Current Topics in Developmental Biology, Volume 64 (Current Topics in Developmental Biology)

Current Topics in Developmental Biology Volume 64 Series Editor Gerald P. Schatten Director, PITTSBURGH DEVELOPMENTA...

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Current Topics in Developmental Biology

Volume 64

Series Editor Gerald P. Schatten Director, PITTSBURGH DEVELOPMENTAL CENTER Deputy Director, Magee-Women’s Research Institute Professor and Vice-Chair of Ob-Gyn Reproductive Sci. & Cell Biol.-Physiology University of Pittsburgh School of Medicine Pittsburgh, Pennsylvania 15213

Editorial Board Peter Gru¨ss Max-Planck-Institute of Biophysical Chemistry Go¨ttingen, Germany

Phillip Ingham University of Sheffield, United Kingdom

Mary Lou King University of Miami, Florida

Story C. Landis National Institutes of Health National Institute of Neurological Disorders and Stroke Bethesda, Maryland

David R. McClay Duke University, Durham, North Carolina

Yoshitaka Nagahama National Institute for Basic Biology, Okazaki, Japan

Susan Strome Indiana University, Bloomington, Indiana

Virginia Walbot Stanford University, Palo Alto, California

Founding Editors A. A. Moscona Alberto Monroy

Current Topics in Developmental Biology Volume 64 Edited by

Gerald P. Schatten Director, PITTSBURGH DEVELOPMENTAL CENTER Deputy Director, Magee-Women’s Research Institute Professor and Vice-Chair of Ob-Gyn-Reproductive Sci. & Cell Biol.-Physiology University of Pittsburgh School of Medicine Pittsburgh, Pennsylvania 15213

Cover Photo Credit: Xenograft of human airway epithelium demonstrating an expanded retroviral-infected progenitor clone capable of submucosal gland and pluripotent surface airway epithelial differentiation. Photo courtesy of Drs. Xiaoming Liu and John F. Engelhardt, Center for Gene Therapy at the University of Iowa, Iowa City, Iowa. Elsevier Academic Press 525 B Street, Suite 1900, San Diego, California 92101-4495, USA 84 Theobald’s Road, London WC1X 8RR, UK

This book is printed on acid-free paper. Copyright ß 2004, Elsevier Inc. All Rights Reserved.

No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopy, recording, or any information storage and retrieval system, without permission in writing from the Publisher. The appearance of the code at the bottom of the first page of a chapter in this book indicates the Publisher’s consent that copies of the chapter may be made for personal or internal use of specific clients. This consent is given on the condition, however, that the copier pay the stated per copy fee through the Copyright Clearance Center, Inc. (www.copyright.com), for copying beyond that permitted by Sections 107 or 108 of the U.S. Copyright Law. This consent does not extend to other kinds of copying, such as copying for general distribution, for advertising or promotional purposes, for creating new collective works, or for resale. Copy fees for pre-2004 chapters are as shown on the title pages. If no fee code appears on the title page, the copy fee is the same as for current chapters. 0070-2153/2004 $35.00 Permissions may be sought directly from Elsevier’s Science & Technology Rights Department in Oxford, UK: phone: (þ44) 1865 843830, fax: (þ44) 1865 853333, E-mail: [email protected]. You may also complete your request on-line via the Elsevier homepage (http://elsevier.com), by selecting ‘‘Customer Support’’ and then ‘‘Obtaining Permissions.’’ For all information on all Academic Press publications visit our Web site at www.academicpress.com

ISBN: 0-12-153164-3 PRINTED IN THE UNITED STATES OF AMERICA 04 05 06 07 08 9 8 7 6 5 4 3 2 1

Contents

Contributors Preface xi

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1 Stem/Progenitor Cells in Lung Morphogenesis, Repair, and Regeneration David Warburton, Mary Anne Berberich, and Barbara Driscoll I. II. III. IV.

Introduction 1 Background 3 Symposium Presentations and Discussion Summary and Conclusions 10 References 11

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2 Lessons from a Canine Model of Compensatory Lung Growth Connie C. W. Hsia I. II. III. IV.

Structure–Function Considerations during Compensation Dysanaptic Lung Growth 23 Signals for Compensatory Lung Growth 26 Application to Human Lung Growth 29 Acknowledgments 29 References 30

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3 Airway Glandular Development and Stem Cells Xiaoming Liu, Ryan R. Driskell, and John F. Engelhardt I. Introduction and Scope 34 II. Airway Submucosal Gland Structure/Function and Its Relationship to Human Diseases 35 v

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Contents III. Species-Specific DiVerences in Conducting Surface Airway Epithelial Cell Types and Submucosal Gland Abundance 37 IV. Evidence for Human Adult Proximal Airway Stem Cells Capable of Developing Submucosal Glands 41 V. Mouse Tracheal Injury Models Suggest Submucosal Gland Ducts Are a Niche for Proximal Airway Stem Cells 45 VI. Developmental Molecular Markers of Glandular Stem Cell Phenotypes 46 VII. Concluding Remarks and Future Challenges 51 Acknowledgments 52 References 52

4 Gene Expression Studies in Lung Development and Lung Stem Cell Biology Thomas J. Mariani and Naftali Kaminski I. II. III. IV. V. VI. VII. VIII.

Introduction 57 Verifying and Challenging Old Concepts 59 Establishing New Concepts 61 A Model for Lung Alveogenesis 62 Development and Stem Cells 63 Applying Developmental Transcriptional Modules to Adult Disease Lung Cancer 65 Conclusion 66 Acknowledgments 67 References 67

5 Mechanisms and Regulation of Lung Vascular Development Michelle Haynes Pauling and Thiennu H. Vu I. II. III. IV.

Introduction 73 Overview of Pulmonary Vessel Formation 74 Regulation of Lung Vasculature Formation 77 Epithelial–Mesenchymal Interactions during Lung Vascular Development 85 V. Specification of Arterio-Venous Identity 86 VI. Endothelial Stem Cells in Lung Regeneration and Repair VII. Conclusions 91 Acknowledgments 92 References 92

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6 The Engineering of Tissues Using Progenitor Cells Nancy L. Parenteau, Lawrence Rosenberg, and Janet Hardin-Young I. The Parenchyma: An Interactive Cell Population 102 II. A Working Hypothesis for Understanding Parenchymal Cell Interaction and Response 103 III. The Epidermis 105 IV. The Liver 116 V. The Pancreas and Generation of Pancreatic Islets 125 VI. Conclusions and Future Directions 130 References 131

7 Adult Bone Marrow-Derived Hemangioblasts, Endothelial Cell Progenitors, and EPCs Gina C. Schatteman I. II. III. IV. V. VI. VII. VIII. IX.

Introduction 142 Stem and Progenitor Cells 143 Embryonic Hemangioblasts 145 Adult Angioblasts 148 Adult Hemangioblasts 152 Physiological Significance of Bone Marrow-Derived Endothelium Endothelial Cell Progenitors 156 Mobilization 160 Bone Marrow-Induced Functional Improvements in Ischemic Tissue 163 X. Are We Ready for Human Trials? 165 XI. Looking to the Future 167 Acknowledgments 168 References 168

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8 Synthetic Extracellular Matrices for Tissue Engineering and Regeneration Eduardo A. Silva and David J. Mooney I. Introduction 182 II. Inductive and Cell Transplantation Strategies

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Contents III. Engineering Vascular Structures 191 IV. Bone Regeneration 196 V. Conclusions and Future Directions 199 Acknowledgments 200 References 200

9 Integrins and Angiogenesis D. G. Stupack and D. A. Cheresh I. II. III. IV. V.

Introduction 208 Endothelial Cell Integrins 209 Initiation of Angiogenesis 214 Alterations in Integrin–ECM Interactions During Angiogenesis Integrin Antagonists EVects versus the Phenotype of Knockout Animals 227 VI. Perspective and Conclusions 228 References 230

Index 239 Contents of Previous Volumes

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Contributors Numbers in parentheses indicate the pages on which the authors’ contributions begin.

Mary Anne Berberich (1), Developmental Biology Program, Saban Research Institute, Childrens Hospital Los Angeles, Los Angeles, California 90027 D. A. Cheresh (207), Department of Immunology, The Scripps Research Institute, La Jolla, California 92037 Barbara Driscoll (1), Developmental Biology Program, Saban Research Institute, Childrens Hospital Los Angeles, Los Angeles, California 90027 Ryan R. Driskell (33), Department of Anatomy and Cell Biology, College of Medicine, The University of Iowa, Iowa City, Iowa 52242 John F. Engelhardt (33), Department of Anatomy and Cell Biology and Department of Internal Medicine, and Center of Gene Therapy of Cystic Fibrosis and Other Genetic Diseases, College of Medicine, The University of Iowa, Iowa City, Iowa 52242 Janet Hardin-Young (101), Amaranth Bio, Inc., Watertown, Massachusetts 02472 Connie C. W. Hsia (17), Department of Internal Medicine, Pulmonary and Critical Care Medicine, University of Texas Southwestern Medical Center, Dallas, Texas 75390 Naftali Kaminski (57), Dorothy P. and Richard P. Simmons Center for Interstitial Lung Disease, Lung Translational Genomics Center, Pulmonary, Allergy, and Critical Care Medicine, University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania 15260 Xiaoming Liu (33), Department of Anatomy and Cell Biology, College of Medicine, The University of Iowa, Iowa City, Iowa 52242 Thomas J. Mariani (57), Division of Pulmonary and Critical Care Medicine, Brigham and Women’s Hospital, Pulmonary Bioinformatics, The Lung Biology Center, Harvard Medical School, Boston, Massachusetts 02115 David J. Mooney (181), Division of Engineering and Applied Sciences, Harvard University, Cambridge, Massachusetts 02138. Nancy L. Parenteau (101), Amaranth Bio, Inc., Watertown, Massachusetts 02472

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Michelle Haynes Pauling (73), Department of Medicine and Lung Biology Center, University of California, San Francisco, California 94143 Lawrence Rosenberg (101), Division of Surgical Research, McGill University, Montreal, Quebec H3G 1A4, Canada Gina C. Schatteman (141), Department of Exercise Science, University of Iowa, Iowa City, Iowa 52242 Eduardo A. Silva (181), Division of Engineering and Applied Sciences, Harvard University, Cambridge, Massachusetts 02138. D. G. Stupack (207), Department of Immunology, The Scripps Research Institute, La Jolla, California 92037 Thiennu H. Vu (73), Department of Medicine and Lung Biology Center, University of California, San Francisco, California 94143 David Warburton (1), Developmental Biology Program, Saban Research Institute, Childrens Hospital Los Angeles, Los Angeles, California 90027

Preface This volume of Current Topics in Developmental Biology showcases an exciting array of topics in our field. While lung development and regeneration are featured prominently in this issue, the collection as a whole covers important elements of tissue engineering – adult progenitor cells, synthetic polymer extracellular matrices, and integrin dynamics in blood vessel formation – in impressive breadth and depth. Stem/Progenitor Cells in Lung Morphogenesis, Repair, and Regeneration by David Warburton, Mary Anne Berberich, and Barbara Driscoll of Childrens Hospital Los Angeles reports from the NHLBI-sponsored symposium of the same name at the 62nd annual meeting of the Society for Developmental Biology. As summarized here, symposium participants addressed the outstanding questions in lung development and repair with their own exceptional work, and challenged the field to explore new directions, particularly in the therapeutic realm. Lessons from a Canine Model of Compensatory Lung Growth by Connie Hsia of the University of Texas is a comprehensive review of this valuable large-mammal model, and its applications to human lung growth. While canines demonstrate far more vigorous compensatory lung growth than humans, understanding the mechanisms of growth (in canines) and growth inhibition (in humans) may lead to better therapies for patients undergoing pneumonectomy. Airway Glandular Development and Stem Cells by Xiaoming Liu, Ryan Driskell, and John Englehardt of the University of Iowa explores the cell biology of the submucosal glands of the lung, their role in disease, and their relationship to adult surface airway epithelial progenitor cells, with implications for the treatment of hypersecretory diseases such as cystic fibrosis. Gene Expression Studies in Lung Development and Lung Stem Cell Biology by Thomas Mariani of Harvard and Naftali Kaminski of the University of Pittsburgh explores the application of microarray technology to lung development, and its potential for raising exciting new questions and concepts about both normal development and disease. In Mechanisms and Regulation of Lung Vascular Development by Michael Pauling and Thiennu Vu of the University of California, the authors describe current concepts – including genetic and epigenetic factors – in vessel formation. Interestingly, known general vascular growth factors don’t seem to play a part in lung vessel formation in particular, which speaks to the molecular complexity and specificity of developmental cues. xi

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The Engineering of Tissues Using Progenitor Cells by Nancy Parenteau, Lawrence Rosenberg, and Janet Hardin-Young of McGill University examines the potential for developing regenerative disease and injury therapies using adult precursor cells, particularly of the epidermis, the liver, and the pancreas. The authors conclude that in order to generate useful populations of cells from adult progenitors, wrongly characterized as being of limited use, culture conditions must replicate the in vivo environments which encourage new tissue formation. In Adult Bone Marrow-Derived Hemangioblasts, Endothelial Cell Progenitors, and EPCs, Gina Schatteman of the University of Iowa reviews the dynamics and utility of progenitor cells from adult marrow, along with their therapeutic potential, important caveats about their clinical use, and encouragement to researchers to continue their hard work so that the full healing potential of these cells can be safely realized. Synthetic Extracellular Matrices for Tissue Engineering and Regeneration by Eduardo Silva and David Mooney of the University of Michigan discusses the design of laboratory-made, polymer-based ECMs for the guided regrowth of specific cell types. The authors point out that the growth of whole organs, which are composed of more than one cell type, may depend upon a synthetic ECM releasing cascades of signals over time, thereby prompting the correct sequences of tissue generation. Finally, Integrins and Angiogenesis by D.G. Stupack and D.A. Cheresh of the Scripps Research Institute is a comprehensive primer on the molecular biology of blood vessel growth, the short but exciting history of therapeutic integrin antagonists, and the future of translational integrin research. This volume has benefited from the ongoing cooperation of a team of participants who are jointly responsible for the content and quality of its material. The authors deserve the full credit for their success in covering their subjects in depth yet with clarity, and for challenging the reader to think about these topics in new ways. The members of the Editorial Board are thanked for their suggestions of topics and authors. I also thank Leah KauVman for her fabulous editorial insight and Anna Vacca for her exemplary administrative support. Finally, we are grateful to everyone at the Pittsburgh Development Center of Magee-Womens Research Institute here at the University of Pittsburgh School of Medicine for providing intellectual and infrastructural support for Current Topics in Developmental Biology. Jerry Schatten Pittsburgh Development Center, Pennsylvania

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Stem/Progenitor Cells in Lung Morphogenesis, Repair, and Regeneration David Warburton, Mary Anne Berberich, and Barbara Driscoll Developmental Biology Program Saban Research Institute, Childrens Hospital Los Angeles Los Angeles, California 90027

I. II. III. IV.

Introduction Background Symposium Presentations and Discussion Summary and Conclusions References

I. Introduction Ex ovo omnia (William Harvey, circa 1620) The future is in eggs (Eugene Ionesco, circa 1960) Recently, the National Heart, Lung and Blood Institute (NHLBI) sponsored a symposium on stem/progenitor cells in lung morphogenesis, repair, and regeneration, which was held in conjunction with the 62nd annual meeting of the Society for Developmental Biology (SDB). The symposium participants critically examined the evidence for the existence and function of stem/progenitor cells within both the upper and lower airways of the lung and evaluated their contribution to lung development, repair, and regeneration. The key points in the oral presentations and discussion at this symposium are reported in this chapter and are placed within the context of current ideas presented at the main SDB meeting. New directions for future research in this exciting new field are identified. Synopses describing several of the key oral presentations at this symposium are reported elsewhere in this volume and are discussed here. This symposium was dedicated by the SDB to the memory of Merton Bernfield, who was one of the first to appreciate that unraveling the molecular embryology of the lung would yield major insights into lung development and disease (Warburton et al., 1993, 1999). He would have been delighted to know that the proteoglycans he discovered are now increasingly realized to play pivotal roles in such processes as localizing growth factor activation Current Topics in Developmental Biology, Vol. 64 Copyright 2004, Elsevier Inc. All rights reserved. 0070-2153/04 $35.00

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and signaling as well as diapedesis of neutrophils into the airspaces within the lung (Li et al., 2002). Organismal and, indeed, organ development, up to and including senescence, is programmed by the genome. Developmental biologists study the process of functional elaboration of the information stored in the genome, which, taking advantage of physical principles, forms functional structures, including all the three germ layers of the embryo and all of the structures derived thereform. Considerable excitement has been generated by the possibility that human cells, tissues, and perhaps even organs, such as the lung, could be regenerated following reactivation of these or similarly regulated developmental processes. Stem cells can be operationally defined as multipotent, undiVerentiated cells with the capacity to maintain themselves indefinitely as stem cells, while simultaneously dividing to give rise to daughter progenitor cells. Progenitor cells in turn continue to divide, but eventually give rise to diVerentiated cell lineages. These diVerentiated cell lineages in turn give rise to tissues and organs, in a temporospatial collaboration with other diVerentiated cell lineages. The best-studied examples of stem cells include those that give rise to the gonadal germ cells and the hematopoietic and mesenchymal stem cells of the bone marrow. Other somewhat more restricted examples include the stem cells in the shafts of hair follicles and in the intervillous crypts of the gut (Watt and Hogan, 2000). The SDB sponsored several interesting and informative discussions on stem cell biology in such models as the round worm Caenorhabditis elegans, as well as on tissue regeneration in such relatively simple organisms as planaria, which can regenerate the whole organism from a few cells; axolotls, which can regenerate severed limbs; zebrafish embryos, which can regenerate rather substantial portions of their myocardium and other organs; and stags, which regenerate their antlers every spring. Allan Spradling was awarded the Conklin prize for his life’s work on flies, most particularly the germline stem cell populations and the molecular genetic signals that give rise to the germline (Kai and Spradling, 2003; Spradling et al., 2001). The issue of whether resident stem cells continue to exist in the adult pancreas and can account for the limited capacity of pancreatic islet cells to regenerate (apparently they do not and cannot, respectively) was addressed by Doug Melton (Gu et al., 2004; Murtaugh and Melton, 2003). The mature lung is a vast organ that arises (E9 in mice, 4 weeks in humans) from a very few stem/progenitor cells that arrive in the ventral midline surface of the primitive embryonic foregut endoderm at some time prior to the emergence of a posterior pharyngeal pouch structure termed the laryngotracheal groove. The genetic and molecular mechanisms that determine the expansion of the stem/progenitor cells within the primitive laryngotracheal anlagen into the mature lung with a surface area of 70 m2 are

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progressively being unraveled; this has been comprehensively reviewed in Warburton et al. (2003, 2004). It is amusing to note that, were the respiratory gas exchange surface actually planar and not packed into the chest through the processes of branching morphogenesis and alveolarization, humans would have the ‘‘wingspan’’ of a small airplane! Herein we consider the somewhat limited information available about the specific roles contributed by stem/progenitor cells in these processes.

II. Background Mutations of RARs (Mendelsohn et al., 1994; Mollard et al., 2000), Foxf1 (also known as HNF3) (Kalinichenko et al., 2000, 2002, 2004; Lim et al., 2002; Mahlapuu et al., 2001), and the Gli2/Gli3 genes (Motoyama et al., 1998) in mice show clearly that specific transcriptional factors are required for the induction of the laryngotracheal groove from the primitive foregut endoderm. In the Foxf1 null mutants, the primitive foregut fails to close and the laryngotracheal groove therefore never arises, suggesting that the morphogenetic field that should induce the correct stem/progenitor cells to form this organ has also failed to organize. Foxf1 heterozygotes show abnormal lobar and vascular patterning. In Gli2/Gli3 mutants, the esophageal tube forms correctly from the primitive foregut, but the laryngotracheal cleft fails to arise. Therefore, the larynx, trachea, and lungs do not form. Presumably, the stem/progenitor cell population that should give rise to the laryngotracheal groove again was not induced correctly. Mutations of the Nkx2.1 transcriptional factor gene (Iwatani et al., 2000; Kimura et al., 1996, 1999; Krude et al., 2002; Minoo et al., 1999; Yuan et al., 2000), as well as of Shh (Litingtung et al., 1998; Pepicelli et al., 1998; Spilde et al., 2003a,b), not only abrogate correct septation of the esophagus from the trachea but also correct peripheral lung morphogenesis distal to the first bronchial branches. Null mutation of Fg f10 results in a blind ending trachea, while inhibition of global fibroblast growth factor receptor (FGFR) signaling results in abrogation of branching distal to the first bronchial branch (Celli et al., 1998; Sekine et al., 1999). This has led to the suggestion that the larynx and trachea may be formed as an organ genetically separate from the peripheral bronchi and pulmonary parenchyma. Rarely, in human cases of tracheal atresia, the peripheral lung can arise bilaterally from the esophagus, in the apparent absence of a larynx or trachea. The view that the esophago-trachea and the peripheral lung are embryologically and genetically distinct organs is further supported by lineage tracing studies in which cell lineages that form the trachea and proximal bronchi were shown not only to be established well in advance of the

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emergence of a recognizable lung anlagen but also to be distinct from cell lineages of the distal and peripheral airway (Perl et al., 2002). Genetically marked precursor cells of the peripheral lung were also found to be few in number and were located principally at the tips of the proximal airway buds. Presumably, these stem/progrenitor cells require FGFR signaling to undergo developmental expansion to form into the peripheral lung. These lineage-marking studies also identified distinct lineages of cells in the conducting airways, comprising neuroepithelial and tracheo-bronchial gland precursors. However, protein level studies have previously shown that airway epithelial cells can initially co-express markers of several mature cell lineages, suggesting that early epithelial precursor cells may be multipotent and that restriction of lineage-specific gene expression, such as Nkx2.1, may mediate the emergence of distinct cell lineage phenotypes within restricted epithelial niches (Wuenschell et al., 1996). Emergence of distinct pulmonary epithelial cell lineage phenotypes is now well known to be under the control of specific transcriptional factors. For example, null mutation of Mash1 ablates the pulmonary neuroendocrine (PNE) cell lineage, while null mutation of Nkx2.1 negatively impacts SpC expression as a marker of alveolar epithelial type 2 cells (AEC2) (Borges et al., 1997; Minoo et al., 1999; Yuan et al., 2000). Gata6 expression has also been found to be essential for diVerentiation of the alveolar epithelial type 1 cell (AEC1) lineage (Liu et al., 2003). Factors that control proliferation of the putative epithelial stem/ progenitors and derivation of their daughter cells during the upwards of 500 million morphogenetic events that mediate lung morphogenesis remain incompletely understood (reviewed in Warburton et al., 2003). However, in general, peptide growth factors such as epidermal growth factor (EGF), FGFs, hepatocyte growth factor (HGF), and platelet-derived growth factors (PDGFs) control the rate of emergence of epithelial cells from GO into G1 and the transition through S phase of the cell cycle. These growth factors share the common characteristic of signaling through cognate tyrosine kinase receptors, which in turn activate extracellular signal-regulated kinases (ERKs) and Ras, and hence cyclin-dependent kinases. The Sprouty gene family is rapidly emerging as important inducible negative regulators of certain steps in Ras activation involving modulation of components in the juxtamembrane signaling complex, including Grb2, SOS, and the Shp2 tyrosine phosphatase (Warburton and Bellusci, 2004). Members of the transforming growth factor-beta (TGF-) superfamily in general negatively regulate epithelial proliferation through the canonical serine-threonine kinase to the Smad2, -3, and -4 signaling pathway. However, bone morphogenetic protein (BMP4) signaling plays a more complex role than that and can directly inhibit but indirectly activates proliferation of the epithelium in the intact embryonic lung through Smads1 and -5, while

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Smurf, a ubiquitin ligase, negatively modulates signaling by the BMP Smads (Shi et al., 2004). Null mutation of the Pdg f-A chain gene (Bostrom et al., 1996), double null mutation of the Fg fr3/Fg fr4 genes (Weinstein et al., 1998), and null mutation of the Elastin gene (Wendel et al., 2000) give rise to a lack of alveolarization phenotype, which appears to be mediated, at least in part, by the common mechanism of abnormal or absent elastin deposition. This points out the major issue that proliferation and diVerentiation of the putative alveolar epithelial stem/progenitor cells also depend on the availability of the correct matrix substrates, in the correct orientation. This is the concept of the inductive niche. It is also very interesting to note that bioavailability of TGF- family ligands within the matrix may play a very important role, as has been suggested to be the case in the alveolar hypoplasia seen in Marfan syndrome. Mice null mutant for the Fibulin-1 (Fbn-1) gene, which is also mutated in Marfan, fail to secure latent TGF- binding protein (LTBP) within the matrix, allowing excess freely activated TGF- to accumulate (Kaartinen and Warburton, 2003; Neptune et al., 2003). This is associated with alveolar hypoplasia, presumably caused by the adverse eVects of excess TGF- on alveolar epithelial cell (AEC) proliferation. Alveolar hypoplasia is also a key pathobiological feature of the disease of human premature lung, termed bronchopulmonary dysplasia (BPD) (Jobe, 1999). Excess TGF-1 expression, activation, and signaling are strongly implicated in the alveolar hypoplasia, emphysema, and interstitial fibrosis that are characteristic pathobiological features of BPD (Gauldie et al., 2003). Emphysema in the adult lung is also associated with alveolar atrophy. However, Smad3 null mutation demonstrates that intact TGF- signaling is required to preserve the alveolar architecture, since these mice get progressive centrilobular emphysema during their adulthood (Bonniaud et al., 2004). It is tempting to speculate that the rate of recovery or decline in alveolar function in all of these important lung diseases and disease models could be determined by proliferation versus ablation or senescence and atrophy of the putative alveolar stem/progenitor cell population. Thus, the rate of proliferation versus apoptosis versus senescence clearly plays an important role in fine-tuning the AEC population. Correct comorphogenesis of the epithelial, vascular, and lymphatic trees is also essential for the lung to function correctly as a gas exchange organ. Vascular morphogenesis in the lung is modulated by several alternatively spliced and processed vascular endothelial growth factor (VEGF) isoforms, which are expressed and secreted from the epithelium to activate cognate VEGFs 1 and 2 on the endothelium (Galambos et al., 2002). Lymphatic morphogenesis is mediated through VEGF-C and -D activation of vascular endothelial growth factor receptor 3 (VEGFR3) on the lymphatic endothelium (Karkkainen et al., 2004; McColl et al., 2003). Misexpression of VEGF

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isoforms can lead to both vascular and epithelial dysplasia (Akeson et al., 2003). Proliferation and diVerentiation of the smooth muscle cell precursor population from within the lung mesenchyme is another important factor in lung morphogenesis. We have used mesenchymal cell lineage tracing to discover a novel population of peripheral smooth muscle precursor cells that arises within the Fg f10 expression domain, which lies in the subpleural region of peripheral embryonic lung. These marked cells eventually contribute substantially to smooth muscle surrounding the peripheral airways (Bellusci, unpublished results). The diVerentiation of this novel cell population may also be under the control of Sonic hedgehog (SHH) and BMP4, since misexpression of these ligands under the control of the SpC promoter results in gross dysplasia of the smooth muscle actin expressing cell phenotype in the peripheral embryonic lung mesenchyme (Bellusci et al., 1996, 1997). Four major morphogen signaling pathways, FGF, BMP, Shh, and Wnt, act together in repetitively and interatively expressed morphogenetic centers to determine many of the key aspects both of branching morphogenesis and of cell lineage specification within the lung. We postulate that these signals integrate within the nuclei of pulmonary stem/progenitor cells to determine their relative rates of proliferation, apoptosis, senescence, migration, and diVerentiation within these morphogenetic centers. (The rationale for this idea was put forward in detail in Warburton et al., 2003.) It has long been known from morphometric studies that the alveolar epithelium is capable of rapid regeneration following injury. Whether stem/precursor cells are maintained within the mature lung following the completion of the alveolar phase of lung development during late childhood or early adolescence in humans is controversial. We have shown that in rat, cells expressing markers of putative precursor/progenitor cells such as telomerase can be identified during the recovery phase of hyperoxic lung injury (Driscoll et al., 2000). These cells can be physically sorted from AEC populations isolated from rat lungs during the recovery phase from sublethal hyperoxia, based on the relative expression of -cadherin (Reddy et al., 2003). These telomerase-positive and e-cadherin-reduced AECs are also highly proliferative, and we have also shown that this subpopulation is relatively resistant to injury. As such, it may be responsible for the proliferative phase of repopulation of the injured alveolar epithelium. The pattern of telomerase immunohistochemistry in hyperoxia-injured lungs suggests that if alveolar stem/progenitor cells do in fact exist, they must be widely distributed among the alveoli (Driscoll et al., 2000). This makes some teleological sense, considering that rapid repair of the 70 m2 alveolar surface area, which is moreover highly geographically restricted by folding up into alveoli, is a matter of vital importance and urgency that could be expected to require a large number of widely distributed precursor cells. Evidence is

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emerging, however, that reparative stem/precursor cells may also arrive just in time at the alveolar epithelial surface, somewhat like the cavalry in old Western B movies, from elsewhere in the body, such as the bone marrow or fat, via the pulmonary capillary circulation. This again makes for an attractive teleological hypothesis, since the alveolar capillary bed is perfused by the entire right cardiac output and is very narrow (5–7 microns), making it a perfect sieve for larger, more rigid cells than the standard red or white blood cell. Diapedesis of neutrophils was discovered to be mediated via a CXC cytokine and proteoglycan-mediated call out mechanism (Liu et al., 2003). It is tempting to speculate that similar mechanisms may be involved in calling out circulating stem or precursor cells as part of the lung injury response. In addition, circulating signals may stimulate the bone marrow to release increased numbers of stem cells. It is also tempting to speculate that resident pulmonary stem/progenitor cells may serve as mutational targets for lung carcinogens relatively early in life, so that, because of their relative longevity within the lung as an apoptosis-resistant reservoir of cells that can be activated to conduct essential tissue repairs, they may survive to carry forward their mutations into compound mutants that eventually clonally give rise to cancers.

III. Symposium Presentations and Discussion The following important but unanswered questions for future research about stem/progenitor cell in the lung were considered at this symposium: 1. Are there two or more distinct regenerative cell populations in the lung, one being stem cells defined as a stable population of undiVerentiated cells with unlimited self-renewal potential, and another being progenitor cells with limited self-renewal capacity and some diVerentiated features? Are there other kinds? 2. Are there multipotent progenitor cells within the embryonic lung that function as stem or progenitor cells, and do they persist in the adult lung? Do these same cells contribute to lung repair or regeneration? 3. What are the recently described ‘‘side’’ populations of cells, identified by fluorescent cell sorting of pulmonary cells, that apparently have regenerative capacity? 4. What are the molecular signals that call out exogenous stem or progenitor cells from the circulation into the lung versus those that activate resident cells with regenerative capacity? 5. How far and by what methods can embryonic stem (ES) cells be induced to express lung-specific phenotypes, and is the methodology scalable for engineering, tissue repair, or regeneration?

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6. What are the molecular rules that govern stem/progenitor cell plasticity in the lung, and to what extent are they applicable to gene- and cell-based therapy for lung diseases? The symposium discussion suggested that further critical examination is needed to establish solid evidence for the existence and function of stem/ progenitor cells within both the upper and lower airways of the lung that contribute to lung development, repair, and regeneration. The possibility that ES cells as well as bone marrow-derived stem cells may be induced to contribute to lung repair and regeneration also requires further critical examination. The molecular rules governing stem/progenitor cell plasticity in the lung require further elucidation as do the possibilities for gene- and cell-based therapy for lung diseases. The symposium presentations shed some light on these key issues. Anne Bishop of Imperial College, London, reviewed her work on AEC diVerentiation from human ES cells. She presented intriguing preliminary results suggesting that cells expressing SpC as well as containing primitive lamellar bodies can be derived from human ES cell cultures under certain conditions (Ali et al., 2002). She suggested that this provocative work could in the future lead to scalable derivation of human AECs for cell-based therapy in lung injury as well as perhaps other diseases of the air–gas interface. Although Dr. Bishop discussed the feasibility of diVerentiating AECs from human ES cells, and while some preliminary success has been reported by her group, the process is neither fully understood nor scalable as of yet (Bishop, 2004). Barbara Driscoll of the Saban Research Institute, Childrens Hospital Los Angeles reviewed the latest evidence for the existence of resident alveolar epithelial progenitor/stem cells. She cautioned that it has not been proven that these cells have true stem cell characteristics. However, they do appear to proliferate and repopulate the denuded alveolar epithelium following hyperoxic injury. They do indeed increase in telomerase expression and activity during the repair process. Moreover, she has found that this proliferative, putative progenitor cell population can be further characterized by FACS analysis using the criteria of absence of e-cadherin and -catenin expression as well as relative resistance to apoptosis (Driscoll et al., 2000; Reddy et al., 2003). Connie Hsia of the University of Texas Southwestern discussed her pioneering work on postpneumonectomy lung regeneration in dogs. This occurs over a much longer time period than in lung injury recovery in rodents and is particularly localized in the periphery of the lung. She has also shown quite conclusively that physical forces of breathing, acting within the lung, play a necessary role in the initiation and maintenance of lung regeneration in the dog, because placement of an inert prosthesis within the chest cavity to

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replace the resected lung tissue abrogates the regeneration of the remaining lung (Hsia et al., 2000, 2001, 2003; Takeda et al., 1999). Diane Krause of Yale University presented her latest results on the derivation of type 2 pneumocytes from bone marrow-derived stem cells. Her work has caused considerable excitement as well as controversy within the stem cell field as to whether the integration of marker gene expression she has observed is due to cell fusion. She presented further evidence that cell fusion is unlikely to be a major factor (Grove et al., 2002; Herzog et al., 2003; Krause et al., 2001; Thiese et al., 2002). Taken together with the results reported by Allan Fine of Boston University, who presented novel results supporting uptake of circulating stem cells into the alveolar epithelium following lung injury with bleomycin (Kotton and Fine, 2003; Kotton et al., 2001; Summer et al., 2003), this approach to cell-based therapy in the lung clearly deserves further investigation. Darwin Prockop from Tulane provided further insight into this issue when he presented new data on progenitor cells for the lung, particularly diVerentiation and cell fusion using an ex vivo cell culture system (Prockop, 2002, 2003; Prockop et al., 2003). Scott H. Randell, from the University of North Carolina, presented interesting results supporting the idea that there are tracheal stem cell niches in the upper airway epithelium (Borthwick et al., 2001; Neuringer et al., 2002). This is important because these stem cells would be critical targets for cystic fibrosis gene therapy. Barry Stripp from Pittsburgh also talked about stem/progenitor cells in the intrapulmonary conducting airways as a source for repopulation of the Clara cells following naphthalene-induced injury (Giangreco et al., 2002, 2003; Hong et al., 2003, 2004; Peake et al., 2000; Reynolds et al., 2000). Dr. Stripp discussed the biology of stem cell niches within the conducting airway epithelium and the potential to manipulate them both for cell-based and for gene-based therapy in the airway epithelium. Alice Tarantal of the University of California at Davis highlighted the current high interest in gene delivery to the lung epithelium as a strategy for gene delivery to the regenerative cells of the airway epithelium (Lee et al., 2004; Tarantal et al., 2001). The contributions elsewhere in this volume by Drs. Bishop, Hsia, Sunday, and Stripp provide further details regarding some of these key issues. The audience discussion following these interesting presentations that are not included in this volume, including those by Darwin Prockop, Barbara Driscoll, Allan Fine and Diane Krause, and Scott Randell, brought up some interesting additional points and contexts. Drs. Fine and Krause discussed the increasing evidence for plasticity of systemic stem/progenitor cells and their ability to contribute to the repair of injured lung. The molecular mechanisms remain to be fully elucidated, and there is much to be done in terms of optimization. Dr. Krause discussed the critical issue of cell fusion

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and suggested that this appears to be more of a theoretical than an actual problem. However, even if cell fusion turns out to occur frequently, it was pointed out that this would also be interesting and possibly important as a genome repair mechanism, as has been suggested in Purkinje cell damage models (Alvarez-Dolado et al., 2003; Kozorovitskiy and Gould, 2003; Weimann et al., 2003). Dr. Driscoll discussed the evidence in favor of the existence of a resident population of progenitor cells within adult rat alveoli that can be activated to repair injured alveolar epithelium. Drs. Fine and Stripp have also isolated a ‘‘side’’ population of lung cells using FACS criteria, and this stem/progenitor cell population appears to have pluripotential properties (Giangreco et al., 2004; Summer et al., 2003). Scott Randell discussed the importance and relevance of tracheal stem cell niches to cell-based therapy. Darwin Prockop advocated strongly for increased emphasis on the translational aspects of stem/progenitor cell research in the lung, as he felt that the technology was becoming mature and relatively well established.

IV. Summary and Conclusions The discovery that stem/progenitor cells derived from the bone marrow or fat may sometimes contribute to the repair of the lung, liver, and brain as well as of the blood itself has opened up broad new prospects for therapeutic innovation. Presenters and discussants at this meeting advocated parallel basic and translational eVorts to rapidly advance the field of lung stem/ progenitor cell research. On the basic side, much more work will be required not only to characterize both resident and systemic stem/progenitor cells but also to characterize the signaling mechanisms that regulate their function in lung morphogenesis, injury repair, and regeneration. In parallel, a strong push is needed in translational research to realize the potential benefits of lung stem/progenitor cell therapy for lung repair and regeneration, as well as the considerable potential for gene- and cell-based therapy for genetic lung disease. The possibility that the lung epithelium may contain endogenous populations of stem-like progenitor cells, both in the basal layers of the upper airways, within neuroepithelial bodies, and in the alveolar epithelium, has been suggested to account for the ability of the epithelium to repair following injury. It has further been suggested that these stem/progenitor cells may contribute to lung growth during development and to regeneration of lung tissue following lung injury as well as following lung resection. Reports have further indicated that circulating stem cells arising from the bone marrow or other sites such as fat may contribute to repair and regeneration of

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lung epithelium. Further, the eventual failure of these stem/progenitor cells during senescence or disease has been suggested to account for the decline in pulmonary diVusion capacity with advancing age and disease pathology. The susceptibility of stem/progenitor cells to preserve genetic alterations caused by toxins such as cigarette smoke, because of their relative longevity, has also been postulated to account for the clonal nature of lung cancers. Currently, the therapeutic options available to the increasing number of patients with end stage lung disease are limited to cadaveric heart/lung or living related donor lobar transplantation. Both of these options are now more often than not immediately life-saving but are done relatively infrequently due to donor shortages. Moreover, these transplants require life-long immunosuppression and may remain viable for only a limited period of time. The best results have been obtained with living related donor lobes, but even then the mortality is 50% within 5 years, and the longest reported survival so far is for 10 years (Bowdish et al., 2003; Sritippayawan et al., 2002; Starnes et al., 2004). Thus, autologous lung or well-matched stem cell-based lung regeneration therapy would be a boon to many suVering from primary or secondary lung alveolar epithelial and vascular hypoplasia and hence critical gas diVusion deficiency. Similarly, cell-based therapy for genetic diseases such as cystic fibrosis or a1-antitrypsin deficiency could hold the promise of preventing the accelerated deterioration of lung function. In addition, the appreciation that the lung contains populations of stem/progenitor cells that can repair the respiratory epithelium is complemented by the discovery of so-called side populations of cells that can be isolated from the lung, based on a specific pattern of FACS, and that can repopulate other organs, such as the blood. Conversely, the discovery that stem/progenitor cells from the bone marrow or fat may under certain conditions contribute to repair of the lung, heart, liver, and brain as well as the blood itself has opened up broad and exciting new prospects for therapeutic innovation.

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Lessons from a Canine Model of Compensatory Lung Growth Connie C. W. Hsia Department of Internal Medicine, Pulmonary and Critical Care Medicine University of Texas Southwestern Medical Center Dallas, Texas 75390

I. Structure–Function Considerations During Compensation A. Epiphyseal Closure and Growth B. Complexity, Reserves, and Regenerative Potential C. Mechanisms of Compensatory Alveolar Growth II. Dysanaptic Lung Growth A. DiVerent Growth Rates Between Conducting Structures and Parenchyma B. DiVerent Rates of Septal and Capillary Growth C. Dysanaptic Growth Impairs Functional Compensation III. Signals for Compensatory Lung Growth IV. Application to Human Lung Growth Acknowledgments References

For over a century, canines have been used to study adaptation to surgical lung resection or pneumonectomy (PNX) that results in a quantifiable and reproducible loss of lung units. As reviewed by Schilling (1965), the first successful experimental pneumonectomies were performed in dogs and rabbits in 1881. By the early 1920s, it was appreciated that dogs can function normally with one remaining lung that increases in volume to fill the thoracic cavity (Andrus, 1923; Heuer and Andrus, 1922; Heuer and Dunn, 1920); these pioneering observations paved the way for surgeons to perform major lung resection in patients. Reports in the 1950s (Schilling et al., 1956) detail surprisingly well-preserved work performance in dogs following staged resection of up to 70% of lung mass. Since then, the bulk of the literature on post-PNX adaptation has shifted to rodents, especially for defining molecular mediators of compensatory lung growth. Because rodents are smaller and easier to handle, more animals can be studied over a shorter duration, resulting in time and cost savings. On the other hand, key aspects of lung anatomy, development, and time course of response in the rodent do not mimic those in the human subject, and few rodent studies have related structural adaptation to functional consequences. In larger mammals, anatomical lung development more closely resembles that in Current Topics in Developmental Biology, Vol. 64 Copyright 2004, Elsevier Inc. All rights reserved. 0070-2153/04 $35.00

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humans, and physiological function can be readily measured. Because dogs are natural athletes, functional limits of compensation can be characterized relatively easily by stressing oxygen transport at peak exercise. Thus, the canine model remains useful for relating structure to function, defining sources and limits of adaptation as well as evaluating therapeutic manipulation. This chapter summarizes key concepts of compensatory lung growth that have been consolidated from canine studies: (i) structure–function relationships during adaptation, (ii) dysanaptic (unequal) nature of compensation, and (iii) signals for initiation of cellular growth. C 2004, Elsevier Inc.

I. Structure–Function Considerations During Compensation The major diVerences in lung structure and function between large and small mammals are outlined in Table I. The fundamental anatomical unit of mammalian gas exchange is the acinus, which is subtended by a terminal bronchus and in humans contains several generations of respiratory bronchioles and alveolar ducts eventually leading to the alveolar sac. Acinar architecture is less stratified in the small rodent lung than in large mammalian lungs. For example, respiratory bronchioles in rodents are extremely short (Juhos et al., 1980), and the tracheo-bronchial capacitance vessels in murine lungs do not penetrate into intrapulmonary airways (Mitzner et al., 2000; Widdicombe, 1996). Stratification modulates physiological function such as distribution of ventilation and perfusion as well as deposition of inhaled particles or pathogens. Intra-acinar oxygen transport from the terminal bronchiole to the alveolar blood–gas interface occurs primarily by diVusion. Theoretically, the short pathway length within rodent acini is more eYcient for diVusive gas transport to the lung periphery (Sapoval et al., 2002), but it may impose a less eVective barrier to deposition of inhaled particles. In large athletic species such as dogs, an abundance of interalveolar pores facilitates

Table I Small and Large Animal Models of Compensatory Lung Growth

Lung stratification Physiological reserves Epiphyseal closure Size of rib cage Size of lung Time course of compensation

Rodent

Large mammal

Simple Small Incomplete Never fixed Never fixed Days to weeks

Complex Large Complete Fixed at maturity Fixed at maturity Months

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gas mixing among peripheral lung units to compensate for a longer path length for intra-acinar diVusion. A. Epiphyseal Closure and Growth In the rodent, epiphyses do not close and somatic growth continues through life (Dawson, 1934; van der Eerden et al., 2002). Consequently, the rib cage does not reach a fixed size, and there is no stable upper limit to the size of the normal lung. Compensatory lung growth in the rodent could potentially occur via amplification of developmental pathways that remain active through life. In larger mammals, the rib cage reaches maximum dimensions at maturity when epiphyses close, signaling an end to somatic development; the lung also reaches its stable adult dimensions and stops growing. Postpneumonectomy (PNX) lung growth in the adult large mammal involves reinitiating growth-related pathways to modify an existing highly diVerentiated structural scaVold. Reinitiation may not necessarily engage the same developmental pathways, and higher signal intensity is likely required to trigger quiescent pathways of growth than to perpetuate persistently active pathways (see later discussion). This important interspecies diVerence may explain why lung growth is so easily enhanced in rodents but is much more diYcult to manipulate in large mammals. B. Complexity, Reserves, and Regenerative Potential The rodent lung possesses smaller physiological reserves (or safety factors) for oxygen transport that are required to meet sudden and unexpected increases in metabolic demands. Utilization of physiological reserves depends on the ability to increase eVective surfaces at the alveolar–capillary–erythrocyte interface for gas exchange, via opening and distention of capillaries, unfolding of alveolar wall, or a uniform flow and distribution of capillary erythrocytes. [For a detailed discussion on gas exchange reserves see Hsia (2002).] In small lungs, these physiological reserves are easily exhausted in the face of injury or disease; the only avenue to compensate for the loss of functioning lung units is through growth of additional alveolar-capillary units. A vigorous cellular reaction of the rodent lung to even mild challenges constitutes one reason for its popularity as an experimental model. Following even a modest loss of lung mass (one to two lobes), cellular proliferative activities in the remaining lung escalate immediately to regenerate suYcient new alveolar-capillary tissue within 2–3 weeks to fully reconstitute the initial loss (Burri and Sehovic, 1979; Rannels et al., 1984). The intense post-PNX cellular activities markedly heighten susceptibility of the remaining lung to

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carcinogenesis (Brown et al., 1999) and tumor metastasis (Brown et al., 2002a,b), as well as pharmacological manipulation via a variety of growth factors (Kaza et al., 2000, 2001, 2002; Sakamaki et al., 2002; Yuan et al., 2002). Rapid structural compensation is possible because a relatively simple three-dimensional acinar architecture needs to be reconstituted from the regenerated alveolar tissue. Adaptation in larger mammalian lungs necessarily involves additional tiers of complexity. In adult dogs, a much higher threshold of lung resection (50%) must be exceeded before compensatory lung growth is triggered. Below this threshold, as following resection of the smaller left lung (42–45% of total), large existing physiological reserves for oxygen transport in the remaining lung can easily be recruited to meet metabolic demands without invoking cellular growth (Hsia et al., 1993a). Following removal of the larger right lung (55–58% resection) (Hsia et al., 1994), compensatory alveolar growth is invoked, but its time course spans several months, ultimately restoring approximately half of the alveolar-capillary surface areas and septal tissue volume that had been lost. In contrast, growing young dogs demonstrate more vigorous compensatory growth of alveolar tissue following PNX that completely normalizes alveolar septal tissue volume, gas exchange function, and aerobic capacity upon reaching somatic maturity (Takeda et al., 1999a). Thus, coexisting maturational signals and/or mediators greatly intensify post-PNX compensatory alveolar growth. The relative increase in volumes of septal cells (epithelium, interstitium, endothelium) and capillary blood relative to the same lung in normal animals is consistently larger in animals pneumonectomized as puppies (2.5-fold) than in adult animals (1.5-fold), with the exception that volume of type II pneumocytes increases to the same extent in both mature and immature animals (2.3- and 2.1-fold, respectively) (Takeda et al., 1999a; Table II). Data indirectly support a role for type II pneumocytes as potential progenitor cells in the reinitiated growth response.

C. Mechanisms of Compensatory Alveolar Growth Following PNX in young canines, new alveolar tissue is generated at least partly via cell division, associated with elevated levels of proliferating cell nuclear antigen (PCNA) in lung tissue (Foster et al., 2002), although cell hypertrophy may also occur. Limited evidence in these animals suggests that cellular pathways of compensatory alveolar growth do not represent mere reactivation of developmental events. During normal postnatal maturation, enhanced alveolar cell proliferation parallels an increase in epidermal growth factor (EGF) and receptor (EGFR) levels and correlates inversely

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Table II Fold Increase in Alveolar Septal Volumes in Immature and Mature Dogs Following Right PNX Relative to Respective Sham Controls Fold increase in volume per kilogram body weight Epithelium Type I Type II Interstitium Endothelium Capillary blood

Immature

Mature

2.6 2.7 2.3 2.8 2.6 2.7

1.7a 1.6a 2.1 2.0a 1.6a 1.4a

a p < 0.05 between mature and immature groups. From Takeda et al. (1999a).

with surfactant protein-A (SP-A) and pro-SP-C levels. However, post-PNX alveolar growth has no clear association with EGF or EGFR, but correlates with a markedly increased SP-A level and moderately increased SP-D level, while pro-SP-B and pro-SP-C levels do not change significantly (Foster et al., 2002). How the divergent patterns of cellular response relate to diVerent signals for growth initiation during maturation and compensation remains to be clarified. New alveolar septa may arise from secondary crests along existing alveolar walls and subdivide an alveolus. Other mechanisms to further increase surface area of the blood–gas interface may coexist but are less well understood. For instance, respiratory bronchioles undergo compensatory dilation and proliferation in proportion to the post-PNX increase in alveolar tissue volume (Hsia et al., 2000); new alveoli or alveolar ducts could arise directly from these respiratory bronchioles. Alternatively, the three-dimensional folding pattern of enlarging alveolar sacs could undergo rearrangement with deposition of connective tissue elements at selective sites to create a new branch of alveolar duct. Branching of a terminal alveolar duct or respiratory bronchiole could eVectively account for the near doubling of alveolar volume while maintaining a normal acinar morphology post-PNX. One mechanism for generating new alveolar capillaries in the presence of an already highly organized microvascular network is via the process of intussusception (Djonov et al., 2000), whereby tissue pillars grow into the lumen of an existing capillary, eventually dividing the capillary into two segments. Repetition of this process accompanied by remodeling of tissue components results in the formation of new branching capillaries (Fig. 1). Intussusception has been extensively studied in the chick chorioallantoic membrane (Djonov et al., 2000) and is probably prevalent in the embryonic formation and postnatal maturation of both micro- and macrovasculature

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Figure 1 How capillaries can proliferate by intussusception during compensatory alveolar growth. A tissue pillar extends into an existing capillary (A) and eventually transects the capillary into two (B), resulting in a ‘‘double capillary’’ alveolar profile on two-dimensional micrographs that is more frequently seen in growing lungs. Further tissue remodeling lengthens the septum and reconstitutes the ‘‘single capillary’’ alveolar profile typical of the adult lung (C). See text for discussion.

(CaduV et al., 1986; Kurz et al., 2003). Because intussusception does not require intense endothelial cell proliferation, it is distinct from angiogenesis by sprouting. There is indirect evidence supporting compensatory alveolar capillary growth by intussusception in dogs (Yan et al., 2004). The transcapillary tissue pillar confers a ‘‘double capillary’’ profile to the two-dimensional cross-section of the alveolar septum on micrographs, which is typical of growing or immature lungs, while normal alveolar profile in adult dog lungs consists predominantly of ‘‘single capillaries’’ along the length of the septum. Following right PNX in adult dogs, the prevalence of double capillary profiles is approximately 50% higher compared to sham controls; the prevalence further increases by nearly 100% in pneumonectomized animals treated with all-trans-retinoic acid, which preferentially enhanced the compensatory increase in capillary and endothelial cell volume (Fig. 2; Yan et al., 2004). In the absence of compensatory alveolar growth following less extensive lung resection by left PNX, the prevalence of double capillaries is unchanged from normal.

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Figure 2 Prevalence of double capillary profiles along alveolar septa in adult dogs following left PNX (L-PNX, 42–45% of lung removed), right PNX (R-PNX, 55–58% of lung removed) or R-PNX plus all-trans-retinoic acid treatment (R-PNX-RA) compared to sham controls. Expressed as a percentage (mean SEM) of total alveolar capillary profiles measured using systematic linear intercepts. *, p < 0.0005 vs all other groups.

II. Dysanaptic Lung Growth A. Different Growth Rates Between Conducting Structures and Parenchyma To achieve optimal function, growth rates of structural components within the lung should be matched, that is, there should be equally vigorous adaptation of alveoli, airways, and blood vessels and proportional increases in volumes of constituent cell types within alveolar septa. Matched adaptation is not always possible, as these structures and cells derive from diVerent embryonic origins and their postnatal plasticity is constrained by divergent signals and mediators. Because conducting airways and blood vessels do not branch further after birth, these structures adapt only by elongation and dilatation while alveoli continue to subdivide and increase in surface area through early childhood. Thus, postnatal growth and adaptation of conducting structures normally lag behind those of the alveoli (Green et al., 1974; Hibbert et al., 1995; Hopper et al., 1991). This discrepancy, termed dysanaptic (i.e., unequal) growth, is developmentally accentuated in the highly stratified lungs of large mammals and is further exaggerated during accelerated alveolar growth such as that induced by high-altitude residence (Brody et al., 1977) or major lung resection (Greville et al., 1986; McBride et al., 1980; Werner et al., 1993).

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In small animals after pneumonectomy, volume and cross-sectional area of the remaining conducting airways increase less than the volume of the parenchyma (Burri and Sehovic, 1979; Kirchner and McBride, 1990; McBride, 1985). In pneumonectomized dogs, conducting airways initially elongate with little change in diameter; dilatation occurs slowly beyond 4 months after PNX. Pneumonectomy reduces total airway cross-sectional area by 50% and doubles airflow to the remaining lung at any workload. Given that laminar flow resistance in an airway is directly proportional to its length and inversely proportional to the 2nd power of its cross-sectional area (Poiseuille’s Law), the initial airway lengthening worsens the expected post-PNX increase in airflow resistance, while the subsequent airway dilatation partially mitigates the increase (Dane et al., 2002; Takeda et al., 1999b). As a result of the disparity in plasticity between conducting structures and parenchyma, significantly greater long-term abnormalities persist in airway mechanics and vascular hemodynamics than in alveolar gas exchange, regardless of somatic maturity of the animal at the time of PNX (Takeda et al., 1999b). As more lung units are resected, alveolar growth intensifies, while airway/vascular compensation remains slow and incomplete, thereby widening the functional disparity. In dogs following extensive staged resection of up to 68% of lung mass, exaggerated impairment in lung mechanical and hemodynamic function imposes the major limitation to exercise (D. M. Dane, C. C. W. Hsia, R. L. Johnson, Jr., unpublished observations). Similarly persistent airwayparenchymal dissociation has also been documented in children following major lung resection (McBride et al., 1980; Werner et al., 1993).

B. Different Rates of Septal and Capillary Growth Another type of dysanaptic growth occurs when alveolar septal components grow at diVerent rates (Fig. 3). For example, administration of all-transretinoic acid (RA) for 4 months to adult dogs following right PNX during the period of active alveolar growth selectively enhances compensatory increases in alveolar capillary and endothelial cell volumes associated with an increased prevalence of double capillary profiles in comparison with placebotreated controls; these changes are consistent with RA-enhanced capillary growth (Yan et al., 2004). However, there is no significant increase in volume of the epithelium or interstitium or in alveolar-capillary surface area. As a result, the alveolar surface-to-volume ratio declines, suggesting loss of surface folding or a change in three-dimensional alveolar geometry. Because surface area and the harmonic mean barrier for diVusion are unchanged, diVusing capacity of the alveolar membrane estimated from these structural parameters is not diVerent from that in placebo-treated controls; the latter finding is corroborated by ante mortem measurement of diVusing capacity

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Figure 3 Exogenous all-trans-retinoic acid (RA) treatment enhances compensatory increases in alveolar capillary and endothelial cell volumes relative to placebo treatment without significant enhancement of other septal compartments or gas exchange surface area. *, p < 0.05 RA vs placebo (ratio diVerent from 1.0). From data in Yan et al. (2004).

using physiological methods in the same animals (Dane et al., 2004). DiVusing capacity measured by physiological methods was abnormally low in RAtreated animals at a low lung volume but not at a high lung volume, suggesting altered airway function (i.e., distribution of ventilation) associated with RA treatment, although the eVects of RA on airway resistance or morphology remain to be fully characterized. In the absence of alveolar growth following left PNX in adult dogs, RA treatment has no eVect on lung function, volume, morphology, or ultrastructure (Dane et al., 2003). Thus, RA appears to facilitate existing compensatory growth, but it is unable to initiate compensatory growth in the absence of an independent signal for growth. C. Dysanaptic Growth Impairs Functional Compensation The preceding canine studies exemplify the paramount importance of maintaining a balanced response at all levels of structural organization during compensatory lung growth in order to translate cell growth into enhanced lung function. They point out an inherent caveat when attempting to manipulate lung growth by pharmacological means. Because no single drug or growth factor can synchronously activate all the biochemical pathways

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necessary for the coordinated natural response, selective stimulation of one or even several pathways will likely lead to anatomical and/or physiological distortions that could oVset the expected benefit. Administration of pharmacological doses to induce dramatic short-term increases in a few biochemical mediators is inherently unrealistic, since a sustained mild to modest elevation of a multitude of mediators is the more likely physiological pattern, especially during the extended time course of compensation seen in large animals (Foster et al., 2002). This caveat applies even to the transplantation of pleuripotent stem cells to augment lung growth, as the micro- and macroenvironment surrounding the transplanted cells must provide discriminating signals to appropriately direct their diVerentiation into separate lineages that develop into all the structural components of a regenerated lung as well as perpetuate their subsequent proliferation and growth.

