NADPH oxidase signal transduces angiotensin II in hepatic stellate cells and is critical in hepatic fibrosis (original) (raw)

Chemicals. Ang II, _N_-acetylcysteine (NAC), DPI, and PD123319 were purchased from Sigma-Aldrich (St. Louis, Missouri, USA). Losartan was kindly provided by Merck & Co. (Rahway, New Jersey, USA). 2′,7′-Dichlorofluorescein diacetate (DCFDA) was obtained from Molecular Probes Inc. (Eugene, Oregon, USA). PD98059, SB203580, and LY294002 were purchased from Calbiochem-Novabiochem Corp. (La Jolla, California, USA). SP600125 was obtained from Celgene Corp. (San Diego, California, USA).

Animals and treatments. NADPH oxidase–deficient (p47phox–/–) C57BL/6 mice, which lack a critical cytosolic component required for assembly of an active NADPH oxidase complex, were expanded in parallel with parental inbred WT mice (30, 33). Two-month-old female mice were used for HSC isolations and for experimental liver fibrosis. Liver fibrosis was induced by bile duct ligation. Briefly, mice were anesthetized with sodium pentobarbital. After midline laparotomy, the common bile duct was doubly ligated with 4-0 silk and transected between the two ligations. Sham operation was performed similarly except that the bile duct was not ligated and transected. Mice were randomized to undergo bile duct ligation or sham operation. Ten mice were used in each group. All animals were sacrificed 2 weeks after surgery, and blood and liver samples were obtained. Mice were housed in a pathogen-free barrier facility accredited by the Association for the Accreditation and Assessment of Laboratory Animal Care. All procedures were approved by the Investigation and Ethics Committee and Institutional Animal Care and Use Committee of the University of North Carolina at Chapel Hill.

Cell culture. Human HSCs were isolated from surgical specimens of normal human livers as previously described (34). After isolation, cells were seeded on uncoated plastic tissue-culture dishes and cultured in DMEM (GIBCO BRL; Life Technologies Inc., Grand Island, New York, USA) supplemented with 15% FCS. Experiments were performed in five independent preparations between passages 2 and 5. Rat HSCs were isolated from male Sprague-Dawley rats (>400 g) as previously described (35). Mouse HSCs were isolated from WT (C57BL/6) and p47phox–/– mice as previously described (36). Both rat and mouse HSCs were cultured in DMEM supplemented with 10% FCS. Rat hepatocytes were isolated from male Sprague-Dawley rats (250 g) as described previously (37). Hepatocytes were plated on dishes coated with rat type I collagen in Waymouth’s medium (GIBCO BRL; Life Technologies Inc.) containing 10% FCS, 0.1 mmol/l insulin, and 0.1 mmol/l dexamethasone. After 2 hours, the cultures were washed with PBS and changed to RPMI medium (GIBCO BRL) containing 0.1 mmol/l insulin, 2 mmol/l L-glutamine, 5 mg/ml transferrin, 1 nmol/l selenium, and 1.52 μmol/l FFAs.

Serum biochemical measurements. Serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), and bilirubin levels were measured using standard enzymatic procedures by the Pathology Department of the University of North Carolina at Chapel Hill.

Histochemical studies. Paraffin-embedded sections were stained with H&E and Masson’s trichrome. For immunohistochemical analysis, sections were deparaffinized, rehydrated, and stained using the DAKO EnVision system protocol (DAKO Corp., Carpinteria, California, USA). Sections were incubated with anti–smooth muscle α-actin (1:1,000; DAKO Corp.) and anti–TGF-β1 (1:1,000; Santa Cruz Biotechnology Inc., Santa Cruz, California, USA) for 30 minutes at room temperature. As negative controls, all specimens were incubated with an isotype-matched control antibody. The area of positive staining was measured using a Macintosh-based morphometric analysis system (Apple Computer Inc., Brea, California, USA) with MetaMorph software (Universal Imaging Corp., Downingtown, Pennsylvania, USA).

