All‐trans‐retinoic acid ameliorates carbon tetrachloride‐induced liver fibrosis in mice through modulating cytokine production

Background/Aims: Liver fibrosis with any aetiology, induced by the transdifferentiation and proliferation of hepatic stellate cells (HSCs) to produce collagen, is characterized by progressive worsening in liver function, leading to a high incidence of death. We have recently reported that all‐trans‐retinoic acid (ATRA) suppresses the transdifferentiation and proliferation of lung fibroblasts and prevents radiation‐ or bleomycin‐induced lung fibrosis. Methods: We examined the impact of ATRA on carbon tetrachloride (CCl₄)‐induced liver fibrosis. We performed histological examinations and quantitative measurements of transforming growth factor (TGF)‐β1 and interleukin (IL)‐6 in CCl₄‐treated mouse liver tissues with or without the administration of ATRA, and investigated the effect of ATRA on the production of the cytokines in quiescent and activated HSCs. Results: CCl₄‐induced liver fibrosis was attenuated in histology by intraperitoneal administration of ATRA, and the overall survival rate at 12 weeks was 26.5% without ATRA (n=25), whereas it was 75.0% (n=24) in the treatment group (P=0.0187). In vitro studies disclosed that the administration of ATRA reduced (i) the production of TGF‐β1, IL‐6 and collagen from HSCs, (ii) TGF‐β‐dependent transdifferentiation of the cells and IL‐6‐dependent cell proliferation and (iii) the activities of nuclear factor‐κB p65 and p38mitogen‐activated protein kinase, which stimulate the production of TGF‐β1 and IL‐6, which could be the mechanism underlying the preventive effect of ATRA on liver fibrosis. Conclusions: Our findings indicate that ATRA ameliorates liver fibrosis. As the oral administration of the drug results in good compliance, ATRA could be a novel approach in the treatment of liver fibrosis.     

Cytokine blockade inhibits hepatic tissue inhibitor of metalloproteinase‐1 expression and up‐regulates matrix metalloproteinase‐9 in toxic liver injury

Abstract: Background: Tissue inhibitor of metalloproteinases (TIMP)‐1, the most important endogenous inhibitor of matrix metalloproteinases, plays a pivotal role in the pathogenesis of liver fibrosis and may represent an effective therapeutic target in the design of antifibrotic strategies for chronic liver diseases. Methods: Intraperitoneal application of a single dose of either tumor necrosis factor (TNF)‐α or interleukin (IL)‐1β in mice led to an enhanced expression of hepatic TIMP‐1 after 4–16 h. Male Sprague–Dawley rats were treated with carbon tetrachloride (CCl₄) in the presence and absence of specific TNF‐α and IL‐1β inhibitors. Results: Real‐time PCR revealed a significant increase of TIMP‐1 mRNA in total rat liver 24 h after CCl₄ injection. Repetitive injection of both, etanercept and anakinra, before and after CCl₄ injection effectively inactivated TNF‐α and IL‐1β. Anticytokine pretreatment reduced the increase of TIMP‐1 expression after a single CCl₄ injection by 50% and 75%, respectively. In contrast to CCl₄‐treated rats with and without TNF‐α blockade, IL‐1β inactivation caused a sevenfold increase in matrix metalloproteinases‐9 mRNA levels. Conclusions: In conclusion, TIMP‐1 expression is up‐regulated in the early phase of toxic liver injury by proinflammatory cytokines such as TNF‐α and IL‐1β in rodents. Pharmacological inactivation of these cytokines significantly reduces TIMP‐1 gene expression. Our data provide a potential new antifibrotic approach.     

Effect of human umbilical cord blood‐derived mesenchymal stem cells in a cirrhotic rat model

