Developing an in vitro screening assay platform for evaluation of antifibrotic drugs using precision-cut liver slices
- Satish Kumar Sadasivan†1,
- Nethra Siddaraju†1,
- Khaiser Mehdi Khan1,
- Balamuralikrishna Vasamsetti1,
- Nimisha R Kumar1,
- Vibha Haridas1,
- Madhusudhan B Reddy1,
- Somesh Baggavalli1,
- Anup M Oommen1Email author and
- Raghavendra Pralhada Rao1Email author
© Sadasivan et al.; licensee BioMed Central. 2015
Received: 25 September 2014
Accepted: 5 December 2014
Published: 9 January 2015
Precision-cut liver slices present different cell types of liver in a physiological context, and they have been explored as effective in vitro model systems to study liver fibrosis. Inducing fibrosis in the liver slices using toxicants like carbon tetrachloride is of less relevance to human disease conditions. Our aim for this study was to establish physiologically relevant conditions in vitro to induce fibrotic phenotypes in the liver slices.
Precision-cut liver slices of 150 μm thickness were obtained from female C57BL/6 J mice. The slices were cultured for 24 hours in media containing a cocktail of 10 nM each of TGF-β, PDGF, 5 μM each of lysophosphatidic acid and sphingosine 1 phosphate and 0.2 μg/ml of lipopolysaccharide along with 500 μM of palmitate and were analyzed for triglyceride accumulation, stress and inflammation, myofibroblast activation and extracellular matrix (ECM) accumulation. Incubation with the cocktail resulted in increased triglyceride accumulation, a hallmark of steatosis. The levels of Acta2, a hallmark of myofibroblast activation and the levels of inflammatory genes (IL-6, TNF-α and C-reactive protein) were significantly elevated. In addition, this treatment resulted in increased levels of ECM markers - collagen, lumican and fibronectin.
This study reports the experimental conditions required to induce fibrosis associated with steatohepatitis using physiologically relevant inducers. The system presented here captures various aspects of the fibrosis process like steatosis, inflammation, stellate cell activation and ECM accumulation and serves as a platform to study the liver fibrosis in vitro and to screen small molecules for their antifibrotic activity.
KeywordsLiver slice Fibrosis Screening platform Myofibroblast Stellate cells
Liver fibrosis is a pathological condition that results due to progressive accumulation of extracellular matrix in the liver. Several etiological factors like viral infection, alcohol abuse, insulin resistance and metabolic disorder contribute to the development of fibrotic phenotype . It is a complex process involving various cell types of liver including hepatocytes, several immune cell types and stellate cells [2,3]. Following an initial injury to the liver (mainly to hepatocytes), the hepatic stellate cells get activated and differentiate into myofibroblasts, acquiring a pro-inflammatory and fibrogenic properties , and this event coupled with several other dysregulations leads to excess production of extracellular matrix (ECM). Uncontrolled liver fibrosis can eventually lead to total liver failure and it is one of the top 10 causes of mortality in the western world . An effective cure for liver fibrosis is not available yet, and part of the reason for the slow progress of the pharmaceutical industry in this direction is lack of an effective in vitro model system to screen the small molecules [6,7]. Several research groups are working toward mechanisms underlying the development of disease and to identify potential antifibrotic compounds. The success of these studies would greatly depend on employing a suitable model system that captures various aspects of liver fibrosis as motioned above. Cell lines and isolated primary cultures serve as good model systems to address mechanism-based questions and to understand the cell type-specific biology. However, they fail to represent the liver as a multicellular system in which several cell types and cell-cell interactions contribute toward fibrogenesis . Precision-cut liver slices have recently been evaluated for their use in studies with liver fibrosis [8-10], and they are more promising as model systems when compared to cell line-based systems. One major advantage of employing them as a model system is that they present several cell types of liver in a physiological milieu and they retain crucial interactions between different cell types and between cells and their ECM.
Earlier studies have used carbon tetrachloride (CCL4) as an inducer of liver fibrosis in a liver slice model system. CCL4 captures several endpoints involved in liver fibrosis, and is one of the oldest toxins known to stimulate fibrotic phenotype in the liver. However CCL4 is a nonphysiological challenge, and it has no etiological significance in human disease  but only leads to biochemical and histological changes similar to those of human disease condition . Liver slices prepared from the rats with established fibrosis is a more physiologically relevant model, and this system has been used for screening antifibrotic compounds [8,13]. However, developing this model system can be time consuming, requiring about 3 to 4 weeks for the animals to develop disease.
