Arterial enlargement in response to high flow requires early expression of matrix metalloproteinases to degrade extracellular matrix

This study investigated the effects of high flow and shear stress on the expression of matrix metalloproteinases (MMPs) and tissue inhibitor of metalloproteinase-2 (TIMP-2) during flow-induced arterial enlargement using a model of arteriovenous fistula (AVF) creation on the carotid artery with the corresponding jugular vein in Japanese white male rabbits. Flow increased 8-fold 7 days after AVF. Endothelial cells (EC) and smooth muscle cells (SMC) proliferated with internal elastic lamina (IEL) degradation in response to high flow and shear stress. Expression of MMP-2 mRNA peaked at 2 days (1700-fold) and maintained high level expression. MMP-9 mRNA gave a 10.8-fold increase within 2 days and decreased later. Their proteins were detected in EC and SMC. Membrane type-1-MMP (MT1-MMP) mRNA increased 121-fold at 3 days and maintained high expression. TGF-beta1 was increased after AVF. Two-peak up-regulation of Egr-1 mRNA was recognized at 1 and 5 days of AVF. These results suggest that high flow and shear stress can mediate EC and SMC to express MMP-2 and MMP-9, which degrade cell basement membranes and IEL to induce arterial enlargement. The disproportional increase in MT1-MMP and TIMP-2 might contribute to MMP-2 activation. Egr-1 and TGF-beta1 might play important roles in this process.     

Immunolocalization of membrane-type 1 MMP in human rheumatoid synovium tissues

Membrane-type 1 matrix metalloproteinase (MT1-MMP, also known as MMP14), the best characterized membrane-anchored MMP, is an important matrix-degrading proteinase that could digest a broad spectrum of extracellular matrix proteins and accelerate angiogenesis. We have previously reported that some MMPs involved in the angiogenesis and the pannus formation within the joint, leading to the erosion of articular cartilage and bone in the pathological process of rheumatoid arthritis (RA). In the present study, we used immunohistochemistry assay and con-focal scanning technique to study the detailed immunolocalization of MT1-MMP in human RA synovium tissues as well as the infiltrating immune cell subsets. Our results showed that the positive MT1-MMP immunostaining could be found in synoviocytes, vascular endothelial cells, infiltrating macrophages and monocytes in RA synovium tissues, while weak or negative immunostaining could be found in infiltrating T cells, B cells and NK cells, respectively. Moreover, the Ki-67⁺ highly proliferating synoviocytes also showed higher MT1-MMP expression in RA synoviocytes. Thus, the aberrant expression of MT1-MMP in RA synoviocytes as well as infiltrating immune cells may contribute to the proliferation of the synoviocytes, and the angiogenesis and the pannus formation in RA pathological progression.     

Differential Expression and Origin of Membrane-Type 1 and 2 Matrix Metalloproteinases (MT-MMPs) in Association with MMP2 Activation in Injured Human Livers

Matrix metalloproteinase-2 (MMP2) activation is associated with basement membrane remodeling that occurs in injured tissues and during tumor invasion. The newly described membrane-type MMPs (MT-MMPs) form a family of potential MMP2 activators. We investigated the localization and steady-state levels of MT1-MMP and MT2-MMP mRNA, compared with those of MMP2 and tissue inhibitor of MMP-2 in 22 hepatocellular carcinomas, 12 liver metastases from colonic adenocarcinomas, 13 nontumoral samples from livers with metastases, 10 benign tumors, and 6 normal livers. MMP2 activation was analyzed by zymography in the same series. The expression of MT1-MMP mRNA and the activation of MMP-2 were increased in hepatocellular carcinomas, metastases, and cholestatic nontumoral samples. MT2-MMP mRNA was rather stable in the different groups. MT1-MMP mRNA levels, but not MT2-MMP mRNA, correlated with MMP-2 and tissue inhibitor of MMP-2 mRNA levels and with MMP2 activation. In situ hybridization showed that MT1-MMP mRNA was expressed in stromal cells, and MT2-MMP mRNA was principally located in both hepatocytes and biliary epithelial cells. Consistently, freshly isolated hepatocytes expressed only MT2-MMP mRNA, and culture-activated hepatic stellate cells showed high levels of MT1-MMP mRNA. These results indicate that in injured livers, MMP2 activation is related to a coordinated high expression of MMP2, tissue inhibitor of MMP-2, and MT1-MMP. Furthermore, the finding of a preferential expression of MT2-MMP in hepatocytes, together with our previous demonstration that the activation of stellate cell-derived MMP2 in co-culture requires interactions with hepatocytes (Am J Pathol 1997, 150:51–58), suggests that parenchymal cells might play a pivotal role in the MMP2 activation process.     

