Liver damage leads to an inflammatory response and to the activation and proliferation of mesenchymal cell populations within the liver which remodel the extracellular matrix as part of an orchestrated wound-healing response. Chronic damage results in a progressive accumulation of scarring proteins (fibrosis) that, with increasing severity, alters tissue structure and function, leading to cirrhosis and liver failure. Efforts to modulate the fibrogenesis process have focused on understanding the biology of the heterogeneous liver fibroblast populations. The fibroblasts are derived from sources within and outwith the liver. Fibroblasts expressing α-smooth muscle actin (myofibroblasts) may be derived from the transdifferentiation of quiescent hepatic stellate cells. Other fibroblasts emerge from the portal tracts within the liver. At least a proportion of these cells in diseased liver originate from the bone marrow. In addition, fibrogenic fibroblasts may also be generated through liver epithelial (hepatocyte and biliary epithelial cell)–mesenchymal transition. Whatever their origin, it is clear that fibrogenic fibroblast activity is sensitive to (and may be active in) the cytokine and chemokine profiles of liver-resident leucocytes such as macrophages. They may also be a component driving the regeneration of tissue. Understanding the complex intercellular interactions regulating liver fibrogenesis is of increasing importance in view of predicted increases in chronic liver disease and the current paucity of effective therapies.

INTRODUCTION

The adult human liver typically weighs approx. 1.5 kg. It is the largest internal organ and plays many pivotal roles in intermediary metabolism, and in the metabolism and clearance of xenobiotics. The liver is responsible for the disposal of bile pigments and for the generation of bile acids that are central to the maintenance of cholesterol homoeostasis and the absorption of dietary lipid from the intestine. The liver also plays an important role in lipoprotein metabolism and cholesterol homoeostasis. It is the site of synthesis for major serum proteins, including albumin, complement and clotting factors, and of catabolism of amino acids and the generation of urea. In the normal state, the liver is maintained at a size which provides substantial overcapacity. It also has a remarkable ability to regenerate in response to functional parenchymal loss and can return to normal size and functional state, even after 70% of the parenchyma is lost.

Chronic liver injury, irrespective of cause, is generally associated with the accumulation of matrix proteins, a process referred to as fibrosis. In parallel with this, there is a continued stimulus for regeneration, leading to further distortion of the hepatic architecture and vascular structures (portal veins, hepatic veins). This results in a transformation to a nodular architecture, so-called cirrhosis (Figure 1). Given the normal functional overcapacity of the liver, patients with cirrhosis can have apparently normal (compensated) liver function for long periods of time, but, with many, there is ultimately decompensation with catastrophic effects on the various strands of intermediary metabolism referred to above. In addition, the altered vasculature leads to the development of portal hypertension. To date, most therapies for chronic liver disease have targeted the aetiological agent: for example, there are now several effective antiviral agents for the treatment of hepatitis B and C. Similarly, there are a number of immunosuppressive agents that can be given for immune-driven processes such as autoimmune hepatitis. Although these agents often have a beneficial effect on the degree of fibrosis, there are currently few agents that are specifically developed to interfere with fibrosis. For some patients with end-stage liver disease, the only available option for treatment is orthotopic transplantation.

Appearance of a cirrhotic liver

Figure 1
Appearance of a cirrhotic liver

Scale is in cm.

Figure 1
Appearance of a cirrhotic liver

Scale is in cm.

The present review will examine our current understanding of fibrosis, the experimental models available for its study and the potential therapeutic options currently being tested.

CLINICAL PERSPECTIVE

Accidental (e.g. adverse reaction to a normally therapeutic drug dose [1]) or deliberate (e.g. paracetamol overdose [2]) poisoning can lead to massive liver necrosis, acute liver failure and death. However, chronic liver damage and its associated progressive fibrosis is, quantitatively, a more significant clinical problem [3]. Table 1 outlines the major causes of chronic liver disease in the human population. Viral infections are currently the major global cause of liver fibrosis. However, alcohol abuse is another important cause of chronic liver disease. Furthermore, over the last 25 years, it has become increasingly apparent that a similar spectrum to that observed with alcohol can be seen in individuals who are obese and/or diabetic. This so-called NAFLD (non-alcoholic fatty liver disease) can lead to end-stage liver injury and cirrhosis. The long-term socioeconomic impact of the ‘epidemic’ of NAFLD and obesity is yet to be established.

Table 1
Major causes of chronic liver disease

dsDNA, double-stranded DNA; HBV, hepatitis B virus; HCV, hepatitis C virus; HDV, hepatitis D virus; HFE, human haemochromatosis; LKM, liver/kidney microsomal; PPARγ, peroxisome-proliferatoractivated receptor γ; ssRNA, single-stranded RNA.

Disease Cause Incidence Diagnosis Treatments Additional comments 
Infectious diseases      
 Hepatitis B dsDNA virus (HBV). Passed through infected body fluids [95Estimates of 350 million people infected worldwide [96ELISAs to detect antibody to viral antigens. PCR may also be used to check for the presence of the virus Vaccination available. Patient may be monitored to see whether virus is cleared. If not, antiviral therapy may be used (interferon-α and nucleoside analogue therapies [97])  
 Hepatitis C ssRNA virus (HCV). Passed through infected body fluids [98Estimated 170 million chronic carriers worldwide [99PCR is used to check for the presence of the virus. ELISAs also available No vaccine available. Patient may be monitored to see whether virus cleared with antiviral therapy (interferon-α and nucleoside analogue therapies [100]) Around a third of patients clear the virus without treatment. A third respond to treatment. The remainder become chronically infected and develop fibrosis and cirrhosis [101
 Hepatitis D ssRNA virus (HDV). Passed through infected body fluids [102A proportion of HBV-infected individuals will also be positive for HDV [103ELISAs to detect antibody to viral antigens. PCR may also be used to check for the presence of the virus HBV vaccination. As for HBV HDV requires co-infection with HBV for it to be replicated [104
Autoimmune diseases      
 Primary biliary cirrhosis Immune-mediated attack of the small intrahepatic bile ducts. Unknown cause; may be triggered by an infection or chemical exposure [1051:700 women (Western populations), disease ten times more common in women than in men [106Presence of AMAs (anti-mitochondrial antibodies) primarily to the E2 subunit of the pyruvate dehydrogenase complex [107No curative treatment; symptoms and potentially disease progression treated with ursodeoxycholic acid [105Therapeutic agents that either replace endogenous more toxic bile acids (ursodeoxycholic acid [108]) and/or increase bile acid metabolism through induction of CYP3A [109] (although this is disputed [110]) and/or inhibit fibrogenesis [38,72]. Potential animal models [111113
 Autoimmune hepatitis Immune-mediated attack of hepatocytes [114Female predominant [114Based on a variety of criteria with exclusion of other causes [115Glucocorticoids. Effective, but in the long term, most progress to cirrhosis Type I characterized by antibodies against smooth muscle (SMA) and/or anti-nuclear antibodies (ANA). Type II characterized by antibodies against CYPs such as CYP2D6 (LKM). Others include soluble liver antigen, a tRNA-associated protein [116
Inherited      
 Hereditary haemachromatosis Homozygous mutation in HFE gene [117]: the body absorbs too much iron which is stored in the liver, leading to toxicity 1:400–1:200 of European populations [118Iron can be measured in liver biopsy specimens for overload. Genetic analysis [119Venisection Other disorders can lead to iron accumulation in the liver 
 Wilson's disease Mutations in a P-type copper-transporting ATPase gene, leads to copper accumulation in the liver [1201:5000–1:30000 [121Measurement of blood levels of copper and caeruloplasmin. Also genetic diagnosis [122Administration of copper-chelating agents (e.g. penicillamine) [123Long–Evans Cinnamon rat model [124
 Cystic fibrosis Homozygous mutation in the CFTR (cystic fibrosis transmembrane conductance regulator) [1251:2500. Approx. 30% suffer liver disease [125Genotyping PCR [126Ursodeoxycholate [127The CTFR gene is expressed in the apical membranes of biliary epithelial cells of the branching hepatic bile ducts [128
Dietary      
 NAFLD and non-alcoholic steatohepatitis (NASH) Fat accumulation (steatosis) in the liver in the absence of alcohol consumption. Obesity with an additional damaging component such as hepatic inflammation is termed NASH Prevalence of up to 30%. Associated with obesity. NAFLD considered benign in the absence of other aetiologies [129Liver biopsy Weight loss and exercise. Drugs used to treat Type 2 diabetes are also considered, such as the biguanide metformin [130] and thiazolidinediones (PPARγ agonists) such as pioglitazone [131NASH probably develops on a background of NAFLD via ‘two-hit’ theory [132
Chemical      
 Alcohol Increased gut permeability to endotoxin leading to a pro-inflammatory liver environment. Elevation of ROS with hypoxia production of acetaldehyde by metabolism (alcohol dehydrogenase/CYP2E1) Commonest cause of cirrhosis in the Western world and one of the ten most common causes of death [133Liver biopsy Abstinence. Drugs designed to ameliorate inflammation in the liver include glucocorticoids, and drugs which reduce TNFα expression or activity [133]. Coffee consumption is associated with reduced liver damage with alcohol consumption [134Steatosis and steatohepatitis are both features of alcoholic liver disease 
Other      
 Gallstones Accumulation of solid material/stones in gall bladder Increases with age: present in up to 20% of 60–69-year-olds [135Imaging techniques Surgery Blockage of gall bladder outlet or bile duct leads to inflammation of the bile ducts (cholangitis) 
 Budd–Chiari syndrome Obstruction of hepatic venous outflow normally due to thrombosis 1:100000 [136Anti-coagulants. Stents to redirect blood flow to heart [137  
 Primary sclerosing cholangitis Progressive loss of bile ducts due to inflammation. Cause unknown 1:100000 per year. More common in males [138Cholangiography [138No curative treatment. Symptoms treated with agents that reduce build up of systemic bile acids, e.g. ursodeoxycholic acid [138Often linked with inflammatory bowel diseases, particularly chronic ulcerative colitis [139
Disease Cause Incidence Diagnosis Treatments Additional comments 
Infectious diseases      
 Hepatitis B dsDNA virus (HBV). Passed through infected body fluids [95Estimates of 350 million people infected worldwide [96ELISAs to detect antibody to viral antigens. PCR may also be used to check for the presence of the virus Vaccination available. Patient may be monitored to see whether virus is cleared. If not, antiviral therapy may be used (interferon-α and nucleoside analogue therapies [97])  
 Hepatitis C ssRNA virus (HCV). Passed through infected body fluids [98Estimated 170 million chronic carriers worldwide [99PCR is used to check for the presence of the virus. ELISAs also available No vaccine available. Patient may be monitored to see whether virus cleared with antiviral therapy (interferon-α and nucleoside analogue therapies [100]) Around a third of patients clear the virus without treatment. A third respond to treatment. The remainder become chronically infected and develop fibrosis and cirrhosis [101
 Hepatitis D ssRNA virus (HDV). Passed through infected body fluids [102A proportion of HBV-infected individuals will also be positive for HDV [103ELISAs to detect antibody to viral antigens. PCR may also be used to check for the presence of the virus HBV vaccination. As for HBV HDV requires co-infection with HBV for it to be replicated [104
Autoimmune diseases      
 Primary biliary cirrhosis Immune-mediated attack of the small intrahepatic bile ducts. Unknown cause; may be triggered by an infection or chemical exposure [1051:700 women (Western populations), disease ten times more common in women than in men [106Presence of AMAs (anti-mitochondrial antibodies) primarily to the E2 subunit of the pyruvate dehydrogenase complex [107No curative treatment; symptoms and potentially disease progression treated with ursodeoxycholic acid [105Therapeutic agents that either replace endogenous more toxic bile acids (ursodeoxycholic acid [108]) and/or increase bile acid metabolism through induction of CYP3A [109] (although this is disputed [110]) and/or inhibit fibrogenesis [38,72]. Potential animal models [111113
 Autoimmune hepatitis Immune-mediated attack of hepatocytes [114Female predominant [114Based on a variety of criteria with exclusion of other causes [115Glucocorticoids. Effective, but in the long term, most progress to cirrhosis Type I characterized by antibodies against smooth muscle (SMA) and/or anti-nuclear antibodies (ANA). Type II characterized by antibodies against CYPs such as CYP2D6 (LKM). Others include soluble liver antigen, a tRNA-associated protein [116
Inherited      
 Hereditary haemachromatosis Homozygous mutation in HFE gene [117]: the body absorbs too much iron which is stored in the liver, leading to toxicity 1:400–1:200 of European populations [118Iron can be measured in liver biopsy specimens for overload. Genetic analysis [119Venisection Other disorders can lead to iron accumulation in the liver 
 Wilson's disease Mutations in a P-type copper-transporting ATPase gene, leads to copper accumulation in the liver [1201:5000–1:30000 [121Measurement of blood levels of copper and caeruloplasmin. Also genetic diagnosis [122Administration of copper-chelating agents (e.g. penicillamine) [123Long–Evans Cinnamon rat model [124
 Cystic fibrosis Homozygous mutation in the CFTR (cystic fibrosis transmembrane conductance regulator) [1251:2500. Approx. 30% suffer liver disease [125Genotyping PCR [126Ursodeoxycholate [127The CTFR gene is expressed in the apical membranes of biliary epithelial cells of the branching hepatic bile ducts [128
Dietary      
 NAFLD and non-alcoholic steatohepatitis (NASH) Fat accumulation (steatosis) in the liver in the absence of alcohol consumption. Obesity with an additional damaging component such as hepatic inflammation is termed NASH Prevalence of up to 30%. Associated with obesity. NAFLD considered benign in the absence of other aetiologies [129Liver biopsy Weight loss and exercise. Drugs used to treat Type 2 diabetes are also considered, such as the biguanide metformin [130] and thiazolidinediones (PPARγ agonists) such as pioglitazone [131NASH probably develops on a background of NAFLD via ‘two-hit’ theory [132
Chemical      
 Alcohol Increased gut permeability to endotoxin leading to a pro-inflammatory liver environment. Elevation of ROS with hypoxia production of acetaldehyde by metabolism (alcohol dehydrogenase/CYP2E1) Commonest cause of cirrhosis in the Western world and one of the ten most common causes of death [133Liver biopsy Abstinence. Drugs designed to ameliorate inflammation in the liver include glucocorticoids, and drugs which reduce TNFα expression or activity [133]. Coffee consumption is associated with reduced liver damage with alcohol consumption [134Steatosis and steatohepatitis are both features of alcoholic liver disease 
Other      
 Gallstones Accumulation of solid material/stones in gall bladder Increases with age: present in up to 20% of 60–69-year-olds [135Imaging techniques Surgery Blockage of gall bladder outlet or bile duct leads to inflammation of the bile ducts (cholangitis) 
 Budd–Chiari syndrome Obstruction of hepatic venous outflow normally due to thrombosis 1:100000 [136Anti-coagulants. Stents to redirect blood flow to heart [137  
 Primary sclerosing cholangitis Progressive loss of bile ducts due to inflammation. Cause unknown 1:100000 per year. More common in males [138Cholangiography [138No curative treatment. Symptoms treated with agents that reduce build up of systemic bile acids, e.g. ursodeoxycholic acid [138Often linked with inflammatory bowel diseases, particularly chronic ulcerative colitis [139

LIVER STRUCTURE

Liver tissue is composed of functional units that contain all the cells of the liver arranged around point(s) of entry (portal tract) and exit (central veins) of blood. There remains a debate about the structure of the functional hepatic unit with several models proposed. In part, this is because there is no impermeable barrier between the units in most species, including humans and rodents. A review of the various proposed functional units is beyond the scope of this review (for more information, see [4]); however, the most favoured in their simplest terms are referred to here as the liver lobule (first proposed by Kiernan) and the liver acinus of Rappaport (Figures 2A and 2B). Zonal expression of many genes fits with a lobular functional structure for the liver. However, the acinus is the unit of choice for histopathologists because it aids in the explanation of many pathological lesions (Figure 2C).

Commonly used liver unit structures and their relationship to liver damage

Figure 2
Commonly used liver unit structures and their relationship to liver damage

(A) Schematic diagrams of the hepatic lobule of Kiernan (upper) and liver acinus of Rappaport (lower). The position of the lobule has been superimposed on to the acinus to aid in their comparison. Note that, in the lobular model, blood from the portal tract (PT) containing a periportal venule (PV) and bile duct (BD) and periportal arteriole (PA) may flow into other adjacent lobules with nutrient and oxygen levels falling as blood flows from the portal tracts to various central veins (CV). In contrast, blood entering the acinus via the portal tract is thought to remain within the acinar unit, with nutrient and oxygen levels falling as the blood flows from zone 1 to 3. Dye-injection studies [263] demonstrated that portal tracts should not be considered as terminal afferent vasculature since vessels extend and meet and terminate in regions termed the nodal point of mall (NPM). (B) Immunohistochemical staining for CYP2E expression in mouse liver. This gene is highly expressed in hepatocytes, but it can clearly be seen that expression is restricted to hepatocytes surrounding the central veins (CV). Scale bars, 100 μm (left) and 50 μm (right). (C) Haematoxylin- and eosin-stained liver sections from a normal and CCl4-treated mouse. It can be seen that damage is restricted primarily to hepatocytes surrounding the central vein (CV). PT, portal tract.

Figure 2
Commonly used liver unit structures and their relationship to liver damage

(A) Schematic diagrams of the hepatic lobule of Kiernan (upper) and liver acinus of Rappaport (lower). The position of the lobule has been superimposed on to the acinus to aid in their comparison. Note that, in the lobular model, blood from the portal tract (PT) containing a periportal venule (PV) and bile duct (BD) and periportal arteriole (PA) may flow into other adjacent lobules with nutrient and oxygen levels falling as blood flows from the portal tracts to various central veins (CV). In contrast, blood entering the acinus via the portal tract is thought to remain within the acinar unit, with nutrient and oxygen levels falling as the blood flows from zone 1 to 3. Dye-injection studies [263] demonstrated that portal tracts should not be considered as terminal afferent vasculature since vessels extend and meet and terminate in regions termed the nodal point of mall (NPM). (B) Immunohistochemical staining for CYP2E expression in mouse liver. This gene is highly expressed in hepatocytes, but it can clearly be seen that expression is restricted to hepatocytes surrounding the central veins (CV). Scale bars, 100 μm (left) and 50 μm (right). (C) Haematoxylin- and eosin-stained liver sections from a normal and CCl4-treated mouse. It can be seen that damage is restricted primarily to hepatocytes surrounding the central vein (CV). PT, portal tract.

The liver receives a dual blood supply: there is a portal circulation which brings partially oxygenated blood from the gut, rich in compounds absorbed following digestion, and, in addition, some 30% of the hepatic blood comes via the hepatic artery. The two blood inputs become mixed at the edge of the portal tracts where the liver sinusoids are found. These, in essence, are the liver's capillaries and are lined by specialized fenestrated endothelial cells, controlling the flow of materials to and from the space between hepatocytes and endothelial cells (space of Dissé) [5]. The space of Dissé contains a low density of extracellular matrix proteins and the hepatic stellate cell. Hepatocytes constitute the major epithelial cell type of the liver and extend along the sinusoids to the centrilobular region (lobule) or zone 3 (acini) as polarized epithelial cells. Liver macrophages (Kupffer cells) reside within the sinusoids. The major cell types of the liver and their functions are summarized in Table 2.

