Reglare CSH

Wound healing and local
neuroendocrine regulation in the

injured liver
Mohammad R. Ebrahimkhani, Ahmed M. Elsharkawy and Derek
A. Mann*
The hepatic wound-healing response is a complex process involving many
different cell types and factors. It leads to the formation of excessive matrix
and a brotic scar, which ultimately disrupts proper functioning of the liver and
establishes cirrhosis. Activated hepatic myobroblasts, which are derived
from cells such as hepatic stellate cells (HSCs), play a key role in this process.
Upon chronic liver injury, there is an upregulation in the local neuroendocrine
system and it has recently been demonstrated that activated HSCs express
specic receptors and respond to different components of this system.
Neuroendocrine factors and their receptors participate in a complex network
that modulates liver inammation and wound healing, and controls the
development and progression of liver brosis. The rst part of this review
provides an overview of the molecular mechanisms governing hepatic wound
healing. In the second section, we explore important components of the
hepatic neuroendocrine system and their recently highlighted roles in HSC
biology and hepatic brogenesis. We discuss the therapeutic interventions
that are being developed for use in antibrotic therapy.
Theliver, whichisthelargest solidorganinthebody,
plays a central role in the regulation of metabolic
homeostasis and participates in many important
immunological functions. Hepatocytes comprise
about 70% of hepatic cells (Ref. 1). The remaining
30% is made up of nonparenchymal cells,
including hepatic stellate cells (HSCs), Kupffer
cells, endothelial cells, cholangiocytes and several
subsets of resident lymphocytes (Ref. 1) (Fig. 1).
Hepatocytes are organised into epithelial
plates and are primarily responsible for
metabolic functions in the liver. Between the
plates are the sinusoids, which are distensible
vascular channels lined with sinusoidal
endothelial cells (Fig. 1). Small fenestrations in
the sinusoidal linings allow direct cell-to-cell
contact and free diffusion of many substances.
Kupffer cells are resident liver macrophages
Cell signalling, Liver Group, Institute of Cellular Medicine, Newcastle University, Newcastle upon
Tyne, UK.
* Corresponding author: Prof. Derek A. Mann, Liver Group, Institute of Cellular Medicine, 4thFloor,
Cookson Building, Medical School, University of Newcastle, Newcastle upon Tyne, NE2 4HH, UK.
Tel: + 44 191 2223851; Fax: + 44 191 2225455; E-mail: derek.mann@ncl.ac.uk
expert reviews
http://www.expertreviews.org/ in molecular medicine
1
Accession information: doi:10.1017/S146239940800063X; Vol. 10; e11; April 2008
&2008 Cambridge University Press
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that reside within the hepatic sinusoids and act
as a rst line of defence against antigens
passing through the gastrointestinal barrier.
Between the endothelium and hepatocytes lies
the space of Disse where lymph is collected for
delivery to lymphatic capillaries. HSCs are
present within this space, store 80% of body
vitamin A and participate in hepatic wound
healing (Ref. 2), regulation of sinusoidal blood
ow (Ref. 2), angiogenesis (Ref. 3) and
hepatocyte growth (Ref. 4). The space of Disse
also contains basement-membrane-like matrix,
which is essential for the differentiated and
normal functions of all the hepatic cellular
compartments (Ref. 2).
Wound healing is the normal response of tissue
to an injury, and liver brosis occurs as a result of
repeated cycles of injury and repair. The cascade
of events that establishhepatic brosis is complex,
andis inuencedby howdifferent cell types inthe
liver interact inresponse to injury. Hepatic brosis
andits end-stage cirrhosis are a major cause of
mortality and morbidity worldwide. The most
common causes of cirrhosis are alcohol abuse,
hepatitis B, hepatitis C and, increasingly,
obesity, which leads to the metabolic syndrome
that can be complicated by non-alcoholic fatty
liver disease (NAFLD). Other less-common
causes include primary biliary cirrhosis,
haemochromatosis, autoimmune hepatitis and
primary sclerosing cholangitis.
Research over the past decade has greatly
improved our knowledge of the cellular and
molecular biology of brosis in the liver.
Several high-prole papers have shed new light
on the important roles that hepatic
neuroendocrine pathways play in the processes
of wound healing and regeneration in the liver.
These functions are also being increasingly
recognised in different organs (Refs 5, 6, 7). The
hepatic neuroendocrine system is upregulated
in the liver following injury and can regulate
the pattern of wound healing and hepatic
regeneration in different ways. In this review,
we will highlight some of the molecular
mechanisms underlying hepatic wound healing
and address recent advances in understanding
the role of the neuroendocrine system in liver
brosis. The fact that many modulators of
neuroendocrine factors are already in
widespread clinical use makes this eld
particularly exciting. There is now, more than
ever, a need to generate effective antibrotic
therapies in a world where mortality from liver
disease will see exponential growth as a result
of the current obesity epidemic, as well as
increasing burdens from alcohol abuse and
viral hepatitis.
Hepatic sinusoid and hepatocytes in the liver
Expert Reviews in Molecular Medicine 2008 Cambridge University Press
Quiescent hepatic stellate cells
Endothelial cells
Hepatocytes
Sinusoid
Kupffer cells
T cells
Space of Disse
Figure1. Thehepaticsinusoidandhepatocytesintheliver. Sinusoids(distensiblevascular channels) arelined
with sinusoidal endothelial cells. Kupffer cells (resident liver macrophages) reside within the hepatic sinusoids.
The space between the endothelium and hepatocytes is called the space of Disse. Hepatic stellate cells are
present in this space.