In the present study, we report on developing liver fibrosis in liver slices using physiological signals that will activate key signaling pathways effectively and finally result in important end points relevant to NAFLD/fibrosis - triglyceride accumulation, hepatocyte dysfunction and inflammation, hepatic stellate cell activation, and ECM remodeling with increased collagen production.
Results and discussion
Several signaling pathways are activated during pathogenesis of fibrosis, and each of these pathways contributes at various stages of the pathology finally leading to hepatic stellate cell activation and ECM production. The key pathways that contribute can be broadly categorized into inflammatory pathway, growth factor signaling and lipid signaling pathway. Most important among these pathways are the inflammatory pathway and the growth factor signaling mediated by TGF-β and PDGF signaling [2,10].
TGF-β is one of the potent inducers of fibrogenesis . It plays a major role in the transformation of hepatic stellate cells into myofibroblasts and stimulates the synthesis of extracellular matrix proteins while inhibiting their degradation . TGF-β signaling pathways have been explored as a target for fibrosis therapy . PDGF is another potent proliferative factor for hepatic stellate cells and myofibroblasts during liver fibrogenesis . During the process of fibrogenesis, it is secreted by a variety of cell types such as hepatocytes, kupffer cells and activated hepatic stellate cells, and many pro-inflammatory cytokines mediate their mitogenic effects via the autocrine release of PDGF .
Sphingosine 1 phosphate is well known for its diverse biological roles . In the context of tissue fibrosis, S1P influences various aspects of fibroblast migration, stellate cell activation, myofibroblast differentiation and vascular permeability . Several studies have established a causal connection between S1P and fibrosis of various organs like liver, lung and heart [20-22].
Phospholipid growth factors like lysophosphatidic acid (LPA) are known for their growth factor-like activity [23,24]. LPA exerts its action through well-characterized membrane receptors and has been found to promote cell division and migration and to inhibit apoptosis . Relevant to fibrosis, LPA is shown to facilitate myofibroblast differentiation and ECM generation through activation of Rho-ROCK pathway [26,27].
Lipopolysaccharides (LPS), the cell wall derivatives of gram negative bacteria, activate toll-like receptor (TLR) pathways. The TLRs are expressed on variety of liver cell types that are central to the process of fibrosis like hepatocytes, kupffer cells and HSCs . TLR pathways play critical role in fibrogenesis [28,29].
An inflammatory, growth factor and lipid mediator cocktail (IGL) system captures the aspects of steatosis and inflammation
Increased inflammation in these slices was evident with increased expression levels of CRP, IL6 and TNF-α (Figure 2B, C and D) following treatment with the IGL cocktail. CRP is a well-known marker of inflammation and is also proposed as a marker of nonalcoholic fatty liver disease [34,35]. Animal model and clinical studies indicate that TNF-α is involved in mediating both initial and advanced stages of liver damage . IL-6 is a pleiotropic inflammatory cytokine and is involved synthesis of broad spectrum of acute phase proteins, chronic inflammation and fibrogenesis . Monocyte chemoattractant protein 1 (MCP-1) plays an important an role in inflammation, liver injury and NASH [38,39] and is used as a reliable marker for inflammation. However, in our study, MCP-1 levels did not significantly increase following treatment, either with the IGL cocktail or CCL4 (Figure 2E). The reason behind this could be kinetics of expression of MCP-1 during the process of in vitro fibrosis (as in the current study), and we speculate that MCP-1 expression is a very early event during fibrosis. This is further supported by a study that reports that following CCL4 treatment rat liver shows increased expression of MCP-1 between 6 to 48 hours, but is not detectable after 60 hours . Oxidative stress is known to significantly contribute to fibrogenesis, and reactive oxygen species and the lipid peroxides are shown to enhance inflammation and cellular damage, stellate activation and production of collagen [40-42]. To assess if the IGL cocktail treatment influences the oxidative stress in the slices, we estimated the oxidative stress in the liver slices. As shown in the Figure 2F, the IGL cocktail treatment resulted in about a 50% increase in oxidative stress levels. CCL4 treatment, in comparison, had higher levels of oxidative stress compared to the cocktail treatment.
