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Fibroblast growth factors and notch signaling in a diethoxycarbonyl dihydrocollidine-induced hepatic progenitor cell liver injury model

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Fibroblast growth factors and notch signaling in a diethoxycarbonyl dihydrocollidine-induced hepatic progenitor cell liver injury model

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FIBROBLAST GROWTH FACTORS AND NOTCH SIGNALING IN A
DIETHOXYCARBONYL DIHYDROCOLLIDINE-INDUCED HEPATIC
PROGENITOR CELL LIVER INJURY MODEL

by
Christopher Lee Vendryes

A Thesis Presented to the
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
MASTER OF SCIENCE
(BIOCHEMISTRY AND MOLECULAR BIOLOGY)

December 2010

Copyright 2010 Christopher Lee Vendryes
ii

ACKNOWLEDGEMENTS

I would like to thank the faculty and friends I have made during my time in the lab:
Frederic Sala, PhD, Denise Al Alam, PhD, Jamil Matthews MS, MD, Sha-Ron Jackson,
MD, Yigit Guner, MD, Michael Petrosyan, MD, Cindy Tai, MD, Shannon Castle, MD,
Alison Speer, MD, Gianluca Turcatel, PhD, Caterina Tiozzo MD, PhD, Saverio Bellusci,
PhD, David Warburton, MD, Deidra Garret, MD, PhD, Gerald Gracia, MD, Dean
Anselmo, MD, Akemi Kawaguchi, MD, Cathy Shin, MD, Nam Nguyen, MD, Tracy
Grikscheit, MD, Jeffrey Upperman, MD, and Henri Ford, MD, for indirectly assisting in
this research.

Most importantly, the laboratory has provided insurmountable help through my time in
the laboratory. I would like to especially recognize Tove Berg, PhD, Steven “Mike”
Salsibury, BS, Jennifer Phan, BS, David James, BS, Jun Wu, MD, PhD, and Nirmala
Mavila, PhD. I would be remiss if I did not recognize my partner in crime in the lab,
Sarah Utley, BS (and to be PhD). My opportunity to study the liver and encouragement
to purse the Master’s in Science degree would be naught, without the guidance of my
mentor Kasper Wang, MD.

My pursuit for knowledge comes from my family (“Daddy,” “Mommy,” Shaun, and
Brittany), and they are thanked for their continued love and support. Last, but far from
least, is my beloved fiancé, Courtney Lockhart, MD, PhD. You have been with me
iii

through it all, it seems, and your words of wisdom and guidance are unmatched. I pray,
time and time again, that our distance will close, but until then, I’m lucky to have you as
my light house beckoning me home.

iv

TABLE OF CONTENTS

ACKNOWLEDGMENTS ii
LIST OF TABLES v
LIST OF FIGURES vi
ABBREVIATIONS viii
ABSTRACT xii
INTRODUCTION
Anatomy and Physiology 1
Liver Development 5
Liver Stem Cells and Regeneration 8
Fibroblast Growth Factors 14
Notch Signaling 17
Biliary Atresia, a bile duct pathology 19
Hypothesis 20
CHAPTER 1: MATERIALS AND METHODS
Mice and Liver Harvest 22
Immunostaining 23
Western Blot Sample Preparation and Analysis 25
Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) 27
Fluorescent Activated Cell Sorting (FACS) 30
CHAPTER 2: RESULTS
DDC stimulates oval-shaped cell and bile ductule expansion 31
FGFR1 expression is associated with DDC injury 32
Notch1 and HES1 are expressed after DDC injury 36
Dominant-negative FGFR2b inhibition decreases CD49f
+
CD45
-
cells 38
Dominant-negative FGFR2b inhibition results in decrease AKT activation 39
Dominant-negative FGFR2b inhibition results in decreased proliferation, 40
FGFR1 expression, and Notch activation
CHAPTER 3: DISCUSSION 42
REFERENCES 47

v

LIST OF TABLES
Table 1: Fibroblast growth factor receptors, its ligands, and its specificites 16
Table 2: Antibodies for Immunostaining 24
Table 3: Primers used for RT-PCR 29

vi

LIST OF FIGURES

Figure 1: Liver Segments (I-VIII) 1
Figure 2: Liver lobule 2
Figure 3: Liver bud formation under the control of cardiac mesoderm 5
Figure 4: Fibroblast growth factors influence liver bud outgrowth 6
Figure 5: Fgf10 and Fgfr2b KOs result in small, misshapen livers 7
Figure 6: Mechanism of hepatocyte regeneration 9
Figure 7: Differentiation of HPC or oval cells after chronic injury 10
Figure 8: Survival curve describing the percentage of cirrhosis 12
seen after liver injury with DDC, through diet or intra-peritoneal injection
Figure 9: Heme biosynthesis pathway 13
Figure 10: Activated FGFR1 15
Figure 11: FGFR signaling pathway 16
Figure 12: Pleiotropic effects of Notch signaling for stem cells 18
and transactivating cells
Figure 13: Kasai hepatoportoenterostomy 20
Figure 14: BSA-Bradford standard curve 26
Figure 15: DDC leads to progressive ductular proliferation 32
Figure 16: Fgfr1 and Fgf10 mRNA transcripts are expressed after DDC injury 33

Figure 17: FGFR1 is expressed after DDC injury 34
Figure 18: Proliferation of cells after DDC injury 35
Figure 19: % Total cells expressing FGFR1 and PCNA after DDC injury 36
vii

Figure 20: Notch receptors and ligand mRNA expression after DDC injury 37
Figure 21: NICD is expressed after 14 days of DDC injury 37
Figure 22: HES-1 is expressed after DDC injury 38
Figure 23: Percent CD49f
Pos
CD45
Neg
cells in dominant negative transgenic mice 39
after 14 days of DDC injury
Figure 24: Phosphorylated AKT expression is reduced with dominant negative 40
expression of soluble FGFR2b

Figure 25: HES and FGFR1 expression decreases in dominant negative 41
transgenic expression of soluble FGFR2b after 14 days of DDC injury.

viii

ABBREVIATIONS

˚C degree Centigrade
2-Acetylaminofluorene 2-AAF
3,5-diethoxycarbonyl-1,4-dihydrocollidine DDC
Alpha 
Alpha Feto Protein AFP
Alpha-naphthyl-isothiocyanate ANIT
Beta 
Biliary Atresia BA
Bone Morphogenic Protein BMP
Bovine serum albumin BSA
Carbon tetrachloride CCl
4

Cluster of Differentiation CD
Common bile duct ligation CBDL
Complementary DNA cDNA
Cyclization recombination enzyme Cre
Cytidine-cytidine-adenosine-adenosie-thymidine C/EBP
Enhancer Binding Protein
Cytokeratin CK
Days d
Delta like ligand Dll
Delta like protein dlk
ix

Deoxynucleic Nucleic Acid DNA
Embryonic day E
End-stage liver disease ESLD
Epithelial cell adhesion molecule EPCAM
Example Ex
Extracellular domain ECD
Fibroblast Growth Factor FGF
Fibroblast Growth Factor Receptor FGFR
Fibroblast Growth Factor Receptor substrate FRS2
Forkhead FOXL
Gamma 
Growth factor receptor-bound GRB2
Hairy and enhancer of split HES
Hematopoietic Stem Cells HemSC
Hepatic Progenitor Cell HPC
Hepatic Stellate Cell HSC
Hepatocyte Growth Factor HGF
Hepatocyte Growth Factor Receptor HGFR
Hepatocyte Nuclear Factor HNF
Intracellular domain ICD
KO knock out
Messenger RNA mRNA
x

Microliters L
Micrometers m
Milligrams mg
Milliliters mL
Millimolar mM
Mitogen activated protein kinase MAPK
Nanomolar nM
Nanogram ng
Non-parenchymal fraction NPF
Para-formaldehyde PFA
Partial Hepatectomy PHx
Phosphate buffered saline PBS
Phospholipase C PLC
Polymerase chain reaction PCR
Proliferating Cell Nuclear Antigen PCNA
Rat Sarcoma Ras
Reverse tetracycline transactivator rtTA
Reverse Transcriptase Polymerase Chain Reaction RT-PCR
Stem Cell Antigen SCA
Tetracycline Tet
Thymocyte differentiation antigen THY
Tris buffered saline with 20% Tween TBST
xi

