Expression of matrix metalloproteinases and tissue inhibitors of metalloproteinases in human chondrosarcomas

The aim of the present study was to characterise the ability of malignant chondrosarcomas to invade normal bone by analysing their production of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs). For this purpose 12 chondrosarcomas were investigated for the expression of mRNAs for several MMPs and all 4 TIMPs by Northern hybridisation, and for immunohistochemical localisation of the proteins. A characteristic finding of these analyses was increased expression of MMP-13, MMP-14 and TIMP-2 mRNAs in chondrosarcomas when compared with nonmalignant control samples. Individual chondrosarcomas also exhibited elevated levels of MMP-1, MMP-7 and MMP-9 mRNAs. The results of Northern hybridisations were supported by immunohistochemical stainings of the corresponding tumour areas for MMP-2, MMP-14 and TIMP-2, further suggesting that these may have prognostic value for determining whether individual chondrosarcomas are locally aggressive or have a probability of recurrence. Another finding of the present study was a marked heterogeneity in histologic appearance and gene expression of the chondrosarcomas, emphasising the importance of analysing several areas of these tumours to get representative results. These findings suggest that analysis of MMPs could be a useful diagnostic indicator in patients with cartilaginous tumours and could help in differentiating between a low-grade malignant chondrosarcoma and a benign growing enchondroma.     

The matrix metalloproteinases and their inhibitors.

A number of metalloproteinases that degrade the extracellular matrix of connective tissues and two specific tissue inhibitors of metalloproteinases (TIMPs) have now been isolated, characterized, and cloned. Comparison of the enzyme sequences has allowed the delineation of domain structures, and initial studies have been carried out to assess the contribution of these domains to their biochemical and biologic properties, including activation, inhibition by TIMPs, and matrix binding. Such events represent the major levels of extracellular regulation of metalloproteinase activity, which is thought to be an important aspect of their control. Activation is probably a cell surface phenomenon, involving the plasminogen activator cascade or other membrane-associated mechanisms. The inhibitory action of TIMPs is postulated to be as important in activation as in the subsequent regulation of enzyme degradation of the matrix.     

The identification of matrix metalloproteinases and their tissue inhibitors in broiler chickens by immunohistochemistry.

The aim of this study was to investigate the distribution of matrix metalloproteinase (MMP)-2, MMP-9, tissue inhibitor of matrix metalloproteinase (TIMP)-1 and TIMP-2 using immunohistochemistry in the ascites syndrome of broiler chickens in a salt-induced experimental model. The presence of the enzymes in the lung, heart, liver, kidney and brain was evaluated semi-quantitatively with the streptavidin-biotin-peroxidase (Strep-ABC) method using commercially available primary monoclonal antibodies. Immunostaining of MMP-2 and MMP-9 was more intense and extensive in ascitic broilers than in the controls, although a decrease was seen with increasing age both in normal and ascitic chickens. The presence of MMP-9 enzyme was negatively correlated with the presence of TIMP-1 enzyme. It is suggested that MMP-2 and MMP-9 enzymes might play a role in the permeability increase of vessel walls by the destruction of the basement membranes in the salt-induced experimental ascites syndrome in broiler chickens.     

Dysregulation of matrix metalloproteinases and their tissue inhibitors by S100A4

The S100A4 protein as well as the matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) are associated with diseases such as arthritis and cancer. This mini review focuses on in vitro and in vivo studies indicating S100A4 involvement in regulation of MMPs and TIMPs, and the biological and pathobiological consequences of this regulation.     

Involvement of matrix metalloproteinases (MMPs) in cutaneous melanoma progression

Among skin cancers, melanoma is probably the most highly invasive and metastasizing, with a poor outcome. During melanoma progression, tumor cells must across the dermal-epidermal junction, and invade the dermis, its principal site of propagation. Therefore, degradation of matrix proteins constituting dermal-epidermal junction and dermis by proteolytic enzymes is an essential step of melanoma invasion. Serines proteinases and Matrix Metalloproteinases (MMPs) families are the main degrading substances involved in this process. Among MMPs, the expression of MMP-1, -2, -3, -9, -14, 15, -16 by melanoma cells was shown in vitro and in vivo, and correlated with the invasive phenotype. In addition to disrupt matrix proteins, MMPs can also cleave non matrix components such as cytokines, and growth factors. The modifications generated by the remodeling of matrix and non-matrix components can influence melanoma cells proliferation, adhesion, vascularization, survival, proteases expression, and migration. Thus, using inhibitors in order to control expression, activation and activity of MMPs could regulate cellular process which led to melanoma progression.     

