserved in all cases of AAH. MMP-2, but not MT-1-MMP, was expressed in two of the five cases of AAH. Immunostaining for type IV collagen MMP-2 is useful in evaluating the prognosis of the lung.     

Matrix Metalloproteinases 2, 7, 9 and Tissue Inhibitor of Matrix Metalloproteinase-1 in the Sera of Patients with Bone Tumors

Comparative enzyme immunoassay of matrix metalloproteinases (MMP-2, -7, -9) and their tissue inhibitor-1 (TIMP-1) in the sera of 26 healthy individuals and 54 patients with primary osteal tumors before therapy revealed elevated TIMP-1 levels in the patients with classical central and periosteal osteosarcomas in comparison with the control. In patients, the level of MMP-9 signifi cantly decreased compared to that in healthy individuals, while the levels of MMP-2 and MMP-7 remained unchanged. No differences in serum levels of MMP and TIMP-1 associated with gender, age, primary osteal tumor location and size were detected. Overall 3-year survival of patients with classical central osteosarcoma with serum level of MMP-9 below its median was higher than that of patients with MMP-9 level equal to above the median (90.9±8.7 and 50.8±23%, respectively).     

Matrix metalloproteinase (MMP)-2 and MMP-9 activities in human seminal plasma.

We report on the existence of two kinds of matrix metalloproteinases (MMPs), MMP-2 and MMP-9, in human seminal plasma. Partial purification of the proteinases was achieved by two steps, consisting of chromatography on a gel-filtration column and then on a gelatin affinity column. Proteinase activities in the chromatography extracts were shown to hydrolyse a fluorescent substrate specific to MMPs (Dnp-Pro-Leu-Gly-Leu-Trp-Ala-D-Arg-NH2). The proteinases were detected using gelatin-zymography, but were not detected using casein-zymography, and were also inhibited by EDTA, EGTA and o-phenanthroline. Molecular weights of the proteinases were determined by SDS-PAGE, gelatin-zymography and Western blot to be approximately 92, 84, 72, 67, 52 and 45 kDa. Gelatin-zymography showed three major bands of activity at 72, 67 and 52 kDa and minor bands at 92, 84 and 45 kDa. Apart from the two smallest bands, these proteinases were all recognized by the polyclonal antibodies for MMP-2 or MMP-9. These results indicate that two kinds of pro-form and active-form matrix metalloproteinases, MMP-2 and MMP-9, and their degradation products, are present in human seminal plasma.     

Relationship between the Expression of VEGF Signal Components and Matrix Metalloproteinases in Ovarian Tumors

The content of vascular endothelium growth factor is significantly increased, while the level of matrix metalloproteinase-2 is 2-fold reduced in ovarian cancer tissue compared to benign tumors. A trend to an increase in the levels of matrix metalloproteinases 7 and 9 and reduction of vascular endothelial growth factor type 2 receptors in tumor tissue was also detected. A highly significant negative correlation between the levels of vascular endothelial growth factor and matrix metalloproteinase 2 and positive correlations between vascular endothelial growth factor and matrix metalloproteinase 7, vascular endothelial growth factor and matrix metalloproteinase 9, matrix metalloproteinase 2 and vascular endothelial growth factor type 2 receptors were revealed. In the tumors assayed after preoperative therapy, relative normalization of the studied parameters was observed: the level of vascular endothelial growth factor decreased significantly, while the levels of matrix metalloproteinase 2 and vascular endothelial growth factor type 2 receptors increased. The levels of the markers differed significantly in ovarian tumors of different histological types, and the levels of vascular endothelial growth factor type 2 receptors were higher in patients with stage III compared to stage I and the content of matrix metalloproteinase 7 was higher in stage III compared to stage II cancer.     

Expression of MT-1 MMP, MMP2, MMP9 and TIMP2 mRNAs in ductal carcinoma in situ and invasive ductal carcinoma of the breast

