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Lectures
Path. Res. Pract. 191, 182-279 (1995)
Abstracts Lectures
1
3
THE LIVER FIBROGENIC RESPONSE IS FOCUSED ON A CASCADE MECHANISM OF FAT STORING CELL ACTIVATION A.M. Gressner (a. G.), Department of Clinical Chemistry and Central Laboratory, Philipps University, Baldingerstr., 35033 Marburg
MOLECULAR BASIS OF ALCOHOLIC HEPATITIS K. Zatloukal (a. G.), 1. Kenner (a.G.), K.H. Preisegger (a. G.), H. Denk
Aims: Ample evidence has been provided that fat storing cells (FSC) are the major source of matrix components (MC), if these precursor cells are activated in areas of necroinflammation to proliferate and to switch from the retinoid storing to the MC-producing phenotype termed myofibroblast (MFB). The mechanisms of FSC activation, which are crucial to fibrogenesis, have been studied. Methods: FSC, hepatocytes (PC), and Kupffer cells (KC) were isolated and cultured to collect conditioned media, which were added to FSC. Alternatively, cocultures of FSC with other cell types were used .. The response of FSC was studied with regard to MC-production, mitotic activation and phenotypic transformation to MFB (sm-a-actin). Mediators involved in paracrine and autocrine activation (TGF-~, TGF-a, IGF-I etc.) were identified by Northern blots and RIA. Results: FSC are strongly activated by cytokines from KC and platelets, of which TGF-~ is of great importance. It stimulates MC gene expression but inhibits FSC proliferation and stimulates phenotypic transformation. It is activated in cooperation with endothelial cells. TGF-a, also secreted by KC, stimulates FSC proliferation. In addition, PC also stimulate proliferation of FSC but MC production is not affected. The mitogenic effect is mediated by TGF-a, IGF-I and other cytokines. The paracrine signals of PC are increased by PC damage. During transformation MFB increasingly express TGF-~ and TGF-a, which are involved in autocrine stimulation of myofibroblasts and paracrine activation of still untransformed FSC. Conclusion: Cellular crosstalk let us suggest a three-step cascade model of FSC activation. A preinflammatory phase initiates FSC activation by discharge of mitogenic cytokines (TGF-a, IGF-I) from damaged PC followed by the inflammatory phase based on cytokines (TGF-~ etc.) from activated KC. In the postinflammatory phase MFB are stimulated by autocrine mechanisms contributing potentially to a perpetuation of the fibrogenic process even after cessation of the primary event (PC damage).
Institute of Pathology, University of Graz, Austria. Occurrence of alcoholic hepatitis, which reveals characteristic morphologic alterations (i.e. appearence of Mallory bodies [MB], disruption of the keratin cytoskeleton, ballooning of hepatocytes, steatosis, granulocytic inflammation, chicken wire-like fibrosis and perivenular fibrosis) dramatically accelerates the development ofliver cirrhosis and significantly correlates with poor prognosis of alcoholic liver disease. In contrast to the pathogenesis of alcoholic fatty liver, which can be explained by direct effects of ethanol oxidation on cellular metabolism, the molecular events leading to alcoholic hepatitis are unknown. MB and a disturbance of the hepatocytic keratin cytoskeleton as well as the ballooning of hepatocytes can be experimentally induced by chronic intoxication of mice with griseofulvin (GF) or 3,5diethoxycarbonyl-I,4-dihydrochollidine (ODC). MB consist of aggregates of filamentous material containing keratin polypeptides and non-keratin components, such as ubiquitin, tau and the MM120-1 antigen. The formation of MB filaments involves posttranslational modification of MB components by hyperphosphorylation, transglutaminase-induced cross-linking and partial proteolysis. The accumulation of the hyperphosphorylated microtubule-associated protein tau in MB could be responsible for the alteration of microtubule function, which has been discussed to cause a disturbance of secretory processes and the ballooning of hepatocytes in alcoholic hepatitis. There is evidence that activation of protein kinases and Ca++dependent enzymes initiate the cytoskeletal alterations. These initiators themselves might be activated by cytokines, such as TNFalpha which is increased in the serum of patients with alcoholic hepatitis and accumulates in affected hepatocytes. Moreover, TNFalpha is a good candidate for the mediator of the granulocytic inflammatory response.
