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Pathogenesis and reversal of liver fibrosis: Effects of genes and environment
Sokolović, A.
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Citation for published version (APA):Sokolovi, A. (2013). Pathogenesis and reversal of liver fibrosis: Effects of genes and environment.
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Download date: 05 Sep 2020
Pathogenesis and reversal of liver fibrosis
– effects of genes and environment
PATHOGENESIS AND REVERSAL OF LIVER FIBROSIS
– EFFECTS OF GENES AND ENVIRONMENT
Author:
Aleksandar Sokolović/PhD Thesis/Faculty of Medicine, University of Amsterdam, 2013
Cover and illustrations:
Tomislav Sokolović
Printed by:
Ipskamp Drukkers BV
ISBN: 978-94-6191-623-5
Copyright © 2013 Aleksandar Sokolović
The research described in this thesis was performed at Tytgat Institute for Liver and Intestinal
Research Academic Medical Center, University of Amsterdam, The Netherlands.
The work was supported by Dutch MLDS grant CG 102001.
The study presented in Chapter 4 of the thesis was partly performed at Division of Hepatology and
Gene Therapy, CIMA University of Navarra, and CIBERehd, Pamplona, Spain. The research
described in Chapter 5 was done in collaboration with Department of Medical Biochemistry,
Academic Medical Center, University of Amsterdam, The Netherlands.
Printing of this thesis was financially supported by The Netherlands Federation for Hepatology, and
Tytgat Institute for Liver and Intestinal Research Academic Medical Center, University of
Amsterdam, The Netherlands.
PATHOGENESIS AND REVERSAL OF LIVER FIBROSIS
– EFFECTS OF GENES AND ENVIRONMENT
ACADEMISCH PROEFSCHRIFT
ter verkrijging van de graad van doctor aan de Universiteit van Amsterdam
op gezag van de Rector Magnificus prof.dr. D.C. van den Boom
ten overstaan van een door het college voor promoties ingestelde commissie,
in het openbaar te verdedigen in de Agnietenkapel
op donderdag 28 februari 2013, te 10:00 uur
door
Aleksandar Sokolović
geboren te Leskovac, Serviё
Promotiecommissie:
Promotor Prof.dr. R.P.J. Oude Elferink
Co-promotor Dr. P.J. Bosma
Overige leden: Prof.dr.em. W.H. Lamers
Prof.dr. U.H.W. Beuers
Prof.dr. J. Prieto
Prof.dr. A.J. Moshage
Prof.dr. K. Poelstra
Prof.dr. J. Rothuizen
Faculteit der Geneeskunde
za mamu, tatu, Ducu, Milku i Miu
TABLE OF CONTENTS
Chapter 1 Intro: Scope of the thesis 8
Introduction
Outline of the thesis
Chapter 2 IGFBP5 enhances survival of LX2 human hepatic stellate cells 30
Fibrogenesis & Tissue Repair 2010, 3:3
Chapter 3 Unexpected reduction of murine liver fibrosis by IGFBP5 46
Biochimica et Biophysica Acta (BBA) - Molecular Basis of Disease;
2012 Jun;1822(6):996-1003.
Chapter 4 IGF1 enhances bile-duct proliferation and fibrosis in Abcb4-/- mice 64
accepted for publication in:
Biochimica et Biophysica Acta (BBA) - Molecular Basis of Disease, 2013
Chapter 5 Fasting reduces liver fibrosis in mouse model 80
for chronic cholangiopathies
under revision
Chapter 6 Closure: Summarizing discussion and conclusions 96
Summaries
Abbreviations
Personalia Acknowledgements 116
Curriculum vitae
List of publications
c h a p t e r 1
intro:
scope of the thesis
introduction
outline of the thesis
chapter 1
10
SCOPE OF THE THESIS
Hepatic fibrosis results from repeated liver tissue damage, caused by chronic viral hepatitis, parasitic
diseases, inborn errors of metabolism, toxic damage and non-alcoholic fatty liver disease (NAFLD).
Fibrosis is a generic wound healing response to chronic insults, regardless of their mechanism. In the
liver, resulting progressive accumulation of fibrillar extracellular matrix (ECM) is associated with
ECM degradation and remodeling that can result either in restoration of normal hepatic structure
and function, or in cirrhosis, conversion of normal liver architecture into structurally abnormal
nodules.
Efforts to elucidate the cellular and molecular basis of fibrogenesis have generated a
comprehensive picture of progression and regression of liver fibrosis, essential for developing
antifibrotic therapies. Due to the dynamic evolving nature of the disease, it seems that interventions
even as late as in cirrhosis could improve clinical outcomes. Though translation of these findings into
diagnostic tools and treatments does not seem too far away, there are still no effective antifibrotics.
There is also a requirement for better markers of the stage and activity of fibrosis, predicting if
patients will progress to cirrhosis or reverse.
The objective of this thesis was therefore to make use of multiple complementary
experimental model systems, molecular techniques and metabolic challenges, to study liver fibrosis,
with the final aim to create new opportunities for antifibrotic therapies.
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INTRODUCTION
Fibrotic diseases account for up to 45% of deaths in the developed world (1). They are the most
common non-neoplastic cause of death among hepatobiliary and digestive diseases in the USA and
Europe, even though only 25-30% of patients affected by chronic liver diseases are likely to develop
significant fibrosis and cirrhosis, with an inevitably poor prognosis, where 1-year mortality ranges
from 1% in early cirrhosis to 57% in decompensated disease (2). The main causes of liver fibrosis in
developed countries are chronic viral hepatitis, alcohol and drug abuse. The association of
fibrosis/cirrhosis with primary liver cancer further increases the relative mortality rate (3, 4). The
worldwide burden is equally compelling, with chronic liver diseases affecting hundreds of millions of
individuals (5). In addition, the explosive growth in obesity worldwide has led to a dramatic increase
in the prevalence of non-alcoholic steatohepatitis (NASH) (6), which also confers a risk of
hepatocellular carcinoma (HCC), (fastest rising cancer incidence of any neoplasm in the USA and
Western Europe) once cirrhosis develops (7, 8). The large number of people affected worldwide and
the lack of effective anti-fibrotic treatment necessitates further studies of the pathophysiology in
order to deliver novel therapies.
HEPATIC FIBROGENESIS
Liver fibrosis results from perpetuation of the normal wound healing response. Repeated cycles of
hepatocyte injury and repair, accompanied with inflammation and excessive deposition of ECM
proteins, result in an abnormal continuation of fibrogenesis. Its progression depends on the etiology
of liver disease and is influenced by environmental and genetic factors (9-11).
During hepatic fibrogenesis, significant changes in quality, quantity and distribution of ECM
components occur in the periportal and perisinusoidal space, increasing the content of collagens and
non-collagenous components (12).
In normal liver, the subendothelial space of Disse that separates the epithelium (hepatocytes)
from the sinusoidal endothelium, contains both an interstitial (high-density) and a basement
membrane–like, perisinusoidal (low density) ECM (Figure 1a). During fibrosis, perisinusoidal ECM is
replaced by an interstitial (scar-type) matrix with bundles of collagen fibrils and an electron-dense
basement membrane (13). This is tied in with a loss of hepatocyte microvilli and a disappearance of
endothelial fenestrations (Figure 1b). The dominant event in fibrogenic cascade is activation of
hepatic stellate cells (HSCs) (14), remarkably adaptable mesenchymal cells that are vital to
hepatocellular function and the liver’s response to injury. These cells resident in the space of Disse
are the major storage of vitamin A in the body. They represent one-third of the nonparenchymal
cells, and ∼15% of the total number of resident cells in normal liver (Figure 1a). HSCs are primary
effector cells that play a pivotal role in activating the immune response through secretion of
cytokines and chemokines and interacting with immune cells, contributing thereby to angiogenesis
and the regulation of oxidant stress, and orchestrating the deposition of ECM in normal and fibrotic
liver (15). In the injured liver, numbers of activated hepatic stellate cells/myofibroblasts
(HSCs/MFs) dramatically increase at sites of infection, inflammation and injury, promoting the
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deposition of scar-tissue (Figure 1b). These cells are generated locally, by transdifferentiation of
resident perisinusoidal HSCs and periportal fibroblasts (16, 17), or via the recruitment and
differentiation of bone-marrow-derived mesenchymal stem cells (18, 19). Activated HSCs secrete
large amount of interstitial collagen, accompanied by matrix metalloproteinases (MMPs), which
induce three-dimensional changes in the ECM. They degrade the normal basement membrane
matrix, whilst having a mild effect on the neo-matrix rich in fibrillar collagen. On the other hand, the
production of tissue inhibitor of metalloproteinase (TIMPs) by HSCs also increases, promoting
ECM accumulation by inhibiting matrix degradation, improving survival of activated HSCs and
preventing their clearance, all delaying the regression of liver fibrosis (20-22).
The changes in ECM impair the flow of metabolites between the hepatocytes and the
perfusing plasma, exposing hepatocytes to hypoxia (particularly in pericentral zones). In unopposed
fibrosis this causes distortion of hepatic architecture, formation of septae (broad bands that divide
the parenchyma into regenerative nodules), increase in intrahepatic vascular resistance, angiogenesis
and sinusoidal remodeling, inducing portal hypertension (23). This ultimately leads to
pathophysiological damage and cirrhosis as the final stage (24) . Advanced fibrotic changes are
strongly associated with hepatocellular carcinoma, which further reduces chances of survival (25).
Potential reversibility of hepatic fibrosis varies depending on the duration of injury, the
degree of angiogenesis, and the composition, spatial distribution, and cellularity of scar. Cirrhotic
areas resistant to degradation are fairly acellular and rich in elastin and highly cross-linked collagen,
suggesting that ECM cross-linking might represent a “point of no return” in fibrosis (22). Fibrotic
patients have a good short-term prognosis, as it usually requires several months to years of ongoing
insult to reach chirrosis (26). Cirrhosis, however, as present evidence indicates, is not completely
reversible.
SIGNALS FOR PATHOGENESIS IN LIVER FIBROSIS
Similar to fibrotic disorders in other organs, chronic activation of the wound-healing process is the
mechanism behind hepatic fibrogenesis. Upon hepatic injury, a fibrogenic response is provoked by
activation of key signals in resident and infiltrating liver cells – reactive oxygen species (ROS),
hypoxia, inflammation and apoptosis. As a consequence, ECM proteins accumulate in the liver due
to imbalance between the fibrogenesis and fibrinolysis.
Oxidative stress in chronic liver diseases (CLD) results from an increased generation of
ROS (and other reactive intermediates) and from a decreased efficiency of antioxidant defenses. This
stress contributes to excessive tissue remodeling and fibrogenesis by stimulating the production of
profibrogenic mediators by Kupffer cells and other resident and circulating inflammatory cells. It
directly affects the behavior of HSCs/MFs by induction of critical profibrogenic genes, like
procollagen type 1, monocyte chemoattractant protein 1, and tissue inhibitor of metalloproteinase 1
(TIMP1). This is likely enabled by the activation of transcription factors, such as c-jun N-terminal
kinases (JNKs), transcription factor AP1 and nuclear factor κB (NFκB) (27).
Hypoxia is a critical, early fibrogenic stimulus, caused by capillarisation of sinusoids,
intrahepatic shunting, vasoconstriction, compression and thrombosis (28). It impairs mitochondrial
function, produces oxidative stress, induces vascular endothelial cell growth factor (VEGF) and its
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receptors, and stimulates synthesis of collagen 1 in HSCs (29). Hypoxia also potentiates expression
of transforming growth factor, beta 1 (TGFβ1) (30), contributing to both autocrine and paracrine
loops that drive angiogenesis, chemotaxis and fibrogenesis.
Figure 1: Hepatic liver cells and the hepatic sinusoid in normal and injured liver
Inflammation initiates and regulates fibrosis by the release of soluble mediators and the
secretion of products that remodel/degrade the normal basement membrane matrix within the liver
(31). Inflammatory cells, resident (e.g. NK cells and macrophages) or recruited (e.g. T and B cells),
are involved in pathogen elimination, cell removal (e.g. hepatocytes damage during anti-viral immune
reaction), recruitment and activation of MFs, and spontaneous recovery of fibrosis (32, 33).
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Apoptosis, a common feature of chronic liver disease, results in the generation of apoptotic
bodies, which phagocytotic clearance creates pro-inflammatory and fibrogenic stimuli. After
engulfing the apoptotic bodies, Kupffer cells secrete death ligands and tumor necrosis factor alpha
(TNFα). Similarly, the engulfment of apoptotic bodies by HSCs triggers a profibrogenic response
with production of oxidative radicals, and upregulation of expression of TGFβ1 and collagen 1 (34-
37).
HEPATIC STELLATE CELLS AND FIBROGENESIS
In response to liver injury, HSC undergo rapid activation, functional and morphological changes.
Activation, which occurs in two phases, initiation and perpetuation, is, if liver injury has subsided,
followed by resolution (Figure 2) (38). Initiation of HSCs activation comprises early changes in
gene expression and phenotype, which make them responsive to cytokines and other stimuli released
from affected cells, like damaged hepatocytes, bile duct cells, Kupffer cells and inflammatory cells
(39). Perpetuation encompasses cellular series of events that amplify the activated phenotype
through enhanced growth factor expression and responsiveness. It results from autocrine and
paracrine stimulation, and accelerated ECM remodeling.
In response to acute and chronic liver injury, HSCs proliferation is promoted by strongly
increased mitogens. They include platelet-derived growth factor (PDGF), VEGFA, thrombin and its
receptor, epidermal growth factor (EGF), transforming growth factor α (TGFα), keratinocyte growth
factor and fibroblast growth factors 2 (FGF2) and its receptors (FGFR1) (11). The most potent
fibrogenic factor for HSCs is TGFβ1 (40, 41). Activated TGFβ1 released from the local ECM binds
to an increased number of TGFβ receptors on HSCs. In response, signaling via the SMAD pathway
enhances proliferation and survival of activated HSCs, increases collagen synthesis, upregulates
TIMPs, and decreases MMPs expression (42). HSCs migrate and accumulate in zones of
injury/repair (43) secreting large amounts of cytokines (amplifying the inflammatory and fibrogenic
tissue responses), and MMPs (accelerating the replacement of normal with "scar" matrix). Activated
HSCs accumulated in the collagenous bands impede portal blood flow by constricting individual
sinusoids and entire organ (44, 45).
Resolution of fibrosis refers to the fate of activated HSCs when the primary insult is
withdrawn or attenuated, and refers to pathways that cause apoptosis, senescence, or quiescence of
HSCs (Figure 2) (46-48).
ANTIFIBROTIC THERAPIES
Current treatments of cirrhosis include withdrawal of the causative agent and treatment of
complications, but the only curative treatment for advanced cirrhosis is liver transplantation. Its
clinical applicability is, however, limited by shortage of donors, by the commitment of recipients to
life-long toxic immunosuppression, and by poor condition of the potential recipient.
intro
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Figure 2: The role of hepatic stellate cells in recurrent liver injury
Since the progress of fibrosis is basically similar in all liver diseases regardless of the etiology,
the development of antifibrotic therapies should benefit all patients with fibrosing liver injury. The
liver's ability to regenerate and its first-pass metabolism allow lower doses of orally administered
compounds to achieve a therapeutic response, minimizing systemic distribution and non-hepatic side
effects, which makes liver fibrosis an attractive target for therapy. The progress in clinical research
and basic science and improved diagnostic tools promise to halt the progression of liver fibrosis
and/or promote its reversion. Potential therapeutic approaches for liver fibrosis include: treatment
of the primary disease, suppression of hepatic inflammation, inhibition of HSCs activation,
promotion of matrix degradation, stimulation of HSCs apoptosis, and targeted antifibrotic therapies
(Figure 3). These approaches will be addressed individually.
Treatment of the primary disease. Clearing the primary cause of liver disease is still the
most effective intervention in the treatment of liver fibrosis. Removal of excess iron or copper in
haemochromatosis or Wilson’s disease, respectively, abstinence in alcoholic liver disease, anti-
helminthic therapy in parasitic infections, clearance of HBV or HCV in chronic viral hepatitis, and
biliary decompression in bile duct obstruction, or, most recently, weight loss in patients with NASH,
lead to reduced fibrogenesis (49-53) This approach can be highly effective when applied early,
resulting in a near-normal restitution of hepatic histology.
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Figure 3: Targets for antifibrotic therapies.
Suppression of hepatic inflammation. In sustained liver injury, persistent inflammation
perpetuates the fibrogenic HSCs phenotype and leads to fibrosis. Therefore, a number of anti-
inflammatory agents have been evaluated in vitro and in vivo. Treatment with corticosteroids in
patients with severe autoimmune hepatitis leads to hepatic fibrosis only in a minority of patients (54).
Blocking interleukin 1 by expressing the interleukin-1 receptor antagonist, also reduced liver damage
and pro-inflammatory cytokine levels in an in vivo model of ischaemia-reperfusion injury (55).
Recombinant interleukin 10 reduces inflammation and fibrosis in patients with chronic hepatitis C,
but results in an increased viral load (56). Local expression of interferon alpha (IFNα) improved liver
fibrosis in a dimethylnitrosamine-induced model of cirrhosis (57). Finally, the beneficial effects of
ursodeoxycholic acid in the treatment of primary biliary cirrhosis (PBC) are thought to be in part via
anti-inflammatory mechanisms (58).
Inhibition of HSCs activation. HSCs activation, proliferation and fibrogenesis are pivotal
steps in the development of liver fibrosis, making the mechanisms that mediate them attractive
targets for therapeutic intervention. Oxidative stress, which stimulates HSCs activation, is one such
target. Several therapeutic agents have been tested in animal models to counteract the activation of
HSCs by oxidative stress, like vitamin E and C (59), and S-adenosyl- L- methionine (60) but the
effects were minor. A number of other agents have been shown to inhibit the HSC activation. They
include interferon gamma (IFNγ) (61) and ligands for peroxisomal proliferator activated receptor
(PPAR) gamma.(62). TGFβ antagonists are extensively tested, since neutralizing of this potent
cytokine would have the dual effect: inhibition of matrix production and acceleration of its
degradation. Adenoviral expression of Tgfβ1 antisense mRNA, and gene transfer of Smad7, which
blocks TGFβ intracellular signaling, also had positive effect. Gene transfer of soluble TGFβR2 that
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inhibits liver fibrogenesis in rats is shown to attenuate apoptosis, injury, and fibrosis in bleomycin-
induced lung fibrosis in mice. Blocking TGFβ activity by synthetic peptide reduces fibrosis in mice in
carbon tetrachloride (CCL4)-induced liver fibrosis, and bleomycin-induced skin and lung fibrosis (41,
63). Although inhibitors of TGFβ1 are effective in short-term animal models, they are not suitable
for long term therapy because of the significant role of TGFβ1 in homeostasis and repair (47).
Promotion of matrix degradation. Remodeling of ECM occurs through the action of
MMPs that efficiently cleave collagens and other matrix components, and the TIMPs, critical
determinants of fibrosis reversal, which inhibit their activity. During the regression, decreased TIMP-
1 levels are associated with clearance of activated HSCs through apoptosis (64). Fibrinolysis and
matrix rearrangement can be induced by a number of agents, as shown by studies in animal models
of liver fibrosis. For example, direct adenoviral administration of Mmp1 mRNA attenuated
established liver fibrosis in thioacetamide treated rats, while administration of an anti-TIMP1
antibody decreased HSCs activation and attenuated fibrosis in a CCl4 rat model (65, 66).
Downregulation of TIMPs by the reproductive hormone relaxin also inhibits liver fibrosis in vitro
(67). Furthermore, adenoviral delivery of urokinase-type plasminogen activator (uPA), which initiates
the matrix proteolysis and upregulates hepatocyte growth factor (HGF) (68), results in enhanced
collagenase activity, fibrosis regression and hepatocyte regeneration (69). MMPs gene delivery in
advanced liver fibrosis shows that even transient shifts in the imbalance between MMPs and TIMPs
are sufficient for the resolution - if the underlying causative stimuli have been successfully removed
(70). Dietary supplementation with zinc, an essential co-factor for MMP activity, has also been
shown to promote collagen degradation in CCl4-injured rats (71). Collagen synthesis is also sensitive
to changes in food intake, and malnutrition may have profound effects on its production (72).
Stimulation of HSCs apoptosis. Apoptosis is a key event in the spontaneous recovery
from liver fibrosis (73) and can be induced by two general mechanisms (34). The first, ”extrinsic”
pathway, works via stimulation of specific cell surface death receptors, which trigger an intracellular
cascade of caspases, ensuing cellular apoptosis. Second, ”intrinsic” pathway, operates at the level of
the mitochondria. Stimuli like toxins, UV radiation, and reactive oxygen species stimulate the
proapoptotic factors in the mitochondrial membrane, resulting in leakage of cytochrome C into the
cytosol, caspase activation, and apoptosis. In addition, upon activation of HSCs, the expression of a
cell surface death receptors CD95 (APO-1/Fas) and CD95L (APO-1-/Fas-ligand) increases, which
may explain the increased apoptosis seen upon their activation in vitro and in vivo (74). Also, activation
of TNFα receptor, and low-affinity nerve growth factor (NGF) receptor (p75) increases HSCs
apoptosis in vitro (75). Transformed HSCs also express peripheral benzodiazepine receptors, which
render the cells sensitive to peripheral benzodiazepine receptor-ligand-induced apoptosis (76).
NFκB, on the other hand, protects HSCs from apoptosis, and its inhibition with gliotoxin accelerates
recovery from drug-induced liver fibrosis in rats (77), proving that HSCs pro-apoptotic agents could
abrogate liver fibrosis. The approaches mentioned in this section may sound attractive for in vitro
work, but the selectivity may be too low to use this approach in vivo.
Targeted antifibrotic therapy. Wound healing is an important universal process, hence
liver-specific targeting of antifibrotic therapies minimizes the potential harmful effects on other
tissues. Furthermore the extensive first pass metabolism makes liver an attractive target for directed
therapy with orally administered drugs. Still, hepatic delivery needs to be optimized via cell-specific
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targeting (of hepatocytes, Kupffer cells, or HSCs). It has been shown, for instance, that
administration of recombinant insulin-like growth factor 1 (rIGF1) or HSCs-targeted induction of its
expression using a viral vector in CCl4-treated mice and rats, significantly reduced liver fibrosis (78-
80). Treatment of cirrhotic rats with rIGF1 promotes weight gain, nitrogen retention, and intestinal
absorption of nutrients and was able to exert hepatoprotective effect (78, 81). Concerns regarding
the safety of gene therapy with regard to genotoxicity and effects on the immune system remain.
However, with improved delivery techniques and the conditional expression systems, the anti-
fibrotic trials in humans are no longer a matter of (distant) future.
IGF AXIS IN LIVER FIBROSIS
Insulin-like growth factor 1 (IGF1) is a potent cytoprotective and anabolic hormone, synthesized
mainly in the liver. It circulates bound to a set of binding proteins (IGFBPs) that prolong its half-life
and, by modulating its interaction with the IGF1 receptor (IGF1R), control its biological activity
(82). Detachment from IGFBPs releases free IGF1, which can bind to and activate the IGF1R.
Interaction with IGF1R leads to activation of mitogen activated protein (MAP) kinase and
phosphatidylinositol 3-kinases (PI3) cascades that regulate genes involved in cell survival, growth,
and differentiation (83). In advanced liver fibrosis, the IGF1 axis is severely impaired mostly due to a
reduced number of healthy, IGF1 producing hepatocytes. The decrease in IGF1 signalling can
disturb systemic metabolism (e.g. in liver cirrhosis), and provide a pro-fibrotic environment, since
the progression of liver fibrosis could be delayed by IGF1 administration (79, 84).
As mentioned, the action of IGF1 depends on six IGFBPs of multifunctional nature, which
can act both via IGF-dependent and independent mechanisms. Among them, insulin-like growth
factor binding protein 5 (IGFBP5) that binds to IGFs with high affinity (85) is strongly induced in
the fibrotic livers of Abcb4-/- mice (86), suggesting a potential role in the pathogenesis of chronic
cholangiopathy. Its expression is also high in skin and lung fibrosis (SSc and IPF; (87, 88)). IGFBP5
induces collagen and fibronectin production in fibroblasts, and their transdifferentiation to
myofibroblast in vitro and in vivo (89, 90). In IGFBP5-induced dermal fibrosis, an increase in the
number of fibroblasts expressing proliferating cell nuclear antigen PCNA is accompanied by
increased vimentin and α-SMA expression (suggesting an IGFBP5-induced myofibroblast
differentiation). This leads to an excessive production of ECM and the development of fibrosis.
IGFBP5 seems to play an important role in other aspects of fibrosis, such as epithelial injury.
IGFBP5 expression is dramatically increased in the involuting mammary gland and prostate during
apoptosis, and in the brain after ischaemia (91, 92). IGFBP5 induces epithelial–mesenchymal
transition in pulmonary epithelial cells in vitro (90). Senescence, a process in which cells lose the
ability to proliferate, is also associated with increased IGFBP5 expression in human dermal
fibroblasts and endothelial cells (93, 94). A knockdown of IGFBP5 partially reverses, whereas its
induction causes a premature senescence in human umbilical-vein endothelial cells (95).
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LIVER FIBROSIS AND DIET
As the prevalence of obesity is rapidly increasing, the prevalence of NASH is also on the rise .
NASH has been recognized as a major cause of liver fibrosis (96), ranging from steatosis to cirrhosis,
eventually leading to HCC. NASH is a component of metabolic syndrome and type 2 diabetes,
characterized by obesity, dyslipidemia and insulin resistance (97). Insulin resistance is a prominent
mechanism that links steatosis and fibrogenesis (98), hence adipokines that play a role in insulin
resistance but also steatosis and inflammation are considered to be involved in liver fibrosis.
Fibrogenic action of insulin itself works via the release of TGFβ1, which stimulates type 1 collagen
production in HSC (99). Leptin promotes stellate cell mitogenesis and cell survival, two seminal
events that promote liver fibrosis (100). In contrast, administration of recombinant adiponectin and
its agonists reduced steatosis, attenuated inflammation and the elevated levels of serum alanine
aminotransferase (ALT) in (non)alcoholic fatty liver diseases in mice, indicating potential clinical
application (101, 102).
Treatment options in obesity-linked liver fibrosis are limited so lifestyle changes like
introducing exercise and healthy diet are advised. Weight loss in morbidly obese patients is associated
with a reduction in the prevalence of hepatic fibrosis (103). In a mouse model of metabolic
syndrome, stimulation of weight loss by blocking the endocannabinoid receptor CB1 prevents
progression of fibrosis (104). Still, beneficial effects of insulin sensitizers on progression of liver
fibrosis need to be documented in therapeutic trials.