We investigated the expression of membrane type-1 (MT1)-MMP, MMP2, MMP9 and TIMP2 mRNAs and their roles in ductal carcinoma in situ (DCIS) and T1 and T2 invasive ductal carcinoma of the breast. We further compared these two types of carcinomas for differences in microvessel density, and expression of angiogenic factors and CD44std. MT1-MMP, MMP2, MMP9 and TIMP2 mRNA were expressed in both DCIS and invasive ductal carcinomas. Expression rates of MT1-MMP, MMP2, MMP9 and TIMP2 mRNAs were not statistically different between DCIS and invasive ductal carcinomas, nor did they differ statistically when grouped by tumor size, histologic grade or nuclear grade of invasive ductal carcinoma. Microvessel density and expression of VEGF and TGF-beta1 were not statistically different between DCIS and invasive ductal carcinoma. CD44std expression was significantly increased in DCIS compared to invasive ductal carcinoma (p < 0.05) and it was also significantly increased in lower clinical stage, histologic grade and nuclear grade of invasive ductal carcinoma (p < 0.05). Axillary node metastasis was significantly correlated with MT1-MMP mRNA, VEGF and TGF-beta1 expression (p < 0.05) and MT1-MMP mRNA was positively correlated with VEGF expression and TIMP2 mRNA (p < 0.05). In summary, patterns of MMP mRNA expression in DCIS and invasive ductal carcinoma suggest that the invasive potential of breast carcinoma is already achieved before morphologically overt invasive growth is observed. As MT1-MMP mRNA expression is significantly correlated with axillary nodal metastasis, it may be useful as a prognostic indicator of invasive ductal carcinoma. Considering the positive correlation of MT1-MMP mRNA and TIMP2mRNA expression, our finding supports a role for TIMP2 in tumor growth, as well as the utility of CD44std as a prognostic indicator of breast cancer.     

Association between matrix metalloproteinase (MMP)-2 polymorphisms and MMP-2 levels in hypertensive disorders of pregnancy

We examined whether two functional polymorphisms (g.-1306C>T and g.-735C>T) in matrix metalloproteinase (MMP)-2 gene are associated with preeclampsia (PE) or gestational hypertension (GH), and whether they modify MMP-2 or tissue inhibitor of metalloproteinase (TIMP)-2 plasma concentrations in these hypertensive disorders of pregnancy. We studied 130 healthy pregnant (HP), 130 pregnant with GH, and 133 pregnant with PE. Genomic DNA was extracted from whole blood and genotypes for g.-1306C>T and g.-735C>T polymorphisms were determined by Real Time-PCR, using Taqman allele discrimination assays. Haplotypes were inferred using the PHASE program. Plasma MMP-2 and TIMP-2 concentrations were measured by ELISA. The main findings were that pregnant with PE have higher plasma MMP-2 and TIMP-2 concentrations than HP (P<0.05), although the MMP-2/TIMP-2 ratios were similar (P>0.05). Moreover, pregnant with GH have elevated plasma MMP-2 levels and MMP-2/TIMP-2 ratios compared to HP (P<0.05). While MMP-2 genotypes and haplotypes are not linked with hypertensive disorders of pregnancy, MMP-2 genotypes and haplotypes are associated with significant alterations in plasma MMP-2 and TIMP-2 concentrations in preeclampsia (P<0.05). Our findings may help to understand the relevance of MMP-2 and its genetic polymorphisms to the pathophysiology of hypertensive disorders of pregnancy. It is possible that patients with PE and the MMP-2 haplotype combining the C and T alleles for the g.-1306C>T and g.-735C>T polymorphisms may benefit from the use of MMPs inhibitors such as doxycycline. However, this possibility remains to be determined.     

Expression of MMP2, MMP9, MT1-MMP, TIMP1, and TIMP2 mRNA in valvular lesions of the heart

Matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) play an important role in several diseases. This study was undertaken to investigate the mRNA synthesis of MMP2, MMP9, membrane-type 1 (MT1)-MMP, and matrix metalloproteinase inhibitors TIMP1 and TIMP2 by in situ hybridization in a set of heart mitral and aortic valves operatively removed due to degenerative or inflammatory valvular diseases. The material consisted of 21 valves, eight with endocarditis and 13 with a degenerative valvular disease. The samples were studied by in situ hybridization with specific probes for MMP2, MMP9, MT1-MMP, TIMP1, and TIMP2. Synthesis of MMP2 mRNA was found in seven valves, five with endocarditis and two with degenerative valvular disease. Signals for MMP9 mRNA were found in two cases with endocarditis and five cases with degenerative valvular disease. No signal for MT1-MMP mRNA was found in the lesions. TIMP1 mRNA, on the other hand, was found in 17 cases, both endocarditis and degenerative valvular disease. TIMP2 mRNA was found in three cases of endocarditis. The signals for MMP2, MMP9, TIMP1, and TIMP2 mRNA were localized in endothelial cells and in fibroblast-like cells expressing alpha-smooth muscle actin, thus showing myofibroblast-type differentiation. The results show that matrix metalloproteinases MMP2 and MMP9, and matrix metalloproteinase inhibitors TIMP1 and TIMP2 mRNAs are synthesized in diseased valves and suggest that they may contribute to matrix remodelling in valvular disease.     

