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HEPATIC FIBROSIS AND CIRRHOSIS
Don C. Rockey and Scott L. Friedman
Abbreviations ALT alanine aminotransferase AST aspartate aminotransferase AUROC area under the receiver operator characteristic BMI body mass index CINC cytokine-induced neutrophil chemoattractant CTGF connective tissue growth factor DDR discoidin domain receptors ECM extracellular matrix EGF epidermal growth factor ELF European liver fibrosis ET-1 endothelin-1 FGF fibroblast growth factor FPI fibrosis probability index GGT g-glutamyl transferase

6
MMP-9 NASH NGFR NO PDGF PELD PIIINP PPAR QTL ROC TGF-b1 TIMPs ULN VEGF matrix metalloproteinase 9 non-alcoholic steatohepatitis nerve growth factor receptor nitric oxide platelet-derived growth factor pediatric end-stage liver disease propeptide of type III collagen peroxisomal proliferator-activated receptor quantitative trait loci receiver operating characteristic transforming growth factor beta 1 tissue inhibitors of metalloproteinases upper limit of normal vascular endothelial growth factor

GnT-III HA HBV HCV HGF HIV HOMA-IR IL-10 JI LPS MCP-1 MEGX MELD MMPs MMP-2

N-acetylglucosaminyl transferase III hyaluronic acid hepatitis B virus hepatitis C hepatocyte growth factor human immunodeficiency virus insulin resistance by the homeostasis model assessment interleukin-10 jejuno-ileal lipopolysaccharide monocyte chemotactic protein-1 monoethylglycinexylidide model for end-stage liver disease matrix metalloproteinases matrix metalloproteinase 2

INTRODUCTION
Hepatic fibrosis has emerged as a highly relevant aspect of liver biology because of the significant progress in uncovering its mechanisms, combined with a growing realization that effective antifibrotic therapies may soon alter the natural history of chronic liver disease. Thus, liver fibrosis can now be viewed as a clinical problem whose diagnosis and treatment will soon have rational, evidence-based approaches. This progress is very timely, as the continued ‘aging’ of the HCV-infected cohort and the growing prevalence of obesityrelated liver diseases are leading to precipitous increases in the prevalence of advanced liver disease.1 With these issues in mind, this chapter will review clinical aspects of hepatic fibrosis, including natural history, pathophysiologic mechanisms, current and future tools for diagnosis, and emerging antifibrotic strategies. In addition, several recent reviews highlight many of these aspects in greater detail.2–6 Hepatic fibrosis is the accumulation of extracellular matrix, or scar, in response to acute or chronic liver injury. Fibrogenesis represents a wound healing response to injury (Figure 6-1), and ultimately leads to cirrhosis. Cirrhosis is the end-stage consequence of fibrosis of the hepatic parenchyma, resulting in nodule formation that may lead to altered hepatic function and blood flow. Both fibrosis and cirrhosis are the consequences of a sustained wound-healing response to chronic liver injury from a range of causes, including viral, autoimmune, drug induced, cholestatic and metabolic diseases. The clinical manifestations of cirrhosis vary widely, from no symp-

toms at all to liver failure, and are determined by both the nature and severity of the underlying liver disease as well as the extent of hepatic fibrosis. Up to 40% of patients with cirrhosis are asymptomatic and may remain so for long periods, but progressive deterioration leading to death or liver transplantation is typical once complications (such as ascites, variceal hemorrhage or encephalopathy) develop. In such patients there is a 50% 5-year mortality, with approximately 70% of these deaths directly attributable to liver disease.7 In asymptomatic individuals cirrhosis may be first suggested during routine examination, although histologic analysis may be required to establish the diagnosis. Cirrhosis affects hundreds of millions of patients worldwide. The overall burden of liver disease in the United States – the vast majority of which is due to chronic disease with fibrosis – continues to expand, exacting an increasing economic and social cost.1 Indeed, in the US cirrhosis is the most common non-neoplastic cause of death among hepatobiliary and digestive diseases, accounting for approximately 30 000 deaths per year. In addition, 10 000 deaths are due to liver cancer, the majority of which arise in cirrhotic livers, consistent with a steadily rising mortality rate from hepatic cancer.8 Notably, hepatocellular carcinoma is the most rapidly increasing neoplasm in the US and western Europe.9 Initial studies of hepatic fibrosis focused on the composition of extracellular matrix in liver, and continued incremental progress in this area is still anticipated. However, attention has gradually shifted towards exploring the cellular basis of fibrosis and the cellular mediators that drive fibrosis progression and regression (see Pathophysiology, below). In general, the molecular composition of the scar

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Normal liver Hepatocytes

Liver injury Loss of Hepatocyte microvilli

Space of Disse

Quiescent stellate cell Kupffer cell Hepatic sinusoid Endothelial cell

Activated stellate cell Deposition of scar matrix

Loss of fenestrae

Kupffer cell activation

Figure 6-1. Hepatic liver cells and the hepatic sinusoid in normal and injured liver. On the left panel is shown the multiple key liver-specific cellular elements in the normal liver, including hepatocytes, endothelial cells, Kupffer cells, and stellate cells. Stellate cells are located within the subendothelial space of Disse (i.e. between the sinusoidal endothelium and hepatocytes). The figure emphasizes the close physical relationships between the various cellular elements in the liver. After liver injury, changes in numerous cells occur; for example, stellate and Kupffer cells become activated (see Figure 6-3), hepatocytes lose their microvilli, and endothelial cells lose their characteristic fenestrae. All of these features contribute to continued cell activation and injury, as well as dysfunction at the whole organ level.

tissue in cirrhosis is similar regardless of etiology, and resembles that of other parenchymal scarring (e.g. kidney), consisting of the extracellular matrix constituents, collagen types I and III (i.e. ‘fibrillar’ collagens), sulfated proteoglycans, and glycoproteins.10 However, some isoforms of extracellular matrix constituents, for example fibronectin11 and proteoglycans,12 may be relatively enriched during progressive injury. These scar constituents accumulate from a net increase in their deposition in liver and not simply from the collapse of existing stroma.

CLINICAL ASPECTS OF HEPATIC FIBROSIS
NATURAL HISTORY AND RISK FACTORS
Fibrosis leading to cirrhosis can accompany virtually any chronic liver disease that is characterized by the presence of architectural disruption and/or inflammation. The vast majority of patients with liver disease worldwide have chronic viral hepatitis, or steatohepatitis associated with either alcohol or obesity; other etiologies of liver disease include parasitic infestation (e.g. schistosomiasis), autoimmune attack on hepatocytes or biliary epithelium, neonatal liver disease, metabolic disorders including Wilson’s disease,

hemochromatosis and a variety of storage diseases, chronic inflammatory conditions (e.g. sarcoidosis), drug toxicity (e.g. methotrexate or hypervitaminosis A), and vascular derangements, either congenital or acquired. Of the many causes of chronic liver disease, our understanding of natural history of fibrosis is most complete in HCV, with some information about HBV and steatohepatitic diseases, including alcoholic liver disease and NASH. Information about fibrosis progression in other diseases is largely anecdotal, but the development of cirrhosis typically requires many years to decades. There are, however, some notable exceptions in which the development of cirrhosis can be greatly accelerated, possibly occurring within months rather than years: (1) neonatal liver disease – infants with biliary atresia may present at birth with severe fibrosis and marked parenchymal distortion; (2) HCV-infected patients after liver transplantation – a subset of patients who undergo liver transplantation for HCV cirrhosis may develop rapidly progressive cholestasis and recurrent cirrhosis within months, requiring retransplantation;13 (3) patients with HIV/HCV co-infection – these patients have relatively rapid fibrosis compared to those with HCV alone,14 especially if the HIV is untreated (see below); (4) severe delta hepatitis;15 and (5) some cases of drug-induced liver disease. These examples of ‘fulminant fibrosis’ probably reflect dysregulation of several pathways, including defective immunity, massive inflammation and necrosis, and/or

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altered matrix resorption. Together, they highlight the highly dynamic nature of scar accumulation and degradation. Moreover, when matrix accumulation is unopposed because degradation is ineffective, more rapid fibrosis may ensue. Once cirrhosis and its complications develop the prognosis is predicted by widely used systems, including Child–Pugh, PELD16 and MELD,17 which are predictive independent of the etiology of liver disease.

Hepatitis C Virus
The risk and natural history of fibrosis associated with HCV have been greatly clarified as a result of several large clinical studies incorporating standardized assessments of fibrosis that combine detailed historical and clinical information.18 The disease can run a remarkably variable course, from decades of viremia with little fibrosis to a rapid onset of cirrhosis within 10–15 years. It appears to be host factors rather than viral factors that correlate with fibrosis progression in HCV. The data supporting this conclusion include the following: (1) there is no relationship between viral load or genotype and severity of fibrosis even though these former factors affect the response to antiviral therapy; (2) human promoter polymorphisms (e.g. TGF-b1 and angiotensin) appear to correlate with fibrosis risk,19 with large-scale efforts currently under way to identify additional genetic markers of fibrosis risk;20 (3) host immune phenotype may be critical, as there is more rapid progression in immunosuppressed patients, whether due to HIV or to immunosuppressive drugs.14 In mice, a Th2 phenotype strongly correlates with fibrogenic potential,21 which has led to successful efforts to use quantitative trait loci (QTL) mapping to identify specific fibrosis risk genes in these animals.22 Other identified host risk factors for more rapid progression of HCV include: (1) older age at the time of infection; (2) concurrent liver disease due to HBV or alcohol (>50 g/day); it is uncertain, however, whether lesser amounts of alcohol intake are additive towards fibrosis progression: recent studies suggest that less than 50 g/day of alcohol results in a neglible increased risk of hepatic fibrosis;23 (3) male gender; (4) increased body mass index (BMI), associated with hepatic steatosis;24 (5) HIV infection or immunosuppression following liver transplantation. Because standard clinical indices cannot distinguish between minimal and even advanced fibrosis, knowledge about these risk factors and duration of infection can greatly inform clinical management. Thus, for chronic HCV, if the time of infection is known and a biopsy obtained at any time thereafter, the rate of progression per year based on either Ishak or METAVIR scoring can be estimated.25 Although initial analyses of this type suggested that fibrosis progression is truly linear, it is now increasingly clear that the progression rate accelerates as the disease advances,26 such that it takes less time to progress between Metavir stages 3 and 4, than from stage 1 to 2, for example. Assessment of fibrosis stage and rate of fibrosis progression can be valuable for at least three reasons: (1) the actual stage of fibrosis will indicate the likelihood of response to a-interferon or ainterferon/ribavirin, as the advanced stages of fibrosis (F3 or F4) generally have a lower response rate to antiviral therapy;27,28 (2) if little fibrosis progression has occurred over a long interval, then treatment with antiviral therapy may be deemed to be less urgent

and it may be safe to await more effective and/or better-tolerated therapy; (3) the approximate time to the development of cirrhosis can be estimated. This would not, however, indicate if or when clinical liver failure would occur, as the complications of liver disease may be delayed for up to a decade or more after the establishment of cirrhosis. As genetic risk markers that predict a rapid fibrosis progression rate are developed, this information, combined with the absolute stage of fibrosis, may enable more accurate identification of patients at risk for disease and thus in need of antifibrotic therapy.