MODELS OF LIVER FIBROSIS
Although fibrosis occurs as a part of normal wound healing, excessive (dysregulated) deposition of
ECM leads to severe organ dysfunction and is a feature of a variety of diseases. Due to its insidious
onset, fibrosis tends to go undetected in its early stages. This is, in part, why these diseases remain so
poorly understood. To improve our understanding of development and reversal of liver fibrosis, a
number of experimental models have been established.
The first are in vitro models - purified primary cells from normal or experimentally injured
livers, or stable cell lines representative of specific hepatic cell types. These models enhance the
understanding of the molecular pathogenesis of liver fibrosis (15), and permit high-throughput
testing and improvement of potential antifibrotic agents, thanks to low cost and high reproducibility.
However, they cannot recapitulate the in vivo situation that involves the complex interplay of resident
and incoming cells in a dynamic microenvironment.
The alternative are the ex vivo models - human hepatic tissue obtained from biopsies,
resections or explants. This type of models is vital for validating observations made in animal and
cell culture models (105). However, ethical reasons prevent multiple liver biopsies in humans,
resulting in limited amount of data, usually at the advanced end of the disease.
In vivo experimental animal models of fibrosis are used to address the limitations of the
other two types, and to facilitate the study of a highly dynamic process (106, 107). In spite their
questionable relevance to human disease, they allow consecutive sampling of tissue in the volumes
required for detailed studies of cellular and molecular pathogenesis. These models provide a chance
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to examine the early stages of fibrosis and changes in signaling pathways, chemokines, and cytokines.
The two most frequently used models of experimental fibrosis are repetitive toxic damage by carbon
tetrachloride (CCl4) and bile duct ligation (BDL) (107). CCl4 induces zone III necrosis, hepatocyte
apoptosis and, with iterative dosing, can be used to generate bridging hepatic fibrosis, early cirrhosis
and advanced micronodular cirrhosis (4, 8 and 12 weeks of twice weekly dosing, respectively) (73,
108). BDL stimulates the proliferation of biliary epithelial cells and oval cells (hepatocyte
progenitors), resulting in proliferating bile ductules and an accompanying portal inflammation and
fibrosis. Fibrosis can also be established using CCl4 or BDL in specific transgenic mice (over-
expressing pro- or anti-fibrotic factors) and knock-out mice (in which the expression of a pro or anti
fibrotic factor is abolished) (79, 109). This allows addressing the molecular mechanisms and/or
importance of these factors in hepatic functions and diseases.
In vitro and in vivo models used in this thesis will be described in more detail in the coming
paragraphs. The LX2 cell and MFs were used to study the mechanism of action of IGFBP5 in
(activated) HSCs. LX2 human HSCs lines are generated by spontaneous immortalisation in low
serum culture conditions. They retain key features of HSCs, like expression of α-SMA, vimentin, and
glial fibrillary acid protein. Similarly to primary HSCs, LX2 cells express key receptors regulating liver
fibrosis, like platelet derived growth factor receptor β (PDGFβ-R), as well as proteins involved in
matrix remodelling; MMP2, TIMP1 and 2 (110).
Abcb4-/- mice used in this thesis are a well characterized non-surgical mouse model for
chronic cholangiopathies. These mice deficient in the canalicular phospholipid flippase are created
on FVB background (FVB.129P2-Abcb4tm1Bor; (111)). They are characterized by a lack of Mdr2 P-
glycoprotein (multidrug resistance transporter 2, Abcb4), a phosphatidylcholine transporter. This
results in absent phospholipid secretion into bile and spontaneous bile duct injury, with macroscopic
and microscopic features closely resembling human sclerosing cholangitis (PSC) (112). These animals
are an animal model for human MDR3 deficiency (progressive familial intrahepatic cholestasis type
3) which progresses to liver cirrhosis (113). The hepatic pathogenesis in Abcb4-/- mice include
disruption of tight junctions and basement membranes of bile ducts, and bile leakage to the portal
tract. This triggers a multistep process that upregulates fibrogenesis and downregulates fibrinolysis,
leading to the formation of periportal biliary fibrosis (114), which makes these mice an attractive
model for liver fibrosis. As a consequence of chronic inflammation and progressing fibrosis, Abcb4-/-
mice develop HCC within 12 to 15 months of age (115), and, at later age, they serve as a model for
studying HCC. The effect of genetic and environmental factors on the development of liver fibrosis
was studied in this thesis by exposing these mice either to liver specific overexpression of genes
(belonging to IGF axis), or to metabolic challenges (like food deprivation).
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OUTLINE OF THE THESIS
The aim of the research described in this thesis was to identify novel approaches and therapeutic
targets for the treatment of liver fibrosis. The initial experiments were based on our previous finding
that IGFBP5 was strongly upregulated upon HSC activation and transdifferentiation into MFs in vitro
and in vivo (86). In Chapter 2, we therefore investigated the role of IGFBP5 in HSCs using gain- and
loss-of-function approaches. We used the human LX2 cell line (110) that recapitulates many features
of the activated HSCs phenotype. To assess its role in more advanced stages of fibrosis, the response
to IGFBP5 was also dissected in human primary liver MFs. To clarify the role of IGFBP5 in the
pathogenesis of liver fibrosis caused by chronic cholangiopathies (Chapter 3), we overexpressed this
gene in hepatocytes of Abcb4-/- mice using an adeno-associated viral (AAV) vector large amounts of
IGFBP5 were delivered to the hepatic tissue including HSCs. To substantiate the findings in the
Abcb4-/- model and relate them to the physiological role of hepatic IGFBP5 expression, a systems
genetics approach was applied to a mouse genetic reference population, using an open source
database. Since the biliary epithelium expresses a receptor for IGF1 (116), and insulin-like growth
factor 1 (IGF1) protects cholangiocytes against cholestatic injury in vitro (117), overexpression of
IGF1 could be beneficial in cholestatic liver diseases. Therefore, in Chapter 4 we aimed to study if a
prolonged increase of hepatic IGF1 expression in a model for chronic cholangiopathy, the Abcb4-/-
mouse, was beneficial. Using a rat IGF1 gene behind an α-Sma promoter the expression of this
growth factor was enhanced in activated HSCs.
Intermittent fasting and caloric restriction have beneficial effect on health status of
laboratory animals (118). In NASH and NAFLD patients, histological improvements of liver
pathology positively correlate with the size of weight loss (119-121), but the mechanism is not fully
understood. Weight loss has beneficial effects on fibrosis induced by nutrient overload in men and
mice (103, 104). We therefore postulated that the complex adaptive response to nutrient deprivation
would also benefit the fibrotic pathology in cholestatic liver injury. In Chapter 5 we therefore tested
the hypothesis that food deprivation could positively influence pathology in fibrotic Abcb4-/- mouse
liver. Finally, in Chapter 6 the work described in this thesis is discussed, and future perspectives are
given.
chapter 1
22
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intro
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c h a p t e r 2
igfbp5 enhances survival
of lx2 human hepatic stellate cells
chapter 2
32
ABSTRACT
Background: Expression of insulin-like growth factor binding protein 5 (IGFBP5) is strongly
induced upon activation of hepatic stellate cells and their transdifferentiation into myofibroblasts in
vitro. This was confirmed in vivo in an animal model of liver fibrosis. Since IGFBP5 has been shown
to promote fibrosis in other tissues, the aim of this study was to investigate its role in the progression
of liver fibrosis.
Methods: The effect of IGFBP5 was studied in LX2 cells, a model for partially activated hepatic
stellate cells, and in human primary liver myofibroblasts. IGFBP5 signalling was modulated by the
addition of recombinant protein, by lentiviral overexpression, and by siRNA mediated silencing.
Furthermore, the addition of IGF1 and silencing of the IGF1R was used to investigate the role of
the IGF-axis in IGFBP5 mediated effects.
Results: IGFBP5 enhanced the survival of LX2 cells and myofibroblasts via a >50% suppression of
apoptosis. This effect of IGFBP5 was not modulated by the addition of IGF1, nor by silencing of
the IGF1R. Additionally, IGFBP5 was able to enhance the expression of established pro-fibrotic
markers, such as collagen Iα1, TIMP1 and MMP1.
Conclusion: IGFBP5 enhances the survival of (partially) activated hepatic stellate cells and
myofibroblasts by lowering apoptosis via an IGF1-independent mechanism, and enhances the
expression of profibrotic genes. Its lowered expression may, therefore, reduce the progression of
liver fibrosis.
Fibrogenesis & Tissue Repair 2010, 3:3
Authors
Aleksandar Sokolović1, Milka Sokolović2, Willem Boers1, Ronald P.J. Oude Elferink1 and Piter J.
Bosma1
Affiliations
1Tytgat Institute for Liver and Intestinal Research,
2Department of Medical Biochemistry,
Academic Medical Centre, University of Amsterdam, Amsterdam, The Netherlands
IGFBP5 in LX2
33
INTRODUCTION
The extensive accumulation of extracellular matrix (ECM) produced by activated hepatic stellate cells
(HSCs), which normally reside in the space of Disse as the major vitamin A storage site, is a hallmark
of liver fibrosis (1, 2). Liver damage induces HSCs activation and, upon repeated and/or chronic
injury, they transdifferentiate into myofibroblast-like cells (1, 3). These cells migrate to the damaged
regions of the liver (4-6) where they play a central role in the pathogenic deposition of ECM (7, 8).
In order to identify novel therapeutic targets, we used gene expression profiling at different
stages of the pathogenic transdifferentiation of HSCs (9). One of the factors found to be upregulated
upon HSCs activation and further enhanced upon transdifferentiation into myofibroblasts was
IGFBP5 (insulin-like growth factor binding protein 5). This strong induction of IGFBP5 expression
was confirmed during the development of liver fibrosis in the Mdr2-/- mice, a well established animal
model of liver fibrosis (10). Expression of IGFBP5 in HSCs has been reported to be enhanced by
insulin-like growth factor 1 (IGF1) via a post-translational mechanism, while its novel synthesis was
decreased by TGFβ1 (transforming growth factor beta 1) (11).
IGFBP5 is a member the IGFBPs that bind IGF1 (12). IGF1 is mainly synthesized by the
liver and gets secreted into the circulation bound to IGFBPs, which prolong its half-life and, by
modulating its interaction with the IGF1 receptor (IGF1R), control its biological activity (12-14). In
advanced liver fibrosis, the IGF1 axis is severely impaired mostly due to a reduced number of healthy
IGF1 producing hepatocytes (15). The decrease in IGF1 signalling seems to provide a pro-fibrotic
environment, since the progression of liver fibrosis could be delayed by IGF1 administration (16,
17). As IGFBP5 impairs the binding of IGF1 to the cell-surface receptor IGF1R (18), its increased
expression in activated HSCs and myofibroblasts may reduce IGF1 signalling and, thus, promote
liver fibrosis. In contrast, in another study IGF1 has been reported to exert pro-fibrotic activity (19).
In that case the inhibition of IGF1 signalling by IGFBP5 would impair the pathogenesis of liver
fibrosis.
In lung and skin, IGFBP5 has also been shown to induce fibrosis upon epithelial injury (20-
22). Induction of IGFBP5 expression initiated the activation and transdifferentiation of resident
fibroblasts into myofibroblasts, causing increased ECM production and deposition in these tissues.
Moreover, it seemed to cause cellular senescence and epithelial-mesenchymal transition (23).
The aim of this study was to investigate the role of IGFBP5 in liver fibrosis by employing
both gain and loss of function approaches. We focused on the effect of IGFBP5 on HSCs, using the
human LX2 cell line (24), which recapitulates many features of the activated HSCs phenotype. In
addition, to see if IGFBP5 could play a role in more advanced stages of fibrosis, we analysed its
effect on human primary liver myofibroblasts.
MATERIALS AND METHODS
Cell culturing
LX2 cells (kindly provided by Prof. dr. S. Friedman) and human myofibroblasts (obtained as
described (9)) were cultured in Dulbecco’s modified Eagle’s medium (Lonza, Verviers, Belgium),
supplemented with 10% fetal calf serum (FCS), 1 mmol/l L-glutamine, 100 IU/ml penicillin and
chapter 2
34
streptomycin. Human recombinant IGFBP5 and IGF1 (rIGFBP5 and rIGF1; Gro-Pep, Reutlingen,
Germany) were added in concentration 0.1 ng/μl (25) and 1ng/μl, respectively, at 24 and 45 h of cell
culturing. The cells were used for additional assays 3 h after the last protein additions.
In order to induce apoptosis, the cells were serum deprived for 48 h. For drug induced
apoptosis, the cells grown in 10% FCS were incubated with 0.5 μM gliotoxin (Alexis Biochemicals,
Lausen, Switzerland) for 3 h. This dose induced caspase activity sixfold 3h after gliotoxin
administration (data not shown).
Lentiviral transduction
A lentiviral vector encoding human IGFBP5 behind the constitutive PGK promoter was generated
using the self-inactivating cppt-PGKiresGFP-PRE vector (26). Sequencing was performed to
exclude mutations.
siRNA mediated silencing
IGFBP5, IGF1R and negative control small interfering RNAs (siRNAs) were purchased from
Invitrogen (Breda, Netherlands). siRNA was introduced to the cells using Lipofectamine 2000
(Invitrogen, Breda, Netherland) in Optimem (Gibco, Invitrogen, Paisley, UK), according to the
manufacturer’s instructions. For the downstream applications siRNA transfected cells were grown in
10% FCS and used 48 h after transfection. When apoptosis was induced by serum starvation, the
cells were first grown o/n (16 h) in serum containing medium, which was then replaced for 48 h by
serum depleted medium.
Cell viability test
Cell Proliferation Reagent WST-1 (Roche Applied Science, Mannheim, Germany) was added (1/10th
of the culture volume) to cells grown in 96-well plates. Absorbance was measured at 450 nm
(reference at 630 nm) immediately and after 1h of incubation at 37°C.
Bromo-2’-deoxy-uridine (BrdU) assay (Roche applied Science, Penzberg, Germany) was
performed according to the manufacturer’s protocol. The cells grown in 96 well plates were
incubated with BrdU during the last 16 h of culturing.
Apoptosis assay
The Apo-One homogeneous Caspase 3/7 assay (Promega, Medison, WI, USA) was performed
according to the manufacturer’s protocol. The Apo-One Caspase 3/7 reagent was added in a 1:1
ratio with medium and fluorescence was measured using Novostar plate reader at 521 nm.
Reverse transcription and quantitative polymerase chain reaction (qPCR)
Total RNA was isolated using Trizol (Invitrogen, Breda, The Netherlands). RNA concentration was
determined spectrophotometrically and the integrity was checked by gel electrophoreses. cDNA was
synthesized using an oligo-dT primer and Superscript III (Invitrogen, Breda, The Netherlands).
qPCR was performed on a LightCycler using FastStart DNA Master Plus SYBR Green I (Roche,
Mannheim, Germany). The relative expression levels were calculated using the LinReg program (27)
IGFBP5 in LX2
35
and for each sample normalized by the expression of housekeeping gene 36B4 (acidic ribosomal
phosphoprotein P0). Primer sequences are shown in Table 1.
Western blot analysis
Cell extracts were analysed by Western blotting as described (28). We used antisera against IGFBP5
(1:250; Abcam, Cambridge, UK), IGF1R (1:200; Santa Cruz Biotechnology, CA, USA), poly (ADP-
ribose) polymerase (PARP; 1:100; Promega, Madison, USA) and β-actine (1:3000; Sigma, St Louis,
USA). Goat anti-mouse (1:2500; Bio-Rad Laboratories, Veenendaal, The Netherlands) and goat anti-
rabbit IgG horseradish peroxidase conjugated (1:5000; Santa Cruz Biotechnology, CA, USA) were
used as secondary antibodies. Chemiluminescence was quantified on the Lumi-Imager F1 using
CDP-Star (Roche, Mannheim, Germany). Amido-black staining and β-actine immuno-blot verified
similar protein loading.
Statistical analysis
All cell culturing experiments were performed in quadruplo, and were repeated at least three times. In
order to assess the significance of the data, ANOVA analysis and Student’s t test were employed.
The error bars in the figures represent the standard deviation. Significance threshold was set to
P<0.05 (denoted by the asterisks in all the Figures).
RESULTS
IGFBP5 expression in LX2 cells
In human HSCs in vitro, IGFBP5 expression is enhanced twofold upon activation and 200-fold upon
transdifferentiation into myofibroblasts (9). LX2 cells also express IGFBP5 (Table 2). The 30%
higher expression in these cells compared to resting HSCs confirms reports demonstrating that it is a
model for partially activated HSCs (24).
Table 2. Relative gene expression of IGF1 and IGFBP5 in LX2 cells, activated HSCs and myofibroblasts given
as fold changes of basal expression in freshly isolated human HSCs.
LX2 cells HSCs Activated HSCs Myofibroblasts
IGFBP5 1.3 1 2.3 236
IGF1 0.7 1 1.2 0.3
In order to investigate a potential role of IGFBP5 in liver fibrosis, we developed methods to
modulate its expression in LX2 cells. The endogenous expression of IGFBP5 mRNA was doubled
using lentiviral transduction (Figure 1A). This resulted in a 2.5-fold increase in protein levels (Figure
1B). Transfection with IGFBP5 siRNA reduced IGFBP5 mRNA 80% and led to complete absence
of detectable IGFBP5 signal on a Western blot (Figure 1C and D, respectively)
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36
Figure 1. Modulation of IGFBP5 expression in LX2 cells. A) IGFBP5 mRNA levels in control cells (LX2) and in
cells with lentiviral overexpression of IGFBP5 (LX2(BP5)). RNA levels normalized to 36B4 are shown as a % of control
cells. B) IGFBP5 protein levels in control (LX2) and transduced (LX2(BP5)) cells as demonstrated on a Western blot
and C) as quantified using the LumiImager. D) Silencing of IGFBP5 expression. Proteins were isolated from the cells
and medium 48 h after small interfering RNA (sRNA) transfection. The presence of IGFBP5 protein was visualized by
Western blotting in LX2 and LX2(BP5) cells transfected with control (siCtrl) and IGFBP5 siRNA (siBP5) and in medium
obtained from transfected LX2(BP5). E) Cells were transfected with small interfering (si) RNA for IGFBP5 (siBP5) or
with a control siRNA (siCtrl). RNA was isolated 48 h later and IGFBP5 mRNA levels were quantified and given as a
percentage of control cells. The results are given as a % of the control cells. The error bars represent standard deviations,
with asterisks denoting significant difference (P<0.05).
IGFBP5 enhances survival of LX2 cells
Several studies have shown that IGFBP5 can affect cell survival (29-32). We therefore compared the
viability of control LX2 cells with cells over-expressing IGFBP5. When grown with 10% FCS, cell
growth did not differ (data not shown). However, upon culturing in serum free medium for 48 h, a
WST assay (for cell viability) indicated 100% higher viability of the transduced cells (Figure 2A). The
similar 60% increase in viability seen upon addition of recombinant IGFBP5 (rIGFBP5) confirmed
the effect of IGFBP5 on LX2 survival (Figure 2B). In serum free medium, the siRNA mediated
reduction of IGFBP5 expression resulted in a >40% decrease in cell viability. This reduction in
viability was prevented by the addition of rIGFBP5 to the silenced cells (Figure 2C).
In order to investigate if IGFBP5 affected proliferation, we studied its effect on the BrdU
incorporation. In the cells grown in serum depleted medium the addition of 0.1 ng/µl rIGFBP5 at 24
h and 45 h of culturing did not effect the incorporation of BrdU (Figure 2D). This indicates that
IGFBP5 does not enhance LX2 cell proliferation.
IGFBP5 in LX2
37
Figure 2. IGFBP5 increases survival of LX2 cells. A) The viability of IGFBP5 overexpressing (LX2(BP5)) and
control cells (LX2) cultured in serum-free medium for 48 h was measured using WST. (B) The viability of LX2 cells
cultured in serum-free medium for 48 h was determined using WST. At 24 and 45 h, recombinant IGFBP5 was added
(rBP5). C) LX2 cells were transfected with control (siCtrl) or IGFBP5 siRNA (siBP5). At 16 h after transfection the
medium was replaced by serum-free medium and 48 h later the viability of the cells was measured using WST. rIGFBP5
was added at 24 and 45 h of serum-free culturing (siBP5+rBP5). D) BrdU incorporation was used to determine the
proliferation of LX2 cells cultured in serum-free medium for 48 h. rIGFBP5 was added at 24 and 45 h (rBP5). BrdU was
added at t=32 h. The results are shown as % of the control cells.
Impact of IGFBP5 on apoptosis in LX2 cells
In order to investigate if the effect of IGFBP5 on viability was caused by decreased apoptosis, we
determined the caspase activity using a Caspase 3/7 assay. The addition of rIGFBP5 to LX2 cells
cultured in serum free medium did reduce the caspase activity by 40% (Figure 3A). In order to
substantiate this protective effect, we also tested it in a model of drug induced apoptosis by gliotoxin
administration (33). The addition of rIGFBP5 3 h prior to the addition of 0.5 μM of gliotoxin
resulted in a significant (40%) lowering of caspase activity (Figure 3B). In order to investigate if
lowering of IGFBP5 would increase apoptosis, we studied the effect of siRNA mediated silencing on
its expression. Compared to control-siRNA transfected cells, the caspase activity in silenced cells was
increased by 30%. This increase was seen when apoptosis was induced both by serum starvation and
gliotoxin. Administration of rIGFBP5 reduced caspase activity in silenced cells to a level significantly
lover than that in control cells (Figure 3C and D, respectively). The increase in apoptosis in silenced
cells was further confirmed by Western blot analysis, which demonstrated a 150% increase of poly
(ADP-ribose) polymerase (PARP) protein expression in silenced compared to control cells (Figure
3E).
Altogether, these data indicate that IGFBP5 enhances the survival of this model for partially
activated HSCs by arresting apoptosis.
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38
Figure 3. IGFBP5 impact on apoptosis in LX2 cells. A) LX2 cells were cultured in serum-free medium for 48 h.
Recombinant IGFBP5 (rBP5) was added at 24h and 45 h and, at 48 h, a Caspase 3/7 assay was performed. B) Cells
grown in 10% FCS were treated with rBP5 as above, with the addition of 0.5 μM gliotoxin after 45 h. C) LX2 cells were
transfected with control (siCtrl) or IGFBP5 siRNA (siBP5). 16 h after transfection, the medium was replaced by serum-
free medium and cells were cultured for additional 48 h before performing the caspase assay. To siIGFBP5 transfected
cells, rBP5 was added at 24 h and 45 h (siBP5+rBP5). D) To cells treated with si- and rBP5 as in C, 0.5 µM gliotoxin was
added 3 h before performing the caspase assay. All results are shown as a % of the control cells. E) Apoptosis was
induced in LX2 cells transfected with siCtrl or siBP5 by the addition of 0.5 uM gliotoxin at 45 h. Proteins were isolated at
48 h and Western blotting was performed for PARP detection (left panel). Quantification is shown in the right panel.
IGFBP5 also enhances survival of human myofibroblasts
Activated HSCs transdifferentiate into myofibroblasts in advanced stages of liver fibrosis. This
transdifferentiation results in a further induction of IGFBP5 expression (9). Given the effects on
LX2 cells, IGFBP5 may also affect the survival of liver myofibroblasts. In order to investigate this,
we performed siRNA mediated silencing of IGFBP5 expression in primary human myofibroblasts
(Figure 4A). Forty-eight hours after transfection, IGFBP5 mRNA levels in primary myofibroblasts
were decreased by 60%. Although the silencing was not as effective as that in LX2 cells, it did cause
a small, but significant, decrease in survival, which could be prevented by the addition of both rIGF1
and rIGFBP5 (Figure 4B). As in LX2 cells, this decrease in survival was due to an increase in caspase
activity that could be avoided by the addition of rIGF1 and rIGFBP5 (Figure 4C). Thus, IGFBP5
also increases the survival of human liver myofibroblasts by reducing apoptosis.
Involvement of BCL2
In order to investigate the mechanism involved in the anti-apoptotic effect of IGFBP5 we studied its
effect on the expression of several pro- and anti-apoptotic genes. The enhanced expression of
IGFBP5 in transduced cells resulted in a significant induction of B-cell CLL/lymphoma 2 (BCL2)
mRNA levels (Figure 5). Also, the addition of rIGFBP5 to IGFBP5 silenced LX2 cells resulted in a
clear increase in BCL2 expression. The lack of effect of IGFBP5 silencing on BCL2 expression is
unexpected and seems to suggest that, in presence of low levels of IGFBP5, other factors do
determine (basal) BCL2 expression. BCL2 mRNA induction by high IGFBP5 levels seems to suggest
IGFBP5 in LX2
39
that it could play a role in the anti-apoptotic effect of IGFBP5, but additional studies such as BCL2
silencing are needed in order to establish this possibility.
Figure 4. In human myofibroblasts IGFBP5 silencing decreases survival and promotes apoptosis. A) Primary
human myofibroblasts were transfected with siRNA for IGFBP5 (siBP5) or with a control siRNA (siCtrl). At 16 h after
transfection the medium was replaced by serum-free medium, and cultured for an additional 48 h. Then RNA was
isolated and IGFBP5 mRNA level determined by qPCR. B) Recombinant IGFBP5 (rBP5), IGF1 (rIGF1), or both
(rIGF1+ rBP5), where added at 24 h and 45 h, after replacing the medium, to siBP5 transfected cells. At 48 h the WST
assay was performed. C) To cells treated as above but grown in 10% FCS, 0,5 µM gliotoxin was added at 45 h. Caspase
3/7 assay was performed at 48 h. Results are given as a % of control cells.
Figure 5. IGFBP5 influences BCL2 expression. LX2 cells were
transfected with control (siCtrl) or IGFBP5 siRNA (siBP5). 16 h after
transfection the medium was replaced by serum-free medium.
Recombinant IGFBP5 was added at 24 h and 45 h, after replacing the
medium, to siIGFBP5 transfected cells (siBP5+rBP5). After 48 h RNA
was isolated from control (siCtrl), silenced (siBP5), silenced treated with
rIGFBP5 (siBP5+rBP5) and LX2 cells overexpression IGFBP5
(LX2(BP5)) and used for qPCRin order to determine BCL2 mRNA. The
BCL2 mRNA levels were given as % of control cells.