2
4
FIBROGENESIS AND FIBROLYSIS IN THE LIVER H. HERBST, S. MILANI' (a.G.), D. SCHUPPAN+ (a.G.) Institut flir Pathologie and +Abteilung flir Gastroenterologie, Klinikum Benjamin Franklin, Freie Universitiit Berlin, Germany; 'Dipartimento di Fisiopatologia Clinica, Universita di Fierenze, Italy
Neoductular metaplasia in neoplastic and regenerative liver diseases
Aims: Chronic liver disease is frequently associated with fibrosis characterized by quantitative and qualitative changes in the extracellular matrix. To assess the relative contributions of fibrogenesis and fibrolysis in the generation of liver fibrosis, we analyzed the expression of the major procollagens and non-collagenous glycoproteins, matrix-degrading enzymes, their activators and inhibitors as well as growth factors with direct or indirect fibrogenic potential such as platelet-derived growth factor (PDGF) and transforming growth factors (TGF)-B. Methods: Gene expression was analyzed by irmnunohistology and by ill situ hybridization on isolated liver cells, normal and fibrotic rat liver with toxic or biliary fibrosis, human fibrotic/cirrhotic livers, and liver tumors. Results: Activated Ito cells and portal fibroblasts were the predominant source of interstitial matrix molecules, whereas basement membrane constituents were also derived from endothelial and bile duct epithelial cells. Expression of matrix-metalloproteinase uthibitors was upregulated in fibrotic liver tissue. Gene transcripts of matrix-metalloproteinases were found in isolated Ito cells, but some of these enzymes were undetectable ill vivo or showed distinct anatomical and kUletic patterns of expression. PDGF and TGF-B were expressed in epithelial and mesenchymal cells of fibrotic livers. Conclusions: Fibrogenesis is a function predomiIlantiy of the mesenchymal compartment of the liver. However, expression of matrixmetalloproteinases and their inhibitors is attributable to mesenchymal and to epithelial cell types, pointing to the unportance of epithelialmesenchymal interactions for the removal of extracellular matrix. Damage to one compartment may thus lead to unbalance between fibrogenesis and fibrolysis. The deduced patterns of matrixmetalloproteinase activity iII fibrotic liver suggest inlpaiI·ed degradation of fibrillar interstitial collagen, thus providing an explanation for the observed reanangements in the molecular composition of fibrotic liver matrix. The expression patterns of TGF-B and PDGF and their receptors pOUlt to epithelial-mesenchymal interactions iII triggering of fibrogenesis and modulation of fibrolysis, and suggest a pivotal role for members of the TGF-B gene family in the process of hepatic fibrosis.
H.-P. Fischer and M•. Meybehm Department of Pathology, University Bonn, FRG Proliferation of preexisting bile ducts, ductular metaplasia of hepatocytes as well as proliferation and differentiation of liver stem cells are discussed in the pathogenesis of neoductules in the liver .. We investigated the role of ductular metaplasia in the neoductogenesis in regenerative and neoplastic liver diseases. Yet in embryogenesis intrahepatic bile ducts develop from hepatoblasts sited close to the mesenchyme around t:he portal veins. In an"logy, ilepatoblastol8ab may contain small ductular elements, which sometimes even form ductal plate-like aggregates. Ductal structures in otherwise classical hepatocellular carcinomas and especially in combined hepato-cholangiocellular carcinomas demonstrate the potency of malignant hepatocytes for a ductular differentiation. In liver cirrhosis, chronic active hepatitis and in focal nodular hyperplasia (FNH) neoductules can be found in continuity with hepatocytes in periportal and periseptal fibrous stroma. The ductular differentiation of parenchymal cells is characterized by a gradual change of the cellular marker profile towards that of bile duct epithelia (positivity for keratin 7 and 19, CA 19-9). In alpha-l-AT-deficiency, hemochromatosis, and chronic active hepatitis B cellular deposites typically localized in hepatocytes such as alpha-I-antitrypsin, stderin, HBsAg and HBcAg can also be found in neoductular cells close to hepatocytes. These deposites seem to be retained during ductular transformation of hepatocytes. In alcoholic cirrhoses, FNH and in later stage of submassive liver necrosis hepatocytes have a 2 to 4 times higher Ki 67-index than neoductular cells and bile duct epithelia. This finding suggests that under these pathologic conditions the mature hepatocytes constitute the main proliferative cell compartment, and that the formation of neoductules lying in line with hepatocytes seems t:o be more a metaplast:ic than a proliferative process.
