In silico scaffold evaluation and solid phase approach to identify new gelatinase inhibitors

Among matrix metalloproteinases (MMPs), gelatinases MMP-2 (gelatinase A) and MMP-9 (gelatinase B) play a key role in a number of physiological processes such as tissue repair and fibrosis. Many evidences point out their involvement in a series of pathological events, such as arthritis, multiple sclerosis, cardiovascular diseases, inflammatory processes and tumor progression by degradation of the extracellular matrix. To date, the identification of non-specific MMP inhibitors has made difficult the selective targeting of gelatinases. In this work we report the identification, design and synthesis of new gelatinase inhibitors with appropriate drug-like properties and good profile in terms of affinity and selectivity. By a detailed in silico protocol and innovative and versatile solid phase approaches, a series of 4-thiazolydinyl-N-hydroxycarboxyamide derivatives were identified. In particular, compounds 9a and 10a showed a potent inhibitory activity against gelatinase B and good selectivity over the other MMP considered in this study. The identified compounds could represent novel potential candidates as therapeutic agents.     

Matrix metalloproteinase-9 deficiency results in enhanced allergen-induced airway inflammation

Matrix metalloproteinases (MMPs) are a large family of endopeptidases that proteolytically degrade extracellular matrix. Many different cells produce MMP-9, and levels have been shown to be up-regulated in patients with allergic asthma. The aim of this study was to investigate the in vivo role of MMP-9 during allergen-induced airway inflammation. Acute allergic pulmonary eosinophilia was established in MMP-9 knockout (KO) and wild-type (WT) control mice by sensitization and challenge with OVA. Cell recruitment was significantly increased in both bronchoalveolar lavage (BAL) and lung tissue compartments in MMP-9 KO mice compared with WT mice. This heightened cell recruitment was primarily due to increased eosinophils and Th2 cells in the BAL and lung tissue of MMP-9 KO mice in comparison with WT controls. Moreover, levels of the Th2 cytokines, IL-4 and IL-13, and the chemokines eotaxin/CCL11 and macrophage-derived chemokine/CCL22 were substantially increased in MMP-9 KO mice compared with WT after OVA challenge. Resolution of eosinophilia was similar between MMP-9 KO and WT mice, but Th2 cells persisted in BAL and lungs of MMP-9 KO mice for longer than in WT mice. Our results indicate that MMP-9 is critically involved in the recruitment of eosinophils and Th2 cells to the lung following allergen challenge, and suggest that MMP-9 plays a role in the development of Th2 responses to allergen.     

Polarized release of matrix metalloproteinase-2 and -9 from cultured human placental syncytiotrophoblasts

The large increase in placental surface area and fetal villous vascular development in the third trimester of pregnancy requires degradation and reformation of the placental basal lamina. Degradation is carried out by matrix metalloproteinases (MMPs) secreted by adjacent cells. Although the gelatinases, MMP-2 and MMP-9, which are released by extravillous cytotrophoblasts (CTs) are believed to play crucial roles in early placental expansion, neither has been reported in third trimester villous trophoblasts nor has appropriate (basolateral) release of any MMP by the highly polarized syncytiotrophoblast (ST) been demonstrated. We demonstrated villous trophoblast expression of both MMP-2 and MMP-9 by in situ immunohistochemistry and by Western blot analysis and zymography of lysates and culture supernatants of highly purified villous CTs. We also found that epidermal growth factor (EGF)-stimulated CT differentiation into ST and stimulation by the phorbol diester, PMA, both increase MMP-9 secretion. The direction of MMP release was determined with confluent cultures of ST on porous membranes. We found that >90% of MMP-2 and MMP-9 were released from the basolateral surface. We conclude that villous STs express and release gelatinases from their basolateral surfaces in a regulated manner and suggest that such polarized release may be important to villous tissue remodeling.     