Hepatitis B Virus
Very few studies have assessed the progression rate of fibrosis in chronic HBV infection. In general, inflammatory activity, as influenced by viral factors, including e Ag status, that indicate active viral replication, correlates with fibrosis.29,30 Fibrosis progression has been correlated with HBV genotype in at least one study.31 In a subset of patients a rapidly progressive ‘fibrosing cholestatic hepatitis’ may occur,32 but there are neither definitive risk factors for this condition nor unique etiologic, cellular or molecular determinants identified. In addition, delta hepatitis superinfection or co-infection may greatly accelerate the risk of advanced fibrosis and cirrhosis.15 What is striking, however, is that virologic suppression in response to potent antiviral regimens can effect remarkable improvement not only in serum alanine aminotransferase (ALT) levels and histologic inflammation, but also in fibrosis.15,33–35 Indeed, dramatic resolution of cirrhosis in a 10-year follow-up has been reported in patients with delta hepatitis who were successfully treated with a-interferon.15

Alcoholic Liver Disease
The clearest clinical determinant of fibrosis is continued alcohol abuse: patients with fibrosis who continue to drink are virtually assured of progression. In addition, two clinical features commonly seen in steatohepatitis, elevated BMI and serum glucose, also confer an increased risk of fibrosis in alcoholic liver disease.36 Pathologically, the presence of pericentral fibrosis (central hyaline sclerosis) carries a high risk of eventual panlobular cirrhosis, which is almost certain if alcohol intake continues.

Non-Alcoholic Steatohepatitis
There is a critical need for better data about natural history, risk factors for fibrosis, and rate of fibrosis progression in NASH, issues now being addressed in several multicenter studies. Patients with only steatosis and no inflammation appear to have a benign course when followed for up to 19 years;37 however, it is unclear whether this lesion is completely distinct from steatohepatitis, or instead represents a precursor of NASH. It is instructive to remember that HCV fibrosis progression rates were underestimated shortly after the virus was first identified, as many patients had a relatively early fibrosis stage. With continued infection, however, a sizeable fraction eventually have progressed to more advanced stages. In a parallel situation, the obesity epidemic in the US and the developed world is only now being fully appreciated, and a threshold level of obesity may have only begun to confer a risk of liver disease that will become clinically significant in the next decade. In patients with sustained

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NASH spontaneous histologic improvement is very uncommon, but better longitudinal data are needed to understand the natural history of this disease; for example, data examining the evolution of NASH in patients undergoing bariatric surgery who have extensive weight loss and improvement in their metabolic syndrome are awaited. In three combined studies of 26 patients followed with sequential biopsies for up to 9 years, 27% had progression of fibrosis and 19% advanced to cirrhosis, but none had reversal of fibrosis.37 Of interest is the recurrence of NASH following liver transplantation in some patients with cryptogenic cirrhosis, implicating an underlying metabolic defect that may account for liver disease in both the native and the transplanted organs. Risk of fibrosis and rate of progression are critical issues that will influence risk stratification and patient selection for clinical trials, as progression to cirrhosis is the most important clinical consequence of NASH. Recently developed systems to grade and stage liver disease in NASH38 should allow for improved, prospective collection of standardized data that can further address these vital questions. In general, increasing obesity (BMI) >28 kg/m2) correlates with severity of fibrosis and risk of cirrhosis. Other risk factors include necroinflammatory activity with ALT >2¥ normal and/or AST/ALT >1, age, elevated triglycerides, insulin resistance and/or diabetes mellitus, and systemic hypertension.39 It is uncertain whether these features are comparable across the spectrum of disorders associated with NASH, including obesity with insulin-resistance, JI (jejuno-ileal) bypass, total parenteral nutrition and rapid weight loss, among others. Whether these factors represent surrogates for other risk factors (i.e. reduced antioxidant levels in older patients, increased renin–angiotensin activity in hypertensives) is unknown. Ratziu and colleagues40 have reported a clinicobio-logical score that combines age, BMI, triglycerides and ALT and which reportedly has 100% negative predictive value for excluding significant fibrosis.

It remains unclear what distinguishes those patients whose cirrhosis is reversible from those in whom it is fixed. Potential factors influencing reversibility probably include: (1) a prolonged period of established cirrhosis, which could reflect a longer period of crosslinking of collagen, rendering this collagen less sensitive to degradation by enzymes over time. Animal studies now clearly support this possibility;47 (2) total content of collagen and other scar molecules, which might lead to a large mass of scar that is physically inaccessible to degradative enzymes; (3) reduced expression of enzymes that degrade matrix, or sustained elevation of proteins that inhibit the function of these degradative enzymes, in particular elevated levels of tissue inhibitors of metalloproteinases (TIMPs), which block matrix proteases and also prevent apoptosis of activated stellate cells.48,49 All three scenarios highlight the dynamic process of collagen deposition and degradation.

PATHOPHYSIOLOGY OF HEPATIC FIBROSIS AND CIRRHOSIS
EXTRACELLULAR MATRIX (ECM) IN THE NORMAL AND THE FIBROTIC LIVER
Extracellular matrix refers to the array of macromolecules that comprise the scaffolding of normal and fibrotic liver. These macromolecules consist of three main families: collagens, glycoproteins and proteoglycans (see 10 for review). The number of collagens identified in liver is rapidly growing, and includes collagen XVIII, which is a precursor to the molecule angiostatin. Glycoproteins include fibronectin, laminin, merosin, tenascin, nidogen, and hyaluronic acid, among others. Proteoglycans include heparan, dermatan sulfates, chondroitin sulfates, perlecan dystroglycan syndecan, biglycan and decorin. There is tremendous heterogeneity of these matrix macromolecules with respect to their different isoforms, variable combinations within different tissue regions, and changes related to age. In normal liver the subendothelial space of Disse separates the epithelium (hepatocytes) from the sinusoidal endothelium. This space contains a basement membrane-like matrix which, unlike the typical basement membrane, is not electron dense. The hepatic basement membrane is composed of non-fibril-forming collagens, including types IV, VI and XIV, glycoproteins and proteoglycans. This normal subendothelial ECM is critical for maintaining the differentiated functions of resident liver cells, including hepatocytes, stellate cells and sinusoidal endothelium. In contrast to basement membrane-type matrix, in normal liver the so-called interstitial ECM is largely confined to the capsule, around large vessels, and in the portal areas. It is composed of fibrilforming collagens (e.g. types I and III) together with cellular (EDA) fibronectin, undulin, and other glycoconjugates. As the liver becomes fibrotic, the total content of collagens and non-collagenous components increases three- to fivefold, accompanied by a shift in the type of ECM in the subendothelial space from the normal low-density basement membrane-like matrix to interstitial-type matrix (see 10 for review). This ‘capillarization’ leads to the loss of hepatocyte microvilli and the disappearance of endothelial fenestration (Figure 6-1).

REVERSIBILITY OF FIBROSIS AND CIRRHOSIS
There is now clear evidence that fibrosis and even cirrhosis can be reversible. The feature common to all cases of cirrhosis improvement is the elimination of the underlying cause of liver disease, whether due to eradication of HBV,41 delta hepatitis15 or HCV,42 decompression of biliary obstruction in chronic pancreatitis,43 or to immunosuppressive treatment of autoimmune liver disease.44 Moreover, there is ample evidence of reversibility in animal models, which provide vital clues to underlying mechanisms.45 Earlier studies demonstrated that fibrosis improves with treatment of HCV,46 and even cirrhosis can regress following HCV eradication with a-interferon/ribavirin.42 Among a large cohort of patients successfully treated with this combination there were 150 with cirrhosis, half of whom had a reduction in their fibrosis score according to METAVIR staging, with several regressing by two or more stages. Because fibrosis in HCV typically progresses over three decades, one might anticipate an equally slow but steady regression of fibrosis following viral clearance.

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The outcome of fibrogenesis is the conversion of normal lowdensity basement membrane-like matrix to high-density interstitialtype matrix. A number of components are responsible for ECM remodeling (see 49,50 for reviews) (Figure 6.2). These include a family of zinc-dependent enzymes matrix metalloproteinases (MMPs),51 their inhibitors (tissue inhibitor of metalloproteinases, TIMP), and several converting enzymes (MT1-MMP , and stromelysin, for example). In human liver diseases there is down-regulation of MMP1 (interstitial collagenase, collagenase I) and up-regulation of MMP2 (gelatinase A) and MMP9 (gelatinase B). Based on the differing substrate specificities of these enzymes, the result is increased degradation of basement membrane collagen and decreased degradation of interstitial collagens. These activated MMPs are regulated in part by their tissue inhibitors, the so-called TIMPs. TIMP1 and TIMP2 are upregulated relative to MMP1 in progressive experimental liver fibrosis, which may explain the decreased degradation of interstitial-type matrix observed in experimental and human liver injury. In contrast, during the resolution of experimental liver injury TIMP-1 and TIMP-2 expression is decreased whereas collagenase expression is unchanged, resulting in a net increase in collagenase activity and increased resorption of scar matrix. Stellate cells are a key source of MMP-2 and stromelysin. They also express TIMP-1 and TIMP-2 mRNAs and produce TIMP-1 and MT1-MMP MMP-9, which is a type IV collagenase locally secreted by Kupffer cells, and may also be produced by stellate cells in

response to interleukin-1.52 The source of MMP-1, which plays a crucial role in degrading the excess interstitial matrix in advanced liver disease, is still uncertain.53 However, interstitial collagenase activity in liver may be attributable to either MT1-MMP or even MMP-2, although further studies are required.

