Liver Regeneration (De Gruyter Textbook)

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Liver Regeneration Edited by Dieter Häussinger

Liver Regeneration Edited by Dieter Häussinger

DE GRUYTER

Editor Prof. Dr. Dieter Häussinger Department of Internal Medicine Gastroenterology, Hepatology and Infectious Diseases University Hospital Düsseldorf Heinrich-Heine-University Moorenstrasse 5 D-40225 Düsseldorf Germany This book has 46 figures 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 Häussinger 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 Häussinger. p. ; cm. Includes bibliographical references. ISBN 978-3-11-025078-7 (alk. paper) 1. Liver—Regeneration. 2. Liver—Diseases. 3. Stem cells. I. Häussinger, D. (Dieter), 1951[DNLM: 1. Liver Regeneration—physiology. 2. Stem Cells—metabolism. WI 702] QP185.L57 2011 611'.36—dc22 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 information 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 any errors 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, Göttingen U Printed on acid-free paper Printed in Germany www.degruyter.com

Preface

The liver has a high capacity to regenerate, which was already known in ancient Greece, as exemplified 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 field 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 inflammation 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. Düsseldorf, May 2011 Dieter Häussinger

Contents

Author Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Abbreviations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1

2

3

xi xv

Liver Regeneration and Partial Hepatectomy: Process and Prototype . . . . . . . Marie C. DeFrances and George K. Michalopoulos

1

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: Definition 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 Marrow–Derived Stem Cells in Hepatology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

17 21 25 30

Inflammation and Liver Regeneration. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Johannes G. Bode

39

3.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2 Liver Regeneration and Inflammation: General Aspects. . . . . . . . . . . . . 3.3 Liver Macrophages and Their Relevance for Liver Regeneration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.4 Inflammatory Mediators Are Required to Promote Liver Regeneration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.5 Inappropriate Inflammation 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 β 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 LTβR and Liver Regeneration. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

53 56 57 58

5 The Hepatic Stem Cell Niches . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Iris Sawitza, Claus Kordes, and Dieter Häussinger

63

4

5.1 5.2 5.3 5.4 5.5 6

7

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Secreted Factors in the Stem Cell Niche . . . . . . . . . . . . . . . . . . . . . . . . Physical Contacts of Stem Cells with Their Niche . . . . . . . . . . . . . . . . . Identification 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 Häussinger

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 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Claus Kordes, Iris Sawitza, and Dieter Häussinger

99

7.1 7.2 7.3 7.4 8

Definition 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 Häussinger 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

Definition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Methods to Quantify Spatial–Temporal 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

x

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, Günter Fürst, 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 Department of Internal Medicine Gastroenterology, Hepatology and Infectious Diseases University Hospital Düsseldorf Heinrich-Heine-University Moorenstrasse 5 D-40225 Düsseldorf Germany Tobias Buschman Institute of Metabolic Physiology Heinrich-Heine-University Universitätsstrasse 1 D-40225 Düsseldorf 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 Institute of Metabolic Physiology Heinrich-Heine-University Universitätsstrasse 1 D-40225 Düsseldorf Germany Günter Fürst, MD Department of Diagnostic and Interventional Radiology University Hospital Düsseldorf Heinrich-Heine-University Moorenstrasse 5 D-40225 Düsseldorf Germany

xii

Author Index

Antonio Gasbarrini, PhD Gastrointestinal and Liver Stem Cell Research Group (GILSteR) Department of Internal Medicine Gemelli Hospital Catholic University of Rome (Italy) Largo A. Gemelli 8 – 00168 Roma Italy Rolf Gebhardt, PhD Institute of Biochemistry Faculty of Medicine University of Leipzig Johannisallee 30 D-04103 Leipzig Germany Dieter Häussinger, MD Department of Internal Medicine Gastroenterology, Hepatology and Infectious Diseases University Hospital Düsseldorf Heinrich-Heine-University Moorenstrasse 5 D-40225 Düsseldorf 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 University of Leipzig D-04103 Leipzig Germany Amalya Hovhannisyan, MD, PhD Institute of Biochemistry Faculty of Medicine University of Leipzig Johannisallee 30 D- 04103 Leipzig Germany

Wolfram Trudo Knoefel, MD Department of General-, Visceral- and Pediatric Surgery University Hospital Düsseldorf Heinrich-Heine-University Moorenstrasse 5 D-40225 Düsseldorf Germany Claus Kordes, PhD Department of Internal Medicine Gastroenterology, Hepatology and Infectious Diseases University Hospital Düsseldorf Heinrich-Heine-University Moorenstrasse 5 D-40225 Düsseldorf Germany Eckhard Lammert, PhD Institute of Metabolic Physiology Heinrich-Heine-University Universitätsstrasse 1 D-40225 Düsseldorf 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 Heinrich-Heine-University Universitätsstrasse 1 D-40225 Düsseldorf Germany Anna C. Piscaglia, PhD Gastrointestinal and Liver Stem Cell Research Group (GILSteR) Department of Internal Medicine Gemelli Hospital Catholic University of Rome (Italy) Largo A. Gemelli 8 – 00168 Roma Italy Roland Reinehr, MD Department of Internal Medicine Gastroenterology, Hepatology and Infectious Diseases University Hospital Düsseldorf Heinrich-Heine-University Moorenstrasse 5 D-40225 Düsseldorf Germany Iris Sawitza, PhD Department of Internal Medicine Gastroenterology, Hepatology and Infectious Diseases University Hospital Düsseldorf Heinrich-Heine-University Moorenstrasse 5 D-40225 Düsseldorf Germany

Moritz Schmelzle Department of General-, Visceral- and Pediatric Surgery University Hospital Düsseldorf Heinrich Heine University Moorenstrasse 5 D-40225 Düsseldorf Germany Jan Schulte am Esch, MD Department of General-, Visceral- and Pediatric Surgery University Hospital Düsseldorf Heinrich-Heine-University Moorenstrasse 5 D-40225 Düsseldorf Germany Ursula R. Sorg, PhD Institute of Medical Microbiology and Hospital Hygiene Heinrich-Heine-University Universitätsstrasse 1 D-40225 Düsseldorf 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-acetylaminofluorene 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 cell–cell 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-deficient extracellular matrix epidermal growth factor enhanced green fluorescent protein epithelial-to-mesenchymal transition epithelial neutrophil-activating protein endoplasmic reticulum embryonic stem cell Fas-associated death domain free fatty acid fibroblast 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 fibrillary 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 inflammatory protein matrix metalloproteinases mesenchymal stem cells non-alcoholic steatohepatitis neighboring cell Notch intracellular domain NF͑B 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 modifier 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

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; 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 deficits 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).

2

Liver Regeneration and Partial Hepatectomy

It also figured prominently in Hesiod’s myth of Prometheus—a Greek god who, having stolen fire from Zeus to give as a gift to humans, was punished by daily consumption of his liver (which regrew overnight) by Zeus’s eagle—the 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 reflects 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 sufficient enough to prompt early researchers to test its scientific 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 scientific investigation. As surgical techniques underwent refinement 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 first performed on the rat. Following lapartomy, the anterior lobes (i.e., the large medial lobe and the left lateral lobe) of the rat liver—consisting 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 (SECs—specialized 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 36–44 hours post-PHx.) A second smaller surge of hepatocyte DNA synthetic activity typically occurs about 48 hours later (at 60–72 hours postsurgery). The remaining hepatic cells types replicate subsequently: DNA synthesis in Kupffer cells, stellate cells, and cholangiocytes reaches a maximum at about 48–72 hours post-PHx, while SEC DNA replication peaks at 3–4 days after surgery (Michalopoulos and DeFrances, 1997).

1.3

Partial Hepatectomy as a Means to Study Liver Regeneration

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portal venous pressure ≠ NO release HGF/Met activation beta-catenin translocation notch signaling cytokine release EGFR activation Tx factor binding ≠ TGFbeta Ø £ 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 Ø 4 –7+ days

1–3 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 7–14 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 specific 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 modified 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 Swaffield, 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.

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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, inflammation, 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-acetylaminofluorene (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 inflammation 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 efficiently after partial hepatectomy?” The answer is understandably complex. The entire process can be roughly divided into three phases: 1)

2)

3)

initiation/priming—the 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 12–18 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). proliferation—hepatocytes 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 12–18 hours to about 4 days after PHx in rodents. termination—the 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

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Phase One: Initiation/Priming

During the initiation/priming phase of liver regeneration after PHx, the very first event to transpire following excision of liver tissue is an immediate induction of sheer stress in 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 (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 NOS—iNOS) (Hortelano et al., 2007). Animals engineered to lack NOS-2 show reduced liver mass beginning at 36–48 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 1–1.5 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 betacatenin’s 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 (Köhler 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 protease responsible 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 abundance in the liver shows a small uptick at 15 minutes and a major peak at about 3–6 hours 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 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 fibrin in liver regeneration continue, HGF’s 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 receptor’s 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 60–120 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 (Efimova et al., 2005). A series of studies have solidified 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 sufficiently potent to prompt a “regeneration-like” response in the liver in the absence of tissue damage or loss. Meanwhile, regeneration is significantly impaired in mice in which the Met tyrosine kinase domain is deleted specifically 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).


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EGF is produced by exocrine glands of the alimentary tract (the salivary glands and Brunner’s 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 significantly 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 finding that animals treated with anti-EGFR monoclonal antibodies regenerate their livers normally after surgery (Van Buren et al., 2008). Within the 30–60 minutes after surgical resection, the liver’s 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 15–60 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 3–4 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 130—gp130—which mediates signaling) is deleted specifically 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

8


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-jun’s 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-specific 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 significant 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 1–2 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 modified in a precise manner. Within minutes of PHx in rats, the protein abundances of fibronectin, entactin, and laminin fall, with fibronectin and entactin levels returning to near pre-PHx levels by 18–24 hours after surgery (Kim et al., 1997). Later after surgery, the activity profiles of metalloproteinases MMP-9 and MMP-2 rise (at about 3–6 hours and 6–12 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 (6–18 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 confined to the nonparenchymal compartment ( Jakowlew et al., 1991). To transmit inhibitory signals, TGFbeta binds a heterodimeric receptor composed of TGFbeta receptor I (TGFBRI—a serine threonine kinase involved in 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 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 significant 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 first with peak DNA synthetic activity ranging from 24–44 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 2–3 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 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 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 specifically 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

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at about 4–6 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 defined 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 liver’s 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 significantly 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 define the impact of inflammation, steatosis, and other physiologic complications on regeneration in order to improve recovery after surgery. Exciting challenges have been defined; we must now square our shoulders and meet them.

References

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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 regeneration—a 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 cell–matrix 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, 457–65. Haber, B.A., Mohn, K.L., Diamond, R.H., and Taub, R. (1993). Induction patterns of 70 genes during nine days after hepatectomy define the temporal course of liver regeneration. J. Clin. Invest. 91, 1319–26. Martinez-Hernandez, A., and Amenta, P.S. (1995). The extracellular matrix in hepatic regeneration. FASEB J. 9, 1401–10. Palmes, D., and Spiegel, H.-U. (2004). Animal models of liver regeneration. Biomaterials 25, 1601–11.

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 following changes induced by hepatocyte-specific genetic ablation of integrin-linked kinase. Hepatology 50, 844–51. Bell, A., Chen, Q., DeFrances, M.C., Michalopoulos, G.K., and Zarnegar, R. (1999). The five amino acid-deleted isoform of hepatocyte growth factor promotes carcinogenesis in transgenic mice. Oncogene 18, 887–95. Bergmann, O., Bhardwaj, R.D., Bernard, S., Zdunek, S., Barnabe-Heider, F., Walsh, S., Zupicich, J., Alkass, K., Buchholz, B.A., Druid, H. et al. (2009). Evidence for Cardiomyocyte Renewal in Humans. Science 324, 98–102. 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, 768–71.

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Borowiak, M., Garratt, A.N., Wüstefeld, T., Strehle, M., Trautwein, C., and Birchmeier, C. (2004). Met provides essential signals for liver regeneration. Proc. Natl. Acad. Sci. USA 101, 10608–13. Boulton, R., Woodman, A., Calnan, D., Selden, C., Tam, F., and Hodgson, H. (1997). Nonparenchymal cells from regenerating rat liver generate interleukin-1α and -1β: A mechanism of negative regulation of hepatocyte proliferation. Hepatology 26, 49–58. Brill, M.S., Ninkovic, J., Winpenny, E., Hodge, R.D., Ozen, I., Yang, R., Lepier, A., Gascon, S., Erdelyi, F., Szabo, G. et al. (2009). Adult generation of glutamatergic olfactory bulb interneurons. Nat. Neurosci. 12, 1524–33. Bucher, N.L., and Swaffield, M.N. (1964). The Rate of Incorporation of Labeled Thymidine into the Deoxyribonucleic Acid of Regenerating Rat Liver in Relation to the Amount of Liver Excised. Cancer Res. 24, 1611–25. Bucher, N.L.R., Scott, J.F., and Aub, J.C. (1951). Regeneration of the liver in parabiotic rats. Cancer Res. 11, 457–65. Chen, J., Ishac, E.J., Dent, P., Kunos, G., and Gao, B. (1998). Effects of ethanol on mitogenactivated protein kinase and stress-activated protein kinase cascades in normal and regenerating liver. Biochem. J. 334, 669–76. Chen, T.S., and Chen, P.S. (1994). The myth of Prometheus and the liver. J.R. Soc. Med. 87, 754–5. Cressman, D.E., Greenbaum, L.E., Haber, B.A., and Taub, R. (1994). Rapid activation of post-hepatectomy factor/nuclear factor kappa B in hepatocytes, a primary response in the regenerating liver. J. Biol. Chem. 269, 30429–35. Daoust, R., Cantero, A. (1959). The numerical proportions of cell types in rat liver during carcinogenesis by 4-dimethylaminoazobenzene (DAB). Cancer Res. 19, 757–62. DeAngelis, R.A., Kovalovich, K., Cressman, D.E., and Taub, R. (2001). Normal liver regeneration in p50/nuclear factor kappaB1 knockout mice. Hepatology 33, 915–24. DeFrances, M. (2010). Molecular Mechanisms of Hepatocellular Carcinoma: Insights to Therapy. Totowa, NJ: Humana Press. Diehl, A.M., and Rai, R. (1996). Review: Regulation of liver regeneration by pro-inflammatory cytokines. J. Gastroenterol. Hepatol. 11, 466–70. Diehl, A.M., and Yang, S.Q. (1994). Regenerative changes in C/EBPα and C/EBPß expression modulate binding to the C/EBP site in the c-fos promoter. Hepatology 19, 447–56. Diehl, A.M., Yin, M., Fleckenstein, J., Yang, S.Q., Lin, HZ, Brenner, D.A., Westwick, J., Bagby, G., and Nelson, S. (1994). Tumor necrosis factor-alpha induces c-jun during the regenerative response to liver injury. Am. J. Physiol.-Gastroint. Liver Physiol. 267, G552–61. Dong, J., Feldmann, G., Huang, J., Wu, S., Zhang, N., Comerford, S.A., Gayyed, M.F., Anders, R.A., Maitra, A., and Pan, D. (2007). Elucidation of a universal size-control mechanism in Drosophila and mammals. Cell 130, 1120–33. Efimova, E.A., Glanemann, M., Nussler, A.K., Schumacher, G., Settmacher, U., Jonas, S., Nussler, N., and Neuhaus, P. (2005). Changes in Serum Levels of Growth Factors in Healthy Individuals After Living Related Liver Donation. Transplant. Proc. 37, 1074–5. Evarts, R.P., Nagy, P., Marsden, E., Thorgeirsson, S.S. (1987). A precursor-product relationship exists between oval cells and hepatocytes in rat liver. Carcinogenesis 8, 1737–40. Gkretsi, V., Bowen, W.C., Yang, Y., Wu, C., and Michalopoulos, G.K. (2007). Integrin-linked kinase is involved in matrix-induced hepatocyte differentiation. Biochem. Biophys. Res. Commun. 353, 638–43. Greenbaum, L.E., Cressman, D.E., Haber, B.A., and Taub, R. (1995). Coexistence of C/EBP alpha, beta, growth-induced proteins and DNA synthesis in hepatocytes during liver regeneration. Implications for maintenance of the differentiated state during liver growth. J. Clin. Invest. 96, 1351–65.

