Browsing by Author "Neubauer, K."

The hepatic stellate cell (HSC), the pericyte of the liver sinusoids belongs to the mesenchymal cells of the liver. Damaging noxae induce a transformation from the quiescent (vitamin A-storing cell) to the activated (connective tissue-producing cell) state. The balance between proapoptotic and surviving factors decides about the fate of the activated HSC. Interferon-alpha (IFN-alpha) has been shown to elicit antiproliferative and/or antifibrogenic effects in various cell types of mesenchymal origin. We therefore investigated the effect of IFN-alpha on primary cultured rat HSC in their quiescent (day 2) and activated state (day 7). IFN-alpha significantly inhibited spontaneous apoptosis in activated HSC in vitro and simultaneously inhibited cell cycle progression by inducing a G, arrest. The effect of IFN-alpha is not accompanied by a modulation of CD95, CD95L, p53, p21(WAF1), p27, bcl-2, bcl-X-L, bax, NFkappaB, or IkappaB gene expression. Surprisingly, the IFN-alpha effect could be abolished completely by blocking JAK2 activity or JAK2 translation. The downregulating effect of IFN-alpha on the activity of caspase-8 and caspase-3 could also be neutralized using tyrphostin AG490 or JAK-2 antisense. Taken together IFN-alpha inhibits apoptosis of activated HSC by activation of JAK2 which inhibits the caspase-8 apoptosis pathway.

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 CCl4-treated animals indicated, that in the areas of necrosis 24-45 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 CCl4 treatment within the pericentral region. Immunocytology, flow cytometry, and Northern blot analysis of MCs and SECs isolated at different times after the administration of CCl4 revealed a decrease of PECAM-1 gene expression in MCs and in SECs, whereas ICAM-1 expression increased. As TNF alpha has been shown to be upregulated early after CCl4 administration, the influence of TNF alpha on PECAM-gene-expression was analyzed. TNF alpha 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 TNF alpha 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. (C) 2000 Elsevier Science B.V. All rights reserved.

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 KB 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 rumours might promote the development of uncontrolled growth and gene expression favouring the neoplastic process.

Decorin is a small extracellular matrix proteoglycan. It binds and modulates transforming growth factor (TGF)-beta1 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-beta1 and to inflammatory activity. Liver fibrosis progressed despite high temporospatial expression of decorin with TGF-beta1. Neither decorin nor TGF-beta1 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.

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. (C) 2002 European Association for the Study of the Liver. Published by Elsevier Science B.V. All rights reserved.

Gelsolin, a 90-kDa protein, was suggested to be involved in cell motility, to inhibit apoptosis and to have a protective role for tissue. This study intends to analyse the modulation of cytoplasmic gelsolin expression in damaged rat and human livers and to identify its cellular sources. In the normal liver gelsolin-immunoreactive cells could be identified along vessel walls and along the sinusoids. In cultured rat hepatic stellate cells (HSCs), liver myofibroblasts (MFs), mononuclear cells (MCs) and sinusoidal endothelial cells (SECs), but not in hepatocytes, gelsolin expression could be detected by immunostaining and Northern blot analysis. In acute CCl4-induced liver damage there was no gelsolin positivity detectable in necrotic areas. However, in human fulminant hepatic failure positivity in the necrotic areas was detected. In chronically damaged rat and human livers gelsolin-immunoreactive cells could be identified within the fibrotic septa. Northern blot analysis revealed an increase of the gelsolin-specific transcript level under conditions of acute and chronic human or rat liver damage. The amount of gelsolin-specific transcripts in SECs and large MCs isolated from damaged rat livers increased in comparison to cells obtained from normal rats. However, the amount of gelsolin-specific transcripts in small MCs (representing recruited inflammatory cells) decreased. In conclusion, SECs, MCs, MFs and HSCs, but not hepatocytes, express gelsolin. In the damaged liver all tested cell populations but the inflammatory cells and the hepatocytes are responsible for the enhanced gelsolin expression.

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, bc1-2 upregulation and bax downregulation are effected downstream of ERK, whereas downregulation of NFkappaB and consecutive of bcl-X-L is mediated upstream. In the rMF upregulation of both, the antiapoptotic bcl-2 and bcl-x(L) 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.

