Profibrogenic Gene Expression Research Articles

Astaxanthin prevents TGFβ1-induced pro-fibrogenic gene expression by inhibiting Smad3 activation in hepatic stellate cells

BackgroundNon-alcoholic steatohepatitis (NASH) is a subset of non-alcoholic fatty liver disease, the most common chronic liver disease in the U.S. Fibrosis, a common feature of NASH, results from the dysregulation of fibrogenesis in hepatic stellate cells (HSCs). In this study, we investigated whether astaxanthin (ASTX), a xanthophyll carotenoid, can inhibit fibrogenic effects of transforming growth factor β1 (TGFβ1), a key fibrogenic cytokine, in HSCs. MethodsReactive oxygen species (ROS) accumulation was measured in LX-2, an immortalized human HSC cell line. Quantitative realtime PCR, Western blot, immunocytochemical analysis, and in-cell Western blot were performed to determine mRNA and protein of fibrogenic genes, and the activation of Smad3 in TGFβ1-activated LX-2 cells and primary mouse HSCs. ResultsIn LX-2 cells, ROS accumulation induced by tert-butyl hydrogen peroxide and TGFβ1 was abolished by ASTX. ASTX significantly decreased TGFβ1-induced α-smooth muscle actin (α-SMA) and procollagen type 1, alpha 1 (Col1A1) mRNA as well as α-SMA protein levels. Knockdown of Smad3 showed the significant role of Smad3 in the expression of α-SMA and Col1A1, but not TGFβ1, in LX-2 cells. ASTX attenuated TGFβ1-induced Smad3 phosphorylation and nuclear translocation with a concomitant inhibition of Smad3, Smad7, TGFβ receptor I (TβRI), and TβRII expression. The inhibitory effect of ASTX on HSC activation was confirmed in primary mouse HSCs as evidenced by decreased mRNA and protein levels of α-SMA during activation. ConclusionTaken together, ASTX exerted anti-fibrogenic effects by blocking TGFβ1-signaling, consequently inhibiting the activation of Smad3 pathway in HSCs. General significanceThis study suggests that ASTX may be used as a preventive/therapeutic agent to prevent hepatic fibrosis.

Alcohol directly stimulates epigenetic modifications in hepatic stellate cells

Alcohol is a primary cause of liver disease and an important co-morbidity factor in other causes of liver disease. A common feature of progressive liver disease is fibrosis, which results from the net deposition of fibril-forming extracellular matrix (ECM). The hepatic stellate cell (HSC) is widely considered to be the major cellular source of fibrotic ECM. We determined if HSCs are responsive to direct stimulation by alcohol. HSCs undergoing transdifferentiation were incubated with ethanol and expression of fibrogenic genes and epigenetic regulators was measured. Mechanisms responsible for recorded changes were investigated using ChIP-Seq and bioinformatics analysis. Ethanol induced changes were confirmed using HSCs isolated from a mouse alcohol model and from ALD patient's liver and through precision cut liver slices. HSCs responded to ethanol exposure by increasing profibrogenic and ECM gene expression including elastin. Ethanol induced an altered expression of multiple epigenetic regulators, indicative of a potential to modulate chromatin structure during HSC transdifferentiation. MLL1, a histone 3 lysine 4 (H3K4) methyltransferase, was induced by ethanol and recruited to the elastin gene promoter where it was associated with enriched H3K4me3, a mark of active chromatin. Chromatin immunoprecipitation sequencing (ChIPseq) revealed that ethanol has broad effects on the HSC epigenome and identified 41 gene loci at which both MML1 and its H3K4me3 mark were enriched in response to ethanol. Ethanol directly influences HSC transdifferentiation by stimulating global changes in chromatin structure, resulting in the increased expression of ECM proteins. The ability of alcohol to remodel the epigenome during HSC transdifferentiation provides mechanisms for it to act as a co-morbidity factor in liver disease.