ECM–CELL INTERACTIONS
Changes in the microenvironment of the space of Disse result in phenotypic changes in all resident liver cells. Hepatic stellate cells are activated by the surrounding increase in interstitial matrix.54 Sinusoidal endothelial cells produce cellular fibronectin in very early liver injury, which also contributes to stellate cell activation. In addition, endothelial cells produce type IV collagen, proteoglycans and factors (e.g. urokinase-type plasminogen activator) that participate in the activation of latent cytokines such as TGF-b1. Activated Kupffer cells release cytokines and reactive oxygen intermediates that may stimulate stellate cells in a paracrine manner.55 Platelets are also an abundant source of cytokines upon injury, producing a rich array of important growth factors. Hepatocytes, the most abundant cells in the liver, generate lipid peroxides following injury that lead to stellate cell activation, a prerequisite for fibrogenesis (see below). The dynamic interactions between fibrogenic cells in liver and the ECM is an important determinant of fibrogenesis. The ECM is a reservoir for growth factors, for example platelet-derived growth factor (PDGF).10 Like all cytokines, PDGF signals by binding to

Activated stellate cell Early pathological degradation Regression Apoptotic stellate cell Normal ECM MT1-MMP MMP-2 TIMP-2

Kupffer cell MMP-1 MMP-13 Other MMPs Progression

TIMP-1 TIMP-2

Figure 6-2. Emerging Mechanisms of Early Pathologic Matrix Degradation, Fibrosis Progression & Fibrosis Resolution in Chronic Liver Disease. Activation of stellate cells (top left panel) is a key event in hepatic fibrosis, and is associated with pathologic matrix degradation due to increased production of membrane type matrix metalloproteinase 1 (MT1-MMP), matrix metalloproteinase-2 (MMP-2), and tissue inhibitors of metalloproteinases (TIMPs), leading to replacement by interstitial collagen or scar matrix. As fibrosis progresses (middle panel), sustained expression of TIMPs prevents matrix degradation and apoptosis of activated stellate cells. Regression of fibrosis (upper right panel) is associated with increased apoptosis of activated stellate cells. Apoptosis requires decreased expression of tissue inhibitor of metalloproteinase-1 (TIMP-1), yielding a net increase in protease activity. These events may occur coincident with production of matrix metalloproteinases, which could include MMP-1 (in humans) and/or MMP-13 (in rodents), although cellular sources of these enzymes (possibly including Kupffer cells), and clear evidence of their induction in vivo are still lacking. Validation of these events and further elucidation of mechanisms underlying fibrosis regression represent key challenges for future studies.147a

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membrane receptors. The PDGF receptor belongs to a receptor family known as receptor tyrosine kinases, which collectively are key transducers for many important cytokines, including hepatocyte growth factor (HGF), epidermal growth factor (EGF), vascular endothelial growth factor (VEGF) and fibroblast growth factor (FGF). Interestingly, a new subclass of receptor tyrosine kinases, socalled discoidin domain receptors (DDR), has been identified; this group of receptors signal in response to fibrillar collagens rather than peptide ligands.56 Indeed, stellate cell activation is accompanied by up-regulation of DDR2 receptors, and increased signaling is associated with altered MMP-2 expression.57,58 Intracellular signaling cascades downstream of receptor tyrosine kinases and other receptors are pervasive (see 59 for review). Integrins are another type of membrane receptor that transduce extracellular signals in liver. These are heterodimeric transmembrane proteins composed of an a and a b subunit whose ligands are matrix molecules rather than cytokines. Several integrins and their downstream effectors have been identified in stellate cells, including a1b1, a2b1, a5b1, avb1, avb3 and a6b4.6,60,61 Integrins may also complex with other receptor families in mediating cell motility and fibrogenesis, for example the tetraspanin family of receptors.62

HEPATIC STELLATE CELL ACTIVATION – THE COMMON PATHWAY LEADING TO HEPATIC FIBROSIS
The identification of stellate cells as the key cellular source of extracellular matrix in liver has been a major advance. This distinct cell population, located in subendothelial space of Disse between hepatocytes and sinusoidal endothelial cells (Figure 6-1), represents onethird of the non-parenchymal population or about 15% of the total number of resident cells in normal liver.63 In normal liver they are the principal storage site for retinoids (vitamin A metabolites),

which accounts for 40–70% of retinoids in the body. Most of the retinoids are in the form of retinyl esters and are confined to cytoplasmic droplets. Preferential expression of ECM genes in stellate cells has been confirmed in mechanistically distinct experiment models of injury. Recent studies have emphasized the heterogeneity of mesenchymal populations in the liver, with variable expression of neural,64 angiogenic,65 contractile,66 and even bone marrow-derived67 markers. Moreover, experimental genetic ‘marking’ of stellate cells by the expression of fluorescent proteins downstream of either fibrogenic or contractile gene promoters illustrates the plasticity of fibrogenic populations in vivo.68 In view of this capacity for ‘transdifferentiation’ between different mesenchymal cell lineages, and possibly even from epithelium,69 the key issue is whether fibrogenic cells express target molecules such as receptors or cytokines in sufficient concentrations in vivo to merit their targeting by diagnostic agents or antifibrotic compounds. Following liver injury of any etiology, stellate cells undergo a process known as ‘activation’, which is characterized by the transition of quiescent vitamin A-rich cells into proliferative, fibrogenic, and contractile myofibroblasts.54 Stellate cell activation is typically a result of complex interplay among ECM (Figure 6.2) and cellular (Figure 6.3) elements found in the local environment. It should be noted that activation most often occurs in the setting of hepatocellular injury and subsequent inflammation. Activation can be conceptually viewed as a two-stage process: initiation (also referred to as ‘preinflammatory’) and perpetuation54 (Figure 6-4). Initiation refers to early changes in gene expression and phenotype that render the cells responsive to other cytokines and stimuli, whereas perpetuation results from the effects of these stimuli on maintaining the activated phenotype and generating fibrosis. Initiation is largely due to paracrine stimulation, whereas perpetuation involves autocrine as well as paracrine loops.

Inciting injury Recruitment of inflammatory cells T-cell NK cell Hepatocyte Stellate cell Kupffer cell

Expression of cytokines

Figure 6-3. Cellular response to wound healing. Most forms of liver injury result in hepatocyte injury followed by inflammation, which in turn leads to activation of hepatic stellate cells. Inflammatory effectors are multiple and include T cells, NK and NKT cells as well as Kupffer cells. These cells produce growth factors, cytokines, and chemokines that play an important role in stellate cell activation. Additionally, injury leads to disruption of the normal cellular environment, and also to stellate cell activation (right upper panel). Once activated, stellate cells themselves produce a variety of compounds, including growth factors, cytokines, chemokines, and vasoactive peptides. These substances have pleotropic effects in the local environment, including many which have autocrine effects on stellate cells themselves. One of the major results of stellate cell activation is extracellular matrix synthesis, as well as the production of matrix degrading enzymes.

Activated stellate cell

Stellate cell activation

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PERPETUATION PROLIFERATION INITIATION CONTRACTILITY

ET-1 PDGF TGF-β1 FIBROGENESIS

RESOLUTION MMP-2 MATRIX DEGRADATION PDGF MCP-1 REVERSION? MCP-1 PDGF

CHEMOTAXIS APOPTOSIS LEUKOCYTE CHEMOTAXIS RETINOID LOSS

Figure 6-4. Stellate cell activation. Stellate cell activation is a key pathogenic feature underlying liver fibrosis and cirrhosis. Multiple and varied stimuli contribute to the induction and maintenance of activation, including (but not limited to) cytokines, peptides, and the extracellular matrix itself. Key phenotypic features of activation include the production of extracellular matrix, loss of retinoids, proliferation, of up-regulation of smooth muscle proteins, secretion of peptides and cytokines (which have autocrine effects), and up-regulation of various cytokine and peptide receptors. Reprinted with permission from ref 54a.

Initiation
Oxidant stress may be an early determinant of stellate cell activation. In hepatic injury, whether subclinical or overt, there is a perturbation of normal liver homeostasis, with extracellular release of either free radicals (i.e. ‘oxidant stress’), intracellular constituents, and/or cytokines and signaling molecules. Sources of these mediators may be circulating (i.e. endocrine), paracrine or autocrine. In particular, oxidant stress-mediated necrosis leading to stellate cell activation may underlie a variety of liver diseases, including hemachromatosis, alcoholic liver disease, viral hepatitis and nonalcoholic steatohepatitis (NASH).55,70,71 Liver injury is typically associated with infiltration of inflammatory cells, but even in their absence the liver contains sufficient resident macrophages (Kupffer cells) and natural killer cells (pit cells) to initiate local inflammation prior to the arrival of extrahepatic cells. In addition to oxidant stress, following early injury endothelial cells produce a splice variant of cellular fibronectin that is able to stimulate stellate activation.

Endothelial cells in early injury may also participate in the conversion of latent TGF-b1 to its active, profibrogenic form through the activation of plasmin. Whereas necrosis is considered a classic inflammatory and fibrogenic stimulus, recent findings also suggest that apoptosis may provoke a fibrogenic response in stellate cells. Apoptotic fragments released from hepatocytes are fibrogenic towards cultured stellate cells,72 and Fas-mediated hepatocyte apoptosis in vivo in experimental animals is also fibrogenic.73 Platelets in injured liver are a potent source of paracrine stimuli by generating multiple potentially important mediators, including PDGF, TGF-b1, and epidermal growth factor (EGF). Additionally, activated stellate cells have also been observed in primary and metastatic human tumors, as well as a murine model of metastatic melanoma to liver.74 In recent years, increasing interest has been focused on the molecular regulation of gene expression during early stellate cell activa-

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tion. There have been many advances in dissecting pathways of membrane and intracellular signaling and transcriptional gene regulation in activated hepatic stellate cells that are too numerous to detail here.75 A growing number of transcription factors may regulate stellate cell behavior, including peroxisomal proliferator-activated receptors (PPAR) a, b and g,76 retinoid receptors,77 NF-kB,78,79 Jun D,75 Krüppel-like factor 6 (previously called ‘Zf9’),80 Foxf1,81 and CRP282 among others.

The expression of smooth muscle a actin is increased during stellate cell activation. ET-1 and other vasoactive mediators increase their expression.83 Thus studies of contractile proteins in stellate cells may yield a therapeutic target for the treatment of intrahepatic portal hypertension.

Fibrogenesis
Fibrogenesis is perhaps the key component of the stellate cell’s contribution to hepatic fibrosis. TGF-b1 is the most potent fibrogenic factor, with some fibrogenic activity documented for interleukin-1b, TNF, lipid peroxides, acetaldehyde, and others (see 2,6 for reviews). Because of its importance, TGF-b1 regulation has received considerable attention. TGF-b1 is up-regulated in experimental and human hepatic fibrosis. Although sources of this cytokine are many, autocrine expression is among the most important (see 2 for review). Several mechanisms underlie the increase in TGF-b1 expression by stellate cells during liver injury, including TGF-b transcriptional upregulation, activation of latent TGF-b1, increased TGF-b receptor expression, and up-regulation of TGF-b signaling components.92–96

Perpetuation
After initiation, activated stellate cells undergo a series of phenotypic changes that collectively lead to the accumulation of ECM. These include proliferation; contractility; fibrogenesis; chemotaxis; matrix degradation; retinoid loss; and proinflammatory responses and cytokine release. The following sections detail the mechanisms underlying each of these events.