Table 2
Cell types of the liver

GFAP, glial fibrillary acidic protein; vWF, von Willebrand factor

Cell type Functions Useful markers in adult liver tissue 
Hepatocyte Most functions of the liver. Intermediary metabolism Albumin, cytokeratin 8 and 18 [140
Kupffer cell Liver-resident macrophage. Phagocytosis of ingested pathogens and particles ED-1 (orthologue of human CD68; also labels monocytes and peripheral macrophages) and ED-2 (orthologue of human CD163 [141]) 
Endothelial cell Fenestrated endothelial cell lining liver sinusoids CD31 and vWF [142
Hepatic stellate cell Storage of vitamin A, production of myofibroblasts in injury Quiescent phenotype: GFAP, desmin in experimental animals [143]. Myofibroblast phenotype: α smooth muscle actin 
Biliary epithelial cell Line bile ducts Cytokeratin 7 and 19 [144,145
Portal tract fibroblast Integrity of portal tracts; supporting function Vimentin [146
‘Oval cell’ Bi-potential progenitor cell for hepatocytes and biliary epithelial cells α-Fetoprotein [147], cytokeratin 19 [148], cytokeratin 8 [147] and OV-6 [149
Liver-associated lymphocyte (NK (Pit) cell, γδT cell, NKT cell) [150Kill infected or tumorigenic liver cells CD3 [150
Vascular endothelial cell Line blood vessels CD34 and CD31 [151
Lymphatic endothelial cell Line lymphatic vessels Podoplanin [152
Cell type Functions Useful markers in adult liver tissue 
Hepatocyte Most functions of the liver. Intermediary metabolism Albumin, cytokeratin 8 and 18 [140
Kupffer cell Liver-resident macrophage. Phagocytosis of ingested pathogens and particles ED-1 (orthologue of human CD68; also labels monocytes and peripheral macrophages) and ED-2 (orthologue of human CD163 [141]) 
Endothelial cell Fenestrated endothelial cell lining liver sinusoids CD31 and vWF [142
Hepatic stellate cell Storage of vitamin A, production of myofibroblasts in injury Quiescent phenotype: GFAP, desmin in experimental animals [143]. Myofibroblast phenotype: α smooth muscle actin 
Biliary epithelial cell Line bile ducts Cytokeratin 7 and 19 [144,145
Portal tract fibroblast Integrity of portal tracts; supporting function Vimentin [146
‘Oval cell’ Bi-potential progenitor cell for hepatocytes and biliary epithelial cells α-Fetoprotein [147], cytokeratin 19 [148], cytokeratin 8 [147] and OV-6 [149
Liver-associated lymphocyte (NK (Pit) cell, γδT cell, NKT cell) [150Kill infected or tumorigenic liver cells CD3 [150
Vascular endothelial cell Line blood vessels CD34 and CD31 [151
Lymphatic endothelial cell Line lymphatic vessels Podoplanin [152

A striking feature of the liver is the heterogeneous expression of many genes along the sinusoid. A classic example is the expression of CYP (cytochrome P450) genes which are expressed at their highest levels in hepatocytes surrounding central veins [6] (see also Figure 2B). Xenobiotics requiring metabolism by CYP cause predominantly centrilobular/zone 3 damage, despite the fact that periportal/zone 1 hepatocytes are exposed first to ingested xenobiotics. The majority of exposure to toxins is through ingestion of pro-toxins that require enzyme-mediated conversion into a cytotoxic product (many directly cytotoxic substances are obviously avoided). Thus pro-toxin damage is located in the regions where the activating enzyme is expressed. Additionally, oxygen levels are also at their lowest in the centrilobular/zone 3 regions, which may have an impact on the ability of cells to respond to and survive injury. Hepatocytes are therefore more often the cell type that is damaged by hepatotoxins (although they are not intrinsically sensitive and have a number of active defence mechanisms, such as a capacity to maintain high levels of reduced glutathione to defend against oxidative stress [7]).

Enzyme-mediated toxicity has particular relevance to acute mechanisms of xenobiotic hepatotoxicity, but is the cause of only a minor proportion of chronic liver disease cases. Indeed, the major chemically induced cause of liver disease, alcohol, induces changes in gut permeability to bacterial membrane components, leading to a pro-inflammatory hepatic environment as a major part of its mechanisms of action [8,9]. Most experimental models of fibrosis employ pro-toxin/enzyme-mediated mechanisms to cause liver damage. An understanding of how toxins work in experimental models of fibrosis is therefore essential to avoid misinterpretation of data, particularly when the efficacy of a potential anti-fibrogenic agent is being examined.

CURRENT VIEWS OF LIVER FIBROGENESIS

Fibrogenesis is predominantly viewed as a repercussion of the wound-healing response that occurs after persistent liver tissue damage. These events are therefore discussed (Figure 3 and Animation 1 at http://www.BiochemJ.org/bj/411/0001/bj4110001add.htm).

Schematic diagram of the events that occur in the liver in response to acute liver damage around the central veins

Figure 3
Schematic diagram of the events that occur in the liver in response to acute liver damage around the central veins

Top to bottom: sequential events from normal structure, localized cell death, inflammation and regeneration. BD, bile duct; CV, central vein; PA, periportal arteriole; PV, periportal venule. An animated version of this Figure can be seen at http://www.BiochemJ.org/bj/411/0001/bj4110001add.htm

Figure 3
Schematic diagram of the events that occur in the liver in response to acute liver damage around the central veins

Top to bottom: sequential events from normal structure, localized cell death, inflammation and regeneration. BD, bile duct; CV, central vein; PA, periportal arteriole; PV, periportal venule. An animated version of this Figure can be seen at http://www.BiochemJ.org/bj/411/0001/bj4110001add.htm

Liver injury and the inflammatory response

In most cases of acute liver injury, hepatocytes are damaged and undergo both necrosis and apoptosis. Figure 2(C) demonstrates the typical effects observed shortly after treatment with the hepatotoxin CCl4. Hepatocytes located mainly around the central veins are damaged because CCl4 is a pro-toxin that requires metabolism by CYP for toxicity (mainly by CYP2E [10], see Table 4). Cell death is primarily via necrosis, although a proportion of hepatocytes may undergo an apoptotic mechanism of cell death, particularly following chronic exposure [11]. Kupffer cells in damaged areas detect the release of intracellular contents and/or apoptotic cells and release a range of cytokines and chemokines that activate local resident leucocytes and promote the recruitment of circulating leucocytes to the region of damage [12,13]. Sinusoidal endothelial cells play a complementary role in this process through changes in the expression of adhesion proteins that promote the tethering of passing leucocytes, leading to their trans-endothelial migration to the site of injury [14]. Major factors contributing to fibrogenesis include cytokines, of which many are released from Kupffer cells, but some are derived from myofibroblasts themselves (Table 3).

Table 3
The major endogenous factors that regulate liver fibrogenesis

Many other factors have been shown to modulate fibrosis via a direct role in myofibroblasts, including adiponectin [179], angiotensin [180], cannabinoids [181,182], leptin [183] and opioids [184]. FGF, fibroblast growth factor; gp130, glycoprotein 130; HBV, hepatitis B virus; HCV, hepatitis C virus; IFN, inteferon; PDGF, platelet-derived growth factor.

Factor Cell source Effect on fibrosis Experimental evidence 
IFNα Many cell types ↓ PEGylated IFNα is used clinically (see Table 1) to reduce HBV and HCV viraemia, but may also inhibit fibrosis [153,154]. In animals, IFNα inhibited liver fibrosis in a CCl4 model, but not in a bile duct ligation model [155
IFNγ Lymphocytes ↓ IFNγ treatment inhibits hepatic stellate cell activation and fibrogenesis in vitro and in vivo [156,157]. Peripheral blood mononuclear cells from HCV patients with low fibrosis had more IFNγ-producing cells than patients with higher fibrosis scores [158
IL-1β Leucocytes, including Kupffer cells ↑ An IL-1 receptor antagonist inhibits liver fibrosis in rats [159]. IL-1β induces collagen type 1 expression in liver myofibroblasts in vitro [160]. IL-1 induced matrix metalloproteinase expression by quiescent hepatic stellate cells as part of their activation to myofibroblasts [161
IL-6 Leucocytes, including Kupffer cells, and myofibroblasts ↓ Conditional gp130 (IL-6 receptor protein partner) knockout in liver non-parenchymal cells had no effect on CCl4 toxicity, but resulted in increased fibrosis in a chronic model [162]. Note that IL-6 is pivotal in priming hepatocyte proliferation/regeneration [163] and this complicates interpretation of direct IL-6-knockout effects on liver fibrosis. Also, other studies have suggested that IL-6 promotes fibrosis, but, in these studies, there was evidence that IL-6 treatment also gave rise to greater levels of liver damage [164,165
IL-10 Leucocytes, including Kupffer cells ↓ Reduced liver fibrosis in IL-10−/− mice administered CCl4 without significant effects on toxicity [166,167]. IL-10 treatment led to reductions in liver fibrosis in HCV patients (IFN-non-responders) [168
PDGF Megakaryocytes and platelets in blood. Expressed by many cell types, however, including myofibroblasts ↑ PDGF is the most potent mitogen for hepatic stellate cell-derived myofibroblasts (EGF, TGFα and FGF-2 are also mitogens) [169]. PDGF levels increase in liver disease [170,171]. Dominant-negative soluble PDGF receptor β inhibits fibrosis [172
TGFβ1 Expressed by many cell types, including myofibroblasts ↑ TGFβ1 transcriptionally up-regulates collagen 1AI in hepatic stellate cells in vitro [173]. Albumin promoter (hepatocyte) overexpression of TGFβ1 in transgenic mice increases fibrosis in vivo [174]. Soluble TGFβ receptor type II treatment inhibits fibrosis in vivo [175]. Adenovirus encoding antisense TGFβ mRNA inhibits fibrogenesis in vivo [176
TNFα Leucocytes, including Kupffer cells ↑ TNFα receptor 1−/− (but not TNFα receptor 2−/−) had reduced fibrosis in a CCl4in vivo model [177]. Note that TNFα receptor 1 also primes hepatocyte proliferation/regeneration [178
Factor Cell source Effect on fibrosis Experimental evidence 
IFNα Many cell types ↓ PEGylated IFNα is used clinically (see Table 1) to reduce HBV and HCV viraemia, but may also inhibit fibrosis [153,154]. In animals, IFNα inhibited liver fibrosis in a CCl4 model, but not in a bile duct ligation model [155
IFNγ Lymphocytes ↓ IFNγ treatment inhibits hepatic stellate cell activation and fibrogenesis in vitro and in vivo [156,157]. Peripheral blood mononuclear cells from HCV patients with low fibrosis had more IFNγ-producing cells than patients with higher fibrosis scores [158
IL-1β Leucocytes, including Kupffer cells ↑ An IL-1 receptor antagonist inhibits liver fibrosis in rats [159]. IL-1β induces collagen type 1 expression in liver myofibroblasts in vitro [160]. IL-1 induced matrix metalloproteinase expression by quiescent hepatic stellate cells as part of their activation to myofibroblasts [161
IL-6 Leucocytes, including Kupffer cells, and myofibroblasts ↓ Conditional gp130 (IL-6 receptor protein partner) knockout in liver non-parenchymal cells had no effect on CCl4 toxicity, but resulted in increased fibrosis in a chronic model [162]. Note that IL-6 is pivotal in priming hepatocyte proliferation/regeneration [163] and this complicates interpretation of direct IL-6-knockout effects on liver fibrosis. Also, other studies have suggested that IL-6 promotes fibrosis, but, in these studies, there was evidence that IL-6 treatment also gave rise to greater levels of liver damage [164,165
IL-10 Leucocytes, including Kupffer cells ↓ Reduced liver fibrosis in IL-10−/− mice administered CCl4 without significant effects on toxicity [166,167]. IL-10 treatment led to reductions in liver fibrosis in HCV patients (IFN-non-responders) [168
PDGF Megakaryocytes and platelets in blood. Expressed by many cell types, however, including myofibroblasts ↑ PDGF is the most potent mitogen for hepatic stellate cell-derived myofibroblasts (EGF, TGFα and FGF-2 are also mitogens) [169]. PDGF levels increase in liver disease [170,171]. Dominant-negative soluble PDGF receptor β inhibits fibrosis [172
TGFβ1 Expressed by many cell types, including myofibroblasts ↑ TGFβ1 transcriptionally up-regulates collagen 1AI in hepatic stellate cells in vitro [173]. Albumin promoter (hepatocyte) overexpression of TGFβ1 in transgenic mice increases fibrosis in vivo [174]. Soluble TGFβ receptor type II treatment inhibits fibrosis in vivo [175]. Adenovirus encoding antisense TGFβ mRNA inhibits fibrogenesis in vivo [176
TNFα Leucocytes, including Kupffer cells ↑ TNFα receptor 1−/− (but not TNFα receptor 2−/−) had reduced fibrosis in a CCl4in vivo model [177]. Note that TNFα receptor 1 also primes hepatocyte proliferation/regeneration [178

The response to tissue damage and infection is therefore analogous with activation of the innate immune system and the potential activation of the adaptive immune system. In some cases, the latter is followed by a breakdown in tolerance to self-antigens and therefore to the generation of T-cells and antibodies to self-antigens [15,16]. In cases where liver damage is drug-induced, it is probable that the drug and/or a metabolite(s) reacts with a protein(s), which is then seen by the immune system as foreign. Once the drug and neo-antigen are cleared, immune tolerance to unmodified protein is lost, resulting in continued attack of the self-antigen and a chronic autoimmune hepatitis. Typically, antibodies against smooth muscle proteins and/or a nuclear antigen are detected in affected individuals (in type 1 autoimmune hepatitis) or to microsomal xenobiotic-metabolizing enzymes such as CYPs (in type 2 autoimmune hepatitis) [16]. Ticrynafen (tienillic acid) is converted into a metabolite which reacts with the generating enzyme (CYP2C9), against which antibodies are produced [17,18].

The inflammation associated with hepatic injury forms part of a regulated response to cell damage resulting in the removal of necrotic material. The liver, presumably because of its precarious function as a protector from endobiotics and ingested xenobiotics, has evolved the ability to regenerate [19]. The cytokines involved in marshalling the inflammatory response to tissue damage also play an important role in the replacement of hepatocytes. Cytokines, such as TNFα (tumour necrosis factor α) and IL (interleukin)-6, prime remaining hepatocytes to be responsive to growth factors such as HGF (hepatocyte growth factor) and EGF (epidermal growth factor) [19]. The stimulus for liver growth appears to be through a sense of its own functional output. The surgical removal of part of the liver (partial hepatectomy) in which there may be relatively minimal cell death, results in regeneration of the remaining liver to the required size, as if responding to the rise in the levels of a factor normally metabolized by the liver. This factor may be serotonin [20,21], which is primarily synthesized by the gut and would therefore pass directly to the liver via the hepatic portal vein.

Hepatocyte regeneration in most diseased states therefore comes from existing viable hepatocyte mitosis [19]. Previous work suggesting that hepatocytes can be derived from the bone marrow [22,23] has now been shown, at best, to be a rare event (the presence of donor genetic material in recipient hepatocytes is now thought to occur via donor bone-marrow-derived macrophage fusion with recipient hepatocytes [24,25]). Liver-resident leucocytes (e.g. Kupffer cells) are derived from the bone marrow in addition to a proportion of other non-parenchymal liver cells [26,27]. A hepatocyte/bile duct epithelial cell progenitor cell, termed ‘oval cells’ in some species, is present in portal tract regions (at the level of the so-called ‘canal of Hering’), but only gives rise to hepatocytes when existing hepatocytes are prevented from replicating [28,29]. In this respect, the ‘streaming’ hypothesis in which hepatocytes are normally replenished (potentially from an oval cell progenitor) from replication in the periportal region [30], which could replace hepatocytes after acute centrilobular injury, is not now widely accepted [31,32]. Many of the factors which drive the innate immune system to clear away the damage also act in an apparent co-ordinate fashion to prime hepatocyte renewal. A less well understood component of this process is the remodelling of the extracellular matrix that occurs in damaged parenchyma. An additional factor, however, is that myofibroblasts may be essential for effective liver regeneration. Plasminogen (Plg)-deficient mice spontaneously develop liver fibrosis [33]. The neurotrophin receptor p75NTR is expressed in myofibroblasts and appears to function in the transdifferentiation from hepatic stellate cells [34]. Plg−/− p75NTR−/− mice have exacerbated pathology compared with Plg−/− p75NTR+/+ mice due to inhibited hepatocyte proliferation [34].

(Myo)Fibroblast proliferation

Fibroblasts were hitherto considered to be a relatively inert ‘space-filling’ cell type secreting extracellular matrix proteins. However, there is increasing evidence in the liver to suggest that they play a role in tissue regeneration; they are now known to express a range of cytokines and chemokines [3538]. In generic terms, a population of fibroblast-like cells is activated by (or activation is significantly enhanced by) the release of cytokines and ROS (reactive oxygen species) from Kupffer cells and other leucocytes [3941] to proliferate and secrete proteases, extracellular matrix proteins and other factors. In the acute setting and under some chronic conditions in which fibrosis may be reversible, the fibroblast-like cells undergo apoptosis and Kupffer cells release factors which remodel the extracellular matrix through secretion of matrix metalloproteinases [42,43]. The end result is that the liver sinusoidal structure returns to normal, and the current theory is that the process of fibroblast-like cell activation is an essential component of this process, although this remains to be experimentally tested.

If damage to the liver persists, either continuously or through repeated acute episodes within a timeframe such that resolution has not had a chance to occur, fibroblast numbers increase and fibrosis develops (see Animation 2 at http://www.BiochemJ.org/bj/411/0001/bj4110001add.htm). As the liver disease progresses, the scarring becomes more extensive, impeding hepatocyte regeneration, and the function of the organ is compromised. For a period of time, fibrosis remains reversible if the primary cause of liver damage is removed or suppressed [44,45].

Early work on liver fibrosis originally proposed that hepatocytes were responsible for the production of scarring extracellular matrix protein in liver fibrosis [46]. It was subsequently shown that hepatic stellate cells were a major source [47,48]. In a normal liver, the cells are referred to as ‘quiescent’ and function to store much of the body's vitamin A [49]. Hepatocytes esterify vitamin A with fatty acids which are then stored by quiescent hepatic stellate cells [49,50]. In response to liver damage, quiescent hepatic stellate cells lose their vitamin A and transdifferentiate into a myofibroblast phenotype expressing α-smooth muscle actin [5153], an actin isoform restricted to smooth muscle cells.

Heterogeneity of hepatic stellate cell-derived myofibroblasts

Under the appropriate in vitro culture conditions (see below) quiescent hepatic stellate cells undergo a phenotypically similar process of transdifferentiation to myofibroblasts as that which occurs in vivo in response to liver damage [54]. It has recently emerged that quiescent hepatic stellate cells are not the only fibrogenic cell in the liver. Quiescent hepatic stellate cells can be found in the centrilobular and perisinusoidal regions of the liver lobule (Figure 4). Injury to the centrilobular region (e.g. in alcoholic liver disease) results in their activation. However, Ramadori and co-workers first suggested that portal tract fibroblasts rather than stellate cells were predominantly responsible for fibrogenesis in cases where damage was located in the periportal regions of the liver lobule (e.g. cholestasis) [55], a view that is now broadly accepted [5658]. Figure 5 compares the fibrosis that occurs in rats treated with CCl4 (a centrilobular hepatotoxin) and bile duct ligation, which preferentially damages hepatocytes in the periportal region of the lobule. Both injuries result in fibrosis, but extensive α-smooth muscle actin myofibroblast immunostaining is only seen in the former, with cells positive for vimentin in the latter. Thus it is probable that a different population of vimentin-positive hepatic fibroblasts located in the periportal region is responsible for fibrogenesis in cases where periportal damage occurs. However, with more extensive injury, the disease progresses, and populations of cells from other regions probably contribute to fibrogenesis.

Sources of liver myofibroblasts in the liver

Figure 4
Sources of liver myofibroblasts in the liver

BD, bile duct; CV, central vein; NASH, non-alcoholic steatohepatitis; PA, periportal arteriole; PBC, primary biliary cirrhosis; PSC, primary sclerosing cholangitis; PV, periportal vein.

Figure 4
Sources of liver myofibroblasts in the liver

BD, bile duct; CV, central vein; NASH, non-alcoholic steatohepatitis; PA, periportal arteriole; PBC, primary biliary cirrhosis; PSC, primary sclerosing cholangitis; PV, periportal vein.

Different populations of fibroblasts proliferate in the liver depending on the location of the damage

Figure 5
Different populations of fibroblasts proliferate in the liver depending on the location of the damage

Typical views of rat liver sections from control animals or animals after chronic liver injury via repeated CCl4 injection or through ligation of the bile duct (BDL). The stain employed is as indicated. α-SMA, α-smooth muscle actin. Scale bars, 100 μm.

Figure 5
Different populations of fibroblasts proliferate in the liver depending on the location of the damage

Typical views of rat liver sections from control animals or animals after chronic liver injury via repeated CCl4 injection or through ligation of the bile duct (BDL). The stain employed is as indicated. α-SMA, α-smooth muscle actin. Scale bars, 100 μm.

The complexity of establishing liver myofibroblast identity is compounded by the fact that each population of hepatic fibroblasts may be heterogeneous in its expression of fibrotic marker genes (and potentially other genes). In an elegant study from Brenner and co-workers, transgenic mice were generated in which red and green fluorescent protein reporter gene expression were driven by the α-smooth muscle actin and collagen IA1 promoters respectively [59]. Both reporter genes were expressed in culture-activated hepatic stellate cells, but they were not co-expressed in all cells. In bile duct-ligated mice, pericentral and perisinusoidal myofibroblasts were both α-smooth muscle actin- and collagen IA1 marker gene-positive, whereas periportal fibroblasts only expressed the collagen IA1 reporter gene.