Increased cell survival by IGFBP5 seems independent of IGF1
IGFBP5 binds IGF1 with high affinity. Therefore, its protective role in LX2 cells and myofibroblasts
could be due to modulation of the pro-survival effects of IGF1 signalling (18, 34). In order to
investigate if IGFBP5 exerts its action via IGF1, we also determined the effect of IGF1 on LX2 cells
viability. As with IGFBP5, the addition of IGF1 induced a 70% increase in survival of LX2 cells
(Figure 6A), and lowering of apoptosis by 40% (Figure 6B). In order to investigate if IGF1 effect is
modulated by IGFBP5, we studied the effect of the two factors added together. The response in
viability and apoptosis were similar when IGF1 and IGFBP5 were added, alone or in combination
(Figure 6A and B). However, in contrast to IGFBP5, IGF1 was able to enhance proliferation of LX2
cells for about 40% (Figure 6C), indicating that the two factors exert different effects on LX2 cells.
chapter 2
40
In order to establish a possible IGF1-independent effect of IGFBP5, we chose to silence
IGF1R, the receptor that mediates IGF1 signalling. Transfection of LX2 cells with siRNA for
IGF1R resulted in a 90% drop of IGF1R protein expression after 48 h (Figure 6D). This large
decrease effectively inhibited the pro-survival effect of IGF1, but not that of IGFBP5 (Figure 6E).
The possibility of IGF1-independent action of IGFBP5 was further confirmed by the addition of
rIGFBP5 to IGF1R-silenced LX2 cells, which caused a decrease in caspase activity, even to the level
below that in the control cells. The addition of IGF1, on the other hand, was not able to overcome
the effect of IGF1R silencing and its effect on caspase activity was lacking (Figure 6F). Thus, in
contrast to IGF1, the effect of IGFBP5 on LX2 cells survival is not mediated by the IGF1R,
suggesting that the effect it exerts on partly activated stellate cells is not by modulation of IGF1
signalling.
Figure 6. Cell survival by IGFBP5 is not affected by IGF1 or mediated by IGF1R. A) LX2 cells were transfected
with IGFBP5 siRNA (siBP5). 16 h after transfection the medium was replaced by serum-free medium for 48 h and then
the viability was determined by WST assay. Recombinant IGFBP5 (rBP5), IGF1 (rIGF1), or both (rIGF1+ rBP5), were
added at 24 h and 45 h. B) To cells treated as above, but cultured in 10% FCS, 0.5 μM gliotoxin was added at 45 h
followed by a Caspase 3/7 assay 3 h later. C) LX2 cells were cultured in serum-free medium for 48 h. rIGF1 was added
in different concentrations at 24 h and 45 h, BrdU was added at 32 h. Cells were lysed 16 h later (at 48 h) and assayed for
BrdU incorporation. D) LX2 cells were transfected with control (siCtrl) or siRNA for IGF1R (siIGF1R). 16 h after
transfection the medium was replaced by serum-free medium and cells were cultured for additional 48 h, and then lysed
and used for Western blot analysis in order to detect IGF1R. E) Cells were transfected and cultured as in D. rIGF1 or
rIGFBP5 (rBP5) was added at 24 h and 45 h after replacing medium. After 48 h a WST assay was performed. F) 0.5 µM
gliotoxin was added at 45 h, after replacing the medium. Caspase activity was measured 3 h later. All results are given as a
% of control cells.
IGFBP5 and fibrotic markers
In tissues such as lung and skin, IGFBP5 was reported to enhance the expression of established pro-
fibrotic markers, such as αSMA, collagen and fibronectin. As changed expression of these markers
could further establish the (pro-fibrotic) role of IGFBP5 in liver fibrosis, we examined its influence
on their expression in LX2 cells. As shown in Figure 7, overexpression of IGFBP5 induced the
mRNA level of collagen, type I, alpha 1 (COL1A1) by 70%, tissue inhibitor of metalloproteinase
(TIMP) metallopeptidase inhibitor 1 (TIMP1) by 70% and matrix metallopeptidase 1 (MMP1) by
100% in LX2 cells (Figure 7A-C). Silencing of IGFBP5 reduced the expression of these genes, which
IGFBP5 in LX2
41
could be recuperated by the addition of recombinant IGFBP5. This indicates that IGFBP5 may not
only enhance fibrosis by promoting the survival of activated HSCs and myofibroblasts, but also by
stimulating the expression of pro-fibrotic genes in these cells.
Figure 7. IGFBP5 influences the expression of genes involved in fibrogenesis. LX2 cells were transfected with
small interfering control (siCtrl) or IGFBP5 siRNA (siBP5). Sixteen hours after transfection, medium was replaced by
serum-free medium. Recombinant IGFBP5 (rIGFBP5) was added at 24 h and 45 h, after replacing the medium, to
siIGFBP5 transfected cells (siBP5+rBP5). After 48 h of culturing on serum-free medium RNA was isolated from
control, transfected and transfected treated with rIGFBP5. For comparison, RNA was isolated from LX2 cells
overexpressing IGFBP5 cultured in serum-free medium (LX2(BP5)). A) Collagen 1α1, B) TIMP1 and C) MMP1 mRNA
levels were measured with quantitative polymerase chain reaction, normalized to 36B4 and given as a percentage of
control cells.
DISCUSSION
We have previously shown that IGFBP5 expression was strongly upregulated during HSCSs
transdifferentiation in vitro and during development of liver fibrosis in vivo (9). The aim of this study
was to scrutinize the role of IGFBP5 in (partially) activated and transdifferentiated hepatic stellate
cells.
IGFBP5 expression is increased in fibrotic lung, skin and liver (21). The effects of IGFBP5
depend on cell type and tissue. For instance, in the process of mammary gland involution, IGFBP5
promotes apoptosis of epithelial cells in vivo and in vitro (35). In contrast, an anti-apoptotic role of
IGFBP5 has been reported, for instance, in myogenesis, in breast cancer cells grown in vitro, and in
gingival epithelial cells (36-38).
In liver fibrosis, the level of apoptosis is reduced in activated HSCs that play a pivotal role in
the initiation and perpetuation of this pathological process (39). Since IGFBP5 is induced in these
cells upon activation and can promote survival, it may play a role in increasing the numbers of these
activated cells seen in fibrotic liver. In order to study the effects of IGFBP5 in vitro, we used primary
human myofibroblasts and LX2 cells, an established human model cell line for partially activated
HSCs (40). The expression of IGFBP5 in LX2 cells was 30% higher than in quiescent normal HSCs.
For comparison, in cultured (that is, partially activated) HSCs, IGFBP5 expression was doubled,
while in hepatic myofibroblasts expression went up more than 200-fold. We used lentiviral
transduction of LX2 cells to enhance the IGFBP5 expression to the levels seen in activated HSCs.
Upon serum depletion, both IGFBP5 over-expression and the addition of rIGFBP5 promoted the
survival of LX2 cells by lowering the apoptosis. This suggests that the increased IGFBP5 expression
chapter 2
42
could play a role in the reduction of apoptosis seen in activated HSCs in the fibrotic liver. In
accordance, lowered IGFBP5 expression by RNA silencing decreased the viability of LX2 cells.
IGFBP5 silencing also decreased survival in human primary liver myofibroblasts. In both cell types
this was due to increased apoptosis, demonstrating that IGFBP5 functions as an anti-apoptotic, pro-
survival factor in these two pro-fibrotic cell types. Two recent studies reported a high expression of
IGFBP5 in hepatocellular carcinoma (HCC) and intra-hepatic cholangiocarcinoma (CC), suggesting
that it has a similar role in vivo by promoting the survival of cancer cells [48, 49]. We demonstrated
that IGFBP5 expression strongly increased during development of liver fibrosis in Mdr2 -/- mice (9).
This model of liver fibrosis not only spontaneously develops portal fibrosis, but is also prone to
HCC formation (41). The presence of IGFBP5 in this animal model and in patients with HCC or CC
suggests that, in addition to a role in the pathogenesis of liver fibrosis, IGFBP5 may also contribute
to tumour formation. Lowering IGFBP5 expression may, therefore, not only impair the
development of liver fibrosis but also reduce the risk of tumour formation.
The role of IGFBP5 in IGF1 signalling is well established. IGFBP5 binds IGF1 with high
affinity and protects it from rapid degradation (18) but, at the same time, prevents induction of the
IGF1R and thereby inhibits pro-survival signalling by IGF1. The observed effects of IGFBP5 may
therefore be due to its role in IGF1 signalling – that is to its disturbance of the IGF1 axis observed
in liver fibrosis (17). Our in vitro studies show, however, that, in contrast to IGFBP5, IGF1 increases
proliferation of LX2 cells. Moreover, no effect of IGF1 on IGFBP5 action, nor of IGFBP5 on IGF1
signalling, was detected. Therefore, it seems that the two factors exert their effect on LX2 cells via
different routes. In line with this, silencing of IGF1R resulted in loss of the pro-survival effects of
IGF1 but did not interfere with the pro-survival effects of IGFBP5. This strongly supports the
notion that IGFBP5 promotes survival of activated HSCs by lowering apoptosis in an IGF1-
independent manner. Several mechanisms, such as activation of TGFβ1, modulation of the
coagulation cascade and integrin activation may be involved in the IGF1-independent effect of
IGFBP5 (42). In addition, the existence of an IGFBP5 receptor has been proposed to explain the
IGF1-independent actions of IGFBP5 (43, 44).
The finding that IGFBP5 promoted survival without inducing proliferation suggests that it
may induce cellular senescence. Overexpression of IGFBP5 is shown to induce senescence in
HUVEC cells by induction of the tumour suppressor p53 (25). The induction of senescence plays a
promoting role in the development of pulmonary fibrosis (23). On the other hand, senescence in
activated HSCs was associated with impediment of liver fibrosis (45), leaving the role of IGFBP5 in
the development of senescence in liver fibrogenesis opened for further inquiries. Additionally,
adenovirally mediated overexpression of IGFBP5 induced the expression pro-fibrotic genes and
ECM deposition in lung and skin (20, 21). We showed that IGFBP5 also enhanced the expression of
genes directly involved in liver fibrogenesis such as collagen1α1, TIMP1 and MMP1 in LX2 cells. Its
effect on these pro-fibrotic genes in a model for activated HSCs indicates that IGFBP5 may also
have a direct effect on the ECM deposition in liver fibrosis. Increase of both TIMP1 and MMP1
expression, and thus exertion of both fibrotic and matrix-degrading effects, could be seen in the light
of a necessity to establish a balance between matrix deposition and restructuring during the process
of fibrogenesis. However, since our data only demonstrate changes on the mRNA level, it seems
possible that the changes in the expression of these opposing factors may not have occurred on the
protein level due to posttranscriptional and posttranslational modifications.
IGFBP5 in LX2
43
In conclusion, our data show that IGFBP5 improves the survival of (partially) activated
HSCs and liver myofibroblasts by lowering the level of apoptosis via an IGF1-independent
mechanism. In addition, IGFBP5 increases the expression of genes involved in ECM deposition.
Accordingly, lowering expression of this apparently pro-fibrotic factor may impair the progression of
liver fibrosis.
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c h a p t e r 3
unexpected reduction of
murine liver fibrosis by igfbp5
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ABSTRACT
Background: The ATP-binding cassette, sub-family B member 4 knock-out mouse (Abcb4-/-) is a
relevant model for chronic cholangiopathy in man. Due to the lack of this P-glycoprotein in the
canalicular membrane of hepatocytes, the secretion of phospholipids into bile is absent, resulting in
increased bile toxicity. Expression of insulin like growth factor binding protein 5 (Igfbp5) increases in
time in the livers of these mice. It is unclear whether this induction is a consequence of or plays a
role in the progression of liver pathology. Aim of this study was therefore to investigate the effect of
IGFBP5 induction on the progression of liver fibrosis caused by chronic cholangiopathy.
Methods: IGFBP5 and, as a control, green fluorescent protein were overexpressed in the
hepatocytes of Abcb4-/- mice, using an adeno-associated viral vector (AAV). Progression of liver
fibrosis was studied 3, 6, and 12 weeks after vector injection by analyzing serum parameters, collagen
deposition, expression of pro-fibrotic genes, inflammation and oxidative stress.
Results: A single administration of the AAV vectors provided prolonged expression of IGFBP5 and
GFP in the livers of Abcb4-/-mice. Compared to GFP control, fractional liver weight, ECM
deposition and amount of activated HSCs significantly decreased in IGFBP5 overexpressing mice
even 12 weeks after treatment. This effect was not due to a change in bile composition, but driven by
reduced inflammation, oxidative stress, and proliferation.
Conclusion: Overexpression of IGFBP5 seems to have a protective effect on liver pathology in this
model for chronic cholangiopathy.
Biochimica et Biophysica Acta (BBA) - Molecular Basis of Disease
Volume 1822, Issue 6, June 2012, Pages 996–1003
Authors
Aleksandar Sokolović1, Paula S. Montenegro-Miranda1, Dirk Rudi de Waart1, Radha M.N. Cappai1,
Suzanne Duijst1, Milka Sokolović2 and Piter J. Bosma1
Affiliations
1 Tytgat Institute for Liver and Intestinal Research, 2 Department of Medical Biochemistry,
Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands
Acknowledgments
We are indebted to Profs. M. Kotb and R.W. Moyer for permission to use their unpublished
phenotypic data deposited in the GeneNetwork database. We thank Prof. Dr. R.P.J. Oude Elferink
for constructive discussions, Dr. J. Ruijter for help with quantification in Scion Image, and J. K.
Hiralall, L. ten Bloemendaal and C. Kunne for technical assistance.
IGFBP5 in Abcb4-/-
49
INTRODUCTION
Abcb4-/- (ATP-binding cassette, subfamily b, member 4) mice are the model for progressive familial
intrahepatic cholestasis type 3. In humans, ABCB4 deficiency results in spontaneous development of
biliary fibrosis shortly after birth, due to the lack of biliary phospholipids essential for neutralizing
the toxic effects of the high concentrations of bile acids (1). Since murine bile acids are less toxic, the
phenotype is milder in mice than in humans, which also qualifies Abcb4-/- mice as a model for other
forms of progressive cholangiopathies, such as progressive sclerosing cholangitis (2, 3). Recurrent
chronic injury in this mouse model, caused by leakage of toxic bile into the portal area leads to
progressive accumulation of extracellular matrix (ECM) components, mainly produced by activated
hepatic stellate cells (HSCs) in an attempt to counter hepatic damage (4-6). This results in an
imbalance between fibrogenesis and fibrolysis, leading to liver fibrosis, architectural distortion,
cirrhosis and ultimately liver failure (7, 8).
Insulin like growth factor binding protein 5 (Igfbp5) is strongly induced in the livers of Abcb4-
/- mice suggesting a potential role in the pathogenesis of chronic cholangiopathy (9). This seems to
be supported by its high expression in patients with cholangiocarcinoma (10). We have shown
previously that overexpression of IGFBP5 increases the survival of partially activated HSCs and
myofibroblasts (MF) (11). This pointed to its profibrotic role in liver, as also reported for skin and
lung where IGFBP5 overexpression stimulates the progression of fibrosis (12, 13).
To clarify the role of IGFBP5 in the pathogenesis of chronic cholangiopathies, we overexpressed it
in the Abcb4-/- mice, using an adeno-associated viral (AAV) vector. This in vivo AAV delivery that
efficiently provides expression in hepatocytes, should result in the supply of large amounts this
excretory protein to the intracellular space and to the HSCs (14).To substantiate the experimental
findings in this model, and to relate them to the physiological role of hepatic IGFBP5 expression, a
systems genetics approach was applied to a mouse genetic reference population, using the open
source GeneNetwork database. The results have pointed to a beneficial role of IGFBP5 in liver
fibrosis caused by chronic cholangiopathy in Abcb4-/- mouse model.
MATERIALS AND METHODS
scAAV vectors
scAAV8-LP1-Igfbp5 and scAAV8-LP1-eGFP were generated by replacing the factor IX cDNA from
scAAV-LP1-IX by the cDNA of mouse Igfbp5 or eGFP, using the EcoR1 and Bspm1 sites (Figure
1A) (15). Correctness of constructs was checked by sequencing. scAAV8 pseudotyped vector was
produced and titrated as described (16).
Animals
The animal studies were reviewed and approved by the AMC committee for animal care and use.
Three months after birth, a single dose of 5x1012 genomic copies (gc)/kg body weight of scAAV8-
LP1- Igfbp5 or scAAV8-LP1-eGFP was injected in the tail vein of male FVB and Abcb4-/- mice (FVB
background). Animals were sacrificed 3 (n=5), 6 (n=7) and 12 (n=7) weeks thereafter. In FVB
controls (n=5) the effect was studied only after 3 weeks. Five animals per treatment group were used
at 3 weeks, and 7 animals per group at weeks 6 and 12.
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Tissue collection and biochemical analyses
Upon sacrificing, blood was collected, livers were snap-frozen in liquid nitrogen and stored at -80oC,
or fixed in 4% formaldehyde in PBS. Total liver collagen was determined by measuring
hydroxyproline content as described (17). GSH/GSSG ratio in liver and bile was determined (18).
ASAT, ALAT and alkaline phosphatase (ALP) concentrations in blood were determined using
standard clinical chemistry methods.
RNA isolation, reverse transcription and quantitative PCR
These analyses were performed and analyzed as previously described (11). We used 5–7 animals for
qRT-PCR, depending on the time point—5 animals per group at 3 weeks, and 7 animals per group at
6 and 12 weeks.
Western blot analysis
They were performed as described (11), using the following antibodies: IGFBP5 (Abcam,
Cambridge, UK), GFP, PCNA and p21 (Santa Cruz biotechnology, CA, USA). Goat anti-mouse
(Bio-Rad Laboratories, Veenendaal, The Netherlands) and goat anti-rabbit IgG horseradish
peroxidase conjugated (Santa Cruz Biotechnology, CA, USA) were used as secondary antibodies.
Chemiluminescence was quantified by Lumi-Imager F1, using CDP-Star (Roche, Mannheim,
Germany) and β-actin as a reference. For detection of IGFBP5 in serum, the same amount of (10x
diluted) serum was loaded for each sample.
Histology and immunohistochemistry
Formaldehyde-fixed liver sections were stained with Sirius red (morphometrical connective tissue
assessment), or immunohistochemically, as described (19). Antibodies directed against PCNA (Santa
Cruz biotechnology, CA, USA) and α-SMA (Sigma, Zwijndrecht, The Netherlands) were visualized
with goat anti-mouse or goat anti-rabbit IgG coupled to horse-radish peroxidase (Sigma,
Zwijndrecht, The Netherlands), or alkaline phosphatase, respectively. Anti-α-F4/80 (AbDSerotec,
Dusseldorf, Germany) and anti-MAC-1 (R&D systems, Abingdon UK) were both visualized using
rabbit-anti-rat-biotin as secondary antibody (DAKO, Glostrup, Denmark).
Correlation analyses and QTL interval mapping in GeneNetwork
These analyses were performed as described (20). Analysis of Igfbp5 expression in liver of the BxD
genetic reference population. The BxD mouse genetic reference population (GRP) is a set of
recombinant inbred (RI) mouse strains, derived by crossing F2 mice obtained from an intercross of
C57BL/6J (B6) and DBA/2J (D2) mice, and inbreeding the resulting progeny for at least 20
generations to generate the BxD GRP (21, 22). RI panels, comprised of a number of lines with
limited intra- and large inter-line phenotypic variation, are widely used to determine genotype-
phenotype associations with quantitative trait locus (QTL) mapping techniques, or eQTL mapping,
when mRNA levels are used as phenotypes (23-27). The relationships between the phenotypes and
genotypes are calculated with a likelihood ratio statistic (LRS), a measure of the probability that a
given genetic marker explains the variation in the phenotype. This genetic reference panel contains
data for hundreds of phenotypic traits, including clinical and molecular ones (i.e. microarray
expression studies in multiple tissues) acquired over 25-years. The data are publically available at
GeneNetwork (www.genenetwork.org). The web-based software further allows extraction of sets of
IGFBP5 in Abcb4-/-
51
transcripts or clinical phenotypes that tightly co-vary with a gene of interest across genetically
different populations. Patterns of co-variations can be studied in multiple experimental crosses and
multiple tissues.
For this study we used data from BxD published phenotypes database
(www.genenetwork.com), and gene expression data from:
liver (http://www.genenetwork.org/dbdoc/LV_G_0106_B.html),
kidney (http://www.genenetwork.org/dbdoc/MA_M2_0806_R.html)
brain (http://www.genenetwork.org/dbdoc/BR_M2_1106_R.html).
Statistical analysis
ANOVA analysis and Student’s t-test were used. The error bars represent the standard deviation, and
significance threshold was set to P<0.05.
RESULTS
Figure 1: Overexpression of IGFBP5 in liver using AAV. A) Time course and genome structure of scAAV-LP1-Igfbp5 and scAAV-LP1-GFP. B) Western blot of GFP (left panel) and IGFBP5 (30kD; right panel) present in liver homogenates obtained 3, 6 and 12 weeks after injection, and of IGFBP5 in plasma after 3 weeks (right panel). In both panels, the GFP-overexpressing animals are on the left (“g”) and IGFBP5-overexpressing mice (“i”) on the right side. For detection, the Western blots in the left panel were incubated with anti-GFP, while those in the right panel were incubated with an anti-IGFBP5 antibody. Every lane represents an individual animal. In total, 4–6 animals of each treatment group were analyzed. C–D) Relative gene expression of Igfbp5 in Abcb4−/− mice 3, 6 and 12 weeks after administration of scAAV-LP1-Igfbp5 (right panel) or scAAV-LP1-GFP as a control (left panel) (n=5-7). Asterisks denote significance (p<0.05) in comparison with control treatment.
IGFBP5 overexpression reduces hepatocyte damage and proliferation in Abcb4-/- mice
To investigate the effect of IGFBP5 on the liver pathogenesis in Abcb4-/-mice, its expression
was increased in hepatocytes using the liver specific scAAV8-LP1-IGFBP5 vector and scAAV8-LP1-
GFP as a control (Figure 1A). qPCR and Western blot confirmed the hepatic overexpression of both
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52
transgenes 3, 6 and 12 weeks after injection (Figure 1B). During this period, the expression declined,
but was still significantly increased for Igfbp5 at 12 weeks (~15-fold) compared to mock transduction
(Figure 1C). Because of the ongoing hepatocyte proliferation in the Abcb4-/- mice this loss of
episomal AAV vectors was expected (28, 29). The lower expression of GFP seen at 3 weeks after
injection while the vector dose was equal, suggested a more rapid decline of GFP expression during
this period, which could be due to a higher hepatocyte proliferation rate in these control mice
(Figure 1D).
Therefore, the expression of proliferation markers PCNA and Ki67 was determined (Figure
2A-C). The 2-3 fold lower PCNA expression level after three weeks, and the lower expression level
of Ki67 at both later time points, confirmed that proliferation was reduced in mice overexpressing
IGFBP5.
Figure 2: IGFBP5 overexpression decreases liver cell proliferation in Abcb4-/- mice. A) Representative PCNA
immunostaining in liver at 3 weeks after injection of scAAV-LP1-GFP (control) or scAAV-LP1-Igfbp5 (IGFBP5). B)
Quantification of PCNA expressing nuclei in control (GFP) or IGFBP5 overexpressing mice per mm2 at 3 weeks after
treatment. C) mRNA level of Ki67 in liver after 3, 6 and 12 weeks determined by qPCR expressed as % of the control.
Asterisks denote significance (p<0.05).
This may be due to enhanced expression of p21, involved in repair of DNA damage by
arresting cell cycle and proliferation. Both p21 mRNA and protein expression level were significantly
upregulated in Igfbp5 injected animals (Figure 3A-B). This was accompanied by an increased presence
of senescence-associated β-galactosidase (a widely used biomarker for senescent and aging cells),
especially in fibrotic regions (Figure 3C). Lower ALP (alkaline phosphatase) and ASAT (aspartate
transaminase) levels in these mice 6, 9 and 12 weeks after injection, suggested that IGFBP5 alleviates
hepatocyte damage (Figure 3D–E).
IGFBP5 in Abcb4-/-
53
Figure 3: IGFBP5 overexpression reduces hepatocyte damage in Abcb4-/- mice. A) Gene expression of p21 in liver after 3, 6 and 12 weeks after vector administration. B) p21 level 12 weeks after vector administration as detected by Western blot, and quantified as percentage of the control (GFP). C) Representative SA-β-gal staining of control (GFP) and IGFBP5-treated animals 12 weeks after vector injection. D–E) Plasma concentrations of ALP and ASAT in GFP- and IGFBP5-treated Abcb4−/−mice 1, 3, 6, 9, and 12 weeks upon vector administration.
Overexpression of IGFBP5 reduces liver fibrosis in Abcb4-/-mice
A protective role of IGFBP5, as suggested by decreased hepatocyte proliferation and lowered liver
enzymes in serum, may also slow down the progression of fibrosis. The 20% lowered fractional liver
weight, the reduced presence of ECM shown by hydroxyproline content (Figure 4A-B) and Sirius red
staining (Figure 4C) all confirmed that IGFBP5 overexpression impairs the progression of liver
fibrosis in this model.
Figure 4: IGFBP5 overexpression
reduces liver fibrosis in Abcb4-/- mice.
A) Average fractional liver weight of
control and IGFBP5 overexpressing mice 6
and 12 weeks after vector administration.
B) Hydroxyproline amount in liver 6 and
12 weeks after viral treatment. C)
Representative Sirius red staining in control
(GFP) and IGFBP5 overexpressing livers
12 weeks after experiment started.
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Figure 5: IGFBP5 overexpression decreases expression of fibrotic markers in Abcb4-/- mice. A) Representative α-SMA staining in control (GFP; left and middle panel; 5X and 20X magnified respectively) and IGFBP5 overexpressing livers (right panel) 12 weeks after vector administration. B) Number of α-SMA expressing cells per mm2 in liver 12 weeks after vector administration as determined by a custom written counting macro in Scion Image (Scion Corp, Frederick, MD). C–F) Relative mRNA level for procollagen, collagens 1, 3, 4 and Timp1, after 3, 6 and 12 weeks. All results are given as a percentage of the control. Asterisks denote significance (p < 0.05).
This was supported by 70% reduction of α-SMA (marker for activated HSCs/MFs)
expressing HSCs/MFs 12 weeks after vector administration (Figure 5A-B), and, as a consequence,
reduced expression of procollagen, collagens 1, 3, 4 and Timp1 (Figure 5C-G).
Since the pathogenesis in the Abcb4-/- mouse results from toxic bile, an effect of IGFBP5 on
bile composition could provide an alternative explanation of its protective role in this model. The
absence of significant differences in bile composition 12 weeks after vector administration however
renders this explanation highly unlikely (Table 1).
Table 1: Bile salt composition of GFP control and IGFBP5-treated Abcb4-/- mice and FVB.
