Molecular and Cellular Basis of Hepatic Failure

Molecular and Cellular Basis of Hepatic Failure HAL F. YEE, JR TIMOTHY J. DAVERN II

Definitions

44

Acute liver failure 44 Clinical manifestations Etiology 45 Pathogenesis 45 Cirrhosis 48 Clinical manifestations Etiology 50 Pathogenesis 50

44

48

Perspectives and Future Directions

55

Hepatic failure is the principal indication for liver transplantation. It arises from the loss of functional hepatic parenchyma that can result from either acute liver failure or cirrhosis. In the United States, acute liver failure affects approximately 2000 to 4000 individuals per year and is the indication for approximately 5 % of liver transplants. Cirrhosis, which kills approximately 25,000 Americans per year, is responsible for the vast majority of liver transplants performed. Although impaired hepatic function characterizes both acute liver failure and cirrhosis, the mechanisms underlying the pathogenesis of these two disorders are, in general, distinct. In this chapter, we briefly review the molecular and cellular basis of acute liver failure and cirrhosis, recognizing that the separation of liver failure into acute and chronic is somewhat artificial. Many patients with chronic liver disease succumb to acute on chronic liver failure when an acute insult (e.g., hepatitis A) is superimposed on hitherto compensated cirrhosis (e.g., hepatitis C). In addition, many of the clinical manifestations (Table 3-1) and causes (Table 3-2) of acute and chronic liver failure are shared. Nonetheless, we separate these two ends of a spectrum in this chapter for clarity. Space limitations dictate that many important areas of research in this field are not addressed. Similarly, worthy contributions from many laboratories are not cited. Accordingly, references to several recent reviews are provided for readers interested in a more detailed treatment. 43

I

I-General Considerations

Table 3-1 •. CLINICAL MANIFESTATlONS OF ACUTE LIVER FAILURE AND DECOMPENSATED CIRRHOSIS

Fluid retention

Acute Liver Failure

Decompensated Cirrhosis

+

+++

Portal hypertensive bleeding

+++

Coagulopathy

+++

++

Jaundice

++

+++

Hepatic encephalopathy

+++

++

Cerebral edema

+++

Infection

+++

++

Renal failure

+++

++

Hepatocellular carcinoma

++

-, unusual; +, Infrequent: ++, common; +++, characteristic of the syndrome.

8 weeks of the onset of symptoms, which are often nonspecific and flu-like, or within 2 weeks of the onset of jaundice. A variant of ALF with a more insidious onset, called subfulminant hepatic failure, is usually defined by liver failure with hepatic encephalopathy that develops between 2 weeks and 3 months from the onset of jaundice. Cirrhosis is a pathological diagnosis that is defined by the presence of extensive hepatic fibrosis associated with derangement of the liver's normal lobular and vascular architecture. It is characterized by the presence of nodules of regenerating hepatocytes surrounded by exuberant extracellular matrix CECM) in the form of fibrotic bands. Hepatic function can be impaired to a clinically significant degree when the structural abnormalities that distinguish cirrhosis are sufficiently advanced. This potentially life-threatening condition is the final common pathway through which nearly all forms of chronic liver disease cause morbidity and mortality.

Definitions

Acute Liver Failure

Acute liver failure CALF) is a clinical syndrome characterized by severe liver injury complicated by encephalopathy. Essential for the diagnosis of ALF are the absence of clinically overt chronic liver disease and the presence of encephalopathy not caused by sedation or some other nonhepatic cause. Most studies of ALF include patients that develop encephalopathy within

Clinical Manifestations Hepatic encephalopathy is, by definition, present to some degree in all patients with ALF. Other common clinical manifestations of ALF include cerebral edema, infection, renal failure, hypotension, hypoglycemia, jaundice, and electrolyte abnormalities, whereas portal

Table 3-2. ETIOLOGIES THAT LEAD TO LIVER FAILURE Acute Liver Failure

Decompensated Cirrhosis

Drugs/toxins

Acetaminophen Isoniazid

Ethanol Methotrexate Excess vitamin A

Infections

Hepatitis A Hepatitis B (± Hepatitis 1\) Hepatitis E

Hepatitis B Hepatitis C Schistosomiasis

Vascular

Shock (I.e" acute Ischemia) Hepatic vein occlusion (Budd-Chiarl syndrome)

Congestive heart failure Hepatic vein occlusion (Budd-Chlari syndrome)

Metabolic and genetic disorders

Wilson's disease Reye's syndrome Tyrosinemia Pregnancy-associated (Acute fatty liver/HELLP syndrome)

Nonalcoholic steatohepatitis Hereditary hemochromatosis ai-antitrypsin deficiency Wilson's disease Tyrosinemia

Autoimmune

Autoimmune hepatitis

Autoimmune hepatitis Primary biliary cirrhosis Primary sclerosing cholangitis Chronic obstruction of the biliary tract Byler disease

Biliary disorders Unknown

Indeterminate acute liver failure

HELLP, hemolysis, elevated liver enzymes, and low platelet (count).

Cryptogenic cirrhosis

3-Molecular and Cellular Basis of Hepatic Failure

hypertensive bleeding and severe fluid retention are distinctly unusual (see Table 3-1). The syndrome of ALF is associated with high mortality, with most patients dying from cerebral edema and sepsis. However, some causes of ALF are associated with a better prognosis than are other causes. In general, the more rapid onset forms of ALF, such as that associated with acetaminophen hepatotoxicity, have a higher incidence of cerebral edema but an overall better prognosis, probably reflecting the lack of liver architectural derangement and, thus, more favorable conditions for hepatic regeneration. In contrast, ALF caused by idiosyncratic drug reactions, Wilson's disease, and indeterminate cause, which tend to follow a subfulminant course, carries a particularly poor prognosis. 1

Etiology ALF results from the abrupt loss of liver function secondary to severe injury from a variety of causes that may be grouped into several general categories (see Table 3-2). The most common causes of ALF in the United States currently are drugs and toxins, in particular, acetaminophen, and ALF of indeterminate cause, presumably a heterogeneous group of occult viral infections, autoimmune disorders, toxic exposures, and metabolic insults. The incidence of ALF from viral hepatitis A and B in the United States appears to be decreasing, perhaps in part the result of an active vaccination program, and together they now account for less than 10 % of ALF cases per year. 2