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Hepatic wound healing
The normal resolution of tissue injury involves a
series of precise orchestrated phases: (1)
inammation; (2) production of cytokines and
growth factors; (3) myobroblast activation; (4)
extracellular matrix (ECM) production; (5)
angiogenesis; (6) maturation; and (7)
remodelling, which eventually leads to scar
elimination and a return of injured tissue to the
normal state (Refs 8, 9) (Fig. 2). Inappropriate
tissue repair and pathological scarring occurs if
any element of this intricate process becomes
interrupted or overactivated. An imbalance
between ECM formation and degradation can
lead to the accumulation of ECM: an outcome
known as brosis (Ref. 9) (Fig. 2). Fibrosis is the
nal general consequence of uncontrolled
repair processes in many organs in response to
a wide variety of chronic insults and ultimately
can disable proper functioning of the organ. It
is known to occur in the liver, pancreas, lungs,
heart, kidneys, eyes and skin. Over the past
decade, there has been extensive investigation
of the molecular events underlying tissue
brogenesis. In the following sections, we
explore the mechanism of hepatic wound
healing and describe the cellular compartments
in which it occurs.
The inammationbrosis pathway
Chronic persistent inammation typically
precedes brosis (Refs 10, 11, 12). Following
injury to the liver, hepatocytes release factors
that start an inammatory process by
recruitment of leukocytes to the site of injury.
Local production of matrix metalloproteinases
(MMPs) at the site of injury results in the
disruption of the basement membrane and
favours inammatory cell inltration (Ref. 13).
Neutrophils are the most abundant
inammatory cell in the early stages of wound
healing. Their granulation is followed by
macrophage inltration and subsequently by
lymphocyte recruitment (Refs 8, 12). The
primary roles of leukocytes are to eliminate any
invading organisms and to remove dead cells
(Ref. 12). Inammation also produces
probrogenic cytokines and chemokines, which
activate myobroblasts and induce wound-
healing responses, which, if unchecked, drive
brogenesis (Fig. 2). However, it has also been
proposed that brosis is not always driven by
inammation, suggesting that the mechanisms
that regulate brogenesis partly differ from
those regulating inammation (Ref. 8). This
might explain the lack of efcacy of some anti-
inammatory agents in the treatment of brotic
disease and the need to identify targeted
antibrotic therapies.
Myobroblasts: the key players in hepatic
brosis
In many organs, including the liver,
myobroblasts are the key cellular effectors
during wound contraction and repair, and
inappropriate myobroblast activation is the
central pathogenic mechanism of brotic
disorders (Ref. 14). Upon tissue injury,
myobroblasts become activated and migrate
toward the site of damage where they
proliferate. Myobroblasts produce ECM
proteins, such as type I and type III collagen,
and control ECM remodelling through the
expression of several MMPs and tissue
inhibitors of metalloproteinases (TIMPs). The
balance between ECM degradation and
production is a critical factor that determines
the normal wound-healing response and
returns the tissue to its preinjury state (Ref. 15).
This process is inuenced by the number of
activated myobroblasts at the site of injury,
which is controlled by the rate of production
and proliferation as well as apoptosis of these
cells (Refs 9, 16). Chronic injury induces a
marked alteration in the normal healing process
and prevents return of tissue to the preinjury
state. In fact, constant inammation and/or
infection leads to permanent myobroblast
activation, either directly, by acting on HSCs or
indirectly, through paracarine-dependent factors
(Fig 2). This activated phenotype of
myobroblasts promotes the inappropriate
regulation of tissue repair and leads to
extensive scar formation, and nally results in
brosis (Ref. 17) (Fig. 2).
Developmentally, broblasts are mesenchymal
in origin (Ref. 18). However, it is now widely
believed that myobroblasts are derived from a
number of different sources in injured adult
tissues (Ref. 19). In the context of the liver, there
are four different cellular sources (Fig. 3). HSCs
are well-characterised cells that have been
shown to contribute signicantly to activated
myobroblasts in various types of hepatic
injury. The other potential cellular origins are
bone-marrow-derived mesenchymal cells
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Activated
Kupffer cells
Paracrine signals
Transdifferentiation
of q-HSCs
Apoptosis of
myofibroblasts
Cessation of
injury
Persistent
injury/
infection
Proliferation and survival of
myofibroblasts, increased
TIMPs and ECM
Regeneration and remodelling
a-HSCs
a-HSCs
Other sources of myofibroblasts
(e.g. portal fibroblasts and fibrocytes)
Activated
myofibroblasts
Autocrine signals
ROS and RNS
Apoptotic and
necrotic bodies
Chemokines
Cytokines
Inflammatory
infiltration
Normal hepatic architecture
q-HSCs
Injury
1
2
3
5
6 7
4
Fibrosis
Hepatic wound healing
Figure 2. Hepatic wound healing. Normal hepatic structure (1) is disrupted following injury to the liver (2) when
hepatocytes release factors that start an inammatory process via recruitment of leukocytes to the site of injury.
Thisoccursinparallel tolocal activationof Kupffer cells, whichproducecytokinesandtoxicfreeradicals(Reactive
oxygen and nitrogen species, RNSand ROS). (3) Probrogenic cytokines and chemokines, and free radicals, as
well as apoptotic and necrotic bodies, together establish a paracrine signal leading to transdifferentiation of
quiescent hepatic stellate cells (q-HSCs) into activated myobroblasts [activated hepatic stellate cells
(a-HSCs)]. This step in HSC activation is known as the initiation phase, which renders the cells susceptible
to cytokines, growth factors and other stimuli. Following this phase, perpetuation occurs a phase in which
the differentiated phenotype of the cell is amplied by mediators via autocrine signals. (4) In addition to
a-HSCs, other cells, such as portal broblasts, bone-marrow-derived cells and epithelial compartments
within the liver, can contribute to the activated pools of myobroblasts during liver injury. (5) After cessation
of injury, activated myobroblasts undergo apoptosis, collagen bres become more organised and scar
tissue is removed. (6) This occurs in parallel to hepatocyte regeneration and remodelling, which restores the
damaged tissue to the normal state. However, in the case of persistent injury, the normal healing process is
interrupted (7) and persistent inammation and/or infection results in chronic myobroblast activation and
proliferation, as well as excessive accumulation of extracellular matrix (ECM) components, which promotes
and establishes hepatic brosis.
