Reversal of hepatic fibrosis: pathophysiological basis of antifibrotic therapies

Abstract

Chronic liver injuries of different etiologies eventually lead to fibrosis, a scarring process associated with increased and altered deposition of extracellular matrix in the liver. Progression of fibrosis has a major worldwide clinical impact due to the high number of patients affected by chronic liver disease which can lead to severe complications, expensive treatment, a possible need for liver transplantation, and death. Liver fibrogenesis is characterized by activation of hepatic stellate cells and other extracellular matrix producing cells. Liver fibrosis may regress following specific therapeutic interventions. Other than removing agents causing chronic liver damage, no antifibrotic drug is currently available in clinical practice. The extent of liver fibrosis is variable between individuals, even after controlling for exogenous factors. Thus, host genetic factors are considered to play an important role in the process of liver scarring. Until recently it was believed that this process was irreversible. However, emerging experimental and clinical evidence is starting to show that even cirrhosis in its early stages is potentially reversible.

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Open Access Full Text Article
Reversal of hepatic fibrosis: pathophysiological
basis of antifibrotic therapies
http://dx.doi.org/10.2147/HMER.S9051
Mona H Ismail1
Massimo Pinzani2
1Department of Internal Medicine,
Division of Gastroenterology, King
Fahad University Hospital, Al-Khobar,
Saudi Arabia; 2Dipartimento di
Medicina Interna Center for
Research, High Education and Transfer,
Università degli Studi di Firenze,
Florence, Italy
Correspondence: Mona H Ismail
College of Medicine, University
of Dammam, Department of Internal
Medicine, Division of Gastroenterology,
King Fahad Hospital, PO Box 40149,
Al-Khobar 31952, Saudi Arabia
Tel +966 3896 6741
Fax +966 3896 6741
Email mismail.md@gmail.com
Abstract: Chronic liver injuries of different etiologies eventually lead to fibrosis, a scarring
process associated with increased and altered deposition of extracellular matrix in the liver.
Progression of fibrosis has a major worldwide clinical impact due to the high number of patients
affected by chronic liver disease which can lead to severe complications, expensive treatment,
a possible need for liver transplantation, and death. Liver fibrogenesis is characterized by
activation of hepatic stellate cells and other extracellular matrix producing cells. Liver fibrosis
may regress following specific therapeutic interventions. Other than removing agents causing
chronic liver damage, no antifibrotic drug is currently available in clinical practice. The extent
of liver fibrosis is variable between individuals, even after controlling for exogenous factors.
Thus, host genetic factors are considered to play an important role in the process of liver
scarring. Until recently it was believed that this process was irreversible. However, emerging
experimental and clinical evidence is starting to show that even cirrhosis in its early stages is
potentially reversible.
Keywords: liver fibrosis, cirrhosis, fibrogenesis, antifibrotic agents
Introduction
Chronic liver disease is a major cause of mortality and morbidity worldwide. Most
chronic liver diseases progress from mild inflammation to more severe inflammation,
leading to fibrosis or cirrhosis. This can result in liver failure and portal hypertension,
and is associated with an increased risk of liver cancer.1 The development of advanced
fibrosis, and particularly cirrhosis, is associated with significant and life-threatening
complications, with liver transplantation being the only available treatment. However,
transplantation is not always possible, due to limited organ availability and the pres-
ence of contraindicating comorbidities.
Fibrosis is a progressive pathological process in which the body’s wound healing
and tissue remodeling mechanisms respond to liver injury by promoting replacement
of normal hepatic tissue with a scar-like matrix composed of cross-linked collagen.2
Until recently, it was believed that this process was irreversible.3 However, emerg-
ing experimental and clinical evidence is starting to show that even early stages of
cirrhosis are potentially reversible.
The cascade of events leading to hepatic fibrosis is complex, and is influenced by
how different cell types in the liver interact in response to injury. The main cell type
responsible for the development of fibrosis in chronic liver disease is the activated
hepatic stellate cell. Chronically activated hepatic stellate cells proliferate and
synthesize extracellular matrix proteins to produce the fibrous scar (Figure 1).4
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Ismail and Pinzani
Following any type of liver injury, hepatic stellate cells
differentiate into myofibroblasts and acquire the typical
“ myofibroblast-like” phenotype characterized by a multifunc-
tional profibrogenic, proinflammatory, and proangiogenic profile.
