Homozygous mice with a null mutation in the MMP-9/gelatinase B gene exhibit an abnormal pattern of skeletal growth plate vascularization and ossification. Although hypertrophic chondrocytes develop normally, apoptosis, vascularization, and ossification are delayed, resulting in progressive lengthening of the growth plate to about eight times normal. After 3 weeks postnatal, aberrant apoptosis, vascularization, and ossification compensate to remodel the enlarged growth plate and ultimately produce an axial skeleton of normal appearance. Transplantation of wild-type bone marrow cells rescues vascularization and ossification in gelatinase B-null growth plates, indicating that these processes are mediated by gelatinase B-expressing cells of bone marrow origin, designated chondroclasts. Growth plates from gelatinase B-null mice in culture show a delayed release of an angiogenic activator, establishing a role for this proteinase in controlling angiogenesis.

No abstract

Interleukin (IL)-13 is a key mediator of tissue fibrosis caused by T helper cell type 2 inflammation. We hypothesized that the fibrogenic effects of IL-13 are mediated by transforming growth factor (TGF)-β. To test this hypothesis we compared the regulation of TGF-β in lungs from wild-type mice and CC10-IL-13 mice in which IL-13 overexpression causes pulmonary fibrosis. IL-13 selectively stimulated TGF-β1 production in transgenic animals and macrophages were the major site of TGF-β1 production and deposition in these tissues. IL-13 also activated TGF-β1 in vivo. This activation was associated with decreased levels of mRNA encoding latent TGF-β–binding protein-1 and increased mRNA encoding urinary plasminogen activator, matrix metalloproteinase (MMP)-9, and CD44. TGF-β1 activation was abrogated by the plasmin/serine protease antagonist aprotinin. It was also decreased in progeny of crosses of CC10-IL-13 mice and MMP-9 null mice but was not altered in crosses with CD44 null animals. IL-13–induced fibrosis was also significantly ameliorated by treatment with the TGF-β antagonist soluble TGFβR-Fc (sTGFβR-Fc). These studies demonstrate that IL-13 is a potent stimulator and activator of TGF-β1 in vivo. They also demonstrate that this activation is mediated by a plasmin/serine protease- and MMP-9–dependent and CD44-independent mechanism(s) and that the fibrogenic effects of IL-13 are mediated, in great extent, by this TGF-β pathway.

The molecular mechanism of tissue-specific metastasis in tumors endogenously expressing members of the vascular endothelial growth factor (VEGF) family is not yet clear. Here we demonstrate that MMP9 is specifically induced in premetastatic lung endothelial cells and macrophages by distant primary tumors via VEGFR-1/Flt-1 tyrosine kinase (TK) and that it significantly promotes lung metastasis. In a genetic approach using mice, suppression of MMP9 induction by deletion of either VEGFR-1TK or MMP9 markedly reduced lung metastasis. Furthermore, the MMP9 levels in endothelial cells of normal lung lobes from patients carrying distant tumors were significantly elevated as compared with those from patients without tumors. Thus, a block of MMP9 induction via VEGFR-1 inhibition could be useful for the prevention of tumor metastasis in lung.

Macrophages secrete a variety of proteinases that are thought to participate in remodeling of the extracellular matrix associated with inflammatory processes. We have eliminated expression of the macrophage metalloelastase (MME) gene by targeted disruption to assess the role of this protein in macrophage-mediated proteolysis. We found that the macrophages of MME-deficient (MME -/-) mice have a markedly diminished capacity to degrade extracellular matrix components. In addition, MME -/-macrophages are essentially unable to penetrate reconstituted basement membranes in vitro and in vivo. MME is therefore required for macrophage-mediated extracellular matrix proteolysis and tissue invasion.Macrophages produce a variety of cysteine, serine, and metalloproteinases that are involved with the physiologic tissue remodeling associated with inflammation and wound repair. However, proteolytic activity released by macrophages can also lead to pathologic tissue destruction. Metalloproteinases constitute a family of structurally related matrix-degrading proteinases including the collagenases, stromelysins, gelatinases, matrilysin, membrane-type metalloproteinases, and macrophage metalloelastase. These enzymes require zinc for catalytic activity and are inhibited by the tissue inhibitors of metalloproteinases (1, 2). Macrophage metalloelastase (MME) is characterized by macrophage-specific expression and the capacity to hydrolyze a broad spectrum of substrates (3,4). To determine the contribution of metalloelastase to macrophage-mediated proteolysis, we generated mice deficient in metalloelastase (MME -/-) by targeted mutagenesis. MATERIALS AND METHODSGeneration of the Targeting Construct. A genomic clone encoding MME was isolated from a mouse 129/SvJ library (Stratagene) by using the cDNA as a probe. The

