My Lifeeeeee Oviis

Liver Regeneration
Edited by Dieter Hussinger
Liver Regeneration
Edited by Dieter Hussinger
DE GRUYTER
Editor
Prof. Dr. Dieter Hussinger
Department of Internal Medicine
Gastroenterology, Hepatology and Infectious Diseases
University Hospital Dsseldorf
Heinrich-Heine-University
Moorenstrasse 5
D-40225 Dsseldorf
Germany
This book has 46 gures and 7 tables.
The cover image shows a section through a regenerating rat liver 5 days after partial hepatectomy.
Sprouting blood vessels are shown in red color, and nuclei in blue color. The image was produced
by the Lammert and Hussinger laboratories.
ISBN 978-3-11-025078-7
e-ISBN 978-3-11-025079-4
Library of Congress Cataloging-in-Publication Data
Liver regeneration / edited by Dieter Hussinger.
p. ; cm.
Includes bibliographical references.
ISBN 978-3-11-025078-7 (alk. paper)
1. LiverRegeneration. 2. LiverDiseases. 3. Stem cells.
I. Hussinger, D. (Dieter), 1951[DNLM: 1. Liver Regenerationphysiology. 2. Stem Cellsmetabolism. WI 702]
QP185.L57 2011
611'.36dc22
2011009091
Bibliograc information published by the Deutsche Nationalbibliothek
The Deutsche Nationalbibliothek lists this publication in the Deutsche Nationalbibliograe; detailed
bibliographic data are available in the Internet at http://dnb.d-nb.de.
2011 Walter de Gruyter GmbH & Co. KG, Berlin/Boston.
The publisher, together with the authors and editors, has taken great pains to ensure that all information presented in this work (programs, applications, amounts, dosages, etc.) reects the standard of
knowledge at the time of publication. Despite careful manuscript preparation and proof correction,
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Preface
The liver has a high capacity to regenerate, which was already known in ancient
Greece, as exemplied in the Prometheus saga. Although liver regeneration has been
paradigmatic for organ repair and renewal for more than 2,000 years, only during the
past decades has much effort been devoted to the understanding of the molecular and
cell biological mechanisms underlying liver regeneration. Such knowledge is of crucial
importance for clinical medicine not only regarding liver physiology and pathology,
but also for the use of stem cells for cell therapy and liver surgery. This graduate-level
text book provides an overview of the current state of knowledge about the molecular mechanisms of liver regeneration. The chapters were written by renowned experts
and active researchers in the eld of liver regeneration; some of them members of the
Collaborative Research Center 575 Experimental Hepatology. Hepatic stem cells are
introduced, and the important players involved in regeneration, such as oval cells, bone
marrow, and stellate cells, are reviewed. Also, the cell-signaling pathways that initiate
liver regeneration and regulate the switch between proliferation and apoptosis are presented. The book also treats the epigenetic regulation of liver stem cells and the roles of
inammation and angiogenesis in liver regeneration. This compact overview of the fascinating regenerative capacity of the liver will be of interest to both, graduate students
and postdoctorate scientists in molecular biology, biochemistry, and medicine, and it is
hoped that this survey on the various aspects of liver regeneration will stimulate further
research in this area and help young scientists develop their research strategies. The
topics treated are central to the biomedical curriculum, including stem cell research,
cancer biology, cell signaling, and epigenetics.
I would like to express my sincere thanks not only to the authors for their excellent
contributions but also to my collaborators, Mrs. Katrin Nagel, editor for science, technology, and medicine, from de Gruyter Publishers for her excellent collaboration and
professional help in preparing and producing this book project, and Dr. Martin Lay for
the artwork and beautiful illustrations.
Dsseldorf, May 2011
Dieter Hussinger
Contents
Author Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Abbreviations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
xi
xv
Liver Regeneration and Partial Hepatectomy: Process and Prototype . . . . . . .

Marie C. DeFrances and George K. Michalopoulos
1.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1.2 Liver Regeneration: Historical Perspective . . . . . . . . . . . . . . . . . . . . . . .
1.3 Partial Hepatectomy as a Means to Study Liver Regeneration . . . . . . . .
1.4 Three Phases of Liver Regeneration after Partial Hepatectomy . . . . . . . .
1.5 Future Directions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1
1
2
4
10
Oval Cells, Bone Marrow, and Liver Regeneration . . . . . . . . . . . . . . . . . . . . .

Anna C. Piscaglia, Antonio Gasbarrini, and Bryon E. Petersen
17
2.1 Stem Cells: Denition and Properties . . . . . . . . . . . . . . . . . . . . . . . . . .

2.2 Liver Stem Cells and Their Role in Hepatic Regeneration. . . . . . . . . . . .
2.3 Extrahepatic Stem Cells with Hepatogenic Potential:
The Blood of Prometheus. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.4 Clinical Applications of Bone MarrowDerived Stem Cells
in Hepatology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
17
21
25
30
Inammation and Liver Regeneration. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Johannes G. Bode
39
3.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.2 Liver Regeneration and Inammation: General Aspects. . . . . . . . . . . . .
3.3 Liver Macrophages and Their Relevance for
Liver Regeneration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.4 Inammatory Mediators Are Required to Promote
Liver Regeneration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.5 Inappropriate Inammation Impairs Liver Regeneration . . . . . . . . . . . .
3.6 Role of NK and NKT-cells for Liver Regeneration:
Negative Regulators of Regeneration. . . . . . . . . . . . . . . . . . . . . . . . . . .
39
40
41
44
46
47
viii
Contents
Lymphotoxin Receptor and Tumor Necrosis Factor Receptor p55 in Liver

Regeneration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Ursula R. Sorg and Klaus Pfeffer
53
4.1 The TNF/TNFR Superfamily . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

4.2 Liver Regeneration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.3 TNFRp55 and Liver Regeneration . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.4 LTR and Liver Regeneration. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
53
56
57
58
5 The Hepatic Stem Cell Niches . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Iris Sawitza, Claus Kordes, and Dieter Hussinger
63
5.1
5.2
5.3
5.4
5.5
6
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Secreted Factors in the Stem Cell Niche . . . . . . . . . . . . . . . . . . . . . . . .
Physical Contacts of Stem Cells with Their Niche . . . . . . . . . . . . . . . . .
Identication of Stem Cell Niches . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Stem Cell Niches in the Liver . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
63
64
71
72
74
Stellate Cells in the Regenerating Liver. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Claus Kordes, Iris Sawitza, and Dieter Hussinger
85
6.1
6.2
6.3
Characterization of Stellate Cells. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Plasticity of Hepatic Stellate Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Stellate Cells in Liver Regeneration . . . . . . . . . . . . . . . . . . . . . . . . . . . .
85
90
90
Epigenetics during Liver Regeneration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

99
7.1
7.2
7.3
7.4
8
Denition and Mechanisms of Epigenetics . . . . . . . . . . . . . . . . . . . . . .

Methods to Investigate Epigenetic Mechanisms . . . . . . . . . . . . . . . . . . .
Epigenomics in Liver Regeneration . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Epigenetics During Stellate Cell Activation . . . . . . . . . . . . . . . . . . . . . .
99
103
104
105
Hedgehog Signaling and Liver Regeneration . . . . . . . . . . . . . . . . . . . . . . . . .

Steve S. Choi and Anna Mae Diehl
111
8.1
8.2
8.3
8.4
8.5
111
112
112
113
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Liver Regeneration after Partial Hepatectomy . . . . . . . . . . . . . . . . . . . .
Fetal Development of the Liver . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Overview of Hedgehog Signaling Pathway . . . . . . . . . . . . . . . . . . . . . .
Reactivation of the Hedgehog Pathway
after Partial Hepatectomy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
8.6 Hedgehog Pathway Activation during Repair of
Chronic Liver Injury: General Concepts . . . . . . . . . . . . . . . . . . . . . . . .
8.7 Hedgehog Pathway Activation and Liver Progenitors
in Chronic Injury Models . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
115
116
117
Contents
8.8
8.9
8.10
9
Hedgehog Pathway Activation and Liver Fibrosis . . . . . . . . . . . . . . . .

Hedgehog Pathway Activation and Vascular Remodeling in
Injured Livers. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Hedgehog Pathway Activation and Hepatocarcinogenesis . . . . . . . . .
EGFR, CD95, and the Switch between Proliferation and

Apoptosis in Hepatic Stellate Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Roland Reinehr and Dieter Hussinger
9.1 Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
9.2 Liver Cell Proliferation Involves Ligand-dependent
EGFR Activation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
9.3 Liver Cell Apoptosis Involves EGFR-dependent
CD95 Activation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
9.4 EGFR Activation Can Couple to Both Proliferation and
Apoptosis in Hepatic Stellate Cells. . . . . . . . . . . . . . . . . . . . . . . . . . . .
ix
118
121
121
129
129
130
132
135
10 Angiogenesis and Liver Regeneration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Tobias Buschmann, Jan Eglinger, and Eckhard Lammert
145
10.1 Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
10.2 Blood Flow and Cell Types in the Adult Liver . . . . . . . . . . . . . . . . . . .
10.3 Angiogenesis in Liver Regeneration . . . . . . . . . . . . . . . . . . . . . . . . . .
10.4 Importance of VEGF for Liver Regeneration . . . . . . . . . . . . . . . . . . . .
10.5 Role of Angiogenesis in Liver Damage/Disease . . . . . . . . . . . . . . . . .
10.6 Questions and Problems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
145
145
148
150
151
153
11 A Quantitative Mathematical Modeling Approach to

Liver Regeneration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Dirk Drasdo, Stefan Hoehme, and Jan G. Hengstler
159
11.1
11.2
11.3
11.4
11.5
11.6
11.7
Denition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Methods to Quantify SpatialTemporal Information
in Liver Lobules . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Normal Liver Lobule: The Reference State . . . . . . . . . . . . . . . . . . . . .
Quantifying the Regeneration Process: Process Parameters . . . . . . . .
Mathematical Model . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Simulation Results with the Mathematical Model. . . . . . . . . . . . . . . .
Limitations. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
12 Animal Models for Studies on Liver Regeneration . . . . . . . . . . . . . . . . . . . .

Amalya Hovhannisyan and Rolf Gebhardt
12.1
12.2
Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Different Types of Regenerative Processes . . . . . . . . . . . . . . . . . . . . .
159
161
163
164
164
169
171
175
175
175
Contents
12.3 Different Types of Animal Models . . . . . . . . . . . . . . . . . . . . . . . . . . .

12.4 Surgical Animal Models . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
12.5 Pharmacological Models . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
12.6 Transgenic Models . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
12.7 Immunological Models . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
177
179
180
181
186
13 Therapeutic Potential of Bone Marrow Stem Cells in Liver Surgery . . . . . . . .

Jan Schulte am Esch, Moritz Schmelzle, Gnter Frst, and
Wolfram Trudo Knoefel
191
13.1
13.2
13.3
13.4
Clinical Scenario . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Mechanisms of Hepatic Regeneration . . . . . . . . . . . . . . . . . . . . . . . .
Stem Cells in Liver Regeneration . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Mesenchymal or Hematopoietic Stem Cells to
Support Liver Regeneration?. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
13.5 BMSC as External Conductors of Liver Regeneration . . . . . . . . . . . . .
13.6 Stem Cell Treatment in Chronic Liver Disease in Humans . . . . . . . . .
13.7 BMSC to Support Liver Proliferation Prior to Hepatectomy. . . . . . . . .
191
192
192
193
194
194
195
Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
207
Author Index
Johannes G. Bode, MD
Gastroenterology, Hepatology and
Infectious Diseases
Moorenstrasse 5
D-40225 Dsseldorf
Germany
Tobias Buschman
Institute of Metabolic Physiology
Universittsstrasse 1
D-40225 Dsseldorf
Germany
Steve S. Choi, PhD
Division of Gastroenterology
Duke Liver Center, Duke University
DUMC 3256
595 LaSalle Street, Suite 1073
Durham, NC 27710
USA
and
Section of Gastroenterology
Durham Veterans Affairs Medical Center
Durham, NC 27710
USA
Marie C. DeFrances, MD, PhD
Department of Pathology,
McGowan Institute for Regenerative
Medicine
and
University of Pittsburgh Cancer Institute
University of Pittsburgh
200 Lothrop Street
Pittsburgh, PA 15261
USA
Anna Mae Diehl, MD

Division of Gastroenterology
Duke Liver Center, Duke University
DUMC 3256
595 LaSalle Street, Suite 1073
Durham, NC 27710
USA
Dirk Drasdo, PhD
Institute National de Recherche
en Informatique et en Automatique
Paris-Rocquencourt
France
and
Interdisciplinary Centre for Bioinformatics
University of Leipzig
D-04103 Leipzig
Germany
Jan Eglinger, PhD
D-40225 Dsseldorf
Germany
Gnter Frst, MD
Department of Diagnostic and
Interventional Radiology
Moorenstrasse 5
D-40225 Dsseldorf
Germany
xii
Author Index
Antonio Gasbarrini, PhD

Gastrointestinal and Liver Stem Cell
Research Group (GILSteR)
Gemelli Hospital
Catholic University of Rome (Italy)
Largo A. Gemelli
8 00168 Roma
Italy
Rolf Gebhardt, PhD
Institute of Biochemistry
Faculty of Medicine
Johannisallee 30
D-04103 Leipzig
Germany
Dieter Hussinger, MD
Infectious Diseases
Moorenstrasse 5
D-40225 Dsseldorf
Germany
Jan G. Hengstler, MD
Leibniz Research Centre for Working
Environment and Human Factors
Ardeystrasse 67
D-44139 Dortmund
Germany
Stefan Hoehme, PhD
Interdisciplinary Centre for Bioinformatics
D-04103 Leipzig
Germany
Amalya Hovhannisyan, MD, PhD
Institute of Biochemistry
Faculty of Medicine
Johannisallee 30
D- 04103 Leipzig
Germany
Wolfram Trudo Knoefel, MD

Department of General-, Visceral- and
Pediatric Surgery
Moorenstrasse 5
D-40225 Dsseldorf
Germany
Claus Kordes, PhD
Infectious Diseases
Moorenstrasse 5
D-40225 Dsseldorf
Germany
Eckhard Lammert, PhD
D-40225 Dsseldorf
Germany
George K. Michalopoulos, MD, PhD
Department of Pathology,
McGowan Institute for Regenerative
Medicine
and
University of Pittsburgh Cancer Institute
University of Pittsburgh
200 Lothrop Street
Pittsburgh, PA 15261
USA
Bryon E. Petersen, PhD
Organogenesis Program
Department of Regenerative Medicine
Institute of Regenerative Medicine
Wake Forest University Baptist Medical
Center
Medical Center Boulevard
Winston-Salem, NC 27157-1094
USA
xiii
Klaus Pfeffer, MD
Institute of Medical Microbiology and
Hospital Hygiene
D-40225 Dsseldorf
Germany
Anna C. Piscaglia, PhD
Gastrointestinal and Liver Stem Cell
Research Group (GILSteR)
Gemelli Hospital
Catholic University of Rome (Italy)
Largo A. Gemelli
8 00168 Roma
Italy
Roland Reinehr, MD
Infectious Diseases
Moorenstrasse 5
D-40225 Dsseldorf
Germany
Iris Sawitza, PhD
Infectious Diseases
Moorenstrasse 5
D-40225 Dsseldorf
Germany
Moritz Schmelzle
Pediatric Surgery
Heinrich Heine University
Moorenstrasse 5
D-40225 Dsseldorf
Germany
Jan Schulte am Esch, MD
Pediatric Surgery
Moorenstrasse 5
D-40225 Dsseldorf
Germany
Ursula R. Sorg, PhD
Institute of Medical Microbiology and
Hospital Hygiene
D-40225 Dsseldorf
Germany
Abbreviations
2-AAF
2/3 PHx
AFP
AP-1
APAP
APP
ASC
ASH
ASM
ATSCs
BLP
BM
Bmp/BMP
BMSCs
BTLA
BV
CCC
CCl4
ChIP
CHX
CoH
CR
CRD
CT
DcR3
DD
Dhh
DISC
Dpp
DPPIV
ECM
EGF
EGFP
EMT
ENA-78
ER
ESC
FADD
FFA
FGF
2-acetylaminouorene
two-thirds partial hepatectomy
alpha-fetoprotein
activator protein 1
acetaminophen
acute phase proteins
adult stem cell
alcoholic steatohepatitis
acidic sphingomyelinase
adipose tissue stromal cells
basal lamina proteins
bone marrow
bone morphogenetic protein
bone marrow stem cells
B and T lymphocyte attenuator
blood vessels
cellcell contacts
carbon tetrachloride
Chromatin immunoprecipitation
cycloheximide
canal of Hering
cysteine rich
cysteine-rich domain
computed tomography
decoy receptor 3
death domains
Desert hedgehog
death-inducing signaling complex
Decapentaplegic
dipeptidyl-peptidase-IV-decient
extracellular matrix
epidermal growth factor
enhanced green uorescent protein
epithelial-to-mesenchymal transition
epithelial neutrophil-activating protein
endoplasmic reticulum
embryonic stem cell
Fas-associated death domain
free fatty acid
broblast growth factors
xvi
Abbreviations
FLRV
Fsrp
FXR
G-CSF
G-CSFR
GF
GFAP
GS
HB-EGF
HCC
Hep
HGF
Hh
HHIP
HIFs
HPCs
HSA
HSCs
HUVEC
HVEM
IBD
ICAM
IGFBP1
Ihh
iPSs
IKK
ILK
INR
JNK
KCs
LGLs
L-NAME
LPS
LSECs
LSCs
LT
MAP
MAPCs
M-CSF
MELD
MIP
MMP
MSCs
NASH
NC
NICD
NIK
future liver remnant volume

Follistatin related protein
farnesoid-X-receptor
granulocyte-colony stimulating factor
G-CSF receptor
growth factor
glial brillary acidic protein
glutamine synthetase
heparin-binding EGF
hepatocellular carcinomas
hepatocytes
hepatocyte growth factor
hedgehog
hedgehog interacting protein
hypoxia-inducible factors
hepatic progenitor cells
hepatocyte sinusoid alignment
hepatic stellate cells
human umbilical vein endothelial cells
herpes virus entry mediator
intralobular bile duct
intercellular adhesion molecule
insulin growth factor binding protein 1
Indian hedgehog
inducible-pluripotent stem cells
IKB kinase
integrin linked kinase
international normalized ratio
Jun Kinase
Kupffer cells
large granular lymphocytes
NG-nitro-L-arginine methyl ester
lipopolysaccharide
Liver sinusoidal endothelial cells
liver stem cells
liver transplantation
mitogen-activated protein
multipotent adult progenitor cells
macrophage colony stimulating factor
model for end-stage liver disease
macrophage inammatory protein
matrix metalloproteinases
mesenchymal stem cells
non-alcoholic steatohepatitis
neighboring cell
Notch intracellular domain
NFB inducing kinase
Abbreviations
NK
NKT
NO
NOS-2
OCs
OSM
PCI
PCNA
PCR
PDGF
PHx
PI3K
PRR
PUMA
PVE
PVP
RLGS
ROS
SC
SCF
SECs
SERPIN
sFRP
Shh
SNS
SUMO
tBDL
TGFalpha
TGFbeta
TGFBRI / TGFBRII
TIMs
TLR
TLV
TNF
TNFalpha
TNFRI
TRADD
TRAF
TRE
TUDC
TWEAK
Tx
uPA
uPAR
Wnt
YAP
xvii
natural killer
natural killer T
nitric oxide
nitric oxide synthase 2
oval cells
oncostatin M
Protein C inhibitor
proliferating cell nuclear antigen
polymerase chain reaction
platelet-derived growth factor
partial hepatectomy
phosphatidylinositol 3-kinase
pathogen recognition receptors
p53 up-regulated modulator of apoptosis
portal venous embolization
portal venous pressure
restriction landmark genomic scanning
reactive oxygen species
stem cell
stem cell factor
sinusoidal endothelial cells
serine protease inhibitor
soluble frizzled related peptide
Sonic hedgehog
sympathetic nervous system
small ubiquitin-related modier
Total bile duct ligation
transforming growth factor alpha
transforming growth factor beta 1
TGFbeta receptor I / TGFbeta receptor II
TRAF-interacting molecules
Toll like receptor
total liver volume
tumor necrosis factor
tumor necrosis factor alpha
TNF receptor I
TNF-receptor associated death domain
TNF-receptor associated factor
tetracycline response element
tauroursodeoxycholate
transforming growth factor like weak inhibitor of apoptosis
transcription
urokinase plasminogen activator
urokinase plasminogen activator receptor
wingless type
Yes-associated protein
Liver Regeneration and Partial Hepatectomy:

Process and Prototype
Marie C. DeFrances and George K. Michalopoulos
Learning Targets
1. Recognize the three phases of liver regeneration after partial hepatectomy: initiation/
priming, proliferation, and termination
2. Understand the utility and drawbacks of the partial hepatectomy technique to study
the process of liver regeneration
3. Describe the major cellular and molecular events that characterize each phase of
liver regeneration
1.1
Introduction
The liver is characterized by a unique and extraordinary capacity for self-renewal; it

is the only internal solid organ in the mammal to fully regenerate after injury or loss.
This occurs through organized proliferation of all resident cell types resulting in restored function. Other organs, such as cardiac muscle (Bergmann et al., 2009) or central
nervous system (Brill et al., 2009), may demonstrate some endogenous propensity for
regeneration, particularly after an insult, but complete organ restoration and functional
recovery (as seen with the liver) are not the norm. In fact, liver tissue decits are readily
and rapidly replenished (in just a matter of 1 or 2 weeks in rodents), even following
extensive loss of up to ~75% of liver mass. Such a remarkable competence for renewal
has been capitalized upon by surgeons to cure patients of resectable hepatic tumors and
cysts as well as to safely and effectively provide a source of transplantable tissue in the
case of living related liver donation.
1.2
Liver Regeneration: Historical Perspective
Although a fairly clear understanding of what drives hepatic cells to regenerate has been
established during the past several decades, the concept of liver regeneration may have
originated thousands of years earlier. Of all internal organs, the liver appears to be the
most revered by ancient civilizations who bestowed upon it mystical properties. Among
them, the liver was believed to house the soul of the individual (Chen and Chen, 1994),
and by virtue of its subcapsular scars and other peculiarities, to harbor insights into the
future that could be divined by soothsayers (i.e., hepatoscopy) (Power and Rasko, 2008).
Liver Regeneration and Partial Hepatectomy
It also gured prominently in Hesiods myth of Prometheusa Greek god who, having
stolen re from Zeus to give as a gift to humans, was punished by daily consumption of
his liver (which regrew overnight) by Zeuss eaglethe tale embodies the phenomenon
of endless hepatic renewal that we embrace today. Some argue, however, that the reference to liver regeneration in the story of Prometheus is not one based on the ancient
Greeks having any direct knowledge of the process, per se, but merely reects their
assignation of immortality to the gods, and by extension, to their gods livers (Power
and Rasko, 2008)! Regardless of which of these possibilities is true, the mere mention of
liver renewal in a work of classical literature familiar to so many over the centuries may
have been sufcient enough to prompt early researchers to test its scientic merit.
1.3
Partial Hepatectomy as a Means to Study Liver

Regeneration
It is only in the relatively recent past that liver regeneration has become the focus of
systematic, rigorous scientic investigation. As surgical techniques underwent renement and survival following surgery improved in the late 1800s, surgeons and scientists
alike began to experiment with hepatic resections in animals (Power and Rasko, 2008).
By 1931, Higgins and Anderson (1931) had devised the classic surgical model that is
still widely in use today. It is referred to as two-thirds partial hepatectomy (2/3 PHx)
and was rst performed on the rat. Following lapartomy, the anterior lobes (i.e., the
large medial lobe and the left lateral lobe) of the rat liverconsisting of approximately
68% of the liver mass (i.e., 2/3)are ligated at the hilus and resected. As the animal
recovers, the excised anterior lobes of the liver do not regrow; rather, the remaining
lobes undergo compensatory hyperplasia via replication of the cells, therein restoring
the liver to its original mass in about one to two weeks (Higgins and Anderson, 1931)
(fFigure 1.1).
The liver is mainly composed of hepatocytes, which account for approximately 60%
of the cellular constituents (Daoust and Cantero, 1959) (but roughly 80%90% of liver
mass, underscoring the fact that hepatocytes are rather large cells [about 30 uM in
diameter]). Stellate cells (hepatic stromal cells that produce and secrete growth factors
and extracellular matrix and store lipids and fat-soluble vitamins), Kupffer cells (resident
hepatic macrophages), sinusoidal endothelial cells (SECsspecialized endothelia that
display punctate membrane conduits, or fenestrae, which permit certain blood-borne
nutrients, metabolites, and toxins direct access to hepatocytes) and cholangiocytes (biliary epithelial cells) contribute the remaining hepatic cell numbers and add to tissue
mass.
In response to partial hepatectomy in mammals, an orderly progression in DNA synthetic activity and replication is observed among the different hepatic cell types. In the
rat, for example, hepatocytes begin to enter DNA synthesis at about 12 hours post-PHx
with a robust peak observed at 24 hours after surgery. (For mice, the pinnacle of DNA
synthetic activity is slightly later at 3644 hours post-PHx.) A second smaller surge of
hepatocyte DNA synthetic activity typically occurs about 48 hours later (at 6072 hours
postsurgery). The remaining hepatic cells types replicate subsequently: DNA synthesis
in Kupffer cells, stellate cells, and cholangiocytes reaches a maximum at about 4872
hours post-PHx, while SEC DNA replication peaks at 34 days after surgery (Michalopoulos and DeFrances, 1997).
1.3
Partial Hepatectomy as a Means to Study Liver Regeneration
portal venous pressure

NO release
HGF/Met activation
beta-catenin translocation
notch signaling
cytokine release
EGFR activation
Tx factor binding
TGFbeta
60 minutes
5 minutes
G1 progression
collagenase activation
3 ~12 hours
immed early gene expression
sinusoidalization
ILK activation
TGFbeta redeposition
glypican-3, IL-1
Yap, GF expression
4 7+ days
13 hours
termination
initiation /
priming
2/3 PHx
proliferation
all cell constituents
proliferate
ECM deposition
~12 hours 4 days
Figure 1.1 Liver Regeneration after 2/3 Partial Hepatectomy

Notes: Following surgical resection of the two anterior hepatic lobes of rodents accounting for
~68% (2/3) of liver tissue, the remaining lobes undergo compensatory hyperplasia restoring
the liver to its original presurgical mass. Liver regeneration, which reaches completion in
about 714 days, can be divided into three phases: Initiation/priming (which lasts ~12 hours
after surgery), proliferation (extending from ~12 hours to 4 days post-PHx), and termination
(accounting for the remainder of the time). Each phase is characterized by specic events as
indicated. NO = nitric oxide, Tx = transcription, ECM = extracellular matrix, PHx = partial
hepatectomy.
Historically, 2/3 PHx in rodents has been a heavily utilized method to study liver
regeneration. It is rather simple to perform with a fairly high survival rate (Palmes and
Spiegel, 2004). The procedure can be easily modied so that more or less tissue (than
~70%) is excised, although surgically removing greater than ~75% of hepatic mass
compromises survival of the animal due to, among other reasons, hepatic hyperperfusion associated with ischemia/reperfusion injury and acute liver failure. Otherwise, it
has been shown that the degree of ensuing hepatic cell replication is proportional to
the amount of liver mass excised (Bucher and Swafeld, 1964). It seems that a hepatic
rheostat (or hepatostat), the exact nature of which remains to be resolved, is at play to
delicately regulate initiation and termination of the regenerative response, thus ensuring
that it is wholly adequate and appropriate.
Other compensatory hyperplasia models have been developed to study the process of
liver regeneration. For example, toxins (such as carbon tetrachloride [CCl4]) that cause
hepatocyte necrosis, inammation, cytokine release, and liver regeneration can be administered to rodents (Palmes and Spiegel, 2004). Another method induces bipotential
liver stem cells (oval cells) to replicate and differentiate into hepatocytes; in one version
of this model, rodents are treated with the chemical 2-acetylaminouorene (2-AAF) to
inhibit hepatocyte proliferation and then subjected to partial hepatectomy to stimulate
oval cell replication, differentiation, and, ultimately, liver repair (Evarts et al., 1987).
A downside of the PHx model may be that it lacks direct applicability to most common clinical scenarios. For example, patients who must regenerate liver mass after hepatic surgery often have cirrhosis, hepatic viral infection, steatosis, or liver metastases,
or are liver transplant recipients. The standard PHx model does not recapitulate the
physiologic complexity of these types of cases. In addition, wild animals that undergo
endogenous liver regeneration do so because of exposure to environmental hepatotoxins or suffer from hepatic infections (i.e., woodchuck hepatitis virus in the case of the
groundhog; Snyder et al., 1982), not as a result of a sterile and precise excision of pristine hepatic tissue. Despite these acknowledged drawbacks, the 2/3 PHx model remains
a uniquely valuable system to delineate the mechanisms underlying liver regeneration:
its relative simplicity, its reproducibility among different laboratories, the fact that hazardous chemicals need not be handled nor administered to animals, and a relative lack
of tissue inammation or necrosis (as seen in some other models, the extent of which
can be variable among animals impacting the regenerative response and thus muddling
data interpretation) make its use compelling.
1.4 Three Phases of Liver Regeneration after Partial

Hepatectomy
An obvious question to ask is, Why does the liver regenerate so rapidly and efciently
after partial hepatectomy? The answer is understandably complex. The entire process
can be roughly divided into three phases:
1)
2)
3)
initiation/primingthe majority of hepatocytes exit a quiescent state (G0), enter

the cell cycle (G1), and cross the G1/S checkpoint. Dissolution of the extracellular
matrix (ECM) begins. In the rat, this phase lasts about 1218 hours. Although it is
the shortest of the three phases, it has been perhaps the most intensely analyzed in
order to identify the primary event that triggers liver regeneration. Studies reveal that
rapid and pronounced alterations in a multitude of signaling pathways and other
tissue functions occur simultaneously and no single alteration likely predominates
(Michalopoulos, 2010).
proliferationhepatocytes synthesize DNA, complete the remainder of the cell
cycle, and reenter G0 ; a small proportion of hepatocytes engage in a subsequent
round of mitosis. Remodeling of the ECM proceeds. Other hepatic cell types such
as cholangiocytes and SECs divide. This phase extends from 1218 hours to about
4 days after PHx in rodents.
terminationthe remainder of the regenerative period (day 4 to day 7) is devoted
to diminishment of progrowth cues, recommencement of inhibitory signaling,
replenishment of liver mass, and return of hepatic homeostasis (fFigure 1.1).
1.4 Three Phases of Liver Regeneration after Partial Hepatectomy
1.4.1
Phase One: Initiation/Priming
During the initiation/priming phase of liver regeneration after PHx, the very rst event
to transpire following excision of liver tissue is an immediate induction of sheer stress
in the portal circulation reected by an increase in portal venous pressure (PVP)
(Schoen et al., 2001). The liver is fed by two blood supplies: (1) the portal vein (which
provides the liver about 75% of its blood) carries to the liver nutrients, toxins, bile
acids, and other substances absorbed or produced by the gastrointestinal tract for
further metabolism, if necessary; and (2) the hepatic artery, although contributing less
blood by volume, supplies the liver with, among other things, a necessary source
of oxygen, hormones, cytokines, and immune surveillants (lymphocytes, monocytes,
etc.). Increased PVP is accompanied by release of nitric oxide (NO) in the liver, likely
by endothelial cells (Schoen et al., 2001). Blocking NO synthase by NG-nitro-L-arginine
methyl ester (L-NAME) administration inhibits c-fos mRNA expression typically induced 15 minutes after PHx (Schoen et al., 2001) and prevents liver enlargement at
48 hours after surgery (Wang and Lautt, 1998). NO may also be produced later in
regeneration by Kupffer cells, hepatocytes, or other liver constituents through induction of nitric oxide synthase 2 (NOS-2, also referred to as inducible NOSiNOS)
(Hortelano et al., 2007). Animals engineered to lack NOS-2 show reduced liver mass
beginning at 3648 hours after PHx (Kumamoto et al., 2008; Rai et al., 1998) (although
liver mass of mice in one of the studies reached control levels by day 7; Kumamoto
et al., 2008).
SECs react to changes in PVP by increasing the diameter of fenestrae and overall
porosity at 5 minutes post-PHx (Wack et al., 2001). At the same time (5 min. after surgery), the hepatocyte plasma membrane depolarizes (Zhang et al., 1996), but preventing depolarization does not diminish the gene expression signature usually observed
within 11.5 hours after surgery, suggesting that depolarization has little impact on
the early stages of regeneration (Minuk et al., 1997). Beta-catenin, a transcriptional
regulator normally bound to E-cadherin at the hepatocyte plasma membrane, migrates
to the hepatocyte nucleus to activate target genes within 5 minutes of resection. This
is accompanied by E-cadherin downregulation, which may account in part for betacatenins rapid subcellular redistribution (Monga et al., 2001). Proper hepatic development is regulated by the Notch/Jagged signaling system; mutation of either the Jagged-1
or Notch-2 gene is associated with a paucity of intrahepatic bile ducts (referred to as
Alagille Syndrome) in humans. Jagged is a cell surface ligand that binds and activates
Notch, its transmembrane receptor expressed on adjacent cells. Following interaction,
Notch undergoes enzymatic cleavage, and its intracellular domain (NICD) moves to
the nucleus to regulate gene transcription. Fifteen minutes after PHx, NICD appears in
the nuclei of hepatoctyes (and possibly other hepatic constituents such as endothelial
cells) peaking at 15 minutes postsurgery. Injection of Jagged-1 siRNA to rats prior to
PHx blunts DNA synthesis particularly at the day 2 post-PHx time point, suggesting that
the Jagged/Notch paradigm is active during hepatic repair in addition to development
(Khler et al., 2004).
Within 1 minute after PHx, interaction of the urokinase plasminogen activator (uPA)
and its cell surface receptor (uPAR) expressed by hepatocytes promotes increased uPA
activity (Mars et al., 1995), which is a signicant event because uPA is a serine protease
responsible for cleaving and activating a variety of proteins. For example, uPA converts
plasminogen into plasmin, which in turn cleaves brinogen into brin. Plasmin abundance in the liver shows a small uptick at 15 minutes and a major peak at about 36
hours after PHx, while brinogen levels fall in the liver 15 minutes following surgery
(Kim et al., 1997). uPA is also one of the main enzymes responsible for activating hepatocyte growth factor (HGF) into its biologically functional form (Mars et al., 1993).
Interestingly, mice lacking uPA, but not uPAR, in hepatocytes show delayed regeneration after PHx (Roselli et al., 1998), suggesting that uPA functions normally in response
to surgery without the aid of its receptor.
While investigations of plasmin and brin in liver regeneration continue, HGFs role
in the process is well-documented. Studies in the 1950s had shown that when the circulatory systems of two rats are joined and one of the pair is subjected to PHx, not only
does the liver of the animal that underwent liver resection regenerate, but the liver of
the unoperated rat responds with increased DNA synthesis. This led to the postulate that
a soluble blood-borne factor was responsible for inciting liver regeneration after PHx
(Bucher et al., 1951). In the late 1980s, three groups described HGF, a protein isolated
from plasma or serum capable of stimulating isolated hepatocytes in culture to undergo
DNA synthesis (Miyazawa et al., 1989; Nakamura et al., 1989; Zarnegar et al., 1989).
HGF and the protein epidermal growth factor (EGF) have since been proven to be the
most potent mitogenic stimuli for hepatocytes in culture (Michalopoulos and DeFrances, 1997). EGF family members, such as transforming growth factor alpha (TGFalpha)
(Luetteke et al., 1988) and heparin-binding EGF (HB-EGF) (Ito et al., 1994), also act as
inducers of DNA synthesis in cultured hepatocytes.
During the very early time points in liver regeneration, HGF sequestered by proteoglycans is released and cleaved by active uPA bound to uPAR. Biologically functional
HGF then binds its transmembrane receptor Met stimulating the receptors tyrosine
kinase activity (within 1 min. of surgery but reaching a maximum level at 60 min.)
(Stolz et al., 1999), while the majority of active HGF is subsequently released into
the circulation with a peak plasma level seen at 60120 minutes following surgery
(Lindroos et al., 1991). Interestingly, healthy patients undergoing right hepatectomy
as part of living related liver donation also show a prominent spike in serum levels
of HGF (but not EGF, VEGF, nor TGFalpha) at 2 hours postsurgery (Emova et al.,
2005).
A series of studies have solidied HGF and Met as key regulators of liver growth and
regeneration. Enforced expression of HGF in mouse hepatocytes hastens recovery after
PHx (Bell et al., 1999; Shiota et al., 1994) up to 3-fold over controls (Shiota et al., 1994).
In fact, if HGF is infused into the portal vein of mice over a 5-day period, livers increase
in size by 140% via a hyperplastic response and return to normal upon terminating
HGF treatment (Patijn et al., 1998). This observation highlights a remarkable phenomenon: that HGF is sufciently potent to prompt a regeneration-like response in the
liver in the absence of tissue damage or loss. Meanwhile, regeneration is signicantly
impaired in mice in which the Met tyrosine kinase domain is deleted specically in the
liver (Borowiak et al., 2004) or hepatocytes (Huh et al., 2004), while delivery of a Met
shRNA to the hepatocytes of rats delays DNA synthesis after PHx by 24 hours (Paranjpe
et al., 2007). In addition to regulating regeneration after PHx, this growth factor/receptor system is also necessary for proper embryonic liver development (Bladt et al., 1995;
Schmidt et al., 1995; Uehara et al., 1995), and both are dysregulated in hepatocellular
carcinoma (DeFrances, 2010).
EGF is produced by exocrine glands of the alimentary tract (the salivary glands and
Brunners glands of the duodenum, in particular); its availability to the liver and ultimately to its cell surface receptor (EGFR) expressed on hepatocytes may be enhanced
by virtue of altered portal circulation after liver surgery (Skov Olsen et al., 1988). While
EGFR shows low level constitutive tyrosine phosphorylation in the unoperated liver, at
60 minutes post-PHx maximum EGFR phosphorylation is observed (Stolz et al., 1999).
In experimental models, mice lacking EGFR in hepatocytes have a lower liver-to-body
weight ratio at day 7 after PHx as compared to controls (Natarajan et al., 2007), and
rats treated with EGFR shRNA respond to PHx with signicantly reduced mitotic activity
and appear to restore liver mass (which ultimately reaches control levels), at least in part
through hepatocyte hypertrophy (Paranjpe et al., 2010). Of note, however, is the nding that animals treated with anti-EGFR monoclonal antibodies regenerate their livers
normally after surgery (Van Buren et al., 2008).
Within the 3060 minutes after surgical resection, the livers reticuloendothelial
compartment, and perhaps other nonparenchymal cells such as cholangiocytes, are
stimulated to produce cytokines, including tumor necrosis factor alpha (TNFalpha) and
interleukin-6 (IL-6). These cytokines likely act in an autocrine manner on Kupffer cells
(particularly with respect to TNFalpha), in a paracrine fashion on neighboring cells such
as hepatocytes, and possibly by an endocrine route because their plasma levels peak
at 1 hour after PHx. TNFalpha and IL-6 are responsible at least in part for the surge
in DNA binding activity of an established set of transcriptional activators in the liver
in response to hepatic surgery (Diehl and Rai, 1996). They include AP-1 (DNA binding at 1560 min. post-PHx and involve c-jun, c-fos, and other AP-1 partners; Diehl
et al., 1994), c-myc (mRNA expression increasing at 15 min. post-PHx; Thompson
et al., 1986), NFkappaB (DNA binding appearing at 30 min. and disappearing 60 min.
following PHx; Cressman et al., 1994), STAT-3 (DNA binding beginning at 1 hour and
peaking at 34 hours after surgery; Yamada et al., 1997), and C/EBPbeta (2- to 3-fold
higher DNA binding than presurgical values at 3 hours post-PHx returning to normal by
24 hours after surgery; Diehl and Yang, 1994).
If signaling of the cytokines or the transactivators they induce is experimentally perturbed, a range of postsurgical outcomes (from no effect to moderate delay in liver
regeneration) is observed. For example, TNF receptor I (TNFRI) knock-out mice show
moderate reductions in DNA synthesis following hepatic resection (Shimizu et al.,
2009; Yamada et al., 1997); however, by 14 days after surgery, their liver weights reach
control levels (Shimizu et al, 2009). In one report, IL-6 injection restores the mitotic
response in these animals, suggesting that IL-6 lies downstream of TNFalpha signaling
(Yamada et al., 1997). Performing PHx in mice in which one partner of the IL-6 receptor (glycoprotein 130gp130which mediates signaling) is deleted specically in
hepatocytes results in reduced STAT-3, c-jun, NFkappaB, and c-myc expression but no
overt difference in liver regeneration (Wuestefeld et al., 2003). Mice that lack the p50
subunit of NFkappaB in hepatocytes show no change in DNA synthesis or overall liver
weight (DeAngelis et al., 2001). This may be due to the fact the NFkappaB is activated
predominately in Kupffer cells and SECs, and not in hepatocytes, after PHx (Sakuda
et al., 2002). It should be noted that the addition of cytokines such as TNFalpha to
cultured hepatocytes does not stimulate appreciable DNA synthetic activity, but when
coupled with a known hepatic mitogen, synergistic effects on DNA synthesis are observed (Diehl and Rai, 1996). Thus, they are best referred to as auxillary mitogens or
priming factors. Substances that augment the effect of hepatic growth factors akin to
TNFalpha and IL-6 include catacholamines, thyroxine, insulin, and others (Michalopoulos and DeFrances, 1997).
Several signal transduction pathways are upregulated early after PHx. These pathways
are stimulated by a combination of activated growth factor receptors (such as Met)
and cytokine signaling. For example, within 15 minutes after PHx, TNFalpha appears
to upregulate Jun Kinase ( JNK, also known as MAPK8), which phosphorylates c-jun to
enhances c-juns transcriptional activity (Diehl et al., 1994). Blocking the PI3K pathway
by injection of wortmannin, a potent PI3K inhibitor, results in reduced liver-to-body
weight ratio as compared to controls at 48 and 72 hours post-PHx, returning to presurgical levels by 7 days after surgery in mice ( Jackson et al., 2008), while liver-specic
loss of PDK1, a serine threonine kinase that binds PI3K-generated PIP3 at the plasma
membrane and phosphorylates AKT, causes death of mice when 32 PHx is performed.
However, when 30% hepatectomy is carried out, no signicant difference in mitotic
rate is noted between the mice lacking PDK1 or controls (Haga et al., 2009). The p42/44
MAPK pathway is upregulated in a biphasic pattern in rats after surgery: a peak is seen
at 1 hour post-PHx, remains elevated, and peaks again at about 5 hours before falling
to presurgical levels by 16 hours (Chen et al., 1998).
The net effect of the concerted stimulation of hepatocytes by growth factors, cytokines, and mechanical distortion is increased expression of 70 genes, some of
which are described as immediate early genes because their abundances rise about
12 hours after surgery. They encode for transcription factors such as early growth
response-1 (egr-1), growth factor modulators like insulin growth factor binding protein 1 (IGFBP1), and other proteins necessary for the hepatocyte to exit quiescence,
engage in DNA synthesis and undergo cell replication hours to days later (Haber
et al., 1993). As hepatocytes traverse the G0 /G1 boundary, it is necessary for the contact
between cells and their extracellular environment to be modied in a precise manner.
Within minutes of PHx in rats, the protein abundances of bronectin, entactin, and
laminin fall, with bronectin and entactin levels returning to near pre-PHx levels by
1824 hours after surgery (Kim et al., 1997). Later after surgery, the activity proles
of metalloproteinases MMP-9 and MMP-2 rise (at about 36 hours and 612 hours,
respectively), and perhaps as a safeguard to prevent excessive matrix dissolution, the
protein level of a key metalloproteinase inhibitor (TIMP-1) accumulates at roughly the
same time (618 hours following surgery) (Kim et al., 2000). A nadir in heparan sulfate
proteoglycan content is also observed in the rat liver at 12 hours after surgery (Matsuya
et al., 2001).
Given the surge in pro-growth signaling and matrix remodeling required to induce a
regenerative response in the liver following surgery, it seems intuitive that the activity
of hepatic mitoinhibitors would be simultaneously suppressed. Transforming growth
factor beta 1 (TGFbeta) added to cultures of hepatocytes exposed to mitogens prevents
cells from entering DNA synthesis ( Jakowlew et al., 1991) and are arrested at the G1/S
checkpoint. After PHx in rats, TGFbeta mRNA levels in the liver show a biphasic pattern
rising sharply early after surgery (2 hours) then falling and reaching a nadir at 24 hours
before climbing again; expression is conned to the nonparenchymal compartment
( Jakowlew et al., 1991). To transmit inhibitory signals, TGFbeta binds a heterodimeric
receptor composed of TGFbeta receptor I (TGFBRIa serine threonine kinase involved
in intracellular signal transduction) and TGF beta receptor II (TGFBRII). The levels of
these two receptors decrease immediately after PHx hitting a low point at 24 hours after
surgery. TGFBRII expression returns by 5 days post-PHx, but the level of TGFBRI does
not fully rebound during that timeframe (Ravi et al., 1995). From this, it is apparent that
at the peak of hepatocyte DNA synthesis, a coordinated maximal downregulation of
both TGFbeta and its receptors occurs, presumably to allow hepatocyte replication to
proceed unimpeded. Of note, when active TGFbeta is injected into rats 24 hours prior
to performing PHx, the animals die 24 hours after surgery with a signicant increase in
apoptotic hepatocytes as compared to controls (Schrum et al., 2001).
1.4.2
Phase Two: Proliferation
The middle (or proliferation) phase of liver regeneration is mainly characterized by the
replication of all constituent cell types in the liver. As mentioned, among the cell types,
hepatocytes replicate rst with peak DNA synthetic activity ranging from 2444 hours
in rodents; a subsequent smaller boost in hepatocyte DNA synthesis is observed ~48
hours later. The hepatic plates thicken with the replicating hepatocytes and compress
sinusoids; in addition, SEC porosity decreases at 72 hours after surgery (Wack et al.,
2001). Cholangiocyte replication reaches a maximum at approximately 23 days following PHx, while hepatocyte tight junctions dissolved during the initiation phase are
reconstituted allowing bile secretion to recommence at day 3. The volume density
of bile ducts is restored by about day 10 (Lesage et al., 1996). Substantial paracrine
communication occurs between hepatic cell types during this period. Hepatocytes upregulate expression and secretion of VEGF at 24 hours peaking at 72 hours after PHx,
increasing its availability to SECs to stimulate endothelial replication at about 3 4 days
after surgery (Shimizu et al., 2001). SECs (Ping et al., 2006) and stellate cells (Takeishi
et al., 1999) secrete HGF, which may prompt the latter DNA synthetic peak seen in
hepatocytes. At about day 4 after PHx, stellate cells upregulate synthesis and deposition
of extracellular matrix proteins, which is likely to help the liver segue to the nal stage
(Martinez-Hernandez and Amenta, 1995).
1.4.3
Phase Three: Termination
During the termination phase of liver regeneration (i.e., day 4 to day 7 after PHx),
SECs re-establish the sinusoids by migrating among the hepatocytes that have accumulated into avascular clusters as a result of robust cell division (Martinez-Hernandez and
Amenta, 1995). This in turn allows hepatocytes to reorient into one-to-two cell thick
plates and to reform contacts with the extracellular environment. Impeding proper hepatocyte-ECM interaction can drastically alter the termination of liver regeneration. For
example, integrin linked kinase (ILK), an intracellular signal transducer that associates
with integrins, appears to be particularly important to ending the regenerative response
after surgery. When mice that lack ILK specically in hepatocytes are subjected to PHx,
their livers respond by becoming roughly 1.5-fold larger at 14 days post-PHx than prior
to surgery. In addition, hepatocyte DNA synthetic activity in these mice is more robust
and prolonged (Apte et al., 2009). This may be due to altered expression of integrins
in the liver (Gkretsi et al., 2007) and upregulated Met signaling (Apte et al., 2009).
Glypican-3, a heparan sulfate proteoglycan known to be overexpressed in HCC and to
underlie a genetic syndrome with an overgrowth phenotype in humans, is upregulated
10
at about 46 days after PHx. When subjected to PHx, mice engineered to overexpress
glypican-3 in hepatocytes show delayed and reduced DNA synthesis and do not regain
proper liver mass at day 6 post-PHx, suggesting that the upswing in endogenous glypican-3 expression after surgery promotes termination of regeneration (Liu et al., 2009).
Changes in the relative abundance of transcription activators and secreted mitoinhibitors also help dictate the end of regeneration. The DNA binding activity of C/EBPalpha
transcription factor initially rises then falls precipitously after PHx; however, it climbs
again in the latter stages of the regenerative response. This is noteworthy because it is
antiproliferative toward hepatocytes (Wang et al., 2001) and promotes their differentiation and quiescence (Greenbaum et al., 1995). Apte et al. (2009) demonstrated that
the abundance of Yes-associated protein (YAP), a transcription coactivator and member
of the newly dened mammalian Hippo kinase pathway that controls liver size and
induces liver cancer (Dong et al., 2007), is downregulated late in liver regeneration after
PHx. With regard to secreted factors, non-parenchymal cells begin to release IL-1 alpha
and beta (which inhibit mitogen-induced hepatocyte DNA synthesis) after the proliferative phase of liver regeneration (Boulton et al., 1997). As mentioned earlier, TGFbeta
and its receptors are downregulated during the early period after surgery, but levels rise
subsequently after the peak of hepatocyte DNA synthesis. When mice lacking TGFBRII
in hepatocytes are subjected to PHx, they have a pronounced increase in the number
of hepatocytes entering the S-phase (Oe et al., 2004; Romero-Gallo et al., 2005) and a
higher liver-to-body weight ratio at 1 week and 1 month after surgery (Romero-Gallo
et al., 2005). Activin-A is a mitoinhibitor related to TGFbeta. Infusion of follistatin, an
endogenous Activin-A inhibitor, results in increased liver weight and DNA content
more than 5 days post-PHx, suggesting that Activin-A acts during the termination phase
of liver regeneration (Kogure et al., 1995). It seems likely that downregulation of hepatic
mitogens such as HGF accounts at least in part for bringing about termination of the
livers regenerative response. This is supported by the data mentioned earlier demonstrating that hyperplastic liver induced by HGF infusion returns to normal size in a few
weeks after infusion is stopped (Patijn et al., 1998). To summarize, the data suggest that
the elusive hepatostat actively drives the liver to quiescence by promoting perpetual
antagonism between growth promoters, such as HGF and EGF, and growth inhibitors,
including TGFbeta, ILK, IL-1, and C/EBPalpha, with the inhibitors predominating during
the termination phase (Michalopoulos and DeFrances, 2005).
1.5
Future Directions
Several lapses in our knowledge of liver regeneration persist: (1) We need a better
understanding of the events that terminate the regenerative response. While some
fore-runners in this process have been described, it seems likely that termination is
signicantly more complex. (2) Acute liver failure continues to take a global toll on
human life. Are there certain molecular pathways mediating liver regeneration that can
be exploited to promote survival in this scenario? (3) Patients undergoing hepatic resection usually have underlying liver disease. We must more fully dene the impact of
inammation, steatosis, and other physiologic complications on regeneration in order
to improve recovery after surgery. Exciting challenges have been dened; we must now
square our shoulders and meet them.
References
11
Summary
What has emerged from nearly a century of research on liver regeneration is that the liver is
remarkably resilient. Genetic manipulation of specic genes in animals has demonstrated that
perturbation of a single signaling pathway is usually insufcient to block liver regenerationa
delay in the regenerative response may result, but rarely is the process completely inhibited
ending in acute hepatic failure or death. It seems that, when hepatic tissue is lost or damaged,
a collection of primary and auxiliary pathways are activated en masse in the liver during
the initiation/priming phase to ensure an adequate entry into the proliferation phase; these
pathways encompass physical distortion of the portal vasculature, growth factor signaling
primarily through the Met and EGF receptors and TNFalpha/IL-6 stimulation, which together
activate signal transduction molecules and ultimately lead to gene transcription and entry into
the cell cycle. Then, a second set of signaling mechanisms as diverse and numerous as those
that initiate regeneration bring about closure of the process in the nal termination phase
and include reestablishing cellmatrix contacts; reappearance of mitoinhibitory molecules
such as TGFbeta family members, IL-1 cytokine, and C/EBPalpha; and minimization of the
pro-stimulatory effects of growth factors. The nal outcome is a liver that is fully functional,
thus sustaining the animal.
Further Reading
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Invest. 91, 131926.
Martinez-Hernandez, A., and Amenta, P.S. (1995). The extracellular matrix in hepatic regeneration. FASEB J. 9, 140110.
Palmes, D., and Spiegel, H.-U. (2004). Animal models of liver regeneration. Biomaterials 25,
160111.
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12
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Oval Cells, Bone Marrow, and Liver

