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Liver Regeneration

Edited by Dieter Hussinger


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Liver RegenerationEdited by Dieter Hussinger

DE GRUYTER


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Editor

Prof. Dr. Dieter Hussinger

Department of Internal Medicine

Gastroenterology, Hepatology and Infectious Diseases

University Hospital Dsseldorf

Heinrich-Heine-UniversityMoorenstrasse 5

D-40225 Dsseldorf

Germany

This book has 46figures and 7tables.

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 2011611'.36dc22 2011009091

Bibliografic information published by the Deutsche Nationalbibliothek

The Deutsche Nationalbibliothek lists this publication in the Deutsche Nationalbibliografie; 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 informa-

tion presented in this work (programs, applications, amounts, dosages, etc.) reflects the standard of

knowledge at the time of publication. Despite careful manuscript preparation and proof correction,

errors can nevertheless occur. Authors, editors and publisher disclaim all responsibility and for anyerrors or omissions or liability for the results obtained from use of the information, or parts thereof,

contained in this work.

The citation of registered names, trade names, trade marks, etc. in this work does not imply, even in

the absence of a specific statement, that such names are exempt from laws and regulations protecting

trade marks etc. and therefore free for general use.

Typesetting: Apex CoVantage, LLC

Graphic designer: Dr. Martin Lay, Breisach a. Rh., Germany; [email protected]

Printing and binding: Hubert & Co. GmbH & Co. KG, Gttingen

UPrinted on acid-free paperPrinted in Germany

www.degruyter.com


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Preface

The liver has a high capacity to regenerate, which was already known in ancientGreece, as exemplified in the Prometheus saga. Although liver regeneration has beenparadigmatic for organ repair and renewal for more than 2,000 years, only during thepast decades has much effort been devoted to the understanding of the molecular andcell biological mechanisms underlying liver regeneration. Such knowledge is of crucialimportance 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-leveltext book provides an overview of the current state of knowledge about the molecu-

lar mechanisms of liver regeneration. The chapters were written by renowned expertsand active researchers in the field of liver regeneration; some of them members of theCollaborative Research Center 575 Experimental Hepatology. Hepatic stem cells areintroduced, and the important players involved in regeneration, such as oval cells, bonemarrow, and stellate cells, are reviewed. Also, the cell-signaling pathways that initiateliver regeneration and regulate the switch between proliferation and apoptosis are pre-sented. The book also treats the epigenetic regulation of liver stem cells and the roles ofinflammation and angiogenesis in liver regeneration. This compact overview of the fas-cinating regenerative capacity of the liver will be of interest to both, graduate studentsand postdoctorate scientists in molecular biology, biochemistry, and medicine, and it is

hoped that this survey on the various aspects of liver regeneration will stimulate furtherresearch in this area and help young scientists develop their research strategies. Thetopics 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 excellentcontributions but also to my collaborators, Mrs. Katrin Nagel, editor for science, tech-nology, and medicine, from de Gruyter Publishers for her excellent collaboration andprofessional help in preparing and producing this book project, and Dr. Martin Lay forthe artwork and beautiful illustrations.

Dsseldorf, May 2011

Dieter Hussinger


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Contents

Author Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xi

Abbreviations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xv

1 Liver Regeneration and Partial Hepatectomy: Process and Prototype . . . . . . . 1

Marie C. DeFrances and George K. Michalopoulos

1.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1

1.2 Liver Regeneration: Historical Perspective. . . . . . . . . . . . . . . . . . . . . . . 1

1.3 Partial Hepatectomy as a Means to Study Liver Regeneration . . . . . . . . 2

1.4 Three Phases of Liver Regeneration after Partial Hepatectomy . . . . . . . . 4

1.5 Future Directions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10

2 Oval Cells, Bone Marrow, and Liver Regeneration . . . . . . . . . . . . . . . . . . . . . 17

Anna C. Piscaglia, Antonio Gasbarrini, and Bryon E. Petersen

2.1 Stem Cells: Definition and Properties . . . . . . . . . . . . . . . . . . . . . . . . . . 17

2.2 Liver Stem Cells and Their Role in Hepatic Regeneration. . . . . . . . . . . . 21

2.3 Extrahepatic Stem Cells with Hepatogenic Potential:The Blood of Prometheus. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25

2.4 Clinical Applications of Bone MarrowDerived Stem Cells

in Hepatology. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30

3 Inflammation and Liver Regeneration. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39

Johannes G. Bode

3.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39

3.2 Liver Regeneration and Inflammation: General Aspects. . . . . . . . . . . . . 40

3.3 Liver Macrophages and Their Relevance forLiver Regeneration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41

3.4 Inflammatory Mediators Are Required to Promote

Liver Regeneration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44

3.5 Inappropriate Inflammation Impairs Liver Regeneration . . . . . . . . . . . . 46

3.6 Role of NK and NKT-cells for Liver Regeneration:

Negative Regulators of Regeneration. . . . . . . . . . . . . . . . . . . . . . . . . . . 47


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viii Contents

4 Lymphotoxin Receptor and Tumor Necrosis Factor Receptor p55 in LiverRegeneration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53

Ursula R. Sorg and Klaus Pfeffer

4.1 The TNF/TNFR Superfamily . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53

4.2 Liver Regeneration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 564.3 TNFRp55 and Liver Regeneration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57

4.4 LTR and Liver Regeneration. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58

5 The Hepatic Stem Cell Niches . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63

Iris Sawitza, Claus Kordes, and Dieter Hussinger

5.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63

5.2 Secreted Factors in the Stem Cell Niche . . . . . . . . . . . . . . . . . . . . . . . . 64

5.3 Physical Contacts of Stem Cells with Their Niche . . . . . . . . . . . . . . . . . 71

5.4 Identification of Stem Cell Niches . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 725.5 Stem Cell Niches in the Liver . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 74

