Marianna D. A. Gaça

Basement membrane-like matrix inhibits proliferation and collagen synthesis by activated rat hepatic stellate cells: evidence for matrix-dependent deactivation of stellate cells

During liver fibrosis hepatic stellate cells become activated, transforming into proliferative myofibroblastic cells expressing type I collagen and alpha-smooth muscle actin. They become the major producers of the fibrotic neomatrix in injured liver. This study examines if activated stellate cells are a committed phenotype, or whether they can become deactivated by extracellular matrix. Stellate cells isolated from normal rat liver proliferated and expressed mRNA for activation markers, alpha-smooth muscle actin, type I procollagen and tissue inhibitor of metalloproteinases-1 following 5-7 day culture on plastic, but culture on Matrigel suppressed proliferation and mRNA expression. Activated stellate cells were recovered from plastic by trypsinisation and replated onto plastic, type I collagen films or Matrigel. Cells replated on plastic and type I collagen films proliferated and remained morphologically myofibroblastic, expressing alpha-smooth muscle actin and type I procollagen. However, activated cells replated on Matrigel showed <30% of the proliferative rate of these cells, and this was associated with reduced cellular expression of proliferating cell nuclear antigen and phosphorylation of mitogen-activated protein kinase in response to serum. Activated HSC replated on Matrigel for 3-7 days progressively reduced their expression of mRNA for type I procollagen and alpha-smooth muscle actin and both became undetectable after 7 days. We conclude that basement membrane-like matrix induces deactivation of stellate cells. Deactivation represents an important potential mechanism mediating recovery from liver fibrosis in vivo where type I collagen is removed from the liver and stellate cells might re-acquire contact with their normal basement membrane-like pericellular matrix.

Smads 2 and 3 are differentially activated by TGF-β in quiescent and activated hepatic stellate cells: Constitutive nuclear localization of Smads in activated cells is TGF-β 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-β (TGF-β). We have studied activation of the TGF-β 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-β superfamily signaling pathways, do not change as HSC transdifferentiate, and the protein is found in both nucleus and cytoplasm, independent of treatment with TGF-β or the nuclear export inhibitor leptomycin B. TGF-β 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-β-neutralizing antibodies, a type I TGF-β receptor kinase inhibitor, or activin-neutralizing antibodies. These results demonstrate essential differences between TGF-β-mediated signaling pathways in quiescent and in vitro transdifferentiated hepatic stellate cells.

Increased expression of connective tissue growth factor in fibrotic human liver and in activated hepatic stellate cells

BACKGROUND/AIMS Connective tissue growth factor is a recently described mitogenic protein implicated in a variety of fibrotic disorders. Connective tissue growth factor may be a downstream mediator of the pro-fibrotic and mitogenic actions of transforming growth factor-beta, promoting extracellular matrix deposition and fibrogenesis. As transforming growth factor-beta is considered important to the pathogenesis of hepatic fibrosis, we examined the possible contribution of connective tissue growth factor to this process. METHODS Connective tissue growth factor expression was examined in normal and fibrotic human and rat livers using RT-PCR and ribonuclease protection assays, and in primary cultures of rat hepatic stellate cells by Northern and Western blotting. RESULTS Ribonuclease protection assays demonstrated connective tissue growth factor mRNA was increased 3-5-fold in human fibrotic liver compared with normal. RT-PCR showed this mRNA was increased in carbon-tetrachloride-treated rat liver. Northern analysis showed connective tissue growth factor mRNA was increasingly expressed during progressive activation of cultured rat hepatic stellate cells. Western analysis confirmed that freshly isolated hepatic stellate cells secreted relatively little connective tissue growth factor compared with hepatic stellate cells activated in culture. Hepatic stellate cells stimulated with transforming growth factor-beta showed increased expression of connective tissue growth factor mRNA and protein. CONCLUSIONS Connective tissue growth factor mRNA is consistently upregulated in human liver cirrhosis of various aetiologies, supporting a role for this growth factor in hepatic fibrogenesis. Our studies suggest that hepatic stellate cells may be an important source of hepatic connective tissue growth factor in vivo, particularly following stimulation with transforming growth factor-beta.

Regulation of hepatic stellate cell proliferation and collagen synthesis by proteinase-activated receptors

BACKGROUND/AIMS Thrombin and MC tryptase, which are agonists for proteinase-activated receptors-1 and -2, respectively, are both increased in injured liver. We have examined if rat stellate cells express these receptors and if receptor agonists influence stellate cell activation. METHODS Expression of mRNA for proteinase activated receptors-1 and -2 were examined by RT-PCR and Northern blotting in lysates of cultured stellate cells and receptor protein examined by Western blotting. The effects of receptor agonists on cell proliferation and collagen synthesis were examined by 3H-thymidine and 3H-proline incorporation assays, respectively. RESULTS Rat stellate cells activated by culture on plastic showed a progressive increase in expression of proteinase-activated receptor-1 and -2 mRNA and proteinase-activated receptor-2 protein as they transformed to a myofibroblastic phenotype. Proteinase-activated receptor-1 agonists thrombin and the peptide SFFLRN, and proteinase-activated receptor-2 agonists tryptase and the peptide SLIGRL induced stellate cell proliferation and the rapid phosphorylation of 44 and 42 kDa mitogen-activated protein kinases. PD98059, an inhibitor of these kinases, inhibited this proliferative response. Both tryptase and SLIGRL increased collagen secretion by stellate cells. CONCLUSIONS This study indicates that the natural proteinase-activated receptor agonists thrombin and MC tryptase might sustain liver fibrosis by promoting stellate cell proliferation and collagen synthesis.

