Journal of Leukocyte Biology BioLegend: Treg, Th17, Stem Cell
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published online as doi:10.1189/jlb.1204747 on July 20, 2005

Published online before print July 20, 2005
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
jlb.1204747v1
78/4/871    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Titos, E.
Right arrow Articles by Rodés, J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Titos, E.
Right arrow Articles by Rodés, J.
(Journal of Leukocyte Biology. 2005;78:871-878.)
© 2005 by Society for Leukocyte Biology

Inhibition of 5-lipoxygenase-activating protein abrogates experimental liver injury: role of Kupffer cells

Esther Titos*, Joan Clària*,1, Anna Planagumà*, Marta López-Parra*, Ana González-Périz*, Joan Gaya*, Rosa Miquel{dagger}, Vicente Arroyo{ddagger} and Joan Rodés{ddagger}

* DNA Unit,
{dagger} Pathology Laboratory, and
{ddagger} Liver Unit, Hospital Clínic, Institut d’Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), University of Barcelona School of Medicine, Spain

1Correspondence: DNA Unit, Hospital Clínic, Institut d’Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), University of Barcelona School of Medicine, Villarroel 170, Barcelona 08036, Spain. E-mail: jclaria{at}clinic.ub.es


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Activation of Kupffer cells is a prominent feature of necro-inflammatory liver injury. We have recently demonstrated that 5-lipoxygenase (5-LO) and its accessory protein, 5-LO-activating protein (FLAP), are essential for the survival of Kupffer cells in culture, as their inhibition drives these liver resident macrophages to programmed cell death. In the current study, we explored whether the potent FLAP inhibitor, Bay-X-1005, reduces the number of Kupffer cells in vivo and whether this pharmacological intervention protects the liver from carbon tetrachloride (CCl4)-induced damage. Rats treated with CCl4 showed an increased number of Kupffer cells, an effect that was abrogated by the administration of Bay-X-1005 (100 mg/Kg body weight, per oral, daily). Consistent with a role for Kupffer cells in necro-inflammatory liver injury, partial depletion of Kupffer cells following FLAP inhibition was associated with a remarkable hepatoprotective action. Indeed, Bay-X-1005 significantly reduced the intense hepatocyte degeneration and large bridging necrosis induced by CCl4 treatment. Moreover, Bay-X-1005 induced a reduction in the gelatinolytic activity of matrix metalloproteinase-2 (MMP-2) and a decrease in mRNA expression of tissue inhibitor of MMP-2. The FLAP inhibitor reduced leukotriene (LT)B4 and cysteinyl LT levels and down-regulated 5-LO and FLAP protein expression in the liver. It is interesting that a significant increase in the hepatic formation of lipoxin A4, an endogenous, anti-inflammatory lipid mediator involved in the resolution of inflammation, was observed after the administration of Bay-X-1005. These findings support the concept that modulation of the 5-LO pathway by FLAP inhibition may be useful in the prevention of hepatotoxin-induced necro-inflammatory injury.

Key Words: liver macrophages • necro-inflammatory injury • 5-lipoxygenase pathway


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Kupffer cells, the resident macrophages of the liver, have been widely implicated in hepatic injury [1 ]. Indeed, activation of Kupffer cells and the subsequent release of proinflammatory mediators such as cytokines, reactive oxygen species (ROS), and eicosanoids are considered to be an early step in the pathogenesis of liver damage and tissue remodeling, as they stimulate inflammatory and fibrogenic events in the liver [1 2 3 4 ]. Moreover, in animals with experimental liver disease, the number of macrophages is increased consistently and correlates closely with the degree of hepatic injury [5 6 7 8 ]. Evidence for the role of liver macrophages in hepatic injury has been provided by attenuating hepatotoxicity in terms of steatosis, inflammation, necrosis, and collagen content in animals, which have been depleted of Kupffer cells by treatment with gadolinium chloride [9 10 11 12 ], liposomal clodronate [13 ], or by a unique, conditional ablation system mediated by the diphtheria toxin receptor [14 ]. Thus, selective inactivation of Kupffer cells represents a potential mechanism aimed to disrupt the sequence of events leading to liver injury.

The five lipoxygenase (5-LO) pathway is essential for cell survival [15 ], and we have recently demonstrated that inhibition of this pathway stops growth-related signals and induces programmed cell death in Kupffer cells in culture [16 ]. As expression of 5-LO in the liver is basically restricted to macrophages [16 , 17 ], 5-LO inhibition provides a selective strategy for depleting Kupffer cells. In the current investigation, we assessed in vivo whether inhibition of the 5-LO pathway is able to reduce the number of Kupffer cells and protect the liver from necro-inflammatory damage. To this end, we administered the compound Bay-X-1005, an indirect inhibitor of the 5-LO pathway, which binds 5-LO-activating protein (FLAP), a 18-kDa integral membrane protein that presents arachidonic acid (AA) to 5-LO in the nuclear membrane [18 19 20 ], to rats with carbon tetrachloride (CCl4)-induced liver injury.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Materials
Adult male Wistar rats were from Criffa S. A. (Santa Perpètua de Mogoda, Spain). Aprotinin, leupeptin, pepstatin A, phenylmethylsulfonyl fluoride (PMSF), bovine serum albumin, gelatin, trypsin, indomethacin, and poly-L-lysine slides were purchased from Sigma Chemical Co. (St. Louis, MO). Dulbecco-Vogt phosphate-buffered saline (DPBS)++ was from BioWhittaker (Cambrex Co., East Rutherford, NJ). Hyperfilm MP and the enhanced chemiluminescence (ECL) Western blotting system were from Amersham-Pharmacia (Buckinghamshire, UK). The Micro bicinchoninic acid (BCA) protein assay reagent kit was from Pierce (Rockford, IL). Immobilon-P transfer membranes were from Millipore Co. (Bedford, MA). Bio-Safe Coomassie (Coomassie 6250 staining) was from Bio-Rad (Hercules, CA). The Vectastain elite ABC kit, avidin-biotin-blocking kit, 3,3'-diaminobenzidine (DAB) substrate kit, and VectaMount were from Vector Laboratories (Burlingame, CA). 8-Isoprostane affinity columns and enzyme immunoassay (EIA) kits for 8-isoprostanes, leukotriene (LT)B4, and cysteinyl LTs were purchased from Cayman Chemical Co. (Ann Arbor, MI). Lipoxin (LX)A4 was from Neogen Co. (Lansing, MI). Sep-Pak C18 cartridges were from Waters Associates (Milford, MA). Mouse anti-rat ED-2 antibody was from Serotec (Oxford, UK). The RNAqueous kit was from Ambion Inc. (Austin, TX). The SuperScript II reverse transcriptase (RT) kit was from Invitrogen (Carlsbad, CA). TaqMan gene expression assays and universal TaqMan 2x polymerase chain reaction (PCR) mastermix were from Applied Biosytems (Foster City, CA). Dr. Jilly Evans (Merck Research Laboratories, Rahway, NJ) generously provided the polyclonal rabbit antisera specific for 5-LO (LO-32) and FLAP (H4). Bay-X-1005 was kindly provided by Dr. Reiner Müller-Peddinghaus (Pharma Research Center, Bayer AG, Wuppertal, Germany).