Expression of matrix metalloproteinases and their inhibitors in human brain tumors

Sixty human brain tumors, classified according to the New World Health Organization (WHO) classification including, grade I schwannomas, meningiomas and pilocytic astrocytomas, grade II astrocytomas, grade III anaplastic astrocytomas, grade IV glioblastomas, grade III anaplastic oligodendrogliomas and grade IV glioblastomas and lung and melanoma metastases were analyzed for the expression of three matrix metalloproteinases (MMPs), two tissue inhibitors of MMPs (TIMPs) and for MMP activity. Some correlation was found between MMP expression and the degree of malignancy. Western blotting analysis revealed a more uniform pattern of distribution of MMP-2 (gelatinase A) than of MMP-9 (gelatinase B) and MMP-12 (metalloelastase) among tumors. MMP-9 levels were found to be significantly higher in grade III anaplastic astrocytomas and anaplastic oligodendrogliomas than those in grade I schwannomas and meningiomas. Anaplastic astrocytomas and Grade IV glioblastomas expressed significantly higher levels MMP-12 than grade I meningiomas. All sixty tumors showed a similar pattern of activity in zymography, proMMP-9 being the major species detected. Interestingly, TIMP-1 and TIMP-2 expression levels were especially low in tumors of grade II and grade III but significantly higher in tumors of grade I, particularly in schwannomas. Taken together, these data suggest that: 1) a balance between MMPs and TIMPs has an important role to play in human brain tumors; 2) TIMP expression may be valuable markers for tumor malignancy. This revised version was published online in July 2006 with corrections to the Cover Date.     

Expression of matrix metalloproteinases and tissue inhibitors of metalloproteinases define the migratory characteristics of human monocyte-derived dendritic cells

Dendritic cells (DCs) have an essential role in the initiation of immune responses as they deliver antigen/epitope and the appropriate signals to activate naïve T cells and thus start an immune response. In order to fulfil their function, DCs have to patrol different part of the body, thus migrating through the extracellular matrix to sample the local ‘antigenic’ environment. In the present study, we have investigated which enzymes might be involved in this process using the Matrigel trans‐well migration assay, an in vitro model of extracellular matrix migration. In this assay we analysed the migratory ability of interleukin‐4 (IL‐4)/granulocyte macrophage–colony‐stimulating factor (GM‐CSF)‐derived immature DCs as well as mature DCs, induced by tumour necrosis factor‐α (TNF‐α) and modified vaccinia virus Ankara (MVA). The ‘mature’ DCs showed an increased migration through Matrigel, which was significantly inhibited by inhibitors of matrix metalloproteinases (MMP). We also observed that the dominant MMP involved in this process was MMP‐9, and a concomitant decrease of the endogenous tissue inhibitors of metalloproteinases (TIMP)‐1 and TIMP‐2 was also observed. Collectively these data suggest that the balance between MMP/TIMP determines the net migratory capacity of human DCs. Surprisingly, TIMP‐3 was significantly increased in mature DC. Our data thus indicate that MMP and TIMP play a role in the migratory ability of human DCs. Our results also suggest that TIMP‐3 expression might represent a new marker of maturation of human DCs.     

Expression of matrix metalloproteinases and tissue inhibitors of metalloproteinases during normal human pulmonary development

AIMS : Matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) are thought to be involved in lung development because they play an important role in the turnover of the extracellular matrix. Although limited data on MMP and TIMP expression are available from animal studies during prenatal pulmonary development, little is known about their expression during human fetal lung development. The aim of this study was to investigate the expression of MMP-1, -2, -9, TIMP-1, -2 and -3 in human fetal lungs from 9 to 42 weeks of gestation. METHODS AND RESULTS : Forty-five normal human fetal lung samples were analysed by immunohistochemistry. MMP-1, -9, TIMP-1, -2 and -3, but not MMP-2, were expressed in the epithelium at all gestational ages. The endothelium of all vessels and the arterial smooth muscle cells expressed MMP-1, -2, -9, TIMP-2 and -3, but not TIMP-1, at all developmental stages. CONCLUSION : The extensive distribution of MMPs and TIMPs throughout all stages of human lung development suggests that they play a significant role in the remodelling that occurs in the interstitium and epithelial basement membrane during lung development and in pulmonary vascular development. These data will serve as a base line for comparison with neonatal lung pathology, including pulmonary hypertension.     

