Adenoviral expression of a transforming growth factor-β1 antisense mRNA is effective in preventing liver fibrosis in bile-duct ligated rats
© Arias et al; licensee BioMed Central Ltd. 2003
Received: 14 July 2003
Accepted: 18 October 2003
Published: 18 October 2003
Transforming growth factor-β (TGF-β) is a key mediator in establishing liver fibrosis. Therefore, TGF-β as a causative agent may serve as a primary target for antifibrotic gene therapy approaches. We have previously shown that the adenoviral delivery of a transgene constitutively expressing a TGF-β1 antisense mRNA blocks TGF-β synthesis in culture-activated hepatic stellate cells and effectively abolishes ongoing fibrogenesis in vitro.
Ligature of the common bile duct was used to induce liver fibrosis in rats. The effect of the TGF-β1 antisense on fibrogenesis was analyzed in this model of liver injury.
In the present study, we demonstrate that the adenoviral vector directs the synthesis of mRNA quantities that are approximately 8000-fold more abundant than endogenous TGF-β1 mRNA. In experimentally injured rat livers induced by ligature of the common bile duct, a model for persistent fibrogenesis and cirrhosis, administration of the adenoviral vector abrogates TGF-β-enhanced production of collagen and α-smooth muscle actin. Furthermore, the number of cells positive for α-smooth muscle actin resulting from active recruitment of activated hepatic stellate cells around the bile ductular structures was significantly reduced in animals after application of Ad5-CMV-AS-TGF-β1. However, the observed elevated serum levels of aspartate aminotransferase, alanine aminotransferase, and bilirubin induced in this obstructive liver injury model were not significantly altered in the presence of the TGF-β antagonist.
Taken together, our data provides in vivo evidence that the delivery of TGF-β1 antisense mRNA specifically abolishes the diverse effects of direct TGF-β function in ongoing liver fibrogenesis. Therefore, we conclude that the expressed transgene is therapeutically useful for inhibition of TGF-β effects in diverse applications, ranging from clarification of TGF-β function in the course of liver injury to the development of novel gene therapeutic approaches.
Transforming growth factor-β1 (TGF-β1) is a multifunctional cytokine involved in the regulation of cell proliferation, differentiation, extracellular matrix production, wound healing and tissue repair . In liver fibrogenesis, TGF-β is of crucial importance triggering excessive formation and deposition of connective tissue matrix molecules . Typically, during hepatic injury resting hepatic stellate cells (HSC) undergo cellular activation which in term is associated with proliferation, increased contractile activity, fibrogenesis, changes in matrix protease activity, loss of intracellular retinoid storage, production of cytokines, and phenotypic transformation to a myofibroblast-like morphology . TGF-β binds and signals through distinct heteromeric transmembrane receptors, including type I (TβRI) and type II (TβRII) serine/threonine kinase receptors . Activation of this complex is initiated by binding of TGF-β to TβR-II triggering heteromerization with and transphosphorylation of TβRI. The signal is then propagated through phosphorylation of receptor associated Smad2 and 3 and oligomerization with the common mediator Smad4. Complexes of phosphorylated Smad2 or 3 and Smad4 translocate into the nucleus, where they affect transcription of target genes via direct DNA binding or by association with numerous DNA binding proteins [5, 6]. Aberrant expression of TGF-β is involved in a number of disease processes, including fibrosis and inflammation. This is demonstrated in transgenic mice, which develop multiple tissue lesions including hepatic fibrosis as a consequence of elevated levels of TGF-β [7–9]. This evidence provides a rationale for targeting TGF-β as an antifibrotic agent. In the last decade, significant advances in cell biology have opened several ways to inhibit TGF-β action. One experimental approach to block TGF-β signaling is the local expression of a soluble, dominant negative TβRII . During liver injury, this strategy is appropriate to prevent progression of fibrosis, to inhibit matrix synthesis and to decrease cell proliferation [11–13] indicating that prevention of fibrosis through anti-TGF-β treatment could have some future therapeutic value. Treatment with short DNA antisense oligonucleotides was shown to suppress TGF-β1 function in an interstitial fibrosis model and in balloon catheter injury [14, 15]. Impressively, overexpression of antagonistic Smad7, a natural antagonist of TGF-β signaling was sufficient to prevent bleomycin-induced pulmonary fibrosis in mice .
