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Am J Physiol Gastrointest Liver Physiol 291: G902-G911, 2006. First published June 22, 2006; doi:10.1152/ajpgi.00124.2006
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LIVER AND BILIARY TRACT

Regulation of peroxisome proliferator-activated receptor-{gamma} in liver fibrosis

Liu Yang,1 Che-Chang Chan,1 Oh-Sang Kwon,1 Songling Liu,1 Jason McGhee,1 Stephen A. Stimpson,2 Lihong Z. Chen,2 W. Wallace Harrington,2 William T. Symonds,2 and Don C. Rockey3

1Departments of Cell Biology and Medicine, Duke University Medical Center, Durham, North Carolina; 2GlaxoSmithKline, Research Triangle Park, North Carolina; and 3University of Texas Southwestern Medical Center, Dallas, Texas

Submitted 18 March 2006 ; accepted in final form 1 June 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
The peroxisome proliferator-activated receptors (PPARs) impart diverse cellular effects in biological systems. Because stellate cell activation during liver injury is associated with declining PPAR{gamma} expression, we hypothesized that its expression is critical in stellate cell-mediated fibrogenesis. We therefore modulated its expression during liver injury in vivo. PPAR{gamma} was depleted in rat livers by using an adenovirus-Cre recombinase system. PPAR{gamma} was overexpressed by using an additional adenoviral vector (AdPPAR{gamma}). Bile duct ligation was utilized to induce stellate cell activation and liver fibrosis in vivo; phenotypic effects (collagen I, smooth muscle {alpha}-actin, hydroxyproline content, etc.) were measured. PPAR{gamma} mRNA levels decreased fivefold and PPAR{gamma} protein was undetectable in stellate cells after culture-induced activation. During activation in vivo, collagen accumulation, assessed histomorphometrically and by hydroxyproline content, was significantly increased after PPAR{gamma} depletion compared with controls (1.28 ± 0.14 vs. 1.89 ± 0.21 mg/g liver tissue, P < 0.03). In isolated stellate cells, AdPPAR{gamma} overexpression resulted in significantly increased adiponectin mRNA expression and decreased collagen I and smooth muscle {alpha}-actin mRNA expression compared with controls. During in vivo fibrogenesis, rat livers exposed to AdPPAR{gamma} had significantly less fibrosis than controls. Collagen I and smooth muscle {alpha}-actin mRNA expression were significantly reduced in AdPPAR{gamma}-infected rats compared with controls (P < 0.05, n = 10). PPAR{gamma}-deficient mice exhibited enhanced fibrogenesis after liver injury, whereas PPAR{gamma} receptor overexpression in vivo attenuated stellate cell activation and fibrosis. The data highlight a critical role for PPAR{gamma} during in vivo fibrogenesis and emphasize the importance of the PPAR{gamma} pathway in stellate cells during liver injury.

stellate cell; cirrhosis; biliary; adenovirus; wound healing

THE RESPONSE TO RECURRENT injury, in liver and other organs, is one of wound healing. Many different types of injury (i.e., chronic hepatitis, ethanol, biliary tract disease, cystic fibrosis, iron or copper overload, etc.) lead to injury and ultimately to hepatic fibrosis and cirrhosis. Cirrhosis in turn leads to hepatocellular dysfunction and portal hypertension, each of which have grave clinical consequences.

Extensive investigation has now established that a key effector in the liver wound healing process is the hepatic stellate cell (HSC). A central feature of the wounding response to liver injury is the transformation of resident stellate cells from a "quiescent" (normal) to an "activated" (injured liver) state. Characteristics of this transition include morphological and functional changes. For example, morphological changes include loss of vitamin A, acquisition of stress bundles, and development of prominent rough endoplasmic reticulum (14, 25, 33, 40). Functional changes include the production of increased quantities of extracellular matrix, including types I, III, and IV collagens, fibronectin, laminin, and proteoglycans, some of which are increased by >50-fold compared with normal liver (33). Furthermore, the available evidence now indicates that the overall increase in extracellular matrix protein deposition typical of cirrhosis can largely be ascribed to excess production by stellate cells (42). Thus a great deal of emphasis has been placed on elucidating mechanisms underlying stellate cell fibrogenesis. A number of events, typically acting in concert, play a role in stimulating stellate cell fibrogenesis (3, 4, 11, 12, 26, 37, 39, 41). A notable feature recently described for stellate cells is their expression of peroxisome proliferator-activated receptors (PPARs) (19, 35).

Stellate cells possess each of the three classes of PPARs ({alpha}, {gamma}, and {delta}/beta). PPAR{gamma} appears to be unique in that it appears to be markedly downregulated during stellate cell activation (19, 35). Interestingly, despite the fact that PPAR{gamma} is downregulated, stimulation of this receptor with PPAR{gamma} ligands reverses the activation process and phenotype (18, 34, 35). In this study, we hypothesized that PPAR{gamma} expression plays a pivotal role in stellate cell-mediated hepatic fibrogenesis; furthermore, we postulated that modulation of its expression both in vitro and in vivo would lead to altered effects of endogenous PPAR{gamma} ligands and have important phenotypic effects during injury-mediated hepatic fibrogenesis.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Adenovirus, in vivo infection, and animal model of fibrosis. Adenoviral vectors, AdPPAR{gamma} (encoding mouse PPAR{gamma}1, kindly provided by Dr. Janardan K. Reddy) and AdCre (encoding Cre recombinase), were amplified and purified as described (21).

Liver injury and fibrosis were induced by ligation of the common bile duct as previously described (27). In brief, male Sprague-Dawley rats weighing 500–600 g were anesthetized, and the common bile duct was ligated and sectioned using aseptic technique.

