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Journal of Pediatric Gastroenterology & Nutrition:
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Liver Cell Injury and Fibrosis

Sokol, Ronald J.

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University of Colorado School of Medicine and The Children's Hospital, Denver, Colorado, U.S.A.

The cellular, biochemical, and molecular mechanisms that cause injury to hepatocytes and the biliary tree play a role in virtually all childhood liver diseases. The necrotic hepatocyte releases its contents, inducing an acute inflammatory response; communicates with nonparenchymal cells involved in fibrogenesis; and releases toxic products that may injure other cells or may distort normal liver architecture. As a consequence of hepatocyte injury, decreased functional hepatocyte mass eventually leads to hepatic synthetic failure and the possible need for liver transplantation. Therefore, understanding the processes by which the hepatocyte and the biliary tree are injured not only will shed light on the pathophysiology of pediatric liver disease but also will be essential in developing and applying new therapeutic strategies.

Recent studies show clearly that hepatocytes and bile duct cells may have two separate fates if they fail to recover from injury: cellular necrosis and apoptosis (programmed cell death) (1). Fibrosis in the liver results from synthesis and secretion of collagen by hepatic stellate cells under the control of cytokines, growth factors, and products of lipid peroxidation (2). In part, other cell types present in the liver determine and modify the degree and type of injury. These processes must occur in pediatric liver disorders, but little is known about their unique features in pediatric hepatobiliary disorders and in the developing liver. Because new interventional strategies to treat childhood liver diseases will depend to a great extent on reversing or preventing these processes, better understanding of the underlying mechanisms that lead to necrosis, apoptosis, and fibrogenesis in the fetal, neonatal, and pediatric liver is necessary.

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AREAS OF EMPHASIS

Cell Types in the Developing Liver

The liver is composed of six major cell types: hepatocytes, bile duct epithelial cells (cholangiocytes), Kupffer cells, hepatic stellate cells, sinusoidal endothelium, and pit cells. In addition, arterial and venous structures are lined by vascular endothelium and contain vascular smooth muscle cells. Each of the major cell types plays a specific role in the liver during normal function and during injury.

Hepatocytes have several hundred functions and are a frequent target in infectious, toxic, metabolic, and immunologic injury to the liver. When injured, hepatocytes may produce and release oxygen free radicals, lipid peroxide products, proteases, cytokines, and growth factors that injure adjacent cells and stimulate synthesis of collagen.

Bile duct epithelial cells are important in forming bile and are targets in infectious, immunologic, and congenital forms of injury to the liver. These cells also are capable of synthesizing and releasing a number of cytokines, chemokines, and growth factors. Injury may activate Kupffer cells, as the hepatic macrophage, resulting in increased phagocytosis. In addition, they can elaborate a large number of secretory products, including tumor necrosis factor, interferon alfa-1 and beta-1, interleukin (IL)–1, IL-6, prostaglandin 2, prostaglandin D2, prostaglandin F2α, thromboxane B2, leukotrienes, superoxide, proteases, nitric oxide, transforming growth factor, and many other proinflammatory compounds (3). This may result in activation of T cells, cytotoxicity, magnification of the inflammatory response, and stimulation of fibrogenesis.

Hepatic stellate cells store vitamin A and, on activation, are the major producers of hepatic extracellular matrix, including collagens type I and III. Therefore, stellate cells are responsible for hepatic fibrogenesis and the development of cirrhosis, under the direction of growth factors, oxidants, and additional stimuli released from injured hepatocytes, bile duct epithelia, Kupffer cells, or other inflammatory cells. In addition, stellate cells synthesize and release dozens of secretory products, including transforming growth factor β, endothelin, nitric oxide, insulinlike growth factor, endothelial growth factor, and matrix metalloproteinase 2 and its inhibitor (3). Because these cells are contractile, they also may regulate blood flow and sinusoidal tone.

Sinusoidal endothelia have a specialized, highly permeable pore system that allows ready access of circulating molecules to the hepatocyte. These cells also scavenge soluble compounds and can phagocytose small particles; in addition they synthesize and secrete hepatocyte growth factor, insulinlike growth factor-2, transforming growth factor β, endothelin, and matrix components. During inflammation, these cells express intracellular adhesion molecule 1, leading to adhesion of neutrophils and magnification of the inflammatory response that can lead to tissue damage. If sinusoidal endothelia become damaged during this process, the resulting impaired blood flow can lead to further ischemic injury.

The pit cell is a large granular lymphocyte that has natural killer cell activity and is found within the sinusoidal lumen. Pit cells release soluble mediators that lead to rapid killing of tumor cells and virally infected hepatocytes.

