Role of the Ets-1 transcription factor during activation of rat hepatic stellate cells in culture.

During liver tissue repair, hepatic stellate cells (HSCs), a pericyte-like nonparenchymal liver cell population, transform from a quiescent status (resting HSCs) into myofibroblast like cells (activated HSCs); the latter is the principal matrix-synthesizing cell of the liver. Although several factors have been shown to be involved in this important process, the molecular mechanisms regulating HSC activation are still under investigation. To identify key regulatory proteins involved in the HSC activation process, we used different mRNA display technologies, with cDNAs prepared from HSCs at different stages of in vitro activation. With the latter technique, the transcription factor Ets-1 was detected through its down-regulation during activation. As confirmed by Northern blot and reverse transcriptase-polymerase chain reaction (RT-PCR) analysis, mRNAs coding for Ets-1 were present in the highest amounts in freshly isolated HSCs and in HSCs 2 days after plating (classified as resting HSCs/early activated HSCs) and were diminished in HSCs 7 days after plating (activated cells). Ets-1 protein was present in HSC-lysates, as assessed by Western blot, and bound to an oligonucleotide containing the Ets-1 consensus cis-acting motif, as demonstrated by electrophoretic mobility shift assay. Ets-1 binding activity peaked in nuclear extracts prepared from resting/early activated cells and was diminished in extracts derived from fully activated cells. In contrast, binding activity of the transcription factors TFIID, AP-1, and SP-1 was highest in activated HSCs and only barely detectable in resting/early activated HSCs. By Northern blot and RT-PCR analysis, Ets-1-specific transcripts were present in parenchymal and other nonparenchymal liver cells too, illustrating that hepatic Ets-1 expression is not specific or restricted to HSCs. However, the unique pattern of Ets-1 binding activity present in resting versus activated HSCs and its known implications for cellular differentiation and tissue remodeling suggest that Ets-1 could be of crucial importance for HSC activation and hepatic tissue repair.

Localization of liver myofibroblasts and hepatic stellate cells in normal and diseased rat livers: distinct roles of (myo-)fibroblast subpopulations in hepatic tissue repair.

Previous in vitro studies indicated that hepatic stellate cells (HSC) and rat liver myofibroblasts (rMF) have to be regarded as different cell populations of the myofibroblastic lineage with fibrogenic potential. Employing the discrimination features defined by these studies the localization of HSC and rMF was analyzed in diseased livers. Normal and acutely as well as chronically carbon tetrachloride-injured livers were analyzed by immunohistochemistry and by in situ hybridization. In normal livers HSC [desmin/glial fibrillary acid protein (GFAP)-positive cells] were distributed in the hepatic parenchyma, while rMF (desmin/smooth muscle alpha actin-positive, GFAP-negative cells colocalized with fibulin-2) were located in the portal field, the walls of central veins, and only occasionally in the parenchyma. Acute liver injury was characterized almost exclusively by an increase in the number of HSC, while the amount of rMF was nearly unchanged. In early stages of fibrosis, HSC and rMF were detected within the developing scars. In advanced stages of fibrosis, HSC were mainly present at the scar-parenchymal interface, while rMF accounted for the majority of the cells located within the scar. At every stage of fibrogenesis, rMF, in contrast to HSC, were only occasionally detected in the hepatic parenchyma. HSC and rMF are present in normal and diseased livers in distinct compartments and respond differentially to tissue injury. Acute liver injury is followed by an almost exclusive increase in the number of HSC, while in chronically injured livers not only HSC but also rMF are involved in scar formation.

Rat liver myofibroblasts and hepatic stellate cells: different cell populations of the fibroblast lineage with fibrogenic potential.