PPAR{gamma} Loxp –/– and strain-matched control mice were from Jackson Laboratory. Either bile duct ligation (BDL) or sham operation was performed in wild-type (Wt) or PPAR{gamma} Loxp –/– mice. During surgery, AdCre (2.4 x 1010 pfu/kg) was injected into the portal vein of PPAR{gamma} Loxp –/– mice and control Wt mice. Twelve mice were included in each experimental group. Animals were killed 10 days after surgery, and blood and liver samples were obtained.

In other experiments, during BDL, AdPPAR{gamma} was injected into the inferior vena cava in 500 µl of PBS at a concentration of 1 x 1010 pfu/kg. A virus containing an identical adenovirus backbone expressing green fluorescent protein (AdGFP) was used as control virus.

BDL in each species (mouse and rat) resulted in periportal expansion of biliary duct cells with concomitant periductular and lobular matrix deposition. Controls were sham operated by exposing the common bile duct but without ligation and/or scission.

Animal care and surgical procedures were approved by the Duke University Medical Center Institutional Animal Care and Use Committee as set forth in the "Guide for the Care and Use of Laboratory Animals" published by the National Institutes of Health.

Cell isolation and culture. Liver cells were isolated from normal rats and mice as previously described (8, 13, 44). Briefly, after in situ perfusion of the liver with pronase (Boehringer Mannheim, Indianapolis, IN) followed by collagenase (Crescent Chemical, Hauppauge, NY), dispersed cell suspensions were layered on a discontinuous density gradient of 8.2 and 15.6% Accudenz (Accurate Chemical and Scientific, Westbury, NY). The resulting upper layer consisted of >95% stellate cells. Hepatocytes were isolated by collagenase perfusion of the liver and centrifugal elutriation as described (32). The viability of all cells was verified by phase-contrast microscopy as well as the ability to exclude propidium iodide. The viability of all cell cultures utilized for study was >95%. Isolated stellate cells were seeded at a density of 3 x 102 cells/mm2 with DMEM supplemented with 10% FBS, 100 units/ml streptomycin, and 100 units/ml penicillin. Primary stellate cell cultures were allowed to grow to confluence and then subcultured by trypsinization. In some experiments, cells after one passage (only) were utilized.

Transient transfection and reporter gene assay. To determine whether PPAR{gamma} expressed by the adenoviral vector induces the PPAR response element (PPRE) promoter activity, HSC were transiently transfected with liver fatty acid binding protein (DR1)4-TK-GL3 reporter vector (containing four copies of the liver fatty-acid binding PPRE upstream of a luciferase reporter driven by a thymidine kinase promoter), which was a gift from GlaxoSmithKline (Welwyn Garden City, UK), using Fugene 6 (Roche, Indianapolis, IN). For transfection, 10-day cultures of HSC were used in six-well plates (70,000 cells/well; 2 days after infection with a viral vector). One microgram of DNA and 6 µl of Fugene 6.0 reagent were mixed in 150 µl of OptiMEM-I medium and incubated at room temperature for 15 min. During the 15-min incubation, the cells were washed three times with OptiMEM-I medium. The volume of the mixture was diluted to 1.5 ml with OptiMEM-I, and the contents of the tube were added to the well of the cells. At 48 h posttransfection, the luciferase assay system with Luciferase cell lysis buffer (BD Pharmingen, San Diego, CA) was used to measure PPRE transcriptional induction according to the manufacturer's protocol. All measurements of luciferase activity (relative light units) were normalized to the protein concentration.

Immunoblot. Frozen liver samples and cells were homogenized in 150 µl of Dignam C buffer (9) containing protease and phosphatase inhibitors as described (30). The homogenized samples were rotated on a tumbler for 30 min at 4°C and then centrifuged at 14,000 rpm for 5 min at 4°C. The protein concentration of samples was determined by the Bio-Rad protein assay (Bio-Rad Laboratories, Hercules, CA). Proteins (50 µg from whole liver; 10 µg from cell) were separated by SDS-PAGE and then transferred to nitrocellulose (Schleicher and Schuell, Keene, NH) in a buffer containing 20 mmol/l Tris, pH 8.3, 150 mmol/l glycine, 0.01% SDS, and 20% methanol. Equal loading of the gel was confirmed by staining nitrocellulose membranes with Ponceau S. After blocking with 5% nonfat milk (Carnation, Swampscott, MA) in Tris-buffered saline (20 mmol/l Tris, pH 7.5, 150 mmol/l NaCl) containing 0.1% Tween 20 (TBS-T) for 1 h, nitrocellulose membranes were incubated with primary antibody smooth muscle {alpha}-actin antibody (Sigma, St. Louis, MO), anti-PPAR{gamma} (Santa Cruz, Santa Cruz, CA), anti-Cre antibody (EMD Biosciences, San Diego, CA) or beta-actin antibody (Sigma), all diluted 1:1,000 in 5% nonfat milk for 1 h (or in the case of immunoblotting involving whole liver extracts, for 12 h), and then washed three times in TBS-T. Secondary antibody (horseradish peroxidase-conjugated anti-mouse IgG from Amersham, UK) was incubated with nitrocellulose membranes at a dilution of 1:1,000 in 5% nonfat milk for 30 min. After four washes in TBS-T, antibody complexes were detected by using the Amersham ECL system in linear range. Specific signals were scanned and quantitated by scanning densitometry.

