Takeshi Itoh

Matrix metalloproteinase-9 triggers the angiogenic switch during carcinogenesis.

During carcinogenesis of pancreatic islets in transgenic mice, an angiogenic switch activates the quiescent vasculature. Paradoxically, vascular endothelial growth factor (VEGF) and its receptors are expressed constitutively. Nevertheless, a synthetic inhibitor (SU5416) of VEGF signalling impairs angiogenic switching and tumour growth. Two metalloproteinases, MMP-2/gelatinase-A and MMP-9/gelatinase-B, are upregulated in angiogenic lesions. MMP-9 can render normal islets angiogenic, releasing VEGF. MMP inhibitors reduce angiogenic switching, and tumour number and growth, as does genetic ablation of MMP-9. Absence of MMP-2 does not impair induction of angiogenesis, but retards tumour growth, whereas lack of urokinase has no effect. Our results show that MMP-9 is a component of the angiogenic switch.

Experimental metastasis is suppressed in MMP-9-deficient mice

Matrix metalloproteinases (MMPs) are thought to play a key role in tumor invasion and metastasis. The role of MMP-9 (gelatinase B) in tumor metastasis was examined in MMP-9-deficient mice produced by gene targeting using embryonic stem cells. MMP-9-deficient mice develop normally and are fertile. In these mice, the number of metastatic colonies of B16-BL6 melanoma cells or Lewis lung carcinoma cells that were implanted intravenously fell by 45% for B16-BL6 melanoma and 59% for Lewis lung carcinoma (p=0.03 and p=0.0043, respectively). Gelatin zymography showed that both tumor cell lines did not secrete MMP-9 by themselves but the host cells surrounding the tumor cells secrete MMP-9 in vivo. These results indicated that host-derived MMP-9 plays an important role in the process of tumor metastasis.

Mice devoid of the glial fibrillary acidic protein develop normally and are susceptible to scrapie prions

Glial fibrillary acidic protein (GFAP) is an intermediate filament protein specifically expressed in astrocytes in the CNS. To examine the function of GFAP in vivo, the Gfap gene was disrupted by gene targeting in embryonic stem cells. Mice homozygous for the mutation were completely devoid of GFAP but exhibited normal development and showed no obvious anatomical abnormalities in the CNS. When inoculated with infectious scrapie prions, the mutant mice exhibited neuropathological changes typical of prion diseases. Infectious prions accumulated in brains of the mutant mice to a degree similar to that in control littermates. These results suggest that GFAP is not essential for the morphogenesis of the CNS or for astrocytic responses against neuronal injury. The results argue against the hypothesis that GFAP plays a crucial role in the pathogenesis of prion diseases.

Inhibition of matrix metalloproteinases blocks lethal hepatitis and apoptosis induced by tumor necrosis factor and allows safe antitumor therapy

Acute and fulminant liver failure induced by viral hepatitis, alcohol or other hepatotoxic drugs, are associated with tumor necrosis factor (TNF) production. In a mouse model of lethal hepatitis induced by TNF, apoptosis and necrosis of hepatocytes, but also lethality, hypothermia and influx of leukocytes into the liver, are prevented by a broad-spectrum matrix metalloproteinase (MMP) inhibitor, BB-94. Mice deficient in MMP-2, MMP-3 or MMP-9 had lower levels of apoptosis and necrosis of hepatocytes, and better survival. We found induction of MMP-9 activity and fibronectin degradation. Our findings suggest that several MMPs play a critical role in acute, fulminant hepatitis by degrading the extracellular matrix and allowing massive leukocyte influx in the liver. BB-94 also prevented lethality in TNF/interferon-γ therapy in tumor-bearing mice. A broad-spectrum MMP inhibitor may be potentially useful for the treatment of patients with acute and perhaps chronic liver failure, and in cancer therapies using inflammatory cytokines.

