A Review of Liver Fibrosis and Emerging Therapies

Abstract

With the increasing burden of liver cirrhosis, the most advanced stage of hepatic fibrosis, there is a need to better understand the pathological processes and mechanisms to target specific treatments to reverse or cease fibrosis progression. Antiviral therapy for hepatitis B and C has effectively treated underlying causes of chronic liver disease and has induced fibrosis reversal in some; however, this has not been targeted for the majority of aetiologies for cirrhosis including alcohol or nonalcoholic steatohepatitis. Fibrosis, characterised by the accumulation of extracellular matrix proteins, is caused by chronic injury from toxic, infectious, or metabolic causes. The primary event of fibrogenesis is increased matrix production and scar formation mediated by the hepatic stellate cell, which is the principal cell type involved. Experimental models using rodent and human cell lines of liver injury have assisted in better understanding of fibrogenesis, especially in recognising the role of procoagulant factors. This has led to interventional studies using anticoagulants in animal models with reversal of fibrosis as the primary endpoint. Though these trials have been encouraging, no antifibrotic therapies are currently licenced for human use. This literature review discusses current knowledge in the pathophysiology of hepatic fibrosis, including characteristics of the extracellular matrix, signalling pathways, and hepatic stellate cells. Current types of experimental models used to induce fibrosis, as well as up-to-date anticoagulant therapies and agents targeting the hepatic stellate cell that have been trialled in animal and human studies with antifibrotic properties, are also reviewed.

Full text

Creative Commons Attribution-Non Commercial 4.0 December 2019 • EUROPEAN MEDICAL JOURNAL
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A Review of Liver Fibrosis and Emerging Therapies
Authors: Rooshi Nathwani, Benjamin H. Mullish, David Kockerling, Roberta
Forlano, Pinelopi Manousou, *Ameet Dhar
Integrative Systems Medicine and Digestive Disease, Imperial College London,
London, UK
*Correspondence to a.dhar@imperial.ac.uk
Disclosure: The authors have declared no conflicts of interest.
Received: 03.05.19
Accepted: 01.08.19
Keywords: Anticoagulation, antifibrotic therapy, fibrosis, hepatic fibrosis, hepatic stellate cell.
Citation: EMJ. 2019;4[4]:105-116.
Abstract
With the increasing burden of liver cirrhosis, the most advanced stage of hepatic fibrosis, there is a
need to better understand the pathological processes and mechanisms to target specific treatments
to reverse or cease fibrosis progression. Antiviral therapy for hepatitis B and C has eectively treated
underlying causes of chronic liver disease and has induced fibrosis reversal in some; however, this
has not been targeted for the majority of aetiologies for cirrhosis including alcohol or nonalcoholic
steatohepatitis. Fibrosis, characterised by the accumulation of extracellular matrix proteins, is caused
by chronic injury from toxic, infectious, or metabolic causes. The primary event of fibrogenesis is
increased matrix production and scar formation mediated by the hepatic stellate cell, which is the
principal cell type involved. Experimental models using rodent and human cell lines of liver injury have
assisted in better understanding of fibrogenesis, especially in recognising the role of procoagulant
factors. This has led to interventional studies using anticoagulants in animal models with reversal of
fibrosis as the primary endpoint. Though these trials have been encouraging, no antifibrotic therapies
are currently licenced for human use. This literature review discusses current knowledge in the
pathophysiology of hepatic fibrosis, including characteristics of the extracellular matrix, signalling
pathways, and hepatic stellate cells. Current types of experimental models used to induce fibrosis,
as well as up-to-date anticoagulant therapies and agents targeting the hepatic stellate cell that have
been trialled in animal and human studies with antifibrotic properties, are also reviewed.
