Activation of peroxisome proliferator-activated receptor-gamma contributes to the inhibitory effects of curcumin on rat hepatic stellate cell growth

Hepatic fibrogenesis occurs as a wound-healing process after many forms of chronic liver injury. Hepatic fibrosis ultimately leads to cirrhosis if not treated effectively. During liver injury, quiescent hepatic stellate cells (HSC), the most relevant cell type, become active and proliferative. Oxidative stress is a major and critical factor for HSC activation. Activation of peroxisome proliferator-activated receptor-gamma (PPAR-gamma) inhibits the proliferation of nonadipocytes. The level of PPAR-gamma is dramatically diminished along with activation of HSC. Curcumin, the yellow pigment in curry, is a potent antioxidant. The aims of this study were to evaluate the effect of curcumin on HSC proliferation and to begin elucidating underlying mechanisms. It was hypothesized that curcumin might inhibit the proliferation of activated HSC by inducing PPAR-gamma gene expression and reviving PPAR-gamma activation. Our results indicated that curcumin significantly inhibited the proliferation of activated HSC and induced apoptosis in vitro. We demonstrated, for the first time, that curcumin dramatically induced the gene expression of PPAR-gamma and activated PPAR-gamma in activated HSC. Blocking its trans-activating activity by a PPAR-gamma antagonist markedly abrogated the effects of curcumin on inhibition of cell proliferation. Our results provide a novel insight into mechanisms underlying the inhibition of activated HSC growth by curcumin. The characteristics of curcumin, including antioxidant potential, reduction of activated HSC growth, and no adverse health effects, make it a potential antifibrotic candidate for prevention and treatment of hepatic fibrosis.     

Neuregulin-1 suppresses cardiomyocyte apoptosis by activating PI3K/Akt and inhibiting mitochondrial permeability transition pore

In order to explore the effects of fat-specific protein 27 (Fsp27) on regulation of hepatic stellate cell (HSC) activation and liver fibrosis. HSCs were isolated from rat liver tissues and cultivated in vitro for gene expression and lentivirus infection. CCK-8 cell viability assay, immunofluorescence staining, qRT-PCR, and western blot assays were used to assess phenotypic changes and gene expression in HSCs. The rat liver fibrosis model was produced by intraperitoneal injection of carbon tetrachloride for assessing the effects of Fsp27 in the rat liver. Gene expression was then detected by immunohistochemistry and ELISA assays. The results of the study showed that Fsp27 was constitutively expressed in primary quiescent HSCs, but was absent in activated HSCs. Ectopic expression of Fsp27 significantly inhibited HSC proliferation and activation, as well as expression of α-smooth muscle actin. Fsp27 expression also significantly reduced collagen I production and matrix metalloproteinases 2 protein levels, and to a lesser degree, reduced tissue inhibitors of metalloproteinases 1 expression. In vivo data showed that ectopic expression of Fsp27 protein significantly reduced levels of hydroxyproline in liver tissue, and decreased serum levels of collagen III and hyaluronic acid, which in turn, suppressed liver fibrosis in rats. From these findings, it can be concluded that Fsp27 expression suppressed HSC activation in vitro and liver fibrogenesis in vivo. Further studies are needed to explore whether expression of Fsp27 can be selected as a potential novel strategy for anti-fibrotic therapy against liver fibrosis.     

Regulation of CCN2 mRNA expression and promoter activity in activated hepatic stellate cells

The matricellular protein connective tissue growth factor (CCN2) is considered a faithful marker of fibroblast activation in wound healing and in fibrosis. CCN2 is induced during activation of hepatic stellate cells (HSC). Here, we investigate the molecular basis of CCN2 gene expression in HSC. Fluoroscence activated cell sorting was used to investigate CCN2 expression in HSC in vivo in mice treated with CCl(4). CCN2 and TGF-beta mRNA expression were assessed by polymerase chain reaction as a function of culture-induced activation of HSC. CCN2 promoter/reporter constructs were used to map cis-acting elements required for basal and TGFbeta-induced CCN2 promoter activity. Real-time polymerase chain reaction analysis was used to further clarify signaling pathways required for CCN2 expression in HSC. CCl(4) administration in vivo increased CCN2 production by HSC. In vitro, expression of CCN2 and TGF-beta mRNA were concommitantly increased in mouse HSC between days 0 and 14 of culture. TGFbeta-induced CCN2 promoter activity required the Smad and Ets-1 elements in the CCN2 promoter and was reduced by TGFbeta type I receptor (ALK4/5/7) inhibition. CCN2 overexpression in activated HSC was ALK4/5/7-dependent. As CCN2 overexpression is a faithful marker of fibrogenesis, our data are consistent with the notion that signaling through TGFbeta type I receptors such as ALK5 contributes to the activation of HSC and hence ALK4/5/7 inhibition would be expected to be an appropriate treatment for liver fibrosis.     

