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Neuroregulation of the neuroendocrine compartment of the liver.

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Neuroregulation of
the Neuroendocrine Compartment of
the Liver
Department of Pathology, University of Leuven, Leuven, Belgium
Department of Hepatology, University of Leuven, Leuven, Belgium
Liver progenitor cells as well as hepatic stellate cells have neuroendocrine
features. Progenitor cells express chromogranin-A and neural cell adhesion
molecule, parathyroid hormone-related peptide, S-100 protein, neurotrophins,
and neurotrophin receptors, while hepatic stellate cells express synaptophysin,
glial fibrillary acidic protein, neural cell adhesion molecule, nestin, neurotrophins, and their receptors. This phenotype suggests that these cell types form
a neuroendocrine compartment of the liver, which could be under the control of
the central nervous system. We recently showed that the parasympathetic
nervous system promotes progenitor cell expansion after liver injury, since
selective vagotomy reduces the number of progenitor cells after chemical injury
in the rat. Similarly, after transplantation, which surgically denervates the
liver, human livers that develop hepatitis have fewer progenitor cells than
native, fully innervated livers with similar degrees of liver injury. There is also
accumulating experimental evidence linking the autonomic system, in particular the sympathetic nervous system (SNS), with the pathogenesis of cirrhosis
and its complications. Recently, it has been shown that hepatic stellate cells
themselves respond to neurotransmitters. Moreover, inhibition of the SNS
reduced fibrosis in carbon tetrachloride-induced liver injury. In view of the
denervated state of transplanted livers, it is very important to unravel the
neural control mechanisms of regeneration and fibrogenesis. Moreover, since
there is a shortage of donor organs, a better understanding of the mechanisms
of regeneration could have therapeutic possibilities, which could even obviate
the need for orthotopic liver transplantation. © 2004 Wiley-Liss, Inc.
Key words: liver progenitor cell; hepatic stellate cell; myofibroblast; sympathetic nervous system; neuroendocrine
The liver consists of two mature epithelial cell compartments, hepatocytes and bile duct epithelial cells, and also
harbors an epithelial progenitor cell compartment
(Roskams et al., 2003a). Progenitor cells are activated
when the mature cell compartments are damaged or inhibited in their replication by, e.g., toxic agents. Progenitor cells can differentiate into hepatocytes and bile duct
epithelial cells and hence contribute to the regeneration of
these cell compartments in diseased liver (Roskams et al.,
2003a). Hepatic stellate cells (HSCs) after activation differentiate into myofibroblasts and play an important role
in repair and cicatrization (fibrogenesis, development of
cirrosis) (Reeves and Friedman, 2002; Safadi and Friedman, 2002). We have previously shown that the human
hepatic progenitor cell compartment displays neural/neu©
roendocrine features such as chromogranin-A, neural cell
adhesion molecule, parathyroid hormone-related peptide,
Louis Libbrecht is a postdoctoral researcher of the “FWOVlaanderen.”
*Correspondence to: Tania Roskams, Department of Morphology and Molecular Pathology, University of Leuven, Minderbroederstraat 12, B-3000 Leuven, Belgium. Fax: 32-16-336548. Email:
Received 28 June 2004; Accepted 28 June 2004
DOI 10.1002/ar.a.20096
Published online 24 August 2004 in Wiley InterScience
S-100 protein, and neurotrophins and neurotrophin receptors (Sciot et al., 1986; Roskams et al., 1991, 1993a, 1993b;
Cassiman et al., 2001a, 2001b; Pirenne et al., 2001). In
addition to that, we have extended the list of neural/
neuroendocrine features expressed by HSCs (Reynaert et
al., 2000). Activation and proliferation of HSCs and hepatic progenitor cells are often observed in close anatomical and temporal relationship (Dabeva and Shafritz,
1993; Miyazaki et al., 1993; Yin et al., 1999, 2002). HSCs
are proposed to interact with hepatic progenitor cells, for
instance by secreting hepatocyte growth factor (HGF),
stem cell factor (SCF), and brain-derived nerve growth
factor (BDNF), while the receptors for these growth factors are expressed on hepatic progenitor cells (Hu et al.,
1993; Fujio et al., 1994). HSCs have been shown to have
close contacts with nerve endings in normal liver (Akiyoshi, 1989; Ueno et al., 1990; Akiyoshi and Terada, 1998).
In cirrhotic liver, the innervation of the parenchyma is
decreased and the contacts between nerve endings and
activated intranodular HSCs are less numerous (Akiyoshi, 1989; Ueno et al., 1990; Akiyoshi and Terada, 1998). A
clear effect of the hepatic innervation on regeneration has
been shown previously (Ohtake et al., 1993; Kiba et al.,
1994, 1995; LeSage et al., 1999; Cassiman et al., 2002b;
Oben et al., 2003a). All these facts lead us to hypothesize
that progenitor cells and HSCs/myofibroblasts form a neural/neuroendocrine compartment in the liver. In this neural/neuroendocrine compartment, HSCs and hepatic progenitor cells might be considered the effector cells of
repair and regeneration, working under control of the
central nervous system. Conversely, HSCs and hepatic
progenitor cells may also be necessary to create the right
environment for reinnervation of diseased or renewed tissue. Understanding the control mechanisms of the central
nervous system over the neural/neuroendocrine compartment of the liver is important, since after liver transplantation, the transplanted liver is denervated (the nerves
are dissected) and as such has no connection with the
nervous system of the recipient. Since progenitor cells and
hepatic stellate cells/myofibroblasts play an important
role in regeneration and cicatrization, the transplanted
liver might have a disturbed regeneration potential compared to the nontransplanted liver.
This review describes the liver progenitor cell compartment and hepatic stellate cells/myofibroblasts and their
neuroendocrine features. In addition, the current knowledge on the neural control mechanisms of progenitor cells
and stellate cells/myofibroblasts is discussed.