Tumor Necrosis Factor TNF
TNF-weak enhancer of apoptosis TWEAK

xii

ABSTRACT

INTRODUCTION: The liver is a multi-purposeful organ, susceptible to injury.
Following acute injury, cells such as hepatocytes, regenerate, while latent hepatic
progenitor cell (HPC) become active after chronic liver injury. Some studies suggest
Fibroblast growth factors (FGF) may activate HPCs similarly to embryonic liver stem
cells. In addition, Notch signaling is critical in bile duct development and injury. With
progenitor cell transplantation as an alternative to tissue transplantation, we hypothesize
that FGF and Notch signaling pathways induce proliferation of HPC after chronic liver
injury. MATERIALS AND METHODS: Wild-type mice were fed either regular or 0.1%
3,5-diethoxycarbonyl dihydrocollidine (DDC) chow to induce injury. Transgenic,
dominant-negative Rosa
rtTA
;tet(o)sFGFR2b
+/-
mice were given doxycycline 2 days prior
and throughout DDC injury. mRNA and immunostaining of FGF and Notch receptors
and ligands were performed. Cells isolated from transgenic mice were fluoresecently
sorted for stem cell marker CD49f. In addition, western blot analyses of downstream
targets were studied. RESULTS: After confirming DDC injury, FGF signaling was
identified, with expression of Fgf1, 10, and receptor Fgfr1b and c. Similarly, FGFR1,
proliferating cell nuclear antigen (PCNA), Notch-1, delta like ligand 1, and Notch-1
intracellular domain (NICD), were expressed after DDC injury. With dominant negative
transgenic mice, we saw a reduction of CD49f
pos
cells, FGFR1, phosphorylated-AKT, and
Notch downstream mediator Hes-1 between induced and control groups. DISCUSSION:
We believe that FGF and Notch signaling are involved in the induction of HPC after
xiii

chronic liver injury. Expression of FGFR1 suggests that chronic liver injury may work
through this receptor. Dominant-negative soluble FGFR2b analysis suggests FGF may
have an influence on a HPC population with a reduction in FGFR1, CD49f
pos
cells,
PCNA, and Hes-1. Notch-1 expression after chronic injury may associate with prior data
describing Notch-2 as a marker for bile duct disease.
1

Figure 1: Liver segments (I-VIII). HA= hepatic
artery, MPV=middle portal vein, RPV=right
portal vein, LPV=left portal vein, MHV=middle
hepatic vein, RHV=right hepatic vein, LHV=left
hepatic vein. Modified from Lee. Press Med,
1953

INTRODUCTION

Anatomy and Physiology
The liver is a multifunctional organ that helps maintain the homeostatic balance of
the body. Located in the right upper
quadrant of the abdomen in humans, the
liver is divided into 8 segments (13).
Divided by longitudinal and transverse
planes, liver anatomical segments receive
blood from an afferent portal vein and
hepatic artery. The liver drains into the
inferior vena cava via a middle, left, and
right hepatic vein (Figure 1).
The functional unit of the liver is
the liver lobule (Figure 2). The lobules are
plates of hepatocytes approximately hexagonal in shape outlined by a triad of vessels: a
branch of the portal vein, hepatic artery, and bile duct. At the center of the lobule is the
central vein. Blood supplied by the hepatic artery and portal vein sequester in sinusoids.
The blood flows toward the center of the lobule and drains into the central vein. The
central veins coalesce into hepatic veins that return blood to the general circulation.

2

Figure 2: Liver lobule. Modified from
Cunningham, Alcohol Res & Health, 2003.
The liver has many functions. One of the principle functions of the liver is to
metabolize dietary supplements that are absorbed by the intestines. The liver receives
medium chain fatty acids, carbohydrate
derivatives such as glucose, fructose, and
galactose, and di- and tripeptides from the
portal vein for processing and synthesis.
Protein synthesis for multiple blood-borne
cytokines and metabolic cycles (ex.Cori
Cycle) occurs in the liver. During states of
starvation, the liver undergoes catabolism
and maintains nutritional homeostasis.
Whether undergoing gluconeogenesis or
ketone synthesis, the liver breakdowns
sugars or proteins to provide nutritional
supplements for the entire body. However, after a meal, the liver also undertakes
anabolism to prepare the body for sugar, protein, and fat synthesis. Bile salts, produced
by the liver, emulsify fats in the intestine lumen. Soluble triglycerides are cleaved by the
liver-synthesized enzyme lipase to form free fatty acids and di-acylglycerol.
Carbohydrate and protein derivatives are stored and packaged by the liver for use by
other organs during times of stress or starvation. Although a key organ in nutritional
balance, the liver has a cellular diversity that broadens its functions.
3

Hepatocytes are the primary cells of the liver. Hepatocytes carry a polarity with
different functions at the superior and inferior poles. At their apical surface, hepatocytes
synthesize material that is secreted into bile canaliculi. Bile acids are secreted into the
canaliculus, where numerous biliary canaliculi congregate and stream outward toward the
portal triad. Along the basolateral membrane, hepatocytes communicate with sinusoids
that collect and direct blood from the incoming portal vein and hepatic artery to the
central vein. Hepatocytes metabolize the dietary supplements drained from the intestine,
detoxify drug compounds, and synthesize exocrine and endocrine solutes for the body.
Although hepatocytes account for 78% of liver volume (5), they function with a
host of cells that diversify liver function. Bililary epithelial cells (cholangiocytes),
Kupffer cells, and hepatic stellate cells form a complex network of cells that manage a
variety of process that is key in maintaining homeostatsis. These constitute the “non-
parenchymal” fraction (NPF) of the liver, which makes up only 6.3% of liver volume.
The Kupffer cell resides in the liver sinusoids and provides a systemic and local
immune response. Although hepatocytes do secrete some cytokines (C-reactive protein,
-1-antitrypsin, haptoglobin), it is in response to Kupffer cell release of interleukin-6
(56). Kupffer cells are the first cells exposed to foreign antigen through the blood stream.
They are important mediators of antigen phagocytosis and pro-inflammatory cytokine
release.
Hepatic stellate cells (HSC) are largely responsible for the pro-inflammatory
molecules released in the liver after injury. Located in the space of Disse, HSC behave
similarly to the other antigen presenting cells, such as T cells. In addition, HSC secrete
4

profibrogenic signals, such as TGF- , which induces hepatic fibrosis during chronic
states of injury (85). During this, HSCs transdifferentiate into myofibroblasts, and there is
a congruent increase in extracellular matrix components and immune surveillance (49,
109), suggesting the activated myofibroblast is the cell responsible in fibrogenic disease
states such as chronic liver disease (9).
The liver emulsifies fats and eliminates fat-soluble metabolites via the bile acids
within bile. Hepatocytes secrete bile into bile canaliculi, which is lined by biliary
epithelium. Bile canaliculi terminate in the periphery of the hepatic lobule at the bile
duct. The bile ducts are composed of biliary epithelium and direct bile flow toward the
extrahepatic biliary tree. At the junction of the biliary canaliculi and the peripheral bile
ducts, a special area, known as the Canals of Herring, house postnatal, pluripotent, stem
cells, which may be key in liver recovery after severe disease (102).
As a major homeostatic organ, the liver becomes essential during states of
disease. The liver is a large organ congested with both portal (enteric) and arterial
(systemic) vascular circulations, making it vulnerable to many exogenous and
endogenous pathogens, including toxins such as ethanol and hepatitis B or C viruses. In
pediatrics, congenital pathologies, such as Biliary Atresia (BA), are primary etiologies
that require liver transplantation. BA, in particular, is the most common cause for
pediatric liver transplantation in the world (18, 61, 88). Although a temporizing surgical
procedure can extend the life of a child with BA by as much as 30% (88), whole liver
transplantation is the most successful therapy for this end-stage liver disease (ESLD),
which carries inherent morbidities and a high financial burden. Our current
5

Figure 3: Liver bud formation under the control of cardiac mesoderm.
Modified from Zaret, K. Mech Dev, 2000.
understanding of stem cell biology provides a framework for investigating the use of liver
stem cells in treating children afflicted with BA.
Liver Development
As early as embryonic day 8.5 (E8.5), the liver bud sprouts from the anterior
portion of definitive endoderm superiorly toward the heart. Early studies describe the
developing cardiac mesoderm as an important structure that secretes factors to assist in
liver bud formation from the developing foregut (22) (Figure 3) .