Embryological origin of hepatic stellate cells and liver myofibroblasts

Establishing the embryological origin of hepatic stellate cells (i.e. the germ layer from which the cells are derived) is complicated by their apparent heterogeneity as a myofibroblast. To date, it has not been formally determined. Given that adipose, smooth muscle cells and fibroblasts are derived from the mesoderm and that stellate cells are lipid-storing cells with an ability to transdifferentiate into myofibroblasts, it is likely that at least a proportion of both hepatic stellate cells (and myofibroblasts) and liver fibroblasts are mesodermal in embryological origin. However, hepatic stellate cells and liver myofibroblasts express several genes normally associated with neural tissue, such as synaptophysin, nestin and glial fibrillary acidic protein [60]. This supports the possibility that hepatic stellate cells (or a proportion of them) have an ectodermal origin, perhaps from the neural crest given its ability to generate a range of mesenchymal-like cells [61]. However, this has recently been tested in transgenic mice [61a]. A transgenic mouse expressing the Cre recombinase transgene under control of a WNT-1 promoter /enhancer sequence (specific to neural crest cells) was crossed with a transgenic for a fluorescent protein whose expression was dependent on removal of a lox P-flanked stop cassette by Cre recombinase [61a]. Although fluorescent protein was found in all tissues known to be derived from the neural crest, there was no expression in desmin-positive perisinusoidal cells of the liver [61a]. This suggests that hepatic stellate cells are not derived from neural crest cells.

In the adult mouse, recent work suggests that some hepatic stellate cells are derived from the bone marrow [62]. When bone marrow cells expressing GFP (green fluorescent protein) were transplanted into normal mice, hepatic stellate cells in the recipient mice were found to express GFP. In culture, the GFP-positive stellate cells transdifferentiated into α-smooth muscle actin-positive myofibroblasts [62]. In CCl4-treated fibrotic animals, GFP also co-localized with α-smooth muscle actin [62]. This observation has been confirmed in irradiated female mice receiving bone marrow from male donor mice. Myofibroblasts containing a Y chromosome were detected in approx. two-thirds of recipient fibrotic liver myofibroblasts, with little evidence of cell fusion [27]. More recently, the scar-producing cells associated with bile duct ligation (periportal myofibroblasts) have also been shown to originate from the bone marrow in an animal model, but yet remain distinct from hepatic stellate cells in that they are CD45-positive [63]. Examination of human liver sections from non sex-matched recipients of either bone marrow or liver transplants confirmed that bone marrow contributed to myofibroblast populations in patients who subsequently developed liver disease [64].

These data suggest that hepatic stellate cells and liver fibroblasts at least in part originate from a mesenchymal stem cell within the bone marrow in the liver disease state. However, there remain a number of observations which suggest that there are other sources from which fibrogenic cells may be generated. For example, Zeisberg et al. [65] reported that liver fibroblasts are derived from hepatocytes in vivo during fibrosis through an “epithelial-to-mesenchymal transition”, although most of these fibroblasts were not α-smooth muscle actin-positive. Similarly, Robertson et al. [66] have shown biliary epithelial cell “epithelial-to-mesenchymal transition” in a patient with primary biliary cirrhosis, which may account for the bile ductopenia and portal tract fibrosis that occurs with this disease.

There is an increasing body of literature concerning the plasticity of cells from several tissues which have normally been considered mature and terminally differentiated. In this respect, quiescent hepatic stellate cells (specifically the proportion that are CD133-positive) have been proposed as a progenitor cell not only for liver myofibroblasts, but also for hepatocytes and endothelial cells [67]. Mesenchymal stem cells from the adult bone marrow have also been stimulated to form multiple cell types (including hepatocytes) in vitro [68,69]. However, it is possible that the in vitro environment imparts an unusually potent plasticity to many cells that enables them to differentiate in a way that would not occur in vivo (since bone marrow cells do not differentiate into hepatocytes appreciably in vivo [23,24]).

Pivotal role for Kupffer cells in fibrogenesis

Although the status of hepatic myofibroblasts (their number and degree of activation) may directly control fibrosis severity, increasing attention is now been given to the role of other cells in the process. Macrophages within the liver or monocytes/macrophages recruited to the tissue are now known to exert a considerable influence. Their number increases in damaged liver and they are principally located around the regions of damage and fibrosis (Figure 6).

Macrophages and liver fibrosis

Figure 6
Macrophages and liver fibrosis

ED1 (CD68) immunostain for monocytes, macrophages and Kupffer cells in rat liver sections from a control or CCl4-treated rat. Scale bars, 100 μm. Right-hand panel, high-power view. Scale bar, 10 μm. Arrows indicate Kupffer cells.

Figure 6
Macrophages and liver fibrosis

ED1 (CD68) immunostain for monocytes, macrophages and Kupffer cells in rat liver sections from a control or CCl4-treated rat. Scale bars, 100 μm. Right-hand panel, high-power view. Scale bar, 10 μm. Arrows indicate Kupffer cells.

Thurman's group showed that inhibition of Kupffer cell function (using gadolinium chloride), reduced fibrosis in a CCl4 model of liver fibrosis [70]. More recently, Duffield et al. [71], using a transgenic mouse model in which CD11b-positive cells can be conditionally stimulated to undergo cell death, have shown that liver-resident monocytes and macrophages not only promote fibrogenesis, but also are instrumental in the removal of fibrosis during any recovery phase.

The promotion of fibrogenesis by Kupffer cells is associated with the release of a range of pro-fibrogenic cytokines and ROS as part of their inflammatory response to liver damage. It is notable that the presence of Kupffer cells (normally present in low numbers in most isolations) in hepatic stellate cell cultures promotes a myofibroblast phenotype closely similar to the cells in vivo [54], but macrophages, in response to the milieu of cytokines and other factors, respond by releasing different mixtures of cytokines. It may be appropriate to suggest that macrophages differentiate into a variety of states. With regard to recovery from fibrosis, macrophages may differentiate into an anti-inflammatory phenotype and/or secrete proteinases that promote the degradation of scarring extracellular matrix proteins.

RESEARCH MODELS OF LIVER FIBROSIS

In vivo models of liver fibrosis

In view of the complex intercellular communication between cell types within the liver, and that extrahepatic cells may contribute to fibrogenesis, animal models remain an important experimental tool. In studies where the aim is to test the efficacy of a potential anti-fibrogenic agent, it is generally considered that drugs should be tested in at least two animal models (where the region of lobular damage is different so that effects on hepatic myofibroblasts derived from different regions of the lobule can be determined). Table 4 summarizes animal models of liver fibrosis most commonly employed. A vital issue with regard to the use of these models is that their use should be carefully matched to the mode of action of potential anti-fibrogenic agents. It is clear that treating the primary cause of liver damage in the human population would be the mode of therapeutic choice when available. The use of a potential anti-fibrogenic agent would be indicated when the primary cause of liver damage cannot be treated and/or fibrosis has progressed to cirrhosis. Accordingly, it is essential to ensure that the potential anti-fibrogenic agent does not modulate the hepatotoxicity of the experimental agent used to generate liver damage (i.e. that a potential anti-fibrogenic agent was not a CYP2E inhibitor if using the CCl4 model of liver fibrosis [72]) when using an animal model. Reductions in fibrosis could therefore be associated with reduced levels of liver damage, and not due to action of an anti-fibrogenic agent. Commonly used markers of liver fibrosis are given in Table 5.

Table 4
In vivo models of liver fibrosis

In general, rats generate a more robust fibrosis and/or tolerate liver fibrosis more readily than mice. However, the utility of transgenic mice in biological research and widespread use and availability of mouse transgenics often results in the use of mice in studies. ALP, alkaline phosphatase; ALT, alanine aminotransferase; CV, central vein; FMO, flavin-containing mono-oxygenase; i.p., intraperitoneally; i.v., intravenously; LPS, lipopolysaccharide; n/a, not applicable; NASH, non-alcoholic steatohepatitis.

Hepatotoxin or causativeagent (typical dose) Model species System of activation Acute mechanism of damage Histological lobular effects Fibrosis Further comments 
CCl4 (1 ml/kg of body weight injected i.p. twice weekly from 4 to 12 weeks) [185Rat, mouse CYP, primarily CYP2E1 [186Lipid peroxidation [187,188Centrilobular hepatocyte death [188Fibrosis emerging from the CVs eventually leading to central–central bridging fibrosis. Most commonly employed means to generate liver fibrosis in animal models. Primarily α-smooth muscle actin-positive myofibroblasts 
Thioacetamide (300 mg/l of drinking water or 200 mg/kg of body weight injected i.p. two or three times per week for up to 12 weeks) [189Rat, mouse CYP, primarily CYP2E1 [190]. Also FMO [191Generation of reactive thioacetamide sulfoxide, leading to non-specific covalent binding of cell constituents [192Centrilobular hepatocyte death [189Fibrosis emerging from the CVs eventually leading to central–central bridging fibrosis Primarily α-smooth muscle actin-positive myofibroblasts 
Dimethylnitrosamine. Various protocols: 10 mg/kg of body weight injected i.p. for 3 consecutive days/week for up to 2–4 weeks; single 50 mg/kg of body weight injected i.p. Endpoints up to 10 months [193Rat (mouse [194]) CYP, primarily CYP2E1 [195Metabolism by CYP to produce reactive formaldehyde: methylation of cell constituents [196]. Hepatocyte apoptosis [197Haemorrhagic centrilobular necrosis, destruction of sinusoidal endothelial cells leads to coagulation [198Fibrosis emerging from the CVs eventually leading to central–central bridging fibrosis Haemorrhagic necrosis results in macrophage phagocytosis of erythrocytes and necrotic debris which accumulate high levels of iron for extended periods after injury, giving rise to localized high levels of oxidative stress [193]. Primarily α-smooth muscle actin-positive myofibroblasts 
Pig serum 0.5 ml twice weekly for 4–16 weeks [199Rat n/a Portal vein thickening, haemodynamic effects, cell damage/apoptosis [200No overt damage to hepatocytes (no increase in ALT or ALP [200]) Fibrosis emerging from the CVs eventually leading to central–central bridging fibrosis. Hepatitis probably stimulated. Tolerized rats have marked reduced fibrosis [201]. Primarily α-smooth muscle actin-positive myofibroblasts 
Bile duct ligation: surgical procedure in which the bile duct is ligated, normally for up to 3–4 weeks [202Rat, mouse n/a Raised liver bile acids stimulate hepatocyte apoptosis [203,204] and, at higher levels, necrosis (cell membrane disruption [205]) Periportal hepatocyte death [206Fibrosis emerging from the portal tract eventually bridging fibrosis Primarily vimentin-positive myofibroblasts [207,208
Methionine- and choline-deficient diet [209]. Up to 12–15 weeks [210,211Rat, mouse Choline deficiency results in reduced fatty acid β-oxidation and reduced triacylglycerol clearance by the liver [209, 212]. Methionine deficiency leads to reductions in liver glutathione and increases in ROS (e.g. via CYP2E1) [209Lipid accumulates in hepatocytes. Oxidative stress results in hepatocyte necrosis and inflammation Lipid accumulation occurs in the centrilobular regions of the liver [213]. In humans, lipid accumulation is in the portal tract region [214Fibrosis Model of NASH that includes fibrogenesis although mice lose weight in contrast with NASH in humans. Note that in the leptin-deficient (ob/ob) mouse, there is lipid accumulation within the liver, but that a ‘second hit’ is required to generate inflammation (e.g. administration of LPS) [215]. Despite this, little fibrosis is seen in the ob/ob mouse, probably because of leptin deficiency [216
Alcohol: intragastric enteral feeding model [217Rat, mouse Metabolism of alcohol to acetaldehyde unlikely to be the major mechanism in the absence of acetaldehyde dehydrogenase inhibition (e.g. disulfiram or genetic mutations [218]) Increased gut permeability to endotoxin resulting in hepatic exposure to reactive oxygen species and inflammation [8Lipid accumulation occurs in the centrilobular regions of the liver. Inflammation and necrosis in the centrilobular region at 2–4 weeks [220Liver fibrosis at 8–10 weeks [220This model enables calorific pair feeding where control animals receive the same diet with alcohol substituted for glucose. For other models, see [221
Concanavalin A (up to 20 mg/kg of body weight injected i.v. once per week for up to 20 weeks [222Mouse The lectin concanavalin A stimulates a cytokine secretion syndrome. CD4+ T-cells infiltrate the liver Hepatocyte necrosis observed only after initial treatments. Inflammatory infiltrate persists, particularly centrilobular region Centrilobular and perisinusoidal fibrosis [223  
Hepatotoxin or causativeagent (typical dose) Model species System of activation Acute mechanism of damage Histological lobular effects Fibrosis Further comments 
CCl4 (1 ml/kg of body weight injected i.p. twice weekly from 4 to 12 weeks) [185Rat, mouse CYP, primarily CYP2E1 [186Lipid peroxidation [187,188Centrilobular hepatocyte death [188Fibrosis emerging from the CVs eventually leading to central–central bridging fibrosis. Most commonly employed means to generate liver fibrosis in animal models. Primarily α-smooth muscle actin-positive myofibroblasts 
Thioacetamide (300 mg/l of drinking water or 200 mg/kg of body weight injected i.p. two or three times per week for up to 12 weeks) [189Rat, mouse CYP, primarily CYP2E1 [190]. Also FMO [191Generation of reactive thioacetamide sulfoxide, leading to non-specific covalent binding of cell constituents [192Centrilobular hepatocyte death [189Fibrosis emerging from the CVs eventually leading to central–central bridging fibrosis Primarily α-smooth muscle actin-positive myofibroblasts 
Dimethylnitrosamine. Various protocols: 10 mg/kg of body weight injected i.p. for 3 consecutive days/week for up to 2–4 weeks; single 50 mg/kg of body weight injected i.p. Endpoints up to 10 months [193Rat (mouse [194]) CYP, primarily CYP2E1 [195Metabolism by CYP to produce reactive formaldehyde: methylation of cell constituents [196]. Hepatocyte apoptosis [197Haemorrhagic centrilobular necrosis, destruction of sinusoidal endothelial cells leads to coagulation [198Fibrosis emerging from the CVs eventually leading to central–central bridging fibrosis Haemorrhagic necrosis results in macrophage phagocytosis of erythrocytes and necrotic debris which accumulate high levels of iron for extended periods after injury, giving rise to localized high levels of oxidative stress [193]. Primarily α-smooth muscle actin-positive myofibroblasts 
Pig serum 0.5 ml twice weekly for 4–16 weeks [199Rat n/a Portal vein thickening, haemodynamic effects, cell damage/apoptosis [200No overt damage to hepatocytes (no increase in ALT or ALP [200]) Fibrosis emerging from the CVs eventually leading to central–central bridging fibrosis. Hepatitis probably stimulated. Tolerized rats have marked reduced fibrosis [201]. Primarily α-smooth muscle actin-positive myofibroblasts 
Bile duct ligation: surgical procedure in which the bile duct is ligated, normally for up to 3–4 weeks [202Rat, mouse n/a Raised liver bile acids stimulate hepatocyte apoptosis [203,204] and, at higher levels, necrosis (cell membrane disruption [205]) Periportal hepatocyte death [206Fibrosis emerging from the portal tract eventually bridging fibrosis Primarily vimentin-positive myofibroblasts [207,208
Methionine- and choline-deficient diet [209]. Up to 12–15 weeks [210,211Rat, mouse Choline deficiency results in reduced fatty acid β-oxidation and reduced triacylglycerol clearance by the liver [209, 212]. Methionine deficiency leads to reductions in liver glutathione and increases in ROS (e.g. via CYP2E1) [209Lipid accumulates in hepatocytes. Oxidative stress results in hepatocyte necrosis and inflammation Lipid accumulation occurs in the centrilobular regions of the liver [213]. In humans, lipid accumulation is in the portal tract region [214Fibrosis Model of NASH that includes fibrogenesis although mice lose weight in contrast with NASH in humans. Note that in the leptin-deficient (ob/ob) mouse, there is lipid accumulation within the liver, but that a ‘second hit’ is required to generate inflammation (e.g. administration of LPS) [215]. Despite this, little fibrosis is seen in the ob/ob mouse, probably because of leptin deficiency [216
Alcohol: intragastric enteral feeding model [217Rat, mouse Metabolism of alcohol to acetaldehyde unlikely to be the major mechanism in the absence of acetaldehyde dehydrogenase inhibition (e.g. disulfiram or genetic mutations [218]) Increased gut permeability to endotoxin resulting in hepatic exposure to reactive oxygen species and inflammation [8Lipid accumulation occurs in the centrilobular regions of the liver. Inflammation and necrosis in the centrilobular region at 2–4 weeks [220Liver fibrosis at 8–10 weeks [220This model enables calorific pair feeding where control animals receive the same diet with alcohol substituted for glucose. For other models, see [221
Concanavalin A (up to 20 mg/kg of body weight injected i.v. once per week for up to 20 weeks [222Mouse The lectin concanavalin A stimulates a cytokine secretion syndrome. CD4+ T-cells infiltrate the liver Hepatocyte necrosis observed only after initial treatments. Inflammatory infiltrate persists, particularly centrilobular region Centrilobular and perisinusoidal fibrosis [223  
Table 5
Commonly used tests and markers for liver fibrosis

AST, aspartate transaminase; ELF, European Liver Fibrosis group; GGT, γ-glutamyl transpeptidase; HCV, hepatitis C virus.

Marker Comments 
Non-invasive tests  
 Diffusion weight MRI Measures the apparent diffusion coefficient (ADC) of water. Assessed for the diagnosis of liver fibrosis in patients with chronic HCV [224
 Elastography (Fibroscan) Ultrasound (5 MHz) and low-frequency (50 Hz) elastic wave detection: propagation velocity through tissues is related to elasticity [225
Tests requiring blood analysis  
 APRI Aspartate aminotransferase to platelets ratio index [226
 ELF Serum hyaluronic acid, N-terminal propeptide of type III collagen and tissue inhibitor of matrix metalloproteinase 1 plus age [227
 Fibrometer Hyaluronate, prothrombin time, platelets, AST, α2-macroglobulin, urea and age [228
 FIBROSpect II Hyaluronic acid, tissue inhibitor of metalloproteinases 1 and α2-macroglobulin [229
 FibroTest FibroTest is used for the assessment of fibrosis, ActiTest for the assessment of liver necrosis [230]. The test determines α2-macroglobulin, haptoglobin, GGT, total bilirubin, apolipoprotein A1 and alanine aminotransferase 
 Forns test Combining age, GGT, cholesterol and platelet count [231
 Hepascore Bilirubin, GGT, hyaluronic acid, α2-macroglobulin, age and sex [232
 MP3 Procollagen type III N-terminal peptide (PIIINP) and matrix metalloproteinases (MMP)-1 [233
Test requiring liver tissue*  
 Parameter associated with pro-fibrogenic cell numbers  
  α-Smooth muscle actin Primarily via immunohistochemical staining. Detects myofibroblasts (see Figure 5). Smooth muscle cells within blood vessels are also positive, therefore interpretation is required to assess the degree of fibrosis. Western blotting liver extracts will not distinguish myofibroblast-associated α-smooth muscle actin 
  Vimentin Primarily via immunohistochemical staining. Detects fibroblasts associated with portal tract fibrosis (see Figure 5
 Direct determination of fibrosis  
  Sirius Red stain Relatively simple histochemical stain primarily for collagen type I (see Figure 5). Vessels are positive in normal liver, therefore interpretation is required to assess the degree of fibrosis. Also Masson's trichrome stain [238
  Collagen 1A1 Immunohistochemical staining [239]. Vessels are positive in normal liver, therefore interpretation is required to assess the degree of fibrosis 
  Hydroxyproline Collagens have a high proportion of hydroxyproline residues. Biochemical assay [240] to detect total liver hydroxyproline but high control tissue values result in small overall fold changes in fibrotic liver 
  Reticulin Stains collagen type III, which is highly glycosylated and therefore visible through silver staining techniques [241
  Shikata's orcein Detects elastic fibres: this is a marker of long-standing fibrosis 
Marker Comments 
Non-invasive tests  
 Diffusion weight MRI Measures the apparent diffusion coefficient (ADC) of water. Assessed for the diagnosis of liver fibrosis in patients with chronic HCV [224
 Elastography (Fibroscan) Ultrasound (5 MHz) and low-frequency (50 Hz) elastic wave detection: propagation velocity through tissues is related to elasticity [225
Tests requiring blood analysis  
 APRI Aspartate aminotransferase to platelets ratio index [226
 ELF Serum hyaluronic acid, N-terminal propeptide of type III collagen and tissue inhibitor of matrix metalloproteinase 1 plus age [227
 Fibrometer Hyaluronate, prothrombin time, platelets, AST, α2-macroglobulin, urea and age [228
 FIBROSpect II Hyaluronic acid, tissue inhibitor of metalloproteinases 1 and α2-macroglobulin [229
 FibroTest FibroTest is used for the assessment of fibrosis, ActiTest for the assessment of liver necrosis [230]. The test determines α2-macroglobulin, haptoglobin, GGT, total bilirubin, apolipoprotein A1 and alanine aminotransferase 
 Forns test Combining age, GGT, cholesterol and platelet count [231
 Hepascore Bilirubin, GGT, hyaluronic acid, α2-macroglobulin, age and sex [232
 MP3 Procollagen type III N-terminal peptide (PIIINP) and matrix metalloproteinases (MMP)-1 [233
Test requiring liver tissue*  
 Parameter associated with pro-fibrogenic cell numbers  
  α-Smooth muscle actin Primarily via immunohistochemical staining. Detects myofibroblasts (see Figure 5). Smooth muscle cells within blood vessels are also positive, therefore interpretation is required to assess the degree of fibrosis. Western blotting liver extracts will not distinguish myofibroblast-associated α-smooth muscle actin 
  Vimentin Primarily via immunohistochemical staining. Detects fibroblasts associated with portal tract fibrosis (see Figure 5
 Direct determination of fibrosis  
  Sirius Red stain Relatively simple histochemical stain primarily for collagen type I (see Figure 5). Vessels are positive in normal liver, therefore interpretation is required to assess the degree of fibrosis. Also Masson's trichrome stain [238
  Collagen 1A1 Immunohistochemical staining [239]. Vessels are positive in normal liver, therefore interpretation is required to assess the degree of fibrosis 
  Hydroxyproline Collagens have a high proportion of hydroxyproline residues. Biochemical assay [240] to detect total liver hydroxyproline but high control tissue values result in small overall fold changes in fibrotic liver 
  Reticulin Stains collagen type III, which is highly glycosylated and therefore visible through silver staining techniques [241
  Shikata's orcein Detects elastic fibres: this is a marker of long-standing fibrosis 
*

Routine liver function tests do not give an indication of liver fibrosis severity. At present, the gold standard for assessing the stage of liver disease and direct assessment of fibrosis uses a general haematoxylin and eosin stain and some of the stains outlined below. Scoring systems have been developed for liver disease severity such as those referred to as METAVIR [234], Scheuer [235], Ishak [236] and Knodell [237] systems.