Quantification of hepatic collagen content. Collagen content was assessed both by morphometric analysis of Sirius red staining of liver sections and by hydroxyproline concentration. The area of positive Sirius red staining was measured using MetaMorph software. Hydroxyproline content was quantified colorimetrically from 0.2-g liver samples. Tissue was homogenized in 6N HCl and hydrolyzed at 110°C for 16 hours. The hydrolysate was filtered, aliquots were evaporated under vacuum, and the sediment was redissolved in 50% isopropanol. Samples were then incubated in a solution containing 0.84% chloramine-T 42 mM sodium acetate, 2.6 mM citric acid, and 39.5% (vol/vol) isopropanol (pH 6.0) for 10 minutes at room temperature. Next, samples were incubated in a solution containing 0.248 g _p_-dimethylaminobenzaldehyde dissolved in 0.27 ml of 60% perchloric acid and 0.73 ml of isopropanol for 90 minutes at 50°C. Hydroxyproline content was quantified photometrically at 558 nm. The results were expressed as micrograms hydroxyproline per gram liver.

Measurement of intracellular ROS. Cells cultured in 24-well plates were loaded with the redox-sensitive dye DCFDA (10 μM) for 20 minutes at 37°C. Cells were then rinsed twice with DMEM and stimulated with agonist. DCFDA fluorescence was detected at excitation and emission wavelengths of 488 nm and 520 nm, respectively. ROS formation was measured using a multiwell fluorescence scanner (CytoFluor 2300; Millipore Corp., Bedford, Massachusetts, USA) and a Zeiss LSM 510 confocal laser-scanning microscope (Carl Zeiss, Oberkochen, Germany).

RT-PCR. The presence of p47phox mRNA in human HSCs was investigated by semiquantitative RT-PCR. Total RNA was extracted from cultured cells and normal human livers using TRIzol reagent (Invitrogen Corp., Carlsbad, California, USA). One microgram of RNA was reverse-transcribed using dT15-oligonucleotide and Moloney murine leukemia virus reverse transcriptase (Perkin-Elmer Applied Biosystems, Foster City, California, USA) in 25 μl of volume. Specific primers were designed as follows: p47phox: sense 5′-GTACCCAGCCAGCACTATG-3′, antisense 5′-GTCTGGTTGTCTGTGGGGAG-3′; gp91phox: sense 5′-GAATGGGGAAAAATAAAGGAATG-3′, antisense 5′-ACCCTTCTTCATCTGTAGC-3′; Nox1: sense 5′-CCAGGATTGAAGTGGATGGT-3′, antisense 5′-CGGTGAGGAAGAGACGGTAG-3′. Reactions were performed using 1 μM primers in a 50-μl total volume containing 1.5 mM MgCl2, 50 mM KCl, and 10 mM Tris (pH 8.3). PCR amplification was carried out by 30 cycles of denaturation (96°C, 30 seconds), annealing (62°C, 45 seconds), and extension (72°C, 20 seconds). PCR products were analyzed by electrophoresis in a 1.5% agarose gel. GAPDH amplification was used to demonstrate equal RNA load.

RNase protection assay. Rat-liver total RNA was obtained using the TRIzol. RNase protection assay for procollagen α1(I) was performed as previously described (35). Ten micrograms of total RNA was hybridized with 5.105 cpm of riboprobe. Riboprobes were derived from the 375-bp PstI-AvaI fragment from rat procollagen α1(I) cDNA, cloned into HincII-PstI sites of the pGEM 3zf+ vector, and from the plasmid pTRI-GAPDH-rat (Ambion Inc., Austin, Texas, USA). Twenty micrograms of yeast transfer RNA(tRNA) was hybridized as a negative control. The protected riboprobes were visualized by autoradiography and quantitated by PhosphorImager analysis (Molecular Dynamics, Sunnyvale, California, USA).

