Liver fibrosis

Ramón Bataller 1 and David A. Brenner 2

1 Liver Unit, Institut de Malalties Digestives i Metabòliques, Hospital
Clinic, Institut d’Investigació Biomèdiques August Pi i Sunyer
(IDIBAPS), Barcelona, Catalonia, Spain.

2Department of Medicine, Columbia University, New York, New York, USA.

Address correspondence to:
David A. Brenner, Department of Medicine, Columbia University Medical
Center, College of Physicians and Surgeons, 622 West 168th Street, PH
8E-105J, New York, New York 10032, USA. Phone: (212) 305-5838; Fax:
(212) 305-8466; E-mail: dab2106@columbia.edu.

Abstract

Liver fibrosis is the excessive accumulation of extracellular matrix
proteins including collagen that occurs in most types of chronic liver
diseases. Advanced liver fibrosis results in cirrhosis, liver failure,
and portal hypertension and often requires liver transplantation. Our
knowledge of the cellular and molecular mechanisms of liver fibrosis
has greatly advanced. Activated hepatic stellate cells, portal
fibroblasts, and myofibroblasts of bone marrow origin have been
identified as major collagen-producing cells in the injured liver.
These cells are activated by fibrogenic cytokines such as TGF-ß1,
angiotensin II, and leptin. Reversibility of advanced liver fibrosis in
patients has been recently documented, which has stimulated researchers
to develop antifibrotic drugs. Emerging antifibrotic therapies are
aimed at inhibiting the accumulation of fibrogenic cells and/or
preventing the deposition of extracellular matrix proteins. Although
many therapeutic interventions are effective in experimental models of
liver fibrosis, their efficacy and safety in humans is unknown. This
review summarizes recent progress in the study of the pathogenesis and
diagnosis of liver fibrosis and discusses current antifibrotic
strategies.

Historical perspective

Liver fibrosis results from chronic damage to the liver in
conjunction with the accumulation of ECM proteins, which is a
characteristic of most types of chronic liver diseases (1).
The main causes of liver fibrosis in industrialized countries include
chronic HCV infection, alcohol abuse, and nonalcoholic steatohepatitis
(NASH). The accumulation of ECM proteins distorts the hepatic
architecture by forming a fibrous scar, and the subsequent development
of nodules of regenerating hepatocytes defines cirrhosis. Cirrhosis
produces hepatocellular dysfunction and increased intrahepatic
resistance to blood flow, which result in hepatic insufficiency and
portal hypertension, respectively (2).

Hepatic fibrosis was historically thought to be a passive and
irreversible process due to the collapse of the hepatic parenchyma and
its substitution with a collagen-rich tissue (3, 4). Currently, it is considered a model of the wound-healing response to chronic liver injury (5). Early clinical reports in the 1970s suggested that advanced liver fibrosis is potentially reversible (6).
However, liver fibrosis received little attention until the 1980s, when
hepatic stellate cells (HSCs), formerly known as lipocytes, Ito cells,
or perisinusoidal cells, were identified as the main collagen-producing
cells in the liver (7).
This cell type, first described by von Kupffer in 1876, undergoes a
dramatic phenotypic activation in chronic liver diseases with the
acquisition of fibrogenic properties (8). Methods to obtain HSCs from both rodent and human livers were rapidly standardized in the 1980s (9, 10), and prolonged culture of HSCs on plastic was widely accepted as a model for the study of activated HSCs (11). Key signals that modulate HSCs’ fibrogenic actions were delineated (12).
Experimental models for studying liver fibrogenesis in rats and in
transgenic mice were developed, which corroborated the cell culture
studies and led to the identification of key fibrogenic mediators (13).
Besides HSCs, portal myofibroblasts and cells of bone marrow origin
have been recently shown to exhibit fibrogenic potential (14, 15).
At the clinical level, the natural history of liver fibrosis, from
early changes to liver cirrhosis, was delineated in patients with
chronic HCV infection (16, 17).
Rapid and slower fibrosers were identified, and genetic and
environmental factors influencing fibrosis progression were partially
uncovered (18).
Since the demonstration, in the 1990s, that even advanced liver
fibrosis is reversible, researchers have been stimulated to identify
antifibrotic therapies (19).
Biotechnology and pharmaceutical companies are increasingly interested
in developing antifibrotic programs, and clinical trials are currently
underway. However, the most effective therapy for treating hepatic
fibrosis to date is still to remove the causative agent (20).
A number of drugs are able to reduce the accumulation of scar tissue in
experimental models of chronic liver injury. Renin-angiotensin system
blockers and antioxidants are the most promising drugs, although their
efficacy has not been tested in humans. Lack of clinical trials is due
to the requirement of long follow-up studies and to the fact that liver
biopsy, an invasive procedure, is still the gold-standard method for
detecting changes in liver fibrosis. The current effort to develop
noninvasive markers to assess liver fibrosis is expected to facilitate
the design of clinical trials.