Melatonin limits the expression of profibrogenic genes and ameliorates the progression of hepatic fibrosis in mice

We investigated whether melatonin ameliorates fibrosis and limits the expression of fibrogenic genes in mice treated with carbon tetrachloride (CCl4). Mice in treatment groups received CCl4 5μL/g body weight intraperitoneally twice a week for 4 or 6weeks. Melatonin was given at 5 or 10mg/kg/d intraperitoneally, beginning 2weeks after the start of CCl4 administration. Treatment with CCl4 resulted in fibrosis evidenced by the staining of Van Gieson and α-smooth muscle actin (α-SMA) positive cells in the liver. At both 4 and 6weeks, CCl4 induced an increase in the messenger RNA levels of collagens I and III, transforming growth factor (TGF)-β, platelet-derived growth factor (PDGF), connective tissue growth factor (CTGF), amphiregulin, matrix metalloproteinase (MMP)-9, and tissue inhibitor of metalloproteinase (TIMP)-1. Protein concentrations of CTGF, amphiregulin, MMP-9, TIMP-1, and phospho-Smad3 were also significantly augmented in fibrotic mice. Melatonin successfully attenuated liver injury, as shown by histopathology and decreased levels of serum transaminases. Immunohistochemical staining of α-SMA indicated an abrogation of hepatic stellate cell activation by the indol. Furthermore, melatonin treatment resulted in significant inhibition of the expression of collagens I and III, TGF-β, PDGF, CTGF, amphiregulin, and phospho-Smad3. The MMP-9 activity decreased and the expression of nuclear factor erythroid-2-related factor 2 (Nrf2) increased in mice receiving melatonin. Data obtained suggest that attenuation of multiple profibrogenic gene pathways contributes to the beneficial effects of melatonin in mice with CCl4-induced liver fibrosis.

Vitamin D counteracts fibrogenic TGF-β signalling in human hepatic stellate cells both receptor-dependently and independently

ObjectiveNon-alcoholic fatty liver disease (NAFLD) is closely linked to obesity and constitutes part of the metabolic syndrome, which have been associated with low serum vitamin D (VD). Due to known...

Enhanced expression of BMP6 inhibits hepatic fibrosis in non-alcoholic fatty liver disease

ObjectiveBone morphogenetic protein 6 (BMP6) has been identified as crucial regulator of iron homeostasis. However, its further role in liver pathology including non-alcoholic fatty liver disease (NAFLD) and its advanced...

Lack of efficacy of mTOR inhibitors and ACE pathway inhibitors as antifibrotic agents in evolving and established fibrosis in Mdr2−/− mice

Mammalian target of rapamycin and angiotensin-converting enzyme inhibition has been shown to have antifibrotic activity in models of liver fibrosis. The aim of our study was to determine the efficacy of rapamycin, everolimus, irbesartan and captopril, alone and in combination, as antifibrotic agents in the Mdr2(-/-) model of cholestasis both in early injury and established disease. Mdr2(-/-) mice were treated for 4 weeks with vehicle, rapamycin (1 mg/kg) or everolimus (5 mg/kg) every second day or with captopril (30 mg/kg/day), irbesartan (10 mg/kg/day) or vehicle. Further groups of 3-week-old Mdr2(-/-) mice were treated with rapamycin and irbesartan in combination (1 mg/kg/day and 10 mg/kg/day) or with rapamycin (2 mg/kg/day) for 4 weeks. Liver injury and fibrosis were compared between treated and untreated animals. There were no significant improvements in liver injury, histology, hepatic hydroxyproline or profibrogenic gene expression following treatment with rapamycin, everolimus, captopril or irbesartan at any time point studied. Likewise, there were no improvements in liver histology or profibrogenic gene expression following combination therapy or high-dose rapamycin treatment. The antifibrotic effects of rapamycin, everolimus, captopril and irbesartan seen in other models of fibrosis were not replicated in the Mdr2(-/-) model in this study. This highlights the clear need to test specific antifibrotic agents in a number of different animal models. We believe this animal model is ideal to study usefulness of antifibrotic agents in cholestatic liver disease because of the similarity in genetics and hepatic histopathology to human cholestatic liver disease.

Role of TWEAK in coregulating liver progenitor cell and fibrogenic responses

Failure of fibrotic liver to regenerate after resection limits therapeutic options and increases demand for liver transplantation, representing a significant clinical problem. The mechanism underlying regenerative failure in fibrosis is poorly understood. Seventy percent partial hepatectomy (PHx) was performed in C57Bl/6 mice with or without carbon tetrachloride (CCl4)- induced liver fibrosis. Liver function and regeneration was monitored at 1 to 14 days thereafter by assessing liver mass, alanine aminotransferase (ALT), mRNA expression, and histology. Progenitor (oval) cell mitogen tumor necrosis factor-like weak inducer of apoptosis (TWEAK) and TWEAK-neutralizing antibody were used to manipulate progenitor cell proliferation in vivo. In fibrotic liver, hepatocytes failed to replicate efficiently after PHx. Fibrotic livers showed late (day 5) peak of serum ALT (35426355 IU/L compared to 93665 IU/L in nonfibrotic livers), which coincided with progenitor cell expansion, increase in profibrogenic gene expression and de novo collagen deposition. In fibrotic mice, inhibition of progenitor activation using TWEAK-neutralizing antibody after PHx resulted in strongly down-regulated profibrogenic mRNA, reduced serum ALT levels and improved regeneration. Failure of hepatocyte-mediated regeneration in fibrotic liver triggers activation of the progenitor (oval) cell compartment and a severe fibrogenic response. Inhibition of progenitor cell proliferation using anti-TWEAK antibody prevents fibrogenic response and augments fibrotic liver regeneration. Targeting the fibrogenic progenitor response represents a promising strategy to improve hepatectomy outcomes in patients with liver fibrosis.

