Targeted disruption of Smad3 confers resistance to the development of dimethylnitrosamine‐induced hepatic fibrosis in mice

Background: Hepatic fibrosis is characterized by a progressive accumulation of fibrillar extracellular matrix (ECM) proteins including collagen, which occurs in most types of chronic liver diseases. Transforming growth factor‐β (TGF‐β)/Smad3 signalling plays a central role in tissue fibrogenesis, acting as a potent stimulus of ECM accumulation. Aim: To evaluate the potential protective role of Smad3 deficiency in the pathogenesis of liver fibrosis induced by dimethylnitrosamine (DMN) in Smad3 null mice. Methods: Chronic hepatitis‐associated fibrosis was induced in 13 Smad3 null and 13 wild‐type (WT) mice by intraperitoneal DMN administration (10 μg/g body weight/day) for three consecutive days per week for 6 weeks. The liver was excised for macroscopic examination and histological, morphometric and immunohistochemical (IHC) analyses. For IHC, α‐smooth muscle actin (α‐SMA), collagen types I–III, TGF‐β1, connective tissue growth factor (CTGF), Smad3, Smad7 and CD3 antibodies were used. Results: At macroscopic examination, the liver of DMN‐treated Smad3 WT appeared harder with a dark brown colouring and necrotic areas compared with that from null mice. Histological and morphometric evaluation revealed a significantly higher degree of hepatic fibrosis and accumulation of connective tissue in the Smad3 WT compared with null mice. IHC evaluation showed a marked increase in α‐SMA, CTGF, collagen I‐III, TGF‐β and Smad3 staining in the liver of Smad3 WT compared with that in null mice, whereas Smad7 was increased only in null mice. Conclusions: The results indicate that Smad3 loss confers resistance to the development of DMN‐induced hepatic fibrosis. The reduced fibrotic response appears to be due to a reduction of fibrogenic myofibroblast activation and ECM production and accumulation. Smad3 could be a novel target for potential treatment of fibrosis complicating chronic hepatitis.     

Transforming growth factor‐α attenuates hepatic fibrosis: possible involvement of matrix metalloproteinase‐1

Background: The effect of transforming growth factor (TGF)‐α on fibrosis varies between cell types and the role of TGF‐α in hepatic fibrosis has not been fully elucidated. Methods: We examined the effect of TGF‐α on hepatic fibrosis using TGF‐α‐expressing transgenic mice fed a methionine‐ and choline‐deficient (MCD) diet and human hepatic stellate cells (HSCs) line LX‐2, rat and human primary HSCs. Results: Although the expression levels of the tissue inhibitor of metalloproteinases‐1 and α1(I) collagen mRNA were unchanged, feeding the TGF‐α transgenic mice the MCD diet resulted in greater expression of the murine functional analogue of matrix metalloproteinase‐1 (MMP‐1), MMP‐13 mRNA and protein and attenuated hepatic fibrosis compared with wild‐type mice. TGF‐α overexpression did not affect the extent of the steatosis, oxidative stress and hepatic inflammation in the MCD diet‐fed mice. The effect of TGF‐α on the fibrogenic and anti‐fibrogenic gene expressions varied between cell types in vitro. TGF‐α increased MMP‐1 mRNA expressions that were completely blocked by gefitinib in LX‐2 cells. The extracellular signal‐regulated kinase (ERK) 1/2, c‐Jun N‐terminal kinase and p38 pathways were involved in MMP‐1 mRNA expression in LX‐2 cells. Although TGF‐α increased the phosphorylation of p38, the p38 inhibitor activated the RAS‐ERK pathway and increased TGF‐α‐induced MMP‐1 mRNA expression, which suggested that there may be a crosstalk between the RAS‐ERK and the p38 pathways in LX‐2 cells. Conclusions: The TGF‐α may attenuate hepatic fibrosis in part because of upregulation of the expression of MMP‐1. The balance between fibrogenic and anti‐fibrogenic gene expression and between the activity of the RAS‐ERK and the p38 pathways may be crucial for the fibrotic process.     