Experimental model of liver injury
Chronic liver injury was induced in male adult Wistar rats by repeated inhalation of CCl4 (twice weekly, Monday and Friday), as described elsewhere [21 ]. Animals were fed ad libitum with standard chow and distilled water containing phenobarbital (0.3 g/l) as drinking fluid and were distributed randomly into two groups receiving Bay-X-1005 [n=20, 100 mg/Kg body weight (b.w.), per oral (p.o.)] or placebo (n=15, 0.5% carboxymethylcellulose, p.o.). Animals were dosed daily starting 2 days before the first administration of CCl4 and killed in a staggered manner under ketamine anesthesia after 4, 6, 8, and 10 weeks of CCl4 administration. A group of control animals (rats not treated with CCl4) was also included in the investigation. Liver specimens were obtained from each animal and fixed in 4% formaldehyde or snap-frozen in N2 for further analysis. All animal studies were conducted in accordance with the criteria of the Investigation and Ethics Committee of the Hospital Clínic (Barcelona, Spain) and the European Community laws governing the use of experimental animals.

Histological analysis
Liver samples fixed in 4% formaldehyde were embedded in paraffin and stained with hematoxylin-eosin (H&E) for histological analysis. Hepatocellular necrosis was assessed by a registered pathologist unaware of the treatments and scored by examining randomly chosen fields of view per tissue section as follows: grade 0 (absent), grade I (spotty necrosis; one or few necrotic hepatocytes), and grade II (bridging necrosis).

Assessment of Kupffer cell number by ED-2 immunohistochemistry
Samples of liver tissue fixed in 4% formaldehyde were embedded in paraffin, cut in 5 µm sections, and mounted on poly-L-lysine slides. ED-2-positive cells (Kupffer cells) were detected immunohistochemically by the avidin-biotin complex method using a Vectastain elite ABC kit as described previously [22 ] with modifications. Briefly, liver sections were deparaffinized and incubated with preheated (37°C) 0.1% trypsin solution for 15 min and then with 3% hydrogen peroxide for 10 min to quench endogenous peroxidase. To block nonspecific reactions, sections were first incubated with horse-blocking serum for 30 min and then with avidin-biotin-blocking solution according to the manufacturer’s instructions. Following blocking steps, sections were incubated overnight at 4°C with mouse monoclonal anti-ED-2 (1:200) as the primary antibody and then incubated for 30 min with a biotinylated horse anti-mouse antibody. Sections were incubated for 30 min with the avidin-horseradish peroxidase (HRP) complex, then treated with DAB for 5 min, and finally, counterstained with hematoxylin. Sections were dehydrated, cleared, and mounted in VectaMount mounting medium. For quantitative analysis, the number of ED-2-positive cells was counted in a total of 25 high-power fields (HPFs) per tissue section under a light microscope (Model BH-2, Olympus Optical Co., Tokyo, Japan) at 400x magnification, and results were given as number of positive cells/HPF.

Measurement of hepatic levels of LTB4, cysteinyl LTs, and LXA4
Liver samples of ~120 mg were homogenized individually with an Ultra-Turrax T 25 basic homogenizer (IKA-Werke, Staufen, Germany) in 4 ml DPBS++ and kept with 2 vol ice-cold methanol (MeOH). Homogenates were centrifuged at 2000 rpm for 10 min at 4°C, and eicosanoids present in the supernatants were extracted with Sep-Pak C18 cartridges. The eluted methylformate and MeOH fractions were taken for further analysis of LTB4 and LXA4 and cysteinyl LT levels, respectively, by specific EIA kits.

Analysis of 5-LO and FLAP protein expression by Western blot
Frozen rat liver tissue stored in N2 was homogenized in 8 vol 10 mM Hepes/KOH, pH 7.4, containing 0.25% (w/v) sucrose, 2 mM EDTA, aprotinin (1 µg/ml), leupeptin (1 µg/ml), pepstatin A (1 µg/ml), and 10 µM PMSF. Tissue debris was removed by centrifugation at 1000 g for 10 min at 4°C. After centrifugation, the total tissue protein in the supernatant fraction was measured spectrophotometrically at 562 nm using the BCA method (protein assay) following the manufacturer’s protocol. Western blot analysis for 5-LO and FLAP was performed as described previously [23 ]. Briefly, 100–150 µg total protein in sodium dodecyl sulfate (SDS)-containing Laemmli sample buffer was heated for 5 min at 95°C and separated through 12.5% or 16% SDS-polyacrylamide gel electrophoresis (PAGE) for 5-LO and FLAP detection, respectively. Proteins were electroblotted onto Immobilon-P membranes, and the efficiency of the transfer was visualized by Ponceau S staining. Membranes were subsequently soaked for 1 h at 25°C in Tris-buffered saline (20 mM Tris/HCl, pH 7.5, and 0.5 M NaCl) containing 0.05% (v/v) Tween 20 (T-TBS; 0.05%) and 5% (w/v) nonfat dry milk. Blots were washed twice for 5 min each with 0.05% T-TBS and subsequently treated for 2 h at 25°C with anti-5-LO polyclonal antibody (dilution 1:1000) or anti-FLAP polyclonal antibody (dilution 1:500) in 0.05% T-TBS containing 1% dry milk. After washing the blots two times for 5 min each with 0.05% T-TBS, membranes were incubated for 1 h at 25°C with a HRP-linked donkey anti-rabbit antibody (dilution 1:2000) in 0.05% T-TBS containing 1% dry milk. The blots were washed again twice for 5 min each with 0.05% T-TBS and subsequently developed using the ECL Western blot analysis system. Total protein from rat lung tissue was used as a positive control for 5-LO and FLAP expression.

Analysis of metalloproteinase-2 (MMP-2) activity by zymography
Liver samples were homogenized at 4°C in 50 mM Tris-HCl (pH 7.5) containing 0.15 mM NaCl, 10 mM CaCl2, 1 µg/ml aprotinin, 10 µg/ml leupeptin, and 10 µg/ml pepstatin. The homogenates were centrifuged at 10,000 g for 20 min at 4°C, the supernatants collected, and the protein content determined by the Bradford method. Proteins were analyzed for gelatinolytic activity by zymography. Briefly, the supernatants of liver homogenates (50 µg protein) were subjected to 7% SDS-PAGE in a 0.13% gelatin-containing gel. After electrophoresis, the gel was washed twice for 15 min in 2.5% Triton X-100 to remove the SDS. The gel was subsequently incubated at 37°C during 22 h in 5 mM CaCl2, 50 mM Tris-HCl (pH 8), 0.02% NaN3, and 2 µM ZnCl and thereafter, stained in Coomassie blue and destained in equilibrated buffer (3% glycerol, 10% acetic acid, and 40% MeOH). Bands of 62 and 72 kDa, which correspond to the active and latent forms of MMP-2, respectively, were scanned, and activities in the gel slabs were quantified using the Phoretix 1D image analysis software (Phoretix International, Newcastle upon Tyne, UK).

