American Journal of Transplantation 2006; 6: 12–19
Blackwell Munksgaard
Minireview
Antifibrotic Agents for Liver Disease
E. Albanis∗ and S. L. Friedman
Division of Liver Diseases, Mount Sinai Medical Center,
New York, New York, USA
∗ Corresponding author: E. Albanis,
efsevia.albanis@msnyuhealth.org
Complications from chronic hepatitis C (HCV) and re-
current HCV post-transplant are responsible for sig-
nificant morbidity and mortality in the United States
and Europe. Current antiviral therapies are at best, ef-
fective in up to 50% of patients in the pre-transplant
setting, and in the post-transplant setting are associ-
ated with more limited efficacy and increased toxicity.
With this reduced efficacy of antiviral strategies in the
post-transplant setting, new approaches are urgently
needed. Substantial progress has been made in under-
standing the pathogenesis of hepatic fibrosis over the
last 20 years, which has yielded potential new thera-
peutic targets. The prospect of antifibrotic therapies is
nearing reality in order to reduce progression to cir-
rhosis, thereby reducing morbidity, mortality and the
need for re-transplantation. Current and evolving ap-
proaches primarily target the activated hepatic stellate
cells, which are the main source of extracellular matrix,
along with related fibrogenic cell types. Key issues yet
to be clarified include the optimal duration of antifi-
brotic therapies, endpoints of clinical trials, indications
in clinical practice and whether combination therapies
might yield synergistic activity.
Key words: Extracellular matrix, fibrosis, hepatitis C,
stellate cell
Received 20 May 2005, revised 29 August 2005 and ac-
cepted for publication 7 September 2005
Introduction
There has been great progress made over the past 20 years
in understanding hepatic fibrosis, the liver’s wound healing
response and in particular the importance of the hepatic
stellate cell (HSC) as the central component in the fibro-
genic process. When fibrosis advances to cirrhosis the life
threatening complications associated with liver disease oc-
cur, including variceal bleeding, ascites formation and hepa-
torenal syndrome, among others. These complications cre-
ate a substantial burden on healthcare resources and are
expected to increase dramatically in the near future (1). Ef-
forts are underway to target the HSC in an effort to attenu-
ate the fibrotic response. Such approaches may effectively
12

C 2005 The American Society of Transplantation
and the American Society of Transplant Surgeons
doi: 10.1111/j.1600-6143.2005.01143.x
slow fibrosis progression, or could even reverse it. Ideally,
treatment with antifibrotic compounds could also lead to
cirrhosis regression.
This review will highlight fibrosis progression in patients
with chronic hepatitis C (HCV) pre-orthotopic liver trans-
plantation (OLT) and recurrent HCV post-OLT, review the
pathogenesis of fibrosis and discuss targeted antifibrotic
therapies directed against parts of the HSC activation
cascade.
Chronic HCV Infection in the Pre- and
Post-Transplant Setting
The development of antifibrotic therapy is especially im-
portant for patients with chronic HCV and those with re-
current HCV and fibrosis post-OLT. HCV is the most preva-
lent blood-borne viral disease in the United States: approx-
imately 4,000,000 people are seropositive for HCV and
270 000 are chronically infected with HCV (2). Seventy four
percent of patients with HCV in the United States are in-
fected with Genotype 1, which is least likely to respond
(up to 50%) to the only drugs approved by the FDA for
treatment of this infection: pegylated interferon with rib-
avirin (3). End-stage liver disease associated with chronic
HCV infection is the leading cause for decompensated liver
disease and is the leading indication for OLT in the United
States. Within the next 4 years, the projected need for
liver transplantation will increase by 528%, mainly due to
the HCV epidemic (1). While the need for OLT is increasing,
the number of deceased donor organs available has effec-
tively plateaued. Living donor liver transplantation has not
significantly impacted on mortality in patients with end-
stage liver disease. Thus, there is a dire need for new ap-
proaches to treatment; antifibrotic therapies are amongst
the most promising.