We have recently demonstrated that adenoviral delivery of an antisense RNA complementary to the 3' coding sequence of rat TGF-β1 is able to suppress the synthesis of TGF-β1 in culture-activated rat HSC . The adenoviral vehicle directs high-level expression of the transgene and the transduced antisense was found to block TGF-β synthesis as assessed by immunoprecipitation, western blot analysis, quantitative TGF-β1 ELISA, and cell proliferation assays. Moreover, we found that the construct was able to induce differential activity of TGF-β1 responsive genes indicating that the delivery of this mRNA, complementary to endogenous TGF-β transcript, offers a feasible approach to block TGF-β1 signaling in this experimental in vitro model for liver fibrogenesis .
In the present study, we demonstrate that infection with Ad5-CMV-AS-TGF-β1 induces cellular mRNA quantities that are approximately 8000-fold abundant over endogenous TGF-β1 mRNA. In rats with ligature of the common bile duct (BDL), an experimental model of liver fibrogenesis, the administration of the adenoviral vector abrogates the production of collagen and α-smooth muscle actin (α-SMA) but has no significant impact on serum levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), or bilirubin. Taken together, our data gives evidence that the transfer of the TGF-β1 antisense is sufficient to specifically abolish ongoing liver fibrogenesis but does not interfere with the injury per se.
Isolation and culture of liver cells
Hepatic stellate cells (HSC) were isolated from male Sprague-Dawley rats following a standard procedure with slight modifications [18, 19]. Briefly, livers were perfused with pronase and collagenase and the resulting cell suspensions were filtered through a nylon mesh, centrifuged and washed in ice cold Hanks buffered standard saline (HBSS; PAA Laboratories GmbH, Linz, Austria) containing 0.25% (w/v) BSA. HSC were further purified by a single-step density gradient centrifugation with 8.25% (w/v) Nycodenz® (Nycomed Pharma, Oslo, Norway) as described in detail elsewhere [20, 21] and seeded in Dulbeccos's modified Eagle medium (DMEM; Bio Whittaker Europe, Verviers, Belgium) supplemented with 10% (v/v) fetal calf serum (FCS; Seromed, Biochrom KG, Berlin, Germany), and 4 mM L-glutamine (ICN Biomedicals Inc., Aurora, Ohio). Additionally, the culture medium was supplemented with penicillin (100 IU/ml) and streptomycin (100 μg/ml).
RNA isolation and northern blot analysis
Isolation and Northern blot analysis of total cellular RNA from HSC was carried out as described previously . Briefly, equal amounts (5 μg) of total RNA were separated by electrophoresis on a 1.2% (w/v) denaturing agarose gel, transferred to a Hybond-N membrane (Amersham Pharmacia, Braunschweig, Germany), and fixed by baking for 2 hours at 80°C. Blots were hybridized with [α-32P]-dCTP-labeled random primed probes (Amersham) and autoradiographs were exposed to Kodak X-OMAT AR films using intensifying screens. As an internal standard for equal gel loading the blots were re-hybridized with a cDNA specific for glyceraldehyde-3-phosphate dehydrogenase (GAPDH).
SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting
Whole cell protein extracts were prepared in RIPA buffer [20 mM Tris-HCl (pH 7.2), 0.15 M NaCl, 2% (v/v) NP-40, 0.1% (w/v) SDS, 0.5% sodium deoxycholate] and concentrations were quantified using the Micro BCA protein assay reagent kit (Pierce, Rockford, IL). Equal amounts of protein were resolved by reducing SDS-PAGE (Novex, Groningen, The Netherlands). For immunoblotting, proteins were electroblotted onto nitrocellulose membranes. Membrane blocking and incubation with antibodies were performed as described previously . α-SMA was detected with the monoclonal mouse antibody clone asm-1 (Roche Diagnostics, Mannheim, Germany) followed by incubation with horseradish peroxidase (HRP)-conjugated anti-mouse IgG (Santa Cruz Biotech., Santa Cruz, CA) and the supersignal chemiluminescent substrate (Pierce).
Adenoviral vector construction and virus purification
The recombinant E1-deleted adenoviral vectors constitutively expressing the enhanced green fluorescent protein (EGFP) or rat antisense TGF-β1 were generated as described previously [17, 22]. Adenoviral particles were purified by a standard CsCl density protocol followed by a membrane based ion exchange chromatography for purification of adenoviral particles (BD Bioscience, CLONTECH, Palo Alto, CA). Adenoviral titers were spectrometrically determined and appropriate aliquots were stored at -80°C until use.