BACKGROUND & AIMS: Hepatic stellate cells (HSCs) are considered the principal matrix-producing cells of the damaged liver. However, other cell types of the fibroblast lineage that have not yet been characterized are also involved in liver tissue repair and fibrogenesis. METHODS: We established cultures of cells of the fibroblast lineage, termed rat liver myofibroblasts, and analyzed their phenotypical and functional properties in comparison with HSCs. RESULTS: HSCs and rat liver myofibroblasts were discernible by morphological criteria and growth behavior. Prolonged subcultivation of rat liver myofibroblasts was achieved, but HSCs were maintained in culture at maximum until second passage. HSCs were characterized by expression of glial fibrillary acidic protein, desmin, and vascular cell adhesion molecule 1, which were almost completely absent in rat liver myofibroblasts. For synthetic properties, HSCs and rat liver myofibroblasts displayed mostly overlapping properties with 4 striking differences. The complement-activating protease P100 and the protease inhibitor alpha(2)-macroglobulin were preferentially expressed by HSCs, whereas interleukin 6-coding messenger RNAs and the extracellular matrix protein fibulin 2 were almost exclusively detectable in rat liver myofibroblasts. CONCLUSIONS: The data show that morphologically and functionally different fibroblastic populations, HSCs and rat liver myofibroblasts, can be derived from liver tissue. HSCs may not represent the single matrix-producing cell type of the fibroblast lineage in the liver.

Cellular localization of hepatic cytochrome 1B1 expression and its regulation by aromatic hydrocarbons and inflammatory cytokines.

Cytochrome P450 1B1 (CYP1B1) is an activator of several xenobiotics and is induced in the liver upon experimental exposure to aromatic hydrocarbons. Since its cellular localization and regulation are incompletely clarified, Cyp1B1 expression and inducibility by 9,10-dimethyl-1,2-benzanthracene (DMBA) and inflammatory cytokines were investigated in different rat liver cell populations in vitro and in the liver during hepatocellular injury. Expression of Cyp1B1 was studied by Northern blot analysis in hepatic stellate cells (HSCs), myofibroblasts (MFs), Kupffer cells (KCs), and hepatocytes at various time points of primary cultures and in acutely damaged rat liver (carbon tetrachloride model). Enzyme inducibility was assessed by incubation of cells with DMBA as well as, in the case of HSCs, with tumor necrosis factor-alpha (TNF-alpha) and transforming growth factor beta1 (TGFbeta1). Cyp1B1 messengers were expressed at high levels by HSCs and MFs, whereas constitutive expression was not detectable in KCs or in hepatocytes. Cyp1B1-specific mRNA were expressed at highest levels in HSCs at an early stage of activation (2 days after plating) and were diminished upon further activation. DMBA strongly enhanced Cyp1B1 gene expression in HSCs, MFs, and in hepatocytes at day 3 of primary cultures, but not in hepatocytes at day 1, or in KCs. The inflammatory cytokine TNF-alpha enhanced the Cyp1B1 gene expression in HSCs, either when administered alone or in addition to DMBA, while TGFbeta1 did not affect Cyp1B1 expression, even after DMBA induction. We conclude that HSCs and MFs seem to be the major cellular sources of hepatic Cyp1B1 expression and that the constitutive expression of the Cyp1B1 gene and the responsiveness to DMBA stimulation differ between mesenchymal and parenchymal liver cells, indicating a cell-specific regulation of Cyp1B1 gene expression. Interestingly, TNF-alpha is a potent stimulator of the Cyp1B1 gene in HSCs and acts in concert with DMBA.

Expression patterns of matrix metalloproteinases and their inhibitors in parenchymal and non-parenchymal cells of rat liver: regulation by TNF-alpha and TGF-beta1.