Reported phenotypic changes garnered from hepatic stellate cell
culture studies include increased proliferation, increased expres-
sion levels of platelet-derived growth factor-β and smooth muscle
α-actin (α-SMA), and enhanced collagen secretion.5–8 More
recent evidence implicates a role for leptin in hepatic stellate
cell transdifferentiation by activating the hedgehog pathway, and
galectin-3 for hepatic stellate cell activation in vivo.9,10
In addition to hepatic stellate cells, other resident
liver mesenchymal cells, such as portal fibroblasts and
myofibroblasts, contribute to hepatic fibrogenesis, par-
ticularly in chronic liver diseases characterized by primary
involvement of the portal and periportal areas (ie, chronic
viral hepatitis and cholestatic diseases).11
There is some evidence that bone marrow-derived
fibrocytes or circulating mesenchymal cells can migrate
through the injured liver and become myofibroblasts, which
participate in the fibrotic process.12,13 In addition, there is
controversial evidence that hepatocytes and cholangiocytes
may undergo epithelial–mesenchymal transition to become
activated myofibroblasts.14 Likewise, phenotypic agility
associated with epithelial–mesenchymal transition allows
for the reverse process whereby mesenchymal cells convert
into epithelial derivatives.14
Among the three types of proposed epithelial–
mesenchymal transitions, type 2 epithelial–mesenchymal
transition refers to the process occurring in chronic fibro-
genic disorders. In the context of fibrogenesis, synthesis
and deposition of type I collagens most appropriately signal
myofibroblast function in vivo.14 However, despite in vitro
evidence supporting 1α1 gene activation in cultured epithe-
lial cells, including hepatocytes, these cells fail to generate
collagen 1α1 transcripts in vivo.14–16 In addition, some cells,
particularly macrophages, internalize the collagen fibrillar
extracellular matrix that leads to mistaken association of
collagen accumulation with collagen synthesis. These con-
tradictions beg the question of whether epithelial cells really
play a role in fibrogenesis.14,15,17–19
Many cellular markers have been wrongly defined as
myofibroblast-specific based on gene expression patterns.
For example, certain hepatocytes and cholangiocytes
express “mesenchymal markers” typical of cell motility
and survival. Hepatocytes when cultured in vitro and
chronically stimulated with transforming growth factor-β1
or serum factors exhibit genetic expression patterns
analogous to myofibroblasts in vivo and connective
tissue at the development stage.14,20–24 Some of the genes
expressed include α-SMA, Slug, Twist, Snail, vimentin,
and fibroblast-specific protein implicated in cell motility
in general, although these genes do not mark myofibro-
blast or epithelial cell lineages specifically.14,15,18,25–28
HSC activation
by several factors:
• HBV
• HCV
• Alcohol
• Others
Quiescent hepatic
stellate cell (HSC)
Activated HSC
(myofibroblast)
Collagen
deposition
Over several years
Liver fibrosis
Liver cirrhosis
Liver
Vitamin A
droplet
Scar matrix
If process unresolved
Figure 1 Schematic presentation of liver fibrogenesis.

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Reversal of hepatic fibrosis
Oddly, α-SMA, that forms part of the cell’s contractile
machinery, constitutes a poor lineage marker for fibro-
genesis, although it is the most widely used marker for
recognizing myofibroblasts. Activation of this protein
expression during disease or as part of an adaptive
response to injury, compounded by the fact that it contrib-
utes minimally, if at all, to the synthesis and deposition of
fibrillar extracellular matrix, explains the ambiguous role
of α-SMA in marking myofibroblastic activity.14
A direct contribution of cholangiocytes to fibrosis via
an epithelial–mesenchymal transition pathway has been
proposed by Omenetti et al who demonstrated that immature
cholangiocytes undergo a complete epithelial–mesenchymal
transition when treated with activated hepatic stellate
cell medium.29 Others have shown coexpression of epithelial
and mesenchymal markers in human liver cholangiocytes
extracted from patients with cholestatic disease.30,31 However,
recent reports have challenged the concept of cholangiocyte
epithelial–mesenchymal transition, with conclusions pointing
towards an absence of this process in liver fibrosis models.32
Specifically, fluorescently labeled cholangiocytes express-
ing the bile ductular cell-specific marker, K19, failed to
coexpress myofibroblast markers in either bile duct-ligated
or CCL4-induced fibrosis mouse models.33 Clearly, further
explorations beyond intuitive speculation are needed to
unravel the role of epithelial cells and cholangioctyes in
fibrogenesis, if any.