The development of antifibrotic therapies is additionally im-
portant in patients with HCV recurrence post-OLT. Approx-
imately 50% of liver transplants in the United States and in
Europe are performed for patients with chronic HCV. Re-
currence is virtually universal in those transplanted with ac-
tive infection. In a majority of patients, disease recurrence
is mild or slowly progressive. However, many patients de-
velop progressive injury to their liver allograft with fibro-
sis progression post-transplant, advancing at a rate that
is almost 10-fold higher than fibrosis progression in the
pre-transplant setting. Approximately 25–33% of these pa-
tients develop cirrhosis within 5 years of undergoing OLT

(4–6) contrasted with the pre-OLT setting where cirrhosis
requires ∼20–40 years to develop. Thus, progression to cir-
rhosis in patients with recurrent HCV post-OLT is acceler-
ated compared to the pre-OLT setting (6,7). Progression to
cirrhosis post-OLT is near-linear in a significant percentage
of patients with a median rate of fibrosis progression of 0.3
Metavir units/year, ranging from 0.6 to 0.8 stage/year (6–8).
A subgroup of patients develop fibrosing cholestatic hepati-
tis, characterized by very aggressive recurrence, and lead-
ing to graft failure in 50% of patients within a few months
(6).
Once cirrhosis occurs, the rate of decompensation in
transplanted patients is accelerated compared to non-
transplanted cirrhotic patients. At the first and third years,
the rate of decompensation is 42% and 63%, respec-
tively, in transplanted patients, compared to 3% and 18%
of non-transplanted cirrhotic patients who decompensate
(9). Ultimately, this rapid fibrosis progression leads to re-
duced graft and patient survival when compared to pa-
tients who are transplanted for non-viral causes (11,12).
The 5-year survival rates of 60–70% for patients who have
been transplanted for HCV-associated liver disease are less
in those patients who have undergone transplantation for
other causes, in whom survival is 75–80% (10,11). Antiviral
therapy for recurrent HCV post-OLT has met with limited
efficacy and substantial toxicity (11).
Factors that may be associated with the more rapid pro-
gression of fibrosis post-OLT include donor age, immuno-
suppression, cytomegalovirus infection, race, with non-
Caucasians doing worse than Caucasians, HCV RNA levels
Figure 1: With ongoing liver
injury, HSC become activated,
leading to the deposition of scar
matrix which, in turn, leads to
changes in neighbouring cell
types. Endothelial cells lose their
fenestrae and nepatocytes their
microvilli.
American Journal of Transplantation 2006; 6: 12–19
Antifibrotic Agents for Liver Disease
pre- and post-OLT and genetic background of the recipient
(10).
What Is Fibrosis?
Fibrosis is the liver’s scarring response to injury that oc-
curs in most chronic inflammatory liver diseases, including
hepatitis B (HBV) and HCV, autoimmune or metabolic, for
example, hemochromatosis. The ultimate result of chronic
injury is the accumulation of extracellular matrix (ECM) and
replacement of low density type IV collagen with high den-
sity type I collagen within the subendothelial space of Disse
(12) (Figure 1). With prolonged chronic injury cirrhosis may
develop, which connotes the distortion of normal architec-
ture with nodule formation surrounding regenerative hepa-
tocytes. The major complications of end-stage liver disease
occur almost exclusively in patients with cirrhosis, because
as more scar matrix is deposited within the subendothelial
space of Disse there is disruption of the intercellular milieu,
eventually leading to disruption of the cellular homeosta-
sis and culminating in hepatocellular dysfunction. This dys-
function is manifested clinically by complications of portal
hypertension (ascites, variceal hemorrhage, portosystemic
encephalopathy) and liver synthetic dysfunction. The hope
is that if antifibrotic therapy can reconstitute the normal
microenvironment of liver, normal function can be restored
and clinical manifestations may regress.
Fibrosis requires many years to decades of injury to de-
velop in most patients (13), but there are at least three set-
tings where progression is typically more rapid: infants with
13
Albanis and Friedman
congenital hepatic fibrosis may present at birth with ad-
vanced fibrosis, patients with recurrent HCV post-OLT, pa-
tients co-infected with HCV and HIV. Mechanisms underly-
ing rapid fibrosis development in these cases are unknown,
but may reflect profound alterations in matrix degradation
and/or significant immune dysfunction, which is increas-
ingly recognized to regulate fibrosis independent of effects
on injury.
