High Levels Of Matrix Metalloproteinases Research Articles

Upregulation of tissue inhibitor of metalloproteinases (TIMP)-2 promotes matrix metalloproteinase (MMP)-2 activation and cell invasion in a human glioblastoma cell line

Local invasiveness is a characteristic feature of glioblastoma that makes surgical resection nearly impossible and accounts in large part for its poor prognosis. To identify mechanisms underlying glioblastoma invasion and motility, we used Transwell invasion chambers to select for a more potently invasive subpopulation of U87MG human glioblastoma cells. The stable population of tumor cells (U87-C1) obtained through this in vitro selection process were three times more invasive than parental U87MG cells and demonstrated faster monolayer wound healing and enhanced radial motility from cell spheroids. This enhanced invasiveness was associated with an 80% increase in matrix metalloproteinase 2 (MMP-2) activation. No differences in expression levels of pro-MMP-2, membrane-type matrix metalloproteinase I (MT1-MMP), or integrin αvβ3 (mediators of MMP-2 activation) were detected. However, U87-C1 cells exhibited two-fold elevation of tissue inhibitor of metalloproteinases (TIMP)-2 mRNA and protein relative to parental cells. Exogenous addition of comparable levels of purified TIMP-2 to parental U87MG cells increased MMP-2 activation and invasion. Similarly, U87MG cells engineered to overexpress TIMP-2 at the same levels as U87-C1 cells also demonstrated increased MMP-2 activation, indicating that an increase in physiological levels of TIMP-2 can promote MMP-2 activation and invasion in glioblastoma cells. However, exogenous administration or recombinant overexpression of higher amounts of TIMP-2 in U87MG cells resulted in inhibition of MMP-2 activation. These results demonstrate that the complex balance between TIMP-2 and MMP-2 is a critical determinant of glioblastoma invasion, and indicate that increasing TIMP-2 in glioblastoma patients may potentially cause adverse effects, particularly in tumors containing high levels of MT1-MMP and MMP-2.

Effects of periodate and chondroitin 4-sulfate on proteoglycan stabilization of ostrich pericardium. Inhibition of calcification in subcutaneous implants in rats

Chemical modification of biological materials used in the manufacture of cardiac valves tends to reduce the relatively high degree of biodegradation and calcification of the implanted bioprostheses. The most widely used treatment to reduce biodegradability of the valves is glutaraldehyde fixation. However, this treatment is potentially toxic and induces tissue calcification. In order to minimize these undesirable effects, we have analyzed the effect of a pre-fixation of endogenous proteoglycans and exogenous glycosaminoglycans, as well as the borohydride reduction influence on the different modified ostrich pericardium implants after subcutaneous implantation in rats. The presence of calcific deposits was detected in all implanted GA-fixed samples; however, calcification was highly reduced in both groups of periodate-prefixed materials, which showed also a very low Ca/P molar ratio. Borohydride post-treatment of these biomaterials resulted in a significant increase in calcium phosphate precipitation, with the appearance of calcium deposits mainly in an amorphous form even though X-ray diffraction allowed the detection of brushite- and apatite-like crystals. Regarding tissue stability, no significant differences were found among the borohydride-untreated implants but higher levels of matrix metalloproteinases were observed by gelatin zymography in the periodate pre-fixed materials. This increase was partially reduced by pre-fixation of exogenous chondroitin 4-sulfate. On the other hand, borohydride post-treatment not only increased calcification, but also reduced tissue stability and increased the presence of matrix-degrading activities.

ATP Potentiates Interleukin-1β-induced MMP-9 Expression in Mesangial Cells via Recruitment of the ELAV Protein HuR

