Axel M. Gressner

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.

Modern pathogenetic concepts of liver fibrosis suggest stellate cells and TGF-β as major players and therapeutic targets

Hepatic fibrosis is a scarring process that is associated with an increased and altered deposition of extracellular matrix in liver. At the cellular and molecular level, this progressive process is mainly characterized by cellular activation of hepatic stellate cells and aberrant activity of transforming growth factor‐β1 and its downstream cellular mediators. Although the cellular responses to this cytokine are complex, the signalling pathways of this pivotal cytokine during the fibrogenic response and its connection to other signal cascades are now understood in some detail. Based on the current advances in understanding the pleiotropic reactions during fibrogenesis, various inhibitors of transforming growth factor‐β were developed and are now being investigated as potential drug candidates in experimental models of hepatic injury. Although it is too early to favour one of these antagonists for the treatment of hepatic fibrogenesis in human, the experimental results obtained yet provide stimulatory impulses for the development of an effective treatment of choice in the not too distant future. The present review summarises the actual knowledge on the pathogenesis of hepatic fibrogenesis, the role of transforming growth factor‐β and its signalling pathways in promoting the fibrogenic response, and the therapeutic modalities that are presently in the spotlight of many investigations and are already on the way to take the plunge into clinical studies.

Complement factor 5 is a quantitative trait gene that modifies liver fibrogenesis in mice and humans

Fibrogenesis or scarring of the liver is a common consequence of all chronic liver diseases. Here we refine a quantitative trait locus that confers susceptibility to hepatic fibrosis by in silico mapping and show, using congenic mice and transgenesis with recombined artificial chromosomes, that the gene Hc (encoding complement factor C5) underlies this locus. Small molecule inhibitors of the C5a receptor had antifibrotic effects in vivo, and common haplotype-tagging polymorphisms of the human gene C5 were associated with advanced fibrosis in chronic hepatitis C virus infection. Thus, the mouse quantitative trait gene led to the identification of an unknown gene underlying human susceptibility to liver fibrosis, supporting the idea that C5 has a causal role in fibrogenesis across species.

Activation of rat liver perisinusoidal lipocytes by transforming growth factors derived from myofibroblastlike cells. A potential mechanism of self perpetuation in liver fibrogenesis.

Rat liver perisinusoidal lipocytes (PL) cultured on uncoated plastic transform spontaneously within 6-10 d to myofibroblastlike cells (MFBlC). Parallel to the transformation the TGF alpha- and TGF beta 1-mRNA expression increased and was highest in MFBlC. Competitive radioligand binding assays demonstrated that in contrast to untransformed PL the MFBlC synthesize and secrete transforming growth factor (TGF)-alpha (15 fmol/cell per 24 h) and predominantly the latent form of TGF beta 1 (0.2 fmol/cell per 24 h). Medium conditioned by MFBlC (MFBcM) significantly stimulated PL proliferation with little effect on PL proteoglycan synthesis. By transient acidification of the MFBcM, known to activate the latent form of TGF beta 1, the stimulatory effect on PL proteoglycan synthesis was enhanced and furthermore PL transformation (measured by expression of iso-alpha smooth muscle actin and loss of retinylpalmitate) was accelerated. Preincubation of this medium with neutralizing antibodies to TGF beta resulted in (a) the growth inhibitory effect was converted to a growth stimulation and (b) the stimulatory effect on proteoglycan synthesis was abolished. In summary our data indicate that progressive activation of PL on plastic (transformation to MFBlC) leads to an enhanced expression of the TGF alpha- and TGF beta 1-mRNAs and secretion of the corresponding proteins. Medium conditioned by MFBIC stimulates proliferation, transformation, and PG synthesis of untransformed PL. These mechanisms are suggested to be relevant in self perpetuation of liver fibrogenesis.

Connective tissue growth factor: a fibrogenic master switch in fibrotic liver diseases.

