Bernhard Saile

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.

Expression patterns of matrix metalloproteinases and their inhibitors in parenchymal and non-parenchymal cells of rat liver: regulation by TNF-α and TGF-β1

Abstract 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-α stimulated both MMP and TIMP expression of hepatic stellate cells, while TGF-β1 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-α, while diminished matrix degradation during chronic tissue injury might be due to the action of TGF-β1 through TIMP induction.

Portal tract fibrogenesis in the liver.

The portal area is the ‘main entrance’ and one of the two main exits of the liver lobule. Through the main entrance portal and arterial blood reach the liver sinusoids. Through the exit the bile flows towards the duodenum. The three main structures, portal vein and artery with their own wall (and vascular smooth muscle cells) and bile duct with its basal membrane, are surrounded by loose myofibroblasts and by the first layer of hepatocytes and non-parenchymal cells. Chronic diseases of the liver can lead to development of liver cirrhosis, characterized by formation of fibrotic septa which can be portal–portal in the case of the chronic biliary damage or portal–central in the case of the chronic viral hepatitis. Central–central septa can also be observed under other pathological conditions. When damaging noxae are introduced to the liver, inflammatory cells are first recruited to the portal field, the first layer of hepatocytes may be destroyed (enlargement of the portal field) and portal (myo)fibroblasts become activated. A similar reaction may take place when the target of inflammation is the bile duct with consecutive reduction of the bile flow, activation of the portal (myo)fibroblasts, proliferation of bile ducts and destruction of the hepatocytes around the portal field. Increased matrix deposition may be the consequence. During the past years several publications dealt with the pathomechanisms of portal fibrogenesis as well as with its resolution. One of the most intriguing observations was that it is not hepatic stellate cells of the hepatic sinusoid, but portal (myo)fibroblasts which rapidly acquire the phenotype of ‘activated’ (myo)fibroblastas in the early stages of cholestatic fibrosis. These may also become the main mesenchymal cells of the porto-portal or porto-central fibrotic septa. This article reviews the similarities as well as differences between the mesenchymal cells of the portal tract and of the fibrotic septa vs ‘activated’ stellate cells of the hepatic sinusoids, and discusses the debate over their relative contributions to liver fibrogenesis.

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

Abstract 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.

The bcl, NFκB and p53/p21WAF1 systems are involved in spontaneous apoptosis and in the anti-apoptotic effect of TGF-β or TNF-α on activated hepatic stellate cells

Summary Activated hepatic stellate cells (HSC) are thought to play a pivotal role in development of liver fibrosis which takes place in chronic liver diseases. Previous studies have shown that “activated” rat HSC undergo spontaneous apoptosis probably through the CD95/CD95L pathway. TGF-β as well as TNF-α reduced spontaneous apoptosis and CD95L expression. The aim of this study was to investigate the possible mechanisms responsible for the spontaneous apoptosis and for the anti-apoptotic effect of TGF-β and TNF-α on activated HSC. While bcl-2, bax, NFκB and p53 gene expression were spontaneously upregulated, bcl-x L and p21 WAF1 gene expression decreased and IκB remained unchanged during the activation process in vitro. TGF-β as well as TNF-α induced activation of NFκB and upregulated bcl-x L . The latter was inhibited by overexpression of IκB. By suppressing spontaneous apoptosis TGF-β as well as TNF-α inhibited p53 gene expression while that of the p21 WAF1 gene was increased. We conclude that TGF-β as well as TNF-α may act as surviving factors for activated rat HSC not only through reduction of CD95L gene expression but also by upregulating the anti-apoptotic factors NFκB, bcl-x L and p21 WAF1 and by downregulating the proapoptotic factor p53. The interaction with these factors may lead to the generation of new antifibrotic drugs.

IGF-I induces DNA synthesis and apoptosis in rat liver hepatic stellate cells (HSC) but DNA synthesis and proliferation in rat liver myofibroblasts (rMF).

Several lines of evidence suggest a role of insulin-like growth factor I (IGF-I) in the regulation of apoptosis. Up to now its impact on many specific cells is unknown. We therefore studied the effect of IGF-I on two similar mesenchymal matrix-producing cell types of the liver, the hepatic stellate cells (HSC) and the myofibroblasts (rMF). The present study aimed to reveal the influence of IGF-I on cell cycle and apoptosis of HSC and rMF and to elucidate responsible signaling. While IGF-I significantly increased DNA synthesis in HSC, cell number decreased and apoptosis increased. In rMF IGF-I also increased DNA synthesis, which is, however, followed by proliferation. Blocking extracellular signal regulating kinase (ERK) revealed that in HSC, bcl-2 upregulation and bax downregulation are effected downstream of ERK, whereas downregulation of NFκB and consecutive of bcl-xL is mediated upstream. In the rMF upregulation of both, the antiapoptotic bcl-2 and bcl-xL is mediated upstream of ERK. The expression of the proapoptotic bax is not regulated by IGF-I in rMF. The studies demonstrate a completely different effect and signaling of IGF-I in two morphologically and functionally similar matrix-producing cells of the liver.