INTRODUCTION
The burden of chronic liver disease continues
to grow, with 0.1% of the European population
aected by cirrhosis, the most advanced stage
of hepatic fibrosis.1 Although the aetiology of
the disease varies between countries,
fibrogenesis is the common pathological
mechanism that causes cirrhosis. Fibrosis
occurs following chronic liver injury from a
range of insults including toxins (alcohol),
infections (hepatitis B [HBV] and C viruses [HCV]),
and metabolic disease (nonalcoholic fatty liver
disease). Such insults drive inflammation, resulting
in increased synthesis and altered deposition
of extracellular matrix (ECM) components,
and impaired regeneration and wound healing
responses.2 This is a complex, dynamic process,
involving recruitment and activation of platelets,
inflammatory cells, hepatic stellate cells (HSC),
and other ECM-producing cells including portal
fibroblasts, hepatocytes, cholangiocytes, and

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bone marrow-derived cells.3 The end result,
cirrhosis, is defined by profound distortion of
hepatic microarchitecture, ultimately resulting
in the development of portal hypertension.4
Histologically, cirrhosis is characterised by
regenerative nodules of liver parenchyma
separated by, and encapsulated in, fibrotic
septa, with a clinical consequence of increased
mortality, morbidity, complications of portal
hypertension, and diminished quality of life.2,5 The
presence of hepatic fibrosis is a key predictor of
prognosis in chronic liver disease, independent
of aetiology.6 Generally this process evolves over
decades (usually 20–40 years), but it can be
rapidly progressive, as seen in children aected
by biliary atresia, drug-induced liver injury, HCV
co-infection with HIV, or HCV infection post
liver transplantation.7
Management of chronic liver disease has largely
focussed on aetiology-specific treatments;
however, significant progress has been made in
understanding the pathophysiology of fibrosis,
which has identified targets for potential
antifibrotic agents to either halt progression or
reverse fibrosis.
A search of the existing literature up to June
2019 was conducted using electronic databases
PubMed, Medline, and the Cochrane library, as well
as relevant guidelines, to present this literature
review, an overview of the current understanding
of the pathogenesis of hepatic fibrosis, in vitro and
in vivo models used in exploring pathogenesis,
and an update on proposed therapies.
PATHOPHYSIOLOGY
Extracellular Matrix
The ECM is a dynamic structural component of
the normal liver.8 It contains macromolecules
that provide the scaolding of the liver and act
as transducers of extracellular signals. In normal
liver tissue the ECM is composed of collagens,
glycoproteins, fibronectin, laminin, tenascin,
von Willebrand factor, and proteoglycans.9 It
is a component of Glisson’s capsule, portal
tracts, central veins, and the subendothelial
space of Disse.10 Various cellular sources of the
ECM have been identified but the main source
is the HSC. When liver injury is not severe,
neighbouring hepatocytes regenerate and
replace apoptotic and necrotic cells. However,
in chronic insult, the mechanism contributing
to fibrosis, this process fails and ECM proteins
take on the role of hepatocytes.11 The quantity
and quality of the ECM changes, increasing
up to 8-fold compared to a normal liver. There
is significant increase in collagen content and
proteoglycans, resulting in a higher density
interstitial type matrix.12 The ECM composition
also transforms, from one predominantly made
up of Type IV and VI collagen, glycoproteins,
proteoglycans, and laminin, to a matrix
consisting of Type I and III collagen and
fibronectin.10 These adaptations alter the local
microenvironment leading to subsequent
functional and physical restrictions on plasma
flow between sinusoids and hepatocytes, causing
impaired hepatic function.9
Signalling in Fibrosis
Fibrogenesis is a complex mechanism
involving an array of cellular and extracellular
signalling. Cytokine, chemokine, adipokine,
neuroendocrine, angiogenic, and nicotinamide
adenine dinucleotide phosphate-oxidase
(NADPH) signalling have all been found to play
important functions.13 The release of cytokines
in the context of fibrogenesis is controlled
in part by activation of metalloproteinases.
TGF-β1, platelet-derived growth factor (PDGF),
connective tissue growth factor (CTGF), TNF-α,
and vascular endothelial growth factor (VEGF)
are all involved in key mechanisms of fibrosis.9,1 4
TGF-α is particularly important in mediating
stellate cell activation to myofibroblasts and
stellate cell collagen production.14 VEGF and
PDGF control angiogenesis, which contributes
to ECM production, portal hypertension, and
regeneration postinjury.15 During acute or chronic
liver injury, platelets are the first cells recruited to
the injury site. They form aggregates in damaged
vasculature by converting fibrinogen into fibrin
and platelet α-granules rich in PDGF, TGF-α,
and VEGF.16
Chemokines, CCL2 (produced by Kuper cells and
HSC), and CCL5 are best recognised in fibrosis.17
CCL2 promotes HSC activation, facilitating
macrophage and monocyte recruitment into the
liver. Inhibition of CCL2, or its receptor CCR2, is
associated with reduced fibrosis in experimental
models.18 CCL5 and its receptors, CCR1 and CCR5,
are associated with fibrogenesis promotion.19

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Other signalling molecules involved in
fibrogenesis include the adipokines, leptin, and
adiponectin, synthesised by HSC, as well as
cannabinoids and their respective receptors.