Pathogenesis

•

The lack of suitable animal models has hampered research on the mechanisms of ALF and, as a result, the pathophysiological basis for the syndrome of ALF is incompletely understood. Nevertheless, based on the available data, it appears that ALF usually reflects the effects of inflammatory cytokines and cells initiating a cascade of events culminating in massive liver cell death while inhibiting cell replication. Although the hepatocyte is often the focus of attention in ALF, all of the various liver cell types (Table 3-3) undoubtedly play important roles. Liver injury usually results from a complex interaction between the injurious agent (e.g., virus, chemical, self-reactive immune cells) and the response of host cells to the insult, including activation of both liver parenchymal and nonparenchymal cells with cytokine release. Acute infection with hepatitis B (HBV) illustrates the importance of the host cell response in the pathogenesis of ALE Under most circumstances, HBV is a noncytopathic virus, although overexpression of certain viral proteins in cultured cells may cause toxicity. In vivo liver damage from HBV appears to result largely from the host immune responses to virally infected cells rather than from a direct result of viral injury per se. For example, in a transgenic mouse model expressing HBV proteins, severe acute liver damage occurs only after HBV-specific cytotoxic T-lymphocytes (CTLs) are infused that target and destroy HBV surface antigen (HBsAg)-positive hepatocytes. Prior to such a challenge, the mice have little or no demonstrable liver pathology.

Table 3-3. THE ROLES OF THE MAJOR CELL POPULATIONS IN THE HEALTHY LIVER Cell Type

Approximate Fraction in the Healthy Liver

Roles in the Healthy Liver

Hepatocytes

60%

Stellate cells

5%

Storage of vitamin A Synthesis of extracellular matrix Support of homeostasis of hepatocytes and endothelial cells

Cholangiocytes

3%

Fluid and electroyte secretion/resorption Protein translocation

Uptake, storage, metabolism, and release of carbohyrates, proteins, lipids, and vitamins Synthesis of plasma proteins, lipoproteins, fatty acids, cholesterol. phospholipids, and glucose Bile synthesis and secretion Degradation and detoxification of exogenous and endogenous compounds

Kupffer cells

15%

Phagocytosis and clearance of microrganisms, endotoxins, tumor cells, particulate matter Immune defense Tumor cell surveillance

Endothelial cells

15%

Endocytlc uptake of glycoproteins Scavenging of denatured circulating proteins

Immune cells

2%

Cytotoxicity toward virus-infected and tumor cells

I

I-Gon,,,1 Con,Id,,,II,n,

Full recovery from ALF is possible, and this suggests that outcomes may be improved if cell death can be curtailed and hepatic repair enhanced. Ultimately, prognosis in ALF depends on the balance of liver cell death with liver repair and regeneration. Indeed, survival critically depends upon rapid and robust recovery of liver cell function before the life-threatening complications, such as cerebral edema and sepsis, of ALF supervene. Liver Regeneration and Repair

The adequacy of liver repair and regeneration following acute liver injury appears to be as important as the extent of the injury in determining outcome. The molecular mechanisms underlying hepatic regeneration have been elucidated primarily in the partial hepatectomy rodent model, in which two thirds of the liver, including the left lateral and medial lobes, are removed intact. 3 However, relatively little is known about liver regeneration in the setting of ALF, particularly in humans, and it is likely that this process differs in many important ways from regeneration associated with partial hepatectomy because, for example, with partial hepatectomy the remaining liver is intact and normal, whereas in ALF the entire liver is involved. Illustrating this point, the roles of hepatocyte growth factor (HGF) and transforming growth factor-~ (TGF-~), which are relatively well-characterized positive and negative regulators of hepatocyte proliferation following partial hepatectomy, are unclear in the setting of ALF. Indeed, paradoxically, serum HGF levels, which rise within an hour of partial hepatectomy, appear to correlate inversely with prognosis in the setting of ALF. Hepatic regeneration represents the culmination of a complex interaction among liver cells, matrix, cytokines, and hormones. 4 ,s Under normal conditions, only a small fraction of hepatocytes (rv 1/20,000) are in mitosis. When hepatocytes are injured and die, they are usually replaced by mature hepatocytes. Experiments in several different murine models suggest that mature hepatocytes have the ability to undergo multiple rounds of replication. 6 Whether this is also true of fully differentiated human hepatocytes, which (unlike mice) lack active telomerase-the enzyme responsible for maintaining chromosome end length with DNA replication-is currently unclear. Under certain situations, including when protein synthesis is inhibited, perisinusoidal oval cells, which have some stem cell properties, also participate in hepatic regeneration. Furthermore, recent experiments both in murine models of liver injury and histological studies of liver transplant explants suggest that extrahepatic cells derived from the bone marrow also appear to have the ability to contribute to hepatic

regeneration, but the molecular mechanisms of this process are currently controversial. 7 After partial hepatectomy, the onset of liver cell replication is rapid, with the peak of hepatocyte DNA synthesis occurring within approximately 24 hours, and the peak of nonparenchymal cell DNA synthesis occurring approximately 24 hours later. 4 Amazingly, normal liver mass is restored after only 7 to 10 days following 50 % partial hepatectomy in rats, although the regenerative capacity of hepatocytes is in part related to the age of the animal. 8 Liver regeneration, encompassing both hypertrophy and hyperplasia, is characterized by the activation of more than 100 genes encoding cytokines, growth factors, transcription factors, and cellular constituents. 8 HGF, epidermal growth factor (EGF), TGF-~, tumor necrosis factor-a (TNF-a), and interleukin (IL)-6 appear to have particularly important roles in hepatic regeneration. The plasma concentration of HGF, produced primarily by stellate cells, increases dramatically within 1 hour of a partial hepatectomy, and it acts through its receptor, comet, which is highly expressed on hepatocytes. 3 The role of TGF-~, a potent growth inhibitor factor produced by sinusoidal endothelial cells and hepatocytes, in possibly terminating hepatic regeneration once complete is still unclear. During the so-called priming phase of replication, normally quiescent hepatocytes enter the cell cyclemoving from the Go to the G1 phase-and become replication competent. This early phase, which lasts 4 to 6 hours following partial hepatectomy, is marked by increased circulating levels of TNF-a and IL-6, as well as other cytokines, matrix remodeling in the remaining liver, and the activation of a series of immediate early genes including the proto-oncogenes c-{os, c-jun, and c-myc. 8 Activation of these and other genes ultimately leads to progression through the early to mid-G 1 phase of the cell cycle. 3 Progression through the cell cycle (G 1 through S phases) is regulated by growth factors and activation of early (D and E) and late (A and B) cyclins and their associated cyclindependent kinases (CDKs). Inactivation of cell cycle inhibitory proteins, retinoblastoma (Rb) protein and pl30, is also required to release the transcription factor E2F that stimulates cell cycle progression and DNA replication (S phase). TNF-a, released primarily from Kupffer cells, appears to play a critical role in the initiation of the transcriptional cascade contributing to hepatocyte replication. Thanscription factors activated as part of this cascade include, among others8 : • Nuclear factor-K B (NF-KB) • Signal transducer and activator of transcription 3 (STAB)