Inflammatory, growth factor and lipid mediator cocktail treatment results in stellate cell activation
Inflammatory, growth factor and lipid mediator cocktail system captures the aspects of extracellular matrix accumulation and remodeling
Our assay system indeed captures critical aspects of the pathology-like inflammation and oxidative stress, hepatic stellate cell activation and extracellular matrix overproduction. Although this cocktail is not exhaustive in representing all the signaling pathways, it nevertheless corresponds to diverse arms of signaling networks involved in fibrogenesis. The fact that the IGL cocktail treatment results in a steatotic phenotype in the slices as measured in terms of triglyceride accumulation makes it very suitable for use in studying fibrosis in the background of steatosis. It should be noted, however, that this system does not represent progression of fibrosis pathology from steatosis to steatohepatitis and fibrogenesis, in which case one would expect development of fibrogenesis in the slices following incubation with palmitic acid alone. We feel that this would not be practically possible in a liver slice system given that progression from steatosis to fibrosis requires a long time, at least in vivo , and translating this in an ex vivo set-up such as liver slice may be limited due to viability issues. Nevertheless, triglyceride accumulation in the slices sets up a suitable background of steatosis that contributes to key aspects of liver fibrosis.
The William’s E Media, GlutaMAX, fetal bovine serum for the cell culture, ATP estimation kit, recombinant TGF-β and the PDGF were purchased from Life Technologies USA. Lysophosphatidic acid, lipopoly saccharide and palmitic acid were purchased from Sigma Aldrich. The cDNA synthesis kit was from BioRad, the qPCR kit was from KAPA Biosystems, and the triglyceride estimation kit (TAG reagent) was from Diasys.
C57BL/6 J female mice were housed at 22 ± 3°C, with a relative humidity of 50 to 70% on a 12 h light and 12 h dark cycle with artificial fluorescent tubes. Animals were fed ad libitum with normal chow diet. Mice aged between 8 to 12 weeks were used for preparation of liver slices. In order to minimize any possible variations emanating from sex differences, only female mice were used throughout the study. The study protocol, animal maintenance, and experimental procedures were all approved by the Institutional Animal Ethics Committee (IAEC) of Connexios Life Sciences, which is approved by CPCSEA (Committee for the Purpose of Control and Supervision of Experiments on Animals, government of India).
Preparation of liver slices
Williams E media was prepared with 15% FBS and 1% GlutaMAX. Five milliliters of media was dispensed to each T25 flask. 8 to 12 week old C57BL/6 J animals were euthanized using isoflurane, and the liver was collected in a Petri dish containing pre-warmed Williams E media The lobes of the liver were separated and were cut into small pieces of about 10 mm3. Precision-cut liver slices of 150 μm thickness was obtained using automated vibrating blade microtome (Leica VT 1200S), and the slices were collected under aseptic conditions into pre-warmed media. About 8 to 10 precision-cut liver sections were then distributed to each T25 flask on a random basis. The thickness of the liver slices influences the viability of the cells and oxygen diffusion during incubations . Using the slices of greater thickness would result in reduced oxygen diffusion into the slices, while using slices of lesser thickness can affect the viability of the cells in the outer layer of the slices. In literature people have successfully used thicknesses as low as 100 μm  and also the slices up to about 250 μm . In the current study, we use slices of 150-μm thickness, and this thickness was good enough to retain viability of the slices for up to 24 hours as discussed in results section.
Liver slice culture
Liver slices from mouse (8- to 12-week-old C57BL/6 J) were cultured in William’s E Media supplemented with 15% Fetal Bovine serum (FBS) and 1% GlutaMAX . Cultures were maintained in a humidified atmosphere of 95% air and 5% CO2 at 37°C. In order to induce a fibrotic phenotype, the slices were cultured for 24 hours in the media with a cocktail containing 10nM each of TGF-β, PDGF, 5 μM each of lysophosphatidic acid and sphingosine 1 phosphate and 0.2 μg/ml of lipopolysaccharide. Where mentioned, CCL4 was used at a concentration of 0.1%.
