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GASTROINTESTINAL, HEPATIC, PULMONARY, AND RENAL
Department of Biochemistry and Molecular Genetics (R.H., A.P., A.G.-P., N.F., E.T., M.L.-P., J.C.), Pathology Laboratory (R.M.) and Liver Unit (V.A.), Hospital Clínic, Centro de Investigación Biomédica Esther Koplowitz and Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas, Universitat de Barcelona, Barcelona, Spain; and Pfizer Global Research & Development, St. Louis Missouri (J.L.M.)
Received July 6, 2007; accepted August 30, 2007.
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Abstract |
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). In particular, COX-2, the inducible COX isoform, is a key executor of uncontrolled inflammation and its inhibition represents a major target in the treatment of inflammatory disorders (Fitzgerald and Patrono, 2001). On the other hand, emerging information has indicated the contribution of another pathway of arachidonic acid metabolism, the 5-lipoxygenase (5-LO) pathway, in developing and sustaining inflammation (Samuelsson et al., 1987; Funk, 2001). Through the 5-LO pathway, arachidonic acid is converted into leukotrienes (LTs), including LTB4, a potent chemotactic agent for leukocytes, and cysteinyl-LTs (LTC4, LTD4, and LTE4), which are potent mediators of allergic and hypersensitivity reactions (Samuelsson et al., 1987; Funk, 2001). The fact that COX-2- and 5-LO-derived products are directly implicated in inflammation has fostered the development of drugs that directly target the formation of PGs and LTs or their binding to specific receptors. The current availability of these pharmacological tools provides new opportunities in inflammation therapy.
Recent evidence indicate that the COX-2 and 5-LO pathways have converging functions in liver inflammation, tissue remodeling, and fibrosis (Titos et al., 2000, 2003, 2005; Yamamoto et al., 2003; Núñez et al., 2004; Planagumà et al., 2005). Indeed, COX-2 expression is up-regulated in patients with chronic liver disease and closely correlates with progressive fibrosis in patients with hepatitis C infection (Cheng et al., 2002a; Mohammed et al., 2004; Núñez et al., 2004). COX-2 expression is also up-regulated in rats with carbon tetrachloride (CCl4)-induced liver injury and in experimental models of alcoholic liver disease and steatohepatitis (Nanji et al., 1997; Planagumà et al., 2005; Yu et al., 2006). Recently, Yu et al. (2007) have shown that the expression of a human COX-2 transgene in murine liver causes hepatitis. Importantly, COX-2 inhibition reduces cell growth and triggers apoptosis in hepatic stellate cells and exerts antifibrogenic actions in vivo (Cheng et al., 2002b; Yamamoto et al., 2003; Planagumà et al., 2005). Similar to COX-2, the up-regulation of 5-LO has been reported in patients with chronic liver disease and in experimental models of liver injury (Uemura et al., 1994; Titos et al., 2000). 5-LO-derived products have been shown to activate hepatic stellate cells, and inhibition of their formation induces apoptosis in Kupffer cells, the major inflammatory cell type in the liver (Titos et al., 2000, 2003). Finally, blockade of the 5-LO pathway with a 5-lipoxygenase-activating protein (FLAP) inhibitor protects the liver from experimental necroinflammatory damage and fibrosis (Titos et al., 2003, 2005).
Although both COX-2 and 5-LO seem to be implicated in liver injury, at present the exact contribution of each pathway to the initiation and progression of liver inflammation and fibrosis is uncertain. In the current study we assessed the relative contribution of COX-2 and 5-LO pathways to liver inflammation and fibrosis by administering a selective COX-2 inhibitor (SC-236) and a nonredox-type 5-LO inhibitor (CJ-13,610), either alone or in combination, to mice with CCl4-induced liver injury. Additional studies were performed in 5-LO-deficient mice, as a genetic model of 5-LO inhibition, as well as in murine macrophages, as a relevant model of inflammatory cell type.
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Materials and Methods |
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-actin assays-on-demand were from Applied Biosystems (Foster City, CA).