Abstracts . 183
5
7
HEPATIC EPITHELIAL STEM-LIKE CELLS
CONTROL OF LIVER CELL PROLIFERATION: STRUCTURAL, FUNCTIONAL AND MOLECULAR ASPECTS HARTMUT M. RABES, Pathologisches Institut der Universitat Mlinchen (LMU), Mlinchen, BRD After a period of active growth during embryonic and postnatal development, the liver attains a steady-state. In the adult organ, cell proliferation occurs only after cell loss or upon increased functional demand. It is triggered by an altered balance between growth inhibitory and stimulatory factors. The sensitivity to respond differs widely among hepatocytes. It is dependent on cellular receptor expression and on the microenvironment, the extracellular matrix, intercellular communication, intralobular distribution as well as DNA ploidy, nuclearity, age and specific differentiation status and function, all of which play an important part in modifying the actual hepatocellular reaction. Negative growth by apoptosis may result from cessation of functional load. Dying single cells are replaced by adjacent hepatocytes. Major resection of liver parenchyma evokes a partially synchronized wave of systemic cell proliferation in a typical intralobular pattern, hepatocytes in the periportal zone being the first to enter the cell cycle. They show the shortest cycle times. Coordinated proliferation of non-parenchymal cells warrants the preservation of the organ-specific structure. Destruction of the lobular pattern by toxic or inflammatory processes may end up in structural and functional deficiency, as in cirrhosis. Multicellular organ-specific growth response is in contrast to monocellular escape from growth control in the clonal development of early neoplasia. Recent comparative studies on genetic alterations during hepatocarcinogenesis provide insight in the role of various genes involved in the control of cell proliferation under normal conditions and in malignant transformation.
J. W. GRISHAM University of North Carolina at Chapel Hill Chapel Hill, NC USA ." Poorly differentiated epithelial cells With stem-likepropertles can be isolated from either normal or pathologically altered livers of rats or mice and cultured. Genetically tagged stem-like cells insert into hepatic plates and acquire the differentiated properties of host hepatocytes when transplanted into livers of syngeneic. animals. Pathologically proliferated "oval" cells, which resemble stem-like liver epithelial cells Isolated from normal organs, differentiate into hepatocytes under some conditions. Differentiation of "oval" cells into hepatocytes may contnbute to the repair of the liver following injuries which cause their proliferation, . although many of the proliferated "oval" cells die by apoptosls. Both hepatocytes and bile duct epithelial cells arise from blpotentlal stem cells in embryos, but the role of stem-like cells In normal adult liver is obscure. Recent hypotheses propose that new hepatocytes and biliary epithelial cells are derived normally from stem cells located In generative zones situated in or near portal tracts. Newly formed stem cell progeny are posited to migrate along hepatic plates or bile ducts, respectively, . undergo age-dependent differentiation and, ultimately, death. According to this theory, hepatocyte popUlation kinetics is a drastically slowed analog of the intestinal mucosa. Much evidence indicates that thiS theory IS not correct. Proliferation of hepatocytes in genetically mosaic rats and mice is a fractal process during both embryonic and postnatal growth. Fractal growth is quasi-clonal, with random cycling of cells and nondlfectlOnal siting of the progeny, demonstrating that hepatic plates are not pathways along which hepatocytes migrate. Hepato~ytes In all parts of hepatic . plates proliferate repeatedly when appropnately stimulated. More rapid cycling of periportal hepatocytes results from a short G I phase, possibly reflecting the cells' location near the points of entry of perfUSing blood. Apparent movement of tagged hepatocytes along hepatic plates may result from the dynamic reconstruction of plates which occurs dunng liver growth, and from reutilization of radioactive DNA tags. . . In summary, livers contain epithelial stem-like cells which take part In embryogenesis but do not participate in the normal generatIOn of new hepatocytes or biliary epithelial cells in adults. Hepatic stem-like cells may be involved in pathological responses to inJury. TransplantatIOn of hepatic stem-like cells may ultimately be applied to replace lost hepatocytes or defective genes.
