Expression of Smads during in vitro transdifferentiation of hepatic stellate cells to myofibroblasts

TGFbeta is of crucial importance during transdifferentiation of resting retinoid-storing hepatic stellate cells (HSC) to extracellular matrix producing myofibroblasts (MFB) and consequently, inhibition of TGFbeta signal transduction is an effective means for preventing experimental fibrosis. We have shown that isolated HSC lose TGFbeta-dependent growth control during in vitro activation and that alpha2 (I) collagen production in transdifferentiated MFB is TGFbeta-independent. Furthermore, Smad complexes with SBE binding activity were only detected in early cultures of HSC, although TGFbeta receptor types I and II were significantly expressed in HSC and MFB. In the present report, we compared the expression pattern of TGFbeta downstream mediators, i.e., the Smads, in TGFbeta responsive HSC versus nonresponding MFB. The transdifferentiation process was monitored by morphology and increasing expression of TGFbeta and alpha-smooth muscle actin, and TGFbeta signaling was investigated by (CAGA)(9)-MLP-Luc. The expression level of all Smads remained essentially unchanged both during the activation process and after TGFbeta-treatment. Smad7 was transiently upregulated upon TGFbeta stimulation in quiescent HSC, indicating a negative feed back loop in responsive cells. In contrast, MFB neither displayed TGFbeta-inducible nor constitutively upregulated Smad7 expression. Instead, Smad3 mRNA was increased in MFB. Our data indicate that abrogation of the TGFbeta response in MFB versus HSC is not based on different regulation of Smad expression.     

Immunomodulation of activated hepatic stellate cells by mesenchymal stem cells

Bone marrow-derived mesenchymal stem cells (MSCs) have been reported to prevent the development of liver fibrosis in a number of pre-clinical studies. Marked changes in liver histopathology and serological markers of liver function have been observed without a clear understanding of the therapeutic mechanism by which stem cells act. We sought to determine if MSCs could modulate the activity of resident liver cells, specifically hepatic stellate cells (SCs) by paracrine mechanisms using indirect cocultures. Indirect coculture of MSCs and activated SCs led to a significant decrease in collagen deposition and proliferation, while inducing apoptosis of activated SCs. The molecular mechanisms underlying the modulation of SC activity by MSCs were examined. IL-6 secretion from activated SCs induced IL-10 secretion from MSCs, suggesting a dynamic response of MSCs to the SCs in the microenvironment. Blockade of MSC-derived IL-10 and TNF-alpha abolished the inhibitory effects of MSCs on SC proliferation and collagen synthesis. In addition, release of HGF by MSCs was responsible for the marked induction of apoptosis in SCs as determined by antibody-neutralization studies. These findings demonstrate that MSCs can modulate the function of activated SCs via paracrine mechanisms provide a plausible explanation for the protective role of MSCs in liver inflammation and fibrosis, which may also be relevant to other models of tissue fibrosis.     

Thrombin inhibits migration of human hepatic myofibroblasts

Several lines of data recently pointed out a role of the serine proteinase thrombin in liver fibrogenesis, but its mechanism of action is unknown. The aim of this study was to evaluate the effect of thrombin on the migration of human liver myofibroblasts. We show here that thrombin inhibits both basal migration and platelet-derived growth factor (PDGF)-BB-induced migration of myofibroblasts. By using a thrombin antagonist, a protease-activated receptor (PAR)-1 mimetic peptide, and a PAR-1 antibody, we show that this effect is dependent on the catalytic activity of thrombin and on PAR-1 activation. Thrombin's effect on basal migration was dependent on cyclooxygenase 2 (COX-2) activation because it was blocked by the COX-2 inhibitors NS-398 and nimesulide, and pharmacological studies showed that it was relayed through prostaglandin E(2) and its EP(2) receptor. On the other hand, thrombin-induced inhibition of PDGF-BB-induced migration was not dependent on COX-2. We show that thrombin inhibits PDGF-induced Akt-1 phosphorylation. This effect was consecutive to inhibition of PDGF-beta receptor activation through active dephosphorylation. Thus thrombin, through two distinct mechanisms, inhibits both basal- and PDGF-BB-induced migration of human hepatic liver myofibroblasts. The fine tuning of myofibroblast migration may be one of the mechanisms used by thrombin to regulate liver fibrogenesis.     