Follistatin attenuates early liver fibrosis: effects on hepatic stellate cell activation and hepatocyte apoptosis

Activin A, a member of the transforming growth factor-beta superfamily, is constitutively expressed in hepatocytes and regulates liver mass through tonic inhibition of hepatocyte DNA synthesis. Follistatin is the main biological inhibitor of activin bioactivity. These molecules may be involved in hepatic fibrogenesis, although defined roles remain unclear. We studied activin and follistatin gene and protein expression in cultured rat hepatic stellate cells (HSCs) and in rats given CCl4 for 8 wk and examined the effect of follistatin administration on the development of hepatic fibrosis. In activated HSCs, activin mRNA was upregulated with high expression levels, whereas follistatin mRNA expression was unchanged from baseline. Activin A expression in normal lobular hepatocytes redistributed to periseptal hepatocytes and smooth muscle actin-positive HSCs in the fibrotic liver. A 32% reduction in fibrosis, maximal at week 4, occurred in CCl4-exposed rats treated with follistatin. Hepatocyte apoptosis decreased by 87% and was maximal at week 4 during follistatin treatment. In conclusion, activin is produced by activated HSCs in vitro and in vivo. Absence of simultaneous upregulation of follistatin gene expression in HSCs suggests that HSC-derived activin is biologically active and unopposed by follistatin. Our in vivo and in vitro results demonstrate that activin-mediated events contribute to hepatic fibrogenesis and that follistatin attenuates early events in fibrogenesis by constraining HSC proliferation and inhibiting hepatocyte apoptosis.     

Loss of expression of miR-335 is implicated in hepatic stellate cell migration and activation

Activation and migration of resident stellate cells (HSCs) within the hepatic space of Disse play an important role in hepatic fibrosis, which accounts for the increased numbers of activated HSCs in areas of inflammation during hepatic fibrosis. Currently, microRNAs have been found to play essential roles in HSC differentiation, proliferation, apoptosis, fat accumulation and collagen production. However, little is known about microRNA mediated HSC activation and migration. In this study, the miRNA expression profiles of quiescent HSCs, partially activated HSCs and fully activated HSCs were compared in pairs. Gene ontology (GO) and GO-Map network analysis indicated that the activation of HSCs was regulated by microRNAs. Among them miR-335 was confirmed to be significantly reduced during HSC activation by qRT-PCR, and restoring expression of miR-335 inhibited HSC migration and reduced α-SMA and collagen type I. Previous study revealed that tenascin-C (TNC), an extracellular matrix glycoprotein involved in cell migration, might be a target of miR-335. Therefore, we further studied the TNC expression in miR-335 over-expressed HSCs. Our data showed that exogenous TNC could enhance HSC migration in vitro and miR-335 restoration resulted in a significant inhibition of TNC expression. These results demonstrated that miR-335 restoration inhibited HSC migration, at least in part, via downregulating the TNC expression.     