Structural insights into the binding mode of flavonols with the active site of matrix metalloproteinase-9 through molecular docking and molecular dynamic simulations studies

Cartilage degradation in rheumatoid arthritis is mediated principally by the collagenases and gelatinases. Gelatinase B (also called matrix metalloproteinase 9 – MMP-9), is a valid target molecule which is known to participate in cartilage degradation as well as angiogenesis associated with the disease and inhibition of its activity shall prevent cartilage damage and angiogenesis. The focus of this study is to investigate the possibilities of MMP-9 inhibition by flavonol class of bioflavonoids by studying their crucial binding interactions at the active site of MMP 9 using molecular docking (Glide XP and QPLD) and further improvisation by post-docking MM-GBSA and molecular dynamic (MD) simulations. The results show that flavonols can convincingly bind to active site of MMP-9 as demonstrated by their stable interactions at the S1′ specificity pocket and favourable binding energies. Gossypin has emerged as a promising candidate with a docking score of ?14.618 kcal/mol, binding energy of ?79.97 kcal/mol and a stable MD pattern over 15 ns. In addition, interaction mechanisms with respect to catalytic site zinc are also discussed. Further, the drug-like characters of the ligands were also analysed using ADME analysis.     

Identifying specific matrix metalloproteinase-2-inhibiting peptides through phage display-based subtractive screening

Gelatinases A and B, which are members of the matrix metalloproteinase (MMP) family, play essential roles in cancer development and metastasis, as they can break down basal membranes. Therefore, the determination and inhibition of gelatinases is essential for cancer treatment. Peptides that can specifically block each gelatinase may, therefore, be useful for cancer treatment. In this study, subtractive panning was carried out using a 12-mer peptide library to identify peptides that block gelatinase A activity (MMP-2), which is a key pharmacological target. Using this method, 17 unique peptide sequences were determined. MMP-2 inhibition by these peptides was evaluated through zymogram analyses, which revealed that four peptides inhibited MMP-2 activity by at least 65%. These four peptides were synthesized and used for in vitro wound healing using human umbilical vein endothelial cells, and two peptides, AOMP12 and AOMP29, were found to inhibit wound healing by 40%. These peptides are, thus, potential candidates for MMP-2 inhibition for cancer treatment. Furthermore, our findings suggest that our substractive biopanning screening method is a suitable strategy for identifying peptides that selectively inhibit MMP-2.     

Control of type IV collagenase activity by components of the urokinase-plasmin system: a regulatory mechanism with cell-bound reactants.

The urokinase-type plasminogen activator (uPA) and the matrix-degrading metalloproteinases MMP-2 and MMP-9 (type IV collagenases/gelatinases) have been implicated in a variety of invasive processes, including tumor invasion, metastasis and angiogenesis. MMP-2 and MMP-9 are secreted in the form of inactive zymogens that are activated extracellularly, a fundamental process for the control of their activity. The physiological mechanism(s) of gelatinase activation are still poorly understood; their comprehension may provide tools to control cell invasion. The data reported in this paper show multiple roles of the uPA-plasmin system in the control of gelatinase activity: (i) both gelatinases are associated with the cell surface; binding of uPA and plasmin(ogen) to the cell surface results in gelatinase activation without the action of other metallo- or acid proteinases; (ii) inhibition of uPA or plasminogen binding to the cell surface blocks gelatinase activation; (iii) in soluble phase plasmin degrades both gelatinases; and (iv) gelatinase activation and degradation occur in a dose- and time-dependent manner in the presence of physiological plasminogen and uPA concentrations. Thus, the uPA-plasmin system may represent a physiological mechanism for the control of gelatinase activity.     

Selective ablation of matrix metalloproteinase-2 exacerbates experimental colitis: contrasting role of gelatinases in the pathogenesis of colitis