Analysis of tissue inhibitor of MMP-1 (TIMP-1) and TIMP-2 mRNA expression by RT and real-time quantitative PCR
Total RNA was isolated from rat liver tissue using the RNAqueous kit and reverse-transcribed into cDNA with the SuperScript II RT kit. Briefly, the RT reaction mixture (25 µl final volume) contained 2 µg total RNA, 5 µl 5x first-strand buffer, 2.5 µl 0.1 M dithiothreitol, 1.25 µl 10 mM deoxy-unspecified nucleoside 5'-triphosphates, 1.25 µl Oligo dT15 (500 µg/ml), and 1.25 µl SuperScript II RT (200 U). First-strand cDNA synthesis was performed in a MJ Research PTC-100 thermal cycler (Bio-Rad) at the following reaction conditions: 70°C 10 min, 25°C 10 min, 42°C 50 min, 47°C 10 min, 52°C 10 min, and 97°C 10 min to inactivate the enzyme. Real-time quantitative PCR was performed with an ABI Prism 7900 sequence detection system (Applied Biosytems) using the fluorescent Taqman methodology. Ready-to-use primer and probe sets predeveloped by Applied Biosystems (TaqMan gene expression assays) were used to quantify TIMP-1 (ID: Rn00587558_m1) and TIMP-2 (ID:Rn00573232_m1) gene expression using ß-actin as endogenous control (ID: Rn00667869_m1). Briefly, PCR reactions were performed in duplicate using the Universal TaqMan 2x PCR mastermix in a volume of 25 µl containing 1 µl cDNA. The thermal profile included 2 min at 50°C and 10 min at 95°C followed by 40 cycles of 95°C for 15 s and 60°C for 1 min. PCR results were analyzed with the sequence detector software Version 2.1 (Applied Biosystems). Relative quantitation of gene expression was performed using the standard curve method. Every set of reactions included four serial twofold dilutions of the same liver cDNA sample, which was used to generate standard curves for the target genes (TIMP-1 and TIMP-2) and the endogenous reference (ß-actin). The amount of each target was then calculated from the standard curve and divided by the amount of ß-actin to obtain a normalized target value.

Measurement of hepatic levels of 8-isoprostanes and malondialdehyde (MDA) and serum concentrations of alanine aminotransferase (ALT) and aspartate aminotransferase (AST)
Total (free and esterified) hepatic 8-isoprostane levels were determined by EIA after extraction of samples in affinity columns. Briefly, liver samples were homogenized in 0.1 M KH2PO4, pH 7.4, containing 1 mM EDTA and 10 µM indomethacin and subsequently, hydrolyzed by the addition of an equal volume of 15% KOH for 1 h at 40°C. After adding 2 vol ethanol containing 0.01% butylated hydroxytoluene, proteins were removed by centrifugation, and the supernatant was dried under a stream of N2. For 8-isoprostane purification, samples were reconstituted in 1 M KH2PO4 buffer, acidified by adding 35% HCl (pH 7.4), and applied to the 8-isoprostane affinity column according to the manufacturer’s instructions. Measurement of 8-isoprostanes in the sample eluant was performed by a specific EIA kit, which allows detection from 3.9 to 500 pg/ml 8-isoprostane standard. The concentration of MDA was measured by high-pressure liquid chromatography (HPLC) as described elsewhere [24 ]. Briefly, liver samples were homogenized in 0.3% trichloracetic acid and centrifuged at 8000 g for 3 min at 4°C. Supernatants were subsequently exposed to phosphoric and thiobarbituric acids and heated at 80°C for 30 min, prior to analysis by HPLC. MDA levels were expressed as pmol MDA/100 mg protein after measuring the total protein concentration in the pellets by means of the BCA protein assay. Serum ALT and AST levels were measured by standardized, routine techniques.

Statistical analysis of the results was performed using the ANOVA and unpaired Student’s t-test. Results were expressed as mean ± SEM, and differences were considered significant at a Pvalue <0.05.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Comparable body weight gains were observed in CCl4-treated rats receiving placebo and Bay-X-1005 (data not shown). As expected, histological examination of livers from rats treated with CCl4 for more than 4 weeks revealed massive and severe hepatocyte necrosis at the centrilobular zone, and bridging of necrosis severely disrupted the sinusoidal and lobular architecture of the liver (Fig. 1 , left panels). All animals included in the placebo group had grade II hepatocellular necrosis. In contrast, administration of the FLAP inhibitor Bay-X-1005 to CCl4-treated rats exerted a remarkable, protective effect on hepatocellular necrosis in such a way that at the 4th week of the study, these animals showed no evidence of centrilobular necrosis or hepatocyte ballooning degeneration (Fig. 1 , right panels). In fact, all animals included in the Bay-X-1005 group had a grade 0 necrosis score. A reduction in serum ALT (from 1114±55 to 362±20 UI/L, P<0.05) and AST (from 956±173 to 489±16 UI/L, P<0.05) concentrations was observed in rats receiving Bay-X-1005.



View larger version (181K):
[in this window]
[in a new window]
  		Figure 1. Bay-X-1005 prevents hepatocellular necrosis in CCl4-treated rats. Representative photomicrographs of liver sections stained with H&E from 4-week CCl4-treated rats receiving placebo (left panels) and Bay-X-1005 (right panels).

 
To evaluate the potential implication of Kupffer cells in the progression of CCl4-induced liver injury, liver sections were stained with the ED-2 monoclonal antibody, a specific marker of liver resident macrophages, and the number of ED-2-positive cells quantified by optical microscopy. As shown in Figure 2 and as compared with control animals, the number of ED-2-positive cells per HPF was increased significantly in liver sections from CCl4-treated rats. In these animals, ED-2-positive cells were preferentially located in the mid-zone area surrounding the damaged perivenular area (Fig. 2 , upper panels). The number of ED-2-positive cells per HPF in CCl4-treated rats was reduced significantly by the administration of the FLAP inhibitor Bay-X-1005 (Fig. 2) .



