Katrin Neubauer

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.

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.

Glial fibrillary acidic protein--a cell type specific marker for Ito cells in vivo and in vitro.

BACKGROUND/AIMS Glial fibrillary acidic protein is an intermediate filament first identified in the brain in astrocytes. This study examines glial fibrillary acidic protein immunoreactivity in normal and damaged rat livers. Glial fibrillary acidic protein-gene-expression in Ito cells, endothelial cells, Kupffer cells, and hepatocytes is also analyzed. METHODS Sequential cryostat sections from normal, as well as acutely or chronically CC14 damaged rat livers were analyzed by immunostaining for the presence of glial fibrillary acidic protein and desmin. Glial fibrillary acidic protein-expression in isolated liver cells was studied by immunocytology, Western blot, Northern blot analysis, and reverse-transcription polymerase chain reaction. The specificity of polymerase chain reaction products was tested by Southern blot hybridization and partial sequencing. RESULTS In the normal liver, glial fibrillary acidic protein-positive cells were detected in the perisinusoidal area. These cells were also desmin-immunoreactive as determined by immunostaining. In contrast, cells of the vessel walls were desmin-positive, but glial fibrillary acidic protein-negative. In the acutely damaged livers glial fibrillary acidic protein-positivity was detectable along the non-damaged sinusoids as well as in the necrotic areas. In chronically damaged livers glial fibrillary acidic protein was more detectable at the margins of the fibrotic septa, less inside the septa. All glial fibrillary acidic protein-positive cells were desmin-positive, but several desmin-positive cells were glial fibrillary acidic protein-negative (especially inside the septa). Among the different liver cell subpopulations tested in vitro, glial fibrillary acidic protein-gene expression was only detectable in Ito cells. During primary culture, glial fibrillary acidic protein-expression decreased in parallel to Ito cell activation. CONCLUSIONS Glial fibrillary acidic protein is a new cell type specific marker for Ito cells, which might allow distinction between Ito cells and other fibroblastic liver cells (cells of the vessel walls). Cells located at the margins of fibrotic septa definitely represent Ito cells.

Expression and Regulation of Cell Adhesion Molecules by Hepatic Stellate Cells (HSC) of Rat Liver : Involvment 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.

Expression of reelin in hepatic stellate cells and during hepatic tissue repair: a novel marker for the differentiation of HSC from other liver myofibroblasts

BACKGROUND/AIMS Hepatic stellate cells (HSC) and rat liver myofibroblasts (rMF), two similar but not identical cell populations, play a major role during hepatic tissue repair. METHODS To identify marker proteins for the different fibroblastic cell populations, m-RNA-profiling technology was employed using c-DNAs prepared from HSC and rMF. RESULTS/CONCLUSIONS The extracellular matrix protein reelin was identified through its presence in HSC and absence in rMF derived samples. As confirmed by Northern blot analysis and by immunoprecipitation, reelin expression was present in similar amounts in resting and activated HSC and was not detectable in rMF. Therefore reelin is the only marker presently available to distinguish HSC at any stage of differentiation from rMF. Following a single CCl4 mediated liver injury, reelin specific mRNAs were induced early, were elevated up to 24 h following CCl4 dosage and were diminished afterwards. Hepatocytes and non-parenchymal liver cells located in the damaged areas were identified as the main cellular source of enhanced reelin expression. Although reelin expression was upregulated during liver injury, reelin deficient mice recovered completely suggesting either a more distinct role in tissue repair reactions or a case of redundancy through the action of related proteins.

Effect of heparin and liver heparan sulphate on interaction of HepG2-derived transcription factors and their cis-acting elements: altered potential of hepatocellular carcinoma heparan sulphate.

