Inhibition Of MMP-9 Activity Research Articles

Implication of matrix metalloproteinases (MMPs) in transmural inflammation induced by bacteroides fragilis

Background: Anaerobic bacteria, such as Bacteroides fragilis (Bf), may be implicated in the pathophysiology of intestinal inflammation and can induce transmural inflammatory responses with serosal involvement. Bf produce a zinc-dependent MMP, that could be responsible of damage to intestinal cells and bacterial translocatinn, since MMPs are endopeptidases capable to degrade extracelfular matrix. Aim: To investigate the impact of inhibition of MMP-activity in a model of colitis induced by intramural inyection of Bf in male Sprague Dawley rats (220-250 g). Methods: Bf and E. coli (Ec) were grown in liquid media during 24 h under appropriate conditions. Under anesthesia, a midline abdominal incision was performed, and the left colon was identified. Rats were inoculated into the colonic wall at 2-4 cm from the distal end with either Bf or Ec suspensions, and controls were inoculated with the respective liquid medium without bacteria. The MMP-inhibitor, phenantroline, was administered to test groups by oral gavage at 20 mg/Kg 1 h before, and 6 and 24 h after bacteria inoculation, whereas matched groups received oral vehicle. Colons were removed on day 7 after inoculation for blind microscopic assessment of lesions. A score was obtained by grading inflammation (0-6), fibrosis (0-3), presence of grannlomas (0-3), and counts of myofibroblasts (alpha-actin staining, 0-3) in the mucosa and the serosa. Results: Control rats inoculated with sterile media did not show any histological change in the colonic wall (score <: 1). Transmural inflammatory lesions were observed in most rats inoculated with bacteria. Treatment with phenantroline sigmficauily decreased total histological score and serosal involvement in rots inoculated with Bf, and reduced mucosal but not serosal involvement in rats inoculated with Ec (table) Conclusion: A MMP-inhibitor substantially nuttgates intestinal injury in this experimental model of colitis. Our data suggest that bacterial MMPs may be implicated in transmural migration of Bacteroides fragilis.

N-Myc and Bcl-2 coexpression induces MMP-2 secretion and activation in human neuroblastoma cells

Neuroblastoma is a peripheral nervous system tumor that accounts for 8-10% of all solid childhood tumors. N-Myc is the most reliable prognostic indicator for neuroblastoma. Bcl-2 is detected in 40-60% of primary neuroblastoma tumors and demonstrates anti-apoptotic action by conferring resistance to chemotherapy and radiation treatment. In neuroblastoma cell lines, the coexpression of N-Myc and Bcl-2 leads to increased tumorigenic properties. Matrix metalloproteinases (MMPs) are endopeptidases that degrade a wide range of basement membrane components, a process important for tumor invasion. This study investigates the effect of N-Myc and Bcl-2 on MMP expression and activation. MMP-2 expression and secretion are increased in SHEP neuroblastoma cells expressing Bcl-2 alone (SHEP/Bcl-2 cells) or both N-Myc and Bcl-2 (SHEP/N-Myc/Bcl-2 cells). MMP-2 activity is increased in the SHEP/N-Myc/Bcl-2 cells yet remains unchanged in SHEP/Bcl-2 cells. TIMP-2 expression is high in SHEP/Bcl-2 cells, which likely inhibits MMP-2 activity, and absent in SHEP/N-Myc/Bcl-2 cells, allowing MMP-2 activity. Invasion is increased in SHEP/N-Myc/Bcl-2 cells and prevented by the use of a pharmacologic MMP-2 inhibitor. These data imply that N-Myc and Bcl-2 cooperate to increase the expression, secretion, and activation of MMP-2, which likely leads to a more tumorigenic phenotype due to increased MMP-2 mediated invasion.