Background/Aim: Cirrhosis is a long‐term consequence of chronic hepatic injury and no effective therapy is currently available for this disease. Recent reports have shown that the mesenchymal stem cells (MSCs) have the capacity to differentiate into hepatocytes, and umbilical cord blood is a rich source of MSCs. Hence, we investigated the effect of infusing of human umbilical cord blood‐derived MSCs (HMSCs) in carbon tetrachloride (CCl₄)‐induced cirrhosis in a rat model. Methods: The effect of HMSCs on cirrhosis was evaluated using haematoxylin and eosin and Masson's trichrome staining. To evaluate cirrhosis‐related factors, we measured protein and mRNA expression of transforming growth factor β₁ (TGF‐β₁), collagen type I and α‐smooth muscle actin (α‐SMA). Results: Histological findings showed that liver fibrosis in rats was alleviated by HMSCs infusion. Interestingly, CM‐DiI‐labelled HMSCs expressed the hepatocyte‐specific markers, human albumin and α‐fetoprotein. Infusion of HMSCs significantly inhibited TGF‐β₁, collagen type I and α‐SMA expressions in CCl₄‐induced cirrhotic rats. Conclusion: Our results showed that HMSCs infusion could improve liver fibrosis in rats with CCl₄‐induced cirrhosis, raising the possibility for clinical use of HMSCs in the treatment of cirrhosis.     

Inhibition of hepatic tumour necrosis factor‐α attenuates the anandamide‐induced vasoconstrictive response in cirrhotic rat livers

Background: Increased anandamide, an endocannabinoid that interacts with both cannabinoid CB₁ and CB₂ receptors, can induce hepatic vasoconstrictive responses that contribute to the increased intrahepatic resistance (IHR) in cirrhotic rats. Chronic endotoxaemia and the subsequent release of tumour necrosis factor‐α (TNF‐α) are suggested to result in increased anandamide in cirrhotic livers. Thalidomide, which inhibited TNF‐α effectively, has been used clinically in states of chronic TNF‐α elevation with encouraging results. Aims: This study explores the possible effects of thalidomide on hepatic endocannabinoids and microcirculation of cirrhotic rats. Methods: Portal venous pressure (PVP), superior mesenteric arterial blood flow (SMA BF), hepatic TNF‐α, interleukin (IL‐6), protein expression of CB₁ and CB₂ receptor and thromboxane synthase (TXS) were measured in bile duct‐ligated (BDL) rats receiving 1‐month of vehicle (BDL‐V) or thalidomide (BDL‐thalido). The degree of hepatic fibrosis was also assessed. In the liver perfusion system, IHR and concentration–response curves of the portal perfusion pressure to anandamide were evaluated. Results: In BDL‐thalido rats, PVP, IHR and hepatic levels of TNF‐α and IL‐6, protein expression of CB₁ receptors, TXS and hepatic fibrosis were lower than in BDL‐V rats. In BDL‐thalido rat livers, the attenuation of the vasoconstrictive response to anandamide was associated with an upregulation of the CB₂ receptor and a downregulation of the CB₁ receptor. Nevertheless, SMA BF was not different between BDL‐thalido and BDL‐V rats. Conclusions: Thalidomide decreased the PVP and IHR through the attenuation of anandamide‐induced constrictive response, decreasing the production of TNF‐α, IL‐6 and TXA₂ in the liver and the suppression of hepatic fibrogenesis of rats with biliary cirrhosis of this study.     

Pentoxifylline prevents pig serum‐induced rat liver fibrosis by inhibiting interleukin‐6 production

Background/Aim: Pig serum‐induced rat liver fibrosis is a model of liver fibrosis in the absence of obvious hepatocyte injury. Penoxifylline (PTX), a xanthine derivative, which is a well‐known suppressor of tumor necrosis factor‐α (TNF‐α) production from inflammatory cells, has also been shown to inhibit the growth of hepatic stellate cells and to inhibit collagen synthesis in these cells in vitro. We investigated the effect of PTX on pig serum‐induced liver fibrosis in vivo, and assessed the mechanisms of prevention of fibrogenesis by this drug. Methods: Male Wistar rats were given intraperitoneal injections of 0.5 ml normal pig serum twice a week for 10 weeks with or without concomitant oral administration of PTX (20 mg/kg). Results: Rats that received pig serum showed significant liver fibrosis, and their serum interleukin‐6 (IL‐6) and hyaluronic acid levels were significantly increased. The serum levels of IL‐6 were well correlated with the serum levels of hyaluronic acid, and increased as the liver fibrosis progressed. Penoxifylline prevented the development of fibrosis in this animal model and reduced the serum levels of IL‐6 in a dose‐dependent manner. In vitro, by the addition of PTX to the culture medium of the rat hepatic stellate cells (HSCs), the proliferation of the HSCs was significantly inhibited and IL‐6 in the culture supernatant was also reduced significantly. Exogenous addition of IL‐6 partially restored the proliferation. Conclusion: Penoxifylline prevents pig serum‐induced rat liver fibrosis by inhibiting the proliferation of HSCs and by inhibiting the production of IL‐6 from HSCs.     