Connective tissue growth factor (CTGF=CCN2), one of six members of cysteine‐rich, secreted, heparin‐binding proteins with a modular structure, is recognized as an important player in fibrogenic pathways as deduced from findings in non‐hepatic tissues and emerging results from liver fibrosis. Collectively, the data show strongly increased expression in fibrosing tissues and transforming growth factor (TGF‐β)‐stimulated expression in hepatocytes, biliary epithelial cells and stellate cells. Functional activity as a mediator of fibre–fibre, fibre–matrix and matrix–matrix interactions, as an enhancer of profibrogenic TGF‐β and several secondary effects owing to TGF‐β enhancement, and as a down‐modulator of the bioactivity of bone morphogenetic protein‐7 has been proposed. By changing the activity ratio of TGF‐β to its antagonist bone‐morphogenetic protein‐7, CTGF is proposed as a fibrogenic master switch for epithelial–mesenchymal transition. Consequently, knockdown of CTGF considerably attenuates experimental liver fibrosis. The spill‐over of CTGF from the liver into the blood stream proposes this protein as a non‐invasive reporter of TGF‐β bioactivity in this organ. Indeed, CTGF‐levels in sera correlate significantly with fibrogenic activity. The data suggest CTGF as a multifaceted regulatory protein in fibrosis, which offers important translational aspects for diagnosis and follow‐up of hepatic fibrogenesis and as a target for therapeutic interventions. In addition, CTGF‐promoter polymorphism might be of importance as a prognostic genetic marker to predict the progression of fibrosis.

THE CELL BIOLOGY OF LIVER FIBROGENESIS: AN IMBALANCE OF PROLIFERATION, GROWTH ARREST AND APOPTOSIS OF MYOFIBROBLASTS

Abstract Fibrosis following liver damage and factors influencing this process are discussed with special reference to hepatic stellate cells and their transformation to myo- fibroblasts.

Transforming growth factor β signal transduction in hepatic stellate cells via Smad2/3 phosphorylation, a pathway that is abrogated during in vitro progression to myofibroblasts

To current knowledge, transforming growth factor β (TGFβ) signaling is mandatory to establish liver fibrosis and various molecular interventions designed to affect the TGFβ system were successfully used to inhibit fibrogenesis. Activated hepatic stellate cells (HSC), which are one important source of TGFβ, are the major producers of extracellular matrix proteins in liver injury. We have previously shown that the TGFβ response of this cell type is modulated during the transdifferentiation process. This work delineates the activation of TGFβ downstream mediators, the Smads, in quiescent HSC and transdifferentiated myofibroblasts (MFB). The expression level of all Smads remained largely unchanged during this process. The response of HSC to TGFβ, leading to, e.g., induction of α2 (I) collagen expression, is mediated by phosphorylation of Smad2 and Smad3 and subsequent nuclear translocation of a Smad containing complex. Neither TGFβ‐dependent nor endogenously phosphorylated Smad2/3 was detectable in comparable amounts in transdifferentiated MFB, indicating loss of TGFβ sensitivity. Ectopic expression of Smad7 in HSC led to inhibition of Smad2 phosphorylation and abrogated TGFβ response. In transdifferentiated MFB, expression of a constitutively active TGFβ receptor I, but not treatment with TGFβ1, resulted in transcriptional activation of a TGFβ responsive promoter, thereby demonstrating completely restored TGFβ signal transduction. Our data indicate that in contrast to a postulated mechanism of enduring autocrine TGFβ signal transduction, early and late stages of HSC activation have to be distinguished, which is of importance for antifibrotic therapies.

Participation of Smad2, Smad3, and Smad4 in transforming growth factor beta (TGF-beta)-induced activation of Smad7. THE TGF-beta response element of the promoter requires functional Smad binding element and E-box sequences for transcriptional regulation.