Renal mesangial cells express high levels of matrix metalloproteinase 9 (MMP-9) in response to inflammatory cytokines such as interleukin (IL)-1 beta. We demonstrate here that the stable ATP analog adenosine 5'-O-(thiotriphosphate) (ATP gamma S) potently amplifies the cytokine-induced gelatinolytic content of mesangial cells mainly by an increase in the MMP-9 steady-state mRNA level. A Luciferase reporter gene containing 1.3 kb of the MMP-9 5'-promoter region showed weak responses to ATP gamma S but conferred a strong ATP-dependent increase in Luciferase activity when under the additional control of the 3'-untranslated region of MMP-9. By in vitro degradation assay and actinomycin D experiments we found that ATP gamma S potently delayed the decay of MMP-9 mRNA. Gel-shift and supershift assays demonstrated that three AU-rich elements (AREs) present in the 3'-untranslated region of MMP-9 are constitutively bound by complexes containing the mRNA stabilizing factor HuR. The RNA binding of these complexes was markedly increased by ATP gamma S. Mutation of each ARE element strongly impaired the RNA binding of the HuR containing complexes. Reporter gene assays revealed that mutation of one ARE did not affect the stimulatory effects by ATP gamma S, but mutation of all three ARE motifs caused a loss of ATP-dependent increase in luciferase activity without affecting IL-1 beta-inducibility. By confocal microscopy we demonstrate that ATP gamma S increased the nucleo cytoplasmic shuttling of HuR and caused an increase in the cytosolic HuR level as shown by cell fractionation experiments. Together, our results indicate that the amplification of MMP-9 expression by extracellular ATP is triggered through mechanisms that likely involve a HuR-dependent rise in MMP-9 mRNA stability.

Upregulation of tissue inhibitor of metalloproteinases (TIMP)-2 promotes matrix metalloproteinase (MMP)-2 activation and cell invasion in a human glioblastoma cell line.

Local invasiveness is a characteristic feature of glioblastoma that makes surgical resection nearly impossible and accounts in large part for its poor prognosis. To identify mechanisms underlying glioblastoma invasion and motility, we used Transwell invasion chambers to select for a more potently invasive subpopulation of U87MG human glioblastoma cells. The stable population of tumor cells (U87-C1) obtained through this in vitro selection process were three times more invasive than parental U87MG cells and demonstrated faster monolayer wound healing and enhanced radial motility from cell spheroids. This enhanced invasiveness was associated with an 80% increase in matrix metalloproteinase 2 (MMP-2) activation. No differences in expression levels of pro-MMP-2, membrane-type matrix metalloproteinase I (MT1-MMP), or integrin alphavbeta3 (mediators of MMP-2 activation) were detected. However, U87-C1 cells exhibited two-fold elevation of tissue inhibitor of metalloproteinases (TIMP)-2 mRNA and protein relative to parental cells. Exogenous addition of comparable levels of purified TIMP-2 to parental U87MG cells increased MMP-2 activation and invasion. Similarly, U87MG cells engineered to overexpress TIMP-2 at the same levels as U87-C1 cells also demonstrated increased MMP-2 activation, indicating that an increase in physiological levels of TIMP-2 can promote MMP-2 activation and invasion in glioblastoma cells. However, exogenous administration or recombinant overexpression of higher amounts of TIMP-2 in U87MG cells resulted in inhibition of MMP-2 activation. These results demonstrate that the complex balance between TIMP-2 and MMP-2 is a critical determinant of glioblastoma invasion, and indicate that increasing TIMP-2 in glioblastoma patients may potentially cause adverse effects, particularly in tumors containing high levels of MT1-MMP and MMP-2.

Alpha 1-antitrypsin and ilomastat inhibit inflammatory proteases present in human middle ear effusions.

Proteases of both the serine and metalloproteinase families have been shown to play a role in the pathogenesis of otitis media (OM). Inhibitors of proteases from each of these families have been shown to beneficially impact disease progression in a number of related chronic inflammatory conditions, but their use has not been studied in OM. The purpose of this study was to assess the activity of the protease inhibitors recombinant alpha 1-antitrypsin (rAAT) and ilomastat on inflammatory proteases present in human middle ear effusions (MEEs), with a view to their potential utility in the treatment of OM. Prospective and ex vivo. MEEs were collected from 100 patients presenting for middle ear surgery, most commonly tympanostomy tube placement or treatment of acute posttympanostomy otorrhea (APTO). MEEs were analyzed for the presence of matrix metalloproteinases (MMPs) and human neutrophil elastase (HNE) and the inhibitory activity of rAAT and ilomastat on these proteases, respectively. MMP levels were highest in APTO, and HNE was highest in chronic suppurative OM and APTO. High levels of MMP and HNE (>3 mAU/min) were found in 52% and 37% of MEEs, respectively. Ilomastat and rAAT demonstrated significant inhibition of MMP and HNE activity (>30% reduction), respectively, in 80% and 82% of MEEs with high levels of activity. Proteases are commonly found in OM. Ilomastat and rAAT are potent inhibitors of proteases in MEEs across a wide range of OM in humans. Investigation into the potential therapeutic benefits of these protease inhibitors is warranted.