3-Molecular and Cellular Basis of Hepatic Failure

• Activating protein-l (AP-l) • CAAT enhancer binding protein ~ (C/EBP-~) • Extracellular signal-regulated kinase (ERK) • c-Jun N-terminal kinase (JNK) kinases The early activation of NF-KB by a rapid posttranscriptional mechanism activates expression of IL-6, which in turn activates STAB and other genes. When NF-KB activity is blocked after partial hepatectomy, the residual liver undergoes massive apoptosis. 4 Genetically modified mice that lack IL-6 or the receptor for TNF-a have deficient liver regeneration and develop liver failure following partial hepatectomy that is ameliorated by recombinant IL-6 administration, strongly suggesting that IL-6 is acting downstream of TNF-a in the regeneration cascade. In the setting of severe ALE hepatic regeneration is impaired despite high serum levels of IL-6, TNF-a, and HGF. Liver Cell Death

Like other cells, liver cells die from apoptosis and necrosis, two pathways of cell death that are morphologically distinct but interrelated and that probably should be viewed as two ends of a cell death continuum. The cell death pathway taken, either apoptosis or necrosis, appears to be related to the nature and severity of the inciting insult, the cell type, its metabolic status, and the integrity of the cell death machinery. Both types of cell death probably occur simultaneously in most forms of ALF.9 Necrosis involves severe depletion of cellular adenosine triphosphate (ATP) and results in cell swelling, loss of cell membrane integrity, and lysis, which invariably elicits a secondary immune response. In contrast, apoptosis, or programmed cell death, is characterized by a more orderly process of nuclear and cytoplasmic shrinkage, condensation, and blebbing without loss of cell membrane integrity or release of intracellular contents; thus it allows cellular debris to be removed without intense secondary inflammation and marked perturbation of neighboring cells. Again, both processes (apoptosis and necrosis) may, and probably often do, occur simultaneously, and the same stimuli in varying intensities can trigger both processes. In general, liver cell necrosis rather than apoptosis tends to predominate, with extensive oxidative damage to mitochondria because this depletes cellular ATP stores and also may inhibit caspase activity, both of which are necessary for the successful execution of the apoptosis pathway. Apoptosis is a highly conserved process essential to organogenesis and immune cell homeostasis that was first recognized pathologically in liver 3 decades ago as acidophilic (Councilman) bodies. However, fundamental insights into the molecular details of the apoptosis

I

pathway are more recent, initially gleaned from experiments in the worm Caenorhabditis elegans and only later in mammalian cells. The apoptosis cascade involves the sequential activation of a series of cysteine proteases called caspases. This cascade can be triggered by a variety of insults that may be extrinsic or intrinsic to the cell undergoing apoptosis. Extrinsic triggers involve activation of cell surface death receptors, whereas intrinsic triggers signal apoptosis via oxidative stress of mitochondria and possibly other cellular organelles. 10 Diverse factors trigger liver cell death, such as hypoxia (e.g., with ischemia-reperfusion), reactive oxygen species (e.g., generated during drug metabolism), viral infection, and autoimmune injury; however, they generally induce cell death via cell surface death receptors or by injuring mitochondria, although both processes often occur simultaneously. Indeed, participation of mitochondria appears to be essential to death receptor-mediated apoptosis in hepatocytes. II Oxidative injury to mitochondria secondary to TNF-a, for example, results in opening of mitochondrial permeability transition (MPT) pores at the junction of the inner and outer mitochondrial membranes. Opening of the MPT pores, in turn, leads to release of intramitochondrial cytochrome c and apoptosis-inducing factor (AIF) and to initiation of the apoptosis cascade via caspase-9. Loss of the mitochondrial membrane potential also results in disruption of oxidative phosphorylation and ATP depletion, and this may also contribute to liver cell death. 12 Focus on Fas. Perhaps the best-studied extrinsic trigger of hepatocyte apoptosis is engagement of the cell surface receptor, Fas (CD95/APO-l), a member of the tumor necrosis/nerve growth factor receptor family that is highly expressed on activated lymphocytes, and also constitutively expressed on a variety of nonlymphoid cells, including hepatocytes. The ligand for Fas, Fas ligand (FasL/CD95L), is a cell surface protein that is expressed by activated T cells in which it mediates lymphocyte homeostasis and, together with the perforin/ granzyme system, T-cell cytotoxicity. In addition to lymphocytes, hepatocytes also appear to be capable of expressing FasL in certain situations. Binding of FasL or agonist antibodies (e.g., J02) to Fas causes the latter to trimerize, resulting in the recruitment of a series of intracellular molecules in a signaling cascade that activates caspases responsible for degrading cellular components and ultimately results in the morphological features of apoptosis. A physiological role for Fas in liver homeostasis is suggested by the observation that mice deficient in Fas develop, among other abnormalities, significant liver hyperplasia. 13 Based on immunohistological studies,

Fas is expressed at low levels in a normal human liver, but expression appears to be upregulated in the setting of both acute and chronic liver disease. 14 In particular, Fasmediated apoptosis plays a major role in development of liver failure from Wilson's disease and viral hepatitis 8. 10 ,15 Hepatocytes constitutively express a lower level of certain antiapoptotic proteins (e.g., Bcl-2 and Bcl-xL) than most other cells, which may partly explain their special sensitivity to Fas-mediated apoptosis. In addition, FasL expression on hepatocytes has also given rise to the idea that under certain circumstances hep.atocytes may actively induce apoptosis in neighbormg cells, a process termed fratricide. The expression of death receptors, including Fas, on hepatocytes is relatively well established, but expression of these receptors on nonparenchymal cells is less well defined. Fas expression has been demonstrated on murine endothelial cells, stellate cells, and cholangiocytes: 16 When it was reported more than a decade ago that mtravenous administration of an activating antiFas antibody to mice results in ALF secondary to massive hepatocyte apoptosis and death, it was initially assumed that direct engagement and activation of hepatocyte Fas was responsible. However, injury to sinusoidal endothelial cells appears to playa predominant role in the development of FasL-induced ALF in this model, highlighting that injury and death of nonparenchymal cells, as opposed to hepatocytes, may be critical to the development of some forms of ALFY Recent studies in murine models suggest that inhibiting Fas expression in the liver may prevent or ameliorate ALF (Fig. 3-1). For example, a recent report showed that liver Fas expression could be reduced by RNA interference (RNAi) , now a popular method of experimentally knocking down gene expression in cultured cells and in mouse models. 18 Knocking down expression of Fas in this fashion largely protected mice against an otherwise lethal challenge with either an apoptosis-inducing anti-Fas antibody, or concavalin A, which causes immune-mediated liver damage. 19 This work not only directly implicates Fas-mediated apoptosis in liver injury but also suggests that selectively inhibiting this process, in this case by RNAi, may be therapeutic. A similar study using RNAi to decrease expression of caspase-8, a key enzyme in death receptor-mediated apoptosis, also demonstrates a significant therapeutic effect even if the RNAi was initiated after liver injury, in this case by a viral (adenovirus) infection. 20 Likewise, prior studies show that expression of antiapoptotic proteins (e.g., Bcl-2) in hepatocytes may also prevent liver cell apoptosis secondary to a variety of noxious agents. Caution must be exercised with any of these potential therapeutic approaches, however, as inhibition of the apoptosis pathway may redirect cells to the necrosis pathway and also may predispose to neoplasia.

