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CARDIOVASCULAR
Departments of Vascular Research (C.V., A.G.-H., E.S.-G., J.E.), Biochemistry (J.A.G.-G.), and Cardiology (A.G., J.T.), Fundación Jiménez Díaz, Autónoma University, Madrid, Spain; and Department of Pathology, Hospital San Carlos, Madrid, Spain (L.O.)
Received July 5, 2006; accepted September 29, 2006.
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Abstract |
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B in rabbit atheroma. Moreover, licofelone inhibited COX-2 and 5-LOX protein expression in vascular lesions. Rofecoxib only diminished COX-2 protein expression and MCP-1 gene expression in vascular atheroma. Prostaglandin E2 in rabbit plasma was attenuated by both drugs. Licofelone almost abolished 5-LOX activity by inhibiting leukotriene B4 generation in rabbit neutrophils and prevented platelet thromboxane B2 production from whole blood. Licofelone reduces neointimal formation and inflammation in an atherosclerotic rabbit model more markedly than rofecoxib. This effect, together with the antiplatelet activity of licofelone, suggests that this drug may have a favorable cardiovascular profile.
). These inflammatory cells express metalloproteinases that weaken the fibrous cap of the lesions making them prone to rupture and subsequent thrombosis, causing an acute coronary syndrome (Nomoto et al., 2003).
COX-2 is expressed mainly in inflammatory disorders, such as atherosclerosis, and converts arachidonic acid to PGG2 (Linton and Fazio, 2004). Depending on the enzymes working downstream, PGG2 may yield a variety of PGs and TXA2, with opposite effects on vascular wall biology. COX-2 is coexpressed frequently with PGE synthase, yielding PGE2 that possesses proinflammatory and chemoattractant properties. Then, theoretically, COX-2 blockade could be beneficial in atherothrombosis. However, clinical trials have shown different viewpoints about the protector or hazardous effect of selective COX-2 inhibitors.
Given that TXA2 is produced in platelets via COX-1, selective COX-2 inhibition may leave this pathway unblocked. Therefore, it has been suggested that COX-2 inhibitors would lead to an imbalance between prothrombotic TXA2 and vasodilatory prostacyclins (PGI2), which may be synthesized via COX-2, favoring platelet aggregation and vasoconstriction.
In the Vioxx gastrointestinal outcomes research study, the COX-2 inhibitor rofecoxib (ROF) increased the risk of cardiovascular events in patients with rheumatoid arthritis (Bombardier et al., 2000). Although in a similar study celecoxib did not increase the cardiovascular risk (Silverstein et al., 2000), and even some favorable effects were reported with this drug and with meloxicam (Altman et al., 2002; Chenevard et al., 2003), recent data from patients with colon adenoma suggest that both rofecoxib and celecoxib may increase cardiovascular risk at long-term (Bresalier et al., 2005; Solomon et al., 2005). As a result, the clinical evidence of an increased risk of cardiovascular events in patients taking COX-2 inhibitors remains controversial (Mukherjee et al., 2001).
5-Lipoxygenase (LOX) is another enzyme involved in the metabolism of arachidonic acid that catalyzes leukotriene (LT) production, inflammation, apoptosis, proliferation, and atherogenesis (Spanbroek et al., 2003). 5-LOX is expressed in monocytes and macrophages, and it contributes to the development and rupture of atherosclerotic plaques (Steinhilber, 1999). Polymorphisms in the 5-LOX gene promoter and certain 5-LOX-activating protein haplotypes have been linked to an increased risk of infarction and stroke (Zhao et al., 2004).
In the last years, several new 5-LOX and COX-2 inhibitors have been developed (Martel-Pelletier et al., 2003) in a search for an anti-inflammatory compound with higher gastrointestinal safety than the classic nonsteroidal anti-inflammatory drugs and without the cardiovascular risk associated to COX-2 inhibitors. Licofelone (LIC) is one of these potent anti-inflammatory drugs, which inhibits COX-2, COX-1, and 5-LOX. In in vitro studies, licofelone has been reported to suppress both 5-LOX (with an IC50 of 0.18 µM) and COX-2 (with an IC50 of 0.21 µM) (Singh et al., 2006).
Licofelone has shown anti-inflammatory, analgesic, and antiasthmatic effects in several experimental models at dosages between 10 and 100 mg/kg that do not cause any gastrointestinal damage (Rotondo et al., 2002). The anti-inflammatory action of this drug has been shown in animal models of osteoarthritis (Jovanovic et al., 2001; Pelletier et al., 2005), and it is currently being evaluated in phase III clinical trials in this disorder. Moreover, antithrombotic effects of licofelone due to inhibition of COX-1-mediated platelet function have been reported in mice and rat models (Tries et al., 2002) and in human platelets (Rotondo et al., 2004). Licofelone, compared with classic nonsteroidal anti-inflammatory drugs, have been found to possess a unique ability to inhibit leukocyte rolling and adhesion to endothelium (Ulbrich et al., 2005). Its analgesic, anti-inflammatory, and antiplatelet properties are present at doses, which are safe for gastrointestinal tract (Cicero et al., 2005). The results from a randomized trial in healthy human volunteers indicate that licofelone has a potential gastrointestinal safety advantage over conventional nonsteroidal anti-inflammatory drug therapy, because 200 or 400 mg b.i.d. licofelone was associated with a lower incidence of ulcers compared with 500 mg b.i.d. naproxen (Bias et al., 2004).
