Wiley

ABSTRACT— Hepatic stellate cells (HSC) are presently regarded as one of the key cell types involved in the progression of liver fibrosis and in the related pathophysiological and clinical complications. Following acute or chronic liver tissue damage, HSC undergo a process of activation towards a phenotype characterised by increased proliferation, motility, contractility and synthesis of extracellular matrix (ECM) components. Several factors have been shown to play a key role in the promotion of the full‐blown picture of activated HSC. These include extensive changes in the composition and organisation of the ECM, the secretion of several growth factors, cytokines, chemokines, products of oxidative stress and other soluble factors. It is evident that each cellular response to extracellular stimuli must be framed in a scenario where different forces modulate one another and result in a prevalent biological effect. Along these lines, the identification and characterisation of intracellular signalling pathways activated by different stimuli in HSC represent a mandatory step. In this review article we have made an attempt to summarise recent acquisitions to our knowledge of the involvement of different intracellular signalling pathways in key aspects of HSC biology.

ABSTRACT— The appearance of α‐smooth‐muscle‐actin (α‐smA)‐positive cells during hepatic fibrosis was studied immunohistochemically in rat and human livers. In the normal rat liver, α‐smA was observed only in vascular smooth muscle cells. With the progression of fibrosis induced by CCl₄ injection, α‐smA‐positive cells appeared in the perisinusoidal space and the fibrous septa, and ultimately surrounded regenerative nodules. An increase of desmin‐positive cells was recognized in the fibrotic areas and the perisinusoidal area. In the human liver, α‐smA‐positive cells appeared in the fibrotic area, whereas no desmin‐positive cells were observed, except in vascular walls of the central vein and the portal tract. α‐smA is a good marker for the detection of myofibroblast‐like cells, and the appearance of α‐smA in liver mesenchymal cells seems closely related to the process of hepatic fibrosis in both rat and man.

Abstract: The liver has long been known to respond to exposure to certain chemicals with hyperplasia and proliferation of the peroxisomal compartment. This response is now known to be mediated by specific receptors. The peroxisome proliferator‐activated receptors (PPARs) were cloned 10 years ago, and in that interval, have been found to serve as receptors for a number of endogenous lipid compounds, in addition to the peroxisome proliferators that originally led to their study. Three receptors, designated the α, δ, and γ receptors, have been found in mammals. PPARα is the most abundant form found in the liver, with smaller amounts of the δ and γ forms also expressed there. Kupffer cells, like other macrophages, appear to express the α and γ isoforms. Hepatic stellate cells are reported to express the γ isoform. PPARα knock‐out mice fail to undergo peroxisome proliferation when challenged with the proliferators. Moreover, they have severe derangements of lipid metabolism, particularly during fasting, indicating that normal function of the alpha receptors is needed for lipid homeostasis. This in turn suggests that inadequate PPAR‐mediated responses may contribute to abnormal fatty acid metabolism in alcoholic and non‐alcoholic steatohepatitis. Recent information suggests that PPARγ receptors may be important in control of the activation state of the stellate cells, and their repression or inactivation may predispose to hepatic fibrosis. The first approved drug that specifically activates PPARγ, troglitazone, has rarely been found to cause serious liver injury. Although this is likely to represent an idiosyncratic reaction, the medical community will need to be alert to the possibility that activation or blockade of these receptors may cause hepatic dysfunction.