Characterization of metalloproteinases and tissue inhibitors of metalloproteinases in human plasma.

In this study, we have identified and characterized metalloproteinases and tissue inhibitors of metalloproteinases (TIMPs) in human plasma. Treatment of plasma with trypsin or aminophenylmercuric acetate resulted in activation of latent gelatinolytic activity. Fractionation of plasma by gelatin Sepharose chromatography resulted in the isolation of 72 kDa and 92 kDa gelatinases/type IV collagenases. The 72 kDa gelatinase was purified by gel filtration chromatography. Stromelysin-1 was isolated from plasma by Matrex green A affinity chromatography. Immunoblotting of plasma fractions with antibodies to unique peptide regions of human gelatinases differentiated the 72 kDa gelatinase from the 92 kDa gelatinase. Antibodies to the amino terminal peptides of each enzyme were used to determine that plasma gelatinases circulate as latent proenzymes. Immunoblotting with antibodies directed against human stromelysin identified a 57 kDa stromelysin. TIMP-1 (28 kDa) and TIMP-2 (21 kDa) were also identified by immunoblotting of gelatin Sepharose bound plasma proteins using non-crossreacting antibodies to each protein.     

Coordinate regulation of matrix metalloproteinase-1 and tissue inhibitor of metalloproteinase-1 expression in human vascular smooth muscle cells

The expression of matrix metalloproteinases (MMPs) and their inhibitors (TIMPs) by human vascular smooth muscle cells (SMC) was monitored as a function of the phenotypic modulation in vitro. Cell phenotype was manipulated by varying serum concentration and cell density. Synthetic phenotype was characterized by a minimum expression of the contractile proteins and a maximal proliferation rate. Contractile phenotype was quiescent and expressed a maximal level of contractile proteins. Synthetic cells expressed the highest levels of both MMP-1 and TIMP-1 and displayed maximal collagenolytic activity. No significant change was detected in MMP-2 expression or catalytic activity. Enzyme immunoassays revealed that MMP-1 expression fell by 77+/-2.4-95+/-0.5%, and that of TIMP-1 by 34+/-0.5-59+/-1.9%, as the cells acquired a contractile phenotype. The level of the MMP-1/TIMP-1 complex was similarly reduced by 78+/-2.9-85+/-1.6%. These data demonstrate that the expression of MMP-1 and TIMP-1 are coordinately regulated with SMC phenotype.     

Matrix metalloproteinases, tissue inhibitors of MMPs and TACE in experimental cerebral malaria

Cerebral malaria (CM) is a life-threatening disorder and a major medical problem in developing countries. It is caused by the sequestration of malaria-infected erythrocytes onto brain endothelia, followed by blood-brain barrier (BBB) damage and neurological deficit. In the present study, matrix metalloproteinases (MMPs) were analysed in a mouse model of CM with Plasmodium berghei ANKA. Increased numbers of gelatinase B (MMP-9)-positive cells, which were also CD11b(+), were detected in the brain. In addition, activation of gelatinase B occurred in CM brains, and not in brains of mice with non-CM. However, selective genetic knockout of gelatinase B did not alter the clinical evolution of experimental CM. To study other protease balances, the mRNA expression levels of nine matrix metalloproteinases (MMPs), five membrane-type MMPs, TNF-alpha converting enzyme (TACE) and the four tissue inhibitors of metalloproteinases (TIMPs) were analysed during CM in different organs. Significant alterations in expression were observed, including increases of the mRNAs of MMP-3, -8, -13 and -14 in the spleen, MMP-8, -12, -13 and -14 in the liver and MMP-8 and -13 in the brain. Net gelatinolytic activity, independent of gelatinase B and inhibitable with EDTA, was detected in situ in the endothelia of blood vessels in CM brains, but not in brains of mice with non-CM, suggesting that metalloproteases, different from gelatinase B, are active in the BBB environment in CM. The increase in MMP expression in the brain was significantly less pronounced after infection of C57Bl/6 mice with the noncerebral strain P. berghei NK65, but it was similar in CM-susceptible C57Bl/6 and CM-resistant Balb/C mice upon infection with P. berghei ANKA. Furthermore, in comparison with C57Bl/6 mice, a larger increase in TIMP-1 and a marked, >30-fold induction in MMP-3 were found in the brains of Balb/C mice, suggesting possible protective roles for TIMP-1 and MMP-3.     

