J. Michael Shipley

MMP-9/Gelatinase B Is a Key Regulator of Growth Plate Angiogenesis and Apoptosis of Hypertrophic Chondrocytes

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.

Targeted gene disruption of matrix metalloproteinase-9 (gelatinase B) suppresses development of experimental abdominal aortic aneurysms

Abdominal aortic aneurysms represent a life-threatening condition characterized by chronic inflammation, destructive remodeling of the extracellular matrix, and increased local expression of matrix metalloproteinases (MMPs). Both 92-kD gelatinase (MMP-9) and macrophage elastase (MMP-12) have been implicated in this disease, but it is not known if either is necessary in aneurysmal degeneration. We show here that transient elastase perfusion of the mouse aorta results in delayed aneurysm development that is temporally associated with transmural mononuclear inflammation, increased local production of several elastolytic MMPs, and progressive destruction of the elastic lamellae. Elastase-induced aneurysmal degeneration was suppressed by treatment with a nonselective MMP inhibitor (doxycycline) and by targeted gene disruption of MMP-9, but not by isolated deficiency of MMP-12. Bone marrow transplantation from wild-type mice prevented the aneurysm-resistant phenotype in MMP-9-deficient animals, and wild-type mice acquired aneurysm resistance after transplantation from MMP-9-deficient donors. These results demonstrate that inflammatory cell expression of MMP-9 plays a critical role in an experimental model of aortic aneurysm disease, suggesting that therapeutic strategies targeting MMP-9 may limit the growth of small abdominal aortic aneurysms.

MMP9 induction by vascular endothelial growth factor receptor-1 is involved in lung-specific metastasis

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.

Targeted Disruption of the Matrix Metalloproteinase-9 Gene Impairs Smooth Muscle Cell Migration and Geometrical Arterial Remodeling

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.

The Serpin α1-Proteinase Inhibitor Is a Critical Substrate for Gelatinase B/MMP-9 In Vivo

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.

Overlapping and enzyme-specific contributions of matrix metalloproteinases-9 and -12 in IL-13–induced inflammation and remodeling

IL-13 potently stimulates eosinophilic and lymphocytic inflammation and alveolar remodeling in the lung, effects that depend on the induction of various matrix metalloproteinases (MMPs). Here, we compared the remodeling and inflammatory effects of an IL-13 transgene in lungs of wild-type, MMP-9-deficient, or MMP-12-deficient mice. IL-13-induced alveolar enlargement, lung enlargement, compliance alterations, and respiratory failure and death were markedly decreased in the absence of MMP-9 or MMP-12. Moreover, IL-13 potently induced MMPs-2, -12, -13, and -14 in the absence of MMP-9, while induction of MMPs-2, -9, -13, and -14 by IL-13 was diminished in the absence of MMP-12. A deficiency in MMP-9 did not alter eosinophil, macrophage, or lymphocyte recovery, but increased the recovery of total leukocytes and neutrophils in bronchoalveolar lavage (BAL) fluids from IL-13 transgenic mice. In contrast, a deficiency in MMP-12 decreased the recovery of leukocytes, eosinophils, and macrophages, but not lymphocytes or neutrophils. These studies demonstrate that IL-13 acts via MMPs-9 and -12 to induce alveolar remodeling, respiratory failure, and death and that IL-13 induction of MMPs-2, -9, -13, and -14 is mediated at least partially by an MMP-12-dependent pathway. The also demonstrate that MMPs-9 and -12 play different roles in the generation of IL-13-induced inflammation, with MMP-9 inhibiting neutrophil accumulation and MMP-12 contributing to the accumulation of eosinophils and macrophages.

Matrix Metalloproteinases 9 and 2 Are Necessary for the Migration of Langerhans Cells and Dermal Dendritic Cells from Human and Murine Skin

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.