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Mars, W.M., Liu, M.L., Kitson, R.P., Goldfarb, R.H., Gabauer, M.K., and Michalopoulos, G.K. (1995). Immediate early detection of urokinase receptor after partial hepatectomy and its implications for initiation of liver regeneration. Hepatology 21, 1695–1701. Mars, W.M., Zarnegar, R., and Michalopoulos, G.K. (1993). Activation of hepatocyte growth factor by the plasminogen activators uPA and tPA. Am. J. Pathol. 143, 949–58. Martinez-Hernandez, A., and Amenta, P.S. (1995). The extracellular matrix in hepatic regeneration. FASEB J. 9, 1401–10. Matsuya, H., Takagaki, K., Yoshihara, S., Ishido, K., Sasaki, M., and Endo, M. (2001). Changes in glycosaminoglycan, galactosyltransferase-I, and sialyltransferase during rat liver regeneration. Tohoku J. Exp. Med. 193, 187–95. Michalopoulos, G.K. (2010). Liver regeneration after partial hepatectomy: critical analysis of mechanistic dilemmas. Am. J. Pathol. 176: 2–13. Michalopoulos, G.K., and DeFrances, M.C. (1997). Liver regeneration. Science 276, 60–6. Michalopoulos, G.K., and DeFrances, M. (2005). Liver regeneration. Adv. Biochem. Eng. Biotechnol. 93, 101–34. Minuk, G.Y., Kren, B.T., Xu, R., Zhang, X., Burczynski, F., Mulrooney, N.P., Fan, G., Gong, Y., and Steer, C.J. (1997). The effect of changes in hepatocyte membrane potential on immediate-early proto-oncogene expression following partial hepatectomy in rats. Hepatology 25, 1123–7. Miyazawa, K., Tsubouchi, H., Naka, D., Takahashi, K., Okigaki, M., Arakaki, N., Nakayama, H., Hirono, S., Sakiyama, O., et al. (1989). Molecular cloning and sequence analysis of cDNA for human hepatocyte growth factor. Biochem. Biophys. Res. Commun. 163, 967–73. Monga, S.P.S., Pediaditakis, P., Mule, K., Stolz, D.B., and Michalopoulos, G.K. (2001). Changes in WNT/β-catenin pathway during regulated growth in rat liver regeneration. Hepatology 33, 1098–1109. Nakamura, T., Nishizawa, T., Hagiya, M., Seki, T., Shimonishi, M., Sugimura, A., Tashiro, K., and Shimizu, S. (1989). Molecular cloning and expression of human hepatocyte growth factor. Nature 342: 440–3. Natarajan, A., Wagner, B., and Sibilia, M. (2007). The EGF receptor is required for efficient liver regeneration. Proc. Natl. Acad. Sci. U.S.A. 104, 17081–6. Oe, S., Lemmer, E.R., Conner, E.A., Factor, V.M., Levéen, P., Larsson, J., Karlsson, S., and Thorgeirsson, S.S. (2004). Intact signaling by transforming growth factor β is not required for termination of liver regeneration in mice. Hepatology 40, 1098–1105. Palmes, D., and Spiegel, H.-U. (2004). Animal models of liver regeneration. Biomaterials 25, 1601–11. Paranjpe, S., Bowen, W.C., Bell, A.W., Nejak-Bowen, K., Luo, J.-H., and Michalopoulos, G.K. (2007). Cell cycle effects resulting from inhibition of hepatocyte growth factor and its receptor c-Met in regenerating rat livers by RNA interference. Hepatology 45, 1471–7. Paranjpe, S., Bowen, W.C., Tseng, G.C., Luo, J.H., Orr, A., and Michalopoulos, G.K. (2010). RNA interference against hepatic epidermal growth factor receptor has suppressive effects on liver regeneration in rats. Am. J. Pathol. 176, 2669–81. Patijn, G.A., Lieber, A., Schowalter, D.B., Schwall, R., and Kay, M.A. (1998). Hepatocyte growth factor induces hepatocyte proliferation in vivo and allows for efficient retroviralmediated gene transfer in mice. Hepatology 28, 707–16. Ping, C., Xiaoling, D., Jin, Z., Jiahong, D., Jiming, D., and Lin, Z. (2006). Hepatic Sinusoidal Endothelial Cells Promote Hepatocyte Proliferation Early after Partial Hepatectomy in Rats. Arch. Med. Res. 37, 576–83. Power, C., and Rasko, J.E.J. (2008). Whither Prometheus’ Liver? Greek Myth and the Science of Regeneration. Ann. Intern. Med. 149, 421–6.

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Rai, R.M., Lee, F.Y., Rosen, A., Yang, S.Q., Lin, H.Z., Koteish, A., Liew, F.Y., Zaragoza, C., Lowenstein, C., and Diehl, A.M. (1998). Impaired liver regeneration in inducible nitric oxide synthasedeficient mice. Proc. Natl. Acad. Sci. U.S.A. 95, 13829–34. Ravi, S.C., David, T.P., Sean, R.S., William, C.M., and Randy, L.J. (1995). Down-regulation of transforming growth factor beta receptor type I, II, and III during liver regeneration. Am. J. Surg. 169, 126–32. Romero-Gallo, J., Sozmen, E.G., Chytil, A., Russell, W.E., Whitehead, R., Parks, W.T., Holdren, M.S., Her, M.F., Gautam, S., Magnuson, M. et al. (2005). Inactivation of TGF-beta signaling in hepatocytes results in an increased proliferative response after partial hepatectomy. Oncogene 24, 3028–41. Roselli, H.T., Su, M., Washington, K., Kerins, D.M., Vaughan, D.E., and Russell, W.E. (1998). Liver regeneration is transiently impaired in urokinase-deficient mice. Am. J. Physiol. 275, G1472–9. Sakuda, S., Tamura, S., Yamada, A., Miyagawa, J.-I., Yamamoto, K., Kiso, S., Ito, N., Higashiyama, S., Taniguchi, N., Kawata, S. et al. (2002). NF-kappaB activation in non-parenchymal liver cells after partial hepatectomy in rats: possible involvement in expression of heparinbinding epidermal growth factor-like growth factor. J. Hepatol. 36, 527–33. Schmidt, C., Bladt, F., Goedecke, S., Brinkmann, V., Zschiesche, W., Sharpe, M., Gherardi, E., and Birchmeier, C. (1995). Scatter factor/hepatocyte growth factor is essential for liver development. Nature 373, 699–702. Schoen, J.M., Wang, H.H., Minuk, G.Y., and Lautt, W.W. (2001). Shear stress-induced nitric oxide release triggers the liver regeneration cascade. Nitric Oxide 5, 453–64. Schrum, L.W., Bird, M.A., Salcher, O., Burchardt, E.-R., Grisham, J.W., Brenner, D.A., Rippe, R.A., and Behrns, K.E. (2001). Autocrine expression of activated transforming growth factor-B1 induces apoptosis in normal rat liver. Am. J. Physiol.-Gastroint. Liver Physiol. 280, G139–48. Shimizu, H., Miyazaki, M., Wakabayashi, Y., Mitsuhashi, N., Kato, A., Ito, H., Nakagawa, K., Yoshidome, H., Kataoka, M., and Nakajima, N. (2001). Vascular endothelial growth factor secreted by replicating hepatocytes induces sinusoidal endothelial cell proliferation during regeneration after partial hepatectomy in rats. J. Hepatol. 34, 683–9. Shimizu, T., Togo, S., Kumamoto, T., Makino, H., Morita, T., Tanaka, K., Kubota, T., Ichikawa, Y., Nagasima, Y., Okazaki, Y. et al. (2009). Gene expression during liver regeneration after partial hepatectomy in mice lacking type 1 tumor necrosis factor receptor. J. Surg. Res. 152, 178–88. Shiota, G., Wang, T.C., Nakamura, T., and Schmidt, E.V. (1994). Hepatocyte growth factor in transgenic mice: effects on hepatocyte growth, liver regeneration and gene expression. Hepatology 19, 962–72. Skov Olsen, P., Boesby, S., Kirkegaard, P., Therkelsen, K., Almdal, T., Poulsen, S.S., and Nex, E. (1988). Influence of epidermal growth factor on liver regeneration after partial hepatectomy in rats. Hepatology 8, 992–6. Snyder, R.L., Tyler, G., and Summers, J. (1982). Chronic hepatitis and hepatocellular carcinoma associated with woodchuck hepatitis virus. Am. J. Pathol. 107, 422–5. Stolz, D.B., Mars, W.M., Petersen, B.E., Kim, T.H., and Michalopoulos, G.K. (1999). Growth factor signal transduction immediately after two-thirds partial hepatectomy in the rat. Cancer Res. 59, 3954–60. Takeishi, T., Hirano, K., Kobayashi, T., Hasegawa, G., Hatakeyama, K., and Naito, M. (1999). The role of Kupffer cells in liver regeneration. Arch. Histol. Cytol. 62, 413–22. Thompson, N.L., Mead, J.E., Braun, L., Goyette, M., Shank, P.R., Fausto, N. (1986). Sequential Protooncogene Expression during Rat Liver Regeneration. Cancer Res. 46, 3111–7.

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Uehara, Y., Minowa, O., Mori, C., Shiota, K., Kuno, J., Noda, T., Kitamura, N. (1995). Placental defect and embryonic lethality in mice lacking hepatocyte growth factor/scatter factor. Nature 373, 702–5. Van Buren, G., Yang, A.D., Dallas, N.A., Gray, M.J., Lim, S.J., Xia, L., Fan, F., Somcio, R., Wu, Y., Hicklin, D.J. et al. (2008). Effect of Molecular Therapeutics on Liver Regeneration in a Murine Model. J. Clin. Oncol. 26, 1836–42. Wack, K.E., Ross, M.A., Zegarra, V., Sysko, L.R., Watkins, S.C., and Stolz, D.B. (2001). Sinusoidal ultrastructure evaluated during the revascularization of regenerating rat liver. Hepatology 33, 363–78. Wang, H., Iakova, P., Wilde, M., Welm, A., Goode, T., Roesler, W.J., and Timchenko, N.A. (2001). C/EBPalpha arrests cell proliferation through direct inhibition of Cdk2 and Cdk4. Mol. Cell. 8, 817–28. Wang, H.H., Lautt, W.W. (1998). Evidence of nitric oxide, a flow-dependent factor, being a trigger of liver regeneration in rats. Can. J. Physiol. Pharmacol. 76, 1072–9. Wuestefeld, T., Klein, C., Streetz, K.L., Betz, U., Lauber, J., Buer, J., Manns, M.P., Müller, W., and Trautwein, C. (2003). Interleukin-6/Glycoprotein 130-dependent Pathways Are Protective during Liver Regeneration. J. Biol. Chem. 278, 11281–8. Yamada, Y., Kirillova, I., Peschon, J.J., and Fausto, N. (1997). Initiation of liver growth by tumor necrosis factor: deficient liver regeneration in mice lacking type I tumor necrosis factor receptor. Proc. Natl. Acad. Sci. U.S.A. 94, 1441–6. Zarnegar, R., Muga, S., Enghild, J., and Michalopoulos, G. (1989). NH2-terminal amino acid sequence of rabbit hepatopoietin A, a heparin-binding polypeptide growth factor for hepatocytes. Biochem. Biophys. Res. Commun. 163, 1370–6. Zhang, X.K., Gauthier, T., Burczynski, F.J., Wang, G.Q., Gong, Y.W., and Minuk, G.Y. (1996). Changes in liver membrane potentials after partial hepatectomy in rats. Hepatology 23, 549–51.

2

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 marrow–derived stem cell therapies for the treatment of liver diseases: from bench to bedside

2.1

Stem Cells: Definition 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 defined by two fundamental properties: self-maintenance and multipotency. v Self-maintenance represents a cell’s 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 (non–self-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).

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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 modifications 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-specific, 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 cell–cell and cell–matrix connections as well as the secretion of soluble factors, influence 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: Definition 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 firstly to the yolk sac, then to the fetal liver and spleen, and finally 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 specific 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-fibroblastic 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 hepatocytes—have been shown both in vitro and in vivo, as reviewed elsewhere (Alvarez-Dolado et al., 2003). Overall, cell fusion is a

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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 beneficial 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 specific 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 cell’s 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

Liver Stem Cells and Their Role in Hepatic Regeneration

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 efficient 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

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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 specific 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-deficient diet followed by ethionine exposure, galactosamine, 2-acetylaminofluorene (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 confirmed 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 identifies 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 identified by one specific 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 specific for OC response. Differential activity was observed on normal liver cells and at different stages of OC activation, indicating potential utility for progenitor cell identification (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

Liver Stem Cells and Their Role in Hepatic Regeneration

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 amplification 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 identification of a specific LSC marker awaits further investigation, as reviewed elsewhere (Duncan et al., 2009; Piscaglia, 2008a). Overall, whether OCs/HPCs fulfill 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 Dissé, 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 difficulty 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 identified: (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.

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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, fibroblast 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 inflammatory cells within the niche, producing a range of cytokines and chemokines (such as TWEAK, TGFbeta, and INF-gamma) that could influence the LSC response (Duncan et al., 2009). Another molecule of growing interest in the field 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 myelofibrosis and Cooley’s 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 significant 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 significant 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).

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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 identified 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 identifiable 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 fluorescent protein or genetic markers, or by sex-mismatching, so that their progenies within the recipient’s 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 first 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-deficient (DPPIV) rats received the mitotic inhibitor monocrotaline, followed by DPPIV BM transplantation, and finally 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 significant 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 sufficient per se to achieve a significant 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 modifications 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, detoxification, tissue repair, and remodeling (Piscaglia, 2005). In this context, it must be noted that BM cells significantly 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.

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cells within the liver, such as Kupffer cells, endothelial cells, and myofibroblasts. 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 marrow–derived stem cells as a final attempt to restore liver homeostasis.

2.4

Clinical Applications of Bone Marrow–Derived 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 fibrosis 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 cell–cell 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 specific growth factors, such as basic fibroblastic 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 beneficial 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 profile 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 profiles of ATSC-derived hepatocyte-like cells were compared with undifferentiated ATSCs. We identified 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).

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2.4

2



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 defined 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 confined to preclinical studies, given the scarce tissue-specific 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 2.2–2.4). 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 efficacy of these SC-based treatments, as well as their pro-fibrogenic 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):463–70.

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

Fürst G, et al. Radiology. 2007 Apr;243(1):171–9.

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):2292–8. Epub 2006 Jun 15.

Autologous BMSC transplantation prior to surgery in patients with cirrhosis and hepatocellular carcinoma

Significant increase of liver function post-LR

Ismail A, et al. J Gastrointest Cancer. 2010 Mar;41(1):17–23.

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):3359–63.

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Table 2.3

Oval Cells, Bone Marrow, and Liver Regeneration 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):1583–7.

Intraportal administration of mobilized CD34 BMSCs following G-CSF exposure in one patient affected by drug-induced acute liver failure

Significant biochemical and histopathological improvement

Gasbarrini A, et al. Dig Liver Dis. 2007 Sep;39(9):878–82. 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 significant side effects during a follow-up of 60 days

Gordon MY, et al. Stem Cells. 2006 Jul;24(7):1822–30. 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 beneficial effects lasted around 12 months

Levicar N, et al. Cell Prolif. 2008 Feb;41 Suppl 1:115–25.

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):1952–8.

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 significant biochemical and clinical improvement was observed in both groups (G-CSF plus SC infusion obtained the best beneficial effects)

Han Y, et al. Cytotherapy. 2008;10(4):390–6.

2.4 Table 2.4


33

G-CSF Treatment

G-CSF 5 μg/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):13–9. 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):1071–6.

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):932–9.

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 efficacy

Spahr L, et al. Hepatology. 2008 Jul;48(1):221–9.

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 identification of a specific 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 sufficient to achieve a significant contribution to hepatic repopulation. However, the few bone marrow cells that do engraft may play

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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 Dollé, 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, 117–29. 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, 1608–16. 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, 178–89. Kisseleva, T., Gigante, E., and Brenner, D.A. (2010). Recent advances in liver stem cell therapy. Curr Opin Gastroenterol. 26, 395–402. 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, 61–74. Piscaglia, A.C., Shupe, T.D., Petersen, B.E., and Gasbarrini, A. (2007). Stem cells, cancer, liver, and liver cancer stem cells: finding a way out of the labyrinth . . . Curr Cancer Drug Targets. 7, 582–90. Quante, M., and Wang, T.C. (2009). Stem cells in gastroenterology and hepatology. Nat Rev Gastroenterol Hepatol. 6, 724–37. Scadden, D.T. (2006). The stem-cell niche as an entity of action. Nature 441, 1075–1079. Sell, S. (2004). Stem cell origin of cancer and differentiation therapy. Crit. Rev. Oncol. Hematol. 51, 1–28. 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, 1598–607.