III. Signals for Compensatory Lung Growth A logical approach is to identify the endogenous signals involved in initiating compensatory lung growth and attempt to amplify those signals at a susceptible stage during growth. Mechanical stress (force) causing tissue strain (deformation) is believed to be a major general signal triggering a cascade of biochemical and molecular alterations that mediate compensatory cellular growth in many organs. Cellular growth in turn relieves stress and ultimately limits further organ growth. Following PNX, the negative intrathoracic pressure imposes greater mechanical stress on the remaining lung as it gradually expands across the midline, increasing its volume by 90% over several days (Fig. 4). Intrathoracic stress across the remaining lung is not uniformly distributed, resulting in diVerent degrees of expansion in diVerent lobes. As early as 1939, Cohn reported that plombage material inserted into the empty thoracic space prevented post-PNX lung expansion in rats (Cohn, 1939). Plombage impairs compensatory increase in DNA synthesis (Brody et al., 1978) and mitotic index (Fisher and Simnett, 1973) in rodents but causes the remaining lung in rabbits to change shape and elongate in the caudal direction (Olson and HoVman, 1994), suggesting that mechanical strain may not be the only signal for adaptation. To quantify the contribution of strain-related compensatory response, we inserted inflatable silicone prosthesis in the shape and volume of the normal canine right lung into the empty hemithorax of adult dogs at the time of right PNX and kept the prosthesis either inflated or deflated for approximately one year (Hsia et al., 2001, 2003; Wu et al., 2000). The inflated prosthesis kept the mediastinum at the midline, minimizing alveolar septal strain, and lateral expansion of the remaining lung. The deflated prosthesis allowed mediastinal shift across the midline, increased alveolar septal strain, and lateral expansion of the remaining lung.

Figure 4 Thoracic image obtained by computerized tomographic scan at the level of tracheal bifurcation from adult dogs after left (left) or right PNX (right) in comparison with a normal animal (sham, center). From Ravikumar et al. (2004).

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Subsequent evaluation showed significantly greater impairment in exercise performance, maximal oxygen uptake, and lung-diVusing capacity in animals with inflated prosthesis compared to pneumonectomized control animals with deflated prosthesis (Hsia et al., 2003; Wu et al., 2000). At postmortem, the expected compensatory increase of alveolar septal cell volumes was blunted in the remaining left lung of animals with inflated prosthesis, although they remained significantly higher than that in a normal left lung (Hsia et al., 2001; Fig. 5). Thus, minimizing alveolar septal strain impaired but did not completely abolish compensatory growth of alveolar tissue. Though unable to expand across the midline in the presence of inflated prosthesis, the remaining lung enlarged 20% via caudal displacement of the left hemidiaphragm and outward displacement of the left lower rib cage (Wu et al., 2000). Overall, minimizing lung strain consistently reduced absolute physiological and anatomical indices by 30%, that is, diminishing the compensatory increase in these indices per unit of remaining lung by 70%. These data illustrate the importance of chronic alveolar septal strain as a signal for compensatory growth. However, the prosthesis did not prevent the post-PNX increase in tidal expansion of the remaining lung with respiration or the increase in alveolar capillary blood flow that leads to capillary distention and shear in the remaining lung. These remaining mechanical forces could potentially explain the residual 20–30% compensation not abolished by the inflated lung prosthesis. Alternatively, nonmechanical signals, such as intermittent exercise-induced alveolar hypoxia and humoral or

Figure 5 Minimizing alveolar septal strain by inflated (INF) lung prosthesis impairs septal tissue volume (left) and alveolar-capillary surface area (right) in the remaining lung of adult dogs following right PNX relative to control animals with deflated (DEF) prosthesis following right PNX or sham controls. p < 0.05: *, vs sham left lung; #, vs sham both lungs, {, vs deflated prosthesis. From data in Wu et al. (2000), and reprinted with permission from Hsia et al. (2001).

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local growth factors, could modulate compensation, but these factors are unlikely to be the main instigator of the growth response.

IV. Application to Human Lung Growth Compensatory lung growth probably does not occur to any significant degree in adult patients following PNX; a possible explanation is suggested by comparison of human and canine studies. In adult patients following pneumonectomy, expansion of the remaining lung is more limited than in pneumonectomized canines, which can be attributed to the persistence of significant postoperative pleuro-mediastinal fibrosis and adhesions in patients that restrict mobility of the lung, heart, diaphragm, and rib cage (Hijazi et al., 1998; Hsia et al., 1993b). Thus, the anticipated increase in chronic alveolar septal strain in the remaining lung is also limited and probably never exceeds the threshold required for reinitiation of alveolar cellular growth. In patients with relatively normal remaining lungs, functional compensation of the heart, lung, and respiratory muscles lags behind that observed in pneumonectomized canines. Development of fibrous adhesions in patients is likely multifactorial, related to postoperative accumulation of bloody fluid in the thorax, poor lymphatic drainage, or active inflammation and possibly exacerbated by physical inactivity. However, the potential for compensatory alveolar growth clearly exists in humans as in quadrupeds, shown by a relatively normal lung volume and diVusing capacity in long-term follow-up of patients who had undergone lung resection at a young age (Giammona et al., 1966; Laros and Westermann, 1987; Werner et al., 1993) and by the accelerated improvement of lung function in pediatric patients whose lung disease is no longer active (Gerhardt et al., 1987; Marven et al., 1998). It remains possible that compensatory growth could be reinitiated or accelerated in the human lung by (i) amplifying endogenous mechanical signals, for example, enlarging the space available for lung expansion or tidal excursion or by (ii) enhancing the response to mechanical signals, for example, via accelerated activation of resident progenitor cells across a spectrum of lineages. Although these goals continue to challenge investigators, understanding the factors governing structure– function integration in large mammalian lungs should help us formulate rational approaches to augment compensatory responses as well as to translate and apply such strategies to patients with lung disease.

Acknowledgments This work was supported by National Heart, Lung and Blood Institute Grants R01 HL-40070, HL-54060, HL-45716, and HL62873.

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Hibbert, M., Lannigan, A., Raven, J., Landau, L., and Phelan, P. (1995). Gender diVerences in lung growth. Pediatr. Pulmonol. 19, 129–134. Hijazi, O. M., Ramanathan, M., Estrera, A. S., Peshock, R. M., and Hsia, C. C. W. (1998). Fixed maximal stroke index in patients after pneumonectomy. Am. J. Respir. Crit. Care Med. 157, 1623–1629. Hopper, J. L., Hibbert, M. E., Macaskill, G. T., Phelan, P. D., and Landau, L. I. (1991). Longitudinal analysis of lung function growth in healthy children and adolescents. J. Appl. Physiol. 70, 770–777. Hsia, C. C. W. (2002). Recruitment of lung diVusing capacity: Update of concept and application. Chest 122, 1774–1783. Hsia, C. C. W., Fryder-DoVey, F., Stalder-Navarro, V., Johnson, R. L., Jr., and Weibel, E. R. (1993a). Structural changes underlying compensatory increase of diVusing capacity after left pneumonectomy in adult dogs. J. Clin. Invest. 92, 758–764 [Published erratum in J. Clin. Invest. 93(2), 913, 1994.]. Hsia, C. C. W., Herazo, L. F., Fryder-DoVey, F., and Weibel, E. R. (1994). Compensatory lung growth occurs in adult dogs after right pneumonectomy. J. Clin. Invest. 94, 405–412. Hsia, C. C. W., Johnson, R. L., Jr., Wu, E. Y., Estrera, A. S., Wagner, H., and Wagner, P. D. (2003). Reducing lung strain after pneumonectomy impairs diVusing capacity but not ventilation-perfusion matching. J. Appl. Physiol. 95, 1370–1378. Hsia, C. C. W., Peshock, R. M., Estrera, A. S., McIntire, D. D., and Ramanathan, M. (1993b). Respiratory muscle limitation in patients after pneumonectomy. Am. Rev. Respir. Dis. 147, 744–752. Hsia, C. C. W., Wu, E. Y., Wagner, E., and Weibel, E. R. (2001). Preventing mediastinal shift after pneumonectomy impairs regenerative alveolar tissue growth. Am. J. Physiol. Lung Cell. Mol. Physiol. 281, L1279–L1287. Hsia, C. C. W., Zhou, X. S., Bellotto, D. J., and Hagler, H. K. (2000). Regenerative growth of respiratory bronchioles in dogs. Am. J. Physiol. Lung Cell. Mol. Physiol. 279, L136–L142. Juhos, L. T., Green, D. P., Furiosi, N. J., and Freeman, G. (1980). A quantitative study of stenosis in the respiratory bronchiole of the rat in NO2-induced emphysema. Am. Rev. Respir. Dis. 121, 541–549. Kaza, A. K., Kron, I. L., Kern, J. A., Long, S. M., Fiser, S. M., Nguyen, R. P., Tribble, C. G., and Laubach, V. E. (2001). Retinoic acid enhances lung growth after pneumonectomy. Ann. Thorac. Surg. 71, 1645–1650. Kaza, A. K., Kron, I. L., Leuwerke, S. M., Tribble, C. G., and Laubach, V. E. (2002). Keratinocyte growth factor enhances post-pneumonectomy lung growth by alveolar proliferation. Circulation 106, I120–I124. Kaza, A. K., Laubach, V. E., Kern, J. A., Long, S. M., Fiser, S. M., Tepper, J. A., Nguyen, R. P., Shockey, K. S., Tribble, C. G., and Kron, I. L. (2000). Epidermal growth factor augments postpneumonectomy lung growth. J. Thorac. Cardiovasc. Surg. 120, 916–921. Kirchner, K. K., and McBride, J. T. (1990). Changes in airway length after unilateral pneumonectomy in weanling ferrets. J. Appl. Physiol. 68, 187–192. Kurz, H., Burri, P. H., and Djonov, V. G. (2003). Angiogenesis and vascular remodeling by intussusception: From form to function. News Physiol. Sci. 18, 65–70. Laros, C. D., and Westermann, C. J. J. (1987). Dilatation, compensatory growth, or both after pneumonectomy during childhood and adolescence. A thirty-year follow-up study. J. Thorac. Cardiovasc. Surg. 93, 570–576. Marven, S. S., Smith, C. M., Claxton, D., Chapman, J., Davies, H. A., Primhak, R. A., and Powell, C. V. (1998). Pulmonary function, exercise performance, and growth in survivors of congenital diaphragmatic hernia. Arch. Dis. Child 78, 137–142. McBride, J. T. (1985). Postpneumonectomy airway growth in the ferret. J. Appl. Physiol. 58, 1010–1014.

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McBride, J. T., Wohl, M. E., Strieder, D. J., Jackson, A. C., Morton, J. R., Zwerdling, R. G., Griscom, N. T., Treves, S., Williams, A. J., and Schuster, S. (1980). Lung growth and airway function after lobectomy in infancy for congenital lobar emphysema. J. Clin. Invest. 66, 962–970. Mitzner, W., Lee, W., Georgakopoulos, D., and Wagner, E. (2000). Angiogenesis in the mouse lung. Am. J. Pathol. 157, 93–101. Olson, L. E., and HoVman, E. A. (1994). Lung volumes and distribution of regional air content determined by cine x-ray CT of pneumonectomized rabbits. J. Appl. Physiol. 76, 1774–1785. Rannels, D. E., Burkhart, L. R., and Watkins, C. A. (1984). EVect of age on the accumulation of lung protein following unilateral pneumonectomy in rats. Growth 48, 297–308. Ravikumar, P., Yilmaz, C., Dane, D. M., Johnson, R. L., Jr., Estrera, A. S., and Hsia, C. C. W. (2004). Regional lung growth following pneumonectomy assessed by computed tomography. J. Appl. Physiol. 97, 1567–1574. Sakamaki, Y., Matsumoto, K., Mizuno, S., Miyoshi, S., Matsuda, H., and Nakamura, T. (2002). Hepatocyte growth factor stimulates proliferation of respiratory epithelial cells during postpneumonectomy compensatory lung growth in mice. Am. J. Respir. Cell. Mol. Biol. 26, 525–533. Sapoval, B., Filoche, M., and Weibel, E. R. (2002). Smaller is better—but not too small: A physical scale for the design of the mammalian pulmonary acinus. Proc. Natl. Acad. Sci. USA 99, 10411–10416. Schilling, J. A. (1965). Pulmonary resection and sequelae of thoracic surgery. In ‘‘Handbook of Physiology. Section 3: Respiration.’’ (W. O. Fenn and H. Rahn, Eds.), pp. 1531–1563. American Physiological Society, Washington, D.C. Schilling, J. A., Harvey, R. B., Balke, B., and Rattunde, H. F. (1956). Extensive pulmonary resection in dogs: Altitude tolerance, work capacity, and pathologic-physiologic changes. Ann. Surg. 144, 635–646. Takeda, S., Hsia, C. C. W., Wagner, E., Ramanathan, M., Estrera, A. S., and Weibel, E. R. (1999a). Compensatory alveolar growth normalizes gas exchange function in immature dogs after pneumonectomy. J. Appl. Physiol. 86, 1301–1310. Takeda, S., Ramanathan, M., Estrera, A. S., and Hsia, C. C. W. (1999b). Postpneumonectomy alveolar growth does not normalize hemodynamic and mechanical function. J. Appl. Physiol. 87, 491–497. van der Eerden, B. C., Gevers, E. F., Lowik, C. W., Karperien, M., and Wit, J. M. (2002). Expression of estrogen receptor alpha and beta in the epiphyseal plate of the rat. Bone 30, 478–485. Werner, H. A., Pirie, G. E., Nadel, H. R., Fleisher, A. G., and LeBlanc, J. G. (1993). Lung volumes, mechanics, and perfusion after pulmonary resection in infancy. J. Thorac. Cardiovasc. Surg. 105, 737–742. Widdicombe, J. (1996). The tracheobronchial vasculature: A possible role in asthma. Microcirculation 3, 129–141. Wu, E. Y., Hsia, C. C., Estrera, A. S., Epstein, R. H., Ramanathan, M., and Johnson, R. L., Jr. (2000). Preventing mediastinal shift after pneumonectomy does not abolish physiologic compensation. J. Appl. Physiol. 89, 182–191. Yan, X., Bellotto, D. J., Foster, D. J., Johnson, R. L., Jr., Hagler, H. K., Estrera, A. S., and Hsia, C. C. W. (2004). Retinoic acid induces non-uniform alveolar septal growth after right pneumonectomy. J. Appl. Physiol. 96, 1080–1089. Yuan, S., Hannam, V., Belcastro, R., Cartel, N., Cabacungan, J., Wang, J., Diambomba, Y., Johnstone, L., Post, M., and Tanswell, A. K. (2002). A role for platelet-derived growth factor-BB in rat postpneumonectomy compensatory lung growth. Pediatr. Res. 52, 25–33.

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Airway Glandular Development and Stem Cells Xiaoming Liu,* Ryan R. Driskell,* and John F. Engelhardt *,{ *Department of Anatomy and Cell Biology College of Medicine, The University of Iowa Iowa City, Iowa 52242 { Department of Internal Medicine, and Center for Gene Therapy of Cystic Fibrosis and Other Genetic Diseases College of Medicine, The University of Iowa Iowa City, Iowa 52242

I. Introduction and Scope II. Airway Submucosal Gland Structure/Function and Its Relationship to Human Diseases III. Species-Specific DiVerences in Conducting Surface Airway Epithelial Cell Types and Submucosal Gland Abundance IV. Evidence for Human Adult Proximal Airway Stem Cells Capable of Developing Submucosal Glands V. Mouse Tracheal Injury Models Suggest Submucosal Gland Ducts Are a Niche for Proximal Airway Stem Cells VI. Developmental Molecular Markers of Glandular Stem Cell Phenotypes VII. Concluding Remarks and Future Challenges Acknowledgments References

Submucosal glands in the lung play important roles in several hypersecretory lung disease processes, including chronic bronchitis, asthma, and cystic fibrosis. In this context, submucosal glands undergo abnormal growth and diVerentiation through processes that are poorly understood. To better understand the pathophysiological mechanisms that lead to submucosal gland hypertrophy and hyperplasia in the adult human lung, eVorts have been made to dissect the molecular signals and cell types responsible for normal submucosal gland development in the airway. Such studies have revealed a close relationship between progenitor/stem cell phenotypes in the surface airway epithelia and submucosal glands, and thus it has been suggested that submucosal glands serve as a protective niche for surface airway epithelial stem cells. Furthermore, the pluripotent progenitor cells that exist in the surface airway epithelium, which have the capacity to Current Topics in Developmental Biology, Vol. 64 Copyright 2004, Elsevier Inc. All rights reserved. 0070-2153/04 $35.00

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diVerentiate into ciliated, secretory, intermediate, and basal cells, also have a developmental capacity for submucosal glands. This putative adult stem cell compartment of the airway epithelium has been the focus of research attempting to identify molecular markers for signaling pathways that control stem cell phenotypes and their capacity for proliferation and diVerentiation following airway injury. C 2004, Elsevier Inc.

I. Introduction and Scope Submucosal glands in the tracheobronchial airways play potentially important roles in both innate immunity of the lung and cell biology of the proximal airways. Submucosal glands are major secretory structures that reside in the interstitium beneath the cartilaginous airways of many mammalian species (JeVery, 1983) and that have a continuous epithelium with the surface airway. Several types of epithelial cells are found within airway submucosal glands; some are unique to glands, and others are similar to those found in the surface airway epithelium. However, both the epithelial cell types of the conducting airways and the abundance of submucosal glands that exist in the proximal cartilaginous conducting airways significantly diVer between rodents and humans (JeVery, 1983; Pack et al., 1981; Plopper et al., 1980; Widdicombe et al., 2001). This chapter reviews the functional roles submucosal glands are thought to play in disease processes of the lung and the changes in cell biology of glands associated with these disease processes. Important aspects of speciesspecific diVerences in submucosal gland abundance and the models used to study cell biology of submucosal glands are also addressed. For example, submucosal glands have been suggested to be a protective niche for proximal airway stem cells in the mouse (Borthwick et al., 2001; Engelhardt, 2001). However, because mice have relatively few submucosal glands in their tracheal airways compared to humans, the significance of this finding remains obscure. We review various approaches and models used to study adult surface airway epithelial progenitors and their relationship to submucosal glands. Such models have begun to shape current theories regarding stem cell niches and their molecular phenotypes. For the purposes of this chapter, we refer to candidate adult stem cells in the proximal airway as a cell type that retains the ability to diVerentiate into all surface airway and submucosal gland epithelial cell types. The major goal of this chapter is to provide a foundation for the scientific investigation and future dialogue required to better understand the diversity of the adult stem cell in the proximal airway of diVerent species.

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II. Airway Submucosal Gland Structure/Function and Its Relationship to Human Diseases Airway submucosal glands secrete the fluid, mucous, and bacteriocidal proteins critical for maintaining normal lung function. For example, submucosal glands produce a number of antibacterial proteins, including lactoperoxidase (Wijkstrom-Frei et al., 2003), LL-37 (Bals et al., 1998b; Kim et al., 2003), beta-defensin 1 (Bals et al., 1998a), lysozyme (Klockars and Reitamo, 1975; Wang et al., 2001), and lactoferrin (Raphael et al., 1989), which are all thought to bolster the immunity of the airways against bacterial infection. Furthermore, ex vivo models of airways with and without submucosal glands suggest that the presence of submucosal glands also significantly influences bioelectric and fluid transport properties of the airway (Ballard and Inglis, 2003; Wang et al., 2001). Submucosal glands are also thought to play an important role in the pathogenesis of a number of hypersecretory lung diseases, such as cystic fibrosis (CF), chronic bronchitis, and asthma (Fahy, 2001; Finkbeiner, 1999; JeVery, 1992, 2001; Vignola et al., 2001). A common feature encountered in these diseases is the increased expansion of submucosal glands, which leads to abnormally high levels of mucous production in the airways. The potential involvement of submucosal glands in the etiology of CF pathogenesis is suggested by several findings, including the high level of cystic fibrosis transmembrane conductance regulator (CFTR) expression in submucosal glands (Engelhardt et al., 1992) and the severe hypertrophy and hyperplasia of submucosal glands that is characteristic of the progressing disease (Oppenheimer and Esterly, 1975; Welsh and Smith, 1995). In the normal human airway, submucosal glands are restricted to the cartilaginous airways (the trachea and bronchi), while in CF, the distribution of submucosal glands extends more distally into bronchioles (i.e., hyperplasia) (Oppenheimer and Esterly, 1975). Similar observations of submucosal gland hyperplasia in CF mouse models have also been reported (Borthwick et al., 1999). Alterations in the cellular architecture of submucosal glands also accompany CF airway disease, leading to the increased cell mass of glands (i.e., hypertrophy) with increased numbers of mucous-secreting cell types. Anatomically, submucosal glands are composed of a series of interconnecting tubules and ducts that are localized in the interstitium beneath the surface epithelium (Fig. 1). The most distal regions of the glandular network are composed of serous acini and tubules. Secretory products move vectorially from the distal serous tubules through mucous tubules and then accumulate in collecting ducts. At the proximal end of submucosal glands, collecting and ciliated ducts connect the glandular tubules to

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Figure 1 Cellular diversity of distinct epithelia in the human adult conducting airways of the lung. When discussing progenitor/stem cells in adult airways, the diversity of cell phenotypes that exist in spatially distinct epithelia of the lung must always be considered. Three main levels of conducting airways exist in the lung, including the trachea, bronchi, and bronchioles. Alveoli, which represent distinct regions and epithelia in the lung that function in gas exchanges, are not discussed in this chapter. Predominant cells types in the human pseudostratified, columnar tracheal and bronchial epithelia include basal (B), intermediate (I), goblet (G), and ciliated (C) cells; less abundant nonciliated and neuroendocrine cells are not shown. Submucosal glands, predominantly composed of mucous (Mt) and serous tubules (St), are present in the interstitium of cartilaginous airways (trachea and bronchi). They secrete their contents to the surface airway epithelium (SAE) through a centrally positioned collecting duct (Cd). More distally, the simple columnar epithelia of bronchioles contain two main cell types in most species, Clara (Cl) and ciliated cells (C). (See Color Insert.)

the airway lumen (Meyrick et al., 1969). Each of these spatially distinct regions of submucosal glands has specific cell types both controlling the content and viscosity of secretory products and timing the expulsion of secretions in response to airway irritation and infection (Meyrick et al., 1969; Nadel and Davis, 1980; Tom-Moy et al., 1983; Widdicombe et al., 1997).

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III. Species-Specific Differences in Conducting Surface Airway Epithelial Cell Types and Submucosal Gland Abundance The conducting airways of the adult lung can be subdivided into two functionally and structurally distinct epithelial domains that likely contain unique types of adult epithelial progenitor/stem cells (Fig. 1). These domains are (1) proximal cartilaginous airways (trachea and bronchi) and (2) distal bronchioles (bronchioles, terminal bronchioles, and respiratory bronchioles). Each of these domains has historically been classified by morphological criteria that define the unique type of airway epithelium resident within each domain of the lung (Fig. 1). In the proximal airway epithelium, the major cell types found are ciliated cells, basal cells, intermediate cells, Clara cells, goblet cells, serous cells, and neuroendocrine cells. The distribution and abundance of each of these cell types vary from species to species. In human and nonhuman primate proximal cartilaginous airways, a pseudostratified columnar epithelium is composed of ciliated cells, goblet cells, nonciliated columnar cells, intermediate cells, neuroendocrine cells, and basal cells. Proximal cartilaginous airways also contain an additional anatomical structure contiguous with the surface airway epithelium, the submucosal gland (Fig. 1). Because studies evaluating the role of submucosal glands in stem cell biology in the airway are highly dependent on animal models, consideration of the anatomical and cellular diVerences between rodent and primate conducting airways is critical. In terms of the morphology of cell types residing in the conducting airways of small mammals, mouse airways perhaps diVer the most from human airways. In mice, Clara cells reside throughout the tracheobronchial and bronchiolar epithelium, whereas in humans, Clara cells reside only in the bronchioles (Pack et al., 1980, 1981). Hence, Clara cells in mice are the predominant secretory cell type throughout the conducting airways. In contrast, the predominant secretory cell type in rat conducting airways is the serous cell (JeVery and Reid, 1975). Goblet cells in mice and rats can be induced by specific cytokine stimuli but are not usually present in the pristine mouse airway (Basbaum et al., 1990). Intermediate cells are also very visible in rat and human but are much less visible in mouse proximal conducting airways. (Lee et al., 2002; Zuhdi Alimam et al., 2000). A summary of the cellular diVerences in mouse, rat, human, and ferret conducting airways is given in Table I. Although rodent and primate species significantly diVer in terms of the cell biology of their conducting airway epithelia, they also share many features, including basal cells, ciliated cells, and neuroendocrine cells. It is important to recognize that classification of cell types has historically been based on electron microscopic

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Table I Summary of Predominant Epithelial Cell Types and Submucosal Glands within Various Regions of the Conducting Airways in DiVerent Species Species

Basal

Mouse Rat Human Ferret

T, T, T, T,

B B B B

Intermediate

Goblet

Serous

Clara

NCC

Ciliated

SMG

— T, B T, B T, B

—a —a T, B T, B

— T, B — —

T, B, Br Br Br Br

— T, B T, B T, B

T, T, T, T,

T T T, B T, B

B, B, B, B,

Br Br Br Br

a Summary for rodents applies to pathogen-free animals; the abundance of goblet cells may increase in the setting of infection or cytokine simulation. Abbreviations: T, trachea; B, bronchi; Br, bronchioles; NCC, nonciliated, nonsecretory columnar cells; SMG, submucosal glands.

morphology. This method of determining cell phenotype is only beginning to change as more molecular markers become available for classification. As the field advances in this area, it has become evident that many of the secretory cell type diVerences between human (goblet), mice (Clara), and rat (serous) proximal airways can be modified by inducing injury or creating an inflammatory state. Both of these methods promote changes in secretory cell phenotypes; the induction of goblet cells in mouse and rat airways is one example. Whether this induction occurs through alterations in cell phenotypes of resident secretory cells or through the induction of diVerent progenitor cell diVerentiation profiles remains to be formally proven. The development of submucosal glands in the airway has been examined in the mouse, rat, hamster, guinea pig, rabbit, ferret, and human. The abundance and distribution of submucosal glands throughout the conducting airways vary greatly between species and between diVerent inbred strains of mice (Innes and Dorin, 2001; Widdicombe et al., 2001). The density and distribution of glands in the trachea of diVerent species are summarized in Table II. In the mouse, airway submucosal glands reside in the larynx and proximal trachea, but do not extend below the distal trachea. Mouse nasal submucosal glands begin to develop at E14–15, while tracheal gland development starts during the first postnatal weeks of life. Furthermore, the distribution of submucosal glands in the mouse trachea varies between inbred stains, appearing to be linked to a common genetic determination on chromosome 9 (Innes and Dorin, 2001). Development of hamster submucosal glands begins prenatally, following the same progression as for humans, but glands in the hamster do not achieve the same degree of complexity and density as human submucosal glands (Emura and Mohr, 1975). Submucosal glands in rats are also much less frequent than in human proximal airways, and they have much less extensive tubule network structures (Widdicombe et al., 2001).

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3. Glandular Stem Cells Table II Comparison of the Density and Distribution of Glands in the Airways Density of Glands in Airway (glands per mm 2) Species

Larynx

Trachea

Mouse

0.7

0a

Rat

1.8

0.3

Guinea pig

2.3

0.2

Hamster

ND

0.03 b

Rabbit

4.10–4.70 c

0.8–5.4 d

Human (cartilaginous wall) Human (membranous wall) Ferret

ND

ND

ND

36 26 13 31 10

(GW (GW (GW (GW (GW

ND e

15) 20) 31) 17) 31)

Distribution

Reference

Most beneath the cricoid cartilage in the larynx Ventrolateral side in the larynx, ventral aspect in the trachea Evenly distributed in the larynx, mostly in the ventral side, less in the dorsal edge between the cartilage rings of the trachea Evenly dispersed along the trachea Evenly distributed in the larynx; declined in number from the proximal to the distal portion of the trachea Evenly distributed along the trachea

Widdicombe et al., 2001 Widdicombe et al., 2001

Evenly distributed along trachea

Widdicombe et al., 2001

Widdicombe et al., 2001 Widdicombe et al., 2001

Tos, 1966

Leigh et al., 1986

a The gland number in the mouse trachea may vary between inbred strains (Innes and Dorin, 2001). b Assumes tracheal surface area of 1.6 cm 2 (Widdicombe et al., 2001). c Data from two rabbits (Widdicombe et al., 2001). d Data from two rabbit tracheas; the distribution of glands varied between diVerent segments of the trachea (Widdicombe et al., 2001). e The density of submucosal glands in the ferret trachea was not evaluated; the pattern of diVerentiation and complexity of the glands is similar to that in the human (Leigh et al., 1986). Abbreviations: ND, not evaluated; GW, gestational week.

Morphological analysis of submucosal gland development during in utero development of the human airway has been extensively studied (Tos, 1966, 1968). In humans, airway submucosal gland development initiates when clusters of surface epithelial cells invade the lamina propria within the membranous portion of the proximal trachea. This takes place between the

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10th and 15th week of gestation. Development then extends more distally down the trachea and bronchi until the 17th gestational week. There is no diVerence in glandular density between the upper and lower trachea at this stage. Further expansion of submucosal glands occurs by lateral growth and dichotomous branching within the submucosal regions of the airways until approximately the 23rd week of gestation; de novo gland formation ceases thereafter (Smolich et al., 1978; Tos, 1968). Tos described five stages of tracheal submucosal gland development in humans (Tos, 1966). The earliest stages of gland formation begin when surface airway epithelium buds, presumably composed of basal cells, invade the lamina propria and extend into cylinders (stage 1). The cylinders then form lumens to communicate with the airway surface (stage 2). The lumens develop two later ducts by branching oV their tubules during stage 3. In stages 4 and 5, the ducts develop more extensive dichotomous branches and line the main duct with a columnar, ciliated epithelium. By the 24th week of gestation, glands arborize, forming the characteristics of tubuloacinar mucous and serous components of adult submucosal glands. After all five stages, the glands have increased in size but not in number, suggesting that submucosal glands are formed during gestation (Smolich et al., 1978; Tos, 1966, 1968). Although airway submucosal glands exist in humans, mice, and rats, their abundance, developmental patterns, and anatomical structures significantly diVer. Submucosal glands in rodents, for example, appear to be evolutionary remnants not required for normal lung function. Presumably, alterations in surface airway cell biology have caused the functions of submucosal glands in rodents to adapt. Increased abundance of Clara cells or serous cells in the proximal airways has likely altered the functions of submucosal glands in mice and rats, respectively. Because of the shortage of small animal models that can be used to study airway gland biology, the ferret has emerged as a useful surrogate animal model to study submucosal gland function. Ferrets, unlike rodents, have an abundance of submucosal glands throughout their cartilaginous airways, with distribution and cellular composition similar to those found in the human airway (Basbaum et al., 1981; Duan et al., 1999; Ekstrom and Helander, 2002; Leigh et al., 1986; Robinson et al., 1986, 1989). The development of ferret submucosal glands initiates within the trachea during the first few postnatal weeks of life, closely resembling the in utero human airway at 15 weeks of gestation (Leigh et al., 1986; Tos, 1968). The anatomy and cellular constituents of diVerentiated ferret submucosal glands are also identical to those found in adult human airways (by morphological criteria) (Robinson et al., 1986) and express several similar genes (Sehgal et al., 1996; Tom-Moy et al., 1983). The ferret is the only known placental mammal in which substantial development of both airway epithelium and submucosal glands occurs postnatally. These morphological and developmental features of the ferret airway make it uniquely

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suited as a model for studies focusing on the development of tracheal submucosal glands.

IV. Evidence for Human Adult Proximal Airway Stem Cells Capable of Developing Submucosal Glands One historical approach that has been used to determine the existence of adult airway stem cells and to study progenitor/progeny relationships is the reconstitution of epithelia onto denuded tracheal xenografts. This model seeds isolated airway epithelial cells onto graft tracheas that have been denuded of all endogenous airway epithelia by freeze-thawing. After the airway stem/progenitor cells are seeded, the tracheal grafts are implanted subcutaneously into immunocompromised hosts, such as nu/nu or severe combined immunodeficiency (SCID) mice. After approximately 2 weeks of transplantation, xenografted airways vascularize, and stem/progenitor cells expand to reconstitute the grafted airway epithelium. A major advantage of this approach is that it can be applied to airway epithelia from multiple species. Two generalized approaches have used this model of studying airway biology. The first includes purification or enrichment of specific cell phenotypes, followed by reconstitution of denuded rat tracheal grafts. This procedure has been used to study the ability of basal and secretory cells to reconstitute a fully diVerentiated surface airway epithelium in multiple species (Hook et al., 1987; Inayama et al., 1988, 1989; Johnson and Hubbs, 1990; Johnson et al., 1990; Randell, 1992; Randell et al., 1991). Because these studies do not discuss relevant aspects of progenitor cell biology in relationship to submucosal glands in the airway, they are not discussed further. The second approach used for evaluating progenitor/progeny relationships and the existence of a stem cell compartment in the adult human proximal airway has combined tracheal xenograft reconstitution with ex vivo retroviral marking (Engelhardt et al., 1995). Results using this approach are discussed further below. Integrating retroviral vectors are an eVective way to genetically tag epithelial progenitors with transgene markers and follow lineage relationships using histochemical staining. This technique has been applied in the context of human proximal airway epithelia using the xenograft model (Engelhardt et al., 1995). In these studies, researchers reconstituted the xenograft airway epithelium with primary human airway cells infected with a variety of retroviral vectors that were encoding either -galactosidase or alkaline phosphatase genes. They then examined the expansion of retrovirally tagged epithelia clones to determine the size and types of epithelial cells contained within each clone. The number of cells in each clone (i.e., its size) was used as an index of proliferative capacity, with the notion that clones arising from

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Figure 2 Retroviral lineage analysis in the human proximal airway epithelium using a tracheal xenograft model. Retroviral vectors encoding histochemical reporter genes, such as a galactosidase transgene (as shown), were used to infect primary human tracheobronchial airway epithelial progenitors in vitro prior to seeding into denuded rat tracheas and transplantation subcutaneously into nu/nu mice. (A) Four to five weeks posttransplantation, a diVerentiated epithelium reconstitutes the denuded rat trachea, and retrovirally infected clones (arrows) expand within the xenograft epithelium. Each of these clones is derived from a single retroviral integration event, as judged by co-infection with two viral vectors encoding diVerent histochemical markers (Engelhardt et al., 1995). (B–D) Analysis of the cellular phenotype of these clones demonstrated that diVerent progenitors with either pluripotent (B) or limited capacity for diVerentiation (C and D) existed. Pluripotent clones composed of basal, intermediate, goblet, and ciliated cells (B) were most abundant as well as the largest in size. In contrast, the clones with a more limited capacity to diVerentiate into only basal cells (C) or ciliated and intermediate cells (D) were smaller in size. The abundance of these clone phenotypes is shown as a percentage in the right-hand corner of each panel; other clone types not shown are discussed in Table III. (E) Only pluripotent, surface airway epithelial, clone phenotypes were associated with transgene-expressing submucosal glands. These findings

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adult stem cell fractions would have a larger proliferative capacity. Furthermore, the phenotyping of clones (with regard to the type of transgeneexpressing cells in each clone—basal, ciliated, goblet, or intermediate cells) was used to assess the repertoire of progenitor cell types in the airway with either limited or pluripotent capacity for diVerentiation. It was critical in this approach to demonstrate that the majority of expanded transgene-expressing clones had indeed arisen from a single retrovirally infected progenitor/ stem cell. To assess the percentage of error in clonal assignment, these researchers performed studies using co-infected cells with two retroviruses expressing diVerent histochemical markers (LacZ and alkaline phosphatase) before reconstituting the xenografts. The percentage of overlap in clones expressing both histochemical markers was used to demonstrate that the error of clonal assignment was less than 2%. Results from the phenotypic analysis of retrovirally tagged clones demonstrated incredible diversity in the cell types contained within each clone. For example, pluripotent clones containing basal, intermediate, ciliated, and goblet cells were the most abundant in reconstituted epithelia, composing 43 2.4% of all clones (Engelhardt et al., 1995; Fig. 2). These clones were also seen to have the largest proliferative capacity, as shown by clone size. The second most abundant clone phenotype observed consisted of basal, intermediate, and ciliated cells, with an average appearance in 26 3.0% of all clones. Concordant with a reduced frequency of appearance, this clone phenotype with limited capacity for diVerentiation contained half as many cells as pluripotent clones. In contrast, clones containing only basal cells or basal and intermediate cells were much less frequent (2–6%) and were the smallest of all clones observed. The results from this study, as well as other types of clones observed (summarized in Table III), suggested that a diverse repertoire of progenitor cells likely exists in the adult human proximal airway, with diVering capacities for proliferation and diVerentiation. Furthermore, these studies also imply that progenitor cells with a higher capacity for proliferation also have a higher capacity for diVerentiation. The preceding study provided insight into the potential existence of stem cells and the diversity of transient amplifying cell populations in the human adult proximal airway. First, the complexity of clone phenotypes originating from ex vivo retrovirally infected cells favors a directed lineage relationship in the human airway. A second interesting finding was the existence of transgene-marked submucosal glands that infrequently developed in these suggest that the cell type giving rise to transgene-expressing glands also has pluripotent capacity to diVerentiate into all surface airway epithelial cell types. Such an airway phenotype is a good candidate for an adult proximal airway stem cell. SAE, surface airway epithelium; SMG, submucosal glands. Data derived from previously published work (Engelhardt et al., 1995). (See Color Insert.)

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Table III Clonal Analysis of Progenitor/Progeny Relationships in the Human Proximal Airway Clone Phenotype a B/I/G/C B/I/C I/G/C B/I/G B/I I/C B

Frequency b 43 26 10 6.6 6.8 5.7 2.5

2.4 3.0 2.0 2.4 1.3 1.8 0.4

Size c 21 10 10 6 4 6 3

10d 4 5 2 1 3 0.7

a Clone phenotypes are based on morphological analyses of glycol methacrylate (GMA) sections from retroviral expressing clones within fully diVerentiated human bronchial xenografts (n ¼ 8). B, basal cell; I, intermediate cell; G, goblet cell; C, ciliated cell. Two retroviral reporter constructs that express the -galactosidase transgene driven by the long terminal repeat (LTR) and cytomegalovirus (CMV) promoters were used. b The frequency of clonal phenotypes was assessed in eight independent xenografts from 1115 independent clones. Values represent the mean percent distribution of each clone type SEM. c The mean size [(number of nuclei)/(cross-section)] of transgene-expressing clones. Nonoverlapping clones were evaluated by quantitating xenograft sections at 60 m intervals. Values represent the mean SEM. d Clones containing basal, intermediate, goblet, and ciliated cells are statistically twofold larger in size than all other clone types seen ( p < 0.001), as measured by the Student’s t-test. Data derived from previously published work (Engelhardt et al., 1995).

xenograft airways. When observed, transgene-expressing glands were always associated with pluripotent transgene-expressing clones in the surface airway epithelium (Fig. 2). Because this clone phenotype also had the highest proliferative capacity of all clones observed, it is hypothesized that a subset of progenitors with pluripotent capacity for surface airway epithelial diVerentiation and for submucosal gland development comprises a candidate adult proximal airway stem cell. This adult airway stem cell appears to be derived from a subset of basal cells, since studies have shown that retrovirally infected basal cells expanded in vitro by serial passage gave rise to only pluripotent basal/intermediate/ciliated/goblet-containing clones in vivo within xenografts (Engelhardt et al., 1995). Studies evaluating the number and size of expanded pluripotent progenitor cell clones required to reconstitute a xenograft airway epithelium have also been useful in estimating the abundance of this cell phenotype in the adult human airway. These studies have estimated that as few as 100 progenitors can reconstitute a 1 cm3 diVerentiated xenograft epithelium following seeding with 5 104 basal-like cells that had been expanded in vitro (Zepeda et al., 1995; J. F. Engelhardt, unpublished data). This has led to the conclusion that a subset of basal cells

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(1 in 500, or 0.2%) has a pluripotent capacity for diVerentiation into all surface airway epithelial cell types. If the ability to diVerentiate into submucosal glands is then superimposed as a stem cell phenotype, and approximately five glands per xenograft form on average (Engelhardt et al., 1995), it can be estimated that approximately 0.01% of in vitro expanded basal-like cells may be stem cells with the capacity to diVerentiate into both surface airway and glandular epithelial cell types. Such findings of a primordial adult stem cell in the proximal airway with pluripotent capacity for both surface airway epithelial and submucosal gland development have also been supported by in vivo retroviral infection studies in ferret tracheal xenografts (Duan et al., 1998). In this model, newborn ferret tracheas were transplanted subcutaneously in nu/nu mice and were retrovirally infected in vivo with an alkaline phosphatase-expressing retrovirus. During the first few postnatal weeks of ferret tracheal development, submucosal glands developed from the surface airway epithelium. Retroviral infection studies have demonstrated an unusually high eYciency of retroviral transduction in submucosal glands using this model, which suggests that the cell phenotype that gives rise to glands has a relatively high proliferative index and is hence very susceptible to retroviral infection. Furthermore, clones expanded from retroviral infection demonstrated similar pluripotent cell phenotypes, as seen in ex vivo xenograft reconstitution studies, with contiguous, transgene-expressing cells in the surface airway epithelium and submucosal glands. These studies also support the possibility that the proximal airway stem cell may have capacities for diVerentiation into both surface airway and glandular epithelial cells.

V. Mouse Tracheal Injury Models Suggest Submucosal Gland Ducts Are a Niche for Proximal Airway Stem Cells Because the airway epithelium turns over at a relatively low rate, the analysis of stem cells responsible for normal maintenance of the airway epithelium in the in vivo lung has been challenging. It is generally accepted that stem cells are most often slow cycling. They give rise to transient-amplifying (TA) progenitor cells, which impart the majority of tissue renewal in the setting of an injury. The heterogeneity of lineage-restricted clone phenotypes in human xenograft reconstitution models also suggests a diverse repertoire of TA cell types in the proximal airway. Unlike stem cells, which by definition have unlimited proliferative capacity, TA cells are restricted in their capacity to divide and cannot proliferate indefinitely. This limitation has been reflected in human xenograft reconstitution models, as shown by the smaller size of clones derived from lineage-restricted progenitors and the ability of expanded basal cell clones to reconstitute an airway epithelium

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(Engelhardt et al., 1995). Although stem cells and TA cells are both selfrenewing populations, TA cells are primarily thought to be the major progenitor population, which gives rise to the terminally diVerentiated cell types of an adult organ. Due to the low abundance of stem cells and their relatively slow cycling time, the use of injury models has been required to characterize stem cell populations in the airway. Xenograft reconstitution models are one example of artificially imposed airway injury. Recent studies have taken advantage of the slow-cycling phenotype of stem cells in the airway to track their location in vivo following the incorporation of nucleotide analogs, such as BrdU. Because stem cells divide relatively slowly, even in the setting of airway injury, the label is maintained within genomic DNA for long periods of time, giving rise to a label-retaining cell (LRC) characteristic. Several laboratories have used this LRC phenotype to assess the location of stem cell niches in the mouse lung (Borthwick et al., 2001; Giangreco et al., 2002; Hong et al., 2001; Reynolds et al., 2000a,b). This review discusses the findings pertinent to progenitors associated with glandular structures (Borthwick et al., 2001). Using a mouse tracheal model of SO2 injury, Borthwick and colleagues identified specific niches of stem cell expansion that are marked by distinct zonal boundaries. In the proximal glandular-containing trachea, LRC-expanding zones were confined to the ducts of submucosal glands (Borthwick et al., 2001; Fig. 3). Interesting similarities between the location of these stem cell niches and gland ducts can be seen from human xenograft reconstitution models that associate retrovirally tagged stem cell phenotypes with the ability to diVerentiate into both surface airway and glandular epithelial cell types (Duan et al., 1998; Engelhardt et al., 1995). Further evidence of the regenerative capacity of stem cells in the gland ducts was demonstrated using xenograft reconstitution models for which the surface airway epithelium was removed by pronase digestion (Borthwick et al., 2001). In these studies, airways predominantly devoid of surface airway epithelial cells demonstrated a regenerating surface airway epithelium emerging from within gland ducts when subcutaneously transplanted in nude mice. Such findings support the notion that this region may be a stem cell niche in the proximal airway.

VI. Developmental Molecular Markers of Glandular Stem Cell Phenotypes Several studies have suggested an association between candidate adult stem cells in the proximal airways and submucosal gland biology. First, human bronchial xenograft studies have revealed that there are surface airway epithelial progenitors with pluripotent capacity for both submucosal gland

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Figure 3 Stem cell niche associated with submucosal gland ducts. Studies using an SO2 tracheal injury model in the mouse have demonstrated label-retaining cell (LRC) expansion at submucosal gland ducts following BrdU labeling (Borthwick et al., 2001). LRCs (marked in red) indicate these cells are putative stem cells that give rise to transient amplifying (TA) progenitors (not shown) that migrate into the airway and diVerentiate to repopulate the injured epithelium. (See Color Insert.)

and surface airway epithelial diVerentiation. Second, LRCs have been shown to expand from submucosal gland ducts following airway injury. These studies suggest that elucidating molecular events controlling the commitment of airway stem cells to form submucosal glands might help to further define stem cell phenotypes in the airway. During proliferation and diVerentiation, stem cells must coordinate a variety of intracellular and extracellular signals. Changes in gene expression play a key role in the regulating processes that allow stem cells to react to changes in their environment during development and in the setting of injury. Studies identifying cellspecific molecular markers in airway epithelium and submucosal glands have in this way begun to define the molecular characteristics of airway progenitor/stem cells. The development of glandular organs is a tightly regulated process that requires complex interactions between the epithelium and mesenchyme. Although relatively little is known about the epithelial–mesenchymal interactions that regulate submucosal gland developmental processes in the airway, better-studied developmental biology of other bud-forming organs has presented some recurrent themes. These reciprocal interactions may be mediated by intercellular molecular signals or by signals from neighbor cells and are characteristic of epithelial bud formation and tubulogenesis in the hair follicle, kidney, liver, lung, pancreas, and mammary gland. It is likely

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that the progressive invagination, branching, and arborization of submucosal gland tubules and ducts are also controlled by these reciprocal interactions. To date, a number of signaling pathways and transcription factors have been identified as playing important roles in the morphogenesis of glands. For example, the high mobility group (HMG) box transcription factor lymphoid enhancing factor-1 (Lef-1) is involved in the initiation of submucosal gland development and a wide range of other epithelial, budforming organs, including mammary glands, teeth, and hair follicles (Duan et al., 1998, 1999; van Genderen et al., 1994). As with Lef-1, the involvement of other transcription factors, such as Msx1 and Msx2, in bud formation during mammary gland and tooth development has been shown. In the absence of Msx1 and Msx2, mammary gland and tooth development halts at the bud stage, shortly after initiation (Hennighausen and Robinson, 2001; Peters and Balling, 1999). Sonic hedgehog (Shh)/Ptch (Motoyama et al., 1998a) and transforming growth factors, such as bone morphogenic proteins (BMPs) 2 and 4, are also required for the morphogenesis of mammary glands, teeth, and follicles (Jamora et al., 2003). Expression of Shh and Ptch mRNA has also been detected in the mouse nasal gland, suggesting that Shh may be involved in gland formation (Motoyama et al., 1998a). Shh and its encoding downstream intracellular regulator, Gli1, are also expressed in regenerating airway epithelium, implicating Shh signaling in airway progenitor cell function (Motoyama et al., 1998b; Watkins et al., 2003). It has also been suggested that the extracellular matrix (ECM) greatly influences the invagination of the airway epithelium during submucosal gland formation (Infeld et al., 1993). Cell-specific expression of cytokeratins has also been a useful marker for evaluating diVerentiated components of submucosal glands. In a human airway xenograft model, cytokeratin 7 (K7) is expressed by gland duct cells, ciliated cells, and secretory cells (Delplanque et al., 2000). In contrast, K13, K14, and K18 expression is restricted to myoepithelial cells in submucosal glands and basal cells in the surface epithelium. Of these molecular markers identified with submucosal glands, Lef-1, Shh, and Ptch appear to be the most specific markers linked to bud formation of submucosal glands. Lef-1 is thus far the earliest transcriptional factor known to be induced during submucosal gland bud formation. Hence, knowledge of its transcriptional regulation could provide significant insight into the molecular cues that regulate stem cell commitment to form glands in the airway. Studies using Lef-1 knockout models have demonstrated that expression of the Lef-1 gene is required for a wide range of inductive epithelial/mesenchymal interactions involved in mammary gland, tooth, vibrissa, hair, and airway/nasal submucosal gland development (Duan et al., 1999; van Genderen et al., 1994). Lef-1-deficient mice lack both mammary glands and airway submucosal glands. In the context of airway submucosal gland development, Lef-1

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mRNA expression is induced at high levels in early stage buds, in which three to five cells aggregate in the surface epithelium prior to invagination (Fig. 4; Duan et al., 1998). During later stages of gland development, Lef-1 expression is restricted to cells in the most distal invading tips of tubules. In the ferret tracheal xenograft airway model, inhibition of Lef-1 expression with antisense oligonucleotides decreases the abundance and branching

Figure 4 Molecular analysis of Lef-1 expression during submucosal gland development in the airway. Studies in ferret and mouse tracheal and nasal airways have demonstrated a requirement for Lef-1 expression during the initial stages of submucosal gland development (Duan et al., 1998, 1999). (A) The first three stages of gland development are schematically shown to originate from a cuboidal epithelium. Morphological sections of stages 1 and 2 from newborn ferret tracheas are given above the schematic representations. Regions of Lef-1 expression at the mRNA level in ferret trachea are indicated schematically by red highlighted cells. (B and C) In situ hybridization with antisense ferret Lef-1 mRNA probes showing expression at stages 1 (B) and 2 (C) in the ferret trachea (Duan et al., 1998). Bright and dark field photomicrographs are given at the top and bottom of each panel, respectively. (D) Nasal submucosal gland buds from transgenic mice (þ/ ) harboring a 2.5-kb Lef-1 promoter fragment that drives expression of a -galactosidase reporter gene demonstrates X-gal staining in a subset of gland bud cells (Driskell et al., 2004). (E) X-gal staining was not observed in nontransgene-containing littermates ( / ) or in transgenic mice harboring Wnt-responsive element (WRE)-deleted reporter constructs (data not shown). The transgene reporter used in these studies is indicated in panels D and E. These studies suggest that Wnt induction of the Lef-1 promoter likely plays a role in mouse nasal gland development. (See Color Insert.)

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morphogenesis of submucosal glands (Duan et al., 1999). These characteristics, together with the fact that submucosal gland development can be rescued in Lef-1-deficient mice by airway-specific expression of a Lef-1 cDNA under the direction of the CC10 promoter, strongly suggest the need for Lef-1 expression in airway gland development. However, ectopic expression of Lef-1 in the proximal airways of these CC10-Lef-1 transgenic mice was insuYcient to increase submucosal gland abundance. Hence, signals in addition to Lef-1 are required for airway glandular development. The morphogenesis of bud-forming organs, such as hair follicles and glands, causes changes in the polarity of stem cells that necessitate cell–cell contacts. This process is controlled by highly regulated interactions between bud-forming epithelia and the underlying mesenchymal layers that stimulate signal transduction cascades and the transcription of genes important to epithelial cell movement, proliferation, and diVerentiation. The Wnt/-catenin/Lef-1-signaling pathway is one of the most extensively studied transcriptional cascades involved in various types of organogenesis (Duan et al., 1999; Galceran et al., 2001; Yasumoto et al., 2002). In this context, Wnt proteins bind to transmembrane-frizzled receptors and activate signaling cascades by stabilizing -catenin and its interactions with transcription factors of the T-cell factor (TCF)/Lef-1 family. Following translocation to the nucleus, -catenin/Lef-1 and -catenin/TCF complexes modulate transcription of target genes through the assembly of multiprotein complexes. In the absence of Wnt signaling, Tcf/Lef factors associate with Groucho/ HDAC (histone deacetylase) corepressor complexes to potently block expression of Wnt target gene programs (Clevers, 2002; Galceran et al., 2001; Hsu et al., 1998; McKendry et al., 1997; Novak et al., 1998; Riese et al., 1997). Studies beginning to establish signals that regulate the Lef-1 gene have begun to identify candidate pathways that might influence stem cell commitment in airway submucosal gland formation. In vitro studies dissecting the human Lef-1 promoter have demonstrated transcriptional responsiveness to Wnt3A and -catenin (Filali et al., 2002). These studies identify a 110-bp Lef-1 promoter segment that was required for responsiveness to Wnt3A/catenin (WRE). When this segment was deleted from the Lef-1 promoter, basal levels of transcription were significantly increased, and Wnt responsiveness was lost. Furthermore, this WRE was able to convey Wnt3A/-catenin responsiveness to a heterologous minimal promoter in a context-independent fashion (Filali et al., 2002). Other groups have also demonstrated that isolated segments of the Lef-1 promoter are responsive to specific Tcf isoforms in a -catenin-dependent manner (Atcha et al., 2003). Given the apparent complexities of Lef-1 promoter regulation by the Wnt/-catenin/Tcf cascade and its diverse roles in the developmental processes of multiple organ systems, in vivo studies of the Lef-1 promoter are required to more clearly evaluate its

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regulatory components. Lef-1-deficient mice develop normal lungs and airways, with the exception of absent submucosal glands. Hence, a better understanding of the molecular regulation of the Lef-1 gene could provide insights into transcriptional processes that control adult airway progenitor cell phenotypes responsible for submucosal gland development. To this end, studies in transgenic mice harboring a 2.5-kb Lef-1 promoter fragment driving expression of a LacZ transgene have indicated that this segment alone can regulate expression in nasal submucosal gland buds (Fig. 4D) (Driskell et al., 2004). Interestingly, deletion of the 110-bp WRE from the Lef-1 promoter led to a lack of expression in nasal gland buds in this model system (data not shown). These findings suggest the interesting hypothesis that Wnt/-catenin pathways may play an important role in regulation of Lef-1 expression during the early stages of gland development. Although the field has yet to identify a single gene or set of genes required for the maintenance of airway stem cells, several interesting comparisons to Lef-1 regulation in airway glandular precursors can be drawn with other organ systems. For example, several intrinsic factors that influence stem cell fates in epithelial organs, such as the intestine and skin, include the Wnt/Lef/ Tcf/-catenin pathway. This pathway has been shown to play a critical role in controlling stem cell maintenance and/or cell commitment to diVerentiation (Brittan and Wright, 2002; DasGupta and Fuchs, 1999; Korinek et al., 1998; Koster et al., 2002). Wnt3A has also been shown to stimulate proliferation and self-renewal of hematopoietic stem cells (Willert et al., 2003). This finding is of particular interest to those conducting studies evaluating the regulation of the Lef-1 gene in glandular progenitors, as well as those researching the requirement of Wnt-responsive sequences in the Lef-1 promoter for proper activation in vivo. Whether Lef-1 or an upstream signal controlling the transcriptional activation of the Lef-1 gene is required for stem cell maintenance and/or activation following airway injury remains to be proven. However, given the similarities between Wnt/Tcf/-catenin involvement in stem cell maintenance and/or stem cell commitment to progenitor cells in other organs, this pathway may be a good candidate for controlling stem cell niches in the submucosal glands.

VII. Concluding Remarks and Future Challenges Defining the intrinsic and extrinsic cues required for the proliferation and diVerentiation of stem cells in airways that form submucosal glands may ultimately provide new avenues for treatment of hypersecretory lung disease involving glandular abnormalities. The adult lung has been one of the more challenging organs in which to study adult stem cells due to the anatomical and functional complexities of distinct spatially separated airway epithelia.

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Furthermore, diVerences in airway biology between many species have complicated the development of a generalized consensus on stem cell phenotypes. Despite these formidable hurdles, recent studies demonstrating unique aspects of stem cell niches associated with submucosal gland ducts in the airways have begun to clarify the field’s understanding of extrinsic factors that may influence stem cell renewal and proliferation following airway injury. Equally important to the extrinsic features of stem cell niches are the intrinsic properties of stem cells that define their unique characteristics. Regulatory pathways that direct Lef-1 expression in submucosal gland precursors are currently good candidates for ultimately understanding the regulatory circuits at work in airway stem cells. These pathways will likely produce some common themes important in both adult airway biology and pathophysiology.