Although the various animal models developed previously have served an important function in identifying basic mechanisms of liver fibrogenesis and testing new anti-fibrotic agents, it must be appreciated that all show some significant morphological differences with their human ‘counterparts’. This is perhaps least so with cholestatic models, particularly common bile duct ligation where the pattern of fibrosis is very similar to that seen when there is interruption of bile flow in humans. On the other hand, the commonly used CCl4 model is probably the most frequently used tool for investigating regression of fibrosis and cirrhosis, but the fibrous septa observed in animals, even on long-term CCl4, are generally much less developed compared with most human cirrhoses. Probably one of the most important differences between human and animal model diseases is the lack of significant alteration of vascular relationships in the latter; human cirrhosis is often confounded by liver cell loss through secondary hypoxic events, leading to parenchymal extinction. This does not occur in the commonly used animal models.

In vitro models of liver fibrosis

The most commonly used in vitro model of liver fibrosis is the isolation of quiescent hepatic stellate cells and their culture on plastic culture dishes in serum-containing culture medium. Under these conditions, cells commence a phenotypically similar process of transdifferentiation to the activated myofibroblast phenotype apparent in the liver in response to chronic liver damage (Figure 7), particularly when Kupffer cells are present (as a minor impurity) within the culture [54]. The cells are amenable to trypsin subculture, although, once the cells have activated to a myofibroblast phenotype, the cells remain in the myofibroblast phenotype in subsequent passages. Where possible, cells should be used in primary culture since this period is the only time to examine effects on transdifferentiation.

Transdifferentiation of quiescent hepatic stellate cells to myofibroblasts in vitro

Figure 7
Transdifferentiation of quiescent hepatic stellate cells to myofibroblasts in vitro

Human hepatic stellate cells were isolated and cultured as outlined [38]. Top-left-hand panel: soon after isolation as cells attach. Top-right-hand panel: after 17 days of culture. Lower panel: Western blot for α-smooth muscle actin (α-SMA) expression in cultured cells at the indicated time. Each lane contains 10 μg of total cell protein. Scale bars, 10 μm.

Figure 7
Transdifferentiation of quiescent hepatic stellate cells to myofibroblasts in vitro

Human hepatic stellate cells were isolated and cultured as outlined [38]. Top-left-hand panel: soon after isolation as cells attach. Top-right-hand panel: after 17 days of culture. Lower panel: Western blot for α-smooth muscle actin (α-SMA) expression in cultured cells at the indicated time. Each lane contains 10 μg of total cell protein. Scale bars, 10 μm.

There is a concern that differences in laboratory protocols may result in differences in the nature of hepatic myofibroblasts, and that hepatic stellate cell-derived myofibroblasts may be overgrown by portal tract myofibroblasts [5557]. Indeed, at present, there is no universally agreed diagnostic marker for quiescent hepatic stellate cell populations that could be used to establish cell identity and purity. Nevertheless, the in vitro culture system will remain a valuable tool for studying liver fibrosis and can readily be used in high-throughput screening assays for potential anti-fibrogenic agents.

A technically more challenging in vitro model is the culture of liver slices [73,74]. Liver slices from normal or diseased liver will retain their native extracellular matrix, cell–cell contacts and cell density and may be a valuable model for short-term studies. However, even the thinnest tissue slices (∼200 μm thick, ∼10 cells thick) prevent effective access of nutrients to cells, and a large proportion of the slice, particularly hepatocytes, die after 24 h [75,76].

Hepatic stellate cells from rodents are readily subcultured for many passages and therefore, for studies using myofibroblasts in their pro-fibrogenic state only, immortalized cell lines are often not required. To study the process of transdifferentiation from the quiescent state, cells must be isolated repeatedly from animals, as myofibroblasts do not readily revert to the quiescent state. The value of a rodent hepatic stellate cell line (see Table 6) lies with their propensity for being transfected with plasmid constructs, because cells isolated from rodents are difficult to transfect with high efficiency. Human hepatic stellate cell lines are more common because of difficulties in most laboratories of accessing human tissue. Furthermore, in our hands, most human myofibroblast cultures appear to senesce at passage 2–5 (from 58 human preparations, only two cultures have remained proliferative for more than five passages). This may be due to isolation of cells primarily from elderly patients (mainly from partial hepatectomy specimens from individuals with a secondary tumour in the liver). Human myofibroblasts are also resistant to transfection, and, in our hands, only nucleofection was sufficiently effective (with repeatable high efficiency) for the transfection of a range of reporter gene plasmid constructs [38]. Viral vectors have therefore been investigated from time to time for their ability to infect cells with high efficiency, including adenoviral [77,78], retroviral [79] and baculoviral [80] vectors. Although reported to be effective, the resources required to generate recombinant vectors has probably contributed to their lack of widespread use.

Table 6
Hepatic stellate cell lines

HSC, hepatic stellate cell; SV40, simian virus 40.

Cell line Species Comments 
LX-1 Human SV40 T-antigen immortalized [242
LX-2 Human [242
LI-90 Human [243
TWNT-4 Human LI-90 cells with retrovirally induced human telomerase reverse transcriptase (hTERT) [244
HSC T6 Rat Clone from cells transfected with the large T-antigen of SV40 [245
PAV1 Rat [246
CFSC-2G Rat [247
HSCs from metallothienein+/+ and −/− mice Mouse SV40-transformed [248
Cell line Species Comments 
LX-1 Human SV40 T-antigen immortalized [242
LX-2 Human [242
LI-90 Human [243
TWNT-4 Human LI-90 cells with retrovirally induced human telomerase reverse transcriptase (hTERT) [244
HSC T6 Rat Clone from cells transfected with the large T-antigen of SV40 [245
PAV1 Rat [246
CFSC-2G Rat [247
HSCs from metallothienein+/+ and −/− mice Mouse SV40-transformed [248

The in vitro model systems outlined above remain an effective way to screen a large number of potential compounds at an early stage for their ability to modulate the fibrogenic process using a variety of endpoints. However, as with many in vitro systems, there are limitations that prevent the identification of anti-fibrogenic agents with 100% accuracy. In vitro systems are closed artificial units that cannot allow for the absorption, distribution, metabolism and excretion of drugs that you see in whole-body studies. Liver myofibroblasts also reside next to the major drug-metabolizing cells of the body in vivo, but these are arguably functionally absent from most in vitro systems. Thus the replacement of in vivo animal models with in vitro models has yet to be achieved, and the former will probably always have a role to play in studies of liver fibrosis.

CLINICAL TREATMENTS FOR LIVER FIBROSIS

A number of reviews have appeared on approaches to the treatment of liver fibrosis [8183] and so only brief discussion is given here. Currently, there is no therapeutic agent indicated and licensed for the primary treatment of liver fibrosis, although a number of therapies are in clinical trials (Table 7). A variety of potential drug targets have been shown to exist within hepatic myofibroblasts in experimental systems, but many of these are associated with other cell types which presents problems of drug specificity. Indeed, the surprisingly varied number of targets available suggests that there may be redundancy for each pathway in myofibroblasts. Thus the drug-mediated block of a pathway promoting fibrogenesis may be overcome by the remaining unrelated fibrogenesis-promoting pathways. For these reasons and because myofibroblasts produce a number of factors which act in an autocrine fashion to promote fibrogenesis, the efficacy of drug treatments that lead to myofibroblast apoptosis have been examined [84,85].

Table 7
Recent and current clinical trials for anti-fibrogenic agents for the liver

Trials data are freely available at http://clinicaltrials.gov/ct, a service provided by the U.S. National Institute for Health. AT1, angiotensin II receptor 1; FGF, fibroblast growth factor; FKBP12, FK506-binding protein 12; FXR, farnesoid X receptor; HCV, hepatitis C virus; PBC, primary biliary cirrhosis; PPAR, peroxisome-proliferator-activated receptor.

Drug Company Pharmacology Comments 
Cyclosporin A Novartis Anti-rejection (immunosuppressive) drugs. Cyclosporin A targets cyclophilin, FK506 (tacrolimus) targets FKBP12 which then inhibits calcineurin and T-cell function [249Cyclosporin A has been shown to inhibit the severity of liver disease progression (fibrosis) in transplanted PBC patients compared with those given an alternative anti-rejection drug FK506 [250]. This study will compare cyclosporin A and FK506 for their abilities to inhibit fibrosis in HCV patients after liver transplantation. Trial NCT00260208 
Farglitazar (GI262570) GlaxoSmithKline PPARγ agonist [251]. Hepatic stellate cells express the PPARγ, activators inhibit fibrogenicity [252,253]. Trial NCT00244751 
INT-747 (6-ethyl chenodeoxycholic acid) Intercept FXR agonist [254Currently being tested in Type 2 diabetics with presumed NAFLD for safety and tolerability. Trial NCT00501592. Hepatic stellate cells express the FXR and activators inhibit fibrogenicity in vitro [255]. Therefore potential anti-fibrogenic agent 
Interferon γ1b Intermune Interferon γ1b agonist Trial NCT00043303 completed. Did not reverse fibrosis in HCV patients with advanced liver disease for 1 year [256
Irbesartan Sanofi-Synthelabo/French National Agency for Research on AIDS and Viral Hepatitis AT1 receptor antagonist Angiotensin II is secreted by activated hepatic stellate cells, which induces fibrogenic actions through the activation of NADPH oxidase. Trial NCT00265642 
Peginterferon α-2a Hoffmann–La Roche Interferon α agonist Interferon α may reduce portal pressure through a direct affect on the hepatic vasculature and may prevent complications of cirrhosis regardless of its effects on HCV RNA. Study to determine whether drug will lower portal pressure in patients with hepatitis C virus infections and advanced fibrosis or cirrhosis. Trial NCT00252642 
Peginterferon α-2b Schering-Plough Interferon α agonist Potentially may inhibit fibrosis in HCV patients not responding to treatment with respect to viraemia [257]. Potential effects with and without ribavirin to be examined. Trial NCT00323804 
Pentoxifylline Assistance Publique – Hôpitaux de Paris Phosphodiesterase inhibition leading to increases in cAMP; inhibition of cytokine expression such as TNFα [258Survival rate in cirrhotic patients. Trial NCT00162552 
Pirfenidone Marnac Inc., Cell Therapy and Technology, and Intermune FGF/TGFβ1/TNFα antagonist [259Fibrosis was reduced in 30% of patients by the end of 12 months of treatment [260
Warfarin Imperial College, London Anti-coagulant (vitamin K antagonist) Possession of the Factor V Leiden polymorphism associated with increased fibrosis in HCV, suggests a role for coagulation [261]. An anti-thrombin drug inhibits fibrosis in an animal model [262]. Trial NCT00180674 
Drug Company Pharmacology Comments 
Cyclosporin A Novartis Anti-rejection (immunosuppressive) drugs. Cyclosporin A targets cyclophilin, FK506 (tacrolimus) targets FKBP12 which then inhibits calcineurin and T-cell function [249Cyclosporin A has been shown to inhibit the severity of liver disease progression (fibrosis) in transplanted PBC patients compared with those given an alternative anti-rejection drug FK506 [250]. This study will compare cyclosporin A and FK506 for their abilities to inhibit fibrosis in HCV patients after liver transplantation. Trial NCT00260208 
Farglitazar (GI262570) GlaxoSmithKline PPARγ agonist [251]. Hepatic stellate cells express the PPARγ, activators inhibit fibrogenicity [252,253]. Trial NCT00244751 
INT-747 (6-ethyl chenodeoxycholic acid) Intercept FXR agonist [254Currently being tested in Type 2 diabetics with presumed NAFLD for safety and tolerability. Trial NCT00501592. Hepatic stellate cells express the FXR and activators inhibit fibrogenicity in vitro [255]. Therefore potential anti-fibrogenic agent 
Interferon γ1b Intermune Interferon γ1b agonist Trial NCT00043303 completed. Did not reverse fibrosis in HCV patients with advanced liver disease for 1 year [256
Irbesartan Sanofi-Synthelabo/French National Agency for Research on AIDS and Viral Hepatitis AT1 receptor antagonist Angiotensin II is secreted by activated hepatic stellate cells, which induces fibrogenic actions through the activation of NADPH oxidase. Trial NCT00265642 
Peginterferon α-2a Hoffmann–La Roche Interferon α agonist Interferon α may reduce portal pressure through a direct affect on the hepatic vasculature and may prevent complications of cirrhosis regardless of its effects on HCV RNA. Study to determine whether drug will lower portal pressure in patients with hepatitis C virus infections and advanced fibrosis or cirrhosis. Trial NCT00252642 
Peginterferon α-2b Schering-Plough Interferon α agonist Potentially may inhibit fibrosis in HCV patients not responding to treatment with respect to viraemia [257]. Potential effects with and without ribavirin to be examined. Trial NCT00323804 
Pentoxifylline Assistance Publique – Hôpitaux de Paris Phosphodiesterase inhibition leading to increases in cAMP; inhibition of cytokine expression such as TNFα [258Survival rate in cirrhotic patients. Trial NCT00162552 
Pirfenidone Marnac Inc., Cell Therapy and Technology, and Intermune FGF/TGFβ1/TNFα antagonist [259Fibrosis was reduced in 30% of patients by the end of 12 months of treatment [260
Warfarin Imperial College, London Anti-coagulant (vitamin K antagonist) Possession of the Factor V Leiden polymorphism associated with increased fibrosis in HCV, suggests a role for coagulation [261]. An anti-thrombin drug inhibits fibrosis in an animal model [262]. Trial NCT00180674 

In animal models recovering from chronic liver damage (i.e. the cause of damage is stopped), fibrosis reversal is associated with the apoptosis of liver myofibroblasts [44]. NF-κB (nuclear factor κB) is a transcription factor that regulates the expression of a range of genes associated with inflammation [86]. NF-κB becomes constitutively active in myofibroblasts in vitro and induces the expression of several pro-inflammatory genes [e.g. ICAM (intercellular adhesion molecule) and nitric oxide synthase 2] [87]. NF-κB is also constitutively active in myofibroblasts in vivo, suggesting that it is not an artefact of the in vitro culture system (F. Oakley, personal communication). NF-κB functions to prevent the apoptosis of myofibroblasts [84,87] as it does in other cells (e.g. hepatocytes) when they are exposed to pro-inflammatory mediators such as TNFα [89,90]. Inhibition of NF-κB therefore leads to the apoptosis of myofibroblasts in vitro [87] and in vivo [84,85]. The stimulation of liver myofibroblast apoptosis using gliotoxin (a fungal toxin that inhibits NF-κB [91], but which also works directly on the mitochondria to promote apoptosis [92]), resulted in a rapid recovery from fibrosis, and using this compound provided the first evidence in vivo that myofibroblast apoptosis may be an effective therapeutic approach to liver fibrosis treatment [84]. Using existing licensed NF-κB inhibitor drugs such as sulfasalazine to achieve the same ends may be a realistic treatment option [85]. More recently, targeting gliotoxin to hepatic myofibroblasts using a recombinant single-chain antibody to a surface antigen on the cells (thereby retaining functional liver macrophages), reduced fibrosis in vivo in a sustained injury model in contrast with free gliotoxin [93]. The specific removal of liver myofibroblasts in a ‘non-inflammatory’ manner while leaving macrophages in place therefore appears to be an effective anti-fibrogenic approach. By removing the liver myofibroblasts, the potential of specific drug targets within these cells becoming redundant with time is avoided. The converse, however, is that employing cell-death mechanisms as a therapeutic approach requires careful targeting to avoid adverse effects. Furthermore, it remains to be seen whether myofibroblast reductions as part of an anti-fibrogenic approach has any deleterious effects on liver regeneration.

Treatments in which drug targets have opposite effects in hepatocytes and liver myofibroblasts may turn out to be safer options, particularly when disease progression is to be slowed (in contrast with stimulating a reversal of cirrhosis). The PXR (pregnane X receptor) is a nuclear receptor transcription factor that regulates the expression of genes associated with endobiotic and xenobiotic clearance in hepatocytes [94]. PXR function is normally activated through contact with a range of ligands, including existing licensed drugs (e.g. rifampicin) and endogenous compounds (steroids, bile acids) [94]. Recent work from our laboratory has shown that the PXR is expressed in myofibroblasts, is capable of binding to its DNA enhancer response element in myofibroblast nuclear extracts and is transcriptionally functional on transfected reporter gene constructs [38]. The PXR regulates a specific set of genes in myofibroblasts. Ligand activators of the PXR reduce the expression of pro-fibrogenic cytokines such as TGF (transforming growth factor) β and inhibit proliferation [38]. PXR activators therefore act in an anti-fibrogenic manner in human liver myofibroblasts in vitro [38]. Using mice with a disrupted PXR gene, the role of the PXR in mediating an anti-fibrogenic effect was unequivocally established [72].

CONCLUSIONS

The fibrotic response in the liver is dependent on multiple cell types, both resident and recruited to the liver, and is regulated by a vast array of factors in vivo. A full understanding of the response remains a goal at present, particularly the benefit of fibrosis to tissue function and viability. The ability to specifically ‘knock out’ cell types in a tissue should help to resolve many questions in the same way that transgenic technology has resolved questions surrounding a specific gene's function. Clinically employed anti-fibrogenic agents may then become available.