References Alison, M.R., Poulsom, R., Jeffrey, R., Dhillon, A.P., Quaglia, A., Jacob, J., Novelli, M., Prentice, G., Williamson, J., and Wright, N.A. (2000). Hepatocytes from non-hepatic adult stem cells. Nature 406, 257. Alison, M.R., and Islam, S. (2009). Attributes of adult stem cells. J Pathol. 217, 144–60. Alvarez-Dolado, M., Pardal, R., Garcia-Verdugo, J.M., Fike, J.R., Lee, H.O., Pfeffer, K., Lois, C., Morrison, S.J., and Alvarez-Buylla, A. (2003). Fusion of bone-marrow-derived cells with Purkinjie neurons, cardiomyocytes and hepatocytes. Nature 425, 968–73.

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Kordes, C., Sawitza, I., Müller-Marbach, A., Ale-Agha, N., Keitel, V., Klonowski-Stumpe, H., and Häussinger, D. (2007). CD133 hepatic stellate cells are progenitor cells. Biochem Biophys Res Commun. 352, 410–17. Kordes, C., Sawitza, I., and Häussinger, D. (2009). Hepatic and pancreatic stellate cells in focus. Biol Chem. 390, 1003–12. Krause, D.S., Theise, N.D., Collector, M.I., Henegariu, O., Hwang, S., Gardner, R., Neutzel, S., and Sharkis, S.J. (2001). Multi-organ, multi-lineage engraftment by a single bone marrowderived stem cell. Cell. 105, 369–77. Kucia, M., Reca, R., Campbell, F.R., Zuba-Surma, E., Majka, M., Ratajczak, J., and Ratajczak, M.Z. (2006). A population of very small embryonic-like (VSEL) CXCR4()SSEA-1() Oct-4 stem cells identified in adult bone marrow. Leukemia. 20, 857–69. Kuwahara, R., Kofman, A.V., Landis, C.S., Swenson, E.S., Barendswaard, E., and Theise, N.D. (2008). The hepatic stem cell niche: identification by label-retaining cell assay. Hepatology 47, 1994–2002. Lagasse, E., Connors, H., Al-Dhalimy, M., Reitsma, M., Dohse, M., Osborne, L., Wang, X., Finegold, M., Weissman, I.L., and Grompe, M. (2000). Purified haematopoietic stem cells can differentiate into hepatocytes in vivo. Nat Med 6, 1229–34. Liongue, C., Wright, C., Russell, A.P., and Ward, A.C. (2009). Granulocyte colony-stimulating factor receptor: stimulating granulopoiesis and much more. Int J Biochem Cell Biol. 41, 2372–5. Marshall, A., Rushbrook, S., Davies, S.E., Morris, L.S., Scott, I.S., Vowler, S.L., Coleman, N., and Alexander, G. (2005). Relation between hepatocyte G1 arrest, impaired hepatic regeneration, and fibrosis in chronic hepatitis C virus infection. Gastroenterology 128, 33–42. Michalopoulos, G.K. (2011). Liver regeneration: alternative epithelial pathways. Int J Biochem Cell Biol. 43, 173–9. Mimeault, M., Hauke, R., and Batra, S.K. (2007). Stem cells: a revolution in therapeuticsrecent advances in stem cell biology and their therapeutic applications in regenerative medicine and cancer therapies. Clin Pharmacol Ther. 82, 252–64. Moore, K.A., and Lemischka, I.R. (2006). Stem cells and their niches. Science 311, 1880–5. Murase, N., Starzl, T.E., Ye, Q., Tsamandas, A., Thomson, A.W., Rao, A.S., and Demetris, A.J. (1996). Multilineage hematopoietic reconstitution of supralethally irradiated rats by syngeneic whole organ transplantation. Transplantation 61, 1–4. Nierhoff, D., Horvarth, H.C., Mytilineos, J., Golling, M., Bud, O., Klar, E., Opelz, G., Voso, M.T., Ho, A.D., Haas, R., and Hohaus, S. (2000). Microchimerism in bone marrow derived CD34() cells of patients after liver transplantation. Blood 96, 763–7. Nowak, M.A., Bonhoeffer, S., Hill, A.M., Boehme, R., Thomas, H.C., and McDade, H. (1996). Viral dynamics in hepatitis B virus infection. Proc Natl Acad Sci USA 93, 4398–402. Oh, S.H., Witek, R.P., Bae, S.H., Zheng, D., Jung, Y., Piscaglia, A.C., and Petersen, B.E. (2007). Bone marrow-derived hepatic oval cells differentiate into hepatocytes in 2-acetylaminofluorene/partial hepatectomy-induced liver regeneration. Gastroenterology 132, 1077–87. Overturf, K., al-Dhalimy, M., Ou, C.N., Finegold, M., and Grompe, M. (1997). Serial transplantation reveals the stem-cell-like regenerative potential of adult mouse hepatocytes. Am J Pathol. 151, 1273–80. Petersen, B.E., Bowen, W.C., Patrene, K.D., Mars, W.M., Sullivan, A.K., Murase, N., Boggs, S.S., Greenberger, J.S., and Goff, J.P. (1999). Bone marrow as a potential source of hepatic oval cells. Science 284, 1168–70. Petersen, B., and Shupe, T. (2008). Location is everything: the liver stem cell niche. Hepatology 47, 1810–2. Piscaglia, A.C., Zocco, M.A., Di Campli, C., Sparano, L., Rutella, S., Monego, G., Bonanno, G., Michetti, F., Mancuso, S., Pola, P., Leone, G., Gasbarrini, G., and Gasbarrini, A. (2005).

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How does human stem cell therapy influence gene expression after liver injury? Microarray evaluation on a rat model. Dig Liver Dis. 37, 952–63. Piscaglia, A.C., Shupe, T., Gasbarrini, A., and Petersen, B.E. (2007a). Microarray RNA/DNA in different stem cell lines. Curr Pharm Biotechnol. 8, 167–75. Piscaglia, A.C., Shupe, T.D., Oh, S.H., Gasbarrini, A., and Petersen, B.E. (2007b). Granulocyte-colony stimulating factor promotes liver repair and induces oval cell migration and proliferation in rats. Gastroenterology 133, 619–31. Piscaglia, A.C., Novi, M., Campanale, M., and Gasbarrini, A. (2008a). Stem cell-based therapy in gastroenterology and hepatology. Minim Invasive Ther Allied Technol. 17, 100–18. Piscaglia, A.C. (2008b). Stem cells, a two-edged sword: risks and potentials of regenerative medicine. World J Gastroenterol. 14, 4273–9. Piscaglia, A.C., Zocco, M.A., Giuliante, F., Arena, V., Novi, M., Rinninella, E., Tortora, A., Rumi, C., Nuzzo, G., Vecchio, F.M., Bombardieri, G., and Gasbarrini, A. (2011). CD133 stem cell mobilization after partial hepatectomy depends on resection extent and underlying disease. Dig Liver Dis. 43, 147–54. Puglisi, M.A., Saulnier, N., Piscaglia, A.C., and Gasbarrini, A. (2011). Adipose tissue-derived mesenchymal stem cells and hepatic differentiation: old concepts and future perspectives. Eur Rev Med Pharmacol Sci. (in press) Ratajczak, M.Z., Kucia, M., Reca, R., Majka, M., Janowska-Wieczorek, A., and Ratajczak, J. (2004). Stem cell plasticity revisited: CXCR4-positive cells expressing mRNA for early muscle, liver and neural cells ‘hide out’ in the bone marrow. Leukemia 18, 29–40. Roskams, T. (2006). Different types of liver progenitor cells and their niches. J Hepatol. 45, 1–4. Sackett, S.D., Li, Z., Hurtt, R., Gao, Y., Wells, R.G., Brondell, K., Kaestner, K.H., and Greenbaum, L.E. (2009). Foxl1 is a marker of bipotential hepatic progenitor cells in mice. Hepatology 49, 920–9. Saji, Y., Tamura, S., Yoshida, Y., Kiso, S., Iizuka, A.S., Matsumoto, H., Kawasaki, T., Kamada, Y., Matsuzawa, Y. and Shinomura, Y. (2004). Basic fibroblast growth factor promotes the trans-differentiation of mouse bone marrow cells into hepatic lineage cells via multiple liver-enriched transcription factors. J. Hepatol. 41, 545–50. Saulnier, N., Piscaglia, A.C., Pani, G., Puglisi, M.A., Barba, M., Alfieri, S., and Gasbarrini, A. (2010). Molecular mechanisms underlying human adipose tissue-derived stromal cells differentiation into a hepatocyte-like phenotype. Dig Liver Dis. 42, 890–901. Sawitza, I., Kordes, C., Reister, S., and Häussinger, D. (2009). The niche of stellate cells within rat liver. Hepatology 50,1617–24. Shupe, T.D., Piscaglia, A.C., Oh, S.H., Gasbarrini, A., and Petersen, B.E. (2009). Isolation and characterization of hepatic stem cells, or “oval cells,” from rat livers. Methods Mol Biol. 482, 387–405. Spee, B., Carpino, G., Schotanus, B.A., Katoonizadeh, A., Vander Borght, S., Gaudio, E., and Roskams, T. (2010). Characterisation of the liver progenitor cell niche in liver diseases: potential involvement of Wnt and Notch signalling. Gut. 59, 247–57. Spees, J.L., Olson, S.D., Ylostalo, J., Lynch, P.J., Smith, P., Perry, A., Peister, A., Wang, M.Y., and Prockop, D.J. (2003). Differentiation, cell fusion, and nuclear fusion during ex vivo repair of epithelium by human adult stem cells from bone marrow stroma. PNAS, 100, 2397–402. Tanabe, Y., Tajima, F., Nakamura, Y., Shibasaki, E., Wakejima, M., Shimomura, T., Murai, R., Murawaki, Y., Hashiguchi, K., Kanbe, T., Saeki, T., Ichiba, M., Yoshida, Y., Mitsunari, M., Yoshida, S., Miake, J., Yamamoto, Y., Nagata, N., Harada, T., Kurimasa, A., Hisatome, I., Terakawa, N., Murawaki, Y. and Shiota, G. (2004). Analyses to clarify rich fractions in hepatic progenitor cells from human umbilical cord blood and cell fusion. Biochem. Biophys. Res. Com. 324, 711–18.

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Theise, N.D., Badve, S., Saxena, R., Henegariu, O., Sell, S., Crawford, J.M., and Krause, D.S. (2000a). Derivation of hepatocytes from bone marrow in mice after radiation induced myeloablation. Hepatology 31, 235–40. Theise, N.D., Nimmakajalu, M., Gardner, R., Illei, P.B., Morgan, G., Teperman, L., Henegariu, O., and Krause, D.S. (2000b). Liver from bone marrow in humans. Hepatology 32, 11–6. Theise, N.D., and Krause, D.S. (2002). Bone marrow to liver: the blood of Prometheus. Semin Cell Dev Biol. 13, 411–7. Theise, N.D. (2006). Gastrointestinal stem cells. III. Emergent themes of liver stem cell biology: niche, quiescence, self-renewal, and plasticity. Am J Physiol Gastrointest Liver Physiol. 290, G189–93. Thorgeirsson, S.S., and Grisham, J.W. (2006). Hematopoietic cells as hepatocyte stem cells: a critical review of the evidence. Hepatology 43, 2–8. Tocci, A., and Forte, L. (2003). Mesenchymal stem cell: use and perspectives. Hematol J. 4, 92–6. Vassilopoulos, G., Wang, P.R., and Russel, D. (2003). Transplanted bone marrow generates liver by cell fusion. Nature 422, 901–4. Wang, X., Willenbring, H., Akkari, Y., Torimaru, Y., Foster, M., Al-Dhalimy, M., Lagasse, E., Finegold, M., Olson, S., and Grompe, M. (2003). Cell fusion is the principal source of bone-marrow-derived hepatocytes. Nature 422, 897–901. Zhou, H., Rogler, L.E., Teperman, L., Morgan, G., and Rogler, C.E. (2007). Identification of hepatocytic and bile ductular cell lineages and candidate stem cells in bipolar ductular reactions in cirrhotic human liver. Hepatology 45, 716–24.

3

Inflammation and Liver Regeneration Johannes G. Bode

Learning Targets 1. Regulatory role of the acute phase response and of macrophages for liver regeneration 2. The inflammatory 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 detoxification 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 salt–dependent 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 influx 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 inflammatory response and innate and adaptive immunity as it integrates inflammatory 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 structural–functional organization with the different non-parenchymal cells of the liver, such as sinusoidal endothelial cells, hepatic stellate cells, and liver macrophages

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Inflammation and Liver Regeneration

(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 inflammatory process by releasing a variety of inflammatory 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 inflammatory mediators released upon their activation. This chapter focuses on the role of inflammation for the course of liver regeneration and, in particular, on those inflammatory mediators and immune cells involved in liver regeneration.

3.2

Liver Regeneration and Inflammation: 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 structural–functional organization of the sinusoidal unit required to fulfill 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 defined inflammatory response is central to regulation of liver regeneration subsequent to liver injury. Thereby, the release of inflammatory cytokines, such as IL-6, OSM, or TNFα, 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 inflammatory 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 inflammatory 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 inflammatory reactions that occurs upon tissue injury caused by trauma, infection, or noninfectious inflammatory 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 inflammatory reactions varies and largely depends on the eliciting pathogenic cause. It becomes clear from this that a misdirected and inappropriate inflammatory response would unequivocally result in an imbalance of tissue destruction, repair, and regeneration, leading to insufficient 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 inflammation, alterations of the inflammatory 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 reflects the activation of innate immunity in response to liver injury. Although the source of inflammatory cytokines released during liver regeneration is not fully clarified 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 conflicting 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 first 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 findings because liposomeencapsulated dichloromethylene diphosphonate, which is used by the majority of the aforementioned reports, is nontoxic to hepatocytes, and upon intracellular release of

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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 specifically 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 inflammation and regeneration and are reported to be the most important source of TNFα 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 TNFα 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 TNFα 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 inflammatory 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 significant decrease in hepatocyte proliferation and reduced tissue levels of TNFα and IL-6 ( Jackson et al., 2008). These data corroborate the relevance of macrophage recruitment being required for inflammatory 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 deficient 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 deficient 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 deficient animals underscores the relevance of the inflammatory cytokine response generated by these inflammatory 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, Inflammatory 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 TNFα or LTα. 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)-κB. 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 (inflammatory) mediators, including chemokines, which mediate recruitment of cells from the circulation; monocytes; and other immune competent cells or even bone marrow–derived stem cells.

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Inflammatory 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 inflammatory cytokines such as TNFα 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 TNFα 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 inflammatory cytokines in the regulation of hepatocyte proliferation (Abshagen et al., 2007; Selzner et al., 2003). The important role of TNFα for liver regeneration is supported by the observations that injection of TNFα 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 TNFα 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 deficient 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 TNFα signaling and decreased release of IL-6, the activation of nuclear-factor (NF)-κB 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 deficient 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 confirmed by the demonstration that regeneration after partial hepatectomy was substantially delayed in TNFR1 deficient 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 deficient 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-κB binding in the regenerating liver (Yamada et al., 1997). Thereby, sustained activation of NF-κB 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 TNFα that TNFR1-dependently mediates NF-κB 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 TNFα prevents IL-6 from activating STAT3 in macrophages but not in hepatocytes (Bode et al., 1999).

