Oral imatinib treatment reduces early fibrogenesis but does not prevent progression in the long term

BACKGROUND/AIMS: Transactivated hepatic stellate cells (HSCs) represent the key source of extra cellular matrix (ECM) in fibrotic liver. Imatinib, a potent inhibitor of the PDGF receptor tyrosine kinase, reduces HSC proliferation and fibrogenesis when treatment is initiated before fibrosis has developed. We tested the antifibrotic potential of imatinib in ongoing liver injury and in established fibrosis. METHODS: BDL-rats were gavage fed with 20 mg/kg/d imatinib either early (days 0-21) or late (days 22-35) after BDL. Untreated BDL-rats served as controls. ECM and activated HSCs were quantified by morphometry. Tissue activity of MMP-2 was determined by gelatin zymography. mRNA expression of TIMP-1 and procollagen alpha1(I) were measured by RT-PCR. Liver tissue concentration of imatinib was measured by tandem mass spectrometry. RESULTS: Early imatinib reduced ECM formation by 30% (P=0.0455) but left numbers of activated HSCs and procollagen I expression unchanged. MMP-2 activity and TIMP-1 expression were reduced by 50%. Late imatinib treatment did not alter histological or molecular markers of fibrogenesis despite high imatinib tissue levels. CONCLUSIONS: The antifibrotic effectiveness of imatinib is limited to the early phase of fibrogenesis. In ongoing liver injury other mediators most likely compensate for the inhibited PDGF effect.     

The nuclear receptor SHP mediates inhibition of hepatic stellate cells by FXR and protects against liver fibrosis

BACKGROUND AND AIMS: The farnesoid X receptor (FXR) is an endogenous sensor for bile acids and inhibits bile acid synthesis by inducing small heterodimer partner (SHP) gene expression. The aim of this study was to investigate whether FXR is expressed by and modulates function of hepatic stellate cells (HSCs). METHODS: The antifibrotic activity of FXR ligand was tested in 2 rodent models: the porcine serum and bile duct ligation (BDL). RESULTS: Twelve-week administration of 1-10 mg/kg 6-ethyl chenodeoxycholic acid (6-ECDCA), a synthetic FXR ligand, to porcine serum-treated rats prevented liver fibrosis development and reduced liver expression of alpha1(I) collagen, TGF-beta1 and alpha-SMA mRNA by approximately 90%. Therapeutic administration of 6-ECDCA, 3 mg/kg, to BDL rats reduced liver fibrosis and alpha1(I) collagen, transforming growth factor (TGF)-beta1, alpha-SMA, and tissue metalloproteinase inhibitor (TIMP)-1 and 2 messenger RNA (mRNA) by 70%-80%. No protection was observed in BDL rats treated with CDCA, 3 mg/kg, and ursodeoxycholic acid, 15 mg/kg. FXR expression was detected in HSCs. Exposure of HSCs to FXR ligands caused a 3-fold increase of SHP, reduced alpha1(I)collagen and TGF-beta1 by approximately 60%-70% and abrogates alpha1(I) collagen mRNA up-regulation induced by thrombin and TGF-beta1. By retrovirus infection and small interference RNA, we generated SHP overexpressing and SHP-deficient HSC-T6. Using these cell lines, we demonstrated that SHP binds JunD and inhibits DNA binding of adaptor protein (AP)-1 induced by thrombin. CONCLUSIONS: By demonstrating that an FXR-SHP regulatory cascade promotes resolution of liver fibrosis, this study establish that FXR ligands might represent a novel therapeutic option to treat liver fibrosis.     