Cannabinoid receptor 1 is a profibrotic mediator;
however, cannabinoid receptor 2 has antifibrotic,
but proinflammatory, properties.13
Reactive oxygen species mediate fibrogenesis
by stimulating profibrogenic mediator release
from Kuper cells and activating HSC. Emerging
evidence suggests that NADPH also plays a role
in this process.20
Hepatic Stellate Cells
The HSC is the principal cell type involved in
fibrogenesis21 and its activation is the common
pathway leading to fibrosis. Developing a clear
understanding of factors that stimulate or inhibit
activation of HSC has helped guide development
of antifibrotic therapies. HSC lie in a quiescent
state in the subendothelial space of Disse and
comprise 15% of liver cells.8 In the normal liver,
they are the primary storage site for retinoids
and are derived from neural crest tissue because
of expression of neural crest markers, including
glial fibrillary acidic protein and nestin.22 These
cells are activated by proinflammatory cytokines
and protease activated receptor-1 ligation,
resulting in a myofibroblastic phenotype which is
proliferative, fibrogenic, and contractile.
Activation of HSC proceeds through an initiation
and perpetuation phase.13
Initiation refers to early changes that occur in
gene expression and phenotype, initiated by
mediators that render quiescent HSC responsive
to other stimuli. Characteristic to this phase is the
production of a fibrogenic, contractile phenotype
with induction of PDGF receptors.22 Initiation is
stimulated by oxidant stress signals, apoptotic
hepatocytes, lipopolysaccharides, and paracrine
stimuli from neighbouring cells including
cholangiocytes, Kuper cells, injured sinusoidal
endothelial cells, or other stellate cells.13 Initiation
activates stellate cells, while perpetuation is the
response to stimuli that maintains the activated
stellate cell in its myofibroblastic state to
allow fibrotic scar formation.23 These changes
include stellate cell proliferation, chemotaxis,
fibrogenesis, increased contractility, altered
matrix degeneration, and cytokine signalling.22
The release of a variety of mitogenic factors
causes proliferation of stellate cells. PDGF is a
potent mitogen upregulated during liver injury.24
Other molecules with mitogenic activity include
VEGF, thrombin, epidermal growth factor, TGF-α,
keratinocyte growth factor, and fibroblast growth
factor.25 Matrix metalloproteinase-2 (MMP-2)
acts as a stellate cell mitogen via activation
by discoidin domain receptor-2 binding to
fibrillar collagen.26
Activated stellate cells migrate towards areas of
hepatic injury during chemotaxis. Growth factors
within the liver which have chemoattractant
properties include PDGF, insulin growth factor-1,
endothelin-1, monocyte attractant protein-1, and
the chemokine receptor CXCR3;27 in contrast,
adenosine inhibits chemotaxis, allowing cells to
fix at injury sites.28
The primary event of fibrogenesis is increased
matrix production and scar formation mediated
by stellate cells.10 Type I collagen, the major
constituent of scar tissue, is upregulated post-
transcriptionally in stellate cells by the actions
of TGF-α via Smads, pivotal intracellular eector
proteins that mediate TGF-α signalling.29 CTGF
also promotes fibrogenesis.30 Other less potent
mediation of Type I collagen production include
angiotensin II, IL-1β, and TNF. 31
On activation the HSC contracts, which is
characterised by the increased expression of
α-smooth muscle actin, a contractile filament
protein.32 This results in impendence of sinusoidal
blood flow increasing portal resistance, and
eventually increasing portal pressure once
advanced fibrosis has developed. Progressive
development of intrahepatic shunting occurs
in conjunction with the constriction of
hepatic sinusoids. The process is mediated
by endothelin-1, as well as angiotensinogen
II and atrial natriuretic peptide. Nitric
oxide has an opposing eect, resulting in
sinusoidal relaxation.33
The concept of matrix degradation evolved
following evidence demonstrating that fibrosis
is reversible. This propagated the theory that
fibrosis is a dynamic balance between matrix
production and degradation.34 Initial matrix
degradation may be termed pathological,
corresponding to disruption of normal low-
density matrix of the subendothelial basement

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membrane, occurring in early stages of fibrosis.
