Monocyte Matrix Metalloproteinase-9 Research Articles

Specialized regulatory T cells control venous blood clot resolution through SPARC

The cells and mechanisms involved in blood clot resorption are only partially known. We show that regulatory T cells (Tregs) accumulate in venous blood clots and regulate thrombolysis by controlling the recruitment, differentiation and matrix metalloproteinase (MMP) activity of monocytes. We describe a clot Treg population that forms the matricellular acid– and cysteine-rich protein SPARC (secreted protein acidic and rich in cysteine) and show that SPARC enhances monocyte MMP activity and that SPARC+ Tregs are crucial for blood clot resorption. By comparing different treatment times, we define a therapeutic window of Treg expansion that accelerates clot resorption.

Ureaplasma isolates stimulate pro-inflammatory CC chemokines and matrix metalloproteinase-9 in neonatal and adult monocytes.

Being generally regarded as commensal bacteria, the pro-inflammatory capacity of Ureaplasma species has long been debated. Recently, we confirmed Ureaplasma–driven pro-inflammatory cytokine responses and a disturbance of cytokine equilibrium in primary human monocytes in vitro. The present study addressed the expression of CC chemokines and matrix metalloproteinase-9 (MMP-9) in purified term neonatal and adult monocytes stimulated with serovar 8 of Ureaplasma urealyticum (Uu) and serovar 3 of U. parvum (Up). Using qRT-PCR and multi-analyte immunoassay, we assessed mRNA and protein expression of the monocyte chemotactic proteins 1 and 3 (MCP-1/3), the macrophage inflammatory proteins 1α and 1β (MIP-1α/β) as well as MMP-9. For the most part, both isolates stimulated mRNA expression of all given chemokines and MMP-9 in cord blood and adult monocytes (p<0.05 and p<0.01). These results were paralleled by Uu and Up-induced secretion of MCP-1 protein in both cells (neonatal: p<0.01, adult: p<0.05 and p<0.01). Release of MCP-3, MIP-1α, MIP-1β and MMP-9 was enhanced upon exposure to Up (neonatal: p<0.05, p<0.01 and p<0.001, respectively; adult: p<0.05). Co-stimulation of LPS-primed monocytes with Up increased LPS-induced MCP-1 release in neonatal cells (p<0.05) and aggravated LPS-induced MMP-9 mRNA in both cell subsets (neonatal: p<0.05, adult: p<0.01). Our results document considerable expression of pro-inflammatory CC chemokines and MMP-9 in human monocytes in response to Ureaplasma isolates in vitro, adding to our previous data. Findings from co-stimulated cells indicate that Ureaplasma may modulate monocyte immune responses to a second stimulus.

Monocyte Adhesion, Migration, and Extracellular Matrix Breakdown Are Regulated by Integrin αVβ3 in Mycobacterium tuberculosis Infection.

In tuberculosis (TB), the innate inflammatory immune response drives tissue destruction, morbidity, and mortality. Monocytes secrete matrix metalloproteinases (MMPs), which have key roles in local tissue destruction and cavitation. We hypothesized that integrin signaling might regulate monocyte MMP secretion in pulmonary TB during cell adhesion to the extracellular matrix (ECM). Adhesion to type I collagen and fibronectin by Mycobacterium tuberculosis–stimulated monocytes increased MMP-1 gene expression by 2.6-fold and 4.3-fold respectively, and secretion by 60% (from 1208.1 ± 186 to 1934.4 ± 135 pg/ml; p < 0.0001) and 63% (1970.3 ± 95 pg/ml; p < 0.001). MMP-10 secretion increased by 90% with binding to type I collagen and 55% with fibronectin, whereas MMP-7 increased 57% with collagen. The ECM did not affect the secretion of tissue inhibitors of metalloproteinases-1 or -2. Integrin αVβ3 surface expression was specifically upregulated in stimulated monocytes and was further increased after adhesion to type I collagen. Binding of either β3 or αV integrin subunits increased MMP-1/10 secretion in M. tuberculosis–stimulated monocytes. In a cohort of TB patients, significantly increased integrin β3 mRNA accumulation in induced sputum was detected, to our knowledge, for the first time, compared with control subjects (p < 0.05). Integrin αVβ3 colocalized with areas of increased and functionally active MMP-1 on infected monocytes, and αVβ3 blockade markedly decreased type I collagen breakdown, and impaired both monocyte adhesion and leukocyte migration in a transwell system (p < 0.0001). In summary, our data demonstrate that M. tuberculosis stimulation upregulates integrin αVβ3 expression on monocytes, which upregulates secretion of MMP-1 and -10 on adhesion to the ECM. This leads to increased monocyte recruitment and collagenase activity, which will drive inflammatory tissue damage.