Support for the liver progenitor cell hypothesis has
come mainly from cell culture data (Fausto, 1994) and
from rodent models of chemical hepatocarcinogenesis
(Opie, 1944; Farber, 1956; Grisham and Hartroft, 1961;
Fausto, 1990; Sell, 1990) and liver cell regeneration after
chemical injury (Wilson and Leduc, 1958; Lemire et al.,
1991). In these models, a periportal population of small
primitive epithelial cells proliferates in association with or
before hepatocyte multiplication. These cells were called
oval cells because of their shape (Opie, 1944; Farber,
1956), and this name is widely used for hepatic progenitor
cells in animal liver. An extensive review of the currently
available data on oval cells is beyond the scope of this
review. Oval cells are related to terminal biliary ductules
and the so-called canals of Hering, which represent the
terminal branches of the biliary tree that connect the
interhepatocytic bile canaliculi with the bile ducts in the
portal tract (Grisham, 1980; Germain et al., 1988; Lemire
et al., 1991; Lenzi et al., 1992; Paku et al., 2001). Oval cells
express phenotypic markers of both (immature) hepatocytes (such as ␣-fetoprotein) and bile duct cells (such as
bile duct-type cytokeratins) (Germain et al., 1985; Hixson
and Allison, 1985; Dunsford et al., 1989; Dabeva et al.,
1993). They constitute a heterogeneous cell population
(Germain et al., 1985; Hixson and Allison, 1985; Dunsford
and Sell, 1989; Dabeva and Shafritz, 1993; Dabeva et al.,
1993; Mandache et al., 2002), but at least a subset of oval
cells is pluripotent and has the capacity to differentiate
toward hepatocytes, bile ductular cells, and intestinal epithelium and can give rise to hepatocellular carcinoma
and cholangiocellular carcinoma (Grisham, 1980; Evarts,
1987, 1996; Tsao, 1987; Sell, 1990; Yasui, 1997; Alison,
2001). A new light was shed on the liver progenitor cell
field with studies suggesting that bone marrow-derived
stem cells can give rise to hepatocytes, oval cells, and bile
duct cells (Petersen et al., 1999; Alison et al., 2000; Theise
et al., 2000a; Korbling et al., 2002). Similar mechanisms
were described in human liver (Theise et al., 2000b). At
least in certain animal models, liver repair by bone marrow-derived cells takes place by fusion (Vassilopoulos et
al., 2003; Wang et al., 2003). This new evolution in the
field needs further study, since fusion of cells could induce
genome instability and loss of chromosomes potentiating
malignant transformation (Holden, 2003).
Although a heresy for a long time, it is now generally
accepted that in human liver also, progenitor cells (the
equivalents of rat oval cells) exist and are activated in
different liver diseases (Haque et al., 1996; Roskams et al.,
1998, 2003a; Sell, 1998; Theise et al., 1999; Libbrecht and
Roskams, 2002). Human liver progenitor cells have
mainly been studied in regeneration after severe hepatocellular necrosis (Gerber, 1983; Roskams et al., 1991,
1998; Haque et al., 1996; Lowes et al., 1999; Fujita, 2000),
but recent studies show that this cell compartment is also
activated in chronic viral hepatitis (Sakamoto et al., 1975;
Libbrecht et al., 2000; Xiao et al., 2003), alcoholic liver
disease (Ray, 1993; Roskams, 2003), and nonalcoholic
fatty liver disease (Roskams, 2003), the most frequent
chronic liver diseases in the Western world.
The only possible therapy for end-stage chronic liver
diseases is liver transplantation. The shortage of donor
organs necessitates the search for alternative treatment
strategies. Cell therapy could be a possible alternative in
the future. Because of their bipotential differentiation
abilities, progenitor cells are ideal candidates for cell therapy. Therefore, studies on their activation and differentiation mechanisms are of high priority.
Activation of hepatic progenitor cells (oval cells in rodents) is a term that is used for an increase in the number
of progenitor cells and their differentiation toward the
hepatocytic and/or biliary lineage (Dabeva et al., 1993;
Alison, 1998; Alison and Sarraf, 1998). In human liver,
differentiation toward the biliary lineage leads to formation of reactive ductules, while differentiation toward the
hepatocytic lineage occurs via intermediate hepatobiliary
cells. Reactive ductules are anastomosing strands of immature biliary cells with an oval nucleus and a small rim
of cytoplasm, located at the mesenchymal (portal/septal)-
parenchymal interface (Roskams et al., 1996a, 1998;
Roskams and Desmet, 1998; Theise et al., 1999; Libbrecht
et al., 2001). Intermediate hepatobiliary cells are polygonal cells with a size and phenotype intermediate between
progenitor cells and hepatocytes (Roskams et al., 1991,
1998; Demetris et al., 1996; Theise et al., 1999). Different
subtypes of progenitor cells have also been recognized by
electron microscopy: the most immature progenitor cell
type besides cells already featuring some biliary or hepatocytic features (De Vos and Desmet, 1992; Xiao et al.,
1999, 2003; Mandache et al., 2002). This spectrum of cells
ranging from the most immature phenotype to ductules
and intermediate hepatocytes forms a cell compartment
with a specific phenotype and is often referred to as the
progenitor cell compartment.
From animal models, we have learned that progenitor
cells (oval cells) are activated when damage or loss of
hepatocytes and/or cholangiocytes is combined with impaired regeneration of the mature cell types involved (Alison, 1998). Virtually every acute and chronic human liver
disease is associated with damage to and loss of hepatocytes and/or cholangiocytes, the two major epithelial cell
compartments of the liver. Injury can be caused by numerous triggers, including viruses, alcohol, toxic substances,
metabolic errors, and unknown factors. Hence, it is not
surprising that hepatic progenitor cell activation has been
described in a variety of human liver diseases (Libbrecht
and Roskams, 2002; Roskams et al., 2003a).
We have previously shown that the hepatic progenitor
cell compartment has neuroendocrine features such as
expression of chromogranin-A, neural cell adhesion molecule, parathyroid hormone-related peptide, and neuronspecific enolase (Fig. 1) (Fukuda et al., 1989; Roskams et
al., 1990, 1991, 1993a; Roskams and Desmet, 1997).