The development of significant tissue fibrosis usually
requires several years of ongoing insult. However, not
all patients exposed to a similar causal agent develop the
same degree of liver fibrosis, ie, patients with similar risk
factors have some variability in progression of liver fibrosis,
which also may reflect host genotypic polymorphisms.33
Evidence of fibrotic regression has now been documented
in the entire spectrum of chronic liver diseases, including
autoimmune hepatitis, biliary obstruction, iron overload,
nonalcoholic steatohepatitis,34 and viral hepatitis B (HBV)
and C (HCV).35,36
Patterns of fibrosis progression have been described on
the basis of their disease origin. Among the contributors,
chronic HCV poses a significant risk for developing fibrotic
tissue, but only becomes lethal following cirrhosis onset.
Factors affecting the rate of fibrosis progression in HCV-
infected patients include duration of infection, age, male
gender, consumption of alcohol, human immunodeficiency
(HIV) coinfection, and low CD4 count.37–41 Metabolic con-
ditions, including obesity, steatosis, and diabetes, are also
surfacing as independent risk factors.42
Both male gender and age strongly influence the speed
of disease transition from infection to cirrhosis, with a 300-
fold higher progression rate seen in men aged 61–70 years
versus those 20–40 years of age.41 In contrast, HCV-infected
women progress to cirrhosis at a much slower rate compared
with their male counterparts, regardless of age. A multicenter
study aimed at evaluating the natural history of liver fibrosis
progression in HCV and associated risk factors reported a
median 39% higher annual rate of fibrosis progression in
men compared with women.43 The significance of this result
was partially rationalized by the greater alcohol consumption
associated with men in this trial.
Antifibrotic effects from estrogen may also explain the
fibrosis progression disparity seen between the genders.
Long-term estrogen exposure reduced liver fibrosis progres-
sion in HCV-infected women who had either been pregnant,
taken oral contraceptives, or received hormone replacement
therapy in one retrospective study conducted across two
centers in France.44 Infected postmenopausal women not
receiving hormone replacement therapy showed the highest
rate of fibrosis progression.44
Some speculative data have emerged demonstrating a
correlation between insulin resistance and increased rate
of fibrosis progression in nondiabetic individuals infected
with chronic HCV, although a longitudinal study is needed
to confirm this interesting observation.45
Patients coinfected with HIV and HCV have more rapid
progression of cirrhosis that may be partially curbed with
highly active antiretroviral therapies.46–50 In a recent study,
Berenguer et al examined the effects of exposing coinfected
patients to nonnucleoside reverse transcriptase inhibitor and
protease inhibitor therapy, and reported a successful outcome
in reducing fibrosis progression with nonnucleoside reverse
transcriptase inhibitors but not with protease inhibitors.46
Other special populations on the radar for developing
fibrosis-related complications include those with hemo-
chromatosis or nonalcoholic steatohepatitis. Cirrhotic
patients with nonalcoholic steatohepatitis have a reduced life
expectancy and a concomitant 67% 5-year survival rate.51,52
Evaluation of pooled data from histological studies identified
inflammation on the initial biopsy and age as independent
predictors of progression of fibrosis to advanced nonalcoholic
steatohepatitis.52
Individuals suffering from hemochromatosis, char-
acterized by iron deposition within tissues and related
end-organ damage, develop cirrhosis, which may or may
not present with symptoms or elevated liver enzymes.53–55
Some important risk factors identified for increased rate

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Ismail and Pinzani
of progression of fibrosis in this patient population include
alcohol consumption, hepatic steatosis, HCV coinfection,
male gender, oxidative injury, and genetic polymorphisms,
particularly implicating transforming growth factor-β1,
tumor necrosis factor-β, and MCP1.53
Although there is no doubt that liver tissue fibrosis can
regress, the issue of regression/reversibility of cirrhosis is
controversial and requires considerable clarification. This
is particularly relevant after several claims of “cirrhosis
reversal” in patients with chronic HBV and HCV infection
after antiviral treatment.35,56 The concept of cirrhosis
reversibility originates from evidence obtained in animal
models upon the discontinuation of the cause of liver damage
or following treatment with antifibrotic agents. Although
cirrhosis tends to be defined as one stage, it is quite evident,
both morphologically and clinically, that there are several
stages of evolution following the initial morphological
evidence of cirrhotic transformation of liver tissue. “Early”
cirrhosis is often macronodular, with a limited thickness of
fibrotic septa and a scarcity of neoangiogenesis. In addition,
at this stage, the fibrotic tissue is still characterized by the
presence of profibrogenic cells and an abundant inflammatory
infiltrate. The evolution of this picture is characterized
by increased thickness of the septa that become progres-
sively acellular and by the development of micronodular
cirrhosis. From the biochemical point of view, collagen fibers
undergo extensive crosslinking and are wrapped in filaments
of elastin. At this advanced stage, fibrosis becomes largely
irreversible. Therefore, claims of “cirrhosis reversal” need
to be framed in the stage of evolution of a cirrhotic liver and
the term “reversal” used to denote a return to near-normal
liver structure.