FVB Abcb4-/- ctrl Abcb4-/- BP5
tauro β-muricholate 223.52 ± 67.90 277.96 ± 142.05 253.98 ± 172.18 taurocholate 94.97 ± 22.84 145.85 ± 68.17 180.53 ± 162.50 tauro α-muricholate 1.92 ± 0.84 6.73 ± 4.86 9.33 ± 7.36 tauroursodeoxycholate 0.35 ± 0.14 0.74 ± 0.36 1.18 ± 0.89 taurochenodeoxycholate 1.56 ± 0.59 4.36 ± 1.66 5.55 ± 3.14 ∑ (bile salts) 322.32 ± 85.73 435.65 ± 209.62 450.58 ± 334.74
IGFBP5 overexpression reduces inflammation and oxidative stress in the liver of Abcb4-/-
mice
IGFBP5 has been reported to impair the influx of proinflammatory cells (30). Influx of these cells
into the liver will result in the release of inflammatory mediators that enhance fibrosis and cause
oxidative stress (31). Inhibition of this influx by IGFBP5 may therefore explain the protective effect
observed in this model. To investigate this, markers for inflammatory cells were studied. Presence of
F4/80, a marker for infiltrating macrophages and liver-resident Kupffer cells, was significantly lower
IGFBP5 in Abcb4-/-
55
6 and 12 weeks (mRNA) and 12 weeks (protein) after vector administration (Figure 6A-C).
Expression of Mpo, a marker for infiltrating neutrophils, was reduced at all three time points (Figure
6B). Furthermore, the presence of MAC1-expressing activated macrophages was clearly reduced in
IGFBP5 overexpressing animals (Figure 6D). In addition, the expression of Mcp1, which plays a role
in the recruitment of monocytes to sites of injury and infection, was reduced 3 and 6 weeks upon
IGFBP5 administration (Figure 6E) Finally, Tgfβ1 mRNA level was also reduced in Abcb4-/- mice
overexpressing IGFBP5 (Figure 6F).
Figure 6: IGFBP5 reduces expression of inflammatory markers. A, B, E and F) Relative mRNA levels of F4/80,
Mpo, Mcp1 and Tgfβ1, respectively, in livers of control (GFP) and IGFBP5 overexpressing mice, 3, 6 and 12 weeks
upon vector administration. All results are given as a percentage of the control (GFP). Asterisks denote significance (P <
0.05). C and D) Representative pictures of immunostainings for F4/80 and MAC1, respectively, 12 weeks after vector
administration.
The reduced influx caused by IGFBP5 overexpression will also lower the release of
proinflammatory cytokines in liver and as such may explain the lower the oxidative stress in these
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56
livers as indicated by the significant increased ratio of glutathione (GSH) and its oxidized form
glutathione-disulfide (GSSG; Figure 7A). In support, mRNA expression level of heme oxygenase 1
was significantly upregulated upon injection of IGFBP5 (Figure 7B)
Figure 7: IGFBP5 overexpression lower the oxidative stress. A) The GSH/GSSG ratio determined at 6 weeks (liver) and 12 weeks (liver and bile). B) Relative mRNA level for HO-1 at 6 and 12 weeks. All results are given as a percentage of the control. Asterisks denote significance (P < 0.05). C) Absence of anti-GFP antibodies in plasma of GFP- and IGFBP5-expressing mice demonstrated by an ELISA.
Specific expression of GFP in hepatocytes did not induce an immune response, as shown by
the lack of anti-GFP antibodies in serum of control mice and the comparable degree of liver fibrosis
in non-treated and GFP-treated Abcb4-/-mice (Figure 7C). This confirms previous findings of our
group and others that hepatocyte specific expression provided by an AAV vector does not result in
an immune response towards the transgene (16).
Altogether these data support an anti-inflammatory effect of IGFBP5 in the liver of Abcb4-/- mice.
Co-variation of Igfbp5 expression with phenotypic traits in a mouse genetic reference
population supports its anti-inflammatory role
To gain insight into the physiological role of Igfbp5 expression in adult liver, its co-variation with
published phenotypic traits in the BxD genetic reference population (GRP) was analyzed. This
experimental model that incorporates the natural range of genetic variation is obtained by 20
generation inbreeding in the F2 mice from an intercross between C57BL/6J and DBA/2J strains
(www.genenetwork.org) (24, 26).The analysis revealed a significant negative correlation between liver
Igfbp5 expression and plasma concentrations of ALAT and ASAT (Figure 8A and B) (32). These
relationships underline a hepato-protective role of IGFBP5. The negative correlation between Timp1
and Igfbp5 expression in the BxD GRP livers (5) provides further support for a possible physiological
role of IGFBP5 in ECM remodeling (Figure 8C). In the reference population, the expression of pro-
fibrotic cytokine Tgfβ1 positively correlated with that of F4/80 (Figure 8D), which corroborated the
reduction of inflammatory response by IGFBP5.
Furthermore, the correlation of IGFBP5 expression in liver of BxD GRP with clinical
phenotypes was calculated. A number of phenotypes were related to immune function
(Supplementary file 2 in a published manuscript), which was consistent with the anti-inflammatory
effect of IGFBP5 in the liver of Abcb4-/- mice. Igfbp5 expression in the livers of BxD mice negatively
IGFBP5 in Abcb4-/-
57
correlated with inflammation in spleen, bone marrow and with a general inflammatory response, the
exception being a positive correlation with TBC infection in the lung (Figure 8E).
Figure 8: Igfbp5 expression in liver correlates with suppressed immune response in the BxD reference population. Correlation between Igfbp5 mRNA in liver and ASAT A) and ALAT B) serum levels (n=7) as found in (GeneNetwork database). C) Correlations between Igfbp5 and Timp1 mRNA levels liver of BxD recombinant inbred strains (as determined in GeneNetwork database). D) Expression of Tgfβ in liver correlates with that of F4/80 in BxD genetic reference population. E) A correlation network between Igfbp5 expression level (blue rectangle) in livers of BxD mice and immune-linked phenotypic features measured in the same animals. The phenotypic features (depicted in green rectangles) are as follows: “IrradSpleen” (10407)- Antigenic activity of irradiated BXD spleen cells for Thy-1+CD3+CD4+CD8- T-cell clone E4.3 [Thymidine uptake cpm]; (33); “CpvInDMCs” (12678) - Infectious disease, immune function: Cowpox virus measure of disease onset measured by the day of maximum clinical score over two weeks after 10^6 pfu intranasal inoculation (Rice AD, Moyer RW, 2010, unpublished data); “ProgCellMob” (10206)- Immune function: Granulocyte colony stimulating factor (G-CSF) induced progenitor cell mobilization from bone marrow to blood [PB-CFC/ul]; (34); “BCGPulmGranInflm” (10695) - Immune function: Lung inflammatory response, pulmonary granulomatous inflammation induced by BCG [units] (35).
Tissue specific regulation of Igfbp5 expression
Given the unanticipated effect of IGFBP5 on liver fibrosis, we addressed the tissue-specificity of its
expression. Notably, a direct analysis of variation in Igfbp5 mRNA levels in different organs - liver,
kidney, (whole) brain, hippocampus and cartilage - did not reveal any correlation (not shown),
underscoring organ specific regulation of Igfbp5 expression. The correlations between Igfbp5
expression in different organs and phenotypic features in the BxD GRP were distinct for liver,
kidney and brain, (as published in Supplementary file 2). Unsurprisingly, the variation of Igfbp5
expression in other two organs did not correlate with ALAT and ASAT levels in serum.
The systems genetic approach was further employed to identify the quantitative trait loci
(QTLs) that specifically modulate the expression of Igfbp5 in mouse liver. No cisQTLs were
determined on Chr.1, where Igfbp5 resides (Figure 9A). A putative trans-regulatory region that
modulates Igfbp5 expression in liver was identified on Chr.7. The QTL peak (135-142Mb) comprised
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83 positional candidates that may modulate hepatic Igfbp5 expression (Figure 9B, as published in
Supplementary table 1).
Figure 9: Interval mapping, a WebQTL analysis of upstream controllers of liver Igfbp5 expression. A) The X-axis represents 19 mouse autosomes and the X chromosome. The small purple triangle marks the location of the Igfbp5 gene. The thick blue line summarizes the strength of association between variation in Igfbp5 expression and the two genotypes of all markers and the intervals between markers. The height of the peak (primary Y-axis) provides the likelihood ratio statistic (LRS; which can be read as a chi-square-like statistic). The additive coefficient (green line, secondary Y-axis) indicates that DBA/2J alleles increase trait values. In contrast, a negative additive coefficient (red line) indicates that C57BL/6J alleles increase trait values. The yellow histogram bars summarize the results of a whole-genome bootstrap of the trait performed 1000 times, supporting thereby the robustness of the results. The horizontal dashed lines at 10.5 and 17.3 are the LRS values associated with the suggestive and significant genome-wide probabilities that were established by permutations of phenotypes across genotypes. B) Physical map of variation in Igfbp5 expression in liver on distal Chr 7 (a blow up of the whole-genome map in panel A). A physical scale in megabases (Mb) is shown on the X-axis. The small irregular colored blocks and marks at the top of the map indicate the locations of genes superimposed on the physical map. The orange hash marks along the X-axis represent the number of single nucleotide polymorphisms that distinguish the two parental strains (C57BL/6J and DBA/2J) from each other.
To narrow them down to a biologically relevant subset, genes were scrutinized by analyzing
their co-expression, by QTL mapping (to establish if they were cisQTLs per se), by examining the
number of SNPs, and by studying the literature. Among the four most promising candidates (Tacc2,
Nkx1-2, Zranb1 and Ctbp) Nkx1-2 appeared the most likely one, since it was a cisQTL, it was
modulated by the same transQTL on Chr.14 that possibly also regulates the expression of Igfbp5, and
IGFBP5 in Abcb4-/-
59
its expression positively correlated with that of Igfbp5 (Figure 10A-C). The almost linear relationship
between Nkx1-2 expression in the liver and the survival coefficient in infectious diseases suggests its
potential important role in orchestrating inflammatory responses, possibly including Igfbp5
expression (Figure 10D; (Kotb M., 2010, unpublished data)). Although this analysis reveals Nkx1 as an
interesting candidate, the role of other candidate genes cannot be excluded.
Figure 10: NKX1-2 is a likely modulator of Igfbp5 expression in liver. A) Interval mapping for Nkx1-2. (For explanation see Supplemtary figure 6.) B) QTL heatmap for Chrs. 1, 7 and 14 for Igfbp5 (left column) and Nkx1-2 (right column). The yellow triangles indicate the location of the two genes (Igfbp5 on Chr.1 and Nkx1-2 on Chr.7). The intensity of color indicates the likelihood ratio statistic (LRS) values associated with the suggestive and significant genome-wide probabilities established by permutations of phenotypes across genotypes for both of the parental strains. C) Correlation of Igfbp5 and Nkx1-2 expression in BxD GRP. D) Correlation between the expression level of Nkx1-2 and the coefficient of survival in infectious diseases (M. Kotb, 2010, unpublished data).
DISCUSSION
The aim of this study was to investigate the role of IGFBP5 in the liver pathogenesis in the Abcb4-/-
mice, a model for chronic cholangiopathy. Prolonged overexpression of IGFBP5 in hepatocytes
decreased their proliferation, reduced inflammation and oxidative stress, and lowered number of
activated HSCs, all leading to reduced development of liver fibrosis.
IGFBP5 has cell- and tissue-type specific effects. It induces apoptosis in the involuting
mammary gland (36) and inhibits cell proliferation in the involuting prostate gland (37), but supports
the survival of skeletal myoblast (38). To clarify the differential role and expression of Igfbp5 in liver
and other organs, its steady-state expression was analyzed in the BxD genetic reference population.
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The correlations with clinical features not only indicated an organ specific regulation of IGFBP5, but
also a role that differed between organs. This seems to explain why IGFBP5 was found a profibrotic
factor in lung and skin (12, 13), while the current study revealed its protective effect in liver fibrosis.
The latter is underscored by the negative correlation between Igfbp5 expression and serum
parameters of liver damage in IGFBP5 overexpressing Abcb4-/- mice, and in the BxD genetic
reference population. The systems genetic approach made it clear that the relation between these
liver enzyme levels and IGFBP5 goes beyond the issue of damage in the Abcb4-/- model. In addition,
a protective effect of IGFBP5 on hepatocytes in vivo is consistent with its prosurvival effect seen in
activated HSCs in vitro (11). Reduced liver fibrosis after 12 weeks of IGFBP5 expression, as
demonstrated by considerably reduced portal bridging compared to that seen in control animals at
the time of vector administration and compared to GFP-expressing controls (Figure 4C), indicated
involvement of IGFBP5 not only in impediment of ongoing fibrogenesis, but in reduction of the
existing scar tissue.
The most likely mechanism of the anti-fibrotic effect of IGFBP5 expression in the Abcb4-/-
mice is a reduction of inflammation. The portal inflammation, caused by leakage of toxic bile is one
of the most prominent pathological features in this model for chronic cholangiopathy (3).
Overexpression of IGFBP5 lowers the influx of inflammatory cells into this area, possibly by
impairing their migration (39). This would also lower the production of inflammatory cytokines,
known to stimulate collagen synthesis and to enhance hepatocyte damage. The negative correlation
of Igfbp5 expression with inflammation in the BxD genetic reference population further supports
such an anti-inflammatory role. In addition, the systems genetics approach indicated Nkx1-2 as the
putative modulator of liver Igfbp5 expression, possibly in response to an inflammatory assault, which
would then explain the Igfbp5 upregulation in Abcb4-/-mice upon cholate feeding (9). Damaged
hepatocytes and activated inflammatory cells release reactive oxygen species (ROS) and other
reactive intermediates (40, 41), which enhance the expression of profibrotic genes in human HSCs
and MFs (42-44). The reduced oxidative stress in IGFBP5 overexpressing livers is in agreement with
the observed decrease in inflammation, and may additionally alleviate liver fibrosis. However, the
lower influx may also reduce the immune surveillance in these livers and as such may provide
protection of cancer cells. Such a mechanism could explain the high IGFBP5 expression reported in
patients with cholangiocarcinoma (10).
IGFBP5, however, may have a more direct effect. The increased amount of p21 in IGFBP5
overexpressing livers may be instrumental for the reduced proliferation. p21 regulates PCNA binding
to damaged DNA (45), reducing its nuclear presence as seen in this study (Figure 2A-B). In addition,
the increased presence of p21 may play a role in senescence (46), particularly that in the fibrotic
regions of IGFBP5 treated livers. Changes in gene expression profiles of senescent HSCs point to
the cell cycle standstill, decreased synthesis of ECM, increased release of ECM remodeling enzymes
(47). In this case, the increased expression of IGFBP5 seems aimed at reducing the pathology in this
model of chronic cholangiopathy.
In conclusion, this study reveals that expression of IGFBP5 in the Abcb4-/-mice reduces
inflammation, oxidative stress, ECM deposition and hepatocyte proliferation, thereby evidently
reducing liver fibrosis. Furthermore, the systems genetics approach reveals correlation between
IGFBP5 in Abcb4-/-
61
endogenous hepatic Igfbp5 expression and decrease of inflammation in a reference population,
underscoring its role in ameliorating pathology in the model for chronic cholangiopathy.
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c h a p t e r 4
igf1 enhances
bile-duct proliferation and fibrosis
in abcb4-/- mice
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ABSTRACT
Background and Aims: Adamant progression of chronic cholangiopathies towards cirrhosis and
limited therapeutic options leave a liver transplantation the only effective treatment. Insulin-like
growth factor 1 (IGF1) effectively blocks fibrosis in acute models of liver damage in mice, and a
phase I clinical trial suggested an improved liver function. IGF1 targets the biliary epithelium, but its
potential benefit in chronic cholangiopathies has not been studied.
Methods: To investigate the possible therapeutic effect of increased IGF1 expression, we crossed
Abcb4-/- mice (a model for chronic cholangiopathy), with transgenic animals that overexpress IGF1.
The effect on disease progression was studied in the resulting IGF1-overexpressing Abcb4-/- mice,
and compared to that of Abcb4-/- littermates. The specificity of this effect was further studied in an
acute model of fibrosis.
Results: The overexpression of IGF1 in transgenic Abcb4-/- mice resulted in stimulation of
fibrogenic processes - as shown by increased expression of Tgfß, and collagens 1, 3 and 4, and
confirmed by Sirius red staining and hydroxyproline measurements. Excessive extracellular matrix
deposition was favoured by raise in Timp1 and Timp2, while a reduction of tPA expression indicated
lower tissue remodelling. These effects were accompanied by an increase in expression of
inflammation markers like Tnfα, and higher presence of infiltrating macrophages. Finally, increased
number of Ck19-expressing cells indicated proliferation of biliary epithelium.
Conclusion: In contrast to liver fibrosis associated with hepatocellular damage, IGF1
overexpression does not inhibit liver fibrogenesis in chronic cholangiopathy.
accepted for publication in Biochimica et Biophysica Acta (BBA) - Molecular Basis of Disease
Authors
Aleksandar Sokolović1, Carlos M Rodriguez-Ortigosa2, Lysbeth ten Bloemendaal1, Ronald P.J. Oude
Elferink1, Jesús Prieto2, Piter J Bosma1
Affiliations
1Tytgat Institute for Liver and Intestinal Research, Academic Medical Center, University of
Amsterdam, Amsterdam, The Netherlands
2Division of Hepatology and Gene Therapy, CIMA University of Navarra, and CIBERehd,
Pamplona, Spain
Acknowledgements:
We thank Nerea Juanarena and Sara Arcelus for their excellent technical assistance, and dr. Dirk
Rudi de Waart for hydroxyplroline quantification. We would also like to thank dr. Milka Sokolović
for her assistance and advice during preparation of the manuscript.
IGF1 in Abcb4-/-
67
INTRODUCTION
Primary sclerosing cholangitis and primary biliary cirrhosis are chronic diseases of the bile duct
characterized by progressive inflammation that leads to the development of biliary fibrosis. There is
no effective therapy to halt the disease progression and most patients progress to cirrhosis and liver
failure (1, 2). The lack of sufficient donor organs and surgical contraindications render development
of therapeutic interventions that stop progression of liver fibrosis urgently needed.
Insulin-like growth factor 1 (IGF1) regulates the proliferation and differentiation of many
cell types (3). The liver is responsible for the abundant presence of IGF1 in the circulation (4) where
it is present attached to binding proteins (IGFBPs) that prolong its half-life and regulate its biological
activity (3, 5). Once released from IGFBPs, IGF1 can bind and activate the receptor (IGF1R), and
by activation of mitogen activated protein (MAP) kinase and PI3 kinase induce genes involved in cell
survival, growth, and differentiation (6). Liver fibrosis reduces the IGF1 expression in the liver (7, 8).
Administration of recombinant IGF1 or enhancing IGF1 expression using a viral vector significantly
reduced liver fibrosis in rats with CCl4-induced liver cirrhosis (9, 10). In the follow-up phase I-II
clinical trials, IGF1 administration increased the serum albumin level, suggesting an improved liver
function in patients suffering from this devastating disease (11).
The potential use of IGF1 in chronic cholangiopathies has not been studied. Since the biliary
epithelium expresses IGF1R (12), and IGF1 protects cholangiocytes against cholestatic injury in vitro
(13), overexpression of IGF1 could be beneficial in these diseases. Therefore the aim of this study
was to investigate if a prolonged increase of hepatic IGF1 expression in a model for chronic
cholangiopathy, the Abcb4-/- mouse, was beneficial.
The Abcb4-/- mouse is a model for primary familial cholestasis type 3 (14). ABCB4 is a
flippase essential for the excretion of phospholipids into bile, needed to neutralize the harmful
effects of bile acids. Bile lacking phospholipids is toxic and causes hepatocyte and cholangiocyte
injury (15). Since murine bile acids are less toxic, the damage in the Abcb4-/- is smaller compared to
that in PFIC type 3 patients, making this mouse also a model for other chronic cholangiopathies,
more specifically, primary sclerosing cholangitis (PSC) (16).To study the long term effect of IGF1 on
chronic cholangiopathy we crossbred the Abcb4-/- with the transgenic SMP8-IGF1 mouse. This
transgenic mouse expresses the rat IGF1 behind an α-SMA promoter (17). The increased α-SMA
expression in Abcb4-/- livers (15) should make this cross-bred model well suited to study long-term
effects of IGF1 on PSC.
MATERIALS AND METHODS
Animals.
This study was approved by the Ethical Committee for Animal Research of the University of
Navarra with the number: 008/08.
Abcb4-/- IGF1 model
Homozygous Abcb4-/- mice (FVB background; (18)) were cross-bred with transgenic SMP8-IGF1
mice, with a germline integration of a transgene composed of the murine α-Sma promoter linked to a
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68
full length rat Igf1 cDNA (17). The resulting heterozygotes for Abcb4 were back-crossed with Abcb4-
/- mice to obtain Abcb4-/- SMP8-Igf1+/- transgenic mice (TG) and their nontransgenic (NT)
littermates as a control. After weaning, the mice WT (FVB; n=4), Abcb4-/- (n=7) and Abcb4-/-
SMP8-Igf1+/- (n=7) were fed a 0.03% cholate diet to augment fibrosis, and were sacrificed after 3, 6
and 12 weeks (WT control for 3 weeks).
Bile duct ligation (BDL) model
SMP8-IGF1 (n=5) and WT (FVB; n=5) mice were bile duct ligated. After a 1 cm abdominal midline
incision, the common bile duct was isolated, double ligated close to the liver hilus (immediately
below the bifurcation) with a silk suture, and transected between ligatures. In sham operated mice
(SMP8-IGF1 and WT; n=5), the same operation was performed, but neither ligatures nor bile duct
section were carried out. All the mice used in the protocol were 12 week old at the beginning of the
experiment. The mice were sacrificed a week after BDL.
Tissue collection and biochemical analyses
Upon sacrificing, blood was collected, livers were snap-frozen in liquid nitrogen and stored at -80oC,
or fixed in 4% formaldehyde in PBS. Total liver collagen was determined by measuring
hydroxyproline content as described (19) Serum transaminases (ALT and AST), alkaline phosphatase
and bilirubin were determined in retro-orbital collected blood, using standard clinical chemistry
methods in a Cobas C311 autoanalyzer (Roche Diagnostics, Mannheim, Germany).
Reverse transcription, quantitative PCR and data analyses
The qPCR analysis was performed and the data analyzed as previously described (20). Shortly, total
liver RNA was extracted from snap-frozen livers using TRIzol reagent (Invitrogen, Breda, The
Netherlands) following the manufacturer’s instructions. 4M LiCl was used to specifically precipitate
RNA. All RNA isolates had A260/A280 ratio around 2, as assessed by NanoDrop ND-1000
spectrophotometer. The RNA integrity was confirmed by denaturing agarose gel electrophoresis.
The absence of DNA contamination was established by including RT- controls in cDNA synthesis
reactions. Gene specific primers (for Igf1, Hgf, aSma, Tgfb, Col1, Col3, Col4, Timp1, Timp2, tPa,
Tnfa, Ki67 and Ck19) were designed using Primer3Plus program and their sequences are available
upon request. qPCR was performed using a Light Cyler 480 (Roche) as described (21). The
expression level for each of the studied genes in liver was calculated using the LinRegPCR software
(22, 23), thereby avoiding the assumption of equal amplification efficiency between the reactions.
Gene expression was normalized by dividing the expression levels of tested genes by those of a
reference gene (36B4), and calculating the average of normalized values per condition. 36B4 was
used only after having validated it against three reference genes in our previous studies (24). In BDL
model, qPCR was performed as previously described (25). To assess the significance of the data,
ANOVA analysis and Student’s t test were employed. The error bars in the Figures represent the
standard deviation. Significance threshold was set to P<0.05 (denoted by the asterisks in the Figures
and Tables).
Histology and immunohistochemistry
Formaldehyde-fixed liver sections were stained with Sirius red (morphometrical connective tissue
assessment), or immunohistochemically, as described (26). Liver collagen content was quantified by
IGF1 in Abcb4-/-
69
Sirius red staining, using an Axioplan 2 Imaging microscope (Zeiss, Jena, Germany); random
acquisition of images was performed with Metamorph software (Molecular Devices, Sunnyvale, CA,
USA), and scoring of areas was carried out with Matlab software and the imaging processing libraries
of DIPlib (MathWorks, Natick, MA, USA). Ck19 (Eurogentec, Koln, Germany) was detected using
GAR-AP (DAKO, Glostrup, Denmark) as a secondary antibody. F4/80 (AbD Serotec, Dusseldorf,
Germany) was visualized using rabbit anti Rat-Ig (DAKO, Glostrup, Denmark) as a secondary
antibody.
Double staining for Ck19 and Ki67 was performed on frozen sections, fixed for 5 minutes in ice
cold acetone and air dried for 30 minutes. Slides were rinsed in PBS, blocked for 30 minutes in
TengT and incubated o/n at 4oC with the primary anti-Ki67 Ab (Abcam, Cambridge, UK; 1:500).
Washing was followed by 30 minutes incubation at RT with the secondary Alexa Fluor488 goat anti
rabbit IgG (Invitrogen, Paisley, UK; 1:1000). Sections were then rinsed and incubated with the
primary anti-Ck19 Ab (Eurogentec, Koln, Germany; 1:1000) for 1 hour at RT, washed again, and
incubated with Alexa Fluor594 goat anti rabbit IgG (Invitrogen, Paisley, UK; 1:1000) for 30 minutes
at RT. After washing, the slides were covered with vectashield + DAPI and visualized by confocal
microscopy. Quantification of Ki67-positive cholangiocytes after 12 weeks of cholate diet was
perfomed in 5 animals per phenotype, 5 images per mouse. The results are expressed as a percentage
of Ki67-positive in the total number of cholangiocytes.
In BDL model, bromodeoxyuridine (BrdU) method was used for counting proliferating
cholangiocytes as previously described (27). Shortly, formalin-fixed and paraffin-embedded liver
sections were stained with mouse monoclonal anti-BrdU antibody (1:1000 GE Healthcare,
Barcelona, Spain), at 4°C, overnight. Secondary peroxidase-labeled goat anti-mouse antibody (Dako
Envision System, Carpinteria, CA) was incubated at room temperature for 30 min. Two persons
unaware of the study groups scored BrdU positive cholangiocytes in SMP8-IGF1 BDL (n=5) and
WT BDL (n=5), analyzing 6 panels per animal.