Chromogranin-A is a molecule present in the matrix of
neuroendocrine granules where it is thought to play a role
in the packaging and processing of hormones and neuropeptides and in stabilizing the granule content. Chromogranin-A may have extracellular roles as well, since the
intact protein or proteolytic fragments derived thereof
exert biological effects on target cells, e.g., as a prohormone of pancreastatin (Simon and Aunis, 1989). In contrast to other molecules, chromogranin-A has been found
to be a highly reliable marker for neuroendocrine cells
(Simon and Aunis, 1989; Wiedenmann and Huttner,
The neuroendocrine character of the progenitor cell
compartment was furthermore supported by their reactivity for N-CAM (Roskams et al., 1991, 1996a, 1998). Differential expression of NCAM on either a temporal or a
topographic basis is of fundamental importance to neuroontogenesis. Although the cell distribution of N-CAM is
widespread in early embryonic life, it shows a more restricted distribution in adult tissues, as it is limited
mainly to neural cells and endocrine cells (Crossin et al.,
1985; Murray et al., 1986; Langley et al., 1989). N-CAM is
a cell surface adhesion molecule that is known to play an
important regulatory role in development through cell-cell
and cell-matrix interactions. It works through homotypic
binding of two N-CAM molecules. Therefore, N-CAM could
play an important role in modulating the extent of progenitor cells/reactive ductules in the surrounding matrix.
Fig. 1. a: Chromogranin-A staining in a liver biopsy of a patient with
primary biliary cirrhosis. A large network of reactive ductules is present.
b-c: Electron micrograph showing neuroendocrine granules (arrows) in
the cytoplasm of hepatocytes (Fig. 1b) and epithelial cells with features
of both hepatocytes and progenitor cells (Fig. 1c).
This could be through cell-cell interaction, via binding
with N-CAM on hepatic stellate cells, which have been
shown to express N-CAM. On the other hand, N-CAM has
been shown to have binding sites for extracellular matrix
components such as heparan sulfate (Werz and
Schachner, 1988), which is present around the progenitor
cell compartment; we showed strong reactivity for heparan sulfate proteoglycan perlecan around activated progenitor cells and reactive ductules in different diseases
(Roskams et al., 1996b).
Neuron-specific enolase (NSE) is a glycolytic enzyme
found in neural and neuroendocrine cells. It is used as a
broad-range marker that reacts with most neuroendocrine
cells and neoplasms. Reactive bile ductules have been
shown to express NSE (Fukuda et al., 1989)
The immunohistochemical evidence for a neuroendocrine phenotype was confirmed by the electron microscopic
demonstration of dense-cored secretory granules in the
progenitor cell compartment (Fig. 1b and c). Previous electron microscopic studies focused mainly on the effect of
biliary constituents on the ultrastructural appearance of
reactive bile ductules but have failed to identify densecored granules in these cells. This may be due to the
relative scarcity of these granules. The existence of a
well-developed peribiliary capillary plexus led already to
the hypothesis that biliary epithelial cells subserve an
endocrine function and that these cells might release biologically active peptides in the surrounding liver tissue or
in the general circulation (Ohtani, 1979).
In addition to progenitor cells/reactive bile ductules,
clusters of periportal hepatocytes express diffuse cytoplasmic positivity for chromogranin-A, OV-6, and cytokeratin
7 (biliary cytokeratin, also marking progenitor cells).
These hepatocytes are smaller than normal hepatocytes
and seemed in continuity with bile ductular structures.
They lack N-CAM reactivity. These intermediate hepatobiliary cells are seen in livers with long-standing cholestasis, in regenerating livers after submassive necrosis, in
chronic hepatitis, and in alcoholic and nonalcoholic steatohepatitis (Roskams et al., 1990, 1991, 1998, 2003a, 2003b;
Crosby et al., 1998; Roskams and Desmet, 1998; Lowes et
al., 1999). Electron microscopy confirmed the presence of
neuroendocrine granules in the cytoplasm of hepatocytes
(Fig. 1b). Moreover, epithelial cells presenting features of
both hepatocytes and progenitor cells/bile ductular cells in
variable proportions were found in the parenchyma. Hepatocyte characteristics included glycogen rosettes, abundant mitochondria with short cristae, and many endoplasmatic reticulum cisternae; progenitor cell/bile duct cell
characteristics included interdigitations of their adjoining
lateral membranes, the presence of basement membranes,
and many thick bundles of tonofilaments. These cells contained one or more small dense-cored granules, localized
preferentially near the peripheral membrane (Fig. 1b and
c). The presence of cells intermediate between hepatocytes
and progenitor cells/bile duct cells supports the concept of
differentiation of progenitor cells into hepatocytes. We
found chromogranin-A-positive hepatocytes never without
the presence of immunoreactive bile ductules/progenitor
cells. These results hint at a (bile ductule-related) progenitor cell origin of the intermediate cells and small hepatocytes (Roskams et al., 1990, 1991, 1996a, 1998, 2003a).
The presence of cells with neuroendocrine features in
regenerating and cholestatic liver tissue suggested that
the substance(s) in the dense-cored granules might play a
role in the growth and/or differentiation of liver epithelial
cells through an autocrine and/or paracrine pathway. In
search of the substance(s) presumed to be present in the
granules, initially we performed immunohistochemistry
for the following hormones and growth factors: somatostatin, gastrin, vasoactive intestinal peptide, protein gene
product, glucagon, secretin, growth hormone, pancreatic
polypeptide, motilin, metenkephalin, neurotensin, and
parathyroid hormone. All these immunohistochemical
stainings were negative in the progenitor cell compartment, at least with the techniques available at that time
(the 1990s).
Parathyroid hormone-related peptide (PTHrP), a peptide that has been purified and sequenced in 1987 (Suva et
al., 1987), is the major factor responsible for humoral
hypercalcemia of malignancy (HHM). PTHrP has been
demonstrated in neoplasms associated with HHM, but
also in tumors not associated with HHM (Rabbani et al.,
1986; Ikeda et al., 1988; Deftos et al., 1989; Drucker et al.,
1989; Asa et al., 1990). The detection of PTHrP mRNA
transcripts and peptide in many tissues (Roskams and
Desmet, 1997) raised the possibility that PTHrP may play
a role in the biology of nonneoplastic cells, different from
its role in the regulation of calcium balance. Current evidence supports an important role for PTHrP in growth
and differentiation of neoplastic as well as nonneoplastic
cells (Roskams and Desmet, 1997). Moreover, the gene
encoding PTHrP shares several features with members of
the immediate early gene family. In addition, PTHrP was
detected in fetal but not in normal adult rat liver. This
prompted us to examine the expression of PTHrP in conditions associated with progenitor cell activation and to
compare its distribution with the results obtained with
neuroendocrine markers.