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(Refs 20, 21), brocytes (Ref. 22) and portal
broblasts (Refs 23, 24). Recent evidence also
suggests that biliary epithelial cells and
hepatocytes may contribute to the hepatic
myobroblast pool by undergoing epithelial
mesenchymal transition (EMT) (Refs 25, 26, 27,
28). However, there is a need for further
lineage-specic studies to explore EMT
processes in liver brogenesis. The contribution
of each cellular source to activated hepatic
myobroblasts varies according to the type of
liver injury. For instance, myobroblasts
derived from portal broblasts the broblasts
surrounding the biliary tree are important
brogenic cells in the early stages of biliary
brosis (Refs 23, 29, 30). In cases of rapid biliary
injury, such as that seen in biliary atresia,
biliary epithelial cells play an important role in
biliary brosis via an EMT process (Ref. 26). It
is possible that the different potential sources of
brogenic cells in various liver diseases or
different lobular regions of the diseased liver
could lead to antibrotic therapies that are
specically targeted only to certain
subpopulations or diseases.
Hepatic stellate cell transdifferentiation
After hepatic injury of any aetiology, HSCs
undergo a highly regulated transdifferentiation
process from quiescent cells into proliferative
and brogenic myobroblasts. This change in
the phenotype of HSCs is also known as HSC
activation and has been divided into two
phases (Ref. 2). First, there is an initiation
phase, which renders the cells susceptible to
cytokines, growth factors and other stimuli, and
occurs in parallel with transcriptional
alterations in early response genes. Following
this, perpetuation occurs a phase in which the
differentiated phenotype of the cell is amplied
by soluble and insoluble mediators (Ref. 2).
Initiation of HSC transdifferentiation is largely
due to paracrine stimulation, whereas the
perpetuation of the differentiated state involves
autocrine as well as paracrine loops (Fig. 2).
Ultimately HSC transdifferentiation is
characterised by the development of activated
hepatic myobroblasts with proliferative,
contractile, migratory, brogenic and
inammatory properties (Ref. 2).
Current knowledge suggests that the initial
signals driving HSC transdifferentiaion are
produced by injured cells, pathogens,
inammatory mediators and alterations in the
local ECM. Injured hepatocytes, Kupffer cells
and other nonparenchymal cells produce
necrotic cell debris, apoptotic bodies (Refs 31,
32, 33), reactive oxygen and nitrogen species
(ROS and RNS) (Refs 34, 35), as well as other
active mediators, all of which signal through
specic activation pathways and can be sensed
by HSCs. In addition, early alterations in the
mechanical stiffness of the whole tissue precede
matrix deposition and can trigger
transdifferentiation of HSCs (Ref. 36).
Pattern-recognition receptors, which recognise
conserved pathogen or host-associated molecular
patterns, are expressed by different cells of the
immune system and act as a rst line of defence
against injury or infection (Ref. 10). Recent
advances in the eld have identied the
expression of pattern-recognition receptors such
The origins of liver myofibroblasts
Expert Reviews in Molecular Medicine
2008 Cambridge University Press
Epithelial cells in
the liver
Quiescent hepatic
stellate cells
Portal
fibroblasts
Activated
myofibroblasts
Bone-marrow-
derived cells
Figure 3. The origins of liver myobroblasts.
During the development of hepatic brosis,
myobroblasts are derived from a number of
different sources. Hepatic stellate cells are the
best characterised source. Other potential cellular
origins are bone-marrow-derived mesenchymal
cells, portal broblasts and epithelial
compartments in the liver, such as cholangiocytes
and hepatocytes. Biliary epithelium and
hepatocytes can contribute to the activated
myobroblast pool by epithelial mesenchymal
transition.
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as toll-like receptor (TLR) 4 and TLR9 in hepatic
myobroblasts (Refs 33, 37). Subsequently, it has
been shown that DNA from apoptotic
hepatocytes, which are rich in cytosine-
phosphate-guanine (CpG), can activate TLR9 in
HSCs and contribute to the activated phenotype
in these cells (Ref. 33). This observation may
explain why apoptosis of hepatocytes results in
activation of HSCs and development of hepatic
brosis (Ref. 38, 39). In addition, a recent study
using TLR4 mutant mice has conrmed a role
for TLR4 in liver brogenesis (Ref. 40). It
therefore appears that activation of several
pattern-recognition receptors can act as a
primary signal for HSC transdifferentiation
(Ref. 10). In addition, HSCs have recently been
shown to act as professional antigen-presenting
cells (Ref. 41). The ability of these cells to
process and present antigens to immune cells
can itself help to activate immune-dependent
mechanisms, which subsequently inuence the
transdifferentiation process in HSCs. However,
the impact of various immune cells on
transdifferentiation of HSCs warrants further
study. It is clear, therefore, that there is a
network of multiple signals, as well as different
cell types, that are inuenced by liver injury
and drive transdifferentiation in quiescent HSCs.
Maintaining the activated phenotype
Transdifferentiation of quiescent HSCs to a
myobroblast phenotype does not appear to be
a transient event. Instead, the activated
myobroblasts state is maintained by paracrine
signalling (e.g. ROS and RNS) from other types
of liver cell, including hepatocytes,
cholangiocytes and Kupffer cells (Refs 2, 35, 42).
HSCs themselves develop new autocrine
pathways, including those involving
transforming growth factor b (TGF-b) (Refs 43,
44), angiotensin II (Refs 45, 46), platelet-derived
growth factor (PDGF) (Ref. 47), monocyte
chemoattractant protein 1 (MCP1) (Refs 48, 49)
and vascular endothelial growth factor (VEGF)
(Ref. 50), which maintain the activated state of
the myobroblasts and drive brogenesis. One
of the critical events in this regard, is that HSCs
upregulate new membrane receptors and
signalling proteins that prime these cells to
respond to inammatory mediators and growth
factors, such as interleukin 6 (Ref. 51), TGF-b
(Refs 43, 44) and PDGF (Ref. 47). The alteration
in ECM components during this phase actively
regulates HSC behaviour. Different matrix-
associated molecules, such as TIMP1, integrins
and other adhesion molecules, contribute to
HSC survival and perpetuation of the activated
myobroblast phenotype (Ref. 52, 53). Constant
signals from a stable chronic infection and/or
injury through pattern-recognition receptors
may also directly regulate the survival and
function of HSC-derived myobroblasts (Ref. 10).