Western blotting. Whole-cell extracts were obtained in Triton lysis buffer, and liver extracts were obtained in modified radioimmunoprecipitation buffer as previously described (37). Western blotting was performed under standard conditions. Antibodies against 4-hydroxy-2-nonenal (HNE; Alpha Diagnostic International Inc., San Antonio, Texas, USA), phosphoserine (Zymed Laboratories Inc., San Francisco, California, USA), phospho-ERK, phospho–p38 MAPK, phospho-AKT (Cell Signaling Technology Inc., Beverly, Massachusetts, USA), heme oxygenase-1, α-tubulin, inhibitor of κB-α (IκBα), p47phox, and phospho-Jun (Santa Cruz Biotechnology Inc.) were used at a dilution of 1:1,000. For detection of collagen I secretion, cells were cultured in 100-mm culture dishes (106 cells per dish). Cells were stimulated with agonists for 20 hours, and 4 ml of cell media were precipitated with 0.76 g of sodium sulfite at 4°C for 3 hours and then centrifuged at 10,000 g for 30 minutes. Pellets were resuspended in 0.5 M acetic acid, and 40-μl aliquots were subjected to electrophoresis on a 7.5% acrylamide gel. After blotting, membranes were probed with anti–human collagen type I antibody (1:1,000; BIODESIGN International, Saco, Maine, USA), which recognizes the procollagen 1(I) chain, the mature 1(I) chain, and the heterotrimer of type I collagen. Prior to electrophoresis, some samples were digested at room temperature for 30 minutes with pepsin (1,000 U; Sigma) at pH 2.5 or with bacterial collagenase (7.5 U; Roche Molecular Biochemicals, Indianapolis, Indiana,USA) as controls for antibody specificity. Treatment with collagenase resulted in complete loss of signal, whereas treatment with pepsin resulted in reduction of the molecular mass from 170 to 120 kDa (not shown).

Kinase assays. Kinase reactions were performed as previously described (38). For IκB kinase (IKK) assay, 300 μg proteins were immunoprecipitated with 2 μl anti-IKKγ (a gift from F. Mercurio, Celgene Corp.) for 2 hours followed by 20 μl protein A/G-agarose (Santa Cruz Biotechnology Inc.) for 1 hour. Kinase reaction was performed using glutathione-_S_-transferase–IκB (GST-IκB) as a substrate (a gift from H. Sakurai, Tanabe Seiyaku Co., Osaka, Japan). For the JNK assay, 50 μg protein was incubated with 1 μl GST–c-Jun bound to GST beads washed, and subjected to a kinase reaction. For the extracellular signal–regulated kinase (ERK) assay, 200 μg proteins were immunoprecipitated with 2 μl anti–ERK-2 (Santa Cruz Biotechnology Inc.) for 2 hours followed by 20 μl protein A/G-agarose for 1 hour. The kinase reaction was performed using myelinic basic protein (Sigma) as a substrate. For the p38 MAPK assay, 250 μg proteins were immunoprecipitated with 5 μl of immobilized anti-p38 antibody (Santa Cruz Biotechnology Inc.), and the kinase reaction was performed using myelinic basic protein as a substrate. For the AKT assay, 250 μg protein was immunoprecipitated with 4 μl of immobilized AKT antibody (Santa Cruz Biotechnology Inc.). The kinase reaction was performed using a GSK-3 fusion protein as a substrate.

Electrophoretic mobility shift assay. Cell nuclear proteins were extracted as described previously (39). Eight micrograms of nuclear proteins were incubated with 100 pg of a 32P-labeled probe containing the AP-1 consensus site (5′-GTAAAGCATGAGTCAGACACCTC-3′) in buffer containing 10 mM HEPES (pH 7.8), 2 mM MgCl2, 50 mM KCl, 1 mM DTT, 0.1 mM EDTA, and 20% glycerol in the presence of single-stranded oligonucleotide (25 μg/ml) and Poly(dI-dC) (Amersham Biosciences Corp., Piscataway, New Jersey, USA) (25 μg/ml) for 20 minutes at room temperature. For competition assay, one sample was incubated with 10 ng unlabeled probe.