6 Stellate Cells in the Regenerating Liver. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85

Claus Kordes, Iris Sawitza, and Dieter Hussinger

6.1 Characterization of Stellate Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85

6.2 Plasticity of Hepatic Stellate Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 90

6.3 Stellate Cells in Liver Regeneration . . . . . . . . . . . . . . . . . . . . . . . . . . . . 90

7 Epigenetics during Liver Regeneration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 99

Claus Kordes, Iris Sawitza, and Dieter Hussinger

7.1 Definition and Mechanisms of Epigenetics . . . . . . . . . . . . . . . . . . . . . . 99

7.2 Methods to Investigate Epigenetic Mechanisms . . . . . . . . . . . . . . . . . . . 103

7.3 Epigenomics in Liver Regeneration . . . . . . . . . . . . . . . . . . . . . . . . . . . . 104

7.4 Epigenetics During Stellate Cell Activation . . . . . . . . . . . . . . . . . . . . . . 105

8 Hedgehog Signaling and Liver Regeneration . . . . . . . . . . . . . . . . . . . . . . . . . 111

Steve S. Choi and Anna Mae Diehl

8.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111

8.2 Liver Regeneration after Partial Hepatectomy . . . . . . . . . . . . . . . . . . . . 112

8.3 Fetal Development of the Liver . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 112

8.4 Overview of Hedgehog Signaling Pathway . . . . . . . . . . . . . . . . . . . . . . 113

8.5 Reactivation of the Hedgehog Pathway

after Partial Hepatectomy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 115

8.6 Hedgehog Pathway Activation during Repair of

Chronic Liver Injury: General Concepts . . . . . . . . . . . . . . . . . . . . . . . . 116

8.7 Hedgehog Pathway Activation and Liver Progenitorsin Chronic Injury Models . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 117


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Contents ix

8.8 Hedgehog Pathway Activation and Liver Fibrosis . . . . . . . . . . . . . . . . 118

8.9 Hedgehog Pathway Activation and Vascular Remodeling in

Injured Livers. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 121

8.10 Hedgehog Pathway Activation and Hepatocarcinogenesis . . . . . . . . . 121

9 EGFR, CD95, and the Switch between Proliferation and

Apoptosis in Hepatic Stellate Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 129

Roland Reinehr and Dieter Hussinger

9.1 Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 129

9.2 Liver Cell Proliferation Involves Ligand-dependent

EGFR Activation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 130

9.3 Liver Cell Apoptosis Involves EGFR-dependent

CD95 Activation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 132

9.4 EGFR Activation Can Couple to Both Proliferation andApoptosis in Hepatic Stellate Cells. . . . . . . . . . . . . . . . . . . . . . . . . . . . 135

10 Angiogenesis and Liver Regeneration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 145

Tobias Buschmann, Jan Eglinger, and Eckhard Lammert

10.1 Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 145

10.2 Blood Flow and Cell Types in the Adult Liver. . . . . . . . . . . . . . . . . . . 145

10.3 Angiogenesis in Liver Regeneration . . . . . . . . . . . . . . . . . . . . . . . . . . 148

10.4 Importance of VEGF for Liver Regeneration . . . . . . . . . . . . . . . . . . . . 150

10.5 Role of Angiogenesis in Liver Damage/Disease . . . . . . . . . . . . . . . . . 15110.6 Questions and Problems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 153

11 A Quantitative Mathematical Modeling Approach to

Liver Regeneration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 159

Dirk Drasdo, Stefan Hoehme, and Jan G. Hengstler

11.1 Definition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 159

11.2 Methods to Quantify SpatialTemporal Information

in Liver Lobules . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 161

11.3 Normal Liver Lobule: The Reference State . . . . . . . . . . . . . . . . . . . . . 163

11.4 Quantifying the Regeneration Process: Process Parameters . . . . . . . . 164

11.5 Mathematical Model . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 164

11.6 Simulation Results with the Mathematical Model. . . . . . . . . . . . . . . . 169

11.7 Limitations. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 171

12 Animal Models for Studies on Liver Regeneration . . . . . . . . . . . . . . . . . . . . 175

Amalya Hovhannisyan and Rolf Gebhardt

12.1 Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17512.2 Different Types of Regenerative Processes . . . . . . . . . . . . . . . . . . . . . 175


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12.3 Different Types of Animal Models . . . . . . . . . . . . . . . . . . . . . . . . . . . 177

12.4 Surgical Animal Models . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 179

12.5 Pharmacological Models . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 180

12.6 Transgenic Models . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 181

12.7 Immunological Models . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 186

13 Therapeutic Potential of Bone Marrow Stem Cells in Liver Surgery . . . . . . . . 191

Jan Schulte am Esch, Moritz Schmelzle, Gnter Frst, and

Wolfram Trudo Knoefel

13.1 Clinical Scenario . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 191

13.2 Mechanisms of Hepatic Regeneration . . . . . . . . . . . . . . . . . . . . . . . . 192

13.3 Stem Cells in Liver Regeneration . . . . . . . . . . . . . . . . . . . . . . . . . . . . 192

13.4 Mesenchymal or Hematopoietic Stem Cells to

Support Liver Regeneration?. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19313.5 BMSC as External Conductors of Liver Regeneration . . . . . . . . . . . . . 194

13.6 Stem Cell Treatment in Chronic Liver Disease in Humans . . . . . . . . . 194

13.7 BMSC to Support Liver Proliferation Prior to Hepatectomy. . . . . . . . . 195

Index. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 207


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Author Index

Johannes G. Bode, MD


Gastroenterology, Hepatology and

Infectious Diseases


Heinrich-Heine-University

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


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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 LeipzigGermany

Dieter Hussinger, MD



Infectious Diseases



Moorenstrasse 5

D-40225 DsseldorfGermany

Jan G. Hengstler, MD

Leibniz Research Centre for Working

Environment and Human Factors

Ardeystrasse 67

D-44139 Dortmund

Germany

Stefan Hoehme, PhDInterdisciplinary Centre for Bioinformatics


D-04103 Leipzig

Germany

Amalya Hovhannisyan, MD, PhD

Institute of Biochemistry

Faculty of Medicine


Johannisallee 30D- 04103 Leipzig

Germany

Wolfram Trudo Knoefel, MD

Department of General-, Visceral- and

Pediatric Surgery



Moorenstrasse 5D-40225 Dsseldorf

Germany

Claus Kordes, PhD



Infectious Diseases




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


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xiii

Klaus Pfeffer, MD

Institute of Medical Microbiology and

Hospital Hygiene




Anna C. Piscaglia, PhD

Gastrointestinal and Liver Stem Cell

Research Group (GILSteR)