Acknowledgments We gratefully acknowledge National Institute of Diabetes and Digestive and Kidney Diseases research funding (DK47967) for John F. Engelhardt’s laboratory in the area of this review and the editorial assistance of Leah Williams.

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Gene Expression Studies in Lung Development and Lung Stem Cell Biology Thomas J. Mariani* and Naftali Kaminski{ *Division of Pulmonary and Critical Care Medicine Brigham and Women’s Hospital Pulmonary Bioinformatics The Lung Biology Center Harvard Medical School Boston, Massachusetts 02115 { Dorothy P. and Richard P. Simmons Center for Interstitial Lung Disease Lung Translational Genomics Center Pulmonary, Allergy and Critical Care Medicine University of Pittsburgh Medical Center Pittsburgh, Pennsylvania 15260

I. II. III. IV. V. VI. VII. VIII.

Introduction Verifying and Challenging Old Concepts Establishing New Concepts A Model for Lung Alveogenesis Development and Stem Cells Applying Developmental Transcriptional Modules to Adult Disease Lung Cancer Conclusion Acknowledgments References

I. Introduction Lung development is regulated by a complex network of molecular events that determine the temporal and spatial formation of structures that in due time become a fully functional respiratory apparatus. It has long been known that this morphogenic process is regulated by mesenchymal–epithelial interactions, with a significant role for the extracellular matrix (ECM). In the past decade, studies using traditional molecular biology methods, model organisms, and genetically modified animals have greatly improved our understanding of every stage in lung development. It is now well established that lung bud initiation is regulated by the sonic hedgehog (Shh) signaling pathway (Pepicelli et al., 1998), by fibroblast growth factor (FGF) receptor Current Topics in Developmental Biology, Vol. 64 Copyright 2004, Elsevier Inc. All rights reserved. 0070-2153/04 $35.00

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signaling (Peters et al., 1994), and probably also by retinoid-related signaling (Malpel et al., 2000). Branching morphogenesis, the dichotomous branching process involved in defining the proximal–distal structure of the conducting airway prior to the saccular stage, is dependent on the integrated eVects of multiple growth factors including bone morphogenetic protein (BMP) and FGF signaling pathways, with FGF-10 being the major coordinating factor for the spatio-temporal regulation of branching (Bellusci et al., 1997). The characteristics of terminal development and maturation include distal airway diVerentiation and alveolization or alveogenesis. The diVerentiation and flattening of distal airway epithelia is regulated by multiple factors, including GATA-6, Nkx2.1, HNF3, C/EBP, glucocorticoid hormones, and FGFs (Cardoso, 2001), while alveolization is at least in part regulated by retinoids and growth factors such as platelet-derived growth factor (PDGF) and FGF (Cardoso, 2001). One amazing feature of lung development is this coordinated interaction of cells, molecules, and genes that is temporally and spatially integrated with very little room for error. Naturally, one would like to be able to profile all the genes that are expressed at every stage to gain a more complete understanding of this process at the systems biology level. The availability of the complete sequence of the human genome (Lander et al., 2001), as well as complete genomes of multiple model organisms such as the fruit fly (Rubin and Lewis, 2000), the mouse (Waterston et al., 2002) and the dog (O’Brien and Murphy, 2003), together with the emergence of high throughput gene expression profiling technologies, make this wish attainable. Microarrays allow for the simultaneous profiling of the steady-state levels of thousands of mRNAs, currently even complete genomes. They have been extensively used in almost every field of biomedical research, including numerous developmental processes (see Smith and Greenfield, 2003). Arguably, the most successful applications have been in lower eukaryotic systems, including Caenorhabditis elegans and Drosophila melanogaster. In these cases, it has been clearly shown that the technology is capable of extracting meaningful biology from these complex organisms and complex systems. Kim et al. (2001) compiled data from more than 500 microarray experiments describing gene expression in C. elegans. Using this information, they were able to assign putative function to groups of coregulated genes. Arbeitman et al. (2002) used comprehensive expression profiling in order to characterize regulation throughout development in D. melanogaster. Using whole organisms as a source for RNA, the investigators were able to identify regulatory networks acting in specific organs. These studies revealed the capabilities of microarray technology as well as specific biological insights of the model systems studied. Microarray technology has also been successfully applied to organ development in mammals. Novel insights have resulted from the analysis of

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genome-wide expression patterns during the development of kidney (Dekel, 2003; Schwab et al., 2003), hippocampus (Mody et al., 2001), cerebellum, adipose tissue (Burton et al., 2002), and breast (Master et al., 2002), to name but a few. While these insights have not necessarily been as paradigmatic as those derived from invertebrate data, one can speculate that this is due to the complexity of the biological system and not the inadequacy of the technology. If true, it is likely that the clearly evident pace of advancement in the technology will surmount the remaining obstacles in the very near future. In this chapter, we explore what we have learned from the application of this technology to lung development, as an example of its accomplishments and future potential. As with most vertebrate organs, expression profiling data sets concerning lung development come in two types: temporal and interventional. Again, each experimental design has strengths and weaknesses, and when appropriately applied, can define answers to specific types of questions.

II. Verifying and Challenging Old Concepts A review of the microarray literature relevant to lung development suggests that the microarray data universally verified things we already know. This, in and of itself, may be boring from the perspective of lung biology, but is exciting from the perspective of genomics. Genomically speaking, we do not know much. It is highly satisfying that an extremely small proportion of these data confirms previous knowledge, as it speaks to the reliability of the vast majority of the data, which is novel. Is it interesting that a temporal profile of mouse lung development shows that type I and type III collagens are coregulated (Mariani et al., 2002)? It is not, if one reads the literature and sees that these two proteins are the primary constituents of the collagenous ECM of the lung. It is, if one hypothesizes that this coregulation may be a sign of functional relatedness (a basic tenet of functional genomics) and uses this information to learn about other genes that behave similarly. One major advantage of expression profiling data is that it often forces us to reconsider common truths and concepts. In the case of lung development, information from microarray data required us to reconsider basic aspects of gene expression behavior (the notion of ‘‘housekeeping genes’’) or biological concepts. For many years, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was the steady-state mRNA endogenous control ‘‘gold standard.’’ Hundreds, if not thousands, of papers reported the use of GAPDH as a housekeeping gene and the results of other genes normalized to GAPDH. Interestingly, expression profiling showed that GAPDH expression is regulated during normal lung development (Fig. 1) and is probably representative of the overall metabolic activity of the organ (Mariani et al.,

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Figure 1 Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression is not constitutive during mouse lung development. GAPDH gene expression has been considered, for the most part, to be constitutive. Upon generating a complete expression profile of normal mouse lung development, we noted that GAPDH expression changes considerably during gestation, while postnatal GAPDH expression is constitutive. This modified concept of GAPDH expression has been validated in the developing mouse lung by Northern blot analysis and in numerous other expression profiling studies. Reprinted from Mariani et al. (2002), Am. J. Resp. Cell Mol. Biol. 26, 541–548, with permission.

2002). These results were confirmed by Northern blot. Such an observation, not directly related to the mechanisms of lung development, is critical to molecular biology in general, and to evaluation of studies that use GAPDH as a housekeeping gene for normalization of real-time polymerase chain reaction (PCR) and Northern blot experiments. The concept that GAPDH is constitutively expressed has now been thoroughly disputed by numerous expression profiling studies. In all elastic organs studied to date (none using microarrays), elastin gene expression showed a typical pattern: initiating prior to birth, peaking during maximal growth, and subsiding after maturation (Mariani et al., 1997). Expression profiling identified a bimodal expression pattern for elastin in lung development (Mariani et al., 2002). This pattern of elastin gene expression during lung development has been interpreted as truly a modification of an old concept. The two peaks likely represent distinct regions of the lung undergoing elastogenesis at diVerent times: first large vessels, then parenchyma. This result was confirmed by Northern blot and is consistent with previous studies showing a majority of elastin expression in the vessels during embryonic development and in the parenchyma in the postnatal lung (Koh et al., 1996; Mariani et al., 1997; Pierce et al., 1995a,b, 1997; Swee et al., 1995). Interestingly, -smooth muscle actin (ASMA) exhibits an expression pattern very similar to that of elastin, but preceding elastin expression

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in time. This likely reflects the establishment of a population of ASMAexpressing elastogenic-competent cells, which was already known to be established prior to elastin expression during lung development (Bostrom et al., 1996; Lindahl et al., 1997). The retinoic acid (RA) and glucocorticoid signaling pathways have long been appreciated as major contributors to prenatal and postnatal lung maturation, and some evidence exists for their coordination or antagonism during lung development (Xu et al., 1995). Through microarray analysis of lung tissue from the glucocorticoid receptor-deficient mouse, Kaplan et al. (2003) identified Midkine as a gene regulated by both retinoids and glucocorticoids. They concluded that this ECM-associated regulatory molecule, and its binding partner versican, may coordinate these two essential pathways. Clerch and colleagues (2004) found that the vascular endothelial growth factor (VEGF) pathway is aberrantly regulated in an animal model of pharmacologically inhibited lung maturation. Given the recent appreciation for the relationship between vascularization and alveogenesis in the lung, this finding is not entirely surprising. However, this report provides additional justification for the significance of the role of the vasculature in lung maturation and may further define a specific molecule key to this process.

III. Establishing New Concepts In addition to verifying old concepts and modifying preconceptions, microarray experiments are fulfilling their promise of uncovering novel insights leading to new concepts in the regulation of lung development. Analysis of microarray data sets describing normal mouse lung development identified intriguing expression patterns for previously unconsidered transcription factors. The Sox family of transcription factors includes key regulators of mammalian tissue and organ development, but almost nothing is known regarding their role in the lung. Expression profiling data indicate that many Sox genes show regulated expression during lung development. Further, cluster analysis implicates diVerent family members in distinct roles, particularly in the regulation of ECM formation; Sox4 and -12 gene expression was similar to that for many interstitial ECM genes, such as type I and III collagen, while Sox18 expression was similar to that for many basement membrane-type ECM genes, such as type IV collagen, SPARC, and matrix Gla protein (T. Mariani et al., unpublished data). Similarly, cluster analysis of this data set strongly suggested a role for the transcription factor P311 in the regulation of interstitial ECM (type I and III collagen) expression. This relationship was confirmed when Schuger and colleagues reported a role for P311 in lung myofibroblast diVerentiation (Pan et al., 2002).

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IV. A Model for Lung Alveogenesis Perhaps the greatest benefit of expression profiling is that it provides comprehensive information regarding gene expression in a cell, tissue, or organ, thus allowing a systems biology outlook on the process studied. This allows experienced users to integrate a tremendous amount of information (in addition to the published data, but often times free of the bias of previous information) and to generate global hypotheses or models of biological processes. These models can be tested both computationally and, most importantly, experimentally. This use of microarray technology is often referred to as ‘‘hypothesis generating.’’ Some might say that this is the only ‘‘real’’ use for the technology, but this is no more true for data derived from a microarray experiment than for data derived from any other individual experiment. One could argue that, if forced to have a hypothesis, it is best to use as much of the available information as possible to generate that hypothesis. Lung development is recognized as occurring through discrete stages, primarily defined histologically (e.g., descriptively). The past decade has seen an enormous expansion in our understanding of the regulation of this process, almost entirely without the insight provided by expression profiling (reviewed in Cardoso, 2000; Costa et al., 2001; Mariani, 2004; Perl and Whitsett, 1999; Warburton and Lee, 1999; Warburton et al., 2000). A majority of this data has revealed the regulation of lung bud initiation, elongation, and branching morphogenesis prior to maturation of the gas– exchange unit—the alveolus. The past 3–4 years has seen an increasing gain in the understanding of the regulation of lung maturation, in particular. Regulation of alveolar formation—or alveogenesis—must control two overlapping processes: capillary bed reorganization and alveolar wall (secondary crest) elongation. We know a small number of pathways that contribute to the regulation of alveogenesis, including PDGF, RA, and FGF signaling. However, a satisfactory model of this morphogenetic event has been elusive (Pierce and Shipley, 2000). Expression profiling data of normal lung development has provided for an integrated, unifying hypothesis of the regulation of alveogenesis—the balloon model of secondary crest elongation (Fig. 2) (Mariani, 2004). This concept was derived, in large part, from two separate observations in the normal lung development data set, concerning two signaling pathways previously shown to be essential specifically for alveogenesis. First, the coregulation of FGF receptor (FGFR) 3, FGFR4, and numerous BM genes indicated a potential mechanism for the control of alveogenesis by FGF signaling, via the regulation of BM gene expression (Mariani et al., 2002). Second, the expression pattern of Meis genes suggested that they might play a part in alveogenesis by promoting RA signaling and countering FGF

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Figure 2 A proposed ‘‘balloon’’ model for regulating secondary crest elongation by concerted fibroblast growth factor (FGF) and retinoic acid (RA) signaling. (Left) Prior to alveogenesis, pre-proximal(/distal) mesenchymal ‘‘stem cells’’ are located at sites of initial secondary crests. Gradients of both FGF and RA are established along a proximo-distal axis. (Right) This morphogen gradient promotes the diVerentiation of the stem cells into distinct proximal and distal cells with distinct phenotypes, in part driving secondary crest elongation. Septae elongate away from a fixed proximal location (e.g., trachea), analogous to the growth of a balloon, away from the fixed opening, upon inflation.

signaling (Mariani, unpublished data). This balloon model predicts that secondary crest elongation occurs as a result of the establishment of two overlapping and competing morphogen gradients. In this model, a gradient of RA signaling is established at the proximal position within the alveolar wall and promotes a ‘‘phenotype’’ distinguished by elastin expression, a known target of RA, both of which are essential for normal alveogenesis. An analogous gradient of FGF signaling, which is also essential for normal alveogenesis, is established at the distal position in the alveolar wall and promotes a phenotype distinguished by an absence of elastin expression. These counterregulatory gradients function to promote distinct phenotypes along the proximo-distal axis from a naı¨ve, pre-proximal (/distal) ‘‘stem cell’’ population. Clearly, this model is not exhaustive and does not specify the eVects on the individual cell populations within the alveolar septa. However, it does provide a framework for future studies aiming to define the specific molecular mechanisms contributing to alveogenesis.

V. Development and Stem Cells What about lung stem cells? Many separate stem cell populations have been isolated and subjected to expression profiling, including those derived from embryonic, neural, hematopoietic, and dermal epithelial cells (Ivanova et al., 2002; Ramalho-Santos et al., 2002; Tumbar et al., 2004). In these analyses, there appear to be a number of common stem cell ‘‘markers.’’ So, is there a

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stem cell signature present in normal lung development, and if so what does it tell us? An analysis of the expression of these stem cell marker genes during lung development shows some surprising data (Fig. 3). Many stem cell markers are expressed at high levels in the normal lung, such as ak1, ctbp2, efnb2, fhl1, idb1, idb2, lgals1, myo1b, ndn, peg3, prdx2, and txn1. As expected, some of the marker genes, such as ctbp2, ndn, peg3, and prx2, decrease over time of development. However, these genes all show complex patterns of expression, including an induction subsequent to an initial decline. For instance, ctbp2, ndn, and prx2 show higher expression at postnatal day 7 (D7) than D1. Unexpectedly, many of the stem cell markers show increasing expression during lung development, such as ak1, efnb2, fhl1, idb1, and myo1b. Fhl1 is more highly expressed in adult lung than at embryonic day 12 (E12) but has two distinct peaks of maximal expression at E18 and then at D7. Many of the stem cell markers, such as idb2, lgals1 and txn1, are highly expressed throughout normal lung development. Again, the expression patterns for these genes are complex. Lgals1, in particular, shows a biphasic expression pattern with maximal expression at E14 and D7–D10, reminiscent of the patterns for elastin and ASMA expression. It is exceedingly diYcult at present to interpret the expression patterns of these

Figure 3 Expression of common stem cell markers during normal lung development. Gene expression for 12,000 genes was determined using AVymetrix Mu11K GeneChips. Patterns of expression are displayed as raw (A) or median-normalized signal intensity from embryonic day (E) 12–18, postnatal day (D) 1–21 to adult. (A) Many common stem cell markers are highly expressed, but show complex patterns during normal lung development. (B) A subset of these genes showing decreasing expression across development. (C) A subset of these genes showing persistent expression. (D) A subset of these genes showing increasing expression.

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genes within the context of identifying lung stem cells. However, the complexity of expression patterns for these markers would support the existence of multiple, independent stem cell populations within the growing and adult lung. With rapid advances in the identification of resident and circulating stem cells capable of repopulating the lung, it is likely that this knowledge will improve dramatically in the immediate future.

VI. Applying Developmental Transcriptional Modules to Adult Disease If it is worthwhile to understand lung development for its own sake, then it may be more beneficial than one might think. It has been postulated that biological processes are defined by regulatory networks or modules of interrelated groups of gene products (Alon, 2003). In fact, this is merely a modification of the classical biochemical pathways we all studied. Further, these biological modules are likely to be limited in number, and so ‘‘normal’’ modules may be co-opted during abnormal states. Essentially, understanding regulation under normal conditions may help us understand regulation in diseased states. An intuitive example is the involvement of developmental modules in lung cancer. It is plausible that some transcriptional modules that tightly regulate branching morphogenesis, epithelial diVerentiation, or alveogenesis may have detrimental eVects when abnormally activated in adult life. Another example is the pathogenesis of emphysema, where abnormal alveolar repair and elastolysis probably play a role. Identification of endogenous programs/modules that regulate the formation of alveoli during normal development may contribute to the understanding of the cause of airspace enlargement in patients with emphysema. Even more excitingly, they may help identify targets for promoting or reinitiating alveolar development in adults.

VII. Lung Cancer Lung cancer is probably the area of lung biology in which microarray analysis has been most applied and accepted for its ability to provide molecular detail at a level previously impossible. The application of microarrays to tumor samples led to identification of genes associated with diVerent biological behavior of tumors and with prognosis prediction in both hematological malignancies and in solid tumors. (Alizadeh et al., 2000; Beer et al., 2002; Huang et al., 2003; Ramaswamy et al., 2003; Sandusky et al., 2002; van’t Veer et al., 2002). Interestingly, many of these studies did not

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directly test whether a developmental signature could be identified in the tumor characteristic gene expression patterns, although many genes known to be involved in developmental pathways were observed in these data sets (N. Kaminski and T. Mariani, unpublished data). Powell and colleagues (Borczuk et al., 2003) compared gene expression patterns in non-small-cell lung cancer with those from lung development (Borczuk et al., 2003). They found that large-cell carcinoma-related genes were enriched in those showing prominent expression patterns during the pseudoglandular and canalicular stages of mouse lung development. Conversely, adenocarcinoma-related genes were similar to those expressed primarily during the saccular and alveolar stages of normal lung development. These data may provide clues to the cellular origins and molecular etiology of these specific tumor types. Analyzing our own microarray data and published data (Bhattacharjee et al., 2001), we have identified SIL (Stem Cell Leukemia–interrupting locus) as a gene that was associated with tumors with increased mitotic rates (Erez et al., 2004). Although SIL is not directly related to lung development, it is associated with the sonic hedgehog pathway (Izraeli et al., 2001) and with regulation of cell fate in early development, and is required for mouse embryonic axial development (Izraeli et al., 1999). We believe that systematic searches for development-related genes in lung cancer data sets, and, probably, searches in other chronic non-remitting lung disease data sets, will reveal similar results.

VIII. Conclusion In this chapter we described some of the applications of microarrays to lung development, highlighting how this technology has impacted our knowledge of the regulation of this complex process. Microarray data has verified and provided incremental gain to previous studies, caused us to modify some pre-existing beliefs about gene regulation during lung development, and allowed the formulation of novel integrated models to shape future experimentation. We are just beginning to appreciate the existence of stem cells within and/or for the lung. An initial glimpse of the expression patterns of stem cell markers during normal lung development indicates a sobering complexity. Yet the significant level of expression for many of these genes oVers guarded optimism. To many, the importance of understanding lung development and the related biological processes is purely for insight into the aberration of these processes during diseased states. Expression profiling of lung development is already paying dividends in that respect, by providing a novel perspective with which to characterize abnormal gene expression in diseases such as lung cancer. It is important to remember that

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microarray experiments are just experiments like any other (e.g., histology, Western). However, the future for the application of this technology toward understanding lung development and, potentially, lung regeneration is very bright and has plenty of room for growth.

Acknowledgments This work was supported in part by National Institutes of Health grants HL 073745-01 (N.K.) and HL 071885 (T.J.M.). N.K is the Dorothy P. and Richard P. Simmons Chair of Interstitial Lung Diseases.

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Mechanisms and Regulation of Lung Vascular Development Michelle Haynes Pauling and Thiennu H. Vu Department of Medicine and Lung Biology Center University of California, San Francisco San Francisco, California 94143

I. Introduction II. Overview of Pulmonary Vessel Formation A. Development of the Gas Exchange Capillary Network III. Regulation of Lung Vasculature Formation A. Genetic Factors Involved in Lung Vasculature Development B. Genetic Factors Likely to Be Involved in Lung Vasculature Development IV. Epithelial–Mesenchymal Interactions During Lung Vascular Development V. Specification of Arterio-Venous Identity A. Notch Signaling, Shh, and VEGF B. Ephrins and Eph Receptors C. Angiopoietin Ligands and Tie Receptors D. Environmental Regulation of Arterio-Venous Fate VI. Endothelial Stem Cells in Lung Regeneration and Repair VII. Conclusions Acknowledgments References

I. Introduction The developing lung vasculature is a quite complicated yet beautiful orchestration of diVerent cell types, cell–cell interactions, and yet-unknown processes that occur in a reproducible fashion to form the conduit carrying deoxygenated blood to the gas exchange surface of the lungs and oxygenated blood back to the heart to be distributed to the body. We study the mechanisms and regulation of the development of the lung vasculature both to elucidate the secrets of its formation and to discover therapeutic insights into lung diseases. Dissection of the cellular and molecular mechanisms of lung vasculature development will not only teach us about the development of the lungs and other organs, but also may help to design treatment for both impaired lung development in premature infants and lung injuries in adults. In this chapter we summarize current concepts in the formation of the lung Current Topics in Developmental Biology, Vol. 64 Copyright 2004, Elsevier Inc. All rights reserved. 0070-2153/04 $35.00

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vasculature and review the roles of genetic and epigenetic factors that have been implicated in regulating lung vessel development.

II. Overview of Pulmonary Vessel Formation The vasculature is essential for the survival of an organism. Thus, it must develop in a precise and fastidious manner. The conventional hypothesis regarding the development of the lung vasculature is that it arises from two distinct origins (reviewed in Stenmark and Gebb, 2003). The endothelium of the proximal macrovasculature is derived from the pulmonary truncus and develops through angiogenesis, the process by which new vessels branch from existing ones. The endothelium of the peripheral microvasculature is derived through vasculogenesis, the process by which endothelial cells develop in situ from progenitors derived from lung mesenchyme and coalesce to form a primitive vascular plexus surrounding the branching epithelial tubes. The primitive primary plexus then remodels into a mature branching vascular system containing vessels with diVerent sized lumens. This process is usually referred to as angiogenic remodeling and involves pruning of extraneous vessels, lumen enlargement, and recruitment of mural support cells. The proximal and the peripheral vasculatures are subsequently connected to form a complete vascular circuit through development of communications between the central and peripheral systems. The hypothesis of separate vasculogenesis and angiogenesis joined together by fusion was drawn from observations on mouse and human fetuses studied by light microscopy, angiograms, transmission electron microscopy, and scanning electron microscopy of vascular casts. These studies show the anatomy of the developing proximal and distal lung vasculature (deMello and Reid, 2000; deMello et al., 1997; Stenmark and Mecham, 1997). Through analysis of microscopic images it was concluded that peripheral lung vasculature arises in the mesenchyme through vasculogenesis, as evidenced by the observation of isolated vascular lakes around the budding lung epithelial tubes. Central lung vessels were observed to arise as outgrowths from the aortic sac and left atrial chamber, with no initial connection to the peripheral vessels. ‘‘Communications’’ between distal and proximal vessels were observed only at embryonic day 13–14 in the mouse and gestation week 10–11 in the human. In the mouse embryonic lungs, these were visualized as small protrusions from two adjacent vascular segments that appeared to have migrated toward each other and become almost or completely contiguous (deMello et al., 1997). In the human, structures resembling communications between a proximal muscular vessel and a peripheral nonmuscular vessel were seen in tissue sections of 10–11 week gestation lungs (deMello and Reid, 2000).

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Support for vasculogenic formation of pulmonary vessels was initially demonstrated through classical avian embryological studies. Quail lung rudiments were grafted onto chick hosts, and the vessels that developed in the graft were analyzed for their origin by immunostaining for chicken- and quail-specific endothelial cell markers (Pardanaud et al., 1989). The vessels that developed in the grafts were found to be of graft origin, demonstrating that they formed by vasculogenesis from angioblasts intrinsic to the grafts. Similar findings were reported in mice. Subcutaneous allografts of lung rudiments from transgenic mice expressing the -galactosidase gene in endothelial cells into wild-type mice showed that vessels that subsequently developed in the grafts expressed -galactosidase and therefore were of graft origin (Schwarz et al., 2000b). Additional studies using a model of lung development with renal capsule allografts of mouse embryonic lungs confirmed that vessels in the lung grafts develop from endogenous endothelial cells and further demonstrated that host vessels made connections to graft vessels but did not vascularize the grafts independently (Vu et al., 2003). The hypothesis that proximal lung vessels arise by angiogenesis has been called into question. Studies of mice carrying the -galactosidase transgene recombined into the flk-1 (KDR) gene locus, thereby marking endothelial cells by the expression of -galactosidase activity, showed the presence of endothelial cells forming the pulmonary arteries in apparent continuity with both the aortic sac proximally and the peripheral primitive vascular network surrounding the branching lung buds distally (Schachtner et al., 2000). This would favor the model that the central vessels also form by vasculogenesis. This conclusion was further supported by studies of human fetal lungs using immunohistochemical staining to identify blood vessels and serial reconstruction to trace the vessels from proximal to distal regions of the lung (Hall et al., 2000, 2002). These studies showed that pulmonary arteries in human fetal lungs at early as 38 days gestation were continuous both proximally with the heart and peripherally with the plexus of capillaries found around the terminal buds. Alongside airways, capillaries coalesced and formed progressively larger vessels in a distal-to-proximal direction (Hall et al., 2000). Similar findings were observed for the pulmonary veins (Hall et al., 2002). The apparent contradictions in the current models of how proximal vessels form may be due to diVerences in the techniques employed in the studies as well as to terminology. It is clear that there is early continuity between the heart and the proximal lung vessels. The question is whether continuity between the proximal vessels and the peripheral primitive vascular plexus is always present or only develops at a later time. The absence or presence of these connections was used to support angiogenic versus vasculogenic origin of the central vessels. There are several points to consider. One

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is that it may not be possible to detect connections between vascular beds by vascular castings if there are no lumens in these connections. Another point is that what appears to be connections developing between the central and peripheral vessels may actually represent connections developing within the peripheral vascular plexus. Which model of proximal vessel formation is correct remains to be determined.

A. Development of the Gas Exchange Capillary Network During development of the distal lung, a large and intricate capillary network develops surrounding the terminal epithelial sacs to form the gas exchange surface. After birth, the surface area of the distal lung vasculature expands more than 30-fold to accommodate the growth and development of the alveolar surface. The mechanism of the development of this vast capillary network has not been completely defined, but may involve both sprouting angiogenesis and intussusceptive angiogenesis. The latter is an interesting mechanism of capillary network expansion proposed by Burri and colleagues (Burri and Djonov, 2002). It was initially believed that sprouting angiogenesis accounts for the expansion of the postnatal distal lung vasculature, yet repeated observations of lung micrographs were disappointingly lacking in capillary sprouts. Instead, tiny holes in the vasculature were observed in the postnatal rat lung. These holes were not randomly placed, but seemed to occur where the vessels were slightly wider. The reader is referred to the original literature for electron micrographs of the described observations (Burri and Tarek, 1990; CaduV et al., 1986). Review of the literature dating back to 1893 showed that these holes were present in the casts of many diVerent vascular beds, and that Short had proposed that these holes may be linked with vessel growth (Burri and Djonov, 2002; Short, 1950). Burri and colleagues proposed that these holes represented transcapillary pillars and were a mechanism of capillary network growth, and coined the term ‘‘intussusceptive angiogenesis’’ to describe this process. Electron micrographs of consecutive sections of the lung vasculature showed that the holes indeed corresponded to slender transcapillary tissue pillars (Burri and Tarek, 1990). Once the pillar is formed, it can widen to the size of a normal intercapillary mesh and is stabilized by collagen fibrils within its core. Thus, repeated insertions of these slender transcapillary tissue pillars, and their subsequent increase in size, allow the capillary network to grow ‘‘within itself.’’ Intussusceptive angiogenesis was subsequently found to occur in many other vascular beds besides the lungs (Patan et al., 1992). Whether it also occurs in the fetal lung during the early development of the distal gas exchange surface (saccular stage) remains to be determined.

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III. Regulation of Lung Vasculature Formation Many factors, both genetic and epigenetic, act together to accomplish the fidelity of the formation of the lung vasculature essential for the survival of the organism. Genes involved in the development and maintenance of the lung vasculature include transcription factors, peptide growth factors and receptors, extracellular matrix (ECM) components, cell adhesion receptors, and intercellular adhesion molecules (Post and Copland, 2002). These regulating molecules can be secreted, cytoplasmic, or cell surface-associated. They can act through a variety of mechanisms, as an intrinsic switch (on/oV) of cellular behavior, through gradients, giving positional information based on local concentration, or as initiators of cellular cascades in which responder cells in turn influence other cells (for a brief review, see Post and Copland, 2002). Even though many molecules have been described as playing important roles in vessel formation in general, relatively few factors have been shown to actually function in the development of the lung vasculature. It is likely that there are tissue-specific factors that guide the development and patterning of the vasculature bed specific to each organ. In the following sections, we describe the few genetic factors that are known to function in lung vessel development and then describe some of the many factors that are likely to function in the formation of the lung vasculature due to their implication in vascular development in general and their expression in the lungs.

A. Genetic Factors Involved in Lung Vasculature Development 1. Vascular Endothelial Growth Factor Vascular endothelial growth factor (VEGF-A) signaling is an important regulator of embryonic vascular development as well as of postnatal angiogenesis (Ferrara et al., 1992). Targeted disruption of the VEGF-A gene resulted in severe defects in the formation of blood vessels and midgestational embryonic lethality even in the heterozygous state, indicating an important gene dosage eVect (Carmeliet et al., 1996; Ferrara et al., 1996). Development of endothelial cells was deficient, and those that developed were disorganized and scattered. VEGF-A acts through two tyrosine kinase receptors, Flt-1 (VEGFR-1) and Flk-1 (VEGR-2). Both receptors are important for embryonic vascular formation, as targeted inactivation of either gene resulted in embryonic lethality due to defective vascular development. Flk-1 is expressed in the vascular endothelium and is the earliest known marker for endothelium and endothelial precursors (Flamme et al., 1995; Millauer et al., 1993; Terman et al., 1992). Null alleles of the Flk-1 gene in

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mice resulted in lack of a vasculature and very few endothelial cells, suggesting that it functions in the diVerentiation and/or proliferation of these cells (Shalaby et al., 1997). In contrast, mice deficient in Flt-1 had excess endothelial cells that were not organized into normal tubular networks (Fong et al., 1995). Thus, Flt-1 may function to downregulate VEGF-A activity. The reader is referred to the review by Ferrara and colleagues for a comprehensive review of the biology of VEGF-A and its receptors (Ferrara et al., 2003). A role for VEGF-A signaling in lung vascular development is suggested by the pattern of expression of VEGF-A and its receptors. Epithelial cells express VEGF-A during the process of lung morphogenesis, and the expression of its receptors is found in mesenchymal cells (Greenberg et al., 2002; Millauer et al., 1993; Ng et al., 2001; Peters et al., 1993; Schachtner et al., 2000). The complementary expression of this ligand–receptor system suggests the involvement of VEGF-A in the regulatory interactions between epithelial and vascular cells during lung morphogenesis. Because mice with a null allele of the VEGF-A gene die in early gestation, analysis of lung vascular development in VEGF-A deficiency awaits conditional and/or tissue-specific gene inactivation. However, several published studies using both gain- and lossof-function approaches in diVerent in vitro and in vivo models showed that VEGF-A did indeed play a role in lung vascular development. Treatment of cultured embryonic lung explants with VEGF-A-coated beads stimulated angiogenesis in the mesenchyme surrounding the grafted beads (Healy et al., 2000). Constitutive overexpression of VEGF-A in lung epithelium during embryogenesis in transgenic mice resulted in abnormal lung development with dilated epithelial tubes and increased peritubular vascularity (Zeng et al., 1998). Even though these studies may not be physiological, they showed that VEGF-A is capable of stimulating endothelial cell development in lung mesenchyme. Particular isoforms of VEGF-A expressed are also critical. At least three diVerent isoforms of VEGF resulting from alternative splicing are expressed in the mouse embryonic lungs: VEGF120, VEGF164, and VEGF188 (Ng et al., 2001). These isoforms diVer in their ability to bind to the ECM. VEGF120 does not bind ECM and is freely diVusible; VEGF188 is normally bound to the cell surface or the ECM; and VEGF164 has intermediate properties. All of these three isoforms are expressed in the mouse embryonic lungs. However, the expression of VEGF188 increases significantly after E16, during the period of distal lung development. Mice genetically engineered to express only the VEGF120 isoform (VEGF120/120 mice) showed impaired distal lung development, with reduced number of distal airspaces and capillaries (Ng et al., 2001). Scanning electron microscopic study of lung vascular casts from these mice confirmed that the density of the peripheral lung vasculature was reduced, whereas the proximal vessels were not aVected (Galambos et al., 2002). Thus, the ECM-bound isoforms of VEGF are important in the development of the distal lung

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vasculature. Using tetracycline-regulated tissue-specific promoters to temporally and spatially direct the overexpression of VEGF164 during embryonic lung development, Akeson and colleagues showed that the lung vasculature developmental response to VEGF164 stimulation was time- and spacedependent (Akeson et al., 2003). Overexpression of VEGF164 in proximal epithelial cells caused capillary ingrowths, resulting in evaginations of proximal airways epithelium. Overexpression of VEGF164 in distal lung epithelium caused decreased peripheral capillary network formation and decreased distal airspace branching. Thus, the temporal and spatial pattern of expression of VEGF during lung development is critical for both normal development and patterning of the lung vasculature, as endothelial cells at diVerent locations and times during lung development respond diVerently to the same factor. VEGF-A signaling is also important for the capillary network growth accompanying alveolarization. Treatment of newborn rats with a VEGF receptor tyrosine kinase inhibitor resulted in decreased capillary density and impaired alveolar formation (Le Cras et al., 2002). Further research identifying the function and mechanisms of action of VEGF and VEGF receptors in lung vascular development is needed to further our understanding of the role of this important angiogenic factor in the development of the lung vasculature. Studies utilizing conditional and tissue-specific gene inactivation models will be very informative.

2. Endothelial Monocyte Activating Polypeptide II Endothelial monocyte activating polypeptide II (EMAP II) was initially identified as a tumor-derived cytokine with pro-inflammatory properties (Kao et al., 1992, 1994). It was subsequently found to have antiangiogenic properties (Schwarz et al., 1999b). EMAPII is expressed in the developing lungs, and its expression level decreases in the late fetal stage, a period of increased vascular formation (Schwarz et al., 1999a), suggesting that it may act to negatively regulate lung vessel development. Indeed, treatment of subcutaneous embryonic lung allografts with exogenous EMAP II resulted in decreased vascularity in the lung grafts and alteration of epithelial development. Epithelial morphology was abnormal, and there was increased cell apoptosis and decreased expression of the distal epithelial cell diVerentiation marker SP-C (Schwarz et al., 2000a). In contrast, treatment with an EMAP II neutralizing antibody resulted in increased vascularity and increased SP-C expression (Schwarz et al., 2000a). Because the precise patterning of the lung vasculature would require both positive and negative regulation, EMAP II may be a critical negative regulator of lung vessel development. However, its role in vivo remains to be determined.

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3. Transforming Growth Factor-b Family The transforming growth factor- (TGF-) family of cytokines is composed of secreted proteins that signal through serine/threonine kinase receptors. TGF- signaling is important in the development of many tissues, including the vasculature (Mummery, 2001). TGF- family members, including TGF1, TGF-2, and TGF-3, as well as TGF- receptors, are expressed in the developing lungs, but their roles in lung development, especially in lung vascular development, have not been completely defined. Overexpression of TGF-1 in developing lung epithelium resulted in altered vascular and epithelial development (Zeng et al., 1998; Zhou et al., 1996). Epithelial development was arrested at the pseudoglandular stage. Expression of VEGF and its receptor, FLK-1, was decreased, vascular development was altered with reduced number of vessels, and capillaries were not found in proximity to the epithelium. Because TGF-1 may aVect the development of epithelial cells, endothelial cells, or both, it was unclear from this study whether TGF-1 had a direct eVect on lung vascular development or a secondary eVect through an eVect on epithelial development. Targeted inactivation of TGF-1 resulted in either early embryonic lethality with defective vascular and hematopoietic development (Martin et al., 1995) or an inflammatory phenotype in those that survive (Kulkarni et al., 1993; Shull et al., 1992). A lung developmental phenotype has not been described. Targeted disruption of other TGF- ligands, TGF-2 and TGF-3, resulted in abnormalities in lung development (Kaartinen et al., 1995; Sanford et al., 1997). However, vascular development was not analyzed in these mouse mutants. Further studies are needed to clarify the role of TGF- family in lung vascular formation.

4. Wnt Signaling Pathway The Wnt family of proteins are conserved, secreted signaling molecules that regulate cell–cell interactions during embryogenesis. Wnt proteins bind to receptors of the Frizzled family on the cell surface. A majority of Wnt ligands act through the canonical pathway involving beta-catenin, which mediates transcription of Wnt target genes (Wodarz and Nusse, 1998). Two members of the Wnt family, Wnt5a and Wnt7b, have been shown to function in lung development. Wnt5a null mice demonstrated a perinatal lethality phenotype with impaired distal lung development (Li et al., 2002). There was delayed maturation of distal lung epithelial cells and increased proliferation of both epithelial and mesenchymal compartments, and the walls of the distal airspaces were thickened. Pulmonary capillaries developed normally in these mice, but because of the thickened mesenchyme, the capillaries were not apposed to the epithelium, resulting in a poor gas exchange surface.

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Wnt7b null mice also died perinatally from respiratory failure (Shu et al., 2002). The lungs of these mice were poorly inflated and had hemorrhages. Endothelial cell development was not aVected; however, the large pulmonary vessels were dilated and had defective smooth muscle cell layers. There were holes in the smooth muscle layers with herniation of the endothelial lining, which was probably the cause of the hemorrhages. Thus, Wnt7b may regulate the development and/or recruitment of the mural support cells during maturation of the lung vessels. 5. Forkhead Box Transcription Factors Forkhead box (Fox) proteins are a family of transcription factors that play diverse roles in development due to their roles in the regulation of cell cycle progression, cell survival, expression of diVerentiated genes, and cell metabolism (Carlsson and Mahlapuu, 2002). Mice homozygous for a targeted allele of the Forkhead Box f1 (Foxf1) transcription factor showed midgestational embryonic lethality and defects in mesodermal diVerentiation and cell adhesion (Mahlapuu et al., 2001b). Interestingly, heterozygous mice developed further but showed a variety of foregut developmental defects, including malformed lungs and perinatal lethality (Kalinichenko et al., 2001; Mahlapuu et al., 2001a). Lung vascular development was found to be impaired in the heterozygous mice expressing low levels of Foxf1 protein. In the lungs of these mice, the number of distal lung capillaries was decreased, and tight junctions between endothelial and distal lung epithelial cells were disrupted, leading to hemorrhage (Kalinichenko et al., 2001). Interestingly, these lungs also showed decreased expression of VEGF-A, suggesting that Foxf1 may regulate pulmonary vascular development through regulation of VEGF-A expression. 6. Insulin-like Growth Factors Insulin-like growth factors I and II (IGF-I, IGF-II) are polypeptide growth factors related to proinsulin. These factors can regulate cell proliferation, survival, and diVerentiation (Rechler, 1988). IGFs signal through the tyrosine kinase type I IGF receptor. IGFs and the receptors are expressed in the lungs during development in epithelial and endothelial cells, as well as in other mesenchymal cells (Han et al., 2003; Retsch-Bogart et al., 1996). Treatment of cultured lung explants with an IGF-I neutralizing antibody resulted in decreased lung growth, reduced branching of the epithelium, and inhibition of endothelial cell development (Han et al., 2003). It was unclear whether IGF-I acts directly on endothelial cells to aVect their development or indirectly through its actions on other cell types, as the type I receptor was expressed in a wide variety of cells in the embryonic lungs (Han et al., 2003;

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Retsch-Bogart et al., 1996). However, IGFs have been shown to have angiogenic properties (Grant et al., 1993), suggesting that they may stimulate lung vascular development directly. Mice homozygous for a targeted null mutation in the type I IGF receptor died at birth due to respiratory failure (Liu et al., 1993), supporting a role for this receptor in lung development. Deficiency of either IGF-I or IGF-II also caused perinatal lethality in some of the homozygous mice (Baker et al., 1993; Liu et al., 1993). In light of the results by Han et al. (2003) on the role of IGF-I in lung endothelial cell development in organ culture, it may be informative to analyze lung vascular development in mice with null mutations in the IGF signaling pathway. B. Genetic Factors Likely to Be Involved in Lung Vasculature Development There are many genes that have not been directly shown to function in lung vascular development, yet are likely to do so by virtue of their roles in vascular development in other systems and their pattern of expression in the developing lungs. We review some of these genes in the following sections. 1. Notch Signaling Notch signaling is used by both vertebrates and invertebrates to control cell fates and thus regulate the development of many tissues through local cell– cell interactions (Artavanis-Tsakonas et al., 1999). Notch signaling has been implicated in the development of the vasculature (reviewed in Iso et al., 2003). The role of Notch signaling in the vasculature may not be in the formation of endothelial cells but in their morphogenesis, especially in angiogenic remodeling. Mice homozygous for a targeted null mutation of the Notch ligand Jagged-1 gene died around E10.5 with hemorrhage and defects in vascular remodeling in both the yolk sac and the embryos (Xue et al., 1999). Notch-1 null mice also had vascular defects in many tissues and lack of angiogenic remodeling of the primary vascular plexus in the yolk sac. These defects were worsened when combined with null mutations in Notch-4 (Krebs et al., 2000). Mice with a hypomorphic mutation of the Notch-2 gene had abnormal development of the capillary tuVs of the kidney glomeruli (McCright et al., 2001) Many Notch ligands and receptors are expressed in the developing lungs, both in epithelial and mesenchymal cells and in endothelial cells (Beckers et al., 1999; Post et al., 2000; Rao et al., 2000; Taichman et al., 2002). Thus, this signaling pathway may play an important role in the development of the lung vasculature. Unfortunately, targeted inactivation of many of the Notch ligands and receptors reported so far resulted in early embryonic lethality, precluding analyses of lung development in these mice.

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2. Ephrins and Eph Receptor Ephrins and their tyrosine kinase receptors Eph are cell surface ligands and receptors with bidirectional signal transduction properties. Ephrins are divided into two subclasses, ephrinA and ephrinB, based on whether they are associated with the cell membrane by GPI anchor (ephrinA) or transmembrane domains (ephrinB). Members of the ephrinA subclass bind nonselectively to EphA receptors, and those of the ephrinB subclass bind to EphB receptors. The ephrins and their receptors play critical roles in many developmental processes, including vascular development (Cheng et al., 2002; Gauthier and Robbins, 2003). They provide migration cues for cells and cellular processes such as nerve growth cones, as well as cues for the establishment of tissue borders and spatial organization. EphrinA1 can stimulate angiogenesis in vivo and is chemotactic for endothelial cells in vitro (Pandey et al., 1995). Targeted gene inactivation of several ephrins and Eph receptors, including ephrinB2, EphB4, and Eph2/Eph3, resulted in defects in angiogenic remodeling and sprouting (Adams et al., 1999, 2001; Gerety et al., 1999; Wang et al., 1998). In the mutant mice, remodeling of the primary capillary plexus in the yolk sac and the head was impaired, and angiogenic ingrowths of vessels destined to vascularize the neural tube did not occur. Few studies have reported the expression pattern of this important ligand/ receptor system in the developing lungs. EphrinA1 was reported to be expressed in rat lung epithelial cells in late gestation (Takahashi and Ikeda, 1995). EphrinB2 was expressed in endothelium of the pulmonary arteries of human fetal lungs, and EphB4 was expressed in both arteries and veins (Hall et al., 2002). A comprehensive study of the expression pattern of ephrins and Eph in the developing lungs would be very informative.

3. Roundabouts and Slits Slit proteins and their receptors Roundabout (Robo) were initially identified as negative regulators of neuronal migration (Wong et al., 2002). Using bioinformatics data mining to identify endothelium-specific genes a robo receptor, magic roundabout or robo4, was shown to be expressed mainly on endothelial cells, and particularly at the site of active angiogenesis in the adult (Huminiecki et al., 2002). Robo4 was subsequently found to be expressed in the endothelium of the embryonic vasculature as well as in the adult; it was also found that interaction of Robo4 with its ligand Slit inhibited endothelial cell migration in vitro (Park et al., 2003). Several members of the Robo and Slit families are expressed in the embryonic mouse lung in a temporally and spatially regulated manner (Anselmo et al., 2003), suggesting that they may play a role in lung vascular development.

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4. Semaphorins and Neuropilins Semaphorins are a family of proteins initially identified by their ability to provide axonal repulsive guidance signals by causing growth cone collapse. They were subsequently found to have diverse functions, including the activation of immune cells, regulation of tissue morphogenesis, and vascular development (Goshima et al., 2002). The semaphorins act through two families of receptors, the neuropilins and the plexins. Neuropilins bind to the class 3 subfamily of semaphorins as well as to certain isoforms of vascular endothelial growth factor (VEGF-A). Neuropilins also function in the development of the nervous and vascular systems (Neufeld et al., 2002). Semaphorin 3A (Sema3A) inhibited endothelial cell migration in vitro and also inhibited capillary sprouting from cultured rat aortic rings (Miao et al., 1999). Targeted inactivation of the Sema3A gene resulted in disrupted vascular development in the CD-1 strain background (Serini et al., 2003). Sema3A acts on endothelial cells through inhibition of integrin function. Sema3A regulated integrinmediated cell adhesion and migration in cultured endothelial cells, as well as vascular remodeling in chick embryos (Serini et al., 2003). Targeted inactivation of the Neuropilin-1 gene also resulted in abnormal vascular development (Kawasaki et al., 1999). Capillary angiogenesis into the neural tube was absent, formation of the great vessels was defective, and the yolk sac vasculature had abnormal morphology. The vascular developmental eVect of Neuropilin-1 may be mediated through its being a receptor for VEGF-A (Gu et al., 2003). Semaphorins and Neuropilins are expressed in the developing lungs in a temporally and spatially regulated manner (Kagoshima and Ito, 2001). The semaphorins were shown to regulate branching morphogenesis of cultured embryonic lung explants (Ito et al., 2000; Kagoshima and Ito, 2001). However, the roles of the semaphorins and neuropilins in lung vascular development are not known.

5. Angiopoietins and Tie Receptors The angiopoietins are angiogenic growth factors that specifically act upon vascular endothelium (Davis and Yancopoulos, 1999; Loughna and Sato, 2001a). Their specificity is due to the fact that their receptors, the tyrosine kinases Tie-1 and Tie-2, are expressed on endothelial cells. The angiopoietins function in the angiogenic remodeling of the primitive vascular plexus. A null mutation of either Angiopoietin-1 or Tie-2 causes embryonic lethality in mice, with defects in vascular development. The primitive vascular plexus developed in the mutant embryos, but was not remodeled into mature vessels and tended to regress (Dumont et al., 1994; Sato et al., 1995; Suri et al., 1996). Null mutation in the receptor Tie-1 caused perinatal lethality due to respiratory distress (Sato et al., 1995). The basic defects in these mice appear

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to be defective vessel integrity leading to hemorrhage and edema. Null mutation in the angiopoietin-2 gene did not cause defects in embryonic vascular development, but aVected lymphatic development and postnatal angiogenesis in the eye (Gale et al., 2002). The current model states that the angiopoietins mediate the interactions between endothelial cells and the underlying support cells that are important for vessel maturation and stabilization. A role for the angiopoietins in lung vascular development is suggested by their expression in the embryonic lungs. Expression of Ang-1 and Ang-2 in the developing mouse lung between gestational day E9.5 and postnatal day 1 was demonstrated by reverse transcription polymerase chain reaction (RT-PCR) and southern blot analysis (Colen et al., 1999). However, detailed temporal and spatial expression patterns of the angiopoietins and their receptors in the developing lungs have not been reported.

IV. Epithelial–Mesenchymal Interactions During Lung Vascular Development In the lungs, blood vessels have a very precise relationship with the epithelium. Pulmonary arteries run alongside and branch with the conducting airways. Pulmonary capillaries surround and are apposed to the alveolar epithelium to form an eVective gas exchange surface. Pulmonary veins initially run along the interlobular septum, then continue along the bronchovascular bundles. The precise alignment of airways, respiratory epithelium, and blood vessels requires exquisite coordinated development of the diVerent tissue components. During distal lung development, it has been noted that distal epithelial cells that are found closely apposed to endothelial cells tended to have a flattened morphology. It was thus postulated that endothelial cells induce morphological diVerentiation of the apposing epithelial cells to form the gas exchange surface. The requirement for tissue interactions is obvious, but the molecular and cellular mechanisms underlying these interactions are far from being clearly defined. Gebb and Shannon showed that lung epithelium was necessary for the development of endothelial cells in lung mesenchyme (Gebb and Shannon, 2000). When intact E13 mouse lungs were placed in organ culture, endothelial cells, assayed by their expression of VEGFR-2 (Flk-1), developed in the mesechyme. When lung epithelium was removed, the cultured lung mesenchyme deteriorated and few Flk-1-positive cells developed. However, when isolated mesenchyme was recombined with lung epithelium prior to culture, mesenchyme grew and Flk-1-positive cells developed. Thus, lung epithelium was necessary for the growth of lung mesenchyme and for the development of endothelial cells. However, it was not clear from these experiments whether epithelium was necessary for the

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development of endothelial cells specifically or for the growth and survival of the mesenchyme in general. The expression pattern of some ligand–receptor systems involved in the development of blood vessels in the lungs suggests direct paracrine or cell– cell interactions between epithelial and endothelial cells during their development. For example, VEGF-A is expressed by epithelial cells and the receptors are expressed by mesenchymal cells, presumably endothelial cells or their precursors. Thus, epithelium-derived VEGF-A may stimulate endothelial cell development in the surrounding mesenchyme to coordinate blood vessel development with epithelial development. The developing vasculature will in turn support epithelial growth by providing circulating oxygen and nutrients. Whether endothelial cells also signal back via paracrine growth, survival, or morphogenetic factors to influence epithelial development is an attractive hypothesis and remains to be determined. Another signaling system that may play a role in epithelial–mesenchymal interactions during lung vascular development is the sonic hedgehog (Shh) signaling pathway. Shh acts as a morphogen in the development of many organs, including the lungs. During lung development, Shh is expressed by epithelium, and its receptor Patch-1 (Ptc) is expressed in the surrounding mesenchyme. Targeted null mutations in the Shh gene resulted in hypoplastic lungs secondary to impaired epithelial branching morphogenesis, coupled with increased cell death and decreased cell proliferation in the mescenchyme (Litingtung et al., 1998; Pepicelli et al., 1998). Shh’s eVects on branching morphogenesis may be secondary to its regulation of mesenchymal cell growth and/or its regulation of the spatial restriction of FGF-10 expression in the mesenchyme. Because Shh is important for mesenchymal cell growth, it may also regulate endothelial cell development. It is of interest that Shh has been shown to induce angiogenesis in vivo due to its ability to induce the expression of VEGF-A and angiopoietins (Pola et al., 2001). In addition, Shh induces the expression of Foxf1 in the developing lung. Foxf1 mRNA was absent in Shh null lungs, and exogenous Shh stimulates transcription of Foxf1 in cultured lung mesenchyme (Mahlapuu et al., 2001a). Because Foxf1 may regulate lung vessel formation (see previous discussion), this could be another means by which epithelium-derived Shh coordinates vascular development in the mesenchyme.

V. Specification of Arterio-Venous Identity Arteries and veins are both formed from vascular endothelial tissue, yet must perform diVerent functions of critical importance to blood flow. Arteries in general carry blood away from the heart under high pressure, while veins return blood to the heart under low pressure. Arteries and veins are

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morphologically distinct as well, with diVerent supporting cells ensheathing their endothelial tubes. In early development, before recruitment of supporting cells, vessels destined to be either arteries or veins are morphologically indistinguishable, and it has long been thought that arterio-venous diVerentiation depended on hemodynamic forces with the beginning of circulation. However, studies showed that there are molecular diVerences between arterial and venous endothelial cells even before final blood vessel identity (Wang et al., 1998). Several signaling pathways have also been found to play a role in arterio-venous diVerentiation (Torres-Vazquez et al., 2003). At present, many questions about arterio-venous specification remain unanswered. What are the roles of environmental and genetic factors in arterio-venous specification? What are these factors? At what point in lung development does this specification occur? The regulation of the specification of arteries and veins in lung vascular development is unknown. In this section, we discuss some of the signaling pathways that are involved in arterio-venous identity in general, as these factors may also play a role in arterio-venous specification in lung vascular development. We also describe studies that suggest that environmental eVects may also be required for arterio-venous identity.

A. Notch Signaling, Shh, and VEGF Notch signaling pathways are involved in directing arterial versus venous cell fate (reviewed in Torres-Vazquez et al., 2003). Notch-1, Notch-4, Delta-4, Jagged-1, and Jagged-2 are expressed by arterial but not venous endothelial cells (Shutter et al., 1995; Villa et al., 2001). Studies in zebrafish have demonstrated that Notch signaling promoted arterial endothelial cell fate (Lawson et al., 2001). When Notch signaling was inhibited by the expression of a dominant-negative mutant of Xenopus laevis Suppressor of Hairless protein, a downstream eVector of the Notch signaling pathway, there was loss of arterial endothelial cell marker expression and ectopic expression of venous endothelial cell marker. Similar findings were noted in the Notch signaling-deficient zebrafish mutant mindbomb. In contrast, constitutive activation of Notch signaling resulted in increased expression of arterial endothelial cell marker and suppression of venous endothelial marker. Another study in zebrafish demonstrated that Shh activity induced VEGF expression, which acted upstream of Notch to determine arterial cell fate (Lawson et al., 2002). Inhibition of VEGF expression by antisense morpholino oligos resulted in loss of arterial endothelial cell marker, ectopic expression of venous endothelial cell markers, and malformation of the aorta and cardinal vein. In contrast, exogenous VEGF induced ectopic expression of arterial markers. Notch signaling was found to be downstream of the VEGF eVect.

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Exogenous VEGF had no eVect in the Notch signaling-deficient mutant mindbomb, and activation of Notch signaling rescued the defects caused by inhibition of VEGF. VEGF signaling may also play a role in arteriovenous diVerentiation in mammals. In mice, VEGF has been shown to guide arterial specification and patterning of arteries (Mukouyama et al., 2002). Additionally, overexpression of VEGF in the mouse heart resulted in increased arterial vessels and a decrease in venous endothelial cells (Visconti et al., 2002). Whether the VEGF/Shh/Notch pathways regulate arterio-venous diVerentiation in lung vascular development remains to be determined.

B. Ephrins and Eph Receptors In early vascular development, the expression of Ephrin B2 and its receptor EphB4 shows arterial and venous specificity. EphrinB2 is expressed in arterial endothelial cells, and its receptor EphB4 is expressed in venous endothelial cells (Gale and Yancopoulos, 1999; Wang et al., 1998). That diVerential EphB4 and EphrinB2 expression is established prior to the formation of arteries and veins suggests that the Eph/Ephrin system may induce or maintain arterio-venous diVerentiation. Targeted disruption of either EphrinB2 or EphB4 resulted in normal vasculogenesis but disruption of angiogenic remodeling of both veins and arteries (Adams et al., 2001; Gerety et al., 1999; Wang et al., 1998). These results are intriguing, since EphrinB2 is not expressed in veins and EphB4 is not expressed in arteries. Thus, the development of arteries and veins must be dependent upon each other. Because the ephrins and Eph receptors are capable of bidirectional signaling, interactions between ligand and receptor may lead to signal transduction in both cell types. Signaling through the ligand may lead to a diVerent response compared to signaling through the receptor. In vitro studies showed that forward arterial-ephrinB2 to venous-EphB4 signaling may suppress sprouting and proliferation of venous endothelial cells, whereas reverse venous-EphB4 to arterial-ephrinB2 signaling may promote angiogenic sprouting and proliferation of arterial endothelial cells (Hamada et al., 2003; Kim et al., 2002; Palmer et al., 2002). Whether ephrins and their receptors play a role in arterio-venous specification in lung vascular development is unknown. Notably, EphrinB2 and EphB4 do not display arterial and venous specific expression in the human fetal lung vasculature as seen in early mouse embryonic vascular development. Instead, EphrinB2 is found in arteries, but EphB4 is found in both arteries and veins and throughout the capillary bed (Hall et al., 2002).