Summary of the Pathogenesis of Acute Liver Failure The preceding discussion is by necessity incomplete and largely ignores several important areas of research relevant to the pathogenesis of ALF. For example, both pro- and anti-inflammatory cytokines play critical roles in the pathogenesis of ALF. Interferon-y, a proinflammato~ cy~okine in~olved in macrophage and T-Iymphocyte activatIon, medIates liver cell injury in a mouse model of hepatitis B. Similarly, by acting through interferon-y, IL-12 appears to playa role in liver injury in some murine models of ALF.IO A variety of cytokines, including IL-lO, IL-11, IL-13, and IL-4, protect against liver injury when administered to mice, presumably by downregulating proinflammatory cytokines, nitric oxide, and reactive oxygen species. Preliminary immunocytochemical analysis of livers from patients with ALF suggests that an imbalance of proinflammatory (interferon-y) and anti-inflammatory (lL-12 and IL-lO) cytokines may, in fact, contribute to the pathogenesis of liver failure. 21 Nitric oxide-a gas that is generated during enzymatic conversion of L-arginine to L-citrulline by hepatocytes, Kupffer cells, and endothelial cells-is both constitutively expressed and induced by proinflammatory cytokines (e,g., TNF-a) in the liver and may contribute to oxidative stress in certain situations (e.g., acetaminophen toxicity). 10 However, nitric oxide may also have protective effects, and its role in liver injury is still incompletely defined. The relative rarity of ALF speaks to the resiliency of the liver, which is normally capable of withstanding tremendous insults caused by an impressive array or protective, repair, and regenerative mechanisms. It is only in the rare situations, when these mechanisms are critically impaired or have been overwhelmed, that clinically overt liver failure becomes manifest. Despite its relative rarity, ALF represents an important medical problem because it typically affects young, otherwise healthy individuals and is associated with high mortality. A more complete understanding of the fundamental molecular mechanisms underlying development of ALF, particularly those responsible for liver cell death and regeneration, is clearly needed before rational therapeutics can be developed. Until that time liver transplantation must continue to be considered for any patient developing ALP.

Cirrhosis Clinical Manifestations The majority of individuals whose liver biopsies demonstrate cirrhosis exhibit no symptoms or signs of

3-Molecular and Cellular Basis of Hepatic Failure

•

FIGURE 3-1 Knocking down Fas expression improves outcome of mice with acute liver failure (ALF). RNA interference (RNAi) is an evolutionarily conserved, posttranscriptional, homology-dependent gene-silencing mechanism used by eukaryotic cells to target destruction of mRNA. RNAi has been exploited as a powerful and popular experimental method to knock down gene expression with great precision both in cultured cells and in mice. 18 Within cells, RNAi is initiated by small interfering RNA (siRNA), a double-stranded form of RNA that is 21 to 23 bases in length, usually generated by cleavage of larger double-stranded transcripts by an endonuclease complex (Dicer). Experimentally, RNAi can also be accomplished by expressing siRNA precursors (small. hairpin RNAs) from DNA templates (1) or by introducing synthetic siRNA directly into cells (2) by transfection. siRNAs introduced into cells by either route assemble with a multiprotein complex, termed RNA-inducing silencing complex (RISC), (3) that uses the siRNA as a gUide to identify and degrade homologous mRNA target sequence, thus acting as a sequence-specific nuclease (4). In the study by Song and colleagues,19 investigators used a technique called hydrodynamic transfection to deliver and express anti-Fas siRNAs in a mouse liver to specifically decrease Fas expression. Mice treated in this fashion were largely resistant to the subsequent administration of an activating anti-Fas antibody (l02), which otherwise results in uniformly lethal ALF by inducing massive hepatocyte apoptosis.

liver disease, and their tests for liver synthetic function are intact. Clinically silent cirrhosis, however, can progress, eventually compromising hepatocyte function and hepatic circulation. If cirrhosis becomes sufficiently severe, liver failure and portal hypertension can occur. The first signs of advanced cirrhosis are commonly laboratory abnormalities, which can include thrombocytopenia, prolonged prothrombin time, hyperbilirubinemia, or hypoalbuminemia. When cirrhosis causes hepatic

decompensation, any or all of the following clinical manifestations can occur (see Table 3-1): • Encephalopathy • Variceal bleeding • Peripheral edema, ascites, and spontaneous bacterial peritonitis • Hepatorenal and hepatopulmonary syndromes • Muscle atrophy

•

I-General Considerations

• Coagulopathy

• Jaundice Although some of these complications (e.g., variceal bleeding, spontaneous bacterial peritonitis, hepatorenal syndrome) are in themselves life-threatening, the prognosis for any patient with decompensated cirrhosis is poor and warrants consideration for liver transplantation.

architecture (Le., cirrhosis). If sufficiently severe, fibrosis can result in compromised hepatocyte function. The alterations in hepatic structure and function associated with cirrhosis are similar regardless of the cause of chronic liver injury, which indicates that the general mechanisms underlying fibrosis of the liver are shared. Two populations of fibrogenic cell types, hepatic stellate cells and hepatic myofibroblasts, mediate hepatic fibrosis.