Quantitative real-time PCR
Sequences of the primers used in this study
Forward TGG TGG GAG ACA TCG GAG AT
Reverse GCC CGC CAG TTC AAA ACA TT
Forward CTG ATG CTG GTG ACA ACC AC
Reverse CAG AAT TGC CAT TGC ACA AC
Forward TAG CCA GGA GGG AGA ACA GAA A
Reverse CCA GTG AGT GAA AGG GAC AGA A
Forward TTC TTC GGA GAG CAC CTG TT
Reverse CCC CAG AAC CTT GAC TTT GA
Forward ATG GCC AAC CTG GTG CGA AAG G
Reverse ACC AAC GTTA CCA ATG GGG CCG
Forward TGC AGT GGC TCA TTC TTG AC
Reverse GGA CTC GGT CAG GTT GTT GT
Forward GAA CTT CTC GGA TGC TGA GG
Reverse CAA CTG GCC AGG GTG TTA CT
Forward CAG CCC TTG CTT GCC TCA T
Reverse CCG AGG ACA CGC CAT AGG
Forward AGC ACC AGC CAA CTC TCA CT
Reverse TCA TTG GGA TCA TCT TGC TG
Forward GTT TCT TGG GAC AGG CAG GAG
Reverse GCC TGC CTT TTT CAT TCT GGG C
Forward TCG CGG CTA AGA ACA TCT CT
Reverse TCG GTA TTC CAT CAT CTC CTG
Liver slices were bead lysed in 100 μl lysis buffer ( 50 mM Tris, 150 mM NaCl, 0.1% Triton X 100, pH 7.4), at 25 Hz for 5 minutes. The lysed samples were centrifuged at 10,000 rpm for 10 minutes, and the supernatant was taken for analysis. Next, 200 μl of TAG reagent (Triacyl glycerol reagent, supplied with the kit) was added to 10 μl of the sample or standard and incubated at 37°C for 10 min and absorbance was read at 500 nm. The TAG was normalized to total cellular protein.
Soluble collagen estimation
Liver slices were bead lysed in 100 μl lysis buffer ( 50 mM Tris, 150 mM NaCl, 0.1% Triton X 100, pH 7.4) at 25 Hz for 5 minutes. The lysed samples were centrifuged at 10,000 rpm for 10 minutes and the supernatant was taken for analysis. Next, 200 μl of Sirius red dye was added to 40 μl of sample and incubated at room temperature for 2 h. The samples were centrifuged at 12,000 rpm for 15 minutes. The pellet was washed with 500 μl of phosphate buffered saline (PBS) and then with 500 μl of 0.05 N hydrochloric acid. Pellet was dissolved in 100 μl of 0.2 N Sodium hydroxide and absorbance read at 540 nm. The collagen levels were normalized to total cellular protein content.
Assay for viability
Immediately following termination of the experiment, liver slices were lysed in 100 μl lysis buffer (0.1 N NaOH, 0.1% Triton X100). The samples were centrifuged at 10,000 rpm for 10 minutes, and the supernatant was used for estimation of ATP using ATP determination kit following manufacturer’s instructions (Life Technologies).
platelet derived growth factor
transforming growth factor beta
tumor necrosis factor alpha
The authors sincerely thank Dr.Jagannath MR, Dr.Yogananda Moolemath, Dr.Mahesh Verma and Dr.Anil Mathew, for valuable comments and helpful discussions. These studies were supported by Connexios Life Sciences PVT LTD, a Nadathur Holdings Company.
