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.

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 deletion of MMP-2 attenuates early LV rupture and late remodeling after experimental myocardial infarction. Am J Physiol Heart Circ Physiol 285: H1229-H1235, 2003. First published May 29, 2003 10.1152/ ajpheart.00207.2003.-Matrix metalloproteinase-2 (MMP-2) is prominently overexpressed both after myocardial infarction (MI) and in heart failure. However, its pathophysiological significance in these conditions is still unclear. We thus examined the effects of targeted deletion of MMP-2 on post-MI left ventricular (LV) remodeling and failure. Anterior MI was produced in 10-to 12-wk-old male MMP-2 knockout (KO) and sibling wild-type (WT) mice by ligating the left coronary artery. By day 28, MI resulted in a significant increase in mortality in association with LV cavity dilatation and dysfunction. The MMP-2 KO mice had a significantly better survival rate than WT mice (56% vs. 85%, P Ͻ 0.05), despite a comparable infarct size (50 Ϯ 3% vs. 51 Ϯ 3%, P ϭ not significant), heart rate, and arterial blood pressure. The KO mice had a significantly lower incidence of LV rupture (10% vs. 39%, P Ͻ 0.05), which occurred within 7 days of MI. The KO mice exerted less LV cavity dilatation and improved fractional shortening after MI by echocardiography. The LV zymographic MMP-2 level significantly increased in WT mice after coronary artery ligation; however, this was completely prevented in KO mice. In contrast, the increase in the LV zymographic MMP-9 level after MI was similar between KO and WT mice. MMP-2 activation is therefore considered to contribute to an early cardiac rupture as well as late LV remodeling after MI. The inhibition of MMP-2 activation may therefore be a potentially useful therapeutic strategy to manage post-MI hearts. matrix metalloproteinase; cardiac rupture; heart failure; myocyte; extracellular matrix; mouse MYOCARDIAL INFARCTION (MI) leads to complex structural alterations (remodeling) involving both the infarcted and noninfarcted left ventricular (LV) myocardium (21). Early remodeling as LV cavity dilatation occurs during the early phase of MI, which is likely due to wall thinning in the infarct region. This might lead to a cardiac rupture, thereby accounting for the 5-30% of in-hospital mortality after acute MI (1). During the first several days, LV enlargement follows, and, thereafter, a progressive dilatation of the noninfarcted LV occurs over weeks (21). These progressive changes in LV geometry contribute to the development of depressed cardiac function, clinical heart failure, and increased mortality. Accordingly, it is of critical importance to explore the mechanisms of LV remodeling and develop therapeutic strategies that will effectively inhibit this deleterious process.The dynamic synthesis and breakdown of extracellular matrix (ECM) proteins play an important role in post-MI LV remodeling. In particular, the increased expression and activation of matrix metalloproteinases (MMPs) have been implicated in this process (4,5). Several studies have demonstrated that MMPs are involved not...

The ␤-amyloid peptide, which forms extracellular cerebral deposits in Alzheimer's disease, is derived from a large membrane-spanning glycoprotein referred to as the ␤-amyloid precursor protein (APP). The APP is normally cleaved within the ␤-amyloid region by a putative proteinase (␣-secretase) to generate large soluble amino-terminal derivatives of APP, and this event prevents the ␤-amyloid peptide formation. It has been suggested that the gelatinase A (matrix metalloproteinase 2, a 72-kDa type IV collagenase) may act either as ␣-secretase or as ␤-secretase. Mice devoid of gelatinase A generated by gene targeting develop normally, except for a subtle delay in their growth, thus providing a useful system to examine the role of gelatinase A in the cleavage and secretion of APP in vivo. We show here that APP is cleaved within the ␤-amyloid region and secreted into the extracellular milieu of brain and cultured fibroblasts without gelatinase A activity. The data suggest that gelatinase A does not play an essential role in the generation and release of soluble derivatives of APP at physiological conditions. Amyloid precursor protein (APP) 1 is an integral membrane protein that is produced by most cells (1). Several isoforms ranging from 365 to 770 amino acids are generated by alternative splicing of transcripts from the APP gene on the long arm of chromosome 21 (2). Proteolytic cleavage of APP by enzymes termed ␣-, ␤-, and ␥-secretases generates various APP fragments that are released from APP expressing cells (3). ␤-Amyloid peptides (A␤) of 40 -43 amino acids are released by the action of ␤-and ␥-secretases cleaving at or near residues 671 and 713 (numbers refer to APP 770 ), respectively (3). Cleavage of APP at a membrane proximal site by an ␣-secretase releases larger APP fragments (sAPP) and prevents generation of A␤. The absolute and relative amounts of various APP fragments that are released in the brain are thought to be of importance in the formation of first amorphous (diffuse) and then filamentous (amyloid) plaques that are characteristic of Alzheimer's disease. Two larger forms sAPP also known as protease nexin II have a Kunitz type serine protease inhibitor domain (4, 5). In addition all forms of sAPP have in the carboxyl-terminal region a domain that inhibits gelatinase A (matrix metalloproteinase 2, a 72-kDa type IV collagenase) activity (6). On the other hand, gelatinase A has been suggested to act on APP either as ␣-secretase (6) or as ␤-secretase (7), although the hypothesis is controversial (8, 9). To clarify the putative role of this enzyme, we studied APP fragmentation and release in gelatinase A knockout mice. MATERIALS AND METHODSGeneration of Gelatinase A Gene-deficient Mice-A genomic DNA clone of mouse gelatinase A (Clg4a) was isolated from a 129/Sv genomic library. The fragments used for constructing the targeting vector were a 2.0-kb HindIII fragment of the distal region of the promoter and a 4.5-kb XbaI-SacI fragment. In the resulting targeting construct, 5.9 kb containing the exon 1 were r...