Gemelli Hospital

Catholic University of Rome (Italy)

Largo A. Gemelli

8 00168 RomaItaly

Roland Reinehr, MD



Infectious Diseases



Moorenstrasse 5


Iris Sawitza, PhD



Infectious Diseases



Moorenstrasse 5


Moritz Schmelzle


Pediatric Surgery


Heinrich Heine University


Germany

Jan Schulte am Esch, MD


Pediatric Surgery



Moorenstrasse 5


Ursula R. Sorg, PhD

Institute of Medical Microbiology and

Hospital Hygiene



D-40225 Dsseldorf

Germany


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Abbreviations

2-AAF 2-acetylaminofluorene2/3 PHx two-thirds partial hepatectomyAFP alpha-fetoproteinAP-1 activator protein 1APAP acetaminophenAPP acute phase proteinsASC adult stem cellASH alcoholic steatohepatitisASM acidic sphingomyelinaseATSCs adipose tissue stromal cellsBLP basal lamina proteinsBM bone marrowBmp/BMP bone morphogenetic proteinBMSCs bone marrow stem cellsBTLA B and T lymphocyte attenuatorBV blood vesselsCCC cellcell contactsCCl4 carbon tetrachlorideChIP Chromatin immunoprecipitationCHX cycloheximideCoH canal of HeringCR cysteine richCRD cysteine-rich domainCT computed tomographyDcR3 decoy receptor 3DD death domainsDhh Desert hedgehogDISC death-inducing signaling complexDpp DecapentaplegicDPPIV dipeptidyl-peptidase-IV-deficientECM extracellular matrixEGF epidermal growth factorEGFP enhanced green fluorescent proteinEMT epithelial-to-mesenchymal transitionENA-78 epithelial neutrophil-activating proteinER endoplasmic reticulumESC embryonic stem cellFADD Fas-associated death domainFFA free fatty acidFGF fibroblast growth factors


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xvi Abbreviations

FLRV future liver remnant volumeFsrp Follistatin related proteinFXR farnesoid-X-receptorG-CSF granulocyte-colony stimulating factorG-CSFR G-CSF receptor

GF growth factorGFAP glial fibrillary acidic proteinGS glutamine synthetaseHB-EGF heparin-binding EGFHCC hepatocellular carcinomasHep hepatocytesHGF hepatocyte growth factorHh hedgehogHHIP hedgehog interacting proteinHIFs hypoxia-inducible factors

HPCs hepatic progenitor cellsHSA hepatocyte sinusoid alignmentHSCs hepatic stellate cellsHUVEC human umbilical vein endothelial cellsHVEM herpes virus entry mediatorIBD intralobular bile ductICAM intercellular adhesion moleculeIGFBP1 insulin growth factor binding protein 1Ihh Indian hedgehogiPSs inducible-pluripotent stem cells

IKK IKB kinaseILK integrin linked kinaseINR international normalized ratioJNK Jun KinaseKCs Kupffer cellsLGLs large granular lymphocytesL-NAME NG-nitro-L-arginine methyl esterLPS lipopolysaccharideLSECs Liver sinusoidal endothelial cellsLSCs liver stem cells

LT liver transplantationMAP mitogen-activated proteinMAPCs multipotent adult progenitor cellsM-CSF macrophage colony stimulating factorMELD model for end-stage liver diseaseMIP macrophage inflammatory proteinMMP matrix metalloproteinasesMSCs mesenchymal stem cellsNASH non-alcoholic steatohepatitisNC neighboring cell

NICD Notch intracellular domainNIK NFB inducing kinase


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Abbreviations xvii

NK natural killerNKT natural killer TNO nitric oxideNOS-2 nitric oxide synthase 2OCs oval cells

OSM oncostatin MPCI Protein C inhibitorPCNA proliferating cell nuclear antigenPCR polymerase chain reactionPDGF platelet-derived growth factorPHx partial hepatectomyPI3K phosphatidylinositol 3-kinasePRR pathogen recognition receptorsPUMA p53 up-regulated modulator of apoptosisPVE portal venous embolization

PVP portal venous pressureRLGS restriction landmark genomic scanningROS reactive oxygen speciesSC stem cellSCF stem cell factorSECs sinusoidal endothelial cellsSERPIN serine protease inhibitorsFRP soluble frizzled related peptideShh Sonic hedgehogSNS sympathetic nervous system

SUMO small ubiquitin-related modifiertBDL Total bile duct ligationTGFalpha transforming growth factor alphaTGFbeta transforming growth factor beta 1TGFBRI / TGFBRII TGFbeta receptor I / TGFbeta receptor IITIMs TRAF-interacting moleculesTLR Toll like receptorTLV total liver volumeTNF tumor necrosis factorTNFalpha tumor necrosis factor alpha

TNFRI TNF receptor ITRADD TNF-receptor associated death domainTRAF TNF-receptor associated factorTRE tetracycline response elementTUDC tauroursodeoxycholateTWEAK transforming growth factor like weak inhibitor of apoptosisTx transcriptionuPA urokinase plasminogen activatoruPAR urokinase plasminogen activator receptorWnt wingless type

YAP Yes-associated protein


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1 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; itis 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 re-stored function. Other organs, such as cardiac muscle (Bergmann et al., 2009) or centralnervous system (Brill et al., 2009), may demonstrate some endogenous propensity forregeneration, particularly after an insult, but complete organ restoration and functionalrecovery (as seen with the liver) are not the norm. In fact, liver tissue deficits are readilyand rapidly replenished (in just a matter of 1 or 2 weeks in rodents), even followingextensive loss of up to ~75% of liver mass. Such a remarkable competence for renewalhas been capitalized upon by surgeons to cure patients of resectable hepatic tumors andcysts as well as to safely and effectively provide a source of transplantable tissue in thecase of living related liver donation.

1.2 Liver Regeneration: Historical Perspective

Although a fairly clear understanding of what drives hepatic cells to regenerate has beenestablished during the past several decades, the concept of liver regeneration may haveoriginated thousands of years earlier. Of all internal organs, the liver appears to be themost revered by ancient civilizations who bestowed upon it mystical properties. Amongthem, 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).