Protective effect of Bifidobacterium pseudocatenulatum CECT7765 against induced bacterial antigen translocation in experimental cirrhosis

Intervention in the gut ecosystem is considered as a potential strategy to treat liver diseases and their complications. We have evaluated the effects of Bifidobacterium pseudocatenulatum CECT7765 on bacterial translocation and the liver status in experimental cirrhosis. Liver damage was induced in Balb/c mice by weight-controlled oral administration of carbon tetrachloride. Laparotomies were performed at week 12. One week prior to laparotomy, animals received B. pseudocatenulatum CECT7765 (10(9) cfu/daily) or placebo intragastrically. All animals received Escherichia coli (10(7) cfu/single dose) intragastrically 24 hours before laparotomy. A group of naïve non-treated animals was included as control. Liver tissue specimens, mesenteric lymph nodes, intestinal content and blood were collected. Liver histology, profibrogenic genes expression, bacterial DNA translocation, serum endotoxaemia and liver cytokine levels were measured. Bifidobacterium pseudocatenulatum CECT7765 showed no significant effect on structural liver damage, as determined by histological evaluation, alpha-smooth muscle actin distribution, profibrogenic gene expression levels, total hydroxyproline levels and malon dialdehyde production compared with mice receiving placebo. Interestingly, bacterial DNA translocation and serum endotoxin levels were significantly decreased in mice receiving the Bifidobacterium strain compared with placebo. Gut barrier integrity markers were up-regulated in mice receiving B. pseudocatenulatum CECT7765 and quantitatively correlated with intestinal gene copy numbers of the bifidobacterial strain. Gene expression levels of several anti-inflammatory mediators were also increased in mice receiving B. pseudocatenulatum CECT7765 compared with placebo. Oral administration of B. pseudocatenulatum CECT7765 is associated with improved gut barrier integrity and shows a beneficial effect against induced bacterial antigen translocation in the CCl4 -model of cirrhosis.

Olive Oil Attenuates the Cholesterol-induced Development of Nonalcoholic Steatohepatitis Despite Increased Insulin Resistance in a Rodent Model

It is indefinite whether nonalcoholic steatohepatitis (NASH) results as by-product from general metabolic perturbations and adipokine dysregulations or whether defined dietary factors also play a pathogenetic role. Here, we examine the effects of a modification of dietary lipids in a NASH inducing diet on metabolic changes as well as hepatic steatosis, inflammation, and fibrosis in rats. Male Wistar rats were fed with variations of the atherogenic diet (AD), which induces pathophysiological changes resembling human NASH. Dietary variants (AD without cholesterol, cholate, or choline; change of neutral fat to olive oil or coconut oil) were fed for 8 weeks. Insulin resistance, adipokine profile, liver histology, and lipid content as well as expression of proinflammatory and profibrogenic genes were examined. AD led to clear signs of hepatic steatosis and inflammation together with an increase in TNF and collagen type 1 expression. AD without cholesterol showed markedly less liver damage without changes of insulin action and adipokine profile. AD with olive oil and AD without cholate clearly attenuated hepatic inflammation, whereas fat deposition and features of the metabolic syndrome were increased in these animals. Insulin resistance and hepatic fat deposition per se do not cause significant hepatic inflammation in this rodent model. However, dietary cholesterol is an important causal agent for the development of NASH. Olive oil plays a protective role in this respect, which might be due to the high content of monounsaturated fatty acids.