Peroxisome proliferator-activated receptors gamma reverses hepatic nutritional fibrosis in mice and suppresses activation of hepatic stellate cells in vitro

Nonalcoholic steatohepatitis with fibrosis is a more severe form of nonalcoholic fatty liver disease, one of the most common liver diseases. We have previously shown that peroxisome proliferator-activated receptors gamma (PPARγ) ligand, rosiglitazone, prevented the development of the methionine choline deficient (MCD) diet-induced fibrosing steatohepatitis. We have now tested whether overexpression of PPARγ ameliorates established steatohepatitis and fibrosis. Male C57BL6 mice fed with MCD diet for 8 weeks developed hepatic fibrosis with increased hepatic expression of collagen1α(I), inhibitors of fibrosis reversal-1, regulator involved in matrix degradation-9 and connective tissue growth factor. After 2 weeks of transduction of PPARγ through an adenovirus-expressing PPARγ (Ad-PPARγ), expression of these genes was reduced in a manner that paralleled the reduction in activated hepatic stellate cells (HSCs) and resolution of liver fibrosis. On the in vitro study, PPARγ is expressed in primary quiescent HSC, but depleted in culture activated HSC. Conversely, ectopic expression of PPARγ in activated HSC achieved the phenotypic reversal to the quiescent cell. Such induction markedly suppressed cell viability and cell proliferation, downregulated proliferating cell nuclear antigen, and caused cell cycle arrest at G0/G1 phase. Further, introduction of PPARγ in HSC increased cell apoptosis, this was confirmed by enhanced expression of FasL, cleaved caspase-3, cleaved caspase-7 and poly ADP-ribose polymerase, indicating an extrinsic apoptosis pathway. In conclusion, the present study shows that MCD diet-induced fibrosing steatohepatitis can be reversed by overexpression of PPARγ. It is likely that PPARγ reverses fibrosis by reducing HSCs proliferation, inducing cell cycle arrest and apoptosis.     

Induction of cell death in activated hepatic stellate cells by targeted gene expression of the thymidine kinase/ganciclovir system

Liver fibrosis is the result from a relative imbalance between synthesis and degradation of matrix proteins. Following liver injury of any etiology, hepatic stellate cells undergo a response known as activation, which is the transition of quiescent cells into proliferative, fibrogenic, and contractile myofibroblasts. Upon this cellular transdifferentiation the effector cell becomes the major source of fibrillar and non-fibrillar matrix proteins resulting in excessive scar formation and cirrhosis, the end stage of fibrosis. Concomitant with progressive liver fibrosis, the tissue inhibitor of metalloproteinases-1 (TIMP-1) is strongly activated in hepatic stellate cells. We have developed a recombinant replication-defective adenovirus in which the TIMP-1 promoter is coupled to the herpes simplex virus thymidine kinase gene rendering activated hepatic stellate cells susceptible to ganciclovir. This novel targeted suicide gene approach was validated in a culture model considered to reflect an accelerated time course of the cellular and molecular events that occur during liver fibrosis. We demonstrate that transfer of the suicide gene to culture-activated hepatic stellate cells results in a strong expression of the respective transgene as assessed by Northern blot and Western blot analyses. The enzyme catalyzed the proper conversion of its prodrug subsequently initiating programmed cell death as estimated by caspase-3 assay and Annexin V-Fluos staining. Altogether, these results indicate that induction of programmed cell death is a promising approach to eliminate fibrogenic HSC.     

Peroxisome proliferator-activated receptor gamma induces a phenotypic switch from activated to quiescent hepatic stellate cells

Depletion of peroxisome proliferator-activated receptor gamma (PPARgamma) accompanies myofibroblastic transdifferentiation of hepatic stellate cells (HSC), the primary cellular event underlying liver fibrogenesis. The treatment of activated HSC in vitro or in vivo with synthetic PPARgamma ligands suppresses the fibrogenic activity of HSC. However, it is uncertain whether PPARgamma is indeed a molecular target of this effect, because the ligands are also known to have receptor-independent actions. To test this question, the present study examined the effects of forced expression of PPARgamma via an adenoviral vector on morphologic and biochemical features of culture-activated HSC. The vector-mediated expression of PPARgamma itself is sufficient to reverse the morphology of activated HSC to the quiescent phenotype with retracted cytoplasm, prominent dendritic processes, reduced stress fibers, and accumulation of retinyl palmitate. These effects are abrogated by concomitant expression of a dominant negative mutant of PPARgamma that prevents transactivation of but not binding to the PPAR response element. PPARgamma expression also inhibits the activation markers such as the expression of alpha-smooth muscle actin, type I collagen, and transforming growth factor beta1; DNA synthesis; and JunD binding to the activator protein-1 (AP-1) site and AP-1 promoter activity. Inhibited JunD activity by PPARgamma is not due to reduced JunD expression or JNK activity or to a competition for p300. But it is due to a JunD-PPARgamma interaction as demonstrated by co-immunoprecipitation and glutathione S-transferase pull-down analysis. Further, the use of deletion constructs reveals that the DNA binding region of PPARgamma is the JunD interaction domain. In summary, our results demonstrate that the restoration of PPARgamma reverses the activated HSC to the quiescent phenotype and suppresses AP-1 activity via a physical interaction between PPARgamma and JunD.     