Smad7 has recently been identified as a player that antagonizes transforming growth factor β (TGF-β) signals by acting downstream of TGF-β receptors. TGF-β rapidly induces expression of Smad7 mRNA in a variety of cell types, suggesting participation in a negative feedback loop to control TGF-β responses. We have previously described the genomic locus of rat Smad7 including the promoter region. Here we report polymerase chain reaction cloning of the corresponding promoter regions of human and murine Smad7 genes and functional characterization of the rat Smad7 promoter. Using transient transfection experiments of HepG2 cells, we identified the TGF-β response element within a strongly conserved region, containing a perfect Smad binding element (SBE; GTCTAGAC). Performing electrophoretic mobility shift assay and cotransfection experiments, we were able to delineate DNA-binding complexes and identified Smad3, Smad4, and Smad2. Mutation of the SBE completely abolished TGF-β inducibility of Smad7 in HepG2 cells, indicating that this sequence is necessary for TGF-β-induced transcription. Furthermore, a 3-base pair adjacent E-box is additionally essential for TGF-β-dependent promoter activation and an overlapping AP1 site is also involved. We conclude that regulation of Smad7 transcription by TGF-β is mediated via a specific constellation of recognition motifs localized around the SBE, which is conserved in human, rat, and murine genes.

Dominant-negative soluble PDGF- β receptor inhibits hepatic stellate cell activation and attenuates liver fibrosis

Hepatic fibrogenesis is a consequence of hepatic stellate cells that become activated and transdifferentiate into a myofibroblastic phenotype with the ability to proliferate and synthesize large quantities of extracellular matrix components. In this process, platelet-derived growth factor (PDGF) is the most potent stimulus for hepatic stellate cell proliferation and migration, and is overexpressed during active hepatic fibrogenesis. This cytokine binds to the PDGF receptor type β, activates Ras and sequentially propagates the stimulatory signal sequentially via phosphorylation of Raf-1, MEK and the extracellular-signal regulated kinases ERK1/ERK2. Hepatic injury is associated with both increased autocrine PDGF signaling and upregulation of PDGF receptor. In this study, we report that a dominant-negative soluble PDGF-β receptor consisting of a chimeric IgG containing the extracellular portion of the PDGF receptor type β blocks HSC activation and attenuates fibrogenesis induced by ligation of the common bile duct in rats. In culture-activated hepatic stellate cells, the soluble receptor blocks phosphorylation of endogenous PDGF receptor, phosphorylation of the ERK1/EKR2 signal and reduces proliferative activities of HSC. In vivo, both the delivery of the purified soluble PDGF antagonist and the administration of adenoviruses expressing the artificial transgene were able to reduce significantly the expression of collagen and α-smooth muscle actin. Our results demonstrate that PDGF plays a critical role in the progression and initiation of experimental liver fibrogenesis, and suggest that early anti-PDGF intervention should have a therapeutical impact on the treatment of liver fibrogenesis.

Tumor necrosis factor alpha (TNFα) and transforming growth factor β1 (TGFβ1) stimulate fibronectin synthesis and the transdifferentiation of fat-storing cells in the rat liver into myofibroblasts

SummaryTransforming growth factor-β (TGFβ1) and tumor necrosis factor alpha (TNFα) stimulate the trans-differentiation of fat-storing cells (FSC) in the rat liver into highly active and “synthetic” myofibroblast-like cells (MFBIC). This activation has been documented by differential-interference contrast and light microscopy using morphologic criteria (a reduction in the number and size of fat droplets, cell flattening and the development of long cytoplasmic extensions), by the loss of retinyl-palmitate (measured by HPLC) and by the enhanced expression of iso-α smooth muscle actin (demonstrated by immunofluorescence microscopy). Furthermore, while cell growth measured by the cell count and DNA content is slightly inhibited by TGFβ1 (0.81 of the control), the combination of TGFβ1 with TNFα stimulates cell proliferation to 1.44 times of the control. In addition the combination of TGFβ and TNFα potentiated the stimulatory effect on fibronectin synthesis (TGFβ alone: 1.4 times control; TNFα alone: 2.2 times control; TGFβ plus TNFα: 4.7 times control). The total protein synthesis was not altered by TGFβ or TNFα. In summary the results obtained identify TGFβ and TNFα as mediators stimulating key events in liver fibrogenesis (i.e. FSC proliferation, FSC transdifferentiation into MFBIC, and fibronectin synthesis).