We showed that reactive bile ductules, but not portal
bile ducts of all sizes, stained positive for chromogranin-A
and neural cell adhesion molecule, providing further evidence for their neuroendocrine phenotype. In liver biopsies characterized by intermittent or partial obstruction,
regeneration after submassive necrosis, as well as in primary biliary cirrhosis and primary sclerosing cholangitis,
the majority of reactive bile ductular cells expressed
PTHrP. In focal nodular hyperplasia, smaller numbers of
ductular cells expressed PTHrP and unstained cells were
found among immunoreactive cells. Bile ductular cells and
hepatocytes in normal human liver tissue showed no immunoreactivity for PTHrP or for neuroendocrine markers
(Roskams et al., 1993a).
Our finding that PTHrP is present in reactive human
bile ductules/progenitor cell compartment that have a
neuroendocrine phenotype suggested that PTHrP may
play a role in the early cellular response of progenitor cells
to growth factor stimulation in an autocrine and/or paracrine manner. Since PTHrP stimulates DNA synthesis in
a variety of cells only in the presence of epidermal growth
factor (EGF) or transforming growth factor-␤ (TGF-␤),
PTHrP has been suggested to contribute to a multifactorial growth factor loop. Also in the liver, PTHrP may be
part of such a multifactorial growth factor loop. We suggested that PTHrP may act as a possible autocrine growth
factor for progenitor cells. PTHrP induces EGF-dependent
transformation of fibroblast cell lines with concomitant
significant increase in the production of fibronectin, a
property it shares with TGF-␤. Therefore, it is possible
that PTHrP plays also a role in the fibrogenesis that
accompanies ductular reaction by acting in an paracrine
way on matrix-producing cells (Roskams and Desmet,
Which factors induce the neuroendocrine phenotype of
the progenitor cell compartment? An in vitro study of de
Bruine et al. (1993) showed that the endocrine phenotype
of cells in culture (a colon tumor cell line with endocrine
differentiation) could be induced by adding basic fibroblast
growth factor (FGF) to the culture medium. A similar
effect was obtained by seeding them on plates coated with
both collagen type IV and matrix heparan sulfate proteoglycan (HSPG) perlecan, while each of these matrix components separately did not have an inducing effect. Interestingly, collagen type IV and perlecan are basement
membrane components and reactive bile ductules are always surrounded by a basement membrane (Abdel-Aziz et
al., 1990).
Therefore, we tested the hypothesis that collagen type
IV and basement membrane-type HSPG perlecan could
induce the neuroendocrine phenotype of bile ductular epithelial cells. HepG2 cells (cell line with a hepatocyte
phenotype) and A16 and H1 cells (cell lines with a bile
duct phenotype) were cultured on basement membrane
matrigel, a medium that contains these components, and
on minimal essential medium.
As expected, A16 and H1 cells were clearly immunoreactive for chromogranin-A when cultured on basement
membrane matrigel, while HepG2 cells remained negative
for this neuroendocrine marker. When cultured on minimal essential medium, all cell lines remained unreactive
for chromogranin-A (data not shown).
This in vitro study indicates that interaction of bile duct
epithelial cells with the surrounding matrix and in particular with collagen type IV and perlecan has important
implications on their phenotype. In a recent in vitro study,
Shiojiri and Mizuno (1993) used primary organ cultures of
fetal mouse liver fragments without evidence of bile ducts
to demonstrate that the addition of dexamethasone to the
medium stimulated the development of both mature hepatocytes and bile duct epithelium. When the immature liver
fragments were cultured on substrata such as collagen
gel, Millipore filter, or Spongel, the presence of dexamethasone dramatically stimulated glycogen storage, which is
a feature of hepatocytes, but did not induce bile duct
differentiation. On the other hand, when the fragments
were cultured on basement membrane matrigel, dexamethasone stimulated the expression of bile duct markers.
This study confirms our results that basement membrane
components are important for the phenotype of bile duct
epithelial cells.
Since it was unknown whether matrix HSPG perlecan is
present in human liver and whether its expression is
enhanced in progenitor cell activation, we studied the
expression of perlecan and integral membrane HSPGs in
both normal and diseased human liver. We showed expression of perlecan in normal liver around the bile ducts,
around progenitor cells, and also in the space of Disse. We
confirmed this expression by immunoelectron microscopy
(Roskams et al., 1995, 1996b). The in vivo expression fits
very nicely with our in vitro experiments.
In multicellular organisms, the maintenance of tissue
architecture, cellular polarization, and processes such as
embryogenesis, proliferation, differentiation, inflammation, and wound healing are dependent on interactions of
the cells with the components of the extracellular matrix.
Extracellular matrix molecules interact with cell surface
receptors that signal cells to change behavior. In many
instances, signaling by these highly specific protein receptors requires the assistance of a complex carbohydrate,
principally heparan sulfate (HS). This HS exists as proteoglycan (PG), as it is covalently bound to a variety of
core proteins. These HSPGs are ubiquitous and are found
inside cells, principally within storage vesicles of various
secretory cells and possibly in the nucleus, at the cell
surface as part of integral membrane PGs, and extracellularly in the pericellular matrix and basement membrane. According to the structure of the core proteins and
their location, different families of HSPGs have been recognized: HSPGs of the extracellular matrix/basement
membrane, related to the high-molecular-weight proteoglycan perlecan, and two families of integral membrane
HSPGs. The first family of membrane HSPGs include
syndecan-like proteoglycans, with core proteins that span
the membrane and that share sequence motifs in highly
conserved cytoplasmic domains. The second is made up by
glypican-related integral membrane proteoglycans that
are linked to the cell surface via glycosyl phosphatidyl
inositol (David and Bernfield, 1998).