Etiology Nearly all causes of chronic liver injury can produce fibrosis and lead to the development of cirrhosis (see Table 3-2). Hepatitis B is the most common cause of cirrhosis worldwide. Cirrhosis will develop in 25 % to 33 % of the estimated 400 million individuals chronically infected with hepatitis B throughout the world. In the United States, the most common causes of cirrhosis are nonalcoholic fatty liver (NAFL), alcoholism, and hepatitis C. NAFL was recently recognized as a major cause of cirrhosis in industrialized nations, in which up to 5 % of the population has NAFL. The proportion of those with NAFL that progresses to cirrhosis is not known, but emerging data indicate that NAFL may be the principal cause of cryptogenic cirrhosis among those undergoing evaluation for liver transplantation. Alcoholism is reported to contribute to 40 % to 90 % of cases of cirrhosis in North America and Europe. Alcohol-associated cirrhosis is a leading indication for this surgery. Hepatitis C is the primary indication for liver transplantation. One hundred million persons around the world are chronically infected with hepatitis C, with approximately 4 million cases in the United States. Of those with hepatitis C, 15% to 20% of livers are believed to progress to cirrhosis. Currently, approximately 60 % of those receiving liver transplants are chronically infected with hepatitis C. Table 3-2 lists other less common causes of cirrhosis.

Hepatic Stellate Cells and Myofibroblasts Mediate the Liver's Response to Injury Hepatic stellate cells, also called Ito cells or hepatic lipocytes, occupy the space of Disse (Le., perisinusoidal space). In a normal liver, these cells have a star-like appearance radially extending numerous cytoplasmic protrusions that contact the basal face of the hepatocytes and run along and encircle the endothelial cells that line the sinusoids (Fig. 3-2). It is partly the anatomic location of stellate cells, along with circumstantial experimental evidence, that suggests these cells modulate sinusoidal blood flow and enhance solute exchange within the perisinusoidal space. Stellate cells also synthesize small amounts of extracellular matrix proteins, including laminin and type IV collagen, which make up the basement membrane. A notable attribute of this cell type is that it displays prominent cytosolic vesicles in which retinoids, including vitamin A, are stored. In addition, stellate cells release soluble growth factors, cytokines, and peptides that contribute to liver

Pathogenesis Over the past 2 decades, substantial effort has been made to elucidate the molecular and cellular mechanisms underlying the development of cirrhosis. Because space constraints only permit us to provide an overview, the reader is directed to a number of excellent reviews for a deeper examination of the pathogenesis of cirrhosis. 22 -32 In the discussion to follow, primary references are provided for data that are not already found in these comprehensive review articles. Cirrhosis is distinguished by increased deposition and altered composition of ECM components in the portal tracts, around the central veins, or in the perisinusoidal spaces of the liver. This pathological surplus of ECM, termed fibrosis, can progress to distort the liver's lobular and microvascular

FIGURE 3-2 The three-dimensional microanatomy of the liver. The stellate cell occupies the perisinusoidal space between the hepatocytes and sinusoidal endothelial cells. Note the defining star-like shape with protrusions extending around the sinusoid. (From Friedman SL, Arthur MlP: Targeting hepatic fibrosis. Sci Med 8:194-205, 2002.)

3-Molecular and Cellular Basis of Hepatic Failure

cell development, differentiation, and survival. Thus, under normal conditions, stellate cells store vitamin A, support the homeostasis of hepatocytes and the endothelium, and may contribute to regulation of the microcirculation. Hepatic injury provokes the release of diverse soluble and insoluble mediators generated by hepatocytes, sinusoidal endothelia, biliary epithelia, Kupffer cells and other leukocytes, stellate cells and hepatic myofibroblasts, and platelets. These injury-induced factors stimulate a wound-healing response in which stellate cells migrate to the site of insult, proliferate at that site, produce ECM (fibrogenesis), and place tension across the ECM. These stellate cell responses facilitate parenchymal restitution after an acute hepatic insult. If liver injury resolves, stellate cell chemotaxis and proliferation end, excess stellate cells undergo apoptosis, and surplus ECM is broken down (fibrolysis) by extracellular matrix metalloproteinases (MMPs). In this way, the wound-repair response is terminated once injury has resolved and tissue healing has been accomplished. However, if liver injury persists, hepatic myofibroblasts (mesenchymally derived cells located primarily adjacent to the portal triads and central veins) are also recruited to affected sites. Chronic hepatic injury stimulates both hepatic myofibroblasts and stellate cells to proliferate, lay down ECM, and mediate contractiondependent remodeling of ECM. Clearly, synthesis of extracellular matrix components (e.g., collagens and fibronectins) is essential for the development of fibrosis, but other properties of these fibrogenic cells are also necessary. For example, chemotaxis and proliferation augment the number of stellate cells and hepatic myofibroblasts located within areas of liver injury, which intensifies the synthesis and remodeling of ECM. Remodeling of ECM also requires regulation of extracellular MMP activity and the contractile tension generated by the fibrogenic cells. Accumulation of excess ECM in the form of contracted fibrotic bands can result from chronic liver injury. Thus, fibrosis occurs when injury-induced stimuli persist and keep the homeostatic balance tipped toward migration, proliferation, fibrogenesis, and contraction and away from apoptosis, fibrolysis, and relaxation. From a molecular perspective, fibrosis is the combined result of the sustained effects of a series of diverse extracellular stimuli on many interconnected signaling pathways that differentially modulate critical dynamic and well-coordinated behaviors of the fibrogenic cells of the liver (Fig. 3:-'3). Fibrosis Is a Consequence of the Liver's Response to Chronic Injury

During injury, the behavior of stellate cells and hepatic myofibroblasts is regulated by paracrine interactions with damaged hepatocytes and endothelial cells; activated

•

I

Apoptosis

H

Proliferation

Fibrolysis

H

Fibrogenesis

I

Relaxation

H

Contraction

I

FIGURSW In this proposed model for the liver's injury response, fibrosis is the combined result of the effects of a series of diverse extracellular mediators of injury on many interconnected signaling pathways that differentially modulate dynamic and well-coordinated behaviors of the fibrogenic cells of the liver. Whether normal healing or fibrosis occurs depends on the location, duration, and intensity of the injury response.

platelets, Kupffer cells, and infiltrating leukocytes; and other stellate cells and hepatic myofibroblasts. These interactions are mediated by growth factors, regulatory peptides and lipids, cytokines, extracellular matrix components, and toxic metabolites (Table 3-4). Injured hepatocytes can release highly toxic compounds, such as reactive oxygen intermediates (ROJ) and lipid peroxides, that can induce activation of Kupffer cells and leukocytes. Hepatocytes can also produce inflammatory mediators including insulin-like growth factor-l and vascular endothelial growth factor. Activated Kupffer cells and other leukocytes can also generate ROI and lipid peroxides, as well as soluble agents such as TGF-~, plateletderived growth factor (PDGF), TNF-a, interferon-y, and IL-I, IL-2, IL-6, IL-lD, and IL-B. Sinusoidal endothelial cells can influence the injury response by producing laminin and a splice variant of fibronectin (EIIIA isoform), converting latent TGF-~ to the active form through the activation of plasmin, and secreting ET-l and nitric oxide. Platelets also produce TGF-~ and PDGF and are the principal source of regulatory lipids,