PAI-1 promotes extracellular matrix deposition in the airways of a murine asthma model

Dysregulation of matrix metalloproteinases (MMPs) and ineffective fibrinolysis are associated with the deposition of extracellular matrix (ECM). We hypothesized that elevated plasminogen activator inhibitor (PAI)-1 promotes ECM deposition in the asthmatic airway by inhibiting MMP-9 activity and fibrinolysis. Degree of airway inflammation was similar in PAI-1 −/− and wild type (WT) mice after ovalbumin (OVA) challenge. PAI-1 production, deposition of collagen and fibrin, and MMP-9 activity in the lung tissue or airways were greater after OVA challenge compared with saline challenge. However, in PAI-1 −/− mice, collagen deposition was 2-fold less, fibrin deposition was 4-fold less, and MMP-9 activity was 3-fold higher. This is the first direct evidence that the plasmin system regulates ECM deposition in the airways of a murine asthma model, independently of the effect of PAI-1 on inflammatory cells. The results suggest that the PAI-1-dependent inhibition of MMP-9 activity and fibrinolysis is a major mechanism by which ECM deposition occurs.

Up-regulation of MMP-2 and MMP-9 Leads to Degradation of Type IV Collagen During Skeletal Muscle Reperfusion Injury; Protection by the MMP Inhibitor, Doxycycline

Objectives: to determine the role of matrix metalloproteinases, MMP-2 and MMP-9, in reperfusion injury following skeletal muscle ischaemia and whether inhibition of MMPs by doxycycline protects against tissue damage.Methods : rats were anaesthetised and a tourniquet applied to the proximal thigh to occlude blood flow. Four hours of ischaemia was followed by reperfusion for 0, 4, 24 or 72 h. Two further groups received doxycycline for 7 days prior to bilateral ischaemia and 24 h reperfusion. Skeletal muscle from both limbs, kidneys and lungs were harvested for zymography and immunohistochemical staining for type IV collagen. Results: upregulation of MMP-2 and MMP-9 was detected by zymography in the ischaemic leg and lung but not in the kidney. Quantitative immunohistochemical analysis showed marked degradation of type IV collagen in reperfused muscle, lung and kidney. Doxycycline-treated rats showed significant preservation of type IV collagen in skeletal muscle and a trend towards preservation in kidney and lung. Conclusions: MMP-2 and MMP-9 are strongly upregulated in skeletal muscle ischaemia/reperfusion injury and are also upregulated in remote organs, leading to degradation of basement membranes. Inhibition of MMP activity may therefore be potentially therapeutically useful in reducing the severity of reperfusion injury.

Pirfenidone Inhibits Early Myointimal Proliferation but has No Effect on Late Lesion Size in Rats

Aims: intimal hyperplasia is mediated by smooth muscle cell proliferation, migration and deposition of extracellular matrix. The anti-fibrotic agent pirfenidone has been shown to inhibit pro-fibrotic growth factors in non-vascular inflammatory models. This study investigated the effect of the novel anti-fibrotic agent pirfenidone on the development of neointima. Methods: male Sprague–Dawley rats received either standard diet or diet supplemented with pirfenidone (250, 500, 1000 mg/kg/day). Animals underwent left common carotid balloon angioplasty and were explanted at 4, 8, 14 and 28 days and analysed for intimal thickening, pro-fibrotic gene expression, extracellular matrix deposition and metalloproteinase activity. Results: neointimal thickness was significantly reduced in a dose-dependent manner at 14 days; pirfenidone 250 mg/kg (p<0.005), pirfenidone 500 mg/kg (p<0.001), pirfenidone 1 g/kg (p <0.001). There were no significant differences in intimal thickening at 28 days. Expression of MMP-2, MMP-9, TIMP-1, collagen III and TGF-beta were all significantly inhibited at 14 days. Both collagen III expression and ECM deposition were reduced at 28 days ( p<0.05 and <0.002 respectively). Conclusion: pirfenidone reduces expression of MMPs governing smooth muscle cell proliferation and migration (MMP-2 and 9), and genes favouring ECM accumulation (TIMP-1 and collagen III). This study shows that inhibition of MMP activity is not sufficient to inhibit late lesion size.