The biological roles of HSPGs are highly diversified,
ranging from relatively simple mechanical support functions to more intricate effects on various cellular processes
such as cell adhesion, motility, proliferation, differentiation, and tissue morphogenesis. Most of these effects
mainly depend on binding of ligands to the HS chains
(David and Bernfield, 1998).
Although the ubiquitous presence of HSPGs has long
been apparent, recent interest in these molecules stems
from increasing awareness of the functional implications
of their interactions. This applies especially to their function as essential cofactors in receptor-growth factor interactions (e.g., as coreceptor for acidic and basic fibroblast
growth factor), in cell-cell recognition systems, and in
cell-matrix adhesion processes (e.g., by binding different
collagen types, fibronectin, tenascin) (David and Bernfield, 1998; De Cat and David, 2001).
HSCs are described to activate in all pathological conditions affecting the liver (hepatitis, fibrosis, cirrhosis).
HSC activation is characterized by the loss of lipid droplets and the acquisition of ␣-smooth muscle actin (␣SMA)
expression (Hautekeete and Geerts, 1997). Based on their
ultrastructural characteristics and their expression of vimentin, desmin (in rats), and ␣SMA, activated HSCs are
referred to as myofibroblast-like cells or myofibroblasts
(MFs). HSC activation is accompanied by a major switch
in expression profile, which is amply documented at the
level of intermediate filament expression, extracellular
matrix production, and degradation. Activated human
and rat HSCs express ␣SMA (Hautekeete and Geerts,
1997), activated human HSCs express GFAP (Levy et al.,
1999), activated rat HSCs express N-CAM (Fig. 2a) (Knit-
Fig. 2. a: Reactive ductules at the edge of a portal tract (PT) and
hepatic stellate cell in the parenchyma (P) of a cirrhotic liver are positive
for NCAM. b-c: Expression of synaptophysin (Fig. 2b) and neurotrophin
3 (Fig. 2c) by hepatic stellate cells in a cirrhotic nodule. d: GFAP-positive
myofibroblasts at the interface between a septum and the parenchyma
in the liver of a rat with carbon-tetrachloride-induced cirrhosis. e: Elec-
tron micrograph of normal human liver biopsy showing a stellate cell in
the Disse space, flanked by part of two hepatocytes (H). The stellate cell
contains fat droplets. Inset: Detail of a cytoplasmic processus containing
many small clear vesicles, comparable to the synaptic vesicles found in
tel et al., 1996) and intermediate filament nestin (Niki et
al., 1999).
The expression of ␣SMA in activated HSCs is held responsible for the contractile properties of these cells. This
contractility, together with the unique localization of
HSCs around the sinusoids, is currently held responsible
for the active component of portal hypertension (Rockey
and Weisiger, 1996).
HSCs are known to proliferate and migrate toward areas of necrosis and areas of regeneration in varying pathological conditions (Johnson et al., 1992; Dabeva and Shafritz, 1993). In acute hepatitis with activation of the stem
cell compartment and in bile ductular reaction, coproliferation of HSCs with hepatic progenitor cells has been
described (Dabeva et al., 1993; Miyazaki et al., 1993).
Interactions between HSC and stem cells have been suggested. For instance, HSCs produce SCFs, while oval cells
carry the c-kit SCF receptor; HSCs also produce HGFs,
while oval cells carry the c-met HGF receptor (Hu et al.,
1993; Fujio et al., 1994).
Human and Rat Hepatic Stellate Cells Express
Synaptophysin (SYN) is a major transmembrane glycoprotein of small (30 – 80 nm) electron-translucent (SET)
vesicles. This class of vesicles includes the presynaptic
vesicles in neuronal cells and the somewhat larger synaptic-like microvesicles (SLMVs) in neuroendocrine cells
(Edelmann et al., 1995). The SYN protein is implicated in
the control of exocytosis (Edelmann et al., 1995) and neurotransmitter release, e.g., in the neuromuscular synapse
(Alder et al., 1992, 1995). Immunohistochemical detection
of SYN is commonly used in conjunction with chromogranin-A, neuron-specific enolase, and Leu-7 immunohistochemistry to determine neuroendocrine origin of or differentiation in tissues and tumors throughout the body
(Schurmann et al., 1990; Poola and Graziano, 1998). To
the best of our knowledge, only neural and neuroendocrine
cell types have been demonstrated to express SYN (Bargou and Leube, 1991), with the exception of rabbit thrombocytes (Bahler et al., 1990).
Since SYN staining is a well-established diagnostic tool
in clinical practice and the presence of SYN protein is
clearly linked to neural/neuroendocrine differentiation,
we decided to check the expression profile of SYN in the
liver (Cassiman et al., 1999).
We showed that quiescent as well as activated human
and rat HSCs express synaptophysin (Fig. 2b) (Cassiman
et al., 1999). The SYN protein is known to be present in
membranes of neuronal synaptic vesicles containing neurotransmitters. Functionally, SYN is most probably involved in membrane fusion leading to exocytosis (Edelmann et al., 1995). Ultrastructurally, synaptic vesicles
were shown to be present in both quiescent and activated
stellate cells (Fig. 2e).
Based on the expression of vimentin, desmin, and ␣SMA
(Yokoi et al., 1984; Burt et al., 1986; Ballardini et al.,
1988; Takase et al., 1988; Schmitt-Gräff et al., 1991),
HSCs have been considered to be of mesenchymal origin.