View larger version (71K):
[in this window]
[in a new window]
  		Figure 2. Bay-X-1005 significantly abrogates the increased number of ED-2-positive cells in livers from CCl4-treated rats. (Upper panels) Representative photomicrographs (original magnification, x250) of liver sections immunostained with the ED-2 antibody from control (upper left panel) and 6-week CCl4-treated rats receiving placebo (upper middle panel) or Bay-X-1005 (upper right panel). A representative number of ED-2-positive cells (Kupffer cells) are denoted by arrows. In CCl4-treated rats, ED-2-positive cells are located mainly in the mid-zonal area surrounding the damaged perivenular area. (Lower panel) Number of ED-2-positive cells per HPF in liver tissue sections from control (open bar) and CCl4-treated animals receiving placebo (hatched bar) or Bay-X-1005 (solid bar) for 6 weeks. Results are expressed as mean ± SEM.

 
As Bay-X-1005 binds FLAP and prevents the interaction between 5-LO and the substrate AA [20 ], the hepatoprotective effects of this compound presumably reflect a reduction in LT formation. In fact, we observed a significant decrease in the hepatic levels of LTB4 and cysteinyl LTs in CCl4-treated rats receiving Bay-X-1005 (Fig. 3 , left and middle panels). It is interesting that at the 6th week of CCl4 treatment, Bay-X-1005 significantly increased the hepatic formation of LXA4, a biologically active eicosanoid, in which the biosynthetic circuit is initiated by the interaction with 5-LO [25 ] (Fig. 3 , right panel). As shown in Figure 4 , Bay-X-1005 not only decreased LT formation but also reduced 5-LO and FLAP protein expression in the liver of CCl4-treated rats.



View larger version (15K):
[in this window]
[in a new window]
  		Figure 3. Bay-X-1005 significantly reduces LTB4 and cysteinyl LT levels while increasing LXA4 formation in livers from CCl4-treated rats. Liver tissue was obtained after 6 and 8 weeks of CCl4 treatment from animals receiving placebo (open bars) or Bay-X-1005 (solid bars). Samples were homogenized and extracted in C18-silica, reverse-phase cartridges, and LTB4, cysteinyl LT, and LXA4 levels were determined by highly specific EIAs. Results represent the mean ± SEM.

 


View larger version (41K):
[in this window]
[in a new window]
  		Figure 4. Bay-X-1005 significantly decreases hepatic 5-LO and FLAP protein expression in CCl4-treated animals. (A) Representative Western blot analysis of 5-LO protein expression in liver samples at the 4th, 6th, 8th, and 10th weeks of CCl4 treatment in animals receiving placebo or Bay-X-1005. Equal quantities of total protein (150 µg/lane) were separated on a 12.5% SDS-PAGE gel and electrotransferred onto Immobilon-P membranes before the analysis with a specific anti-5-LO antibody. C+, Lung tissue total protein. (B) Animals were treated as described in A, and FLAP protein expression was analyzed in equal quantities of total protein (100 µg/lane), separated on a 16% SDS-PAGE gel, and electrotransferred onto Immobilon-P membranes. (C) Densitometric analysis of 5-LO (left panel)- and FLAP (right panel)-positive bands from rats receiving placebo (open bars) or Bay-X-1005 (hatched bars)-treated animals. Data are expressed as mean ± SEM of three to five different experiments. *, P < 0.05, versus placebo.

 
We next determined the effects of Bay-X-1005 on key enzymes involved in the regulation of matrix remodeling in the liver. We specifically analyzed MMP-2 activity by gelatin zymography and TIMP-1 and TIMP-2 mRNA expression by RT and real-time PCR in liver tissue samples from rats receiving placebo and Bay-X-1005 along the different weeks of CCl4 administration. As shown in Figure 5A , Bay-X-1005 significantly reduced MMP-2 gelatinolytic activity, an effect that reached statistical significance at the 6th week of CCl4 treatment. Consistent with this finding, a significant decrease in TIMP-2 mRNA expression was observed at this time-point in animals treated with Bay-X-1005 (Fig. 5B) .



View larger version (23K):
[in this window]
[in a new window]
  		Figure 5. Bay-X-1005 reduces MMP-2 activity and TIMP-2 mRNA expression in liver samples from CCl4-treated rats. (A, Upper panel) Representative gelatin zymography analysis of total protein extracts isolated at the 4th, 6th, 8th, and 10th weeks of CCl4 treatment in rats receiving placebo (–) or Bay-X-1005 (+). Equal protein quantities (50 µg) were loaded onto 7% SDS-PAGE containing 0.13% gelatin and processed as described in Materials and Methods. (A, Lower panel) Densitometric analysis of digitized photographs obtained from the gels (placebo, open bars; Bay-X-1005, hatched bars). Data are expressed as mean ± SEM of three different experiments. (B) TIMP-1 (left panel) and TIMP-2 (right panel) mRNA expression was determined by RT real-time quantitative PCR in liver tissue from 6-week CCl4-treated rats receiving placebo (open bars) or Bay-X-1005 (solid bars). Results are expressed as mean ± SEM with duplicate determinations.

 
We finally assessed the effects of Bay-X-1005 on hepatic lipid peroxidation and oxidative stress levels in CCl4-treated rats. To this end, we used two different, specific markers of lipid peroxidation, namely MDA and 8-isoprostanes. MDA is a stable end by-product of the metabolism of lipid peroxides [24 ], whereas 8-isoprostanes are AA-derived products, initially formed in situ in the phospholipid domain by free, radical-mediated peroxidation [26 ]. MDA levels significantly increased over the course of the induction of liver injury, although values did not significantly differ between animals receiving Bay-X-1005 and placebo (Table 1 ). Moreover, there were no differences in hepatic 8-isoprostane concentrations between CCl4-treated rats receiving Bay-X-1005 and placebo (8.24±0.77 vs. 8.49±1.75 ng/g liver tissue at the 8th week of CCl4 treatment).


View this table:
[in this window]
[in a new window]
  		Table 1. MDA Levels in Livers from CCl4-Treated Rats Receiving Placebo (0.5% Carboxymethylcellulose) or Bay-X-1005 (100 mg/Kg b.w., p.o., Daily)

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The results of the current investigation show, for the first time, that oral administration of the potent and selective FLAP inhibitor, Bay-X-1005, prevents hepatic necro-inflammatory damage in rats with CCl4-induced liver injury. This beneficial effect was associated with a depletion of Kupffer cells, where their population increases in response to CCl4. A similar strategy involving macrophage depletion has been reported in other disease models, such as accelerated arteriopathies in rats and hypercholesterolemic rabbits [27 ], endotoxin-induced lung inflammation [28 ], and lung ischemia-reperfusion injury [29 ].