Recently, NASH has been recognized as a major cause of liver fibrosis (21). First described by Ludwig et al., it is considered part of the spectrum of nonalcoholic fatty liver diseases (22).
These range from steatosis to cirrhosis and can eventually lead to
hepatocellular carcinoma. NASH is a component of the metabolic
syndrome, which is characterized by obesity, type 2 diabetes mellitus,
and dyslipidemia, with insulin resistance as a common feature. As the
prevalence of obesity is rapidly increasing, a rise in the prevalence
of NASH is anticipated.

This review outlines recent progress in the pathogenesis, diagnosis,
and treatment of liver fibrosis, summarizes recent data on the
mechanisms leading to fibrosis resolution, and discusses future
prospects aimed at developing effective antifibrotic therapies.

Natural history and diagnosis

The onset of liver fibrosis is usually insidious, and most of the
related morbidity and mortality occur after the development of
cirrhosis (16).
In the majority of patients, progression to cirrhosis occurs after an
interval of 15–20 years. Major clinical complications of cirrhosis
include ascites, renal failure, hepatic encephalopathy, and variceal
bleeding. Patients with cirrhosis can remain free of major
complications for several years (compensated cirrhosis). Decompensated
cirrhosis is associated with short survival, and liver transplantation
is often indicated as the only effective therapy (23).
Cirrhosis is also a risk factor for developing hepatocellular
carcinoma. Liver fibrosis progresses rapidly to cirrhosis in several
clinical settings, including repeated episodes of severe acute
alcoholic hepatitis, subfulminant hepatitis, and fibrosing cholestasis
in patients with HCV reinfection after liver transplantation (24). The natural history of liver fibrosis is influenced by both genetic and environmental factors (Table 1).
Epidemiological studies have identified polymorphisms in a number of
candidate genes that may influence the progression of liver fibrosis in
humans (18).
These genetic factors may explain the broad spectrum of responses to
the same etiological agent found in patients with chronic liver
diseases. However, some studies have yielded contradictory results due
to poor study design, and further research is required to clarify the
actual role of genetic variants in liver fibrosis.

Liver biopsy is considered the gold-standard method for the assessment of liver fibrosis (25).
Histologic examination is useful in identifying the underlying cause of
liver disease and assessing the necroinflammatory grade and the stage
of fibrosis. Fibrosis stage is assessed by using scales such as Metavir
(stages I–IV) and Ishak score (stages I–V). Specific staining of ECM
proteins (e.g., with Sirius red) can be used to quantify the degree of
fibrosis, using computer-guided morphometric analysis. Liver biopsy is
an invasive procedure, with pain and major complications occurring in
40% and 0.5% of patients, respectively (26).
Sampling error can occur, especially when small biopsies are analyzed.
Histologic examination is prone to intra- and interobserver variation
and does not predict disease progression (27).
Therefore, there is a need for reliable, simple, and noninvasive
methods for assessing liver fibrosis. Scores that include routine
laboratory tests, such as platelet count, aminotransferase serum
levels, prothrombin time, and serum levels of acute phase proteins have
been proposed (28, 29).
Serum levels of proteins directly related to the hepatic fibrogenic
process are also used as surrogate markers of liver fibrosis (30),
including N-terminal propeptide of type III collagen, hyaluronic acid,
tissue inhibitor of metalloproteinase type 1 (TIMP-1), and YKL-40.
Although these scores are useful in detecting advanced fibrosis
(cirrhosis) in patients, as well as minimal or no fibrosis, they are
not effective for differentiating intermediate grades of fibrosis.
Also, fibrosis-specific markers may reflect fibrogenesis in other
organs (i.e., pancreatic fibrosis in alcoholic patients). Finally,
hepatic fibrosis can be estimated by imaging techniques.
Ultrasonography, computed tomography, and MRI can detect changes in the
hepatic parenchyma due to moderate to severe fibrosis (31).
Due to its low cost, ultrasonography is an appealing technique. It is
able to detect liver cirrhosis based on changes in liver echogenicity
and nodularity as well as signs of portal hypertension. However,
ultrasound is highly operator-dependent, and the presence of increased
liver echogenicity does not reliably differentiate hepatic steatosis
from fibrosis. Noninvasive methods currently in development include
blood protein profiling using proteomic technology and new clinical
glycomics technology, which is based on DNA sequencer/fragment
analyzers able to generate profiles of serum protein N-glycans (32). As the technology becomes validated, the noninvasive diagnosis of liver disease may become routine clinical practice.