Abbreviations

     
  • CYP

    cytochrome P450

  •  
  • EGF

    epidermal growth factor

  •  
  • GFP

    green fluorescent protein

  •  
  • IL

    interleukin

  •  
  • NAFLD

    non-alcoholic fatty liver disease

  •  
  • NF-κB

    nuclear factor κB

  •  
  • Plg

    plasminogen

  •  
  • PXR

    pregnane X receptor

  •  
  • ROS

    reactive oxygen species

  •  
  • TGF

    transforming growth factor

  •  
  • TNF

    tumour necrosis factor

References

References
1
Park
K.
Williams
D. P.
Naisbitt
D. J.
Kitteringham
N. R.
Pirmohamed
M.
Investigation of toxic metabolites during drug development
Toxicol. Appl. Pharmacol.
2005
, vol. 
207
 (pg. 
425
-
434
)
2
Larson
A. M.
Polson
J.
Fontana
R. J.
Davern
T. J.
Lalani
E.
Hynan
L. S.
Reisch
J. S.
Schiodt
F. V.
Ostapowicz
G.
Shakil
A. O.
, et al. 
Acetaminophen-induced acute liver failure: results of a United States multicenter, prospective study
Hepatology
2005
, vol. 
42
 (pg. 
1364
-
1372
)
3
Williams
R.
Global challenges in liver disease
Hepatology
2006
, vol. 
44
 (pg. 
521
-
526
)
4
MacSween
R. N.
Desmet
V. J.
Roskams
T.
Scothorne
R. J.
MacSween
R. N. M.
Burt
A. D.
Portman
B. C.
Ishak
K. G.
Scheuer
P. J.
Anthony
P. P.
Developmental anatomy and normal structure
Pathology of the Liver
2002
4th edn
Edinburgh
Churchill-Livingstone
(pg. 
1
-
66
)
5
Braet
F.
Wisse
E.
Structural and functional aspects of liver sinusoidal endothelial cell fenestrae
Comp. Hepatol.
2002
, vol. 
1
 (pg. 
1
-
17
)
6
Oinonen
T.
Lindros
K. O.
Zonation of hepatic cytochrome P-450 expression and regulation
Biochem. J.
1998
, vol. 
329
 (pg. 
17
-
35
)
7
Ookhtens
M.
Kaplowitz
N.
Role of the liver in interorgan homeostasis of glutathione and cyst(e)ine
Semin. Liver Dis.
1998
, vol. 
18
 (pg. 
313
-
329
)
8
Wheeler
M. D.
Kono
H.
Yin
M.
Nakagami
M.
Uesugi
T.
Arteel
G. E.
Gabele
E.
Rusyn
I.
Yamashina
S.
Froh
M.
, et al. 
The role of Kupffer cell oxidant production in early ethanol-induced liver disease
Free Radical Biol. Med.
2001
, vol. 
31
 (pg. 
1544
-
1549
)
9
Bode
C.
Bode
J. C.
Activation of the innate immune system and alcoholic liver disease: effects of ethanol per se or enhanced intestinal translocation of bacterial toxins induced by ethanol?
Alcohol Clin. Exp. Res.
2005
, vol. 
29
 (pg. 
166S
-
171S
)
10
Wong
F. W.
Chan
W. Y.
Lee
S. S.
Resistance to carbon tetrachloride-induced hepatotoxicity in mice which lack CYP2E1 expression
Toxicol. Appl. Pharmacol.
1998
, vol. 
153
 (pg. 
109
-
118
)
11
Bansal
M. B.
Kovalovich
K.
Gupta
R.
Li
W.
Agarwal
A.
Radbill
B.
Alvarez
C. E.
Safadi
R.
Fiel
M. I.
Friedman
S. L.
Taub
R. A.
Interleukin-6 protects hepatocytes from CCl4-mediated necrosis and apoptosis in mice by reducing MMP-2 expression
J. Hepatol.
2005
, vol. 
42
 (pg. 
548
-
556
)
12
Bilzer
M.
Roggel
F.
Gerbes
A. L.
Role of Kupffer cells in host defense and liver disease
Liver Int.
2006
, vol. 
26
 (pg. 
1175
-
1186
)
13
Canbay
A.
Feldstein
A. E.
Higuchi
H.
Werneburg
N.
Grambihler
A.
Bronk
S. F.
Gores
G. J.
Kupffer cell engulfment of apoptotic bodies stimulates death ligand and cytokine expression
Hepatology
2003
, vol. 
38
 (pg. 
1188
-
1198
)
14
Jaeschke
H.
Hasegawa
T.
Role of neutrophils in acute inflammatory liver injury
Liver Int.
2006
, vol. 
26
 (pg. 
912
-
919
)
15
Dansette
P. M.
Bonierbale
E.
Minoletti
C.
Beaune
P. H.
Pessayre
D.
Mansuy
D.
Drug-induced immunotoxicity
Eur. J. Drug Metab. Pharmacokinet.
1998
, vol. 
23
 (pg. 
443
-
451
)
16
Manns
M. P.
Vogel
A.
Autoimmune hepatitis, from mechanisms to therapy
Hepatology
2006
, vol. 
43
 (pg. 
S132
-
S144
)
17
Beaune
P.
Dansette
P. M.
Mansuy
D.
Kiffel
L.
Finck
M.
Amar
C.
Leroux
J. P.
Homberg
J. C.
Human anti-endoplasmic reticulum autoantibodies appearing in a drug-induced hepatitis are directed against a human liver cytochrome P-450 that hydroxylates the drug
Proc. Natl. Acad. Sci. U.S.A.
1987
, vol. 
84
 (pg. 
551
-
555
)
18
Lecoeur
S.
Andre
C.
Beaune
P. H.
Tienilic acid-induced autoimmune hepatitis: anti-liver and -kidney microsomal type 2 autoantibodies recognize a three-site conformational epitope on cytochrome P4502C9
Mol. Pharmacol.
1996
, vol. 
50
 (pg. 
326
-
333
)
19
Fausto
N.
Campbell
J. S.
Riehle
K. J.
Liver regeneration
Hepatology
2006
, vol. 
43
 (pg. 
S45
-
S53
)
20
Lesurtel
M.
Graf
R.
Aleil
B.
Walther
D. J.
Tian
Y.
Jochum
W.
Gachet
C.
Bader
M.
Clavien
P. A.
Platelet-derived serotonin mediates liver regeneration
Science
2006
, vol. 
312
 (pg. 
104
-
107
)
21
Nocito
A.
Georgiev
P.
Dahm
F.
Jochum
W.
Bader
M.
Graf
R.
Clavien
P. A.
Platelets and platelet-derived serotonin promote tissue repair after normothermic hepatic ischemia in mice
Hepatology
2007
, vol. 
45
 (pg. 
369
-
376
)
22
Theise
N. D.
Badve
S.
Saxena
R.
Henegariu
O.
Sell
S.
Crawford
J. M.
Krause
D. S.
Derivation of hepatocytes from bone marrow cells in mice after radiation-induced myeloablation
Hepatology
2000
, vol. 
31
 (pg. 
235
-
240
)
23
Alison
M. R.
Poulsom
R.
Jeffery
R.
Dhillon
A. P.
Quaglia
A.
Jacob
J.
Novelli
M.
Prentice
G.
Williamson
J.
Wright
N. A.
Hepatocytes from non-hepatic adult stem cells
Nature
2000
, vol. 
406
 pg. 
257
 
24
Wang
X.
Willenbring
H.
Akkari
Y.
Torimaru
Y.
Foster
M.
Al-Dhalimy
M.
Lagasse
E.
Finegold
M.
Olson
S.
Grompe
M.
Cell fusion is the principal source of bone-marrow-derived hepatocytes
Nature
2003
, vol. 
422
 (pg. 
897
-
901
)
25
Vassilopoulos
G.
Wang
P. R.
Russell
D. W.
Transplanted bone marrow regenerates liver by cell fusion
Nature
2003
, vol. 
422
 (pg. 
901
-
904
)
26
Forbes
S. J.
Russo
F. P
Rey
V.
Burra
P.
Rugge
M.
Wright
N. A.
Alison
M. R.
A significant proportion of myofibroblasts are of bone marrow origin in human liver fibrosis
Gastroenterology
2004
, vol. 
126
 (pg. 
955
-
963
)
27
Russo
F. P.
Alison
M. R.
Bigger
B. W.
Amofah
E.
Florou
A.
Amin
F.
Bou-Gharios
G.
Jeffery
R.
Iredale
J. P.
Forbes
S. J.
The bone marrow functionally contributes to liver fibrosis
Gastroenterology
2006
, vol. 
130
 (pg. 
1807
-
1821
)
28
Menthena
A.
Deb
N.
Oertel
M.
Grozdanov
P. N.
Sandhu
J.
Shah
S.
Guha
C.
Shafritz
D. A.
Dabeva
M. D.
Bone marrow progenitors are not the source of expanding oval cells in injured liver
Stem Cells
2004
, vol. 
22
 (pg. 
1049
-
1061
)
29
Vig
P.
Russo
F. P.
Edwards
R. J.
Tadrous
P. J.
Wright
N. A.
Thomas
H. C.
Alison
M. R.
Forbes
S. J.
The sources of parenchymal regeneration after chronic hepatocellular liver injury in mice
Hepatology
2006
, vol. 
43
 (pg. 
316
-
324
)
30
Zajicek
G.
Oren
R.
Weinreb
M.
Jr
The streaming liver
Liver
1985
, vol. 
5
 (pg. 
293
-
300
)
31
Kennedy
S.
Rettinger
S.
Flye
M. W.
Ponder
K. P.
Experiments in transgenic mice show that hepatocytes are the source for postnatal liver growth and do not stream
Hepatology
1995
, vol. 
22
 (pg. 
160
-
168
)
32
Sarraf
C. E.
Horgan
M.
Edwards
R. J.
Alison
M. R.
Reversal of phenobarbital-induced hyperplasia and hypertrophy in the livers of lpr mice
Int. J. Exp. Pathol.
1997
, vol. 
78
 (pg. 
49
-
56
)
33
Bugge
T. H
Kombrinck
K. W.
Flick
M. J.
Daugherty
C. C.
Danton
M. J.
Degen
J. L.
Loss of fibrinogen rescues mice from the pleiotropic effects of plasminogen deficiency
Cell
1996
, vol. 
87
 (pg. 
709
-
719
)
34
Passino
M. A.
Adams
R. A.
Sikorski
S. L.
Akassoglou
K.
Regulation of hepatic stellate cell differentiation by the neurotrophin receptor p75NTR
Science
2007
, vol. 
315
 (pg. 
1853
-
1856
)
35
Kariv
R.
Enden
A.
Zvibel
I.
Rosner
G.
Brill
S.
Shafritz
D. A.
Halpern
Z.
Oren
R.
Triiodothyronine and interleukin-6 (IL-6) induce expression of HGF in an immortalized rat hepatic stellate cell line
Liver Int.
2003
, vol. 
23
 (pg. 
187
-
193
)
36
Tomiya
T.
Nishikawa
T.
Inoue
Y.
Ohtomo
N.
Ikeda
H.
Tejima
K.
Watanabe
N.
Tanque
Y.
Omata
M.
Fujiwara
K.
Leucine stimulates HGF production by hepatic stellate cells through mTOR pathway
Biochem. Biophys. Res. Commun.
2007
, vol. 
358
 (pg. 
176
-
180
)
37
Smart
D. E.
Vincent
K. J.
Arthur
M. J.
Eickelberg
O.
Castellazzi
M.
Mann
J.
Mann
D. A.
JunD regulates transcription of the tissue inhibitor of metalloproteinases-1 and interleukin-6 genes in activated hepatic stellate cells
J. Biol. Chem.
2001
, vol. 
276
 (pg. 
24414
-
24421
)
38
Haughton
E. L.
Tucker
S. J.
Marek
C. J.
Durward
E.
Leel
V.
Bascal
Z.
Monaghan
T.
Koruth
M.
Collie-Duguid
E.
Mann
D. A.
, et al. 
Pregnane X receptor activators inhibit human hepatic stellate cell trans-differentiation in vitro
Gastroenterology
2006
, vol. 
131
 (pg. 
194
-
209
)
39
Simeonova
P. P.
Gallucci
R. M.
Hulderman
T.
Wilson
R.
Kommineni
C.
Rao
M.
Luster
M. I.
The role of tumor necrosis factor-α in liver toxicity, inflammation, and fibrosis induced by carbon tetrachloride
Toxicol. Appl. Pharmacol.
2001
, vol. 
177
 (pg. 
112
-
120
)
40
Casini
A.
Ceni
E.
Salzano
R.
Biondi
P.
Parola
M.
Galli
A.
Foschi
M.
Caligiuri
A.
Pinzani
M.
Surrenti
C.
Neutrophil-derived superoxide anion induces lipid peroxidation and stimulates collagen synthesis in human hepatic stellate cells: role of nitric oxide
Hepatology
1997
, vol. 
25
 (pg. 
361
-
367
)
41
Bataller
R.
Schwabe
R. F.
Choi
Y. H.
Yang
L.
Paik
Y. H.
Lindquist
J.
Qian
T.
Schoonhoven
R.
Hagedorn
C. H.
Lemasters
J. J.
Brenner
D. A.
NADPH oxidase signal transduces angiotensin II in hepatic stellate cells and is critical in hepatic fibrosis
J. Clin. Invest.
2003
, vol. 
112
 (pg. 
1383
-
1394
)
42
Hironaka
K.
Sakaida
I.
Matsumura
Y.
Kaino
S.
Miyamoto
K.
Okita
K.
Enhanced interstitial collagenase (matrix metalloproteinase-13) production of Kupffer cell by gadolinium chloride prevents pig serum-induced rat liver fibrosis
Biochem. Biophys. Res. Commun.
2000
, vol. 
267
 (pg. 
290
-
295
)
43
Fallowfield
J. A.
Mizuno
M.
Kendall
T. J.
Constandinou
C. M.
Benyon
R. C.
Duffield
J. S.
Iredale
J. P.
Scar-associated macrophages are a major source of hepatic matrix metalloproteinase-13 and facilitate the resolution of murine hepatic fibrosis
J. Immunol.
2007
, vol. 
178
 (pg. 
5288
-
5295
)
44
Iredale
J. P.
Benyon
R. C.
Pickering
J.
McCullen
M.
Northrop
M.
Pawley
S.
Hovell
C.
Arthur
M. J.
Mechanisms of spontaneous resolution of rat liver fibrosis: hepatic stellate cell apoptosis and reduced hepatic expression of metalloproteinase inhibitors
J. Clin. Invest.
1998
, vol. 
102
 (pg. 
538
-
549
)
45
Issa
R.
Zhou
X.
Constandinou
C. M.
Fallowfield
J.
Millward-Sadler
H.
Gaca
M. D.
Sands
E.
Suliman
I.
Trim
N.
Knorr
A.
, et al. 
Spontaneous recovery from micronodular cirrhosis: evidence for incomplete resolution associated with matrix cross-linking
Gastroenterology
2004
, vol. 
126
 (pg. 
1795
-
1808
)
46
Diegelmann
R. F.
Guzelian
P. S.
Gay
R.
Gay
S.
Collagen formation by the hepatocyte in primary monolayer culture and in vivo
Science
1983
, vol. 
219
 (pg. 