LPS-mediated NFkappaB activation varies between activated human hepatic stellate cells from different donors

The activation of hepatic stellate cells (HSC) is recognized as the key event of hepatic fibrosis [Virchows Arch. 430 (1997) 195; Semin. Liver Dis. 21 (2001) 437; Front. Biosci. 7 (2002) d808]. NFkappaB has been associated with the development of the activated phenotype, the expression of proinflammatory genes, and with promoting survival of activated HSC. High levels of circulating endotoxin are observed in liver fibrosis and several lines of evidence indicate that LPS plays an important role in chronic liver disease. Here, we investigated the LPS-induced NFkappaB activation in activated HSC from different human donors. HSC were isolated from liver specimens obtained during surgical liver resection and were activated by culturing on plastic. LPS-induced NFkappaB activity and IL-8 expression revealed a significant correlation but differed significantly comparing HSC from individual donors. These variations seen in LPS mediated NFkappaB activation and chemokine secretion between HSC from different donors in vitro may contribute to differences seen in vivo between patients in the progression of fibrosis and the degree of inflammation during chronic liver disease.     

Asiatic acid inhibits liver fibrosis by blocking TGF-beta/Smad signaling in vivo and in vitro

Liver fibrosis is a major cause of liver failure, but treatment remains ineffective. In the present study, we investigated the mechanisms and anti-hepatofibrotic activities of asiatic acid (AA) in a rat model of liver fibrosis induced by carbon tetrachloride (CCl(4)) and in vitro in TGF-beta1-stimulated rat hepatic stellate cell line (HSC-T6). Treatment with AA significantly attenuated CCl(4)-induced liver fibrosis and functional impairment in a dosage-dependent manner, including blockade of the activation of HSC as determined by inhibiting de novo alpha smooth muscle actin (a-SMA) and collagen matrix expression, and an increase in ALT and AST (all p<0.01). The hepatoprotective effects of AA on fibrosis were associated with upregulation of hepatic Smad7, an inhibitor of TGF-beta signaling, thereby blocking upregulation of TGF-beta1 and CTGF and the activation of TGF-beta/Smad signaling. The anti-fibrosis activity and mechanisms of AA were further detected in vitro in HSC-T6. Addition of AA significantly induced Smad7 expression by HSC-T6 cells, thereby inhibiting TGF-beta1-induced Smad2/3 activation, myofibroblast transformation, and collagen matrix expression in a dosage-dependent manner. In contrast, knockdown of Smad7 in HSC-T6 cells prevented AA-induced inhibition of HSC-T6 cell activation and fibrosis in response to TGF-beta1, revealing an essential role for Smad7 in AA-induced anti-fibrotic activities during liver fibrosis in vivo and in vitro. In conclusion, AA may be a novel therapeutic agent for liver fibrosis. Induction of Smad7-dependent inhibition of TGF-beta/Smad-mediated fibrogenesis may be a central mechanism by which AA protects liver from injury.     