Transforming growth factor‐β induces epithelial to mesenchymal transition by down‐regulation of claudin‐1 expression and the fence function in adult rat hepatocytes

Background/Aims: Transforming growth factor‐β (TGF‐β) initiates and maintains epithelial–mesenchymal transition (EMT), which causes disassembly of tight junctions and loss of epithelial cell polarity. In mature hepatocytes during EMT induced by TGF‐β, changes in the expression of tight junction proteins and the fence function indicated that epithelial cell polarity remains unclear. Methods: In the present study, using primary cultures of adult rat hepatocytes at day 10 after plating, in which epithelial cell polarity is well maintained by tight junctions, we examined the effects of 0.01–20 ng/ml TGF‐β on the expression of the integral tight junction proteins, claudin‐1, ‐2 and occludin, as well as the fence function. Results: In adult rat hepatocytes, TGF‐β induced EMT, which was indicated as upregulation of Smad‐interacting protein‐1 (SIP1) and Snail and down‐regulation of E‐cadherin. Down‐regulation of claudin‐1 and upregulation of occludin were observed beginning from a low dose of TGF‐β, whereas upregulation of claudin‐2 was observed at a high dose of TGF‐β. Furthermore, treatment with TGF‐β caused disruption of the fence function, which was closely associated with the expression of claudin‐1 via p38 mitogen‐activated protein kinase (MAPK), phosphoinositide‐3 kinase and protein kinase C but not MAPK signalling pathways. Conclusion: These results suggest that in mature hepatocytes in vitro, TGF‐β induces EMT by down‐regulation of claudin‐1 and the fence function via distinct signalling pathways.     

Identification of paraxanthine as the most potent caffeine‐derived inhibitor of connective tissue growth factor expression in liver parenchymal cells

Background: Recently, we identified hepatocytes as the major cellular source of profibrogenic connective tissue growth factor (CTGF/CCN2) in the liver. Based on reports of a hepatoprotective effect of coffee consumption, we were the first to provide evidence that caffeine suppresses transforming growth factor (TGF)‐β dependent and ‐independent CTGF expression in hepatocytes in vitro and in vivo, thus suggesting this xanthine‐alkaloid as a potential therapeutic agent. Aim: This study aims at comparing the inhibitory capacities of caffeine and its three demethylated derivates paraxanthine, theophylline and theobromine on CTGF expression in hepatocytes and hepatic stellate cells (HSC). Results: Our data suggest paraxanthine as the most important pharmacological repressor of hepatocellular CTGF expression among the caffeine‐derived metabolic methylxanthines with an inhibitory dosage (ID)₅₀ of 1.15 mM, i.e. 3.84‐fold lower than what is observed for caffeine. In addition, paraxanthine displayed the least cell toxicity as proven by the water‐soluble tetrazolium‐1 cell vitality assay. However, caffeine or any of the metabolites did not inhibit CTGF expression in HSC. At the toxicological threshold concentration of 1 mM for paraxanthine, we observed an inhibition of hepatocellular CTGF synthesis by 44%, which was strongly reverted in the presence of the specific competitive cyclic adenosine monophosphate inhibitor Rp‐adenosine 3′,5‐cyclic monophosphorothioate triethylammonium salt. Furthermore, CTGF protein expression induced by various concentrations of TGF‐β (0.13–1 ng/ml) is still reduced by, on average, 27%/45% in the presence of paraxanthine (1.25 mM/2.5 mM). Conclusion: Our data provide an evidence‐based suggestion of the caffeine‐derived primary metabolite paraxanthine as a potentially powerful antifibrotic drug by its inhibitory effect on (hepatocellular) CTGF synthesis.     