Loss of MMP 13 attenuates murine hepatic injury and fibrosis during cholestasis

Cholestasis occurs in a variety of clinical settings and often results in liver injury and secondary biliary fibrosis. Several matrix metalloproteinases (MMPs) are upregulated in the liver during cholestasis. The function of the major interstitial collagenase, MMP-13, in the initial phase of liver fibrosis is unknown. The aim of this study was to evaluate the role of MMP-13 during the development of cholestasis-induced liver fibrosis by comparing wild-type and MMP-13-deficient mice. Cholestasis was induced by bile duct ligation (BDL) for 5 days or 3 weeks. Activation and proliferation of hepatic stellate cells (HSCs) were detected by immunohistochemistry. Expression of MMP-13 mRNA increased significantly in BDL livers of WT mice. After BDL for 3 weeks liver fibrosis was suppressed in MMP-13-deficient mice versus WT animals. Activation and proliferation of HSCs were also suppressed in livers of MMP-13-deficient mice after BDL. To clarify the mechanism of this suppression, samples from 5-day BDL mice were used for evaluation of liver injury. Compared with those in WT animals, serum ALT and the number of hepatic neutrophils were reduced in MMP-13-deficient mice. Increased expression of the mRNA of inflammatory mediators such as tumor necrosis factor-alpha (TNF-alpha) was significantly suppressed in livers of MMP-13-deficient mice. Upregulation of fibrogenic markers, for example, transforming growth factor beta1 (TGF-beta1), was also significantly suppressed in livers of MMP-13-deficient mice versus in WT mice. In conclusion, distinct from the known function of interstitial collagenase to reduce liver fibrosis by degrading the extracellular matrix, MMP-13 contributes to accelerating fibrogenesis in cholestatic livers by mediating the initial inflammation of the liver.     

Effect of selective cyclooxygenase-2 inhibitor meloxicam on liver fibrosis in rats with ligated common bile ducts.

Aim: Cholestasis triggers fibrogenesis in the liver. Hepatic cyclooxygenase-2 (COX-2) expression increases in various chronic liver diseases caused either by viruses or toxins. We hypothesized that selective COX-2 inhibitor meloxicam could suppress inflammation and fibrogenesis in a rat model of cholestasis induced by bile duct ligation (BDL). Methods: Forty-three Sprague-Dawley rats were assigned to one of four treatment groups (sham-operation, BDL, daily meloxicam injections following BDL, and daily meloxicam injection without BDL). Liver histopathology was analyzed with hematoxylin-eosin and Masson's trichrome staining. The expression of alpha-smooth muscle actin (alpha-SMA), transforming growth factor-beta1 (TGF-beta1), and COX-2 were measured with immunohistochemical staining. The levels of COX-2, TGF-beta1, and matrix metalloproteinase-9 (MMP-9) production were measured with the Western blot method and an enzyme immunoassay. Results: Meloxicam treatment attenuated the expression of alpha-SMA, TGF-beta1, and COX-2 in rats that were treated with BDL for 3 weeks. This was associated with a marked reduction in collagen accumulation and histological improvement. In addition, meloxicam treatment was found to downregulate the levels of hepatic COX-2, TGF-beta1, and MMP-9 production. Conclusion: Cholestasis in BDL rats induces hepatic COX-2 expression. Selective COX-2 inhibitor meloxicam reduces BDL-induced hepatic fibrosis, and this is associated with reduced hepatic TGF-beta1 expression as well as decreased cyclooxygenase activity in the liver.     

Antifibrotic effects of interleukin-10 on experimental hepatic fibrosis

BACKGROUND/AIMS: To study the effects of interleukin-10 on hepatic stellate cells and liver tissue in experimental rats hepatic fibrosis. METHODOLOGY: Rat hepatic fibrosis model induced by carbon tetrachloride was established. Liver tissues were harvested from the rats administered CCl4 with or without IL-10 treatment and the animals of the control group. The expression of TGF-beta1, MMP-2 and TIMP-1 in the liver tissues was measured by S-P immunohistochemistry. In addition, another model was established; HSCs in rats in each group were isolated. RT-PCR was employed to analyze TGF-beta1, MMP-2 and TIMP-1 mRNA expression in cells and immunocytochemistry was performed to detect protein expression of alpha-SMA, NF-kappaB, TGF-beta1, MMP-2 and TIMP-1 in HSCs. RESULTS: Rat hepatic fibrosis was developed successfully. The fibrosis changes were partially reversed by simultaneous administration of IL-10. The positive signals of TGF-beta1, MMP-2 and TIMP-1 were observed more frequently (P<0.05) in the CCl4-treated group compared to those in the IL-10-treated group and the control group. HSCs were successfully isolated. TGF-beta1, MMP-2 and TIMP-1 mRNA in HSCs increased obviously during the course of hepatic fibrosis, and their levels were decreased after the treatment with IL-10 (P<0.05). The immunocytochemistry positive levels for TGF-beta1, MMP-2, TIMP-1, alpha-SMA and NF-kappaB in the fibrogenesis group were increased significantly compared to the normal group (P<0.01). The positive signals decreased significantly (P<0.05) after the treatment with IL-10. CONCLUSIONS: The expression of TGF-beta1, MMP-2 and TIMP-1 increased in liver or in HSC of hepatic fibrosis rats and decreased after treatment with IL-10. The IL-10 could inhibit the activation of HSCs and make an antifibrogenic process come into effect in this way.     