This allows for normal matrix to be replaced by
a higher density pathological matrix, containing
fibrils. Matrix degrading collagenases, including
the MMP, break down Type IV collagen and are
central to this paradigm.35,36 Stellate cells and
Kuper cells are the primary sources of MMP-2
and MMP-9, respectively.35 Conversely, MMP-
1 degrades Type I collagen and is involved in
the restorative degradation of the pathological
matrix, thus, upregulation of this enzyme
would favour resolution of scar tissue. Tissue
inhibitors of metalloproteinases (TIMP) inhibit
the actions of collagenases, resulting in reduced
matrix degradation, and inhibit stellate cell
apoptosis, which prevents fibrosis resolution
and favours matrix accumulation.36 TIMP-1 and
2 are upregulated in progressive fibrosis, and
their sustained release from stellate cells is a key
determinant of progressive fibrosis.36,37
HSC are not only eectors of fibrosis but also
have a central role in amplifying inflammatory
signaling via toll-like receptors and cytokines
including monocyte chemotactic protein-1, CCL21,
CCR5, and Regulated upon Activation, Normal
T-cell Expressed, and Secreted (RANTES). They
demonstrate antigen presenting capabilities and
produce neutrophil chemoattractants.38,39
EXPERIMENTAL MODELS
OF LIVER INJURY
Techniques isolating and cultivating HSC have
represented a major advance in exploring the
complex mechanisms involved in fibrogenesis.
Rodent stellate cell lines have been characterised
but have now been superseded by human cell
lines.23 The most utilised human HSC lines include
the LX1 and LX2 cell lines. LX1 cell line is generated
by transformation with SV40 T antigen.40 The
LX2 cell line is generated by isolating stellate
cells from normal human livers and immortalising
them by culturing in low serum conditions.
When activated, these cells bear close similarity
to in vivo human activated HSC.41 The LX2 cell
line has been extensively validated and used
in a number of studies exploring the eects of
mediators on stellate cell activity.25 Other human
HSC lines are summarised in Table 1.40 Utilising
these cell lines therefore allows us the unique
opportunity to study factors that both inhibit
and activate HSC.
Animal models oer the opportunity to study
interactions of dierent cell types, gene
activation, protein-expressing profiles, and
signalling pathways, but as yet no animal model
exactly reproduces human hepatic fibrosis.42
Table 1: Summary table of human stellate cell lines used.
Human hepatic stellate cell line Derivation
LI90 Human hepatic epithelioid haemangioendothelioma.
TWNT-1
TWNT-4
Retrovirally-induced human telomerase reverse
transcriptase into LI90 cell line.
Human telomerase reverse transcriptase gene Normal human liver.
HSC-Li Normal human liver.
GREF-X Cirrhotic human liver.
LX1 Transformation with SV40 T antigen.
LX2 Isolating stellate cells from normal human livers and
immortalising them by culturing in low serum conditions.

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A variety of agents, often carcinogenic, can be
employed to chemically induce fibrosis. The most
commonly used are carbon tetrachloride (CCl4)
and thioacetamide (TAA). Fibrosis generated in
this manner has both a degree of reproducibility
and similarity with human mechanisms and
pathways involved in hepatic fibrosis.43 They
also have the added advantage that they can
be used in conjunction with either transgenic or
wild-type mice to explore underlying molecular
mechanisms involved in hepatic fibrosis, or
can be used to evaluate the potential of novel
antifibrotic agents using strictly controlled
environmental and genetic conditions.43
Carbon Tetrachloride
CCL4 is the oldest and most common method of
inducing liver fibrosis in rodent models.44 It is a
halothane and induces hepatic injury when given
at repetitive low doses. It can be administered
in a variety of ways, but is most commonly
administered by intraperitoneal injection ≤3
times per week. CCl4 is bioactivated by oxidases.