Proliferation
An increase in the number of stellate cells has been documented after both human and experimental liver injury, in large part due to local proliferation. Following liver injury, many mitogenic factors as well as their cognate tyrosine kinase receptors are unregulated, primariliy through receptor tyrosine kinases.59 PDGF is the bestcharacterized and most potent mitogen towards stellate cells. Upregulation of PDGF receptor following liver injury enhances the responsiveness to autocrine PDGF, whose expression is also increased. The downstream signaling pathways involve ERK/MAP kinase, phosphoinositol 3 kinase (PI 3-kinase) and STAT-1 (signal transducers and activators of transcription) (see 3 for review). PDGF-induced proliferation correlates with increased intracellular Ca2+ and pH, raising the possibility that calcium channel blockers might modulate stellate cell mitogenesis or activation. Other stellate cell mitogens include endothelin-1 (ET-1),83,84 thrombin,85 FGF,86 and IGF,87,88 among others (see 89,90 for reviews). A recent study has documented increased sensitivity to ET-1 during activation,91 suggesting potentiation of autocrine/paracrine stimulation.

Chemotaxis
Stellate cells may accumulate both through proliferation and via directed migration into regions of injury, or chemotaxis. PDGF, the leukocyte chemoattractant MCP-1, and a growing family of chemokines have been identified as key stellate cell chemoattractants.97 In addition to tyrosine kinase receptors, new agents have been implicated in stellate cell migration, in particular tetraspanin receptors.3,62

Matrix Degradation
A greater understanding of matrix degradation in liver is emerging. Quantitative and qualitative changes in the activity of MMPs and their inhibitors play a vital role in extracellular matrix remodeling in liver fibrogenesis (see ECM in the normal and fibrotic liver and Figure 6.2 above). As noted above, the net effect of changes in matrix degradation is the conversion of the low-density subendothelial matrix to one rich in interstitial collagens.

Retinoid Loss Contractility
Contraction by stellate cells may be a major determinant of early and late increases in portal resistance during liver fibrosis. Activated stellate cells impede hepatic blood flow both by constricting individual sinusoids and by contracting the cirrhotic liver, as the collagenous bands typical of end-stage cirrhosis contain large numbers of activated stellate cells (see 66 for review). A key contractile stimulus towards stellate cells is ET-1.66 Other contractile agonists include arginine vasopressin, adrenomedullin, and eicosanoids.66 The regulation of stellate cell contraction is complex. The endothelium-derived relaxing factor nitric oxide (NO) appears to be an important relaxing factor in the sinusoid (although other factors, such as carbon monoxide, also play a role). The net contractile activity of stellate cells in vivo therefore reflects the relative strength of each of these opposing activities. Current evidence suggests that intrahepatic portal hypertension probably results from diminished NO (and/or other vasodilators) activity as well as increased stimulation by ET-1 (or other constrictors).66 Stellate cell activation is accompanied by loss of their characteristic perinuclear retinoid (vitamin A) droplets. Although the intracellular form is largely retinyl esters, when retinol is exported from the cell during activation it is primarily as retinol, suggesting the possibility of intracellular hydrolysis of esters before being exported. Several nuclear retinoid receptors that bind intracellular retinoid ligands have been identified and their effects characterized in stellate cells.77,98

Proinflammatory Responses and Cytokine Release
Hepatic stellate cells and sinusoidal endothelial cells have emerged as inflammatory effectors. Sinusoidal endothelial cells, normally fenestrated to allow rapid bidirectional transport of solutes between sinusoidal blood and parenchymal cells, may rapidly lose their fenestrations upon injury and express proinflammatory molecules, including ICAM-1, VEGF and adhesion molecules.74,99 Together with stellate cells, they activate angiogenic pathways in response to hypoxia associated with local injury or malignancy.74,97,100,101

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Key inflammatory pathways converge on stellate cells, leading to fibrosis (see Figure 6-3). Thus, the cell type is a central mediator in inflammation, rather than just a passive target. Upon activation, they release chemokines97,102 and other leukocyte chemoattractants, proteinase-activated receptors,103 and up-regulate expression of key inflammatory receptors, including ICAM-1,104 chemokine receptors,105 and those mediating lipopolysaccharide (LPS) signaling, including Toll-like receptor 4.78 Stellate cells may also contribute to intrahepatic apoptosis of T lymphocytes.106 Remarkably, little attention has focused on the contribution of different lymphocyte subsets to hepatic fibrogenesis. Interest has increased recently, in part because of the observation that patients with HCV who are co-infected with HIV, as well as those who are immunosuppressed following liver transplantation, have accelerated fibrosis rates, implicating the immune system as a determinant of fibrogenesis. These observations have been supported by animal studies demonstrating that the immune phenotype regulates fibrogenesis independent of effects on injury, which in turn have led to efforts to map the genetic loci accounting for these differences.22 Most recently, CD8 lymphocytes have emerged as potential profibrogenic cells, based on their ability to induce early fibrogenesis following adoptive transfer to naïve SCID mice from animals with liver injury.107 Autocrine cytokines play vital roles in regulating the activation process of stellate cells. These cytokines include TGF-b1, PDGF, FGF, HGF, PAF, stem cell factor and ET-1, among others.59,90,97,108 Furthermore, stellate cells release neutrophil and monocyte chemoattractants, which can amplify inflammation in liver injury. These chemokines include colony-stimulating factor, monocyte chemotactic protein-1 (MCP-1), and cytokine-induced neutrophil chemoattractant (CINC).97 Anti-inflammatory cytokines produced by stellate cells have also been identified. Up-regulation of interleukin-10 (IL-10) occurs in early stellate cell activation. The anti-inflammatory effects of this cytokine are demonstrated by its ability to down-regulate TNF-a production from macrophages. Knockout mice lacking IL-10 have more severe hepatic fibrosis following CCl4 administration, and transgenic mice expressing IL-10 in liver have reduced fibrosis.107 Based on the consistent antifibrotic effect of IL-10 in experimental liver disease, a clinical trial was undertaken which failed to show an antifibrotic effect in patients with HCV infection, possibly because of marked increases in HCV replication109 (see Therapy of hepatic fibrosis, below).

The increasing prevalence of obesity in the US and western Europe is associated with an alarming increase in NASH,39 leading to advanced fibrosis and cirrhosis. Leptin, a circulating adipogenic hormone that is proportionate to adipose mass in circulating blood, has been clearly linked to stellate cell fibrogenesis.113–115 Sources are likely to be both endocrine and autocrine, associated with enhanced signaling through the leptin receptor, which is up-regulated during stellate cell activation.113 Concurrently, down-regulation of adiponectin, a counterregulatory hormone, in obesity may amplify the fibrogenic activity of leptin. This possibility is supported by findings in mice lacking adiponectin, which have enhanced fibrosis following toxic liver injury.116

RESOLUTION OF LIVER FIBROSIS AND THE FATE OF ACTIVATED STELLATE CELLS
During recovery from acute human and experimental liver injury the number of activated stellate cells decreases as tissue integrity is restored. Either reversion of stellate cell activation, or selective clearance of activated stellate cells by apoptosis, may explain the loss of activated cells in resolving liver injury. To date, evidence is strongest for stellate cell apoptosis in this setting. Apoptosis of stellate cells probably accounts for the decrease of activated stellate cells during resolution of hepatic fibrosis.49 Following injury, apoptosis may be inhibited by soluble factors and matrix components that are present during injury, whereas an apoptotic pathway otherwise represents a ‘default’ mode. Furthermore, cell death ligands, including TRAIL and fas, are expressed in liver injury, and activated stellate cells are more susceptible to TRAILmediated apoptosis.73,117,118 Another death receptor, nerve growth factor receptor (NGFR), is also expressed by activated stellate cells, and its stimulation with ligand drives apoptosis.119 Survival factors also regulate the net activity of stellate cell apoptosis. IGF-I promotes stellate cell survival via the PI3-K/c-Akt pathway and TNF-a has the same effect, but utilizes the NF-kB pathway.120,121 Molecules regulating matrix degradation appear closely linked to survival and apoptosis. Active MMP2 correlates closely with apoptosis, and in fact may be stimulated by it.122 Inhibition of MMP2 activity by TIMP-1 blocks apoptosis in response to a number of apoptotic stimuli.123 Interactions between stellate cells and the surrounding matrix also influence their propensity towards apoptosis, and this might partly explain the antiapoptotic activity of TIMP-1. Moreover, the fibrotic matrix may provide important survival signals to activated stellate cells. For example, animals expressing a mutant collagen I resistant to degradation have more sustained fibrosis and less stellate cell apoptosis following liver injury,48 and transgenic animals expressing TIMP-1 in liver have delayed resolution of fibrosis.124 Studies using gliotoxin,125 a fungal toxin that induces apoptosis in stellate cells, emphasize the role of this pathway in stellate cell removal during resolution of liver fibrosis. It is unknown whether an activated stellate cell can revert to a quiescent state in vivo, although it has been observed in culture. When stellate cells are grown on a basement membrane substratum (Matrigel) they remain quiescent, and plating of highly activated cells on this substratum down-regulates stellate cell activation.58,126

DISEASE-SPECIFIC MECHANISMS REGULATING HEPATIC FIBROSIS – HCV AND NASH
In addition to generic mechanisms of fibrogenesis common to all experimental and human liver disease, there has been progress in elucidating disease-specific mechanisms, in particular in hepatitis C (HCV) and NASH (non-alcoholic steatohepatitis). In HCV, stellate cells might be infectable by the virus because they express putative HCV receptors.104,110 Moreover, adenoviral transduction of HCV non-structural and core proteins induces stellate cell proliferation and the release of inflammatory signals.104 In HCV-infected liver chemokines and their receptors are up-regulated, stimulating lymphocyte recruitment.111 HCV proteins may also interact directly with sinusoidal endothelium.112

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METHODS TO MEASURE FIBROSIS
OVERVIEW
Measurement of fibrosis not only helps to stage the severity of disease, it allows serial determination of disease progression. The level of fibrosis may play an important role in clinical management and determine patients’ prognosis. For example, aggressive therapy is more appropriate in HCV-infected patients with advanced fibrosis. Further, the fibrosis progression rate is an important predictor of the time to develop cirrhosis.18 It is essential to measure fibrosis accurately, given the growing prospect of antifibrotic therapies and the need to track their efficacy. Moreover, with growing evidence that fibrosis is reversible, methods will need to assess both progression and regression accurately. For example, specific therapy leads to a reduction in fibrosis in a number of diseases, including autoimmune liver disease, hepatitis C, hepatitis B, and others.34,35,42,44,127–129 Percutaneous liver biopsy has traditionally been considered to be the gold standard test to assess liver fibrosis. However, a variety of non-invasive tests have been advanced as potential alternatives to biopsy. These include clinical signs, routine laboratory tests, quantitative assays of liver function, markers of extracellular matrix synthesis and/or degradation, and radiologic imaging studies. In addition to individual indicators of fibrosis, combination tests, and a number of models for predicting liver fibrosis have been developed. Individual and combination tests are discussed below. The ideal method to measure fibrosis would be simple, noninvasive, reproducible, inexpensive, accurate, and readily available. Unfortunately, none of the currently available approaches fulfills all of these criteria.