From Kupffer’s first description of Sternzellen (HSC) in
liver, using gold chloride staining in search of nerve fibers
onward (Kupffer, 1876), indications of neural/neuroendo-
crine differentiation of HSCs are accumulating. HSCs
show GFAP reactivity (Neubauer et al., 1996; Niki et al.,
1996, 1999), N-CAM expression (Knittel et al., 1996), nestin reactivity (Niki et al., 1999), and SYN expression
(Cassiman et al., 1999). All these can be considered as
arguments in favor of a neural/neuroendocrine differentiation or origin. Neural tissue originates from the ectodermal layer. Neuroendocrine tissue is thought to originate
from either the neural crest, migrating out of the ectodermal layer, or from the endodermal layer. Expression of
so-called mesenchymal features (vimentin, desmin,
␣SMA) is not contradictory to the suggested neural/neuroendocrine origin, since for example in the aorticopulmonary septum, smooth muscle cells supposedly originating
from the neural crest also express ␣SMA (Beall and
Rosenquist, 1990) and desmin (Sumida et al., 1987). In
view of the evidence mentioned above, unavoidable questions about the mesenchymal origin and functional differentiation of HSCs rise. As a consequence, new studies
focusing on the expression of neural and neuroendocrine
markers in embryonic as well as adult liver become necessary.
Human and Rat Hepatic Stellate Cells Express
Neurotrophins and Their Receptors
The family of neurotrophins (NTs) consists of nerve
growth factor (NGF), BDNF, neurotrophin 3 (NT-3), and
neurotrophin 4/5 (NT-4/5). Their known receptors (NTr)
are the high-affinity tyrosine kinases Trk-A, Trk-B, and
Trk-C and the low-affinity nerve growth factor receptor
p75. The NTs are known to bind to the Trks in a specific
manner, NGF binding to the Trk-A receptor, BDNF to
Trk-B, NT-3 to Trk-C and, to a lesser degree, Trk-B, and
NT-4/5 to Trk-B. All NTs bind to p75, a member of the
tumor necrosis factor receptor family, which lacks an intracellular signal transduction domain. P75 modulates
survival and death decisions (Chao et al., 1998; CasacciaBonnefil et al., 1999).
Originally, research on NTs focused on their role in the
development and pathology of the central and peripheral
nervous system (Barde, 1989). The expression of NTs by
organs or systems other than the central or peripheral
nervous system was considered to be aimed at stimulation
of outgrowth and maintenance of the peripheral nervous
system (Shalizi et al., 2003).
On the other hand, a rapidly growing body of literature
is suggesting a more fundamental and more widespread
role for the different NTs in varying nonneural organ
systems in normal and pathological conditions. For instance, stromal smooth muscle cells in human prostate
express NGF, BDNF, and Trk-C, while the prostate epithelial tumor cell line LNCaP expresses Trk-A, Trk-B, and
Trk-C. These findings were considered indicative of a local
paracrine interaction between stromal smooth muscle
cells and tumor cells (Dalal and Djakiew, 1997). Secondly,
rat vascular smooth muscle cells express NGF, BDNF,
NT-3, and NT-4/5 as well as Trk-A, Trk-B, and Trk-C. A
dramatic increase in NGF, BDNF, Trk-B, and Trk-C expression in areas of vascular injury was noted, as well as
a migratory influence, exerted by NGF, on vascular
smooth muscle cells in vitro (Donovan et al., 1995). In
murine skin, finally, epidermal keratinocytes were shown
to express Trk-B and to proliferate in response to stimulation with BDNF, NT-3, and NT-4/5 (Botchkarev et al.,
1999). In accordance with the cited evidence, a fundamen-
tal role for NTs outside the nervous system (a role in
differentiation, tissue remodeling, proliferation, and migration) is becoming widely accepted.
We showed that human and rat HSCs express the neurotrophins NGF, BDNF, NT-3, and NT-4/5 as well as the
neurotrophin receptors p75 and Trk-C in normal and various pathological conditions (Fig. 2) (Cassiman et al.,
2001a, 2002a). In addition, HSCs express Trk-A mRNA
and Trk-B protein. The functional significance of the
present data is supported by the finding that expression of
NT and NTr in HSCs is maintained during activation,
migration, and proliferation of HSCs in various pathological and experimental conditions. For instance, the numbers of HSCs immunoreactive to NGF, NT-3, Trk-C, and
p75 following galactosamine (Gal) intoxication increased
significantly over the first 48 hr by a factor of 5, 9, 2, and
7, respectively. The proliferation of HSCs in the Gal intoxication model, measured by bromodeoxyuridine incorporation, has been described to reach a maximum around
48 hr postintoxication, the number of desmin-reactive
HSCs reaching a maximum at the same point. The number of HSCs was described to increase by a factor of 2–3
over the first 48 hr, with the increase of desmin-reactive
cells ascribed, at least in part, to proliferation. Combination of our quantitative data with these published data
strongly suggests upregulation of NT-3 and p75 expression in HSCs following Gal intoxication, since their respective increases (factors of 9 and 7) clearly exceed the
expected increase in HSC number (factors of 2 to 3).
The immunohistochemical expression of both NTs and
their receptors by HSCs suggests an autocrine and/or
paracrine growth factor action, in analogy with findings in
other organs and systems (Chalazonitis et al., 1994; Lamballe et al., 1994). RT-PCR results and results from immunohistochemistry (also at lower antibody dilutions),
however, suggest additional expression sites. Low titers of
BDNF, NT-4/5, and Trk-A mRNA transcripts and high
titers of NT-3 transcripts were found in rat hepatocytes as
well as immunoreactivity to BDNF, NT-3, and NT-4/5.
Thus, a growth factor action by NTs, emanating from
hepatocytes, could be exerted on HSCs and vice versa. We
also observed staining of bile duct epithelial cells for NT4/5 and Trk-B. Since coproliferation of reactive bile
ductules/progenitor cells with HSCs is a well-known feature in regenerating liver, after submassive necrosis and
in animal models of bile ductular proliferation (Miyazaki
et al., 1993), a paracrine interaction between HSCs and
the progenitor cell compartment is probable. Since NT-4/5
binds to Trk-B, autocrine action of NT-4/5 in progenitor
cells and bile duct epithelial cells is also a possibility.
There were significant differences in the number of
HSCs reactive for the different NT/NTr, as demonstrated
for NGF, NT-3, p75, and Trk-C. The question as to
whether every single HSC produces every single NT or
carries every single NTr cannot be answered, however.
HSCs are a heterogeneous population of cells, as was
already shown for desmin (Niki et al., 1996) and ␣SMA
(Schmitt-Gräff et al., 1991; Hautekeete and Geerts, 1997).