I

I-General Considerations

Table 3-4. EFFECTS OF SELECTED MEDIATORS OF HEPATIC FIBROSIS Molecule

Source

Effects on Fibrogenic Cell Functions

RECEPTOR TYROSINE KINASE LIGANDS

K, F, E, P

(+) flbrogenesls, migration: (-) proliferation, fibrolysis

Platelet-derived GF

B, K, F, P

(+) proliferation: (±) migration: (-) contraction

Insulin-like GF-1

H, E, P

(+) proliferation

Epidermal GF

P

(+) proliferation, migration

Vascular endothelial GF

H, F, E, P

(+) proliferation: (-) contraction

E, F

(+) migration, contraction; (±) proliferation

Transforming

GF-~

G-PROTEIN-COUPLED RECEPTOR LIGANDS

Endothelin-1 Lysophosphatidic acid

P

(+) migration, contraction

Angiotensin II

F

(+) proliferation, flbrogenesls, contraction

Thrombin

F

(+) proliferation, contraction

Leptin

F

(+) flbrogenesls; (-) fibrolysls

Tumor necrosis factor-a (TNF-a)

K

(+) apoptosis

Interleukln-1

K, E

(+) flbrogenesls

Interleukin-4

K

(+) fibrogenesls

Interleukln-6

K

(+) fibrogenesls

Interleukin-10

K

(-) fibrogenesis

Interleukln- 13

K

(+) flbrogenesls

Interferon-y

K

(-) fibrogenesis, migration

Monocyte chemotactic proteln-1

F

(+) migration

Collagen I

F

(+) proliferation, migration, flbrolysls

Collagen III

F

(+) proliferation

Collagen IV

F

(+) proliferation, fibrolysls; (-) fibrogenesis

Fibronectin

E, F

(+) fibrogenesis

Reactive oxygen Intermediates

H, K, E

(+) fibrogenesls

Lipid peroxides

H, K

(+) flbrogenesis

Nitric oxide

E, H. K

(-) proliferation, contraction

INTEGRIN RECEPTOR LIGANDS

MISCELLANEOUS FACTORS

B, biliary epithelium; E, sinusoidal endothelium; F, stellate cells and myoflbroblasts; GF, growth factor; H, hepatocytes; K, Kuppfer and other Inflammatory cells; P, platelets; (+), stimulate; (-), Inhibit.

including lysophosphatidic acid and sphingosine-Iphosphate. Stellate cells and hepatic myofibroblasts themselves can secrete soluble and insoluble factors that can act in paracrine or autocrine fashion, including: • TGF-~ • PDGF • Vascular endothelial growth factor (VEGF)

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Endothelin-I Leptin IL-B Monocyte chemotactic protein

• Cytokine-induced neutrophil chemoattractant • Fibronectin • Laminin • Collagens I, III, IV, VI, XIV, and XVIII In addition, significant amounts of certain factors, including PDGF, HGF, VEGF, and TNF-a, can bind to the ECM and be released, particularly during fibrolysis. The effects of these injury-associated extracellular mediators are primarily transduced by plasma membrane receptors (Le., receptor tyrosine kinases, G-proteincoupled receptors, and integrins) or intracellular receptors (Le., nuclear receptors). These receptors in turn act through intracellular signaling pathways that control

3-Molecular and Cellular Basis of Hepatic Failure

protein expression or directly regulate the physical behavior of stellate cells and hepatic myofibroblasts. It has become clear that no single mediator or signaling pathway is sufficient to trigger hepatic fibrosis. Moreover. the functional consequence of any given mediator or signaling pathway is not stereotypical. but depends on the timing and subcellular localization of the signal, as well as cross-talk from other pathways. The emerging model for wound healing in the liver is one in which diverse stimuli orchestrate the activation and inhibition of multiple interconnected signal transduction pathways that regulate distinct cellular responses (e.g.• chemotaxis-chemostasis. proliferation-apoptosis. fibrogenesis-fibrolysis. contraction-relaxation). In this dynamic model, acute hepatic damage leads to injuryinduced signaling that regulates a wound-healing response that requires accumulation of the liver's fibrogenic cells and ECM at the site of injury. If this damage is transient. the injury response ceases once healing has occurred. and normal homeostatic mechanisms result in the removal of surplus fibrogenic cells and ECM. Conversely, during chronic liver disease injury-induced signaling persists, causing a continuing wound-healing response that results in the pathological accumulation of fibrogenic cells and ECM at sites of injury. With time, this sustained wound-healing response can result in the development of fibrosis and, subsequently, cirrhosis. In other words, fibrosis occurs when the net balance of injury-induced signaling is tipped toward the woundhealing response for too long.

Fibrosis Results From a Complex Cascade in Interconnected Signaling Events Current knowledge is insufficient to provide a complete picture of the pathogenesis of fibrosis. However, a plethora of studies over the past 1S years provides a glimpse into the intricate signaling pathways that govern the wound-healing response. Much of this research has depended on well-characterized stellate cell and hepatic myofibroblast culture models. The relevance of this work is not entirely certain, but key elements have been validated by animal and human studies of liver injury. It is impossible in a short chapter to detail even a modest portion of the myriad molecular and cellular signals that have already been shown to participate in the development of fibrosis. However, it is instructive to discuss the elaborately regulated pleiotropic effects of three injuryassociated mediators that play important roles in the liver's wound-healing response.

Effects of Platelet-Derived Growth Factor. PDGF, particularly PDGF-BB, is the strongest chemotactic and mitogenic agent for the fibrogenic cells of the liver. During liver injury, expression of this growth factor and its cognate receptor are highest in areas of greatest damage.