Adenoviral gene transfer of connective tissue growth factor in the lung induces transient fibrosis

Connective tissue growth factor (CTGF) is felt to be one of the key profibrotic factors and is a downstream effector molecule mediating the action of transforming growth factor (TGF)-beta1, a cytokine known to induce severe and progressive fibrosis. However, the in vivo fibrogenic effect of isolated CTGF expression is not well described. We used adenoviral gene transfer to transiently overexpress CTGF in rat lungs after intratracheal administration and compared it with transient overexpression of active TGF-beta1 delivered by a similar adenovirus vector. This high expression of CTGF over 6-10 days induced a moderate but reversible fibrosis. We observed an increase of fibronectin, procollagen 1a2, and endogenous CTGF gene expression at 14 days, which suggested an indirect activation by CTGF. Tissue inhibitor of metalloproteinase-1 was weakly and transiently upregulated after CTGF exposure. These same genes were robustly and persistently stimulated by TGF-beta1 from Day 3 to Day 21. This data suggested that CTGF may act as a TGF-beta1 cofactor rather than a direct fibrogenic factor. We demonstrate that CTGF overexpression can initiate fibrogenic activity but likely requires the presence of additional factors, such as tissue inhibitor of metalloproteinase-1, to maintain a nonfibrolytic environment and to cause progression of fibrosis.     

Oral application of 1,7-dimethylxanthine (paraxanthine) attenuates the formation of experimental cholestatic liver fibrosis

Aim: Several epidemiological studies suggest a beneficial effect of coffee consumption on the formation and progression of fibrogenic diseases, particularly of the liver. Recent data now point to a modulation of transforming growth factor‐β (TGF‐β) signaling by paraxanthine (1,7‐dimethylxanthine [1,7‐DMX]), the demethylated primary metabolite of caffeine Methods: Twenty adult Sprague–Dawley rats were bile duct ligated (BDL) or sham operated with or without concomitant oral 1,7‐DMX (1 mM) application. Serum was investigated by standard biochemical analysis, in‐house connective tissue growth factor (CTGF), enzyme linked immunosorbent assay (ELISA) or liquid chromatography‐mass spectrometry analysis. Liver tissue was stained using hematoxylin‐eosin (HE) and Sirius‐red staining. Whole liver lysates, primary rat hepatocytes (PC) and hepatic stellate cells (HSC) were investigated by CTGF, and total Smad2/3 Western blot analysis, CTGF reporter gene assay or an in‐house malondialdehyde ELISA. Results: The in vitro 50% inhibitory dose (ID50) of 1,7‐DMX was 0.95 mM by for CTGF promoter activity and protein expression in PC and 1.25 mM for protein expression in HSC. Oral 1,7‐DMX application (1 mM) attenuated cholestatic hepatocellular injury in vivo as determined by biochemical serum analysis and reduced intercellular collagen deposition in the cholestatic rat liver (HE‐ and Sirius‐red staining). Western Blot analysis of whole liver lysates revealed a reduction of intrahepatic concentrations of Smad2/3 and CTGF following oral 1,7‐DMX intake. However, serum CTGF concentrations were not reduced in 1,7‐DMX treated BDL rats. Oral 1,7‐DMX furthermore led to a reduction of intrahepatic lipid peroxidation (malondialdehyde concentrations) as markers of oxidative stress in BDL rats. Conclusion: Our pilot study warrants further studies of 1,7‐DMX as a potential new drug to fight fibrotic processes, not just of the liver.     

Selective expression of connective tissue growth factor in fibroblasts in vivo promotes systemic tissue fibrosis

Objective

Connective tissue growth factor (CTGF) is a cysteine‐rich secreted matricellular protein involved in wound healing and tissue repair. Enhanced and prolonged expression of CTGF has been associated with tissue fibrosis in humans. However, questions remain as to whether CTGF expression alone is sufficient to drive fibrosis. This study was undertaken to investigate whether CTGF alone is sufficient to cause fibrosis in intact animals and whether its effects are mediated through activation of transforming growth factor β (TGFβ) signaling or through distinct signal transduction pathways.

Methods

We generated mice overexpressing CTGF in fibroblasts under the control of the fibroblast‐specific collagen α2(I) promoter enhancer. Tissues such as skin, lung, and kidney were harvested for histologic analysis. Mouse embryonic fibroblasts were prepared from embryos (14.5 days postcoitum) for biochemical analysis.