Pathogenesis of liver fibrosis

Hepatic fibrosis is the result of the wound-healing response of the liver to repeated injury (1) (Figure 1).
After an acute liver injury (e.g., viral hepatitis), parenchymal cells
regenerate and replace the necrotic or apoptotic cells. This process is
associated with an inflammatory response and a limited deposition of
ECM. If the hepatic injury persists, then eventually the liver
regeneration fails, and hepatocytes are substituted with abundant ECM,
including fibrillar collagen. The distribution of this fibrous material
depends on the origin of the liver injury. In chronic viral hepatitis
and chronic cholestatic disorders, the fibrotic tissue is initially
located around portal tracts, while in alcohol-induced liver disease,
it locates in pericentral and perisinusoidal areas (33). As fibrotic liver diseases advance, disease progression from collagen bands to bridging fibrosis to frank cirrhosis occurs.

Figure 1

Changes in the hepatic architecture (A) associated with advanced hepatic fibrosis (B).
Following chronic liver injury, inflammatory lymphocytes infiltrate the
hepatic parenchyma. Some hepatocytes undergo apoptosis, and Kupffer
cells activate, releasing fibrogenic mediators. HSCs proliferate and
undergo a dramatic phenotypical activation, secreting large amounts of
extracellular matrix proteins. Sinusoidal endothelial cells lose their
fenestrations, and the tonic contraction of HSCs causes increased
resistance to blood flow in the hepatic sinusoid. Figure modified with
permission from Science & Medicine (S28).

Liver fibrosis is associated with major alterations in both the quantity and composition of ECM (34).
In advanced stages, the liver contains approximately 6 times more ECM
than normal, including collagens (I, III, and IV), fibronectin,
undulin, elastin, laminin, hyaluronan, and proteoglycans. Accumulation
of ECM results from both increased synthesis and decreased degradation (35). Decreased activity of ECM-removing MMPs is mainly due to an overexpression of their specific inhibitors (TIMPs).

HSCs are the main ECM-producing cells in the injured liver (36).
In the normal liver, HSCs reside in the space of Disse and are the
major storage sites of vitamin A. Following chronic injury, HSCs
activate or transdifferentiate into myofibroblast-like cells, acquiring
contractile, proinflammatory, and fibrogenic properties (37, 38) (Figure 2A).
Activated HSCs migrate and accumulate at the sites of tissue repair,
secreting large amounts of ECM and regulating ECM degradation. PDGF,
mainly produced by Kupffer cells, is the predominant mitogen for
activated HSCs. Collagen synthesis in HSCs is regulated at the
transcriptional and posttranscriptional levels (39).
Increased collagen mRNA stability mediates the increased collagen
synthesis in activated HSCs. In these cells, posttranscriptional
regulation of collagen is governed by sequences in the 3′ untranslated
region via the RNA-binding protein {alpha} CP2 as well as a stem-loop structure in the 5′ end of collagen mRNA (40).
Interestingly, HSCs express a number of neuroendocrine markers (e.g.,
reelin, nestin, neurotrophins, synaptophysin, and glial-fibrillary
acidic protein) and bear receptors for neurotransmitters (8, 41, 42). Quiescent HSCs express markers that are characteristic of adipocytes (PPAR{gamma}, SREBP-1c, and leptin), while activated HSCs express myogenic markers ({alpha} smooth muscle actin, c-myb, and myocyte enhancer factor–2).