Another major category of test includes those that are based specifically on the pathogenesis of fibrosis (see above). For example, proteins that are produced as a result of the fibrogenic process itself that have been studied as markers of fibrosis include procollagen I, fibronectin, tenascin, laminin, hyaluronic acid and others. Other markers have included cytokines (i.e. TGF-b1), connective tissue growth factor (CTGF), PDGF and others, matrix degrading enzymes (i.e. TIMP1), and others (Table 6-1). Finally, groups of tests, including those that utilize markers of fibrosis in combination with each other or in combination with other types of test, have been advanced in an attempt to detect and measure fibrosis. Ideally, a blood-based test should have both high sensitivity and high specificity. Many of the available tests have a high specificity (>95%) for advanced fibrosis. However, few (including algorithms) have great sensitivity to detect moderate levels of fibrosis. Moreover, a serum-based assay ideally should be linear over the full range of fibrosis, follow the natural history, and accurately reflect the effect of treatment.

Routine Laboratory Tests
A number of studies have used routine laboratory tests in an attempt to determine whether a patient may have advanced liver disease, in particular to exclude or confirm portal hypertension and/or esophageal varices.130,131 Although tests such as the prothrombin time, albumin level, and portal vein diameter (measured by ultrasound) have all been associated with varices, studies have been remarkably consistent in their identification of the platelet count as

Table 6-1. Cytokines, Growth Factors, Peptides, Proteases, and other Components Important in Hepatic Fibrogenesis Cytokines Transforming growth factor-b Transforming growth factor-a Interleukin-1 Interleukin-4 *Interleukin-6 Interleukin-10 Interleukin-13 *Monocyte chemotactic factor Growth factors Transforming growth factor-b Transforming growth factor-a *Insulin-like growth factor (I, II) *Platelet-derived growth factor *Fibroblast growth factor Vascular endothelial growth factor Hepatocyte growth factor Connective tissue growth factor Peptides Endothelin-1 Norepinephrine Angiotensin II

BEDSIDE DIAGNOSTIC TOOLS
Clinical signs and symptoms of liver disease are frequently highlighted in assessing patients with liver disease, but these are of little value in detecting early, precirrhotic stages of liver fibrosis. In contrast, a number of clinical features can be utilized to assess whether cirrhosis with portal hypertension may be present. Signs of cirrhosis include spider angiomata, distension of abdominal wall veins, ascites, splenomegaly, muscle wasting, Dupuytren’s contractures (especially with ethanol-associated cirrhosis), gynecomastia and testicular atrophy in males, and palmar erythema. However, it is important to emphasize that even in patients with histologic cirrhosis, and in those with portal hypertension, these physical signs may not be present.

NON-INVASIVE MARKERS OF FIBROSIS Blood-Based Markers – Overview
A wide variety of blood, serum, or plasma ‘markers’ for fibrosis have been proposed. There are several categories of marker or test. For example, some detect abnormalities in serum chemistries. Included in these types of test are aspartate aminotransferase (AST), alanine aminotransferase (ALT), g-glutamyl transferase (GGT), bilirubin, albumin, and a2-macroglobulin, among others. Moreover, some of these individual tests have been incorporated into simple and/or complex mathematical models or algorithms (see below).

Proteases and their inhibitors Matrix-metalloproteinase-1 (interstitial collagenase) Matrix-metalloproteinase-2 (gelatinase A) Matrix-metalloproteinase-3 (stromelysin-1) Matrix-metalloproteinase-7 (matrilysin) Matrix-metalloproteinase-8 Matrix-metalloproteinase-9 (gelatinase B) Matrix-metalloproteinase-10 (stromelysin-2) Tissue inhibitor of metalloproteinase-1

Miscellaneous Thrombospondin (1,2) Leptin Activin A *Thrombin Osteopontin

Agents may have direct effects on hepatic stellate cells, or indirect effects in the wounding environment. *Compounds whose effect is largely via stimulation of proliferation.

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the best single predictor of esophageal varices. For example, in one study, cirrhotics without splenomegaly on physical examination and with a platelet count >88 000/mm3 had a risk of large esophageal varices of 7.2%, whereas the risk was 28% if the platelet count was less than 88 000/mm3.130 An AST/ALT ratio >1 has been proposed to indicate the presence of cirrhosis.132 In one study of patients with HCV, a ratio >1 had 100% specificity and positive predictive value for distinguishing cirrhotic from non-cirrhotic patients, with a 53.2% sensitivity and 80.7% negative predictive value.133 In addition, the ratio correlated positively with the stage of fibrosis, but not with the grade of activity or other biochemical indices. Of cirrhotic patients, 17% had no clinical or biochemical evidence of chronic liver disease except for an elevated AST/ALT ratio. In another study, the AST/ALT ratio had 81.3% sensitivity and 55.3% specificity in identifying cirrhotic patients who died within 1 year of follow-up.132,134 In a further attempt to develop non-invasive tools for the measurement of liver fibrosis, Forns and coworkers developed a model using data from HCV patients that included age, GGT, cholesterol, and platelet count.135 This model was developed with the intention to differentiate patients with significant fibrosis from those without. The sensitivity for detecting METAVIR F2–F4 fibrosis was 94%, and the presence of significant F2–F4 fibrosis could be excluded with high accuracy (negative predictive value of 96%).135 Likewise, Wai et al.136 constructed a simple model utilizing routine laboratory data (Table 6-2). The authors devised a novel index, termed the AST to platelet ratio index, or APRI, which is the AST level/upper limit of normal (ULN) divided by the platelet count (109/l) multiplied by 100. The sensitivity and specificity for fibrosis of the APRI value depended on the cut-offs used. Using an APRI value of 1.50, the positive and negative predictive values for significant fibrosis (Ishak score = 3) were 91% and 65%, respectively, whereas for cirrhosis and an APRI of 2.00, the positive and negative predictive values were 65% and 95%, respectively. Thus for a hypothetical patient, if the AST was 90 IU/l (and the ULN 45) and platelet count was 100 ¥ 109/l, then the APRI would be 2.00. This means that the patient has essentially a 90% chance of having significant fibrosis, and somewhat less likelihood of having cirrhosis. However, cirrhosis could not be excluded with certainty. Although the APRI is attractive because of its simplicity, it can neither definitively diagnose nor exclude cirrhosis, and it will not identify patients with early fibrosis. Other simple quantitative systems based on routine laboratory values have been developed. One early example was the ‘PGA index’, which combined prothrombin time, GGT and apolipoprotein A1 (Table 6-2); this test was examined in patients with alcoholic cirrhosis.137 The diagnostic accuracy of this index was later improved by the addition of a2-macroglobulin (and hence termed the ‘PGAA index’).138 The test characteristics of many of these indirect assays have been derived from datasets, but have not been validated on independent datasets. More complicated algorithms based on commonly available laboratory tests include the ‘Fibrotest,’ reported by the French MULTIVIRC group.25 This group used mathematical modeling to develop an algorithm including five different markers to predict fibrosis (the markers selected were a2-macroglobulin, haptoglobin, GGT, apolipoprotein A1, and total bilirubin). This index predicted a spe-

Table 6-2. Combined Panels of Blood Markers used to Detect Liver Fibrosis Panel AST/ALT § Forns APRI PGA index Fibrotest Fibrospect *ELF FPI Components AST/ALT Platelets, GGT, cholesterol AST, Platelets Platelets, GGT, apolipoprotein A GGT, haptoglobin, bilirubin, apolipoprotein A, a2-macroglobulin Hyaluronic acid, TIMP-1, a2-macroglobulin ECM proteins AST, cholesterol, HOMA-IR References 132–134 135 136 137–138 25, 139–142 146 147

ALT, alanine aminotransferase; AST, aspartate aminotransferase; GGT, g-glutamyl transpeptidase; APRI, AST to platelet ratio index; TIMP-1, tissue inhibitor of metalloproteinase 1; ECM, extracellular matrix; ELF, European liver fibrosis; FPI, fibrosis probability index; HOMA-IR, insulin resistance by the homeostasis model assessment. § Also includes age in the panel. *Components tested include collagen IV, collagen VI, amino terminal propeptide of type III collagen (PIIINP), matrix metalloproteinase 2 (MMP-2), matrix metalloproteinase 9 (MMP-9), tissue inhibitor of matrix metalloproteinase 1 (TIMP-1), tenascin, laminin, and hyaluronic acid (HA).

cific biopsy category in 46% of patients139 and has been validated in a number of hepatitis C patient cohorts, having been found to have an area under the receiver operator characteristic (AUROC) curve of 0.73–0.87.140 The addition of ALT to the marker panel allows for prediction of METAVIR necroinflammatory activity.140 The panel has also been examined in other liver disease cohorts.141,142 Limitations of this panel in fibrosis include false positive results due to increases in bilirubin or decreases in haptoglobin, for example from hemolysis secondary to ribavirin therapy. Likewise, false positive results may also occur in situations where there is hyperbilirubinemia, such as Gilbert’s disease and cholestasis. Acute inflammation may also affect the results of the test owing to changes in a2macroglobulin or increases in haptoglobin. Currently, it is unclear whether the ‘fibrotest’ assay meets sufficiently rigorous criteria, given a predictive value of only 46%, for routine clinical use.

Tests Using Extracellular Matrix/Fibrosis Markers
Analyses of serum markers of extracellular matrix/fibrosis include many proteins important in fibrogenesis, ECM constituents (i.e. fibronectin, collagen I, collagen IV, collagen VI, amino terminal propeptide of type III collagen (PIIINP), tenascin, and hyaluronic acid, metalloproteinases (including many of those listed in Table 6-1), inhibitors of matrix metalloproteinases (i.e. TIMP-1, TIMP-2), and other proteins, peptides, and cytokines, as highlighted in Table 6-1. Although many tests have been studied individually, they are generally not sensitive for detection of fibrosis143,144 (see 145 for review).