Animal Studies. 129S2/SvPasCrl mice were assigned to four different groups (nine mice each) that received placebo, a COX-2 inhibitor (SC-236), a nonredox-type 5-LO Inhibitor (CJ-13,610), or a combination of SC-236 plus CJ-13,610. SC-236 was given in the drinking water at a dose of 6 mg/kg (Kishi et al., 2000). To achieve this dose, SC-236 was dissolved in a stock solution of 5% Tween 20 and 95% polyethylene glycol and diluted in distilled water to achieve a final SC-236 concentration of 0.045 mg/ml. CJ-13,610 was given in the diet at a dose of 10 mg/kg. The CJ-13,610-containing diet was prepared by PMI Nutrition International (Brentwood, MO). After 1 week of drug administration, mice received i.p. injections of CCl4 (1 ml/kg in olive oil, twice a week) for 8 weeks. Thereafter, animals were sacrificed, and liver samples were either fixed in 10% formalin or snap-frozen in N2.
Additional studies were performed in 5-LO-deficient (129-Alox5tm1Fun, n = 15) and wild-type (129S2/SvPasCrl, n = 14) mice (The Jackson Laboratory), that were divided in four experimental groups: wild-type plus placebo, wild-type plus SC-236, 5-LO-deficient plus placebo, and 5-LO-deficient plus SC-236. Mice were sacrificed after 6 weeks, and liver samples collected as described above.
All animal studies were conducted in accordance with the criteria of the Investigation and Ethics Committee of the Hospital Clínic and the European Community laws governing the use of experimental animals.
Histological Analysis. Liver samples were formalin-fixed, embedded in paraffin, sectioned (5 µm), and stained with hematoxylineosin. Necroinflammation was scored by a registered pathologist (R.M.) unaware of the treatments as grade 0 (absent), grade 1 (spotty necrosis), grade 2 (confluent necrosis), and grade 3 (bridging necrosis). Liver fibrosis was assessed by Sirius Red staining. In brief, liver sections were incubated for 10 min with thiosemicarbazide (0.5%), stained in Sirius Red F3B (0.1%) in saturated picric acid for 1 h, and subsequently washed with acetic acid (0.5%). Sections were visualized under a microscope (Eclipse E600; Nikon, Kawasaki, Japan), and the fibrosis area quantified by morphometry using a computerized system (AnalySIS, Munster, Germany).
TUNEL Assay. Apoptosis in liver sections (5 µm) was determined by TUNEL assay (Roche Diagnostics). Enzymatic labeling was performed after blocking the endogenous peroxidase in 3% H2O2 for 20 min and after proteinase K treatment (20 µg/ml in HCl-Tris) for 20 min. The fluorescent signal was converted into a chromogenic signal by adding diaminobenzidine. Finally, slides were counterstained with hematoxylin-eosin. The number of nonparenchymal TUNEL-positive cells was counted in a total of 20 fields/tissue section under the microscope (200x magnification).
Analysis of Eicosanoids. PGE2 and LTB4 concentrations in cell supernatants were determined in unextracted samples by EIAs.
Cell Incubations. Raw 264.7 cells were grown in 150 cm2 flasks in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 50 U/ml penicillin, and 50 µg/ml streptomycin. All incubations were performed in cells under the 16th passage. Cells were transferred to 12-well plates at a density of 200,000 cells/well in complete medium, and incubations were performed under a humidified 5% CO2 atmosphere at 37°C. In experiments assessing the effects of compounds on PGE2 biosynthesis, cells were primed with LPS (500 ng/ml) to induce a sustained expression of COX-2 mRNA and protein, as described previously for macrophages (Barrios-Rodiles et al., 1999). Twenty-four hours later, cells were washed and incubated with vehicle (0.2% DMSO) or increasing concentrations of the selective COX-2 inhibitor, SC-236 (0.1, 1, and 10 µM) and the dual COX/5-LO inhibitor, licofelone (1, 10, and 30 µM) for 15 min. Thereafter, cells were exposed to ionophore A23187
[GenBank]
(2–5 µM) for an additional 15 min to stimulate the production of arachidonic acid metabolites (Kouzan et al., 1985). To test the effects on LTB4 biosynthesis, cells were directly incubated with vehicle (0.2% DMSO), increasing concentrations of the 5-LO inhibitors CJ-13,610 (0.1, 1, and 10 µM) and AA-861 (1, 10, and 15 µM) or licofelone (1, 10, and 30 µM) for 15 min and subsequently stimulated with ionophore A23187
[GenBank]
(2–5 µM) for an additional 15 min. In some experiments, CJ-13,610 (1 µM) or AA-861 (10 µM) were combined with SC-236 (1 µM).