Regeneration
Anna C. Piscaglia, Antonio Gasbarrini, and Bryon E. Petersen
Learning Targets
1. Stem cell properties and role in homeostasis maintenance
2. Liver stem cells in rodents and humans: mechanisms of activation, phenotype, and
involvement in liver regeneration
3. Bone marrow as a source of liver stem cells: experimental models and human
observations
4. Bone marrowderived stem cell therapies for the treatment of liver diseases: from
bench to bedside
2.1
Stem Cells: Denition and Properties
The existence of hepatic stem cells has been controversial for decades, and it was
thought that if such cells existed, they would reside within the liver. However, there is
now consensus that not only do putative liver stem/progenitor cells exist within the liver,
but also that circulating stem cells from extra-hepatic sites, in particular the bone marrow (BM), can contribute to liver repopulation, although the physiological importance
and therapeutic utility of this phenomenon are still debated.
Stemness can be dened by two fundamental properties: self-maintenance and

multipotency.
v Self-maintenance represents a cells ability to preserve its own population. During
mitosis, a stem cell can divide asymmetrically, to produce one daughter stem cell
and one daughter cell that leaves the stem cell compartment as a transit-amplifying or
progenitor cell. The latter actively proliferates and differentiates, ultimately generating
mature cells within the tissue of origin. Alternatively, stem cells can produce two identical daughter cells that could be either stem cells (self-renewing division) or progenitor cells (nonself-renewing division); this process is called symmetrical division, and
it respectively expands or reduces the stem cell compartment. It is generally accepted
that stem cells have the ability to switch between these various options in response to
environmental conditions in order to regulate their own number (fFigure 2.1).
18
Oval Cells, Bone Marrow, and Liver Regeneration
v Multipotency is the capacity to produce mature cell-lineages from a relatively undifferentiated element. The process that ultimately leads a stem cell to become a mature
and functional cell is named differentiation and results from progressive phenotypic
modications due to dynamic changes in the gene expression pattern.
asymmetric division
quiescence
SC pool maintenance
asymmetric division:
one daughter stem cell
and one daughter
transit-amplifying cell
Figure 2.1
symmetric division
expansion
exhaustion
self-renewing
symmetric division:
two daughter
stem cells
non-self-renewing
symmetric division:
two daughter transitamplifying cells
Mechanisms for the Maintenance of Stem Cell Numbers
Notes:
Abbreviation: SC = stem cell.
Stem cells (SCs) exist in all multicellular organisms and play a central role in tissue
genesis, regeneration, and homeostasis by providing new elements to increase tissue
mass during pre- and postnatal growth and by replacing cell loss due to senescence or
damage.
SCs possess a hierarchy of potentialities: from the totipotency of the zygote and
its immediate progeny, to the pluripotency of embryonic stem cells (ESCs), to the
multi/unipotency of tissue-specic, adult SCs (ASCs). The latter persist in terminally
differentiated tissues, allowing for their renewal and regeneration (Alison and Islam,
2009).
SCs colocalize with supporting cells in a physiologically limited and specialized
microenvironment, or niche, that varies in nature and location depending upon the
tissue type. The reciprocal interactions between SCs and their microenvironment,
through cellcell and cellmatrix connections as well as the secretion of soluble factors,
inuence SC behavior (Moore, 2006).
Despite the paradigm of unidirectional cell determination, recent studies have shown
that ASCs are endowed with an unexpected plasticity, as circulating ASCs have been
demonstrated to differentiate into mature cells of other tissue types, a process called
transdifferentiation (Piscaglia et al., 2008a). A particularly high degree of plasticity is
2.1
Stem Cells: Denition and Properties
19
shown by hematopoietic stem cells and mesenchymal stem cells (MSCs), which can
give rise to a wide range of phenotypes. Hematopoietic SCs are responsible for the
renewal of blood cells. During embryogenesis, hematopoietic SCs arise from the dorsal
and ventral mesenchyme and migrate rstly to the yolk sac, then to the fetal liver and
spleen, and nally to the bone marrow (BM), which remains the only hematopoietic
organ from birth throughout life. In addition to BM and embryos obtained through
techniques of either nuclear transplantation or in vitro fertilization, two additional
sources of hematopoietic SCs are available: peripheral blood and umbilical cord
blood. The different hematopoietic SC sources are distinguished in terms of accessibility and ethical issues, as well as biological properties such as immunogenicity
and clonogenicity (Piscaglia et al., 2007a). The membrane phosphoglycoprotein CD34
is considered a valid hematopoietic SC marker, although some studies suggested the
existence of CD34/Lin hematopoietic SCs capable of producing CD34 cells in vitro.
AC133 (CD133, or prominin1 in rodents), a glycoprotein transmembrane, present on
progenitors belonging to neuronal, epithelial, and endothelial lineages, is common to
both CD34 and CD34 hematopoietic SCs (Guo et al., 2003). It is generally accepted
that the most primitive and long-term human hematopoietic SCs are characterized
by the expression of CD133, Thy1 (CD90), and VEGFR2 and by a variable expression of CD34 and CD38 (Bryder et al., 2006). BM-resident hematopoietic SCs can
be mobilized into the peripheral blood under specic stimuli such as tissue injury or
administration of mobilizing agents. Hematopoietic SCs may be used in autologous or
allogeneic transplantations for the treatment of hematopoietic disorders, autoimmune
diseases, and aggressive cancers to reconstitute the hematopoietic SC lineages and
the immune system integrity. Additionally, in vitro culture and in vivo transplantation
assays have demonstrated that hematopoietic SCs are able to give rise to a wide array
of phenotypes, including blood, cartilage, fat, tendon, lung, liver, muscle, brain, heart,
and kidney cells (Mimeault et al., 2007). Mobilized hematopoietic SCs can colonize
extramedullar sites and contribute to their regeneration by promoting the immune
response and/or by converting into ASCs within peripheral tissues. Moreover, it has
been demonstrated that the number of circulating hematopoietic SCs expressing early
markers for muscle, nerve, and hepatic differentiation increases following treatment
with mobilizing agents. This phenomenon has led to speculation about the existence
of BM-derived circulating pluripotent SCs, which could migrate from the peripheral
blood into every tissue and participate in normal turnover and repair following injury
(Ratajczak et al., 2004). MSCs, also called stromal stem cells, stromal precursors, mesenchymal progenitors, and colony-forming unit-broblastic cells, are highly proliferating, adherent cells that reside in a perivascular niche within the BM and also in the
wall of blood vessels within most organs. MSCs have been shown to differentiate into a
variety of mesodermal cell lineages, including osteoblasts, chondroblasts, adipocytes,
myocytes, and cardiomyocytes, as well as non-mesodermal cells, such as hepatocytes
and neurons (Tocci and Forte, 2003).
Regarding the mechanisms of SC plasticity, it has been observed that in some
models of apparent transdifferentiation, SCs may actually be fusing with cells in the
host tissue, generating elements with multiple and genetically variant nuclei called
heterokaryons. Fusion phenomena between hematopoietic SCs and other cells
Purkinjie cells, cardiomyocytes, and hepatocyteshave been shown both in vitro and
in vivo, as reviewed elsewhere (Alvarez-Dolado et al., 2003). Overall, cell fusion is a
20
physiological phenomenon in certain districts, such as liver and muscle, and it may or
may not play a prominent role in SC plasticity, depending on the model of injury and
the host phenotype. It has been also proved that fusion and transdifferentiation can
coexist and produce therapeutically benecial results: Spees et al., (2003) showed the
coexistence of fusion and transdifferentiation in co-cultures of MSCs and pulmonary
epithelial cells.
fTable 2.1 enlists a glossary of stem cell terminology; fFigure 2.2 depicts the
properties of adult stem cells.
Table 2.1 Glossary of Stem Cell Terminology

CELL FUSION
A mechanism of plasticity that consists in the melting of

one stem cell and one mature cell into one cell termed
heterokaryon
MULTIPOTENCY
The ability to divide and produce a limited number of other

cell types, usually within a specic germ layer
PLASTICITY
The ability of a stem cell to give rise to a cell type outside

its established differentiation capabilities, often across cell
lineage
PLURIPOTENCY
The ability to divide and produce cells of any of the three

germ layers (ectoderm, mesoderm, endoderm)
PROGENITOR
(TRANSIT-AMPLIFYING)
CELL
An immature cell with a restricted differentiation potential

and no self-renewal capacity
STEM CELL
Undifferentiated cell that is able to self-renew and

differentiate into a wide range of specialized
cell types
STEM CELL NICHE
The environment of stem cells, formed mainly by the

surrounding cells and the extracellular matrix, that regulates
stem cell behavior
TOTIPOTENCY
The ability to divide and produce all the differentiated cells

in an adult organism plus all of the cell types that make up
the extraembryonic tissues
TRANSDIFFERENTIATION
A mechanism of plasticity that consists in the switch of

differentiation program toward mature phenotypes outside a
stem cells established capabilities
UNIPOTENCY
The ability to divide and produce only one type of tissue or

cell
2.2
Liver Stem Cells and Their Role in Hepatic Regeneration

matrix
basement
membrane
support cell
21
stem cell niche

adhesion
molecule
stem cell
asymmetric
division
mobilization
quiescence
symmetric
division
stem cell
fate
differentiation
progenitor
cells
circulating
stem cell
self-renewal
mature cell
proliferation
maturation
transdifferentiation
host tissue
progenitor
cell
cell
fusion
Figure 2.2
2.2
proliferation
mature cell
maturation
Adult Stem Cells and Their Properties
As discussed in chapter 1, the liver has an extensive regenerative potential in response

to parenchymal loss, mainly granted by mature hepatocytes, which can re-enter the
cell cycle to restore the liver mass. This is a very efcient system and, after 2/3 partial
hepatectomy (PHx) in rats, proliferation of hepatocytes and cholangiocytes, followed by
stellate and endothelial cells, can regrow the remnant to the original mass in less than
2 weeks (Michalopoulos, 2011). Consequently, despite their quiescent state, hepatocytes
may be considered functional liver stem cells, with very high clonogenic potential, as
demonstrated by serial transplantation experiments (Overturf et al., 1997). In chronic
viral hepatitis, mathematical modeling of viral infection demonstrated that an increased
proliferation of hepatocytes compensates for increase in parenchyma loss due to disease
or injury (Nowak et al., 1996). However, when the cirrhotic stage is reached, the hepatocyte proliferation rate tends to fall, probably due to replicative senescence, telomere
shortening, and/or metabolic exhaustion, also related to the architectural distortion of
the organ (Marshall et al., 2005). In such circumstances, and whenever the replication
22
ability of hepatocytes is impaired or experimentally inhibited, regeneration can be accomplished by the activation, expansion, and differentiation of liver stem cells (LSCs)
putatively located within the canal of Hering. LSCs are thought to be responsible for the
human ductular reaction, which corresponds to the oval cell activation seen in specic
models of liver injury in mice and rats (Duncan et al., 2009; Roskams, 2006).
Oval cells represent a heterogeneous population of rodent bipotential LSCs originating from the canal of Hering and activate in the damaged liver when the proliferative
potential of hepatocytes is impaired or inhibited.
The term oval cells (OCs) was introduced by Farber in 1956 to describe the small
oval cells with scant lightly basophilic cytoplasm and pale blue-staining nuclei observed in rat livers following certain carcinogenic regimens. While several methods are
available for the activation of OCs, they all involve the same basic principle: inhibition
of the proliferative potential of mature hepatocytes followed by liver injury. Chemical inhibition of hepatocyte proliferation is made possible by the unique expression of
mixed function oxidases (P450s) within the parenchymal cells of the liver. Popular OC
induction models in the rat include choline-decient diet followed by ethionine exposure, galactosamine, 2-acetylaminouorene (2AAF)/CCl4, 2AAF/PHx, and allyl alcohol
(Shupe et al., 2009).
OCs are an extremely heterogeneous population that includes various fractions with
different stemness potential, depending upon the experimental protocol and the animal
model under investigation ( Jelnes et al., 2007). OCs are capable of extensive proliferation and studies of lineage tracing have conrmed that they are bipotent because they
can give rise to both cholangiocytes and hepatocytes (Sackett et al., 2009). Moreover,
OCs coexpress biliary and hepatocytic markers, such as CK19 and albumin, respectively, and they also express hematopoietic markers (such as CD34, CD133, and c-kit).
In rats, OCs are OV6 positive (where OV6 identies epitopes shared on cytokeratins 14
and 19 and marks OCs, bile duct cells, and nodular hepatocytes) and alpha-fetoprotein
(AFP) positive, thus resembling hepatoblasts in their gene expression pattern. Mouse
OCs differ from rat OCs by not expressing AFP or OV-6, while they can be identied
by one specic antibody, termed A6 (Duncan et al., 2009). Recently, a new collection
of monoclonal antibodies has been generated by immunization of Fischer rats with
enzymatically dispersed nonparenchymal cells from the livers of adult mice treated with
3,5-diethoxycarbonyl-1,4-dihydrocollidine to produce cell surface reactive reagents
more specic for OC response. Differential activity was observed on normal liver cells
and at different stages of OC activation, indicating potential utility for progenitor cell
identication (Dorrell et al., 2008).
The hepatic progenitor cells, also known as intermediate hepatobiliary cells, or ductular
hepatocytes, represent the counterpart of OCs in humans.
2.2
23
Hepatic progenitor cells (HPCs) can be seen in several hepatopathies, such as

chronic cholestasis, sub-massive necrosis, alcoholic liver disease, focal nodular hyperplasia, and liver allograft failure (Zhou et al., 2007). In such conditions, intermediates
between hepatic SCs and hepatoblasts and between hepatoblasts and adult parenchyma are observed, and amplication of one or both pluripotent cell subpopulations
can occur. A frequent trigger of ductular reaction is the presence of chronic damage,
resulting in hepatocyte senescence. Ductular reactions are not due to metaplastic
hepatocytes but conversely represent a true progenitor/stem cell response. Progenitor
cells seem to be able to survive when hepatocytes are lost due to viral infections or
toxic damage, possibly because of their ability to express protecting factors, such as
ABC-transporters, multidrug resistance genes, and antioxidant enzymes. Like OCs,
HPCs are bipotent, coexpress biliary and hepatocytic markers (including CK19, OV6,
AFP, and albumin), and also express hematopoietic progenitor cell antigens (Spee
et al., 2010).
In the last years, numerous studies have been published on the isolation, characterization, and differentiation of the putative liver stem cells. A variety of surface antigens
(including CD44 and EpCAM, CD34, CD133, c-kit, and Thy-1) have been used to identify and isolate OCs/HPCs, but the identication of a specic LSC marker awaits further
investigation, as reviewed elsewhere (Duncan et al., 2009; Piscaglia, 2008a).
Overall, whether OCs/HPCs fulll the criteria to be considered true LSCs is still controversial. Some authors believe that OCs and HPCs may represent transit-amplifying
cells derived from a more primitive LSC (Theise, 2006). Moreover, other cell populations within the liver might function as hepatic stem/progenitor cells. In particular,
it has been demonstrated that the hepatic stellate cells (HSCs), located in the space
of Diss, express stem/progenitor cell markers, such as CD133 and Oct4, and have
stem cell properties (Kordes et al., 2007; Kordes et al., 2009; Sawitza et al., 2009) (see
chapters 6, 7, and 8 for further discussion on stem cell properties of HSCs and their
niche). Given the difculty of identifying unique LSC markers, it has been suggested to
consider stemness as a function instead of an entity: marking resident SCs based upon
their quiescence might differentiate true SCs from their rapidly dividing derivatives
(Piscaglia et al., 2008a). By using a label-retention assay following acetaminopheninduced liver injury in mice, 4 possible LSC populations have been identied: (a)
replicating hepatocytes at the parenchymal/stromal interface; (b) ductular cells of the
canal of Hering (CoH); (c) cholangiocytes of the intralobular bile ducts; and (d) periductular null cells (devoid of hepatocytic and biliary markers) (Kuwahara et al., 2008).
The asymmetrically dividing cells that populate these sites might represent some form
of lineage hierarchy within the LSC population: periductular null cells might give
rise to the cytokeratin-positive cells of the CoH, which in turn could give rise to the
intraductular cells and periductular hepatocyte-like cells (Petersen and Shupe, 2008).
De Alwis and colleagues have demonstrated that regenerating hepatocytes arise from
a LSC population in the CoH and move outward into the liver parenchyma, as in the
streaming liver hypothesis. Interestingly, this observation seems to indicate that the
LSC population is active also in healthy livers and contributes to the hepatic turnover
(De Alwis et al., 2009). In conclusion, LSC participation to hepatic repair is probably
more complicated than in organs with normally rapid cell turnover, and multiple
liver cell populations might function as LSCs, depending on severity, location, and
chronicity of injury.
24
The existence of multiple populations of liver cells with stemness potential implies the existence of multiple LSC niches that can be activated depending upon the
mechanism and location of injury. Presumably, the damaged liver releases molecules
that stimulate the activation of OCs/HPCs and mediate their subsequent proliferation,
migration, and differentiation into mature hepatic phenotypes. Up to date, an extensive number of growth factors and cytokines that regulate the various phases of the
OCs/HPCs response have been described, including stem cell factor (SCF), HGF, EGF,
broblast growth factor (FGF), Hedgehog, and Wnt signaling pathways, SDF-1/CXCR4
axis, tumor necrosis factor, interleukin-6, interferons, transforming growth factors, and
transforming growth factor like weak inhibitor of apoptosis (TWEAK) (see chapter 5
for further discussion about the hepatic stem cell niche) (fFigure 2.3). Noteworthy,
the responses of OCs/HPCs to HGF via cMet and the potential autocrine loops with
TGFalpha, EGF, and FGF are similar to the patterns expressed by hepatocytes during
liver regeneration, despite the fact that hepatocytes and LSCs do not tend to proliferate contemporaneously. This might be due to the modulatory effects of inammatory cells within the niche, producing a range of cytokines and chemokines (such as
TWEAK, TGFbeta, and INF-gamma) that could inuence the LSC response (Duncan
et al., 2009). Another molecule of growing interest in the eld of liver regeneration is
the granulocyte-colony stimulating factor (G-CSF), a cytokine involved in mediating
hematopoietic SC mobilization from the BM into the peripheral blood (Liongue et
al., 2009). In 2007, we elucidated the double mechanism of action of G-CSF during
activation
null
cell
early
HPC
proliferation
migration
differentiation
intermediate
HPCs
late HPCs
hepatocytes
cholangiocytes
WNT pathway
WNT pathway
LIF
OSM
SCF
IL-6
TNF
Hedgehog
pathway
INF-gamma
TNF
TWEAK
Figure 2.3
SDF-1
HGF
G-CSF
HGF
FGF
EGF
G-CSF
plasmid activator
cascade
TGF-beta
MMPs
LIF
OSM
Dlk /Pref-1
Cellular and Molecular Factors Involved in Liver Stem Cell Response
Note:
Abbreviations: HPC = hepatic stem/progenitor cell.
2.3
Extrahepatic Stem Cells with Hepatogenic Potential
25
OC-mediated liver regeneration in rats: G-CSF is able to contribute to liver repair by

increasing the BM-derived liver repopulation (vide infra), and also by activating the
endogenous OCs, that express G-CSF receptor (G-CSFR) (Piscaglia et al., 2007b).
2.3
Extrahepatic Stem Cells with Hepatogenic Potential:

The Blood of Prometheus
Bone marrow harbors various populations of stem cells with hepatogenic potential:
hematopoietic stem cells, mesenchymal stem cells, and multipotent adult progenitor
cells.
Liver regeneration is mainly an endogenous process driven by hepatocytes and

resident hepatic stem/progenitor cells. However, it has been observed that certain
populations of extrahepatic ASCs can migrate into the liver and contribute to its repopulation and turnover. A particularly high degree of plasticity has been shown by
bone marrow stem cells (BMSCs), which can give rise to a wide range of phenotypes,
including hepatic cells (Krause et al., 2001; Theise and Krause, 2002). Adult BM comprises two main populations of ASCs able to convert into hepatic cells, either by
fusion or transdifferentiation: hematopoietic SCs and MSCs.
The relationship between the liver and the hematopoietic stem cells begins during
embryogenesis, being the fetal liver directly involved in hemopoiesis. Most hematopoietic SCs leave the liver afterbirth, but some CD34 cells persist during postnatal life
and might reacquire their hematopoietic potential, as it happens in patients suffering
from hematologic disorders (such as myelobrosis and Cooleys disease) (Crosbie et al.,
1999). It has been shown that after liver transplantation (LT) into lethally irradiated rats,
donor-derived hematopoietic SCs migrate from the graft to the recipient BM and reconstitute the hemolymphoid system (Murase et al., 1996). Passenger liver donor-derived
hematopoietic SCs are thought to be the cause of the stable donor micro-chimerism
seen in patients after LT and might play a role in the induction of tolerance (Nierhoff
et al., 2000). Moreover, hematopoietic SCs seem to be physiologically involved in liver
repair in humans. A spontaneous mobilization of CD34 hematopoietic SCs has been
reported following liver resection in patients with primary liver cancer or metastasis
(De Silvestro et al., 2004). Similarly, a signicant increase in the percentage of CD133
cells has been found in blood samples of healthy living liver donors and further in vitro
investigations have demonstrated that the mobilized cells were indeed liver-committed
(Gehling et al., 2005). Moreover, liver cirrhosis is associated with an intermittent mobilization of various populations of liver-committed cells of putative BM origin into
the circulation (Gehling et al., 2010). We have recently demonstrated that a signicant
CD133 hematopoietic SC mobilization can be seen in patients undergoing major liver
resection, especially in the presence of underlying hepatic disease, and that HGF and
G-CSF are involved in the dynamics underlying hepatic regeneration and hematopoietic
SC recruitment (Piscaglia et al., 2011). The hypothesis that hematopoietic SCs can participate in liver regeneration and renewal is substantiated by several studies on animal
models and in humans, and it has offered a rationale for the development of SC-based
therapies in hepatology (discussed later).
26
Mesenchymal stem cells might become an even more suitable source for SC-based
therapies than hematopoietic SCs because of their immunological properties: MSCs are
less immunogenic and can induce tolerance upon transplantation. Moreover, MSCs
showed the highest potential for liver regeneration compared with other BM cell subpopulations in an animal model of hepatic injury (Cho et al., 2009).
Another identied SC population within the BM, the multipotent adult progenitor
cells (MAPCs), seems to be endowed with an impressive plasticity and has shown liver
differentiation potential both in culture and in animal models (Kallis et al., 2007). These
cells could potentially co-purify with hematopoietic SCs or MSCs and contaminate these
cell populations investigated in liver repopulation studies. According to this hypothesis,
rather than being a source of liver-committed SCs, BM could act as a hide out for recirculating pluripotent SCs that might be deposited early during development in BM and
could be a source for tissue/organ regeneration (Kucia et al., 2006). Therefore, the present distinction between hematopoietic SCs and MSCs may become obsolete, given the
heterogeneity and possible overlaps of these various BMSC populations, which could
share a common stemness core.
In order to study the BM contribution to hepatic regeneration, it is essential that the
BM-derived cells be identiable within the liver. This can be achieved by the construction of chimeric animals in which traceable BM cells are implanted, typically after lethal
irradiation. Donor-derived BM cells can be marked by labeling with green uorescent
protein or genetic markers, or by sex-mismatching, so that their progenies within the
recipients tissues can be detected. The chimeric animals can be subsequently subjected
to a reproducible form of liver injury, and the BM contribution to hepatic repair can be
easily studied by evaluating the donor-derived cells within the liver (see chapter 12 for
a detailed discussion of animals models for studies on liver regeneration). In contrast
to animal models, in humans the only way to identify BM-derived cells within the
liver is to study patients who received a sex-mismatched liver or a BM transplant and
subsequently developed a hepatic injury.
The rst demonstration that BMSCs transplanted into lethally irradiated recipients
can migrate to the liver and differentiate, via oval cells, into either hepatocytes and
biliary duct cells was given 11 years ago (Petersen et al., 1999). Similar results were
obtained shortly after in other animal models (Lagasse et al., 2000; Theise et al., 2000a)
and in clinical settings (Alison et al., 2000; Korbling et al., 2002; Theise et al., 2000b).
Since then, numerous reports and reviews have been published on this exciting topic,
often with contradictory conclusions (Gilchrist and Plevris, 2010; Thorgeirsson and
Grisham, 2006). As we clearly demonstrated in 2007, BMSCs may or may not play
a critical role in liver regeneration, depending upon the experimental setting (Oh et al.,
2007). In this study, mutant F344 dipeptidyl-peptidase-IV-decient (DPPIV) rats received the mitotic inhibitor monocrotaline, followed by DPPIV BM transplantation,
and nally PHx (group A) or 2AAF/PHx (group B). The last group (C) was used to
analyze the effects of monocrotaline on transplanted BM cells: these rats underwent
BM-transplantation prior to monocrotaline and were then treated with 2AAF/PHx. In
group A, DPPIV hepatocytes were found in the liver. Group B showed that approximately 1/5 of the oval cell population was BM-derived, while animals in group C failed
to show a signicant BM contribution to liver regeneration. Based on the already cited
model of lineage hierarchy within the LSC population (Petersen and Shupe, 2008),
we can postulate that the periductular null cells might, at least in part, originate from
2.3
Extrahepatic Stem Cells with Hepatogenic Potential
27
extrahepatic SCs of BM origin and then give rise to the ductular cells of the CoH. These
cells, in turn, can differentiate into intraductular cells and periductular hepatocyte-like
cells (fFigure 2.4).
It is generally agreed that BM represents a possible source of LSCs, even if the
frequency of colonization, in the absence of a strong selective pressure, is very low,
it is unlikely sufcient per se to achieve a signicant contribution to hepatic repopulation. However, the few BM-derived cells that do engraft may play an important
role in modulating the endogenous repair mechanisms within the hepatic stem cell
niche. We have reported gene expression modications induced by human cord
blood hematopoietic SC therapy in allyl alcohol-treated rats. In this model, cordonal
hematopoietic SCs were able to colonize the liver, differentiate into hepatocytes,
and also enhance the endogenous process of hepatic regeneration by means of an
up-regulation of factors involved in OC activation, resistance to oxidative stress,
detoxication, tissue repair, and remodeling (Piscaglia, 2005). In this context, it must
be noted that BM cells signicantly contribute to the genesis of non-parenchymal
circulating liver-committed
stem cells
bone marrow
stem cells
periductular
null cells
hepatic
artery
ductular
cells
portal
triad
IBD
ductule
CoH
bile canaliculus
portal
vein
cholangiocytes
replicating
hepatocytes
limiting plate
Figure 2.4 Hepatic Cell Populations with Stemness Potentials and Their Location within
the Liver
Note:
Abbreviations: IBD = intralobular bile duct; CoH = canal of Hering.
28
cells within the liver, such as Kupffer cells, endothelial cells, and myobroblasts.
These non-parenchymal cells play a central role in the liver microenvironment supporting the hepatocyte functions and contribute to the modulation of the hepatic
stem cell niche (Kallis, 2007).
Presumably, the contribution of BMSCs to liver regeneration grows with the extent
of liver injury because the damaged liver releases molecules that stimulate the recruitment of stem cells and mediate differentiation to the mature phenotype. These chemical
signals stimulate the recruitment of endogenous LSCs and, under certain circumstances,
circulating pluripotent BM-derived SCs, which migrate to the portal spaces and contribute to the regenerative processes (fFigure 2.5). Indeed, in order to initiate a BM
response, the injured liver must signal to the responding cell types. A pivotal role in
BMSC recruitment is played by SDF-1. BM-derived liver-committed SCs expressing
circulating liver-committed
stem cells
severe injury
bone marrow
stem cells
ongoing injury
periductular
null cells
hepatic
artery
ductular
cells
portal
triad
IBD
ductule
CoH
bile canaliculus
portal
vein
cholangiocytes
replicating
hepatocytes
minor injury
Figure 2.5
The Degree of Liver Injury can Dictate the Cellular Response of the Liver
Notes:
Minor insults result in mitotic division of hepatocytes and cholangiocytes. Failure to restore
the liver microenvironment during repeated injury sees the activation and proliferation of
liver stem/progenitor cells (oval cells in rodents, or hepatic progenitors in humans). Finally,
a highly altered liver microenvironment can encourage homing and engraftment of bone
marrowderived stem cells as a nal attempt to restore liver homeostasis.
2.4
Clinical Applications of Bone MarrowDerived Stem Cells in Hepatology
29
SDF-1 receptor (CXCR4) are present in the peripheral blood and may colonize the damaged liver by following a SDF-1 gradient (Hatch et al., 2002). Other molecules secreted
by the injured hepatic milieu that can contribute to BMSC recruitment and homing
into the liver are the hepatocyte growth factor, some brosis mediators, such as matrix
metalloproteinase-9, and the G-CSF. The mechanisms underlying the adhesion and
retention of BMSCs to human liver compartments have been only in part elucidated
(Piscaglia, 2008b).
Regarding the mechanisms underlying BMSC plasticity, upon engraftment BMSCs
might either transdifferentiate into parenchymal cells or fuse with resident cells in the
host tissue. In two independent studies published in 2003, Wang and Vassilopoulos
proved that the regenerating liver nodules in tyrosinemic mice following hematopoietic SC transplantation were generated by fusion between the transplanted cells and
resident hepatocytes (Vassilopoulos et al., 2003; Wang et al., 2003). However, other
researchers remain convinced that liver regeneration is possible without fusion. Indeed, hematopoietic SCs cocultured with damaged liver tissue and separated by a
membrane that prevents direct cellcell contact, were able to transdifferentiate into
hepatocytes. In addition, after transplantation into mice subjected to liver damage
by CCl4, cells differentiated into hepatocytes without any evidence of fusion, leading
to functional recovery within 7 days ( Jang et al., 2004). In a sophisticated murine
transgenic Cre-lox model, cell fusion was not necessary to induce hematopoietic SC
differentiation into epithelial cells (Harris et al., 2004). Hematopoietic SCs differentiated into hepatocytes in vitro without fusion when provided specic growth factors,
such as basic broblastic growth factor (Saji et al., 2004). In our already mentioned
study, upon transplantation into rats subjected to 2AAF/PHx, approximately 20% of
the OC population derived from BMSCs without fusion phenomena (Oh et al., 2007).
Previously discussed, fusion and transdifferentiation are possible mechanisms of plasticity under a particular set of experimental conditions, and they can also coexist
and produce therapeutically benecial results: it has been shown that hematopoietic
SCs from human umbilical cord blood, transplanted into NOD/SCID mice, are able
to colonize the liver by means of both fusion and transdifferentiation (Tanabe et al.,
2004).
As a closing remark on other possible extra-hepatic sources of stem cells for liver
regeneration, it is worth a note that adipose tissue has been reported as a rich source
of easily accessible MSCs (adipose tissue stromal cells [ATSCs]) capable of hepatic
differentiation in vitro and in vivo. ATSCs are similar to bone marrow mesenchymal
stem cells (BM-MSCs) in terms of surface antigen marker prole and differentiation
potential, and ATSCs have been reported to exert an even higher proliferative capacity
in vitro (Puglisi et al., 2010). We have recently achieved the hepatogenic conversion
of ATSCs using a two-step protocol with sequential addition of growth factors. In order
to understand the molecular events involved in ATSC hepatic transdifferentiation, the
full genome expression proles of ATSC-derived hepatocyte-like cells were compared
with undifferentiated ATSCs. We identied several targets that depict the numerous
biological functions exerted by the liver, including protein metabolism, innate immune
response regulation, and biodegradation of toxic compounds. Moreover, we showed
that ATSC differentiation into hepatocyte-like cells might be caused by a mesenchymalto-epithelial transition (Saulnier et al., 2010).
30
2.4
Clinical Applications of Bone MarrowDerived Stem Cells

in Hepatology
Liver pathologies affect hundreds of millions of patients worldwide. There are currently
more than 5 million people in the United States suffering from end-stage liver pathologies, for which the only curative therapy is LT. Given the donor organ shortage, various alternatives have been evaluated, such as split-liver and related living-donor liver
transplantation. These procedures are still limited by the donor scarcity, the high costs,
and the lifelong immunosuppressive treatments that they all require. Thus, the development of cell therapies for the treatment of end-stage hepatic diseases is currently under
investigation all over the world.
A cell therapy can be dened as the use of living cells to restore, maintain, or enhance
tissue and organ function.
Cell therapies in hepatology have numerous potential advantages when compared

to LT because transplantable cells can be: (1) in vitro expanded and cryopreserved,
abolishing the limit of organ shortage; (2) genetically manipulated to correct inborn
errors of metabolism; (3) cryopreserved for future use; (4) infused without major surgery;
and (5) obtained from the same patient, avoiding risk of rejection and need for lifelong
immunosuppression.
SCs are already leaving the bench and reaching the bedside, despite an incomplete knowledge of the genetic control program driving their fate and plasticity. Different types of SCs with hepatic differentiation potential are theoretically eligible for
liver cell replacement. These include ESCs, inducible-pluripotent stem cells (IPSs), hepatoblasts, and ASCs, both resident (HPCs) and extrahepatic (hematopoietic SCs and
MSCs). Despite encouraging results in vitro, the use of hepatocyte-like cells derived
from these stem/progenitor cell populations is still conned to preclinical studies, given
the scarce tissue-specic functionality and, up to now, the lack of evidence of strong
liver repopulation levels in animal models. The most promising source for SC-based
therapies is represented by the intraportal or intrahepatic infusion of freshly isolated or
in vitro expanded hematopoietic SCs. Another appealing option is represented by the
administration of mobilizing/proliferating agents, such as G-CSF, which are able to both
enhance the hematopoietic SC mobilization into the peripheral blood and facilitate the
endogenous LSC activation (Piscaglia et al., 2008a).
Few clinical trials have been published regarding hematopoietic SCs transplantation and/or G-CSF infusion for the treatment of end stage liver pathologies (fTables
2.22.4). These studies share common limits, being conducted on small groups of patients, without controls, and using outcome parameters easily subjected to be biased.
Nowadays, the major role for stem cell therapy is as a bridge to transplantation or as
a way of maintaining those patients who are not eligible for LT. Nonetheless, prior to
large-scale clinical application of BMSCs for the treatment of liver pathologies, critical
aspects need to be further addressed, including the long-term safety, tolerability, and
efcacy of these SC-based treatments, as well as their pro-brogenic and carcinogenic
potential, as reviewed elsewhere (Piscaglia, 2008b) (see also chapters 5 and 13 for
further discussion).
2.4
Table 2.2
31
BMSC Transplantation
Intraportal autologous
transplantation of CD133
BMSCs in patients with liver
cancer undergoing portal
embolization before extensive
liver resection (LR)
Clinical improvement
Schulte am Esch, et al.

Stem Cells. 2005
Apr;23(4):46370.
Combination of portal vein

embolization and CD133
BMSC administration in
patients with malignant liver
lesions
Increased degree of hepatic

regeneration in comparison
with embolization alone
Frst G, et al. Radiology.

2007 Apr;243(1):1719.
Portal vein infusion of

unsorted autologous BMSCs
in 9 patients with cirrhosis
Improvement in Child-Pugh
score and albumin levels
Terai S, et al. Stem Cells.

2006 Oct;24(10):22928.
Epub 2006 Jun 15.
Autologous BMSC
transplantation prior to
surgery in patients with
cirrhosis and hepatocellular
carcinoma
Signicant increase of liver

function post-LR
Ismail A, et al. J
Gastrointest Cancer. 2010
Mar;41(1):1723.
Phase-I clinical trial on

decompensated cirrhotic
patients who received
infusion of autologous CD34
BM cells through the hepatic
artery
The treatment was unsafe

and ineffective in improving
the liver function
Mohamadnejad M, et al.
World J Gastroenterol. 2007
Jun 28;13(24):335963.
32
Table 2.3

Circulating Hematopoietic Stem Cell Mobilization, Collection, and Reinfusion
Boost infusions of mobilized

CD34 cells after a standard
G-CSF regimen in two
patients
Safe, well-tolerated, and

associated with a lasting
amelioration in the clinical
course of the disease during
the follow-up
(1 month)
Yannaki E, et al.
Exp Hematol. 2006
Nov;34(11):15837.
Intraportal administration of
mobilized CD34 BMSCs
following G-CSF exposure
in one patient affected by
drug-induced acute liver
failure
Signicant biochemical
and histopathological
improvement
Gasbarrini A, et al.
Dig Liver Dis. 2007
Sep;39(9):87882. Epub
2006 Jul 27.
Phase-I clinical trial on

5 patients with acute or
chronic liver failure: G-CSF
administration, followed by
collection and intraportal
or intrahepatic reinfusion of
circulating CD34 cells
Improvement of the hepatic

function in more than
50% of the cases, without
signicant side effects
during a follow-up of 60
days
Gordon MY, et al.

Stem Cells. 2006
Jul;24(7):182230. Epub
2006 Mar 23
Same patients of the

previous study by Gordon
et al., monitored for up to
18 months
The procedure was safe in

short and over long term, by
absence of tumor formation
and the benecial effects
lasted around 12 months
Levicar N, et al. Cell

Prolif. 2008 Feb;41 Suppl
1:11525.
Reinfusion into the hepatic

artery of CD34 BM-derived
cells, collected after G-CSF
mobilization and in vitro
expanded, in 9 patients with
alcohol-related cirrhosis
Well-tolerated and
produced a clinical and
biochemical improvement
Pai M, et al. Am J
Gastroenterol. 2008
Aug;103(8):19528.
40 patients with HBVrelated cirrhosis randomized

to receive G-CSF alone
or in combination with
leukapheresis and reinfusion
of peripheral blood
monocytes into the hepatic
artery
During a follow-up of
6 months, a signicant
biochemical and clinical
improvement was observed
in both groups (G-CSF plus
SC infusion obtained the
best benecial effects)
Han Y, et al. Cytotherapy.

2008;10(4):3906.
2.4
Table 2.4
33
G-CSF Treatment
G-CSF 5 g/Kg bid for 3

days in 8 patients affected by
severe liver cirrhosis
Well-tolerated in all
patients during a follow-up
of 8 months, and a
mobilization of BMSCs
co-expressing epithelial and
stem markers was noted
Gaia S, et al. J Hepatol.

2006 Jul;45(1):139. Epub
2006 Apr 6.
G-CSF (5 or 15 microgr/Kg/
day) for 6 days in 24 patients
with severe liver cirrhosis
Safe, dose-dependent
mobilization of BMSCs;
absence of clinical
improvement
Di Campli C, et al.
Dig Liver Dis. 2007
Dec;39(12):10716.
G-CSF treatment in 18 nondecompensated cirrhotic

patients
Good CD34/CD133
cell mobilization, despite
the absence of clinical
improvement
Lorenzini S, et al. Aliment

Pharmacol Ther. 2008
May;27(10):9329.
Largest randomized trial,

conducted on 24 patients
with alcoholic cirrhosis,
randomized to standard care
associated with G-CSF or
standard care alone.
G-CSF was safe and able to

mobilize CD34 cells and
increase HGF; however, the
study was too small to make
any comment regarding
survival or efcacy
Spahr L, et al. Hepatology.

2008 Jul;48(1):2219.
Summary
In a normal adult liver, the main source of repair is represented by the mature hepatocytes,
which can re-enter the cell cycle and restore the liver mass in response to parenchymal loss.
However, whenever the proliferation potential of hepatocytes is impaired or inhibited, hepatic
regeneration can be accomplished by the activation, expansion, and differentiation of liver
stem cells, which have been named oval cells in rodents and hepatic progenitor cells in
humans. Liver stem cells are capable of extensive proliferation and are bipotent because they
can give rise to both cholangiocytes and hepatocytes. In recent years, numerous studies have
been published on the isolation, characterization, and differentiation of putative liver stem
cells, but the identication of a specic marker awaits further investigation. Moreover, some
authors believe that oval cells and hepatic progenitor cells may represent transit-amplifying
cells derived from a more primitive liver stem cell and that multiple hepatic cell populations
might function as liver stem cells, depending on severity, location, and chronicity of the
damage.
Although liver regeneration is mainly an endogenous process, it has been observed that
circulating stem cells of bone marrow origin can migrate into the liver and contribute to its
repopulation and turnover. In the last 11 years, numerous reports and reviews have been
published on this exciting topic. It is generally agreed that bone marrow represents a possible source of liver stem cells, even if the frequency of colonization, in the absence of a
strong selective pressure, is very low and unlikely sufcient to achieve a signicant contribution to hepatic repopulation. However, the few bone marrow cells that do engraft may play
34
an important role in modulating the endogenous repair mechanisms within the hepatic stem
cell niche.
Despite an incomplete knowledge of the nature of the liver-committed bone marrow
stem cells, of the mechanisms underlying their plasticity, and of the physiological importance of this phenomenon in humans, bone marrow stem cells have been already used
in clinical settings for the treatment of end-stage hepatic diseases, mainly as a bridge to
transplantation or as a way of maintaining those patients who are not eligible for liver
transplant.
Further Reading
Doll, L., Best, J., Mei, J., Al Battah, F., Reynaert, H., van Grunsven, L.A., and Geerts, A. (2010).
The quest for liver progenitor cells: a practical point of view. J Hepatol. 52, 11729.
Eckersley-Maslin, M.A., Warner, F.J., Grzelak, C.A., McCaughan, G.W., and Shackel, N.A.
(2009). Bone marrow stem cells and the liver: are they relevant? J Gastroenterol Hepatol.
24, 160816.
Gennero, L., Roos, M.A., Sperber, K., Denysenko, T., Bernabei, P., Calisti, G.F., Papotti, M.,
Cappia, S., Pagni, R., Aimo, G., Mengozzi, G., Cavallo, G., Reguzzi, S., Pescarmona, G.P.,
and Ponzetto, A. (2010). Pluripotent plasticity of stem cells and liver repopulation. Cell
Biochem Funct. 28, 17889.
Kisseleva, T., Gigante, E., and Brenner, D.A. (2010). Recent advances in liver stem cell therapy.
Curr Opin Gastroenterol. 26, 395402.
Kung, J.W., Currie, I.S., Forbes, S.J., and Ross, J.A. (2010). Liver development, regeneration,
and carcinogenesis. J Biomed Biotechnol. 2010, 984248.
Oertel, M., and Shafritz, D.A. (2008). Stem cells, cell transplantation and liver repopulation.
Biochim Biophys Acta 1782, 6174.
Piscaglia, A.C., Shupe, T.D., Petersen, B.E., and Gasbarrini, A. (2007). Stem cells, cancer,
liver, and liver cancer stem cells: nding a way out of the labyrinth . . . Curr Cancer Drug
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Quante, M., and Wang, T.C. (2009). Stem cells in gastroenterology and hepatology. Nat Rev
Gastroenterol Hepatol. 6, 72437.
Scadden, D.T. (2006). The stem-cell niche as an entity of action. Nature 441, 10751079.
Sell, S. (2004). Stem cell origin of cancer and differentiation therapy. Crit. Rev. Oncol. Hematol. 51, 128.
Zhang, L., Theise, N., Chua, M., and Reid, L.M. (2008). The stem cell niche of human livers:
symmetry between development and regeneration. Hepatology 48, 1598607.
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Inammation and Liver Regeneration

Johannes G. Bode
Learning Targets
1. Regulatory role of the acute phase response and of macrophages for liver regeneration
2. The inammatory cytokine and chemokine network induced upon liver injury and its
relevance for the regulation of the regenerative response in the liver
3. Relevance of natural killer cells and natural killer T cells for the course of liver
regeneration
3.1
Introduction
The liver has multiple functions in the organism, which all in all make it indispensable
for survival. It is central for regulation of metabolism of carbohydrate, lipid, and protein, as well as for biotransformation and detoxication of endogenous metabolites or
xenobiotics, and is the major site for the biosynthesis of important serum constituents
such as albumin, coagulation factors, components of the complement system, secreted
pathogen recognition receptors (PRR), and other acute phase proteins (APP). Furthermore, it has an important function in the regulation of pH and ammonia-homeostasis
and is crucial for synthesis and secretion of bile salts and bile saltdependent absorption
of nutrients.
Apart from this, the liver is crucial for regulation of the acute phase response and
innate and adaptive immunity. The liver receives 80% of its blood supply from the gut
and the spleen, which is rich in gut-derived bacterial products, environmental toxins,
and food antigens, as well as in spleen-derived immunological signals, including the
inux of immune-competent cells that leave the spleen via the portal venous blood. This
makes the liver an important intersection point for regulation of systemic inammatory
response and innate and adaptive immunity as it integrates inammatory or immunological signals from endogenous (spleen, arterial blood) as well as exogenous (gutderived) sources. Increasing evidence indicates that the liver is of particular importance
for induction of immunological tolerance. The notion that hepatocytes are important
constituents of innate immunity is further substantiated by the observation that patients
who received livers from donors with a genetic predisposition to lowered production of
secreted PRR had a higher risk for bacterial infections (Bouwman et al., 2005).
All these different functions of the liver are tightly linked to the complex assembly of
highly specialized cell types organized in the sinusoidal unit, embedding hepatocytes
into a structuralfunctional organization with the different non-parenchymal cells of the
liver, such as sinusoidal endothelial cells, hepatic stellate cells, and liver macrophages
40
(also termed Kupffer cells). The latter are present in the sinusoids and under homeostatic
conditions represent about 15% of total liver cell population. The fact that the liver
harbors almost 80% to 90% of all tissue macrophages in the body, located in a strategic
position for screening of pathogens, which enter the liver via the portal-venous blood,
underscores the relevance of the liver for systemic acute phase response and innate immunity. Central to innate immunity, liver macrophages are responsible for clearance of
exogenous material that is perceived as foreign and harmful and also sense endogenous
molecular signals that may result from perturbed homeostasis of the host (Kolios et al.,
2006). Liver macrophages recognize potential danger from both sources and undergo
activation, enabling them to launch biochemical attack and to involve the other parenchymal and non-parenchymal cells of the liver in the inammatory process by releasing
a variety of inammatory mediators.
Macrophages and other cells of the innate immune system populating the liver, such
as natural killer (NK) cells, have been demonstrated to play a regulatory role for liver
regeneration, and this function is closely connected to the inammatory mediators released upon their activation. This chapter focuses on the role of inammation for the
course of liver regeneration and, in particular, on those inammatory mediators and
immune cells involved in liver regeneration.
3.2
Liver Regeneration and Inammation: General Aspects
The wide array of functions provided by the liver to the organism is safeguarded by its
phenomenal capacity to regenerate. Thereby, the term regeneration should be better
conceived as compensation of lost function as it is not the anatomically correct regrowth of a lost part of an organ or a limb, but rather a reconstitution of lost functional
tissue. Nevertheless, the process of liver regeneration is highly complex and at the end
leads to the reconstitution not only of the mass of hepatocytes but also of the complex
structuralfunctional organization of the sinusoidal unit required to fulll the whole
spectrum of functions the liver provides to the body. This process is controlled by the
activation of a multitude of different signals that do not act independent from each other
but are rather multifariously interlinked with a high redundancy existing between the
different components of the intracellular networks. Accordingly, as far as can be judged
from the current literature, there is no single gene or pathway that can be considered as
the essential gene or pathway that drives liver regeneration.
Although the molecular regulation of liver regeneration is far from being understood, liver regeneration can be viewed as a multistep process roughly dividable into
priming pathways rendering hepatocytes sensitive to growth factors and subsequent
induction of growth promoting pathways leading to enhanced DNA synthesis and
hepatocyte proliferation. Finally, regeneration is terminated by growth inhibitory
pathways.
During the past it became increasingly evident that induction of a dened inammatory
response is central to regulation of liver regeneration subsequent to liver injury. Thereby,
the release of inammatory cytokines, such as IL-6, OSM, or TNF, is considered to be
an important constituent of the priming pathways. These pathways render hepatocytes
sensitive to those signals that control growth promoting pathways, including the action
of growth factors, such as hepatocyte growth factor (HGF), which is considered to be
3.3
Liver Macrophages and Their Relevance for Liver Regeneration
41
most important for liver regeneration, or the different ligands of the epidermal growth
factor (EGF) receptor. The inammatory response, which can be observed during liver
regeneration after liver injury in humans or in animal models, displays characteristics of
cytokine driven acute phase response. In accordance with this, liver regeneration is also
accompanied by a systemic response characterized by increased temperature, neutrophilia, derangement in energy metabolism, enhanced release of inammatory cytokines,
and up-regulation of the serum concentrations of acute phase proteins and components
of the complement system, as well as of growth factors.
Generally, the acute phase response represents an immediate set of inammatory
reactions that occurs upon tissue injury caused by trauma, infection, or noninfectious
inammatory causes of tissue injury. The central aim of these reactions is, on the one
hand, the isolation and neutralization of the eliciting pathogen, or the prevention of
further pathogen entry, and on the other hand, the minimization of tissue damage and
promotion of repair processes to permit the homeostatic mechanisms of the organism
to rapidly restore normal physiological function. Considering this, the acute phase response is an integral constituent of every type of wound healing processes, including
liver regeneration subsequent to liver injury. Thereby, the set of inammatory reactions
varies and largely depends on the eliciting pathogenic cause. It becomes clear from this
that a misdirected and inappropriate inammatory response would unequivocally result
in an imbalance of tissue destruction, repair, and regeneration, leading to insufcient or
blemished restoration of functional tissue, which in turn causes chronic disease or, as the
worst case scenario, fulminant organ failure. Hence, as a consequence of this closed
interrelationship between liver regeneration and inammation, alterations of the inammatory response would result in impaired liver regeneration or even liver failure.
3.3
Liver Macrophages and Their Relevance for

Liver Regeneration
As discussed previously, it is widely accepted that liver regeneration following partial

hepatectomy is accompanied by an acute phase response, which mainly reects the
activation of innate immunity in response to liver injury. Although the source of inammatory cytokines released during liver regeneration is not fully claried several lines
of evidence strongly indicate that macrophages are the most relevant source and that
macrophage activation promotes liver regeneration. This view is supported by the vast
majority of studies investigating liver regeneration after macrophage-depletion using
liposome-encapsulated dichloromethylene diphosphonate. These studies provide strong
evidence that activation of macrophages promotes the early phase of liver regeneration,
mainly through its supportive effects on hepatocyte proliferation (Abshagen et al., 2007;
Meijer et al., 2000; Selzner et al., 2003). There are only a few conicting reports that
do not support this perception and suggest that the activation of macrophages compromises rather than supports liver regeneration (Rai et al., 1996). These latter studies used
gadolinium chloride for macrophage depletion, which at rst activates macrophages to
secrete biologically active substances (Rai et al., 1997) and is considered to be retained
in and toxic to hepatocytes. This may explain the diverging ndings because liposomeencapsulated dichloromethylene diphosphonate, which is used by the majority of the
aforementioned reports, is nontoxic to hepatocytes, and upon intracellular release of
42
the substance, macrophages are selectively eliminated without activation (Van Rooijen
and Sanders, 1994).
The inhibition of hepatocyte proliferation observed upon macrophage depletion prior
to liver resection is paralleled by a markedly decreased expression of the proliferation
marker PCNA and of cyclin B1 (Abshagen et al., 2007; Selzner et al., 2003). As this
marker is thought to be specically up-regulated during the meta-phase, this may suggest that depletion of liver macrophages interferes with the cell cycle of hepatocytes at
the transition from the S to the G2/M phases.
Liver macrophages are known to produce a variety of different mediators involved
in the regulation of inammation and regeneration and are reported to be the most
important source of TNF in the liver (Decker, 1998). However, with respect to the
question, whether liver resection induces the release of cytokines and the role of macrophages for this, the data are in part controversial as some reports could not detect
an up-regulation of TNF or IL-6 expression subsequent to liver resection (Enayati et
al., 1994) or did not support the view that this increase in cytokine expression is due
to activation of liver macrophages (Loffreda et al., 1997). These differences may depend on the animal models used, the technique employed for macrophage depletion,
the operation technique, and in part, also on the samples (tissue or serum) analyzed
and how (detection of transcript or protein) cytokine production has been determined.
Nevertheless, the majority of reports indicate that, while control animals show an increase in serum concentrations of TNF and/or IL-6 or of hepatic transcript or protein
levels of these cytokines, macrophage depleted animals failed to mount a comparable
cytokine response upon liver resection (Abshagen et al., 2007; Meijer et al., 2000;
Selzner et al., 2003). All in all, these data strongly support the view that liver resection and successional initiating liver regeneration results in an increased expression of
inammatory cytokines and that this, to an important part, is due to the activation of
liver macrophages (fFigure 3.1).
This notion is further supported by the observation that the lack of functional macrophage colony stimulating factor (M-CSF) results in impaired liver regeneration, which
is probably due to a substantial reduction of the number of liver macrophages in these
animals (Amemiya et al., 2011). Thereby, activation of phosphatidylinositol 3-kinase
(PI3K)-dependent signaling may further be relevant for macrophage recruitment to
the regenerating liver as inhibition of its activity results in a reduced number of macrophages. This in turn is associated with a signicant decrease in hepatocyte proliferation and reduced tissue levels of TNF and IL-6 ( Jackson et al., 2008). These data
corroborate the relevance of macrophage recruitment being required for inammatory
cytokine elaboration mediating hepatocyte priming for replication and indicate that
PI3K-dependent signaling is substantially involved in this process.
That recruitment of leucocytes is indeed a prerequisite for undisturbed liver regeneration is further substantiated by the observation that animals decient for intercellular
adhesion molecule (ICAM)-1 show an impaired regenerative response upon partial
hepatectomy. In these animals, disturbed liver regeneration and cytokine release is
demonstrated to be due to their impaired ability to recruit leukocytes and, in particular,
macrophages and neutrophils to the regenerating liver. Correspondingly, both depletion
of macrophages or of neutrophils were able to mimic the defect of the regenerative
response observed in ICAM-1 decient animals. Thereby, the observation that pretreatment with recombinant IL-6 completely reversed the failure of hepatocytes to replicate
3.3
Liver Macrophages and Their Relevance forLiver Regeneration
43
in ICAM-1 decient animals underscores the relevance of the inammatory cytokine

response generated by these inammatory cells for liver regeneration (Selzner et al.,
2003).
chemotactic factors:
e.g. CXCL2
priming factors:
e.g. IL-6, TNFa,
OSM, SCF
growth factors:
e.g. insulin, HGF,
EGF, TGFa
proliferation
c-Kit
chemotaxis
IL-6
OSM
OSMR /gp130
inhibitory factors:
e.g. TGFb,
IL-1b, IFNg
STAT3
IL-6R /gp130
IL-6
ICAM-1
IL-6R /gp130
TNFR1
NF-kB
TNFa /LTa
release of other
(inflammatory)
mediators, or
induction of their
production through
other cell types
- chemotaxis
- release of GF
- tissue remodeling
- termination
Figure 3.1 Schematic Summary of the Interrelationship between Macrophage Activation,

Inammatory Mediator Release, and Liver Regeneration
Notes:
Liver injury leads to ICAM-1 dependent chemotactic recruitment of macrophages and to the
release of macrophage-derived mediators such as TNF or LT. Autocrine activation of TNF
receptor (TNFR)1 results in activation of intracellular signal transduction also involving the
activation and nuclear-translocation of nuclear factor (NF)-B. This mediates the production
and release of other mediators, including IL-6 and oncostatin M (OSM), which, like other
factors such as stem cell factor (SCF), contribute to hepatocyte priming. This results in the sensitization of hepatocytes toward the action of growth factors (GF) such as hepatocyte growth
factor (HFG) or epidermal growth factor (EGF). Apart from macrophages and hepatocytes,
liver regeneration in response to liver injury also involves other non-parenchymal cells of the
liver, such as hepatic stellate cells, sinusoidal endothelial cells, or cells of the innate immune
system such as natural killer cells. All these cells contribute to the intracellular signaling network controlling liver regeneration through the release of (inammatory) mediators, including
chemokines, which mediate recruitment of cells from the circulation; monocytes; and other
immune competent cells or even bone marrowderived stem cells.
44
3.4
Inammatory Mediators Are Required to Promote

