Metalloproteinases in juvenile angiofibroma--a collagen rich tumor

Matrix metalloproteinases (MMPs) act in diverse physiological and pathological conditions such as tumor growth and angiogenesis by cleaving extracellular matrix and nonmatrix substrates. MMPs with gelatinase/collagenase activity have not yet been studied in juvenile angiofibroma, a unique fibrovascular tumor with prominent collagen expression. Quantitative real-time polymerase chain reaction studies, Western blot analysis, immunofluorescence studies, gel zymography, and in situ zymography were used to analyze MMP-1, MMP-2, MMP-9, MMP-13, MMP-14, TIMP-1, and TIMP-2 in 9 juvenile angiofibromas and 2 inferior nasal turbinate specimens. Quantitative real-time polymerase chain reaction found significantly elevated expression of MMP-2, MMP-9, and MMP-14 (P < .05) in tumor tissue compared with the inferior nasal turbinate specimens. Western blot analysis detected more prominent MMP-1, MMP-2, and MMP-9 protein levels in juvenile angiofibromas compared with inferior nasal turbinates, but not MMP-13, MMP-14, TIMP-1, and TIMP-2. Immunofluorescent staining proved a mainly stromal localization of the analyzed MMPs. Only MMP-9 and MMP-14 were also detected in vessel walls. MMP-1, MMP-2, and MMP-13 also stained mast cells. Gel zymography indicated increased MMP-2 and MMP-9 gelatinase activity in juvenile angiofibromas compared with inferior nasal turbinates. Finally, in situ zymography detected very high stromal gelatinase/collagenase activity. This study indicates significant expression of MMPs with gelatinase/collagenase activity in juvenile angiofibromas with evidence of a disturbed balance of MMPs to TIMPs toward enhanced MMP activity. These MMPs are assumed to be involved in tumor pathology with an influence on tumor growth and angiogenesis.     

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.     

Role of metalloproteinases and tissue inhibitors of metalloproteinases during cardiopulmonary bypass in rats

Matrix metalloproteinases (MMPs) and the tissue inhibitors of matrix metalloproteinases (TIMPs) regulate matrix remodeling in the heart. Changes in synthesis and release of MMPs and TIMPs are observed after extracorporeal circulation (ECC). Thus, MMPs and TIMPs are supposed to be involved in ECC-mediated cardiac dysfunction. The aim was to examine the role of MMPs and TIMPs in ECC-mediated cardiac dysfunction. Extracorporeal circulation was instituted in rats for 60 min at a flow rate of 120 ml/kg/min. Three groups (n = 10) were studied: group CAO: 60 min ECC without aortic cross-clamping, group CAC: 60 min ECC including 30 min aortic cross-clamping (crystalloid Inzolen(?) cardioplegia), and group CAB: 60 min ECC including 30 min aortic cross-clamping (blood cardioplegia). Left ventricular (LV) function was measured with conductance catheter. Matrix metalloproteinase-activity was determined by zymography and TIMP activity was determined by reverse zymography. Gene expression of MMPs and TIMPs was determined by real-time polymerase chain reaction. Sixty minutes after weaning from bypass, there was a preserved LV function in the CAO and CAB group and an impaired LV function in the CAC group. We observed an increased myocardial activity and an increased myocardial messenger RNA expression of MMP-2, MMP-9, TIMP-1, and TIMP-4 in all ECC groups, when compared with sham animals. With regard to enzyme activity, there was an imbalance of MMP/TIMP ratio leading to an increased activity of MMP in the CAC group. In terms of gene expression, there was an imbalance of MMP-2/TIMP-4 ratio leading to an increased expression of MMP-2 in the CAC group. MMP-2 contributes to myocardial reperfusion injury in this in vivo model of ECC with cardioplegic arrest.     

Analyses of matrix metalloproteinases and their inhibitors in cyst fluid of serous ovarian tumors.