The matrix metalloproteinases (MMPs), MMP-2 and MMP-9, share structural and substrate similarities and are up-regulated during human as well as animal models of inflammatory bowel disease. We recently demonstrated that epithelial-derived MMP-9 is an important mediator of inflammation and tissue damage in colitis. In this study, we examined the role of MMP-2 in acute colitis. Colitis was induced using two models, administration of dextran sodium sulfate (DSS) and Salmonella enterica subsp. serovar Typhimurium (S.T.). Bone marrow chimeras were performed using bone marrow cells from wild-type (WT) and MMP-2(-/-) mice. Colitis was evaluated by clinical symptoms, myeloperoxidase assay, and histology. MMP-2 protein expression and activity were up-regulated in WT mice treated with DSS or S.T. MMP-2(-/-) mice were highly susceptible to the development of colitis induced by DSS (or S.T.) compared with WT. During inflammation, MMP-2 expression was increased in epithelial cells as well as in the infiltrating immune cells. Bone marrow chimera demonstrated that mucosa-derived MMP-2 was required for its protective effects toward colitis. Furthermore, we demonstrate that severe colitis in MMP-2(-/-) is not due to a compensatory increase in MMP-9. Finally, we show that MMP-2 regulates epithelial barrier function. In contrast to MMP-9, mucosa-derived MMP-2 may be a critical host factor that is involved in the prevention or cessation of the host response to luminal pathogens or toxins, an important aspect of healing and tissue resolution. Together, our data suggest that a critical balance between the two gelatinases determines the outcome of inflammatory response during acute colitis.     

Modulation of gelatinase activity correlates with the dedifferentiation profile of regenerating salamander limbs.

Remodeling of extracellular matrix (ECM) is one of the key events in many developmental processes. In the present study, a temporal profile of gelatinase activities in regenerating salamander limbs was examined zymographically. In addition, the effect of retinoic acid (RA) on these enzyme activities was examined to relate the pattern-duplicating effect of RA in limb regenerates with gelatinase activities. During regeneration, various types of gelatinase activities were detected, and these activities were at their maximum levels at the dedifferentiation stage. Upon treatment with chelating agents EDTA and 1,10-phenanthroline, the enzyme activities were inhibited indicating that those enzymes are likely matrix metalloproteinases (MMPs). Considering the molecular sizes and the decrease of molecular sizes by treatment with p-aminophenylmercuric acetate, an artificial activator of proMMP, some of the gelatinases expressed during limb regeneration are presumed to be MMP-2 and MMP-9. In RA-treated regenerates, overall gelatinase activities increased, especially the MMP-2-like gelatinase activity which increased markedly. These results suggest that MMP-2-like and MMP-9-like gelatinases play a role in ECM remodeling during regeneration, and that gelatinases are involved in the excessive dedifferentiation after RA treatment.     

Statins for the prevention of vein graft stenosis: a role for inhibition of matrix metalloproteinase-9

Saphenous vein (SV) grafts are commonly used to bypass coronary arteries that are diseased due to atherosclerosis. However, the development of intimal hyperplasia in such grafts can lead to patency-threatening stenosis and re-occlusion of the vessel. The proliferation and migration of smooth muscle cells (SMC) play key roles in the development of intimal hyperplasia, and an agent that inhibits both of these processes therefore has therapeutic potential. A prerequisite for SMC proliferation and migration in vivo is degradation of the basement membrane, achieved by secretion of the matrix-degrading gelatinases matrix metalloproteinase-2 (MMP-2) and MMP-9. Statins are cholesterol-lowering drugs that also have direct effects on SMC function. Here we report that neointima formation in organ-cultured human SV segments is inhibited by simvastatin, an effect that is associated with reduced MMP-9 activity. Additionally, our work shows that simvastatin not only inhibits proliferation, but importantly also inhibits invasion (migration through a matrix barrier), of cultured human SV SMC. Thus simvastatin treatment appears to inhibit neointima formation as a result of combined inhibition of SMC proliferation and invasion. The potential intracellular mechanisms by which statins affect SMC proliferation and migration, and thus attenuate intimal hyperplasia, are discussed, with particular emphasis on the role of MMP-9.     