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C. Angiopoietin Ligands and Tie Receptors Angiopoietins may regulate arterio-venous diVerentiation by modulating arterial development induced by VEGF-A. Overexpression of VEGF in cardiac muscle in transgenic mice caused increased capillary growth. Some of these were arterial vessels, as they expressed the arterial marker ephrinB2 (Visconti et al., 2002). The ratio of arterial to venous vessels induced depended upon whether Angiopoietin-2 was also overexpressed. Concomitant overexpression of Angiopoietin-2 with VEGF-A decreased the number of vessels expressing arterial markers. Notably, lack of both Ang-1 and the receptor Tie-1 resulted in specific ablation of the right hand side venous system (Loughna and Sato, 2001b). Additional studies are necessary to further elucidate the role of angiopoietins in regulation of arterio-venous fate and vascular symmetry.

D. Environmental Regulation of Arterio-Venous Fate Whether arterio-venous diVerentiation is controlled by genetics or environment remains controversial. The studies described previously suggest that genetic factors are necessary and suYcient for arterio-venous fate decisions. However, these studies did not take into account the roles of physical forces. Several studies point to the environment as a potential influence on arterio-venous determination. In the first study, embryonic artery or vein segments were grafted from quail donors into chick hosts (Moyon et al., 2001). The arterial and venous identity of donor cells was assessed through detection of Tie-2 (vein-specific) or ephrinB2 and Neuropilin-1 (artery-specific). Cells from younger donors were able to populate both recipient arteries and veins and assumed the appropriate molecular identity despite their origin. This plasticity progressively decreased when vascular segments were grafted from older donors. After E11, arterial donor grafts contributed only to recipient arteries and venous donor grafts contributed only to recipient veins. However, when isolated from the vessel wall, quail aortic endothelium from older embryos was able to colonize both host arteries and veins (Moyon et al., 2001; Othman-Hassan et al., 2001). Thus, the endothelium remains plastic with regard to arterio-venous diVerentiation. In another study using time-lapse video microscopy to study arteriovenous diVerentiation in chick embryo yolk sacs, Le Noble and colleagues observed small arterial vessels disconnecting from the arterial tree and reconnecting to the venous system (Le Noble et al., 2004). This suggested

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that endothelial plasticity is needed for normal vein development. When blood flow through the yolk sac vascular bed was disturbed by various means, formation of the primitive vascular plexus occurred normally. However, arterio-venous patterning was perturbed. Arterio-venous diVerentiation did not occur in the absence of flow. When flow was reversed, veins form in areas normally occupied by arteries and vice versa. Thus, arteriovenous identity is plastic and can be determined by environmental factors such as hemodynamic forces.

VI. Endothelial Stem Cells in Lung Regeneration and Repair It has long been held that the adult lungs do not regenerate. After significant injury, the lungs may become either emphysematous or fibrotic instead of regenerating functional tissues. It is possible that the capacity for growth of the adult lung epithelial and endothelial cells is limited, and therefore a large area of injury cannot be repaired. If this is the case, the supply of epithelial or endothelial stem cells may help regenerate or repair lung tissues. Some studies suggested that bone marrow stem cells might contribute to lung regeneration and repair. Studies in mice showed that bone marrow-derived stem cells can be incorporated into lung epithelium (Kotton et al., 2001; Krause et al., 2001). Similar findings were found in humans following hematopoietic stem cell transplantation. Lung specimens from female patients who had been treated with stem cell transplants from male donors were examined for the presence of male cells containing the Y chromosome (Suratt et al., 2003). Significant rates of both epithelial (2.5–8.0%) and endothelial (37.5–42.3%) chimerism were found. However, in these studies, it was not determined whether the bone marrow cells diVerentiated into lung epithelial and endothelial cells or only fused with existing lung cells. If bone marrow stem cells can diVerentiate into lung epithelial and endothelial cells, it suggests that bone marrow stem cells may be able to repair or regenerate lungs. Recent studies suggested that this might be a possibility. In a model of elastase-induced lung damage, treatment with all-trans retinoic acid (ATRA) and/or granulocye colony stimulating factor (G-CSF) resulted in reduced emphysematous changes and an associated increase in the number of bone marrow-derived cells in the alveoli (Ishizawa et al., 2004). The authors suggested that ATRA and G-CSF mobilized bone marrow progenitor cells, which subsequently contributed to lung regeneration. In a similar study, there was an increased number of bone marrow-derived progenitors both in the circulation and at sites of inflammation in the lungs following lipopolysaccharide (LPS)-induced lung injury (Yamada et al., 2004). In the lungs, these cells display epithelial and endothelial cell markers, suggesting that the

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bone marrow-derived cells diVerentiated into lung epithelial and endothelial cells. Suppression of the bone marrow by sublethal irradiation prior to the administration of LPS resulted in emphysematous changes. Thus, bone marrow-derived progenitor cells may contribute to lung repair after LPSinduced lung injury. However, another study reported that bone marrowderived cells did not contribute to compensatory lung growth after pneumonectomy (Voswinckel et al., 2003). In mice, left-sided pneumonectomy resulted in growth of the right lung. Studies of lung sections did not show the presence of bone marrow-derived cells contributing to the right lung parenchyma. There were bone marrow-derived cells in the lungs, but they were determined to be leukocytes. The diVerences in these studies may be dependent upon the diVerences in the models, compensatory lung growth versus lung repair after injury. These preliminary studies give us hope for using epithelial and endothelial stem cells for the repair of damaged lungs. Further studies identifying resident stem cells in the adult lungs and the conditions to promote their growth, as well as further studies in the use of bone marrow-derived stem cells, will be necessary before this hope can become a reality.

VII. Conclusions We have reviewed in this chapter part of our current understanding of the mechanisms and regulation of lung vascular development. Many questions still remain. The cellular and molecular mechanisms of actions of factors known to influence lung vascular formation remain to be defined. The role in lung vascular development of other factors shown to function in vascular formation in general needs to be characterized. In addition, since the patterning of the vasculature is specific to each tissue, there may be organspecific factors that direct the formation of the vasculature in that organ. Such factors, if they exist in the lungs, remain to be identified. The mechanism by which the pattern of vessel formation is coordinated with airway development also needs to be characterized. The question of how arterio-venous diVerentiation is determined precisely so that a functional circuit is formed and arterio-venous malformations are avoided also needs to be answered. Organ-related diVerences in endothelial cells may also exist. Lung endothelium may need to acquire specific characteristics that are critical for its function. The regulation of the expression of organ-specific endothelial cell phenotypes is not well defined. Last, whether adult endothelium is capable of reiterating developmental pathways to eVect lung regeneration and repair is another unanswered question. If endothelial stem cells are necessary, it will be important to determine if there are such cells in the adult lungs and how

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to stimulate their growth, or to determine if bone marrow-derived stem cells can assume the function. With recent advances in investigational approaches and rise in interest in development of the lung vasculature, it is to be hoped that these questions will be answered in the near future.

Acknowledgments We thank the authors referenced in this chapter for their inspirational work and observations and oVer our apologies to the authors whose work we have not been able to cite. This work was supported by National Institutes of Health grants to T.H.V. (HL69925, HL075680, and HL073823).

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The Engineering of Tissues Using Progenitor Cells Nancy L. Parenteau,* Lawrence Rosenberg,{ and Janet Hardin-Young * *Amaranth Bio, Inc. Watertown, Massachusetts 02472 { Division of Surgical Research McGill University Montreal, Quebec H3G 1A4, Canada

I. The Parenchyma: An Interactive Cell Population II. A Working Hypothesis for Understanding Parenchymal Cell Interaction and Response III. The Epidermis A. Properties of the Epidermal Keratinocyte Population B. Modulation of the Population C. Regulation of the Epidermal Cell Compartments D. When Skin Fails to Heal IV. The Liver A. The Liver Cell Population B. The Development and Regulation of the Hepatocyte Phenotype C. The Importance of Tissue Architecture D. Liver Repopulation Studies E. When Pancreas Turns to Liver F. Models of Liver Injury and Disease and Activation of the Progenitor Pool G. Why Hepatocytes Are Hard to Handle In Vitro V. The Pancreas and Generation of Pancreatic Islets A. Evidence of Neogenesis in the Pancreas B. When Regeneration Is Not Neogenesis C. Stimulation of the Progenitor Pool D. Islet Neogenesis During Development VI. Conclusions and Future Directions References

The ‘‘engineering’’ of a tissue implies that it can be constructed by assembling the necessary components. However, tissues are formed through an evolving, interactive process, not through a collection of parts. This chapter focuses on the biology of the progenitor cell, the native precursor to new tissue, and its role in neogenesis, or the de novo generation of functional tissue. We present a working hypothesis for the generation of parenchymal cell populations and use this hypothesis as a basis for analysis of three parenchymal populations, epidermal cells, hepatocytes of the liver, and Current Topics in Developmental Biology, Vol. 64 Copyright 2004, Elsevier Inc. All rights reserved. 0070-2153/04 $35.00

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pancreatic islets, with a view toward what impact this information will have on the development of cell therapies. By comparing developmental processes, response to injury and disease, and behavior in vitro, we conclude that the adult progenitor cell retains the potential for substantial growth and organ neogenesis and that its biological properties make it the cell of first choice for the engineering of tissues. C 2004, Elsevier Inc.

I. The Parenchyma: An Interactive Cell Population Virtually all parenchyma, or functional tissues of an organ, manifest as populations of cells: mature, multipotent, highly diVerentiated, slow-cycling, proliferative, and apoptotic. The cell population is dynamic and, as we will present, remarkably self-regulated through both autocrine and paracrine parenchymal interaction. This is not to say that accessory components such as connective tissue cells, extracellular matrix, or infiltrating inflammatory cells do not play a role, but in many cases in the adult, it will be a modulatory one. We review the evidence for a progenitor cell compartment in the mature organ and the potential of the adult progenitor cell. Our analysis examines progenitor regulation and parenchymal interaction in three adult organ systems, skin, liver, and pancreas, with a view toward what impact this information has on the generation of epidermis, hepatocytes, and pancreatic islets for clinical therapy. Also, by examining more than one tissue, we arrive at some common principles that are likely to govern the ‘‘engineering’’ of a living therapy or the ability to aVect organ regeneration in situ. The body uses mechanisms of repair, regrowth, and neogenesis to reestablish tissues. Analysis of cell populations requires that we distinguish between growth and neogenesis. Regeneration, as the term is often used, does not distinguish between these processes. However, this distinction is important in our interpretation of data and in our strategy for engineering tissues. Most adult parenchyma are capable of both processes, and the cell response depends on the physiology of the remaining parenchyma and the extent of damage or loss of function. Repair and regrowth most often first involves the action of mature cells in concert with inflammatory cells, and other ‘‘accessory’’ cells such as dermal fibroblasts in skin and stellate cells in the liver. However, mature parenchymal cells are the result of neogenesis, and are neither the natural creators nor the sustainers of the organ parenchyma. While they have a limited and, in the case of the liver, somewhat impressive ability to reconstitute and maintain a functional parenchyma (Overturf et al., 1997), it is the stem and progenitor cell compartments that are naturally able to form tissues anew.

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The extent to which each process plays a role can be diVerent in each organ system based on the functional demands of that organ. However, some degree of neogenesis is always needed to maintain tissue homeostasis. Failure to fully appreciate that each of these processes can and will manifest themselves under certain circumstances has sometimes led to confusion and misinterpretation of data concerning adult stem and progenitor cells and their potential. Although our organs harbor adult cells capable of growth and neogenesis, many times our organs do not regenerate. By removing these cell populations from the body and providing a permissive environment, there is an opportunity to help the adult progenitor cell re-establish a functional parenchymal population, i.e., undergo organ neogenesis free of the inhibitory constraints of disease and confounding and competing physiological factors. The more knowledge we have about how cell populations respond and are regulated, the more likely we are to make the right decisions in the development of enabling technology and the design of clinical strategy. The progenitor cell is the natural precursor to parenchymal tissue formation. Therefore, our success in engineering tissues will heavily rely on our ability to identify, cultivate, and enable the progenitor cell. If more primitive cells or more distantly related cells are used as a starting cell source, such as embryonic stem cells, the goal should still be to arrive at a progenitor cell. However, as this chapter illustrates, the mechanisms of progenitor and population regulation are at once elegant and complex, exquisitely controlled (Artavanis-Tsakonas et al., 1999; Odom et al., 2004) yet confusingly malleable (Ber et al., 2003; Dabeva et al., 1997; Kojima et al., 2004; Krakowski et al., 1999; Marek et al., 2003; Wang et al., 2001b; Zulewski et al., 2001). Therefore, the most straightforward strategy to ensure that neogenesis will occur with the high fidelity needed for clinical applications is to enable native capability in the progenitor cells of the tissue in question wherever possible.

II. A Working Hypothesis for Understanding Parenchymal Cell Interaction and Response The use of the terms stem cell and progenitor cell varies in the literature, but in order to discuss these cell populations, we will define them within the context of a working hypothesis of progenitor cell regulation (Fig. 1). Our working hypothesis is that progenitor cells are the proliferative oVspring of slow-cycling stem cells resident in adult organs. The progenitor cells are the minimally diVerentiated cells that make up the proliferative cell compartment responsible for organ regeneration. Other models of stem cell regulation have also hypothesized a transition between the quiescent and

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Figure 1

A working hypothesis of parenchymal generation.

‘‘activated’’ stem cell (Roeder et al., 2003). It is unclear whether this is a bidirectional process, although it is interesting to speculate on the return of an activated progenitor to quiescence as a possible mechanism for the renewal of the slow-cycling stem cell during recovery from injury. Based on developmental studies, gene expression, and apparent regulation by transcription factors, we propose that the progenitor cell compartment should be viewed as distinct from the stem cell, which, by our definition, is slow-cycling. It is logical that neogenesis should involve several parallels to processes of organogenesis during development where there is a clear progenitor cell population. In development, for example, an inadequate progenitor pool results in severe hypoplasia of the organ (Jensen et al., 2000b; Mills

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et al., 1999; Yang et al., 1999). The progenitor compartment or pool gives rise to transit amplifying (TA) cells. The TA compartment exhibits limited replication and is committed to functional (sometimes terminal) diVerentiation. It is this compartment that directly gives rise to the mature functional parenchyma. In the past, many analyses have not distinguished between the slowcycling stem cell compartment and the progenitor pool, while others have not distinguished between the activated progenitor and the TA compartment, yet appreciating these distinctions, clearly at work in development and response to injury and disease, will be important to enabling the engineering of new tissues. Also, by distinguishing between the stem, progenitor, and TA cell, it becomes easier to unify the interpretation of existing data and derive important practical concepts for parenchymal regulation that we will use to develop our technologies. Therefore, there are three levels of population regulation that are particularly important to regenerative technology: (1) activation of the progenitor cell, (2) control of progenitor pool size, and (3) regulation of the TA cell toward acquisition of advanced function. The best way to advance our working hypothesis is to examine the progenitor cell compartment in vivo under conditions of tissue homeostasis, injury, disease, and development and compare these conditions to cell behavior in vitro. We focus on three cell populations in our analysis: epidermal keratinocytes, liver hepatocytes, and pancreatic islets.

III. The Epidermis A. Properties of the Epidermal Keratinocyte Population The epidermis has been of interest to many cell and developmental biologists because of its comparatively clear process of cell diVerentiation. The stratified epidermal cell layers show evidence of a linear progression of diVerentiation leading to terminal diVerentiation and are easily distinguished morphologically and biochemically (Bilbo PR et al., 1993; Johnson et al., 1992). Terminal diVerentiation leading to cell death is a necessity in skin, for it is needed to form the cornified layer of the epidermis, which is vital for barrier function. Barrier function is the primary purpose of the epidermis, and it is reasonable to expect that cell population regulation in the mature tissue will be biased toward mechanisms that will rapidly establish and maintain this barrier. Therefore, the keratinocyte population has a strong genetic predisposition to progress to terminal diVerentiation. The proliferative cells are normally limited to the basal layer. The basal layer is heterogeneous, consisting of slow-cycling stem cells, progenitor cells, and proliferating transit amplifying cells. Normal transit of an epidermal cell

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from the basal to cornified layer is approximately 14 days (Fuchs and Raghavan, 2002). The epidermal keratinocyte was the first human cell to be recognized as a heterogeneous population in vitro. The bias toward diVerentiation made the adult population very diYcult to cultivate. A breakthrough came with the development of the 3T3 feeder cell method, which allowed the serial propagation of the normal human keratinocyte (Rheinwald and Green, 1975). Importantly, the feeder system supported a prolonged period of growth and the clonal expansion of the population, which made further analysis of diVerences within the population possible (Barrandon and Green, 1985, 1987). The in vitro behavior of the keratinocyte population during serial expansion and how it relates to epidermal neogenesis has been covered in detail elsewhere (Hardin-Young and Parenteau, 2004); however, there are a few aspects of the in vitro keratinocyte population that are relevant to the biology of the progenitor cell. Study of the colonies formed from individual keratinocytes revealed cells with widely diVerent proliferative capacity (Barrandon and Green, 1987). Clones with abundant growth potential were thought to derive from an epidermal ‘‘stem’’ cell, clones with limited potential from transit amplifying cells, and abortive clones from late-stage transit amplifying cells. High growth potential in the feeder system was the dominant criterion for confirmation of ‘‘stemness’’ for many years. Slow-cycling cells isolated from the epidermis and hair shaft in man and rodents by fluorescence-activated cell sorting (FACS) (Li et al., 1998; Tani et al., 2000) or microdissection (Oshima et al., 2001) were selectively cultured and found to correspond to a high frequency of proliferative cell colonies. This was interpreted as direct activation of the slow-cycling cell to a rapidly dividing cell due to stimulatory in vitro conditions. Based on the colony assay, it was estimated that the human neonatal epidermis contained less than 0.1% of cells that were ‘‘stem’’ cells (Rheinwald, 1980). In the feeder system, the calcium content permitted stratification and limited diVerentiation; cells left the proliferative compartment and underwent diVerentiation, although incomplete. However, at confluence, a coherent, stratified epithelium could be removed using dispase and grafted onto burn patients (Gallico et al., 1984). These autologous-derived cultured epithelial sheets were able to restore an epidermis on severely burned patients (Compton et al., 1989). Cultured epidermis was the first laboratory-grown tissue for clinical use. Despite the widespread use of the feeder cell method in the study of human keratinocyte biology and its advance to clinical use, there was little understanding of what was actually going on in the population beyond assessment of proliferation on one end and the regulation of diVerentiation markers on the other. The gradual decline in colony growth and increase in modestly growing colonies and abortive colonies was viewed as a result of a linear progression of cell proliferation as the ‘‘stem’’ cell of high generative

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capacity simply depleted its potential to give rise to TA cells (Barrandon and Green, 1987; Pellegrini et al., 1998). The advent of defined culture systems (Boyce and Ham, 1983; Johnson et al., 1992) allowed population behavior to be analyzed in a diVerent way. Parenchymal regulation could now be viewed in a simpler form. Stratification was inhibited by a low calcium environment, yet diVerentiation still proceeded and the population shifted to a preponderance of transit amplifying cells with limited growth capacity over time (Johnson et al., 1992). What was discovered, however, was that the makeup of the population could not be explained simply by the growth and depletion of a stem cell. In the defined system, keratinocytes developed from explant outgrowth showed a high (60–100%) plating eYciency and colony formation in first passage from primary (Johnson et al., 1992; Parenteau, 1998, unpublished observations). Beyond this, heterogeneity developed gradually, resulting in increasing population doubling time and a shift in the size of the population to a point where the enlarged, diVerentiated cells made up the majority of the population in late passage. Small proliferative keratinocytes (presumed to be of ‘‘basal’’ character) became negligible. If cultures were serially propagated to senescence, enlarged cells eventually underwent apoptosis and released from the culture surface. With the removal of the mature cells, the occasional springing up of new colonies of small, very proliferative cells was observed. Although the colony proliferated and looked much like an earlier passage colony, we hypothesize that conditions favored the progression to TA in this instance and that the generation of these diVerentiated cells inhibited reestablishment of a proliferative pool capable of a new round of serial cultivation. Although the reasons behind the result may be speculation at this point, this observation did indicate that certain cells retained regenerative potential. A better understanding of what activates and inhibits the size of the progenitor cell pool will be important to our ability to arrive at large numbers of adult progenitor cells for therapy. Work using defined serial cultures generated several interesting pieces of information. One was that the gradual decline in the proliferative capacity of the population was due to feedback from the development of more diVerentiated cells, not merely a progression of all cells to senescence. A second was that colony formation in very late-stage keratinocyte cultures indicated that a proliferative cell was at the very least carried along in passage but was inhibited from proliferating by repressive feedback from the diVerentiated cells. A third was that the early, near-uniform cell behavior indicated a lack of heterogeneity of the population in the early stages of the cultures. Today, we believe that those early colonies most likely represented activated progenitor cells, which were the dominant cell type in early cultures. The late-stage culture gave us glimpse of a possible slow-cycling stem cell, preserved but not renewed in the cultured population, which became

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activated or released from inhibition upon depletion of repressive signaling. It was also evident that as we inhibited the progress toward terminal diVerentiation in the mature cells, we may have exacerbated repressive signaling by inhibiting diVerentiation just enough to allow them to remain viable in culture longer than they normally would, something that is probably a common failure in most culture systems of mature cell populations. Although the cultures became gradually more diVerentiated with time, the keratinocyte cell populations generated using the defined system were more than suYcient to support commercial-scale expansion of the neonatal epidermal keratinocytes (Johnson et al., 1992). The population was then challenged to reform epidermal tissue as part of a skin construct (Wilkins et al., 1994). The transition from proliferating single cells to formation of an organized epidermis has been covered in detail elsewhere (Bilbo PR et al., 1993; Parenteau et al., 1992; Wilkins et al., 1994). Despite increasing heterogeneity in the cultures, the epidermis was able to last at least 1 year when engrafted onto the back of an athymic mouse (Hardin-Young and Parenteau, 2004) and 6 months under certain clinical circumstances (Falabella et al., 2000).

B. Modulation of the Population Using diVerences in 6 integrin and transferrin receptor expression (Li et al., 1998; Tani et al., 2000), Li et al. (2004) were able to sort basal keratinocytes into a stem/progenitor fraction and a TA fraction. Skin constructs were then made with the fractionated cells. As expected, these epidermal cells were easily able to give rise to a diVerentiated epidermis. Surprisingly, the TA fraction was able to form a full epidermis, albeit thinner than the one generated by the stem cell fraction on its own. This potential for limited neogenesis appeared to be further modulated by the status of the dermal component of the construct. If it contained primary dermal fibroblasts rather than the standard serially passaged dermal fibroblasts, the TA keratinocytes grew and formed an epidermis that was now indistinguishable to the one generated by the stem cell fraction, and once engrafted, this epidermis was able to maintain for at least 10 weeks. This led to the discovery that laminin 10/11 produced by the isolated dermal population was responsible for this enhanced regenerative capacity. Although the presence of contaminating stem and activated progenitor cells might account for the long-term maintenance of the tissue in vivo, these results illustrate the ability of conditions to modulate the activity of a cell population and its compartments. Additional evidence of TA cell modulation comes from recombination experiments using embryonic dermis. Central corneal epithelium, which has been shown to consist of only TA and diVerentiated cells (Cotsarelis et al.,

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1989; Lavker et al., 1991; Pellegrini et al., 1999) presumably committed to a corneal lineage, has been reprogrammed toward epidermal, pilosebaceous (hair and sebaceous gland), and sweat gland lineages by recombination with embryonic dermis (Ferraris et al., 2000), confirming the classic embryological influence of epithelial/mesenchymal interaction as a powerful modulator of epithelial diVerentiation. It also gives us evidence that when the cells are removed from their native context, they can undergo metaplasia and the acquisition of alternative, but often genetically related, phenotypic character that some term ‘‘transdiVerentiation.’’ Instances of significant modulation between pancreas and liver phenotype have also been reported (Dabeva et al., 1997; Krakowski et al., 1999; Wang et al., 2001b). However, despite the ability of the TA compartment to behave impressively under certain circumstances, the progenitor cell remains the most important cell for neogenesis and a necessity for long-term maintenance of the tissue implant. What we now appreciate is that the population of keratinocytes is malleable, the lines between progenitor, TA cell, and diVerentiated cell can blur, and the progression to terminal diVerentiation is controlled by circumstances and is not merely a steady progression to apoptosis and terminal diVerentiation. Additional data on the behavior of the epidermal stem, progenitor, and TA compartments is now being rapidly gathered through the use of in vivo experiments using normal and transgenic animals. From this, we can construct several clues as to how the keratinocyte population is further regulated.

C. Regulation of the Epidermal Cell Compartments There is now clear evidence for the presence of slow-cycling stem cells in the epidermis (Braun et al., 2003; Cotsarelis et al., 1989; Lavker et al., 1993; Schneider et al., 2003). Much of this work has been done in rodents, but the general principles should apply to human skin as well. Slow-cycling cells are present in the midfollicular region along the hair shaft, sometimes called the ‘‘bulge’’ region due to its histological appearance sitting just below the sebaceous gland (Cotsarelis et al., 1990), within the interfollicular epidermis, and within the sebaceous gland (Braun et al., 2003; Gandarillas and Watt, 1997). It was first proposed that the bulge stem cells were the primary stem cells of the epidermis and that activity in the interfollicular space, and presumably the sebaceous gland, were progeny, that is, progenitor cells of the bulge stem cell. In support of this were two common observations: first, that skin, which lost its epidermal covering while retaining the deeper hair follicles, could regenerate epidermis and second, that murine cutaneous carcinomas resulting from topical application of carcinogens initiated from

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the hair follicle (Lavker et al., 1993). Labeling in vitro has demonstrated that the epidermal cells residing in the midfollicular region preferentially give rise to hair and that those of the interfollicular space preferentially give rise to epidermis (Ghazizadeh and Taichman, 2001). Despite this natural preference, there is indication that the slow-cycling cells retain a potential for multipotency to form hair, sebaceous glands, and epidermis (Fuchs and Raghavan, 2002; Niemann et al., 2002; Oshima et al., 2001), and thus it is not surprising that stem cells present in the hair follicle can generate epidermis when needed. It also implies that regenerating epidermis must retain some ability to give rise to hair; however, in severaly burned patients where the dermal elements no longer remain, this does not happen—the healed skin is devoid of hair (and associated sebaceous glands) and apocrine glands, another epithelial subpopulation (Compton et al., 1989). It therefore appears that epidermal regeneration is the default pathway and that modulation by accessory cells such as the cells of the dermal papilla is needed for hair formation (Ferraris et al., 2000; Reynolds and Jahoda, 1991, 1996). Information concerning the interparenchymal signaling involved in this regulation is derived from the analysis of development and the behavior of various transgenes and their eVect on the formation of these structures. The slow-cycling stem cells of the epidermis and the hair and sebaceous gland (pilosebaceous unit) appear to be related, if not essentially the same, in their capacity to generate these three tissue types, although as noted, under normal circumstances they preferentially contribute to the tissue they are most closely associated with, in part due to the diVerent signaling needed to aVect each lineage (Fig. 2). The cells of the bulge region have been the most extensively studied to date (Fuchs and Raghavan, 2002). Cell sorting and cell cycle analysis as well as labeling of label-retaining cells and their response to cMyc and phorbol ester (Gandarillas and Watt, 1997) clearly indicate that there are subpopulations within this ‘‘stem’’ cell compartment. Cells of the bulge region express Tcf3, which acts as a repressor for cMyc and cyclin D (Merrill et al., 2001). It is believed that Tcf3 expression helps maintain the bulge cells. Wnt signaling is crucial to progression of the progenitor through a hair lineage (Alonso and Fuchs, 2003; Fuchs and Raghavan, 2002). Tcf3 expression switches to related Lef1 in this pathway (Merrill et al., 2001). Wnt signaling stabilizes ß-catenin, which then accumulates, translocates to the nucleus, and activates Lef1. Activation of Lef1 is needed for the

Figure 2 The progenitor cell and orchestration of cell lineage in the epidermis and pilosebaceous unit. Regulation of the progenitor and transit amplifying cell (TA) compartments is evidenced by their diVerences in regulation. References: Braun et al., 2003; Fuchs and Raghavan, 2002; Gandarillas and Watt, 1997; Li et al., 2004; Lowell et al., 2000; Merrill et al., 2001; Niemann et al., 2002, 2003; Oshima et al., 2001; Pellegrini et al., 2001; Rangarajan et al., 2001; Reynolds and Jahoda, 1996; Sasaki et al., 2002; Yang et al., 1999.

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generation of hair cortical cells and ultimately hair morphogenesis. Blocking the interaction of Lef1 and ß-catenin using a transgene of Lef1 that lacks a binding site for ß-catenin, NLef1 blocks hair development, giving rise to hair follicle cysts with epidermal character and sebaceous gland tumors (Niemann et al., 2002). In contrast, expression of NLef1 in interfollicular epidermis has a relatively minor eVect, leading to a twofold stimulation of proliferation and an increase in the number of diVerentiated layers. Further examination of the signaling involved in the progenitor pools of both the hair and the sebaceous gland indicates a role for hedgehog signaling in the regulation of committed progenitor cells (Niemann et al., 2003). The size of the Lef1þ/ß-catenin responsive progenitor cell pool in mice appears to be regulated by Sonic hedgehog (SHH), while the size of the sebaceous progenitor pool appears to be regulated by Indian hedgehog (IHH), which is produced by the mature sebocyte in a feedback loop on the sebocyte progenitor pool. Development of an epidermal lineage will normally arise from slowcycling stem cells within the basal layer of the interfollicular epidermis. Labeling of mouse skin using retroviral transduction of ß-galactosidase indicates that the epidermal layers arise in distinct columns (Mackenzie, 1997). It has been hypothesized that each epidermal column represents the transit amplifying progeny of a single basal cell and has been termed an epidermal proliferative unit (EPU). But how are these columns generated and maintained? The slow-cycling stem cell has been determined to be present at a frequency of 1 in 104 basal cells, a frequency similar to the hematopoietic stem cell and significantly lower than clonal assays would suggest. Further analysis of the slow-cycling stem cell and the generation of the EPU has revealed that the size and extent of the EPU can vary widely from fifty to thousands of cells (Schneider et al., 2003). We postulate that the wide variation of EPU size and extent might be due to the existence of a varying progenitor cell pool that responds to the need and physiological state of the skin [an increase in proliferative TA cells results in a thickening or hyperproliferation of the epidermis (Rangarajan et al., 2001), not necessarily an increase in the extent of the EPU]. In development, the progenitor pool, but not the more primitive stem cell or the proliferation of the committed cell (similar to a TA), defines tissue mass (Jensen et al., 2000b; Yang et al., 1999). While cell compartments can be modulated, it is reasonable to expect that this mechanism of neogenesis is not going to be appreciably diVerent in the adult organ. Studies in vivo (Mills et al., 1999; Yang et al., 1999) and in vivo (Pellegrini et al., 2001) point to the transcription factor p63, from the p53 family, as being associated with the biology of the basal progenitor cell pool. Transcription of p63 appears essential for regenerative proliferation through the maintenance of the progenitor cell population. It has been postulated that

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p63 preserves important self-renewal capacity in the epidermal progenitor cell while still allowing generation of transit amplifying cells (Yang et al., 1999). In cell culture, expression of p63 is associated with highly proliferative colonies in the feeder cell system (Pellegrini et al., 2001). In the developing mouse, ablation of p63 in vivo results in nonregenerative squamous epithelia (Mills et al., 1999; Yang et al., 1999). Analysis of the embryos revealed the presence of diVerentiation markers within the stunted epithelium, indicating a severe hypoplasia of the tissue. There was a distinct absence of basal cell markers such as keratin 5, while late-stage diVerentiation markers and clear apoptosis could be detected in the desquamating remnants of epithelium (Yang et al., 1999). This result implies a severe depletion of the progenitor cell compartment and rapid, if not immediate, progress of the TA to diVerentiation. A similar outcome is seen in the development of pancreatic islets, where block of Hes-1 expression causes a reduction in the progenitor cell pool and rapid progress through the islet diVerentiation program. Failure of Hes-1 to laterally inhibit the progress of neurogenin3 (ngn3) pancreatic cells (the islet progenitor) results in a loss of any appreciable ngn3 positive pool (Fig. 3). The result is severe hypoplasia of the endocrine component and failure of the exocrine component to develop at all (Jensen et al., 2000b). Interestingly, p63 is known to stimulate the expression of Jagged 1 (JAG1) Notch ligand. JAG1 expression in a cell line (Saos2) transfected with p63 has been shown to stimulate Notch signaling and subsequent activation of Hes-1 in a co-cultured cell line (Jurkat cells) (Sasaki et al., 2002). Whether a similar role for Hes-1 exists in the regulation of the epidermal progenitor pool remains to be determined. However, the Notch signaling pathway appears to be active in these cells, and parallels might be expected. Studies on the eVects of cMyc overexpression on keratinocytes in vitro (Gandarillas and Watt, 1997) and on the retention of label-retaining cells (slow-cycling cells) in vivo (Braun et al., 2003) indicate that the action of cMyc likely occurs in the progression of the progenitor to transit amplifying cell. Stimulation of cMyc in vivo results in a hyperproliferation of the epidermis without the loss of diVerentiation or depletion of label retention in the slow-cycling stem cell, indicating a lack of activation of this compartment. This suggested that the action of cMyc involved the progenitor and/or TA compartments. Additional clues to its action come from the observation that in vitro overexpression of cMyc leads to an increase in abortive colonies using the feeder cell system (Gandarillas and Watt, 1997). This narrows the likely action of cMyc to the TA compartment. Increased generation of, or conversion to, TA cells will lead to inhibition of the progenitor cell compartment and to an increase in abortive colonies characteristic of late-stage TA (Barrandon and Green, 1987). If the action of cMyc was on the proliferation of the progenitor cell, this would result in increased or sustained proliferation of actively dividing colonies rather than

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Figure 3 The islet progenitor and development of pancreatic lineage. Development of the pancreatic cell population illustrates the important distinction between control of the progenitor pool and progress to diVerentiation. It also illustrates the cascade of factors needed for islet neogenesis, which must include glucose response as well as insulin production for a clinical therapy. References: Ahlgren et al., 1996, 1998; Apelqvist et al., 1999; Boj et al., 2001; Edlund, 2001; Gradwohl et al., 2000; Gu et al., 2002; Jensen et al., 2000a,b; Kawaguchi et al., 2002; Lee et al., 2001; Murtaugh et al., 2003; Schwitzgebel et al., 2000; Smith et al., 2003, 2004; St-Onge et al., 1997. Figure copyright Amaranth Bio, Inc.

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an increase in abortive colonies. These results indicate a role for cMyc in the control of TA proliferation and perhaps in the transition from progenitor to TA. It is unclear whether the loss of Tcf3, a repressor of cMyc and cyclin D (Merrill et al., 2001), occurs gradually, similar to ß-catenin, or whether it is still present in cells of the progenitor pool. It appears, however, that the progenitor pool and the TA compartment are subject to distinct regulation (Fig. 2). Notch signaling causes the TA to leave the cell cycle. Notch1 stimulates the expression of p21, one of the earliest indicators of a basal cell’s commitment to leave the cell cycle and begin terminal diVerentiation (Rangarajan et al., 2001). Notch / mice have a hyperproliferative epidermis with suprabasal cells expressing ß1 and ß4 integrins, normally associated with the basal layer. It was determined that in addition to Notch1’s ability to stimulate the expression of p21 through an RDP-J pathway, Notch1 and Notch2 also influence the expression of early diVerentiation markers through an RBP-J-independent pathway. In humans, it appears that a Notch/Delta negative feedback mechanism also influences commitment of cells to the TA compartment (Lowell et al., 2000).

D. When Skin Fails to Heal Impairments in wound healing clearly exist, although the etiology of these conditions is still vague. In acute wounds, the loss of an epidermal covering leads to an inflammatory reaction and a dermal response. There is contraction of the underlying connective tissue through the action of fibroblasts that take on myofibroblastic properties, to help reduce the surface area of the wound. Wound contraction is particularly active in loose-skinned animals but less so in humans, although contraction of the scarred dermis does occur. The next step is recruitment of the suprabasal or late-stage transit amplifying cells to migrate over the wound bed to help rapidly establish wound closure (Garlick and Taichman, 1994). For many years, it was thought that these suprabasal cells were irreversible in their commitment to terminal diVerentiation; however, the intriguing in vitro work by Li et al. indicates that the transit amplifying cell compartment might be much more responsive than originally thought. In the case of chronic wounds, the epidermis fails to regenerate and migrate over the surface of the wound bed. This failure has been attributed to a number of factors, including an imbalance of growth factors, bacterial contamination and associated protease activity, inappropriate extracellular matrix, cells at the wound edge that have become refractory, or any and all of the above (Hasan et al., 1997; Phillips et al., 2000). One approach to dealing with this problem was to form skin in vitro and deliver it to the wound. Application of bilayered skin constructs (Brem et al., 2001; Falanga et al., 1998) or epidermis (Phillips, 1994) does promote

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healing even without the ready ability to take as a graft on the chronic wound bed. Stimulation of the patient’s own tissue is seen, and wound healing ability that may have been lost for years appears to be restored (Sabolinski et al., 1996). What is the mechanism for this stimulation of regeneration? Although specifics are still unknown, it appears that the cultivated tissue is able to restore signaling that then stimulates the generation of new tissue (Sabolinski et al., 1996). The epidermis at the wound margins clearly changes, as does the underlying connective tissue of the wound bed. Based on what we now know, interparenchymal signaling and modulation by changes in the connective tissue are probably both important to the outcome. Ironically, despite the relative ease in observing diVerentiation in the epidermis, aside from an appearance of hyperplasia seen with some dermatological conditions, identification of definitive changes in the basal cell populations has been diYcult to detect morphologically and distinguish biochemically. However, better understanding of cell signaling during morphogenesis now gives us evidence of their existence and regulation and a better understanding of their importance in tissue neogenesis. The pancreas and liver, two developmentally related organs, provide further information on the role and potential of the progenitor cell in adult tissues.

IV. The Liver A. The Liver Cell Population The liver is a paradox. It is a highly developed organ containing hepatocytes able to carry out a number of important functions, ranging from biosynthesis of several serum proteins and regulation of glycogen stores to detoxification. These cells normally exhibit a very slow turnover, which can extend to 400 days with little apoptosis (Fausto and Campbell, 2003), yet these highly diVerentiated cells remain capable of substantial proliferation when needed, able to regenerate almost the entire liver mass within a few days (Michalopoulos and DeFrances, 1997). It is one of the organs best able to regenerate, yet in vitro cultivation of the hepatocyte has been fraught with diYculty. It appears that the diVerentiated function of the hepatocyte is highly modulated based on position within the hepatic cord or plate (Fausto and Campbell, 2003; Nagy et al., 1994), which extends from the portal triad to the central vein. Therefore, this exquisitely capable cell population appears to also be a highly flexible one (Gupta et al., 1999). Flexibility is not something that has been traditionally associated with advanced function, but the liver may represent the human’s best example of regeneration by

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being able to modulate between an activated and quiescent state without loss of diVerentiated function. In skin, the primary purpose was to maintain barrier; this required terminal diVerentiation. In the liver, the goal is to maintain a number of biosynthetic and metabolic functions. Functional cell mass is important in the liver, and therefore the liver has mechanisms that maintain its ability to generate that mass rapidly. The fact that liver regeneration appeared through the division of virtually all existing hepatocytes led many to question the need for or existence of a progenitor cell. It was believed that all hepatocytes contributed to growth when needed and therefore were, by loose definition, all ‘‘progenitor’’ cells. However, it is now well-accepted that while the mature liver cell retains its ability to enter the cell cycle even though normally quiescent, a progenitor population does reside in the liver, and it is hypothesized to be possibly related to the original fetal ductal plate cells (Dabeva and Shafritz, 2003). These cells are multipotent, able to generate cholangeocytes for the generation of bile ducts as well as hepatocytes. Although what is usually thought of as the progenitor population is somewhat heterogeneous based on the expression of bile duct, hepatocyte, and fetal cell markers (Yang et al., 1993), these cells are often referred to collectively as oval cells in the rat based on their appearance (Strain et al., 2003). Several studies have attempted to establish the relationship of the oval/ progenitor cell to the hepatoblast in development (Zheng and Taniguchi, 2003), the bile duct, and the hepatocyte using markers (Weiss and Strick-Marchand, 2003). However, analysis based on expression of surface markers or keratins is not definitive and can be misleading, particularly outside the context of functional behavior. Therefore, our analysis focuses on cell behavior and what is known about gene regulation. The progenitor cell is best studied in development and in certain pathological conditions where the proliferative capability of the mature parenchymal is compromised.

B. The Development and Regulation of the Hepatocyte Phenotype The liver develops from the primitive endoderm induced by the paracrine action of fibroblast growth factor (FGF) produced by the adjacent cardiac mesoderm (Deutsch et al., 2001; Zaret, 2002). Without this signaling, the endoderm defaults to a pancreatic lineage. FGF induces the local expression of SHH, which was shown to repress Pdx-1, important in pancreas determination, thereby favoring the development of the hepatoblast (Deutsch et al., 2001). Development of the fetal hepatoblast, diVerentiation of the progenitor, and maintenance of the functional hepatocyte are controlled by transcriptional regulation of several hepatocyte nuclear factors (HNFs) (Duncan et al., 1998; Nagy et al., 1994; Odom et al., 2004; Fig. 4).

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Figure 4 A simplified scheme of hepatocyte nuclear factor (HNF) interaction and its relationship to hepatocyte diVerentiation. Illustration of the complex interaction of the HNFs in hepatocyte development and their relationship to hepatocyte diVerentiation. A much more thorough analysis of HNF function and mechanisms of interaction in both liver and islet can be found elsewhere (Odom et al., 2004). References: Bailly et al., 1998; Duncan et al., 1998; Hatzis and Talianidis, 2001; Jacob et al., 1999; Jacquemin et al., 2000; Li et al., 2000; Nagy et al., 1994; Parviz et al., 2003; Qian et al., 2000; Rausa et al., 1997; Samadani and Costa, 1996; Trautwein et al., 1996.

HNFs work cooperatively to direct cell-specific transcription and can also act as negative regulators (Odom et al., 2004). In the liver, these transcription factors work during development to control the gradual progression from hepatoblast to either hepatocyte or cholangeocyte. HNF3 and HNF1ß are thought of as ‘‘establishment’’ factors and are proposed to be important in oval cell activation (Nagy et al., 1994). Fetoprotein transcription factor (FTF) is also considered to be an establishment factor through its activation of HNF3ß, HNF4, and HNF1 (refer to Fig. 4). HNF3’s target genes include genes for serum carrier proteins, albumin, -fetoprotein, -1 antitrypsin, and complement protein cytochrome P450 2C6, among others (Odom et al., 2004; Samadani and Costa, 1996). HNF4 appears to be particularly important to liver organogenesis. It is required for commitment to the hepatocyte phenotype, working cooperatively with HNF1 to regulate several hepatocyte genes (Bailly et al., 1998). HNF1 requires HNF4 for its expression (Li et al., 2000). Putative target genes of HNF4 (or the HNF4/HNF1 loop) include those encoding for apolipoproteins, blood coagulation factors, and enzymes involved in glucose metabolism (Li et al., 2000) and the maintenance of lipid homeostasis (Hayhurst et al., 2001). Loss of HNF4 function does not eliminate the expression of -1 antitrypsin, -fetoprotein, or the apolipoproteins that are under the regulation of HNF3 (Nagy et al., 1994). However, the HNF4/ HNF1 loop appears to be able to influence, either directly or indirectly, the regulation of HNF3ß and CCAAT/enhancer binding protein (C/EBP) (Bailly et al., 1998). In addition to having a primary role in enabling glycogen storage, HNF4 appears to regulate genes that control epithelial morphogenesis of the mature hepatocyte by activating cell adhesion and cell

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junction molecules (Parviz et al., 2003). Early embryonic morphogenesis is unaVected by the lack of HNF4. Together, HNF4, HNF3ß, HNF1, and C/EBP play an interactive role in the acquisition, maintenance, and modulation of the hepatocyte phenotype. A much more extensive analysis of the diVerent ways in which HNFs interact and the proteins they influence in both the liver and pancreas can be found in a recent paper by Odom et al. (2004).

C. The Importance of Tissue Architecture Architecture of the liver appears particularly important to the expression of hepatocyte functions. Functional zones are established during development (Fausto and Campbell, 2003), and the lobular architecture appears to be required for their development (Notenboom et al., 1996). Periportal hepatocytes specialize in glycogenolysis and gluconeogenesis and central cells in glycolysis and glycogen synthesis, while the cells closest to the central vein express glutamine synthetase, which is important in ammonia metabolism (Fausto and Campbell, 2003). The establishment of glutamine synthetase function appears to be one of the most sensitive to lobular structure (Notenboom et al., 1996). The zonal distribution of functional hepatocytes was once thought to represent a cell lineage resulting from the proliferation and movement of hepatocytes from the portal triad to the central vein (Sigal et al., 1992). This hypothesis was particularly attractive since the oval cell was known to reside in the canal of Hering, which is associated with the biliary ductules of the portal triad. Cells were thought to originate from oval progenitors diVerentiating/maturing as they moved through the hepatic cord toward the central vein. In this model, it was proposed that the hepatocyte ended in a terminally diVerentiated hepatocyte next to the central vein. This model of the ‘‘streaming’’ liver has since been abandoned. It is now generally accepted that diVerences in hepatocyte function are derived through modulation of function by position within the hepatic cord rather than a progression of cell lineage. For example, although cell movement toward the central vein can be seen in progressive rounds of ‘‘wound healing’’ using labeled transplanted hepatocytes, diVerences in resulting function appear to be related to position, not the result of the prior status of diVerentiation of the transplanted cells (Gupta et al., 1999). The modulation of the HNFs and the multiple ways in which they interact (Duncan et al., 1998; Odom et al., 2004; Fig. 4) suggest a way in which hepatocytes could be modulated based on physiological demands transmitted via the blood and the influence of growth factor, retinoid, and matrix signaling from the associated stellate cells (Geerts, 2001; Pinzani and Marra, 2001; Rockey, 2001; Sato et al., 2003). Progression of the mature

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hepatocyte through the cell cycle requires both autocrine and paracrine growth factors, such as the hepatocyte-produced transforming growth factor , hepatoctye growth factor provided by the stellate cells, and interleukin 6 (IL6) provided by inflammatory cells (Fausto and Campbell, 2003; Michalopoulos and DeFrances, 1997). Although much is known regarding the stellate cell because of its links to liver fibrosis (Bataller and Brenner, 2001; Benyon and Arthur, 2001; Iredale, 2001), by comparison, relatively little is known regarding possible parenchymal signaling in the modulation of the diVerentiated hepatocyte population. One reason for this is the inability to appreciably cultivate and manipulate the hepatocyte in vitro.

D. Liver Repopulation Studies Liver repopulation studies have been used to assess the proliferation and diVerentiation potential (Dabeva and Shafritz, 2003; Gupta et al., 1995). Cells are delivered into the portal vein or are injected into the splenic pulp (Gupta and Rogler, 1999; Gupta et al., 1995). The injected hepatocytes then migrate through the liver sinusoids, enter the liver plates, and integrate with host hepatocytes (Dabeva et al., 1997; Gupta et al., 1995). The proliferation of the transplanted cell is limited in the normal liver. However, the more compromised the organ, the more proliferation is seen in the transplanted cells (Dabeva and Shafritz, 2003; Laconi et al., 1998; Overturf et al., 1999), that is, the transplanted hepatocytes must have a selective growth advantage. In the fumarylacetoacetate hydrolase-deficient (FAH) mouse model of tyrosinemia type I, normal hepatocytes positive for FAH are preferentially selected over the donor FAH-deficient ones. As few as 1000 donor cells are able to rescue a FAH-deficient mouse and repopulate the liver within 6 weeks, making this a good model for studying the proliferation potential of transplanted hepatocytes, which are examined using serial transplantation (Overturf et al., 1997). It has been estimated that the engrafted cells can undergo an impressive 69–100 population doublings (Overturf et al., 1997, 1999). To rule out the possibility of proliferation due to a stem or progenitor population, hepatocytes were fractionated based on size and were transplanted into the cells in competition with unfractionated hepatocytes in the FAH mouse model (Overturf et al., 1999). While some quantitative diVerences were seen in the ability of the cell fraction to repopulate the liver (the middle fraction being preferable), all fractions were able to repopulate the liver, indicating that the majority of mouse hepatocytes had this ability and that it was retained upon serial transplantation. Liver repopulation experiments might invite the question of whether a cell therapy incorporating liver progenitor cells is even needed. Modest scale

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repopulation using liver cells and direct transplantation of harvested cells from a human liver donor is considered a clinical option (Fox et al., 1998), but the ability to cultivate and manipulate the progenitor cell will likely be a favored commercial option to better achieve in vitro expansion of a tested uniform cell population with which a true neogenesis process can be stimulated. In both cases, the status of the organ and the ability to safely manipulate the donor hepatocyte population to improve incorporation and acceptance of the transplanted tissue will be key.

E. When Pancreas Turns to Liver In the case of copper deficiency, the rat pancreas develops cells similar to hepatic oval cells (Gordon et al., 2000b), and overexpression of keratinocyte growth factor leads to hepatic diVerentiation around the perimeter of the islet, a place where pancreatic stem cells are thought to reside (Krakowski et al., 1999). Therefore, it appears that the pancreas harbors a stem or progenitor cell capable of some degree of hepatocyte diVerentiation. This is not entirely surprising, given their close developmental relationship (Deutsch et al., 2001). Pancreatic cells from copper-deficient animals and normal pancreatic cells have also been shown to incorporate into hepatic plates (Dabeva et al., 1997) and diVerentiate into liver cells upon transplantation, albeit at a lower frequency (Wang et al., 2001b). Although the latter study was more extensive in its characterization, both of these studies used limited parameters to demonstrate hepatocyte diVerentiation. The diVerences or similarities between hepatocytes and hepatocyte-like cells must be further substantiated, with an emphasis on regulation and response, before the pancreatic and hepatic progenitor cells can be considered equivalent. Distinguishing diVerentiation from modulation entails much more than what is present in the literature to date for any cell type (Hardin-Young and Parenteau, 2004). While additional work may confirm the pancreatic cell’s ability to acquire a mature hepatocyte phenotype, as a cell therapy, any cell source outside the normal parameters of the organ in questions will require the following: (1) the natural cell source has been ruled out as a viable option, (2) the ‘‘transdiVerentiated’’ cell demonstrates bioequivalence to the native cell showing sensitivity to regulation of the host cell population, and (3) it demonstrates stability within the population with the capacity for renewal. For these reasons, although important from a biological and developmental perspective, the use of cells other than those of the organ in question, and the additional work in scientific and clinical validation it will entail, will rarely be justified for clinical and commercial applications if the true organ progenitor can be used.

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F. Models of Liver Injury and Disease and Activation of the Progenitor Pool Liver progenitor cells become activated in cases of severe hepatocellular necrosis (Roskams et al., 2003; Strain et al., 2003) but can also be activated in chronic viral hepatitis, alcoholic liver disease, and non-alcoholic fatty liver disease (Fausto and Campbell, 2003; Roskams et al., 2003). The requirement appears to be a substantial loss of hepatocyte function combined with an inability of the remaining hepatocytes to proliferate. These cells appear as small epithelial cells with an oval nucleus and little cytoplasm, hence the term oval cell in rats (Brill et al., 1993). The hepatic stem cell is thought to preferentially reside in the canal of Hering associated with the terminal biliary ductules, which would be consistent with the hypothesis that these cells are related to the embryonic ductal plate cells (Roskams et al., 2003) that give rise to hepatocytes and cholangeocytes in development. This location is generally accepted. However, others have described the appearance of small hepatocyte-like cells arising in the hepatocyte parenchyma not associated with the periportal area in the rat when retrorsine treatment is combined with partial hepatectomy (Gordon et al., 2000a,b; Laconi et al., 1998). Retrorsine, a pyrrolizidine alkaloid, inhibits both hepatocyte replication and appears to inhibit any significant growth from the periportal area where oval cells are thought to exclusively reside, leading to formation of foci of small hepatocytes in the liver parenchyma that express a combination of phenotypic characteristics of hepatoblasts, oval cells, and mature hepatocytes (Gordon et al., 2000b). The relationship of this population to the progenitor arising from the periportal stem cell is unclear; however, it would not be unusual for phenotypic markers to be diVerent in progenitors in diVerent locations. Interesting small cells have also been described in the pancreas expressing stem cell markers, alphafetoprotein associated with fetal hepatocytes, and islet hormones (Petropavlovskaia and Rosenberg, 2002). In addition, Dabeva et al. (1997) have reported on the potential of small pancreatic ‘‘oval’’ cells to diVerentiate into hepatocytes upon transplantation into the liver. The relationship between these ‘‘small cells’’ in pancreas and liver and their relationship to the progenitor population is not yet known. The ability to undergo liver diVerentiation and the expression of the major liver transcription factors in the retrorsine/PH model would indicate a potential to generate functional tissue, which appears committed to hepatocyte lineage (Gordon et al., 2000a), although position in the organ cannot be ruled out as a limiting influence. Although somewhat puzzling, the presence of this type of cell in more than one organ indicates that their presence has a purpose. Progenitor cells are activated when there is severe damage or loss of hepatocytes or the cholangeocytes (cells of the bile duct) and when compensatory hyperplasia of the liver parenchyma is impaired. In experimental animal models, partial hepatectomy will fail to activate the progenitor cell

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without concomitant impairment of the remaining mature liver cells (Alison, 2003; Fausto and Campbell, 2003; Roskams et al., 2003). The activated progenitor pool can give rise to reactive ductules of primitive cholangeocytes or basophilic small hepatocyte-like cells termed intermediate hepatocytes (Alison, 2003). The degree to which reactive ductules or intermediate hepatocytes form will depend on the need for each tissue (Roskams et al., 2003). To fit our working hypothesis, we can think of the reactive ductules and intermediate hepatocytes as transit amplifying cells, but because hepatocytes always maintain the ability to proliferate and contribute to the organ mass, they too might be considered potential TA cells. The transitory nature of the reactive ductules and intermediate hepatocytes, however, indicates that they are more analogous to this compartment. Because we know that hepatocyte diVerentiation is sensitive to tissue architecture (Fausto and Campbell, 2003; Notenboom et al., 1996), we might imagine that diVerentiation cannot be achieved without enough cell mass to aVect some structural organization. In addition, during development, the organ is formed through these intermediate steps, although it has been suggested that progenitor diVerentiation during regeneration is more direct in the adult and a gradual process in the embryo (Zaret, 2002). Intermediate hepatocytes express albumin and alpha1-antitrypsin (Dabeva et al., 1997; Zheng and Taniguchi, 2003). The number of intermediate hepatocytes increases with the stage of liver disease in both alcoholic cirrhotic liver disease and fatty liver disease (Roskams et al., 2003). In the cirrhotic liver, intermediate hepatocytes and ‘‘tubular arrangements’’ of hepatocytes are seen in continuity with immature progenitor cells. Progress toward hepatocyte diVerentiation is based on the need for new hepatocytes. DiVerentiation of the hepatocytes and progress of this compartment is likely influenced by the hepatic stellate cells, as the degree of progenitor cell activation correlates with the degree of fibrosis (Fausto and Campbell, 2003). This signaling will be important to any cell transplant as well. While we may be able to provide an advantage to the neogenesis process by separating the process of progenitor cell activation and neogenesis from a hostile, degenerating environment, eventually the tissue or cells must be returned to the body. Therefore, understanding the good and bad environments, the eVect of inflammation, and the action of accessory cells will be vital to the generation of a successful transplant.