For gene expression analysis, Raw 264.7 cells were grown in six-well plates (500,000 cells/well) in serum-free medium. Twentyfour hours later, cells were incubated with vehicle (0.1% DMSO), SC-236 (3 µM), CJ-13,610 (1 µM), AA-861 (10 µM), licofelone (30 µM), the LTB4 receptor (LTB1) antagonist CP-105,696 (0.1 µM), or the LTD4 receptor (Cys-LT1) antagonist MK-571 (1 µM) for 2 h at 37°C and then stimulated with PMA (50 nM) and ionophore (2 µM) for additional 4 h. In some incubations, the compounds were combined with SC-236 (3 µM). The concentrations of SC-236, CJ-13,610, and AA-861 were selected from previous studies demonstrating effective inhibition of either COX-2 or 5-LO activity (Titos et al., 2003; Fischer et al., 2004; López-Parra et al., 2005). The concentrations of CP-105,696 and MK-571 were selected from previous publications demonstrating selective blockade of LTB4 and LTD4 receptors, respectively (Titos et al., 2000; Huang et al., 2004). The licofelone concentration was selected from Tries et al. (2002), Marcouiller et al. (2005), and Vidal et al. (2007).
Cell Viability Assay. Raw 264.7 macrophages were seeded in 24-well plates (100,000 cells/well) and incubated with vehicle (0.2% DMSO), SC-236 (0.1, 1, 10, and 50 µM), or CJ-13,610 (0.1, 1, 10, and 50 µM) for 1 h at 37°C. Cell viability was tested by addition of 100 µl of MTT (5 mg/ml stock solution) to each well for 3 h and subsequent lysis with isopropyl alcohol and shaking for 20 min. The absorbance at 570 nm was measured in a multiwell plate reader (BMG Labtech, Offenburg, Germany) and cell number was calculated from a standard curve.
Gene Profiling by RT-PCR. RNA was isolated using the RNAqueous kit. RNA concentration was assessed in a UV spectrophotometer, and its integrity was tested in a 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA). Samples were digested with DNase and retrotranscribed with the high-capacity cDNA archive kit. PCR amplification of COX-1, COX-2, 5-LO, FLAP, LTC4 synthase (LTC4S), LTA4 hydrolase (LTA4H), and 12/15-LO was performed with specific oligonucleotides (Table 1). The specificity of primers was confirmed in the GenBank database using the Basic Local Alignment Search Tool (BLAST) and by direct sequencing of the amplified PCR products in an ABI Prism 3130xl Genetic Analyzer using a Big Dye Terminator version 3.1 Cycle Sequencing Kit (Applied Biosystems). PCR products were analyzed by electrophoresis in 1.5% agarose gels and visualized by ethidium bromide staining.
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Analysis of Gene Expression by RT and Real-time PCR. Real-time quantitative PCR was performed in an ABI Prism 7900 Sequence Detection System (Applied Biosystems). Ready-to-use primer and probe sets (TaqMan Gene Expression Assays) were used to quantify MCP-1 and IL-6 gene expression using -actin as an endogenous control. PCR results were analyzed with Sequence Detector Software (version 2.1; Applied Biosystems). Relative quantification of gene expression was performed using the comparative Ct method. The amount of target gene, normalized to -actin and relative to a calibrator, was determined by the arithmetic formula 2–
Ct described in the Comparative Ct Method (User Bulletin #2; http://docs.appliedbiosystems.com/pebiodocs/04303859.pdf).
Statistical analysis of the results was performed using analysis of variance and unpaired Student's t test. Results are expressed as means ± S.E.M., and differences were considered significant at P < 0.05.