Dexamethasone Inhibits Vascular Smooth Muscle Cell Migration via Modulation of Matrix Metalloproteinase Activity

Background. Dexamethasone (DEX) has been shown to inhibit development of neointimal hyperplasia in rats. We hypothesize that DEX inhibits neointimal hyperplasia by altering matrix metalloproteinase (MMP) activity, resulting in inhibition of smooth muscle cell migration.Methods. Rat aortic smooth muscle cells (RASMC) were harvested and cultured for two to four passages. A migration assay was performed in a Boyden chamber with chemoattractant (platelet-derived growth factor) and varying concentrations of DEX (10−9 to 10−5 M). The number of migrated cells was counted under light microscopy. Zymography was performed on culture media to assess MMP activity, and Western blotting was performed to assay MMP and levels of tissue inhibitors of MMPs (TIMPs).Results. DEX progressively inhibited RASMC migration in a dose-dependent fashion. This effect was statistically significant for concentrations of 10−7 to 10−5 M (P < 0.0005). Zymography showed that DEX inhibits MMP-2 activity in a dose-dependent manner. Western blots indicated that total MMP-2 secretion was inhibited and that TIMP-2 secretion was increased by DEX.Conclusions. DEX inhibits platelet-derived growth factor-induced migration of RASMCs and MMP-2 activity in vitro. Our data suggest that DEX suppresses MMP activity and secretion, resulting in the inhibition of smooth muscle cell migration. This may explain the mechanism by which DEX inhibits neointimal hyperplasia.

Matrix metalloproteinase inhibitor development for the treatment of heart failure

AbstractMatrix metalloprotease inhibitor (MMPi) drug discovery initially focused on the therapeutic use of these compounds as antineoplastic and antiarthritis agents to reduce metastases and joint matrix degradation, respectively. However, increased MMP expression has been shown to occur after myocardial ischemia (MI) and during progressive heart failure (HF). Inhibition of MMP activity has been shown to limit left ventricular aneurysm, slow atherosclerotic plaque progression, and reduce left ventricular dilation after MI in mice as well as preserving left ventricular geometry and function in several animal HF models. A growing literature indicates that MMPi treatment may represent a novel therapeutic strategy in the treatment for a variety of cardiovascular diseases, especially prevention of heart failure. The objective of this article is to define the role of MMPs in HF, identify which MMPs are upregulated, and outline potential limitations of MMPi therapy in this disease. Drug Dev. Res. 55:29–44, 2002. © 2002 Wiley‐Liss, Inc.

Inactivation of tissue inhibitor of metalloproteinases-1 (TIMP-1) by Porphyromonas gingivalis

In response to periodontal inflammation, host cells release matrix metalloproteinases (MMPs) that contribute to periodontal tissue breakdown unless the tissue inhibitors of metalloproteinases (TIMPs) neutralize their activity. In this study, the capacity of Porphyromonas gingivalis to inactivate TIMP-1 was investigated. Proteolytic digestion of TIMP-1 was monitored by SDS–PAGE and Western immunoblotting. Planktonic cells and biofilms of P. gingivalis degraded TIMP-1 with production of several lower molecular mass fragments. Incorporation of human serum in the assay mixture had no effect on the degradation of TIMP-1 by P. gingivalis, whereas a cysteine proteinase inhibitor caused a complete inhibition. Using a fluorogenic assay, it was found that TIMP-1 treated with P. gingivalis lost its capacity to inhibit MMP-9 activity. This study revealed the potential of P. gingivalis to inactivate TIMP-1 through proteolytic degradation. This phenomenon may contribute to increasing significantly the level of active MMPs in affected periodontal sites and subsequently favor tissue destruction.

Inactivation of tissue inhibitor of metalloproteinases-1 (TIMP-1) by Porphyromonas gingivalis.

In response to periodontal inflammation, host cells release matrix metalloproteinases (MMPs) that contribute to periodontal tissue breakdown unless the tissue inhibitors of metalloproteinases (TIMPs) neutralize their activity. In this study, the capacity of Porphyromonas gingivalis to inactivate TIMP-1 was investigated. Proteolytic digestion of TIMP-1 was monitored by SDS-PAGE and Western immunoblotting. Planktonic cells and biofilms of P. gingivalis degraded TIMP-1 with production of several lower molecular mass fragments. Incorporation of human serum in the assay mixture had no effect on the degradation of TIMP-1 by P. gingivalis, whereas a cysteine proteinase inhibitor caused a complete inhibition. Using a fluorogenic assay, it was found that TIMP-1 treated with P. gingivalis lost its capacity to inhibit MMP-9 activity. This study revealed the potential of P. gingivalis to inactivate TIMP-1 through proteolytic degradation. This phenomenon may contribute to increasing significantly the level of active MMPs in affected periodontal sites and subsequently favor tissue destruction.