Contribution of host MMP-2 and MMP-9 to promote tumor vascularization and invasion of malignant keratinocytes

The matrix metalloproteinases (MMPs) play a key role in normal and pathological angiogenesis by mediating extracellular matrix degradation and/or controlling the biological activity of growth factors, chemokines, and/or cytokines. Specific functions of individual MMPs as anti‐ or proangiogenic mediators remain to be elucidated. In the present study, we assessed the impact of single or combined MMP deficiencies in in vivo and in vitro models of angiogenesis (malignant keratinocyte transplantation and the aortic ring assay, respectively). MMP‐9 was predominantly expressed by neutrophils in tumor transplants, whereas MMP‐2 and MMP‐3 were stromal. Neither the single deficiency of MMP‐2, MMP‐3, or MMP‐9, nor the combined absence of MMP‐9 and MMP‐3 did impair tumor invasion and vascularization in vivo. However, there was a striking cooperative effect in double MMP‐2:MMP‐9‐deficient mice as demonstrated by the absence of tumor vascularization and invasion. In contrast, the combined lack of MMP‐2 and MMP‐9 did not impair the in vitro capillary outgrowth from aortic rings. These results point to the importance of a cross talk between several host cells for the in vivo tumor promoting and angiogenic effects of MMP‐2 and MMP‐9. Our data demonstrate for the first time in an experimental model that MMP‐2 and MMP‐9 cooperate in promoting the in vivo invasive and angiogenic phenotype of malignant keratinocytes.

MMP-2 and MMP-9 synergize in promoting choroidal neovascularization

Matrix metalloproteinase 2 (MMP‐2) and MMP‐9 are increased in human choroidal neovascularization (CNV) occurring during the exudative most aggressive form of age‐related macular degeneration (AMD), but their precise role and potential interactions remain unclear. To address the question of MMP‐2 and MMP‐9 functions, mice deficient in the expression of MMP‐ 2 (MMP‐2 KO), MMP‐9 (MMP‐9 KO), and both MMP‐2 and MMP‐9 (MMP‐2,9 KO) with their corresponding wild‐type mice (WT) underwent CNV induction by laser‐induced rupture of the Bruchs membrane. Both the incidence and the severity of CNV were strongly attenuated in double deficient compared with single gene deficient mice or corresponding WT controls. The reduced neovascularization was accompanied by fibrinogen/fibrin accumulation. Furthermore, overexpression of the endogenous MMP inhibitors TIMP‐1 or TIMP‐2 (delivered by adenoviral vectors) in WT mice or daily injection of a synthetic and gelatinase selective MMP inhibitor (Ro 26‐2853) significantly decreased the pathological reaction. These findings suggest that MMP‐2 and MMP‐9 may cooperate in the development of AMD and that their selective inhibition represents an alternative strategy for the treatment of choroidal neovascularization.

A crucial role for matrix metalloproteinase 2 in osteocytic canalicular formation and bone metabolism

Extracellular matrix production and degradation by bone cells are critical steps in bone metabolism. Mutations of the gene encoding MMP-2, an extracellular matrix-degrading enzyme, are associated with a human genetic disorder characterized by subcutaneous nodules, arthropathy, and focal osteolysis. It is not known how the loss of MMP-2 function results in the pathology. Here, we show that Mmp2-/- mice exhibited opposing bone phenotypes caused by an impaired osteocytic canalicular network. Mmp2-/- mice showed decreased bone mineral density in the limb and trunk bones but increased bone volume in the calvariae. In the long bones, there was moderate disruption of the osteocytic networks and reduced bone density throughout life, whereas osteoblast and osteoclast function was normal. In contrast, aged but not young Mmp2-/- mice had calvarial sclerosis with osteocyte death. Severe disruption of the osteocytic networks preceded osteocyte loss in Mmp2-/- calvariae. Successful transplantation of wild-type periosteum restored the osteocytic canalicular networks in the Mmp2-/- calvariae, suggesting local roles of MMP-2 in determining bone phenotypes. Our results indicate that MMP-2 plays a crucial role in forming and maintaining the osteocytic canalicular network, and we propose that osteocytic network formation is a determinant of bone remodeling and mineralization.