Syncoilin is an intermediate filament protein in activated hepatic stellate cells

Hepatic stellate cells (HSCs) play an important role in several (patho)physiologic conditions in the liver. In response to chronic injury, HSCs are activated and change from quiescent to myofibroblast-like cells with contractile properties. This shift in phenotype is accompanied by a change in expression of intermediate filament (IF) proteins. HSCs express a broad, but variable spectrum of IF proteins. In muscle, syncoilin was identified as an alpha-dystrobrevin binding protein with sequence homology to IF proteins. We investigated the expression of syncoilin in mouse and human HSCs. Syncoilin expression in isolated and cultured HSCs was studied by qPCR, Western blotting, and fluorescence immunocytochemistry. Syncoilin expression was also evaluated in other primary liver cell types and in in vivo-activated HSCs as well as total liver samples from fibrotic mice and cirrhotic patients. Syncoilin mRNA was present in human and mouse HSCs and was highly expressed in in vitro- and in vivo-activated HSCs. Syncoilin protein was strongly upregulated during in vitro activation of HSCs and undetectable in hepatocytes and liver sinusoidal endothelial cells. Syncoilin mRNA levels were elevated in both CCl₄- and common bile duct ligation-treated mice. Syncoilin immunocytochemistry revealed filamentous staining in activated mouse HSCs that partially colocalized with α-smooth muscle actin, β-actin, desmin, and α-tubulin. We show that in the liver, syncoilin is predominantly expressed by activated HSCs and displays very low-expression levels in other liver cell types, making it a good marker of activated HSCs. During in vitro activation of mouse HSCs, syncoilin is able to form filamentous structures or at least to closely interact with existing cellular filaments.     

Changes of sphingolipid species in the phenotype conversion from myofibroblasts to lipocytes in hepatic stellate cells

Sphingolipids play a relevant role in cell-cell interaction, communication, and migration. We studied the sphingolipid content in the murine hepatic stellate cell line GRX, which expresses the myofibroblast phenotype, and can be induced in vitro to display the fat-storing phenotype. Lipid modifications along this induction were investigated by labeling sphingolipids with [(14)C]galactose, [(14)C]serine, or [(14)C]choline, and determination of fatty acid composition of sphingomyelin. The total ganglioside content and the GM2 synthase activity were lower in myofibroblasts. Both phenotypes presented similar gangliosides of the a-pathway: GM2, GM1, and GD1a as well as their precursor GM3. Sphingomyelin and all the gangliosides were expressed as doublets; the upper/lower band ratio increased in lipocytes, containing more long-chain fatty acids in retinol-induced lipocytes as compared to the insulin/indomethacin induced ones. Time-course experiments indicated a transfer of metabolic precursors from phosphatidylcholine to sphingomyelin in the two phenotypes. Taken together, these results indicate that myofibroblast and lipocytes can use distinct ceramide pools for sphingolipid synthesis. Differential ganglioside expression and presence of the long-chain saturated fatty acids suggested that they may participate in formation of distinct membrane microdomains or rafts with specific functions on the two phenotypes of GRX-cells.     

Thy-1 is expressed in myofibroblasts but not found in hepatic stellate cells following liver injury

Thy-1 (CD90) is an adhesion molecule induced in fibroblast populations associated with wound healing and fibrosis. In this study the question whether Thy-1-gene-expression can be induced in hepatic stellate cells (HSC) in vivo, under conditions of liver injury or liver regeneration was addressed. Acute and chronic rat liver injury was induced by the administration of CCl₄. For comparison, cirrhotic human liver, and rat 67% partial hepatectomy (PH) was studied as well. Thy-1-gene-expression was examined also in isolated human liver myofibroblasts. Thy-1-mRNA expression was significantly upregulated in chronic liver injury. Thy-1⁺ cells were detected in the periportal area of rat liver specimens in normal-, injured- and regenerative-conditions. In chronic human and rat liver injury, Thy-1⁺ cells were located predominantly in scar tissue. In the pericentral necrotic zone after CCl₄-treatment, no induction of Thy-1 was found. Gremlin and Thy-1 showed comparable localization in the periportal areas. Thy-1 was not detected in either normal or capillarized sinusoids, in isolated rat HSC, and was neither inducible by inflammatory cytokines in isolated HSC, nor upregulated in treated myofibroblasts. Based upon these data Thy-1 is not a marker of “activated” sinusoidal HSC, but it is a marker of “activated” (myo)fibroblasts found in portal areas and in scar tissue. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Comparative evaluation of gene delivery devices in primary cultures of rat hepatic stellate cells and rat myofibroblasts

Background  

The cell surface undergoes continuous change during cell movement. This is characterized by transient protrusion and partial or complete retraction of microspikes, filopodia, and lamellipodia. This requires a dynamic actin cytoskeleton, moesin, components of Rho-mediated signal pathways, rearrangement of membrane constituents and the formation of focal adhesion sites. While the immunofluorescence distribution of endogenous moesin is that of a membrane-bound molecule with marked enhancement in some but not all microextensions, the C-terminal fragment of moesin co-distributes with filamentous actin consistent with its actin-binding activity. By taking advantage of this property we studied the spontaneous protrusive activity of live NIH3T3 cells, expressing a fusion of GFP and the C-terminal domain of moesin.     