Subsequent culturing studies demonstrated that the lateral plate of the septum
transversum mesenchyme may contribute to hepatocyte lineage differentiation and
proliferation (21, 33, 37, 52).
The factors involved in hepatic cell fate in developing endoderm were believed to
be short distance, paracrine, molecules expressed by the precardiac mesoderm (21, 33).
Fibroblast growth factors (FGFs), mitogens known for inducing cell migration,
angiogenesis and epithelial morphogenesis (2, 92, 107), were investigated, due to their
limited range of action and high affinity for the extracellular matrix (15, 42, 72, 95, 117).
In vitro studies of exogenous FGF1 and 2 demonstrated differentiation of ventral
6

Figure 4: Fibroblast growth factors influence liver bud outgrowth. Modified
from Zaret. K. Mech Dev, 2000.
endoderm with expression of the hepatocyte gene albumin, one of the earliest indicators
of liver specification. Moreover, FGF8, a factor not involved in endoderm-hepatic
differentiation, was necessary for liver bud development (42) (Figure 4).
Although FGF is one of the first regulatory proteins described to be essential in
liver bud formation, there are other proteins that work with FGF to coordinate liver
development. Bone morphogenic protein (BMP) 4 is another expressed growth factor
that assists in the outgrowth of hepatoblasts (nascent liver stem cells) with FGFs (78).
The Wnt/ -catenin canonical pathway is a pathway implicated in progenitor cell survival
and differentiation. Our lab has previously shown that by E9.5, FGF10 is another key
ligand in activating the WNT/ -cantenin signaling pathway and maintaining the
hepatoblast population during early murine hepatogenesis. In addition, we demonstrated
small, misshaped liver develop with a lack of Fgf10 or FGF receptor Fgfr2IIIb gene
expression, suggesting it is key in HPC maintenance (Figure 5) (4).
7

With FGFs and other growth factors identified as key early molecules in the
proliferation of nascent hepatic stem cells, other molecules were studied to understand
hepatoblast differentiation. As the initial liver stem cells, hepatoblasts transform into
either hepatocytes or cholaniocytes, which is based on the type of signaling pathways or
transcription factors expressed. Recently, it has been described that cholangiocyte
specific transcription factor HNF1  is expressed when hepatocyte transcription factor
HNF4  is downregulated in a model studying heptoblast differentiation (55). Additional
evidence supports Notch signaling as one mechanism by which hepatoblasts differentiate
toward a biliary lineage. Notch activation decreases expression of hepatocyte
8

transcription factors HNF4 , HNF1 , and c/EBP, while upregulating biliary epithelial
marker HNF1  (97). During liver development, Notch signaling has been well described
as a guide for cholangiocyte differentiation from hepatoblasts. For example, cultured
hepatoblasts with intracellular activation of the Notch receptor were differentiated toward
a bile duct lineage rather than a hepatocyte phenotype (98).
Liver Stem Cells and Regeneration
All stem cells have the unique property to undergo self-renewal and/or
differentiate to a determined cell state in order to develop tissue during development or
restore injured tissue after damage (65). During development, hepatoblasts differentiate
into either cholangiocytes or hepatocytes. Hepatoblasts typically are identified with
antigens present during early embryogenesis, including alpha fetal protein (AFP) (24,
86), hepatocyte growth factor receptor (HGFR) (39), and delta-like protein (dlk) (41, 99).
However, with the liver being one of few organs that can regenerate after injury, there is
clear evidence that postnatal cell behavior that includes proliferation and differentiation
resides in the liver and is activated after injury.
Although not considered a traditional stem cell, hepatocytes are the functional cell
involved in regeneration after acute injury. Within the normal murine liver, the
hepatocyte turnover rate is low: 1 out of every 3000 hepatocytes divides to maintain
homeostatic equilibrium. After injury, this rate increases. For example, after a 70%
partial hepatectomy of the mouse liver, restoration of removed liver mass can be gained
within a week (62). Mechanistically, hepatocytes release autocrine factors (ex.
Hepatocyte Growth Factor, HGF), early after injury to induce self-proliferation (Figure
9

Figure 6: Mechanism of hepatocyte
regeneration. G=G phase of the cell cycle,
S=synthesis phase of the cell cycle Modified
from Michalopoulos, GK, Faseb J, 1990.
6). This is a highly conserved model for liver regeneration and has a documented pattern
for DNA synthesis within murine non-parenchymal fractions and parenchymal cells at 12
and 40 hours post hepatectomy, respectively.
Under states of chronic liver injury,
hepatic regeneration can become
exhausted; unsurprisingly, the liver adapts
with a “backup” stem cell source to restore
non-functional tissue. The exact origin of
these adult progenitor cells is unclear. For
instance, there is evidence of progenitor
cells arising from a hematopoietic stem cell
(HemSC) lineage and carrying markers
common to mesenchymal stem cells, such
as c-kit, SCA-1, and THY-1 (50). In fact,
HemSC have the potential to derive
hepatocytes and rescue terminal mice,
suggesting a possible future for bone
marrow transplantation as a treatment for liver failure (100, 101). Unfortunately, the
concept of differentiated HemSC supplying lost hepatocytes is not steadfast. In 2003
evidence of stem cell-host cell fusion was delineated through lineage tracing (1, 104,
106), questioning the hypothesis of a mesenchymal derived liver rescue.
10

Other adult liver stem cells are hepatic progenitor cells (HPC) that appear after
the liver is chronically injured. Also termed oval cells in the murine literature due to
their shape, HPC arise from the periportal region of the liver and expand transiently and
infiltrate into the parenchyma along bile duct canaliculi once hepatocytes achieve
senescence (26). Characteristic of these stem cells is their importance in potentiality:
after their proliferation, HPC differentiate into hepatocytes and cholangiocytes (Figure 7).
Within rodent models, these cells are transplantable and effective in repopulating
recipient livers (27, 105, 112).

Figure 7: Differentiation of HPC or oval cells after chronic injury. Modified
from Michalopoulos GK,J Cellu Physio 213 (2) 2007.
11

With HPCs possessing great potentiality and ability to regenerate diseased murine
liver, efforts have been used to identify oval cell equivalents in human tissue. Because
HPC appear after hepatocyte regeneration becomes exhausted, chronic liver injury
diseases become more interesting due to these latent progenitor cells. Human, adult and
pediatric liver disease such as severe hepatic necrosis (19), hepatoblastoma (80), and
biliary diseases such as primary billiary cirrhosis and BA (14) have described a distinct
small or oval shaped cell commonly seen in rodent chronic liver injury studies.
Unfortunately, the antigens used to identify HPCs are numerous and species
specific. In addition to co-expression of proteins found on both cholangiocytes (CK8,
CK18, CK19) and hepatocytes (albumin, -fetoprotein), HPC have unique factors used
for recognition. In particular, the antigens A6 (76), EPCAM (69, 96), CD133 (79),
FOXL1 (81) and integrin 6 (CD49f) (30, 110) exclusively tag HPC after chronic liver
injury in murine models, while other markers particularly identify humans or rats. With
our ability to identify HPCs, and use them to galvanize liver regeneration within the end-
stage liver, more attention must be made on how these cells are stimulated and which
factors are significant in their activation.
Various murine liver injury models mimic fibrosis and biliary pathology seen in
ESLD patients. A key feature to each model is hepatocyte senescence due to excessive
fibrosis, and therefore, HPC activation. Many models use hepatocyte injury drugs such as
hepatotoxins, surgical procedures, or both. Common hepatotoxins include, but not limited
to, silica, carbon tetrachloride (CCl
4
), and 3,5-diethoxycarbonyl-1,4-dihydrocollidine
(DDC). Surgical techniques include common bile duct ligation (CBDL), or partial
12

hepatectomy (PHx,70% liver mass removal) administered with the toxin 2-
acetylaminofluorene (2-AAF). In 2005, a comparison between various hepatotoxins and
CBDL was done, and pathology scores and murine survival was assessed. Although
CBDL resulted in a shorter time to cirrhosis, hepatotoxins, such as DDC and alpha-
naphthyl-isothiocyanate (ANIT), resulted in longer survival and just as significant
cirrhotic scores (11). With its unique properties of biliary cirrhosis and high survival
score (Figure 8), DDC was chosen as the appropriate injury model to simulate ESLD in
our study.

