Pieter De Bleser

Transforming growth factor-β gene expression in normal and fibrotic rat liver

BACKGROUND/AIMS: Transforming growth factor-beta (TGF-beta) is considered to be an important mediator in the development of fibrosis in several chronic liver diseases. To understand the mechanism(s) by which TGF-beta exerts its action(s), we investigated the cellular distribution of TGF-beta(1,2,3) transcripts in normal and carbon tetrachloride (CCl4)-induced fibrotic rat liver. METHODS: Parenchymal, sinusoidal endothelial, Kupffer and stellate cells were isolated and purified. The exact cellular composition of each isolate was determined by transmission electron microscopy. Expression of TGF-beta(1,2,3) transcripts was investigated using Northern hybridization analysis. Hybridization signals were quantified by scanning densitometry and corrected for: (i) differences in extractable RNA per cell type, (ii) signal contribution from contaminating cells, and (iii) differences in loading, capillary transfer and hybridization. RESULTS: In normal liver, TGF-beta1 mRNA was predominantly expressed in Kupffer cells, exhibiting values approximately 9-fold higher than those in stellate cells. No expression was found in endothelial and parenchymal cells. Signals for TGF-beta2 and TGF-beta3 were much weaker when compared to TGF-beta1. In Kupffer cells, the level of TGF-beta2 was approximately 4-fold higher than in stellate cells. Little expression was found in endothelial cells. TGF-beta3 expression could only be detected in stellate cells. TGF-beta2 and TGF-beta3 was not expressed in parenchymal cells. In fibrotic liver, TGF-beta1 mRNA was strongly expressed in all the sinusoidal cells. TGF-beta2 and TGF-beta3 could no longer be detected. When compared to the level of expression in normal stellate cells, the level of TGF-beta1 increased 12-fold in stellate cells from fibrotic livers, and 6-fold in endothelial cells. In Kupffer cells, the level of expression remained unchanged. CONCLUSIONS: (i) In both normal and fibrotic liver, TGF-beta1 is the most abundant isoform, (ii) in normal liver, TGF-beta1 is expressed strongly by Kupffer cells and moderately by stellate cells, TGF-beta2 expression is highest in Kupffer cells, followed by stellate cells and endothelial cells. TGF-beta3 is expressed by stellate cells, (iii) in fibrotic liver, the level of TGF-beta1 expression increases selectively in stellate cells and endothelial cells. This suggests an important role, not only for stellate, but also for endothelial cells in fibrogenesis.

Glutathione levels discriminate between oxidative stress and transforming growth factor-beta signaling in activated rat hepatic stellate cells.

Reactive oxygen species are implicated in the pathogenesis of several diseases, including Alzheimers disease, multiple sclerosis, human immunodeficiency virus, and liver fibrosis. With respect to liver fibrosis, we have investigated differences in antioxidant enzymes expression in stellate cells (SCs) and parenchymal cells from normal and CCl4-treated rat livers. We observed an increase in the expression of catalase in activated SCs. Treatment with transforming growth factor-β (TGF-β) increased the production of H2O2. Treatment with catalase decreased TGF-β expression. Addition of H2O2resulted in increased TGF-β production. 3-Amino-1,2,4-triazole abolished the capacity of SCs to remove H2O2. A paradoxical increase in capacity was observed when the cells were pretreated with diethyl maleate. Treatment with 3-amino-1,2,4-triazole increased TGF-β production. A paradoxical decrease of TGF-β production was observed with diethyl maleate. Treatment of the cells with N-acetylcysteine resulted in increased TGF-β production. TGF-β decreased the capacity of the SCs to remove H2O2. An increase in the capacity to remove H2O2 was observed when TGF-β was removed by neutralizing antibodies. In conclusion, our results suggest: 1) a link between cellular GSH levels and TGF-β production and 2) that cellular GSH levels discriminate whether H2O2 is the result of oxidative stress or acts as second messenger in the TGF-β signal transduction pathway.

Actin filament formation, reorganization and migration are impaired in hepatic stellate cells under influence of trichostatin A, a histone deacetylase inhibitor

BACKGROUND/AIMS Previously, trichostatin A (TSA), a histone deacetylase inhibitor, has been shown to exhibit strong antifibrotic characteristics in hepatic stellate cells (HSC), which are known to play a central role in chronic liver diseases. TSA retained a more quiescent phenotype in spite of culture conditions that favor transdifferentiation into activated HSC. METHODS To identify TSA-sensitive genes, differential mRNA display, Northern and Western blot analysis were used and genes were functionally validated by using contraction and motility assays. RESULTS TSA prevented new actin filament formation by down-regulation of two nucleating proteins, actin related protein 2 (Arp2) and Arp3, and by up-regulation of adducin like protein 70 (ADDL70) and gelsolin, two capping proteins. RhoA, a key mediator in the development of the actin cytoskeleton, decreased following TSA exposure. Expression of proteins of Class III intermediate filaments was affected by TSA. Furthermore, F-actin and G-actin were expressed heterogeneously under influence of TSA. Functionally, TSA treatment abrogated migration of quiescent HSC, while migration was reduced in transitional HSC. The endothelin-1-induced contractility properties of HSC was not affected by TSA. CONCLUSIONS These data indicate that TSA affects the development of the actin cytoskeleton in quiescent HSC and thereby abrogates the process of HSC transdifferentiation.