Liver Regeneration
The cytokine pattern released during liver regeneration must be considered as largely
unmapped, and the exact role of the different components of the cytokine network
activated after liver injury is unclear. However, it is meanwhile widely accepted that the
release of cytokines during the early phase of liver regeneration is important and that
macrophage-derived mediators play a role. The expression of inammatory cytokines
such as TNF or IL-6 is up-regulated in the liver within hours after partial hepatectomy
at the transcript and protein levels, and increased serum levels of TNF and IL-6 remain
detectable for at least 3 to 5 days (Abshagen et al., 2007; Akerman et al., 1992; Meijer
et al., 2000; Selzner et al., 2003). Depletion of the major cellular sources impedes
proliferation of hepatocytes during liver regeneration, further corroborating the role of
inammatory cytokines in the regulation of hepatocyte proliferation (Abshagen et al.,
2007; Selzner et al., 2003).
The important role of TNF for liver regeneration is supported by the observations that
injection of TNF antagonistic antibodies prior to partial hepatectomy strongly reduced
proliferation of hepatocytes and non-parenchymal liver cells (Akerman et al., 1992). This
was associated with an impaired up-regulation of IL-6, suggesting that IL-6 release is
regulated by TNF in an autocrine manner, which was also in line with the observation
that the release of IL-6 in response to liver injury is reduced in TNF-receptor (TNFR)-1
decient animals. Congruently, DNA synthesis after partial hepatectomy or tetrachloride
mediated liver injury was severely impaired in mice lacking the TNFR1, resulting in
delayed liver regeneration and an increased mortality (Yamada et al., 1997; Yamada et
al., 1998). As a result of impaired TNF signaling and decreased release of IL-6, the
activation of nuclear-factor (NF)-B and signal-transducer and activator of transcription
(STAT)-3 normally observed in response to partial hepatectomy (Cressman et al., 1995)
was likewise blocked in TNFR1 decient animals (Yamada et al., 1997; Yamada et al.,
1998), whereas ablation of the TNFR2 gene had no effect on the course of liver regeneration (Yamada et al., 1998). The relevance of TNFR1 has further been conrmed by the
demonstration that regeneration after partial hepatectomy was substantially delayed in
TNFR1 decient mice and that this was accompanied by a decreased expression of
cell-cycle-regulated genes such as cyclin D1 (Shimizu et al., 2009). Interestingly, administration of stem cell factor (SCF) to mice lacking the TNFR1 in the context of partial
hepatectomy restores hepatocyte proliferation to normal (Ren et al., 2008). Additionally,
injection of IL-6 in TNFR1 decient animals prior to partial hepatectomy also corrected
the defect in DNA synthesis and restored STAT3 and AP-1 binding to normal levels but
had no effect on NF-B binding in the regenerating liver (Yamada et al., 1997). Thereby,
sustained activation of NF-B in response to liver injury is considered to mainly occur
in non-parenchymal cells of the liver and, in particular, in liver macrophages and only
fugaciously occurs in hepatocytes (Abshagen et al., 2007; Yang et al., 2005). In summary
these observations led to the assumption that liver injury leads to an up-regulation of
TNF that TNFR1-dependently mediates NF-B activation in macrophages. This results
in the release of macrophage-derived mediators such as SCF or IL-6 which in turn activate
STAT3 in an autocrine and paracrine manner (fFigure 3.1). Thereby, STAT3 activation
should mainly occur in hepatocytes because TNF prevents IL-6 from activating STAT3
in macrophages but not in hepatocytes (Bode et al., 1999).
3.4
Inammatory Mediators Are Required to PromoteLiver Regeneration
45
The up-regulation of cytokine expression after liver resection has been suggested to
be due to an increased exposure of the remaining liver macrophages toward entericderived bacterial products such as lipopolysaccharide (LPS) that reach the liver via the
portal vein (Cornell, 1985). However, cytokine release and transcriptional activation of
STAT-3 dependent genes after liver resection was severely depressed in mice lacking
MyD88 but not in animals decient for Toll like receptor (TLR)4, TLR2, or CD14. These
data indicate that the adapter molecule MyD88 is required to launch cytokine expression upon liver injury but argue against a critical role of enteric-derived bacterial products such as LPS for this process. Of note, hepatocyte proliferation was neither impaired
in MyD88-decient animals nor in those lacking CD14, TLR2, or TLR4 (Campbell et al.,
2006). Hence, these data may also suggest that TNF and IL-6 are not as important for
regulation of hepatocyte proliferation as originally presumed, a view that is also suggested from studies analyzing TNF-decient animals (Hayashi et al., 2005). Considering the well-documented role of macrophage activation and TNFR1-mediated signaling
for liver regeneration, these data further indicate that other macrophage-derived mediators acting via TNFR1 are more important than TNF itself. In this context, it should be
noted that in contrast to mice decient for TNF, hepatocyte DNA replication after partial hepatectomy is down-regulated in animals lacking both TNF and lymphotoxin
(Knight and Yeoh, 2005).
As for TNF, there is also controversy concerning the role of IL-6 for liver regeneration and, in particular, for the induction or propagation of hepatocyte proliferation.
Similar to TNF, IL-6 is thought to be mainly produced by liver macrophages, and its
expression is regulated by MyD88 and ICAM-1 (Campbell et al., 2006; Selzner et al.,
2003). In addition, components of the complement system, such as C3A and C5A, are
also involved in mediating IL-6 expression (Strey et al., 2003). IL-6 has been originally
suggested to mediate hepatocyte proliferation (Cressman et al., 1996). Congruently,
injection of IL-6 was able to rescue impaired liver regeneration in IL-6-decient animals
but also in mice lacking TNFR1 or ICAM-1 (Cressman et al., 1996; Selzner et al., 2003;
Yamada et al., 1997). Hence, one may conclude that an important effect of leukocyte recruitment and activation of TNFR1 is the up-regulation of cytokines such as
IL-6, which in turn promote hepatocytes to proliferate (fFigure 3.1). In line with this
view, studies with bone marrow chimeric mice indicated that IL-6 derived from intrahepatic sources of bone marrow origin, most likely liver macrophages, is required for
undisturbed liver regeneration (Aldeguer et al., 2002).
The appraisal of the role of IL-6 has been complicated as some studies analyzing
liver regeneration in mice decient for IL-6 or gp130, the signal transducing subunit
of the IL-6 receptor complex, could not conrm the reported defect in DNA replication (Wuestefeld et al., 2003) or observed a much less severe phenotype (Sakamoto
et al., 1999). These discrepancies may be due to lacking standardization of experimental conditions, such as different genetic background of the mice used or differences in
the operation technique employed. Apart from this, the level of IL-6 present after partial hepatectomy may be critical in determining its effects on hepatocyte proliferation
(Blindenbacher et al., 2003; Zimmers et al., 2003). A protective rather than a mitogenic
role of IL-6 is suggested by studies demonstrating that mice lacking gp130 display minor
defects in hepatocyte proliferation after partial hepatectomy but showed massive apoptosis of hepatocytes if treated with LPS prior to hepatectomy (Wuestefeld et al., 2003).
Consistently, IL-6 was reported to mediate protective effects in a mouse model of severe
46
liver injury after 87% hepatectomy ( Jin et al., 2007) as well as upon warm ischemia/
reperfusion mediated injury where it also promotes regeneration (Camargo et al., 1997).
Whereas the role of TNF and IL-6 has been comparably broadly addressed, the role
of other inammatory mediators was only subject of some scattered reports. However,
there is an increasing body of evidence that several other cytokines and chemokines are
also up-regulated during liver regeneration and play a role in liver regeneration. Thus,
studies on oncostatin M receptordecient animals indicate that OSM is important for
liver regeneration, promoting hepatocyte proliferation, and being involved in regulation
of tissue remodeling (Nakamura et al., 2004). Likewise IL-22 has been suggested to
support liver regeneration, in particular, by interaction with STAT3-dependent signaltransduction and with the release of IL-6 and TGF in response to liver resection (Ren
et al., 2010).
Another group of inammatory mediators that has been implicated in the regulation
of liver regeneration are members of the so termed CXC chemokine family, characterized by a conserved cysteine-acid containing motif CXC (where X is any amino acid)
located within their N-terminus. CXC chemokines play an essential role for chemotaxis
of neutrophils, and induction of proliferation and chemotaxis of endothelial cells in a
manner that facilitates angiogenesisfunctions that suggest an involvement of these
chemokines in regeneration and tissue repair. Correspondingly, recent reports indicated
that expression of members of this chemokine family is increased after hepatic resection and that CXC chemokines mediate proliferative effects on hepatocytes in vitro
and in vivo. Thus, blocking of the CXC chemokine epithelial neutrophil-activating protein (ENA-78) retards hepatic regeneration after resection. Furthermore, CXCL2 (also
termed macrophage inammatory protein [MIP]-2) is important for hepatocyte proliferation after partial hepatectomy, and pharmacological MIP-2 doses accelerate hepatic
regeneration after hepatic injury (Colletti et al., 1998).
In addition to cytokines and chemokines, components of the complement system
appear to be likewise important for liver regeneration because mice lacking C3 or C5
exhibited high mortality, parenchymal damage, and impaired liver regeneration after
partial hepatectomy. This phenotype was even more exacerbated in mice with combined ablation of the C3 and the C5 gene and was reversed by reconstitution of C3a
and C5a (Strey et al., 2003).
3.5
Inappropriate Inammation Impairs Liver Regeneration
The increased release of the different inammatory mediators, which regulate liver
regeneration, is the result of a controlled inammatory response. Imbalanced or misdirected induction of this inammatory response, for example, because of infectious
disorders acquired prior to or during liver regeneration, would inevitably result in insufcient tissue repair, tissue destruction, and impaired organ/hepatocyte functionality, and
therefore, might be fatal for a patients recovery following partial hepatectomy. Thereby,
cytokines such as IL-1 released during an inammatory response toward pathogens act
as potent inhibitors of hepatocyte proliferation and liver regeneration and can delicately
disturb the regeneration process (Boermeester et al., 1995; Boulton et al., 1997). Other
mediators such as TNF seem to play a dual role as they support the priming phase but
are also involved in mediating hepatotoxicity of perturbating pathogen-derived factors
3.6
Role of NK and NKT-cells for Liver Regeneration
47
such as LPS (Nowak et al., 2000). Likewise, CXC receptor (CXCR)2-transmitted signals
mediate supportive effects on liver regeneration but have been also identied as important mediators of ischemia/reperfusion-induced liver injury. The molecular mechanisms
enabling factors such as TNF and CXCL2 to mediate liver injury on the one hand
and to promote tissue repair and regeneration on the other hand are not clear. One
potential mechanism may be that the controversial effects are related to the amount
of the respective mediators produced. Thus, in a recent review Clarke et al. (2009)
reported that levels of CXC chemokines are increased 3 to 5 fold in response to partial
hepatectomy, whereas liver injury upon ischemia/reperfusion resulted in an increase
of CXC chemokines up to 25 to 50 fold. Consistently, low concentrations of CXCL2
mediated hepato-protective effects in vitro, whereas high concentration had signicant
cytotoxicity. These observations suggest that moderate increase in CXCR2 ligands promotes liver regeneration, while an excessive increase mediates hepatotoxic effects and
impairs liver regeneration. According to this model, the protective and pro-regenerative
effects observed for IL-6 in ischemia/reperfusion-mediated liver injury may be, in part,
due to the modulatory effects of IL-6 on the inammatory response and, in particular,
on TNF concentrations, which were signicantly decreased in animals pretreated with
IL-6 (Camargo et al., 1997). Another point that may be decisive for whether a signal is
supportive for liver regeneration or acts as a hepatotoxic signal is the pattern of co-acting
mediators, which may inuence the nal effect. Thus, for example, in hepatocytes IL-1
prevents IL-6 from activating STAT3 (Albrecht et al., 2007; Bode et al., 1999), which
may contribute to the antiproliferative effects of IL-1 on hepatocytes.
3.6
Role of NK and NKT-cells for Liver Regeneration:

Negative Regulators of Regeneration
Natural killer (NK) cells and natural killer T (NKT)-cells constitute the predominant
lymphoid cell population in human and mouse liver and, apart from macrophages,
represent important components of innate immune cells prevalent in the liver. There
is increasing evidence that, apart from macrophages, in particular, NK cells accumulate in the remnant liver and inuence the regeneration process. Thereby, the existing
data indicate that, in contrast to macrophages, NK cells mediate inhibitory rather than
supportive effects on liver regeneration and that this is likely a result of the biological
activity of IFN produced by NK cells (Sun and Gao, 2004) and IFN mediated activation of the transcription factor STAT1. Hence, depletion of NK cells or inhibition by
immunosuppressive drugs such as FK506 or cyclosporine A enhances liver regeneration
(Francavilla et al., 1991), whereas liver regeneration is impaired upon activation of
NK cells by poly I:C or viral infection. Evidence for IFN as the central mediator of the
effects of NK-cells on liver regeneration came from the observation that in NK cell
depleted animals attenuation of liver regeneration by poly I:C was restored by adoptive
transfer of NK cells isolated from wild type mice but not from IFN-decient mice. The
fact that IFN or IFN receptordecient animals show an enhanced liver regeneration
upon partial hepatectomy, whereas administration of IFN resulted in impaired liver
regeneration, further supports the notion that IFN is central for the inhibitory effects of
NK cells on liver regeneration (Sun and Gao, 2004). Apart from this, NK cells have been
further demonstrated to hamper liver regeneration by expressing direct cytotoxicity
48
against regenerating hepatocytes (Itoh et al., 1988). Most interestingly, hepatotrophic

factors such as augmenter of liver regeneration, hepatocyte growth factor, and insulin
like growth factor specically suppress lytic activity of liver resident NK cells being
associated with suppression of IFN expression in vivo (Francavilla et al., 1997). Hence
suppression of NK cell activity and down-regulation of NK cellderived mediators may
be part of the pro-regenerative effects of factors such as augmenter of liver regeneration.
Apart from this, recent evidence suggests that recombinant human G-CSF facilitates
liver regeneration by immunoregulation through down-regulation of intra-hepatic IL-12
levels and evacuation of sinusoidal NK cells (Oishi et al., 2006).
NKT cells have been originally named as such because they co-express surface markers typical for conventional T cells as well as those that characterize NK cells. Like
NK cells, these cells accumulate in the regenerating liver. However, their role for liver
regeneration is still a matter of debate as liver regeneration is comparable between mice
decient for NKT cells and wild type controls, suggesting that they are of minor importance. This view has become relativized, as activation of NKT cells in hepatitis B virus
(HBV) transgenic animals impedes liver regeneration, suggesting that under dened
conditions NKT cells may also negatively inuence liver regeneration, an effect that has
also been mainly attributed to the release of IFN. This assumption is further supported
by the fact that activation of NKT cells by IL-12 or -galactosylceramide enhances liver
injury during liver regeneration.
All together these data suggest that upon their activation NK cells and, under certain
conditions, NKT cells impede liver regeneration. Although this view is supported by the
majority of reports, there are some reports that suggest that under dened conditions
NK cells may also stimulate oval cell expansion or enhance hepatocyte mitosis (Gao
et al., 2009).
Summary
The liver is irreplaceable for whole body metabolism, biotransformation, and detoxication
and plays a critical role for acute phase response and innate and adaptive immunity. All of
these functions are tightly linked to the complex assembly of highly specialized cell types
organized in the sinusoidal unit. To safeguard this wide array of functions the liver owns
a phenomenal capacity to regenerate. During the past it became increasingly evident that
macrophage-derived mediators released in the context of a controlled inammatory response
play an important role for the early phase of liver regeneration, particularly regulating hepatocyte proliferation. In this context, not only cytokines such as TNF, IL-6, IL-22, and OSM
or chemokines such as CXCL2 but also other inammatory mediators such as components
of the complement system are of importance. Disturbance of this inammatory response can
have detrimental effects on the regenerative response, resulting in impaired liver regeneration
or liver failure.
References
49
Further Reading
Bode, J.G., and Heinrich, P.C. (2001). Interleukin-6 signaling during the acute -phase response
of the liver. In: The Liver: Biology and Pathobiology, 4th edition, Arias, I.M., Boyer, J.L.,
Chisari, F.V., Fausto, N., Schachter, D., and Shafritz, D.A., eds. (Philadelphia: Lippincott
Williams Wilkins), pp. 56580.
Crispe, I.N. (2009). The liver as a lymphoid organ. Annu. Rev. Immunol. 27, 14763.
Hussinger, D., Kubitz, R., Reinehr, R., Bode, J.G., and Schliess, F. (2004). Molecular aspects
of medicine: from experimental to clinical hepatology. Mol. Aspects Med. 25, 221360.
mechanistic dilemmas. Am. J. Pathol. 176, 213.
Tiegs, G., and Lohse, A.W. (2010). Immune tolerance: what is unique about the liver. J. Autoimmun. 34, 16.
References
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activation in Kupffer cell-depleted mice impairs liver regeneration after partial hepatectomy. Am. J. Physiol. Gastrointest. Liver Physiol. 292, G15707.
Akerman, P., Cote, P., Yang, S.Q., McClain, C., Nelson, S., Bagby, G.J., and Diehl, A.M.
(1992). Antibodies to tumor necrosis factor-alpha inhibit liver regeneration after partial
hepatectomy. Am. J. Physiol. 263, G57985.
Albrecht, U., Yang, X., Asselta, R., Keitel, V., Tenchini, M.L., Ludwig, S., Heinrich, P.C.,
Hussinger, D., Schaper, F., and Bode, J.G. (2007). Activation of NF-kappaB by IL-1beta
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Aldeguer, X., Debonera, F., Shaked, A., Krasinkas, A.M., Gelman, A.E., Que, X., Zamir, G.A.,
Hiroyasu, S., Kovalovich, K.K., Taub, R., and Olthoff, K.M. (2002). Interleukin-6 from intrahepatic cells of bone marrow origin is required for normal murine liver regeneration.
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S.P., Daha, M.R., Frolich, M., van der Slik, A.R., Doxiadis, I.I., Roep, B.O., and Schaapherder, A.F. (2005). Mannose binding lectin gene polymorphisms confer a major risk for
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50
Camargo, C.A. Jr., Madden, J.F., Gao, W., Selvan, R.S., and Clavien, P.A. (1997). Interleukin-6 protects liver against warm ischemia/reperfusion injury and promotes hepatocyte
proliferation in the rodent. Hepatology 26, 151320.
Campbell, J.S., Riehle, K.J., Brooling, J.T., Bauer, R.L., Mitchell, C., and Fausto, N. (2006).
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32634.
Lymphotoxin Receptor and Tumor Necrosis

Factor Receptor p55 in Liver Regeneration
Ursula R. Sorg and Klaus Pfeffer
Learning Targets
1. The receptors TNFRp55 and LTR, members of the TNFR superfamily, play a central
role in the early phase of liver regeneration after loss of liver mass.
2. Signaling via TNFRp55 and LTR leads to activation of the transcription factor NFB.
4.1 TNF/TNFR Superfamily

The tumor necrosis factor receptor p55 (TNFRp55) and the lymphotoxin receptor
(LTR) are prototypic members of the TNF/TNFR superfamily, a large group of approximately 50 molecules of receptors and their respective ligands (Hehlgans and Pfeffer,
2005). The members of the TNF/ TNFR superfamily are expressed on many cell types.
Their biological functions encompass a broad spectrum of activities: benecial effects
in inammation and protective immune responses to infectious diseases, a crucial
role in the organogenesis of secondary lymphoid tissues, and the maintenance of the
structural architecture of lymphoid organs in the mature organism, but also detrimental
actions for instance in sepsis, fever syndromes, cachexia, and immune disorders such
as rheumatoid arthritis, psoriasis, and inammatory bowel disease.
Some years ago it became apparent that most members of the TNF ligand superfamily
can interact with more than one member of the TNFR superfamily. First, it was thought
that this reected a certain redundancy in their biological functions. Now, however, it
is clear that most of the individual ligand-receptor interactions within the TNF/TNFR
superfamily engage in a unique and non-redundant function (Hehlgans and Pfeffer,
2005; Ware, 2008).
Most of the TNF superfamily ligands are type II transmembrane proteins. They are
biologically active as self assembling, non-covalently bound homotrimers. Exceptions
are the secreted LT3 homotrimer, which does not contain a transmembrane region, and
the heterotrimeric LT12, which contains one LT and two LT subunits. Some of the
ligands (e.g., TNF and LT) are active not only in the membrane bound form but also
act as soluble ligands that are released from the cell membrane after the appropriate
stimuli, commonly through proteolytic cleavage by metalloproteases.
The members of the TNFR superfamily are type I transmembrane proteins and are also
usually active as trimers. They are characterized by the presence of one to six cysteine
rich domains (CRDs) within their extracellular regions. Structurally, they can be divided
54
Lymphotoxin B Receptor and Tumor Necrosis Factor Receptor p55
into three distinct groups. The rst group contains death domains (DD) within their
cytoplasmic regions. Upon binding of the appropriate ligand and subsequent activation,
intracellular adaptor molecules containing death domains (such as TNFR-associated
death domain [TRADD] or Fas associated death domain [FADD]) are recruited (Hehlgans and Pfeffer, 2005). The second group of receptors contains TNF-receptor associated
factor (TRAF)-interacting motifs (TIMs), which can bind members of the TRAF family.
The third group of receptors apparently does not contain signaling motifs; its members
compete with the other groups of receptors for their respective ligands, possibly providing another level of regulation (Hehlgans and Pfeffer, 2005).
LTR and TNFRp55, together with the receptors TNFRp75, herpes virus entry mediator (HVEM) and the soluble decoy receptor 3 (DcR3) as well as the ligands TNF, LT3 ,
LT12 , LIGHT (lymphotoxin homolog exhibits inducible expression and competes with
herpes simplex virus glycoprotein D for HVEM, a receptor expressed on T cells), and
the B and T lymphocyte attenuator (BTLA) comprise the core or immediate members of
the TNF/TNFR superfamily (fFigure 4.1) (Hehlgans and Pfeffer, 2005). TNF and LIGHT
can be expressed either in a membrane bound form or as soluble factors, when they are
cleaved from the cell surface. LT3 lacks the membrane anchoring domain and is always
secreted. LT12 is anchored to the membrane via the LT subunits and, as it lacks cleavage sites, cannot be found as a soluble factor. The TNF receptors TNFRp55, TNFRp75,
TNF
LTa 3
LTa 1b 2
LIGHT
BTLA
TNFRp55
TNFRp75
LTbR
HVEM
DcR3
TRADD
TRAF
TRAF
TRAF
cleavage site
CRD
DD
apoptosis
Figure 4.1
activation
TIM
The Core TNF/TNFR Family
Notes:
Arrows indicate interactions between trimeric ligands and their cognate receptors, which
trimerize only after ligand binding. CRD: cysteine-rich domain; TRAF: TNF-receptor associated factor; TIM: TRAF-interacting motifs; DD: death domain; TRADD: TNF receptor associated death domain.
4.1 TNF/TNFR Superfamily
55
LTCR, and HVEM are expressed on the cell surface as monomers, and ligand binding
initiates receptor clustering, which is the initial step in signaling activation (Hehlgans
and Pfeffer, 2005).
Upon ligand binding and activation, the intracellular DD of TNFRp55 recruits the
adaptor protein TRADD (Ware, 2008). In the canonical pathway of nuclear factor B
(NFB) activation (fFigure 4.2), formation of the TNFRp55/TRADD complex induces
phosphorylation and activation of the inhibitor of NFB (IB) kinase (IKK) complex,
consisting of IKK, and G. This complex is then responsible for the phosphorylation
and subsequent ubiquitinylation and, ultimately, the proteasomal degradation of IB.
NFB is a family of transcription factors that exists in various isoforms: homo- or
cytoplasm
LTa 1b 2
TNF
TNFRp55
cytoplasm
LTbR
DD
TRADD
TIM
TRAF
NIK
IKKa
internalization
IKKb
IKKg
IKKa
canonical
NFkB activation
caspase
activation
IkBa
p100
relB
relA p50
ub
i
ny quiti
lat
ion
apoptosis
IKKa
alternative
NFkB activation
Ub
IkBa
relA p50
relA p50
Ub
itiiqu on
b
u ati
l
ny
degradation
degradation
e.g. p100
activation of
transcription
p52 relB
p52 relB
nucleus
Figure 4.2 NFB Activation Via TNFRp55 and LTR

Note:
Shown are the canonical and the alternative pathways.
activation of
transcription
56
heterodimers of a family of ve related proteins (relA, relB, c-rel, p50/NFB1, and p52/
NFB2). The degradation of IB releases the active NFB(RelA:p50) complex, unmasking its nuclear translocation sequence, whereupon RelA:p50 relocates into the nucleus,
binds to its appropriate recognition sequences, and initiates gene transcription. When
NFB signaling is not available, the TNFRp55/TRADD complex is internalized and
subsequent activation of the caspase cascade leads to apoptosis of the cell. Thus, TNF
has proapoptotic as well as promitogenic effects. It has been demonstrated for other
members of the TNF/TNFR superfamily (Fas/FasL) that higher order structures (clustering
of more than one ligand/receptor unit) may be required for death receptor activation.
Possibly, this could also play a role in deciding which signaling pathway is activated
after TNF/TNFRp55 binding.
The canonical pathway of NFB activation is also induced through LTR after binding
of its ligands LT12 or LIGHT. The LTR contains an intracellular TRAF-interacting motif
(TIM), which recruits TRAFs 2,3, and 5. TRAF2 and 5 are able to activate the IKK complex, again leading to the translocation of the active NFB(RelA:p50) complex into the
nucleus. In an alternative (non-canonical) pathway of NFB activation (fFigure 4.2),
the TNFRp55/TRAF2 complex can activate the NFB inducing kinase (NIK), which activates the IKK subunit, which is bound to and stabilizes the inactive NFB(RelB:p100)
dimer. Activation of IKK leads to ubiquitinylation and partial degradation of p100
into the active p52 subunit. Subsequently, active NFB(RelB:p52) translocates into the
nucleus and initiates gene transcription. The transcription factor NFB induces (among
many other genes) the expression of p100, linking the TNFR with the LTR signaling
pathway. Together, the NFB family can activate the expression of several hundred target genes (Ware, 2008). LTR/TRAF signaling can also lead to activation of the JNK
signaling cascade, resulting in the activation of activator protein 1 (AP-1), a transcription
factor, and can induce the release of reactive oxygen species (ROS).
4.2
Liver Regeneration
The liver fullls a number of important functions, such as the synthesis of blood proteins
(e.g., coagulation factors, albumin) and bile, the regulation of the homeostasis of many
plasma constituents (e.g., glucose, amino acids, hormones, vitamins, lipoproteins),
metabolization of endogenous and exogenous substances, detoxication, and defense
against infection.
Liver parenchymal cells (hepatocytes) and the cholangiocytes, the epithelial cells
lining the bile ducts, make up about 70% of the cells in the adult liver. The sinusoidal
endothelial cells (up to 15%) form sinusoids that carry the oxygen-rich blood from the
hepatic artery and the nutrient-rich blood from the hepatic veins. Kupffer cells (up to
15%) are the liver resident macrophages and phagocytose particulate matter (e.g., antigens, detritus) that is brought into the liver via the portal vein. Stellate cells / Ito cells
(approx. 5%) are responsible for the storage of vitamin A, play a role in brotic/cirrhotic
processes of the liver, and may also represent a novel stem/progenitor cell compartment
in the liver (Kordes et al., 2007).
Liver mass is tightly regulated so that in mammals the liver makes up around 5% of the
total body weight. Loss or gain of body weight leads to the reduction or increase of liver
mass through apoptosis or division of hepatocytes, respectively. After decrease of liver
4.3 TNFRp55 and Liver Regeneration
57
mass, either through liver resection, after small for size liver transplantation (less than
0.8%1% of total body weight) or due to acute poisoning, as well as after loss of hepatocyte function caused by chronic infections or chronic poisoning, the liver shows
a remarkable capacity to regenerate. In the case of loss of liver mass, regeneration is
achieved by division of mature hepatocytes (Michalopoulos, 2007; see also Chapter 1),
a process also referred to as compensatory hyperplasia. When the ability of mature
hepatocytes to undergo mitosis is lost or blocked, restoration of liver function occurs
via oval cell mediated regeneration. Oval cells expressing biliary as well as hepatocellular markers appear in the liver and are able to differentiate into hepatocytes as well
as cholangiocytes. Currently, stellate cells are also considered as a stem cell population
with an important role in liver regeneration because they have recently been shown to
express stem cell markers on their surface (Kordes et al., 2009; Kordes et al., 2007).
Within the mature healthy liver, most hepatocytes reside in the G0 phase of the cell
cycle, and only a very small percentage of mitotic hepatocytes can be found at any
given point of time. A loss of at least 10% of liver mass leads to the division of mature
hepatocytes. In the case of liver tissue loss in the range of 10%30%, hepatocytes enter
mitosis in a slow and non-synchronous manner. When at least 30%40% of liver tissue is lost, hepatocytes divide in a rapid and synchronized manner. A loss of more
than 75%85% of liver tissue leads to inefcient regeneration accompanied by high
mortality. In rodents, 70% or 2/3 partial hepatectomy (PHx) is the accepted in vivo model
to study liver regeneration. In mice, this is achieved by surgically removing the three
largest liver lobes, leading to a peak of DNA replication in hepatocytes 4044 hours
after PHx (2226 hours in rats), followed by hepatocyte mitosis 68 hours later. Cell
division of the non-parenchymal liver cells occurs 1224 hours after hepatocyte mitosis.
A second, less synchronized wave of hepatocyte division can be detected several days
later. In rodents, up to 95% of all mature hepatocytes enter the mitotic cycle at least
once, and in order to regenerate physiologic liver mass after 70% hepatectomy, statistically each hepatocyte has to divide 1.3 times. It has been demonstrated that in older
animals the percentage of hepatocytes undergoing mitosis after PHx decreases to about
70%, and time needed for complete liver regeneration increases. In mice, physiologic
liver mass is regenerated 710 days after PHx.
Gene expression in rodents after 70% PHx shows a characteristic pattern (Fausto
et al., 2006). Expression of the so-called immediate early genes can be detected within
minutes after PHx and lasts for about 4 hours. Protooncogenes such as c-fos, c-myc,
and c-jun can be detected, together with transcription factors (e.g., NFB, AP-1, STAT3,
CEBP), tyrosine phosphatases, and metabolic proteins. During this phase, hepatocytes
enter the G1 phase of the cell cycle but do not progress further. In the next phase, 4 8
hours after PHx, transcription of the delayed early genes is detectable, among which
antiapoptotic genes, such as Bcl-XL, a well-known antiapoptotic protein of the liver,
can be found. In the subsequent stage (from 8 hours post-PHx), many cell cycle related
genes such as cyclins (e.g., cyclin D1) and cyclin dependent kinases are expressed.
4.3 TNFRp55 and Liver Regeneration

The TNFRp55 is expressed by many cells of the hematopoietic lineage, including almost
all T cells at some point in their development. It binds two ligands of the immediate
58
TNF/TNFR superfamily: TNF, expressed by many somatic, antigen presenting and also
T cells, as well as LT3, expressed on T, B, and NK cells with similar functions as TNF
(Apostolaki et al., 2010). TNF/TNFRp55 signaling plays a prominent role in proinammatory processes during the early immune response (Croft, 2009). It is not yet clearly
understood what links molecules of the innate immune response to liver regeneration,
but after PHx, levels of TNFRp55 increase rapidly (within 30 minutes) and administration
of anti-TNF antibodies inhibits liver regeneration. Mice lacking a functional TNFRp55
(TNFRp55-/- mice) show severely inhibited DNA replication and increased mortality
after PHx. NFB and STAT3 activation is inhibited, and AP-1 activation is decreased
(Yamada et al., 1997). In contrast, mice decient in TNFRp75 show normal hepatocyte
proliferation and gain of liver mass after PHx, although expression of the transcription
factors AP-1 and CEBP is delayed, demonstrating that TNF/TNFRp55 signaling but not
TNF/TNFRp75 signaling is essential for efcient liver regeneration. IL-6 levels also increase shortly after PHx in WT mice but not in TNFRp55-/- animals, where, interestingly,
injection of IL-6 can restore efcient liver regeneration (Yamada et al., 1997). From
these data, the following pathway for TNF signaling after PHx can be constructed: TNF
binds to TNFRp55 on Kupffer cells and hepatocytes and induces activation of the NFB
transcription factors via the canonical pathway. NFB is known to induce transcription
of IL-6, which in turn binds to its receptor (IL6R) and, ultimately, via JAK-signaling, leads
to activation of the transcription factor STAT3. This network of cytokine signaling promotes entry of the hepatocytes into the G1-phase and renders them responsive to growth
factors (priming), such as hepatocyte growth factor (HGF) and the EGFR-ligands that
initiate the transition of hepatocytes from the G1 into the S-phase. While it is clear that
the primary source of TNF lies within the hepatic Kupffer cells, it is still unclear which
signals induce TNF secretion after PHx. A potent inducer of TNF is bacterial lipopolysaccharide (LPS), which is a component of the cell wall of Gram-negative bacteria in
the gut and is continually transported in low amounts into the liver via the portal vein
(Crispe, 2009). By an as yet not completely understood mechanism, these basal levels of
LPS do not lead to an immune response but induce a tolerogenic state in the liver. It was
found that mice that are poor responders to LPS or germ-free rodents show a delayed
peak of DNA replication after PHx. On the other hand, mice decient in the LPS receptors (toll like receptors [TLR] 4) and CD14 (involved in TLR/LPS binding) do not show
any defects in TNF and IL-6 signaling immediately after PHx. Because mice decient in
Myd88, an adaptor molecule in the LPS signaling pathway, do show these defects, this
possibly argues for TNF induction not by LPS but by an as yet unknown sensor/receptor
interaction that engages the same signaling pathway(s) as LPS.
4.4
LTBR and Liver Regeneration
The LTR is expressed on non-hematopoietic cells and has two ligands: LT12, which is
expressed in high amounts by nave B cells in the spleen but also by CD4 T cells; and
LIGHT, which is inducible in resting T cells. LT12/LTR signaling plays an important
role during organogenesis of the secondary lymphoid organs and in maintenance of
secondary lymphoid organ structure (Futterer et al., 1998). LIGHT/LTR signaling is
also involved in dendritic cell homeostasis. Transgenic mice overexpressing LIGHT (Tg
LIGHT mice), show signicantly increased liver size compared to WT litter mates, and
4.4
LTBR and Liver Regeneration
59
histology shows enlarged hepatocytes and an increase of mitotic gures (Anders et al.,
2005). When Tg LIGHT mice were crossed with LTR decient (LTR-/-) mice, offspring
showed normal liver size and histology, indicating that constitutive LIGHT/LTR signaling
stimulates hepatocyte growth and induces hepatomegaly. The abnormal liver phenotype
was not abrogated when Tg LIGHT mice were crossed with mice that were decient for
HVEM (HVEM-/-), the other receptor for LIGHT, demonstrating that LIGHT/LTR but not
LIGHT/HVEM signaling is important for liver homeostasis (Anders et al., 2005). When
bone marrow from Tg LIGHT mice was transplanted into lethally irradiated WT mice,
these mice developed a phenotype comparable to the Tg LIGHT mice. A similar effect
was observed when thymocytes from Tg LIGHT mice were adoptively transferred into
RAG1-/- mice, which are decient in the lymphocyte compartment, conrming that
T-cell derived LIGHT is involved in regulation of liver homeostasis (Tumanov et al.,
2009). When 70% PHx was performed on LTR-/- mice, the animals showed increased
mortality (survival rates of around 30% compared to 100% in WT animals), and histology showed signicantly increased rates of apoptosis and signicantly decreased
hepatocyte proliferation, documenting the necessity of LTR signaling for efcient hepatocyte proliferation in the context of liver regeneration. Hepatectomized RAG1-/- mice
show a marked increase in mortality (15% survival compared to 100% in WT animals),
suggesting that the presence of B and/or T cells is necessary for liver regeneration (Tumanov et al., 2009). Further analysis of mice decient for LT12 in either the B or
T cell compartment clearly demonstrated that LT12 delivered by T cells to the liver is
essential for efcient liver regeneration (as opposed to LIGHT/LTR signaling in liver homeostasis). Treatment of RAG1-/- mice with an agonistic anti-LTR antibody immediately
after PHx increased serum TNF levels. Also, the addition of TNF and LIGHT, but not of
TNF or LIGHT alone, to cultures of mouse embryonic broblasts led to induction of IL-6
expression, indicating that LTR dependent activation of TNF signaling may play a role
in liver regeneration (Tumanov et al., 2009).
Even though TNFRp55 and LTR obviously play a major role in liver regeneration as
demonstrated by signicantly decreased survival rates after 70% PHx in the respective
knockout mouse strains, it is important to note that a stable percentage of these knockout animals survive the procedure without any apparent difference to WT animals.
Therefore, neither receptor alone is absolutely required for liver regeneration. Possibly,
a certain overlap in the signaling pathways and resulting redundancy of function of the
immediate TNF/TNFR supergene family could explain this, and it should be interesting
to observe the survival rates after 70% PHx in LTR/TNFRp55 double knockout mice.
Clearly, the TNF/TNFRp55 and the LT12/LTR pathways play an important role in
liver regeneration. The next task is to gain a molecular understanding of the cellular,
transcriptional, and functional effects of these ligand and receptor systems within liver
regulation. Precisely understanding these complex interactions will, hopefully, lead to
the ability to access this regulatory network at dened junctions and intervene in a
clinical context (Hehlgans and Pfeffer, 2005).
60
Summary
The TNF/TNFR superfamily comprises a large group of about 50 ligands and their receptors, which play a benecial role, for example, in inammation and protective immune
responses but are also responsible for detrimental actions such as in sepsis or certain immune disorders. In general, the ligands as well as the receptors are active as non-covalently
bound trimers. The TNFRp55 and the LTR belong to the core members of the family, and
signaling through TNFRp55 as well as the LTR can lead to activation of NFB, an important
transcription factor, via the canonical activation pathway. The LTR is also able to activate
NFB via an alternative pathway. Liver mass is tightly regulated in relation to body weight,
and, in contrast to other organs, the liver is capable of regeneration either when hepatocytes
become dysfunctional, for example, through chronic poisoning, or when liver mass is lost,
for example, after tumor resection. Loss of 30%70% of liver mass leads to synchronous division of mature hepatocytes until physiologic liver mass is regained. It has been determined
in animal models that TNF/TNFRp55 signaling is an early event in liver regeneration after
70% hepatectomy. Presumably, this leads to NFB activation and subsequent transcription
of IL6, which in turn initiates STAT3 activation via JAK-signaling. This promotes entry of
the hepatocytes into the G1-phase of the cell cycle and renders them responsive to growth
factors (priming). It is still unclear what triggers initial production of TNF. Signaling via
the LTR pathway is known to play an important role in the organogenesis of secondary
lymphoid organs. Experiments with transgenic and knockout animals, which constitutively
express or are decient in certain genes, respectively, have shown that signaling through
the LTR is involved not only in liver homeostasis (via LIGHT) but also in the early stages of
liver regeneration (via LT12): LTR decient animals show signicantly reduced survival
after 70% hepatectomy.
Further Reading
Bohm, F., Kohler, U.A., Speicher, T., and Werner, S. (2010). Regulation of liver regeneration by
growth factors and cytokines. EMBO Mol Med 2, 294305.
Capecchi, M.R. (2005). Gene targeting in mice: functional analysis of the mammalian genome
for the twenty-rst century. Nat Rev Genet 6, 50712.
Iimuro, Y., and Fujimoto, J. (2010). TLRs, NF-kappaB, JNK, and Liver Regeneration. Gastroenterol Res Pract. Published electronically. doi: 10.1155/2010/598109
Reinehr, R., and Hussinger, D. (2009). Epidermal growth factor receptor signaling in liver cell
proliferation and apoptosis. Biol Chem 390, 10337.
References
Anders, R.A., Subudhi, S.K., Wang, J., Pfeffer, K., and Fu, Y.X. (2005). Contribution of the
lymphotoxin beta receptor to liver regeneration. J. Immunol. 175, 1295300.
Apostolaki, M., Armaka, M., Victoratos, P., and Kollias, G. (2010). Cellular mechanisms of
TNF function in models of inammation and autoimmunity. Curr. Dir. Autoimmun. 11,
126.
Crispe, I.N. (2009). The liver as a lymphoid organ. Annu. Rev. Immunol. 27, 14763.
References
61
Croft, M. (2009). The role of TNF superfamily members in T-cell function and diseases. Nat.
Rev. Immunol. 9, 27185.
Fausto, N., Campbell, J.S., and Riehle, K.J. (2006). Liver regeneration. Hepatology 43,
S4553.
Futterer, A., Mink, K., Luz, A., Kosco-Vilbois, M.H., and Pfeffer, K. (1998). The lymphotoxin
beta receptor controls organogenesis and afnity maturation in peripheral lymphoid tissues. Immunity 9, 5970.
Hehlgans, T., and Pfeffer, K. (2005). The intriguing biology of the tumour necrosis factor/
tumour necrosis factor receptor superfamily: players, rules and the games. Immunology
115, 120.
focus. Biol. Chem. 390, 100312.
Kordes, C., Sawitza, I., Muller-Marbach, A., Ale-Agha, N., Keitel, V., Klonowski-Stumpe, H.,
and Hussinger, D. (2007). CD133 hepatic stellate cells are progenitor cells. Biochem
Biophys Res. Commun. 352, 4107.
Michalopoulos, G.K. (2007). Liver regeneration. J. Cell. Physiol. 213, 286300.
Tumanov, A.V., Koroleva, E.P., Christiansen, P.A., Khan, M.A., Ruddy, M.J., Burnette, B., Papa,
S., Franzoso, G., Nedospasov, S., Fu, Y.X., and Anders, R.A. (2009). T Cell-Derived Lymphotoxin Regulates Liver Regeneration. Gastroenterology 136, 694704.
Ware, C.F. (2008). Targeting lymphocyte activation through the lymphotoxin and LIGHT pathways. Immunol. Rev. 223, 186201.
5 The Hepatic Stem Cell Niches

Iris Sawitza, Claus Kordes, and Dieter Hussinger
Learning Targets
1. Components of a stem cell niche are the stem cells, neighboring cells, secreted
factors, cellcell contacts, basal lamina proteins, blood vessels, and the innervation
by the sympathetic nervous system.
2. Oval cells are hepatic progenitor cells that may originate from stem cells in the canals
of Hering.
3. Hepatic stellate cells are undifferentiated cells that maintain their characteristics in
their niche, the space of Diss.
5.1
Introduction
Stem cells are found in all multicellular organisms. Broadly dened, two types of mammalian stem cells are known: embryonic stem cells, which can be isolated from the
inner cell mass of blastocysts; and adult stem cells, which are maintained in many tissues throughout life. Embryonic stem cells can differentiate into all specialized cells of
the developing embryo and are, therefore, pluripotent. In adult organisms, the somatic
stem and progenitor cells act as a repair system for the body by replenishing damaged
cells after tissue injury, they but also maintain the regular turnover of regenerative organs such as blood, skin, or intestinal tract. Stem cells are characterized by the ability
to renew themselves, a process of cell duplication without loss of the developmental potential. They can also differentiate into diverse specialized effector cells. Three
principles exist to ensure that the stem cell population is maintained during replication:
s Asymmetric replicationone stem cell divides into one daughter cell that is
identical to the original stem cell and another daughter cell that undergoes further
development
s Symmetric replicationone stem cell divides into two daughter cells that remain
identical to the original stem cell
s Stochastic replicationone stem cell divides and develops into two differentiated
daughter cells, and another stem cell passes through mitosis and produces two
daughter cells identical to the original stem cell
Research in this stem cell eld emanates from ndings by Ernest A. McCulloch and
James E. Till at the University of Toronto in the 1960s. Under healthy conditions of an
64
SC
Notes:
Essential components of the niche are the
stem cells (SC), different neighboring cell
types (NC), secreted factors (yellow dots),
cellcell contacts (CCC), basal lamina proteins (BLP), blood vessels with erythrocytes
(BV), and the sympathetic nervous system
(SNS).
CC
Figure 5.1 Model of Endothelial Stem

Cell Niches
NC
CC
C
NC
BLP
NC
BV
NC
SNS
organism most stem cells reside in a quiescent state without replication or differentiation and are therefore difcult to detect. The behavior of stem cells, especially the balance of self-renewal and differentiation, is controlled by external signals and their cues,
which make up the stem cell microenvironment known as the stem cell niche. The stem
cell niche hypothesis was developed by Raymond Schoeld in 1978, who proposed that
stem cells reside within xed compartments or niches that are conducive to the maintenance of denitive stem cell properties. The microenvironment of stem cells modulates
their proliferation, inuences symmetric versus asymmetric cell division, controls cell
differentiation, protects the stem cells from physiological stresses, and helps them to
contribute to tissue formation in development and regeneration during life time.
Components of the niche are the stem cells themselves and different neighboring
cells that interact directly with the stem cells through secreted factors and direct physical cellcell contacts. Stem cell niches are mainly found close to basal laminas that
provide structure, organization, and mechanical signals to the niche. They are often
located in the vicinity to blood vessels that provide nutrient supply and carry systemic
signals or other circulating cells into the niche. Finally, the innervation by the sympathetic nervous system is also required for distant communication and recruitment of
stem cells out of their niche (fFigure 5.1).
5.2
Secreted Factors in the Stem Cell Niche
The communication within the niche is essential for the maintenance of the stem
cell function and to dene the rate of stem cell self-renewal. Secreted factors can act
locally or diffuse throughout the niche to direct stem cell fate decisions. In vitro systems
were developed to support proliferation, differentiation, and survival of distinct stem/
progenitor cell populations. It turned out that the developmental fate of, for instance,
hematopoietic stem cells depends on factors secreted by supportive or stromal cells,
which are adjacent to hematopoietic stem cells in the bone marrow. A wide range of
secreted factors regulate stem cell proliferation and fate. It is widely accepted that the
formation of tissues and structures during embryonic development and the regeneration of tissues in adults is controlled by intracellular signal transduction pathways. Two
signaling molecule families, the transforming growth factors (TGF) and wingless type
(Wnt), show remarkable evolutionary conservation among species and are capable of
directing stem cell fate.
5.2
65
TGF family members of signaling proteins are TGF, Nodal, Activin, and bone morphogenetic protein (Bmp), which regulate diverse cellular functions such as growth arrest, apoptosis, migration, and differentiation. TGF2 is present at high concentrations
in hair bulge stem cell niches. Interestingly, TGF2 is produced by hair bulge stem cells
and is one of the key factors that promote quiescence of adjacent mesenchymal stem
cells within the hair follicle (Tumbar et al., 2004). In Drosophila the Bmp 2/4 homolog
Decapentaplegic (Dpp) is required to maintain female germ line stem cells and promote
their replication. In mammals, Bmp4 supports the self-renewal of embryonic stem cells
and is required for hematopoiesis. TGF and Bmp signaling are mediated by phosphorylation of different intracellular Smads (Smad 2/3 or Smad 5/8) after binding to their cell
surface receptors. Phosphorylated Smads can inuence gene expression through binding
to Smad responsive elements of the DNA and act as transcription factors (fFigure 5.2).
TGF
TGF betaglycan
activin
activin
nodal
nodal EGF-CFC
TGFRI
ActRIB
(Alk4) P
Smad 7
ActRII
TGFRII
ActRII
P
Smad 4
P
Smad 2/3
ActRIB
(Alk4)
Sara
Smad 2/3
Smad 4
nucleus
cytoplasma
Smad 4
P
Smad 2/3
SmadRE gene expression
Figure 5.2A Schemes of Transforming Growth Factor- Superfamily Mediated Pathways

Notes:
The TGF/Activin/Nodal signaling pathway. TGF binding to the transforming growth factor
receptors type I and type II (TGFRI/II) or binding of Activin or Nodal to the activin receptors
IB and II (ActRIB/II) leads to receptor oligomerization and activation. Co-receptors such as
betaglycan and endoglin are described for TGF signaling, whereas the co-receptor epidermal
growth factor like-Cripto/FRL-1/Cryptic (EGF-CFC) is involved in signaling of Nodal. Recruitment of the receptor-regulated (R-) Smads 2/3 to the cell membrane by Sara (Smad anchor
for receptor activation) leads to a phosphorylation of Smad 2/3, which then form a heterocomplex with common Smads (Co-Smad, Smad 4). Subsequently, the complex translocates
into the nucleus and regulates the transcription of target gens through binding to Smad
responsive elements (SmadRE). The inhibitory Smad (I-Smad, Smad 7) negatively regulates
Smad signaling by blocking the binding of Smad 2/3 to type I receptors, hetero-complex
formation between Smad 2/3 and Smad 4, and the transcriptional regulation by Smad 2/3 in
the nucleus.
66

dan/cerberus
sclerostin
follistatin
follistatin-related protein
Bmp
Bmp
noggin
chordin
chordin-like
BmpRI
BmpRII
Smad 6
Smad 7
Smad 4
P
Smad 1/5/8
Smad 1/5/8
Smad 4
nucleus
cytoplasma
Smad 4
P
Smad 1/5/8
SmadRE gene expression
Figure 5.2B Schemes of Transforming Growth Factor- Superfamily Mediated Pathways

Notes:
Bone morphogentic protein (Bmp) signal transduction. Signaling by members of the Bmpsubfamily of ligands is initiated by binding to a heteromeric complex of type I receptors
(BmpRI) as well as type II receptors (BmpRII). The constitutively active kinase domains of type
II receptors phosphorylate type I receptors by binding Bmp so that a heterotetramer is formed
with two receptors of each type. This in turn activates the Smad signaling pathway through
phosphorylation of R-Smads (Smad 1, Smad 5, and Smad 8). These associate with Smad4 to
form a heteromeric complex that translocates to the nucleus and stimulates a wide range of
target genes. Inhibitory Smad 6 or Smad 7 act in an autoregulatory negative feedback loop
of the Bmp signal. Other known inhibitors of this signaling pathway are Noggin, Chordin,
Chordin-like, differential screening-selected gene aberrant in neuroblastoma (Dan)/Cerberus,
Sclerostin, Follistatin, or Follistatin related protein (Fsrp).
The -catenin-dependent or canonical Wnt signaling pathway is highly conserved

throughout the animal kingdom. The central mediator of this signaling pathway is
-catenin, which is also an integral part of the cytoskeleton. Wnt ligand binding to the
seven-span transmembrane receptor Frizzled leads to accumulation of free -catenin in
the cytoplasm and subsequently its translocation into the nucleus where it acts as an activator of gene transcription after binding to the nuclear T-cell factor/lymphoid enhancer
factor 1 (Tcf/Lef1). In the absence of Wnt ligands, cytoplasmic -catenin is phosphorylated by the glycogen synthase kinase-3 (Gsk3) and subsequently degraded through
the proteasom (fFigure 5.3).
The Wnt/ B-catenin signaling was identied as a pathway that critically regulates
various postnatal stem cell compartments. The expression of many stem cell factors is
controlled by active Wnt/ B-catenin signaling and required for self-renewal of stem cells
(Reya et al., 2003; Sato et al., 2004). This signaling pathway is further required to keep
5.2
Fzd
Frat
Dvl
Apc
F-actin
Ck1
67
Lrp
Ck1g
CRD
axin
b-cat Gsk3
P
b-cat ubiquitin
proteolysis
a-cat
cytoplasma
nucleus
cadherin b-cat
Tle
Tcf/Lef1
gene expression
Figure 5.3A Scheme of Canonical Wnt Signaling via -Catenin

Notes:
In this diagram the inactive canonical Wnt signaling pathway with elevated proteolysis of
-catenin and function of -catenin as an integral part of the cytoskeleton is depicted. In the
absence of Wnt ligands, -catenin is sequestered in a multiprotein degradation complex containing the scaffold protein Axin, the tumor supressor gene product adenomatous polyposis coli
(Apc), as well as casein kinase 1 (Ck1) and glycogen synthase kinase 3 (Gsk3). Upon sequential phosphorylation, -catenin is ubiquitylated and subsequently degraded by the proteasome
machinery. As a result, no free -catenin enters the nucleus to form a transcriptional complex
with T-cell-specic transcription factor (Tcf )/lymphoid enhancer binding protein 1 (Lef1) to
regulate downstream gene expression.
hematopoietic stem cells quiescent in their niche (Fleming et al., 2008). However, the
effects of canonical Wnt signaling seem to be context-dependent because progenitor
cells proliferate with activated Wnt/-catenin signaling. Constitutively active canonical
Wnt signaling has a carcinogenic potential and accounts for the majority of intestinal
tumors. This signal transduction pathway also has a role in specifying stem cell selfrenewal in hematopoietic stem cells. Being expressed by surrounding stromal cells of
the bone marrow, Wnts may be also secreted from hematopoietic stem cells themselves
and could act in an autocrine loop. Although the Wnt signaling pathway is important
for stem cell self-renewal in the intestine and hematopoietic system, it can direct tissuespecic differentiation in other contexts. For example, in the mammalian epidermis
Wnt ligands assist the differentiation of hair follicle precursors. Wnt/-catenin signaling
can also direct the maturation of specic cells such as the Paneth cells at the base of
the crypts within the small intestine (van Es et al., 2005). In the liver, there is growing
evidence for an involvement of the Wnt/-catenin cascade in various aspects, such as
the maintenance of zonal organization of the liver tissue. There is also evidence that
-catenin-mediated Wnt signaling is crucial for cell division during embryonic liver
development and regeneration after partial hepatectomy.