Experimental model of liver fibrosis
Male Sprague-Dawley rats (245.6 ± 10.8 g) were injected twice (at day 0 and day 7) with recombinant adenoviruses (1 × 1010 pfu/kg) or phosphate buffered saline via the tail vein. At day two, the common bile ducts were double ligated under halothane anesthesia. Rats were sacrificed after 12 days, and pieces of the livers were fixed in 10% formalin for histological examination or frozen immediately in liquid nitrogen and stored at -80°C for RNA isolation. Measurements of AST, ALT, and bilirubin were performed from blood samples following standard protocols. The study as presented is in compliance with the German Animal Protection Act, and was approved by the local committee for care and use of laboratory animals at the RWTH-University Hospital. Experiments applying adenoviral constructs to cells or animals are covered by permission of the Landesumweltamt Nordrhein-Westfalen (Az. 521-K-1.59/99).
Histological scoring of liver fibrosis in rats
Morphological evaluation of induced liver fibrogenesis was performed using the semi-quantitative fibrotic focus score proposed for staging and grading of histopathological lesion of chronic hepatitis in humans . Briefly, pieces of left liver lobules were fixed in 10% formalin and stained with hematoxylin/eosine for identification of lesions. In the stained sections, the lesions were defined as follows: absence of lesions = 0; occasional small localized lesions with fibrous expansion of some portal areas = 1; thickening of liver septa with fibrous expansion of most portal areas = 2; and thickened continuous fibrous areas with periportal rounding and occasional portal to portal bridging = 3. The liver sections were coded and independently examined in a blinded manner by two different pathologists.
Liver tissues were fixed in 4% paraformaldehyde and embedded in paraffin and 1.5-μm sections were prepared. For immunohistochemistry, the sections were treated with xylene and rehydrated with decreasing graded ethanol washes. For α-SMA staining, the slides were treated with a polyclonal anti-rat α-SMA (Roche Diagnostics) followed by HRP-conjugated secondary anti-rabbit antibody (DAKO, Hamburg, Germany). α-SMA staining was detected using the DAB substrate (Vector Laboratories, Burlingame, CA) and sections were counterstained with eosine.
Sirius red staining
Collagen staining was performed as described previously . Briefly, liver sections were deparaffinized and the slides were incubated for 30 min in a solution of saturated picric acid containing 0.1% direct red 80 and 0.1% Fast Green FCF. Stained slides were washed in running distilled water, dehydrated, mounted, and examined by light microscopy.
Quantitative real-time PCR
Total RNA (2 μg) isolated from untreated HSC, and HSC infected with Ad5-CMV-EGFP or Ad5-CMV-AS-TGF-β1 was reversed transcribed in 20 μl using the SuperscriptII Reverse Transcriptase (Invitrogen, Karlsruhe, Germany) and random hexamer primer according to the manufacturer's instructions. 2-μl aliquots of first strand cDNA samples were subjected to PCR in a volume of 20 μl using 2 μM forward and reverse primer, 2 μl dNTPs (each 10 mM dATP, dCTP, dGTP, dTTP), 1x PCR reaction buffer, and 2.5 U Taq DNA polymerase (Roche Diagnostics). All PCR-assays were conducted in capillaries on a LightCycler system (20 μl-reaction volume) using the LC-FastStart DNA Master SYBR Green I kit (Roche Diagnostics). Conditions were: 95°C for 10 min (initial denaturation), 95°C for 10 s, 66°C for 5 s, 72°C for 15 s (45 cycles) and 72°C for 1 min (final extension). Primer P1 [5'-d-(TGG CGT TAC CTT GGT AAC C)-3'] and P2 [5'-d(GGT GTT GAG CCC TTT CCA G)-3'] directed the amplification of a 277-bp fragment of endogenous TGF-β1. The same PCR conditions with primer P3 [5'-d(CAA GGT CCT TGC CCT CTA)-3'] and P4 [5'-d(GCG CAC AAT CAT GTT GGA CA)-3'] annealing to both endogenous TGF-β1 and antisense TGF-β1 were taken to amplify a 145-bp fragment. For amplification of AS-TGF-β1 specific transcripts we used primer P5 [5'-d(CGG TGA TGC GGA AGC ACC CG)-3'] and P6 [5'-d(CCT CTA CAA ATG TGG TAT GG)-3'] directing the amplification of a hybrid amplicon containing sequences specific for TGF-β1 and the downstream SV40-polyadenylation signal. After PCR, products were melted in a temperature transition procedure from 65°C to 95°C in steps of 0.1°C/s and fluorescence signals were measured and plotted online against the temperature. The second derivative maximum method was taken to determine the crossing points automatically for individual samples and relative amounts of target gene was calculated based on the crossing point analysis (LC-software, version 5.32).