Differential effects of GdCl3- or MDP treatment on rat liver microcirculation and gene expression in the hepatic non-parenchymal cell fraction in LPS shock

Background: Dichloromethylenebisphosphonate (MDP) and gadolinium chloride (GdCl₃) are substances frequently used for experimental depletion of Kupffer Cells (KC) in models of endotoxin shock. The aim was to determine whether depletion of KC through pretreatment with GdCl₃ or MDP alters the hepatic microcirculation during lipopolysaccharide (LPS)‐induced shock in rats and to test if there are substance‐specific differences. Methods: Rats received either MDP or GdCl₃ or saline prior to induction of LPS shock. Hepatic microcirculation was evaluated by intravital microscopy (sinusoidal diameter, sinusoidal bloodflow, leukocyte adhesion), and the gene expression in the hepatic non‐parenchymal cell fraction was determined by RT‐PCR. Results: GdCl₃ pretreatment prevented sinusoidal narrowing but did not restore sinusoidal blood flow and did not normalize leukocyte‐endothelial interaction time after LPS. In contrast, MDP pretreatment improved hepatic microcirculation consistently for all parameters measured compared to GdCl₃ pretreated animals. In the non‐parenchymal cell fraction, eNOS gene expression was preserved and gene expression of TNF‐α was blocked after MDP but not after GdCl₃ application prior to LPS shock. Conclusions: The results show that GdCl₃ and MDP cannot be used equivalently for experimental KC depletion in the condition of LPS‐induced shock. These findings should be taken into consideration in studies that evaluate the role of Kupffer cells in models of endotoxin‐induced shock.     

Exogenous melatonin protects small‐for‐size liver grafts by promoting monocyte infiltration and releases interleukin‐6

Defective regeneration of small‐for‐size (SFS ) liver remnants and partial grafts remains a key limiting factor in the application of liver surgery and transplantation. Exogenous melatonin (MLT ) has protective effects on hepatic ischemia‐reperfusion injury (IRI ), but its influence on graft regeneration is unknown. The aim of the study is to investigate the role of MLT in IRI and graft regeneration in settings of partial liver transplantation. We established three mouse models to study hepatic IRI and regeneration associated with partial liver transplantation: (I) IR +PH group: 60 minutes liver ischemia (IR ) plus 2/3 hepatectomy (PH ); (II ) IR +exPH group: 60 minutes liver IR plus extended hepatectomy (exPH ) associated with the SFS syndrome; (III ) SFS ‐LT group: Arterialized 30% SFS liver transplant. Each group was divided into MLT or vehicle‐treated subgroups. Hepatic injury, inflammatory signatures, liver regeneration, and animal survival rates were assessed. MLT reduced liver injury, enhanced liver regeneration, and promoted interleukin (IL ) 6, IL 10, and tumor necrosis factor‐α release by infiltrating, inflammatory Ly6C+ F4/80+ monocytes in the IR +PH group. MLT ‐induced IL 6 significantly improved hepatic microcirculation and survival in the IR +exPH model. In the SFS ‐LT group, MLT promoted graft regeneration and increased recipient survival along with increased IL 6/GP 130‐STAT 3 signaling. In IL 6 ^?/? mice, MLT failed to promote liver recovery, which could be restored through recombinant IL 6. In the IR +exPH and SFS ‐LT groups, inhibition of the IL 6 co‐receptor GP 130 through SC 144 abolished the beneficial effects of MLT . MLT ameliorates SFS liver graft IRI and restores regeneration through monocyte‐released IL 6 and downstream IL 6/GP 130‐STAT 3 signaling.     