Modulation of airway remodeling-associated mediators by the antifibrotic compound, pirfenidone, and the matrix metalloproteinase inhibitor, batimastat, during acute lung injury in mice

Matrix metalloproteinases (MMPs) are potent to degrade basement membrane collagen associated with acute lung injury in inflammatory processes. We have investigated effects of pirfenidone, antifibrotic agent, and batimastat, inhibitor of MMPs, on gelatinase activities, on release of tumor necrosis factor-α (TNF-α) and transforming growth factor-β (TGF-β), as well as on recruitment of inflammatory cells in bronchoalveolar lavage (BAL) fluid after aerosol administration of lipopolysaccharide (LPS) in mice. Pretreatment with pirfenidone reduced neutrophil recruitment, TNF-α and TGF-β levels, and MMP-9 secretion. In contrast, pretreatment with batimastat (30 or 60 mg/kg, i.p.) only reduced TNF-α and TGF-β levels. Batimastat did not reduce MMP secretion in BAL fluid but inhibited MMP-9 activity. The increase in tissue inhibitor of matrix metalloproteinase (TIMP)-1 induced by LPS was not modified by the two drugs. These findings demonstrate that the two drugs can inhibit the in vivo increase in MMP induced by LPS, batimastat with a direct inhibitor effect on MMP activity and pirfenidone as a consequence of its antiinflammatory effect.

CD40L Induces Matrix-Metalloproteinase-9 but Not Tissue Inhibitor of Metalloproteinases-1 in Cervical Carcinoma Cells: Imbalance between NF-κB and STAT3 Activation

Matrix-metalloproteinases (MMPs) are essentially required for tumor cell invasion and metastasis. Production of precursor enzymes is regulated on transcriptional level, while activation of the pro-enzymes is tightly controlled by posttranscriptional mechanisms. The enzyme activity can be blocked by specific tissue inhibitors of MMPs (TIMPs). In cervical carcinomas strong up-regulation of the type IV collagenase MMP-9 had been demonstrated. We show that activation of CD40, a receptor highly expressed on cervical carcinomas, induces MMP-9 in cervical carcinoma cells, whereas TIMP-1 production inhibiting MMP-9 activity was not affected. This gene induction pattern corresponded to the differential activation of the transcription factor nuclear factor κB (NF-κB) regulating MMP-9, but not signal transducer and activator of transcription 3 (STAT3), which is involved in TIMP-1 gene regulation. Transient expression of the CD40-inducible NF-κB subunit p65 was sufficient for MMP-9 induction. Agents that suppressed CD40-mediated NF-κB activation also reduced MMP-9 induction, further supporting an important role of NF-κB in CD40-mediated MMP-9 induction. Our data suggest that CD40 expression in carcinoma cells might convert a CD40L-dependent immunological defense signal into a tumor-promoting signal. Selective CD40-mediated signaling through NF-κB but not STAT3 correlates to a shift of the balance between MMP-9 and TIMP-1 toward the protease.

Doxazosin inhibits monocyte chemotactic protein 1-directed migration of human monocytes.

Monocyte chemotactic protein 1 (MCP-1)-directed transendothelial migration of monocytes plays a key role in the early development of atherosclerosis. Migration of monocytes requires degradation of extracellular matrices, a process that involves matrix metalloproteinases (MMP) and tissue inhibitors of MMPs (TIMP). Recent studies suggest that the alpha1-adrenergic receptor antagonist doxazosin (Dox) might have antiatherosclerotic effects, although the underlying mechanisms are poorly understood. The purpose of the present study was to determine the effects of Dox on MCP-1-directed monocyte migration, MMP-9 activity, and TIMP-1 expression. MCP-1 (50 ng/ml) stimulated migration of human peripheral blood monocytes (HPBM) 2.7+/-0.42-fold and THP-1 human monocytes 5.9+/-0.83-fold compared with unstimulated control. Dox inhibited MCP-1-induced migration in a dose-dependent manner, with a maximal reduction at 10 microM of 69.5+/-5.9% in HPBM and 72.2+/-3.2% in THP-1 cells. Dox blocked migration even after pretreatment with phenoxybenzamine, an irreversible alpha1-adrenergic receptor antagonist (HPBM: phenoxybenzamine 1 microM + Dox 10 microM, 71.9+/-2.2% inhibition; THP-1 cells: phenoxybenzamine 1 microM + Dox 10 microM: 78+/-7.7% inhibition), suggesting that the antimigratory activity of Dox is mediated through a novel mechanism unrelated to its blocking of the alpha1-adrenergic receptor. Dox (10 microM) inhibited MMP-9 activity by 67.6+/-10.5%, whereas MMP-9 protein levels were not affected. Also, Dox increased PMA-induced-tissue inhibitor of MMPs-1 (TIMP-1) expression by 134.4+/-6.6%. Dox 10 microM. The present study demonstrates a potential novel antiatherosclerotic action of Dox by blocking MCP-1-directed monocyte migration, which might be partly mediated by inhibition of MMP-9 activity.