High-level expression and antifibrotic capacity of the antisense TGF-β1 device
Ligature of the common bile buct induces fibrosis in rat
Gene transfer of the antisense construct directed against TGF-β1 prevents liver fibrosis in the common bile duct ligature model
In previous reports, the elimination of TGF-β signaling by adenovirus-mediated local expression of a dominant-negative type II TGF-β receptor in the liver of rats treated with dimethylnitrosamine was associated with serum levels of liver specific transaminases comparable to normal controls . When we measured AST and ALT in the serum of sacrificed rats which received BDL and gene transfer, the levels of these hepatic enzymes were the same regardless of whether the BDL-treated rats were infused with saline, Ad5-CMV-EGFP, or Ad5-CMV-AS-TGF-β1 indicating that the hepatic injury induced by BDL per se is not influenced by TGF-β under the chosen experimental conditions.
In liver fibrogenesis TGF-β is the most potent profibrogenic signaling factor triggering expression, accumulation and deposition of collagen . Elevated levels of TGF-β are associated with altered lobular organisation, increase of hepatocyte turnover and apoptosis [7–9]. Therefore, mechanisms antagonizing TGF-β function or blocking TGF-β synthesis are primary targets for directing antifibrotic therapies. In this view several approaches were identified to abolish TGF-β signaling. These include administration of antioxidants, specific drugs, herbal compounds, neutralizing antibodies, TGF-β binding proteins (scavengers), antagonistic cytokines, suppressors of apoptosis, or specifically designed oligonucleotides . Another promising approach is the inhibition of proteolytic release and activation of latent TGF-β. Representative, the serine protease inhibitor camostat mesilate was recently found to suppress HSC activation by inhibiting hepatic plasmin activity and thereby preventing hepatic fibrosis . Presently, potential gene therapies using dominant negative or soluble TβR-II are under close investigation. Because TβR-II is the primary binding receptor for TGF-β, overexpression of an inactive TβR-II construct counters TGF-β actions. The development of hepatic fibrosis by dimethylnitrosamine (DMN) in rats was markedly reduced by adenoviral vectors expressing either a truncated human TβR-II injected via the portal vein  or soluble human TGF-β receptors (a chimeric protein between an entire ectodomain of human TβR-II and the Fc portion of human immunoglobulin G) injected intramuscularly . Impressively, a single injection of adenovirus expressing the truncated receptor, given prior to DMN administration, appeared to prevent both hepatic injury and the development of hepatic fibrosis. In a subsequent study, the same adenoviral vector was administered to animals with ongoing fibrosis after 3 weeks of DMN in order to determine whether reversal of fibrosis occurs with this agent. The results were similar with lack of progression and possibly some regression of hepatic fibrosis in rats that received the dominant negative receptor . The antifibrogenic potential of soluble TβR-II was also demonstrated in the rat bile duct ligature model by slow infusion of the chimeric proteins into the femoral vein . Another option to inhibit TGF-β function is to interfere with postreceptor signaling. Overexpression of Smad7, a natural antagonist of TGF-β signaling, prevents bleomycin induced pulmonary fibrosis in mice  and was recently shown to prevent activation of hepatic stellate cells and liver fibrosis in rats .