- Friedman SL. Liver fibrosis – from bench to bedside. J Hepatol. 2003;38 Suppl 1:S38–53.PubMedView ArticleGoogle Scholar
- Bataller R, Brenner DA. Liver fibrosis. J Clin Invest. 2005;115:209–18.PubMed CentralPubMedView ArticleGoogle Scholar
- Kmiec Z. Cooperation of liver cells in health and disease. Adv Anat Embryol Cell Biol. 2001;161:III–XIII. 1–151.PubMedGoogle Scholar
- Marra F. Hepatic stellate cells and the regulation of liver inflammation. J Hepatol. 1999;31:1120–30.PubMedView ArticleGoogle Scholar
- Van de Bovenkamp M, Groothuis GM, Meijer DK, Olinga P. Liver fibrosis in vitro: cell culture models and precision-cut liver slices. Toxicol In Vitro. 2007;21:545–57.PubMedView ArticleGoogle Scholar
- Chen CZ, Raghunath M. Focus on collagen: in vitro systems to study fibrogenesis and antifibrosis state of the art. Fibrogenesis Tissue Repair. 2009;2:7.PubMed CentralPubMedView ArticleGoogle Scholar
- Chen CZ, Peng YX, Wang ZB, Fish PV, Kaar JL, Koepsel RR, et al. The Scar-in-a-Jar: studying potential antifibrotic compounds from the epigenetic to extracellular level in a single well. Br J Pharmacol. 2009;158:1196–209.PubMed CentralPubMedView ArticleGoogle Scholar
- van de Bovenkamp M, Groothuis GM, Meijer DK, Olinga P. Precision-cut fibrotic rat liver slices as a new model to test the effects of anti-fibrotic drugs in vitro. J Hepatol. 2006;45:696–703.PubMedView ArticleGoogle Scholar
- van de Bovenkamp M, Groothuis GM, Draaisma AL, Merema MT, Bezuijen JI, van Gils MJ, et al. Precision-cut liver slices as a new model to study toxicity-induced hepatic stellate cell activation in a physiologic milieu. Toxicol Sci. 2005;85:632–8.PubMedView ArticleGoogle Scholar
- Westra IM, Oosterhuis D, Groothuis GM, Olinga P. The effect of antifibrotic drugs in rat precision-cut fibrotic liver slices. PLoS One. 2014;9:e95462.PubMed CentralPubMedView ArticleGoogle Scholar
- Constandinou C, Henderson N, Iredale JP. Modeling liver fibrosis in rodents. Methods Mol Med. 2005;117:237–50.PubMedGoogle Scholar
- Perez Tamayo R. Is cirrhosis of the liver experimentally produced by CCl4 and adequate model of human cirrhosis? Hepatology. 1983;3:112–20.PubMedView ArticleGoogle Scholar
- Westra IM, Oosterhuis D, Groothuis GM, Olinga P. Precision-cut liver slices as a model for the early onset of liver fibrosis to test antifibrotic drugs. Toxicol Appl Pharmacol. 2014;274:328–38.PubMedView ArticleGoogle Scholar
- Gressner AM, Weiskirchen R, Breitkopf K, Dooley S. Roles of TGF-beta in hepatic fibrosis. Front Biosci. 2002;7:d793–807.PubMedView ArticleGoogle Scholar
- Liu Y, Wen XM, Lui EL, Friedman SL, Cui W, Ho NP, et al. Therapeutic targeting of the PDGF and TGF-beta-signaling pathways in hepatic stellate cells by PTK787/ZK22258. Lab Invest. 2009;89:1152–60.PubMed CentralPubMedView ArticleGoogle Scholar
- Liu X, Hu H, Yin JQ. Therapeutic strategies against TGF-beta signaling pathway in hepatic fibrosis. Liver Int. 2006;26:8–22.PubMedView ArticleGoogle Scholar
- Bonner JC. Regulation of PDGF and its receptors in fibrotic diseases. Cytokine Growth Factor Rev. 2004;15:255–73.PubMedView ArticleGoogle Scholar
- Pralhada Rao R, Vaidyanathan N, Rengasamy M, Mammen Oommen A, Somaiya N, Jagannath MR. Sphingolipid metabolic pathway: an overview of major roles played in human diseases. J Lipids. 2013;2013:178910.PubMed CentralPubMedView ArticleGoogle Scholar
- Shea BS, Tager AM. Sphingolipid regulation of tissue fibrosis. Open Rheumatol J. 2012;6:123–9.PubMed CentralPubMedView ArticleGoogle Scholar