BACKGROUND/AIMS: Although matrix metalloproteinases (MMPs) and their specific inhibitors (TIMPs) play an essential role in liver injury associated with tissue remodeling, the cellular origin of MMPs/TMPs within the liver remains to be clarified. METHODS: Different liver cell populations were analysed with respect to their expression by reverse transcription-polymerase chain reaction, Northern blot analysis and zymography. RESULTS: MMP and TIMP coding transcripts were detectable in all liver cell types by reverse transcription-polymerase chain reaction; however, the cellular expression levels were markedly different as assessed by Northern blot analysis. Gelatinase-B was predominantly expressed in Kupffer cells, gelatinase-A in hepatic stellate cells and rat liver myofibroblasts and stromelysins-1, -2 as well as collagenase in hepatic stellate cells. Membrane type-1 MMP (MMP-14) was found in significant amounts in all liver cells. TIMP-1 coding m-RNAs were present mainly in hepatic stellate cells and rat liver myofibroblasts, TIMP-2 additionally in Kupffer cells, while TIMP-3 expression was detectable only in hepatocytes. During in vitro activation of hepatic stellate cells, MMP expression was mostly downregulated, while TIMP expression was enhanced, thereby providing an explanation for matrix accumulation co-localised with these cells during chronic liver injury. In general, TNF-alpha stimulated both MMP and TIMP expression of hepatic stellate cells, while TGF-beta1 induced TIMP expression only. CONCLUSIONS: Collectively these data demonstrate that all resident liver cells are involved in matrix degradation to some extent and that hepatic stellate cells play an important role in matrix breakdown in addition to matrix synthesis. The cytokine-specific regulation of MMP/TIMP expression in hepatic stellate cells suggests that the initial matrix breakdown following liver injury might be enhanced by TNF-alpha, while diminished matrix degradation during chronic tissue injury might be due to the action of TGF-beta1 through TIMP induction.

Expression and regulation of cell adhesion molecules by hepatic stellate cells (HSC) of rat liver: involvement of HSC in recruitment of inflammatory cells during hepatic tissue repair.

Hepatic stellate cells (HSC), a pericyte-like nonparenchymal liver cell population, are regarded as the principal matrix-synthesizing cells of fibrotic liver. They might also play a role during liver inflammation. The present study analyzed (i) expression of cell adhesion molecules (CAMs) mediating cell infiltration, like intercellular adhesion molecule-1 (I-CAM-1) and vascular cell adhesion molecule-1 (V-CAM-1), by HSC, (ii) CAM regulation in HSC by growth factors and inflammatory cytokines, and (iii) CAM expression in situ during liver inflammation, using immunochemistry and Northern blot analysis. I-CAM-1 and V-CAM-1 expression was present in HSC in vitro and in cells located in the sinusoidal/perisinusoidal area of normal liver. Growth factors, eg, transforming growth factor-beta1, down-regulated I-CAM-1- and V-CAM-1-coding mRNAs and stimulated N-CAM expression of HSC. In contrast, inflammatory cytokines like tumor necrosis factor-alpha reduced N-CAM-coding mRNAs, whereas induction of I-CAM-1- and V-CAM-1-specific transcripts increased several fold. In situ, messengers specific for I-CAM-1 and V-CAM-1 were induced 3 hours after CCl4 treatment (thereby preceding mononuclear cell infiltration starting at 12 hours), were expressed at maximal levels 9-12 hours after CCl4 application, and decreased afterwards. I-CAM-1 and V-CAM-1 immunoreactivity increased in a linear fashion starting 3 hours after CCl4-induced liver injury, was detected in highest amounts at 24-48 hours characterized by maximal cell infiltration, and returned to baseline values at 96 hours. Interestingly, the induction/repression of CAM-specific messengers paralleled the time kinetics of tumor necrosis factor-alpha transforming growth factor-beta1 expression in injured liver. HSC might be important during the onset of hepatic tissue injury as proinflammatory elements and might interact with I-CAM-1 and V-CAM-1 ligand-bearing cells, namely lymphocyte function-associated antigen-1- or Mac-1/very late activation antigen-4-positive inflammatory cells, thereby modulating the recruitment and migration of mononuclear cells within the perisinusoidal space of diseased livers.