Consequently, since licofelone shares the anti-inflammatory effect and gastric safety of COX-2 inhibitors (Lehmann and Beglinger, 2005) but also inhibits COX-1-mediated platelet function, thereby avoiding the prothrombotic state, this drug may have a better cardiovascular profile than COX-2 inhibitors. The aim of this study was to investigate the effects of licofelone, in comparison with the selective COX-2 inhibitor rofecoxib, on the inflammatory response in vascular lesions in a rabbit atherosclerosis model as well as in rabbit neutrophils and platelet-rich plasma isolated from whole blood. Additional in vitro studies were also performed in vascular smooth muscle cells (VSMCs).
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Materials and Methods |
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). After 1 week, the animals were randomized to receive either 10 mg/kg/day LIC (n = 10) (provided by Merckle, Ulm, Germany and Lacer, Barcelona, Spain) or 5 mg/kg/day ROF (n = 10) mixed with chow. A third group did not receive any treatment (NT; n = 10). The atherogenic diet was maintained in all cases. Four weeks later, they were sacrificed. Ten rabbits feeding standard chow without intervention were included in the study as healthy controls. At sacrifice, the animals were anesthetized and the femoral arteries were exposed. Then, they were sacrificed with an overdose of pentobarbital (Abbott, Madrid, Spain), and one of the femoral arteries was fixed by perfusion of the descending aorta with 4% buffered formaldehyde at 100 mm Hg, removed, and kept for 24 h in 4% buffered formaldehyde and afterward in 70% ethanol until it was paraffin-embedded.
Serum Chemistry. Rabbits were bled from a marginal ear vein 24 h postmeal on day 0 and at the end of weeks 2 (randomization) and 6 (sacrifice) of the study. Total cholesterol, chylomicrons, low-density lipoproteins, high-density lipoproteins (HDLs), very low-density lipoproteins (VLDLs), intermediate-density lipoproteins, and triglycerides (TGs) were measured with enzymatic assays (Sigma Diagnostics, Madrid, Spain).
PGE2 Plasma Levels. Ten milliliters of blood was drawn from ear vein at sacrifice. Plasma was obtained from blood by centrifugation (2500 rpm for 10 min) and frozen at –80°C. PGE2 levels were measured by a competitive immunoassay (R&D Systems, Minneapolis, MN). Total concentration of PGE2 was expressed as picograms per milliliter.
Histological Analysis
Morphometric Analysis. The morphometric analysis was performed on Masson-stained femoral artery preparations. Intima and media area were measured, and the results were expressed as intima/media ratio.
Immunohistochemistry. Paraffin-embedded arteries were cross-sectioned into 4-µm-thick pieces at 5-mm intervals, dewaxed, and rehydrated. For macrophage identification, a monoclonal anti-rabbit macrophage antibody (RAM11; Dako Denmark A/S, Glostrup, Denmark) was applied as described previously (Hernández-Presa et al., 2002). COX-1 and COX-2 were detected with polyclonal goat anti-human antibodies (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). 5-LOX were immunolocalized using polyclonal rabbit anti-human antibody (Cayman Chemical, Ann Arbor, MI). As secondary antibody for COX-2 and COX-1, a donkey anti-goat IgG was biotin-labeled (GE Healthcare, Little Chalfont, Buckinghamshire, UK), a goat anti-mouse IgG was biotin-labeled (Dako Denmark A/S) for RAM11, and anti-rabbit IgG was biotin-labeled for 5-LOX. The secondary antibodies were applied for 1 h at 1:200 dilution. Then, ABComplex/horseradish peroxidase (Dako Denmark A/S) was added for an additional 30-min period. Then, ABComplex/horseradish peroxidase (Dako Denmark A/S) was added for an additional 30-min period. The sections were stained for 10 min at room temperature with 3,3'-diaminobenzidine tetrahidroclorure (Dako Denmark A/S) and then counterstained with hematoxylin and mounted in Pertex (Medite, Burgdorf, Germany). In each experiment, negative controls without the primary antibody or using a nonrelated antibody were included to check for nonspecific staining.
Southwestern Histochemistry. The distribution and DNA-binding activity of NF-B in situ was detected as described previously (Hernández-Presa et al., 2002), using a digoxigenin-labeled double-stranded DNA probe with a specific consensus sequence that binds to NF-B (5'-AGTTGAGGGGACTTTCCCAGGC-3'). Preparations without probe were used as negative controls and to test the specificity of the technique, a mutant NF-B probe was used (5'-AGTTGAGGCTCCTTTCCCAGGC-3').
Image Analysis. Preparations were digitized via a BH-2 microscope (Olympus) connected to a charge-coupled device videocamera as described previously (Hernández-Presa et al., 2002). Image analysis was performed using the Olympus software. For morphometric analysis, intima and media areas were measured. For immunohistochemistry specimens, the percentage of neointima staining positive per square millimeter was evaluated. In Southwestern preparations, nuclei staining positive per square millimeter were assessed.