Results

Mice overexpressing CTGF in fibroblasts were susceptible to accelerated tissue fibrosis affecting the skin, lung, kidney, and vasculature, most notably the small arteries. We identified a marked expansion of the myofibroblast cell population in the dermis. RNA analysis of transgenic dermal fibroblasts revealed elevated expression of key matrix genes, consistent with a fibrogenic response. CTGF induced phosphorylation of p38, ERK‐1/2, JNK, and Akt, but not Smad3, in transgenic mouse fibroblasts compared with wild‐type mouse fibroblasts. Transfection experiments showed significantly increased basal activity of the CTGF and serum response element promoters, and enhanced induction of the CTGF promoter in the presence of TGFβ.

Conclusion

These results demonstrate that selective expression of CTGF in fibroblasts alone causes tissue fibrosis in vivo through specific signaling pathways, integrating cues from the extracellular matrix into signal transduction pathways to orchestrate pivotal biologic responses relevant to tissue repair and fibrosis.

     

Antifibrotic and fibrolytic properties of celecoxib in liver damage induced by carbon tetrachloride in the rat

Background: Transforming growth factor‐β (TGF‐β) plays a pivotal role in liver fibrosis, because it activates hepatic stellate cells, stimulating extracellular matrix deposition. Cyclooxygenase‐2 (COX‐2) has been associated with TGF‐β because its inhibition decreases TGF‐β expression and collagen production in some cultured cell types. Aim: The aim of this work was to evaluate the ability of celecoxib (a selective COX‐2 inhibitor) to prevent and to reverse the liver fibrosis induced by CCl₄. Methods: We established experimental groups of rats including vehicle and drug controls, damage induced by chronic CCl₄ administration and CCl₄ plus pharmacological treatment in both prevention and reversion models. We determined: alanine aminotransferase, alkaline phosphatase, γ‐glutamyl transpeptidase, COX and metalloproteinase‐2 and ‐9 activities, lipid peroxidation, glutathione levels, glycogen and collagen content and TGF‐β expression. Results: Celecoxib prevented and aided to the recovery of livers with necrotic and cholestatic damage. Celecoxib exhibited anti‐oxidant properties by restoring the redox equilibrium (lipid peroxidation and glutathione levels). Glycogen was decreased by CCl₄, while celecoxib partially prevented and reversed this effect. Celecoxib inhibited COX‐2 activity, decreased TGF‐β expression, induced metalloproteinase‐2 activity and, consequently, prevented and reversed collagen accumulation. Conclusion: Our findings indicate that celecoxib exerts strong antifibrogenic and fibrolytic effects in the CCl₄ model of cirrhosis.     

糖基化终末产物对活化肝星状细胞合成前胶原及致纤维化细胞因子的影响

目的探讨不同浓度的糖基化终末产物（AGEs）对肝星状细胞（HSC）活性的影响。方法体外合成糖基化牛血清白蛋白（AGE-BSA）,以不同浓度的AGE-BSA刺激HSC,观察AGE受体（RAGE）、转化生长因子（TGFβ1）,结缔组织生长因子（CTGF）、Ⅰ、Ⅲ型前胶原mRNA;ELISA法检测细胞上清液TGFβ1及Ⅰ、Ⅲ型前胶原蛋白的水平。结果高浓度的AGE-BSA（100μg/ml）培养24 h便能促进RAGE、TGFβ1、CTGF和Ⅰ、Ⅲ型前胶原mRNA的表达,并随着时间延长而升高,细胞上清液蛋白水平有同样改变。结论一定浓度的AGEs能促进HSC前胶原及致纤维化细胞因子的表达,并呈时效性改变。AGEs对肝纤维化可能起到促进作用。     

