Published online before print July 20, 2005
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* DNA Unit,
Pathology Laboratory, and
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
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Key Words: liver macrophages • necro-inflammatory injury • 5-lipoxygenase pathway
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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.
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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.
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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).
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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.
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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.
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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.
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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.
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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)
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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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 
), 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.
Received December 23, 2004; revised May 17, 2005; accepted June 16, 2005.
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