G. Why Hepatocytes Are Hard to Handle In Vitro Knowledge of the various factors and influences on liver neogenesis gives us some clues as to why hepatocytes have been notoriously hard to handle in vitro. First, they are a highly diVerentiated population that has a normal

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predisposition toward quiescence, and second, they are cells that are highly modulated. Expression of functional proteins will be highly malleable, and therefore it is easy to unwittingly modulate the population away from certain diVerentiated parameters. Third, the population relies on architecture and organization to regulate some of its diVerentiated function; therefore, it should not be surprising that conditions that inhibit hepatic cord formation will inhibit expression of certain aspects of diVerentiation, and those that enable it will improve this (Dunn et al., 1989). Block et al. (1996) demonstrated some expansion and modulation of diVerentiation of adult rat hepatocytes induced by growth factors such as hepatocyte growth factor (paracrine) and transforming growth factor (autocrine) in a defined culture environment. Their results indicated that virtually all the cells are stimulated to divide and that expression of HNF3, HNF4, and HNF1 is maintained throughout this process. Significantly, when conditions were changed to promote a return to a quiescent state, a return of biosynthesis and hepatocyte-like morphology did not require DNA synthesis, ruling out neogenesis. It appears from these results and those of others (Brill et al., 1993; Malhi et al., 2002; Yin et al., 2002) that the process of hepatocyte compensatory growth can occur in vitro to some extent and that the mature hepatocyte appears primed for modulation at any time, not requiring a change in gene expression for modulation. Unfortunately, to our knowledge, no one has yet been able to demonstrate a sustained ‘‘compensatory growth’’ reaction in serial passage. Interestingly, C/EBP has been implicated in the downregulation of albumin synthesis and preparation of the hepatocyte to enter the cell cycle through influence on genes that regulate the cell cycle (Trautwein et al., 1996; Fig. 4). It is also understandable that the hepatocyte stem and progenitor population in a healthy liver will be very small and therefore hard to uncover in a mixture of adult cells. A strategy to search for specific markers to specifically select these cells is being pursued by some. The problem with that approach is that the progenitor cell most often, if not always, requires activation to exist in any appreciable number, and even if selection of a population is successful, if we are devoid of the ability to activate the population, our selection will lead to abortive cultures. To put it another way, the progenitor is identified more by its behavior and the outcome of its behavior than by any marker it might express in any given time or location. Some have turned to the use of fetal cell cultures to allow nature to provide a substantial progenitor cell pool and improve on the growth potential of the culture (Alison, 2003; Brill et al., 2002; Kubota and Reid, 2000; Lazaro et al., 2003; Malhi et al., 2002), but control of development still poses a problem, and enabling the process of neogenesis must still be addressed. Keratinocyte culture has been successful because it is able to support the survival of the infrequent stem and progenitor cell while enabling regenerative signaling.

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V. The Pancreas and Generation of Pancreatic Islets A. Evidence of Neogenesis in the Pancreas Pancreatic islets are often cited as one of the greatest opportunities and greatest challenges in cell therapy. It has been shown that islet transplants can restore normoglycemia in the severe insulin-dependent diabetic, yet there is not nearly enough tissue to transplant. If we are able to generate human islet tissue, we have a chance to provide a therapy with the potential to cure. The key to islet regeneration in situ and islet neogenesis in vitro lies once again in the behavior of cell populations. Unlike the skin, the pancreas provides ample evidence for a progenitor population, and in the last few years, we have learned a great deal regarding the development and relationship of the cell types within the pancreas. That said, the cell source for islet regeneration within the pancreas is still debated. In vitro results demonstrating insulin production in a variety of cell types and the lack of clear growth of islet tissue led to many questions as to potential cell sources for a diabetes therapy (Bonner-Weir et al., 2000; Dor et al., 2004; Ianus et al., 2003; Kojima et al., 2004; Petropavlovskaia and Rosenberg, 2002; Rooman et al., 2002; Shapiro et al., 2000; Soria et al., 2001; Zulewski et al., 2001). However, analysis of the pancreas during development, experimental injury, disease, and in vitro culture illustrates that under certain conditions, the pancreas clearly harbors islet progenitor cells capable of islet neogenesis and that the seemingly varying results often can be explained in the context of cell populations. The pancreas consists of three tissues: the exocrine or acinar component, which makes up the majority of the pancreas and is responsible for the production of digestive enzymes; the ductal component, which is responsible for transport of the digestive enzymes to the gut; and the endocrine component or Islets of Langerhans, which appear as islands of cells within the acinar parenchyma. The islet contains endocrine cells that produce somatostatin, glucagon, pancreatic polypeptide (PP), and insulin. Insulin is produced by the beta cells, which are therefore the focus of any diabetes therapy. The islet is able to respond to physiological demands with compensatory growth of existing islet tissue (Bonner-Weir, 2000a,b; Grapin-Botton et al., 2001), and mice appear able to generate new beta cells exclusively through division of pre-existing insulinþ cells after partial pancreatectomy (Dor et al., 2004). But can cells in the pancreas generate islets de novo? Experimental evidence points to three potential niduses of islet neogenesis in the pancreas: within the outer mantle of the islet, within the pancreatic ducts, and among the acinar component (Bonner-Weir et al., 1993; Guz et al., 2001; Rosenberg and Duguid, 1996; Wang et al., 2001a). Slow-cycling stem cells

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can be found scattered within the acinar component and in the outer mantle of the islet, suggesting the possible presence of stem cells in these locations (Duvillie´ et al., 2003). Based on our working hypothesis, the progenitor cell arises from the unequal division of the pancreatic stem cell. The extent of subsequent proliferation of the progenitor cell appears aVected by the degree of regenerative signaling from the surrounding tissue and infiltrating inflammatory cells (Dembinski et al., 2001). In cases of moderate injury, the progenitor population rapidly generates diVerentiated islet tissue without significant proliferation of the minimally diVerentiated progenitor cell compartment. This is clearly seen in the pancreas. For example, in the partial duct obstruction model (Rosenberg et al., 1989), new islet tissue can be observed appearing to arise from ducts in the absence of extensive damage, disruption, or hyperplasia in the organ (Rosenberg and Duguid, 1996). Several factors have been identified as playing a role in islet regeneration, such as islet neogenesis-associated protein (INGAP) produced by islet cells, which was identified using the partial obstruction model (RafaeloV et al., 1997), as well as gastrin (Rooman et al., 2002), regeneration protein (Reg) (Terazono et al., 1988), and glucagon-like peptide-1 (GLP-1) and its long-acting agonist exendin-4 (Drucker, 2003). All are endogenously produced. Several of these factors are being studied clinically for their ability to enhance regenerative signaling to improve islet regeneration or increase islet cell mass.

B. When Regeneration Is Not Neogenesis Exposure to streptozotocin (STZ), a beta cell toxin, results in the near total destruction of the islet beta cells. When administered to adult rats, some regeneration occurs, but the number of beta cells is small and the animals remain hypoglycemic. Attempted regeneration by the remaining cells appears to manifest as growth of cells from the outer region of the islet (apparently spared during the STZ treatment). It appears that a few insulinþ cells can survive in the outer region (Fernandes et al., 1997). Recovery of beta cell mass can reach 40% with concomitant insulin administration to prevent severe hyperglycemia (Guz et al., 2001). Characterization of these cells showed cells co-expressing somatostatin and insulin with expression of Pdx-1 (Fernandes et al., 1997), an additional cell type expressing GLUT2 (Guz et al., 2001). The number of cells coexpressing somatostatin and insulin is low, and the expression of insulin in these cells may be attributed to the developmental observation of transient co-expression of insulin during normal islet endocrine development and should be not considered an indicator of beta cell diVerentiation (Guz

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et al., 1995). In the adult studies, it appears that metaplasia and limited compensatory growth are at work rather than neogenesis. If STZ is administered to neonatal rats shortly after birth, the animals are able to recover approximately 60% of the beta cell mass and become normoglycemic for a time (Bonner-Weir et al., 1981). However, recovery from STZ appears to produce beta cells that are deficient in their ability to respond to glucose (Bonner-Weir et al., 1981; Tourrel et al., 2001). If GLP-1 or exendin-4 is administered during the regenerative period, there appears to be a selective stimulation of islet neogenesis appearing as an increase in single beta cells and beta cell clusters associated with pancreatic ducts (Tourrel et al., 2001). Surprisingly, there is no improvement in restoring glucose responsiveness. While the reason for this remains unanswered, the result would indicate that the observed stimulation in neogenesis observed in the neonate with or without GLP-1 or exendin-4 is incomplete or abridged. One factor important to the acquisition and maintenance of glucose response is HNF1ß (Boj et al., 2001). Insulin gene expression can occur without acquisition of glucose responsiveness. HNF1ß appears to be essential to stimulate and maintain the genes required for glucose responsiveness, e.g., GLUT2 (Fig. 3). What may also be important is the fact that there is little associated distress to the surrounding acinar and ductal cell population, and although there is apparent stimulation of neogenesis (from what appear to be ‘‘ductal’’ cells), similar to that seen in partial duct obstruction, either the stimulation of GLP-1 receptor alone is insuYcient to set up conditions that allow self-directed completion of neogenesis or there is inhibition from the surrounding parenchyma that interferes with islet diVerentiation. Support for either or both of these possibilities is provided by the observation that if partial duct obstruction (a condition known to stimulate neogenesis via production of the pro-neogenesis factor INGAP while causing a reduction in acinar gland output) is used in the STZ-treated animal, 50% of the animals will be able to achieve normal serum glucose and insulin levels compared to 12% in control STZ animals (Rosenberg et al., 1989). Factor interaction and tissue context will be important issues in any therapy that attempts to use single or multiple factors to interfere with normal parenchymal regulation. C. Stimulation of the Progenitor Pool Significant proliferation of the progenitor cell compartment can be seen in vivo when damage and functional tissue loss is more severe, such as in experimental duct ligation (Rooman et al., 2001), ischemia-reperfusion (Dembinski et al., 2001), or partial pancreatectomy in rats (Bonner-Weir et al., 1997; Dor et al., 2004). Severe damage to the acinar component

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results in the appearance of a new population of simple epithelium, appearing as tubular or duct-like complexes reminiscent of the simple pancreatic epithelium during development (Bonner-Weir et al., 1993; Dembinski et al., 2001; Rooman et al., 2002). The appearance of duct-like tissue led researchers to hypothesize that this occurred due to transdiVerentiation of the acinar cells (Rooman et al., 2002) and/or the proliferation of ductal cells (BonnerWeir et al., 1993). However, analysis of pancreatic development revealed that the islet progenitor and the ductal cell are developmentally distinct populations (Gu et al., 2002) (Fig. 3). Islet neogenesis does occur from these tubular, duct-like complexes. Neogenesis can be further stimulated by gastrin, with an increase in small islets appearing throughout the acinar parenchyma as well (Rooman et al., 2002). The data suggest that it is possible to activate the adult progenitor pool and that neogenesis is related to, but regulated separately from, progenitor cell proliferation.

D. Islet Neogenesis During Development Recently, developmental biologists have deciphered some of the important transcription factors involved in pancreatic organogenesis and have been able to map pancreatic lineage based in large part on transgenic studies in mice (Fig. 3). This lineage enables a better understanding of the genetic relationship among the primitive pancreatic epithelium, the endocrine progenitor cells, and the mature endocrine cells of the islet. The transcription factors identified as important regulators of this process can be used to identify and understand the cell populations generated in vitro. One of the most significant transcription factors in this developmental program is neurogenin3 (ngn3), which is the earliest marker of the committed islet progenitor cell (Gradwohl et al., 2000; Jensen et al., 2000a; Schwitzgebel et al., 2000). Pdx-1 influences early pancreatic organogenesis (Ahlgren et al., 1996; Holland et al., 2002) and later becomes important in the regulation of the insulin-producing beta cell (Ahlgren et al., 1998). Early in development, it distinguishes endoderm that will become pancreas from that which will contribute to the formation of the liver or gut (Edlund, 1999; Zaret, 2002). Developmental studies examining influence of Notch signaling on proliferation (Apelqvist et al., 1999; Murtaugh et al., 2003), notch influence on the expression of Hes-1 (Jensen et al., 2000b) and Ptf1a (Kawaguchi et al., 2002), and their relationship to generation of the ngn3þ progenitor and diVerentiated hormone-producing cells (Lee et al., 2001) support the notion that proliferation of a progenitor pool and progress to a diVerentiated phenotype are distinct events. Expression of Hes-1 requires Notch signaling, whereas in the absence of active notch signaling, the Pdx-1þ pancreatic epithelial cell progresses to an islet progenitor and the expression of ngn3 (Apelqvist et al.,

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1999). Hes-1 controls the advance of the ngn3 pool through lateral specification (Jensen et al., 2000b). The presence of Ki67 in the ngn3-positive cells indicates that the islet progenitor is proliferative during development. However, loss of Hes-1 expression results in hypoplasia of the islet component, although diVerentiation appears to proceed (akin to that seen in the development of skin in p63 / animals), and the exocrine component fails to develop entirely, presumably through the lack of generation of Ptf1a-p48 progenitor population (Kawaguchi et al., 2002) (Fig. 3). Insight as to how this might work in the adult is provided by in vitro studies using both human and canine islets. When isolated islets are suspended in collagen I with the addition of cyclic AMP elevating agents, the mature islet cells undergo apoptosis, leaving behind a population of proliferative duct-like cells that now grow as a duct-like epithelial cyst (DEC) in the matrix (Wang et al., 2001a; Yuan et al., 1995, 1996). This process of biological cell selection and growth takes 10 days. The cells of the cystic structures appear somewhat heterogeneous but are devoid of insulin, somatostatin, and glucagon, label almost uniformly with BrdU, and express cytokeratin 19, an indicator of a proliferative epithelial cell (often used as a ‘‘ductal’’ cell marker). If the DECs are removed from the solid matrix and exogenous cAMP elevation, they undergo what appears to be neogenesis in a way somewhat reminiscent of the islet buds from duct-like structures in vivo and develop islet-like clusters within 4 days (Jamal et al., 2003). Importantly, the generated neo-islet clusters produce insulin at a level indistinguishable from freshly isolated islets and are glucose responsive (L. Rosenberg, unpublished observations). It is unclear whether all or a subpopulation of the cystic cells directly contribute to islet neogenesis (apoptosis is low during the generation of the neo-islet cluster); however, the heterogeneity of the cystic cells would indicate that at the very least, the population contains the islet progenitor. Ongoing analysis of the expression of key developmental factors indicates that this is likely (L. Rosenberg, unpublished observations). There is a lack of progress in islet diVerentiation in the cysts (without a change in culture conditions) (Wang et al., 2001a; Yuan et al., 1995). However, DECs do respond to neogenesis stimulation by the proneogenesis factor INGAP but not GLP-1 or exendin-4 (L. Rosenberg, unpublished observations), which is particularly interesting in light of GLP-1’s failure to establish glucose-responsive islet tissue in the STZ animal model (Tourrel et al., 2001). Early observations of the cystic conversion led to speculation about the possible transdiVerentiation of the mature islet cells to ductal cells; however, further analysis clearly indicates that generation of the DEC is through apoptosis of the mature cells and stimulation of a subpopulation (Jamal et al., 2003). DECs can be subcultivated, but serial propagation of islet precursor cells with demonstrated ability to undergo neogenesis has not yet been reported.

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Culture of cells from islet and ductal isolations has met with limited success in part due to lack of ability to select and foster the appropriate cell. Basic principles governing pancreatic cells are no diVerent from those governing the keratinocyte or hepatocyte, i.e., the presence of diVerentiated cells (be they islet or ductal) limits the ability to uncover the progenitor cell and promote its selective proliferation. We believe the answer is to improve conditions for cell signaling, and not, as has been the case in the last few years, to digress to more primitive or more distantly related cell types (Liang and Bickenbach, 2002; Lumelsky et al., 2001; Soria et al., 2001; Zulewski et al., 2001). Ideally, all roads should lead to the generation of the islet progenitor cell, which will include expression of ngn3 and the ability to undergo the cascade of events leading to robust beta cell diVerentiation (Fig. 3).

VI. Conclusions and Future Directions Comparison of the three tissues yields several important guidelines for future progress. The first is that parenchymal tissue is made up of a population of cells even within a particular parenchymal cell type. Second, progenitor cells exist in the adult organ parenchyma, but their activation and subsequent behavior, quantity, and appearance are dictated by the needs of the tissue. Third, normal surrounding tissue is naturally inhibitory to the generation of a substantial progenitor pool. Distress of the surrounding parenchymal tissue appears to be required in order to observe substantial progenitor pool activation in vivo, and this may be the case in vitro as well. Therefore, cell culture, which most often exclusively focused on getting cells, usually mature cells, to grow and/or maintain diVerentiated function, must now be thought of diVerently. Cultivation of the mature cell will lead to constant inhibition of the progenitor population, poor growth, and usually abridged diVerentiation, if it is enabled at all. Interparenchymal regulation can work for or against us in the engineering and clinical application of cell therapy. It therefore requires that we gather as much information as possible to be able to design the proper tissue implant and formulate a realistic clinical strategy. We must appreciate that tissue regulation will work against the stem and progenitor cell without a very good reason to do otherwise. Therefore, we must consider whether in some cases neogenesis is better done outside the body. Organ transplantation would suggest that in some cases this indeed is what will work best. For example, we already know that skin grafts work and that islet transplantation works (Shapiro et al., 2000) (albeit with limitations), which make them logical goals. In the case of the liver, clinical and experimental evidence suggests that delivery of individual hepatocytes can have clinical benefit (Fox et al., 1998)

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and will be biologically preferable given the need to integrate them into the existing tissue architecture. The challenge will come in overcoming the natural competition of the recipient’s hepatocytes for improved incorporation of the transplanted cells, and in the case of advanced disease, overcoming the limitations of the pathological, fibrotic condition in a way that will still ensure safe regulation of the progenitor cells (Roskams et al., 2003). From this we can get the sense that not all clinical applications will be equally approachable. This is particularly true for skin and liver, which may be used to treat a number of conditions. The argument against the use of normal adult human cells has stated that they are too limiting, incapable, and unyielding. We hope that this chapter has done its part to debunk those views by demonstrating that the adult parenchyma retains remarkable regenerative potential and that the key to that potential lies with the progenitor cell. It will be our job to continue to uncover and enable that potential.

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Adult Bone Marrow-Derived Hemangioblasts, Endothelial Cell Progenitors, and EPCs Gina C. Schatteman Department of Exercise Science University of Iowa Iowa City, Iowa 52242

I. II. III. IV. V. VI. VII. VIII. IX. X. XI.

Introduction Stem and Progenitor Cells Embryonic Hemangioblasts Adult Angioblasts Adult Hemangioblasts Physiological Significance of Bone Marrow-Derived Endothelium Endothelial Cell Progenitors Mobilization Bone Marrow-Induced Functional Improvements in Ischemic Tissue Are We Ready for Human Trials? Looking to the Future Acknowledgments References

Long before their existence was proven, work with blood islands pointed to the existence of hemangioblasts in the embryo, and it was widely accepted that such cells existed. In contrast, though evidence for adult hemangioblasts appeared at least as early as 1932, until quite recently, it was commonly assumed that there were no adult hemangioblasts. Over the past decade, these views have changed, and it is now generally accepted that a subset of bone marrow cells or their progeny can and do function as adult hemangioblasts. This chapter will examine the basic biology of bone marrow-derived hemangioblasts and endothelial cell progenitors (angioblasts) and the relationship of these adult cells to their embryonic counterparts. EVorts to define the endothelial cell progenitor phenotype will also be discussed, though to date, there is no consensus on the definitive adult phenotype, probably because there are multiple phenotypes and because the cells are plastic. Also examined are the putative roles of bone marrow-derived cells in vascular homeostasis and repair, including both their ability to diVerentiate and contribute directly to vascular repair, as well as to promote vascular growth by secreting pro-angiogenic factors. Finally, the use of bone marrow cells as therapeutic tools will be addressed. C 2004, Elsevier Inc. Current Topics in Developmental Biology, Vol. 64 Copyright 2004, Elsevier Inc. All rights reserved. 0070-2153/04 $35.00

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I. Introduction Certainly much of the lay public ‘‘knows’’ that nerves cannot regenerate, and undergraduate physiology students will tell you that cardiomyocytes do not proliferate and cannot be replaced. As for ova, until recently, you would have been hard pressed to find disagreement with the notion that women are born with a fixed number of eggs that die gradually over time, never to be replaced. Yet in the past decade, all of these ‘‘facts’’ have been called into question. There has been a revolution in the way science looks at tissue maintenance and repair, due to the recognition and better understanding of a wide variety of adult somatic stem cells. The first modern evidence of adult somatic cell pluripotency was reported in 1961 in the hematopoietic system (Till, 1961). Hematopoietic stem cells were shown to diVerentiate into all of the mature circulating blood cells: erythrocytes, granulocytes, and cells of the myeloid and lymphoid lineages (Lemischka et al., 1986; Uchida et al., 1993). The idea that hematopoietic cells derived from a pluripotent precursor had long been postulated, so its discovery was not particularly startling to the scientific community. In contrast, the 1976 discovery of adult mesenchymal stem cells was a rather unexpected finding. These cells, located in the bone marrow stroma, could be induced to diVerentiate into a variety of tissues of the mesodermal lineage, although it has taken decades to appreciate the full extent of their pluripotency (Friedenstein et al., 1976; Grigoriadis et al., 1988; Pereira et al., 1995; Pittenger et al., 1999; Umezawa et al., 1992). Pluripotent intestinal crypt stem cells were also described in the 1980s and were shown to provide all four cell types of the small intestine (paneth, enteroendocrine, goblet, and intestinal epithelial cells) (Potten and LoeZer, 1990). In retrospect, it seems surprising that the identification of these three pluripotent adult stem cells did not lead to a stampede to search for other stem cells. If three types existed, why not others? The year 1997 marked the beginning of a dramatic acceleration in the pace of adult stem cell biology. In this year it was reported that hemangioblasts, a common hematopoietic and endothelial cell precursor, might be present in the blood (Asahara et al., 1997). Shortly thereafter, a bevy of papers described a variety of adult stem cells. Conjunctiva stem cells diVerentiate into epithelial and goblet cells of the eye (Pellegrini et al., 1999). Brain contains neural stem cells that can diVerentiate into neurons and blood (Bjornson et al., 1999; Johansson et al., 1999; Kuhn and Svendsen, 1999). Mesenchymal stem cells give rise to tissues of nonmesodermal origin (Pittenger et al., 1999). Cells of the liver and skeletal and cardiac muscle derive from bone marrow (Bittner et al., 1999; Ferrari et al., 1998; Makino et al., 1999; Petersen et al., 1999). Cells in the muscle can act as hematopoietic cells (Jackson et al., 1999). Finally, in 2001 it was demonstrated that single bone marrow cells are

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capable of multiorgan multilineage engraftment (Krause et al., 2001). Moreover, these hematopoietic cells may be the source of other tissue-specific stem cells, such as satellite cells of skeletal muscle and liver (McKinney-Freeman et al., 2002; Petersen et al., 1999). The preponderance of current adult stem cell research focuses on the use of stem cells as therapeutic tools. Clinical trials involving bone marrow and mesenchymal stem cells are already underway around the world. Less attention has been paid to the fundamental properties of the cells and to the role they play in normal and pathological physiology processes. Nevertheless, using the experience of hematopoietic stem cell biologists as a guide, we are gaining a rudimentary understanding of normal stem cell function in the body. This chapter examines the basic biology of bone marrow-derived cells and their progeny in the context of maintaining homeostasis of the vascular endothelium via diVerentiation and proliferation. It also discusses the ability of bone marrow-derived cells to promote vascular growth, a function possibly distinct from their ability to diVerentiate into endothelial cells (ECs). Finally, the use of bone marrow cells as therapeutic tools is addressed.

II. Stem and Progenitor Cells Though we have used the term stem cell, we have not defined a stem cell or its relationship to progenitor cells. As will become clear, distinguishing between stem and progenitor cells is critical for understanding the role of bone marrow cells in the endothelium. Classically, all stem cells share two characteristics. First, they self-renew. That is, they can make identical copies of themselves indefinitely. Second, they give rise to one or more mature cell types with characteristic morphologies and specialized functions. True stem cells are thought to be rare; hematopoietic stem cells probably represent less than 0.01% of bone marrow cells (Weissman, 2000). It is also generally believed that stem cells divide relatively infrequently. That is, even in tissues such as the blood and skin, which are constantly and rapidly replacing themselves, it is the more diVerentiated progeny of the stem cells that divide many times rather than the stem cells themselves (Albert et al., 2001; Bickenbach and Mackenzie, 1984; Cheng et al., 2000; Dormer and Ucci, 1984; LoeZer et al., 1984; Mackenzie and Bickenbach, 1985; Suda et al., 1983). If a stem cell must self-renew for the life of the organism, it may then be inappropriate to refer to many adult pluripotent cells as stem cells. Because long-term self-renewal is diYcult to prove in vivo, it has not been demonstrated for many adult ‘‘stem cells.’’ This limitation has been skirted for bone marrow cells through serial transplantation studies wherein the bone marrow of a recipient mouse is reconstituted from a single donor mouse bone marrow cell (Dick et al., 1997; Fig. 1). The recipient is aged,

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Figure 1 Serial Bone Marrow Transplantation. Bone marrow transplantation has been used widely for bone marrow lineage tracing and to verify self-renewal capabilities. (Top) Genetically tagged cells (eg., lacZ or GFP) are isolated from a donor mouse and clonally expanded. (Middle) Cells are injected into a recipient whose bone marrow has been destroyed by irradiation. After 6 weeks the bone marrow is reconstituted, and mice can be used for lineage tracing. To establish that stem cells were present in the original donor population and have repopulated the bone marrow, bone marrow cells are isolated from the chimeric mouse. (Bottom) These cells are used to reconstitute a second recipient. The bone marrow is examined 6 weeks later to determine if full-lineage reconstitution has occurred.

and its bone marrow is then transplanted to yet another recipient mouse. All hematopoietic lineages can be reconstituted in the serially transplanted mice, demonstrating long-term self-renewal of stem cells (Iscove and Nawa, 1997; Migliaccio et al., 1999). More recently, serial transplantation resulted not only in hematopoietic system reconstitution, but also in multiorgan, multiembryonic lineage engraftment (Grant et al., 2002; Krause et al., 2001). The dominant theory of how stem cells achieve self-renewal states that the cells divide asymmetrically. That is, the parent stem cell produces an identical copy as well as a subtly more diVerentiated copy, and this model of diVerentiation has been recently confirmed (Takano et al., 2004; Yamashita et al., 2003). In some cases, daughter cell orientation and/or cell contact with its niche during cell division retains the stemness (Yamashita et al., 2003), though orientation is not an absolute predictor of stemness (Lyons et al., 2003; Saito et al., 2003). Further, in a study of embryonic neuronal stem cells, the cells did not divide ‘‘in the manner expected of a classic stem cell; that is, one that repeatedly

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self-renews and generates a diVerentiated cell type by asymmetric division’’ (Lyons et al., 2003). Instead, the majority of neurons arose from divisions that generated two neurons, and most lineage trees contained no asymmetric stem cell-like divisions, suggesting that the molecular determinants that control whether a cell will become a neuron may not be linked to a mechanism that generates asymmetric divisions, at least in zebrafish (Lyons et al., 2003). It is not known if this ability to lose and reacquire stemness is unique to the embryo. If present in the adult, it could provide support for reports of transdiVerentiation (BadorV et al., 2003; McKinney-Freeman et al., 2002) and explain why defining the hemangioblast phenotype is so problematic. While proving that a cell is long-term self-renewing is diYcult, on the surface it would seem less tricky to show multi- or pluripotency, at least in vitro. Yet the unique dependence of the stem cell on the microenvironment, or ‘‘niche,’’ to maintain their stemness makes this diYcult as well (Lemischka, 1997; Poulsom et al., 2002; Schofield, 1983; Spradling et al., 2001; Williams et al., 1992; Zieske, 1994). Thus, culturing a single stem cell and coaxing its progeny to diVerentiate into multiple lineages can be a problem. In practice, this is commonly accomplished by co-culturing the stem cell with other diVerentiated cells that provide a suitable microenvironment (Dick et al., 1997; Sutherland et al., 1989; Terstappen et al., 1991). Whether created through asymmetric cell division or another mechanism, many stem cells produce one or more progenitor cells before the final diVerentiated cell is generated. In the epidermis, for example, these progenitors are represented by transient amplifying cells, while in the blood, myeloid, lymphoid, and other progenitor types have been identified (Janes et al., 2002; Nakano, 2003; Uher et al., 2003). Classically, progenitor or precursor cells are considered partially diVerentiated cells that can proliferate extensively and give rise to identical progenitors, more diVerentiated subprogenitors, or fully diVerentiated cells (Fig. 2). Progenitors have been regarded as ‘‘committed’’ to diVerentiating along a particular cellular development pathway, but the complexities in adult cell lineages call this notion into question (Robey, 2000). The concept of a progenitor cell is important to a discussion of EC lineage in the adult, because ECs that arise from the bone marrow probably derive principally from progenitors rather than directly from a stem cell population (Harraz et al., 2001).

III. Embryonic Hemangioblasts Historically, it was thought that blood and ECs arise from a common embryological precursor, the hemangioblast found in blood islands. These small clusters of round antigenically indistinguishable mesenchymal cells begin to change morphologically over time such that cells on the periphery

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Figure 2 Potential Pathways of Stem Cell DiVerentiation. Stem cells may diVerentiate directly into a (A) single or (B) multiple diVerentiated cell types. More typically, stem cells give rise to progenitor cells, which then diVerentiate into (C) a single or (D) multiple diVerentiated cell types, or (E) multiple progenitor cells, which can themselves diVerentiate into one or more diVerentiated cell types.

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become spindle shaped, while those in the interior remain round. The interior cells ultimately diVerentiate into blood cells, while the peripheral spindle-shaped cells coalesce into blood vessels via the process of vasculogenesis (Risau and Flamme, 1995). The discovery and cloning of vascular endothelial growth factor (VEGF) and its receptors VEGFR1 and VEGFR2 turned out to be the keys to proving the existence of hemangioblasts and to understanding how they might be distinguished from other cells (Breier et al., 1992; de Vries et al., 1992; Leung et al., 1989; Matthews et al., 1991; Quinn et al., 1993; Shibuya et al., 1990; Terman et al., 1991). Analysis of mouse and quail embryos suggested that VEGFR2/quek-1 expression characterized the transition from epiblast into hemangioblasts (Eichmann et al., 1993; Flamme et al., 1995). VEGFR2 mRNA also was present in ECs at all stages of mouse development, including the blood islands in the yolk sac of day 8.5–10.5 embryos (Millauer et al., 1993). VEGFR1 was shown to be expressed in embryonic cells from which endothelium is derived, including early yolk sac mesenchyme (Peters et al., 1993). In the quail, quek-1 was detected in the quail mesoderm at the onset of gastrulation, while quek-2 (the VEGFR1 homologue) was found on QH1 expressing ECs (Eichmann et al., 1993). These suggested that the initial diVerentiation of hemangioblasts is characterized by expression of VEGFR2/quek-1, while VEGFR1/quek-2 is an indicator of a partially or fully diVerentiated ECs. If hemangioblasts express VEGFR2, they should respond to VEGF, and if dependent on it, the lack of VEGF or VEGFR2 should aVect both embryonic hematopoiesis and vascular development. In fact, VEGFR2 / mice lack both ECs and hematopoietic cells, supporting the idea that not only is VEGFR2 expressed by the hemangioblast but it also is essential for their diVerentiation along both the endothelial and hematopoietic lineages (Shalaby et al., 1995, 1997). Definitive proof of hemangioblasts was finally achieved when a single VEGFR2þ cell from the chick gastrula was induced to give rise to both hematopoietic and EC colonies in vitro (Eichmann et al., 1997). Binding of two diVerent VEGFR2 ligands directed cells to either the hematopoietic or the EC lineage, and in the absence of the VEGFR2, neither hematopoietic nor EC progenitors survived. Further, while EC diVerentiation required VEGF, a second VEGFR2 ligand appeared to be important for hematopoietic cell development. At about the same time, hemangioblasts were identified in mouse inner cell mass-derived embryoid bodies. These cells appeared early in embryoid body development but were rapidly lost. In response to VEGF, they gave rise to both hematopoietic blast colonies and adherent cells that eventually diVerentiated into ECs (Choi et al., 1998). These studies provided the elusive proof of the existence of hemangioblasts and demonstrated a role for VEGF in their development, a role that has been further elucidated through work with VEGFR2 / mice.

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VEGFR2 mice initially develop a normal complement of hematopoietic progenitors, and VEGFR2 / embryonic stem cells diVerentiate normally into hematopoietic and endothelial cells in vitro, indicating that VEGF is not required for hemangioblast formation (Schuh et al., 1999). Subsequently, embryos are deficient in progenitors, cultures have few blast colonies (Argraves et al., 2002; Schuh et al., 1999), and blocking VEGF signaling inhibits vasculogenesis in embryos and in cultures of 7.5-day prevascular mesoderm (Argraves et al., 2002). On the other hand, VEGF induces VEGFR2 expressing (VEGFR2þ) embryonic cells to diVerentiate into ECs (Yamashita et al., 2000). It appears, then, VEGFR2 signaling is required for hemangioblast migration and/or expansion. There appears to be no critical dependence of ECs on VEGF after vascular assembly, since inhibition of VEGF-mediated activity has no discernible eVect on established embryonic vasculature or vascularized mesodermal cultures (Argraves et al., 2002).

IV. Adult Angioblasts In the embryo blood vessel, growth occurs by two processes. The first, vasculogenesis, is the coalescence of angioblasts into tubes that ultimately form large vessels or vascular plexuses (Risau and Flamme, 1995). The second process, angiogenesis, involves EC proliferation and migration leading to capillary sprouting from pre-existing vessels and intussusception (Kurz and Christ, 1998; Tomanek and Schatteman, 2000). It is generally accepted that adult tissue repair processes, to some extent, mimic ontogeny. Yet, although the existence of embryonic hemangioblasts was long accepted without proof, until recently, it was generally assumed that there were neither hemangioblasts nor angioblasts in the adult, and that vascular growth in the adult occurred only via angiogenesis. Another tenet of faith was that the turnover rate of ECs in the uninjured vasculature is extremely low because proliferative rates in the intact endothelium are low (Gerrity et al., 1975, 1976). As noted, the first publication in the recent wave of adult stem cell papers provided evidence that peripheral blood, that is, bone marrow-derived cells, contained angioblasts, if not hemangioblasts (Asahara et al., 1997). This finding forced the rethinking of long-held assumptions. If angioblasts are present in the circulation, could they not coalesce to form new blood vessels, as in the embryo? Could they not also replace damaged ECs in the vessel wall, implying a higher turnover rate of the ‘‘quiescent’’ endothelium than previously thought? In retrospect, it seems somewhat surprising that the existence of adult angioblasts was ignored or denied for so long. There was no compelling data to rule out their existence, but rather the opposite. In 1932, Hueper and Russell found capillary-like formations in leukocyte cultures; in the

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following year organized vessels were observed in cultures of blood cells; and, somewhat later, adult chicken bone marrow cells were shown to form blood vessels in vitro (Hueper and Russell, 1932; Parker, 1933; White and Parshley, 1950). In the 1980s, two studies of thrombi concluded that the components of the thrombi, including myofibroblastic cells and ECs, were derived from blood cells, not cells from the vessel wall (Feigl et al., 1985; Leu et al., 1987). All of these studies suggested that angioblasts and/or hemangioblasts were present in the blood and bone marrow. Analysis of synthetic arterial grafts beginning in the 1960s also provided evidence for angioblasts, or at least circulating ECs. Grafts were re-endothelialized through ‘‘fallout endothelialization,’’ that is, by adherent blood-derived cells (Mackenzie et al., 1968; Scott et al., 1994; Shi et al., 1994; Stump et al., 1963). This reendothelialization occurred even when ingrowth from the anastomoses and the vaso vasorum was prevented (Shi et al., 1994; Wu et al., 1995). Further, infiltration of bone marrow cells into synthetic vascular grafts led to a more rapid re-endothelialization of grafts than uninfiltrated controls (Noishiki et al., 1996). Data suggesting that graft materials were colonized by EC, smooth muscle cells, macrophages, and monocytes led others to propose in 1994 that stem cells are present in the blood (Scott et al., 1994). A potential source of cells integrating into thrombi and seeding vascular grafts is circulating ECs. ECs are present in the blood, and the number of these cells increases with vascular injury and conditions associated with endothelial dysfunction, such as angina, myocardial infarct, endotoxin damage, smoking, and sickle cell anemia (George et al., 1992; Grefte et al., 1993; Hladovec, 1978; Hladovec et al., 1978; Mutin et al., 1999; Prerovsky and Hladovec, 1979; Sbarbati et al., 1991; Sinzinger et al., 1996; Solovey et al., 1997, 1999; Sowemimo-Coker et al., 1989). These cells could be participating in repair of EC damage. However, in all of these studies the number of ECs was small (less than 10 ECs per milliliter of blood). Most of the ECs were enucleated, and one report indicated that most circulating ECs were apoptotic under normal conditions (Hladovec, 1978; Hladovec et al., 1978; Solovey et al., 1999). Thus, it seemed unlikely that circulating diVerentiated ECs were the source of cells in the thrombi or on the grafts. My group became involved in the study of circulating adult angioblasts through discussions with JeVrey Isner and colleagues, who were studying the eVect of VEGF on re-endothelialization of injured arteries. After denuding both carotid arteries, they delivered VEGF protein locally to one carotid via a double balloon catheter. As expected, VEGF markedly increased reendothelialization of the treated artery, but re-endothelialization of the untreated contralateral artery also improved relative to arteries in animals that received no treatment, albeit to a lesser extent (Asahara et al., 1996). Because only small amounts of VEGF were administered, the insignificant increase in systemic levels of VEGF could not have accounted for the eVect

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even if none of the VEGF remained at the site of administration. My laboratory became interested in the question of what, then, could account for the distal increased re-endothelialization. Might a subset of blood cells maintain hemangioblastic potential? If so, were the circulating cells stimulated, as they passed through the region of high VEGF concentration in the vicinity of the balloon, to take on a more EC-like phenotype? This phenotypic change might also induce the circulating cells to adhere preferentially to sites of denudation, where they would fully diVerentiate into ECs. CD34 is an antigen routinely used to enrich for human hematopoietic stem and primitive progenitor cells that is expressed in blood islands, early embryonic ECs, and activated adult ECs (Civin, 1984; Fritsch et al., 1995; Krause et al., 1996; Siena et al., 1989; Young et al., 1995). Because of this, we focused first on CD34-expressing (CD34þ) peripheral blood mononuclear cells (PBMCs) as a possible source of adult angioblasts. Using standard culture conditions for human umbilical vein EC, cultures of CD34þ, but not CD34 , PBMCs produced EC-like cells (Asahara et al., 1997). The diVerentiated cells made endothelial nitric oxide synthase (eNOS) mRNA (Lamas et al., 1992), expressed CD31 (PECAM) and tie-2 protein (Newman, 1997; Newman et al., 1990; Sato et al., 1993; Schnurch and Risau, 1993), took up acetylated lowdensity lipoprotein (acLDL) (Stein and Stein, 1980; Voyta et al., 1984), bound Ulex lectin (JaVe et al., 1973), formed blood island and tube-like structures, and produced NO in response to acetylcholine and VEGF in a dose-dependent manner. Since freshly isolated CD34þ PBMCs exhibited none of these properties, the data suggested that the cells were not circulating ECs but rather a true adult angioblast population (Asahara et al., 1997). Because VEGFR2 is expressed on embryonic hemangioblasts (Eichmann et al., 1997), it was also a potential marker for adult hemangioblasts, and cultured VEGFR2þ PBMCs diVerentiated into endothelial-like cells in vitro (Asahara et al., 1997). More convincing evidence of CD34þ and VEGFRþ PBMC angioblast activity came when CD34þ, VEGFR2þ, CD34 , or VEGFR2 cells were injected intravenously into mice, rats, and rabbits undergoing neovascularization due to hindlimb ischemia. CD34þ and VEGFR2þ cells, but rarely CD34 or VEGFR2 PBMCs, incorporated into the endothelium of the neovasculature and expressed the EC antigens tie-2 and CD31 (Asahara et al., 1997). Because the CD34þ fraction of human blood is highly enriched for hematopoietic stem cells (Siena et al., 1989) and further enrichment is achieved when cells are co-selected for CD34 and VEGFR2 expression (Ziegler et al., 1999), the ischemic limb data suggested that CD34þVEGFR2þ PBMCs might represent adult human hemangioblasts. The presence of circulating re-endothelializing cells was confirmed when Sauvage and colleagues extended their earlier studies on fallout endothelialization, using dogs whose bone marrow had been replaced by genetically distinct bone marrow (Shi et al., 1998). An implanted Dacron aortic graft,

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impervious to ingrowth from perigraft tissue and the vaso vasorum, was reendothelialized by cells derived from the bone marrow transplant donor. That is, the ECs were bone marrow-derived cells. In the same study, CD34þ cells from human bone marrow, umbilical cord, fetal liver, and mobilized peripheral blood were cultured in the presence of VEGF, basic fibroblast growth factor (FGF-2), and insulin-like growth factor-1 (IGF-1). Cells from all four sources diVerentiated into EC-like cells that internalized acLDL and expressed CD34þ, von Willebrand factor (vWF), and VEGFR2. This study demonstrated that at least some ECs could be marrow-derived, yet the possibility remained that diVerentiated circulating ECs could be responsible for at least some of the observed data. Another study that took advantage of bone marrow transplant mismatch made this less likely. P1H12þ, which is thought to be an EC-specific antigen (Solovey et al., 1997), enriched circulating cells from sex-mismatched transplant patients were cultured. Initially, recipient-derived cells, presumably circulating ECs, proliferated rapidly (Lin et al., 2000), but they expanded only approximately 20-fold. As they died, they were replaced by more slowly growing donor bone marrow-derived cells. These cells ultimately expanded more than 1000-fold, suggesting that they were the more physiologically important cell. Significantly, P1H12 had never been reported to be expressed by angioblasts, and we have never detected the antigen on angioblasts or their progeny in culture. It would appear, then, that in the absence of a procedure that actively selects for circulating ECs, angioblast cultures contain few circulating ECs. These studies made it impossible to ignore the possibility that angioblasts and hemangioblasts might be present in the adult blood and bone marrow, though many remained skeptical that the cultured cells were truly ECs. Others suggested that cells that appeared to be integrated into the endothelium in vivo were not actually part of the endothelium, or if they were, that they were not actually endothelial cells (ZiegelhoeVer et al., 2003). Further, even if the presence of adult angioblasts was accepted, there was no proof for hemangioblasts. Nevertheless, a number of investigators began to work to identify both cell types. Because CD34þ and VEGFR2þ PBMCs appeared to be enriched for adult angioblasts, and both are expressed on most human hematopoietic stem cells (Civin et al., 1984; Krause et al., 1996; Ziegler et al., 1999), it seemed reasonable that hematopoietic stem cells and angioblasts were in fact the same cell, that is, hemangioblasts. In support of this, over the next several years, evidence accumulated that cells expressing hematopoietic stem and progenitor cell antigens including CD34 (human), sca-1 (mouse Ly 5.2), CD117 (c-kit), VEGFR2, and CD133 (AC133) are all enriched for angioblasts, as are side population (SP) cells (Asahara et al., 1997; Gehling et al., 2000; Jackson et al., 2001; Lin et al., 2000; Orlic et al., 2001; Peichev et al., 2000; Quirici et al., 2001; Shi et al., 1998). Work with mesangioblasts also suggested

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that the most primitive precursors express VEGFR2 and that the lack or presence of the transcription factor tal-1 distinguishes between mesangioblasts (which make mesodermal cells and ECs) and hemangioblasts (Ema et al., 2003; Minasi et al., 2002; Robertson et al., 2000). With the exception of Sca-1, cells that express any of these antigens are rare, and there is extensive overlap among the populations, suggesting that hemangioblasts/angioblasts are a rare cell. Still, as in the embryo, these antigens can be found both on subsets of hematopoietic cells and ECs, so that it could not be definitively concluded that the presumed progenitors were not circulating ECs. However, because CD133 is expressed on hematopoietic stem cells but not progenitors and is not known to be expressed on diVerentiated ECs, the ability of CD133þ cells to diVerentiate into ECs provided compelling evidence for the existence of adult angioblasts and hemangioblasts (Gehling et al., 2000). Though initial attention focused on hematopoietic stem cell-related cells as the source of angioblasts, studies began to emerge suggesting that other cells might serve the same purpose. Among these, one purported that a small population of brain cells could act as hematopoietic stem cells, indirectly suggesting that they might also act as hemangioblasts (Bjornson et al., 1999). While this data remains controversial (Yusta-Boyo et al., 2004), it does appear that murine neuronal stem cells injected into stage 4 chick embryos can contribute to tissues of ectodermal, mesodermal, and endodermal origins, indicating that the cells are indeed highly plastic (Clarke et al., 2000). Further, multiple blood lineages were derived from CD34 CD45 CD133þ neural cells in culture (Jay et al., 2002). Muscle-derived cells were also reported to be capable of functioning as hematopoietic stem cells (Jackson et al., 1999), but a subsequent study by the same group showed that the muscle-derived angioblasts actually originated in the bone marrow and came to reside in the muscle, where they lost their blood-like phenotype (Issarachai et al., 2002; McKinney-Freeman et al., 2002). This was consistent with work of Cossu and colleagues suggesting that some satellite cells are bone marrow derived (Ferrari et al., 1998).

V. Adult Hemangioblasts As in the embryo, to prove the existence of adult hemangioblasts it must be demonstrated that a single cell can produce both blood cells and ECs, and ideally this would be done in vivo. In 2001, an exciting study showed that a single lineage-depleted (lin ) mouse bone marrow cell was capable of multiorgan, multilineage engraftment. The experiment demonstrated that hematopoietic stem cells (since blood was reconstituted) are multipotent, but whether ECs specifically were bone marrow derived was not examined (Krause et al., 2001). In the following year, a single cell bone marrow

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transplantation experiment designed specifically to search for hemangioblasts provided definitive proof of their existence. Mouse bone marrow was reconstituted with a single enhanced green fluorescent protein (EGFP) expressing nonadherent Sca-1þ lin cell, and subsequent hypoxia-induced retinal neovascularization led to significant diVerentiation and integration of EGFP-expressing cells into the retinal vasculature (Grant et al., 2002). A recent study showing that single Sca-1þ c-kitþ Lin cells both reconstituted the bone marrow and contributed to the endothelium in irradiated mice confirmed this finding, although bone marrow irradiation was required to induce bone marrow cell population of the endothelium (Bailey et al., 2004). The hemangioblastic potential of human cells has yet to be definitively proven, but data from a patient with chronic myelogenous leukemia carrying the BCR/ABL fusion gene in their bone marrow cells strongly support the hypothesis. After bone marrow transplant from a nonleukemic donor, both BCR/ABL positive and donor-derived ECs were found in the myocardial endothelium (Gunsilius, 2003). In addition, engraftment of 2 105 CD34þ umbilical cord blood cells into NOD/scid mice resulted in bone marrow reconstitution and bone marrow-derived retinal EC integration after induction of retinal hypoxia (Cogle et al., 2004). It is interesting that a diVerent single cell transplants experiment led investigators to conclude that adult bone marrow lacks hemangioblasts. After transplantation of single Sca-1þ c-kitþ Thy1.1lo Lin bone marrow cells, no bone marrow-derived ECs were detected in the chimeric mice (Wagers et al., 2002). However, the cells used were a subpopulation of those used in the other two studies, so the hemangioblasts may have been inadvertently discarded. Further, diVerences in isolation procedures and cell handling could have been suYcient to drive the Sca-1þ lin cells down a nonhemangioblastic pathway. Moreover, since the cells are likely to depend very strongly on environmental cues, the precise transplantation procedure could have modulated cell potential. Probably, however, the answer is much simpler. Both the Grant and Cogle studies used ischemic insult to induce neovascularization, and the Bailey group required bone marrow irradiation to induce diVerentiation of bone marrow into ECs (Bailey et al., 2004; Cogle et al., 2004; Grant et al., 2002). Tissue damage apparently ‘‘pushed’’ the bone marrow cells to become ECs, though why irradiation did not induce a similar eVect in the Wagers study is unclear (Wagers et al., 2002). Possibly it is related to the method of irradiation. It has been argued that the hemangioblastic potential of the bone marrow cells was created artificially and is nonphysiological. Perhaps, but because unusual physiological conditions exist during disease, and retinal hypoxia is reminiscent of what occurs in diabetic retinopathy, these studies seem reasonable. This is especially true because in the absence of injury or disease, vascular growth rarely occurs in the adult. In any case, these studies

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demonstrate clearly that hemangioblasts are present in the bone marrow, but when or if they are normally used can be questioned.

VI. Physiological Significance of Bone Marrow-Derived Endothelium Although the existence of bone marrow-derived hemangioblasts and angioblasts has become widely accepted, as noted, skepticism as to their physiological relevance remains. A number of mismatched bone marrow transplantation experiments have been and continue to be performed, but each seems to arrive at a diVerent conclusion. In the first such study, low-density bone marrow mononuclear cells were implanted into nude (immunocompromised) mice (Asahara et al., 1999a). The transplanted bone marrow was derived from transgenic mice expressing lacZ gene under control of either the flk-1 or Tie-2 promoters. The data showed -galactosidase positive cells integrated into the vasculature in injury models as well as in physiological neovascularization in the ovaries and uterus. (-galactosidase is transcribed from the LacZ gene.) The study did not, however, quantitate the contribution of -galactosidase-expressing cells. Using a related approach, bone marrow was reconstituted using murine fetal liver cells in nonimmunocompromised mice (Crosby et al., 2000). To avoid the requirement for gene expression in order to detect the donorderived cells, donor cells carried 50–100 copies of a pBR322/-globin gene segment (Lo, 1986). Cells carrying the transgene could be detected by in situ hybridization with a pBR322 probe. When gelfoam sponges were implanted in these chimeric mice to induce neovascularization, 4 weeks later, rates of incorporation into the neovessels of the implanted sponge ranged from 8.3 to 11.2%. In other pBR322/-globin chimeric mice, neovascularization was induced by hindlimb ischemia, and rates of bone marrow cell integration into the neovasculature were similar to those seen in the sponges (D. Bowen-Pope and G. C. Schatteman, unpublished). This was the first report to suggest that bone marrow might be a significant physiological source of ECs. It is important to note, however, that the donor bone marrow cells were embryonic; this could have influenced the levels of subsequent tissue engraftment. In the same study, bone marrow cells were found in the uninjured vasculature of the brain, aorta, and skin, but the rates of integration varied among vascular beds (Crosby et al., 2000). DiVerentiation and integration of bone marrow-derived cells into the quiescent endothelium was also reported in the study of patients with chronic myelogenous leukemia mentioned previously (Gunsilius, 2003). Thus, the ‘‘quiescent endothelium’’ may be less quiescent than previously thought, and replacement by circulating cells rather

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than proliferation of adjacent ECs may be a significant means of repairing micro-damage to the endothelium. Shortly thereafter, hematopoietic stem cells containing SP cells were used to reconstitute bone marrow and study the contribution of bone marrow to the vasculature of the infarcted heart in mice. Bone marrow cells were present in 3.3% of vessels in the infarcted hearts, but the cells were localized principally adjacent to the infarct (Jackson et al., 2001). One might wonder what the percentage would have been if vessels only in the peri-infarct region were examined. This question is germane since 20–25% of capillaries in nude rats injected intravenously with human CD34þ cells after myocardial infarct contained injected cells, but the human-derived ECs were located exclusively within the central infarct zone (Kocher et al., 2001). Thus, bone marrowderived EC progenitors can make physiologically significant contributions to neovascularization in the rodent, but the extent of their participation probably depends on physiological context. Female-to-male heart transplants make it possible to examine human chimerism in the heart by Y chromosome labeling. In one such study, 20% of arterioles and 15% of capillaries were principally male derived (Quaini et al., 2002). In another study in human kidney transplant patients, Y chromosome labeling or HLA typing revealed that in some grafts, most of the ECs were recipient derived, while in others less than one-third were (Lagaaij et al., 2001). Interestingly, the highest levels of recipient-derived ECs were associated with the greatest vascular rejection. When aortic allografts were performed in rats, the endothelium of the grafts was replaced by recipient ECs, but if transplantation was accompanied by immunosuppression, the ECs were donor derived (Hillebrands et al., 2001). Though it is not known if the cells were bone marrow derived, the correlation between inflammatory response and recipient-derived ECs suggests that the ECs may originate from the inflammatory cells in the blood. However, transplants of aortas from Dark Agouti rats into Brown Norway rats whose bone marrow had been reconstituted with Lewis rat bone marrow do not appear to support this hypothesis. These mice developed marked transplant atherosclerosis with a neointima that was formed exclusively by host Brown Norway-derived cells. In the reciprocal experiment of transplanting Brown Norway bone marrow into Lewis rats, virtually no transplant atherosclerosis developed, and both donor-derived (Dark Agouti) and host-derived ECs were found lining the aorta. Of the host ECs, only 1–3% were bone marrow derived (Brown Norway) (Hillebrands et al., 2002). While this suggests a non-bone marrow source for ECs in the lesions, the authors suggest that the ECs may be delivered via the circulation because of the dispersed clonal distribution of host-derived cells in the grafts. Further, the introduction of three diVerent sets of histocompatibility antigens into a single animal complicates interpretation of the data.

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Muddying the waters even further, replacement of donor endothelium by recipient cells was not observed in cardiac allografts, even in the presence of a robust inflammatory response (Hillebrands et al., 2001). In the final analysis, the significance of host bone marrow or other types of cells in tissue repair may be organ (or endothelium type) and injury type dependent. The use of supraphysiological growth factors to induce angiogenesis may give an unrealistic picture of the normal contribution of bone marrow cells to the vasculature, but it can give insight into the maximal potential of bone marrow-derived cells to contribute to the endothelium. In the mouse retinal neovascularization model described previously, although not specifically quantitated, large bone marrow-derived foci of neovascularization were observed (Grant et al., 2002). In another such study, in mice whose bone marrow was reconstituted with cells from transgenic mice expressing the lacZ gene under control of the Tie-2 promoters, 26.5% of ECs in vessels in subcutaneously implanted matrigel plugs impregnated with FGF-2 were bone marrow derived (Murayama et al., 2002). Also, implantation of VEGF-impregnated pellets into the cornea resulted in 17.7% of ECs in the neovasculature being bone marrow derived (Murayama et al., 2002). Curiously, though, the same group reported incorporation of only 7.3% in the same model in the preceding year, but found that simvastatin markedly increased the percentage of bone marrow-derived ECs to 25.7% (Llevadot et al., 2001). Their own data suggest that the discrepancy is attributable to diVerences in methodologies used to detect lacZ activity in the two studies, but which method more accurately reflects bone marrow contribution to the endothelium is debatable (Murayama et al., 2002). Nevertheless, clearly, bone marrow cells can be coaxed to make substantial contributions to the growing vasculature, and this has tremendous implications for the understanding of tumor growth and pro-angiogenic therapies. The broadest distribution and most robust integration of ECs into the vasculature may occur when bone marrow is reconstituted with a phenotypically diverse population. All bone marrow progenitors may not be created equal, and a mixed repertoire of cells may be required for any single cell to reach its full multipotent potential. This leads to the question of whether specific subpopulations of bone marrow cells might be lost due to disease or perhaps aging, and if that loss could contribute to a decreased ability to repair the endothelium despite no detectable hematopoietic impairment.

VII. Endothelial Cell Progenitors Like most stem cells, hematopoietic stem cells do not diVerentiate directly into terminally or ‘‘fully’’ diVerentiated cells but proceed through a series of intermediaries, many of which are multipotent progenitors. Because

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hematopoietic stem cells and angioblasts are derived from the same precursor, it is reasonable to think that diVerentiation of hemangioblasts into ECs might also be mediated through progenitor cells. Further, the high proportion of bone marrow-derived cells in the neo-endothelium in some circumstances seems remarkable if hemangioblasts directly produce all ECs, since hemangioblasts appear to be an extremely rare cell. In addition, various studies have used highly heterogeneous adherent peripheral blood mononuclear cells and assumed the rare CD34þ cells in the population were the source of the EC precursors. Yet the number of ECs generated in the very short time periods in these studies would have required extraordinary proliferation rates for the CD34þ cells. A more plausible interpretation of the data is that there is another source of angioblasts, that is, a progenitor cell. Studies on thrombi gave clues as to the possible identity of circulating EC progenitors. As early as 1967, morphological studies suggested that monocytes could form intrathrombus capillaries (Feigl et al., 1985; Leu et al., 1987; Prathap, 1972; Tsapogas et al., 1967). Because monocytes are found at all sites of injury, they would be present where and when needed for vascular repair, and an intimate relationship between monocytes and the endothelial surface during collateral artery growth has long been recognized (Schaper et al., 1976; Scholz et al., 2000). Additionally, Waltenberger and colleagues reported that VEGF-A-induced chemotaxis is attenuated in monocytes derived from diabetic patients (Waltenberger et al., 2000). This is significant because of the association of diabetes with vascular damage and impaired neovascularization. Finally, monocytes are a highly plastic cell type that can diVerentiate into dendritic cells, various types of tissue macrophages, and KupVer cells (Eiermann et al., 1989; Hausser et al., 1997; Randolph et al., 1998; Wisse, 1974). Not surprisingly, in 2000 and 2001, a group of reports appeared demonstrating that monocytes could diVerentiate into ECs (Fernandez Pujol et al., 2000, 2001; Harraz et al., 2001; Schmeisser et al., 2001). The ECs derived from cultured monocytes were phenotypically distinct from those derived from cultures of more ‘‘primitive’’ cells (i.e., CD133þ or CD34þ cells), simultaneously expressing EC antigens including CD31, Tie-2, and vWF as well as the macrophage/monocyte antigens CD68, CD80, CD45, and CD36 (Fernandez Pujol et al., 2001; Schmeisser and Strasser, 2002; Schmeisser et al., 2001). Yet, the cells also expressed VE-cadherin and made the enzyme eNOS, suggesting that they could perform physiological functions associated with ECs, and with time in culture, downregulated expression of monocytic antigens (Harraz et al., 2001). In long-term highdensity monocyte cultures, many cells formed large vacuoles, taking on the appearance of a capillary in cross-section. Over time, small rings tended to fuse, creating larger rings of the size of small arterioles in a novel ‘‘vasculogenic’’ process not previously reported for CD34þ cell cultures (Harraz et al., 2001).