Evaluation of stromal metalloproteinases and vascular endothelial growth factors in a spontaneous metastasis model

This study aims to investigate MMP2 and MT1-MMP protein as well as VEGF-C and VEGF-D mRNA expression in tumor cells and distant organs considered to be targets for metastasis in a tumor spontaneous metastasis model previously described. Cultured tumor cells, able to express pro-MMP2, MMP2, pro-MMP9, and MT1-MMP, develop tumor growth and metastasis, mainly in the liver and spleen, when they are injected in the mammary pad gland of Wistar rats. Immunohistochemical studies of tumor masses showed small groups of tumor cells staining for MT1-MMP but not for MMP2. In the liver, tumor metastatic foci and a stromal positive staining for both MMP2 and MT1-MMP were shown. The spleen and lymph nodes, with only scattered metastatic cells, did not show MMPs immunostaining. Using RT-PCR, a significantly higher VEGF-C and VEGF-D gene expression was shown in the liver of tumor-bearing rats respect to normal rats, whereas spleen and lymph nodes did not show significant differences in mRNA VEGF-C/D levels. Taken together, our results suggest that the stroma microenvironment of target organs for metastasis has the ability to produce MMPs and VEGFs that facilitate the anchorage of tumor cells and promote tumor cell growth and angiogenesis.     

Up-regulation of matrix metalloproteinase-2 and membrane-type 1-matrix metalloproteinase were coupled with that of type I procollagen in granulation tissue response after the onset of aortic dissection

The pathophysiological significance of matrix metalloproteinases (MMPs) in aortic dissection remains poorly understood. The purpose of the present study is to clarify the significance of MMPs in aortic dissection. The activities and distributions of MMP-2, membrane-type 1-MMP (MT1-MMP), and MMP-9 were evaluated by gelatin zymography, immunohistochemistry, and in situ hybridization in 29 patients and seven autopsy cases. To assess if these MMPs are related to a tissue remodeling process, we compared the expression of these MMPs with that of type I procollagen and platelet-derived growth factor receptor β chain (PDGF Rβ). Patients were divided into three groups based on histological findings: acute, intermediate, and healed groups. The most remarkable changes were observed in the intermediate group, in which MMP-2 activity peaked and tissue expression of mRNAs for MMP-2 and MT1-MMP were observed in spindle-shaped cells in the neointima, organizing thrombus, and the adventitia. These expression patterns were essentially coupled with those of type I procollagen mRNA and PDGF-Rβ protein. The association of MMP-2, MT1-MMP, type I procollagen, and PDGF-Rβ suggests that MMP-2 and MT1-MMP could be involved not only in the degradation of aortic tissue but also in tissue remodeling, which may be associated with the healing process.     

Rapid trafficking of membrane type 1-matrix metalloproteinase to the cell surface regulates progelatinase a activation

Pericellular matrix degradation during cancer invasion and inflammation is dependent on activation of progelatinase A by membrane type 1-matrix metalloproteinase (MT1-MMP); a stoichiometric concentration of tissue inhibitor of metalloproteinase-2 (TIMP-2) is required. Activation of progelatinase A has generally been considered to be a slow process occurring as a result of enhanced expression of MT1-MMP. We herein report that ConA treatment of HT1080 fibrosarcoma cells is followed by MT1-MMP-induced activation of progelatinase A on the cell surface within 1 hour. Cell surface biotinylation, immunohistochemistry, and (125)I-labeled TIMP-2 binding to cell surface MT1-MMP were used to characterize the appearance and function of MT1-MMP on the plasma membrane. Treatment of HT1080 cells with ConA resulted in increased specific binding of (125)I-labeled TIMP-2 to cell surface receptors within 5 minutes. TIMP-2 binds almost exclusively to activated MT1-MMP on the surface of HT1080 cells. MT1-MMP function at the cell surface was also accelerated by treatment of cells with cytochalasin D, an inhibitor of actin filaments, PMA, a stimulator of protein kinase C, and bafilomycin A(1), an inhibitor of lysosome/endosome function. A functional pool of intracellular MT1-MMP available for trafficking to the cell surface was demonstrated by repetitive ConA stimulation. ConA-induced expression of MT1-MMP mRNA (Northern blot analysis) in HT1080 cells was a delayed event (>6 hours). These data suggest that presynthesized MT1-MMP is sorted to a transient storage compartment (trans-Golgi network/endosomes), where it is available for rapid trafficking to the plasma membrane and cell surface proteolytic activity.     