Deleterious effect of oltipraz on extrahepatic cholestasis in bile duct-ligated mice

Oltipraz (4-methyl-5(pyrazinyl-2)-1-2-dithiole-3-thione), a promising cancer preventive agent, has an antioxidative activity and ability to enhance glutathione biosynthesis, phase II detoxification enzymes and multidrug resistance-associated protein-mediated efflux transporters. Oltipraz can protect against hepatotoxicity caused by carbon tetrachloride, acetaminophen and alpha-naphthylisothiocyanate. Whether oltipraz has hepato-protective effects on obstructive cholestasis is unknown. We administered oltipraz to mice for 5 days prior to bile duct ligation (BDL) for 3 days. Liver histology, liver function markers, bile flow rates and hepatic expression of profibrogenic genes were evaluated. Mice pretreated with oltipraz prior to BDL demonstrated higher levels of serum aminotransferases and more severe liver damage than in control mice. Higher bile flow and glutathione secretion rates were observed in unoperated mice treated with oltipraz than in control mice, suggesting that liver necrosis in oltipraz-treated BDL mice may be related partially to increased bile-acid independent flow and biliary pressure. Oltipraz treatment in BDL mice enhanced α-smooth muscle actin expression, consistent with activation of hepatic stellate cells and portal fibroblasts. Matrix metalloproteinases (Mmp) 9 and 13 and tissue inhibitors of metalloproteinases (Timp) 1 and 2 levels were increased in the oltipraz-treated BDL group, suggesting that the secondary phase of liver injury induced by oltipraz might be due to excessive Mmp and Timp secretions, which induce remodeling of the extracellular matrix. Oltipraz treatment exacerbates the severity of liver injury following BDL and should be avoided as therapy for extrahepatic cholestatic disorders due to bile duct obstruction.

Failure of Fibrotic Liver Regeneration in Mice Is Linked to a Severe Fibrogenic Response Driven by Hepatic Progenitor Cell Activation

Failure of fibrotic liver to regenerate after resection limits therapeutic options and increases demand for liver transplantation, representing a significant clinical problem. The mechanism underlying regenerative failure in fibrosis is poorly understood. Seventy percent partial hepatectomy (PHx) was performed in C57Bl/6 mice with or without carbon tetrachloride (CCl4)-induced liver fibrosis. Liver function and regeneration was monitored at 1 to 14 days thereafter by assessing liver mass, alanine aminotransferase (ALT), mRNA expression, and histology. Progenitor (oval) cell mitogen tumor necrosis factor-like weak inducer of apoptosis (TWEAK) and TWEAK-neutralizing antibody were used to manipulate progenitor cell proliferation in vivo. In fibrotic liver, hepatocytes failed to replicate efficiently after PHx. Fibrotic livers showed late (day 5) peak of serum ALT (3542 ± 355 IU/L compared to 93 ± 65 IU/L in nonfibrotic livers), which coincided with progenitor cell expansion, increase in profibrogenic gene expression and de novo collagen deposition. In fibrotic mice, inhibition of progenitor activation using TWEAK-neutralizing antibody after PHx resulted in strongly down-regulated profibrogenic mRNA, reduced serum ALT levels and improved regeneration. Failure of hepatocyte-mediated regeneration in fibrotic liver triggers activation of the progenitor (oval) cell compartment and a severe fibrogenic response. Inhibition of progenitor cell proliferation using anti-TWEAK antibody prevents fibrogenic response and augments fibrotic liver regeneration. Targeting the fibrogenic progenitor response represents a promising strategy to improve hepatectomy outcomes in patients with liver fibrosis.

1132 EFFECT OF ADENOSINE 2B RECEPTOR ANTAGONIST MRS1754 ON FIBROSIS PROGRESSION AND REGRESSION IN MICE

during cancer and cirrhosis, but it also participates in the pathogenesis of metabolic syndrome. Recently, myostatin levels have been associated with fibrosis during NASH. However, no information is available on the possible direct role of myostatin in the biology of HSC. Aim of the study was to evaluate the effect of myostatin on biological actions and intracellular signaling in human HSC. Methods: We employed both an immortalized human HSC line (LX-2) and primary HSC isolated from human livers. Cell proliferation was evaluated by MTT. Cell migration was assessed with modified Boyden chambers. Gene expression and secretion of cytokines or ECM proteins were measured by qRT-PCR and ELISA, respectively. Intracellular signaling pathways were evaluated using phosphorylation-specific antibodies. Results: Transcripts for ActRIIB were expressed by HSC. Exposure to myostatin (50ng/ml) induced a significant increase in cell migration in both LX-2 and primary HSC, and time-dependently reduced cell proliferation. In addition, myostatin increased mRNA expression of TGF-beta and procollagen type1. We tested the ability of myostatin to activate intracellular signaling pathways in HSC. Myostatin rapidly and markedly induced phosphorylation of Smad3 and JNK. In contrast, p38MAPK and ERK1/2 were only modestly activated. Pretreatment of both LX-2 or primary HSC with the selective JNK inhibitor, SP600125 (20mM), caused a marked inhibition of myostatin-induced cell migration. Transcripts for myostatin and ActRIIB were measurable in normal liver. Induction of steatohepatitis by administration of a methionine and choline-deficient diet significantly increased the expression of both molecules. Conclusions: HSC express ActRIIB and respond to myostatin with increased chemotaxis, reduced proliferation, and increased expression of profibrogenic genes. At least some of these require JNK activation. Expression of myostatin and its receptor is present in the liver and up-regulated following chronic liver injury, indicating a possible role for this molecule in liver fibrosis. The first two Authors contributed equally to the present study.