MMP/TIMP production by HSCs
Activated hepatic myobroblasts proliferate and
migrate at the sites of liver injury, secreting
large amounts of ECM and regulating ECM
degradation. They express a combination of
MMPs and their specic TIMPs. In the early
phases of liver injury, HSCs transiently express
MMP3 (stromelysin 1), MMP13 (collagenase 3)
and uroplasminogen activator, and exhibit a
matrix-degrading phenotype, all of which
enables them to migrate toward the site of
injury (Ref. 54). In the later stages of liver injury
and HSC activation, the expression pattern
changes, and the cells express a combination of
MMPs, which have the ability to degrade
normal liver matrix while inhibiting
degradation of the brillar collagens that
accumulate in liver brosis. This new
expression pattern is characterised by the
combination of pro-MMP2 and membrane type
1 (MT1) MMP expression, which drives
pericellular generation of active MMP2 and
local degradation of subendothelial matrix,
facilitating replacement with high-density
interstitial matrix (Ref. 54). In addition, there is
a marked increase in the expression of TIMP1,
leading to a more global inhibition of
degradation of brillar liver collagens by
interstitial collagenases (MMP1/MMP13). As a
consequence, normal matrix homeostasis is
severely disrupted in favour of the net
deposition of brillar collagen, non-collagen
ECM molecules and integrin ligands.
Neuroendocrine differentiation and
cancer: implications for wound healing
Neuroendocrine phenotype
Neuroendocrine cells are scattered in various
organs of the body and have endocrine
phenotypes that share some of the structural,
functional and metabolic properties of neurons.
Advances in molecular pathology have led to
the identication of several markers in these
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cells. Neuroendocrine cells typically contain
secretory granules, called large dense-core vesicles
because of their characteristic appearance upon
electron microscopy. In addition to peptides, these
granules also contain one or more chromogranin/
secretogranin proteins. They express markers such
as chromogranin A, glycolipid A2-B4, S-100
protein, neural cell adhesion molecule, neuron-
specic enolase and synaptophysin (Refs 55, 56,
57). These markers have different specicities and
sensitivities in identifying the neuroendocrine
phenotype of a cell; many participate in various
intracellular excretory activities of cells, such as
packaging of hormones and neuropeptides, or
modulation of exocytosis and neurotransmitter
release in synapses.
Parathyroid-hormone-related peptide was
previously identied as the factor responsible
for the syndrome of humoral hypercalcaemia of
malignancy and is also a classical
neuroendocrine peptide that is involved in
growth, differentiation and angiogenesis in
various tissues (Ref. 58). In addition, a wide
range of neuropeptides are produced by
neuroendocrine cells or tissues, including
serotonin, neurotrophins, endogenous opioid
peptides, somatostatin, cannabinoids and
calcitonin (Refs 55, 57, 59, 60, 61). These factors
commonly regulate growth and survival in
various cells or tissues.
Little is known of the functional role of
neuroendocrine cells in many organs. It has been
suggested that they probably serve a paracrine or
local regulatory role. Neuroendocrine
differentiation has been found in a subgroup of
various carcinomas, including prostate, breast,
stomach, colorectal and non-small-cell lung
cancer (Refs 62, 63, 64, 65, 66, 67, 68). In many of
these tumours, neuroendocrine differentiation
has adverse prognostic effects, suggesting an
integral role for neuroendocrine factors in the
regulation of the malignant phenotype (Refs 62,
63, 64). The detailed molecular mechanisms
underlying the observed behaviour are mainly
undened but the neuroendocrine cells of the
tumour probably play a signicant role during
tumour growth, angiogenesis and metastasis
(Refs 62, 64, 69, 70). The histogenesis and origins
of neuroendocrine cells in the tumour
environment are not clear. However, it may
involvealocal transdifferentiationprocess (Ref. 62).
Cancers have beendescribedas wounds that do
not heal (Ref. 71), suggesting that both tumours
and wounds may be part of a continuum.
Indeed, there are similarities between tumour
stroma generation and wound healing. This
includes various features that can regulate
growth, differentiation or angiogenesis in both
the wound and tumour environment. In fact,
persistent injuring stimuli result in
inammation and continuous wound healing
two mechanisms that lead to accentuated scar
tissue formation or brosis and have frequently
been linked to cancer formation (Refs 71, 72).
We have recently coined the phrase hepatic
inammationbrosiscancer axis to reect
this phenomenon (Ref. 73).
During chronic hepatic injury, different types of
liver cells acquire a neuroendocrine phenotype.
Given the putative role of the neuroendocrine
system in the regulation of growth and survival
in various conditions, and the shared
similarities between tumours and wounds, the
neuroendocrine compartment of the inamed
liver might be expected to regulate cell growth,
migration and angiogenesis during wound
healing. In fact, there is increasing evidence that
local neuroendocrine factors do indeed
inuence wound healing and brogenesis in
many organs (Refs 5, 6, 74, 75, 76, 77).
Neuroendocrine compartments in the
chronic-injured liver
In the diseased liver, atypically proliferating
cholangiocytes acquire a neuroendocrine
phenotype (Ref. 55). These cells, also known as
reactive bile ductules, have been identied as
one of the major contributors to the production
of various neuroendocrine factors in diverse
liver pathologies, and appear at interface zones
where maximal cell death and inammation
occurs (Ref. 55). Proliferation of cholangiocytes
precedes the development of cirrhosis in most
liver diseases, including chronic cholestasis and
biliary cirrhosis, alcoholic liver disease and
chronic toxic liver injuries. This cholangiocyte
component of the local neuroendocrine system
in the injured liver has been widely studied in
recent years (Refs 55, 57, 59, 61).