Western Blot Analysis. The protein levels of COX-1, COX-2, and 5-LOX in femoral arteries were determined by Western blot analysis with specific antibodies (Santa Cruz Biotechnology, Inc. for COX-1/2 and Cayman Chemical for 5-LOX). Fifty micrograms of whole proteins were separated on a 10 to 15% SDS-polyacrylamide gel electrophoresis and transferred to a polyvinylidene difluoride membrane (Immobilon P; Millipore Corporation, Billerica, MA). The membrane was blocked in washing solution with 7% nonfat dried milk for 60 min at 37°C and then incubated with 1 mg/ml primary antibody overnight at 4°C and later with a peroxidase-conjugated secondary antibody for 60 min at 37°C. The bands were detected with a chemiluminescent system (ECL; GE Healthcare) and exposed to X-ray film.
RNA Extraction and Real-Time Polymerase Chain Reaction. Total RNA was extracted from femoral arteries by TRIzol method (Invitrogen, Barcelona, Spain) and quantified by absorbance at 260 nm in duplicate. One microgram of RNA was necessary to perform the reverse transcription reaction, for 15 min at 25°C and 2 h at 37°C, with the High Capacity cDNA Archive kit (Applied Biosystems, Foster City, CA). Oligonucleotide primers/probe for rabbit MCP-1 and COX-2 were designed using the Primer Express program and were synthesized by Applied Biosystems. Amplification of Eukaryotic 18S RNA was used in the same reaction of all samples as an internal control. Gene-specific mRNA was subsequently normalized to 18S RNA. Quantitative reverse transcription-PCR was performed by 7500 Real-Time PCR system, and the relative quantification was performed with the Prism 7000 system SDS software (Applied Biosystems).
Ex Vivo Studies
Isolation of Rabbit Neutrophils and LTB4 Measurement. Heparinized peripheral blood was collected from a total of six healthy rabbits, and neutrophils were isolated by using a lymphocyte separation medium gradient, gelatin sedimentation, and hypotonic lysis of erythrocytes as described previously (Lee et al., 2005). In brief, the sedimentation of the cellular portion was developed by mixing with 2.5% gelatin in phosphate-buffered saline (PBS) and kept over 20 min at 37°C. The supernatant of the suspension was harvested and centrifuged at 2500 rpm for 15 min and then the residual erythrocytes from the pellet obtained were removed by hypotonic lysis, and the neutrophils population was isolated with a purity of more than 95%. Neutrophils were resuspended in serum-free RPMI 1640 medium (25 x 104/300 µl) and preincubated 60 min at 37°C with 10 µM licofelone, 10 µM rofecoxib, and MK-886, a 5-lipoxygenase inhibitor, at 0.1 µM concentration. Calcium ionophore (10 µM) A23187
[GenBank]
(calcimycin) (10 min at 37°C) was used to stimulate the LTB4 release in the natural population of neutrophils isolated from whole blood. After stimulation, cell-free supernatants were collected and centrifuged (1000 rpm for 5 min), and LTB4 concentration was measured by using the commercial enzyme-linked immunoassay (EIA) (Cayman Chemical) according to the manufacturer's instructions. This kit is highly specific for LTB4. The intra- and interassay variability has been determined at multiple points on the standard curve. LTB4 intra-assay variation is between 13 and 18% and interassay variation is between 7 and 18%. Total concentration of LTB4 was expressed as picograms per milliliter.
Isolation of Rabbit Platelets and TXB2 Levels Measurement. To assess platelet TXB2 levels, the stable metabolite of TXA2, venous blood from healthy rabbits (n = 4) was collected in sodium citrate solution 3.8%. Platelet-rich plasma (PRP) was isolated from whole blood by centrifugation (600 rpm for 30 min), and washed platelet suspension was distributed in samples of 1 ml of PRP. The PRP was incubated for 60 min at 37°C with different drugs: licofelone, rofecoxib, and SC560, a COX-1 inhibitor for 1 h, at 37°C in agitation. After pretreatment with these drugs, platelet-rich plasma was incubated with 0.5 U/ml thrombin for 10 min at 37°C in agitation. Finally, we measured platelet TXB2 production by the EIA method. This kit is highly specific for TXB2 measurement. The intraand interassay variability has been determined at multiple points on the standard curve. TXB2 intra-assay variation is more than 18%, and interassay variation is between 15 and 20%. Total concentration of TXB2 was expressed as picograms per milliliter.
In Vitro Studies
Cell Cultures. Rat VSMCs were isolated and cultured as explained previously (Hernández-Presa et al., 1997). Cells were growth-arrested by incubation in serum-free medium for 48 h and then incubated with the corresponding stimuli. For inhibition studies, licofelone, rofecoxib, SC560, and MK-866 were added to the culture medium 1 h before the stimuli.
RNA Extraction and Real-Time Polymerase Chain Reaction. Total RNA extraction of different experiments in VSMCs was performed as described above. Oligonucleotide primers/probe for rat MCP-1 were designed using the Primer Express program and were synthesized by Applied Biosystems. Amplification of eukaryotic 18S RNA was used in the same reaction of all samples as an internal control.
Statistical Analysis
Statistical analysis was performed with GraphPad InStat (GraphPad Software Inc., San Diego, CA). Lipid values, morphometric analysis, immunohistochemistry, Western blot, real-time PCR, enzyme-linked immunosorbent assay, and EIA data are presented as mean ± S.E.M. and were analyzed by the Mann-Whitney U test. When multiple comparisons were needed, the Kruskal-Wallis test was used.