Wnt
CRD
Wnt
Ck1g
Sfrp
Wif
Dkk
Fzd
Lrp
68
P Gsk3
axin
Dvl
Frat
Apc
b-cat
Nkd
b-cat
Gsk3
Ck1
b-cat
reduced proteolysis
b-cat
b-cat
b-cat
nucleus
Tle
cytoplasma
b-cat
b-cat
Tcf/Lef1
gene expression
Figure 5.3B Scheme of Canonical Wnt Signaling via -Catenin

Notes:
The canonical Wnt signaling pathway is active in the presence of Wnt ligands. Wnt ligands
associate with a cysteine-rich domain (CRD) of the receptors frizzled (Fzd) and the lipoprotein receptor-related proteins (Lrp 5/6) co-receptors. This leads to translocation of Axin to the
plasma membrane and dissociation of the multiprotein complex. Gsk3 becomes displaced
from this complex through Dvl and the Gsk3 binding protein Frat (frequently rearranged
in advanced T-cell lymphomas). -catenin is then released from the multiprotein complex,
accumulates in the cytoplasm in a non-phosphorylated form, and subsequently translocates
into the nucleus. Nuclear -catenin alters expression of Wnt target genes such as c-Myc by
binding to Tcf/Lef1, converting them from repressors to activators of gene transcription after
replacing the co-repressor groucho/transducin-like enhancer protein (Tle). The initiation of
gene transcription involves also accessory factors such as legless, pygopus, brahma-related
gene 1, and CREB-binding protein (not shown). Natural regulatory elements of Wnt signaling
are secreted frizzled-related protein (Sfrp15), Wnt inhibitory factor 1 (Wif1), and dickkopf
(Dkk13). Sfrp and Wif1 interact directly with Wnt ligands, whereas Dkk occupies Wnt binding sites of co-receptors like Lrp to prevent signaling. The activity of Wnt signaling is also
controlled by cytoplasmic factors such as the Wnt-induced naked cuticle (Nkd12), which
interacts with Dvl. The targets of Wnt inhibitors are indicated as broken lines in this scheme.
Additionally, hedgehog (Hh) signaling is critical for embryonic development and

involved in differentiation, proliferation, and maintenance of multiple adult tissues. It
is involved in patterning the neural tube, lung, skin, and gastrointestinal tract. Dysregulation of the Hh pathway is associated with birth defects and cancer. There are three
ligands of Hh signaling in mammals: Sonic hedgehog (Shh), Indian hedgehog (Ihh), and
Desert hedgehog (Dhh). All Hh ligands bind with similar afnity to a twelve-span transmembrane receptor called Patched (Ptch) to activate Hh signaling. This signaling pathway is otherwise repressed by Ptch through inhibition of the seven-span transmembrane
5.2
69
receptor Smoothened (Smo). The cytoplasmic tail of Smo serves as a nidus for the accumulation of the glioblastoma transcription factor protein family (Gli) held in a complex
with regulatory proteins such as kinesin family member 7 (Kif7/Costal2/Cos2), Fused
(Fu), and suppressor of Fused (Sufu). Upon binding of the ligand to Ptch, the inhibition
of Smo is removed and processed forms of Gli migrate to the nucleus to bind Hh target
genes (fFigure 5.4).
Especially Gli1 seems to be important for the regulation of stem cell fate. If Hh signaling is silenced in the adult stem cell niche through deletion of Smo, quiescent B
stem cells (glial brillary acidic protein/GFAP stem cells) and transit amplifying C cells
become depleted in the subventricular zone of the brain (Balordi and Fishell, 2007). Hh
signaling seems to be required for both maintenance of neuronal stem cell and their
A non-responding cell
Ptch
Boc
Cdon
primary
cilium
Kif7 Gsk3
Fused
endosome
PKA
Ck1
Gli
P
Ac
Sufu
microtubule
processing
Smo
cytoplasma
nucleus
Gli
rep
gene expression
Figure 5.4A Scheme of Hedgehog Signaling

Notes:
How signals are transmitted from hedgehog (Hh) to patched (Ptch) and smoothened (Smo) is
still unclear and apparently not fully conserved between vertebrates and invertebrates. In the
absence of the Hh ligands, Ptch is located in the primary cilium and blocks the entry of Smo
to the cilium. A cytosolic complex comprising kinesin family member 7 (Kif7), Fused, protein
kinase A (PKA), casein kinase I (Ck1), and glycogen synthase kinase 3 (Gsk3) phosphorylates
the transcription factor glioma-associated oncogene homolog (Gli) for proteolytic processing
to convert it into a transcriptional repressor (Gli rep). This process might be regulated by Smocontrolled inhibitory G proteins and subsequent changes in adenylate cyclase (Ac) activity as
demonstrated in Drosophila.
70

B responding cell
Ihh
Ptch
primary
cilium
Boc
Cdon
Smo
Shh
Dhh
Kif7
Fused
Sufu
Gli
PKA
Ck1
microtubule
act
cytoplasma
Ac
Gsk3
nucleus
Gli
act
gene expression
Figure 5.4B Scheme of Hedgehog Signaling

Notes:
In responding cells, Hh binds to the receptor Ptch and the co-receptors cell adhesion molecule-related, down-regulated by oncogenes (Cdon) and brother of Cdon (Boc). Three mammalian homologs of Hh (Shh, Ihh, Dhh) bind to Ptch and allow it to move out of the primary
cilium. Smo is then no longer repressed by Ptch and moved into the cilium, which results in
release of the transcription factor Gli from the cytosolic complex. During this process, the Gli
transcription factors are processed and activated (Gli act) to facilitate their translocation into
the nucleus and to induce the transcription of target genes.
differentiation (Ahn and Joyner, 2005). In the gastrointestinal tract, the Hh ligands Shh
and Ihh as well as the receptor Ptch are all expressed during development and persist
at the base of the crypts also in the adult intestine. Moreover, intestinal injury is accompanied by a dramatic induction of both ligand and receptor expression, suggesting
that the Hh pathway plays a role in the stem cellbased repair of the damaged intestinal
mucosa during adulthood. Hh signaling is obviously also involved in liver regeneration
after partial hepatectomy (Ochoa et al., 2010).
Another soluble factor released by supportive cells such as endothelial cells,
osteoblasts, or other stromal cells in the bone marrow is stromal cellderived factor-1 (SDF1). The chemokine cysteine-X-cysteine motive receptor 4 (CXCR4) is a
G-protein-coupled seven-span transmembrane protein, which binds SDF1 exclusively. CXCR4/SDF1 axis plays an essential role in directing hematopoietic stem cells
along a SDF1 gradient to their nal niche in the bone marrow during ontogenesis
(homing), but also in their mobilization to the peripheral blood. The recruitment
5.3
of
of
et
to
Physical Contacts of Stem Cells with Their Niche
71
stem cells from the bone marrow is partly mediated by local down regulation
SDF1, which facilitates the release of stem cells into the blood stream (Lapidot
al., 2005). Therefore, the interaction of SDF1 and CXCR4 is an essential process
maintain stem cell niches.
5.3
Physical Contacts of Stem Cells with Their Niche
Cell adhesion to supporting stromal cells or to the basal lamina is important to control
stem cell behavior and maintain them within their niche. Although secreted factors
can potentially act over distance, other signals that control stem cell fate require direct
cellcell contact. Adherens junctions are cellcell contacts that are formed by interactions between transmembrane proteins called cadherins. First evidence for an essential
function of cadherins in stem cell niches came from Drosophila. Germline stem cells in
the ovary are associated with neighboring cap cells via Drosophila epithelial cadherin
(DE-cadherin), and removal of this cadherin results in stem cell loss (Song et al., 2002).
Based on expression studies, cadherin-mediated cell adhesion has also been suggested
to facilitate hematopoietic stem cell association with osteoblasts (through neural or
N-cadherin) and has been implicated in determining the correct positioning of muscle
satellite cells along the muscle ber (through myotuble or M-cadherin). Integrins are
required to connect stem cells with the basal lamina and hold them at the right place
in their niche. A loss or alteration of integrin expression enables recruitment of stem
cells. Within mammalian tissues, high levels of integrin expression can be used as a
marker to identify stem cells. High expression levels of 1 integrin appear to be a characteristic of stem cells in multiple tissues, including multipotent stem cells in the hair
bulge, hematopoietic stem cells of the bone marrow, and satellite cells of the skeletal
muscle. The specic importance of cell adhesion mediated by 1 integrin for the stem
cell maintenance varies among tissues. In the skin, 1 integrins regulate the differentiation of epidermal stem cells into keratinocytes through mitogen-activated protein (MAP)
kinase signaling (Zhu et al., 1999). However, hematopoietic stem cells are retained
in the bone marrow and showed overall hematopoietic function despite 1 integrin
deletion, suggesting that hematopoietic stem cell activity is not only regulated by this
integrin. Also extracellular matrix proteins can modulate the expression and activation
of 1 integrins. A local variation in the composition of basement membranes could play
a role in initiating stem cell activation and migration. Basement membranes typically
underlie the epithelium or endothelium and are mainly composed of reticular collagen
type IV, laminin, nidogen-1, and heparan sulphate proteoglycans. Stem cells are often
found on basal laminas (Fuchs et al., 2004). For example, muscle-specic stem cells
termed satellite cells stay in close contact to the basement membrane of myobers. The
interaction of stem cells with proteins of the extracellular matrix can suppress the beginning of their terminal differentiation. In addition, the extracellular matrix can potentially
sequester and modulate the local concentration of secreted factors available within the
stem cell niche.
Another excellent example of signaling that requires physical cellcell contacts is
presented by Notch receptors and their ligands Serrate/Jagged and Delta/Delta-like.
Four Notch receptors are known in mammals: Notch14. The Notch receptors and their
ligands are single-pass transmembrane proteins. To activate Notch signaling, a direct
physical contact of signal sending and signal receiving cells is essential. The intracellular
72
domain of the Notch receptors is then cleaved by the enzyme -secretase and translocates into the cell nucleus to inuence gene expression (fFigure 5.5).
Especially Notch1 signaling is important to facilitate self-renewal in stem cell niches
as demonstrated for neuronal stem cells of mice (Nyfeler et al., 2005), whereas Notch3
is reported to be involved in differentiation of hematopoietic stem/progenitor cells
(Karanu et al., 2003). In Drosophila, Notch activity is required for the progeny of sensory organ precursor cells to assume their correct fate. During each cell division within
the sensory organ lineage, Numb, an inhibitory protein of Notch signaling, appears to
bias Notch-mediated cellcell interaction so that the stem cell division becomes asymmetric. Numb protein amounts after asymmetric stem cell division are higher in the
daughter cell that preserves stem cell characteristics than in the second daughter cell
that initiated further development ( Jan and Jan, 1998). Moreover, the numb isoforms 1
and 3 are associated with the undifferentiated state of stem cells, whereas the smaller
isoforms 2 and 4 of numb appear during their differentiation.
In the bone marrow, at least two types of niches are known for hematopoietic stem/
progenitor cells: one is associated with osteoblasts and the other with endothelial cells.
The basement membranes of blood vessels seem to provide suitable niches for stem
cells. Blood vessels are further important for the nutrient supply and can carry systemic
signals from distant tissues to the stem cell niche. Nerves are other essential elements
for a distant communication of cells and organs with stem cell niches in other tissues.
First evidence for an important function of the sympathetic nervous system for stem
cell niches came from the hematopoietic system. The sympathetic nervous system can
initiate stem cell recruitment into the blood stream (Katayama et al., 2006). The relevance of a stem cell niche innervation in other tissues remains speculative up to now,
even though anatomic juxtapositions of nerves and epithelial stem cell niches in the
intestinal crypts and the bulge of the hair follicle have been demonstrated.
5.4
Identication of Stem Cell Niches
One approach for the identication of stem cell niches is the histological detection of
stem cell niche elements described previously at sites where cells with stem cell characteristics occur. Label retention assays are also suitable to identify the location of possible stem cell niches within organs. This approach is applicable for organs with rapid
cell turnover such as the small intestine. A standard assay exploits the fact that during
DNA synthesis through asymmetric or symmetric stem cell division both daughter cells
are labeled with tritiated thymidine or bromodeoxyuridine (BrdU). If a stem cell becomes quiescent after division, the incorporated label is retained. Quiescent stem cells
are normally slow cycling cells in the stem cell niche, and this facilitates label retention.
Progenitor cells, which are transient amplifying cells and divide rapidly, dilute the label
to undetectable levels in organs with a rapid turnover (e.g., skin or intestinal tract).
However, label retention assays are unable to detect quiescent stem cells. Therefore, the
experimental setup selected to induce a stem cell response is crucial for the outcome
of this assay. A third approach could be the in situ detection of transplanted stem cells
labeled by mutation, uorescence, or magnetism. Only within suitable niches transplanted stem cells can survive and maintain their characteristics for a long time.
5.4
Identication of Stem Cell Niches
73
signal sending cell

endosome
ub
ligand
degradation
or recycling
e
tiv
ac d
in igan
l
ub
de
lta
-li
ja
ke
gg
ed
Neur/Mib
ub
active ligand
ub
Nb
Dsl
g-secretase
complex
S3/S4 cleavage
inhibition
Fringe
Nicd
ub
endosome
Nicd
Ni
nucleus
cytoplasma
Nicd
glycosylation
cd
Ni
cd
Furin
S1
cleavage
ER
Go
lg
ic
om
pl
ex
c
Ni
S2 cleavage
Nicd
Nicd
ch
t
No
Numb
Next
Adam/
Tace
Mam
Csl
gene expression
signal receiving cell
Figure 5.5 The Notch-signaling Pathway

Notes:
Notch receptors are synthesized as single precursor proteins that are glycosylated in the endoplasmic reticulum (ER) and cleaved in the Golgi complex by a Furin-like convertase (S1) during their
transport to the cell surface. Fringe glycosyltransferases can modify EGF-like repeats by adding
N-acetylglucosamine within the Golgi complex. Notch signaling is initiated through interaction of
the membrane bound ligands Jagged or Delta-like at the Delta/Serrate/Lag2 (Dsl) domain with the
Notch receptors. This ligandreceptor interaction induces two sequential proteolytic cleavages. The
rst cleavage (S2) within the extracellular domain of Notch receptors is mediated by a desintegrin
and metalloproteinase (Adam) and/or the metalloproteinase tumor necrosis factor -converting
enzyme (Tace). The cleaved extracellular subunit of the receptor is endocytosed by the neighboring
ligand-expressing or signal sending cell. This process seems to be controlled by Neuralized (Neur)
and/or Mind bomb (Mib) E3 ubiquitin ligases. The second cleavage (S3) of the Notch extracellular
truncation (Next) occurs within the transmembrane domain and is mediated by the -secretase
complex. An additional cleavage (S4) of the transmembrane domain of Notch receptors results in
the release of -amyloid precursor proteins (N). The Notch intracellular domain (Nicd) translocates
into the nucleus and binds to the transcription factor C-promotor binding factor, RBP-jk/supressor
of hairless/Lag-1 (Csl). This interaction leads to transcriptional activation of Notch target genes by
displacement of corepressors and simultaneous recruitment of coactivators including Mastermind
proteins (Mam). Notch-target genes are hairy/enhancer of split (Hes) and hairy/enhancer of split
related with YRPW (Hey). Notch signaling can be regulated by Numb and/or by endocytosis. Nicd
becomes monoubiquitylated (ub), targeting the receptor to the lysosome for degradation.
74
5.5
Stem Cell Niches in the Liver
Different amounts of information about the stem cell niches of various organs are available thus far. Some principles appear to be organ specic, whereas others apply across
organ boundaries. Organs with reasonably dened stem cell niches are those that have
a high rate of cell turnover. These are, for example, the hematopoietic system and gastrointestinal tract. Other organs with less cell turnover also appear to have dened stem
cell niches as demonstrated for the subventricular zone of the brain. In contrast to these
organs, the identity of liver stem cells as well as their niches is still an open question.
Reiichiro Kuwahara and colleagues (2008) found BrdU label retention by cells in the
canals of Hering, cells in the intralobular bile ducts, periductular cells, and peribiliary
hepatocytes. In a recent study the space of Diss was also described to possess essential
elements of stem cell niches at sites where hepatic stellate cells reside (Sawitza et al.,
2009). Thus, several putative stem cell niches have been described in the liver thus far.
In 1958, Walter Wilson and Elizabeth Leduc described a cell population in the distal
biliary ducts of the liver capable of hepatocyte and cholangiocyte differentiation. These
bipotential liver cells were termed oval cells in rodents in recognition of their morphologic appearance. Later, similar cells were also found in human liver (De Vos and
Desmet, 1992) and named hepatic progenitor cells (HPC). Numerous in vivo and in vitro
studies have documented a central role of oval cells in liver biology and carcinogenesis
(Fausto et al., 1992). The population is heterogeneous and contains cells that may differ
in their developmental state. The oval cell compartment is also used to describe the cells
invading the parenchyma after administration of certain carcinogens. Those cells may
originate from cells present in the canals of Hering (Theise et al., 1999) or derive from
blast-like cells located next to bile ducts (Haruna et al., 1996). However, oval cells are
widely considered to be liver progenitor cells that can regenerate the parenchyma when
hepatocyte proliferation is overwhelmed by persistent or severe liver injury.
After injury or intoxication of the murine liver, BrdU-positive/cytokeratin (panCK)positive cells are found in the canals of Hering that seem to retain the BrdU label for
more than 56 days (Kuwahara et al., 2008). The canals of Hering are the most proximal
branches of the biliary tree that comprise the smallest cholangiocytes on the one side and
hepatocytes on the other side. The hepatocytes and cholangiocytes could represent the
typical asymmetrical composition of a niche required for maintaining stem cell characteristics. Cholangiocytes release SDF1 (Kollet et al., 2003), and hepatocytes secrete canonical Wnt ligands and present Notch ligands on their cell surface (Sawitza et al., 2009) that
potentially can interact with oval cells precursors to inuence their proliferation, migration, and development. Oval cells express CXCR4 and migrate along SDF1 gradients,
suggesting the relevance of SDF1/CXCR4 interaction in oval cell migration (Hatch et al.,
2002). For yet unknown reasons oval cells are apparently also able to synthesize SDF1
(Kollet et al., 2003). The previously mentioned Wnt/-catenin and Notch signaling pathways are further important for stem cells in their niche, and there are hints for a possible
activity of each in oval cells. It has been reported that the autocrine and paracrine Wnt secretion is involved in the hepatic progenitor response in mice, rats, and humans. After the
application of 2-(N-Acetyl)-aminouorene (2-AAF) with subsequent partial hepatectomy
in rats, a noteworthy increase in total and nuclear -catenin was observed, indicating
active Wnt/ -catenin signaling in oval cells (Hu et al., 2007). In addition, an increase of
Wnt-1 expression in hepatocytes along with increased expression of the Wnt receptor
5.5
75
Frizzled-2 in oval cells was observed. This mechanism coincides with a decrease in Wnt
inhibitory factor-1 (Wif1) and Gsk3 down-regulation, leading to -catenin stabilization
(Williams et al., 2010). There is further evidence for the activation of canonical Wnt
signaling in oval cells in response to Wnt ligands. It could be suggested that the activation of Wnt/-catenin signaling via Tcf/Lef1 regulates the proliferative response of hepatic
oval cells (Hu et al., 2007). However, a detailed analysis of the function of Wnt/-catenin
signaling and its relevance to maintain oval cell precursors in their niche has not yet been
provided. Also, little information is available about Notch signaling in oval cells and if
Notch signaling is relevant for their precursors in the canals of Hering. In hematopoietic,
intestinal, and neuronal stem cell niches, the modulation of Notch activity is fundamental
in inuencing the fate of progenitor cells. Its dysfunction is associated with several severe
human pathologies, including developmental defects of the liver. Point mutations in the
Jagged-1 gene are the cause for the Alagille syndrome, which is associated with paucity
of intrahepatic bile ducts. Interactions between nerves and bile ducts are known (Terada
and Nakanuma, 1989), but a direct innervation of the stem cell niche within the canals
of Hering has not been demonstrated thus far. The nerves sometimes cross the basement
membrane, appearing to make direct contact with cholangiocytes and, thereby, also may
inuence the stem cell activation in the canals of Hering. Taken together, all these ndings support the canals of Hering as being one possible hepatic stem cell niche for stem
cells that are precursors of more committed oval cells.
Periportal hepatocytes are also identied as BrdU-positive label-retaining cells (Kuwahara et al., 2008). They can be found during liver regeneration after intoxication with
acetaminophen adjacent to panCK-positive cells. Examination of serial sections and
relative proportions of label-retaining cells in the canals of Hering to label-retaining
peribiliary hepatocytes suggests that the label-retaining hepatocytes eventually derive
from stem cells of the canals of Hering through differentiation. Thus, label-retaining
cells in the canals of Hering and peribiliary label-retaining hepatocytes may represent
different developmental states of the same cell population. Preliminary studies show
hepatic stem/progenitor cells within interlobular bile ducts of human livers on the basis
of c-kit expression (Baumann et al., 1999). In line with these ndings, label-retaining
cholangiocytes are detected in the bile ducts (Kuwahara et al., 2008), but evidence for
a contribution of these cells to liver regeneration has not yet been provided.
The space of Diss in the liver is lined by hepatocytes and fenestrated sinusoidal
endothelial cells (SEC) that form discontinuous capillaries (sinusoids) as known from
the spleen, lymph nodes, and bone marrow. Within this intercellular space basal lamina
proteins such as laminin and collagen type IV are deposited in normal liver. Hepatic
stellate cells express many factors associated with stem/progenitor cells and possess the
capacity to differentiate as documented in vitro and in vivo (Kordes et al., 2007; Sawitza
et al., 2009; Yang et al., 2008). Hepatic stellate cells, which can be identied within rat
liver through their expression of the lamentous protein GFAP, typically reside within
the space of Diss embedded between these basal lamina proteins (fFigure 5.6A, B).
Although stellate cells are principally able to synthesize basal lamina proteins after their
activation (Friedman et al., 1985), hepatic stellate cells have a less evolved endoplasmic
reticulum in their quiescent stage. For this reason, endothelial cells and hepatocytes are
apparently the main producers of basal lamina proteins in the space of Diss of normal
liver. Extracellular matrix proteins such as laminin and collagen type IV promote the quiescence of stellate cells (Davis et al., 1988; Friedman et al., 1989) and, therefore, represent
76
Figure 5.6A,B Basal Lamina Proteins Collagen Type IV and Laminin in Sinusoids of Normal Rat Liver
Notes:
A Immunostaining of reticular collagen type IV (red) and the rat stellate cell marker glial
brillary acidic protein (GFAP; green) by uorochrome-labeled antibodies on liver tissue
sections.
B Simultaneous detection of laminin (red) and GFAP (green) by uorescent antibodies. The
cell nuclei were counterstained by 4,6-diamidino-2-phenylindole (DAPI).
important elements of their niche. In acute liver injury, the synthesis of extracellular matrix
protein synthesis is increased by endothelial cells, hepatocytes, and stellate cells (Ueno
et al., 1993). With respect to stem/progenitor cell characteristics of stellate cells, the expression of extracellular matrix proteins is not unusual for undifferentiated cells because human
mesenchymal stem cells are known to express, for example, laminin-5 (Klees et al., 2005).
The architecture of the hepatic stellate cells niche is further completed by the sympathetic nervous system. Stellate cells are highly innervated, especially in the human liver.
Nerve endings are found in the liver close to stellate cells (Biolac-Sage et al., 1990), which
respond to perivascular nerve stimulation through the release of the osmolyte myoinositol
and exhibit Ca2 inux in response to phenylephrin (vom Dahl et al., 1999). Stimulation
of freshly isolated hepatic stellate cells with noradrenaline, the neurotransmitter of the
peripheral sympathetic nervous system, leads to rapid secretion of the prostaglandins F2
and D2, which can activate the glycogenolysis in neighboring hepatocytes (Athari et al.,
1994; Hussinger et al., 1987). Obviously, stellate cells can integrate signals from distant
cells or organs to affect the behavior of neighboring cells in their niche.
Hepatic stellate cells possess signaling pathways that are important for the maintenance of stemness and required for cell differentiation such as hedgehog, Wnt/-catenin,
and Notch signaling. Isolated rat hepatic stellate cells express multiple components of
the Hh pathway, such as Shh, Ihh, Ptch, Smo, and Gli13. Hh signaling seems to be
required for both stem cell maintenance in their niche and their differentiation. The
inuence of Hh signaling on murine hepatic stellate cell activation is demonstrated by
cyclopamine treatment in vitro and in vivo. Cyclopamine is a pharmacologic inhibitor of the Hh signaling and able to counteract the activation of stellate cells (Sicklick
et al., 2005). In contrast to this, mimicking of the Wnt/-catenin signaling pathway can
preserve the quiescent stage of rat hepatic stellate cells, which is in agreement with the
effects of the canonical Wnt cascade on hematopoietic stem cells (Fleming et al., 2008).
5.5
77
Quiescent stellate cells display nuclear -catenin and express the Wnt target genes
paired-like homeodomain transcription factor 2 (Pitx2), c-Myc, and axin2 (conductin),
indicating an active canonical Wnt/-catenin signaling. In their quiescent state stellate
cells also express Wnt ligands known to activate -catenin-dependent Wnt signaling,
such as Wnt1, Wnt2, Wnt3/3a, Wnt7a/b, Wnt8a, and Wnt10b. During culture-dependent activation and development into myobroblast-like cells, -catenin becomes
increasingly associated with the cytoskeleton, and a remarkable change in Wnt ligand
expression from canonical to noncanonical Wnt ligands (e.g., Wnt 4, Wnt5a, Wnt11)
occurs (Kordes et al., 2008). A similar switch from canonical to noncanonical Wnt
signaling is observed during differentiation of mesenchymal stem cells (Davis and zur
Nieden, 2008). The -catenin-dependent Wnt signaling might persist in stellate cellderived myobroblast-like cells, but at a lower level compared to quiescent stellate
cells. A decrease of the expression of stem/progenitor cell markers such as CD133 and
Notch1 is associated with this process (Sawitza et al., 2009).
Parenchymal cells seem to be one major source of canonical Wnt ligands in the
liver. Wnt7a/b, especially, is highly synthesized by hepatocytes compared to the low
levels observed in stellate cells. In co-culture experiments of hepatic stellate cells with
hepatocytes, in which the cells are separated by a membrane that allowed permeation
of soluble factors only, an increased level of nuclear -catenin and up-regulation of the
Wnt target gene c-Myc is found in stellate cells, indicating a release of Wnt ligands by
hepatocytes. In addition, the morphology of quiescent stellate cells is preserved, and also
the synthesis of -smooth muscle actin, as a marker of activated stellate cells, remains
low. Under these conditions further development of stellate cells is largely prevented.
For this reason, Wnt/-catenin signaling is apparently an essential element of the stellate
cell niche. In contrast to the ndings obtained with quiescent cells, culture-activated
hepatic stellate cells, which are co-cultured for 7 days with hepatocytes, display the
expression of hepatocyte markers such as -fetoprotein, albumin, hepatocyte nuclear
factor 1 (Hnf1), Hnf4, Hnf6, and multidrug resistance protein 2 (Mrp2). Obviously,
the effects of hepatocytes on stellate cell fate decisions mainly depend on their stage of
activation or development (Sawitza et al., 2009).
Notch1 expression is controlled by canonical Wnt signaling (Reya et al., 2003; Sawitza
et al., 2009), and Notch signaling is known to guide stem cell fate decisions also. The
expression of Notch1 is crucial to maintain neuronal stem cells in their niche and is expressed by hepatic stellate cells, along with the Notch target genes hairy and enhancer
of split 1 (Hes1) as well as hairy/enhancer of split-related with YRPW motif-like (HeyL).
Neighboring hepatocytes synthesize the Notch ligand Jag1 and, therefore, constitute the
basis of physical cellcell contacts for stellate cells in their niche (fFigure 5.7A, B).
Although little information is available about the function of Notch1 in stellate cells,
its presence in their quiescent state supports the concept of a stellate cell niche in the
space of Diss (fFigure 5.8A).
Another hint for the space of Diss as a niche for stellate cells is their CXCR4 expression.
Hepatic stellate cells are able to migrate in response to SDF1 or SDF1. Neighboring
SEC release SDF1 and strongly attract stellate cells as investigated with modied Boyden
chambers, which allow stellate cell migration through pores of a membrane into a second chamber with SEC. This stellate cell migration can be completely antagonized by
addition of SDF1 antibodies (Sawitza et al., 2009). Because CXCR4/SDF1 axis plays an
essential role in maintaining the hematopoietic stem cell niche of the bone marrow, it
78
HSC 1d
PC 1d
150 kDa
250 kDa
Jag1
150 kDa
100 kDa
100 kDa
75 kDa
Notch1
Figure 5.7A,B
Notch Signaling in Hepatic Stellate Cells (HSC)
Notes:
A Fluorochrome-labeled antibodies highlight the Notch ligand Jagged1 ( Jag1) in the cell
membrane of hepatocytes (red). Immunouorescence staining of glial brillary acidic protein
(GFAP) indicates hepatic stellate cells (green). The cell nuclei were counterstained by DAPI.
B Western blot analysis of Notch1 in freshly isolated hepatic stellate cells (HSC cultured for
1 day) and its ligand Jag1 in liver parenchymal cells (PC cultured for 1 day)
might be also required for the retention of stellate cells in the space of Diss (fFigure
5.8A).
The cell interactions in stem cell niches described thus far are mainly narrowed to
functions of nonstem cell neighbors and their effects on stem cells, but there is also
evidence that stem cells create their niches. One example for stem cells that inuence
their niche is provided by hair bulge stem cells that release TGF2 to inuence neighboring mesenchymal stem cells. At this point another level of complexity is reached
because two types of stem cells can reside in one niche. In the bone marrow Nestinpositive mesenchymal stem cells act as supportive cell neighbors for hematopoietic
stem/progenitor cells. A depletion of Nestin-positive mesenchymal stem cells in vivo
rapidly reduces the number of hematopoietic stem/progenitor cells and lowers their
homing in the bone marrow (Mndez-Ferrer et al., 2010). Mesenchymal stem cells
can release soluble factors such as hepatocyte growth factor (HGF/hepatoprotein-A/
scatter factor) that inuence proliferation, adhesion, and survival of hematopoietic
progenitor cells. Stellate cells from liver and pancreas synthesize HGF, which is also
detectable in other undifferentiated cells, such as umbilical cord blood stem cells,
and may support c-Met expressing cells like hepatocytes in their niche within the
space of Diss (fFigure 5.8A, B). Moreover, stellate cells synthesize several neurotrophic growth factors, such as brain-derived neurotrophic growth factor (BDNF),
nerve growth factor (NGF), and neurothrophins (NT3, NT4, NT5). These factors could
facilitate nerve and blood vessel sprouting and are probably important to initiate and
sustain the stellate cell niche.
5.5
79
A
PC
Wnt
Jag1
HSC
BLP
HGF
NA
SEC
sympathetic
nervous system
SDF1

time series
umbilical cord pancreatic stellate cells

blood stem cells cultured for 7 days
83 kDa
79 kDa
HGFa
g-tubulin
0
14
HGFa
83 kDa
79 kDa
51 kDa g-tubulin
51 kDa
21 days
Figure 5.8A,B Architecture and Cytokine Crosstalk Within the Hepatic Stellate Cell
Niche
Notes:
A Scheme of the hepatic stellate cell niche. The release of SDF1 by sinusoidal endothelial
cells (SEC) attracts hepatic stellate cells (HSC) and may retain them within the space of Diss.
Liver parenchymal cells (PC) synthesize paracrine factors such as canonical Wnt ligands that
affect the stellate cells. The parenchymal cells synthesize also the membrane-bound Jag1 to
stimulate Notch signaling in stellate cells. Hepatic stellate cells produce hepatocyte growth
factor (HGF). HGF released by stellate cells probably maintains neighboring cells such as
hepatocytes and endothelial cells. By this means stellate cells could create their own niche.
The space of Diss contains the basal lamina proteins (BLP) laminin (blue cross) and collagen
type IV (purple grid). Hepatic stellate cells can respond to noradrenaline (NA) released by the
sympathetic nervous system to integrate signals from distant cells.
B Western blot analysis of HGF in a time course of cultivated rat hepatic stellate cells.
The HGF precursor protein (83 kDa) and processed HGF (79 kDa) is mainly synthesized by
quiescent hepatic stellate cells. HGF synthesis is also detectable by Western blot in clonally expanded umbilical cord blood stem cells and primary cultures of pancreatic stellate cells of rats.
Obviously, HGF expression is a feature of stem cells. The Western blot analysis of -tubulin
serves as a loading control.
80
Summary
Stem cell niches are microenvironments organized as structural units to protect stem cells
and to inuence their developmental fate in a suitable manner. Elements of a stem cell niche
are the stem cell itself, different neighboring cell types, secreted factors, physical cellcell
contacts, basal lamina proteins, blood vessels, and the sympathetic nervous system. The stem
cell niches are able to maintain stem cells or to direct their further development through
signaling pathways, such as Wnt/-catenin, Notch, TGF, Bmp, and hedgehog. The CXCR4/
SDF1 signaling is essential to control stem cell mobilization and homing. Stem cells niches
can contain different types of stem/progenitor cells that interact with each other. There is
evidence for several stem/progenitor cells and different stem cell niches in the liver. Oval
cells apparently originate from stem cells within the canals of Hering, and hepatic stellate
cells maintain their characteristics in the space of Diss. Both microenvironments meet
requirements of stem cell niches.
Further Reading
Lapidot, T., Dar, A., and Kollet, O. (2005). How do stem cells nd their way home? Blood,
106, 190110.
Moore, K.A., and Lemischka, L. (2006). Stem cells and their niches. Science 31, 18805.
Morrison, S.J., and Spradling, A.C. (2008). Stem cells and niches: Mechanisms that promote
stem cell maintenance throughout life. Cell 132, 598611.
Purton, L.E., and Scadden, D.T. (2008). The hematopoietic stem cell niche. StemBook, ed. The
Stem Cell Research Community, StemBook, doi/10.3824/stembook.1.28.1, http://www.
stembook.org.
Rueger, M.A., Backes, H., Walberer, M., Neumaier, B., Ullrich, R., Simard, M.L., Emig, B.,
Fink, G.R., Hoehn, M., Graf, R., and Schroeter, M. (2010). Noninvasive imaging of endogenous neural stem cell mobilization in vivo using positron emission tomography.
J. Neurosci. 30, 645460.
Sato, T., Vries, R.G., Snippert, H.J., van de Wetering, M., Barker, N., Stange,. D.E., van Es, J.H.,
Abo, A., Kujala, P., Peters, P.J., and Clevers, H. (2009). Single Lgr5 stem cells build
crypt-villus structures in vitro without a mesenchymal niche. Nature 459, 2626.
Scadden, D.T. (2006). The stem-cell niche as an entity of action. Nature 441, 10759.
Theise, N. (2006). Gastrointestinal stem cells. III Emergent themes of liver stem cells biology:
niche, quiescence, self-renewal, and plasticity. Am. J. Physiol. Gastrointest. Liver Physiol.
290, G18993.
Voog, J., and Jones, D.L. (2010). Stem cells and their niche: a dynamic duo. Cell Stem Cell
6, 10315.
Watt, F.M., and Hogan, B.L.M. (2000). Out of Eden: stem cells and their niches. Science 287,
142730.
References
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U.S.A. 96, 672833.
Stellate Cells in the Regenerating Liver

Learning Targets
1. Hepatic stellate cells are undifferentiated cells that are activated during liver
regeneration.
2. An early and reliable indicator of the stellate cell activation in vitro and in vivo is
Nestin.
3. Environmental conditions such as culture surfaces and soluble factors inuence the
activation of isolated hepatic stellate cells.
4. Only activated hepatic stellate cells are able to undergo further development.
5. Activated stellate cells are zonally distributed in stem cellbased liver regeneration.
6. Activated stellate cells are detectable on basal laminas that surround ducts of cytokeratin 19 expressing cells and blood vessels during stem cellbased liver regeneration.
6.1
Characterization of Stellate Cells
Hepatic stellate cells occur in primitive and higher vertebrates, ranging from lampreys
(primitive jawless sh) to humans. This high prevalence among vertebrates indicates a
major importance of this cell type for the liver. Stellate cells display a stellate-shaped
morphology in the liver tissue and contain vitamin A in their quiescent stage. Apart
from the liver, vitamin A storing cells have been found in many organs of vertebrates
such as the pancreas, kidney, intestine, lung, spleen, uterus, and skin. Among cells
that contain retinoids, the stellate-shaped cells of the liver are studied best. The hepatic stellate cells store vitamin A mainly as biologically inactive retinyl palmitate in
membrane coated lipid vesicles, but also retinol, which can initiate signal transduction, is detectable to a lower degree. The retinoids are involved in the preservation of
the quiescent stage of stellate cells. In rat hepatic stellate cells the lipid vesicles are
located around the cell nucleus (fFigure 6.1), whereas hepatic stellate cells isolated
from mice contain vesicles that strongly differ in size and are mainly located on one
side of the cell nucleus. Due to their high lipid content, hepatic stellate cells can be
easily isolated in high purity (>95%) by density gradient centrifugation after enzymatic
digestion of the liver tissue. Owing to the relative size of the liver, rats are a suitable
organism to isolate stellate cells and often preferred as a model to study the stellate
cell biology.
The cellular vitamin A content of hepatic stellate cells increases with age as investigated in rats and is visible after excitation with UV light at 350 nm through rapidly
86
Figure 6.1 Characterization of Stellate Cells from Rat Liver

Notes:
The stellate-shaped morphology and lipid droplets are preserved in freshly isolated hepatic
stellate cells after 1 day (1d) of culture as documented by phase contrast transmission light
microscopy (center). Stellate cells typically contain the vitamin A derivate retinyl palmitate,
which can be visualized after excitation with UV light by emitted uorescence light (light
blue). Quiescent hepatic stellate cells of rats express the lamentous proteins glial brillary
acidic protein (GFAP), desmin, and vimentin at the rst day of culture, whereas cultureactivated stellate cells start to express -smooth muscle actin (-SMA) as documented by
immunouorescence staining (red) at the 7th day (7d) of culture. One of the earliest markers
that indicate the activation of hepatic stellate cells is the intermediate lament protein Nestin,
which is already detectable at the second day (2d) of culture (red). Nestin is expressed by
activated somatic stem/progenitor cells and points toward an undifferentiated state of stellate
cells. This is further conrmed by their expression of CD133. Especially the cell protrusions
and the Golgi complex are stained by the CD133 antibodies (red). The cell nuclei are counterstained by DAPI (4',6-diamidino-2-phenylindole; blue).
6.1
87
fading uorescence light (fFigure 6.1). This vitamin A uorescence is one of the best
markers to identify hepatic stellate cells, but also some intermediate lament proteins
are useful to detect them. In rodents, quiescent hepatic stellate cells synthesize the lamentous protein glial brillary acidic protein (GFAP; Gard et al., 1985) (fFigure 6.1),
but quiescent stellate cells of the normal human liver are mainly devoid of GFAP expression. The synthesis of GFAP can be only observed in a small population of stellate cells
close to the portal tracts (Hautekeete and Geerts, 1997). In addition, quiescent stellate
cells of the normal human liver lack the intermediate lament protein desmin (SchmittGrff et al., 1991). Desmin expressing stellate cells are mainly found in the periportal
zone of the normal rat liver, whereas stellate cells of the pericentral zone are negative
for desmin. This implies that hepatic stellate cells of a single organism are heterogenous,
displaying a differential gene expression, which may depend on their relative position
from periportal to pericentral zones of the liver (Geerts, 2001). Desmin is a lamentous
protein that is expressed by contractile cells such as muscle cells. Other intermediate
lament proteins of contractile cells congruently found in hepatic stellate cells of humans and rodents are vimentin (fFigure 6.1) and synemin (Ahmed et al., 1991; Casini
et al., 1993; Schmitt-Grff et al., 2006; Uyama et al., 2006). This expression prole
indicates that hepatic stellate cells are contractile.
Isolated stellate cells from humans and rodents cultured on plastic dishes lose their
vitamin A stores and develop into contractile myobroblast-like cells that synthesize
-smooth muscle actin (-SMA), a process called culture-dependent stellate cell activation. These culture-activated hepatic stellate cells start to synthesize extracellular
matrix proteins such as collagen type I, type III, and type IV as well as laminin and bronectin. For this reason, activated stellate cells are regarded to be involved in formation of liver brosis (Friedman et al., 1985). The activation of isolated stellate cells is
mainly dependent on environmental conditions or signals such as the culture surface
(fFigure 6.2) or soluble factors (Sawitza et al., 2009). However, with the exception
of GFAP all intermediate lament proteins aforementioned as well as -SMA are also
expressed by other liver cell types and, therefore, are not suitable to reliably identify
hepatic stellate cells. Moreover, hepatocytes, endothelial cells, and portal broblasts
of the liver can also synthesize extracellular matrix proteins, and there is increasing
evidence that hepatic myobroblasts may emerge from more than one cellular origin.
Thus, reliable markers are required to monitor the behavior of stellate cells in liver
diseases or regeneration. In rodents, hepatic stellate cells seem to be negative for cytokeratins, which are synthesized by hepatocytes, cholangiocytes, and liver progenitor
cells called oval cells, but they are positive for GFAP and Nestin expression. These
two lament proteins can be used to reliably identify quiescent or activated hepatic
stellate cells, respectively. The sole expression of GFAP is typical for quiescent stellate
cells, whereas the simultaneous expression of GFAP and Nestin can be observed early
during activation of stellate cells. When the activation process proceeds, the stellate
cells maintain their Nestin synthesis but decrease their GFAP expression. In this case,
a combination of Nestin and desmin can be used to detect stellate cells.
The intermediate lament protein Nestin is induced during culture-dependent activation of hepatic stellate cells (Niki et al., 1999) and is already detectable on the second
day of culture (fFigure 6.1). Moreover, Nestin expression is also up-regulated in stellate
cells during early liver regeneration after partial hepatectomy and, thus, also suitable as
an indicator of their activation in vivo. Interestingly, Nestin represents a characteristic
88
Figure 6.2AF The Activation of Isolated Rat Hepatic Stellate Cells Depends on Culture
Conditions
Notes:
The activation of cultured stellate cells on plastic surfaces is commonly used to investigate
mechanisms of brosis.
A Freshly isolated hepatic stellate cells with lipid droplets are attached to the plastic dishes
at the rst day (1d) of culture.
B Within 7 days (7d) of culture on plastic the stellate cells develop into enlarged myobroblast-like cells that frequently possess two or more nuclei per cell. This activation process is
accompanied by the loss of lipid vesicles and induction of -SMA and extracellular matrix
protein expression.
6.1
89
marker of multilineage stem/progenitor cells (Wiese et al., 2004), and its expression
is highly elevated in stem cell-based liver regeneration after partial hepatectomy in
the presence of 2-acetylaminouorene (2-AAF) (Reister et al., 2011). The synthesis of
Nestin points toward an undifferentiated state of hepatic stellate cells, which is further conrmed by their expression of CD133, also called prominin-1 (Kordes et al.,
2007). CD133 is a glycosylated cell surface protein that was initially discovered in
hematopoietic and neuronal stem/progenitor cells. The functions of CD133 are hitherto unknown, but it is apparently able to repress cell differentiation as demonstrated
for neuroblastoma cells. In line with this, CD133 is detectable on the cell membrane
protrusions mainly of quiescent hepatic stellate cells (fFigure 6.1). Further analysis of
stem and progenitor cell markers in isolated hepatic stellate cells of rats revealed the
expression of multiple genes typically found in undifferentiated cells. They express, for
example, Oct4 (octamer binding factor 4), slain1, c-kit, and breast cancer resistance
protein 1 as well as other factors that are suitable to characterize them as stem/progenitor cells (Kordes et al., 2007). The transcription factors Oct4 and nanog are well-known
from embryonic stem cells, and their expression is sustained in germ cells during adulthood. The occurrence of the pluripotency associated Oct4 is thought to be restricted to
these cells, but there are also convincing reports about an expression of Oct4 in some
adult somatic stem cells. Experimental evidence exists that bone marrow stem cells
can express Oct4 because transplanted eGFP (enhanced green uorescent protein)
are reported to constitute eGFP oocytes in female host animals of the wild type ( Johnson et al., 2005). Transplantation studies using eGFP bone marrow cells revealed also
that hepatic stellate cells can originate from the bone marrow (Baba et al., 2004) and
obviously maintain their undifferentiated state in the liver.
Figure 6.2
(continued)
C Quiescent hepatic stellate cells attach to thick layers of collagen obtained from rat tail
within the rst day of culture.
D After a few days, the stellate cells partly lose their lipids; display an elongated, branched
morphology; and primarily possess one nucleus per cell on rat tail collagen. About 3 ml
of gelatinous rat tail collagen, mainly consisting of collagen type 1, is digested by a nearly
conuent stellate cell population of a 6-well culture plate in less than 14 days through the
release of matrix metalloproteinases (MMP). Migrating hepatic stellate cells are known to
secrete MMP2 und MMP9.
E Freshly isolated hepatic stellate cells with lipid droplets attach to matrigel, which represents
a gelatinous protein mixture mainly composed of the basal lamina proteins laminin and collagen type IV secreted by Engelbreth-Holm-Swarm mouse sarcoma cells, within the rst day
of culture.
F The morphology and lipid droplets of freshly isolated hepatic stellate cells are preserved
during 7 to 14 days of culture on matrigel. Thus, matrigel efciently inhibits the activation of
cultured hepatic stellate cells. The stellate cells tend to form small cell aggregates on matrigel
during culture time.
90
6.2
Plasticity of Hepatic Stellate Cells
Stem and progenitor cells can differentiate into specialized cell types that fulll welldened functions within organs. The differentiation potential of stem/progenitor cells can
be analyzed in vitro using appropriate culture conditions and cytokines, which initiate
their further development. When hepatic stellate cells of rats are cultured on rat tail collagen they become activated and start to proliferate. The simultaneous exposure of these
activated stellate cells to cytokines that either favor the differentiation of stem/progenitor
cells into endothelial cells (e.g., vascular endothelial growth factor164) or hepatocytes
(e.g., broblast growth factor4 , hepatocyte growth factor) induce molecular markers of
endothelial cells (vascular endothelial cadherin, endothelial nitric oxide synthase) or
hepatocytes (albumin, -fetoprotein, hepatocyte nuclear factor 4), respectively (Kordes
et al., 2007). The expression of hepatocyte markers is also induced when isolated rat
hepatic stellate cells were activated on plastic for 7 days and subsequently co-cultured
with liver parenchymal cells, which are separated from the stellate cells by a membrane
that allowed permeation of soluble factors only. If hepatic stellate cells from the same cell
preparation are kept in monoculture, hepatocyte markers remain undetectable, indicating
that neither parenchymal cells nor liver progenitor cells, as potential contaminants in the
stellate cell preparations, contributed to this hepatocyte-specic expression prole. These
experiments demonstrate that hepatic stellate cells possess a differentiation potential.
However, quiescent hepatic stellate cells that contain retinoids and maintain their characteristics in co-cultures with liver parenchymal cells lack any signs of cell differentiation (Sawitza et al., 2009). The induction of cell differentiation obviously depends on the
developmental stage of stellate cells, and their activation is a prerequisite for their further
development. Owing to this plasticity, a direct contribution of hepatic stellate cells to liver
regeneration can be expected. Indeed, cell fate-mapping experiments using a GFAP reporter construct documented a possible contribution of stellate cells to liver regeneration
in vivo because cells that initially express GFAP were found among hepatocytes and cholangiocytes (Yang et al., 2008). However, due to the fact that several other cell types in the
body also synthesize GFAP, the transplantation of highly enriched stellate cell preparations
into the regenerating liver is also necessary to prove their differentiation potential. Indeed,
when eGFP hepatic stellate cells from rats are transplanted via tail vein injections into
wild type rats that underwent partial hepatectomy in the presence of 2-AAF, they reached
the liver and partly differentiated into hepatocytes in vivo. The transplanted eGFP stellate
cells can be clearly detected in the host liver by RT-PCR and eGFP uorescence colocalized with parenchymal cell markers such as hepatocyte nuclear factor 4 in cells displaying hepatocyte morphology. Therefore, hepatic stellate cells fulll another feature of stem/
progenitor cells: they are transplantable and can contribute to tissue repair.
6.3
Stellate Cells in Liver Regeneration
A contribution of stellate cells to liver regeneration requires their activation as outlined

previously. The Nestin synthesis is early induced in stellate cells within the sinusoids
after partial hepatectomy. In this model of rat liver injury, hepatocytes proliferate on
the rst day of regeneration, which leads to rearrangements of the stellate cell niche
in the space of Diss. Due to changes in their microenvironment, hepatic stellate cells
are transiently activated from the second to the sixth day of liver regeneration. When
6.3
91
the proliferation of hepatocytes is inhibited through exposure of rats with 2-AAF, which
can be applied by daily injections or implantation of pellets (e.g., 14 day release of
70 mg 2-AAF) under the skin of the neck, the Nestin expression is elevated throughout
the regeneration and displays a maximum around the 7th day during the ductular reaction in the portal eld (Reister et al., 2011). In this model of stem cellbased liver regeneration, in which liver progenitor cells or oval cells contribute to the restoration of liver
mass, proliferating cells are visible by incorporation of BrdU (5-brome-2-deoxyuridine)
into the DNA of the cell nuclei mainly in the portal elds, but also in the liver sinusoids
close to the central vein (fFigure 6.3). Thus, stellate cells are activated and proliferate
early during stem cellbased liver regeneration.
Immunouorescence stainings of cytokeratins, thymocyte antigen-1 (Thy-1), -SMA,
desmin, and Nestin in the regenerating liver display a zonal distribution of cells 7 days
after partial hepatectomy in the presence of 2-AAF. With the exception of cytokeratins,
these proteins are expressed by activated stellate cells and mainly located in the portal
eld, where oval cells are thought to originate (fFigure 6.4AE). This model of stem cell
based liver regeneration is also characterized by the strong deposition of basal lamina
proteins such as laminin and collagen type IV in the portal eld, whereas collagen type
I deposition remains weak in the vicinity of portal tracts despite the presence of activated
hepatic stellate cells (fFigure 6.5AC). In contrast to this, GFAP is typically expressed by
quiescent hepatic stellate cells and mainly restricted to the remaining tissue around the
central vein (fFigure 6.4F). A liver zonation is also detectable in the normal liver and
was initially described for the hepatic glutamine and ammonia metabolism (Hussinger,
1983; 1990). The exclusive expression of the enzyme glutamine synthetase by hepatocytes that surround the central vein is typical for the normal liver and is maintained during
liver regeneration (fFigure 6.5D). In nodules of the cirrhotic liver, no such zonation
is observed, and the glutamine synthetase is hardly detectable (Racine-Samson et al.,
Figure 6.3A,B BrdU Incorporation of Cells During Early Rat Liver Regeneration After
Partial Hepatectomy in the Presence of 2-AAF
Notes:
A Immunouorescence staining of BrdU in the nuclei of cells close to the portal eld.
B Detection of BrdU by immunouorescence in the nuclei of cells within liver sinusoids
close to a central vein. Parafn sections of rat liver (5 m thickness) were analyzed with
antibodies against BrdU after antigen retrieval with acid.
92
Figure 6.4AF Zonal Distribution of Cells Involved in Stem CellBased Liver Regeneration After Partial Hepatectomy in the Presence of 2-AAF
Notes:
Cryosections of rat liver were analyzed on the 7th day of regeneration by immunouorescence
stainings of marker proteins (red), and all images were combined with a DAPI cell nucleus
staining (blue). (A) Cytokeratin (panCK) and (B) Thy-1 expressing cells are zonal distributed
mainly around the portal elds. Molecular markers of activated hepatic stellate cells, such as
(C) -smooth muscle actin (-SMA), (D) desmin, and (E) Nestin were detected primarily in the
portal elds. (F) Hepatic stellate cells that still express glial brillary acidic protein (GFAP) are
mainly restricted to areas around the central veins.
6.3
93
Figure 6.5AD Zonal Distribution of Cells Involved in Stem CellBased Liver Regeneration
After Partial Hepatectomy in the Presence of 2-AAF
Notes:
Cryosections of rat liver were analyzed on the 7th day of regeneration by immunouorescence
stainings of marker proteins (red), and all images were combined with a DAPI cell nucleus
staining (blue). The deposition of the basal lamina proteins (A) laminin and (B) collagen type
IV is highly elevated in the portal eld, whereas (C) collagen type I is not elevated in the portal
eld despite the presence of activated hepatic stellate cells. (D) As in the normal rat liver, the
expression of the enzyme glutamine synthetase (GS) is detectable in a small rim of perivenous
hepatocytes surrounding the central veins and indicates the maintenance of liver zonation in
the stem cellbased liver regeneration.
1996). It can be presumed, that a functional tissue zonation is required for a proper liver
regeneration.
The zonal organization of the liver can at least in part be attributed to -catenindependent or canonical Wnt signaling that inversely controls the genetic programs
of perivenous and periportal cells. This signaling pathway is maximally active in cells
close to the central veins (Benhamouche et al., 2006). Although previous reports suggest that canonical Wnt signaling is associated with the proliferation of stem/progenitor
cells, a recent study provides evidence that quiescence of, for instance, hematopoietic
stem cells is maintained by this signaling pathway (Fleming et al., 2008). In addition,
-catenin-independent or non-canonical Wnt signaling via the ligand Wnt5a inhibits
the canonical Wnt cascade in hematopoietic stem cells (Nemeth et al., 2007) and is
associated with further development of stem/progenitor cells. The activation of isolated
94
Figure 6.6AG Activated Hepatic Stellate Cells are Associated with Duct Formation in the
Stem CellBased Liver Regeneration and in Culture
Notes:
Cryosections of rat liver were analyzed on the 7th day of regeneration after partial hepatectomy
in the presence of 2-AAF by immunouorescence stainings of marker proteins (red), and all
images were combined with a DAPI cell nucleus staining (blue).
A Activated hepatic stellate cells that express Nestin (green) surround cells that express cytokeratins (panCK; red) and spread through duct-like structures in the vicinity to the portal
elds. The three-dimensional (3D) rendering of image sections made by confocal laser scanning microscopy reveals that the expressions of Nestin and panCK are distinct. Cytokeratin
expressing cells are covered by activated Nestin positive stellate cells.
B Activated stellate cells that cover the ducts in the portal eld can be identied by the
simultaneous detection (yellow) of Nestin (green) and desmin (red).
6.3
95
hepatic stellate cells on plastic is prevented in part by stimulation of the canonical Wnt
signaling pathway (Kordes et al., 2008) and may explain the prevalence of GFAP expressing stellate cells close to the central vein during stem cellbased liver regeneration.
Interestingly, activated hepatic stellate cells secrete Wnt4 and Wnt5a that may inuence
the developmental fate of adjacent progenitor cells. In line with this, stellate cells can apparently also promote the differentiation of liver progenitor cells into hepatocytes when
they are used as feeder cells (Wang et al., 2010). On the other hand, the typical expression pattern of liver parenchymal cells can be induced in stellate cells, and therefore,
they possess the intrinsic capacity to differentiate into hepatocytes. In both cases hepatic
stellate cells act like Nestin expressing mesenchymal stem cells, which are known to
inuence the developmental fate of neighboring hematopoietic stem/progenitor cells
and can differentiate into hepatocytes, osteoblasts, chondocytes, and lipocytes, also.
Activated hepatic stellate cells are in close proximity to cytokeratin expressing cells
that spread in duct-like structures, mainly without cavities in the portal eld during the
stem cellbased liver regeneration (Evarts et al., 1990) (fFigure 6.6A). Nestin stellate
cells can clearly be identied by the simultaneous detection of desmin (fFigure 6.6B)
and are located on the surface of the ducts formed by cytokeratin 19 expressing cells,
which in turn are covered by the basal lamina proteins laminin and collagen type IV
(fFigure 6.6C, D). The basal lamina of the ducts may support the maintenance of the undifferentiated state of stellate cells, which is denoted by the expression of Nestin, on the
one hand and may also provide a structure for their further dispersion within the remodeling liver tissue on the other hand. Ductular structures are also formed by isolated hepatic
stellate cells of rats that express Nestin (Kordes et al., 2007) (fFigure 6.6EG). These
duct-like structures are instable and nally develop into cell aggregates, which are placed
Figure 6.6AG (continued)

C The duct-like structures harbor cytokeratin 19 (CK19) expressing cells (green) in the portal
eld and are covered by laminin (red). Collagen type IV is another basal lamina protein that
is highly elevated around these ducts.
D Scheme of the spatial association of activated hepatic stellate cells and CK19 expressing
cells that spread in ducts during stem cellbased liver regeneration.
E Isolated hepatic stellate cells of rats cultured in a 6-well plate on rat tail collagen form ductlike structures within 7 days when they are treated with the cytokines vascular endothelial
growth factor164, basic broblast growth factor, erythropoietin, and interleukin-6 in conjunction with fetal calf serum.
F The ducts formed by cultured hepatic stellate cells contain discontinuous cavities as observed after staining of xed cells by phalloidin-tetramethyl rhodamine isothiocyanate and 3D
rendering of image series made by confocal laser scanning microscopy.
G The ducts formed in vitro are composed of Nestin expressing hepatic stellate cells as investigated by immunouorescence staining (red).
96
nearly equispaced in the culture dishes. This transient duct formation is apparently used
by stellate cells for their migration. The basal laminas of preexisting or sprouting blood
vessels may also serve as a basis for migrating stellate cells as indicated by the appearance
of Nestin expressing cells at the blood vessels during liver regeneration, which otherwise
remain undetectable in the uninjured liver. Although the exact function of hepatic stellate
cells in the restoration of the injured liver still remains to be elucidated, their denite
activation in this process indicates that they are major players in liver regeneration.
Summary
Quiescent hepatic stellate cells store retinoids (vitamin A), which prevent their activation. The
activation of stellate cells largely depends on environmental conditions and can be indicated
by the induction of Nestin. The intermediate lament protein Nestin is known to be expressed
by stem/progenitor cells such as mesenchymal stem cells and points toward an undifferentiated state of hepatic stellate cells. The expression of CD133 and their capacity to differentiate
further supports the classication of stellate cells as stem/progenitor cells. After liver injury
of rats through partial hepatectomy in the presence of 2-acetylaminouorene, stellate cells
are activated and display a distinct zonal distribution in the portal eld. Nestin stellate cells
are located on basal laminas that surround duct-like structures formed by cytokeratin 19
expressing cells and blood vessels in this model of stem cellbased liver regeneration. Isolated
hepatic stellate cells are also able to form duct-like structures, which may facilitate their
migration and maintenance of their undifferentiated state as denoted by a sustained Nestin
expression. The function of activated stellate cells during stem cellbased liver regeneration
is hitherto unknown. Evidence exists that activated hepatic stellate cells can inuence the
development of liver progenitor cells (oval cells) and possess the potential to differentiate
into hepatocytes. The relationship between hepatic stellate cells and oval cells remains an
interesting research topic.
Further Reading
Friedman, S.L. (2008). Hepatic stellate cells: protean, multifunctional, and enigmatic cells of
the liver. Physiol. Rev. 88, 12572.
Desmet, V.J. (2009). The amazing universe of hepatic microstructure. Hepatology 50, 333 44.
Gebhardt, R., and Hovhannisyan, A. (2009). Organ patterning in the adult stage: the role of
Wnt/beta-catenin signaling in liver zonation and beyond. Dev. Dyn. 239, 4555.
Higgins, G.M., and Anderson, R.M. (1931). Experimental pathology of the liver. I. Restoration
of the liver of the white rat following partial surgical removal. Arch. Pathol. Lab. Med. 12,
186202.
Tatematsu, M., Ho, R.H., Kaku, T., Ekem, J.K., and Farber, E. (1984). Studies on the proliferation and fate of oval cells in the liver of rats treated with 2-acetylaminouorene and partial
hepatectomy. Am. J. Pathol. 114, 41830.
References
Ahmed, Q., Hines, J.E., Harrison, D., and Burt, A.D. (1991). Expression of muscle-associated
cytoskeletal proteins by human sinusoidal liver cells. In: Cells of the hepatic sinusoid,
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quiescent stage of hepatic stellate cells. Biochem. Biophys. Res. Commun. 367, 11623.
Nemeth, M.J., Topol, L., Anderson, S.M., Yang, Y., and Bodine, D.M. (2007). Wnt5a inhibits
canonical Wnt signaling in hematopoietic stem cells and enhances repopulation. Proc.
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Niki, T., Pekny, M., Hellemans, K., De Bleser, P., Van Den Berg, K., Vaeyens, F., Quartier, E.,
Schutt, F., and Geerts, A. (1999). Class VI intermediate lament protein nestin is induced
during activation of rat hepatic stellate cells. Hepatology 29, 520 7.
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alpha smooth muscle actin and desmin expression in perisinusoidal cells of normal and
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expression is widespread in liver brosis and is induced in proliferating and malignant
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Robson, R., Pekny, M., and Geerts, A. (2006). Hepatic stellate cells express synemin, a
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Epigenetics during Liver Regeneration