3.4

Inflammatory Mediators Are Required to PromoteLiver Regeneration

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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 deficient 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-deficient animals nor in those lacking CD14, TLR2, or TLR4 (Campbell et al., 2006). Hence, these data may also suggest that TNFα and IL-6 are not as important for regulation of hepatocyte proliferation as originally presumed, a view that is also suggested from studies analyzing TNFα-deficient 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 TNFα itself. In this context, it should be noted that in contrast to mice deficient for TNFα, hepatocyte DNA replication after partial hepatectomy is down-regulated in animals lacking both TNFα and lymphotoxin α (Knight and Yeoh, 2005). As for TNFα, 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 TNFα, 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-deficient 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 deficient for IL-6 or gp130, the signal transducing subunit of the IL-6 receptor complex, could not confirm 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

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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 TNFα and IL-6 has been comparably broadly addressed, the role of other inflammatory 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 receptor–deficient 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 TGFα in response to liver resection (Ren et al., 2010). Another group of inflammatory 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 angiogenesis—functions 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 inflammatory 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 Inflammation Impairs Liver Regeneration

The increased release of the different inflammatory mediators, which regulate liver regeneration, is the result of a controlled inflammatory response. Imbalanced or misdirected induction of this inflammatory response, for example, because of infectious disorders acquired prior to or during liver regeneration, would inevitably result in insufficient tissue repair, tissue destruction, and impaired organ/hepatocyte functionality, and therefore, might be fatal for a patient’s recovery following partial hepatectomy. Thereby, cytokines such as IL-1β released during an inflammatory 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 TNFα 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 identified as important mediators of ischemia/reperfusion-induced liver injury. The molecular mechanisms enabling factors such as TNFα 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 significant 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 inflammatory response and, in particular, on TNFα concentrations, which were significantly 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 influence the final effect. Thus, for example, in hepatocytes IL-1β prevents IL-6 from activating STAT3 (Albrecht et al., 2007; Bode et al., 1999), which may contribute to the antiproliferative effects of IL-1β 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 influence 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 IFNγ produced by NK cells (Sun and Gao, 2004) and IFNγ 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 IFNγ 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 IFNγ-deficient mice. The fact that IFNγ or IFNγ receptor–deficient animals show an enhanced liver regeneration upon partial hepatectomy, whereas administration of IFNγ resulted in impaired liver regeneration, further supports the notion that IFNγ 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

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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 specifically suppress lytic activity of liver resident NK cells being associated with suppression of IFNγ expression in vivo (Francavilla et al., 1997). Hence suppression of NK cell activity and down-regulation of NK cell–derived 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 deficient 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 defined conditions NKT cells may also negatively influence liver regeneration, an effect that has also been mainly attributed to the release of IFNγ. This assumption is further supported by the fact that activation of NKT cells by IL-12 or α-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 defined 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 detoxification 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 inflammatory response play an important role for the early phase of liver regeneration, particularly regulating hepatocyte proliferation. In this context, not only cytokines such as TNFα, IL-6, IL-22, and OSM or chemokines such as CXCL2 but also other inflammatory mediators such as components of the complement system are of importance. Disturbance of this inflammatory response can have detrimental effects on the regenerative response, resulting in impaired liver regeneration or liver failure.

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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. 565–80. Crispe, I.N. (2009). The liver as a lymphoid organ. Annu. Rev. Immunol. 27, 147–63. Häussinger, 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, 221–360. Michalopoulos, G.K. (2010). Liver regeneration after partial hepatectomy: critical analysis of mechanistic dilemmas. Am. J. Pathol. 176, 2–13. Tiegs, G., and Lohse, A.W. (2010). Immune tolerance: what is unique about the liver. J. Autoimmun. 34, 1–6.

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Meijer, C., Wiezer, M.J., Diehl, A.M., Schouten, H.J., Meijer, S., van Rooijen, N., van Lambalgen, A.A., Dijkstra, C.D., and van Leeuwen, P.A. (2000). Kupffer cell depletion by CI2MDPliposomes alters hepatic cytokine expression and delays liver regeneration after partial hepatectomy. Liver 20, 66–77. Nakamura, K., Nonaka, H., Saito, H., Tanaka, M., and Miyajima, A. (2004). Hepatocyte proliferation and tissue remodeling is impaired after liver injury in oncostatin M receptor knockout mice. Hepatology 39, 635–44. Nowak, M., Gaines, G.C., Rosenberg, J., Minter, R., Bahjat, F.R., Rectenwald, J., MacKay, S.L., Edwards, C.K., and Moldawer, L.L. (2000). LPS-induced liver injury in D-galactosaminesensitized mice requires secreted TNF-alpha and the TNF-p55 receptor. Am. J. Physiol. Regul. Integr. Comp. Physiol. 278, R1202–9. Oishi, K., Hayamizu, K., Aihaiti, X., Itamoto, T., Arihiro, K., and Asahara, T. (2006). G-CSFinduced evacuation of sinusoidal NK cells and the facilitation of liver regeneration in a partial hepatectomy. Cytokine 34, 66–75. Rai, R.M., Loffreda, S., Karp, C.L., Yang, S.Q., Lin, H.Z., and Diehl, A.M. (1997). Kupffer cell depletion abolishes induction of interleukin-10 and permits sustained overexpression of tumor necrosis factor alpha messenger RNA in the regenerating rat liver. Hepatology 25, 889–95. Rai, R.M., Yang, S.Q., McClain, C., Karp C.L., Klein, A.S., and Diehl, A.M. (1996). Kupffer cell depletion by gadolinium chloride enhances liver regeneration after partial hepatectomy in rats. Am. J. Physiol. 270, G909–18. Ren, X., Hu, B., and Colletti, L. (2008). Stem cell factor and its receptor, c-kit, are important for hepatocyte proliferation in wild-type and tumor necrosis factor receptor-1 knockout mice after 70% hepatectomy. Surgery 143, 790–802. Ren, X., Hu, B., and Colletti, L.M. (2010). IL-22 is involved in liver regeneration after hepatectomy. Am. J. Physiol. Gastrointest. Liver Physiol. 298, G74–80. Sakamoto, T., Liu, Z., Murase, N., Ezure, T., Yokomuro, S., Poli, V., and Demetris, A.J. (1999). Mitosis and apoptosis in the liver of interleukin-6-deficient mice after partial hepatectomy. Hepatology 29, 403–11. Selzner, N., Selzner, M., Odermatt, B., Tian, Y., Van Rooijen, N., and Clavien, P.A. (2003). ICAM-1 triggers liver regeneration through leukocyte recruitment and Kupffer celldependent release of TNF-alpha/IL-6 in mice. Gastroenterology 124, 692–700. Shimizu, T., Togo, S., Kumamoto, T., Makino, H., Morita, T., Tanaka, K., Kubota, T., Ichikawa, Y., Nagasima, Y., Okazaki, Y., Hayashizaki, Y., and Shimada, H. (2009). Gene expression during liver regeneration after partial hepatectomy in mice lacking type 1 tumor necrosis factor receptor. J. Surg. Res. 152, 178–88. Strey, C.W., Markiewski, M., Mastellos, D., Tudoran, R., Spruce, L.A., Greenbaum, L.E., and Lambris, J.D. (2003). The proinflammatory mediators C3a and C5a are essential for liver regeneration. J. Exp. Med. 198, 913–23. Sun, R., and Gao, B. (2004). Negative regulation of liver regeneration by innate immunity (natural killer cells/interferon-gamma). Gastroenterology 127, 1525–39. Van Rooijen, N., and Sanders, A. (1994). Liposome mediated depletion of macrophages: mechanism of action, preparation of liposomes and applications. J. Immunol. Methods 174, 83–93. Wuestefeld, T., Klein, C., Streetz, K.L., Betz, U., Lauber, J., Buer, J., Manns, M.P., Muller, W., and Trautwein, C. (2003). Interleukin-6/glycoprotein 130-dependent pathways are protective during liver regeneration. J. Biol. Chem. 278, 11281–8. Yamada, Y., Kirillova, I., Peschon, J.J., and Fausto, N. (1997). Initiation of liver growth by tumor necrosis factor: deficient liver regeneration in mice lacking type I tumor necrosis factor receptor. Proc. Natl. Acad. Sci. U.S.A. 94, 1441–6.

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Yamada, Y., Webber, E.M., Kirillova, I., Peschon, J.J., and Fausto, N. (1998). Analysis of liver regeneration in mice lacking type 1 or type 2 tumor necrosis factor receptor: requirement for type 1 but not type 2 receptor. Hepatology 28, 959–70. Yang, L., Magness, S.T., Bataller, R., Rippe, R.A., and Brenner, D.A. (2005). NF-kappaB activation in Kupffer cells after partial hepatectomy. Am. J. Physiol. Gastrointest. Liver Physiol. 289, G530–8. Zimmers, T.A., McKillop, I.H., Pierce, R.H., Yoo, J.Y., and Koniaris, L.G. (2003). Massive liver growth in mice induced by systemic interleukin 6 administration. Hepatology 38, 326–34.

4

Lymphotoxin β Receptor and Tumor Necrosis Factor Receptor p55 in Liver Regeneration Ursula R. Sorg and Klaus Pfeffer

Learning Targets 1. The receptors TNFRp55 and LTβR, 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 LTβR leads to activation of the transcription factor NFκB.

4.1 TNF/TNFR Superfamily The tumor necrosis factor receptor p55 (TNFRp55) and the lymphotoxin β receptor (LTβR) 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: beneficial effects in inflammation 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 inflammatory 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 reflected 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 LTα3 homotrimer, which does not contain a transmembrane region, and the heterotrimeric LTα1β2, which contains one LTα and two LTβ subunits. Some of the ligands (e.g., TNF and LTα) 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

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Lymphotoxin B Receptor and Tumor Necrosis Factor Receptor p55

into three distinct groups. The first 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). LTβR 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, LTα3 , LTα1β2 , 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. LTα3 lacks the membrane anchoring domain and is always secreted. LTα1β2 is anchored to the membrane via the LTβ 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 κB (NFκB) activation (fFigure 4.2), formation of the TNFRp55/TRADD complex induces phosphorylation and activation of the inhibitor of NFκB (IκB) kinase (IKK) complex, consisting of IKKα, β and G. This complex is then responsible for the phosphorylation and subsequent ubiquitinylation and, ultimately, the proteasomal degradation of IκBα. NFκB 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

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ub i ny quiti lat ion apoptosis

IKKa

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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 NFκB Activation Via TNFRp55 and LTβR Note: Shown are the canonical and the alternative pathways.

activation of transcription

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heterodimers of a family of five related proteins (relA, relB, c-rel, p50/NFκB1, and p52/ NFκB2). The degradation of IκBα releases the active NFκB(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 NFκB 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 NFκB activation is also induced through LTβR after binding of its ligands LTα1β2 or LIGHT. The LTβR 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 NFκB(RelA:p50) complex into the nucleus. In an alternative (non-canonical) pathway of NFκB activation (fFigure 4.2), the TNFRp55/TRAF2 complex can activate the NFκB inducing kinase (NIK), which activates the IKKα subunit, which is bound to and stabilizes the inactive NFκB(RelB:p100) dimer. Activation of IKKα leads to ubiquitinylation and partial degradation of p100 into the active p52 subunit. Subsequently, active NFκB(RelB:p52) translocates into the nucleus and initiates gene transcription. The transcription factor NFκB induces (among many other genes) the expression of p100, linking the TNFR with the LTβR signaling pathway. Together, the NFκB family can activate the expression of several hundred target genes (Ware, 2008). LTβR/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 fulfills 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, detoxification, 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 fibrotic/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

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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 inefficient 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 40–44 hours after PHx (22–26 hours in rats), followed by hepatocyte mitosis 6–8 hours later. Cell division of the non-parenchymal liver cells occurs 12–24 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 7–10 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., NFκB, AP-1, STAT3, CEBPβ), 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

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TNF/TNFR superfamily: TNF, expressed by many somatic, antigen presenting and also T cells, as well as LTα3, expressed on T, B, and NK cells with similar functions as TNF (Apostolaki et al., 2010). TNF/TNFRp55 signaling plays a prominent role in proinflammatory 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. NFκB and STAT3 activation is inhibited, and AP-1 activation is decreased (Yamada et al., 1997). In contrast, mice deficient in TNFRp75 show normal hepatocyte proliferation and gain of liver mass after PHx, although expression of the transcription factors AP-1 and CEBPβ is delayed, demonstrating that TNF/TNFRp55 signaling but not TNF/TNFRp75 signaling is essential for efficient 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 efficient 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 NFκB transcription factors via the canonical pathway. NFκB 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 deficient 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 deficient 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 LTβR is expressed on non-hematopoietic cells and has two ligands: LTα1β2, which is expressed in high amounts by naïve B cells in the spleen but also by CD4 T cells; and LIGHT, which is inducible in resting T cells. LTα1β2/LTβR 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/LTβR signaling is also involved in dendritic cell homeostasis. Transgenic mice overexpressing LIGHT (Tg LIGHT mice), show significantly increased liver size compared to WT litter mates, and

4.4

LTBR and Liver Regeneration

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histology shows enlarged hepatocytes and an increase of mitotic figures (Anders et al., 2005). When Tg LIGHT mice were crossed with LTβR deficient (LTβR-/-) mice, offspring showed normal liver size and histology, indicating that constitutive LIGHT/LTβR signaling stimulates hepatocyte growth and induces hepatomegaly. The abnormal liver phenotype was not abrogated when Tg LIGHT mice were crossed with mice that were deficient for HVEM (HVEM-/-), the other receptor for LIGHT, demonstrating that LIGHT/LTβR 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 deficient in the lymphocyte compartment, confirming that T-cell derived LIGHT is involved in regulation of liver homeostasis (Tumanov et al., 2009). When 70% PHx was performed on LTβR-/- mice, the animals showed increased mortality (survival rates of around 30% compared to 100% in WT animals), and histology showed significantly increased rates of apoptosis and significantly decreased hepatocyte proliferation, documenting the necessity of LTβR signaling for efficient 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 deficient for LTα1β2 in either the B or T cell compartment clearly demonstrated that LTα1β2 delivered by T cells to the liver is essential for efficient liver regeneration (as opposed to LIGHT/LTβR signaling in liver homeostasis). Treatment of RAG1-/- mice with an agonistic anti-LTβR 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 fibroblasts led to induction of IL-6 expression, indicating that LTβR dependent activation of TNF signaling may play a role in liver regeneration (Tumanov et al., 2009). Even though TNFRp55 and LTβR obviously play a major role in liver regeneration as demonstrated by significantly 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 LTβR/TNFRp55 double knockout mice. Clearly, the TNF/TNFRp55 and the LTα1β2/LTβR 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 defined junctions and intervene in a clinical context (Hehlgans and Pfeffer, 2005).

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Summary The TNF/TNFR superfamily comprises a large group of about 50 ligands and their receptors, which play a beneficial role, for example, in inflammation 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 LTβR belong to the core members of the family, and signaling through TNFRp55 as well as the LTβR can lead to activation of NFκB, an important transcription factor, via the canonical activation pathway. The LTβR is also able to activate NFκB 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 NFκB 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 LTβR 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 deficient in certain genes, respectively, have shown that signaling through the LTβR is involved not only in liver homeostasis (via LIGHT) but also in the early stages of liver regeneration (via LTα1β2): LTβR deficient animals show significantly 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, 294–305. Capecchi, M.R. (2005). Gene targeting in mice: functional analysis of the mammalian genome for the twenty-first century. Nat Rev Genet 6, 507–12. 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 Häussinger, D. (2009). Epidermal growth factor receptor signaling in liver cell proliferation and apoptosis. Biol Chem 390, 1033–7.

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, 1295–300. Apostolaki, M., Armaka, M., Victoratos, P., and Kollias, G. (2010). Cellular mechanisms of TNF function in models of inflammation and autoimmunity. Curr. Dir. Autoimmun. 11, 1–26. Crispe, I.N. (2009). The liver as a lymphoid organ. Annu. Rev. Immunol. 27, 147–63.

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Croft, M. (2009). The role of TNF superfamily members in T-cell function and diseases. Nat. Rev. Immunol. 9, 271–85. Fausto, N., Campbell, J.S., and Riehle, K.J. (2006). Liver regeneration. Hepatology 43, S45–53. Futterer, A., Mink, K., Luz, A., Kosco-Vilbois, M.H., and Pfeffer, K. (1998). The lymphotoxin beta receptor controls organogenesis and affinity maturation in peripheral lymphoid tissues. Immunity 9, 59–70. 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, 1–20. Kordes, C., Sawitza, I., and Häussinger, D. (2009). Hepatic and pancreatic stellate cells in focus. Biol. Chem. 390, 1003–12. Kordes, C., Sawitza, I., Muller-Marbach, A., Ale-Agha, N., Keitel, V., Klonowski-Stumpe, H., and Häussinger, D. (2007). CD133 hepatic stellate cells are progenitor cells. Biochem Biophys Res. Commun. 352, 410–7. Michalopoulos, G.K. (2007). Liver regeneration. J. Cell. Physiol. 213, 286–300. 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, 694–704. Ware, C.F. (2008). Targeting lymphocyte activation through the lymphotoxin and LIGHT pathways. Immunol. Rev. 223, 186–201. Yamada, Y., Kirillova, I., Peschon, J.J., and Fausto, N. (1997). Initiation of liver growth by tumor necrosis factor: deficient liver regeneration in mice lacking type I tumor necrosis factor receptor. Proc. Natl. Acad. Sci. U.S.A. 94, 1441–6.

5 The Hepatic Stem Cell Niches Iris Sawitza, Claus Kordes, and Dieter Häussinger

Learning Targets 1. Components of a stem cell niche are the stem cells, neighboring cells, secreted factors, cell–cell 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 Dissé.