In our study, Bay-X-1005 induced a partial depletion of Kupffer cells, indicating that this drug is able to reduce the excessive number of Kupffer cells without threatening vital functions for these macrophages such as the regulation of the host defense and immune responses. Previous work from our laboratory has provided evidence that the mechanism by which Bay-X-1005 depletes Kupffer cells is related to the induction of apoptosis [16 , 30 ]. In fact, we have reported that FLAP and 5-LO inhibitors induce major changes in cell morphology and DNA content frequency distribution in Kupffer cells, which together with DNA fragmentation, are characteristic features of cells undergoing apoptosis [16 , 30 ]. The apoptotic effects of FLAP and 5-LO inhibitors in Kupffer cells are consistent with previous studies reporting similar effects in inflammatory and cancer cells expressing the 5-LO pathway [15 , 31 , 32 ]. It is also important to note that pharmacological intervention of the 5-LO pathway is a rather selective strategy to induce programmed cell death, as it only triggers apoptosis in cells displaying an active 5-LO biosynthetic pathway [16 , 30 31 32 ]. In the liver, Kupffer cells are indeed the only sinusoidal cell type apparently endowed with the complete enzymatic machinery for the formation of 5-LO products [16 , 17 , 30 , 33 ].

Depletion of Kupffer cells appears to confer a protective role in the liver by reducing the bulk production of potent, hepatic, tissue-toxic mediators such as inflammatory cytokines (i.e., tumor necrosis factor {alpha}), ROS, proteases, and eicosanoids [1 , 2 , 34 ]. Among the eicosanoids, we observed a significant decrease in the hepatic formation of LTB4 and cysteinyl LTs following the administration of Bay-X-1005. LTB4 is a potent chemoattractant to neutrophils, whereas cysteinyl LTs are potent vasoconstrictors, increase vascular permeability, and are involved in tissue remodeling [35 , 36 ]. A number of studies in 5-LO- and FLAP-deficient mice have clearly established the important role of these 5-LO-derived eicosanoids in inflammation and organ injury [37 , 38 ]. Regarding the liver, there is a large body of evidence implicating 5-LO products in the pathogenesis of hepatocellular injury, specifically, as mediators of inflammation and cell damage in experimental hepatitis induced by D-galactosamine and in liver injury induced by the administration of endotoxin and Propionibacterium acnes to rats [39 40 41 42 43 ]. The hepatoprotective actions of Bay-X-1005 in our experimental model appear to be mediated by a combination of mechanisms involving not only inhibition of LT biosynthesis but also down-regulation of 5-LO and FLAP protein expression. Nevertheless, given that Kupffer cells are the major cell type carrying 5-LO expression and activity in the liver, these inhibitory actions are likely to be secondary to Kupffer cell depletion. The hepatoprotective effects of Bay-X-1005 are not mediated by changes in hepatic lipid peroxidation, as MDA and the 8-isoprostane levels were not modified by this FLAP inhibitor.

An interesting finding of our study was that we observed a switching from the biosynthesis of LTs to LXs in the liver of animals treated with Bay-X-1005. LXs are conjugated, trihydroxytetraene-containing eicosanoids generated by transcellular biosynthesis during cell–cell interactions [25 ]. The two major routes of LX biosynthesis include the conversion of 5-LO-derived LTA4 by 12-LO and the conversion of 15(S)-hydroxy-6,8,11,14-eicosatetraenoic acid (15-HETE) by 5-LO [25 ]. As Bay-X-1005 inhibits LT biosynthesis by its binding to FLAP without directly affecting 5-LO, we speculate that the increased hepatic formation of LXA4 following FLAP inhibition probably reflects an enhanced conversion of 15-HETE by this enzyme. Indeed, previous work from our laboratory has demonstrated that liver cells are a rich source of 15-HETE and that substantial amounts of LXA4 are generated during the interaction of hepatocytes with 5-LO-containing, nonparenchymal liver cells [44 ]. Conversely, Bay-X-1005 not only binds with high affinity to a specific site of FLAP but also inhibits LTC4 synthase [45 ], thus favoring the formation of 5-LO-derived products (i.e., LXA4) other than cysteinyl LTs. Unlike LTs, which are proinflammatory [35 , 36 ], LXs work as endogenous, anti-inflammatory lipid mediators of resolution attenuating in vivo renal ischemia-reperfusion injury and glomerulonephritis [46 ], airway hyper-reactivity and inflammation [47 ], and hapten-induced colitis [48 ]. Therefore, formation of these anti-inflammatory compounds during Bay-X-1005 treatment may contribute to explain the hepatoprotective actions associated with FLAP inhibition.

Another interesting finding of the current study was that FLAP inhibition in CCl4-treated rats was associated with a reduction in MMP-2 activity and TIMP-2 mRNA expression. MMP-2, also called gelatinase A, belongs to the growing family of calcium zinc-dependent MMPs, which degrade type IV collagen, denaturated interstitial collagens (gelatin), and a variety of other matrix proteins [49 ]. MMP-2, and also TIMPs, increase during liver injury and contribute to liver fibrogenesis by degrading normal liver architecture, allowing for increased hepatic stellate cell activation [50 51 52 53 ]. As liver fibrosis resolves, their expression decreases [50 51 52 53 ]. Thus, modulation of MMP-2 and TIMP-2 by Bay-X-1005 represents an additional protective action of the pharmacological intervention of the 5-LO pathway in the liver. Our results are consistent with those reported in an experimental model of atherosclerosis, in which 5-LO-deficient mice display a decreased MMP-2 activity [54 ]. The mechanisms by which Bay-X-1005 modulates MMPs remain unknown at present, but inhibition of LT formation and stimulation of LXA4 biosynthesis are probably implicated. In fact, in fibroblasts, LTs stimulate collagen production, whereas LXA4, at nanomolar concentrations, significantly inhibits MMP-1 and -3 levels [55 , 56 ].

In summary, our study identifies a novel, potential, therapeutic target against the progression of liver injury based on the pharmacological intervention of the 5-LO pathway with a potent, orally active FLAP inhibitor. It is important to note that Bay-X-1005 has been tested in clinical studies for the treatment of asthma [57 , 58 ] and has recently attracted much attention for the treatment of patients with cardiovascular disease, in whom linkage analysis has shown that the gene encoding for FLAP confers a higher risk [59 ]. MK-591, another orally active FLAP inhibitor, has been tested in humans for the treatment of glomerulonephritis and active ulcerative colitis [60 , 61 ]. Further studies, however, are needed to fully establish the therapeutic potential of FLAP inhibitors.


   ACKNOWLEDGEMENTS
 
This investigation was supported in part by a grant from Ministerio de Ciencia y Tecnología (03/0586) and Instituto de Salud Carlos III (C03/02). E. T. and A. P. were supported by IDIBAPS and M. L-P. and A. G-P., by Ministerio de Ciencia y Tecnología. We thank Dr. Jilly Evans (Merck Research Laboratories, Rahway, NJ) for providing the 5-LO and FLAP antibodies.