Smads 2 and 3 are differentially activated by transforming growth factor-beta (TGF-beta ) in quiescent and activated hepatic stellate cells. Constitutive nuclear localization of Smads in activated cells is TGF-beta-independent

Hepatic stellate cells are the primary cell type responsible for matrix deposition in liver fibrosis, undergoing a process of transdifferentiation into fibrogenic myofibroblasts. These cells, which undergo a similar transdifferentiation process when cultured in vitro, are a major target of the profibrogenic agent transforming growth factor-beta (TGF-beta). We have studied activation of the TGF-beta downstream signaling molecules Smads 2, 3, and 4 in hepatic stellate cells (HSC) cultured in vitro for 1, 4, and 7 days, with quiescent, intermediate, and fully transdifferentiated phenotypes, respectively. Total levels of Smad4, common to multiple TGF-beta superfamily signaling pathways, do not change as HSC transdifferentiate, and the protein is found in both nucleus and cytoplasm, independent of treatment with TGF-beta or the nuclear export inhibitor leptomycin B. TGF-beta mediates activation of Smad2 primarily in early cultured cells and that of Smad3 primarily in transdifferentiated cells. The linker protein SARA, which is required for Smad2 signaling, disappears with transdifferentiation. Additionally, day 7 cells demonstrate constitutive phosphorylation and nuclear localization of Smad 2, which is not affected by pretreatment with TGF-beta-neutralizing antibodies, a type I TGF-beta receptor kinase inhibitor, or activin-neutralizing antibodies. These results demonstrate essential differences between TGF-beta-mediated signaling pathways in quiescent and in vitro transdifferentiated hepatic stellate cells.     

Embryonic liver fodrin involved in hepatic stellate cell activation and formation of regenerative nodule in liver cirrhosis

Transforming growth factor (TGF) β(1) plays a critical role in liver fibrosis. Previous studies demonstrated embryonic liver fodrin (ELF), a β-spectrin was involved in TGF-β/Smad signalling pathway as Smad3/4 adaptor. Here we investigate the role of ELF in pathogenesis of liver cirrhosis. In carbon tetrachloride (CCl(4))-induced mice model of liver cirrhosis, ELF is up-regulated in activated hepatic stellate cells (HSCs), and down-regulated in regenerative hepatocytes of cirrhotic nodules. In activated HSCs in vitro, reduction of ELF expression mediated by siRNA leads to the inhibition of HSC activation and procollagen I expression. BrdU assay demonstrates that down-regulation of ELF expression does not inhibit proliferation of activated HSCs in vitro. Immunostaining of cytokeratin 19 and Ki67 indicates that regenerative hepatocytes in cirrhotic liver are derived from hepatic progenitor cells (HPC). Further study reveals that HPC expansion occurs as an initial phase, before the reduction of ELF expression in regenerative hepatocytes. Regenerative hepatocytes in cirrhotic liver show the change in proliferative activity and expression pattern of proteins involved in G1/S transition, which suggests the deregulation of cell cycle in regenerative hepatocytes. Finally, we find that ELF participates in TGF-β/Smad signal in activated HSCs and hepatocytes through regulating the localization of Smad3/4. These data reveal that ELF is involved in HSC activation and the formation of regenerative nodules derived from HPC in cirrhotic liver.     

MicroRNA-145 induces the senescence of activated hepatic stellate cells through the activation of p53 pathway by ZEB2

Activation of quiescent hepatic stellate cells (HSCs) is the major event in liver fibrosis, along with enhancement of cell proliferation and overproduction of extracellular matrix. Recent findings suggest that senescence of activated HSCs might limit the development of liver fibrosis. The p53, a guardian of the genome is associated with liver fibrosis, has been shown to regulate HSCs senescence. In this study, we report that microRNA-145 (miR-145) and p53 were downregulated in vivo and in vitro, concomitant with the enhanced expression of zinc finger E-box binding homeobox 2 (ZEB2). In addition, overexpression of miR-145 and p53 led to upregulation of the number of senescence-associated β-galactosidase-positive HSCs and the expression of senescence markers p16 and p21, along with the reduced abundance of HSC activation markers α-smooth muscle actin and type I collagen in activated HSCs. Furthermore, silencing of ZEB2 promoted senescence of activated HSCs. Moreover, we also demonstrated that miR-145 specifically targeted the 3′-untranslated regions of ZEB2. In vitro promoter regulation studies show that ZEB2 could bind to the E-box of the p53 promoter as well as inhibit its promoter activity and thus suppress the expression of p53, which in turn repressed activated HSCs senescence. Taken together, our results describe a novel miR-145-ZEB2-p53 regulatory line might participate in the senescence of activated HSCs and might carry potential therapeutic targets for restraining liver fibrosis.