Fibrosis is generally reversible if the primary injury is re-
moved. In contrast, cirrhosis has historically been viewed
as irreversible; however, recent animal and human clinical
data in patients with cirrhosis due to HBV (14) and HCV
(15) have demonstrated that cirrhosis may be reversible
when the underlying chronic injury is removed. At some
point, it is clear that cirrhosis becomes truly irreversible,
as manifested clinically by refractory ascites, encephalopa-
thy, hyponatremia and renal dysfunction, but the feature(s)
which distinguish irreversible fibrosis are yet to be identi-
fied. Recent animal studies have suggested that cirrhosis
becomes irreversible when collagen cross-linking by tis-
sue transglutaminase occurs, which probably leads to an
insoluble hepatic matrix characterized by scar that is rela-
tively resistant to degradation (16). Additional factors which
may influence the reversibility of cirrhosis might include (a)
total collagen content, which may coalesce into broad fi-
brotic bands that are inaccessible to collagen degrading en-
zymes; (b) duration of cirrhosis, which could reflect a longer
period of collagen cross-linking, rendering it less sensitive
to degradation by enzymes over time and (c) reduced ex-
pression of matrix-degrading enzymes, or increased ex-
pression of proteins which inhibit matrix-degrading enzyme
function.
14
Figure 2: Following liver in-
jury, HSC undergo activation,
transforming from quiescent,
vitamin-A-rich cells to cells ca-
pable of proliferation, contrac-
tion, fibrogenesis, matrix degra-
dation and WBC chemoattrac-
tion, in response to cytokine
stimulation.
Fibrogenesis and the Role of the Hepatic
Stellate Cell (HSC)
Central to fibrogenesis is the activation of hepatic stellate
cells (HSC). HSC reside in the space of Disse between hep-
atocytes and sinusoidal endothelial cells. In normal liver,
HSC are quiescent and store vitamin A. In injured liver, HSC
become ‘activated’, in which quiescent, vitamin A-rich cells
transit into myofibroblasts capable of proliferation, fibroge-
nesis, contractility and matrix degradation, among other
functions (12,13) (Figure 2). Activated HSC are also a key
source of mediators, matrix molecules, proteases and their
inhibitors that together lead to the formation of the liver
scar. Each step of the ‘activation’ cascade may be targeted
by an antifibrotic compound, and is detailed below.
Proliferation
PDGF-BB (platelet-derived growth factor beta) is the most
potent proliferative stimulus toward HSC. Both PDGF-BB
and its receptor are upregulated following liver injury (17).
Other mitogenic factors and their cognate tyrosine kinase
receptors which increase HSC numbers during liver injury
include thrombin, insulin-like growth factor, endothelin-1,
fibroblast growth factor and vascular endothelial growth
factor (VEGF) (18,19). Thus, targeting activated stellate cell
proliferation with an inhibitor of tyrosine kinases may atten-
uate fibrosis.
Fibrogenesis
HSC, Kupffer cells and platelets secrete transforming
growth factor beta-1 (TGFb-1), the most potent fibrogenic
factor for HSC (20,21). Both quiescent and activated HSC
express TGFb-1 receptors (22).
American Journal of Transplantation 2006; 6: 12–19

Contractility
Activated HSC are contractile, primarily in response to
endothelin-1 (ET-1) (23). Net contractile activity of HSC is
determined by the balance between ET-1 and its antago-
nist nitric oxide (NO) (24). In advanced chronic liver disease,
there is increased ET-1 and decreased NO activity. Further-
more, because of their location in the subendothelial space
of Disse, activated HSC may constrict individual sinusoids,
thereby increasing portal resistance.
Matrix degradation
In normal liver, the rate of ECM production equals that of
its degradation, resulting in no net accumulation of matrix.
Fibrogenesis occurs when there is an imbalance between
ECM degradation and production. Proteinases, including
the matrix metalloproteinases (MMP), are responsible for
matrix breakdown and include five categories: interstitial
collagenases (MMP-1, -8, -13), gelatinases (MMP-2, -9),
stromelysins (MMP-3, -7, -10, -11), membrane type (MMP-
14, -15, -16, -17, -24, -25) and a metalloelastase (MMP-12)
(25). Regulation of metalloproteinases is complex. Inac-
tive MMPs may be activated through proteolytic cleavage
and inhibited by binding to specific inhibitors known as tis-
sue inhibitors of metalloproteinases (TIMPs). Downregu-
lation of MMPs by a reduction in gene expression or an
increase in their tissue inhibitors favors the accumulation
of ECM. Progressive fibrosis is associated with increases
in TIMP-1 and TIMP-2, which leads to net decrease in MMP
(protease) activity, and therefore more unopposed matrix
accumulation (26,27). HSC are the major source of TIMPs.
