Oral Methylthioadenosine Administration Attenuates Fibrosis and Chronic Liver Disease Progression in Mdr2−/− Mice

BACKGROUND: Inflammation and fibrogenesis are directly related to chronic liver disease progression, including hepatocellular carcinoma (HCC) development. Currently there are few therapeutic options available to inhibit liver fibrosis. We have evaluated the hepatoprotective and anti-fibrotic potential of orally-administered 5'-methylthioadenosine (MTA) in Mdr2(-/-) mice, a clinically relevant model of sclerosing cholangitis and spontaneous biliary fibrosis, followed at later stages by HCC development. METHODOLOGY: MTA was administered daily by gavage to wild type and Mdr2(-/-) mice for three weeks. MTA anti-inflammatory and anti-fibrotic effects and potential mechanisms of action were examined in the liver of Mdr2(-/-) mice with ongoing fibrogenesis and in cultured liver fibrogenic cells (myofibroblasts). PRINCIPAL FINDINGS: MTA treatment reduced hepatomegaly and liver injury. α-Smooth muscle actin immunoreactivity and collagen deposition were also significantly decreased. Inflammatory infiltrate, the expression of the cytokines IL6 and Mcp-1, pro-fibrogenic factors like TGFβ2 and tenascin-C, as well as pro-fibrogenic intracellular signalling pathways were reduced by MTA in vivo. MTA inhibited the activation and proliferation of isolated myofibroblasts and down-regulated cyclin D1 gene expression at the transcriptional level. The expression of JunD, a key transcription factor in liver fibrogenesis, was also reduced by MTA in activated myofibroblasts. CONCLUSIONS/SIGNIFICANCE: Oral MTA administration was well tolerated and proved its efficacy in reducing liver inflammation and fibrosis. MTA may have multiple molecular and cellular targets. These include the inhibition of inflammatory and pro-fibrogenic cytokines, as well as the attenuation of myofibroblast activation and proliferation. Downregulation of JunD and cyclin D1 expression in myofibroblasts may be important regarding the mechanism of action of MTA. This compound could be a good candidate to be tested for the treatment of (biliary) liver fibrosis

Insulin-like growth factor I gene transfer to cirrhotic liver induces fibrolysis and reduces fibrogenesus leading to cirrhosis reversion in rats

Weinvestigated whether gene transfer of insulin-like growth factor I (IGF-I) to the hepatic tissue was able to improve liver histology and function in established liver cirrhosis. Rats with liver cirrhosis induced by carbon tetrachloride (CCl4) given orally for 8 weeks were injected through the hepatic artery with saline or with Simian virus 40 vectors encoding IGF-I (SVIGF-I), or luciferase (SVLuc). Animalsweresacrificed8weeksafter vector injection. In cirrhotic ratsweobserved that, whereas IGF-I was synthesized by hepatocytes, IGF-I receptor was predominantly expressed by nonparenchymal cells, mainly in fibrous septa surrounding hepatic nodules. Rats treated with SVIGF-I showed increased hepatic levels of IGF-I, improved liver function tests, and reduced fibrosis in association with diminished -smoothmuscle actin expression, up-regulation of matrix metalloproteases(MMPs)and decreased expression of the tissue inhibitors of MMPs TIM-1 and TIM-2. SVIGF-I therapy induced down-regulation of the profibrogenic molecules transforming growth factor beta (TGF ), amphiregulin, platelet-derived growth factor (PDGF), connective tissue growth factor (CTGF), and vascular endotheliumgrowthfactor(VEGF)andinduction of the antifibrogenicandcytoprotective hepatocyte growth factor (HGF). Furthermore, SVIGF-I-treated animals showed decreased expression of Wilms tumor-1 (WT-1; a nuclear factor involved in hepatocyte dedifferentiation) and up-regulation of hepatocyte nuclear factor 4 alpha (HNF4 ) (which stimulates hepatocellular differentiation). The therapeutic potential of SVIGF-I was also tested in rats with thioacetamide-induced liver cirrhosis. Also in this model, SVIGF-I improved liver function and reduced liver fibrosis in association with up-regulation of HGF and MMPs and down-regulation of tissue inhibitor of metalloproteinase 1 (TIMP-1). Conclusion: IGF-I gene transfer to cirrhotic livers induces MMPs and hepatoprotective factors leading to reversion of fibrosis and improvement of liver function. IGF-I gene therapy may be a useful alternative therapy for patients with advanced cirrhosis without timely access to liver transplantation