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Because of their ‘‘schizophrenic’’ phenotype, initially there was a great deal of skepticism as to whether these monocytic cells represent true EC progenitors. Yet, in vivo data demonstrate that ECs with a mixed phenotype are present in many tissues. For example, liver sinusoidal cells express the classical monocytic markers CD32, CD16, CD14, and CD80, activated microvascular cells can express class II major histocompatibility complex molecules, and CD86 is expressed in vessels of patients with vascular neuropathy (Schmeisser and Strasser, 2002). Studies from a number of laboratories have confirmed and extended these initial findings (Fujiyama et al., 2003; Nakul-Aquaronne et al., 2003; Rehman et al., 2003; Urbich et al., 2003; Zhao et al., 2003). Interestingly, one of these found that monocytes derived from the bone marrow and those from the circulation yield antigenically distinct EC-like cells (Fujiyama et al., 2003). Also of note, another provided evidence that monocytes are multipotent stem or progenitor cells (Zhao et al., 2003). That is, not only can highly purified monocyte populations diVerentiate into lymphocytic, epithelial, neuronal, endothelial, and hepatocytic-like cells in vitro, but also a colony derived from expansion of a single such cell could produce diVerentiated cells in all of these lineages. Thus, the monocyte appears to represent a highly plastic reserve cell that can modulate its phenotype according to local conditions and needs. Whether the replacement cells act as temporary ‘‘placeholders,’’ or if they remain, ultimately diVerentiating into fully functional replacement cells, is unclear. At the same time that work was progressing on monocytes as a source of EC progenitors, a number of investigators began using more heterogeneous cell populations as sources of EC progenitors. These mixed populations are now commonly referred to as EPCs and have been studied widely as potential therapeutic agents. The term EPC, meaning endothelial progenitor cell, was coined in 1999 by Isner and colleagues (Asahara et al., 1999b; Takahashi et al., 1999), but this group used EPC to refer to vitronectin adherent mouse bone marrow mononuclear cells in one publication (Asahara et al., 1999b), to Sca-1þ bone marrow mononuclear cells in a second (Takahashi et al., 1999), and to human PBMCs that were adhered to fibronectin after 4 days in culture in a third (Kalka et al., 2000c). Other cells, including EC derived from late outgrowth colonies of blood mononuclear cells and mouse Sca-1þKDRþ (presumably VEGFR2þ) circulating cells, have also been referred to as EPCs (Simper et al., 2003). This nonstandardized use of the term EPC has made reading and interpretation of the literature diYcult. Recently, the term has been applied more consistently and most commonly to adherent circulating or bone marrow mononuclear cells cultured for 4 days on fibronectin or gelatin (Kalka et al., 2000b; Tepper et al., 2002; Vasa et al., 2001b). The recent relative uniformity in the definition of EPCs is helpful for beginning to understand their origins and phenotype. The most consistently described feature of the cells is that they take up acLDL and bind Ulex

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lectin, characteristics that are present after the initial 4-day plating period, a time at which cells are routinely harvested for in vivo applications. These cells have been shown to take on an EC phenotype in vitro, expressing VE-cadherin, CD34þ, CD31þ, VEGFR2, and P1H12 (Dimmeler et al., 2001; Loomans et al., 2004; Tepper et al., 2002), yet after the initial 4 days in culture, few cells express VE-cadherin, E-selectin, or CD34, but they do express CD45, CD14, CD163, and CD11c (Kalka et al., 2000b; Rehman et al., 2003). This phenotype is characteristic of monocyte/macrophages but not ECs. With time in culture, EPCs co-express monocytic and EC antigens (Kalka et al., 2000b), which is consistent with studies demonstrating that monocyte-derived ECs initially co-express EC and monocyte antigens but lose monocytic characteristics over time (Fernandez Pujol et al., 2001; Harraz et al., 2001; Schmeisser et al., 2001). It appears, then, that the majority of, if not all, EPCs are monocyte derived. Although we were unable to derive ECs from a CD14 fraction of peripheral blood, a study suggests that expression of CD14 is not requisite for EPCs (Harraz et al., 2001; Urbich et al., 2003). The expression of vWF was evaluated on CD14þ and CD14 cells after 4 days in culture, and levels of the EC antigen were similar in CD14þ and CD14 cell cultures. Since not all monocytes express CD14þ and the CD14 cells exhibited other characteristics of monocytes, including uptake of acLDL and Ulex lectin binding, the CD14 EPCs may be derived from CD14 monocytes. Monocytes as EC progenitors in vivo is only beginning to be studied, but what data are available indicate that understanding monocyte participation in vascular growth and repair will not be a simple task. It is apparent that vascular growth per se may not be enough to induce monocytes to integrate into the vasculature, but that additional extrinsic stimuli may be required. One factor that can coax monocytes into an EC phenotype may be MCP-1. In mice overexpressing monocyte chemoattractant protein-1 (MCP-1) in the heart, Moldovan and colleagues found that monocytes can drill channels in the myopathic heart and these channels carry blood (Moldovan et al., 2000). In another study, MCP-1 was required to induce infused PKH2-GL (a green fluorescent dye) labeled monocytes or CD34þ cells to adhere to denuded vessels. Once adhered, cells diVerentiated and covered the denuded surface with a neo-endothelium (Fujiyama et al., 2003). We labeled CD14þ monocytes with the lipophilic dye DiI and injected the cells into ischemic mouse muscle. Five days and 2 weeks later, few labeled cells were present in the wall of the neovasculature of the muscle. When the experiment was repeated, this time co-injecting unlabeled CD34þ peripheral blood mononuclear cells and labeled CD14þ cells, many DiI-labeled cells were observed in the neo-endothelium (Harraz et al., 2001). More recently, we have found that co-injection of CD34þ cells and monocytes, but not injection of either alone, resulted in formation of arteriole-like structures

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Figure 3 Diabetic Sca-1þ Bone Marrow Cells Inhibit Vessel Growth. Micrographs of 7m sections of mouse limb muscle 5 days after induction of ischemia and co-injection of human CD34þ and CD14þ PBMCs. At the time of sacrifice, the limb was perfused via the abdominal aorta with India ink. (A) Phase contrast micrograph showing capillaries and arterioles filled with black India ink. (B) Fluorescence micrograph of same section as in A labeled with Ulex lectin to label human-derived ECs. Arrow in (A) and (B) indicate a human-derived arteriolesized vessel filled with India ink demonstrating its connection to the systemic vasculature.

that are connected to the systemic vasculature (Fig. 3). Thus, again extrinsic cues provided appear to be required for the diVerentiation or vascular integration of monocytes. In this case, VEGF and angiopoietin-1 could be the relevant factors, since CD34þ cells secrete VEGF and angiopoietin-1, both of which may be critical inducers of monocyte diVerentiation into the EC lineage (Bautz et al., 2000; Majka et al., 2001; Takakura et al., 2000).

VIII. Mobilization To make a significant impact on healing, circulating EC progenitors must be present at the site of injury in suYcient numbers to eVect a repair, but this could be problematic if rare stem-like cells, such as human CD133þ or CD34þ or mouse Sca-1þ c-kitþ cells, need to be recruited. One means by which the body might recruit progenitors would be to mobilize cells from the bone marrow and then recruit them to the site of injury through a homing mechanism. Many agents that can mobilize hematopoietic stem cells have already been identified, and since the stem cells that produce ECs cannot be distinguished from hematopoietic stem cells, it is thought that these factors also mobilize circulating EC stem cells. Other nonclassical mobilizing agents have been examined, particularly as mobilizers of EPCs (Asahara et al., 1999b; Kalka et al., 2000a,c; Moore et al., 2001). One nonclassical potential mobilizer is VEGF. VEGF induced expression of EC antigens on circulating cells, evidence of EC progenitor mobilization, or alternatively, an increase in circulating ECs (Asahara et al., 1999b; Kalka et al., 2000a; Moore et al., 2001). VEGF also induced increases in circulating VEGFR2þ cells, suggestive of primitive EC progenitor mobilization

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(Asahara et al., 1999b). In addition, when whole PBMCs were plated, cells from VEGF-treated mice yielded more than twice as many EPCs as untreated animals. However, the increase in EPCs was of the same magnitude as the increase in total monocytes, so putative mobilization does not appear to be specific to EPC progenitors (Asahara et al., 1999b; Moore et al., 2001). VEGF also induced an increase in the uptake of acLDL by circulating cell populations. It has been suggested that this indicates mobilization of EC progenitors, but a relationship between uptake of acLDL in circulating mononuclear cells and EC progenitors has not been established (Asahara et al., 1999b). Like VEGF, angiopoietin-1 mobilizes VEGFR2þ cells, and the two factors appear to act synergistically to enhance mobilization (Moore et al., 2001). Further, late outgrowth colonies, EC colonies that are thought to derive from the most primitive of EC progenitors, are more numerous in cultures of cells mobilized with both factors than with either VEGF or angiopoietin-1 alone. Surprisingly, late outgrowth cells are more numerous in cultures of angiopoietin-1 than in VEGF-mobilized blood (Moore et al., 2001). In vivo VEGF mobilization led to an increase in the number of bone marrow-derived cells present in injured corneas, though cells were present in the stroma, so it is not clear how many of the recruited cells were ECs. Because the identified cells did express lacZ driven by the tie-2 promoter, it is likely that they were at least EC progenitors. In another study of patients undergoing VEGF gene therapy, the number of EPCs 4 days after plating of whole PBMCs rose with the rise in circulating levels of VEGF (Kalka et al., 2000a). Hence, VEGF does appear to potentiate the ability of circulating cells to produce EPCs, possibly through mobilization, possibly through other mechanisms. Other potential nonclassical mobilizing agents are placental growth factor and stromal cell-derived factor 1. Although these ligands have not been shown directly to mobilize EC progenitors, they do mobilize primitive hematopoietic progenitors as well as monocytes, and could well mobilize both primitive and monocytic EC progenitors (Hattori et al., 2001, 2002; Moore et al., 2001). Statins also may mobilize EC progenitors. Atorvastatin treatment increased the number of circulating VEGFR2þ cells (Vasa et al., 2001a), and EPC production by cultured mononuclear cells increased in patients treated with the statins simvastatin, atorvastatin, and mevastatin (Dimmeler et al., 2001; Llevadot et al., 2001). Simvastatin treatment also increased bone marrow-derived ECs or EC progenitors (i.e., cells that expressed lacZ driven by the tie-2 promoter) in the injured cornea (Llevadot et al., 2001). Among classical hematopoietic stem cell mobilizers, granulocyte colonystimulating factor (G-CSF) mobilizes CD34þ cells in baboons. Mobilization led to increased vascularization of ischemic myocardium but only very modest functional improvements (Noro1 et al., 2003). To examine the eVects of granulocyte-macrophage colony-stimulating factor (GM-CSF) on EC

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progenitor mobilization, the number of Sca-1 cells in the circulation was measured before and after mobilization and ‘‘the frequency of EPC-enriched population marked by Sca-1 in the circulation was 10.7 1.0%. . . ’’ (Takahashi et al., 1999). Unfortunately, since the majority of Sca-1þ cells (typically >95%) in the blood are normally T-lymphocytes, Sca-1 alone is not likely to be a meaningful marker for mobilization of EC progenitors (Kimura et al., 1984; Spangrude et al., 1988a,b). However, GM-CSF is known to mobilize primitive Sca-1þ hematopoietic progenitors, so the conclusion may still be correct (Brasel et al., 1997; Robinson et al., 2000). Further, GM-CSF treatment increased the ability of circulating cells to produce EPCs in culture and accelerated re-endothelialization of denuded vessels in rabbits (Cho et al., 2003). As with VEGF mobilization, GM-CSF treatment led to an increase in the number of bone marrow-derived ECs or EC progenitors (i.e., cells that expressed lacZ driven by the tie-2 promoter) in injured corneas (Takahashi et al., 1999). GM-CSF, simvastatin, and VEGF all promoted neovascularization in injured corneas, and the number of bone marrow-derived cells in the tissue increased. An intriguing question is if the increase in bone marrow-derived cells is due to a higher rate of incorporation of progenitor cells into the vasculature or if it is due simply to the fact that there were more blood vessels. In the early postnatal period, VEGF stimulates a 10-fold increase in bone marrow-derived liver sinusoidal ECs, with no concomitant increase in total sinusoidal endothelium, suggesting a VEGF-stimulated increase in the rate of EC precursor integration (Young et al., 2002). In contrast, in the early postnatal heart, the increase in bone marrow-derived EC precisely parallels increases in vascularity (Young et al., 2002). Thus, there appears to be tissue-to-tissue variability in progenitor function, at least in the young. The mobilization described thus far involves the use of exogenous mediators. Does the body have endogenous mechanisms for mobilizing EC progenitors? Evidence is accumulating that it does. Hindlimb ischemia, myocardial infarction, vascular trauma, and exercise mobilize bone marrow EC progenitors as assessed in vitro, and this mobilization correlates with increased vascularization in tissues undergoing neovascularization (Adams et al., 2004; Gill et al., 2001; Laufs et al., 2004; Shintani et al., 2001a; Takahashi et al., 1999). It is important to remember that despite the evidence that these various molecular and physiological mediators can mobilize EC progenitors or EPCs, the data to date have not proven this. Increases in EPC numbers in culture could be due to activation of EC progenitors, such that they are more likely to survive (e.g., by upregulating adhesion molecules), proliferate, or diVerentiate. In support of this, simvastatin both induced proliferation and increased survival under stress of EPCs in culture (Llevadot et al., 2001), and VEGF is a stimulator of EC progenitor growth and diVerentiation (Fernandez Pujol et al., 2000; Wang et al.,

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2004). Careful analysis of plating eYciencies, apoptosis, proliferation, and related parameters will be needed before more definitive conclusions can be reached. Even then, since the most likely scenario is that more than one of these mechanisms will come into play in response to a given stimulus, sorting mechanisms out may be diYcult. Numerous studies have shown that EC progenitors tend to localize at sites of injury (Asahara et al., 1997; Takahashi et al., 1999). We and others have attributed the relative abundance of EC progenitors in ischemic or damaged tissue to ‘‘homing’’ (Asahara et al., 1997; Schatteman et al., 2000; Takahashi et al., 1999). However, chemokines and other factors released at the wound site recruit inflammatory cells. Because EC progenitors appear to be inflammatory cells that have a capacity to diVerentiate into a variety of cell types, one of which is ECs, it is unlikely that EC progenitor homing is specific. Rather, factors at the site of injury may induce blood-borne cells to move into the EC lineage, and presumed mobilization agents could act by promoting this diVerentiation. This seems likely, particularly in the case of VEGF, which is essential for diVerentiation of ECs (Eichmann et al., 1997; Shalaby et al., 1995, 1997) and is upregulated at many sites of tissue repair (Banai et al., 1994; Hashimoto et al., 1994; Pierce et al., 1995).

IX. Bone Marrow-Induced Functional Improvements in Ischemic Tissue The ability to harvest EC progenitors from the blood and bone marrow has generated tremendous interest in their therapeutic potential. Numerous studies have been performed in just the past 5 years examining this potential in therapeutically relevant animal models and in clinical trials in humans. In the first of these studies, freshly isolated and cultured bone marrow mononuclear cells were injected into cryo-injured myocardium, and both fresh and cultured cells more than doubled capillary density in the scar relative to controls (Tomita et al., 1999). Improvements in functional parameters, including left ventricular developed pressure, ejection fraction, and muscle perfusion, also have been observed after injecting whole bone marrow mononuclear cells or human CD34þ into the rat heart (Kamihata et al., 2001; Kawamoto et al., 2001; Kobayashi et al., 2000). Significantly, freshly isolated whole bone marrow and nonadherent CD31þ cells also improve flow and myocardial function after ameroid constriction in the pig, a large animal (Fuchs et al., 2001; Kawamoto et al., 2003). The ability of blood-derived cells to improve vascularization of ischemic hindlimbs has been examined as well. Whole bone marrow injection elevated muscle blood flow and rat limb functional capacity (Ikenaga et al., 2001), and introduction of EPCs into the systemic circulation of mice with ischemic

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limbs resulted in dramatic improvement in blood flow to the limb as well as limb salvage (Kalka et al., 2000b). Limb salvage is relevant because control mice regularly auto-amputated. In contrast, when CD34þ-enriched cells (20% pure) were injected directly into ischemic muscle, little improvement in blood flow after treatment was reported for nondiabetic mice, but in these mice auto-amputation does not occur and the mice normally recover rapidly from the ischemia (Schatteman et al., 2000). However, when diabetic mice received the same treatment, profound improvement in flow was observed. Injection of circulating CD34þ cells into immunodeficient mice and rats also resulted in significant improvements in limb blood flow, arteriolar density, and capillary density (Murohara et al., 2000; Pesce et al., 2003). Improvements in angiographic score and capillary density also were seen in rabbit ischemic hindlimbs injected with fresh whole bone marrow mononuclear cells (Shintani et al., 2001b). Collectively, these studies found that injected cells were present in the vasculature of the ischemic limb of mice, rats, and rabbits, but not in the unoperated contralateral limb. The myriad of in vivo studies, including these, have used diVerent subsets of bone marrow cells, which have contained hematopoietic stem and progenitor cells, mesenchymal stem cells, and/or monocytes. While many of the subsets are mutually exclusive, each has been reported to induce vascularization of ischemic tissue. Thus, though it is clear that administration of bone marrow cells can improve blood flow, its eVects cannot be restricted to a single cell type, and which cells mediate its eVects is a mystery. In fact, injection of platelets alone has been found to be suYcient to promote vascularization of an ischemic rat limb (Norol et al., 2003). Interestingly, large-scale integration of exogenous cells into the neovasculature was not observed in the majority of these studies, and certainly not on the scale that would have been suYcient to produce huge increases in blood flow and capillary density. Thus, injection of bone marrow cells probably induces changes in the local environment that enhance angiogenesis or endogenous cell-mediated vasculogenesis. This makes sense because, in the embryo at least, angiogenesis and hematopoiesis are interdependent. Hematopoietic stem cells are critical for vascular growth, an eVect that appears to be mediated in large part through secretion of angiopoietin-1 (Takakura et al., 2000). Primitive EC progenitors have also been used to induce heart repair. Human peripheral blood CD34þ cells transplanted into the ischemic myocardium of nude rats resulted in improved ventricular systolic function and increased capillary density. Integration of the cells into the endothelium was not investigated (Kawamoto et al., 2003). When highly purified CD34þ cells isolated from human G-CSF-mobilized pheresis product were injected intravenously, large improvements in left ventricular ejection fraction, wall motion, capillary density, and myocardial morphology as well as reduced apoptosis in nude rat hearts after LAD occlusion were observed (Kocher

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et al., 2001). Surprisingly, although exogenous blood-derived ECs were localized exclusively in the central infarct zone, they comprised 20–25% of capillaries in the infarct. Vessels of rat origin were not found in this region but were abundant in the peri-infarct region. Similarly, injection of GFP expressing c-kitþlin bone marrow cells into the peri-infarct region in mice improved heart function and resulted in essentially all ECs within the repairing infarct being donor derived (Orlic et al., 2001). With the exception of our study using human CD34þ cells in nude mice (Schatteman et al., 2000), all studies using primitive EC progenitors led to relatively large contributions of the exogenous cells to the vascular endothelium. Thus, it appears that adult vasculogenesis may require primitive progenitors and that EPCs may not be capable of this function, at least not without other extrinsic support. In addition, the primary means of neovascularization in the ischemic heart may well be bone marrow cell-mediated vasculogenesis. Interestingly, because injection of primitive progenitors into the ischemic limb leads to somewhat lower levels of engraftment than in the heart, the heart could be a particularly nurturing environment for bone marrow-derived stem and progenitor cells.

X. Are We Ready for Human Trials? Clinical trials are under way to test the therapeutic potential of bone marrow cells in various settings, and data from these trials are somewhat encouraging. For example, in a 20-patient study wherein autologous bone marrow cells were infused 5 to 9 days after myocardial infarct, the size of the wall defect decreased, and wall movement increased in the 10 treated patients relative to controls (Strauer et al., 2002). In similar studies of 8–21 patients each, intracoronary or ventricular bone marrow cell infusion modestly improved cardiac function, including injection fraction and coronary reserve after infarct (Assmus et al., 2002; Perin et al., 2003; Tse et al., 2003). More recently, 26 patients were infused locally with bone marrow cells or EPCs via intracoronary injection after myocardial infarction, and functional improvements were observed in the patients (Dobert et al., 2004). Finally, CD133þ bone marrow cells were injected around the circumference of the infarct border following coronary artery bypass graft (CABG). Only a small number of cells were implanted and no eVect on contractility was noted, but perfusion improved dramatically (Stamm et al., 2003). However, because no control data was presented in the latter two studies, it is impossible to know the significance, if any, of these findings. For example, in the Stamm (2003) study, since CABG was also performed, one would expect improvements in perfusion, so the eVect of the implanted cells cannot be judged. These studies were characterized by rather small improvements in ejection fraction or end systolic and diastolic volumes, but there

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seemed to be consistent findings of wall motion improvements. This suggests that the benefits of the therapy may not be readily apparent at the 2- to 4-month time points examined but may be significant with respect to slowing further deterioration of the muscle over longer periods. Bone marrow cell therapy to improve healing and revascularization of ischemic limbs also has shown some promise. Perfusion in a patient’s saline-injected nonischemic limb and bone marrow-treated contralateral limb was examined. In the nonischemic limbs, perfusion was unchanged, but in the treated limb, transcutaneous oxygen pressure (TcO2) increased from a mean of 28 to 46 mm Hg at 4 weeks and remained there at 24 weeks. Data from patients with two ischemic limbs were somewhat surprising, based on EPC data in animals. In the limb treated with blood-derived cells, TcO2 improved slightly (31 to 36 mm Hg), while in the other limb treated with bone marrowderived cells, TcO2 improvement was dramatic (29 to 46 mm Hg). Again, these levels of TcO2 persisted for 24 weeks (Tateishi-Yuyama et al., 2002). These data are consistent with the finding that monocytes derived from the bone marrow, but not the circulation, can promote re-endothelialization in rats (Fujiyama et al., 2003). Hence, nuanced choices of bone marrow-derived cells may be critical to therapeutic eYcacy. Do these studies coupled with animal data mean that we should press forward with large-scale human trials? Unfortunately, a series of studies suggest that we should not. First, cardiomyocytes were derived from mouse embryonic stem cells in culture. When the electrical properties of the cells were examined, it was found that they had surprising and worrisome arrhythmic potential (Zhang et al., 2002). Cells showed persistent automaticity, slow conduction, and triggered activity. In light of this, it is worth noting that in the Stamm (2003) study, which used primitive CD133þ cells as the therapeutic agent, two of six patients had supraventricular arrythmia after transplant. Another concern has to do with the use of autologous cells in diabetic patients, in whom EC progenitors may be dysfunctional. There is evidence that the number of total blood-derived ECs may be reduced by both type 1 and type 2 diabetes. The apparent reduction correlates with the number of years postdiagnosis in type 2 diabetes and inversely correlates with HbA1c levels in type 1 diabetes (Loomans et al., 2004; Schatteman et al., 2000; Tepper et al., 2002). This suggests that local application of autologous EC progenitors might be beneficial. However, there appears to be a reduced ability of type 2 diabetic-derived EPCs to proliferate, adhere, and integrate into tubes in vitro, suggesting that autologous bone marrow-derived cells may not substantially enhance vascularization in diabetic patients (Tepper et al., 2002). Studies from our laboratory go one step further. We have found that treatment with mouse Sca-1þ bone marrow cells from Leprdb mice, a mouse model of type 2 diabetes, actually inhibits vascularization of skin wounds and ischemic limbs of both nondiabetic and diabetic mice

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(Stepanovic et al., 2003; G. C. Schatteman, unpublished). That is, autologous bone marrow transplantation in type 2 diabetic patients could be harmful. Other possible roles of EC progenitor dysfunction in vascular disease have come to light. For example, the number and migratory activity of EPCs obtained from peripheral blood inversely correlate with risk factors for coronary artery disease, and bone marrow-derived ECs are found in atherosclerotic plaques (Campbell et al., 2001; Sata et al., 2002; Vasa et al., 2001b). Recently, G-CSF therapy in conjunction with intracoronary infusion of autologous mobilized peripheral blood mononuclear cells was found to improve cardiac function, but the trial was stopped because of an unexpectedly high rate of instent restenosis (Kang et al., 2004). Do these data mean that impaired EC progenitors cause coronary artery disease by diVerentiating down an inappropriate pathway, or simply that the dysfunctional cells cannot repair lesions, or something else? Until questions such as these are resolved, except in exceptional cases, EC progenitor therapy may be a somewhat dangerous gamble. Finally, as with other pro-angiogenic therapies, it is important to consider the potential of bone marrow-derived cells to exacerbate or even induce tumor growth. This is highlighted by the finding that bone marrow cells contribute to tumor neovascularization, and when modified to express angiogenesis inhibitors can decrease tumor growth (DavidoV et al., 2001). Further, data from chronic myelogenous leukemia patients indicate that malignant blood cells and ECs arise from a common malignant bone marrow-derived hemangioblastic progenitor, and the ECs contribute to the vascular endothelium in vivo (Gunsilius, 2003). While normally the blood appears to inhibit growth and diVerentiation of EC progenitors (Wang et al., 2004), tissue ischemia and tumors provide a pro-angiogenic environment. After tissue damage, increases in local levels of reactive oxygen species can lead to cellular DNA damage. Thus, transplanting a quiescent but potentially malignant bone marrow or blood cell into a reactive oxygen species rich pro-angiogenic environment could significantly increase the risk of development of a malignancy. This might take the form of a classic tumor or, perhaps, an atherosclerotic plaque.

XI. Looking to the Future With the realization that adult hemangioblasts are present in the bone marrow, blood, and tissues, old ideas about tissue repair are being rethought, novel mechanisms for disease processes are being proposed, and new ways of thinking about vascular growth are emerging. Bone marrow cell dysfunction may be involved in the etiology of a number of vascular conditions, but whether dysfunction is the cause or eVect remains to be established. This will be important if we are to learn to harness the cells to safely prevent and treat a variety of disorders. Though cell-based therapies are not without risk and

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must be approached with caution, it is likely that the cells by themselves, in combination with angiogenic factors or as gene therapy vectors, are likely to become important therapeutic tools in the not-too-distant future. Exactly what the contribution of bone marrow-derived cells is to tissue repair and what physiological and molecular factors govern their diVerentiation and incorporation into the endothelium remain unclear. Nevertheless, bone marrow-derived cells clearly play a pivotal role in neovascularization and remodeling in at least some physiological settings, probably both by releasing factors that promote vascular growth and by acting as a source of ECs. There appear to be at least two distinct classes of EC progenitors. The first, related to hematopoietic stem cells, is likely to be a true stem cell, while the second, related to monocytes, is probably a progenitor cell, capable of diVerentiating into multiple phenotypes, including dendritic cells, macrophages, and ECs. A great deal of work remains to be done to better understand and define these two types of EC progenitors, though there may be a continuum of phenotypes that will make definitive characterization of the cells impossible (Eisenberg and Eisenberg, 2003). Moreover, the environment in which a cell finds itself may dictate its potential to become an EC as much as its intrinsic properties.

Acknowledgments The author thanks Raven Twitchell for preparation of figures and Joan Seye for assistance in manuscript preparation. This work was supported by grants from the National Institutes of Health (DK55965 and DK52293) and the Juvenile Diabetes Research Foundation (2001-534).

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Voyta, J. C., Via, D. P., Butterfield, C. E., and Zetter, B. R. (1984). Identification and isolation of endothelial cells based on their increased uptake of acetylated-low density lipoprotein. J. Cell Biol. 99, 2034–2040. Wagers, A. J., Sherwood, R. I., Christensen, J. L., and Weissman, I. L. (2002). Little evidence for developmental plasticity of adult hematopoietic stem cells. Science 297, 2256–2259. Waltenberger, J., Lange, J., and Kranz, A. (2000). Vascular endothelial growth factor-A-induced chemotaxis of monocytes is attenuated in patients with diabetes mellitus: A potential predictor for the individual capacity to develop collaterals. Circulation 102, 185–190. Wang, C., Jiao, C., Hanlon, H. D., Zheng, W., Tomanek, R. J., and Schatteman, G. C. (2004). Mechanical, cellular, and molecular factors interact to modulate circulating endothelial cell progenitors. Am. J. Physiol. Heart Circ. Physiol. 286, H1985–H1993. Weissman, I. L. (2000). Stem cells: Units of development, units of regeneration, and units in evolution. Cell 100, 157–168. White, G. C., and Parshley, M. S. (1950). Growth of in vitro blood vessels from bone marrow of adult chickens. Am. J. Anat. 89, 321–345. Williams, E. D., Lowes, A. P., Williams, D., and Williams, G. T. (1992). A stem cell niche theory of intestinal crypt maintenance based on a study of somatic mutation in colonic mucosa. Am. J. Pathol. 141, 773–776. Wisse, E. (1974). Observations on the fine structure and peroxidase cytochemistry of normal rat liver KupVer cells. J. Ultrastruc. Res. 46, 393–426. Wu, M. H., Shi, Q., Wechezak, A. R., Clowes, A. W., Gordon, I. L., and Sauvage, L. R. (1995). Definitive proof of endothelialization of a Dacron arterial prosthesis in a human being. J. Vasc. Surg. 21, 862–867. Yamashita, J., Itoh, H., Hirashima, M., Ogawa, M., Nishikawa, S., Yurugi, T., Naito, M., and Nakao, K. (2000). Flk1-positive cells derived from embryonic stem cells serve as vascular progenitors. Nature 408, 92–96. Yamashita, Y. M., Jones, D. L., and Fuller, M. T. (2003). Orientation of asymmetric stem cell division by the APC tumor suppressor and centrosome. Science 301, 1547–1550. Young, P. E., Baumhueter, S., and Lasky, L. A. (1995). The sialomucin CD34 is expressed on hematopoietic cells and blood vessels during murine development. Blood 85, 96–105. Young, P. P., Hofling, A. A., and Sands, M. S. (2002). VEGF increases engraftment of bone marrow-derived endothelial progenitor cells (EPCs) into vasculature of newborn murine recipients. Proc. Natl. Acad. Sci. USA 99, 11951–11956. Yusta-Boyo, M. J., Gonzalez, M. A., Pavon, N., Martin, A. B., De La Fuente, R., GarciaCastro, J., De Pablo, F., Moratalla, R., Bernad, A., and Vicario-Abejon, C. (2004). Absence of hematopoiesis from transplanted olfactory bulb neural stem cells. Eur. J. Neurosci. 19, 505–512. Zhang, Y. M., Hartzell, C., Narlow, M., and Dudley, S. C., Jr. (2002). Stem cell-derived cardiomyocytes demonstrate arrhythmic potential. Circulation 106, 1294–1299. Zhao, Y., Glesne, D., and Huberman, E. (2003). A human peripheral blood monocyte-derived subset acts as pluripotent stem cells. Proc. Natl. Acad. Sci. USA 100, 2426–2431. ZiegelhoeVer, T., Fernandez, B., Kostin, S., Heil, M., Voswinckel, R., Helisch, A., and Schaper, W. (2003). Bone marrow-derived cells do not incorporate into the adult growing vasculature. Circ. Res. 94, 230–238. Ziegler, B. L., Valtieri, M., Porada, G. A., De Maria, R., Muller, R., Masella, B., Gabbianelli, M., Casella, I., Pelosi, E., Bock, T., Zanjani, E. D., and Peschle, C. (1999). KDR receptor: A key marker defining hematopoietic stem cells. Science 285, 1553–1558. Zieske, J. D. (1994). Perpetuation of stem cells in the eye. Eye 8(pt2), 163–169.

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Synthetic Extracellular Matrices for Tissue Engineering and Regeneration Eduardo A. Silva and David J. Mooney Division of Engineering and Applied Sciences Harvard University Cambridge, Massachusetts 02138

I. Introduction A. Design of Synthetic Mimics of Extracellular Matrix II. Inductive and Cell Transplantation Strategies A. Inductive Approaches B. Cell Transplantation III. Engineering Vascular Structures A. Mimicking Vasculogenesis B. Therapeutic Angiogenesis C. Large Blood Vessel Engineering IV. Bone Regeneration A. Osteoconduction B. Osteoinduction C. Cell Transplantation V. Conclusions and Future Directions Acknowledgments References

The need for replacement tissues or organs requires a tissue supply that cannot be satisfied by the donor supply. The tissue engineering and regeneration field is focused on the development of biological tissue and organ substitutes and may provide functional tissues to restore, maintain, or improve tissue formation. This field is already providing new therapeutic options to bypass the limitations of organ/tissue transplantation and will likely increase in medical importance in the future. This interdisciplinary field accommodates principles of life sciences and engineering and encompasses three major strategies. The first, guided tissue regeneration, relies on synthetic matrices that are conductive to host cells populating a tissue defect site and reforming the lost tissue. The second approach, inductive strategy, involves the delivery of growth factors, typically using drug delivery strategies, which are targeted to specific cell populations in the tissues surrounding the tissue defect. In the third approach, specific cell populations, typically multiplied in culture, are Current Topics in Developmental Biology, Vol. 64 Copyright 2004, Elsevier Inc. All rights reserved. 0070-2153/04 $35.00

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directly delivered to the site at which one desires to create a new tissue or organ. In all of these approaches, the knowledge acquired from developmental studies often serves as a template for the tissue engineering approach for a specific tissue or organ. This article overviews the development of synthetic extracellular matrices (ECMs) for use in tissue engineering that aim to mimic functions of the native ECM of developing and regenerating tissues. In addition to the potential therapeutic uses of these materials, they also provide model systems for basic studies that may shed light on developmental processes. C 2004, Elsevier Inc.

I. Introduction Advances in medicine over the past several decades have led to significant improvements in the quality and quantity of life. Despite these advances, it is still necessary to develop alternative therapies to treat patients who suVer from the loss or failure of organs and tissues (Langer and Vacanti, 1993). The numbers of individuals willing or able to donate their organs and tissues do not nearly match those that require transplantation therapies. For example, the number of putative candidates for heart transplantation is approximately 16,000, but only 2,202 patients received heart transplants in the United States in the year 2001 (United Network of Organ Sharing, 2002) due to the lack of donor organs. More generally, every 16 minutes, another new patient requires an organ transplantation. The fields of tissue engineering and regenerative medicine are attempting to address this significant number of patients who need a new or improved organ or tissue. In these fields, a variety of life sciences and engineering disciplines is integrated with the goal of promoting and controlling tissue regeneration. The ultimate goal is to develop synthetic constructs that restore and enhance the functions of healthy tissues. Developmental studies provide crucial information about how the interactions of cells with the extracellular matrix (ECM) regulate cell fate and function. In particular, studies regarding how tissues and organs grow during embryogenesis and how they remodel and maintain certain functionalities are prime sources of information for designing synthetic ECMs used in tissue engineering and regeneration. Development signals are mimicked in tissue engineering approaches, using the developmental mechanisms and events as templates to design new synthetic ECMs. The evolution in the design of synthetic ECMs parallels the development of our knowledge of the ECM. Historically, the ECM was viewed as a structural support for tissues, but it is now clear that the ECM plays a crucial role in controlling cell adhesion, migration, growth, and

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diVerentiation via intracellular signaling pathways. Similarly, the main function of the early synthetic ECMs was to provide mechanical support. Current approaches are more ambitious, as the synthetic ECMs are designed to regulate cell function via presentation of various receptor-binding ligands, in addition to providing structural and mechanical information regulating tissue configuration. Tissue engineering approaches can be divided into three basic strategies, conductive, inductive, and cell transplantation strategies (Fig. 1), and synthetic ECMs typically play a key role in all three. In conductive approaches, synthetic ECMs are used to maintain a space and passively allow host cell infiltration into the tissue defect site. A diVerent stratagem for new tissue formation involves the delivery of bioactive signaling molecules (e.g., growth

Figure 1 Schematic representation of tissue engineering approaches using synthetic extracellular matrices (ECMs). Conductive approaches exploit the ability of the host cell populations to infiltrate ECMs and guide the tissue regeneration at the defect site. Inductive bioactive molecules may also be delivered from ECMs to target specific cell populations to migrate and direct tissue regeneration. Cell transplantation strategies utilize the delivery of specific cell population cultured in vitro to the defect site in order to create new tissues or organs.

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factors, adhesive peptides) that bind to target host cell populations and induce them to migrate into the defect site and participate in new tissue formation. In a manner similar to the sequestration and presentation to cells of growth factors by ECM molecules, the synthetic ECMs are often used as both depots and controlled release vehicles for growth factors. Again similar to native ECM molecules, adhesive signals (e.g., cell adhesion peptides) can be incorporated into the matrix to control cell migration from the defect interface with the surrounding tissue. The third strategy involves the transplantation of cells that can participate in tissue formation. The transplanted cells can be derived either from the patient or from a donor, and the multiplication of the cells in vitro prior to transplantation allows a small cell supply to be greatly expanded. In this chapter, we discuss the design parameters for synthetic ECMs used in inductive and cell transplantation strategies of tissue engineering and regeneration. Synthetic EMCs used in conductive approaches are not reviewed, as their design in mainly dictated by materials science principles. A variety of diVerent types and sources of synthetic ECMs is currently used for induction and cell transplantation, but this chapter focuses on polymerbased ECMs. Specific examples of the use of these synthetic ECMs in bone regeneration and therapeutic angiogenesis are provided, as well as a discussion of potential research directions for the future.

A. Design of Synthetic Mimics of the Extracellular Matrix The ECM of developing tissues regulates development through multiple mechanisms, including the binding of cells to the ECM molecules via specific receptors, conveyance of mechanical signals, and presentation of growth factors and cytokines. These signals vary in a temporally dynamic manner due to the constant remodeling of the ECM. Synthetic mimics of the ECM are currently being designed to incorporate these signaling features, and all except growth factor presentation, which is discussed in the next section, is overviewed in this section. One function of the ECM in tissue development is providing a foundation for cell attachment, as cell anchorage plays an important role in the regulation of cell growth, diVerentiation, and apoptosis. The adhesion of cells to native ECM is mediated by specific cell surface receptors, such as integrins that interact with short amino acid sequences presented in the ECM molecules (Hubbell, 1999). This signaling is frequently mimicked in synthetic ECMs by the presentation of cell-binding motifs from the polymers used to fabricate the synthetic ECM. The amino acid sequence arginine-glycineaspartic acid (RGD), a ubiquitous cell-binding domain derived from fibronectin and laminin, is the peptide most frequently used to promote cellular

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attachment to synthetic ECMs (Drury and Mooney, 2003; Hubbell, 1995; Shin et al., 2003), and the density of these ligands presented to cells from the material has been shown to regulate the cellular response in vitro and in vivo (Alsberg et al., 2003; Healy et al., 1999; Hubbell, 1999; Rezania and Healy, 1999). As diVerent cell populations exhibit varying patterns of integrin expression, it is possible to use distinct peptide sequences in order to bind specific cell types to the synthetic ECM (Table I). For example, the amino acid sequence REDV (notation derived from the amino acid code), derived from fibronectin, has been reported to specifically bind endothelial cells (ECs) (Hubbell et al., 1991), and thus may be useful in designing vascular grafts (see Section IV.C). Several diVerent materials have been used as synthetic ECMs for peptide presentation. An important criterion used to choose an appropriate material is minimal nonspecific cell adhesion to the material, in order to maximize the specificity of cell adhesion via the incorporated peptide. Alginate, a naturally derived polysaccharide, and polyethylene glycol (PEG), a synthetic polymer, both exhibit little native cell adhesion and are widely used as synthetic ECMs for this reason. DiVerent techniques are employed to present peptides from synthetic ECMs solely at the surface or throughout the bulk, and these include absorption and covalent coupling. Surface covalent coupling is a simple way to present peptides with defined orientation and density. For example, carbodiimide chemistry has been successfully used with alginate hydrogels to promote covalent coupling of cell adhesion peptides (Rowley et al., 1999). Bulk modification of the material may present advantages as compared to surface modifications, as this approach allows cells throughout the material to contact the peptide. Bulk peptide immobilization in synthetic ECMs is typically achieved by physical, chemical, photochemical, or ionic crosslinking (Shin et al., 2003). For example, it has recently been demonstrated that alginate hydrogels covalently coupled with G4RGD peptide sequence throughout its bulk promote the proliferation of diVerent cell types in vitro and in vivo (Hirano and Mooney, 2004). In addition to signaling resulting directly from receptor–ligand binding, the integrin–ECM bonds form a physical link between the cells and the ECM through which mechanical signals may be conveyed to cells. It will likely be critical to the success of tissue engineering approaches of designing synthetic ECMs that allow the passage of stresses and strains from the matrix to the cells in order to direct cellular gene expression and the structure and function of the resultant tissue. For example, the presence of cyclic mechanical strain can be responsible for the maintenance of a diVerentiated phenotype, cell organization, and development of superior mechanical properties in engineered smooth muscle tissues (Mitchell and Niklason, 2003; Niklason et al., 1999; Nikolovski et al., 2003). In addition, the ability of synthetic ECMs to accommodate cell-generated forces is important to the ECM’s ability to

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Table I Representative Cell Adhesion Peptides Used to Modulate Cell Adhesion to Synthetic ECMs Peptide sequence RGD

REDV YIGSR

KQAGDV

Material Alginate PEG PLGA; PLA Glass Fluorinated ethylene propylene (FEP) PEG Polyethylene terephthalate (PET), PTFE PEG

Representative reference Alsberg et al., 2003 Hubbell, 1999 Shin et al., 2003 Hubbell et al., 1991; Massia and Hubbell, 1992 Ranieri et al., 1995

Dai et al., 1994 Massia and Hubbell, 1991

Mann et al., 2001

control various cellular events, such as adhesion, migration, and diVerentiation (Ingber, 2003a,b; Ingber et al., 1994; Pelham and Wang, 1997; Wang et al., 2000). The surface density of binding peptides may be used to control the cellular force balance and switch cells from a diVerentiated phenotype to a proliferative state (Mooney et al., 1992). Controlling the remodeling of synthetic ECMs is crucial for modulating the development of engineered tissues. Two diVerent approaches for regulating synthetic ECM remodeling can be pursued. One is based on the concept of pre-defining the degradation rate to occur at a specific rate, independent of the environment. This approach has been exploited to demonstrate that increasing the degradation rate of alginate hydrogels leads to an increase in the quality and quantity of regenerated bone formed with cell transplantation (Alsberg et al., 2003). This study supports the idea that matching the degradation rate of the material to the rate of tissue formation can improve tissue regeneration. An alternative approach involves allowing the cells themselves to mediate the remodeling process. In this approach, proteolytic degradation sites are built into the matrix to allow cellular enzymes to degrade the material. These materials mimic the substrates for matrix metalloproteinases (MMPs), and when associated with integrinbinding sites can result in an improvement of cell ingrowth in vivo (Lutolf et al., 2003a). Further, it has been demonstrated that combining RGD peptides and MMP substrates in a matrix can enhance new bone formation (Lutolf et al., 2003b).

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II. Inductive and Cell Transplantation Strategies Growth factors have an important role in regulating cell migration, diVerentiation, and proliferation during development, and the action mechanism of growth factors is typically time and concentration dependent. In therapeutic approaches, growth factors are usually delivered either by systemic administration or by bolus injection. However, these approaches present some disadvantages, such as the low targeting for the specific cell population (systemic administration) and the short half-life of many growth factors in the body. The use of synthetic ECMs to locally deliver either the growth factors or DNA encoding the factors can be used to bypass these disadvantages. Moreover, the polymeric materials used for inductive factor delivery can be simultaneously used for cell transplantation at the desired site in order to provide additional cell populations that can participate in new tissue formation. This section overviews the general principles used with inductive and cell transplantation strategies, and specific applications of these approaches are described in the next sections.

A. Inductive Approaches Synthetic ECMs can provide growth factors to desired cell populations in a localized and sustainable manner. The growth factors are usually encapsulated into the polymer, preventing their denaturation, and their release can be controlled by the degradation rate of the polymer or by their diVusion through the polymer. Similarly, the synthetic ECMs can be used to locally provide DNA encoding the desired factor in order to transfect cells and increase the local production of the factor. 1. Protein Delivery The simplest approach to providing a desired growth factor(s) at the site at which one desires tissue regeneration is to directly deliver recombinant versions of the factor. The first, and still dominant, approach involves the injection of solutions containing the factor either into the systemic circulation or directly at the desired site. Supraphysiologic quantities of proteins are typically delivered in an attempt to maintain the required concentration of the factor in the tissue of interest for the desired time frame. However, this approach has significant limitations, including the limited lifetime of many of these proteins in the body and the possibility of side eVects at distant sites due to the presence of the factors in undesired tissues.

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An alternative approach that more closely mimics the normal process of presentation for many factors in tissue development and regeneration is to locally provide growth factor gradients at a desired site using synthetic ECMs as a carrier. This approach allows physiologically relevant concentrations of the factors to be delivered in a sustainable and localized manner. Polymeric materials are attractive candidates for this approach, as the protein can be slowly and controllably released from the polymer while the polymer protects the incorporated protein from denaturation. Polymeric delivery vehicles may be either implantable or injectable, and can be fabricated from a variety of synthetic and natural polymers. Implantable materials require invasive procedures in order to be placed at the desired site, while injectable materials may be delivered using minimally invasive technologies. A variety of polymers, including poly(glycolide) (PGA), poly(L-lactide) (PLA), and their copolymer (poly[lactide-co-glycolide]) (PLG) has been widely used for protein delivery (Hubbell, 1999; Richardson et al., 2001a; Robey et al., 2000; Sheridan et al., 2000; Shin et al., 2003). These materials are generally considered to be biocompatible and have been used in biomedical applications for over 30 years. These polymers can be formed into a variety of physical structures relevant to drug delivery using several processes (Mikos et al., 1994; Mooney et al., 1996; Sheridan et al., 2000), but a key issue is maintenance of the bioactivity of the encapsulated growth factors. A high-pressure gas foaming process has been developed that allows factor incorporation without the use of organic solvents or high temperatures (both can lead to protein denaturation) (Sheridan et al., 2000), and this system can be used for the delivery of growth factors in vivo in a bioactive form, resulting in sustainable release that can lead to new tissue formation (Chen and Mooney, 2003). Both synthetic and naturally derived hydrogel forming materials are used in growth factor delivery due to their ability to be injected. Synthetic materials (e.g., poly[ethyleneoxide] [PEO], poly[vinyl alcohol] [PVA]) are advantageous in that their chemistry and physical properties can be readily controlled (Drury and Mooney, 2003; Sakiyama-Elbert and Hubbell, 2001). Naturally derived gel-forming polymers (e.g., alginate, chitosan) typically exhibit excellent biocompatibility. Some physical properties, such as the degradation rate, can be diYcult to control with naturally derived materials. However, covalent modifications can be used to allow control over features such as degradation (Drury and Mooney, 2003). 2. Local Gene Therapy An alternative inductive approach to direct protein delivery is to instead use gene therapy approaches to locally produce the desired factor(s) from host cell populations at the site. Gene therapy approaches may bypass some

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limitations presented by protein formulations, mainly related to the glycosylation of recombinantly produced proteins and protein instability in polymeric delivery vehicles. Viral gene therapy approaches (e.g., adenovirus) can be highly eYcient and have been pursued in clinical trials (Epstein et al., 2001). However, there are still are a number of concerns (e.g., immune response to virus) associated with this approach. On the other hand, nonviral approaches that utilize the delivery of plasmid DNA encoding the desired factor may circumvent some of the concerns related to viral strategies (Levy et al., 1998). The main limitation associated with nonviral approaches is the very low transfection eYciency. Synthetic ECMs may be used to deliver plasmid DNA in order to obtain high expression levels from host cells (Shea et al., 1999). This expands the function of synthetic ECMs beyond the normal functions of native ECMs, but is a natural extension of the concept of using synthetic ECMs to regulate cell gene expression in vivo. Polymer-based DNA delivery using various physical forms of the polymer, including microspheres and macroporous matrices (Levy et al., 1998; Murphy and Mooney, 1999), has been pursued. Microspheres can be delivered in a minimally invasive manner. Macroporous matrices, which are typically implanted using an invasive surgical procedure, allow cell invasion and thus serve to bring host cells to the site of DNA release as well as serve as a sustained release device for the plasmid DNA. This approach to produce growth factors locally has led to promising results (Levy et al., 1998; Park and Healy, 2003; Shea et al., 1999). Plasmid DNA released from collagen sponges has led to the transfection of 20–50% of available cells, and porous PLG matrices and PLG microspheres similarly have been used to obtain high levels of local gene expression (Richardson et al., 2001a). Condensation of the plasmid DNA with polycations to form electropositive nanoparticles has been combined with synthetic ECM in a sustained and localized delivery to further enhance gene expression (Huang et al., 2003). Electropositive nanoparticles demonstrate enhanced cellular uptake and can be designed to escape lysosomal degradation (Godbey et al., 2000; Lemkine and Demeneix, 2001).

B. Cell Transplantation Limitations (e.g., unpredictable and uncontrolled cell type recruitment, time gap between matrix delivery, and tissue development) associated with inductive strategies could be circumvented by directly transplanting the desired cell population to the target site. Cell populations may be delivered without the use of a synthetic ECM (Traggiai et al., 2004), but the use of a synthetic ECM provides an opportunity to both regulate the gene expression of the

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transplanted cells and control the structure and function of the tissues formed either in vitro or in vivo. The identification of the specific types and sources of cells to be transplanted is critical to the success of this approach. Most often, diVerentiated cell types related to the target tissue have been used in past work (e.g., dermal fibroblasts were used to engineer dermal tissues), as this provides more certainty regarding the functionality of the transplanted cell population and the possibility that they may have the genetic pre-programming to reassemble appropriately into the desired tissue or organ structure (Alsberg et al., 2002; Hubbell, 1995; Oberpenning et al., 1999; Uyama et al., 2001). The cells may potentially be obtained from autologous, allogenic, or xenogenic sources. Autologous tissues are the most direct source of cells and reduce problems related with compatibility and immune reactions to the transplanted cells. Allogenic cells are typically used when there exists a limited supply of autologous cells and/or banking and when use of a constant cell source is desirable. Xenogeneic cells are attractive when the autologous and allogeneic sources are insuYcient to achieve the necessary cell mass. Advances in stem cell research provide encouragement that either embryonic stem cells or adult tissue-derived multipotent cells could be used for tissue repair or formation (Caplan and Bruder, 2001; Passier and Mummery, 2003; Verfaillie, 2002a,b). Stem cell populations oVer the possibility of significant expansion in culture from a small starting tissue mass and the ability to form multiple cell types from a single starting source. Synthetic ECMs are frequently used to deliver cells to the desired tissue site, provide the space for tissue development, and evoke specific cellular responses to modulate tissue architecture and function (Hirano and Mooney, 2004; Parenteau and Hardin-Young, 2002). Their design is based on the criteria discussed earlier, in terms of biological and physical characteristics, and several of the same biodegradable polymers used for protein/plasmid DNA delivery have also been used for cell transplantation. These include synthetic macroporous scaVolds fabricated from collagen and PLG (Ameer et al., 2002; Carrier et al., 1999; Park et al., 2002) and hydrogels formed from synthetic and naturally derived polymers (e.g., alginate) (Dar et al., 2002). Due to their structural similarity to the ECM native to many developing tissues (e.g., highly hydrated, space-filling gel), hydrogels are appealing for cell transplantation approaches. Alginate and PGA have been used extensively for new cartilage regeneration via the transplantation of autologous chondrocyte (Fuchs et al., 2003; Koch and Gorti, 2002). Combining inductive approaches with cell transplantation may allow the modulation of the fate of transplanted cells and is being pursued to more precisely regulate new tissue development and remodeling (Smith et al., 2004).

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III. Engineering Vascular Structures Cardiovascular diseases cause over 15 million deaths in the world each year (Al-Radi et al., 2003). In the western countries, coronary artery disease is a principal cause of mortality despite enormous improvements in prevention and development of therapies for ischemic heart disease. Current treatment approaches consist of pharmacologic agents (anti-anginal medications) or the use of invasive surgical procedures (e.g., coronary artery bypass grafting) aimed at re-establishing perfusion. However, there still remains a major need for new approaches to treat patients who are not candidates for the current approaches. The insuYcient and incomplete revascularization associated with residual symptoms of myocardial ischemia, demonstrated by many patients after these procedures, also motivates interest in new therapeutic approaches (Epstein et al., 2001). A promising alternative method to treat heart ischemia involves the delivery of appropriate therapeutic agents that result in activation of the angiogenesis process or a partial activation of the vasculogenic process. Developmental studies are responsible for many insights that have led to our current understanding of the molecular basis of blood vessel formation (Darland and D’Amore, 2001). The identification of key factors associated with blood vessel formation allows the replication of aspects of this signaling to drive new blood vessel formation in emerging therapies. For example, the identification of signals that recruit or diVerentiate endothelial cell precursors (Fig. 2) oVers opportunities to mimic and maneuver the contributions of these cells to vascular growth. Blood vessel formation occurs by several mechanisms and occurs with distinct chronological phases. Vasculogenesis refers to the initial development of vascular structures in the embryo by endothelial cell precursors. Angiogenesis, in contrast, refers to new blood vessel formation by sprouting and subsequent stabilization of new vessels from existing vessels (Carmeliet and Conway, 2001; Conway et al., 2001). Both processes are complex, and occur in a stepwise fashion. Despite recent advances in delineating molecular, genetic, and cellular mechanisms of vasculogenesis and angiogenesis, many aspects of these processes are unclear. Tissue engineering systems may provide useful models to study this process while also potentially providing new therapies. A. Mimicking Vasculogenesis Vasculogenesis refers to the in situ growth of vessels from progenitor endothelial cells (ECs) or angioblasts. Under the influence of specific molecular cues, these cells self-organize and assemble into a primitive network

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Figure 2 Schematic illustration of the role of certain growth factors in the angiogenesis process. Under the influence of factors such as vascular endothelial growth factor (VEGF), the endothelial cells are recruited and form an immature vessel; upregulation of platelet-derived growth factor (PDGF) may be responsible for the recruitment of mural cells necessary for the maturation of the blood vessel. The absence of VEGF destabilizes the immature vessels, and the endothelial cells are susceptible to apoptosis. The presence of transforming growth factor- (TGF-) may be required for the further maturation of blood vessels by the deposition of extracellular matrix.

(Conway et al., 2001). There are a number of genes involved in vasculogenesis, including Hex, Vezf, and basic helix-loop-helix (bHLH) (Carmeliet, 1999). Some genes that play important roles in this process are, as well, upregulated during blood vessel formation in the adult (e.g., the vegf gene) (Luttun and Carmeliet, 2003). Until recently, it was believed that the process of vasculogenesis was restricted to embryonic development, while blood vessel formation in the adult was an exclusive result of angiogenesis, but it was recently demonstrated that processes related to vasculogenesis can occur in the adult. The identification of angioblasts circulating in adults suggests that not only can these cells migrate from vessels in the embryo, but they can also be recruited to form capillaries in the adult (Risau, 1995). The isolation of circulating stem cells in peripheral blood followed by their diVerentiation by local signals may allow vasculogenesis to be directed in a therapeutic sense to treat tissue ischemia. However, opposite to vessel formation in the embryo, where the activity and proliferation of endothelial cell precursors is high, in adults these cells remain in an extraordinary latent phase. Studies estimate that the interval for the entrance of ECs to the circulation is less than 1000 days (Isner and Takayuki, 1998). One strategy for bypassing the limitation of EC precursors is to instead transplant mature ECs. A key issue in attempts to mimic vasculogenesis via delivery of mature ECs or precursors is the development of systems that eYciently allow the

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delivery of these cells to the desired site and simultaneously provide an environment conductive to the formation of functional vessel networks. Polymeric scaVolds are appealing for this type of cell delivery, as they can deliver cells in an eYcient manner and provide the necessary biochemical and mechanical signals to the cells and forming vessels. Three-dimensional porous polymeric scaVolds formed from PLG present high porosity and surface area to readily allow cell introduction and have been used to transplant ECs. Subcutaneously implanted sponges seeded with ECs led to a significant number of new capillaries formed in vivo from the transplanted ECs (Nor et al., 1999, 2001). Surfaces that specifically allow EC adhesion and discourage adhesion of other cell types could potentially be achieved by the incorporation of specific cell-binding peptides via surface modification (Shin et al., 2003) and used in this approach to revascularization.