6
8
ROLE OF GROWTH FACTORS IN LIVER REGENERATION AND HEPATOCARCINOGENESIS P. Schirmacher (a. G.), HP. Dienes Institut fur Pathologie der Universitiitskliniken Mainz Growth factors are multifunctional pleiotropic and secreted polypeptides. They interact with specific high affinity receptors that are in general members of the membrane anchored tyrosinekinase family. These receptors are able to activate a number of intracellular signal transduction pathways after ligand induced autophosphorylation of their cytoplasmic part. Beside their mitogenic activity, growth factors frequently exert motogenic and angiogenic/morphogenic effects. In most cases their activity is modulated by a number of serogenic binding factors. In the liver growth factors are essential for a number of different processes: fetal organ development, organ regeneration after injury and tumorigenesis. Some growth factors, like IGF-2, TGFa and HGF-SF, are highly expressed in fetal liver and are turned off or significantly reduced in expression after birth. After acute liver injury growth factor expression is temporarily activated. HGF-SF (peak expression - 12 h after liver injury) and TGFa (peak expression -24 h) are suspected to be key stimulators, while TGF131 (peak expression 48 - 72 h after injury) has growth suppressive activity on hepatocytes. In the case ofHGF-SF basal expression is derived from Ito cells, early on activation of expression from Ito cells and sinusoidal endothelial cells and maintainance of high expression additionally from infiltrating inflammatory cells. In hepatocarcinogenesis overexpression of autocrine growth factors is thought to be relevant for the establishment or maintainance of the transformed phenotype. Especially overexpression of IGF-2 and TGFa has been reported and both factors significantly increase the frequency of hepatic neoplasia in transgenic mice, while inactivation of these factors can slow down tumor cell growth. In the future hepatic growth factors may gain therapeutic relevance either in supporting liver regeneration or as a target for cytostatic therapy.
SEQUENTIAL MOLECULAR AND CELLULAR CHANGES DURING HEPATOCARCINOGENESIS P. BANNASCH, Abteilung fUr Cytopathologie, Deutsches Krebsforschungszentrum, 69120 Heidelberg, Germany The appearance of hepatocellular neoplasms induced by chemicals, radiation or viruses in different species or developing in transgenic rodent strains is regularly preceded by focal metabolic and morphological alterations which emerge in the liver parenchyma long before the neoplasms become manifest. The predominant sequence of metabolic changes leads from a focal excessive storage of glycogen (hepatocellular glycogenosis) through various intermediate stages, in which the glycogenosis may be replaced by an accumulation of lipids (lipidosis), to glycogen-poor hepatocellular carcinomas. The early hepatocellular glycogenosis is due to a disturbance in glycogen breakdown, which is associated with a dysfunction of signal transduction and glucose transport. During progression from the preneoplastic hepatocellular glycogenosis to glycogen-poor hepatocellular neoplasms a fundamental shift in carbohydrate metabolism takes place, gradually directing metabolites such as glucose-6-phosphate toward the pentose phosphate pathway and glycolysis, and frequently passing an intermediate stage of increased lipogenesis. This metabolic shift is accompanied by an ever increasing cell proliferation and overexpression of several oncogenes. Studies on about 70 resected or explanted livers from patients bearing hepatocellular carcinomas or suffering from cirrhosis provided evidence for focal changes in glycogen metabolism during human hepatocarcinogenesis which are in principle similar to those observed in laboratory animals. Our observations suggest that the molecular changes underlying the hepatocellular glycogenosis induced by carcinogenic agents are causally related to neoplastic conversion of the hepatocytes.