Hepatic progenitor cells, also known as liver
oval cells, lie within or immediately adjacent to
the canal of Herring (Ref. 78) and express
neuroendocrine proteins, such as chromogranin-
A, neural-cell-adhesion molecule, parathyroid-
hormone-related peptide, S-100 protein,
neurotrophins and neurotrophin receptors
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(Refs 56, 57, 79). These cells are activated to
proliferate upon chronic liver damage in
situations where the proliferation of hepatocytes
is inhibited, which includes non-alcoholic
steatohepatitis, chronic cholestatic liver disease,
chronic alcoholic hepatitis and viral hepatitis.
Progenitor cells differentiate into hepatocytes
and contribute to the pool of reactive bile
ductules during the course of liver disease. In
addition to progenitor cells and reactive bile
ductules, small hepatocytes in periportal regions
express chromogranin A (Ref. 79). These newly
formed intermediate hepatocytes are observed in
chronic conditions where there is activation of
progenitor components (Ref. 79).
HSCs, themainplayersintheliverwound-healing
process, share a number of different markers with
neuroendocrine cells and the cells of the nervous
system (Ref. 80). They express synaptophysin,
which is one of the factors primarily correlated
with neuroendocrine differentiation (Refs 56, 81,
82). HSCs also express neutrophins and neural
cell adhesion molecules (Ref. 83). Generation
of neuroendocrine factors by HSCs may help to
maintain the activated myobroblast phenotype
through autocrine loops. However, what is
potentially more central to the process is that
upon transdifferentiation HSCs upregulate
many receptors of neuroendocrine factors, which
renders them susceptible to neuroendocrine
regulation in wound healing. This issue will be
discussed in more detail below.
Which factors trigger neuroendocrine
differentiation in the injured liver?
Neuroendocrine differentiation in the liver is often
associated with the presence of cellular stress
and inammation (Refs 55, 84). For instance,
ductular reactions in cholangiocytes are usually
accompanied by periductal inammation, which
is presumed to trigger the reaction (Refs 55, 85,
86). The possible role of hepatic inammation in
the development of neuroendocrine differentiation
is also supported by studies of cancer (Refs 70, 84).
It has been shown that interleukin 6 (IL-6) and
tumour necrosis factor (TNF) a two important
cytokines in the liver regulate both growth and
neuroendocrine differentiation in different types of
experimental cells, as well as human cancer cells
(Refs 70, 84). Several recent in vitro studies with
cancer cells have demonstrated that IL-6 can
promote neuroendocrine differentiation through
different intracellular signal-transduction
pathways, including the phosphatidylinositol 3-
kinase, signal transducers and activators of
transcription 3 (STAT3), and mitogen-activated
protein kinase (MAPK) pathways (Refs 70, 87, 88).
However, there is clearly a need to study how
much of this data can be extrapolated to liver
inammation and brosis.
In addition, signals derived from the ECM can
also inuence neuroendocrine functions in bile
duct epithelial cells. It has been reported that
interaction of these cells with the surrounding
matrix in particular collagen type IV and
matrix heparan sulfate proteoglycan perlecan
induces neuroendocrine differentiation
(Ref. 56). This is consistent with the fact that
reactive bile ductules are always surrounded by
basement membrane containing these ECM
components. Therefore, the neuroendocrine
phenotype in cholangiocytes and other cellular
populations in the liver can be regulated by
signals from both the matrix and inammatory
factors. However, the specic cellular and
molecular players that promote neuroendocrine
differentiation in the liver remain to be identied.
Neuroendocrine regulation of
myobroblast differentiation and wound
healing
As discussed earlier, transdifferentiation of HSCs
into activated myobroblasts is one of the pivotal
events during wound healing and brogenesis in
the injured liver, and can be subdivided into an
initiation phase and a perpetuation phase. After
liver injury, inammatory cells may directly
contribute to this transdifferentiation; but they
are not likely to provide all the growth factors
necessary for maintaining or accentuating the
activated phenotype at later stages. However,
induction of neuroendocrine differentiation in
cholangiocytes and oval cells in response to
inammation and/or other mechanisms can
contribute to maintenance of the activated state
of HSCs. This may occur in parallel to the
expression of various neuroendocrine receptors
during HSC differentiation, which renders these
cells responsive to regulation (Fig. 4). Many of
these factors can further affect hepatic wound
healing by inuencing neoangiogenesis,
inammation and hepatocyte regeneration. It
should also be considered that HSCs have their
own neuroendocrine features that may
contribute to their brogenic activities via
autocrine mechanisms. Here we discuss some
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of the recently identied neuroendocrine factors
that regulate the production and function of
HSC-derived myobroblasts in the liver
(Table 1). It is interesting that many medications
that could potentially modulate the effect of
neuroendocrine factors and prevent brogenesis
are already in routine clinical practice in other
contexts and have established safety records,
making them very attractive candidates for
rapid translation into clinical trials. However, it
is important to stress that the regulation of HSC
biology by these factors has not yet been fully
dened and may be more complex than initially
anticipated. Furthermore, there is no evidence
that neuroendocrine factors regulate the
functions of HSC-derived myobroblasts
exclusively. In fact, the role of these factors in
the differentiation of other cellular sources of
liver myobroblasts is possible and warrants
further study.
Neutrophins and their receptors
Neutrophins were originally reported to regulate
growth and development in the nervous system.
A rapidly growing body of evidence now
suggests that neutrophins also play profound
Local neuroendocrine regulation of stellate cell transdifferentiation
q-HSC
Inflammation
Matrix
Transdifferentiation
Activated myofibroblasts
a-HSC
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p
rod
uction
Induction of
neuroendocrine phenotype
in cells in the liver
Neuroendocrine components of the injured liver
(proliferating bile ducts, oval cells etc)
?
Different neuroendocrine
factors help to maintain
the activated phenotype
in a-HSCs
Figure 4. Local neuroendocrine regulation of stellate cell transdifferentiation. Normal hepatic structure is
disrupted following injury to the liver, subsequently, quiescent stellate cells (q-HSCs) transdifferentiate into
activated myobroblasts (a-HSCs). At the same time, inammation and altered matrix components induce
neuroendocrine differentiation in proliferating bile ducts, oval cells and even HSCs. These cells produce
various neuropeptides endocannabinoids, serotonin, opioids, neutrophins which contribute to
maintenance and amplication of the differentiated phenotype in activated myobroblasts. Neuroendocrine
factors contribute to various aspects of probrogenic activity in activated myobroblasts, such as
contraction, migration, proliferation and extracellular matrix production.