Learning Targets
1. Epigenetic mechanisms in eukaryotes are DNA methylation, histone modications,
and non-coding RNA.
2. Epigenetic gene regulation is different in stem cells and mature effector cells.
3. Hepatic stellate cells display major changes in their epigenome during culturedependent activation.
4. Epigenetic gene regulation is transiently altered during liver regeneration.
7.1
Denition and Mechanisms of Epigenetics
Epigenetics can be dened as the study of mitotically and/or meiotically heritable

changes in gene function that can not be explained by changes in DNA sequences
(Riggs and Porter, 1996). Although the genetic information is identical in all somatic
cells of a single individual, about 200 different cell types develop during embryogenesis, indicating the presence of epigenetic mechanisms that guide developmental
fate of somatic cells. The powerful inuence of epigenetic mechanisms on cellular
development is best illustrated by experiments in which frog cell nuclei of advanced
blastula cells and, later on, of adult somatic cells are transplanted into enucleated
oocytes of frogs. Components of the oocyte cytoplasm are able to reprogram the
transplanted nucleus and to initiate the development of functional tadpoles (Briggs
and King, 1952; Lasky and Gurdon, 1970). Nowadays, this method was also adopted
to clone mammals such as the sheep Dolly. Obviously, the genome of a transplanted
cell nucleus can respond to environmental signals, which derive in this case from egg
cytoplasm. Epigenetic mechanisms apparently allow also the whole organism to respond to changes in the environment through altered gene expression. Environmental
signals are different and can derive, for example, from qualitative and quantitative
changes of the nutrition status. Altered epigenetic gene regulation through the nourishment can affect the expression of key genes linked to the development of type
2 diabetes.
The epigenome undergoes profound changes during development from an undifferentiated totipotent state, which is only represented by the zygote, to more differentiated or mature states of cells. Therefore, embryonic or adult stem cells should display
a unique epigenome that differs from somatic effector cells. Until now there are three
100
principle mechanisms of epigenetic gene regulation discovered in eukaryotes: (1) DNA

methylation, (2) histone modications, and (3) non-coding RNA.
1. The DNA methylation is restricted to cytosine phosphatidyl guanosine (5'-CpG3') dinucleotide sequences in mammals and is associated with gene silencing. The
methylation of cytosine to 5-methylcytosine is catalyzed by specic DNA methyl transferases (Dnmt; fFigure 7.1A). It was thought to be the only naturally occurring base
modication in vertebrate DNA until 5-hydroxymethylcytosine was discovered in some
mammalian tissues such as the brain in 2009 (fFigure 7.1B). There are two different
mechanisms of DNA methylation, the de novo and maintenance methylation. The de
novo DNA methylation selectively transfers the methyl group to non-methylated CpG,
and this is mainly catalyzed by the enzymes Dnmt3a and Dnmt3b, especially during
embryonic development. The maintenance methylation of DNA, which involves the
Dnmt1, is required during mitosis of cells to transfer the methylation pattern of the
original DNA strand to the newly synthesized DNA strand (fFigure 7.1A). The Dnmt2,
in contrast, modies tRNA and does not contribute to DNA methylation.
Regulatory CpG are normally clustered in islands (>500 bp), which contain a high
frequency of CpG (>55%) within a promoter region and are usually not methylated, for
example, in housekeeping genes. The expression of other genes can be repressed by
methylation within CpG islands. Proteins with methyl-CpG binding domain (MBD) such
as MeCP2 (methyl-CpG binding protein 2) interact with methylated DNA to control the
gene expression in conjunction with other proteins. MeCP2 may generally act as a gene
repressor but is also associated with methylated CpG of active promoters. MeCP2 and
Dnmts seem to be dispensable in embryonic stem cells but are required for their further
development. An adequate DNA methylation is essential for imprinting, X chromosome
silencing in female mammals, and proper embryogenesis. Mice that lack one of the
Dnmt genes die early during development, demonstrating the importance of correct
DNA methylation. An unusual DNA methylation status is frequently seen in tumors.
During progression of tumor cells a global demethylation of the DNA can be observed
NH 2
+ HN
NH 2
maintenance
methylation
CH 3
+ HN
Dnmt1
NH 2
CH 2 OH
+ HN
Dnmt3a/b
O
N
R
cytosine
de novo
methylation
5-methylcytosine
5-hydroxymethylcytosine
Figure 7.1A,B 5-methylcytosine and 5-hydroxymethylcytosine are Naturally Occurring

Modications of Cytosine in Vertebrate DNA
Notes:
A The enzyme Dnmt1 methylates cytosine to 5-methylcytosine during mitosis, when the DNA
methylation pattern of the original DNA strand is conferred to the newly synthesized DNA
strand (maintenance methylation). Newly established DNA methylation patterns during cell
development are transferred by the Dnmt3a/b to cytosines (de novo methylation).
B An additional modication of cytosine is 5-hydroxymethylcytosine, which is mainly detectable in embryonic stem cells and the brain.
7.1
Denition and Mechanisms of Epigenetics
101
that leads to expression of stem cell factors such as CD133. The expression of CD133
was described in hepatocellular carcinoma and liver cancer stem cells. In contrast to
this, hypermethylation of DNA affects the promoters of tumor suppressor genes in many
cancers. A hypermethylation of the tumor suppressor gene P16ink4a, for instance, protects
the tumor cells from cell cycle arrest. Other examples for hypermethylation of genes in
liver tumors that can counteract their growth are deleted in liver cancer 1 (DLC-1) and
hedgehog interacting protein (HHIP). In contrast to tumor cells, a global increase of
DNA methylation is seen during differentiation of stem cells.
2. The DNA is associated with nucleosomes, which are composed of an octamer of
the core histones H2A, H2B, H3, and H4 (including variants of histone H2A and H3) to
control condensation of chromatin. Linker histones H1 are responsible for higher order
chromatin structure. The chromatin is present as open euchromatin, often associated
with active gene transcription, or as highly condensed heterochromatin, mainly associated with gene silencing (fFigure 7.2A). Embryonic stem cells display less abundant
gene transcription
gene transcription
HP1
HP1
HP1
HP1
HP1
HP1 HKMT HKMT HKMT HP1
HP1
HP1
HP1
HP1
HP1
euchromatin
S
P
H2A
H2B
H3
H2A
H4
acetylation
H4
AC
H2B
H3
phosphorylation
Me
H2A
H2B
H3
H4
methylation
Me
H2A
H2B
H3
H4
Ub
ubiquitylation
H2A
H2B
H3
H2A
H4
H2B
SUMO
Rib
P
H3
DN
heterochromatin
AC
euchromatin
H4
Ade
Rib
P
Rib
P
Ade
Rib
P
SUMOylation ADP-ribosylation
Figure 7.2A,B Histone Modications Contribute to Regulation of Chromatin Condensation and Gene Transcription
Notes:
A The gene transcription is mainly active in open euchromatin and often repressed in
condensed heterochromatin. Factors such as the heterochromatin protein 1 (HP1) and histone lysine methyltransferases (HKMT) are required for heterochromatin formation. Polycomb repressive complexes are further associated with silenced gene regions, whereas RNA
polymerase II and Trithorax group proteins are found in euchromatin during active gene
transcription.
B The DNA is associated with octamers of core histones (nucleosomes) that are involved in
chromatin condensation. The accessibility of the DNA is inuenced by histone modications
such as acetylation (AC), phosphorylation (P), methylation (Me), ubiquitylation (Ub), SUMOylation (SUMO) and ADP-ribosylation (Ade, adenine; Rib, ribose; P, phosphate) at amino acid
residues of N-terminal tails (E, glutamate; K, lysine; R, arginine; S, serine; T, threonine).
102
heterochromatin compared to adult soma cells, reecting their high developmental potential. Post-translational histone modications at N-terminal tails through acetylation,
phosphorylation, methylation, ubiquitylation, SUMOylation, and ADP-ribosylation can
affect the structure of chromatin and can alter binding of effector proteins to the chromatin (fFigure 7.2B). Histone modications can thus contribute to regulation of gene
transcription. The modication of histones by acetylation is long known and catalyzed
by histone acetylases. A histone hyperacetylation of specic lysine residues is associated with increased gene transcription. The histone acetylation is reversed by histone
deacetylases (HDAC), leading to gene silencing. The phosphorylation of specic serine
and threonine residues of histones is mediated by histone kinases and their dephosphorylation by phosphatases. Histone H3 phosphorylation is associated with euchromatin
and heterochromatin, depending on the sites of phosphorylation. For ubiquitylation,
the small peptide ubiquitin is linked by E3 ubiquitin ligase to lysine residues of target
proteins to tag them for proteasomal degradation. Ubiquitylated histone H2B, however,
is not degraded but is associated with active gene transcription and is crucial for development and cell cycle progression. Other histones such as H1, H2A, and H3 can also be
ubiquitylated at lysine. The ubiquitylation is reversible through ubiquitin-specic proteases. SUMOylation is a covalent modication of target proteins such as histone H4 at
lysine residues through small ubiquitin-related modier (SUMO)-conjugating enzymes
and plays a central role in coordinating histone modications and chromatin structure.
SUMOylation has been correlated mainly with transcriptional repression. SUMO moieties, which share 18% amino acid sequence identity with ubiquitin, can be removed
by SUMO-specic proteases. ADP-ribosyl transferases transfer ADP-ribose moieties to
glutamate or aspartate residues of histones, resulting in poly(ADP-ribose) chains that
affect the chromatin condensation. The modication of histones by poly(ADP-ribose)
is reversible through poly(ADP-ribose) glycohydrolases. Methyl groups are coupled to
arginine or lysine of histones by methyltransferases. Lysine can bind one, two, or three
methyl groups, which inhibit or facilitate gene transcription depending on the position
of the modied amino acid. For instance, methylation of histone H3, which is highly
conserved among many species, at lysine 9 (H3K9) and lysine 27 (H3K27) is associated
with silenced heterochromatin, whereas methylation of lysine 4 (H3K4) is observed in
activated promoters. The methylation can be reversed from H3K4 by a lysine specic
demethylase. The activating trimethylation of histone H3 at lysine 4 (H3K4me3) can be
combined with acetylation of histones H3 at lysine 9 to alter histone conformation and
enable transcription factor binding to DNA, showing that different histone modications are intertwined to regulate gene expression. Alterations in chromatin structure
are energy consuming and involve also ATP-dependent chromatin-remodeling factors, which are protein complexes with ATPase activity and play an additional role in
regulating DNA accessibility.
3. Non-coding RNA or RNA interference (RNAi) is generated from exogenous (virus)
or endogenous (e.g. centromeric repeats) double-strand RNA, which is processed by
the enzyme Dicer to small interference RNA (siRNA) or microRNA (miRNA) through
different pathways. One strand of the siRNA or miRNA then becomes part of the RNAinduced silencing complex and enables destruction of mRNA with complementary
nucleotide sequences through the enzyme argonaute as a part of this protein complex. Through this mechanism gene transcription is silenced at a post-transcriptional
level. However, there is also evidence that RNAi can initiate heterochromatin assembly,
7.2
Methods to Investigate Epigenetic Mechanisms
103
thereby leading to RNA-induced transcriptional gene silencing. The importance for

RNA-induced transcriptional silencing during development of mammals becomes evident in Dicer-decient embryonic stem cells, which are able to proliferate but fail to
differentiate correctly (Kanellopoulou et al., 2005). This nding indicates that RNAi are
important to guide mammalian development through epigenetic mechanisms, but their
exact mode of action remains to be established.
7.2
Methods to Investigate Epigenetic Mechanisms
The earliest methods evolved to study gene-specic methylation used methylation

specic restriction enzymes to digest DNA. The resulting DNA fragments are then
analyzed by Southern blot (electrophoresis of DNA fragments followed by probe
hybridization) or polymerase chain reaction (PCR). The DNA methylation of specic
genes can be investigated further by sodium bisulte conversion of cytosine to uracil
in single-stranded DNA. The bisulte conversion of cytosine through deamination does
not occur with 5-methylcytosine, which remains as cytosine. The correct position of
methylated CpG is then detectable by sequencing of PCR products of the gene region
of interest (fFigure 7.3). In another approach, antibodies against 5-methylcytosine can
be used for immunoprecipitation of methylated DNA fragments. The enriched methylated DNA sequences can be identied through PCR. To nd unknown methylated
CpG islands, several genome-wide screen methods such as DNA methylation arrays
and restriction landmark genomic scanning (RLGS) were developed. RLGS consists
of a two-dimensional electrophoretic separation of radio-labeled DNA restriction
A
deamination
sulfonation
NH 2
desulfonation
NH 2
O
H
HSO3
+ HN
+ HN
OH
O
H
O
O
H
SO3
HN
H2O
NH4+
H
H
SO3
HN
OH
HSO3
cytosine
cytosine sulfonate
uracil sulfonate
uracil
GGCCCGT TGCGATCC
CH3
CH3
DNA
Figure 7.3A,B
GGUUCGT TGCGATUU
CH3
GGT TCGT TGCGAT T T
CH3
bisulfite conversion of DNA
PCR product
Analysis of DNA Methylation by Bisulte Sequencing
Notes:
A Sodium bisulte treatment of genomic DNA leads to deamination of cytosine that converts into uracil.
B Cytosine (red) but not 5-methylcytosine (blue) is converted into uracil. The DNA methylation status of a given gene can be analyzed by sequencing after PCR amplication using
specic primers. An incomplete bisulte conversion of cytosine residues will be detected as
false 5-methylcytosine.
104
fragments. Selected DNA fragments can be cloned and sequenced for further analysis.
For the investigation of histone modications specic antibodies against the modied amino acid residues are usually applied. Chromatin immunoprecipitation (ChIP)
enriches modied histones with DNA fragments, and genes of interest can be further
identied by PCR. This method is also suitable for genome-wide analysis of histone
modications through combination with DNA arrays: the ChIP on chip arrays. To unravel the epigenetic gene regulation of non-coding RNA species, isolated RNAi must
be sequenced, and computational approaches with appropriate algorithms are used to
predict possible targets based on sequence similarities. This search is hampered by the
fact that in animals the miRNA target binding is only loosely complementary. In a rst
approach the gene regulative function of a limited number of miRNA can be assessed
by miRNA transcriptome PCR arrays, where the transcripts of a given cell (line) pretreated with miRNA is analyzed by PCR using specic primers for the genes of interest
to identify target genes.
7.3
Epigenomics in Liver Regeneration
Epigenetic mechanisms regulate gene expression and direct cell differentiation to provide a stable acquisition of cell fate during organogenesis. The methylation of total liver
DNA was found to be considerably increased during normal development of the rat,
apparently owing to changes in the cellular composition of the liver (Gama-Sosa et al.,
1983). It is likely that epigenetic mechanisms are also involved in the regeneration of
damaged organs. The analysis of the 5-methylcytosine status of the regenerating rat liver
after partial hepatectomy, a standardized method with surgical removal of 2/3 of the
liver, has a long history; rst experiments were already carried out in the late 1960s.
There are several reports about changes in global DNA methylation after liver injury
using this model in rats, showing variable results ranging from no detectable alterations
(Gama-Sosa et al., 1983) to a transient decrease of 5-methylcytosine 24 hours after
partial hepatectomy by 11% compared to the uninjured liver (Kanduc et al., 1988). In
addition, it has been shown that the histone acetylation pattern is signicantly altered
within hours after partial hepatectomy in rats. Acetylated arginine residues of histones
are maximal after 34 hours, whereas lysine residues of histones display elevated acetylation 16 hours after partial hepatectomy in rats (Pogo et al., 1968). In other reports the
highest lysine methylation of histones is observed 30 hours after hepatic regeneration
of rats, while the rate of DNA synthesis is maximal after 24 hours (Tidwell et al., 1968).
Although the exact timing of lysine methylation is variable in these studies, the histone
modication by either acetylation or methylation are early events after partial hepatectomy as observed for the transient hypomethylation of the DNA. A transient decrease
in nuclear matrix associated poly(ADP-ribose) polymerase activity is also detectable
after partial hepatectomy, showing a minimum of 40% compared to control livers after
24 hours (Alvarez-Gonzalez and Ringer, 1988). Rat liver parenchymal cells display their
maximal DNA synthesis at the rst day after surgical intervention, and this is most
likely responsible for the observed changes in the epigenome during liver regeneration.
In mice, ubiquitylated histone H2A (ubH2A) decreases after partial hepatectomy and
displays low levels 24 hours after removal of liver lobes. The decrease in ubH2A is
associated with elevated expression of the ubiquitin-specic protease USP21 that can
7.4
Epigenetics during Stellate Cell Activation
105
catalyze the hydrolysis of ubH2A. Lowered ubH2A amounts are accompanied by increased H3K4 di- and trimethylations, which are histone modications associated with
activation of gene expression and seem to be repressed by ubH2A (Nakagawa et al.,
2008). Although the maximal DNA synthesis of hepatocytes after partial hepatectomy is
at the second day of liver regeneration in mice, the decrease in ubH2A might be also referred to the early response of liver parenchymal cells to injury. The same apparently applies for the limited number of studies that analyze the functions of miRNA after partial
hepatectomy. During early liver regeneration the miR-21 was signicantly up-regulated
in the liver parenchyma after partial hepatectomy in mice and rats (Castro et al., 2010;
Song et al., 2010). The transcription factor forkhead box M1 (FoxM1) is necessary for
DNA synthesis of hepatocytes and inhibited by Btg2 (B-cell translocation gene 2), which
controls cell cycle progression. The miR-21 in turn apparently inhibits Btg2 and probably also signaling of transforming growth factor family members, which then enables
proliferation of parenchymal cells that is otherwise repressed through these mechanisms
(Song et al., 2010).
Little information is available about changes of epigenetic mechanisms in animal
models of liver injury that promote a stem cellbased liver regeneration. Although oval
cells, a non-homogenous cell population of facultative stem/progenitor cells of the liver,
are well accepted to contribute to liver regeneration through differentiation into hepatocytes and cholangiocytes, their epigenome has not been analyzed in detail thus far. The
presence of undifferentiated cells in the liver tissue might be indicated by local DNA
hypomethylation and their differentiation through the activity of Dnmt3a/ b followed by
increased de novo DNA methylation. A global hypomethylation of DNA is known from
embryonic stem cells, but also from dysplastic nodules induced by carcinogens such as
ethionine. These nodules display signicantly reduced DNA methylation compared to
the surrounding tissue, and this effect is even more pronounced after partial hepatectomy (Kanduc et al., 1988). Hypomethylation is an important process in carcinogenesis
and could be enhanced by treatment with the cytosine analogue 5-aza-2'-deoxycytidine
(Denda et al., 1985), which is implemented in the DNA of proliferating cells instead
of cytosine and cannot be methylated by Dnmts. In contrast to this, the application of
S-adenosylmethionine, a donor of methyl groups, can counteract the growth of hepatic
nodules (Garcea et al., 1989). Our current knowledge about epigenetic mechanisms in
liver tumor cells could help to gain access to the epigenome of liver stem/progenitor
cells and may contribute to a better understanding of regenerative processes in the
liver.
7.4
Very few studies exist about the function of epigenetic mechanisms in hepatic stellate
cells. Histone acetylation seems to play a signicant role in activation of HSC because
the HDAC inhibitor trichostatin A can counteract their culture-dependent activation as
indicated by decreased synthesis of collagen type I and III as well as -smooth muscle
actin. Obviously, the interference with the acetylation status of histone H4 inhibits their
activation (Niki et al., 1999). Ethanol can induce an increase in histone H3 acetylation
at lysine 9 (H3K9) in hepatic stellate cells and hepatocytes, which is elevated earlier in
hepatocytes than in hepatic stellate cells (Kim and Shukla, 2005). The H3K9 acetylation
106
is associated with increased gene transcription and obviously reects a response of stellate cells to toxic environmental changes through epigenetic mechanisms. Apparently,
different gene products controlled by histone acetylation are required for either quiescence or activation of hepatic stellate cells. The relevance of epigenetic changes during
activation of hepatic stellate cells is further documented by altered DNA methylation
patterns. The treatment of rat hepatic stellate cells with 5-aza-2-deoxycytidine can
counteract their culture-dependent activation. The activation process is accompanied
by increased expression of MeCP2, which otherwise remains undetectable in quiescent
hepatic stellate cells (Mann et al., 2007). Obviously, genes are also silenced during
stellate cell activation through DNA methylation, and this is actually the case as demonstrated for CD133 (prominin-1) and Notch1. These molecular markers of stem/progenitor cells are required to maintain the undifferentiated state of stem cells, and their
DNA remains non-methylated in quiescent hepatic stellate cells of rats, which further
conrms their undifferentiated state. During culture-dependent activation of hepatic
stellate cells the methylation of CD133 and Notch1 DNA increases gradually, and this
is in agreement with decreased mRNA and protein levels of these proteins. In contrast
to this, the DNA of the Notch3 gene is methylated in quiescent hepatic stellate cells and
becomes demethylated and detectable on mRNA level during their activation (Reister
et al., 2011). Notch3 is involved in cell differentiation as demonstrated for hematopoietic stem/progenitor cells and may indicate a rst sign of cell differentiation in hepatic
stellate cells. CD133, Notch1, and Notch3 are examples for a differential regulation of
gene expression through epigenetic mechanisms such as DNA methylation in quiescent
and activated stellate cells (fFigure 7.4).
Another marker of activated rat hepatic stellate cells is the class VI intermediate lament protein Nestin, which is typically expressed by activated somatic stem/progenitor
cells. Although Nestin DNA is not methylated in quiescent stellate cells, Nestin is only
detectable in activated stellate cells at protein level. The Nestin expression is apparently
inhibited in quiescent hepatic stellate cells by trimethylation of histone H3 at lysine
9 (H3K9me3), whereas the activating methylation of histone 3 at lysine 4 (H3K4me3)
is low. The same inhibitory regulation of Nestin expression occurs in embryonic stem
cells of rats, which display no Nestin expression at any time despite the presence of
non-methylated Nestin DNA. The histone modication pattern of the Nestin DNA locus
turns to the opposite during stellate cell activation. The levels of inhibiting H3K9me3 decreased and the amount of activating H3K4me3 increases, which is in accordance with
the appearance of the protein Nestin in activated hepatic stellate cells (fFigure 7.4).
The lack of Nestin DNA methylation suggests that the expression of Nestin is generally
not controlled through this epigenetic mechanism, but the rat hepatoma cell line H4IIE
as well as rat hepatocytes show Nestin DNA methylation. These cells are devoid of Nestin synthesis as expected. Treatment with the cytosine analogue 5-aza-2-deoxycytidine
induces Nestin synthesis in H4IIE cells and proves the principal regulation of Nestin
expression via DNA methylation. Thus, the expression of Nestin is apparently controlled
by different epigenetic mechanisms in undifferentiated and differentiated cells by either
histone modications or DNA methylation, respectively (Reister et al., 2011). All epigenetic mechanisms described herein characterize the hepatic stellate cells as a cell with
the potential to undergo developmental processes. The DNA methylation and expression pattern of quiescent hepatic stellate cells display strikingly similarities with other
stem/progenitor cells.
7.4
quiescent
107
culture-activated
MeCP2 protein synthesis
CD133 DNA methylation
Notch1 DNA methylation
Notch3 DNA methylation
Nestin DNA methylation
Nestin histone H3K9 methylation
Nestin histone H3K4 methylation
Figure 7.4 Epigenetic Changes During Culture-Dependent Activation of Rat Hepatic

Stellate Cells
Notes:
Only culture-activated hepatic stellate cells synthesize MeCP2 (methyl-CpG binding protein
2). The DNA of CD133 and Notch1 is methylated during activation of stellate cells, whereas
the Notch3 DNA is methylated in quiescent stellate cells. The Nestin expression is not controlled by DNA methylation in stellate cells. They suppress the expression of Nestin through
histone H3 methylation at lysine 9 (H3K9me3). Activated hepatic stellate cells apparently
enhance the Nestin transcription by histone H3 methylation at lysine 4 (H3K4me3).
108
Summary
Important mechanisms of epigenetic gene regulation in eukaryotes are DNA methylation,
histone modications, and non-coding RNA. The DNA methylation is mainly associated
with silenced gene expression, whereas histone modications by acetylation, methylation,
phosphorylation, ubiquitylation, SUMOylation, and ADP-ribosylation inuence chromatin
condensation to enable or repress gene expression. Non-coding RNA can initiate heterochromatin assembly and leads to RNA-induced transcriptional gene silencing. Embryonic stem
cells display less condensed heterochromatin than differentiated soma cells. Methyl-CpG
binding protein 2 (MeCP2) and DNA methyl transferases (Dnmt) seem to be dispensable in
embryonic stem cells but are required for their further development. Quiescent hepatic stellate cells are negative for MeCP2 synthesis but seem to require MeCP2 during their activation.
This process is characterized by major alterations in the epigenome of stellate cells. Only few
studies are available about epigenetic changes in hepatocytes and other cell types of the liver
that contribute to reconstitution of liver mass after injury, including stem/progenitor cells.
Early alterations in epigenetic gene regulation during liver regeneration involve acetylation,
methylation, and deubiquitylation of histones as well as transient decrease in DNA methylation and up-regulation of specic miRNA such as miR-21.
Further Reading
Allis, C.D., Jenuwein, T., and Reinberg, D. (2006). Epigenetics (Cold Spring Harbor, NY, USA:
Cold Spring Harbor Laboratory Press).
De Smet, C., and Loriot, A. (2010). DNA hypomethylation in cancer: Epigenetic scars of a
neoplastic journey. Epigenetics 5, 20613.
Lee, J.S., Smith, E., and Shilatifard, A. (2010). The language of histone crosstalk. Cell 142,
6825.
Marquardt, J.U., Factor, V.M., and Thorgeirsson, S.S. (2010). Epigenetic regulation of cancer stem cells in liver cancer: Current concepts and clinical implications. J. Hepatol. 53,
56877.
Meshorer, E., and Misteli, T. (2006). Chromatin in pluripotent embryonic stem cells and differentiation. Nat. Rev. Mol. Cell. Biol. 7, 5406.
Pinney, S.E., and Simmons, R.A. (2010). Epigenetic mechanisms in the development of type 2
diabetes. Trends Endocrinol. Metab. 21, 2239.
Saladi, S.V., and de la Serna, I.L. (2010). ATP dependent chromatin remodeling enzymes in
embryonic stem cells. Stem Cell Rev. 6, 6273.
Shukla, A., Chaurasia, P., and Bhaumik, S.R. (2009). Histone methylation and ubiquitination
with their cross-talk and roles in gene expression and stability. Cell. Mol. Life Sci. 66,
141933.
Snykers, S., Henkens, T., De Rop, E., Vinken, M., Fraczek, J., De Kock, J., De Prins, E., Geerts,
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Hedgehog Signaling and Liver Regeneration

Steve S. Choi and Anna Mae Diehl
Learning Targets
1. Adult liver regeneration requires replenishment of numerous cell types, including
stromal cells and liver epithelial cells such as hepatocytes and cholangiocytes to
restore organ function.
2. Adult liver regeneration following partial hepatectomy involves activation of the
Hedgehog (Hh) signaling pathway, suggesting that adult liver regeneration may
recapitulate certain features of fetal liver development.
3. Many types of liver cells are capable of producing and/or responding to Hh ligands to
promote reparative and regenerative responses to acute and chronic liver injury.
4. Sustained Hh pathway activation occurs as a response to chronic injury, and the level
of Hh pathway activation is typically proportional to the severity and duration of liver
injury.
8.1
Introduction
Liver regeneration describes a process of reconstructing healthy liver tissue after liver
injury with the purpose of restoring tissue-specic functions. Adult livers become
damaged through the course of normal day-to-day wear and tear and as a result
of superimposed stresses imposed by various insults, such as trauma, exposure to endogenous or environmental toxins, infectious agents and immune system-mediated attack, disturbed perfusion, and processes that obstruct bile ow. Numerous models of
chronic liver injury have been used to study the reparative and regenerative responses
that accompany chronic liver diseases; however, research on liver regeneration after
acute liver injury has typically focused on mechanisms that regulate the proliferative
activity of mature hepatocytes following 70% partial hepatectomy. In both settings, it
is important to recognize that successful repair and regeneration of injured liver tissue
requires much more than the mere replacement of one type of liver epithelial cell (i.e.,
hepatocytes) because the healthy adult liver is a tissue that comprises different cell
types working in concert to maintain normal function. In addition to the mature liver
epithelial cells (e.g., hepatocytes and cholangiocytes) and their progenitors, the liver
comprises different types of stromal cells, including hepatic stellate cells, endothelial
cells, and various types of immune cells, such as macrophages and lymphocytes. Liver
regenerative processes must replenish all of these cells and then re-establish normal
112
cell-to-cell and cell-to-matrix relationships amongst various cell populations so that the
tissue can resume its normal biosynthetic, metabolic, and excretory functions.
8.2
Liver Regeneration after Partial Hepatectomy
In the partial hepatectomy model, resection of 70% of healthy adult liver triggers regenerative mechanisms that result in complete restoration of liver mass and function
typically within a week in adult rodents and within months in adult humans. Although
this process involves hyperplasia of residual mature hepatocytes, other cell types that
were removed during resection must be replaced in order to restore the ow of blood
and bile within the segment of newly formed liver. Without the latter, full recovery of
liver-specic functions would not be possible. Therefore, liver regeneration after partial
hepatectomy requires reconstruction of stromal elements (e.g., hepatic stellate cells,
resident immune cells), vasculature, and biliary channels that support the viability and
function of mature hepatocytes. Unfortunately, unlike mechanisms regulating hepatocyte proliferation (which have been extensively researched) (Michalopoulos, 2007),
mechanisms for replacing other types of liver cells and for re-establishing appropriate
cellcell interactions amongst various liver cell populations after partial hepatectomy
have not been well studied and are thus poorly understood.
We postulated that regeneration of adult liver after partial hepatectomy may resemble
certain aspects of fetal liver development because both situations require global construction of a complex tissue. To better appreciate the concept that adult liver regeneration recapitulates fetal development, a brief review of pertinent aspects of fetal liver
development is presented.
8.3
Fetal Development of the Liver
The liver bud is formed through a process that requires paracrine signaling and cell
migration between the developing heart (mesoderm) eld, septum transversum mesenchyme (mesoderm and ectoderm), and the ventral endoderm. In mice, this typically
occurs between days E8.59.5. Bone marrowderived cells subsequently inltrate the
liver bud and provide signals that are critical for further growth of the developing liver.
Hence, liver morphogenesis involves movement of, and interactions among, cells that
originated in mesoderm, ectoderm, and endoderm.
Briey, work in fetal explants has shown that broblast growth factors (FGF ) and bone
morphogenetic proteins(BMPs) released from the developing heart eld are required for
hepatic specication of the ventral endoderm. Furthermore, it was demonstrated that
hepatic specication is accompanied by induction of Sonic hedgehog (Shh) expression
in endodermal cells, with the latter not occurring unless FGF is provided. Those studies,
therefore, suggest that Hedgehog (Hh) pathway activation is amongst the earliest events
that occurred during construction of fetal liver. Due to the pleiotropic actions of the Hh
pathway during development, and functional redundancies among different Hh ligands
and Hh-regulated transcription factors, the precise roles of Hh signaling in hepatic
morphogenesis have been difcult to demonstrate. Nevertheless, the following data
support a role for the Hh pathway in fetal liver morphogenesis: rst, pluripotent embryonic stem cells, endoderm-committed multipotent progenitors, and some committed
8.4
Overview of Hedgehog Signaling Pathway
113
hepatic progenitors from fetal livers produce and respond to Hh ligands (Hirose et al.,
2009, Sicklick et al., 2006b); second, FoxA1 and FoxA2 (transcription factors that have
been proven to be essential for hepatic specication of endodermal progenitors) are
Hh target genes (Katoh, 2004); third, Hh signaling promotes viability and growth of
Hh-responsive fetal liver progenitors (Sicklick et al., 2006b); fourth, the Hh pathway is
known to regulate growth and differentiation of endothelial progenitors, thymic-derived
lymphocyte progenitors, and bone marrowderived hematopoietic progenitors (El Andaloussi et al., 2006; Fu et al., 2006; Williams et al., 2010) (all of which inltrate the
developing liver bud at some point during fetal life). Such cells also generally produce
Hh ligands. Finally, activation of Hh signaling promotes migration in multiple types of
Hh-responsive cells ( Jenkins, 2009). Thus, many of the cell types that migrate into and
accumulate within the fetal liver are capable of producing and responding to Hh ligands, and these factors regulate important cell fate decisions, as well as the net growth,
of those Hh-responsive cell populations.
The Hh pathway, however, seems to be gradually repressed during hepatocyte differentiation because mature hepatocytes in healthy adult livers neither produce, nor
respond to, Hh ligands. On the other hand, many other types of resident cells in healthy
adult livers are capable of producing and responding to Hh ligands. In uninjured adult
livers, however, only rare cells in and around portal tracts exhibit appreciable Hh pathway activity. The mechanisms that silence Hh signaling during hepatocyte differentiation,
as well as those that more generally repress Hh pathway activation in Hh-responsive
populations of other resident liver cells, remain to be discovered.
8.4
Overview of Hedgehog Signaling Pathway
The hedgehog pathway, originally identied in Drosophila (Hooper and Scott, 2005;
Lee et al., 1992; Schuske et al., 1994), is a highly conserved signaling pathway that
orchestrates multiple aspects of embryogenesis, development, and tissue remodeling
(Beachy et al., 2004; Berman et al., 2003; Ingham and McMahon, 2001; van den Brink,
2007). Hh ligands typically transduce local and long distance autocrine and/or paracrine signals that control the size and localization of Hh-responsive cell populations
(Ingham and McMahon, 2001; Ingham and Placzek, 2006). Hh pathway activation
usually enhances the growth and viability of Hh-responsive cells, whereas abrogating
Hh signaling usually triggers apoptosis in such cells, unless other locally available differentiating factors expedite cellular differentiation to a more mature phenotype that no
longer requires Hh viability signals (Beachy et al., 2004; Ingham and Placzek, 2006).
Thus, depending upon the context, up-regulation and down-regulation of the Hh pathway can provide selective growth advantages for cell types that are capable of responding to Hh ligands. This in turn leads to either expansion or contraction, respectively, of
Hh-responsive cells, thereby orchestrating the cellular composition of various tissues
(Beachy et al., 2004; Ingham and McMahon, 2001; van den Brink, 2007).
In certain conditions, Hh-producing cells (which may or may not be Hh-responsive
themselves) release Hh ligands into the extracellular environment. Hh ligands (Sonic,
Shh; Indian, Ihh; Desert, Dhh) are soluble, lipid-modied morphogens (Chamoun et al.,
2001; Lee and Treisman, 2001; Pepinsky et al., 1998; Porter et al., 1996; Varjosalo and
Taipale, 2008) that may be secreted in two different forms: a short-range acting, poorly
114
diffusible type, and a second form for long-range transport, packaged in membranous
structures (Ingham and McMahon, 2001; Porter et al., 1995; Varjosalo and Taipale,
2008). Hh proteins interact with Patched (Ptch), a membrane-spanning receptor on the
surface of Hh-responsive cells (Carpenter et al., 1998). In the absence of Hh ligands,
Ptch keeps the co-receptor Smoothened (Smo) in its inactive form, thereby silencing
the Smo-dependent down-stream intracellular signaling (Ingham and McMahon, 2001;
Varjosalo and Taipale, 2008). Hence, when Smo-signaling is inhibited by free-Ptch,
Hh-regulated transcription factors (which typically reside in the cytosol) undergo phosphorylation by glycogen synthase kinase 3 (GSK3), protein kinase A (PKA), and casein
kinase (CSK); the phosphorylated (inactive) forms become target for proteasome degradation, and their nuclear translocation is prevented (Pan et al., 2006; Pan et al., 2009).
In contrast, when the extracellular microenviroment is enriched with soluble Hh
ligands, ligand-receptor interaction de-represses Smo, and its activation, in turn, inhibits
Hh transcription factor phosphorylation, leading to an intracellular signaling cascade
that ultimately drives the activation and nuclear translocation of Glioblastoma (Gli)
family zinc-nger transcription factors (Pan et al., 2006; Pan et al., 2009). In vertebrates,
pathway off
pathway on
Shh
Smo
Ptch
Shh
Smo
Ptch
cell
membrane
Gli
P
nucleus
Gli
PKA CSK
PKA CSK
GSK3
GSK3
nucleus
P
DNA
Gli
degradation
Figure 8.1
Gli
DNA
target gene
transcription
Hedgehog Ligands Activate Hh Pathway Signaling
Notes:
Interaction between Hh ligands such as Sonic hedgehog (Shh) and its receptor, Patched
(Ptch) liberates Smoothened (Smo) from the normal repressive actions of Ptch. This results in
eventual inhibition of factors that promote Gli phosphorylation/degradation and permits cellular accumulation of Gli. Nuclear accumulation of Gli factors, in turn, inuences transcriptional activity of Gli-target genes. Gli1 and Gli2 generally increase gene transcription, while
Gli3 can either increase or decrease gene transcription depending on its post-translational
modication.
8.5
Reactivation of the Hedgehog Pathway after Partial Hepatectomy
115
three distinct Gli proteins have been described (Gli1, Gli2, and Gli3) (Hui et al., 1994),
and the binding of Gli proteins to their cognate cis-acting elements regulates the expression of Hh target genes. The latter include several components of the Hh pathway
itself, such as Ptch, Smo, and Glis. Gli1 and Gli2 are mostly responsible for providing
prolonged cellular responses to Hh ligands, while Gli3 is thought to primarily act as
signaling repressor (Ingham and McMahon, 2001; van den Brink, 2007; Varjosalo and
Taipale, 2008). Thus, Hh pathway activity is autoregulated by complex positive and
negative feedback mechanisms that are conserved across species (Hooper and Scott,
2005; Ingham and McMahon, 2001; van den Brink, 2007; Varjosalo and Taipale, 2008)
(fFigure 8.1).
Despite the conservation of the Hh pathway between invertebrates and vertebrates,
Hh pathway regulation diverges and differentiates at some point (Varjosalo et al., 2006;
Varjosalo and Taipale, 2008), as only vertebrates have an additional transmembrane
protein, Hh-interacting protein (HHIP, also a Hh target gene), that competes with Ptch
for binding to Hh soluble ligands (Chuang and McMahon, 1999; Jeong and McMahon,
2005). Thus, in vertebrates, when levels of Ptch exceed those of the Hh ligands, or when
HHIP sequesters Hh ligands and subtracts activating signals to Ptch, the Hh pathway is
silenced.
8.5
Reactivation of the Hedgehog Pathway after Partial

Hepatectomy
Because many types of cells (not merely mature hepatocytes) must be replaced after
partial hepatectomy, and as mentioned previously, most of these are derivatives of
Hh-responsive progenitors, we examined the possibility that the Hh pathway might
become reactivated in the remnant liver following partial hepatectomy. Healthy adult
mice were subjected to partial hepatectomy and sacriced at various time points ranging from 6 hours to 10 days post-partial hepatectomy so that Hh pathway activity could
be examined in whole liver and primary liver cells that were harvested from regenerating liver remnants. Partial hepatectomy was followed by an immediate and sustained
reduction in hepatic expression of Hh interacting protein (HHIP), a soluble inhibitor of
Hh ligands, and sequential increases in mRNA and protein levels of the Hh ligands,
Indian hedgehog (Ihh) and Sonic hedgehog (Shh). Subsequently, expression of the Hhregulated transcription factors, Gli1 and Gli2, also increased, followed by signicant
up-regulation of well-established Gli-target genes, such as soluble frizzled related peptide (sFRP)-1. These ndings indicated that partial hepatectomy resulted in Hh pathway
activation in regenerating livers.
Immunohistochemistry and complementary cell isolation studies provided further
proof that the hepatocyte and bile duct populations became particularly enriched with
Hh-responsive cells (i.e., cells that expressed Hh-regulated transcription factors) around
the times when these cells were maximally proliferative following partial hepatectomy.
Moreover, mice treated with cyclopamine, a specic inhibitor of Hh signal transduction,
demonstrated signicant inhibition of normal post-partial hepatectomy increases in both
hepatocyte and cholangiocyte proliferation. Cyclopamine treatment also dramatically
reduced post-partial hepatectomy survival in mice during the initial 72 hours after partial
hepatectomy. When regenerating hepatocytes that had been harvested from mice 24 hours
116
after partial hepatectomy were treated in vitro with cyclopamine, Hh signaling and proliferation were also reduced, suggesting that Hh pathway activation contributed directly to
post-partial hepatectomy increases in hepatocyte proliferation. Given that healthy mature
hepatocytes are not Hh-responsive, this nding was somewhat surprising.
One potential explanation for this observation is that Hh pathway activation promoted
the outgrowth of hepatic progenitors that quickly differentiated and then proliferated.
This concept contradicts conventional wisdom that posits that liver regeneration following partial hepatectomy results from replication of mature hepatocytes in the liver remnant, but it is supported by several lines of evidence from recent studies by our group.
s #YCLOPAMINE TREATMENT ALSO INHIBITED CHOLANGIOCYTE PROLIFERATION FOLLOWING PARTIAL
hepatectomy, and both hepatocytes and cholangiocytes are the progeny of bipotent
hepatic progenitors.
s 0OPULATIONS OF REGENERATING HEPATOCYTES THAT WERE HARVESTED HOURS AFTER PARTIAL
hepatectomy were enriched with cells expressing progenitor markers.
s 0ARTIAL HEPATECTOMY RESULTED IN A STRIKING UP REGULATION OF PROGENITOR ASSOCIATED GENE
expression in whole liver tissue, leading to a 40-fold induction of Fn14 (a TNF superfamily receptor that is thought to specically mark bipotent hepatic progenitors)
and a more than 160-fold induction of alpha-fetoprotein (AFP), a well-established
marker of immature liver epithelial cells.
s )MMUNOHISTOCHEMISTRY FOR SEVERAL OTHER PROGENITOR MARKERS CONlRMED THAT PARTIAL
hepatectomy stimulated accumulation of liver progenitors in regenerating livers.
s #YCLOPAMINE TREATMENT SIGNIlCANTLY INHIBITED NORMAL POST PARTIAL HEPATECTOMY
induction of Fn14, AFP, and several other progenitor markers in mice.
s #YCLOPAMINE VIRTUALLY ELIMINATED PROGENITOR ACCUMULATION FOLLOWING PARTIAL HEPAtectomy, as assessed by immunohistochemistry. Similar approaches were used to
prove that Hh pathway activation plays a signicant role in stromal cell responses
that follow partial hepatectomy, including transient expansion of myobroblast
populations and hepatic matrix remodeling.
Together, these ndings support the concept that normal regenerative responses in
adult livers involve transient reactivation of mechanisms, such as Hh, that are likely to
regulate liver morphogenesis during fetal development.
8.6
Hedgehog Pathway Activation during Repair of Chronic

Liver Injury: General Concepts
Like regeneration after partial hepatectomy, adult hepatic damage evokes an intricate
wound-healing response aimed to reconstitute the normal structure and function of
chronically injured livers. As in many other tissues, the complex repair processes involve
the postnatal reactivation of mechanisms that regulate tissue construction during development. As described previously, expression of Hh ligands and Hh-target genes, such as
Gli1 or Gli2, is barely detectable in healthy adult livers (Sicklick et al., 2006b); however,
chronic liver injury increases mRNA and protein levels of Hh ligands and target genes.
The level of Hh pathway activation is typically proportional to the severity and duration
of liver injury in both rodents and humans (Fleig et al., 2007). This is explained, at least
8.7
Hedgehog Pathway Activation and Liver Progenitors in Chronic Injury Models
117
in part, by recent evidence that injured/dying mature hepatocytes produce Shh and
Ihh ligands ( Jung et al., 2010). However, because mature hepatocytes do not express
all of the proteins that are necessary to transduce Hh ligand-initiated signals, they are
not capable of responding to the Hh ligands that they produce (Sicklick et al., 2006b).
On the other hand, many neighboring cells are Hh-responsive, including resident hepatic
cell populations that are most engaged in liver remodeling (e.g., liver myobroblasts,
hepatic progenitors, hepatic stellate cells, immature cholangiocytes, endothelial cells,
and T lymphocytes) (Choi et al., 2009; Fleig et al., 2007; Jung et al., 2008; Jung et al.,
2007; Lowrey et al., 2002; Omenetti et al., 2008a; Omenetti et al., 2008b; Omenetti
et al., 2007; Sicklick et al., 2005; Sicklick et al., 2006b; Stewart et al., 2002; Witek
et al., 2009; Yang et al., 2008). In response to the increased Hh ligands generated by
neighboring injured hepatocytes, these cells activate Hh pathway signaling.
Many of these Hh-responsive cells can also produce Hh ligands in response to other
injury-associated factors. For example, platelet-derived growth factor (PDGF )-BB, transforming growth factor (TGF )-1, and epidermal growth factor (EGF ) have been shown to
stimulate immature ductular cells and hepatic stellate cells to up-regulate their synthesis
and release of Hh ligands ( Jung et al., 2008; Omenetti et al., 2008a; Omenetti et al.,
2008b; Omenetti et al., 2007; Witek et al., 2009; Yang et al., 2008). This then further
enriches the injured liver with Hh ligands and amplies the stimulus for Hh pathway activation. The proximity of various types of Hh-producing and Hh-responding cell types
in injured livers suggests that Hh pathway activation coordinates complex autocrine
and paracrine signaling loops that help to orchestrate remodeling and reconstruction of
the injured liver.
Because mature hepatocytes are not capable of responding to Hh ligands, enrichment of the microenvironment with these factors provides a selective survival advantage
for cell types that are Hh-responsive, leading to the outgrowth of these populations as
long as injury persists. When the insult abates and injury subsides, the Hh pathway turns
off (Omenetti et al., 2008a), and other factors promote the differentiation of the progeny
of Hh-responsive cells toward one cell population or another. Because PDGF-BB, EGF,
and IGF-1 activate AKT-dependent post-translational mechanisms that stabilize Gli transcription factors in Hh-responsive cells (Riobo et al., 2006) (in addition to stimulating
production of Hh ligands), Hh signaling may modulate the actions of multiple growth
factors, while the converse may be true as well. Thus, variations in tissue remodeling
during liver injury probably ultimately reect differences in: local cytokine/growth factor accumulation, the dose and duration of Hh ligand exposure, the balance between
Hh-responsive and unresponsive cell types, and the presence/absence of other factors
that regulate cell differentiation when these injury-related signals wane (fFigure 8.2).
8.7
Hedgehog Pathway Activation and Liver

Progenitors in Chronic Injury Models
Progenitor populations in many tissues are enriched with Hh-responsive cells, and
Hh ligands generally enhance the growth of such progenitors by inhibiting apoptosis
and/or enhancing proliferative activity ( Jung et al., 2007; Sicklick et al., 2006b; Yang
et al., 2008). We reported that human embryonic stem cells that had been specied to
undergo hepatic differentiation, as well as more-differentiated EpCam-expressing liver
118
healthy liver
canal of
Hering
Progenitors
hepatocytes
space of
Diss
bile canaliculus
cholangiocytes
bile
duct
HSC
NKT cells
space of
Diss
Figure 8.2A
sinusoidal endothelial cells
Differential Activity of the Hedghog Pathway in Healthy and Injured Livers
Notes:
Healthy liver. There is very little evidence of Hedgehog (Hh) pathway activity in healthy adult
liver, although this tissue harbors a number of different cell types that are capable of producing and/or responding to Hedgehog ligands. Two mechanisms seem to account for these low
basal levels of Hh activity: (1) relative lack of Hh ligand production, and (2) high expression
of the Hh ligand antagonist HHIP by sinusoidal lining cells, such as quiescent hepatic stellate
cells (HSC) and sinusoidal endothelial cells.
progenitors from human fetal livers, were Hh-responsive. Both cell types relied upon Hh
pathway activity to retain optimal viability (Sicklick et al., 2006b). Progenitor populations in adult rodent and human livers, including oval cells, immature ductular cells,
and small hepatocytic cells, are also Hh responsive. Cell culture studies demonstrate
that Hh ligands inhibit apoptosis and enhance proliferation in such cells (Omenetti
et al., 2007), which complement our recent studies that used cyclopamine to manipulate Hh signaling in post-partial hepatectomy liver remnants, conrming that Hh signaling plays a major role in promoting progenitor growth in intact liver tissue (Ochoa et al.,
2010). This concept is supported by other evidence in various rodent models of chronic
liver injury. For example, in rodents, the numbers of cells expressing various progenitor
markers increase in parallel with the level of Hh ligand production during liver injury
and regress in parallel with the disappearance of Hh ligands as injury abates (Fleig et al.,
2007). Furthermore, liver injuryrelated accumulation of Hh-responsive progenitor cells
is enhanced in transgenic mice with an impaired ability to silence Hh signaling (Omenetti et al., 2007; Syn et al., 2009). A similar relationship between the hepatic progenitor
content and level of Hh pathway activity has been documented in different human liver
diseases, including primary biliary cirrhosis ( Jung et al., 2007; Omenetti et al., 2008b),
alcoholic steatohepatitis ( Jung et al., 2008), nonalcoholic fatty liver disease (Syn et al.,
2009), chronic hepatitis B, and chronic hepatitis C (Pereira et al., 2010).
8.7
Hedgehog Pathway Activation and LiverProgenitors in Chronic Injury Models
injured liver
Hedgehog ligand
canal of
Hering
Progenitors
119
ductular
proliferation
hepatocytes
space of
Diss
myofibroblastic HSC
chemokines
neutrophils
space of
Diss
monocytes
T/B lymphocytes
Figure 8.2B
Differential Activity of the Hedghog Pathway in Healthy and Injured Livers
Notes:
Liver injury. Injury to liver epithelial cell unleashes a cascade that results in progressive activation of the Hh pathway and consequent expansion of cell populations that are involved in liver
inammation, regeneration, and brogenesis. (1) Injury stimulates mature hepatocytes and
cholangiocytes to produce and release Hh ligands into the bile, space of Diss, and liver sinusoids. (2) Hh ligands stimulate quiescent hepatic stellate cells to undergo transition to myobroblastic hepatic stellate cells, enhance myobroblastic HSC proliferation and survival, and
stimulate further production of Hh ligands by myobroblastic HSC. (3) Hh ligands generated
by injured liver epithelial cells and myobroblastic HSC enhance the proliferation and survival
of liver progenitors and cholangiocytes, both of which also produce Hh ligands. (4) Hh ligands
stimulate cholangiocytes to undergo EMT, thereby providing another source of myobroblasts
in injured livers. (5) Hh-activated cholangiocytes also produce various chemokines that recruit
different types of bone marrowderived cells and immune cells to liver, such as brocytes,
monocytes, neutrophils, T and B lymphocytes, and NKT cells. (6) Hh ligands are viability
factors for lymphocytes, including NKT cells. Thus, they promote the accumulation of NKT
cells within a microenvironment that is enriched with CD1d-expressing cells that function
as antigen-presenting cells for NKT cells. Thus, injury-related activation of the Hh pathway
contributes to many of the repair responses that typify chronic liver injury, including inammation, brogenesis, vascular remodeling, and accumulation of progenitor populations that
might provide the seeds for subsequent liver cancers.
120
8.8
Hedgehog Pathway Activation and Liver Fibrosis
Myobroblasts are the major source of brous matrix that accumulates during chronic
liver injuries that result in cirrhosis. Liver myobroblasts may be derived from several
sources, including circulating bone marrowderived monocytes/brocytes, epithelialto-mesenchymal transition (EMT) of certain types of liver epithelial cells, and myobroblastic transformation of resident hepatic stellate cells. The latter process is generally
believed to be the predominant source of myobroblasts in most types of adult liver
injury (Friedman, 2008).
In healthy adult livers, most hepatic stellate cells are quiescent and exhibit a fatstoring, non-myobroblastic phenotype; however, factors released during liver injury
stimulate quiescent hepatic stellate cells to undergo transition to myobroblastic hepatic
stellate cells. Other injury-related factors then promote hepatic accumulation of myobroblastic hepatic stellate cells by enhancing proliferation and/or inhibiting apoptosis of
myobroblastic hepatic stellate cells (Friedman, 2008). Quiescent hepatic stellate cells
produce large amounts of the Hh inhibitor HHIP, but quickly down-regulate HHIP when
exposed to conditions that promote their transition to myobroblastic hepatic stellate
cells (Choi et al., 2009). These situations also activate PI3K/AKT-dependent mechanisms
that induce hepatic stellate cells production of Hh ligands (Yang et al., 2008). Hepatic stellate cellderived Hh ligands, in turn, increase expression of Hh-target genes,
such as Gli2 (Choi et al., 2009). Hh neutralizing antibodies or specic pharmacologic
inhibitors of smoothened that abrogate down-stream Hh signaling drastically reduce
myobroblastic hepatic stellate cell viability and virtually eliminate the proliferative
effects of known myobroblastic hepatic stellate cell mitogens, such as PDGF-BB (Yang
et al., 2008). In addition, pharmacologic inhibition of Hh pathway with the smoothened
antagonist cyclopamine caused surviving myobroblastic hepatic stellate cells to revert
to a less myobroblastic and more quiescent phenotype (Choi et al., 2009; Choi et al.,
2010; Sicklick et al., 2005).
Hh pathway activation may also promote accumulation of myobroblasts that are
derived from non-conventional sources, such as bone marrow and EMT. Activation of
Hh signaling in immature ductular cells caused such cells to produce MCP-1, which is
a known chemokine for circulating monocytes and brocytes (Omenetti et al., 2009).
Moreover, Hh pathway activation increases local production of IL-13, and IL-13 has
been shown to promote the differentiation of monocytes into brocytes (Shao et al.,
2008). Finally, Hh signaling induces EMT in immature ductular cells (Omenetti et al.,
2008b). Thus, the aggregate ndings predict that injury-related activation of the Hh
pathway would play a major role in hepatic accumulation of myobroblasts.
The concept that Hh pathway activation promotes accumulation of myobroblasts during repair of chronic liver injury is supported by several lines of experimental evidence:
s -YOlBROBLAST NUMBERS AND MATRIX ACCUMULATION IN REGENERATING LIVERS AFTER PARtial hepatectomy parallel Hh pathway activation and are virtually abolished by
pharmacologic inhibition of Hh signaling (Ochoa et al., 2010).
s (EPATIC ACCUMULATION OF MYOlBROBLASTS AND COLLAGEN MATRIX INCREASE IN PARALLEL
with cholestatic liver injury following chronic bile duct ligation, and resolve in parallel with the progressive down-regulating of Hh signaling that occurs once biliary
obstruction is relieved (Omenetti et al., 2008a).
8.9
Hedgehog Pathway Activation and Vascular Remodeling in Injured Livers
121
s -YOlBROBLAST ACCUMULATION AND LIVER lBROGENIC ACTIVITY ARE ENHANCED IN TRANSGENIC

mice with an overly active Hh pathway following bile duct ligation (Omenetti et al.,
2008a) or exposure to hepatotoxic diets (Syn et al., 2009).
s -YOlBROBLAST ACCUMULATION AND THE SEVERITY OF LIVER lBROSIS PARALLEL THE LEVEL OF (H
pathway activity in patients with different types of liver disease, including chronic
viral hepatitis (Pereira et al., 2010) and nonalcoholic fatty liver disease (Syn et al.,
2009).
8.9
Hedgehog Pathway Activation and Vascular Remodeling in