Cytokine release and chemotaxis
HSC release cytokines including TGFb-1, PDGF-BB, fi-
broblast growth factor, hepatocyte growth factor (HGF),
platelet activating factor, ET-1 and neutrophil and mono-
cyte chemoattractants which can amplify inflammation in
liver injury. HSC also release anti-inflammatory cytokines
like interleukin-10. HSC may also migrate directly to areas
of injury in response to PDGF (the most potent chemotac-
tic factor), endothelin and monocyte chemotactic protein 1
(28,29).
Loss of vitamin A (retinoids)
Activated HSC lose their vitamin A droplets, but it is not
known if vitamin A loss is required for HSC activation.
Resolution of liver fibrosis and the fate
of activated HSC
It is unclear what happens to HSC when fibrosis and cirrho-
sis resolve. There is a decrease in the number of activated
HSC as fibrosis resolves, but it is uncertain if HSC return
to a quiescent state or undergo apoptosis (30). Cell culture
has demonstrated that the HSC can revert to a quiescent
cell type, but it is unknown if this occurs in vivo (28). Induc-
ing apoptosis of activated stellate cells is possibly another
targeted antifibrotic.
American Journal of Transplantation 2006; 6: 12–19
Antifibrotic Agents for Liver Disease
Potential Antifibrotic Therapies
Properties of an antifibrotic compound
Since hepatic fibrosis pathways are largely similar in all liver
diseases regardless of their etiology, the development of
antifibrotics should benefit all patients with fibrosing liver
injury. Because of the liver’s great ability to regenerate, liver
fibrosis is an attractive target for antifibrotic therapy com-
pared to fibrosing diseases in other organs like the lung
and kidneys, which tend to have less regenerative capac-
ity. Furthermore, because of first-pass metabolism through
the liver, lower doses of orally available compounds may be
necessary to achieve a therapeutic response, thereby min-
imizing systemic distribution of the agent and non-hepatic
side effects.
Desirable properties of an antifibrotic compound include
the following features: (a) HSC-specific and non-toxic to
neighboring hepatic cells; (b) no effect on the metabolism
of immunosuppressive agents, to avoid increasing the tox-
icity of immunosuppressive agents on the kidneys or ner-
vous system by increasing their relative activity or acceler-
ating the chance of rejection if relative drug levels are re-
duced;(c) orally available and easily delivered; (d) non-toxic
to other organs, such as the kidneys and (e) safe when
used over long periods of time.
Obstacles to antifibrotic drug development
There are still some obstacles to the development of an-
tifibrotic therapies. Since fibrogenesis is a slow process
that requires years or decades to develop, assessment of
changes over a short interval is problematic. Furthermore,
prospective human clinical trials of potential antifibrotic
agents may be costly because of long duration. What is the
optimal duration of therapy? Typically, pharmaceutical com-
panies appear willing to sponsor 1-year ‘proof of concept’
trials. However, is 1 year enough to demonstrate an an-
tifibrotic effect of a putative compound, given that fibrosis
takes years to develop? Furthermore, an anti-fibrotic trial
would probably require a pre- and post-treatment biopsy to
assess the antifibrotic efficacy, since current non-invasive
tests for liver fibrosis cannot replace biopsy.
Nonetheless, there are limitations to a liver biopsy, even
though this technique is considered the gold standard
for assessing hepatic fibrosis. The procedure is invasive
and adverse events including pain, hemorrhage necessi-
tating intervention, rupture of adjacent organs (0.3–0.5%)
and death (0.01–1%) occur (31–33). As a result, many pa-
tients are reluctant to undergo this procedure. Historically,
about 33% of patients in clinical trials have refused post-
treatment liver biopsies (34,35). The ability to perform re-
peated measures of fibrosis to assess treatment efficacy
is vital to the success of any antifibrotic trial.