Hepatic cell types other than HSCs may also have fibrogenic
potential. Myofibroblasts derived from small portal vessels proliferate
around biliary tracts in cholestasis-induced liver fibrosis to initiate
collagen deposition (43, 44) (Figure 2B). HSCs and portal myofibroblasts differ in specific cell markers and response to apoptotic stimuli (45).
Culture of CD34 +CD38 – hematopoietic stem cells with various growth
factors has been shown to generate HSCs and myofibroblasts of bone
marrow origin that infiltrate human livers undergoing tissue remodeling
(15, 46).
These data suggest that cells originating in bone marrow can be a
source of fibrogenic cells in the injured liver. Other potential
sources of fibrogenic cells (i.e., epithelial-mesenchymal transition
and circulating fibrocytes) have not been demonstrated in the liver (47, 48).
The relative importance of each cell type in liver fibrogenesis may
depend on the origin of the liver injury. While HSCs are the main
fibrogenic cell type in pericentral areas, portal myofibroblasts may
predominate when liver injury occurs around portal tracts.

A complex interplay among different hepatic cell types takes place during hepatic fibrogenesis (Figure 3) (49). Hepatocytes are targets for most hepatotoxic agents, including hepatitis viruses, alcohol metabolites, and bile acids (50).
Damaged hepatocytes release ROS and fibrogenic mediators and induce the
recruitment of white blood cells by inflammatory cells. Apoptosis of
damaged hepatocytes stimulates the fibrogenic actions of liver
myofibroblasts (51). Inflammatory cells, either lymphocytes or polymorphonuclear cells, activate HSCs to secrete collagen (52). Activated HSCs secrete inflammatory chemokines, express cell adhesion molecules, and modulate the activation of lymphocytes (53). Therefore, a vicious circle in which inflammatory and fibrogenic cells stimulate each other is likely to occur (54). Fibrosis is influenced by different T helper subsets, the Th2 response being associated with more active fibrogenesis (55). Kupffer cells are resident macrophages that play a major role in liver inflammation by releasing ROS and cytokines (56, 57).
In chronic cholestatic disorders (i.e., primary biliary cirrhosis [PBC]
and primary sclerosis cholangitis), epithelial cells stimulate the
accumulated portal myofibroblasts to initiate collagen deposition
around damaged bile ducts (43).
Finally, changes in the composition of the ECM can directly stimulate
fibrogenesis. Type IV collagen, fibrinogen, and urokinase type
plasminogen activator stimulate resident HSCs by activating latent
cytokines such as TGF-ß1 (58).
Fibrillar collagens can bind and stimulate HSCs via discoidin domain
receptor DDR2 and integrins. Moreover, the altered ECM can serve as a
reservoir for growth factors and MMPs (59).

Genetic studies in rodents and humans

Extensive studies using models of hepatic fibrosis in transgenic mice have revealed key genes mediating liver fibrogenesis (1, 18).
Genes regulating hepatocellular apoptosis and/or necrosis (e.g.,
Bcl-xL, Fas) influence the extent of hepatic damage and the subsequent
fibrogenic response (60, 61). Genes regulating the inflammatory response to injury (e.g., IL-1ß, IL-6, IL-10, and IL-13, IFN-{gamma}, SOCS-1, and osteopontin) determine the fibrogenic response to injury (55, 62-65). Genes mediating ROS generation (e.g., NADPH oxidase) regulate both inflammation and ECM deposition (66).
Fibrogenic growth factors (e.g., TGF-ß1, FGF), vasoactive substances
(angiotensin II, norepinephrine), and adipokines (leptin and
adiponectin) are each required for the development of fibrosis (67-70). Finally, removal of excess collagen after cessation of liver injury is regulated by TIMP-1 and TGF-ß1 (71, 72).