The primary mitogen (TCPOBOP)‐induced hepatocyte proliferation is resistant to transforming growth factor‐ β‐1 inhibition

Background: Transforming growth factor (TGF)‐β‐1 is a very efficient inhibitor of hepatocyte proliferation in various in vivo and in vitro experimental systems. However, there are no data on whether it can influence the mitogenic response induced by primary hepatocyte mitogens. Aims: In this study, we compared the proliferative response in the liver between wild‐type and transgenic mice, overexpressing active TGF‐β‐1 in their liver following the treatment by a primary hepatocyte mitogen TCPOBOP (1,4‐bis[2‐(3,5‐dichloropyridyloxy)]benzene). Methods: The proliferative response was characterized by the immunohistochemical examination of pulse and cumulative bromodeoxyuridine labelling and by quantitative real‐time polymerase chain reaction analysis of cell cycle‐related genes. Results: Neither of the applied techniques revealed significant differences between the two groups of mice; furthermore, we observed the upregulation of TGF‐β‐1 expression following the mitogenic treatment. Conclusions: TGF‐β‐1 does not inhibit the primary mitogen‐induced proliferative response of the hepatocytes. This observation may provide an explanation for the divergent consequences of hepatic proliferations induced by partial hepatectomy or primary mitogenic treatment.     

Elevated concentrations of 15‐deoxy‐Δ12,14‐prostaglandin J2 in chronic liver disease propose therapeutic trials with peroxisome proliferator activated receptor γ‐inducing drugs

Background/Aims: Current knowledge confers a crucial role to connective tissue growth factor (CTGF/CCN2) in hepatic fibrogenesis. Hepatocytes are likely to be the major cellular source of CTGF in the liver in which CTGF is sensitively upregulated by TGF‐β. Recently, we demonstrated that the methylxanthine derivate caffeine leads to an upregulation of peroxisome proliferator activated receptor γ (PPARγ) expression in hepatocytes, thus sensitizing these cells to the well‐known inhibitory effect of 15‐deoxy‐Δ^12,14‐prostaglandin J₂ (15‐d‐PGJ₂) on CTGF expression. However, upregulation of the receptor alone is not sufficient per se; its physiological ligand 15‐d‐PGJ₂ is required to exert an inhibitory effect on transforming growth factor‐β (TGF‐β) target genes such as CTGF. Methods: This study compared serum concentrations of 15‐d‐PGJ₂ in Caucasian patients with fibrotic liver diseases (n=289), Caucasian controls (n=136) and Caucasian non‐liver disease (NLD) sick (n=307), as well as of Chinese patients with hepatocellular carcinoma (HCC) (n=43) and Chinese healthy controls (n=63) in order to characterize their suitability for therapeutic approaches with PPARγ‐inducing (i.e. CTGF inhibitory) drugs such as caffeine. Results: The presented data showed that Caucasian patients with ongoing hepatic fibrogenesis (mean 6.2±5.9 μg/L) displayed strikingly higher serum concentrations of 15‐d‐PGJ₂ than healthy probands (mean 2.3±1.0) and Caucasian patients with NLD (mean 2.7±1.4 μg/L). Similar results were found in Chinese patients with fully developed HCC (mean 1.3±0.7 μg/L) compared with Chinese healthy controls (mean 0.4±0.2 μg/L). Conclusions: In conclusion, our data thus proposed an increased suitability of these patient groups for therapeutic approaches with drugs inducing PPARγ expression, such as methylxanthine derivates.     

RNA interference targeting the platelet‐derived growth factor receptor β subunit ameliorates experimental hepatic fibrosis in rats

Background/Aims: Platelet‐derived growth factor (PDGF) is the strongest stimulator of the proliferation of hepatic stellate cells (HSCs). PDGF receptor β subunit (PDGFR‐β) is acquired on HSCs proliferation induced by PDGF. In this study, we aim to investigate the effect of PDGFR‐β small interference RNA (siRNA) on experimental hepatic fibrosis. Methods: We constructed a PDGFR‐β siRNA expression plasmid and investigated its effect on the activation of HSCs. Bromodeoxyuridine incorporation was performed to investigate the effect of PDGFR‐β siRNA on HSCs proliferation. A hydrodynamics‐based transfection method was used to deliver PDGFR‐β siRNA to rats with hepatic fibrosis. The distribution of transgenes in the liver was observed by immunofluorescence. The antifibrogenic effect of PDGFR‐β siRNA was investigated pathologically. Results: Platelet‐derived growth factor receptor‐β subunit siRNA could significantly downregulate PDGFR‐β expression, suppress HSCs activation, block the mitogen‐activated protein kinase signalling pathway and inhibit HSCs proliferation in vitro. PDGFR‐β siRNA expression plasmid could be delivered into activated HSCs by the hydrodynamics‐based transfection method, and remarkably improve the liver function of the rat model induced by dimethylnitrosamine and bile duct ligation. Furthermore, the progression of fibrosis in the liver was significantly suppressed by PDGFR‐β siRNA in both animal models. Conclusions: Platelet‐derived growth factor receptor‐β subunit siRNA may be presented as an effective antifibrogenic gene therapeutic method for hepatic fibrosis.     