PKCδ as a Regulator for TGFβ1-Induced α-SMA Production in a Murine Nonalcoholic Steatohepatitis Model

The precise mechanism of TGFβ1 signaling in the progression of non-alcoholic steatohepatitis (NASH) has remained unclear. In particular, a potential regulatory mechanism by which PKCδ affects profibrogenic gene expression had never been explored. In this study, therefore, the role of PKCδ in TGFβ1 mediated α-SMA expression was investigated using NASH model mice. In preparation of the NASH model, male C57BL6/J mice were fed a methionine-choline-deficient (MCD) diet for 3 weeks, after which time they were intraperitoneally injected with lipopolysaccharide (LPS). In addition, Tlr4Lps-d (CH3/HeJ) mice were used to demonstrate the TGFβ1 signaling’s dependency on TLR4 induction. Liver histology and hepatic hepatitis markers were investigated, and hepatic gene expression levels were determined by real-time PCR. Acute liver injury by LPS injection specifically elevated not only α-SMA expression but also phospho-PKCδ in this model. In contrast, Tlr4Lps-d (CH3/HeJ) and blockade of TGFβ1 receptor by SB431542 resulted in a significant reduction of PKCδ activation and α-SMA expression. Moreover, the TGFβ1-induced α-SMA production was significantly reduced by a specific PKCδ inhibitor. These findings suggested that PKCδ plays a critical role in TGFβ1-induced α-SMA production in a NASH model. Thus, this was the first demonstration of the involvement of PKCδ in the regulation of α-SMA expression in NASH liver tissues, and the impaired induction of PKCδ phosphorylation by LPS in a steatohepatitis condition. Interestingly, treatment by PKCδ inhibitor caused dramatic reduction of myofibroblast activation, indicating that PKCδ represents a promising target for treating NASH.

Intramuscular administration of MMP-8 adenoviral vector reduces inflammation up-grade induced by vector delivery and allows prevention of liver fibrosis