Figure 8: Survival Curve describing the percentage of cirrhosis seen
after liver injury with DDC, through diet or intra-peritoneal
injection. Modified from Chang ML, World J Gastroenterol 2005
13

DDC is a well-established model to study chronic cholestatic liver disease with
Mallory body formation (20, 29), biliary cholangiopathies (29), and HPC proliferation
(40) in mice. DDC disrupts proper cytochrome p450 synthesis by removing a ferrous iron
from the heme moiety resulting in protoporhyrin XI accumulation (Figure 9) (16). With
indirect inhibition of ferrocheatalase, DDC reliably reproduces a ductular proliferation
with increased periductal fibrosis and bile acid levels (29) commonly seen in BA and
other ESLDs. With fibrosis and hepatocyte exhaustion, DDC-related intrahepatic
destruction progresses to HPC activation (105); however, the mechanisms that induce
HPC proliferation are unknown and remain an interest for studying.
The mechanisms that stimulate hepatocyte regeneration after acute injury differ
from the signaling pathways responsible for galvanizing the HPC compartment after
Figure 9: Heme biosynthesis pathway. Modified from De Matteis Can
J Physiol Pharmacol, 1996
14

chronic injury. The pathways involved are dependent upon model used (mouse vs. rat),
duration, magnitude, and cellular environment. For example, the tumor necrosis factor
family has data describing HPC expansion in mice directly related to TWEAK (TNF-
weak enhancer of apoptosis) factor (40). With HPCs carrying the important receptor
Fn14, TWEAk induces a HPC response, while its inhibition results in an attenuated
reaction (40). Primary growth factors, such as hepatocyte growth factor (HGF) and
fibroblast growth factor (FGF) have been largely studied in rats and are described as
potent receptors found on HPC capable of promoting expansion (35, 38, 67). However,
with FGF signaling integral in embryonic hepatic stem cell activation and liver
regeneration, much remains to be discovered about FGF signaling in HPC activation.
Fibroblast Growth Factors
Fibroblast growth factors signal through the FGF receptors (FGFRs) to coordinate
embryonic development (17, 46) and regulate angiogenesis and wound repair in the adult
organism. There are 23 members of the FGF family with only 18 being FGFR ligands.
As secreted glycoproteins, FGF target 4 conserved transmembrane tyrosine kinase
receptors (FGFR1-4). Through their various downstream mediators, FGFR exert
different effects on the embryonic and adult tissue.
FGF and FGFR interaction and activation are dependent on ligand-heparin
(sulfate)-receptor binding. Heparin and FGF binding is specific and reliant on specific
heparin binding domains found on FGFs (75). Moreover, the binding of FGFs to heparin
serves to protect FGFs from proteases and to house a pool of FGF to release at
appropriate times (12, 32, 54, 82, 89). For example, the concentration gradient of FGFs
15

established during limb development is specific, as saturation of FGF does not result in
limb growth (12).
FGFR function and signaling relies not only on FGF
ligand and heparin (sulfate) binding, but also on receptor
alternative splicing, dimerization, and tyrosine residue
phosphorylation. Once the FGF-heparin structure binds to
FGFR, the receptor dimerizes and is stabilized (84); thereafter,
the intracellular portion of the phosphotyrosine kinase receptor
becomes active (83) (Figure 10). The function of FGFR
depends largely on the different isoforms of the receptors. The
alternatively spliced FGFR isoforms allow binding of various
FGFs (23, 63, 113). FGFR1, 2, and 3 have exons within the
third Ig-like domain. This domain is spliced so that the receptor
is designated either form IIIb or IIIc. The different isoforms of
the receptors allow promiscuous binding of FGF ligands to
receptors (Table 1).
After dimerization and autophosphorylation, FGFR signaling effects downstream
targets through 2 major pathways: Ras/MAPK pathway and PI-3 kinase/Akt pathway
(Figure 11). Direct stimulation of FGFR with FGFs lead to either Phospholipase C
(PLC) activation, with eventual Protein kinase C activity, or activate the MAPK pathway
directly via phosphorylation of docking protein FGFR substrate 2 (FRS2). FRS2
activation draws other docking proteins such as growth factor receptor-bound 2 (GRB2).
Figure 10: Activated
FGFR1. HSPG=
heparin sulfate
proteoglycans.
Modified from Groth,
C,Int J Dev Biol 2002
16

However, GRB2 is divergent based on the physiology of the cell: GRB2 can activate the
RAS/RAF/MAPK pathways or the Akt-dependent anti-apoptotic pathway (23).

Table 1 Fibroblast growth factor receptors, its isoforms, and their ligands.
Modified from Eswarakumar ,et al.(23).
Figure 11: FGFR signaling pathway. The 2 main tributaries to
downstream activation include the RAS/MAPK and PI-3K/Akt
pathways. Modified from Eswarakumar, Cytokine & Growth Factor
17

Because FGFR signaling assists in endoderm development (8) and promotes
epithelial healing during injury (64, 70) studies have considered the role of FGFR
signaling in liver injury. As previously described, hepatoblast proliferation is activated
partially by the ligands FGF1, 2, 8, and 10, and the receptor FGFR2IIIb. Postnatal
studies have examined the role of FGF and their receptors in the context of liver
regeneration. Believed to be a comparable model to hepatogenesis, liver regeneration
uses adult resident progenitor cells to reconstitute damaged liver parenchyma. Recently
it has been described that liver regeneration is impaired without FGF ligand signaling
after acute injury (93). With significant differences between acute liver injury and
chronic injury, we are studying the role of FGFs in chronic liver injury.
Notch Signaling
The Notch pathway is decisive in mapping cell differentiation within the embryo
and particularly in the hepatobiliary system. It is a mediator of short-range cell-cell
communication and functions to either promote or suppress proliferation, cell death, stem
cell differentiation, or stem cell maintenance, depending on the cell state (Figure 12).
Outlined by 4 transmembrane receptors (Notch 1-4) and 5 ligands (Delta like ligand [Dll]
1, 3, 4, and Jagged 1 and 2), the Notch pathway is detailed by division of an intracellular
domain (ICD) from an extracellular portion (ECD). The separation of the
ICD from the ECD occurs after ligand binding activates the γ-secretase enzyme. The
Notch ICD translocates into the nucleus and influences transcription.
18

Although Notch signaling is present during biliary development, the pathway is
also active during liver recovery after injury. Notch-1 and its ligand Jagged-1 are
expressed with liver regeneration after PHx (47). After HPC induction in AAF/PHx
chronic liver injury models, Notch ligand, dlk, demonstrated increased expression
relative to acute injury models such as a partial hepatectomy alone. Injuries involving
biliary pathology, including intrahepatic ductopenia syndrome Alagille’s, are well
documented to have mutations in Notch receptors or its ligands, such as Jagged 1or
Notch-2 (53, 59, 68). In fact it has been documented that defective Notch signaling leads
to increased reactive ductular cells and decreased differentiation of HPCs to biliary
epithelium in human BA and Alagille patients (25).