OBJECTIVES: Expression of matrix metalloproteinases (MMPs) and tissue inhibitors of MMPs (TIMPs) in serous tumors of the ovary was investigated to determine whether and how these proteolytic enzymes are associated with the progression of these tumors. METHODS: Cyst fluid of 24 serous ovarian tumors (8 adenocarcinomas, 2 borderline tumors and 14 adenomas) was analyzed using gelatin/casein zymography and enzyme-linked immunosorbent assay. RESULTS: Concentrations of MMP-9 and MMP-2 were statistically higher in serous adenocarcinomas than in serous adenomas (p < 0.01, p < 0.05, respectively), while the concentrations of TIMP-1 and TIMP-2 showed no significant difference between adenocarcinomas and adenomas. The molar ratio of TIMP-2/MMP-2 was lower in adenocarcinomas than in adenomas (p < 0.05). With gelatin zymography, the MMP-9 band was detected in all serous adenocarcinomas, but only in 8 of 14 serous adenomas (p = 0.05). Using casein zymography, MMP-7 was more frequently detected in serous adenocarcinomas (7/8) than in serous adenomas (4/14; p < 0.05). CONCLUSIONS: These observations indicate that matriolytic enzymes such as MMP-2, MMP-7 and MMP-9 are secreted into cyst fluid from serous adenocarcinoma tissues. In part, the aggressive invasion of serous carcinoma cells may be explained by the expression of matriolytic enzymes.     

Expression of metalloproteinases and its inhibitor in later stage of rabbit neointima development

Neointima formation after arterial de-endothelialization refers not only to smooth muscle cell (SMC) migration and proliferation, but also involves extracellular matrix (ECM) metabolism. Most studies regarding the role of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) in neointima have focused on the early phase of vascular remodeling. In this study, we examined the expression of MMP and TIMP in rabbit aortic neointima at a relatively late stage of lesion development, between 4 and 12 weeks after initial de-endothelialization. Northern blot analysis revealed expression of steady-state MMP-9 mRNA was increased up to the 4th week and MMP-2 mRNA to the 12th week after de-endothelialization. In situ hybridization shown that MMP positive cells were predominantly distributed in arterial neointima. Expression of TIMP-1 mRNA was continuously up-regulated up to the 12th week and TIMP-1 positive cells, primarily SMCs, were also localized to the neointimal tissue. Alteration at mRNA level was accompanied by that at protein level, as assessed by SDS-PAGE zymography for MMPs and immunoblotting for TIMP-1. The profile of alteration at protein level correlated well with that at mRNA level. These data suggest that synthesis of MMPs and TIMP is a prolonged process and arterial SMC is a major source of MMP production in arterial neointima. Enhanced synthesis of MMPs and TIMPs at late stage of neointimal development may contribute to arterial ECM metabolism.     

Expression and role of matrix metalloproteinases MMP-2 and MMP-9 in human spinal column tumors

Matrix metalloproteinases (MMPs) have been implicated in the process of tumor invasion and metastasis formation. Thus, we determined the expression of MMPs in various primary and metastatic spinal tumors in order to assess the role of these enzymes in spinal invasion. MMP expression was examined by immuno-histochemical localization, and quantitative evaluation of MMP protein content was determined by enzyme-linked immunosorbant assay (ELISA) and Western blotting. MMP enzyme activity was determined by gelatin zymography. Lung carcinomas and melanomas metastatic to the spine were shown to have higher levels of MMP-9 activity than those of breast, thyroid, renal metastases and primary spinal tumors. Immunohistochemical analysis revealed similar difference in expression of MMP-9 in tissue samples. When the tissue samples were subjected to gelatin zymography for examination of MMP-2 and MMP-9 activity and to ELISA and Western blotting for quantitative estimation of protein content, the most striking results were obtained for lung carcinomas and melanomas relative to the other tumors. Lung carcinomas and melanomas metastatic to the spine had considerably higher levels of MMP-9 activity than those of primary spinal tumor or breast, thyroid, and renal carcinoma metastases. Within the metastatic tumor category, neoplasms that are known to be associated with the shortest overall survival rates and most aggressive behavior, such as lung carcinomas and melanomas, had the highest levels of MMP-2 and MMP-9 activity compared to those less aggressive metastatic tumors such as breast, renal cell, and thyroid carcinomas. Our results suggest that MMPs may contribute to the metastases to the spinal column, and overexpression of these enzymes may correlate with enhanced invasive properties of both primary and metastatic spinal tumors.© Kluwer Academic Publishers 1998     