Insulin-like growth factor I gene transfer to cirrhotic liver induces fibrolysis and reduces fibrogenesus leading to cirrhosis reversion in rats

Weinvestigated whether gene transfer of insulin-like growth factor I (IGF-I) to the hepatic tissue was able to improve liver histology and function in established liver cirrhosis. Rats with liver cirrhosis induced by carbon tetrachloride (CCl4) given orally for 8 weeks were injected through the hepatic artery with saline or with Simian virus 40 vectors encoding IGF-I (SVIGF-I), or luciferase (SVLuc). Animalsweresacrificed8weeksafter vector injection. In cirrhotic ratsweobserved that, whereas IGF-I was synthesized by hepatocytes, IGF-I receptor was predominantly expressed by nonparenchymal cells, mainly in fibrous septa surrounding hepatic nodules. Rats treated with SVIGF-I showed increased hepatic levels of IGF-I, improved liver function tests, and reduced fibrosis in association with diminished -smoothmuscle actin expression, up-regulation of matrix metalloproteases(MMPs)and decreased expression of the tissue inhibitors of MMPs TIM-1 and TIM-2. SVIGF-I therapy induced down-regulation of the profibrogenic molecules transforming growth factor beta (TGF ), amphiregulin, platelet-derived growth factor (PDGF), connective tissue growth factor (CTGF), and vascular endotheliumgrowthfactor(VEGF)andinduction of the antifibrogenicandcytoprotective hepatocyte growth factor (HGF). Furthermore, SVIGF-I-treated animals showed decreased expression of Wilms tumor-1 (WT-1; a nuclear factor involved in hepatocyte dedifferentiation) and up-regulation of hepatocyte nuclear factor 4 alpha (HNF4 ) (which stimulates hepatocellular differentiation). The therapeutic potential of SVIGF-I was also tested in rats with thioacetamide-induced liver cirrhosis. Also in this model, SVIGF-I improved liver function and reduced liver fibrosis in association with up-regulation of HGF and MMPs and down-regulation of tissue inhibitor of metalloproteinase 1 (TIMP-1). Conclusion: IGF-I gene transfer to cirrhotic livers induces MMPs and hepatoprotective factors leading to reversion of fibrosis and improvement of liver function. IGF-I gene therapy may be a useful alternative therapy for patients with advanced cirrhosis without timely access to liver transplantation

Potential mechanisms of action of MTA in <i>Mdr2^−/−</i> mice.

<p>Our <i>in vivo</i> and <i>in vitro</i> observations indicate that MTA displays different actions that may underlie its beneficial effects on the course of liver injury and fibrosis in <i>Mdr2^−/−</i> mice. MTA may exert a direct cytoprotective effect on hepatocytes by reducing JNK activity, preventing hepatocellular death and further inflammation. MTA also inhibits the production of cytokines by inflammatory cells, likely through interference with NFκB activity as described before <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0015690#pone.0015690-Hevia1" target="_blank">[16]</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0015690#pone.0015690-IglesiasAra1" target="_blank">[18]</a>. In addition, enhanced expression of adenosine A2B receptors may contribute to the anti-inflammatory pharmacological profile of MTA. This compound can also exert direct effects on extracellular matrix (ECM) producing cells (myofibroblasts), reducing their activation, proliferation and the production of pro-fibrogenic factors. Inhibition of JunD expression may be an important event in the antifibrogenic action of MTA.</p

Effects of MTA on liver myofibroblast activation and cytokine expression.