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Results |
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Licofelone Reduces Neointimal Formation of Rabbit Atheroma. Licofelone significantly reduced intima/media ratio in injured femoral arteries compared with nontreated animals (0.2 ± 0.1 versus 0.7 ± 0.2; p < 0.05), whereas rofecoxib did not modify the neointimal size in the animal model (0.5 ± 0.2 versus 0.7 ± 0.2; p = N.S. versus NT) (Fig. 1).
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Licofelone Diminishes NF-B Activity in Vascular Lesions of Atherosclerotic Rabbits. The activation of the nuclear factor NF-B in the area of vascular lesion of the artery was markedly reduced by LIC compared with NT group of rabbits (1967 ± 483 versus 3548 ± 324 positive nuclei staining/mm2; p < 0.05), whereas no significant inhibition was observed in ROF animals (2987 ± 583 nuclei staining/mm2; p = N.S. versus NT). Control animals did not show NF-B activation (Fig. 3).
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Licofelone and Rofecoxib Reduce MCP-1 Expression in Vascular Lesions and in Cultured VSMCs. In rabbit vascular lesions, both licofelone and rofecoxib markedly reduced MCP-1 mRNA expression compared with those values of nontreated animals (60 ± 0.8 and 47 ± 1.9% inhibition, respectively; p < 0.01 versus NT) (Fig. 4A).
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caused an increase of MCP-1 mRNA expression. LIC at 10 and 1 µM concentration was able to reduce MCP-1 mRNA (79 ± 0.1 and 63 ± 0.4% inhibition, respectively; p < 0.01 versus stimulus). At the same concentration (10 and 1 µM), ROF inhibited MCP-1 mRNA expression (66 ± 0.3% inhibition, p < 0.01 and 68 ± 0.4% inhibition, p < 0.05, respectively) (Fig. 4B). To confirm the 5-LOX pathway implication in MCP-1 mRNA expression, we performed additional experiments with the 5-LOX inhibitor MK-866 (1 µM). This compound inhibited MCP-1 expression (66 ± 0.2% inhibition; p < 0.01) elicited by interleukin-1 in VSMCs (Fig. 4B). Atheroma 5-LOX, COX-1, and COX-2 Expression in LIC- and ROF-Treated Rabbits. The effect of licofelone and rofecoxib on 5-LOX, COX-1, and COX-2 protein expression was measured in the neointimal area of rabbit arteries by Western blot (Fig. 5) and immunohistochemistry (Fig. 6).
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Licofelone reduced markedly 5-LOX protein levels by Western blot (0.8 ± 0.12 versus 4.7 ± 2, p < 0.01 versus NT and 0.8 ± 0.12 versus 5.5 ± 1.8, p < 0.05 versus ROF) and by immunohistochemistry (19 ± 6.4 versus 47 ± 5% of positive staining/mm2; p < 0.05 versus NT) in the neointimal area, whereas, as expected, RFC did not reduce 5-LOX protein levels.
As expected, there were no differences between licofelone and rofecoxib on COX-2 and COX-1 expression. Licofelone and rofecoxib showed a trend to inhibit COX-2 protein expression (Fig. 5) and mRNA expression (data not shown), although it did not reach statistical significance. This tendency was verified by immunohistochemistry, where COX-2 expression in the neointima was significantly inhibited by licofelone (21 ± 5 versus 39 ± 6% of positive staining/mm2; p < 0.05 versus NT) and rofecoxib (23 ± 10 versus 39 ± 6% of positive staining/mm2; p < 0.05 versus NT). COX-1 expression showed a significant reduction in the neointima by licofelone (28 ± 5 versus 37 ± 4% of positive staining/mm2; p < 0.05 versus NT).
PGE2 Plasma Concentration Was Decreased by Both Licofelone and Rofecoxib in Rabbits. The concentration of PGE2 in rabbit plasma was increased in NT compared with healthy animals (5300 ± 66 pg/ml; p < 0.001 versus healthy). These levels returned to the normal values of healthy animals by licofelone (1057.5 ± 191.3 pg/ml, p < 0.01 versus NT) and rofecoxib (1603.1 ± 344.6 pg/ml, p < 0.01 versus NT) treatment (Fig. 7A).
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Licofelone Prevents TXB2 Production in Rabbit Platelets. To assess whether licofelone treatment affects platelet COX-1 expression and TXA2 production in rabbits, we isolated PRP from whole blood of healthy rabbits (n = 4), and we measured platelet TXB2, as an index of TXA2 production, in vivo. Platelets were pretreated with licofelone, rofecoxib, and SC560 for 1 h before stimulation with thrombin for 10 min. Thrombin (0.5 U/ml) significantly increased platelet TXB2 production (669 ± 85 versus 41 ± 13 pg/ml, p < 0.05 versus control). Licofelone at 10 µmol and SC560 at 10 µmol inhibited the COX-1 pathway, reducing noticeably platelet TXB2 production elicited by thrombin (162 ± 50 and 224 ± 77 pg/ml; p < 0.01 in both cases versus stimulus). Rofecoxib failed to modify platelet TXB2 production induced by thrombin (468 ± 113 pg/ml; p = N.S. versus stimulus) (Fig. 6C) (Fig. 7).