Sampling error is the major limitation of liver biopsy (36–
40). In a study of 124 patients with HCV who underwent
15
Albanis and Friedman
a laparoscopic biopsy, 33% had a difference of at least
one fibrosis stage between biopsies from the right and left
hepatic lobes (41). Clearly, a measure of fibrosis that more
accurately reflects fibrogenic events occurring within the
liver as a whole is needed. In addition to sampling error, liver
biopsy is prone to subjectivity in pathological interpretation.
Furthermore, despite fibrogenesis being an active process
characterized by deposition and degradation of matrix, the
liver biopsy reveals only static information about fibrosis in
a small fraction of the organ. In contrast, there may be early
changes in fibrotic marker gene expression that precede
changes in fibrosis as assessed using scoring systems that
include Metavir or Knodell scales, and could be used as the
basis for novel diagnostic approaches.
Which group of patients would be suitable for antifibrotic
therapies? Currently, the main focus is on patients with
chronic HCV who have failed antiviral therapy, since they
represent a very large cohort with a well characterized nat-
ural history. Other target population include those with re-
current HCV and fibrosis post-OLT.
Targeted antifibrotic therapies
There is a tremendous interest in the field of drug develop-
ment and testing for hepatic fibrosis. To date however, no
drugs are approved as antifibrotic therapy. Removing the
primary injury is the best antifibrotic therapy. For example,
in patients with chronic HCV, treatment with an antiviral in
an effort to eradicate the virus is the optimal approach to
reversing fibrosis. If the primary disease cannot be cured,
then an antifibrotic compound can potentially target any
part of the HSC activation cascade described above: (a)
downregulating HSC activation; (b) neutralizing prolifera-
tive, fibrogenic and contractile responses of HSC; (c) in-
creasing the degradation of scar matrix and (d) stimulating
apoptosis of HSC.
Downregulating HSC activation: Attenuating HSC acti-
vation is an attractive antifibrotic target. Both gamma in-
terferon (c -IFN) and HGF have demonstrated inhibitory ef-
fects on HSC activation in animal models of liver fibrosis
(42,43). A recent double blind controlled clinical trial assess-
ing the antifibrotic efficacy of c -IFN showed no benefit,
however.
Peroxisome proliferator activated nuclear receptor gamma
(PPARc ) is expressed in HSC, and synthetic PPARc lig-
ands, or thiazolidinediones, downregulate HSC activa-
tion (44–46). There are trials underway assessing the ef-
fect of these ligands in patients with non-alcoholic fatty
liver disease, and fibrosis is a major endpoint of such
trials.
Oxidant stress stimulates HSC activation, and thus reduc-
ing oxidant stress is a possible antifibrotic target. In studies
of experimental or human fibrogenesis, antioxidants, in-
cluding vitamin E, suppress fibrogenesis (47). Herbal prod-
16
ucts like Silymarin, the active ingredient in milk thistle, as
well as the traditional Chinese/Japanese plant extract Sho-
saiko-to have been noted to have free radical-scavenging
ability, Sho-saiko-to has antifibrotic properties in vitro and
in rat models of porcine-serum induced fibrosis (48).
Neutralizing proliferative, fibrogenic and contractile
responses of HSC: Neutralizing the proliferative, fibro-
genic and contractile responses of HSC is another tar-
geted approach of a potential antifibrotic compound. Cy-
tokine receptor antagonists may be used as potential an-
tifibrotic agents. In activated HSC, the most potent pro-
liferative factor is PDGF-BB, binds to its tyrosine kinase
receptor, bb-PDGF, which is upregulated during activation.
Glivec, a targeted tyrosine kinase receptor antagonist, al-
ready in use against chronic myelogenous leukemia and
gastrointestinal stromal tumors (49), has attenuated the
fibrotic response in animal fibrotic models (unpublished
data).