The present work evaluates the potency of an antisense mRNA complementary to the 3' coding sequence of TGF-β1 in preventing liver fibrogenesis during obstructive bile duct ligature in rat. The efficacy of the expressed antisense to block TGF-β synthesis in culture-activated HSC was previously shown by immunoprecipitation, Western blot, quantitative TGF-β1 ELISA, and cell proliferation assays . We here demonstrate that the in vitro synthesized quantities of the expressed transgene delivered by an adenoviral vector exceed endogenous levels of TGF-β1 mRNAs by several orders of magnitude. To verify the efficacy of the antisense we determined collagen and α-SMA as markers in an experimental in vivo model of liver fibrogenesis. In this experimental model of obstructive hepatic injury, damage was induced by ligature of the common bile duct leading to a drastic accumulation of collagen content in the liver. Delivery of the antisense complementary to TGF-β1 mRNA prevented the deposition of collagen as assessed by histochemistry using Sirius red staining. Further, in animals receiving the transgene, we found also reduced expression of α-SMA in histological sections and protein extracts analyzed by western blot. Previous studies examined the role of TGF-β inhibition on hepatic fibrogenesis induced by bile duct ligation, using soluble TβRII constructs sequestering TGF-β. In one report it was demonstrated that the soluble receptor was able to reduce collagen and α-SMA quantities when given at the time of injury or given at day 4 after injury . The authors concluded that soluble TGF-β receptor is an effective inhibitor in vivo and merits clinical evaluation as a novel agent for controlling hepatic fibrosis in chronic liver injury . However, the chimeric fusion consisting of the extracellular domain of TβRII with the Fc portion of an immunoglobulin might create potent epitopes and thereby preventing its repetitive application for longer time periods. Therefore, the finding that an antisense complementary to endogenous TGF-β1 mRNA has the same beneficial antifibrotic capacity may offer considerable promise to alleviate TGF-β induced damages during chronic liver diseases and increase the safety of future intervention strategies by diminishing the risk of avoidable immune responses.
However, we have not yet analyzed the consequences of long-term systemic exposure to the TGF-β antisense, particular in regard to systemic effects like body weight, deterioration of a clinical status, or the likelihood of increased spontaneous tumorigenesis and immune system dysfunctions. Moreover, we have no estimation of how much active TGF-β is still present in liver cells of Ad5-CMV-AS-TGF-β1-infected animals. Complementary future studies are necessary to address these critical issues.
We conclude that our data gives strong evidence that the transfer of the designed TGF-β1 antisense is sufficient to specifically abolish the diverse effects of TGF-β in ongoing hepatic fibrogenesis. Therefore, the antisense strategy is attractive as a therapeutic agent for preventing stellate cell activation and may help clarify the role of TGF-β in this process both in vitro and in vivo. Moreover, the antisense technology may provide opportunities for the treatment of chronic liver diseases.
This work was supported in part by grants from the Federal Ministry of Education and Research of Germany (BMBF, IZKF Biomat) and Aachen University (START project Identification of Molecular Markers and Gene Therapy of Fibrosis and Wound Healing) to RW. We thank H. P. Fischer and C. Esch for help with the immunohistochemistry.
List of abbreviations
adenovirus serotype 5,
α-smooth muscle actin,
bile duct ligature,
Dulbecco's modified Eagle medium,
fetal calf serum,
- GAPDH :
enhanced green fluorescent protein,
Hanks buffered standard saline,
hepatic stellate cell(s),
polymerase chain reaction,
transforming growth factor-β1.