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Discussion |
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). COX is the rate-limiting enzyme in the biosynthesis of prostanoids, which exert a variety of actions on the vascular wall. COX-2 isoform is a key mediator of inflammation, up-regulated in activated monocyte/macrophages, suggesting that COX-2 inhibition might reduce atherogenesis through its anti-inflammatory effects (Linton and Fazio, 2004). However, in the past few years there has been great interest in the possible antiatherogenic effects of COX-2 inhibitors. Clinical studies have raised concern about the safety of these drugs because not only a reduction but also a lack of effect or even an increase in the incidence of cardiovascular events was observed (Bombardier et al., 2000; Silverstein et al., 2000; Altman et al., 2002; Chenevard et al., 2003; Bresalier et al., 2005; Solomon et al., 2005). It was assumed that COX-2 inhibitors would cause an imbalance between thromboxane A2 and prostacyclins, favoring platelet aggregation, vasoconstriction, and thrombosis. However, PGs are not the only metabolites of arachidonic acid. The enzyme 5-LOX, localized in macrophages and other inflammatory cells, converts arachidonic acid into proinflammatory LTs (Steinhilber, 1999).
In the few past years, several new 5-LOX and COX-2 inhibitors have been developed (Martel-Pelletier et al., 2003) in a search for an anti-inflammatory compound with higher gastrointestinal safety than the classic nonsteroidal anti-inflammatory drugs and without the cardiovascular risk associated to COX-2 inhibitors. Licofelone is one of these potent anti-inflammatory drugs.
In our experimental model, licofelone was able to reduce intima/media ratio in injured rabbit arteries, whereas the selective COX-2 inhibitor rofecoxib did not show a significant reduction of the neointimal size in this animal model. Therefore, licofelone could be able to interfere with the process of neointimal growth linked to vascular injury. Indeed, lipoxygenase products of arachidonic acid, especially hydroxyeicosatetraenoic acids, have been shown to play an important role in neointimal formation after vascular injury (Fujita et al., 1999).
Neointimal macrophage infiltration in the atherosclerotic lesions in rabbits was attenuated by licofelone but not by rofecoxib. We have also demonstrated that the action of licofelone on reducing the inflammatory cells in the atherosclerotic process could also be mediated through its inhibition of MCP-1, which participates in the atherosclerotic process by the recruitment of monocytes into the arterial wall. We showed that licofelone inhibits MCP-1 expression in cultured VSMCs in an inflammatory state probably via COX-2 inhibition, because it has been reported that COX-2 inhibition decreases MCP-1 expression (Wang et al., 2005), as well as by 5-LOX blocking, since the inhibitor of this enzyme, MK-866, also reduced MCP-1 expression in the presence of proinflammatory cytokines, although other factors must be interfering in this process. Therefore, it is tempting to suggest that the enhanced inhibitory effect of licofelone on MCP-1 gene expression could be mediated not only via COX-2 but also through 5-LOX. In this regard, LTB4 derived from 5-LOX activity binds to its high-affinity receptor, BLT1, promoting monocyte adhesion by increasing MCP-1 expression (Friedrich et al., 2003; Huang et al., 2004).
The nuclear factor NF-B is a key mediator of inflammation that regulates many genes involved in cell recruitment (Barnes and Karin, 1997; Martín-Ventura et al., 2004). Since licofelone inhibited NF-B in vascular lesions and the modulation of NF-B regulates the transcription of a number of genes as COX-2 and 5-LOX, among others, we assessed the effect of licofelone and rofecoxib on protein expression of COX-2, COX-1 and 5-LOX in rabbit vascular atheroma. Licofelone and rofecoxib showed a similar inhibitory effect on COX-1 and COX-2 protein expression. Conversely, 5-LOX expression was only altered by licofelone, which exerted a profound lessening of the levels of this enzyme compared with nontreated animals and rofecoxib group. The inhibitory effect of licofelone on 5-LOX expression in vascular lesions was associated with the lessening of the activated NF-B observed in vascular atheroma of licofelone-treated rabbits. In contrast, licofelone could have an inhibitory effect on NF-B through the inhibition of 5-LOX by a feedback mechanism. In this regard, it has been reported that arachidonic acid-derived metabolites as LTB4, acting through its G protein-coupled receptor in a feedback mechanism, contribute to the activation of NF-B in response to tumor necrosis factor-
and interleukin-1 (Anthonsen et al., 2001).
Therefore, although there is an important contribution of COX-2 and 5-LOX protein inhibition in the anti-inflammatory action of licofelone, we also characterized the impact of this drug on the generation of the eicosanoids derived from the activity of COX and 5-LOX enzymes under proinflammatory conditions, as a measure of the activity of these enzymes.
The proinflammatory metabolite PGE2, a product of COX-2 activity, was reduced in the same way by both licofelone and rofecoxib in rabbit plasma, which is in agreement with the lessening of COX-2 expression by both drugs in rabbit atheroma. The most important eicosanoids resulting from 5-LOX activity is LTB4, which exhibits strong proinflammatory activity in cardiovascular tissues and enhances vascular permeability and endothelial dysfunction. LTB4, a potent leukocyte chemoattractant, is also involved in cytokine synthesis, regulation of lymphocyte proliferation, and macrophage cytotoxic and natural killer cell activities (Vila, 2004). LTB4 is also an important regulator of neutrophil function and causes chemotaxis, degranulation, phagocytosis, and superoxide generation in neutrophils (Tager et al., 2003). In this sense, we have studied LTB4 generation by neutrophils isolated from whole blood of rabbits as a measure of 5-LOX activity. The results obtained in our study are of potential interest, because licofelone at 10 µM concentration almost abolished the production of the proinflammatory LTB4 after stimulation with calcium ionophore. This fact confirms that licofelone is able to interfere with the enzymatic activity of 5-lipoxygenase under proinflammatory conditions.