Tests Using Combinations of Extracellular Matrix and/or Routine Markers
A combination test including hyaluronic acid, TIMP1, and a2macroglobulin was examined in a cohort of 294 patients with HCV infection and subsequently validated in a second cohort of 402 patients146 (‘Fibrospect’, Table 6-2). This had a combined AUROC of 0.831 for METAVIR F2–F4 fibrosis. The positive and negative predictive values were 74.3% and 75.8%, respectively, with an accuracy of 75%. This three-marker panel thus may help differentiate

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patients with HCV infection with moderate/severe fibrosis from those with no/mild fibrosis, although it was not possible to differentiate specific stages accurately. Another combination test was developed by the European Liver Fibrosis (ELF) Study Group.147 This group examined collagen IV, collagen VI, PIIINP , matrix metalloproteinase 2 (MMP-2), matrix metalloproteinase 9 (MMP-9), TIMP-1, tenascin, laminin, and hyaluronic acid (HA). The study was unique in that it examined patients with a wide variety of liver diseases, including those with chronic hepatitis C virus infection (n = 496), alcoholic liver disease (n = 64), non-alcoholic fatty liver disease (n = 61), chronic hepatitis B virus infection (n = 61), primary biliary cirrhosis or primary sclerosing cholangitis (n = 53), recurrent liver disease post orthotopic liver transplantation (n = 48), autoimmune hepatitis (n = 45), hemochromatosis (n = 32), cryptogenic cirrhosis (n = 19), both hepatitis B and C (n = 4), and other or no known diagnosis (n = 138); the cohort also had a wide distribution of fibrosis stages (Scheuer fibrosis stages were as follows: stage 0 = 24.6%; stage 1 = 35.5%; stage 2 = 13.4%; stage 3 = 14.9%; and stage 4 = 11.8%). An algorithm was developed that detected the upper third of fibrosis groups (Scheuer stages 2, 3, and 4) with a sensitivity of 90% and accurately detected the absence of fibrosis (Scheuer stages 0, 1), with a negative predictive value for this level of fibrosis of 92%. The AUC of a receiver operating characteristic (ROC) plot was 0.804. Interestingly, the addition of clinical chemistry tests including liver function tests, or hematological indices including platelet count and prothrombin time, did not improve test performance. The test appeared to be best in patients with hepatitis C, non-alcoholic fatty liver disease and alcoholic liver disease. The inclusion of patients with multiple etiologies of liver disease, although appealing, has the potential to limit the accuracy of these and other panels, as the characteristics of specific assays may be disease specific. Another model, including AST, cholesterol, and insulin resistance (as well as age and an estimate of past alcohol intake) in patients with HCV147a found that the sensitivity for detection of advanced fibrosis depended on the index value used. At a low probability index, the sensitivity for predicting significant fibrosis was high, but specificity was low, while at a high probability index, sensitivity for significant fibrosis was low, but specificity was high.

this study, patients with advanced fibrosis had elevated levels of a2macroglobulin, haptoglobin, and albumin, but apolipoprotein AI, apolipoprotein A-IV, complement C4, and serum retinol-binding protein were reduced. Another approach has included measurement of labeled Nglycans found in serum.151 The technique exploits the ability to analyze the desialylated total serum N-glycome on a DNA analyzer. The authors focused on cirrhosis (primarily ethanol induced), demonstrating unique patterns of serum N-glycans in those with cirrhosis compared to those with chronic liver disease alone. It was postulated that in cirrhotic livers characteristic N-glycans with a bisecting GlcNAc residue were prominent. In normal liver, the enzyme responsible for this modification, N-acetylglucosaminyl transferase III (GnT-III), is found only in non-parenchymal cells, but in regenerating liver (two-thirds partial hepatectomy) this enzyme is produced in hepatocytes. Thus, GnT-III expression is presumably a manifestation of hepatocellular regeneration, reflected by regenerative nodules. This approach was most sensitive for the detection of cirrhosis and was also able to exclude cirrhosis with great accuracy. When combined with the commercially available Fibrotest this test had 100% specificity and 75% sensitivity for diagnosing compensated cirrhosis.151

Summary of Blood-based Markers
A key advantage of serum markers to detect fibrosis is their noninvasiveness. Additionally, it has been argued that serum markers overcome sampling problems associated with liver biopsy. However, these approaches have several drawbacks. First, most of the studies examining serum markers have been performed in cohorts of patients that have been biased toward advanced fibrosis/cirrhosis. A further problem is that the currently proposed serum marker algorithms use dichotomous rather than continuous scales. The dichotomous nature of these variables would be less problematic if there were clear clinical associations, for example if prognosis or treatment response were highly linked to stage 0–1 versus stages 2–4. In the absence of clinical correlates between dichotomous variables and outcomes, it remains important to diagnose the different stages of fibrosis accurately (0–4). Unfortunately, current tests and algorithms are unable to do this, and perhaps most importantly, the tests do not differentiate between intermediate levels of fibrosis. Thus, although assessments of fibrosis with approaches that use serum markers have great appeal, and indeed, in some areas the tests have begun to replace liver biopsy. Further investigation is required to optimize these tests.

Proteomics
With the recent explosion in proteomics, proteomic approaches have attempted to identify unique protein fingerprints in patients with liver disease. Various platforms are available, including those that measure protein expression, protein–protein interactions, or even enzymatic activity. The majority of approaches have used highthroughput technologies to identify novel protein expression patterns. For example, a recent study in 46 patients with chronic hepatitis B identified 30 proteomic features predictive of significant fibrosis (Ishak stage = 3) and cirrhosis. The AUROC for this analysis was 0.906 and 0.921, for advanced fibrosis and cirrhosis, respectively.148 Another study in 193 patients with chronic hepatitis C identified eight peaks that differentiated METAVIR fibrosis stages with an AUROC of 0.88; this was compared to an AUROC 0.81 for the Fibrotest.149 Another report in patients with HCV fibrosis identified several serum proteins to be differentially regulated.150 In

Imaging Tests
A wide variety of radiographic tests have been used to image patients with fibrosis/cirrhosis. Included in this group are ultrasound, CT, and MRI. In general, these tests are capable of detecting evidence of portal hypertension, thus they have the ability to detect advanced disease. As currently used in clinical practice, however, they are insensitive for the detection of moderate degrees of fibrosis. Transient elastography, which uses pulse-echo ultrasound acquisitions to measure liver stiffness and predict fibrosis stage, has gained interest as a method to quantify fibrosis as it appears that liver ‘stiffness’ may accompany the fibrogenic response.152 In a prospective multicenter study of 327 chronic HCV patients, the AUROCs for

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METAVIR stage F2–F4 and cirrhosis were 0.79 and 0.97, respectively.153 In a separate study of 183 chronic HCV patients, transient elastography compared favorably with the Fibrotest and APRI (AUROC for F2–F4 = 0.83, 0.85 and 0.78, for transient transient elastography, Fibrotest and APRI, respectively).154 When transient elastography was combined with the Fibrotest, the predictive value for fibrosis stage F2–F4 was improved, with an AUROC of 0.88.154 Transient elastography (Fibroscan) reportedly offers good reproducibility with low inter- and intraobserver variability. The procedure is performed by obtaining multiple validated measurements in each patient, further reducing the potential for sampling errors. The depth of measurement from the skin surface is between 25 and 65 mm, raising the possibility that this technique may be difficult to use in obese patients or those with ascites. However, newer probes are being developed for obese patients, and further investigation is expected. Finally, it would theoretically be desirable to utilize advances in the molecular understanding of liver fibrosis to image the liver. For example, the number of activated stellate cells, which reflect fibrogenic activity, might be identified by tagging them with cell-specific markers.155 Alternatively, matrix or matrix turnover could be labeled using molecular tools. Although such approaches are appealing, they remain experimental at present.

in the other. Finally, in 10% of subjects, stage 0–2 disease was identified in one lobe and stage 3–4 fibrosis was found in the other. Similar variability was reported in another study in patients with fatty liver disease.161 There are several other limitations of liver biopsy. Quantification of fibrosis in biopsies is subject to significant interobserver variation. In chronic hepatitis C, for example, standardized grading systems, including Knodell, METAVIR, Scheuer or Ishak, are concordant in only 70–80% of samples. Specimen quality is very important, with smaller samples leading to an underestimation of disease severity.162 A recent study created digitized virtual image biopsy specimens of varying length from large liver sections, and revealed that 75% of 25-mm biopsy specimens were correctly classified using the METAVIR staging system, compared to only 65% for biopsies 15 mm long.163 Interestingly, a recent study noted that the experience of the pathologist may have more influence on interobserver agreement than specimen length.164 Another major problem with using liver biopsy or serum markers to quantify fibrosis is that all of the currently utilized grading systems use a simple linear numerical scoring approach, implying that they represent linear changes in fibrosis content. Such an inference is highly inaccurate, as METAVIR stage 4 fibrosis does not represent twice as much fibrosis as stage 2, but rather a 5–20-fold difference.

Tests of Liver Function
A variety of bona fide liver function tests have been used to assess liver fibrosis and cirrhosis. Such tests generally measure advanced disease and several depend on perfusion, such as indocyanine green, sorbitol and galactose clearance tests, or tests such as the 13C– galactose breath test and the 13C–aminopyrine breath test that depend on the functional capacity of the liver.156–158 Another test, the MEGX test, which measures monoethylglycinexylidide (MEGX) formation after the administration of lidocaine, depends upon the activity hepatic cytochrome P450 3A4 isoenzyme (which catalyzes oxidative N-de-ethylation of lidocaine to MEGX.159 The MEGX test has a sensitivity and specificity in the 80% range for distinguishing chronic hepatitis from cirrhosis in comparison to standard liver tests.159 Unfortunately, although the MEGX test and other function tests may predict prognosis in cirrhotic patients, they are insensitive for quantifying fibrosis in patients with less advanced disease.156–158

TREATMENT OF FIBROSIS
Specific therapy for the treatment of liver fibrosis is attractive because the scarring response leads to many if not all of the complications of chronic liver disease, in particular impaired synthetic function, liver failure, and perhaps hepatocellular cancer. Fibrosis, particularly in its advanced stages, may also contribute to portal hypertension, by preventing blood flow through fibrotic nodules. Although attempts have been made previously to treat specifically the ‘fibrosis’ component of liver disease, these approaches have generally been unsuccessful. Thus, there remains a major unmet need for novel and effective antifibrotic therapy. Advances in elucidating the pathogenesis of fibrosis have led to renewed efforts in this area. Additionally, data indicating that fibrosis is reversible have helped fuel this effort (Figure 6.5). Although fibrosis is commonly accepted as the precursor to cirrhosis, it is not clear that mortality risk increases directly with the stage of fibrosis, until the patient actually becomes cirrhotic. Even with established cirrhosis, in a cohort of patients with chronic HCV infection Fattovich and colleagues demonstrated that complications of cirrhosis developed over prolonged periods, and only when complications occurred was mortality increased.7 The most effective ‘antifibrotic’ therapies are currently those that treat or remove the underlying stimulus to fibrogenesis (Table 6-3). In addition, preclinical and human clinical studies have highlighted a number of therapies that may abrogate fibrogenesis without affecting the underlying disease, by targeting specific steps in the fibrogenic response. Anti-inflammatory therapies have been based on the knowledge that inflammation drives the fibrogenic cascade. Other treatments have attempted to inhibit cellular injury or focused on stellate cell activation, whereas others have targeted collagen syn-