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We next compared the effects of pharmacological inhibition of 5-LO with those exerted by the genetic inhibition of this pathway. In 5-LO-deficient mice, we confirmed that inhibition of a single pathway is not enough for preventing hepatic necroinflammation induced by CCl4. Indeed, hepatocellular damage was significantly reduced only in 5-LO-deficient mice receiving SC-236 but not in wild-type mice receiving SC-236 or in mice lacking the 5-LO gene (Fig. 2, left panels). Consistent with these findings, expression of hepatic MCP-1, a potent chemoattractant protein that contributes to the maintenance of the inflammatory infiltrate during liver injury (Efsen et al., 2001), was only significantly down-regulated in 5-LO-deficient mice receiving SC-236 (Fig. 2, middle panels). Hepatic fibrosis was reduced to a similar extent by SC-236 in both wild-type and 5-LO-deficient mice (Fig. 2, right panels). 5-LO-deficient mice were not resistant to CCl4-induced fibrosis (Fig. 2, right panels). Given that COX-2-deficient mice exhibit decreased fertility and survival, we were unable to perform similar studies in these mice.
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). As shown in Fig. 3A, Raw 264.7 macrophages expressed all key enzymes of eicosanoid biosynthesis, namely COX-1, COX-2, 5-LO, FLAP, LTC4S, LTA4H, and 12/15-LO. Consistent with these findings, these cells generated PGE2 and LTB4, two proinflammatory products derived from COX and 5-LO pathways, respectively (Fig. 3, B and C). SC-236 inhibited in a concentration-dependent manner PGE2 biosynthesis (Fig. 3B), whereas CJ-13,610 markedly blocked LTB4 formation (Fig. 3C). SC-236 and CJ-13,610 only affected macrophage viability at concentrations higher than those necessary to inhibit PGE2 and LTB4 formation (Fig. 3D). In addition, we compared the effects of SC-236 and CJ-13,610 with those of the dual COX/5-LO inhibitor, licofelone. As shown in Fig. 3E, licofelone inhibited PGE2 and LTB4 formation in a concentration-dependent manner. However, the inhibitory profile of this drug on PGE2 biosynthesis was lower than that produced by the combination of SC-236 and CJ-13,610 (Table 2). Finally, given that CJ-13,610 is a nonredox-type 5-LO inhibitor of recent development poorly characterized in macrophages, we compared its inhibitory properties with those of a well known inhibitor (i.e., AA-861) (Titos et al., 2003). As shown in Table 3, CJ-13,610 inhibited 5-LO activity, estimated by LTB4 production, to an extent similar to that of AA-861.
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We next explored the effects of drugs on macrophage IL-6 expression, as COX-2 and 5-LO are known to regulate cytokine secretion in these cells (Sipe et al., 1992; Marcouiller et al., 2005). As shown in Fig. 4A, SC-236 and CJ-13,610 had opposite effects on the expression of this cytokine. On one hand, SC-236 further stimulated IL-6 expression, whereas on the other hand, CJ-13,610 significantly down-regulated its expression (Fig. 4A). AA-861 reproduced the inhibitory actions seen with CJ-13,610 (Fig. 4A). SC-236 abrogated the inhibitory effect on IL-6 expression exerted by CJ-13,610 but not that exerted by AA-861 (Fig. 4A). The modulation of IL-6 expression by 5-LO appeared to be mediated by LTB4 as CP-105,696, a BLT1 receptor antagonist, but not MK-571, a Cys-LT1 receptor antagonist, significantly reduced IL-6 expression (Fig. 4B). Of note, licofelone significantly up-regulated IL-6 mRNA expression (Fig. 4C).
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; Cheng et al., 2002a; Mohammed et al., 2004; Núñez et al., 2004) and in experimental models including CCl4-induced liver injury, alcoholic liver disease, and diet-induced steatohepatitis (Nanji et al., 1997; Titos et al., 2000, 2003, 2005; Planagumà et al., 2005; Yu et al., 2006). Moreover, our results underscore previous studies showing protective effects of selective COX-2 and FLAP inhibitors in the liver (Endoh et al., 1996; Yamamoto et al., 2003; Planagumà et al., 2005; Titos et al., 2005).