1343
-
1345
)
47
Friedman
S. L.
Roll
F. J.
Boyles
J.
Bissell
D. M.
Hepatic lipocytes: the principal collagen-producing cells of normal rat liver
Proc. Natl. Acad. Sci. U.S.A.
1985
, vol. 
82
 (pg. 
8681
-
8685
)
48
Maher
J. J.
Bissell
D. M.
Friedman
S. L.
Roll
F. J.
Collagen measured in primary cultures of normal rat hepatocytes derives from lipocytes within the monolayer
J. Clin. Invest.
1988
, vol. 
82
 (pg. 
450
-
459
)
49
Burt
A. D.
Pathobiology of hepatic stellate cells
J. Gastroenterol.
1999
, vol. 
34
 (pg. 
299
-
304
)
50
Blaner
W. S.
Hendriks
H. F.
Brouwer
A.
de Leeuw
A. M.
Knook
D. L.
Goodman
D. S.
Retinoids, retinoid-binding proteins, and retinyl palmitate hydrolase distributions in different types of rat liver cells
J. Lipid Res.
1985
, vol. 
26
 (pg. 
1241
-
1251
)
51
Nouchi
T.
Tanaka
Y.
Tsukada
T.
Sato
C.
Marumo
F.
Appearance of α-smooth-muscle-actin-positive cells in hepatic fibrosis
Liver
1991
, vol. 
11
 (pg. 
100
-
105
)
52
Rockey
D. C.
Boyles
J. K.
Gabbiani
G.
Friedman
S. L.
Rat hepatic lipocytes express smooth muscle actin upon activation in vivo and in culture
J. Submicrosc. Cytol. Pathol.
1992
, vol. 
24
 (pg. 
193
-
203
)
53
Yamaoka
K.
Nouchi
T.
Marumo
F.
Sato
C.
α-Smooth-muscle actin expression in normal and fibrotic human livers
Dig. Dis. Sci.
1993
, vol. 
38
 (pg. 
1473
-
1479
)
54
De Minicis
S.
Seki
E.
Uchinami
H.
Kluwe
J.
Zhang
Y.
Brenner
D. A.
Schwabe
R. F.
Gene expression profiles during hepatic stellate cell activation in culture and in vivo
Gastroenterology
2007
, vol. 
132
 (pg. 
1937
-
1946
)
55
Knittel
T.
Kobold
D.
Saile
B.
Grundmann
A.
Neubauer
K.
Piscaglia
F.
Ramadori
G.
Rat liver myofibroblasts and hepatic stellate cells: different cell populations of the fibroblast lineage with fibrogenic potential
Gastroenterology
1999
, vol. 
117
 (pg. 
1205
-
1221
)
56
Cassiman
D.
Libbrecht
L.
Desmet
V.
Denef
C.
Roskams
T.
Hepatic stellate cell/myofibroblast subpopulations in fibrotic human and rat livers
J. Hepatol.
2002
, vol. 
36
 (pg. 
200
-
209
)
57
Guyot
C.
Lepreux
S.
Combe
C.
Doudnikoff
E.
Bioulac-Sage
P.
Balabaud
C.
Desmouliere
A.
Hepatic fibrosis and cirrhosis: the (myo)fibroblastic cell subpopulations involved
Int. J. Biochem. Cell Biol.
2006
, vol. 
38
 (pg. 
135
-
151
)
58
Beaussier
M.
Wendum
D.
Schiffer
E.
Dumont
S
Rey
C.
Lienhart
A.
Housset
C.
Prominent contribution of portal mesenchymal cells to liver fibrosis in ischaemic and obstructive cholestatic injuries
Lab. Invest.
2007
, vol. 
87
 (pg. 
292
-
303
)
59
Magness
S. T.
Bataller
R.
Yang
L.
Brenner
D. A.
A dual reporter gene transgenic mouse demonstrates heterogeneity in hepatic fibrogenic cell populations
Hepatology
2004
, vol. 
40
 (pg. 
1151
-
1159
)
60
Roskams
T.
Cassiman
D.
De Vos
R.
Libbrecht
L.
Neuroregulation of the neuroendocrine compartment of the liver
Anat. Rec. Part A Discov. Mol. Cell Evol. Biol.
2004
, vol. 
280
 (pg. 
910
-
923
)
61
Nieto
M. A.
The early steps of neural crest development
Mech. Dev.
2001
, vol. 
105
 (pg. 
27
-
35
)
61a
Cassiman
D.
Barlow
A.
Vander Borght
S.
Libbrecht
L.
Pachnis
V.
Hepatic stellate cells do not derive from the neural crest
J. Hepatol.
2006
, vol. 
44
 (pg. 
1098
-
1104
)
62
Baba
S.
Fujii
H.
Hirose
T.
Yasuchika
K.
Azuma
H.
Hoppo
T.
Naito
M.
Machimoto
T.
Ikai
I.
Commitment of bone marrow cells to hepatic stellate cells in mouse
J. Hepatol.
2004
, vol. 
40
 (pg. 
255
-
260
)
63
Kisseleva
T.
Uchinami
H.
Feirt
N.
Quintana-Bustamante
O.
Segovia
J. C.
Schwabe
R. F.
Brenner
D. A.
Bone marrow-derived fibrocytes participate in pathogenesis of liver fibrosis
J. Hepatol.
2006
, vol. 
45
 (pg. 
429
-
438
)
64
Forbes
S. J.
Russo
F. P.
Rey
V.
Burra
P.
Rugge
M.
Wright
N. A.
Alison
M. R.
A significant proportion of myofibroblasts are of bone marrow origin in human liver fibrosis
Gastroenterology
2004
, vol. 
126
 (pg. 
955
-
963
)
65
Zeisberg
M.
Yang
C.
Martino
M.
Duncan
M. B.
Rieder
F.
Tanjore
H.
Kalluri
R.
Fibroblasts derive from hepatocytes in liver fibrosis via epithelial to mesenchymal transition
J. Biol. Chem.
2007
, vol. 
282
 (pg. 
23337
-
23347
)
66
Robertson
H.
Kirby
J. A.
Yip
W. W.
Jones
D. E.
Burt
A. D.
Biliary epithelial–mesenchymal transition in posttransplantation recurrence of primary biliary cirrhosis
Hepatology
2007
, vol. 
45
 (pg. 
977
-
981
)
67
Kordes
C.
Sawitza
I.
Muller-Marbach
A.
Ale-Agha
N.
Keitel
V.
Klonowski-Stumpe
H.
Haussinger
D.
CD133+ hepatic stellate cells are progenitor cells
Biochem. Biophys. Res. Commun.
2007
, vol. 
352
 (pg. 
410
-
417
)
68
Schwartz
R. E.
Reyes
M.
Koodie
L.
Jiang
Y.
Blackstad
M.
Lund
T.
Lenvik
T.
Johnson
S.
Hu
W. S.
Verfaillie
C. M.
Multipotent adult progenitor cells from bone marrow differentiate into functional hepatocyte-like cells
J. Clin. Invest.
2002
, vol. 
109
 (pg. 
1291
-
1302
)
69
Jiang
Y.
Jahagirdar
B. N.
Reinhardt
R. L.
Schwartz
R. E.
Keene
C. D.
Ortiz-Gonzalez
X. R.
Reyes
M.
Lenvik
T.
Lund
T.
Blackstad
M.
, et al. 
Pluripotency of mesenchymal stem cells derived from adult marrow
Nature
2002
, vol. 
418
 (pg. 
41
-
49
)
70
Rivera
C. A.
Bradford
B. U.
Hunt
K. J.
Adachi
Y.
Schrum
L. W.
Koop
D. R.
Burchardt
E. R.
Rippe
R. A.
Thurman
R. G.
Attenuation of CCl4-induced hepatic fibrosis by GdCl3 treatment or dietary glycine
Am. J. Physiol. Gastrointest. Liver Physiol.
2001
, vol. 
281
 (pg. 
G200
-
G207
)
71
Duffield
J. S.
Forbes
S. J.
Constandinou
C. M.
Clay
S.
Partolina
M.
Vuthoori
S.
Wu
S.
Lang
R.
Iredale
J. P.
Selective depletion of macrophages reveals distinct, opposing roles during liver injury and repair
J. Clin. Invest.
2005
, vol. 
115
 (pg. 
56
-
65
)
72
Marek
C. J.
Tucker
S. J.
Konstantinou
D. K.
Elrick
L. J.
Haefner
D.
Sigalas
C.
Murray
G. I.
Goodwin
B.
Wright
M. C.
Pregnenolone-16α-carbonitrile inhibits rodent liver fibrogenesis via PXR (pregnane X receptor)-dependent and PXR-independent mechanisms
Biochem. J.
2005
, vol. 
387
 (pg. 
601
-
608
)
73
Verrill
C.
Davies
J.
Millward-Sadler
H.
Sundstrom
L.
Sheron
N.
Organotypic liver culture in a fluid-air interface using slices of neonatal rat and adult human tissue: a model of fibrosis in vitro
J. Pharmacol. Toxicol. Methods
2002
, vol. 
48
 (pg. 
103
-
110
)
74
Hagens
W. I.
Olinga
P.
Meijer
D. K.
Groothuis
G. M.
Beljaars
L.
Poelstra
K.
Gliotoxin non-selectively induces apoptosis in fibrotic and normal livers
Liver Int.
2006
, vol. 
26
 (pg. 
232
-
239
)
75
Wright
M. C.
Paine
A. J.
Evidence that the loss of rat liver cytochrome P450 in vitro is not solely associated with the use of collagenase, the loss of cell–cell contacts and/or the absence of an extracellular matrix
Biochem. Pharmacol.
1992
, vol. 
43
 (pg. 
237
-
243
)
76
Wright
M. C.
Paine
A. J.
Resistance of precision-cut liver slices to the toxic effects of menadione
Toxicol. In Vitro
1992
, vol. 
6
 (pg. 
475
-
481
)
77
Abriss
B.
Hollweg
G.
Gressner
A. M.
Weiskirchen
R.
Adenoviral-mediated transfer of p53 or retinoblastoma protein blocks cell proliferation and induces apoptosis in culture-activated hepatic stellate cells
J. Hepatol.
2003
, vol. 
38
 (pg. 
169
-
178
)
78
Kinoshita
K.
Iimuro
Y.
Fujimoto
J.
Inagaki
Y.
Namikawa
K.
Kiyama
H.
Nakajima
Y.
Otogawa
K.
Kawada
N.
Friedman
S. L.
Ikeda
K.
Targeted and regulable expression of transgenes in hepatic stellate cells and myofibroblasts in culture and in vivo using an adenoviral Cre/loxP system to antagonise hepatic fibrosis
Gut
2007
, vol. 
56
 (pg. 
396
-
404
)
79
Olaso
E.
Ikeda
K.
Eng
F. J.
Xu
L.
Wang
L. H.
Lin
H. C.
Friedman
S. L.
DDR2 receptor promotes MMP-2-mediated proliferation and invasion by hepatic stellate cells
J. Clin. Invest.
2001
, vol. 
108
 (pg. 
1369
-
1378
)
80
Gao
R.
McCormick
C. J.
Arthur
M. J.
Ruddell
R.
Oakley
F.
Smart
D. E.
Murphy
F. R.
Harris
M. P.
Mann
D. A.
High efficiency gene transfer into cultured primary rat and human hepatic stellate cells using baculovirus vectors
Liver
2002
, vol. 
22
 (pg. 
15
-
22
)
81
Friedman
S. L.
Liver fibrosis: from bench to bedside
J. Hepatol.
2003
, vol. 
38
 (pg. 
S38
-
S53
)
82
Pinzani
M.
Rombouts
K.
Liver fibrosis: from the bench to clinical targets
Dig. Liver Dis.
2004
, vol. 
36
 (pg. 
231
-
242
)
83
Muddu
A. K.
Guha
I. N.
Elsharkawy
A. M.
Mann
D. A.
Resolving fibrosis in the diseased liver: translating the scientific promise to the clinic
Int. J. Biochem. Cell Biol.
2007
, vol. 
39
 (pg. 
695
-
714
)
84
Wright
M. C.
Issa
R.
Smart
D. E.
Trim
N.
Murray
G. I.
Primrose
J. N.
Arthur
M. J.
Iredale
J. P.
Mann
D. A.
Gliotoxin stimulates the apoptosis of human and rat hepatic stellate cells and enhances the resolution of liver fibrosis in rats
Gastroenterology
2001
, vol. 
121
 (pg. 
685
-
698
)
85
Oakley
F.
Meso
M.
Iredale
J. P.
Green
K.
Marek
C. J.
Zhou
X.
May
M. J.
Millward-Sadler
H.
Wright
M. C.
Mann
D. A.
Inhibition of inhibitor of κB kinases stimulates hepatic stellate cell apoptosis and accelerated recovery from rat liver fibrosis
Gastroenterology
2005
, vol. 
128
 (pg. 
108
-
120
)
86
Elsharkawy
A. M.
Mann
D. A.
Nuclear factor-κB and the hepatic inflammation–fibrosis–cancer axis
Hepatology
2007
, vol. 
46
 (pg. 
590
-
597
)
87
Elsharkawy
A. M.
Wright
M. C.
Hay
R. T.
Arthur
M. J.
Hughes
T.
Bahr
M. J.
Degitz
K.
Mann
D. A.
Persistent activation of nuclear factor-κB in cultured rat hepatic stellate cells involves the induction of potentially novel Rel-like factors and prolonged changes in the expression of IκB family proteins
Hepatology
1999
, vol. 
30
 (pg. 
761
-
769
)
88
Reference deleted
89
Bradham
C. A.
Qian
T.
Streetz
K.
Trautwein
C.
Brenner
D. A.
Lemasters
J. J.
The mitochondrial permeability transition is required for tumor necrosis factor α-mediated apoptosis and cytochrome c release
Mol. Cell. Biol.
1998
, vol. 
18
 (pg. 
6353
-
6364
)
90
Chang
L.
Kamata
H.
Solinas
G.
Luo
J. L.
Maeda
S.
Venuprasad
K.
Liu
Y. C.
Karin
M.
The E3 ubiquitin ligase itch couples JNK activation to TNFα-induced cell death by inducing c-FLIP(L) turnover
Cell
2006
, vol. 
124
 (pg. 
601
-
613
)
91
Pahl
H. L.
Krauss
B.
Schulze-Osthoff
K.
Decker
T.
Traenckner
E. B.
Vogt
M.
Myers
C.
Parks
T.
Warring
P.
Muhlbacher
A.
, et al. 
The immunosuppressive fungal metabolite gliotoxin specifically inhibits transcription factor NF-κB
J. Exp. Med.
1996
, vol. 
183
 (pg. 
1829
-
1840
)
92
Orr
J. G.
Leel
V.
Cameron
G. A.
Marek
C. J.
Haughton
E. L.
Elrick
L. J.
Trim
J. E.
Hawksworth
G. M.
Halestrap
A. P.
Wright
M. C.
Mechanism of action of the antifibrogenic compound gliotoxin in rat liver cells
Hepatology
2004
, vol. 
40
 (pg. 
232
-
242
)
93
Douglass
A.
Wallace
K.
Parr
R.
Park
J.
Durward
E.
Broadbent
I.
Barelle
C.
Porter
A. J.
Wright
M. C.
Antibody-targeted myofibroblast apoptosis reduces fibrosis during sustained liver injury
J. Hepatol.
2008
 