Identification of connective tissue gene transcripts in freshly isolated parenchymal, endothelial, Kupffer and fat-storing cells by Northern hybridization analysis

The aim of the present study was to identify the cell types that express collagen alpha 1(I), alpha 1(III) and alpha 1(IV), fibronectin and laminin B1 genes in normal rat liver. Parenchymal, sinusoidal endothelial, Kupffer and fat-storing (Ito) cells were isolated and purified. Total RNA of the freshly isolated cells was subjected to Northern hybridization analysis. We also compared the steady state levels of specific mRNAs in freshly isolated fat-storing cells to the levels in myofibroblast-like cells obtained from purified fat-storing cells cultured for two passages. The average purity of each cell preparation, and the percentage of contaminating cells, were determined by transmission electron microscopy and by examining the presence of vitamin A-autofluorescent cells. Fibronectin and collagen alpha 1(III) mRNAs were detected in total RNA of purified parenchymal cells. In poly(A)+ enriched RNA, small amounts of collagen alpha 1(I) mRNA were also present. In total RNA of freshly isolated fat-storing cells, collagen alpha 1(III), alpha 1(IV), and laminin B1 transcripts were found, whereas collagen alpha 1(I) and fibronectin mRNAs were not detected. Cultured fat-storing cells, however, did contain high levels of collagen alpha 1(I) and fibronectin mRNAs. The molecular size of the latter transcript was larger than the fibronectin transcript found in parenchymal cells and the whole liver. Endothelial cells contained small amounts of alpha 1(IV) mRNA. Kupffer cells did not contain the investigated transcripts. We conclude that normal parenchymal, fat-storing and endothelial cells each express a typical pattern of connective tissue molecules. When fat-storing cells are allowed to differentiate into myofibroblast-like cells, they express high levels of collagen alpha 1(I) and fibronectin mRNAs, in addition to collagen alpha 1(III) and alpha 1(IV), and laminin B1 chain mRNAs.

Gene expression and synthesis of fibronectin isoforms in rat hepatic stellate cells. Comparison with liver parenchymal cells and skin fibroblasts.

Fibronectins are multifunctional glycoproteins that are important components of the extracellular matrix in normal and fibrotic liver. Multiple fibronectin isoforms are generated from a single gene by alternative splicing of the primary transcript at the domains EIIIA, EIIIB, and V. The aim of this study was to investigate the fibronectin isoforms expressed by activated hepatic stellate cells, the most important connective tissue‐producing cells in injured liver. Hepatocytes and skin fibroblasts were also studied for comparison. Activation of hepatic stellate cells in vivo was induced by injecting CCl4 twice weekly for 3 weeks. Activation in vitro was achieved by culturing cells on plastic. The level of activation was evaluated by α‐smooth muscle actin immunocytochemistry. Steady‐state levels of fibronectin isoform messenger RNA were examined by Northern hybridization analysis using specific cDNA probes for the EIIIA, EIIIB, and V domains. The de novo synthesis of fibronectin isoforms was examined by metabolic labelling and immunoprecipitation using domain‐specific monoclonal antibodies. Fibronectin transcripts were not detectable in freshly isolated hepatic stellate cells from normal liver. Cultured hepatic stellate cells, as well as skin fibroblasts, expressed EIIIA+, EIIIB+ and V95+ transcripts. They were detectable as early as day 3 and increased with time in culture. At 3 days in culture, more than 90 per cent of stellate cells were α‐smooth muscle actin‐positive. In vivo activated hepatic stellate cells expressed EIIIA+ and V95+ transcripts; EIIIB+ fibronectin mRNA was absent. Less than 20 per cent of in vivo activated stellate cells expressed α‐smooth muscle actin. Freshly isolated parenchymal cells from normal liver as well as from CCl4‐treated liver expressed V95+ transcripts, but were negative for EIIIA or EIIIB fibronectin mRNA. Immunoprecipitation results were in accordance with Northern hybridization analysis. Hepatic stellate cells in culture synthesized and secreted fibronectin molecules that contained EIIIA, EIIIB, and V fragments. Our results indicate that hepatic stellate cells synthesize and secrete fibronectin isoforms that are distinct from those of parenchymal cells.

Renal antioxidant enzymes and fibrosis-related markers in the rat adriamycin model

Excessive generation of reactive oxygen intermediates can induce changes in the cellular antioxidant defence system. In this study we examine the antioxidant enzyme status and the expression of fibrosis-related marker proteins in the Adriamycin model of chronic renal failure in the rat. Twenty weeks after Adriamycin treatment, rats have overt nephrotic syndrome and renal failure with development of tubulo-interstitial fibrosis and glomerulosclerosis. Lipids accumulate in blood and in both glomeruli and tubulo-interstitial tissue. Desmin and α-smooth muscle actin expression increases in glomeruli and in the tubulo-interstitial area. Renal cortex antioxidant enzyme activities are decreased 20 weeks after Adriamycin injection (to 41% for catalase, to 56% for total superoxide dismutase and to 69% for glutathione peroxidase). The mRNA levels of catalase, Cu/Zn-superoxide dismutase and glutathione peroxidase-1 evaluated by Northern blot are decreased by more than 50% for catalase, Cu/Zn-superoxide dismutase and glutathione peroxidase-1. We conclude that in the rat Adriamycin-induced model of chronic renal failure with fibrosis, the combination of decreased antioxidant enzyme status in renal cortex with high concentrations of lipids in blood and renal tissue facilitates oxidative damage. Development of fibrosis is paralleled by increased expression of desmin and α-smooth muscle actin.