Little is known about the specific roles and interactions of these cell types in the pathogenesis of pediatric hepatobiliary disorders. Moreover, in the developing liver, the actions and roles of these cells, their regulation and response to mediators, and the substances elaborated by the cells may have unique aspects that have not been explored. Therefore, the complex interactions of these cell types in models of adult liver disorders may not be applicable to conditions that injure the liver in the fetus or the neonate. Characterization of the genetic control of these cells and understanding the functions and responses of these cell types in the developing liver are essential if certain processes must be interrupted to prevent and treat childhood liver disorders. Moreover, polymorphisms or mutations in genes that control important functions of these cells may modify or predispose the infant's liver to more significant injury during pathologic states. The differences between the infant's liver and that of the adult in these areas must be studied.

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Role of Cellular Necrosis and Apoptosis in Pediatric Liver Diseases

Cell necrosis is characterized by injury that causes irreversible loss of metabolic functions and of structural integrity of the cell's plasma membrane. This results in dissipation of ion gradients, failure to synthesize proteins, mitochondrial dysfunction leading to adenosine triphosphate depletion, and loss of plasma membrane integrity with blebbing or rupture and cytolysis. The rupture of hepatocytes leads to a characteristic increase in circulating aminotransferase levels and to the release of cell constituents that stimulate an inflammatory response and, potentially, fibrogenesis.

Apoptosis is a highly regulated form of programmed cell death characterized by cell shrinkage, fragmentation of the cell into membrane-bound fragments called apoptotic bodies, and disposal of these fragments by phagocytosis, macrophages, or neighboring cells (1). This results in the removal of dying cells without the release of enzymes and cell constituents, therefore without initiating an inflammatory response. Apoptosis is important in development, in remodeling of bile ducts, and in normal disposal of aging or malignant cells.

External agents or chemicals generally cause necrosis, whereas in apoptosis the cell actively participates in its own death. Recent studies suggest that mitochondria are a key organelle in both these types of cell death (4). Indeed, genetic deficiencies in mitochondrial respiratory chain enzymes may cause fatal liver failure in infants (5,6). In addition, a group of intracellular proteases, termed caspases, participate actively in the apoptotic process (7). Two signaling pathways activate caspases, either through cell-surface “death receptors” or through intracellular stress signals (7). The intracellular stresses involved may include oxidative stress (8) and its effect on opening a pore in the mitochondrial membrane that releases cytochrome c, which in turn participates in activating caspases (9). Interestingly, ursodeoxycholic acid, an agent used in treating cholestatic liver disease, seems to block the opening of this pore, which may explain its cytoprotective effect (10).

An important group of cytosolic proapoptotic proteins, the BCL2 family, may be activated by intracellular stresses and may translocate to the mitochondria and activate the permeability transition pore of the mitochondria, which results in release of cytochrome c and activation of caspases (11). Cellular necrosis and apoptosis may exist simultaneously; however, the former is more prominent in acute liver injury. Apoptosis may remove injured or poorly functioning hepatocytes, with hepatocyte regeneration and repopulation with more viable cells. However, apoptosis of bile duct epithelia may lead to irreversible loss of intrahepatic bile ducts.

Finally, an expanding set of cell survival genes and signals have been identified in recent years that counteract the apoptotic and necrotic cellular machinery. For example, activation of NFkB may lead to enhanced activity of nitric oxide synthases, PI3 kinases, and other factors that protect the hepatocyte from apoptosis.

The relative roles of these two types of cell death in pediatric hepatobiliary disorders have not been investigated. Moreover, polymorphisms of genes that regulate cell death processes, cell survival signals, and hepatic regeneration may significantly influence the response of the pediatric liver to injury. Whether developing hepatocytes and bile ducts are more susceptible or resistant to these processes is unknown, but the clarification of these mechanisms may add to understanding the pathogenesis of individual disorders. Differing pharmacologic strategies may be necessary to treat diseases, depending on the primary mode of cell injury and death. The capacity of the neonatal and childhood liver to resist injury and regenerate also require investigation.

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Fibrogenesis

Ultimately, the degree to which fibrosis develops is a major determinant of outcome in many pediatric hepatobiliary disorders. The resulting portal hypertension and interference with normal blood flow to hepatocytes become critical factors that affect growth, metabolic derangement, complications, and survival. The hepatic stellate cell is responsible for most extracellular matrix synthesis that contributes to fibrosis (3). Various factors activate the quiescent stellate cell from one that stores retinoids to one that synthesizes collagen type I and inflammatory mediators—these include transforming growth factor β, IL-1, IL-4, endothelin, cellular fibronectin, prostaglandin F, and lipid peroxide products that may be released from surrounding cells undergoing injury or participating in the inflammatory reaction (2,3). Stellate cells also may participate in degrading collagen by secreting metalloproteinases. A number of antifibrogenic agents are effective in decreasing hepatic stellate cell activation or collagen synthesis in vitro and in animal models, although few have been shown effective in humans.