Injured Livers
Advanced brotic injury (cirrhosis) is characterized by changes in hepatic sinusoidal

architecture together with extrahepatic vasculature rearrangement (Friedman, 2008).
Several types of cells that reside near sinusoidal endothelial cells are capable of generating Hh ligands, including injured hepatocytes, activated hepatic stellate cells, liver
progenitors, and certain types of resident lymphocytes. The Hh pathway is a key regulator of vascular remodeling during development (Vokes et al., 2004), while PDGF-BB
(which activates Hh signaling in adult liver cell populations) (Omenetti et al., 2008a;
Yang et al., 2008) has also been demonstrated to regulate hepatic vascular structure
and function (Semela et al., 2008). A potential role for Hh-mediated activation by other
angiogenic factors that could impact vascular remodeling has also been demonstrated
in other tissues (Lavine et al., 2006; Pola et al., 2001; Ueda et al., 2010; Yamazaki et al.,
2008). Hence, Hh signaling might regulate vascular remodeling during repair of acute
and chronic liver damage.
In support of this concept, biologically active Hh ligands were identied in exosomes that were released from myobroblasts and immature cholangiocytes after these
cells were exposed to PDGF-BB (Witek et al., 2009). Moreover, treatment of other cells
that contained Hh-reporter constructs with exosomes puried from myobroblast- or
cholangiocyte- conditioned medium activated Hh transcriptional activity, proving that
Hh-containing exosomes are capable of initiating Hh signaling in distant Hh-target cells
(Witek et al., 2009). Consistent with the in vitro data, BDL-induced brosis/cirrhosis
elicited the release of membrane-associated Hh ligands into both plasma and bile
(Witek et al., 2009). Even more interestingly, when exposed to either plasma- or bilederived exosome-enriched Hh-containing membrane particles, sinusoidal endothelial
cells were stimulated to undergo phenotypic changes that are known to occur during the capillarization process that accompanies cirrhosis-related vascular remodeling
(Witek et al., 2009). These ndings identify a potentially novel mechanism for vascular
remodeling during cirrhosis, namely, Hh-induced phenotypic changes in endothelial
cells.
8.10
Hedgehog Pathway Activation and Hepatocarcinogenesis
Hh pathway activation has been demonstrated in many types of cancer (Beachy et al.,
2004). In some tumors, this results from activating mutations in smoothened or Gli family members (Daya-Grosjean and Couve-Privat, 2005; Romer and Curran, 2005; Saldanha, 2001; Toftgard, 2000). However, in others enhanced Hh signaling is explained
122
by epigenetic events that silence HHIP or that increase production of Hh ligands (Freeman et al., 2009). Beachy et al. (2004) demonstrated excessive Hh signaling in cholangiocarcinomas. Our group was the rst to report increased Hh pathway activity in
human hepatocellular carcinomas (HCC) and certain human HCC-derived hepatoma
cell lines. Moreover, we showed that smoothened inhibitors signicantly reduced the
growth of Hh-responsive hepatoma cells (Sicklick et al., 2006a). These ndings were
quickly validated by several other research teams (Cheng et al., 2009; Fu et al., 2008;
Huang et al., 2006; Patil et al., 2006; Tada et al., 2008). Subsequent microarray analyses
of human HCC banks suggest that HHIP may be hypermethylated and silenced in as
many as two-thirds of human hepatocellular carcinomas (Wang et al., 2007; Villanueva
et al., 2007), identifying the Hh pathway as a potentially important therapeutic target in
hepatocarcinogenesis.
To date, however, little information has been published to clarify the cellular sources
and targets of Hh ligand in HCC. We are using immunohistochemistry to address this
issue. Preliminary results in an HCC that developed in a patient with HCV-related cirrhosis suggest that the malignant stroma is particularly enriched with cells that produce
Hh ligand, as well as Hh responsive cells. This nding is particularly intriguing given a
recent report that demonstrated that a pharmacologic Hh inhibitor eliminated most of
the tumor stroma and improved the outcomes in a mouse model of pancreatic cancer
(Olive et al., 2009).
Summary
Recent evidence suggests that regeneration and repair of adult liver, like many tissues, involves the coordinated response of a number of different cell types. In adult livers, broblastic
cells, ductular cells, inammatory cells, and progenitor cells contribute to this process. The
fates of such cells are dictated, at least in part, by fetal morphogenic pathways that were once
thought to be active mainly during embryogenesis, such as Hh. Emerging data from studies
of injured adult human and rodent livers demonstrate that injury-related activation of the Hh
pathway modulates several important aspects of regeneration and repair, including the growth
of hepatic progenitor populations, hepatic accumulation of myobroblasts, repair-related inammatory responses, vascular remodeling, liver brosis, and hepatocarcinogenesis. These
ndings identify the Hh pathway as a potentially important target for further study and potential therapeutic manipulation, while emphasizing the need to advance knowledge about how
this pathway is regulated by and interacts with other signals that regulate the regenerative and
reparative processes after acute and chronic liver injury.
Further Reading
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Duncan, A.W., Dorrell, C., and Grompe, M. (2009). Stem cells and liver regeneration. Gastroenterology 137, 46681.
S4553.
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from human pancreatic cancer cells augments angiogenic function of endothelial progenitor cells. Cancer Sci. 99, 11318.
Yang, L., Wang, Y., Mao, H., Fleig, S., Omenetti, A., Brown, K.D., Sicklick, J.K., Li, Y.X., and
Diehl, A.M. (2008). Sonic hedgehog is an autocrine viability factor for myobroblastic
hepatic stellate cells. J. Hepatol. 48, 98106.
EGFR, CD95, and the Switch between

Proliferation and Apoptosis in Hepatic
Stellate Cells
Roland Reinehr and Dieter Hussinger
Learning Targets
1. Hepatic stellate cells (HSC) represent a hepatic stem/progenitor cell compartment
and are fairly resistant toward apoptotic cell death.
2. In hepatocytes, CD95 (Fas, APO-1) is activated upon treatment with CD95 ligand
(CD95L) or pro-apoptotic bile acids in an epidermal growth factor receptor (EGFR)and c-Jun-N-terminal-kinase (JNK)-dependent manner.
3. CD95 tyrosine nitration prevents CD95 activation and subsequent apoptosis.
4. In quiescent HSC, CD95L leads to CD95 tyrosine nitration and fails to induce a
sustained JNK activation. Therefore, CD95 ligand-induced EGFR activation triggers
cell proliferation but no apoptotic cell death.
5. In quiescent HSC, pro-apoptotic bile acids also induce proliferation in an EGFRdependent fashion. However, EGFR activation can be coupled to CD95-mediated
apoptosis if a sustained JNK signal is introduced by a second stimulus. Thus, JNKs
provide a switch between proliferation and cell death under these conditions.
9.1
Introduction
Recent evidence suggests that signaling pathways toward cell proliferation and cell
death are much more interconnected than previously thought. Whereas death receptors, for example, CD95 (Fas, APO-1), can couple to both cell death and proliferation (Budd, 2002; Reinehr et al., 2008b), also growth factor receptors, for example,
epidermal growth factor receptor (EGFR), are involved in both cell proliferation and
cell death (for review see Reinehr and Hussinger, 2009 and Michalopoulos, 2010).
This chapter summarizes different ways of EGFR- and CD95-dependent signaling in
the liver. Here, depending on the hepatic cell type and the respective signaling context
EGFR-/ CD95-mediated signaling ends up in either liver cell proliferation or apoptotic
cell death. First, EGFR is discussed as a growth factor receptor involved in liver cell
proliferation during liver regeneration. Then, the role of EGFR in activating CD95 death
receptor in hepatocytes is described. In hepatocytes, the complex and c-Jun-N-terminalkinase (JNK)-dependent interplay between EGFR and CD95 leading to EGFR-mediated
CD95 tyrosine phosphorylation and subsequent apoptosis has been studied in detail
130
EGFR, CD95, and the Switch between Proliferation and Apoptosis in HSCs
(for review see Reinehr and Hussinger, 2007a, 2009). In contrast to hepatocytes, in
hepatic stellate cells (HSC) pro-apoptotic stimuli like CD95 ligand (CD95L) or hydrophobic bile acids fail to induce a sustained JNK-activation and apoptotic HSC death
(Reinehr et al., 2008a; Sommerfeld et al., 2009). Thus EGFR activation by pro-apoptotic
stimuli, that is, CD95L or bile acids, leads to HSC proliferation. However, when JNKs
are activated by a second stimulus, EGFR couples to CD95 activation and subsequent
apoptosis (Sommerfeld et al., 2009) unless apoptosis resistance was generated by CD95
tyrosine nitration, which occurs regularly upon CD95L treatment in quiescent HSC
(Reinehr et al., 2008a).
9.2
Liver Cell Proliferation Involves Ligand-dependent

EGFR Activation
Growth factors, such as the epidermal growth factor (EGF), the transforming growth
factor (TGF), and the hepatocyte growth factor (HGF), are known to stimulate DNA
synthesis, cell cycle progression, and proliferation in hepatocytes in vivo and in
culture and are thought to have major effects on liver growth (for review see Fausto
et al., 1995, 2006; Michalopoulos and DeFrances, 1997; Michalopoulos, 2007). EGF
and TGF share 35% homology and both can bind to the EGFR, whereas the HGF,
whose -chain shares 40% homology with plasminogen, binds to the HGF receptor (c-Met). EGF functions as an endocrine factor, is produced by a variety of cells,
and is essential for liver regeneration (Fausto et al., 1995, 2006; Michalopoulos and
DeFrances, 1997; Michalopoulos, 2007; Natarajan et al., 2007). In contrast, TGF
exerts its effect on hepatocytes mainly via an autocrine loop. Interestingly, this autocrine loop is stimulated by EGF or TGF itself indicating an (auto-)amplication
mechanism (Webber et al., 1993). On the other hand, HGF is not produced by hepatocytes, but by liver endothelial cells, hepatic stellate cells (HSC), and Kupffer cells
(Maher 1993).
Most of our knowledge about the action of growth factors in liver is based on studies after partial hepatectomy (PH). These revealed that after PH the following occurs:
(a) immediate-early gene activation is triggered by a rise in levels of circulating growth
factors, and (b) hepatocytes have to enter a state of replicative competence or priming
before growth factors can exert their full efcacy (Fausto et al., 1995, 2006; Michalopoulos and DeFrances, 1997, Michalopoulos, 2007). This priming step involves the
activation and DNA binding of nuclear factor (NF)-b and other transcription factors,
which are induced by tumor necrosis factor (TNF), and other cytokines such as interleukin (IL) 6 (FitzGerald et al., 1995; Fausto et al., 1995, 2006; Iwai et al., 2001,
Michalopoulos, 2007). Apart from cell proliferation, these growth factors, that is, EGF,
TGF, or HGF, respectively, also exert effects on cell morphogenesis, angiogenesis,
cell motility, differentiation, and cell survival (for review see Fausto et al., 1995, 2006;
Michalopoulos and DeFrances, 1997, Michalopoulos, 2007).
Plasma EGF concentrations rise only very slightly (less then 30%) after PH (Jones
et al., 1995; Michalopoulos and DeFrances, 1997, Michalopoulos, 2007). Because
EGF is already present in the blood under control conditions, it has been discussed that
EGF might act as a hepatocyte mitogen only in the regenerating and therefore primed
liver and that a change in the EGFR during this priming process is required to permit
9.2
Liver Cell Proliferation Involves Ligand-dependent EGFR Activation
131
ligand binding and effective activation of signal transduction under these conditions
(Fausto et al., 1995, 2006; Jones et al., 1995, Michalopoulos and DeFrances, 1997,
Michalopoulos, 2007; Mullhaupt et al., 1994). Webber et al. (1994) demonstrated
in an in vivo-model with continuous growth factor infusion into rat mesenteric veins
that neither EGF, TGF, nor HGF had detectable effects on DNA synthesis. When,
however, a so-called minimal hepatectomy (i.e., 30% liver resection) preceded the
growth factor infusion, a signicant increase in DNA synthesis occurred, suggestive for
a priming process under these conditions (Webber et al., 1994). This priming process
was attributed to an increase in c-myc expression and transcription factor binding
involving NF-b, activator protein (AP)1 and signal transducer, and activator of transcription (STAT)3 (Cressman et al., 1995; Webber et al., 1994; Westwick et al., 1995).
On the other hand, Skarpen and coworkers (2005) demonstrated that a 70% PH is
followed by increased EGFR ubiquitination, EGFR internalization, down-regulation
of the receptor protein, and a down-regulation in EGF-mediated EGFR (auto)phosphorylation, whereas an increased mitogenic signaling via the Ras-Raf-Erk signaling
cascade occurred. Therefore, the authors concluded that hepatocyte priming following
PH involves modulation of EGFR itself, which enhances its ability to mediate growth
factor responses without an increase of its receptor tyrosine kinase-activity (Skarpen
et al., 2005). Further studies are required to elucidate the suggested changes in receptor susceptibility and/or signal transduction in the intact liver and after PH at the
molecular level.
Recently, evidence has been presented that EGFR-dependent signaling is important not
only for cell growth and development but also for tumorigenesis (for review see Sibilia
et al., 2007). Jo et al. (2000) demonstrated in the human hepatoma cell lines HepG2,
AKN-1, and Huh6 as well as in the human epidermoid carcinoma cell line A431, that
c-Met (HGF-receptor) is constitutively activated due to an autocrine TGF expression
and subsequent TGF-induced EGFR activation and EGFR/c-Met heterodimerization as
shown by TGF- and EGFR-neutralizing antibodies as well as EGFR and c-Met immunoprecipitation studies, respectively. Therefore, cross-talk between the TGF/EGFR and
HGF/c-Met pathways might induce mitogenic and/or motogenic signal amplication
in many tumor cell lines and might explain how overexpression of TGF in those cells
alters cell growth and contributes to tumor cell transformation and tumorigenesis. Also,
the complexity of EGFR signaling after PH is most likely attributable to the number and
type of EGFR ligands involved, their specicity for different receptor heterodimers, and
the nuances of subsequent activation of intracellular signaling cascades (Carver et al.,
2002; Fausto et al., 2006).
Apart from ligand-dependent EGFR activation, ligand-independent EGFR activation
in the liver has been described, also. In hepatocytes, hydrophobic bile acids induce
a reactive oxygen species (ROS)-dependent EGFR tyrosine phosphorylation and activation of downstream kinases, that is, mitogen-activated protein (MAP) kinases (Qiao
et al., 2001; Reinehr et al., 2003a; Schliess et al., 1997). Furthermore, bile acids were
shown to induce extracellular signal regulated kinase (Erk)-dependent HSC proliferation
via ROS-dependent activation of the EGFR (Svegliati-Baroni et al., 2005). However, in
human cholangiocyte cell lines hydrophobic bile acids can induce a c-Src-dependent
TGF shedding resulting in a ligand-dependent EGFR activation and EGFR tyrosine
kinase activity-dependent cell proliferation (Werneburg et al., 2003).
132
9.3
Liver Cell Apoptosis Involves EGFR-dependent

CD95 Activation
Apart from hepatocytes, also hepatic stellate cells (HSC), cholangiocytes, sinusoidal
endothelial cells, and Kupffer cells express CD95 (Fas/APO-1) (Cardier et al., 1999;
Mschen et al., 1998; Ueno et al., 2000), a member of the tumor necrosis factor (TNF)death receptor family. CD95 has rst been described by the groups of Yonehara and
Krammer reporting on extensive apoptotic cell death induction upon treating cells
with CD95-specic monoclonal antibodies (Yonehara et al., 1989; Trauth et al., 1989).
CD95 is not only activated upon binding of membrane-bound or soluble CD95 ligand
(CD95L), mainly expressed on cytotoxic T lymphocytes and natural killer (NK) cells (reviewed by Berke, 1995), but also by hydrophobic bile acids (Qiao et al., 2001; Reinehr
et al., 2003a; Sodeman et al., 2002) and hyperosmolarity (Reinehr et al., 2002). CD95
plays an important role in liver physiology and pathophysiology (reviewed in Mahli and
Gores, 2008). Mice genetically decient for CD95 exhibit liver hyperplasia (Adachi
et al., 1996) and hepatic neoplasms (Park et al., 2008). CD95-induced cell death was
reported to lead to a removal of virus-infected or malignant transformed hepatocytes
by NK cells, NK T cells, and T lymphocytes (Lapinski et al., 2004; Mochizuki et al.,
1996; Song et al., 2004). The exceptional sensitivity of the liver to CD95 was shown
by the induction of fulminant hepatic failure on injection of a CD95 agonistic antibody
in mice (Ogasawara et al., 1993). In patients with acute liver failure, such as druginduced liver injury, signicant elevations of soluble CD95L occur (Rutherford et al.,
2007; Ryo et al., 2000). Both CD95 receptor and CD95L expression is up-regulated in
many chronic liver diseases such as alcoholic steatohepatitis (ASH; Taieb et al., 1998)
and non-alcoholic steatohepatitis (NASH; Feldstein et al., 2003) or chronical hepatitis
B- and C- virus infection (Kiyici et al., 2003; Lapinski et al., 2004).
Different ways of ligand-independent activation of the EGFR have been described in
hepatocytes during the last years, that is, EGFR transactivation by either CD95 ligand
(CD95L) (Reinehr et al., 2003b), hydrophobic bile acids (deoxycholate, glycochenodeoxycholate, taurolithocholate-3-sulfate, and taurochenodeoxycholate) (Qiao et al.,
2001; Reinehr et al., 2003a), or hyperosmolarity (Reinehr et al., 2003b). CD95L induces a caspase 8-dependent increase of the intracellular chloride concentration [Cl]i
(Reinehr et al., 2008b), whereas hydrophobic bile acids induce an increase in [Cl]i in a
caspase 8-independent way. This increase in [Cl[]i is thought to enhance vacuolar-type
H-ATPase-dependent acidication of an early endosomal compartment (Faundez and
Hartzell, 2004; Moriyama and Nelson, 1987; Pazoles et al., 1980). Upon acidication
of these acidic sphingomyelinase (ASM)-containing endosomes, ASM gets activated followed by an increased ceramide formation (Becker et al., 2007; Reinehr et al., 2005a,
2005b, 2006). Ceramide activates protein kinase C(PKC) followed by a serine phosphorylation of p47phox, a regulatory subunit of the NADPH oxidase (NOX), which results
in a NOX-driven generation of reactive oxygen species (ROS) (Reinehr et al., 2005a,
2005b, 2006). This ROS formation has two functional consequences: (i) inhibition of
phosphatase activities triggering an activation of the Src family-kinase Yes (Reinehr
et al., 2004a, 2004b), and (ii) activation of the c-Jun-N-terminal kinase (JNK) (Reinehr
et al., 2003a, 2003b). Activated Yes kinase associates with the EGFR resulting in a Yesmediated EGFR transactivation by tyrosine phosphorylation at EGFR-Y845 within the
EGFR-tyrosine kinase domain followed by an AG1478-sensitive autophosphorylation
9.3
Liver Cell Apoptosis Involves EGFR-dependent CD95 Activation
133
of its C-terminal residue EGFR-Y1173 (Reinehr et al., 2004a, 2004b). However, CD95L,
hydrophobic bile acids, or hyperosmolarity fail to induce phosphorylation at EGFR-Y1045
(Reinehr et al., 2004a, 2004b), a tyrosine residue that represents a cbl-binding site (Ravid
et al., 2002) associated with receptor internalization and subsequent phosphatasedependent receptor deactivation at the endoplasmic reticulum (ER) (Haj et al., 2002).
The activated EGFR then associates with the death receptor CD95 in a JNK-dependent
way (Eberle et al., 2005; Reinehr et al., 2003a, 2003b). Indeed, JNK couples EGFR
activation toward apoptotic cell death. CD95 is a substrate for the EGFR tyrosine kinase
activity and EGFR-dependent phosphorylation of the CD95 tyrosine residues within
the death domain, that is, CD95-Y232 and CD95-Y291, was identied as a crucial step
for both intracellular CD95 receptor oligomerization and translocation of the CD95/
EGFR protein complex to the plasma membrane (Eberle et al., 2005, 2007). After CD95
translocation to the plasma membrane, the death-inducing signaling complex (DISC) is
formed, that is, association of Fas-associated death domain (FADD) and caspase 8 to the
death receptor, followed by caspase 8-dependent mitochondrial amplication of proapoptotic signaling, which nally leads to apoptotic cell death (Reinehr et al., 2003a,
2003b). fFigure 9.1 summarizes the signaling events leading to CD95 activation in
hepatocytes.
The CD95/EGFR heteromer was shown to be fairly stable in CD95-YFP/EGFR-CFP
cotransfected Huh7 and MEF cells, and the CD95/EGFR protein complex was detectable
even in apoptotic blebs (Eberle et al., 2005). In contrast to bile acidinduced hepatocyte
apoptosis, recent evidence suggests that nuclear receptor-dependent bile acid signaling
is required for normal liver regeneration. In a model of PH it has been demonstrated that
elevated bile acid levels accelerate liver regeneration via the nuclear bile acid receptor
farnesoid-X-receptor (FXR) (Huang et al., 2006). It is proposed that FXR activation by
increased bile acid levels may signal loss of functional capacity of the liver (Huang
et al., 2006), which thereby could counteract the pro-apoptotic effect on hepatocytes.
However, the nature of the bile acid is also decisive: hydrophobic bile acids induce hepatocyte apoptosis, whereas hydrophilic ones, such as tauroursodeoxycholate (TUDC),
exhibit even anti-apoptotic properties (Mahli and Gores, 2008).
Apart from many sites of pharmacological inhibition of EGFR-dependent CD95 tyrosine phosphorylation and thus inhibition of apoptosis induction, also posttranslational
modications of the CD95 receptor associated with apoptosis resistance have been described: that is, CD95 tyrosine nitration (Reinehr et al., 2004c; Shrivastava et al., 2004)
and CD95 serine/threonine phosphorylation (Reinehr and Hussinger, 2004). Addition
of peroxynitrite (ONOO-) in vitro as well as lipopolysaccharide in vivo induce CD95
tyrosine nitration (Reinehr et al., 2004c).
CD95 tyrosine nitration does not affect CD95/EGFR association but prevents CD95
tyrosine phosphorylation, because CD95 tyrosine nitration and phosphorylation are
mutually exclusive (Reinehr et al., 2004a). Therefore, CD95 tyrosine nitration leads
to resistance toward CD95-dependent and EGFR-mediated apoptosis. Interestingly,
Saito and coworkers (2010) recently showed, that acetaminophen (APAP) intoxicationinduced hepatic peroxynitrite formation is sensitive to JNK inhibition. In addition, JNK
inhibition attenuated APAP-induced liver injury (Saito et al., 2010). It is an interesting
speculation, whether the observed preponderance of hepatocyte necrosis versus apoptosis (Saito et al., 2010) is due to APAP-induced CD95 tyrosine nitration and subsequent
apoptosis resistance.
134
CD95 ligand, pro-apoptotic bile acids,
hyperosmotic cell shrinkage
cell membrane
EGFR
FADD
P
Casp8
P
Cl
vH+-ATPase
Bafilomycin
endosomes
AY9944
colchicine
EGFR
P
pH
PPP
CD95-oligomerization
ASM
ceramide
PKCzinhibitor
PKCz
knockdown
p47phox
Apocynin
microtubules
NADPHoxidase
L-JNKI
EGFR
P
CD95
DIDS
DISC
formation
EGFR
P
ROS
P
AG1478
JNK
YES
knockdown
SU6656
knockdown
NAC
Figure 9.1 Signaling events and respective inhibitors (red) involved in CD95 ligand-,
pro-apoptotic bile acid- and hyperosmotic cell shrinkage-induced CD95 activation in
hepatocytes.
In hepatocytes, cyclic AMP (cAMP) does not interfere with the oxidative stress response, JNK activation, and the EGFR/CD95 association induced by pro-apoptotic bile
acids. However, cAMP prevents EGFR activation by inhibiting Yes/EGFR association
and subsequent Yes-mediated EGFR transactivation (Reinehr and Hussinger, 2004).
Therefore, cAMP also inhibits CD95 tyrosine phosphorylation and subsequent apoptosis (Reinehr and Hussinger, 2004). Furthermore, in both cultured rat hepatocytes and
perfused liver, cAMP triggers CD95 serine/threonine phosphorylation via activation of
protein kinase A (PKA). This CD95 serine/threonine phosphorylation acts as a signal for
CD95 internalization (Reinehr and Hussinger, 2004). Interestingly, prolonged exposure
of hepatocytes to pro-apoptotic bile salts also activates PKA as a late response, which
is then followed by CD95-serine/threonine phosphorylation and CD95 internalization
(Reinehr and Hussinger, 2004). This may reect a late compensatory anti-apoptotic
counter-regulation of the hepatocyte with protective potential in prolonged cholestasis.
fFigure 9.2 depicts CD95 serine, threonine, and tyrosine residues, which are targets for
EGFR- (tyrosine) or PKA- (serine/threonine) mediated phosphorylation.
JNK has not only been reported to play a critical role in CD95L- or bile acid-induced
apoptosis, respectively, but also in free fatty acid (FFA)-induced hepatic lipoapoptosis
EGFR Activation Can Couple to Both Proliferation and Apoptosis in HSCs
135
N-terminal
Figure 9.2 Schematic Illustration of the CD95

Death Receptor
Tyr 91
cell membrane
Ser 209, 212
Thr 214, 219
death domain
Tyr 232
Ser 322
YFP
C-terminal
Tyr 291
Notes:
Tyrosine residues CD95-Y232 and CD95-Y291
are critical for CD95(-YFP) oligomerization,
membrane translocation, DISC formation, and
apoptosis (Eberle et al., 2005 and 2007). CD95
serine/threonine phosphorylation provides a
signal for CD95 internalization (Reinehr and
Hussinger, 2004). In order to view CD95-YFP
membrane translocation in living cells, please
see www.jbc.org/content/283/4/2211/suppl/
DC1 mmovie 1 (Reinehr et al., 2008b).
(Cazanave et al., 2009). Here, the saturated FFA palmitate induces a JNK1-dependent
increase of the p53 up-regulated modulator of apoptosis (PUMA) protein expression.
PUMA protein knockdown resulted in an attenuated Bax activation, caspase 3/7 activities, and cell death upon palmitate exposure in murine hepatocytes (Cazanave et al.,
2009). Thus, apart from coupling EGFR activation toward CD95 phosphorylation, JNKs
also fulll a pro-apoptotic function in FFA-induced lipoapoptosis.
9.4
EGFR Activation Can Couple to Both Proliferation and

Apoptosis in Hepatic Stellate Cells
9.4.1 Quiescent Hepatic Stellate Cells: CD95 Ligand-induced EGFR

Activation, Proliferation, and Apoptosis Resistance
Quiescent HSC (qHSC), that is, cells that have been in culture for 2448 hours only and
do not express detectable amounts of -smooth muscle actin, represent a hepatic stem/
progenitor cell compartment (Kordes et al., 2007; Sawitza et al., 2009) and are fairly
resistant towards apoptotic cell death (Cariers et al., 2002; Reinehr et al., 2008a). In
qHSC, CD95L also induces EGFR tyrosine phosphorylation and activation of the receptor tyrosine kinase activity (Reinehr et al., 2008a), but in contrast to liver parenchymal
cells, in qHSC CD95L induces an EGF-dependent EGFR activation, which involves
phosphorylation of EGFR-Y845, -Y1045, and -Y1173 (Reinehr et al., 2008a). This is due to a
c-Src-dependent activation of MMP9 leading to EGF shedding and subsequent liganddependent EGFR activation (Reinehr et al., 2008a). In a model of PH, pro-MMP2 and
pro-MMP9 were elevated at 30 min and activated at 6 to 12 h and 3 to 6 h, respectively,
after partial hepatectomy but not after sham operation suggesting an involvement of
MMP2 and MMP9 in early liver regeneration (Fausto et al. 2006; Kim et al., 2000).
Therefore, it is an interesting speculation whether an up-regulation of MMP9 during
early phase of liver regeneration might lead to an increased EGFR-mediated proliferation in quiescent HSC because those cells represent a hepatic stem/progenitor cell
compartment (Kordes et al., 2007; Sawitza et al., 2009).
136
CD95L-induced EGFR activation couples to an Erk-mediated HSC proliferation,

as reected by an increase in bromodeoxyuridine (BrdU)-incorporation and in cell
number. In addition to CD95L-induced EGFR activation and subsequent proliferation,
CD95L also triggers CD95 tyrosine nitration in qHSC (Reinehr et al., 2008a), which is
associated with apoptosis resistance (Reinehr et al., 2004c). Apoptosis resistance is a
common feature of stem/progenitor cells. Therefore, ndings on CD95L-induced CD95
tyrosine nitration again relate to the fact that quiescent HSC represent a hepatic stem/
progenitor cell compartment (Kordes et al., 2007; Sawitza et al., 2009), which requires
protection from apoptosis in a hostile cytokine milieu during liver injury. In contrast to
liver parenchymal cells, CD95L failed to induce a sustained JNK-activation in qHSC
(Cariers et al., 2002; Reinehr et al., 2008a), which is a prerequisite for CD95/EGFR
association (Eberle et al., 2005; Reinehr et al., 2003a, 2003b).
9.4.2 Quiescent Hepatic Stellate Cells: Bile Acid-induced EGFR Activation

Can Couple to Both Proliferation and Apoptosis
Similar to ndings in liver parenchymal cells (Reinehr et al., 2004b), hydrophobic bile
acids were shown to induce an antioxidant-sensitive EGFR tyrosine phosphorylation
in passaged HSC (Svegliati-Baroni et al., 2005). In contrast to hepatocytes in quiescent hepatic stellate cells (qHSC), bile acidinduced EGFR activation is coupled to
cell proliferation (Sommerfeld et al., 2009). The underlying mechanisms were recently
studied in primary qHSC cultures and involve an ASM-dependent and NADPH oxidase (NOX)-driven ROS-formation, which nally leads to a Yes-mediated EGFR transactivation (Sommerfeld et al., 2009). However, as reported for CD95L (Reinehr et al.,
2008a), also hydrophobic bile acids fail to induce a sustained JNK-activation in qHSC
and therefore CD95/EGFR association does not occur upon bile acid administration
in those cells. This might also contribute to the recently published observation that
increased bile acid levels accelerate liver regeneration after PH (Huang et al., 2006).
If, however, CHX or hydrogen peroxide (H2O2), well-known inducers of a sustained
JNK signal, were coadministered with hydrophobic bile acids, CD95/EGFR association, EGFR-dependent CD95 tyrosine phosphorylation, membrane translocation, DISC
formation, and apoptotic cell death occurred (Sommerfeld et al., 2009). In contrast
to CD95L, no CD95 tyrosine nitration became detectable upon bile acid exposure in
qHSC. Therefore, depending on the signaling context, that is, presence of a transient or
a sustained JNK signal, respectively, EGFR activation by hydrophobic bile acids in HSC
can couple to both cell proliferation and apoptosis. Therefore, JNKs provide the switch
between EGFR-triggered proliferation and apoptosis.
9.4.3 Activated Hepatic STELLATE Cells: CD95 Ligand- and Bile

Acidinduced Signaling
In activated HSC (aHSC), that is, cells cultured for 714 days and high expression of
-smooth muscle actin reecting a myobroblast phenotype, CD95L-induced CD95
tyrosine nitration no longer occurs, whereas CD95L-induced EGFR activation is mediated via the c-Src/MMP9/EGF/EGFR cascade, similar to the situation found in qHSC
(Reinehr et al., 2008b). Therefore, upon coadministration of CD95L and cycloheximide
(CHX), a stimulus leading to a sustained JNK activation (Reinehr et al., 2008a), CD95/
EGFR association, subsequent EGFR-dependent CD95 tyrosine phosphorylation,
EGFR Activation Can Couple to Both Proliferation and Apoptosis in HSCs

quiescent HSC
quiescent HSC
CD95L
bile acid
quiescent HSC
bile acid + CHX
c-Src
NOX
NOX
MMP9
ROS
ROS
EGF
YES
YES
EGFR
EGFR
EGFR
Erk-1/-2
Erk-1/-2
CD95-Tyr-NO2
proliferation
apoptosis
resistance
CD95L
+ CHX
EGFR /CD95
DISC formation
apoptosis
activated HSC
bile acid + CHX
hepatocytes
CD95L or bile acid
c-Src
NOX
NOX
MMP9
ROS
ROS
EGF
JNK-1/-2
EGFR
JNK-1/-2
CD95-Tyr-P
proliferation
activated HSC
137
YES
JNK-1/-2
EGFR
YES
JNK-1/-2
EGFR
EGFR /CD95
EGFR /CD95
EGFR /CD95
CD95-Tyr-P
CD95-Tyr-P
CD95-Tyr-P
DISC formation
DISC formation
DISC formation
apoptosis
apoptosis
apoptosis
Figure 9.3 CD95 ligand- and pro-apoptotic bile acid-induced signaling pathways in hepatocytes, quiescent and activated hepatic stellate cells.
membrane translocation, and DISC formation occur and nally end up in CD95mediated aHSC apoptosis (Reinehr et al., 2008a). Thus, depending on the HSC activation state (quiescent vs. activated) and the underlying signaling context (transient vs.
sustained JNK activation) CD95L-mediated EGFR activation can couple to both HSC
proliferation and apoptotic cell death.
Recently it has been reported that in two different in vivo models, that is, bile duct
ligation (BDL) and CCl4 administration, respectively, hepatic brosis induction was
sensitive to pan-JNK inhibition and was ameliorated in JNK1-decient mice (Kluwe
et al., 2010). Furthermore, choline-decient L-amino acid-dened (CDAA) diet-induced
138
hepatic inammation and brosis was attenuated in jnk1 mice despite a similar degree of liver steatosis (Kodama et al., 2009). Thus JNK may not only play in liver cell
apoptosis but also in brogenesis. Further studies are necessary to settle this issue.
In aHSC, bile acid-induced EGFR activation is also coupled to proliferation (SvegliatiBaroni et al., 2005) and follows the same mechanisms as described in liver parenchymal cells (Reinehr et al., 2004b) and qHSC (Sommerfeld et al., 2009). The underlying
mechanisms involve an ASM-dependent and NADPH oxidase (NOX)-driven ROS-formation, which nally leads to a Yes-mediated EGFR transactivation (Sommerfeld et al.,
2009). However, hydrophobic bile acids fail to induce a sustained JNK-activation in
aHSC, and therefore, CD95/EGFR association does not occur upon bile acid administration in those cells. When, however, inducers of a sustained JNK signal come into play,
CD95/EGFR association, EGFR-dependent CD95 tyrosine phosphorylation, membrane
translocation, DISC formation, and apoptotic cell death together induced by hydrophobic bile acids occurred (Sommerfeld et al., 2009). Thus, as described in qHSC, also in
aHSC JNK provides the switch between EGFR-triggered proliferation and apoptosis.
fFigure 9.3 summarizes the different signaling pathways in hepatocytes, quiescent and
activated hepatic stellate cells upon addition of either CD95 ligand (CD95L) or hydrophobic bile acids, such as taurolithocholic-3-sulfate (TLCS) or glycochenodeoxycholate
(GCDC).
Summary
s 0ROLIFERATIVE STIMULI SUCH AS %'& AS WELL AS PRO APOPTOTIC ONES SUCH AS #$, HYdrophobic bile acids, or hyperosmolarity, induce EGFR activation in different liver cell
types. Whereas ligand-dependent EGFR activation leads to phosphorylation of EGFRY845, -Y1045, and -Y1173, ligand-independent but Src family-kinase Yes-mediated EGFR
transactivation involves EGFR-Y845- and -Y1173-phosphorylation only.
s 5PON ADDITION OF A PRO APOPTOTIC STIMULUS AND DEPENDING ON A SUSTAINED *.+ ACTIVATION
EGFR associates with the death receptor CD95 followed by an EGFR tyrosine kinasemediated CD95 tyrosine phosphorylation at position CD95-Y232 and -Y291, which has
been shown to be a prerequisite for CD95 oligomerization, membrane translocation,
DISC formation, and execution of apoptotic cell death.
s #$ TYROSINE NITRATION WHICH OCCURS DURING A ,03 INDUCED INmAMMATORY RESPONSE IN
hepatocytes and regularly occurs in quiescent HSC upon CD95L addition, is associated
with apoptosis resistance and couples CD95L-triggered EGFR activation to cell proliferation in quiescent HSC, which may be important during liver regeneration because HSC
were recently identied as a liver stem/progenitor cell compartment.
s )N ACTIVATED (3# #$, INDUCED AND 3RC--0%'& MEDIATED %'&2 ACTIVATION AS
well as bile acid-induced ASM/NOX/Yes-mediated EGFR transactivation in both quiescent and activated HSC can be coupled to CD95-mediated apoptotic cell death, including EGFR/CD95 association, subsequent CD95 tyrosine phosphorylation, membrane
translocation, and DISC formation if a sustained JNK activation is introduced by use
of an additional stimulus. Therefore, JNK provides a switch between proliferation and
apoptosis in hepatic stellate cells.
References
139
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98111.
Malhi, H., and Gores, G.J. (2008). Cellular and molecular mechanism of liver injury. Gastroenterology 134, 164154.
Michalopoulus, G.K. (2010). Liver regeneration after partial hepatectomy. Am. J. Pathol. 176,
213.
Michalopoulus, G.K., and DeFrances, M.C. (1997). Liver regeneration. Science 276, 606.
Peter, M.E., and Krammer, P.H. (2003). The CD95(APO-1/Fas) DISC and beyond. Cell Death
Differ. 10, 2635.
Reinehr, R., and Hussinger, D. (2007). Hyperosmotic activation of the CD95 system. Methods Enzymol. 428, 14560.
Wajant, H., Pzenmaier, K., and Scheurich P. (2003). Non-apoptotic Fas signaling. Cytokine
Growth Factor Rev. 14, 5366.
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10 Angiogenesis and Liver Regeneration

Tobias Buschmann, Jan Eglinger, and Eckhard Lammert
Learning Targets
1. Blood ow in liver sinusoids is particularly slow.
2. Liver sinusoidal endothelial cells contain large fenestrae and have little basement
membrane.
3. Angiogenesis contributes to liver regeneration.
4. Angiogenesis involves sprouting and intussusception.
5. During angiogenesis, the endothelial cells lose their fenestrae.
6. Hepatic stellate cells play an important role during angiogenesis in the regenerating
liver.
7. During chronic liver diseases, a pathogenic rather than regenerative angiogenesis
takes place.
10.1
Introduction
Angiogenesis is the process of blood vessel growth out of pre-existing vessels, contrasting vasculogenesis, the de novo formation of blood vessels from endothelial progenitor
cells. A physiological role of vascularization is to supply tissues with blood and its
nutrients and gases. In addition, angiogenesis also contributes to wound healing and regeneration. There are two main kinds of angiogenesis: sprouting angiogenesis, in which
a new vessel, guided by a tip cell and elongated by stalk cell proliferation, branches out
of an existing vessel toward an angiogenic stimulus (Gerhardt, 2008); and intussusceptive angiogenesis, in which transcapillary pillows are formed, which split a vessel in two
(Djonov et al., 2003). In this chapter, we rst want to describe the hepatic blood ow,
from large blood vessels to microvasculature, and vice versa. We will then introduce the
cell types contributing to the vasculature of the liver. Finally, we will discuss the role of
angiogenesis in liver regeneration and liver diseases.
10.2
Blood Flow and Cell Types in the Adult Liver
The liver receives a dual blood supply (fFigure 10.1). On the one hand, the hepatic
portal vein provides the liver with nutrient-rich, but oxygen-decient (venous) blood
from the abdominal organs, i.e. stomach, small intestine, colon, pancreas, and spleen.
On the other hand, the hepatic artery provides the liver with oxygenated blood without
large amounts of nutrients. Together, these two blood vessels carry about 25% of the
146

heart
hepatic lobule
hepatic vein
liver lobe
Figure 10.1 Overview of the Blood Flow

in the Liver
Note:
The blood supplying vessels are the portal
vein and hepatic artery; the blood draining
vessel is the hepatic vein.
gall
bladder
bile duct
hepatic portal vein
hepatic artery
cardiac output to the liver, with the portal vein contributing two-thirds and the hepatic
artery contributing one-third of the blood ow (Fernandez et al., 2009). The blood vessels enter the liver between the lobus caudatus and the lobus quadratus via the hilum or
porta hepatis at the bottom of the liver. The large blood vessels branch into venules and
arterioles before they converge in portal triads or portal tracts and deliver blood to the
hepatic lobules. The portal triad is the microscopic region where branches of the hepatic
artery, hepatic portal vein, and bile duct pervade the liver parenchyma. The hepatic lobule is the functional unit of the liver parenchyma and is 12 mm in diameter. It consists of
roughly hexagonal arrangements of plates of hepatocytes, which are 1525 hepatocytes
in length (Guyot et al., 2006; Ishibashi et al., 2009). On every edge of the hepatic lobule, a portal triad is positioned, draining blood into the hepatic lobule (fFigure 10.2).
The blood ows from the portal triads to the middle of the hepatic lobule, where a
central venule drains the blood. The central venules coalesce to the hepatic vein, which
exits the liver on its anterior side and transfers the blood back to the heart. On the way
through the hepatic lobules, the blood passes through vascular sinusoids, a fenestrated
type of capillaries. In contrast to most other capillaries, sinusoids have a discontinuous
basement membrane (BM) and harbor intercellular gaps between the endothelial cells
(fFigure 10.2).
In general, there is a slow blood ow in capillaries to optimize the time for diffusion
and ltration of blood (Aird, 2007). In the liver sinusoids, the blood ow is even slower
compared to other vascular beds. For example, the blood ow velocity in liver sinusoids
is about 0.40.45 mm/sec (Oda et al., 2003), compared to 1.14 mm/sec in muscle or
0.79 mm/sec in the brain (Ivanov et al., 1981). The fenestrated endothelium of the liver
sinusoids functions as a selective sieve that allows uid and small particles to pass from
the vascular lumen to the hepatocytes (Aird, 2007). The liver sinusoids are lined by
hepatocyte plates (Ishibashi et al., 2009) and receive blood from the portal triad (Oda
et al., 2006). As a result, the blood near the portal triad is more oxygenated than the
blood near the central venule because the hepatocytes in the sinusoids consume most
of the oxygen (Gumucio, 1989).
Liver sinusoidal endothelial cells (LSECs) line the blood vessel lumen and harbor
large pores, so-called fenestrae. These fenestrae are about 150175 nm in diameter and
10.2
Blood Flow and Cell Types in the Adult Liver

hepatocytes
bile
duct
hepatic
arteriole
central
venule
portal
venule
portal
triad
147
space of Diss
hepatocytes
LSEC
Kupffer cell
SEC
central venule
HSC
portal
triad
hepatic lobule
Figure 10.2A,B
Notes:
A A macroscopic overview over a hepatic lobule and the blood ow in a schematic sinusoid
from the portal triad to the central venule.
B The sinusoidal structure in detail on the cellular level. The blood ow direction is marked
with arrows.
comprise about 6%8% of the endothelial cell surface (Braet and Wisse, 2002). In general,
2050 fenestrae are arranged in sieve plates, which are approximately 0.1 m in diameter
(Aird, 2007). The sinusoids show micro-heterogeneity, as shown by scanning-electronmicroscopy (Wisse et al., 1983). Periportally (near the portal triad), the endothelial fenestrae have a larger diameter (110.7 0.2 nm) compared with the fenestrae in the center
of the lobule (104.8 0.2 nm). However, the number of fenestrae per square micrometer
and, therefore, the porosity (percentage of open area in the endothelial cell surface) is
higher in sinusoids near the central venule (7.94% vs 5.96%). This heterogeneity might
have something to do with the different oxygen levels in the blood, and the larger porosity
in areas exposed to less oxygenated blood might improve the oxygen diffusion to the hepatocytes (DeLeve, 2007). Consistent with this notion, loss of endothelial fenestrae during
liver cirrhosis coincides with a decrease in oxygen-dependent hepatocyte functions, such
as oxidative drug metabolism (Le Couteur et al., 1999). Finally, LSECs also have scavenger functions, as they can endocytose macromolecular waste from the blood, including
denatured albumin and other denatured or modied plasma proteins (Aird, 2007).
Hepatic stellate cells (HSCs) are located in the space of Diss (or perisinusoidal
space), which lies between the hepatocytes and LSECs. HSCs are liver-specic contractile pericytes (Bosch et al., 2010). HSCs are also known as lipocytes, fat storing cells,
or Ito cells and store about 80% of the vitamin A in the body in the form of retinyl
palmitate located in fat droplets (Senoo et al., 2007). The cell processes or cell protrusions of HSCs adhere to LSECs, and HSCs are likely to also adhere to each other (Imai
et al., 2004).
HSCs express several stem cell markers, such as CD133 (prominin-1) and Oct-4
(Kordes et al., 2009). On the one hand, HSCs contribute to liver regeneration (Sawitza
et al., 2009), but on the other hand, HSCs also contribute to liver diseases through
excessive ECM deposition.
148
Kupffer cells (KCs) are macrophages that adhere to the luminal side of the sinusoids
and move in an amoeboid fashion. They are the rst cells to get exposed to nutrients
and all substances absorbed in the gastro-intestinal tract. KCs might also regulate the
blood ow in the sinusoids to some extent (McCuskey, 2000) and represent the largest
population of resident tissue macrophages in the body (Ishibashi et al., 2009; Naito
et al., 2004). KCs remove pathogens (Wick et al., 2002) as well as old and damaged
erythrocytes from the blood (Terpstra and van Berkel, 2000). In general, KCs express
macrophage markers, such as ED1, ED2, and Ki-M2R in rats, and F4/80 in mice (Naito
et al., 2004).
Hepatocytes (Hep) are the parenchymal cells of the liver, and their population accounts for approximately 78% of the liver tissue volume. All other cell types constitute about 6.3% of the liver volume, in which about 2.8% are endothelial cells, 2.1%
Kupffer cells, and 1.4% HSCs. The extracellular space constitutes the remaining 16%
(Ishibashi et al., 2009). Hepatocytes line the sinusoids and are separated by the space
of Diss from the LSECs (fFigure 10.2). Solutes, uid, and small particles can diffuse to
the hepatocytes through the fenestrated endothelium (Aird, 2007), and lipoproteins are
taken up by the hepatocytes via receptor-mediated endocytosis (Carpenter et al., 2005).
Hepatocytes are responsible for the essential functions of the liver, including detoxication, synthesis of coagulation factors, and many serum proteins as well as regulation of
the bodys metabolism. Along the sinusoids, hepatocytes are heterogeneous. Periportally,
they have a high degree of oxygen uptake, gluconeogenesis, glycogenesis, cholesterol
biosynthesis, -oxidation, and ureogenesis, whereas pericentrally, they have a high rate
of bile acid synthesis, glycolysis, liponeogenesis, ketogenesis, and glutamine synthesis
(Jungermann, 1987). This heterogeneity was described as metabolic zonation (Gebhardt
and Mecke, 1983; Hussinger, 1983). Markers for hepatocytes are, for example, hepatocyte parafn 1 (Hep Par 1) (Kanitakis et al., 2004), hepatocyte antigen (Hep) (Chu et al.,
2002), or intestinal leucine aminopeptidase (LAP) (Roman and Hubbard, 1983).
Pit cells are liver-associated natural killer (NK) cells and are morphologically classied as large granular lymphocytes (LGLs) (Bouwens et al., 1987; Wisse et al., 1976). The
pit cells occur in the sinusoidal lumen, where they adhere to LSECs and KCs (Nakatani
et al., 2004), but they also occur in terminal branches of the portal vein (Wisse et al.,
1976). Natural killer cells are cellular components of the innate immune system and
are functionally dened by their ability to kill certain tumor cells and virus infected
cells without prior sensitization (Trinchieri, 1989). In rats, NK-cell markers like the one
stained with the monoclonal antibody 2.3.2 are used to identify pit cells because no
specic pit cell marker has been discovered yet (Wisse et al., 1997).
10.3 Angiogenesis in Liver Regeneration

Regeneration of the liver already played an important role in ancient Greek mythology,
namely in the myth of Prometheus. As a punishment for stealing the re from Zeus,
Prometheus was chained to a rock, and every day the big eagle Ethon came to eat his
liver, which regenerated until the next day. Liver regeneration probably arose during
evolution as a protection against toxins, which are taken up with food and deposited in
liver cells. Today, the regenerative capacity of the liver is challenged by alcohol abuse,
viral hepatitis, trauma, or partial hepatectomy (PH) for treatment of liver tumors, such
10.3 Angiogenesis in Liver Regeneration
149
as hepatocellular carcinoma (HCC). During liver regeneration, the vascularization has

to be restored, and this involves angiogenesis, the process of blood vessel growth out of
pre-existing blood vessels.
Partial hepatectomy (PH) is the surgical removal of liver parts. In rodents the 32 partial
hepatectomy (PH) is a common method to study liver regeneration. To this end, whole
liver lobes get pinched off by silk sutures and are removed. The removed lobes do not
grow back. Instead the remaining lobes massively increase their mass to compensate for
the tissue loss.
Hepatocellular carcinoma (HCC) is a malignant tumor of the liver. It develops directly
from dysplastic hepatocytes but may occasionally also arise from oval cells. HCCs usually develop in a cirrhotic liver, regardless the underlying cause for liver disease. HCCs
receive their blood supply almost exclusively via the hepatic artery.
Liver regeneration after partial hepatectomy (PH) represents a standard model for liver
regeneration. At around 16 hours after PH, the hepatocytes begin to proliferate periportally (fFigure 10.3A). Subsequently all other cell types start to periportally proliferate.
This process continues in a wavelike manner to the pericentral areas of the liver lobules
(Michalopoulos and DeFrances, 1997; Rabes et al., 1976). It is remarkable that during
liver regeneration, all cell types in the tissue proliferate. Three days after PH, clusters
of 1014 hepatocytes devoid of extracellular matrix (ECM) and sinusoids are observed
(Martinez-Hernandez and Amenta, 1995; Michalopoulos and DeFrances, 1997). Four
days after PH, delicate cell processes of the HSCs penetrate the hepatocyte clumps, and
laminins are detected in the HSCs (fFigure 10.3B) (Martinez-Hernandez et al., 1991;
Michalopoulos and DeFrances, 1997). Importantly, HSCs might regulate hepatocyte aggregation (Thomas et al., 2006). Subsequently, the hepatocytes arrange into hepatocyte
plates, consisting of two cell layers (as opposed to the normal phenotype of only one cell
layer) (fFigure 10.3C) (Michalopoulos and DeFrances, 1997). Finally, LSECs follow the
HSC processes and form angiogenic sprouts (Martinez-Hernandez and Amenta, 1995).
In this manner the hepatocyte plates get vascularized, and capillaries are established
(fFigure 10.3C). The BM of the penetrating capillaries slowly changes from the typical
capillary BM with a high laminin content to a discontinuous BM with a low laminin
content. Mature liver sinusoids display a very scant extracellular matrix, primarily containing bronectin, collagen type IV and I, and small amounts of glycosaminoglycans
(Michalopoulos and DeFrances, 1997). After 58 days post PH, the regenerating liver
almost restored its preoperative mass, and some LSECs start to undergo apoptosis (Greene
et al., 2003; Shimizu et al., 2005).
In addition to sprouting angiogenesis, intussusceptive angiogenesis might also play
a role in liver regeneration as formation of transvascular pillars are observed in the
liver sinusoids 12 hours after PH by using electron microscopy (Stroka et al., 2007).
This intussusceptive angiogenesis is followed by endothelial cell division, and thus, the
microvascular surface increases after partial hepatectomy.
150

cell division
hepatocytes
hepatic stellate cell
sinusoidal endothelial cell
bile
duct
hepatic
arteriole
portal
triad
portal
venule
hepatocyte plate
cell process
endothelial cell proliferation
intussusceptive angiogenesis
C
angiogenic
sprout
endothelial cell
tip cell
Figure 10.3AC
Notes:
A The beginning of angiogenesis during liver regeneration. First hepatocytes proliferate and
form hepatocyte clumps.
B After 34 days, HSCs send delicate cell processes between the hepatocyte clumps, which
can regulate the hepatocyte aggregation. Later endothelial cells proliferate and align to these
processes.
C The endothelial cells establish capillaries, which are penetrating the hepatocyte clumps.
Later the capillaries get rebuilt to sinusoids by changing the BM and gaining fenestrae.
10.4
Importance of VEGF for Liver Regeneration
VEGF (or VEGF-A) deletion in homozygous or heterozygous mice is embryonic lethal