in the press
94
Kliewer
S. A.
Willson
T. M.
Regulation of xenobiotic and bile acid metabolism by the nuclear pregnane X receptor
J. Lipid. Res.
2002
, vol. 
43
 (pg. 
359
-
364
)
95
Tiollais
P.
Pourcel
C.
Dejean
A.
The hepatitis B virus
Nature
1985
, vol. 
317
 (pg. 
489
-
495
)
96
Lok
A. S.
Hepatitis B infection: pathogenesis and management
J. Hepatol.
2000
, vol. 
32
 (pg. 
89
-
97
)
97
Pardo
M.
Bartolome
J.
Carreno
V.
Current therapy of chronic hepatitis B
Arch. Med. Res.
2007
, vol. 
38
 (pg. 
661
-
677
)
98
Choo
Q. L.
Kuo
G.
Weiner
A. J.
Overby
L. R.
Bradley
D. W.
Houghton
M.
Isolation of a cDNA clone derived from a blood-borne non-A, non-B viral hepatitis genome
Science
1989
, vol. 
244
 (pg. 
359
-
362
)
99
Anon
Hepatitis C: global prevalence
Wkly. Epidemiol. Rec.
1997
, vol. 
72
 (pg. 
341
-
344
)
100
Firpi
R. J.
Nelson
D. R.
Current and future hepatitis C therapies
Arch. Med. Res.
2007
, vol. 
38
 (pg. 
678
-
690
)
101
Koike
K.
Antiviral treatment of hepatitis C: present status and future prospects
J. Infect. Chemother.
2006
, vol. 
12
 (pg. 
227
-
232
)
102
Rizzetto
M.
Canese
M. G.
Arico
S.
Crivelli
O.
Trepo
C.
Bonino
F.
Verme
G.
Immunofluorescence detection of new antigen–antibody system (delta/anti-delta) associated to hepatitis B virus in liver and in serum of HBsAg carriers
Gut
1977
, vol. 
18
 (pg. 
997
-
1003
)
103
Flodgren
E.
Bengtsson
S.
Knutsson
M.
Strebkova
E. A.
Kidd
A. H.
Alexeyev
O. A.
Kidd-Ljunggren
K.
Recent high incidence of fulminant hepatitis in Samara, Russia: molecular analysis of prevailing hepatitis B and D virus strains
J. Clin. Microbiol.
2000
, vol. 
38
 (pg. 
3311
-
3316
)
104
Jacobson
I. M.
Dienstag
J. L.
The delta hepatitis agent: “viral hepatitis, type D”
Gastroenterology
1984
, vol. 
86
 (pg. 
1614
-
1617
)
105
Ueno
Y.
Moritoki
Y.
Shimosegawa
T.
Gershwin
M. E.
Primary biliary cirrhosis: what we know and what we want to know about human PBC and spontaneous PBC mouse models
J. Gastroenterol.
2007
, vol. 
42
 (pg. 
189
-
195
)
106
Jones
D. E.
Pathogenesis of primary biliary cirrhosis
Gut
2007
, vol. 
56
 (pg. 
1615
-
1624
)
107
Fussey
S. P.
Guest
J. R.
James
O. F.
Bassendine
M. F.
Yeaman
S. J.
Identification and analysis of the major M2 autoantigens in primary biliary cirrhosis
Proc. Natl. Acad. Sci. U.S.A.
1988
, vol. 
85
 (pg. 
8654
-
8658
)
108
Poo
J. L.
Feldmann
G.
Erlinger
S.
Braillon
A.
Gaudin
C.
Dumont
M.
Lebrec
D.
Ursodeoxycholic acid limits liver histologic alterations and portal hypertension induced by bile duct ligation in the rat
Gastroenterology
1992
, vol. 
102
 (pg. 
1752
-
1759
)
109
Schuetz
E. G.
Strom
S.
Yasuda
K.
Lecureur
V.
Assem
M.
Brimer
C.
Lamba
J.
Kim
R. B.
Ramachandran
V.
Komoroski
B. J.
, et al. 
Disrupted bile acid homeostasis reveals an unexpected interaction among nuclear hormone receptors, transporters, and cytochrome P450
J. Biol. Chem.
2001
, vol. 
276
 (pg. 
39411
-
39418
)
110
Dilger
K.
Denk
A.
Heeg
M. H.
Beuers
U.
No relevant effect of ursodeoxycholic acid on cytochrome P450 3A metabolism in primary biliary cirrhosis
Hepatology
2005
, vol. 
41
 (pg. 
595
-
602
)
111
Jones
D. E.
Palmer
J. M.
Kirby
J. A.
de Cruz
D. J.
McCaughan
G. W.
Sedgwick
J. D.
Yeaman
S. J.
Burt
A. D.
Bassendine
M. F.
Experimental autoimmune cholangitis: a mouse model of immune-mediated cholangiopathy
Liver
200
, vol. 
20
 (pg. 
351
-
356
)
112
Palmer
J. M.
Robe
A. J.
Burt
A. D.
Kirby
J. A.
Jones
D. E.
Covalent modification as a mechanism for the breakdown of immune tolerance to pyruvate dehydrogenase complex in the mouse
Hepatology
2004
, vol. 
39
 (pg. 
1583
-
1592
)
113
Irie
J.
Wu
Y.
Wicker
L. S.
Rainbow
D.
Nalesnik
M. A.
Hirsch
R.
Peterson
L. B.
Leung
P. S.
Cheng
C.
Mackay
I. R.
, et al. 
NOD.c3c4 congenic mice develop autoimmune biliary disease that serologically and pathogenetically models human primary biliary cirrhosis
J. Exp. Med.
2006
, vol. 
203
 (pg. 
1209
-
1219
)
114
Czaja
A. J.
Autoimmune liver disease
Curr. Opin. Gastroenterol.
2007
, vol. 
23
 (pg. 
255
-
262
)
115
Alvarez
F.
Berg
P. A.
Bianchi
F. B.
Bianchi
L.
Burroughs
A. K.
Cancado
E. L.
Chapman
R. W.
Cooksley
W. G.
Czaja
A. J.
Desmet
V. J.
, et al. 
International Autoimmune Hepatitis Group Report: review of criteria for diagnosis of autoimmune hepatitis
J. Hepatol.
1999
, vol. 
31
 (pg. 
929
-
938
)
116
Wies
I.
Brunner
S.
Henninger
J.
Herkel
J.
Kanzler
S.
Meyer zum Buschenfelde
K. H.
Lohse
A. W.
Identification of target antigen for SLA/LP autoantibodies in autoimmune hepatitis
Lancet
2000
, vol. 
355
 (pg. 
1510
-
1515
)
117
Feder
J. N.
Gnirke
A.
Thomas
W.
Tsuchihashi
Z.
Ruddy
D. A.
Basava
A.
Dormishian
F.
Domingo
R.
Jr
Ellis
M. C.
Fullan
A.
, et al. 
A novel MHC class I-like gene is mutated in patients with hereditary haemochromatosis
Nat. Genet.
1996
, vol. 
13
 (pg. 
399
-
408
)
118
Motulsky
A. G.
Beutler
E.
Population screening in hereditary hemochromatosis
Annu. Rev. Public Health
2000
, vol. 
21
 (pg. 
65
-
79
)
119
Tavill
A. S.
Adams
P. C.
A diagnostic approach to hemochromatosis
Can. J. Gastroenterol.
2006
, vol. 
20
 (pg. 
535
-
540
)
120
Ferenci
P.
Wilson's disease
Clin. Gastroenterol. Hepatol.
2005
, vol. 
3
 (pg. 
726
-
733
)
121
Das
S. K.
Ray
K.
Wilson's disease: an update
Nat. Clin. Pract. Neurol.
2006
, vol. 
2
 (pg. 
482
-
493
)
122
Butler
P.
McIntyre
N.
Mistry
P. K.
Molecular diagnosis of Wilson disease
Mol. Genet. Metab.
2001
, vol. 
72
 (pg. 
223
-
230
)
123
Friedman
M.
Chemical basis for pharmacological and therapeutic actions of penicillamine
Adv. Exp. Med. Biol.
1977
, vol. 
86B
 (pg. 
649
-
673
)
124
Li
Y.
Togashi
Y.
Sato
S.
Emoto
T.
Kang
J. H.
Takeichi
N.
Kobayashi
H.
Kojima
Y.
Une
Y.
Uchino
J.
Spontaneous hepatic copper accumulation in Long–Evans Cinnamon rats with hereditary hepatitis: a model of Wilson's disease
J. Clin. Invest.
1991
, vol. 
87
 (pg. 
1858
-
1861
)
125
Colombo
C.
Russo
M. C.
Zazzeron
L.
Romano
G.
Liver disease in cystic fibrosis
J. Pediatr. Gastroenterol. Nutr.
2006
, vol. 
43
 (pg. 
S49
-
S55
)
126
Burn
J.
Magnay
D.
Claber
O.
Curtis
A.
Population screening in cystic fibrosis
J. R. Soc. Med.
1993
, vol. 
86
 (pg. 
2
-
6
)
127
Brigman
C.
Feranchak
A.
Liver involvement in cystic fibrosis
Curr. Treat. Options Gastroenterol.
2006
, vol. 
9
 (pg. 
484
-
496
)
128
Cohn
J. A.
Strong
T. V.
Picciotto
M. R.
Nairn
A. C.
Collins
F. S.
Fitz
J. G.
Localization of the cystic fibrosis transmembrane conductance regulator in human bile duct epithelial cells
Gastroenterology
1993
, vol. 
105
 (pg. 
1857
-
1864
)
129
Day
C. P.
Natural history of NAFLD: remarkably benign in the absence of cirrhosis
Gastroenterology
2005
, vol. 
129
 (pg. 
375
-
378
)
130
Marchesini
G.
Brizi
M.
Bianchi
G.
Tomassetti
S.
Zoli
M.
Melchionda
N.
Metformin in non-alcoholic steatohepatitis
Lancet
2001
, vol. 
358
 (pg. 
893
-
894
)
131
Belfort
R.
Harrison
S. A.
Brown
K.
Darland
C.
Finch
J.
Hardies
J.
Balas
B.
Gastaldelli
A.
Tio
F.
Pulcini
J.
, et al. 
A placebo-controlled trial of pioglitazone in subjects with nonalcoholic steatohepatitis
N. Engl. J. Med.
2006
, vol. 
355
 (pg. 
2297
-
2307
)
132
Day
C. P.
James
O. F.
Steatohepatitis: a tale of two “hits”?
Gastroenterology
1998
, vol. 
114
 (pg. 
842
-
845
)
133
Tilg
H.
Day
C. P.
Management strategies in alcoholic liver disease
Nat. Clin. Pract. Gastroenterol. Hepatol.
2007
, vol. 
4
 (pg. 
24
-
34
)
134
Ruhl
C. E.
Everhart
J. E.
Coffee and tea consumption are associated with a lower incidence of chronic liver disease in the United States
Gastroenterology
2005
, vol. 
129
 (pg. 
1928
-
1936
)
135
Bates
T.
Harrison
M.
Lowe
D.
Lawson
C.
Padley
N.
Longitudinal study of gall stone prevalence at necropsy
Gut
1992
, vol. 
33
 (pg. 
103
-
107
)
136
Valla
D. C.
The diagnosis and management of the Budd–Chiari syndrome: consensus and controversies
Hepatology
2003
, vol. 
38
 (pg. 
793
-
803
)
137
Senzolo
M.
Cholongitas
E. C.
Patch
D.
Burroughs
A. K.
Update on the classification, assessment of prognosis and therapy of Budd–Chiari syndrome
Nat. Clin. Pract. Gastroenterol. Hepatol.
2005
, vol. 
2
 (pg. 
182
-
190
)
138
LaRusso
N. F.
Shneider
B. L.
Black
D.
Gores
G. J.
James
S. P.
Doo
E.
Hoofnagle
J. H.
Primary sclerosing cholangitis: summary of a workshop
Hepatology
2006
, vol. 
44
 (pg. 
746
-
764
)
139
Olsson
R.
Danielsson
A.
Jarnerot
G.
Lindstrom
E.
Loof
L.
Rolny
P.
Ryden
B. O.
Tysk
C.
Wallerstedt
S.
Prevalence of primary sclerosing cholangitis in patients with ulcerative colitis
Gastroenterology
1991
, vol. 
100
 (pg. 
1319
-
1323
)
140
Moll
R.
Franke
W. W.
Schiller
D. L.
Geiger
B.
Krepler
R.
The catalog of human cytokeratins: patterns of expression in normal epithelia, tumors and cultured cells
Cell
1982
, vol. 
31
 (pg. 
11
-
24
)
141
Polfliet
M. M.
Fabriek
B. O.
Daniels
W. P.
Dijkstra
C. D.
van den Berg
T. K.
The rat macrophage scavenger receptor CD163: expression, regulation and role in inflammatory mediator production
Immunobiology
2006
, vol. 
211
 (pg. 
419
-
425
)
142
Lalor
P. F.
Lai
W. K.
Curbishley
S. M.
Shetty
S.
Adams
D. H.
Human hepatic sinusoidal endothelial cells can be distinguished by expression of phenotypic markers related to their specialised functions in vivo
World J. Gastroenterol.
2006
, vol. 
12
 (pg. 
5429
-
5439
)
143
Zhao
L.
Burt
A. D.
The diffuse stellate cell system
J. Mol. Histol.
2007
, vol. 
38
 (pg. 
53
-
64
)
144
van Eyken
P.
Sciot
R.
van Damme
B.
de Wolf-Peeters
C.
Desmet
V. J.
Keratin immunohistochemistry in normal human liver
Cytokeratin pattern of hepatocytes, bile ducts and acinar gradient
Virchows Arch. A. Pathol. Anat. Histopathol
1987
, vol. 
412
 (pg. 
63
-
72
)
145
Shimonishi
T.
Miyazaki
K.
Nakanuma
Y.
Cytokeratin profile relates to histological subtypes and intrahepatic location of intrahepatic cholangiocarcinoma and primary sites of metastatic adenocarcinoma of liver
Histopathology
2000
, vol. 
37
 (pg. 
55
-
63
)
146
Libbrecht
L.
Cassiman
D.
Desmet
V.
Roskams
T.
The correlation between portal myofibroblasts and development of intrahepatic bile ducts and arterial branches in human liver
Liver
2002
, vol. 
22
 (pg. 
252
-
258
)
147
Alison
M. R.
Vig
P.
Russo
F.
Bigger
B. W.
Amofah
E.
Themis
M.
Forbes
S.
Hepatic stem cells: from inside and outside the liver?
Cell Prolif.
2004
, vol. 
37
 (pg. 
1
-
21
)
148
He
Z. P.
Tan
W. Q.
Tang
Y. F.
Zhang
H. J.
Feng
M. F.
Activation, isolation, identification and in vitro proliferation of oval cells from adult rat livers
Cell Prolif.
2004
, vol. 
37
 (pg. 
177
-
187
)
149
Crosby
H. A.
Hubscher
S. G.
Joplin
R. E.
Kelly
D. A.
Strain
A. J.
Immunolocalization of OV-6, a putative progenitor cell marker in human fetal and diseased pediatric liver
Hepatology
1998
, vol. 
28
 (pg. 
980
-
985
)
150
Doherty
D. G.
O'Farrelly
C.
Innate and adaptive lymphoid cells in the human liver
Immunol. Rev.
2000
, vol. 
174
 (pg. 
5
-
20
)
151
Pusztaszeri
M. P.
Seelentag
W.
Bosman
F. T.
Immunohistochemical expression of endothelial markers CD31, CD34, von Willebrand factor, and Fli-1 in normal human tissues
J. Histochem. Cytochem.
2006
, vol. 
54
 (pg. 
385
-
395
)
152
Kono
T.
Shimoda
M.
Takahashi
M.
Matsumoto
K.
Yoshimoto
T.
Mizutani
M.
Tabata
C.
Okoshi
K.
Wada
H.
Kubo
H.
Immunohistochemical detection of the lymphatic marker podoplanin in diverse types of human cancer cells using a novel antibody
Int. J. Oncol.
2007
, vol. 
31
 (pg. 
501
-
508
)
153
Manabe
N.
Chevallier
M.
Chossegros
P.
Causse
X.
Guerret
S.
Trepo
C.
Grimaud
J. A.
Interferon-α 2b therapy reduces liver fibrosis in chronic non-A, non-B hepatitis: a quantitative histological evaluation
Hepatology
1993
, vol. 
18
 (pg. 
1344
-
1349
)
154
Guerret
S.
Desmouliere
A.
Chossegros
P.
Costa
A. M.
Badid
C.
Trepo
C.
Grimaud
J. A.
Chevallier
M.
Long-term administration of interferon-α in non-responder patients with chronic hepatitis C: follow-up of liver fibrosis over 5 years
J. Viral Hepat.
1999
, vol. 
6
 (pg. 
125
-
133
)
155
Fort
J.
Pilette
C.
Veal
N.
Oberti
F.
Gallois
Y.
Douay
O.
Rosenbaum
J.
Cales
P.
Effects of long-term administration of interferon α in two models of liver fibrosis in rats
J. Hepatol.
1998
, vol. 
29
 (pg. 
263
-
270
)
156
Baroni
G. S.
D'Ambrosio
L.
Curto
P.
Casini
A.
Mancini
R.
Jezequel
A. M.
Benedetti
A.
Interferon γ decreases hepatic stellate cell activation and extracellular matrix deposition in rat liver fibrosis
Hepatology
1996
, vol. 
23
 (pg. 
1189
-
1199
)
157
Sakaida
I.
Uchida
K.
Matsumura
Y.
Okita
K.
Interferon γ treatment prevents procollagen gene expression without affecting transforming growth factor-β1 expression in pig serum-induced rat liver fibrosis in vivo
J. Hepatol.
1998
, vol. 
28
 (pg. 
471
-
479
)
158
Watson
M. W.
Jaksic
A.
Price
P.
Cheng
W.
McInerney
M.
French
M. A.
Fisher
S.
Lee
S.
Flexman
J. P.
Interferon-γ response by peripheral blood mononuclear cells to hepatitis C virus core antigen is reduced in patients with liver fibrosis
J. Infect. Dis.
2003
, vol. 
188
 (pg. 
1533
-
1536
)
159
Mancini
R.
Benedetti
A.
Jezequel
A. M.
An interleukin-1 receptor antagonist decreases fibrosis induced by dimethylnitrosamine in rat liver
Virchows Arch.
1994
, vol. 
424
 (pg. 
25
-
31
)
160
Tiggelman
A. M.
Boers
W.
Linthorst
C.
Sala
M.
Chamuleau
R. A.
Collagen synthesis by human liver (myo)fibroblasts in culture: evidence for a regulatory role of IL-1β, IL-4, TGFβ and IFNγ
J. Hepatol.
1995
, vol. 
23
 (pg. 
307
-
317
)
161
Han
Y. P.
Zhou
L.
Wang
J.
Xiong
S.
Garner
W. L.
French
S. W.
Tsukamoto
H.
Essential role of matrix metalloproteinases in interleukin-1-induced myofibroblastic activation of hepatic stellate cell in collagen
J. Biol. Chem.
2004
, vol. 
279
 (pg. 
4820
-
4828
)
162
Streetz
K. L.
Tacke
F.
Leifeld
L.
Wustefeld
T.
Graw
A.
Klein
C.
Kamino
K.
Spengler
U.
Kreipe
H.
Kubicka
S.
, et al. 
Interleukin 6/gp130-dependent pathways are protective during chronic liver diseases
Hepatology
2003
, vol. 
38
 (pg. 
218
-
229
)
163
Cressman
D. E.
Greenbaum
L. E.
DeAngelis
R. A.
Ciliberto
G.
Furth
E. E.
Poli
V.
Taub
R.
Liver failure and defective hepatocyte regeneration in interleukin-6-deficient mice
Science
1996
, vol. 
274
 (pg. 
1379
-
1383
)
164
Choi
I.
Kang
H. S.
Yang
Y.
Pyun
K. H.
IL-6 induces hepatic inflammation and collagen synthesis in vivo
Clin. Exp. Immunol.
1994
, vol. 
95
 (pg. 
530
-
535
)
165
Natsume
M.
Tsuji
H.
Harada
A.
Akiyama
M.
Yano
T.
Ishikura
H.
Nakanishi
I.
Matsushima
K.
Kaneko
S.
Mukaida
N.
Attenuated liver fibrosis and depressed serum albumin levels in carbon tetrachloride-treated IL-6-deficient mice
J. Leukocyte Biol.
1999
, vol. 
66
 (pg. 
601
-
608
)
166
Louis
H.
Van Laethem
J. L.
Wu
W.
Quertinmont
E.
Degraef
C.
Van Den Berg
K.
Demols
A.
Goldman
M.
Le Moine
O.
Geerts
A.
Devière
J.
Interleukin-10 controls neutrophilic infiltration, hepatocyte proliferation, and liver fibrosis induced by carbon tetrachloride in mice
Hepatology
1998
, vol. 
28
 (pg. 
1607
-
1615
)
167
Thompson
K.
Maltby
J.
Fallowfield
J.
McAulay
M.
Millward-Sadler
H.
Sheron
N.
Interleukin-10 expression and function in experimental murine liver inflammation and fibrosis
Hepatology
1998
, vol. 
28
 (pg. 
1597
-
1606
)
168
Nelson
D. R.
Lauwers
G. Y.
Lau
J. Y.
Davis
G. L.
Interleukin 10 treatment reduces fibrosis in patients with chronic hepatitis C: a pilot trial of interferon nonresponders
Gastroenterology
2000
, vol. 
118
 (pg. 
655
-
660
)
169
Pinzani
M.
Gesualdo
L.
Sabbah
G. M.
Abboud
H. E.
Effects of platelet-derived growth factor and other polypeptide mitogens on DNA synthesis and growth of cultured rat liver fat-storing cells
J. Clin. Invest.
1989
, vol. 
84
 (pg. 
1786
-
1793
)
170
Shiraishi
T.
Morimoto
S.
Koh
E.
Fukuo
K.
Ogihara
T.
Increased release of platelet-derived growth factor from platelets in chronic liver disease
Eur. J. Clin. Chem. Clin. Biochem.
1994
, vol. 
32
 (pg. 
5
-
9
)
171
Pinzani
M.
Milani
S.
Herbst
H.
DeFranco
R.
Grappone
C.
Gentilini
A.
Caligiuri
A.
Pellegrini
G.
Ngo
D. V.
Romanelli
R. G.
Gentilini
P.
Expression of platelet-derived growth factor and its receptors in normal human liver and during active hepatic fibrogenesis
Am. J. Pathol.
1996
, vol. 
148
 (pg. 
785
-
800
)
172
Borkham-Kamphorst
E.
Herrmann
J.
Stoll
D.
Treptau
J.
Gressner
A. M.
Weiskirchen
R.
Dominant-negative soluble PDGF-β receptor inhibits hepatic stellate cell activation and attenuates liver fibrosis
Lab. Invest.
2004
, vol. 
84
 (pg. 
766
-
777
)
173
Armendariz-Borunda
J.
Katayama
K.
Seyer
J. M.
Transcriptional mechanisms of type I collagen gene expression are differentially regulated by interleukin-1β, tumor necrosis factor α, and transforming growth factor β in Ito cells
J. Biol. Chem.
1992
, vol. 
267
 (pg. 
14316
-
14321
)
174
Sanderson
N.
Factor
V.
Nagy
P.
Kopp
J.
Kondaiah
P.
Wakefield
L.
Roberts
A. B.
Sporn
M. B.
Thorgeirsson
S. S.
Hepatic expression of mature transforming growth factor β1 in transgenic mice results in multiple tissue lesions
Proc. Natl. Acad. Sci. U.S.A.
1995
, vol. 
92
 (pg. 
2572
-
2576
)
175
George
J.
Roulot
D.
Koteliansky
V. E.
Bissell
D. M.
In vivo inhibition of rat stellate cell activation by soluble transforming growth factor β type II receptor: a potential new therapy for hepatic fibrosis
Proc. Natl. Acad. Sci. U.S.A.
1999
, vol. 
96
 (pg. 
12719
-
12724
)
176
Arias
M.
Sauer-Lehnen
S.
Treptau
J.
Janoschek
N.
Theuerkauf
I.
Buettner
R.
Gressner
A. M.
Weiskirchen
R.
Adenoviral expression of a transforming growth factor-β1 antisense mRNA is effective in preventing liver fibrosis in bile-duct ligated rats
BMC Gastroenterol.
2003
, vol. 
3
 pg. 
29
 