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roles in various non-neuronal tissues. The
neurotrophin family consists of nerve growth
factor (NGF), brain-derived neurotrophin
(BDNF), neurotrophin 3 and neurotrophin 4/5.
The neutrophin receptors are high-afnity
neurotrophin tyrosine kinase receptors TrkA,
TrkB and TrkC, as well as the low-afnity pan-
neurotrophin receptor p75NTR, which belongs
to the TNF-receptor superfamily (Table 1).
Activated myobroblasts, cholangiocytes and
hepatocytes secrete NGF and express the
receptors for different neutrophins (Refs 83, 89,
90). A recent study has shown that HSCs
derived from p75-knockout mice showed
signicantly reduced differentiation into
activated myobroblasts, as well as reductions
in the protein expression of smooth muscle
actin and collagen I (Ref. 91). This effect was
ameliorated by transfection with the
intracellular portion of p75, demonstrating a
critical role for this receptor in HSC activation.
Interestingly, an exon-3 knockout of p75, which
retains an intact intracellular domain but lacks
the structural requirements for interaction with
its ligand, supports the activation of HSCs and
the development of liver brosis in a
predictable manner after carbon tetrachloride
(CCl
4
) intoxication (Iredale, J.P. and Kendall,
T.J., University of Edinburgh, UK, pers.
commun.). It has been suggested that p75
Table 1. Local neuroendocrine regulation of hepatic brosis
Neuroendocrine
factor
Producer cell type Known
Receptors
Effect on HSCs and brosis
Nerve growth factor
and other
neurotrophins
Myobroblasts
Cholangiocytes
Hepatocytes
Tyrosine kinase
receptors (TrkA,
TrkB, TrkC)
Pan-
neurotrophin
receptor p75NTR
p75NTR induces HSC
differentiation or apoptosis
during brogenesis;
NGF regulates migration and
differentiation of broblasts
by TrkA-dependent mechanisms
Serotonin Platelets
Myobroblasts?
(Storage/release)
5HT15HT7 Expression of 5HT1B, 5HT2A
and 5HT2B is induced with HSC
activation. 5-HT synergises with
PDGF to stimulate increased
HSC proliferation. 5-HT
signicantly increases TGF-b1
and Smad4 in HSCs
Opioids Cholangiocytes
Hepatocytes
d, m, k Activation of d opioid receptor
increases TIMP1 and
procollagen I. m-opioid receptor
activationinduces proliferationof
HSCs. In vivo inhibition of opioid
system reduces brosis in
cholestatic and
dimethylnitrosamine-induced
liver injury
Cannabinoids Probably at site of
injury (on demand) by
receptor-stimulated
cleavage of lipid
precursors (Specic
cells?)
CB1, CB2,
vanilloid
Genetic ablation or
pharmacological blockade of the
CB1 receptor attenuates hepatic
brosis in different models.
However, CB2 signalling is
antibrogenic in vivo and in vitro
Abbreviations: 5-HT, 5-hydroxytryptamine; CB-1, cannabinoid receptor 1; CB-2, cannabinoid receptor 2; HSC,
hepatic stellate cell; NGF, nerve growth factor; PDGF, platelet-derived growth factor; RNS, Reactive nitrogen
species; ROS, reactive oxygen species; TIMP-1, Tissue inhibitor of metalloproteinase 1.
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signalling through Rho promotes HSC
differentiation into active myobroblasts
(Ref. 91). However, the regulation of HSCs by
neutrophin receptors appears to be more
complicated and warrants additional research.
NGF induces apoptosis in activated HSCs via a
p75-dependent mechanism (Ref. 90). p75NTR
may therefore function not only as a regulator
of HSC transdifferentiation but may also limit
the brogenic response by exerting a negative
inuence on the life span of the activated
myobroblasts. A similar regulatory system has
been observed in broblastmyobroblast
differentiation in other situations. Quiescent
broblasts express TrkA constitutively, whereas
myobroblats express both TrkA and p75, and
all produce NGF (Refs 92, 93). NGF regulates
the migration and differentiation of broblasts
by a TrkA-dependent mechanism, and it also
induces apoptosis of activated myobroblats
via the p75 receptor. Thus, it appears that
different temporal expression patterns of TrkA/
p75 might regulate the nal pathological
process (Ref. 93). Together, these data suggest
an important role for neutrophins and their
receptors during liver wound healing.
The serotonin system
Serotonin or 5-hydroxytryptamine (5-HT) has
been recognised for more than 50 years as an
effector on various types of smooth muscle cell
and subsequently as an agent that enhances
platelet aggregation and as a neurotransmitter
in the nervous system. Despite the critical role
of serotonergic mechanisms in the central
nervous system, the brain actually contains very
little serotonin in relative terms. About 95% of
serotonin is produced in enterochromafn cells
throughout the gut. Enterochromafn cells
produce far more serotonin than is required by
the gut and it overows into the blood, where it
is then taken up and concentrated in platelets,
the only source of blood serotonin. After its
release by platelets, serotonin is absorbed
rapidly by various cell types via the specic
membrane-bound serotonin transporter (Ref. 94).
Molecular cloning has revealed an unexpected
diversity of receptor subtypes in serotonin
signalling (5HT15HT7), which are coupled to
different, but overlapping, transmembrane-
signalling mechanisms (Table 1). The expression
of serotonin receptors has been reported in
hepatocytes, and the authors suggested a key
role for serotonin in liver regeneration (Ref. 95).