Aspirin Inhibits Matrix Metalloproteinase-2 Activity, Increases E-Cadherin Production, and Inhibits in Vitro Invasion of Tumor Cells

Aspirin (acetylsalicylic acid) is a widely used anti-inflammatory drug. Recently, aspirin was shown to reduce the risk of development of cancer and mortality from it. Tumor metastasis is the most important cause of cancer death. The aim of the present study was to investigate if aspirin affects the invasion of cancer cells. Matrix metalloproteinases (MMPs) and cell adhesion molecules play important roles in the modulation of tumor invasion. Gelatin-based zymography assay showed that aspirin inhibited MMP-2 activity of SK-Hep-1 cancer cells. Matrigel-based chemoinvasion assay showed that aspirin inhibited in vitro invasion of SK-Hep-1 cancer cells. Aspirin treatment also increased the production of the cell adhesion molecule, E-cadherin, in Hep G2 cancer cells. Aspirin is a cyclooxygenase (COX) inhibitor. Treatment of cells with another COX inhibitor, sulindac, also inhibited MMP-2 activity and increased E-cadherin production of cells. These results indicate that aspirin can modulate both MMP-2 and E-cadherin production and therein may possess antimetastatic effect.

Identification of a Novel Class of Small-Molecule Antiangiogenic Agents through the Screening of Combinatorial Libraries Which Function by Inhibiting the Binding and Localization of Proteinase MMP2 to Integrin αVβ3

The process of new blood vessel growth from existing vasculature, known as angiogenesis, is critical to several pathological conditions, most notably cancer. Both MMP2, which degrades the extracellular matrix (ECM), and integrin alpha(V)beta(3), which contributes to endothelial cell attachment to the ECM, are critically involved in this process. Recent findings have shown that MMP2 is localized in an active form on the surface of invasive endothelial cells based on its ability to directly bind integrin alpha(V)beta(3), suggesting that disrupting this protein--protein interaction may represent a new target for the development of angiogenesis inhibitors. The screening of small molecule libraries led to the identification of compounds which disrupt the MMP2--alpha(V)beta(3) interaction in an in vitro binding assay. A prototypical inhibitor was further found to prevent the degradation of the protein matrix without directly inhibiting MMP2 activity or disrupting the binding of alpha(V)beta(3) to its classical ECM ligand, vitronectin. The synthesis and screening of analogues and substructures of this lead compound allowed the identification of requisite structural features for inhibition of MMP2 binding to alpha(V)beta(3). This led to the synthesis of a more water-soluble derivative which maintains the in vitro biological properties and has potent antiangiogenic and antitumor activity in vivo, validating the target as one useful for therapeutic intervention.

Thioredoxin alters the matrix metalloproteinase/tissue inhibitors of metalloproteinase balance and stimulates human SK‐N‐SH neuroblastoma cell invasion

Thioredoxin (Trx) inhibited tissue inhibitor of metalloproteinase (TIMP)-1 and TIMP-2 activity with an approximate IC50 of 0.3 microM, matrix metalloproteinase (MMP)-2 activity with an approximate IC50 of 2 microM but did not inhibit MMP-9 activity. This differential capacity of Trx to inhibit TIMP and MMP activity resulted in the promotion of MMP-2 and MMP-9 activity in the presence of molar TIMP excess. Inhibition of TIMP and MMP-2 activity by Trx was dependent upon thioredoxin reductase (TrxR), was abolished by Trx catalytic site mutation and did not result from TIMP or MMP-2 degradation. HepG2 hepatocellular carcinoma cells induced to secrete Trx inhibited TIMP activity in the presence of TrxR. SK-N-SH neuroblastoma cells secreted TrxR, which inhibited TIMP and MMP-2 activity in the presence of Trx. Trx stimulated SK-N-SH invasive capacity in vitro in the absence of exogenous TrxR. This study therefore identifies a novel extracellular role for the thioredoxin/thioredoxin reductase redox system in the differential inhibition of TIMP and MMP activity and provides a novel mechanism for altering the TIMP/MMP balance that is of potential relevance to tumor invasion.