This leads to a combination of eects including
CCl3 radical formation in the liver, which causes
hepatocyte damage via lipid peroxidation;
HSC activation; Kuper cell activation; TGF-β-1
upregulation; and increased oxidative stress.45,46
Histological alterations result in fatty change
with necrosis and intense necroinflammation in
centrilobular areas. This progresses to both septal
and nonseptal fibrosis resulting in extensive
fibrosis and cirrhosis. The fibrous septa are
classically thin and can regress with CCl4
withdrawal. The reproducibility and ease of
induction of fibrosis are its main advantages.
Disadvantages include heterogeneity in the
amounts of fibrosis produced between animals.32
Thioacetamide
TAA is a selective hepatotoxin. Chronic
administration not only induces liver fibrosis,
but can result in carcinogenesis, including
cholangiocarcinoma and hepatocellular
carcinoma. TAA is usually given in drinking
water over a period of 8–18 weeks to induce
liver fibrosis.47 Histologically, mild to moderate
amounts of fibrosis develop by 8 weeks
with elevated transaminases. By 12 weeks,
parenchymal damage occurs, with hepatocyte
swelling, necrosis, and proliferation. This results
in fibrous enlargement of the portal tracts, with
portal–portal and portal–central septa developing
resulting in cirrhosis. Histological similarities with
human viral hepatitis have led to its use as an
indirect model of both fibrosis secondary to HBV
and HCV infection. Withdrawal of TAA results in
resolution of fibrosis over an 8-week period. A
longer period of resolution, compared with CCl4
models, makes it a more suitable model when
evaluating potential antifibrotic therapies, and
the occurrence of regenerative nodules make it
more comparable to human cirrhosis.48
Dimethylnitrosamine
Dimethylnitrosamine (DMN) is a hepatotoxin
and a carcinogenic and mutagenic agent. DMN
is typically administered intraperitoneally three
times a week, with centrilobular and periportal
fibrosis characteristically developing after 3
weeks. Microscopically, this pattern of fibrosis is
seen in cirrhosis.49 DMN models induce HSC and
Kuper cells to express profibrotic cytokines
resulting in deposition of excessive ECM, the
primary pathogenesis of fibrosis, making it a
potentially useful animal model.50 However, DMN
has mutagenic and carcinogenic properties and
exposure can cause hepatocellular carcinoma.
This makes understanding fibrosis pathways
dicult to interpret but does allow the
pathogenesis of fibrosis to hepatocellular
carcinoma to be better understood.51
BILE DUCT LIGATION
Surgical ligation of the common bile duct causes
cholestasis and periportal inflammation. This
technique causes proliferation of biliary epithelial
cells and increases expression of fibrogenic
markers such as TIMP-1, Alpha-SMA, Type I
collagen, and TGF-β1.52 Although this model
aids portal fibrosis and portal myofibroblast
interrogation, bile duct ligation model use is
restricted due to the high frequency of gall
bladder perforation and bilio-peritoneum in
mice.53 Mortality risk is therefore significant and
this model is more suitable for short-term studies
investigating cholestasis-induced fibrosis.52,53

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Table 2: Summary table of anti-fibrotic strategies targeting hepatic stellate cell.
Agent Target Mechanisms
1. Reducing inflammation and immune responses before HSC activation.
Silymarin Oxidative stress mechanisms, NFκB
pathways, and PDGF signalling.
Inhibits oxidative stress and
subsequently stellate cell activation.
Glucocorticoids Immune mediators Reduction of inflammation.
Caeine A2A adenosine receptor Inhibition of A2A adenosine
reception expressed by
myofibroblasts and linked to matrix
production.
Curcumin Cannabinoid receptors Downregulation of cannabinoid 1
receptor, a profibrotic mediator.
Ursodeoxycholic acid Cholangiocytes Reduction in the cytotoxic eects of
bile acids and protects hepatocytes
against apoptosis.
2. Inhibition of HSC activation
Vitamin E Oxidative stress mechanisms Prevention of oxidative stress
implicated in fibrogenesis.