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2 Liver Regeneration and Partial Hepatectomy

It also figured prominently in Hesiods myth of Prometheusa Greek god who, havingstolen fire from Zeus to give as a gift to humans, was punished by daily consumption ofhis liver (which regrew overnight) by Zeuss eaglethe tale embodies the phenomenonof endless hepatic renewal that we embrace today. Some argue, however, that the refer-ence 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 reflects theirassignation of immortality to the gods, and by extension, to their gods livers (Powerand Rasko, 2008)! Regardless of which of these possibilities is true, the mere mention ofliver renewal in a work of classical literature familiar to so many over the centuries mayhave been sufficient enough to prompt early researchers to test its scientific merit.

1.3 Partial Hepatectomy as a Means to Study LiverRegeneration

It is only in the relatively recent past that liver regeneration has become the focus of

systematic, rigorous scientific investigation. As surgical techniques underwent refine-ment and survival following surgery improved in the late 1800s, surgeons and scientistsalike 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 isstill widely in use today. It is referred to as two-thirds partial hepatectomy(2/3 PHx)and was first performed on the rat. Following lapartomy, the anterior lobes (i.e., thelarge medial lobe and the left lateral lobe) of the rat liverconsisting of approximately68% of the liver mass (i.e., 2/3)are ligated at the hilus and resected. As the animalrecovers, the excised anterior lobes of the liver do not regrow; rather, the remaininglobes undergo compensatory hyperplasiavia replication of the cells, therein restoring

the liver to its original mass in about one to two weeks (Higgins and Anderson, 1931)(Figure 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 livermass, underscoring the fact that hepatocytes are rather large cells [about 30 uM indiameter]). Stellate cells(hepatic stromal cells that produce and secrete growth factorsand extracellular matrix and store lipids and fat-soluble vitamins), Kupffer cells(residenthepatic macrophages), sinusoidal endothelial cells(SECsspecialized endothelia thatdisplay punctate membrane conduits, or fenestrae,which permit certain blood-bornenutrients, metabolites, and toxins direct access to hepatocytes) and cholangiocytes(bil-

iary epithelial cells) contribute the remaining hepatic cell numbers and add to tissuemass.

In response to partial hepatectomy in mammals, an orderly progression in DNA syn-thetic activity and replication is observed among the different hepatic cell types. In therat, for example, hepatocytes begin to enter DNA synthesis at about 12 hours post-PHxwith a robust peak observed at 24 hours after surgery. (For mice, the pinnacle of DNAsynthetic activity is slightly later at 3644 hours post-PHx.) A second smaller surge ofhepatocyte DNA synthetic activity typically occurs about 48 hours later (at 6072 hourspostsurgery). The remaining hepatic cells types replicate subsequently: DNA synthesisin 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 (Michalo-poulos and DeFrances, 1997).


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1.3 Partial Hepatectomy as a Means to Study Liver Regeneration 3

initiation/

priming

termination

2/3 PHxproliferation

immed early gene expression

13 hours

sinusoidalizationILK activation

TGFbeta redepositionglypican-3, IL-1

Yap, GF expression

4 7+ days

G1progressioncollagenase activation

3 ~12 hours

all cell constituents

proliferate

ECM deposition

~12 hours 4 days

notch signalingcytokine release

EGFR activationTx factor binding

TGFbeta

60 minutes

portal venous pressure

NO releaseHGF/Met activation

beta-catenin translocation

5 minutes

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 restoringthe liver to its original presurgical mass. Liver regeneration, which reaches completion inabout 714 days, can be divided into three phases: Initiation/priming(which lasts ~12 hoursafter 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 specific events asindicated. NO = nitric oxide, Tx = transcription, ECM = extracellular matrix, PHx = partialhepatectomy.

Historically, 2/3 PHx in rodents has been a heavily utilized method to study liverregeneration. It is rather simple to perform with a fairly high survival rate (Palmes andSpiegel, 2004). The procedure can be easily modified so that more or less tissue (than~70%) is excised, although surgically removing greater than ~75% of hepatic masscompromises survival of the animal due to, among other reasons, hepatic hyperperfu-sion associated with ischemia/reperfusion injury and acute liver failure. Otherwise, ithas been shown that the degree of ensuing hepatic cell replication is proportional tothe amount of liver mass excised (Bucher and Swaffield, 1964). It seems that a hepaticrheostat (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 ensuringthat it is wholly adequate and appropriate.


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Other compensatory hyperplasia models have been developed to study the process ofliver regeneration. For example, toxins (such as carbon tetrachloride [CCl4]) that causehepatocyte necrosis, inflammation, cytokine release, and liver regeneration can be ad-ministered to rodents (Palmes and Spiegel, 2004). Another method induces bipotentialliver stem cells (oval cells) to replicate and differentiate into hepatocytes; in one version

of this model, rodents are treated with the chemical 2-acetylaminofluorene (2-AAF) toinhibit hepatocyte proliferation and then subjected to partial hepatectomy to stimulateoval 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 com-mon clinical scenarios. For example, patients who must regenerate liver mass after he-patic surgery often have cirrhosis, hepatic viral infection, steatosis, or liver metastases,or are liver transplant recipients. The standard PHx model does not recapitulate thephysiologic complexity of these types of cases. In addition, wild animals that undergoendogenousliver regeneration do so because of exposure to environmental hepatotox-ins 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 pris-tine hepatic tissue. Despite these acknowledged drawbacks, the 2/3 PHx model remainsa uniquely valuable system to delineate the mechanisms underlying liver regeneration:its relative simplicity, its reproducibility among different laboratories, the fact that haz-ardous chemicals need not be handled nor administered to animals, and a relative lackof tissue inflammation or necrosis (as seen in some other models, the extent of whichcan be variable among animals impacting the regenerative response and thus muddlingdata interpretation) make its use compelling.