Liver Biopsy
Percutaneous liver biopsy has traditionally been considered to be the gold standard test to measure fibrosis. Although there is great experience with liver biopsy, this procedure is time consuming, inconvenient, uncomfortable, invasive, and makes both patients and physicians anxious. Further, liver biopsy can be associated with substantial sampling-error (see Chapter 12 for further details about liver biopsy). In a recent study in which 124 patients with chronic HCV infection underwent laparoscopy-guided biopsy of each the right and left hepatic lobes, 33.1% had a difference of at least one histologic stage (modified Scheuer system) between the two lobes.160 Furthermore, in 18 study subjects a stage consistent with cirrhosis was found in one lobe, whereas stage 3 fibrosis was reported

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Inset in A

B

Figure 6-5. Reversal of fibrosis. An example of reversal of advanced fibrosis (cirrhosis in this situation) is depicted. A liver biopsy prior to lamivudine treatment is shown (upper panel and left panel). After treatment with lamivudine, liver biopsy was repeated and reveals almost complete dissolution of fibrosis. Data similar to these have been published in autoimmune liver disease, hepatitis C, alcoholic hepatitis, hepatitis B, and others. (Reprinted with permission, Wanless, et al: Arch Pathol Lab Med 2001;124:1599–1607.)

A

C

Table 6-3. Approaches to Treat Liver Fibrosis Approach Remove injurious agent Anti-inflammatory agents Antioxidants Cytoprotective agents Inhibit stellate cell activation Inhibit stellate cell activation phenotypes (fibrogenesis) Example Eradication of HBV Corticosteroids in AIH PPC in alcoholic hepatitis Ursodeoxycholic acid Interferon-g Colchicine

Table 6-4. Diseases and Therapies in which there is Strong Evidence that Treatment Reduces Liver Fibrosis Disease Hepatitis B Hepatitis C Bile duct obstruction Autoimmune hepatitis Hemochromatosis Alcoholic hepatitis Therapy Lamivudine Interferon-a* Surgical decompression Corticosteroids Iron depletion Corticosteroids References 33–35,129 42 43 44 165,166 168,169

Note: some approaches have not been demonstrated to be successful. AIH, autoimmune hepatitis; PPC, polyenylphosphatidylcholine.

*or PEG-interferon-a, with or without ribavirin. MTX, methotrexate; PPAR, peroxisomal proliferator-activated receptor.

thesis and matrix deposition. The following section highlights human studies in these areas.

THERAPIES DIRECTED AT THE UNDERLYING DISEASE
In many forms of liver disease treatment of the underlying inciting lesion leads to an improvement in fibrosis (Table 6-4). For example, eradication or inhibition of HBV33,34,129 or HCV replication42 leads to reversion of fibrosis, even in patients with histological cirrhosis. Fibrosis reverts in patients with hemochromatosis during iron depletion,165,166 after corticosteroid therapy in autoimmune hepatitis,165,166 and in patients with secondary biliary cirrhosis after decompression of bile duct obstruction.43 In a preliminary report in patients with non-alcoholic steatohepatitis (NASH) treated with the peroxisomal proliferator active receptor (PPAR)-g agonist rosiglitazone both steatosis and fibrosis were reduced.167

atitis who respond to medical treatment (prednisone or equivalent) advanced fibrosis and cirrhosis are reversible.44 Fibrosis may improve in patients with alcoholic liver disease who respond to corticosteroids.168,169 Thus, corticosteroids appear to have antifibrotic effects in patients with certain liver disorders.

Interleukin-10 (IL-10)
Interleukin (IL)-10 has both anti-inflammatory and immunosuppressive effects. IL-10 has been shown to reduce the production of proinflammatory cytokines, such as TNF-a, IL-1, interferon-g, and IL-2 from T cells. These cytokines belong to the Th1 family. Endogenous IL-10 reduces the intrahepatic inflammatory response, shifts the cytokine milieu towards a Th2 predominance, and reduces fibrosis in several in vivo models of liver injury.170 It was hypothesized that in vivo administration of IL-10 in patients with hepatitis C virus infection may have an anti-inflammatory and hence an antifibrotic effect.109 Therefore, 30 patients with advanced HCV-mediated fibrosis who had failed standard interferon-a-based antiviral therapy were enrolled in a 12-month treatment trial of IL-10 given daily or thrice weekly subcutaneously. In 13 of 28 of these patients the hepatic inflammation score decreased by at least two points (Ishak score) and 11 of 28 had a reduction in fibrosis score (mean change from 5.0 ± 0.2 to 4.5 ± 0.3, p < 0.05). However, serum HCV RNA levels increased during therapy (mean HCV RNA at day 0: 12.3 ± 3.0 mEq/ml; and at 12 months: 38 mEq/ml; p < 0.05). Changes in liver histology and HCV RNA levels were accompanied by an apparent shift in toward a Th2-predominant lymphocyte phenotype, as had been originally hypothesized. Long-term therapy with

ANTI-INFLAMMATORY COMPOUNDS
Many liver diseases, such as HCV disease, have an important inflammatory component. Inflammation in these disorders typically drives stellate cell activation and fibrogenesis, and it is these diseases in particular that have been studied in order to evaluate the efficacy of anti-inflammatory drugs.

Corticosteroids
Classic examples of the benefits of steroids include autoimmune hepatitis and alcoholic hepatitis. In patients with autoimmune hep-

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IL-10 decreased hepatic inflammatory activity and appeared to have an inhibitory overall effect on fibrosis. Thus, although IL-10 appears to reduce inflammation and fibrosis, it has not been pursued as an antifibrotic compound because of putative detrimental virologic effects.

Miscellaneous Anti-inflammatory Drugs
A number of other anti-inflammatory approaches have gained attention as therapies for fibrosis. Because TNF-a drives inflammation in many diseases, and because TNF-a is up-regulated in liver diseases (such as alcoholic hepatitis), an anti-TNF-a compound should theoretically reduce inflammation and hence the stimulus for fibrosis.171–174 Preliminary analyses from a study of anti-TNF-a therapy suggest an improvement in inflammation which presumably precedes fibrosis in patients with alcoholic hepatitis,173 although there was little effect of anti-TNF-a on hepatic fibrosis. Such data, in addition to the favorable effects of the TNF-a inhibitor pentoxifylline on mortality, provide the rationale for future study in patients with alcoholic hepatitis. These approaches and others that broadly inhibit inflammation must be considered cautiously because of the concern about disruption of the immune system, with increased risk of infection. Penicillamine is a heavy metal chelating compound that has been proposed to have anti-inflammatory and thus antifibrogenic effects.175 However, this compound had no effect on fibrogenesis in patients with primary biliary cirrhosis.176,177 Metrothrexate is thought to have anti-inflammatory properties, but interestingly has typically been considered to be profibrogenic in the liver for patients receiving methotrexate for treatment of rheumatologic diseases178 (although it is noteworthy that the risk of fibrosis progression may be less prominent than typically believed178,179). Metrothrexate has been studied in patients with primary biliary cirrhosis. Although some investigators have reported highly favorable effects in this disease, including improvement of the disease and reversion of fibrosis,180 the majority of the data on methotrexate have either been negative181,182 or show that the drug’s effects have been marginal, either alone181 or in combination with colchicine.183 It is important to emphasize that if methotrexate is used to treat patients with primary biliary cirrhosis, this must be undertaken by an experienced hepatologist.

A VA cooperative multicenter clinical trial examined the effect of polyenylphosphatidylcholine in 789 patients with alcoholic hepatitis who had a very high daily average alcohol intake (16 drinks/day).185 Subjects were randomized to either polyenylphosphatidylcholine or placebo for 2 years. The long period of treatment is noteworthy as it is likely that long periods of treatment will be required to effect changes in the liver fibrosis. Many subjects substantially reduced their ethanol consumption during the trial, which probably accounted for improvement in fibrosis in the control group, making it difficult to demonstrate an improvement in fibrosis in the polyenylphosphatidylcholine group. Thus, overall, polyenylphosphatidylcholine failed to lead to significant improvement in fibrosis.

Silymarin
Silymarin is derived from the milk thistle Silybum marianum. This extract has been shown to reduce lipid peroxidation and inhibit fibrogenesis in rodent animal models,186,187 as well as in baboons.188 It has been tested in several carefully performed human clinical trials, although fibrosis was not used as an endpoint. The compound has been found to be safe, but reportedly has mixed effects.189,190 In one study examining silymarin in alcoholics189 mortality was reduced; in addition, patients with early stages of cirrhosis also appeared to benefit. However, in another study in alcoholics no survival benefit could be identified.190 In both of these trials, silymarin appeared to be safe. Thus, although the agent is safe and is commonly used by patients with fibrosing liver disease, there is limited evidence of its efficacy.

Other Antioxidants
Antioxidants such as vitamin E have been examined in animal models191 as well as in humans.192–195 A vitamin E precursor, D-atocopherol (1200 IU/day for 8 weeks), was studied in six patients with hepatitis C virus infection who failed to respond to interferon therapy,192 and was found to inhibit stellate cell activation but did not affect fibrosis. A randomized controlled trial examined vitamin E in patients with mild to moderate alcoholic hepatitis and found that vitamin E reduced serum hyaluronic acid, but did not lead to a change in type III collagen.194 Combined antioxidant therapy, including vitamin E, had no effect on outcome in patients with severe alcoholic hepatitis, although fibrosis was not specifically addressed.195 Malotilate is another potential cytoprotective agent, perhaps acting via inhibition of cytochrome P450 2E1; in addition, this compound may have anti-inflammatory properties. In patients with primary biliary cirrhosis, although it was found to diminish plasma cell and lymphocytic infiltrate and piecemeal necrosis, it had no significant effect on fibrogenesis.196 Another agent used to antagonize oxidative stress is S-adenosylmethionine; this compound is important in the synthesis of the antioxidant glutathione. The enzyme (methionine adenosyltransferase) responsible for its synthesis is reduced in the injured liver;197 thus it has been hypothesized that if S-adenosylmethionine were replaced, then injury and fibrogenesis might be slowed. S-adenosylmethionine has been tested in a large randomized trial in patients with alcoholic cirrhosis.198 There was an improvement in overall mortality/need for liver transplantation in the treatment arm, espe-

ANTIOXIDANT AGENTS
Oxidative stress is thought to play an important role in injury, stellate cell activation, and the stimulation of extracellular matrix production, as discussed above. Thus, a wide variety of antioxidants have received attention as potential antifibrotics.