Abstracts Lectures
1
3
THE LIVER FIBROGENIC RESPONSE IS FOCUSED ON A CASCADE MECHANISM OF FAT STORING CELL ACTIVATION A.M. Gressner (a. G.), Department of Clinical Chemistry and Central Laboratory, Philipps University, Baldingerstr., 35033 Marburg
MOLECULAR BASIS OF ALCOHOLIC HEPATITIS K. Zatloukal (a. G.), 1. Kenner (a.G.), K.H. Preisegger (a. G.), H. Denk
Aims: Ample evidence has been provided that fat storing cells (FSC) are the major source of matrix components (MC), if these precursor cells are activated in areas of necroinflammation to proliferate and to switch from the retinoid storing to the MC-producing phenotype termed myofibroblast (MFB). The mechanisms of FSC activation, which are crucial to fibrogenesis, have been studied. Methods: FSC, hepatocytes (PC), and Kupffer cells (KC) were isolated and cultured to collect conditioned media, which were added to FSC. Alternatively, cocultures of FSC with other cell types were used .. The response of FSC was studied with regard to MC-production, mitotic activation and phenotypic transformation to MFB (sm-a-actin). Mediators involved in paracrine and autocrine activation (TGF-~, TGF-a, IGF-I etc.) were identified by Northern blots and RIA. Results: FSC are strongly activated by cytokines from KC and platelets, of which TGF-~ is of great importance. It stimulates MC gene expression but inhibits FSC proliferation and stimulates phenotypic transformation. It is activated in cooperation with endothelial cells. TGF-a, also secreted by KC, stimulates FSC proliferation. In addition, PC also stimulate proliferation of FSC but MC production is not affected. The mitogenic effect is mediated by TGF-a, IGF-I and other cytokines. The paracrine signals of PC are increased by PC damage. During transformation MFB increasingly express TGF-~ and TGF-a, which are involved in autocrine stimulation of myofibroblasts and paracrine activation of still untransformed FSC. Conclusion: Cellular crosstalk let us suggest a three-step cascade model of FSC activation. A preinflammatory phase initiates FSC activation by discharge of mitogenic cytokines (TGF-a, IGF-I) from damaged PC followed by the inflammatory phase based on cytokines (TGF-~ etc.) from activated KC. In the postinflammatory phase MFB are stimulated by autocrine mechanisms contributing potentially to a perpetuation of the fibrogenic process even after cessation of the primary event (PC damage).
Institute of Pathology, University of Graz, Austria. Occurrence of alcoholic hepatitis, which reveals characteristic morphologic alterations (i.e. appearence of Mallory bodies [MB], disruption of the keratin cytoskeleton, ballooning of hepatocytes, steatosis, granulocytic inflammation, chicken wire-like fibrosis and perivenular fibrosis) dramatically accelerates the development ofliver cirrhosis and significantly correlates with poor prognosis of alcoholic liver disease. In contrast to the pathogenesis of alcoholic fatty liver, which can be explained by direct effects of ethanol oxidation on cellular metabolism, the molecular events leading to alcoholic hepatitis are unknown. MB and a disturbance of the hepatocytic keratin cytoskeleton as well as the ballooning of hepatocytes can be experimentally induced by chronic intoxication of mice with griseofulvin (GF) or 3,5diethoxycarbonyl-I,4-dihydrochollidine (ODC). MB consist of aggregates of filamentous material containing keratin polypeptides and non-keratin components, such as ubiquitin, tau and the MM120-1 antigen. The formation of MB filaments involves posttranslational modification of MB components by hyperphosphorylation, transglutaminase-induced cross-linking and partial proteolysis. The accumulation of the hyperphosphorylated microtubule-associated protein tau in MB could be responsible for the alteration of microtubule function, which has been discussed to cause a disturbance of secretory processes and the ballooning of hepatocytes in alcoholic hepatitis. There is evidence that activation of protein kinases and Ca++dependent enzymes initiate the cytoskeletal alterations. These initiators themselves might be activated by cytokines, such as TNFalpha which is increased in the serum of patients with alcoholic hepatitis and accumulates in affected hepatocytes. Moreover, TNFalpha is a good candidate for the mediator of the granulocytic inflammatory response.