mRNA quantification by real-time RT-PCR. mRNAs were quantified by real-time RT-PCR per the manufacturer's specifications (Stratagene, Mx3000P real-time PCR). The sequences of primers for rat 18S, type I collagen, transforming growth factor-beta1 (TGF-beta1), extra domain A fibronectin, PPAR{gamma}, and liver X receptor (LXR){alpha} are as follows: 18S, sense, 5'-TTGACGGAAGGGCACCACCAG-3', antisense, 5'-GCACCACCACCCACGGAATCG-3', product size = 131 bp; collagen I {alpha}1, sense, 5'-TTCCCTGGACCTAAGGGTACT-3', antisense, 5'-TTGAGCTCCAGCTTCGCC-3', product size = 114 bp; TGF-beta1, sense, 5'-TTGCCCTCTACAACCAACACAA-3', antisense, 5'-GCTTGCGACCCACGTAGTA-3', product size = 103 bp; tissue inhibitor of metalloproteinase-1 (TIMP-1), sense, 5'-CCTTGCAAACTGGAGAGTGACA-3', antisense, 5'-AGGCAAAGTGATCGCTCTGGT-3', product size = 91 bp; PPAR{gamma}:, sense, 5'-CACAATGCCATCAGGTTTGG-3', antisense, 5'-GCTGGTCGATATCACTGGAGATC-3', product size = 82 bp; adiponectin, sense, 5'-ACAAGGCCGTTCTCTTCACCTA-3', antisense, 5'-GGTCCACATTTTTTTCCTGATACTG-3', product size = 51 bp; LXR{alpha}, sense, 5'-TCAGCATCTTCTCTGCAGACCGG-3', anti-sense, 5'-TCATTAGCATCCGTGGGAACA-3', product size = 144 bp. The sequences of primers for mouse 18S and type I collagen were as follows: 18S sense: 5'-TTGACGGAAGGGCACCACCAG-3', antisense, 5'-GCACCACCACCCACGGAATCG-3; collagen I {alpha}1, sense, 5'-GAGCGGAGAGTACTGGATCG-3', antisense, 5'-GCTTCTTTTCCTTGGGGTTC-3'.

Total RNA was extracted from cells or whole livers using TRIzol (Invitrogen, Carlsbad, CA). One microgram of RNA was reverse-transcribed by using random primer and Superscript RNase H-reverse transcriptase (Invitrogen, Carlsbad, CA). Samples were incubated at 20°C for 10 min, 42°C for 30 min; reverse transcriptase was inactivated by heating at 99°C for 5 min and cooling at 5°C for 5 min. Amplification reactions were performed with a SYBRgreen PCR master mix (Applied Biosystems). Five microliters of diluted cDNA samples (1 to 5 dilution) were used for quantitative two-step PCR (a 10-min step at 95°C, followed by 50 cycles of 15 s at 95°C and 1 min at 65°C) in the presence of 400 nM specific forward and reverse primers, 5 mM MgCl2, 50 mM KCl, 10 mM Tris buffer (pH 8.3), 200 µM dATP, dCTP, dGTP, and 400 µM dUTP and 1.25 U of AmpliTaq Gold DNA polymerase (Perkin-Elmer Applied Biosystems). Each sample was analyzed in triplicate.

Serum biochemical measurements. Serum alanine aminotransferase, aspartate aminotransferase, and bilirubin levels were measured by standard enzymatic procedures by the Pathology Department, University of North Carolina.

Immunohistochemistry. Liver tissue was fixed in formalin and embedded in paraffin. Immunohistochemical staining to detect smooth muscle {alpha}-actin was performed by using the DAKO Envision System (DAKO) according to the manufacturer's protocol. Specimens were incubated with the peroxidase-labeled polymer conjugated to goat anti-mouse immunoglobulins (diluted 1:2 in phosphate-buffered saline) for 5 min. The tissue was counterstained with Aqua Hematoxylin-INNOVEX (Innovex Biosciences, Richmond, CA). Controls included specimens exposed to 1% bovine serum albumin instead of primary antibody.

BrdU proliferation assay. Five thousand rat hepatic stellate cells were resuspended in 100 µl of culture medium and dispensed in each well of a 96-culture plate. Stellate cells were serum starved (0.2% serum) for 48 h and infected with AdGFP and AdPPAR{gamma}. Proliferation was induced by 10% FBS for 24 h, and DNA synthesis was assessed by bromodeoxyuridine (BrdU) incorporation. The BrdU assay was performed according to the manufacturer's protocol (Amersham, Little Chalfont, UK).

ELISA for TGF-beta1. Liver tissues ({approx}0.1 g) were homogenized in lysis buffer containing 25 mM HEPES, 0.1% CHAPS, 5 mM MgCl2, 1.3 mM EDTA, 1 mM EGTA, and phosphatase and protease inhibitors. A sandwich ELISA for mouse TGF-beta1 (R&D Systems, Minneapolis, MN) was performed.

Morphometry. Livers were fixed in 10% phosphate-buffered formalin for 48 h at 4°C, washed twice with water, stored in 70% ethanol at 4°C, and embedded in paraffin. Five-micrometer sections were stained with picrosirius red (Sigma) and counterstained with fast green (Sigma). The proportion of tissue stained with picrosirius red content was assessed by morphometric analysis with MetaView software (Universal Imaging, Downingtown, PA) as described (44). Collagen stained with Sirius red was quantitated in the sections that were randomly chosen (under x20 magnification, 10 fields each from sample).