B. Therapeutic Angiogenesis Angiogenesis involves a cascade of processes, including EC activation, recruitment, and proliferation followed by interactions with mural cells for the stabilization of the initially immature new vasculature. A number of angiogenic and antiangiogenic signals have been identified, and these factors are presented locally in a well-regulated way to control angiogenesis. Angiogenesis may be described as a process first involving the vasodilation of existing vessels, an event that is mediated by vascular endothelial growth factor (VEGF), accompanied by an increase of the vascular permeability. Associated with these processes is degradation of the ECM, which is necessary for the subsequent EC migration. These processes are regulated by angiopoietin-1 (Ang1) and its antagonist angiopoetin-2 (Ang2). Degradation of ECM is mainly orchestrated by MMPs secreted by the ECs. EC proliferation and migration is followed by the assembly of immature vessels networks. It was reported that monocyte chemotatic protein (MCP)-1 can also provoke EC proliferation (Belperio et al., 2003). The immature EC assemblies are susceptible to regression due to EC apoptosis, or maturation. VEGF, Ang1, and Ang2 are key players in all steps of these processes (Holash et al., 1999; Kockx and Knaapen, 2000). Finally, the vessels mature to form stable vascular networks via the recruitment of smooth muscle cells (SMC), and this process is regulated by platelet-derived growth factor (PDGF) (Conway et al., 2001; Fig. 2). Therapeutic angiogenesis involves the delivery of various angiogeneic factors to drive new blood vessel formation in an ischemic tissue. Currently, the number of growth factors used in therapeutic angiogenesis is considerable (Table II); they include VEGF, PDGF, and basic fibroblast growth factor (b-FGF). Moreover, VEGF and bFGF might be involved in the

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Table II Representative Growth Factors Delivered with Synthetic ECMs to Drive Tissue Regeneration

Growth factor

Synthetic ECM

Regenerated tissue

VEGF

Alginate PEG PLG PLG Alginate PEG Collagen PGA

Blood Blood Blood Blood Blood Bone Bone Bone

PDGF bFGF BMP-2 BMP-4 BMP-7

vessels vessels vessels vessels vessels

Representative reference Lee et al., 2003 Hubbell, 1999 R]ichardson et al., 2001b Richardson et al., 2001b Lee et al., 2003 Lutolf et al., 2003a Fang et al., 1996 Breitbart et al., 1999

recruitment and diVerentiation of bone marrow-derived angioblasts (Rossant and Howard, 2002). Therapeutic angiogenesis can be realized either by using growth factor formulations or by the delivery of genes encoding these proteins. VEGF and b-FGF have been extensively used in phase I/II clinical trials, but no results for phase III trials have been reported to date. Phase I trials typically have reported promising results (Rosengart et al., 1999; Schumacher et al., 1998). However, the results obtained in the larger phase II trials have not shown the expected benefit to patients (Simons et al., 2002). One potential limitation of current approaches in therapeutic angiogenesis is the mode of delivery, which involves the introduction of large doses of the potent angiogenic molecules in solution form. This may not allow suYcient levels of the factors to be present in the target tissue for the necessary time frame and may lead to severe side eVects. Synthetic ECMs can potentially be used for the controlled delivery of angiogenic molecules with low systemic exposure. Hydrogels may be particularly useful, as they can supply growth factors using minimally invasive procedures, and several angiogenic molecules, including PDGF, VEGF, and b-FGF, have been incorporated into hydrogels (Lee and Mooney, 2001; Zisch et al., 2003). Growth factor release is typically controlled by diVusion and/or polymer degradation. However, the mechanical environment of the local site may dramatically influence the release rate (Lee et al., 2000). This eVect could be exploited to deliver bioactive molecules in locations under mechanical stress (e.g., the heart). VEGF bioactivity is maintained in these systems, and in some cases it seems to be enhanced, perhaps due to monomers associating with VEGF and protecting the protein even after its release (Peters et al., 1998). Polymeric materials can be also useful for investigating the role of combinations of growth factors in angiogenesis, since a synergy between diVerent

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growth factors has been demonstrated in angiogenesis (Pepper et al., 1992). For example, a three-dimensional PLG scaVold has been developed that allows the release of sequences of growth factors (Richardson et al., 2001b). Release of VEGF followed by PDGF led to both a significant increase in the local blood vessel density and maturation of the newly formed vessels. Simultaneous delivery of the two factors led to little to no revascularization.

C. Large Blood Vessel Engineering In addition to angiogenesis and vasculogenesis approaches to form new blood vessel networks, there is often a need for immediate replacement of large blood vessels. Autologous veins or arteries are used in cardiac or peripheral bypass surgery. However, many individuals are not candidates for this clinical procedure due to the lack of appropriate blood vessels for use as replacements. In these cases it is necessary to use synthetic vascular grafts, and polymers have been used as a blood vessel substitute (RatcliVe, 2000). However, these synthetic vessels have a lower patency rate when compared with natural vessels due to their susceptibility to thrombus formation as well as to a lack of mechanical conformity with adjacent native tissues. The failure of synthetic polymers to successfully replace blood vessels is more accentuated in small diameter grafts (<6 mm internal diameter) (Niklason et al., 1999). Cardiovascular tissue engineering strategies have been developed to provide an alternative supply of vessels to replace diseased arteries. Ideally, the three diVerent structures (intima, media, and adventia) present in blood vessels would be mimicked in the tissue-engineered vascular graft and could provide function immediately on implantation. Seeding ECs on the luminal surface of synthetic polymers is one approach to circumvent the thrombogenic events associated with synthetic grafts (Daly et al., 2004). However, this strategy is limited by EC detachment from the surface following initiation of blood flow (Daly et al., 2004). EC attachment on synthetic ECMs can be increased by surface modifications with adhesion peptides. For example, vascular grafts of expanded polytetrafluoroethylene (ePTFE) layered with fibronectin and RGD peptides led to an increased EC attachment and retention (RatcliVe, 2000). However, this approach does not address the mismatch in mechanical properties between synthetic and natural vessels. To address this issue, investigators have attempted to engineer complete new vessels. For example, polyglycolic acid scaVolds have been seeded with smooth muscle cells alone or combined with ECs, and maintained under cyclic strain or pulsatile flow for use as cardiovascular graft (Kim et al., 1999; Sodian et al., 1999). Over time, the polymer undergoes degradation,

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resulting in a completely natural new tissue. Application of an appropriate regimen of mechanical stimulation can greatly enhance the mechanical properties of these ex vivo engineered vessels and improve their success following implantation (Niklason et al., 1999).

IV. Bone Regeneration More than 800,000 bone grafting procedures are completed each year in the United States (Laurencin et al., 1999), and restoring or enhancing the repair of bone is a crucial problem in orthopedics and dentistry. Bone graft procedures often utilize autograft or allograft bone. While autografts are often highly successful, their availability is limited and the bone harvest creates another defect. In contrast, allografts are more available but are susceptible to viral contamination and immune responses by the host. Synthetic bone replacements are widely used, but typically fail over time and may have issues with biocompatibility. Engineering new bone tissue may bypass the limitations associated with the standard techniques. All three major tissue engineering approaches, osteoconduction, osteoinduction, and cell transplantation strategies, have been investigated as approaches for regenerating bone (Alsberg et al., 2001). The following sections briefly review the process of bone formation and the approaches presently under investigation for bone tissue regeneration. The vertebrate skeleton is composed of craniofacial, axial, and appendicular (i.e., limb skeleton) bone, and the cells that constitute the vertebrate skeleton are derived from three distinct embryonic lineages, cranial neural crest, paraxial mesoderm, and lateral mesoderm. The cranial neural crest cells are responsible for the craniofacial skeleton, paraxial mesoderm (somites) cells give rise to the axial skeleton, and the limb skeleton is derived from lateral plate mesodermal cells (Olsen et al., 2000). The bone cell precursors present in these three sections migrate to the precise location of future bones and diVerentiate into osteoblasts. Several transcription factors regulate the migration events involved in the morphogenesis of bone (Olsen et al., 2000; Zelzer and Olsen, 2003). Organogenesis, diVerentiation of these cells into bone-forming cells called osteoblasts, is known to be controlled by a variety of signals, including the ECM, transcription factors, cytokines, and growth factors (Zelzer and Olsen, 2003). The available knowledge regarding the molecular and cellular basis of bone development during embryo formation has been exploited for therapeutic goals. The signaling molecules and cells responsible for bone morphogenesis and organogenesis are often combined with synthetic matrices mimicking certain functions of the ECM to promote regeneration.

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A. Osteoconduction Osteoconductive approaches to bone regeneration, also called guided tissue or bone regeneration, are used to allow the infiltration of osteoprogenitors from bone marrow to the local defect, while providing temporary mechanical support. The physical properties of the synthetic ECM significantly aVect its osteoconduction properties. For example, the pore size distribution, total porosity, and pore interconnectivity can be controlled to regulate cell infiltration. The pore size typically expected to yield successful cell permeation is greater than 10 m (Wald et al., 1993; White et al., 1981), as is the connectivity between the pores to obtain space-filling new bone tissue. In certain situations, the material is used to block undesired cells types (i.e., fibroblasts) from accessing the defect site, while allowing cell types that can form bone to migrate into the site from a diVerent location. This more selective cell conduction can be achieved by having the surface in contact with bone cells containing large pores, while the surface in contact with fibroblasts presents a pore size prohibitive for cell conduction. ePTFE has been successfully used as a selective membrane for periodontal guided tissue regeneration (Hermann and Buser, 1996). However, a second surgical procedure is necessary for removal of the ePTFE construct after tissue formation, due to the absence of ePTFE degradation. In order to circumvent this limitation, degradable materials have been developed, including PLA, PLG, and collagen-based materials (Alsberg et al., 2001; Christgau et al., 2002; Trejo et al., 2000). Alginate also has potential in bone conduction strategies (Tonnesen and Karlsen, 2002). An important requirement for engineered bone constructs in many applications is high mechanical strength and stiVness. Synthetic ECMs formed from polymeric materials often have poor mechanical properties compared to native bone (Orban et al., 2002). Ceramic materials have been used for bone regeneration due to their better mechanical and osteoconduction properties. However, they can be too stiV and lead to stress shielding, and they also typically present poor degradation properties (e.g., slow degradation rates) when compared with polymers. One way of bypassing these limations may be to combine the advantageous features of polymers and ceramics. The presence of a bone-like mineral on a polymer scaVold may enhance osteoconduction and mechanical integrity while allowing a readily controlled biodegradation rate (Laurencin et al., 1999). The formation of a bone-like mineral film coating on three-dimensional PLG scaVolds has been demonstrated to enhance bone regeneration in cranial defects (Murphy et al., 2004). In spite of the relative successes of osteoconductive strategies in forming new bone in certain situations, this approach exhibits modest control over the molecular mechanisms of bone regeneration. Regeneration may be greatly improved with more active approaches.

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B. Osteoinduction Osteoinductive approaches involve the delivery of inductive signals aimed to provoke the migration, proliferation, and diVerentiation of desired cell types into a bone defect site in order to actively control bone regeneration. Inductive signals can be delivered both by direct introduction of specific growth factors and by using gene therapy techniques. In both cases, materials are typically used as delivery vehicles for these agents. A large number of growth factors have been implicated in bone formation and adaptation, but bone morphogenetic proteins (BMPs) appear to be crucially important in the regulation of bone development and repair (Seeherman et al., 2002; Wozney, 2002). BMPs are members of the transforming growth factor- (TGF-) superfamily, which is responsible for controlling the phenotype and apoptosis of a variety of cell types, including osteoblasts, chondroblasts, neural cells, and epithelial cells (Wozney, 2002). Recombinant and naturally derived BMPs have been combined with several carrier materials, resulting in enhanced bone regeneration. For example, recombinant human BMP-2 (rhBMP-2) has been associated with sponges of PLG to allow a sustainable and localized delivery of the factor (Howell et al., 1997), and this promotes the migration and proliferation of bone-forming cells into the defect. Vascularization represents a critical step in the formation and remodeling of bone, and a number of studies have demonstrated that angiogenic factor delivery using synthetic ECMs regulates bone regeneration (Uchida et al., 2003). It is possible to use osteoconductive scaVolds to delivery angiogenic factors; this may enhance the eVectiveness of the osteoconductive material. For example, mineralized PLG scaVolds capable of localized and sustained VEGF delivery promote new blood vessel formation at bone defect sites and increase bone regeneration (Murphy et al., 2004). Another approach to osteoinduction involves the immobilization of specific peptides on the surface of polymer materials placed in the defect in order to enhance migration of the desired cell populations into the defect. PEG gels have been adapted to allow cellular invasion by coupling cell adhesion peptides to the polymer chains and providing proteolytic degradation sites for cell-based degradation. Delivery of rhBMP-2 from these gels has led to promising results in bone regeneration (Lutolf et al., 2003b). Gene therapy approaches can also be applied to provide inductive factors that can drive bone regeneration. In this situation, the synthetic ECM is directly altering gene expression in cells, and plasmid DNA encoding osteogenic proteins is incorporated into implantable polymer constructs for local and sustained delivery. Delivery of plasmid DNA encoding for BMP-4 from natural based hydrogels, such as collagen, has been reported to eVectively induce bone regeneration (Fang et al., 1996). The DNA may also be condensed prior to incorporation into three-dimensional PLG scaVolds to

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increase transfection levels, and this may further enhance bone regeneration with small doses of DNA (Huang et al., 2003). C. Cell Transplantation An alternative approach to osteoconduction/induction strategies for bone regeneration is direct transplantation of desired cell populations into the bone defect. This approach at least partially bypasses the requirement for host cell migration, and may accelerate the process of bone regeneration, and/or make this possible in conditions where insuYcient numbers of host cells are available to migrate into the site to regenerate the tissue. The transplanted cells are typically expanded in vitro before transplantation, to permit for a large cell supply to be generated from a small initial tissue mass (Langer and Vacanti, 1993). Synthetic ECMs are typically used to deliver bone forming cells (e.g., bone marrow stroma cells [BMSCs]) as they can localize the cells to a desired site, and provide cues regulating bone formation from the transplanted cells and host cells (Alsberg et al., 2001). Combining cell transplantation with inductive approaches may provide a faster or greater extent of bone regeneration, by more closely mimicking the multifactor nature of the normal regenerative process. For example, the transplantation of human BMP-7 gene-enhanced BMSCs on PGA scaVolds into osteochondral knee defects significantly increased bone regeneration as compared to the cells or gene delivery alone (Breitbart et al., 1999).

V. Conclusions and Future Directions The field of tissue engineering and regeneration has developed in response to the critical lack of tissues and organs available for transplantation. Progress has been rapid, and several tissue engineering therapies are now available for treating patients (e.g., Carticel, Genzyme Corporation; Apligraf, Organogenesis Inc.). The field of tissue engineering has had considerable success to date in the development of small tissue masses. However, a critical challenge is to create large tissue masses and entire organs. Approaches to rapidly promote blood vessel networks will clearly be critical to achieve this goal. Advances in the understanding of development biology will lead to improved approaches to tissue regeneration; on the other hand, tissue engineering systems will provide novel models to study developmental events by controlling the environment signals. In spite of the tremendous progress to date with approaches focused on single factor or cell type delivery, tissue development is not regulated by a single factor or accomplished by a single cell type. Optimal tissue regeneration may in the future be accomplished by integrating a temporally

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synchronized cascade of signals and cell populations to create new tissues. Synthetic ECMs that provide multiple key signals with distinct temporal and spatial availability will be critical to this approach. A combination of cell types may also be useful in engineering organs comprised of multiple tissues types. The use of embryonic or adult stem cells may provide an ideal approach to create these types of tissues structures, as one cell population could lead to multiple tissue types. However, it will be necessary to obtain a better understanding of the biology of these cell populations for their successful use in tissue engineering. Synthetic ECMs may be ideal systems for providing the local cues required to tightly regulate the diVerentiation of these cell populations. Further, these systems may make ideal model systems for determining the relation between microenvironment signals and stem cell fate.

Acknowledgments The authors acknowledge funding from the National Institutes of Health. E. A. S. is a student of the Gulbenkian PhD Program in Biomedicine, Portugal and is supported by the Portuguese Foundation for Technology and Science.

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Integrins and Angiogenesis D. G. Stupack and D. A. Cheresh Department of Immunology The Scripps Research Institute La Jolla, California 92037

I. Introduction II. Endothelial Cell Integrins A. Integrin Structure B. Alterations in Endothelial Cell Integrins During Angiogenesis III. Initiation of Angiogenesis A. MAP Kinases Are Critical for Angiogenesis B. Integrins and Other MAPKs During Angiogenesis IV. Alterations in Integrin–ECM Interactions During Angiogenesis A. The ECM is Dynamically Altered by Proteolysis During Angiogenesis B. Deposition of New Integrin Ligands into the ECM C. Alterations in Integrin Expression May Influence Endothelial Survival D. Integrin v3 as a Functional Marker of Angiogenic Endothelium V. Integrin Antagonist EVects versus the Phenotype of Knockout Animals VI. Perspective and Conclusions A. Current Clinical Perspective B. Future Directions C. Conclusions References

The growth of new blood vessels is a dynamic yet highly regulated process that depends on coordinated signaling by growth factor and cell adhesion receptors. As part of the molecular program regulating angiogenesis, endothelial cells acquire a proliferative and invasive phenotype but also show increased susceptibility to apoptotic stimuli. Integrins are the principle adhesion receptors used by endothelial cells to interact with their extracellular microenvironment, and integrin-mediated interactions play a critical role in regulating cell proliferation, migration, and survival. Alterations in the repertoire and/or activity of integrins, as well as the availability and structural property of their ligands, regulate the vascular cell during the growth or repair of blood vessels. C 2004, Elsevier Inc.

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I. Introduction Blood vessels develop from at least two processes, termed vasculogenesis and angiogenesis (Isner and Asahara, 1999). Vasculogenesis is the process by which blood vessels form de novo, during development, as the nascent vascular bed arises from hematopoietic precursor angioblasts (Drake et al., 1995). By contrast, angiogenesis occurs as the growth or sprouting of new blood vessels from a pre-existing vascular bed. Although angiogenesis is generally accepted to be the principle mechanism of blood vessel growth within the adult, it has recently become clear that the recruitment of hematopoietic precursors is a critical event during angiogenesis, suggesting a previously unsuspected vasculogenic component (Ribatti et al., 2001). Thus, the distinction between these processes has blurred somewhat, and many of the mechanisms that regulate angiogenesis may be common to both processes (Rupp et al., 2003). Neovascularization is a tightly regulated process during development and wound repair but appears considerably less regulated during pathological angiogenesis associated with cancer and inflammatory disease. However, normal or ‘‘unactivated’’ blood vessels in the adult are relatively quiescent and nonproliferative. The endothelial cells lining the inner surface of the blood vessels maintain tight cell–cell junctions and stable interactions with the underlying extracellular matrix (ECM), or basement membrane. These endothelial cells exhibit a very low mitotic index. Cell cycle entry occurs in only about 1 in 1000 cells, replacing endothelial cells lost through routine attrition (Folkman and Klagsbrun, 1987). In contrast to this resting state, endothelial cells stimulated with angiogenic growth factors such as vascular endothelial cell growth factor (VEGF) (Dvorak et al., 1995; Ferrara et al., 2003) or basic fibroblast growth factor (bFGF) (Folkman et al., 1988) become highly proliferative, achieving mitotic indices similar to growing tumors. Concomitant with accelerated proliferation, increased transcription and protein synthesis also occur, as endothelial cells prepare for cell migration/invasion and neovessel outgrowth. In addition to the increased expression of cell cycle proteins, angiogenic endothelial cells express a repertoire of new proteins, including transcription factors such as Hox (Myers et al., 2000) and Id genes (Benezra et al., 2001), which in turn regulate the expression of integrins and production of new provisional ECM components, matrix-proteolyzing enzymes (Heissig et al., 2003), their inhibitors (Mannello and Gazzanelli, 2001), as well as growth factors and apoptosis-regulating proteins (reviewed in Dimmeler and Zeiher, 2000). Physiologically, angiogenic endothelial cells exhibit an increased capacity to proliferate and invade tissue, but perhaps somewhat surprisingly, demonstrate an increased predisposition to undergo apoptosis (Brooks et al.,

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1994a,b). Previous reports suggested that proper blood vessel formation depends on a balance between endothelial cell proliferation and apoptosis (reviewed in Stupack and Cheresh, 2003). The regulation of these processes is closely tied to remodeling events in the local ECM and to integrins that enable endothelial cells to respond to the ECM.

II. Endothelial Cell Integrins A. Integrin Structure Integrins are heterodimeric receptors that mediate divalent cation-dependent cell attachment to the ECM, but that can also interact with cell surface and soluble ligands. Nascent integrin and subunits are paired in the endoplasmic reticulum to form functional heterodimers, which then traYck to the cell surface (Cheresh, 1992). While there are at least 18 diVerent subunits and 8 diVerent subunits, only 24 diVerent / combinations have been observed. Each / heterodimer has its own ligand subset and specificity (some integrins bind many diVerent ligands, while others bind very few). Thus, the repertoire of integrins present on the surface of a given cell will enable that cell to biologically respond to a particular ECM and microenvironment. All integrins (except 4) have small cytosolic domains, and none have intrinsic kinase activity. Despite this, integrins are important mediators of cell signaling in addition to anchoring the cell to the ECM (Giancotti and Ruoslahti, 1999). Higher-order structuring of the integrins (i.e., clustering and associating with other proteins) likely plays a major role in regulating integrin signaling, which requires the recruitment of cytosolic nonreceptor kinases, such as focal adhesion kinase (FAK) (Hauck et al., 2002), Src (Parsons and Parsons, 1997), phosphoinositide 30 kinase (PI3K) (Franke et al., 1997), and p21-dependent kinases (PDKs) (Attwell et al., 2000). Endothelial cells have been reported to express up to 10 diVerent integrins depending on the location and activation state of the endothelial cell (Fig. 1). The major integrins on quiescent endothelial cells are 11, 21, 31, 51, 64, 61, and v5. It is possible that v1 may be expressed as well, as both v and 1 subunits are present. These integrins tend to be receptors for basal ECM components (such as collagen and laminin), with the exception of integrins v5 and 51, which tend to bind provisional matrix ligands such as vitronectin and fibronectin, respectively. Cellular fibronectin may be a ligand for both of these integrins on quiescent endothelial cells, although integrin v5 appears to act as an endocytosis receptor and also to interact with depositions of vitronectin found at endothelial cell– cell junctions (Preissner and Potzsch, 1995), while 51 may interact with the endothelial cell surface adhesion molecule L1-CAM (Voura et al., 2001)

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Figure 1 Known integrin heterodimers. The 24 known integrin heterodimers are shown, grouped into broad families. The families are generalizations based on structure and common function. For example, the collagen-binding integrins 11 and 21 can bind laminin under some circumstances, and similarly, the laminin-binding integrin 31 may bind collagen. The RGD-binding family of integrins all bind in an RGD-dependent manner to at least some of their ligands. The leukocyte integrins, also called the 2 integrins, are expressed principally on leukocytes and lymphocytes and mediate cell–cell and cell–ECM interactions involved in immune function (but have been described on a few other cell types, including tumors and keratinocytes). Similarly, the VCAM-binding, or 4-like integrins, were initially discovered on lymphocytes but have been reported on other cell types, and 91 in particular is not expressed on hematopoietic cells. Those integrins expressed on endothelial cells are boxed, while those that are induced during angiogenesis are underlined. The dashed box around v1 indicates that although both v and 1 subunits are present, it is not certain whether they form a heterodimer in endothelial cells.

or with matrix-immobilized angiopoietin-1 (Carlson et al., 2001). During angiogenesis, remodeling of the perivascular ECM provides additional ligands for both v5 and 51, as well as for new integrins that are expressed on the endothelial cell surface. B. Alterations in Endothelial Cell Integrins During Angiogenesis Angiogenic endothelial cells alter their cell surface integrins through two major mechanisms. The first is at the level of transcription of individual integrin subunits, resulting in alterations in the relative expression of individual integrins subunits. Although integrins that bind to ECM components such as collagen and laminin tend to be downregulated, and integrins that bind to provisional ECM components such as fibrinogen, fibronectins,

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osteopontin, vitronectin, and proteolyzed forms of collagen tend to be upregulated, an overall increase in integrin expression is observed. This underscores the critical nature of integrin–ECM interactions in regulating angiogenesis. The second modification to the integrins is at the level of function. Endothelial cells stimulated with angiogenic growth factors alter the ligand binding characteristics of cell surface integrins to suit the new roles required during neovascularization. 1. Expression of New Integrins Angiogenic endothelial cells express an altered repertoire of integrin heterodimers relative to quiescent endothelium, including several heterodimers not found on quiescent endothelial cells. The most significant increase in integrin expression on the surface of angiogenic endothelial cells involves the de novo expression of v3, an integrin that binds a wide range of provisional ECM components, including fibrinogen, vitronectin von Willebrand factor (Cheresh, 1987), fibronectin (Charo et al., 1990), and osteopontin (Ross et al., 1993), yet can interact with basal ECM components such as laminin (Clyman et al., 1992) and proteolyzed collagen (Davis, 1992) (Fig. 2). Integrin v3 is expressed on a number of diVerent cell types during embryogenesis, yet its expression is primarily limited to angiogenic endothelial and smooth muscle cells and a few subpopulations of hematopoietic cells (myeloid subsets, including osteoclasts) in the adult. Integrin v3 appears to be a critical regulator of angiogenesis, since blockade of this integrin with small molecules or antibodies disrupts neovascularization in a number of animal models (Brooks et al., 1994a,b; Drake et al., 1995; Friedlander et al., 1995; MacDonald et al., 2001; Nemeth et al., 2003; Penta et al., 1999; Storgard et al., 1999; Varner et al., 1999). In addition to v3, the de novo expression of integrin 4 has been reported on angiogenic endothelial cells (Vanderslice et al., 1998). Integrin 41 is a receptor for the CS1 domain of fibronectin as well as VCAM-1, an immunoglobulin superfamily cell adhesion molecule expressed on endothelium and smooth muscle cells during angiogenesis and inflammation. Integrin 41 can also bind thrombospondins (Calzada et al., 2004). In the adult, integrin 4 is generally present only on hematopoietic cells or tumors, but it has been described on cytotrophoblasts and endothelial cell precursors during development (Sheppard et al., 1994) and on cultured endothelial cells in vitro (Vanderslice et al., 1998). Integrin 4 pairs with either 1 or 7; both integrins have been reported on cultured endothelial cells (Brezinschek et al., 1996). The 41 ligands VCAM and thrombospondin (at relatively low concentrations) are both proangiogenic. These forms of angiogenesis are blocked by the administration of antibodies directed against integrin 41 (Calzada et al., 2004; Koch et al., 1995).

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Figure 2 Ligands of integrin v3. A partial list of ligands bound by integrin v3 is shown. Integrin v3 binds to a wide array of ligands, including components of the basal ECM, provisional ECM, cell surface adhesion molecules (CAMs), and soluble fragments of all of these ligand types, as well as soluble fragments of proteases themselves. The binding of soluble fragments to integrins interferes with their binding to matrix-immobilized ligands and may promote apoptosis. Integrin v3 may also interact with ECM-bound cytokines such as bFGF and CTGF, although the purpose of these interactions in vivo is not fully understood. Several v integrins, including v3, bind to LAP1 and LAP3, the latency activating peptides of TGF-. Finally, a number of pathogenic proteins also target integrin v3, including a number of snake venoms as well as proteins that mediate internalization and delivery of viral genetic material into the cell. These latter proteins provide a solid rationale for targeting integrin v3 as a therapeutic target during angiogenesis.

2. Altered Expression of Pre-Existing Integrins The relative expression of the integrins already present on endothelial cells is also altered during angiogenesis. Integrins such as 11 and 64 are downregulated during angiogenesis (Hiran et al., 1998). This occurs at the transcriptional level (Tagaya et al., 2001) but may also occur through endocytosis via association with the tetraspanin PETA-3 (Sincock et al., 1999). It is not clear whether internalization, or ligation, of 64 is necessary for cell survival. Although monoclonal antibodies directed against 64 increase cell survival in response to stress (Tang et al., 1999), ligation of this integrin with laminin 5 is suYcient to permit the survival of transformed cells in the absence of other ligands (Zahir et al., 2003).

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Endothelial cells bordering regions of ischemia dramatically upregulate transcription of integrin 1 (Tagaya et al., 2001). The increased 1 subunits pair with increased levels of 6 and 5 but also pair with 2 and 1 subunits. Integrin 2 expression is maintained on angiogenic endothelial cells and plays an important role, together with 1, in maintaining a repertoire of integrins that can interact with both proteolyzed and native forms of interstitial collagens. Abrogation of this interstitial collagen binding capability using antibodies directed to these two integrins blocks angiogenesis (Senger et al., 1997b). Integrin 51 is also significantly upregulated on angiogenic endothelial cells (Boudreau and Varner, 2004). This integrin is principally a receptor for fibronectin but also mediates cell adhesion to the proangiogenic cytokine/ ECM component angiopoietin-1 and the provisional ECM component fibrin. Antagonism of integrin 51 promotes apoptosis among angiogenic endothelial cells in vitro and in vivo (Kim et al., 2000), suggesting a significant role for this integrin in regulating angiogenesis. In fact, there appear to be vascular defects in 51-deficient mice, although these mice (as well as mice deficient in fibronectin) exhibit a number of developmental defects and die quite early during embryogenesis. Thus, it is diYcult to conclude whether the vascular defects observed are directly due to the absence of 51 from the endothelium or whether they arise as secondary eVects. However, these results collectively indicate an increased capacity for endothelial cells to interact with provisional ECM components while maintaining a capacity to interact with basal/interstitial ECM components as well. 3. Alterations to Endothelial Integrin Function During Angiogenesis Prior to the alterations in transcription just described, functional changes occur among the pre-existing integrins on the endothelial cell surface. These alterations occur on the order of seconds to minutes, and they serve to modify the role of integrins from substrate anchorage structures to signaling and invasion receptors. During this process, endothelial integrins (including 11, 21, 51, and v5) release pre-existing tight contacts with other cells or the underlying ECM (Naik et al., 2003; Sincock et al., 1999) and localize in clusters with other endothelial cell surface molecules (including growth factor receptors or other integrins) on the endothelial cell membrane (Eliceiri et al., 2002). These complex molecular structures recruit cytosolic signaling molecules, including Src (Eliceiri et al., 2002) and FAK, which aid in the turnover of focal contacts required for cell migration and invasive behavior (Hauck et al., 2002). One of the best characterized integrins to undergo ‘‘functional’’ activation in response to growth factors is integrin v5. Integrin v5 is expressed in a number of cells and tissues and mediates tight adhesion to substrate ECM,

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but does not normally associate with focal adhesions (Wayner et al., 1991). In response to activation with VEGF, v5 on endothelial cells becomes mobilized and potentiates cell migration and tissue invasion (Eliceiri et al., 2002). These events may be dependent upon proteases such as urokinase plasminogen activator (UPA) (Yebra et al., 1996) that modify the underlying ECM, as well as on the cytokine-initiated recruitment of FAK to the cytoplasmic tail of integrin 5 (Brooks et al., 1997). VEGF and integrin v5 form a functional partnership on angiogenic endothelial cells in vivo. VEGF, by binding to VEGFR2 (Flk), appears to cosignal with integrin v5. Antagonism of v5 blocks angiogenesis initiated by VEGF (Friedlander et al., 1995). In contrast, bFGF-induced angiogenesis is not influenced by antibodies directed against v5; rather, it depends on ligation of integrin v3 (Friedlander et al., 1995; Hood et al., 2003). These observations have led to the identification of two distinct pathways of angiogenesis, each dependent upon somewhat diVerent downstream signaling elements (Eliceiri et al., 2002; Hood et al., 2003).

III. Initiation of Angiogenesis The transition from resting to angiogenic endothelium requires a trigger to activate the cellular programs of proliferation and protein synthesis required for neovessel formation. In addition to bFGF and VEGF, mentioned previously, a growing list of proangiogenic factors have now been identified, including glycoproteins (Zhong et al., 2003), complex carbohydrates (Presta et al., 2003), and bioactive lipids (Hla et al., 2000), in addition to wellestablished proangiogenic growth factors such as bFGF and VEGF. Some of these growth factors act directly on endothelial cells, while others stimulate ‘‘bystander’’ cells that, in turn, produce factors that act on the endothelium. Regardless of the angiogenesis-inducing agent, certain common features can be found within all stimulated blood vessels.

A. MAP Kinases Are Critical for Angiogenesis Among the common downstream integrin-dependent signaling molecules required for angiogenesis, the mitogen-activated protein kinases (MAPKs) act as central regulators of transcription, migration, and cell survival. Several MAPKs have been implicated, or demonstrated, to be involved in regulating endothelial cell proliferation, invasion, or survival, including Extracellular Regulated Protein Kinase 1/2 (ERK1/2), Jun amino-terminal kinase (JNK), p38, and ERK5/BMK-1. In particular, the activation of ERK1/2 is an important factor in inducing proliferation and survival (Wada

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and Penninger, 2004) as well as migration (Klemke et al., 1997). Thus, it is perhaps not surprising that pharmacological inhibition of ERK1/2 activation blocks angiogenesis in vivo (Eliceiri et al., 1998). 1. Integrins Are Required for Activation of ERK1/2 ERK1 and ERK2 become phosphorylated and activated as part of the downstream signaling cascade initiated by angiogenic growth factors, irrespective of whether signaling is initiated by a factor that binds to G proteincoupled receptors (Hla et al., 2000) or cell surface receptor-tyrosine kinases (Cabrita and Christofori, 2003). ERK1 and ERK2 are also activated by integrin-mediated adhesion and spreading of endothelial cells upon an ECM substrate (Zhu and Assoian, 1995). Although ERK1/2 activation by integrins does not require growth factors in vitro, growth factors do generally require integrin ligation to elicit ERK1/2 activation. Accordingly, integrin antagonists that prevent endothelial cell activation of ERK downstream of growth factor binding block angiogenesis in vivo (Eliceiri et al., 1998). 2. Cross-talk between Integrins and Growth Factor Receptors A number of integrins have now been shown to directly associate with specific growth factor receptors (Eliceiri, 2001). This association has been linked to signaling events mediated by the growth factor receptors themselves. Alternatively, integrins have been shown to co-signal with growth factor receptors without necessarily being physically linked (Eliceiri et al., 1999; Friedlander et al., 1995; Hood et al., 2003). In either case, it is possible that integrins functionally regulate growth factor signals to enable cells to diVerentially respond to distinct tissues or microenviroments. During angiogenesis, one or more growth factors initiate a program of cell proliferation and migration followed by cell maturation. This biological cascade may be regulated by diVerential integrin growth factor pairs that activate distinct signaling cascades as seen with v3 and v5 in their regulation of bFGF and VEGF signaling, respectively. Endothelial cells are anchorage dependent. As such, they remain unresponsive to growth factors such as VEGF or bFGF when denied integrin ligation in vitro (Meredith et al., 1993). However, if the endothelial cells are subsequently replated on an integrin ligand, such as fibronectin or collagen, they will activate ERK1/2 and begin to proliferate (Renshaw et al., 1999). Moreover, growth factors added to cells that are actively attaching to the ECM lead to a further increase in the intensity and longevity of ERK signaling, significantly above that induced by integrin ligation alone. Thus, it appears that integrins and growth factor receptors signal cooperatively (Renshaw et al., 1997). The mechanism of cooperation between integrins and growth factors is not clear but likely involves both co-localization in

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signaling complexes within the membrane and cross-talk with common cytosolic eVectors. Even among a specific type of receptor, whether they be integrin or growth factor receptors, the intermediate signaling elements required for ERK1/2 activation may be quite distinct. For example, the receptors for both VEGF and bFGF receptors are tyrosine kinases that promote downstream signaling via Src family kinases (SFKs) (Thomas and Brugge, 1997). However, while Src activation is essential for VEGFmediated signaling to ERK1/2, it is dispensable for bFGF-mediated signaling (Eliceiri et al., 2002). 3. Molecular Events Governing ERK1/2 Activation in Angiogenesis The activation of the several ‘‘typical’’ MAPKs involves two immediate upstream kinases: the specific MAPK-kinase (MAPKK) and its respective MAPK kinase-kinase (MAPKKK). These elements are not directly activated by transmembrane receptors; rather, the activation of these proteins is regulated by adaptor proteins and intermediate nonreceptor kinases. Each MAPKK/MAPKKK combination acts to amplify the initial stimulus and to add specificity to these signaling events. In the case of the ERK1/2 pathway, the MAPKK is MEK1 and the MAPKKK is Raf (Fig. 3). Integrins are thought to activate Raf kinases via activation of the eVector small GTPase, Ras (Schlaepfer et al., 1994). There are at least two distinct mechanisms by which integrins can activate Ras: either via the FAK pathway (Schlaepfer et al., 1994) or via the combined action of Shc and Fyn (Barberis et al., 2000). The particular integrin, and the cell it is expressed on, appear to dictate which pathway may be used to activate ERK1/2, although some integrins appear incapable of activating one or the other pathway (Giancotti and Ruoslahti, 1999). In either case, the adaptor protein Grb-2 is recruited, and in turn recruits SOS, which presents GTP-bound Ras to Raf. Angiogenic growth factors such as bFGF and VEGF activate a number of common general upstream signaling mechanisms, not all of which are essential for ERK1/2 activation. Surprisingly, diVerent growth factors appear to depend on distinct upstream signaling proteins to activate Raf, and thus Mek1 and ERK1/2 in vivo. For example, the activation of ERK1/2 by bFGF requires activation of an intermediate serine/threonine kinase, p21-activated protein kinase (PAK1) (Kiosses et al., 2002) which is activated by small GTPases of the Rho family, in particular Rac and Cdc42. PAKs phosphorylate the activation loop of c-Raf on serine residues 338 and 339, displacing it from the active site of the kinase domain. Antagonism of v3 disrupts this pathway, possibly by uncoupling v3-mediated activation of Rac (Bialkowska et al., 2000) that may be required for subsequent PAK activation (Hood et al., 2003). In contrast, although VEGF promotes activation

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Figure 3 DiVerent mechanisms of ERK activation during angiogenesis. The cytokines bFGF and VEGF activate ERK by diVerent mechanisms. The activation of the MAPKKK c-Raf by bFGF (left side) requires the activation of the intermediate kinase PAK-1 and the phosphorylation of the activation loop on serine at residues 338/339. In contrast, VEGF-mediated activation is dependent upon the tyrosine kinase Src (right side), and results in the phosphorylation of this loop at the 340/341 residues. Activated c-Raf then phosphorylates MEK-1 and MEK-2, the MAPKKs that phosphorylate ERK1 and ERK2. ERK1/2 phosphorylates several cytosolic targets, including transcription factors such as Elk-1 and motility-promoting proteins such as myosin light chain kinase.

of PAK, it is not required for VEGF-mediated angiogenesis. Rather, VEGFmediated angiogenesis requires Src activation, an event dependent upon integrin v5 (Alavi et al., 2003). In this case, angiogenesis requires the formation of a Src/FAK/v5 complex that assembles within 5 min of VEGF stimulation (Eliceiri et al., 2002). Src kinases phosphorylate tyrosine residues (340/341) within the activation loop of c-Raf immediately adjacent to the PAK site, providing an alternative means of activating the kinase domain (Alavi et al., 2003) (Fig. 3). These distinct signaling pathways appear to promote endothelial cell survival in diVerent manners. While bFGFdependent Raf activation drives Raf to the mitochondria and protects cells from stress-mediated death, VEGF activity promotes cell survival in an ERK-dependent manner and selectively protects endothelial cells from receptor-mediated apoptosis (Alavi et al., 2003). B. Integrins and Other MAPKs During Angiogenesis The activity of other MAPKs may be important in regulating angiogenesis as well. In general, the overt activation of JNK1/2 and p38 has been implicated in the induction of apoptosis among angiogenic endothelial cells.

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However, JNK1 may be recruited to focal adhesion complexes with integrins (Damsky and Ilic, 2002) and has been implicated in cell migration and invasion (Hsia et al., 2003), suggesting that some level of JNK activation may be necessary for angiogenesis. Similary, Src-dependent activation of p38 appears to be required for endothelial cell migration in response to VEGF (Nakatsu et al., 2003; Zhu et al., 2002) Recently, ERK-5, also known as big map kinase 1, was demonstrated to be critical for maintenance of endothelial cell survival and vascular integrity (Hayashi et al., 2004). Unlike ERK1/2, which are dependent upon integrins and Ras for activation, ERK-5 is activated by VEGF in a Ras-independent manner. The dependence of other ERK1/2 on integrin-mediated activation of Ras suggests that the activation of ERK-5 could be independent of integrins. However, ERK5 is strongly activated by the application of shear stresses (a mechanical form of stimulation in which integrins are critical) that mimic blood flow, suggesting that it is an integrin-regulated MAPK (Pi et al., 2004). While ERK5 plays an important role in maintaining the integrity of the resting vasculature, it will be important to determine whether this MAPK also plays an important role in regulating angiogenesis. The activation of the MAPKs leads to the phosphorylation of nuclear and cytosolic target proteins, and thereby leads to the acquisition of a proliferative and invasive phenotype, two primary characteristics of angiogenic endothelial cells. However, within the nucleus, a cascade of transcription factors, including Hox D3 (Boudreau et al., 1997) and Id1 and Id3 (Lyden et al., 1999), is activated. These events are critical not only for proliferation, but also for alterations in the expression of proteases, ECM components, and integrins that play a major role in governing angiogenic events.

IV. Alterations in Integrin–ECM Interactions During Angiogenesis The transcriptional programs activated by proangiogenic growth factors result in the synthesis and release of new ECM components as well as ECM-digesting enzymes from endothelial cells and in some cases also from the surrounding tumor and/or stromal cells themselves. A number of proteases become associated with the endothelial cell surface, and upon activation, cleave ECM components, process other protease zymogens, or do both. However, proteases such as MMPs may release growth factors such as VEGF from the ECM, or process procytokines into their active forms, thereby inducing angiogenesis (Egeblad and Werb, 2002). These changes in the ECM influence the way endothelial cell integrins become ligated and thus the way cells process molecular signals from the local microenvironment.

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A. The ECM is Dynamically Modified by Proteolysis During Angiogenesis 1. UPA and Plasminogen UPA becomes localized to the surface of endothelial cells via binding to UPA receptor (UPAR). UPAR localizes on the cell surface with integrins, thus focusing the proteolytic activity of bound UPA at points of integrin– ECM contact (Sidenius and Blasi, 2003). While UPA can act directly against some ECM components, its principal activity is likely to process the plasminogen zymogen to plasmin (Ellis, 2003), which has significantly greater activity against a variety of diVerent ECM proteins, including fibrin and fibronectins. Plasmin also cleaves and activates zymogen forms of other proteases, including a subset of the matrix metalloproteases (MMPs), as well as proUPA. The reciprocal activation of plasmin by UPA and UPA by plasmin is controlled by antiplasmin and plasminogen activator inhibitors (PAI), which largely restrict protease activity to the cell surface (or pericellular regions). Among the inhibitors of UPA, PAI-1 also exhibits a significant de-adhesive activity, downregulating integrin-mediated attachment to the ECM and decreasing cell survival (Al-Fakhri et al., 2003). The tight association of integrins, adhesion, and proteases underscores the co-dependency of these elements during angiogenesis.

2. Matrix Metalloproteinases The basal lamina surrounding the endothelium consists principally of type IV collagen, laminin, and proteoglycans. These components are among the extensive list of substrates proteolyzed by MMPs (Egeblad and Werb, 2002). The degradation of the perivascular ECM has been observed by the accumulation of denatured collagen in angiogenic, but not quiescent, endothelium, as detected with monoclonal antibodies specific for epitopes on denatured collagen (Brooks et al., 1996). Protein cleavage events mediated by the MMPs are site specific and selective. MMP-mediated cleavage events act to modify the physical nature of the perivascular ECM rather than to simply eliminate it. The modified ECM continues to act as a scaVold but acquires the capacity to bind new provisional ECM components. Additionally, in many ECM proteins, MMP cleavage leads to the ‘‘unveiling’’ of cryptic adhesion sites that appear to interact with integrin v3 on angiogenic endothelial cells (Davis, 1992; PfaV et al., 1994; Xu et al., 2001). This limited proteolysis of the ECM also plays a critical role in the outgrowth of neovessels into the surrounding tissue. The invasion of angiogenic blood vessels into type I collagen gels in vivo requires proteolytic cleavage of the collagen and is prevented by mutation of the MMP-2 cleavage site in type I collagen (Seandel et al., 2001). Cell migration within a collagen gel

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occurs independent of proteolysis in vitro, suggesting that the requirements for proteolysis in vivo may relate to requirements for vessel growth or maturation, rather than simply migration or invasion. In this case, collagen cleavage may be required to clear suYcient quantities of ECM to provide adequate space for vessel maturation. Alternatively, denatured ECM may provide important cues to endothelial cells that guide angiogenesis. The cleavage of type IV collagen by MMPs eliminates the sites recognized by integrin 11 while unmasking cryptic sites recognized by integrin v3 (Petitclerc et al., 2000). These cryptic sites appear to be important for regulating angiogenesis, since administration of site-directed monoclonal antibodies that recognize these cryptic sites will block angiogenesis in vivo. The cleavage of laminins by MMP-2 during tissue remodeling similarly reveals cryptic sites that promote cell migration and/or invasive events. MMP-2-mediated laminin cleavage of the ‘‘2X’’ arm of laminin produces an 80 kDa fragment that increases migration mediated by integrin 31 yet does not change in the overall adhesive potential of proteolyzed laminin (Giannelli et al., 1997). Thus, proteolytic cleavage of the laminin may also alter existing sites or produce novel sites, which are important in regulating cell invasion and migration during neovessel formation (Seftor et al., 2001). 3. Other Proteases In addition to matrix metalloproteinases, UPA, and plasminogen, there is evidence that kalikrein and high molecular weight kininogen (Al-Fakhri et al., 2003), as well as tissue plasminogen activator, may play roles in regulating angiogenesis. Additional proteases, including emmprin and even cathepsins, may also contribute to an ongoing angiogenic response, especially if they are overproduced within a local tumor environment. However, a specific role for these additional enzymes remains to be firmly established. 4. Byproducts of Protease Activity The cleavage of ECM components, cell surface molecules, and protease zymogens during these events creates a large number of fragments that are unanchored to the ECM. Angiostatin (O’Reilly et al., 1994), a byproduct of plasminogen processing as well as a cleavage product of MMP-2 comprising the hemopexin domain, termed PEX (Brooks et al., 1998), together with soluble fragments of cell surface molecules such as L1-CAM (Silletti et al., 2000) and ECM components including endostatin (O’Reilly et al., 1997), tumstatin (Maeshima et al., 2001), and other NC1 domain fragments (Petitclerc et al., 2000), are available to ‘‘ligate’’ integrins. Each of these has been shown to block angiogenesis, all are produced during angiogenesis,

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and many can be detected in animals or human patients with an ongoing angiogenic response (Brooks et al., 1998; Cao et al., 2000; Gerstmeier et al., 1988). These protein fragments can function as soluble agonists or antagonists of integrins, thereby allowing them to possibly modulate integrinmediated function during angiogenesis. Integrin occupancy with soluble ligands may alter their interactions with the substrate ECM, which may have a variety of biological eVects, ranging from increased focal adhesion turnover and cell migration (Huttenlocher et al., 1996) to programmed cell death (Stupack and Cheresh, 2003). Thus, ECM remodeling does not simply occur to remove an ECM barrier. Rather, these proteases act cooperatively with new ECM deposition to present an environment capable of supporting and regulating cell invasion and survival.

B. Deposition of New Integrin Ligands into the ECM Coordinated with the proteolytic changes to the ECM, alterations are also made in the production of ECM proteins by the endothelial cells themselves. Moreover, changes may also occur in endothelial cell morphology and/or barrier function. This provides at least two separate mechanisms by which endothelial cells can promote the deposition of new proteins during ECM remodeling: (1) de novo production of ECM proteins by angiogenic endothelial cells and/or local tumor/stromal elements and (2) deposition of plasma-borne proteins. 1. The Role of avb5 and Src in Vascular Permeability and Fibrin Deposition The deposition of new ECM requires access to the existing subendothelial ECM. This event is not possible in quiescent endothelium, where barrier function is intact. However, one angiogenic growth factor, VEGF, mediates a vascular permeability response via disassembly of cell–cell junctions, thus permitting deposition of blood-borne provisional ECM proteins upon the underlying ECM. This permeability response may account for the fibrin barrier typically found around primary tumors and wounds (Dvorak et al., 1983). In fact, VEGF was initially described as vascular permeability factor (Dvorak et al., 1995; Senger et al., 1997a), and it is the only angiogenic factor that induces vascular permeability. VEGF binding to Flk-1 (VEGFR2) triggers cytosolic signaling events dependent upon the ligation of integrin v5 and the activation of the tyrosine kinase Src (Eliceiri et al., 1999; Weis et al., 2004). This results in the phosphorylation of the cytoplasmic domain of vascular endothelial cadherin, promoting the disassembly of the catenin/ cadherin complex and resulting in breakdown of the cell–cell junction

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(Weis et al., 2004). Accordingly, genetic deficiency in integrin 5 or in Src compromises VEGF-mediated vascular permeability (Eliceiri et al., 1999). However, deficiency in either Src (Lowe et al., 1993) or integrin 5 (Huang et al., 2000) does not appear to have any overt eVect upon neovascularization during development. 2. New Integrin Ligands Produced by Endothelial Cells Endothelial cells produce a variety of new ECM components that serve specialized roles in regulating angiogenesis. Much like the provisional ECM components (fibronectin, fibrinogen, and vitronectin) deposited from stores in the plasma, these new proteins are ligands for integrins such as 51, v3, and v5. Provisional ECM proteins produced by endothelial cells include tenascin, thrombospondins, osteonectin, and oncofetal proteins such as Del-1 and B-fibronectin. Some of these proteins are tightly associated with the provisional ECM, while others interact in a low-aYnity manner and may act to modulate interactions between endothelial cells and the provisional matrix. In this respect, Del-1, a discoidin and EGF domain-containing protein, is known to act contextually. It can promote cell attachment when substrate-immobilized via a binding site for integrins v3 and v5 or promote endothelial detachment from ECM substrates when present in solution (Penta et al., 1999). Thus, it is perhaps not surprising that Del-1 can promote angiogenesis when present at low concentrations, while blocking angiogenesis at higher concentrations (Zhong et al., 2003). In contrast, the reasons for producing oncofetal forms of fibronectin are less well understood. Oncofetal fibronectins are composed of all the domains that are present in adult fibronectin but also contain an alternatively spliced designated ED-A or ED-B (Kosmehl et al., 1996). In particular, B-fibronectin is a selective marker of angiogenic endothelium. However, it is not yet clear what role the ‘‘additional’’ alternatively spliced type III fibronectin domain present in A or B fibronectin plays (if any) in regulating angiogenesis. These domains do not appear to promote or disrupt integrin-mediated binding to the cell-binding domain (the 10th type III repeat) mediated by v3, v5, and 51 and are distal to the known sites of cleavage by proteases.

C. Alterations in Integrin Expression May Influence Endothelial Survival As mentioned in Section II, specific integrin expression may be up- or downregulated during angiogenesis. While integrin 11 (a collagen receptor) and the 64 (a laminin receptor) are downregulated, integrin 51 and

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v3 are upregulated or expressed de novo, respectively. These changes are consistent with the degradation of a pre-existing basal ECM and its replacement with a provisional ECM. The ligation of endothelial cell integrins is important not only for ECM attachment, but also to facilitate invasion and survival. Among these events, the regulation of survival appears to be the most critical role played by integrins 51 and avb3. Ligation of integrin 51 is known to protect other cells against apoptosis initiated via both caspase-dependent (Matter and Ruoslahti, 2001) and -independent (Jan et al., 2004) pathways. Antagonism of integrin 51, like antagonism of integrin v3, will selectively block angiogenesis induced by bFGF in vivo. In vivo, caspase inhibitors are suYcient to rescue angiogenesis induced by 51 blockade (Kim et al., 2000). These results indicate that the major mechanism of angiogenesis inhibition by 51 antagonism is caspase-dependent apoptosis in vivo. Moreover, the studies show that denying a single integrin ligation (in this case, 51) with the provisional ECM is insuYcient for blocking endothelial cell tissue invasion and neovessel growth. Interestingly, although integrins have been implicated in cellular resistance to death initiated by exogenous insults, such as radiation and chemotherapeutics, apoptosis in this case occurs in the absence of an apoptotic insult and in the presence of well-characterized growth/survival factors (e.g., bFGF). This argues that the accumulation of a given integrin, in an antagonized or unligated state, may itself be suYcient to induce apoptosis. In the case of integrin 51, this has been shown to occur downstream of protein kinase A activation in a caspase 8-dependent manner (Kim et al., 2000). In vitro data suggest a similar mechanism for integrin 3 and perhaps other 1 integrins (Stupack et al., 2001). While ligation of these upregulated integrins during neovascularization represents a potential mechanism to promote angiogenesis, the downregulation of integrins whose ligands are eliminated or proteolyzed may also represent a mechanism that promotes endothelial cell survival during angiogenesis (Cheresh and Stupack, 2002). 1. ECM Fragments ‘‘Occupy’’ Integrins and Block Integrin Function The regulation of integrin-mediated adhesion is, however, more complex than providing or removing integrin ligands from the local ECM. Small molecule inhibitors of integrins act as integrin agonists, incorporating into the ligand binding site of the integrin heterodimer. Nevertheless, like antagonists that allosterically inhibit ligand binding, these small molecule inhibitors block angiogenesis (Brooks et al., 1994, 1995; Kim et al., 2000). Thus, the diVerentiation between what has been classically thought of as an agonist and an antagonist is not entirely clear when considering their eVects on integrin function. For example, when presented in soluble form, neither

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an agonist nor an antagonist promotes survival, whereas the immobilized form of these ligands does. The precise mechanism governing integrin suppression of death is unclear. However, it is important to consider that integrin signaling is influenced by ligand form, valency, geometry, and presentation to the cell (Stupack et al., 1999). Perhaps more important is the fact that integrins couple to the actin cytoskeleton and act as mechanoreceptors (Ingber, 1992). Accordingly, the application of mechanical forces alone is suYcient to initiate integrinmediated signaling (Jalali et al., 2001). Small, soluble ligands that oVer no mechanical resistance will only abortively engage peripheral integrinmediated signaling events, and sustained signaling does not occur. Larger soluble particles that are bound by integrins, such as viruses, activate signaling events only transiently (although this is suYcient to permit internalization as a result of the mechanical forces engaged) (Nemerow, 2000). Therefore, although soluble integrin agonists interact with the ligand binding site, they nevertheless prevent it from engaging other matrix-immobilized ligands. Physiologically, this requirement for mechanical force provides a mechanism by which integrins gauge the relative integrity of the local ECM and convey this information to the cell via contractile forces applied to the cytoskeleton. In turn, this interaction with the cytoskeleton influences integrin-mediated signals as well as signaling by elements that require integrins, such as growth factors (Ingber, 2002). At least a subset of integrins acts as biosensors, assessing the form and integrity of the local ECM microenvironment. Integrins such as v3 and 51 may play a somewhat diVerent role relative to that of simple ECMbinding receptors mediating tissue invasion. Rather, they govern decisions about cell life and death in response to a myriad of factors that influence the dynamically remodeling ECM. As described earlier, the balance between proteolysis and new ligand deposition provides a constant flux of ligand forms that interact with endothelial cell integrins. Many of the fragments produced by ongoing proteolysis are soluble, having been cleaved free of the underlying ECM. Because soluble integrin ligands can induce apoptosis, it is perhaps not surprising to find that many of these soluble proteolytic fragments are also anti-angiogenic. Within a highly proteolytic environment, some integrins (such as v3) may be ligated principally with soluble ligands, which serve as antagonists of integrin function, thereby promoting cell death. 2. Proteolytic ECM Fragments Suppress Angiogenesis by Interfering with Integrin Ligation to a Substrate ECM Recently, it has become clear that soluble fragments of ECM proteins suppress angiogenesis. Many of these appear to bind to, and suppress, the function of endothelial cell integrins. These include, but are not limited to,

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proteolytic cleavage fragments of plasminogen (angiostatin), MMP-2 (PEX), collagen 18 (endostatin), and the NC domains from the alpha 2, alpha 3 (tumstatin), and alpha 6 chains of type IV collagen. These proteins may act to block interactions with immobilized ECM components and may therefore elicit apoptosis through a variety of mechanisms. Small molecule-mediated blockade of integrin v3 and v5 can prevent integrin-mediated suppression of the p53-mediated apoptotic pathway in vitro and in vivo (Stromblad et al., 1996). Tumstatin binds to integrin v3 and disrupts Akt signaling, suppressing mTOR activity (Maeshima et al., 2002) as well as other Aktdependent processes (Sudhakar et al., 2003). Importantly, tumstatin does not bind to the RGD-binding site of integrins (Sudhakar et al., 2003), suggesting it may interfere with integrin ligation by an allosteric mechanism, much like the LM609 antibody, which also does not bind to the RGD ligand-binding site on integrin v3 (Marcinkiewicz et al., 1996).