177
Simeonova
P. P.
Gallucci
R. M.
Hulderman
T.
Wilson
R.
Kommineni
C.
Rao
M.
Luster
M. I.
The role of tumor necrosis factor-α in liver toxicity, inflammation, and fibrosis induced by carbon tetrachloride
Toxicol. Appl. Pharmacol.
2001
, vol. 
177
 (pg. 
112
-
120
)
178
Yamada
Y.
Fausto
N.
Deficient liver regeneration after carbon tetrachloride injury in mice lacking type 1 but not type 2 tumor necrosis factor receptor
Am. J. Pathol.
1998
, vol. 
152
 (pg. 
1577
-
1589
)
179
Kamada
Y.
Tamura
S.
Kiso
S.
Matsumoto
H.
Saji
Y.
Yoshida
Y.
Fukui
K.
Maeda
N.
Nishizawa
H.
Nagaretani
H.
, et al. 
Enhanced carbon tetrachlorideinduced liver fibrosis in mice lacking adiponectin
Gastroenterology
2003
, vol. 
125
 (pg. 
1796
-
1807
)
180
Yoshiji
H.
Kuriyama
S.
Yoshii
J.
Ikenaka
Y.
Noguchi
R.
Nakatani
T.
Tsujinoue
H.
Fukui
H.
Angiotensin-II type 1 receptor interaction is a major regulator for liver fibrosis development in rats
Hepatology
2001
, vol. 
34
 (pg. 
745
-
750
)
181
Julien
B.
Grenard
P.
Teixeira-Clerc
F.
van Nhieu
J. T.
Li
L.
Karsak
M.
Zimmer
A.
Mallat
A.
Lotersztajn
S.
Antifibrogenic role of the cannabinoid receptor CB2 in the liver
Gastroenterology
2005
, vol. 
128
 (pg. 
742
-
755
)
182
Teixeira-Clerc
F.
Julien
B.
Grenard
P.
Tran Van Nhieu
J.
Deveaux
V.
Li
L.
Serriere-Lanneau
V.
Ledent
C.
Mallat
A.
Lotersztajn
S.
CB1 cannabinoid receptor antagonism: a new strategy for the treatment of liver fibrosis
Nat. Med.
2006
, vol. 
12
 (pg. 
671
-
676
)
183
Ikejima
K.
Takei
Y.
Honda
H.
Hirose
M.
Yoshikawa
M.
Zhang
Y. J.
Lang
T.
Fukuda
T.
Yamashina
S.
Kitamura
T.
Sato
N.
Leptin receptor-mediated signaling regulates hepatic fibrogenesis and remodeling of extracellular matrix in the rat
Gastroenterology
2002
, vol. 
122
 (pg. 
1399
-
1410
)
184
Ebrahimkhani
M. R.
Kiani
S.
Oakley
F.
Kendall
T.
Shariftabrizi
A.
Tavangar
S. M.
Moezi
L.
Payabvash
S.
Karoon
A.
Hoseininik
H.
, et al. 
Naltrexone, an opioid receptor antagonist, attenuates liver fibrosis in bile duct ligated rats
Gut
2006
, vol. 
55
 (pg. 
1606
-
1616
)
185
Constandinou
C.
Henderson
N.
Iredale
J. P.
Modeling liver fibrosis in rodents
Methods Mol. Med.
2005
, vol. 
117
 (pg. 
237
-
250
)
186
Wong
F. W.
Chan
W. Y.
Lee
S. S.
Resistance to carbon tetrachloride-induced hepatotoxicity in mice which lack CYP2E1 expression
Toxicol. Appl. Pharmacol.
1998
, vol. 
153
 (pg. 
109
-
118
)
187
Plaa
G. L.
Chlorinated methanes and liver injury: highlights of the past 50 years
Annu. Rev. Pharmacol. Toxicol.
2000
, vol. 
40
 (pg. 
42
-
65
)
188
Cheeseman
K. H.
Albano
E. F.
Tomasi
A.
Slater
T. F.
Biochemical studies on the metabolic activation of halogenated alkanes
Environ. Health Perspect.
1985
, vol. 
64
 (pg. 
85
-
101
)
189
Li
X.
Benjamin
I. S.
Alexander
B.
Reproducible production of thioacetamide-induced macronodular cirrhosis in the rat with no mortality
J. Hepatol.
2002
, vol. 
36
 (pg. 
488
-
493
)
190
Ramaiah
S. K.
Apte
U.
Mehendale
H. M.
Cytochrome P4502E1 induction increases thioacetamide liver injury in diet-restricted rats
Drug Metab. Dispos.
2001
, vol. 
29
 (pg. 
1088
-
1095
)
191
Lee
J. W.
Shin
K. D.
Lee
M.
Kim
E. J.
Han
S. S.
Han
M. Y.
Ha
H.
Jeong
T. C.
Koh
W. S.
Role of metabolism by flavin-containing monooxygenase in thioacetamide-induced immunosuppression
Toxicol. Lett.
2003
, vol. 
136
 (pg. 
163
-
172
)
192
Chilakapati
J.
Korrapati
M. C.
Hill
R. A.
Warbritton
A.
Latendresse
J. R.
Mehendale
H. M.
Toxicokinetics and toxicity of thioacetamide sulfoxide: a metabolite of thioacetamide
Toxicology
2007
, vol. 
230
 (pg. 
105
-
116
)
193
Guo
L.
Enzan
H.
Hayashi
Y.
Miyazaki
E.
Jin
Y.
Toi
M.
Kuroda
N.
Hirió
M.
Increased iron deposition in rat liver fibrosis induced by a high-dose injection of dimethylnitrosamine
Exp. Mol. Pathol.
2006
, vol. 
81
 (pg. 
255
-
261
)
194
Kitamura
K.
Nakamoto
Y.
Akiyama
M.
Fujii
C.
Kondo
T.
Kobayashi
K.
Kaneko
S.
Mukaida
N.
Pathogenic roles of tumor necrosis factor receptor p55-mediated signals in dimethylnitrosamine-induced murine liver fibrosis
Lab. Invest.
2002
, vol. 
82
 (pg. 
571
-
583
)
195
Yoo
J. S.
Ishizaki
H.
Yang
C. S.
Roles of cytochrome P450IIE1 in the dealkylation and denitrosation of N-nitrosodimethylamine and N-nitrosodiethylamine in rat liver microsomes
Carcinogenesis
1990
, vol. 
11
 (pg. 
2239
-
2243
)
196
Pegg
A. E.
Perry
W.
Alkylation of nucleic acids and metabolism of small doses of dimethylnitrosamine in the rat
Cancer Res.
1981
, vol. 
41
 (pg. 
3128
-
3132
)
197
Pritchard
D. J.
Butler
W. H.
Apoptosis: the mechanism of cell death in dimethylnitrosamine-induced hepatotoxicity
J. Pathol.
1989
, vol. 
158
 (pg. 
253
-
260
)
198
Hirata
K.
Ogata
I.
Ohta
Y.
Fujiwara
K.
Hepatic sinusoidal cell destruction in the development of intravascular coagulation in acute liver failure of rats
J. Pathol.
1989
, vol. 
158
 (pg. 
157
-
165
)
199
Baba
Y.
Uetsuka
K.
Nakayama
H.
Dot
K.
Rat strain differences in the early stage of porcine-serum-induced hepatic fibrosis
Exp. Toxicol. Pathol.
2004
, vol. 
55
 (pg. 
325
-
330
)
200
Shiga
A.
Shirota
K.
Ikeda
T.
Nombra
Y.
Morphological and immunohistochemical studies on porcine serum-induced rat liver fibrosis
J. Vet. Med. Sci.
1997
, vol. 
59
 (pg. 
159
-
167
)
201
Bhunchet
E.
Eishi
Y.
Wake
K.
Contribution of immune response to the hepatic fibrosis induced by porcine serum
Hepatology
1996
, vol. 
23
 (pg. 
811
-
817
)
202
Boker
K.
Schwarting
G.
Kaule
G.
Gunzler
V.
Schmidt
E.
Fibrosis of the liver in rats induced by bile duct ligation: effects of inhibition by prolyl 4-hydroxylase
J. Hepatol.
1991
, vol. 
13
 (pg. 
S35
-
S40
)
203
Yerushalmi
B.
Dahl
R.
Devereaux
M. W.
Gumpricht
E.
Sokol
R. J.
Bile acid-induced rat hepatocyte apoptosis is inhibited by antioxidants and blockers of the mitochondrial permeability transition
Hepatology
2001
, vol. 
33
 (pg. 
616
-
626
)
204
Higuchi
H.
Gores
G. J.
Bile acid regulation of hepatic physiology. IV. Bile acids and death receptors
Am. J. Physiol. Gastrointest. Liver Physiol.
2003
, vol. 
284
 (pg. 
G734
-
G738
)
205
Palmeira
C. M.
Rolo
A. P.
Mitochondrially-mediated toxicity of bile acids
Toxicology
2004
, vol. 
203
 (pg. 
1
-
15
)
206
Miyoshi
H.
Rust
C.
Roberts
P. J.
Burgart
L. J.
Gores
G. J.
Hepatocyte apoptosis after bile duct ligation in the mouse involves Fas
Gastroenterology
1999
, vol. 
117
 (pg. 
669
-
677
)
207
Tuchweber
B.
Desmouliere
A.
Bochaton-Piallat
M. L.
Rubbia-Brandt
L.
Gabbiani
G.
Proliferation and phenotypic modulation of portal fibroblasts in the early stages of cholestatic fibrosis in the rat
Lab. Invest.
1996
, vol. 
74
 (pg. 
265
-
278
)
208
Ramadori
G.
Saile
B.
Portal tract fibrogenesis in the liver
Lab. Invest.
2004
, vol. 
84
 (pg. 
153
-
159
)
209
Anstee
Q. M.
Goldin
R. D.
Mouse models in non-alcoholic fatty liver disease and steatohepatitis research
Int. J. Exp. Pathol.
2006
, vol. 
87
 (pg. 
1
-
16
)
210
Hirose
A.
Ono
M.
Saibara
T.
Nozaki
Y.
Masuda
K.
Yoshioka
A.
Takahashi
M.
Akisawa
N.
Iwasaki
S.
Oben
J. A.
Onishi
S.
Angiotensin II type 1 receptor blocker inhibits fibrosis in rat nonalcoholic steatohepatitis
Hepatology
2007
, vol. 
45
 (pg. 
1375
-
1381
)
211
Sahai
A.
Malladi
P.
Melin-Aldana
H.
Green
R. M.
Whitington
P. F.
Upregulation of osteopontin expression is involved in the development of nonalcoholic steatohepatitis in a dietary murine model
Am. J. Physiol. Gastrointest. Liver Physiol.
2004
, vol. 
287
 (pg. 
G264
-
G273
)
212
Kulinski
A.
Vance
D. E.
Vance
J. E.
A choline-deficient diet in mice inhibits neither the CDP-choline pathway for phosphatidylcholine synthesis in hepatocytes nor apolipoprotein B secretion
J. Biol. Chem.
2004
, vol. 
279
 (pg. 
23916
-
23924
)
213
McCuskey
R. S.
Ito
Y.
Robertson
G. R.
McCuskey
M. K.
Perry
M.
Farrell
G. C.
Hepatic microvascular dysfunction during evolution of dietary steatohepatitis in mice
Hepatology
2004
, vol. 
40
 (pg. 
386
-
393
)
214
Koteish
A.
Diehl
A. M
Animal models of steatosis
Semin. Liver Dis.
2001
, vol. 
21
 (pg. 
89
-
104
)
215
Yang
S. Q.
Lin
H. Z.
Lane
M. D.
Clemens
M.
Diehl
A. M.
Obesity increases sensitivity to endotoxin liver injury: implications for the pathogenesis of steatohepatitis
Proc. Natl. Acad. Sci. U.S.A.
1997
, vol. 
94
 (pg. 
2557
-
2562
)
216
Honda
H.
Ikejima
K.
Hirose
M.
Yoshikawa
M.
Lang
T.
Enomoto
N.
Kitamura
T.
Takei
Y.
Sato
N.
Leptin is required for fibrogenic responses induced by thioacetamide in the murine liver
Hepatology
2002
, vol. 
36
 (pg. 
12
-
21
)
217
French
S. W.
Miyamoto
K.
Tsukamoto
H.
Ethanol-induced hepatic fibrosis in the rat: role of the amount of dietary fat
Alcohol Clin. Exp. Res.
1986
, vol. 
10
 (pg. 
13S
-
19S
)
218
Petersen
E. N.
The pharmacology and toxicology of disulfiram and its metabolites
Acta Psychiatr. Scand. Suppl.
1992
, vol. 
369
 (pg. 
7
-
13
)
219
Reference deleted
220
Kono
H.
Bradford
B. U.
Rusyn
I.
Fujii
H.
Matsumoto
Y.
Yin
M.
Thurman
R. G.
Development of an intragastric enteral model in the mouse: studies of alcoholinduced liver disease using knockout technology
J. Hepatobiliary Pancreat. Surg.
2000
, vol. 
7
 (pg. 
395
-
400
)
221
Hall
P. de la M.
Lieber
C. S.
DeCarli
L. M.
French
S. W.
Lindros
K. O.
Jarvelainen
H.
Bode
C.
Parlesak
A.
Bode
J. C.
Models of alcoholic liver disease in rodents: a critical evaluation
Alcohol Clin. Exp. Res.
2001
, vol. 
25
 (pg. 
254S
-
261S
)
222
Tiegs
G.
Hentschel
J.
Wendel
A.
A T cell-dependent experimental liver injury in mice inducible by concanavalin A
J. Clin. Invest.
1992
, vol. 
90
 (pg. 
196
-
203
)
223
Louis
H.
Le Moine
A.
Quertinmont
E.
Peny
M. O.
Geerts
A.
Goldman
M.
Le Moine
O.
Deviere
J.
Repeated concanavalin A challenge in mice induces an interleukin 10-producing phenotype and liver fibrosis
Hepatology
2000
, vol. 
31
 (pg. 
381
-
390
)
224
Lewin
M.
Poujol-Robert
A.
Boelle
P. Y.
Wendum
D.
Lasnier
E.
Viallon
M.
Guechot
J.
Hoeffel
C.
Arrive
L.
Tubiana
J. M.
Poupon
R.
Diffusion-weighted magnetic resonance imaging for the assessment of fibrosis in chronic hepatitis C
Hepatology
2007
, vol. 
46
 (pg. 
658
-
665
)
225
Sandrin
L.
Fourquet
B.
Hasquenoph
J. M.
Yon
S.
Fournier
C.
Mal
F.
Christidis
C.
Ziol
M.
Poulet
B.
Kazemi
F.
, et al. 
Transient elastography: a new noninvasive method for assessment of hepatic fibrosis
Ultrasound Med. Biol.
2003
, vol. 
29
 (pg. 
1705
-
1713
)
226
Wai
C. T.
Greenson
J. K.
Fontana
R. J.
Kalbfleisch
J. D.
Marrero
J. A.
Conjeevaram
H. S.
Lok
A. S.
A simple noninvasive index can predict both significant fibrosis and cirrhosis in patients with chronic hepatitis C
Hepatology
2003
, vol. 
38
 (pg. 
518
-
526
)
227
Rosenberg
W. M.
Voelker
M.
Thiel
R.
Becka
M.
Burt
A.
Schuppan
D.
Hubscher
S.
Roskams
T.
Pinzani
M.
Arthur
M. J.
Serum markers detect the presence of liver fibrosis: a cohort study
Gastroenterology
2004
, vol. 
127
 (pg. 
1704
-
1713
)
228
Cales
P.
Oberti
F.
Michalak
S.
Hubert-Fouchard
I.
Rousselet
M. C.
Konate
A.
Gallois
Y.
Ternisien
C.
Chevailler
A.
Lunel
F.
A novel panel of blood markers to assess the degree of liver fibrosis
Hepatology
2005
, vol. 
42
 (pg. 
1373
-
1381
)
229
Patel
K.
Gordon
S. C.
Jacobson
I.
Hezode
C.
Oh
E.
Smith
K. M.
Pawlotsky
J. M.
McHutchison
J. G.
Evaluation of a panel of non-invasive serum markers to differentiate mild from moderate-to-advanced liver fibrosis in chronic hepatitis C patients
J. Hepatol.
2004
, vol. 
41
 (pg. 
935
-
942
)
230
Poynard
T.
McHutchison
J.
Manns
M.
Myers
R. P.
Albrecht
J.
Biochemical surrogate markers of liver fibrosis and activity in a randomized trial of peginterferon alfa-2b and ribavirin
Hepatology
2003
, vol. 
38
 (pg. 
481
-
492
)
231
Forns
X.
Ampurdanes
S.
Llovet
J. M.
Aponte
J.
Quinto
L.
Martinez-Bauer
E.
Bruguera
M.
Sanchez-Tapias
J. M.
Rodes
J.
Identification of chronic hepatitis C patients without hepatic fibrosis by a simple predictive model
Hepatology
2002
, vol. 
36
 (pg. 
986
-
992
)
232
Adams
L. A.
Bulsara
M.
Rossi
E.
DeBoer
B.
Speers
D.
George
J.
Kench
J.
Farrell
G.
McCaughan
G. W.
Jeffrey
G. P.
Hepascore: an accurate validated predictor of liver fibrosis in chronic hepatitis C infection
Clin. Chem.
2005
, vol. 
51
 (pg. 
1867
-
1873
)
233
Leroy
V.
Hilleret
M. N.
Sturm
N.
Trocme
C.
Renversez
J. C.
Faure
P.
Morel
F.
Zarski
J. P.
Prospective comparison of six non-invasive scores for the diagnosis of liver fibrosis in chronic hepatitis C
J. Hepatol.
2007
, vol. 
46
 (pg. 
775
-
782
)
234
Anon
Intraobserver and interobserver variations in liver biopsy interpretation in patients with chronic hepatitis C. The French METAVIR Cooperative Study Group
Hepatology
1994
, vol. 
20
 (pg. 
15
-
20
)
235
Scheuer
P. J.
Classification of chronic viral hepatitis: a need for reassessment
J. Hepatol.
1991
, vol. 
13
 (pg. 
372
-
374
)
236
Ishak
K.
Baptista
A.
Bianchi
L.
Callea
F.
de Groote
J.
Gudat
F.
Denk
H.
Desmet
V.
Korb
G.
MacSween
R. N.
, et al. 
Histological grading and staging of chronic hepatitis
J. Hepatol.
1995
, vol. 
22
 (pg. 
696
-
699
)
237
Knodell
R. G.
Ishak
K. G.
Black
W. C.
Chen
T. S.
Craig
R.
Kaplowitz
N.
Kiernan
T. W.
Wollman
J.
Formulation and application of a numerical scoring system for assessing histological activity in asymptomatic chronic active hepatitis
Hepatology
1981
, vol. 
1
 (pg. 
431
-
435
)
238
Bancroft
J.
Stevens
A.
In Theory and Practice of Histological Techniques
1982
2nd
New York
Churchill-Livingston
(pg. 
131
-
135
)
239
Jonker
A. M.
Dijkhuis
F. W.
Boes
A.
Hardonk
M. J.
Grond
J.
Immunohistochemical study of extracellular matrix in acute galactosamine hepatitis in rats
Hepatology
1992
, vol. 
15
 (pg. 
423
-
431
)
240
Bergman
I.
Loxley
R.
Two improved and simplified methods for the spectrophotometric determination of hydroxyproline
Anal. Chem.
1963
, vol. 
35
 (pg. 
1961
-
1965
)
241
Biempica
L.
Morecki
R.
Wu
C. H.
Giambrone
M. A.
Rojkind
M.
Immunocytochemical localization of type B collagen: a component of basement membrane in human liver
Am. J. Pathol.
1980
, vol. 
98
 (pg. 
591
-
602
)
242
Xu
L.
Hui
A. Y.
Albanis
E.
Arthur
M. J.
O'Byrne
S. M.
Blaner
W. S.
Mukherjee
P.
Friedman
S. L.
Eng
F. J.
Human hepatic stellate cell lines, LX-1 and LX-2: new tools for analysis of hepatic fibrosis
Gut
2005
, vol. 
54
 (pg. 
142
-
151
)
243
Murakami
K.
Abe
T.
Miyazawa
M.
Yamaguchi
M.
Masuda
T.
Matsuura
T.
Nagamori
S.
Takeuchi
K.
Abe
K.
Kyogoku
M.
Establishment of a new human cell line, LI90, exhibiting characteristics of hepatic Ito (fat-storing) cells
Lab. Invest.
1995
, vol. 
72
 (pg. 
731
-
739
)
244
Shibata
N.
Watanabe
T.
Okitsu
T.
Sakaguchi
M.
Takesue
M.
Kunieda
T.
Omoto
K.
Yamamoto
S.
Tanaka
N.
Kobayashi
N.
Establishment of an immortalized human hepatic stellate cell line to develop antifibrotic therapies
Cell Transplant.
2003
, vol. 
12
 (pg. 
499
-
507
)
245
Vogel
S.
Piantedosi
R.
Frank
J.
Lalazar
A.
Rockey
D. C.
Friedman
S. L.
Blaner
W. S.
An immortalized rat liver stellate cell line (HSC-T6): a new cell model for the study of retinoid metabolism in vitro
J. Lipid Res.
2000
, vol. 
41
 (pg. 
882
-
893
)
246
Sauvant
P.
Sapin
V.
Abergel
A.
Schmidt
C. K.
Blanchon
L.
Alexandre-Gouabau
M. C.
Rosenbaum
J.
Bommelaer
G.
Rock
E.
Dastugue
B.
, et al. 
PAV-1, a new rat hepatic stellate cell line converts retinol into retinoic acid, a process altered by ethanol
Int. J. Biochem. Cell Biol.
2002
, vol. 
34
 (pg. 
1017
-
1029
)
247
Rojkind
M.
Novikoff
P. M.
Greenwel
P.
Rubin
J.
Rojas-Valencia
L.
de Carvalho
A. C.
Stockert
R.
Spray
D.
Hertzberg
E. L.
Wolkoff
A. W.
Characterization and functional studies on rat liver fat-storing cell line and freshly isolated hepatocyte coculture system
Am. J. Pathol.
1995
, vol. 
146
 (pg. 
1508
-
1520
)
248
Miura
N.
Kanayama
Y.
Nagai
W.
Hasegawa
T.
Seko
Y.
Kaji
T.
Naganuma
A.
Characterization of an immortalized hepatic stellate cell line established from metallothionein-null mice
J. Toxicol. Sci.
2006
, vol. 
31
 (pg. 
391
-
398
)
249
Rovira
P.
Mascarell
L.
Truffa-Bachi
P.
The impact of immunosuppressive drugs on the analysis of T cell activation
Curr. Med. Chem.
2000
, vol. 
7
 (pg. 
673
-
692
)
250
Neuberger
J.
Gunson
B.
Hubscher
S.
Nightingale
P.
Immunosuppression affects the rate of recurrent primary biliary cirrhosis after liver transplantation
Liver Transplant.
2004
, vol. 
10
 (pg. 
488
-
491
)
251
Willson
T. M.
Brown
P. J.
Sternbach
D. D.
Henke
B. R.
The PPARs: from orphan receptors to drug discovery
J. Med. Chem.
2000
, vol. 
43
 (pg. 
527
-
550
)
252
Marra
F.
Efsen
E.
Romanelli
R. G.
Caligiuri
A.
Pastacaldi
S.
Batignani
G.
Bonacchi
A.
Caporale
R.
Laffi
G.
Pinzani
M.
Gentilini
P.
Ligands of peroxisome proliferator-activated receptor γ modulate profibrogenic and proinflammatory actions in hepatic stellate cells
Gastroenterology
2000
, vol. 
119
 (pg. 
466
-
478
)
253
Zhao
C.
Chen
W.
Yang
L.
Chen
L.
Stimpson
S. A.
Diehl
A. M.
PPARγ agonists prevent TGFβ1/Smad3 signaling in human hepatic stellate cells
Biochem. Biophys. Res. Commun.
2006
, vol. 
350
 (pg. 
385
-
391
)
254
Fiorucci
S.
Clerici
C.
Antonelli
E.
Orlandi
S.
Goodwin
B.
Sadeghpour
B. M.
Sabatino
G.
Russo
G.
Castellani
D.
Willson
T. M.
, et al. 
Protective effects of 6-ethyl chenodeoxycholic acid, a farnesoid X receptor ligand, in estrogen-induced cholestasis
J. Pharmacol. Exp. Ther.
2005
, vol. 
313
 (pg. 
604
-
612
)
255
Fiorucci
S.
Rizzo
G.
Antonelli
E.
Renga
B.
Mencarelli
A.
Riccardi
L.
Morelli
A.
Pruzanski
M.
Pellicciari
R.
Cross-talk between farnesoid-X-receptor (FXR) and peroxisome proliferator-activated receptor γ contributes to the antifibrotic activity of FXR ligands in rodent models of liver cirrhosis
J. Pharmacol. Exp. Ther.
2005
, vol. 
315
 (pg. 
58
-
68
)
256
Pockros
P. J.
Jeffers
L.
Afdhal
N.
Goodman
Z. D.
Nelson
D.
Gish
R. G.
Reddy
K. R.
Reindollar
R.
Rodriguez-Torres
M.
Sullivan
S.
, et al. 
Final results of a double-blind, placebo-controlled trial of the antifibrotic efficacy of interferon-γ1b in chronic hepatitis C patients with advanced fibrosis or cirrhosis
Hepatology
2007
, vol. 
45
 (pg. 
569
-
578
)
257
Poynard
T.
McHutchison
J.
Manns
M.
Trepo
C.
Lindsay
K.
Goodman
Z.
Ling
M. H.
Albrecht
J.
Impact of pegylated interferon alfa-2b and ribavirin on liver fibrosis in patients with chronic hepatitis C
Gastroenterology
2002
, vol. 
122
 (pg. 
1303
-
1313
)
258
Windmeier
C.
Gressner
A. M.
Pharmacological aspects of pentoxifylline with emphasis on its inhibitory actions on hepatic fibrogenesis
Gen. Pharmacol.
1997
, vol. 
29
 (pg. 
181
-
196
)
259
Dosanjh
A.
Pirfenidone: anti-fibrotic agent with a potential therapeutic role in the management of transplantation patients
Eur. J. Pharmacol.
2006
, vol. 
536
 (pg. 
219
-
222
)
260
Armendariz-Borunda
J.
Islas-Carbajal
M. C.
Meza-Garcia
E.
Rincon
A. R.
Lucano
S.
Sandoval
A. S.
Salazar
A.
Berumen
J.
Alvarez
A.
Covarrubias
A.
, et al. 
A pilot study in patients with established advanced liver fibrosis using pirfenidone
Gut
2006
, vol. 
55
 (pg. 
1663
-
1665
)
261
Wright
M.
Goldin
R.
Hellier
S.
Knapp
S.
Frodsham
A.
Hennig
B.
Hill
A.
Apple
R.
Cheng
S.
Thomas
H.
Thursz
M.
Factor V Leiden polymorphism and the rate of fibrosis development in chronic hepatitis C virus infection
Gut
2003
, vol. 
52
 (pg. 
1206
-
1210
)
262
Duplantier
J. G.
Dubuisson
L.
Senant
N.
Freyburger
G.
Laurendeau
I.
Herbert
J. M.
Desmouliere
A.
Rosenbaum
J.
A role for thrombin in liver fibrosis
Gut
2004
, vol. 
53
 (pg. 
1682
-
1687
)
263
Rappaport
A. M.
The microcirculatory acinar concept of normal and pathological hepatic structure
Beitr. Pathol.
1976
, vol. 
157
 (pg. 
215
-
243
)