5.1

Introduction

Stem cells are found in all multicellular organisms. Broadly defined, 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 replication—one 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 replication—one stem cell divides into two daughter cells that remain identical to the original stem cell s Stochastic replication—one 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 field emanates from findings by Ernest A. McCulloch and James E. Till at the University of Toronto in the 1960s. Under healthy conditions of an

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5 The Hepatic Stem Cell Niches

SC

C

Notes: Essential components of the niche are the stem cells (SC), different neighboring cell types (NC), secreted factors (yellow dots), cell–cell 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 difficult 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 Schofield in 1978, who proposed that stem cells reside within fixed compartments or niches that are conducive to the maintenance of definitive stem cell properties. The microenvironment of stem cells modulates their proliferation, influences 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 cell–cell 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 define 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 β (TGFβ) and wingless type (Wnt), show remarkable evolutionary conservation among species and are capable of directing stem cell fate.

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TGFβ family members of signaling proteins are TGFβ, Nodal, Activin, and bone morphogenetic protein (Bmp), which regulate diverse cellular functions such as growth arrest, apoptosis, migration, and differentiation. TGFβ2 is present at high concentrations in hair bulge stem cell niches. Interestingly, TGFβ2 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. TGFβ 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 influence 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

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P

ActRII P

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Sara Smad 2/3

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nucleus cytoplasma

Smad 4 P Smad 2/3 SmadRE gene expression

Figure 5.2A Schemes of Transforming Growth Factor-β Superfamily Mediated Pathways Notes: The TGFβ/Activin/Nodal signaling pathway. TGFβ 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.

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5 The Hepatic Stem Cell Niches 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

P

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-β 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 β-catenin-dependent or canonical Wnt signaling pathway is highly conserved throughout the animal kingdom. The central mediator of this signaling pathway is β-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 β-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 β-catenin is phosphorylated by the glycogen synthase kinase-3β (Gsk3β) and subsequently degraded through the proteasom (fFigure 5.3). The Wnt/ B-catenin signaling was identified 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

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Figure 5.3A Scheme of Canonical Wnt Signaling via β-Catenin Notes: In this diagram the inactive canonical Wnt signaling pathway with elevated proteolysis of β-catenin and function of β-catenin as an integral part of the cytoskeleton is depicted. In the absence of Wnt ligands, β-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 β (Gsk3). Upon sequential phosphorylation, β-catenin is ubiquitylated and subsequently degraded by the proteasome machinery. As a result, no free β-catenin enters the nucleus to form a transcriptional complex with T-cell-specific 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/β-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 tissuespecific differentiation in other contexts. For example, in the mammalian epidermis Wnt ligands assist the differentiation of hair follicle precursors. Wnt/β-catenin signaling can also direct the maturation of specific 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/β-catenin cascade in various aspects, such as the maintenance of zonal organization of the liver tissue. There is also evidence that β-catenin-mediated Wnt signaling is crucial for cell division during embryonic liver development and regeneration after partial hepatectomy.

5 The Hepatic Stem Cell Niches Wnt CRD

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Figure 5.3B Scheme of Canonical Wnt Signaling via β-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 Gsk3β binding protein Frat (frequently rearranged in advanced T-cell lymphomas). β-catenin is then released from the multiprotein complex, accumulates in the cytoplasm in a non-phosphorylated form, and subsequently translocates into the nucleus. Nuclear β-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 (Sfrp1–5), Wnt inhibitory factor 1 (Wif1), and dickkopf (Dkk1–3). 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 (Nkd1–2), 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 affinity 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

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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 fibrillary 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

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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.

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5 The Hepatic Stem Cell Niches B responding cell Ihh

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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 cell–based 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 cell–derived 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 final niche in the bone marrow during ontogenesis (homing), but also in their mobilization to the peripheral blood. The recruitment

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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 cell–cell contact. Adherens junctions are cell–cell 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 fiber (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 β1 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 specific importance of cell adhesion mediated by β1 integrin for the stem cell maintenance varies among tissues. In the skin, β1 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 β1 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 β1 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-specific stem cells termed satellite cells stay in close contact to the basement membrane of myofibers. 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 cell–cell contacts is presented by Notch receptors and their ligands Serrate/Jagged and Delta/Delta-like. Four Notch receptors are known in mammals: Notch1–4. 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

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domain of the Notch receptors is then cleaved by the enzyme γ-secretase and translocates into the cell nucleus to influence 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 cell–cell 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

Identification of Stem Cell Niches

One approach for the identification 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, fluorescence, or magnetism. Only within suitable niches transplanted stem cells can survive and maintain their characteristics for a long time.

5.4

Identification of Stem Cell Niches

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signal sending cell endosome ub

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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 ligand–receptor interaction induces two sequential proteolytic cleavages. The first cleavage (S2) within the extracellular domain of Notch receptors is mediated by a desintegrin and metalloproteinase (Adam) and/or the metalloproteinase tumor necrosis factor α-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 γ-secretase complex. An additional cleavage (S4) of the transmembrane domain of Notch receptors results in the release of β-amyloid precursor proteins (Nβ). 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.

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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 specific, whereas others apply across organ boundaries. Organs with reasonably defined 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 defined 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 Dissé 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 influence their proliferation, migration, and development. Oval cells express CXCR4 and migrate along SDF1α 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/β-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)-aminofluorene (2-AAF) with subsequent partial hepatectomy in rats, a noteworthy increase in total and nuclear β-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

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Frizzled-2 in oval cells was observed. This mechanism coincides with a decrease in Wnt inhibitory factor-1 (Wif1) and Gsk3β down-regulation, leading to β-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/β-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/β-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 influencing 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 influence the stem cell activation in the canals of Hering. Taken together, all these findings 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 identified 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 findings, 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 Dissé 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 identified within rat liver through their expression of the filamentous protein GFAP, typically reside within the space of Dissé 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 Dissé 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

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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 fibrillary acidic protein (GFAP; green) by fluorochrome-labeled antibodies on liver tissue sections. B Simultaneous detection of laminin (red) and GFAP (green) by fluorescent 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 influx 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 F2α and D2, which can activate the glycogenolysis in neighboring hepatocytes (Athari et al., 1994; Häussinger 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/β-catenin, and Notch signaling. Isolated rat hepatic stellate cells express multiple components of the Hh pathway, such as Shh, Ihh, Ptch, Smo, and Gli1–3. Hh signaling seems to be required for both stem cell maintenance in their niche and their differentiation. The influence 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/β-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).

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Quiescent stellate cells display nuclear β-catenin and express the Wnt target genes paired-like homeodomain transcription factor 2 (Pitx2), c-Myc, and axin2 (conductin), indicating an active canonical Wnt/β-catenin signaling. In their quiescent state stellate cells also express Wnt ligands known to activate β-catenin-dependent Wnt signaling, such as Wnt1, Wnt2, Wnt3/3a, Wnt7a/b, Wnt8a, and Wnt10b. During culture-dependent activation and development into myofibroblast-like cells, β-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 β-catenin-dependent Wnt signaling might persist in stellate cellderived myofibroblast-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 β-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 α-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/β-catenin signaling is apparently an essential element of the stellate cell niche. In contrast to the findings 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 α-fetoprotein, albumin, hepatocyte nuclear factor 1α (Hnf1α), Hnf4α, 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 cell–cell 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 Dissé (fFigure 5.8A). Another hint for the space of Dissé as a niche for stellate cells is their CXCR4 expression. Hepatic stellate cells are able to migrate in response to SDF1α or SDF1β. Neighboring SEC release SDF1 and strongly attract stellate cells as investigated with modified 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

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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). Immunofluorescence staining of glial fibrillary 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 Dissé (fFigure 5.8A). The cell interactions in stem cell niches described thus far are mainly narrowed to functions of non–stem 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 influence their niche is provided by hair bulge stem cells that release TGFβ2 to influence 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 (Méndez-Ferrer et al., 2010). Mesenchymal stem cells can release soluble factors such as hepatocyte growth factor (HGF/hepatoprotein-A/ scatter factor) that influence 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 Dissé (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.

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A PC

Wnt

Jag1

HSC

BLP

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SEC sympathetic nervous system

SDF1

hepatic stellate cells time series

B

umbilical cord pancreatic stellate cells blood stem cells cultured for 7 days 83 kDa 79 kDa

HGFa g-tubulin 0

1

7

14

HGFa

83 kDa 79 kDa

51 kDa g-tubulin

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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 Dissé. 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 Dissé 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 HGFα in a time course of cultivated rat hepatic stellate cells. The HGFα precursor protein (83 kDa) and processed HGFα (79 kDa) is mainly synthesized by quiescent hepatic stellate cells. HGFα 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 γ-tubulin serves as a loading control.

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Summary Stem cell niches are microenvironments organized as structural units to protect stem cells and to influence 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 cell–cell 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/β-catenin, Notch, TGFβ, 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 Dissé. Both microenvironments meet requirements of stem cell niches.

Further Reading Kordes, C., Sawitza, I., and Häussinger, D. (2009). Hepatic and pancreatic stellate cells in focus. Biol. Chem. 390, 1003–12. Lapidot, T., Dar, A., and Kollet, O. (2005). How do stem cells find their way home? Blood, 106, 1901–10. Moore, K.A., and Lemischka, L. (2006). Stem cells and their niches. Science 31, 1880–5. Morrison, S.J., and Spradling, A.C. (2008). Stem cells and niches: Mechanisms that promote stem cell maintenance throughout life. Cell 132, 598–611. 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, 6454–60. 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, 262–6. Scadden, D.T. (2006). The stem-cell niche as an entity of action. Nature 441, 1075–9. 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, G189–93. Voog, J., and Jones, D.L. (2010). Stem cells and their niche: a dynamic duo. Cell Stem Cell 6, 103–15. Watt, F.M., and Hogan, B.L.M. (2000). Out of Eden: stem cells and their niches. Science 287, 1427–30.

References Ahn, S., and Joyner, A.L. (2005). In vivo analysis of quiescent adult neural stem cells responding to Sonic hedgehog. Nature 437, 894–7.

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6

Stellate Cells in the Regenerating Liver Claus Kordes, Iris Sawitza, and Dieter Häussinger

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 influence 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 cell–based liver regeneration. 6. Activated stellate cells are detectable on basal laminas that surround ducts of cytokeratin 19 expressing cells and blood vessels during stem cell–based liver regeneration.

6.1

Characterization of Stellate Cells

Hepatic stellate cells occur in primitive and higher vertebrates, ranging from lampreys (primitive jawless fish) 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

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Stellate Cells in the Regenerating Liver

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 fluorescence light (light blue). Quiescent hepatic stellate cells of rats express the filamentous proteins glial fibrillary acidic protein (GFAP), desmin, and vimentin at the first day of culture, whereas cultureactivated stellate cells start to express α-smooth muscle actin (α-SMA) as documented by immunofluorescence 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 filament 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 confirmed 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).

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fading fluorescence light (fFigure 6.1). This vitamin A fluorescence is one of the best markers to identify hepatic stellate cells, but also some intermediate filament proteins are useful to detect them. In rodents, quiescent hepatic stellate cells synthesize the filamentous protein glial fibrillary 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 filament protein desmin (SchmittGräff 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 filamentous protein that is expressed by contractile cells such as muscle cells. Other intermediate filament 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-Gräff et al., 2006; Uyama et al., 2006). This expression profile 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 myofibroblast-like cells that synthesize α-smooth muscle actin (α-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 fibronectin. For this reason, activated stellate cells are regarded to be involved in formation of liver fibrosis (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 filament proteins aforementioned as well as α-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 fibroblasts of the liver can also synthesize extracellular matrix proteins, and there is increasing evidence that hepatic myofibroblasts 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 filament 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 filament 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

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Figure 6.2A–F 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 fibrosis. A Freshly isolated hepatic stellate cells with lipid droplets are attached to the plastic dishes at the first day (1d) of culture. B Within 7 days (7d) of culture on plastic the stellate cells develop into enlarged myofibroblast-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 α-SMA and extracellular matrix protein expression.

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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-acetylaminofluorene (2-AAF) (Reister et al., 2011). The synthesis of Nestin points toward an undifferentiated state of hepatic stellate cells, which is further confirmed 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 fluorescent 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 first 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 confluent 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 first 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 efficiently inhibits the activation of cultured hepatic stellate cells. The stellate cells tend to form small cell aggregates on matrigel during culture time.

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Plasticity of Hepatic Stellate Cells

Stem and progenitor cells can differentiate into specialized cell types that fulfill welldefined 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., fibroblast growth factor4 , hepatocyte growth factor) induce molecular markers of endothelial cells (vascular endothelial cadherin, endothelial nitric oxide synthase) or hepatocytes (albumin, α-fetoprotein, hepatocyte nuclear factor 4α), 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-specific expression profile. 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 fluorescence colocalized with parenchymal cell markers such as hepatocyte nuclear factor 4α in cells displaying hepatocyte morphology. Therefore, hepatic stellate cells fulfill 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 first day of regeneration, which leads to rearrangements of the stellate cell niche in the space of Dissé. Due to changes in their microenvironment, hepatic stellate cells are transiently activated from the second to the sixth day of liver regeneration. When

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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 field (Reister et al., 2011). In this model of stem cell–based 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 fields, but also in the liver sinusoids close to the central vein (fFigure 6.3). Thus, stellate cells are activated and proliferate early during stem cell–based liver regeneration. Immunofluorescence stainings of cytokeratins, thymocyte antigen-1 (Thy-1), α-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 field, where oval cells are thought to originate (fFigure 6.4A–E). 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 field, whereas collagen type I deposition remains weak in the vicinity of portal tracts despite the presence of activated hepatic stellate cells (fFigure 6.5A–C). 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 (Häussinger, 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 Immunofluorescence staining of BrdU in the nuclei of cells close to the portal field. B Detection of BrdU by immunofluorescence in the nuclei of cells within liver sinusoids close to a central vein. Paraffin sections of rat liver (5 μm thickness) were analyzed with antibodies against BrdU after antigen retrieval with acid.

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Figure 6.4A–F Zonal Distribution of Cells Involved in Stem Cell–Based 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 immunofluorescence 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 fields. Molecular markers of activated hepatic stellate cells, such as (C) α-smooth muscle actin (α-SMA), (D) desmin, and (E) Nestin were detected primarily in the portal fields. (F) Hepatic stellate cells that still express glial fibrillary acidic protein (GFAP) are mainly restricted to areas around the central veins.

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Figure 6.5A–D Zonal Distribution of Cells Involved in Stem Cell–Based 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 immunofluorescence 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 field, whereas (C) collagen type I is not elevated in the portal field 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 cell–based 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 β-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, β-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

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Figure 6.6A–G Activated Hepatic Stellate Cells are Associated with Duct Formation in the Stem Cell–Based 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 immunofluorescence 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 fields. 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 field can be identified by the simultaneous detection (yellow) of Nestin (green) and desmin (red).

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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 cell–based liver regeneration. Interestingly, activated hepatic stellate cells secrete Wnt4 and Wnt5a that may influence 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 influence 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 field during the stem cell–based liver regeneration (Evarts et al., 1990) (fFigure 6.6A). Nestin stellate cells can clearly be identified 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 6.6E–G). These duct-like structures are instable and finally develop into cell aggregates, which are placed

Figure 6.6A–G (continued) C The duct-like structures harbor cytokeratin 19 (CK19) expressing cells (green) in the portal field 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 cell–based 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 fibroblast 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 fixed 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 immunofluorescence staining (red).

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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 definite 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 filament 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 classification of stellate cells as stem/progenitor cells. After liver injury of rats through partial hepatectomy in the presence of 2-acetylaminofluorene, stellate cells are activated and display a distinct zonal distribution in the portal field. 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 cell–based 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 cell–based liver regeneration is hitherto unknown. Evidence exists that activated hepatic stellate cells can influence 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, 125–72. 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, 45–55. 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, 186–202. 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-acetylaminofluorene and partial hepatectomy. Am. J. Pathol. 114, 418–30.