1.4 Three Phases of Liver Regeneration after PartialHepatectomy

An obvious question to ask is, Why doesthe liver regenerate so rapidly and efficientlyafter partial hepatectomy? The answer is understandably complex. The entire processcan be roughly divided into three phases:

1) initiation/primingthe majority of hepatocytes exit a quiescent state (G0), enterthe cell cycle (G1), and cross the G1/S checkpoint. Dissolution of the extracellularmatrix (ECM) begins. In the rat, this phase lasts about 1218 hours. Although it isthe shortest of the three phases, it has been perhaps the most intensely analyzed in

order to identify the primary eventthat triggers liver regeneration. Studies reveal thatrapid and pronounced alterations in a multitude of signaling pathways and othertissue functions occur simultaneously and no single alteration likely predominates(Michalopoulos, 2010).

2) proliferationhepatocytes synthesize DNA, complete the remainder of the cellcycle, and reenter G0 ; a small proportion of hepatocytes engage in a subsequentround of mitosis. Remodeling of the ECM proceeds. Other hepatic cell types suchas cholangiocytes and SECs divide. This phase extends from 1218 hours to about4 days after PHx in rodents.

3) 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 (Figure 1.1).


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1.4 Three Phases of Liver Regeneration after Partial Hepatectomy 5

1.4.1 Phase One: Initiation/Priming

During the initiation/priming phase of liver regeneration after PHx, the very first eventto transpire following excision of liver tissue is an immediate induction of sheer stressin the portal circulation reflected by an increase in portal venous pressure (PVP)

(Schoen et al., 2001). The liver is fed by two blood supplies: (1) the portal vein (whichprovides the liver about 75% of its blood) carries to the liver nutrients, toxins, bileacids, and other substances absorbed or produced by the gastrointestinal tract forfurther metabolism, if necessary; and (2) the hepatic artery, although contributing lessblood by volume, supplies the liver with, among other things, a necessary sourceof oxygen, hormones, cytokines, and immune surveillants (lymphocytes, monocytes,etc.). Increased PVP is accompanied by release of nitric oxide (NO) in the liver, likelyby endothelial cells (Schoen et al., 2001). Blocking NO synthase by NG-nitro-L-argininemethyl ester (L-NAME) administration inhibits c-fosmRNA expression typically in-duced 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 inregeneration by Kupffer cells, hepatocytes, or other liver constituents through induc-tion 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 massbeginning at 3648 hours after PHx (Kumamoto et al., 2008; Rai et al., 1998) (althoughliver mass of mice in one of the studies reached control levels by day 7; Kumamotoet al., 2008).

SECs react to changes in PVP by increasing the diameter of fenestrae and overallporosity at 5 minutes post-PHx (Wack et al., 2001). At the same time (5 min. after sur-gery), the hepatocyte plasma membrane depolarizes (Zhang et al., 1996), but prevent-

ing depolarization does not diminish the gene expression signature usually observedwithin 11.5 hours after surgery, suggesting that depolarization has little impact onthe early stages of regeneration (Minuk et al., 1997). Beta-catenin, a transcriptionalregulator normally bound to E-cadherin at the hepatocyte plasma membrane, migratesto the hepatocyte nucleus to activate target genes within 5 minutes of resection. Thisis accompanied by E-cadherin downregulation, which may account in part for beta-catenins rapid subcellular redistribution (Monga et al., 2001). Proper hepatic develop-ment is regulated by the Notch/Jagged signaling system; mutation of either the Jagged-1or Notch-2 gene is associated with a paucity of intrahepatic bile ducts (referred to asAlagille 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 tothe nucleus to regulate gene transcription. Fifteen minutes after PHx, NICD appears inthe nuclei of hepatoctyes (and possibly other hepatic constituents such as endothelialcells) peaking at 15 minutes postsurgery. Injection of Jagged-1 siRNA to rats prior toPHx blunts DNA synthesis particularly at the day 2 post-PHx time point, suggesting thatthe 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 significant event because uPA is a serine proteaseresponsible for cleaving and activating a variety of proteins. For example, uPA converts


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plasminogen into plasmin, which in turn cleaves fibrinogen into fibrin. Plasmin abun-dance in the liver shows a small uptick at 15 minutes and a major peak at about 36hours after PHx, while fibrinogen levels fall in the liver 15 minutes following surgery(Kim et al., 1997). uPA is also one of the main enzymes responsible for activating he-patocyte growth factor (HGF) into its biologically functional form (Mars et al., 1993).

Interestingly, mice lacking uPA, but not uPAR, in hepatocytes show delayed regenera-tion after PHx (Roselli et al., 1998), suggesting that uPA functions normally in responseto surgery without the aid of its receptor.

While investigations of plasmin and fibrin in liver regeneration continue, HGFs rolein the process is well-documented. Studies in the 1950s had shown that when the cir-culatory systems of two rats are joined and one of the pair is subjected to PHx, not onlydoes the liver of the animal that underwent liver resection regenerate, but the liver ofthe unoperated rat responds with increased DNA synthesis. This led to the postulate thata 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 undergoDNA 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 themost potent mitogenic stimuli for hepatocytes in culture (Michalopoulos and DeFran-ces, 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 asinducers of DNA synthesis in cultured hepatocytes.

During the very early time points in liver regeneration, HGF sequestered by proteo-glycans is released and cleaved by active uPA bound to uPAR. Biologically functionalHGF 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 intothe circulation with a peak plasma level seen at 60120 minutes following surgery(Lindroos et al., 1991). Interestingly, healthy patients undergoing right hepatectomyas part of living related liver donation also show a prominent spike in serum levelsof HGF (but not EGF, VEGF, nor TGFalpha) at 2 hours postsurgery (Efimova et al.,2005).

A series of studies have solidified HGF and Met as key regulators of liver growth andregeneration. Enforced expression of HGF in mouse hepatocytes hastens recovery afterPHx (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 increasein size by 140% via a hyperplastic response and return to normal upon terminatingHGF treatment (Patijn et al., 1998). This observation highlights a remarkable phenom-enon: that HGF is sufficiently potent to prompt a regeneration-like response in theliver in the absence of tissue damage or loss. Meanwhile, regeneration is significantlyimpaired in mice in which the Met tyrosine kinase domain is deleted specifically in theliver (Borowiak et al., 2004) or hepatocytes (Huh et al., 2004), while delivery of a MetshRNA to the hepatocytes of rats delays DNA synthesis after PHx by 24 hours (Paranjpeet al., 2007). In addition to regulating regeneration after PHx, this growth factor/recep-tor 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 hepatocellularcarcinoma (DeFrances, 2010).