D. Integrin avb3 as a Marker of Angiogenic Endothelium In the adult, integrin v3 is generally not expressed on blood vessels, yet it is expressed on endothelium during development (Drake et al., 1995) and in pathological angiogenesis in a number of diVerent vertebrates. In addition, v3 is found on blood vessels within 2–3 hours following ischemic stroke prior to vascular remodeling (Tagaya et al., 2001). The v3 heterodimer recognizes a wide array of ligands. In fact, v3 is the most promiscuous integrin by far. In addition to recognizing many intact ECM proteins, v3 binds to a number of cleaved or denatured ECM components present during angiogenesis (Fig. 4). Most major provisional ECM components secreted or deposited during angiogenesis possess binding sites for this integrin, and cryptic sites for v3 are revealed on both laminins and collagens through the action of proteases. Thus, integrin v3 provides endothelial cells with the capability of attaching to, migrating on, or responding to the ECM in most every organ or tissue, or to those undergoing remodeling. Antagonism of integrin v3 blocks angiogenesis in a number of in vivo models, suggesting an important role for this integrin in regulating angiogenesis. Paradoxically, although upregulation of v3 during angiogenesis is conserved among the vertebrates, humans or mice lacking this integrin develop an apparently normal vasculature (Coller et al., 1991; Hodivala-Dilke et al., 1999). This may be related to the fact that other integrins can perform the functions of v3 when it is absent, yet the universal expression of this integrin on angiogenic endothelial cells across species suggests a specific role. Animals lacking v3 are refractory to inhibition of angiogenesis mediated by agents that target v3, such as tumstatin (Maeshima et al., 2002). Interestingly, while animals lacking v3 exhibit normal developmental

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Figure 4 Alterations to the endothelial microenvironment during angiogenesis. Quiescent endothelial cells form tight junctions with each other through the action of cadherins, immunoglobulin superfamily CAMs, and zona adherens proteins (left side). There, cells are transcriptionally active but proliferate very slowly. They are anchored to an underlying ECM composed principally of proteoglycan, type IV collagen, and laminin. In response to stimulation with VEGF, endothelial cells disassemble the cell–cell junctions in a manner dependent upon the kinases Src and Yes, thereby inducing vascular permeability. This permits deposition of provisional ECM components present in the plasma in a precursor state. Concomitantly, the activation of cellular signaling cascades leads to changes in the integrins present on the endothelial cells, as well as to changes in ECM and protease production by both the endothelium and the underlying cells. This dramatically alters the composition of the ECM around the endothelium and is thought to play a major role in regulating angiogenesis in vivo.

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angiogenesis, they exhibit increased pathological forms of angiogenesis (Reynolds et al., 2002). Because blockade of v3 integrins can actively promote apoptosis via activation of caspase 8 (Kim et al., 2000; Stupack et al., 2001), it is likely that these v3-deficient mice fail to limit or control angiogenesis via an apoptotic mechanism. The abundance of ligands for v3 suggests that in the milieu of a remodeling ECM, this integrin will be highly occupied on the cell surface. It is possible that proteolytic environments may simply eliminate ECM ligands for integrins such as v3, rendering it unoccupied and promoting apoptosis.

V. Integrin Antagonists Effects versus the Phenotype of Knockout Animals Allosteric antagonists or small molecule agonists of v integrins block angiogenesis in multiple species (reviewed in Rupp et al., 2003) and disrupt vasculogenesis during avian development (Drake et al., 1995). In contrast with these results, mice lacking v3 show no obvious vascular defect during development (Hodivala-Dilke et al., 1999). This is, however, consistent with the observation that this integrin is known to be deficient in some forms of Glanzmann’s thrombaesthenia in human disease (Coller et al., 1991), yet these patients have normal-appearing vasculature. The murine disorder resembles that found in humans, at least superficially, as the mice are aZicted with a profound platelet-clotting disorder. These adult 3-deficient animals show increased angiogenesis during tumor growth. On the surface this may seem paradoxical. Nevertheless, several explanations might account for this. As mentioned previously, these endothelial cells would be refractory to the presence of soluble integrin ligands, and thus would lack a proapoptotic response. It has been suggested that the soluble v3 ligands provide a ‘‘negative’’ (or anti-angiogenic) signal to endothelial cells. However, it is important to point out that several v3-targeted anti-angiogenic agents, including monoclonal antibody LM609 and tumstatin, do not bind to the ligand binding site of this integrin. At least in the case of LM609, no signaling occurs. Moreover, recent studies have demonstrated that antagonism of v3 blocks the extension of pseudopodia from hemangioblasts, preventing the initial contacts necessary for the assembly of the nascent vascular bed (Rupp et al., 2004). In this case, v3 blockade is not apoptogenic in these embryonic endothelial precursors. Nevertheless, the results illustrate how an antagonist that does not elicit signaling (LM609) can nevertheless act to misregulate a given receptor and, indeed, an entire biological process critical for neovessel formation. While the ‘‘absence of a receptor’’ has sometimes

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been equated with the presence of a ‘‘misregulated receptor,’’ any number of biological systems (ranging from the action of toxins to oncogenic transformation) indicate that this is generally not true. Further studies will reveal that tumstatin acts similarly to LM609. However, since these antagonists do not compete with RGD ligands, they would appear to allosterically inhibit v3 rather than activate a negative signaling program. It is also quite conceivable that mice lacking v3 have somehow compensated for the loss of the gene product, as often happens in knockout animals. While alternative integrins do not appear to be altered in these animals, both VEGF and its receptor are upregulated in mice lacking v3 (Reynolds et al., 2002). This is significant for a couple of reasons. The titration of VEGF is a critical event during development, and relatively modest alterations in this gene are lethal during development (Ferrara, 2002). The upregulation of VEGF or Flk-1 might represent an amplification of a signaling pathway (v5/VEGF) complementary to that dependent upon v3 (v3/bFGF). Clearly, an increase in VEGF expression or its receptor would be consistent with the increased tumor-induced angiogenesis in these 3-deficient animals. In contrast to the v3-deficient mice, those lacking integrin 51 (or its principal ligand, fibronectin) die from a number of defects early in development (Yang et al., 1993), implying a crucial role for this integrin in regulating primary morphogenesis. This finding recapitulates the finding that antibodies directed against 1 integrin block the lumen formation by nascent clusters of angioblasts during quail development (Drake et al., 1992). It will be important to determine whether a conditional endothelial knockout of 51 will disrupt angiogenesis and mimic the block in tumor angiogenesis eVected by antagonist antibodies or small molecule in chick embryos and adult mice (Kim et al., 2002). However, this may not be the case. Although antibodies to integrins 11 and 21 can block angiogenesis in collagen-rich tissues such as skin, the 21- and 11-deficient mice do not demonstrate a vascular phenotype (Chen et al., 2002; Gardner et al., 1996). These results underscore a general principle of cell biology: the presence of a disregulated receptor is not the same as the absence of that receptor.

VI. Perspective and Conclusions A. Current Clinical Perspective There are at lease three integrin antagonists currently undergoing clinical trials for a number of diseases associated with angiogenesis. The first of these agents to enter clinical testing was Vitaxin, the fully humanized form of

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monoclonal antibody LM609 directed against integrin v3. In an early phase I trial, Vitaxin was shown to promote disease stabilization in more than half of the patients (Gutheil et al., 2000). All of the patients had progressive disease and had failed multiple treatment regimens. One patient with end stage sarcoma remained on the drug for over 2 years, with stable disease. Importantly, Vitaxin was not associated with any significant side eVects, and phase II trials are underway to support these initial, anecdotal studies. Based on promising early results, Vitaxin is also being tested in patients with rheumatoid arthritis and psoriasis in blinded placebocontrolled phase II trials. Celengitide, a cyclic peptide antagonist of v3 and v5, has been tested in cancer patients. This agent is currently being tested in patients with glioblastoma, a disease that does not respond to any chemotherapeutic strategy. The rationale for treating this patient population is based on preclinical studies in which Celengitide produced significant tumor regression in an orthotopic glioblastoma model of glioblastoma (MacDonald et al., 2001). It is important to point out that these drugs targeting v3 show little or no toxicity on the patients treated thus far, even after chronic and prolonged treatment. More recently, the first 51 antagonist entered phase I clinical trials for cancer (http://www.pdl.com/index.cfm?navId¼114). In this case, late-stage cancer patients are being treated with a weekly infusion of a humanized anti51 monoclonal antibody. Unlike v3, integrin 51 is also expressed on a number of immune, stromal, and tumor cells. It will be important to establish the safety and eYcacy of this approach.

B. Future Directions The fact that integrin v3 is selectively expressed on angiogenic endothelial cells makes it a good candidate for the targeted delivery of drugs and imaging agents. In fact, v3-targeted nanoparticles coupled to gadolinium or radionuclides selectively bind to tumor blood vessels in rabbits, allowing for magnetic resonance or radiographic imaging of the tumor vasculature noninvasively (Anderson et al., 2000; Sipkins et al., 1998). More recently, a similar targeted nanoparticle was able to deliver a death-promoting mutant form of the C-Raf gene to tumor blood vessels (Hood and Cheresh, 2002). This initially lead to apoptosis of the tumor vasculature followed by tumor cell apoptosis, leading to the regression of large established primary and metastatic tumors in mice. Lanza and colleagues have been successful at using this nanoparticle approach both to image early angiogenesis (Anderson et al., 2000) and to deliver cytotoxic drugs selectively to tumor endothelium. Conventional drugs targeted to endothelial integrins, such as doxorubicin, exhibit increased eYcacy (Arap et al., 1998), thus it is likely

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that nanoparticle approaches will increase eYcacy and decrease tolerated dose and side eVects to a much greater degree. Ultimately, the nanoparticle approaches may provide an eVective strategy not only to identify neovasculature noninvasively, but also as a surrogate marker (via targeted imaging) of the eYcacy of these, or other, anti-angiogenic agents being tested in the clinic. In the future, it is likely that multiple anti-angiogenic agents will be used in combination, possibly with conventional chemotherapeutic regimens. Therefore, it will be critical to establish a reliable approach to monitor how these agents influence angiogenesis in various disease states. Moreover, using these nanoparticletargeted radiation, chemotherapeutic, or death-promoting genes, it may be possible to maximize the anti-angiogenic eVect while minimizing side eVects.

C. Conclusions Integrin-mediated interactions with the local ECM play a critical role in a number of events in neovessel formation. Early in vasculogenesis, integrins play a critical role in defining the physical form and characteristics of the emerging vascular plexus. In mature endothelium, integrins anchor the quiescent endothelial cells and provide resiliency to the vasculature, possibly even maintaining endothelial viability via mechanical stimulation provided by the shear forces of blood flow. During neovascularization, integrins are required for the transmission of the initial signaling events that trigger the angiogenic cascade and are subsequently altered to permit a sensory and invasive role during endothelial cell invasion of the dynamically remodeling ECM. Collectively, integrins are thus involved in the regulation of almost every facet of blood vessel maintenance, growth, and remodeling. This may explain why integrins have been such attractive targets for the development of anti-angiogenic therapies. In particular, the specific expression of integrin v3 on angiogenic endothelial cells, and its role in regulating endothelial viability, give this integrin the potential to be a selective target for a number of anti-angiogenesis therapies.

References Alavi, A., Hood, J. D., Frausto, R., Stupack, D. G., and Cheresh, D. A. (2003). Role of Raf in vascular protection from distinct apoptotic stimuli. Science 301(5629), 94–96. Al-Fakhri, N., Chavakis, T., Schmidt-Woll, T., Huang, B., Cherian, S. M., Bobryshev, Y. V., Lord, R. S., Katz, N., and Preissner, K. T. (2003). Induction of apoptosis in vascular cells by plasminogen activator inhibitor-1 and high molecular weight kininogen correlates with their anti-adhesive properties. Biol. Chem. 384(3), 423–435.

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Xu, J., Rodriguez, D., Petitclerc, E., Kim, J. J., Hangai, M., Yuen, S. M., Davis, G. E., and Brooks, P. C. (2001). Proteolytic exposure of a cryptic site within collagen type IV is required for angiogenesis and tumor growth in vivo. J. Cell Biol. 154(5), 1069–1079. Yang, J. T., Rayburn, H., and Hynes, R. O. (1993). Embryonic mesodermal defects in alpha 5 integrin-deficient mice. Development 119, 1093–1105. Yebra, M., Parry, G. C., Stromblad, S., Mackman, N., Rosenberg, S., Mueller, B. M., and Cheresh, D. A. (1996). Requirement of receptor-bound urokinase-type plasminogen activator for integrin alphavbeta5-directed cell migration. J. Biol. Chem. 271(46), 29393–29399. Zahir, N., Lakins, J. N., Russell, A., Ming, W., Chatterjee, C., Rozenberg, G. I., Marinkovich, M. P., and Weaver, V. M. (2003). Autocrine laminin-5 ligates alpha6beta4 integrin and activates RAC and NFkappaB to mediate anchorage-independent survival of mammary tumors. J. Cell. Biol. 163, 1397–1407. Zhong, J., Eliceiri, B., Stupack, D., Penta, K., Sakamoto, G., Quertermous, T., Coleman, M., Boudreau, N., and Varner, J. A. (2003). Neovascularization of ischemic tissues by gene delivery of the extracellular matrix protein Del-1. J. Clin. Invest. 112(1), 30–41. Zhu, W. H., MacIntyre, A., and Nicosia, R. F. (2002). Regulation of angiogenesis by vascular endothelial growth factor and angiopoietin-1 in the rat aorta model: Distinct temporal patterns of intracellular signaling correlate with induction of angiogenic sprouting. Am. J. Pathol. 161(3), 823–830. Zhu, X., and Assoian, R. K. (1995). Integrin-dependent activation of MAP kinase: A link to shape-dependent cell proliferation. Mol. Biol. Cell 6(3), 273–282.

Index A Acetylated low-density lipoprotein (acLDL) bone marrow-derived hemangioblasts with, 150, 158–161 acLDL. See Acetylated low-density lipoprotein AEC. See Alveolar epithelial cell Alveolar epithelial cell (AEC) lung morphogenesis with, 5, 8 Alveolar epithelium lung morphogenesis with, 6, 8 Angioblasts adult bone marrow with, 148–152 Angiogenesis, 148 integrins with, 207–230 altered endothelial function of, 210–214 angiogenesis initiation of, 214–218 antagonist effects in, 227–228 v3 as marker in, 225–227 v5 role with, 221–222 conclusion to, 228–230 ECM blocked integrin function in, 223–225 ECM fragments in, 223–225 ECM interactions with, 218–227 ECM modified by proteolysis in, 219–221 endothelial cell produced ligands of, 222 endothelial microenvironment alterations of, 226 endothelial survival in, 222–225 ERK for, 214–218 expression alteration in, 222–225 fibrin deposition in, 221–222 future direction to, 229–230 growth factor receptor/integrin cross-talk in, 215–216 JNK for, 214, 217–218 knockout animal phenotype in, 227–228 MAP kinases for, 214–218 MMP in, 218–220, 225 new ligand deposition into ECM with, 221–222

other protease in, 220 p38 for, 214, 217–218 perspective on, 228–229 protease activity byproducts in, 220–221 src role with, 221–222 vascular permeability with, 221–222 tissue engineering with, 193–195 Angiopoietins arterio-venous identity differentiation with, 89 genetic factors in lung development with, 84–85, 89 ASMA. See -smooth muscle actin B b-FGF. See Basic fibroblast growth factor Basic fibroblast growth factor (b-FGF) integrins with, 208, 212, 214–217, 223, 228 tissue engineering with, 193–194 BMP. See Bone morphogenetic protein Bone marrow-derived hemangioblasts adult angioblasts with, 148–152 adults with, 152–153 CD34 with, 147–150, 154, 155, 156–161, 163–165 ECP with, 156–160 embryo with, 145–147 eNOS with, 150 FGF with, 150 future of, 167–168 G-CSF with, 161, 164, 167 GM-CSF with, 161–162 human trials for, 165–167 IGF with, 150 introduction to, 141–142 ischemic tissue improvements with, 163–165 mobilization with, 160–163 PBMC with, 149–151, 158, 160, 161 physiological significance of, 154–156 progenitor cells with, 143–145 simvastatin with, 156, 161–162

239

240 Bone marrow-derived hemangioblasts (cont.) stem cells with, 143–145, 146 transplantation with, 143, 144, 153–154 VEGF with, 145, 147, 149–150, 156, 157, 159–163 Bone morphogenetic protein (BMP) glandular stem cells with, 48 lung development with, 58 lung morphogenesis with, 4 tissue engineering with, 194, 198–199 Bone regeneration tissue engineering with, 196–199 osteoconduction in, 197–198 osteoinduction in, 198–199 BPD. See Bronchopulmonary dysplasia Bronchopulmonary dysplasia (BPD) lung morphogenesis with, 5 C C. elegans. See Caenorhabditis elegans Caenorhabditis elegans lung development in, 58 stem cell biology in, 2 Canine model lung compensatory growth in, 17–29 alveolar growth in, 20–23 alveolar septal component growth rate in, 24–25 application to human lung growth of, 29 complexity in, 19–20, 21 dysanaptic growth impairing functional compensation in, 25–26 dysanaptic lung growth with, 23–26 EGF in, 20 epiphyseal closure in, 19 parenchyma in, 23–24 PCNA in, 20 physiological reserves in, 19–20 post PNX lung growth in, 17, 19–22, 23, 24–26, 27, 28, 29 regenerative potential in, 19–20 signals in, 26–29 SP-A in, 21 structure/function considerations in, 18–23 CD34 bone marrow-derived hemangioblasts with, 149–152, 153, 154, 156–161, 163–165 CFTR. See Cystic fibrosis transmembrane conductance regulator

Index Cystic fibrosis transmembrane conductance regulator (CFTR) airway submucosal glands and, 35 D D. melanogaster. See Drosophila melanogaster DEC. See Duct-like epithelial cyst Dogs. See Canine model Drosophila melanogaster lung development in, 58 Duct-like epithelial cyst (DEC) pancreas with, 129 E ECM. See Extracellular matrices EGF. See Epidermal growth factor EMAP II. See Endothelial monocyte activating polypeptide II Endothelial cell progenitors (EPC) adult angioblasts with, 148–152 adults hemangioblasts with, 152–153 embryonic hemangioblasts with, 145–147 future of, 167–168 human trials for, 165–167 introduction to, 141–142 ischemic tissue improvements with, 163–165 marrow derived hemangioblasts’ significance with, 153–156 mobilization with, 160–163 origins of, 156–160 progenitor cells with, 143–145 stem cells with, 143–145, 146 Endothelial monocyte activating polypeptide II (EMAP II) genetic factors in lung development with, 80 Endothelial nitric oxide synthase (eNOS) bone marrow-derived hemangioblasts with, 150 eNOS. See Endothelial nitric oxide synthase EPC. See Endothelial cell progenitors Eph receptor arterio-venous identity specificity with, 88–89 genetic factors in lung development with, 83, 88–89 Ephrins arterio-venous identity specificity with, 88–89

241

Index genetic factors in lung development with, 83, 88–89 Epidermal growth factor (EGF) lung compensatory growth with, 20 lung morphogenesis with, 4 Epidermal proliferative unit (EPU) epidermis cell regulation with, 112 Epidermis progenitor cells with, 105–116 cell compartment regulation in, 109–115 defined culture systems in, 107–108 EPU in, 112 FACS in, 106 healing failure in, 115–116 IHH in, 112 keratinocyte population properties in, 105–108 population modulation in, 108–109 SHH in, 112 TA cell modulation in, 108–109 EPU. See Epidermal proliferative unit ERK. See Extracellular signal-regulated kinases Extracellular matrices (ECM) endothelial cell integrins with, 208–214 angiogenesis related alterations in, 210–214 structure of, 209–210 fragments of, 223–225 angiogenesis suppressed by, 224–225 integrin function blocked by, 223–224 integrin interactions during angiogenesis of, 218–227 v3 as marker in, 225–227 v5 role with, 221–222 conclusions to, 230 endothelial microenvironment alterations of, 226 endothelial survival in, 222–225 fibrin deposition in, 221–222 MMP with, 219–220 other proteases with, 220 plasminogen with, 219 proteases activity byproducts with, 220–221 proteolysis influencing, 219–221 src role with, 221–222 UPA with, 219 vascular permeability with, 221–222 lung vasculature formation with, 77–78 tissue engineering with, 181–200

b-FGF in, 193–194 blood vessel formation in, 191–192 BMP in, 194, 198–199 bone regeneration in, 196–199 cell adhesion peptides in, 186 cell transplantation for bone defect in, 199 cell transplantation strategy in, 183–184, 187–191 conclusion of, 199–200 conductive strategy in, 183 future direction in, 199–200 growth factors in, 184 inductive strategy in, 183–184, 187–191 introduction to, 182–187 large blood vessel in, 195–196 mimicking vasculogenesis in, 192–193 osteoconduction in, 197–198 osteoinduction in, 198–199 PDGF in, 184, 193–195 schematic representation in, 183 synthetic mimic design in, 185–187 TGF- in, 184, 198 therapeutic angiogenesis in, 193–195 three strategies in, 183–184 vascular structures in, 191–196 VEGF in, 184, 192, 193–195, 198 Extracellular signal-regulated kinases (ERK) angiogenesis initiation with, 214–218 lung morphogenesis with, 4 F FACS. See Fluorescence-activated cell sorting FAK. See Focal adhesion kinase Ferret surface airway epithelial cells in, 37–38, 39, 40 Fetoprotein transcription factor (FTF) hepatocyte phenotype regulation with, 118 FGF. See Fibroblast growth factor FGFR. See Fibroblast growth factor receptor Fibroblast growth factor (FGF) bone marrow-derived hemangioblasts -with, 150 hepatocyte phenotype regulation with, 116 lung development with, 57–58, 62–63 Fibroblast growth factor receptor (FGFR) lung morphogenesis with, 3 Fluorescence-activated cell sorting (FACS) epidermis with, 106

242 Focal adhesion kinase (FAK) cytosolic nonreceptor kinases with, 209, 213–214, 216, 217 Forkhead box (Fox) genetic factors in lung development with, 81 Fox. See Forkhead box FTF. See Fetoprotein transcription factor G G-CSF. See Granulocyte colony-stimulating factor GAPDH. See Glyceraldehyde-3-phosphate dehydrogenase Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) lung development in, 59–60 GM-CSF. See Granulocyte-macrophage colony-stimulating factor Granulocyte colony-stimulating factor (GCSF) bone marrow-derived hemangioblasts with, 161, 164, 167 Granulocyte-macrophage colony-stimulating factor (GM-CSF) bone marrow-derived hemangioblasts with, 161–162 H Hamster surface airway epithelial cells in, 38, 39 Hemangioblasts. See Bone marrow-derived hemangioblasts Hepatocyte growth factor (HGF) lung morphogenesis with, 4 Hepatocyte nuclear factors (HNF) hepatocyte phenotype regulation with, 117, 118–119, 123 Human surface airway epithelial cells in, 37–41 I Indian hedgehog (IHH) epidermis cell regulation with, 112 INGAP. See Islet neogenesis-associated protein Insulin-like growth factors (IGF) bone marrow-derived hemangioblasts with, 150

Index genetic factors in lung development with, 81–82 Integrins angiogenesis with, 207–230 alterations from, 210–214 altered endothelial integrin function during, 213–214 altered pre-existing integrin expression in, 212–213 v3 as marker in, 225–227 v5 role during, 221–222 conclusion to, 228–230 ECM blocked integrin function in, 223–225 ECM fragments in, 223–225 ECM modified by proteolysis during, 219–221 endothelial cell produced ligands during, 222 endothelial microenvironment alterations during, 226 endothelial survival in, 222–225 ERK for, 214–218 fibrin deposition during, 221–222 future direction to, 229–230 growth factor receptor/integrin cross-talk in, 215–216 initiation of, 214–218 integrin antagonist effects in, 227–228 integrin-ECM interactions altered during, 218–227 integrin expression alteration in, 222–225 JNK for, 214, 217–218 knockout animal phenotype in, 227–228 MAP kinases for, 214–218 MMP in, 218–220, 225 new integrin expression in, 211, 212 new ligand deposition into ECM during, 221–222 other protease in, 220 p38 for, 214, 217–218 perspective on, 228–229 protease activity byproducts in, 220–221 src role during, 221–222 vascular permeability during, 221–222 bFGF with, 208, 212, 214–217, 223, 228 endothelial cells as, 209–214 alterations in, 210–214 cytosolic nonreceptor kinases with, 209 structure of, 209–210 24 heterodimers of, 210

Index FAK with, 209, 213–214, 216, 217 introduction to, 208–209 PDK with, 209 VEGF with, 208, 214–218, 221, 222, 226, 228 Ischemic tissue bone marrow induced improvements in, 163–165 Islet neogenesis-associated protein (INGAP) pancreas regeneration with, 126, 129 J JNK angiogenesis initiation with, 214, 217–218 L Label-retaining cell (LRC) glandular stem cells with, 46, 47 Latent TGF- binding protein (LTBP) lung morphogenesis with, 5 Lef-1. See Lymphoid enhancing factor-1 Liver progenitor cells in, 116–124 cell population with, 116–117 disease with, 122–123 FTF with, 118 hepatocyte phenotype development/ regulation and, 117–119 HNF with, 117, 118–119, 124 injury models with, 122–123 pancreas turning into liver with, 121 progenitor pool activation with, 122–123 repopulation studies with, 120–121 tissue architecture with, 118, 119–120 in vitro hepatocyte handling with, 119, 123–124 LRC. See Label-retaining cell LTBP. See Latent TGF- binding protein Lung cancer gene expression studies with, 65–66 Lung compensatory growth canine model for, 17–29 alveolar growth in, 20–23 alveolar septal component growth rate in, 24–25 application to human lung growth of, 29 complexity in, 19–20, 21 dysanaptic growth impairing functional compensation in, 25–26

243 dysanaptic lung growth with, 23–26 EGF in, 20 epiphyseal closure in, 19 parenchyma in, 23–24 PCNA in, 20 physiological reserves in, 19–20 post PNX lung growth in, 17, 19–22, 23, 24–26, 27, 28, 29 regenerative potential in, 19–20 signals in, 26–29 SP-A in, 21 structure/function considerations in, 18–23 large/small animal models of, 18 rodent with, 19 Lung development gene expression studies in, 57–67 adult disease with, 65 ASMA with, 60–61, 64 BMP with, 58 conclusion on, 66–67 developmental transcription modules with, 65 FGF with, 57–58, 62–63 GAPDH with, 59–60 introduction to, 57–59 lung alveogenesis model with, 62–63 lung cancer with, 65–66 new concepts with, 61 old concepts with, 59–61 PCR with, 60 PDGF with, 58, 62 SHH with, 57 stem cells with, 63–65 VEGF with, 61 vasculature in, 73–92 angiopoietins with, 84–85, 89 arterio-venous identity specification with, 86–90 conclusion to, 91–92 EMAP II with, 79 endothelial stem cells with, 90–91 environmental regulation of arteriovenous fate with, 89–90 Eph receptor with, 83, 88–89 ephrins with, 83, 88–89 epithelial-mesenchymal interactions during, 85–86 Forkhead box proteins with, 81 gas exchange capillary network with, 76 genetic factors with, 77–85

244 Lung development (cont.) IGF with, 81–82 introduction to, 73–74 neuropilins with, 84 Notch signaling with, 82, 87–88 pulmonary vessel formation overview with, 74–77 regeneration/repair with, 90–91 regulation of, 77–85 Roundabouts with, 83 RT-PCR with, 85 semaphorins with, 84 SHH with, 87–88 Slit proteins with, 83 TGF- with, 80 Tie receptors with, 84–85, 89 VEGF with, 77–81, 84–89 Wnt signaling pathway with, 80–81 Lung morphogenesis AEC with, 5, 8 alveolar epithelium with, 6, 8 BMP4 with, 4 BPD with, 5 EGF with, 4 ERK with, 4 FGFR with, 3 HGF with, 4 LTBP with, 5 morphogen signaling pathways with, 6 PDGF with, 4 PNE with, 4 stem cells in, 1–11 background on, 3–7 conclusions on, 10–11 introduction to, 1–3 summary of, 10–11 symposium presentations on, 7–10 TGF- with, 4, 5 VEGF with, 5 Lymphoid enhancing factor-1 (Lef-1) glandular stem cells with, 48–52 M MAPK. See Mitogen-activated protein kinases Matrix metalloproteinase (MMP) integrins with, 218–220, 225 Mitogen-activated protein kinases (MAPK) angiogenesis initiation with, 214–218 ERK activation in, 216–217

Index MMP. See Matrix metalloproteinase Mouse Lef-1 expression with, 48 SHH with, 48 submucosal gland hyperplasia in, 35 surface airway epithelial cells in, 34, 37–38 tracheal injury model with, 45–46, 47 N National Heart, Lung and Blood Institute (NHLBI), 1 Neuropilins genetic factors in lung development with, 84 Notch signaling arterio-venous cell fate with, 87–88 genetic factors in lung development with, 82, 87–88 O Osteoconduction tissue engineering with, 197–198 Osteoinduction tissue engineering with, 198–199 P P38 angiogenesis initiation with, 214, 217–218 P21-dependent kinases (PDK) cytosolic nonreceptor kinases with, 209 Pancreas progenitor cells in, 125–130 DEC with, 129 developmental islet neogenesis with, 117, 128–130 INGAP with, 126, 129 neogenesis with, 125–126 non-neogenesis regeneration with, 126–127 pancreas turning into liver with, 121 progenitor pool stimulation with, 127–128 STZ with, 126–127, 129 Parenchyma progenitor cells with, 102–103 cell interaction and, 103–105 PBMC. See Peripheral blood mononuclear cells PCNA. See Proliferating cell nuclear antigen

245

Index PCR. See Polymerase chain reaction PDGF. See Platelet-derived growth factor PDK. See P21-dependent kinases Peripheral blood mononuclear cells (PBMC) bone marrow-derived hemangioblasts with, 149–151, 158, 160, 161 Platelet-derived growth factor (PDGF) lung development with, 58, 62 lung morphogenesis with, 4 tissue engineering with, 184, 193–195 PNE cell. See Pulmonary neuroendocrine cell Pneumonectomy (PNX) lung compensatory growth after, 17, 19–22, 23, 24–26, 27, 28, 29 Polymerase chain reaction (PCR) lung development with, 60 Progenitor cells bone marrow-derived hemangioblasts with, 143–145, 146, 149, 151, 156–158, 162–165, 168 epidermis with, 105–115 cell compartment regulation in, 109–114 defined culture systems in, 107–108 EPU in, 112 FACS in, 106 healing failure in, 114–115 IHH in, 112 keratinocyte population properties in, 105–108 population modulation in, 108–109 SHH in, 112 TA cell modulation in, 108–109 liver with, 116–124 cell population of, 116–117 disease in, 122–123 FTF in, 118 hepatocyte phenotype development/ regulation and, 117–119 HNF in, 117, 118–119, 124 injury models of, 122–123 pancreas turning into, 121 progenitor pool activation in, 122–123 repopulation studies of, 120–121 tissue architecture in, 118, 119–120 in vitro hepatocyte handling and, 118, 124–125 lung morphogenesis with, 1–11 background on, 3–7 conclusions on, 10–11 introduction to, 1–3

summary of, 10–11 symposium presentations on, 7–10 pancreas with, 125–130 DEC in, 129 developmental islet neogenesis in, 117, 128–130 INGAP in, 126, 129 neogenesis in, 125–126 non-neogenesis regeneration in, 126–127 progenitor pool stimulation in, 127–128 STZ in, 126–127, 129 parenchyma cell interaction with, 103–105 parenchyma with, 102–103 tissue engineering with, 101–131 conclusion/future directions of, 130–131 working hypothesis of, 103–105 Proliferating cell nuclear antigen (PCNA) lung compensatory growth with, 20 Pulmonary neuroendocrine cell (PNE cell) lung morphogenesis with, 4 R Rat surface airway epithelial cells in, 37–38, 39 Reverse transcription polymerase chain reaction (RT-PCR) genetic factors in lung development with, 84–85 Roundabouts (Robo) genetic factors in lung development with, 83 RT-PCR. See Reverse transcription polymerase chain reaction S SCID. See Severe combined immunodeficiency SDB. See Society for Developmental Biology Semaphorins genetic factors in lung development with, 84 Severe combined immunodeficiency (SCID) mice airway submucosal glands with, 41 SHH. See Sonic hedgehog Simvastatin bone marrow-derived hemangioblasts with, 156, 161–162 Slit proteins genetic factors in lung development with, 83 -smooth muscle actin (ASMA) lung development with, 60–61, 64

246 Society for Developmental Biology (SDB), 1–2 Sonic hedgehog (SHH) arterio-venous identity specificity with, 87–88 epidermis cell regulation with, 112 glandular stem cells with, 48 lung development with, 57 lung morphogenesis with, 5 SP-A. See Surfactant protein-A Stem cells airway glandular development with, 33–52 BMP with, 48 concluding remarks on, 51–52 developmental molecular markers with, 46–51 future challenges with, 51–52 human adult proximal airway with, 41–45 Lef-1 with, 48–52 LRC with, 46, 47 mouse tracheal injury model with, 45–46 SCID mice with, 41 SHH with, 48 species-specific epithelial domains with, 36, 37–41 bone marrow-derived hemangioblasts with, 142–145, 146, 147, 149–152, 155, 156, 160, 164 epidermis with, 105–109, 111–113 lung development with, 63–65 lung morphogenesis with, 1–11 background on, 3–7 conclusions on, 10–11 introduction to, 1–3 summary of, 10–11 symposium presentations on, 7–10 lung vasculature development with, 90–91 pancreas with, 127 parenchyma with, 103–105 tissue engineering with, 103–110, 111–113, 121, 122, 126 Streptozotocin (STZ) pancreas regeneration with, 126–127, 129 STZ. See Streptozotocin Submucosal glands (airway) CFTR and, 35 function of, 35–37 human diseases related to, 35–36 introduction to, 34 species-specific differences with, 36, 37–41

Index stem cells and, 33–52 BMP with, 48 concluding remarks on, 51–52 developmental molecular markers with, 46–51 future challenges with, 51–52 human adult proximal airway with, 41–45 Lef-1 with, 48–52 LRC with, 46, 47 mouse tracheal injury model with, 45–46 SCID mice with, 41 SHH with, 48 species-specific epithelial domains with, 36, 37–41 structure of, 35–37 Surfactant protein-A (SP-A) lung compensatory growth with, 20 T TA cells. See Transit amplifying cells TGF-. See Transforming growth factor-beta Tie receptors arterio-venous identity differentiation with, 89 genetic factors in lung development with, 84–85, 89 Tissue engineering cell transplantation strategy for, 183–184, 187, 190–191 conclusion to, 130–131 epidermis in, 105–115 cell compartment regulation in, 109–114 defined culture systems in, 107–108 EPU in, 112 FACS in, 106 healing failure in, 114–115 IHH in, 112 keratinocyte population properties in, 105–108 population modulation in, 108–109 SHH in, 112 TA cell modulation in, 108–109 future directions of, 130–131 inductive strategy for, 183–184, 187–190 local gene therapy in, 189–190 polymeric delivery vehicles with, 188 protein delivery in, 188–189 liver in, 116–124 cell population of, 116–117

247

Index disease in, 122–123 FTF in, 118 hepatocyte phenotype development/ regulation and, 115–119 HNF in, 117, 118–119, 124 injury models of, 122–123 pancreas turning into, 121 progenitor pool activation in, 122–123 repopulation studies of, 120–121 tissue architecture in, 118, 119–120 in vitro hepatocyte handling and, 118, 124–125 pancreas with, 125–130 DEC in, 129 developmental islet neogenesis in, 117, 128–130 INGAP in, 126, 129 neogenesis in, 125–126 non-neogenesis regeneration in, 126–127 progenitor pool stimulation in, 127–128 STZ in, 126–127, 129 parenchyma cell interaction with, 102–105 progenitor cells in, 101–131 synthetic extracellular matrices for, 181–200 b-FGF with, 193–194 blood vessel formation and, 191–192 BMP with, 194, 198–199 bone regeneration with, 196–199 cell adhesion peptides with, 186 cell transplantation for bone defect with, 199 cell transplantation strategy with, 183–184, 187–191 conclusion of, 199–200 conductive strategy with, 183 engineering vascular structures with, 191–196 future direction in, 199–200 growth factors with, 184 inductive strategy with, 183–184, 187–191 introduction to, 182–187 large blood vessel engineering with, 195–196

mimicking vasculogenesis with, 192–193 osteoconduction with, 197–198 osteoinduction with, 198–199 PDGF with, 184, 193–195 schematic representation with, 183 synthetic mimic design with, 185–187 TGF- with, 184, 198 therapeutic angiogenesis with, 193–195 three strategies with, 183–184 VEGF with, 184, 192, 193–195, 198 working hypothesis of, 103–105 Transforming growth factor-beta (TGF-) genetic factors in lung development with, 80 lung morphogenesis with, 4, 5 tissue engineering with, 184, 198 Transit amplifying cells (TA cells) epidermis with, 107–109, 110 progenitor cell tissue engineering with, 105, 107–109, 110, 112–114, 123 V Vascular endothelial growth factor (VEGF) arterio-venous identity specificity with, 87–88 bone marrow-derived hemangioblasts with, 145, 147, 149–150, 156, 157, 159–163 genetic factors in lung development with, 77–81 integrins with, 208, 214–218, 221, 222, 226, 228 lung development with, 61 lung morphogenesis with, 5 tissue engineering with, 184, 192, 193–195, 198 Vasculogenesis, 148 tissue engineering mimicking of, 192–193 VEGF. See Vascular endothelial growth factor W Wnt signaling pathway genetic factors in lung development with, 80–81

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Contents of Previous Volumes Volume 47 1 Early Events of Somitogenesis in Higher Vertebrates: Allocation of Precursor Cells during Gastrulation and the Organization of a Moristic Pattern in the Paraxial Mesoderm Patrick P. L. Tam, Devorah Goldman, Anne Camus, and Gary C. Shoenwolf

2 Retrospective Tracing of the Developmental Lineage of the Mouse Myotome Sophie Eloy-Trinquet, Luc Mathis, and Jean-Franc¸ois Nicolas

3 Segmentation of the Paraxial Mesoderm and Vertebrate Somitogenesis Olivier Pourqule´

4 Segmentation: A View from the Border Claudio D. Stern and Daniel Vasiliauskas

5 Genetic Regulation of Somite Formation Alan Rawls, Jeanne Wilson-Rawls, and Eric N. Olsen

6 Hox Genes and the Global Patterning of the Somitic Mesoderm Ann Campbell Burke

7 The Origin and Morphogenesis of Amphibian Somites Ray Keller

8 Somitogenesis in Zebrafish Scott A. Halley and Christiana Nu¨sslain-Volhard

9 Rostrocaudal Differences within the Somites Confer Segmental Pattern to Trunk Neural Crest Migration Marianne Bronner-Fraser

249

250

Contents of Previous Volumes

Volume 48 1 Evolution and Development of Distinct Cell Lineages Derived from Somites Beate Brand-Saberi and Bodo Christ

2 Duality of Molecular Signaling Involved in Vertebral Chondrogenesis Anne-He´le`ne Monsoro-Burq and Nicole Le Douarin

3 Sclerotome Induction and Differentiation Jennifer L. Docker

4 Genetics of Muscle Determination and Development Hans-Henning Arnold and Thomas Braun

5 Multiple Tissue Interactions and Signal Transduction Pathways Control Somite Myogenesis Anne-Gae¨lle Borycki and Charles P. Emerson, Jr.

6 The Birth of Muscle Progenitor Cells in the Mouse: Spatiotemporal Considerations Shahragim Tajbakhsh and Margaret Buckingham

7 Mouse–Chick Chimera: An Experimental System for Study of Somite Development Josiane Fontaine-Pe´rus

8 Transcriptional Regulation during Somitogenesis Dennis Summerbell and Peter W. J. Rigby

9 Determination and Morphogenesis in Myogenic Progenitor Cells: An Experimental Embryological Approach Charles P. Ordahl, Brian A. Williams, and Wilfred Denetclaw

Volume 49 1 The Centrosome and Parthenogenesis Thomas Ku¨ntziger and Michel Bornens

2 g-Tubulin Berl R. Oakley

Contents of Previous Volumes

251

3 g-Tubulin Complexes and Their Role in Microtubule Nucleation Ruwanthi N. Gunawardane, Sofia B. Lizarraga, Christiane Wiese, Andrew Wilde, and Yixian Zheng

4 g-Tubulin of Budding Yeast Jackie Vogel and Michael Snyder

5 The Spindle Pole Body of Saccharomyces cerevisiae: Architecture and Assembly of the Core Components Susan E. Francis and Trisha N. Davis

6 The Microtubule Organizing Centers of Schizosaccharomyces pombe Iain M. Hagan and Janni Petersen

7 Comparative Structural, Molecular, and Functional Aspects of the Dictyostelium discoideum Centrosome Ralph Gra¨f, Nicole Brusis, Christine Daunderer, Ursula Euteneuer, Andrea Hestermann, Manfred Schliwa, and Masahiro Ueda

8 Are There Nucleic Acids in the Centrosome? Wallace F. Marshall and Joel L. Rosenbaum

9 Basal Bodies and Centrioles: Their Function and Structure Andrea M. Preble, Thomas M. Giddings, Jr., and Susan K. Dutcher

10 Centriole Duplication and Maturation in Animal Cells B. M. H. Lange, A. J. Faragher, P. March, and K. Gull

11 Centrosome Replication in Somatic Cells: The Significance of the G1 Phase Ron Balczon

12 The Coordination of Centrosome Reproduction with Nuclear Events during the Cell Cycle Greenfield Sluder and Edward H. Hinchcliffe

13 Regulating Centrosomes by Protein Phosphorylation Andrew M. Fry, Thibault Mayor, and Erich A. Nigg

14 The Role of the Centrosome in the Development of Malignant Tumors Wilma L. Lingle and Jeffrey L. Salisbury

15 The Centrosome-Associated Aurora/IpI-like Kinase Family T. M. Goepfert and B. R. Brinkley

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Contents of Previous Volumes

16 Centrosome Reduction during Mammalian Spermiogenesis G. Manandhar, C. Simerly, and G. Schatten

17 The Centrosome of the Early C. elegans Embryo: Inheritance, Assembly, Replication, and Developmental Roles Kevin F. O’Connell

18 The Centrosome in Drosophila Oocyte Development Timothy L. Megraw and Thomas C. Kaufman

19 The Centrosome in Early Drosophila Embryogenesis W. F. Rothwell and W. Sullivan

20 Centrosome Maturation Robert E. Palazzo, Jacalyn M. Vogel, Bradley J. Schnackenberg, Dawn R. Hull, and Xingyong Wu

Volume 50 1 Patterning the Early Sea Urchin Embryo Charles A. Ettensohn and Hyla C. Sweet

2 Turning Mesoderm into Blood: The Formation of Hematopoietic Stem Cells during Embryogenesis Alan J. Davidson and Leonard I. Zon

3 Mechanisms of Plant Embryo Development Shunong Bai, Lingjing Chen, Mary Alice Yund, and Zinmay Rence Sung

4 Sperm-Mediated Gene Transfer Anthony W. S. Chan, C. Marc Luetjens, and Gerald P. Schatten

5 Gonocyte–Sertoli Cell Interactions during Development of the Neonatal Rodent Testis Joanne M. Orth, William F. Jester, Ling-Hong Li, and Andrew L. Laslett

6 Attributes and Dynamics of the Endoplasmic Reticulum in Mammalian Eggs Douglas Kline

7 Germ Plasm and Molecular Determinants of Germ Cell Fate Douglas W. Houston and Mary Lou King

Contents of Previous Volumes

253

Volume 51 1 Patterning and Lineage Specification in the Amphibian Embryo Agnes P. Chan and Laurence D. Etkin

2 Transcriptional Programs Regulating Vascular Smooth Muscle Cell Development and Differentiation Michael S. Parmacek

3 Myofibroblasts: Molecular Crossdressers Gennyne A. Walker, Ivan A. Guerrero, and Leslie A. Leinwand

4 Checkpoint and DNA-Repair Proteins Are Associated with the Cores of Mammalian Meiotic Chromosomes Madalena Tarsounas and Peter B. Moens

5 Cytoskeletal and Ca2+ Regulation of Hyphal Tip Growth and Initiation Sara Torralba and I. Brent Heath

6 Pattern Formation during C. elegans Vulval Induction Minqin Wang and Paul W. Sternberg

7 A Molecular Clock Involved in Somite Segmentation Miguel Maroto and Olivier Pourquie´

Volume 52 1 Mechanism and Control of Meiotic Recombination Initiation Scott Keeney

2 Osmoregulation and Cell Volume Regulation in the Preimplantation Embryo Jay M. Baltz

3 Cell–Cell Interactions in Vascular Development Diane C. Darland and Patricia A. D’Amore

4 Genetic Regulation of Preimplantation Embryo Survival Carol M. Warner and Carol A. Brenner

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Contents of Previous Volumes

Volume 53 1 Developmental Roles and Clinical Significance of Hedgehog Signaling Andrew P. McMahon, Philip W. Ingham, and Clifford J. Tabin

2 Genomic Imprinting: Could the Chromatin Structure Be the Driving Force? Andras Paldi

3 Ontogeny of Hematopoiesis: Examining the Emergence of Hematopoietic Cells in the Vertebrate Embryo Jenna L. Galloway and Leonard I. Zon

4 Patterning the Sea Urchin Embryo: Gene Regulatory Networks, Signaling Pathways, and Cellular Interactions Lynne M. Angerer and Robert C. Angerer

Volume 54 1 Membrane Type-Matrix Metalloproteinases (MT-MMP) Stanley Zucker, Duanqing Pei, Jian Cao, and Carlos Lopez-Otin

2 Surface Association of Secreted Matrix Metalloproteinases Rafael Fridman

3 Biochemical Properties and Functions of Membrane-Anchored Metalloprotease-Disintegrin Proteins (ADAMs) J. David Becherer and Carl P. Blobel

4 Shedding of Plasma Membrane Proteins Joaquı´n Arribas and Anna Merlos-Sua´rez

5 Expression of Meprins in Health and Disease Lourdes P. Norman, Gail L. Matters, Jacqueline M. Crisman, and Judith S. Bond

6 Type II Transmembrane Serine Proteases Qingyu Wu

7 DPPIV, Seprase, and Related Serine Peptidases in Multiple Cellular Functions Wen-Tien Chen, Thomas Kelly, and Giulio Ghersi

Contents of Previous Volumes

255

8 The Secretases of Alzheimer’s Disease Michael S. Wolfe

9 Plasminogen Activation at the Cell Surface Vincent Ellis

10 Cell-Surface Cathepsin B: Understanding Its Functional Significance Dora Cavallo-Medved and Bonnie F. Sloane

11 Protease-Activated Receptors Wadie F. Bahou

12 Emmprin (CD147), a Cell Surface Regulator of Matrix Metalloproteinase Production and Function Bryan P. Toole

13 The Evolving Roles of Cell Surface Proteases in Health and Disease: Implications for Developmental, Adaptive, Inflammatory, and Neoplastic Processes Joseph A. Madri

14 Shed Membrane Vesicles and Clustering of Membrane-Bound Proteolytic Enzymes M. Letizia Vittorelli

Volume 55 1 The Dynamics of Chromosome Replication in Yeast Isabelle A. Lucas and M. K. Raghuraman

2 Micromechanical Studies of Mitotic Chromosomes M. G. Poirier and John F. Marko

3 Patterning of the Zebrafish Embryo by Nodal Signals Jennifer O. Liang and Amy L. Rubinstein

4 Folding Chromosomes in Bacteria: Examining the Role of Csp Proteins and Other Small Nucleic Acid-Binding Proteins Nancy Trun and Danielle Johnston

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Contents of Previous Volumes

Volume 56 1 Selfishness in Moderation: Evolutionary Success of the Yeast Plasmid Soundarapandian Velmurugan, Shwetal Mehta, and Makkuni Jayaram

2 Nongenomic Actions of Androgen in Sertoli Cells William H. Walker

3 Regulation of Chromatin Structure and Gene Activity by Poly(ADP-Ribose) Polymerases Alexei Tulin, Yurli Chinenov, and Allan Spradling

4 Centrosomes and Kinetochores, Who needs ‘Em? The Role of Noncentromeric Chromatin in Spindle Assembly Priya Prakash Budde and Rebecca Heald

5 Modeling Cardiogenesis: The Challenges and Promises of 3D Reconstruction Jeffrey O. Penetcost, Claudio Silva, Maurice Pesticelli, Jr., and Kent L. Thornburg

6 Plasmid and Chromosome Traffic Control: How ParA and ParB Drive Partition Jennifer A. Surtees and Barbara E. Funnell

Volume 57 1 Molecular Conservation and Novelties in Vertebrate Ear Development B. Fritzsch and K. W. Beisel

2 Use of Mouse Genetics for Studying Inner Ear Development Elizabeth Quint and Karen P. Steel

3 Formation of the Outer and Middle Ear, Molecular Mechanisms Moise´s Mallo

4 Molecular Basis of Inner Ear Induction Stephen T. Brown, Kareen Martin, and Andrew K. Groves

5 Molecular Basis of Otic Commitment and Morphogenesis: A Role for Homeodomain-Containing Transcription Factors and Signaling Molecules Eva Bober, Silke Rinkwitz, and Heike Herbrand

Contents of Previous Volumes

257

6 Growth Factors and Early Development of Otic Neurons: Interactions between Intrinsic and Extrinsic Signals Berta Alsina, Fernando Giraldez, and Isabel Varela-Nieto

7 Neurotrophic Factors during Inner Ear Development Ulla Pirvola and Jukka Ylikoski

8 FGF Signaling in Ear Development and Innervation Tracy J. Wright and Suzanne L. Mansour

9 The Roles of Retinoic Acid during Inner Ear Development Raymond Romand

10 Hair Cell Development in Higher Vertebrates Wei-Qiang Gao

11 Cell Adhesion Molecules during Inner Ear and Hair Cell Development, Including Notch and Its Ligands Matthew W. Kelley

12 Genes Controlling the Development of the Zebrafish Inner Ear and Hair Cells Bruce B. Riley

13 Functional Development of Hair Cells Ruth Anne Eatock and Karen M. Hurley

14 The Cell Cycle and the Development and Regeneration of Hair Cells Allen F. Ryan

Volume 58 1 A Role for Endogenous Electric Fields in Wound Healing Richard Nuccitelli

2 The Role of Mitotic Checkpoint in Maintaining Genomic Stability Song-Tao Liu, Jan M. van Deursen, and Tim J. Yen

3 The Regulation of Oocyte Maturation Ekaterina Voronina and Gary M. Wessel

4 Stem Cells: A Promising Source of Pancreatic Islets for Transplantation in Type 1 Diabetes Cale N. Street, Ray V. Rajotte, and Gregory S. Korbutt

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Contents of Previous Volumes

5 Differentiation Potential of Adipose Derived Adult Stem (ADAS) Cells Jeffrey M. Gimble and Farshid Guilak

Volume 59 1 The Balbiani Body and Germ Cell Determinants: 150 Years Later Malgorzata Kloc, Szczepan Bilinski, and Laurence D. Etkin

2 Fetal–Maternal Interactions: Prenatal Psychobiological Precursors to Adaptive Infant Development Matthew F. S. X. Novak

3 Paradoxical Role of Methyl-CpG-Binding Protein 2 in Rett Syndrome Janine M. LaSalle

4 Genetic Approaches to Analyzing Mitochondrial Outer Membrane Permeability Brett H. Graham and William J. Craigen

5 Mitochondrial Dynamics in Mammals Hsiuchen Chen and David C. Chan

6 Histone Modification in Corepressor Functions Judith K. Davie and Sharon Y. R. Dent

7 Death by Abl: A Matter of Location Jiangyu Zhu and Jean Y. J. Wang

Volume 60 1 Therapeutic Cloning and Tissue Engineering Chester J. Koh and Anthony Atala

2 a-Synuclein: Normal Function and Role in Neurodegenerative Diseases Erin H. Norris, Benoit I. Giasson, and Virginia M.-Y. Lee

3 Structure and Function of Eukaryotic DNA Methyltransferases Taiping Chen and En Li

4 Mechanical Signals as Regulators of Stem Cell Fate Bradley T. Estes, Jeffrey M. Gimble, and Farshid Guilak

Contents of Previous Volumes

259

5 Origins of Mammalian Hematopoiesis: In Vivo Paradigms and In Vitro Models M. William Lensch and George Q. Daley

6 Regulation of Gene Activity and Repression: A Consideration of Unifying Themes Anne C. Ferguson-Smith, Shau-Ping Lin, and Neil Youngson

7 Molecular Basis for the Chloride Channel Activity of Cystic Fibrosis Transmembrane Conductance Regulator and the Consequences of Disease-Causing Mutations Jackie F. Kidd, Ilana Kogan, and Christine E. Bear

Volume 61 1 Hepatic Oval Cells: Helping Redefine a Paradigm in Stem Cell Biology P. N. Newsome, M. A. Hussain, and N. D. Theise

2 Meiotic DNA Replication Randy Strich

3 Pollen Tube Guidance: The Role of Adhesion and Chemotropic Molecules Sunran Kim, Juan Dong, and Elizabeth M. Lord

4 The Biology and Diagnostic Applications of Fetal DNA and RNA in Maternal Plasma Rossa W. K. Chiu and Y. M. Dennis Lo

5 Advances in Tissue Engineering Shulamit Levenberg and Robert Langer

6 Directions in Cell Migration Along the Rostral Migratory Stream: The Pathway for Migration in the Brain Shin-ichi Murase and Alan F. Horwitz

7 Retinoids in Lung Development and Regeneration Malcolm Maden

8 Structural Organization and Functions of the Nucleus in Development, Aging, and Disease Leslie Mounkes and Colin L. Stewart

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Contents of Previous Volumes

Volume 62 1 Blood Vessel Signals During Development and Beyond Ondine Cleaver

2 HIFs, Hypoxia, and Vascular Development Kelly L. Covello and M. Celeste Simon

3 Blood Vessel Patterning at the Embryonic Midline Kelly A. Hogan and Victoria L. Bautch

4 Wiring the Vascular Circuitry: From Growth Factors to Guidance Cues Lisa D. Urness and Dean Y. Li

5 Vascular Endothelial Growth Factor and Its Receptors in Embryonic Zebrafish Blood Vessel Development Katsutoshi Goishi and Michael Klagsbrun

6 Vascular Extracellular Matrix and Aortic Development Cassandra M. Kelleher, Sean E. McLean, and Robert P. Mecham

7 Genetics in Zebrafish, Mice, and Humans to Dissect Congenital Heart Disease: Insights in the Role of VEGF Diether Lambrechts and Peter Carmeliet

8 Development of Coronary Vessels Mark W. Majesky

9 Identifying Early Vascular Genes Through Gene Trapping in Mouse Embryonic Stem Cells Frank Kuhnert and Heidi Stuhlmann

Volume 63 1 Early Events in the DNA Damage Response Irene Ward and Junjie Chen

2 Afrotherian Origins and Interrelationships: New Views and Future Prospects Terence J. Robinson and Erik R. Seiffert

3 The Role of Antisense Transcription in the Regulation of X-Inactivation Claire Rougeulle and Philip Avner

Contents of Previous Volumes

261

4 The Genetics of Hiding the Corpse: Engulfment and Degradation of Apoptotic Cells in C. elegans and D. melanogaster Zheng Zhou, Paolo M. Mangahas, and Xiaomeng Yu

5 Beginning and Ending an Actin Filament: Control at the Barbed End Sally H. Zigmond

6 Life Extension in the Dwarf Mouse Andrzej Bartke and Holly Brown-Borg

Current Topics in Developmental Biology, Volume 64 (Current Topics in Developmental Biology)

Current Topics Developmental Biology (Current Topics in Developmental Biology)

Current Topics Developmental Biology (Current Topics in Developmental Biology)