Received December 23, 2004; revised May 17, 2005; accepted June 16, 2005.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Laskin, D. L. (1990) Nonparenchymal cells and hepatotoxicity Semin. Liver Dis. 10,293-304[Medline]
  2. Decker, K. (1990) Biologically active products of stimulated liver macrophages (Kupffer cells) Eur. J. Biochem. 192,245-261[Medline]
  3. Friedman, S. L. (2000) Molecular regulation of hepatic fibrosis, an integrated cellular response to tissue injury J. Biol. Chem. 275,2247-2250[Free Full Text]
  4. Winwood, P. J., Arthur, M. J. (1993) Kupffer cells: their activation and role in animal models of liver injury and human liver disease Semin. Liver Dis. 13,50-59[Medline]
  5. Alric, L., Orfila, C., Carrere, N., Beraud, M., Carrera, G., Lepert, J. C., Dufaut, M., Pipy, B., Vinel, J. P. (2000) Reactive oxygen intermediates and eicosanoid production by Kupffer cells and infiltrated macrophages in acute and chronic liver injury induced in rats by CCl4 Inflamm. Res. 49,700-707[CrossRef][Medline]
  6. Geerts, A., Schellinck, P., Bouwens, L., Wisse, E. (1988) Cell population kinetics of Kupffer cells during the onset of fibrosis in rat liver by chronic carbon tetrachloride administration J. Hepatol. 6,50-56[CrossRef][Medline]
  7. Luckey, S. W., Petersen, D. R. (2001) Activation of Kupffer cells during the course of carbon tetrachloride-induced liver injury and fibrosis in rats Exp. Mol. Pathol. 71,226-240[CrossRef][Medline]
  8. Thompson, W. D., Jack, A. S., Patrick, R. S. (1980) The possible role of macrophages in transient hepatic fibrogenesis induced by acute carbon tetrachloride injury J. Pathol. 130,65-73[CrossRef][Medline]
  9. Andrés, D., Sánchez-Reus, I., Bautista, M., Cascales, M. (2003) Depletion of Kupffer cell function by gadolinium chloride attenuates thioacetamide-induced hepatotoxicity. Expression of metallothionein and HSP70 Biochem. Pharmacol. 66,917-926[CrossRef][Medline]
  10. Edwards, M. J., Keller, B. J., Kauffman, F. C., Thurman, R. G. (1993) The involvement of Kupffer cells in carbon tetrachloride toxicity Toxicol. Appl. Pharmacol. 119,275-279[CrossRef][Medline]
  11. Muriel, P., Escobar, Y. (2003) Kupffer cells are responsible for liver cirrhosis induced by carbon tetrachloride J. Appl. Toxicol. 23,103-108[CrossRef][Medline]
  12. Rivera, C. A., Bradford, B. U., Hunt, K. J., Adachi, Y., Schrum, L. W., Koop, D. R., Burchardt, E. R., Rippe, R. A., Thurman, R. G. (2001) Attenuation of CCl(4)-induced hepatic fibrosis by GdCl(3) treatment or dietary glycine Am. J. Physiol. Gastrointest. Liver Physiol. 281,G200-G207[Abstract/Free Full Text]
  13. Sturm, E., Havinga, R., Baller, J. F., Wolters, H., van Rooijen, N., Kamps, J. A., Verkade, H. J., Karpen, S. J., Kuipers, F. (2005) Kupffer cell depletion with liposomal clodronate prevents suppression of Ntcp expression in endotoxin-treated rats J. Hepatol. 42,102-109[Medline]
  14. Duffield, J. S., Forbes, S. J., Constandinou, C. M., Clay, S., Partolina, M., Vuthoori, S., Wu, S., Lang, R., Iredale, J. P. (2005) Selective depletion of macrophages reveals distinct, opposing roles during liver injury and repair J. Clin. Invest. 115,56-65[CrossRef][Medline]
  15. Romano, M., Clària, J. (2003) Cyclooxygenase-2 and 5-lipoxygenase converging functions on cell proliferation and tumor angiogenesis: implications for cancer therapy FASEB J. 17,1986-1995[Abstract/Free Full Text]
  16. Titos, E., Clària, J., Planagumà, A., López-Parra, M., Villamor, N., Párrizas, M., Carrió, A., Miquel, R., Jiménez, W., Arroyo, V., Rivera, F., Rodés, J. (2003) Inhibition of 5-lipoxygenase induces cell growth arrest and apoptosis in rat Kupffer cells: implications for liver fibrosis FASEB J. 17,1745-1747[Abstract/Free Full Text]
  17. Titos, E., Clària, J., Bataller, R., Bosch-Marcé, M., Ginès, P., Jiménez, W., Arroyo, V., Rivera, F., Rodés, J. (2000) Hepatocyte-derived cysteinyl leukotrienes modulate vascular tone in experimental cirrhosis Gastroenterology 119,794-805[CrossRef][Medline]
  18. Miller, D. K., Gillard, J. W., Vickers, P. J., Sadowski, S., Leveille, C., Mancini, J. A., Charleson, P., Dixon, R. A. F., Ford-Hutchinson, A. W., Fortin, R., Gauthier, J. Y., Rodkey, J., Rosen, R., Rouzer, C., Sigal, I. S., Strader, C. D., Evans, J. F. (1990) Identification and isolation of a membrane protein necessary for leukotriene production Nature 343,278-281[CrossRef][Medline]
  19. Peters-Golden, M., Brock, T. G. (2003) 5-Lipoxygenase and FLAP Prostaglandins Leukot. Essent. Fatty Acids 69,99-109[CrossRef][Medline]
  20. Muller-Peddinghaus, R., Kohlsdorfer, C., Theisen-Popp, P., Fruchtmann, R., Perzborn, E., Beckermann, B., Buhner, K., Ahr, H. J., Mohrs, K. H. (1993) BAY X1005, a new inhibitor of leukotriene synthesis: in vivo inflammation pharmacology and pharmacokinetics J. Pharmacol. Exp. Ther. 267,51-57[Abstract/Free Full Text]
  21. Clària, J., Jiménez, W. (1999) Renal dysfunction and ascites in carbon tetrachloride-induced cirrhosis in rats Arroyo, V. Ginès, P. Rodés, J. Schrier, R. W. eds. Ascites and Renal Dysfunction in Liver Disease ,379-396 Blackwell Science Malden, MA.
  22. Yamate, J., Kumagai, D., Tsujino, K., Nakatsuji, S., Kuwamura, M., Kotani, T., Sakuma, S., LaMarre, J. (1999) Macrophage populations and apoptotic cells in the liver before spontaneous hepatitis in Long-Evans Cinnamon (LEC) rats J. Comp. Pathol. 120,333-346[CrossRef][Medline]
  23. Woods, J. W., Evans, J. F., Ethier, D., Scott, S., Vickers, P. J., Hearn, L., Heibein, J. A., Charlesson, S., Singer, I. I. (1993) 5-Lipoxygenase and 5-lipoxygenase-activating protein are localized in the nuclear envelope of activated human leukocytes J. Exp. Med. 178,1935-1946[Abstract/Free Full Text]
  24. Halliwell, B., Chirico, S. (1993) Lipid peroxidation: its mechanism, measurement, and significance Am. J. Clin. Nutr. 57(Suppl. 5),715S-724S[Abstract/Free Full Text]
  25. Serhan, C. N. (2002) Lipoxins and aspirin-triggered 15-epi-lipoxin biosynthesis: an update and role in anti-inflammation and pro-resolution Prostaglandins Other Lipid Mediat. 68-69,433-455
  26. Morrow, J. D., Hill, K. E., Burk, R. F., Nammour, T. M., Badr, K. F., Roberts, L. J. (1990) A series of prostaglandin F2-like compounds are produced in vivo in humans by a non-cyclooxygenase, free radical-catalyzed mechanism Proc. Natl. Acad. Sci. USA 87,9383-9387[Abstract/Free Full Text]
  27. Danenberg, H. D., Fishbein, I., Gao, J., Monkkonen, J., Reich, R., Gati, I., Moerman, E., Golomb, G. (2002) Macrophage depletion by clodronate-containing liposomes reduces neointimal formation after balloon injury in rats and rabbits Circulation 106,599-605[Abstract/Free Full Text]
  28. Koay, M. A., Gao, X., Washington, M. K., Parman, K. S., Sadikot, R. T., Blackwell, T. S., Christman, J. W. (2002) Macrophages are necessary for maximal nuclear factor-{kappa} B activation in response to endotoxin Am. J. Respir. Cell Mol. Biol. 26,572-578[Abstract/Free Full Text]
  29. Naidu, B. V., Krishnadasan, B., Farivar, A. S., Woolley, S. M., Thomas, R., Van Rooijen, N., Verrier, E. D., Mulligan, M. S. (2003) Early activation of the alveolar macrophage is critical to the development of lung ischemia-reperfusion injury J. Thorac. Cardiovasc. Surg. 126,200-207[Abstract/Free Full Text]
  30. Titos, E., Clària, J., Planagumà, A., López-Parra, M., Villamor, N., Miquel, R., Jiménez, W., Arroyo, V., Rivera, F., Rodés, J. (2004) 5-Lipoxygenase (5-LO) is involved in Kupffer cell survival. Possible role of 5-LO products in the pathogenesis of liver fibrosis Comp. Hepatol. 3(Suppl. 1),S19