The biology of the hepatic stellate cell in the developing liver has not been characterized. Because of the importance of retinoids in the growing child, the dual role of the stellate cell may have greater importance in pediatric liver disorders than in adult liver disorders.

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RESEARCH GOALS

1. To characterize the unique developmental features of the six cell types in the liver of the fetus, the preterm and term infant, and the child: These features should include morphology; differentiation and maturation; response to growth factors and cytokines; effect of and role in hepatic injury; and elaboration of cytokines, growth factors, and other humoral mediators.

2. To determine the role of hepatocyte and cholangiocyte apoptosis and necrosis in pediatric hepatobiliary disorders: Understanding the mechanisms of cellular injury, the regulatory factors, and the signal pathways involved will provide a rationale for therapeutic strategies that must be developed in coming years.

3. To characterize the role of the hepatic stellate cell in the developing liver and its regulation and role in hepatic fibrogenesis in pediatric liver disorders.

4. To determine the effect of antifibrogenic agents in animal and cellular models for pediatric liver disorders.

5. To conduct randomized controlled studies of suitable antifibrogenic agents in children with prototypic liver diseases associated with predictable fibrogenesis: Improved noninvasive markers of fibrogenesis also must be developed to monitor clinical effects on fibrosis.

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RESEARCH STRATEGIES

Cell Types in the Developing Liver

Developmental studies in mammalian models must be conducted to understand the developmental role of stem cells that are the progenitors of the liver cell types. In addition, factors that regulate and stimulate the appearance and function of the various cell types in the liver during mammalian and human development from the fetus through childhood must be characterized. Moreover, investigation should center on the developmental regulation of cellular secretory products, interactions, and cell signaling among these cell types during development and on the role of extracellular matrix in these processes.

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Role of Cellular Necrosis and Apoptosis in Pediatric Liver Diseases

The mechanisms involved in and the relative roles of necrosis and apoptosis of hepatocytes, biliary epithelia, and hepatic stellate cells in liver diseases that affect the infant and child require characterization in representative animal models and, ultimately, in children. The regulatory factors and genetic control of apoptosis of bile duct epithelia in the pathogenesis of bile duct paucity disorders of childhood require exploration. The importance and interactions among the following interrelated biologic processes must be determined, particularly in cholestatic liver disorders, metal storage diseases, viral infections, circulatory disorders, and organ preservation: loss of intracellular ion homeostasis, mitochondrial dysfunction and depletion of cellular adenosine triphosphate, opening of the mitochondrial permeability pore with release of cytochrome c and apoptosis-inducing factors, generation of oxygen free radicals, depletion of antioxidant defenses, activation of intracellular proteases and caspases and cell survival genes, role of BCL2 family of intracellular proapoptotic factors, and alterations of plasma membrane structure and integrity. When the relevant processes are defined, strategies to prevent cellular necrosis and apoptosis should be developed and tested in animal and cellular models, with the goal of using these strategies to treat pediatric liver disease.

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Role of Hepatic Stellate Cell

Mediators secreted from inflammatory cells, Kupffer cells, endothelium, and injured hepatocytes and bile duct epithelia stimulate activation of hepatic stellate cells and stimulate the synthesis of collagen and other extracellular matrix components. The regulatory factors and mediators of fibrogenesis in pediatric disorders may differ from those in adult liver diseases and may, therefore, lead to novel and age-specific strategies to decrease or prevent fibrogenesis and, ultimately, cirrhosis. Therefore, cell culture techniques, whole animal models, and transgenic strategies should be used to determine cell types, soluble factors, and genetic factors that regulate extracellular matrix synthesis and degradation. In pediatric patients, this would be of particular importance in regard to congenital hepatic fibrosis, disorders of bile acid metabolism and other metabolic liver diseases, biliary obstruction, and autoimmune disorders.

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Antifibrogenic Agents

A variety of strategies should be used to develop and test ways to decrease extracellular matrix synthesis and to stimulate matrix degradation. Besides techniques for inhibiting hepatocellular and bile duct epithelial injury and inflammation, both of which would decrease fibrogenesis, specific strategies should be used to modify hepatic stellate cell activation, recruitment, proliferation, and collagen synthesis.