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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Sawitza, I., Kordes, C., Reister, S., and Häussinger, D. (2009). The niche of stellate cells within rat liver. Hepatology 50, 1617–24. Schmitt-Gräff, A., Krüger, S., Bochard, F., Gabbiani, G., and Denk, H. (1991). Modulation of alpha smooth muscle actin and desmin expression in perisinusoidal cells of normal and diseased human livers. Am. J. Pathol. 138, 123 3– 42. Schmitt-Gräff, A., Jing, R., Nitschke, R., Desmoulière, A., and Skalli, O. (2006). Synemin expression is widespread in liver fibrosis and is induced in proliferating and malignant biliary epithelial cells. Hum. Pathol. 37, 1200 –10. Uyama, N., Zhao, L., Van Rossen, E., Hirako, Y., Reynaert, H., Adams, D.H., Xue, Z., Li, Z., Robson, R., Pekny, M., and Geerts, A. (2006). Hepatic stellate cells express synemin, a protein bridging intermediate filaments to focal adhesions. Gut 55, 1276 –89. Wang, Y., Yao, H.L., Cui, C.B., Wauthier, E., Barbier, C., Costello, M.J., Moss, N., Yamauchi, M., Sricholpech, M., Gerber, D., Loboa, E.G., and Reid, L.M. (2010). Paracrine signals from mesenchymal cell populations govern the expansion and differentiation of human hepatic stem cells to adult liver fates. Hepatology 52, 1443–54. Wiese, C., Rolletschek, A., Kania, G., Blyszczuk, P., Tarasov, K.V., Tarasova, Y., Wersto, R.P., Boheler, K.R., and Wobus, A.M. (2004). Nestin expression—a property of multi-lineage progenitor cells? Cell. Mol. Life Sci. 61, 2510 –22. Yang, L., Jung, Y., Omenetti, A., Witek, R.P., Choi, S.S., Vandongen, H.M., Huang, J., Alpini, G.D., and Diehl, A.M. (2008). Fate-Mapping evidence that hepatic stellate cells are epithelial progenitors in adult mouse livers. Stem Cells 26, 2104 –13.

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Epigenetics during Liver Regeneration Claus Kordes, Iris Sawitza, and Dieter Häussinger

Learning Targets 1. Epigenetic mechanisms in eukaryotes are DNA methylation, histone modifications, 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.

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Definition and Mechanisms of Epigenetics

Epigenetics can be defined 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 influence 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

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principle mechanisms of epigenetic gene regulation discovered in eukaryotes: (1) DNA methylation, (2) histone modifications, 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 specific DNA methyl transferases (Dnmt; fFigure 7.1A). It was thought to be the only naturally occurring base modification 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, modifies 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

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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

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Figure 7.2A,B Histone Modifications 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 influenced by histone modifications 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).

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heterochromatin compared to adult soma cells, reflecting their high developmental potential. Post-translational histone modifications 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 modifications can thus contribute to regulation of gene transcription. The modification of histones by acetylation is long known and catalyzed by histone acetylases. A histone hyperacetylation of specific lysine residues is associated with increased gene transcription. The histone acetylation is reversed by histone deacetylases (HDAC), leading to gene silencing. The phosphorylation of specific 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-specific proteases. SUMOylation is a covalent modification of target proteins such as histone H4 at lysine residues through small ubiquitin-related modifier (SUMO)-conjugating enzymes and plays a central role in coordinating histone modifications 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-specific 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 modification 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 modified 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 specific 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 modifications 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,

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thereby leading to RNA-induced transcriptional gene silencing. The importance for RNA-induced transcriptional silencing during development of mammals becomes evident in Dicer-deficient embryonic stem cells, which are able to proliferate but fail to differentiate correctly (Kanellopoulou et al., 2005). This finding indicates that RNAi are important to guide mammalian development through epigenetic mechanisms, but their exact mode of action remains to be established.

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Methods to Investigate Epigenetic Mechanisms

The earliest methods evolved to study gene-specific methylation used methylation specific 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 specific genes can be investigated further by sodium bisulfite conversion of cytosine to uracil in single-stranded DNA. The bisulfite 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 identified through PCR. To find 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

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Notes: A Sodium bisulfite 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 amplification using specific primers. An incomplete bisulfite conversion of cytosine residues will be detected as false 5-methylcytosine.

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fragments. Selected DNA fragments can be cloned and sequenced for further analysis. For the investigation of histone modifications specific antibodies against the modified amino acid residues are usually applied. Chromatin immunoprecipitation (ChIP) enriches modified histones with DNA fragments, and genes of interest can be further identified by PCR. This method is also suitable for genome-wide analysis of histone modifications 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 first 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 specific 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; first 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 significantly altered within hours after partial hepatectomy in rats. Acetylated arginine residues of histones are maximal after 3–4 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 modification 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 first 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-specific protease USP21 that can

7.4

Epigenetics during Stellate Cell Activation

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catalyze the hydrolysis of ubH2A. Lowered ubH2A amounts are accompanied by increased H3K4 di- and trimethylations, which are histone modifications 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 significantly 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 β 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 cell–based 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 significantly 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 significant 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 α-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

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is associated with increased gene transcription and obviously reflects 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 confirms 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 first 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 filament 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 modification 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 modifications 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 hepatic stellate cells


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culture-activated hepatic stellate cells

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).

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Summary Important mechanisms of epigenetic gene regulation in eukaryotes are DNA methylation, histone modifications, and non-coding RNA. The DNA methylation is mainly associated with silenced gene expression, whereas histone modifications by acetylation, methylation, phosphorylation, ubiquitylation, SUMOylation, and ADP-ribosylation influence 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 specific 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, 206–13. Lee, J.S., Smith, E., and Shilatifard, A. (2010). The language of histone crosstalk. Cell 142, 682–5. 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, 568–77. Meshorer, E., and Misteli, T. (2006). Chromatin in pluripotent embryonic stem cells and differentiation. Nat. Rev. Mol. Cell. Biol. 7, 540–6. Pinney, S.E., and Simmons, R.A. (2010). Epigenetic mechanisms in the development of type 2 diabetes. Trends Endocrinol. Metab. 21, 223–9. Saladi, S.V., and de la Serna, I.L. (2010). ATP dependent chromatin remodeling enzymes in embryonic stem cells. Stem Cell Rev. 6, 62–73. 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, 1419–33. Snykers, S., Henkens, T., De Rop, E., Vinken, M., Fraczek, J., De Kock, J., De Prins, E., Geerts, A., Rogiers, V., and Vanhaecke, T. (2009). Role of epigenetics in liver-specific gene transcription, hepatocyte differentiation and stem cell reprogrammation. J. Hepatol. 51, 187–211. Tost, J. (2008). Epigenetics (Norfolk, UK: Caister Academic Press).

References Alvarez-Gonzalez, R., and Ringer, D.P. (1988). Nuclear matrix associated poly(ADP-ribose) metabolism in regenerating rat liver. FEBS Lett. 236, 362–6.

References

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Briggs, R., and King, T.J. (1952). Transplantation of living nuclei from blastula cells into enucleated frogs’ eggs. Proc. Natl. Acad. Sci. 38, 455–63. Castro, R.E., Ferreira, D.M., Zhang, X, Borralho, P.M., Sarver, A.L., Zeng, Y., Steer, C.J., Kren, B.T., Rodrigues, C.M. (2010). Identification of microRNAs during rat liver regeneration after partial hepatectomy and modulation by ursodeoxycholic acid. Am. J. Physiol. Gastrointest. Liver Physiol. 299, 887–97. Denda, A., Rao, P.M., Rajalakshmi, S., and Sarma, D.S. (1985). 5-azacytidine potentiates initiation induced by carcinogens in rat liver. Carcinogenesis 6, 145–6. Gama-Sosa, M.A., Midgett, R.M., Slagel, V.A., Githens, S., Kuo, K.C., Gehrke, C.W., and Ehrlich, M. (1983). Tissue-specific differences in DNA methylation in various mammals. Biochim. Biophys. Acta. 740, 212–9. Garcea, R., Daino, L., Pascale, R., Simile, M.M., Puddu, M., Ruggiu, M.E., Seddaiu, M.A., Satta, G., Sequenza, M.J., and Feo, F. (1989). Protooncogene methylation and expression in regenerating liver and preneoplastic liver nodules induced in the rat by diethylnitrosamine: effect of variations of S-adenosylmethionine:S-adenosylhomocysteine ratio. Carcinogenesis 10, 1183–92. Kanduc, D., Ghoshal, A., Quagliariello, E., and Farber, E. (1988). DNA hypomethylation in ethionine-induced rat preneoplastic hepatocyte nodules. Biochem. Biophys. Res. Commun. 150, 739–44. Kanellopoulou, C., Muljo, S.A., Kung, A.L., Ganesan, S., Drapkin, R., Jenuwein, T, Livingston, D.M., and Rajewsky, K. (2005). Dicer-deficient mouse embryonic stem cells are defective in differentiation and centromeric silencing. Genes Dev. 19, 489–501. Kim, J.S., and Shukla, S.D. (2005). Histone h3 modifications in rat hepatic stellate cells by ethanol. Alcohol 40, 367–72. Lasky, R.A., and Gurdon, J.B. (1970). Genetic content of adult somatic cells tested by nuclear transplantation from cultured cells. Nature 228, 1332–4. Mann, J., Oakley, F., Akiboye, F., Elsharkawy, A., Thorne, A.W., and Mann, D.A. (2007). Regulation of myofibroblast transdifferentiation by DNA methylation and MeCP2: implications for wound healing and fibrogenesis. Cell Death Differ. 14, 275–85. Nakagawa, T., Kajitani, T., Togo, S., Masuko, N., Ohdan, H., Hishikawa, Y., Koji, T., Matsuyama, T., Ikura, T., Muramatsu, M., and Ito T. (2008). Deubiquitylation of histone H2A activates transcriptional initiation via trans-histone cross-talk with H3K4 di- and trimethylation. Genes Dev. 22, 37–49. Niki, T., Rombouts, K., De Bleser, P., De Smet, K., Rogiers, V., Schuppan, D., Yoshida, M., Gabbiani, G., and Geerts, A. (1999). A histone deacetylase inhibitor, trichostatin A, suppresses myofibroblastic differentiation of rat hepatic stellate cells in primary culture. Hepatology 29, 858–67. Pogo, B.G., Pogo, A.O., Allfrey, V.G., and Mirsky, A.E.(1968). Changing patterns of histone acetylation and RNA synthesis in regeneration of the liver. Proc. Natl. Acad. Sci. U.S.A. 59, 1337–44. Reister, S., Kordes, C., Sawitza, I., and Häussinger, D. (2011). The epigenetic regulation of stem cell factors in stellate cells. Stem Cells Dev. doi: 10.1089/scd.2010.0418. Riggs, A.D., and Porter, T.N. (1996). Overview of genetic mechanisms. In: Epigenetic mechanisms of gene regulation, V.E.A. Russo et al., eds. (Cold Spring Harbor, New York, USA: Cold Spring Harbor Laboratory Press) pp. 29–45. Song, G., Sharma, A.D., Roll, G.R., Ng, R., Lee, A.Y., Blelloch, R.H., Frandsen, N.M., and Willenbring, H. (2010). MicroRNAs control hepatocyte proliferation during liver regeneration. Hepatology 51, 1735–43. Tidwell, T., Allfrey, V.G., and Mirsky, A.E. (1968). The methylation of histones during regeneration of the liver. J. Biol. Chem. 243, 707–15.

8

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-specific 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 flow. 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

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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 flow of blood and bile within the segment of newly formed liver. Without the latter, full recovery of liver-specific 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 cell–cell 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) field, septum transversum mesenchyme (mesoderm and ectoderm), and the ventral endoderm. In mice, this typically occurs between days E8.5–9.5. Bone marrow–derived cells subsequently infiltrate 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. Briefly, work in fetal explants has shown that fibroblast growth factors (FGF ) and bone morphogenetic proteins(BMPs) released from the developing heart field are required for hepatic specification of the ventral endoderm. Furthermore, it was demonstrated that hepatic specification 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 difficult to demonstrate. Nevertheless, the following data support a role for the Hh pathway in fetal liver morphogenesis: first, pluripotent embryonic stem cells, endoderm-committed multipotent progenitors, and some committed

8.4

Overview of Hedgehog Signaling Pathway

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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 specification 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 marrow–derived hematopoietic progenitors (El Andaloussi et al., 2006; Fu et al., 2006; Williams et al., 2010) (all of which infiltrate 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 identified 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-modified 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

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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-finger 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

P

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, influences 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 modification.

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Reactivation of the Hedgehog Pathway after Partial Hepatectomy

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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 sacrificed 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 significant up-regulation of well-established Gli-target genes, such as soluble frizzled related peptide (sFRP)-1. These findings 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 specific inhibitor of Hh signal transduction, demonstrated significant 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

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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 finding 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 specifically 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 significant role in stromal cell responses that follow partial hepatectomy, including transient expansion of myofibroblast populations and hepatic matrix remodeling. Together, these findings 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

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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 myofibroblasts, 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 )-β1, 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 amplifies 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 reflect 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 specified to undergo hepatic differentiation, as well as more-differentiated EpCam-expressing liver

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healthy liver

canal of Hering Progenitors

hepatocytes

space of Dissé

bile canaliculus

cholangiocytes

bile duct

HSC

NKT cells

space of Dissé

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, confirming 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 injury–related 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 Dissé

myofibroblastic HSC chemokines neutrophils

space of Dissé

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 inflammation, regeneration, and fibrogenesis. (1) Injury stimulates mature hepatocytes and cholangiocytes to produce and release Hh ligands into the bile, space of Dissé, and liver sinusoids. (2) Hh ligands stimulate quiescent hepatic stellate cells to undergo transition to myofibroblastic hepatic stellate cells, enhance myofibroblastic HSC proliferation and survival, and stimulate further production of Hh ligands by myofibroblastic HSC. (3) Hh ligands generated by injured liver epithelial cells and myofibroblastic 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 myofibroblasts in injured livers. (5) Hh-activated cholangiocytes also produce various chemokines that recruit different types of bone marrow–derived cells and immune cells to liver, such as fibrocytes, 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 inflammation, fibrogenesis, vascular remodeling, and accumulation of progenitor populations that might provide the seeds for subsequent liver cancers.

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Hedgehog Pathway Activation and Liver Fibrosis

Myofibroblasts are the major source of fibrous matrix that accumulates during chronic liver injuries that result in cirrhosis. Liver myofibroblasts may be derived from several sources, including circulating bone marrow–derived monocytes/fibrocytes, epithelialto-mesenchymal transition (EMT) of certain types of liver epithelial cells, and myofibroblastic transformation of resident hepatic stellate cells. The latter process is generally believed to be the predominant source of myofibroblasts in most types of adult liver injury (Friedman, 2008). In healthy adult livers, most hepatic stellate cells are quiescent and exhibit a fatstoring, non-myofibroblastic phenotype; however, factors released during liver injury stimulate quiescent hepatic stellate cells to undergo transition to myofibroblastic hepatic stellate cells. Other injury-related factors then promote hepatic accumulation of myofibroblastic hepatic stellate cells by enhancing proliferation and/or inhibiting apoptosis of myofibroblastic 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 myofibroblastic 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 cell–derived Hh ligands, in turn, increase expression of Hh-target genes, such as Gli2 (Choi et al., 2009). Hh neutralizing antibodies or specific pharmacologic inhibitors of smoothened that abrogate down-stream Hh signaling drastically reduce myofibroblastic hepatic stellate cell viability and virtually eliminate the proliferative effects of known myofibroblastic 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 myofibroblastic hepatic stellate cells to revert to a less myofibroblastic and more quiescent phenotype (Choi et al., 2009; Choi et al., 2010; Sicklick et al., 2005). Hh pathway activation may also promote accumulation of myofibroblasts 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 fibrocytes (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 fibrocytes (Shao et al., 2008). Finally, Hh signaling induces EMT in immature ductular cells (Omenetti et al., 2008b). Thus, the aggregate findings predict that injury-related activation of the Hh pathway would play a major role in hepatic accumulation of myofibroblasts. The concept that Hh pathway activation promotes accumulation of myofibroblasts 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).

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Hedgehog Pathway Activation and Vascular Remodeling in Injured Livers

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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 fibrotic 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 identified in exosomes that were released from myofibroblasts 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 purified from myofibroblast- 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 fibrosis/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 findings 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

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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 first 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 significantly reduced the growth of Hh-responsive hepatoma cells (Sicklick et al., 2006a). These findings 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 finding 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, fibroblastic cells, ductular cells, inflammatory 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 myofibroblasts, repair-related inflammatory responses, vascular remodeling, liver fibrosis, and hepatocarcinogenesis. These findings 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 Acloque, H., Adams, M.S., Fishwick, K., Bronner-Fraser, M., and Nieto, M.A. (2009). Epithelial mesenchymal transitions: the importance of changing cell state in development and disease. J. Clin. Investig. 119, 1438–49. Duncan, A.W., Dorrell, C., and Grompe, M. (2009). Stem cells and liver regeneration. Gastroenterology 137, 466–81. Fausto, N., Campbell, J.S., and Riehle, K.J. (2006). Liver regeneration. Hepatology 43, S45–53.