Event Abstract Back to Event Intramuscular administration of MMP-8 adenoviral vector reduces inflammation up-grade induced by vector delivery and allows prevention of liver fibrosis Jesus García-Bañuelos1*, Eden Oceguera-Contreras1, Daniela Gordillo-Bastidas1, Ana Sandoval-Rodríguez1, Blanca E. Bastidas-Ramírez2, Jaime González-Cuevas1, Jose Macias-Barragan1, 3, Belinda Gómez-Meda1 and Juan Armendariz-Borunda1, 4 1 Universidad de Guadalajara, Instituto de Biología Molecular en Medicina y Terapia Génica, CUCS., Mexico 2 Universidad de Guadalajara, Instituto de Enfermedades Crónico Degenerativas, CUCS., Mexico 3 Universidad de Guadalajara, Departamento de Ciencias Naturales y Exactas , CUValles., Mexico 4 OPD Hospital Civil de Guadalajara, Mexico BACKGROUND. Multiple protocols regarding liver fibrosis gene therapy using adenoviral vectors systemically delivered, have been reported. Unfortunately, a decrease in the efficiency of liver transduction and increase in proinflammatory cytokines have been exhibited when utilizing these delivering strategies. AIM. To establish a model to deliver therapeutic genes, focused in the prevention of liver fibrosis without promoting an inflammatory process by intramuscular administration using an adenoviral vector containing MMP8 gene (AdMMP8). MATERIAL AND METHODS. Experimental liver fibrosis was induced in male Wistar rats by TTA administration for 7 weeks. Four groups were included: control (no fibrosis), TAA induced-cirrhosis (TAA), TAA+AdGFP (irrelevant gene) and TAA+AdMMP8 (therapeutic gene). At the fifth week of TAA intoxication, administration of vectors in soleum muscle was accomplished. Subgroups of rats (n=5) at first, second and third week after vector administration were sacrificed. Percentage of fibrosis, liver function, MMP8 gene expression, proinflammatory (IL1β, TNFα), profibrogenic (COLIA1, TGFβ) and antifibrogenic (MMP1, MMP9) genes were determined. RESULTS. Liver and serum expression of MMP8 protein was sustained, fibrosis decreased up to 48%, proinflammatory genes expression was not modified, profibrogenic gene expression decreased, antifibrogenic genes expression increased, reduction of liver function was not statistically significant. According to Knodell score, intramuscular administration of AdMMP8, shows a diminishment of inflammatory cells infiltration in comparison with animals treated with AdGFP. CONCLUSION. AdMMP8 delivered in muscle did not generate liver inflammation in fibrotic animals, MMP8 gene was expressed in muscle with satisfactory functions in the liver. Acknowledgements This work was supported by a grant from CONACYT to Jesus Garcia-Bañuelos No. 25480 and CONACYT to Juan Armendáriz- Borunda No. 25474. Keywords: liver inflammation, Gene Therapy, viral vectors, inflammatory cytokines, TNFa Conference: 15th International Congress of Immunology (ICI), Milan, Italy, 22 Aug - 27 Aug, 2013. Presentation Type: Abstract Topic: Immune-mediated disease pathogenesis Citation: García-Bañuelos J, Oceguera-Contreras E, Gordillo-Bastidas D, Sandoval-Rodríguez A, Bastidas-Ramírez BE, González-Cuevas J, Macias-Barragan J, Gómez-Meda B and Armendariz-Borunda J (2013). Intramuscular administration of MMP-8 adenoviral vector reduces inflammation up-grade induced by vector delivery and allows prevention of liver fibrosis. Front. Immunol. Conference Abstract: 15th International Congress of Immunology (ICI). doi: 10.3389/conf.fimmu.2013.02.00235 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 13 Mar 2013; Published Online: 22 Aug 2013. * Correspondence: Dr. Jesus García-Bañuelos, Universidad de Guadalajara, Instituto de Biología Molecular en Medicina y Terapia Génica, CUCS., Guadalajara, Mexico, bgarcia@cucs.udg.mx Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Jesus García-Bañuelos Eden Oceguera-Contreras Daniela Gordillo-Bastidas Ana Sandoval-Rodríguez Blanca E Bastidas-Ramírez Jaime González-Cuevas Jose Macias-Barragan Belinda Gómez-Meda Juan Armendariz-Borunda Google Jesus García-Bañuelos Eden Oceguera-Contreras Daniela Gordillo-Bastidas Ana Sandoval-Rodríguez Blanca E Bastidas-Ramírez Jaime González-Cuevas Jose Macias-Barragan Belinda Gómez-Meda Juan Armendariz-Borunda Google Scholar Jesus García-Bañuelos Eden Oceguera-Contreras Daniela Gordillo-Bastidas Ana Sandoval-Rodríguez Blanca E Bastidas-Ramírez Jaime González-Cuevas Jose Macias-Barragan Belinda Gómez-Meda Juan Armendariz-Borunda PubMed Jesus García-Bañuelos Eden Oceguera-Contreras Daniela Gordillo-Bastidas Ana Sandoval-Rodríguez Blanca E Bastidas-Ramírez Jaime González-Cuevas Jose Macias-Barragan Belinda Gómez-Meda Juan Armendariz-Borunda Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. 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Selective activation of Toll-like receptor 7 in activated hepatic stellate cells may modulate their profibrogenic phenotype

Cholestatic liver injury may activate HSCs (hepatic stellate cells) to a profibrogenic phenotype, contributing to liver fibrogenesis. We have previously demonstrated the involvement of TLR (Toll-like receptor) 7 in the pathogenesis of biliary atresia. In the present study we investigated the ability of TLR7 to modulate the profibrogenic phenotype in HSCs. Obstructive jaundice was associated with significant down-regulation of TLR7. Primary HSCs isolated from BDL (bile duct ligation) rats with obstructive jaundice exhibited reduced expression of TLR7 and increased expression of α-SMA (α-smooth muscle actin) and collagen-α1 compared with sham rats, reflecting HSC-mediated changes. Treatment of primary activated rat HSCs and rat T6 cells with CL075, a TLR7 and TLR8 ligand, significantly decreased expression of MCP-1 (monocyte chemotactic protein-1), TGF-β1 (transforming growth factor-β1), collagen-α1 and MMP-2 (matrix metalloproteinase-2), and inhibited cell proliferation and migration. In contrast, silencing TLR7 expression with shRNA (short hairpin RNA) in T6 cells effectively blocked the effects of CL075 stimulation, reversing the changes in MCP-1, TGF-β1 and collagen-α1 expression and accelerating cell migration. Our results indicate that obstructive jaundice is associated with down-regulation of TLR7 and up-regulation of profibrogenic gene expression in HSCs. Selective activation of TLR7 may modulate the profibrogenic phenotype in activated HSCs associated with cholestatic liver injury.