- Shea BS, Brooks SF, Fontaine BA, Chun J, Luster AD, Tager AM. Prolonged exposure to sphingosine 1-phosphate receptor-1 agonists exacerbates vascular leak, fibrosis, and mortality after lung injury. Am J Respir Cell Mol Biol. 2010;43:662–73.PubMed CentralPubMedView ArticleGoogle Scholar
- Li C, Jiang X, Yang L, Liu X, Yue S, Li L. Involvement of sphingosine 1-phosphate (SIP)/S1P3 signaling in cholestasis-induced liver fibrosis. Am J Pathol. 2009;175:1464–72.PubMed CentralPubMedView ArticleGoogle Scholar
- Takuwa N, Ohkura S, Takashima S, Ohtani K, Okamoto Y, Tanaka T, et al. S1P3-mediated cardiac fibrosis in sphingosine kinase 1 transgenic mice involves reactive oxygen species. Cardiovasc Res. 2010;85:484–93.PubMed CentralPubMedView ArticleGoogle Scholar
- Sugiura T, Nakane S, Kishimoto S, Waku K, Yoshioka Y, Tokumura A. Lysophosphatidic acid, a growth factor-like lipid, in the saliva. J Lipid Res. 2002;43:2049–55.PubMedView ArticleGoogle Scholar
- Tokumura A, Iimori M, Nishioka Y, Kitahara M, Sakashita M, Tanaka S. Lysophosphatidic acids induce proliferation of cultured vascular smooth muscle cells from rat aorta. Am J Physiol. 1994;267:C204–10.PubMedGoogle Scholar
- Birgbauer E, Chun J. New developments in the biological functions of lysophospholipids. Cell Mol Life Sci. 2006;63:2695–701.PubMedView ArticleGoogle Scholar
- Yin Z, Watsky MA. Chloride channel activity in human lung fibroblasts and myofibroblasts. Am J Physiol Lung Cell Mol Physiol. 2005;288:L1110–6.PubMedView ArticleGoogle Scholar
- Akhmetshina A, Dees C, Pileckyte M, Szucs G, Spriewald BM, Zwerina J, et al. Rho-associated kinases are crucial for myofibroblast differentiation and production of extracellular matrix in scleroderma fibroblasts. Arthritis Rheum. 2008;58:2553–64.PubMedView ArticleGoogle Scholar
- Yang L, Seki E. Toll-like receptors in liver fibrosis: cellular crosstalk and mechanisms. Front Physiol. 2012;3:138.PubMed CentralPubMedGoogle Scholar
- Cong M, Iwaisako K, Jiang C, Kisseleva T. Cell signals influencing hepatic fibrosis. Int J Hepatol. 2012;2012:158547.PubMed CentralPubMedGoogle Scholar
- Rosso N, Chavez-Tapia NC, Tiribelli C, Bellentani S. Translational approaches: from fatty liver to non-alcoholic steatohepatitis. World J Gastroenterol. 2014;20:9038–49.PubMed CentralPubMedView ArticleGoogle Scholar
- Day CP, James OF. Steatohepatitis: a tale of two “hits”? Gastroenterology. 1998;114:842–5.PubMedView ArticleGoogle Scholar
- Wobser H, Dorn C, Weiss TS, Amann T, Bollheimer C, Buttner R, et al. Lipid accumulation in hepatocytes induces fibrogenic activation of hepatic stellate cells. Cell Res. 2009;19:996–1005.PubMedView ArticleGoogle Scholar
- Berlanga A, Guiu-Jurado E, Porras JA, Auguet T. Molecular pathways in non-alcoholic fatty liver disease. Clin Exp Gastroenterol. 2014;7:221–39.PubMed CentralPubMedGoogle Scholar
- Yeniova AO, Kucukazman M, Ata N, Dal K, Kefeli A, Basyigit S, et al. High-sensitivity C-reactive protein is a strong predictor of non-alcoholic fatty liver disease. Hepatogastroenterology. 2014;61:422–5.PubMedGoogle Scholar
- Fierbinteanu-Braticevici C, Baicus C, Tribus L, Papacocea R. Predictive factors for nonalcoholic steatohepatitis (NASH) in patients with nonalcoholic fatty liver disease (NAFLD). J Gastrointestin Liver Dis. 2011;20:153–9.PubMedGoogle Scholar
- Manco M, Marcellini M, Giannone G, Nobili V. Correlation of serum TNF-alpha levels and histologic liver injury scores in pediatric nonalcoholic fatty liver disease. Am J Clin Pathol. 2007;127:954–60.PubMedView ArticleGoogle Scholar