Transgenic expression of matrix metalloproteinase-9 causes adult-onset emphysema in mice associated with the loss of alveolar elastin

Matrix metalloproteinase (MMP)-9 has been consistently identified in the lungs of patients with chronic obstructive pulmonary disease (COPD). However, its role in the development of the disease remains undefined. Mice that specifically express human MMP-9 in their macrophages were generated, and morphometric, biochemical, and histological analyses were conducted on the transgenic and littermate control mice over 1 yr to determine the effect of macrophage MMP-9 expression on emphysema formation and lung matrix content. Lung morphometry was normal in transgenic mice at 2 mo of age (mean linear intercept = 50+/-3 littermate mice vs. 51+/-2 transgenic mice). However, after 12 mo of age, the MMP-9 transgenic mice developed significant air space enlargement (mean linear intercept = 53+/-3 littermate mice vs. 61+/-2 MMP-9 transgenic mice; P<0.04). Lung hydroxyproline content was not significantly different between wild-type and transgenic mice, but MMP-9 did significantly decrease alveolar wall elastin at 1 yr of age (4.9+/-0.3% area of alveolar wall in the littermate mice vs. 3.3+/-0.3% area of alveolar wall in the MMP-9 mice; P<0.004). Thus these results establish a central role for MMP-9 in the pathogenesis of this disease by demonstrating that expression of this protease in macrophages can alter the extracellular matrix and induce progressive air space enlargement in mice.     

Involvement of fibronectin type II repeats in the efficient inhibition of gelatinases A and B by long-chain unsaturated fatty acids

The matrix metalloproteinases gelatinase A (MMP-2) and gelatinase B (MMP-9) are implicated in the physiological and pathological breakdown of several extracellular matrix proteins. In the present study, we show that long-chain fatty acids (e.g. oleic acid, elaidic acid, and cis- and trans-parinaric acids) inhibit gelatinase A as well as gelatinase B with K(i) values in the micromolar range but had only weak inhibitory effect on collagenase-1 (MMP-1), as assessed using synthetic or natural substrates. The inhibition of gelatinases depended on fatty acid chain length (with C18 > C16, C14, and C10), and the presence of unsaturations increased their inhibitory capacity on both types of gelatinase. Ex vivo experiments on human skin tissue sections have shown that micromolar concentrations of a long-chain unsaturated fatty acid (elaidic acid) protect collagen and elastin fibers against degradation by gelatinases A and B, respectively. In order to understand why gelatinases are more susceptible than collagenase-1 to inhibition by long-chain fatty acids, the possible role of the fibronectin-like domain (a domain unique to gelatinases) in binding inhibitory fatty acids was investigated. Affinity and kinetic studies with a recombinant fibronectin-like domain of gelatinase A and with a recombinant mutant of gelatinase A from which this domain had been deleted pointed to an interaction of long-chain fatty acids with the fibronectin-like domain of the protease. Surface plasmon resonance studies on the interaction of long-chain fatty acids with the three individual type II modules of the fibronectin-like domain of gelatinase A revealed that the first type II module is primarily responsible for binding these compounds.     

Lack of matrix metalloproteinase-9 worsens ventilator-induced lung injury

Matrix metalloproteinase-9 (MMP-9) is released by neutrophils at the sites of acute inflammation. This enzyme modulates matrix turnover and inflammatory response, and its activity has been found to be increased after ventilator-induced lung injury. To clarify the role of MMP-9, mice lacking this enzyme and their wild-type counterparts were ventilated for 2 h with high- or low-peak inspiratory pressures (25 and 15 cmH2O, respectively). Lung injury was evaluated by gas exchange, respiratory mechanics, wet-to-dry weight ratio, and histological analysis. The activity of MMP-9 and levels of IL-1beta, IL-4, and macrophage inflammatory protein (MIP-2) were measured in lung tissue and bronchoalveolar lavage fluid (BALF). Cell count and myeloperoxidase activity were measured in BALF. There were no differences between wild-type and Mmp9-/- animals after low-pressure ventilation. After high-pressure ventilation, wild-type mice exhibited an increase in MMP-9 in tissue and BALF. Mice lacking MMP-9 developed more severe lung injury than wild-type mice, in terms of impaired oxygenation and lung mechanics, and higher damage in the histological study. These effects correlated with an increase in both cell count and myeloperoxidase activity in the BALF, suggesting an increased neutrophilic influx in response to ventilation. An increase in IL-1beta and IL-4 in the BALF only in knockout mice could be responsible for the differences. There were no differences between genotypes in MMP-2, MMP-8, or tissue inhibitors of metalloproteinases. These results show that MMP-9 protects against ventilator-induced lung injury by decreasing alveolar neutrophilic infiltration, probably by modulation of the cytokine response in the air spaces.     