Figure 12: Pleiotropic effects of Notch signaling for stem cells
and transactivating cells (TA). Modified from Radtke, F, FEBS
Letters (580) 2006.
19

Biliary Atresia, a bile duct pathology
BA is a biliary developmental pathology diagnosed in children. Even with an
early diagnosis and treatment, children are at an increased risk of developing ESLD and
needing liver transplantation. Characterized by obliteration of the hepatic ducts
connecting the liver to the small intestine, BA has an incidence of 1 in 8,000 live births.
It is the most common case of liver transplant in the pediatric population. Although the
diagnosis is initially made clinically with patients presenting with jaundice and acholic
stools, confirmatory liver biopsy and intraoperative cholangiogram details bile stasis and
plugging, widening of the portal tracts with inflammation, bile duct proliferation, and
inadequate drainage of injected contrast material (73).
Surgery is the only treatment option available for patients diagnosed with BA. If
the patient has a distal atretic segment of the common bile duct, then a
choledochojejunostomy procedure is appropriate, where the remaining bile duct is joined
to a segment of bowel. More commonly, the atretic segment is close to the liver hilum,
and a formal Kasai hepato-portoenterostomy must be performed (Figure 13). Described
by Dr. Kasai in 1968 (45), this surgical technique allows up to 80% adequate bile
drainage after correction (44, 51). However, without correction, these children are
certain for death prior to the age of 2 (43). Unfortunately, patients who have undergone
Kasai hepatoportoenterostomy have a 10 year survival around 33% (44). Children who
develop progressive cholestasis, liver failure, or failure of the Kasai Procedure, are likely
(80%) to undergo liver transplantation following Kasai procedure (51, 57, 73).
20

Figure 13: Kasai hepatoportoenterostomy.
Modified from http://digestive.niddk.nih.gov
Although the field of liver transplantation expanded with the addition of living-
donor transplants, there remains a greater burden for liver demand than supply. Within
the last few decades, cellular liver transplantation using hepatocytes has become a
popular means for treating liver failure (36, 66, 87) and as a bridge to orthotopic liver
transplantation for patients with fulminant
hepatic failure (34, 90, 94). However,
study concerning transplantation of HPC
for liver regeneration is limited.
To date, however, little data exists
describing the relationship between FGF
and Notch pathways and the atypical
ductular proliferation commonly seen in
fibrotic lesions of the liver, such as BA.
Neurodevelopmental studies hint toward
some Notch signaling downstream of
FGFR1 activity (25). The mitogenic
properties of FGF and differentiating characteristics of Notch, we believe, help define the
regenerative characteristics of postnatal HPC after chronic liver injury by inducing HPC
proliferation and later transforming into biliary epithelium.
Hypothesis
The liver is a key organ critical for homeostatic balance of the body, including
drug detoxification, glucose metabolism, and clotting regulation. Unfortunately, during
21

ESLD or with improper development, the liver becomes fibrotic and fails to function
appropriately.
Moreover, little is known about the cellular mechanisms controlling HPC
proliferation and differentiation. Because an HPC population may exist in humans (102)
and FGF and Notch signaling are a significant pathway in HPC activation after acute
liver injury, we hypothesize that FGF and Notch signaling are associated with the HPC
population in liver diseases such as BA, and an understanding of their signaling
mechanisms can enhance our potential stem cell pool for transplantation to these patients.

22

CHAPTER 1: MATERIALS AND METHODS

Mice and Liver Harvest
Livers were harvested from 6 week old, male, wild type C57BL6J mice purchased
from Jackson Laboratories (Bar Harbor, ME), and transgenic mice derived on the a
mixed C57BL6 and CD1 background. Transgenic mice housing the cyclization
recombination enzyme (Cre) under the Rosa26 promoter (Rosa) were mated with a
reverse tetracycline transactivator (rtTA) linked Tet-On system that controlled the
expression of a truncated, soluble form of fibroblast growth factor receptor 2b
(sFGFR2b). sFGFR2b is a dominant negative receptor that lacks the anchoring
transmembrane domain of the protein, which competitively inhibits FGF ligand from
binding to membrane bound FGFR (10). Mice were genotyped for
Rosa
,rtTA+/+
tet(o)sFGFR2b
+/-
(inducible) and Rosa26
rtTA+/+
tet(o)sFgfr2b
-/-
(littermate
controls).
Mice were fed either a standard diet (Test Diet 300) or 0.1% 3,5-
diethoxycarbonyl-1,4-dihydroxylcarbonyl (DDC) and housed in a temperature-controlled
facility with 12 hour light/dark cycles. Wild type C57BL6 mice were fed DDC and
injured at intervals ranging from as short as 12 hours (0.5d) to daily and weekly intervals
of 3 days (3d), 7 days (7d), 14 days (14d), or 42 days (42d). Inducible and littermate
control transgenic mice were given doxycycline infused water 2 days prior to DDC injury
and were continued on the doxycycline water after the start of 2 week DDC treatment.
23

All procedures were done in compliance with Childrens Hospital Los Angeles/Saban
Research Institute guidelines for the use of laboratory animals.
After carbon dioxide euthanasia, mice had their livers flushed with 1x phosphate
buffered saline (PBS) through the portal vein via a 25-gauge needle and 10 mL syringe.
The heart was cut to allow an outflow of perfusate. Portions of the right lobe were taken
for histology, total RNA, and protein collection. The remaining lobes of liver were
bluntly minced in 1x PBS and digested with 0.1% collagenase, 0.1% DNAse, and 0.01%
protease (Sigma-Aldrich, St. Louis, MO) at 37 C for 45 minutes. Enzyme digestion was
stopped with 10% fetal calf serum (FCS; Gibco Carlsbad, CA) diluted in PBS. Digested
tissue was strained through a 70-micrometer mesh filter (BD; Franklin Lakes, New
Jersey) twice. Serial centrifugation was done to isolate hepatocytes and the non-
parenchymal fraction (NPF) layer. After 1 minute at 50xg (Eppendorf; Hamburg, GER),
the hepatocyte cell suspension was isolated. Sequential centrifugation steps at 50xg
(Eppendorf) for 1 minute three times, and a final centrifugation at 100xg for 8 minutes
was done to isolate the NPF. Cells were immediately frozen at -80 C or prepared for
fluorescent assorted cell sorting analysis.
Immunostaining
Harvested tissue was immediately placed in 15 mL of 4% formaldehyde
(Polysciences; Warrington, PA) and 30% sucrose overnight for fixation. PFA fixed
tissue was sequentially dehydrated with 15 mL of 70% ethanol and 100% ethanol (3
changes of each solution for an hour with final solution overnight). After removing the
ethanol solution, fifteen mL of 100% toluene was placed on each tissue twice an hour at a
24

time. Half of the toluene was removed and replaced with heated paraffin (60 C) for a
total volume of 15 mL for 1 hour at 60 C. This was exchanged for one volume of
paraffin (15 mL) and stored overnight at 60 C. Paraffin blocks were freeze-mounted
(0 C) onto plastic. Five-micron sections were taken from each block (Leica RM2235
microtome) and placed on frosted
coated glass slides (Ted Pella;
Redding, CA) while warmed in 42 C
deionized water. Slides were warmed
overnight at 37 C prior to staining.
Slides were prepared for
staining using a standard protocol for
deparaffinization and tissue hydration.
Slides were washed in 0.6%
hematoxylin (Fischer Scientific; Fair
Lawn, New Jersy) for 2 minutes twice.
The slides were then sequentially
moved through a series of dehydration
steps with ethanol (range from 30%-
100%) for one minute at a time. Slides were finally washed in water for 15 seconds.
Slides were either stained with hematoxylin and eosin (Harleco, Lawrence, KS) or
prepared for immunofluorescence.
Antibody Host
Pan-Cytokeratin (PCK)
Sigma (1:200)
Rabbit
Proliferating Cell Nuclear
Antigen (PCNA)
Santa Cruz (1:200)
Goat
Fibroblast Growth Factor
Receptor 1 (FGFR1/FLG)
Santa Cruz (1:200)
Rabbit
Notch1 intra-cellular
domain (NICD)
AB CAM
Rabbit
Akt (AKT)
Cell Signaling
Rabbit
Phospho-Akt (P-AKT)
Cell Signaling
Rabbit
Glyceraldehyde-3-
Phosphate Dehydrogenase
(GAPDH)
Meridian Life Science
Mouse
Hairy and enhancer of split
1 (HES1)
Santa Cruz (1:200)
Rabbit
Table 2 Antibodies for Immunostaining
25