(Carmeliet et al., 1996; Ferrara et al., 1996), showing that VEGF is required for blood
vessel formation and embryonic growth. In the regenerating liver, hepatocytes are the
main source of VEGF, and thus, hepatocyte proliferation is followed by LSEC proliferation (Shimizu et al., 2001). At the end of liver regeneration, when VEGF levels decline,
Angiopoietin-2 (Ang-2) might trigger LSEC apoptosis (Greene et al., 2003).
10.5
Role of Angiogenesis in Liver Damage/Disease
151
Vascular endothelial growth factor (VEGF) is an important signaling molecule for blood
vessel growth. VEGF contributes to endothelial cell migration, mitosis, and vascular
lumen formation, and it contributes to liver vascularization.
Experiments in rat PH models suggested that VEGF signicantly increases LSEC proliferation (Shimizu et al., 2005), vessel density, and vessel diameter (Bockhorn et al.,
2007). In addition, VEGF also increases hepatocyte and HSC proliferation (Bockhorn
et al., 2007), either directly or indirectly via LSECs. Finally, other vascular growth factors
besides VEGF are likely to trigger angiogenesis during liver regeneration (Shergill et al.,
2010).
10.5
Role of Angiogenesis in Liver Damage/Disease
Chronic liver disease (CLD) is the process of gradual liver destruction. Many liver
diseases fall under this category, including fatty liver disease or viral hepatitis. CLDs
can lead to liver brosis and can result in liver cirrhosis.
The regenerative capacity of the liver normally allows a total recovery after injury
and/or inammation (Guyot et al., 2006). However, chronic liver diseases (CLDs) do
not allow the liver to completely restore because the regeneration process is disturbed
due to a chronic inammation. The main consequence is brosis, which can develop
into cirrhosis. During the brogenic process, an excessive amount of extracellular matrix is deposited (Medina et al., 2004). Because brotic tissue inhibits blood ow and
oxygen delivery, the liver becomes hypoxic. Stimulation of hypoxia-inducible factors
(HIFs) subsequently lead to an angiogenic switch, to the up-regulation of proangiogenic
factors, like VEGF, and the formation of new vessels. The latter can disturb normal blood
ow and hepatic functions.
During the brogenic process granulation tissue develops within the inamed or
injured tissue. This tissue is the perfused, brous connective tissue that replaces a brin clot in wound healing. Usually, broblasts rst proliferate, then angiogenesis takes
place, and nally, ECM is deposited. During tissue repair and granulating tissue formation, cells, such as broblasts and HSCs, acquire features of smooth muscle cells
and are often called myobroblasts (Guyot et al., 2006; Serini and Gabbiani, 1999).
Myobroblasts are the main cell type in the granulation tissue and the main ECM depositioning cells. They contain stress bers, which play a role in contraction during wound
closure. A possible outcome of ECM deposition is the formation of scar tissue, which
can irreversibly disrupt liver physiology.
Fibrosis is currently viewed as a dynamic process related to the extent and duration of
parenchymal injury and hepatic cell death (Pinzani, 1999). It is mainly caused by viral
hepatitis, alcohol abuse, or fatty or autoimmune liver disease, and it is characterized
by the replacement of liver tissue with dense brous tissue (Guyot et al., 2006). During
152
brosis, sinusoids show a progressive loss of fenestrae and develop a continuous basement membrane (BM) (Aird, 2007). This process is called capillarization (Aird, 2007).
Fibrosis can take place in several patterns. For example, there is the so-called portal
to central brosis, which develops from the portal triad to the central venule. Another
kind of brosis is the portal to portal brosis, which develops between two portal triads. The brous ECM leads to a partial loss of liver function because the ECM forms
brotic strands, which disturb the blood ow in the liver lobule. As a consequence,
the cells die and get replaced by even more ECM, thus introducing a vicious cycle. The
wound healing process in CLDs is characterized by hypoxia, and overexpression of
growth factors (TGF-1, FGF and VEGF), matrix metalloproteinases (MMPs), and cytokines (Fernandez et al., 2009). In the new architectural environment of the brotic liver,
sinusoidal blood supply mainly derives from branches of the hepatic artery (arterialization) (Pinzani, 1999). Because LSECs loose their fenestrae (capillarization) during the
course of the disease, oxygen diffusion to the hepatocytes subsequently decreases, and
pro-angiogenic pathways become up-regulated to counteract the developing hypoxia
(angiogenic switch). Thus, brogenesis and angiogenesis could be regarded as crucial in
disease progression and in the search for therapeutic targets (Fernandez et al., 2009).
Cirrhosis is the incurable and nal stage of CLDs, and it develops over years or
decades. Cirrhosis is responsible for a signicant morbidity and mortality in the human
population, even though the exact worldwide prevalence of cirrhosis is unknown (Schuppan and Afdhal, 2008). In Germany, 4 to 5 million people (about 5%) suffer from
hepatic diseases, such as fatty liver or chronic viral hepatitis, which can develop into
cirrhosis with all its complications such as ascites, variceal bleeding, hepatic encephalopathy, or hepatocellular carcinoma (Bundesgesundheitsbericht fr Deutschland,
1998). In the USA, the prevalence of cirrhosis was estimated to be 0.15% (Everhart,
1994). A cirrhotic liver can harbor up to 50% scar tissue, whereas a healthy liver harbors less than 0.6% ECM (Gressner and Bachem, 1990). Cirrhosis is characterized by
the formation of regenerative nodules in the liver parenchyma, encapsulated by septae
made of brotic tissue (Guyot et al., 2006).
Portal hypertension is the syndrome of high blood pressure in the portal vein and its
tributaries (incoming vessels), generating a gradient in the blood pressure between the
portal vein and the hepatic veins of more than 5mm Hg. Portal hypertension can be a
serious complication of advanced CLDs and can result in the formation of esophageal
varices, ascites, renal dysfunction, and hepatic encephalopathy.
Portal hypertension is a main complication of liver cirrhosis and a leading cause of

death in CLDs (Bosch et al., 2010). It is dened as a portal pressure gradient (the difference in pressure between the portal vein and the hepatic veins) of 5 mm Hg or greater.
CLDs can restrict blood ow through the liver and, therefore, force the blood to ow
to the heart through alternative blood vessels, that is, portasystemic collaterals. As a
consequence, the collateral blood vessels pathologically enlarge to bypass the blockage
(Sanyal et al., 2008). A common location for such vessels is at the gastroesophageal
junction at which they lie, immediately subjacent to the mucosa and present as gastric
and esophageal varices. These varices can get instable over the time and start to bleed.
10.6
Questions and Problems
153
0h
6h
growth
medium
cell layer
(hepatocytes,
HSCs, ...)
12 h
18 h
lumen
fibrin gel
microcarrier
bead
endothelial
cell
24 h
30 h
B
Figure 10.4A,B The Fibrin Bead Sprouting Assay Can Be Used to Identify Liver Cell
Derived Angiogenic Factors
Notes:
A Scheme of the angiogenesis assay: endothelial cells on beads are embedded into
a brin gel, which is overlaid with liver cells to investigate their effects on sprouting
angiogenesis.
B 30 h time series of a vascular sprout growing from a bead into the brin gel. Scale
bar: 50 m.
Ascites (free uid within the peritoneal cavity), hepatic encephalopathy, splenomegaly
(enlarged spleen), and an increased risk of spontaneous bacterial peritonitis and of the
hepatorenal syndrome can be the outcome of portal hypertension. Possible reasons for
the development of portal hypertension are brotic areas, in which pathologic angiogenesis results in an abnormal vascular system, disturbing hepatic blood ow.
Angiogenesis assays have been used to investigate the pro- and anti-angiogenic potential of proteins and drugs in vitro. In addition, by creating a dened environment,
they are valuable to elucidate the interaction between individual cell types and their
distinct roles in promoting or inhibiting angiogenesis. An assay suitable for this kind
of investigation has been developed by Nehls and Drenckhahn (1995) and involves
endothelial cells coated on microcarrier beads, which are embedded into brin gels
(fFigure 10.4A). A modication of this assay uses human umbilical vein endothelial
cells (HUVEC) and a feeder layer of skin broblasts (Nakatsu et al., 2003).
The endothelial cells in this assay form multicellular lumenized angiogenic sprouts
(fFigure 10.4B), which are surrounded by a basement membrane (Nikolova et al.,
2007). Using this assay, liver cell types or liver-derived factors can be identied that
induce physiologic or pathologic blood vessels.
10.6
Questions and Problems
The specic features of healthy and unhealthy angiogenesis are not well understood in liver regeneration and liver damage, respectively. However it is important
154
to understand the molecular differences in order to guide liver angiogenesis into a

healthy process, thus facilitating liver regeneration and preventing CLDs. Thus, molecular studies on the role of angiogenesis during liver regeneration and CLD development are warranted.
Summary
The functional units of the liver are the hepatic lobules, vascularized by branches of two large
blood vessels, the hepatic portal vein and hepatic artery. The liver receives about 25% of the
cardiac output, which passes the hepatic lobules via sinusoids. During liver regeneration, a
new vascular network is generated by angiogenesis in the regenerating tissue. During partial
hepatectomy, a model for liver regeneration, hepatocytes proliferate and form cell clumps.
After inltration by hepatic stellate cells, the hepatocytes form multicellular plates. These
hepatocyte plates consist of two hepatocyte cell layers instead of one found in the normal
liver. Consequently, regenerated tissue is less vascularized than normal liver tissue. However,
during regeneration, endothelial cells form capillaries surrounded by hepatic stellate cell
processes, develop fenestrae, and become fully functional. In contrast, during chronic liver
diseases, the characteristic features of the hepatic vascular network are severely changed and
may thus contribute to liver failure.
Further Reading
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beds. Circ. Res. 100, 17490.
Braet, F., and Wisse, E. (2002). Structural and functional aspects of liver sinusoidal endothelial
cell fenestrae: a review. Comp. Hepatol. 1, 1.
Fernandez, M., Semela, D., Bruix, J., Colle, I., Pinzani, M., and Bosch, J. (2009). Angiogenesis
in liver disease. J. Hepatol. 50, 60420.
Ferrara, N. (1999). Role of vascular endothelial growth factor in the regulation of angiogenesis. Kidney Int. 56, 794814.
Gerhardt, H. (2008). VEGF and endothelial guidance in angiogenic sprouting. Organogenesis
4, 2416.
Guyot, C., Lepreux, S., Combe, C., Doudnikoff, E., Bioulac-Sage, P., Balabaud, C., and Desmouliere, A. (2006). Hepatic brosis and cirrhosis: the (myo)broblastic cell subpopulations involved. Int. J. Biochem. Cell Biol. 38,13551.
Lammert, E., Cleaver, O., and Melton, D. (2003). Role of endothelial cells in early pancreas
and liver development. Mech. Dev. 120, 5964.
Pinzani, M. (1999). Liver brosis. Springer Semin Immunopathol 21, 47590.
Shergill, U., Das, A., Langer, D., Adluri, R., Maulik, N., and Shah, V.H. (2010). Inhibition of
VEGF- and NO-dependent angiogenesis does not impair liver regeneration. Am. J. Physiol.
Zeeb, M., Strilic, B., and Lammert, E. (2010). Resolving cell-cell junctions: lumen formation
in blood vessels. Curr. Opin. Cell. Biol. 22, 62632.
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11 A Quantitative Mathematical Modeling

Approach to Liver Regeneration
Dirk Drasdo, Stefan Hoehme, and Jan G. Hengstler
Learning Targets
1. Iterative application of a process chain consisting of experiments, image analysis,
and mathematical modeling to identify spatialtemporal mechanisms during liver
regeneration
2. Quantitative characterization of image information
3. Mathematical model abstractions for multicellular tissue models
4. Parameter sensitivity analysis to explore the systems behavior of mathematical
multicellular tissue models
5. Hepatocyte alignment along sinusoids as order principle to restore liver architecture
11.1
Denition
Hepatic parenchyma is organized in repetitive functional units called liver lobules

(fFigure 11.1AC), which, besides its main constituents, hepatocytes, consists of sinusoidal endothelial cells, Kupffer, stellate, and bile duct cells (Michalopoulos and
DeFrances, 1997). Branches of the hepatic artery and the portal vein guide blood to the
periportal regions of the lobules. From there, it ows through microvessels, the sinusoids, along hepatocyte columns that are lined with endothelial cells (generally known
as sinusoidal cells), and drains into the central vein. This complex lobule architecture
ensures a maximal exchange area for metabolites between blood and hepatocytes in
healthy liver. The shape of a lobule can be approximated by a polyhedron. Mouse liver
has about 1,000, and human liver about 1 million lobules. Drug damage can lead to
massive cell death compromising liver architecture and function. For example, overdosage of paracetamol (acetaminophen) causes massive death of hepatocytes close to
the central vein. In mice the same effect can be mimicked by intoxication with carbon
tetrachloride (CCl4). Within only 10 days the liver is able to completely restore mass and
architecture of damage where about 50% of hepatocytes have been destroyed (Hoehme
et al., 2010) (fFigure 11.1D). Sinusoids are almost not affected by the drug (Hoehme
et al., 2010).
160
11 A Quantitative Mathematical Modeling Approach to Liver Regeneration
t = 2 days
t = 4 days
t = 10 days
Figure 11.1AD (A), (B) Confocal Laser Scanning; (C) Bright Field Micrograph of Liver
Lobules
Notes:
A Sinusoids can be identied by the overlap (yellow) of ICAM (red) and DPPIV (green)
staining. DAPI (blue) denotes the cell nuclei.
B CD31, EGFP were used for sinusoid staining, DAPI for the cell nuclei.
C Bright eld micrograph. The lines serve as an orientation to identify the approximate
shape of a lobule. blue nuclei: DAPI. The yellow arrows denotes the central veins, the
red arrows (only in C) the portal veins.
D Time sequence of bright eld micrographs showing the regeneration process 2, 4,
and 10 days after injection of CCl4 (at day t = 0). Brown nuclei: BrdU, The bright area
denotes a necrotic zone caused by CCl4. After about 10 days, the necrotic area has
disappeared. Note that each image is from a different mouse as the mouse has to be
sacriced for the analysis (Hoehme, et al., 2010).
A systems biology analysis for a multicellular tissue organization process with signicant spatial changes, as this is the case for liver regeneration after massive cell death in
localized liver regions, requires a number of fundamental steps.
1)
2)
The characterization of the liver lobule architecture prior to drug administration,

serving as a reference state, as well as during the regeneration process should not
be purely descriptive to ensure that the observations are objectied. Hence, tissuelevel parameters should be dened that permit to cast the observed regeneration
pattern into numbers (or distributions) and thereby to objectify the experimental
observation.
Identication of relevant cell-level parameters. A natural rst step for processes
involving small populations of cells spatially organized in a complex architecture,
11.2 Methods to Quantify Spatial-Temporal Information in Liver Lobules
3)
4)
5)
161
as in a liver lobule, is to set up a mathematical model down to the single-cell level

to study which cell-level mechanisms are necessary and sufcient to explain the
observed tissue organization (here: regeneration) process. As cells have only a limited
number of degrees of freedom, the number of possible cell-level parameters that have
to be controlled is much smaller than given by the possible complexity of genetic,
intracellular signal transduction and metabolic network states. The relevant cell-level
parameters need to be considered if the regeneration process should be understood
and will later serve to parameterize the mathematical model properly. Such cell-level
parameters within a liver lobule are, for example, the cell size, shape, cell material
properties, whether a cell enters the cell cycle (i.e., proliferates or whether it dies by
necrosis or apoptosis), the cell micromotility, the cell type, and so forth.
Image processing and analysis. In order to reliably collect information on the
parameters denoted under (1) and (2), labeling experiments utilizing markers
specic for the described parameters should be performed. The information needs
to be sampled over several mice and over several lobules within each mouse to
obtain statistically signicant results. Specic spatial features of the lobules as
its shape or the architecture of its blood vessel network requires 3D information
in order to be correctly quantied. 2D information can partly be collected using
bright eld micrographs, while 3D information requires the use of confocal laser
scanning micrographs. As explained later, only confocal micrographs after image
processing and analysis were able to guarantee an image quality permitting to
identify spatial structures down to a few micrometers. The latter is necessary as the
blood microvessels (called sinusoids) are only about 5 micrometers thick.
The development of a mathematical model able to represent both the liver lobule
level as well as the cell-level parameters. The cell-level parameters are used to
dene the mathematical model on the cell-level, and the tissue-level model
parameters are used to dene the initial spatial arrangement of the mathematical
model components as well as to compare simulation results with this model to the
experimentally observed regeneration scenario.
Mathematical model predictions to validate the mathematical model and exclude
other equally likely mechanisms.
11.2 Methods to Quantify Spatial-Temporal Information in

Liver Lobules
A thorough quantitative analysis of the tissue architecture, including information about
ongoing processes and functional ngerprints prior to the liver regeneration process,
is necessary to dene the liver lobule state prior to drug administration. Subsequently,
a careful characterization of the parameter changes during regeneration after drug administration is required. For spatialtemporal tissue organization processes down to the
cell scale, it is natural and important to collect functional information at the cell level.
Such information can, in principle, be obtained either from bright eld micrographs or
confocal scanning micrographs in combination with appropriate cell and tissue specic
markers.
Bright eld micrographs provide purely 2D information. In order to infer 3D information from 2D serial sections the structural elements on neighboring micrographs have
162
to be identied. This is called image registration. As sinusoids can have any direction
in 3D and are only a few micrometers in diameter, the thickness of each tissue slice cut
from a tissue block would have to be not larger than about one micrometer. However,
the cutting process leads to distortions and rupture of the tissue. For proper registration
the resulting artifacts would need to be eliminated by mathematical processing of the
image. This requires stable structures or pattern (such as landmarks) to match two neighboring images by proper global or local transformations. Extensive attempts have shown
that bright eld micrographs of liver lobules contained too many artifacts to permit a
reliable image registration.
Confocal laser scanning microscopy turned out to be the method of choice. Confocal
microscopy is able to acquire in-focus images from selected depths. This is called optical sectioning. It permits a choice of the focal plane. Moreover, the optical resolution is
slightly better than for bright eld microscopy. Most of the parameters were calculated
after processing and analysis of confocal scanning micrographs (fFigure 11.2).
Figure 11.2 Image Processing Chain

Notes:
Different channels of each image of a stack of consecutive confocal micrographs (upper left)
obtained from a vibratome slice of about 70150 m are analyzed. The blue channel contains
information on the hepatocytes (upper row) from DAPI staining and the overlap of the green
and red channel (lower row) on sinusoids from ICAM (red) and DPPIV (green) staining. Each
of the two elements are separately processed and nally overlaid to the nal image (rightmost
upper image). Lower row: The 1st image shows the quality prior to image processing. For the
sinusoidal structures, the overlap of the green and red channel information was extracted (2nd
image). Adaptive histogram equalization was then used to increase contrast and equalize
brightness (3rd image), general erosion ltering was utilized to remove noise (4th image),
and eventually dilatation operators used to strengthen the remaining pattern (5th image). The
result of this processing, after further renement by more complex morphological operators,
is shown in the 6th image Upper row: To identify the hepatocytes for each confocal laser
scanning micrograph (1st picture), the blue channel information was extracted (2nd image).
Then, adaptive histogram equalization (3rd image), general erosion operators (4th image), and
dilatation operators (5th image) were used resulting in a fully segmented 3D volume dataset
(last image in upper row). One of the helpful technical features of a confocal microscope
is that the images in this volume are already perfectly aligned and thus no registration is
required (modied after Hoehme et. al., 2010).
11.3
Normal Liver Lobule: The Reference State
11.3
Normal Liver Lobule: The Reference State
11.3.1
Image Processing
163
The main aim of the image processing step is to reconstruct the three dimensional
architecture of the liver lobule by identifying the different sub-elements (Acharya and
Ray, 2005). This is called image segmentation. The analysis was limited on identifying hepatocytes and the sinusoidal network. Starting with a stack of optical sections
generated with a confocal laser scanning microscope (Olympus, Germany, FV1000),
the information for the cell nuclei and the blood vessel structures were separated to
different channels: DAPI for the cell nuclei on the blue channel, and ICAM/DPPIV for
the sinusoids on the overlap of the red and green channels.
In order to reconstruct and analyze the sinusoidal network from the confocal images,
adaptive histogram equalization (to increase contrast and equalize brightness; Stark,
2000), generalized erosion (to remove noise), dilatation (to strengthen remaining structures), and other, more complex morphological operators, were applied (fFigure 11.2).
These more complex operators, for example, analyze the local environment of each
pixel by sampling along Bresenham lines in 3D emanating from that pixel (Bresenham,
1996). Depending on the information gathered along these rays, pixels are classied
either as within or outside a vessel. Depending on that classication, the intensity of the
pixels is changed in order to ll the lumina of the veins and sinusoids. We employed a
medial axis transform-like process to geometrically represent the sinusoidal network as
an undirected graph in three-dimensions and used this to investigate properties of the
sinusoidal network. We basically inscribed a chain of linked spheres of maximum size
into the voxels labeled as belonging to the vascular network starting from the central
vein (e.g., Rohrschneider et al., 2007).
Adaptive histogram equalization, median ltering (reduces image noise), and cell
shape reconstruction (based on Voronoi space decomposition) were used to investigate hepatocyte properties (fFigure 11.2). The Erosion lter is a morphological lter
that changes the shape of objects in an image by eroding (reducing) the boundaries
of objects that belong to one pixel class and enlarging the boundaries of pixels of
other classes. It is often used to reduce, or eliminate, small bright objects. The Dilation lter is a morphological lter that changes the shape of objects in an image by
dilating (enlarging) the boundaries of bright objects and reducing the boundaries of
dark ones. The decision, which points are removed during erosion ltering and which
points are added by dilatation ltering, depends on setting threshold values and the
selected kernels. By using further morphologic lters that, for example, remove interconnected pixel clusters below and above given size thresholds, cell nuclei that
are outside the volume range known for hepatocytes (and therefore are likely to be
assigned with other cells e.g. macrophages) can be eliminated. The centers of the
hepatocyte nuclei are used to generate a Voronoi tessellation where each point that
is closer to a given hepatocyte nucleus than to any other hepatocyte nucleus is assigned to this nucleus. From this procedure a polyhedron (with on average 12 faces)
around each nucleus emerges that is subsequently corrected for partial volumes occupied by the sinusoidal network. The resulting corrected polyhedrons approximate
the hepatocyte shape.
164
11.3.2
Image Analysis
The undirected graph obtained from medial axis transform-like processes and the
Voronoi tessellation corrected for the partial volumes occupied by the sinusoidal
network formed the basis to parameterize the image information. For the sinusoidal
network within a liver lobule, the parameters measured were the radii of the sinusoid
vessels, the orthogonal minimal vessel distance, the non-branched segment length, the
mean branching angles, and the vessel volume in the lobule. For the hepatocytes, the
measured parameters were the hepatocyte volume, size, density, the next neighbor distance, and the diameter of the hepatocyte nucleus. For the central vein, the length, the
radii, and the inclination to the viewing plane were measured (Hoehme et al., 2010).
By sampling over sufciently many (in our case: 26) different lobules, we obtained
distributions over these parameters. By sampling from these distributions a representative statistical liver lobule could be investigated that served as a reference state of the
mathematical model (fFigure 11.3A).
11.4 Quantifying the Regeneration Process:

Process Parameters
As shown in the time sequence after administration of CCl4 in fFigure 11.1, the hepatocytes close to the central vein are destroyed by the drug. To quantify the regeneration process, four process parameters have been measured (fFigure 11.3BE): (i) the
BrdU incorporation resolved in time and space, (ii) the local average of hepatocytes
density, (iii) the necrotic area around the central vein and, (iv) the fraction of contact
area a hepatocyte shares with sinusoids. After CCl4-induced damage, cell proliferation
is maximal at the border between the pericentral lesion and the viable hepatocytes in
the periportal zones 23 days after drug intoxication.
The central necrotic lesion has its maximum size after 1 day and has largely closed
after 4 days. The maximum of the necrotic area occurs about 1 day after administration
of CCl4 (fFigure 11.3A). The contact area between hepatocytes and sinusoids is a good
parameter to characterize liver architecture. It reaches a minimum 4 days after drug
injection and has nearly returned to its original value 16 days after intoxication.
Interestingly, the sinusoidal network was almost not affected by CCl4, so we did not
need to dene any process parameter for the sinusoidal network.
Notice that the tissue-level parameters obtained after image processing and subsequent image analysis serve to cast the spatialtemporal functional information into
numbers (distributions of those markers) permitting to objectify the image information.
The data obtained in this way can be used to develop and calibrate a mathematical
model and to compare computer simulations with that mathematical model to experimental ndings.
11.5
Mathematical Model
The image analysis permits to describe the regeneration process on the tissue level by
quantitative parameters, but it provides only little information on which mechanisms
on the cell-level are responsible for the observation. From an experimentalists point
11.5
B
hepatocytes per mm2
2200
1800
1400
1000
0
2 4 6 8
16
time after CCl4
administration (days)
165
7
6 n
io
es
5
l
ic
4 crot )
e
s
3 om n layer
fr
l
2 nce (cel
a
t
dis
E
0.08
0.06
0.04
0.02
0
0
2 4 6 8
16
time after CCl4
experimental data
without HSA
hepatocyte-sinusoid contact area (%)
D
area of necrotic lesion (in mm2)
16
s)
14 ay
(d
12 on
ti
10 tra
s
8 ini
m
6 ad
l
4 CC 4
r
2 fte
a
20
10
0 me
30
i
BrdU positive hepatocytes (%) t
Mathematical Model
55
50
45
40
35
0
2 4 6 8
16
time after CCl4
HSA
Figure 11.3AE Process Parameters for Quantifying the Regeneration Process

Notes:
A Statistical liver lobule after image analysis and processing calculated from tissue level
parameters to quantify the liver lobule architecture. The periportal triads denoted in blue,
the sinusoids and central vein in red. The necrotic zone is in light brown, while the viable
hepatocytes are in dark brown. The yellow arrow denotes the border between necrotic zone
and viable hepatocytes.
BE Parameters to quantify the regeneration process (Hoehme, et al., 2010).
B The BrdU incorporation in percent vs. time since administration of CCl4 and with increasing distance from the border between necrotic zone and viable hepatocytes (arrow in A).
C Hepatocytes per mm2.
D Size of the central necrotic area (light brown zone in A; the area is calculated from the
section).
E Fraction of the surface area of a hepatocyte in common with sinusoids. All values were
averaged over many lobules. The symbols show experimental results, while the lines denote
model simulations. Both are explained later in the text. The simulations show that only if
the daughter cells of dividing hepatocytes align along the closest sinusoids (assumption IVa,
HSA), a quantitative agreement between model and experimental results could be found.
166
of view it might look tempting to set up a number of hypotheses about the underlying molecular and cell-level parameters and test them subsequently. However, each of
these hypotheses would have needed to be experimentally tested in vivo, which is time
consuming, challenging, and costly.
Mathematical modeling provides a complementary tool to experiments and data
analysis. If properly set up, possible mechanisms can be tested in silico (on the computer). Mechanisms disagreeing with the experimental observations can be abolished
before any experimental testing such that only those mechanisms agreeing with the
experimental observations needs to be experimentally validated. Moreover, the mathematical model can summarize biological, physical, and chemical aspects, which lead to
a complexity that cannot easily be controlled without mathematical model simulations.
However, different alternative mechanisms might account for a correct regeneration
process. In this case, it is possible to search by computer simulations for those experimental settings for which these different alternative mechanisms would lead to different
outcomes, that is, to use simulated predictions of the outcome of new experiments to
stepwise identify the correct out of the different possible mechanisms by iterations of
model predictions and experimental testing.
The model needs to fulll several requirements: (1) It needs to allow for cell displacements much smaller than the size of an individual cell because cells must be able to rearrange in the small space between the sinusoids. The complex liver lobule architecture
and the small displacements of cells and sinusoids during liver regeneration after CCl4induced damage favor a model type capable of representing each single cell (known
as individual-cellbased, agent-based, or single-cellbased models; Anderson et al.,
2007). (2) Cells must be able to grow and divide, avoiding physically unrealistic local
cues of mechanical pressure. (3) Cell shape has been found to be largely cubical and
compatible with the assumption that hepatocyte shape mainly emerges by deformation
of a sphere. Hence, one can assume that hepatocyte shape in isolation is approximately
spherical. (4) The model needs to represent active cell migration which, besides passive
shifts by external forces, can be the second cause of cell position changes.
The model we chose to fulll these requirements is a so-called center-based
(single-cellbased) model (Anderson et al., 2007). It bases on the following model
assumptions:
(I) Hepatocytes can be mimicked as homogeneous, isotropic elastic and adhesive,
intrinsically spherical, and objects capable of migration, growth, division, and death.
A suitable model for the pair-wise force between interacting sticky isotropic, homogeneous elastic spheres is the Johnson-Kendall-Roberts (JKR) model. It has been shown
by Chu et al. (2005) to apply to the pair-wise interaction force of S180 cells if compression and pulling of one cell with respect to the other cell is sufciently fast, and it has
independently been proposed by Drasdo and Hoehme (2005) to mimic the interaction
forces between cells in monolayer and multicellular spheroid growth dynamic simulations. This suggests using the JKR model to mimic hepatocytehepatocyte forces and
hepatocyteblood vessel forces. The JKR model shows a hysteresis behavior depending on whether two objects approach each other or are pulled apart, that is, cells
stick together beyond the distance at which they came into contact when they were
approached.
Mathematically, the force can be expressed as a vector function that describes the
strength of the force F between hepatocytes (or a hepatocyte and a sinusoid) with the
11.5
Mathematical Model
167
hepatocytehepatocyte distance dij (or the hepatocytesinusoid distance), F(dij). Here

the underscore denotes that the force is a vector and not a scalar.
(II) Migration of each hepatocyte can be calculated using an equation of motion. An
equation of motion is an equation that permits to calculate the position of an object
(here a hepatocyte) with time.
To understand the concept of an equation of motion, consider a classical example:
Newtons equation of motion of an object of mass m falling on a oor as a consequence
of the attraction by the earth: ma = F. Here, a = dv/dt is the acceleration of the center
of mass of the object. The left hand side, ma, describes the inertia of the object and is
called inertia term. The acceleration describes the increment of the velocity v within an
arbitrarily short period of time: a =
dv
v
where $v = v(t + $t) - v(t). Knowing the
= lim
0
dt
t
velocity and the current position permits calculation of the new position of the object
from dr/dt = v. Acceleration, force, velocity, and position are vectors, that is, they have
an absolute value and a direction. If the acting force on the object is due to the attraction between the object and the earth, then F = mg, where g is the earth acceleration.
For hepatocytes, the equation of motion is more complicated. It is assumed that hepatocyte movement results from the superposition of (i) hepatocytehepatocyte interactions by friction and adhesion and repulsion between them, (ii) hepatocyte-extracellular
matrix (ECM) friction (ECM can be found in the up to about one micrometer large
spacecalled the space of Dissbetween the endothelial cells making up the sinusoids and the hepatocytes), (iii) hepatocytesinusoidal interactions by friction and repulsion, and (iv) the active movement of the hepatocytes. The active movement includes
the hepatocyte micromotility and possible directed movements of hepatocytes specied
later. The repulsive forces emerge mainly from cell deformation (and to a small extent,
from compression) and are mimicked by elastic and adhesive central forces of the JKRtype, introduced previously, among cells, between cells and sinusoids, and between
cells and the substrate. The sinusoidal network can be modeled as network of chains of
linked spheres (sub-elements), each chain characterized by its extensibility. The equation of motion for hepatocyte (superscript H) i reads as follows:
H
HH H
HE H
H
HH
H dv i
+ 6 v i (t ) = 6 (v j (t ) v i (t )) + F ij
m
N
iE
ij
dt

jNNi

hepatocyte-
hepatocyte
inertia
hepatocytehepatocyte
substrate
hepatocyte
adhesion &
friction
friction
repulsion
+ F i active ,H + 6 HS (v j H (t ) v i S (t )) + F ij HS

jNNi ij
N
hepatocytemicro-motility
hepatocytesinusoid
sinusoid
adhesion &
friction
repulsion
(11.1)
168
X
In this equation, m is the mass of a hepatocyte, v i (t ) is the velocity of object i of type X
where X = H denotes a hepatocyte, object S a sub-element of the sinusoid. 6 ij
HX
with X = H,
S denotes the friction tensor (here a 3 x 3 matrix) describing the friction of hepatocyte (H) i
and object j of type X (X = H, S, E (extracellular matrix)). The friction tensor may be decomAX
posed into a perpendicular and a parallel component: 6 ij = G (u ij uij ) + G ||(I u ij u ij ).
Here, uij=(rj-ri)/|rj-ri| with ri denoting the position of cell i. denotes the dyadic product.
F ij HX denotes the JKR-force between hepatocyte i and object j of type X. It includes
adhesion if X = H and does not consider any adhesion if X = S (i.e., between hepatocytes
and sinusoids). I is the unity matrix (here a 3 x 3 matrix with 1 on the diagonal and
0 on the off-diagonal). G , G || are the perpendicular and parallel friction coefcients,
HE
respectively. F iactive ,H denotes the active movement force. The model assumes 6 iE = G I ,
that is, isotropic friction with the extracellular matrix in the space of Diss, G =0
(neglecting friction inside the cell that emerges from intracellular reorganization if cells
are deformed or compressed). F i active ,H = (1 [pi H active , A ]) 2DG 2 H i (t ). H (t ) denotes a
i
Gaussian-distributed random variable with H (t ) = 0. H (t ')H (t ) = D (t ' t ) . Here, X

i
i
j
denotes the expectation value obtained by averaging the random variable X over many
of its realizations. As each component of H is Gaussian distributed, each realization is
sampled from a Gaussian distribution. D is the cell diffusion constant and assumed to
be a scalar, pi a quantity by which the cell can sense the position of their neighbors. The
active movement described by F iactive ,H has the effect that hepatocytes have the tendency
to move into regions of locally smaller cell density (for details, see Hoehme et al.,
2010). Alternatively, the directed contribution to the active hepatocyte motion may result from chemotaxis triggered by a morphogene secreted by the necrotic cells. In this
active ,H
= C c + 2DG 2 H i (t )(C is the chemotaxis coefcient, c(r,t) the morphogene
case, F i
concentration). Both active directed motion terms have the same effect. Within tissues
the friction between cells and the extracellular matrix components and the sinusoids is
large so that the inertia term, the rst term in equation (11.1), can be neglected and be
set to zero.
The sinusoids obey an equivalent equation of motion as Eqn. (11.1) for the hepatocytes (Hoehme et al. 2010) with some modications: the neighboring spheres belonging to the same sinusoid are linked by linear springs, sinusoidal objects do not move
actively, so, F i active ,S = 0 and sinusoidal sub-elements not belonging to the same sinusoid
(blood vessel) do not adhere.
Note that for all cells an equation of the type (11.1) has to be solved simultaneously.
As the velocity is a vector in 3D space having three components (a component in x,
y, z direction), for N cells 3N equations of motion have to be solved simultaneously.
Before the system of 3N equations can be solved, the forces and friction matrices have
to be calculated from the current position of the cells. Then the new velocities are
calculated by solving the systems of equations, followed by calculating the new cell
positions. Finally, it is important to notice that dealing with stochastic differential equations needs particular caution because standard calculus is not valid (for details, see
Gardiner, 2000; Iacus, 2008).
11.6
Simulation Results with the Mathematical Model
169
(III) Cell orientation changes can be mimicked by an optimization principle using the
Metropolis algorithm for the energy change in case of a cell orientation change (Drasdo
et al. 2007) or an equation for the angular momentum (Drasdo, 2005). Here we used
the Metropolis algorithm for convenience as the equations for the angular momentum
lead to very complicated equations of motion (Drasdo, 2005). The concept behind the
Metropolis algorithm is to perform a trial step (here: a small rotation) and subsequently
evaluate whether this step is accepted or rejected (in which case the step is taken back).
The change of total energy of the whole cell conguration is used to evaluate the step.
As the orientation change of a hepatocyte only affects the next and maybe next-next
neighbors, only those neighbors need to be considered. To calculate the orientation
change, within each time interval $t for each hepatocyte a rotation trial around three
space-xed axes by angles DBi with i = 1, 2, 3, DBi [0, DBmax), with DBmax << 0/2
was performed, using the algorithm of Barker and Watts (Allen and Tildersley, 1987).
The energy can be calculated by integration of the equation F ij =
Vij
r i
where only the
JKR-force contributions were considered. The energy difference is then calculated from
Vij (t ) = Vij (t + t ) V ij (t ) ,and the probability that a step is accepted is calculated using
p = min(1, e
Vij / FT
) where FT z10-16J is a reference energy (comparable to the kbT in uids
or gases were kb is the Boltzmann factor, T the temperature).

(IV) The cell division orientation was assumed to be random. We performed simulations
assuming that (a) either after a hepatocyte division its daughter cells align along the closest
sinusoid triggered by chemotaxis of a short range diffusive morphogen secreted by the
sinusoids (we call this process hepatocyte sinusoid alignment [HSA]) within a short period of time, or (b) that such an alignment does not occur. The cell division frequency was
chosen according to the experimentally determined values depicted in fFigure 11.3B.
(V) The model only considers sinusoids and hepatocytes, the main constituents in a
liver lobule. Other cell types are neglected.
11.6
Simulation Results with the Mathematical Model
The mathematical model is completely parameterized by measurable biophysical and

cell-biological parameters, which makes each of its components testable. Selected
simulation scenarios are depicted in fFigures 11.4 and 11.5.
If HSA is absent (IVb), regeneration is incomplete (fFigure 11.4). In order to exclude
that this result depends on the parameter choices we have performed a parameter sensitivity analysis, where each mathematical model parameter was varied within its physiologically compatible range. The process parameters depicted in fFigure 11.3BE were
used to quantify the regeneration process in the simulation and to directly compare the
simulation result with the experimental ndings (lines in fFigures 11.3CE). Without
HSA, for no parameter combination a complete regeneration could be found within the
experimentally observed period of 10 days.
Only if HSA was present (IVa) and if the cell micromotility was sufciently large, a
quantitative agreement with the experimental ndings could be found (fFigure 11.5
and green line in fFigure 11.3CE). The model predicted that the daughter cells of a
dividing hepatocyte should align within 2 hours along the closest sinusoid in order
170
t = 0 days
t = 1 day
t = 2 days
t = 4 days
t = 6 day
t = 10 days
Figure 11.4 Regeneration Scenario Without Alignment of the Daughter Cells along the
Closest Sinusoids, that is, Without HSA
Note:
The scenario shows a section through the sinusoid. Sinusoids and the central vein are denoted
in red, hepatocytes in brown, the necrotic zone in light grey. Shown are days 0, 1, 2, 4, 6, and
10. Here, the necrotic zone is not closed after 10 days.
to recover the lobule architecture within the experimentally observed time period. It
further predicted that without HSA, the angle distribution between the orientation of the
daughter cells and the closest sinusoid should be uniform, while with HSA it should be
peaked as a small angle.
In total we performed several hundred simulations and tested a number of alternative
model variants until we arrived at the model depicted in fFigure 11.5. Generally, for
those model parameters for which only ranges but not the precise parameter values
are known, the best possible parameter combinations for different models should be
compared to the experimental data in order to exclude bias by unfavorable parameter
choices. For the given model of liver regeneration, the prediction, namely the alignment
of daughter cells along the closest sinusoids (HSA), could be experimentally validated
in vivo by inference of the angle distribution between pairs of daughter cells and their
closest sinusoids from 3D liver lobule reconstructions from confocal scanning laser
micrographs. In these micrographs BrdU was used to label the proliferating cells and
ICAM/DPPIV to identify the sinusoids, as explained in fFigures 11.1 and 11.2 (Hoehme
et al., 2010). The experiment validated the non-isotropic angle distribution predicted by
the computer simulations.
It is one of the rare cases where a mathematical tissue model was able to correctly
predict a spatial organization principle in vivo.
11.7
Figure 11.5
Limitations
171
t = 0 days
t = 1 day
t = 2 days
t = 4 days
t = 6 day
t = 10 days
Regeneration Scenario With HSA
Note:
After about 6 days the lesion is almost closed. The quantitative comparison of experiment
and simulation using the process parameters shows an excellent agreement (green lines in
fFigure 11.3CE).
The same process chain used here can be applied to many spatialtemporal tissues.
The center-based model permits simulation of up to about 1Mio cells.
11.7
Limitations
The precise cellcell interaction forces over long time periods are not known. The JKRmodel does not take into account viscous effects and deviations of the cell shape from
a sphere, even though large deviations from spherical shapes could not be observed
(Hoehme et al., 2010). The precise molecular mechanisms involved are not known.
Staining for the mitotic spindle suggests that hepatocytes in mitosis may be randomly
oriented. The precise mechanism for the alignment is not yet validated. However, within
in vitro experiments, short range attraction (not adhesion) between endothelial cells and
hepatocytes could be observed, supporting the model predictions. The model used the
experimentally determined cell proliferation pattern as input parameter. Its mechanistic
origin has not yet been explored.
172
Summary
The mechanisms underlying spatialtemporal tissue organization processes can be inferred
by a strategy involving the iterative application of experiments, image processing and analysis, and mathematical modeling. Images serve to visualize the spatial distribution of cells and
other tissue components. If 3D information at the spatial resolution of a few micrometers is
needed, confocal laser scanning microscopy is a suitable method. Parameters are used in order
to objectify the experimental image information. Their value is tracked over the considered
process of interest in sufciently short time intervals. The parameters are dened using image
processing and analysis. Besides quantitative characterization of the image information, they
serve to set up a quantitative mathematical model and subsequently compare simulation results with that model to the experimentally ndings. The mathematical model permits to take
into account biological, chemical, and physical aspects. For models involving sufciently
small cell populationsup to about 1 million cellssingle-cellbased models resolving each
individual cell are often the method of choice as they permit a direct comparison of the
experimental image information at cell resolution with that of the computer simulations. In
liver regeneration after CCl4 administration, this strategy could be used to explain how the
drug-induced pericentral cell death within each liver lobule was regenerated within a time
period of about 10 days. The experiments could validate the model prediction of the alignment of the daughter cells of a dividing hepatocyte along the nearest sinusoid (HSA) within at
most 2 hours after the cell has divided. Alternative, simpler mechanisms not using HSA could
be excluded by parameter sensitivity analysis.
Further Reading
Drasdo, D. (2007). Center-based Single-cell Models: An Approach to Multi-cellular Organization Based on a Conceptual Analogy to Colloidal Particles. In: Anderson, A.R.A., Chaplain, M.A.J., and Rejniak, K.A. (2007). Single-Cell-Based Models in Biology and Medicine
(Basel: Birkhuser).
Drasdo, D., Hoehme, S., and Block, M. (2007). On the role of physics in the growth and pattern formation of cellular systems: What can we learn from individual-cell-based models?
J. Stat. Phys. 128, 287345.
Hoehme, S., Brulport, M., Bauer, A., Bedawy, E., Schormann, W., Gebhardt, R., Zellmer, S.,
Schwarz, M., Bockamp, E., Timmel, T.G., Hengstler, J.G., and Drasdo, D. (2010). Prediction and validation of cell alignment along microvessels as order principle to restore tissue
architecture in liver regeneration. Proc. Natl. Acad. Sci. U.S.A. 107, 103716 (and its 58
pages supplementary information).
References
Anderson, A.R.A., Chaplain, M.A.J., and Rejniak, K.A. (2007). Single-Cell-Based Models in
Biology and Medicine (Basel: Birkhuser).
Acharya, T., and Ray, A. (2005). Image Processing: Principles and Applications (Hoboken,
New Jersey: Wiley-Interscience).
Allen, M.P. and Tildersley, D. J. (1987) Computer Simulation of Liquids. (Oxford: Oxford
Science).
References
173
Bresenham, J. (1996). Pixel-processing fundamentals. IEEE Computer Graphics and Applications 16, 7482.
Chu, Y.S., Dufour, S., Thiery, J.P., Perez, E., and Pincet, F. (2005). Johnson-Kendall-Roberts
theory applied to living cells. Phys. Rev. Lett. 94, 028102.
Drasdo, D., Hoehme, S., Block, M. (2007). On the role of physics in the growth and pattern
formation of cellular systems: What can we learn from individual-cell-based models? J.
Stat. Phys. 128, 287345.
Drasdo, D. and Hoehme, S. (2005). A single-cell-based model of tumor growth in vitro:
monolayers and spheroids. Phys. Biol. 2, 13347.
Drasdo, D. (2005). Coarse Graining in Simulated Cell Populations. Adv. Complex Syst. 2/3,
31963.
Gardiner, C. (2009). Handbook of Stochastic Methods (New York: Springer).
Hoehme, S., Brulport, M., Bauer, A., Bedawy, E., Schormann, W., Gebhardt, R., Zellmer, S.,
Schwarz, M., Bockamp, E., Timmel, T.G., Hengstler, J.G., and Drasdo, D. (2010). Prediction and validation of cell alignment along microvessels as order principle to restore tissue
architecture in liver regeneration. Proc. Natl. Acad. Sci. U.S.A. 107, 103716.
Iacus, S.M. (2008). Simulation and inference for stochastic differential equations (New York:
Springer).
Michalopoulos, G.K., and DeFrances, M. (1997). Liver regeneration. Science 276, 606.
Rohrschneider, M., Scheuermann, G., Hoehme, S., and Drasdo, D. (2007). Shape Characterization of Extracted and Simulated Tumor Samples using Topological and Geometric
Measures. IEEE Engineering in Medicine and Biology Society, 2007, 62717.
Stark, J.A. (2000). Adaptive image contrast enhancement using generalizations of histogram
equalization. IEEE Trans Image Process 9, 88996.
12 Animal Models for Studies on

Liver Regeneration
Amalya Hovhannisyan and Rolf Gebhardt
Learning Targets
1.
2.
3.
4.
5.
Different types of animal models for liver regeneration

To proliferate or not: how the choices of the hepatocyte inuence regeneration
Regenerative processes vary in different types of models
How transgenic mice can aid in unraveling regenerative mechanisms
Applications of animal models in transplantation studies
12.1
Introduction
The liver is an essential organ for the maintenance of body homeostasis. Throughout the
whole life it is constantly exposed to various types of insults, including environmental
toxic substances. Liver regeneration is an evolutionary adaptive response to the constant
exposure to toxic compounds, viral infection, ischemia, and other types of damage. In
most circumstances, the regenerative process leads to full recovery of the structural and
functional capacity. However, in the event of ineffective or completely absent liver regeneration, the life-threatening scenario of acute liver failure may supervene. In other cases,
incomplete regeneration may occur characterized by replacement of large parts of liver
parenchyma with excessive deposition of matrix proteins and connective tissue, conditions
known as hepatic brosis and cirrhoses. These pathological states are associated with the
clinical picture of chronic liver failurea condition with a notoriously bad prognosis.
Understanding the mechanisms that control hepatocyte division and survival has
broad implications for the treatment of acute and chronic liver diseases as well as for
increasing the feasibility of split liver transplantation. This chapter provides an overview
on various types of animal models that are in use for investigating liver regeneration.
Because there is no single regenerative process, but a plethora of cellular reactions
nally leading to regeneration that may be different in different models of liver injury,
it seems appropriate to focus briey on the contribution of the few cell types present in
liver tissue before discussing the animal models.
12.2
Different Types of Regenerative Processes
The hepatocyte, though being a highly differentiated cell type, still has the unique
ability to proliferate for several rounds. This is remarkable because replication may
176
12 Animal Models for Studies on Liver Regeneration
occur in undamaged tissue and without intermediary loss of biliary polarity. Thus,
under all conditions where hepatocytes are only modestly affected and maintain the
capacity to replicate, proliferation of these cells is the primary choice for liver regeneration (fFigure 12.1A). As long as the lobular structure of the liver is not disturbed,
proliferation of sinusoidal cells such as endothelial cells follows hepatocyte proliferation without much delay, in order to maintain the sinusoidal structure. In cases
where hepatocyte proliferation is hampered or blocked, alternative pathways for liver
regeneration are used (fFigure 12.1BD). These start either from ultimate bipolar
precursor cells (oval cells) or, apparently, from different types of (liver) stem cells (see
other chapters of this book) that may give rise of oval cells. It appears that there is no
A partial hepatectomy
B toxic injury
differentiation
regeneration
undamaged
hepatocyte
high proliferative potential
proliferation
C trangene expression
damaged
hepatocyte
unable to
proliferate
D gene knockout
alternative
mechanisms
of regeneration
alternative
mechanisms
of regeneration
additional proliferative stimulus

variable influence
on hepatocyte
depending on
transgene
activation of
stem /progedifferent
nitor cells
sources
modified
proliferation
additional proliferative stimulus

variable influence
on hepatocyte
depending on
target gene
modified
proliferation
Figure 12.1AD Schematic Depiction of Regenerative Phenomena in Different Model of

Liver Injury or Genetic Manipulation
Notes:
A After partial hepatectomy regeneration is based solely on hepatocyte proliferation.
B In toxic injury, where hepatocytes are damaged and unable to proliferate, stem or progenitor cells (from various sources) are activated and nally differentiate into new hepatocytes.
C Hepatocyte-specic expression of a transgene may modify the proliferative response and
sometimes lead to hepatocyte damage and death. If regeneration is based on alternative
mechanisms (e.g., activation of stem cells), new hepatocytes may occur that do not express
the transgene.
D Knockout of a target gene may lead to a similar scenario as in C.
12.3
Different Types of Animal Models
177
unique lineage leading from liver stem cells to the differentiated hepatocytes. This
scenario is further complicated by the fact that usually all types of non-parenchymal
cells are activated simultaneously, leading to their proliferation and changing their
phenotypic feature (Ueberham et al., 2010). Because these changes often result in
the novel expression of cellular markers, which are traditionally considered as stem
cell and/or oval cell markers, it is almost impossible to draw rm conclusions on
lineage relationships solely based on such markers. Sorting out which kind of cellular
reactions of non-parenchymal cells are associated with establishing proper stem cell
niches or are directly involved in stem cell activation and further differentiation remains a challenge for future investigations. Obviously, the diversity of animal models
of liver regeneration may aid in this endeavor because the various diverse routes for
cell renewal in the liver reect the unmatched capacity of this organ to cope with a
large variety of insults.
12.3
Different Types of Animal Models
Experimentally, liver regeneration can be triggered in different ways. Surgical models

(fTable 12.1) are based on the unique feature of the liver to respond to physical damage
(e.g., loss of whole parts or complete lobes, altered vascularization) by initiating hepatocyte proliferation that leads to complete regeneration of the organ. Pharmacological
models (fTable 12.1) reect the response of the liver to intoxication by endogenous or
exogenous compounds and drugs. Usually, intoxication leads to the loss of hepatocytes
or hampers their regenerative capacity. As pointed out previously, alternative mechanisms of regeneration predominate, liver stem cells or progenitor cells are activated,
proliferate, and differentiate into new hepatocytes or bile duct cells. Viral models (not
Surgical
Table 12.1 Surgical and pharmacological animal models for studying liver regeneration.
Models discussed in the text are marked in bold.
Animal model
Specication
Partial hepatectomy (PHx)
30%; 40%; 70%; 90%; 95%
Bile duct ligation
Cholestasis
Altered vascularization
e.g., Porto-caval shunt; arterialization
Pharmacological
Ischemia/reperfusion
Carbon tetrachloride
Pericentral damage
Acetaminophen
Pericentral damage
Thioacetamide
Primarily periportal damage
D-galactosamine
General damage
Azoxymethane
Fulminant hepatic failure
Choline-decient, Ethionine
(CDE)-diet
General damage
2-Acetylaminouorene
Periportal damage
Retrorsine
General damage
178
discussed in detail) take advantage of the fact that many viruses trigger a regenerative
response because the infected hepatocytes are severely stressed or even prone to die.
To some extent, the regenerative response resembles that in pharmacological models.
Transgenic models (fTable 12.2) are based on the overexpression or the knockout of a
certain gene in hepatocytes. Depending on whether this gene affects the viability of these
cells or simply modies the regenerative response, these models may serve as ultimate
models of liver injury or need to be further challenged by additional insults like those
mentioned previously in order to trigger the regenerative process. The latter type enables
Table 12.2 Transgenic animal models for studies of liver regeneration. Models discussed
in the text are marked in bold.
Targeted genes
Existing TG or KO mouse models
Growth factors, morphogens
HGF/SF KO; IGF-1R KO; Wnt/beta-catenin KO; TGF2R

KO; FGF1/FGF2 KO
Cytokines, chemokines,
adipocytokines
IL6 KO; TNF KO; TNFR1 KO; TNFR2 KO; Fn14 KO;
OSMR KO, LTR KO, LT KO, TNF/LT KO;
LIGHT TG; CXCR2 KO; Adiponectin KO
Transcription factors
STAT3 KO; NFB KO; IB TG; CREM KO; C/EBP KO;

NFAT KO, EGR1 KO; FoxM1B TG
Nuclear receptors
PPAR KO; FXR KO; CAR KO; PXR KO; RXR KO
Regulator molecules for

cytokines, growth factors, etc.
PCI-TG; PAI-KO; SOCS3 KO; p21 TG; bi-1 KO
Proteases
uPA TG; TTR-Casp3 TG
ECM components, their

regulators and effectors
MMP-9 KO; TIMP-1 KO; ILK-liver- KO; GPC3-TG
Various enzymes
iNOS KO; AT1KO; Cox2-TG; GNMT-KO, PDK1 KO;

PDK1/STAT3 KO, Atm KO; Aurora-A TG
Table 12.3 Models of immune-mediated liver injury. Models discussed in the text are
marked in bold.
Animal model
Mediating cells
Concanavalin A
NK cells; NKT cells;

Neutrophils; Eosinophils; T-cells
Lipopolysaccharide (LPS)
Kupffer cells; NKT cells
Poly I:C
NK cells; Kupffer cells
Alcohol
NKT cells; Neutrophils
Carrageenan
NK cells; NKT cells
12.4
Surgical Animal Models
179
the researcher to study the molecular function of the gene product in the framework of
the regenerative response. Immunological models (fTable 12.3) emphasize the fact that
resident macrophages, the Kupffer cells, or blood-derived immune cells can inuence
liver regeneration. Depending on the type of stimulus, the response may range from
rapid and severe loss of hepatocytes to minor changes of the regenerative process.
In the following, several distinguished examples of these animal models will be considered in more detail in order to illustrate their advantages and limits, to emphasize
important methodological aspects, and to highlight mechanistic results obtained with
these models.
12.4
Surgical Animal Models
Mass ligation, also called en-bloc ligation, is an easy technique for partial hepatectomy
by which the lobes to be resected are lifted, while one single suture loop is placed
around the lobe pedicles and tied by hand. The ischemic lobes are then cut by ne
scissors close to the ligature. Depending on which and how many lobes are removed
(particularly when including the median lobe) this method may result in vena cava stenosis and/or outow obstruction of parts of the remaining tissue because of unfavorable
branching of the hepatic vein. The vessel-oriented approach avoids such complications
by microscope-assisted separate ligation of the individual vessel branches of each lobe,
thus not including elements of other pedicles inside the suture loops.
12.4.1
Partial Hepatectomy
One of the most effective surgical models is partial hepatectomy (PHx) in rodents. This
technique, which was rst described in 1931 by Higgins and Anderson in rats, can be
modied to be safely and reproducibly performed in mice. In PHx, resection of about
2/3 of liver tissue (usually the median lobe and the left lateral lobe) by mass ligation
forces quiescent hepatocytes to rapidly re-enter the cell cycle. This highly regulated process is primed by different cytokines and growth factors but also cytokine-independent
mechanisms (Zellmer et al., 2010) that activate downstream kinases and transcription
factors. As a result, the hepatocytes initiate the transcription of many (>100) early genes
and accumulate triglyceride and cholesterol to supply the energy and materials required
for restoring the liver mass. After one or two rounds of replication of hepatocytes, the
original liver mass is restored within 57 days.
It has to be emphasized that mass ligation surmounting the sole resection of the left
lateral lobe in rats and mice (approx. 30% PHx) is frequently accompanied by outow
obstruction within the remaining tissue. This may have considerable consequences
upon functional integrity and regeneration, one of which may be retardation of the
regenerating process indicated by BrdU-incorporation (Dirsch et al., 2008). To avoid
such unwanted complications a vessel-oriented approach needs to be followed. With
this improvement, 90% PHx was achieved with 100% survival after 1 week, and even
95% PHx could be performed with a 1-week survival rate of 66% in rats (Madrahimov
et al., 2006). The vessel-oriented surgical procedure also enables efcient PHx in mice
without risk of outow obstruction.
180
12.4.2
Bile Duct Ligation
Total bile duct ligation (tBDL) is the standard model for research in cholestasis and has
been extensively used in rats and mice. Similar to humans, mice develop bile ductule
and septal proliferations leading to brosis. Because total bile duct obstruction does
rarely occur in humans, a model of partial BDL was established for mice that better
reects the human situation and is not associated with massive tissue injury as observed
in tBDL in mice (Heinrich et al., 2010), which should be ideal for research in resolved
acute cholestasis.
12.5
Pharmacological Models
12.5.1
Carbon Tetrachloride (CCl4)-induced Hepatotoxicity
CCl4 is a hepatotoxin causing severe centrilobular necrosis due to formation of highly

reactive CCl3-radicals that are formed by the mixed function oxidase system during
CCl4 metabolism. The trichloromethyl radicals initiate lipid peroxidation and lead to
hepatocellular membrane damage. Recently, it has been shown that CCl4 causes not
only primary liver necrosis but also hepatocyte apoptosis (Simeonova et al., 2001).
CCl4-induced liver injury is also associated with increased levels of cytokines, including
TNF, which is thought to enhance CCl4-mediated injury (Sudo et al., 2005) but is also
important for hepatocyte proliferation, acting as a mitogen (discussed later).
Chronic intermittent administration of CCl4 induces brotic changes after marked
inltration of inammatory cells, especially mononuclear cells, such as neutrophils,
thus mimicking the changes seen in chronic viral hepatitis-associated brosis, and has
therefore been widely used to experimentally induce liver brosis and cirrhosis.
12.5.2 Acetaminophen Hepatotoxicity

Acetaminophen (APAP) hepatotoxicity is the leading cause of drug-induced liver failure
in the United States and other industrialized nations. The mechanism of toxicity includes
the formation of a reactive metabolite (N-acetyl-p-benzoquinoneimine [NAPQI]),
which is initially detoxied by cellular glutathione. However, after the GSH levels are
exhausted, NAPQI binds to cellular proteins, especially mitochondrial proteins. This
results in mitochondrial dysfunction with inhibition of mitochondrial respiration, reactive oxygen and peroxynitrite formation, and declining ATP levels. The mitochondrial
dysfunction eventually leads to mitochondrial membrane permeability transition pore
opening and collapse of the mitochondrial membrane potential.
12.5.3
Choline-decient, Ethionine-supplemented Diet
Feeding a choline-decient, ethionine-supplemented (CDE) diet is a preferred model for

studying alternative cellular regenerating mechanisms. The CDE diet damages the liver
parenchyma and prevents hepatocyte division, leading to the activation and rapid induction of large numbers of oval cells in mice. In parallel, all types of non-parenchymal
cells appear to be activated in this model as well (Ueberham et al., 2010) and may
differentially inuence the regenerative process based on stem cell and precursor cell
populations.
12.6 Transgenic Models
181

Transgenic mice (TG) carry genetic manipulations allowing the (over)expression of endogenous or foreign genes coding for any type of protein, in particular of enzymes. In the
case of the Cre-recombinase, which is able to trigger gene recombination at loxP sites
placed at both ends of (part of ) a target gene, the recombination results in the removal
(knockout [KO]) of the oxed (part of ) gene. Thus, the KO mouse cannot produce a
functional protein of the targeted gene resulting in a loss-of-function phenotype. In order
to avoid interference of the transgenic phenotype with embryonic processes (which may
lead to embryonic lethality) and/or to restrict it to a certain cell type conditional, cellspecic activation of the transgenic phenotype must be established by further genetic
manipulation. fFigure 12.2 illustrates an example for doxycycline-dependent liverspecic knockout of a gene.
PLap
Dox
+Dox
tetO7
rtetR
Pmin
Cre
Cre recombinase
Cre
Figure 12.2A,B Strategy for Conditional and Hepatocyte-Specic Disruption of a

Target Gene in Mice
Notes:
A Scheme of doxycycline (Dox)-induced activation of Cre recombinase in tripletransgenic mice expressing the synthetic transactivator variant (rtetR) of the tet-repressor
driven by the liver-specic LAP-promoter (PLAP). In the presence of Dox, rtetR binds to an
array of seven tet operator sequences (tetO7) activating transcription of the Cre gene.
B Structure of the target gene with exon 3 anked by loxP sites (black triangles). The
mutated target gene lacking exon 3 is formed by the active Cre recombinase.
12.6.1 Advantages and Limitations