- Moustakas A, Pardali K, Gaal A, Heldin CH: Mechanisms of TGF-β signalling in regulation of cell growth and differentiation. Immunol Lett. 2002, 82: 85-91. 10.1016/S0165-2478(02)00023-8.View ArticlePubMedGoogle Scholar
- Gressner AM, Weiskirchen R, Breitkopf K, Dooley S: Roles of TGF-β in hepatic fibrosis. Front Biosci. 2002, 7: D793-D807.View ArticlePubMedGoogle Scholar
- Friedman SL: Molecular regulation of hepatic fibrosis, an integrated cellular response to tissue injury. J Biol Chem. 2000, 275: 2247-2250. 10.1074/jbc.275.4.2247.View ArticlePubMedGoogle Scholar
- Heldin CH, Miyazono K, ten Dijke P: TGF-β signalling from cell membrane to nucleus through SMAD proteins. Nature. 1997, 390: 465-471. 10.1038/37284.View ArticlePubMedGoogle Scholar
- Piek E, Heldin CH, ten Dijke P: Specificity, diversity, and regulation in TGF-beta superfamily signaling. FASEB J. 1999, 13: 2105-2124.PubMedGoogle Scholar
- Miyazono K, ten Dijke P, Heldin CH: TGF-β signalling by Smad proteins. Adv Immunol. 2000, 75: 115-157.View ArticlePubMedGoogle Scholar
- Sanderson N, Factor V, Nagy P, Kopp J, Kondaiah P, Wakefield L, Roberts AB, Sporn MB, Thorgeirsson SS: Hepatic expression of mature transforming growth factor beta 1 in transgenic mice results in multiple tissue lesions. Proc Natl Acad Sci USA. 1995, 92: 2572-2576.View ArticlePubMedPubMed CentralGoogle Scholar
- Clouthier DE, Comerford SA, Hammer RE: Hepatic fibrosis, glomerulosclerosis, and a lipodystrophy-like sindrome in PEPCK-TGF-β1 transgenic mice. J Clin Invest. 1997, 100: 2697-2713.View ArticlePubMedPubMed CentralGoogle Scholar
- Kanzler S, Lohse AW, Keil A, Henninger J, Dienes HP, Schirmacher P, Rose-John S, zum Buschenfelde KH, Blessing M: TGF-beta 1 in liver fibrosis: an inducible transgenic mouse model to study liver fibrogenesis. Amer J Physiol. 1999, 276: G1059-G1068.PubMedGoogle Scholar
- Komesli S, Vivien D, Dutartre P: Chimeric extracellular domain of type II transforming growth factor (TGF)-β receptor fused to the Fc region of human immunoglobulin as a TGF-β antagonist. Eur J Biochem. 1998, 254: 505-513. 10.1046/j.1432-1327.1998.2540505.x.View ArticlePubMedGoogle Scholar
- Qi Z, Atsuchi N, Ooshima A, Takeshita A, Ueno H: Blockade of type β transforming growth factor signaling prevents liver fibrosis and dysfunction in the rat. Proc Natl Acad Sci USA. 1999, 96: 2345-2349. 10.1073/pnas.96.5.2345.View ArticlePubMedPubMed CentralGoogle Scholar
- Nakamura T, Sakata R, Ueno T, Sata M, Ueno H: Inhibition of transforming growth factor β prevents progression of liver fibrosis and enhances hepatocyte regeneration in dimethylnitrosamine-treated rats. Hepatology. 2000, 32: 247-255.View ArticlePubMedGoogle Scholar
- Ueno H, Sakamoto T, Nakamura T, Qi Z, Astuchi N, Takeshita A, Shimizu K, Ohashi H: A soluble transforming growth factor β receptor expressed in muscle prevents liver fibrogenesis and dysfunction in rats. Hum Gene Ther. 2000, 11: 33-42. 10.1089/10430340050016139.View ArticlePubMedGoogle Scholar
- Isaka Y, Tsujie M, Ando Y, Nakamura H, Kaneda Y, Imai E, Hori M: Transforming growth factor-β1 antisense oligodeoxynucleotides block interstitial fibrosis in unilateral ureteral obstruction. Kidney Int. 2000, 58: 1885-1892. 10.1046/j.1523-1755.2000.00360.x.View ArticlePubMedGoogle Scholar
- Merrilees M, Beaumont B, Scott L, Hermanutz V, Fennessy P: Effects of TGF-β (1) antisense S-oligonucleotide on synthesis and accumulation of matrix proteoglycans in balloon catheter-injury neointima of rabbit carotid arteries. J Vasc Res. 2000, 37: 50-60. 10.1159/000025713.View ArticlePubMedGoogle Scholar
- Nakao A, Fujii M, Matsumura R, Kumano K, Saito Y, Miyazono K, Iwamoto I: Transient gene transfer and expression of Smad7 prevents bleomycin-induced lung fibrosis in mice. J Clin Invest. 1999, 104: 5-11.View ArticlePubMedPubMed CentralGoogle Scholar
- Arias M, Lahme B, Van de Leur E, Gressner AM, Weiskirchen R: Adenoviral delivery of an antisense RNA complementary to the 3' coding sequence of transforming growth factor-β1 inhibits fibrogenic activities of hepatic stellate cells. Cell Growth Differ. 2002, 13: 265-273.PubMedGoogle Scholar