RESULTS
Overexpression of IGF1 in Abcb4-/- mice
To study the long-term effect of IGF1 on the pathology of chronic cholangiopathy, Abcb4-/- mice
were crossbred with a transgenic mouse expressing rat Igf1 behind an α-Sma promoter (17). This
restricts rat Igf1 hepatic expression to the α-SMA expressing, activated hepatic stellate cells (HSC)
(27). To aggravate the pathology, after weaning the animals were given a cholate-supplemented diet
(0.03%) (15, 18). The expression of rat Igf1 was confirmed by qPCR in livers of the transgenic (TG)
Abcb4-/-IGF1 mice (Fig.1A). Since rat Igf1 expression in TG mice is controlled by the α-Sma
promoter, its stable expression reflects the α-Sma expression, that remained comparable at all three
time points (Fig.1B). IGF1 induces HGF expression in HSC (28). The significant increase of Hgf
expression at 6 and 12 weeks in TG animals compared to non-transgenic (NT) Abcb4-/- littermates
confirmed that IGF1 signaling was indeed enhanced (Fig.1C).
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Fig.1: Functional expression of IGF-1 in livers of Abcb4-/- mice. After weaning, WT, Abcb4-/- and Abcb4-/- IGF1 mice - WT (n=4; for 3 weeks), Abcb4-/- (n=7) and Abcb4-/- IGF1 (n=7) - were fed a 0.03% cholate diet. (A-C) mRNA levels of transgenic Igf1, α-Sma and Hgf in livers of WT, Abcb4-/- and Abcb4-/- IGF1 mice fed a cholate supplemented diet for 3, 6 and 12 weeks. D) Average fractional liver weight of Abcb4-/- and Abcb4-/- IGF1 mice, after 3, 6 and 12 weeks of cholate diet. Asterisks denote significance (P<0.05).
Overexpression of IGF1 induced liver fibrosis in Abcb4-/- mice
Liver function tests were performed to investigate if IGF1 could protect this mouse model against
the toxicity of bile salts, thereby reducing chronic bile duct proliferation. Serum levels of ALT, AST,
ALP and bilirubin significantly increased in Abcb4-/-IGF1 and Abcb4-/- mice compared to WT,
indicating liver damage in both models (Table 1). The initially high ALP serum levels, a result of
osteoblast activity in growing bones (29), decreased to normal after weaning (66-260 U/L). After 12
weeks of cholate diet, however, ALP and bilirubin concentration significantly increased in the IGF1
transgenics compared to their Abcb4-/- littermates, suggesting cholestasis and possibly an enhanced
progression of liver fibrosis (Table 1).
Table 1: The influence of IGF1 overexpression on liver function shown by clinical parameters in plasma.
ALT (U/L)
AST (U/L)
ALP (U/L)
tBil (mg/dl)
TP (g/dl)
Chol (mg/dl)
TG (mg/dl)
WT
3 weeks 76.1±16.9 126.9±29.5 188.2±36.6 0.08±0.01 5.2±0.2 183.5±9.9 83.50±17.4
Abcb4-/-
3 weeks 355.4±125.8 502.5±186.9 1992.6±1257.6 0.2±0.2 5.2±0.3 107.2±25.9 137.3±24.7
6 weeks 185.3±95.4 238.7±74.8 448.8±319.5 0.2±0.1 5.3±0.8 103.6±28.7 144.9±12.9
12 weeks 356.6±220.3 591.1±460.4 285±136.4 0.3±0.3 5.9±0.5 80.1±16.6 89.14±34.2
Abcb4-/-IGF1
3 weeks 320.7±86.2 502.2±231.2 1020.5±657.4 0.2±0.1 5.9±0.5 112.9±15.9 116.6±29.1
6 weeks 238.5±127.7 314.6±152.9 533.4±323.2 0.2±0.1 5.4±1.4 101.5±29.8 132.1±56.7
12 weeks 388.8±191.2 600.9±322.7 1712.9±1095.5* 1.9±1.6* 5.0±1.2 103.3±24.6 128.3±42.9
The table shows mean values and standard deviation of serum parameters measured in plasma of WT (n=4), Abcb4 -/- (n=7) and Abcb4-/-IGF1 (n=7) mice. The differences between Abcb4-/- and Abcb4-/-IGF1 mice were tested by Student’s t-test, with asterisks denoting significant difference (P<0.05). The values for WT are shown as a reference. ALT (alanine transaminase), AST (aspartate transaminase), ALP (alkaline phosphatase), tBil (total bilirubin), TP (total protein), Chol (cholesterol), Tg (triglycerides).
After 3 weeks of cholate diet, the fractional liver weight in Abcb4-/- mice was significantly
higher than in Abcb4-/- IGF1 group. At 6 weeks this difference had disappeared, while at 12 weeks
there was even a tendency toward an increased liver/body weight ratio in Abcb4-/- IGF1 mice
(Fig.1D). Sirius red staining and hydroxyproline measurement revealed a significant increase in the
IGF1 in Abcb4-/-
71
amount of connective tissue in the Abcb4-/- IGF1 group, indicating increased liver fibrosis in
transgenic mice after 12 weeks of cholate diet (Fig.2A-C). The increased expression of the pro-
fibrotic transforming growth factor beta 1 (Tgfβ1), which stimulates trans-differentiation of HSC into
myofibroblasts and enhances TIMP and collagen production (30), may also support the increased
fibrosis in IGF1 expression Abcb4-/- mice at twelve weeks (Fig.3A). Also the mRNA levels of Col1, 3
and 4 at 12 weeks were significantly increased in these mice (Fig.3B-D), providing additional support
that IGF1 enhanced the progression of liver fibrosis in the Abcb4-/- mouse model.
Fig.2: IGF1 overexpression promotes fibrosis Abcb4-/- livers. (A) Sirius red staining of liver of WT, Abcb4-/- mice and transgenic Abcb4-/-IGF1 mice with representative pictures of large bile duct structures 12 weeks of cholate diet. (B) Percentage of fibrotic area estimated by image analysis of Sirius red stainings in Abcb4-/- and Abcb4-/-IGF1 mice after 12 weeks of cholate diet. Y axe represents the ratio between collagen area and total tissue area. (C) Hydroxyproline amount at 3, 6 and 12 weeks of cholate diet in livers of Abcb4-/- and Abcb4-/-IGF1 mice. Asterisks denote significance (P<0.05).
In addition to enhanced synthesis, the increased ECM deposition in mice overexpressing
IGF1 may also in part be due to reduced matrix remodeling. Inhibition of matrix metalloproteinases
(MMP) by their tissue inhibitors (TIMPs) plays an important role in this process (31, 32). The
significantly increased expression of Timp1 and 2 after 12 weeks suggested that tissue remodeling was
reduced in the Abcb4-/- IGF1 mice (Fig.3E, F). Additional support for this was provided by the
decreased expression of tPa, an activator of plasminogen that plays an essential role in the activation
of MMP needed for tissue remodeling (33).
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Fig.3: IGF1 overexpression hinders matrix remodeling in Abcb4-/- livers. (A-G) Relative mRNA level of Tgfβ1, Col1, 3, 4, tPA, Timp1, 2 and Tnfα in livers of WT, Abcb4-/-IGF1 and Abcb4-/- mice after 3, 6 and 12 weeks of cholate diet. Asterisks denote significant difference (P<0.05).
Overexpression of IGF1 enhances inflammation in Abcb4-/- liver
Inflammation due to regurgitation of bile acids plays an important role in the development of
pathology in the Abcb4-/- mice. The increased pathology in Abcb4-/- IGF1 animals was accompanied
by an increased inflammation, as shown by the enhanced expression of Tnfa (Fig.4A).
Immunostaining for F4/80, a marker for infiltrating macrophages and liver-resident Kupffer cells,
was higher at 12 weeks in Abcb4-/- IGF1 animals, which suggested an increased influx of
macrophages compared with non-transgenic controls (Fig.4B).
Fig.4: IGF1 overexpression induces expression of inflammatory markers in Abcb4-/- mice. Relative mRNA level of Tnfα in livers of WT, Abcb4-/-IGF1 and Abcb4-/- mice after 3, 6 and 12 weeks of cholate diet. Asterisks denote significant difference (P<0.05). (B) The representative pictures of F4/80 immunostaining in non-transgenic (left panel) and Abcb4-/-IGF1 mice (right panel) after 12 weeks of cholate diet are shown. (C) Representative CK19 immunostaining of the same livers.
IGF1 in Abcb4-/-
73
Overexpression of IGF1 increases cholangiocyte proliferation in Abcb4-/- mice
Since bile duct proliferation is one of the major characteristics of the pathology in Abcb4-/- mice, and
cholangiocytes are target of IGF1 signaling (12), the increased pathology could be due to an
enhanced cholangiocyte proliferation in response to the IGF1 overexpression. Therefore, the level
of, the cholangiocyte marker Ck19 was determined in these livers. A significant increase in mRNA
expression level and an increased presence of Ck19-expressing cells detected by
immunohistochemistry confirmed an enhanced presence of cholangiocytes in the transgenic animals
(Fig.5C and Fig.4C).
Subsequently, the expression of the proliferation marker Ki67 was determined to investigate if this
effect was caused by a higher proliferation. Although a higher Ki67 level was observed, the
difference did not reach significance (Fig.5B). This lack of significance could be due to the fact that
the proliferation was not limited to cholangiocytes, hence the proliferation of biliary epithelium was
determinated by fluorescent cell labelling and co-localisation for Ck19 and Ki67. This indicated an
ongoing proliferation of Ck19 positive cells in the transgenic animals (Fig.5A). Quantification of
Ki67-positive cholangiocytes with respect to the total number of cholangiocytes demonstrated a
significant increase in bile duct proliferation in IGF1 overexpressing animals after 12 weeks of
cholate diet (Fig.5D). This enhanced proliferation of biliary epithelium could have resulted in the
formation of numerous large bile duct structures seen in the IGF1 overexpressing mice (Fig.2A).
Table 2: Plasma liver function parameters, hepatic mRNA expression levels, and percentage of BrdU-positive cholangiocyte in WT and SMP8-IGF1 mice in response to BDL
liver function tests WT ctrl WT BDL SMP8-IGF1 ctrl SMP8-IGF1 BDL
AST 133.30 ± 59.90 730.70 ± 186* 113.30 ± 47.72 507.20 ± 158.70*
ALT 77 ± 48.08 777 ± 262*# 73.67 ± 38.84 386.20 ± 105.40*
ALP 83 ±10.17 1543 ± 332.90* 112.30 ± 14.47 1333 ± 523.60*
bilirubin 0.08 ± 0.01 10.07 ± 0.65* 0.10 ± 0.02 11.23 ± 2.09*
mRNA expression
transgen Igf1 no expression no expression 2.28 ±0.63 5.03 ± 2.13*
αSma 1.56 ± 0.69 23.55 ± 7.69*# 2.53 ± 0.33 11.65 ± 5.44*
Ck19 1.22 ± 0.54 7.04 ± 3.09* 1.47 ± 0.83 10.63 ± 4.70*
Tnfα 6.83 ± 3.63 214.35 ± 88.56* 9.22 ± 7.08 115.68 ± 56.46*
Col1 0.73 ± 0.82 1.18 ± 0.79 0.31 ± 0.19 0.78 ± 0.17*
Tgfβ 1.34 ± 0.89 1.73 ± 1.07 1.35 ± 0.01 1.34 ± 0.61
Asbt 19.16 ± 13.64 25.01 ± 15.68 20.89 ±4.72 49.07 ± 20.57*
BrdU+ cholangiocytes (%) 11.69 ± 4.30 12.70 ± 5.89
The table shows mean values and standard deviations of liver function parameters and hepatic mRNA expression levels (relative to housekeeping 36B4) in WT and SMP8-IGF1 mice and one week after BDL treatment and in sham-operated controls (n=5 per group). Percentage of BrdU-positive cholangiocytes was assessed in WT and SMP8-IGF1 mice one week after BDL. Asterisks denote significant difference between sham-operated animals and BDL counterparts; hashtags indicate significant difference between BDL groups (P<0.05).
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Fig.5: IGF1 overexpression in Abcb4-/- mice enhances proliferation of cholangiocytes: (A) Representative pictures
of double staining for Ck19, Ki67 in Abcb4-/- (left panels) and Abcb4-/-IGF1 (right panels) after 12 weeks of cholate diet.
(B-C) Relative gene expression of Ki67 and Ck19 in control, Abcb4-/- and Abcb4-/-IGF1 mice. (D) Percent of Ki67-
positive cholangiocytes with respect to the total number of cholangiocytes, as detected by immunohistochemistry.
The number of a-Sma expressing activated HSC increased, and was accompanied by an increased scar
formation reflected by higher Col1 levels, by increased cholangiocyte proliferation demonstrated by
higher Ck19 levels, and more inflammation based on Tnfα expression.
Overexpression of IGF1 in BDL model
Though Abcb4-/- is a relevant model for chronic cholangiopathy, it does not reproduce all features of
acute models of cholestasis, e.g. CCl4- and BDL-induced. To compare the effect of IGF1 in Abcb4-/-
mice with that in an acute model, we used BDL to induced acute cholestasis in SMP8-IGF1 mice.
Sirius red staining of livers in BDL-treated SMP8-IGF1 and WT animals demonstrated fibrosis in
peribiliary areas (data not shown). Upon BDL, the expression of IGF1 was significantly higher in
transgenic SMP8-IGF1 mice in comparison to sham-operated animals indicating transgenic
upregulation by cholestatic injury. Changes in gene expression profiles in BDL treated animals and
IGF1 in Abcb4-/-
75
liver function test (ALT, AST, ALP and bilirubin) confirmed the development of liver fibrosis in
both SMP8-IGF1 and WT mice (Table 2).
The gene expression data one week after BDL did not reveal an increased development of
fibrosis in SMP8-IGF1 mice compared to WT mice. In fact, the only significant difference was a
decreased expression of α-Sma in transgenic compared to WT (Table 2), while that of Tnfα, Tgfβ, and
Col1 did not differ between the models. In contrast to the Abcb4-/- model, IGF1 overexpression did
not cause an increased Ck19 level in BDL-treated mice (Table 2). This absence of an effect on
proliferation was confirmed by the similar percentage of BrdU-positive cholangiocytes (Table 2). In
conclusion, in contrast to the chronic model for bile duct damage, increased IGF expression did not
affect pathology in the acute BDL model, but did not display a protective effect either.
DISCUSSION
Prolonged overexpression of IGF1 increased fibrosis in the Abcb4-/- mouse, a model for primary
sclerosing cholangitis. The enhanced IGF1 signaling resulted in stimulated cholangiocyte
proliferation, one of the main pathological features of the liver damage in these mice.
The expression of transgenic Igf1, controlled by the α-Sma promoter, was stable during the
entire experiment. It was previously reported that in Abcb4-/- mice expression of α-Sma (also of pro-
fibrotic Tgfβ and Pdgfr) reaches maximum at 4 weeks of age and then gradually decreases (34). This
was due to the generation of fibrous septa that shield the healthy liver from the toxic bile, thereby
limiting the increase in pro-fibrotic factors. In our study, the expression of pro-fibrotic factors in the
Abcb4-/- mice remained high, resulting in a hydroxyproline level about 2-fold higher than previously
reported. The difference between the studies is most likely due to the cholate diet, which enhances
the pathology in this model, resulting in prolonged high expression of pro-fibrotic factors (15).
IGF1 induces the expression of hepato-protective HGF in HSC (28). Confirming our
previous observation (27), Hgf mRNA levels also increased in the IGF1 overexpressing Abcb4-/- mice
compared to the non-transgenic litter-mates. The extent of damage in this model is much less severe
than in the previously used acute CCl4 model (100-fold lower levels of liver enzymes in serum; (9,
10)). The increase of AST and ALT in serum of the Abcb4-/- mice was comparable to the data
reported before for this model (15), and indicated an ongoing liver damage. The elevation of serum
ALP and bilirubin after 12 weeks of cholate diet points to an increase in progression of bile duct
damage in mice that overexpress IGF1. This worsening of pathology was subsequently confirmed by
the increased level of hydroxyproline and collagen, indicating an enhanced deposition of ECM. Thus,
in contrast to our previous study in animals with CCl4- induced parenchymal damage, in the present
work IGF1 overexpression enhanced the damage and increased portal fibrosis in the cholangiopathic
livers of Abcb4-/- mice.
Fibrogenesis depends on tissue remodeling, composed of strictly controlled processes. A
subtle balance of MMPs (mainly involved in the ECM degradation) and TIMPs (their endogenous
inhibitors) modulates liver fibrogenesis (35). MMPs are regulated by uPA/tPA/plasmin proteases,
which directly contribute to degradation of collagens, and so play a pivotal role in the maintenance of
ECM and tissue homeostasis (36-38). IGF1 overexpression increased the expression of Timp1, which
could reduce MMP activity and ECM degradation in Abcb4-/-IGF1 animals. Moreover, lower tPA
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expression points to a less active tissue remodeling resulting in a more prominent fibrosis in IGF1
overexpressing mice, which may further promote liver fibrosis.
Several studies have shown that bile-duct epithelium cells, in contrat to hepatocytes, express
the IGF1R and directly respond to IGF1. IGF1 enhances proliferation and survival of
cholangiocytes via IRS1/2 and ERK and PI3-kinase pathways (12). The proliferative effect of IGF1
has also been reported for smooth muscle cells in IGF1-overexpressing mice (17). Activation of the
IGF1 system improves the survival of cholangiocytes in PBC patients suffering from chronic bile
duct damage (39). In the acute CCl4 model, IGF1 did not reduce the initial damage, but caused a
more rapid DNA synthesis, resulting in liver regeneration (27). Depletion of IGF1R reduces ductular
and fibrogenic responses and increased cholestasis tolerance in BDL mice (40). The damage in the
Abcb4-/- mouse is chronic, less severe, and localized to the portal areas (16). This results in a
persistent increase in IGF1 signaling in the portal area, enhancing cholangiocyte proliferation (12). In
addition, IGF1 enhances the proliferation of liver cystic epithelium and reduction of its expression
reduces the size of liver cysts in a mouse model (41). A similar action may explain the formation of
extremely large, but ill-developed bile-duct structures observed in those mice, leading to the
formation of strictures. Presence of strictures may explain the cholestasis that, according to serum
bilirubin and ALP level, developed eventually in our transgenic mice. Alternatively, the increased
proliferation of bile ducts could promote the cholehepatic shunt (42), thereby increasing the amount
of bile salts reaching the hepatocytes.
Submitting SMP8-IGF1 and WT mice to BDL revealed an absence of protective effect of
overexpression of IGF1 also in this model. However, no difference in bile duct proliferation was
seen between the WT and the SMP8-IGF1 mice. BDL did induce a-Sma mRNA expression in both
models, suggesting that one week was enough to enhance IGF1 signaling. Nevertheless, 1 week BDL
protocol seemed insufficient to induce the extent of liver damage needed to reveal an effect of
enhanced IGF1 signaling.
Bile duct proliferation increased in IGF1 overexpressing Abcb4-/- and showed a tendency to
increase in the SMP8-IGF1 mice submitted to BDL based on Ck-19 expression and BrdU staining.
Increased Asbt expression in SMP8-IGF1 BDL mice suggests an increased cholehepatic shunt of bile
salts, probably in an attempt to reduce the presence of damaging bile salts in biliary ducts. Likewise,
Abcb4-/- mice showed an increased expression of Asbt (43). The augmented expression of IGF1 in
Abcb4-/-IGF1 mice could result in an even higher increase of Asbt that would enhance the excessive
return of bile salts to the hepatocyte. Thus although these responses are aimed at the protection and
repair of the bile duct epithelium, the additional increase of IGF1 to supra-normal levels induced
extensive proliferation resulting in more bile duct abnormalities.
A difference in pathogenesis between the models may explain the discrepancy of the effect of
IGF1 seen between Abcb4-/- mice and other mouse models of liver fibrosis (e.g. BDL- and CCl4-
induced). Ligation of the common bile duct results in an acute interstitial (biliary) fibrosis, associated
with massive proliferation of bile ducts that is only rarely observed in man. On the other hand,
progression of fibrosis in Abcb4-/- mice is spontaneous due to cholangiocyte proliferation and
massive up-regulation of profibrogenic genes, and bears more resemblance to human biliary fibrosis
(34).
IGF1 in Abcb4-/-
77
Our data demonstrate that rise in IGF1 expression in Abcb4-/- mouse, model for primary
sclerosing cholangitis, worsens the pathology by increasing cholangiocyte proliferation leading to the
formation of bile duct strictures, comparable to those in patients (44). This indicates that, in contrast
to chronic parenchymal live damage, IGF1 administration does not seem an option for treating
fibrosis caused by chronic cholangiopathies, and should be taken into account in the design of IGF1
replacement therapy for cirrhotic patients.
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33. Hsiao Y, Zou T, Ling CC, Hu H, Tao XM, Song HY. Disruption of tissue-type plasminogen activator gene in mice aggravated liver fibrosis. J Gastroenterol Hepatol 2008 Jul;23(7 Pt 2):e258-e264.
34. Popov Y, Patsenker E, Fickert P, Trauner M, Schuppan D. Mdr2 (Abcb4)-/- mice spontaneously develop severe biliary fibrosis via massive dysregulation of pro- and antifibrogenic genes. J Hepatol 2005 Dec;43(6):1045-1054.
35. Hemmann S, Graf J, Roderfeld M, Roeb E. Expression of MMPs and TIMPs in liver fibrosis - a systematic review with special emphasis on anti-fibrotic strategies. J Hepatol 2007 May;46(5):955-975.
36. Li YY, McTiernan CF, Feldman AM. Interplay of matrix metalloproteinases, tissue inhibitors of metalloproteinases and their regulators in cardiac matrix remodeling. Cardiovasc Res 2000 May;46(2):214-224.
37. Nagase H, Woessner JF, Jr. Matrix metalloproteinases. J Biol Chem 1999 Jul 30;274(31):21491-21494. 38. Visse R, Nagase H. Matrix metalloproteinases and tissue inhibitors of metalloproteinases: structure, function,
and biochemistry. Circ Res 2003 May 2;92(8):827-839. 39. Onori P, Alvaro D, Floreani AR, Mancino MG, Franchitto A, Guido M, et al. Activation of the IGF1 system
characterizes cholangiocyte survival during progression of primary biliary cirrhosis. J Histochem Cytochem 2007 Apr;55(4):327-334.
40. Cadoret A, Rey C, Wendum D, Elriz K, Tronche F, Holzenberger M, et al. IGF-1R contributes to stress-induced hepatocellular damage in experimental cholestasis. Am J Pathol 2009 Aug;175(2):627-635.
41. Spirli C, Okolicsanyi S, Fiorotto R, Fabris L, Cadamuro M, Lecchi S, et al. Mammalian target of rapamycin regulates vascular endothelial growth factor-dependent liver cyst growth in polycystin-2-defective mice. Hepatology 2010 May;51(5):1778-1788.
42. Hulzebos CV, Voshol PJ, Wolters H, Kruit JK, Ottenhof R, Groen AK, et al. Bile duct proliferation associated with bile salt-induced hypercholeresis in Mdr2 P-glycoprotein-deficient mice. Liver Int 2005 Jun;25(3):604-612.
43. Hulzebos CV, Voshol PJ, Wolters H, Kruit JK, Ottenhof R, Groen AK, et al. Bile duct proliferation associated with bile salt-induced hypercholeresis in Mdr2 P-glycoprotein-deficient mice. Liver Int 2005 Jun;25(3):604-612.
44. Trauner M, Fickert P, Wagner M. MDR3 (ABCB4) defects: a paradigm for the genetics of adult cholestatic syndromes. Semin Liver Dis 2007 Feb;27(1):77-98.
IGF1 in Abcb4-/-
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c h a p t e r 5
fasting reduces liver fibrosis
in mouse model
for chronic cholangyopathies
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ABSTRACT
Background: Chronic cholangiopathies often lead to fibrosis, as a result of a perpetuated wound
healing response, characterized by increased inflammation and excessive deposition of proteins of
the extracellular matrix. Our previous studies have shown that food deprivation suppresses the
immune response, which led us to postulate its beneficial effects on pathology when liver fibrosis
driven by portal inflammation.
Methods: We investigated the consequences of fasting on liver fibrosis in Abcb4-/- mice that
spontaneously develop it due to a lack of phospholipids in bile. The effect of up to 48 hours of food
deprivation was studied by gene expression profiling, (immuno)histochemistry, and biochemical
assessments of biliary output, and hepatic and plasma lipid composition.
Results: Bile composition in Abcb4-/- mice remained unchanged with fasting, in contrast to
increased biliary output in the wild type counterparts. Markers of inflammation and cell proliferation
dramatically decreased in livers of Abcb4-/- mice already after 12 hours of fasting. Reduced presence
of activated hepatic stellate cells and actively increased tissue remodeling further propelled a decrease
in parenchymal fibrosis in fasting.
Conclusion:This study is the first to show that food deprivation positively influences liver pathology
in a fibrotic mouse model, opening a door for new strategies to improve liver regeneration in chronic
disease.
under revision
Authors
Aleksandar Sokolović1, Cindy P.A.A. van Roomen2, Roelof Ottenhoff2, Saskia Scheij2, Johan K.
Hiralall1, Nike Claessen3, Jan Aten3, Ronald P.J. Oude Elferink1, Albert K. Groen4 and Milka
Sokolović2
Affiliations
1Tytgat Institute for Liver and Intestinal Research, 2Department of Medical Biochemistry, 3Pathology
Department, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands;
4Department of Paediatrics, Center for Liver, Digestive and Metabolic Diseases, University Medical
Centre, Groningen, The Netherlands
Acknowledgements
The authors would like to thank dr. D.R. de Waart for hydroxyproline measurement.
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INTRODUCTION
Chronic cholangiopathies, like primary sclerosing cholangitis (PSC), are caused by progressive
inflammation and scarring of the bile ducts in the liver (1). The inflammation impedes the flow of
bile to the gut, which can lead to liver fibrosis (and cirrhosis as its ultimate stage), the final common
phase of all chronic liver diseases. In general, liver injury is followed by a wound healing response,
characterized by compensatory proliferation, inflammation and deposition of extracellular matrix
(ECM) (2). Initially, fibrosis is reversible - normal architecture is restored by fibrolysis, and ECM
producing cells are removed by apoptosis (3, 4). Recurrent chronic injury, however, results in an
imbalance between fibrogenesis and fibrolysis, leading to scar formation, architectural distortion,
cirrhosis and liver failure (5, 6). Liver transplantation is the only effective treatment, but surgical
contraindications and the lack of donors urge for interventions that halt disease progression.