Regulation of pancreatic stellate cell function in vitro: biological and molecular effects of all-trans retinoic acid

Pancreatic stellate cells (PSCs) are essentially involved in the development of pancreatic fibrosis, a constant feature of chronic pancreatitis and pancreatic cancer. Profibrogenic mediators, such as ethanol metabolites and cytokines, induce a PSC activation process that involves proliferation, enhanced production of extracellular matrix proteins and a phenotypic transition towards myofibroblasts which includes a loss of the characteristic retinoid-containing fat droplets. Here, we have analysed how exogenous all-trans retinoic acid (ATRA) affects activation of rat PSCs induced by sustained culture. Bromodeoxyuridine-incorporation assays indicated an ATRA-dependent inhibition of DNA synthesis. In contrast, ATRA did not affect expression of α-smooth muscle actin, a protein typical for myofibroblasts. Quantification of [3H]proline incorporation revealed a diminished collagen production in ATRA-treated PSCs. Furthermore, zymography experiments showed that supernatants of ATRA-exposed PSC cultures contained higher levels of matrix metalloproteinase-9 but not of matrix metalloproteinase-2 than untreated controls. At the level of intracellular signalling, ATRA had no effect on extracellular signal-regulated kinase activation after incubation of PSCs with the mitogen platelet-derived growth factor (PDGF). In addition, PDGF-induced DNA binding of activator protein-1 (AP-1) transcription factors was not inhibited by ATRA treatment. Luciferase reporter gene assays, however, revealed an ATRA-dependent transrepression of AP-1 in PDGF-stimulated PSCs. Together, the results indicate that exogenous ATRA displays inhibitory effects on PSC proliferation and collagen synthesis but does not block phenotypic transition towards myofibroblasts. We hypothesise that inhibition of AP-1 signalling may be involved in the mediation of biological effects of ATRA on PSCs.

Nitric Oxide Increases the Decay of Matrix Metalloproteinase 9 mRNA by Inhibiting the Expression of mRNA-Stabilizing Factor HuR

Dysregulation of extracellular matrix turnover is an important feature of many inflammatory processes. Rat renal mesangial cells express high levels of matrix metalloproteinase 9 (MMP-9) in response to inflammatory cytokines such as interleukin-1 beta. We demonstrate that NO does strongly destabilize MMP-9 mRNA, since different luciferase reporter gene constructs containing the MMP-9 3' untranslated region (UTR) displayed significant reduced luciferase activity in response to the presence of NO. Moreover, by use of an in vitro degradation assay we found that the cytoplasmic fractions of NO-treated cells contained a higher capacity to degrade MMP-9 transcripts than those obtained from control cells. An RNA electrophoretic mobility shift assay demonstrated that three of four putative AU-rich elements present in the 3' UTR of MMP-9 were constitutively occupied by the mRNA-stabilizing factor HuR and that the RNA binding was strongly attenuated by the presence of NO. The addition of recombinant glutathione transferase-HuR prevented the rapid decay of MMP-9 mRNA, whereas the addition of a neutralizing anti-HuR antibody caused an acceleration of MMP-9 mRNA degradation. Furthermore, the expression of HuR mRNA and protein was significantly reduced by exogenously and endogenously produced NO. These inhibitory effects were mimicked by the cGMP analog 8-bromo-cGMP and reversed by LY-83583, an inhibitor of soluble guanylyl cyclase. These results demonstrate that NO acts in a cGMP-dependent mechanism to inhibit the expression level of HuR, thereby reducing the stability of MMP-9 mRNA.