Resistance to Endotoxic Shock in Phospholipase A2 Receptor-deficient Mice

Mammals possess various types of secretory phospholipase A2, which differ in the primary structure and tissue distribution. The phosholipase A2receptor (PLA2R) recognizes group IB phospholipase A2 (PLA2-IB) and mediates the PLA2-IB-induced biological responses in non-digestive organs, including eicosanoid production and contraction of airway smooth muscles. In this study, we generated PLA2R-deficient mice to define its biological roles further. These mice are viable, fertile, and without evident histopathological abnormalities. There was no difference in the clearance of circulating PLA2-IB between wild-type and mutant mice. After challenge with bacterial lipopolysaccharide (LPS), PLA2R-deficient mice exhibited longer survival than wild-type mice. The mutant mice were also resistant to lethal effects of exogenous PLA2-IB after sensitization with sublethal dose of LPS. The plasma levels of tumor necrosis factor-α and interleukin-1β elevated after LPS treatment were significantly reduced in mutant mice compared with wild-type mice. These findings suggest a potential role of PLA2R in the progression of endotoxic shock.

Mutations in two matrix metalloproteinase genes, MMP-2 and MT1-MMP, are synthetic lethal in mice

The matrix metalloproteinase (MMP) family (∼25 members in mammals) has been implicated in extracellular matrix remodeling associated with embryonic development, cancer formation and progression, and various other physiological and pathological events. Inactivating mutations in individual matrix metalloproteinase genes in mice described so far, however, are nonlethal at least up to the first few weeks after birth, suggesting functional redundancy among MMP family members. Here, we report that mice lacking two MMPs, MMP-2 (nonmembrane type) and MT1-MMP (membrane type), die immediately after birth with respiratory failure, abnormal blood vessels, and immature muscle fibers reminiscent of central core disease. In the absence of MMP-2 and MT1-MMP, myoblast fusion in vitro is also significantly retarded. These findings suggest functional overlap in mice between the two MMPs with distinct molecular natures.

Stroma-Derived Matrix Metalloproteinase (MMP)-2 Promotes Membrane Type 1-MMP–Dependent Tumor Growth in Mice

Matrix metalloproteinase-2 (MMP-2) is a stroma-derived MMP belonging to the type IV collagenase family. It is believed to mediate tumor cell behavior by degrading deposits of type IV collagen, a major component of the basement membrane. The membrane type 1-MMP (MT1-MMP) is a highly potent activator of MMP-2 and is expressed in many tumor and stromal cells. However, the roles played by stromal MMP-2 in tumor progression in vivo remain poorly understood. We established a colon epithelial cell line from an Mt1-mmp(-/-) mouse strain and transfected these cells with an inducible expression system for MT1-MMP (MT1rev cells). Following s.c. implantation into Mmp-2(+/+) mice and induction of MT1-MMP expression, MT1rev cells grew rapidly, whereas they grew very slowly in Mmp-2(-/-) mice, even in the presence of MT1-MMP. This MT1-MMP-dependent tumor growth of MT1rev cells was enhanced in Mmp-2(-/-) mice as long as MMP-2 was supplied via transfection or coimplantation of MMP-2-positive fibroblasts. MT1rev cells cultured in vitro in a three-dimensional collagen gel matrix also required the MT1-MMP/MMP-2 axis for rapid proliferation. MT1rev cells deposit type IV collagen primarily at the cell-collagen interface, and these deposits seem scarce at sites of invasion and proliferation. These data suggest that cooperation between stroma-derived MMP-2 and tumor-derived MT1-MMP may play a role in tumor invasion and proliferation via remodeling of the tumor-associated basement membrane. To our knowledge, this is the first study demonstrating that MT1-MMP-dependent tumor growth in vivo requires stromal-derived MMP-2. It also suggests that MMP-2 represents a potential target for tumor therapeutics.