MT2-MMP expression associates with tumor progression and angiogenesis in human lung cancer

Matrix metalloproteinases (MMPs) are a family of important proteolytic enzymes that play an important role in the remodeling of the tumor microenvironment and associate with tumorigenesis and metastasis. We previously reported that membrane type-2 MMP (MT2-MMP) is highly expressed in human esophageal cancer tissues, and its expression level is positively correlated to tumor size and intratumoral angiogenesis. In order to reveal whether MT2-MMP expression is operative in human lung cancer and its underlying physio-pathological role, in the present study, we examined both mRNA and protein expression levels of MT2-MMP in non-small cell lung caner (NSCLC) tissues and in adjacent normal tissues by using real-time RT-PCR and immunohistochemistry respectively, which showed that both MT2-MMP mRNA (P=0.0359) and protein (P<0.0001) expression levels were significantly increased in cancer tissues in contrast to adjacent normal tissues. Moreover, we also found that the MT2-MMP protein level in cancer tissues positively correlated to lymph node metastasis (P=0.0483), tumor stage (P=0.0483), intra-tumoral microvessel density (MVD) (P=0.0445). We have not found statistically significant correlation between MT2-MMP expression and patients’ prognoses, but we found that the patients with both higher MT2-MMP protein expression and higher intra-tumoral microvessel density showed better prognoses than that of the patients with either higher MT2-MMP protein expression or higher intra-tumoral microvessel density (P=0.0311). Thus, our data suggest that MT2-MMP expression positively involves in NSCLC, and might play an important role in promoting the tumor progression and intra-tumoral angiogenesis in NSCLC.     

Thrombin rapidly and efficiently activates gelatinase A in human microvascular endothelial cells via a mechanism independent of active MT1 matrix metalloproteinase

Thrombin has been shown previously to activate gelatinase A in human umbilical vein endothelial cells. The activation is thought to be mediated by membrane-type 1 matrix metalloproteinase (MT1-MMP) on the cell surface, which generates the 62-kd intermediate and the 59-kd fully active forms. We used microvascular endothelial cells derived from human neonatal foreskin to investigate the mechanism of gelatinase A activation by thrombin. Gelatinase A was measured using zymography. Whereas activation by PMA generated both the 62-kd intermediate and the 59-kd fully active forms of gelatinase A after 24 hours, activation by thrombin produced only the 59-kd species rapidly (within 2 hours). Four findings indicate that MT1-MMP was not involved in thrombin-induced activation: (1) there was no up-regulation of MT1-MMP after 2 hours stimulation by thrombin, even though there was activation of gelatinase A; (2) the 62-kd intermediate species was never detected in response to thrombin; (3) tissue inhibitor of matrix metalloproteinase-2 completely prevented gelatinase A activation induced by PMA but not by thrombin; and (4) the metalloproteinase inhibitor 1,10-phenanthroline did not inhibit thrombin-induced activation. Together, these data demonstrate that activation of gelatinase A by thrombin is different from PMA and operating via a pathway independent of MT1-MMP. The ability of thrombin to rapidly and efficiently activate gelatinase A is likely to be a major contributing factor to its potent angiogenic activity.     