Association genetic studies have investigated the role of gene
polymorphisms in the progression of liver fibrosis in patients with
chronic liver diseases (18).
In alcoholic liver disease, candidate genes include genes encoding for
alcohol-metabolizing enzymes and proteins involved in liver toxicity (73).
Polymorphisms in genes encoding alcohol-dehydrogenase,
aldehyde-dehydrogenase, and cytochrome P450 are involved in individual
susceptibility to alcoholism, yet their role in the progression of
liver disease remains controversial. Variations in genes encoding
inflammatory mediators (e.g., TNF-{alpha},
IL-1ß, Il-10, and cytotoxic T lymphocyte antigen–4 [CTLA-4]), the
lipopolysaccharide receptor CD14, and antioxidants (e.g., superoxide
dismutase) may influence the progression of alcohol-induced liver
disease (74, 75). In chronic cholestatic disorders such as PBC, polymorphisms in IL-1ß, IL-1 receptor antagonists, and TNF-{alpha} genes are associated with faster disease progression (76).
Some alleles of the apolipoprotein E gene influence the response to
therapy of PBC with ursodeoxycholic acid, which suggests that genetic
polymorphisms may predict therapeutic response (77).
In HCV liver disease, genetic variations are involved in susceptibility
to persistent HCV infection, response to antiviral therapy, and
progression of liver disease (78).
Polymorphisms in genes involved in the immune response to HCV infection
(e.g., transporter associated with antigen processing 2,
mannose-binding lectin, and specific HLA-II alleles) and fibrogenic
agonists (angiotensinogen and TGF-ß1) influence fibrosis progression (79-81).
The fibrogenic effect of heterozygosity in the C282Y mutation of the
hemochromatosis gene in patients with chronic hepatitis C is
controversial (82, 83). Finally, little is known about genetic factors and NASH (84),
and polymorphisms in fibrogenic mediators such as angiotensinogen and
TGF-ß1 may be associated with more severe liver disease.

Key cytokines involved in liver fibrosis

Cytokines regulating the inflammatory response to injury modulate hepatic fibrogenesis in vivo and in vitro (85). Monocyte chemotactic protein type 1 and RANTES stimulate fibrogenesis while IL-10 and IFN-{gamma} exert the opposite effect (55, 86). Among growth factors, TGF-ß1 appears to be a key mediator in human fibrogenesis (58).
In HSCs, TGF-ß favors the transition to myofibroblast-like cells,
stimulates the synthesis of ECM proteins, and inhibits their
degradation. Strategies aimed at disrupting TGF-ß1 synthesis and/or
signaling pathways markedly decreased fibrosis in experimental models (87). PDGF is the most potent mitogen for HSCs and is upregulated in the fibrotic liver (12); its inhibition attenuates experimental liver fibrogenesis (88).

Cytokines with vasoactive properties also regulate liver
fibrogenesis. Vasodilator substances (e.g., nitric oxide, relaxin)
exert antifibrotic effects while vasoconstrictors (e.g.,
norepinephrine, angiotensin II) have opposite effects (67, 89). Endothelin-1, a powerful vasoconstrictor, stimulates fibrogenesis through its type A receptor (90).
Among vasoactive cytokines, angiotensin II seems to play a major role
in liver fibrogenesis. Angiotensin II is the effector peptide of the
renin-angiotensin system, which is a major regulator of arterial
pressure homeostasis in humans. Key components of this system are
locally expressed in chronically injured livers, and activated HSCs de
novo generate angiotensin II (91, 92).
Importantly, pharmacological and/or genetic ablation of the
renin-angiotensin system markedly attenuates experimental liver
fibrosis (70, 93-98).
Angiotensin II induces hepatic inflammation and stimulates an array of
fibrogenic actions in activated HSCs, including cell proliferation,
cell migration, secretion of proinflammatory cytokines, and collagen
synthesis (66, 99, 100).
These actions are largely mediated by ROS generated by a nonphagocytic
form of NADPH oxidase. Unlike the phagocytic type, NADPH oxidases
present in fibrogenic cell types are constitutively active, producing
relatively low levels of ROS under basal conditions and generating
higher levels of oxidants in response to cytokines, stimulating
redox-sensitive intracellular pathways. NADPH oxidase also plays a key
role in the inflammatory actions of Kupffer cells (101).
Disruption of an active NADPH oxidase protects mice from developing
severe liver injury following prolonged alcohol intake and/or bile duct
ligation (66, 102).

Adipokines, which are cytokines mainly derived from the adipose
tissue, regulate liver fibrogenesis. Leptin is required for HSC
activation and fibrosis development (103, 104). In contrast, adiponectin markedly inhibits liver fibrogenesis in vitro and in vivo (69). The actions of these cytokines may explain why obesity influences fibrosis development in patients with chronic hepatitis C (105).