Fluorescently stained slides were prepared for antigen retrieval with 1 mM Tris-1
mM EDTA-0.05%Tween (pH 9.0) buffer. Slides were washed in 1x PBS for 10 minutes.
Tissue was then blocked with 5% goat serum in 1x 0.1% Tris buffered saline with 20%
Tween (TBST) at 25 C in a humidified box. After a 10 minute wash in 1x PBS, thirty μL
of primary antibody diluted in 5% goat serum in 0.1% TBST was added per tissue section
and incubated overnight at 4 C. The following day the primary antibody was removed
with a 30 minute 0.05% TBST wash. Secondary antibody, diluted in 5% goat serum and
0.1% TBST, was incubated in a humidified box for 1 hour. A final wash in 0.05% TBST
for 30 minutes was performed prior to imaging. Microscopy was performed with Leica
DM5500, using the LAS-AF software (Leica; Wetzlar, GER). Table 2 lists the antibodies
used for immunostaining.
Western Blot Sample Preparation and Analysis
Tissue lysate buffer preparation (Rippa buffer) using stock solutions of 1 molar
Tris (pH 7.4), NaOH, Deoxycholate, 10% Sodium dodecyle sulfate, 0.5 molar EDTA,
PhosphoSTOP phosphotase inhibitor (Roche, GER), and protease inhibitor was prepared.
Tissue was individually homogenized (Omni Inc; Kennesaw GA) in 2 milliliter
Eppendorf tubes with 250 milliliters of buffer. Samples were then centrifuged at 1000xg
(4 C) for 10 minutes. One hundred microliters of sample was mixed with 2x Lammeli
sample buffer containing 20mM of -mercaptoethanol (BIORAD, Hercules, CA).
Samples were boiled in a water bath (100 C) for 10 minutes. The remaining sample was
stored for Bradford protein concentration assay.
26

For the Bradford micro-protein assay, a standard curve was prepared using 0, 2, 4,
6, 8, and 10 μg of BSA. One milliliter of 1x Bradford protein assay reagent was mixed
with serial concentrations of BSA, and absorbance readings were done at 595nm on a
spectrophotometer (Pharmacia Ultraspec III). Each sample was measured in duplicates
and the average was taken to construct a BSA standard curve.
Protein concentration of tissue lysates was measured in the same way using 1x
Bradford reagent and quantified using the calculated BSA standard curve (Figure 14).
Fifty micrograms of lysate protein was separated on a 8% SDS-polyacrylamide gel. Gel
electrophoresis was conducted in BIORAD Mini-PROTEAN Tetra Cell gel
electrophoresis kit at 100V for 2hours in 1x Tris/Glycine/SDS “running” buffer
(BIORAD). Separated proteins were transferred onto 0.2 μm nitrocellulose membrane
(BIORAD), using 1x Tris/Glycine “transfer” buffer (BIORAD) with 200 mL of methanol
(Sigma Aldrich) in the Mini-Protean Tetra Cell (BIORAD) kit at 200mAmps for 2 hours
and 30 minutes.

BSA Bradford Standard Curve
y = 0.0506x + 0.0027
R
2
= 0.9883
0
0.1
0.2
0.3
0.4
0.5
0.6
02468 10 12
BSA concentration (mg)
Figure 14: BSA-Bradford Standard Curve.
27

Membranes were blocked with 5% non-fat milk or 5%BSA in TBS-0.05%
Tween-20 for non-specific binding for 1 hour at 25 C. Membranes were incubated with
primary antibody at 4 C overnight. Horse raddish peroxidase (HRP)-conjugated
secondary antibodies were incubated at 25 C for 1 hour (Table 2). Washings done in
between antibody incubations consisted of 0.05% TBST (Sigma Aldrich) for 10 minutes
at 25 C. Signals were detected on an AutoRad radiographic film (ISC Bioexpress) using
a chemiluminescent reagent (Denville Scientific). Exposure times ranged from less than
5 seconds to no longer than 1 minute, and all were developed using the Konica SRX-
101A developer.
Reverse Transcriptase Polymerase Chain Reaction (RT-PCR)
Harvested tissue was stored at -80 C until preparation for RNA extraction began.
Total RNA was isolated by using the Quigen RNeasy Mini kit. In brief, approximately
30mg of tissue was used for RNA extraction. The tissue was disrupted and
homgenization using 25 gauge needle with the supplied lysis buffer containing -
mercaptoethanol. An equal volume of 50% ethanol was added to the lysate. The sample
was added to a supplied, filtered, centrifuge tube and spun for 15 seconds at 8000xg.
DNase digestion (Qiagen; Valencia, CA) was used on the spin column with the supplied
solution and sequential centrifugation steps. RNA was collected in samples volumes of
30 μL in RNase free water (Qiagen). mRNA concentration and purity were determined
by Nanodrop spectrophotometry (ND-1000, Thermo Scientific, Wilmington, DE)
Complementary DNA (cDNA) was prepared with use of iScript cDNA synthesis
kit (BIORAD). In short, a master mix solution was made using the supplied nuclease-
28

free water, iScript reaction mix consisting of deoxynucleotides and random primer
sequences, and iScript reverse transcriptase. After the addition of RNA, the PCR solution
was incubated for 5 minutes at 25 C, then 30 minutes at 42 C. To heat-inactivate the
reverse transcriptase, the reaction was raised to 85 C for 5 minutes. Synthesized cDNA
was prepared at a concentration of 50 ng per μL.
Qualitative RT-PCR was performed using cDNA harvest from the NPF and whole
liver samples. PCR was performed using Taq polymerase and nuclease free water
(Qiagen). PCR reactions were heated to 94˚C, cooled to a specific annealing
temperature, and finally heated again to 72˚C at 30 seconds intervals each. Every PCR
went for 35 cycles. Two percent, ethidium bromide mixed, agarose gel electrophoresis
was done loading 20μL of PCR solution diluted in 5x sample buffer. Band detection was
done on a BIORAD transluminescene system. Primer sequences and annealing
temperatures are listed on Table 3.

29

Gene
Forward Primer Reverse Primer length Ta
FGF Signaling
Fgfr1b TCTGGCCTCTACGCTTGC AGGATGGGAGTGCATCTG
A
140bp 62
C
Fgfr1c TGCTGGAGTTAATACCAC
CG
TTCCAGAACGGTCAACCA
TG
141bp 58
C
Fgfr2b CCTACCTCAAGGTCCTGA
AGC
CATCCATCTCCGTCACATT
G
180bp 62
C
Fgfr2c AACGGGAAGGAGGTTTA
AGCAG
TGGCAGAACTGTCAACCA
TGC
503bp 58
C
Fgf1 CCGAAGGGCTTTTATACG
G
TCTTGGAGGTGTAAGTGTT
ATAATGG
244bp 55
C
Fgf7 ACTATCTGCTTATAAAAT
GGCTGCT
GTGGGGCTTGATCATCTG
AC
548bp 55
C
Fgf10 CGGGACCAAGAATGAAG
ACT-
AACAACTCCGATTTCCACT
GA
546 60
House Keeping Genes
β-actin TGTTACCAACTGGGACG
ACA
GGGGTGTTGAAGGTCTCA
AA
165bp 55
C
Notch Pathway
Jagged1 ATCTGTCCACCTGGCTAT
GCAG
ATCACTTCGCAGGTGGTG
GTAC
64
C
Jagged2 GTCGTCATTCCCTTTCAG
TTCG
AGTTCTCATCACAGCGTA
CTCG
217bp
Delta1 TAATAGGCCTGCGAAGG
AAG
GTCCACGGAGAGGTGAGT
GT
159bp 60
Delta2 GGGCAGGAAAGTTCTGT
GACAAAGAT
CAAACACCACCAGGCAAC
CAGGCTA
690bp 64
Notch1 CAATCAGGGCACCTGTG
AGCCCACAT
TAGAGCGCTTGATTGGGT
GCTTGCGC
710bp 69
C
Notch2 ACATCATCACAGACTTGG
TC
ATTATTGACAGCAGCTGC
C
399bp 60
C
Table 3: Primers used for RT-PCR
30

Fluorescent Activated Cell Sorting (FACS)
Isolated NPF cells were sorted fluorescently to detect the HPC population after
DDC injury. Cells underwent red blood cell lysis with RBC lysis buffer. Cells were
counted, and approximately 1x10
6
cells were re-suspended in 400 μl of 1x PBS with10%
FCS. Non-specific binding was prevented with the use of a rat anti-mouse-Fc-blocker AB
(BD Pharmigen) for 10 minutes at 4
o
C. Cells were stained with antibodies (1:20) to
identify HPCs. Cells were washed in 1x PBS and re-suspended in 1x PBS with 1% FCS
for FACS analysis. The antibodies used to sort the cell suspension are listed on Table 5.
Control samples include CD49f-PE, CD45-APC-Cy7, CD-133-APC, unstained control,
and cells that were heated at 37 C (control for dead cells) stained with Dapi.