β2 Glycoprotein I Recognition Drives Th1 Inflammation in Atherosclerotic Plaques of Patients with Primary Antiphospholipid Syndrome.

Antiphospholipid syndrome (APS) is characterized by recurrent arterial/venous thrombosis and miscarriages in the persistent presence of autoantibodies against phospholipid-binding proteins (aPLs), such as β2 glycoprotein I (β2GPI). In addition to the aPL thrombophilic effect, arterial thrombosis was related to accelerated atherosclerosis in animal models; however, contrasting findings were reported in primary APS patients with regard to the increased number of plaques or abnormal arterial wall thickness. We investigated the cytokine production induced by β2GPI in activated T cells that infiltrate in vivo atherosclerotic lesions of primary APS patients with atherothrombosis. We also examined the helper function of β2GPI-specific T cells for monocyte matrix metalloproteinase-9 and tissue factor production, as well as their cytolytic potential and their helper function for Ab production. APS patients with atherothrombosis harbor in vivo-activated CD4+ T cells that recognize β2GPI in atherothrombotic lesions. β2GPI induces T cell proliferation and IFN-γ expression in plaque-derived T cell clones. β2GPI-specific T cells display helper function for monocyte matrix metalloproteinase-9 and tissue factor production and promote Ig production in autologous B cells. Moreover, plaque-derived β2GPI-specific CD4+ T lymphocytes express perforin-mediated and Fas/Fas ligand-mediated cytotoxicity. β2GPI, and especially the DI domain, drive a local Th1 inflammatory response, with subsequent plaque instability that eventually favors atherothrombosis. This finding may explain the association between aPLs and arterial thrombosis, despite the lack of evidence of surrogate markers for atherosclerosis in primary APS.

Natural Haemozoin Induces Expression and Release of Human Monocyte Tissue Inhibitor of Metalloproteinase-1

Recently matrix metalloproteinase-9 (MMP-9) and its endogenous inhibitor (tissue inhibitor of metalloproteinase-1, TIMP-1) have been implicated in complicated malaria. In vivo, mice with cerebral malaria (CM) display high levels of both MMP-9 and TIMP-1, and in human patients TIMP-1 serum levels directly correlate with disease severity. In vitro, natural haemozoin (nHZ, malarial pigment) enhances monocyte MMP-9 expression and release. The present study analyses the effects of nHZ on TIMP-1 regulation in human adherent monocytes. nHZ induced TIMP-1 mRNA expression and protein release, and promoted TNF-α, IL-1β, and MIP-1α/CCL3 production. Blocking antibodies or recombinant cytokines abrogated or mimicked nHZ effects on TIMP-1, respectively. p38 MAPK and NF-κB inhibitors blocked all nHZ effects on TIMP-1 and pro-inflammatory molecules. Still, total gelatinolytic activity was enhanced by nHZ despite TIMP-1 induction. Collectively, these data indicate that nHZ induces inflammation-mediated expression and release of human monocyte TIMP-1 through p38 MAPK- and NF-κB-dependent mechanisms. However, TIMP-1 induction is not sufficient to counterbalance nHZ-dependent MMP-9 enhancement. Future investigation on proteinase-independent functions of TIMP-1 (i.e. cell survival promotion and growth/differentiation inhibition) is needed to clarify the role of TIMP-1 in malaria pathogenesis.