TIMP-2、MMP-2和MT1-MMP在活化肝星状细胞中的表达及对细胞外基质合成分泌的影响

目的 研究金属蛋白酶组织抑制剂-2(TIMP-2)、基质金属蛋白酶-2(MMP-2)和膜型基质金属蛋白酶-1(MT1-MMP)在活化肝星状细胞(HSCs)中的表达,观察其对细胞外基质(ECM)合成分泌的影响.方法 原代分离培养大鼠HSCs活化后,分别给予40～160 pmol化学合成经修饰抗TIMP-2 siRNA进行干预,检测培养细胞上清液透明质酸(HA)、Ⅲ型前胶原(PCⅢ)和羟脯氨酸(Hyp)的含量,采用荧光实时定量PCR法检测TIMP-2、MMP-2、MT1-MMP、MMP-13、COL Ⅰ和COL Ⅲ mRNA的表达,western印迹检测TIMP-2、MT1-MMP和MMP-13蛋白表达及明胶酶谱法检测MMP-2蛋白表达.结果 应用化学合成经修饰抗TIMP-2 siRNA后,TIMP-2、MMP-2、MT1-MMP、COL Ⅰ和COL Ⅲ的表达明显降低,而MMP-13的表达则明显增加,培养细胞上清液中HA、PCⅢ和Hyp的含量也明显减少.结论 TIMP-2通过MT1-MMP介导MMP-2的活化,抑制TIMP-2的表达,MT1-MMP和MMP-2的表达随之降低,而HSCs合成分泌ECM也相应减少.     

IFN-gamma abrogates profibrogenic TGF-beta signaling in liver by targeting expression of inhibitory and receptor Smads

BACKGROUND/AIMS: In a randomized open-labeled multicenter trial with patients suffering from chronic HBV infection, we recently identified a benefit of 9-month IFN-gamma treatment resulting in decreased fibrosis scores and a reduced number of alpha-smooth muscle actin-positive hepatic stellate cells (HSCs). Approaches opposing profibrogenic activities of TGF-beta may be amenable in chronic liver disease. According to experimental models, IFN-gamma counteracts several TGF-beta effects. METHODS: The crosstalk of IFN-gamma and TGF-beta signaling relevant for fibrogenesis was investigated in primary cultured rat HSCs and a cell line representing activated HSCs. RESULTS: In vitro studies with HSCs demonstrate that TGF-beta-dependent activation of (CAGA)9-MLP-Luc, a Smad3/4 responsive reporter construct, was significantly decreased by IFN-gamma, indicating a TGF-beta antagonizing function. IFN-gamma induced the activity of the Smad7 promoter and Smad7 protein expression via STAT-1 signaling. In contrast to TGF-beta, IFN-gamma was able to induce Smad7 expression in activated HSCs providing increased protein levels for at least 12h. In addition, expression of Smad2/3 was reduced by IFN-gamma and activation of Smads2/3 was abrogated. CONCLUSIONS: IFN-gamma displays antifibrotic effects in liver cells via STAT-1 phosphorylation, upregulation of Smad7 expression and impaired TGF-beta signaling.     

Regulation of matrix metallo-proteinase expression by extracellular matrix components in cultured hepatic stellate cells

Hepatic stellate cells (HSC) changed their morphology and function including production of matrix metalloproteinases (MMPs) in response to extracellular matrix (ECM) component used as a substratum in culture. We examined in this study the regulatory role of ECM component on expression of MMPs and tissue inhibitor of metalloproteinase (TIMP) in rat HSCs cultured on polystyrene, type I collagen-coated surface, type I collagen gel, or Matrigel, respectively. When cultured on type I collagen gel, HSCs showed the asteroid cell shape and MMP-1 activity, as detected by in situ zymography. Expression of MMP-1 protein and mRNA were examined by using immunofluorescence staining and RT-PCR analysis in HSCs cultured on type I collagen gel. Active form of MMP-2 was detected by gelatin zymography in the conditioned medium of HSCs cultured on type I collagen gel, whereas it was not detected when HSCs were cultured on polystyrene, type I collagen-coated surface, or Matrigel. Increased MMP-2 mRNA was detected by RT-PCR in HSCs cultured on type I collagen gel. Increased MT1-MMP proteins were shown to localize on the cell membrane by using immunofluorescence staining in HSCs cultured on type I collagen gel. Elevated expression of membrane-type matrix metallproteinase-1 (MT1-MMP) mRNA and tissue inhibitor of metalloproteinase-2 (TIMP-2) mRNA was detected by RT-PCR in HSCs cultured on type I collagen-coated surface or type I collagen gel. These results indicate that expression of MMPs and TIMP-2 is regulated by ECM components in cultured HSCs, suggesting an important role of HSCs in the remodeling of liver tissue.     