Liver fibrosis represents the uniform response of liver to toxic, infectious or metabolic agents. The process leading to liver fibro sis resembles the process of wound healing, including the three phases following tissue injury: inflammation, synthesis of collagenous and noncollagenous extracellular matrix components, and tissue remodelling (scar formation). While a single liver tissue injury can be followed by an almost complete restitution ad integrum, the persistence of the original damaging nora results in tissue damage. During the establishment of liver fibrosis, the basement membrane components collagen type IV, entactin and laminin increase and form a basement membrane-like structure within the space of Disse. The number of endothelial fenestrae of the sinusoids decreases. These changes of the sinusoids are called 'capillarization' because the altered structure of the sinusoids resembles that of capillaries. At the cellular level, origin of liver fibrogenesis is initiated by the damage of hepatocytes, resulting in the recruitment of inflammatory cells and platelets, and activation of Kupffer cells, with subsequent release of cytokines and growth factors. The hepatic stellate cells seem to be the primary target cells for these inflammatory stimuli, because during fibrogenesis, they undergo an activation process to a myofibroblast-like cell, which represents the major matrix producing cell. Based on this pathophysiological mechanism, therapeutic methods are developed to inhibit matrix synthesis or stimulate matrix degradation. A number of substances are currently being tested that either neutralize fibrogenic stimuli and prevent the activation of hepatic stellate cells, or directly modulate the matrix metabolism However, until now, the elimination of the hepatotoxins has been the sole therapeutic concept available for the treatment of liver fibrogenesis in humans.

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.

Hepatic stellate cells (HSC), particularly activated HSC, are thought to be the principle matrix-producing cell of the diseased liver. However, other cell types of the fibroblast lineage, especially the rat liver myofibroblasts (rMF), also have fibrogenic potential. A major difference between the two cell types is the different life span under culture conditions. Although nearly no spontaneous apoptosis could be shown in rMF cultures, 18 +/- 2% of the activated HSC (day 7) were apoptotic. Compared with activated HSC, CD95R was expressed in 70% higher amounts in rMF. CD95L could only be detected in activated HSC. Stimulation of the CD95 system by agonistic antibodies (1 ng/ml) led to apoptosis of all rMF within 2 h, whereas activated HSC were more resistant (5.3 h/ 40% of total cells). Although transforming growth factor-beta downregulated apoptosis in both activated HSC and rMF, tumor necrosis factor-alpha (TNF-alpha) upregulated apoptosis in rMF. Lack of spontaneous apoptosis and CD95L expression in rMF and the different reaction on TNF-alpha stimulation reveal that activated HSC and rMF belong to different cell populations.

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-beta as well as TNF-alpha 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-beta and TNF-alpha on activated HSC. While bcl-2, bax, NF kappaB and p53 gene expression were spontaneously upregulated, bcl-x(L) and p21(WAF1) gene expression decreased and I kappaB remained unchanged during the activation process in vitro. TGF-P as well as TNF-a induced activation of NF kappaB and upregulated bcl-x(L). The latter was inhibited by overexpression of I kappaB. By suppressing spontaneous apoptosis TGF-beta as well as TNF-alpha inhibited p53 gene expression while that of the p21(WAF1) gene was increased. We conclude that TGF-beta as well as TNF-alpha 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 NF kappaB, 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.

Background/Aims: Von Willebrand factor (vWf) is found in high levels in plasma of patients with acute and chronic liver disease. The role of vWf in liver injury and repair is unknown. We studied the effect of liver mass and remodeling on plasma and tissue vWf after partial hepatectomy. Methods: Rats were sacrificed postoperatively at intervals ranging from 60 min to 5 days, and vWf plasma levels were measured by enzyme-linked inummosorbent assay, using rabbit anti-human vWf, and by immunoperoxidase on cryosections, using rabbit anti-vWf/factor VIII. Northern blot hybridization was prepared with a complementary DNA specific to human vWf. Results: vWf plasma levels increased early after sham operation and after 70% partial hepatectomy. The highest levels were reached at 24 h, remaining high for 5 days. Immunostaining showed intense staining of sinusoidal lining cells 4 h after partial hepatectomy, remaining so for 5 days. Non-significant changes in overall liver messenger RNA expression of vWf were seen over 5 days in sham operation and partial hepatectomy. Conclusions: After partial hepatectomy, plasma vWf is increased, probably due to both acute-phase reaction and decreased degradation. An increase in sinusoidal vWf immunostaining may suggest a role for this factor in tissue remodeling. (C) 2002 European Association for the Study of the Liver. Published by Elsevier Science B.V. All rights reserved.

Background/Aims: von Willebrand factor (vWf) is an adhesive glycoprotein known to play a role in hemostasis and in tissue injury. It is found in high levels in plasma of patients with acute hepatic failure and chronic liver disease. The aim of this study was to investigate the pattern of tissue vWf in acute liver failure in humans. Methodology: We studied vWf immunostaining and mRNA expression in the liver of three patients with fulminant liver failure, two patients with chronic liver disease, and two controls. PECAM-1 (CD31) immunostaining and mRNA expression were used as an additional endothelial marker. Results: In chronic liver cirrhosis, vWf deposits were strongly detected at the scar-parenchyma interface. In fulminant hepatic failure, intense deposits were seen in tissue sections in the area of necrosis. A similar pattern of immunostaining was seen with PECAM-1. vWf transcripts were abundant in the liver of patients with chronic disease and minimally expressed in patients with acute hepatic failure and in controls. Conclusions: vWf is deposited within the liver sinusoids early after liver damage. The factor is only partially produced locally during the acute phase of the disease, but is overproduced in chronic disease states. These changes may suggest a role for vWf in liver injury and repair.