罗格列酮阻止非酒精性脂肪性肝纤维化进展作用机制的研究

目的观察PPARγ靶向性激动剂罗格列酮对高脂、蛋氨酸-胆碱缺乏（MCD）饮食诱导的非酒精性脂肪性肝纤维化模型肝组织中转化生长因子（TGFβ1）及其下游效应因子结缔组织生长因子（CTGF）表达的影响，以明确其阻止脂肪性肝纤维化进展的作用及作用机制。方法采用MCD饮食8周建立C57BL6／J小鼠非酒精性脂肪性肝纤维化模型，以蛋氨酸-胆碱充足饮食设立对照组，干预组小鼠采用MCD饮食加罗格列酮（每日50mg／kg）喂养。血清丙氨酸氨基转移酶（ALT）采用全自动生化仪测定。HE染色、Masson染色观察肝脂肪变、炎症及纤维化程度。TGFβ1、CTGFmRNA及蛋白表达分别应用RT—PCR、免疫组织化学染色方法检测。结果模型组肝组织出现重度肝细胞脂肪变，伴有点、灶状肝细胞坏死及炎细胞浸润、汇管区纤维组织增生及窦周纤维化，ALT水平和TGFβ1、CTGFmRNA及蛋白表达均较对照组明显升高（P〈0．05和P值均〈0．01），罗格列酮干预组肝组织学改变较模型组明显减轻，ALT水平及TGFβ1、CTGF表达明显下降（P值均〈0．05）。结论在MCD饮食诱导的小鼠非酒精性脂肪性肝纤维化模型中，罗格列酮可通过靶向激活PPARγ下调TGFβ1及其下游效应因子CTGF表达，从而缓解或阻止疾病的进展。     

TGF⁃β1 p38MAPK及BMP⁃7蛋白在肝多房棘球蚴病患者肝组织中的表达及意义

目的　检测肝多房棘球蚴病患者肝组织中转化生长因子⁃β1（transforming growth factor⁃β1, TGF⁃β1）、p38MAPK及骨形态发生蛋白⁃7（bone morphogenetic protein⁃7, BMP⁃7）表达水平,探讨其在肝多房棘球蚴病肝纤维化中的潜在作用。方法　以20例肝多房棘球蚴病患者为研究对象,分别采集距肝脏病灶0.5 cm内（A组）、距肝脏病灶0.5～1.5 cm（B组）肝组织及距肝脏病灶2 cm及以上的正常肝组织（C组）。肝组织标本分别行HE和Masson染色观察纤维化病理变化,采用Western blotting检测肝组织中TGF⁃β1、p38MAPK及BMP⁃7蛋白表达水平,分析TGF⁃β1、p38MAPK及BMP⁃7蛋白表达与肝纤维化的相关性。结果　HE染色结果显示,A组和B组肝组织中肝细胞结构排列紊乱、肝小叶结构有不同程度破坏,可见不同程度肝细胞变性、萎缩、坏死及纤维组织增生,并有嗜酸性粒细胞浸润;C组肝组织无异常病理改变,肝细胞结构形态正常、大小均匀,未见明显排列紊乱,肝小叶结构清晰,无或轻度细胞变性、坏死及炎性细胞浸润。Masson染色结果显示,A组和B组肝组织可见汇管区较多纤维结缔组织增生,出现不同程度小叶内纤维化;C组肝组织无明显异常病理改变。A、B、C组肝组织中TGF⁃β1（P < 0.001）、p38MAPK（P < 0.01）及BMP⁃7蛋白（P < 0.05）表达水平差异均有统计学意义,A组和B组TGF⁃β1、p38MAPK及BMP⁃7蛋白表达水平均显著高于C组（P均< 0.05）,B组TGF⁃β1、p38MAPK及BMP⁃7蛋白表达水平亦显著高于C组（P均< 0.05）。TGF⁃β1、p38MAPK及BMP⁃7蛋白表达水平均与肝纤维化程度呈正相关（r = 0.866、0.702、0.801,P均< 0.05）,不同纤维化程度肝组织中TGF⁃β1（F = 72.580,P < 0.01）、p38MAPK（[χ2] = 31.705,P < 0.01）及BMP⁃7蛋白（[χ2] = 48.388,P < 0.01）表达水平差异均有统计学意义。TGF⁃β1蛋白与p38MAPK、BMP⁃7蛋白表达水平均呈显著正相关（r = 0.607、0.702,P均 < 0.001）,BMP⁃7与p38MAPK蛋白表达水平亦呈显著正相关（r = 0.456,P < 0.001）。结论　TGF⁃β1、p38MAPK和BMP⁃7蛋白通过相互作用、共同介导了肝多房棘球蚴病肝纤维化发生。     