Relaxin inhibits effective collagen deposition by cultured hepatic stellate cells and decreases rat liver fibrosis in vivo

BACKGROUND: Following liver injury, hepatic stellate cells (HSC) transform into myofibroblast-like cells (activation) and are the major source of type I collagen and the potent collagenase inhibitors tissue inhibitors of metalloproteinases 1 and 2 (TIMP-1 and TIMP-2) in the fibrotic liver. The reproductive hormone relaxin has been reported to reduce collagen and TIMP-1 expression by dermal and lung fibroblasts and thus has potential antifibrotic activity in liver fibrosis. AIMS: To determine the effects of relaxin on activated HSC. METHODS: Following isolation, HSC were activated by culture on plastic and exposed to relaxin (1-100 ng/ml). Collagen deposition was determined by Sirius red dye binding and radiolabelled proline incorporation. Matrix metalloproteinase (MMP) and TIMP expression were assessed by zymography and northern analysis. Transforming growth factor beta1 (TGF-beta1) mRNA and protein levels were quantified by northern analysis and ELISA, respectively. RESULTS: Exposure of activated HSC to relaxin resulted in a concentration dependent decrease in both collagen synthesis and deposition. There was a parallel decrease in TIMP-1 and TIMP-2 secretion into the HSC conditioned media but no change in gelatinase expression was observed. Northern analysis demonstrated that primary HSC, continuously exposed to relaxin, had decreased TIMP-1 mRNA expression but unaltered type I collagen, collagenase (MMP-13), alpha smooth muscle actin, and TGF-beta1 mRNA expression. CONCLUSION: These data demonstrate that relaxin modulates effective collagen deposition by HSC, at least in part, due to changes in the pattern of matrix degradation.     

Role of the spleen in liver fibrosis in rats may be mediated by transforming growth factor beta-1

BACKGROUND: The effect of the spleen on the cirrhotic liver is unknown. Transforming growth factor-beta 1 (TGF-beta 1), which plays a crucial role in the matrix production during liver fibrosis, is an inhibitory factor regarding the regeneration of hepatocytes. In this study, we investigated the TGF-beta 1 production in the spleen of cirrhotic rats and the effects of a splenectomy on the healing process from liver fibrosis. METHODS: Thirty-six Wistar male rats were used. Thioacetamide (TAA) was administered intraperitoneally for 24 weeks. The rats underwent either a sham operation (TAA + Sham) or a splenectomy (TAA + SPL). The improvements in liver fibrosis and liver regeneration were investigated 10, 30 and 60 days after the operations in each group. The effect of a splenectomy on the plasma concentration of TGF-beta 1 in the portal vein was investigated by ELISA. The TGF-beta 1 expressions in the spleen were measured using immunohistochemical staining and the degree of such expression was measured using RT-PCR. The activity of TGF-beta 1 in the portal vein of TAA + Sham and TAA + SPL was assessed by the inhibiting effect of rat parenchymal hepatocyte proliferation in primary culture. RESULTS: Liver regeneration (PCNA-labeling index) in the TAA + SPL rats was stimulated more at 10 and 30 days after the operation (P < 0.05) than in the TAA + Sham rats, and the improvement of liver fibrosis (fibrosis rate) in the TAA + SPL rats was higher at 60 days (P < 0.05) than in the TAA + Sham rats. The plasma concentration of TGF-beta1 of the portal vein in TAA + SPL rats was significantly lower than in the TAA + Sham rats for each period. Immunohistochemically, TGF-beta1-positive stained cells were recognized in the spleen macrophages in the red pulp of cirrhotic rats. The plasma of the TAA + Sham rats at 10 and 30 days after the operation was significantly stronger than that of the TAA + SPL rats in inhibiting the proliferation of rat hepatocytes of primary culture. Inhibitory effects were then dose-dependently neutralized by monoclonal TGF-beta 1 antibody. CONCLUSION: Spleen-derived TGF-beta 1 may thus play an inhibitory role in the healing of liver cirrhosis by inhibiting the regeneration of the damaged liver.     