Gelatinase B Is Required for Alveolar Bronchiolization after Intratracheal Bleomycin

Increased expression of matrix metalloproteinases, particularly gelatinase B (MMP-9), has been described in the lungs in pulmonary fibrosis. Intratracheal bleomycin is often used experimentally to produce lesions resembling human fibrosing alveolitis. To assess the role of gelatinase B in bleomycin-induced fibrosing alveolitis, we instilled bleomycin intratracheally into gelatinase B-deficient mice and gelatinase B+/+ littermates. Twenty-one days after bleomycin the two groups of mice were indistinguishable in terms of pulmonary histology and total lung collagen and elastin. However, the lungs of gelatinase B-deficient mice showed minimal alveolar bronchiolization, whereas bronchiolization was prominent in the lungs of gelatinase B+/+ mice. Gelatinase B was identified immunohistochemically in terminal bronchiolar cells and bronchiolized cells 7 and 14 days after bleomycin in gelatinase B+/+ mice, and whole lung gelatinase B mRNA was increased at the same times. Many bronchiolized cells displayed Clara cell features by electron microscopy. Some bronchiolized cells stained with antibody to helix transcription factor 4, a factor associated with the ciliated cell phenotype. Thus, fibrosing alveolitis develops after intratracheal bleomycin irrespective of gelatinase B. However, gelatinase B is required for alveolar bronchiolization, perhaps by facilitating migration of Clara cells and other bronchiolar cells into the regions of alveolar injury.

Matrix Metalloproteinase-9 Deficiency Results in Enhanced Allergen-Induced Airway Inflammation

Matrix metalloproteinases (MMPs) are a large family of endopeptidases that proteolytically degrade extracellular matrix. Many different cells produce MMP-9, and levels have been shown to be up-regulated in patients with allergic asthma. The aim of this study was to investigate the in vivo role of MMP-9 during allergen-induced airway inflammation. Acute allergic pulmonary eosinophilia was established in MMP-9 knockout (KO) and wild-type (WT) control mice by sensitization and challenge with OVA. Cell recruitment was significantly increased in both bronchoalveolar lavage (BAL) and lung tissue compartments in MMP-9 KO mice compared with WT mice. This heightened cell recruitment was primarily due to increased eosinophils and Th2 cells in the BAL and lung tissue of MMP-9 KO mice in comparison with WT controls. Moreover, levels of the Th2 cytokines, IL-4 and IL-13, and the chemokines eotaxin/CCL11 and macrophage-derived chemokine/CCL22 were substantially increased in MMP-9 KO mice compared with WT after OVA challenge. Resolution of eosinophilia was similar between MMP-9 KO and WT mice, but Th2 cells persisted in BAL and lungs of MMP-9 KO mice for longer than in WT mice. Our results indicate that MMP-9 is critically involved in the recruitment of eosinophils and Th2 cells to the lung following allergen challenge, and suggest that MMP-9 plays a role in the development of Th2 responses to allergen.

Neutrophils from MMP‐9‐ or neutrophil elastase‐deficient mice show no defect in transendothelial migration under flow in vitro

Recent evidence has suggested a role for neutrophil proteases during certain inflammatory responses. We demonstrated previously that neutrophil proteases can degrade components of the adherens junctions during neutrophil‐endothelial adhesion. We tested the hypothesis that degradation of VE‐cadherin at lateral junctions by elastase or MMP‐9 facilitates neutrophil transendothelial migration. Neutrophils from MMP‐9 or elastase null mice and strain‐matched control mice expressed high levels of LFA‐1, Mac‐1, and L‐selectin on their cell surface. Under flow conditions, wild‐type and deficient neutrophils rolled, arrested, and transmigrated activated murine endothelium. There was no difference in the total numbers of interacting neutrophils or in the percentage of transmigrated cells. In addition, deficient neutrophils remained capable of degrading murine endothelial VE‐cadherin. These results indicate that although neutrophil proteases may play a role in the acute inflammatory response, neutrophil elastase or MMP‐9 is not essential for neutrophil transendothelial migration in this murine system.