Fibrogenic cell phenotype modifications during remodelling of normal and pathological human liver in cultured slices

Background: The debate concerning the potential remodelling and/or reversibility of cirrhotic lesions and biliary fibrosis is still open. Aims/Methods: In this work, we have used the precision‐cut liver slice (PCLS) model, which maintains cell–cell and cell–matrix interactions to study, by immunohistochemistry, the behaviour of the different fibrogenic cells, i.e. hepatic stellate cells (HSC) and portal fibroblasts, in cultured (for 1 week) PCLS derived from normal and fibrotic human livers. Results: In normal liver, before and after culture, α‐smooth muscle (SM) actin was present only in the vessel walls. Platelet‐derived growth factor (PDGF) receptor‐β was expressed before and after culture by portal fibroblasts, and appeared after culture in HSC. Before culture, CD 34 was not expressed in parenchyma, but appeared after culture in sinusoidal endothelial cells. In cirrhotic lesions, before culture, α‐SM actin, PDGF receptor‐β and Thy‐1 were expressed in septa; after culture, α‐SM actin expression disappeared but the expression of the PDGF receptor‐β and Thy‐1 was maintained. In cholestatic liver specimens, α‐SM actin, PDGF receptor‐β and Thy‐1 expression, which was present before culture in enlarged portal areas, disappeared after culture, and apoptosis was detected. In the parenchyma of both cirrhotic and cholestatic livers, the expression of the PDGF receptor‐β and of CD 34, which was not observed before culture, was present in HSC and sinusoidal endothelial cells, respectively, after culture. Conclusions: These results indicate that during remodelling of pathological tissues in cultured liver slices, the myofibroblastic cells derived from HSC or from portal fibroblasts show different behaviours, suggesting different mechanisms of activation/deactivation.     

Bezafibrate prevents hepatic stellate cell activation and fibrogenesis in a murine steatohepatitis model,and suppresses fibrogenic response induced by transforming growth factor‐β1 in a cultured stellate cell line

Aim: The aim of this study was to investigate the preventive actions of bezafibrate against non‐alcoholic steatohepatitis (NASH), the activation of hepatic stellate cells (HSC), and fibrogenesis by using a model of NASH and an in vitro model. Methods: Male KK‐A^y/TaJcl (KK‐A^y) mice were fed a methionine and choline‐deficient (MCD) diet or a MCD diet containing bezafibrate or pioglitazone for 7 weeks, after which biochemical parameters, pathological changes, and hepatic mRNA levels were assessed. An in vitro HSC model was designed by using a previously described RI‐T cell line stimulated by transforming growth factor‐β1 (TGF‐β1). Results: MCD diet‐fed KK‐A^y mice developed hepatic steatosis, oxidative stress, inflammation, and hepatic fibrosis. Bezafibrate markedly decreased the hepatic content of triglyceride accumulation of fatty droplets within hepatocytes, and increased the expression of hepatic fatty acid β‐oxidative genes in MCD diet‐fed KK‐A^y mice. Bezafibrate markedly inhibited the increases in the plasma alanine aminotransferase level and hepatic content of thiobarbituric acid‐reactive substances in this model. Moreover, it dramatically reduced hepatic inflammatory changes and fibrosis concomitantly with marked reductions in the mRNA levels for inflammatory cytokine, chemokine, and profibrogenic genes. Importantly, both bezafibrate and pioglitazone markedly reduced the mRNA levels of profibrogenic and fibrogenic genes in TGF‐β1‐stimulated cells. Conclusion: Bezafibrate improved hepatic steatosis and potently prevented inflammation, oxidative stress, HSC activation, and fibrogenesis in the liver. Moreover, this study was the first to demonstrate that bezafibrate directly inhibits hepatic fibrogenic response induced by TGF‐β1 in vitro. Hence bezafibrate may be a new therapeutic strategy against NASH and hepatic fibrosis.     