Gene manipulations in mice are powerful tools for generating murine models for human
pathologies and for understanding their pathophysiological mechanisms. The overexpression or functional inactivation/deletion (knockout [KO]) of a particular gene in
transgenic animals (TG) directly aids in highlighting its role in normal and pathological
states. However, some concerns have been raised about their intrinsic value as an ideal
animal model for a given disease/pathophysiological process, and particular attention
must be paid to a number of facts that inuence the success and value of the generated
TG models: First, the genetic background plays an important role, as some of the null
182
mutants have demonstrated a surprisingly high degree of phenotypic variability between

individual mouse lines. Thus, inbred lines are preferable compared to those with a mixed
genetic background. However, the properties of the inbred strain, in which gene manipulations will be generated, must be considered as well. Unlinked genes in the background
strain can signicantly affect the disease phenotype. On the other hand, this variability
in phenotypes in related genetic backgrounds could potentially help to identify modier
loci that affect the phenotype of interest. Second, gene-targeting strategies need to be
selected and performed carefully as they can result in different, sometimes even opposite, pathophysiological conclusions. Here, an interference with a selection marker
gene could play a role, as targeted disruption of a gene of interest is usually carried out
by the introduction of a selection marker at the site of disruption of the coding sequence.
Likewise, targeting of a point mutation into a gene cluster can result in misexpression
of a neighboring gene. Furthermore, expression of a transgene is frequently driven by a
potent promoter that may not normally be present in the cells that are being examined.
Thus, overexpression of the transgene is generally many times higher than in physiological or even pathological conditions. Third, the existence of compensatory genetic loci
and redundant or interactive pathways, particularly for critical cell functions, are of high
importance. The lack of an abnormal phenotype in a KO mouse does not necessarily
mean that the gene in question is not important. Its role might only become apparent by
functional inactivation of another gene in a double KO model. Moreover, development
and growth can be normal in some KOs, but they may fail to respond to or survive certain
types of stresses such as PHx, carcinogen application, or toxic injury. Fourth, attention
should be paid on possible changes in the expression of other genes, as overexpression
of the transgene or functional inactivation of gene(s) throughout the life of the animal
may modify the expression of other genes, which may themselves be suppressed or overexpressed to compensate for the high and nonphysiological expression of the transgene
and/or lead to the development of another pathophysiological conditions.
Last, but not least, embryonic or early lethality of some TG animals creates problems
for further studies. This could happen due to interruption of physiological processes
critical for embryogenesis, as well as due to exhibition of more severe symptoms than
expected, leading to a shortened life span. Usually, strategies using conditional activation of the TG phenotype (fFigure 12.2) may overcome these problems and have
successfully applied to studies on liver regeneration.
In general, the TG animal models can be classied into two major groups: (1) transgenic models simulating liver injury, and (2) transgenic models for studying regenerative
mechanisms.
12.6.2 Transgenic Models to Simulate Liver Injury

Song and coworkers (2009) generated a TG model with inducible expression of urokinase-type plasminogen activator (uPA), inducible Alb-rtTA2S-M2 mice. In this model,
Alb-rtTA mice (containing the reverse tetracycline transactivator [rtTA] driven by the
mouse albumin promoter) were backcrossed onto severe combined immunodecient
(SCID)/TG mice to generate immunodecient rtTA/SCID mice. Then mice were infected
with recombinant adenoviruses Ad.TRE-uPA, in which the urokinase was located
downstream of the tetracycline response element (TRE), to establish an inducible liver
injury mouse model. In the presence of doxycycline, uPA was exclusively expressed in
183
endogenous hepatocytes and caused extensive liver injury. These mice created excellent opportunities for studies of cell transplantation. It was shown that enhanced green
uorescent protein (EGFP)-labeled mouse hepatocytes selectively repopulated the rtTA/
SCID mouse liver and replaced over 80% of the recipient liver mass after repeated
administration of Ad.TRE-uPA. Compared with the other models of uPA mice, rtTA/SCID
mice did not exhibit problems regarding breeding efciency, high mortality, or the time
window for transplantation. In addition, the extent of liver injury could be controlled to
facilitate transplantation surgery by regulating the dose of Ad.TRE-uPA.
The development of a TTR-Casp 3 transgenic mouse model, where inducible hepatocyte ablation and hepatic injury is caused via hepatocyte-specic expression based
on a 3 kilobase mouse transthyretin (TTR) promoter sequence combined with an inducible, dimerizable procaspase-3, was reported by Mallet et al. (2002). This interestingly
engineered TG mouse offers new opportunities to study liver regeneration and stem
cell plasticity. Advantages of this model are no detectable cytotoxicity in uninduced
controls and the absence of mortality in heterozygous animals even in the case of 85%
hepatocyte destruction.
12.6.3 Transgenic Models for Studying Regenerative Mechanisms

The number of TG animals for revealing mechanistic details of liver regeneration was
signicantly growing during last years, allowing increasingly better understanding of
the role of different cytokines, growth factors and their receptors, negative and positive
regulators, transcription factors, nuclear receptors, enzymes, and other proteins in the
process of liver regeneration (fTable 12.2).
Hepatocyte growth factor/scatter factor (HGF/SF) is produced by mesenchymal cells
and acts predominantly in a paracrine or endocrine manner as a potent mitogen on a
variety of cell types. Conditional hepatocyte-specic ablation of HGF/SF in mice signicantly reduced the regenerative capacity of hepatocytes after CCl4 injection (Phaneuf
et al., 2004). Likewise, hepatocyte-specic deletion of HGF/SF receptor c-Met showed
increased liver cell damage and brosis in a chronic cholestatic liver injury model due
to bile duct ligation (Giebeler et al., 2009).
Studies on liver regeneration induced by PHx in liver-specic insulin growth factor-1
(IGF-1) receptor KO mice revealed that intact IGF-1/IGF-1R signaling is required for
normal liver regeneration, where IRS-1, rather than IRS-2, seems to be responsible for
transduction of the proliferative response downstream from IGF-1R (Desbois-Mouthon
et al., 2006). Interestingly, the absence of IGF-1R caused a signicant decrease in hepatocyte proliferation in males, but not in females, suggesting that sex-dimorphism in liver
regeneration may involve signal transduction via IGF-1R.
Wnt/beta-catenin morphogen signaling plays a considerable role in liver development. Although Wnt/beta-catenin signaling is rather low in adult hepatocytes and shows
a gradient declining from the pericentral to periportal zone (Gebhardt and Hovhannisyan, 2010), its activity shows a short transient increase during PHx. Hepatocytespecic beta-catenin KO in adult mice resulted in attenuated liver regeneration after
PHx, while mice overexpressing a beta-catenin mutant accelerated liver regeneration
(Nejak-Bowen et al., 2010).
Transforming growth factor (TGF-) is a multifunctional cytokine with diverse effects on development, growth, and homeostasis in most tissues. Studies on hepatocyte-
184
specic ablation of TGF- type II receptor in mice showed that TGF- signaling appears
to limit the proliferative response of regenerating hepatocytes after PHx through inhibition of G1 to S phase cell cycle transition, but is not required for normal termination
of liver regeneration probably due to a compensatory activation of activin signaling
(Oe et al., 2004). Likewise, in conditional hepatocyte-specic TGF-1 expressing mice
proliferation of hepatocytes almost disappeared, while withdrawal of TGF-1 resulted
in a proliferative burst possibly due to enhanced matrix degradation by matrix metalloproteinase 13 (Arendt et al., 2005).
Generation of FGF1(/)/FGF2(/) double-KO mice has revealed an interesting role
of broblast growth factor (FGF) 1 and 2 in matrix deposition and hepatic brogenesis
in response to acute or chronic exposure to CCl4, but not in restoration of liver after PHx
(Yu et al., 2003). In contrast, expression of a dominant-negative form of FGF receptor1
in zebrash revealed that FGF signaling is crucial in early proliferative response to PHx
(Kan et al., 2009).
The proinammatory cytokine Interleukin 6 (IL-6) has long been recognized to play
diverse roles in various models of liver regeneration ranging from directly affecting
hepatocyte proliferation to mediation of inammatory reactions. IL-6 KO mice have
mainly conrmed these functions demonstrating pronounced liver failure and defective regeneration after PHx. However, concerning the contribution of IL-6 in mediating toxicity-induced liver injury and brosis particularly in cases of chronic exposure
to the toxin (e.g., CCl4), stimulating as well as dampening effects have been reported
(Kovalovich et al., 2000; Rio et al., 2008).
Tumor necrosis factor-alpha (TNF-) has been recognized as a cell-death mediator
and is produced from immune cells after liver damage. TNF- gene decient mice
revealed little changes in liver regeneration after PHx (Hayashi et al., 2005). However,
TNFR-1 mice, but not TNFR-2 KO mice, showed high mortality, severely impaired
DNA synthesis, and delayed gain in liver mass, as well as lower binding of the NFB, STAT3, and AP-1 transcription factors after PHx (Yamada et al., 1998). The same
receptor preference was seen in CCl4-induced liver regeneration as well without,
however, causing mortality. Also, liver brosis formation after chronic exposure to
CCl4 depended on TNFR1 rather than TNFR2.
Tumor necrosis factor-like weak inducer of apoptosis (TWEAK) is a TNF superfamily
ligand and regulates a diverse range of cellular processes, including proliferation, differentiation, migration, cell survival, and cell death, and has also been shown to act as
a proangiogenic and proinammatory factor. Knockout of its receptor, a 14-kD type I
transmembrane receptor termed broblast growth factor-inducible 14 (Fn14), considerably changed the response to CDE feeding within 2 weeks. Besides a signicantly reduced number of liver progenitor cells, attenuated inammation, cytokine production,
and expression of key brogenesis mediators were found (Tirnitz-Parker et al., 2010).
Adiponectin is the most abundant adipocytokine produced by adipocytes. Adiponectin KO mice (129S1 background) exhibit impaired liver regeneration and increased hepatic steatosis after PHx. Increased expression of Socs3 and reduced activation of STAT3
seemed to contribute to these alterations (Shu et al., 2009). Similar changes were shown
in adiponectin KO mice on the C57BL/6 background. Here, the expression levels of peroxisome proliferator-activated receptor (PPAR) and carnitine palmitoyltransferase-1
were decreased, suggesting a possible contribution of altered fat metabolism to the
impaired liver regeneration in adiponectin KO mice.
185
As already mentioned in some cases presented previously, several transcription factors are activated after PHx or toxic liver injury, including STAT3, NFB, and C/EBP.
The contribution of these transcription factors to liver regeneration was further demonstrated in various studies using conditional KO technology, conrming their importance
in regulating cell cycle progression, preventing apoptosis, and expression of cyclins
E and B, respectively, just to name a few of their functions. Further transcription factors identied by this approach include EGR-1 and FoxM1B. EGR-1 is a zinc nger
transcription factor induced as part of the immediate-early gene expression program in
the regenerating liver. Egr-1 null mice showed impaired liver regeneration after PHx,
associated with increased activation of the p38 mitogen-activated protein kinase and
reduced expression of the cell division cycle 20 gene (Cdc20), a key regulator of the
mitotic anaphase-promoting complex (Liao et al., 2004). Forkhead box M1B (FoxM1B)
is a ubiquitously expressed member of the Fox transcription factor family whose expression is restricted to proliferating cells mediating hepatocyte entry into DNA synthesis
and mitosis during liver regeneration (Wang et al., 2001). Interestingly, it was shown
that age-related defects in cellular proliferation are associated with diminished expression of FoxM1B and that constitutive expression of FoxM1B mRNA in hepatocytes of
TTR-FoxM1B transgenic CD-1 mice restored the young regenerating liver phenotype
after PHx. Thus, FoxM1B controls the transcriptional network of genes that are essential
for cell division and exit from mitosis, and it plays a role in the decline of cellular
proliferation observed during aging.
Also, many nuclear receptor-mediated signals have been implicated in the proliferative response of regenerating liver. Peroxisome proliferator-activated receptor- mediates the effects of peroxisome proliferator chemicals, which are potent hepatic mitogens
and carcinogens in mice and rats. Studies on Ppar-null mice revealed transient impairment in liver regeneration, associated with altered expression of genes involved in cell
cycle control, cytokine signaling, and fat metabolism (Anderson et al., 2002).
After PHx, bile acid levels in blood tend to be increased. It has been shown that modestly elevated bile acid levels are sufcient to drive hepatocyte DNA replication in vivo.
Bile acids activate the primary bile acid receptor farnesoid X receptor (FXR), as well as
constitutive androstane receptor (CAR) and pregnane X receptor (PXR). FXR/ mice
showed an increased mortality and strong inhibition in the early stage of liver regeneration after PHx. However, at later stages, the FXR/livers showed relatively rapid growth,
and the weights of the livers from WT and KO mice did not differ signicantly at 7 days
(Huang et al., 2006). CAR/mice showed a modest decrease in liver growth in the early
stages of liver regeneration after PHx, which was combined with delayed hepatocyte
replication.
Control of cell-cycle progression, cellular proliferation, and liver regeneration requires
a balance between positive and negative regulatory pathways of extra- and intracellular
signals. Protein C inhibitor (PCI) is a member of the serine protease inhibitor (SERPIN)
superfamily, which inhibits activated protein C as well as other serine proteases, including HGF activator (HGFA). The latter is necessary for the activation of proHGF released
after liver injury. Experiments on hPCI-Tg mice establish the regulatory role of PCI in
liver regeneration after PHx by forming an HGFA-PCI complex (Hamada et al., 2008).
Plasminogen activator inhibitor-1 (PAI-1) is an inhibitor of tissue-type plasminogen activator (tPA) (stimulates brinolysis in blood vessels) and urokinase-type plasminogen
activator (uPA) (mediates extracellular matrix turnover). In addition to their role in blood
186
coagulation, both PAs are able to cleave the inactive single chain hepatocyte growth
factor (HGF) to its active two-chain form. Studies on PAI-KO mice revealed an increased
liver injury after Acetaminophen toxicity, massive intrahepatic hemorrhage, and high
mortality (Bajt et al., 2008). In the surviving animals expression of the cell cycle inhibitor p21 was increased, and liver regeneration was signicantly delayed. The CDK inhibitor p21 complexes with and thereby regulates the activity of a wide range of cyclins
and cyclin-dependent kinases. In addition, p21 binds proliferating cell nuclear antigen
(PCNA), a processing factor for DNA polymerase 8, and inhibits PCNA-dependent DNA
replication in vitro. Transgenic mice that abundantly express p21 specically in hepatocytes failed to stimulate hepatocytes proliferation after PHx (Wu et al., 1996).
Liver regeneration depends on timely restoration of cellular mass and proper orchestration of matrix remodeling. Extracellular matrix formation and degradation during
liver regeneration is poorly understood. Some transgenic mouse models were generated
to shed light on these processes. Matrix metalloproteinases (MMPs) are a family of
zinc-containing neutral proteinases that are controlling both extracellular matrix (ECM)
remodeling and growth factor bioactivity in normal and pathophysiological states. It
has been shown that pro-MMP-9 is activated after PHx and may contribute to priming
hepatocytes for proliferation by modulation of the matrix environment in the remnant
liver. Accordingly, gelatinase Bdecient animals (MMP-9/) showed a delayed regenerative response after 70% PHx (Olle et al., 2006). Integrin-linked kinase (ILK) is a
protein involved in transmitting extracellular matrix signals upon integrin binding. Apte
and coworkers (2009), using liver-specic ILK-ablated mice, demonstrated an important
role of ILK in the termination of liver regeneration after PHx. The increased post-PHx
liver mass was due to sustained cell proliferation driven in part by increased signaling
through HGF and the -catenin and Hippo pathways.
Glypican 3 (GPC3) is a heparan sulfate proteoglycan of ECM that is bound to the cell
surface through a glycosylphosphatidylinositol anchor. GPC3 TG mice with hepatocytetargeted, overexpressed GPC3 had a diminished rate of hepatocyte proliferation and
liver regeneration after PHx accompanied by an altered gene expression of potential
cell cyclerelated proteins and other signalings (Liu et al., 2010).
12.7
Immunological Models
The innate immune system is the rst line of defense against initial environmental challenges and insults. It plays a decisive role in different types of liver injury and participates
in orchestrating of liver regeneration. In the liver, the innate immune system comprises
NK cells, NKT cells, Kupffer cells (KC), as well as neutrophils and complement components. Further, also cells of the adaptive immune system such as T-cells and B-cells
were shown to considerably inuence liver regeneration (Tumanov et al., 2009). Though
much has been learned about the impact and role of all these cells and components
in liver regeneration, there is still much uncertainty about the mutual interdependence
between regeneration and inammation. Concerning decisive immunological models
only Concanavalin A-induced liver injury will be mentioned here, because others (e.g.,
CXC chemokine receptor KO, or lymphotoxin alpha receptor KO) fall into the category
of TG models already presented previously, but are too specialized to be discussed here
in detail.
12.7
12.7.1
Immunological Models
187
Concanavalin A Hepatotoxicity
Intravenous administration of Concanavalin A (ConA) is an excellent model resembling

immune-mediated fulminant hepatic failure in humans. ConA rapidly induces clinical and histological evidence of hepatitis, including elevation of transaminases, T-cell
inltration, and necrosis. ConA-induced liver injury is mediated by the activation of the
innate and adaptive immune cells, including NK cells, Kupffer cells, and CD4 T-cells,
and their production of inammatory cytokines, such as TNF- and IFN-. Interestingly,
tolerance develops toward ConA within 8 days (Erhardt and Tiegs, 2010).
Summary
Animal models represent the core experimental systems in any type of research of liver regeneration because it is impossible to maintain the injured organ outside the body long enough
to allow for its full recovery. Beyond these technical limitations there is increasing evidence
showing that regeneration is dependent on extrahepatic signals on various levels up to the
point of restricting liver growth to the optimal size tting to gender and age of the respective
animal. Because of this importance, we have tried to provide an overview about existing
models and to discuss their advantages and limitations as well as some of their contributions
to the understanding of cellular and molecular mechanism of regenerative processes. In addition, important technical improvements made over time with some of these models were
emphasized in order to help the reader to prot as much as possible from existing models or
to design new ones avoiding already identied drawbacks for the benet of future research.
Further Reading
Bockamp, E., Sprengel, R., Eshkind, L., Lehmann, T., Braun, J.M., Emmrich, F., and Hengstler
J.G. (2008). Conditional transgenic mouse models: from the basics to genome-wide sets of
knockouts and current studies of tissue regeneration. Regen. Med. 3, 21735.
Bhm, F., Kller, U.A., Speicher, T., and Werner, S. (2010). Regulation of liver regeneration by
growth factors and cytokines. EMBO Mol. Med. 2, 294305.
Chu, J., and Sadler, K.C. (2009). New school in liver development: lessons from zebrash.
deGraaf, W., Bennink, R.J., Vetelinen, R., and van Gulik, T.M. (2010). Nuclear Imaging
Techniques for the Assessment of Hepatic Function in Liver Surgery and Transplantation. J.
Nucl. Med. 51, 74252.
Dong, Z., Wei, H., Sun, R., and Tian, Z. (2007). The Roles of Innate Immune Cells in Liver
Injury and Regeneration. Cell. Mol. Immun. 4, 24152.
Liu, H., and Zhu, S. (2009). Present status and future perspectives of preoperative portal vein
embolization. Am. J. Surg. 197, 68690.
Martins, P.N., Theruvath, T.P., and Neuhaus, P. (2008). Rodent models of partial hepatectomies. Liver Int. 28, 311.
Weber, A., Groyer-Picard, M.T., Franco, D., and Dagher, I. (2009). Hepatocyte transplantation
in animal models. Liver Transpl. 15, 714.
Zaret, K.S., and Grompe, M. (2008). Generation and regeneration of cells of the liver and
pancreas. Science 322, 14904.
188
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13 Therapeutic Potential of Bone Marrow

Stem Cells in Liver Surgery
Jan Schulte am Esch, Moritz Schmelzle, Gnter Frst,
and Wolfram Trudo Knoefel
Learning Targets
1.
2.
3.
4.
5.
Clinical background for stem cell therapy in resective liver surgery

Role of intrahepatic stem cell niches and extrahepatic progenitor cells for liver
Regeneration before and after hepatic resection
Mechanisms of stem cell contribution to hepatic regeneration
Status quo of clinical application of extrahepatic autologous stem cells to support
liver regeneration
6. Initial experience with bone marrow stem cell therapy to support liver proliferation
concepts
13.1
Clinical Scenario
Complete resection of hepatic tumor remains the rst choice for curative treatment
in patients with primary or secondary hepatobiliary malignant tumors. In up to 45%
of patients extended hepatectomy (more than four segments) is necessary to achieve
margin negative resection. Until now the minimal hepatic volume required to support
postoperative liver function has not been clearly dened. However, it is generally accepted that patients with an anticipated future liver remnant volume (FLRV) below 25%
of the total liver volume (TLV) have an increased risk of post-operative morbidity and
mortality (Bozzetti et al., 1992; Brancatisano et al., 1998; Cunningham et al., 1994;
Hemming et al., 2003). In these patients, the concept of preoperative expansion of the
left-lateral FLRV (segments II and III), utilizing selective portal venous embolization
(PVE) of contra-lateral liver segments I and IV to VIII is increasingly performed as a
safe and effective concept to provide a proliferation stimulus (Broering et al., 2002;
Hemming et al., 2003).
High regeneration rates up to 20 ml/day and relative volume gains of more than
30% have been reported for patients without cirrhosis or diabetes mellitus subsequent
to PVE prior to standard right hepatectomy (de Baire et al., 1996; Lee et al., 1993).
However, patients eligible for extended liver resection frequently suffer from large and
fast progressing liver lesions, limiting the waiting time after PVE to reach an adequate
left lateral liver mass. Time to surgery may be unacceptably long (observed to be up to
150 days), particularly if the left lateral liver segments determining the FLRV are small
and quality of hepatic parenchyma is limited by cirrhosis, brosis, severe steatosis, or
192
13 Therapeutic Potential of Bone Marrow Stem Cells in Liver Surgery
hepatotoxic chemotherapy (Broering et al., 2002). These patients, in particular, may be

initially considered unsuitable for resection due to lack of sufcient remaining normal
parenchyma.
To further accelerate hepatic proliferation, we consequently followed a growing
body of evidence suggesting that extrahepatic stem cells (SCs) and hematopoietic and
mesenchymal progenitor cells participate in the concert of liver regeneration (Alison
et al., 2000; Kollet et al., 2003; Petersen et al., 1999; Theise et al., 2000a). This triggered
the exploration of bone marrow stem cells (BMSC) as therapeutic modality to further
accelerate liver augmentation after PVE.
13.2
Mechanisms of Hepatic Regeneration
Liver regeneration is an organized response of the liver to injury and involves incremental changes in morphologic structure, gene expression, and growth factor production (Fausto et al., 2006; Michalopoulos et al., 1997). A great number of mediators
is involved in hepatic regeneration, including cytokines, growth factors, and various
hormones (Fausto et al., 2006; Krieg et al., 2006; Michalopoulos and DeFrances, 1997).
However, the specic cell types involved and the relevant biochemical pathways are
still poorly understood (Lautt, 2007). Several cell types are thought to be responsible
for the self-renewing and regenerative potential of the liver. Hepatocytes themselves
fulll many requirements of self-renewal by showing the capacity for almost limitless
proliferation. These differentiated cells play a major role in liver regeneration, particularly in the surgical context (Fausto et al., 2006; Michalopoulos and DeFrances, 1997).
Beside these differentiated, mature hepatocytes, the liver also seems to contain distinct
separate SC compartments (Dunkelberg et al., 2001). Kuwahara et al. postulated four
possible hepatic stem cell niches: viz. the canal of Hering (proximal biliary tree), intralobular bile ducts, peribiliary hepatocytes, and periductular mononuclear cells that are
located in the space of Mall (Greene et al., 2003; Kuwahara et al., 2008). Furthermore,
the space of Diss was shown to have stem cell niche properties (Sawitza et al., 2009).
13.3
Stem Cells in Liver Regeneration
The ability to dene and then manipulate hepatic stem/progenitor cells promises signicant advances in our treatment of liver disease. For this reason, it is critical to dene
the basic parameters of resident liver stem/progenitor cells: their size, precise location
within the liver, morphology, and their interaction with other cells in the liver (Cantz
et al., 2008). Hepatic oval cells, assumed to be representative of hepatic progenitor cells,
can be induced by specic hepatic toxins. These cells have been shown to differentiate into both hepatocytes and cholangiocytes. Oval cells gain importance particularly
in chronic liver disease with decreased or perturbed replenishment of differentiated
hepatocytes (Murphy and Iredale, 2003; Wiemann et al., 2002). Interestingly, oval cell
proliferation is mediated by activation of granulocyte-colony-stimulating factor (G-CSF)
receptors (Piscaglia et al., 2007). Cells positive for the hematopoietic marker CD133
proliferate in response to liver injury, and these represent a subgroup of the population
of oval cells (Rountree et al., 2007). Recently, the space of Diss was demonstrated to
share features of a SC niche (see Chapter 5). Evidence is growing to support a role for
13.4
Mesenchymal or Hematopoietic Stem Cells to Support Liver Regeneration?
193
SCs in the treatment of acute and chronic liver diseases. Several in vivo and in vitro
studies demonstrate a signicant BM-derived contribution to hepatocyte turnover after
injury, which can lead to an improvement in liver function (Alison et al., 2000; Avital
et al., 2001; Korbling et al., 2002; Krause et al., 2001; Mallet et al., 2002; Newsome
et al., 2003; Theise et al., 2000a; Theise et al., 2000b). However, low numbers of hepatocytes are thought to develop as a consequence of cellular fusion between putative stem cells with hepatocytes in vivo that complicate interpretation of these studies.
Despite these caveats, several lines of evidence suggest promising clinical potential of
administered bone marrow cells (BMSC) and exogenous stimuli in the augmentation of
liver regeneration (Frst et al., 2007; Schulte am Esch et al., 2005).
Beyond hepatocytes and intrahepatic-based SC compartments an extrahepatic source
of liver- and hepatic sinusoid repopulating cells seems to ensconce itself in bone marrow. These bone marrowderived circulating cells have been shown experimentally to
participate in hepatic proliferation after liver resection (Fujii et al., 2002). Consequently,
a certain relevance of BMSC for liver regeneration has been postulated.
13.4 Mesenchymal or Hematopoietic Stem Cells to Support

Liver Regeneration?
Two types of adult extrahepatic SCs, hematopoietic stem cells and mesenchymal stem
cells (MSC), are found predominantly in the BM and have been investigated in vitro and
in several clinical trials.
Human hematopoietic stem cells like CD133+/CD14+ leukocytes, bearing the capacity to differentiate in vitro into a hepatic lineage, demonstrated peripheral mobilization after partial loss of liver tissue subsequent to clinical hepatectomy (that was
not observed for other major abdominal surgery) (Gehling et al., 2005). The latter was
similarly observed for CD34+ hematopoietic stem cells (De Silvestro et al., 2004). Bone
marrowderived hepatocytes may populate the regenerating liver and transdifferentiate
without fusion ( Jang et al., 2004; Newsome et al., 2003). Also, conversion to liver
cells via cellular fusion (bone marrow cell with hepatocyte) was reported (Vassilopoulos
et al., 2003; Wang et al., 2003). Hematopoietic stem cells to substitute intrahepatic SC
compartments were hypothesized as another way to support liver regeneration (Murphy
et al., 2003). Although now disputed it was suggested that oval cells can express the
hematopoietic stem cell markers CD34+ (Blakolmer et al., 1995; Lemmer et al., 1998),
Thy1+ (Fiegel et al., 2003), and c-kit (Monga et al., 2001). However, a growing body
of work suggests that liver progenitor cells (oval cells) are an independent stem cell
population, distinct from hematopoietic stem cells (Nierhoff et al., 2005).
MSC are promising candidates for a cell-based treatment of liver diseases because
they can easily be harvested from adult bone marrow and expanded in vitro (Fiegel
et al., 2006) Recently, bone marrowderived MSC were demonstrated to provide rescue
in experimental liver failure and were discussed to offer a potentially alternative therapy
to organ transplantation (Kuo et al., 2008). Co-cultured with liver cells MSC express
albumin-, CK-18, CK-19, and AFP at the transcriptional level over 3 weeks (Lange et al.,
2006). MSC were given to rats, subsequent to toxic (CCl4) liver damage. Attenuation of
the liver damage and improved recovery was demonstrated (Oyagi et al., 2006; Yannaki
et al., 2005). However, transplanted MSC were found to have a propensity to form
194
myobroblast-like cells (scar-forming cells) in the areas of hepatic injury (di Bonzo
et al., 2008), and MSC are the major source of BM-derived myobroblasts (Russo et al.,
2006). These data demonstrate the advantages and disadvantages of MSC. Especially
if given in vivo to damaged organs, these characteristics of MSC should be taken into
consideration for clinical trials of MSC-therapy for liver cirrhosis (Forbes et al., 2008).
13.5
BMSC as External Conductors of Liver Regeneration
BMSC have been demonstrated to contribute to the non-parenchymal cells within the
liver, such as neutrophils, lymphocytes, and other inammatory cells, and may play a
role in the immunoregulation of the liver. Others hypothesized that BMSC may serve
as a source for the replacement of endothelial cells and may provide crucial factors
required for efcient healing of the damaged liver (Grompe, 2003).
Two basic hypotheses are discussed regarding how extrahepatic BMSC may contribute to the regeneration of the damaged liver subsequent to therapeutic application. One
concept is that adding hepatocytes and hepatocyte precursors to the regenerating liver
can reconstitute the local body of primary liver cells (Almeida-Porada et al., 2004; Fiegel et al., 2006, Okumoto et al., 2006; Thorgeirsson and Grisham, 2006). Alternatively
extrahepatic BMSC could remodel the local regenerating capacity. The latter may optimize the high potential of the liver for self-renewal via direct and/or humoral interaction or horizontal gene transfer with (to) liver-based actors and infrastructure in hepatic
healing processes (Brulport et al., 2007; Forbes et al., 2008; Grompe et al., 2003).
Although both phenomena may occur, the second hypothesis solves the contradiction that bone marrow was demonstrated to participate in extensive forms of liver
regeneration (Theise et al., 2000b; Theise et al., 2002), but homing of BMSC as primary
liver cells in the regenerating liver is rare (Cantz et al., 2004; Fausto et al., 2003).
Whether BMSC contribute to regeneration indirectly by coordinating the local regeneration process or directly by transdifferentiation to (modied) hepatocytes (formed by
transdifferentiation or fusion) remains unclear and needs to be further evaluated. Both
may occur simultaneously, independently, or as an alliance in hepatic regeneration.
13.6 Stem Cell Treatment in Chronic Liver Disease in Humans

Experimental data have stimulated a fast growing number of clinical trials. In cardiology, there are now many studies with controlled and some double-blinded trials using
BMSC therapeutically to promote recovery of left ventricular systolic function in patients
with myocardial infarction. They show variable success. In contrast, clinical trials using
BMSC to treat patients with liver disease are still mostly uncontrolled and small-scale
feasibility and safety studies. Most of the trials investigated whether these procedures
led to clinical benet in patients with chronic liver disease. Serum albumin level, bilirubin level, international normalized ratio (INR), Child-Pugh, and/or model for end-stage
liver disease (MELD) score or other scores were measured at baseline and after various
follow-up periods.
There are several published clinical studies on stem cellbased treatment of liver
disease (Gaia et al., 2006; Gordon et al., 2006; Levicar et al., 2008; Lyra et al., 2007;
Lyra et al., 2007; Mohamadnejad et al., 2007, Mohamadnejad et al., 2007, Terai et al.,
13.7
BMSC to Support Liver Proliferation Prior to Hepatectomy
195
2006; Yannaki et al., 2006). A critical review has been given by Houlihan and Newsome (2008). In one study mobilization of BMSC with G-CSF was used in patients with
chronic liver disease. Results were encouraging in so far as the Child-Pugh score improved by 2 points or more in 4 of 8 patients, whereas it deteriorated in 1 patient and
remained unchanged in the remaining 3 patients (Gaia et al., 2006). In a second type of
trials autologous bone marrow cells were collected and administered either into a peripheral vein or the hepatic artery (Lyra et al., 2007a; Lyra et al., 2007b; Mohamadnejad
et al., 2007a, Mohamadnejad et al., 2007b, Terai et al., 2006). Patients with established
cirrhosis or decompensated cirrhosis on a waiting list for liver transplantation were
enrolled. Except for one study that was prematurely terminated after the death of two
of the patients (Mohamadnejad et al., 2007), all studies revealed benets with respect
to the evaluated parameters. A third category of trials investigated effects of collection
( ex vivo manipulation) and reinfusion of mobilized BM cells (Gordon et al., 2006;
Levicar et al., 2008; Yannaki et al., 2006). In a preliminary, uncontrolled study in ve
patients with cirrhosis, Gordon et al. (2006) showed a transient improvement in serum
bilirubin and albumin levels after portal vein or hepatic artery infusion of autologous
CD34+ BMSC. In one patient complete resolution of ascites was observed. Outcomes
of these patients were published recently indicating a trend toward reduced serum bilirubin and increased albumin levels (Levicar et al., 2008). Unsorted peripheral blood
SCs collected after G-CSF administration were used to treat two patients with decompensated alcoholic cirrhosis by Yannaki et al. (2006). Improvements in Child-Pugh
and MELD scores were observed in both patients. Also, cytokines interleukin-6 (IL-6)
and tumor necrosis factor--receptor (TNFR), known to correlate with the outcome in
alcoholic cirrhosis, decreased.
Although these trials provide encouraging results in the treatment of patients with
chronic liver disease, the proof that BMSC robustly induce in vivo organ regeneration is
still lacking. Randomized, controlled, and double-blinded stem cell trials are needed to
conrm these data. Also, in none of the trials so far has colonization or engraftment of
transplanted cells been demonstrated in the recipient liver.
13.7
13.7.1
Patients
Growing evidence suggests the existence of a bone marrowliver axis. Therefore, autologous CD133 BMSCs were used to stimulate liver regeneration in patients scheduled for extended right hepatectomy (Frst et al., 2006; Schulte am Esch et al., 2005).
In this approach, CD133+ BMSC are applied after PVE of contra lateral liver segments,
the latter representing a strong stimulus for liver proliferation of nonembolized hepatic
segments (fFigure 13.1). Until now, 11 patients with large central malignant tumors
of the liver scheduled for extended right hepatectomy underwent PVE and intraportal
administration of CD133+ BMSC. Eleven patients underwent PVE alone and served as
a control group. Patients in both groups were characterized by a future liver remnant
volume of less than 25% of the total liver volume minus tumor volume, except one
patient with liver cirrhosis. In the stem cell group, PVE as an isolated technique was
questionably sufcient to induce adequate proliferation of the left lateral liver segments
within a reasonable time. A panel of criteria was used indicating compromised hepatic
196
parenchyma or co-morbidity, possibly impairing liver regeneration capacity, including

liver cirrhosis (Child-Pugh score A), brosis following chronic replicating hepatitis, severe liver steatosis, and prior hepatotoxic chemotherapy (Nagasue et al., 1987). Other
patients showed unusually low basal volume of segments II and III and fast progressing
liver lesions. None of the control patients had comparable hepatic co-morbidities.
(A)
right, middle and left
hepatic vein
VII
II
VIII
I
III
IV
VI
inferior
vena cava
portal vein
(B)
Figure 13.1A,B
Concept of PVE and Portal Application of BMSC
Notes:
A Autologous BM is aspirated and processed by a GMP-grade cell-separation unit to enrich
for CD133+ cells.
B PVE of liver segments I and IVVIII
13.7
197
left hepatic vein
right, middle and left

hepatic vein
II
VII
II
VIII
III
III
IV
VI
inferior
vena cava
inferior
vena cava
portal vein
portal vein
(C)
(D)
Figure 13.1C,D Concept of PVE and Portal Application of BMSC

Notes:
C Selective readministration of selected cells to the nonoccluded portal branches of liver
segments II and III. The whole procedure from harvesting bone marrow until readministration
is performed in a closed system.
D Extended right hepatectomy with resection of segments I and IVVIII about 3 weeks later.
13.7.2
Preparation, Characterization, and Administration of BMSCs
The procedure of harvesting bone marrow for readministration of selected cells was
performed in a closed system and general anesthesia. Autologous bone marrow aspirated from the posterior iliac crest was drawn in heparin-coated syringes. Bone
marrow cells were prepared simultaneously with PVE. The cell suspension was rst
ltered to remove bone spicula and was then processed by using a cell-separation
unit (ClinMACS; Miltenyi Biotech, Bergisch-Gladbach, Germany) according to GMP
(good-manufacturing-practice) standards to immunomagnetically enrich CD133+
cells as previously described (Ghodsizad et al., 2004). The protocol was certied
by the relevant authorities (Paul Ehrlich Institute). After approximately 2 12 hours
of preparation, the cells were ready for intraportal application. Cells were resuspended in a phosphate-buffered solution. Aliquots from the BM aspirate and the
injected cell fraction were collected for cytouorometric analyses (Frst et al., 2006)
(fFigure 13.2).
For readministration of the cell suspension a 5-F cobra catheter was introduced into
the nonoccluded left lateral portal vein system under uoroscopic guidance (fFigure 13.1). The time needed for the entire procedure ranged between 3.5 and 5.5 hours.
No special medication was required after BMSC administration.
Even though the optimal technique for SC application has not been dened (peripheral venous or hepatic arterial application are alternatively possible), we hypothesized
198

native bone marrow cells
after selection for CD133
1000
side scatter
800
600
400
97.2 %
99.3 %
200
0
100
101
102
CD45
103
104
100
101
102
CD45
103
104
105
104
Pl
103
102
94.8 %
87.7 %
101
100
100
101
102
CD34
103
104
100
101
102
CD34
103
104
105
0.45 %
69.7 %
CD133
104
103
102
101
100
100
101
102
CD34
103
104
100
101
102
CD34
103
104
Figure 13.2 Representative Cytouometric Analyses of Applied CD133+BMSCs (taken

from Schulte am Esch et al., StemCells 2005)
Notes:
Aliquots of unselected bone marrow (left panels) harvested from patients, and the readministered positive fractions after enrichment for stem cell marker CD133 (right panels) were
analyzed by FACS. Upper panels demonstrate the concentration of leucocytes detected by
anti-CD45 antibodies. DNA-stain propidium iodide was employed to assess rate of cell viability (middle panels). Utilizing anti-CD133 and anti-CD34 antibodies the concentration for
CD133+ cells was evaluated (lower panels).
that the direct portal administration of high concentrations of CD133+ BMSC may ease
the homing to the target segments II and III. The rationale for this application mode was
supported by a study in which a high percentage of rst-pass entrapment of BMSC to the
liver when applied to the portal vein was reported (Fan et al., 2001). In the same study
it was suggested that interaction of SCs with stromal cells in the liver is a crucial step for
successful engraftment.
13.7
199
13.7.3 Hepatic Proliferation Subsequent to Autologous

BMSC Application
All patients underwent helical computed tomography (CT) to estimate liver volumes
prior to PVE, 2 weeks after PVE, and then in 12 weeks intervals to determine the degree
of induced hypertrophy until the prospectively remaining hepatic volume was adequate
for extended right hepatectomy. CT examinations were performed using multisection
CT scanners. Transverse scans were obtained in the portal venous phase to measure
the total liver volume, the future liver remnant volume (segments II and III), and the
intrahepatic tumor volume. We found a 2.4-fold higher mean daily hepatic growth rate
compared with patients who underwent PVE alone (p < 0.004) (fFigure 13.3). Also,
the relative volume gain of hepatic segments II/III was signicantly higher in the patients
additionally treated with BMSC (70.8% versus 41.3%; p < 0.005). The increased proliferation rate resulted in a signicant reduction of the waiting time from PVE to liver
resection (46 days versus 30 days; p < 0.05) in our early experience (Frst et al., 2006;
Schulte am Esch et al., 2005).
p = 0.004
p = 0.005
200
14
absolute change in volume

after 14 days (ml)
change in volume
after 14 days (% of initial TLV)
16
12
10
8
6
4
2
0
150
100
50
0
PVE + BMSCs
PVE
PVE + BMSCs
PVE
Figure 13.3 Total (ml) and relative (% of initial total liver volume [TLV]) gain in liver volume (left segments II and III) by day 14 subsequent to portal venous embolization (PVE)
with (n = 11) vs. without (n = 11) portal CD133+ bone marrow stem cell application (BMSC)
in patients with large and/or central liver tumors prior to resection surgery. *p values determined by students t test.
13.7.4
Safety and Clinical Outcome
No complications or side effects linked to the BMSC-treatment were observed. Solely,

minimal transient elevations of the routinely assessed markers (total bilirubin level [INR]
and aspartate aminotransferase [AST] and alanine aminotransferase levels [ALT]) normalized to their pre-interventional levels 4 or 5 days after PVE, which indicated no lasting effect on liver metabolism, hepatic synthetic capacity, and hepatocellular integrity.
There were no differences concerning patient and oncological characteristics between the two groups. In three patients with preoperative PVE alone right hepatic trisectionectomy was not feasible due to advanced disease. Only one patient in the group
200
of additional portal administration of autologous CD133+ BMSC was ultimately not

eligible for curative resection due to tumor progress. One other patient in the latter
treatment group, suffering from Child Pugh A liver cirrhosis, was not scheduled for liver
resection subsequent to a signicant impairment of physical condition with marked
limitation of activity (NYHA III).
Within the rst post-operative week following extended liver resection, no differences
in markers of liver damage or function were observed for BMSC plus PVE treatment if
contrasted to PVE alone. Those markers included AST (p = 0.533), bilirubin concentrations in serum (p = 0.474), and the coagulation marker international normalized ratio
(INR; p = 0.122). Kaplan Meier survival was statistically not different among groups
(p = 0.929). No differences concerning the rate of tumor relapse after a median follow-up
of 39 months have been observed among patient groups (p = 0.819) (unpublished data).
These data suggest, that the faster proliferated liver tissue subsequent to PVE and portal
administration of autologous CD133+ BMSC is functional comparable to liver tissue
proliferated subsequent to PVE alone. The shorter time of hepatic proliferation seems to
be without negative impact on function and outcome following large liver resection.
The data currently available suggest that portal administration of autologous CD133+
BMSC is a safe and a promising additive of inducing preoperative hepatic proliferation.
It seems to be superior to PVE alone in preparation for extensive liver resection in
particular for patients with very small left lateral segments, limited quality of hepatic
parenchyma, or a large and fast-progressing tumor mass.
Summary
In patients scheduled for extensive hepatectomies, the functional future liver remnant capacity may be insufcient as adequate liver function reserve, due to small remnant volume or
compromised parenchymal quality. Standard measures, like portal venous embolization (PVE)
of hepatic segments to be resected as an isolated modality, may fail to induce an adequate
hepatic volume response within a reasonable period of time. In particular, patients with very
small future liver remnant volume or hepatic co-morbidity impairing liver regeneration capacity may be initially considered unsuitable for resection. Hematopoietic stem cells and MSC are
promising candidates for cell-based approaches for the treatment of liver diseases. Possible
stem cell interactions with the liver include stimulation of endogeneous hepatocyte proliferation, transdifferentiation to hepatocytes, fusion of stem cells and hepatocytes, and antibrotic
and immunomodulatory effects. In a new concept autologous CD133+ hematopoietic stem
cells are used to augment left lateral liver volume prior to extended liver resection. PVE
was used as a strong proliferation stimulus to the nonembolized liver segments. Signicantly
increased hepatic growth rates and reduction of waiting time from PVE to liver resection were
found compared to a control group. No complications or side effects linked to the stem cell
treatment were observed. Clinical follow-up revealed no signicant differences in tumor free
survival times and recurrence rates. Beyond a signicant impact on the surgical treatment of
oncological patients requiring extensive liver resection, such concepts bear the potential to
open novel therapeutic options to promote organ regeneration in various scenarios of acute
and chronic liver damage.
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Xu, Y. Q. and Z. C. Liu (2008). Therapeutic Potential of Adult Bone Marrow Stem Cells in Liver
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Index
Acetaminophen, 180
Activin-A, 10
Acute phase response, 41
Adiponectin, 184
Adipose tissue, 29
AKT, 8
A-smooth muscle actin, 91
Angiogenesis, 145, 149
Angiogenesis assays, 153
Angiopoietin-2, 150
Animal models, 175
Apoptosis, 135, 136
ATP-dependent chromatin-remodelling
factors, 102
Basal lamina, 91
Basement membrane, 71, 146, 153
-catenin, 5, 66, 93, 159, 160, 164, 165,
166, 172, 180
Bile duct ligation, 180
Bisulte conversion, 103
Blood ow, 146, 152
Blood vessels, 145, 146, 149, 152, 153,
154
Bmp signalling, 65
Cadherins, 71
Canal of Hering, 192
Capillaries, 146
Capillarization, 152
Carbon tetrachloride, 4
CD95 (Fas/APO-1), 129, 132, 133
CD95 ligand, 130, 132, 135, 136
CD95 phosphorylation, 129, 133, 134
CD95 tyrosine nitration, 130, 133, 136
CD133, 89, 192
C/EBPbeta, 7
Cell therapy, 30
Central venule, 146, 147
chemokine cysteine-X-cystein motif

receptor 4 (CXCR4), 70
Chemokines, 46
Chemotaxis, 46
Cholangiocytes, 2
Choline-decient, 180
Chronic liver disease, 151
Cirrhosis, 91, 152
C-Jun, 7
C-Jun-N-terminal kinase, 8, 129, 132,
133, 136, 138
C-myc, 7
Collagen, 91
Compensatory hyperplasia, 2, 57
Concanavalin A, 178, 187
Constitutive androstane receptor,
185
CpG islands, 100
Cyclopamine, 115
Cytokeratin 19, 91, 95
Cytokines, 178, 179, 180, 183, 187
Cytosine phosphatidyl guanosine (CpG),
100
Desmin, 91
DNA methylation, 100
DNA methyl transferases (Dnmt), 100
E-cadherin, 5
Endothelial cell, 147, 153
Epidermal growth factor (EGF), 6,135
Epidermal growth factor receptor (EGFR),
129, 130, 132, 133, 135137
Epigenetic
denition, 99
hepatic stellate cells, 105
histone modications, 102
liver regeneration, 104
208
Index
mechanisms, 100
non-coding RNA, 102
stem cell-based liver regeneration,
105
Epithelial-to-mesenchymal, 120
Euchromatin, 101
Extracellular matrix (ECM), 149, 152
Farnesoid X receptor, 185
Fenestrated endothelium, 146, 147
Fibroblast growth factor, 184
Fibrosis, 151
5-brome-2-deoxyuridine, 91
Fusion, 19, 34
G-CSF, 29
Gene-targeting strategies, 182
Glioblastoma (Gli), 114
Glutamine synthetase, 91
Glycoprotein 130, 7
Glypican-3, 9, 186
Granulation tissue, 151
Growth factors, 179
Growth inhibitory pathways, 40
Growth promoting pathways, 40
Hedgehog signalling, 68, 112
Hematopoietic stem cells, 25, 193
Heparin-binding EGF (HB-EGF), 6
Hepatectomy, 191
Hepatic artery, 145, 146
Hepatic progenitor cells, 22, 33, 37
Hepatic stellate cells, 120, 136, 192
5-aza-2-deoxycytidine, 106
activation, 87
bone marrow, 89
CD133, 89
CD133 DNA methylation, 106
characterization, 85
CXCR4/SDF1, 77
desmin, 87
differentiation, 90
duct-like structures, 95
brosis, 87
glial brillary acidic protein, 87
hedgehog signalling, 76
HSC, 130, 135
isolation, 85
Nestin, 87
Nestin DNA methylation, 106
Nestin histone modications,
106
Notch3 DNA methylation, 106
Notch signalling, 77
plasticity, 90
space of Diss, 75
synemin, 87
transplantation, 90
trichostatin A, 105
vimentin, 87
vitamin A, 85
Wnt signalling, 76
Hepatocellular carcinoma (HCC), 149
Hepatocyte division, 180
Hepatocyte growth factor (HGF), 6, 29,
183
Hepatocyte plates, 146
Hepatocytes, 2, 148, 192
Hering
canal of, 192
Heterochromatin, 101
HHIP, 120
Hh-responsive, 115
Hierarchy of potentialities, 18
Histone modications, 102
acetylation, 102
ADP-ribosylation, 102
chromatin immunoprecipitation,
104
methylation, 102
Nestin, 106
phosphorylation, 102
SUMOylation, 102
ubH2A, 104
ubiquitylation, 102
Hydrophobic bile acids, 132, 136, 138
Hyperosmolarity, 132
Image analysis, 161
Image processing, 161
Immunological models, 186
Indian hedgehog (Ihh), 115
Inammatory response, 40
Initiation/priming, 4
Index
Innate immunity, 40
Integrin-linked kinase (ILK), 9, 186
Integrins, 71
Intercellular adhesion molecule (ICAM)1, 42
Interleukin-1, 10
Interleukin-6 (IL-6), 7, 58, 184
Intracellular networks, 40
Intussusceptive angiogenesis, 145
NADPH oxidase, 132

Nestin, 91
histone modications, 106
NFKB, 7, 55, 56
Niche, 18, 24, 63
Nitric oxide (NO), 5
Notch signalling, 5, 71
Nuclear receptors, 183
Jagged-1, 5
JNK, 8, 129, 132, 133, 136, 138
Johnson-Kendall-Roberts model, 166
Outow obstruction, 179

Oval cells, 22, 91, 180, 192,
193
canals of Hering, 74
Notch signalling, 75
Wnt signalling, 74
Kupffer cells, 2, 58, 148, 186

Laminin, 91
Leukocyte recruitment, 45
LIGHT, 58
Lipopolysaccharide, 58
Liver cirrhosis, 194
Liver injury, 186
Liver macrophages, 39
Liver regeneration, 111, 175, 176
Liver sinusoidal endothelial cells,
146
Liver stem cells, 17, 21, 22, 23, 33, 35
Liver zonation, 91
Lobular structure, 176
Lymphotoxin receptor, 58
Macrophages, 42
Malignant tumor
of the liver, 195
MAPK, 8
Mass ligation, 179
Mathematical modeling, 166
Matrix metalloproteinases, 8, 29,
186
Mesenchymal stem cells, 26, 193
Methyl-CpG binding protein 2 (MeCP2),
100
Methylcytosine, 100
Microenvironment, 117
Multipotency, 18
Multipotent adult progenitor cells, 26
Myobroblasts, 151, 194
209
Partial hepatectomy (PH), 2, 57, 87, 112,

130, 148, 149, 179
PDK1, 8
Peroxisome proliferator-activated
receptor-, 185
Pharmacological models, 177
PI3K, 8
Pit cells, 148
Plasminogen activator inhibitor-1,
185
Plasticity, 18, 19, 20, 25, 26, 30, 34, 37,
38
Portal hypertension, 152, 153
Portal tracts, 146
Portal triad, 146, 152
Portal vein, 145, 146
Portal venous embolization, 191
Pregnane X receptor, 185
Priming pathways, 40
Proliferation, 4, 129, 136, 137
Protein C inhibitor, 185
Recruitment, 42
RNA
interference RNA (RNAi), 102
microRNA (miRNA), 102
miR-21, 105
non-coding RNA, 102
small interference RNA (siRNA),
102
210
Index
SDF-1, 28
Self-maintenance, 17
Sieve plates, 147
Sinusoidal endothelial cells, 2
Sinusoids, 146, 149, 152
Sonic hedgehog, 112
Space of Diss, 90, 147
Sprouting angiogenesis, 145
STAT-3, 7
Stem cell niche, 24, 72, 74, 192
basal lamina, 71
blood vessels, 72
canals of Hering, 74
cell-cell contacts, 71
identication, 72
neighbouring cells, 64
secreted factors, 64
space of Diss, 75
stem cells, 78
sympathetic nervous system, 72
Stem cells
asymmetric replication, 63
hematopoietic, 193
liver, 17, 21, 22, 23, 33, 35, 64, 90
mesenchymal, 193
stochastic replication, 63
symmetric replication, 63
Stemness, 17, 27
Stromal cell-derived factor-1 (SDF1), 70
Surgical models, 177
Toxic injury, 182

Transcription factors, 179
Transforming growth factor A (TGFA), 6
Transforming growth factor (TGF), 8,
183
Transforming growth factor (TGF)
receptor I, 8
receptor II, 8
signalling, 65
Transgenic mice, 175, 181, 189
Transgenic models, 178
Tumorigenesis, 131
Tumor necrosis factor-alpha (TNFalpha),
7, 58, 184
Tumor necrosis factor (TNF) receptor, 7,
44, 53, 54, 55, 57
2-acetylaminouorene, 4, 89
Urokinase plasminogen activator, 5
Varices, 152
Vascular endothelial growth factor
(VEGF), 9, 151
Vasculature, 145
Viral models, 177
Wnt signalling, 66, 67, 76, 93, 183
Yes-associated protein (YAP), 10
Termination, 4
Thymocyte antigen-1 (Thy-1), 91

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Liver Regeneration

Edited by Dieter Hussinger

Liver Regeneration and Partial Hepatectomy: Process and Prototype . . . . . . .

Oval Cells, Bone Marrow, and Liver Regeneration . . . . . . . . . . . . . . . . . . . . .

2.1 Stem Cells: Denition and Properties . . . . . . . . . . . . . . . . . . . . . . . . . .

Inammation and Liver Regeneration. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Lymphotoxin Receptor and Tumor Necrosis Factor Receptor p55 in Liver

4.1 The TNF/TNFR Superfamily . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

5 The Hepatic Stem Cell Niches . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Stellate Cells in the Regenerating Liver. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Characterization of Stellate Cells. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Epigenetics during Liver Regeneration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Denition and Mechanisms of Epigenetics . . . . . . . . . . . . . . . . . . . . . .

Hedgehog Signaling and Liver Regeneration . . . . . . . . . . . . . . . . . . . . . . . . .

Hedgehog Pathway Activation and Liver Fibrosis . . . . . . . . . . . . . . . .

EGFR, CD95, and the Switch between Proliferation and

10 Angiogenesis and Liver Regeneration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

11 A Quantitative Mathematical Modeling Approach to

12 Animal Models for Studies on Liver Regeneration . . . . . . . . . . . . . . . . . . . .

12.3 Different Types of Animal Models . . . . . . . . . . . . . . . . . . . . . . . . . . .

13 Therapeutic Potential of Bone Marrow Stem Cells in Liver Surgery . . . . . . . .

Anna Mae Diehl, MD

Antonio Gasbarrini, PhD

Wolfram Trudo Knoefel, MD

future liver remnant volume

Liver Regeneration and Partial Hepatectomy:

The liver is characterized by a unique and extraordinary capacity for self-renewal; it

Liver Regeneration: Historical Perspective

Liver Regeneration and Partial Hepatectomy

Partial Hepatectomy as a Means to Study Liver

Partial Hepatectomy as a Means to Study Liver Regeneration

portal venous pressure

Figure 1.1 Liver Regeneration after 2/3 Partial Hepatectomy

Liver Regeneration and Partial Hepatectomy

1.4 Three Phases of Liver Regeneration after Partial

initiation/primingthe majority of hepatocytes exit a quiescent state (G0), enter

1.4 Three Phases of Liver Regeneration after Partial Hepatectomy

Phase One: Initiation/Priming

Liver Regeneration and Partial Hepatectomy

1.4 Three Phases of Liver Regeneration after Partial Hepatectomy

Liver Regeneration and Partial Hepatectomy

1.4 Three Phases of Liver Regeneration after Partial Hepatectomy

Phase Two: Proliferation

Phase Three: Termination

Liver Regeneration and Partial Hepatectomy

Liver Regeneration and Partial Hepatectomy

Liver Regeneration and Partial Hepatectomy

Liver Regeneration and Partial Hepatectomy

Oval Cells, Bone Marrow, and Liver

Stem Cells: Denition and Properties

Stemness can be dened by two fundamental properties: self-maintenance and

Oval Cells, Bone Marrow, and Liver Regeneration

Mechanisms for the Maintenance of Stem Cell Numbers

Stem Cells: Denition and Properties

Oval Cells, Bone Marrow, and Liver Regeneration

Table 2.1 Glossary of Stem Cell Terminology

A mechanism of plasticity that consists in the melting of

The ability to divide and produce a limited number of other

The ability of a stem cell to give rise to a cell type outside

The ability to divide and produce cells of any of the three

An immature cell with a restricted differentiation potential

Undifferentiated cell that is able to self-renew and

STEM CELL NICHE

The environment of stem cells, formed mainly by the