Induction of lipocalin‐2 expression in acute and chronic experimental liver injury moderated by pro‐inflammatory cytokines interleukin‐1β through nuclear factor‐κB activation

Background: Lipocalin‐2 (LCN2) belongs to the lipocalin superfamily, sharing a barrel‐shaped tertiary structure with a hydrophobic pocket and an ability to bind lipophilic molecules. LCN2 has recently emerged as an important modulator of cellular homeostasis in several organs, i.e. heart, lung and kidney, but little is known about the expression of LCN2 in acute and chronic liver injury. Aims: In this study, we wanted to analyse the expression and regulation of LCN2 in models of acute and chronic experimental liver injury. Materials and methods: We analysed LCN2 expression in livers of rats subjected to bile duct ligation or repeated doses of carbon tetrachloride and tested the impact of various pro‐inflammatory cytokines in cultured primary liver cells. Results: By using primary cultures of hepatic stellate cells and hepatocytes isolated from normal and injured rat livers, we found a significant LCN2 expression in early hepatic stellate cell cultures, a lower expression in fully transdifferentiated myofibroblasts and no expression in freshly isolated hepatocytes. However, LCN2 expression and secretion in hepatocytes increased dramatically during culturing. In addition, chronic in vivo liver injury resulting from both bile duct ligation and repeated application of carbon tetrachloride resulted in rapid and well‐sustained induction of LCN2 expression. Immunohistochemistry and primary liver cell isolation identified injured hepatocytes as the main source of LCN2 production. LCN2 is strongly induced in both primary hepatocytes and immortalized hepatocellular carcinoma cell line HepG2 by the pro‐inflammatory cytokine interleukin‐1β via nuclear factor‐κB activation, but not by the profibrotic cytokines platelet‐derived growth factor and transforming growth factor‐β. Conclusion: LCN2 expression shows clear correlation to liver damage and resulting inflammatory responses, rather than to the degree of liver fibrosis, which in fact may imply a distinct diagnostic value as an early biomarker of liver inflammation.     

The role of IL‐1β in reduced IL‐7 production by stromal and epithelial cells: a model for impaired T‐cell numbers in the gut during HIV‐1 infection

Abstract. Thang PH, Ruffin N, Brodin D, Rethi B, Cam PD, Hien NT, Lopalco L, Vivar N, Chiodi F (Karolinska Institutet, Stockholm, Sweden; National Institute of Hygiene and Epidemiology, Hanoi, Vietnam; San Raffaele Scientific Institute, Milan, Italy). The role of IL‐1β in reduced IL‐7 production by stromal and epithelial cells: a model for impaired T‐cell numbers in the gut during HIV‐1 infection. J Intern Med 2010; 268 : 181–193. Objectives. Interleukin (IL)‐7 is a key cytokine in T‐cell homeostasis. Stromal cells, intestinal epithelial cells and keratinocytes are known to produce this cytokine. The mechanisms and cellular factors regulating IL‐7 production are still unclear. We assessed whether IL‐1β and interferon (IFN)‐γ, cytokines produced during inflammatory conditions, may impact on IL‐7 production. Design. We used human intestinal epithelial cells (DLD‐1 cell line) and bone marrow stromal cells (HS27 cell line), known to produce IL‐7; IL‐7 production was evaluated at the mRNA and protein levels. To assess whether treatment of HS27 cells with IL‐1β and/or IFN‐γ leads to changes in the gene expression of cytokines, Toll‐like receptors (TLRs) and chemokines, we analysed gene expression profiles using the whole‐genome microarray Human Gene 1.0 ST. Results. We found that IFN‐γ enhanced the expression of IL‐7 mRNA (P < 0.001) in both cell lines. IL‐1β treatment led to a significant down‐regulation (P < 0.001) of IL‐7 mRNA expression in both cell lines. The IL‐7 concentration in supernatants collected from treated DLD‐1 and HS27 cell cultures reflected the trend of IL‐7 mRNA levels. The gene profiles revealed dramatic changes in expression of cytokines and their receptors (IL‐7/IL‐7Rα; IL‐1α,IL‐1β/IL‐1R1; IFN‐γ/IFN‐γR1), of IFN regulatory factors (IRF‐1 and 2), of TLRs and of important chemo‐attractants for T cells. The microarray results were verified by additional methods. Conclusions. Our results are discussed in the setting of inflammation and T‐cell survival in the gut compartment during HIV‐1 infection where stromal and epithelial cells may produce factors that contribute to impaired IL‐7 homeostasis and homing of T cells.     