The strong inhibitory effect of the 5-LOX inhibitor MK-886 at 10 µM concentration was similar to that produced by licofelone. Actually, it has been reported that the effects of MK-886 are associated with a profound inhibition of ex vivo LTB4 synthesis in blood and a significant reduction of neutrophil aggregation in whole blood (Lee et al., 2005). The significant effects of MK-886 are mainly due to inhibition of neutrophil function, and this suggests an important modulatory role for leukotrienes, and obviously for 5-LOX, in the pathology of inflammatory disease. In contrast, when neutrophils were incubated in presence of rofecoxib, the levels LTB4 remained elevated compared with the stimuli. Consequently, the use of a selective inhibitor of COX-2 similar to rofecoxib, in vascular inflammatory states would lead to a decrease in antithrombotic prostacyclin synthesized by arachidonate flux through COX-2, and more arachidonic acid would be available for leukotriene synthesis. Indeed, it has been published that the inhibition of COX and PGE2 synthesis could be responsible for the increased levels of LTB4 production by human osteoarthritis joint tissues (Martel-Pelletier et al., 2004). As a result, the overproduction of LTB4 would cause inflammatory responses that may result in the migration and adhesion of inflammatory cells by increasing MCP-1 expression (Friedrich et al., 2003; Huang et al., 2004), which is in agreement with the results.
The present study has also confirmed the protective anti-thrombotic effect of licofelone, since the concentration of TXB2, the degradation product of the prothrombotic TXA2, was diminished in platelets isolated from peripheral blood of rabbits. This effect was mainly due to COX-1 inhibitory activity of this drug, since the COX-1-specific inhibitor SC560 had similar results. In contrast, the selective inhibition of COX-2 by rofecoxib was not able to reduce TXB2; thus, its levels remained elevated, disrupting the physiological balance between thromboxane and prostacyclin, and, consequently, augmenting thrombogenesis and the risk of cardiovascular complications (Linton and Fazio, 2004).
In a rabbit model of atherosclerosis as well as in cultured cells, we have demonstrated the protective effect of licofelone on the atherosclerotic process in comparison with rofecoxib as shown by others (Moreau et al., 2005). We have also confirmed the anti-inflammatory and antithrombotic properties of licofelone in neutrophils and platelets isolated from whole blood.
On the whole, our findings disclose that licofelone, in addition to exerting a beneficial anti-inflammatory effect on the atherosclerotic lesions, also avoids the cardiovascular risk associated to selective COX-2 inhibitors due to its inhibitory effect on prothrombotic TXA2. Therefore, licofelone could have a safer cardiovascular profile than COX-2 inhibitors in inflammatory diseases, although clinical trials are needed to confirm this issue.
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Footnotes |
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C.V., A.G.-H., and E.S.-G. contributed equally to this work.
Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
ABBREVIATIONS: MCP, monocyte chemoattractant protein; COX, cyclooxygenase; PG, prostaglandin; TX, thromboxane; ROF, rofecoxib; LOX, lipoxygenase; LT, leukotriene; VSMC, vascular smooth muscle cell; LIC, licofelone; NT, nontreated; HDL, high-density lipoprotein; VLDL, very low-density lipoprotein; TG, triglyceride; NF-B, nuclear factor-B; PCR, polymerase chain reaction; EIA, enzyme-linked immunoassay; PRP, platelet-rich plasma; SC560, 5-(4-chloro-phenyl)-1-(4-methoxyphenyl)-3-trifluoromethylpyrazole; MK-886, 1-[(4-chlorophenyl)methyl]-3-3-[(1,1-dimethylethyl)thio-,-dimethyl-5-(1-methylrthyl)-14-indole-2-propanoic acid, sodium salt; A23187
[GenBank]
, calcium ionophore (calcimycin).
Address correspondence to: Dr. Cristina Vidal, Vascular Research Laboratory, Fundación Jiménez Díaz, Avda Reyes Católicos 2, Madrid 28040, Spain. E-mail: cvidal{at}fjd.es
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References |
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Altman R, Luciardi HL, Muntaner J, Del Rio F, Berman SG, Lopez R, Gonzalez C (2002) Efficacy assessment of meloxicam, a preferential cyclooxygenase-2 inhibitor, in acute coronary syndromes without ST-segment elevation: the nonsteroidal anti-inflammatory drugs in unstable angina treatment-2 (NUT-2) pilot study. Circulation 106: 191–195.
Anthonsen MW, Andersen S, Solhaug A, and Johansen B (2001) Atypical lambda/iota PKC conveys 5-lipoxygenase/leukotriene B4-mediated cross-talk between phospholipase A2s regulating NF-kappa B activation in response to tumor necrosis factor- and interleukin-1. J Biol Chem 21: 35344–35351.
Barnes PJ and Karin M (1997) Nuclear factor-kappaB: a pivotal transcription factor in chronic inflammatory diseases. N Engl J Med 336: 1066–1071.