Bcl‐2 overexpression in hepatic stellate cell line CFSC‐2G,induces a pro‐fibrotic state

Background and Aim: Development of hepatic fibrosis is a complex process that involves oxidative stress (OS) and an altered balance between pro‐ and anti‐apoptotic molecules. Since Bcl‐2 overexpression preserves viability against OS, our objective was to address the effect of Bcl‐2 overexpression in the hepatic stellate cells (HSC) cell‐line CFSC‐2G under acetaldehyde and H₂O₂ challenge, and explore if it protects these cells against OS, induces replicative senescence and/or modify extracellular matrix (ECM) remodeling potential. Methods: To induce Bcl‐2 overexpression, HSC cell line CFSC‐2G was transfected by lipofection technique. Green fluorescent protein‐only CFSC‐2G cells were used as a control. Cell survival after H₂O₂ treatment and total protein oxidation were assessed. To determine cell cycle arrest, proliferation‐rate, DNA synthesis and senescence were assessed. Matrix metalloproteinases (MMP), tissue‐inhibitor of MMP (TIMP), transglutaminases (TG) and smooth muscle a‐actin (α‐SMA) were evaluated by western blot in response to acetaldehyde treatment as markers of ECM remodeling capacity in addition to transforming growth factor‐β (TGF‐β) mRNA. Results: Cells overexpressing Bcl‐2 survived ≈ 20% more than control cells when exposed to H₂O₂ and ≈ 35% proteins were protected from oxidation, but Bcl‐2 did not slow proliferation or induced senescence. Bcl‐2 overexpression did not change α‐SMA levels, but it increased TIMP‐1 (55%), tissue transglutaminases (tTG) (25%) and TGF‐β mRNA (49%), when exposed to acetaldehyde, while MMP‐13 content decreased (47%). Conclusions: Bcl‐2 overexpression protected HSC against oxidative stress but it did not induce replicative senescence. It increased TIMP‐1, tTG and TGF‐β mRNA levels and decreased MMP‐13 content, suggesting that Bcl‐2 overexpression may play a key role in the progression of fibrosis in chronic liver diseases.     

New mechanism of transforming growth factor‐β signaling in hepatoma: Dramatic up‐regulation of tumor initiating cells and epidermal growth factor receptor expression

Aim: Transforming growth factor‐β (TGF‐β) has dual activity in tumor cells. We studied the effect of TGF‐β on tumor‐initiating cells (TICs), which are similar in self‐renewal and differentiation features to normal adult stem cells. Methods: We used side population (SP) cells that exclude DNA binding dye Hoechst 33342 to obtain TICs, studied the differences in the kinetics of the SP cell response to TGF‐β treatment between hepatic tumor cell lines, and performed gene analysis. Results: SP cells from all cell lines have higher proliferative ability compared to non‐SP cells and they are drug resistant. TGF‐β treatment increased the percentage of SP cells (%SP) and the survival rate; chemotherapeutic drug resistance developed only in K‐251 SP cells. Gene analysis showed that TGF‐β up‐regulated epidermal growth factor receptor (EGFR) only in K‐251 cells. There were no EGFR mutations in K‐251, which had been reported in lung cancer. Knockdown of Smad4 using the small‐interfering RNA technique in K‐251 cells inhibited EGFR overexpression and significantly decreased the %SP. In contrast, the JNK inhibitor had little effect on EGFR expression or the %SP. Conclusion: TGF‐β treatment of K‐251 cells causes tumor progression and the anti‐cancer drug resistant phenotype by increasing SP.     

Cellular sources of extracellular matrix in hepatic fibrosis

The deposition of increased and abnormal extracellular matrix is the hallmark of liver fibrosis. Hepatic stellate cells are well known as the major source of the fibrillar collagens and other components of the liver scar, but are now appreciated to be only one of many potentially fibrogenic cell populations in the diseased liver. Portal fibroblasts and circulating mesenchymal cells derived from the bone marrow are also important sources of matrix proteins in fibrosis. Recent data suggest that hepatocytes and biliary epithelial cells undergo an epithelial to mesenchymal transition, similarly assuming a fibrogenic phenotype. Sinusoidal endothelial cells and hepatocytes produce specific matrix proteins important in liver health and disease. The future challenge will be to define more explicitly the roles of these different fibrogenic cell populations in fibrosis in a disease-specific way.