ASSA13-03-13 Inhibitory Effects of Vinpocetine on the Progression of Atherosclerosis Are Mediated by Akt/NF-kB Dependent Mechanisms

<h3>Objective</h3> Recent studies have found additional role for vinpocetine, a potent phosphodiesterase type I inhibitor, in anti-proliferation and anti-inflammation of vascular smooth muscle cells and cancer cells via different mechanisms. In this study, we attempted to investigate whether vinpocetine protected against atherosclerotic development in apoE^-/- mice and explore the underlying anti-atherogenic mechanisms in macrophages. <h3>Methods and Results</h3> Vinpocetine markedly decreased atherosclerotic lesion size in apoE^-/- mice measured by oil red O. Masson’s trichrome staining and immunohistochemical analyses revealed that vinpocetine significantly increased the thickness of fibrous cap, reduced the size of lipid-rich necrotic core and attenuated the expression of TNF-alpha and matrix metalloproteinase-9 (MMP-9) within plaque area. <i>In vitro</i> experiments exhibited a significant decrease in monocyte adhension treated with vinpocetine. In parallel with few changes in ox-LDL uptake and foam cell formation, the expression levels of scavenger receptor A and CD 36 were not altered after treatment with vinpocetine. Further, active TNF-alpha, IL-6, monocyte chemoattractant protein-1 and MMP-9 expression induced by ox-LDL was attenuated by vinpocetine in a dose-dependent manner. Similarly, ox-LDL-induced reactive oxygen species were significantly repressed by vinpocetine. Both western blot and luciferase activity assay showed that vinpocetine restored the enhanced Akt, IKK-alpha/beta, IkappaB-alpha phosphorylation and NF-kappaB activity induced by ox-LDL. Cotreatment with LY294002, a specific Akt inhibitor, augmented the inhibitory effects of vinpocetine on IKK-alpha/beta and IkappaB-alpha phosphorylation, suggesting that inhibition of NF-kappaB activity was partly caused by Akt dephosphorylation. Knockdown of PDE1B resulted in an increase of intracellular cGMP contents, however, did not affect Akt, IKK-alpha/beta and IkappaB-alpha phosphorylation. <h3>Conclusions</h3> Vinpocetine exerts anti-atherogenic effects through inhibition of monocyte adhension, oxidative stress and inflammatory response, which are mediated by Akt/NF-kappaB dependent pathway but independent of PDE1 blockade in macrophages.

Involvement of p38 MAPK in haemozoin‐dependent MMP‐9 enhancement in human monocytes

The lipid moiety of natural haemozoin (nHZ, malarial pigment) was previously shown to enhance expression and release of human monocyte matrix metalloproteinase-9 (MMP-9), and a major role for 15-(S,R)-hydroxy-6,8,11,13-eicosatetraenoic acid (15-HETE), a nHZ lipoperoxidation product, was proposed. Here, the underlying mechanisms were investigated, focusing on the involvement of mitogen-activated protein kinases (MAPKs). Results showed that nHZ promoted either early or late p38 MAPK phosphorylation; however, nHZ did not modify basal phosphorylation/expression ratios of extracellular signal-regulated kinase-1/2 and c-jun N-terminal kinase-1/2. 15-HETE mimicked nHZ effects on p38 MAPK, whereas lipid-free synthetic (s)HZ and delipidized (d)HZ did not. Consistently, both nHZ and 15-HETE also promoted phosphorylation of MAPK-activated protein kinase-2, a known p38 MAPK substrate; such an effect was abolished by SB203580, a synthetic p38 MAPK inhibitor. SB203580 also abrogated nHZ-dependent and 15-HETE-dependent enhancement of MMP-9 mRNA and protein (latent and activated forms) levels in cell lysates and supernatants. Collectively, these data suggest that in human monocytes, nHZ and 15-HETE upregulate MMP-9 expression and secretion through activation of p38 MAPK pathway. The present work provides new evidence on mechanisms underlying MMP-9 deregulation in malaria, which might be helpful to design new specific drugs for adjuvant therapy in complicated malaria.