Thiazolindinediones PPAR Anti-inflammatory and antifibrotic
eects by inhibiting PDGF
expression. Antifibrotic eect has
been demonstrated in patients with
nonalcoholic fatty liver disease.
Oleoylethanolamide PPAR Same mechanism as
thiazolidinediones and but also
initiation of α-smooth muscle
expression.
Imatinib mesylate PDGF Suppression of PDGF and HSC
activation.
ACE inhibitors Renin–angiotensin system Downregulation of angiotensin II
receptors on HSC, responsible for
proliferation and contraction. Though
readily available, no large randomised
trials have been conducted in
humans.
Recombinant IL-22 Th22 receptors Inhibit HSC activation and suppresses
inflammatory cytokine release.
Thrombin and FXa PAR1 and 2 stellate cell activation Inhibition of PAR 1 and 2 stellate
mediated activation.
ROCK inhibitor GTP-binding protein Rho Inhibition of stellate cell activation.
3. Inhibiting response after HSC activation
GW6604 TGF-α Inhibit TGF-β1 signalling pathways.
Cytosporone B
NR4A1 gene agonist
TGF-α Inhibit TGF-β1 signalling pathways.
Bosentan Endothelin Endothelin antagonism reduces
stellate cell activation and
extracellular matrix production.
Halofuginone Collagen synthesis Inhibition of collagen Type I
synthesis by inhibition of SMAD3
phosphorylation.

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Agent Target Mechanisms
Caspase inhibitors Caspase Inhibits eectors of apoptosis
signalling in hepatocytes.
Obeticholic acid Farnesoid-X receptor Improved integrity of hepatocytes,
reduction in HSC contractility and
reduction of collagen.
4. Promoting activated HSC into apoptosis
Gliotoxin NFκB Inhibition of NFκB pathways,
suppressing chronic hepatic
inflammation.
Sulfasalazine NFκB Inhibition of NFκB pathways,
suppressing chronic hepatic
inflammation.
Thalidomide NFκB Inhibition of NFκB pathways,
suppressing chronic hepatic
inflammation.
Melatonin NFκB Inhibition of NFκB pathways,
suppressing chronic hepatic
inflammation.
Cannabinoid 1 antagonist Cannabinoid 1 receptor Inhibition of collagen Type I
synthesis by inhibition of SMAD3
phosphorylation. Reduces cellular
proliferation and promotes
myofibroblast apoptosis.
Cannabinoid 2 agonist Cannabinoid 2 receptor Inhibits myofibroblast proliferation
and induces apoptosis.
Interferon NK Cells Promotes NK cell activity and
promotes HSC death.
Hepatocyte growth factor Myofibroblast Inhibits extracellular matrix producing
myofibroblasts.
Table 2 continued.
ATP-Binding Cassette Subfamily B
Member 4
The ATP-binding cassette subfamily B
member 4 (Abcb4-/-) gene encodes multidrug
resistance 3 (MDR3) protein, the canalicular
phosphatidylcholine lipid transporter. Altering
this gene by rendering it deficient causes
intrahepatic cholestasis and disease similar to
that of primary biliary cirrhosis. It functions to
protect cellular membranes facing the biliary
tree against bile acids and without
phosphatidylcholine there is biliary epithelial
and ductular damage, portal inflammation and
proliferation, and progressive portal fibrosis.54
Abcb4-/-knockout mice develop fibrosis between
4 and 8 weeks with TGF-β1 expression, HSC
activation at 4 weeks with collagen deposition,
and scarring at 8 weeks.55
These models have been used to study
primary biliary cirrhosis, cholestasis of
ACE: angiotensin-converting enzyme; FXa: Factor Xa; GTP: guanosine-5'-triphosphate; HSC: hepatic stellate cell;
NK: natural killer; NR4A1: nuclear receptor subfamily 4 group A member 1; PAR1: protease-activated receptor 1;
PAR2: protease-activated receptor 2; PDGF: platelet-derived growth factor; PPAR: peroxisome proliferator activated
receptors; ROCK: Rho kinase; SMAD3: SMAD Family Member 3.
Adapted from Ebrahimi H et al.58

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pregnancy drug-induced cholestasis, and
therapeutic interventions.56
ANTIFIBROTIC THERAPY
Trials of antifibrotic therapies in humans
have been limited compared to experimental
models in animals. This is partly due to the long
duration antifibrotic agents would need to be
administered to demonstrate a treatment
eect because of the time taken for the agent
to systemically accumulate. Trials are limited
by funding and concerns over side eects.