Smad7 prevents activation of hepatic stellate cells and liver fibrosis in rats

BACKGROUND & AIMS: Numerous studies implicate transforming growth factor (TGF)-beta signaling in liver fibrogenesis. To perturb the TGF-beta pathway during this process, we overexpressed Smad7, an intracellular antagonist of TGF-beta signaling, in vivo and in primary-cultured hepatic stellate cells (HSCs). METHODS: Ligation of the common bile duct (BDL) was used to induce liver fibrosis in rats. Animals received injections of an adenovirus carrying Smad7 cDNA into the portal vein during surgery and via the tail vein at later stages. The effect of Smad7 on TGF-beta signaling and activation of HSC was further analyzed in primary-cultured cells. RESULTS: Smad7-overexpressing BDL rats displayed reduced collagen and alpha-SMA expression and reduced hydroxyproline content in the liver, when compared with animals administered AdLacZ. Such a beneficial effect was also observed when Smad7 was expressed in animals with established fibrosis. Accordingly, Smad7 arrested transdifferentiation of primary-cultured HSCs. AdSmad7 infected cells remained in a quiescent stage and retained storage of vitamin A droplets. Smad7 expression totally blocked TGF-beta signal transduction, shown by inhibiting Smad2/3 phosphorylation, nuclear translocation of activated Smad complexes, and activation of (CAGA)(9)-MLP-Luc, resulting in decreased collagen I expression. Smad7 also abrogated TGF-beta-dependent proliferation inhibition of HSC. Smad7 did not decrease expression of alpha-SMA, but immunofluorescent staining with anti alpha-SMA antibodies displayed destruction of the fibrillar organization of the actin cytoskeleton. CONCLUSIONS: In summary, gene transfer of Smad7 inhibits experimental fibrogenesis in vivo. Studies with isolated HSC suggest that the underlying mechanisms involve inhibition of TGF-beta signaling and HSC transdifferentiation.     

Id1 is a critical mediator in TGF-beta-induced transdifferentiation of rat hepatic stellate cells

Transforming growth factor (TGF)-beta is critically involved in the activation of hepatic stellate cells (HSCs) that occurs during the process of liver damage, for example, by alcohol, hepatotoxic viruses, or aflatoxins. Overexpression of the TGF-beta antagonist Smad7 inhibits transdifferentiation and arrests HSCs in a quiescent stage. Additionally, bile duct ligation (BDL)-induced fibrosis is ameliorated by introducing adenoviruses expressing Smad7 with down-regulated collagen and alpha-smooth muscle actin (alpha-SMA) expression. The aim of this study was to further characterize the molecular details of TGF-beta pathways that control the transdifferentiation process. In an attempt to elucidate TGF-beta target genes responsible for fibrogenesis, an analysis of Smad7-dependent mRNA expression profiles in HSCs was performed, resulting in the identification of the inhibitor of differentiation 1 (Id1) gene. Ectopic Smad7 expression in HSCs strongly reduced Id1 mRNA and protein expression. Conversely, Id1 overexpression in HSCs enhanced cell activation and circumvented Smad7-dependent inhibition of transdifferentiation. Moreover, knock-down of Id1 in HSCs interfered with alpha-SMA fiber formation, indicating a pivotal role of Id1 for fibrogenesis. Treatment of HSCs with TGF-beta1 led to increased Id1 protein expression, which was not directly mediated by the ALK5/Smad2/3, but the ALK1/Smad1 pathway. In vivo, Id1 expression and Smad1 phosphorylation were co-induced during fibrogenesis. In conclusion, Id1 is identified as TGF-beta/ALK1/Smad1 target gene in HSCs and represents a critical mediator of transdifferentiation that might be involved in hepatic fibrogenesis. Supplementary material for this article can be found on the HEPATOLOGY website (http://interscience.wiley.com/jpages/0270-9139/suppmat/index.html).