Chlamydophila pneumoniae phospholipase D (CpPLD) drives Th17 inflammation in human atherosclerosis

Phospholipases are produced from bacterial pathogens causing very different diseases. One of the most intriguing aspects of phospholipases is their potential to interfere with cellular signaling cascades and to modulate the host-immune response. Here, we investigated the role of the innate and acquired immune responses elicited by Chlamydophila pneumoniae phospholipase D (CpPLD) in the pathogenesis of atherosclerosis. We evaluated the cytokine and chemokine production induced by CpPLD in healthy donors' monocytes and in vivo activated T cells specific for CpPLD that infiltrate atherosclerotic lesions of patients with C. pneumoniae antibodies. We also examined the helper function of CpPLD-specific T cells for monocyte matrix metalloproteinase (MMP)-9 and tissue factor (TF) production as well as the CpPLD-induced chemokine expression by human venular endothelial cells (HUVECs). We report here that CpPLD is a TLR4 agonist able to induce the expression of IL-23, IL-6, IL-1β, TGF-β, and CCL-20 in monocytes, as well as CXCL-9, CCL-20, CCL-4, CCL-2, ICAM-1, and VCAM-1 in HUVECs. Plaque-derived T cells produce IL-17 in response to CpPLD. Moreover, CpPLD-specific CD4(+) T lymphocytes display helper function for monocyte MMP-9 and TF production. CpPLD promotes Th17 cell migration through the induction of chemokine secretion and adhesion molecule expression on endothelial cells. These findings indicate that CpPLD is able to drive the expression of IL-23, IL-6, IL-1β, TGF-β, and CCL-20 by monocytes and to elicit a Th17 immune response that plays a key role in the genesis of atherosclerosis.

Cilostazol inhibits matrix invasion and modulates the gene expressions of MMP-9 and TIMP-1 in PMA-differentiated THP-1 cells

The invasion of monocytes into the subendothelium space plays an important role in the early stage of atherosclerosis. Cilostazol, a specific phosphodiesterase type III (PDE3) inhibitor, has been shown to exhibit anti-atherosclerotic effect. The present study aimed to investigate the modulating effects of cilostazol on monocyte invasion and the gene expressions of matrix metalloproteinase-9 (MMP-9) and tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) in phorbol 12-myristate 13-acetate (PMA)-differentiated THP-1. We found that PMA significantly increased the invasive ability and the MMP-9 activity of THP-1 cells, as analyzed by matrix invasion assay and gelatin zymography, respectively. The increased expression of MMP-9 was demonstrated at both the RNA and protein levels by RT/real-time PCR and western blot analysis. These changes were markedly inhibited by cilostazol in a dose-dependent manner, which also could be observed when cAMP analog was used. On the contrary, the expression of TIMP-1, an inhibitor of MMP-9, was significantly upregulated by cilostazol dose dependently at both the RNA and protein levels. Reverse zymography further confirmed the increase of TIMP-1 activity after cilostazol treatment. The increase of TIMP-1 by cilostazol, however, was not cAMP-dependent. Cilostazol reduced the MMP-9 promoter activity and suppressed the nuclear translocation of NF-κB, indicating that the inhibitory effect of cilostazol is at the transcriptional level. In conclusion, the present study provides an additional mechanism underlying the anti-atherosclerotic effect of cilostazol by inhibiting the monocyte invasion and modulating the gene expressions of MMP-9 and TIMP-1 in monocytes upon differentiating to macrophages.

Role of the NF-κB transcription pathway in the haemozoin- and 15-HETE-mediated activation of matrix metalloproteinase-9 in human adherent monocytes