Establishing cirrhosis reversal in humans has been ham-
pered by a lack of supportive evidence, compounded by the
fact that few relevant experiments were carried out using
animal models.57,58 It should be stressed that in the large
majority of the available animal models (mostly in rodents),
cirrhosis develops within weeks and is rather different both
in morphological and biochemical (ie, composition and
three-dimensional arrangement) terms from the cirrhosis
observed in chronically ill patients who develop liver disease
over several decades.
Mechanisms of fibrosis reversal
The first documented evidence for the reversal of liver fibro-
sis derived from studies showing a progressive decrease of
fibrosis associated with apoptosis of hepatic stellate cells
in rat models of chronic intoxication with CCl4 following
suspension of toxic and bile duct ligation after recanalization
of the bile duct.59,60 Altogether, these studies suggested that
apoptosis of hepatic stellate cells plays a critical role in the
recovery from biliary as well as toxic-induced liver fibrosis.
In addition, hepatic stellate cell survival and apoptosis are
regulated by growth factors expressed during fibrotic liver
injury. Issa et al58 went further to show that reversal was
obtained in a chronic CCl4 model, even when an advanced
stage of micronodular cirrhosis was obtained after 12 weeks
of administration of CCl4. Indeed, 1 year after the cessation
of administration of CCl4, micronodular cirrhosis underwent
remodeling to macronodular cirrhosis. In addition, expres-
sion of collagen type 1 and tissue inhibitor of metallopro-
teinases 1 (TIMP-1) mRNA decreased significantly, and
active metalloproteinases were shown in liver tissue during
fibrosis remodeling. This was paralleled by a significant loss
of more recently formed fibrils, decreased perisinusoidal
fibrosis, and decreased thickness of fibrotic septa. Once
again, resolution was characterized by apoptosis of hepatic
stellate cells, predominantly at the edges of fibrotic septa.
Residual septa, not remodeled after 1 year, were character-
ized by transglutaminase-mediated crosslinking and relative
hypocellularity. This experimental study highlighted a lim-
ited potential for the cirrhotic liver to engage in spontaneous
regression of the mature matrix in the absence of a therapeutic
intervention.57,58
The relationship between fibrosis reversal and hepatic
stellate cell apoptosis highlighted by these studies in animal
models suggested that induction of hepatic stellate cell
apoptosis could represent a potential antifibrogenic target.
This process would necessarily remove the primary hepatic
source of newly synthesized collagens as well as TIMP-1.61
Hepatic TIMP-1 inhibits metalloproteinases implicated in
matrix degradation, and both collagen and TIMP-1 promote
myofibroblast survival.62,63 Reverse differentiation of the
myofibroblasts back to their original phenotype occurs
in vitro and therefore constitutes an alternative mechanism
for fibrosis regression, although as yet there is no in vivo evi-
dence supporting this claim.57,64,65 In fact, Kim et al reported
decreases in desmin-positive cells in rats recovering from
dimethylnitrosamine-induced fibrosis.66 This result argues for
the apoptotic pathway because desmin marks both activated
myofibroblasts and their hepatic stellate cell precursors.66
However, the concept of hepatic stellate cell apoptosis in
animal models is not likely to be completely reproducible in the
human setting. Despite what we have learned about antifibrotic
mechanisms from animal studies, the evidence is at best specu-
lative and needs further support particularly with relevance to

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Reversal of hepatic fibrosis
clinical evidence. Although apoptosis seems to play a role in
fibrosis regression, it may simply represent a symptom of extra-
cellular matrix remodeling. This explanation garners validity
from experimental evidence showing that expression levels of
both collagen 1 and TIMP-1 ramp up during fibrogenesis, but
then decrease in the recovering injured liver.57,63,67
Different studies performed in human hepatic stellate cells
and in liver tissue obtained from patients with chronic viral hep-
atitis have suggested that human profibrogenic cells, and par-
ticularly hepatic stellate cells, are characterized by a resistance
to apoptosis once activated and fully involved in the fibrogenic
process.68,69 It is therefore plausible that long-term fibrogenesis
is characterized, in addition to biochemical evolution of scar
tissue and lack of an appropriate degradation machinery, by the
immovability of a critical mass of profibrogenic cells.