Proteoglycan assembly in malignant tumours is subject to profound changes. The significance of these alterations is not well understood; especially, their role in nuclear regulation is a topic for debate. The capacity of heparin and liver carcinoma heparan sulphate (HS) to alter DNA-transcription factor interactions has been studied to provide further evidence concerning the regulatory potential of glycosaminoglycan (GAG) in the nucleus. Experiments both in vitro and in vivo indicated that heparin and HS are capable of inhibiting the interaction of transcription factors with their consensus oligonucleotide elements. Among five transcription factors studied, AP-1, SP-1, ETS-1 and nuclear factor kappaB proved to be sensitive to heparin and heparan sulphate, whereas TFIID was hardly inhibited in either in vitro or in vivo systems. Interestingly, HS from peritumoral liver was five times more effective than heparin. Liver carcinoma HS was less effective than liver HS, but its activity was comparable with that of heparin. These results indicate that the structural differences of GAG chains strongly influence their biological behaviour. The loss of their recognized functional activity in malignant tumours might promote the development of uncontrolled growth and gene expression favouring the neoplastic process.

Expression of decorin, transforming growth factor-beta1, tissue inhibitor metalloproteinase 1 and 2, and type IV collagenases in chronic hepatitis

Decorin is a small extracellular matrix proteoglycan. It binds and modulates transforming growth factor (TGF)-beta 1 action, the major stimulator of fibrogenesis. Its role in the pathogenesis of human liver cirrhosis is unknown. Therefore, we studied the relationship of the 2 proteins in normal human liver and in 43 chronic hepatitis and liver cirrhosis specimens. To understand the mechanism that maintains matrix deposition in stage IV hepatitis, we studied expression of tissue inhibitor of metalloproteinase (TIMP)-1 and TIMP-2, as well as the activities of type IV collagenases. Gene expression was analyzed on messenger RNA and protein level by morphologic and biochemical approaches. Decorin proved to be an early marker of fibrogenesis, and its deposition increased parallel to that of TGF-beta 1 and to inflammatory activity. Liver fibrosis progressed despite high temporospatial expression of decorin with TGF-beta 1. Neither decorin nor TGF-beta 1 protein deposition increased further in cirrhosis with low inflammatory activity, suggesting that impaired extracellular matrix catabolism rather than active production plays a role in this stage. This possibility was supported by high message levels of metalloproteinase inhibitors, no 72-kd collagenase activities, and low 92-kd collagenase activities.

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.

Platelet-endothelial cell adhesion molecule-1 gene expression in liver sinusoidal endothelial cells during liver injury and repair

BACKGROUND/AIMS Platelet-endothelial cell adhesion molecule (PECAM)-1 is suggested to be critical for transmigration processes. It is a matter of debate whether PECAM-1 is expressed in liver sinusoids and whether it is involved in liver inflammation. METHODS Indirect immunostaining and in situ hybridization was used to analyze PECAM-1 gene expression in normal and diseased rat and human livers as well as in isolated rat sinusoidal endothelial cells (SECs), hepatic stellate cells and hepatocytes. At various time points after the administration of CCl4 (6 h, 24 h, 48 h, and 72 h), PECAM-1 gene expression was analyzed in livers and in SECs by immunostaining, and Northern blot analysis. RESULTS In normal rat or human livers PECAM-1 immunoreactivity was detected along the sinusoids in a pattern similar to ICAM-1 staining. PECAM-1 specific transcripts were detected in freshly isolated and cultured SECs. After a single CCl4-administration, PECAM-1 immunoreactivity did not increase along the sinusoids in contrast to the early increase of ICAM-1. Northern blot analysis indicated that PECAM-1 expression in liver tissue and in isolated SECs does not increase after a single administration of CCl4, whereas ICAM-1 steady-state level increased after 6 h. In diseased human livers PECAM-1 was detectable along the sinusoids, within inflammatory infiltrates and within fibrotic septa. Neither in acutely nor chronically diseased human livers was an obvious increase of PECAM-1 immunoreactivity detectable. CONCLUSIONS PECAM-1 is expressed by SECs. In contrast to ICAM-1, PECAM-1 transcript level is not enhanced during liver damage.

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.