Haemozoin (HZ, malarial pigment) is a crystalline ferriprotoporphyrin IX polymer derived from undigested host haemoglobin haem, present in late stages of Plasmodium falciparum-parasitized RBCs and in residual bodies shed after schizogony. It was shown previously that phagocytosed HZ or HZ-containing trophozoites increased monocyte matrix metalloproteinase-9 (MMP-9) activity and enhanced production of MMP-9-related cytokines TNF and IL-1beta. Here we show that in human monocytes the HZ/trophozoite phagocytosis effects and their recapitulation by 15(S,R)-hydroxy-6,8,11,13-eicosatetraenoic acid (15-HETE), a potent lipoperoxidation derivative generated by HZ from arachidonic acid via haem catalysis, were mediated via activation of NF-κB transcription pathway. After phagocytosis of HZ/trophozoites or treatment with 15-HETE, the NF-κB complex migrated to the nuclear fraction while the inhibitory cytosolic IκBalpha protein was phosphorylated and degraded. All HZ/trophozoite/15-HETE effects on MMP-9 activity and TNF/IL-1beta production were abrogated by quercetin, artemisinin and parthenolide, inhibitors of IκBalpha phosphorylation and subsequent degradation, NF-κB nuclear translocation, and NF-κB-p65 binding to DNA respectively. In conclusion, enhanced activation of MMP-9, and release of pro-inflammatory cytokines TNF and IL-1beta, a triad of effects involved in malaria pathogenesis, elicited in human monocytes by trophozoite and HZ phagocytosis and recapitulated by 15-HETE, appear to be causally connected to persisting activation of the NF-κB system.

Monocyte CD147 is induced by advanced glycation end products and high glucose concentration: possible role in diabetic complications

CD147 is a highly glycosylated transmembrane protein that is known to play a role in regulation of many protein families. It has the unique ability to maintain functional activity in both the membrane bound state and in the soluble form. CD147 is known to play a role in regulation of matrix metalloproteinase (MMP) expression, but whether its expression is affected by the diabetic milieu is not known, and its role in regulation of monocyte MMPs in this environment has not been investigated. Therefore, in this study we investigated the effect of advanced glycation end products (AGEs) and high glucose (HG; 25 mM), on monocyte CD147 expression. Culture of THP-1 monocytes in the presence of AGEs or HG significantly increased CD147 at the gene and protein level. THP-1 cell results were confirmed using freshly isolated monocytes from human volunteers. The effect of AGEs and HG on CD147 expression was also mimicked by addition of proinflammatory cytokines. Addition of AGEs or HG also increased expression of monocyte MMP-1 and MMP-9 but not MMP-2. This increase in MMPs was significantly attenuated by inhibition of CD147 using either a small interfering RNA or an anti-CD147 antibody. Inhibition of NF-κB or addition of antibodies to either TNF-α or the receptor for AGE (RAGE) each significantly prevented in a dose-dependent manner the induction of CD147 gene and protein by AGE and also decreased MMP-1 and MMP-9. This novel result shows that AGEs can induce monocyte CD147 expression, an effect mediated by inflammatory pathways and RAGE. Because MMPs play a role in monocyte migration, inhibition of their regulator CD147 may assist in the prevention of diabetic complications, particularly those where monocyte infiltration is an early initiating event.

Influence of atorvastatin on lipids and human monocyte matrix metalloproteinase-9 in acute cerebral infarction

Objective To observe the influence of atorvastatin on levels of lipids and human monocyte matrix metalloproteinase-9 (MMP-9) in acute cerebral infarction. Methods 60 patients with acute cerebral infarction receive oral atorvastatin of 20 mg daily for 4 weeks. The changes of serum lipids and MMP-9 after treatment were determined. Results 4 weeks after atorvastatin therapy, levels of TC and LDL-C were significantly lower while HDL-C was significantly higher; the changes of the lipid levels were much more obvious in the treatment group than in the control group (P<0.05 and P<0.01). MMP-9 levels were reduced significantly (P<0.01). Conclusions Atorvastatin not only has effects on lipid levels but also on a marked reduction in MMP-9 level. Key words: Atorvastatin; Cerebral infarction; Lipids; Human monocyte matrix metalloproteinase-9

Phagocytosis of haemozoin (malarial pigment) enhances metalloproteinase-9 activity in human adherent monocytes: Role of IL-1beta and 15-HETE