- de Leeuw AM, McCarthy SP, Geerts A, Knook DL: Purified rat liver fat-storing cells in culture divide and contain collagen. Hepatology. 1984, 4: 392-403.View ArticlePubMedGoogle Scholar
- Fehrenbach H, Weiskirchen R, Kasper M, Gressner AM: Up regulated expression of the receptor for advanced glycation end products in cultured rat hepatic stellate cells during transdifferentiation to myofibroblasts. Hepatology. 2001, 34: 943-952. 10.1053/jhep.2001.28788.View ArticlePubMedGoogle Scholar
- Schafer S, Zerbe O, Gressner AM: The synthesis of proteoglycans in fat-storing cells of rat liver. Hepatology. 1987, 7: 680-687.View ArticlePubMedGoogle Scholar
- Gressner AM, Zerbe O: Kupffer cell-mediated induction of synthesis and secretion of proteoglycans by rat liver fat-storing cells in culture. J Hepatol. 1987, 5: 299-310.View ArticlePubMedGoogle Scholar
- Weiskirchen R, Kneifel J, Weiskirchen S, van de Leur E, Kunz D, Gressner AM: Comparative evaluation of gene delivery devices in primary cultures of rat hepatic stellate cells and rat myofibroblasts. BMC Cell Biol. 2000, 1: 4-10.1186/1471-2121-1-4.View ArticlePubMedPubMed CentralGoogle Scholar
- Brunt EM: Grading and staging the histopathological lesions of chronic hepatitis: the Knodell histology activity index and beyond. Hepatology. 2000, 31: 241-246.View ArticlePubMedGoogle Scholar
- Lopez-De Leon A, Rojkind M: A simple micromethod for collagen and total protein determination in formalin-fixed paraffin-embedded sections. J Histochem Cytochem. 1985, 33: 737-743.View ArticlePubMedGoogle Scholar
- Accatino L, Contreras A, Fernandez S, Quintana C: The effect of complete biliary obstruction on bile flow and bile acid excretion: postcholestatic choleresis in the rat. J Lab Clin Med. 1979, 93: 706-717.PubMedGoogle Scholar
- Accatino L, Contreras A, Berdichevsky E, Quintana C: The effect of complete biliary obstruction on bile secretion. Studies on the mechanisms of postcholestatic choleresis in the rat. J Lab Clin Med. 1981, 97: 525-534.PubMedGoogle Scholar
- Kountouras J, Billing BH, Scheuer PJ: Prolonged bile duct obstruction: a new experimental model for cirrhosis in the rat. Br J Exp Pathol. 1984, 65: 305-311.PubMedPubMed CentralGoogle Scholar
- Yu Q, Que LG, Rockey DC: Adenovirus-mediated gene transfer to nonparenchymal cells in normal and injured liver. Am J Physiol Gastrointest Liver Physiol. 2002, 282: G565-G572.View ArticlePubMedGoogle Scholar
- Yu Q, Shao R, Qian HS, George SE, Rockey DC: Gene transfer of the neuronal NO synthase isoform to cirrhotic rat liver ameliorates portal hypertension. J Clin Invest. 2000, 105: 741-748.View ArticlePubMedPubMed CentralGoogle Scholar
- Garcia-Trevijano ER, Iraburu MJ, Fontana L, José A, Dominguez-Rosales JA, Auster A, Covarrubias-Pinedo A, Rojkind M: Transforming growth factor β1 induces the expression of a α1(I) procollagen mRNA by a hydrogen peroxide-C/EBPβ-dependent mechanism in rat hepatic stellate cells. Hepatology. 1999, 29: 960-970.View ArticlePubMedGoogle Scholar
- Okuno M, Akita K, Moriwaki H, Kawada N, Ikeda K, Kaneda K, Suzuki Y, Kojima S: Prevention of rat hepatic fibrosis by the protease inhibitor, camostat mesilate, via reduced generation of active TGF-β. Gastroenterology. 2001, 120: 1784-1800.View ArticlePubMedGoogle Scholar
- George J, Roulot D, Koteliansky VE, Bissell DM: In vivo inhibition of rat stellate cell activation by soluble transforming growth factor beta type II receptor: A potential new therapy for hepatic fibrosis. Proc Natl Acad Sci USA. 1999, 96: 12719-12724. 10.1073/pnas.96.22.12719.View ArticlePubMedPubMed CentralGoogle Scholar
- Dooley S, Hamzavi J, Breitkopf K, Wiercinska E, Said HM, Lorenzen J, ten Dijke P, Gressner AM: Smad7 prevents activation of hepatic stellate cells and liver fibrosis in rats. Gastroenterology. 2003, 125: 178-191. 10.1016/S0016-5085(03)00666-8.View ArticlePubMedGoogle Scholar
- The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-230X/3/29/prepub
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