<p>Primary myofibroblasts were serum starved (12 h) and then treated (12 h) with 10% FCS and MTA as indicated. mRNA levels of α1(I)procollagen (A), αSMA (B), TGFβ1 (C), TGFβ2 (D), Mcp-1 (E) and IL6 (F) were measured at the end of treatments. *<i>P</i><0.05 <i>vs</i> FCS, #<i>P</i><0.05 <i>vs</i> FCS+MTA 200 µM.</p

Increased hepatocellular proliferation in <i>Mdr2^−/−</i> mice is attenuated by MTA administration.

<p>Immunohistochemical staining of Ki-67 in liver sections from <i>Mdr2^+/+</i>, control <i>Mdr2^−/−</i>, and MTA-treated <i>Mdr2^−/−</i> mice (<i>Mdr2^−/−</i>+MTA)(A). Ki-67 positive hepatocytes were counted in 30 high-power fields per mouse, n = 5 per group (B). *<i>P</i><0.01 <i>vs Mdr2^+/+</i> mice, #<i>P</i><0.05 <i>vs</i> untreated <i>Mdr2^−/−</i> mice. Western blot analysis of cyclin-D1 protein in liver extracts from <i>Mdr2^+/+</i> mice, untreated <i>Mdr2⁻</i>^/<i>−</i> and MTA-treated <i>Mdr2^−/−</i> mice (C).</p

Expression of fibrogenic activation and cellular proliferation-related genes in myofibroblasts isolated from the liver of control and MTA-treated <i>Mdr2−/−</i> mice.

<p><i>Mdr2−/−</i> mice were treated for three weeks with MTA or vehicle as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0015690#s2" target="_blank">Methods</a> section. At the end of the treatments hepatic myofibroblasts were isolated and the mRNA levels of the indicated genes were measured. Data are means ±SEM of three independent cell preparations per condition. *<i>P</i><0.05 <i>vs</i> vehicle-treated <i>Mdr2−/−</i> mice.</p

MTA modulates intracellular signalling pathways activated in <i>Mdr2^−/−</i> mouse liver.

<p>Phosphorylation levels of Smad1/5/8 (A), Smad2 (B), JNK and c-Jun (C) and Erk1/2 (D) were analyzed by western blotting in liver extracts from <i>Mdr2^+/+</i>, control <i>Mdr2^−/−</i>, and MTA-treated <i>Mdr2^−/−</i> mice. *<i>P</i><0.05 <i>vs Mdr2^+/+</i> mice, #<i>P</i><0.05 <i>vs</i> untreated <i>Mdr2^−/−</i> mice.</p

Effect of MTA on liver myofibroblast intracellular signalling pathways.

<p>Liver myofibroblasts were pre-treated with MTA for 30 min in the absence of serum and then stimulated with 10% FCS for 30 min or PDGF (20 ng/ml) for 60 min. Phosphorylation levels of Erk1/2 and c-Jun upon FCS stimulation (A), and S6 ribosomal protein after PDGF treatment (B) were analyzed by western blotting. Representative blots are shown.</p

Effect of MTA on the expression of inflammatory cytokines and iNOS in <i>Mdr2^−/−</i> mouse liver.

<p>mRNA levels of TNFα (A), IL6 (B), iNOS (C), Mcp-1 (D), TGFβ1 (E) and TGFβ2 (F) were measured in the liver of <i>Mdr2^+/+</i>, control <i>Mdr2^−/−</i>, and MTA-treated <i>Mdr2^−/−</i> mice. *<i>P</i><0.05 <i>vs Mdr2^+/+</i> mice, #<i>P</i><0.05 <i>vs</i> untreated <i>Mdr2^−/−</i> mice.</p