An interesting finding of the current study was that in addition to reducing fibrosis, the simultaneous inhibition of the COX-2 and 5-LO pathways significantly reduced necroinflammatory liver injury. This effect was not observed after the separate administration of each inhibitor. This result is somewhat surprising because previous studies have shown that necroinflammatory liver injury, assessed by transaminases levels, is reduced after the administration of a COX-2 inhibitor to rats (Yamamoto et al., 2003). Whether species differences and/or differential drug properties may explain these divergent findings remains unknown at present. In any event, the protective effects of combined inhibition of COX-2 and 5-LO pathways were confirmed by administering the COX-2 inhibitor to 5-LO-deficient mice. Unfortunately, these results could not be confirmed in COX-2-deficient mice because these animals exhibit markedly decreased fertility and survival. Overall, these findings open new avenues for the application of dual inhibitors with the ability to inhibit both the COX-2 and 5-LO pathways. In fact, dual inhibitors, such as the COX/5-LO inhibitor licofelone, have been shown to be efficacious in the treatment of inflammatory disorders such as arthritis, osteoarthritis, asthma, and inflammatory bowel disease (Martel-Pelletier et al., 2003; Vidal et al., 2007). Whether dual inhibitors have superior efficacy compared with inhibition of a single pathway has not been completely proven, but dual inhibitors may prevent the observed shunting of the arachidonic acid metabolism toward the 5-LO pathway after COX inhibition (Martel-Pelletier et al., 2003). In addition, dual inhibitors appear to exert some disease-modifying activity and, for example, they may stop disease progression by reducing the expression of matrix metalloproteinase-13 and IL-1 (Celotti and Durand, 2003). Moreover, dual inhibitors have an excellent gastrointestinal profile, much better than that for conventional nonsteroidal antiinflammatory drugs and equivalent to that of selective COX-2 inhibitors (Laufer et al., 1994; Wallace et al., 1994).
Another interesting finding of our study was that pharmacological COX-2 inhibition in 5-LO-deficient mice resulted in a reduction of hepatic MCP-1 expression. MCP-1, also known as CCL2, is a prototype of the C-C chemokine subfamily and exhibits potent chemotactic activity for monocytes, lymphocytes, and mesenchymal cells, including hepatic stellate cells (Marra et al., 1999; Muller, 2001). Mounting evidence indicates that 5-LO products, particularly LTB4, strongly induce expression of MCP-1 mRNA and protein and that COX-derived products modulate MCP-1 expression in an agonist-specific fashion (Efsen et al., 2001; Huang et al., 2004). Considering that increased MCP-1 expression contributes to the development of the inflammatory response in patients with active fibrogenesis (Marra et al., 1998), down-regulation of this proinflammatory chemokine may be regarded as an additional protective action associated with the simultaneous inhibition of the COX-2 and 5-LO pathways. In fact, a recent study has demonstrated that dual COX/5-LO inhibition attenuates monocyte recruitment into the arterial wall by mechanisms related to MCP-1 inhibition (Vidal et al., 2007). Along these lines, a reduction in MCP-1 expression after selective COX-2 inhibition consistently ameliorates the severity of inflammation and fibrosis in chronic pancreatitis (Reding et al., 2006).
Our findings support the concept that COX-2 and 5-LO play opposite roles in the regulation of expression of IL-6, a primary proinflammatory cytokine and a determinant factor in triggering the process of hepatic inflammation (McClain et al., 1999). Indeed, in our study we found that COX-2 inhibition amplified IL-6 expression in macrophages, whereas 5-LO inhibition down-regulated IL-6 expression in these cells. These findings are consistent with previous studies showing the potential of COX-2- and 5-LO-derived products to modulate the expression and synthesis of proinflammatory cytokines in monocytes and macrophages (Sipe et al., 1992; Marcouiller et al., 2005). Interestingly, an inhibitory effect similar to that exerted by the 5-LO inhibitor was observed with a BLT1 receptor antagonist, suggesting the direct involvement of LTB4 in the regulation of IL-6 expression in macrophages. This finding is in agreement with the view that among the different eicosanoids, 5-LO products and, in particular, LTB4, are important positive signals for cytokine expression and synthesis in inflammatory cells (Sipe et al., 1992; Marcouiller et al., 2005).