  31. Romano, M., Catalano, A., Nutini, M., D’Urbano, E., Crescenzi, C., Clària, J., Libner, R., Davì, G., Procopio, A. (2001) 5-Lipoxygenase regulates malignant mesothelial cell survival: involvement of vascular endothelial growth factor FASEB J. 15,2326-2336[Abstract/Free Full Text]
  32. Catalano, A., Caprari, P., Soddu, S., Procopio, A., Romano, M. (2004) 5-Lipoxygenase antagonizes genotoxic stress-induced apoptosis by altering p53 nuclear trafficking FASEB J. 18,1740-1742[Abstract/Free Full Text]
  33. Shimada, K., Navarro, J., Goeger, D. E., Mustafa, S. B., Weigel, P. H., Weinman, S. A. (1998) Expression and regulation of leukotriene-synthesis enzymes in rat liver cells Hepatology 28,1275-1281[CrossRef][Medline]
  34. Jaeschke, H., Gores, G. J., Cederbaum, A. I., Hinson, J. A., Pessayre, D., Lemasters, J. J. (2002) Mechanisms of hepatotoxicity Toxicol. Sci. 65,166-176[Abstract/Free Full Text]
  35. Samuelsson, B., Dahlen, S. E., Lindgren, J. A., Rouzer, C. A., Serhan, C. N. (1987) Leukotrienes and lipoxins: structures, biosynthesis, and biological effects Science 237,1171-1176[Abstract/Free Full Text]
  36. Funk, C. D. (2001) Prostaglandins and leukotrienes: advances in eicosanoid biology Science 294,1871-1875[Abstract/Free Full Text]
  37. Chen, X. S., Sheller, J. R., Johnson, E. N., Funk, C. D. (1994) Role of leukotrienes revealed by targeted disruption of the 5-lipoxygenase gene Nature 372,179-182[CrossRef][Medline]
  38. Goulet, J. L., Snouwaert, J. N., Latour, A. M., Coffman, T. M., Koller, B. H. (1994) Altered inflammatory responses in leukotriene-deficient mice Proc. Natl. Acad. Sci. USA 91,12852-12856[Abstract/Free Full Text]
  39. Farzaneh-Far, R., Moore, K. (2003) Cysteinyl-leukotrienes and the liver Prostaglandins Other Lipid Mediat. 72,35-50[CrossRef][Medline]
  40. Keppler, D., Huber, M., Baumert, T. (1988) Leukotrienes as mediators in diseases of the liver Semin. Liver Dis. 8,357-366[Medline]
  41. Keppler, D., Lesch, R., Reutter, W., Decker, K. (1968) Experimental hepatitis induced by D-galactosamine Exp. Mol. Pathol. 9,279-290[CrossRef][Medline]
  42. Kawada, N., Ueda, N., Mizoguchi, Y., Kobayashi, K., Monna, T., Morisawa, S., Ishimura, K., Suzuki, T., Yamamoto, S. (1992) Increased 5-lipoxygenase activity in massive hepatic cell necrosis in the rat correlates with neutrophil infiltration Hepatology 16,462-468[Medline]
  43. Decker, K. (1985) Eicosanoids, signal molecules of liver cells Semin. Liver Dis. 5,175-190[Medline]
  44. Titos, E., Chiang, N., Serhan, C. N., Romano, M., Gaya, J., Pueyo, G., Clària, J. (1999) Hepatocytes are a rich source of novel aspirin-triggered 15-epi-lipoxin A(4) Am. J. Physiol. 277,C870-C877
  45. Sala, A., Folco, G., Henson, P. M., Murphy, R. C. (1997) Pharmacological modulation of human platelet leukotriene C4-synthase Biochem. Pharmacol. 53,905-908[CrossRef][Medline]
  46. Kieran, N. E., Maderna, P., Godson, C. (2004) Lipoxins: potential anti-inflammatory, proresolution, and antifibrotic mediators in renal disease Kidney Int. 65,1145-1154[CrossRef][Medline]
  47. Levy, B. D., De Sanctis, G. T., Devchand, P. R., Kim, E., Ackerman, K., Schmidt, B. A., Szczeklik, W., Drazen, J. M., Serhan, C. N. (2002) Multi-pronged inhibition of airway hyper-responsiveness and inflammation by lipoxin A(4) Nat. Med. 8,1018-1023[CrossRef][Medline]
  48. Fiorucci, S., Wallace, J. L., Mencarelli, A., Distrutti, E., Rizzo, G., Farneti, S., Morelli, A., Tseng, J. L., Suramanyam, B., Guilford, W. J., Parkinson, J. F. (2004) A ß-oxidation-resistant lipoxin A4 analog treats hapten-induced colitis by attenuating inflammation and immune dysfunction Proc. Natl. Acad. Sci. USA 101,15736-15741[Abstract/Free Full Text]
  49. Arthur, M. J. (2000) Fibrogenesis II. Metalloproteinases and their inhibitors in liver fibrosis Am. J. Physiol. Gastrointest. Liver Physiol. 279,G245-G249[Abstract/Free Full Text]
  50. Milani, S., Herbst, H., Schuppan, D., Grappone, C., Pellegrini, G., Pinzani, M., Casini, A., Calabro, A., Ciancio, G., Stefanini, F. (1994) Differential expression of matrix-metalloproteinase-1 and -2 genes in normal and fibrotic human liver Am. J. Pathol. 144,528-537[Abstract]
  51. Takahara, T., Furui, K., Funaki, J., Nakayama, Y., Itoh, H., Miyabayashi, C., Sato, H., Seiki, M., Ooshima, A., Watanabe, A. (1995) Increased expression of matrix metalloproteinase-II in experimental liver fibrosis in rats Hepatology 21,787-795[CrossRef][Medline]
  52. Parsons, C. J., Bradford, B. U., Pan, C. Q., Cheung, E., Schauer, M., Knorr, A., Krebs, B., Kraft, S., Zahn, S., Brocks, B., Feirt, N., Mei, B., Cho, M. S., Ramamoorthi, R., Roldan, G., Ng, P., Lum, P., Hirth-Dietrich, C., Tomkinson, A., Brenner, D. A. (2004) Antifibrotic effects of a tissue inhibitor of metalloproteinase-1 antibody on established liver fibrosis in rats Hepatology 40,1106-1115[CrossRef][Medline]
  53. Préaux, A. M., Mallat, A., Nhieu, J. T., D’Ortho, M. P., Hembry, R. M., Mavier, P. (1999) Matrix metalloproteinase-2 activation in human hepatic fibrosis regulation by cell-matrix interactions Hepatology 30,944-950[CrossRef][Medline]
  54. Zhao, L., Moos, M. P., Grabner, R., Pedrono, F., Fan, J., Kaiser, B., John, N., Schmidt, S., Spanbroek, R., Lotzer, K., Huang, L., Cui, J., Rader, D. J., Evans, J. F., Habenicht, A. J., Funk, C. D. (2004) The 5-lipoxygenase pathway promotes pathogenesis of hyperlipidemia-dependent aortic aneurysm Nat. Med. 10,966-973[CrossRef][Medline]
  55. Phan, S. H., McGarry, B. M., Loeffler, K. M., Kunkel, S. L. (1988) Binding of leukotriene C4 to rat lung fibroblasts and stimulation of collagen synthesis in vitro Biochemistry 27,2846-2853[CrossRef][Medline]
  56. Sodin-Semrl, S., Taddeo, B., Tseng, D., Varga, J., Fiore, S. (2000) Lipoxin A4 inhibits IL-1 ß-induced IL-6, IL-8, and matrix metalloproteinase-3 production in human synovial fibroblasts and enhances synthesis of tissue inhibitors of metalloproteinases J. Immunol. 164,2660-2666[Abstract/Free Full Text]
  57. Fischer, A. R., Rosenberg, M. A., Roth, M., Loper, M., Jungerwirth, S., Israel, E. (1997) Effect of a novel 5-lipoxygenase-activating protein inhibitor, BAYx 1005, on asthma induced by cold dry air Thorax 52,1074-1077[Abstract]
  58. Hamilton, A. L., Watson, R. M., Wyile, G., O’Byrne, P. M. (1997) Attenuation of early and late phase allergen-induced bronchoconstriction in asthmatic subjects by a 5-lipoxygenase activating protein antagonist, BAYx 1005 Thorax 52,348-354[Abstract]
  59. Helgadottir, A., Manolescu, A., Thorleifsson, G., Gretarsdottir, S., Jonsdottir, H., Thorsteinsdottir, U., Samani, N. J., Gudmundsson, G., Grant, S. F., Thorgeirsson, G., Sveinbjornsdottir, S., Valdimarsson, E. M., Matthiasson, S. E., Johannsson, H., Gudmundsdottir, O., Gurney, M. E., Sainz, J., Thorhallsdottir, M., Andresdottir, M., Frigge, M. L., Topol, E. J., Kong, A., Gudnason, V., Hakonarson, H., Gulcher, J. R., Stefansson, K. (2004) The gene encoding 5-lipoxygenase activating protein confers risk of myocardial infarction and stroke Nat. Genet. 36,233-239[CrossRef][Medline]
  60. Guasch, A., Zayas, C. F., Badr, K. F. (1999) MK-591 acutely restores glomerular size selectivity and reduces proteinuria in human glomerulonephritis Kidney Int. 56,261-267[CrossRef][Medline]
  61. Roberts, W. G., Simon, T. J., Berlin, R. G., Haggitt, R. C., Snyder, E. S., Stenson, W. F., Hanauer, S. B., Reagan, J. E., Cagliola, A., Tanaka, W. K., Simon, S., Berger, M. L. (1997) Leukotrienes in ulcerative colitis: results of a multicenter trial of a leukotriene biosynthesis inhibitor, MK-591 Gastroenterology 112,725-732[CrossRef][Medline]