BackgroundIt has been shown previously that human monocytes fed with haemozoin (HZ) or trophozoite-parasitized RBCs displayed increased matrix metalloproteinase-9 (MMP-9) enzyme activity and protein/mRNA expression and increased TNF production, and showed higher matrix invasion ability. The present study utilized the same experimental model to analyse the effect of phagocytosis of: HZ, delipidized HZ, beta-haematin (lipid-free synthetic HZ) and trophozoites on production of IL-1beta and MMP-9 activity and expression. The second aim was to find out which component of HZ was responsible for the effects.MethodsNative HZ freshly isolated from Plasmodium falciparum (Palo Alto strain, Mycoplasma-free), delipidized HZ, beta-haematin (lipid-free synthetic HZ), trophozoites and control meals such as opsonized non-parasitized RBCs and inert latex particles, were fed to human monocytes. The production of IL-1beta by differently fed monocytes, in presence or absence of specific MMP-9 inhibitor or anti-hIL-1beta antibodies, was quantified in supernatants by ELISA. Expression of IL-1beta was analysed by quantitative real-time RT-PCR. MMP-9 activity and protein expression were quantified by gelatin zymography and Western blotting.ResultsMonocytes fed with HZ or trophozoite-parasitized RBCs generated increased amounts of IL-1beta and enhanced enzyme activity (in cell supernatants) and protein/mRNA expression (in cell lysates) of monocyte MMP-9. The latter appears to be causally related to enhanced IL-1beta production, as enhancement of both expression and enzyme activity were abrogated by anti-hIL-1beta Abs. Upregulation of IL-1beta and MMP-9 were absent in monocytes fed with beta-haematin or delipidized HZ, indicating a role for HZ-attached or HZ-generated lipid components. 15-HETE (15(S,R)-hydroxy-6,8,11,13-eicosatetraenoic acid) a potent lipoperoxidation derivative generated by HZ from arachidonic acid via haem-catalysis was identified as one mediator possibly responsible for increase of both IL-1beta production and MMP-9 activity.ConclusionResults indicate that specific lipoperoxide derivatives generated by HZ may play a role in modulating production of IL-1beta and MMP-9 expression and activity in HZ/trophozoite-fed human monocytes. Results may clarify aspects of cerebral malaria pathogenesis, since MMP-9, a metalloproteinase able to disrupt the basal lamina is possibly involved in generation of hallmarks of cerebral malaria, such as blood-brain barrier endothelium dysfunction, localized haemorrhages and extravasation of phagocytic cells and parasitized RBCs into brain tissues.

Low density lipoprotein from patients with Type 2 diabetes increases expression of monocyte matrix metalloproteinase and ADAM metalloproteinase genes

AimsType 2 diabetes is characterised by increased plasma concentrations of pro-inflammatory cytokines [such as tumour necrosis factor – alpha; TNF-α] and soluble forms of adhesion molecules involved in leukocyte – endothelial interactions. These molecules are synthesised as transmembrane proteins and the plasma soluble forms are generated by ectodomain cleavage from the cell surface by members of the ADAM [adisintegrin and metalloproteinase] proteinase family. We hypothesised that plasma low density lipoprotein [LDL] from subjects with Type 2 diabetes would influence in vitro monocytic ADAM and matrix metalloproteinase [MMP] gene expression differently compared to control LDL.MethodsWe examined relative mRNA expression by real time PCR in a monocytic cell line [THP-1] cultured for 4, 8 and 24 hrs with human plasma LDL derived from subjects with [n = 5] or without [n = 4] Type 2 diabetes. Gene expression for MMP-1 and 9, and ADAM – 8, 15, 17 and 28 was studied.ResultsType 2 diabetes LDL significantly increased gene expression of MMP – 1 [p < 0.01] MMP – 9 [p < 0.001], and ADAM 17 [p < 0.05], – 28 [p < 0.01] and – 15 [p < 0.01] compared to control LDL. Type 2 diabetes LDL had disparate effects on inhibitors of MMP.ConclusionThese data suggest that Type 2 diabetes LDL could lead to increased adhesion molecule and TNF alpha cell surface shedding, and vascular plaque instability, by promoting increased expression of ADAM and MMP genes.