Human and rat hepatic stellate cells produce stem cell factor: a possible mechanism for mast cell recruitment in liver fibrosis

BACKGROUND/AIMS Mast cell numbers are markedly increased in advanced liver fibrosis. Stem cell factor may recruit mast cells to the liver following injury as it induces mast cell proliferation, survival and differentiation from resident tissue precursors. This study examines stem cell factor production in human fibrotic liver and by hepatic stellate cells during culture in vitro. METHODS Stem cell factor production was examined in human fibrotic livers by ELISA and in human and rat hepatic stellate cell cultures using reverse transcription-polymerase chain reaction (RT-PCR), Northern blotting, Western blotting and immunocytochemistry. Co-culture studies examined adhesion between hepatic stellate cells and purified mast cells. RESULTS RT-PCR showed stem cell factor mRNA was more consistently expressed in fibrotic human livers relative to normal, and ELISA confirmed this by showing stem cell factor protein was significantly increased 2-3-fold in homogenates of human cirrhotic liver (primary biliary cirrhosis, primary sclerosing cholangitis) relative to normal. RT-PCR detected stem cell factor mRNA in human and rat hepatic stellate cells activated by culture on plastic. This was confirmed by Western blotting, which showed that freshly isolated hepatic stellate cells expressed relatively little 30 kD stem cell factor compared to late primary culture activated hepatic stellate cells (14 day) and passaged hepatic stellate cells. As assessed by fluorescence immunocytochemistry, stem cell factor protein was homogeneously expressed by populations of culture-activated rat hepatic stellate cells. During co-culture, purified human skin mast cells adhered to hepatic stellate cell monolayers on plastic, and this adherence was inhibited >50% by addition of antibodies against stem cell factor. CONCLUSIONS Hepatic stellate cells activated in vitro produce stem cell factor. These cells may play an important role in recruiting mast cells to liver during injury and fibrosis.

The activation of synovial mast cells: modulation of histamine release by tryptase and chymase and their inhibitors

Mast cells have been implicated as having pivotal roles in arthritis, but little is known of the processes leading to the activation of synovial mast cells or their potential for pharmacological control. We have investigated the ability of tryptase and chymase, and inhibitors of these major mast cell proteases to modulate the activation of mast cells from human synovial tissue. The tryptase inhibitor drug N-(1-hydroxy-2-naphthoyl)-L-arginyl-L-prolinamide hydrochloride (APC366) inhibited immunoglobulin E (IgE)-dependent histamine release in a dose-dependent manner, with about 70% inhibition being achieved at a dose of 300 microM. Histamine release stimulated by calcium ionophore A23187 was also inhibited by this compound. The chymase inhibitor chymostatin inhibited IgE-dependent histamine release by approximately 60% at 1 microg/ml. Tryptase at concentrations of 3.0 microg/ml and greater stimulated histamine release from synovial cells, which was dependent on catalytic activity, whereas chymase had little effect on these cells. The activation of mast cells by tryptase may represent an amplification process in the synovium. The mast cell stabilising properties of inhibitors of tryptase and chymase could be of therapeutic value in arthritis.

Extracellular matrix regulates hepatic stellate cell phenotype and survival

During liver fibrosis, hepatic stellate cells (HSC) phenotypically alter from quiescent compact, vitamin A rich non-fibrogenic cells into proliferative myofibroblasts (activated HSC) with reduced vitamin A and expressing abundant extracellular matrix components (ECM). Modifications to the ECM during liver injury may initiate and support HSC activation. Integrins may play a role in transducing these ECM signals and their expression on HSC alters dunng cell activation. In this study we have examined if ECM can influence HSC transformation and survival in vitro. HSC were isolated from normal rat liver and activated by culturing for 14 days on plastic. Cells were recovered by trypsinisation and transferred back onto plastic or 0.5 mm layers of Matrigel. Dunng culture over the next 7 days, HSC on plastic maintained a myofibroblastic appearance, expressing abundant ~-smooth muscle actin (SMA) and collagen I by a Northern and Western blotting. They continued to proliferate by H thymidine incorporation, but also underwent a high rate of apoptosis assessed by nuclear acridine orange staining when subjected to serum deprivation for 6 hours. In contrast, HSC raplated on Matrigel over the same period adopted a quiescent phenotypa, with minimal proliferation, reacquired vitamin A droplets and stopped expressing SMA. These cells also showed enhanced bcl-2 expression and a 3-fold reduction in rate of apoptosis dunng serum deprivation compared to plastic cultures. The disintegdn echistatin increased HSC apoptosis on plastic or Matrigel. Our studies show that ECM profoundly influences HSC phenotypa, proliferation and apoptosis. We suggest that, dunng liver fibrosis, remodelling of the non~al basement membrane-like ECM underlying HSC might sustain the activated fibrogenic phenotype of these cells. Molecular and cell biology (gene expression, signalling, fibrosis)