Intracellular signaling pathways mediating liver fibrogenesis

Data on intracellular pathways regulating liver fibrogenesis are
mainly derived from studies using cultured HSCs, while understanding of
their role in vivo is progressing through experimental fibrogenesis
studies using knockout mice (106).
Several mitogen-activated protein kinases modulate major fibrogenic
actions of HSCs. Extracellular-regulated kinase, which is stimulated in
experimentally induced liver injury, mediates proliferation and
migration of HSCs (107).
In contrast, c-Jun N-terminal kinase regulates apoptosis of hepatocytes
as well as the secretion of inflammatory cytokines by cultured HSCs (66, 108, 109). The focal adhesion kinase PI3K-Akt–signaling pathway mediates agonist-induced fibrogenic actions in HSCs (107). The TGF-ß1–activated Smad-signaling pathway stimulates experimental hepatic fibrosis and is a potential target for therapy (110, 111). The PPAR pathway regulates HSC activation and experimental liver fibrosis. PPAR-{gamma} ligands inhibit the fibrogenic actions in HSCs and attenuate liver fibrosis in vivo (112, 113). NF-{kappa}B may have an inhibitory action on liver fibrosis (114, 115). Other transcription factors are involved in HSC activation and may participate in liver fibrogenesis (116). Recent studies suggest a role for intracellular pathways signaled by Toll-like receptors and ß-cathepsin (117, 118).

Pathogenesis of fibrosis in different liver diseases

The pathogenesis of liver fibrosis depends on the underlying
etiology. In alcohol-induced liver disease, alcohol alters the
population of gut bacteria and inhibits intestinal motility, resulting
in an overgrowth of Gram-negative flora. Lipopolysaccharide is elevated
in portal blood and activates Kupffer cells through the CD14/Toll-like
receptor–4 complex to produce ROS via NADPH oxidase (101). Oxidants activate Kupffer cell NF-{kappa}B, causing an increase in TNF-{alpha} production. TNF-{alpha}
induces neutrophil infiltration and stimulates mitochondrial oxidant
production in hepatocytes, which are sensitized to undergo apoptosis.
Acetaldehyde, the major alcohol metabolism product, and ROS activate
HSCs and stimulate inflammatory and fibrogenic signals (119).
The pathogenesis of HCV-induced liver fibrosis is poorly understood due
to the lack of a rodent model of persistent HCV infection (78).
HCV escapes surveillance of the HLA-II–directed immune response and
infects hepatocytes, causing oxidative stress and inducing the
recruitment of inflammatory cells. Both factors lead to HSC activation
and collagen deposition. Moreover, several HCV proteins directly
stimulate the inflammatory and fibrogenic actions of HSCs (120). In chronic cholestatic disorders such as PBC, T lymphocytes and cytokines mediate persistent bile duct damage (14).
Biliary cells secrete fibrogenic mediators activating neighboring
portal myofibroblasts to secrete ECM. Eventually, perisinusoidal HSCs
become activated, and fibrotic bands develop. The pathogenesis of liver
fibrosis due to NASH is poorly understood. Obesity, type 2 diabetes
mellitus, and dyslipidemia are the most common associated conditions (121).
A 2-hit model has been proposed: hyperglycemia and insulin resistance
lead to elevated serum levels of free fatty acids, resulting in hepatic
steatosis. In the second hit, oxidative stress and proinflammatory
cytokines promote hepatocyte apoptosis and the recruitment of
inflammatory cells, leading to progressive fibrosis.

Is liver fibrosis reversible?

In contrast with the traditional view that cirrhosis is an
irreversible disease, recent evidence indicates that even advanced
fibrosis is reversible (122). In experimentally induced fibrosis, cessation of liver injury results in fibrosis regression (123).
In humans, spontaneous resolution of liver fibrosis can occur after
successful treatment of the underlying disease. This observation has
been described in patients with iron and copper overload,
alcohol-induced liver injury, chronic hepatitis C, B, and D,
hemochromatosis, secondary biliary cirrhosis, NASH, and autoimmune
hepatitis (19, 122, 124, 125, S1, S2) (Figure 4).
It may take years for significant regression to be achieved; the time
varies depending on the underlying cause of the liver disease and its
severity. Chronic HCV infection is the most extensively studied
condition, and therapy (IFN-{alpha}
plus ribavirin) with viral clearance results in fibrosis improvement.
Importantly, nearly half of patients with cirrhosis exhibit reversal to
a significant degree (90).
Whether this beneficial effect is associated with improvements in
long-term clinical outcome, including decreased portal hypertension, is
unknown.