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PDGF is secreted in response to injury by platelets, Kupffer cells, stellate cells, and hepatic myofibroblasts. Moreover, it is sequestered by the ECM and can be released during fibrolysis. An early response to injury is the upregulation of PDGF receptors, which enhances the sensitivity of stellate cells to this growth factor. PDGF receptors are members of the receptor tyrosine kinase superfamily that acts via protein phosphorylation cascades. It has been reported that PDGF has an unusual bell-shaped effect on the migration of hepatic myofibroblasts. 33 At lower concentrations of PDGF, migration is stimulated, but at higher concentrations migration is inhibited. PDGF induces migration through signaling pathways that involve phosphatidylinositol-3 (PI3) kinase, p38 mitogen-activated protein (MAP) kinase, and focal adhesion proteins, including focal adhesion kinase (FAK) and paxillin. Specifically, the effects of p38 MAP kinase on migration are transduced by actin-dependent membrane ruffling and cell spreading. 34 At higher concentrations, PDGF inhibits migration by provoking depolymerization of the actin cytoskeleton and reducing the activity of focal adhesion proteins. Hence, we suggest that early in the injury response, or distant from the injured region, PDGF concentrations are relatively low and stimulate chemotaxis toward the site of damage. Later, during the injury response after fibrogenic cells have migrated into the affected area, PDGF concentrations would be relatively high and the migratory response should be attenuated. Thus, the bell-shaped PDGF dose-response relationship for migration suggests a novel mechanism for accumulating fibrogenic cells within injured areas. PDGF is also a potent stimulus for the proliferation of fibrogenic cells in the liver. However, PDGF-induced proliferation is mediated primarily by pathways that signal through Ras/MEK (mitogen-activating protein ERK kinase)/ERK, rather than p38 MAP kinase. 34 Additionally, Ca2+ and W signaling pathways appear to be required for the mitogenic response to PDGF. Involvement by phosphoinositol-3 kinase has also been suggested, possibly by enhancing the activity of ERK. It is notable that PDGF does not have a bell-shaped effect on proliferation. One might predict that fibrogenic cells would continue to proliferate within injured areas where the PDGF concentration is relatively high, even though chemotaxis would have been shut down at those concentrations. To make matters even more complex, PDGF increases the synthesis of prostaglandin E2, which inhibits proliferation through a cyclic 3',S'-adenosine monophosphate (cAMP)-dependent mechanism. This implies the possibility of a PDGF-triggered negative feedback loop that would self-limit the growth of these cells. Taken together, these data suggest that PDGF facilitates accumulation of fibrogenic cells within injured areas of the liver through distinct effects on chemotaxis and proliferation that are mediated by discrete signal transduction pathways.

I

I-General Considerations

Effects of lfansforming Growth Factor-~. TGF-~ is the principal stimulus for ECM accumulation. In the cirrhotic livers of humans, the expression of TGF-~ is greatest in areas where ECM is most abundant. This cytokine, which is produced by Kupffer cells, platelets, and sinusoidal endothelial cells in response to injury, has paracrine actions on the endothelial cells and fibrogenic cells of the liver. Moreover, TGF-~ induces sinusoidal endothelial cells to express a fibronectin splice variant that stimulates stellate cell fibrogenesis. In fibrogenic cells, TGF-~ stimulates its own expression, which permits the development of a powerful autocrine-positive feedback loop. TGF-~ signaling can also be modulated by the conversion of TGF-~ from its latent to its active form by sinusoidal endothelial cells and by augmenting the expression and ligand affinity of TGF-~ receptors in the fibrogenic cells of the liver. TGF-~ induces the accumulation of ECM by enhancing ECM synthesis and reducing ECM degradation. First, TGF-~ enhances the transcription of collagen I, the predominant ECM component observed in cirrhosis, probably by reducing the expression of negative regulators of transcription and through putative TGF-~ responsive elements in the gene encoding for collagen I. TGF-~ also upregulates synthesis of other ECM components, including fibronectin and proteoglycan. Second, TGF-~ inhibits ECM degradation by reducing synthesis of important matrix metalloproteinases (e.g., MMP-l, MMP-2, MMP-3) and by upregulating plasminogen activator inhibitor (PAl) and tissue inhibitors of metalloproteinases (TIMPs), which are proteins that inhibit the breakdown of ECM. In addition to regulating the accumulation of ECM, TGF-~ also modulates other processes important for the development of fibrosis. TGF-~ stimulates the migration of stellate cells and inhibits apoptosis. Surprisingly, in different studies, TGF-~ stimulated, inhibited, or had no effect on proliferation. It is uncertain whether this phenomenon has physiological importance or is simply a technical artifact. It is significant, however, that TGF-~ upregulates the expression of PDGF receptors, which playa fundamental role in fibrosis, as described previously. Although the molecular mechanisms linking TGF-~ to its observed effects on fibrogenic cells are incompletely understood, evidence suggests that they involve the regulation of transcription by pathways that signal through Sma and Mad (SMAD)-related proteins, MEK and ERK, and hydrogen peroxide and C/EBP-~. Effects of Endothelin-I. Endothelin-l (ET-l) is a vasoactive peptide that strongly stimulates generation of contractile tension by the fibrogenic cells of the liver. Sinusoidal endothelial cells and fibrogenic cells secrete this peptide in response to hepatic injury. ET-l binds to ETA and ETB receptors, which are G-protein-coupled seven-transmembrane receptors. Binding of ET-l to its cognate receptors causes an augmentation of myosin

light-chain phosphorylation through G-protein-coupled activation of Ca2+-dependent myosin light-chain kinase and rho-dependent inhibition of myosin phosphatase. 35 Phosphorylation of the myosin light chain activates myosin, which interacts with bundles of polymerized actin, resulting in the generation of tension. The tension generated by these fibrogenic cells permits orientation and remodeling of the ECM. Evidence also suggests that alterations in the tension generated by stellate cells, which encircle the sinusoids, modulates hepatic blood flow. 30,36 In addition to its role in the regulation of contractile tension, ET-l modulates the migration and proliferation of fibrogenic cells in the liver. The effect of ET-l on migration is predicted by the essential role that retrograde contraction plays in cellular locomotion. As expected, ET-l stimulates migration through a rho-associated, kinasedependent pathway. The role that this peptide plays in the regulation of proliferation is more complex. ETA stimulates proliferation through Ras/MEK/ERK-signaling pathways, whereas ETB inhibits proliferation through a prostaglandin/cAMP-signaling pathway, Since the relative ratio of ETB:ETAincreases with time after injury, the effects of ET-l on cell growth change with the duration of injury. As discussed, PDGF, TGF-~, and ET-l each act via multiple signal transduction pathways to regulate patterns of cellular behavior that are essential for the development of cirrhosis: • PDGF is a powerful regulator of chemotaxis and proliferation. • TGF-~ strongly induces the accumulation of ECM, but also facilitates migration and inhibits apoptosis. • ET-l is a strong agonist for contraction, but also affects chemotaxis and proliferation. Yet, PDGF, TGF-~, and ET-l represent only three of the numerous soluble and insoluble molecules that are produced in response to hepatic injury (see Table 3-4). All of these other injury mediators also have pleiotropic effects that are mediated by signal transduction pathways that work in a coordinated manner. Thus, the molecular and cellular mechanisms underlying the development of cirrhosis are incredibly complex, Despite this complexity, there have been advances to develop preventive and therapeutic strategies for the management of cirrhosis. Indeed, pharmacological antagonists of each of the three injury mediators discussed here prevent or reduce fibrosis in animal models of chronic liver injury.23,26,32 Summary of the Pathogenesis of Cirrhosis It is increasingly clear that fibrosis of the liver is mediated

by the same molecular signals and cellular processes that govern the normal wound-healing response. It is the location, duration, and intensity of liver injury that