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9

EGFR, CD95, and the Switch between Proliferation and Apoptosis in Hepatic Stellate Cells Roland Reinehr and Dieter Häussinger

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 Häussinger, 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

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EGFR, CD95, and the Switch between Proliferation and Apoptosis in HSCs

(for review see Reinehr and Häussinger, 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)α, 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 TGFα share 35% homology and both can bind to the EGFR, whereas the HGF, whose α-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, TGFα exerts its effect on hepatocytes mainly via an autocrine loop. Interestingly, this autocrine loop is stimulated by EGF or TGFα itself indicating an (auto-)amplification 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 efficacy (Fausto et al., 1995, 2006; Michalopoulos and DeFrances, 1997, Michalopoulos, 2007). This priming step involves the activation and DNA binding of nuclear factor (NF)-κb and other transcription factors, which are induced by tumor necrosis factor (TNF)α, 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, TGFα, 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

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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, TGFα, 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 significant 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-κb, 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 TGFα expression and subsequent TGFα-induced EGFR activation and EGFR/c-Met heterodimerization as shown by TGFα- and EGFR-neutralizing antibodies as well as EGFR and c-Met immunoprecipitation studies, respectively. Therefore, cross-talk between the TGFα/EGFR and HGF/c-Met pathways might induce mitogenic and/or motogenic signal amplification in many tumor cell lines and might explain how overexpression of TGFα 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 specificity 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 TGFα shedding resulting in a ligand-dependent EGFR activation and EGFR tyrosine kinase activity-dependent cell proliferation (Werneburg et al., 2003).

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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; Müschen et al., 1998; Ueno et al., 2000), a member of the tumor necrosis factor (TNF)death receptor family. CD95 has first been described by the groups of Yonehara and Krammer reporting on extensive apoptotic cell death induction upon treating cells with CD95-specific 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 deficient 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, significant 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 acidification of an early endosomal compartment (Faundez and Hartzell, 2004; Moriyama and Nelson, 1987; Pazoles et al., 1980). Upon acidification 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)ζ 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 identified 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 amplification of proapoptotic signaling, which finally 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 acid–induced 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 modifications 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 Häussinger, 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.

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EGFR, CD95, and the Switch between Proliferation and Apoptosis in HSCs 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 Ø

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 Häussinger, 2004). Therefore, cAMP also inhibits CD95 tyrosine phosphorylation and subsequent apoptosis (Reinehr and Häussinger, 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 Häussinger, 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 Häussinger, 2004). This may reflect 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

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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 Häussinger, 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 fulfill 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 24–48 hours only and do not express detectable amounts of α-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).

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CD95L-induced EGFR activation couples to an Erk-mediated HSC proliferation, as reflected 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, findings 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 findings 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 acid–induced 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 finally 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 Acid–induced Signaling In activated HSC (aHSC), that is, cells cultured for 7–14 days and high expression of α-smooth muscle actin reflecting a myofibroblast 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

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MMP9

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YES

EGFR

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Erk-1/-2 CD95-Tyr-NO2

proliferation apoptosis resistance

CD95L

+ CHX

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DISC formation apoptosis

activated HSC bile acid + CHX

hepatocytes CD95L or bile acid

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EGFR /CD95

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CD95-Tyr-P

CD95-Tyr-P

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DISC formation

DISC formation

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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 finally 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 fibrosis induction was sensitive to pan-JNK inhibition and was ameliorated in JNK1-deficient mice (Kluwe et al., 2010). Furthermore, choline-deficient L-amino acid-defined (CDAA) diet-induced

138


hepatic inflammation and fibrosis 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 fibrogenesis. 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 finally 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 identified 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

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Further Reading Czaja, M.J. (2007). Cell signaling in oxidative stress-induced liver injury. Semin Liver Dis. 27, 378–89. Jones, D.P., Lemasters, J.J., Han, D., Boelsterli, U.A., and Kaplowitz, N. (2010). Mechanisms of pathogenesis in drug hepatotoxicity putting the stress on mitochondria. Mol Interv. 10, 98–111. Malhi, H., and Gores, G.J. (2008). Cellular and molecular mechanism of liver injury. Gastroenterology 134, 1641–54. Michalopoulus, G.K. (2010). Liver regeneration after partial hepatectomy. Am. J. Pathol. 176, 2–13. Michalopoulus, G.K., and DeFrances, M.C. (1997). Liver regeneration. Science 276, 60–6. Peter, M.E., and Krammer, P.H. (2003). The CD95(APO-1/Fas) DISC and beyond. Cell Death Differ. 10, 26–35. Reinehr, R., and Häussinger, D. (2007). Hyperosmotic activation of the CD95 system. Methods Enzymol. 428, 145–60. Wajant, H., Pfizenmaier, K., and Scheurich P. (2003). Non-apoptotic Fas signaling. Cytokine Growth Factor Rev. 14, 53–66.

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10 Angiogenesis and Liver Regeneration Tobias Buschmann, Jan Eglinger, and Eckhard Lammert

Learning Targets 1. Blood flow 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 first want to describe the hepatic blood flow, 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-deficient (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

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10 Angiogenesis and Liver Regeneration 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 flow (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 1–2 mm in diameter. It consists of roughly hexagonal arrangements of plates of hepatocytes, which are 15–25 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 flows 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 flow in capillaries to optimize the time for diffusion and filtration of blood (Aird, 2007). In the liver sinusoids, the blood flow is even slower compared to other vascular beds. For example, the blood flow velocity in liver sinusoids is about 0.4–0.45 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 fluid 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 150–175 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 Dissé hepatocytes

LSEC Kupffer cell SEC

central venule

HSC

portal triad hepatic lobule

A

B

Figure 10.2A,B Notes: A A macroscopic overview over a hepatic lobule and the blood flow in a schematic sinusoid from the portal triad to the central venule. B The sinusoidal structure in detail on the cellular level. The blood flow direction is marked with arrows.

comprise about 6%–8% of the endothelial cell surface (Braet and Wisse, 2002). In general, 20–50 fenestrae are arranged in sieve plates, which are approximately 0.1 μm 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 modified plasma proteins (Aird, 2007). Hepatic stellate cells (HSCs) are located in the space of Dissé (or perisinusoidal space), which lies between the hepatocytes and LSECs. HSCs are liver-specific 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.

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10 Angiogenesis and Liver Regeneration

Kupffer cells (KCs) are macrophages that adhere to the luminal side of the sinusoids and move in an amoeboid fashion. They are the first cells to get exposed to nutrients and all substances absorbed in the gastro-intestinal tract. KCs might also regulate the blood flow 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 Dissé from the LSECs (fFigure 10.2). Solutes, fluid, 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 detoxification, synthesis of coagulation factors, and many serum proteins as well as regulation of the body’s metabolism. Along the sinusoids, hepatocytes are heterogeneous. Periportally, they have a high degree of oxygen uptake, gluconeogenesis, glycogenesis, cholesterol biosynthesis, ß-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; Häussinger, 1983). Markers for hepatocytes are, for example, hepatocyte paraffin 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 classified 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 defined 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 specific 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 fire 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 10–14 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 fibronectin, collagen type IV and I, and small amounts of glycosaminoglycans (Michalopoulos and DeFrances, 1997). After 5–8 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.

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10 Angiogenesis and Liver Regeneration cell division

A

hepatocytes hepatic stellate cell sinusoidal endothelial cell

bile duct hepatic arteriole portal triad

B

portal venule hepatocyte plate

cell process endothelial cell proliferation

intussusceptive angiogenesis

C angiogenic sprout

endothelial cell tip cell

Figure 10.3A–C Notes: A The beginning of angiogenesis during liver regeneration. First hepatocytes proliferate and form hepatocyte clumps. B After 3–4 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 significantly 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 fibrosis and can result in liver cirrhosis.

The regenerative capacity of the liver normally allows a total recovery after injury and/or inflammation (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 inflammation. The main consequence is fibrosis, which can develop into cirrhosis. During the fibrogenic process, an excessive amount of extracellular matrix is deposited (Medina et al., 2004). Because fibrotic tissue inhibits blood flow 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 flow and hepatic functions. During the fibrogenic process granulation tissue develops within the inflamed or injured tissue. This tissue is the perfused, fibrous connective tissue that replaces a fibrin clot in wound healing. Usually, fibroblasts first proliferate, then angiogenesis takes place, and finally, ECM is deposited. During tissue repair and granulating tissue formation, cells, such as fibroblasts and HSCs, acquire features of smooth muscle cells and are often called myofibroblasts (Guyot et al., 2006; Serini and Gabbiani, 1999). Myofibroblasts are the main cell type in the granulation tissue and the main ECM depositioning cells. They contain stress fibers, 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 fibrous tissue (Guyot et al., 2006). During

152


fibrosis, 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 fibrosis, which develops from the portal triad to the central venule. Another kind of fibrosis is the portal to portal fibrosis, which develops between two portal triads. The fibrous ECM leads to a partial loss of liver function because the ECM forms fibrotic strands, which disturb the blood flow 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-ß1, FGF and VEGF), matrix metalloproteinases (MMPs), and cytokines (Fernandez et al., 2009). In the new architectural environment of the fibrotic 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, fibrogenesis 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 final stage of CLDs, and it develops over years or decades. Cirrhosis is responsible for a significant 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 für 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 fibrotic 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 defined 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 flow through the liver and, therefore, force the blood to flow 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

A

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 fibrin 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 fibrin gel. Scale bar: 50 μm. Ascites (free fluid 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 fibrotic areas, in which pathologic angiogenesis results in an abnormal vascular system, disturbing hepatic blood flow. Angiogenesis assays have been used to investigate the pro- and anti-angiogenic potential of proteins and drugs in vitro. In addition, by creating a defined 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 fibrin gels (fFigure 10.4A). A modification of this assay uses human umbilical vein endothelial cells (HUVEC) and a feeder layer of skin fibroblasts (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 identified that induce physiologic or pathologic blood vessels.

10.6

Questions and Problems

The specific features of healthy and unhealthy angiogenesis are not well understood in liver regeneration and liver damage, respectively. However it is important

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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 infiltration 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 Aird, W.C. (2007). Phenotypic heterogeneity of the endothelium: II. Representative vascular beds. Circ. Res. 100, 174–90. 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, 604–20. Ferrara, N. (1999). Role of vascular endothelial growth factor in the regulation of angiogenesis. Kidney Int. 56, 794–814. Gerhardt, H. (2008). VEGF and endothelial guidance in angiogenic sprouting. Organogenesis 4, 241–6. Guyot, C., Lepreux, S., Combe, C., Doudnikoff, E., Bioulac-Sage, P., Balabaud, C., and Desmouliere, A. (2006). Hepatic fibrosis and cirrhosis: the (myo)fibroblastic cell subpopulations involved. Int. J. Biochem. Cell Biol. 38,135–51. Kordes, C., Sawitza, I., and Häussinger, D. (2009). Hepatic and pancreatic stellate cells in focus. Biol. Chem. 390, 1003–12. Lammert, E., Cleaver, O., and Melton, D. (2003). Role of endothelial cells in early pancreas and liver development. Mech. Dev. 120, 59–64. Pinzani, M. (1999). Liver fibrosis. Springer Semin Immunopathol 21, 475–90. 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. Regul. Integr. Comp. Physiol. 298, R1279–87. Zeeb, M., Strilic, B., and Lammert, E. (2010). Resolving cell-cell junctions: lumen formation in blood vessels. Curr. Opin. Cell. Biol. 22, 626–32.

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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 spatial–temporal 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

Definition

Hepatic parenchyma is organized in repetitive functional units called liver lobules (fFigure 11.1A–C), 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 flows 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).

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11 A Quantitative Mathematical Modeling Approach to Liver Regeneration

t = 2 days

t = 4 days

t = 10 days

Figure 11.1A–D (A), (B) Confocal Laser Scanning; (C) Bright Field Micrograph of Liver Lobules Notes:

A Sinusoids can be identified 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 field 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 field 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 sacrificed for the analysis (Hoehme, et al., 2010).

A systems biology analysis for a multicellular tissue organization process with significant 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 objectified. Hence, tissuelevel parameters should be defined that permit to cast the observed regeneration pattern into numbers (or distributions) and thereby to objectify the experimental observation. Identification of relevant cell-level parameters. A natural first 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 sufficient 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 specific 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 significant results. Specific spatial features of the lobules as its shape or the architecture of its blood vessel network requires 3D information in order to be correctly quantified. 2D information can partly be collected using bright field 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 define the mathematical model on the cell-level, and the tissue-level model parameters are used to define 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 fingerprints prior to the liver regeneration process, is necessary to define the liver lobule state prior to drug administration. Subsequently, a careful characterization of the parameter changes during regeneration after drug administration is required. For spatial–temporal 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 field micrographs or confocal scanning micrographs in combination with appropriate cell and tissue specific markers. Bright field 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 identified. 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 field 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 field 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 70–150 μm 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 finally overlaid to the final 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 filtering 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 refinement 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 (modified 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 classified either as within or outside a vessel. Depending on that classification, the intensity of the pixels is changed in order to fill 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 filtering (reduces image noise), and cell shape reconstruction (based on Voronoi space decomposition) were used to investigate hepatocyte properties (fFigure 11.2). The Erosion filter is a morphological filter 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 filter is a morphological filter 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 filtering and which points are added by dilatation filtering, depends on setting threshold values and the selected kernels. By using further morphologic filters 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 sufficiently 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 11.3B–E): (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 2–3 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 define 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 spatial–temporal 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 findings.

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)

1

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


experimental data

without HSA

hepatocyte-sinusoid contact area (%)

D area of necrotic lesion (in mm2)

C

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

A

Mathematical Model

55 50 45 40 35 0


HSA

Figure 11.3A–E 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. B–E 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 fulfill 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-cell–based, agent-based, or single-cell–based 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 fulfill these requirements is a so-called center-based (single-cell–based) 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 sufficiently 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 hepatocyte—hepatocyte forces and hepatocyte—blood 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

hepatocyte–hepatocyte distance dij (or the hepatocyte–sinusoid 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: Newton’s equation of motion of an object of mass m falling on a floor 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 Δv where $v = v(t + $t) - v(t). Knowing the = lim Δ t → 0 dt Δt

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) hepatocyte–hepatocyte 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 space—called the “space of Dissé”—between the endothelial cells making up the sinusoids and the hepatocytes), (iii) hepatocyte–sinusoidal 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 specified 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)

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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 coefficients, 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 Dissé, G ⊥ =0 (neglecting friction inside the cell that emerges from intracellular reorganization if cells are deformed or compressed). F i active ,H = (1− Θ[∇pi H active , A ]) 2DG 2 H i (t ). H (t ) denotes a i

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 coefficient, 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 first 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 modifications: 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

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(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 configuration 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-fixed axes by angles DBi with i = 1, 2, 3, DBi є [0, DBmax), with DBmax 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 5–7 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 outflow 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 efficient PHx in mice without risk of outflow obstruction.

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12.4.2

12 Animal Models for Studies on Liver Regeneration

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 fibrosis. Because total bile duct obstruction does rarely occur in humans, a model of partial BDL was established for mice that better reflects 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 fibrotic changes after marked infiltration of inflammatory cells, especially mononuclear cells, such as neutrophils, thus mimicking the changes seen in chronic viral hepatitis-associated fibrosis, and has therefore been widely used to experimentally induce liver fibrosis 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 detoxified 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-deficient, Ethionine-supplemented Diet

Feeding a choline-deficient, 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 influence the regenerative process based on stem cell and precursor cell populations.

12.6 Transgenic Models

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12.6 Transgenic Models 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 “floxed” (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, cellspecific activation of the transgenic phenotype must be established by further genetic manipulation. fFigure 12.2 illustrates an example for doxycycline-dependent liverspecific knockout of a gene.