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Clinical Trials of Antifibrogenic Agents and Developing Noninvasive Markers of Fibrogenesis

Clinical testing of new therapies must be conducted in a controlled fashion for pediatric liver disorders associated with progressive fibrosis. Current cytoprotective agents (e.g., antioxidants, ursodeoxycholic acid) and antiinflammatory drugs (e.g., interferons) have variable effects in adult fibrosing hepatic disorders and require evaluation in children. Some of these agents include herbal compounds that seem safe and may be effective; however, current federal regulations make it difficult to test these agents in children. New federal regulatory policies must be developed that allow testing of these potentially valuable treatments. New agents that down-regulate the inflammatory response; that directly decrease or inhibit stellate cell activation, proliferation, and collagen synthesis; and that stimulate extracellular matrix degradation must be tested for safety in adults and then used in pediatric multicenter controlled trials. The development of a pediatric liver disease network of centers in the United States will provide the infrastructure for these important clinical trials.

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PROJECTED TIMETABLE AND FUNDING REQUIREMENTS

Investigations of the biology and regulation of cell types in the developing liver, of stellate cell biology, and of the role of apoptosis and cellular necrosis in pediatric liver disorders should be investigated through the RO1 and program project granting during the next 5 years. Basic researchers funded by RO1 National Institutes of Health grants and investigators in industry should partner to develop new treatment strategies and agents. Funding should be sought through the Food and Drug Administration Orphan Drug Program when applicable. A network of pediatric liver disease centers should be established and funded federally to provide the infrastructure to test new therapeutic agents and to test the strategies designed to decrease hepatocellular and bile duct cell injury and to decrease/prevent hepatic fibrosis, specifically in pediatric disorders. An National Institutes of Health consortium contract should supply funding.

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HEALTH AND ECONOMIC OUTCOMES

Better understanding of the mechanisms that cause liver injury and fibrosis in childhood liver diseases and the translation of this knowledge into new therapies will have obvious benefits for children, their families, and society. Other sections of this agenda outline the health and economic impact of the individual liver disorders. Progress in improving the lives of children with liver disorders will benefit other family members and siblings. Understanding the mechanisms that underlie liver injury and fibrosis in pediatric disorders and new strategies for treating pediatric liver disorders may also have an impact on adults with other liver diseases.

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CONCLUSION

Acquiring better understanding of the processes involved in liver injury and fibrogenesis in the developing infant and child will influence the impetus to develop new therapeutic strategies that may benefit adults and children with liver diseases. Establishing a national network of pediatric liver disease centers funded by the National Institutes of Health will enhance our ability to conduct clinical trials and to test interventions in sufficient numbers of infants and children affected by liver disease.

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REFERENCES

1. Patel T, Roberts LR, Jones BA, Gores GJ. Dysregulation of apoptosis as a mechanism of liver disease: an overview. Semin Liver Dis 1998; 18:105–114.

2. Olaso E, Friedman SL. Molecular regulation of hepatic fibrogenesis. J Hepatol 1998; 29:836–847.

3. Albanis E, Friedman SL. Hepatic fibrosis. Pathogenesis and principles of therapy. Clin Liver Dis 2001; 5:315–34.

4. Lemasters JJ. The mitochondrial permeability transition: from biochemical curiosity to pathophysiological mechanism. Gastroenterology 1998; 115:783–86.

5. Bioulac-Sage P, Parrot-Rouland F, Mazat JP, Lamireau T, et al. Fatal neonatal liver failure and mitochondrial cytopathy (oxidative phosphorylation deficiency): a light and electron microscopy study of the liver. Hepatology 1993; 18:839–46.

6. Sokol RJ, Treem W. Mitochondrial hepatopathies. In: Suchy F, Sokol RJ, Balistreri WF (eds). Liver Disease in Children, 2nd Edition, Lippincott, Williams & Wilkins, Philadelphia, 2001; pp. 787–810.

7. Salvesen GS, Dixit VM. Caspases: intracellular signaling by proteolysis. Cell 1998; 94:695–98.

8. Yerushalmi B, Dahl R, Gumpricht E, Yerushalmi B, Devereaux MW, Sokol RJ. Bile acid-induced rat hepatocyte apoptosis is inhibited by antioxidants and blockers of the mitochondrial permeability transition. Hepatology 2001; 33:616–26.

9. Rodrigues CM, Ma X, Linehan-Stieers C, Fan G, Kren BT, Steer CJ. Ursodeoxycholic acid prevents cytochrome c release in apoptosis by inhibiting mitochondrial membrane depolarization and channel formation. Cell Death Differ 1999; 6:842–54.

10. Rodrigues CMP, Fan G, Xiaomong M, Kren BT, Steer CJ. A novel role for ursodeoxycholic acid in inhibiting apoptosis by modulating mitochondrial membrane perturbation. J Clin Invest 1998; 101:2790–99.

11. Green DR. Apoptotic pathways: the roads to ruin. Cell 1998; 94:695–98.

© 2002 Lippincott Williams & Wilkins, Inc.

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