Role of angiotensin‐1 receptor blockade in cirrhotic liver resection

Background: The regeneration capacity of cirrhotic livers might be affected by angiotensin‐1 (AT1) receptors located on hepatic stellate cells (HSC). The effect of AT1 receptor blockade on microcirculation, fibrosis and liver regeneration was investigated. Materials and methods: In 112 Lewis rats, cirrhosis was induced by repetitive intraperitoneal injections of CCl₄. Six hours, 3, 7 and 14 days after partial hepatectomy or sham operation, rats were sacrificed for analysis. Animals were treated with either vehicle or 5 mg/kg body weight losartan pre‐operatively and once daily after surgery by gavage. Microcirculation and portal vein flow were investigated at 6 h. The degree of cirrhosis was assessed by Azan Heidenhein staining, activation of HSC by desmin staining, apoptosis by ssDNA detection and liver regeneration by Ki‐67 staining. Changes in expression of various genes important for liver regeneration and fibrosis were analysed at 6 h and 3 days. Haemodynamic parameters and liver enzymes were monitored. Results: Losartan treatment increased sinusoidal diameter, sinusoidal blood flow and portal vein flow after partial hepatectomy (P<0.05), but not after sham operation. AT1 receptor blockade resulted in increased apoptosis early after resection. HSC activation was reduced and after 7 days, a significantly lower degree of cirrhosis in resected animals was observed. Losartan increased the proliferation of hepatocytes at late time‐points and of non‐parenchymal cells early after partial hepatectomy (P<0.05). Tumour necrosis factor (TNF)‐α was significantly upregulated at 6 h and stem cell growth factor (SCF) was downregulated at 3 days (P<0.05). Conclusion: Losartan increased hepatic blood flow, reduced HSC activation and liver fibrosis, but interfered with hepatocyte proliferation after partial hepatectomy in cirrhotic livers.     

Effect of mutant β‐catenin on liver growth homeostasis and hepatocarcinogenesis in transgenic mice

Background: Mutations in the Wnt signalling pathway molecule β‐catenin are associated with liver cancer. Aims: Our aim was to confirm the effects of stabilized β‐catenin on liver growth, identify whether those effects were reversible and cell autonomous or non‐cell autonomous and to model β‐catenin‐induced liver cancer in mice. Methods: Using a liver‐specific inducible promoter, we generated transgenic mice in which the expression of mutant β‐catenin can be induced or repressed within hepatocytes in mice of different ages. Results: Similar to other models, the hepatic expression of mutant β‐catenin in our model beginning in utero or induced in quiescent adult liver resulted in a two‐fold liver enlargement and development of disease with a latency of 1–5 months, and mice displayed elevated blood ammonia and altered hepatic gene expression. Our model additionally allowed us to discover that molecular and phenotypic abnormalities were reversible following the inhibition of transgene expression. Hepatocyte transplant studies indicated that mutant β‐catenin could not increase the growth of transgene‐expressing foci in either growth‐permissive or ‐restrictive hepatic environments, but still directly altered hepatocyte gene expression. Mice with continuous but focal transgene expression developed hepatic neoplasms after the age of 1 year. Conclusions: Our findings indicate that hepatocyte gene expression is directly affected by mutant β‐catenin in a cell autonomous manner. However, hepatomegaly associated with diffuse hepatocyte‐specific expression of mutant β‐catenin is secondary to liver functional alteration or non‐cell autonomous. Both phenotypes are reversible. Nevertheless, some foci of transgene‐expressing cells progressed to carcinoma, confirming the association of mutant β‐catenin with liver cancer.