The most potent fibrogenic factor for HSC is TGFb-1 and
targeting TGFb-1 might greatly down-regulate matrix pro-
duction. The latent form of TGFb-1 must be cleaved by
proteases in order to become active. Different strategies
have been used to block activation of TGFb and to prevent
its binding to specific receptors, which demonstrate de-
creased fibrosis in vivo. Camostate mesilate, a protease in-
hibitor, inhibits the release of active TGFb-1 and attenuates
liver fibrosis in rats (50). Attempts to prevent the binding of
TGFb to its receptors have successfully attenuated fibro-
sis in rats. Studies including use of a dominant-negative
type II TGFb receptor, the expression of truncated type
II receptor and the construction of a soluble type II recep-
tor have demonstrated antifibrotic efficacy (51,52). There is
concern, however, that with long-term TGFb antagonism in
humans, the modulation of inflammation and the immune
response and the loss of TGFb-mediated growth suppres-
sion could stimulate hepatocellular growth, thereby pro-
moting cancer.
Neutralizing the effects of the renin-angiotensin system
(RAS) is an additional antifibrotic approach. The RAS plays
a role in disease states where chronic injury, inflamma-
tion and tissue remodeling occur (53). Its main effector,
angiotensin II stimulates collagen deposition (54) mainly
through the induction of TGFb-1 (55). ATII probably plays
a role in liver fibrosis. The systemic and hepatic and HSC
RAS are active in patients with chronic liver disease (56–
58). The mechanism(s) by which ATII is profibrogenic are
not fully known in liver, but this mediator binds ATI recep-
tors in HSC and induces contraction and proliferation (58).
ATI receptor stimulation may also increase oxidant stress
through increased NADPH oxidase. In liver injury, increas-
ing circulating ATII levels accelerates inflammation and fi-
brosis (59). Moreover, inhibiting the RAS attenuates fibro-
sis development in rats (60); RAS antagonism with losar-
tan, an angiotensin converting enzyme inhibitor, attenuates
fibrosis in rats (61,62). Losartan has reversed fibrosis in
American Journal of Transplantation 2006; 6: 12–19

patients with renal fibrosis, whose pathogenesis is very
similar to patients with liver fibrosis (63). Because pa-
tients post-OLT often develop systemic hypertension, use
of losartan is particularly attractive compared to other an-
tihypertensives, since it may have both hypotensive and
antifibrotic activities.
Hepatocyte growth factor (HGF) can attenuate fibrosis in
animal models (43). HGF modulates HSC proliferation, col-
lagen formation and TGFb expression. Similar to TGFb an-
tagonists, there is concern about the use of HGF over the
long term as it may induce or promote the growth of hep-
atocellular carcinoma (HCC), particularly in cirrhotics, since
they already have an increased HCC risk.
Endothelin-A receptor (ET-AR) antagonists are another po-
tential targeted therapy. ET-AR mediates HSC contraction,
proliferation and may also stimulate collagen synthesis. In
murine models of fibrosis, ET-AR antagonists have both an-
tifibrotic efficacy and can decrease portal pressure, either
by reducing endothelin or by increasing nitric oxide (64).
Promoting matrix degradation: Targeting matrix degra-
dation is another antifibrotic target, given the need to re-
sorb matrix in patients with established fibrosis or cirrhosis.
TGFb antagonists stimulate matrix degradation by down-
regulating TIMPs and increasing activity of interstitial col-
lagenase. Expressing metalloproteinases mRNA via gene
therapy in animal fibrotic models has confirmed, in princi-
ple, that matrix can be resorbed (65).
Promoting apoptosis: Targeting HSC specifically in the
liver for apoptosis is another antifibrotic target. Use of
gliotoxin, a fungal toxin that induces apoptosis specifically
in HSC, has attenuated the fibrotic response in a rat model
of CCl 4 induced fibrosis (66).
Conclusion
Significant progress has been made in understanding the
role of the HSC in fibrogenesis, which has led to many
potential therapies that may improve fibrosis in the pre-
and post-OLT setting. Continued refinement in clinical trial
design, including optimal choice of subjects, duration of
therapy and trial endpoints is anticipated in the immediate
future. As the list of agents grows and the determinants of
success are more clearly defined, inclusion of antifibrotic
therapies is likely to emerge as an important option in pa-
tients with fibrosing liver disease, particularly in patients
at high risk for progression such as those with HCV post-
transplantation.
Acknowledgment
This study is supported by AASLD Sheila Sherlock Translational Research
Award; Wasserman Scholar Award.
American Journal of Transplantation 2006; 6: 12–19
Antifibrotic Agents for Liver Disease
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