31

CHAPTER 2: RESULTS

DDC stimulates oval-shaped cell and bile ductule expansion
3,5-diethoxycarbonyl-1,4-dihydroxylcarbonyl (DDC) is a hepatotoxin that
reduces hepatocyte regeneration after injury while stimulating latent HPCs to proliferate.
We began by recapitulating DDC injury by feeding 6- week old wild type mice 0.1%
DDC mouse chow at various intervals. Light microscopy of hematoxylin and eosin
stained tissues confirms that in comparison to a regular chow fed mouse stains (0d), oval-
shaped cells devoid of a large cytoplasm appear adjacent to bile ducts as early as 3 days
(Figure 15, panel A and B). By the second week of injury (14d), more oval-shaped cells
appeared adjacent to bile ductules. In addition, ductular proliferation occurs, with more
than one bile duct per portal vein. (Figure 15 arrows). These abnormal ducts spread into
the periphery and do not associate with one portal vein.
32

FGFR1 expression is associated with DDC injury
Analysis of mRNA from the NPF identified the FGF signaling receptors and
ligands involved in DDC related liver injury. Figure 16 shows the qualitative PCR bands
demonstrating expression of FGFR and ligands, after various points of injury. When
investigating FGF signaling, we found that Fgf1 is consitiuitively expressed during DDC
injury in comparison to control (Figure 16), while Fgf10 is expressed within the first 12
hours and after 10 weeks of DDC injury. Surprisingly, Fgf7 was not expressed after
Figure 15: DDC leads to progressive ductular proliferation.
WT liver portal tract structures after (A) regular chow feed
[0d]. (B) 3d of 0.1% DDC chow feed. (C) 7d of 0.1% DDC
chow feed, (D) 14d of 0.1% DDC chow feed. PV=portal vein,
arrows = bile ducts
33

Figure 16: Fgfr1 and Fgf10 mRNA transcripts are expressed after DDC
injury. + (positive) control=E18.5 lung RNA, Neg Control=no reverse
transcriptase
DDC injury. The FGF receptors involved with DDC injury appear to be subtypes 1b and
1c (Fgfr1b and Fgfr1c). In contradiction to embryonic HPC activation, Fgfr2 mRNA is
not involved in HPC activation after DDC. With high mRNA expression of Fgfr1 and its
ligands, Fgf1 and Fgf10, after DDC injury, we investigated the protein translation of the
receptor, and any change in proliferation that may accompany its expression. FGFR1 is
expressed adjacent to bile ducts shortly after DDC injury (Figure 17, panel B). Staining
the cell membrane, FGFR1 expression greatly increases with successive injury of DDC.
34

Cells expressing the receptor are not adjacent to the bile ducts after 2 weeks of injury but
permeate the liver parenchyma (Figure 17, panel D). Congruent with an increase in
receptor expression is

Figure 17: FGFR1 is expressed after DDC injury. WT liver after (A)
regular chow feed, 0.1% of DDC chow (B) for 3d, (C) for 7d, and (D) for
14d. PCK, FGFR1, DAPI. PV=portal vein, 20x magnification
35

the increase in proliferating cell nuclear antigen (PCNA, Figure 18). As early as 3 days
after DDC injury, there is presence of PCNA expression within cell nuclei. In fact,
similar to FGFR1 expression, PCNA continues to be rapidly expressed with longer
periods of DDC exposure. (Figure 18). Quantitatively, the number of cells expressing
FGFR1 pleateaus after 14 days of injury (Figure 19-red). PCNA also plateaus after 2
week of injury, as well (Figure 19-green). Interestingly, the lack of co-expression of
these cells may explain a signaling mechanism between the two different cell
populations.

Figure 18: Proliferation of cells after DDC injury. WT liver after (A)
regular chow feed, 0.1% DDC chow for (B) 3d, (C) 7d, (D) 14d. PV=portal
vein, bile ducts outlined, PCNA, DAPI, 20x magnification
36

Notch1 and HES1 are expressed after DDC injury
To investigate if the Notch pathway is involved in atypical ductular proliferation
after DDC injury, whole liver mRNA transcripts were analyzed for Notch receptors and
ligands. Notch1 is expressed early in DDC injury with a peak of mRNA expression after
one week of injury. Notch-2 and ligands Jagged1, 2, and delta like ligand 2 (Dll2) are
not transcribed. Dll1, however, is transcribed as early as 0.5 days after injury and
appears maximally expressed after 14 days of injury (Figure 20).
Protein expression of Notch receptor and activation are seen as well through
immunofluorescence and histochemistry staining. After two weeks of DDC injury,
Notch-1 intracelluar domain (NICD) is expressed in the nucleus of adjacent periportal
cells (Figure 21). These dark stained nuclei are in close proximity to the expanded
periportal regions (Figure 21, panel B and D). While Notch downstream mediator, Hes-
1, is expressed adjacent to bile ducts as well (Figure 22). Hes-1 expression increases
Percent Total Cells Expressing FGFR1 or PCNA after
DDC Injury
0
5
10
15
20
0d 0.5d 3d 7d 14d
Days of Injury
% total cells
FGFR1
PCNA
Figure 19: % Total cells expressing FGFR1 and PCNA after DDC injury
37

after 7 days of DDC injury and remains high after 14 days of injury. (Figure 22, panel B
and D).

Figure 21: NICD is expressed after 14 days of DDC injury. WT liver after 14
days of 0.1% DDC chow. (A-B) 20x magnification, (C-D) 40x magnification,
(A-C) control without primary antibody. PV=portal vein, *=bile duct
Figure 20: Notch receptor and ligand mRNA expression after DDC
injury. Positive Control= E18.5 lung RNA Neg Control= no reverse
transcriptase
38

Dominant-negative FGFR2b inhibition decreases CD49f
+
CD45
-
cells
Transgenic mouse models are powerful tools important in detailing the necessity
of a particular gene of interest in forming a phenotype. Using mice with the fibroblast
growth factor dominant-negative gene construct, Rosa
rtTA
;tet(o)sFGFR2b
+/-
, DDC injury
resulted in a change in number of CD49f
+
CD45
-
cells in comparison to littermate
controls. FACS for HPC markers CD49f shows a 40% reduction in the stem cell marker,
relative to control DDC-fed mice (Figure 23).

Figure 22: HES-1 is expressed after DDC injury. Wt liver after (A) regular
chow feed, 0.1% DDC chow for (B) 3d, (C) 7d, (D) 14d. 40x magnification.
HEs-1, PCK, DAPI
39

Figure 23: Percent CD49f
Pos
CD45
Neg
cells in dominant negative
transgenic mice after 14 days of DDC injury. Control=littermante
controls Induced=Rosa
rtTA
;tet(o)sFgfr2b
+/-
Percent CD49f+ CD45- cells in
RosartT A;tet(o)sFgfr2b+/- mice after 14 days of
DDC injury
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
Control Induced
% total cells

Dominant-negative FGFR2b inhibition results in decrease AKT activation
Fibroblast growth factor receptors are tyrosine kinase receptors with many downstream
targets that include MAPK pathway and the AKT pathway. Evidence of MAPK or AKT
pathway activation is the phosphorylation of downstream transcription factors ERK and
AKT, respectively. Western blot of whole tissue lysates from transgenic mice injured
with DDC chow demonstrated presence of phosphorylated AKT. However, littermate
controls have increased expression of phosphorylated AKT in comparison to dominant-
negative soluble FGFR2b (Figure 24). Although not statistically significant, we believe
there is a trend that suggests a decrease in AKT pathway activation.

40

Dominant-negative FGFR2b inhibition results in decreased proliferation, FGFR1
expression, and Notch activation
FGFs are mitogens that are commonly associated with proliferation. With
decreased activity of FGF ligands through soluble FGFR2b expression, dominant
negative inhibition of FGF signaling demonstrated a reduction in FGFR1 surface (Figure
25, panel A) and HES1 nuclear expression (Figure 25, panel B) in comparison to
littermate controls, suggesting a relationship between the two pathways .