2
4
FIBROGENESIS AND FIBROLYSIS IN THE LIVER H. HERBST, S. MILANI' (a.G.), D. SCHUPPAN+ (a.G.) Institut flir Pathologie and +Abteilung flir Gastroenterologie, Klinikum Benjamin Franklin, Freie Universitiit Berlin, Germany; 'Dipartimento di Fisiopatologia Clinica, Universita di Fierenze, Italy
Neoductular metaplasia in neoplastic and regenerative liver diseases
Aims: Chronic liver disease is frequently associated with fibrosis characterized by quantitative and qualitative changes in the extracellular matrix. To assess the relative contributions of fibrogenesis and fibrolysis in the generation of liver fibrosis, we analyzed the expression of the major procollagens and non-collagenous glycoproteins, matrix-degrading enzymes, their activators and inhibitors as well as growth factors with direct or indirect fibrogenic potential such as platelet-derived growth factor (PDGF) and transforming growth factors (TGF)-B. Methods: Gene expression was analyzed by irmnunohistology and by ill situ hybridization on isolated liver cells, normal and fibrotic rat liver with toxic or biliary fibrosis, human fibrotic/cirrhotic livers, and liver tumors. Results: Activated Ito cells and portal fibroblasts were the predominant source of interstitial matrix molecules, whereas basement membrane constituents were also derived from endothelial and bile duct epithelial cells. Expression of matrix-metalloproteinase uthibitors was upregulated in fibrotic liver tissue. Gene transcripts of matrix-metalloproteinases were found in isolated Ito cells, but some of these enzymes were undetectable ill vivo or showed distinct anatomical and kUletic patterns of expression. PDGF and TGF-B were expressed in epithelial and mesenchymal cells of fibrotic livers. Conclusions: Fibrogenesis is a function predomiIlantiy of the mesenchymal compartment of the liver. However, expression of matrixmetalloproteinases and their inhibitors is attributable to mesenchymal and to epithelial cell types, pointing to the unportance of epithelialmesenchymal interactions for the removal of extracellular matrix. Damage to one compartment may thus lead to unbalance between fibrogenesis and fibrolysis. The deduced patterns of matrixmetalloproteinase activity iII fibrotic liver suggest inlpaiI·ed degradation of fibrillar interstitial collagen, thus providing an explanation for the observed reanangements in the molecular composition of fibrotic liver matrix. The expression patterns of TGF-B and PDGF and their receptors pOUlt to epithelial-mesenchymal interactions iII triggering of fibrogenesis and modulation of fibrolysis, and suggest a pivotal role for members of the TGF-B gene family in the process of hepatic fibrosis.
H.-P. Fischer and M•. Meybehm Department of Pathology, University Bonn, FRG Proliferation of preexisting bile ducts, ductular metaplasia of hepatocytes as well as proliferation and differentiation of liver stem cells are discussed in the pathogenesis of neoductules in the liver .. We investigated the role of ductular metaplasia in the neoductogenesis in regenerative and neoplastic liver diseases. Yet in embryogenesis intrahepatic bile ducts develop from hepatoblasts sited close to the mesenchyme around t:he portal veins. In an"logy, ilepatoblastol8ab may contain small ductular elements, which sometimes even form ductal plate-like aggregates. Ductal structures in otherwise classical hepatocellular carcinomas and especially in combined hepato-cholangiocellular carcinomas demonstrate the potency of malignant hepatocytes for a ductular differentiation. In liver cirrhosis, chronic active hepatitis and in focal nodular hyperplasia (FNH) neoductules can be found in continuity with hepatocytes in periportal and periseptal fibrous stroma. The ductular differentiation of parenchymal cells is characterized by a gradual change of the cellular marker profile towards that of bile duct epithelia (positivity for keratin 7 and 19, CA 19-9). In alpha-l-AT-deficiency, hemochromatosis, and chronic active hepatitis B cellular deposites typically localized in hepatocytes such as alpha-I-antitrypsin, stderin, HBsAg and HBcAg can also be found in neoductular cells close to hepatocytes. These deposites seem to be retained during ductular transformation of hepatocytes. In alcoholic cirrhoses, FNH and in later stage of submassive liver necrosis hepatocytes have a 2 to 4 times higher Ki 67-index than neoductular cells and bile duct epithelia. This finding suggests that under these pathologic conditions the mature hepatocytes constitute the main proliferative cell compartment, and that the formation of neoductules lying in line with hepatocytes seems t:o be more a metaplast:ic than a proliferative process.