Histone methyltransferase ASH1 orchestrates fibrogenic gene transcription during myofibroblast transdifferentiation.

Transdifferentiation of hepatic stellate cells (HSCs) to a myofibroblast-like phenotype is the pivotal event in liver fibrosis. The dramatic change in phenotype associated with transdifferentiation is underpinned by a global change in gene expression. Orchestrated changes in gene expression take place at the level of chromatin packaging which is regulated by enzymatic activity of epigenetic regulators that in turn affect histone modifications. Using expression profiling of epigenetic regulators in quiescent and activated primary HSCs we found a number of histone methyltranferases including MLL1, MLL5, Set1 and ASH1 to be highly up-regulated during transdifferentiation of HSCs. All of these histone methyltranferases regulate methylation of lysine 4 of histone H3, which is a signature of actively transcribed genes. We therefore postulated that one or more of these enzymes may be involved in positively influencing expression of profibrogenic genes. Conclusion: We find that ASH1 directly binds to the regulatory regions of alpha smooth muscle actin (αSMA), collagen I, tissue inhibitor of metalloproteinase-1 (TIMP1) and transforming growth factor beta1 (TGFβ1) in activated HSCs while depletion of ASH1 caused broad suppression of fibrogenic gene expression. We also discovered that MeCP2 positively regulates ASH1 expression and therefore identify ASH1 as a key transcriptional activator component of the MeCP2 epigenetic relay pathway that orchestrates coordinated induction of multiple profibrogenic genes. (Hepatology 2012;56:1129–1139)

HAb18G/CD147 promotes activation of hepatic stellate cells and is a target for antibody therapy of liver fibrosis

Activated hepatic stellate cells (HSCs) located in the Disse's space play a crucial role in liver fibrosis. HAb18G/CD147, a tumor-related glycoprotein, is highly expressed in hepatocellular carcinoma cells and fibroblasts. Whether HAb18G/CD147 plays an important role in the hepatic fibrogenesis is unknown. Immunohistochemistry for HAb18G/CD147 and α-smooth muscle actin expression in diseased liver tissues was used for correlation analysis. The function of HAb18G/CD147 in fibrogenesis was evaluated with the human HSCs LX-2 cell line and carbon tetrachloride-induced mouse liver fibrosis model. The specific antibody HAb18 targeting HAb18G/CD147 was injected intravenously into the mouse to investigate whether HAb18G/CD147 could be a potential target for liver fibrosis treatment. HAb18G/CD147 is highly expressed on activated HSCs in the sinusoid. The positive rates of HAb18G/CD147 expression in human HBV-related liver cirrhosis, liver biopsy with HBV and liver adjacent to hemangioma were 95.6% (65/68), 14.8% (8/54) and 6.4% (8/125), respectively. HAb18G/CD147 expression was significantly correlated with the Child-Pugh grade (r=0.2848, p=0.0186) and with the expression of α-smooth muscle actin in HSCs (r=0.4434, p=0.0002) in liver cirrhosis. Transforming growth factor-β1 upregulated HAb18G/CD147 expression in LX-2 cells. Transfection of HAb18G/CD147 promoted the profibrogenic genes expression. In mouse liver fibrosis model, HAb18G/CD147 expression increased with the development of fibrogenesis and decreased during the liver fibrosis spontaneous recovery. The HAb18 targeting HAb18G/CD147 could attenuate liver fibrosis. These data suggest that HAb18G/CD147 plays a role in HSC activation and is a potential therapeutic target in fibrosis/cirrhosis.

Development of liver fibrosis is critically regulated by phosphodiesterase 4 (PDE4) sub‐family

The causal role of PDE4 sub‐families in the pathogenesis of lung disease has been extensively studied and PDE4 inhibitors have been demonstrated to be beneficial in the treatment of lung inflammation and fibrosis. However, mechanistic studies examining the role of PDEs in liver disease are lacking. Therefore, we examined potential pathogenic role of PDE4 in the development of liver inflammation and fibrosis using animal model of cholestatic liver injury. The obtained data demonstrate that initiation and progression of liver fibrosis is accompanied by a significant up‐regulation of PDE4A, B and D isoforms. Notably, treatment with the specific PDE4 inhibitor, rolipram, significantly decreased liver PDE4 activity, inflammatory cytokine expression, fibrosis and liver injury. Moreover, we show for the first time that primary hepatic stellate cells, which are major contributors to liver fibrosis, require PDE4B and D activity for their activation and production of extracellular matrix proteins. In summary, our results demonstrate that PDE4 isoforms are critically involved in the induction of expression of pro‐inflammatory and pro‐fibrogenic genes during development of liver inflammation and fibrosis. These data strongly suggest that inhibition of PDE4 activity represents a relevant therapeutic strategy for the treatment of hepatic inflammation, injury and fibrosis. The work was supported by NIH grants 1P01AA07103 and R01AA018869.