Role of matrix metalloproteinase-9 in endothelial apoptosis in chronic heart failure in mice.

Accumulation of oxidized extracellular matrix between endothelium and muscle is an important risk factor in the endothelium-myocytes uncoupling in congestive heart failure. Although ventricular remodeling is accompanied by increased matrix metalloproteinase (MMP)-9 activity, it is unclear whether MMP-9 plays a role in endothelial apoptosis in chronic volume overload congestive heart failure. We tested the hypothesis that, in chronic volume overload, myocardial dysfunction involves endocardial endothelial (EE) apoptosis in response to MMP-9 activation, extracellular matrix accumulation, and endothelium-myocytes uncoupling. Arteriovenous fistula (AVF) was created in control (FVB/NJ) and MMP-9 knockout (MMP-9KO; FVB.Cg-MMP9(tm1Tvu)/J) mice. Sham surgery was used as control. Mice were grouped as follows: wild type, n = 3 (sham control); MMP-9KO, n = 3 (sham); AVF, n = 3; and MMP-9KO + AVF (n = 3). Heart function was analyzed by M-mode and Doppler echocardiography, and with a pressure-tipped Millar catheter placed in the left ventricle of anesthetized mice 8 wk after AVF. Apoptosis was detected by measuring caspase-3, transferase-mediated dUTP nick-end labeling (TUNEL), and CD-31 by immunolabeling. Protease-activated receptors-1, connexin-43, and a disintegrin and MMP-12 (ADAM-12) expression were measured by Western blot analyses. MMP-2 and MMP-9 expression were measured by quantitative RT-PCR. Compared with control, AVF caused an increase in left ventricle end diastolic pressure and decrease in -dP/dt. In contrast, in the MMP-9KO + AVF group, these variables were changed toward control levels. Increased EE apoptosis (caspase-3 activation and TUNEL/CD-31 colabeling) in AVF mice was prevented in the MMP-9KO + AVF group. Protease-activated receptor-1, connexin-43, and ADAM-12 were induced in AVF. MMP-9 gene ablation ameliorated the induction. The results suggest that impaired cardiac function in volume overload is associated with EE apoptosis, cardiac remodeling, and endothelium-myocytes uncoupling in response to MMP-9 activation.     

Structural basis for potent slow binding inhibition of human matrix metalloproteinase-2 (MMP-2)

The zinc-dependent gelatinases belong to the family of matrix metalloproteinases (MMPs), enzymes that have been shown to play a key role in angiogenesis and tumor metastasis. These enzymes are capable of hydrolyzing extracellular matrix (ECM) components under physiological conditions. Specific and selective inhibitors aimed at blocking their activity are highly sought for use as potential therapeutic agents. We report herein on a novel mode of inhibition of gelatinase A (MMP-2) by the recently characterized inhibitors 4-(4-phenoxphenylsulfonyl)butane-1,2-dithiol (inhibitor 1) and 5-(4-phenoxphenylsulfonyl) pentane-1,2-dithiol (inhibitor 2). These synthetic inhibitors are selective for MMP-2 and MMP-9. We show that the dithiolate moiety of these inhibitors chelates the catalytic zinc ion of MMP-2 via two sulfur atoms. This mode of binding results in alternation of the coordination number of the metal ion and the induction of conformational changes at the microenvironment of the catalytic zinc ion; a set of events that is likely to be at the root of the potent slow binding inhibition behavior exhibited by these inhibitors. This study demonstrates a distinct approach for the understanding of the structural mechanism governing the molecular interactions between potent inhibitors and catalytic sites of MMPs, which may aid in the design of effective inhibitors.     