Abstracts . 183
5
7
HEPATIC EPITHELIAL STEM-LIKE CELLS
CONTROL OF LIVER CELL PROLIFERATION: STRUCTURAL, FUNCTIONAL AND MOLECULAR ASPECTS HARTMUT M. RABES, Pathologisches Institut der Universitat Mlinchen (LMU), Mlinchen, BRD After a period of active growth during embryonic and postnatal development, the liver attains a steady-state. In the adult organ, cell proliferation occurs only after cell loss or upon increased functional demand. It is triggered by an altered balance between growth inhibitory and stimulatory factors. The sensitivity to respond differs widely among hepatocytes. It is dependent on cellular receptor expression and on the microenvironment, the extracellular matrix, intercellular communication, intralobular distribution as well as DNA ploidy, nuclearity, age and specific differentiation status and function, all of which play an important part in modifying the actual hepatocellular reaction. Negative growth by apoptosis may result from cessation of functional load. Dying single cells are replaced by adjacent hepatocytes. Major resection of liver parenchyma evokes a partially synchronized wave of systemic cell proliferation in a typical intralobular pattern, hepatocytes in the periportal zone being the first to enter the cell cycle. They show the shortest cycle times. Coordinated proliferation of non-parenchymal cells warrants the preservation of the organ-specific structure. Destruction of the lobular pattern by toxic or inflammatory processes may end up in structural and functional deficiency, as in cirrhosis. Multicellular organ-specific growth response is in contrast to monocellular escape from growth control in the clonal development of early neoplasia. Recent comparative studies on genetic alterations during hepatocarcinogenesis provide insight in the role of various genes involved in the control of cell proliferation under normal conditions and in malignant transformation.
J. W. GRISHAM University of North Carolina at Chapel Hill Chapel Hill, NC USA ." Poorly differentiated epithelial cells With stem-likepropertles can be isolated from either normal or pathologically altered livers of rats or mice and cultured. Genetically tagged stem-like cells insert into hepatic plates and acquire the differentiated properties of host hepatocytes when transplanted into livers of syngeneic. animals. Pathologically proliferated "oval" cells, which resemble stem-like liver epithelial cells Isolated from normal organs, differentiate into hepatocytes under some conditions. Differentiation of "oval" cells into hepatocytes may contnbute to the repair of the liver following injuries which cause their proliferation, . although many of the proliferated "oval" cells die by apoptosls. Both hepatocytes and bile duct epithelial cells arise from blpotentlal stem cells in embryos, but the role of stem-like cells In normal adult liver is obscure. Recent hypotheses propose that new hepatocytes and biliary epithelial cells are derived normally from stem cells located In generative zones situated in or near portal tracts. Newly formed stem cell progeny are posited to migrate along hepatic plates or bile ducts, respectively, . undergo age-dependent differentiation and, ultimately, death. According to this theory, hepatocyte popUlation kinetics is a drastically slowed analog of the intestinal mucosa. Much evidence indicates that thiS theory IS not correct. Proliferation of hepatocytes in genetically mosaic rats and mice is a fractal process during both embryonic and postnatal growth. Fractal growth is quasi-clonal, with random cycling of cells and nondlfectlOnal siting of the progeny, demonstrating that hepatic plates are not pathways along which hepatocytes migrate. Hepato~ytes In all parts of hepatic . plates proliferate repeatedly when appropnately stimulated. More rapid cycling of periportal hepatocytes results from a short G I phase, possibly reflecting the cells' location near the points of entry of perfUSing blood. Apparent movement of tagged hepatocytes along hepatic plates may result from the dynamic reconstruction of plates which occurs dunng liver growth, and from reutilization of radioactive DNA tags. . . In summary, livers contain epithelial stem-like cells which take part In embryogenesis but do not participate in the normal generatIOn of new hepatocytes or biliary epithelial cells in adults. Hepatic stem-like cells may be involved in pathological responses to inJury. TransplantatIOn of hepatic stem-like cells may ultimately be applied to replace lost hepatocytes or defective genes.