Thrombospondin Type 1 Repeats Interact with Matrix Metalloproteinase 2: REGULATION OF METALLOPROTEINASE ACTIVITY

Thrombospondins are thought to function as inhibitors of angiogenesis. However, the mechanism(s) of this activity is not well understood. In this study, we have used the yeast two-hybrid system to identify proteins that interact with the thrombospondins 1 (TSP1) and 2 (TSP2) properdin-like type 1 repeats (TSR). One of the proteins identified that interacted with both TSR was matrix metalloproteinase 2 (MMP2). The isolated MMP2 cDNA clone encoded amino acid residues 237-633, which include the fibronectin-like gelatin binding region flanking the catalytic center and the carboxyl hemopexin-like region. Further testing of this clone demonstrated that the TSR interacted with the NH(2)-terminal region of the MMP2 that contains the catalytic domain. The protein interaction observed in yeast was further demonstrated by immunoprecipitation and Western blotting using purified intact TSP1, TSP2, MMP2, and MMP9. Although MMP2 interacted with TSP1 and TSP2 via its gelatin-binding domain or a closely mapping site, neither TSP1 nor TSP2 was degraded by MMP2 in vitro. Tissue culture and in vitro assays demonstrated that the presence of purified TSR and intact TSP1 resulted in inhibition of MMP activity. The ability of TSP1 to inhibit MMP3-dependent activation of pro-MMP9 and thrombin-induced activation of pro-MMP2 suggests that the TSPs may inhibit MMP activity by preventing activation of the MMP2 and MMP9 zymogens.

Doxazosin Inhibits MCP-1 Directed Migration of Human Monocytes

P65 Monocyte chemotactic protein 1 (MCP-1) - directed transendothelial migration of monocytes plays a key role in the early development of atherosclerosis. Migration requires degradation of extracellular matrices, a process that involves matrix metalloproteinases (MMP) and tissue inhibitors of MMPs (TIMP). Recent studies suggest that the alpha1-adrenergic receptor (alpha1-AR) antagonist doxazosin (Dox) might have antiatherosclerotic effects by inhibiting proliferation and migration of vascular cells. The purpose of the present study was to determine the effects of Dox on MCP-1 directed monocyte migration, MMP-9 and TIMP-1 regulation, and to identify signalling pathways mediating these processes. MCP-1 (50ng/ml) stimulated migration of human peripheral blood monocytes (HPBM) by 2.8 +/-0.7 fold and of THP-1 human monocytes by 7 +/- 2.7 - fold (both p&lt;0.05 vs. unstimulated cells). Dox inhibited MCP-1 induced migration in a dose dependent manner, with a maximal reduction at 10μM of 50.8 +/- 1.9% in HPBM and 73.5 +/- 3.2 % in THP-1 cells (both p&lt;0.01 vs. MCP-1 alone). Dox blocked migration even after pretreatment with phenoxybenzamine (PBA), an irreversible alpha1-AR antagonist (THP-1 cells: PBA 1μM + Dox 10μM: 76 +/- 11.6% inhibition, p&lt;0.01 vs. MCP-1 alone), suggesting that the antimigratory activity of Dox is mediated through a novel mechanism unrelated to its blocking the alpha1-AR. Pretreatment of monocytes with SB203580 (10μM), a p38 MAPK inhibitor, also attenuated MCP-1 directed migration ( HPBM: 55.3 +/- 0.3% inhibtion; THP-1 cells: 55.4 +/- 13.9% inhibition, both p&lt; 0.05 vs. MCP-1 alone), indicating that activation of this pathway is required for migration towards MCP-1 by these cells. Dox (10μM) inhibited MMP-9 activity by 62% +/- 3.15% (p&lt;0.05) measured by ELISA, whereas MMP-9 protein levels were not affected. Dox also increased phorbol 12-myristate 13-acetate (PMA) induced TIMP-1 expression, which may account for its inhibition of MMP activity. The present study demonstrates a potential novel antiatherosclerotic action of Dox by blocking MCP-1 directed-monocyte migration, mediated, in part, through an inhibtion of MMP-9 activity and upregulation of TIMP-1 production.