Although platelets are the main source of
serotonin in the injured liver, proliferating
cholangiocytes and HSCs can also secrete
serotonin (Ref. 59, 74). Rat and human HSCs
express the 5HT1B, 5HT1F 5HT2A 5HT2B and
5HT7 receptors, with expression of 5HT1B,
5HT2A and 5HT2B receptors induced upon
HSC activation (Ref. 74). Serotonin signicantly
increases the expression of TGF-b1 and Smad4
in HSCs (Ref. 96) and synergises with PDGF to
stimulate increased HSC proliferation (Ref. 74).
In addition, HSCs express a functional
serotonin transporter (SERT) and can
potentially regulate the concentration of
serotonin in the vicinity of injured liver cells
(Ref. 74). Therefore, serotonin may actively
regulate hepatic brogenesis.
Endogenous opioid peptides
Opiates have been known for decades for their
role in pain management. They have been
shown to be produced endogenously in the
body and to regulate cell growth, differentiation
and survival in neuronal and non-neuronal
cells. Endogenous opioid peptides act by
interacting with three classical opioid receptors:
the m, d and k receptors (Table 1). Three
endogenous families of classical opioid
peptides have been identied in the body: the
enkephalins, endorphins and dynorphins. Each
family is derived from a distinct precursor
polypeptide and has a characteristic anatomical
distribution (Ref. 97).
Studies fromthe 1980s in patients with primary
biliary cirrhosis represent the earliest evidence of
a correlation between endogenous opioids and
liver disease (Ref. 98). It has been demonstrated
that bile duct epithelium and hepatocytes
express preproenkephalin mRNA following
injury, which encodes Met- and Leu-enkephalin
peptides (Refs 99, 100). Recent studies on the
role of opioids in the pathophysiology of
chronic liver injury demonstrate potentially
novel targets for antibrotic therapies (Refs 75,
97, 101). Recently, we identied d-opioid-
receptor expression following transdifferentiation
of HSCs. Activation of this receptor by different
d-opioid agonists increases expression of the
genes encoding TIMP1 and procollagen I and
inhibits apoptosis of activated myobroblasts
(Ref. 75). Subsequent work has demonstrated that
stimulation of opioid receptors activates calcium-
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dependent protein kinase C, extracellular-signal-
regulated kinase, phosphatidylinositol 3-kinase
(PKCERKPI3K) signalling, which then
mediates the effect of endogenous opioids on
HSC proliferation and collagen synthesis.
Furthermore, in vivo inhibition of the opioid
system in a model of chronic cholestatic liver
disease and in dimethylnitrosamine (DMN)-
induced liver injury signicantly prevented the
development of hepatic brosis (Ref. 75, 102). It
has been suggested that during chronic liver
injury, HSC-derived myobroblasts express
opioid receptors rendering them susceptible to
opioid peptides produced by epithelial
compartments in the liver, which can then
contribute to maintenance of the activated
phenotype in these cells.
Endocannabinoids
The role of the endogenous cannabinoidsystemin
the pathophysiology of liver disease has attracted
a great deal of recent attention. Endogenous
cannabinoids (endocannabinoids) are lipid
mediators that include amides and esters of
long-chain polyunsaturated fatty acids. Two
arachidonic acid derivatives,
arachidonoylethanolamide (anandamide) and 2-
arachidonoylglycerol (2-AG) are the most
biologically active endocannabinoids described
so far (Ref. 103). These molecules bind to two
G-protein-coupled receptors, CB1 and CB2,
although other targets, such as vanilloid
receptors have been identied (Table 1). Unlike
classical neurotransmitters and neuropeptides,
endocannabinoids are not stored in intracellular
compartments but are produced on demand
by receptor-stimulated cleavage of lipid
precursors (Ref. 104).
Following transdifferentiation, HSCs express
both CB1 and CB2 receptors (Ref. 105). It has
been reported that CB2 signalling in HSCs is
antibrogenic (Ref. 106) and that stimulation of
activated HSCs by anandamide or 2-AG
provokes cell death by redox-sensitive
mechanisms, not via cannabinoid receptors
(Ref. 105, 107). However, epidemiological
studies have demonstrated that daily cannabis
smoking is an independent risk factor for rapid
progression of brosis in chronic hepatitis C
patients (Ref. 108). A further study, carried out
in three mouse models of hepatic brogenesis,
identied that genetic ablation or
pharmacological blockade of the CB1 receptor
signicantly attenuated development of hepatic
brosis (Ref. 109). This inhibition of CB1
receptor signalling was accompanied by
decreased expression of TGF-b an important
probrogenic cytokine in the injured liver and
occurred in parallel to the reduced proliferation
and increased apoptosis of activated
myobroblasts in vivo and in vitro. Taken
together, these studies suggest that CB1
signalling is the dominant pathway in hepatic
brogenesis in response to factors that can
activate both cannabinoid receptors (Ref. 110)
and propose an intriguing role for this system in
the regulation of hepatic wound healing.
Antagonism of the CB1 receptor, along with
potentiation of the CB2 receptor, may prove to
be an effective antibrotic therapy in the future.
This may be realised soon, with clinical trials of
the CB1 antagonist rimonabant already under
way in the study of obesity.
Future perspectives and therapeutic
potentials
The role of the neuroendocrine system in liver
disease is an important emerging theme that
has already shed new light on the molecular
mechanisms that regulate hepatic wound
healing and brogenesis. However, much
remains to be learned before this new
information can be exploited for therapies that
promote liver regeneration and limit brosis. In
particular, the presence of different receptors
with counteracting signalling events means that
brosis of the liver is a far from simple event.
For instance, serotonin induces probrogenic
behaviour in HSCs, while at the same time
accelerating hepatocyte regeneration and
inhibiting cholangiocyte proliferation (Refs 74,
95). An ideal strategy to inhibit brogenesis
would be to inactivate the probrogenic
serotonin receptors and simultaneously stimulate
those serotonin receptors that promote
hepatocyte regeneration. However, the identities
of the receptors responsible for mediating the
brogenic and regenerative properties of
serotonin are unknown. At different stages in
the time course of liver disease, the distribution
of neuroendocrine receptors can vary from one
cell type to another in a dynamic fashion. For
example, with bile duct ligation, d-opioid-
receptor expression gradually diminishes in
cholangiocytes (Ref. 111). By contrast, the same
receptor is not expressed in quiescent HSCs but
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is expressed following transdifferentiation in
response to cholestasis (Ref. 74). Such opposite
trends for expression of d-opioid receptor in
these two liver cell types may account for the
heterogenic effects of endogenous opioid
peptides on cholangiocytes and HSCs (Ref. 111).