Abstract-Matrix remodeling plays an important role in the physiological and pathological remodeling of blood vessels.We specifically investigated the role of matrix metalloproteinase (MMP)-9, an MMP induced during arterial remodeling, by assessing the effects of genetic MMP-9 deficiency on major parameters of arterial remodeling using the mouse carotid artery flow cessation model. Compared with remodeling of matched wild-type (WT) arteries, MMP-9 deficiency decreased intimal hyperplasia, reduced the late lumen loss, eliminated the correlation between intimal hyperplasia and geometric remodeling, and led to significant accumulation of interstitial collagen. Biochemical analysis of MMP-9 knockout (KO) arterial tissue and isolated smooth muscle cells (SMCs) confirmed the lack of MMP-9 expression or compensation by other gelatinases. To investigate potential mechanisms for the in vivo observations, we analyzed in vitro effects of MMP-9 deficiency on the migration, proliferation, and collagen gel contracting capacity of aortic SMCs isolated from MMP-9 KO and WT mice. Although proliferation was comparable, we found that MMP-9 -deficient cells had not only decreased migratory activity, but they also had decreased capacity to contract collagen compared with WT cells. Thus, MMP-9 appears to be involved not only in degradation, but also in reorganization of a collagenous matrix, both facets being essential for the outcome of arterial remodeling. Our results also establish MMP-9 as an attractive therapeutic target for limiting the effects of pathological arterial remodeling in restenosis and atherosclerosis. Key Words: matrix degradation Ⅲ cell migration Ⅲ restenosis Ⅲ atherosclerosis P hysiological and pathological vascular remodeling in response to a variety of stimuli, including hemodynamic changes, inflammation, and injury, leads to reshaping of the vessel wall. Inappropriate remodeling is currently thought to be the main cause of most prevalent vascular pathologies of arteries, including atherosclerosis and restenosis. 1 Degradation of the matrix scaffold enables cell movement and general tissue reorganization, making specialized enzymes called matrix metalloproteinases (MMPs) 2 prime candidates for agents of vascular remodeling. 3 Although many studies have addressed and endorsed a role for MMPs in pathological remodeling, suggesting these as potential therapeutic targets in restenosis and atherosclerosis, due to the current lack of specific MMP inhibitors, such studies could not resolve the identity of the specific relevant MMP(s). We thus decided to explore the effects of MMP-9 genetic deficiency on the remodeling of carotid arteries in the flow cessation murine model, 4 which allows investigation of both formation of intimal hyperplasia and arterial geometrical remodeling, 5 the two main processes implicated in the stenotic remodeling of human arteries. Materials and Methods Animal ModelMMP-9 expression was disrupted by deletion of most of the exon 2 of the MMP-9 gene in the 129/SvEv genetic background (Jackson La...

Dendritic cells migrate from the skin to the draining lymph nodes. They transport immunogenic MHC-peptide complexes, present them to Ag-specific T cells in the T areas, and thus generate immunity. Migrating dendritic cells encounter physical obstacles, such as basement membranes and collagen meshwork. Prior work has revealed that matrix metalloproteinase-9 (MMP-9) contributes to mouse Langerhans cell migration. In this study, we use mouse and human skin explant culture models to further study the role of MMPs in the migration and maturation of skin dendritic cells. We found that MMP-2 and MMP-9 are expressed on the surface of dendritic cells from the skin, but not from other sources. They are also expressed in migrating Langerhans cells in situ. The migration of both Langerhans cells and dermal dendritic cells is inhibited by a broad spectrum inhibitor of MMPs (BB-3103), by Abs to MMP-9 and -2, and by the natural tissue inhibitors of metalloproteinases (TIMP), TIMP-1 and TIMP-2. Inhibition by anti-MMP-2 and TIMP-2 define a functional role for MMP-2 in addition to the previously described function of MMP-9. The importance of MMP-9 was emphasized using MMP-9-deficient mice in which Langerhans cell migration from skin explants was strikingly reduced. However, MMP-9 was only required for Langerhans cell migration and not maturation, since nonmigrating Langerhans cells isolated from the epidermis matured normally with regard to morphology, phenotype, and T cell stimulatory function. These data underscore the importance of MMPs, and they may be of relevance for therapeutically regulating dendritic cell migration in clinical vaccination approaches.

We have identified the key protein substrate of gelatinase B/MMP-9 (GB) that is cleaved in vivo during dermal-epidermal separation triggered by antibodies to the hemidesmosomal protein BP180 (collagen XVII, BPAG2). Mice deficient in either GB or neutrophil elastase (NE) are resistant to blister formation in response to these antibodies in a mouse model of the autoimmune disease bullous pemphigoid. Disease develops upon complementation of GB -/- mice with NE -/- neutrophils or NE -/- mice with GB -/- neutrophils. Only NE degrades BP180 and produces dermal-epidermal separation in vivo and in culture. Instead, GB acts upstream to regulates NE activity by inactivating alpha1-proteinase inhibitor (alpha1-PI). Excess NE produces lesions in GB -/- mice without cleaving alpha1-PI. Excess alpha1-PI phenocopies GB and NE deficiency in wild-type mice.