基质金属蛋白酶及其组织抑制因子在皮肤增生性瘢痕中的作用

目的探讨基质金属蛋白酶（MMPs）MMP2、MMP9及其抑制物（TIMPs）TIMP-1在皮肤增生性瘢痕中的作用。方法取3-6个月、6-12个月皮肤增生性瘢痕，以正常皮肤组织为对照，ELISA方法测定其MMP2、MMP9以及TIMP-1的含量，采用SPSS统计软件，以成组设计的单因素方差分析，分析它们在不同时段瘢痕组织中变化的意义。结果（1）3-6月瘢痕组织和6-12月瘢痕组织的MMP2，均较正常皮肤显著增高（110.70±5.23ng／ml VS 54.59±3.01，P〈0.01；77.23±7.10ng／ml VS 54.59±3.01，P〈0.01），而3-6月瘢痕组织的MMP2较6-12月瘢痕组织的MMP2亦有显著增高（110.70±5.23 VS 77．23±7．10，P〈0．01）；3-6月瘢痕组织和6-12月瘢痕组织的MMP9之间（3.85±0.88 VS 3.61±0.43，P〉0.05）以及它们与正常皮肤组织的MMP9相比（3.85±0.88 VS 4.13±0.33，P〉0.05；3.61±0.43 VS 4.13±0.33，P〉0.05）均无显著性差异；（2）3-6月瘢痕组织和6-12月瘢痕组织的TIMP-1与正常皮肤组织相比有显著增高（4．74±0．35 VS 3．01±0．11，P〈0．01；5．12±0．34 VS 3.01±0．11，P〈0．01），6-12月瘢痕组织较3-6月瘢痕组织的TIMP-1有显著增高（5.12±0.34 VS 4.74±0．35，P〈0．05）；（3）3-6月瘢痕组织和6-12月瘢痕组织的MMP2与TIMP-1的比值与正常皮肤组织相比均有显著增高（23．38±1．01 VS 18．15±0．58，P〈0.01；15.10±1.45 VS 18．15±0．58，P〈0．01），3-6月瘢痕组织较6-12月瘢痕组织的MMP2与TIMP-1的比值有显著降低（23.38±1.01 VS 15.10±1.45，P〈0.01）。结论MMPs以及TIMPs参与了皮肤瘢痕的增生过程，MMP2、TIMP-1含量及其比值可作为瘢痕生长和预后的指标。     

Regulation of matrix metalloproteinases in human endometrium

Considerable evidence supports a role for matrix metalloproteinases (MMP) in menstruation, but their focal pattern of expression within perimenstrual and menstrual endometrium suggests local rather than hormonal regulation. Menstruation shares a number of features with inflammatory responses, with leukocyte infiltration, proliferation and activation, occurring in the endometrium prior to menstruation. We propose that the leukocytes release MMP at this time and also that interactions between leukocytes and the stromal and epithelial cells of the endometrium induce and activate MMP. Co-culture studies using mast cells or neutrophils with endometrial stromal cells support this hypothesis. How leukocytes enter the endometrium is not understood but a role for chemokines has been proposed. The expression patterns of eotaxin and its receptor CCR3 in endometrium support a role in chemoattraction of eosinophils but expression of monocyte chemotactic proteins 1 and 2 does not correlate with macrophage numbers. Nothing is known of how the leukocytes become activated. Nevertheless, the overall result is a tissue in which an inflammatory-type reaction occurs with release of a myriad of potent regulators. These induce production and activation of MMP and alter the ratio between these and their tissue inhibitors, resulting in tissue breakdown.     

Matrix metalloproteinases and tissue inhibitors of metalloproteinases in human fetal testis and ovary.

Matrix metalloproteinases (MMP) and tissue inhibitors of metalloproteinases (TIMP) are major regulators of tissue remodelling of the extracellular matrix (ECM) and may also be involved in the control of growth factor availability. We have investigated their production and localization in the developing human gonad during mid-gestation using zymographic techniques and immunohistochemistry. The secretion of MMP-2, MMP-9 and all four TIMP was demonstrated from both testis and ovary, with the predominant gelatinase produced by both being MMP-2. In the testis, MMP-1, MMP-2, MMP-9 and all TIMP family members were localized to the interstitium and to varying degrees within the tubules. MMP-9 and TIMP-4 were abundant in both Sertoli cells and gonocytes and MMP-1 and TIMP-1 were localized in particular to Sertoli cells. In the ovary, all TIMP and MMP-1, MMP-2 and MMP-9 were localized to the oogonium/oocyte cytoplasm with varying intensities and MMP-1, TIMP-2 and TIMP-3 were also detected in the ovarian stroma. This study demonstrates that MMP-1, MMP-2, MMP-9 and all TIMP family members are secreted by the developing ovary and testis and are localized to specific cell and tissue sites. MMP and TIMP are likely to play a role in ECM remodelling during gonadal development and also in the cell and matrix interactions that control a range of cellular functions.     