Therefore, inhibition of the same signalling
event at different time points in liver disease
may result in distinct outcomes.
Infuture, weneedtofurtherdissect theexpression
and function of different neuroendocrine factors
and the related receptors in each cell population
of the liver with precise molecular biology
approaches. For example, the increasing
availability of cell-specic knockouts of different
serotonin receptors will enable a careful dissection
of the role of the various receptors in vivo.
Furthermore, there is an urgent need to conrm
the results in human cells and both normal and
diseased human tissues.
Serotonin antagonists and reuptake inhibitors
are used routinely for the management of
psychiatric disorders such as depression.
Naltrexone (a nonselective opioid-receptor
blocker) is clinically administered for the
treatment of cholestatic pruritis in primary
biliary cirrhosis and also in the management of
addiction and alcohol dependence (Refs 112,
113). CB1 antagonists are currently under trial
for the treatment of obesity (Ref. 114). The
antagonist used in a recent study on the role of
CB1 in liver brosis in mice is SR141716A
(rimonabant) (Ref. 108); this drug is currently
an available therapeutic option. Although there
have been no studies on the role of these drugs
in the progression of human liver disease, their
availability in clinical practice makes it likely
that clinical trials of these agents in chronic
brogenic hepatic disease will not be far off.
Portal hypertension, variceal bleeding and
systemic arterial vasodilatation are among the
main reasons for morbidity and mortality in
cirrhotic patients. It has been demonstrated that
some of the neuroendocrine factors, such as
endogenous cannabinoids and opioids,
contribute to the systemic vasodilatory state
seen in cirrhosis (Ref. 115). Therefore,
modulation of these systems can potentially
extend the life of cirrhotic individuals, not only
by slowing or reversing brogenesis, but also
by improving the associated hyperdynamic
circulatory state. CB1 receptor activation has
been linked to obesity and energy homeostasis,
and its inhibition can exert enhanced
antibrotic effects by modulation of metabolic
risk factors (Ref. 116). An important study has
shown that serotonin plays a role in the
pathogenesis of steatohepatitis, and therefore
might represent a novel target for the
prevention and treatment of nonalcoholic
steatohepatitis (NASH) and its associated
brosis (Ref. 117). Opioid-receptor blockade by
naltrexone is an approved strategy for treatment
of alcohol dependence by reducing alcohol
craving and relapse in heavy drinkers, and it
may also prove to be a more effective
antibrotic candidate in alcoholic patients.
Conclusion
It is clear that local neuroendocrine systems play a
critical role in the development of hepatic brosis.
Various modulators of different components of this
system are available clinically and can be
potentially useful in patients with liver disease.
However, to achieve further successful
therapeutic goals through neuroendocrine
targeting in hepatic brosis, we need to consider
the timing of treatment in the course of disease
and the duration of the therapy, as well as cell-
and receptor-specic targeting. In addition,
appropriate pharmacokinetics and safety
margins of the drugs should be considered
when treating patients with liver disease.
Acknowledgements and funding
We recognise many outstanding published
contributions of investigators in the eld that
could not be included in this review owing to
space limitations. We would like to express our
thanks to the peer reviewers of the article.
Funding in the authors laboratory is provided
by the Wellcome Trust, National Institute on
Alcohol Abuse and Alcoholism (NIAAA),
European Association for the Study of the Liver
(EASL), Medical Research Council (MRC) and
British Liver Trust. M.R.E. is an EASL Sheila
Sherlock Research Fellow and A.M.E. is a
Wellcome Trust Clinical Research Fellow.
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Further reading, resources and contacts
Publications
Iredale, J.P. (2007) Models of liver brosis: exploring the dynamic nature of inammation and repair in a solid
organ. J Clin Invest 117, 539-548
An interesting review looking at progress in the eld of liver brosis and specically concentrating on the
different cell and animal models used to study the disease.
Friedman, S.L. and Bansal, M.B. (2006) Reversal of hepatic brosis fact or fantasy? Hepatology 43, S82-S88
A provocative review that critically evaluates the increasingly accumulating evidence that liver brosis is a
reversible process in both animals and humans.
( continued on next page)
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Further reading, resources and contacts (continued)
Weiskirchen, R. and Gressner, A.M. (2005) Isolation and culture of hepatic stellate cells. Methods Mol Med 117,
99-113
A complete paper that explains methods for isolation of hepatic stellate cells from liver.
Mann, J. et al. (2007) Regulation of myobroblast transdifferentiation by DNA methylation and MeCP2:
implications for wound healing and brogenesis. Cell Death Differ 14, 275-285
An excellent paper that presents the rst evidence of epigenetic regulation that underlies myobroblastic
differentiation of hepatic stellate cells.
Websites
The British Liver Trust website provides various resources for both patients and health professionals and
includes links to other websites of interest:
http://www.britishlivertrust.org.uk
The American Association for the Study of Liver Disease (AASLD) website provides guidelines on the
management of various liver diseases for professionals as well as numerous patient resources and links:
https://www.aasld.org
Features associated with this article
Figures
Figure 1. The hepatic sinusoid and hepatocytes in the liver.
Figure 2. Hepatic wound healing.
Figure 3. The origins of liver myobroblasts.
Figure 4. Local neuroendocrine regulation of stellate cell transdifferentiation.
Table
Table 1. Local neuroendocrine regulation of hepatic brosis.
Citation details for this article
Mohammad R. Ebrahimkhani, Ahmed M. Elsharkawy and Derek A. Mann (2008) Wound healing and local
neuroendocrine regulation in the injured liver. Expert Rev. Mol. Med. Vol. 10, e11, April 2008,
doi:10.1017/S146239940800063X
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