Hydroxyproline assay. Hydroxyproline content in whole liver specimens was quantified colorimetrically. Liver specimens were weighed, and 30 mg of freeze-dried sample were hydrolyzed in 6 N HCl at 110°C for 16 h. The hydrolysate was evaporated under vacuum, and the sediment was redissolved in 1 ml distilled water. Samples were filtered then incubated with 0.5 ml of chloramine-T solution, containing 1.41 g of chloramine-T dissolved in 80 ml of acetate-citrate buffer and 20 ml of 50% isopropanol, for 20 min at room temperature. To this were added 0.5 ml of Ehrlich's solution, including 7.5 g of dimethylaminobenzaldehyde dissolved in 13 ml of 60% perchloric acid and 30 ml isopropanol, and the mixture was incubated at 65°C for 15 min. After cooling, the absorbance was read at 561 nm. Hydroxyproline concentration was calculated from a standard curve prepared with high-purity hydroxyproline (Sigma) and expressed as milligrams hydroxyproline per gram liver.

Statistical analysis. Results are expressed as means ± SE. Significance was established using the Student's t-test and analysis of variance when appropriate. Differences were considered significant when P < 0.05.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
PPAR{gamma} expression in liver cells. We initially examined PPAR{gamma} mRNA levels in different hepatic cell types isolated from both normal and injured rat livers. After BDL, PPAR{gamma} mRNA levels in stellate cells were decreased compared with normal animals (Fig. 1A). We found no significant changes in PPAR{gamma} mRNA levels in hepatocytes or endothelial cells (minor changes were noted in Kupffer cells). To further characterize changes in PPAR{gamma} expression during stellate cell activation, we examined a culture model of activation in which cells from normal livers were isolated and grown over a time course of days after isolation. Real-time PCR and Western blotting were used to assess PPAR{gamma} mRNA and protein levels, respectively. PPAR{gamma} mRNA and protein expression were reduced progressively during culture-induced activation (Fig. 1, B and C).


Figure 1
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  		Fig. 1. Peroxisome proliferator-activator receptor-{gamma} (PPAR{gamma}) expression in liver cells. A: stellate cells were isolated from normal and injured rat livers (BDL). Total RNA was isolated from individual cell pellets, and PPAR{gamma} mRNA levels were assessed by real-time PCR as in MATERIALS AND METHODS (*P < 0.05 compared with sham, n = 3). HSC, hepatic stellate cells. B: stellate cells were isolated and placed in culture; PPAR{gamma} mRNA expression was measured at the indicated time points in culture (*P < 0.05 compared with day 0, n = 3 for each day). C: PPAR{gamma} was detected in stellate cells under the same conditions as in B by immunoblotting; beta-actin was detected in the same lysates to highlight protein loading as an internal control. The immunoblot is representative of 3 other independent experiments.

 
PPAR{gamma} inhibition in vivo leads to enhanced fibrogenesis. At the time of BDL, AdCre (2.4 x 1010 pfu/kg) was injected into the portal vein as in MATERIALS AND METHODS (we injected adenovirus at the time of surgery for two reasons: first to ensure adequate Cre gene expression through the major portion of liver injury and secondly to allow us to perform only one invasive procedure). As predicted, AdCre infection resulted in Cre recombinase expression in the liver (Fig. 2A), leading to a significant reduction in PPAR{gamma} expression in stellate cells from these livers compared with those from control livers (Fig. 2B). Aminotransferase and bilirubin levels were elevated after BDL (but not in sham-operated mice), and there was no difference in aminotransferase levels among mice receiving AdGFP and AdCre mice (data not shown).


Figure 2
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  		Fig. 2. PPAR{gamma} depletion leads to enhanced fibrogenesis in vivo. A: adenoviral vectors AdGFP and AdCre were injected into the portal vein of PPAR{gamma} Loxp –/– mice as described in MATERIALS AND METHODS. A: whole liver specimens were lysed and subjected to immunoblot analysis to detect Cre recombinase as described in MATERIALS AND METHODS (30 mg tissue). B: stellate cells were isolated from PPAR{gamma} Loxp –/– mice 5 days after AdGFP or AdCre administration; total cell lysates were subjected to immunoblot analysis to detect PPAR{gamma}, and quantitative data are presented (*P < 0.05 for AdGFP compared with AdCre). C and D: AdCre (2.4 x 1010 pfu/kg) was administered as above, and liver injury was induced by BDL in wild-type (Wt) and PPAR{gamma} Loxp –/– mice. Ten days later, total RNA from liver tissue was extracted as in MATERIALS AND METHODS, and real-time PCR was performed to detect collagen I {alpha}1 (C) (n = 12, *P < 0.05 for BDL vs. sham-operated mice; #P < 0.05 PPAR{gamma} Loxp –/– BDL vs. Wt BDL mice). D: TGF-beta1 was quantitated in liver tissues by ELISA (n = 12, *P < 0.01 for BDL vs. sham-operated mice; #P < 0.05 PPAR{gamma} for Loxp –/– BDL vs. Wt BDL mice).

 
We then investigated the effect of PPAR{gamma} depletion on liver fibrosis. PPAR{gamma} was depleted during liver injury as above; this led to significant increases in collagen I {alpha}1 mRNA and bioactive TGF-beta1 protein expression in BDL compared with sham-operated mice (Fig. 2, C and D). Controls in which AdGFP was administered to Wt and PPAR{gamma} Loxp –/– mice instead of AdCre revealed that there were no changes in collagen I {alpha}1 mRNA and bioactive TGF-beta1 protein expression compared with non-adenovirus-treated mice (data not shown). We further hypothesized that AdPPAR{gamma} depletion in vivo may increase stellate cell activation. Therefore, we examined the marker of activation, smooth muscle {alpha}-actin (40), in liver sections. After liver injury induced by BDL, Wt mouse livers were found to have periductal fibrosis, lobular fibrosis, and disruption of the hepatic architecture (Fig. 3). Additionally, we found that, compared with AdCre-treated Wt mice subjected to BDL (Fig. 3A), smooth muscle {alpha}-actin expression was markedly increased in AdCre-treated PPAR{gamma} Loxp –/– mice (Fig. 3B).