Starvation provokes a massive response of the body and an interorgan integration of the
activities to prevent an irreversible loss of resources (7). When the reserves are low, there is necessity
for a trade-off between the risk of starvation and disease (8, 9). It is not surprising therefore that the
suppression of the immune response is also one of the major transcriptomics adaptations to food
deprivation (10, 11). Given inflammation as a driving force in the pathogenesis of biliary fibrosis
(12), we postulated that starvation-induced immune suppression could alleviate the pathology in
sclerosing cholangitis. To assess the effect of fasting on liver fibrosis, we used the Abcb4-/- mice in
which the lack of ABCB4 (ATP-binding cassette, subfamily b, member 4) leads to an absence of
phosphatidylcholine in the bile, making it toxic. (13). Bile salts accumulate in the intrahepatic biliary
system disrupting tight junctions and basement membranes of bile ducts, and lead to non-
suppurative inflammatory cholangitis, periductal fibrosis shortly after birth, and ductular proliferation
(14). Results of our study of fibrotic Abcb4-/- mouse livers, indeed, confirmed the hypothesis that
food deprivation influenced pathology, and revealed an ameliorating effect on fibrosis of as short as
12 hours of fasting.
MATERIALS AND METHODS
Animals
We used 3 months old male Abcb4-/- (FVB background), and WT FVB mice as a control (Charles
River, Maastricht, The Netherlands). The animals were kept separately in regular cages, and in grid
bottom cages 24 h prior to and during fasting, to prevent coprophagia and intake of bedding. The
study was approved by the AMC Animal Experiments Committee.
Plasma and tissue collection and analytical procedures
The gallbladders in anesthetized mice (Hypnorm and Diazepam, 1 ml/kg and 10 mg/kg respectively)
were cannulated; bile was collected for 15 minutes and stored at –20°C. Blood sample was collected
by cardiac puncture and plasma was stored at -20°C. The liver was quickly excised and parts were
snap-frozen in liquid nitrogen and stored at -80°C, or fixed in 4% buffered formalin and embedded
in paraffin. Total liver collagen was determined by measuring hydroxyproline content (15). Plasma
concentrations of total cholesterol, triglycerides, FFA and choline-containing PL were determined
using the equivalent colourimetric enzymatic kits (Wako Chemicals GmbH, Neuss, Germany and
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bioMérieux Benelux, Boxtel, The Netherlands). Biliary BS and PL content were analyzed as
described (16). Biliary cholesterol was measured fluorescently (17). Tissue lipids were extracted using
a chloroform–methanol-based (2:1 by volume) method (18); cholesterol and triglycerides were
measured using the CHOD-PAP (Ecoline 25 cholesterol, Merck, Darmstadt, Germany) and GPO-
PAP method (Ecoline S+, DiaSys Diagnostic Systems, Holzheim, Germany), and PL was measured
with an enzymatic colourimetric assay (Biolabo, Maizy, France). To correct the obtained lipid values
for the amount of tissue, the protein content of the liver was measured using the BCA method
(Pierce, Perbio Science Nederland BV, Etten-Leur, The Netherlands).
Histology and immunohistochemistry
Four animals per time point and five sections per animal were analyzed. Paraffin sections were
dewaxed and stained with hematoxylin and eosin (HE) for general histology, or with 0.2% picro-
sirius red (PSR) to detect fibrillar collagen. Immunohistology was performed as described (19).
Sections were incubated with rat IgG2b anti-mouse F4/80 monoclonal antibody (AbD Serotec,
Oxford, UK) and Ki67 (Dako, Glostrup, Denmark). Primary antibodies were detected using
appropriate secondary horseradish peroxidase-conjugated polyclonal goat IgG antibodies, directed
against mouse IgG2a, or rat IgG2a (Southern Biotech, Birmingham, AL). Bound peroxidase activity
was visualized using H2O2 and 3,3’-diaminobenzidine as chromogen. Sections were counterstained
with hematoxylin.
Reverse transcription, quantitative PCR and data analyses
Total RNA was extracted from frozen livers with TRIzol reagent (Invitrogen, Breda, The
Netherlands), and its quality assessed with RNA 6000 Nano LabChip® Kit in an Agilent 2100
bioanalyzer (Agilent Technologies, Palo Alto, USA). qPCR was performed as described (20). The
average of three house-keeping genes (36b4, Hprt and cyclophilin b) was used to normalize the
expression of the studied genes. mRNA concentration was calculated using the LinRegPCR program
(21, 22).
Western blot analysis
was performed as described (20), using antibodies against MMP2 and MMP9 (goat anti-mouse; Bio-
Rad Laboratories, Veenendaal, The Netherlands), with goat anti-rabbit IgG horseradish peroxidase
conjugated (Santa Cruz Biotechnology) as secondary antibody. Chemiluminescence was quantified by
Lumi-Imager F1 using CDP-Star (Roche, Mannheim, Germany), using β-actine as a reference.
Statistical analysis
ANOVA and two-tailed Student’s t-test were used to determine statistical significance. P<0.05 is
denoted by asterisks in all figures, with error bars representing the standard deviation.
RESULTS
Fasting reduces ECM deposition in the livers of Abcb4-/- mice
To investigate the effects of fasting on the fibrotic livers in Abcb4-/- mice, we assessed the
connective tissue by picro-Sirius red staining (Fig.1A). Parenchymal fibrosis was clearly reduced
already after 12h of fasting, causing a decrease of inter-portal bridging.
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85
Figure 1: Food deprivation ameliorates parenchymal fibrosis in the livers of Abcb4-/- mice. A) Representative Picro-sirius red immunostaining of liver sections of wild-type (WT) and Abcb4-/- mice, fed or starved for 12, 24 and 48h. Scale bar represents 100 µm. B) Hydroxyproline concentration (expressed in μg/mg wet tissue) in Abcb4-/- and WT livers, 0, 12, 24h and 48h after starvation started. C-I) Relative mRNA expression level of collagens 1, 3, 4 and fibronectin 1 (components of ECM), αSma and vimentin (markers of activated HSC), and Gfap (marker of quiescent HSC), in livers at 0, 12, 24 and 48h of fasting. mRNa expression is normalized by 3 house-keeping genes and shown as fold change compared with WT control at 0h. Asterisks denote significance compared to fed condition in the corresponding strain (P<0.05), and error bars represent SD.
Total liver collagen, responsible for the structural integrity of the ECM and used as a marker for liver fibrogenesis (23), was determined by measuring hydroxyproline content. In Abcb4-/- mice it decreased 40, 70 and 80% at 12, 24 and 48h of fasting, respectively, and was not affected in WT animals (Fig.1B).mRNA expression of collagens 1 and 3 was lower in fasting in both Abcb4-/- and WT mice (Fig.1C-D). Expression of Col4 and fibronectin decreased at 12 and 24h (Fig.1E-F). These data strongly suggest a decrease in matrix deposition in response to fasting in Abcb4-/- mice.
Hepatic stellate cells (HSC) play a central role in ECM remodeling by production of MMPs
and TIMPs (24, 25), and by deposition of fibrillar collagens type 1 and 3 (25). The impact of fasting
was analyzed by measuring the markers for activated (α-Sma and vimentin) and quiescent HSC (Gfap).
While quiescent were more abundant in the WT (Fig.1I), activated HSC were dominating in fibrotic
Abcb4-/- livers (Fig.1G-H). A significant decrease in expression levels was observed for all three
markers in Abcb4-/- mice, regardless of the duration of fasting (Fig.1F-I). In WT mice, these markers
were downregulated after 12 and 24h of fasting. In support, the expression of fibronectin, which
decreases starvation-induced apoptosis in rat HSC (26), was downregulated.
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Clearly different response at 48h compared to previous time points does not come as a
surprise (27), since such a long stretch in mice (comparable to several weeks in humans) provokes a
complex survival response. Although we mainly focus on the effect of short and moderate fasting,
we provide the later time point to complete the picture of the response in fibrotic livers.
Figure 2: Starvation causes an
active matrix remodeling in
Abcb4-/- livers. A-D) Gene
expressions of Plat, Plau, Timp1 and
Tgfβ1 at 0, 12, 24h and 48h of fasting
in Abcb4-/- and WT mice. mRNa
expression is normalized by 3 house-
keeping genes and shown as fold
change compared with WT control at
0h. Asterisks denote significance
compared to fed condition in the
corresponding strain (P<0.05), and
error bars represent SD.
Fibrotic Abcb4-/- livers respond to fasting by matrix remodeling
To assess the mechanism behind the reduced amount of ECM, we studied the components of the
matrix remodeling machinery.
The expression levels of Plau and Plat, which activate MMPs (28), increased in prolonged
fasting in Abcb4-/- mice (80% Plat and 30% Plau; Fig.2A-B), after an initial decrease in short fasting.
In WT animals, food deprivation did not affect their gene expression. Timp1, which inhibits most
MMP activity (29) and promotes the survival of HSC (30), was significantly downregulated by fasting
in fibrotic livers at all time points (Fig.2C). Tgfβ1, which promotes hepatic fibrosis by prompting
HSC differentiation into myofibroblasts, was 2-fold higher in fed fibrotic than in WT livers (Fig.2D).
Compared to fed Abcb4-/-, Tgfβ1 expression was 75, 70 and 50% lower at three fasting time points,
respectively. Matrix remodeling in fasting, therefore, seems to be regulated by reduced expression of
profibrotic cytokine Tgfβ1 and thereby reduced number of activated HSC/MFs.
Hepatic cell turnover is strongly reduced by fasting
Abcb4-/- mice have an intrinsically high hepatocyte proliferation (31). We therefore analyzed the
effect of fasting on proliferation markers Pcna and Ki67. They decreased in both Abcb4-/- and WT
mice (Fig.3B-C; Pcna returned to control values in prolonged fasting). Immunostaining (Fig.3A)
confirmed that the fibrotic livers of Abcb4-/- mice contained cycling hepatocytes and Ki67-positive
cells in the portal tract. Ki67-positive cells were not found in fasted Abcb4-/- livers at any time point,
or in any WT livers.
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Figure 3: Fasting reduces originally high cell turnover in fibrotic livers of Abcb4-/- mice. A) Immunostaining for
Ki67 in the liver sections of fed and 12h fasted WT and Abcb4-/- mice. Scale bar represents 100 µm. B-D) Gene
expression of Ki67, Pcna and p21 upon 0, 12, 24 and 48h of food deprivation. mRNa expression is normalized by 3
house-keeping genes and shown as fold change compared with WT control at 0h. Asterisks denote significance
compared to fed condition in the corresponding strain (P<0.05), and error bars represent SD.
Fasting, therefore, was a strong enough stimulus to reduce proliferation in Abcb4-/- livers.
This was apparently supported by the increased cell-cycle arrest, since the expression of p21,
involved in repair of DNA damage, was strongly upregulated in Abcb4-/- mice (even 700% at 48h;
Fig.3D).
The frequency of apoptotic changes (Councilman bodies) did not change dramatically in
Abcb4-/- mice upon fasting, especially in acinar zones 2 (Fig.4A).
Similarly, mRNA concentrations of proapoptotic markers Bax and Apaf barely changed in Abcb4-/-
mice (Fig.4B-C), while in the WT they significantly decreased with fasting (40-60%). Expression of
antiapoptotic Bcl-xl was unaffected by fasting in fibrotic livers, though it has strongly increased in
prolonged fasting in WT mice (Fig.4D). This suggests a possible different mechanism behind
apoptosis between the highly proliferative livers of fed Abcb4-/-mice, and those affected by fasting
Biliary output and hepatic and plasma lipid composition in fasted Abcb4-/- mice
In contrast to the earlier reported increased biliary output in fasted WT mice (32), bile flow in
Abcb4-/- mice tended to decrease after 12h (40%, P<0.06; Table 1). As expected, PL were barely
measurable in Abcb4-/- animals. Cholesterol concentration was 2.7-6.5 fold lower during fasting
compared to WT, while none of the measured biliary secretion rates – BS, cholesterol or PL- was
affected by fasting, indicating that the Abcb4-/- mice had lost the adaptive biliary response to fasting
seen in WT animals.
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Figure 4: Apoptosis in fasted fibrotic livers. A) Hematoxylin-eosin staining of liver sections of fed and fasted WT and
Abcb4-/- mice. Scale bar represents 100 µm. B-D) Relative mRNA expression level of Bax, Apaf and Bcl-xl, after 0, 12,
24h and 48h of fasting. mRNa expression is normalized by 3 house-keeping genes and shown as fold change compared
with WT control at 0h. Asterisks denote significance compared to fed condition in the corresponding strain (P<0.05),
and error bars represent SD.
Cholesterol concentration in the liver of Abcb4-/- mice increased during fasting (Table 1),
hepatic triglyceride concentration was strongly increased, while that of PL did not change. Hepatic
TG and cholesterol concentrations were somewhat lower in fed Abcb4-/- mice than in their WT
counterparts, but fasting altered them so that both lipids were present in higher concentration in
Abcb4-/- mice.
To assess the lipid status in the systemic circulation of fasted Abcb4-/- mice, we measured the
plasma concentrations of cholesterol, TG, FFA and PL (Table 1). Similarly to WT, plasma
cholesterol concentration increased after 12 and 24h of fasting, TG concentration strongly decreased
in longer fasting, PL concentrations remained stable, while FFA concentration increased (50 and
25% after 12 and 24h). Initially, plasma cholesterol level in fed Abcb4-/- mice was two-fold lower
than in the WT, and remained such also in fasting.
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89
Table 1: Biliary output and hepatic and plasma lipid composition in up to 48 hours fasted Abcb4-/- and wild type mice. The values are given as mean ± S.D. Asterisks denote significance (P<0.05)
Fasting reduces hepatic inflammation in Abcb4-/- mice
Bile duct proliferation and portal inflammation (13) are the most prominent histopathological
features of Abcb4-/- mice and are caused by secretion of toxic bile (33, 34). To assess the effect of
fasting on portal inflammation, we analyzed the presence of inflammatory cells in livers of Abcb4-/-
and WT mice.
Markers for activated monocytes and macrophages (Cd11b; Cd11c), neutrophils (Mpo), circulating monocites (Mcp1), and liver-resident Kupffer cells (F4/80), as well as inflammatory marker Tnfα, were clearly (and expectedly) higher in fibrotic than in control livers (Fig.5A-E; Mpo and Mcp1 not shown). Inflammation seemed attenuated in fasted fibrotic livers by significantly lowered expression of general markers F4/80, Cd11b and Cd11c. In fibrotic livers of fed Abcb4-/- mice, immunostaining for F4/80 showed areas with abundant periportal inflammation, with large numbers of F4/80 positive macrophages and relatively few F4/80 positive Kupffer cells in the adjacent lobules (Fig.5A, top right panel). More Kupffer cells were present in the areas where periportal inflammation was less abundant. A striking shift in localization of F4/80 macrophages was observed at 12 and 24h of fasting (Fig.5A, two mid-right panels). The periportal tracts contained far less F4/80 macrophages, whereas their number was increased in acinar zone 2. Upon 24 and 48h of fasting the number of macrophages was further reduced, with adherent F4/80 macrophages now often present in central veins (Fig.5A, two bottom-right panels). In WT mice, the relative number of F4/80 positive Kupffer cells decreased only after 48h of starvation (Fig.5A, left panels). A decrease in expression of proinflammatory marker Irf5 that promotes inflammatory macrophage polarization
Abcb4-/- WT
0 h 12 h 24 h 48 h 0 h 12 h 24 h 48 h
biliary lipids
bile flow (µl/min.100g)
9.5 ± 1.64 5.6 ± 0.290.06 6.6 ± 1.00 7.4 ± 1.14 3.7 ± 0.9 6.5 ± 0.4* 6.2 ± 0.8* 5.1 ± 0.6
bile salts (nmol/min.100g)
272 ± 87 197 ± 30 260 ± 50 398 ± 85 168 ± 36 267 ± 27* 228 ± 7 367 ± 107*
cholesterol
(nmol/min.100g) 0.5 ± 0.16 0.3 ± 0.04 0.5 ± 0.15 0.8 ± 0.14 1.3 ± 0.2 2.2 ± 0.2* 2.1 ± 0.1 3.0 ± 0.5*
phospholipids (nmol/min.100g)
0.16 ± 0.12 0.01 ± 0.01 0.08 ± 0.04 0.22 ± 0.04 3.0 ± 0.7 8.0 ± 1.3* 6.5 ± 0.4* 14.4 ± 2.6*
hepatic lipids
triglycerides
(nmol/mg) 5.7 ± 0.8 15.2 ± 0.8* 17.6 ± 1.2* 12.8 ± 2.4* 7.9 ± 1.1 11.7 ± 1.6* 13.5 ± 2.3* 12.2 ± 1.0*
cholesterol (nmol/mg)
8.1 ± 0.2 11.9 ± 0.5* 12.7 ± 1.0* 13.2 ± 0.8* 10.6 ± 0.3 10.5 ± 0.4 9.5 ± 0.4 17.4 ± 1.1*
phospholipids (nmol/mg)
19.2 ± 1.2 21.2 ± 0.8 23.0 ± 1.2 21.8 ± 1.2 20.6 ± 0.6 19.2 ± 0.6 21.5 ± 0.6 21.0 ± 0.5
plasma lipids
cholesterol
(mmol/L) 2.2 ± 0.03 2.5 ± 0.1* 2.5 ± 0.1* 2.2 ± 0.2 3.8 ± 0.2 4.3 ± 0.1* 4.5 ± 0.1* 4.9 ± 0.2*
triglicerides (mmol/L)
1.7 ± 0.1 1.8 ± 0.1 1.1 ± 0.07* 0.55 ± 0.04* 1.4 ± 0.2 0.9± 0.1* 0.6 ± 0.1* 0.8 ± 0.1*
free fatty acids (mmol/L)
0.53 ± 0.02 0.77 ± 0.05* 0.66 ± 0.04* 0.64 ± 0.11 1.3 ± 0.3 1.0 ± 0.1 1.0 ± 0.1 1.4 ± 0.3
phospholipids (mmol/L)
2.0 ± 0.03 2.1 ± 0.1 2.0 ± 0.1 1.8 ± 0.1 3.6 ± 0.2 3.5 ± 0.1 3.6 ± 0.1 3.8 ± 0.2
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(35), and an increase of Ym1, a marker for alternatively activated macrophages, indicate a shift towards alternatively activated macrophages in fasted fibrotic livers.
Figure 5: Fasting affects inflammatory status and macrophage localization in fibrotic livers. A) Representative Immunostainings for F4/80 in the livers of fed and fasted WT and Abcb4-/- mice. B-G) Relative mRNA level of F4/80, Tnfα, Cd11b, Cd11c, Irf5 and Ym1 upon 0, 12, 24 and 48h of fasting. mRNa expression is normalized by 3 house-keeping genes and shown as fold change compared with WT control at 0h. Asterisks denote significance compared to fed condition in the corresponding strain (P<0.05), and error bars represent SD.
DISCUSSION
This study reveals that fasting causes a reduction of liver fibrosis in a mouse model for biliary
cholangiopathies. Rapid adaptive response to food deprivation in Abcb4-/- mice (already after 12h)
lowered inflammation, decreased hepatocyte proliferation, lowered the number of activated
HSC/MFs, decreased production of ECM components, and increased expression of genes involved
in tissue remodeling, leading ultimately to amelioration of liver fibrosis.
Collagen, responsible for the structural integrity of the ECM, was strongly affected by
fasting. Within 48 hours, total collagen synthesis in livers of Abcb4-/- mice (measured by
hydroxyproline) decreased to 20%, similarly to the response found in the articular cartilage of fasted
guinea pigs (36). This corresponds with a notion that collagen production is sensitive to changes in
food intake, and that malnutrition may have profound effects on its production (37). Amount and
the types of produced collagen are regulated by the nutritional state of the animal or by the disease
processes - either directly as in wound healing, inflammation, and fibrosis, or indirectly, as in
starvation and diabetes. In advanced starvation, collagen is one of the two major sources of energy
fibrosis in fasting
91
(proteins) in the body (38). The regulation of collagen mass in fasted fibrotic livers occurred,
however, already in early stages of starvation, before the other energy sources were depleted. This
points to an active ECM remodeling, rather than to an ad hoc degradation for mere provision of
amino acids. The most prominent drop was noticed in expression of fibril-forming collagens 1 and 3
and vimentin, predominantly synthesized by activated HSC (39), suggesting that the process of liver
remodeling was specifically induced by food deprivation.
Chronic liver diseases are characterized by aberrant matrix deposition, calling for our
attention to the role of ECM in resolution of liver fibrosis. Tissue remodeling is regulated by MMPs,
involved in the ECM degradation, and TIMPs, their endogenous inhibitors. Their subtle balance
maintains liver fibrogenesis. Tissue homeostasis is further regulated by proteolytic activity of the
PLAU/PLAT/plasmin, responsible for the maintenance of the physiologic levels of ECM (40).
PLAU promotes ECM degradation through activation of MMPs (MMP-2, -3 and -9; (41, 42),
increases the differentiation of hepatic stem cells, and HGF-dependent regeneration of hepatocytes
(43). PLAT protects ECM proteins from proteolytic degradation and helps expedite wound healing
(44). In fed Abcb4-/- mice, hepatic Plau and Plat expression levels were much higher than in their WT
counterparts (3- and 8-fold, respectively). Short and moderate fasting initially reduced the expression
of both, but prolonged fasting posed a demand for higher mRNA concentrations, indicating an
ongoing balancing action between fibrolysis and fibrogenesis. Together with strongly (6-fold)
reduced Timp1 expression, our data indicate that fibrotic livers responded to food deprivation by an
active ECM remodeling. Matrix remodeling and in fasting was further supported by reduced
expression Mmp13. Expression of this collagenase mainly produced by Kupffer cells is high in
collagen 1a1 r/r mouse model that fails to recover from fibrotic liver injury, due to resistance to
MMP degradation of ECM (45). However, given the contradictory findings by other groups (46), the
role of Mmp13 in (resolution of) liver fibrosis remains controversial.
Apart from ECM synthesis and degradation, tissue remodeling comprises cell proliferation,
death, and migration. In general, hepatocyte proliferation is low under normal conditions, except to
compensate for a loss of cells (47) and it gets further reduced when the energy supply is low (48).
Caloric restriction induces apoptosis and decreases hepatocyte proliferation in a murine strain with a
high incidence of spontaneous liver tumors (49). We now show that, as a part of tissue remodeling,
fibrotic livers of Abcb4-/- mice respond to fasting mainly by a decrease in their pathologically high
proliferation. Furthermore, consistently with previously shown increased apoptosis in HSCs in
response to nutrient deprivation (26), this study shows a decrease in number of activated and
quiescent stellate cells, most likely in response to decreased production of fibronectin and TIMP1,
which both prevent HSCs apoptosis (26, 30).
The impressive decrease in inflammation in Abcb4-/- mice, the macrophage migration from the
periportal tract towards the central vein, and the shift from classically to alternatively activated
macrophages, seem a likely driving force for improved pathology (50). Overall, altered
concentrations of cytokines, metabolic and hormonal trophic factors induced by fasting could
decrease inflammation in fibrotic livers. Notably, profibrotic cytokine leptin that increases TIMP1
expression (51) is strongly reduced by fasting (52). Fasting increases circulating corticosterone (53,
54), decreasing thereby the production of cytokines and interleukins (55, 56), with an
immunosuppressive effects in humans (57, 58) and mice (59, 60). In Abcb4-/- mice, persistent hepatic
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inflammation triggers profibrotic signaling via activation of cytokines, promoting the formation of
MFs, which in turn synthetize elevated amounts of ECM proteins (61, 62). TGF-β1, the most potent
fibrogenic cytokine, prompts HSC differentiation into myofibroblasts, by enhancing expression of
TIMPs (that block ECM degradation), and by directly stimulating synthesis of interstitial fibrillar
collagens (12). Liver fibrosis in Abcb4-/- mice was attenuated in fasting, most likely driven by
downregulation of Tgfβ1, which reduced number of activated HSC. Downregulation of a number of
proinflammatory markers, a.o. Tnfα and Irf5 (a transcriptional activator of pro-inflammatory
cytokines and chemokines (35)), clearly points to fasting-induced suppression of immune response.
The change in number, but also localization of resident and infiltrated macrophages, may be causal in
resolution of the parenchymal fibrosis (e.g. by altered production of MMPs (46, 63), especially given
the pivotal but divergent roles of macrophages in matrix remodelling - favoring ECM accumulation
during ongoing injury, and enhancing matrix degradation during recovery (64). In the starved
animals, the high energy demand of the immune response (e.g. for production of acute phase
response proteins) leads to a massive change in hepatic transcriptome directed towards its
suppression (11, 50). The decrease in inflammation in Abcb4-/- mice, is therefore compatible with
our previous notion that the suppression of immune response (subserving energy preservation) is
one of the highlights of the overall body’s fasting response (50).
In conclusion, this study demonstrates that fasting leads to alleviation of biliary fibrosis by
decreased inflammation and by actively increased matrix remodeling. Fasting in the fibrotic Abcb4-/-
model may help improve understanding of the mechanisms of resolution, and inform strategies to
improve liver regeneration in chronic disease.
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c h a p t e r 6
closure:
summarizing discussion and conclusions
summaries
abbreviations
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SUMMARIZING DISCUSSION
SUBJECT AND AIM
Fibrosis is a wound healing response to a variety of acute and/or chronic stimuli (ethanol, viral
infection, drugs and toxins, cholestasis, and metabolic disease) (1, 2). It develops due to an increase
in fibrillar collagen synthesis and deposition, along with insufficient remodeling (3, 4). This process is
associated with a number of pathological and biochemical changes that lead to structural and
metabolic abnormalities, and increased hepatic scarring (5, 6). The progression of liver fibrosis leads
to cirrhosis, characterized by distortion of the normal architecture, formation of septae and nodule,
altered blood flow, portal hypertension, HCC, and ultimately liver failure (7). Understanding the
mechanisms and the pathways involved in the pathogenesis of the fibrogenic response and
identification of potentially new therapeutic agents could provide novel therapeutic approaches for
liver diseases in which fibrosis is a detrimental component.
The overarching aim of the studies described in this thesis, therefore, was to improve the
understanding of the mechanisms underlying liver fibrosis, and to identify ways to slowdown the
development and/or reduce liver fibrosis.
APPROACHES
To live up to the challenge, we used a battery of experimental models and approaches. In our in vitro
studies, we used LX2 cells, a model system for activated HSCs, and primary MFs. In vivo, we studied
Abcb4-/- mice, a model for chronic cholangiopathies that spontaneously develop liver fibrosis, as well
as Abcb4-/- SMP8-Igf1+/-, a transgenic model created in one of the studies. These various models
were challenged either by directly affecting the expression of genes of interest (IGFBP5, IGF1R, and
IGF1) to scrutinize the potential roles of these gene products in the development of liver fibrosis.