Engineering autoactivating forms of matrix metalloproteinase-9 and expression of the active enzyme in cultured cells and transgenic mouse brain

Matrix metalloproteinases (MMPs) are hypothesized to play an important role in the pathogenesis of several central nervous system disorders. Increased levels of expression of MMP-9 (gelatinase B) and MMP-2 (gelatinase A) have been observed in Alzheimer's disease, stroke, multiple sclerosis, and amyotrophic lateral sclerosis. This suggests an aberrant regulation of MMPs that could lead to inappropriate expression of MMP activity. To allow us to evaluate the effect of increased levels of active MMP-9 in the central nervous system, mutant forms of the enzyme were designed to autocatalytically remove the pro domain, yielding active enzyme. This was accomplished by modifying residues in the cysteine switch autoinhibitor region of the propeptide. Stable cell lines and transgenic mice that express G100L and D103N autoactive forms of human MMP-9 were developed to study the role of dysregulation of MMP-9 in disease.     

Matrix metalloproteinase-9 (gelatinase B) deficiency leads to increased severity of Staphylococcus aureus-triggered septic arthritis

Matrix metalloproteinases constitute a family of structurally related endopeptidases that are crucial for the normal turnover of the extracellular matrix. Elevated levels of MMP-9 have been demonstrated in synovial fluids of rheumatoid arthritis patients, and a correlation with the severity of the disease has been described. The aim of this study was to explore the impact of MMP-9 expression on joint inflammation and destruction in a model of bacterially induced septic arthritis. MMP-9 knock-out mice and C57Bl6 congenic controls were inoculated intravenously or intra-articularly with Staphylococcus aureus. Arthritis was evaluated clinically and by means of histology. Zymographic analyses were performed to study ex vivo induction of MMP-9 following exposure to S. aureus. The MMP-9 knock-out mice displayed a significantly higher frequency and severity, but not destructivity, of arthritis than did the wild-type mice. The knock-out mice also proved to harbour an increased number of bacteria locally in joints and systemically in kidneys, possibly by impaired extravasation and recruitment of leukocytes and a deficient early defence against infection. Our findings indicate that deficiency in MMP-9 increases the degree of joint inflammation due to decreased bacterial clearance.     

The role of gelatinases in colorectal cancer progression and metastasis

Various proteases are involved in cancer progression and metastasis. In particular, gelatinases, matrix metalloproteinase-2 (MMP-2) and MMP-9, have been implicated to play a role in colon cancer progression and metastasis in animal models and patients. In the present review, the clinical relevance and the prognostic value of messenger ribonucleic acid (mRNA) and protein expression and proenzyme activation of MMP-2 and MMP-9 are evaluated in relation to colorectal cancer. Expression of tissue inhibitors of MMPs (TIMPs) in relation with MMP expression in cancer tissues and the relevance of detection of plasma or serum levels of MMP-2 and/or MMP-9 and TIMPs for prognosis are also discussed. Furthermore, involvement of MMP-2 and MMP-9 in experimental models of colorectal cancer is reviewed. In vitro studies have suggested that gelatinase is expressed in cancer cells but animal models indicated that gelatinase expression in non-cancer cells in tumors contributes to cancer progression. In fact, interactions between cancer cells and host tissues have been shown to modulate gelatinase expression in host cells. Inhibition of gelatinases by synthetic MMP inhibitors has been considered to be an attractive approach to block cancer progression. However, despite promising results in animal models, clinical trials with MMP inhibitors have been disappointing so far. To obtain more insight in the (patho)physiological functions of gelatinases, regulation of MMP-2 and MMP-9 expression is discussed. Mitogen activated protein kinase (MAPK) signalling has been shown to be involved in regulation of gelatinase expression in both cancer cells and non-cancer cells. Expression can be triggered by a variety of stimuli including growth factors, cytokines and extracellular matrix (ECM) components. On the other hand, MMP-2 and MMP-9 activity regulates bioavailability and activity of growth factors and cytokines, affects the immune response and is involved in angiogenesis. Because of the multifunctionality of gelatinases, it is unpredictable at what stage of cancer development and in which processes gelatinase activity is involved. Therefore, it is concluded that the use of MMP inhibitors to treat cancer should be considered carefully.     