Increased collagenolytic activity is a major mechanism of fibrosis resolution (122).
Fibrillar collagens (I and III) are degraded by interstitial MMPs
(MMP-1, -8, and -13 in humans and MMP-13 in rodents). During fibrosis
resolution, MMP activity increases due to a rapid decrease in the
expression of TIMP-1. Partial degradation of fibrillar collagen occurs,
and the altered interaction between activated HSCs and ECM favors
apoptosis (123).
Removal of activated HSCs by apoptosis precedes fibrosis resolution.
Stimulation of death receptors in activated HSCs and a decrease in
survival factors, including TIMP-1, can precipitate HSC apoptosis (S3).

Several questions remain unanswered: Can we pharmacologically
accelerate fibrosis resolution in humans? Can a fibrotic liver
completely regress to a normal liver? Does fibrosis reverse similarly
in all types of liver diseases? Although isolated cases of complete
fibrosis resolution have been reported, it is conceivable that some
degree of fibrosis cannot be removed (S4). Resolution may be limited by
ECM cross-linking and a failure of activated HSCs to undergo apoptosis.

Therapeutic approaches to the treatment of liver fibrosis

There is no standard treatment for liver fibrosis. Although
experimental studies have revealed targets to prevent fibrosis
progression in rodents (20) (Table 2),
the efficacy of most treatments has not been proven in humans. This is
due to the need to perform serial liver biopsies to accurately assess
changes in liver fibrosis, the necessity of long-term follow-up
studies, and the fact that humans are probably less sensitive to
hepatic antifibrotic therapies than rodents. The development of
reliable noninvasive markers of liver fibrosis should have a positive
impact on the design of clinical trials. The ideal antifibrotic therapy
would be one that is liver-specific, well tolerated when administered
for prolonged periods of time, and effective in attenuating excessive
collagen deposition without affecting normal ECM synthesis. The removal
of the causative agent is the most effective intervention in the
treatment of liver fibrosis. This strategy has been shown effective in
most etiologies of chronic liver diseases (19, 122, 124, 125, S1, S2).
For patients with cirrhosis and clinical complications, liver
transplantation is currently the only curative approach (S5).
Transplantation improves both survival and quality of life. However, in
patients with HCV-induced cirrhosis, viral infection recurs after
transplantation (S6), aggressive chronic hepatitis develops, and
progression to cirrhosis is common.

Because inflammation precedes and promotes the progression of liver
fibrosis, the use of antiinflammatory drugs has been proposed.
Corticosteroids are only indicated for the treatment of hepatic
fibrosis in patients with autoimmune hepatitis and acute alcoholic
hepatitis (S1). Inhibition of the accumulation of activated HSCs by
modulating either their activation and/or proliferation or promoting
their apoptosis is another strategy. Antioxidants such as vitamin E,
silymarin, phosphatidylcholine, and S-adenosyl- L-methionine inhibit
HSC activation, protect hepatocytes from undergoing apoptosis, and
attenuate experimental liver fibrosis (S7). Antioxidants exert
beneficial effects in patients with alcohol-induced liver disease and
NASH (S8, S9). Disrupting TGF-ß synthesis and/or signaling pathways
prevents scar formation in experimental liver fibrosis (58).
Moreover, administration of growth factors (e.g., IGF, hepatocyte
growth factor, and cardiotrophin) or their de livery by gene therapy
attenuates experimental liver fibrosis (S10, S11). However, these
latter approaches have not been tested in humans and may favor cancer
development. Substances that inhibit key signal transduction pathways
involved in liver fibrogenesis also have the potential to treat liver
fibrosis (20).
They include pentoxifylline (phosphodiesterase inhibitor), amiloride
(Na +/H + pump inhibitor), and S-farnesylthiosalicylic acid (Ras
antagonist). Ligands of PPAR{alpha} and/or PPAR{gamma}
such as thiazolindiones exert beneficial effects in experimental liver
fibrosis and in patients with NASH (S12, S13). The inhibition of the
renin-angiotensin system is probably the most promising strategy in
treating liver fibrosis. Renin-angiotensin inhibitors are widely used
as antifibrotic agents in patients with chronic renal and cardiac
diseases and appear to be safe when administered for prolonged periods
of time (S14). Little information is available on the use of this
approach in patients with chronic liver diseases. Preliminary pilot
studies in patients with chronic hepatitis C and NASH suggest that
renin-angiotensin blocking agents may have beneficial effects on
fibrosis progression (S15). Transplanted patients receiving
renin-angiotensin system inhibitors as antihypertensive therapy show
less fibrosis progression than patients receiving other types of drugs
(S16). However, this approach cannot be recommended in clinical
practice until the results of ongoing clinical trials become available.
The blockade of endothelin-1 type A receptors and the administration of
vasodilators (prosta-glandin E2 and nitric oxide donors) exert
antifibrotic activity in rodents, yet the effects in humans are unknown
(90).
Different herbal compounds, many of them traditionally used in Asian
countries to treat liver diseases, have been demonstrated to have
antifibrotic effects (S17). They include Sho-saiko-to, glycyrrhizin, and savia miltiorhiza. An alternative approach is the inhibition of collagen production and/or the promotion of its degradation (20).
Inhibitors of prolyl-4 hydroxylase and halofuginone prevent the
development of experimental liver cirrhosis by inhibiting collagen
synthesis. MMP-8 and urokinase-type plasminogen activator stimulate
collagen degradation in vivo. The efficacy of these drugs in humans is
unknown, and they may result in undesirable side effects. Finally,
infusion of mesenchymal stem cells ameliorates experimentally induced
fibrosis, which suggests a potential for this approach in the treatment
of chronic liver diseases (S18, S19).