What can be stated on the basis of our observations, however, is that each of the NT/NTr is expressed in at least a
subset of HSCs. This is convincingly shown by colocalization of the different NT/NTr in lobular desmin or ␣SMAimmunoreactive cells, while KP-1 and neurofilament
staining showed no lobular colocalization with any of the
NT/NTr in parallel sections, excluding the expression of
NT/NTr in Kupffer cells and lobular nerve endings (Cassiman et al., 2001a).
Based on the new markers for HSCs, three different
subpopulations of HSCs/MFs were described: lobular
HSCs, interface myofibroblasts, and portal/septal myofibroblasts (Knittel et al., 1999a, 1999b; Cassiman and
Roskams, 2002; Cassiman et al., 2002a). These subpopulations can predominate in certain diseases/animal models and are possibly important for drug targeting.
␣-B-Crystallin Expression in HSCs
The list of neural/neuroendocrine features described in
HSCs is extending (Reynaert et al., 2000). Reactive gliosis,
a condition characterized by proliferation of glial cells in
response to injury to the central nervous system, shows
striking morphological similarities with the behavior of
HSCs in varying pathological conditions. Besides apparent morphological parallels, HSCs and glial cells share the
expression of nestin, neural cell adhesion molecule, and
GFAP (Niki et al., 1996, 1999). Reactive glial cells show
marked upregulation of ABCRYS in neurodegenerative
diseases such as Alzheimer’s, multiple sclerosis, and
Creutzfeld-Jacob disease (Oertel et al., 2000; van Rijk and
Bloemendal, 2000). The most abundant alpha-beta-cristallin (ABCRYS) expression in the central nervous system
is found in glial cells, but ABCRYS is also found in neurons (Oertel et al., 2000; van Rijk and Bloemendal, 2000).
In the liver, ABCRYS expression is not restricted to HSCs,
but is also found in hepatocytes, albeit at lower expression
levels (Cassiman et al., 2001b). ABCRYS expression and
induction in the activated/reactive cells constitutes yet
another parallel between HSCs and glial cells.
Although the exact function of ABCRYS protein is not
entirely clear, it is known that ABCRYS is implicated in
the cellular defense against stress, through, for instance,
protection of intermediate filaments (Van den Ijssel et al.,
1999). It is clear, however, that ABCRYS expression is not
merely a common endpoint, induced by all kinds of cellular stress. For instance, in glial cells, ABCRYS could only
be induced by tumor necrosis factor-␣, while other heatshock proteins were in turn induced by other, evenly specific cytokines (Bajramovic et al., 2000).
Hepatic branch vagotomy suppresses liver regeneration
after partial hepatectomy (Ohtake et al., 1993) and causes
an impairment of the ductular reaction after bile duct
ligation (LeSage et al., 1999). Conversely, stimulation of
the nervus vagus by ventromedial hypothalamic lesions
promotes liver regeneration after partial hepatectomy
(Kiba et al., 1994). HSCs are known to coproliferate and
interact with progenitor cells (Dabeva and Shafritz, 1993;
Hu et al., 1993; Miyazaki et al., 1993; Fujio et al., 1994;
Alison et al., 1996; Yin et al., 1999). HSCs show close
contacts with hepatic nerve endings (Ueno et al., 1997;
Akiyoshi and Terada, 1998) and express GFAP (Gard et
al., 1985; Neubauer et al., 1996; Niki et al., 1996), N-CAM
(Knittel et al., 1996; Nakatani et al., 1996), nestin (Niki et
al., 1999), SYN (Cassiman et al., 1999), and neurotrophins
and their receptors (Cassiman et al., 2001). Functional
complexes of myofibroblasts, mast cells, and cholinergic
Fig. 3. a: Double immunostaining for CK7 and M3R in hepatitis C
cirrhosis. Colocalization in HPCs and reactive ductules is shown in
yellow. An intermediate hepatocyte-like cell (arrow) is positive for CK7,
but negative for the M3 receptor. b: Immunohistochemistry for CK7 on a
liver biopsy from a kidney transplant patient with chronic hepatitis C
(innervated) and on a matched biopsy from a transplant liver with recurrent chronic hepatitis C (denervated). The activation of the progenitor cell
compartment is much more pronounced in the innervated compared to
the denervated liver.
nerve terminals have been suggested to play a role in the
development of cirrhosis (Akiyoshi and Terada, 1998). Reactive bile ductules/progenitor cells display neural/neuroendocrine features such as chromogranin-A, neural cell
adhesion molecule, parathyroid hormone-related peptide,
S-100 protein, and neurotrophins and neurotrophin receptors (Sciot et al., 1986; Roskams et al., 1990, 1991, 1993a,
1993b; Cassiman et al., 2001a, 2001b).
In view of the possible function of neural/neuroendocrine factors in the regulation of progenitor cell differentiation and proliferation and the role of the nervus vagus
in liver regeneration, we studied the expression of the five
known muscarinic acetylcholine receptors in normal and
diseased human liver.
We showed that human hepatic progenitor cells express
the M3 receptor (Fig. 3). A minority of intermediate hepatobiliary cells faintly expresses the M3 receptor. These
findings suggest that the expression of the M3 receptor
rapidly decreases upon differentiation of hepatic progenitor cells toward the hepatocytic lineage, while it is re-
tained upon differentiation toward the biliary lineage.
Immunohistochemistry could not demonstrate any other
type of muscarinic receptor (M1, M2, M4, M5) on hepatic
progenitor cells or their progeny. Low abundance of M2 or
M5 receptor expression cannot be excluded, however,
since M2 and M5 messenger RNA transcripts were shown
to be present in human liver homogenates, but protein
could not be localized by immunohistochemistry.
We observed expression of the M3 receptor in septal and
interlobular bile ducts in human liver. Alvaro et al. (1997)
recently showed that cholangiocytes of both large and
small bile ducts in rat liver express the M3 receptor.