Protein expression levels were perturbed in vitro using adenoviral vector for overexpression, or
siRNA for depletion. In vivo, we either used an AAV vector to deliver IGFBP5 to the hepatocytes of
Abcb4-/- mice, or crossbred these mice with a transgenic strain to ensure IGF1-overexpression in
activated HSCs. To test the hypothesis that nutrient deprivation benefits the fibrotic pathology, we
studied the effects of fasting on existing liver fibrosis in Abcb4-/- mice. A broad range of molecular
biology and biochemical techniques were employed, coupled with transcriptomics and system
genetics approaches.
FINDINGS
Igf axis in vitro and in vivo
Our first effort to shed some light on the role of IGFBP5 in liver fibrosis has revealed its
involvement in the survival of HSCs. Earlier studies have indicated cell- and tissue-type specific
effects of IGFBP5. Its increased expression in fibrotic lung, skin and liver (8-11), in myogenesis, in
vitro breast cancer cell growth, and in gingival epithelial cells (12-15), seems to be coupled with
promotion of cell survival. Quite oppositely, in e.g. mammary gland involution IGFBP5 promotes
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apoptosis of epithelial cells in vivo and in vitro (16-19). In liver fibrosis, apoptosis is reduced in
activated HSCs that play a pivotal role in the initiation and perpetuation of pathology (20). Since
IGFBP5 is spontaneously induced in HSCs cells upon activation (8), we hypothesized that it played a
role in proliferation of HSCs in fibrotic liver. Chapter 2 presents a study of the effects of IGFBP5 in
vitro in primary human MFs and LX2 cells, a model for partially activated human HSCs (21-23). In
both cell lines an increased presence of IGFBP5, either by lentiviral overexpression or by addition of
recombinant protein, promoted survival by lowering apoptosis. In support, silencing of endogenous
IGFBP5 decreased the viability of LX2 cells, and of primary hepatic MFs, by an increase in
apoptosis, pointing at IGFBP5 as a pro-survival factor in these two pro-fibrotic cell types.
The same study has addressed another important question raised whenever an IGF-binding
factor is studied, namely what is the net effect of the interplay between IGF1 and IGFBP5? IGFBP5
binds IGF1 with high affinity and protects it from rapid degradation (24). Simultaneously, however,
it prevents activation of the IGF1R and thereby inhibits prosurvival signaling by IGF1. Our in vitro
studies showed that, in contrast to IGFBP5 that acted via lowering apoptosis, IGF1 increased
proliferation of LX2 cells. Moreover, we found no effect of IGF1 on IGFBP5 action, nor of
IGFBP5 on IGF1 signaling, suggesting that in LX2 cells these two factors exert their effect via
different, independent, routes. Several mechanisms may be involved in the IGF1 independent effect
of IGFBP5. It was first shown in vitro that IGFBP5 could increase activation of TGFβ1, a major pro-
fibrotic cytokine, by plasmin via its interaction with tissue plasminogen activator (25). Consistently,
mice overexpressing IGFBP5 had elevated tissue concentrations of plasmin (25). IGFBP5 directly
activates tPA, which in turn activates plasmin. This further leads to activation of several MMPs
involved in cellular migration/invasion, and in activation of TGFβ1 (25, 26). IGFBP-5 can also
interact with TNFR1 (in vivo and in vitro) and block cell proliferation via inactivation of the NFκB
signalling pathway (27). Furthermore, IGFBP5 interacts directly with several proteins from the
matricellular family of proteins (osteopontin, thrombospondin-1 and tenascin-C) (28, 29), which are
implicated in cellular adhesion, migration, wound healing or metastasis. In breast cancer cells
IGFBP5 induces integrin-mediated Cdc42-dependent cell survival pathway, which leads to an
adhesive, anti-migratory, phenotype (involves stimulation of integrin-linked kinase (ILK), Akt and
p53) and restricts epithelial–mesenchymal transgressions. As such, IGFBP5 might play role in
limiting metastasis (30). IGFBP5 provokes formation of a filamin A (FLNa)-based nuclear shuttle
that binds IGFBP5, recruits transcription factors, translocates to the nucleus and induces laminin
(major component of the epithelial basement membrane involved in cell migration) gene
transcription (31). In addition, IGFBP5 contains a nuclear localization sequence that mediates
nuclear translocation of IGFBP5 to influence transcription of genes involved in osteoblast cell
proliferation/differentiation (32). Evidence suggests that IGFBP5 binds and phosphorylates a
putative IGFBP5 receptor on the osteoblast cell surface and stimulates downstream signaling
pathways (33).
Much to our surprise, the following in vivo study (presented in Chapter 3) revealed an
antifibrotic effect of IGFBP5 in the livers of Abcb4-/- mice. Our initial finding that IGFBP5
expression strongly increased during development of liver fibrosis in Abcb4-/- mice (8), that IGFBP5
acts as a prosurvival signal in vitro in LX2 cells, and two recent studies that reported a high expression
of IGFBP5 in HCC and intrahepatic cholangiocarcinoma, suggested that it could have a profibrotic
role in vivo, and promote the survival of cancer cells and tumor formation (34, 35). The current study
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has, however, shown a clearly reduced liver fibrosis in IGFBP5-treated animals, with considerably
reduced portal bridging, and demonstrated the role of IGFBP5 in reduction of the existing scar
tissue. To clarify the different role of IGFBP5 in liver and other organs, we analyzed its steady-state
expression in the BxD genetic reference population (Chapter 3). The gene-phenotype correlations
indicated an organ-specific regulation of IGFBP5, with a role that differed between organs but
possibly protective in the liver. This could explain why IGFBP5 was found a profibrotic factor in
lung and skin (10, 36), as opposed to its protective effect in liver fibrosis. A protective effect of
IGFBP5 on hepatocytes in vivo (as shown by our research) is consistent with its prosurvival effect
seen in activated HSC in vitro (37). Hence, even within the liver the prosurvival signal in stellate cells
vs. hepatocytes may have counteractive effects. Apparently, in the in vivo setting of the Abcb4-/-
mouse the protective effect on hepatocytes prevails over the increased survival of stellate cells.
The most likely mechanism of the anti-fibrotic effect of IGFBP5 expression in the Abcb4-/-
mice is reduction of portal inflammation, which is initially caused by leakage of toxic bile, one of the
most prominent pathological features in this model for chronic cholangiopathy (38). Overexpression
of IGFBP5 lowers the influx of inflammatory cells into the area, lowering the production of
inflammatory cytokines, known to stimulate collagen synthesis and to enhance hepatocyte damage.
Fewer damaged hepatocytes and fewer activated inflammatory cells means less oxidative stress (39,
40) that enhances the expression of profibrotic genes in human HSCs and MFs (41, 42), which may
additionally alleviate liver fibrosis.
IGFBP5, however, also may have a more direct effect. The increased amount of p21 in
IGFBP5-overexpressing livers may be instrumental for the reduced proliferation. p21 serves as a
negative cell cycle regulator under stress conditions caused by various factors (growth factor
deficiency, DNA damage, and exposure to heavy metals or antiproliferative cytokines, particularly
TGFβ). Upon DNA damage, p21 blocks the transition from G1 into S phase or from G2 into
mitosis, enabling the repair of damaged DNA (43). Furthermore it regulates PCNA that binds to
damaged DNA (44) reducing its nuclear presence as seen in our study. In the cytoplasm, p21 protein
has an anti-apoptotic effect through binding to and inhibiting caspase 3, as well as the apoptotic
kinases ASK1 and JNK (45). p21 can act as a tumor suppressor, in particular by participating in the
launch of a senescence program (replicative senescence as well as stress-induced premature
senescence) (46) More and more evidence points to IGFBP5 as an important player in cellular
senescence, process that allows an escape from uncontrolled cell proliferation, thereby halting
tumorigenesis (47, 48). Regardless the cause of senescence, two tumor suppressor pathways, the p53-
p21 and p16-pRB, are typically responsible for the following growth arrest and senescence (48). Also
several cytokines, including IFNα, IFNγ, TGFβ, IGFBP3, IGFBP5 have been reported to regulate
senescence (49-52). Increased IGFBP5 expression is associated with senescence in vitro in pulmonary
fibrosis, human dermal fibroblasts and endothelial cells (53-57). Knockdown of IGFBP5 partially
reverses senescence in aged human umbilical-vein endothelial cells (HUVEC), whereas IGFBP5
induces and accelerates premature senescence in young HUVEC cells. IGFBP5-induced senescence
is associated with induction of the tumour suppressor p53 (51).
Following the path in the IGF-axis upstream of IGFBP5, in Chapter 4 we tested the
influence IGF1 on liver fibrosis in (a model for) chronic cholangiopaties. It has been shown by two
independent studies, that increased expression of IGF1significantly reduced liver fibrosis in CCl4
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treated rats (58, 59). In a follow-up phase I-II clinical trial, IGF1 administration improved liver
function in patients suffering from liver cirrhosis (60). The potential use of IGF1 in chronic
cholangiopathies, however, has not been studied. Since the biliary epithelium expresses IGF1R (61),
and IGF1 protects cholangiocytes against cholestatic injury in vitro (62), we postulated that it could be
beneficial in chronic cholangiopathy. To study it, we crossbred the Abcb4-/- mice with a transgenic
mouse that overexpresses IGF1 in activated HSCs. This cross showed increased expression of pro-
fibrotic factors (Tgfβ and Pdgf), an increased hydroxyproline level (2-fold higher than previously
reported (63)), as well as an enhanced deposition of ECM. IGF1 overexpression also increased the
expression of Timp1, which could reduce MMP activity and ECM degradation in our model.
These observations all point to a more prominent fibrosis in Abcb4-/- in the presence of
IGF1. The increased serum ALP and bilirubin levels after 12 weeks of cholate diet in the current
study indicate that IGF1 could increase proliferation and survival of cholangiocytes, and
independently cause cholestasis in hepatocytes. Though these responses are aimed at repair of the
bile duct epithelium, increase of IGF1 above the normal levels induces extensive proliferation
resulting in bile duct abnormalities. Although unexpected, the different effect of IGF1
overexpression between the previously studied CCl4 model (58, 59) and this model for chronic
cholangiopathy is most likely due to the difference in severity of the models. To clarify this, we have
performed bile duct ligation (BDL) in the SMP8-IGF1. The data revealed an absence of protective
effect of overexpression of IGF1 also in BDL model. In this model, however, no difference in bile
duct proliferation was seen between the WT and the SMP8-IGF1 mice.
A difference in pathogenesis between the models may explain the discrepancy between
Abcb4-/- mice and BDL-induced mouse models of liver fibrosis. Ligation of the common bile duct
results in an acute interstitial (biliary) fibrosis, associated with massive proliferation of bile ducts,
(only rarely observed in man). On the other hand, in Abcb4-/- mice progression of fibrosis is
spontaneous due to cholangiocyte proliferation and massive up-regulation of profibrogenic genes,
and bears more resemblance to human biliary fibrosis (63). The damage in the Abcb4-/- model is
chronic and less severe and localized to the portal areas (64), which results in a persistent increase in
IGF1 signaling enhancing cholangiocyte proliferation (61). In PBC patients suffering from chronic
bile duct damage, activation of the IGF1 system plays a role in the survival of cholangiocytes (65). In
addition, IGF1 enhances the proliferation of liver cystic epithelium and reduction of its expression
reduces the size of liver cysts in a mouse model (66). This may explain the formation of extremely
large, but ill-developed bile-duct structures observed in that model, leading to the formation of
strictures. Presence of strictures (though not specifically demonstrated) could explain the cholestasis
that, according to serum bilirubin and ALP level, has eventually developed in our transgenic mice.
Fasting in abcb4-/- mice
Driven by the data from human and rodent studies (67, 68), in which fibrotic profiles in obese
individuals were improved by weight loss, we postulated that the adaptation to nutrient deprivation
would also be beneficial for fibrosis caused by chronic cholangiopathy. The fasting study in Abcb4-/-
mice (Chapter 5) has indeed exposed a surprisingly quick improvement in pathology of fibrotic liver
- already after 12h of food deprivation. Starvation provokes a massive response of the body to
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prevent an irreversible loss of resources (69). In mice, within hours after the last meal, the organs
respond with changes in gene expression mainly in general metabolism (70). The role of the liver is
to provide energy for glucose-dependent tissues, by glycogenolysis, gluconeogenesis, ketogenesis,
and fatty-acid β-oxidation (71). The basic architecture of the lobules and the zonation are not
affected, but the cell size declines in prolonged fasting, when murine liver restores partly its glycogen
deposits, and much of gene expression returns to control values (72). In Abcb4-/- mice, collagens,
fibronectin and vimentin, responsible for the structural integrity of the ECM, were strongly affected
by fasting. The downregulation of collagen mass in fasted fibrotic livers occurred in early stages of
starvation, before the other energy sources were depleted, pointing to an active ECM remodeling,
rather than to an ad hoc degradation for mere provision of amino acids. As a part of tissue
remodeling, fibrotic livers of Abcb4-/- mice responded to caloric restriction by a decrease in their
pathologically high proliferation. Liver fibrosis was further attenuated by reduced number of
activated HSCs, most likely driven by downregulation of TGFβ1 (73). The impressive decrease in
inflammation in fasted Abcb4-/- mice, and migration of macrophages from the periportal tract
through the acinar zone 2 towards the central vein, seem a likely driving force for improved
pathology. This change in number and localization of resident and infiltrated macrophages may be
causal in resolution of the parenchymal fibrosis (by altered production of MMPs (74, 75)).
A caveat of the studies presented in this thesis could be seen in the fact that liver fibrosis was
addressed mainly in a single in vivo model (of chronic cholangiopathy). Clearly, the effects of IGFBP5
should be further tested in in additional animal models including a viral administration of IGFBP5 to
the newly established IGF1 overexpressing model of Abcb4-/-. In addition, apart from hepatocytes,
other cell types involved in fibrosis should be specifically targeted in Abcb4-/- mice, using different
specific AAV vectors (HSCs by AAV5. Similarly, the effect of fasting should be studied upon
subjecting animals to short(er) and/or alternate day fasting in more than one fibrotic model. The
mechanism behind the antifibrotic effects seen in this thesis should be further elucidated.
CONCLUSIONS
In a nutshell, the studies presented in this thesis have shown that progression of liver fibrosis in
Abcb4-/- mouse model of chronic cholangiopathies can be halted and even reversed by targeted gene
transfer of IGFBP5, or by starvation.
We have demonstrated different, cell-type specific effects of IGFBP5. Firstly by
overexpressing it in vitro, in activated HSCs and MFs, where it improved survival by reducing
apoptosis (in an IGF1-independent manner), suggesting its possible profibrotic role. However,
prolonged expression of IGFBP5 in vivo, in hepatocytes of Abcb4-/- mice, decreased their
proliferation, reduced inflammation and oxidative stress, and lowered number of activated HSCs and
ECM deposition, thereby evidently reducing liver fibrosis.
The star of the IGF-axis, IGF1, did not live up to the expectations in the Abcb4-/- model. In
a new transgenic mouse model created in our study, increased expression of IGF1 in Abcb4-/- mice
aggravated the pathology by increasing cholangiocyte proliferation, opposite to the anti-fibrotic
action of IGF1 in acute parenchymal liver damage caused by BDL or CCl4. In contrast to drug-
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103
induced liver fibrosis, IGF1 administration therefore does not seem an option for treating fibrosis
caused by chronic cholangiopathies.
Furthermore, fasting appeared a strong metabolic prosurvival stimulus that should not be
underestimated. We showed that as short as 12 hours of food deprivation positively influenced
pathology in fibrotic Abcb4-/- mouse liver, by decreasing inflammation, and by actively increasing
matrix remodeling.
These studies were the first to tackle the problem of liver fibrosis by specifically targeting
IGFBP5, and by introducing fasting challenge. Though we have not eliminated the cause of fibrosis
by any of the approaches (i.e. bile composition remained unchanged), we did alleviate its
consequences. This may have opened a door for new therapies of liver fibrosis.
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SUMMARY
As a normal wound-healing response to liver injury, fibrosis is reversible - normal architecture is
restored by fibrolysis, and ECM producing cells are removed by apoptosis. However, as reviewed in
Chapter 1, chronic liver injury increases production of fibrogenic signals by resident and infiltrating
liver cells, causing an imbalance between fibrogenesis and fibrolysis, scar formation, architectural
distortion, cirrhosis, and eventually liver failure. With liver transplantation as the only effective
treatment, the lack of donors and surgical contraindications impel for treatments to halt the
progression of disease.
HSCs, mesenchymal cells vital to hepatic function and its response to injury, play a pivotal
role in the development of liver fibrosis. IGFBP5 was shown to be highly expressed in fibrotic livers
of Abcb4-/- mice. Therefore, in Chapter 2, we examined the influence of IGFBP5 on HSCs and MFs
in vitro. Using gain- and loss-of-function approaches (overexpression by lentiviral transduction, or
silencing by siRNA), we showed that IGFBP5 influenced the survival of human LX2 cells, a model
for (partially) activated HSCs, and of hepatic MFs. Their endurance was improved without enhancing
proliferation, by lowering the level of apoptosis, via an IGF1-independent mechanism. Moreover,
IGFBP5 increased the expression of genes involved in ECM deposition.
The finding that IGFBP5 promotes survival of HSCs and MFs in vitro has led us to
investigate its role in vivo, in Abcb4−/−mice. These mice spontaneously progress to severe biliary
fibrosis, due to absence of biliary phospholipids that leads to primary sclerosing cholangitis. Abcb4-/-
mice are also a model for human MDR3 deficiency, ranging from progressive familial intrahepaic
cholestasis type 3 to adult liver cirrhosis, which makes them an attractive model for testing potential
antifibrotics. In Chapter 3 we demonstrate that prolonged liver-specific overexpression of IGFBP5
alleviated the hepatocyte damage, as demonstrated by improved biomarkers of liver injury, and
decreased their proliferation, possibly by arresting cell cycle, accompanied by senescence.
Furthermore, overexpression of IGFBP5 reduced inflammation, indicated by decreased presence of
markers for infiltrating and resident macrophages, neutrophils and monocytes. Consequential
lowered release of proinflammatory cytokines may explain the decreased oxidative stress in these
livers. The resulting reduced presence of activated HSC/MFs and reduced expression of collagens
led to a decreased amount of ECM, ameliorating pathology in the model for chronic cholangiopathy.
A recent study has indicated that biliary epithelium expresses IGF1R, and that IGF1 protects
cholangiocytes against cholestatic injury in vitro. To establish the effect of IGF1 on the existing
cholestatic injury in vivo, we subjected the Abcb4-/- mice to a prolonged increase in hepatic IGF1
expression, by creating a transgenic animal. Chapter 4 shows that sustained overexpression of IGF1
in fibrotic livers increased cholangiocyte proliferation, enhanced inflammation and reduced matrix
remodeling, bringing about an increase in deposition of scar tissue and progression of liver fibrosis.
IGF1 administration therefore does not seem an option for treating fibrosis caused by chronic
cholangiopathies.
The research presented in Chapters 2-4 has challenged the Abcb4-/- mice directly, by
affecting the expression of genes of interest (IGFBP5 and IGF1), to scrutinize their potential roles in
the development of liver fibrosis. Along the same line, we studied how nutrient deprivation could
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affect liver fibrosis in the same model. In Chapter 5 we show that food deprivation causes a rapid
adaptive response in Abcb4-/- mice, already after 12h. A striking decrease in inflammation in fasted
Abcb4-/- mice seems a likely driving force for a cascade of events, including decreased hepatocyte
proliferation, lowered number of activated HSCs/MFs, decreased production of ECM components,
and increased expression of genes involved in tissue remodeling.
The studies described in this thesis embarked upon the problem of biliary fibrosis by
delineating the roles of specific players in IGF-axis, and by introducing a fasting challenge. Though
we have not eliminated the cause of fibrosis by any of the approaches (i.e. bile composition remained
unchanged), we did alleviate the consequences, which leaves the door to new therapies for liver
fibrosis slightly ajar.
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NEDERLANDSE SAMENVATTING
Door schade aan de lever kan littekenvorming in de lever ontstaan, dit proces noemen we fibrose.
De strengen littekenweefsel in de lever bestaan voor een groot deel uit bindweefsel zoals collageen.
Lever fibrose kan beschouwd worden als een ontstekings of wondhelingsproces en hoeft daarom
niet noodzakelijkerwijs tot permanente leverschade te leiden. Collageen bevattende fibrotische
littekens kunnen ook weer opgeruimd worden.
Wanneer er sprake is van constante leverschade gaat er iets mis en wordt de fibrotische
littekenvorming versterkt. In Hoofdstuk 1 wordt beschreven dat door chronische leverschade de
afbraak van collageen niet goed meer verloopt. Hierdoor gaat het proces van littekenvorming verder
waardoor de leverstructuur verwoest wordt, met als gevolg cirrose en uiteindelijk een complete
teloorgang van de lever.
Het is al lang bekend dat stellaatcellen cruciaal zijn voor het proces van lever fibrose. Omdat
wij vonden dat in fibrotische muizenlevers het IGFBP5 eiwit sterker werd aangemaakt hebben we in
Hoofdstuk 2 onderzocht wat het effect van IGFBP5 op stellaatcellen is. We konden aantonen dat
stellaatcellen gestabiliseerd worden door IGFBP5 zonder dat ze sneller gingen delen. Belangrijk is
ook de vinding dat stellaat cellijnen door IGFBP5 meer collageen gaan produceren.
Deze experimenten zijn allemaal gedaan met stellaatcellen in celkweek, de volgende stap was
het onderzoeken van het effect van IGFBP5 in een diermodel van leverfibrose. In Hoofdstuk 3
laten we zien dat de schade aan de lever van muizen met fibrose verminderd kan worden door de
productie van IGFBP5 in de levers te verhogen. In tegenstelling tot onze vindingen in stellaatcellen
in celkweek in Hoofdstuk 2, blijkt in muizen het IGFBP5 eiwit juist minder ontsteking en
collageenvorming in fibrotische levers te veroorzaken.
In de wetenschappelijk literatuur wordt gesuggereerd dat het IGF1 eiwit een andere
belangrijke factor is die de lever tegen schade door fibrose zou kunnen beschermen. In Hoofdstuk 4
beschrijven we daarom het gevolg van het verhogen van de hoeveelheid IGF1 eiwit in de lever van
muizen met fibrose. Tot onze verassing bleek door IGF1 de fibrose juist erger te worden, er was
meer litteken- en collageenvorming in de lever. Het toedienen van IGF1 is dus geen oplossing voor
het probleem van leverfibrose.
In Hoofdstukken 2-4 hebben we leverfibrose in modelsystemen, cellen en muizen,
bestudeerd en proberen te behandelen door de eiwitten IGFBP5 en IGF1. In Hoofdstuk 5 hebben
we een andere weg behandeld door muizen met leverfibrose te laten vasten. We vonden hierbij dat
het verminderen van de voedselinname in muizen met leverfibrose een snelle vermindering van de
leverontsteking tot gevolg had. Al 12 uur na het begin van het dieet konden we aantonen dat er
minder collageen in de lever werd gevormd.
In dit proefschrift hebben we laten zien dat IGFBP5 leverfibrose vermindert en dat IGF1
leverfibrose juist verergert. We hebben ook gevonden dat vasten leverfibrose kan verminderen.
Alhoewel we met deze vindingen de oorzaak van leverfibrose niet aan hebben kunnen pakken, leven
onze resultaten toch goede aanknopingspunten op waarmee wellicht nieuwe geneesmiddelen voor
fibrose gevonden kunnen worden.
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REZIME
Fibroza kao normalan odgovor na povredu tkiva, je u osnovi reverzibilan proces. Normalna
arhitektura jetre se obnavlja razgradnjom fibroznog matriksa (ožiljačnog tkiva) u procesu zvanom
fibroliza, dok ćelije koje ga produkuju bivaju odstranjene putem apoptoze. Hronično oštećenje jetre,
medjutim, kako je elaborirano u Poglavlju 1, dovodi iznova i iznova do aktiviranja fibrogenih
signala, kako od strane oštećenih parenhimskih ćelija jetre (hepatocita), tako i od strane lokalnih i
infiltrirajućih ćelija imunog odgovora, imajući za posledicu narušavanje fine ravnoteže koja postoji
između fibrogeneze i fibrolize. Taj disbalans izmedju dva procesa dovodi do nagomilavanja
ožiljačnog tkiva, narušavanja normalne arhitekture, ciroza i, na kraju, prestanka rada jetre.
Transplatacija jetre, koja je još uvek jedini efikasni tretman, je suočena sa teško premostivim
izazovima, pre svega manjkom donora, a potom i kontraindikacijama koje prate sam hiruški zahvat,
što nameće potrebu za hitnim razvojem tretmana koji bi zaustavili napredovanje same bolesti.
Poznato je da stelatne ćelije, vitalne za normalno funkcionisanje jetre i njen odgovor na
oštećenje/povredu, imaju vodeću ulogu u razvoju fibroze. Naša prethodna istraživanja su pokazala
da je proizvodnja IGFBP5 proteina značajno povišena u fibrozi kod miševa sa holestazom jetre.
Stoga je istraživanje prikazano u Poglavlju 2 imalo za cilj da prouči specifičan uticaj IGFBP5 na
stelatne ćelije i miofibroblaste. IGFBP5, ispostavilo se, poboljšava njihovo preživljavanje smanjenjem
nivoa apoptoze, ne utičući na ćelijsku deobu. Pokazalo se i da IGFBP5 povećava prisustvo gena
uključenih u proizvodnju proteina ožiljačnog tkiva, ukazujući na njegovu moguću profibrotičku
ulogu.
Ovi rezultati iz eksperimenata u ćelijkoj kulturi su bili uvod u sledeći korak – da se prouči
uloga IGFBP5 u životinjskom modelu fibroze jetre. Korišćeni miševi se karakterišu potpunim
nedostatkom fosfolipida u žuči, što pojačava njena detergentska svojstva, čini je toksičnom i dovodi
do spontanog razvoja fibroze. U Poglavlju 3 smo pokazali da povišeno prisustvo IGFBP5 u jetri,
dovodi do smanjenja oštećenja hepatocita, koje za posledicu ima smirivanje upalnih procesa i,
shodno tome, smanjen oksidativni stres. Kao posledica, došlo je do smanjenja broja aktiviranih
stelatnih ćelija i miofibroblasta, smanjenja količine ekstracelularnog matriksa, što je na kraju dovelo
do neočekivanog ublažavanja patologije, sugerišući da IGFBP5 u jetri, za razliku od izolovanih
stelatnih ćelija, ima antifibrotičku ulogu.