Polyenylphosphatidylcholine
Polyenylphosphatidylcholine is a mixture of polyunsaturated phosphatidylcholines, extracted from soybeans. This compound has antioxidant properties and oxidant stress (see above) is thought to be important in the inflammatory and fibrogenic response to injury, particularly in alcoholic liver disease. As oxidative stress leads to lipid peroxidation, and lipid peroxidation is injurious at the level of the cell membrane, phosphatidylcholine has been proposed to be protective against injury to cell membranes, resulting in reduced cellular injury and fibrogenesis.184

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cially in patients with Child’s A/B cirrhosis, although histologic assessment of fibrosis was not specifically assessed.198 Propylthiouracil is an antithyroid drug that reacts with some of the oxidizing species derived from the respiratory burst and may thus be protective in alcoholic liver disease, a disease in which an increase in hepatic oxygen consumption may predispose the liver to ischemic injury. Thus, propylthiouracil has been tested in a number of randomized clinical trials in patients with alcoholic liver disease. Unfortunately, a systematic review and meta-analysis found that propylthiouracil had no benefit in fibrosis, or in any other outcome measured.199

improve fibrogenesis. Additionally, a small series indicated that seven of 10 patients with cystic fibrosis treated with ursodeoxycholic acid had a reduction in liver fibrosis.204 Although these effects are promising, it should be emphasized that the numbers of patients studied has been small. Finally, in a large randomized controlled trial of ursodeoxycholic acid in patients with non-alcoholic steatohepatitis over 2 years, including 107 subjects who had paired biopsy data, there was no improvement in fibrosis.209

MISCELLANEOUS
Anabolic–androgenic steroids such as oxandrolone have been examined in randomized trials including patients with alcoholic liver disease, but have not been found to have significant effects on fibrosis (or other outcomes).210

CYTOPROTECTIVE AGENTS Ursodeoxycholic Acid
Ursodeoxycholic acid binds to hepatocyte membranes, where it presumably stabilizes them and is thus cytoprotective. This cytoprotective action in turn theoretically reduces inflammation and may in turn have a beneficial effect on fibrogenesis.200 Neither experimental data nor human studies indicate a primary antifibrotic effect of ursodeoxycholic acid in the liver, but the compound has been examined extensively.201–209 Ursodeoxycholic acid has been studied in patients with cystic fibrosis, primary biliary injury (primary biliary cirrhosis, primary sclerosing cholangitis and progressive familial intrahepatic cholestasis), and miscellaneous liver diseases. Results with ursodeoxycholic acid in these conditions have been mixed. Both symptomatic and biochemical improvement has been observed in these diseases, in particular the biliary diseases, but data on histologic improvement (and survival) have not been consistent. For example, in a randomized controlled trial in patients with primary biliary cirrhosis, ursodeoxycholic acid led to reduced fibrosis in those with mild disease but had no effect on those with severe disease.202 In another study, survival was improved in patients treated with ursodeoxycholic acid but fibrosis was not improved.206 Further, in a histopathologic study of 54 patients with primary biliary cirrhosis and paired liver biopsies, 4 years of ursodeoxycholic acid therapy was associated with a significant decrease in the prevalence of florid interlobular bile duct lesions, lobular inflammation, and necrosis. Worsening of fibrosis was observed in 14 patients (the majority had only a onegrade progression in fibrosis score), whereas stabilization was noted in the 40 remaining patients.207 A recent combined analysis of the histologic effect of ursodeoxycholic acid on paired liver biopsies including a total of 367 patients (200 ursodeoxycholic acid and 167 placebo) revealed that subpopulations of patients with initial earlystage disease may benefit from therapy.208 Results of meta-analyses examining ursodeoxycholic acid have been mixed, and have largely reported that ursodeoxycholic acid is not effective in primary biliary cirrhosis.205 The aggregate data suggest that ursodeoxycholic acid may impede the progression of fibrosis in primary biliary cirrhosis via effects on (bile duct) inflammation, particularly if given early in the disease course. It should be emphasized that so far as we know, ursodeoxycholic acid is extremely safe. Thus, although it is also expensive, the available data justify its use as an antifibrotic in patients with primary biliary cirrhosis. Ursodeoxycholic acid has also been studied in children with progressive familial intrahepatic cholestasis,203 where it appeared to

Stellate Cell-Specific Compounds Interferon-g
A wealth of data supports the antifibrotic potential of interferon-g. The interferons consist of a family of three major isoforms, a, b and g. These isoforms are unique, not only in structure but also in their biologic actions. Interferons-a and -b bind to the same receptor, whereas interferon-g binds to a different receptor. Interferon-a has more potent antiviral effects than does interferon-g, and interferong has been shown to specifically inhibit extracellular matrix synthesis in isolated cells, including stellate cells.211,212 Interferon-g potently inhibits multiple aspects of stellate cell activation,211,212 and appears to have antifibrotic effects in patients with pulmonary fibrosis.213 Such data have generated considerable enthusiasm about the use of interferon-g in patients with hepatic fibrosis, although there is theoretical concern about its use because it is proinflammatory, and moreover its overexpression in the liver leads to chronic hepatitis.214 None the less, it has now been tested in humans with fibrosing liver disease, and appears to be safe.215 Although this pilot study provides a firm foundation highlighting the potential use of interferon-g in patients, larger studies are needed to prove a therapeutic benefit.

COMPOUNDS THAT INHIBIT FIBROGENESIS Colchicine
Colchicine is a plant alkaloid that inhibits polymerization of microtubules, a process that in turn is believed to be required for collagen secretion. Based on this concept, colchicine has been advanced as an antifibrotic agent. A sizeable body of literature indicates that colchicine has antifibrotic properties in experimental animal models.216 This work has led to a number of human clinical trials.217–220 A wide variety of liver diseases has been studied, including primary biliary cirrhosis, alcoholic cirrhosis, cryptogenic cirrhosis, and miscellaneous other liver diseases. In a double-blind randomized controlled trial examining colchicine in primary biliary cirrhosis improvements were noted in a number of biochemical markers, but the drug failed to reduce fibrosis.217 In an often-cited popularized double-blind randomized controlled trial of colchicine versus placebo in patients with various liver diseases, colchicine led to improved fibrosis as well as a dramatic improvement in survival.218 However, this study has been extended to clinical practice with great caution because of a variety of methodological concerns. First,

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Table 6-5. New Potential Antifibrotic Targets in Humans Agent Anti-TGF-b Anti-PDGF Interferon-g PPAR ligands Comments Blocks stellate cell fibrogenesis Blocks stellate cell proliferation Inhibits multiple features of stellate cell activation ? Stellate cell-specific effects

thus could evolve into effective antifibrotic compounds. Many others have been highlighted (see 5 for review).

SUMMARY AND FUTURE DIRECTIONS FOR ANTIFIBROTIC THERAPY
The explosion of information about the pathogenesis of fibrogenesis has spawned a field dedicated to antifibrotics focused on the activation of hepatic stellate cells. Stellate cell activation is characterized by a number of important features, including enhanced matrix synthesis and a prominent contractile phenotype, processes that each contribute to the dysfunction of the liver typically found in advanced disease. It should be emphasized that the control of activation is multifactorial, and thus several potential therapeutic interventions are possible. A further critical concept is that fibrosis, in particular the ECM component of fibrosis, is dynamic, and the accumulation of fibrosis may be inhibited. It is likely that fibrosis, including even advanced fibrosis, may be reversible under the appropriate conditions. Currently, effective therapy for hepatic fibrogenesis exists for several diseases in which the cause of the underlying disease is removed. In contrast, specific therapy directed only at the fibrotic lesion is not currently available; the most effective therapies will most likely be directed at the stellate cell. Additionally, approaches that regulate matrix remodeling (i.e. by enhancing matrix degradation or inhibiting factors that prevent matrix breakdown) will be attractive. Thus, multiple potential targets have been identified, and it is highly likely that candidates will emerge. The ideal antifibrotic compound will be specific, effective, safe, and inexpensive.

many patients were lost to follow-up, and in addition there was substantial unexplained excess mortality in the control group (unrelated to liver disease). In a recent large VA cooperative multicenter study involving 549 patients comparing colchicine (0.6 mg orally b.i.d.) to placebo in patients with alcoholic liver disease, there was no apparent effect of active treatment on survival. Histologic data that might have provided information on the anti-inflammatory effects of colchicine were not obtained.219 A meta-analysis including 1138 subjects found that colchicine had no effect on hepatic fibrosis or mortality.220 In summary, the data surrounding colchicine suggest that this compound is safe but likely to be ineffective.

FUTURE ANTIFIBROTICS
Given the major effort in understanding the biology of hepatic fibrogenesis, it is not surprising that numerous pathways have been targeted as having therapeutic potential. Many compounds have been studied in experimental models and have been shown to have antifibrotic properties, including several with great potential in human liver disease (Table 6-5). Several important pathways merit discussion. One of the most important examples is the TGF-b pathway, as it plays a central role in the fibrogenic cascade. Several approaches to inhibit the action of TGF-g have been proposed and include the use of molecules such as decorin, the protein core component of proteoglycan, which binds and inactivates TGF-b,221 antibodies directed against TGF-b1, and soluble receptors which typically encode for sequences that bind active TGF-b and prevent it from binding to its cognate receptors. The concept has been well established experimentally; indeed, the effect of inhibition of TGF-b in animal models of liver injury and fibrogenesis has been striking.222,223 A limitation of approaches that target TGF-b is that the cytokine potently inhibits cellular proliferation, and inhibition of its effects in vivo could predispose to malignant transformation. Another critical pathway involves PDGF. PDGF is the most potent stellate cell mitogen known,3,224 and in addition stimulates stellate cell migration.225 A number of approaches have been used to inhibit the effect of PDGF. For example, kinase inhibitors that specifically inhibit PDGF signaling might be useful,226 as could those with more general effects on tyrosine kinase receptors. Additionally, stellate cells express angiotensin and endothelin receptors and their cognate ligands appear to be overproduced in the liver; further, stimulation of stellate cells with their respective ligands leads to stellate cell activation.66 Thus, inhibition of their binding may be clinically beneficial. Among others agents, compounds such as pirfenidone,227 peroxisomal proliferator-activated receptor (PPAR)-g ligands,76,228 and halofuginone229 appear to have direct effects on stellate cells and

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