肥厚性瘢痕胶原酶和TIMP-1mRNA表达的研究

采用原位杂交技术和图像分析技术观测了ＨＴＳ中ＭＭＰ－１和ＴＩＭＰ－１的基因表达。结果发现ＭＭＰ－１和ＴＩＭＰ－１的表达较正常增加，平均积分光密度值分别增加了１．８（Ｐ＞０．０５）和７倍（Ｐ＜０．００１）。ＨＴＳ中ＴＩＭＰ－１的表达明显高于ＭＭＰ－１（Ｐ＜０．００１）。ＭＭＰ－１和ＴＩＭＰ－１阳性颗粒多分布于胶原结节，这种表达的差异性提示ＨＴＳ胶原酶活性可能受到ＴＩＭＰ－１的抑制，ＴＩＭＰ－１的表达异常可能同ＨＴＳ的形成密切相关     

Expression of MMP2, MMP9 and MMP3 in Breast Cancer Brain Metastasis in a Rat Model

In order to study the expression of MMP2, MMP3 and MMP9 in breast cancer brain metastasis, we used a syngeneic rat model of distant metastasis of ENU1564, a carcinogen-induced mammary adenocarcinoma cell line. At six weeks post inoculation we observed development of micro-metastasis in the brain. Immunohistochemistry and Western Blotting analyses showed that MMP-2, -3 and -9 proteins expressions are consistently significantly higher in neoplastic brain tissue compared to normal brain tissue. These results were confirmed by RT-PCR. In situ zymography revealed gelatinase activity within the brain metastasis. Gel zymography showed increase in MMP2 and MMP3 activity in brain metastasis. Furthermore, we were able to significantly decrease the development of breast cancer brain metastasis in animals by treatment with PD 166793, a selective synthetic MMP inhibitor. In addition, PD 166793 decreased the in vitro invasive cell behavior of ENU1546. Together our results suggest that MMP-2, -3 and -9 may be involved in the process of metastasis of breast cancer to the brain.     

Assessment of Potential Cross-Reactivity of Human Endogenous Matrix Metalloproteinases with Collagenase Clostridium histolyticum Antibodies in Human Sera Obtained from Patients with Dupuytren's Contracture

Collagenase Clostridium histolyticum (CCH) contains a fixed ratio of class I (AUX-I) and class II (AUX-II) collagenases and is used as treatment for Dupuytren''s contracture. These two Zn-dependent enzymes, produced by the Gram-positive bacterium Clostridium histolyticum, are related functionally to matrix metalloproteinases (MMPs) which, among other functions, degrade the extracellular matrix. Since AUX-I and AUX-II exhibit sequence similarities to human MMPs, we assessed MMP-1 (interstitial collagenase), MMP-2 (gelatinase A), MMP-3 (stromelysin 1), MMP-8 (collagenase 2), and MMP-13 (collagenase 3) for cross-reactivity with anti-AUX-I and anti-AUX-II antibodies in patient serum. Serum samples from 71 subjects enrolled in a long-term clinical study (58 males and 13 females; 63 ± 10 years old [mean ± standard error]) were evaluated for cross-reactivity with the five MMPs using the two validated enzyme-linked immunosorbent assays (ELISAs). Inhibition cutoff points for anti-AUX-I and anti-AUX-II antibodies were based on assay inhibition obtained with a nonspecific protein, bovine gamma globulin, which was tested for each clinical sample. No MMP cross-reactivity was found for any of the 71 clinical antibody-positive sera evaluated. Sequence identity assessments indicated minimal, nonmeaningful alignments of the MMPs and AUX-I/AUX-II. Furthermore, clinical adverse event assessments indicated no safety signals related to MMP inhibition. The bioanalytical results, sequence identity, and clinical assessments consistently did not demonstrate cross-reactivity between CCH antidrug antibodies and endogenous human matrix metalloproteinases. The results presented here suggest that treatment of Dupuytren''s contracture patients with CCH does not lead to any clinical adverse events associated with MMP inhibition.     

基质金属蛋白酶1基因多态性与肺癌易感性的关联研究

目的研究我国西北汉族人群基质金属蛋白酶（matrix metalloproteinase 1，MMP1）基因-1607（1G→2G）多态与肺癌发生风险的关系。方法应用聚合酶链反应-限制性片段长度多态性分析的方法，检测了150例肺癌患者和200名正常对照者删1G→2G多态的基因型，比较不同基因型与肺癌发生风险的关系。结果肺癌组2G／2G基因型频率要高于对照组（X^2=5．896，P〈0．05），2G／2G基因型者患肺癌的风险是1G／2G和1G／1G基因型的1．77倍（OR=1．77；95％CI：1．12—2．91）。吸烟者中2G／2G基因型发生肺癌的风险是1G／2G和1G／1G基因型的3．20倍（OR=3．20；95％CI：1．50～6．82）。结论我国西北汉族人群MMP1基因-1607（1G→2G）多态性与肺癌易感性有关，2G／2G基因型可以增加肺癌发生风险。