EMMPRIN-mediated MMP regulation in tumor and endothelial cells

Tumor invasion and metastasis are multistep processes which require extracellular matrix remodeling by proteolytic enzymes such as matrix metalloproteinases (MMPs). The production of these enzymes is stimulated by many soluble or cell-bound factors. Among these factors, extracellular matrix metalloproteinase inducer (EMMPRIN) is known to increase in vitro stromal cell production of MMP-1, MMP-2 and MMP-3. In this study, we demonstrated that EMMPRIN-transfected MDA-MB-436 tumor cells displayed a more invasive capacity than vector-transfected cells in a modified Boyden chamber invasion assay. Using gelatin zymography and protein analyses, we showed that EMMPRIN-transfected cancer cells produced significantly more latent and active MMP-2 and MMP-3 than vector-transfected cancer cells. We found that EMMPRIN did not regulate MMP-1, MMP-9, membrane type-1 MMP (MT1-MMP) expression and had also no effect on the production of the specific tissue inhibitors of MMPs (TIMPs), TIMP-1 and TIMP-2. We also demonstrated that tumor-derived EMMPRIN stimulated MMP-1, -2, and -3 without modification of MMP-9, MT1-MMP, TIMP-1 and TIMP-2 production in human umbilical vein endothelial cells (HUVEC). These data provide support for the role of EMMPRIN in tumor invasion, metastasis, and neoangiogenesis by stimulating extracellular matrix remodeling around tumor cell clusters, stroma, and blood vessels. This revised version was published online in July 2006 with corrections to the Cover Date.     

Expression of matrix metalloproteinases, tissue inhibitors of metalloproteinase, collagens, and Ki67 antigen in pleural malignant mesothelioma: an immunohistochemical and electron microscopic study

Because matrix metalloproteinases (MMPs) degrade extracellular matrix, including basement membrane, and because tissue inhibitors of MMP (TIMPs) suppress MMP activities, MMPs and TIMPs are considered to play important roles in invasion and metastasis in many malignancies. We examined immunohistochemically the expression of MMPs (MMP-1, -2, -3, -7, and -9), TIMPs (TIMP-1 and -2), and collagens (types I, III, and IV) in 16 patients with pleural malignant mesothelioma (PMM; 8 with the epithelial, 4 with the sarcomatous, and 4 with the biphasic type). Electron microscopy revealed that the tumor cells in all types possessed the characteristics of malignant mesotheliomas, including numerous microvilli and moderate amounts of intermediate filaments. Basement lamina was present only focally. The proliferative Ki67 index was at a high level, compared with values reported in various other malignancies. Positive staining for MMP-1 was observed in most tumor cells in all 16 patients (100%). MMP-2 was expressed in most tumor cells in 2 patients (13%). In contrast, MMP-3, -7, and -9 were not detected in any PMM. TIMP-1 and TIMP-2 were expressed in 3 patients (19%) and 2 patients (13%), respectively. The stromal cells were simultaneously positive for MMPs or TIMPs in the patients whose tumor parenchymal cells were positive for each enzyme. These results indicate that the expression of MMP-1 and MMP-2 may be related to PMM invasion and spread. In particular, as MMP-1 was overexpressed in contrast to the lower expression of TIMP-1, MMP-1 is strongly suggested to play an important role in PMM invasion by degrading the tumor stroma. In spite of general agreement that epithelial-type PMM has a better prognosis than other types, there was no significant difference in the Ki67 index among the histological types of PMM.     

Progesterone receptor modulator CDB-2914 induces extracellular matrix metalloproteinase inducer in cultured human uterine leiomyoma cells

Effects of progesterone receptor modulator CDB-2914 on the expression of the extracellular matrix (ECM) components were examined in cultured human uterine leiomyoma and myometrial cells. ECM metalloproteinase inducer (EMMPRIN), matrix metalloproteinases (MMPs), tissue inhibitors of MMP (TIMPs) and collagen levels were assessed by Western blot analysis, MMP activity assay and real-time RT-PCR. RNA interference (RNAi) of EMMPRIN was performed using small interfering mRNA. In cultured leiomyoma cells, CDB-2914 treatment at concentrations greater than or equal to 10(-8) M significantly increased EMMPRIN, MMP-1 and MMP-8 protein contents and MMP-1, MMP-2, MMP-3 and MMP-9 mRNA levels, and activity of MMP-1, MMP-2, MMP-3 and MMP-9 in the medium. TIMP-1 and TIMP-2 were significantly decreased at mRNA and protein levels by CDB-2914 treatment at concentrations > or =10(-7) M in these cells. CDB-2914 treatment decreased types I and III collagen protein contents. However, CDB-2914 treatment did not affect the ECM component expression in cultured myometrial cells. RNAi of EMMPRIN abrogated CDB-2914-mediated both induction of MMPs and reduction of TIMPs and collagens in cultured leiomyoma cells. These results suggest that CDB-2914 modulates the expression of EMMPRIN, MMPs, TIMPs and collagens in cultured leiomyoma cells without comparable effects on myometrial cells.     