- Choi I, Kang HS, Yang Y, Pyun KH. IL-6 induces hepatic inflammation and collagen synthesis in vivo. Clin Exp Immunol. 1994;95:530–5.PubMed CentralPubMedView ArticleGoogle Scholar
- Czaja MJ, Geerts A, Xu J, Schmiedeberg P, Ju Y. Monocyte chemoattractant protein 1 (MCP-1) expression occurs in toxic rat liver injury and human liver disease. J Leukoc Biol. 1994;55:120–6.PubMedGoogle Scholar
- Zimmermann HW, Seidler S, Nattermann J, Gassler N, Hellerbrand C, Zernecke A, et al. Functional contribution of elevated circulating and hepatic non-classical CD14CD16 monocytes to inflammation and human liver fibrosis. PLoS One. 2010;5:e11049.PubMed CentralPubMedView ArticleGoogle Scholar
- Nieto N, Greenwel P, Friedman SL, Zhang F, Dannenberg AJ, Cederbaum AI. Ethanol and arachidonic acid increase alpha 2(I) collagen expression in rat hepatic stellate cells overexpressing cytochrome P450 2E1. Role of H2O2 and cyclooxygenase-2. J Biol Chem. 2000;275:20136–45.PubMedView ArticleGoogle Scholar
- Safadi R, Friedman SL. Hepatic fibrosis–role of hepatic stellate cell activation. MedGenMed. 2002;4:27.PubMedGoogle Scholar
- Poli G, Parola M. Oxidative damage and fibrogenesis. Free Radic Biol Med. 1997;22:287–305.PubMedView ArticleGoogle Scholar
- Rockey DC, Weymouth N, Shi Z. Smooth muscle alpha actin (Acta2) and myofibroblast function during hepatic wound healing. PLoS One. 2013;8:e77166.PubMed CentralPubMedView ArticleGoogle Scholar
- Rockey DC, Boyles JK, Gabbiani G, Friedman SL. Rat hepatic lipocytes express smooth muscle actin upon activation in vivo and in culture. J Submicrosc Cytol Pathol. 1992;24:193–203.PubMedGoogle Scholar
- Cassiman D, Roskams T, van Pelt J, Libbrecht L, Aertsen P, Crabbe T, et al. Alpha B-crystallin expression in human and rat hepatic stellate cells. J Hepatol. 2001;35:200–7.PubMedView ArticleGoogle Scholar
- Friedman SL. Hepatic stellate cells: protean, multifunctional, and enigmatic cells of the liver. Physiol Rev. 2008;88:125–72.PubMed CentralPubMedView ArticleGoogle Scholar
- Clouthier DE, Comerford SA, Hammer RE. Hepatic fibrosis, glomerulosclerosis, and a lipodystrophy-like syndrome in PEPCK-TGF-beta1 transgenic mice. J Clin Invest. 1997;100:2697–713.PubMed CentralPubMedView ArticleGoogle Scholar
- Ghosh AK, Vaughan DE. PAI-1 in tissue fibrosis. J Cell Physiol. 2012;227:493–507.PubMed CentralPubMedView ArticleGoogle Scholar
- Nie QH, Zhang YF, Xie YM, Luo XD, Shao B, Li J, et al. Correlation between TIMP-1 expression and liver fibrosis in two rat liver fibrosis models. World J Gastroenterol. 2006;12:3044–9.PubMed CentralPubMedGoogle Scholar
- Masuda H, Fukumoto M, Hirayoshi K, Nagata K. Coexpression of the collagen-binding stress protein HSP47 gene and the alpha 1(I) and alpha 1(III) collagen genes in carbon tetrachloride-induced rat liver fibrosis. J Clin Invest. 1994;94:2481–8.PubMed CentralPubMedView ArticleGoogle Scholar
- Kanuri G, Bergheim I. In vitro and in vivo models of Non-alcoholic fatty liver disease (NAFLD). Int J Mol Sci. 2013;14:11963–80.PubMed CentralPubMedView ArticleGoogle Scholar
- de Graaf IA, de Kanter R, de Jager MH, Camacho R, Langenkamp E, van de Kerkhof EG, et al. Empirical validation of a rat in vitro organ slice model as a tool for in vivo clearance prediction. Drug Metab Dispos. 2006;34:591–9.PubMedView ArticleGoogle Scholar
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.