Abstract: Background/Aims: Autoimmune hepatitis (AIH) and primary biliary cirrhosis (PBC) are two autoimmune diseases with unknown etiologies that primarily target the liver. In both diseases, liver lesions are accompanied by large infiltrates of mononuclear cells. The purpose of this study was to determine if either the Fas‐mediated or the granule‐exocytosis pathways contribute to apoptosis in these diseases. Methods: To determine the involvement of apoptosis in tissue injury we examined liver tissue for DNA fragmentation and morphological characteristics of apoptosis. The major cytotoxic pathways of activated lymphocytes were compared by quantitating the levels of transcripts for FasL and granzyme B, and expression was confirmed by immunoprecipitation of these molecules. Results: In both diseases, apoptosis was observed. However, the main cell types undergoing apoptosis were hepatocytes in AIH, and biliary epithelial cells in PBC. In AIH the levels of FasL and granzyme B mRNA were increased over the levels detected in normal liver, while in PBC only the levels of granzyme B were elevated. Additionally, in AIH, the ratio of FasL transcripts to granzyme B transcripts was elevated, reflecting a possible increase in the relative contribution of FasL to the progression of the disease. Immunoprecipitation studies further support an increase in FasL protein expression in AIH. Conclusions: These data suggest that both FasL and granzyme B contribute to the apoptosis observed in AIH and PBC. However, FasL appears to play a more prominent role in the induction of hepatocyte apoptosis and tissue destruction in AIH.

Abstract: Background/Aims: The involvement of a direct viral cytopathic effect or an immune‐mediated mechanism in the progression of hepatic damage in chronic hepatitis C is controversial. The type of immune response is itself a matter of controversy, and histological data are lacking. The aim of this study was to identify the factors associated with the progression of liver injury in 30 HCV/RNA‐positive untreated patients with chronic hepatitis. Methods: Necroinflammatory and architectural damage were evaluated using Ishak's score. Activated hepatic stellate cells (HSC) were visualized by immunohistochemistry for α‐smooth muscle actin (αSMA) and quantitated by morphometry. Plasma HCV/RNA was evaluated using a competitive RT‐PCR method. To study the type of immune response involved in the progression of liver injury, interferony (IFNγ)‐positive cells (as expression of a Th1‐like response) were evaluated by immunohistochemistry and quantitated by morphometry. Results: HSC were mostly detected close to areas of lobular necroinflammation or lining fibrotic septa. The αSMA‐ and Sirius Red‐positive parenchyma correlated significantly with necroinflammatory and architectural scores. IFNγ‐positive cells were detected in periportal areas associated with the inflammatory infiltrates and significantly correlated with architectural damage. No relationship was found between the histological features of liver injury and viral load. Conclusions: HSC activation and progression of liver injury are unrelated to viral load but associated with a Th1‐like response, a plausible target for the treatment of chronic hepatitis C.

ABSTRACT— Regression of bile ductular hyperplasia following elimination of the proliferative stimulus is widely recognised but the mode of deletion of the excess biliary epithelial cells (BEC) is not understood. Apoptosis, a process of cell death distinct from coagulative necrosis, is known to be involved in both atrophy and physiological involution of various organs. Therefore, a time course histological study was made of the frequency of apoptosis among BEC following removal of the proliferative stimulus in two rat models of BEC hyperplasia produced by the feeding of α‐naphthyl isothiocy‐anate (ANIT) for 6 weeks or the ligation of the common bile duct for 5 weeks. In the former, the rats were returned to a normal diet after 6 weeks and in the latter, the total biliary obstruction (TBO) was relieved by a Roux‐en‐Y choledochojejunostomy. As it has been reported that apoptotic bodies in glandular tissues may be extruded into acinar lumina to be lysed or washed away, thereby reducing chances of their detection in histological preparations, in a third experiment bile was collected for 22 h following relief of TBO of 7 days' duration. The centrifuged pellet of bile was studied by electron microscopy. In both models, throughout the 21 days of observation following removal of the stimulus, apoptotic bodies were found between or as phagocytosed bodies within BEC. The frequency of apoptotic bodies peaked at 3 days corresponding with a rapid reduction in the number of BEC during the first 3 days followed by a much slower rate of deletion in the succeeding 18 days of observation. Electron microscopy of the centrifuged pellet of bile revealed apoptotic bodies. It is concluded that deletion of excess BEC during regression of BEC hyperplasia occurs principally by apoptosis.