Coordinated Expression of MMPs and TIMPs in Rat Knee Intra-Articular Tissues After ACL Injury

Anterior cruciate ligament (ACL) has poor healing ability and an injured ACL would induce the degeneration of other intra-articular connective tissues. However, the coordinated expression and activities of matrix metalloproteinase (MMPs) in intra-articular tissues induced by ACL rupture were poorly understood. With a rat ACL rotating injury model, we found that after ACL injury, the mRNA levels of MMP-13, TIMP-1, and CD147 were significantly elevated in ACL, posterior cruciate ligament (PCL), synovium, meniscus, and cartilage. Also, MMP-2 activity was also elevated significantly in a time-dependent manner in all intra-articular tissues. Synovium showed the most capability to release MMPs, whereas ACL showed the highest MMP-13/TIMP-1 ratio. Generic MMP activity assay and zymography showed time dependent elevation of MMP activities in synovial fluids (SF). We concluded that the ACL injury would induce a coordinated response of intra-articular tissues to express MMPs, TIMPs, and CD147. The MMP activities in the microenvironment in SF would accumulate, released by all the intra-articular tissues, which would contribute to the knee damage and degeneration induced by ACL injury.     

67ku层黏连蛋白受体促进肝癌细胞MMP-2和MMP-9的表达

目的研究分析67ku层黏连蛋白受体(laminin receptor,67LR)与肝癌细胞基质金属蛋白酶(matrix metalloproteinases,MMPs)及其组织抑制因子(tissue inhibitor of metalloproteinases,TIMPs)表达的关系,探讨67LR促进肝癌细胞体外侵袭能力的分子机制。方法以67LR转染HepG2的稳定细胞株及其对照细胞为材料,采用半定量RT-PCR分析目前已知的23种MMPs和4种TIMPs的表达及变化情况,对表达有变化的基因采用荧光定量PCR进行验证,采用明胶酶谱分析MMP活性的变化。结果半定量RT-PCR和荧光定量PCR发现,67LR高表达的LR4细胞,其MMP2,9的表达比67LR低表达的LR6及对照组pcD-NA-1细胞明显升高,明胶酶谱分析也揭示,LR4细胞分泌的MMP-2和MMP-9的活性明显上升。结论67LR可以促进肝癌细胞MMP-2和MMP-9的表达和分泌,从而促进肝癌细胞体外侵袭能力。     

Matrix metalloproteinases mediate the dismantling of mesenchymal structures in the tadpole tail during thyroid hormone-induced tail resorption.

It has been suggested that a family of tissue remodelling enzymes called matrix metalloproteinases (MMPs) play a causal role in the process of tail resorption during thyroid hormone-induced metamorphosis of the anuran tadpole; however, this hypothesis has never been directly substantiated. We cloned two new Xenopus MMPs, gelatinase A (MMP-2) and MT3-MMP (MMP-16), and the MMP inhibitor TIMP-2. These clones were used along with several others to perform a comprehensive expression study. We show that all MMPs and TIMP-2 are dramatically induced in the resorbing tail during spontaneous metamorphosis and are spatially coexpressed, primarily in the remodelling mesenchymal tissues. By Northern blotting, we show that all the examined MMPs/TIMP-2 are also induced by treatment of organ-cultured tails with thyroid hormone (T(3)). Using the organ culture model, we provide the first direct evidence that MMPs are required for T(3)-induced tail resorption by showing that a synthetic inhibitor of MMP activity/expression can specifically retard the resorption process. By gelatin zymography, we also show T(3) induction of a fifth MMP, preliminarily identified as gelatinase B (GelB; MMP-9). Moreover, T(3) not only induces MMP/TIMP expression but also MMP activation, and we provide evidence that TIMP-2 participates in the latter process. These findings suggest that MMPs and TIMPs act in concert to effect the dismantling of mesenchymal structures during T(3)-induced metamorphic tadpole tail resorption.     