The role of matrix metalloproteinases (MMPs) in the pathophysiology of chronic obstructive pulmonary disease (COPD): a therapeutic role for inhibitors of MMPs?

Chronic obstructive pulmonary disease (COPD) is the collective term describing two separate chronic lung disease diseases: emphysema and chronic bronchitis (1). Initial clinical symptoms are shortness of breath and occasional cough. As the disease progresses difficulties in breathing becomes more pronounced, the cough more persistent and becomes associated with production of a clear sputum. In severe cases there are additional heart complications. The major risk factor for COPD is cigarette smoking. Between 1980 and 1990 there was a 22% increase in the occurrence of the disease with attributed 84,000 deaths in 1990 in the USA (www.nhlbi.nih.gov/health). Current therapies address the symptoms and range from bronchodilators, corticosteroids to oxygen. While there are no effective cures, although the disease can be prevented and progress slowed in many cases by removing the principal risk factor: cigarette smoking. Progression of the disease is associated with degradation of elastin in the walls of the alveoli, resulting in the functional destruction of the these organs. The net increase in proteolytic activity leading to this loss of alveoli function is a growing focus of pharmaceutical efforts for identification of a therapy for the amelioration of this disease. Of specific interest for this review has been the potential roles of members of the MMP family in both the destruction of elastin and the aberrant remodeling of damaged alveoli. An example of such a MMP is Metalloelastase. Metalloelastase (MMP-12) is (as the name suggests) capable of degrading elastin, as well as other extra-cellular matrix components. It is produced predominantly by infiltrating macrophages and appears essential for macrophage migration through extra-cellular matrix (2). Mouse metalloelastase knock-out studies implicate this enzyme as a key mediator in the pathology associated with cigarette smoke induced emhysema (3). There is also associative evidence from human genetic and animal studies suggesting a pathological link with other MMPs, such as MMPs 1,2,3,8 & 9. The evidence for the role of these MMPs in the pathological processes associated with COPD and prospects for MMP inhibitors as the basis for future therapies will be addressed in this review.     

CCNs, fibulin-1C and S100A4 expression in leiomyoma and myometrium: inverse association with TGF-beta and regulation by TGF-beta in leiomyoma and myometrial smooth muscle cells

Connective tissue growth factor (CTGF; CCN2) is considered to serve as downstream midiator of TGF-beta action in tissue fibrosis. We tested this hypothesis in paired leiomyoma and myometrium by evaluating the expression of TGF-beta1/TGF-beta3 and CCN2, the other members of the CCN family, CCN3 and CCN4, as well as fibulin-1C and S100A4, calcium-binding proteins that interact with CCNs. The regulatory function of TGF-beta1 on the expression of these genes was further evaluated using leiomyoma (L) and myometrial (M) smooth muscle cells (SMC). Real-time PCR, Western blotting and immunohistochemistry revealed that leiomyomas and myometrium express CCNs, fibulin-1C and S100A4, whose levels of expression with the exception of fibulin-1C were lower in leiomyomas and inversely correlated with the expression of TGF-beta1 and TGF-beta3 (P<0.05). The expression of these genes was menstrual cycle-independent and GnRHa therapy increased the expression of CCN2 in leiomyomas, while inhibiting CCN3, CCN4 and S100A4 in myometrium (P<0.05). TGF-beta (2.5 ng/ml) in a time- and cell-dependent manner, and through MAPK and Smad pathways, differentially regulated the expression of these genes in LSMC and MSMC. We concluded that CCNs, fibulin-1C and S100A4 are expressed in leiomyomas/myometrium with relative expression levels inversely correlating with TGF-betas and influenced by GnRHa and TGF-beta regulatory actions. The results suggest that unlike other fibrotic disorders, CCN2 (CTGF), at least at tissue level, may not serve as a downstream mediator of TGF-beta action in leiomyomas.