Native and fragmented fibronectin oppositely modulate monocyte secretion of MMP-9

Monocytes remodel the extracellular matrix (ECM) by secreting proteins composing the ECM such as fibronectin (FN) and degrading proteases such as matrix metalloproteinase-9 (MMP-9), which cleaves FN into fragments. The effects of FN and its fragmented products on the expression of monocyte MMP-9 are controversial and largely unknown. We showed that in human monocytes, the proinflammatory cytokine TNF-alpha induced MMP-9 secretion and increased fragmentation of FN into distinct fragments. When primary monocytes or the U937 monocytic cell line were incubated on a plastic substrate, plastic-coated with native FN, and plastic-coated with fragmented FN (frag-FN), native FN inhibited TNF-alpha-induced proMMP-9 secretion by twofold (P<0.01) compared with plastic or frag-FN. Exploration of the dynamics of inflammation by incubating cells sequentially on the three substrates showed that frag-FN opposed the inhibitory effect of native FN. Inhibition of proMMP-9 by native FN was exerted at the translational level, as no change in MMP-9 mRNA, intracellular protein accumulation, or proteomic degradation was observed, and when degradation was blocked, no de novo translation of MMP-9 could be measured. We also showed that the reduction of MMP-9 secretion by native FN was responsible for attenuated migration of U937 cells (P<0.05). We suggest that in the inflammatory tissue, intact, native FN has a homeostatic role in harnessing MMP-9 activity. However, as fragmented products accumulate locally, they alleviate the inhibition and enable faster migration of the monocytes through the degraded ECM.

Phagocytic NADPH Oxidase-Dependent Superoxide Production Stimulates Matrix Metalloproteinase-9

Data suggest that matrix metalloproteinase-9 (MMP-9) has a role in atherosclerosis. The phagocytic NADPH oxidase has been also associated with atherosclerosis. This study aimed to investigate the association between phagocytic NADPH oxidase and MMP-9 in human atherosclerosis. In vitro experiments performed in human monocytes showed that NADPH oxidase activation enhanced MMP-9 secretion and activity, determined by enzyme-linked immunosorbent assay and zymography, respectively. Immunohistochemical study showed that phagocytic NADPH oxidase localized with MMP-9 in endarterectomies from patients with carotid stenosis. In addition, a positive relationship (P<0.001) was found between phagocytic NADPH oxidase-dependent superoxide production determined with lucigenin and plasma MMP-9 levels in 188 asymptomatic subjects free of overt clinical atherosclerosis. In multivariate analysis, this association remained significant after adjustment for cardiovascular risk factors. Interestingly, subjects in the upper quartile of superoxide production exhibited the highest values of MMP-9, oxidized low-density lipoprotein, nitrotyrosine, carotid intima media thickness, and an increased presence of carotid plaques. Enhanced NADPH oxidase-dependent *O2(-) production stimulates MMP-9 in monocytes and this relationship may be relevant in the atherosclerotic process. Moreover, MMP-9 emerges as an important mediator of the phagocytic NADPH oxidase-dependent oxidative stress in atherosclerosis.

Protein tyrosine kinase and p38 MAP kinase pathways are involved in stimulation of matrix metalloproteinase-9 by TNF-α in human monocytes

Matrix metalloproteinase-9 (MMP-9), through its catalytic and non-catalytic activities, plays critical roles in inflammation, tumor invasion and angiogenesis. Human monocytes actively involved in inflammatory and tumoral states secrete proMMP-9 (92 kDa). Endogenous TNF-α stimulates MMP-9 gene transcription in monocytes through NF-κB activation. In this study, we investigated the intracellular signaling pathways underlying TNF-α/NF-κB-dependent expression of MMP-9 in monocytes using chemical inhibitors that specifically inhibit distinct kinase pathways. We confirmed the expression of MMP-9 by reverse transcription chain reaction (RT-PCR), ELISA and gelatin zymography. PGE2/cAMP inhibitor indomethacin, PI-3K inhibitor wortmannin, PKC inhibitor bisindolylmaleimide and PKA inhibitor H-89 did not affect the levels of released MMP-9. In contrast, MMP-9 mRNA and protein expression was down-regulated by p38 MAPK inhibitor SB203580 and protein tyrosine kinase (PTK) inhibitor tyrphostin 25. These inhibitors increased IκB-α levels, which correlate with decreased NF-κB activation. Although SB203580 induced a decrease in TNF-α release, addition of exogenous TNF-α did not reverse the inhibitory effect of SB203580 toward MMP-9 thus suggesting that SB203580 could modulate down-stream effects of TNF-α. In parallel, TIMP-1 levels decreased in the presence of SB203580. Both kinase inhibitors did not influence the maturation pathway of monocytes. Our results indicate that these two inhibitors of p38 MAPK and PTK pathways could be used as combined targets for inhibiting MMP-9 expression in inflamed tissues.