流体剪切应力对大鼠骨髓间充质干细胞MT1-MMP基因表达的影响

目的探讨流体剪切应力对大鼠骨髓间充质干细胞(ratbonemarrow-derived mesenchy malstem cells,rMSCs)中膜型基质金属蛋白酶1(Membrane type-1 matrix metalloproteinase,MT1-MMP)基因表达的影响,为阐明应力诱导rMSCs分化的分子机制提供一些实验依据。方法应用平行平板流动腔系统,给rMSCs施加不同大小和不同加载时间的层流剪切应力,用real-time PCR检测MT1-MMP基因mRNA的表达水平。结果 5,15,30 dynes/cm2剪切应力均能刺激rMSCs的MT1-MMP mRNA表达,且其作用呈时间依赖和强度依赖。p38抑制剂(SB202190)可以明显抑制这种作用,而PI3K抑制剂(wortmannin)却增强了这种效果。结论流体剪切应力可诱导rMSCs的MT1-MMP基因表达,其表达量与刺激时间和剪切应力的强度密切相关,这种作用可能通过p38MAPK和PI3K/Akt信号通路。     

Co-ordinated expression of MMP-2 and its putative activator, MT1-MMP, in human placentation

The spatial expression of mRNA for matrix metalloproteinase 2 (MMP-2), its putative activator, the membrane-type 1 matrix metalloproteinase (MT1-MMP), and the MMP-2 substrate type IV collagen was investigated in human placentas of both normal and tubal ectopic pregnancies and in cyclic endometrium using in-situ hybridization. Cytokeratin staining applied to adjacent sections was used to identify epithelial and trophoblast cells. In both normal and tubal pregnancies MT1-MMP, MMP-2 and type IV collagen mRNA were highly expressed and co-localized in the extravillous cytotrophoblasts of anchoring villi, in cytotrophoblasts that had penatrated into the placental bed and in cytotrophoblastic cell islands. In addition, the decidual cells of normal pregnancies in some areas co-expressed MT1-MMP and MMP-2 mRNA, with moderate signals for both components. Fibroblast-like stromal cells in tubal pregnancies were positive for MMP-2 mRNA but generally negative for MT1-MMP mRNA. The consistent co-localization of MT1-MMP with MMP-2 and type IV collagen in the same subset of cytotrophoblasts strongly suggests that all three components co-operate in the tightly regulated fetal invasion process. The co-expression of MT1-MMP and MMP-2 mRNA in some of the decidual cells indicates that these cells are also actively involved in the placentation process.      

Localization of MT1-MMP, TIMP-1, TIMP-2, and TIMP-3 messenger RNA in normal, hyperplastic, and neoplastic endometrium. Enhanced expression by endometrial adenocarcinomas is associated with low differentiation

We studied membrane-type matrix metalloproteinase-1 (MT1-MMP), tissue inhibitor of metalloproteinase-1 (TIMP-1), TIMP-2, and TIMP-3 messenger RNA (mRNA) using in situ hybridization to elucidate their temporal and spatial expression patterns in normal, hyperplastic, and neoplastic endometrium. All mRNAs studied were expressed weakly in proliferating endometrium but were induced strongly in late secretory endometrium except MT1-MMP. Endometrial hyperplasia samples did not show increased MT1-MMP or TIMP mRNA expression, indicating that the overall expression patterns in hyperplasia are comparable to those in proliferating endometrium under estrogen effect and that synthesis of extracellular matrix proteins, rather than degradation, predominates in this condition. Exceptionally, stromal cells in areas of desquamation were seen to express focally intense MT1-MMP mRNA in hyperplasia samples. All mRNAs investigated were expressed increasingly in endometrial adenocarcinomas, especially in less differentiated carcinomas. Furthermore, gelatin zymography revealed higher functional degradative activities in carcinoma tissues than in normal endometrium. Our results indicate that MT1-MMP expression, together with that of TIMPs, is involved most notably in normal endometrium under progesterone effect and, without being connected to cyclic hormonal levels, has an important role in the invasive growth of endometrial adenocarcinomas.     