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EGF is produced by exocrine glands of the alimentary tract (the salivary glands andBrunners glands of the duodenum, in particular); its availability to the liver and ulti-mately to its cell surface receptor (EGFR) expressed on hepatocytes may be enhancedby virtue of altered portal circulation after liver surgery (Skov Olsen et al., 1988). WhileEGFR 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-bodyweight ratio at day 7 after PHx as compared to controls (Natarajan et al., 2007), andrats treated with EGFR shRNA respond to PHx with significantly reduced mitotic activityand appear to restore liver mass (which ultimately reaches control levels), at least in partthrough hepatocyte hypertrophy (Paranjpe et al., 2010). Of note, however, is the find-ing that animals treated with anti-EGFR monoclonal antibodies regenerate their liversnormally after surgery (Van Buren et al., 2008).

Within the 3060 minutes after surgical resection, the livers reticuloendothelialcompartment, and perhaps other nonparenchymal cells such as cholangiocytes, are

stimulated to produce cytokines, including tumor necrosis factor alpha (TNFalpha) andinterleukin-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 suchas hepatocytes, and possibly by an endocrine route because their plasma levels peakat 1 hour after PHx. TNFalpha and IL-6 are responsible at least in part for the surgein DNA binding activity of an established set of transcriptional activators in the liverin response to hepatic surgery (Diehl and Rai, 1996). They include AP-1 (DNA bind-ing at 1560 min. post-PHx and involve c-jun, c-fos,and other AP-1 partners; Diehlet al., 1994), c-myc (mRNA expression increasing at 15 min. post-PHx; Thompsonet 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 andpeaking at 34 hours after surgery; Yamada et al., 1997), and C/EBPbeta (2- to 3-foldhigher DNA binding than presurgical values at 3 hours post-PHx returning to normal by24 hours after surgery; Diehl and Yang, 1994).

If signaling of the cytokines or the transactivators they induce is experimentally per-turbed, a range of postsurgical outcomes (from no effect to moderate delay in liverregeneration) is observed. For example, TNF receptor I (TNFRI) knock-out mice showmoderate reductions in DNA synthesis following hepatic resection (Shimizu et al.,2009; Yamada et al., 1997); however, by 14 days after surgery, their liver weights reachcontrol 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 re-ceptor (glycoprotein 130gp130which mediates signaling) is deleted specifically inhepatocytes results in reduced STAT-3, c-jun,NFkappaB, and c-mycexpression but noovert difference in liver regeneration (Wuestefeld et al., 2003). Mice that lack the p50subunit of NFkappaB in hepatocytes show no change in DNA synthesis or overall liverweight (DeAngelis et al., 2001). This may be due to the fact the NFkappaB is activatedpredominately in Kupffer cells and SECs, and not in hepatocytes, after PHx (Sakudaet al., 2002). It should be noted that the addition of cytokines such as TNFalpha tocultured hepatocytes does not stimulate appreciable DNA synthetic activity, but when

coupled with a known hepatic mitogen, synergistic effects on DNA synthesis are ob-served (Diehl and Rai, 1996). Thus, they are best referred to as auxillary mitogens or


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priming factors. Substances that augment the effect of hepatic growth factors akin toTNFalpha and IL-6 include catacholamines, thyroxine, insulin, and others (Michalopou-los and DeFrances, 1997).

Several signal transduction pathways are upregulated early after PHx. These pathwaysare stimulated by a combination of activated growth factor receptors (such as Met)

and cytokine signaling. For example, within 15 minutes after PHx, TNFalpha appearsto upregulate Jun Kinase ( JNK, also known as MAPK8), which phosphorylates c-juntoenhances c-junstranscriptional activity (Diehl et al., 1994). Blocking the PI3K pathwayby injection of wortmannin, a potent PI3K inhibitor, results in reduced liver-to-bodyweight ratio as compared to controls at 48 and 72 hours post-PHx, returning to presur-gical levels by 7 days after surgery in mice ( Jackson et al., 2008), while liver-specificloss of PDK1, a serine threonine kinase that binds PI3K-generated PIP3at the plasmamembrane and phosphorylates AKT, causes death of mice when 23 PHx is performed.However, when 30% hepatectomy is carried out, no significant difference in mitoticrate 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 seenat 1 hour post-PHx, remains elevated, and peaks again at about 5 hours before fallingto presurgical levels by 16 hours (Chen et al., 1998).

The net effect of the concerted stimulation of hepatocytes by growth factors, cy-tokines, and mechanical distortion is increased expression of 70 genes, some ofwhich are described as immediate early genes because their abundances rise about12 hours after surgery. They encode for transcription factors such as early growthresponse-1 (egr-1), growth factor modulators like insulin growth factor binding pro-tein 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/G1boundary, it is necessary for the contactbetween cells and their extracellular environment to be modified in a precise manner.Within minutes of PHx in rats, the protein abundances of fibronectin, entactin, andlaminin fall, with fibronectin and entactin levels returning to near pre-PHx levels by1824 hours after surgery (Kim et al., 1997). Later after surgery, the activity profilesof 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, theprotein level of a key metalloproteinase inhibitor (TIMP-1) accumulates at roughly thesame time (618 hours following surgery) (Kim et al., 2000). A nadir in heparan sulfateproteoglycan 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 activityof hepatic mitoinhibitors would be simultaneously suppressed. Transforming growthfactor beta 1 (TGFbeta) added to cultures of hepatocytes exposed to mitogens preventscells from entering DNA synthesis ( Jakowlew et al., 1991) and are arrested at the G1/Scheckpoint. After PHx in rats, TGFbeta mRNA levels in the liver show a biphasic patternrising sharply early after surgery (2 hours) then falling and reaching a nadir at 24 hoursbefore climbing again; expression is confined 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 involvedin intracellular signal transduction) and TGF beta receptor II (TGFBRII). The levels of