Preparation of RNA and cDNA microarrays. Activated human HSCs were cultured in 150-mm dishes until confluent. Cells were serum-starved overnight and then stimulated with buffer or agonists for 24 hours. Total RNA was isolated using TRIzol. Thirty micrograms of RNA from each sample was used to prepare cRNA probes as described in the manufacturer’s protocol (Affymetrix Inc., Santa Clara, California, USA). Probes were hybridized to gene chips (Affymetrix Inc. HGU95AV2; 12,000 genes). Changes in gene expression were assessed using Affymetrix Microarray Suite (version 4.0; Affymetrix Inc.). Microarray experiments were repeated three times with similar results. Two independent hybridizations were technically optimal, and the results shown are the mean of these two experiments. Control experiments (GeneChip Test3 Array; Affymetrix Inc.) were performed to ensure the integrity of the cRNA probes.

DNA synthesis. DNA synthesis was estimated as the amount of methyl-3H-thymidine (ICN Biomedicals Inc., Irvine, California, USA) incorporated into trichloroacetic-precipitable material (36).

Migration studies. Cell migration was assessed both with a modified Boyden chamber and by an in vitro wound-healing assay. For the first approach, 13-mm filters of 8 μm porosity (Whatman Inc., Clifton, New Jersey, USA) were coated with 20 μg/ml collagen I at 37°C for 30–60 minutes. The filters were then placed between the upper and lower chambers of the Boyden system (NeuroProbe Inc., Gaithersburg, Maryland, USA). The lower chamber was filled with serum-free medium (205 μl) containing the substances to be tested. Serum-starved HSCs were trypsinized and placed into the upper chamber (105 cells/ml). After 6 hours of incubation at 37°C, cells adhering to the upper side of the filter were removed with a cotton swab. The filters were then fixed in 96% methanol for 2 minutes and then stained with Harris’ hematoxylin solution for 1–2 minutes. Cells migrated to the lower side of the filter were counted by a light microscope at six to ten randomly chosen high-power fields. For the wound-healing assay, HSCs were cultured until confluence on 40-mm-diameter glass coverslips coated with 20 μg/ml collagen I. Cells were serum-starved for 24 hours, and a 2-mm-wide linear wound was cleared. Cells were then incubated with agonists for 20 hours. Cells migrating into the wound were detected using a phase-contrast microscope.

Determination of TGF-β1, IL-8, and monocyte chemoattractant protein-1 levels by ELISA. For TGF-β1, rat HSCs were cultured in six-well plates at a density of 4.105 cells per well for 4 days. Medium was then removed and cells challenged with agonists for 20 hours. Supernatants were collected, and an ELISA for TGF-β1 (R&D Systems Inc., Minneapolis, Minnesota, USA) was performed. For detection of bioactive TGF-β1, supernatants were acidified for 20 minutes at room temperature with 1N HCl (1:50 vol/vol) and then neutralized with 1N NaOH (1:50 vol/vol) before TGF-β1 determination. For chemokine release, human HSCs were cultured in six-well plates at a density of 4.105 cells per well for 24 hours. Medium was removed, and cells were incubated in serum-free medium for 24 hours in the presence of agonists. Supernatants were collected, and a sandwich ELISA for human IL-8 and monocyte chemoattractant protein-1 (MCP-1) was performed. Results are expressed as fold increase with respect to untreated cells.

Statistical analysis. Results are expressed as mean ± SEM. For Western blot, electrophoretic mobility shift assays, and kinase assays quantitation was carried out by scanning of the intensity of the signals with NIH Image 1.63 software (NIH, Bethesda, Maryland, USA). The results were analyzed using the paired or unpaired Student’s t test or the Newman-Keuls test. A P value of less than 0.05 was considered statistically significant.