38Dimerization of endogenous MT1-MMP is a regulatory step in the activation of the 72 kDa gelatinase, MMP-2, on fibroblasts and fibrosarcoma cells

Matrix metalloproteases (MMPs) are centrally engaged in the processes of extracellular matrix turnover that occur during cancer invasion. An important MMP cascade reaction is initiated by the membrane-anchored matrix metalloprotease, MT1-MMP, which serves to activate the proenzyme form of the secreted gelatinase, matrix metalloprotease-2 (MMP-2). This reaction occurs in an interplay with the matrix metalloprotease inhibitor, TIMP-2, and the proposed mechanism involves two molecules of MT1-MMP in complex with one TIMP-2 molecule. To study this, as well as other roles of MT1-MMP, we have now raised a panel of monoclonal antibodies against the protein. These antibodies have been raised in MT1-MMP knock-out mice and react against conserved epitopes in murine and human MT1-MMP. Using one of these antibodies we provide positive evidence that proMMP-2 activation is governed by dimerization of MT1-MMP on the surface of fibroblasts and fibrosarcoma cells. The antibody in question binds specifically to MT1-MMP on the cell surface, as shown by immunofluorescence experiments. It is directed against the hemopexin domain of MT1-MMP and has no effect on the catalytic activity of the protease domain. The antibody induces dimerization of the endogenous MT1-MMP on the cell surface. Through this reaction, it markedly stimulates the formation of the 62 kDa active MMP-2 and the processing into a 59 kDa product that retains gelatinolytic activity. This effect is indeed a consequence of MT1-MMP dimerization because it requires the divalent monoclonal antibody with no effect being obtained with monovalent Fab fragments. Since only a negligible level of proMMP-2 activation is obtained with MT1-MMP expressing cells in the absence of dimerization, our results identify the dimerization event as a critical level of proteolytic cascade regulation.     

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.     

基质金属蛋白酶及其组织抑制因子在人前列腺组织及各种类型前列腺细胞中的表达

目的：研究基质金属蛋白酶（MMPs）及其组织抑制因子（TIMPs）在人前列腺组织及各种类型细胞中的表达。方法： 用半定量RT－PCR的方法，对癌变和非癌变部分的前列腺组织、原代培养的平滑肌细胞、成纤维细胞、上皮细胞以及4种前列腺上皮细胞系（BPH－1、LNCaP、DU－145和PC－3）中MMP2、MMP7和MMP9、膜型基质金属蛋白酶1和3（MT1－MMP和MT3－MMP）及其组织抑制因子1和2（TIMP－1和TIMP－2）的mRNA 水平进行了测定。结果：MMP-2主要在前列腺基质细胞中表达；MMP-7和MMP-9则在前列腺上皮细胞中有较高的表达；MT1-MMP、MT3-MMP、TIMP-1和TIMP-2在前列腺基质细胞和上皮细胞中均有表达，但MT1-MMP和MT3-MMP在成纤维细胞中的表达量较高；另外，各种基质金属蛋白酶及其组织抑制因子在各种前列腺细胞系中也存在差异表达。结论： MMPs和TIMPs在前列腺组织及其各种类型细胞中的差异表达提示：它们可能在前列腺癌的转移中起着不同的作用。     

Induction of membrane-type matrix metalloproteinase 1 (MT1-MMP) expression in human fibroblasts by breast adenocarcinoma cells

Membrane-type matrix metalloproteinase 1 (MT1-MMP) has been recently described as an activator of proMMP-2 (MMP-2) which is involved in tumor invasion. We have shown by in situ hybridization that MT1-MMP is produced by stromal cells in close contact to preinvasive and invasive tumor cells of breast carcinomas. Of particular interest was the observation that some fibroblasts express this enzyme in focal areas in preinvasive lesions, suggesting that particular tumor cells may stimulate fibroblasts to produce MT1-MMP. We have therefore compared the ability of two different breast cancer cell lines, one non-invasive (MCF7) and one invasive (MDA-MB-231) to stimulate MT1-MMP production in human fibroblasts with consequent proMMP-2-activation. The MDA-MB-231 conditioned medium induced MT1-MMP mRNAs in human fibroblasts and a parallel activation of proMMP-2 whereas MCF7 conditioned medium did not have any effect. These results suggest the existence of soluble factor(s) secreted by invasive or some preinvasive breast tumor cells which stimulate fibroblasts to produce and activate MMPs, and emphasize the cooperation between cancer and stromal cells in tumor invasion.