Figure 3
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  		Fig. 3. PPAR{gamma} depletion in vivo leads to stellate cell activation and enhanced fibrogenesis. PPAR{gamma} Loxp –/– mice and Wt mice were exposed to AdCre during BDL operation as in Fig. 2 and in MATERIALS AND METHODS. A and B: liver sections were fixed, and smooth muscle {alpha}-actin was detected as in MATERIALS AND METHODS (representative images are shown). The bar depicts 100 µm. Controls in which specimens were exposed to 1% bovine serum albumin instead of primary antibody did not reveal specific labeling (not shown). C and D: liver sections were stained with Sirius red as in MATERIALS AND METHODS. C: representative liver section from a Wt mouse. D: representative liver section from a PPAR{gamma} Loxp –/– mouse. Black bar represents 100 µm. E: the area stained by Sirius red in the depicted groups was quantitated as described in MATERIALS AND METHODS (n = 12, *P < 0.01 for BDL vs. sham-operated mice; #P < 0.05 for PPAR{gamma} Loxp –/– BDL mice vs. Wt BDL mice). F: hydroxyproline was measured in the livers of specific groups (n = 12, *P < 0.01 for BDL vs. sham-operated mice; #P < 0.05 for PPAR{gamma} Loxp –/– BDL mice vs. Wt BDL mice).

 
PPAR{gamma}-depleted mouse livers revealed enhanced collagen deposition with prominent fibrosis (Wt BDL mice, and PPAR{gamma} Loxp –/– BDL mice, Fig. 3D). Quantitation of collagen content, by morphometric analysis and by measurement of hydroxyproline content, was markedly increased after BDL in PPAR{gamma}-depleted compared with Wt mice (Fig. 3, E and F).

PPAR{gamma} activation inhibits smooth muscle {alpha}-actin and collagen I{alpha}1 expression during stellate cell activation. Given the effect of PPAR{gamma} knockdown on endogenous PPAR{gamma} signaling and stellate cell phenotypes, we examined the effect of PPAR{gamma} activation on stellate cell activation. We utilized the known expression of smooth muscle {alpha}-actin as a marker of activation (40). Exposure of stellate cells to the PPAR{gamma} agonist ciglitizone for 72 h (from day 2 to day 5 after isolation) substantially reduced smooth muscle {alpha}-actin expression in a dose-dependent fashion (Fig. 4A). Additionally, collagen I {alpha}1 mRNA measured in cellular mRNA by real-time PCR was reduced approximately threefold after cells were exposed to ciglitizone (Fig. 4B).


Figure 4
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  		Fig. 4. Functional effects of PPAR{gamma} activation in isolated stellate cells. A: rat stellate cells were isolated from normal rats and allowed to undergo spontaneous culture-induced activation. Cells were exposed to ciglitizone at the indicated concentrations in DMEM containing 10% FBS. Immunoblot for smooth muscle {alpha}-actin was performed as in MATERIALS AND METHODS; immunoblotting for beta-actin was performed and utilized as an internal loading control. Below the immunoblot shown, specific bands were scanned and quantitated and expressed graphically (*P < 0.05, compared with serum-free medium alone, n = 3). B: cells were isolated from normal rat livers and allowed to undergo spontaneous culture-induced activation. Ciglitizone was added on the 5th day in serum free medium and collagen I {alpha}1 mRNA was measured in cellular mRNA (by real-time PCR) 24 h later (*P < 0.05, compared with serum-free medium alone, n = 3). C: stellate cells were isolated and allowed to undergo spontaneous culture-induced activation. After 5 days in culture, Ad PPAR{gamma} was added to stellate cell cultures at the indicated multiplicity of infection (MOI) for 24 h, and immunoblotting for PPAR{gamma} was performed as in MATERIALS AND METHODS. D: PPAR{gamma}-driven PPRE promoter activity is significantly increased in AdPPAR{gamma}-transduced HSC compared with AdGFP-transduced cells as shown by the transient transfection experiment with the PPRE luciferase construct as described in MATERIALS AND METHODS. E: AdPPAR{gamma} (MOI=40) was added to the 5th day's stellate cell cultures for 24 h, mRNA from the same cells was extracted, and real-time PCR was used to detect LXR{alpha} mRNA (*P < 0.05 for infected compared with uninfected cells, n = 3). PPAR{gamma} overexpression inhibits stellate cell activation in vitro. F: stellate cells were isolated from normal rats as in MATERIALS AND METHODS and were then infected (day 1 in culture) with AdPPAR{gamma} (80 MOI). After a further 4 days in culture (maintained in DMEM with 10% FBS), immunoblotting for smooth muscle {alpha}-actin was performed as in MATERIALS AND METHODS (a total of 10 µg of total protein was used for analysis). The immunoblot shown is representative of 3 others. PPAR{gamma} overexpression inhibits TGF-beta1-induced collagen I {alpha}1 expression in isolated stellate cells. G: stellate cells were isolated from normal rats and allowed to undergo spontaneous culture-induced activation. On the 5th day of culture, cells were infected with AdPPAR{gamma} or AdGFP (both 40 MOI) in DMEM containing 0.2% FBS. After 12 h, cells were washed and maintained in DMEM without serum for another 24 h. Cells were then stimulated with TGF-beta1 (5 ng/ml) for 3 h. Cells were harvested, total mRNA was extracted, and real-time PCR to detect collagen I {alpha}1 RNA was performed (*P < 0.05 for AdPPAR{gamma} compared with AdGFP, n = 3). H: stellate cells were serum starved (0.2% serum) for 48 h to reduce the proliferation rate of cells and infected with AdGFP and AdPPAR{gamma} as in MATERIALS AND METHODS. Proliferation was induced by 10% FBS, and DNA synthesis was assessed by bromodeoxyuridine (BrdU) incorporation. Data represent 3 independent experiments (*P < 0.05 for cells exposed to AdPPAR{gamma} compared with AdGFP).