Matrix metalloproteinases (MMPs) are a large group of enzymes responsible for matrix degradation. Among them, the family of gelatinases (MMP-2/gelatinase A and MMP-9/gelatinase B) is overproduced in the joints of patients with rheumatoid arthritis. Because of their degradative effects on the extracellular matrix, gelatinases have been believed to play an important role in progression and cartilage degradation in this disease, although their precise roles are yet to be defined. To clarify these roles, we investigated the development of Ab-induced arthritis, one of the murine models of rheumatoid arthritis, in MMP-2 or MMP-9 knockout (KO) mice. Surprisingly, the MMP-2 KO mice exhibited severe clinical and histologic arthritis than wild-type mice. The MMP-9 KO mice displayed milder arthritis. Recovery from exacerbated arthritis in the MMP-2 KO mice was possible by injection of wild-type fibroblasts. These results indicated a suppressive role of MMP-2 and a pivotal role of MMP-9 in the development of inflammatory joint disease.

Bone is continuously remodeled to adopt a volume appropriate for the local environment; the amount of bone deposited depends on the balance between bone formation and resorption by bone cells, osteoblasts, osteoclasts, and osteocytes (1). Osteoblasts are bone-forming cells that differentiate from mesenchymal stem cells and secrete extracellular matrix (ECM) 4 proteins, which are subsequently mineralized. Osteoclasts are bone-resorbing cells that differentiate from hematopoietic stem cells and degrade bone ECM proteins after demineralization in the extracellular space (Howship's lacunae) adjacent to the ruffled borders. In contrast to osteoblasts and osteoclasts, which act at bone surfaces, osteocytes, cells of osteoblastic lineage, are embedded in bone and are terminally differentiated. Osteocytes extend their dendritic processes into the bone matrix to constitute a well developed canalicular network with other cells. Although osteocytes are the most abundant cell type in bone tissue, their role in bone metabolism is not firmly established.ECM production and degradation by bone cells are critical steps in bone metabolism (1), and disturbed ECM turnover leads to bone disease. Type I collagen is a major ECM component. Secreted type I collagen molecules are processed by propeptidases and cross-linked by lysyl oxidases into mature collagen. Mutations of genes encoding type I collagen cause the bone disease osteogenesis imperfecta (2). Type I collagens are mainly degraded by matrix metalloproteinases (MMPs), which exert their enzymatic activity at a neutral pH in a zinc ion-dependent manner (3, 4). Several MMPs are expressed in bone tissue (5-9). MMPs may play a role in osteoclastic bone resorption (4, 5). Osteoblasts and osteocytes also produce MMPs such as MMP-2 and MMP-13 (7-9). Recent linkage analysis suggests that a loss of function mutation of MMP2 causes a human autosomal recessive disorder with multicentric nodulosis, arthropathy with joint erosion, and osteolysis, termed NAO syndrome (10, 11). This syndrome also includes facial abnormalities and generalized * This work was supported in part by a grant from the Ministry for Welfare and Health of Japan (to S. I.), by a Sasagawa Scientific Research Grant from the Japan Science Society (to K. I.), by a grant from the Japan Space Forum, National Space Development Agency of Japan (NASDA) (to N. M.), and by a research grant from the National Institutes of Health (to S. M. K.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 4 The abbreviations used are: ECM, extracellular matrix; DMP-1, dentin matrix protein 1; DMEM, Dulbecco's modified Eagle's medium; MMP, matrix metalloproteinase; NAO, nodulosis, arthropathy, and osteolysis; BMD, bone mineral density; pQCT, peripheral quantitative computed tomography; MAR, mineral apposition rate; BFR/BS, ratio of bone formation rate to bone surface.

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.

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-gamma 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.