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these two receptors decrease immediately after PHx hitting a low point at 24 hours aftersurgery. TGFBRII expression returns by 5 days post-PHx, but the level of TGFBRI doesnot fully rebound during that timeframe (Ravi et al., 1995). From this, it is apparent thatat the peak of hepatocyte DNA synthesis, a coordinated maximal downregulation ofboth TGFbeta and its receptors occurs, presumably to allow hepatocyte replication to

proceed unimpeded. Of note, when active TGFbeta is injected into rats 24 hours priorto performing PHx, the animals die 24 hours after surgery with a significant increase inapoptotic 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 thereplication of all constituent cell types in the liver. As mentioned, among the cell types,hepatocytes replicate first with peak DNA synthetic activity ranging from 2444 hoursin rodents; a subsequent smaller boost in hepatocyte DNA synthesis is observed ~48

hours later. The hepatic plates thicken with the replicating hepatocytes and compresssinusoids; in addition, SEC porosity decreases at 72 hours after surgery (Wack et al.,2001). Cholangiocyte replication reaches a maximum at approximately 23 days fol-lowing PHx, while hepatocyte tight junctions dissolved during the initiation phase arereconstituted allowing bile secretion to recommence at day 3. The volume densityof bile ducts is restored by about day 10 (Lesage et al., 1996). Substantial paracrinecommunication occurs between hepatic cell types during this period. Hepatocytes up-regulate 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 daysafter 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 inhepatocytes. At about day 4 after PHx, stellate cells upregulate synthesis and depositionof extracellular matrix proteins, which is likely to help the liver segue to the final 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 7after PHx),SECs re-establish the sinusoids by migrating among the hepatocytes that have accumu-lated 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 thickplates and to reform contacts with the extracellular environment. Impeding proper he-patocyte-ECM interaction can drastically alter the termination of liver regeneration. Forexample, integrin linked kinase (ILK), an intracellular signal transducer that associateswith integrins, appears to be particularly important to ending the regenerative responseafter surgery. When mice that lack ILK specifically in hepatocytes are subjected to PHx,their livers respond by becoming roughly 1.5-fold larger at 14 days post-PHx than priorto surgery. In addition, hepatocyte DNA synthetic activity in these mice is more robustand prolonged (Apte et al., 2009). This may be due to altered expression of integrinsin 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 tounderlie a genetic syndrome with an overgrowth phenotype in humans, is upregulated


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at about 46 days after PHx. When subjected to PHx, mice engineered to overexpressglypican-3 in hepatocytes show delayed and reduced DNA synthesis and do not regainproper liver mass at day 6 post-PHx, suggesting that the upswing in endogenous glypi-can-3 expression after surgery promotes termination of regeneration (Liu et al., 2009).

Changes in the relative abundance of transcription activators and secreted mitoinhibi-

tors also help dictate the end of regeneration. The DNA binding activity of C/EBPalphatranscription factor initially rises then falls precipitously after PHx; however, it climbsagain in the latter stages of the regenerative response. This is noteworthy because it isantiproliferative toward hepatocytes (Wang et al., 2001) and promotes their differentia-tion and quiescence (Greenbaum et al., 1995). Apte et al. (2009) demonstrated thatthe abundance of Yes-associated protein (YAP), a transcription coactivator and memberof the newly defined mammalian Hippo kinase pathway that controls liver size andinduces liver cancer (Dong et al., 2007), is downregulated late in liver regeneration afterPHx. With regard to secreted factors, non-parenchymal cells begin to release IL-1 alphaand beta (which inhibit mitogen-induced hepatocyte DNA synthesis) after the prolifera-

tive phase of liver regeneration (Boulton et al., 1997). As mentioned earlier, TGFbetaand its receptors are downregulated during the early period after surgery, but levels risesubsequently after the peak of hepatocyte DNA synthesis. When mice lacking TGFBRIIin hepatocytes are subjected to PHx, they have a pronounced increase in the numberof hepatocytes entering the S-phase (Oe et al., 2004; Romero-Gallo et al., 2005) and ahigher liver-to-body weight ratio at 1 week and 1 month after surgery (Romero-Galloet al., 2005). Activin-A is a mitoinhibitor related to TGFbeta. Infusion of follistatin, anendogenous Activin-A inhibitor, results in increased liver weight and DNA contentmore than 5 days post-PHx, suggesting that Activin-A acts during the termination phaseof 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 thelivers regenerative response. This is supported by the data mentioned earlier demon-strating that hyperplastic liver induced by HGF infusion returns to normal size in a fewweeks after infusion is stopped (Patijn et al., 1998). To summarize, the data suggest thatthe elusive hepatostatactively drives the liver to quiescence by promoting perpetualantagonism between growth promoters, such as HGF and EGF, and growth inhibitors,including TGFbeta, ILK, IL-1, and C/EBPalpha, with the inhibitors predominating duringthe termination phase (Michalopoulos and DeFrances, 2005).

1.5 Future DirectionsSeveral lapses in our knowledge of liver regeneration persist: (1) We need a betterunderstanding of the events that terminate the regenerative response. While somefore-runners in this process have been described, it seems likely that termination issignificantly more complex. (2) Acute liver failure continues to take a global toll onhuman life. Are there certain molecular pathways mediating liver regeneration that canbe exploited to promote survival in this scenario? (3) Patients undergoing hepatic resec-tion usually have underlying liver disease. We must more fully define the impact ofinflammation, steatosis, and other physiologic complications on regeneration in order

to improve recovery after surgery. Exciting challenges have been defined; we must nowsquare our shoulders and meet them.


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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 specific genes in animals has demonstrated that

perturbation of a single signaling pathway is usually insufficient 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 final 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 final outcome is a liver that is fully functional,

thus sustaining the animal.

Further Reading

Bucher, N.L.R., Scott, J.F., and Aub, J.C. (1951). Regeneration of the liver in parabiotic rats.Cancer Res. 11, 45765.

Haber, B.A., Mohn, K.L., Diamond, R.H., and Taub, R. (1993). Induction patterns of 70 genesduring nine days after hepatectomy define the temporal course of liver regeneration. J. Clin.Invest. 91, 131926.

Martinez-Hernandez, A., and Amenta, P.S. (1995). The extracellular matrix in hepatic regen-eration. FASEB J. 9, 140110.

Palmes, D., and Spiegel, H.-U. (2004). Animal models of liver regeneration. Biomaterials 25,160111.