Therapeutic administration of a direct thrombin inhibitor reduces hepatic inflammation in a mouse model of hypercholesterolemia

Deficiency in the thrombin receptor protease activated receptor‐1 reduces hepatic inflammation and steatosis in mice fed a Western diet. We tested the hypothesis that thrombin inhibition could reverse hepatic steatosis and inflammation in mice with established diet‐induced fatty liver disease. LDLr−/− mice were fed a control diet or a Western diet for 19 weeks. Mice were given either the direct thrombin inhibitor argatroban or its vehicle for the final 4 weeks. Argatroban administration reduced macrophage and neutrophil accumulation in livers of mice fed a Western diet. Similarly, in mice fed a Western diet, argatroban administration reduced hepatic expression of MCP‐1, ICAM‐1, and MIP‐2, three mediators that can contribute to hepatic macrophage and neutrophil accumulation. In addition, argatroban tended to reduce hepatic steatosis, as indicated by hepatic triglyceride levels. Argatroban completely reversed the increase in serum triglycerides and reduced total serum cholesterol levels in mice fed a Western diet. Argatroban also reduced the expression of profibrogenic genes encoding Type 1 collagen and TIMP‐1 in livers of mice fed a Western diet. Overall, this study indicates that therapeutic intervention with a thrombin inhibitor rapidly reduces several features of diet‐induced NAFLD in mice. This work was supported by AHA Grants 0835121G and 11POST7430043 and by NIH Grants R01 ES017537 and DK073566.

P002 The potential role of IL-33 in inflammatory-associated gut fibrosis: Induction of pro-fibrogenic gene expression and myofibroblast hypertrophy

Background: The pathogenesis of human ulcerative colitis (UC) is characterized by the presence of IL-13-producing Natural Killer T cells (NKT cells). One major complication of UC is the development of colitis-associated colorectal cancer. In these studies we determined the role of alternatively activated “M2 macrophages” in a new mouse model of colitis-associated carcinogenesis. Further, we verified these results through immunological analysis of surgical specimens obtained from patients with UC-associated colon cancer. Methods: Studies were performed using a mouse model for colitis-associated carcinogenesis based on chronic Oxazolonecolitis in combination with an initial i.p. azoxymethane (AOM) injection in female BALB/c mice. We observed chronic intestinal inflammation lasting for 12 weeks in mice repeatedly administered Oxazolone. Surgical specimens with UC-associated colon cancer were analyzed by flow cytometry and immunohistochemistry. Results: We could show that Oxazolone-colitis was mediated by colonic alpha-galactosyl-ceramide+CD1+ NKT-cells producing IL-13 and therefore resembles the immunological characteristics of human UC. Colon cancer development in this model was dependent on F4/80+CD11bhighGr1low macrophages producing IL-6 and EGF. Those cells inherited phenotypic characteristics of IL-13-stimulated alternativelyactivated “M2 macrophages”. To elucidate the importance of these “M2 macrophages”, we conducted bone marrow chimera studies and demonstrated that innate immune signaling through MyD88 in “M2 macrophages” is the key event for the production of tumor supporting factors like IL-6 and EGF. In surgical specimens obtained from patients with UC-associated colon cancer we verified those experimental findings. We consistently found a dense accumulation of IL-13-producing NKT cells inside the tumors. In addition, in this IL-13-dominated tumor micromilieu CD14+ and CD68+ antigen-presenting cells accumulated intensively and those antigen-presenting cells demonstrated an alternatively activated “M2 phenotype” by the expression of CD163 and CD205. Conclusions: We could demonstrate that in an experimental UC model, as well as in human UC, pathogenic IL-13producing NKT cells accumulate in the tumor micromilieu of UC-associated colon cancers. This immunologic situation inside the tumor microenvironment of mice and men induces tumor promoting M2 macrophages. Therefore M2 macrophages represent a potential target for future therapeutic strategies.