Our data point to the direction that the hepatic effects exerted by SC-236 and CJ-13,610 were mediated by a combination of mechanisms, involving not only inhibition of proinflammatory 5-LO- and COX-2-derived products but also the induction of apoptosis in nonparenchymal cells. Indeed, in our study we noticed that the decrease in liver fibrosis associated with COX-2 and/or 5-LO inhibition was accompanied by a parallel induction of cell death in nonparenchymal cells (i.e., hepatic stellate cells and liver macrophages). These findings are in agreement with previous studies demonstrating that hepatic stellate cells and Kupffer cells in primary culture undergo apoptosis when exposed to either COX-2 or 5-LO inhibitors (Titos et al., 2003; Planagumà et al., 2005). A wealth of evidence indicates that the process of hepatic stellate cell apoptosis is central to stop the progress of liver fibrosis and represents an important antifibrogenic strategy in the liver (Iredale et al., 1998; Friedman and Bansal, 2006). Moreover, consistent with their role in liver injury, partial depletion of Kupffer cells has been shown to prevent hepatic necroinflammatory damage (Duffield et al., 2005; Titos et al., 2005). Finally, other mechanisms independent of direct COX-2 and 5-LO inhibition may potentially be involved in the hepatoprotective actions exerted by SC-236 and CJ-13,610. For example, SC-236 and other selective COX-2 inhibitors appear to display COX-2-independent actions via activation of peroxisome proliferator-activated receptor-
, a transcription factor that mediates anti-inflammatory and antifibrogenic effects in the liver (Marra et al., 2000; López-Parra et al., 2005). Another potential mechanism by which conventional 5-LO inhibitors may protect against CCl4-induced liver injury is by interfering with the metabolism of CCl4 in the hepatic cytochrome P450 or by exerting antioxidant properties. In our case, this possibility is unlikely because CJ-13,610 is a nonredox-type inhibitor of 5-LO devoid of redox and iron ligand properties (Fischer et al., 2004).
In summary, the current study uncovers the participation of both COX-2 and 5-LO pathways in mounting the inflammatory response and subsequent fibrogenesis in a preclinical model of liver disease. Our results support the notion that the COX-2 and 5-LO pathways have converging functions, not only in cell proliferation and neo-angiogenesis (Romano and Clària, 2003), but also in the progression of liver inflammation and fibrosis. Our findings suggest that inhibition of these two proinflammatory pathways represents a potential strategy for prevention of necroinflammatory liver injury and fibrogenesis.
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Acknowledgements |
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Footnotes |
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
ABBREVIATIONS: COX, cyclooxygenase; PG, prostaglandin; 5-LO, 5-lipoxygenase; LT, leukotriene; CCl4, carbon tetrachloride; FLAP, 5-lipoxygenase-activating protein; SC-236, 4-[5-(4-chlorophenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl] benzenesulfonamide; CJ-13,610, 4-[3-[4-(2-methylimidazol-1-yl)phenylthio]]phenyl-3,4,5,6-tetrahydro-2H-pyran-4-carboxamide; MTT, 3-[4,5-dimethylthiazoyl-2-yl]-2,5-diphenyltetrazolium bromide; DMSO, dimethyl sulfoxide; LPS, lipopolysaccharide; PMA, phorbol 12-myristate 13-acetate; AA-861, (2-(12-hydroxydodeca-5,10-dinyl)-3,5,6-trimethyl-1,4-benzoquinone); MK-571, 3-[[3-[2-(7-chloroquinolin-2-yl)vinyl]phenyl]-(2-dimethylcarbamoylethylsulfanyl)methylsulfanyl] propionic acid; EIA, enzyme immunoassay; IL, interleukin; MCP-1, monocyte chemoattractant protein 1; TUNEL, terminal deoxynucleotidyl transferase dUTP nick-end labeling; CP-105,696, 1-[3-(4-phenyl-benzyl)-4-hydroxy-chroman-7-yl] cyclopentane carboxylic acid; RT, reverse transcription; PCR, polymerase chain reaction; LTC4S, leukotriene C4 synthase; LTA4H, leukotriene A4 hydrolase.
Address correspondence to: Dr. Joan Clària, Department of Biochemistry and Molecular Genetics, Hospital Clínic, Villarroel 170, Barcelona 08036, Spain. E-mail: jclaria{at}clinic.ub.es
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, P < 0.05 with respect to untreated cells. *, P < 0.05; **, P < 0.01; ***, P < 0.001, with respect to cells not exposed to the inhibitor (0 µM concentration).