Bias P, Buchner A, Klesser B, and Laufer S (2004) The gastrointestinal tolerability of the LOX/COX inhibitor, licofelone, is similar to placebo and superior to naproxen therapy in healthy volunteers: results from a randomized, controlled trial. Am J Gastroenterol 99: 611–618.[CrossRef][Medline]
Bombardier C, Laine L, Reicin A, Shapiro D, Burgos-Vargas R, Davis B, Day R, Ferraz MB, Hawkey CJ, Hochberg MC, et al. (2000) Comparison of upper gastrointestinal toxicity of rofecoxib and naproxen in patients with rheumatoid arthritis. VIGOR study group. N Engl J Med 343: 1520–1528.
Bresalier RS, Sandler RS, Quan H, Bolognese JA, Oxenius B, Horgan K, Lines C, Riddell R, Morton D, Lanas A, et al. (2005) Cardiovascular events associated with rofecoxib in a colorectal adenoma chemoprevention trial. N Engl J Med 352: 1092–1102.
Chenevard R, Hurlimann D, Bechir M, Enseleit F, Spieker L, Hermann M, Riesen W, Gay S, Gay RE, Neidhart M, et al. (2003) Selective COX-2 inhibition improves endothelial function in coronary artery disease. Circulation 107: 405–409.
Cicero AF, Derosa G, and Gaddi A (2005) Combined lipoxygenase/cyclo-oxygenase inhibition in the elderly: the example of licofelone. Drugs Aging 22: 393–403.[CrossRef][Medline]
Friedrich EB, Tager AM, Liu E, Pettersson A, Owman C, Munn L, Luster AD, and Gerszten RE (2003) Mechanisms of leukotriene B4-triggered monocyte adhesion. Arterioscler Thromb Vasc Biol 23: 1761–1767.
Fujita H, Saito F, Sawada T, Kushiro T, Yagi H, and Kanmatsuse K (1999) Lipoxygenase inhibition decreases neointimal formation following vascular injury. Atherosclerosis 147: 69–75.[CrossRef][Medline]
Hernández-Presa MA, Bustos C, Ortego M, Tuñón J, Renedo G, Ruiz-Ortega M, and Egido J (1997) Angiotensin-converting enzyme inhibition prevents arterial nuclear factor-kappa B activation, monocyte chemoattractant protein-1 expression, and macrophage infiltration in a rabbit model of early accelerated atherosclerosis. Circulation 95: 1532–1541.
Hernández-Presa MA, Martín-Ventura JL, Ortego M, Gomez-Hernandez A, Tunon J, Hernandez-Vargas P, Blanco-Colio LM, Mas S, Aparicio C, Ortega L, et al. (2002) Atorvastatin reduces the expression of cyclooxygenase-2 in a rabbit model of atherosclerosis and in cultured vascular smooth muscle cells. Atherosclerosis 160: 49–58.[CrossRef][Medline]
Huang L, Zhao A, Wong F, Ayala JM, Struthers M, Ujjainwalla F, Wright SD, Springer MS, Evans J, and Cui J (2004) Leukotriene B4 strongly increases monocyte chemoattractant protein-1 in human monocytes. Arterioscler Thromb Vasc Biol 24: 1783–1788.
Jovanovic DV, Fernandes JC, Martel-Pelletier J, Jolicoeur FC, Reboul P, and Laufer S (2001) In vivo dual inhibition of cyclooxygenase and lipoxygenase by ML3000 reduces the progression of experimental osteoarthritis. Suppression of collagenase 1 and interleukin-1beta synthesis. Arthritis Rheum 44: 2320–2330.[CrossRef][Medline]
Lee HY, Kim MK, Park KS, Shin EH, and Bae YS (2005) Group IB secretory phospholipase A2 stimulates leukotriene B4 production by a unique mechanism in human neutrophils. Biochem Biophys Res Commun 334: 500–508.[CrossRef][Medline]
Lehmann FS and Beglinger C (2005) Impact of COX-2 inhibitors in common clinical practice a gastroenterologist's perspective. Curr Top Med Chem 5: 449–464.[CrossRef][Medline]
Linton MF and Fazio S (2004) Cyclooxygenase-2 and inflammation in atherosclerosis. Curr Opin Pharmacol 4: 116–123.[CrossRef][Medline]
Martel-Pelletier J, Lajeunesse D, Reboul P, and Pelletier JP (2003) Therapeutic role of dual inhibitors of 5-LOX and COX, selective and non-selective non-steroidal anti-inflammatory drugs. Ann Rheum Dis 62: 501–509.
Martel-Pelletier J, Mineau F, Fahmi H, Laufer S, Reboul P, Boileau C, Lavigne M, and Pelletier JP (2004) Regulation of the expression of 5-lipoxygenase-activating protein/5-lipoxygenase and the synthesis of leukotriene B4 in osteoarthritic chondrocytes: role of transforming growth factor beta and eicosanoids. Arthritis Rheum 50: 3925–3933.[CrossRef][Medline]
Martín-Ventura JL, Blanco-Colio LM, Muñoz-Garcia B, Gómez-Hernandez A, Arribas A, Ortega L, Tuñón J, and Egido J (2004) NF-kappaB activation and Fas ligand overexpression in blood and plaques of patients with carotid atherosclerosis: potential implication in plaque instability. Stroke 35: 458–463.