Figure 24: Phosphorylated AKT expression is reduced with dominant negative expression of
soluble FGFR2b. (A) Optical relative intensity of P-AKT compared to AKT in control vs induced
animals, (B) Scanned WB bands in control vs induced mice. P-AKT=Phosphorylated AKT,
GAPDH=glyceraldehydes-3-phosphate-dehydrogenase
41

Figure 25: HES and FGFR1 expression decreases in dominant negative transgenic
expression of soluble FGFR2b after 14 days of DDC injury. (A) Littermate Control, (B)
Rosa
rtTA
;tet(o)sFgfr2b
+/-
, PV=portal vein, Dashed area=bile duct, 40x magnification
42

CHAPTER 3: DISCUSSION

Reportedly, FGF signaling is associated with acute and chronic liver injury
models and HPC activation (91, 114, 115). In addition, Notch signaling is described as a
critical pathway involved in normal bile duct development and intrahepatic bile
ductopenia in humans and mice (58). With pediatric biliary pathologies, such as BA,
potentially associated with a HPC response (14) and Notch signaling (25), we have found
a relationship between FGF and Notch signaling after HPC expansion in a model
resembling BA.
BA is characterized by bile duct (ductular) proliferation, periportal fibrosis, bile
plugging, and in late stages cirrhosis (43). Moreover, DDC injury is associated with
progressive cirrhosis, periportal fibrosis, and HPC activity via ferrochetalase inhibition
(16, 60). Histologically, we have confirmed that progressive injury occurs with reactive
proliferation of the periportal domain. Unfortunately, given the ample data suggesting
DDC is a model for end-stage liver disease (11, 76), little attention is given to its
similarities to BA. Although there is no evidence of extra-hepatic bile duct pathology
with DDC injury, intrahepatic cirrhosis and ductular proliferation is similar to BA, and
DDC may be a useful murine model to study this.
FGFR1 is expressed more than FGFR2 after fibrotic, ductular DDC injury than
acute, hepatocyte injury, such as partial hepatectomy. mRNA and protein expression of
FGFR1 was progressively elevated with DDC injury, but not FGFR2. Currently, it is
believed that both FGFR1 and FGFR2 are essential receptors to promote liver
43

regeneration after PHx and acute injury with CCl
4
(6). The authors also note that FGF7
appears to be the ligand necessary to activate the receptors. This does not appear to be
true for biliary fibrosis and HPC directed regeneration. FGFR1 is the only receptor
expressed, and Fgf1 and 10, not Fgf7, is expressed to varying degrees with injury. An
explanation for this maybe with hepatocyte senescence and chronic liver injury, HPC
express FGFR1, as opposed to FGFR2 seen with hepatocyte expression, and therefore,
bind ligands that do not include Fgf7.
Steiling et al. described FGFs as potential mitogens for activating liver
regeneration after partial hepatectomy, by using a transgenic, membrane-bound
FGFR2IIIb, dominant-negative receptor mouse (93). We showcased similar results using
the Rosa
rtTA
;tet(o)sFGFR2b
+/-
transgenic mouse injured with DDC. However, we believe
the FGF signaling assists with HPC activation in the setting of chronic injury. FGFR2
isoform IIIb binds to FGF1, 3, 7, 10, and 22. Our transgenic mice, that lack the
transmembrane domain of the receptor, bind secreted FGF. By diluting the available
pool of growth factors, induced transgenic mice have limited the activity of FGFR2b and
any FGFR that binds the same ligands. FGFR1, in particular, binds to FGF1, 2, 3, 4, 5, 6,
and 10. Our reduction in FGFR1 expression after secretion of soluble FGFR2IIIb,
confirms this logic. Our evidence of the reduction in the stem cell marker CD49f after
DDC injury with expression of the soluble FGFR2IIIb isoform details a direct link of
FGF signaling in HPC activation in the setting of chronic injury.
Notch signaling is imperative to bile duct development and biliary disease.
Numerous studies outline Notch receptor and ligand deficiencies as inducers of
44

intrahepatic bile duct pathology, such as the ductopenia present in Alagille’s Syndrome
(48, 53). However, the ductular proliferation associated with BA has not been fully
characterized with regards to the Notch signaling pathway. Fabris et al (25) described an
association between Notch and BA, by detailing common cytokeratin expressions in BA
and Alagille’s Syndrome liver samples. Unfortunately, without specific Notch receptor,
ligand, or downstream mediator identification, conclusions about Notch signaling in the
setting of BA is equivocal.
Our data suggest an interaction between Notch-1 and DLL1, which may describe
intrahepatic bile duct proliferation after chronic injury. Notch-1 and NICD expression
with DDC injury strengthens a potential relationship between Notch-1 and abnormal bile
duct development. Geisler in 2008 (31) detailed the importance of Notch-2 in
intrahepatic bile duct development, relative to Notch-1. Using conditionally expressed,
floxed Notch-1 gene, Notch-2 gene, or both, Geisler purposed lack of Notch-2 leads to
portal inflammation and fibrosis. Although our data indicates Notch-1 is involved in
DDC biliary pathology, the low to non-existent expression of Notch-2 after DDC injury
correspond to Geisler’s findings.
Cross talk of the FGF and Notch signaling pathways is previously described in
neurodevelopment models (7, 28, 116). Faux, et al (28) described Notch signaling can be
downstream of FGFR1 expression in inhibition of neuro-differentiation. As described
earlier, FGF signaling is associated with intrahepatic bile duct development (111), hinting
toward an interaction between the two pathways in bile duct development. We, however,
have shown a dramatic decrease of Notch downstream mediator HES-1 with decreased
45

FGF signaling. This suggests Notch activation appears to be under the control of FGF
during bile duct injury and proliferation.
Although we have data to propose a mechanism of HPC activation through FGF
and Notch signaling, there are limitations to our study that have to be addressed prior to
making these conclusions. DDC is a model of chronic injury and has not been proposed
as a model for studying the intrahepatic bile duct proliferation commonly seen in biliary
atresia. As there are many current models for biliary atresia (71, 74, 77), our
observations should be confirmed in another BA animal model or human tissue.
Noteworthy in our data is the constitutive appearance of Fgf1 mRNA and lack of
Fgf7 expression after DDC injury, which differs from conclusions drawn by Bohm et al.
that suggest FGF7 is critical to liver regeneration (6). In addition, we found Fgf10
expressed after 0.5 days and 70 days of injury (not shown). Because we previously
described FGF10 as a key ligand in embryonic HPC activation (4) and Fgf10 expression
precedes the expansion of adult HPC after DDC injury, we believe Fgf10 may be
involved in postnatal HPC activation after injury. An in vitro study comparing FGF10
and FGF7 administration to NPF cells after DDC injury may clarify this relationship.
The dominant negative receptor, soluble fibroblast growth factor receptor 2b,
limits the efficacy of secreted FGFs, by sequestering ligands that traditionally bind to
FGFR2b while maintaining signaling through unaffected receptors (103). Our Rosa-Cre
Tet-on system drives sFGFR2IIIb expression ubiquitously in the mouse. Therefore,
FGFs that bind to FGFR2IIIb have an attenuated effect after secretion. Although this
indirect inhibition of FGFR1 activity can explain our data, a dominant negative FGFR1
46

(108), soluble vs membrane bound, siRNA of Fgfr1, or FGFR1 specific inhibitory
ligands may strengthen our causation data and confirm a mechanism between Notch and
FGFR1.

47

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Abstract (if available)

Abstract INTRODUCTION: The liver is a multi-purposeful organ, susceptible to injury. Following acute injury, cells such as hepatocytes, regenerate, while latent hepatic progenitor cell (HPC) become active after chronic liver injury. Some studies suggest Fibroblast growth factors (FGF) may activate HPCs similarly to embryonic liver stem cells. In addition, Notch signaling is critical in bile duct development and injury. With progenitor cell transplantation as an alternative to tissue transplantation, we hypothesize that FGF and Notch signaling pathways induce proliferation of HPC after chronic liver injury.

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Creator Vendryes, Christopher Lee (author)

Core Title Fibroblast growth factors and notch signaling in a diethoxycarbonyl dihydrocollidine-induced hepatic progenitor cell liver injury model

School Keck School of Medicine

Degree Master of Science

Degree Program Biochemistry and Molecular Biology

Degree Conferral Date 2010-12

Publication Date 12/08/2010

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag cirrhosis,fibroblast growth factors,livers,notch,OAI-PMH Harvest

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Advisor Tokes, Zoltan A. (committee chair), Kalra, Vijay K. (committee member), Wang, Kasper (committee member)

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