Atorvastatin induces apoptosis by a caspase‐9‐dependent pathway: an in vitro study on activated rat hepatic stellate cells

Background: Statins are shown to have cholesterol‐independent properties such as anti‐inflammation and immunomodulation. Activated hepatic stellate cells (HSCs) acquire the capacity to synthesize matrix proteins in damaged liver. We tested the hypothesis that atorvastatin may be capable of inducing apoptosis in HSCs.

Identification of Genes Responsive to Gamma Radiation in Rat Hepatocytes and Rat Liver by cDNA Array Gene Expression Analysis

Abstract Christiansen, H., Batusic, D., Saile, B., Hermann, R. M., Dudas, J., Rave-Frank, M., Hess, C. F., Schmidberger, H. and Ramadori, G. Identification of Genes Responsive to Gamma Radiation in Rat Hepatocytes and Rat Liver by cDNA Array Gene Expression Analysis. Radiat. Res. 165, 318–325 (2006). The mechanisms underlying hepatocellular damage after irradiation are obscure. We identified genes induced by radiation in isolated rat hepatocytes in vitro by cDNA array gene expression analysis and then screened in vivo experiments with those same genes using real-time PCR and Western blotting. Hepatocytes were irradiated and cDNA array analyses were performed 6 h after irradiation. The mRNA of differentially expressed genes was quantitatively analyzed by real-time PCR. cDNA array analyses showed an up-regulation of 10 genes in hepatocytes 6 h after irradiation; this was confirmed by real-time PCR. In vivo, rat livers were irradiated selectively. Treated and sham-irradiated controls were killed humanely 1, 3, 6, 12, 24 and 48 h after irradiation. Liver RNA was analyzed by real-time PCR; expression of in vivo altered genes was also analyzed at the protein level by Western blotting. Up-regulation was confirmed for three of the in vitro altered genes (multidrug resistance protein, proteasome component C3, eukaryotic translation initiation factor 2). Histologically, livers from irradiated animals were characterized by steatosis of hepatocytes. Thus we identified genes that may be involved in liver steatosis after irradiation. The methods shown in this work should help to further clarify the consequences of radiation exposure in the liver.

Decrease of platelet-endothelial cell adhesion molecule 1-gene-expression in inflammatory cells and in endothelial cells in the rat liver following CCl4-administration and in vitro after treatment with TNFα

UNLABELLED Platelet-endothelial cell adhesion molecule (PECAM-1), a member of the Ig superfamily is strongly expressed at endothelial cell-cell junctions, on most leukocytes and on monocytes. PECAM-1 has been implicated as a key mediator of the transendothelial migration of leukocytes and monocytes. To further define the physiological role of PECAM-1, we studied the modulation of PECAM-1-expression in a model of liver inflammation in both mononuclear cells (MCs) and sinusoidal endothelial cells (SECs). In normal rat liver sections, PECAM-1 immunohistology indicated a sinusoidal pattern similar to the ICAM-1 staining. Both, SECs, small and large MCs isolated from control rats express PECAM-1 as demonstrated by immunocytochemistry, flow cytometry, and Northern blot analysis. Immunohistochemical studies on liver sections from CCl(4)-treated animals indicated, that in the areas of necrosis 24-48 h after a single administration of the toxin, there was an accumulation of LFA-1-, ED1- (marker for rat monocytes) and ICAM-1-positive, but ED2-(marker for tissue macrophages)-negative inflammatory cells. Most of these cells were PECAM-1-negative. In situ hybridization indicated that there is no accumulation of PECAM-1 specific transcripts after CCl(4) treatment within the pericentral region. Immunocytology, flow cytometry, and Northern blot analysis of MCs and SECs isolated at different times after the administration of CCl(4) revealed a decrease of PECAM-1 gene expression in MCs and in SECs, whereas ICAM-1 expression increased. As TNFalpha has been shown to be upregulated early after CCl(4) administration, the influence of TNFalpha on PECAM-gene-expression was analyzed. TNFalpha treatment of cultured rat SECs and of small and large MCs from normal liver decreased PECAM-1 specific transcript level in parallel to the increase of ICAM-1 transcript level. CONCLUSIONS Early production of TNFalpha after liver injury could induce an increased ICAM-1-expression and a decreased PECAM-1 expression, which might be essential for the transmigration of inflammatory cells into the parenchyma.

Inflammation and repair in viral hepatitis C.

Hepatitis C viral infection (HCV) results in liver damage leading to inflammation and fibrosis of the liver and increasing rates of hepatic decompensation and hepatocellular carcinoma (HCC). However, the host’s immune response and viral determinants of liver disease progression are poorly understood. This review will address the determinants of liver injury in chronic HCV infection and the risk factors leading to rapid disease progression. We aim to better understand the factors that distinguish a relatively benign course of HCV from one with progression to cirrhosis. We will accomplish this task by discussion of three topics: (1) the role of cytokines in the adaptive immune response against the HCV infection; (2) the progression of fibrosis; and (3) the risk factors of co-morbidity with alcohol and human immunodeficiency virus (HIV) in HCV-infected individuals. Despite recent improvements in treating HCV infection using pegylated interferon alpha (PEGIFN-α) and ribavirin, about half of individuals infected with some genotypes, for example genotypes 1 and 4, will not respond to treatment or cannot be treated because of contraindications. This review will also aim to describe the importance of IFN-α-based therapies in HCV infection, ways of monitoring them, and associated complications.