Increased plasma levels of matrix metalloproteinase-9 and tissue inhibitor of metalloproteinase-1 in lung and breast cancer are altered during chest radiotherapy

Purpose Does the release of plasma matrix metalloproteinase-9 (MMP-9) by radiation-activated airway epithelial cells and infiltrating inflammatory cells play a role in the radiation damage or repair process in the lungs? We evaluated lung damage by ionizing radiation using plasma levels of MMP-9, tissue inhibitor of metalloproteinase-1 (TIMP-1), and MMP-3 as biologic markers of tissue damage, and also their relationship to changes in pulmonary epithelial permeability, clinical signs and symptoms, and lung structural changes. Methods and materials Seven serial studies were conducted in each of 8 patients undergoing chest radiotherapy (RT) for lung or breast cancer, beginning before the first treatment (baseline) and then biweekly to ∼100 days during and after RT. Chest radiographs were monitored for each patient. Sandwich enzyme-linked immunoassays (ELISA) were used to measure plasma MMP-3, MMP-9, and TIMP-1 levels. Lung permeability was evaluated by measuring the rate of epithelial clearance of ∼150 μCi (∼5.6 MBq) inhaled 99mTc diethylenetriamine pentaacetate aerosol (DTPA). Results Lung and breast cancer resulted in very high plasma levels of MMP-9 (126–893 ng/mL) and TIMP-1 (496–8,985 ng/mL) in all subjects studied before initiation of RT. This compares with plasma MMP-9 and TIMP-1 values in healthy volunteers of 29 ± 11 ng/mL and 436 ± 86 ng/mL, respectively. RT was followed by a sharp decrease in plasma MMP-9 within the first 2 weeks, but without a corresponding change in TIMP-1. In contrast, plasma MMP-3 levels, which are generally increased with inflammation, were elevated in only 1 of 5 subjects. Conclusion Lung and breast cancer are associated with high plasma levels of MMP-9 and TIMP-1. These high baseline plasma levels of MMP-9 were reduced in the first 2 weeks of RT in 7 of 8 subjects, and TIMP-1 plasma levels remained high in all subjects. The decrease in plasma MMP-9 after initiation of chest RT appears to reflect a suppressive effect on cancer-induced cellular responses rather than a primary role for MMP-9 in radiation-induced lung damage. Likewise, the lack of a rise in plasma MMP-3 levels does not support a role for MMP-3 in tissue injury or repair in the lung. It remains to be determined whether plasma MMP-9 measurements will serve as a useful parameter in predicting cancer relapse.

Inhibition of Cytokine-induced Matrix Metalloproteinase 9 Expression by Peroxisome Proliferator-activated Receptor α Agonists Is Indirect and Due to a NO-mediated Reduction of mRNA Stability

Rat renal mesangial cells express high levels of matrix metalloproteinase 9 (MMP-9) in response to inflammatory cytokines such as interleukin 1beta (IL-1beta). We tested whether ligands of the peroxisome proliferator-activated receptor (PPARalpha) could influence the cytokine-induced expression of MMP-9. Different PPARalpha agonists dose-dependently inhibited the IL-1beta-triggered increase in gelatinolytic activity mainly by decreasing the MMP-9 steady-state mRNA levels. PPARalpha agonists on their own had no effects on MMP-9 mRNA levels and gelatinolytic activity. Surprisingly, the reduction of MMP-9 mRNA levels by PPARalpha activators contrasted with an amplification of cytokine-mediated MMP-9 gene promoter activity and mRNA expression. The potentiation of MMP-9 promoter activity functionally depends on an upstream peroxisome proliferator-responsive element-like binding site, which displayed an increased DNA binding of a PPARalpha immunopositive complex. In contrast, the IL-1beta-induced DNA-binding of nuclear factor kappaB was significantly impaired by PPARalpha agonists. Most interestingly, in the presence of an inducible nitric-oxide synthase (iNOS) inhibitor, the PPARalpha-mediated suppression switched to a strong amplification of IL-1beta-triggered MMP-9 mRNA expression. Concomitantly, activators of PPARalpha potentiated the cytokine-induced iNOS expression. Using actinomycin D, we found that NO, but not PPARalpha activators, strongly reduced the stability of MMP-9 mRNA. In contrast, the stability of MMP-9 protein was not affected by PPARalpha activators. In summary, our data suggest that the inhibitory effects of PPARalpha agonists on cytokine-induced MMP-9 expression are indirect and primarily due to a superinduction of iNOS with high levels of NO reducing the half-life of MMP-9 mRNA.

Peptide-Retargeted Adenovirus Encoding a Tissue Inhibitor of Metalloproteinase-1 Decreases Restenosis after Intravascular Gene Transfer

In this study we have attached cyclic targeting peptides by way of a poly-lysine spacer on the surface of an adenovirus using a transglutaminase enzymatic reaction to enhance transduction efficiency and to modify tissue tropism in vivo. Nuclear targeted lacZ- and TIMP-1-encoding adenoviruses were coupled to a peptide-motif (HWGF) that can bind to matrix metalloproteinase (MMP)-2 and MMP-9. Modified viruses were used to evaluate gene transfer efficiency, biodistribution, and the effect on neointima formation following balloon denudation injury. In vitro, both rabbit aortic smooth muscle cells and human endothelial hybridoma cells demonstrated significantly increased reporter gene expression with HWGF-modified adenoviruses (AdlacZ(HWGF)) compared with control (AdlacZ) or mismatch peptide-modified (AdlacZ(MM)) adenoviruses. However, in human hepatocellular Hep-G2 cells, both AdlacZ(HWGF) and AdlacZ(MM) produced significantly lower transgene expression compared with the respective control viruses. In vivo, local intravascular catheter-mediated gene transfer of a HWGF-targeted TIMP-1-encoding adenovirus (AdTIMP-1(HWGF)) significantly reduced intimal thickening in a rabbit aortic balloon denudation model (P < 0.05) compared with the control adenovirus. X-Gal staining and biodistribution analyses with TaqMan RT-PCR revealed that the cyclic peptides altered vector tropism and, in particular, reduced transduction of the liver. We found that the HWGF peptide modification increased transduction efficiency of the adenovirus-mediated gene transfer in smooth muscle cells and endothelial cells in in vitro and enhanced gene transfer to the arterial wall in vivo; that peptide modification of adenoviruses beneficially modulated tissue tropism in vivo; and that efficient TIMP-1 gene transfer reduced intimal thickening in an established restenosis model in rabbits.

Loss of p53 accelerates neointimal lesions of vein bypass grafts in mice.

The transcription factor p53 is essentially involved in regulation of cell death and proliferation. Recently, we have established a mouse model for vein graft arteriosclerosis by grafting autologous jugular veins or vena cava to carotid arteries. Using this model, we studied the role of p53 in the development of vein graft arteriosclerosis in p53(-/-) mice. Four weeks after grafting, neointimal hyperplasia of vein grafts in p53(-/-) mice was increased 2-fold compared with that of wild-type controls. Cell component analysis revealed that neointimal lesions in p53(-/-) mice consisted mainly of alpha-actin positive smooth muscle cells (SMCs), whereas the majority of cells in wild-type mice were MAC-1 (CD11b/18)-positive at 4 weeks. Importantly, SMC apoptosis as determined by TUNEL assay was significantly reduced in p53(-/-) vein grafts. TUNEL positive cells in wild-type vein grafts markedly increased from 0.5% to 6.4% of total cells 4 weeks postoperatively, but remained virtually unchanged in p53(-/-) grafts (0.8%). Immunofluorescence analysis revealed that increased p53 expression in neointimal SMCs of wild-type, but not p53(-/-), mice coincided with oxidative DNA damage in vein grafts. Interestingly, SMCs of p53(-/-) mice showed increased apoptosis in response to TNFalpha and decreased apoptosis in response to sodium nitroprusside. Additionally, p53-deficient SMCs showed a higher rate of proliferation and migration and expressed higher levels of matrix metalloproteinases. Thus, p53 deficiency accelerates neointima formation by facilitating SMC proliferation as well as abrogating cell apoptosis.

Bone marrow angiogenesis in patients with active multiple myeloma

Factor VIII-related antigen (FVIII-RA)-positive microvessel areas were measured by both immunohistochemistry and computerized image analysis in patients with active multiple myeloma (MM), nonactive MM, and monoclonal gammopathies of undetermined significance (MGUS). A five-to sixfold larger area was found in patients with active MM compared to the other two groups. Aquaporin I (AQPI)-positive microvessel areas, measured with the same techniques on adjacent tissue sections, were also increased in active MM, and tended to be larger than and closely correlated with the FVIII-RA areas. Numerous mast cells were found in the bone marrow of active MM patients, and counts were strictly correlated with the microvessel density. The conditioned medium (CM) of bone marrow plasma cells from active MM patients stimulated endothelial cell proliferation and chemotaxis, monocyte chemotaxis, and angiogenesis in vivo (assessed by the chick embryo chorioallantoic membrane [CAM] system) more strongly and frequently than the CM of patients with nonactive MM and MGUS. Immunoassay of plasma cell lysates gave significantly higher levels of fibroblast growth factor-2 (FGF-2) in patients with active MM than in the other two groups, and a neutralizing anti-FGF-2 antibody inhibited by 46% to 68% the biological activity exerted by the CM in vitro and in the CAM. In situ hybridization of bone marrow plasma cells and zymography of CM showed that patients with active MM express higher levels of matrix metalloproteinase-2 (MMP-2) mRNA and protein than those with nonactive MM and MGUS, whereas MMP-9 expression and secretion overlapped in all groups. Overall data indicate that patients with active MM represent the vascular phase of plasma cell tumors that is induced, at least partly, through FGF-2 and MMP-2. Mast cells possibly contribute to the vascular phase via angiogenic factors in their secretory granules. Both angiogenesis and MMP-2 secretion can account for intramedullary and extramedullary spreading of plasma cells in patients with active MM.

Alveolar macrophage deficiency in osteopetrotic mice deficient in macrophage colony-stimulating factor is spontaneously corrected with age and associated with matrix metalloproteinase expression and emphysema

AbstractMacrophage colony-stimulating factor (M-CSF) is one of several hematologic growth factors capable of regulating the survival, proliferation, and differentiation of macrophages, but its role in modulation of the accumulation and function of alveolar macrophages (AMs) in vivo is not well defined. Osteopetrotic (Op/Op) mice have no detectable M-CSF and show variable tissue-specific reductions in macrophage numbers. It was hypothesized that AMs would be decreased in number and have altered function in Op/Op mice because of the absence of M-CSF. Lung macrophages identified by Mac-3 staining in lung sections were decreased in number in 20-day-old Op/Op mice (P < .001) but not Op/Op mice older than 4 months (P = .68) compared with findings in age-matched littermate controls. The numbers of AMs recovered by bronchoalveolar lavage (BAL) were also reduced in young but not adult Op/Op mice compared with controls. Expression of interleukin-3 (IL-3) was increased in the lungs of Op/Op mice compared with controls as determined by quantification of IL-3 cytokine levels (P = .04), bioactivity (P = .02), and messenger RNA transcript levels. AMs of Op/Op mice spontaneously released higher levels of matrix metalloproteinases (MMPs) than AMs of controls as determined by immunohistochemical staining of AMs and zymographic assessment of BAL fluid and AM lysates. Consistent with an increased release of MMP, Op/Op mice had abnormal elastin deposition and spontaneously developed emphysema in the absence of molecular or cellular evidence of lung inflammation. These data show that the AM deficiency observed in young Op/Op mice is spontaneously corrected with age and is associated with increased lung levels of IL-3, spontaneous MMP expression by AMs, and destruction of lung tissue.

Augmented metalloproteinase activity and acute lung injury in copper-deficient rats.

Dietary copper is required for normal function of >30 mammalian enzyme systems. Copper deficiency causes a number of cardiovascular defects as well as impaired immune cell function. Little is known regarding the effects of copper deficiency on acute inflammatory responses, but this topic is relevant because many members of the Western population receive less than the recommended dietary allowance of copper. In the current studies, we investigated the effects of dietary copper deficiency on acute lung injury induced by intrapulmonary deposition of IgG immune complexes. Weanling male Long-Evans rats were fed diets either adequate (5.6 microg/g) or deficient (0.3 microg/g) in copper. IgG immune complex lung injury was greatly increased in copper-deficient rats as determined by lung vascular leakage of albumin and histopathology. However, no change was observed in either the lung content of tumor necrosis factor-alpha or lung neutrophil accumulation. Lungs from copper-deficient rats had much higher levels of matrix metalloproteinase (MMP)-2 and MMP-9 than did copper-adequate control animals. This increased activity was not attributable to alveolar macrophages or neutrophils. These data suggest that the augmented lung injury caused by copper deficiency is due to increased pulmonary MMP-2 and MMP-9 activity and not a generalized amplification of the inflammatory response.

TNF-alpha stimulates activation of pro-MMP2 in human skin through NF-(kappa)B mediated induction of MT1-MMP.

Tumor necrosis factor-alpha (TNF-(alpha)) is an important mediator during the inflammatory phase of wound healing. Excessive amounts of pro-inflammatory cytokines such as TNF-(alpha) are associated with inflammatory diseases including chronic wounds. Matrix metalloproteinases (MMPs) are involved in matrix re-modeling during wound healing, angiogenesis and tumor metastasis. As with pro-inflammatory cytokines, high levels of MMPs have been found in inflammatory states such as chronic wounds. In this report we relate these two phenomena. TNF-(alpha) stimulates secretion of active MMP-2, a type IV collagenase, in organ-cultured full-thickness human skin. This suggests a mechanism whereby excess inflammation affects normal wound healing. To investigate this observation at the cellular and molecular levels, we examined TNF-(alpha) mediated activation of pro-MMP-2, induction of MT1-MMP, and the intracellular signaling pathways that regulate the proteinase in isolated human dermal fibroblasts. We found that TNF-(alpha) substantially promoted activation of pro-MMP-2 in dermal fibroblasts embedded in type-I collagen. In marked contrast, collagen or TNF-(alpha) individually had little influence on the fibroblast-mediated pro-MMP-2 activation. One well-characterized mechanism for pro-MMP-2 activation is through a membrane type matrix metalloproteinase, such as MT1-MMP. We report that TNF-(alpha) significantly induced MT1-MMP at the mRNA and protein levels when the dermal fibroblasts were grown in collagen. Although the intracellular signaling pathway regulating mt1-mmp gene expression is still obscure, both TNF-(alpha) and collagen activate the NF-(kappa)B pathway. In this report we provide three sets of evidence to support a hypothesis that activation of NF-(kappa)B is essential to induce MT1-MMP expression in fibroblasts after TNF-(alpha) exposure. First, SN50, a peptide inhibitor for NF-(kappa)B nuclear translocation, simultaneously blocked the TNF-(alpha) and collagen mediated MT1-MMP induction and pro-MMP-2 activation. Secondly, TNF-(alpha) induced I(kappa)B to breakdown in fibroblasts within the collagen lattice, a critical step leading to NF-(kappa)B activation. Lastly, a consensus binding site for p65 NF-(kappa)B (TGGAGCTTCC) was found in the 5'-flanking region of human mt1-mmp gene. Based on these results and previous reports, we propose a model to explain TNF-(alpha) activation of MMP-2 in human skin. Activation of NF(kappa)B signaling in fibroblasts embedded in collagen induces mt1-mmp gene expression, which subsequently activates the pro-MMP-2. The findings provide a specific mechanism whereby TNF-(alpha) may affect matrix remodeling during wound healing and other physiological and pathological processes.

Bone Marrow Angiogenesis and Plasma Cell Angiogenic and Invasive Potential in Patients with Active Multiple Myeloma

Factor VIII-related antigen-positive microvessel areas were measured by both immunohistochemistry and computerized image analysis in patients with active multiple myeloma (MM), nonactive MM and monoclonal gammopathies of undetermined significance (MGUS). A 5- to 6-fold larger area was found in patients with active MM compared to the other two groups. The conditioned medium (CM) of their bone marrow plasma cells stimulated endothelial cell proliferation and chemotaxis, monocyte chemotaxis and angiogenesis in vivo [chick embryo chorioallantoic membrane (CAM) system] more strongly and frequently than the CM of patients with nonactive MM and MGUS. An immunoassay of plasma cell lysates gave significantly higher levels of fibroblast growth factor-2 (FGF-2) in patients with active MM than in the other two groups, and a neutralizing anti-FGF-2 antibody inhibited by 54–68% the biological activity exerted by the CM in vitro and in the CAM. In situ hybridization of bone marrow plasma cells and gelatin zymography of CM showed that patients with active MM express higher levels of matrix metalloproteinase-2 (MMP-2) mRNA and protein than those with nonactive MM and MGUS, whereas MMP-9 expression and secretion overlapped in all groups. Overall data suggest that patients with active MM represent the vascular phase of plasma cell tumors that is triggered by bone marrow plasma cells, at least partly, through FGF-2 and MMP-2. Both angiogenesis and MMP-2 secretion can account for intramedullary and extramedullary spreading of plasma cells during the active MM.

Matrix metalloproteinase and cytokine profiles in monocytes over the course of stroke.

Stroke is a common cause of death and disability in our society. Stroke is associated with changes in immune responses within the central nervous system as well as systemically. The cells contributing to such changes as well as the factors contributing to formation of the inflammatory infiltrate observed in stroke remain to be clarified. In this study, blood monocytes and corresponding mononuclear cells (MNC) were separated and examined in parallel within 4 days and 1-3 months after onset of ischemic stroke. Numbers of TNF-alpha-, IL-12-, IL-6-, and IL-10-secreting cells and of cells expressing mRNA for matrix metalloproteinase (MMP)-1, -2, -7, -9 and tissue inhibitor of MMP (TIMP)-1 were studied. The TNF-alpha-, IL-12-, and IL-6-secreting monocytes and MNC were elevated during the acute phase compared to healthy controls. Such differences were not observed when stroke patients were examined during convalescence. The IL-10-secreting monocytes did not change over the course of stroke. Levels of monocytes expressing MMP-1, MMP-7 and TIMP-1 mRNA were elevated in the acute phase of stroke patients compared to convalescence and healthy controls, as were levels of MMP-1, -2, -7, -9 and TIMP-1 mRNA expressing blood MNC. The MMP-2 and -9 activity as measured by zymography also was higher in MNC supernatants in the acute phase of stroke compared to convalescence. The high levels of proinflammatory cytokines and MMPs in blood monocytes and MNC further demonstrate the presence of systemic aberrations in the acute phase of stroke. Such changes may contribute to the influx of blood-borne cells into the ischemic lesions during the acute phase of stroke.

Matrix metalloproteinases as emerging targets for cancer therapy

The matrix metalloproteinases (MMPs) are a family of zinc-dependent endopeptidases that play a key role in connective tissue-remodeling proceses. The expression and activity of these proteolytic enzymes is highly regulated by growth factors, cytokines, and hormones, as well as by interactions with specific tissue inhibitors of metalloproteinases (TIMPs). These enzymes are frequently overexpressed in many tumors, and in some cases it has been demonstrated a correlation of high MMP levels with local recurrence and increased invasive and metastatic potential of different malignant tumors. Functional studies have provided evidence that MMPs play an important role in the proteolytic destruction of extracellular matrix and basement membranes, thereby facilitating tumor invasion and metastasis. However, recent studies have indicated that MMPs may be also important in other steps of tumor evolution including neoplastic cell proliferation and angiogenesis stimulation at primary and metastatic sites. Based on the functional relevance of MMPs in tumor progression and metastasis, a number of inhibitors have been developed to try to block the activity of these enzymes. Clinical studies currently in progress will contribute to clarify whether these protease inhibitors may be part of future therapeutic strategies to control the metastatic capacity of neoplastic cells.

Matrix metalloproteinase-3-dependent generation of a macrophage chemoattractant in a model of herniated disc resorption.

Herniated disc (HD) is a common health problem that is resolved by surgery unless spontaneous resorption occurs. HD tissue contains abundant macrophage infiltration and high levels of matrix metalloproteinases (MMPs) MMP-3 and MMP-7. We developed a model system in which disc tissue or isolated chondrocytes from wild-type or MMP-null mice were cocultured with peritoneal macrophages and used this system to investigate the role of MMPs and chondrocyte/macrophage interactions in disc resorption. We observed a marked enhancement of MMP-3 protein and mRNA in chondrocytes after exposure to macrophages. Chondrocytic MMP-3, but not MMP-7, was required for disc resorption, as determined by assaying for a reduction in wet weight and proteoglycan content after 3 days of coculture. Surprisingly, chondrocyte MMP-3 was required for the generation of a macrophage chemoattractant and the subsequent infiltration of the disc tissue by proteolytically active macrophages. We conclude that macrophage induction of chondrocyte MMP-3 plays a major role in disc resorption by mechanisms that include the generation of a bioactive macrophage chemoattractant.

High Levels of Matrix Metalloproteinases Regulate Proliferation and Hormone Secretion in Pituitary Cells

High Levels of Matrix Metalloproteinases Regulate Proliferation and Hormone Secretion in Pituitary Cells