Further experiments revealed that acetylcholine secreted
by the hepatic branch of the nervus vagus acts as a trophic
factor for proliferating cholangiocytes via binding to the
M3 receptor (Alvaro et al., 1997). Binding of acetylcholine
to the M3 receptor also stimulates the proliferation of
other epithelial and neural cells such as human colon
cancer cells (Frucht et al., 1999), rat astrocytes, human
astrocytoma cells (Guizzetti and Costa, 1996; Guizzetti et
al., 1996), and human prostate cancer cells (Rayford et al.,
At the moment, the hepatic nerves are the only known
source of acetylcholine in the liver, since only they contain
the necessary choline acetyltransferase enzyme for the
production of acetylcholine and the vesicular acetylcholine
transporter for the packaging of acetylcholine (Houwing et
al., 1996; Schafer et al., 1998; Xue et al., 2000). It is not
impossible that additional sites of acetylcholine production are present in the liver, in nonneural cells, as has
been demonstrated in other tissues (Carmeliet and Denef,
1989; Kummer and Haberberger, 1999; James and Nijkamp, 2000). Because in animal experiments, a clear
trophic influence of the hepatic vagus branch on regeneration and typical ductular reaction was shown (Ohtake et
al., 1993; Kiba et al., 1994, 1995; LeSage et al., 1999), we
can conclude that the acetylcholine secreted by the hepatic
nerves must be of importance and cannot be compensated
by any additional—at the moment hypothetical—local
source. How acetylcholine, secreted by the hepatic nerves,
reaches the M3 receptor expressed on hepatic progenitor
cells is not elucidated at the moment. Electron microscopic
studies to date have not shown any contacts between
nerve endings and hepatic progenitor cells (De Vos and
Desmet, 1992; Scoazec et al., 1993), but acetylcholine is
known to be capable of mediating its effects in a paracrine
manner (Wessler et al., 1999).
Hepatocytes produce and secrete cholinesterase (Brown
et al., 1981; Schuman et al., 1984; Berninsone et al., 1989)
and acetylcholinesterase is present at the cell surface
membrane (Perelman et al., 1989). The concentration of
serum cholinesterase accurately reflects the hepatocyte
mass (Brown et al., 1981) and can be used to assess the
prognosis of patients with liver cirrhosis (Adler et al.,
1997). The pattern of expression of the M3 receptor, in
combination with the absence of direct innervation of the
M3 expressing hepatic progenitor cells and the secretion
of cholinesterase by hepatocytes, has led us to propose the
following model (Fig. 4). In normal liver and after partial
hepatectomy, each hepatic progenitor cell is surrounded
by a normal number of hepatocytes that abrogates binding
of acetylcholine to the M3 receptor on the hepatic progenitor cell by producing and secreting cholinesterase. As a
consequence, the hepatic progenitor compartment is not
activated in these conditions. When there is loss and impaired proliferation of hepatocytes, however, as is the case
in almost all human liver diseases, the presence of cholinesterase activity will decrease proportionally to the localization and severity of hepatocyte loss. This would allow acetylcholine to exert its trophic effects on the hepatic
progenitor cells, until hepatocyte mass is restored again. A
similar mechanism seems to be present in the hematopoietic system. Acetylcholinesterase, which is synthesized by
mature red blood cells, enhances apoptosis and reduces
proliferation of mouse hematopoietic progenitor cells committed to erythroid and other lineages in vitro (Soreq et
al., 1994; Deutsch et al., 2002). Hepatic progenitor cells
have a phenotypic overlap with hematopoietic progenitor
cells (Fujio et al., 1994).
During liver transplantation, the hepatic branch of the
nervus vagus is sectioned and reinnervation of the transplant liver occurs only at the hilum (Boon et al., 1992;
Dhillon et al., 1992). Thus, acetylcholine can no longer
exert its beneficiary effects on hepatic progenitor cells,
neither in normal nor in diseased transplant liver. Since,
Fig. 4. Hypothetical model in which the interplay between acetylcholine secreted by the nervus vagus, the M3 receptor expressed by hepatic
progenitor cells (HPCs), and acetylcholinesterase secreted by hepatocytes explains HPC behavior in different conditions. a: Normal liver or
PHx. b: Liver diseases. c: Liver diseases in transplant liver.
on the one hand, animal experiments strongly indicate a
role for the hepatic vagus branch and cholinomimetic substances in liver regeneration, and, on the other hand, our
present results raise the possibility of a comparable
interaction in human liver (Fig. 3b), further research to
elucidate the function of the M3 receptor expressed by
human hepatic progenitor cells and a possible role for
cholinomimetic substances in patients with denervated
livers is indicated.
Recent reports show that inhibition of the sympathetic
nervous system, either via ␣1-adrenergic antagonism with
prasozin or via chemical sympathectomy with 6-hydroxydopamin, promotes progenitor cell activation and reduces
liver injury in a mouse model of progenitor cell activation
(methionine/choline-deficient diet) (Oben et al., 2003a).
Similar experiments on CCL4-intoxicated rats showed inhibition of fibrosis in chronic CCL4-intoxicated rats by the
sympathetic inhibitors prasozin and 6-hydroxydopamine
(Dubuisson et al., 2002). Oben et al. (2003b) have also
shown that stellate cells themselves produce norepinephrin and react to norepinephrine.
We recently studied both acute and chronic CCL4 intoxication and acute galactosamine intoxication (data not
shown) and showed that in these acute and chronic rat
models, prasozin induced significantly higher numbers of
progenitor cells (marked by OV-6) and significantly lower
number of hepatic stellate cells (marked by GFAP,
desmin, and ␣SMA). The degree of fibrosis was lower in
the prasozin-treated animals than in the control animals,
confirming the results of Dubuisson et al. (2002). Both
progenitor cells and isolated stellate cells express ␣-adrenergic receptors. Since prasozin is a well-tolerated drug,
this opens interesting perspectives for future treatment
In conclusion, progenitor cells and hepatic stellate cells
form a cell compartment with neuroendocrine features in
the liver. There is increasing evidence that both the sympathetic and parasympathetic nervous system influence
this cell compartment. This is important in the development of future therapeutic strategies to stimulate progenitor cell activation and to inhibit stellate cell activation.
Such sustaining therapies could eventually delay or even
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