Differential roles for two gelatinolytic enzymes of the matrix metalloproteinase family in the remodelling cornea.

We have documented changes in collagenolytic/gelatinolytic enzymes of the matrix metalloproteinase family (MMP) in remodelling rabbit cornea. MMP-2 (65 kDa gelatinase) in the proenzyme form is synthesized by the cells of the normal corneal stroma. After keratectomy the level of MMP-2 is increased in the stroma and enzyme appears in both pro- and activated forms. In addition, corneal cells synthesize MMP-9 (92 kDa gelatinase) in the proenzyme form after keratectomy; expression occurs in both the epithelial as well as stromal corneal layers. Changes in expression of both enzymes are precisely localized to the repairing portion of cornea, but demonstrate important differences in timing that correlate with the timing of specific events of matrix remodelling. Our data suggest that each of the gelatinases plays a different role in tissue remodelling after injury. We hypothesize that MMP-2 performs a surveillance function in normal cornea, catalyzing degradation of collagen molecules that occasionally become damaged. After wounding, this enzyme appears to participate in the prolonged process of collagen remodelling in the corneal stroma that eventually results in functional regeneration of the tissue. MMP-9 expression does not correlate with stromal remodelling, but we suggest that the enzyme might play a part in controlling resynthesis of the epithelial basement membrane.     

Effects of nitric oxide on matrix metalloproteinase-2 production by rheumatoid synovial cells

Nitric oxide (NO) is a multifunctional messenger molecule generated from L-arginine by a family of enzymes, including nitric oxide synthase (NOS). This study was performed to examine whether NO modulates the production of matrix metalloproteinases (MMPs), which degrade all components of extracellular matrix (ECM), in rheumatoid synovial cells. We investigated the effects of exogenously generated NO by a NO donor, S-nitroso-N-acetyl-DL-penicillamine (SNAP), on the MMPs production by rheumatoid synovial cells. Culture media conditioned by SNAP-treated synovial cells were examined by gelatin zymography and immunoblot analysis. Incubation of synovial cells with SNAP resulted in gelatinase A production in a dose-dependent fashion. Furthermore, RT-PCR analysis demonstrated that MMP-2 mRNA expression was induced in SNAP-treated synovial cells. In contrast, SNAP did not influence the production of TIMP-1 and TIMP-2, which preferentially inhibit MMP-2, by rheumatoid synovial cells. Our data indicate that NO could modulate MMP production by rheumatoid synovial cells and therefore contribute to ECM degradation of articular components in RA.     

Involvement of 15-lipoxygenase in the inflammatory arthritis

15-Lipoxygenase (15-LOX) is involved in many pathological processes. The aim of this study is to examine the role of 15-LOX in the matrix metalloproteinase (MMP) expression and inflammatory arthritis. It was found that treatment of 15-LOX downstream product of 15-(S)-HETE (15-S-hydroxyeicosatetraenoic acid) increased the mRNA and protein levels of MMP-2 in rheumatoid arthritis synovial fibroblast (RASF) derived from rheumatoid arthritis patients. The enhancement effect of 15-(S)-HETE was antagonized by the addition of LY294002 (PI3K inhibitor) and PDTC (NF-κB inhibitor). Treatment of 15-(S)-HETE increased the phosphorylation of AKT, nuclear translocation of p65 and the breakdown of IκBα. TNF-α and IL-1β are the key cytokines involved in arthritis and also increase the activity of MMP-2 in RASF, which was antagonized by pretreatment with 15-LOX inhibitor PD146176 or knockdown of 15-LOX. It was also found that these two cytokines increased the expression of 15-LOX in RASF. Treatment of glucocorticoid but not NSAIDs inhibited 15-(S)-HETE-induced expression of MMP-2. In comparison with wild-type mice, adjuvant-induced arthritis and MMP-2 expression in synovial membrane were markedly inhibited in 15-LOX knockout (KO) mice. These results indicate that 15-LOX plays an important role in the disease progression of arthritis and may be involved in the inflammatory action induced by TNF-α and IL-1β. 15-LOX is thus a good target for developing drugs in the treatment of inflammatory arthritis.