Abstract: A high prevalence of HCV infection has been reported in patients with hepatocellular carcinoma. The progression from acute transfusion‐associated hepatitis to hepatic cirrhosis and hepatocellular carcinoma has been suggested in several studies to be very long. We have investigated the prevalence of anti‐HCV and the interval between HCV infection and hepatocellular carcinoma among 191 consecutive patients with cirrhosis and liver‐cell carcinoma. Serum samples from 191 patients with cirrhosis and hepatocellular carcinoma, consecutively diagnosed in our hospital between 1988 and 1993, were tested for serological markers of HBV and HCV infection. One hundred and forty‐eight patients (77.5%; 95% confidence interval (c.i): 76% to 80%) were anti‐HCV positive by 2nd generation enzyme immunoassay (confirmed by 2nd generation recombinant immunoblot assay) and 152 patients (79.5%; 95% c.i: 76% to 80%) were anti‐HCV positive by 3rd generation enzyme immunoassay, while only 14 (7.4%; 95% c.i: 5% to 10%) were HBsAg positive. Of the 29 anti‐HCV positive patients with previous transfusion, the interval between the date of blood transfusion and the diagnosis of hepatic cirrhosis was 24±12.5 years and that of hepatocellular carcinoma was 26.8±12.4 years. These results confirm the high prevalence of HCV infection in patients with hepatocellular carcinoma and the slow sequential progression from HCV infection through cirrhosis and hepatocellular carcinoma.

ABSTRACT— Serum concentrations of the aminoterminal propeptide of type III procollagen and of the 7S domain of type IV collagen, presumed to reflect fibrotic activity in liver tissue, and of the glycosamonoglycan hyaluronan, were obtained from 40 alcohol abusers, at the time of liver biopsy. The serological results were related to morphological findings in liver tissue, i.e. no fibrosis, fibrosis without cirrhosis, micronodular cirrhosis and macronodular cirrhosis, and to ultrastructural indications of perisinusoidal fibrosis in the acinar zone 3. All patients with fibrosis and cirrhosis on light microscopy had elevated serum levels of the type III procollagen peptide as well as of the 7S domain of type IV collagen. However, due to a considerable overlap between the groups, no relations could be demonstrated to the severity of the fibrosis, supporting the assumption that these serological markers reflect the current fibrotic activity and not the amount of fibrotic tissue previously deposited. Among patients without fibrosis on light microscopy, a relation between the propeptide levels and ultrastructural perisinusoidal zone 3 fibrosis was observed, suggesting that type III procollagen peptide may be valuable in detecting very early liver fibrosis. A positive correlation was demonstrated between the serum concentrations of type III procollagen peptide and hyaluronan. As hyaluronan is degraded in the liver endothelial cells, it is suggested that the liver is involved, not only in the synthesis, but also in the degradation of the propeptide.

Abstract: Background/Aims: The catalytic subunit of human telomerase (hTERT) is known to be expressed in a variety of malignant tumours, including hepatocellular carcinoma (HCC). We studied hTERT expression in regenerative and precancerous lesions arising in cirrhosis. Methods/Results: As shown by in situ hybridisation, hTERT mRNA was absent in normal liver, but present in varying numbers of hepatocytes and HCC cells of diseased livers, as well as in biliary epithelial cells, lymphocytes, sinusoidal‐lining cells and tumour endothelial cells. RT‐PCR for two hTERT transcript regions demonstrated hTERT expression in 11 out of 15 cirrhotic liver samples, in 20 out of 21 large regenerative nodules/low‐grade dysplastic nodules, in 5 out of 5 high‐grade dysplastic nodules, and in 4 out of 4 HCCs. The beta‐splice variant was identified in all hTERT‐positive cases, while the corresponding full‐length transcript was found only in 13 out of 29 positive large nodular lesions and in 4 out of 11 positive cirrhotic samples. The full‐length transcript was always found in the presence of the beta‐splice variant, usually in low relative levels, and tended to correlate with telomerase activity in the samples, while the beta‐splice variant did not. Conclusions: This study shows that hTERT re‐expression takes place both in hepatic regeneration occurring in cirrhosis and in the early steps of hepatocarcinogenesis, and involves mainly the beta‐splice variant of this molecule. Additional regulatory mechanisms may be required for the expression of the full‐length hTERT transcript.