Furthermore, the historical need for histological
endpoints can limit recruitment. An ideal trial
would be one that has a short duration of
treatment, with a pre-existing clinical need for
routine liver histology.
Though significant progress has been made
in understanding the pathogenesis of fibrosis,
particularly the role of the HSC, no antifibrotic
therapy specifically targeting this cell type
responsible for increased matrix production and
scar formation is currently licensed for human
use. Several potential agents and antifibrotic
molecules such as silymarin, caeine, and
curcumin, summarised in Table 2, have shown
antifibrotic properties via targeting the HSC, but
are not routinely used in clinical practice.57,58
Silymarin, mainly consisting of silibinin, is a
flavonoid complex extracted from milk thistle.
It has been shown to diminish the following:
oxidative stress, lysis of hepatocytes, activation
of Kuper cells, and expression of α-SMA and
TGF-β1, all implicated in the pathogenesis
of fibrosis in rats treated with CCl4 to induce
fibrosis.59 Despite showing promise in animal
models with improved liver function tests, in
humans there is little evidence demonstrating
its clinical benefit. The De Avelar et al.60 meta-
analysis of six human studies demonstrated
that although alanine transaminase and
aspartate transaminase levels were reduced with
silymarin use, there was no clinically significant
benefit associated with this. To date, only one
randomised, double-blinded, placebo-controlled
study with biopsy-proven fibrosis investigating
the eect of silymarin has been conducted. This
showed that silymarin did not significantly reduce
nonalcoholic fatty liver disease activity scores,
although some improvement of fibrosis was
histologically seen compared to placebo. This
study was underpowered and did not include
other aetiologies of fibrosis.61
Several studies have reported the beneficial
eect of caeine against liver disease.62 The
major mechanism by which caeine exerts its
antifibrotic eect is largely attributable to caeine
being a pan antagonist of the adenosine receptor.
Liver myofibroblasts are profibrogenic and
express the A2A adenosine receptor; therefore,
blocking this receptor inhibits fibrogenesis.63 A
second proposed mechanism is that caeine
alters signalling and inflammation pathways in
fibrogenesis by reducing TGF-β expression, as
suggested in rodent models of fibrosis.64
Curcumin, a monomer extract from turmeric,
has not only shown anti-inflammatory and
antiproliferative properties but also antifibrotic
actions, as evidenced by its ability to protect
against fibrosis in CCl4 treated rats.65,66 Curcumin
also interferes with TGF-β signalling pathways
and PDGF receptors, leading to inhibition of
HSC activity. This is further achieved through
modulation of the cannabinoid receptor system
with downregulation of the cannabinoid receptor
1, inhibiting ECM expression by HSC.67
Anticoagulation
Evidence suggests that hepatic fibrogenesis
is associated with prothrombotic tendencies,
including factor V Leiden (FVL); this is
demonstrated by Wright et al.,68 who found that
FVL mutation increased the rate of fibrosis in HCV
infection. The coagulation proteins thrombin and
factor Xa (FXa) are also implicated through their
activation of HSC.69 Activation of the coagulation
system generates FXa, which in turn results in the
production of thrombin from its precursor protein,
prothrombin. Both thrombin and FXa activate
stellate cells via G-protein-coupled receptors
known as protease activated receptors (PAR).70
Duplantier et al.71 evaluated, in a rat model of
CCl4-induced chronic liver injury, the eect of
thrombin inhibition using a synthetic thrombin
antagonist SSR182289. The study demonstrated
a reduction in liver fibrosis and α-smooth
muscle actin expression, a marker of stellate cell
activation. Dhar et al.72 demonstrated that
administration of rivaroxaban, an FXa antagonist,
to mice that had been exposed to TAA for 8
weeks induced milder fibrosis, especially around

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central veins, compared with control mice.