3-Molecular and Cellular Basis of Hepatic Failure

dictate clinical outcome. For example, in most forms of chronic liver injury, including hepatitis C and autoimmune hepatitis, fibrosis is initially most prominent in the portal region, the location most affected by these diseases. In contrast, alcoholic and nonalcoholic steatohepatitis, both of which are characterized by early lobular injury, initially display lobular fibrosis, especially around the sinusoids. If hepatic injury is transient, such as occurs with hepatitis A, complete healing occurs without any evidence of excess accumulation of ECM. Conversely, liver fibrosis occurs only months to decades after onset of chronic hepatic injury. The clinical observation that only a portion of patients suffering from chronic liver diseases-such as hepatitis Band C, alcoholic and nonalcoholic steatohepatitis, and hereditary hemochromatosis-develop cirrhosis suggests that there may be an intensity threshold for a given individual that must be crossed in order for fibrosis to ensue. Finally, it has become generally recognized that if the source of chronic liver injury is removed, fibrosis can be reversed. 27 ,32,37 This has been demonstrated in a number of liver diseases, including biliary obstruction, hepatitis C, and autoimmune hepatitis. Whether cirrhosis itself can be significantly reversed remains controversial. The pathogenesis of cirrhosis is complex and is mediated by the dynamic and multifaceted response of the fibrogenic cells of the liver to chronic injury.

Perspectives and Future Directions Why some patients develop ALF rather than selflimited hepatitis remains an important, but as yet unanswered, question. The same questions can be applied to cirrhosis; a large majority of patients with chronic liver disease never develop cirrhosis. There are undoubtedly genetic polymorphisms that predispose to ALF or cirrhosis. Indeed, the host response to injury is as likely as or even more important than the inciting agent or disease. If these genetic differences can be elucidated, it is likely that novel and therapeutic strategies for ALF and cirrhosis will be developed. At a minimum, an improved ability to assess prognosis would enhance the management of patients with acute and chronic liver disease. A better understanding of the molecular pathogenesis of ALF and cirrhosis will undoubtedly translate into improved therapies in the future. In the case of ALF, such therapy will be directed toward limiting cell death by blocking harmful responses while preserving or even enhancing the liver's innate ability to repair and regenerate. For example, new forms of therapy might focus on modifying the early inflammatory events, interrupting apoptotic- and growth-inhibitory pathways, and providing temporary liver support to allow time for hepatic regeneration and repair. However, as noted earlier,

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specifically inhibiting apoptosis may be problematic in that this may redirect cells toward the generally more destructive necrotic cell death pathway and also potentially promote cancer. Likewise, cytokines may have both detrimental and protective roles in ALF, and anticytokine therapy may thus have unanticipated consequences. For example, in a clinical trial of sepsis, a TNF-a antagonist increased mortality, and it is conceivable that such therapy used for ALF might also inhibit hepatic regeneration and worsen outcomes. Because some of the same molecular pathways critical in liver regeneration are also involved in cell death, therapeutic targets will need to be chosen with great care. In the case of cirrhosis, efforts will be directed toward the prevention or reversal of fibrosis. This will not be a simple task for two major reasons. First, fibrosis results from the liver's response to injury, albeit a sustained and exuberant response. Thus, safe and effective therapies for cirrhosis must blunt the injury response that causes fibrosis without compromising the normal wound-healing response. Second, the large majority of patients with chronic liver disease do not develop cirrhosis, and even those who do, often live many years before developing clinical disease. Therefore, improved strategies for determining which patients have the greatest disposition to progressing to decompensated cirrhosis are critical. Otherwise, any successful therapy for prevention must be very safe, because a large number of patients need to be treated for one to benefit. It is likely that a greatly increased understanding of the molecular and cellular mechanisms underlying fibrosis will be required to overcome the hurdles necessary to create effective and safe therapies for cirrhosis.

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25. Friedman SL: Molecular regulation of hepatic fibrosis, an integrated cellular response to tissue injury. J Bioi Chern 275:2247-2250, 2000. 26. Friedman SL: Hepatic Fibrosis. In Schiff ER, Sorrell MF, Maddrey WC (eds): Schiff's Diseases of the Liver. Philadelphia, Lippincott Williams & Wilkins, 2003, pp 409-427. 27. Iredale JP: Cirrhosis: New research provides a basis for rational and targeted treatments. BMJ 327:143-147, 2003. 28. Pinzani M, Marra F: Cytokine receptors and signaling in hepatic stellate cells. Semin Liver Dis 21:397-416. 2001. 29. Ramadori G, Armbrust T: Cytokines in the liver. Eur J Gastroenterol Hepatol 13:777-784, 2001. 30. Rockey DC: Hepatic blood flow regulation by stellate cells in normal and injured liver. Semin Liver Dis 21(3):337-349, 2001. 31. Schuppan D, Ruehl M, Somasundaram R, et al: Matrix as a modulator of hepatic fibrogenesis. Semin Liver Dis 21:351-372, 2001. 32. Tangkijvanich P, Yee HF Jr: Cirrhosis-Can we reverse hepatic fibrosis? Eur J Surg (Supp!) 587:100-112,2002. 33. Tangkijvanich P, Melton AC, Chitapanarux T, et al: Plateletderived growth factor-B~ and lysophosphatidic acid distinctly regulate hepatic myofibroblast migration through focal adhesion kinase. Exp Cell Res 281:140-147, 2002. 34. Tangkijvanich P, Santiskulvong C, Melton AC, et al: p38 MAP kinase mediates platelet-derived growth factor-stimulated migration of hepatic myofibroblasts. J Cell Physiol 191:351-561, 2002. 35. Saab S, Tam SP, 'Ii'an BN, et al: Myosin mediates contractile force generation by hepatic stellate cells in response to endothelin-1. J Biomed Sci 9(6 Pt 2):607-612, 2002. 36. Thimgan MS, Yee HF Jr: Quantitation of rat hepatic stellate cell contraction: Stellate cells' contribution to sinusoidal resistance. Am J Physiol 277(1 Pt 1):G137-143, 1999. 37. Friedman SL, Arthur MJP: Targeting hepatic fibrosis. Sci Med 8:194-205,2002.