Specific expression of matrix metalloproteinases 1, 3, 9 and 13 associated with invasiveness of breast cancer cells in vitro

Several matrix metalloproteinases (MMPs) and tissue inhibitors of MMPs (TIMPs) were studied in highly invasive (MDA-MB-231) and slightly invasive (MCF-7, T47D, BT-20) breast cancer cell lines. Investigations were carried out at the protein level and/or at the mRNA level, either in cells cultured as monolayers on plastic, or in cells seeded on a thin layer of Matrigel basement membrane matrix. Analysis of MMP expression by RT-PCR showed expression of MMP-1, MMP-3, and MMP-13 in highly invasive MDA-MB-231 cells, but not in slightly invasive cell lines. The extracellular secretion of MMP-1 and MMP-3 by MDA-MB 231 cells could be also shown by ELISA. TIMP-1 and TIMP-2 mRNAs were found in all cell lines, however, the extracellular secretion of both TIMPs was much higher in MDA-MB-231 cells than in the other cell lines. When the cells were cultured on Matrigel matrix, MMP-9 expression was induced in MDA-MB-231 cells only, as assessed by RT-PCR and zymography experiments. The invasive potential of MDA-MB-231 cells evaluated in vitro through Matrigel was significantly inhibited by the MMP inhibitor BB-2516, by 25% and 50% at the concentrations of 2 × 10⁻⁶M and 10⁻⁵M, respectively. In conclusion, our data show that highly invasive MDA-MB-231 cells but not slightly invasive T47D, MCF-7 and BT-20 cells express MMP-1, MMP-3, MMP-9 and MMP-13. MMP-9 which is specifically up-regulated by cell contact to Matrigel, may play a key role in the invasiveness of MDA-MB-231 cells through basement membranes. This revised version was published online in July 2006 with corrections to the Cover Date.     

Monocyte-derived dendritic cell subpopulations use different types of matrix metalloproteinases inhibited by GM6001

Matrix metalloproteinases (MMPs) are endopeptidases with the potential to cleave extracellular matrix, support tissue renewal and regulate cell migration. Functional activities of MMPs are regulated by tissue inhibitors of MMPs (TIMPs) and disruption of the MMP–TIMP balance has pathological consequences. Here we studied the expression and secretion of MMPs and TIMPs in CD1a⁻ and CD1a⁺ monocyte-derived dendritic cell (DC) subpopulations. Our results showed that monocytes express TIMPs but lack MMPs, whereas upon differentiation to moDCs and in response to activation signals the expression of MMPs is increased and that of TIMPs is decreased. MMP-9 is expressed dominantly in the CD1a⁻ subpopulation, while MMP-12 is preferentially expressed in CD1a⁺ cells. Experiments performed with the synthetic MMP inhibitor GM6001 revealed that this drug efficiently inhibits the migration of moDCs through inactivation of MMPs. We conclude that modulation of MMP activity by GM6001 emerges as a novel approach to manipulate DC migration under inflammatory conditions.     

Protein expression of matrix metalloproteinases 2 and 9 and tissue inhibitors of metalloproteinase 1 and 2 in papillary thyroid carcinomas

Matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) play an important role in tumor invasion and metastasis. There have been only a few studies on the protein expression of MMPs and TIMPs in thyroid carcinomas. Therefore, we investigated the protein expression of MMP-2, MMP-9, TIMP-1 and TIMP-2 in 86 papillary thyroid carcinomas using immunohistochemistry, semiquantitative scoring morphometry of immunohistochemistry, gelatin zymography, and western blotting. We also examined the correlations between the immunohistochemical scores and several clinicopathological parameters. The immunoreactivities of MMP-2, MMP-9, TIMP-1, and TIMP-2 were largely located in the tumor cells or non-tumor follicular cells and to a much lesser extent in the fibroblasts and endothelial cells in the tumor and non-tumor regions. Compared with non-tumor regions, these four proteins tended to be overexpressed in the tumor cells; the overexpression was found in 64 of 86 (74%), 80 of 86 (93%), 79 of 86 (92%), and 64 of 86 (74%) cases for MMP-2, MMP-9, TIMP-1, and TIMP-2, respectively. Gelatin zymography showed distinct bands of MMP-2 and MMP-9 in tumor extracts but vague bands in non-tumor extracts. Western blotting revealed the specific bands of MMP-2 and MMP-9 in both tumor and non-tumor extracts. Morphometric scoring revealed that high expression of these proteins significantly correlated with large tumor size, presence of lymph node metastasis, high clinical stage, high intrathyroidal invasion, and high vascular invasion. These data suggest that MMP-2, MMP-9, TIMP-1, and TIMP-2 proteins and activities are increased in tumors cells of papillary thyroid carcinomas and that they play an important role in the invasion and metastasis of papillary thyroid carcinomas.     