 
Stimulation of PPAR{gamma} and PPAR{gamma}-dependent transcription in stellate cells by overexpression of PPAR{gamma}. As predicted, an adenoviral vector, AdPPAR{gamma}, used to infect cultured stellate cells, resulted in enhanced PPAR{gamma} expression in a virus titer-dependent manner (Fig. 4C). We additionally wished to verify functional effects of PPAR{gamma} overexpression and therefore examined transcriptional activation after PPAR{gamma} overexpression. In cotransfection experiments using a PPRE-luciferase reporter construct, PPAR{gamma} overexpression increased PPRE promoter activity by 10-fold (Fig. 4D). Furthermore, we measured expression of LXR{alpha}, a known PPAR{gamma}-regulated gene (5, 28), after overexpression of PPAR{gamma}. LXR{alpha} mRNA levels were increased significantly compared with controls (Fig. 4E).

PPAR{gamma} overexpression inhibits stellate cell activation, collagen I {alpha}1 expression, and stellate cell DNA synthesis in vitro. We next investigated the effect of PPAR{gamma} overexpression on stellate cell activation in an isolated cell-based model. We first examined the effect of PPAR{gamma} overexpression on smooth muscle {alpha}-actin expression. Immunoblot analysis revealed that in AdPPAR{gamma}-transduced stellate cells, smooth muscle {alpha}-actin expression was significantly reduced compared with AdGFP-transduced cells (Fig. 4F). Furthermore, the effect of PPAR{gamma} overexpression on collagen I mRNA expression was also investigated (after stimulation with TGF-beta1). TGF-beta1, at a concentration of 5 ng/ml, significantly stimulated collagen I {alpha}1 mRNA expression; PPAR{gamma} overexpression abrogated this effect (Fig. 4G). To determine whether PPAR{gamma} overexpression has a role in cell proliferation, we assessed BrdU incorporation following infection of stellate cells with AdPPAR{gamma}. After introduction of serum-free conditions (which inhibits proliferation), and AdPPAR{gamma} infection, stellate cell proliferation was induced by 10% FBS. AdPPAR{gamma} inhibited DNA synthesis in culture-activated stellate cells by 49 ± 8% (Fig. 4H).

PPAR{gamma} overexpression in vivo leads to PPAR{gamma} activation, inhibition of stellate cell activation, and attenuation of liver fibrogenesis. Next, we investigated the effect of PPAR{gamma} overexpression during liver injury on liver fibrosis. AdPPAR{gamma} (1 x 1010 pfu/kg) was administered to rats undergoing BDL as in MATERIALS AND METHODS. In vivo infection of the liver with AdPPAR{gamma} led to substantial increases in whole liver PPAR{gamma} and adiponectin mRNA levels compared with AdGFP controls (Fig. 5, A and B). The elevation in adiponectin mRNA was consistent with infection of mouse liver with AdPPAR{gamma} (46). PPAR{gamma} overexpression significantly inhibited collagen I {alpha}1, TIMP-1, and TGF-beta1 mRNA expression in BDL animals compared with AdGFP-treated rats (Fig. 5, C–E), consistent with an effect of PPAR{gamma} ligands on liver fibrosis (16, 24). Notably, aminotransferases were elevated after BDL (but not in sham-operated rats), and there was no difference in aminotransferase levels between AdGFP and AdPPAR{gamma} rats (data not shown).


Figure 5
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  		Fig. 5. In vivo PPAR{gamma} overexpression during liver injury has multiple effects on the fibrogenic cascade. AdPPAR{gamma} (1 x 1010 pfu/kg) was injected via the inferior vena cava to rats during BDL as in MATERIALS AND METHODS. AdGFP was used as a negative control. Fourteen days after BDL, livers were harvested and total RNA was extracted. Real-time PCR was performed to detect PPAR{gamma} (A), adiponectin mRNA (B), collagen I {alpha}1 mRNA (C), tissue inhibitor of metalloproteinase-1 (TIMP-1) mRNA (D), and transforming growth factor-beta (TGF-beta) mRNA level (E). For A and B, *P < 0.05 for AdPPAR{gamma} compared with AdGFP and for adenovirus-infected liver cells compared with controls. For C–E, *P < 0.05 for BDL compared with sham; #P < 0.05 AdPPAR{gamma} BDL compared with AdGFP BDL.

 
Given the data indicating that AdPPAR{gamma} overexpression in the culture model of stellate cell activation was inhibitory, we hypothesized that AdPPAR{gamma} overexpression in vivo may likewise reduce stellate cell activation; thus we investigated the expression of smooth muscle {alpha}-actin [a well-known marker of activation (40)] in injured livers and after overexpression of PPAR{gamma}. Smooth muscle {alpha}-actin expression was markedly increased in AdGFP-treated rats subjected to prolonged BDL (Fig. 6A) but was attenuated in AdPPAR{gamma}-treated rats (Fig. 6B). Immunoblot analysis also demonstrated a marked increase in smooth muscle {alpha}-actin in AdGFP-treated rats and substantial inhibition of this response by overexpression of PPAR{gamma} (Fig. 6C). Additionally, when isolated stellate cells from these rats were analyzed, smooth muscle {alpha}-actin content was specifically reduced in PPAR{gamma}-overexpressing cells (Fig. 6D). These data, in the context of those above demonstrating direct effects of PPAR{gamma} overexpression on isolated stellate cells, suggest that PPAR{gamma} acts in vivo via effects on stellate cells.