References

Apte, U., Gkretsi, V., Bowen, W.C., Mars, W.M., Luo, J.-H., Donthamsetty, S., Orr, A., Monga,S.P.S., Wu, C., and Michalopoulos, G.K. (2009). Enhanced liver regeneration follow-ing changes induced by hepatocyte-specific genetic ablation of integrin-linked kinase.Hepatology 50, 84451.

Bell, A., Chen, Q., DeFrances, M.C., Michalopoulos, G.K., and Zarnegar, R. (1999). Thefive amino acid-deleted isoform of hepatocyte growth factor promotes carcinogenesis intransgenic mice. Oncogene 18, 88795.

Bergmann, O., Bhardwaj, R.D., Bernard, S., Zdunek, S., Barnabe-Heider, F., Walsh, S., Zupi-cich, J., Alkass, K., Buchholz, B.A., Druid, H. et al. (2009). Evidence for CardiomyocyteRenewal in Humans. Science 324, 98102.

Bladt, F., Riethmacher, D., Isenmann, S., Aguzzi, A., and Birchmeier, C. (1995). Essential

role for the c-met receptor in the migration of myogenic precursor cells into the limb bud.Nature 376,76871.


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Borowiak, M., Garratt, A.N., Wstefeld, T., Strehle, M., Trautwein, C., and Birchmeier, C.(2004). Met provides essential signals for liver regeneration. Proc. Natl. Acad. Sci. USA101, 1060813.

Boulton, R., Woodman, A., Calnan, D., Selden, C., Tam, F., and Hodgson, H. (1997). Non-parenchymal cells from regenerating rat liver generate interleukin-1and -1: A mecha-nism of negative regulation of hepatocyte proliferation. Hepatology 26, 4958.

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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 Oval Cells, Bone Marrow, and LiverRegeneration

Anna C. Piscaglia, Antonio Gasbarrini, and Bryon E. Petersen

2.1 Stem Cells: Definition and Properties

The existence of hepatic stem cells has been controversial for decades, and it wasthought that if such cells existed, they would reside within the liver. However, there isnow 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 mar-row (BM), can contribute to liver repopulation, although the physiological importanceand therapeutic utility of this phenomenon are still debated.

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

multipotency.

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 iden-

tical daughter cells that could be either stem cells (self-renewing division) or progeni-

tor 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 (Figure 2.1).


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18 2 Oval Cells, Bone Marrow, and Liver Regeneration

Stem cells (SCs) exist in all multicellular organisms and play a central role in tissuegenesis, regeneration, and homeostasis by providing new elements to increase tissuemass during pre- and postnatal growth and by replacing cell loss due to senescence ordamage.

SCs possess a hierarchy of potentialities: from the totipotency of the zygote andits immediate progeny, to the pluripotency of embryonic stem cells (ESCs), to the

multi/unipotency of tissue-specific, adult SCs (ASCs). The latter persist in terminallydifferentiated tissues, allowing for their renewal and regeneration (Alison and Islam,2009).

SCs colocalize with supporting cells in a physiologically limited and specializedmicroenvironment, or niche, that varies in nature and location depending upon thetissue type. The reciprocal interactions between SCs and their microenvironment,through cellcell and cellmatrix connections as well as the secretion of soluble factors,influence SC behavior (Moore, 2006).

Despite the paradigm of unidirectional cell determination, recent studies have shownthat ASCs are endowed with an unexpected plasticity, as circulating ASCs have been

demonstrated to differentiate into mature cells of other tissue types, a process calledtransdifferentiation(Piscaglia et al., 2008a). A particularly high degree of plasticity is

Multipotency is the capacity to produce mature cell-lineages from a relatively undif-

ferentiated element. The process that ultimately leads a stem cell to become a mature

and functional cell is named differentiationand results from progressive phenotypic

modifications due to dynamic changes in the gene expression pattern.

asymmetric division quiescence symmetric division

SC pool maintenance expansion exhaustion

non-self-renewing

symmetric division:

two daughter transit-

amplifying cells

self-renewing

symmetric division:

two daughter

stem cells

asymmetric division:

one daughter stem cell

and one daughter

transit-amplifying cell

Figure 2.1 Mechanisms for the Maintenance of Stem Cell Numbers

Notes:Abbreviation: SC = stem cell.


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2.1 Stem Cells: Definition and Properties 19

shown by hematopoietic stem cells and mesenchymal stem cells (MSCs), which cangive rise to a wide range of phenotypes. Hematopoietic SCs are responsible for therenewal of blood cells. During embryogenesis, hematopoietic SCs arise from the dorsaland ventral mesenchyme and migrate firstly to the yolk sac, then to the fetal liver andspleen, and finally to the bone marrow (BM), which remains the only hematopoietic

organ from birth throughout life. In addition to BM and embryos obtained throughtechniques of either nuclear transplantation or in vitro fertilization, two additionalsources of hematopoietic SCs are available: peripheral blood and umbilical cordblood. The different hematopoietic SC sources are distinguished in terms of acces-sibility and ethical issues, as well as biological properties such as immunogenicityand clonogenicity (Piscaglia et al., 2007a). The membrane phosphoglycoprotein CD34is considered a valid hematopoietic SC marker, although some studies suggested theexistence of CD34/Linhematopoietic SCs capable of producing CD34cells in vitro.AC133 (CD133, or prominin1 in rodents), a glycoprotein transmembrane, present onprogenitors belonging to neuronal, epithelial, and endothelial lineages, is common to

both CD34

and CD34

hematopoietic SCs (Guo et al., 2003). It is generally acceptedthat the most primitive and long-term human hematopoietic SCs are characterizedby the expression of CD133, Thy1 (CD90), and VEGFR2 and by a variable expres-sion of CD34 and CD38 (Bryder et al., 2006). BM-resident hematopoietic SCs canbe mobilized into the peripheral blood under specific stimuli such as tissue injury oradministration of mobilizing agents. Hematopoietic SCs may be used in autologous orallogeneic transplantations for the treatment of hematopoietic disorders, autoimmunediseases, and aggressive cancers to reconstitute the hematopoietic SC lineages andthe immune system integrity. Additionally, in vitro culture and in vivo transplantationassays have demonstrated that hematopoie

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