Inhibition of matrix metalloproteinase-9 by interferons and TGF-β1 through distinct signalings accounts for reduced monocyte invasiveness

Cytokines may provide signals for regulating human monocyte matrix metalloproteinase-9 (MMP-9) activity. In this study, we investigated the roles of interferons (IFN) type I/II and transforming growth factor-β1 (TGF-β1) in MMP-9-mediated invasiveness. MMP-9 antibody and inhibitor, IFNs and TGF-β1 inhibited monocyte transmigration through Matrigel. IFNs and TGF-β1 downregulated MMP-9 mRNA, protein and activity levels. The inhibitory action of IFNs was associated with the STAT1/IRF-1 pathway since the JAK inhibitor AG490 blocked STAT1 phosphorylation, IRF-1 synthesis and counteracted the blockade of MMP-9 release. TGF-β1-mediated MMP-9 inhibition appeared STAT1/IRF-1-independent but reversed by the protein tyrosine kinase inhibitor tyrphostin 25. Our data point out the importance of IFNs and TGF-β1 in the control of monocyte MMP-9-mediated extravasation.

Chlamydia pneumoniaeEnhances Cytokine-Stimulated Human Monocyte Matrix Metalloproteinases through a Prostaglandin E2-Dependent Mechanism

Exposure of human monocytes to Chlamydia pneumoniae resulted in a significant enhancement of matrix metalloproteinase (MMP) 1 and 9 production following stimulation with tumor necrosis factor alpha and granulocyte monocyte-colony stimulating factor. The effect of C. pneumoniae on monocyte MMPs was mediated through the induction of prostaglandin E(2). These findings may have implications for atherosclerotic plaque rupture.

Interferon-γ Differentially Regulates Monocyte Matrix Metalloproteinase-1 and -9 through Tumor Necrosis Factor-α and Caspase 8

Tumor necrosis factor-alpha (TNFalpha) and granulocyte macrophage colony-stimulating factor (GM-CSF) individually enhance monocyte matrix metalloproteinase-9 (MMP-9) but induce MMP-1 only when added in combination. Because interferon-gamma (IFNgamma) is also found at inflammatory sites, we determined its effect on monocyte MMPs in the presence or absence of TNFalpha and GM-CSF. IFNgamma alone did not stimulate monocyte MMP-9 or MMP-1; however, in the presence of GM-CSF it induced MMP-1 and enhanced MMP-1 stimulated by GM-CSF and TNFalpha. IFNgamma induced MMP-1 in the presence of GM-CSF through the stimulation of TNFalpha production through a mechanism involving both p38 and ERK1/2 MAPKs, in which GM-CSF stimulated ERK1/2 whereas IFNgamma activated p38. In support of this conclusion TNFalpha neutralizing antibody and antibodies against TNF receptor I and -II blocked the induction of MMP-1 by GM-CSF and IFNgamma. In contrast to its effects on MMP-1, IFNgamma inhibited TNFalpha-induced MMP-9 through a caspase 8-dependent pathway as demonstrated by the restoration of MMP-9 with caspase 8 inhibitors. Moreover, the phosphorylation of STAT1 by IFNgamma was blocked by an inhibitor of caspase 8, indicating that STAT1 had a suppressive effect on MMP-9. Caspase 8-mediated phosphorylation of STAT1 through p38 MAPK as shown by the inhibition of IFNgamma-induced phosphorylation of p38 by caspase 8 inhibitors. Activation of caspase 8 by IFNgamma did not result in increased apoptosis. Thus IFNgamma in the presence of GM-CSF and/or TNFalpha differentially regulates monocyte MMPs through induction of TNFalpha and a novel mechanism involving caspase 8 that is independent of apoptosis.