Figure 6
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  		Fig. 6. PPAR{gamma} overexpression in vivo reduces stellate cell activation. Rats were exposed to AdPPAR{gamma} and AdGFP, and BDL was performed as in Fig. 5. Smooth muscle {alpha}-actin immunostaining (A and B) was performed as described in MATERIALS AND METHODS. A: representative liver section from a rat exposed to AdGFP. B: representative liver section from a rat exposed to AdPPAR{gamma}. Controls in which specimens were exposed to 1% bovine serum albumin instead of primary antibody did not reveal specific labeling (not shown). Bar in lower right of B represents 100 µm. C: whole liver specimens were lysed and subjected to immunoblot analysis to detect smooth muscle {alpha}-actin as described in MATERIALS AND METHODS (50 µg tissue); quantitative data are presented (*P < 0.05 for AdPPAR{gamma} compared with AdGFP, n = 6). D: stellate cells were isolated from rats 14 days after BDL and subsequent AdPPAR{gamma} and AdGFP exposure, and total cell lysates were subjected to immunoblot analysis to detect for smooth muscle {alpha}-actin as in MATERIALS AND METHODS; immunoblotting for beta-actin was performed and utilized as an internal loading control (the immunoblot shown is representative of 3 others). Data were scanned and quantitated and expressed graphically (bottom) (*P < 0.05 for AdPPAR{gamma} compared with AdGFP, n = 4).

 
After liver injury induced by BDL, AdGFP-treated rat livers were characterized by severe periductal fibrosis, lobular fibrosis, and disruption of the hepatic architecture (Fig. 7A). AdPPAR{gamma}-treated rats displayed lower collagen deposition with less prominent fibrosis (Fig. 7B). Hepatic collagen content, as assessed by morphometric analysis and by measurement of hepatic hydroxyproline content, was markedly increased after BDL (Fig. 7, C and D). In addition, the increase in hepatic collagen and hydroxyproline content in AdPPAR{gamma}-treated BDL rats was attenuated compared with AdGFP-treated BDL animals (Fig. 7, C and D).


Figure 7
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  		Fig. 7. PPAR{gamma} overexpression in vivo attenuates liver fibrosis. Rats were exposed to AdPPAR{gamma} and AdGFP, and BDL was performed as in Fig. 5 above. Sirius red staining (A and B) was performed as described in MATERIALS AND METHODS. A: representative liver section from a rat exposed to AdGFP. B: representative liver section from a rat exposed to AdPPAR{gamma}. Bar in lower right of B represents 100 µm. C: the area stained by Sirius red in the depicted groups was quantitated as described in MATERIALS AND METHODS (*P < 0.05 for BDL compared with sham; #P < 0.05 for AdPPAR{gamma} BDL compared with AdGFP BDL; n = 10 fields per liver, and 10 livers per group). D: hydroxyproline was measured in the livers of specific groups (*P < 0.05 for BDL compared with sham; #P < 0.05 for AdPPAR{gamma} BDL compared with AdGFP BDL; n = 12).

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
In this report, we have provided definitive evidence that manipulation of PPAR{gamma}, a nuclear receptor transcriptional factor superfamily member, in stellate cells during their activation results in modulation of phenotypic features of activation. We found that PPAR{gamma} inhibition and overexpression had consistent effects both in culture models of activation and during in vivo liver injury (i.e., after BDL). Biological overexpression or depletion of PPAR{gamma} in the present study is an important and specific approach to the investigation of the role of PPAR{gamma} that augments the pharmacological findings of other investigators (16, 24). The pharmacological investigations using high doses of PPAR{gamma}-activating compounds can be potentially misleading because off-target effects of those compounds. However, together with the findings in the present study using specific molecular approaches, a compelling case can be made for the role of PPAR{gamma} in stellate cell activation and fibrosis.

Our data suggest several potential mechanisms whereby differential expression of PPAR{gamma} may affect hepatic fibrogenesis in vivo. First, we demonstrated a specific effect of PPAR{gamma} overexpression on type I collagen mRNA in isolated stellate cells (Fig. 4). Thus it is possible that the PPAR{gamma} pathway has a direct effect on transcriptional regulation of the type I collagen gene in stellate cells (see also below). Alternatively, PPAR{gamma} may have a global effect on stellate cell activation, evidenced by effects on both smooth muscle {alpha}-actin expression and proliferation (Fig. 4). Thus it is possible that PPAR{gamma} overexpression in vivo leads to reduced numbers of activated stellate cells, consistent with the finding of a reduction in the number of smooth muscle {alpha}-actin-positive cells in bile duct-ligated rats overexpressing PPAR{gamma} in liver (Fig. 6). This finding is consistent with previous data in which PPAR{gamma} ligands have been shown to inhibit proliferation of isolated cultured stellate cells (34). Therefore, it is likely that the "antifibrogenic" effect of PPAR{gamma} overexpression in vivo is due, at least in part, to a decreased accumulation of activated/fibrogenic stellate cells.