A

PLap

–Dox

+Dox

tetO7

B

rtetR

Pmin

Cre

Cre recombinase Cre

Figure 12.2A,B Strategy for Conditional and Hepatocyte-Specific 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-specific 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 flanked 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 influence the success and value of the generated TG models: First, the genetic background plays an important role, as some of the null

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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 significantly affect the disease phenotype. On the other hand, this variability in phenotypes in related genetic backgrounds could potentially help to identify modifier 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 classified 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 immunodeficient (SCID)/TG mice to generate immunodeficient 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 fluorescent 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 efficiency, 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-specific 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 significantly 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-specific ablation of HGF/SF in mice significantly reduced the regenerative capacity of hepatocytes after CCl4 injection (Phaneuf et al., 2004). Likewise, hepatocyte-specific deletion of HGF/SF receptor c-Met showed increased liver cell damage and fibrosis 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-specific 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 significant 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. Hepatocytespecific 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 β (TGF-β) is a multifunctional cytokine with diverse effects on development, growth, and homeostasis in most tissues. Studies on hepatocyte-

184


specific ablation of TGF-β type II receptor in mice showed that TGF-β 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-specific TGF-β1 expressing mice proliferation of hepatocytes almost disappeared, while withdrawal of TGF-β1 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 fibroblast growth factor (FGF) 1 and 2 in matrix deposition and hepatic fibrogenesis 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 zebrafish revealed that FGF signaling is crucial in early proliferative response to PHx (Kan et al., 2009). The proinflammatory 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 inflammatory reactions. IL-6 KO mice have mainly confirmed 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 fibrosis 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-α) has been recognized as a cell-death mediator and is produced from immune cells after liver damage. TNF-α gene deficient 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 NFκB, 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 fibrosis 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 proinflammatory factor. Knockout of its receptor, a 14-kD type I transmembrane receptor termed fibroblast growth factor-inducible 14 (Fn14), considerably changed the response to CDE feeding within 2 weeks. Besides a significantly reduced number of liver progenitor cells, attenuated inflammation, cytokine production, and expression of key fibrogenesis 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) α 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, NFκB, and C/EBP. The contribution of these transcription factors to liver regeneration was further demonstrated in various studies using conditional KO technology, confirming 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 identified by this approach include EGR-1 and FoxM1B. EGR-1 is a zinc finger 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-α mediates the effects of peroxisome proliferator chemicals, which are potent hepatic mitogens and carcinogens in mice and rats. Studies on Pparα-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 sufficient 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 significantly 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 fibrinolysis 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 significantly 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 specifically 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 B–deficient 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-specific 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 β-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 cycle–related proteins and other signalings (Liu et al., 2010).

12.7

Immunological Models

The innate immune system is the first 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 influence 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 inflammation. 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

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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 infiltration, 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 inflammatory cytokines, such as TNF-α and IFN-γ. 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 fitting 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 profit as much as possible from existing models or to design new ones avoiding already identified drawbacks for the benefit 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, 217–35. Böhm, F., Köller, U.A., Speicher, T., and Werner, S. (2010). Regulation of liver regeneration by growth factors and cytokines. EMBO Mol. Med. 2, 294–305. Chu, J., and Sadler, K.C. (2009). New school in liver development: lessons from zebrafish. Hepatology 50, 1656–63. deGraaf, W., Bennink, R.J., Veteläinen, 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, 742–52. 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, 241–52. Liu, H., and Zhu, S. (2009). Present status and future perspectives of preoperative portal vein embolization. Am. J. Surg. 197, 686–90. Martins, P.N., Theruvath, T.P., and Neuhaus, P. (2008). Rodent models of partial hepatectomies. Liver Int. 28, 3–11. Weber, A., Groyer-Picard, M.T., Franco, D., and Dagher, I. (2009). Hepatocyte transplantation in animal models. Liver Transpl. 15, 7–14. Zaret, K.S., and Grompe, M. (2008). Generation and regeneration of cells of the liver and pancreas. Science 322, 1490–4.

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References Anderson, S.P., Yoon, L., Richard, E.B., Dunn, C.S., Cattley, R.C., and Corton, J.C. (2002). Delayed liver regeneration in peroxisome proliferator-activated receptor-alpha-null mice. Hepatology 36, 544–54. Apte, U., Gkretsi, V., Bowen, W.C., Mars, W.M., Luo, J.H., Donthamsetty, S., Orr, A., Monga, S.P., Wu, C., and Michalopoulos, G.K. (2009). Enhanced liver regeneration following changes induced by hepatocyte-specific genetic ablation of integrin-linked kinase. Hepatology 50, 844–51. Arendt, E., Ueberham, U., Bittner, R., Gebhardt, R., and Ueberham, E. (2005). Enhanced matrix degradation after withdrawal of TGF-beta1 triggers hepatocytes from apoptosis to proliferation and regeneration. Cell Prolif. 38, 287–99. Bajt, M.L., Yan, H.M., Farhood, A., and Jaeschke, H. (2008). Plasminogen activator inhibitor-1 limits liver injury and facilitates regeneration after acetaminophen overdose. Toxicol Sci. 104, 419–27. Desbois-Mouthon, C., Wendum, D., Cadoret, A., Rey, C., Leneuve, P., Blaise, A., Housset, C., Tronche, F., Le Bouc, Y., and Holzenberger, M. (2006). Hepatocyte proliferation during liver regeneration is impaired in mice with liver-specific IGF-1R knockout. FASEB J. 20, 773–5. Dirsch, O., Madrahimov, N., Chaudri, N., Deng, M., Madrahimova, F., Schenk, A., and Dahmen, U. (2008). Recovery of liver perfusion after outflow obstruction and liver resection. Transplantation 85, 748–56. Erhardt, A., and Tiegs, G. (2010). Tolerance induction in response to Liver Inflammation. Dig. Dis. 28, 86–92. Gebhardt, R., and Hovhannisyan, A. (2010). Organ patterning in the adult stage: the role of Wnt/beta-catenin signalling in liver zonation and beyond. Dev. Dyn. 239, 45–55. Giebeler, A., Boekschoten, M.V., Klein, C., Borowiak, M., Birchmeier, C., Gassler, N., Wasmuth, H.E., Müller, M., Trautwein, C., and Streetz, K.L. (2009). c-Met confers protection against chronic liver tissue damage and fibrosis progression after bile duct ligation in mice. Gastroenterology 137, 297–308. Hamada, T., Kamada, H., Hayashi, T., Nishiok,a J., Gabazza, E.C., Isaji, S., Uemoto, S., and Suzuki, K. (2008). Protein C inhibitor regulates hepatocyte growth factor activator-mediated liver regeneration in mice. Gut. 57, 365–73. Hayashi, H., Nagaki, M., Imose, M., Osawa, Y., Kimura, K., Takai, S., Imao, M., Naiki, T., Kato, T., and Moriwaki, H. (2005). Normal liver regeneration and liver cell apoptosis after partial hepatectomy in tumor necrosis factor-alpha-deficient mice. Liver Int. 25, 162–70. Heinrich, S., Georgiev, P, Weber, A., Vergopoulos, A., Graf, R., and Clavien, P.A. (2010). Partial bile duct ligation in mice: A novel model of acute cholestasis. Surgery, 149, 445–51. Huang, W., Ma, K., Zhang, J., Qatanani, M., Cuvillier, J., Liu, J., Dong, B., Huang, X., and Moore, D.D. (2006). Nuclear receptor-dependent bile acid signaling is required for normal liver regeneration. Science 14, 233–6. Kan, N.G., Junghans, D., and Izpisua Belmonte, J.C. (2009). Compensatory growth mechanisms regulated by BMP and FGF signaling mediate liver regeneration in zebrafish after partial hepatectomy. FASEB J. 23, 3516–25. Kovalovich, K., DeAngelis, R.A., Li, W., Furth, E.E., Ciliberto, G., and Taub, R. (2000). . Increased toxin-induced liver injury and fibrosis in interleukin-6-deficient mice. Hepatology 31, 149–59. Liao, Y., Shikapwashya, O.N., Shteyer, E., Dieckgraefe, B.K., Hruz, P.W., and Rudnick, D.A. (2004). Delayed hepatocellular mitotic progression and impaired liver regeneration in early growth response-1-deficient mice. J. Biol. Chem. 279, 43107–16.

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Liu, B., Bell, A.W., Paranjpe, S., Bowen, W.C., Khillan, J.S., Luo, J.H., Mars, W.M., and Michalopoulos, G.K. (2010). Suppression of liver regeneration and hepatocyte proliferation in hepatocyte-targeted glypican 3 transgenic mice. Hepatology 52, 1060–7. Madrahimov, N., Dirsch, O., Broelsch, C., and Dahmen, U. (2006). Marginal hepatectomy in the rat: from anatomy to surgery. Ann. Surg. 244, 89–98. Mallet, V.O., Mitchell, C., Guidotti, J.E., Jaffray, P., Fabre, M., Spencer, D., Arnoult, D., Kahn, A., and Gilgenkrantz, H. (2002). Conditional cell ablation by tight control of caspase-3 dimerization in transgenic mice. Nat. Biotechnol. 20, 1234–9. Nejak-Bowen, K.N., Thompson, M.D., Singh, S., Bowen, W.C. Jr., Dar, M.J., Khillan, J., Dai, C., and Monga, S.P. (2010). Accelerated liver regeneration and hepatocarcinogenesis in mice overexpressing serine-45 mutant beta-catenin. Hepatology 51, 1603–13. Oe, S., Lemmer, E.R., Conner, E.A., Factor, V.M., Levéen, P., Larsson, J., Karlsson, S., and Thorgeirsson, S.S. (2004). Intact signaling by transforming growth factor beta is not required for termination of liver regeneration in mice. Hepatology 40, 1098–1105. Olle, E.W., Ren, X., McClintock, S.D., Warner, R.L., Deogracias, M.P., Johnson, K.J., and Colletti, L.M. (2006). Matrix metalloproteinase-9 is an important factor in hepatic regeneration after partial hepatectomy in mice. Hepatology 44, 54054–9. Phaneuf, D., Moscioni, A.D., LeClair, C., Raper, S.E., and Wilson, J.M. (2004). Generation of a mouse expressing a conditional knockout of the hepatocyte growth factor gene: demonstration of impaired liver regeneration. DNA Cell Biol. 23, 592–603. Río, A., Gassull, M.A., Aldeguer, X., Ojanguren, I., Cabré, E., and Fernández, E. (2008). Reduced liver injury in the interleukin-6 knockout mice by chronic carbon tetrachloride administration. Eur J Clin Invest. 38, 306–16. Shu, R.Z., Zhang, F., Wang, F., Feng, D.C., Li, X.H., Ren, W.H., Wu, X.L., Yang, X., Liao, X.D., Huang, L., and Wang, Z.G. (2009). Lab Invest. Adiponectin deficiency impairs liver regeneration through attenuating STAT3 phosphorylation in mice. Lab. Invest. 89, 1043–52. Simeonova, P. P., Gallucci, R. M., Hulderman, T., Wilson, R., Kommineni, C., Rao, M., et al. (2001). The Role of tumor necrosis factor-alpha in liver toxicity, inflammation, and fibrosis induced by carbon tetrachloride. Toxicol Appl Pharmacol 177, 112–20. Song, X., Guo, Y., Duo, S., Che, J., Wu, C., Ochiya, T., Ding, M., and Deng, H. (2009). A mouse model of inducible liver injury caused by tet-on regulated urokinase for studies of hepatocyte transplantation. Am. J. Pathol. 175, 1975–83. Sudo, K., Yamada, Y., Moriwaki, H., Saito, K., and Seishima, M. (2005). Lack of tumor necrosis factor receptor type 1 inhibits liver fibrosis induced by carbon tetrachloride in mice. Cytokine. 29, 236–44. Tirnitz-Parker, J.E., Viebahn, C.S., Jakubowski, A., Klopcic, BR., Olynyk, J.K., Yeoh, G.C., and Knight, B. (2010). Tumor necrosis factor-like weak inducer of apoptosis is a mitogen for liver progenitor cells. Hepatology. 52, 291–302. Tumarov, A.V., Koroleva, E.P., Christiansen, P.A., Khan, M.A., Ruddy, M.J., Burnette, B., Papa, S., Franzoso, G., Nedospasov, S.A., Fu, Y.X., Anders, R.A. (2009). T cell-derived lymphotoxin regulates liver regeneration. Gastroenterology. 136, 694–704. Ueberham, E., Böttger, J., Ueberham, U., Grosche, J., and Gebhardt, R. (2010). Response of sinusoidal liver cells in CDE-diet. Comp. Hepatol., Oct. 13;9:8. Wang, X., Quail, E., Hung, N.J., Tan, Y., Ye, H., and Costa, R.H. (2001). Increased levels of forkhead box M1B transcription factor in transgenic mouse hepatocytes prevent age-related proliferation defects in regenerating liver. Proc. Natl. Acad. Sci. U.S.A. 98, 11468–73. Wu, H., Wade, M., Krall, L., Grisham, J., Xiong, Y., and Van Dyke, T. (1996). Targeted in vivo expression of the cyclin-dependent kinase inhibitor p21 halts hepatocyte cell-cycle progression, postnatal liver development and regeneration. Genes Dev. 10, 245–60.

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13 Therapeutic Potential of Bone Marrow Stem Cells in Liver Surgery Jan Schulte am Esch, Moritz Schmelzle, Günter Fürst, 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 first 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 defined. 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, fibrosis, severe steatosis, or

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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 sufficient 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 specific 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 fulfill 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 Dissé was shown to have stem cell niche properties (Sawitza et al., 2009).

13.3

Stem Cells in Liver Regeneration

The ability to define and then manipulate hepatic stem/progenitor cells promises significant advances in our treatment of liver disease. For this reason, it is critical to define 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 specific 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 Dissé 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 significant 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 (Fürst 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 marrow–derived 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 marrow–derived 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 marrow–derived 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

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myofibroblast-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 myofibroblasts (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 inflammatory 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 efficient 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 (modified) 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 benefit 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 cell–based 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

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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 benefits 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 five 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-α-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 confirm 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 marrow–liver axis. Therefore, autologous CD133 BMSCs were used to stimulate liver regeneration in patients scheduled for extended right hepatectomy (Fürst 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 sufficient to induce adequate proliferation of the left lateral liver segments within a reasonable time. A panel of criteria was used indicating compromised hepatic

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parenchyma or co-morbidity, possibly impairing liver regeneration capacity, including liver cirrhosis (Child-Pugh score A), fibrosis 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

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(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 IV–VIII

13.7


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left hepatic vein

right, middle and left hepatic vein

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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 IV–VIII 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 first filtered 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 certified 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 cytofluorometric analyses (Fürst 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 fluoroscopic 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 defined (peripheral venous or hepatic arterial application are alternatively possible), we hypothesized

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13 Therapeutic Potential of Bone Marrow Stem Cells in Liver Surgery native bone marrow cells

after selection for CD133

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Figure 13.2 Representative Cytofluometric 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 first-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 1–2 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 significantly higher in the patients additionally treated with BMSC (70.8% versus 41.3%; p < 0.005). The increased proliferation rate resulted in a significant reduction of the waiting time from PVE to liver resection (46 days versus 30 days; p < 0.05) in our early experience (Fürst 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 significant impairment of physical condition with marked limitation of activity (NYHA III). Within the first 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 insufficient 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 antifibrotic 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. Significantly 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 significant differences in tumor free survival times and recurrence rates. Beyond a significant 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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Further Reading Fausto, N. (2004). Liver regeneration and repair: hepatocytes, progenitor cells, and stem cells. Hepatology 39(6), 1477–87. Shmelkov, S. V., St Clair, R. et al. (2005). AC133/CD133/Prominin-1. Int J Biochem Cell Biol 37(4), 715–9. Walkup, M. H. and D. A. Gerber (2006). Hepatic stem cells: in search of. Stem Cells 24(8), 1833–40. Xu, Y. Q. and Z. C. Liu (2008). Therapeutic Potential of Adult Bone Marrow Stem Cells in Liver Disease and Delivery Approaches. Stem Cell Rev. 2008 Summer; 4(2),101–12.

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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 β-catenin, 5, 66, 93, 159, 160, 164, 165, 166, 172, 180 Bile duct ligation, 180 Bisulfite conversion, 103 Blood flow, 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-deficient, 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, 135–137 Epigenetic — definition, 99 — DNA methylation, 100 — hepatic stellate cells, 105 — histone modifications, 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 — fibrosis, 87 — glial fibrillary acidic protein, 87 — hedgehog signalling, 76 — HSC, 130, 135

— — — —

isolation, 85 Nestin, 87 Nestin DNA methylation, 106 Nestin histone modifications, 106 — Notch3 DNA methylation, 106 — Notch signalling, 77 — plasticity, 90 — space of Dissé, 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 modifications, 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 Inflammatory 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 — DNA methylation, 106 — histone modifications, 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

Outflow 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 β 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 Myofibroblasts, 151, 194

209

Partial hepatectomy (PH), 2, 57, 87, 112, 130, 148, 149, 179 PDK1, 8 Peroxisome proliferator-activated receptor-α, 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 Dissé, 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 — identification, 72 — neighbouring cells, 64 — secreted factors, 64 — space of Dissé, 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 β (TGFβ), 8, 183 Transforming growth factor β (TGFβ) receptor I, 8 Transforming growth factor β (TGFβ) receptor II, 8 Transforming growth factor β (TGFβ) 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-acetylaminofluorene, 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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