Odnedavno je poznato da epitelijalne ćelije u žučnim kanalima (holangiocite) imaju i receptor
za IGF1 protein i da ih, u ćelijskoj kulturi, IGF1 štiti od oštećenja izazvanog toksičnom žuči. Da bi
smo ustanovili uticaj IGF1 na već postojeće ostećenje u jetri, proizveli smo transgenog miša, sa
povišenom količinom ovog proteina u fibrotičkoj jetri. U Poglavlju 4 smo pokazali da stalna
povišena proizvodnja IGF1 u ovim miševima stimuliše deobu holangiocita, pojačava inflamaciju i
smanjuje remodelovanje matriksa, izazivajući povećano deponovanje ožiljačnog tkiva i napredovanje
fibroze. Tako je, nasuprot očekivanjima baziranim na akutnim modelima fibroze, ovo istraživanje
pokazalo da IGF1 nije najbolji kandidat za tretiranje bilijarne fibroze izazvane hroničnom
holangijopatijom.
U studijama opisanim u Poglavljima 2 - 4 direktno smo uticali na ekspresiju gena od interesa
(IGFBP5 i IGF1), da bi proučili njihovu moguću ulogu u razvoju fibroze. Paralelno, proučavali smo i
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kako smanjeno unošenje hrane utiče na fibrozu u istom modelu. Rezultati prikazani u Poglavlju 5
ukazuju na vrlo brz adaptivni odgovor na gladovanje. Upadljivo smirivanje zapaljenjskih procesa u
jetrama izgladnjvanih životinja već posle 12 sati je, čini se, zamajac za kaskadu dogadjaja koja
uključuje smanjenje brze deobe hepatocita, inače karakteristične za ove miševe, smanjenje broja
aktiviranih stelatnih ćelija i miofibroblasta, kao i smanjeno skladištenje komponenata ekstracelularnog
matriksa, i povišenu ekspresiju gena uključenih u remodelovanje tkiva.
Istraživanja prikazana u ovoj tezi su, dakle, bila pokušaj da se rasvetli problem bilijarne
fibroze – bilo odredjujući uloge pojedinih gena, bilo uvodeći gladovanje kao izazov za fibrotičnu
jetru. Iako direktan uzrok fibroze nije eliminisan ni jednim od pristupa (tj. sastav žuči ostao je
nepromenjen), posledice fibroze su ublažene, što ostavlja odškrinuta vrata za razvoj novih terapija.
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ABBREVIATIONS
AAV adeno-associated virus
ABCB4 ATP-binding cassette, sub-family B member 4
ALT alanine aminotransferase
BCL2 B-cell CLL/lymphoma 2
BDL bile duct ligation
BrdU bromo-2’-deoxy-uridine
CB1 endocannabinoid receptor
CC cholangiocarcinoma
CCL4 carbon tetrachloride
CD95 cell surface death receptors
CLD chronic liver diseases
ECM extracellular matrix
EGF epidermal growth factor
F4/80 EGF-like module-containing mucin-like, hormone receptor-like 1
FCS fetal calf serum
FGF2 fibroblast growth factors 2
GFP green fluorescent protein
GSH glutathione
GSSG glutathione disulfide
HCC hepatocellular carcinoma
HGF hepatocyte growth factor
HSCs hepatic stellate cells
IFNα interferon alpha
IFNγ interferon gamma
IGF1 insulin-like growth factor 1
IGF1R insulin-like growth factor 1 receptor
IGFBP5 insulin-like growth factor binding protein 5
ILK integrin-linked kinase
JNKs c-jun N-terminal kinases
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Mac1 integrin alpha M
MFs myofibroblasts
MMPs matrix metalloproteinases
MPO myeloperoxidase
NAFLD non-alcoholic fatty liver disease
NASH non-alcoholic steatohepatitis
NFκB nuclear factor κB
NGF nerve growth factor
p21 cyclin-dependent kinase inhibitor 1A
p53 tumor protein 53
PCNA proliferating cell nuclear antigen
PDGF platelet-derived growth factor
PDGFβ-R platelet derived growth factor receptor β
PI3 phosphatidylinositol 3-kinases
rIGF1 recombinant insulin-like growth factor 1
rIGFBP5 human recombinant insulin-like growth factor binding protein 5
ROS reactive oxygen species
SAβ-gal senescence-associated β-galactosidase
siRNA small interfering RNA
TGFα transforming growth factor α
TGFβ1 transforming growth factor, beta 1
TIMP1 tissue inhibitor of metalloproteinase 1
TNFα tumor necrosis factor alpha
uPA urokinase-type plasminogen activator
VEGF vascular endothelial cell growth factor
α-SMA actin, alpha 2, smooth muscle
closure
115
personalia
acknowledgements
curriculum vitae
list of publications
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ACKNOWLEGEMENTS
The mid-nineties found me in Belgrade, leaving the University at the very last year and setting off to
work, amidst the raging wars, with hyperinflation that made every effort worthless and turned life
into mere survival. The romantic idea from the late eighties of becoming a biologist seemed surreal
and pointless, kind of alien. Ten years later and two thousand kilometers away, hard work and luck
have finally met in the AMC in Amsterdam, and my dream of being a scientist started coming true.
This unexpected adventure would be impossible without special people that I would like to thank –
those that have directly contributed to this thesis, and those who have made the exciting and
eventful road to it worth remembering.
Ronald, thanks for offering me an opportunity to peak into the world of science, for decisive and
proficient guidance, for critical suggestions, for the door always opened for my questions, and for
help in wrapping up this thesis.
Piter, I appreciate your idea to kick off by IGFBP5. Many thanks for daily supervision, for prompt
reviewing of the manuscripts, and for leaving me freedom to grow.
Wout, thanks for showing faith in me by giving me a chance to volunteer, which put a foot in the
door towards the doctorate. Thanks for the constructive suggestions, for contagious enthusiasm, and
for demonstration how to be a Professor (capital letter is not a lapsus clavis).
Distinguished committee members, professors Lamers, Beuers, Prieto, Moshage, Poelstra and
Rothuizen, thanks for your readiness to invest time into reading yet another thesis, and to spend yet
another morning in the Agnietenkapel, as opponents at my defense. Jesus, many thanks for
accepting the invitation to come over from Spain, but also for the successful collaboration and
prompt, useful comments on the manuscript.
Carlos, thanks for the immense effort to organize and perform the experiments in Pamplona and for
all your help during preparation of the manuscript. Thanks for your South European charm and
kindness. I owe my gratitude to all your collaborators from CIMA who took part in IGF1-Abcb4
project.
Milka, my „undercover“ mentor, thanks for the unexpected years of science, for all the techniques
that I have learned, from pipetting to organizing presentations, and, finally, for leading the research
in the last study of this thesis.
Dad, thanks for the countless hours of listening to my (not always clear) ideas, and for transforming
them into drawings that illustrate the thesis. The outcome is amazing! Working together on such an
important project was a special, priceless experience!
Willem, without you there would be a whole different beginning. Thank you for the unforgettable
day crowned by Italian dinner that has marked your retirement and the beginning of my work. I am
glad that I could contribute with a bit of understanding of liver fibrosis in the legendary Mdr2s.
Vassilis, thanks for your endless energy, for the ability to turn ordinary into unusual, for your gift to
make people feel special (if you only want so). Thanks for Nana, moussaka and tzatziki (salad not
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sause!), but most of all thanks for Eirikur! I wonder if my witnessing the phone call from Nana (that
you haven’t answered?!) was to blame for accepting to be my paranymph.
Jurgen, thanks for your delicate talent to be at hand when something goes wrong – in science and in
life. Thanks for the Dutch resume, and thanks for eventually accepting to be exposed to Ulrich’s
artillery fire during the defense. I am glad that Wout’s wild geese have opened your gastronomic
world and have led to pleasant afternoons with our families.
I owe huge gratitude to Susanne, for perfect organization and execution of the animal experiments,
for the long hours of patience finished with her typical “no thanks!” Big thanks to: Cindy – for
delicate surgeries, Lysbeth – for willingness to help, Rudi, the fastest man in the world – for
HPLCs, Kam – for chemicals always appearing where they should, Mona – for all the complicated
bureaucratic actions around the defense, Coen – for the opportunity to work with someone whom I
first saw on TV as a rock-star. Honest and warm Dineke, thanks for becoming a friend, even though
you snatched the position in front of my nose (and became the best young researcher of the ALC). I
do hope you’ll stay in science. To Paula, promoter and defender of Portugal against the rest of the
world (that lacks understanding of “our” South European ways), thanks for the immunostainings.
Johan, apart from thanking you for the evident technical help, I am glad that you, quiet and simple,
with a smile and a good word for everybody, have become my friend. Andy (and Anita) thanks for
the wonderful evening and dinners with flawless attention to detail. And, of course, for
demonstration of proverbial German efficiency!
Though I was officially a part of the ALC’s “downstairs”, I lived and worked (with pleasure)
“upstairs”. Dear special Wil and Jacqueline, thanks for your patience and help with the techniques
that refused to cooperate (they outnumber the compliant ones), for help with the microscope and
histology, for an unusually spacious working place, for… Wil and Ron, thanks for making us feel
(even more) welcome. Jan, thanks for the primers, antibodies and infectively good mood. Theo,
Ingrid, thanks for your suggestions and ideas on Wednesday meetings, and always pleasant
encounters. Farah is acknowledged for a reason to trust her SOPs and solutions that I used routinely
(and every so often secretly). Farah and Marijn, thank you for our warm friendship and a chance to
enjoy the exciting mix of Surinamese and Dutch. Youji, our friendship has started oddly, with a
pounding heart due to a tiny innocent tea-leave, and has grown into something special. Thanks for
every moment we spent with you, Ning and the kids!
Many thanks to the colleagues from Biochemistry, Cindy, Roelof, Saskia and Bert (the latter from
the ALC, Biochemistry and Groningen), for their generous help with the “hungry study”, and Jan
Aten from Pathology for the patience with all the painstaking immunostainings.
It is, obviously, impossible to name everyone who has contributed to me writing these
acknowledgements today – all the family members (inherited and acquired) warm enough to make
the distances shorter; friends who have remained so ever since making my school and faculty days
(more) pleasant; or those that could be classified as „others“, who have, e.g. scanned the illustrations
for this thesis (tens of times!). I have to mention but a few who have left a mark on creation of the
thesis in a very special way. To all the others, simply many thanks!
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Among people who can help, rare and special are the ones that are also willing to. Mile Ivanović is
one of them. Mile, thanks to your help our life is so different than it would be without my
graduation!
Jan, thanks for the cozy months of our Dutch life together, and even cozier years of friendship.
Golub, thanks for your quiet, unpretentious support during these thirtyfive years of friendship,
though I was the one who has “spoiled” you! Siki, our quarter of a century of friendship was neither
quiet not unpretentious, but it was certainly special. Thanks for choosing to spend time with us when
you could have been at more exciting places. Dare, you are a rare soul with enough readiness and
energy to go along with all my actions! Thanks for always having answers, for your friendship and
support for life.
Anne and Arvid, thanks for the warm Norwegian hospitality during my first months abroad, for the
long years of friendship that have followed, and for the countless (unearned) birthday presents. I
wish my gratitude could reach grandma Elsa, for the long talks at the time when my English
vocabulary contained no more than fifty words.
Nočka and Øistein, thanks for pushing that vocabulary to finally start growing in my fourth (?!)
decade. Even more, thanks for being there for us even when we did not think we needed help.
Jojka and Vujo, I wish I could express my pleasure and gratitude for you taking such a special place
in my life. Thanks for becoming such a family!
Mom, thanks for your watchfulness, for all the care and infinite kindness for me. Dad, thanks for
understanding my (extreme) ideas and desires. Thank you both for the uplifting feeling of being
unconditionally loved! Also thanks for high expectations, for always raising the bar, and for the
support for getting past the obstacles. Duca, my dear “little” brother with infinite charm and subtle
sensitivity for the world around you, thanks for all the love and patience that you must have had to
live (and survive) next to me!
Milka, thanks for all the incredibly rich and exciting years since I first saw you in Belgrade’s
Botanical Garden! For all the compassion, understanding, patience and determination that you have
woven into our life, so we could be where we are now. Especially for your courage and persistence to
turn one dream into Mia. Mia, thanks for unveiling an entire new world to me, and for letting me
adore you!
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ZAHVALNICA
Kada sam sredinom devedesetih u Beogradu, na poslednjoj godini fakulteta, u vrtlogu ratova koji su
besneli oko nas, sa hiperinflacijom koja je obesmišljavala rad i svodila život na preživljavanje, odlučio
da ostavim studije i počnem da radim, romantična ideja o bavljenju biologijom s kraja osamdesetih
postala je nerealana i besmislena, nekako tuđa. Desetak godina kasnije i dve hiljade kilometara dalje, u
amsterdamskom AMC-u, naporan rad i sreća su se konačno susreli i moj mladalački san o nauci
počeo da biva realnost. Ova potpuno neočekiva avantura ne bi bila moguća bez posebnih ljudi
kojima bih voleo da se zahvalim - onima koji su direktno doprineli nastanku ove teze, i onima koji su
učinili da ovaj uzbudljiv i pun obrta put postane i ostane vredan pamćenja.
Ronalde, hvala na prilici da zavirim u svet nauke, na odlučnom i stručnom usmeravanju, na
kritičnim sugestijama, na vratima uvek otvorenim za moja pitanja i na pomoći da se teza privede
kraju.
Piter, uz zahvalnost za početnu ideju o IGFBP5, hvala za dnevno rukovođenje, za brzu recenziju
napisanog i za slobodu da rastem.
Vaute, hvala na pruženom poverenju i prilici da volontiram u tvojoj grupi, koja je otškrinula vrata u
doktorat. Hvala za konstruktivne sugestije, za zarazni entuzijazam i pokaznu vežbu kako biti
Profesor (veliko slovo nije omaška).
Uvaženim članovima komisije, profesorima Lamers, Bojers, Prieto, Moshage, Pulstra i
Rothauzen, hvala na spremnosti da ulože vreme u još jedno iščitavanje još jedne teze i da provedu
(još jedno) prepodne u Agnietenkapel, kao oponenti tokom moje odbrane. Hesusu posebno hvala
što je prihvatio poziv da za tu priliku doputuje iz Španije, kao i na uspešnoj saradnji i brzim i
konstruktivnim komentarima tokom pisanja rada.
Karlos, hvala za lavovski trud oko organizacije i izvođenja experimenata u Pamploni i za svu pomoć
prilikom pisanja rada. Hvala za južnoevropski šarm i ljubaznost. Hvala i svima iz CIMA-e koji su
učestvovali u radu na IGF1-Abcb4 projektu.
Milki, mom „tajnom“ mentoru, hvala za neočekivane godine bavljenja naukom, za sve tehnike koje
sam naučio - od pipetiranja do organizovanja prezentacija, i, na kraju, na rukovođenju istraživanima
iz poslednje studije ove teze.
Tata, hvala za bezbrojne sate strpljivog slušanja mojih (ne uvek jasnih) ideja i za njihovo pretakanje u
crteže koji ilustruju tezu. Rezultat je očaravajući! Naš zajednički rad na za mene tako važnom
projektu je bio posebno dragoceno iskustvo.
Vileme, da nije bilo tebe kao prethodnice, ja bih morao da počnem od nekog drugog početka. Hvala
za nezaboravan dan krunisan italijanskom večerom, koji je obeležio tvoj odlazak u penziju i početak
mog rada. Drago mi je što sam bar malo doprineo razumevanju fibroze u famoznim Mdr2-ovima.
Vasili, hvala za neizmernu energiju, za mogućnost da od obicnog napraviš neobično, za sposobnost
da (kad hoćeš) učiniš da se ljudi osećaju posebnima. Hvala za Nanu, musaku i caciki (salatu, ne sos!) i,
najvažnije od svega, za Eirikura! Pitam se da li je moje prisustvo tokom Naninog telefonskog poziva
(na koji nisi odgovorio!?) zaslužno što si prihvatio da mi svedočiš tokom odbrane.
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Jurhen, hvala za delikatan talenat da se nađeš pri ruci kad zapne – u nauci i u životu. Hvala za rezime
na holandskom i, na kraju, za hrabar pristanak da se izložiš Ulrihovoj paljbi tokom odbrane. Drago
mi je što su me Woutove divlje guske uvele u tvoj svet gastronomije i dovele do naših prijatnih
porodičnih poslepodneva.
Suzan dugujem beskrajnu zahvalnost za savršenu organizaciju i izvođenje eksperimenata na
životinjama, za sve duge sate strpljenja završavane famoznim “bez zahvaljivanja!”. Veliko hvala:
Sindi – za delikatne operacije, Lizbet – za spremnost da pomogne, Rudiju, najbržem čoveku na
svetu – za HPLCove, Kam – za to što su se hemikalije uvek stvarale gde treba, Moni – za sve
komplikovane birokratske radnje oko odbrane, Kunu – na prilici da radim sa nekim koga sam prvo
video na TVu kao rok-zvezdu. Iskrena i topla Dineke, hvala sto si postala prijatelj, iako si mi ispred
nosa ugrabila poziciju (i postala najbolji mladi ALC-ovski istrazivač). Nadam se da ćeš u nauci i
ostati. Pauli, braniocu Portugalije od ostatka sveta (kome nedostaje razumevanje za “naš”
juznoevropski način), hvala za immuno bojenja. Johane, osim za evidentnu tehničku pomoć, hvala
što si mi, ćutljiv i jednostavan, sa osmehom i lepom rečju za svakoga, postao prijatelj. Endi (i Anita),
hvala za divne večeri i večere, sa pažnjom posvećenom i najsitnijem detalju. Naravno, i za
demonstraciju poslovične nemačke efikasnosti!
Iako sam zvanično pripadao ALC-ovskom “dole”, živeo sam i sa zadovoljstvom radio “gore”. Drage
i posebne Vil i Žaklin, hvala za strpljenje i pomoć kad god bi tehnika odbila da sarađuje (a takvih je
više od kooperativnih), za pomoć oko mikroskopa i histologije, za neubičajeno prostrano radno
mesto, za... Vil i Ron, hvala što smo se uz vas osećali još dobrodošlije. Janu hvala na prajmerima,
antitelima i zarazno dobrom raspoloženju, Teu i Ingrid, na savetima i idejeama na sredskim
sastancima i na uvek prijatnim susretima. Fari hvala na razlogu za bezgranično poverenje u njene
SOP-ove i rastvore, koje sam (ponekad tajno) nemilo koristio, a njoj i Marajnu na lepom druženju i
prilici da uživam u uzbudljivoj mešavini surinamskog i holandskog. Juđi, naše druženje je započelo
neobično – uzlupavanjem srca od naizgled bezazlenog spiralnog listića čaja, a preraslo u nešto vrlo
posebno. Hvala na svakom trenutku koji smo doživeli sa tobom, Ningom i decom!
Hvala kolegama sa Biohemije, Sindi, Rulofu, Saskiji i Bertu (poslednji iz ALCa, sa Biohemije i iz
Groningena), na nesebičnoj pomoći oko „gladne studije“, i Janu Atenu sa Patologije za strpljenje
tokom mukotrpnih imunobojenja.
Nemoguće je, naravno, imenovati sve koji su uticali na to da danas pišem ovu zahvalnicu – članove
porodice (nasleđene i stečene) koji imaju dovoljno topline da daljine ne budu predaleke, prijatelje koji
su to ostali od kad su mi školu i fakultet činili prijatni(ji)m, ili one koji se vode pod „ostali“, a koji su,
npr. skenirali ilustracije za ovu tezu (na desetine puta!). Pomenuću samo nekoliko njih koji su
nastanak ove teze obeležili na vrlo poseban način. Ostalima, jednostavno, veliko hvala!
Medju ljudima koji mogu da pomognu, retki su i posebni oni koji to i hoće. Mile Ivanović je jedan
od njih. Mile, hvala što je po mom diplomiranju naš život mnogo drugačiji nego što je mogao biti!
Jan, hvala na prijatnim mesecima zajedničkog holandskog života i još lepšim godinama druženja.
Golube, hvala na tihoj, odmerenoj podršci tokom svih 35 godina druženja, iako sam ja bio taj koji te
je „pokvario“! Siki, naših četvrt veka druženja koje nije bilo ni tiho ni odmereno je svakako posebno.
Hvala što si birala da budeš s nama i kad si mogla da budeš na zanimljivijim mestima. Dare, ti si
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jedan od retkih sa dovoljno volje i energije da isprate svaku moju akciju! Hvala na uvek spremnim
odgovorima, na prijateljstvu i osloncu za ceo život.
Ane i Arvide, hvala za toplo norveško gostoprimstvo tokom mojih prvih meseci u inostranstvu, za
duge godine prijateljstva koje su usledile i za nebrojene (ničim zaslužene) rodjendanske poklone.
Voleo bih da moja zahvalnost može da stigne i do bake Else, za duge razgovore u vreme kad moj
rečnik engleskog nije sadžavao više od pedesetak reči.
Nočka i Ejstajne, hvala što je taj rečnik u mojoj četvrtoj deceniji (!?) ipak počeo da raste. Posebno
hvala što ste bili uz nas i kad nismo mislili da nam je pomoć potrebna!
Jojka i Vujo, voleo bih da imam reči da opišem zadovoljstvo i zahvalnost za tako posebno mesto
koje zauzimate u mom životu. Hvala što ste mi postali takva porodica!
Mama, hvala za bdenje nada mnom, za strepnju i beskrajnu nežnost. Tata, hvala na razumevanju za
moje (ekstremne) ideje i želje. Hvala oboma na uspokojavajućem osećaju da sam bezuslovno voljen!
Hvala i za visoka očekivanja, stalno podizanje lestvice i podršku da se hvatam u koštac sa
problemima. Duco, moj dragi „mali” brate sa beskrajnim šarmom i istančanim osećajem za svet oko
sebe, hvala za svu ljubav i strpljenje koje si morao imati da bi živeo (i preživeo) sa mnom!
Milka, hvala za sve ove nestvarno bogate i uzbudljive godine od kada sam te prvi put ugledao u
beogradskoj Botaničkoj bašti! Za svu nežnost, razumevanje, strpljenje i odlučnost koje si utkala u naš
život, da bi smo danas bili ovde gde jesmo. Posebno hvala za hrabrost i upornost da jedan san
postane Mia. Mia, hvala za jedan novi svet koji si mi otkrila i što mi dozvoljvaš da te obožavam!
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CURRICULUM VITAE
Aleksandar Sokolović attended secondary school majoring Biochemistry and Molecular Biology in
his hometown Lebane in Serbia. He graduated as General Biologist at Faculty of Biology, University
of Belgrade, Serbia, focusing on applied genetics. His diploma work on genetic variability of yield
and yield components of soy bean was performed at Maize Research Institute Zemun Polje,
Belgrade, under supervision of Prof. Mile Ivanović. He has worked in a world far distant from
biology for a number of years. Upon coming to the Netherlands, he started volunteering at the AMC
Liver Center in Amsterdam, in the group of Prof. Wout Lamers, on transcriptomics signature of
fasting. A year later, he started his PhD studies under supervision of Prof. Ronald Oude Elferink, at
Tytgat Institute for Liver and Intestinal Research in Amsterdam. His studies on liver fibrosis are
described in this thesis.
Aleksandar Sokolović je srednju školu na smeru Biohemija i molekularna biologija završio u rodnom
Lebanu u Srbiji. Zvanje diplomiranog biologa je stekao na Biološkom fakultetu Univerziteta u
Beogradu, na smeru Primenjena genetika. Diplomski rad na genetičkoj varijabilnosti prinosa i
komponenti prinosa soje je uradio na Institutu za kukuruz u Zemun Polju, pod vođstvom profesora
Mileta Ivanovića. Dugi niz godina je radio u svetu vrlo dalekom od biologije. Po dolasku u
Holandiju, počinje da volontira u grupi prof. Vauta Lamersa na transkriptomskom potpisu
gladovanja, u Centru za jetru Akademskog medicinskog centra u Amsterdamu. Godinu dana kasnije,
započinje doktorske studije pod vođstvom prof. Ronalda Aude Elferinka u Tajthat institutu za
proučavanje jetre i creva u Amsterdamu. Njegova istraživanja na temu fibroze jetre opisana su u ovoj
tezi.
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LIST OF PUBLICATIONS
1) Fasting reduces liver fibrosis in mouse model for chronic cholangyopathies
Sokolović A, van Roomen CP, Ottenhoff R, Scheij S, Hiralall JK, Claessen N, Aten J, Oude Elferink RP, Groen AK, Sokolović M
under revision
2) Insulin-like growth factor 1 enhances bile-duct proliferation and fibrosis in Abcb4-/- mice
Sokolović A, Rodrigues C, ten Bloemendaal L, Prieto J, Oude Elferink RPJ, Bosma PJ
accepted for publication in
Biochimica et Biophysica Acta (BBA) - Molecular Basis of Disease; (2013)
3) Unexpected reduction of murine liver fibrosis by insulin like growth factor binding protein 5
Sokolović A, Miranda PSM, de Waart DR, Cappai RMN, Duijst S, Hiralall JK, ten Bloemendaal L, Sokolović M, Bosma PJ
Biochimica et Biophysica Acta (BBA) - Molecular Basis of Disease; (2012)
4) Interorgan coordination of the murine adaptive response to fasting
Hakvoort TBM, Moerland PD, Frijters R, Sokolović A, Labruyère WT, Vermeulen JLM, Ver Loren van Themaat E, Breit TM, Wittink FR, van Kampen AH , Verhoeven AJ, Lamers WH, Sokolović M
Journal of Biological Chemistry; (2011)
5) Unexpected effects of fasting on murine lipid homeostasis - transcriptomic and lipid profiling
Sokolović M, Sokolović A, van Roomen CP, Gruber A, Ottenhoff R, Scheij S, Hakvoort TB, Lamers WH, Groen AK.
Journal of Hepatology; (2010)
6) Insulin-like growth factor binding protein 5 enhances survival of LX2 human hepatic stellate cells
Sokolović A, Sokolović M, Boers W, Elferink RP, Bosma PJ,
Fibrogenesis and Tissue Repair; (2010)
7) The transcriptomic signature of fasting murine liver
Sokolović M, Sokolović A, Wehkamp D, Ver Loren van Themaat E, de Waart DR, Gilhuijs-Pederson LA, Nikolsky Y, van Kampen AHC, Hakvoort TBM, Lamers WH,
BMC Genomics; (2008)
8) Fasting induces a biphasic adaptive metabolic response in murine small intestine
Sokolović M, Wehkamp D, Sokolović A, Vermeulen J, Gilhuijs-Pederson LA, van Haaften RIM, Nikolsky Y, Evelo CTA, van Kampen AHC, Hakvoort TBM, Lamers WH,
BMC Genomics, (2007)