Moreau M, Daminet S, Martel-Pelletier J, Fernandes J, and Pelletier JP (2005) Superiority of the gastroduodenal safety profile of licofelone over rofecoxib, a COX-2 selective inhibitor, in dogs. J Vet Pharmacol Ther 28: 81–86.[CrossRef][Medline]
Mukherjee D, Nissen SE, and Topol EJ (2001) Risk of cardiovascular events associated with selective COX-2 inhibitors. J Am Med Assoc 286: 954–959.
Nomoto K, Oguchi S, Watanabe I, Kushiro T, and Kanmatsuse K (2003) Involvement of inflammation in acute coronary syndromes assessed by levels of high-sensitivity C-reactive protein, matrix metalloproteinase-9 and soluble vascular-cell adhesion molecule-1. J Cardiol 42: 201–206.[Medline]
Pelletier JP, Boileau C, Boily M, Brunet J, Mineau F, Geng C, Reboul P, Laufer S, Lajeunesse D, and Martel-Pelletier J (2005) The protective effect of licofelone on experimental osteoarthritis is correlated with the downregulation of gene expression and protein synthesis of several major cartilage catabolic factors: MMP-13, cathepsin K and aggrecanases. Arthritis Res Ther 7: R1091–R1102.[CrossRef][Medline]
Rotondo S, Dell Elba G, Krause-Brzosko K, Mnarini S, Martelli N, and Pecce R (2002) Licofelone, a dual lipoxygenase-cyclooxigenase inhibitor, down regulates polymorphonuclear leukocyte and platelet function. Eur J Pharmacol 453: 131–139.[CrossRef][Medline]
Rotondo S, Krauze-Brzosko K, Manarini S, Evangelista V, and Cerletti C (2004) Licofelone, an inhibitor of cyclooxygenase and 5-lipoxygenase, specifically inhibits cyclooxygenase-1-dependent platelet activation. Eur J Pharmacol 488: 79–83.[CrossRef][Medline]
Silverstein FE, Faich G, Goldstein JL, Simon LS, Pincus T, Whelton A, Makuch R, Eisen G, Agrawal NM, Stenson WF, et al. (2000) Gastrointestinal toxicity with celecoxib vs nonsteroidal anti-inflammatory drugs for osteoarthritis and rheumatoid arthritis: the CLASS study: a randomized controlled trial. Celecoxib long-term arthritis safety study. J Am Med Assoc 284: 1247–1255.
Singh VP, Patil CS, and Kulkarni SK (2006) Anti-inflammatory effect of licofelone against various inflammatory challenges. Fundam Clin Pharmacol 20: 65–71.[CrossRef][Medline]
Steinhilber D (1999) 5-Lipoxygenase: a target for antiinflammatory drugs revisited. Curr Med Chem 6: 71–85.[Medline]
Solomon SD, McMurray JJ, Pfeffer MA, Wittes J, Fowler R, Finn P, Anderson WF, Zauber A, Hawk E, and Bertagnolli M (2005) Cardiovascular risk associated with celecoxib in a clinical trial for colorectal adenoma prevention. N Engl J Med 352: 1071–1080.
Spanbroek R, Grabner R, Lotzer K, Hildner M, Urbach A, Ruhling K, Moos MP, Kaiser B, Cohnert TU, Wahlers T, et al. (2003) Expanding expression of the 5-lipoxygenase pathway within the arterial wall during human atherogenesis. Proc Natl Acad Sci USA 100: 1238–1243.
Tager AM, Bromley SK, Medoff BD, Islam SA, Bercury SD, Friedrich EB, Carafone AD, Gerszten RE, and Luster AD (2003) Leukotriene B4 receptor BLT1 mediates early effector T cell recruitment. Nat Immunol 4: 982–990.[CrossRef][Medline]
Tries S, Laufer S, Radziwon P, and Breddin HK (2002) Antithrombotic and platelet function inhibiting effects of ML3000, a new antiinflammatory drug with Cox/5-LOX inhibitory activity. Inflamm Res 51: 129–134.[CrossRef][Medline]
Ulbrich H, Soehnlein O, Xie X, Eriksson EE, Lindbom L, Albrecht W, Laufer S, and Dannhardt G (2005) Licofelone, a novel 5-LOX/COX-inhibitor, attenuates leukocyte rolling and adhesion on endothelium under flow. Biochem Pharmacol 70: 30–36.[CrossRef][Medline]
Vila L (2004) Cyclooxygenase and 5-lipoxygenase pathways in vessel wall: role in atherosclerosis. Med Res Rev 24: 399–424.[CrossRef][Medline]
Wang K, Tarakji K, Zhou Z, Zhang M, Forudi F, Zhou X, Koki AT, Smith ME, Keller BT, Topol EJ, et al. (2005) Celecoxib, a selective cyclooxygenase-2 inhibitor, decreases monocyte chemoattractant protein-1 expression and neointimal hyperplasia in the rabbit atherosclerotic balloon injury model. J Cardiovasc Pharmacol 45: 61–67.[CrossRef][Medline]
Willerson JT and Ridker PM (2004) Inflammation as a cardiovascular risk factor. Circulation 109: II2–II10.
Zhao L, Moos MP, Grabner R, Pedrono F, Fan J, Kaiser B, John N, Schmidt S, Spanbroek R, Lotzer K, et al. (2004) The 5-lipoxygenase pathway promotes pathogenesis of hyperlipidemia-dependent aortic aneurysm. Nat Med 10: 966–973.[CrossRef][Medline]
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