Down-regulation of FoxO-dependent c-FLIP expression mediates TRAIL-induced apoptosis in activated hepatic stellate cells

Activated hepatic stellate cells which contribute to liver fibrosis have represented an important target for antifibrotic therapy. In this study, we found that TRAIL inhibited PI3K/Akt-dependent FoxO phosphorylation and relocated FoxO proteins into the nucleus from the cytosol in activated human hepatic stellate LX-2 cells. The accumulated FoxO proteins in the nucleus led to down-regulation of c-FLIP_L/S expression, resulting in the activation of apoptosis-related signaling molecules including the activation of caspase-8, -3, and Bid, as well as mitochondrial cytochrome c release. These results were supported by showing that siRNA-mediated knockdown of FoxO led to restoration of c-FLIP_L/S expression and resistance to TRAIL-induced apoptosis after treatment of LX-2 cells with TRAIL. Furthermore, c-FLIP_L/S-transfected LX-2 cells showed the decreased sensitivity to TRAIL-induced apoptosis. Collectively, our data suggest that sequential activation of FoxO proteins under conditions of suppressed PI3K/Akt signaling by TRAIL can down-regulate c-FLIP_L/S, consequently promoting TRAIL-induced apoptosis in LX-2 cells. Therefore, the present study suggests TRAIL may be an effective strategy for antifibrotic therapy in liver fibrosis.     

Rat liver myofibroblasts and hepatic stellate cells differ in CD95-mediated apoptosis and response to TNF-alpha

Hepatic stellate cells (HSC), particularly activated HSC, are thought to be the principle matrix-producing cell of the diseased liver. However, other cell types of the fibroblast lineage, especially the rat liver myofibroblasts (rMF), also have fibrogenic potential. A major difference between the two cell types is the different life span under culture conditions. Although nearly no spontaneous apoptosis could be shown in rMF cultures, 18 +/- 2% of the activated HSC (day 7) were apoptotic. Compared with activated HSC, CD95R was expressed in 70% higher amounts in rMF. CD95L could only be detected in activated HSC. Stimulation of the CD95 system by agonistic antibodies (1 ng/ml) led to apoptosis of all rMF within 2 h, whereas activated HSC were more resistant (5.3 h/ 40% of total cells). Although transforming growth factor-beta downregulated apoptosis in both activated HSC and rMF, tumor necrosis factor-alpha (TNF-alpha) upregulated apoptosis in rMF. Lack of spontaneous apoptosis and CD95L expression in rMF and the different reaction on TNF-alpha stimulation reveal that activated HSC and rMF belong to different cell populations.     

Regulatory role of vHL/HIF-1alpha in hypoxia-induced VEGF production in hepatic stellate cells

Activated hepatic stellate cells (HSCs) produce cyclooxygenase-2 (COX-2) protein to induce vascular endothelial growth factor (VEGF) production that participates in angiogenesis in injured liver. To reveal the unknown regulatory mechanism, we used hypoxic atmosphere mimicking injured-tissue microenvironment to induce VEGF expression in a rat hepatic stellate cell line (T6-HSCs). The present study showed that hypoxia up-regulated the protein levels of COX-2 and hypoxia-inducible factor-1-alpha (HIF-1alpha), but rapidly effected degradation of von Hippel-Lindau (vHL) protein. As a result, expression of VEGF in HSCs was markedly elevated; and pretreatment with COX-2 inhibitors (nimesulide or indomethacin) could significantly ameliorate the angiogenic event. Collectively, hypoxic HSCs increased accumulation of HIF-1alpha protein and induced VEGF expression in a time-dependent manner. Inhibition of COX-2 activities would prevent vHL protein from degradation and suppress HIF-1alpha up-regulation. Thus, vHL/HIF-1alpha has a regulatory role in COX-2-mediated VEGF production in hypoxic stellate cells in injured liver.     

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.     

Tenascin-C promotes migration of hepatic stellate cells and production of type I collagen

Tenascin-C (TN-C) is an extracellular matrix glycoprotein markedly upregulated during liver fibrosis. The study is performed to explore the role of TN-C during the growth and activation of hepatic stellate cells (HSCs). We found that TN-C was accumulated accompanying with the HSC activation. Our data on cell migration assay revealed that the rTN-C treatment enhanced HSC migration in a dose- and time-dependent manner, but did not influence their proliferation. HSCs transfected with pTARGET-TN-C overexpression vector displayed increased the type I collagen (Col I) production. TN-C overexpression enhanced the process of HSC activation through TGF-β1 signaling. Moreover, the anti-α9β1 integrin antibody treatment blocked the TN-C-driven Col I increase in rat HSCs. Collectively, TN-C had a positive role in activation of HSCs mediated by TGF-β1 and α9β1 integrin, manifesting elevation of Col I production and promotion of cell migration. Our results provide a potential insight for the therapy of hepatic fibrosis.     

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.