Production of Metalloproteinase-7 (Matrilysin) by Human Myeloma Cells and Its Potential Involvement in Metalloproteinase-2 Activation

Matrix metalloproteinases (MMPs) play a critical role in bone remodeling and tumor spreading. Multiple myeloma (MM) is a plasma cell malignancy primarily localized within the bone marrow and characterized by its capacity to destroy bone matrix and to disseminate. We have reported recently that human myeloma cells were able to induce the conversion of pro-MMP-2 produced by the tumoral environment in its activated form. In the current study, we have investigated the mechanism involved in this process. We demonstrate that a soluble MMP constitutively produced by myeloma cells was responsible for pro-MMP-2 activation. Furthermore, we show that the soluble MMP, MMP-7, also known as matrilysin, was able to activate the MMP-2 produced in its latent form by bone marrow stromal cells. Finally, we demonstrate that myeloma cells constitutively produce MMP-7 with expected proteolytic activity. Our results suggest that MMP-7 produced by myeloma cells could participate in bone destruction and tumor spreading in MM, on one hand by its own proteolytic activity and on the other hand by its capacity to activate pro-MMP-2. These findings strengthen the idea that inhibition of MMP activity could represent an interesting therapeutic approach in MM.

Involvement of the 92-kDa Gelatinase (Matrix Metalloproteinase-9) in the Ceramide-Mediated Inhibition of Human Keratinocyte Growth

Triggering the ceramide pathway by exogenous treatment with neutral sphingomyelinase (Smase) inhibited human keratinocyte growth rate, while having no influence on cell apoptosis. Increasing the ceramide content of keratinocytes with Smase (100 U/ml) or C6-ceramide (1 μM) enhanced matrix metalloproteinase (MMP)-9 production. On the contrary, levels of MMP-2 secretion were unchanged. The inhibition of keratinocyte growth rate induced by ceramide could be annihilated by a peptide hydroxamate MMP inhibitor or an MMP-9 blocking antibody. In addition, inhibiting MMP-9 activity in control keratinocyte culture was found to stimulate keratinocyte proliferation. These data suggest a pivotal function of MMP-9 in the control of keratinocyte growth.

Inhibition of the activities of matrix metalloproteinases 2, 8, and 9 by chlorhexidine.

Matrix metalloproteinases (MMPs) are a host cell-derived proteolytic enzyme family which plays a major role in tissue-destructive inflammatory diseases such as periodontitis. The aim of the present study was to evaluate the inhibitory effect of chlorhexidine (CHX) on MMP-2 (gelatinase A), MMP-9 (gelatinase B), and MMP-8 (collagenase 2) activity. Heat-denatured type I collagen (gelatin) was incubated with pure human MMP-2 or -9 activated with p-aminophenylmercuric acetate (APMA), and the proteolytic degradation of gelatin was monitored by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Coomassie blue staining. The effect of CHX on MMP-8 activity was also studied with a cellular model addressing the ability of phorbol myristate acetate (PMA)-triggered human peripheral blood neutrophils (polymorphonuclear leukocytes [PMNs]) to degrade native type I collagen. CHX inhibited the activities of both gelatinases (A and B), but MMP-2 appeared to be more sensitive than MMP-9. Adding calcium chloride to the assay mixtures almost completely prevented the inhibition of MMP-9 activity by CHX, while the inhibition of MMP-2 activity could be reversed only when CHX was used at a low concentration. This observation suggests that CHX may act via a cation-chelating mechanism. CHX dose-dependently inhibited collagenolytic activity of MMP-8 released by PMA-triggered PMNs. MMP-8 without APMA activation was inhibited clearly more efficiently than APMA-activated MMP-8. Our study suggests that the direct inhibition of the MMPs' activities by CHX may represent a new valuable effect of this antimicrobial agent and explains, at least in part, the beneficial effects of CHX in the treatment of periodontitis.