The molecular mechanisms responsible for the effect of PPAR{gamma} ligands remain controversial. First, an important consideration in stellate cells has to do with the endogenous ligand(s) in stellate cells. That fibrogenesis can be modulated (either up or down) by manipulation of PPAR{gamma} has implications for the biology of PPAR{gamma} in stellate cells and the liver. On one hand, PPAR{gamma} has been shown to have broad ranging biological effects. For example, PPAR{gamma} is intimately involved in lipid metabolism, inflammation, and development (1, 2, 6, 31, 43). On the other hand, a critical point made by our findings is that the PPAR{gamma} receptor itself is important specifically in stellate cell biology and in liver fibrogenesis.

An important issue in PPAR{gamma} biology is that of the effects of PPAR{gamma} ligands on specific cellular signaling pathways. Interestingly, PPAR{gamma} ligands appear to signal via receptor-dependent as well as independent pathways. For example, overexpression of a dominant-negative form of PPAR{gamma} did not alter 15d-PGJ2-induced inhibition of lipopolysaccharide/IFN-{gamma}-mediated inducible nitric oxide synthase and NF-{kappa}B activation (17). In addition, induction of inducible nitric oxide synthase by PPAR{gamma} agonists in RAW 264.7 macrophages was independent of PPAR{gamma} (7). Our results suggest that at least some of the effects of PPAR{gamma} on fibrogenesis are due to endogenous ligands and intrinsic signaling pathways.

In terms of specific molecular mechanisms, it is possible that the PPAR{gamma} system has immediate downstream effects or that its effects are due to cross talk with other systems. For example, recent data suggest that a physical interaction between PPAR{gamma} and JunD in stellate cells suppresses AP-1 activity and that this latter feature may inhibit stellate activation (18). Additionally, PPAR{gamma} appears to bind to SMAD3 and inhibits TGF-beta signaling, an event that could be important in fibrogenesis (15). It is also possible that PPAR{gamma} may act via other pathways. For example, PPAR{gamma} stimulation activates LXR, which has been shown to have anti-inflammatory effects (22). Additionally, there appears to be cross talk between the farnesoid X receptor and PPAR{gamma} systems in liver (10). Finally, PPAR{gamma} induces the expression of adiponectin, a protein that has recently been shown to be involved in hepatic fibrosis (23). Indeed, we found that infection of the liver with AdPPAR{gamma} led to a significant increase in hepatic adiponectin mRNA. Not only did this verify that AdPPAR{gamma} targeted the liver as predicted, but it also raises the possibility that the antifibrotic effect identified could have been through adiponectin's effect on stellate cells. In support of this possibility, we have identified putative adiponectin receptors on stellate cells (unpublished observation).

We did not evaluate in detail whether the biological and molecular approaches used here may lead to reversal of fibrosis. This would be desirable therapeutically in humans. It is noteworthy that pharmacological approaches have not consistently shown that activation of PPAR{gamma} after the induction of liver injury reverses fibrosis (29). For example, when pioglitazone treatment was initiated early after carbon tetrachloride injury, an antifibrotic effect was seen, but when the compound was administered late after injury, no antifibrotic effect was observed (29). Similarly, pioglitazone was associated with a reduced severity of fibrosis induced by a choline-deficient diet when introduced early, whereas delayed treatment with pioglitazone remained ineffective. We have found in preliminary experiments also that manipulation of PPAR{gamma} after the induction of liver injury has little effect on stellate cell fibrogenesis (unpublished observation). These data suggest that if PPAR{gamma} (expression or activation) is to be manipulated in patients with liver disease, the earlier this can be done in the course of the disease, the better.

An important consideration with regard to the in vivo work presented here has to do with the fact that we used adenovirus to target the liver (and presumably stellate cells). We used adenovirus to express Cre recombinase as well as PPAR{gamma} in the injured liver. It is well appreciated that adenovirus infects hepatocytes (20). Additionally, it has also been demonstrated that adenovirus effectively infects nonparenchymal cells (45). Indeed, it has been previously shown in the fibrotic liver that stellate and endothelial cells were infected with greater efficiency than were hepatocytes (45). We cannot exclude the possibility that adenovirus infecting cells other than stellate cells in vivo could have had biological effects on PPAR{gamma} and thus on the overall inflammatory response. Nonetheless, the data support the hypothesis that overexpression of PPAR{gamma} using adenovirus reverses the PPAR{gamma} "depleted" phenotype typical of stellate cells during and perhaps to some extent after their activation and liver injury.

Finally, the data from this study point to the possibility that the PPAR{gamma} system could be manipulated to provide therapeutic benefit in patients with liver fibrosis. In a study of 30 adults with nonalcoholic steatohepatitis, rosiglitazone was administered for 48 wk, leading to a reduction in inflammation, hepatocellular ballooning, and zone 3 perisinusoidal fibrosis (36), although weight gain was noted in a group of patients. A subsequent study in patients with nonalcoholic steatohepatitis using pioglitazone had similar effects (38). Importantly, the thiazolidinediones are known to have a number of important side effects in humans, and these must be carefully considered if used in patients with liver disease. Many questions remain, and future studies will be required to define the optimal target or combination of targets to maximize antifibrotic effects with a minimum of unwanted side effects.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported in part by the Burroughs Welcome Fund (D. C. Rockey is the recipient of a BWF Translational Scientist Award).


   ACKNOWLEDGMENTS
 
We thank Rajender Reddy (Northwestern University) for the kind gift of the PPAR{gamma} adenovirus.


   FOOTNOTES
 

Address for reprint requests and other correspondence: D. C. Rockey, Univ. of Texas Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, TX 75390-8887 (e-mail: don.rockey{at}utsouthwestern.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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