An important aspect of macrophage biology in the fibro-
genic process is the observation that these cells are required
for fibrosis resolution after withdrawal of the damaging agent.
Along these lines, selective depletion of macrophages resulted
in reduced scarring and a lower number of fibrogenic cells in
the active phase of fibrogenesis, as expected based on previ-
ous work.70 However, macrophage depletion during fibrosis
resolution was asso ciated with delayed matrix degradation.
These data indicate the existence of distinct subpopulations of
macrophages and the role of these cells in the recovery phase of
the fibrogenic process. These data have been recently expanded
with the observation that scar-associated macrophages that
mediate fibrosis resolution express matrix metallopeptidase 13
allowing them to degrade complex extracellular matrices and
remodeling of scar.71 The interaction between the chemokine
system and macrophages in the process leading to fibrosis reso-
lution has also been recently investigated. When chronically
intoxicated with CCl4, mice lacking chemokine (C-C motif)
receptor 2 (CCR2) showed lower levels of fibrosis compared
with wild-type animals, and reduced F4/80+, CD11b+, and
CD11c+ populations at the sites of injury.72 However, upon
discontinuation of toxin administration, fibrosis persisted
in CCR2 knockout mice, and was correlated with sustained
expression of TIMP-1 and with reduction in matrix metallopep-
tidase 13 expression. These data suggest that this chemokine
system is one of the molecular effectors of both macrophage
recruitment during active fibrogenesis and of macrophage-
dependent fibrosis resolution. Accordingly, because therapies
that interfere with chemokines or their receptors are being
developed, it may be speculated that a therapy interfering
with CCR2 could possibly retard the regression of fibrosis,
especially when associated with a strategy that reduces the
extent of damage.
Clinical evidence for reversal
Accordingly, when performing an accurate analysis of
results of the clinical studies claiming reversal of cirrhosis
after antiviral treatment, the only prudent conclusion is
that, in most cases, there was a variable degree of fibrosis
regression in cirrhosis but not a reversal of cirrhosis.73,74 For
example, Poynard et al reported fibrosis reduction among
cirrhotic patients with chronic HCV who received pegylated-
interferon (IFN) and ribavirin regimens.75 Of the 153 patients
enrolled in their study, 49% showed downstaging of liver
fibrosis from stage 4 to stage 3. A separate study looked
retrospectively at liver improvements in 113 individuals with
confirmed hepatic cirrhosis and receiving immunosuppres-
sive therapies, including IFN-α, or a combination of IFN-α
and ribavirin in the case of viral hepatitis infection, or steroids
plus azathioprine in autoimmune patients. Among the 113
participants, 14 (12.4%) had biopsy-proven disappearance of
cirrhosis.76 One rare report describing spontaneous recovery
in two young men who had developed HBV-related cirrhosis
as children recently emerged. The investigators attributed
their “cure” to cessation of viral replication.77
Although fibrosis seems to be a reversible process
accompanied by regression of scar tissue, establishing
clear remodeling or an apoptotic mechanism underlying
the regression process remains an elusive goal. However,
the more challenging goal of reversing cirrhosis must
take into account features other than simple regression
alone. Additional factors, including what effects rapid
clearance of the matrix would have on the highly altered
vasculature structure of the cirrhotic liver, warrants special
consideration.13,74,78 The availability of improved diagnostic
techniques for monitoring disease progression should also
enhance the evaluation of potential antifibrotic therapeutics
in the clinic.79
In other words, fibrotic deposition related to recent disease
and characterized by the presence of thin reticulin fibers, often
in the presence of a diffuse inflammatory infiltrate, is likely to
be fully reversible, whereas longstanding fibrosis, character-
ized by extensive collagen crosslinking in a dense acellular/
paucicellular extracellular matrix and decreased expression
and/or activity of specific metalloproteinases, is not.58,80
Assessment of fibrosis regression
Assessment of the fibrotic evolution of chronic liver disease
has relied and still largely relies on histopathological scor-
ing of liver tissue obtained by liver biopsy. However, liver
biopsy is an invasive procedure limited by issues relating
to sampling, cost, and morbidity, and only provides a static