A limitation of the current antifibrotic approaches is that
antifibrotic drugs are not efficiently taken up by activated HSCs and
may produce unwanted side effects. Cell-specific de livery to HSCs
could provide a solution to these problems. Promising preliminary
results have been recently obtained using different carriers (e.g.,
cyclic peptides coupled to albumin recognizing collagen type VI
receptor and/or PDGFR) (S20). Antifibrotic therapy may differ depending
on the type of liver disease. In patients with chronic HCV infection,
current antiviral treatments (pegylated IFN plus ribavirin) clear viral
infection in more than half of the patients (S21). Sustained
virological response is associated with an improvement in liver
fibrosis (122). Patients with no sustained response may also experience improvement of liver fibrosis, which suggests that IFN-{alpha}
has an intrinsic antifibrotic effect (S22). For nonresponder patients,
the use of renin-angiotensin system inhibitors is a promising approach.
Treatment of the metabolic syndrome in patients with chronic hepatitis
C may also decrease fibrosis progression (S23). In patients with
alcohol-induced liver disease, the most effective approach is alcohol
abstinence (124).
Antioxidants (e.g., S-adenosyl- L-methionine and phosphatidylcholine)
and hepatocyte protectors (e.g., silymarin) slow down the progression
of liver fibrosis and can improve survival (S24). For patients with
autoimmune hepatitis, immunosuppressant therapy not only decreases
inflammation but also exerts antifibrotic effects (S25). No
antifibrotic therapy is available for patients with chronic cholestatic
disorders (i.e., primary sclerosing cholangitis and PBC).
Ursodeoxycholic acid improves biochemical tests in these patients, but
its impact on fibrosis is not consistently proven (S26). In patients
with NASH, weight loss and specific treatments of the metabolic
syndrome can reduce fibrosis development (125).
Recent reports have revealed than antioxidants and insulin sensitizers
(e.g., thiazolindiones) may exert antifibrogenic effects in these
patients (S27). Large clinical trials are needed to confirm these
results.

Future directions

The translation of basic research into improved therapeutics for the
management of patients with chronic liver diseases is still poor. The
role of pluripotential stem cells in hepatic wound healing is one of
the most promising fields. Perfusion of these cells may be a potential
approach to promoting fibrosis resolution and liver regeneration.
Approaches to removing fibrogenic cells are being evaluated, including
development of drug de livery systems that target activated HSCs.
Translational research should investigate the molecular mechanisms that
cause fibrosis in different types of human liver diseases in order to
identify new targets for therapy. In the clinical setting, the identity
of the genetic determinants that influence fibrosis progression should
be uncovered. Well-designed large-scale epidemiological genetic studies
are clearly required. Patients at a high risk of progression to
cirrhosis should be identified. Developing simple and reliable
noninvasive markers of hepatic fibrosis is an important goal in
clinical hepatology and will facilitate the design of clinical trials.
Most importantly, the efficacy of antifibrotic drugs known to attenuate
experimental liver fibrosis should be tested in humans.

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