CD40 engagement modulates the production of matrix metalloproteinases by gingival fibroblasts

Chronic periodontitis is a destructive inflammatory disease linked with unbalanced production between matrix metalloproteinases (MMPs), such as interstitial collagenase (MMP-1) and stromelysin-1 (MMP-3) and their endogenous tissue inhibitors of MMPs (TIMPs). In addition to aberrant MMP-1 and MMP-3 expression, periodontal lesions are characterized by dense infiltrations of activated T lymphocytes which may interact with CD40-expressing gingival fibroblasts in the connective tissue via the CD40L-CD40 pathway. In this study we investigated whether CD40 cross-linking influenced MMP production by gingival fibroblasts. Therefore, we analysed the CD40L-induced MMP production by these fibroblasts in the presence of cytokines that are increased in periodontal lesions, such as IL-1β, tumour necrosis factor-alpha (TNF-α) and interferon-gamma (IFN-γ). We show that CD40 ligation on gingival fibroblasts resulted in a decrease of their MMP-1 and MMP-3 production, while MMP-2 and TIMP-1 production were unaffected as determined by Western blot. This down-regulatory effect of CD40 engagement on MMP-1 and MMP-3 production by gingival fibroblasts was also present when MMP production was up-regulated by IL-1β and TNF-α or down-regulated by IFN-γ. These results suggest that CD40 ligation on gingival fibroblasts leads to a restraining of MMP-1 and MMP-3 production by gingival fibroblasts and thereby may be an important mechanism in the retardation of further periodontal tissue damage.     

Gelatinase-B (Matrix Metalloproteinase-9; MMP-9) secretion is involved in the migratory phase of human and murine muscle cell cultures

The remodelling of connective tissue components is a fundamental requirement for a number of pivotal processes in cell biology. These may include myoblast migration and fusion during development and regeneration. In other systems, similar biological processes are facilitated by secretion of the matrix metalloproteinases (MMPs), especially the gelatinases. This study investigated the activity of the gelatinases MMP-2 and 9 by zymography on cell conditioned media in cultures of cells derived from explants of the human masseter muscle and in the murine myoblast cell-line C2C12. Expression of MMP-9 by western blotting and TIMP-1, the major inhibitor of MMPs, by northern blotting, during all phases of myoblast proliferation, migration, alignment and fusion, was also measured. Irrespective of the origin of the cultures, MMP-9 activity was secreted only by single cell and pre-fusion cultures whilst MMP-2 activity was secreted at all stages as well as by myotubes. The loss of MMP-9 activity was due to the loss of MMP-9 protein expression. TIMP-1 mRNA was not detectable at the single cell stage but its expression increased as cells progressed through the pre-fusion and post-fusion stages to reach a maximal in myotube containing cultures. Migration of cells derived from human masseter muscle was inhibited, using a specific anti-MMP-9 blocking monoclonal antibody (6-6B). These data are consistent with the concept that regulation of matrix turnover via MMP-9 may be involved in the events leading to myotube formation, including migration. Loss of expression of this enzyme and expression of TIMP-1 mRNA is associated with myotube containing cultures. Consequently, the ratio between MMPs and TIMPs maybe important in determining myoblast migration and differentiation.     

Promoted activation of matrix metalloproteinase (MMP)-2 in keloid fibroblasts and increased expression of MMP-2 in collagen bundle regions: implications for mechanisms of keloid progression

Aims:  Keloid is characterized by excessive deposition of collagen, resulting from aberrant extracellular matrix (ECM) production and degradation. The aim was to investigate the role of matrix metalloproteinases (MMPs) and tissue inhibitor of metalloproteinases (TIMPs) in pathological wound healing in keloids.
Methods and results:  Semiquantitative analysis of 60 keloid tissue samples and 25 mature scar tissue samples demonstrated significantly increased expression of MMP-2, TIMP-2 and TIMP-3 in keloids compared with mature scars. Within keloid regions, MMP-2 expression was significantly higher in collagen bundle regions than in non-collagen bundle regions. Double immunofluorescence revealed that keloid fibroblasts between collagen bundles exhibited MMP-2, TIMP-2 and membrane-type 1 MMP (MT1-MMP) co-expression, whereas only MMP-2 expression was evident on the edge of collagen bundles. Western blot analysis and gelatin zymography of 13 keloid-derived fibroblasts (KFbs) and six normal skin dermal-derived fibroblasts (NFbs) demonstrated that unstimulated KFbs exhibited significantly increased MMP-2 activity and expression compared with NFbs under the same conditions.
Conclusions:  These results together indicate that MMP-2 activity can be promoted in keloid fibroblasts between collagen bundles in cooperation with TIMP-2 and MT1-MMP. This could contribute to remodelling of collagen bundle regions and invasion of fibroblasts into peripheral normal regions through promoted degradation of ECM.