This article has been cited by other articles:


Home page
 J. Pharmacol. Exp. Ther.Home page
R. Horrillo, A. Planaguma, A. Gonzalez-Periz, N. Ferre, E. Titos, R. Miquel, M. Lopez-Parra, J. L. Masferrer, V. Arroyo, and J. Claria
Comparative Protection against Liver Inflammation and Fibrosis by a Selective Cyclooxygenase-2 Inhibitor and a Nonredox-Type 5-Lipoxygenase Inhibitor
J. Pharmacol. Exp. Ther., December 1, 2007; 323(3): 778 - 786.
[Abstract] [Full Text] [PDF]

Home page
 Eur Respir JHome page
O. Haworth and B. D. Levy
Endogenous lipid mediators in the resolution of airway inflammation
Eur. Respir. J., November 1, 2007; 30(5): 980 - 992.
[Abstract] [Full Text] [PDF]

Home page
 FASEB J.Home page
A. Gonzalez-Periz, A. Planaguma, K. Gronert, R. Miquel, M. Lopez-Parra, E. Titos, R. Horrillo, N. Ferre, R. Deulofeu, V. Arroyo, et al.
Docosahexaenoic acid (DHA) blunts liver injury by conversion to protective lipid mediators: protectin D1 and 17S-hydroxy-DHA
FASEB J, December 1, 2006; 20(14): 2537 - 2539.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
jlb.1204747v1
78/4/871    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Titos, E.
Right arrow Articles by Rodés, J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Titos, E.
Right arrow Articles by Rodés, J.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS