IntroductionMetalloproteinases (MPs) play key roles in the responses of cells to their microenvironment. By effecting proteolytic degradation or activation of cell surface and extracellular matrix (ECM) proteins they can modulate both cell-cell and cell-ECM interactions, which influence cell differentiation, migration, proliferation and survival. Both secreted and membranebound forms of metalloproteinases have been implicated in pericellular proteolysis, including the matrix metalloproteinases (MMPs), the adamalysin-like proteinases with both metalloproteinase and disintegrin-like domains (ADAMs and their counterparts that have a thrombospondin-1-like domain, ADAM-TSs) and the astacins (Werb, 1997). Cells use various strategies to regulate extracellular proteinases: transcriptional regulation, trafficking of membrane-bound forms (secretion and endocytosis), activation of latent proenzyme forms, extracellular binding proteins and the action of endogenous inhibitors. Here we will discuss the role of metalloproteinase inhibitors, from the well-known tissue inhibitors of metalloproteinases (TIMPs) and α2-macroglobulin through to newer and less well-understood putative inhibitors (Fig. 1). We look at the available evidence that their other roles in cell biology do not all relate to their metalloproteinase inhibitory activity. Finally, we discuss the potential for use of such natural metalloproteinase inhibitors as therapeutic agents. Tissue inhibitors of metalloproteinases (TIMPs):basic structure and activity Four mammalian TIMPs have been cloned, purified and characterised. These secreted proteins are thought to regulate MMP activity during tisssue remodelling. One TIMP gene has been identified in Drosophila, and its ablation generates a phenotype similar to that of integrin mutants, which indicates that it has a role in ECM function (Godenschwege et al., 2000). All four mammalian TIMPs have many basic similarities, but they exhibit distinctive structural features, biochemical properties and expression patterns (Table 1). This suggests that each TIMP has specific roles in vivo. As in Drosophila, the mammalian TIMP genes are embedded intragenically in intron 5 of synapsin genes (Edwards, 2000).The TIMPs have molecular weights of ~21 kDa and are variably glycosylated (Table 1). They have six disulphide bonds and comprise a three-loop N-terminal domain and an interacting three-loop C-subdomain. Most of the biological functions of these proteins discovered thus far are attributable to sequences within the N-terminal domain, although the Csubdomains mediate interactions with the catalytic domains of some MMPs and with the hemopexin domains of MMP-2 and MMP-9 . The TIMPs are secreted proteins, but may be found at the cell surface in association with membrane-bound proteins; for example, TIMP-2, TIMP-3 and Many of these MMP inhibitors, including the TIMPs, possess other biological activities which may not be related to their inhibitory capacities. These need to be thoroughly characterized in order to allow informed develop...

Adenoviruses are used extensively as gene transfer agents, both experimentally and clinically. However, targeting of liver cells by adenoviruses compromises their potential efficacy. In cell culture, the adenovirus serotype 5 fiber protein engages the coxsackievirus and adenovirus receptor (CAR) to bind cells. Paradoxically, following intravascular delivery, CAR is not used for liver transduction, implicating alternate pathways. Recently, we demonstrated that coagulation factor (F)X directly binds adenovirus leading to liver infection. Here, we show that FX binds to the Ad5 hexon, not fiber, via an interaction between the FX Gla domain and hypervariable regions of the hexon surface. Binding occurs in multiple human adenovirus serotypes. Liver infection by the FX-Ad5 complex is mediated through a heparin-binding exosite in the FX serine protease domain. This study reveals an unanticipated function for hexon in mediating liver gene transfer in vivo.

Cardiac rupture is a fatal complication of acute myocardial infarction lacking treatment. Here, acute myocardial infarction resulted in rupture in wild-type mice and in mice lacking tissue-type plasminogen activator, urokinase receptor, matrix metalloproteinase stromelysin-1 or metalloelastase. Instead, deficiency of urokinase-type plasminogen activator (u-PA-/-) completely protected against rupture, whereas lack of gelatinase-B partially protected against rupture. However, u-PA-/- mice showed impaired scar formation and infarct revascularization, even after treatment with vascular endothelial growth factor, and died of cardiac failure due to depressed contractility, arrhythmias and ischemia. Temporary administration of PA inhibitor-1 or the matrix metalloproteinase-inhibitor TIMP-1 completely protected wild-type mice against rupture but did not abort infarct healing, thus constituting a new approach to prevent cardiac rupture after acute myocardial infarction.

Atherosclerosis is a chronic inflammatory disease of the vasculature commonly leading to myocardial infarction and stroke. We show that IL-33, which is a novel IL-1–like cytokine that signals via ST2, can reduce atherosclerosis development in ApoE−/− mice on a high-fat diet. IL-33 and ST2 are present in the normal and atherosclerotic vasculature of mice and humans. Although control PBS-treated mice developed severe and inflamed atherosclerotic plaques in the aortic sinus, lesion development was profoundly reduced in IL-33–treated animals. IL-33 also markedly increased levels of IL-4, -5, and -13, but decreased levels of IFNγ in serum and lymph node cells. IL-33 treatment also elevated levels of total serum IgA, IgE, and IgG1, but decreased IgG2a, which is consistent with a Th1-to-Th2 switch. IL-33–treated mice also produced significantly elevated antioxidized low-density lipoprotein (ox-LDL) antibodies. Conversely, mice treated with soluble ST2, a decoy receptor that neutralizes IL-33, developed significantly larger atherosclerotic plaques in the aortic sinus of the ApoE−/− mice compared with control IgG-treated mice. Furthermore, coadministration of an anti–IL-5 mAb with IL-33 prevented the reduction in plaque size and reduced the amount of ox-LDL antibodies induced by IL-33. In conclusion, IL-33 may play a protective role in the development of atherosclerosis via the induction of IL-5 and ox-LDL antibodies.

Tissue inhibitor of metalloproteinases-3 (TIMP3) is one of four members of a family of proteins that were originally classified according to their ability to inhibit matrix metalloproteinases (MMP). TIMP3, which encodes a potent angiogenesis inhibitor, is mutated in Sorsby fundus dystrophy, a macular degenerative disease with submacular choroidal neovascularization. In this study we demonstrate the ability of TIMP3 to inhibit vascular endothelial factor (VEGF)-mediated angiogenesis and identify the potential mechanism by which this occurs: TIMP3 blocks the binding of VEGF to VEGF receptor-2 and inhibits downstream signaling and angiogenesis. This property seems to be independent of its MMP-inhibitory activity, indicating a new function for this molecule.

Matrix metalloproteinase (MMPs) enzymes are implicated in matrix remodelling during proliferative inflammatory processes including wound healing. We report here synergistic upregulation of MMP-9 protein and mRNA by platelet-derived growth factor (PDGF) or basic fibroblast growth factor (bFGF) in combination with interleukin-1K K (IL-1K K) or tumour necrosis factor-K K (TNF-K K) in primary rabbit and human dermal fibroblasts. The synergistic interaction between growth factors and cytokines implies that basement membrane remodelling is maximal physiologically when both are present together. The signalling pathways mediating this synergistic regulation are not understood, although analysis of the MMP-9 promoter has identified an essential proximal AP-1 element and an upstream nuclear factor kappa-B (NF-U UB) site. Using electromobility shift assays, binding to the AP-1 site was only slightly increased by growth factors and cytokines. NF-U UB binding was rapidly induced by IL-1K K or TNF-K K but was neither induced nor potentiated by bFGF or PDGF. Neither AP-1 nor NF-U UB was therefore sufficient on its own for synergistic regulation. Using a recently developed adenovirus that overexpresses the inhibitory subunit, IU UBK K, we demonstrated an absolute requirement for NF-U UB in upregulation of MMP-9. Activation of NF-U UB binding by inflammatory cytokines was therefore necessary but not sufficient for synergistic upregulation of MMP-9.z 1998 Federation of European Biochemical Societies.

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Background-Diabetes mellitus impairs endothelial cell (EC) function and postischemic reparative neovascularization by molecular mechanisms that are not fully understood. microRNAs negatively regulate the expression of target genes mainly by interaction in their 3Ј untranslated region. Methods and Results-We found that microRNA-503 (miR-503) expression in ECs is upregulated in culture conditions mimicking diabetes mellitus (high D-glucose) and ischemia-associated starvation (low growth factors). Under normal culture conditions, lentivirus-mediated miR-503-forced expression inhibited EC proliferation, migration, and network formation on Matrigel (comparisons versus lentivirus.GFP control). Conversely, blocking miR-503 activity by either adenovirus-mediated transfer of a miR-503 decoy (Ad.decoymiR-503) or by antimiR-503 (antisense oligonucleotide) improved the functional capacities of ECs cultured under high D-glucose/low growth factors. We identified CCNE1 and cdc25A as direct miR-503 targets which are downregulated by high glucose/low growth factors in ECs. Next, we obtained evidence that miR-503 expression is increased in ischemic limb muscles of streptozotocin-diabetic mice and in ECs enriched from these muscles. Moreover, Ad.decoymiR-503 delivery to the ischemic adductor of diabetic mice corrected diabetes mellitus-induced impairment of postischemic angiogenesis and blood flow recovery. We finally investigated miR-503 and target gene expression in muscular specimens from the amputated ischemic legs of diabetic patients. As controls, calf biopsies of nondiabetic and nonischemic patients undergoing saphenous vein stripping were used. In diabetic muscles, miR-503 expression was remarkably higher, and it inversely correlated with cdc25 protein expression. Plasma miR-503 levels were also elevated in the diabetic individuals. Conclusions-Our Editorial see p 236 Clinical Perspective on p 291Because of their incapacity to regulate glucose influx, endothelial cells (ECs) represent an important target for diabetes mellitus-induced damage. In particular, it is well established that ECs cultured in high glucose show delayed replication, 3,4 abnormal cell cycling, 5 and increased apoptosis. 6 Progression through the cell cycle is a tightly regulated process that includes multiple checkpoints. An orderly ex- The present study is the first to provide evidence for a role of miRNAs in diabetes mellitus-induced endothelial defects contributing to impaired postischemic angiogenesis. In fact, here we show that in vitro culture conditions mimicking diabetes mellitus and ischemia upregulate miR-503 in ECs and that, in vivo, diabetes mellitus increases miR-503 expression in ECs from ischemic limb muscles. We also show that increased miR-503 is responsible for repressed cdc25A and CCNE1 expression in ECs cultured under conditions mimicking diabetes mellitus and ischemia. Moreover, miR-503-forced expression inhibited EC proliferation, migration, and network formation on Matrigel and it additionally reduced vascular smooth muscle ce...