The overall mean percentage area of fibrosis
was significantly reduced, as was α-smooth
muscle actin expression. This eect is likely
because of FXa antagonists blocking PAR1 and
2-mediated stellate cell activation.72 Prolonged
administration of enoxaparin in a rat model of
cirrhosis, (induced using CCL4 or TAA), resulted
in an improvement in both portal hypertension
and liver fibrosis, possibly by potentiating fibrosis
regression, resulting in reduction of hepatic
vascular resistance and portal pressures.73 The
use of warfarin anticoagulation to reduce liver
fibrosis induced by CCl4 has been tested using a
transgenic mouse model of FVL-activated protein
C resistance. The progression of fibrosis in FVL
mice was compared to the control C57BL/6 mice
which had been anticoagulated with warfarin.
The results confirmed that the thrombophilic
mouse developed fibrosis at a faster rate than
the control mice, but warfarin anticoagulation
significantly reduced the rate of fibrosis seen in
both strains.74
A small study has suggested an ecacy of low-
molecular-weight heparin as an antifibrotic in
humans. Patients with HBV infection who were
treated with 3 weeks of heparin showed improved
serum levels of alanine aminotransferase and
bilirubin, with a reduction in serum hyaluronic
acid and Type IV collagen concentrations.75 Long-
term use of heparin as an antifibrotic agent in
humans may not be practical because of side
eects including osteopaenia, thrombocytopenia,
and idiopathic hepatitis. A multicentre Phase
II study evaluated the antifibrotic eect
of warfarin anticoagulation in transplanted
HCV patients with cirrhosis. Interim results
demonstrated a reduction in fibrosis scores
at 1-year post-transplantation in warfarinised
patients compared to those who were not.76
Angiotensin Converting
Enzyme Inhibitors
The role of angiotensin converting enzyme
(ACE) inhibitors as antifibrotic agents has been
explored with some success. Angiotensinogen
and angiotensin-1 are present in hepatocytes
and are highly activated in chronic liver disease.
Specifically, angiotensin II receptors are
upregulated during chronic liver injury resulting
in HSC activation. Activation of the renin–
angiotensin–aldosterone system also occurs
within the liver, triggering oxidative stress
and inflammatory cell release contributing to
fibrogenesis.77 The use of ACE inhibitors in
preventing HSC, and renin–angiotensin system
activation in preventing fibrogenesis, is a promising
treatment option. This has been supported in
animal studies, which demonstrated that ACE
inhibitor use in CCl4 treated rats ameliorated levels
of oxidative stress, hepatic inflammation, and
hepatic fibrosis.78 A meta-analysis by Kim et al.78
confirmed the benefits of their use in reducing
hepatic fibrosis in humans. This meta-analysis
only included studies in which intervention groups
used ACE inhibitors or angiotensin receptor
blockers and compared their eect to placebo.
Histological changes in fibrosis were the primary
outcome. ACE inhibitors lowered fibrosis scores,
serum fibrosis markers including TGF-β-1,
collagen Types I and IV, TIMP-1, and MMP-2.
Significantly, they were shown to be safe with no
significant dierences in renal function in those
who received ACE inhibitors versus those who
did not.78
Farnesoid X Receptor Agonists
The potent farnesoid X receptor (FXR) agonist
obeticholic acid has been shown to have
antifibrotic eects, especially in those with primary
biliary cirrhosis and nonalcoholic steatohepatitis,
histologically characterised by the presence of
fibrosis. FXR is present on HSC and is involved
in cellular regulation and activation. FXR
agonists, therefore, have the ability to inhibit HSC
activation and hepatic fibrogenesis.79 Obeticholic
acid use in TAA-treated rats decreased hepatic
inflammation and fibrogenesis, as well as portal
pressure and intrahepatic vascular resistance.
There was also decreased profibrotic cytokine
activity, assessed by TGF-β-1, CTGF, and
PDGF.80 The FLINT trial, a Phase IIb nonalcoholic
steatohepatitis study, compared those treated
with 72 weeks of obeticholic acid versus placebo
and fibrosis improvement was the primary
outcome of the study. It assessed for statistically
significant improvement in hepatic fibrosis
and fibrosis scores observed in the treatment
arm compared to those not being treated.79
Although the antifibrotic properties of FXR
agonists are significant, mild-to-moderate
side eects including pruritis, dyslipidaemia,
fatigue, headache, and gall stone disease have
been reported, which may limit their use in
clinical practice.79

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