6
8
ROLE OF GROWTH FACTORS IN LIVER REGENERATION AND HEPATOCARCINOGENESIS P. Schirmacher (a. G.), HP. Dienes Institut fur Pathologie der Universitiitskliniken Mainz Growth factors are multifunctional pleiotropic and secreted polypeptides. They interact with specific high affinity receptors that are in general members of the membrane anchored tyrosinekinase family. These receptors are able to activate a number of intracellular signal transduction pathways after ligand induced autophosphorylation of their cytoplasmic part. Beside their mitogenic activity, growth factors frequently exert motogenic and angiogenic/morphogenic effects. In most cases their activity is modulated by a number of serogenic binding factors. In the liver growth factors are essential for a number of different processes: fetal organ development, organ regeneration after injury and tumorigenesis. Some growth factors, like IGF-2, TGFa and HGF-SF, are highly expressed in fetal liver and are turned off or significantly reduced in expression after birth. After acute liver injury growth factor expression is temporarily activated. HGF-SF (peak expression - 12 h after liver injury) and TGFa (peak expression -24 h) are suspected to be key stimulators, while TGF131 (peak expression 48 - 72 h after injury) has growth suppressive activity on hepatocytes. In the case ofHGF-SF basal expression is derived from Ito cells, early on activation of expression from Ito cells and sinusoidal endothelial cells and maintainance of high expression additionally from infiltrating inflammatory cells. In hepatocarcinogenesis overexpression of autocrine growth factors is thought to be relevant for the establishment or maintainance of the transformed phenotype. Especially overexpression of IGF-2 and TGFa has been reported and both factors significantly increase the frequency of hepatic neoplasia in transgenic mice, while inactivation of these factors can slow down tumor cell growth. In the future hepatic growth factors may gain therapeutic relevance either in supporting liver regeneration or as a target for cytostatic therapy.
SEQUENTIAL MOLECULAR AND CELLULAR CHANGES DURING HEPATOCARCINOGENESIS P. BANNASCH, Abteilung fUr Cytopathologie, Deutsches Krebsforschungszentrum, 69120 Heidelberg, Germany The appearance of hepatocellular neoplasms induced by chemicals, radiation or viruses in different species or developing in transgenic rodent strains is regularly preceded by focal metabolic and morphological alterations which emerge in the liver parenchyma long before the neoplasms become manifest. The predominant sequence of metabolic changes leads from a focal excessive storage of glycogen (hepatocellular glycogenosis) through various intermediate stages, in which the glycogenosis may be replaced by an accumulation of lipids (lipidosis), to glycogen-poor hepatocellular carcinomas. The early hepatocellular glycogenosis is due to a disturbance in glycogen breakdown, which is associated with a dysfunction of signal transduction and glucose transport. During progression from the preneoplastic hepatocellular glycogenosis to glycogen-poor hepatocellular neoplasms a fundamental shift in carbohydrate metabolism takes place, gradually directing metabolites such as glucose-6-phosphate toward the pentose phosphate pathway and glycolysis, and frequently passing an intermediate stage of increased lipogenesis. This metabolic shift is accompanied by an ever increasing cell proliferation and overexpression of several oncogenes. Studies on about 70 resected or explanted livers from patients bearing hepatocellular carcinomas or suffering from cirrhosis provided evidence for focal changes in glycogen metabolism during human hepatocarcinogenesis which are in principle similar to those observed in laboratory animals. Our observations suggest that the molecular changes underlying the hepatocellular glycogenosis induced by carcinogenic agents are causally related to neoplastic conversion of the hepatocytes.